European Society of Mycobacteriology

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    European Society
   of Mycobacteriology

In co-organization with the Instituto Nacional de Saúde Dr. Ricardo Jorge
   •	 Welcome Message
   •	 Introduction to the European Society of Mycobacteriology
   •	 Congress Organization
   •	 Program at a glance
   •	 Programme of Guest Lectures, Oral Presentations and Symposia
   •	 Programme of Poster Presentations
   •	 Abstracts of Guest Lectures (GL)
   •	 Abstracts of Oral Presentations (OP)
   •	 Abstracts of Poster Presentations (PP)
   •	 Author Index

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal   1
    WElCOmE mESSagE

    Dear Colleagues,

        It is with great pleasure that we warmly welcome you to the 30th Annual Congress of the

    European Society of Mycobacteriology, held in the city of Porto in Portugal from July 5-8, 2009.

    The aim of ESM2009 is to promote the exchange of distinguished experts from all around

    the	world	who	will	have	the	opportunity	to	update	information	in	the	front-line	of	scientific	

    achievement,	share	experience	and	ideas,	and	actively	participate	in	contribution	to	the	field	of	


    We have worked hard to make your stay in Porto a pleasant, useful and memorable occasion.

    On behalf of the ESM and the Local Organisers,

    Suzana David

2                                                                                                        ESM 2009
Of myCObaCTEriOlOgy
30Th aNNual CONgrESS
July 5-8, 2009
pOrTO, pOrTugal
The	European	Society	of	Mycobacteriology	(ESM,,	founded	in	1980,	is	a	non-profit	
international	scientific	society	dealing	with	different	aspects	of	mycobacteriology	and	related	diseases.	It	is	considered	
one	of	the	most	active	international	scientific	societies	in	this	area,	being	committed	to:
- Encouraging the highest standards for research to facilitate the discovery of new knowledge;
- Coordinating and providing information and expertise to other organizations worldwide;
-	Disseminating	knowledge	on	all	aspects	of	mycobacteriology	and	related	diseases,	through	scientific
  meetings and publications,
- Encouraging and providing the highest standards of training to interested health care providers;
- Establishing, reviewing and revising guidelines;
- Promoting high quality and cost effective diagnostic procedures;
- Advising, cooperating and participating with government and non-government agencies in matters of
  common interest;
- Participating in activities whose aim is to prevent mycobacterial diseases worldwide.

The ESM meetings are held each year in a different country of Europe. The ESM2009 congress is hosted in the city of
Porto,	 in	 Portugal.	 International	 specialists	 will	 treat	 themes	 at	 the	 front-line	 of	 scientific	 achievement	 in	 the	 field	 of	
mycobacteriology	and	related	diseases.	As	in	previous	meetings,	the	scientific	program	of	the	conferences	covers	a	wide	
range of topics in both applied and fundamental research in areas of priority such as:
- Diagnostics of active and latent tuberculosis
- Diagnostics of atypical mycobacteria
- Molecular epidemiology of tuberculosis
- Antibiotic resistance, MDR, XDR
- Tuberculosis drug development
- Immunology of mycobacterial infections
- Molecular Biology of mycobacteria
- Taxonomy of the genus Mycobacterium
- Veterinarian and environmental Mycobacteriology
- Methods for diagnostics adapted to economically disfavoured settings
- Laboratory safety
- Short and long term programs and recommendations

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                                  3
    CONgrESS OrgaNizaTiON
    EurOpEaN SOCiETy Of myCObaCTEriOlOgy
    Chairman of the ESM 2009 congress   Suzana David (Lisbon, Portugal)

    Steering Committee
    General Secretary                   Enrico Tortoli (Firenze, Italy)
    President                           Todor	Kantardjiev	(Sofia,	Bulgaria)
    Treasurer                           Malcolm Yates (East Dulwich Grove, U.K.)
    Members                             Maria-Jesus Garcia (Madrid, Spain)
                                        Sven Hoffner (Solna, Sweden)
                                        Stefan Niemann (Borstel, Germany)
                                        Gabriela Pfyffer (Luzern, Switzerland)
                                        Nalin Rastogi (Guadeloupe, France)
                                        Veronique Vincent (Geneva, Switzerland)

    hONOrS COmmiTTEE ESm 2009

    Ana Jorge                           Minister of Health
    Mariano Gago                        Minister of Science, Technology and Higher
    Manuel Pizarro                      Secretary of State for Health
    Francisco Ramos                     Secretary of State for Health
    José Pereira Miguel                 President of the National Health Institute
                                        Dr. Ricardo Jorge
    Jorge Sampaio                       UN Secretary General’s Special Envoy to
                                        Stop TB
    Mario Raviglione                    Director of the Stop TB Department,World
                                        Health Organization
    Maria do Céu Machado                High Comissioner for Health
    Francisco George                    Director-General of Health
    Jorge Soares                        Director of the Health and Human de-
                                        velopment Service Calouste Gulbenkian
    Jorge Torgal                        Director do Institute of Hygiene and Tropical
    Rui Rio                             Mayor of the city of Porto
    Fernando Augusto Fiuza de Melo      Director of the Clemente Ferreira Institute,
                                        São Paulo, Brasil

4                                                                                       ESM 2009

                          European Society of mycobacteriology

                          instituto Nacional de Saúde Dr. ricardo Jorge, iNSa


                          foundation for Science and Technology (fCT)

                          fundação Calouste gulbenkian

                          luso-american foundation (flaD)

                          STOp-Tb Working group on New Diagnostics, point of
                          Care sub group

                          The Organization expresses its thanks and appreciation to all those who gener-
                          ously contributed to the success of the 30th Annual Congress of ESM.

                          haiN lifeSciences
                          Platinum Medal Sponsor

                          beckton Dickinson & Quilaban
                          Gold Medal Sponsor

                          Gold Medal Sponsor

                          microsens (logo)



European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal             5
    prOgram aT a glaNCE
    Sunday, July 5th   09h00            REGISTRATION
                       11h00 – 12h00    SYMPOSIUM SPONSORED BY HAIN LIFESCIENCE GMBH:
                                        RAPID MOLECULAR GENETIC DETECTION OF MDR- AND
                                        Chair: Michael Weizenegger
                                        Speakers: Doris Hillemann and Christopher M Gilpin
                       12h00 – 13h30    SYMPOSIUM SPONSORED BY STOP-TB WORKING
                                        GROUP ON NEW DIAGNOSTICS, POINT OF CARE SUB
                                        GROUP: A SYMPOSIUM ON POINT-OF-CARE TESTS FOR
                                        Speakers: Catharina Boehme, Carol Nawina Nyirenda, Rosanna
                                        Peeling, Gerd Michel, Ruth McNerney and Amy P Wong
                       13h30 – 15h30    Break for Lunch
                       15h30 – 16h30    SYMPOSIUM CO-SPONSORED BY BECTON, DICKINSON
                                        AND COMPANY AND QUILABAN - QUÍMICA
                                        AND TREATMENT OF TB IN THE ERA OF MDR-TB AND
                                        XDR-TB. DO WE USE THE RIGHT DRUGS AND TOOLS?
                                        Chair: Francoise Portaels and Salman Siddiqi
                                        Speakers: Stefan Winkler, Andre De Bock, and Virginia Crews
                       17h00 – 19h00    OPENING SESSION
                                        Welcome Address

                                        TB CONTROL PROGRAMS
                                        Chair: Jaime Nina and Cristina Furtado
                                        Guest Lecture-1: Miguel Villar
                                        Guest Lecture-2: Fernando Fiuza de Melo
                       19h15            Welcome Reception

    monday, July 6th   09h00 – 13h15    SCIENTIFIC SESSION ON MOLECULAR EPIDEMIOLOGY
                                        AND DRUG RESISTANCE SURVEILLANCE
                                        Chair: Stefan Niemann and Cristina Gutierez
                       9h00 – 9h45      Guest Lecture-3: Sebastien Gagneux
                       9h45 – 11h00     Oral Presentations
                       11h00 – 11h30    Coffee and tea
                                        Chair: Nalin Rastogi and Dr. Philip Supply
                       11h30 – 12h15    Guest Lecture-4: Dick van Soolingen
                       12h15 – 13h015   Oral Presentations
                       13h15 – 14h00    Lunch

6                                                                                             ESM 2009
                          14h00 – 15h00             POSTER SESSION
                          15h00 – 16h45             SCIENTIFIC SESSION ON NON TUBERCULOUS
                                                    Chair: Enrico Tortoli
                          15h00 – 15h45             Guest Lecture-5: Joseph O. Falkinham, III
                          15h45 – 16h45             Oral Presentations
                          16h45 – 17h15             Coffee and tea
                          17h15 – 19h15             SCIENTIFIC SESSION ON ISSUES IN THE MODERN TB
                                                    Chair: Gabriela Pfyffer and Thomas Shinnick
                          17h15 – 18h00             Guest Lecture-6 : Dr. Jean-Pierre Zellweger
                          18h00 – 18h30             Guest Lecture-7 : Lee W. Riley
                          18h30 – 19h15             Oral Presentations
                          19h30                     Visit and Dinner at the Port Wine Cellars

Tuesday, July 7th         09h00 – 12h30             SCIENTIFIC SESSION ON VACCIN DEVELOPMENT
                                                    AND PATHOGENESIS
                                                    Chair: David Minnikin
                          09h00 – 09h45             Guest Lecture-8: Carlos Martin
                          09h45 – 10h30             Guest Lecture-9 : Lee W. Riley
                          10h30 – 11h00             Coffee and tea
                          11h00 – 12h30             Oral Presentations
                          12h30 – 13h15             Lunch
                          13h15 – 14h15             BUSINESS MEETING
                          14h15 – 18h15             SCIENTIFIC SESSION ON LABORATORY STRENGTHENING
                                                    Chair: Dr. Sven Hoffner and Dr. Mark Perkins
                          14h15 – 15h00             Guest Lecture-10: Thomas Shinnick
                          15h00 – 15h45             Guest Lecture-11: Afrânio Kritski
                          15h45 – 16h30             Guest Lecture-12 : Moisés Palaci
                          16h30 – 17h00             Coffee and tea
                          17h00 – 17h45             Oral Presentations
                          17h45 – 18h15             BEST POSTER
                          18h30                     Cultural Evening Dinner

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal       7
    Wednes-         09h00 – 11h00    SCIENTIFIC SESSION ON DRUG DEVELOPMENT
    day, July 8th
                                     Chair: Dr. Nalin Rastogi
                    09h00-09h45      Guest Lecture-13: Clarice Queico Leite
                    09h45 – 10h30    Guest Lecture-14 : Moisés Palaci
                    10h30 – 11h00    Oral Presentations
                    11h00 – 11h30    Coffee and tea
                    11h30 – 12h20    SESSION ON PRACTICAL ASPECTS AND QUALITY
                                     ASSURANCE IN MOLECULAR EPIDEMIOLOGY
                                     Chair: Dr. Elvira Richter and Prof. Christophe Sola
                    11h30 – 12h00    Guest Lecture-15 : Philip Supply
                    12h00 – 12h20    Oral Presentation
                    12h20 – 12h30    Discussion
                    12h30 – 13h30    SYMPOSIUM SPONSORED BY INSTAND, SOCIETY FOR
                                     RESEARCH PROMOTION OF QUALITY ASSURANCE
                                     IN MEDICAL LABORATORIES, WHO COLLABORATING
                                     CENTRE, DUESSELDORF, GERMANY: EXTERNAL QUALITY
                                     Speakers: Elvira Richter, Girts Skenders, Akos Somoskövy
                    13h:30 – 13h45   Closing Remarks
                    13h45 – 14h15    CLOSING OF CONGRESS
                    14h15            Conference Closes

8                                                                                          ESM 2009
prOgrammE Of guEST lECTurES,
Oral prESENTaTiONS aND SympOSia
 Sunday, July 5th

 9h00 – 11h00                 REGISTRATION
                              Chair: Dr. Michael Weizenegger
                              Dr. Doris Hillemann, National Reference Center for Mycobacteria (Borstel, Germany)
                              rapid detection of XDr-Tb with the genotype mTbDrsl assay
                              Dr. Christopher M Gilpin, PhD MPH, International Organization for Migration (Geneva,
                              implementation of new diagnostic tools and algorithms for enhanced
                              case detection of drug resistant forms of tuberculosis
                              DIAGNOSTICS, POINT OF CARE SUB GROUP
                              Dr. Catharina Boehme, M.D., Foundation for Innovative Diagnostics (FIND) (Geneva,
                              What is a pOC test?
                              Carol Nawina Nyirenda (Zambia)
                              Why do we need rapid tests: a patient’s perspective?
                              Prof. Rosanna Peeling, Ph.D., London School of Hygiene & Tropical Medicine
                              (London, UK)
                              rapid tests for Tb: what is wrong with them?
                              Dr. Gerd Michel, Ph.D., Foundation for Innovative Diagnostics (FIND) (Geneva,
                              biomarker discovery: are we making progress
                              Dr. Ruth McNerney, Ph.D.,          London School of Hygiene & Tropical Medicine
                              (London, UK)
                              Volatile markers for Tb: myth or reality?
                              Dr. Amy P Wong, Ph.D., X PRIZE Foundation (California, U.S.A.)
                              barriers to Tb test development
                              Discussion: The way forward
                              Platform	and	floor
 13h30 – 15h30                Break for Lunch
                              SuCCEpTibiliTyTESTiNg aND TrEaTmENT Of Tb iN ThE Era Of
                              mDr-Tb aND XDr-Tb. DO WE uSE ThE righT DrugS aND TOOlS?
                              Chair: Prof. Francoise Portaels and Dr. Salman Siddiqi
                              Prof. Stefan Winkler, University of Vienna (Vienna, Austria)
                              antibiotic therapy for Tb and new treatment options
European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                       9
                        Dr. Andre De Bock (BD)
                        Extended Drug Susceptibility Testing of m. tuberculosis
                        Virginia Crews (BD)
                        The new BD MGIT TBc Identification Test
     16h30 – 17h00      OPENING SESSION
                        Minister	of	Health	(Portugal)	(to	confirm)
                        Minister	of	Science,	Technology	and	Higher	Education	(Portugal)	(to	confirm)
     17h00 – 17h45      Welcome from the President of the Instituto Nacional de Saúde Dr. Ricardo Jorge,
                        Prof. José Pereira Miguel (Portugal)
                        Welcome from the General Secretary, Dr. Enrico Tortoli (Italy)

                        Welcome address, Dr. Suzana David (Portugal)

                        Tb CONTrOl prOgramS
                        Chair: Prof. Jaime and Prof Cristina Furtado
     17h45 – 18h20      Guest lecture: Dr. Miguel Villar, Consultant or Thuberculosis, Directorate-General
                        of Health (Lisboa, Portugal)
                        Tuberculosis in portugal
     18h20 – 19h00      Guest lecture: Dr. Fernando Fiuza de Mello, Director of the Clemente Ferreira
                        Institute (São Paulo, Brasil)
                        The brazilian experience in the control of multi-drug
                        resistant tuberculosis
                        Welcome reception Welcome Reception

                        RESISTANCE SURVEILLANCE
     9h00 – 9h45        Guest lecture: Dr. Sebastien Gagneux, Ph.D., National Institute for Medical Research    gl-3
                        (London, UK)
                        Evolutionary forces in mycobacterium tuberculosis
                        Chair: Dr. Stefan Niemann and Dr. Cristina Gutierez
     9h45 – 10h00       Honisch C, Mosko M, Arnold C, Gharbia S, Feuerriegel S, Niemann S                       Op-1
                        mass Spectrometry for molecular typing of the mycobacterium
                        tuberculosis complex: One platform and multiple assay formats
     10h00 – 10h15      Borile C, Refrégier G, Labarre M, Franz S, Mézard M, Sola C                             Op-2
                        Clustering of spoligo-patterns: towards an automation of
                        Mycobacterium tuberculosis complex classification
     10h15 – 10h30      Sandoval A, Cubillos A, Reyes A, Correa N, Robledo J, Zambrano MM,                      Op-3
                        Del Portillo P
                        Identification of the insertion element IS6110 in phop promoter
                        in a high transmission mycobacterium tuberculosis strain: a clue
                        to phenotypic variation
     10h30 – 10h45      Oelemann MC, Gomes HM, Willery E, Lima KVB, Possuelo L, Locht C, Goguet de              Op-4
                        la Salmonière YOL, Gutierrez MC, Supply P, Suffys PN

10                                                                                                             ESM 2009
                          genomic interrogation of mycobacterium tuberculosis isolates
                          from brazil
 10h45 – 11h00            Mokrousov I,Valcheva V, Sovhozova N, Aldashev A, Rastogi N, Isakova J                    Op-5
                          penitentiary population of mycobacterium tuberculosis in
                          Kyrgyzstan: Exceptionally high prevalence of the beijing geno-
                          type and its Russia-specific subtype
 11h00 – 11h30            Coffee and Tea
 11h30 – 12h15            Guest lecture: Dr. Dick van Soolingen, Ph.D., National Institute for Public Health and   gl-4
                          the Environment (Bilthoven, Netherlands)
                          advances in the molecular epidemiology of tuberculosis
                          Chair: Dr. Nalin Rastogi and Dr. Philip Supply
 12h15 – 12h30            Millet J, Miyagi-Shiohira C,Yamane N, Mokrousov I, Rastogi N                             Op-6
                          The unique endemic nature of beijing genotype strains in
                          Okinawa, ryukyu islands of Japan as revealed by newly de-
                          scribed 15 and 24-loci miru-VNTr typing schemes
 12h30 – 12h45            Shamputa IC, Lee J, Allix-Béguec C, Cho E-J, Lee J-I, Min JH, Goldfeder LC, Kim JH,      Op-7
                          Kang HS, Hwang SH, Eum SY , Lee H, Park SK, Supply P, Cho SN,Via LE,
                          Barry III CE
                          Mycobacterium tuberculosis genetic diversity in South Korea

 12h45 – 13h00            Feuerriegel S, Homolka S, Post E, Oberhauser B, George AG, Westman L, Dafae F,           Op-8
                          Rüsch-Gerdes S, Niemann S
                          Correlation of molecular resistance mechanisms and phenotypic
                          resistance to first-line drugs in Mycobacterium tuberculosis
                          strains from Sierra leone
 13h00 – 13h15            McNerney R, Mallard K                                                                    Op-9
                          lam and hiV: correlation or co-incidence?
 13h15 – 14h00            Lunch
 14h00 – 15h00            POSTER SESSION
                          Chair: Dr. Enrico Tortoli
 15h00 – 15h45            Guest lecture: Prof. Joseph O. Falkinham, III, Ph.D.,Virginia Tech                        gl-5
                          (Virginia, U.S.A.)
                          Surrounded by mycobacteria
 15h45 – 16h00            Lyberopoulos P, Frangopoulos F, Kontos F, Zerva L, Malagari Ai, Papiris S Op-10
                          Mycobacterium celatum: an emerging pathogen in the immuno-
                          competent. a case report

 16h00 – 16h15            Radomski N, Thibault V, Karoui C, De Cruz K, Cochard T, Gutiérrez C, Supply P,           Op-11
                          Biet F, Boschiroli ML
                          Mycobacterium avium subspecies strains from human
                          and animal origin
 16h15 – 16h30            Leão SC, Tortoli E,Viana-Niero C, Ueki SYM, Lima KVB, Lopes ML,Yubero J,                 Op-12
                          Menendez MC, Garcia MJ
                          The characterization of mycobacteria from an outbreak sug-
                          gests a revision of the taxonomic status of members of the
                          Mycobacterium chelonae-abscessus group

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                             11
     16h30 – 16h45       Santos R, Marques M, Oliveira P, Carvalho F, Carvalho C, Monteiro G, Cabral J,               Op-13
                         Frade R, Silva M, Fernandes P
                         The sunny side of mycobacteria
     16h45 – 17h15       Coffee and Tea
                         Chair: Dr. Gabriela Pfyffer and Dr.Thomas Shinnick
                         Guest lecture: Dr. Jean-Pierre Zellweger, M.D., Swiss Lung Association (Bern, Switzerland)
     17h15 – 18h00       The use of interferon gamma release assays as an aid in the con-                              gl-6
                         trol of tuberculosis
     18h00 – 18h30       Guest lecture: Prof. Lee W. Riley, M.D., Ph.D., University of California at Berkeley          gl-7
                         (California, U.S.A.)
                         a novel diagnostic test to differentiate latent Tb infection and
                         active disease

     18h30 – 18h45       Kaal E, Kolk A, Kuijper S, Janssen HG                                                        Op-14
                         Fast identification of Mycobacterium tuberculosis in sputum and
                         cultures based on thermally-assisted hydrolysis and methylation
                         by gas chromatography-mass spectrometry
     18h45 – 19h00       Ängeby K, Juréen P, Giske C, Chryssanthou E, Werngren J, Hoffner S, Kahlmeter                Op-15
                         G, Sturegård E, Schön T
                         how Wild-type miC distributions can be useful to determine
                         clinical breakpoints in Mycobacterium tuberculosis
     19h00 –19h15        Miotto P, Cirillo DM                                                                         Op-16
                         molecular techniques to monitor Tb patients’ treatment: selec-
                         tive removal of DNa from dead bacteria in mixed populations by
                         use of ethidum monoazide
                         Visit and Dinner at the Port Wine Cellars

                         Chair: Dr. David Minnikin
     9h00 – 9h45         Guest lecture: Prof. Carlos Martin, M.D., Ph.D., University of Zaragoza                       gl-8
                         (Zaragoza, Spain)
                         New live tuberculosis vaccines strategies
     09h45 – 10h30       Guest lecture: Prof. Lee W. Riley, M.D., Ph.D., University of California at Berkeley          gl-9
                         (California, U.S.A.)
                         regulation of Mycobacterium tuberculosis cell wall lipid composi-
                         tion and its effect on in vivo bacterial persistence
     10h30 – 11h00       Coffee and Tea
     11h00 – 11h15       Brzostek A, Pawelczyk J, Rumijowska-Galewicz A, Dziadek B, Dziadek J                         Op-17
                         Mycobacterium tuberculosis is able to accumulate and utilize
     11h15 – 11h30       Rodríguez-Güell E, Alonso C, del Val-Romero B, Clivillé R, Secanella SP, Roura-Mir           Op-18
                         C, Cañete C, Navarro A, de Gispert FX , Luquin M, Julián E
                         mycolic acid-induced ifN-g production by CD1-restricted T cells
                         from tuberculous patients
     11h30 – 11h45       Farnia P, Ali Veleyati A, Masjedi MR, Ibrahim TA, Tabarsei P, Haroun RZ, Kuan HO,            Op-19
                         Omar AR
                         a report on new adapted forms of extensively drug resistance
                         tubercle bacilli : Transmission Electron microscopy analysis
12                                                                                                                    ESM 2009
 11h45 – 12h00            Homolka S, Niemann S, Russell DG, Rohde KH                                          Op-20
                          Growth profile of clinical isolates of Mycobacterium tuberculosis
                          complex in murine macropghages
 12h00 – 12h15            van Ingen J, van der Wel N, Dekhuijzen R, Boeree M, van Soolingen D                 Op-21
                          presence of esat-6 and cfp-10 genes does not lead to phagolyso-
                          some translocation of mycobacterium szulgai
 12h00 – 12h30            Fraga AG, Braga JE, Cruz A, Martins TG, Pereira DR, Meyers WM, Portaels F,          Op-22
                          Castro AG, Pedrosa J
                          Development of an adaptive immune response in the draining
                          lymph node during mycobacterium ulcerans infection
 12h30 – 13h15            Lunch
 13h15 – 14h15            BUSINESS MEETING
                          Chair: Dr. Sven Hoffner and Dr. Mark Perkins

 14h15 – 15h00            guest lecture: Dr. Thomas m. Shinnick, ph.D., Centers for                           gl-10
                          Disease Control and prevention (georgia, u.S.a.)
                          CDC’s global Tb laboratory activities
 15h00 – 15h45            Guest lecture: Prof. Afranio Kritski, M.D., Ph.D., Universidade Federal do Rio de   gl-11
                          Janeiro (Rio de Janeiro, Brazil)
                          Development and validation of new Tb diagnostic tests in brazil:
                          experience of rede-Tb
 15h45 – 16h30            Guest lecture: Prof. Moisés Palaci, Ph.D., Universidade Federal do Espírito Santo gl-12
                          (Vitória, Brazil)
                          Experience of a successful mycobacteriology laboratory network
                          in Espirito Santo- brazil
 16h30 – 17h00            Coffee and Tea
 17h00 – 17h15            Portugal C, Cardoso N, Sancho L, Sousa G                                            Op-23
                          Tuberculosis software in a general hospital, working instrument
 17h15 – 17h30            den Hertog A, Koeleman M, Ingham C, Fey F, Langerak E, Klatser P, Anthony R         Op-24
                          Development of an automated culture system for m. tuberculo-
                          sis with autofluorescence detection
 17h30 – 17h45            Morcillo N, Imperiale B, Di Giulio B                                                Op-25
                          Second-line drug susceptibility testing of mycobacterium tuber-
                          culosis by mgiT 960 system, the microplate colorimetric-based
                          method and the proportion method
 17h45 – 18h15            BEST POSTER
                          Cultural Evening Dinner

 July 8th
                          Chair:Dr. Nalin Rastogi
 9h00 – 9h45              Guest lecture: Prof. Clarice Queico Fujimaro Leite, M.Sc., Ph.D.; Universidade      gl-13
                          Estadual Paulista (Araraquara, Brazil)

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                        13
                     Screening of molecules with anti-Tb activity, from the brazilian
                     cerrado plants, and synthetic metallo- organic compounds
     09h45 – 10h30   Guest lecture: Prof. Moisés Palaci, Ph.D., Universidade Federal do Espírito Santo   gl-14
                     (Vitória, Brazil)
                     Clinical trials of drugs and diagnostic tests: the challenges in
     10h30 – 10h45   van Ingen J, Boeree M, Amaral L, Pando RH, van Soolingen D                          Op-26
                     Thioridazine shows promising activity in a murine model of
                     multidrug-resistant tuberculosis
     10h45 – 11h00   Rodrigues L, Sampaio D, Couto I, Machado D, Kern WV, Amaral L, Viveiros M Op-27
                     Contribution of efflux pump activity for macrolide resistance in
                     m. avium complex
     11h00 – 11h30   Coffee and Tea
                     MOLECULAR EPIDEMIOLOGY
                     Chair: Dr. Elvira Richter and Prof. Christophe Sola
     11h30 – 12h00   Allix-Béguec C, HubansC, Ferreira S, Supply P                                       gl-15
                     New, easy-to-use tools for quality-controlled genotyping of m.
                     tuberculosis complex strains
     12h00 – 12h20   Abadia E, Zhang J, Refrégier G, Sola C                                              Op-28
                     membrane- based versus microbead- based spoligotyping:
                     preliminary results on a quality- insurance study on 10 sites
     12h20 – 12h30   Discussion
                     PD Dr. Elvira Richter, NRL (Borstel, Germany)
                     EQa in a low incidence, high income country
                     Dr. Girts Skenders, State Agency of TB and Lung Disease (Latvia)
                     Organization and EQa of latvian Tb laboratory network
                     Dr. Akos Somoskövy, M.D., Ph.D., D.Sc., Foundation for Innovative Diagnostics
                     (FIND) (Geneva, Switzerland)
                     Quality assurance for new techniques – what is necessary, what
                     is possible?
     13h30 -13h45    Closing remarks
     13h45 – 14h15   CLOSING OF CONGRESS

     14h15           Conference closes

14                                                                                                       ESM 2009
prOgrammE Of pOSTEr
 Miotto P, Baldan R, Cirillo DM                                                                               pp-1
 Evaluation of the high-throughput repetitive-sequence-based pCr diversilab
 system in M. tuberculosis molecular epidemiology studies
 Zhang J, Abadia E, Refrégier G, Ruimy R, Boschiroli ML, Guillard B, Sola C                                   pp-2
 68 spacers Mycobacterium tuberculosis complex spoligotyping : a study using a
 microbead-based high throughput format
 Niemann S, Khechinashvili G, Gegia M, Mdivani N, Tang YW                                                     pp-3
 association between beijing genotype and drug resistance among Mycobacterium
 tuberculosis isolates circulating in the republic of georgia
 Baboolal S, Millet J, Akpaka PE, Ramoutar D, Rastogi N                                                       pp-4
 Mycobacterium tuberculosis epidemiology and genetic diversity in the Twin island
 republic of Trinidad and Tobago
 Mestre O, Luo T, Rauzier J, Golec M, Rastogi N, Rasolofo V, Tonjum T, Sola C, Matic I, Mei J, Gao Q,Vultos   pp-5
 TD, Gicquel B
 Diversity and evolution of M. tuberculosis
 Sharaf-Eldin GS, Elmoula IF, Ali MS, Saaed NS, Ali AB, Mallard K, McNerney R, Algamdi S                      pp-6
 Spoligotype patterns and drug resistant profile of Mycobacterium tuberculosis
 in Sudan
 Valcheva V, Mokrousov I, Panaiotov S, Bachiiska E, Zozio T, Sola C, Markova N, Rastogi N                     pp-7
 Controversial dissemination pattern of the Bulgaria-specific M. tuberculosis
 spoligotype ST125_bgr
 Panaiotov S, Bachiyska E, Brankova N, Levterova V                                                            pp-8
 Mycobacterium tuberculosis beijing genotype and origins of the bulgarians
 Al-Maniri AA, Singh JPN, Al-Rawas O, Al Busaidi S, Al Balushi L, Ahmed I, Al- Mahruqi S, Haile               pp-9
 M, Diwan V, Hoffner S
 a Snapshot on biodiversity and clustering of Mycobacterium tuberculosis among
 nationals and immigrants in Oman using spoligotyping
 David S, Ribeiro JN, Maio JN, João I, Amorim A, Pereira E                                                    pp-10
 The extent of the latin american-mediterranean Mycobacterium tuberculosis
 spoligotype family in portugal
 Von Groll A, Martin A, Felix C, Prata P, Honscha G, Portaels F, Almeida da Silva P, Palomino JC              pp-11
 fitness Study of the rDrio lineage and lam family of Mycobacterium tuberculosis in
 a study population in rio grande, brazil
 Perdigão J, Silva C, Portugal I                                                                              pp-12
 genomic characterization of lisboa family strains by deletion analysis
 Obrovac M, Katalinic-Jankovic V, Grce M, Zmak L                                                              pp-13
 importance of molecular typing in suspected intra-familial transmission
 of tuberculosis
 Oral Zeytinli U, Kayar Mb, Karacali A, Sahan Kipalev A,Yula E, Köksal F                                      pp-14
 Detection of clonal complexity in clinical M. tuberculosis isolates by miru-VNTr in
 Cukurova region, Turkey
 Leite CQF,	Santos	ACB,	Pandolfi	JRC,	Malaspina	AC,	Pavan	FR,	Mendes	NH,	Viana	BHJ                            pp-15
 molecular epidemiology study of tuberculosis patients in a small city of São paulo –
 brazil, from 2002 to 2006
 Leite CQF, Nogutia EN, Malaspina AC, Santos ACB, Hirata RDC, Hirata MH, Cardoso RF                           pp-16
 genotyping of Mycobacterium tuberculosis in northwest of paraná State of brazil

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                        15
     Mello FAF,	Albarral	MIP,	Mendes	NH,	Pandolfi	JRC,	Santos	ACB,	Almeida	EA,	Cardoso	RF               pp-17
     Spoligotyping of Mycobacterium tuberculosis isolated from patients of Clemente
     ferreira ambulatory in São paulo, Sp – brazil
     Tajeddin E, Farnia P, Kargar M, Noroozi J, Ahmadi M, Kazempour M, Hadadi M, Masjedi M,Velayati A   pp-18
     Comparison Of Mycobacterium beijing genotype with VNTr, Spoligotyping and
     Ritacco V, Reniero A, Beltrán M, López B, Kantor I, Barrera L                                      pp-19
     multiply recurrent tuberculosis in a pacient living with hiV: reinfection or
     Tavares Magalhães A,Alves A, BragaR,Valente I, DuarteR, Miranda A                                  pp-20
     molecular epidemiology of tuberculosis in Vila Nova de gaia, portugal
     Alves A, Miranda A                                                                                 pp-21
     molecular study of recurrent tuberculosis cases
     Ehricht R, Slickers P, Monecke S                                                                   pp-22
     genotyping of drug resistance in Mycobacterium tuberculosis using diagnostic
     Al-Hajoj S,Varghese B, Herbawi M, Al-Omari R, Allix-Béguec C                                       pp-23
     genotyping of mono and multi-drug resistance Tb in Saudi arabia
     Machado D,Viveiros M, Rodrigues L, Couto I , Amaral L                                              pp-24
     Early detection of mDrTb by molecular tools in the control of drug resistant
     tuberculosis in portugal: a case of success
     Vladimirov K, Zaitseva E, Ivanov A                                                                 pp-25
     Drug-resistance of Mycobacterium tuberculosis at penitentiary institutions of St.
     petersburg, russian federation
     Stoffels K, Fauville-Dufaux M                                                                      pp-26
     an increase of drug resistance since 2001 in multidrugresistant M. tuberculosis
     isolates from belgium
     Perdigão J, Ferreira A, Malaquias A, Macedo R, Brum L, Portugal I                                  pp-27
     mutational analysis of genes associated with resistance to injectable second-line
     drugs in Mycobacterium tuberculosis clinical isolates from lisbon, portugal
     Nuak J, Ferreira D, Carvalho T, Gomes MH, Sarmento A                                               pp-28
     multidrug-resistant tuberculosis
     Tudó G, Rey E, Alcaide F, Coll P, Codina G, Martín-Casabona N, Montemayor M, Moure R, Salvadó M,   pp-29
     González-Martín J
     Characterisation of streptomycin mutations in Mycobacterium tuberculosis clinical
     isolates in the area of barcelona
     Fattorini L, Pardini M, Cirillo D, Borroni E, Miotto P, Filippini P, Cassone A                     pp-30
     Surveillance of Drug-resistant Tuberculosis in italy
     Sancho L; Portugal C; Tancredo L; Silva M; Dias A; Silva F, Sousa G                                pp-31
     Tuberculosis resistance in a general hospital in portugal – 9 years surveillance
     Yates M, Brown T, Drobniewski F                                                                    pp-32
     Does a mutation in the rpob mean that the M. tuberculosis is resistant
     to rifampicin?
     Zaldumbide MA, Mazarrasa CF, Martinez-Martinez L, Balbin JA                                        pp-33
     genotypic detection of isoniazid and rifampin resistance in Mycobacterium
     tuberculosis clinical isolates
     Chan CYR, Chan WCE, Au TKM, Lai WMR,Yew WW,Yip CW, Kam KM                                          pp-34
     Physiological fitness and transmission potential of multi-drug resistant
     Mycobacterium tuberculosis clinical isolates in hong Kong

16                                                                                                      ESM 2009
 Sousa AS, Pinheiro MD, Carvalho T, Gonçalves H                                                           pp-35
 Mycobacterium tuberculosis: 1999-2008 antituberculosis drugs surveillance in clinical
 isolates from patients in the largest hospital in the North of portugal
 Von Groll A, Martin A, Jureen P, Hoffner S, Portaels F, Palomino JC, Almeida da Silva P                  pp-36
 fitness cost of Mycobacterium tuberculosis clinical isolates resistant to
 Von Groll A, Martin A, Jureen P, Hoffner S, Portaels F, Almeida DA Silva P, Palomino JC                  pp-37
 In vitro activity of ofloxacin, moxifloxacin and gatifloxacin against Mycobacterium
 tuberculosis by the resazurin colorimetric method
 Paasch F, Martin A, Docx S, Fissette K, Portaels F, Palomino JC                                          pp-38
 rapid detection of extensively drug-resistant Mycobacterium tuberculosis by the
 resazurin microtiter assay plate
 Montoro E,Yzquierdo S, Lemus D, Echemendia M, Takiff H                                                   pp-39
 Detection of embb gene codon 306 mutations in ethambutol susceptible and
 resistant Mycobacterium tuberculosis strains
 Yew WW,Yan SW, Fung SL, Chau CH, Chan Chiu Y                                                             pp-40
 Tolerance of moxifloxacin in routine clinical treatment of tuberculosis
 Perdigão J, Sabino A, Milho C, Macedo R, Brum L, Portugal I                                              pp-41
 Characterization of gidB gene in Mycobacterium tuberculosis isolates in lisbon
 health region: role in streptomycin resistance and epidemiological markers
 Gaile I, Skenders G, Leimane V, Jansone I, Bauskenieks M, Pole I, Baumanis V                             pp-42
 fluorquinolone resistant Mycobacterium tuberculosis isolates and their molecular
 Samper S, Millan I, Lopez-Calleja AI, Gavin P, Lezcano MA                                                pp-43
 Design of a rapid method of identification of a highly transmitted strain based on
 the localization of iS6110
 Gutierrez MC, Brosch R, Marceau M, Tap J, Bourdon E, Brisse Smangenot S, Salvignol G, Barbe V, Médigue   pp-44
 C, Supply P
 Driving forces on the evolution of the progenitor of m. tuberculosis
 Ruiz P, Causse M, Zerolo FJ, Gutierrez J, Casal M                                                        pp-45
 resistance, mDr and XDr of M. tuberculosis in Spain in the last years
 Radomski N, Lucas F, Cambau E, Moulin L, Haenn S, Régis M                                                pp-46
 Detection of non tuberculous mycobacteria in surface waters: comparison of
 culture methods
 Spicic S, Cvetnic Z, Pate M, Duvnjak S, Zdelar-Tuk M, Racic I                                            pp-47
 Typing of Mycobacterium avium subsp. avium from different sources using pvuii–
 psti–iS901 restriction fragment length polymorphism (rflp) in Croatia
 Spicic S, Duvnjak S, Obrovac M, Zdelar-Tuk M, Katalinic-Jankovic V, Racic I, Cvetnic Z                   pp-48
 Tuberculosis in pets and wild animals living in urban environment
 Spicic S, Cvetnic Z, Pate M, Katalinic-Jankovic V, Duvnjak S, Ocepek M, Zdelar-Tuk M, Krt B              pp-49
 iS1245-rflp based genetic relatedness of the Mycobacterium avium subsp.
 hominissuis strains isolated from humans, animals and environment in Croatia
 Lucas F, Radomski N, Cambau E, Moulin L, Haenn S, Moilleron R                                            pp-50
 Development of real-time PCR assay for quantification of mycobacteria in
 surface waters
 Amorim A, Macedo R, Pereira E                                                                            pp-51
 Nontuberculous mycobacteria, isolated from patients with lung disease, from
 lisboa e Vale do Tejo region, during 2008

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                    17
     Lima KVB, Lopes ML, Furlaneto IP, Lima EJC, Conceição EC, de Sousa MS, Costa ARF                     pp-52
     Nontuberculous mycobacteria infections in the State of pará, amazon
     region, brazil
     Jahromi NS, Seif S, Farnia P, Kazempour M, Kargar M, Nowroozi J, Kazempour M, Masjedi M,Velayati A   pp-53
     Evaluation of hsp65, Tb ,Sp regions in identifying Mycobacterium Other Than
     Tuberculosis (mOTT); using pCr-rflp
     Svensson E, Ridell M, Åkerström M, Andersson E                                                       pp-54
     Mycobacterium avium alveolitis after cleansing hotel spa whirlpools
     Couto I, Machado D,Viveiros M, Rodrigues L, Amaral L                                                 pp-55
     Identification of nontuberculous mycobacteria in clinical samples using molecular
     methods: a three-year study
     Pate	M,	Ferme	D,	Žolnir	Dovc	M,	Ocepek M                                                             pp-56
     mycobacteria in animals in Slovenia – an overview of the last decade
     Leite SRA, Silva P, Sato DN, Santos ACB, Miyata M, Leite CQF                                         pp-57
     Isolation and identification of Rhodococcus and Nocardia genders in sputum
     samples with tuberculosis suspect
     Neonakis IK, Kontos F, Gitti Z, Baritaki S, Bazigos S, Mihailelis E, Zerva L, Spandidos DA           pp-58
     pCr-rflp of hsp65 for identification of Mycobacterium leprae directly from a
     clinical sample
     Neonakis IK, Kontos F, Gitti Z, Baritaki S, Kosmadakis G, Baritaki M, Zerva L, Spandidos DA          pp-59
     a case-report of Mycobacterium thermoresistibile from greece
     Portugal C, Sancho L, Dias A, Tancredo L, Silva M; Sardinha T, Sousa JG                              pp-60
     isolation and frequency of Mycobacterium sp in a general hospital during
     a 9-year period
     Diogo J, Rodrigues A, Nascimento I, Sardinha E, Raposo A, Figueira R, Monge I, Silva K, Gil MJ,      pp-61
     Rodrigues S
     laboratory microbiology contribution to Mycobacterium spp. Diagnosis in three
     district councils of Setubal (portugal), an area with high mycobacterial
     infection prevalence
     Santos C, Mendes AC, Fernandes SJ, Ramos MH                                                          pp-62
     Mycobacterium lentiflavum as a causative agent of adenopathy
     Watson C, Lockwood D                                                                                 pp-63
     Teaching old bones new tricks; single nucleotide polymorphism analysis of
     european archaeological m. leprae DNa
     Greib C, Lazaro E,Viallard JF, Pellegrin JL, Maugein J                                               pp-64
     Interpretation of positive M. tuberculosis antigen specificifnγ release assays in
     tuberculosis diagnosis
     Wang S, Neo ZY, Mak KX, Quieng MD, Sing LH                                                           pp-65
     Direct Identification of Mycobacterium tuberculosis Complex, Mycobacterium avium
     Complex and Mycobacterium kansasii in Smear-positive Clinical Specimens
     Müllerova M                                                                                          pp-66
     rapid diagnosis and drug susceptibility testing of tuberculosis infection: mTD-
     Test2 and bactec mgiT 960 system
     Levina K, Dementieva A, Saluotsa M                                                                   pp-67
     first experience with genotype mTbDr assai for rapid evaluation of mDr cases
     Fajfar N, Zolnir - Dovc M                                                                            pp-68
     Drug resistant tuberculosis in Slovenia and evaluation of genotype mTbDrpluS
     test in clinical laboratory

18                                                                                                        ESM 2009
 Causse M, Gutierrez-Aroca JB, Casal M                                                               pp-69
 Evaluation of a new real-time pCr kit for the diagnosis of tuberculosis
 inrespiratory specimens
 Karabela S, Papaventsis D, Nikolaou S, Konstantinidou E, Sainti A, Ioannidis P, Kanavaki S          pp-70
 Quantiferon-Tb gold assay (QfT) and tuberculine skin test (TST) clinical
 performance for the diagnosis of active tuberculosis
 Karabela S, Papaventsis D, Nikolaou S, Konstantinidou E, Sainti A, Ioannidis P, Kanavaki S          pp-71
 Clinical performance of Quantiferon-Tb gold assay (QfT) for the diagnosis of
 latent tuberculosis in different patient groups
 Nikolaou S, Karabela S, Papaventsis D, Sainti A, Konstantinidou E, Ioannidis P, Kanavaki S          pp-72
 Tuberculosis diagnosis by Quantiferon Tb gold assay in areas with differences in
 Tb incidence
 Havelkova M, Bartu V, Kubin M                                                                       pp-73
 Quantiferon -Tb gold in-Tube test used in prague patients listed in the National
 Tuberculosis register
 Cacho J, García-Cañas A, González Torralba A, Cano I, Pérez Meixeira A, Ramos Martos, Sánchez-      pp-74
 Concheiro M
 Practical experience of using a DNA amplification assay for rapid detection of
 Mycobacterium tuberculosis complex in respiratory specimens
 Morgan K                                                                                            pp-75
 real-time polymerase chain reaction for the direct detection of Mycobacterium
 tuberculosis in clinical specimens
 Kontos F, Zerva L                                                                                   pp-76
 The utility of molecular testing in routine mycobacteriology diagnosis
 Salas S, Hernández J, Ojeda P, Awad C, de la Hoz F, Murcia M                                        pp-77
 Detection of Mycobacterium tuberculosis DNA in formalin-fixed, paraffin-embedded
 tissue specimens by spoligotyping: application to histopathological diagnosis
 Cardoso S, Coelho R, Paulo C, Abreu C, Silva S, Gomes H, Sarmento A                                 pp-78
 pott´s Disease: an ancient disease?
 Loureiro C, Matos G, Balacó I, Mota M, Nogueira C, Lemos S, Rocha G                                 pp-79
 Osseous tuberculosis at age of 9 months
 Secanella SP, Luquin M, Julián E                                                                    pp-80
 Differences in direct antitumoral capacity among the various Mycobacterium bovis
 bCg substrains
 Anoosheh S, Farnia P, Noruzi J, Kargar M, Kazempour M, Seif S, Masjedi MR,Velayati AA               pp-81
 role of TNf-a gene polymorphisms in host genetic susceptibility to pulmonary
 Torrado E, Fraga AG, Logarinho E, Martins TG, Carmona JA, Gama JB, Carvalho MA, Proença F, Castro   pp-82
 AG, Pedrosa J
 mycolactone interferes with the protective ifN-γ-dependent activation of
 macrophages during infection with Mycobacterium ulcerans
 Montoro E,Valdés I, Aguilar D, Orozco H, Hernández-Pando R                                          pp-83
 Virulence, immunogenicity and protection induced by ´Mycobacterium habana´
 strains in a murine model of pulmonary tuberculosis
 Saraiva M, Sousa C, Carmona JA, Cruz A, Pedrosa J, Castro AG                                        pp-84
 Dendritic cells differentially express il12-family cytokines after infection with
 Mycobacterium tuberculosis or M. bovis bCg
 Simões MF, Jordão L, Teles JMM, Couto S, Moniz-Pereira J, Pimentel M                                pp-85
 analysis of M. smegmatis mutants resistant to ms6 infection

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal               19
     Julián EG, Rodríguez-Güell E, del Val-Romero B, Clivillé R, Cañete C, Navarro A, de Gispert FX,          pp-86
     Luquin M, Alonso C
     humoral response in tuberculous patients against the mycolic acids of
     Mycobacterium tuberculosis
     López AG                                                                                                 pp-87
     Structural, functional and bioinformatic characterization of Tlya protein from
     Mycobacterium tuberculosis
     Ferreira C, Afonso A, Duarte R, Lyashchenko K, Silva A, Rodrigues F, Miranda A, Tavares M, Caldas C,     pp-88
     Valente F,Valente A,Vasconcelos O, Amado J, Correia-Neves M
     Evaluation of the applicability of serodiagnosis for tuberculosis in portugal
     Martin A, Munga Waweru P, Babu Okatch F, Amondi Ouma N, Bonte L, Palomino JC,Varaine F, Portaels F       pp-89
     implementation of the thin layer agar (Tla) for the diagnosis of smear negative
     pulmonary tuberculosis in a high hiV prevalence setting
     Ferro RS, Shikama M-L,Villela G, Sato DN, Giampaglia CS, Martins MC, Martin A, Palomino JC               pp-90
     Direct detection of rifampin resistance in Mycobacterium tuberculosis by the nitrate
     reductase assay applied directly in sputum samples
     Ferro RS, Shikama M-L,Villela G, Sato DN, Giampaglia CS, Martins MC, Martin A, Palomino JC, Telles MAS   pp-91
     Direct detection of rifampin resistance in Mycobacterium tuberculosis by the nitrate
     reductase assay applied directly in sputum samples
     De Haas P, Zenhorst R, Mwamba P, Muvwimi M, Mwanza W, Mbulo G, Kapata N, Ayles H                         pp-92
     mTbDrpluS assay is a useful tool to screen for multi-drug resistant tuberculosis
     in a national survey
     de Haas P, Moyoyeta M, Samutela M, Mwanza W, Musunsa A, Mbulo G, Muvwimi M, Ayles H                      pp-93
     Contribution of laboratory factors to high mgiT culture contamination rate
     in zambia
     Ahmed A, Qazi F, Khan AJ                                                                                 pp-94
     programmatic Community-based management of mDr-Tb: Experience in
     Karachi, pakistan
     Muchwa C, Akol J, Mumbowa F, Orikiriza P, Morgan K, Eisenach K, Joloba M, Etwom A, Mugyenyi P,           pp-95
     Mugerwa R
     Evaluation of Capilia (TAUNS) for rapid identification of Mycobacterium tuberculosis
     complex from cultures
     Muchwa C, Akol J, Orikiriza P, Morgan K, Mumbowa F, Eisenach K, Etwom A, Joloba M                        pp-96
     Comparison of capilia (TAUNS) and IS6110 PCR for rapid identification of
     Mycobacterium tuberculosis complex from cultures in Kampala, uganda
     Bwanga F, Hoffner S, Haile M, Joloba ML                                                                  pp-97
     Direct testing for multi drug resistant tuberculosis with four assays evaluated at
     Kampala, uganda
     McNerney R, Turner C, Mallard K, O’Sullivan D                                                            pp-98
     pEa production by mycobacteria and its application in a rapid drug
     susceptibility test
     Balmoi F                                                                                                 pp-99
     Various strategies to decontaminate acid fast bacilli positive liquid cultures
     from bactec mgiT 960
     Orikiriza P                                                                                              pp-100
     low cost isolation of Mycobacterium tuberculosis (mTb) from blood
     Kayar B, Oral Zeytinli U, Karacali A, Soyal A, Nagiyev T, Köksal F                                       pp-101
     Comparison of rapid Colorimetric method, proportion method and baCTEC460
     Tb System for testing susceptibility of M. tuberculosis to rifampine and isoniaside

20                                                                                                            ESM 2009
 Crews V, Warns M, Pfeltz R, Beaty PS, Rosales J, Kopher K, Joshi S, Hoosen A, Said H                   pp-102
 Evaluation of the mgiT Tbc iD test vs two commercially available rapid
 immunoassays for M. tuberculosis complex organism detection from liquid
 and solid culture
 Montoro E, Milián Y, Lemus D, Echemendía M,Yzquierdo S, Martin A,Van der Stuyft P, Palomino JC         pp-103
 Nitrate reductase assay applied to direct detection of drug resistance in
 Mycobacterium tuberculosis
 Montoro E, Lemus D, Madruga M, Mirabal N, Milián Y,Yzquierdo S, Echemendía M, Martín A,Van der         pp-104
 Stuyft P, Palomino JC
 use of nicotinamide in colorimetric methods for rapid detection of pyrazinamide
 resistance in Mycobacterium tuberculosis
 Hepple P, Novoa-Cain J, Cheruiyot C, Richter E, Ritmeijer K                                            pp-105
 implementation of liquid culture for tuberculosis diagnosis in a remote setting:
 lessons learned
 Ichijo T, Izumi Y,Yamaguchi N, Nasu M                                                                  pp-106
 rapid detection of respiratory active mycobacteria by auramine O-CTC
 double staining
 Rey E, Tudó G, González-Martín J                                                                       pp-107
 Synergistic activity of two antituberculous drug combinations against clinical
 isolates of Mycobacterium tuberculosis resistant to isoniazid
 Stoffels K, Traore H,Van Hoof R, Fauville-Dufaux M                                                     pp-108
 Tobramycin-clarithromycin combination on Mycobacterium tuberculosis
 clinical isolates
 Au-Yeang CKW, Au TK, Chan EWC, Chan RCY                                                                pp-109
 Prevalence of Efflux-Mediated Rifampicin Resistance in Mycobacterium tuberculosis
 Clinical isolates
 Leite CQF,	Pavan	FR,	Maia	PIS,	Deflon	VM,	Sato	DN,	Azevedo	AA,	Poelhsitz	GV,	Leite	SRA,	Franzblau	SG   pp-110
 intra and extracellular activity of ruthenium complexes against Mycobacterium
 tuberculosis and their cytotoxicity
 Leite S,	Pavan	F,	Maia	P,	Deflon	V,	Batista	A,	Sato	D,	Franzblau	S,	Leite	C                            pp-111
 anti-Mycobacterium tuberculosis activity of thiosemicarbazones, semicarbazones
 and hydrazones
 Ramos J, Rodrigues L, Couto I, Amaral L,Viveiros M                                                     pp-112
 methods for assessment of ethidium bromide transport across Mycobacterium
 smegmatis cell wall
 Martins M,Viveiros M, Couto I, Amaral L                                                                pp-113
 The human macrophage as a model to select compounds active against
 Cynamon M, Mookherjee S, Shoen C                                                                       pp-114
 in vitro activities of JpC 2067 alone and in combination with SmX against
 nocardia species
 Nina J
 Nosocomial Tb in a laboratory setting

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                   21
Of guEST
lECTurES (gl)

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal   23

TubErCulOSiS iN pOrTugal
Miguel Villar
Consultant on Tuberculosis, Directorate-General of Health, Lisbon, Portugal

Tuberculosis is a global problem with an estimated number of 9 million cases per year, 83% of which are in Sub-Saharian
Africa	and	South-East	Asia,	where	we	find	many	of	the	high	burden	countries.
Concerning multidrug-resistance tuberculosis (MDR-TB), WHO estimates about half a million cases globally, per year,
including 50.000 extensively drug-resistant tuberculosis (XDR-TB).
In 2007, European Union (EU) had an incidence rate of 17/105, having Portugal one of the highest rates in the EU (27/105).
In	the	last	20	years,	the	incidence	in	Portugal	has	decreased	consistently	more	than	7%	per	year	in	the	last	five	years.
This reduction is mainly in the age group between 25 and 44 years old, leading to a shift to the right of the median age
both in the nationals and in the immigrants.
The foreign born cases have represented about 12% of the cases and the prevalence of HIV has been around 14%.
Most of the cases are pulmonary forms (74.1%) between 2003-2007, 67.5% of which are SS+ and 75.3% are culture positive.
Mixed multidrug-resistance tuberculosis, including XDR-TB, during the same period, represents 1.9% (154 cases) of the
TB cases at the start of treatment, varying from 1.3% (22 cases) in 2006 to 2.4% (38 cases) in 2004, with an average of 31
cases per year (1.9%). These proportions are representative as the coverage of drug sensibility tests (DST) is over 80%.
We	will	address	the	importance	of	the	Micobacteriology	Laboratory	network,	concerning	case	detection,	definition	of	
confirmed	cases,	early	diagnosis	of	MDR-TB	and	1st	and	2nd	line	DST.
As an important complement of the DOTS Strategy, the analysis of the outcomes will be discussed, concerning not only
the general population but also the different risc groups, having as a goal the 85% cure rate proposed by WHO.
We	finish	our	presentation	addressing	the	strategy	for	MDR/XDR-TB	control	in	Portugal,	namely	the	importance	of	the	
reference network for MDR-TB with its national coordination.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                               25

     ThE braziliaN EXpEriENCE CONTrOliNg Tb mulTi-Drug rESiSTaNCE

     Fernando Augusto Fiuza de Melo
        •	 Médico, Diretor do Instituto Clemente Ferreira – Coordenadoria de Controle de Doenças da Secretaria de Estado
           da Saúde de São Paulo – ICF/CCD/SP
        •	 Doutorado em Medicina, área de pneumologia, pela Escola Paulista de Medicina da Universidade Federal de São
           Paulo - EPM/UNIFESP
        •	 Membro	 do	 Comitê	 de	Assessoria	Técnico-Científico	 do	 Programa	 Nacional	 de	 Controle	 da	Tuberculose	 do
           Ministério da Saúde - PNCT/MS

     Rua Santo Estácio, 248 – Cidade Vargas, CEP. 04319-010 - Brasil - São Paulo,SP
     Tele-fax:	0055	11	3218	8653	-	Telemóvel		0055	11	8469	4330	-	e-mail:

     Brazil	was	the	first	developing	country	using	the	short	duration	regimen	of	rifampin	(R)	and	isoniazid	(H)	combined	in	
     one	capsule	for	six	months,	plus	pyrazinamide	(Z)	during	the	first	two	months	after	reorganizing	the	Brazilian	National	
     Tuberculosis Control Program of (BNTBCP, Programa Nacional de Controle da Tuberculose, PNCT) in 1980. At that
     moment Brazil adopted the anti-TB regimen named E-1 (2RHZ/4RH) for all forms of TB with no known previous treat-
     ment. A similar regimen was adopted for Meningitis TB, named E-2, adding corticoids in the intensive phase with the
     recommendation to lengthen the continuation phase of treatment to 7 months (2RHZCort/7RH). For those TB cases of
     relapsing (RC) or re-treatment after defaulting (RA) the anti-TB regimen adopted was E-1R with ethambutol (E) during
     the intensive phase (2RHZE/4RH). The recommended regimen for failure (F-1) cases was named E-3 and included Z and
     E, associated with streptomycin (S) and the ethionamida (Et) during at least 12 months (3SZEEt/9EEt)¹
     The E-1 was evaluated under pragmatic clinical “non-study” conditions during decades 1980/90 and also during half of the
     first	decade	of	the	new	millennium	and	shown	an	efficacy	of	94.6	and	93.9%,	an	effectiveness	of	77.8	and	77.1%,	a	default	
     rate of 13.7 and 13.1%13%, a failure rate of 1.5 and 1.7%%, a serious adverse events rate of 3.1 and 3.3% and a mortality
     rate of 3.9 and 4.8%, respectively. (²,³).The small worsening on mortality rate might be related to high rates of defaulting and
     the HIV co-infection.The good quality of the National Aids Program Control and the increasing rate of TB treatment under
     direct supervision in Brazil are possible reasons for Brazil rates of resistance were not too high. On the other hand, results
     of	E-3	were	not	nice	with	efficacy	and	effectiveness	varying	between	57,5%	-	85,2	and	66,7	-	84.7%,	respectively	(4).
     4%	of	80,000	cases	of	TB	notified	in	Brazil	were	resistant	to	R+H	or	were	unable	to	be	treated	with	these	drugs	for	any	
     other	reason	and	were	defined	as	F-1	case.	These	patients	were	treated	with	E-3	(3SZEEt/9EEt)	and	are	considered	in	
     Brazil as a case of MDR-TB. Brazil estimates a rate between 0,3 and 0,4% of cases of F-1 not responding to E-3. These
     cases	are	defined	in	Brazil	as	a	Multiresistant	TB	(MRTB)	and	there	is	no	well	established	anti-TB	regimen	for	these	pa-
     tients in the BNTBCP5.
     In	1995	an	anti-TB	regimen	including	amicacin	(AM),	ofloxacin	(OFX),	terizidona	(TRZ),	clofazimine	(CFZ)	and	E	was	
     evaluated in some TB Centers in Brazil with reasonable results (6).
     In	2000	the	Monitoring	Program	for	MRTB	was	created,	centralizing	the	notification	of	all	cases	of	TBMR	in	Brazil	and	
     creating	a	work	group	including	health	professionals	involved	with	MRTB	in	order	to	define	and	organize	a	way	to	control	
     MRTB in Brazil. In 2007, Guidelines for MRTB were published presenting the knowledge about TBMR and establishing
     rules for diagnosis, treatment, prevention and biosafety; providing orientation on epidemiologic surveillance, building hu-
     man	and	material	resources,	and	implementing	a	specific	National	Notification	System	for	these	patients.	The	current	
     alternative	regimen	for	MRTB	cases	is	defined	by	the	sensitivity	tests	and	administered	under	supervision,	including:	AM	
     (or S if sensible) for 12 months, OFX, TRZ and E for 18 months and Z (if sensible) for 6 months7. Metronidazole (MTZ)
     replacing Z, especially in cases of intolerance or resistance, is used in some clinics.
     TBMR rates were evaluated in Brazil between 2000 and 2005, showing the following results:
     Between	323	and	334	cases	had	been	annually	notified	from	2000	to	2004,	with	an	increase	in	2005	to	383	cases	notified	
     among a total of 80.000 TB cases.
     Increasing cure rates over time (40 to 62%), with some organized units showing better rates (75 to 85%).

26                                                                                                                        ESM 2009
Default rates between 5 and 7%; failure rates between 10 e 15%; mortality rates had decreased from 33% to 11% over time.
Most of MRTB were post-primary cases (74 to 80%); 6 to 8% were the primary cases, especially contacts and risk groups;
11 to 20% were indeterminate.
TBMR rate among HIV co-infected patients was low, between 1.6 e 3%7,8.

Extensively	multi-drug	resistant	(X-MDR)	TB	cases,	presenting	resistance	to	2	first	line	drugs	and	3	second	line	drugs,	
have been observed since 20009, however for more accurate estimates, a national survey is necessary. In recent survey
at	the	Clemente	Ferreira	Institute,	34	cases	resistant	to	fluoroquinolone	were	notified,	and	16	cases	were	also	resistant	
to AM and 18 to S. The patients were treated with an alternative regimen indicated for MRTB, with 9 cure cases and 25
failure	cases,	including	17	deaths.	An	important	finding	was	the	occurrence	of	3	primary	X-MDR	cases10.

Ministério	da	Saúde/Fundação	Nacional	de	Saúde/Comitê	Técnico-Científico	de	Assessoramento	à	Tuberculose/Comitê	
Assessor para Co-infecção HIV-Tuberculose. Tuberculose: guia de vigilância epidemiológica, Brasília, 2002.
Ministério da Saúde/Fundação Nacional de Saúde/Centro de Referência Prof. Hélio Fraga-Rio de Janeiro, Documento
Básico da Reunião de Avaliação operacional e epidemiológica do PNCT na década de 80. Bol Pneumol Sanit 1993,
Numero Especial.
Ministério da Saúde/Secretaria de Vigilância em Saúde/Centro de Referência Prof. Hélio Fraga-Rio de Janeiro. Análise da
situação da tuberculose no Brasil nos anos 90 e início da década atual. Bol Pneumol Sanit 2005;13:133-179.
Campos HS, Melo FAF. Efetividade do esquema 3 (3sSZEEt/9EEt) no retratamento da tuberculose na rotina das unidades
de saúde. Bol Pneum Sanit 2000;8:7-14.
Melo	FAF,	Ide	Neto	J,	Seiscento	M,	Pinto	JA,	Afiune	JB:	Tuberculose	Multirresistente.	J	Pneumol	1993;19:73-82.
Dalcolmo MP, Fortes A, Melo FAF, Motta R, Ide Neto J, Cardoso N,Andrade M, Barreto AW, Gerhardt G. Estudo de efetivi-
dade de esquemas alternativos para o tratamento da tuberculose multirresistente no Brasil. J Pneumol 1999;25:70-77.
Ministério da Saúde/Secretaria de Vigilância em Saúde/Centro de Referência Prof. Hélio Fraga/Projeto MSH. Tuberculose
multirresistente: guia de vigilância epidemiológica;2005:89pg
Melo	FAF,	Afiune	JB,	Ide	Neto	J,	Almeida	EA,	Spada	DTA,	Antel	ANL,	Cruz	ML.	Aspectos	epidemiológicos	da	tuberculose	
multirresistente em serviço de referência na cidade de São Paulo Rev da Soc Brasil Med Trop 2003;36:733-40.
Emergence of Mycobacterium tuberculosis with extensive resistance to second line drugs worldwide 2000 – 2004.
MMWR 2006;55(11)
Savioli MTG, Melo FAF, Morrone N e Rodrigues DS. Tuberculosis with extensive resistance to drugs in a TB reference
center in Sao Paulo, Brazil. Poster accepted for UICTER 2009;Cancum, Mexico.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                               27

     EVOluTiONary fOrCES iN MyCoBaCTeriuM TuBerCuloSiS

     Sebastien Gagneux
     Division of Mycobacterial Research, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, NW7 1AA,
     London,	United	Kingdom;,	phone:+4420	8816-2399,	fax:	+	4420	8816-2564

     The Mycobacterium tuberculosis complex (MTBC) consists of genetically monomorphic organisms. Studying the genetic
     population structure and evolution of monomorphic bacteria is hindered by the lack of DNA sequence variation; methods
     such as multilocus sequence typing (MLST), which have been well established in other bacteria, are not applicable. Because
     of this limitation, most current genotyping methods for MTBC are based on mobile or repetitive DNA elements (e.g.
     IS6110 RFLP, spoligotyping, MIRU-VNTR). Mobile and repetitive DNA regions change relatively quickly, which makes them
     ideal markers for molecular epidemiological analyses. However, because these markers can exhibit convergent evolution
     leading to homoplasy (similar patterns emerging in unrelated strains), they are less robust to infer phylogenetic relation-
     ships. Furthermore, actual DNA sequence data is preferred for population genetic analyses. To get around this problem,
     we sequenced 89 genes in 108 MTBC strains. We used these DNA sequence data to explore the evolutionary forces that
     have	shaped	the	genetic	diversity	in	MTBC.	Our	findings	show	that	MTBC	is	under	greatly	reduced	selective	constraint	(i.e.	
     purifying selection is reduced in MTBC), and as a result, much of the genetic diversity in MTBC is likely to have functional
     consequences.	These	findings	have	important	implications	for	the	development	of	new	tools	to	control	tuberculosis.

28                                                                                                                    ESM 2009

aDVaNCES iN ThE mOlECular EpiDEmiOlOgy Of TubErCulOSiS

Dick van Soolingen1, Jakko van Ingen1, Philip Supply2, Anita Schürch1, Ida Parwati3, Reinout van Crevel4, Nguyen Van Hung5,
1- Frank Cobelens6, and Kristin Kremer1 National Tuberculosis Reference Laboratory, Nat. Inst. for Public Health and the
Environment	(RIVM),	Bilthoven,	the	Netherlands;
2	-	National	Center	for	Scientific	Research,	Institut	Pasteur,	Lille,	France
3 - Dept. of Clin. Path. Hasan Sadikin Hosp., Med. Fac. Padjadjaran Univ.,Bandung, Indonesia
4 - Dept. Int. Med., Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands
5 - Nat. Hosp. of Tuberculosis and Respiratory Diseases. Hanoi,Vietnam
6 - Center for Poverty-related Comm. Dis., Acad. Medical Center, Amsterdam, the Netherlands

Although elimination of tuberculosis in Europe is not yet in sight, the ECDC held a meeting in Stockholm in April 2009
to	re-define	the	indicators	of	a	successful	TB	control	on	this	continent.	Molecular	epidemiology	was	identified	as	a	key	
component to detect the level of active transmission. In 2009, a new ECDC project has been initiated to re-activate
the molecular surveillance of (MDR/XDR) TB in Europe, with strong focus on a high coverage of MDR/XDR cases in
Central and Eastern Europe. In the previous project transmission of MDR/XDR-TB in Europe was largely caused by
Mycobacterium tuberculosis Beijing genotype strains.
In	a	recent	(2009)	resistance	survey	in	Vietnam,	a	significant	correlation	between	resistance	and	Beijing	strains	was	ob-
served. Moreover, in previous studies in Ho Chi Minh City treatment failures and relapses were more frequently found
in patients infected by Beijing strains. However, in a recent, larger study in Indonesia patients infected with Beijing geno-
type strains also more often had a positive sputum culture after six months treatment (RR:1.95; CI 95%:1.25-3.02), but
this was not correlated with differences in drug resistance. Therefore, this suggests that M. tuberculosis Beijing genotype
strains have a higher capacity to withstand tuberculosis treatment, even in the absence of drug-resistance.
The new European network on molecular epidemiology will implement 24-loci VNTR typing as a standard. Although the
utility of VNTR typing has been shown in multiple studies, a broad and nation wide comparison of IS6110 RFLP typing
and VNTR typing is still missing. In the Netherlands 4400 M. tuberculosis isolates from the period of 2004-2008 have been
subjected to IS6110 RFLP as well as VNTR typing and a concordance of 81% has been observed. Moreover,VNTR typing
showed	a	higher	degree	of	concordance	with	findings	in	the	conventional	contact	tracing	than	RFLP	typing.
To come to the highest resolution of DNA typing, two isolates from the Harlingen tuberculosis outbreak, that have been
isolated with an interval of 12.5 years and which were separated by four person-to-person transmissions were subjected
to whole genome sequencing. Four single nucleotide polymorphisms (SNPs) and one tandem repeat polymorphism
(TRP) and a IS6110 transposition	were	identified.	Typing	of	all	104	isolates	in	the	IS6110 RFLP cluster with the six DNA
polymorphisms	endorsed	the	separate	line	of	transmission	established	by	contact	tracing.	These	findings	suggest	that	the	
microevolution of M. tuberculosis can be used to resolve separate transmission chains in large outbreak clusters.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  29

     SurrOuNDED by myCObaCTEria

     Joseph O. Falkinham, III
     Department of Biological Sciences
     Virginia Tech
     Blacksburg,Virginia 24061-0406
     Phone 1-540-231-5931
     FAX       1-540-231-9307

     Humans, animals, and plants are surrounded by mycobacteria. The environmental opportunistic mycobacteria (also called
     nontuberculous or atypical mycobacteria) include over 100 species; many of which cause disease. Infections include cer-
     vical lymphadenitis in children and pulmonary disease and skin infections in adults. Evidence that the environment was
     the	source	of	human	disease	was	gained	from	the	identity	of	DNA	fingerprints	of	mycobacterial	isolates	from	patients	
     and either their household water or potting soils. Recently, a number of reports have documented a dramatic increase
     in pulmonary disease caused by these mycobacteria amongst elderly and slender men and women who lack all of the
     classic predisposing risk factors (e.g., smoking, exposure to dusts). Although slowly growing with generation times of
     one-half to one day, the environmental opportunistic mycobacteria survive, grow, and persist in a number of habitats
     that are shared with humans and animals. The environmental opportunistic mycobacteria are oligotrophs; able to grow
     in water containing greater than 50 µg AOC/L. Survival and persistence in the environment is due, in part, to the thick,
     impermeable, hydrophobic, lipid-rich envelope of mycobacterial cells. Although the hydrophobic wall reduces the rate of
     transfer	or	hydrophilic	nutrients,	it	promotes	attachment	to	surfaces	where	mycobacteria	form	biofilms.		Hydrophobicity	
     also contributes to disinfectant- (e.g., chlorine and biocides) and antibiotic-resistance. For example, mycobacteria enter-
     ing a water treatment system on particulates survive disinfection and grow during travel in the distribution system in the
     absence of competitors. Hydrophobicity also promotes the aerosolization of mycobacteria from water to air in environ-
     ments such as showers and hot tubs in the home and occupations where aerosols are generated.

30                                                                                                                   ESM 2009

ThE uSE Of iNTErfErON gamma rElEaSE aSSayS aS
aN aiD iN ThE CONTrOl Of TubErCulOSiS

Jean-Pierre Zellweger
Swiss Lung Association, Berne, Switzerland

Interferon Gamma Release Assays (IGRAs) are in vitro tests detecting the presence of latent tuberculosis infection (LTBI)
in asymptomatic persons who may have been infected by M. tbc in	a	recent	or	remote	past	and	who	may		benefit	from	a	
preventive treatment to decrease the risk of later reactivation of tuberculosis.
Basically, the IGRA tests rely on the same immunological phenomenon as the tuberculin skin tests, but they do it in a
much	more	specific	way,	because	the	tests	are	not	influenced	by	a	prior	vaccination	with	BGC	or	by	an	infection	with	
most of the non-tuberculous mycobacteria present in the environment. Therefore, the indications and the use of the
IGRA tests are fundamentally the same as for the tuberculin skin tests :
   •	 Detection of LTBI in persons in contact with an index case of tuberculosis
   •	 Detection of LTBI in persons with a high risk of tuberculsois, if infected (immunosuppressed patients, patients
      receiveing or due to receive immunosuppressive therapy, small children)
   •	 Surveillance of exposed health care workers (as the test can be repeated without risk of inducing a booster effect)
   •	 Aid to the diagnosis of tuberculosis in cases where a bacteriological examination is not feasible or not reliable
      (severe extrapulmonary TB, TB in children)

In spite of their superiority, the IGRAs are not totally devoid of problems in practice and the best use of them is still
a	matter	of	debate.	Some	Guidelines	recommend	their	use	only	for	the	confirmation	of	positive	TST	among	contacts	
(the so-called two-step testing procedure) whereas others recommend the routine replacement of the TST by IGRAs.
Performing	only	one	test	is	easier,	and	avoids	a	possible	influence	of	a	prior	TST	on	the	IGRA	response.
The predictive value of the new IGRAs seems to be superior to the predictive value of TST, reinforcing their usefulness,
if the preventive treatment is corectly precribed and followed. One intriguing phenomenon is the possible reversion of
a positive IGRA after conversion, possibly indicating that some infected contacts may be able to eradicate the mycobac-
teria without treatment.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                              31


     Lee W. Riley
     MD, School of Public Health, University of California, Berkeley

     It is well recognized that the treatment of latent TB infection (LTBI) is a highly effective TB prevention strategy, which is
     still not widely practiced in most parts of the world. Most TB-endemic countries rely on BCG vaccine to prevent TB.
     LTBI treatment requires contact investigation, which is not done in most “BCG countries”. One reason for this reluc-
     tance to practice contact investigation is the lack of a reliable test that can distinguish LTBI from TB. Thus, a test that can
     unequivocally distinguish LTBI from TB could alter the current national prevention programs in TB-endemic countries.
     We	have	identified	a	set	of	M. tuberculosis cell wall proteins that are expressed when the bacilli replicate in vivo, but not
     when they are in a nonreplicative state. Their continued expression is associated with disease progression in infected
     mice, and mouse T cells are sensitized as these proteins are continually expressed in vivo. Exploiting this observation, we
     developed a bioassay that is able to distinguish LTBI from active disease in a mouse model. The assay is based on IFNγ
     induction by T cells exposed to a set of synthetic peptides based on the cell wall protein called Mcep1A. The Cornell
     mouse model was used to study the response of spleen cells exposed ex vivo to these peptides. Cells from untreated
     mice expressed 7-9-fold higher levels of IFNγ than those from treated mice at 24 and 32 weeks of infection, as mea-
     sured by ELISA. Blood cells from healthy tuberculin skin-test positive, QuantiFERON-negative (n=3) and TST-negative,
     QuantiFERON-negative (n=3) human volunteers showed no response to the peptides. These peptides are currently
     under evaluation in newly diagnosed TB patients. If the assay can show a response in these TB patients at levels similar
     to those observed in diseased mice, this assay can be converted into an immunochromatographic (“dip stick”) format.
     Such a test then can be used to readily differentiate those with LTBI and active disease, and could then be incorporated
     as part of National TB Control Programs.

32                                                                                                                       ESM 2009


Jesus Gonzalo Asensio, Ainhoa Arbues and Carlos Martín
Department of Microbiology, University of Zaragoza. Spain

BCG, the current vaccine against tuberculosis (TB), has been used for more than 80 years but is ineffective at providing
protection against adult pulmonary TB. New tuberculosis vaccine candidates and TB vaccination strategies, conferring
better protection against pulmonary tuberculosis than the current vaccine BCG, are needed.
In the recent decade, a global pipeline of novel TB candidates has emerged. Pioneering strategies for the development
of more effective vaccines today have lead to the discovery of subunit vaccines, which have proved ineffective at provid-
ing better protection that BCG in various animal models. Different heterologous prime BCG and boost with subunit
strategies	are	in	clinical	trials	with	the	aim	to	improve	efficacy	of	BCG.	More	recently,	clinical	trials	with	recombinant	
BCG	vaccines	have	started	with	the	aim	to	find	candidates	to	be	used	as	prime,	preventive	vaccines.	Another	innovative	
strategy, live attenuated Mycbacterium tuberculosis vaccines, in late preclinical investigation, are promising new preventive
vaccine candidates to replace BCG.
Based upon the observation that phoP is an essential gene for M. tuberculosis virulence, we rationally attenuated the
tubercle bacillus by inactivating phoP (Perez et al, Mol Micro 2001). The mutant was shown to be strongly attenuated in
cellular and animal models. Moreover, the phoP mutant resulted more attenuated than BCG Pasteur in immunocompro-
mised SCID mice and this vaccine candidate protected guinea pigs and non human primates against tuberculosis infec-
tion (Martin et al Vaccine 2006,Verreck et al PLoS ONE 2009).
Both, the attenuated phenotype and the protective immunity conferred against tuberculosis infection can be accounted
for by the mechanism of action of PhoP, which has been recently shown to be crucial for intricate virulence network of
M. tuberculosis (Gonzalo Asensio et al PLoS ONE 2008). This observation was used to construct a new generation of live
vaccines based on phoP inactivation carrying a second additional mutation which affects the synthesis of a new family of
lipids associated to M. tuberculosis virulence.
It is estimated that at least 20 vaccine candidates should enter phase I safety trials with around half going forward for
immunological	evaluation	in	phase	II	trials	and	leading	to	four	phase	III	efficacy	trials	with	the	goal	to	reach	an	effective	
licensed vaccine in 5-7 years (Young and Dye, Cell 2006). The discovery and use of a new TB vaccine better than BCG is
key to reach the 2050 objective of TB eradication.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                   33

     rEgulaTiON Of MyCoBaCTeriuM TuBerCuloSiS CEll Wall lipiD
     COmpOSiTiON aND iTS EffECT ON iN ViVO baCTErial pErSiSTENCE

     Lee W. Riley
     School of Public Health, University of California, Berkeley

     The hallmark of M. tuberculosis is its ability to survive for many years in an infected host to establish latent tuberculosis
     infection (LTBI). We propose a new model of latent infection that is based on the idea that this organism may simply have
     readapted its “housekeeping” metabolic function to a new environment for its long-term survival. We propose that M.
     tuberculosis, which evolutionarily most likely originated in soil, has readapted a soil-survival strategy its ancestral species
     possessed to the granuloma environment in the human host. Granuloma cells constantly turn over every few days to
     weeks, and after they die, they undergo replacement by new cells that migrate into the granuloma. Hence, M. tuberculosis
     needs to readapt to this constantly changing environment, and we provide evidence that this adaptation is mediated by
     M. tuberculosis remodeling its cell envelope in response to signals produced by dead granuloma cells. This remodeling
     is mediated by a family of operons called mce (mce1,2,3,4). Disruption of the operons results in profound changes in
     lipid	profile	of	the	cell	wall.		The	mce1 operon mutant causes free mycolic acids (MA) to accumulate on its surface, and
     other operon mutants (mce2,3,4)	show	evidence	of	lipid	profile	changes	in	the	cell	wall.		These	operon	products	serve	as	
     energy-dependent lipid importers. Lipid products released from dead host granuloma cells that turnover may be used as
     carbon sources for the resident M. tuberculosis. Thus, the “housekeeping” lipid metabolic function of M. tuberculosis may
     have been readapted in the granuloma environment as a way for this organism to survive, similar to the way its ancestral
     saprophytic organism survived in soil by scavenging dead organic materials as carbon sources. Further elucidation of the
     interaction between M. tuberculosis cell wall and granuloma cell turnover may contribute to a new understanding of the
     mechanism of LTBI.

34                                                                                                                       ESM 2009

CDC’S glObal Tb labOraTOry aCTiViTiES

Thomas M. Shinnick,
Associate Director of Global Laboratory Activities, Division of TB Elimination, Centers for Disease
Control	and	Prevention,	1600	Clifton	Road,	MS-G35,	Atlanta	Georgia	30333	USA.	email:;	FAX:	1-404-
639-1287; Tel: 1-404-639-1474

A key bottleneck in health service delivery is weak laboratory capacity. This is particularly true for drug-resistant
TB — less than 5% of MDR TB cases are currently being detected globally. To meet the 2015 targets of the Stop TB
Global Plan, 60 million culture tests and 5 million drug susceptibility tests will be needed annually.This will require estab-
lishing at least 2,000 new culture laboratories and training of more than 20,000 laboratorians. At least US$ 1 billion will
be	needed	annually	for	building	TB	laboratory	infrastructure	and	recurring	costs.	However,	the	benefit	to	cost	ratio	of	
such investments is estimated to be 9:1 in populations with a high prevalence of HIV infection. Meeting the 2015 goals
could save countries in sub-Saharan Africa alone as much as US$ 52 billion annually.
While	 training	 of	 bench-level	 technicians	 relies	 on	TB-specific	 expertise,	 laboratory	 capacity	 building	 relies	 more	 on	
cross-cutting expertise in infrastructure, biosafety, human resource development, supply chain management, logistics,
quality assurance programs, management principles, information systems, data management, and accreditation processes.
As such, TB laboratory strengthening efforts can build on lessons-learned from the building of laboratory networks for
polio,	measles,	SARS,	influenza,	HIV/AIDS,	and	other	diseases.
The goal of our TB laboratory strengthening efforts is the creation of a network of laboratories that can provide reli-
able, high quality testing and which is based on quality laboratory management principles and integrated across disease
programs, especially HIV and TB. A systems approach is used to optimize laboratory testing and information exchange.
The approach involves understanding the structure, performance, and cost of the network; developing referral processes
to	ensure	prompt	flow	of	specimens	and	information;	and	using	quality-improvement	principles	to	continually	evaluate	
and improve the performance of the network. While TB laboratory strengthening plays the central role in our efforts,
the overriding goal is to ensure that a broader health systems approach is used to maximize the impact and sustainability
of the investments.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                         35

     DEVElOpmENT aND ValiDaTiON Of NEW Tb
     DiagNOSTiC TESTS iN brazil: EXpEriENCE Of rEDE-Tb

     Brazilian Tuberculosis Research Network: A Multi Disciplinary Collaborative Research Project

     In	Brazil,	which	has	an	estimated	124,000	cases	of	TB	per	year,	there	has	been	a	significant	gap	in	communication	and	
     understanding between TB programmatic experts, academics, the community, and nongovernmental organizations. In rec-
     ognition of this gap, a National TB Research Network was established in 2002 to bring these constituencies together to
     promote an integrated, multi-disciplinary and multi-institutional strategy for TB control in Brazil. In the last years, Rede-
     TB has established a solid relationship among the National Tuberculosis Control Program and Oswaldo Cruz Foundation
     that	has	helped	to	foster	Brazilian	leadership	and	competency	in	the	development	and	evaluation	under	field	conditions	
     of new diagnostics for TB.

     To develop new diagnostics, vaccines and drugs for the prevention and cure of TB, including MDR-TB, and develop
     improved, therapeutic alternatives with the renewal of drugs, new formulations, drug associations, use of drugs already
     available in the market, and immunotherapy.
     To perform pre-clinical and clinical studies of new diagnostic tests against TB using adequate ethical standards and to
     capacitate clinical sites for explanatory and pragmatic trials.
     To carried out pragmatic clinical trials and cost-effective analysis of alternative interventions for TB control that include
     diagnostic methods and health service strategies
     To improve case detection by changing health behaviour and mobilizing communities.
     To build a successful research partnership model that has every potential to stimulate changes in national standards and
     practice in ways that serve country needs.

     Through this approach, it is expected that locally algorithms based on clinical features, antibiotic response, and chest ra-
     diography	will	be	validated,	and	clear	guidelines	issued	about	which	patients	might	also	benefit	from	mycobacteria	culture	
     or new phenotypic / molecular diagnostic techniques. Additionally, the incorporation of new tests into clinical practice
     will better planned and regulated, and national policy makers with better decision-making models will be provided.

36                                                                                                                      ESM 2009

EXpEriENCE Of a SuCCESSful myCObaCTEriOlOgy
labOraTOry NETWOrK iN ESpiriTO SaNTO- brazil

Moisés Palaci
Universidade Federal do Espírito Santo,Vitória, Brazil

The State of Espírito Santo occupies an area of approximately 6,750 square miles on the coast of Brazil. The economy
is mainly based on the production of steel, harborage activity, agriculture, a large number of small industries, and tour-
ism. The population of the State is approximately 3.2 million, with the majority living in metropolitan Vitória, the capital,
which is located on an island and connected to the mainland by several bridges. The annual incidence of TB on the island
of Vitória is approximately 70 cases per 100,000 inhabitants. Each year approximately 1,500 new cases of TB (65% smear
positive) are reported for the State of Espírito Santo with 60% occurring in the City of Vitória and its 5 neighboring cities.
The Núcleo de Doenças Infecciosas has organized a local network of mycobacteriology laboratories in the Epírito State,
Brazil, Five local laboratories have been integrated into this network. This network was established by NDI researchers
and is committed to keep a partnership with the City Department of Health of each location and their laboratories. The
first	phase	of	these	partnerships	consisted	in	reforming	and	restructuring	the	laboratories	to	enable	them	to	accomplish	
the technical requirements, data processing, and biosafety regulations involved in these projects. To this extent, basic
equipment and computers were installed to allow for the maintenance of mycobacterial culture and sharing of data over
an Internet database. The second phase consisted of training laboratory staff to properly and safely complete the neces-
sary bench work and data processing procedures. Therefore, a considerable efforts and time has been spent for setting
up and to keep this system working. With the establishment of this network, NDI has gained earlier access to TB patients
and better conditions to conduct several clinical trials, including IND trials. In addition, capacitating local TB laboratories
in the metropolitan region of Vitória to perform mycobacterial cultures, allowed us to increase the detection rate of TB
cases at about 24%.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                    37

     SCrEENiNg Of mOlECulES WiTh aNTi-Tb aCTiViTy, frOm ThE braziliaN
     CErraDO plaNTS, aND SyNThETiC mETallO-OrgaNiC COmpOuNDS

     Clarice Leite
     Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas

     Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death.
     Among all countries in the Americas, Brazil reports the second-highest TB mortality and morbidity, comprising a preva-
     lence of 62/100.000. The global resurgence of TB and the rapid emergence of MDR-TB, underscore the importance of
     the development of new antituberculous drugs.
     Plants have provided many drugs in the past, and they remain a rich source of novel compounds. Plant extracts are among
     the most attractive sources for developing new drugs and have been shown to produce promising results in the treat-
     ment of several diseases. Our research group deals in projects that integrate the chemical and anti-TB activity of plants
     that compose the bioma of the Brazilian Cerrado, a savannah like vegetation. Many of those plants are commonly used
     as natural remedies by people living in these areas to treat many illnesses. To perform the phytochemical step we used
     chromatographic techniques, and to determine the structure of the isolated compounds we used mainly spectrometric
     methods. To evaluate the activity of the extracts, enriched fractions and pure substances against M. tuberculosis we use
     the resazurin microtiter assay (REMA) and M. tuberculosis H37Rv ATCC 27294 strain. In total were studied 77 extracts
     from 39 plants, distributed into 20 families. From all extracts assayed 23% showed promising activity, bellow or equal to
     125 µg/mL.The triterpene bassic acid from B. fagifolia showed strong antitubercular activity with MIC values of 2.5 µg/mL
     comparable	to	MICs	of	some	first-line	tuberculosis	drugs.	The	results	indicated	that	plants	of	"cerrado"	present	fractions	
     and compounds with promising anti tuberculosis activity.
     By	the	way,	the	use	of	natural	compounds	from	plants	is	problematic,	due	to	difficulty	in	obtaining	pure	substances	and	
     their low availability.
     Within the pipeline of new synthetic compounds with potential effectiveness in the treatment of TB, there are 7 novel
     compounds, which are in various stages of clinical development. Inside this group however, there are complexes that
     associate metals to organic compounds. Using the thiosemicarbazones, semicarbazones and hidrazones derivates as li-
     gands, we proposed the complexation with Vanadium, to obtain organo-metallic compounds. We determined the anti-M.
     tuberculosis activity of these compounds using REMA and the study of the citotoxicity of the ligands and complexes was
     performed using murine macrophage cell line J774. We analyzed 37 compounds (14 free ligands and 23 vanadium com-
     plexes) and from of this, 17 (46%) presented promising MIC values varying between 0.97 and 7.80 µg/mL. The vanadium
     complexes of hydrazones, semicarbazones and tiossemicarbazones derivates showed high antiTB activity, most of the
     time this activity was increased from 2 to 10 times when compared with the free ligands. However due to high citotoxic-
     ity of hydrazones, semicarbazones and tiossemicarbazones derivates, the increase in the activity of the complexes didn’t
     compensate the citotoxicity of the ligands.

38                                                                                                                   ESM 2009

CliNiCal TrialS Of DrugS aND DiagNOSTiC TESTS:
ThE ChallENgES iN myCObaCTEriOlOgy

Moisés Palaci
Universidade Federal do Espírito Santo,Vitória, Brazil

Tuberculosis (TB) therapy has three major microbiologic goals: (1) initial killing of actively multiplying organisms in order
to achieve early control of the disease and reduce infectivity (early bactericidal activity [EBA]); (2) eliminating slowly
growing mycobacteria in order to minimize relapses (sterilizing activity); and (3) preventing the emergence of drug
resistance (1). Currently, two month sputum culture conversion on solid medium is the best established predictor of
treatment outcome. Early bactericidal activity (EBA), determined by the serial decline in sputum M. tuberculosis colony
counts	(CFU),	is	a	commonly	used	tool	for	comparing	new	drugs	to	current	anti-TB	drugs	and	dose	finding.	EBA	has	been	
measured as the rate of decrease in colony counts of mycobacteria in quantitative sputum cultures obtained during the
first	days	of	therapy.	Measurement	of	EBA	is	intended	to	be	a	rapid	means	of	assessing	the	relative	potency	of	new	drugs	
during early treatment. The development of new drugs for TB treatment has been hampered by the lack of an early sur-
rogate	marker	that	reflects	long	term	non-relapsing	cure.	Measurement	of	EBA	by	quantitative	culture	however	is	time	
consuming and labor intensive. The ideal marker would measure events early during treatment and be accurate regard-
less of the drug action or regimen being tested. In this lecture we will explore the potential role and the main limitation
of surrogate markers for TB treatment.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  39

     NEW, EaSy-TO-uSE TOOlS fOr QualiTy-CONTrOllED
     gENOTypiNg Of M. TuBerCuloSiS COmplEX STraiNS

     Caroline Allix-Béguec1,2,3, Christine Hubans3, Stéphanie Ferreira3, and Philip Supply1,2,3
     1 - INSERM U629
     2 - Institut Pasteur de Lille, Lille
     3 - Genoscreen, Lille, France, France

     Mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing has become a major
     method for fast and high-resolution genotyping of Mycobacterium tuberculosis complex isolates. A system based on 24
     loci has been proposed for international standardization. Several population-based studies have been published, showing
     similar predictive value of this method compared to the previous gold standard IS6110 RFLP for studying tuberculosis
     transmission in Western European settings. As a result, this method is being internationally adopted, often in combina-
     tion with spoligotyping, as the new standard method for TB molecular epidemiology. New, easy-to-use tools and options
     have recently become available, which facilitate quality-controlled use of this technique and interpretation of the results
     obtained. MIRU-VNTR typing services are already used by international Reference Centers and laboratories, for out-
     sourcing their genotyping (including of M. bovis strains) and/or for QA/QC evaluation. Quality-controlled MIRU-VNTR
     calibration,	validation	and	typing	kits,	as	well	as	on-site	trainings	greatly	facilitate	standardized	set	up	and	efficient	use	of	
     MIRU-VNTR typing in user’s laboratory. Bioinformatic tools, including MIRU-VNTR Data Manager, have been developed
     for further automating and streamlining the genotyping process, as well as ensuring direct compatibility with MIRU-
     VNTRPlus	Database	for	data	interpretation.	We	hope	that	the	availability	of	these	tools	for	easier	and	more	efficient	
     real-time genotyping will contribute to improve molecular-guided TB control and surveillance.

40                                                                                                                          ESM 2009
Of Oral

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal   41

maSS SpECTrOmETry fOr mOlECular TypiNg Of ThE myCoBaCTeriuM
TuBerCuloSiS COmplEX: ONE plaTfOrm aND mulTiplE aSSay fOrmaTS

C. Honisch1, M. Mosko1, C. Arnold2, S. Gharbia2, S. Feuerriegel3, S. Niemann3
1 - SEQUENOM, Inc., San Diego
2 - Health Protection Agency, London
3 - Molecular Mycobacteriology, NRC for Mycobacteria, Forschungszentrum Borstel, Borstel

The analysis of nucleic acids by mass spectrometry has evolved to a user friendly technology for characterizing DNA,
and RNA via SNP genotyping and comparative sequencing in clinical research, agricultural applications, molecular medi-
cine and non-invasive prenatal diagnostics research. Recently, the technology has become a versatile tool for microbial
detection	and	identification	utilizing	comparative	sequence	analysis.	An	example	is	the	successful	application	to	16S	based	
typing of mycobacteria. Here, we adopted the technology to perform high throughput spoligotyping and detection of
resistance conferring SNPs.

Assays were designed in silico for spoligotyping analysis and detection of key resistance mutations. Both assays were evalu-
ated by using well characterized reference collections.

For MassARRAY 43 spacer oligonucleotide probes were designed and grouped into two multiplexed assays (TypePLEXTM).
Over 200 characterized strains from different reference centers representing the major M. tuberculosis complex lineages
were analyzed by the MassARRAY spoligotyping assays. Results were in concordance with classical spoligotyping data.
For detection of resistance mutations, assay were developed based on the MassCLEAVETM system. Resistance regions
are	amplified	by	PCR	with	a	tagged	primer	system	followed	by	in vitro transcription of both DNA strands. Subsequent
endonuclease digests of the RNA transcripts at the bases cytosine and uracil result in four mixtures of RNA cleavage
products.	Resistance	is	identified	by	correlating	acquired	spectra	with	theoretical	peak	patterns	predicted	for in silico
cleavages	of	sequences	contained	in	a	reference	database.	The	first	assays	have	been	successfully	evaluated	in	a	set	of	
reference strains, further analyses are in progress.

Mass	spectrometry	specific	assay	formats	for	genotyping	and	comparative	sequence	analysis	generate	highly	accurate	
qualitative and quantitative data and provide a toolbox for molecular typing of microbes and viruses. Existing typing
schemes can be translated onto the mass spectrometry platform and new typing schemes can easily be developed.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                 43

     CluSTEriNg Of SpOligO-paTTErNS: TOWarDS aN auTOmaTiON
     Of MyCoBaCTeriuM TuBerCuloSiS COmplEX ClaSSifiCaTiON

     Borile C 1, Refrégier G 2, Labarre M 1, Franz S 1, Mézard M 1, Sola C 2
     1	-	LPTMS,	bât.	100,	Université	Paris-Sud,	Centre	scientifique	d’Orsay,	15	rue	Georges	Clémenceau,	91405	Orsay	cedex
     2	-	IGEPE	bât.	400,	Université	Paris-Sud,	Centre	scientifique	d’Orsay,	rue	Gregor	Mendel,	91405	Orsay	cedex

     Spoligotyping	is	a	typing	method	detecting	the	presence	or	absence	of	specific	regions	called	spacers.	These	spacers	
     are grouped on what is called the DR locus (Direct Repeat locus) that belongs to the CRISPR locus family (Clustered
     Regularly Interspaced Palindromic Repeats). The DR locus in Mycobacterium tuberculosis complex is believed to evolve
     solely by deletion.
     Using	the	spoligotyping	technique,	specific	families	of	Mycobacterium tuberculosis complex strains have been recognized,
     showing	that	the	DR	locus	is	phylogenetically	informative.	Specific	signatures	are	recognized	by	experts	so	that	each	
     spoligo-pattern	is	easily	assigned	to	a	specific	family.	Amazingly	however,	when	using	all	available	softwares	for	clustering	
     the	data,	the	strains	of	a	specific	family	are	not	always	clustered	in	the	same	groupe	using	various	methods.
     We implemented a new algorithm to cluster these data. Until now, the single publicly available software to achieve this
     task, SpotClust, provided only sub-optimal results. Our system is based on an evolutionary model taking into account
     that spacers can be deleted as a large group. This is not the case when using the Jaccard distance in the commonly used
     Bionumerics software, that mimicks a one-by-one spacer deletion process.
     We	will	present	data	showing	under	what	conditions	this	algorithm	gives	significantly	better	results	than	the	commonly	
     used one.This studies leads toward an automation of Mycobacterium tuberculosis	complex	classification,	an	otherwise	time	
     consuming and sometimes debatable topic.

44                                                                                                                      ESM 2009

iDENTifiCaTiON Of ThE iNSErTiON ElEmENT iS6110 iN PHoP
prOmOTEr iN a high TraNSmiSSiON MyCoBaCTeriuM
TuBerCuloSiS STraiN: a CluE TO phENOTypiC VariaTiON

Andrea Sandoval1,2, Andrés Cubillos1,2, Alejandro Reyes3, Nidia Correa2,4, Jaime Robledo2,4, Maria Mercedes Zambrano1,
and Patricia Del Portillo1,2
1 - Corporación CorpoGen, Bogotá, Colombia.
2 - Centro Colombiano de Investigación en Tuberculosis CCITB, Bogotá, Colombia.
3 - Center for Genome Sciences, Washington University School of Medicine, St. Louis, Missouri, USA
4 - Corporación para Investigaciones Biológicas CIB, Medellín, Colombia

The insertion element IS6110 can mediate genetic diversity in Mycobacterium tuberculosis (MTB) strains due to its capac-
ity to move and cause rearrangements, deletions and insertions. Transposition of these elements can therefore affect
gene expression and alter the phenotype of MTB. In this study we analyzed the insertion sites of IS6110 in two clinical
MTB Haarlem genotype strains that present differences in transmissibility as demonstrated in a cohort of patients and
household contacts from Colombia.

Restriction Fragment Length Polymorphism (IS6110-RFLP) was performed and revealed genomic differences between
the two strains. DNA was isolated, digested with XmaI	and	ligated	to	specific	adapters	designed	for	this	purpose.	Ligation	
Mediated	PCR	(LM-PCR)	was	carried	out	to	amplify	the	regions	flanking	IS6110 insertion sites. A library containing the
amplified	products	was	constructed	and	sequenced	clones	were	mapped	against	the	annotated	sequenced	genomes.	PCR	
was	used	to	confirm	the	sites	of	insertion.

Twelve	different	insertions	were	identified;	nine	were	common	to	both	strains	(Rv2336,	Rv1754c,	Rv0963c,	Rv0403c,	
Rv1358,	Rv2813/DR,	Rv2254c,	Rv0795	and	PPE34),	two	were	specific	for	the	high	transmission	strain	(DR	region	and	
transcriptional regulator phoP), and one was found just in the low transmission strain (PPE46).

LM-PCR allowed us to identify IS6110	flanking	regions	and	localized	the	genomic	differences	between	two	strains	with	
contrasting transmission pattern. Most of the insertions occur in conserved hypothetical proteins, followed by proteins
involved in cell wall synthesis and cell processes, regulatory proteins and members of the PE/PPE family. The DR region,
considered a hotspot for insertions, had three insertions, two common to both strains and one in the high transmission
strain. Since the phoP gene regulates several functions implicated in virulence, it is possible that the IS6110 insertion in
the phoP promoter could enhance transmission by acting as a portable promoter and inducing gene expression, as has
been reported before in M. bovis.

Consorcio Colombiano de Investigación en Tuberculosis, CCITB. 4312004

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                 45

     gENOmiC iNTErrOgaTiON Of MyCoBaCTeriuM
     TuBerCuloSiS iSOlaTES frOm brazil

     Oelemann, Maranibia C 1, Gomes, Harrison M 1, Willery, Eve 2, Lima, Karla Valéria B 3, Possuelo, Lia 4, Locht, Camille 5,
     Goguet de la Salmonière,Yves-Olivier L 6, Gutierrez, Maria Cristina 6, Supply, Philip 7, Suffys, Philip N 1
     1 - Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, Rio de Janeiro, Brazil
     2 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur
         de Lille, France
     3 - Evandro Chagas Institute, Belém, Brazil
     4	-	Center	of	Scientific	and	Technological	Development,	Porto	Alegre,	Brazil
     5 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur
         de Lille, France
     6 - Department of Infection and Epidemiology, Institut Pasteur, Paris, France
     7 - Laboratory of Molecular Mechanisms of Pathogenesis of Respiratory Pathogens, INSERM U629 and Institut Pasteur
         de Lille, France
     1 - Laboratory of Molecular Biology Applied to Mycobacteria, Oswaldo Cruz Institute, Rio de Janeiro, Brazil

     The Latin-American Mediterranean (LAM) spoligotype“clade” is among the six major M.tuberculosis spoligotype families and is
     particularly	prevalent	in	South	 merica.	In	certain	regions,	there	is	a	dominance	of	geographically	specific	and	genetically	homo-
     geneous strain lineages.Here,we have studied the genetic diversity and the consistency of the LAM and other families,by analyzing
     Mtb isolates from three Brazilian regions including Rio de Janeiro (South East), Belém (North), and Rio Grande do Sul (South).

     methods and findings
     A	 PCR-based	 standardized	 genotyping	 system,	 based	 on	 amplification	 of	 15	 to	 24	 mycobacterial	 interspersed	 re-
     petitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to
     be	 proficient	 for	 molecular-guided	 evaluation	 of	TB	 transmission.	We	 tested	 the	 applicability	 of	 this	 system	 for	 mo-
     lecular epidemiological analysis of 369 M. tuberculosis isolates from three regions of Brazil. Deligotyping, target-
     ing	 multiple	 large	 sequence	 polymorphisms	 (LSPs),	 and	 MIRU-VNTRplus	 identification	 database	 were	 additionally	
     used	 to	 confirm	 phylogenetic	 identification.	The	 high	 congruence	 between	 the	 different	 typing	 results	 showed	 the	
     countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches.
     Nevertheless, by distinguishing 321 genotypes among the 369 isolates, combined MIRU-VNTR typing and spoligo-
     typing	 demonstrated	 the	 presence	 of	 multiple	 distinct	 clones.	 Noteworthy,	 27	 of	 the	 32	 clusters	 identified	 were	 ex-
     clusively composed of patient isolates from a same city, consistent with expected patterns of local TB transmission.

     Notwithstanding the challenges, high-capacity mycobacterial genotyping may now become a usable tool to guide
     TB control efforts, at least on sentinel sites or targeted risk-populations in high TB burden countries. The interro-
     gation of large sequence polymorphisms (LSPs) revealed that certain lineages or clones present genomic fea-
     tures	 that	 could	 change	 the	 phenotype	 and	 play	 a	 role	 in	 the	 clinical	 properties	 of	 specific	 Mtb	 strains.	This	 is	 the	
     first	 countrywide	 report	 of	 an	 in-depth	 analysis	 on	 the	 genomic	 diversity	 of	 M.	 tuberculosis	 isolates	 from	 Brazil.	

     Fiocruz, CAPES, CNPq, FAPERJ, ICOHRTA, NIH, INSERM and Institut Pasteur.

46                                                                                                                                ESM 2009

pENiTENTiary pOpulaTiON Of MyCoBaCTeriuM TuBerCuloSiS
iN KyrgyzSTaN: EXCEpTiONally high prEValENCE Of ThE
bEiJiNg gENOTypE aND iTS ruSSia-SpECifiC SubTypE

Igor Mokrousov 1, 2,Violeta Valcheva 1, 3, Nurmira Sovhozova 4, Almaz Aldashev 4, Nalin Rastogi 1, Jainagul Isakova 4
1 - Institut Pasteur de Guadeloupe, France
2 - St. Petersburg Pasteur Institute, St. Petersburg, Russia
3	-	Institute	of	Microbiology,	Sofia,	Bulgaria
4 - Institute of Molecular Biology and Medicine, Bishkek, Kyrgyz Republic

To identify genotypes and drug resistance properties of M. tuberculosis isolates from Kyrgyzstan’s prison inmates, a
population with high risk for TB; to compare in regional and global context.

56 M. tuberculosis DNA samples from sputum of HIV-negative Kyrgyz prison inmates, 2008, were typed by spoligotyping,
VNTR (12 MIRU and 3 hypervariable [HV] loci), IS6110-inverse-PCR, LAM-PCR. rpoB and katG mutations were detected
using TB-Biochip kit.

Beijing genotype was detected in 42 of 56 samples. 12-locus MIRU-VNTR typing showed 8 of 56 samples to be mixed
cases; 7 of them contained a Beijing strain. MIRU analysis demonstrated a high homogeneity of the studied collection
(HGI=0.66)	while	28	of	56	strains	had	a	profile	223325153533	corresponding	to	Beijing/M2	subtype	highly	prevalent	
in	different	Russian	settings	(Mokrousov,	2004,	2008).	Four	Beijing	strains	belonged	to	types	M33	and	M70	specific	for	
East	Asia.	Regarding	non-Beijing	variants,	a	comparison	of	their	spoligoprofiles	with	SITVIT2	database	(Institut	Pasteur	
de	Guadeloupe)	revealed	a	presence	of	minor	global	and	Eurasia	(Europe/Russia)	specific	types	SIT262/Haarlem,	SIT73,	
SIT254/LAM found in ex-USSR and Europe but very rare in East Asia and global type SIT42/LAM that is also prevalent in
different parts in Eurasia. Three hypervariable loci, QUB-3232, VNTR-3820 and VNTR-4120, permitted to subdivide 28
Beijing	strains	with	MIRU12	profile	223325153533	into	11	subtypes	shared	by	1	to	9	strains.	RIF	and	INH	resistance	was	
detected in 28% and 55% samples. 13 of 15 MDR strains belonged to Beijing genotype. Comparison of the rate of drug
resistance	mutations	in	different	Beijing	subclusters	revealed	no	statistically	significant	difference.

The penitentiary population of M. tuberculosis	in	Kyrgyzstan	shows	a	strong	affinity	to	the	north-west	Eurasia,	especially,	
Russia, and a weak relatedness to East Asia. Beijing genotype constituted 75% of the entire collection while half of the
studied	strains	belonged	to	the	Beijing/M2	MIRU-defined	subtype	that	is	the	major	Beijing	variant	in	Russia.	MDR-TB	was	
detected in 27% samples that is similar to the Kyrgyzstan’s civilian population. IS6110-inverse PCR and HV-VNTR loci
were shown to be useful for detection of and subtyping within the Beijing genotype, directly in sputum-extracted DNA.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                 47

     ThE uNiQuE ENDEmiC NaTurE Of bEiJiNg gENOTypE
     STraiNS iN OKiNaWa, ryuKyu iSlaNDS Of JapaN aS rEVEalED
     by NEWly DESCribED 15 aND 24-lOCi miru-VNTr TypiNg SChEmES

     Julie Millet, 1 Chika Miyagi-Shiohira, 2 Nobuhisa Yamane, 2 Igor Mokrousov, 1,3 Nalin Rastogi 1
     1 - Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe
     2 - Department of Laboratory Medicine, Graduate School and Faculty of Medicine, University of the Ryukyus, Okinawa, Japan
     3 - St. Petersburg Pasteur Institute, St. Petersburg, Russia

     Tuberculosis (TB) in the eastern Asiatic countries is mainly caused by strains of Mycobacterium tuberculosis belonging to
     the Beijing lineage which was identified back in early nineties using IS6110-RFLP and spoligotyping. Associated with mul-
     tiple drug-resistance (MDRTB), this highly homogeneous genogroup is characterized by little molecular diversity. Several
     studies have recently emphasized the utility of using minisatellites in conjunction with IS6110-RFLP or spoligotyping for a
     better discrimination of Beijing strains. In a recent study, we genotyped Beijing TB strains collected in Okinawa (Ryukyu
     Islands, Japan), by using a discriminative selection of 8 MIRU loci (chosen from the classical 12-loci MIRUs), and 7 QUB
     markers. This typing scheme excluded 4 MIRU loci (MIRU2, 4, 20, and 24), that were found to have a too low discrimina-
     tory power within an in-house Beijing database containing 694 strains (Millet et al., J. Clin. Microbiol. 2007, 45:3606–3615).
     In the present study we evaluated the full “classical” 12-loci typing as compared to the newly described 15-loci and 24-
     loci MIRU-VNTR typing schemes, which include 9 and 12 new MIRU loci respectively.We compared the results obtained
     in Okinawa (an insular setting; n=72) with those recently published for Osaka (n=174 strains) and Kobe (n=175) (Wada
     et al., FEMS Microbiol Lett. 2009, 291:35-43). A higher discriminatory power of 15-loci versus 12-loci format was seen
     through percentage of clustered isolates; clustering for 12-loci format in Osaka, Kobe, and Okinawa was 78.3, 81.7, and
     68.1% respectively, as compared to 55.7, 48.0, and 37.1% using 15-loci format. Corresponding discriminatory index (HGI)
     in Osaka, Kobe, Okinawa were 0.901, 0.936, and 0.944 respectively for 12-loci format, as compared to 0.989, 0.989, and
     0.992	for	15-loci	format.	A	finer	comparison	of	15-loci	patterns	in	the	3	settings	revealed	that	contrary	to	Kobe	and	
     Osaka which shared together a high number of similar patterns (25/114 and 25/107 respectively), Okinawa shared a
     single pattern out of 55 with Kobe, and none with Osaka. The full 24-loci format results further reduced the clustering
     observed (from 37.1% to 20%, HGI 0.996). The results analyzed by drawing a minimum spanning tree underlined the
     unique endemic nature of the Beijing genotype strains in the insular setting of Okinawa, and suggest a local evolution of
     M. tuberculosis Beijing genotype in this island starting from a common pool in mainland Japan.

48                                                                                                                      ESM 2009

MyCoBaCTeriuM TuBerCuloSiS gENETiC DiVErSiTy iN SOuTh KOrEa

Isdore Chola Shamputa1, Jongseok Lee2, Caroline Allix-Béguec3, Eun-Jin Cho2, Ji-im Lee2, Jin Hong Min4,
Lisa C. Goldfeder1, Jin Hee Kim4, Hyung Seok Kang4, Soo Hee Hwang4, Seok Yong Eum2 ,Hyeyoung
Lee5, Seung Kyu Park2,4, Philip Supply3,6, Sang Nae Cho7, Laura E.Via1, Clifton E. Barry III1
1 - Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
    Bethesda, Maryland
2 - International Tuberculosis Research Center, Masan South Korea
3 - Genoscreen, Lille, France
4 - Masan National Tuberculosis Hospital, Masan, South Korea
5 - Department of Biomedical Laboratory Sciences,Yonsei University, Wonju, South Korea
6	-	Centre	National	de	la	Recherche	Scientifique,	Institut Pasteur de Lille/Institut de Biologie de Lille, Lille France
7 - Department of Microbiology,Yonsei University College of Medicine, South Korea

South Korea has recorded a nine fold decrease in the incidence of TB in the last four decades, however challenges of TB
control	remain	significant	as	the	Republic	of	Korea	is	among	the	30	countries	with	the	highest	numbers	of	estimated	
MDR-TB cases. Genotypic analysis of M. tuberculosis has greatly contributed to the control of TB by providing information
on transmission dynamics, assessing clonal distribution and expansion of the tubercle bacilli, in investigating of outbreaks
and pseudo-outbreaks, and in identifying laboratory cross contamination. However, there is limited information on the
molecular epidemiology of TB in South Korea.
Genetic diversity of 208 M. tuberculosis isolates from subjects enrolled in a prospective observational cohort study at
National Masan Tuberculosis Hospital in South Korea was determined using spoligotyping, IS6110-RFLP and standardised
MIRU-VNTR typing based on 24 loci. MIRU-VNTR analysis was performed independently and blindly from spoligotyping
and IS6110-RFLP results.
Analysis of MIRU-VNTR typing results use in conjunction with MIRU-VNTRplus database predicted that 202 (97.1%)
isolates	belonged	to	the	Beijing	genotype.	This	prediction	was	fully	confirmed	by	spoligotyping.	Congruence	analysis	indi-
cated the prevalence of 3 branches among Beijing strains respectively named Korea, Masan and China. Preliminary analy-
sis did not show differential distribution of resistant, MDR or XDR strains among the 3 branches. MDR or XDR isolates
were	detected	in	at	least	4	clusters	concordantly	identified	by	the	3	genotyping	methods.	Using	MIRU-VNTR typing and
spoligotyping, 23 clusters of 66 isolates were detected indicating a relatively large diversity of circulating strains despite
the prevalence of the Beijing lineage.
Standardised	 MIRU-VNTR	 typing	 appeared	 efficient	 as	 a	 first	 line	 discriminatory	 method	 for	 this	 country	 with	 high	
prevalence of Beijing strains. However, preliminary analyses suggest that clustered cases may not be epidemiologically
linked and rather correspond to endemic strains circulating in South Korea.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                      49

     COrrElaTiON Of mOlECular rESiSTaNCE mEChaNiSmS
     aND phENOTypiC rESiSTaNCE TO firST-liNE DrugS iN
     MyCoBaCTeriuM TuBerCuloSiS STraiNS frOm SiErra lEONE

     Silke Feuerriegel1, Susanne Homolka1, Erik Post2, Barbara Oberhauser2, Abu Garawani George3, Lars Westman3, Foday
     Dafae4, Sabine Rüsch-Gerdes1, Stefan Niemann1
     1 - Research Center Borstel, National Reference Center for Mycobacteria, Parkallee 18, 23845 Borstel, Germany
     2 - German Leprosy and TB Relief Association, Würzburg, Germany
     3 - National Leprosy/TB Reference Laboratory, Freetown, Sierra Leone
     4 - National Program Manager for Tuberculosis and Leprosy, Freetown, Sierra Leone

     Resistance	to	first-line	drugs	(INH,	RMP,	SM,	EMB	and	PZA)	displays	a	serious	problem	for	the	treatment	of	Mycobacterium
     tuberculosis infections. Resulting MDR-tuberculosis (resistance to at least INH and RMP) implies an enormous threat for
     tuberculosis control worldwide. It is therefore of great importance to analyze the genetic basis of clinical resistance,
     especially in high incidence settings and to correlate molecular resistance data with phenotypic resistance data.

     A total of 97 M. tuberculosis strains from previously treated patients in Sierra Leone which displayed resistance to INH
     (n=32), RMP (n=16), SM (n=39), EMB (n=15) and PZA (n=10), respectively, were sequenced concerning the predomi-
     nant resistance determining regions (katG, rpoB, rrs, rpsL embB and pncA). Strains resistant to INH with no mutation in
     katG were also sequenced in the promoter regions of inhA and ahpC. From all strains analyzed 11 showed resistance to
     INH and RMP and were therefore MDR-TB. Drug susceptibility testing was done by using the proportion method on
     Löwenstein-Jensen medium.

     Among INH resistant strains the most common mutation detected is katG315 (65.6%). From all 32 resistant strains
     3 had mutations in the promoter regions of inhA and ahpC. Among RMP resistant strains 50% displayed mutations at
     rpoB531.	Sensitivity	and	specificity	of	the	DNA	sequencing	of	katG and rpoB for detection of INH and RMP resistance
     were about 90%. Concerning SM resistance none of the resistant strains showed any mutation in rrs, but 46.2 % had rpsL
     mutations, either at codon 43 or 88. Among EMB resistant strains 46.7% showed mutations at embB306 and 13.3% at
     codon 332 and 497 respectively. Strains resistant to PZA displayed a number of different mutations throughout the pncA
     gene.	Specificities	of	sequencing	of	rpsL, embB and pncA for detection of the respective resistance phenotypes were high
     (96-100%), whereas sensitivities were lower (46% for sequencing of rpsL, 60% for embB, 70% for pncA).

     There is a close correlation between data from molecular and phenotypic resistance testing for the determination of
     INH and RMP resistance in strains from Sierra Leone. Sensitivities of sequencing of resistance determining genes for SM,
     EMB and PZA resistance were low due to so far unknown resistance determining regions. Thus it is of great importance
     to gather information on further mechanisms leading to drug resistant MTB strains in different settings.

50                                                                                                                 ESM 2009

lam aND hiV: COrrElaTiON Or CO-iNCiDENCE?

McNerney, Ruth, Mallard, Kim
London School of Hygiene & Tropical Medicine

The deadly synergy between TB and HIV presents a serious challenge to heath and development.Three decades after the
onset of the AIDS pandemic the region with the highest prevalence of HIV infection is sub-Saharan Africa, followed by
the Caribbean and Latin America. Molecular typing methods permit differentiation of M. tuberculosis into strain families
or genotypes. We have undertaken analysis of genotyping data to compare the prevalence of spoligotype lineage with
that of HIV infection. Data was taken from the peer reviewed literature, surveys testing only drug resistant strains or
those not fully describing the genotype population were excluded.The Latin-American-Mediterranean (LAM) lineage are
identified	as	typically	lacking	spoligotype	spacers	corresponding	to	oligonucleotides	21	to	24	and	33	to	36.	They	have	
been observed in many geographic locations and include the F11 family reported in South Africa. Our analysis suggests
that LAM strains are more frequently found in regions with a high prevalence of HIV. Conversely, they are rare in set-
tings where HIV has yet to emerge as a major threat to public health. Interestingly LAM genotypes appear less frequent
in African countries such as Uganda, Tanzania and Cameroon which have lower HIV prevalence’s than countries such as
Malawi, Zimbabwe and South Africa where HIV rates are in excess of 10%. Why tuberculosis of this genotype should be
linked	to	the	HIV	epidemic	remains	a	matter	of	speculation.	Although	socioeconomic	and	political	factors	play	a	signifi-
cant role they do not explain the uneven distribution of HIV in sub Saharan Africa. It is now evident that there is diversity
in	the	interaction	of	M.	tuberculosis	with	its	host	and	strains	of	differing	genotype	appear	to	elicit	subtle	but	significant	
differences in immune response. We present the hypothesis that strains of tuberculosis belonging to the LAM genotype
are associated with enhanced transmission of TB in immunosuppressed populations.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                   51

     MyCoBaCTeriuM CelaTuM: aN EmErgiNg
     paThOgEN iN ThE immuNOCOmpETENT. a CaSE rEpOrT

     Lyberopoulos Panagiotis 1, Frangopoulos, Fragiskos1, Kontos, Fanourios 2, Zerva Loukia 2, Malagari Aikaterini 3, Papiris Spyridon 1
     1 - 2nd Department of Pulmonary Medicine, Attikon University Hospital, Athens, Greece
     2 - Department of Clinical Microbiology, “Attikon” University Hospital, Athens, Greece
     3 - 2nd Department of Radiology, Attikon University Hospital, Athens, Greece

     Mycobacterium celatum is a pathogen for immunocompromised patients but there is little evidence of its pathogenicity
     among immunocompetent individuals. We report the isolation of M. celatum from a middle-aged immunocompetent wom-
     an	with	bronchiectasis	that	was	initially	considered	a	non-significant	finding,	but	eventually	proved	extremely	dangerous.	
     A 55-year-old Caucasian female never smoker, was referred to our hospital complaining of productive cough over the
     preceding two weeks accompanied by general malaise. She lived in an urban area and had no history of alcoholism, use
     of steroids or immunosuppressive drugs. A high resolution computed tomography (HRCT) of the chest showed ill-
     defined	peribronchial	densities	contained	within	the	right	upper	lobe	with	air-bronchograms	and	branching	pattern	with	
     a few opacities of tree-in bud formation. There was no lymphadenopathy or pleural effusion.
      Smears	of	five	sputum	specimens	(before	any	treatment)	were	negative	for	acid-fast	bacilli.	These	specimens	were	pro-
     cessed with the NALC/NaOH method and cultured in the BACTEC MGIT 960 system (Becton Dickinson, USA) and
     on Löwenstein-Jensen medium (BioMerieux, France). Two mycobacterial strains were recovered from these cultures
     and	both	strains	were	identified	as	M. celatum with the use of the Genotype Mycobacterium CM and AS assays (Hain-
     Lifescience). PCR restriction analysis (PRA) of a 439-bp fragment of the hsp65 gene and sequencing of the 16S rRNA
     gene	confirmed	that	they	were	M. celatum type 1.
     From	the	five	aforementioned	sputum specimens Pseudomonas aeruginosa and Serratia liquefaciens were also isolated and
     were	both	sensitive	to	ciprofloxacin.		Ciprofloxacin	(1gr/day,	per-os)	plus	low	dose	azithromycin	(500mg	twice	a	week)	
     were administered. Two months later, a control chest HRCT scan showed a considerable improvement; no pathogens
     were isolated from consecutive sputum samples.
      Eight months later the patient complained for general malaise, night sweats and cough with blood tinged sputum. Imaging
     studies revealed a lung apical cavity.Two bronchial washing and two sputum cultures were smear positives and M. celatum
     was recovered from all.
     In conclusion, when the American Thoracic Society criteria for the diagnosis of NTM infection are met and M. celatum
     is	identified,	it	should be considered as the pathogen causing the pulmonary infection even in patients with apparently
     normal cellular immunity.

52                                                                                                                            ESM 2009

MyCoBaCTeriuM aviuM SubSpECiES STraiNS
frOm humaN aND aNimal OrigiN

Radomski, Nicolas 1, Thibault, Virginie 2, Karoui, Claudine 1, De Cruz, Krystel 1, Cochard, Thierry 1, Gutiérrez, Cristina 3,
Supply, Philip 3, Biet, Frank 2, Boschiroli, María Laura 1
1 - AFSSA-LERPAZ, Maisons Alfort
2 - INRA-UR1282, Nouzilly
3 - INSERM U629-Institut Pasteur, Lille

The Mycobacterium avium sbsp. avium and Mycobacterium avium sbsp. hominissuis are pathogenic emergent bacterial spe-
cies belonging to the Mycobacterium avium complex (MAC). These two subspecies can infect and lead to disease to nu-
merous animal species: birds, pigs, cattle, deer, sheep, goats, horses, cats, dogs, etc. Moreover, Mycobacterium avium subspe-
cies have been isolated in HIV infected patients and in immuno-competent patients with pulmonary pathologies. MAC is
an ubiquitous bacterial group that can be found in water, in the environment, or even in food. A molecular typing study
was undertaken with the primary goal of improving the taxonomic and epidemiological knowledge of MAC. Different
strains of Mycobacterium avium sbsp. avium, Mycobacterium avium sbsp. hominissuis, and also of Mycobacterium avium sbsp.
silvaticum, isolated from animals, humans, and the environment, were typed by two methods: the Restriction Fragments
Length Polymorphism on insertion sequences IS1311 (RFLP1311), which is a standard method to characterise MAC, and
the Variable Number of Tandem Repeats-Mycobacterial Interspersed Repetitive Units (VNTR-MIRUs) characterisation,
which has been recently developed on Mycobacterium avium sbsp. paratuberculosis. Our results demonstrate that the
discrimination power of both methods is comparable (DI of more than 0.92). Therefore, VNTR-MIRUs seems a much
better typing method since it is a PCR based method that requires little genetic material for being performed, which is
easy to standardize in any laboratory and because the deduced numerical patterns do not require special softwares for
being compared in an inter-laboratories fashion.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                   53

     ThE CharaCTErizaTiON Of myCObaCTEria frOm aN
     OuTbrEaK SuggESTS a rEViSiON Of ThE TaXONOmiC STaTuS Of
     mEmbErS Of ThE MyCoBaCTeriuM CHelonae-aBSCeSSuS grOup

     Sylvia Cardoso Leão1, Enrico Tortoli2, Cristina Viana-Niero1, Suely Yoko Mizuka Ueki3, Karla Valeria Batista Lima4, Maria
     Luiza Lopes4, Jesus Yubero5 María Carmen Menendez5, Maria Jesus Garcia5
     1 - Universidade Federal de São Paulo, São Paulo, Brazil
     2 - Centro Regionale di Riferimento per la Diagnostica dei Micobatteri, Ospedale di Careggi, Firenze, Italy
     3 - Instituto Adolfo Lutz, São Paulo, Brazil
     4 - Instituto Evandro Chagas, Belém, Brazil
     5 - Universidad Autonoma de Madrid, Madrid, Spain

     An outbreak of infections by rapidly growing mycobacteria (RGM) related to invasive procedures has been ongoing in
     Brazil since 2004. Isolates from patients submitted to laparoscopic or plastic surgery and to mesotherapy, a cosmetic
     procedure,	were	previously	identified	by	molecular	methods	as	Mycobacterium massiliense and M. bolletii, respectively. The
     similarity of rpoB and hsp65 sequences from the clinical isolates and the corresponding sequences from both M. massil-
     iense and M. bolletii	type	strains	were	above	the	accepted	limit	for	interspecies	variability,	leading	to	conflicting	results.	
     Therefore, an extensive characterization was carried out with six Brazilian clinical isolates from this outbreak study and
     type strains from the members of the M. abscessus-M. chelonae group – M. abscessus, M. chelonae, M. immunogenum, M.
     massiliense and M. bolletii.	Phenotypic	identification	was	performed	by	biochemical	tests,	high	performance	liquid	chro-
     matography	(HPLC)	and	drug	susceptibility	testing.	Molecular	identification	included	PCR-restriction	enzyme	pattern	
     analysis (PRA) of the hsp65 gene, as well as rpoB and hsp65 gene sequencing and analysis of the corresponding phylo-
     genetic trees. DNA-DNA hybridization (DDH) and restriction fragment length polymorphism (RFLP) of the 16S rRNA
     gene were used as the gold standards for RGM speciation. The clinical isolates and the M. abscessus, M. massiliense and
     M. bolletii type strains could not be separated by phenotypic tests and were grouped in the phylogenetic trees obtained.
     Also,	DDH	results	confirmed	>70%	relatedness,	and	indistinguishable	RFLP	16S	rRNA	patterns	were	obtained.	On	the	
     contrary, separation from M. chelonae and M. immunogenum was supported by results from PRA-hsp65, rpoB and hsp65
     phylogenetic trees, DDH and RFLP-16S. Taken together, these results led to the proposition that M. abscesus, M. massil-
     iense and M. bolletii represent a single species, that of M. abscessus. Two subspecies are also proposed, M. abscessus subsp.
     abscessus and M. abscessus subsp. massiliense which can be distinguished by two different PRA-hsp65 patterns, differing in
     a single Hae III band, and by differences in rpoB (3.4%) and hsp65 (1.3%) sequences.

54                                                                                                                       ESM 2009

ThE SuNNy SiDE Of myCObaCTEria

Santos, Ricardo 1, Marques, Marco 2, Oliveira, Pedro 2, Carvalho, Filipe 2, Carvalho, Carla 2, Monteiro, Gabriel 2, Cabral,
Joaquim 2, Frade, Raquel 2, Silva, Maria 3, Fernandes, Pedro 4
1 - Laboratório de Análises
3 - Faculdade de Farmacia, Univ. Coimbra

Mycobacteria are typically seen as cause of morbidity and mortality worldwide. There is however a Jekyll side to these
acid-fast bugs. Taking advantage of some key metabolic pathways, as well as of the particular nature of the hydrophobic
cell wall, that enables operation in aggressive, non-conventional environments, non-pathogenic mycobacteria can be
used for the production of compounds with application in the pharmaceutical, food and environmental areas. The pres-
ent work aims to illustrate this concept, by providing examples of the application of non-pathogenic mycobacteria for
the	production	of	steroid	and	siderophore	molecules,	and	for	the	pinpoint	modification	of	carbocycles.	The	assessment	
of the required biocatalytic activity and the early stages of process characterization have been carried out in miniatur-
ized bioreactors, allowing for a high level of parallelization, hence providing a high throughput platform for bioprocess
development. Going into detail, Mycobacterium sp. NRRL B-3805 was shown to effectively yield androstenedione (AD),
a key intermediate in the production of therapeutic steroids, from phytosterols, recovered from residues of the paper
industry, while operating in phthalate or liquid silicone environments, an approach that enhances process productivity.
Furthermore, this particular strain was shown to also yield the AD out of several polyhydroxylated steroids. Relying on
the high-throughput methodology, a library of non-pathogenic Mycobacterium spp. siderophore producers was devel-
oped in-house. Scaling-up the production process to bench bioreactor using the most promising strains is envisaged.The
same	high-throughput	methodology	has	been	recently	used	to	screen	non-pathogenic	mycobacteria	for	specific	catalytic	
activity, namely hydroxylation, on carbocyclic molecules, with promising results.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                55

     faST iDENTifiCaTiON Of MyCoBaCTeriuM TuBerCuloSiS iN SpuTum
     aND CulTurES baSED ON ThErmally-aSSiSTED hyDrOlySiS aND
     mEThylaTiON by gaS ChrOmaTOgraphy-maSS SpECTrOmETry

     Erwin Kaal 1,2*, Arend Kolk 3, Sjoukje Kuijper 3, Hans-Gerd Janssen 1,4
     1 - Polymer-Analysis group, Van ’t Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht
         166, 1018 WV Amsterdam, The Netherlands.
     2 - ATAS GL International, P.O. Box 17, 5500 AA Veldhoven, The Netherlands.
     3 - KIT Biomedical Research, Royal Tropical Institute, Amsterdam, The Netherlands.
     4 - Unilever Research and Development,Advanced Measurement and Imaging, P.O. Box 114, 3130 AC Vlaardingen,The Netherlands.

     A fast gas chromatography-mass spectrometry (GC-MS) method with minimum sample preparation is described for
     early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are
     then methylated inside the GC-injector using thermally assisted hydrolysis and methylation (THM). The THM-GC-MS
     procedure	was	optimized	for	the	injection	of	sputum	samples.	For	the	identification	of	Mycobacterium tuberculosis the
     known marker tuberculostearic acid (TBSA) and other potentional markers were evaluated. Hexacosanoic acid in com-
     bination	with	TBSA	was	found	to	be	specific	for	the	presence	of	M. tuberculosis. For validation of the method several
     sputum samples with different viscosities spiked with bacterial cultures were analyzed. The detection limit was better
     than 1 x 104 bacteria/ml. 17 methyl octadecanoic acid was used as standard.The detection limit for this compound was 20
     pg/ml. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed
     in Amsterdam by microscopy after decontamination/concentration and using the new THM-GC-MS method. No false
     positives were found by THM-GC-MS and all patients who were diagnosed with TB were also found positive using our
     newly developed THM-GC-MS method. These results show that the new fast and sensitive THM-GC-MS method holds
     great potential for the diagnosis of TB.
     This work was partially supported by the Foundation of New Diagnostics (FIND) and the Optimus Foundation.

56                                                                                                                   ESM 2009

hOW WilD-TypE miC DiSTribuTiONS CaN bE uSEful TO
DETErmiNE CliNiCal brEaKpOiNTS iN MyCoBaCTeriuM TuBerCuloSiS

Ängeby, Kristian 1, Juréen, Pontus 2, Giske, Christian 1, Chryssanthou, Erja 1, Werngren, Jim 2, Hoffner, Sven 2, Kahlmeter,
Gunnar 3, Sturegård, Erik 4, Schön, Thomas 5
1 - Department of Clinical Microbiology, Karolinska University Laboratory and Karolinska Institute, Sweden
2 - Department of Bacteriology, Swedish Institute for Infectious Disease Control, Sweden
3 - Department of Clinical Microbiology,Växjö Hospital, Sweden
4 - Department of Clinical Microbiology, Malmö University Hospital, Sweden
5 - Department of Clinical Microbiology, Kalmar County Hospital, Sweden

The increasing prevalence of multidrug resistant (MDR) and extensively drug resistant (XDR) tuberculosis (TB) un-
derscores the need for accurate drug susceptibility testing (DST) .It is unfortunate that the current critical antibiotic
concentrations	(breakpoints)	are	based	mostly	on	empiry	and	only	to	a	limited	extent	on	scientific	evidence.	This	is	
especially true for second line drugs.
For most other bacterial pathogens, wild-type minimal inhibitory concentration (MIC) distributions has been successfully
applied as one of other tools (such as pharmacokinetic and pharmacodynamic data) to determine clinical breakpoints for
DST.	A	microorganism	is	defined	as	wild-type	by	the	absence	of	acquired	and	mutational	resistance	mechanisms	to	the	
drug in question. In modern breakpoint determination, breakpoints that divide divide wild type distributions of MICs are
avoided. An isolate with a MIC above the wild-type distribution is highly likely to harbor resistance mechanisms and is
considered clinically resistant until there is evidence to the contrary.
At present, wild-type MIC-distribution data for M. tuberculosis is scarce, presumably in part because of the complexity of
DST. In order to establish wild-type MIC distributions for M. tuberculosis we used a 96-stick replicator method, which
allowed	us	to	efficiently	determine	the	MICs	of	95	clinical	isolates		simultaneously	for	four	first	line	drugs	(isoniazid,	
rifampicin, ethambutol and streptomycin) and 15 second line drugs (including four quinolones, four injectables, ethiona-
mide,	prothionamide,	cykloserine,	PAS,	thioacetazone).	Our	findings	clearly	demonstrate	how	wild-type	MIC	distributions	
among	other	tools	can	be	used	to	define	clinical	breakpoints	also	in	M.	tuberculosis.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                 57

     mOlECular TEChNiQuES TO mONiTOr Tb paTiENTS’ TrEaTmENT:
     SElECTiVE rEmOVal Of DNa frOm DEaD baCTEria iN miXED
     pOpulaTiONS by uSE Of EThiDum mONOaziDE

     Miotto Paolo, Cirillo Daniela M.
     Emerging	Bacterial	Pathogens	Unit,	San	Raffaele	Scientific	Institute,	Milan	–	ITALY

     Drug-resistant tuberculosis (TB) represents a public health problem worldwide and is considered a real threat for TB
     control	programs.	Modern	nucleic	acid	amplification	techniques	(NATs)	that	exploit	nucleic	acids	signals	from	clinical	
     samples represent useful tools to allow rapid detection of resistant M. tuberculosis	strains.	Identification	of	resistant	bac-
     teria in clinical samples is fundamental in order to prevent inadequate treatment and further development of resistant
     strains. However, NATs can not be used for patients’ follow-up because DNA-derived signals can originate from nonviable
     bacterial cells and, therefore, generate data that could be misinterpreted. A method developed for microbial food patho-
     gens and already tested on environmental samples is here tested to distinguish between live and dead mycobacteria.
     Ethidium monoazide bromide (EMA) is membrane impermeant that intercalates into both extracellular DNA and DNA
     in nonviable cells and is excluded from viable bacteria. Exposure to a light source renders EMA-DNA incapable of con-
     tributing to PCR.
     Liquid culture of M. tuberculosis H37Rv was heat inactivated by incubation at 95 °C for 30 min and then treated with 6
     µM, 9 µM, 12 µM, respectively, EMA concentrations. EMA treatment consists of 20 min of incubation at +4 °C in the dark
     followed by 30 min of exposure to a 500 Watt light. Controls used in the study were: inactivated bacteria untreated with
     EMA, live bacteria untreated with EMA, and live bacteria treated with the same 3 EMA concentrations. After EMA treat-
     ment, samples and controls were processed for DNA extraction by thermal lysis (95 °C for 30 min) and directly used in
     PCR. Amplicons were analyzed by capillary electrophoresis (Agilent Technologies).
     EMA	treatment	at	all	three	concentrations	tested	suppressed	PCR	amplification	from	heat	inactivated	cultures	without	
     affecting	amplification	from	live	bacteria,	proving	that	EMA	could	be	used	also	for	mycobacterial	samples.
     Preliminary studies were also conducted on mixed live/dead populations using two strains with a different MIRU-VNTR
     genotype. Performing MIRU typing on mixed samples after EMA treatment, only the live strain could be detected.
     Comparison	of	treated	and	untreated	samples	highlighted	the	significant	contribution	that	nonviable	bacteria	can	make	
     to DNA-based diagnostic analysis. EMA approach could be a simple and effective means to monitor patients’ treatment
     allowing the use of NATs during follow-up.

58                                                                                                                      ESM 2009

MyCoBaCTeriuM TuBerCuloSiS iS ablE TO
aCCumulaTE aND uTilizE ChOlESTErOl

Brzostek Anna 1,2, Pawelczyk, Jakub 2, Rumijowska-Galewicz, Anna 2, Dziadek, Bozena 3, Dziadek, Jaroslaw 2
1 - Laboratory of Mycobacterium Genetics and Physiology PAS, Institute for Medical Biology
2 - PAS, Institute for Medical Biology
3 - University of Lodz, Department of Immunoparasitology

Mycobacterium tuberculosis, is the causative agent of tuberculosis that infects one third of the human population. Tubercle
bacilli are able to persist in a dormant state, from which they may reactivate disease state. The presence of lipid me-
tabolism genes in the genome of M. tuberculosis suggests that lipids, including steroids, are important carbon and energy
sources for this pathogen. One potential nutrient that is available in the mammalian host is cholesterol, a major sterol
of the plasma membrane. Cholesterol is essential for the uptake of mycobacteria by macrophages, and it has been found
to accumulate at the site of M. tuberculosis entry. Moreover cholesterol can be utilized by fast-growing, non-pathogenic
mycobacteria,	but	pathogenic	mycobacteria	might	not	be	able	to	use	cholesterol.	Here,	we	show	for	the	first	time	that	M.
tuberculosis grown in media containing carbon source other than cholesterol is able to accumulate cholesterol in the free
lipid zone of its cell wall. This cholesterol accumulation decreases the permeability of the cell wall for the primary anti-
tuberculosis drug, rifampin, and partially masks the mycobacterial surface antigens. Furthermore, M. tuberculosis was able
to grow on mineral media supplemented with cholesterol as the sole carbon source. Targeted disruption of the Rv3537
(kstD) gene inhibited growth due to inactivation of the cholesterol degradation pathway, as evidenced by accumulation of
the	intermediate,	9-hydroxy-4-androstene-3,17-dione.	Our	findings	that	M. tuberculosis is able to accumulate cholesterol
in the presence of alternative nutrients and use it when cholesterol is the sole carbon source in vitro may facilitate future
studies into the pathophysiology of this pathogen.
The	work	was	supported	partially	by	grants	ICGEB	(CRP/POL07-01)	and	State	Committee	for	Scientific	Research	(N302	
035 31/3172).

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  59

     myCOliC aCiD-iNDuCED ifN-γ prODuCTiON by
     CD1-rESTriCTED T CEllS frOm TubErCulOuS paTiENTS

     Rodríguez-Güell, E 1, Alonso, C 2, del Val-Romero, B 2, Clivillé, R 2, Secanella,SP 1, Roura-Mir,C 3, Cañete, C 4, Navarro, A 4,
     de Gispert, FX 4, Luquin, M 1, Julián,E 1
     1. Dept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)
     2. Dept. Microbiologia, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)
     3. Dept. Fisiologia i Immunologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)
     4. Unitat de Respiratori, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)

     The cell wall of Mycobacterium tuberculosis (MTB) has a large number of structurally diverse lipids. Mycolic acids (MAs) merit
     special	interest	because	their	immunogenicity	has	been	demonstrated	in	CD1-restricted	T	cell	clones;	specifically,	they	are	
     presented by CD1b molecules. To date, the recognition of MAs in tuberculous patients (TB) has not been studied.
     The purpose of the study was to determine whether MAs are recognized by the immune system of TB patients and,
     in the event of this being so, to study the evolution of this response throughout anti-TB treatment.

     Immature dendritic cells (iDCs) isolated from 31 TB patients at the time of diagnosis and throughout anti-TB treatment,
     20 PPD-positive and 20 PPD-negative donors were analysed by cytometry for CD1b surface expression. Subsequently,
     the samples were irradiated and cultured with autologous lymphocytes in the presence of phytohemaglutinin, MTB and
     purified	MAs	as	stimuli.	After	48	hours,	IL-10	and	IFN-γ	were	quantified	by	enzyme-linked	immunosorbent	assay.

     No	significant	differences	among	the	surface	expression	of	CD1b	in	iDCs	from	healthy	donors	or	TB	patients	were	ob-
     served at any time during the disease. iDCs from all the individuals were therefore able to present MAs.
     Median levels of stimuli-induced IL-10 in samples obtained at the outset of anti-TB treatment were lower than those
     obtained	at	the	end;	however,	no	significant	differences	were	observed.	Nor	were	any	differences	observed	among	the	
     IL-10 levels obtained from the different groups of individuals.
     In TB patients, MA-induced IFN-γ	median	values	obtained	at	the	end	of	prophylaxis	were	significantly	higher,	statistically,	
     than those elicited at the beginning. Grouping the samples of the 31 TB patients in terms of collection time, IFN-γ median
     levels followed an upward trend throughout anti-TB treatment, reaching maximum levels at the point of disease cure.
     Furthermore, in PPD-negative donors IFN-γ	median	values	were	lower	than	those	from	PPD-positive	donors,	significant	
     differences only being established when MTB was used as antigen.

     The	specific	cellular	immune	response	against	MAs	in	TB	patients	described	here	for	the	first	time	suggests	a	potential	
     immunoprotective role for MAs.

60                                                                                                                        ESM 2009

TubErClE baCilli : TraNSmiSSiON ElECTrON miCrOSCOpy aNalySiS

Parissa Farnia(PhD)1. Ali Akbar Veleyati (MD)1, Mohammal Reza Masjedi (MD)1, 2 Tengku Azmi Ibrahim (PhD)2, Payam
Tabarsei	(MD),	Rafiuz	Zaman	Haroun	(MSC)2,	Ho	Oi	Kuan	(MSC))2,	Abdul	Rahman	Omar	(PhD)1
1 - Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Disease (NRITLD), WHO
    Collaborating Centre, Shahid Beheshti University (Medical Campus), Darabad, Tehran ,19556, P.O: 19575/154, Iran.
2 - Microscopic unit, Institute of Bioscience , University Putra Malaysia ,43400 UPM ,Serdang, Selangor Darul Ehsan , Malaysia

Extensively drug resistance tuberculosis bacilli (XDR-TB), is caused by a strain of M. tuberculosis(MTB) that are resistant to
isoniazid	and	rifampin	(which	defines	MDR	tuberculosis)	in	addition	to	any	fluroquinolone	and	at	least	one	of	the	three	
following injectable drugs: caperomycin, kanamycin and amikacin.Viewed under transmission electron microscopy (TEM),
spore like structure was observed inside the XDR-TB bacilli.

The	susceptibility	testing	against	first	and	second	line	drugs	was	performed	on	isolated	M. tuberculosis strains. Subsequently,
a homogenous thick suspension of 107 to 108 cells at exponential phase was prepared and observed under Transmission
Electron Microscopy (TEM). Five isolates from each group (susceptible, MDR, and XDR TB) were used in this study.

Viewed under the TEM, the XDRTB bacilli at exponential phase had three types of cell division 1) 80-70% of bacilli were
looks as norm one with symmetrical or asymmetrical cell division 2) 5-7% with extra ordinary thick cell wall ( 21 to 26
nm ) which was similar to stationary or dormant phase bacilli. But surprisingly the stationary phase XDR-TB bacilli were
at dividing process 3) 15-20% bacilli had spore formation inside them These spores were different from buds or a polar
division that formed during cell branching of MTB. No spore’s and stationary phase bacilli were detected in susceptible
or multidrug resistant strains of MTB.

Dividing phenomena in XDR-TB bacilli with stationary phase appearance will generate new adapted types of M. tubercu-
losis which can resist the effect of all available anti tuberculosis drugs. At the same time, it is possible that under certain
conditions, the XDR-TB bacilli produce spore to overcome the hostile environment. Spore formation in XDR-TB bacilli
should take very seriously from epidemiological point of view.With these new forms of adaptation that occurs in XDRTB
bacilli , how should we treat and control the diseases.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                    61

     grOWTh prOfilE Of CliNiCal iSOlaTES Of MyCoBaCTeriuM
     TuBerCuloSiS COmplEX iN muriNE maCrOpghagES

     Susanne Homolka1, Stefan Niemann1, David G. Russell2 and Kyle H. Rohde2
     1 - Molecular Mycobacteriology, National Reference Center of Mycobacteria, Borstel Germany
     2 - Department of Microbiology/Immunology, Cornell University,Vet. Med. Center, Ithaca, USA

     Mycobacterium tuberculosis and other members of the Mycobacterium tuberculosis complex (MTC) remain a major cause of
     morbidity and mortality worldwide. Several studies based on spoligotyping, IS6110 fingerprinting	and	MIRU-VNTR	typing
     demonstrated	that	the	global	population	structure	of	MTC	is	defined	by	phylogeographical	lineages	and	genotypes	that	are	
     also	associated	with	pathogenic	differences.	However,	the	influence	of	strain	genomic	variation	on	the	outcome	of	infection	
     and the clinical presentation are not completely understood. In our study, we investigated growth differences of 15 strains
     of	five	different	genotypes	in	comparison	to	the	CDC1551	reference	strains	in	liquid	culture	and	in	macrophages.	

     Murine bone marrow derived macrophages were infected with liquid culture of different clinical isolates (MOI 3:1;
     2x106CFU/ml). Survival of strains in resting and activated macrophages was determined at different time points up to 11
     days post-infection. Additionally, growth kinetics of all strains in 7H9 liquid media were analyzed.

     Growth	analyses	of	clinical	isolates	in	liquid	culture	based	on	OD	measurements	showed	no	significant	differences	in	
     comparison	to	CDC1551.	However,	we	observed	both	strain-	and	genotype-specific	survival	and	growth	profiles	within	
     resting and activated macrophages.

     The	 genomic	 diversity	 of	 clinical	 isolates	 influences	 the	 complex	 interaction	 of	 the	 pathogen	 with	 host	 phagocytes.	
     Further	analyses	to	correlate	intracellular	gene	expression	profiles	with	the	outcome	of	infection	are	in	progress.

62                                                                                                                           ESM 2009

phagOlySOSOmE TraNSlOCaTiON Of MyCoBaCTeriuM Szulgai

Jakko van Ingen1,2, Nicole van der Wel3, Richard Dekhuijzen2, Martin Boeree2, Dick van Soolingen1
1 - National Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment, Bilthoven,
    the Netherlands
2 - Department of Pulmonary diseases, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands
3 - Department of Cell Biology, Netherlands Cancer Institute, Amsterdam, the Netherlands

Bacterial virulence factors in nontuberculous mycobacteria are mostly unknown. In Mycobacterium tuberculosis complex
bacteria, the esat-6 and cfp-10 genes are important virulence factors, which facilitate translocation from the phagolyso-
some to the cytosol of macrophages. Their presence and role among nontuberculous mycobacteria is largely unknown.

We assessed the presence of esat-6 and cfp-10 genes in all 5 M. kansasii subtypes based on 16S-23S internal transcribed
spacer sequencing (n=15), M. szulgai (4), M. marinum (4), M. avium (2), M. conspicuum (4), M. genavense (1), M. bohemicum
(2), M. interjectum (2), M. flavescens (5), M. xenopi (2), M. malmoense (2), “M. riyadhense” (1) and M. tuberculosis H37Rv by
PCR. We used Esa-12 CATGACAGAGCAGCAGTG and Esa-303 5’-GCCCTATGCGAACATCCC-3’ primers for esat-6
GCGAGGACAGC-3’ primers for cfp-10. For PCR negatives, we performed Southern blotting with probes based on the
esat-6 gene of M. tuberculosis H37Rv.
One NTM species with esat-6 and cfp-10 genes was selected for macrophage infection. By cryo-immunogold electron mi-
croscopy we tested whether these genes effect translocation from the phagolysosome to the cytosol of macrophages.

We were able to amplify esat-6 and cfp-10 genes in M. tuberculosis H37Rv, all M. kansasii subtypes, M. szulgai, M. marinum
and “M. riyadhense”. All esat-6 and cfp-10	sequences	among	these	nontuberculous	mycobacteria	were	species-specific;	
multisequence alignment revealed an average of 90% homology with the M. tuberculosis sequences. The remaining spe-
cies were repeatedly negative by both PCR and Soutern blotting. Mycobacterium szulgai was subsequently used for a
macrophage (THP-1) infection experiment, with an M. tuberculosis positive control. Seventy-two hours after infection, no
cytosolic M. szulgai bacteria were found, while 53% of the M. tuberculosis bacteria had translocated to the cytosol.

While some nontuberculous mycobacteria harbor esat-6 and cfp-10 genes, their products, at least for M. szulgai, do not
effect translocation of the bacteria from the phagolysosome to the cytosol in macrophages. ESAT-6 and CFP-10 protein
structure or secretion could be critical factors. While the presence of esat-6 and cfp-10 genes adds an interesting phylo-
genetic marker, the pathogenesis of NTM infections remains unexplained.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  63

     DEVElOpmENT Of aN aDapTiVE immuNE rESpONSE iN ThE DraiNNg
     lymph NODE DuriNg MyCoBaCTeriuM ulCeranS iNfECTiON

     Alexandra G. Fraga; Joana E. Braga; Andrea Cruz; Teresa G. Martins; Daniela R. Pereira; Wayne M. Meyers; Françoise
     Portaels; António G. Castro; Jorge Pedrosa
     1 - Life and Health Sciences Research Institute (ICVS). School of Health Sciences, University of Minho. Braga, Portugal
     2 - Mycobacteriology Unit, Department of Microbiology, Institute of Tropical Medicine, Antwerp, Belgium
     3 - Armed Forces Institute of Pathology, Washington, D. C., USA

     Buruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans. The necrotic cutaneous le-
     sions of BU patients are associated with the cytotoxic properties of the exotoxin mycolactone. Previous results from
     our group show that the protective role of IFN-γ is impaired during infection with the highly virulent strain 98-912. To
     further characterize the development of an adaptive immune response during progressive infection with a highly virulent
     M. ulcerans strain, we decided to study the T cell dynamics in the draining lymph node (DLN) of mice infected with M.
     ulcerans 98-912, as compared to	the	low	virulent,	mycolactone-deficient	strain	5114.
     Subcutaneous infection of mouse footpads with M. ulcerans 5114 induced a modest increase in the number of cells in the
     DLN, prevalent throughout the experimental infection. Surprisingly, infection with strain 98-912 led to a 26-fold increase
     in	the	number	of	cells	in	the	DLN,	followed	by	a	significant	drop	to	basal	levels.	However,	this	increase	in	the	number	of	
     cells did not correlate with a protective response in the infected footpad.
     Following this observation, we explored whether the induction of an adaptive immune response occurs during infection
     with strain 98-912. M. ulcerans	infection	elicited	antigen-specific	T	cells	producing	IFN-γ in the DLN, with the response
     being more prominent in M. ulcerans 98-912 infected mice. Additionally, infection with M. ulcerans followed by adoptive
     transfer	of	OVA-transgenic	T	cells	did	not	impair	the	accumulation,	proliferation	or	activation	of	the	OVA-specific	T	cells	
     in	the	DLN	upon	challenge	with	OVA.	Moreover,	this	T	cell	accumulation	and	activation	was	significantly	increased	in	
     mice infected with strain 98-912. Interestingly, analysis of the DLN at later time points revealed the presence of viable
     bacilli that correlated with the dramatic decrease in the number of cells. This decrease was due to apoptosis, as dem-
     onstrated by the higher number of activated-caspase 3 positive cells. Supporting our observations in the mouse model,
     presence of bacilli and tissue destruction were also observed in DLN of BU patients.
     In summary, the failure of the host to generate a protective response against infection with this highly virulent strain of
     M. ulcerans	is	not	associated	with	an	impaired	development	of	specific	T	cells	in	the	DLN,	but	instead	to	their	destruction	
     in the infection foci and, later, to the destruction of the DLN itself.

64                                                                                                                    ESM 2009

TubErCulOSiS SOfTWarE iN a gENEral hOSpiTal,

Portugal Clara 1; Nuno Cardoso 2, Luísa Sancho 1; Germano Sousa 1
1 - Laboratory of Microbiology, Department of Clinical Pathology
2 - Planning and Management Control Director
Hospital Fernando Fonseca (HFF) - Amadora, Portugal

Our Hospital is located in a Lisbon’s surroundings, its construction was completed in 1995 and, in this same year, it
started the operation.
The HFF was designed to house a population much smaller than that now covers, 750 000.
Our population has a low socioeconomic conditions and a high level of immigrants from Africa and in Eastern Europe,
which may be the main reason for concentrating a large number of tuberculosis’s cases. Portuguese Health Authorities
reported that 66% of TB cases in Portugal were concentrated in Lisbon’s surroundings.
In a few years after the opening of the Hospital, all laboratory’s data relating to tuberculosis became too numerous to be
recorded and cross in the general program.

Given the need to gather and cross all the tuberculosis’s data, in 2000 was developed a software (MYCOHFF) that we
are going to present.

MYCOHFF	results	from	the	interconnection	of	3	Excel	files;	MYCOYEAR	related	to	the	year	in	question,	MYCOZIEHL	
that brings only patient’s data with positive Ziehl-Nielseen (since 1997) and MYCOCULT that joins only the patient’s
data with positive cultural examinations (liquid and/or solid) (since 2000).
For each specimen submitted for mycobacterial culture, we record the name of the patient, process number, analysis
number, product’s type, the origin patient’s Service and product inoculation’s date. Thus takes place an observation’s
weekly list. For each week, we note the medium (liquid and Lowenstein-Jensen) growth or not, the contamination and/
or degradation, during 6 or 8 weeks (sometimes more).
The year’s records are placed in alphabetical order (patient’s name).
The patient’s analysis with positive culture (liquid and/or solid) automatically appears in red.
If the patient had already a positive culture, all the negative analysis appears in purple. If the patient had a positive Ziehl-
Nielseen, then all the analysis will appear in yellow, becomes purple if it has other positive culture or red if this analysis
is positive.
On	patient’s	first	isolation,	we	perform	the	Identification	and	Susceptibility	Test.	This	date	is	noted	as	well	as	the	final	date	
with	the	definitive	results,	which	are	presented	to	the	clinician.

   •	 Warning Signs: When the patient’s registration happens, we know immediately, with different colors (program
      alerts), if the patient has Ziehl-Nielseen positive, culture positive, and when.
   •	 Search: Any search is fast because we are talking about an application that is made in Excel software.
   •	 Weekly	observation’s	list:	The	weekly	sheets	are	easily	created	by	filtering	the	inoculation’s	date	field.
   •	 Statistical indicators and graphics: With only a few records, statistical indicators are automatically calculated and
      graphical analyses are available at any time.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                       65
        Statistical indicators for each year are concerning to different parameters. 1- Concerning the analysis/patient relation-
        ship: Nº of patients, nº of analysis, total positive patients, total positive analysis, analysis/patient ratio, patients positive
        rate, analysis positive rate; 2- Concerning the Ziehl-Nielseen (Z) and liquid/solid medium relationship: Nº of positive
        direct tests (Z+), Nº of positive cultural tests (C+), true Z positive’s (Z+/C+) rate, false Z negative’s (Z-/C+) rate, false
        Z positive’s (Z+/C-) rate, real Z negative’s (Z-/C-) rate, Ziehl-Nielseen’s positivity rate per analysis and per patient;
        3-	Concerning	the	identification	and	TSA:	Nº	of	Mycobacterium sp.	isolated,	total	identified	strains,	total	strains	with	
        the	TSA	and	identification	still	in	study,	Nº	and	rate	of	Mycobacterium tuberculosis complex (CMT), CMT without resis-
        tance, with one resistance, MDR (Multi Drug Resistant - TB), XDR (Extensively Drug Resistant – TB), Nontuberculous
        mycobacteria,	average	response	time	beginning	with	the	isolation	culture	(identification	plus	TSA).
        The	statistics	graphs	show:	1	-	Analysis	requested	/	positive	analysis	by	organic	product;	pleural	biopsy,	pleural	fluid,	
        sputum,	bronchial	secretions,	bronchoalveolar	lavage,	gastric	fluid	(respiratory	specimens),	urine,	pus,	mieloculture,	
        cerebrospinal	fluid,	biopsy	,	ascites	fluid,	fluids,	blood;	and	their	positivity,	2	-	The	isolated	Mycobacterium sp.’s strains,
        including the differentiation in CMT, zero resistance, one resistance, MDR, XDR, 3 - Compare the tuberculosis’s inci-
        dence rate in two populations, MDR and non-MDR, for the age range of patients, sex and HIV.

     For us, MYCOHFF software is an essential tool for the process of gathering and managing tuberculosis data.
     Quick reports of any patient are possible. In a few seconds, we can make the patient laboratory story on tuberculosis.
     Each year a statistical report is communicated to all our hospital’s departments. Monthly, or when requested, for the CDP
     (centre that follows the patients in the community), is sent a list identifying new positive cases and results (preliminary
     and/or	final).

66                                                                                                                            ESM 2009

DEVElOpmENT Of aN auTOmaTED CulTurE SySTEm fOr

den Hertog, Alice 1, Koeleman, Marc 1, Ingham, Colin 2, Fey, Frank 3, Langerak, Edwin 3, Klatser, Paul 1, Anthony, Richard 1
KIT Biomedical Research, Amsterdam, The Netherlands
Microdish BV, Wageningen, The Netherlands
CCM, Nuenen, The Netherlands

Due to the unavailability of rapid sensitive diagnostic methods for tuberculosis (TB), culture remains one of the most
important diagnostic tools - even though it is technically demanding and slow. The time required for culture leads to a
delay in TB diagnosis and treatment with effective drugs. A standardized method for more rapid drug sensitivity testing
(DST) of TB would be valuable.
We	are	developing	a	system	in	which	mycobacterial	(micro-)colonies	are	detected	by	their	autofluorescence	based	on	
the MODS (microscopic observation of drug susceptibility) method, which is much more rapid than the traditional cul-
ture methods, but is laborious.
By	 using	 microscopy	 with	 low-power	 magnification,	 colony	 growth	 could	 be	 determined	 much	 earlier	 than	
when viewed by eye. Studying Mycobacterium smegmatis initially as a model organism, we have made se-
quential	 brightfield	 and	 fluorescence	 microscopy	 photographs	 of	 colonies	 growing	 on	 movable	 solid	 sup-
ports on media. These images were used to develop an image analysis protocol to determine the growth rate.
We found that the time to detection of microcolonies was less than half that required for visual detection. Furthermore,
as soon as the colonies could be visualized, further growth of colonies could be detected by the image analysis protocol
within 1-2 division times, from which the growth rate could be determined. As the bacteria were inoculated on movable
solid	supports,	exposing	the	microcolonies	to	selective	media	after	their	first	detection	could	make	DST	of	many	indi-
vidual colonies possible after only a few additional division times, allowing the rapid determination of the proportion of
antibiotic resistant and sensitive mycobacterial colonies present in a sample.
After	confirmation	of	the	recently	described	autofluorescence	of	mycobacteria,		this	method	was	used	for	the	detection	
and	enumeration	of	viable	mycobacteria.	The	specificity	of	the	autofluorescence	may	be	used	to	confirm	the	presence	
of mycobacteria.
The	use	of	(semispecific-)	autofluorescence	in	combination	with	growth	rate	will	increase	the	feasibility	of	developing	
an automated detection system based on this method, which is very challenging for light microscopy methods (such as
MODS). Additionally, the possibility of performing quick DST without re-inoculation may lead to a reduce time to cor-
rect treatment.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                 67

     SECOND-liNE Drug SuSCEpTibiliTy TESTiNg Of MyCoBaCTeriuM
     TuBerCuloSiS by mgiT 960 SySTEm, ThE miCrOplaTE
     COlOrimETriC-baSED mEThOD aND ThE prOpOrTiON mEThOD

     Nora Morcillo1, Belén Imperiale,1, 2, Beatriz Di Giulio3
     1 - Reference Laboratory of Tuberculosis Control Program of Buenos Aires Province, Dr. Cetrángolo Hospital Buenos
         Aires, Argentina.
     2	-	National	Council	of	Scientific	and	Technological	Research,	Buenos	Aires	City.
     3 - P.V. de Cordero Hospital, San Fernando, Buenos Aires, Argentina.

     The accurate treatment of tuberculosis (TB) cases due to multidrug-resistant and extensively drug-resistant Mycobacterium
     tuberculosis emphasizes the necessity of new tools for rapid detection of these strains in clinical laboratories. Minimal
     inhibitory concentrations (MICs) by MGIT960 and the colorimetric microplate method using dyes as MTT or resarzurin
     (CMM) were determined for the following drugs (µg/ml): amikacin (AMK): 2.0, 4.0, 8.0; kanamycin (KM), capreomycin
     (CPM),	ethionamide	(ETH):	2.5,	5.0,	10.0;	cycloserine	(CS):	15.0;	ofloxacin	(OFX)	and	linezolide	(LZ):	0.5,	1.0,	2.0;	and	
     moxifloxacin	(MOX)	0.25,	0.5,	1.0.	MICs	were	performed	on	94	clinical	isolates.	The	proportion	method	on	Middlebrook	
     7H11 (PM) was used as gold standard. Inoculated MGITs were incubated in the instrument for no longer than 21 days.
     A	strain	tested	by	MGIT960	was	considered	resistant	if	a	positive	signal	flagged	from	the	drug-containing	tube	within	5	
     days of the positive control tube. Microplates of the CMM were incubated for an average of 8 days. Statistical methods
     were	applied	to	define	drug-resistant	strains	on	the	basis	of	the	comparison	between	results	obtained	by	MGIT960	and	
     CMM	with	the	PM.	The	following	critical	concentrations	were	identified	(µg/ml):	AMK:	4.0;	CPM,	ETH	and	KM:	5.0;	CS:	
     30.0; LZ: 1.0; MOX: 0.5; OFX: 2.0. Accuracy of MGIT960 and M-MTT was 100% for AMK, CPM, OFX, MOX and LZ. In this
     study tubes incubation and positivity detection was manually obtained from the MGIT960 instrument which actually can
     be adapted to automatically detect both susceptible and resistant strains to each one of the second-line drugs. Results
     were obtained in less than 10 days for both MGIT960 and CMM. On the other hand CMM, as a complete homemade
     method, was cheaper but more laborious than MGIT960. Our results showed that both methods could be promissory
     implemented as a rapid diagnosis tools to detect MDR and XDR-TB cases in clinical practice.

68                                                                                                                  ESM 2009

ThiOriDaziNE ShOWS prOmiSiNg aCTiViTy iN a
muriNE mODEl Of mulTiDrug-rESiSTaNT TubErCulOSiS

Jakko van Ingen1,2, Martin Boeree1, Leonard Amaral3, Rogelio Hernandez Pando4, Dick van Soolingen2
1 - Department of Pulmonary Diseases, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands
2 - National Mycobacteria Reference Laboratory, National Institute for Public Health and the Environment, Bilthoven,
    the Netherlands
3 - Mycobacteriology Unit, Institute of Hygiene and Tropical Medicine, Universidade Nova de Lisboa, Lisbon, Portugal
4 - Experimental Pathology Section, Department of Pathology, National Institute of Medical Sciences and Nutrition
	 Salvador	Zubiràn,	Mexico	City,	Mexico

Multidrug-resistant tuberculosis (MDR-TB) is a threat to TB control efforts worldwide for which very few active drugs
are currently available. The phenothiazines are antipsychotic agents that have potential as anti-tuberculosis drugs, at least
in vitro.	Within	this	pharmacological	class	thioridazine	is	the	most	efficacious	and	causes	the	mildest	side-effects	when	
used as an antipsychotic agent.Thioridazine is active against mycobacteria by targeting the type II NADH dehydrogenase,
succinate dehydrogenase, the binding of calcium to proteins and disruption of aerobic respiration under micro-aerobic
conditions. We tested its in vivo activity in a murine model.

We infected 3 groups of 40 Balb/c mice with pansusceptibe M. tuberculosis H37Rv. After 60 days, 20 mice in each group
started 2 months of thioridazine monotherapy at 16, 32 and 70 mg/kg dosages; the remaining 20 served as controls. This
experiment was repeated using a clinical multidrug-resistant M. tuberculosis (MDR-TB) isolate and two months daily oral
administration of 32 and 70 mg/kg. In a third experiment, 3 groups of 20 mice were infected with M. tuberculosis H37Rv;
one group received rifampicin, isoniazid and pyrazinamide, another received these 3 drugs and thioridazine, one un-
treated	group	served	as	controls.	In	all	experiments,	groups	of	five	mice	per	group	were	sacrificed	after	2,	4	and	8	weeks	
of treatment; lung tissue was homogenized for quantitative cultures. The bacillary load of the lungs was determined by
colony	forming	units	(CFU)	quantification;	histological	damage	was	observed	by	microscopic	examination.

                            H37Rv	and	MDR-TB	infection,	monotherapy	with	32	and	70mg/kg	thioridazine	lead	to	signifi-
In both the M. tuberculosis	H37Rv	and	MDR-TB	infection,	monotherapy	with	32	and	70mg/kg	thioridazine	lead	to	signifi-
cant reductions in CFU counts from the lung tissue homogenates and in the extent of histological damage, at all time
points. Moreover, when 32 mg/kg of thioridazine was added to a regimen containing rifampicin, isoniazid and pyrazin-
amide	for	susceptible	tuberculosis,	a	significant	synergistic	effect	was	achieved.

Thioridazine shows promising activity in our murine model. It has a potential effect in the treatment of susceptible
and MDR-TB, where few active drugs are available. The low price of this out of patent drug enables extended use in
resource–poor settings where the burden of multidrug-resistance is most grave.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  69

     CONTribuTiON Of EffluX pump aCTiViTy fOr
     maCrOliDE rESiSTaNCE iN M. aviuM COmplEX

     Liliana Rodrigues1,2*, Daniela Sampaio1, Isabel Couto1,3, Diana Machado1,Winfried V. Kern4,5, Leonard Amaral1,2,5 and Miguel Viveiros1,5
     1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal
     2 - UPMM, IHMT/UNL, Lisbon, Portugal
     3 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal
     4 - Center for Infectious Diseases and Travel Medicine, University Hospital, Freiburg, Germany
     5 - COST ACTION BM0701 (ATENS)

     Mycobacterium avium complex (MAC), comprising M. avium and M. intracellulare, is clinically important since it can cause
     severe infections in AIDS patients and other immunocompromised individuals. Therapy of MAC infections is problematic
     due to the intrinsic resistance of these bacteria to many of the available antimicrobial drugs. The use of the macrolides
     clarithromycin and azithromycin has improved the outcome of MAC infections, but therapeutic failure is still a major
     problem.	We	have	recently	shown	that	efflux	pumps	of	MAC	play	an	important	role	on	this	resistance	phenotype.	In	fact,	
     increased	activity	of	efflux	pumps	is	known	to	contribute	to	a	multidrug	resistance	phenotype	by	extruding	a	wide	variety	
     of chemically and structurally unrelated compounds from the cell, preventing them from reaching their cellular targets.
     Thus,	the	characterization	of	such	efflux	pumps	is	crucial	for	the	design	of	new	antimycobacterial	therapeutic	strategies.
     In	this	work,	we	have	studied	the	efflux	pump	activity	in	MAC	clinical	strains	by	a	fluorometric	method	that	detects	efflux	
     activity	on	a	real-time	basis,	and	evaluated	the	contribution	of	active	efflux	to	the	resistance	to	macrolides.
     The	results	to	be	presented	show	that	resistance	to	clarithromycin	was	significantly	reduced	in	the	presence	of	efflux	
     pump	inhibitors	(EPIs)	such	as	the	calcium-channel	inhibitors	thioridazine	or	chlorpromazine	and	the	calcium	ion	influx	
     inhibitor verapamil.	The	same	EPIs	were	effective	in	decreasing	the	efflux	of	ethidium	bromide	(a	common	efflux	pump	
     substrate)	from	MAC	cells,	as	shown	by	fluorometric	analysis.	Moreover,	the	retention	of	[14C]-Erythromycin by the same
     inhibitors	demonstrated	that	active	efflux	contributes	to	MAC	resistance	to	macrolides.
     In	conclusion,	this	study	demonstrates	that	efflux	pumps	play	an	important	role	in	MAC	resistance	to	antibiotics,	par-
     ticularly to macrolides, and opens the possibility to explore the usefulness of these EPIs, already used in clinical practice
     for other purposes, such as thioridazine, as adjuvants to enhance the effectiveness of the therapeutic regimens against
     MAC infections.

70                                                                                                                                ESM 2009

mEmbraNE- baSED VErSuS miCrObEaD- baSED SpOligOTypiNg:
prElimiNary rESulTS ON a QualiTy-

Abadia E1, Zhang J1, Refregier G1, Sola C1.
1 - Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud (Universud), Equipe IGEPE, bât. 400. France.

Spoligotyping have been developed 12 years ago and have been used worlwide for molecular epidemiological or mo-
lecular evolutionary studies. This technique is based on a reverse line- blot hibridization method. Due to it’s wide accep-
tance (458 references in Pubmed on 2009 April 27th) it can be considered as a basic technique in genotyping strains of
Mycobacterium tuberculosis complex together with MLVA (Multi locus variable analysis) typing. However, spoligotyping
has not been the focus of standarization nor of quality insurance studies. The transfer from the membrane- based to a
microbead- based format, through the development of the multiplex Luminex plataform was done in 2004 in the CDC
in Atlanta. We implemented this technique in our team in 2008.
We wanted to evaluate membrane- based spoligotyping results obtained on 10 sites (around 1000 DNA samples from
clinical isolates) comparing them with those from the Luminex microbead- based results obtained on the same samples.
The	 DNA	 samples	 were	 selected	 to	 fulfill	 the	 following	 goals:	 (1)	 Retrospectively	 assess	 the	 quality	 of	 spoligotyping	
results, that have been produced in various laboratories worlwide during a decade (2) Solve inconsistencies, resolve dis-
crepancies, improve quality of hard- to- interprete membrane- based spoligotyping results (3) Study if DNA extraction
procedure (quality, quantity) may have produced errors in spoligotyping patterns production (4) assess the economical
interest of the new Luminex- based technique and it’s contribution (or not) to and increased quality of services in mo-
lecular epidemiological studies.
We will present all results available until today. Our current experience and preliminary results suggest that the Luminex
platform it’s very versatile to produce high quality spoligotypnig results with both, a higher throughput and sensitivity
than the membrane- based spoligotyping and at affordable costs for developed countries.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                           71
prESENTaTiONS (pp)

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal   73

EValuaTiON Of ThE high-ThrOughpuT rEpETiTiVE-SEQuENCE-baSED pCr
DiVErSilab SySTEm iN M. TuBerCuloSiS mOlECular EpiDEmiOlOgy STuDiES

Miotto Paolo, Baldan Rossella, Cirillo Daniela M.
Emerging	Bacterial	Pathogens	Unit,	San	Raffaele	Scientific	Institute,	Milan	–	ITALY

PCR-based methods have been developed to simplify and reduce the time required for genotyping M. tuberculosis by stan-
dard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-VNTR comple-
mented with spoligotyping has been proposed as an alternative. Repetitive-sequence-based PCR (rep-PCR) is useful for
generating	DNA	fingerprints	of	diverse	bacterial	species.	Rep-PCR	amplicon	fingerprints	represent	genomic	segments	
lying between non-coding repetitive sequences. A commercial system (Diversilab, Biomerieux) that electrophoretically
separates	 rep-PCR	 amplicons	 on	 microfluidic	 chips,	 and	 provides	 computer-generated	 readouts	 of	 results	 has	 been	
adapted for use with Mycobacterium species.The ability of this system to type M. tuberculosis was evaluated in comparison
with spoligotyping and MIRU-VNTR in two different panels.
First, we evaluated a 35 strains panel by MIRU-15 complemented with spoligotyping and Diversilab rep-PCR. Results
were analyzed with MIRU-VNTRplus database for spoligo-MIRU, and with Diversilab Software for rep-PCR using two
different	algorithms	(Pearson	Correlation	[PC]	and	Kullback-Leibler	[KL]).	Threshold	for	clusters	was	fixed	at	98%	of	
similarity for rep-PCR. MIRU-15 showed a clustering rate of 11.4% whereas the rep-PCR reported a clustering rate of
28.6% (PC) and 17.1% (KL). The discriminatory power (Hunter-Gaston discriminatory index [HGDI]) for spoligo-MIRU-
15 resulted 0.983 whereas for rep-PCR was 0.978 (PC) and 0.983 (KL).
We also compared the two techniques on a panel composed by 8 closely related strains from a probable outbreak. In
this case the rep-PCR showed a discriminatory power of 0.571 (PC) and 0.679 (KL), compared to a HGDI of 0.643 for
spoligo-MIRU-15. Nevertheless, clustering rate for MIRU-15 was 50.0% whereas rep-PCR algorithms showed a cluster-
ing rate of 100.0% (PC) and 62.5% (KL), respectively. To understand the meaning of the discrepancies still found between
spoligo-MIRU-15 and rep-PCR, analysis of epidemiological data for the clustered patients will be taken in consideration.
Preliminary data obtained by Diversilab suggest that the KL algorithm is more appropriate for M. tuberculosis typing analy-
sis. Nevertheless, even if rep-PCR results analyzed by KL algorithm showed a discriminatory power similar to MIRU-15,
clustering rate remains higher. This new technique could be useful for a routine use in clinical laboratories for real-time
genotyping and laboratory contaminations control.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  75

     68 SpaCErS MyCoBaCTeriuM TuBerCuloSiS COmplEX SpOligOTypiNg :
     a STuDy uSiNg a miCrObEaD-baSED high ThrOughpuT fOrmaT.

     Zhang J1, Abadia E1, Refrégier G1 , Ruimy R2, Boschiroli ML3, Guillard B4 and C. Sola1.
     1 - Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud (Universud), Equipe IGEPE, bât. 400,
     	 Centre	scientifique	d’Orsay,	rue	Gregor	Mendel,	91405	Orsay-Cedex2	-	APHP,	Hôpital	Bichat-Claude	Bernard,	Paris
     3 - Agence française de sécurité sanitaire des aliments AFSSA, Maisons-Alfort
     4 - Institut Pasteur du Cambodge

     New captures probes for the microbead-based spoligotyping assay (Luminex) were designed to test for the presence/
     absence of 25 spacers that are not used in routine spoligotyping. These spacers are expected to provide an improved
     discriminatory power for Major Genetic Group I, including the « ancestral » TbD1+ MTC (Mycobacterium tuberculosis
     complex) clinical isolates.
     The new 68 spacers spoligotyping format developed on a Luminex platform was shown to give excellent results. Around
     400 strains of MTC from 3 different centers (Bichat Hospital, AFSSA, Institut Pasteur of Cambodia) were studied. We
     show that the 68 spacers format is more discriminant to study the strains of the East African Indian family (EAI) : among
     86 strains of the EAI family, it could distinguish 44 clusters compared to 27 clusters by routine 43 spacers spoligotyping.
     Whereas for a total of 210 clinical isolates of Mycobacterium bovis, we reported 31 types instead of 25, and for a total
     of 30 “Beijing” clinical isolates, 4 clusters instead of 3 clusters were found. For Mycobacterium africanum, a total of 17
     strains were assayed and 11 instead of 9 clusters were found. High-thr(East-African Indian - Indo-Oceanic clade) and
     other	Major	Genetic	Group	I	still-undefined	spoligotyping	signatures.	This	observation	often	concerns	the	first	spacer	
     of a string of several missing spacers (border domains). These observations are currently under further investigation by
     sequencing. Extended High-throughput spoligotyping is also a new mean to detect mutational events that could be diag-
     nostics	of	clade-specific	MTC	evolution.	

76                                                                                                                   ESM 2009

rESiSTaNCE amONg MyCoBaCTeriuM TuBerCuloSiS
iSOlaTES CirCulaTiNg iN ThE rEpubliC Of gEOrgia

Stefan Niemann1, G. Khechinashvili2, M. Gegia2, N. Mdivani2, and Y. W. Tang3
Molecular Mycobacteriology, National Reference Center for Mycobacteria, Research Center Borstel, Borstel, Germany
Georgian Foundation against Tuberculosis and Lung Diseases, Tbilisi, Georgia
Vanderbilt University Medical Center, Nashville, TN, USA

Rising tuberculosis (TB) rates and high levels of multidrug-resistant TB (MDR-TB) have become a major public health
problem in several parts of the former Soviet Union. High rates and transmission of MDR-TB have been noticed to be as-
sociated with the presence of Mycobacterium tuberculosis Beijing genotype strains pointing to the importance of pathogen
genetic	factors	for	the	modulation	of	infection	outcome	and	epidemiology.	Here	we	present	the	first	data	on	the	popula-
tion structure of M. tuberculosis strains from the Republic of Georgia, a high incidence setting at the Black Sea Coast.
All strains were analysed by spoligotyping and 24-loci MIRU-VNTR	genotyping.	Identification	of	major	M. tuberculosis
genotypes was carried out by using the MIRU-VNTRPlus database ( and a similarity analysis
performed	to	identify	strains	with	identical	genotyping	profiles	(clusters),	which	are	indicative	for	the	rate	of	recent	
transmission. Anti-tuberculosis drug resistance was determined by in vitro antimicrobial susceptibility testing.
Genotyping	profiles	were	successfully	generated	for	187	M. tuberculosis isolates which were further investigated.The most
prominent genotype found, was Beijing (29%), followed by LAM (18%), Ural (12%), and Haarlem (n=10) strains. Approx
50% of the isolates were grouped in clusters. When the distribution of drug resistance is considered, it was noticed that
MDR-TB was nearly completely restricted to Beijing strains. Further detailed analyses are currently in progress.
Our data underline the importance of Beijing genotype strains for the TB epidemiology in former Soviet Union countries.
However,	Ural,	Haarlem	genotype	and	a	large	variety	of	strains	of	so	far	undefined	lineages	represent	nearly	two	third	of	
the strains found in Georgia. Although Beijing strains are not as dominant as in others Eastern European countries such
as	Kazakhstan,	we	confirm	a	clear	association	between	MDR-TB	and	the	Beijing	genotype	in	Republic	of	Georgia.	

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                               77

     MyCoBaCTeriuM TuBerCuloSiS EpiDEmiOlOgy aND gENETiC
     DiVErSiTy iN ThE TWiN iSlaND rEpubliC Of TriNiDaD aND TObagO

     Shirematee Baboolal, 1, 2 Julie Millet,3 Patrick Eberechi Akpaka, 1 Dottin Ramoutar, 4 Nalin Rastogi 3
     1 - Department of Para-Clinical Sciences, Faculty of Medical Sciences, The University of the West Indies, St. Augustine,
         Trinidad & Tobago
     2 - Caribbean Epidemiology Centre, Jamaica Boulevard, Port of Spain, Trinidad & Tobago
     3 - Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, Abymes, Guadeloupe
     4 - Caura Chest Hospital, Caura, Trinidad & Tobago

     This	work	describes	the	first	application	of	molecular	tools	for	studying	tuberculosis	(TB)	epidemiology,	genetic	diversity,	
     and transmission in the twin island Republic of Trinidad and Tobago (T&T). The study population (n=132) represented
     one year recruitment of all culture positive TB cases from T&T, and was characterized by a high male to female sex-ratio
     of 4 (mean age 42.8 years, range 17 to 78 years), and a HIV/TB coinfection rate of nearly 30%. It mainly occurred among
     African descendants who represented 37.5% of total population but 69.7% of all TB cases (p<0.001). Spoligotyping
     resulted in 25 different patterns and 12 clusters (2 to 74 strains per cluster). In total, 81.3% of the isolates in our
     study	was	defined	as	modern	tubercle	bacilli	belonging	to	the	Principal	Genetic	Groups	2/3.	Five	major	lineages	were	
     observed: East-African Indian (EAI), Latin-American and Mediterranean (LAM), X, Beijing, and Haarlem. A comparison
     with international SITVIT2 database showed that the overall lineage distribution in T&T was completely different from
     other Caribbean neighbors (n=2653 isolates). A high clustering rate of 90% was observed essentially due to a single large
     cluster of 74 strains designated as Spoligotype International Type - SIT566 (T&T clone). Patients harboring this genotype
     were overrepresented in St George Central, the capital city of Port-of-Spain, younger (mean 39.1 years vs. 47.7 years
     for other genotypes, p<0.0005), and more frequently prison inmates and drug users, while those harboring “other geno-
     types” were older and showed diabetes as an associated factor. A study of the evolutionary relationships suggested prob-
     able relatedness of SIT566 with X1 prototype SIT119. A database search localized most of the SIT566 related patterns
     in USA. Second-line typing using Mycobacterial Interspersed Repetitive Units (MIRUs) suggested the highly conserved
     nature	of	SIT566,	its	phylogeographically	specificity	to	T&T.	

     We thank the Caribbean Epidemiology Center, Trinidad & Tobago for support, and the staff of the Chest Clinics at the
     Eric Williams Medical Sciences Complex, the San Fernando General Hospital, and Trinidad Public Health Laboratory for
     assistance	with	data	collection.	SB	is	grateful	to	the	University	of	West	Indies	for	financial	support,	and	JM	to	the	Regional	
     Council of Guadeloupe and European Social Funds for a Ph.D. fellowship. NR and JM thank J. Driscoll for providing infor-
     mation regarding the SIT566 clone in USA.

78                                                                                                                       ESM 2009

SpOligOTypE paTTErNS aND Drug rESiSTaNT
prOfilE Of myCObaCTErium TubErCulOSiS iN SuDaN

Ghada Suliman Sharaf-Eldin 1, Imad F.Elmoula 1, Mohammed S. Ali 1, Nageeb S. Saaed 2,
Ahammed B Ali 1, Kim Mallard 3, Ruth McNerney 3, Saad Algamdi 3
1 - Al Neelain University-Sudan
2 - National Health Laboratory-Sudan
3 - London School of Hygiene & Tropical Medicine.

Sudan has a high burden of tuberculosis with an estimated 93,000 new cases each year. The purpose of this
study was to investigate the genotypic patterns of M. tuberculosis strains circulating in Sudan and to assess
their susceptibly to anti-tuberculosis drugs. Isolates from 237 smear positive tuberculosis patients were col-
lected from different geographic regions of the country. Spoligotyping was performed by the Kamerbeek
method and results were compared with the international SpolDB4 database (Institut Pasteur, Guadeloupe).
Results revealed 28 clusters ranging in size from 12 to 57 isolates. Seventy unique (unclustered) strains were observed,
representing 30% of the strains examined.The most frequently observed spoligotype patterns belonged to the CAS fam-
ily which represented 115 (48.5%) of isolates studied. T1, H3, U and Beijing strains were found in 12 (5.1%), 11 (4.6%),
7 (3%) and 6 (2.5%) patients respectively. Strains belonging to the Beijing family were found mainly in Western Sudan.
Resistance to isoniazid, rifampicin, ethambutol and streptomycin was observed in 18.1, 22.4, 22.2 and 32% of strains re-
spectively. Twenty patients (8.4%) had MDR-TB of which 10 were new cases. Seventeen patients with rifampicin resistant
tuberculosis were infected with CAS1-DELHI strains matching SIT 25 of the SpolDB4 database and 3 were of the SIT
1 Beijing family. 15 loci MIRU-VNTR typing subdivided the 17 CAS strains into one cluster of 5, two clusters of 2 and
8 individual MIRU types. Similarly the 3 Beijing spoligotypes were differentiated into a cluster of 2 and a single strain.
The use of molecular strain typing provides a proactive approach that may be used to initiate, and not just augment,
traditional surveillance outbreak investigation in Sudan. However, caution must be used when interpreting clustered
spoligotype patterns in this region.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                               79

     CONTrOVErSial DiSSEmiNaTiON paTTErN Of ThE bulgaria
     -SpECifiC M. TuBerCuloSiS SpOligOTypE ST125_bgr

     Violeta Valcheva 1, 2, Igor Mokrousov 1, 3, Stefan Panaiotov 4, Elizabeta Bachiiska 4, Thierry Zozio 1, Christophe Sola 1, Nadya
     Markova 2, Nalin Rastogi 1
     1 - Institut Pasteur de Guadeloupe, France
     2	-	Institute	of	Microbiology,	Sofia,	Bulgaria
     3 - St. Petersburg Pasteur Institute, St. Petersburg, Russia
     4	-	National	Center	of	Infectious	and	Parasitic	Diseases,	Sofia,	Bulgaria

     To investigate phylogenetic position and geographic genetic diversity of spoligotype ST125 in Bulgaria.

     material and methods
     Study sample included all available 47 DNA samples belonging to spoligotype ST125 that were taken from two previously
     published M. tuberculosis collections from Bulgaria (Valcheva et al., 2005, 2007, 2008abc; Panaiotov et al., 2005, 2006).
     These DNA were additionally typed using new 24-loci MIRU format, IS6110-RFLP typing and LAM-PCR. Phylogenetic
     analysis was done using PAUP and PHYLIP packages.

     Comparison with SITVIT2 database (Institut Pasteur de Guadeloupe) revealed a high gradient of ST125 in Bulgaria
     (14.3%) compared to its negligible presence in the world. Typing of Bulgarian ST125 strains revealed that they: (i) did
     not	harbor	a	LAM-specific	IS6110 insertion (ii) formed a monophyletic cluster in 24-MIRU tree of Bulgarian strains (iii)
     grouped closely with ST34 that is a prototype of the S family. A similarity of the IS6110-RFLP	profiles	confirmed	a	true	
     relatedness of these ST125 strains whereas a diversity of the MIRU loci suggested a long-term evolution of this spoligo-
     type in Bulgaria. Minimum spanning tree of the 24-MIRU-based subtypes of ST125, and comparison with their geographic
     distribution revealed an enigmatic and complex dissemination pattern of this spoligotype across Bulgaria.

     T125 likely belongs to S family and may have originated from spoligotype ST4 by a deletion of a single spacer #40 in the
     DR	locus.	ST125	is	phylogeographically	specific	for	Bulgaria;	we	propose	its	renaming	as	ST125_BGR.	A	high	diversity	of	
     the MIRU loci suggests a long-term evolution of this spoligotype in Bulgaria.

     This work was supported by NATO grant SFP-982319 “Detect drug-resistant TB”.

80                                                                                                                         ESM 2009

myCObaCTErium TubErCulOSiS bEiJiNg gENOTypE
aND OrigiNS Of ThE bulgariaNS

Panaiotov, Stefan; Bachiyska, Elizabeta; Brankova, Nadia; Levterova,Victoria
National	Center	of	Infectious	and	Parasitic	Diseases,	Sofia	1504,	Bulgaria

Recent	studies	demonstrated	that	exist	genotypes	of	M.	tuberculosis	locally	distributed	to	specific	geographic	region.	
Other genotypes are distributed globally or on vast geographic areas. These facts led to other recent fundamental stud-
ies	and	conclusions	that	exists	certain	genetic	predisposition	of	the	ethnic	groups	to	specific	M.	tuberculosis	genotypes,	
hence	genetic	predisposition	to	tuberculosis	specific	genotypes	is	suspected.	In	the	past	and	nowadays	the	waves	of	hu-
man migration coincide with expansion of tuberculosis genotypes. In our study we associated the global distribution of
MTB Beijing genotype, its’ distribution in Bulgaria, the geographic regions of historical origin of the Bulgarian tribes, and
the	ethnic	affiliation	of	the	Bulgarians.		
For our analysis we used data published in the fourth international spoligotyping database (SpolDB4), publications, per-
sonal	communications	and	official	historical	theories	regarding	origins	of	the	Bulgarians.
From the literature and in SpolDB4 exists about 330 spoligotypes of Bulgarian M. tuberculosis clinical strains collected
from	all	over	the	country.	Beijing	spoligotype	was	not	identified	among	them.	We	concluded	that	this	MTB	genotype	is	
not	(or	very	rare)	distributed	in	Bulgaria.	In	Romania	Beijing	genotype	was	not	identified	too	(personal	communication).	
Other	countries	associated	with	the	origins	and	migration	of	the	Bulgarians	where	Beijing	genotype	is	not	identified	is	
Iran.	Significant	part	of	the	Bulgarian	spoligotypes	are	phylogenetically	related	to	MANU	family,	widely	distributed	in	India.	
These facts correlate with the widely accepted theory that the origins of the Bulgarian tribes are of Indo-Iranian lineage.
In contrary, this fact does not support the theory for the Turkik lineage or more precisely the Turano-Hunnic origins of
the Bulgarian ethnos.
Beiging genotype is widely distributed in Central Asian countries, Kazahstan, Turkmenistan, Russia, Turkey, China etc.
Bulgaria has very active tourist, cultural, political, trade and immigration links with all these countries. Based on these
empiric facts we indirectly conclude that there is genetic predisposed resistance of the Bulgarian and Iranian ethnos to
MTB Beijing genotype.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                    81

     ThE EXTENT Of ThE laTiN amEriCaN-mEDiTErraNEaN
     MyCoBaCTeriuM TuBerCuloSiS SpOligOTypE family iN pOrTugal

     Suzana David 1, João Nuno Ribeiro 1,2, José-Nuno Maio 1,2, Inês João 1, António Amorim 3, Edna Pereira 3
     1 - Unidade de Referência e Vigilância Epidemiológica, Laboratório de Infecções Respiratórias – Micobactérias, Instituto
         Nacional de Saúde Dr. Ricardo Jorge, Portugal
     2 - Grupo de Microbiologia e Imunologia da Infecção, Instituto de Biologia Molecular e Celular,
         Porto, Portugal
     3 - Laboratório de Saúde Pública, Micobacteriologia / Tuberculose, Administração Regional de Lisboa e Vale do Tejo,
         Lisboa, Portugal

     Portugal	 is	 classified	 at	 the	 intermediate	 level	 with	 respect	 to	 the	 incidence	 for	 tuberculosis.	 Geographical	 spread	 is	
     heterogeneous with the majority of cases concentrated in the large urban areas mainly in the Lisbon and Porto districts.
     Previous efforts in the characterization of the population structure of Mycobacterium tuberculosis isolates stressed the
     importance of this approach to tuberculosis control. Spoligotyping data restricted to the metropolitan area of Lisbon,
     revealed a 51% prevalence of the Latin American-Mediterranean (LAM) spoligotype family, and a high proportion of SIT20
     (LAM1) and SIT42 (LAM9) sub-families. In the present study this characterization has been extended to encompass both
     the Lisbon and Porto areas. With the use of complementary data from SpotClust and Ag85C103 RFLP, the proportion of
     the LAM genotype was shown to be as high as 62% of the total number of isolates. The LAM genotypes were further
     characterized for the RDRio deletion detected in up to 60% of the LAM isolates. This study suggests that Portugal may
     have one of the highest global proportions of the LAM family. Monitoring and further characterizing of this relevant spo-
     ligotype family is considered of major importance for the understanding of the dynamics of tuberculosis in Portugal.

82                                                                                                                               ESM 2009

fiTNESS STuDy Of ThE rDriO liNEagE aND lam family Of
MyCoBaCTeriuM TuBerCuloSiS iN a STuDy pOpulaTiON
iN riO graNDE, brazil

Von Groll, Andrea 1;
Martin, Anandi 1; Felix, Carolina 2; Prata, Pedro 2; Honscha, Günther 3; Portaels, Françoise 1; Almeida da Silva, Pedro 2;
Palomino, Juan Carlos 1
1 - Institute of Tropical Medicine, Antwerp, Belgium
2 - Universidade Federal do Rio Grande, Rio Grande, Brazil
3 - Laboratório Municipal de Tisiologia, Rio Grande, Brazil

RDRio is a novel Mycobacterium tuberculosis lineage member of the Latin American-Mediterranean (LAM) family. LAM has
been found worldwide but it is more predominant in South America. The aim of this study was to assess the presence
of the RDRio	lineage	and	LAM	family	in	the	city	of	Rio	Grande,	Brazil,	and	to	investigate	the	fitness	of	these	strains	based	
on the determination of their rate of growth. Fifty clinical isolates of M. tuberculosis were genotyped and 43 different pat-
terns were found by spoligotyping and MIRU-VNTR.The predominant genotypes belonged to the LAM family (54% of the
strains) followed by clade T (22%) and Haarlem (16%). The RDRio lineage represented 38% of the total strains and 70.4%
of	the	LAM	strains	found	in	this	study.	Strains	belonging	to	the	LAM	family	showed	a	fitness	advantage	comparing	their	
rate of growth with that of non-LAM. RDRio	strains	were	predominant	within	the	LAM	family,	but	a	significant	difference	
in	fitness	between	RDRio and the non-RDRio	strains	was	not	confirmed.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  83

     gENOmiC CharaCTErizaTiON Of liSbOa
     family STraiNS by DElETiON aNalySiS

     João Perdigão, Carla Silva and Isabel Portugal
     Centro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa

     Multidrug and extensive drug resistant tuberculosis poses a very serious threat for public health. Lisbon Health Region
     has one of the world’s most serious situations regarding this problem. Such, is the result of a continued circulation of an
     endemic and predominant strains of a particular genetic family – Lisboa family. Little is known regarding the phylogeny,
     relative	virulence	and	genetic	background	of	these	strains.	The	loss	or	deletion	of	specific	genomic	regions	constitutes	
     the most important way by which Mycobacterium tuberculosis diverges and adapts. Several deletions, named Regions
     of Difference (RD), have already been described and associated with phylogeographic lineages. The characterization of
     Lisboa family in this manner may elucidate its origin and perhaps be helpful in explaining its high prevalence.
     Three representative clinical isolates of different genetic clusters of strains circulating in Lisbon Health Region were
     screened	for	the	presence	of	16	distinct	RDs.	Deletion	detection	was	performed	by	PCR	carried	out	using	primers	flank-
     ing	each	RD.	Confirmation	was	performed	by	sequencing	analysis.
     All three isolates were found to possess four of the tested deletions: TbD1, pks15/1, RD174 and RDRIO. It was not pos-
     sible to discriminate between the strains using this deletion typing approach. However, it was possible to infer on the
     phylogeography of these strains. The presence of TbD1 and pks15/1 deletion positions the strains in the modern and
     Euro-American lineage, respectively. On the other hand, RD174 suggests that the analyzed strains are related to the
     West-African sub-lineage.
     The present study point toward the fact that Lisboa strains and others circulating in Lisbon belong to Mycobacterium
     tuberculosis modern lineages of the Euro-American lineage,West-African sub-lineage, therefore revealing more on Lisboa family’s origin.

84                                                                                                                               ESM 2009

impOrTaNCE Of mOlECular TypiNg iN SuSpECTED
iNTra-familial TraNSmiSSiON Of TubErCulOSiS

Obrovac, Mihaela 1, Katalinic-Jankovic,Vera 1, Grce, Magdalena 2, Zmak, Ljiljana 1
1 - Croatian National Institute of Public Health
2 - Rudjer Boskovic Institute

Tuberculosis (TB) is most commonly transmitted from a person suffering from infectious pulmonary TB to another person
by infected droplet nuclei. The chance that a person will be infected with TB depends on the intensity, frequency and dura-
tion of the exposure to tubercle bacilli. Also, numerous studies emphasize the importance of host resistance and hereditary
susceptibility, indicating that the development of TB is a result of a complex interaction between the host and the pathogen
influenced	by	environmental	factors.	Assuming	that	there	is	prolonged	duration	and	frequency	of	contact	between	family	
members and among household contacts, it is estimated that the risk of TB transmission will be high. There are several
studies	confirming	intra-familial	transmission	using	genotyping	of	isolated	M.	tuberculosis	strains.	However,	the	possibility	of	
unsuspected transmission should not be disregarded.We report here of two cases of suspected TB transmission in families
caused	by	M.	tuberculosis	strains	that	were	found	not	to	be	identical	according	to	genotyping	profiles	obtained	by	determin-
ing variabile number of tandem repeats (VNTR) of mycobacterial repetitive interspersed units (MIRU). Genotyping was per-
formed using complete set of 24 VNTR loci. Epidemiological data were collected by contact tracing and interviewing patients.
Our results show that TB cases in family do not necessarily have to be caused by the same M. tuberculosis strain. Epidemiologic
investigations need to be combined with genotyping data for better understanding of transmission dynamics.Transmission of
TB	cannot	be	confirmed	by	contact	investigation	only,	even	when	intra-familial	transmission	is	suspected.	

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                      85

     DETECTiON Of ClONal COmplEXiTy iN CliNiCal M. TuBerCuloSiS
     iSOlaTES by miru-VNTr iN CuKurOVa rEgiON, TurKEy

     University of Cukurova

     Purpose of the study: Tuberculosis remains one of the most prevalent infectious disease in the world . The applica-
     tion of molecular typing methods to the analysis of clinical Mycobacterium tuberculosis (MTB) complex isolates has
     greatly facilitated the understanding of epidemiology of tuberculosis (TB) and revealed that the infection by this patho-
     gen can be clonally complex and reinfection, coinfection. Genotyping using RFLP-IS6110 (Restriction Fragment Length
     Polymorphism) is based on transposable element IS6110 and mycobacterial interspersed repetitive unit-variable number
     of tandem repeat typing (MIRU-VNTR) has become a major method for epidemiological tracking of Mycobacterium
     tuberculosis complex clones. Our aim was to establish the range of applicability of 12 loci MIRU–VNTR genotyping in
     epidemiology of TB and evaluate the discriminatory power obtained with RFLP-IS6110 and MIRU-VNTR used alone or
     in combination.

     In this study, we analyzed 94 clinical MTB complex isolates from sputum in patients with pulmonary TB between February
     2008- February 2009 in Cukurova region, Turkey.

     MIRU–VNTR typing detected 45 different patterns, 61 strains were grouped into 12 clusters and 33 strains had uniqe
     patterns. The largest cluster comprised 9 strains. In addition, 2 clusters contained 5 strains, 6 clusters contained 3 strains
     and 1 cluster contained 2 strains. It is also determined that the loci including MIRU 04, MIRU 10, MIRU 26 and MIRU 40
     have the highest allelic diversities and discriminatory power. On the other hand with IS6110-RFLP typing of same clini-
     cal MTB strains, 33 genotypes were founded. 76 strains of which were gruoped into15 clusters. 18 of the 94 strains had
     unigue RFLP patterns.

     MIRU-VNTR	 is	 more	 discriminative	 methods	 for	 phylogenetic	 studies	 than	 IS6110-RFLP	 and	 could	 define	 from
     reinfections to reactivations important to treatment of MTB complex organisms.

86                                                                                                                       ESM 2009

mOlECular EpiDEmiOlOgy STuDy Of TubErCulOSiS paTiENTS
iN a Small CiTy Of SÃO paulO – brazil, frOm 2002 TO 2006

Leite, Clarice;	Santos,	Adolfo;	Pandolfi,	José	Rodrigo;	Malaspina,	Ana	C;	Pavan,	Fernando;	Mendes,	Natália
Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas

The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology of
tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movements of individual
lines. This project aimed to use the techniques of molecular epidemiology (MIRU) trying to understand more about the
phenomenon of transmission of tuberculosis among patients with pulmonary tuberculosis in a small city of São Paulo
- Brazil, attended the Special Health Service of Araraquara (SESA - clinic of reference in the diagnosis and treatment of
tuberculosis) in the city of Araraquara. From total 163 positive cultures received, the MIRU technique was performed in
74,2% (121/163) of the isolates from patients attended by SESA in the period from 2002 to 2006. 5 isolates were also
identified	as	environmental	mycobacteria	and	4	unidentified	mycobacteria.	From	the	121	isolates	submitted	to	genotyp-
ing, six did not present all alleles among the 12 loci of MIRU. From the 115 isolates submitted to the dendrogram, 29
(25,2%) are grouped into 13 clonal groups with similarity of 100%. 10 groups with two isolates each and 3 groups con-
taining	3	isolates	each.	The	others	86	isolates	(74,8%)	had	a	single	genetic	profile.	About	the	group	of	29	patients,	only	10	
were female and 19 males. Except for one patient, the other 28 were treated with the schedule I having cure in 82,8% of
the cases. Whereas the similarity of 83% or greater, highlighted 3 major clonal groups called A, B and C, involving 86 of
all isolates analyzed. In these large groups were included all 13 clonal groups with 100% similarity. These data suggest the
possibility the tuberculosis in Araraquara is because of the presence of persistent endemic strains responsable for 74,8%
of cases, besides the existence of recent transmission in this work was 25,2%.

Keywords: Epidemiology, Tuberculosis, MIRU
Financial support: FUNDUNESP.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                   87

     gENOTypiNg Of myCObaCTErium TubErCulOSiS
     iN NOrThWEST Of paraNÁ STaTE Of brazil

     Leite, Clarice Queico Fujimura 1, Nogutia, Erika N 2, Malaspina, Ana Carolina 1, Santos,
     Adolfo Carlos Barreto 1, Hirata, Rosáro DC 3, Hirata, Mário H 3, Cardoso, Rosilene Fressatti 2
     1 - São Paulo State University
     2 - Maringá State University
     3 - University of São Paulo

     Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death.
     The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology of
     tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movements of indi-
     vidual	lines.	We	reported	here	the	first	insight	about	the	genetic	diversity	of	M.	tuberculosis	in	northwest	of	Paraná	State,	
     south of Brazil. This knowledge encourages additional prospective epidemiological study for evaluation of the Regional
     Tuberculosis Control Plan in this setting.

     Provide information about the genetic diversity and prevalent genotype of Mycobacterium tuberculosis and compare the
     usefully of two methodologies in epidemiological study of tuberculosis in low endemic area in south of Brazil. Material
     and Methods: We used spoligotyping and MIRU-VNTR typing to genotype M. tuberculosis isolates.

     The 93 isolates analyzed by spoligotyping were divided into 36 different patterns and 25 were described in the SpolDB4.0
     database. Latin American and Mediterranean, Haarlem and T family were responsible for 26.9%, 17.2% and 11.8%, of
     tuberculosis cases respectively. From the 84 isolates analyzed by MIRU-VNTR typing, 58 showed unique pattern and 26
     belonged to 9 clusters. The MIRU loci 40, 23, 10 and 16 were the most discriminatory. MIRU-VNTR and spoligotyping
     combined showed 85.7% of discriminatory power (HGI=0.995).

     Spoligotyping and MIRU-VNTR typing combined are useful tool for epidemiological study in this low endemic setting in
     south of Brazil and tuberculosis predominantly develops through reactivation of latent infection.

88                                                                                                                      ESM 2009

SpOligOTypiNg Of myCObaCTErium TubErCulOSiS iSOlaTED frOm
paTiENTS Of ClEmENTE fErrEira ambulaTOry iN SÃO paulO, Sp – brazil

Mello, Fernado Augusto Fiuza 1, Albarral, Maria Idemar Pedrosa 1, Mendes, Natália Helena 2,	Pandolfi,	José	Rodrigo	
Cláudio 2, Santos, Adolfo Carlos Barreto 2, Almeida, Elisabete Aparecida 1, Cardoso, Rosilene Fressatti 3, Leite, Clarice
Queico Fujimura 2
1 - Clemente Ferreira Institute
2 - São Paulo State University
3 - Maringá State University

The molecular epidemiology study using different techniques revolutionized the understanding of the epidemiology
of tuberculosis allowing comparison between strains of Mycobacterium tuberculosis and tracking the movement of
individual strains. This project aims to use the technique of molecular epidemiology, Spoligotyping, trying to understand
more about the phenomenon of transmission in patients with pulmonary tuberculosis treated at Clemente Ferreira
Ambulatory (ambulatory of reference for the treatment of tuberculosis) in São Paulo city, from August 2006 to July 2008.
The	clinical	isolates	were	re-identified	by	molecular	technique	(PCR	and	PRA),	and	the	strains	identified	as	M.	tuberculo-
sis	conducted	by	the	genotyping	technique	of	Spoligotyping.	From	102	isolates,	the	technique	of	IS	6110-PCR	confirmed	
the	identification	of	M.	tuberculosis	in	96	clinical	isolates	and	the	PRA	in	99,	the	remaining	3,	2	isolates	identified	as	M.	
avium	subtype	2	and	1	unidentified	mycobacteria.	The	results	showed	that	3	clinical	isolates	of	M.	tuberculosis	had	not	
the	IS6110	insertion	sequence	specific	of	M.	tuberculosis,	as	well	as	3	isolates	identified	in	the	clinic	as	M.	tuberculosis	
by	molecular	techniques	were	atypical	mycobacteria.	Of		96	isolates	confirmed	as	M.	tuberculosis,	were	analyzed	by	the	
technique of Spoligotyping, a total of 89 isolates, which revealed the presence of 21 strains (23.6%) with spoligotipes not
yet described in the data base world (spolDB4) and 68 (76.4%) of isolates involved in 7 different families, containing 2 to
30 isolates. The most frequent was T family with 30 isolates), followed by LAM (with 20 isolates) and Haarlem (with 10
isolates),	which	together	accounted	about	67.4%	of	all	isolates.	Were	also	identified	4	genotypes	of	the	Beijing	family,	all	
simultaneously resistant to isoniazid and rifampicin and / or more drugs.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                   89

     COmpariSON Of myCObaCTErium bEiJiNg
     gENOTypE WiTh VNTr, SpOligOTypiNg aND rflp-iS6110

     Elahe Tajeddin , Parissa Farnia, Mohammad Kargar,Jamileh Noroozi, Mojtaba ahmadi,
     Mehdi kazempour, Maryam Hadadi,Mohammadreza Masjedi, Aliakbar Velayati
     Mycobacteriology Research Center (MRC) National Research Institute Of Tuberculosis and
     Lung Disease (NRITLD), Shahid Beheshti University Medical Campus.Tehran,Iran.

     Beijing strains constitute more than 1/4 of Mycobacterium tuberculosis (MTB) genotypes. Beijing genotype is considered
     an important genotype because of its reasonable characteristics such as: association with multi-drugs resistance TB.
     Accordingly these strains are reluctant to conventional TB drugs.Therefore, it is necessary to investigate the transmission
     rate among Beijing strains within the studied communities. In this study, three molecular methods (Spoligotyping,VNTR,
     and RFLP-IS6110) were used to identify transmission among patients infected with Beijing strains.

     materials and methods
     The susceptibility tests were performed on 238 M. tuberculosis culture positive specimens. Thereafter, the isolated Beijing
     genotype was subjected to VNTR and RFLP. The results of Spoligotyping were analysed by using SPOLDB4 database.
     VNTR typing was used to identify alleles diversity in 9 locus (MPTR-A, ETR-A, ETR-B, ETR-C, ETR-D, ETR-E, ETR-F,
     QUB11B, QUB3232) of isolated Beijing strains.The allelic diversity of VNTR was measured by using Hunter Gaston
     Index (HGI).

     The spoligotyping of M. tuberculosis isolates revealed the following 8 patterns: Haarlem (27.7%), CAS1 (25.2%), EAI3
     (21.8%), CAS2 (6.7%), T1 (6.3%), Beijing(5.5%) U(5%), T(0.4), EAI2 (1.2%).
     The	following	VNTR	loci	(QUB3232),	(QUB11b,	ETR-E	and	ETR-F)	and	(other	loci)	were	identified	as	most	(HGI≥ 0.6),
     median (HGI≥0.4-0.6) and weakest (HGI=0) distinctive loci for Beijing families respectively. Whereas the Beijing strains
     demonstrated diverse patterns in RFLP,13/13(100%) and VNTR 10/13(77%).

     Beijing is one of the dominated circulating strains in Iran and interestingly majority of infected cases were due to reactiva-
     tion	rather	than	recent	transmission.The	VNTR	and	spoligotiping	methods	were	more	efficient	to		detect	Beijing	strains	
     than by use VNTR and RFLP allow.

     Spoligotyping / VNTR / RFLP / Mycobacterium tuberculosis Beijing genotype.

90                                                                                                                      ESM 2009

mulTiply rECurrENT TubErCulOSiS iN a paCiENT
liViNg WiTh hiV: rEiNfECTiON Or rEaCTiVaTiON?

Ritacco,Viviana 1, Reniero, Ana 2, Beltrán, Marcelo 2, López, Beatriz 1, Kantor, Isabel 3, Barrera, Lucía 1
1 - ANLIS, CONICET, Argentina
2 - Hospital Municipal de San Isidro
3 - PAHO/WHO consultant

To determine the cause of recurrent clinical episodes of tuberculosis in a patient living with HIV. Patient: Male, 24 year-old
and	illegal	drug	intravenous	user	for	10	years	at	the	time	of	first	consultation,	assisted	between	1995	and	2009	in	our	
outpatient clinic, San Isidro Municipal Hospital, Argentina.

Clinical-epidemiological	 follow	 up.	 Mycobacterial	 culture,	 identification	 and	 drug	 susceptibility	 testing.	 Mycobacterium
tuberculosis IS6110 RFLP and spoligo genotyping within the frame of a population-based study performed in San Isidro,
an outskirt of Buenos Aires City.

Our	patient’s	first	diagnosis	of	tuberculosis	was	confirmed	by	culture	in	1995,	almost	simultaneously	with	HIV	serological	
conversion. At that time, the isolate was found only resistant to isoniazid and its genotype matched that of a isoniazid-
resistant	outbreak	strain	previously	identified	in	two	of	our	patient’s	prison	inmates,	who	were	also	members	of	his	
intravenous-drug-user gang. One year after cure, our patient suffered from a relapse of his tuberculosis due to the same
strain,	which	now	had	added	resistance	to	rifampicin	and	lost	a	band	in	the	IS6110	fingerprint.	In	2002,	the	patient	suf-
fered	from	a	third	episode	of	tuberculosis,	this	time	due	to	a	fully	drug	susceptible	strain,	identified	previously	in	other	
of our patient’s intravenous drug user friends. At that time, given his poor clinical condition, our patient underwent a
prolonged hospitalization in the Muñiz Hospital, the epicentre of a large tuberculosis outbreak caused by the notorious
multidrug-resistant strain “M”. After successful treatment and cure, our patient, who was never compliant with antiretro-
viral treatment, was discharged from the Muñiz Hospital. A few months later, he sought again assistance at our clinic with
active tuberculosis due to the multidrug resistant strain “M”. He is currently struggling with a relapse of disease caused
by this outbreak strain.

 Whether recurrent tuberculosis is due to a newly acquired infection or to reactivation of a previous one is a century-
long controversial question. In our case, both conditions alternated throughout the 14 years of the patient living with
HIV. Work partially funded by FP7 grants 201690 and 223373 of the EC.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                       91

     mOlECular EpiDEmiOlOgy Of TubErCulOSiS
     iN Vila NOVa DE gaia, pOrTugal

     Tavares Magalhães, Ana1,Alves, Adriana1, Braga, Rosário2,Valente, Isabel2, Duarte, Raquel3 and Miranda, Anabela1
     1 - Tuberculosis Reference Laboratory, Department of Infectious Diseases, National Institute of Health, Porto, Portugal
     2 - Central Hospital,Vila Nova de Gaia, Portugal
     3 - Chest Clinic,Vila Nova de Gaia, Portugal

     DNA	fingerprinting	of	Mycobacterium tuberculosis has provided a better understanding of the epidemiology of tubercu-
     losis. Restriction fragment length polymorphism based on IS6110 (RFLP-IS6110) has been the gold standard for typing
     M. tuberculosis since 1993. In recent years, mycobacterial interspersed repetitive units variable number tandem repeat
     (MIRU-VNTR)	has	been	proposed	as	a	first-line	typing	method for M. tuberculosis. This technique generates easily ana-
     lyzed and portable data, has a good discrimination power and has been proven useful for studying the epidemiology of
     tuberculosis and the phylogeography of tuberculosis bacilli.
     In the present study we evaluated the genetic diversity of M. tuberculosis clinical strains, isolated from 115 patients from
     Vila Nova de Gaia, Portugal, during the period of 2004 to 2005, using the standardized MIRU-VNTR typing method based
     on 15 loci, proposed by Supply and collaborators in 2006. Strain lineage designation, allelic diversity and clustering rate
     were determined using the MIRU-VNTRplus	identification	database.	The	discriminative	power	of	the	method	was	ana-
     lysed using the Hunter and Gaston diversity index.
     Based on MIRU-VNTR typing, the 115 strains were divided into 62 types as follows: 69 strains were distributed into 16
     clusters	containing	two	to	eight	isolates,	and	46	strains	had	unique	profiles.	MIRU-VNTR	revealed	a	clustering	rate	of	
     46% in the sample under study. The Hunter-Gaston index was 0.976. The most discriminatory loci were Mtub04, MIRU
     40, MIRU10, QUB11b, Mtub30, Mtub39 and QUB26 showing an allelic diversity higher than 0,6.
     The phylogenetic analysis revealed the presence of two major lineages, LAM and Haarlem, representing 60% and 27% of
     the total isolates, respectively. This fact is in agreement with the M. tuberculosis phylogeography for the South of Europe.
     Fourteen	strains	showed	drug	resistance	but	no	association	was	established	between	drug	resistance	profile	and	phylo-
     genetic family.
     MIRU-VNTR showed a good discriminatory power for typing of M. tuberculosis strains in this setting and the MIRU-
                           is	an	important	tool	for	lineage	identifi
                                                    lineage	identification.	This	typing	approach	offers,	simultaneously,	epidemio-
     VNTRplus database	is	an	important	tool	for	lineage	identification.	This	typing	approach	offers,	simultaneously,	epidemio-
     logic and phylogenetic information, as demonstrated. Overall, this study reveals that recent transmission of tuberculosis
     is high in Vila Nova de Gaia, and that import of strains in this region is not a problem.

92                                                                                                                      ESM 2009

mOlECular STuDy Of rECurrENT TubErCulOSiS CaSES

Alves, Adriana; Miranda, Anabela; Tuberculosis Reference Laboratory Group
Tuberculosis Reference Laboratory, Department of Infectious Diseases, National Institute of Health, Porto, Portugal

Tuberculosis	recurrence	is	frequently	attributed	to	reactivation	of	the	isolate	responsible	for	the	first	episode	of	the	
disease.	Nevertheless,	this	can	also	be	due	to	an	infection	with	another	isolate,	or	to	mixed	infections.	Clarification	of	
the cause of recurrence is very important and can be achieved by molecular typing of serial isolates of M. tuberculosis.The
most appropriate methods to do so are IS6110 RFLP and MIRU-VNTR.
In this work, 39 clinical isolates of M. tuberculosis belonging to nine individuals with recurrent disease were studied
throughout time. Seven of these patients were resistant to isoniazid and rifampicin as well as to most other 1st and 2nd
line drugs. Another patient was resistant to isoniazid and streptomycin, and the last one was resistant to rifampicin only.
For some of these individuals, resistance to drugs worsened during the cause of the disease, which in some cases has
lasted	for	more	than	a	decade.	All	39	strains	were	analyzed	by	IS6110	RFLP.	MIRU-VNTR	was	used	to	type:	(i)	the	first	
strain	of	each	patient	when	serial	isolates	showed	no	change	in	the	IS6110	RFLP	profile;	or	(ii)	each	strain	with	a	pattern	
change in relation to the previous one.
RFLP results showed that only one out of nine patients displayed a change in the pattern of serial isolates with gain of
one IS6110 element. Analysis of the results using Bionumerics grouped these patients in nine clusters, being that: (i) strains
from two patients belong to the same cluster; and (ii) strains of one patient are divided in two clusters. Results of 15
MIRU-VNTR loci	typing	corroborate	the	IS6110	RFLP	findings.	In	other	words,	the	serial	isolate	that	gained	one	IS6110	
element also shows a change in MIRU-VNTR results. In this case, we observed a reduction of one repeat in loci Mtub21
and QUB11b. Finally, 40% of these strains belong to the LAM family, 10% to the Haarlem family, and the remaining 50%
did	not	find	a	match	in	the	database.
This work shows that the cause of recurrent tuberculosis of the nine patients included in this study is due to persistence
of initial M. tuberculosis strain or to reactivation when there is more than one episode of disease. Antibiotic resistance is
the most important cause of chronic tuberculosis in these patients, since all analyzed strains are resistant to the majority
of the 1st and 2nd line drugs.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                   93

     gENOTypiNg Of Drug rESiSTaNCE iN MyCoBaCTeriuM
     TuBerCuloSiS uSiNg DiagNOSTiC miCrOarrayS

     Ehricht, Ralf 1, Slickers, Peter 1, Monecke, Stefan 2
     1 - CLONDIAG, Jena, Germany
     2 - University Hospital Dresden

     Tuberculosis is a disease of worldwide concern. Antimicrobial resistance in Mycobacterium tuberculosis is an increasing
     challenge and, in contrast to other bacteria, not caused by the acquisition of certain genes, but by acquisition of single
     point mutations in genes which are present in all strains. Unfortunately, culturing and subsequent growth inhibition assays
     are still time demanding preventing fast detection and treatment. Genotyping methods as PCR followed by sequencing
     are an alternative. Here the bottlenecks are processing time, overall costs and lack of parallelisation. Hybridisation of
     such PCR products against highly discriminatory oligonucleotide probes is an alternative approach which could solve
     these problems. We developed an assay using a diagnostic oligonucleotide microarray and covering probes for relevant
     mutations in genes rpoB, katG, embA, and embB, for the embC/embA-intergenic region, and the mabA/inhA promoter.
     PCR is employed to amplify these genes and to incorporate biotin 16-dUTP during elongation. These labeled amplicons
     are hybridised to the array which are inserted into ArrayStrips, and hybridisation is visualised using dye precipitation trig-
     gered by streptavidin-peroxidase complexes bound to the biotin labels and the ArrayMate reading device.The procedure
     is currently tested using DNA from previously characterized strains for which conventional susceptibility test results
     and relevant sequences are available.

94                                                                                                                       ESM 2009

gENOTypiNg Of mONO aND mulTi-Drug rESiSTaNCE Tb iN SauDi arabia

Sahal Al-Hajoj1, Bright Varghese1, Mais Herbawi1 , Ruba Al-Omari1 and Caroline Allix-Béguec2
1 - King Faisal Specialist Hospital and Research Centre, Comparative Medicine Tuberculosis Research Unit Saudia Arabia
2 - Genoscreen

A total of 150 isolates collected from different regions in Saudia Arabia were the subject	of	finger	printing	using	GenoScreen	
MIRU-VNTR kit (do you know the timeframe?). Upon genotype the data were compared to the international MIRU-
VNTRplus database (www. The data showed that Saudia Arabia harbors the following major clades EAI
13.07%, Haarlem 12.42%, TUR 13.07%, Beijing 12.42%, unknown12.42%, Dehli/CAS 7.84%, LAM 7.19%, Cameroon 6.54%,
UgandaI 5.23%, S 3.92%, multiple matches1.96%, NEW-1 1.31%, URAL 0.65%, Ghana 0.65%, X, 0.65%, UgandaII, 0.65%. Indicate
the clustering rate for this panel of 150 isolates. Ongoing transmission may be an indication to the need of improving TB
control program. The unknown clades represent a considerable % (12.42%). These could represent unique endogenous
clades. However, further analysis is needed to gain insight into the nature of the unknown clades.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                    95

     Early DETECTiON Of mDrTb by mOlECular TOOlS iN ThE CONTrOl
     Of Drug rESiSTaNT TubErCulOSiS iN pOrTugal: a CaSE Of SuCCESS

     Diana Machado1, Miguel Viveiros1,2, Liliana Rodrigues1,3, Isabel Couto1,4, Leonard Amaral1,2,3
     1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal
     2 - COST ACTION BM0701 (ATENS)
     3 - UPMM, IHMT/UNL, Lisbon, Portugal
     4 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal

     Multidrug resistant tuberculosis (MDRTB) represents a threat to public health and a challenge to tuberculosis (TB) con-
     trol programs. From 1994 to 1997, Portugal had an incidence of 48.3 new cases of TB per 100 000 inhabitants and an
     average of 22.7% of these cases were MDRTB, the highest in Western Europe. In an attempt to assist the National Health
     Authorities in the control of TB, we implemented in 2002 with the support of Fundação Calouste Gulbenkian, the “TB
     Fast-Track” program as part of the TB Task Force of Greater Lisbon, involving 12 Lisbon hospitals and based on the use of
     the BACTECTM	MGIT	960	system,	coupled	with	the	direct	identification	of	M. tuberculosis complex (MTBC) and the de-
     tection of mutations in the rpoB gene, using the INNOLiPA Rif.TB Assay (LiPA) (Innogenetics). Because mutations in the
     rpoB result in resistance to rifampicin (RIF), and resistance to RIF is almost always accompanied by resistance to isoniazid
     (INH),	this	approach	allowed	us	to	identify	the	MDRTB	patient	within	24-48	hours.	A	full	report	confirming	identification	
     and antibiotic susceptibility (AST) of MTBC by conventional methods (BACTEC culture plus AST and Accuprobe ID)
     was issued within additional 12 days.

     From September 2002 to January 2006, the LiPA assay was directly applied to 630 acid fast positive respiratory speci-
     mens. The comparison between data from this assay and conventional methods revealed 84 discrepancies. The 11 false
     positive results corresponded to patients with therapy already established by the time of specimen sampling, whereas
     the	73	false	negative	resulted	from	inhibition	of	amplification.	A	total	of	487	of	the	600	MTBC	positive	isolates	were	
     susceptible	to	all	5	first-line	anti-TB	drugs.	The	frequency	of	MDRTB	(resistant	to	at	least	INH	plus	RIF)	was	10%.	From	
     the 63 MTBC resistant to RIF, 62 were detected by the LiPA as carrying mutations S531L (60 isolates), H526Y and D516V
     (1	isolate	each).	No	mutation	was	detected	by	LiPA	for	one	sample,	repeatedly	identified	as	resistant	by	AST.	Detection	
     of rpoB mutations proved to be a good surrogate marker for MDRTB, since only 2 out of 600 MTBC isolates were
     monoresistant to RIF.

     The early detection of active TB, particularly the detection of MDRTB, is essential for the success of any TB control
     program.	The	application	of	molecular	techniques	for	the	early	identification	of	MDRTB	assisted	the	National	Health	
     Authorities in the reduction of MDRTB rates in Lisbon to less than 8% (average 2003 to 2007).

96                                                                                                                          ESM 2009

Drug-rESiSTaNCE Of MyCoBaCTeriuM TuBerCuloSiS
aT pENiTENTiary iNSTiTuTiONS Of ST. pETErSburg,
ruSSiaN fEDEraTiON.

Vladimirov, Kirill; Zaitseva, Elena; Ivanov, Aleksandr
Institution: State Medical Academy named after I.I. Mechnikov, St. Petersburg, Russia.

Morbidity with tuberculosis (TB) in Russian Federation and the whole world remains high. This index is up to 40 times
above the average level among prison population, with high prevalence of multi-drug resistant (MDR) TB.
Setting. Central hospital for prisoners in St. Petersburg.
Study design. We retrospectively reviewed data of the patients, who were admitted to the hospital for active culture-
positive TB between 2005 and 2008. Between 2005 and 2007, new and re-treatment cases were admitted. In 2008, only
new TB cases were admitted. We studied the results of drug-susceptibility of Mycobacterium to Isoniazid (H), Rifampicin
(R),	Ethambutol	(E),	Streptomycin	(S),	Kanamycin	(K),	Ofloxacin	(O)	in	solid	Lowenstein-Jensen	medium.	Cases	of	pan-
sensitivity,	drug	resistance	(DR),	including	MDR	and	extra	drug	resistance	(XDR)	were	defined.	

As much as 163 cultures were studied. From 52 cases in 2005, 36.5% were pan-sensitive, 25.0% were DR and 38.5%
were MDR. In 2006, 36.0% of 50 cultures were pan-sensitive, while casualty of MDR increased to 48.0%. In 2007, number
of pan-sensitive cultures decreased to 21.4%, while 35.7% were MDR and 7.2% were XDR. In 2008, among 47 cultures
nearly half were MDR (48.9%) and 12.8% were XDR, only 27.7% cultures were pan-sensitive.

Among prisoners in St. Petersburg, the value of the cases of primary MDR and XDR TB increased dramatically. Introduction
of the fast methods of drug resistance detection is required.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                             97

     aN iNCrEaSE Of Drug rESiSTaNCE SiNCE 2001
     iN mulTiDrugrESiSTaNT M. TuBerCuloSiS iSOlaTES frOm bElgium

     Karolien Stoffels, Maryse Fauville-Dufaux
     Reference	Laboratory	of	Tuberculosis	and	Mycobacteria,	Scientific	Institute	of	Public	Health,		642	Rue	Engeland,	1180	
     Brussels, Belgium. Tel. +32-23733210 | Fax. +32-23733281.,	

     Between January 1994 and December 2008, MDR clinical isolates of 174 patients were analyzed in our National Reference
     Laboratory.		They	represent	90%	of	all	the	MDR-TB	patients	identified	in	Belgium	during	this	15-years	period.	Since	2000,	
     the	number	of	MDR	patients	identified	in	our	country	is	stable	(in	average	16	per	year,	i.e.	an	average	of	1,3	%	of	the	
     patients tested for susceptibility to drugs) but the isolates are resistant to more and more second line drugs.We observe
     a	dramatically	increase	in	resistance	to	ethambutol,	rifabutin,	amikacin	and	ofloxacin,	as	well	as	to	pyrazinamid.
     We	divided	the	studied	period	in	2	ranges,	1994	to	2000	and	2001	to	2008.	Only	the	first	MDR	clinical	isolate	of	each	pa-
     tient was taken into account. So these results do not consider the evolution of the isolates during treatment in Belgium,
     but	only	the	initial	MDR	resistance	profile.	
     In the second period (2001 to 2008) 75,6% of the MDR clinical isolates showed resistance to ethambutol versus
     45,5%	in	the	first	period	(increase	of	resistance	of	30,1%);	75,4%	were	resistant	to	rifabutin	versus	70,4%	in	the	first	
     period (increase of 5%). Resistance to pyrazinamid increased from 39,6% to 55,6% (difference of 16%). Resistance
     level to amikacin showed an increase of 12,2% (3,6% to 15,8%) and resistance level to ofloxacin showed an increase
     of 8,6% (3,6% to 12,2%).
     No	primary	XDR	isolate	was	observed	during	the	first	period,	but	5	were	detected	since	2001.	Three	MDR	isolates	developed	
     into	XDR	what	the	total	amount	of	XDR	strains	brought	to	6	and	only	2	for	respectively	the	second	and	first	period.	
     Concerning	the	genetic	families	identified,	14,7%	more	Beijing	strains	were	registered	in	the	second	period	compared	to	
     the	first	period.	A	decrease	of	the	members	of	the	LAM	and	Haarlem	family	was	noted	(16,4%	and	17,3%).
     In	conclusion,	an	important	increase	of	resistance	to	ethambutol,	pyrazinamid,	amikacin	and	ofloxacin	is	observed	in	MDR	
     clinical	isolates	detected	in	Belgium.	This	confirms	the	urgent	need	for	new	anti-tuberculosis	drugs.	

98                                                                                                                  ESM 2009

muTaTiONal aNalySiS Of gENES aSSOCiaTED WiTh
TuBerCuloSiS CliNiCal iSOlaTES frOm liSbON, pOrTugal

João Perdigão1, Ana Ferreira1, Ana Malaquias1, Rita Macedo2, Laura Brum2 and Isabel Portugal.1,2
1 - Centro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa
2 - Laboratório de Micobactérias, Centro de Bacteriologia, Instituto Nacional de Saúde Dr. Ricardo Jorge

Multidrug resistance (MDR) constitutes a serious problem to tuberculosis (TB) control program in Portugal. An even
more serious threat is the one posed by the high rate of extensive drug-resistant TB (XDR-TB). Our laboratory has
already shown that high rates of this form of TB exist in Lisbon. Given the fact that MDR-TB and XDR-TB are currently
associated with a limited number of genetic clusters, mainly Lisboa family clusters, the diversity of genetic polymorphisms
conferring resistance to second-line drugs is also probably limited. In this study we intended to characterize the genetic
polymorphisms associated with resistanace to second-line injectable drugs and to assess the clinical isolates clonality.

We have analyzed 19 MDR-TB strains resistant to one or more second-line injectable drugs, collected from several hos-
pital units across Lisbon Health Region during the year of 2005. All isolates were typed by Mycobacterial Interspersed
Repetitive Units (MIRU-VNTR) and, screened for mutations in tlyA and rrs genes.

Three	different	mutations	were	identified	on	tlyA gene and another three at rrs gene. Overall, 9 isolates had mutations in
tlyA gene and 8 isolates had mutations in rrs gene; two isolates didn’t have any mutation in either gene.The most frequent
mutations found were A1401G in rrs gene (6/19) and 755InsGT in tlyA	gene	(6/19).	We	also	verified	that	there	was	no	
overlapping of mutations from different genes. The genotyping analysis revealed that the isolates could be distributed
through two different MIRU-VNTR genetic clusters: Lisboa3 and Q1. Cluster Q1 contained all clinical isolates bearing the
A1401G mutation in rrs gene, while Lisboa 3 cluster contained all isolates that had the 755InsGT mutation in tlyA gene.

We	have	identified	several	mutations	that	might	be	associated	with	resistance	to	different	but	related	second-line	drugs:	
kanamycin, amikacin and capreomycin. The two most prevalent mutations were associated with different genetic clus-
ters, which suggests recent transmission and, ultimately, that XDR-TB transmission is taking place. The most prevalent
mutations	associated	with	injectable	second-line	drugs	have	therefore	been	defined,	which	opens	the	way	for	molecular	
detection of resistance to second-line drugs in the region.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                99

      mulTiDrug-rESiSTaNT TubErCulOSiS

      Nuak, Joao; Ferreira, Danina; Carvalho, Teresa; Gomes, Maria Helena; Sarmento, Antonio
      Hospital S. Joao, Porto - Portugal

      Multidrug-Resistant Tuberculosis (MDRTB) is caused by M.tuberculosis (MT) resistant to at least Isoniazid (INZ) and
      Rifampin (RIF), and can be due to unsuitable or irregular treatment.

      To know the epidemiological and clinical characteristics of the disease in patients (pts) hospitalized with MDRTB in an
      Infectious Diseases Service.

      patients and methods
      Review of clinical records of pts with MDRTB. Diagnosis was based on drug susceptibility testing.

      Between 1996 and 2007, 16 pts had MDRTB. Eleven were male. Ages ranged between 27-40y(X=32.2±4.33). Fifteen
      (93.8%) had HIV infection (14 drug addicts; 1 sexual risk). One pt had professional contact with MDRTB.The disease was
      only pulmonary in 7(44%) pt, disseminated in 5(31%), pulmonary and extra-pulmonary in 3(19%)-meningeal, lymph nodes
      (LN) and urine in one each pt; another pt had only meningeal disease. Eleven (69%) pts had previous irregular antituber-
      culosis treatment. MT was isolated in sputum in 12/16 pt (75%); CSF in 5/6 pt (83%), bronchoalveolar lavage in 2/6(33%),
      blood in 3/6(50%), urine in 2/6(33%), LN in 2/6(33%), gastric aspirate and feces in one each. Most pts had MT isolated in
      more than one sample. All MT were resistant to INZ and RIF. Thirteen out of 16 pt (81%) were also resistant to strep-
      tomycine, 6/12(50%) to pyrazinamide, 6/16(37%) to ethambutol, 5/12(42%) to rifabutine, 10/15(67%) to ethionamide,
      4/14(28%)	to	kanamycin,	4/13(31%)	to	ofloxacilline,	3/7(43%)	to	PAS,	2/5(40%)	to	kapriomycine	e	1/5(20%)	to	cyclocerin.	
      In 4 pts MT was simultaneously resistant to at least three 2nd line drugs (XDRTB). Fifteen pts had medical treatment ac-
      cording to the drug susceptibilities testing. In two pts pneumectomy was performed. Eight pts died: One before diagnosis
      and 7 between 30-720 days of diagnosis. One pt survived for 6y. maintaining positive cultures of sputum despite medical
      and surgical therapy.Two pts were treated for 18 months with clinical and radiological improvement, without evidence of
      disease 1 and 5y. later. Five pts were lost to follow-up. One is on treatment with clinical and radiological improvement.

      MDRTB is a serious public health problem. HIV, drug addiction and irregular treatment were important factors for its
      development. Prognosis depends on early detection of drug resistance and institution of appropriate therapy.We empha-
      size the very high mortality.

100                                                                                                                  ESM 2009

CharaCTEriSaTiON Of STrEpTOmyCiN muTaTiONS iN MyCoBaCTeriuM
TuBerCuloSiS CliNiCal iSOlaTES iN ThE arEa Of barCElONa

Griselda Tudó1, Emma Rey1, Fernando Alcaide2, Pere Coll3, Gemma Codina4, Núria Martín-Casabona4, Michel Montemayor3,
Raquel Moure2, Margarita Salvadó5, Julià	González-Martín	1
1 - Servei de Microbiologia, CDB. Hospital Clínic de Barcelona-IDIBAPS, Universitat de Barcelona
2 - Servei de Microbiologia, Hospital Universitari de Bellvitge-IDIBELL, Universitat de Barcelona
3 - Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau de Barcelona, Universitat Autònoma de Barcelona
4 - Servei de Microbiologia, Hospital Universitari Vall d’Hebron. Universitat Autònoma de Barcelona
5 - Laboratori de Referència de Catalunya, Barcelona. All the authors are members of Spanish Network for the Research
    in Infectious Diseases (REIPI, RD06/0008).

To determine the proportion and type of mutations in Mycobacterium tuberculosis (Mtb) isolates resistant to streptomycin
(SM) and their relationship with the level of resistance and their genotype.

SM resistant isolates from an Mtb strain collection (1995-2007) were studied. Minimum inhibitory concentrations (MIC)
of SM for each isolate were determined using the proportion method with Middlebrook 7H10 medium. The entire rpsL
gene	and	2	specific	fragments	(loop	530	and	region	912)	of	the	rrs gene were sequenced. IS6110-RFLP and spoligotyping
techniques were used to type Mtb isolates.

Of 69 SM resistant isolates, 36 (52.17%) presented a mutation in either the rpsL gene and/or the rrs530 gene, with no mu-
tation in the rrs912 region. No mutations were found in 33/69 (47.8%) SM resistant isolates (all of them with MIC≤16µg/
ml). Seventeen (24.63%) isolates showed rpsL mutations: 9 (13.04%) at position 88 (7: AAG→AGG, 1: AAG→ACG and 1:
AAG→CAG) and 8 (11.59%) in codon 43 (AAG→AGG). Isolates with mutations in the rpsL gene (94.1%) had a MIC≥512
µg/ml. Among isolates with alterations in the rrs gene (27.53%):10 (14.49%) had a 491 C→T change and low MIC level; 7
(10.1%) had a mutation at position 513 A→T or A→C and 2 (2.89%) had a 516 C→T substitution.These mutation points
were related to intermediate and high MIC levels. One isolate with a codon 88 mutation had a second mutation in the
rrs530 gene at position 491. IS6110-RFLP	typing	identified	4	clusters	(11/69,	13%).	Clusters	I	and	II	were	monoresistant	
to SM, with a low MIC level and a mutation at position 491 in the rrs gene. Cluster III was multidrug-resistant with a
high MIC level and a mutation in codon 88 in the rpsL gene. Cluster IV and V was monoresistant to SM with a low MIC
level and no mutation. Interestingly, all the isolates with a mutation at position 491 in the rrs530	gene	were	identified	as	
LAM3 lineage. All the Beijing family presented mutations in the rpsL gene (2 and 1 at codons 88 and 43, respectively).The
spoligotyping lineageT5-MAD2 was detected in non-mutated isolates.

Mutations in the rpsL and rrs genes were detected in at least 50% of SM resistant isolates. Mutations in the rpsL gene
were associated with high-level resistance while mutations in the rrs530 gene were associated with different MIC levels.
The isolates with no mutations had low-level resistance. Mutations in the rrs530 gene at position 491 were associated
with LAM3 lineage

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  101

      SurVEillaNCE Of Drug-rESiSTaNT TubErCulOSiS iN iTaly

      Fattorini Lanfranco 1, Pardini Manuela 1, Cirillo Daniela 2, Borroni Emanuele 2, Miotto Paolo 2, Filippini Perla 3, Cassone
      Antonio 3, TB-CCM Study Group 4
      1	-	Istituto	Superiore	di	Sanità,	Rome,	Italy
      2	-	Istituto	Scientifico	San	Raffaele,	Milan,	Italy
      3	-	Istituto	Superiore	di	Sanità,	Rome,	Italy
      4 - Italian network of mycobacteriology laboratories

      Drug-resistant TB is an increasing problem worldwide. In order to update the national data on drug resistance, we start-
      ed a surveillance program including the most representative diagnostic centres in Italy. Purpose of the study. The study
      was designed to determine: 1) Accuracy of drug susceptibility testing (DST) for streptomycin (S), isoniazid (I), rifampicin
      (R), ethambutol (E). 2) Drug resistance in new cases (NC), previously treated cases (PTC), patients born in Italy (PBI),
      patients born abroad (PBA). 3) Molecular typing.

      1)	Thirty	laboratories	were	enrolled	in	2006-7	in	18/20	regions	for	proficiency	testing	(PT),	based	on	the	amount	of	
      DST performed every year and geographic location. The laboratories received 20 strains each, and sent DST results to
      the	WHO	Supranational	Reference	Laboratory	(SRL)	of	Istituto	Superiore	di	Sanità	(ISS)	which	compared	them	with	
      the judicial results of the Global Network of SRLs. 2) A questionnaire was returned to ISS with DST results of TB cases
      diagnosed	in	2007;	strains	resistant	to	>1	drugs	and	10%	of	the	susceptibles	were	collected.	3)	molecular	typing	was	
      performed at the SRL of San Raffaele Hospital (Milan) by spoligotyping and MIRU-VNTR. Results. 1) Twenty-nine labora-
      tories	completed	the	PT.	The	average	efficiency	(correct	results/total	results)	was	high	(95.5±2.8%	for	S,	97.6±2.9%	for	I,	
      95.7±2.9% for R, 96.2±4.4% for E). 2) In 2007, a total of 1,698 antibiograms for SIRE were examined. As to NC and PTC,
      total monoresistances were 10.6 and 16.5%, respectively; MDR cases were 2.5 and 26.6%, respectively. As to PBI and
      PBA, total monoresistances were 10.9 and 11.2%, respectively; MDR cases were 2.6 and 5%, respectively. 3) In 257 strains
      collected in 2006-07 and examined for molecular typing, 146 spoligotypes and 29 clusters were found. Beijing genotype
      was detected in 5% of cases. In 44 MDR strains typed by the MIRU-12 technique the clustering rate was 0.023 showing
      low transmission rate.

      1) PT indicated that the DST in this network of laboratories was accurate. 2) MDR rate in Italy consistently increased
      in NC from 1998-2001 (1.1%) to 2007 (2.5%), a phenomenon likely related to immigration. 3) No MDR-TB outbreak
      was detected. Noteworthy, unlike reported from other European countries, MDR strains were not associated with the
      Beijing lineage (This work was supported by the Italian Ministry of Health, CCM Project Surveillance of resistance to
      anti-TB drugs, Grant N92)

102                                                                                                                    ESM 2009

TubErCulOSiS rESiSTaNCE iN a gENEral
hOSpiTal iN pOrTugal – 9 yEarS SurVEillaNCE

Sancho L.; Portugal C.; Tancredo L.; Silva M.; Dias A.; Silva F.; Sousa Germano
Laboratory of Microbiology, Department of Clinical Pathology
Hospital Fernando Fonseca – Amadora, Portugal

Tuberculosis remains a serious public health problem in Portugal. In 2008, the Portuguese Health Authorities reported
a TB incidence of 25,3/100.000 inhbitants (13,6% immigrants). TB Multi Drug Resistant (MDR) were 2,5%, 34% of which
were Extensively Drug Resistant (XDR).
Resistance	 to	 any	 of	 the	 primary	 drugs	 makes	 the	 disease	 more	 difficult	 and	 expensive	 to	 treat.
Our Hospital is located in Lisbon’s surroundings and covers a population of 750.000 inhabitants most of them with poor
socioeconomic level and immigrants from Africa and East Countries. In the Great Lisbon are located 66% of TB cases of Portugal.

The aim of this study was to investigate the frequency of drug resistance of Mycobacterium tuberculosis Complex in a
general Hospital in Amadora, Portugal, during a 9-year period (2000-2008).

A total of 19.417 clinical specimens (15.159 pulmonary and 4.261 extra pulmonary), collected from 9.525 patients, were
cultured for mycobateria.
Molecular	genetic	identification	of	M.tuberculosis Complex and its resistance to Isoniazid and/or Rifampicin was made
with the technology Genotype MTBDR plus (HAIN-Lifecience-Germany).
Antimycobacterial susceptibility test to the primary drugs, Streptomycin (STR), Isoniazid (INH), Rifampicin (RIF),
Ethambutol (ETB) was performed in BACTEC MGIT 960 System

In 19.417 cultured clinical specimens for mycobateria, 1094 (14,2%) were positive by cultural methods.
1029	were	identified	as	M.tuberculosis Complex; 783 (76,1%) were strains without resistance, 150 (14,6%) with one re-
sistance, 73 (7.1%) were MDR, being more than 25% XDR.
The proportion of M.tuberculosis strains resistance rate to antituberculosis drugs during the 9-year period was: Isoniazid
11,1% (114), Streptomycin 20.2% (208)), Rifampicin 7.2% (74), and Ethambutol 4.7% (48); but in 2008 was: Isoniazid 4,8%,
Streptomicin 17%, Rifampicin 4,1% and Etambutol 2%.
On our tuberculosis population (661), in the last 6-years (2003-2008), we have compared the resistance rate related to
3 parameters: sex, age and HIV.
The tuberculosis (661) and MDR (38) populations have the same incidence rate: in male (67%) and in females (33%).
The age distribution in the MDR population (38) was 0% [0-15], 5% [16-25], 29% [26-35], 39% [36-45], 13% [46-55], 11%
[56-65], 0% [66-75], 3% [76-100]; and in patients without resistance (623) was 3% [0-15], 13% [16-25], 30% [26-35], 20%
[36-45], 14% [46-55], 8% [56-65], 7% [66-75], 5% [76-100].
The HIV parameter results were analysed on a 554 tuberculosis population. The MDR incidence rate for the HIV group
(213) was 7%, and for the no HIV group (341) was 4%.

The	level	of	resistance	in	our	population	(MDR	7%)	is	significantly	higher	than	Portugal’s	average	(2,5%)
The Multi Drug Resistance tends to be lower in the last years. The same can be observed in each of the tested drugs.
HIV infection, age and sex patient are factors that contributed to the variation of tuberculosis/MDR incidence rate.
Comparing the resistance rate by sex parameter, we didn’t found differences for tuberculosis or MDR populations; they
both have a bigger incidence in the male sex (67%).
The major incidence of tuberculosis is among active population, between 26 and 45 years old, but, it is between 36 and 45
that we found most of the MDR strains (39% of all), mostly because of drug abuse and HIV infection in this age group.
The incidence of MDR tuberculosis is clearly bigger in HIV positive (7%) than in HIV negative (4%).

In spite of our population have a level of resistance above the Portuguese average, we noted that the number of tuber-
culosis cases, including MDR, decreased 7,8% during this 9-year period analysed, which is comparable to 2008 national
data (-7.2% in the last decade).

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                    103

      DOES a muTaTiON iN ThE rpOb mEaN ThaT ThE
      M.TuBerCuloSiS iS rESiSTaNT TO rifampiCiN?

      Yates, Malcolm; Brown, Tim; Drobniewski, Francis
      HPA National Mycobacterium Reference Unit

      Testing for mutations in the “hot spot” region of the rpoB gene is gaining momentum for the diagnosis of rifampicin
      resistant strains of M.tuberculosis and as a surrogate marker for MDRTB.
      The use of PCR combined with hybridisation to commercial strips (Hain Lifesciences, Innolipa)has decreased time and
      increased ease of use so that these investigations are being performed by more and more laboratories.
      The strips consist of a series of overlapping probes that cover the whole region (S bands) and are all present in Wild Type
      isolates. There are also a series of probes (R bands) covering the commonest mutations which are linked with rifampicin
      resistance and with the deletion of the corresponding S band.
      Occasionally	an	S	band	is	deleted	with	no	R	band	appearing.	We	report	these	as	“unidentified	mutations”.	The	question	
      is whether these isolates are resistant or sensitive to rifampicin.
      Recently	 we	 identified	 three	 patients	 from	 Sao	Tome,	 an	 island	 off	 the	West	 Coast	 of	 Africa	 (population	 55000),	
      with a strains of M.tuberculosis that had the same S band deleted. Sequencing data showed that the strains had the
      same	synonymous	mutation,	VNTR/MIRU	profiles	were	identical,	and	all	strains	were	fully	sensitive	to	first	line	drugs.	
      On	analysis	of	our	database,	466	strains	were	found	to	have	rpoB	mutations,	103	(22%)	of	these	were	unidentified	muta-
      tions of which 13% were sensitive to rifampicin. A further 15 isolates gave a WT result but were rifampicin resistant.
      All strains were sequenced to identify the mutation.
      Before rpoB mutations can be used as a surrogate for MDRTB the rate of mono-resistance to rifampicin in the area must
      be determined: e.g. in the London area 30% of rifampicin resistance is mono.
      Care must, therefore, be taken when reporting these rpoB mutation results to Clinicians as
      1)	unidentified	mutations	do	not	always	correspond	to	rifampicin	resistance	and	2)	rpoB	mutations	may	not	always	cor-
      respond to MDRTB

104                                                                                                                       ESM 2009

gENOTypiC DETECTiON Of iSONiaziD aND rifampiN
rESiSTaNCE iN MyCoBaCTeriuM TuBerCuloSiS CliNiCal iSOlaTES

Maitane Aranzamendi Zaldumbide, Carlos Fernandez Mazarrasa , Luis Martinez-Martinez, Jesus Agüero Balbin.
Hospital Universitario Marques de Valdecilla, Servicio de Microbiologia, Santander, Spain.

The emergence of Mycobacterium tuberculosis	resistant	to	first-line	drugs	underlines	the	urgent	need	for	new	resistance-
profiling	methods	that	would	allow	for	timely	determination	of	proper	treatment.	The	aim	of	this	study	was	to	evaluate	
the	development	of	targeted	and	fast	molecular	diagnostic	method	suitable	for	specific	genome	regions	responsible	for	
isoniazid (INH) and rifampin (RAMP) resistance in M. tuberculosis clinical isolates.

79 strains known to be resistant to INH (n=71) or INH+RAMP (n=8) by the automated system BacT ALERT (bioMérieux)
were selected from a stock collection of clinical isolates (January 2000- March 2009). The genome regions associated
with INH-R (including the codon 315 of the katG gene and the fabG1(mabA)-inhA regulatory region) and RAMP-R (81-bp
hot spot region of the rpoB gene	called	RRDR)	were	amplified	by	PCR	and	the	DNA	sequences	were	studied.

Of the 79 isolates, 22 (27.84%) had the mutation S315T in the katG gene, 5 (6.32%) showed changes at -15 nucleotide of
the fabG-inhA regulatory region and 2 (2.53%) presented both mutations.A	significant	proportion	of	strains, 50 (63.29%), had
no detectable alterations at the studied loci. INH + RAMP-R strains were associated with mutations in the RRDR of the rpoB
gene in all cases. From these, majority (5 of 8, 62.5%) presented the mutation S531L, whereas the others involved changes
at	the	codon	516:	mutations	D516V	and	D516F,	which	were	identified	in	1	(12.5%)	and	in	2	(25%)	strains	respectively.

Our results demonstrated a low sensitivity of this method to detect INH-R strains, and	points	to	the	need	of	finding	
out other mutant regions. On the other side, confirm	the	usefulness	of	this	strategy	for	fast	assessment	of	resistance	to	
RAMP, which in turn is a marker for multiresistance. This analysis can also be done with assays based on reverse line blot
hybridization detecting the same mutations, except D516F, which is not targeted.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                 105

      phySiOlOgiCal fiTNESS aND TraNSmiSSiON pOTENTial Of mulTi-Drug
      rESiSTaNT MyCoBaCTeriuM TuBerCuloSiS CliNiCal iSOlaTES iN hONg KONg

      CHAN Chiu Yeung Raphael 1, CHAN Wai Chi Edward 1, AU Tai Kong Mike 1, LAI Wai Man Raymond 2, YEW Wing Wai 3,
      YIP Chi Wai 4, KAM Kai Man 4.
      1 - Department of Microbiology, the Chinese University of Hong Kong;
      2 - Department of Microbiology, Prince of Wales Hospital
      3 - Tuberculosis & Chest Unit, Grantham Hospital,
      4 - Tuberculosis Reference Laboratory, Department of Health, HKSAR, Hong Kong.

      This study evaluated the infectivity and transmission potential of multi-drug resistant Mycobacterium tuberculosis (MDR-
      MTB) strains by determining (i) whether resistance developed at a physiological cost which rendered them less capable
      of surviving environmental stress and infecting human host, and (ii) the degree of genetic relatedness shared by resistant
      organisms, which indicated the extent by which they spread among humans.The relative growth rates of selected isolates
      were measured using the MGIT system and compared to that of drug-sensitive strains. Our data showed that their aver-
      age initial growth rate, measured within 7 days of inoculation, was inversely proportional to the number of mutations
      they harbored in key resistance genes, with strains carrying 5 mutations growing at a rate 46% slower than that of the
      wild	type.	These	findings	suggested	that	resistance	gene	mutations	in	MTB	imposed	a	range	of	physiological	cost	char-
      acterized	by	reduced	growth	fitness.	However,	results	of	epidemiological	typing	showed	that	34%	of	the	402	MDR-MTB	
      isolates	analyzed	exhibited	genetic	relationship	to	other	strains,	indicating	that	such	fitness	cost	did	not	significantly	affect	
      the	ability	of	MDR-MTB	to	transmit	between	human	hosts	and	cause	infection.	Importantly,	the	identification	of	five	ma-
      jor clusters of 50 strains strongly suggests that infection due to dissemination of ‘parental’ MDR-MTB clones is common.
      On	the	other	hand,	the	majority	of	test	strains	displayed	a	unique	genetic	profile,	indicating	that	MDR-MTB	might	also	
      emerge	independently	through	drug	selection	within	individual	patient.	The	DNA	fingerprints	and	growth	fitness	data	of	
      local resistant isolates may be used for matching the genetic identities, predicting transmission potential, and tracing the
      routes of dissemination of future MDR-MTB isolates in the community.
      This work was supported by the Research Fund for the Control of Infectious Diseases (Project code 6902191 and 6902200)

106                                                                                                                          ESM 2009

myCObaCTErium TubErCulOSiS: 1999-2008 aNTiTubErCulOSiS DrugS
SurVEillaNCE iN CliNiCal iSOlaTES frOm paTiENTS iN ThE
largEST hOSpiTal iN ThE NOrTh Of pOrTugal

Sousa,	Ana	Sofia; Pinheiro, Maria Dolores; Carvalho, Teresa; Gonçalves, Helena
Laboratório de Microbiologia do Serviço de Patologia Clínica. Hospital de S. João, Porto, Portugal

Tuberculosis, whose causal agent is Mycobacterium tuberculosis (MT), is still an important cause of morbidity and a leading
cause of death by infection worldwide. Multidrug resistant (MDR) and extreme resistant drug (XRD) strains of MT are fre-
quently isolated.They have become an emerging global public health problem and an obstacle for tuberculosis (TB) control.
Insights on prevention of disease dissemination and its empirical treatment may be obtained from resistance surveil-
lance and monitorization of its trends.Therefore, in our study we present the susceptibility data of the strains isolated in
Hospital de S. João over the past ten years.

material and methods
A prospective ten year study was done using susceptibility data of MT strains isolated in the hospital. Susceptibility testing
to	first	line	drugs	(streptomycin,	isoniazid,	rifampin	and	ethambutol)	was	made	on	the	first	isolate	of	each	patient,	using	
the proportion method (in Lowenstein-Jensen medium until 2000, from 2001 to 2003 in Bactec 460TB and in Bactec
MGIT thereafter). When MDR strains were present, second line drugs (ethionamide, cycloserine, capreomycin, kanamy-
cin,	amikacin,	ciprofloxacin	and	rifabutin)	were	tested.	

In	these	10	years,	1493	MT	strains	isolated	for	the	first	time	in	the	same	number	of	patients,	including	1103	males,	were	
tested for in vitro susceptibility. Streptomycin was the drug with high index of resistance (8,8%); isoniazid (6,0%) was
the second one; rifampin (2,4%) the third and ethambutol (1,3%) was the less resistant. We found no resistance in 1328
(88,9%) of our patients, but in 29 (1,9%) we isolated MDR strains and 13 (0,8%) patients had XDR ones. From 1999 to
2005, the number of MDR strains isolated was relatively stable (an average of 3 patients/year); in 2006, 2007 and 2008 the
number of patients with MDR was 8, 3 and 1 respectively. In what XDR strains are concerned, we had no isolates from
1999 to 2002; in the following years, 2003-2008, XDR strains were isolated in 1,0,2,7,2 and 1 patients.

The results obtained in our patients are similar to previous reports in Portuguese and international literature, support-
ing the view that tuberculosis is currently a serious public health problem. The knowledge of susceptibility results will
provide evidence in support of preventive health policies. It also emphasizes the role of Laboratory as a cornerstone in
diagnosis, management of individual patients and effective TB control.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                   107

      fiTNESS COST Of MyCoBaCTeriuM TuBerCuloSiS CliNiCal
      iSOlaTES rESiSTaNT TO fluOrOQuiNOlONES

      VON GROLL, Andrea 1; MARTIN, Anandi 1; JUREEN, Pontus 2; HOFFNER, Sven 2; PORTAELS, Françoise 1; PALOMINO,
      Juan Carlos 1; ALMEIDA DA SILVA, Pedro 3
      1 - Institute of Tropical Medicine, Antwerp, Belgium
      2 - Swedish Institute for Infectious Disease Control, Solna, Sweden
      3 - Universidade Federal do Rio Grande, Rio Grande, Brazil

      Fluoroquinolones (FQs) have been used as effective second-line drugs in the treatment of the tuberculosis. However, the
      emergence of M. tuberculosis resistant to FQs has contributed for the occurrence of XDR-TB.This study investigated the
      fitness	cost	related	to	the	mechanism	of	resistance	to	FQs	in	M. tuberculosis clinical isolates. A total of 37 isolates had
      the	ofloxacin,	moxifloxacin	and	gatifloxacin	susceptibility	determined	by	the	proportion	method	and	were	sequenced	
      to look for mutations in gyrA and gyrB.	The	role	of	efflux	pumps	was	evaluated	by	determining	the	minimal	inhibitory	
      concentration of the FQs in the presence and absence of verapamil by resazurin microtiter assay. Growth curves of the
      isolates were obtained using the MGIT960 automated system and the lag phase time and rate of growth were established
      to	compare	the	fitness. On the 25 FQ resistant isolates (FQR), the most frequent mutation was at Ala-90→Val, followed
      by	mutations	at	Asp-94	to	Gly,	Tyr,	Ala	and	Asn.	Some	unusual	mutations	were	identified	at	Asp-89→Asn, Asn-533→Thr
      (gyrB) and deletion of the codon 678 and 679 in gyrB. The isolate with mutation at Asn-533→Thr was the only case of
      no whole cross resistance among the three FQs tested. One FQR isolate was wild type for the region investigated. The
      efflux	mechanism	was	indentified	in	36%	of	the	FQR	isolates,	being	more	frequently	found	in	moxifloxacin	and	gatifloxacin.	
      In	regard	to	the	fitness	parameters,	the	mutation	at	Asp-94	showed	a	longer	lag	phase	while	the	mutation	at	Asn-90	had	
      not	any	significant	difference	related	to	the	wild	type	FQS. The establishment of FQR	isolates	without	fitness	cost	warns	
      for the possibility of a continue emergence of XDR-TB and highlights for a more rational use of FQs, not only for the
      treatment of TB, but also, for other bacteria.

108                                                                                                                   ESM 2009

iN ViTrO aCTiViTy Of OflOXaCiN, mOXiflOXaCiN aND gaTiflOXaCiN
agaiNST MyCoBaCTeriuM TuBerCuloSiS by ThE rESazuriN COlOrimETriC mEThOD

VON GROLL, Andrea 1; MARTIN, Anandi 1; JUREEN, Pontus 2; HOFFNER, Sven 2; PORTAELS, Françoise 1; ALMEIDA DA
SILVA, Pedro 3; PALOMINO, Juan Carlos 1
1 - Institute of Tropical Medicine, Antwerp, Belgium
2 - Swedish Institute for Infectious Disease Control, Solna, Sweden
3 - Universidade Federal do Rio Grande, Rio Grande, Brazil

The	in	vitro	activity	of	ofloxacin,	moxifloxacin	and	gatifloxacin	was	tested	against	41	strains	of	Mycobacterium	tubercu-
losis by the resazurin microtiter assay (REMA) plate and the proportion method on 7H11 agar. A critical concentration
of	2.0	µg/ml	for	ofloxacin	and	0.5	µg/ml	for	moxifloxacin	and	gatifloxacin	was	obtained	by	the	proportion	method	on	
7H11	agar.	For	REMA	we	propose	a	critical	concentration	of	2.0	µg/ml	for	ofloxacin	and	0.25	µg/ml	for	moxifloxacin	and	
gatifloxacin.	Full	cross-resistance	among	the	three	fluoroquinolones	could	not	be	confirmed	since	one	strain	resistant	
to	moxifloxacin	and	gatifloxacin	was	still	susceptible	to	ofloxacin.	This	finding	could	have	important	implications	for	the	
treatment of tuberculosis patients.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                 109

      rapiD DETECTiON Of EXTENSiVEly Drug-rESiSTaNT myCObaCTErium
      TubErCulOSiS by ThE rESazuriN miCrOTiTEr aSSay plaTE

      Paasch, Fabienne; Martin, Anandi; Docx, Sven; Fissette, Kristina; Portaels, Françoise; Palomino, Juan Carlos
      Institute of Tropical Medicine, Antwerp, Belgium

      A major concern for tuberculosis control programs is the emergence of multidrug-resistant (MDR) tuberculosis and
      especially extensively drug-resistant (XDR) tuberculosis. Conventional drug susceptibility testing (DST) requires 3 to 6
      weeks	to	yield	results.	Therefore,	there	is	an	urgent	need	of	new	timely	and	accurate	detection	of	first	and	second	line	
      anti-tuberculosis drug resistance.

      purpose of the study
      The	 aim	 of	 this	 study	 was	 to	 evaluate	 the	 first	 and	 second	 line	 drugs	 rifampicin	 (RIF),	 isoniazid	 (INH),	 ofloxa-
      cin (OFX), kanamycin (KAN), amikacin (AMK) and capreomycin (CAP) with clinical isolates of M. tuberculosis by
      the colorimetric resazurin microtiter assay (REMA) plate in comparison to the indirect proportion method (PM).

      A total of 150 clinical isolates were studied. DST by PM was performed on Löwenstein Jensen for RIF and INH and on
      7H11 agar for the other drugs. The minimal inhibitory concentration obtained by REMA was compared with the PM.

      REMA results were easily determined visually after 8 days compared to 21 to 42 days by the PM. Out of 150 isolates 92
      were	MDR	and	20	were	XDR.	After	defining	the	critical	concentration	for	each	drug	by	the	colorimetric	assay,	excellent	
      results	were	obtained	for	first	and	second	line	drugs	with	levels	of	specificity	and	sensitivity	between	93%	and	100%.	

      In this study drug resistance detection by REMA has shown high level of agreement with the conventional PM.Therefore,
      REMA could be a reliable alternative method for rapid detection of MDR and XDR M. tuberculosis.

110                                                                                                                          ESM 2009

DETECTiON Of Embb gENE CODON 306 muTaTiONS iN EThambuTOl
SuSCEpTiblE aND rESiSTaNT myCObaCTErium TubErCulOSiS STraiNS.

Montoro, Ernesto 1;Yzquierdo, Sergio 1; Lemus, Dihadenys 1; Echemendia, Miguel 1; Takiff, Howard 2
1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba
2	-	Venezuelan	Institute	for	Scientific	Research	(IVIC),	Caracas,	Venezuela

Ethambutol	(EMB)	is	one	of	the	first	line	drugs	in	the	treatment	of	tuberculosis.	The major mechanism of acquisition of
resistance to EMB in Mycobacterium tuberculosis seems to be associated with points mutations in the embCAB operon en-
coding different arabinosyl transferases. In particular, amino acid replacements at embB gene codon 306 occur frequently
in EMB-resistant M. tuberculosis strains. However, this alteration has been also reported in multidrug-resistant (MDR)
strains susceptible to EMB. The aim of this work was to detect the most frequently mutation at codon 306 in EMB-
resistant strains as well as MDR strains. For this purpose, the indirect Proportional Method (PM) on Löwenstein-Jensen
was carried out as susceptibility test to isoniazid (0,2 µg/mL), streptomycin (4 µg/mL), EMB (2 µg/mL) and rifampicin (40
µg/mL) on 86 M. tuberculosis strains from the collection at the National Reference Tuberculosis Laboratory. All strains
that showed resistance to EMB by PM, all MDR strains and a selection of EMB-susceptible strains were extracted the
DNA to obtain a fragment of 803 bp by PCR, corresponding to embB gene codon 306. All this fragments were sequenced
by automatic form using appropriate primers and they data were assembled, edited electronically, and compared with
wildtype gene sequences. From 34 MDR strains, only 18 showed mutations (53%) being 8 resistant and 10 susceptible
strains to EMB. The 61,5% of EMB-resistant strains showed mutations at codon 306 whereas EMB-susceptible strains no
had	alterations	in	this	fragment.	In	conclusion,	the	results	confirm	that	embB gene codon 306 is responsible of majority
EMB-resistant in M. tuberculosis. All mutations were founded in MDR strains and because of this, the simple detection of
changes in this fragment could be considered as multidrug-resistance marker.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                              111

      TOlEraNCE Of mOXiflOXaCiN iN rOuTiNE
      CliNiCal TrEaTmENT Of TubErCulOSiS

      Yew, Wing Wai 1,YAN, See-Wan 1, FUNG, Siu-Leung 1, CHAU ,Chi-Hung 1, CHAN, Chiu-Yeung 2
      1 - Tuberculosis and Chest Unit,Grantham Hospital, Hong Kong, CHINA
      2 - Department of Microbiology. The Chinese University of Hong Koong, Hong Kong, CHINA

      From Sep 07 through Feb 09, in a tertiary centre of pulmonary diseases, 65 patients [all male, age 70.0;16.6 yrs (mean;SD)]
      with	tuberculosis	(TB)	were	commenced	on	moxifloxacin	treatment	as	part	of	their	routine	drug	regimens,	due	to	in-
      tolerance	/contraindication	to	standard	first-line	anti-TB	agents	or	drug-resistant	disease.		96.9%	of	patients	received	
      400mg	moxifloxacin	once	daily.	The	duration	of	moxifloxacin	treatment	for	all	patients	was	51.2;37.3	days,	except	one	
      who	received	the	drug	for	330	days.	28	patients	(43.1%)	had	completed	their	designated	duration	of	moxifloxacin	treat-
      ment. 9 patients had nausea, thrush and headache not necessitating drug withdrawal. 15 patients (23.1%) developed
      probably	moxifloxacin-related	adverse	events	requiring	drug	cessation:	QTc	prolongation	(5),	skin	rash	(3),	arthralgia	(2),	
      neutropenia /thrombocytopenia (2), vomiting (1), angioedema (1) and ECG T-wave inversion (1). Time to occurrence of
      these events was 43.7;42.0 days. A 93-yr old patient died of stroke and myocardial infarct after 3 days of treatment. 2
      other	patients	died	of	pneumonia	during	moxifloxacin	therapy.	A	82-yr	old	patient	who	developed	ECG	T-wave	inversion	
      had sudden death 9 days after drug cessation. Another 6 patients died of serious comorbidities, such as lung carcinoma,
      1–80	days	after	moxifloxacin	cessation.	Otherwise	all	patients	responded	favourably	to	their	anti-TB	therapy,	with	clini-
      cal,	radiographic	and	bacteriologic	improvement.	Univariate	but	not	multivariate	analysis	reveals	that	older	age	(>65	yrs)	
      might	increase	the	risk	of	moxifloxacin	associated	adverse	effects	(P=0.07).	Similarly,	mortality	is	associated	with	the	
      number	of	comorbidities	(P=0.025).	Thus,	patient	tolerance	to	“long-term”	moxifloxacin	use	in	treatment	of	TB	appears	
      reasonable.	However,	in	older	individuals	with	significant	comorbidities,	vigilance	should	be	exercised	regarding	putative	
      drug-associated events of potential severity, especially those of cardiovascular nature.

112                                                                                                                    ESM 2009

CharaCTErizaTiON Of gidB gENE iN MyCoBaCTeriuM
TuBerCuloSiS iSOlaTES iN liSbON hEalTh rEgiON:
rOlE iN STrEpTOmyCiN rESiSTaNCE aND EpiDEmiOlOgiCal marKErS

João Perdigão 1, Ana Sabino 1, Catarina Milho 1, Rita Macedo 2, Laura Brum 2, Isabel Portugal 1,2
1 - Centro de Patogénese Molecular, URIA, Faculdade de Farmácia da Universidade de Lisboa, Portugal
 2 - Departamento de Doenças Infecciosas, Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal

Streptomycin	(STP)	was	the	first	antibacillary	drug	introduced	in	the	treatment	of	tuberculosis	in	1944.	With	the	de-
velopment of further antibacillary drugs, streptomycin has become less used. Development of STP-resistance is usually
explained by the acquisition of mutations in rpsL gene or in the rrs gene. Our laboratory regularly isolates STP-resistant
strains without any mutation in the referred genes. Recently, mutations occurring in a rRNA methyltransferase (encoded
by gidB gene) were shown to be involved in the acquisition and resistance to STP. In this study, we examined the gidB
gene of STP-resistant isolates in search of mutations that may explain the acquisition and STP low-level resistance on
these strains.

We have analyzed by sequencing and/or endonuclease analysis the gidB gene of 57 STP-resistant clinical isolates and 30
STP-susceptible clinical isolates, recovered in 2005 and 2006 from different hospital units. The entire rpsL ORF of all
isolates	was	amplified	and	screened	for	mutations	by	endonuclease	and	sequencing	analysis.	All	clinical	isolates	were	also	
genotyped by MIRU-VNTR.

The gidB gene of 19 STP-resistant isolates was sequenced and two missense mutations, A80P and F12L, were found in 5
and 1 out of 19 isolates, respectively. We have found that these gidB mutations were only present in isolates without rpsL
mutations.The remaining isolates were screened by endonuclease analysis for mutations A80P and K43R in gidB and rpsL
genes, respectively. Overall, mutation A80P in gidB gene was found in 10/57 STP-resistant isolates; 11/14 STP-susceptible
multidrug resistant isolates; and, none of 16 pansusceptible isolates. GidB mutation A80P was also associated with MIRU-
VNTR	genetic	cluster	Q1,	although	an	independent	occurrence	has	been	identified.

We conclude that gidB mutations may in fact explain the high number of STP-resistant strains with no mutation in rpsL
or rrs, isolated in our laboratory. These mutations probably confer STP low-level resistance that may pass undetected in
regular drug susceptibity testing. The independent occurrrence suggests however, that the acquisition of such mutations
present an adaptative advantage.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                113

      fluOrQuiNOlONE rESiSTaNT MyCoBaCTeriuM TuBerCuloSiS
      iSOlaTES aND ThEir mOlECular CharaCTEriSTiCS

      I Gaile 2, G Skenders 1,V Leimane 1, I Jansone 2, M Bauskenieks 2, I Pole 1, V Baumanis 2
      1 - State Agency of Tuberculosis and Lung Diseases, Stopini , Riga District, Latvia, LV 2118
      2 - Latvian Biomedical Research and Study Centre, University of Latvia , Ratsupites 1, Riga LV 1067, Latvia

      Tuberculosis incidence decreased in Latvia from 74 per 100.000 populations in 1998 till 40.3 in 2008. However, multi drug
      resistance (MDR) is still high and ~10% of it is extensive drug resistance (XDR). The goal of present study is analysis of
      fluoro	quinolone	(Q)	resistance	and	genetic	characteristics	of	an	appropriate	isolates.	
      60 MDR cultivated and resistant to Q isolates from 2001-2007 were analysed for mutations in the gyrA gene, 25 of
      them	using	primary	clinical	material	also.	Mutations	were	evaluated	by	sequencing	or	using	in	house	developed	modified	
      (Giannoni reverse hybridisation method, but kanamycine (K) resistance by sequencing of rrs gene fragment.
      48	isolates	were	confirmed	as	typical	XDR	then.		Genotyping	by	PvuII restriction and spoligotyping was performed as
      well retrospective case control studies.
      35 isolates (58%) contained mutations in the codone 94 (D94 to G, A or H), 14 (23%) in the 90 and 3 (5%) in the codone
      91. In 8 cases (13%) mutations were not found. 8 samples (14%) contained whether two mutations or wild type sequenc-
      es also. 15 K resistant isolates (of 48) contained mutation in the codone A1400G, the rest were wild type. Genotyping
      revealed 10 clusters with 2-6 isolates in each. 62% of isolates were of Beijing genotype, but 33% to other typical in Latvia
      MDR genotype – C (related to LAM). The clustering rate (47%) indicates on transmission, however, small cluster size
      shows, that it is more among hospitalised persons or imprisoned ones. 23 patients suffered from primary XDR TB.
      Treatment outcome of XDR patients is 28% cured, failures 55%, the rest defaulted or died.
      More profound biochemical and genetic properties of these MDR and XDR isolates ought to be studied in order to
      improve the cure rate. Typical genotypes mutations in the gyr gene in Q resistant isolates indicate on the necessity to
      search among Q line new drugs affecting other metabolic processes also.

114                                                                                                                     ESM 2009

DESigN Of a rapiD mEThOD Of iDENTifiCaTiON Of a highly
TraNSmiTTED STraiN baSED ON ThE lOCalizaTiON Of iS6110

Sofia	Samper	1, Isabel Millan 2, Ana I. Lopez-Calleja 3, Patricia Gavin 1, M. Antonia. Lezcano 4
1 - Hospital Univesitario Miguel servet / I+CS / CIBER enfermedades respiratorias
2 - Universidad de Zaragoza / Hospital Universitario Miguel Servet / I+CS / CIBER enfermedades respiratorias
3 - Hospital Universitario Miguel Servet / I+CS
4 - Hospital Universitario Miguel Servet / CIBER enfermedades respiratorias

Efficient	molecular	methods	allowed	the	detection	of	a	large	and	unsuspected	tuberculosis	outbreak,	involving	85	pa-
tients in Zaragoza (Spain), caused by a strain named Mycobacterium tuberculosis Zaragoza “MTZ”, representing nearly 20%
of the isolates in 2001 and being still present among the isolates from our tuberculosis population.
We mapped and localized 8 of the insertion sites of IS6110 in its genome observed by RFLP. The insertion sequence
6110 besides being a very useful tool in molecular epidemiology, induces loss of gene activity either by mediating deletion
events or disrupting coding sequences and regulatory domains. It also could modulate expression of neighboring genes
by acting as a promoter sequence, driving or enhancing their expression.
In the present work, we designed a rapid method for identifying this particular M. tuberculosis MTZ. For this purpose,
different	pair	of	primers	which	targeted	the	flanking	sites	of	IS6110 in MTZ were designed. One hundred isolates among
clinical isolates already molecular typed were chosen randomly. and were tested in these isolates. Separate PCR reactions
were performed for these isolates. Among the 8 sites localized, 4 of them were previously described as preferential locus
for IS6110 transposition.
After	testing	with	different	locations	finally	one	intragenic	insertion	in	Rv2823c was selected for rapid diagnosis. Only
4 of the 100 hundred samples tested were positives, being the four positives isolates MTZ. None more of the isolates
were	positive,	the	other	96	showed	the	same	size	of	the	amplified	product	than	the	positive	control	used	H37Rv,	indi-
cating that IS6110 was not present.

the mapping of the IS6110 insertion sites in the genome of “MTZ” strain resulted in both, offers clues for better
understanding of the adaptability and virulence of M. tuberculosis, and the design of a rapid method for identifying this
particular M. tuberculosis MTZ.
Presentations Type: Poster

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                115

      DriViNg fOrCES ON ThE EVOluTiON Of ThE
      prOgENiTOr Of M. TuBerCuloSiS

      M. C. Gutierrez 1,2, R. Brosch2, M. Marceau1, J.Tap2, E. Bourdon2, S. Brisse2, S. Mangenot3, G. Salvignol3,V. Barbe3, C. Médigue3, and P. Supply1
      1 - Institut Pasteur de Lille –INSERM
      2 - Institut Pasteur, Paris
      3 - CEA/DSV/IG/Génoscope, Evry, FRANCE

      Genomic and functional plasticity of agents of tuberculosis (TB) suggest that they are the legacy of extremely long
      evolutionary	fight	for	survival	which	started	with	their	environmental	ancestors	that	evolved	to	the	exclusively	human	
      intracellular parasite of our days. Almost certainly, TB has impacted on humankind through pre-history. Mycobacterium
      tuberculosis and its earlier relatives have probably been co-evolving with Homo sapiens and its earlier relatives for hundred
      of thousand of years. Despite extensive research, the cause of M. tuberculosis speciation and the factors that have led to
      its predominance as a human pathogen are still unknown.
      Previous studies of rare human TB clinical isolates from East-Africa (Van Soolingen et al., Int J Syst Bacteriol 1997; Fabre
      et al., J Clin Microbiol, 2004; Gutierrez et al., PLoS Pathogens 2005) showed that they share many properties of other
      agents of TB,	but	radically	differ	in	terms	of	a	more	diversified	population	structure	and	obvious	traces	of	intra-species	
      horizontal gene transfer (HGT).These features suggest that these smooth TB bacilli are extant representatives of a much
      broader and older progenitor species, named “M. prototuberculosis”.
      We performed comparative genomics on a comprehensive collection of 56 strains of “M. prototuberculosis” to under-
      stand the roles played by various evolutionary processes in shaping the structure of M. tuberculosis genome and of its
      population.	Multi-locus	sequence	typing	of	16	house-keeping	genes	and	three	intein-encoding	sequences	confirmed	the	
      large genetic diversity of the TB bacilli and suggests that the M. tuberculosis complex (MTBC) is just a particularly suc-
      cesful clonal lineage that has emerged from the M. prototuberculosis progenitor pool, probably involving multiple HGT
      episodes (clonal epidemic structure). Preliminary analysis of whole-genome sequences from the four most genetically
      distant strains of smooth TB bacilli indicate extensive chromosomal rearrangements and the existence of multiple ge-
      nomic islands compared to MTBC genomes. Genome downsizing, mutations, HGT of genomic islands, and intra-species
      recombination appear as major driving forces on the evolution of the ancestor of extant M. tuberculosis.

116                                                                                                                                         ESM 2009

rESiSTaNCE, mDr aND XDr Of M. TuBerCuloSiS iN SpaiN iN ThE laST yEarS.

P. Ruiz, M. Causse, F.J. Zerolo, J. Gutierrez, M. Casal
Mycobacteria Reference Center. Department of Microbiology. Faculty of Medicine. HospitL “Reina Sofía” . Córdoba. Spain.

Tuberculosis is among the leading causes of death worldwide. The World Health Organization (WHO) estimates that
32% of the world population is infected with Mycobacterium tuberculosis . The resistance to antituberculous drugs is a big
problem to the control of the illness . The emergence of multidrug-resistant strains (MDR), extensively drug-resistant
(XDR) and extreme drug-resistant (XXDR) strains, is a global problem that has made a considerable alarm. There is an
increasing demand to determinate in vitro susceptibilities of clinical isolates to antimicrobial agents other than those
considered primary drugs.
The purpose of this study, was determinate the resistance, MDR and XDR strains in our Reference Center in the four last years.

material and method
We are studied 624 samples from patients suspects of tuberculosis. 355 strains of M. tuberculosis were isolates , in
BACTEC	MGIT	960	and	Lowenstein	-Jensen	medium	and	identified	using	Accuprobe	and	GENOTYPE	MYCOBACTERIA.	
The susceptibility testing was made for primary drugs and:Amikacin (AK) 1.0 µg/ml; Kanamycin (K) 1 µg/ml; Capreomycin
(CM)	2.5	µg/ml;	Ethionamyde	(ETH)	5.0	µg/ml;	Ofloxacin	(OF)	2.0	µg/ml;	Ciprofloxacin	(CI)	2	µg/ml	;	Moxifloxacin	(MX)	
2	µg/ml;	Levofloxacin	(LE)	4µg/ml;		Rifabutin	(Rb)	0.5	µg/ml	;Rifapentine	5		µg/ml	and	Linezolid	(Lz)	1.0	µg/ml.	The	Bactec	
MGIT technique with standart protocol was strictly followed as recommended,

From 624 samples, in 355 were isolated M. tuberculosis and 55 (15,49 %) of theme were resistant to some of the antimi-
crobial agents studied. The resistance to streptomycin was 3´38 %, to rifampin 7,88%. ethambutol 1,12 %, isoniazid 11.26
% and pyrazinamide 1,97%. A 7,6 % of strains were resistant to some of the second line drugs.The MDR was 5,6 % and
the XDR 1,4 %. No XXDR were isolated.

The resistance to secondary drugs made necessary the in vitro studies.This method, BACTEC MGIT 960 is a reliable and
rapid method to determinate the secondary drugs.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                   117

      DETECTiON Of NON TubErCulOuS myCObaCTEria iN
      SurfaCE WaTErS: COmpariSON Of CulTurE mEThODS

      Radomski, Nicolas 1, Lucas, Francoise 1, Cambau, Emmanuelle 2, Moulin, Laurent 3, Haenn, Sophie 3, Régis, Moilleron 4
      1 - Leesu, Universite Paris-Est AgroParisTech
      2 - CNRMYC, CHU Saint-Louis de Paris
      3 - Crecep, Etude biologie
      4 - Leesu, Universite Paris-Est AgroParisTech

      Since there is no evidence for person-to-person transmission, environment is considered a likely source of non tubercu-
      lous mycobacteria (NTM) infections. Particularly, environmental water, from river, lake, pond or hot spring seems being a
      major source of NTM. NTM has been also isolated from wastewater, from sources of drinking water, from drinking water
      distribution system, from tap water, and even from bottled mineral water. Among human infections caused by NTM from
      water origin, pulmonary infections and cutaneous infections are often described. These NTM human infections linked
      to water could stem from changes in water use, from human vulnerability or from increase of virulence level among
      environmental strains. Also, it seems necessary to determine the origin of these NTM in the environment, in order to
      evaluate the importance of non-clinical habitats and to detect the emergence of virulence. Lack of knowledge about life
      cycle of harmful bacteria from surface water, particularly NTM, require more analytical tools which are not currently
      standardized or adapted to environmental samples. The aim of this study is to propose an improved culture method
      that could be used for counting and isolating NTM from surface water and wastewater. Based on literature, we selected
      different bacteriological methods from medical applications that had previously been applied to water samples. Samples
      from the river Seine (Paris, France) were used to select a culture method that will prevent the growth of interfering
      microbiota, with minimal inhibition of mycobacteria. The effect of antibiotics (polymyxin B, amphotericin B, nalidixic acid,
      triméthoprime, azlocillin, vancomycin) and chemical decontamination process (methods using acids, bases, detergent or
      cetylpyridininium chloride) on have been compared. Results will be discussed and a method adapted to water with high
      densities of interfering bacteria will be proposed.

118                                                                                                                     ESM 2009

TypiNg Of MyCoBaCTeriuM aviuM SubSp. aviuM frOm
DiffErENT SOurCES uSiNg pVuii–pSTi–iS901 rESTriCTiON
fragmENT lENgTh pOlymOrphiSm (rflp) iN CrOaTia

Spicic, Silvio 1, Cvetnic, Zeljko 1, Pate, Mateja 2, Duvnjak, Sanja 1, Zdelar-Tuk, Maja 1, Racic, Ivana 1
1 - Croatian Veterinary Institut, Zagreb
2 - Veterinary Faculty Ljubljana, Ljubljana, Slovenia

A total of 9 M. avium subsp. avium strains isolated from bovines (N=1), pigs (N=2), wild boars (N=2) and poultry (N=4)
were genotyped by PvuII–PstI–IS901 restriction fragment length polymorphism (RFLP) analysis. The isolates were col-
lected	in	the	period	from	2001	to	2006.	Revealed	profiles	were	designated	according	to	the	nomenclature	established	
and used in the OIE reference laboratory for avian tuberculosis in Brno, Czech Republic. Digestion with restriction en-
donuclease	PvuII	resulted	in	3	PvuII	RFLP	profiles	(F,	Q	and	M),	comprising	8-9	bands.	Digestion	with	restriction	endonu-
clease	PstI	was	successfully	accomplished	in	8	isolates	demonstrating	4	different	profiles	of	11-13	bands.	Among	them,	3	
were	found	to	be	new	in	the	database	(A31,	A32	and	A33).	Combination	of	PvuII–PstI	digestion	revealed	4	RFLP	profiles	
(A29/F, A31/F, A32/F, A33/M). No epizootiological connection was found between the isolates expressing the predominant
profile	(A29/F),	found	in	pigs,	wild	boars	and	poultry.	This	is	the	first	research	in	the	field	of	genotyping	M. avium subsp.
avium strains isolated from different animal species in Croatia.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  119

      TubErCulOSiS iN pETS aND WilD aNimalS liViNg iN urbaN ENVirONmENT

      Spicic, Silvio; Duvnjak, Sanja; Obrovac, Mihaela; Zdelar-Tuk, Maja; Katalinic-Jankovic,Vera; Racic, Ivana; Cvetnic, Zeljko
      Croatian Veterinary Institut Zagreb

      Apart from pets and domestic animals humans are often in direct or indirect contact with wild animals, especially
      those living in Zoos. Because of their origin these animals are a possible source of infection with mycobacteria ati-
      pical for certain regions. In that manner, in 2004. M. africanum type I was isolated from organs of a hyrax (Procavia
      capensis) that died in zoological garden in Zagreb. The hyrax had been imported from United Arab Emirates (UAE).
      Also, in the same year, Mycobacterium tuberculosis infection was diagnosed in a dog. This was a pet who lived it’s
      intire life in a city (Zagreb, Croatia) and whose owner was negative on tuberculosis. Both isolates were MIRU typed
      on 12 locuses (2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39 i 40). Sinse no extensive researsch was done in the past with
      regards to molecular typisation of M. africanum with MIRU typing, we compared our results to the ones available
      online (data base www. miru-vntrplus. org )(ALLIX-BÉGUEC et. al., 2008). M. africanum type I isolated from the hy-
      rax had a unique code (235424253422). MIRU type of M. tuberculosis isolated from the dog was identical to 8 hu-
      man isolates originating from all over Croatia in the period from 2004.-2006. Out of these 8 cases, 3 originated from
                                                                             ^               ^            ^
                                                                           c              c          c
      Zagreb	and	County	of	Zagreb;	3	from	neighbouring	counties	(Sisa	 ko	–	moslava		ka,	Karlova	ka	and	Varaždinska)	and	
      2 from more distant counties (Medimurska and Primorsko – Goranska). Considering that the highest tuberculosis in-
      cidence of the same MIRU genotype was localised in a 50 km radius in as many as 6 human cases we deducted that
      it’s very likely that these people were source of infection for this dog. According to these examples, pets and zoo ani-
      mals are important links in the chain of import and spread of pre-existing mycobacteria species in urban environment.

120                                                                                                                        ESM 2009

iS1245-rflp baSED gENETiC rElaTEDNESS Of
ThE MyCoBaCTeriuM aviuM SuBSP. HoMiniSSuiS STraiNS
iSOlaTED frOm humaNS, aNimalS aND ENVirONmENT iN CrOaTia

Spicic, Silvio 1, Cvetnic, Zeljko 1, Pate, Mateja 2, Katalinic-Jankovic,
Vera 1, Duvnjak, Sanja 1, Ocepek, Matjaz 2, Zdelar-Tuk, Maja 1, Krt, Brane 2
1 - Croatian Veterinary Institut, Zagreb, Croatia
3 - Veterinary Faculty Ljubljana, Ljubljana, Slovenia

Significance	of	infections	with	Mycobacterium	avium,	in	particular	subspecies	M.	a.	hominissuis,	in	animals	and	humans	
is constantly increasing. Susceptibility of humans, cattle and swine to various mycobacteria and the prevalence of these
bacteria in the environment, although very important from the aspect of epizootiology and epidemiology, have not been
sufficiently	investigated	in	Croatia.	
This study is based on massive tuberculin skin tests of cattle and swine from large farms, bacteriology and molecular
identification	of	M.	a.hominissuis	isolated	from	domestic	and	wild	animals,	humans	and	environment.	Genotyping	of	the	
23 isolates was conducted by IS1245 RFLP method. The selected cut-off value for cluster designation was similarity level
of	75%.	A	total	of	5	clusters	containing	86.9	%	of	all	genotyped	strains	were	identified.	High	similarity	level	of	the	profiles	
within a cluster was found for the isolates from man, swine from intensive breeding, deer, wild boar and sawdust from
various	counties.	As	no	epizootiological	links	between	animals	and	humans	were	identified,	humans	and	animals	could	be	
considered as dead-end hosts. According to our results, the source of infection for humans and animals is most probably
the environment.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                     121

      DEVElOpmENT Of rEal-TimE pCr aSSay fOr
      QuaNTifiCaTiON Of myCObaCTEria iN SurfaCE WaTErS

      Lucas, Francoise 1, Radomski, Nicolas 1, Cambau, Emmanuelle 2, Moulin, Laurent 3, Haenn, Sophie 3, Moilleron, Regis 1
      1 - Leesu, University Paris-Est
      2 - CNRMYC, CHU Saint-Louis de Paris
      3 - Etude Biologie, Crecep

      Most non-tuberculous mycobacteria (NTM) are saprophytes living in natural environments. However, some species
      are opportunistic pathogens involved in various human diseases. An increase in incidence of mycobacteriosis has been
      recognized worldwide, probably linked to changes in water use, population vulnerability. One important question aris-
      ing from the increasing occurrence of NTM diseases is the origin of these pathogens. Several cases showed that water
      play	a	significant	role	in	the	transmission	of	NTM.	Indeed	NTM	can	be	found	in	various	aquatic	ecosystems,	and	also	in	
      water distribution systems. However their number and diversity in environmental water bodies and in wastewaters are
      often underscored by the actual detection methods and no standard protocol exist. Cultural studies are time consuming
      and	poorly	specific	for	NTM	growth.	Few	quantitative	PCR	have	been	developed	for	NTM	species.	However,	these	PCR	
      methods have only been applied to clinical samples and may not be adapted for environmental samples. The aim of this
      study	is	to	develop	a	real-time	PCR	assay	to	quantify	NTM	in	surface	water	samples.	First	the	specificity	and	sensitiv-
      ity towards NTM of several published target genes, such as 16S rRNA, rpoB, hsp65, ITS and gyrA and gyrB have been
      evaluated	using	the	algorithm	BLAST.	This	first	screening	resulted	in	the	selection	of	8	primer	pairs,	which	specificity	and	
      sensitivity	were	empirically	evaluated	by	DNA	amplification	of	50	microbial	isolates	from	the	river	Seine	(France),	which	
      belong to Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes and Mycetes. This strain library was completed
      with 25 NTM and other 8 Actinobacteria strains from national collections. This second step of screening resulted in the
      selection	of	two	highly	specific	primer	pairs	targeting	gyrB	and	rrs	genes,	showing	respectively	94,83%	et	93,10	%	of	
      specificity.	The	efficiency	of	the	real	time	PCR	was	evaluated	for	both	primer	pairs	using	the	strain	libraries.	

122                                                                                                                     ESM 2009

NONTubErCulOuS myCObaCTEria, iSOlaTED frOm paTiENTS WiTh
luNg DiSEaSE, frOm liSbOa E ValE DO TEJO rEgiON, DuriNg 2008

António Amorim1*, Rita Macedo, Edna Pereira
Laboratório de Saúde Pública - Micobacteriologia/Tuberculose, Administração Regional de Saúde de Lisboa e Vale do Tejo,
I.P., Lisboa, Portugal
 Presenting	author.	Phone:	00351213602520.	E-mail	address:

Despite the clinical relevance of most nontuberculous mycobacteria (NMT) in pulmonary infection in immunocompetent
patients	is	still	unclear,	this	mycobacteria	play	an	increasing	significant	pathogenic	role	in	HIV-positive,	and	other	immuno-
compromised patients. Nevertheless, no data about NTM species isolated from patients with lung disease, from Lisboa e
Vale do Tejo region, are published or available until now.
We carried out a study during the entire year of 2008 with the purpose of identify, determine the incidence of each
specie, and correlate this data with some epidemiological data in patients with lung disease from Lisboa e Vale do Tejo region.
A total of 46 patients with lung disease were detected with NTM infection. Among this patients the isolated nontuber-
culous mycobacteria were M. intracellulare (n=7, 15,2%), M. fortuitum (n=6, 13%), M. kansassi (n=6, 13%), M. chelonae (n=4,
8,7%), M. gordonae (n=4, 8,7%), M. avium (n=3, 6,6%), M. peregrinum (n=3, 6,6%), M. spp (n=3, 6,6%), M. abscessus (n=2,
4,3%), M. mucogenicum (n=2, 4,3%), M. szulgai (n=2, 4,3%), M. triplex (n=2, 4,3%), M. lentiflavum (n=1, 2,2%) and M. simiae
(n=1, 2,2%).The	patients	with	lung	disease	that	we	identified	as	infected	with	NMT	have	a	median	age	of	51,6	years,	45,7%	
(21/46) were male and 54,3% (25/46)were female. Despite 43,5% (20/46) of our patients were HIV-state unknown, in the
group of known HIV-state, 88,5% (23/26) were HIV-negative, and only 11,5% (3/26) were HIV-positive.
Our	results	suggest	that	the	pattern	of	NTM	pulmonary	infection,	in	Lisboa	e	Vale	do	Tejo,	has	no	significant	differences	
from	those	reported	in	the	rest	of	Europe	and	USA,	and	also,	no	significant	correlation	with	HIV	status.	We	carried	out	
the study during only one year, so, further studies are needed to better clarify the NMT infection in patients with lung
disease, in Portugal, in both immunocompromised and immunocompetent patients.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                    123

      NONTubErCulOuS myCObaCTEria iNfECTiONS iN
      ThE STaTE Of parÁ, amazON rEgiON, brazil

      Lima, Karla Valéria Batista 1, Lopes, Maria Luíza 1, Furlaneto, Ismari Perini 2, Lima, Elys Juliane
      Cardoso 2, Conceição, Emilyn Costa 2, Sousa, Maísa Silva de 2, Costa, Ana Roberta Fusco 1
      1 - Instituto Evandro Chagas, Belém, Pará, Brazil
      2 - Universidade Federal do Pará, Belém, Pará, Brazil

      The genus Mycobacterium currently has more than 130 species. This genus includes M. tuberculosis complex and M. le-
      prae and other organisms referred to as nontuberculous mycobacteria (NTM). In recent years, there has been a marked
      increase in the number of cases of human disease due NTM, and the NTM diseases seems to be related to the geo-
      graphic distribution of these species in the environment.

      purpose of the study
      The aim of the present study was to describe the diversity of NTM from clinical isolates received at the Instituto Evandro
      Chagas, Pará, Amazon Region of Brazil, between 2004 and 2008.

      NTM included in this study were isolated from clinical specimens of 95 patients, whom 84 had pulmonary in-
      fection,	 and	 11	 infections	 cases	 related	 to	 other	 sites	 (lymphonod,	 biopsy	 and	 abscess	 fluid).	 Löwenstein-Jensen	
      medium was used for the recovery of mycobacteria from clinical specimens. Genetic characterization to spe-
      cies level was determined by PCR-RFLP analysis of hsp65 gene (PRA), and 16S rDNA and hsp65 sequencing.

      Ninety	five	patients	presented	NTM	infections,	of	whom	88.4%	(84/95)	manifested	pulmonary	symptoms,	1.1%	(1/95)	
      presented lymphadenopathy and 10.5% (10/95) represented cases of healthcare-associated infections. A total of 13 spe-
      cies	were	identified	and	included:	M.	abscessus;	M.	bolletii;	M.	massiliense;	M.	fortuitum;	M.	avium;	M.	intracellulare;	M.	
      scrofulaceum; M. colombiense; M. kansasii; M. simiae; M. interjectum;M. smegmatis and M. szulgai. M. chelonae-M. abscessus
      (26), M. avium-M. intracelullare-M. scrofulaceum (27) and M. simiae (23) complexes were the most frequent in pulmonary
      infections. Lymphadenopathy case was caused by M. fortuitum infection.We encountered M. chelonae-M. abscessus com-
      plex (6), M. smegmatis (2) and M. fortuitum (2) in cases of healthcare-associated infections.

      We showed the diversity of species associates the NTM infections isolated at the Instituto Evandro Chagas and we en-
      countered	high	variety	of	species	in	isolated	from	pulmonary	samples.	A	finding	was	the	presence	of	M.	simiae	complex	
      species in human infections, which is not common.

124                                                                                                                       ESM 2009

EValuaTiON Of hSp65, Tb ,Sp rEgiONS iN
iDENTifyiNg myCObaCTErium OThEr
ThaNTubErCulOSiS( mOTT);uSiNg pCr-rflp

Noorolhoda Saadaee Jahromi ,Shima Seif, Parissa Farnia, Mehdi Kazempour,Mohammad Kargar, JamilehNowroozi, Mehdi
Kazempour, Mohammadreza Masjedi,Aliakbar Velayati Mycobacteriology Research Center (MRC),National Research
Institute Of Tuberculosis and Lung Disease(NRITLD),Shahid Beheshti University Medical Campus.Tehran,Iran.

Mycobacteria Other Than Tuberculosis (MOTT) are frequent causes of pulmonary infections resembling TB, but differ
from MTB complex by being opportunistic pathogens and are acquired mainly from the environment. Recent investiga-
tors	reported	an	increasing	cause	of	pulmonary	infection	by	MOTT	species,	in	Iran.	Identification	of	MOTT	by	conven-
tional biochemical methods is cumbersome and time-consuming. Therefore in the present study,the capabilities of 3
different regions (Tb,Sp,Hsp65) were examined using three primers. We demonstrated that Hsp 65 genotyping would be
very helpful to identify mycobacteria at the species level.

material & method
DNA	were	extracted	from	121	culture	positive	specimens	during	the	year	2007-2009.The		amplification	carried	out	
using following primers ( Tb ; Tb 115’-ACCAACGATGGTGTGTCCAT-3’ ,Tb 12 5’-CTTGTCGAACCGCATACCCT) ,(Sp
The PCR products (Tb: 439bp ,Sp: 250-330 bp ,HSP: 644 bp) were digested using restriction enzymes with BsteII and
HaeIII for Tb ,HaeIII for Sp and AvaII ,HphI,HpaII for HSP regions.The digested patterns were analyzed on 2% agarose gel.

The results demonstrated different sensitivity ratio for various Mycobacterium species by Tb ,Sp and Hsp regions. For
RGM , Rapid Growing Mycobacteria , group(e.g., M. furtitium and M. chelonei ) the sensitivity of Tb primer was the highest
among the other two regions(92%). Although, for slow growing mycobacterium(nonphotochromgen & phtochromgen)
the combination of two primers(i.e., Tb+Sp)	was	required	.	Thereafter	,the	sensitivity	for	identification	of	such	species	
reached upto 94%. Incontrast , scotochromoghen(e.g M. gordonae and M. scrofalceum) were differentiated more precisely
using Sp primer(74.3% ).

The recent increase in MOTT species within the country , underline the need to rapidly distinguish such Mycobacteria
from	tuberculosis	complex	.We	demonstrate		the	use	of	combined	primers	for	accurate	identification	of	mycobacterium	
up to species level.

Key	Word:	Identification,Atypic	Mycobacteria,PCR-RFLP,RGM

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                               125

      myCObaCTErium aVium alVEOliTiS afTEr ClEaNSiNg hOTEl Spa WhirlpOOlS

      Svensson; Erik 1; Ridell; Malin 1; Åkerström; Magnus 2; Andersson; Eva 2
      1 - Institute for Biomedicine, University of Gothenburg
      2 - Department of Occupational and Environmental Medicine, University of Gothenburg

      Hotel	staff	cleaning	spa	whirlpools	and	filters	became	ill	in	a	disease	suspected	to	be	the	so-called	hot	tub	lung,	which	is	an	
      allergic alveolitis-like granulomatous lung disease. In total seven employees at three hotels were involved. Mycobacterium
      sp.	was	suspected	to	be	the	cause.	Cultures	from	patients	and	from	water	and	outlet	filters	were	done.	A	quantitative	
      culture method was developed and water from different parts of the equipment was analysed.
      One	patient	had	definite	allergic	alveolitis	and	M. avium was isolated. Two other employees from the same hotel had
      suspected	alveolitis,	but	no	cultivation	for	mycobacteria	was	done.	In	this	hotel	the	cleansing	of	the	spa	filters	was	done	
      with high-pressure washers.
      Two employees at another hotel had fever, chills and dyspnea related to cleansing the equipment. Their disease was not
      regarded as allergic alveolitis, but both of them were colonized with M. avium
      In the third hotel, two employees were colonized with M. avium, but no one hade symptoms related to work. One patient,
      though,	had	flu	like	symptoms	shortly	after	bathing	in	the	pool.
      In	respiratory	samples	from	five	of	the	seven	patients	M. avium	was	isolated.	The	pool	water	and	the	surface	films	of	the	
      water	filters	contained	M. avium, often mixed with other, rapidly growing mycobacteria, however a pure culture was never
      obtained. In the quantitative water culture 0 - 16000 CFU/mL of M. avium was isolated.
      These	are	the	first	reported	cases	of	hot	tub	lung	alveolitis	(hypersensitivity	pneumonitis)	in	Sweden.	Cultures	from	
      patients,	filters	and	water	have	contained	M. avium. The symptoms of the patients have presently ceased. The cleaning
      practices	have	been	changed	and	the	pool	filter	equipment	has	been	rebuilt.

126                                                                                                                        ESM 2009

iDENTifiCaTiON Of NONTubErCulOuS myCObaCTEria
iN CliNiCal SamplES uSiNg mOlECular mEThODS: a ThrEE-yEar STuDy

Isabel Couto1,2*, Diana Machado1, Miguel Viveiros1,3, Liliana Rodrigues1,4 and Leonard Amaral1,3,4
1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal
2 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal
4 - UPMM, IHMT/UNL, Lisbon, Portugal

Although Mycobacterium tuberculosis, the etiologic agent of human tuberculosis is the main cause of mycobacteriosis in
Man, other species of mycobacteria may also cause infection in humans. The increasing importance of nontuberculous
mycobacteria	(NTM)	is	now	consensually	recognized	and	demands	for	faster	methods	for	their	identification	and	se-
lection	of	appropriate	therapy.	In	this	work	we	report	our	experience	on	the	identification	of	NTM	received	from	12	
hospitals of the Lisbon Health Region (Portugal) over a three-year period using the GenoType Mycobacterium (CM/AS)
assays (HAIN Lifescience). From 1 January 2005 to 31 December 2007, our laboratory received a total of 1192 acid-fast
bacilli (AFB) positive isolates from 1174 patients presenting with presumptive active mycobacteriosis. All isolates were
processed for Ziehl-Neelsen staining and inoculated into MGIT tubes of the BACTEC MGIT 960 system. M. tuberculosis
isolated	from	culture	were	identified	by	the	Accuprobe	system	(Gen-Probe).	Full-grown	AFB	cultures,	negative	for	M.
tuberculosis,	were	identified	by	GenoType	Mycobacterium	(CM/AS)	kits.	Out	of	the	1192	specimen	received,	1181	were	
identified	as	members	of	the	Mycobacterium genus. From these, 1032 cultures (87.4%) were positive for M. tuberculosis
complex. The remaining 149 cultures were NTM, corresponding to 12.6% of the total number of cultures from which
mycobacteria were isolated. During the study period, NTM prevalence increased steadily, starting with 8.7% in 2005 and
rising	to	15.2%	in	2007.	The	joint	use	of	the	CM	and	AS	kits	identified	96.6%	of	all	NTM	isolates	tested.	Among	the	18	
NTM	species	identified,	M. avium complex was the most frequent, although it accounted for only 34% of all NTM. In
countries with high incidence of tuberculosis and, particularly, multidrug resistant tuberculosis (MDRTB) such as Portugal,
therapeutic failure with isoniazid and rifampicin is anticipated to be due to an MDRTB strain. Since many NTM species
are	resistant	to	these	drugs,	the	identification	of	the	mycobacteria	causing	therapeutic	failure	(MDRTB	versus	NTM)	is	of	
major	importance.	The	introduction	of	molecular	methods	for	the	identification	of	NTM	in	our	laboratory	has	resulted	
in an increased awareness of the importance of being able to rapidly identify NTM as potential pathogens and the key
role played by the laboratory in assisting the selection of therapeutic modality.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                     127

      myCObaCTEria iN aNimalS iN SlOVENia –
      aN OVErViEW Of ThE laST DECaDE
      Mateja PATE1, Darja FERME1,	Manca	ŽOLNIR	DOVC2,	Matjaž	OCEPEK1
      1 - Veterinary Faculty Ljubljana, Gerbiceva 60, SI-1115 Ljubljana, Slovenia;
      	 e-mail:	;		fax:	+386	1	4779	352
      2 - University Clinic of Respiratory and Allergic Diseases Golnik, Golnik 36, SI-4204 Golnik, Slovenia

      Successful national control program carried out between 1962 and 1973 contributed to eradication of bovine tubercu-
      losis (BTB) in Slovenia. Since then, infections with the causative agents of BTB were seldom detected. The main role of
      veterinary	mycobacteriologists	has	therefore	become	the	detection	and	identification	of	the	causative	agents	of	avian	
      tuberculosis and opportunistic mycobacterial pathogens. The aim of this study was to summarize the work done in the
      past	ten	years	(1999-2008)	in	the	field	of	veterinary	mycobacteriology	in	Slovenia.	
      Identification	of	the	isolates	was	based	on	the	following	features	and	tests:	colony	morphology	and	growth	characteris-
      tics,	biochemistry,	PCR	and	commercial	identification	kits	AccuProbe	(Gen-Probe)	and	GenoType	(Hain	Lifescience).
      From 1999 to 2008, a total of 292 mycobacteria were isolated from domestic, pet and wild animals in captivity. The vast
      majority of isolates were represented by M. avium	(80.1%),	identified	to	subspecies	level	in	92.7%	(M. a. subsp. avium –
      36.3%, M. a. subsp. hominissuis	–	56.4%),	while	7.3%	isolates	remained	identified	as	M. avium. These mycobacteria were
      found predominantly in pigs, followed by cattle, poultry and exotic birds. M. caprae was found in 1.4% cases and was
      related to an outbreak in a zoo, affecting bisons and camels, and to a single culture-positive case of BTB in cattle in the
      last 15 years. M. tuberculosis was found in one cow (0.3%) as a consequence of human-to-animal transmission proven by
      means of molecular epidemiology. Other species detected included M. terrae (0.7%, pigs), M. fortuitum (0.7%, pig & cattle),
      M. scrofulaceum (0.3%, bison) and M. celatum (0.3%, pig). A relatively large proportion of isolates (16.1%) originating from
      pigs,	cattle,	sheep,	goat,	mouflon,	bison	and	monitor	lizard	were	identified	to	the	genus	level	only.	This	could	be	partly	
      attributed	to	the	lack	of	reliable	identification	methods	in	the	past.	
      Development of diagnostic kits based on molecular tests led to improved and easier diagnostics of mycobacteria, espe-
      cially	of	the	species	commonly	found	in	the	environment,	therefore	reducing	the	burden	of	unidentified	mycobacteria	in	
      a routine laboratory.

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gENDErS iN SpuTum SamplES WiTh TubErCulOSiS SuSpECT

Leite, Sérgio Roberto A; Silva, Paulo; Sato, Daisy Nakamura; Santos, Adolfo; Carlos Barreto; Miyata, Marcelo; Leite, Clarice
Queico Fujimura
São Paulo State University

Species from Rhodococcus and Nocardia genders are partially alcohol acid resistant and could be isolated from clini-
cal samples (sputum, bronchial-alveolar rinsing and lung biopsy). These genders grow successfully in Löwenstein-Jensen
medium and can cause pulmonary infections similar to tuberculosis cases. Our purpose was to evaluate the presence of
these bacteria from 1636 sputum samples of patients with clinic suspect of pulmonary tuberculosis at Ribeirão Preto city,
São Paulo state, Brazil, between the years 2000 and 2002.The samples were analyzed in the laboratory of Instituto Adolfo
Lutz, Ribeirão Preto unit. From 426 acid-fast bacilli positive sputum samples, applying phenotypic methods and chemotax-
onomy,	we	isolated	296	mycobacteria		(identified	as	224	M.	tuberculosis	and	72	NTM),	29	Nocardia	sp.,	51	Rhodococcus	
equi	and	09	non-identified	partially	acid-fast	bacilli	(PAFB).	The	success	of	the	treatment	depends	on	early	diagnosis,	thus	
the differential diagnostic between Mycobacterium, Nocardia and Rhodococcus genders deserves special importance.
Due to the high incidence of tuberculosis and the similarity in symptoms, probably the nocardiosis and rodococosis are
under-notified	in	Brazil.	Therefore	human	health	professionals	need	to	observe	carefully	the	importance	of	Rhodococcus	
equi and/or Nocardia sp., that were found on 12% and 6.8% respectively of patients suspected with tuberculosis, attended
at Ribeirão Preto city between 2000 to 2002.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  129

      pCr-rflp Of HSP65 fOr iDENTifiCaTiON Of
      MyCoBaCTeriuM lePrae DirECTly frOm a CliNiCal SamplE

      Neonakis Ioannis K1, Kontos Fanourios2, Gitti Zoe1, Baritaki Stavroula1, Bazigos Stavros1, Mihailelis Efstratios1, Zerva
      Loukia2, Spandidos Demetrios A1
      1 - Microbiology Labolatory, University Hospital of Heraklion, Heraklion, Greece.
      2 - Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.

      Mycobacterium leprae cannot be cultured in vitro. The application of PCR-RFLP analysis of hsp65	for	identification	of	M.
      leprae had been previously proposed (Rastogi et al., J Clin Microbiol, 1999, 37, 2016-19) and, to our knowledge, the
      method has been used only once.

      The	 identification	 of	 M. leprae directly from a clinical sample by application of PCR- Restriction Fragment Length
      Polymorphism analysis (PCR-RFLP) of hsp65 gene.

      materials and methods
      A sample, taken with a swab from open lesions with exudates from a 51-y old patient suspected of suffering from lep-
      rosy, was suspended in sterile water. Mycobacteria were heat-inactivated at 80o C for 1 h and the DNA was extracted
      using	the	guanidinium	thiocyanate	lysis	buffer	(Casas	et	al.,	J	Med	Virol,	1995,	47,	378-385).	PCR	amplification	of	a	439-bp	
      fragment of hsp65 was performed using the protocol and primers Tb11 and Tb12 as previously described (Telenti et al.,
      J	Clin	Microbiol	,1993,	31,	175-178).	The	PCR	product	was	further	analyzed	by	sequencing	and	RFLP.		The	amplified	PCR	
      product was digested using the Hae III and BsteII (New England Biolabs) restriction enzymes and the mixtures were
      electrophoresed on a 3% Metaphor agarose.

      The BstEII digestion produced two fragments of 315 and 135 bp and the HaeIII digestion produced two fragments of 265
      and	130	bp.	This	profile	matched	the	one	previously	reported	for	M. leprae. Sequencing of the PCR product (GenBank
      accession	number:	FJ497239)	verified	the	identity	of	M. leprae.

      PCR-RFLP could be a useful molecular tool as an adjunct to careful clinical and pathological assessment of patients sus-
      pected of suffering from leprosy.

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a CaSE-rEpOrT Of MyCoBaCTeriuM THerMoreSiSTiBile frOm grEECE.

Neonakis Ioannis K1, Kontos Fanourios2, Gitti Zoe1, Baritaki Stavroula1, Kosmadakis Georgios1, Baritaki Maria1, Zerva
Loukia2, Spandidos Demetrios A1
1 - Microbiology Laboratory, University Hospital of Heraklion, Heraklion, Greece.
2 - Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.

Mycobacterium thermoresistibile	is	a	non-tuberculous	mycobacterium	that	was	first	recovered	in	Japan	by	Tsukamura	in	
1966. Although it is strongly associated with pulmonary and dermal diseases, there have only been six reports of its isola-
tion	from	clinical	samples.	Here	we	report	on	the	first	case	from	Greece.	
The mycobacterium was isolated from the solid culture (Lowenstein-Jensen slant at 37oC) of a sputum sample after 14
days of incubation. The sample was taken from a 67-year-old male, who was a heavy smoker (1pack/day for 30 years)
and had a history of COPD, type II respiratory distress syndrome, diverticulosis and diabetes, and was presented to our
hospital with fever (38o C), productive cough, dyspnea, weakness, and acute purpura. The chest radiograph revealed an
elevated	cardiothoracic	ratio,	as	well	as	chronic	obstructive	lung	disease,	peribronchial	infiltrations,	consolidations	in	the	
right middle and lower lung zones and a small blunt at the left pneumodiaphragmatic angle.
The Accuprobe Mycobacterium tuberculosis complex assay (Gen-Probe, San Diego, CA) was negative. Accuprobe also
yielded negative results for the Mycobacterium avium complex and Mycobacterium gordonae.	 Biochemical	 profile	could	
not distinguish it from other mycobacteria. The banding patterns obtained with GenoType CM and GenoType AS (Hain,
Lifescience,	Nehren,	Germany)	were	not	species-specific	[GenoType	CM:	1,2,3	and	10,	and	GenoType	AS:	1,2,3	and	12].	
The	identification	of	M. thermoresistibile	was	achieved	with	the	amplification	and	sequencing	of	the	16S	rRNA	gene	and	
the 16S-23S internal transcribed region (GenBank accession: FJ236481).
Moreover, a 439-bp fragment of the 65-kDa heat shock protein (hsp65) gene (GenBank accession: FJ236482) was further
used for restriction fragment length polymorphism analysis with Hae III (New England Biolabs) and BsteII (New England
Biolabs). The BstEII digestion produced two fragments of 235 and 210 bp and the HaeIII digestion produced four frag-
ments of 180, 135, 70 and 50 bp.
Although M. thermoresistibile is considered pathogenic, it is rarely isolated from clinical samples. Molecular techniques are
essential	for	its	identification.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                    131

      iSOlaTiON aND frEQuENCy Of MyCoBaCTeriuM Sp iN a
      gENEral hOSpiTal DuriNg a 9-yEar pEriOD

      Portugal C.; Sancho L.; Dias A.; Tancredo L.; Silva M.; Sardinha T.; Sousa Germano
      Laboratory of Microbiology, Department of Clinical Pathology
      Hospital Fernando Fonseca – Amadora, Portugal

      Tuberculosis remains a major public heath problem in Portugal with an incidence rate of 25,3/100.000 inhabitants in 2008,
      being the majority of the cases in the surroundings of the two major cities (Lisbon and Oporto).
      Our Hospital is located in the Lisbon’s surroundings and covers a population of 750.000 inhabitants most of them with
      poor socioeconomic level and immigrants from Africa and East Countries.

      The aim of this study was to investigate the isolation frequency of Mycobacterium sp. in a general Hospital in Amadora,
      Portugal, during a 9-year period (2000-2008).

      A total of 19.417 clinical specimens (15.159 pulmonary and 4.261 extra pulmonary), collected from 9.525 patients, were
      cultured for mycobateria. All specimens were processed by the NaCl-NaOH method as recommended by CDC, stained
      by Ziehl-Neelsen, cultured on Lowenstein-Jensen and liquid medium MGIT. Identification	was	made	with	the	technology
      Genotype MTBDR plus (HAIN-Lifecience- Germany).

      Of the 19.417 cultured specimens for mycobateria, 2751 (14,2%) were positive by cultural methods.
      The positive rates by clinical specimen were: 17% (279/1684) in respiratory specimens and 6% (26/476) in extra pulmo-
      nary tuberculosis; 21% (5/23) in pus, 11% (2/14) in biological liquids, 8% (5/66) in biopsy, 6% (7/112) in blood, 5% (1/22)
      in	ascitics	fluid,	4%	(1/22)	in	mieloculture,	and	3%	in	urine	(4/133)	and	cerebrospinal	fluid	(2/84).
      In 9525 suspected TB patients, 1094 were effective tuberculosis cases (122 TB cases/year average, 97 cases in 2008). The
      incidence rate by patient was 11,5%.
      In comparison with the positive results of the culture, Ziehl-Neelsen stain was positive in 39% of the samples (47% of
      the patients).
      96% (1029) of mycobateria isolated were Mycobacterium tuberculosis Complex, 2% (20) M.avium, 2% (20) other species
      (M.chelonae, M.intracellulare, M.alsiensis / malmoense / szulgai, M.scrofulaceum, M.lentiflavum).

      In spite of the increase of TB suspected patients (+11,3%), the number of effective tuberculosis cases have decreased
      (-7,8%) for a 9-year period, which is comparable to 2008 national data (-7,2%) in the last decade.
      Tuberculosis is a major problem in this area with an recovery rate of 14,2% in the clinical specimens that we receive and
      in 11,5% of the patients, most of them with Mycobacterium tuberculosis Complex (96%).
      We have one positive patient every 3 days.
      In attempt to minimize the impact of this disease, that depend on a large number of factors, we know that the laboratory
      plays	an	important	role	in	making	a	definitive	diagnosis	in	a	short	time	period	(ASAP).	We	also	know	that	is	important	a	
      close relation between the microbiology laboratory and the hospital doctors and also with the centre that follows the
      patients in the community (CDP). This is our policy.

132                                                                                                                    ESM 2009

labOraTOry miCrObiOlOgy CONTribuTiON TO MyCoBaCTeriuM Spp.
DiagNOSiS iN ThrEE DiSTriCT COuNCilS Of SETúbal
(pOrTugal), aN arEa WiTh high myCObaCTErial iNfECTiON prEValENCE.

José Diogo, Ana Rodrigues, Isabel Nascimento, Elisabete Sardinha, Ana Raposo, Rita Figueira, Isabel Monge, Kátia Silva,
Maria José Gil, Susana Rodrigues.
Laboratory Microbiology, Hospital Garcia de Orta (Almada)

The purpose of this work is to evaluate the incidence of Mycobacterium spp. infection in three district councils of Setubal
(Almada, Seixal and Sesimbra) with a high prevalence of this disease, superior to the average in Portugal. The number
of patients with mycobacterial infection and positive mycobacteriological study in Laboratório de Microbiologia (LM),
Serviço de Patologia Clínica, Hospital Garcia de Orta, E. P. E. (HGO) between 1993 and 2008 were evaluated.
Each biological sample was processed as follows: 1) for acid-fast stain with Kinyoun’s carbolfuchsin and microscopic ob-
servation with a 100x immersion oil objective; 2) inoculation in Lowenstein-Jensen medium with aerobic incubation at
37ºC	and/or	inoculation	in	Middlebrock	7H9	and	incubation	in	MGIT	System	(Bactec®);	and	3)	species	identification	and	
first	line	antimycobacterial	susceptibility	testing.
In these sixteen years, LM processed more than 25000 biological products, 3813 were positive and isolated from biologi-
cal products of 1704 patients.
The age, gender, positive samples for BAAR and number of cases of tuberculosis (including variation per year) in Almada,
Seixal and Sesimbra was determinated. A relation was established between the disease localization (pleuro-pulmonar,
extra-pulmonar and disseminated) by clinical and microbiological criteria. The mean age of the patients was 40,6 years,
1199 (70.3%) were male and 505 (29,7%) were female.
Species	identification	(since	2004)	was:	664	(84,1%)	M. tuberculosis, 67 (8,5%) M. gordonae, 20 (2,5%) M. avium-intracelular
and 39 (4,9%) other mycobacteria.
The	first	line	antimycobacterial	resistance	was	determinated	in	649	M. tuberculosis strains. The resistance level was: 116
(17,9%) to streptomycin, 76 (11,7%) to isoniazid, 28 (4,3 %) to rifampicin, 17 (2,6%) to ethambutol and 14 (2,2%) to pyrazi-
namide. The multiresistance (simultaneous resistance to isoniazid and rifampicin) was detected in 27 (4,1 %) strains. Six pa-
tients had extensively drug-resistance tuberculosis (XDR-TB). Susceptibility pattern variation per year was also evaluated.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  133

      MyCoBaCTeriuM lenTiFlavuM aS a CauSaTiVE agENT Of aDENOpaThy

      Santos, Claudia, Mendes, Ana Constança, Fernandes, Sandra João, Ramos, Maria Helena
      Centro Hospitalar do Porto - Hospital Santo António

      We report the case of a 23 year old female patient, HIV positive, presenting with mesenteric adenopathies of unknown
      origin. A biopsy was performed and sent for microbiological study. Results turned out negative for aerobic and anaerobic
      bacterial culture, but with positive AFB examination. Following this information, patient initiated anti-tubercular therapy –
      Isoniazid, rifabutin, pyrazinamide and ethambutol. Molecular detection of Mycobacterium tuberculosis complex, directly
      from the clinical sample, was negative.
      We then performed an in house universal real time PCR targeting a 370 bp region of 16S rDNA bacterial gene, witch
      gave	a	positive	signal.	In	order	to	identify	the	amplified	product,	sequencing	was	performed	using	the	BigDye®	Terminator	
      v3.1 Cycle Sequencing Kit (Applied Biosystems), in ABI PRISM 310 Genetic Analyser (Applied Biosystems). The obtained
      sequences	were	analyzed,	and	identified	as	Micobacterium	lentiflavum.		
      This	information	led	to	treatment	alteration	–	(ciprofloxacin,	clarothromycin,	rifabutin	and	ethambutol).	
      Conventional mycobacterial cultures (MGIT™ and Lowenstein-Jensen medium) turned out negative after incubation time.
      Three months later a new biopsy was sent for microbiological study, with positive AFB examination, but negative cultures.
      Sample	was	insufficient	for	molecular	study.		
      Sequence	based	bacterial	identification	has	been	widely	used	to	identify	microorganisms	isolated	in	clinical	samples,	par-
      ticularly when applied to poorly described, rarely isolated or phenotypically aberrant species.We report sequence based
      bacterial	identification	of	Mycobacterium	lentiflavum	directly	from	a	clinical	sample.	Lack	of	cultural	growth	did	not	allow	
      susceptibility	testing,	and	clinical	specimen	was	insufficient	for	further	molecular	tests,	including	testing	for	mutations	
      associated with antibacterial resistance. However resistance to rifampin has been reported, as has been susceptibility to
      isoniazid,	ethambutol,	clarithromycin,	amikacin	and	ciprofloxacin,	in	vitro	susceptibility	patterns	whose	clinical	relevance	
      remains uncertain.

134                                                                                                                      ESM 2009

TEaChiNg OlD bONES NEW TriCKS; SiNglE NuClEOTiDE pOlymOrphiSm
aNalySiS Of EurOpEaN arChaEOlOgiCal m. lEpraE DNa

Watson, Claire, Lockwood. Diana
LSHTM - London School of Hygiene and Tropical Medicine, Keppel Street, London, UK

Leprosy	was	common	in	Europe	eight	to	twelve	centuries	ago	but	molecular	confirmation	of	this	has	been	lacking.	We	
have extracted M. leprae DNA from medieval bones and SNP typed the DNA, this provides insight into the pattern
of leprosy transmission in Europe and may assist in the understanding of M. leprae evolution.

Methods and findings
Skeletons have been exhumed from 4 European countries (the United Kingdom, France, Denmark and Croatia) and
are	dated	around	the	medieval	period	(476	to	1350	A.D.).	we	tested	for	the	presence	of	5	previously	identified	single	
nucleotide polymorphisms (SNPs) in 50 aDNA extractions chosen at random from the collection. M. leprae aDNA was
extracted from 20 of the 50 bone samples. SNP analysis of these 20 extractions were compared to previously analysed
European SNP data using the same PCR assays. Testing for the presence of SNPs in M. leprae DNA extracted from an-
cient	bone	samples	is	a	novel	approach	to	analysing	European	M.	leprae	DNA	and	the	findings	concur	with	the	previously	
published data that European M. leprae strains fall in to one group (SNP group 3).

These	findings	support	the	suggestion	that	the	M.	leprae	genome	is	extremely	stable	and	show	that	archaeological	M.	
leprae DNA can be analysed to gain detailed information about the genotypic make-up of European leprosy, which may
assist in the understanding of leprosy transmission worldwide.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                            135

      iNTErprETaTiON Of pOSiTiVE M. TuBerCuloSiS aNTigEN
      SpECifiC ifNΓ rElEaSE aSSayS iN TubErCulOSiS DiagNOSiS

      Greib Carine 1, Lazaro, Estibaliz 1,Viallard, Jean-François 1, Pellegrin, Jean-Luc 1, Maugein, Jeanne 2
      1 - Medecine Interne et Maladies Infectieuses CHU Haut-Leveque Bordeaux
      2 - Laboratoire de bactériologie, CHU Haut-Leveque Bordeaux

      QuantiFERON-TB Gold in tube test is an accurate test to detect immune responses against active Mycobacterium
      tuberculosis infection (TB) and has the advantage to eliminate false positive outcomes due to BCG vaccina-
      tion and non-TB mycobacteria. Howevever there are still some false positive tests which remain unexplained.
      In this study we aimed to focus on the prevalence and the potential explanation of false positive tests among a cohort of
      patients accurately screened without TB.
      A total of 250 adults patients with suspicion of active tuberculosis were enrolled in this study between January 2007 and
      December 2008.
      Among them, 88 had positive result in accordance with manufactured interpretation (Nil ≤ 8I/mL, TB antigen ≥ 0.35 IU/
      mL and ≥	25%	of	Nil	value).	For	28	patients,	tuberculosis	was	confirmed	by	culture	of	Mycobacterium	tuberculosis	in	26	
      cases and Mycobacterium bovis in 2 cases. For 9 patients, diagnosis of active tuberculosis was made according to a clinical
      and paraclinical body of arguments. For 10 patients without active tuberculosis, we could suspect a technical mistake to
      explain the positive result of the test (TB antigen near 0.35IU/mL for 4 patients and mitogen < 0.5IU/mL for the 6 others).
      Among the other 41 patients out of 88 with a positive test without any argument for TB, we found that
      12 had previous diagnosis of active tuberculosis in the past and 13 came from tuberculosis endemic areas.
      Finally, 16 positives results out of 88 (18 %) remain without explanation. Errors of diagnosis, bias in collection of medical
      information (previous or latent tuberculosis infection), or technical mistakes could be possible reasons.
      In conclusion, quantiFERON-TB is a useful tool for diagnosis of active TB. However the results have to be carefully analy-
      sed according to the high rate of false positive diagnosed in this study.

136                                                                                                                      ESM 2009

DirECT iDENTifiCaTiON Of MyCoBaCTeriuM TuBerCuloSiS
COmplEX, MyCoBaCTeriuM aviuM COmplEX
aND MyCoBaCTeriuM KanSaSii iN SmEar-pOSiTiVE CliNiCal SpECimENS

Central Tuberculosis Laboratory, Department of Pathology,
Singapore General Hospital

GenoType@	Mycobacteria	Direct	(GTMD)	was	used	for	direct	identification	Mycobacterium tuberculosis complex (MTBC),
M. avium, M. intracellulare, M. kansasii in 136 specimens from 122 patients. All 136 specimens were AFB smear positive with
ranges	from	1+	to	4+.	The	GTMD	correctly	detected	and	identified	134	of	136	of	the	mycobacteria	present.	Compared	
to	results	from	culture,	this	indicates	a	sensitivity	and	specificity	of	GTMD	assay	of	98.5	and	100%,	respectively.	These	
values increased to 100% when specimens with only MTBC isolation were considered. There were two discrepant re-
sults	during	the	study.	The	first	was	sputum	from	which	M. avium complex was isolated which had been kept at –70oC for
nearly 3 years. The test was negative for inhibitors and another sputum collected from same patient at same period was
detected as being positive for M. avium by GTMD. This may have been accounted for by degradation of the RNA or a
sampling issue. The second was a stool specimen from a HIV-positive patient who had M. avium complex isolated from
his	stool.	Both	RNA	isolation	and	amplification	were	repeated	for	the	same	specimen,	and	the	presence	of	inhibitor	was	
confirmed.	Two	stool	specimens	from	one	HIV-positive	patient	were	initially	identified	as	containing	M. avium complex
by AccuProbe. GTMD results showed bands matching M. intracellulae and M. kansasii, indicating possible co-infections in
this	HIV-positive	patient.	This	was	confirmed	when	DNA	probe	was	performed	on	growth	from	the	LJ	slant	even	though	
there were no pigmented colonies and both specimens turned out to be positive for M. kansasii. It was most likely that
the mixed infection was not detected by routine methods as the M. intracellulare in this case, had outgrown M. kansasii as
the predominant organism in broth and solid cultures.
Direct	identification	by	GTMD	takes	about	5	working	hours	compared	to	3	to	5	weeks	required	for	culture	isolation	
and	species	identification	by	conventional	methods.	

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                137

      rapiD DiagNOSiS aND Drug SuSCEpTibiliTy TESTiNg Of
      TubErCulOSiS iNfECTiON: mTD-TEST2 aND baCTEC mgiT 960 SySTEm

      Müllerova, Maria
      Klinlab	Ltd,	U	vojenske	nemocnice	1200,	169	00	Prague	6,	Czech	Republic,

      The Czech Republic is a country situated in the heart of the Europe. It has a low incidence of tuberculosis in the last 10
      years. However, the increasing number of migrants from countries with high incidence of TB is changing the situation.
      Samples were collected from patients in the Central Bohemian Region and in a part of Prague (2 millions inhabitants).
      All samples were tested by MTD-Test2 and conventional methods, partly also by BACTEC MGIT 960, for the detection
      of M.TB complex.
      Drug susceptibility tests were performed by conventional methods and BACTEC MGIT 960 (S.I.R.E.+PZA).
      For the differentiations of separate species of M.TB complex, the GenoType Mycobacteria MTBC test was used.
      MTD-Test2	for	detecting	the	M.TB	complex	takes	only	3.5	hrs	and	its	performance	is	highly	sensitive	and	specific.
      From December 16, 1999 to December 31, 2008 a total of 39,592 different samples were collected for detecting the
      M.TB complex, comprising of 23,582 sputa, 10,910 bronchoalveolar lavages, 1,492 laryngeal swabs and 3,662 non-pulmo-
      nary samples.
      From 35,930 pulmonary samples were MTD-T2 pos., cult. neg. 416 (1.16%), MTD-T2 neg., cult. pos. 116 (0.32%), MTD-T2
      pos., cult. pos. 1,108 (3.08%), and MTD-T2 neg., cult. neg. 34,290 (95.44%).
      Of the 1,296 isolated strains of M.TB tested for susceptibility to basic AT (S.I.R.E.+PZA), 1,109 (85.57%) strains were
      susceptible and 187 (14.43%) were resistant for varying combinations of AT; however, further 78 strains (6.02%) were
      resistant to INH+RIF, i.e. MDR TB.
      The number of multiresistant patients is rising, especially in the last 3 years: in 2006 10.17% patients were resistant, and
      from them 4.81% MDR TB, in 2007 8.78% res., 5.36% MDR TB and in 2008 35.51% res., 13.76% MDR TB. These patients
      are mostly young males, 25-35 years old, from foreign countries.
      The increase in the number of MDR TB is becoming a problem in our country in view of the need of using alternate AT.

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firST EXpEriENCE WiTh gENOTypE mTbDr
aSSai fOr rapiD EValuaTiON Of mDr CaSES

Klavdia Levina, Anna Dementieva, Maret Saluotsa
North Estonia Medical Centre, Tallin

Tuberculosis (TB) incidence in Estonia decreased from 56.6 per 100.000 population in 1998 till 30.7 in 2008. However,
multidrug resistance (MDR) is still very high ~ 12%. Therefore rapid determination of Rifampin (RMP) and Isoniazid
(INH) resistance in M.tuberculosis (MTB) isolates remains actual for the initiation of effective chemotherapy to break
the transmission of the MDR strains.
Drug susceptibility testing (DST) by conventional methods is time-consuming. More rapid results could be achieved by
using molecular methods.
The goal of our study was to investigate the possibility of application of the Hain Lifescience GenoType MBTDRplus assay
as a rapid diagnostic tool for detection of RMP and INH resistance in MTB isolates by comparing the results with those
obtained by conventional phenotypic resistance studies.
61 smear positive clinical material and 97 MTB strains recovered from smear negative patients’ specimens by culture
have been studied by MBTDRplus assay.
Additionally DR was analyzed by standardized DST method on BACTEC MGIT 960 system when culture was later available.
Among tested MTB strains,108 were phenotypically sensitive to INH and RMP,39 were resistant to both drugs,9 and 2 have got
mono resistance to INH and to RMB correspondently.Wild type patterns were found using the MTBDRplus assay in 107 samples.
Specific	mutations	associated	with	resistant	patterns	MTBDRplus	assay	were	displayed	in	48	samples.	
MTBDRplus assay and the DST prepared from strains isolated by cultural methods from smear negative specimens
showed 100% agreement between the two methods.
Concordant results between MTBDRplus test directly from smear positive samples and conventional DST from later
obtained culture strains was found in 95.7 % of cases tested, agreement of the results regarding RMPwas100%.
In two (3.4%) smear positive samples resistance-linked mutations were found, but no phenotypically resistance was de-
tected. One strain recovered from AFB positive specimens was INH resistant, but had not displayed mutations conferring
INH resistance by MTBDRplus assay directly from the same smear positive specimens.
Our results suggest that GenoType MTBDRplus assay is a valuable alternative for rapid detection of resistance and in
agreement with the classical methods gives opportunity to break the transmission of the MDR strains.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                               139

      Drug rESiSTaNT TubErCulOSiS iN SlOVENia aND EValuaTiON
      Of gENOTypE mTbDrpluS TEST iN CliNiCal labOraTOry

      Natasa Fajfar, Manca Zolnir - Dovc
      University Clinic of Pulmonary and Allergic Diseases Golnik, Golnik 36, SI-4204 GOLNIK, SLOVENIA

      Slovenia is one of the countries with relative low rate of drug resistant tuberculosis (TB). In the period 1995-2008 the
      rate was 4.2% (1.6-6.5) and only 0.70 % (0.0-1.8) of Slovenian TB patients had MDR TB in the same period.The aim of our
      retrospective study was to evaluate the performance of MTBDRplus assay (Hain Lifescinece GmbH, Nehren, Germany)
      for the detection of rifampicin (RMP) and isoniazid (INH) resistance in comparison to phenotypic drug susceptibility
      testing method.
      A total of 48 drug-resistant Mycobacterium (M.) tuberculosis isolates were included in the study. M. tuberculosis drug-resis-
      tant strains were obtained from patients living in Slovenia between 1995 and 2008. In total, 20 RMPr/INHr, 27 RMPs/INHr,
      1 RMPr/INHs strains were analysed with MTBDRplus assay. The assay was performed according to the manufacturer’s
      In comparison to conventional drug susceptibility testing MTBDRplus was able to identify RMP resistance in 21 of the 21
      strains (100%), but for INH the accordance of both methods was lower - only in 37 of 47 strains (79%). The most com-
      mon mutation carried in RMP-resistant isolates was rpoB MUT3-S531L (48%) followed by mutation rpoB MUT1-D516V
      (33%). In INH-resistant strains dominating mutation was in the gene katG in 60% (MUT1-S315T1 in 68%, MUT2-S315T2
      in 29%) and the mutations in the inhA promotor gene in 19%.
      Comparative analysis demonstrated very good agreement between molecular and phenotypic drug susceptibility results
      for RMP resistance, but not so for INH. Thus MTBDRplus assay is useful method for the rapid detection of drug resis-
      tance,	but	the	results	should	always	be	confirmed	by	the	phenotypic	methods	as	well.	

140                                                                                                                      ESM 2009

EValuaTiON Of a NEW rEal-TimE pCr KiT fOr
ThE DiagNOSiS Of TubErCulOSiS iNrESpiraTOry SpECimENS

Causse, M; Gutierrez-Aroca, JB; Casal, M
Mycobacteria	Reference	Center.	Microbiology	Department.	Reina	Sofia	University	Hospital,	Cordoba	(Spain)

Early diagnosis of tuberculosis is one of the major objectives of the World Health Organization. In its latest update, the
Center for Disease Control and Prevention (CDC) recommends the use of a rapid molecular diagnostic technique on
at least one sample per patient.

To evaluate a new kit (COBAS Taqman MTB®, Roche) for the diagnosis of tuberculosis in respiratory samples, and com-
pare results with those obtained by the COBAS Amplicor MTB kit. Culturing (Lowenstein-Jensen or BACTEC MGIT
960) was used for reference purposes.

material and methods
A total of 170 respiratory and 19 non-respiratory samples were processed. Specimens decontaminated were stained
with auramine and cultured. A single manual extraction was performed using the AMPLICOR Respiratory Specimen
Preparation Kit; eluate aliquots were then	amplified	using	the	COBAS	Amplicor	MTB	and	the	COBAS	TaqMan	MTB	kit.
Automatic sample extraction was also performed, using the Ampliprep TNAI kit, 200-µl aliquots being previously incu-
bated at 95ºC for 15 minutes.

All	77	smear-positive	samples	were	classified	as	positive	by	both	kits.
Of the 170 respiratory samples tested using the TaqMan, 2 false positives (FP) and one false negative (FN) were recorded.
Of the 169 tested using the Amplicor kit, there were 2 FP (different samples than TaqMan) and 2 FN.
TaqMan	amplification	with	automatic	extraction	yielded	one	false	negative	and	no	false	positives.
Real-time	PCR	sensitivity	and	specificity	were	98.7%	and	97.7%	respectively.	Use	of	TaqMan with automatic extraction
yielded	98.8%	sensitivity	and	100%	specificity.	Kappa	indices	were	0.95	and	0.97	with	respect	to	the	comparator.
For	smear-negative	samples,	sensitivity	declined	to	80%	and	specificity	remained	at	97%	for	the	TaqMan	kit.
For the 19 non-respiratory specimens, all three techniques tested recorded one false negative.

TaqMan MTB kit is a real-time PCR method offering the same reliability as the Amplicor MTB kit. TaqMan MTB kit ap-
peared to display good sensitivity using non-respiratory specimens.
It cuts down time-to-results from 6 to 2.5 hours
Automatic extraction reduced the number of false positives and considerably shortened handling time.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                               141

      QuaNTifErON-Tb gOlD aSSay (QfT) aND TubErCuliNE SKiN TEST (TST)
      CliNiCal pErfOrmaNCE fOr ThE DiagNOSiS Of aCTiVE TubErCulOSiS

      Karabela Simona, Papaventsis Dimitrios, Nikolaou Stavroula, Konstantinidou Efthimia, Sainti Asimina, Ioannidis Panayotis,
      Kanavaki	Sofia	
      National Reference Center for Mycobacteria, “Sotiria” Hospital, Athens, Greece

      The purpose of this study was to evaluate and compare Quantiferon-TB Gold In-Tube (QFT, Cellestis, Australia) and the
      tuberculine skin test (TST) in patients with active TB, with and without previous BCG vaccination.

      Patients with symptoms compatible with active TB were included. The TST was performed according to the Mantoux
      method and the QFT assay according to the manufacturer’s instructions.The cut-off value for a positive result was ≥0.35
      IU/ml interferon-gamma (IFN-γ).	Sensitivity,	specificity,	positive	and	negative	predictive	values	were	calculated	and	com-
      pared for QFT and TST tests. Agreement between QFT and TST was assessed by the kappa (κ) coefficient.

      A total of 296 patients were enrolled in the study. One hundred eighty-nine had a record regarding BCG vaccination.
      Forty-four	(23%)	of	the	189	patients	had	been	vaccinated.	In	total,	the	sensitivity	and	specificity	of	QFT,	excluding	those	
      with indeterminate results, was 79% (52/66; 95% CI: 66-88%) and 66% (153/167; 95% CI: 58-73%), respectively. The sensi-
      tivity	and	specificity	of	TST	was	72%	(46/64;	95%	CI:	58-83%) and 67% (156/232; 95% CI: 59-74%), respectively.The overall
      concordance between the QFT and TST tests was 70.2%, with a kappa value of 0.46 (95% CI: 0.289-0.524). In the BCG-
      vaccinated subgroup, agreement between the two assays was 66%, with a kappa value of 0.350 (95% CI: 0.103-0.597).The
      difference with the non-vaccinated subgroup (κ=0.463; 95% CI: 0.303-0.623) was considered to be not quite statistically
      significant	(p>0.05).	Initial	TST	positive	screening	followed	by	a	QFT	positive	result	was	found	to	have	greater	sensitivity	
      and	specificity	in	the	non-vaccinated	[sensitivity=79%	(95%	CI: 59-92%);	specificity=81%	(95%	CI:	71-88%)] compared to
      the BCC-vaccinated subgroup [sensitivity=67% (95% CI: 30-92%);	specificity=75%	(95%	CI:	57-89%)].

      This	study	confirmed	previous	reports	that	QFT	assay	has	higher	sensitivity	for	detecting	active	TB	compared	to	TST.	
      An overall moderate agreement between TST and QFT was found. The difference in agreement between non-vaccinated
      and	BCG-vaccinated	subgroups	could	be	attributed	to	TST	influence	by	vaccination.	In	patients	with	active	TB	and	no	
      BCG-vaccination history, TST screening followed by subsequent QFT testing proved to present the highest sensitivity
      and	specificity	for	TB	diagnosis.	Larger	prospective	studies	are	needed	to	confirm	our	results.

142                                                                                                                     ESM 2009

CliNiCal pErfOrmaNCE Of QuaNTifErON-Tb gOlD aSSay (QfT) fOr ThE
DiagNOSiS Of laTENT TubErCulOSiS iN DiffErENT paTiENT grOupS

Karabela Simona, Papaventsis Dimitrios, Nikolaou Stavroula, Konstantinidou Efthimia, Sainti Asimina, Ioannidis Panayotis,
Kanavaki	Sofia	
National Reference Center for Mycobacteria, “Sotiria” Hospital, Athens, Greece

The purpose of this study was to evaluate the performance and usefulness of Quantiferon-TB Gold In-Tube (QFT,
Cellestis, Australia) in the diagnosis of latent tuberculosis and to compare it with the tuberculin skin test (TST).

A cohort of 395 high risk adults was prospectively evaluated. Study groups consisted of 139 neoplastic patients, 98 with
autoimmune diseases (SLE, rheumatoid and psoriatic arthritis), 20 immunossupressed (e.g. HIV, hemodialysis) and 26
Intensive Care Unit (ICU) patients, and 112 patients with no underlying disease.The TST was performed according to the
Mantoux method and the QFT assay according to the manufacturer’s instructions. The cut-off value for a positive result
was ≥0.35 IU/ml interferon-gamma (IFN-γ). QFT and TST were simultaneously performed in 297 patients.

Overall, QFT gave a positive result in 72/395 (18.22%) patients. Among the different risk groups, ICU and immuno-
suppressed patients represented the highest positive rates (19% and 20%, respectively). Indeterminate results (7% on
the whole) were more often seen in ICU (34.6%), neoplastic (7.2%) and in patients with autoimmune disease (7.2%).
Indeterminate results represented <1% in patients with no underlying disease. Strength of agreement between GFT and
TST results was poor (agreement=55.44%, kappa=0.171; 95% CI: [0.064-0.278]).

QFT is a very usefully method in TB diagnosis because in contrast to TST, it reduces over diagnosis of latent TB in previ-
ously BCG vaccinated, distinguishing truly tuberculosis cases from BCG vaccinated individuals and/or non-tuberculous
infections. As a consequence, QFT provides valuable information for therapeutical decision-making.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                               143

      TubErCulOSiS DiagNOSiS by QuaNTifErON Tb gOlD
      aSSay iN arEaS WiTh DiffErENCES iN Tb iNCiDENCE

      Nikolaou Stavroula, Karabela Simona, Papaventsis Dimitrios, Sainti Asimina, Konstantinidou Efthimia, Ioannidis Panayotis,
      Kanavaki	Sofia	
      National Reference Center for Mycobacteria, “Sotiria” Hospital, Athens, Greece

      Evaluation of Quantiferon TB Gold in Tube method (QFT) for the latent TB diagnosis in patients originated from countries
      with high (group A) and low (group B) incidence of TB.

      948 whole blood samples from 153 (16,15) individuals belonging to group A and 795 (83,9%) to group B.

      Performance of Quantiferon TB Gold in Tube (Cellestis, Australia) method according to the manufacturers’ instructions.

      QFT positive results was detected in 93/153(60.8%) individuals of group A and 273/795 (29,35) of group B (p<0,0001).
      Clinical information for previous BCG vaccination was available in 351 cases: 65 of group A and 286 of group B, where
      QFT	confirmed	latent	TB	in	36/65(50,8%)	and	46/286(16,1%)	of	group	B	(p<0,0001).	As	it	was	expected,	Tuberculin	Skin	
      Test (TST) was positive in all 351 cases with previous BCG vaccination.

      QFT is a highly diagnostic and useful method, especially in patients originated from areas with high incidence of TB. In
      contrast to widely used TST, it reduces overdiagnosis of latent TB in previously BCG vaccinated.

144                                                                                                                  ESM 2009

QuaNTifErON® -Tb gOlD iN-TubE TEST uSED iN praguE
paTiENTS liSTED iN ThE NaTiONal TubErCulOSiS rEgiSTEr

Havelkova, Marta 1, Bartu,Vaclava 2, Kubin, Milan 3
1 - National Institute of Public Health – NRL for mycobacteria
2 - Charles University , Faculty of Medicine, Faculty Thomayer Hospital
3 - Prague Hygiene Institute

In 2006-2007, 65 (29.0%) of 224 patients registered in the A15, A16 and A18/19 categories (58.5%, 32.3% and 9.2% of all
patients, respectively) were assessed using both the QuantiFERON – TB Gold In-Tube (QFT) method and the tuberculin
skin test (TST) with 2 TU PPD.
After	stimulation	with	tuberculosis-specific	antigens,	a	low	level	of	interferon-gamma	(IFG)	production		(<	0.35	IU)	was	
found	in	18	(27.7%)	patients	and	moderate	or	high	levels	(>	0.35	IU)	in	the	remaining	47	(72.3%)	patients.		After	incuba-
tion	with	a	non-specific	mitogen,	a	low	level	of	IFG	production	was	recovered	in	13	(20%)	individuals,	with	moderate	or	
high levels being found in the remaining 52 (80%) patients.
The	TST	revealed	low	levels	of	skin	infiltrate	reaction	(0-5	mm)	in	25	patients	(39.1%)	and	moderate	or	high	levels	of	skin	
reaction	(range	6	-	>	30	mm)	in	the	remaining	39	individuals	(60.9%).
The high proportion of low reagent levels in both the QFT and tuberculin skin tests can be explained by a high number of
older patients, comorbidity with malignant tumours, diabetes or general fatigue in pre-terminal patients, and the immune
systém	insufficiency	related	to	these	factors.

This presentation was fully supported by project KAN 200520702 of the Grant Agency of Czech Academy of Sciences (GAAV C

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                 145

      praCTiCal EXpEriENCE Of uSiNg a DNa amplifiCaTiON
      aSSay fOr rapiD DETECTiON Of MyCoBaCTeriuM
      TuBerCuloSiS COmplEX iN rESpiraTOry SpECimENS

      J. Cacho1, A. García-Cañas1, A. González Torralba1, I. Cano2, A. Pérez Meixeira3, A. Ramos Martos2, and M. Sánchez-Concheiro1
      1 - Servicio de Microbiología, Hospital Universitario de Getafe, Madrid
      2 - Servicio de Neumología, Hospital Universitario de Getafe, Madrid
      3 - Servicio de Salud Pública, Comunidad de Madrid, Madrid.

      To	evaluate	experience	in	a	clinical	microbiology	laboratory	of	using	a	DNA	amplification	assay	for	routine	detection	
      of Mycobacterium tuberculosis complex (MTC) performed once weekly, and to compare this method with microscopy
      and culture.

      A total of 507 respiratory specimens from 419 patients were screened for tuberculosis (TB). Smear examinations, culture
      and polymerase chain reaction (PCR) were performed on each sample. Specimens were processed according to standard
      laboratory protocols. All samples were processed exactly as described in Cobas Amplicor MTB Methods Manual (Roche
      Molecular Systems, USA). This method was performed once per week.
      The following two groups of samples were considered to be true positive: (1) samples which were culture positive for
      MTC; and (2) all samples which were culture negative for MTC but positive to PCR, provided that one or more of the
      following criteria were met: (i) the samples originated from a patient whose other samples were culture positive; (ii) the
      patient’s	clinical	history	provided	evidence	of	TB	sufficient	to	warrant	initiating	treatment	for	TB.

      Inhibition of PCR was seen in 15 (2.9%) specimens. A total of 37 (7.3%) samples were considered to be true positive:
      33 samples grew MTC in culture and 4 were culture negative for MTC but positive for PCR and met the criteria as de-
      scribed previously. The	overall	sensitivity	and	specificity	of	PCR	as	compared	to	true	positive	samples	was	83.8%	(31/37)	
      and 99.1% (466/470), respectively; that of culture 89.2% (33/37) and 100% (470/470), respectively; and that of direct
      microscopy 64.9% (24/37) and 99.8% (469/470), respectively.
      The average time to reporting for true positive samples was 6 days for positive PCR and 11.4 days for positive culture. Of
      the smear-positive, true positive samples, 91.7% (22/24) were PCR positive. Of the smear-negative, true positive samples,
      69.2%	(9/13)	were	PCR	positive.	The	9	samples	classified	as	true	positive	and	smear	negative	were	from	9	different	pa-
      tients.	Treatment	was	started	earlier	in	44.5%	of	these	patients	because	positive	PCR	findings	were	obtained.	The	average	
      time	to	reporting	was	5.4	days	for	positive	PCR	findings	and	17.4	days	for	positive	culture	findings.

      MTC	infection	was	confirmed	for	PCR	in	91.7%	of	smear-positive	specimens. Although PCR was performed once weekly,
      treatment in 44.5% of patients was initiated earlier because of positive PCR results from smear-negative samples.

146                                                                                                                       ESM 2009

rEal-TimE pOlymEraSE ChaiN rEaCTiON fOr ThE DirECT
DETECTiON Of myCObaCTErium TubErCulOSiS iN CliNiCal SpECimENS

Karen, Morgan
Joint Clinical Research Centre, Kampala, Uganda

The resurgence of tuberculosis is a leading cause of death worldwide. Since M. tuberculosis, is slow growing, methods
of diagnostic testing based on culture are delayed. This study describes the development of a real-time polymerase chain
reaction (RT-PCR) assay and subsequent clinical testing.

Patients were recruited from routine TB suspects in the Monterey County Public Health laboratory, CA, USA. Initially,
using Bactec MGIT 960 cultures as the gold standard, 231 respiratory specimens composed of 76 specimens from cul-
ture-confirmed	tuberculosis	cases	and	155	culture	negative	specimens	were	analyzed	by	RT-PCR.	Over	the	next	2	years	
206	respiratory	patient	specimens	were	tested	from	81	flurochrome	smear	AFB	positive	and	125	negative	sputa.		DNA	
extraction	was	performed	directly	from	patient	specimens	by	the	modified	Qiagen	mini-Amp	kit	(Valencia,	CA).	The		RT-
PCR was performed on the Roche LightCycler instrument (Mannheim, Germany). The assay was designed to target the
ITS region of the 16S rRNA gene of M. tuberculosis using Taqman hybridization probes for detection of amplicons.

The	developed	RT-PCR	assay	yielded	results	in	one	day	and	achieved	a	sensitivity	of	85.5%	and	specificity	of	100%.	In	
subsequent	clinical	use	over	the	two	year	period	the	RT-PCR	assay	achieved	a	sensitivity	of	92.3%	and	specificity	of	100%	
in	direct	detection	of	MTB	from	206	respiratory	patient	specimens,while	in	contrast	fluorochrome	smears	achieved	
only	a	sensitivity	of	60.5%%	for	non-specific	AFB	detection	in	the	same	population.	The	RT-PCR	assay	detected	MTB	in	
64.7%	of	the	smear	negative	but	culture	confirmed	patient	specimens.	The	RT-PCR	assay	costs	$14	per	test,	inexpensive	
compared to commercial MTB assays ($60).

RT-PCR methodolgy can be used to detect MTB directly in patient specimens within eight hours, without waiting for
culture	growth.	This	rapidly	increases	and	enhances	specific	detection	and	treatment	of	MTB.	

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                               147

      ThE uTiliTy Of mOlECular TESTiNg iN
      rOuTiNE myCObaCTEriOlOgy DiagNOSiS

      Fanourios Kontos, Loukia Zerva.
      Clinical Microbiology Laboratory, Medical School of Athens, “Attikon” University Hospital, Athens, Greece.

      Molecular methods increasingly replace phenotypic tests in Mycobacteriology. This study describes a “molecular” strat-
      egy that was developed for routine testing of clinical samples and isolates with the goal of shortening turnaround time
      (TAT) and providing accurate results.

      All samples submitted for mycobacterial cultures (from 12/2006 to 3/2009) were processed and cultured by standard
      methodology.	The	first	smear	(+)	specimen	of	any	patient	and	every	first	(+)	culture	were	tested	by	an	in-house	IS6110-
      PCR in order to differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria
      (NTM) (TAT 4 hours). IS6110-PCR (+) specimens or cultures were tested by Genotype MTBDRplus (Hain-LIFESCIENCE)
      for	MTBC	species	confirmation	and	detection	of	Isoniazid	and	Rifampicin	resistance	(TAT	2	days).	Subsequent	culture	
      (+) specimens from the same patient were tested by Accuprobe MTBC (Biomerieux) (TAT 2 hours). The susceptibility
      testing of MTBC isolates was performed by MGIT960 (Becton Dickinson). If a (-) result was obtained by the IS6110-
      PCR,	identification	of	isolates	proceeded	using	both	Genotype	Μycobacterium CM and AS (Hain-LIFESCIENCE) (TAT
      two	days).	For	NTM	species	confirmation	a	PCR-RFLP	analysis	of	the	hsp65	gene	was	applied	(TAT	3	days);	additionally,	
      sequencing	of	the	16S	rRNA	gene	(TAT	5	days)	represented	the	reference	identification	method	for	selected	isolates.			

      Out of 4.000 specimens, 160 were culture positive (4%) including 85 MTBC positives (63.5% acid fast stain [AFS] posi-
      tive) and 75 NTM positives (26.7% AFS positive). Five samples contained more than one NTM species, which were iden-
      tified	only	by	molecular	testing.	There	was	complete	agreement	between	all	molecular	identification	methods;	however	
      16S rRNA sequencing was more informative (examples: M. fortuitum and M. lentiflavum, M. celatum). All MTBC isolates
      were Rifampicin susceptible, two were high- and one low-level Isoniazid resistant by MGIT960; only the latter was not
      recognized by Genotype MTBDRplus.

      Focusing	on	molecular	methodology	resulted	in	fast	and	accurate	final	reporting.	The	low	incidence	of	MTBC	positivity	
      among	tested	specimens	(2,1%)	justified	the	decision	not	to	use	indiscriminately	a	direct	molecular	test	for	tuberculosis	
      diagnosis. The frequent occurrence of NTM (48,5% of all culture positive samples) necessitates direct molecular testing
      of all AFS (+) specimens.

148                                                                                                                  ESM 2009

DETECTiON Of MyCoBaCTeriuM TuBerCuloSiS DNa iN
fOrmaliN-fiXED, paraffiN-EmbEDDED TiSSuE SpECimENS
by SpOligOTypiNg: appliCaTiON TO hiSTOpaThOlOgiCal DiagNOSiS.

Salas S1, Hernández J1, Ojeda P2, Awad C2, de la Hoz F3, Murcia MI1.
1 - Departamento de Microbiología, Facultad de Medicina. Universidad Nacional de Colombia. Bogotá, Colombia
2 - Hospital Santa Clara E.S.E., Bogotá, Colombia
3 - Departamento de Salud Pública, Facultad de Medicina. Universidad Nacional de Colombia. Bogotá, Colombia

In Colombia, Tuberculosis (TB) remains as a public health problem with an incidence of 25 per 100 000 population while
extra-pulmonary TB has increased. The Spoligotyping method has demonstrated to be a good tool to improve the TB
extra-pulmonary diagnosis.

purpose of study
To identify DNA of Mycobacterium tuberculosis from	Formalin-fixed	paraffin	embedded	Tissues	specimens	(FFPET).		

We examined 160 FFPET storaged for 13 years and with a suspected diagnosis of Tuberculosis infection according to
histopatological	analysis.	Spoligotyping	was	carried	out	as	previously	described	with	some	modifications.	DNA	extraction	
with	CHELEX	was	used	and	human	DNA	was	negative	control.	The	Spoligotype	films	were	scanned	and	classified	by	using	
GeneTools	software	followed	by	manual	editing	and	confirmation.	Statistical	analysis	was	performed	using	SPSS	V.15.0.	

Of the 160 samples analyzed by Spoligotyping, 78 (48.8%) cases showed absence of signal at the spot 33 to 36 compat-
ible with M. tuberculosis. Nevertheless SPOL-DB4 was made with DNA obtained from cultures, we in tented to compare
our patterns obtained and we founded only 4 samples compatible with pattern previously described LAM (2), U (1) and
Haarlem lineage (1). Incomplete patterns were observed in 34 (21.2%) and no patterns were obtained in 48 (30.0%).
Negative	controls	did	not	yield	any	spoligopatterns.	Exact	Fisher’s	statistic	showed	a	significant	difference	between	the	
positivity	of	Spoligotyping	and	storage	time	(P	<0.05)	while	for	the	kind	of	tissue	found	no	differences	(P>	0.05).

In	48.8%	cases	we	confirmed	the	diagnosis	of	M. tuberculosis. The spoligotyping method is a tool that provides some
information about FFPET, the results of this can be affected by storage time. In order to obtain better results is necessary
to examine the samples immediately after her obtention.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                 149

      pOTT´S DiSEaSE: aN aNCiENT DiSEaSE?

      Cardoso, Sara, Coelho, Rui, Paulo, Cristiana, Abreu, Candida, Silva, Susana, Gomes, Helena, Sarmento, António
      Hospital S.João

      Skeletal	tuberculosis	is	a	rare	disease	in	developed	countries	although	it´s	still	a	significant	cause	of	disease	in	Portugal	
      due	to	the	high	prevalence	of	tuberculosis	and	an	insidious	and	non-specific	presentation	of	this	form	of	disease.	Clinical	
      report: We report 3 cases of Pott´s disease.

      Case 1: Male, 62y. History of multiple sclerosis under imunomodulator and leg trauma with disability. He went several
      times	to	the	emergency	room	due	to	intense	lombalgy	without	trauma	and	discharged	with	non-steroidal	anti-inflam-
      matory (NSAIs) medication, without relief. D12-L1 fracture was diagnosed and the patient was discharged under con-
      servative treatment. Fifteen days later, he appeared with paraparesia, fever, asthenia and weight loss and was submitted
      to surgery. Histology of the bone fragments revealed acid fast bacilli. The direct and cultural exam of gastric lavage (GL)
      were positive for M.tuberculosis complex (MTC). Thorax X-ray (XR) was normal.

      Case 2: Female, 88y. History of previous ribs and femur trauma fractures. She had complaints of lombalgy and progressive
      paraparesia during the last year and was chronically medicated with NSAIs. Three months before admission anorexy and
      weight loss appeared. She had no fever.The magnetic ressonance revealed D6-D7 vertebral body fracture with cavitation
      and medular compression. She was submitted to surgery. The bone cultural exam was positive to MTC. The thorax XR
      was suggestive of pulmonary involvement but the GL mycobacteriology exam was negative.

      Case 3: A 77y woman with past history of pulmonary tuberculosis and recent history of dorsal trauma with D12 frac-
      ture, treated conservatively. Later she was admitted with paraplegia and submitted to surgery.The bone culture, broncho-
      alveolar	lavage	culture	and	DNA	(polymerase	chain	reaction)	were	positive	for	MTC.	The	cerebrospinal	fluid	had	24cells/
      uL, high ADA (123U/L), proteins (10,20g/l) and low glucose (0,14g/l), but DNA for MTC and culture were negative. All
      three patients were treated surgically and medically with rifampin, isoniazid, ethambutol and pyrazinamide. All of them
      had come with dorsal or lombar pain and progressed to paraparesia and plegia, which showed some improvement with
      antibiotics and physiotherapy.

      Skeletal tuberculosis is a silent re-emergent disease, for which our doctors are not aware, leading to delayed diagnosis
      and causing severe neurological damage like Pott´s paraplegia as it was observed in our patients.

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OSSEOuS TubErCulOSiS aT agE Of 9 mONThS

Loureiro, Carla 1, Matos, Gabriel 1, Balacó, Inês 1, Mota, Marta 2, Nogueira, Célia 2, Lemos, Sónia 1, Rocha, Graça 1
1. Pediatric Department/H. Pediatrico, CHC
2. Mycrobiology Laboratory, Coimbra Medicine Faculty

background and aims
Osseous tuberculosis is rare, mainly as a primary disease in a previously healthy infant. Several mycobacteria may be
involved such as M. tuberculosis, M. bovis and M. avium. When there is no association with a primary pulmonary disease
in a child vaccinated with Calmet-Guérin Bacillus, M. bovis may be the causal agent.

Case report
 A previously healthy 11 month-old infant BCG vaccinated at birth was admitted with a 2 month evolution of a right elbow
tumefaction. The X-ray presented destruction of the trochea, severe periostic reaction and soft tissue tumefaction, and
the	ecography	a	fluid-filled	cavity.	MRI	suggested	neuroblastoma	or	Ewing	sarcoma.	Sedimentation	rate	and	specific	eno-
lase	were	elevated.	On	surgery	a	whitish	soft	mass	associated	with	necrotic	fluid	compressing	the	cubital	nerve	was	found.	
The histological examination revealed characteristic features of caseum and allowed the diagnosis of osseous tubercu-
losis.	Mycobacterium	tuberculosis	complex	was	identified	by	Real-Time	PCR	in	osseous	biopsy.		Gastric	fluid	and	urine	
were negative. Our patient recovered after surgical debridement and combination drug therapy. He developed a local
cutaneous	fistula	(no	agent	identified	on	fluid	culture)	and	posterior	soft	tissue	calcification.	At	22	months	age	he	remains	
with no other relevant infections.

Skeletal tuberculosis with extravertebral location is rare. Tuberculous osteomyelites of the limb bones requires a high
index of clinical suspicion along with radiological and histopathological investigation in order to establish the diagnosis.
Tuberculosis is still an important differential diagnosis in unusual bone conditions

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                   151

      DiffErENCES iN DirECT aNTiTumOral CapaCiTy amONg
      ThE VariOuS MyCoBaCTeriuM BoviS bCg SubSTraiNS

      SP Secanella, M Luquin, E Julián
      Dept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)

      The administration of Mycobacterium bovis	Bacillus	Calmette-Guérin	(BCG)	is	the	first	treatment	option	for	avoiding	the	
      recurrence of bladder cancer.Various BCG substrains are used. These substrains differ genetically and display differential
      antigenic	determinants,	which	it	has	been	suggested	may	influence	the	efficacy	of	BCG	as	vaccine	in	tuberculosis	studies.	
      In	tuberculosis,	evolutionary	early	strains	are	more	efficacious	than	are	the	more	attenuated	evolutionary	late	strains.	The	
      impact of these differences on BCG antitumoral capacity in bladder-cancer cell lines has not been addressed.
      We aimed to compare the direct antitumoral activity of different BCG substrains by inhibiting cell proliferation and by
      triggering the production of cytokines in bladder-cancer cell lines.
      T24, J82 and RT4 human bladder-cancer cell lines were cultured with different doses of BCGs. We tested three evolutio-
      nary	early	BCG	substrains,	Japan,	Moreau	and	Russia,	and	five	evolutionary	late	strains,	Connaught,	Danish,	Glaxo,	Phipps,	
      and Tice. Inhibition of cell proliferation was assessed by using a colorimetric assay at different time points; the production
      of interleukin (IL)-6 and IL-8 was measured in cell culture supernatants using enzyme-linked immunosorbent assay.
      Among the different BCG substrains, in the case of T24 and J82 cell lines, Connaught and Russia induced both the high-
      est inhibition of proliferation and cytokine production. In contrast, Glaxo and Phipps (for the T24 cell line) and Glaxo
      and	Tice	(for	the	J82)	were	the	least	efficacious	both	in	reducing	cell	viability	and	in	inducing	cytokine	production.	The	
      remaining BCGs behaved differently depending on the cell line.
      Finally, for the RT4 cell line, all BCG strains inhibit cell proliferation at the same level, except for Danish and Glaxo,
      which	were	seen	to	be	less	efficacious.	As	regards	cytokine	production,	IL-6	production	was	not	detected	in	any	culture,	
      whereas low levels of IL-8 production were observed, the lowest being for Danish and Glaxo cultures.
      The	results	showed	that	Connaught	and	Russia	are	the	most	efficient	BCGs	and	that	Glaxo	is	the	least	effective,	both	
      in the inhibition of tumoral-cell proliferation and the induction of cytokine production. No correlation was observed
      between	BCG	antitumoral	efficacy	and	genotypic	evolutionary	classification.

152                                                                                                                      ESM 2009

rOlE Of TNf-a gENE pOlymOrphiSmS iN hOST gENETiC
SuSCEpTibiliTy TO pulmONary TubErCulOSiS

Anoosheh, Saber 1, Farnia, Parissa 1, Noruzi, Jamileh 2, Kargar, Mohammad 3, Kazempour, Mehdi 1, Seif, Shima 1, Masjedi,
Mohammad Reza 4,Velayati, Ali Akbar 4
1 - Mycobacteriology Research Center, NRITLD, Shahid Beheshti University (M.C)
2 - Microbiology Department, Iran University of Medical Science
3 - Microbiology Department, Jahrom Azad University
4 - National Research Institute of Tuberculosis and Lung Disease, Shahid Beheshti University (M.C)

preface and goal
Tuberculosis is one of most common infectious diseases and it causes death of more than 3 million people a year, world-
wide. It caused by Mycobacterium tuberculosis and approximately one - third of world population are infected with this
bacteria, but only 5 - 10 % of them develop active TB.Therefore, individual differences in susceptibility to TB are expected.
These differences might be due to host factors especially genetic diversity between populations. TNF-a	as	a	pro-inflam-
matory	cytokine,	play	a	key	role	in	host	defense	against	tuberculosis.	Presence	of	mutation	in	this	gene	can	influence	the	
effectiveness, performance and capability of immune Responses against infection.The Aim of this study was to investigate
the frequency of TNF-a alleles and relationship between susceptibility to TB and TNF-a gene variations.

materials and methods
A case-control study was conducted and 65 healthy controls and 65 TB patients were enrolled. Genotype of TNF-238,
TNF -244, TNF-308, TNF -857 and TNF-863 were distinguished using by PCR-RFLP method. The results were analyzed by SPSS
v.16, Fisher exact and Hardy-Weinberg tests.

Obtained results showed that,TNF-308,TNF -857 and TNF-863 were as a high frequency mutation regions in population levels,
and	also	we	found		a	significant	differences	at		TNF-308 and TNF -857 between two group of controls and patients ( P-value
< 0.05 ).

presence of mutation in TNF-308 and TNF -857 regions probably increases host susceptibility to mycobacterial infection and
Genotyping of these regions can be used for screening of high risk persons. Also according to high frequency distribution
of mutations in TNF -857 and TNF-863 regions, further studies on association of these regions is suggested.

Key words
Tuberculosis, Genetic Susceptibility, Cytokines, Gene Polymorphism

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  153

      myCOlaCTONE iNTErfErES WiTh ThE prOTECTiVE
      ifN-γ-DEpENDENT aCTiVaTiON Of maCrOphagES
      DuriNg iNfECTiON WiTh MyCoBaCTeriuM ulCeranS

      Egídio Torrado1; Alexandra G. Fraga1; Elsa Logarinho1; Teresa G. Martins1; Jenny A. Carmona1; José B. Gama1; Maria A.
      Carvalho2; Fernanda Proença2; António G. Castro1; Jorge Pedrosa1
      1 - Life and Health Sciences Research Institute (ICVS). School of Health Sciences, University of Minho. Braga, Portugal
      2 - Chemistry Research Center. School of Sciences. University of Minho. Braga, Portugal

      Mycobacterium ulcerans is the etiological agent of a necrotizing cutaneous disease, known as Buruli ulcer. The pathology
      caused by this pathogen is associated with the production of the lipidic exotoxin mycolactone. Following our recent
      demonstration of an intramacrophage growth phase for M. ulcerans, we investigated the biological relevance of interfer-
      on-gamma (IFN-γ), as well as the mechanisms activated by this cytokine in M. ulcerans-infected macrophages.
      We used three different M. ulcerans strains selected based on their virulence for mice and the type of mycolactone
      produced: the low virulent mycolactone-negative strain 5114; the intermediate virulent, mycolactone C-producing strain
      94-1327; and the highly virulent, mycolactone D-producing strain 98-912.
      IFN-γ-deficient	mice	showed	an	increased	susceptibility	to	infection	only	with	strains	5114	and	94-1327,	suggesting	that	
      this cytokine plays a protective role in infections with the low and intermediate virulent strains of M. ulcerans, but not
      with the highly virulent strain. In line with this, IFN-γ-activated mouse primary bone marrow-derived macrophages con-
      trolled the proliferation of the low virulent and the intermediate virulent strains, the latter only at low multiplicities of
      infection. The effector mechanisms induced by IFN-γ in infected macrophages leading to M. ulcerans growth restriction
      involved	both	phagosome	maturation	and	acidification,	as	well	as	increased	nitric	oxide	production.	In	agreement,	the	
      addition	of	mycolactone	D,	purified	from	cultures	of	the	highly	virulent	strain,	led	to	a	dose-dependent	inhibition	of	pha-
      gosome maturation and nitric oxide production in IFN-γ-activated cultured-macrophages infected with the mycolactone-
      negative strain, resulting in an increased bacterial burden.
      Our results suggest that the protection mediated by IFN-γ during the intramacrophage phase of M. ulcerans infection
      depends on the type and amount of mycolactone produced.

154                                                                                                                     ESM 2009

VirulENCE, immuNOgENiCiTy aND prOTECTiON iNDuCED by ´MyCoBaCTeriuM
HaBana´ STraiNS iN a muriNE mODEl Of pulmONary TubErCulOSiS.

Montoro, Ernesto 1;Valdés, Iliana 1; Aguilar, Diana 2; Orozco, Hector 2; Hernández-Pando, Rogelio 2
1. Institute of Tropical Medicine “Pedro Kourí”(IPK), Havana, Cuba
2. National Institute of Medical Science and Nutrition ¨Salvador Zubirán¨. Mexico DF, Mexico

Mycobacterium habana’	was	first	isolated	in	Cuba	by	Valdivia,	in	1971.	Later,	was	demonstrated	its	protection	capacity	
against M. tuberculosis and other mycobacteria. We studied the virulence, immunogenicity and protection of 3 strains
of ‘M. habana’ using	Balb/c	mice.	The	first	experiment	was	done	to	know	the	virulence	potential	of	‘M. habana’ using a
progressive pulmonary TB model. In the 2nd assay mice were vaccinated with 3 doses of bacilli. The grade of immunoge-
nicity was related with the induction of IFNγ by the stimulation of the main organs with antigens of M. tuberculosis. The
best doses that induced immunogenicity were used in the 3rd experiment for animal vaccination. Two months later mice
were challenged with M. tuberculosis H37Rv and Beijing genotype. All the animals infected with M. habana TMC-5135 and
IPK-337 were alive until the end of the experiment. IPK-220 strain showed about 20% of death to the seven week post-
infection.	All	the	strains	had	significative	differences	when	we	compared	with	the	control	group	infected	with	H37Rv	
strain. The values of the colony forming units (CFU) were in correspondence with the survival rate. The percentage of
pneumonia was higher for ‘M. habana’	IPK-220,	showing	final	values	similar	to	mice	infected	with	H37Rv	strain.	Due	to	
the virulent behaviour of ’M. habana’ IPK-220 we discharged it for the coming assays. The most important results of
the 2nd experiment were the statistical differences in the IFNγ production founded in groups vaccinated of ‘M. habana’
IPK-337 and TMC-5135 strains, respectively, with the BCG group. The lung CFU for these doses showed a decreasing
tendency	with	a	total	sterilization	to	the	final	of	the	experiment.	Animal	vaccinated	with	‘M.	habana’	strains	and	chal-
lenged with M. tuberculosis H37Rv had the highest survival. These results are in accordance with the low percentage of
pneumonia	with	both	stains.	Nevertheless	this	differences	was	not	statistical	significative	in	comparison	with	BCG	group.	
With the Beijing challenge we observed differences between vaccination with TMC-5135 and BCG group. The lungs of
the animals that received BCG and challenged with Beijing showed more than 70% of pneumonia and a lower granuloma
area.	The	CFU	reveals	lower	lung	bacilli	load	in	mice	vaccinated	with	‘M.	habana’	strains.	The	final	results	demonstrate	the	
potential of ‘M. habana’ to protect against TB infection.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  155

      DENDriTiC CEllS DiffErENTially EXprESS il12-family CyTOKiNES
      afTEr iNfECTiON WiTh MyCoBaCTeriuM TuBerCuloSiS Or M. BoviS bCg

      Margarida Saraiva, Carole Sousa, Jenny A. Carmona, Andrea Cruz, Jorge Pedrosa, A. Gil Castro
      Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal

      IL-12 family is a group of heterodimeric cytokines, composed of 4 related cytokine members: IL-12, IL-23, IL-27 and IL-35.
      These cytokines, that share some functions and receptor components of IL-12, act on CD4+ T cells modulating the type
      of T helper and T regulatory responses, and initiating the development of the acquired T cell response to intracellular
      pathogens, such as Mycobacterium tuberculosis (MTb) or M. bovis BCG. Bone marrow derived dendritic cells (BMDC)
      originating from wild-type mice were exposed to live MTb strain H37Rv or to BCG for different periods of time. The
      kinetics of mRNA production of each monomer that form the cytokines of the IL-12 family (p19, p28, p35, p40, Ebstein-
      Barr-Virus-induced gene 3 (Ebi-3)) was determined. We show that MTb-stimulated BMDC were strong producers of p40,
      p35 and p19, whereas BMDC exposed to BCG expressed much lower levels of these cytokines. The main TLR involved
      in the recognition of MTb and BCG and activation of BMDC is TLR2, since in its absence the expression of the various
      monomers	was	nearly	abrogated.	A	consequence	of	this	differential	activation	of	BMDC	was	reflected	on	the	distinct	
      type of T helper responses developed when MTb- or BCG-infected BMDC presented OVA peptide to TCR-transgenic
      CD4 T cells. MTb-infected BMDC were able to induce the development of both Th1 and Th17 responses, whereas BCG-
      infected BMDC induced Th17 responses. We are currently addressing the molecular mechanisms that differentiate the
      response of BMDC in the context of an MTb or a BCG infection. Understanding the details of DC activation by MTb or
      BCG and its consequences on the CD4+ T cell arm of the immune response will help to reveal important aspects to be
      improved for a better vaccination strategy.

156                                                                                                                  ESM 2009

aNalySiS Of M. SMegMaTiS muTaNTS rESiSTaNT TO mS6 iNfECTiON

Simões, Marta Filipa; Jordão, Luísa; Teles, JMM; Couto, S; Moniz-Pereira, José; Pimentel, Madalena
Centro de Patogénese Molecular-Unidade dos Retrovirus e Infecções Associadas, Faculty of Pharmacy, University of
Lisbon, Portugal

Mycobacteriophages represent excellent model systems for studying mycobacterial hosts. Ms6 is a temperate myco-
bacteriophage that infects Mycobacterium smegmatis.	The	first	step	of	a	dsDNA	phage	infection	is	adsorption	to	a	host	
receptor followed by injection of DNA into the cytoplasm. Characterization of the phage resistant mutants represents
a	common	genetic	strategy	for	the	identification	of	mycobacterial	genes	involved	in	the	synthesis	of	parietal	phage	re-
ceptors. The study of these genes is of highly importance, as phage receptors are also involved in the entry of others
molecules, such as antibiotics, into the cell. In order to identify the Ms6 receptor we started to select from a mutant
library obtained after a transposition event using the pCG79 system (Guilhot et al, 1994), M. smegmatis mutants to a
Ms6 infection. DNA obtained from these mutants was submitted to an enzymatic restriction analysis, which allowed the
selection	of	four	mutants	with	different	profiles.
In order to understand which step of the phage infection was affected, adsorption and infection assays with DAPI stained
phages were performed.
We	found	different	profiles	among	the	selected	mutants	suggesting	that	different	steps	of	the	infection	are	affected.	With	
these results, our goal is to identify the mycobacterial genes disrupted by the transposition event.
Identification	of	the	affected	genes	is	an	important	achievement	which	may	contribute	to	the	characterization	of	the	
phage receptor.
Guilhot,	C.,	I.	Otal,	I.	Van	Rompaey,	C.	Martín,	and	B.	Gicquel.	1994.	Efficient	transposition	in	mycobacteria:	construction	
of Mycobacterium smegmatis insertional mutant libraries. J. Bacteriol. 176: 535-539.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  157

      humOral rESpONSE iN TubErCulOuS paTiENTS agaiNST
      ThE myCOliC aCiDS Of MyCoBaCTeriuM TuBerCuloSiS

      Julián, Esther Gómez 1; Rodríguez-Güell, E 1; del Val-Romero, B 2; Clivillé, R 2; Cañete, C 3; Navarro, A 3; de Gispert, FX 3;
      Luquin, M 1; Alonso, C 2
      1 - Dept. Genètica i Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Bellaterra (Barcelona)
      2 - Dept. Microbiologia, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)
      3 - Unitat de Respiratori, Consorci Sanitari Integral, L’Hospitalet de Llobregat (Barcelona)

      Although numerous serological tests have been developed for TB serodiagnosis, none of these have shown adequate
      levels of accuracy; in consequence, they have not been widely implemented.
      Diverse mycobacterial antigens have been evaluated in these tests, among which are cell-wall glycolipidic antigens such
      as	cord	factor	(CF).	This	molecule	is	made	up	of	a	trehalose	residue	sterified	to	two	mycolic	acids	(MAs);	it	has	been	
      established that MAs are the epitopes for the anti-CF antibodies recognition. Furthermore, a possible cross-reaction
      between anti-MAs antibodies and cholesterol (COL) in TB patients has recently been published.
      The purpose of this study was to detect and compare the presence of IgG, IgM and IgA antibodies against MAs with
      regard to the presence of anti-CF and anti-COL antibodies in TB patients.
      To	address	this	question,	MAs	and	CF	were	purified	from	M. tuberculosis H37Rv (ATCC 27294T), and commercially avail-
      able COL was used. An ELISA was developed to detect IgG, IgM and IgA antibodies to MAs and COL, and an ELISA
      previously developed for CF was performed.
      The presence of IgG, IgM and IgA anti-MAs, CF and COL in the sera of 31 HIV-negative TB patients at the time of TB
      diagnosis and throughout anti-TB prophylaxis, 20 PPD-positive donors, 20 PPD-negative donors and 20 patients affected
      by other pneumonias were determined.
      No antibodies to MAs were determined in any sera of the groups studied, whether for TB patients at the onset or during
      prophylaxis, or in control groups.
      Anti-CF IgG and IgA antibodies in sera from TB patients were detected, following a downward kinetic from the beginning
      to	the	end	of	the	prophylaxis.	Test	sensitivity	and	test	specificity	were	41%	and	78%,	respectively,	for	IgG	detection,	and	
      19% and 95%, respectively, for IgA detection. Additionally, anti-CF IgM antibodies were detected in all the groups.
      In contrast, neither IgG nor IgA anti-COL antibodies were detected in any of the groups; however, IgM anti-COL antibod-
      ies were also detected in sera from all the groups studied.
      Each serum showing anti-CF and COL antibodies was individually analysed, and their titres against each antigen were
      compared.	Analysis	showed	that	response	profiles	were	different	against	the	two	antigens.	Moreover,	given	that	the	sera	
      reacting against COL or/and CF did not react against MAs, it is therefore possible to rule out a cross-reaction between
      MAs and COL.
      The overall results invalidate MAs as possible antigens for the serodiagnosis of TB.

158                                                                                                                       ESM 2009

MyCoBaCTeriuM TuBerCuloSiS

Arley Gómez López
Infectious Diseases and Tropical Medicine Unit; Universidad del Rosario

TlyA protein has a controversial function as a virulence factor on Mycobacterium tuberculosis (Mtb). Updated studies
have not demonstrated a possible hemolytic activity conferred by TlyA and contrary to recent evidence have suggested
a function enzymatically similar to RNA methyltransferase at ribosomal level which confers antibiotic susceptibility to
capreomycin and viomycin.
Our aim was to determine the In vitro hemolytic activity of Mtb	TlyA	overexpressed	and	purified	from	E. coli BL21-AI and
to carry out an in silico phylogenetic and structural analysis.
 Based on tlyA gene sequence (Rv1694) from Mtb	H37Rv	specific	primers	were	designed.	The	amplification	product	was	
ligated on pEXP5-CT/TOPO vector (Invitrogen), transformed and overexpressed on E. coli	BL-21-AI.	After	purification	by	
affinity	chromatography,	TlyA-His6	recombinant	protein	was	analyzed	by	SDS-PAGE	under	denaturing	conditions	which	
was detected as 28 KDa single band. Recombinant protein as recognized by anti-His monoclonal antibody.
Protein structural characterization by circular dichroism was carried out. On the other hand, hemolytic activity assays
using	TlyA-His6	purified	were	negative	as	well	as	on	bacterial	lysates.	
Hemolytic	assays	with	TlyA-His6	supplemented	with	calcium	and	magnesium	were	negative	suggesting	lack	of	specific	
requirements for this activity.
By using bioinformatics tools, a ribosomal binding called S4 located between 5 and 68 residues and FtsJ among 62 and
247	residues	were	identified	on	TlyA.	These	domains	have	been	described	on	proteins	associated	with	ribosomal	trans-
lational	machinery.	The	Hidrophobicity	profile	was	in	disagreement	with	a	possible	transmembrane	helix	although	non	
polar amino acid composition suggesting that TlyA might not be membrane attachment. Nevertheless, three-dimensional
model (Structural homology with 1L9K model) reveals a consensus structure with a common core, comprising a parallel
β-sheet of six strands, sandwiched between two layers of a-helices corresponding to a RNA methyltransferase structure.
Phylogenetic analyses showed that TlyA is highly conserved among mycobacteria species and it does not exhibit changes
among Mtb complex strains. tlyA gene evolution might operate under purifying selection model. Additionally differences
were observed among TlyA and bacterial pore forming proteins.
This	evidence	supports	a	link	between	ribosomal	modifications	to	posttranslational	level	and	suggests	a	functional	an-
notation error of this family protein at GENBANK as well as missannotation on several genomes as Mtb genome. Studies
on resistance mechanisms of Mtb mediated by ribosomal proteins might be useful for understanding new alternative
therapeutic approaches for tuberculosis control applied to knowledge of second line antibiotics such as capreomycin.

Key words
TlyA, Mycobacterium tuberculosis, hemolysin, Structural modeling, RNA methyltransferase.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                             159

      EValuaTiON Of ThE appliCabiliTy
      Of SErODiagNOSiS fOr TubErCulOSiS iN pOrTugal

      Carina Ferreira 1, Andrea Afonso 2, Raquel Duarte 3, Konstantin Lyashchenko 4, Anabela
      Silva 5, Filomena Rodrigues 5, Anabela Miranda 5, Margarida Tavares 6, Cátia Caldas 6, Fátima
      Valente 2, António Valente 2, Olga Vasconcelos 7, Joana Amado 7, Margarida Correia-Neves 1
      1 - Life and Health Sciences Research Institute (ICVS), University of Minho, Braga, Portugal
      2 - Centro de Saúde de Bragança, Bragança, Portugal
      3 - Centro de Diagnóstico Pneumológico de Vila Nova de Gaia, Portugal
      4 - Chembio Diagnostic Systems, New York, USA
      5 - Centro de Tuberculose e Micobactérias, Instituto Nacional de Saúde Dr Ricardo Jorge, Delegação do Porto, Portugal
      6 - Serviço de Doenças Infecciosas, Hospital de São João, Porto, Portugal
      7 - Hospital Joaquim Urbano, Porto, Portugal

      Diagnosis of tuberculosis requires laboratory techniques to complement clinical information. Among the different tech-
      niques in use, the only accurate diagnostic method is based on the search for Mycobacterium tuberculosis growth in
      cultures of human biological samples, usually sputum. However, this approach is long, lacks sensitivity for samples with
      low bacterial load, and is invasive in the case of non-pulmonary tuberculosis. In order to overcome these constraints,
      several	novel	methodological	approaches	are	under	evaluation.	Among	these	is	the	determination	of	tuberculosis-specific	
      antibodies in the sera - serodiagnostic methods.Validation of serodiagnosis tests in any given country is mandatory, since
      sensitivity	and	specificity	vary	depending	on	factors	such	as	the	composition	and	frequency	of	environmental	mycobac-
      teria, previous vaccination with BCG, prevalence of tuberculosis and other diseases, and host genetic background. We
      evaluated	the	specificity	and	sensitivity	of	two	serodiagnosis	tests:	TB	STAT-PAK	II	-	an	immunochromatographic	test	for	
      the detection of antibodies to Mycobacterium tuberculosis; and MAPIA - Multi-Antigen Print Immunoassay (both devel-
      oped	at	Chembio	Diagnostics,	NY,	USA).	The	specificity	of	the	tests	was	very	high,	ranging	from	94	to	100%	depending	
      on the test and control population analysed. The sensitivity was 37 and 54% for TB STAT-PAK II and MAPIA, respectively,
      and	it	increased	during	the	first	3	months	of	treatment,	for	TB	STAT-PAK	II.	Interestingly,	when	the	smear	results	were	
      combined with the ones from the TB STAT-PAK II, the sensitivity increased from 71 (just smear) to 79%.Thus, taking into
      consideration	the	great	specificity	of	the	TB	STAT-PAK	II,	its	use	for	smear	negative	patients	could	increase	the	rate	of	
      detection in early TB diagnostic.

160                                                                                                                  ESM 2009

implEmENTaTiON Of ThE ThiN layEr agar (Tla) fOr ThE DiagNOSiS Of
SmEar NEgaTiVE pulmONary
TubErCulOSiS iN a high hiV prEValENCE SETTiNg

Martin, Anandi 1, Munga Waweru, Peter 2, Babu Okatch, Fred 2, Amondi Ouma, Naureen
   , Bonte, Laurence 2, Palomino, Juan Carlos 1,Varaine, Francis 2, Portaels, Françoise 1
1 - Institute of Tropical Medicine, Antwerp, Belgium
2 - Médecins Sans Frontières, Paris, France

Early diagnosis of smear-negative pulmonary tuberculosis in countries with high incidence of TB/HIV co-infection is
crucial to limit the mortality and control the disease. The objective of this study was to evaluate the performance of a
low-cost method, the Thin Layer Agar (TLA), for the diagnosis of smear-negative patients compared to the gold standard
Löwenstein-Jensen method. TLA relies on microscopic detection of cording growth that is characteristic of M. tuber-
culosis and is able to differentiate between M. tuberculosis and non-tuberculous mycobacteria. This prospective study
was performed in Homa Bay district Hospital in Kenya. Out of 1584 smear-negative sputum samples, 212 were positive
by LJ (13.5%) and 220 positive by TLA (14%). The sensitivity of LJ and TLA was 71% and 74 % respectively. With further
improvement for decreasing the contamination rate in both methods, TLA could become an affordable method for the
diagnosis	of	smear-negative	tuberculosis	in	resource-limited	settings	allowing	the	simultaneous	detection	and	identifica-
tion of M. tuberculosis, within 2 weeks.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                              161

      DirECT DETECTiON Of rifampiN rESiSTaNCE iN
      MyCoBaCTeriuM TuBerCuloSiS by ThE NiTraTE
      rEDuCTaSE aSSay appliED DirECTly iN SpuTum SamplES

      Regina Ferro e Silva 1, Maria de Lourdes Shikama 1, Gleize Villela 1, Daisy Nakamura Sato 1, Carmen
      Maria Saraiva Giampaglia 1, Maria Conceição Martins 1, Anandi Martin 2, Juan Carlos Palomino 2
      1 - Instituto Adolfo Lutz (IAL), São Paulo, Brazil
      2 - Institut of Tropical Medicine, Antwerpen, Belgium

      Resistance to rifampin (RIF) is an important predictor for the early diagnosis of MDRTB. Conventional methods for
      DST of M. tuberculosis require several weeks to give results.Thus, an alternative in vitro method which can detect drug
      susceptibility directly from sputum and presents quick results and low cost, will be very useful for tuberculosis diagnosis.
      The nitrate reductase assay uses the detection of nitrite as an indication of growth when used as a drug susceptibility test.
      Objective:to compare the nitrate reductase assay (NRA) with the proportion method (PM), considered as gold standard,
      to detect RIF resistance in M. tuberculosis directly from sputum samples. Method: the study was carried out by 4 regional
      laboratories from the state of São Paulo, Brazil. A total of 206 sputum samples tested smear positive from patients with
      pulmonary tuberculosis. The sputum was decontaminated by Petroff method and DST to RIF was carried out using the
      PM and the NRA. Nitrate Reductase Assay: Sputum samples, after decontamination were inoculated in one tube control
      (without drug) and one tube containing 40 µg/ml of rifampicin. Findings: the results of the DST obtained directly from
      sputum were: 6 samples resistant to RIF and 200 samples susceptible. The comparison between NRA and the traditional
      gold	standard	method	showed	agreement	of	100%.	The	sensitivity	and	specificity	of	the	NRA	for	rifampicin	was	100%.	
      Results were available in 10 days for 66 (34%) samples, 15 days for 102 (53%) samples and 20 days for 24 (13%) samples
      while the results of PM took 30 days to be available. Conclusions: the NRA proved to be a promising method for the
      screening of suspect MDRTB cases directly from sputum samples. The simplicity of the method, its low cost and celerity
      to give the results make it a good alternative method for laboratories in resource-poor settings.

162                                                                                                                     ESM 2009

MyCoBaCTeriuM TuBerCuloSiS by ThE NiTraTE rEDuCTaSE
aSSay appliED DirECTly iN SpuTum SamplES

Ferro Regina Silva 1, Shikama Maria de Lourdes 1, Villela Gleize 1, Sato Daisy Nakamura 1, Giampaglia Carmen Saraiva     1,

Martins Maria Conceição 1, Martin Anandi 2,3, Palomino Juan Carlos 2, Telles Maria Alice da Silva 1
1 - Instituto Adolfo Lutz, São Paulo, Brazil
2 - Institute of Tropical Medicine, Antwerp, Belgium
3 - Médecins Sans Frontières, Paris, France

A cost-effective and rapid drug susceptibility testing (DST) method is required to guide TB treatment. Commercially
available systems such as BACTEC MGIT 960 and MB/BacT are faster but demand costly equipment and supplies, there-
fore, are not feasible in most resource-poor settings. Resistance to rifampin (RIF) is an important predictor for the early
diagnosis of MDRTB. To compare the nitrate reductase assay (NRA) with the proportion method (PM) to detect RIF
resistance in M. tuberculosis directly from sputum samples. The study was carried out by 4 regional laboratories from the
state of São Paulo, Brazil. A total of 210 sputum samples tested smear positive from patients with pulmonary tubercu-
losis. The sputum was decontaminated by Petroff method and DST to RIF was carried out using the PM and the NRA.
The results of the DST obtained directly from sputum were: 6 samples resistant to RIF and 204 samples susceptible. No
discordance	was	observed	between	the	two	methods.	The	sensitivity	and	specificity	of	the	NRA	was	100%.	Results	were	
available in 10 days for 75 (36%) samples, 15 days for 107 (51%) samples and 20 days for 28 (13%) samples.The results of
PM took 30 days to be available. The NRA proved to be a promising method for the screening of suspect MDRTB cases
directly from sputum samples. The simplicity of the method, its low cost and celerity to give the results make it a good
alternative method for laboratories in resource-poor settings.

This study was partially funded by INCO-Dev ICA4-CT-2001-10087.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                163

      mTbDrpluS aSSay iS a uSEful TOOl TO SCrEEN fOr
      mulTi-Drug rESiSTaNT TubErCulOSiS iN a NaTiONal SurVEy

      De Haas, Petra 1, Ramona Zenhorst 2, Pike Mwamba 2, Mweemba Muvwimi 3,
      Winnie Mwanza 2, Grace Mbulo 2, Nathan Kapata 2, Helen Ayles 1
      1 - Zambart project, Lusaka, Zambia and LSHTM London
      2 - Zambart project, Lusaka, Zambia
      3 - National Reference laboratory, Lusaka, Zambia

      The	World	Health	Organization	requests	that	countries	conduct	tuberculosis	drug	resistance	surveys	(DRS)	every	five	
      years to monitor trends of drug resistance and to determine rates of multi-drug resistant tuberculosis (MDR-TB). Zambia
      conducted its second nation-wide DRS in 2008. The objective of this study is to investigate whether the MTBDRplus
      assay (HAIN), a new molecular assay performed directly on sputum, is a useful tool in conducting a DRS.

      Throughout Zambia approximately 900 sputum specimens were collected from consecutive smear-posi-
      tive TB patients and transported to the TB Reference Laboratory in Lusaka. Specimens were decontaminat-
      ed and concentrated smears were prepared. MGIT and LJ cultures were inoculated. Drug susceptibility test-
      ing (DST) was performed on positive cultures. Remaining decontaminated sputum was heat-killed, sonicated
      and stored at -80C. The MTBDRplus assay was performed using a 1:5 or 1:10 dilution of decontaminated sputum.
      Results:	Of	the	first	340	specimens	tested	using	the	MTBDRplus	assay,	307	(90.3%)	showed	no	evidence	of	resistance,	
      while thirty-three (9.7%) showed mutations consistent with resistance: 10 were MDR-TB, 20 were isoniazid (INH)
      mono-resistant and 3 were rifampicin (RIF) mono-resistant. MGIT DST results were obtained from 271 (79.7%) of
      340 specimens with an MTBDRplus result. We were unable to obtain MGIT DST results from the remaining 69 (20.3%)
      specimens due to contamination or lack of growth. Thirteen (39.4%) of 33 specimens that showed mutations consistent
      with resistance in the MTBDRplus assay failed to yield a DST result on MGIT. Six out of these 13 samples are according
      to the MTBDRplus assay MDR, 5 are INH mono-resistant and 2 RIF mono-resistant . One sample that showed RIF mono-
      resistance using the MTBDRplus assay, showed both isoniazid and rifampicin resistance uses the MGIT DST.
      In our study, the MTBDRplus assay performed directly on sputum was more rapid and cost-effective than culture to
      screen for MDR-TB.

164                                                                                                                ESM 2009

CONTribuTiON Of labOraTOry faCTOrS TO high mgiT
CulTurE CONTamiNaTiON raTE iN zambia

de Haas, Petra 1, Moyoyeta, Monde 2, Samutela, Mulemba 2, Mwanza, Winnie
   , Musunsa, Alan 3, Mbulo, Grace 2, Muvwimi, Mweemba 3, Ayles, Helen 1
1. Zambart project, Lusaka, Zambia and LSHTM, London
2. Zambart project, Lusaka, Zambia
3. National reference laboratory, Lusaka, Zambia

An evaluation study of the MGIT liquid culture system in Zambia found a high contamination rate. It was suggested that fac-
tors such as delayed sample processing due to long distances, inadequate storage of samples once submitted, poor labora-
tory infrastructure and inexperienced staff may have contributed to the high contamination.The objective of this study was
to investigate the contribution of laboratory factors to the higher than expected rate of MGIT contamination.

As part of the National drug resistance survey, 917 sputum specimens were collected from smear-positive TB patients
and	transported	to	the	TB	Reference	Laboratory	in	Lusaka.	After	decontamination	using	NALC-NAOH	(1,5%	final	con-
centration) MGIT and LJ cultures were inoculated. In addition 584 of the decontaminated samples were inoculated onto
blood agar plates (BA).When the MGIT culture showed growth Ziehl Nielsen staining was performed. If micro-organisms
other	then	acid	fast	bacilli	were	seen,	a	BA	was	inoculated	and	subsequent	colonies	identified	using	biochemical	tools.		

Time between sample submission and processing varied from 1-50 days with a median of 9 days. Increased contamina-
tion in MGIT was found when the time between sample submission and processing was extended; 18.6% for 1-7 days,
27.4% for 8-14 days and 37.5% for more than 15 days. The overall contamination rate was 27.6% (95% CI 24.0-31.4). Of
584 decontaminated sputum 197 (33.7%) showed growth on BA. Out of the 197 samples showing growth 86 (43.6%)
were also contaminated in MGIT whereas for samples that showed no growth on BA, 70 (18%) out of 387 were con-
taminated.	Contaminated	organism	from	the	sputum	and	from	the	contaminated	MGIT	culture	were	identified	for	35	
out	of	the	86.	Identical	species	were	identified	in	only	sixteen	(45.7%)	of	these	35	samples,	whereas	in	19	(54.3%)	cases	
a different contaminanting species was found in the MGIT culture compared to the decontaminated sample.

High contamination on MGIT is a problem in our setting. Delayed processing of samples increases the chance of samples
being contaminated. The contamination of samples that did not show any growth on BA from decontaminated sputum
as well as different species isolated from the contaminated MGIT compared to the decontaminated sputum suggest that
a major part of the contamination may be due to laboratory factors.

Oral Communication

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                165

      prOgrammaTiC COmmuNiTy-baSED maNagEmENT Of
      mDr-Tb: EXpEriENCE iN KaraChi, paKiSTaN

      Ahmed, Altaf; Qazi, Fahad; Khan, Amir Javed
      The Indus Hospital, Karachi, Pakistan

                          T                                     T
      Multidrug	Resistant	 uberculosis	(MDR-TB)	is	defined	as	 B	resistant	to	the	two	most	powerful	anti-TB	drugs,	Isoniazid	(H)	
      and Rifampicin (R). Pakistan is ranked 8th among the high TB burden countries.In November 2007, the Karachi DOTS-Plus
      program was established with an objective to provide free, comprehensive, community-based management of MDR-TB pa-
      tients in Karachi and Hyderabad based on Partners in Health (PIH) and World Health Organization (WHO) guidelines.The
      purpose of this paper is to share our initial experience at programmatic management of MDR-TB in a low income setting.

      At baseline registration, smears, cultures, DST, radiology, and ancillary tests were performed on each patient and each
      patient house was mapped using GPS devices.
      Inclusion criteria were as follows:
      1) Acid Fast Bacilli Culture and Sensitivity (AFBCS) showing MDR-TB or PDR-TB
      2) Clinical, Radiological or Bacteriological (smear and culture) evidence of active disease
      Enrollment was based on availability of funds, clinical judgment and proximity to center. All patients were managed in the
      community. Each enrolled patient received monthly consultation, uninterrupted and monitored second line drug supply,
      laboratory tests as per program guidelines. Social support was also provided to all patients in the form of:
      1) Monthly Food Baskets
      2) Professional Counseling
      3) Treatment Supporters (TS)

      As of May 2009, 106 MDR-TB patients were registered in the program (49 males, 57 females). The mean age was 29.58
      (+/-12.8).	73	patients	were	put	on	treatment.	Their	classification	according	to	previous	treatments	was	as	follows:	
      1) After failure of retreatment ……30 (41%)
      2) Relapse ………………………...14 (19%)
      3)	After	failure	of	first	treatment	…12	(16%)	
      4) New …………………………….6 (8%)
      5) Registered after default…………5 (7%)
      6) Other…………………………….1 (1%)
      7) Transfer in ………………………1 (1%)

      68	(90%)	patients	had	received	first	line	treatment	previously,	1	(2%)	had	received	second	line	treatment	and	6	(8%)	were	
      new patients. HIV testing on 68 of 73 enrolled patients came negative for all. Adverse events recorded were recorded.
      Comorbid conditions included DM,Tobacco use, substance abuse, chronic lung disease, hepatitis, respiratory failure, preg-
      nancy,	hypertension,	gastritis,	renal	failure	and	neuropathy.	Although	treatment	duration	for	the	first	pool	of	p	

166                                                                                                                   ESM 2009

EValuaTiON Of Capilia (TauNS) fOr rapiD iDENTifiCaTiON Of
myCObaCTErium TubErCulOSiS COmplEX frOm CulTurES

C Muchwa2,3, J Akol2,3, F Mumbowa5, P Orikiriza2,3, K Morgan2,3, K Eisenach4 , M Joloba1,3, A Etwom2,3, P Mugyenyi2, R Mugerwa3
1 - Department of Medical Microbiology, Makerere University Medical School, Kampala, Uganda
2 - Joint Clinical Research Centre, Kampala, Uganda
3 - Uganda-CASE Research Collaboration, Kampala, Uganda
4 - University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, USA;.
    5Infectious Diseases Institute (IDI), Kampala, Uganda

The Capilia TB assay is a simple immunochromatographic assay which uses anti-MPB64 monoclonal antibodies to dis-
criminate MTB from non-tuberculosis mycobacteria. Evaluation of Capilia to determine its costs, performance and turn
around time (TAT) was done using PCR IS6110 assay (PCR) as a gold standard.

Respiratory and blood samples specimens were digested and decontaminated using 1.5% NAOH/NALC, concentrated
by centrifugation and inoculated into BACTEC MGIT 960 culture tubes for incubation. Blood was aseptically inoculated
and incubated in the BACTEC 9120 instrument. All BACTEC positive cultures were screened for acid fast bacilli by the
Ziehl-Neelsen method before testing for MTB. Blood cultures were then inoculated on 7H10 agar and incubated for
MTB isolation.The Capilia test was performed according to the manufacturer’s instructions while PCR was done accord-
ing to laboratory protocol. The test included 155 respiratory and 70 blood samples were tested.

Overall agreement between Capilia and PCR IS6110 methods was 98.2%. Capilia achieved a sensitivity of 98.4% and
specificity	of	97.9%.	Initial	PCR	comparison	for	respiratory	cultures	resulted	in	sensitivity	and	specificity	of	97.4%	and	
98.7%	respectively.	Blood	achieved	specificity	of	27.4%	only;	this	may	be	due	to	false	negative	PCR	results	caused	by	PCR	
inhibitors in blood cultures. PCR testing was performed from colonial growth on 7H10 accuracy and reliability of PCR
as	a	gold	standard	and	the	resulting	Capilia	test	sensitivity	increased	to	100%	and	94.4%	specificity.	

The	Capilia	has	an	overall	sensitivity	of	98.4%	and	specificity	of	97.9%	and	is	far	more	accurate	method	of	identifying	
MTB directly in blood cultures. It is less expensive (≈ $5) compared to PCR (≈ $45). It is easier to perform with TAT of
20 minutes while PCR has TAT of 8 hours for respiratory cultures.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                    167

      COmpariSON Of Capilia (TauNS) aND iS6110 pCr fOr
      rapiD iDENTifiCaTiON Of MyCoBaCTeriuM TuBerCuloSiS
      COmplEX frOm CulTurES iN Kampala, ugaNDa.

      C Muchwa2,3, J Akol2,3, P Orikiriza2,3, K Morgan2,3, F Mumbowa5, K
      Eisenach4, A Etwom2,3, M Joloba13
      1 - Department of Medical Microbiology, Makerere University Medical School, Kampala, Uganda
      2 - Joint Clinical Research Centre, Kampala, Uganda
      3 - Uganda-CASE Research Collaboration, Kampala, Uganda
      4 - University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, USA
      5 - Infectious Diseases Institute (IDI), Kampala, Uganda

      The Capilia TB assay, a simple immunochromatographic method which uses anti-MPB64 monoclonal antibodies to dis-
      criminate MTB from non-tuberculous mycobacteria has been successfully evaluated in other settings. Before adopting
      capilia in our laboratory, an evaluation to determine its costs, performance and turn around time (TAT) compared to the
      existing	molecular	identification	method	using	PCR	IS6110	assay	(PCR)	was	done.	

      Sputum/Gastric samples were processed using the 1.5%NAOH/NALC decontamination method and the sediment
      cultured	using	the		BACTEC	MGIT	960	system..	Blood/Pleural	fluids	were	aseptically	inoculated	in	the	BACTEC	9120	
      instrument.	All	 BACTEC	 positive	 cultures,	 (sputum	 and	 body	 fluids)	 were	 screened	 for	 acid	 fast	 bacilli	 by	 the	 Ziehl-
      Neelsen method before testing for MTB. The Capilia test was performed on all ZN positive MGIT 960 tubes according
      to the manufacturer’s (TAUNS ) instructions while the PCR was done according to laboratory protocol.The ZN positive
      blood	and	other	body	fluid	cultures	were		inoculated	on	7H10	agar	 for MTB isolation. Capillia was performed on ZN
      positive	isolates.The	test	included	155	sputum/gastrics	and	70	body	fluid	samples.	The	Capilia	and	PCR	assays	were	done	
      by separate technicians who were blinded to the other test results.

      There was an overall agreement of 98.2% between the Capilia and PCR IS6110 methods. Capilia achieved a sensitivity
      of	98.4%	and	specificity	of	97.9%.	These	results	concurred	with	the	finding	of	Jann	Wang	et	al	publication	of	98.6%	&	
      97.9% respectively. The initial PCR comparison for sputum/gastric cultures resulted in a 97.4% and 98.7% sensitivity and
      specificity	respectively.	When	the	PCR	test	was	performed	from	colonial	growth	on	7H10,	the	accuracy	and	reliability	of	
      PCR	as	a	gold	standard	increased	and	the	resulting	Capilia	test	sensitivity		was	100%	and	94.4%	specificity.	The	sensitivity	
      and	specificity	of	Capilia	on	contaminated	cultures	was	97.0%	and	98.7%	respectively	while	that	on	pure	cultures	was	
      99.0%	and	94.4%.	There	was	no	significant	difference	in	the	test	performance	between	contaminated	and	pure	cultures	
      (p values of 0.12405 and 0.786 respectively). The recurrent cost for capilia was ~$5 and that of PCR ~$45. The average
      TAT for Capilia was 20 minutes while that of PCR 8 hours for sputum/gastric cultures and 22 days for blood cultures.
      Overall Capilia was much easier to learn, had fewer steps and requires no instruments

         •	 The	sensitivity	and	specificity	of	Capilia	is	comparable	to	PCR	for	both	pure	and	contaminated	MTB	cultures.
         •	 Capilia is less expensive, faster and easier to perform than PCR.
         •	 Capilia is capable of identifying MTB directly in blood cultures.

168                                                                                                                            ESM 2009

DirECT TESTiNg fOr mulTi Drug rESiSTaNT TubErCulOSiS WiTh
fOur aSSayS EValuaTED aT Kampala, ugaNDa

Freddie Bwanga1,2,3, Sven Hoffner2,3, Melles Haile2,3, Moses L Joloba1
1 - Department of Medical Microbiology Makerere University College of Health Sciences Kampala, Uganda
2 - Department of Bacteriology, Swedish Institute for Infectious Diseases Control, Solna Sweden
3 - Department of Microbiology, Tumour and Cell Biology (MTC), Karolinska Institute, Stockholm, Sweden

Multi drug resistant tuberculosis (MDR TB) is on the rise worldwide. Early detection of MDR TB is important for effective
control of MDR TB transmission. In most TB high burden countries this remains a challenge due to lack of rapid tests.
This study evaluated four rapid tests for MDR TB detection.

Smear positive re-treatment TB patients were consecutively recruited at Mulago – Uganda’s National referral Hospital.
Samples	were	processed	using	a	final	concentration	of	1.5%	NAOH-NALC	method.	Sediments	were	used	directly	to	
set susceptibility to isoniazid and rifampicin with four rapid tests at the National TB Reference Laboratory Kampala.
The four tests included the Nitrate Reductase Assay (NRA), Microscopic Observation Drug Susceptibility (MODS),
Mycobacterium Growth Indicator Tube (MGIT 960) and Genotype® MTBDRplus (Hain Life Sciences, Nehren, Germany).
Results of the four tests were compared to those of the conventional indirect Lowenstein-Jensen proportion method (L-J PM).

A total of 66 patients were recruited. Interpretable results were obtained for all the samples with the LJ PM and MODS
assay, 64 (97%) with the NRA and MGIT 960, and 62 (94%) with the Genotype® MTBDRplus. Interpretable results across
all	the	five	tests	were	available	for	56	samples	and	results	obtained	on	initial	testing	were	100%,	98%,	91%,	82%	and	68%	
with the Genotype® MTBDRplus, MODS, LJ PM, NRA, and MGIT 960, respectively. Repeat testing with the MGIT 960 was
due to power failure -13 samples, contamination - 4 samples and undergrowth - one sample.
Sensitivity	and	specificity	for	detection	of	resistance	to	isoniazid	was	100%	and	100%	for	NRA,	100%	and	95%	for	MODS,	
93% and 98% with the Genotype® MTBDRplus, and 93% and 100% with the MGIT 960, respectively. For rifampicin it was
100% and 100% with NRA, 91% and 93% with MODS, 100% and 96% with Genotype MTBDR®plus, and 73% and 100%
with the MGIT 960, respectively.
The average time to results was 2 days (range 1-3 days) for Genotype® MTBDRplus, 8 days for MGIT 960 (range 5-13
days), 8 days for MODS (range 7-18 days) and 11 days for NRA (range 10-21 days). Results obtained within 10 days were
91%, 88%, and 75% for the MODS, MGIT 960, and NRA, respectively.

Findings show excellent performance of the direct NRA, MODS, and Genotype® MTBDRplus for MDR TB detection, with
most of the interpretable results obtained on initial testing in <14 day.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                169

      pEa prODuCTiON by myCObaCTEria aND iTS
      appliCaTiON iN a rapiD Drug SuSCEpTibiliTy TEST.

      McNerney, Ruth 1, Turner, Claire 2, Mallard, Kim 1, O’Sullivan, Denise 1
      1 - London School of Hygiene & Tropical Medicine
      2	-	Cranfield	University

      Metabolites produced during bacterial growth may be used to monitor the impact of drugs on bacteria. We have inves-
      tigated volatile organic compounds emitted by mycobacteria growing on Lowenstein Jensen (LJ). Mass spectrometry
      determined one of the major volatile compounds from M. bovis BCG to be phenylethyl alcohol (PEA), a bacteriostatic
      compound that is a reversible inhibitor of DNA synthesis. PEA production was also observed in mycobacteria other than
      tuberculosis (MOTT).That such a compound is produced in quantity by mycobacteria growing on LJ is surprising and may
      explain the limited growth of mycobacteria on this media. PEA is only produced during bacterial growth and monitoring
      production during exposure may provide a means of determining susceptibility to antimicrobial compounds. To test this
      hypothesis, bacteria were placed on LJ slopes containing drug and incubated at 37C. Headspace vapors from the culture
      tubes were analysed using the zNose, an ultra-rapid capillary gas chromatograph coupled to a SAW detector. The test
      required no sample preparation and each slope was tested in less than 2 minutes.To minimize the risk of creating infected
      aerosols culture tube caps incorporated a PTFE/silicone septum enabling air to be drawn from the tube without remov-
      ing the cap. Samples collected were heat sterilized during testing. Headspace samples from drug containing slopes were
      compared to control slopes without drug. The incubation time required for differentiation between positive and negative
      samples or ‘growth’ and ‘no growth’ depended on the size of the inoculum and the species of mycobacteria and ranged
      from hours to a few days. MOTT could be differentiated from M. tuberculosis complex bacilli by continued production of
      PEA in the presence of 500ug/ml para-nitrobenozic acid. We conclude that detection of volatile metabolites emitted by
      mycobacteria growing on Lowenstein Jensen offers a simple, rapid alternative to assess their drug susceptibility.

170                                                                                                                  ESM 2009

VariOuS STraTEgiES TO DECONTamiNaTE aCiD faST baCilli
pOSiTiVE liQuiD CulTurES frOm baCTEC mgiT 960

Balmoi, Faith
Joint Clinical Research Centre, Kampala, Uganda

Though liquid culture is enriched and more sensitive in the recovery of MTB, it yields a greater percent-
age of contaminants as well. It is essential that MTB cultures be isolated pure to permit drug susceptibil-
ity testing which is vital for prognosis of patients besides determining the presence of MDR-TB and XDR-TB.
In this study we evaluated various approaches to reduce or eliminate contamination in Bactec MGIT 960 cultures.

Contaminated AFB PCR positive MGIT cultures were subjected to decontamination methods of:
i) Double PANTA Polymyxin B (750µg/ml); Amphotericin (75µg/ml); Nalidixixic (300µg/ml); Trimethoprim (75µg/ml) and
Azlocillin (75µg/ml)
ii) VAN vancomycin (30.5µg/ml), Amphotericin (106µg/ml) and Nalidixic acid (226.7µg/ml)
iii) 2% NaOH each contaminated culture was divided and subjected to all three decontaminat-
ing procedures. The decontaminated cultures were then inoculated in MGIT tubes and 7H10 se-
lective whole plates and incubated in Bactec MGIT 960 and a 5%-10% CO2 at 37°C respectively.
The	plates	were	read	weekly	until	a	sufficient	colonial	growth	was	achieved.	The	positive	MGIT	was	subjected	to	Ziehl-
Neelsen (ZN) smear and blood agar culture for purity check.

A total of 50 specimens had analyzable results for each method. Double PANTA was able to recover 62.0%,VAN 60.0%
and 2% NaOH 24.4% while 16.0%, 14.0% and 6.7% respectively remained contaminated. However, 22.0%, 26.0% and
68.9% were non-viable after decontamination with double PANTA, VAN and 2% NaOH respectively. Further testing is
being done so as to generate more reliable results.

   •	 VAN and double PANTA were comparable as far as giving more specimens (p value = 0.614). With pure cultures
   when they are used for decontamination.
   •	 More specimens were unrecoverable when 2% NaOH was used than the other two methods.
   •	 The cost of decontaminating a sample using VAN was ~$1 while double PANTA was ~$5.70

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                           171

      lOW COST iSOlaTiON Of myCObaCTErium TubErCulOSiS (mTb) frOm blOOD

      Orikiriza, Patrick
      Uganda-CASE Research Collaboration, Kampala

      Routinely,	identification	of	MTB	from	body	fluids	requires	a	PCR	based	technique	which	has	been	adopted	as	a	gold	
      standard. This technique however has been unsuccessful against blood cultures which are often affected by inhibitors. In
      this study, we evaluated a simple but effective way of isolating and identifying MTB in blood cultures.

      A total of 91 blood cultures positive for acid fast bacilli were sub-cultured onto 7H10 medium and Lowenstein Jensen
      slants and incubated at 37ºc in a 5% carbon dioxide incubator. The isolates that grew on solid media were further tested
      by	PCR	to	confirm	MTB.	The	original	blood	cultures	were	tested	by	diluting	an	aliquot	of	blood	culture	in	PCR	waters	
      and	repeating	the	IS6110	PCR	and	also	by	Capilia	for	identification	of	MTB.	

      The study found 15 cultures PCR positive when diluted in PCR water and 56 positive with Capilia. On the other hand,
      cultures grown on the solid media before performing PCR yielded 53 PCR positives from 7H10, 44 PCR positives from
      LJ and 28 did not grow on either medium.
      The time it took for the cultures to turn positive on the different media was also noted

      Identification	of	MTB	by	PCR	was	more	efficient	blood	cultures	were	sub	cultured	to	solid	media	for	identification	assay	
      Capilia	tests	performed	directly	from	blood	culture	media	gave	comparable	results	(95%)	with	MTB	identification	by	
      PCR from solid growth.
      There was an overall better turn around time using the 7H10 media compared to LJ medium

172                                                                                                                  ESM 2009

COmpariSON Of rapiD COlOrimETriC mEThOD,
prOpOrTiON mEThOD aND baCTEC460 Tb SySTEm fOr TESTiNg
SuSCEpTibiliTy oF M.TuBerCuloSiS TO rifampiNE aND iSONiaSiDE
                                 . .
Begum KAYAR, Ülkü ORAL ZEYTINLI, Ayse KARACALI, Ayben SOYAL, Togrul Nagiyev, Fatih KÖKSAL
Cukurova University, Medical Faculity

Multidrug-resistant (MDR) M. tuberculosis (MTB) strains founded resistant to at least isoniaside [INH] and rifampin [RIF],
the	two	most	important	first-line	drugs	pose	a	serious	threat	to	the	control	of	tuberculosis	(TB)	and	the	spread	of	these	
strains has become a major public health problem. In this study, the performance of antimycobacterial susceptibility test-
ing	for	the	first	line	drugs	(RIF	and	INH)	with	colorimetric	nitrate	reductase-based	antibiotic	susceptibility	(CONRAS)	
and conventional proportion method depend on solid agar culture (LJ) were compared to BACTEC 460 TB system as
the gold standard.

Of the total of 187 MTB strains, when 24 isolates were found as resistant to RIF and INH by Bactec 460 TB other 163
strains were susceptible for these antibiotics. All of strains were isolated in sputum from patient with pulmonary dis-
ease	and	identified	as	MTB	by	NAP	test	in	Bactec	460	system.	The	CONRAS	test	was	performed	with	0.1	mg/mL	of	
INH	and	1	mg/mL	of	RIF	on	LJ	as	modification	of	standart	methods	described	by	H.Syre	et	al	.	Antibiotic	susceptibility	
testing with proportion method was performed on LJ medium according to the standard protocol laid down by WHO.
Results:	The	sensitivity,	specificity	and	overall	agreement	of	the	CONRAS	test	were	95.83%	(23/24),	99.38%(162/163)	and	
97.60% for RIF and 83.33% (20/24), 99.38% (162/163) and 91.35% for INH, respectively. Results for proportion tests were
found as; 91.66% (22/24), 93.25% (152/163) and 92.45% for RIF and 75% (18/24), 97.54% (159/163) and 86.27% for
INH, respectively by Bactec 460TB system as the gold standart.

interpretation & conclusion
CONRAS test showed good agreement with Bactec 460 (the overall agreement of this test was 94.47%) for each of the
antimicrobial tested. CONRAS test is simple, easy to perform, less expensive, reliable and may be used as a preliminary
screen for susceptibility testing of MTB in particularly for resource-poor countries.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                               173

      EValuaTiON Of ThE mgiT TbC iD TEST VS TWO COmmErCially
      aVailablE rapiD immuNOaSSayS fOr M. TuBerCuloSiS
      COmplEX OrgaNiSm DETECTiON frOm liQuiD aND SOliD CulTurE

      Crews, Virginia 1, Warns, Matthew 1, Pfeltz, Richard 1, Beaty, P. Shawn 1, Rosales, Julie 1, Kopher, Ken 1, Joshi, Sudhaunshu 1,
      Hoosen, Anwar 2, Said, Halima 2
      1 - BD Diagnostic Systems, Sparks, Maryland, United States of America
      2 - Department of Medical Microbiology, Faculty of Health Sciences, University of Pretoria and National Health
          Laboratory Service, Tshwane Academic Division, Pretoria, South Africa

      Chromatographic	lateral	flow	immunoassays	for	the	MPT64	protein	antigen	can	provide	a	rapid	and	cost-effective	meth-
      od to qualitatively detect Mycobacterium tuberculosis complex (Mtbc) organisms from acid-fast bacillus (AFB) positive
      cultures.	The	purpose	of	this	study	was	to	compare	the	sensitivity	and	specificity	of	three	such	devices	with	mycobacteria	
      from liquid and solid media cultures.

      Positive liquid (BACTEC MGIT 960 7 mL tubes) and solid (BBL Löwenstein-Jensen slants) culture media seed-
      ed with mycobacteria provided inocula for side-by-side evaluation of two commercially available tests, Capilia
      TB (“Capilia”, Tauns Laboratories, Numazu, Japan) and SD Bioline TB Ag MPT64 Rapid Test (“Bioline”, Standard
      Diagnostics,	 Inc,	 Kyonggi-do,	 Korea),	 and	 the	 BD	 MGIT	TBc	 Identification	Test	 (“TBc	 ID”,	 BD	 Diagnostic	 Systems,	
      Sparks, MD, USA). The MGIT TBc ID test is currently in clinical trials. Devices were inoculated per manufacturer’s in-
      structions and visually assessed as positive for detection (visible test line) and reagent function (visible control line).
      RESULTS. All three devices detected each of the 20 Mtbc organisms tested (100% sensitivity), which included 15 M.
      tuberculosis	strains.	Specificities	for	the	18	non-tuberculous	mycobacteria	(NTM)	tested	were	as	follows:	100%	for	the	
      TBc ID device, 94% for the Capilia device (due to cross-reactivity with M. marinum), and 94% for the Bioline device (due
      to	cross-reactivity	with	M.	gastri).	Additional	testing	with	the	two	cross-reactive	NTM	species	confirmed	these	results.	
      Sensitivity	and	specificity	results	for	a	given	device	were	identical	between	solid	and	liquid	media.	However,	flow	issues	
      after inoculation from solid medium as well as background discoloration after inoculation from liquid medium occurred
      regularly with the Bioline device.

      The	three	rapid	Mtbc	organism	identification	immunoassay	products	exhibited	very	good	sensitivity.	The	specificity	dem-
      onstrated	by	the	TBc	ID	device	was	also	very	good,	but	specificity	was	lower	for	the	Capilia	and	Bioline	devices	due	to	
      cross-reactivity	with	specific	NTMs.	

174                                                                                                                        ESM 2009

Drug rESiSTaNCE iN myCObaCTErium TubErCulOSiS.

Montoro, Ernesto 1, Milián,Yoslaine 1, Lemus, Dihadenys 1, Echemendía, Miguel 1,
Yzquierdo, Sergio 1, Martin, Anandi 2,Van der Stuyft, Patrick 2, Palomino, Juan Carlos 2
1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba
2 - Institute of Tropical Medicine, Antwerp, Belgium

Tuberculosis still represents a major public health problem; especially in low-resource countries were the burden of the
disease is more important. Standard methods for drug susceptibility testing of Mycobacterium tuberculosis, such as the
proportional method (PM), the absolute concentration method, and the resistance ratio method, are used globally but
depend	on	culture	on	solid	media	and	are	therefore	time-consuming.	The	time	lag	is	a	significant	threat	to	the	patient,	
the community, and health care workers. To reduce this period, we have evaluated the nitrate reductase assay (NRA)
on smear-positive sputa for the direct detection of drugs resistance in Mycobacterium tuberculosis. A total of 63 smear-
positive	sputum	were	used	in	this	study.	The	samples	were	decontaminated	using	the	modified	Petroff	method,	a	por-
tion was used to carried out the NRA as susceptibility test to isoniazid (INH, 0,2 µg/mL), streptomycin (SM, 4 µg/mL),
ethambutol (EMB, 2 µg/mL) and rifampicin (RIF, 40 µg/mL). The resulting sample was used to perform the indirect PM
on Löwenstein-Jensen which was used as gold standard method. The NRA results were obtained between 14 and 28
days. However, were necessary 28 or 42 days to obtain PM results.The sensitivity of MNR to INH, SM, EMB and RIF was
88,9%, 75%, 0% and 71,4% respectively. The low sensitivity obtained for all drug evaluated was due to a small amount of
resistance	strains	founded.	On	the	other	hand,	the	specificity	of	MNR	to	each	drugs	was	96,3%	(INH),	96,1%	(SM),	95,2%	
(EMB) and 98,2% (RIF). In general, the concordance between MNR and PM was 95,2% to INH, 92,1% to SM, 93,7 to EMB
and 95,2% to RMP. In conclusion, the MNR applied in sputum samples is more rapid than any conventional method. Two
drugs	that	define	multidrug-resistance	and	are	the	most	potent	in	the	treatment	of	tuberculosis,	INH	and	RIF,	showed	
the higher values of concordance wit PM. The MNR results are easy to performance, use each drug with identical critical
concentration in the same solid media than PM and could be a useful tool for detection of tuberculosis drug resistance
in low-resource countries with limited laboratory facilities.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                             175

      uSE Of NiCOTiNamiDE iN COlOrimETriC mEThODS fOr rapiD
      DETECTiON Of pyraziNamiDE rESiSTaNCE iN
      myCObaCTErium TubErCulOSiS

      Montoro, Ernesto 1, Lemus, Dihadenys 1, Madruga, Mariela 1, Mirabal, Niuris 1, Milián Yoslaine 1, Yzquierdo, Sergio 1,
      Echemendía, Miguel 1, Martín, Anandi 2,Van der Stuyft, Patrick 2, Palomino, Juan Carlos 2
      1 - Institute of Tropical Medicine Pedro Kourí (IPK), Havana, Cuba
      2 - Institute of Tropical Medicine, Antwerp, Belgium

      Pyrazinamide (PZA) is one of the most effective anti-tuberculosis drugs. It is also bactericidal to semidormant
      Mycobacterium tuberculosis and it reduces the total treatment time. The current susceptibility testing methods for this
      drug	are	difficult	due to the poor growth of the bacteria in acid medium which is required for drug activity. One alterna-
      tive has been the use of nicotinamide (NIC), an analogue of PZA that can be converted by PZAase into active form in
      a physiological pH that does not hinder bacterial growth. Recently, the NIC has been applied successfully in inexpensive
      susceptibility testing such as nitrate reductase assay (NRA) for rapid detection of PZA resistance. The purpose of this
      study was to develop the NRA and malachite green indicator (MGI), both with NIC, as rapid susceptibility testing to PZA
      in M. tuberculosis. The NRA and MGI were carried out on 120 M. tuberculosis strains from the collection at Tuberculosis
      National Reference Laboratory. The concentration of NIC applied in both methods was 1 000 µg/mL and all results
      were compared with Wayne method which was used as gold standard, employing 100 µg/mL of PZA.The Wayne method
      results were obtained in 4-7 days whereas MGI and NRA results required 7-14 days. A total of 17 and 85 strains were
      reported as resistant and susceptible respectively by the three methods but MGI had 16 discordant results and NRA only
      4 discrepancies.The MGM showed a	sensitivity	and	specificity	of	80,95%	and	87,88%	respectively	whereas	NRA	provided	
      a	sensitivity	of	90,48%	and	specificity	of	97,98%.	In	general,	the	concordance	of	MGI	and	NRA	were	86,67%	and	96,67%	
      respectively.The MGI employing NIC showed a low sensitivity to detect resistance to PZA. In contrast, the NRA showed
      a high concordance with Wayne method.This assay is rapid, accurate and could be an attractive option for rapid detection
      of PZA resistance, especially in limited-resource countries with high levels of resistance.

176                                                                                                                  ESM 2009

implEmENTaTiON Of liQuiD CulTurE fOr TubErCulOSiS

Pamela Hepple1, Jonathan Novoa-Cain2, Chris Cheruiyot2, Elvira Richter3, Koert Ritmeijer4
1 - Manson Unit, Médecins sans Frontières, London, UK
2 - Médecins sans Frontières OCA South Sudan, Lokichoggio, Kenya
3 - Forschungszentrum Borstel, Nationales Referenzzentrum für Mykobakterien, D-23845 Borstel, Germany
4 - Médecins sans Frontières OCA, Amsterdam, the Netherlands

The diagnosis of tuberculosis (TB) in Médecins sans Frontières (MSF) projects is based on sputum smear micros-
copy, which has low sensitivity. Following WHO recommendations, MSF established a TB liquid culture laboratory in
Lokichoggio, Kenya, processing samples from 4 South Sudan projects, for diagnosis of smear-negative and extra-pulmo-
nary (EP) TB and follow-up of patients.

The manual MGIT (mycobacterial growth indicator tube, Becton Dickinson) system was used with Lowenstein-Jensen
media. One positive culture per patient was sent to Borstel Supranational Reference Laboratory, Germany for speciation
using the Hain Genotype Mycobacteria series and sequencing techniques.
From March 2007 to December 2008, sputum culture was performed for 64 diagnostic and 24 follow-up patients. Ten
EP samples were also cultured.
For diagnostic patients, of two smear-positives, one was culture-positive for both Mycobacterium tuberculosis (MTB) and
M. fortuitum, and one for M. fortuitum.
Eight of 62 (13%) smear-negatives were culture-positive for MTB complex (3 for MTB and 5 for MTBC) with nine (14%)
culture-positive	for	other	identified	mycobacteria;	six	(9%)	grew	unknown,	not	validly-described	mycobacteria,	and	39	
(61%) were culture negative.
For follow-up patients, of seven smear-positives, one was culture-positive for MTB, one for M. intracellulare and one for
M. fortuitum complex, and four (57%) were culture-negative. Among the smear-negatives, eight of 17 (47%) were culture-
positive, four of which were unknown mycobacterial species, and four non-tuberculous mycobacteria (NTM). Nine (53%)
were culture-negative.
Samples	from	three	EP	patients	of	10	grew	mycobacteria,	species	unidentifiable.	
In	total,	only	10	of	36	(28%)	culture-positive	patients	grew	mycobacteria	from	sputum	which	could	be	identified	as	MTB	or	MTBC.	

lessons learned
Due	to	the	long	turn-around	time	between	sample	production	and	species	identification	due	to	shipment	issues	(ap-
proximately 4-6 weeks), clinicians often did not wait for results before initiating or adjusting therapy.The high proportion
of	NTM	was	difficult	to	interpret.	The	culture	results	had	little	clinical	impact,	and	culture	lab	activities	were	suspended	
in February 2009.

Future TB culture programmes require facilities for on-site speciation of mycobacteria or, if working with reference labs,
should minimize turn-around time to results by ensuring timely shipment of samples.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                     177

      rapiD DETECTiON Of rESpiraTOry aCTiVE
      myCObaCTEria by auramiNE O-CTC DOublE STaiNiNg

      Ichijo, Tomoaki; Izumi,Yoko;Yamaguchi, Nobuyasu; Nasu, Masao
      Osaka University

      Clarifying the dynamics of nontuberculosis mycobacterial (NTM) and determining their hot spots in aquatic environ-
      ments are important to prevent their infection to human beings. For this purpose, methods for the detection of active
      mycobacterial cells are required. Culture methods are widely used for detection of mycobacterial cells, but these meth-
      ods	are	rather	difficult	to	detect	mycobacteria	quantitatively	and	rapidly.	Mycobacterial	cells	are	quantifiable	under	a	
      fluorescent	microscope	within	several	hours	with	acid-fast	staining.	In	addition,	several	fluorochromes	are	available	to	
      determine the bacterial activities. In this study, we attempted to detect mycobacteria with physiological activity rapidly
      by combining Auramine O acid-fast staining with 5-cyano-2, 3-ditoryl tetrazolium (CTC) as an indicator of respiratory
      activity (Auramine O-CTC).

      Mycobacterium	smegmatis	was	stained	with	CTC	and	fixed	with	formaldehyde.	Fixed	cells	were	stained	with	Auramine	
      O	and	observed	under	an	epifluorescent	microscope	with	blue	excitation.

      results and Discussion
      We	firstly	optimized	the	condition	of	the	Auramine	O-CTC	double	staining	method	to	address	the	following	problems;	
      (i)	CTC	formazan	dissolved	in	the	decolorization	step	and	(ii)	fluorescent	intensity	of	Auramine	O	was	decreased	by	
      fluorescent	resonant	energy	transfer	(FRET).	Specificity	of	this	method	was	confirmed	by	staining	non-targeted	bacteria.	
      Results can be obtained within 90 min by the optimized procedure, while more than one week is required for the cul-
      ture-dependent approach. In conclusion, the Auramine O-CTC double staining method was useful for rapid detection of
      respiratory active mycobacteria. This method may contribute to identifying dynamics of NTM in aquatic environments.

      This	work	was	supported	by	the	JSPS	Grant-in-Aid	for	Scientific	Research	(A)(20249007).	

178                                                                                                                  ESM 2009

SyNErgiSTiC aCTiViTy Of TWO aNTiTubErCulOuS
Drug COmbiNaTiONS agaiNST CliNiCal iSOlaTES Of
MyCoBaCTeriuM TuBerCuloSiS rESiSTaNT TO iSONiaziD.

Emma Rey,	Griselda	Tudó	and	Julià	González-Martín
Servei de Microbiologia. Hospital Clínic-IDIBAPS. Dept. Microbiologia i Parasitologia Sanitaria, Universitat de Barcelona.
Spanish Network for the Research in Infectius Diseases (REIPI, RD06/0008).

To	determine	the	synergistic	activity	of	2	drug	combinations:	isoniazid	(H)+rifampicin	(R)+ethambutol	(E)	and	ofloxacin	
(O)+R against M. tuberculosis clinical isolates resistant to H versus drug susceptible isolates.

Individual MICs of the strains were studied. Both combinations were studied crossing 7 concentrations of each antibiotic
(including their MIC). The inoculum was of 104CFU/ml. All cultures were performed with the proportional method in
Middlebrook 7H11 medium and incubated 4 weeks. On analysing the results of the combinations, the fractional inhibitory
concentration (FIC) was interpreted: FIC≤0.5,	synergistic	activity;	FIC	from	1-4	indifference	and	FIC>4	antagonistic	activity.

H+R+E Combination: 11 H resistant isolates were studied: 4 (36%) isolates had MIC=52µg/ml and 7 (64%) had MIC=0.8µg/
ml. 9 drug-susceptible isolates were also studied. Among the 20 isolates the individual MIC for R ranged from 1-2 µg/ml,
from 2.5-5 µg/ml for E, being 0.05µg/ml for H in susceptible isolates. In H-resistant isolates, the MIC in combination for
H and R decreased up to 3 dilutions (average) respect to their individual MIC. A lesser decrease (2-3 dilutions) was ob-
served for E. In H-resistant isolates, 9/11 (81.1%) showed synergistic activity while 2/11 (18.18%) of the resistant isolates
indicated indifference (FIC index =0.7). MICs in combination of susceptible strains decreased an average of 2 dilutions
compared to their individual MIC. The susceptible strains had FIC indices ≥ 0.748. O+R Combination: 21 isolates were
studied:11 had MIC≤1µg/ml,	4		MIC=1-6.4	µg/ml	and	6	MIC>6.4.	9	drug-susceptible	isolates	were	studied.	Individual	MICs	
for R ranged from 1-2, with 1 for O. MICs in combination of all strains were the same or decreased up to 1 dilution with
regard to their individual MIC. The combination of OR in 21 strains did not show synergism or antagonism in either the
resistant or the susceptible strains being the FIC values mainly 1.5.

1)These results suggest that in strains resistant to H with an MIC of 0.8µg/ml in vitro, the standard antibiotic regimen, in-
cluding H, would be effective due to the possible compensatory effects of R and E. 2) Although strains resistant to H with
an MIC of 51.2 µg/ml showed synergism, these strains would be within the range of resistance. 3)The combination of HRE
against susceptible strains and that of OR did not show synergism probably because of high individual bactericide action.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                   179

      TObramyCiN-ClariThrOmyCiN COmbiNaTiON ON
      MyCoBaCTeriuM TuBerCuloSiS CliNiCal iSOlaTES

      Karolien Stoffels1, Hamidou Traore2, Raymond Van Hoof3 and Maryse Fauville-Dufaux1
      1	-	National	Reference	Laboratory	of	Tuberculosis	and	Mycobacteria,	Scientific	Institute	of	Public	Health,	Department		
          Institut Pasteur, Rue Engeland 642, 1180 Brussels, Belgium
      2 - Laboratoires SMB, Rue de La Pastorale 26-28, 1080 Brussels, Belgium
      3	-	National	Reference	Laboratory	of	Resistance	to	Aminoglycosides,	Scientific	Institute	of	Public	Health,	Department		
          Institut Pasteur, Rue Engeland 642, 1180 Brussels, Belgium

      This study investigated the in vitro susceptibility of 25 Mycobacterium tuberculosis clinical isolates to two well-known drugs,
      tobramycin (TM) and clarithromycin (CL).The effect of both drugs administered together was also investigated to detect
      a possible synergistic effect.

      MIC	of	isolates	with	variable	resistance	profiles was	determined	by	the	radiometric	BACTEC	460	TB	and	was	defined	as	
      the minimal antibiotic concentration for which at least 99% of the mycobacterial population growth was inhibited. The
      influence	of	TM	on	the	MIC	of	CL	was	interpreted	by	Fractional	Inhibitory	Concentration	(FIC).	

      The median MIC for both TM and CL was 8 µg/ml (range: 2 to 8 µg/ml and ≤2	to	>16	µg/ml	respectively).	For	36%	(9/25)	
      of the tested isolates a decrease of the MIC of CL by a single or twofold dilution was observed (FIC ≤ 0,5) when a sub-
      inhibitory	concentration	of	TM	was	added.	No	antagonistic	effect	(FIC	>	4)	was	observed.	Similar	results	were	observed	
      for	isolates	susceptible	or	resistant	to	first-line	antituberculosis	drugs.

      Although the MICs for CL and TM seem high compared to conventional anti-tuberculosis drugs, these antibiotics should
      not	immediately	be	ruled	out	as	clinically	insignificant.	Indeed,	after	administration	of	300mg	aerosolised	TM,	antibiotic	con-
      centrations	in	the	epithelial	lining	fluid	are	higher	than	10	times	the	median	MIC	observed	for	the	TB	isolates	tested	in	this	
      study (8µg/ml). Oral administration of 500 mg CL twice daily leads to a peak concentration of 13,50 +/- 3,3 µg/g in the lungs,
      505	±	293	µg/ml	in	alveolar	cells	and	34	±	5	µg/ml	in	epithelial	lining	fluid,	thus	all	exceeding	the	MIC	of	8	µg/ml.
      Promising new drug delivery systems such as the Dry Powder Inhaler allow achieving very high local antibiotic concen-
      tration, e.g. 7 times higher for TM compared to aerosol administration and thus far beyond the MIC of resistant isolates.
      These	results	suggest	that	both	drugs	should	be	investigated	further	as	potential	adjuncts	to	the	difficult	treatment	of	
      multi-drug resistant tuberculosis where other alternatives have failed.

180                                                                                                                       ESM 2009

prEValENCE Of EffluX-mEDiaTED rifampiCiN rESiSTaNCE
iN MyCoBaCTeriuM TuBerCuloSiS CliNiCal iSOlaTES

Carrie K. W. Au-Yeang, T. K. Au, Edward W. C. Chan, Raphael C.Y. Chan
Department of Microbiology,The Chinese University of Hong Kong,The Prince of Wales Hospital, Shatin, New Territories, Hong Kong

Rifampicin is one major ingredient in the cocktail-regimen used in treatment of Mycobacterium tuberculosis (MTB) infec-
tion. Although mutations in the rpoB	gene	are	considered	the	basis	of	rifampicin	resistance,	we	noted	that	a	significant	
proportion	of	local	resistant	cases	could	not	be	attributed	to	mutations.		Alternative	mechanisms	such	as	drug	efflux	
have been proposed.		In	order	to	evaluate	the	role	of	drug	efflux	mechanisms	in	mediating	rifampicin	resistance	in	MTB,	
we	examined	the	prevalence	of	efflux	activities	in	rifampicin	resistant	isolates	using	three	efflux	inhibitors:	reserpine,	
carbonyl cyanide chlorophenylhydrazone and verapamil. Forty-two rifampicin resistant and nine drug susceptible MTB
clinical isolates were studied. The minimum inhibitory concentration (MIC) values for rifampicin were determined in the
presence	and	absence	of	efflux	inhibitors.		The	magnitude	of	MIC	reduction	for	each	efflux	inhibitor	and	the	prevalence	
of	efflux-mediated	resistance	were	examined.		We	found	that	among	the	three	efflux	inhibitors	tested,	significant	MIC	
reduction, ≥ 2-fold MIC decrease, was observed only for verapamil. 61% (31/51) of the test isolates had an MIC reduc-
tion	between	2	to	8-fold	in	the	presence	of	verapamil.		This	verapamil-sensitive	efflux-mediated	MIC	reduction	effect	
was much more apparent in rifampicin resistant isolates than the drug susceptible controls: 71% (30/42) versus 11%
(1/9). Likewise, this phenomenon was more prevalent in isolates resistant to 3 - 5 anti-tuberculosis drugs than isolates
resistant	to	1	-	2	drugs:	77%	(24/31)	versus	55%	(6/11).		This	data	support	the	notion	that	drug	efflux	systems	contribute	
significantly	to	rifampicin	resistance	in	MTB	clinical	isolates	and	highlight	the	need	for	determining	the	extent	by	which	
these systems contribute to resistance to other anti-tuberculosis drugs.
This study is supported by a grant (08070292) from Research Fund for the Control of Infectious Diseases.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                    181

      iNTra aND EXTraCEllular aCTiViTy Of ruThENium COmplEXES
      agaiNST MyCoBaCTeriuM TuBerCuloSiS aND ThEir CyTOTOXiCiTy

      Leite, Clarice Queico Fujimura 1, Pavan, Fernando Rogério 1, Maia, Pedro I da S 2,	Deflon,	Victor	M	2, Sato, Daisy Nakamura 3,
      Azevedo, Alzir A 4, Poelhsitz, Gustavo V 4, Leite, Sérgio Roberto Andrade 1, Franzblau, Scott G 5
      1 - São Paulo State University
      2 - University of São Paulo
      3 - Adolfo Lutz Institute
      4 - Federal University of São Carlos
      5 - University of Illinois

      Worldwide, tuberculosis (TB) remains the most frequent and important infectious disease causing morbidity and death.
      One-third of the world’s population is infected with Mycobacterium tuberculosis (MTB), the etiologic agent of TB. The
      World Health Organization estimates that about eight million new TB cases occur annually. The pulmonary TB (most
      common form of TB), is highly contagious and has been increasing in incidence in many areas, not only in developing coun-
      tries but also in industrialized countries. The global resurgence of TB and the rapid emergence of MDR-TB, underscore
      the importance of the development of new antituberculous drugs.

      Objecting	to	find	new	compounds	with	high	activity	against	TB,	we	determined	the	cytotoxicity,	and	intra/extracellular	
      anti-M. tuberculosis activity of 29 new compounds involving different class of ligands such as diimines, phosphines and
      Schiff bases with ruthenium.

      material and methods
      As analytical methods, three standardized techniques were used: 1- in vitro Microplate Rezarurin Assay (REMA) for de-
      tection of minimal concentration of compounds necessary to inhibit 90% of bacillary growth (PALOMINO et al., 2002),
      2- Cytotoxicity (IC50) of compounds against mamarian cells (AHMED et al., 1994) and 3- Determination of compounds
      activity against M. tuberculosis internalized in a macrophage cells (SNEWIN et al., 1999).

      results and Conclusion
      From	the	29	compounds	analyzed,	7	of	them	containing	the	ruthenium,	were	qualified	as	promising	anti-TB	agents.	These	
      complexes presented inhibitory activity ranging of 0.25 – 3.9 µg/mL, whose values are better than some drugs commonly
      used	in	the	TB	treatment,	low	cytotoxicity	(IS	>	10)	and	intracellular	inhibitory	activity	high	than	70%.	

182                                                                                                                       ESM 2009

aNTi-myCObaCTErium TubErCulOSiS aCTiViTy Of
ThiOSEmiCarbazONES, SEmiCarbazONES aND hyDrazONES

Leite; Sergio 1, Pavan; Fernando 2, Maia; Pedro 3,	Deflon;	Victor	3, Batista; Alzir 4, Sato; Daisy 5, Franzblau; Scott 6, Leite; Clarice 2
1 - Universidade Estadual Paulista, Instituto de Química
2 - Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas
3 - Universidade de São Paulo, Instituto de Química de São Carlos
4 - Universidade Federal de São Carlos, Departamento de Química
5 - Instituto Adolfo Lutz, Unidade de Ribeirão Preto
6 - University of Illinois at Chicago, College of Pharmacy

The aim of this study was to identify a candidate drug for anti-tuberculosis therapy development from previously synthe-
sized thiosemicarbazones, semicarbazones and hydrazones, comprising a total of 17 compounds. The Minimal Inhibitory
Concentration (MIC) of these compounds, determined by the resazurin reduction method, was investigated in order to
determine their in vitro antimycobacterial activity against Mycobacterium tuberculosis. In vitro cytotoxicity values (IC50)
of the same compounds were determined on J774 cells to establish a selectivity index (SI = IC50/MIC). Lower values of
MIC were found for four thiosemicarbazones and four hydrazones, namely: 2-acetylpyridine N4 (etil) thiosemicarbazone;
2-acetylpyridine N4 (cyclohexyl) thiosemicarbazone; di-2-pyridyl ketone N4 (phenyl) thiosemicarbazone; 2-acetylpyridine
morpholyl thiosemicarbazone; mono benzoylacetone isonicotinoyl hydrazone; mono-acetylacetone isonicotinoyl hydra-
zone; di-2-pyridyl ketone isonicotinoyl hidrazone; di-2-pyridyl ketone thiophene hidrazone (MIC values ranging from 0.78
to 6.25 µg/mL). All the compounds presented very low cytotoxicity, with the exception of 2-acetylpyridine morpholyl
thiosemicarbazone (IC50 ≤ 3.9 µg/mL and SI ≤ 5).The results obtained with the other 7 compounds (SI ranging from 100
to	800)	qualify	them	as	candidates	for	anti-TB	drugs,	once	their	in	vitro	results	are	comparable	to	some	of	“first	line”	and	
“second line” drugs commonly used in the TB treatment.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                               183

      mEThODS fOr aSSESSmENT Of EThiDium brOmiDE
      TraNSpOrT aCrOSS MyCoBaCTeriuM SMegMaTiS CEll Wall

      Jorge Ramos1*, Liliana Rodrigues1,2, Isabel Couto1,3, Leonard Amaral1,2 and Miguel Viveiros1
      1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisboa, Portugal
      2 - UPMM, IHMT/UNL, Lisboa, Portugal
      3 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal
      *Correspondig author: Jorge Ramos, Unit of Mycobacteriology, Instituto Higiene e Medicina Tropical, Universidade Nova
      de Lisboa (IHMT/UNL), Rua da Junqueira 96, 1349-008 Lisboa, Portugal.
      Tel:	+351	21	3652600;	Fax:	+351	21	3632105;	E-mail:

      Active	efflux	systems	and	reduced	cell	wall	permeability	are	considered	to	be	the	main	causes	of	mycobacterial	intrinsic	
      resistance	to	many	antimicrobials.	Although	several	mycobacterial	efflux	pumps	have	already	been	described,	their	role	in	
      drug	resistance	is	not	yet	fully	understood.	Recent	studies	showed	that	both	LfrA	and	MspA,	the	main	efflux	pump	and	
      porin in M. smegmatis, respectively, are involved in reduced susceptibility to several antimicrobials.
      We have compared the M. smegmatis wild-type strain mc2155 with LfrA and MspA M. smegmatis deleted mutants, for
      their ability to extrude ethidium bromide (EtBr),	 a	 known	 efflux	 pump	 substrate,	 under	 different	 energy	 conditions	
      and	in	the	presence	or	absence	of	efflux	pumps	inhibitors	(EPIs),	by	(i)	a	96	well	microplate	screening	assay	with	the	
      mycobacterial cells grown in Middlebrook 7H9 with 10% of OADC in presence of increasing concentrations of EtBr
      and different concentrations of the EPIs and the plates examined with a UV transilluminator and photographed after
      24 hours of incubation; and (ii)	a	semi-automated	fluorimetric	method	that	detects	efflux	on	a	real	time	basis	during	a	
      period of 30 minutes.The EPIs employed were chlorpromazine, thioridazine, carbonyl cyanide m-chlorophenylhydrazone
      and verapamil.
      The	efflux	activity	detected	for	each	strain	by	these	two	methods	was	then	correlated	with	resistance	to	several	anti-
      biotics (ATBs), by the determination of their minimal inhibitory concentrations in the presence or absence of the EPIs.
      The	ATBs	tested	were	streptomycin,	isoniazid	(INH),	rifampicin	(RIF),	ethambutol	(ETB),	amikacin,	ciprofloxacin	(CIP)	
      and clarithromycin (CLR).
      In the absence of the major porin of M. smegmatis, MspA, it was observed that accumulation of EtBr decreased and the
      cells	became	more	resistant	to	several	ATBs.	On	the	other	hand,	the	mutant	for	the	major	efflux	pump	LfrA	showed	
      increased accumulation of EtBr. This strain also presented increased susceptibility to EtBr, INH, RIF, ETB, CIP and CLR.
      These results show that MspA is an important channel for entrance of quaternary ammonium compounds and ATBs and
      that the pump LfrA is involved in low-level resistance to several ATBs and quaternary ammonium compounds in M. smegmatis.

184                                                                                                                         ESM 2009

ThE humaN maCrOphagE aS a mODEl TO SElECT
COmpOuNDS aCTiVE agaiNST mDr/XDr-Tb

Marta Martins1,2,*, Miguel Viveiros1,3, Isabel Couto1,4 and Leonard Amaral1,2,3.
1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisbon, Portugal
2 - UPMM, IHMT/UNL, Lisbon, Portugal
4 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal
* Corresponding author: Unit of Mycobacteriology and UPMM, Instituto de Higiene e Medicina Tropical, Universidade
Nova de Lisboa (IHMT/UNL), Rua da Junqueira, 96, 1349-008, Lisbon, Portugal;Telf: +351213652600; Fax: +351213632105;

The emergence of Multi- and Extensively-Drug Resistant Mycobacterium tuberculosis (MDR/XDR-TB) represents a major
threat to public health worldwide. Both infections result in high mortality, especially if the patient is co-infected with
HIV. The selection of therapy for these multi-drug resistant infections is limited, and for most situations, ineffective as
many of these strains are untreatable with the available drugs. Thus, there is an urgent need to design and develop new
compounds against drug resistant M. tuberculosis that are effective within the main target of this infection, the human
macrophage.	We	have	recently	demonstrated	that	efflux	pumps	inhibitors	are	active against mycobacteria, by enhancing
the killing activity of the human macrophage and may represent an alternative to the conventional antibiotherapy for the
treatment of the MDR/XDR-TB infections.
From previous studies we have demonstrated that thioridazine (TZ) enhances the killing of MDR-TB phagocytosed by
human macrophages. However, the mechanism of action of TZ on these cells is not fully understood. We have studied
the activity of TZ, several of its derivatives, organosilicon (SILA) compounds and other known inhibitors of K+ and Ca2+
transport (ouabain, reserpine and verapamil) on macrophages infected with MDR-TB and XDR-TB. After phagocytosis,
the compounds were added to the macrophage cultures. Following incubation cells were lysed and the intracellular bac-
terial concentration determined. Our results demonstrate that TZ, three of its derivatives and one SILA compound (SILA
421) enhanced substantially the macrophage killing activity.
The killing activity of neutrophils is correlated with the K+ availability, which is dependent upon transport processes
affected by agents that inhibit Ca2+-activated K+ pumps. Based on this and on our results, we postulate that the enhance-
ment of the macrophage killing activity by these compounds could be due to the inhibition of Ca2+ and K+ transport that
promotes the activation of hydrolases and the killing of intracellular bacteria. A model describing the sequence of events
that lead to the killing of intracellular bacteria will be presented. Moreover, the ex-vivo testing of compounds using pa-
tient’s own macrophages in the clinical TB laboratory might allow screening the most effective compounds against MDR/
XDR-TB providing the basis for the intelligent selection of drugs to be used in the therapy of these infections.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                     185

      in viTro aCTiViTiES Of JpC 2067 alONE aND iN
      COmbiNaTiON WiTh SmX agaiNST NOCarDia SpECiES

      Michael Cynamon, Swagatam Mookherjee, Carolyn Shoen
      Veterans Affairs Medical Center, Syracuse, New York, USA
      E-mail:		FAX:	315-425-4871

      JPC 2056, a biguanide prodrug of JPC 2067, a dihydrotriazine DHFR inhibitor, is being developed as an antimalarial thera-
      peutic. Previously we demonstrated that earlier compounds related to JPC 2067 had promising activities in vitro alone
      and in combination with sulfamethoxazole (SMX) against nocardia species. JPC 2067 is active against M. tuberculosis, M.
      kansasii, and M. marinum at ≤ 1µg/ml. The purpose of the present study was to evaluate the in vitro activities of JPC 2067
      alone and in combination SMX against a group of clinical nocardia isolates.

      JPC 2067 was provided by Jacobus Pharmaceutical Co., Princeton, NJ. SMX was purchased from Sigma Chemical Co, St.
      Louis,	MO.	Each	drug	was	dissolved	in	DMSO	at	a	final	concentration	of	1	mg/ml.	Aliquots	were	frozen	at	-200C. Drugs
      were	thawed	prior	to	testing	and	diluted	in	modified	7H10	broth	(pH	6.6;	7H10	agar	formulation	with	agar	and	malachite	
      green omitted) with 10% OADC enrichment and 0.05% Tween 80. JPC 2067 and SMX were tested alone from 64µg/ml
      –	0.06µg/ml.	When	tested	together	SMX	was	evaluated	at	fixed	concentrations	of	1	µg/ml	and	JPC	at	16µg/ml	–	0.015µg/
      ml. Twenty eight nocardia isolates (from the ATCC and clinical isolates provided by B. Body and B. Forbes) were used in
      the study. An in vitro	broth	dilution	method	similar	to	that	defined	by	CLSI	was	utilized.

      The MIC50 and MIC90	for	JPC	2067,	SMX	and	the	combination	(SMX	fixed	at	1µg/ml)	were	0.125	µg/ml	and	4	µg/ml,	16	
      µg/ml and 32 µg/ml, and 0.03 µg/ml and 2 µg/ml respectively.

      JPC 2067 was more active than the previously tested dihydrotriazine analogs against nocardia. The addition of SMX had a
      modest but consistent effect on lowering the JPC 2067 MIC. It is likely that this effect would be more pronounced if the
      concentration of SMX was increased to perhaps10 µg/ml (a readily achieved serum level for this agent). JPC 2067 alone
      and in combination should be evaluated in animal models of both nocardial and mycobacterial infection to understand
      the clinical potential of these agents.

186                                                                                                                   ESM 2009

NOSOCOmial Tb iN a labOraTOry SETTiNg

Jaime M S Nina1,2,3
1 - Instituto Nacional de Saúde Doutor Ricardo Jorge
2 - Universidade Nova de Lisboa
3 - Hospital Egas Moniz

TB is recognized as a major cause of morbidity and mortality worldwide. Its easily transmissibility is also generally rec-
ognized, both at the family level, in the household, at the place of work and inside health care facilities. This last way of
transmission, properly called nosocomial transmission, has been suspected for long time, and was formally demonstrated
in the ward, both among patients, and health care workers. Several professional bodies and other institutions, both at
the national and international level, produced guidelines trying to minimize TB transmission to health care workers in-
side wards and emergency services. Furthermore several countries produced legislation to protect health care workers
against nosocomial TB, and/or included TB in the list of professional diseases or hazards to health care workers.
However, much less attention has been given to the TB transmission potential to laboratory workers. Even if several
countries moved laboratory work with live TB samples to LSB-3 facilities, the evidence on which to base this decision is
thin, and no systematic study has been published.
Herein are presented three cases of nosocomial transmission inside laboratory settings, in Lisbon. These cases cover all
spectra of professional differentiation, from basic level auxiliary personnel, to a laboratory technician, and to a micro-
biologist physician. In one case the way of transmission was a very common kind of laboratory accident, in another a
common	inattention,	and	in	the	last	one	no	specific	way	of	transmission	was	found.	One	of	the	cases	was	found	to	be	a	
In conclusion, the trend to carry out all routine work with live TB samples only inside a BSL-3 facility seems a right one,
and	so	it	is	fully	justified.	Also	justified	would	be	the	inclusion	of	health	laboratory	workers	in	the	legislation	that	provide	
safety measures and insurance cover to health care workers.

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                      187
auThOr iNDEX

 Abadia E                 Beltrán M               Cubillos A               Furlaneto IP                  Jahromi NS
 Abreu C                  Biet F                  Cvetnic Z                Furtado C                     Jansone I
 Afonso A                 Boehme C                Cynamon M                Gagneux S                     Janssen HG
 Aguilar D                Boeree M                Dafae F                  Gaile I                       João I
 Ahmadi M                 Bonte L                 David S                  Gama JB                       Joloba M
 Ahmed A                  Borile C                De Bock A                Gao Q                         Joloba ML
 Ahmed I                  Borroni E               De Cruz K                Garcia MJ                     Jordão L
 Åkerström M              Boschiroli ML           De Gispert FX            García-Cañas A                Joshi S
 Akol J                   Bourdon E               De Haas P                Gavin P                       Julián E
 Akpaka PE                Braga JE                De la Hoz F              Gegia M                       Julián EG
 Al Balushi L             BragaR                  De Sousa MS              George AG                     Jureen P
 Al Busaidi S             Brankova N              Deflon	V                 Gharbia S                     Kaal E
 Al- Mahruqi S            Brisse Smangenot S      Deflon	VM                Giampaglia CS                 Kahlmeter G
 Albarral MIP             Brosch R                Dekhuijzen R             Gicquel B                     Kam KM
 Alcaide F                Brown T                 Del Portillo P           Gil MJ                        Kanavaki S
 Aldashev A               Brum L                  Del Val-Romero B         Gilpin CM                     Kang HS
 Algamdi S                Brzostek A              Dementieva A             Giske C                       Kantor I
 Al-Hajoj S               Bwanga F                Den Hertog A             Gitti Z                       Kapata N
 Ali AB                   Cabral J                Di Giulio B              Goguet de la Salmonière YOL   Karabela S
 Ali MS                   Cacho J                 Dias A                   Goldfeder LC                  Karacali A
 Ali Veleyati A           Caldas C                Diogo J                  Golec M                       Kargar M
 Allix-Béguec C           Cambau E                Diwan V                  Gomes H                       Karoui C
 Al-Maniri AA             Cañete C                Docx S                   Gomes HM                      Katalinic-Jankovic V
 Almeida da Silva P       Cano I                  Drobniewski F            Gomes MH                      Katalinic-Jankovic V
 Almeida EA               Cardoso N               DuarteR                  Gonçalves H                   Kayar B
 Al-Omari R               Cardoso RF              Duvnjak S                González Torralba A           Kayar Mb
 Alonso C                 Cardoso S               Dziadek B                González-Martín J             Kazempour M
 Al-Rawas O               Carmona JA              Dziadek J                Grce M                        Kern WV
 Alves A                  Carvalho C              Echemendía M             Greib C                       Khan AJ
 Amado J,                 Carvalho F              Ehricht R                Guillard B                    Khechinashvili G
 Amaral L                 Carvalho MA             Eisenach K               Gutiérrez C                   Kim JH
 Amondi Ouma N            Carvalho T              Elmoula IF               Gutierrez J                   Klatser P
 Amorim A                 Casal M                 Etwom A                  Gutierrez MC                  Koeleman M
 Andersson E              Cassone A               Eum SY                   Gutierrez-Aroca JB            Köksal F
 Ängeby K                 Castro AG               Fajfar N                 Hadadi M                      Kolk A
 Anoosheh S               Causse M                Falkinham, III, JO       Haenn S                       Konstantinidou E
 Anthony R                Chan Chiu Y             Farnia P                 Haile M                       Kontos F
 Arnold C                 Chan CYR                Fattorini L              Haroun RZ                     Kopher K
 Au TK                    Chan EWC                Fauville-Dufaux M        Havelkova M                   Kosmadakis G
 Au-Yeang CKW             Chan RCY                Felix C                  Hepple P                      Kritski A
 Awad C                   Chan WCE                Ferme D                  Herbawi M                     Krt B
 Ayles H                  Chau CH                 Fernandes P              Hernández J                   Kuan HO
 Azevedo AA               Cheruiyot C             Fernandes SJ             Hernández-Pando R             Kubin M
 Baboolal S               Cho E-J                 Ferreira A               Hillemann D                   Kuijper S
 Babu Okatch F            Cho SN                  Ferreira C               Hirata MH                     Labarre M
 Bachiiska E              Chryssanthou E          Ferreira D               Hirata RDC                    Lai WMR
 Balacó I                 Cirillo D               Ferreira S               Hoffner S                     Langerak E
 Balbin JA                Cirillo DM              Ferro RS                 Homolka S                     Lazaro E
 Baldan R                 Clivillé R              Feuerriegel S            Honisch C                     Leão SC
 Balmoi F                 Cochard T               Fey F                    Honscha G                     Lee H
 Barbe V                  Codina G                Figueira R               Hoosen A                      Lee J
 Baritaki M               Coelho R                Filippini P              HubansC                       Lee J-I
 Baritaki S               Coll P                  Fissette K               Hwang SH                      Leimane V
 Barrera L                Conceição EC            Fiuza de Melo F          Ibrahim TA                    Leite C
 Barry III CE             Correa N                Frade R                  Ichijo T                      Leite CQF
 Bartu V                  Correia-Neves M         Fraga AG                 Imperiale B                   Leite S
 Batista A                Costa ARF               Frangopoulos F           Ingham C                      Leite SRA
 Baumanis V               Couto I                 Franz S                  Ioannidis P                   Leite SRA
 Bauskenieks M            Couto S                 Franzblau S              Isakova J                     Lemos S
 Bazigos S                Crews V                 Franzblau SG             Ivanov A                      Lemus D
 Beaty PS                 Cruz A                  Fung SL                  Izumi Y                       Levina K

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal                                  189
      Levterova V           Monecke S         Pellegrin JL            Sancho L              Van der Stuyft P
      Lezcano MA            Monge I           Perdigão J              Sandoval A            van der Wel N
      Lima EJC              Moniz-Pereira J   Pereira DR              Santos ACB            Van Hoof R
      Lima KVB              Monteiro G        Pereira E               Santos C              van Ingen J
      Locht C               Montemayor M      Pereira Miguel J        Santos R              Van Soolingen D
      Lockwood D            Montoro E         Pérez Meixeira A        Saraiva M             Varaine F
      Logarinho E           Mookherjee S      Perkins M               Sardinha E            Varghese B
      Lopes ML              Morcillo N        Pfeltz R                Sardinha T            Vasconcelos O
      López AG              Morgan K          Pfyffer G               Sarmento A            Velayati A
      López B               Mosko M           Pimentel M              Sato D                Velayati AA
      Lopez-Calleja AI      Mota M            Pinheiro MD             Sato DN               Via LE
      Loureiro C            Moulin L          Poelhsitz GV            Schön T               Viallard JF
      Lucas F               Moure R           Pole I                  Secanella SP          Viana BHJ
      Luo T                 Moyoyeta M        Portaels F              Seif S                Viana-Niero C
      Luquin M              Muchwa C          Portugal C              Shamputa IC           Villar M
      Lyashchenko K         Mugerwa R         Portugal I              Sharaf-Eldin GS       Villela G
      Lyberopoulos P        Mugyenyi P        Possuelo L              Shikama M-L           Viveiros M
      Macedo R              Müllerova M       Post E                  Shinnick T            Vladimirov K
      Machado D             Mumbowa F         Prata P                 Shinnick TM           Von Groll A
      Madruga M             Munga Waweru P    Proença F               Shoen C               Vultos TD
      Maia P                Murcia M          Qazi F                  Siddiqi S             Wang S
      Maia PIS              Musunsa A         Quieng MD               Silva A               Warns M
      Maio JN               Muvwimi M         Racic I                 Silva C               Watson C
      Mak KX                Mwamba P          Radomski N              Silva F               Weizenegger M
      Malagari AI           Mwanza W          Ramos J                 Silva K               Werngren J
      Malaquias A           Nagiyev T         Ramos Martos            Silva M               Westman L
      Malaspina AC          Nascimento I      Ramos MH                Silva P               Willery E
      Mallard K             Nasu M            Ramoutar D              Silva S               Winkler S
      Marceau M             Navarro A         Raposo A                Simões MF             Wong AP
      Markova N             Neo ZY            Rasolofo V              Sing LH               Yamaguchi N
      Marques M             Neonakis IK       Rastogi N               Singh JPN             Yamane N
      Martín A              Niemann S         Rauzier J               Skenders G            Yan SW
      Martin C              Nikolaou S        Refrégier G             Slickers P            Yates M
      Martín-Casabona N     Nina J            Régis M                 Sola C                Yew WW
      Martinez-Martinez L   Nogueira C        Reniero A               Somoskövy A           Yip CW
      Martins M             Nogutia EN        Rey E                   Sousa AS              Yubero J
      Martins MC            Noroozi J         Reyes A                 Sousa C               Yula E
      Martins TG            Noruzi J          Ribeiro JN              Sousa G               Yzquierdo S
      Masjedi M             Novoa-Cain J      Richter E               Sousa JG              Zaitseva E
      Masjedi MR            Nowroozi J        Ridell M                Sovhozova N           Zaldumbide MA
      Matic I               Nuak J            Riley LW                Soyal A
                                                                                            Zambrano MM
      Matos G               Nyirenda CN       Ritacco V               Spandidos DA
      Maugein J             Oberhauser B      Ritmeijer K             Spicic S              Zdelar-Tuk M
      Mazarrasa CF          Obrovac M         Robledo J               Stoffels K            Zellweger J-P
      Mbulo G               Obrovac M         Rocha G                 Sturegård E           Zenhorst R
      McNerney R            Ocepek M          Rodrigues A             Suffys PN             Zerolo FJ
      Mdivani N             Oelemann MC       Rodrigues F             Supply P              Zerva L
      Médigue C             Ojeda P           Rodrigues L             Svensson E            Zhang J
      Mei J                 Oliveira P        Rodrigues S             Tabarsei P            Zmak L
      Mello FAF             Omar AR           Rodríguez-Güell E       Tajeddin E            Zolnir - Dovc M
      Mendes AC             Oral Zeytinli U   Rohde KH                Takiff H
                                                                                            Žolnir	Dov- M
      Mendes NH             Orikiriza P       Rosales J               Tancredo L
      Mendes NH             Orozco H          Roura-Mir C             Tang YW               Zozio T
      Menendez MC           O’Sullivan D      Ruimy R                 Tap J
      Mestre O              Paasch F          Ruiz P                  Tavares M
      Meyers WM             Palaci M          Rumijowska-Galewicz A   Tavares Magalhães A
      Mézard M              Palomino JC       Rüsch-Gerdes S          Teles JMM
      Michel G              Panaiotov S       Russell DG              Telles MAS
      Mihailelis E          Pando RH          Saaed NS                Thibault V
      Milho C               Pandolfi	JRC      Sabino A                Tonjum T
      Milián Y              Papaventsis D     Şahan	Kipalev	A         Torrado E
      Millan I              Papiris S         Said H                  Tortoli E
      Millet J              Pardini M         Sainti A                Traore H
      Min JH                Park SK           Salas S                 Tudó G
      Miotto P              Pate M            Saluotsa M              Turner C
      Mirabal N             Paulo C           Salvadó M               Ueki SYM
      Miranda A             Pavan F           Salvignol G             Valcheva V
      Miyagi-Shiohira C     Pavan FR          Sampaio D               Valdés I
      Miyata M              Pawelczyk J       Samper S                Valente A
      Moilleron R           Pedrosa J         Samutela M              Valente F
      Mokrousov I           Peeling R         Sánchez-Concheiro M     Valente I

190                                                                                                      ESM 2009
Congress organisers

Av.Antunes Guimarães, nº 554
4100-074 Porto
Tel.: (+351) 226 165 450
Fax: (+351) 226 189 539

European Society of Mycobacteriolog y | 30 th Annual Congress | July 2009 | Por to - Por tugal   191
    Volume XVI                 Suplemento 1 A                   Janeiro 2010

                     DE MICOBACTERIOLOGIA

                     OF MYCOBACTERIOLOGY

                 Editora convidada/Guest editor: Susana David

                                          REVISTA PORTUGUESA DE
                                          P O RT U G U E S E J O U RN A L O F P U L M O N O L O G Y
          Dr.ª Pillar Azevedo
          Dr.ª Fátima Caeiro
           Dr. João Cardoso
                                          DIRETOR: Prof. A. Segorbe Luís        EDITOR: Dr. Renato Sotto-Mayor
         Dr.ª Lurdes Carvalho
        Dr. J. Rosal Gonçalves
          Dr.ª Jessica Jones
         Dr.ª Paula Monteiro
        Dr.ª Fátima Rodrigues
Sociedade Portuguesa de Pneumologia
   REDAÇÃO, ADMINISTRAÇÃO                 Conselho Científico
          Sociedade Portuguesa
            de Pneumologia                Dr.ª Margarida Cancela de Abreu       Prof. J. Agostinho Marques
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 R E V I S T A          P O R T U G U E S A                  D E      P N E U M O L O G I A


                                     Direção Comissões de Trabalho

            Presidente: Prof. António Segorbe Luís     Alergologia Respiratória
         Vice-Presidentes: Dr. Renato Sotto-Mayor      Coordenador: Dra. Luísa Semedo
                          Prof. Henrique Queiroga      Secretário: Dr. Carlos Lopes
                               Prof.ª Lina Carvalho
Secretário-Geral: Dr. José Manuel Rosal Gonçalves      Coordenador: Dra. Aurora Carvalho
     Secretário-Adjunto: Dr. José Miguel Carvalho      Secretário: Dra. Sandra André
               Tesoureiro: Dr. Jorge Roldão Vieira
                                                       Pneumologia Oncológica
                                                       Coordenador: Dra. Ana Figueiredo
                                                       Secretário: Maria de la Salete Valente

                                                       Técnicas Endoscópicas
                                                       Coordenador: Dra. Yvette Martins
                                                       Secretário: Dr. Júlio Semedo

                                                       Reabilitação Respiratória
                                                       Coordenador: Dra. Ana Paula Simão de Oliveira
                                                       Secretário: Dra. Paula Teresa Rodrigues de Almeida
  Mesa da Assembleia Geral                             Fisiopatologia Respiratória
                                                       Coordenador: Dr. Nuno Cortesão
                                                       Secretário: Dra. Maria João Matos
          Presidente: Prof. A. Bugalho de Almeida
          Secretário: Dr. José Manuel Dias Pereira     Doenças do Interstício Pulmonar
                     Vogal: Prof.ª Cristina Bárbara    e Doenças Ocupacionais
                                                       Coordenador: Dr. António Morais
                                                       Secretário: Dra. Cristina Cristóvão

                                                       Cirurgia Torácica
                                                       Coordenador: Dra. Isilda Mendes
                                                       Secretário: Dr. João Bernardo

                                                       Coordenador: Dra. Ivone Pascoal
                                                       Secretário: Dra. Sofia Ravara

                     Conselho Fiscal                   Infecciologia Respiratória
                                                       Coordenador: Dr. Filipe Froes
                                                       Secretário: Dra. Cecília Pardal

                Presidente: Dr. Jorge Branco Pires     Patologia do Sono
                         1.º Vogal: Dr. Júlio Gomes    Coordenador: Dra. Marta Drummond
                     2.º Vogal: Dr. Ulisses de Brito   Secretário: Dra. Paula Pinto

                       DE MICOBACTERIOLOGIA
                       OF MYCOBACTERIOLOGY

        Susana David Introdução                                                                       5

         Miguel Villar A tuberculose em Portugal                                                      7
                       Tuberculosis in Portugal

 Fernando AF de Melo A experiência brasileira de controlo da multidroga-resistência                  11
                     Brazilian experience in the management of multidrug-resistance

   Sebastien Gagneux Forças evolutivas do Mycobacterium tuberculosis                                 21
                     Evolutionary forces in Mycobacterium tuberculosis

Joseph O Falkinham III Epidemiologia e ecologia de micobactérias não tuberculosas                    27
                       Epidemiology and ecology of nontuberculous mycobacteria

 Jean-Pierre Zellweger O uso da análise de libertação de gama interferão como auxiliar no controlo
                       da tuberculose                                                                31
                       The use of interferon gamma release assays as an aid in the control
                       of tuberculosis

          Lee W Riley Regulação da composição lipídica da parede celular do Mycobacterium
                      tuberculosis e o seu efeito na persistência bacteriana in vitro                37
                      Regulation of Mycobacterium tuberculosis cell wall lipid composition and
                      its effects on in vitro bacterial persistence

Jesus Gonzalo Asensio Uma nova vacina viva contra a tuberculose com base na inativação do phoP       43
        Ainhoa Arbues A new live tuberculosis vaccine based on phoP inactivation
       Dessi Marinova
         Carlos Martín

       Ruth NcNerney Simpósio: Testes rápidos para o rastreio preliminar da tuberculose              49
                     Symposium: Point-of-care tests for tuberculosis

   Thomas M Shinnick Criar capacidade laboratorial para a tuberculose: Necessidades e estratégias    57
                     Building tuberculosis laboratory capacity: needs and strategies

 R e v i s t a             P o r t u g u e s a                     d e       P n e u m o l o g i a
                                       Vol XVI Suplemento 1 A Janeiro 2010
           Afranio Kritski Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento
                           e avaliação de novos métodos de diagnóstico da tuberculose                               67
                           The experience of the Brazilian Tuberculosis Research Network in the
                           development and evaluation of new methods of diagnosing tuberculosis

           Moisés Palaci Ensaios clínicos de novas drogas e testes diagnósticos em tuberculose: Desafios
                         micobacteriológicos                                                                        77
                         Clinical trials of new tuberculosis drugs and diagnostic tests: mycobacteriological

        Karina de Prince Avaliação das moléculas com atividade antiTB das plantas do cerrado brasileiro             83
      Fernando R Pavan Screening of molecules with anti-TB activity from the brazilian cerrado plants
           Daisy N Sato
        Wagner Villegas
         Sergio RA Leite
        Clarice QF Leite

    Caroline Allix-Béguec   Novas ferramentas de fácil utilização para genotipagem padronizada e com qualidade
       Christine Hubans     controlada de estirpes do complexo Mycobacterium tuberculosis                           89
      Stéphanie Ferreira    New, easy-to-use tools for standardised and quality-controlled genotyping
            Philip Supply   of Mycobacterium tuberculosis complex strains

           Elvira Richter Simpósio: Avaliação externa da qualidade                                                  95
                          Symposium: External quality assurance

                            Editora convidada/Guest editor: Susana David

                        A edição deste suplemento foi patrocinada com fins educacionais pelo
Instituto Nacional de Saúde Doutor Ricardo Jorge e pela Fundação Luso-Americana para o Desenvolvimento/
                  This supplement is made possíble thanks to an unrestricted educational grant from
        Instituto Nacional de Saúde Doutor Ricardo Jorge and Luso-American Development Foundation

                                   Este suplemento foi escrito ao abrigo do Novo Acordo Ortográfico

4          R e v i s t a              P o r t u g u e s a                       d e         P n e u m o l o g i a
                                                Vol XVI Suplemento 1 A Janeiro 2010

Susana David

A Sociedade Europeia de Micobacteriologia                           The European Society of Mycobacteriology
(ESM,                             (ESM,
é considerada uma das sociedades científicas                        is considered one of the most active interna-
internacionais mais ativas na área da mico-                         tional scientific societies in the area of my-
bacteriologia e doenças relacionadas. As                            cobacteriology and related diseases. The
reuniões da ESM, que têm lugar cada ano                             ESM meetings, held each year in a different
num país europeu diferente, promovem a                              country of Europe, promote the exchange
comunicação de especialistas internacionais                         of distinguished experts from all around the
de renome, criando oportunidades para atua-                         world creating the opportunity to update
lização do conhecimento dos últimos avan-                           information in the front-line of scientific
ços científicos, compartilhar experiências e                        achievement, share experience and ideas,
ideias e participar ativamente no desenvol-                         and actively participate in contribution to
vimento da micobacteriologia.                                       the field of mycobacteriology.
Este número especial da Revista Portuguesa                          This special issue of the Revista Portuguesa de
de Pneumologia é dedicado ao 30.º Con-                              Pneumologia (RPP) is dedicated to the 30th
gresso Anual da Sociedade Europeia de Mi-                           Annual Congress of the European Society of
cobacteriologia (ESM2009), que se realizou                          Mycobacteriology (ESM2009) hosted in Por-
no Porto, Portugal, de 5 a 8 de julho de                            to, Portugal, from July 5-8, 2009. This con-
2009. A organização deste congresso deveu-                          gress was co-organized by the ESM and the
-se à ESM e ao Instituto Nacional de Saúde                          National Health Institute Doutor Ricardo
Doutor Ricardo Jorge (INSA), o braço la-                            Jorge (INSA), the laboratory arm of the Por-
boratorial do sistema português de saúde                            tuguese health system (
(                                               The ESM2009 congress gave particular at-
O Congresso ESM2009 deu particular aten-                            tention to the multi-disciplinary effort
ção ao esforço global e multidisciplinar em                         needed, from mycobacteriologists on a
micobacteriologia necessário na luta contra                         world wide basis, in the fight against tuber-
a tuberculose. As sessões científicas e minis-                      culosis. Scientific sessions and mini-sympo-
simpósios versaram os seguintes temas:                              sia covered the following themes:
• Programas de controlo da tuberculose;                             • Tuberculosis control programs;
• Epidemiologia molecular e vigilância da                           • Molecular epidemiology and drug resis-
     resistência a fármacos;                                             tance surveillance;
• Deteção da resistência a fármacos por                             • Molecular genetic detection of drug re-
     genética molecular;                                                 sistance;

1Laboratório Nacional de Referência das Infecções Respiratórias para as Micobactérias da Unidade de Referência e Vigilância Epidemiológica do Departamento de
 Doenças Infecciosas do Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal

     R e v i s t a                    P o r t u g u e s a                             d e          P n e u m o l o g i a                                    S5
                                                  Vol XVI Suplemento 1 A Janeiro 2010
                                                                                                    Susana David

            •   Testes de susceptibilidade e tratamento       •    Susceptibility testing and treatment of
                da tuberculose;                                    tuberculosis;
            •   Micobactérias não tuberculosas;               •    Non tuberculous mycobacteria;
            •   Novas questões no papel do laboratório        •    Issues in the modern tuberculosis labo-
                de tuberculose;                                    ratory;
            •   Desenvolvimento de vacinas e patogenia;       •    Vaccine development and pathogenesis;
            •   Testes point-of-care para tuberculose;        •    Point-of-care tests for tuberculosis;
            •   Fortalecimento do laboratório;                •    Laboratory strengthening;
            •   Desenvolvimento farmacológico;                •    Drug development;
            •   Aspetos práticos e controlo de qualida-       •    Practical aspects and quality assurance
                de na epidemiologia molecular;                     in molecular epidemiology;
            •   Controlo de qualidade externo.                •    External quality assurance.

            Este número é constituído por uma com-            This issue is comprised of a compilation of
            pilação de breves comunicações baseadas           short communications based on the themes
            nos temas de algumas das principais confe-        of some of the main conferences. The full
            rências. O programa completo pode ser             program may be consulted on the ESM and
            consultado nos websites da ESM e do INSA          INSA websites or at http://www.esm2009.
            ou em                     org.
            Gostaríamos de manifestar o nosso especial        We would like to give special thanks to Dr.
            agradecimento ao Dr. Renato Sotto-Mayor e         Renato Sotto-Mayor and to the RPP for their
            à Revista Portuguesa de Pneumologia pelo seu      interest in putting together this issue and to all
            interesse na publicação deste número e a to-      those who kindly accepted to give their scien-
            dos os que amavelmente deram a sua contri-        tific contribution. The ESM2009 congress
            buição científica. A realização do Congresso      would not have been possible without the
            ESM 2009 não teria sido possível sem o apoio      support of the partners: Fundação Calouste
            das instituições: Fundação Calouste Gulbenkian    Gulbenkian (; Lu-
            (; Fundação Luso-        so-American Foundation (http://www.flad.
            -Americana de Desenvolvimento (http://www.        pt); STOP-TB Working Group on New Di-
  ; STOP-TB Working Group on New            agnostics, Point of Care sub group (www.
            Diagnostics, Point of Care sub group (www. and sponsors: HAIN LifeSciences
            stop e patrocinadores: HAIN LifeScien-    (; Becton
            ces (; Becton     Dickinson (; Quilaban
            Dickinson (; Quilaban           (; BioMerieux (http://
            (; BioMerieux (http://; Cepheid (http://
  ; Cepheid (http://www.; Microsens (http://www.
  ; Microsens (http://www.micro-; Genoscreen (http://www.
  ; Genoscreen (http://www.genoscre-
                                             Susana David                                Susana David
                      Presidente do Congresso ESM2009                Chairman of the ESM2009 congress

S6   R e v i s t a      P o r t u g u e s a                  d e     P n e u m o l o g i a
                                Vol XVI Suplemento 1 A Janeiro 2010
30.º Congresso Anual da Sociedade Europeia de Micobacteriologia
30th Annual Congress of the European Society of Mycobacteriology

Miguel Villar1                                           A tuberculose em Portugal

                                                         Tuberculosis in Portugal

A tuberculose (TB) é um problema glo-                                  Tuberculosis is a global problem with an es-
bal com um número estimado de 9 mi-                                    timated 9 million new cases per year, 83%
lhões de novos casos por ano, 83% dos                                  of which occur in Sub-Saharan Africa and
quais se situam na África subsariana e no                              South-East Asia, where many of the high
Sudeste Asiático, onde se encontram mui-                               burden countries are found (Fig. 1). The
tos dos países com a maior carga de TB                                 World Health Organisation (WHO) esti-
(Fig. 1). A Organização Mundial de Saú-                                mates an emergence of about half a million
de (OMS) estima a emergência global de                                 new cases of multidrug-resistant tuberculo-
cerca de meio milhão de novos casos de                                 sis (MDR-TB), globally, each year, includ-
tuberculose multirresistente (MDR-TB),                                 ing 50 000 of extensively drug-resistant tu-
por ano, incluindo 50 000 casos de tu-                                 berculosis (XDR-TB).
berculose extensamente resistente a fárma-                             In 2007, the European Union (EU) had an
cos (XDR-TB).                                                          incidence rate of 17/100 000, with Portugal
Em 2007, a União Europeia (UE) teve uma                                registering one of the highest rates in the
incidência média de 17/100 000, com Por-                               EU (27/100 000).
tugal a registar um dos mais elevados índices                          Over the last two decades, the incidence in
na UE (27/100 000). Nas últimas duas dé-                               Portugal has decreased consistently, and
cadas, a incidência em Portugal tem vindo a                            more than 7% per year, in the last five years
diminuir consistentemente, e em mais de                                (Fig. 2). This reduction is mainly in the age
7% anualmente nos últimos 5 anos (Fig. 2).                             group between 25 and 44 years old, leading
Esta diminuição é mais notória no grupo                                to a shift to the right of the median age,
etário dos 25 aos 44 anos, o que resulta numa                          both in national citizens and in immigrants
alteração da média de idades, tanto nos do-                            (Fig. 3). The foreign-born have represented
entes nacionais como nos imigrantes (Fig. 3).                          about 12% of the TB cases, and the preva-

1   Em representação do Director-Geral de Saúde/On behalf of the General-Director of Health

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                                                     Vol XVI Suplemento 1 A Janeiro 2010
                                                                                       a tuberculose em portugal
                                                                                                                                      Miguel Villar

                                                                                           100 ou mais/100 or more
                                                                                           Não reportado/No report
                                                                                           Casos de tuberculose notificados (novos
                                                                                           e relapsos) por cada 100 000 habitantes/
                                                                                           Notified TB cases (new and relapse) per
            Fig. 1 – Taxas de incidência da tuberculose por país, 2006. Fonte: OMS         100 000 population

            Fig. 1 – Tuberculosis incidence rates, by country, 2006. Source: WHO

            Fig. 2 – Evolução das taxas de incidência de tuberculose notificada no continente e regiões autónomas (todas as formas,
            10–5 habitantes). Fonte: DGS

            Fig. 2 – Evolution of the tuberculosis incidence rates notified in Mainland Portugal and Autonomous Regions (all forms/10-5
            inhabitants). Source: DGS

            Os estrangeiros têm representado cerca de                      lence of HIV has been around 14%, with a
            12% dos casos, e a prevalência de VIH tem                      34% reduction in the last five years.
            sido de aproximadamente de 14%, com uma                        Between 2003 and 2007, most TB cases
            redução de 34% nos últimos cinco anos.                         were pulmonary forms (74.1%), 67.5% of
            Entre 2003 e 2007, a maioria dos casos de                      which were SS+ and 75.3% were confirmed
            TB foram das formas pulmonares (74,1%),                        by culture. MDR-TB and XDR-TB, during
            dos quais 67,5% SS+ e 75,3% confirmados                        the same period, represents 1.9% (154) of

S8   R e v i s t a         P o r t u g u e s a                          d e          P n e u m o l o g i a
                                      Vol XVI Suplemento 1 A Janeiro 2010
a tuberculose em portugal
Miguel Villar

                 Fig. 3 – Evolução da taxa de incidência de tuberculose notificada, por gru-
                 pos etários (105 habitantes). Fonte: DGS

                 Fig. 3 – Evolution of the tuberculosis incidence rates, according to age
                 groups (105 inhabitants). Source: DGS

por cultura. Neste mesmo período, os casos              the TB cases at the beginning of treatment,
de MDR-TB e XDR-TB representam 1,9%                     varying from 1.3% (22), in 2006, and 2.4%
(154) de todos os casos de tuberculose no               (38), in 2004, with an average of 31 cases
início do tratamento, variando de 1,3%                  per year (1.9%). These proportions are re-
(22), em 2006, e de 2,4% (38), em 2004,                 presentative, since the coverage of drug sen-
com uma média de 31 casos por ano (1,9%).               sibility tests (DST) is over 80%.
Estes números são representativos, já que a             As regards the theme of this Congress, the
cobertura dos testes de sensibilidade a fár-            Mycobacteriology Laboratory Network is
macos (TSF) é superior a 80%.                           an important component in the National
No que respeita ao tema deste congresso, a              Tuberculosis Programme (NTP), with col-
Rede de Laboratórios de Micobacteriologia               laboration in case detection, case defini-
é uma componente importante do Progra-                  tion, early diagnosis of MDR-TB, identifi-
ma Nacional para a Tuberculose (PNT),                   cation of Mycobacterium tuberculosis and
com colaboração na deteção e definição de               performance of 1st and 2nd line DST. Be-
casos, diagnóstico precoce de MDR-TB,                   sides confirmation of TB cases, it also al-
identificação de Mycobacterium tuberculosis             lows us to accompany the negativity rate,
e realização de TSF de 1.ª e 2.ª linha. Para            especially up to two months of treatment.
além da confirmação dos casos de TB,                    This is important for the control of trans-
permite-nos, também, acompanhar o índice                mission and treatment efficacy. In the same
de negatividade, especialmente até aos dois             period mentioned above, we had 34% doc-
meses de tratamento, o que é importante                 umented negativity at two months.
para o controlo da transmissão e da eficácia            The Laboratory is also very useful in the
do tratamento. No mesmo período,                        screening strategy of the tuberculosis sur-
verificou-se um índice de negatividade do-              veys, mainly through the use of nucleic-acid
cumentada de 34% aos dois meses.                        amplification tests (NAAT). Analysing the

     R e v i s t a          P o r t u g u e s a                          d e        P n e u m o l o g i a   S9
                                       Vol XVI Suplemento 1 A Janeiro 2010
                                                                         a tuberculose em portugal
                                                                                                   Miguel Villar

              O laboratório é também muito útil na estra-       outcomes is an important complement of
              tégia de monitorização dos rastreios da tuber-    the DOTS Strategy; we had an 85.6% suc-
              culose, principalmente pelos testes de am-        cess rate in the general population, in ac-
              pliação do ácido nucleico (TAAN). Ao              cordance to the WHO goals. As to risk
              analisar os resultados, como um complemen-        groups, the success rate in the risk-free
              to importante da estratégia DOTS, obtive-         group was of 90%, but under WHO goals,
              mos um índice de sucesso de 85,6% na po-          in the other risk groups, it was namely 69%
              pulação em geral, de acordo com os objetivos      in TB/HIV, 53% in MDR TB and 82% in
              da OMS. No que se refere aos grupos de ris-       immigrants.
              co, o índice de sucesso no grupo sem risco foi    Finally, we finished our presentation ad-
              de 90%, mas, nos outros grupos, e de acordo       dressing the strategy for MDR/XDR-TB
              com os objetivos da OMS, foi nomeadamen-          control in Portugal, through the creation of
              te de 69% no de TB/VIH, de 53% no de              the National Reference Centre for Multi-
              MDR-TB e de 82% no de imigrantes.                 drug-resistant Tuberculosis, in June 2007
              Finalmente, terminámos a nossa apresenta-         (policy document 14-DT, General-Direc-
              ção abordando a estratégia para controlo de       torate of Health). Its main objectives are to
              MDR/XDR-TB em Portugal, através da                reduce prevalence and prevent transmission
              criação, em junho de 2007, do Centro Na-          of MDR-TB, support clinicians in choosing
              cional de Referência da Tuberculose Multi-        the therapeutic regimens, collaborate with
              -Resistente (Circular Informativa 14-DT,          them in the management of adverse effects
              da Direcção-Geral de Saúde). Os seus prin-        and, if needed, in the hospitalization and
              cipais objetivos são os de reduzir a prevalên-    isolation of patients in the best possible
              cia e evitar a transmissão da MDR-TB,             conditions.
              apoiar os médicos na escolha dos regimes          In 2009, one of the high priorities of NTP
              terapêuticos, colaborar com eles no ajuste        was the implementation of a network of re-
              desses regimes por efeitos adversos e, se ne-     gional reference centres for the treatment of
              cessário, na determinação do internamento         MDR/XDR TB cases.
              dos doentes e nas condições de isolamento.
              Em 2009, uma das grandes prioridades do
              PNT foi a implementação de uma rede de
              centros regionais de referência para trata-
              mento de casos de MDR/XDR-TB.

S 10   R e v i s t a      P o r t u g u e s a                  d e    P n e u m o l o g i a
                                  Vol XVI Suplemento 1 A Janeiro 2010
Fernando Augusto Fiuza de Melo1                         A experiência brasileira de controlo da multidroga-

                                                        Brazilian experience in the management of multidrug-

     Resumo                                                                          Abstract
     Neste artigo de revisão, o autor faz uma revisão de                             In this article the author reviews the evolution of the
     como evoluiu a abordagem da tuberculose multirresis-                            approach to multidrug-resistant tuberculosis (MDR-
     tente (MDR) no Brasil, desde a introdução da rifampi-                           TB) in Brazil following the introduction of rifampicin
     cina associada a isoniazida e a pirazinamida (RHZ).                             associated to isoniazid and pyrazinamide (RHZ). It
     Mostra que o país foi um dos primeiros no mundo a                               shows Brazil was one of the world’s first countries to
     aplicar o esquema RHZ dentro de um sistema de tra-                              use the RHZ regimen within a treatment system, with
     tamento com um esquema de primeira linha, outro                                 a first line regimen, another one specific for meningo-
     específico para as formas meningoencefálicas, para re-                          encephalic forms, for re-treatment of recurrences or of
     tratamento para recidivas ou retorno com tuberculose                            patients who returned with active tuberculosis after
     ativa após abandono, e um esquema de reserva. O sis-                            abandoning treatment, and a reserve regimen. The sys-
     tema era de aplicação nacional com garantia de forne-                           tem was applied nationwide with guaranteed cost-free
     cimento gratuito das drogas e autoadministrado. Avalia                          provision of medication, and self-administered. The
     a evolução da resistência aos medicamentos, a emer-                             author evaluates the growth of drug resistance, the
     gência da resistência múltipla e como foi organizado o                          emergence of multidrug-resistance and how manage-
     controlo desta forma da doença.                                                 ment of this form of the disease has been organised.

     Palavras-chave: Tuberculose multirresistente a múl-                             Key-words: Multidrug-resistant tuberculosis (MDR-
     tiplos medicamentos (TB-MDR), tuberculose super-                                TB), extensively drug-resistant tuberculosis (XDR-TB).
     resistente (TB-XDR).

1   Médico, Diretor do Instituto Clemente Ferreira – Coordenadoria de Controle de Doenças da Secretaria de Estado da Saúde de São Paulo – ICF/CCD/SES-SP/Physician,
    Director, Instituto Clemente Ferreira, Coordinator of Disease Control of the Secretary of State for Health of São Paulo – ICF/CCD/SES-SP
    Doutorado em Medicina, área de pneumologia, pela Escola Paulista de Medicina da Universidade Federal de São Paulo – EPM/UNIFESP/PhD, Pulmonology, Escola
    Paulista de Medicina da Universidade Federal de São Paulo – EPM/UNIFESP
    Membro do Comitê de Assessoria Técnico-Científico do Programa Nacional de Controle da Tuberculose do Ministério da Saúde (PNCT/MS)/Member of the Technical-
    -Scientific Committee of the Ministry of Health’s National Tuberculosis Control Programme (PNCT/MS)

Correspondência/Correspondence to:
Rua Santo Estácio, 248
Cidade Vargas, CEP 04319-010 – São Paulo, SP, Brasil
Tele-fax: 0055 11 3218 8653
Telemóvel: 0055 11 8469 4330

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                                                   Vol XVI Suplemento 1 A Janeiro 2010
                       a experiência brasileira de controlo da multidroga-resistência
                                                                                   Fernando Augusto Fiuza de Melo

              Introdução                                       Introduction
              Embora seja um fenômeno mundial, a tu-           Although a worldwide phenomenon, the
              berculose (TB) apresenta características va-     characteristics of tuberculosis (TB) vary in
              riáveis de acordo com a região do mundo,         line with the world region, depending on
              dependente de fatores raciais, ecológicos,       racial, ecological and socioeconomic factors,
              socioeconômicos, interrelações com outras        inter-relationships with other endemics,
              endemias, como a da VIH/SIDA, perfil de          such as HIV/AIDS, the resistance profile of
              resistência às drogas em uso e do desenvolvi-    the drugs in use and the development of
              mento do controlo da doença.                     management of the disease.
              Assim como a TB, também a tuberculose            Similarly to TB, multidrug-resistant tuber-
              multidroga-resistente (MDR-TB), que es-          culosis (MDR-TB), which has alarmed the
              pantou o mundo, apresenta um desenvolvi-         world, has had its own development in
              mento próprio no Brasil1,2,3.                    Brazil1-3.

              O sistema brasileiro                             The Brazilian tuberculosis
              de tratamento da tuberculose                     treatment system
              O Brasil foi o primeiro país não desenvolvi-
              do a usar esquema de curta duração com           Brazil was the first developing country to use
              participação da rifampicina (R) associada a      a short-course regimen involving rifampicin
              hidrazida (H), além da pirazinamida (Z), ao      (R) associated to isoniazid (H) and pyrazi-
              reorganizar em todo o país o Programa Na-        namide (Z), in reorganising a nationwide Tu-
              cional de Controle da Tuberculose, do Mi-        berculosis Control Programme of the Minis-
              nistério da Saúde (PNCT-MS), em 1979.            try of Health (TBCP-MH), in 1979.
              Essa mudança estabelecida, após um ensaio        After a trial comparing the RHZ regimen
              dirigido comparando o esquema RHZ com            with a 12-month long combination regi-
              esquema de longa duração (12 meses) com a        men with the association of H streptomycin
              H associada à estreptomicina (S) e ao etam-      (S) and ethambutol (E)4, this change, once
              butol (E)4, estabelece, não somente altera-      instituted, established changes in medica-
              ções de medicamentos, mas normatiza um           tion and harmonised a treatment system.
              sistema de tratamento.                           This system (Fig. 1) includes a short-course
              Esse sistema (Fig. 1) conta com um esque-        first line regimen, the E-1 (2RHZ/4RH),
              ma de primeira linha, de curta duração, o        suitable for all forms of TB without prior
              E-1 (2RHZ/4RH), indicado para todas as           treatment, except the meningoencephalic
              formas de TB sem tratamento anterior, me-        form, using corticosteroids during the attack
              nos para a forma meningoencefálica, com          stage and prolonging the medication for
              uso de corticosteroides na fase de ataque e      nine months, the E-2 (2RHZCort/7RH).
              prolongando a medicação para nove meses,         The same E-1 was prescribed for recurrences
              o E-2 (2RHZCort/7RH). Para os recidivan-         after cure (RC), or for patients who relapsed
              tes após cura (RC) ou reingresso após aban-      with active disease (RA) after abandoning
              dono com doença ativa (RA), a indicação          treatment, reinforced with ethambutol E

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                                2RHZ/4RH                  E-1R               E-3
           NT                                                         F                  F     TBMR
                                E-2(m)                                     3SZEEt/
                               2RHZ*/7RH                 2RHZE/4RH

                                          ABANDON                         DEATH        F FAILURE

Fig. 1 – O sistema de tratamento da tuberculose no Brasil, 1999

Fig. 1 – Tuberculosis treatment system in Brazil, 1999

do mesmo E-1, reforçado com o etambutol                       during the attack stage, the E-1R. In those
(E) na fase de ataque, o E-1R. Para os que                    for whom the E-1 failed, there was a
apresentassem falência com o E-1, foi pre-                    12-month long second line regimen, which
visto um esquema de segunda linha, com                        associated ethionamide (Et) to S, Z and E;
duração de 12 meses, onde além da S, Z e E,                   the E-3 (3SZEEt/9EEt)5 (Fig. 1).
se associava a etionamida (Et), o E-3                         The mean dosage of H used in Brazil is 10
(3SZEEt/9EEt)5 (Fig. 1).                                      mg/kg/day, substantially higher than that
A dose média da H no país é de 10 mg/kg/                      used internationally. The system’s basic logis-
/dia, bem maior do que a usada internacio-                    tical characteristics were: cost-free application
nalmente. As características operacionais                     nationwide, guaranteed medication, R+H in
básicas do sistema eram a aplicação nacio-                    a single tablet to be self-administered.
nal, a garantia de fármacos, gratuita, com                    Patients whom E-3 failed were referred to
R+H em um único comprimido e de uso                           reference units for evaluation of alternative
autoadministrado.                                             medication, if available, or surgical proce-
Para os fracassos com o E-3, a orientação era                 dures, if possible5.
encaminhar o doente para referências, a fim
de avaliar drogas alternativas, se acessíveis,
ou condutas cirúrgicas, se possíveis5.                        Evolution of the system’s
                                                              resistance and success
                                                              A reference centre cohort study evaluated the
                                                              growth of resistance in three-yearly cohorts in
                                                              the 1960s, 1970s and 1980s. It showed that

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                                                                                                    Fernando Augusto Fiuza de Melo

                   Fig. 2 – Evolução da resistência primária durante três décadas: 1960, 1970 e 1980 – ICF/SP

                   Fig. 2 – Evolution of primary resistance over three decades: 1960’s, 1970’s and 1980’s – ICF/SP

              Evolução da resistência                                     the TBCP organised in the 1960s and the in-
              e do rendimento do sistema                                  troduction of R in its reorganisation in the
              Um estudo de coortes, numa referência,                      1970s provided exceptional protection of the
              avaliou evolutivamente a resistência em co-                 classic drugs with significant reduction in resis-
              ortes trienais nas décadas de 1960, 1970 e                  tance to H and S. The reduction between 1960
              1980, mostrando que o PNCT organizado                       and 1970 (p = 0.002) was more significant
              na década de 1960 e a introdução da R na                    than that between 1970 and 1980 (p = 0.032),
              sua reorganização, na década seguinte, pos-                 suggesting that a good programme offers better
              sibilitaram uma excepcional proteção das                    protection against resistance than the introduc-
              drogas clássicas com significativa redução da               tion of a potent drug such as R (Fig. 2)6.
              resistência à H e à S. A redução entre 1960                 The routine evaluation of the success of E-1
              e 1970 (p=0,002) foi mais significativa que                 in the 1980s, excluding transfers and taking
              entre 1970 e 1980 (p=0,032), sugerindo                      into consideration medication changes due
              que um bom programa protege melhor a re-                    to toxicity in the cohort studies, showed a
              sistência do que a introdução de uma droga                  94.6/77.8 rate of efficacy. It had a 13.7%
              potente, como a R (Fig. 2)6.                                rate of abandonment, a 1.5% rate of failure,
              A avaliação na rotina do rendimento do E-1                  a 3.1% rate of change due to adverse effects
              na década de 1980, excluindo as transferên-                 and a 3.9% mortality rate7. In the 1990s
              cias e considerando as mudanças de drogas                   and early 2000s (1990-2002) these rates
              por toxicidade nos estudos de coortes, apre-                were 93.9, 77.1, 13.1, 1.7, 3.3 and 4.8%,
              sentou uma taxa de eficácia/efetividade de                  respectively8. The success rate of the reserve
              94,6/77,8; com taxa de abandono de 13,7%,                   E-3 was low, with a rate of efficacy ranging

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de falência de 1,5%, troca por efeitos adversos    from 57.5/85.2 to 66.7/84.7, respectively9.
de 3,1% e de mortalidade de 3,9%7. Na déca-        The system’s worsening rate of efficacy was
da de 1990 e início do novo milênio (1990-         related to abandonment, almost certainly a
-2002), estas taxas foram de 93,9, de 77,1, de     result of the self-administration regimen
13,1, de 1,7, de 3,3 e de 4,8%, respetivamen-      and the emergence of the TB-HIV/AIDS
te8. Quanto ao E-3 de reserva, o rendimento        co-infection, tending towards improvement
foi baixo, com a taxa de eficácia/efetividade      with the introduction and expansion of su-
variando entre 57,5/85,2 e 66,7/84,7, respeti-     pervised treatment and the good quality of
vamente9.                                          Brazil’s HIV management programme.
A piora da efetividade do sistema foi rela-
cionada com o abandono, certamente re-
sultante do regime autoadministrado e a            The progress and management
emergência da coinfecção TB-VIH/SIDA,              of multidrug-resistance in Brazil
apresentando uma tendência de melhoria             Based on those numbers, to which are ad-
pela introdução e expansão do tratamento           ded the rates of RC and RA, in 1993 it was
supervisionado e a boa qualidade do pro-           estimated that around 4 – 5% of those noti-
grama de controlo do VIH no país.                  fied as having the disease presented resis-
                                                   tance associated to R+H or the impossibility
                                                   of the use of these drugs due to toxicity,
A evolução e a abordagem                           with indication for E-3. These patients had
resistência múltipla no Brasil                     MDR-TB, and were classified as resistant to
Com base nestes números, acrescentando as          E-1. As E-3 has a low success rate, a number
taxas de RC e RA, em 1993 estimou-se que           of patients ended up leaving the system with
cerca de 4 a 5% dos notificados acabariam          no prospect of treatment with the pro-
por apresentar resistência associada a R+H ou      grammed drugs (R, H, Z, E, S and Et).
impossibilidade de uso destas duas drogas por      These were estimated as ranging from 0.3
toxicidade, com indicação do E-3. Seriam           – 0.4% of the annual number of those noti-
portadores de MDR-TB, chamados entre nós           fied and known to carry multidrug-resistant
resistentes ao E-1. Como o rendimento do           tuberculosis (MDR-TB)10. For these pa-
E-3 é baixo, um número de doentes acaba por        tients, in theory with no perspectives of
sair do sistema sem perspetivas de tratamento      treatment with the programmed drugs, ear-
com as drogas programáticas (R, H, Z, E, S e       lier Ministry norms referred them to refe-
Et), estimado entre 0,3 e 0,4% dos notifica-       rence units without offering the latter the
dos anuais, denominados portadores de tu-          resources and possibilities to treat and ma-
berculose multirresistente (TBMR)10.               nage them.
Para esses doentes, teoricamente sem perspecti-    The first attempts at treatment were put
vas terapêuticas com as drogas programáticas, a    together by state programmes (provincial)
orientação das normas ministeriais no passado      with bigger and better conditions. In the
era que fossem encaminhados para unidades          later 1980s and early 1990s there were se-
de referências sem lhes oferecer recursos e con-   veral trials of alternative regimens. All the
dições para sua atenção e abordagem.               programmed drugs, lung resection surgery

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                                                                                    Fernando Augusto Fiuza de Melo

              As primeiras tentativas de tratamento acaba-      and use of earlier drugs non-standardized
              ram sendo realizadas por programas estaduais      by the TBCP, such as kanamycin (KM), p-
              (provinciais), com maiores e melhores con-        aminosalicylic acid (PAS), thiosemicarba-
              dições. Na segunda metade dos anos de 1980        zone (TSCZ) and others were simultane-
              e início da década de 1990, surgiram diver-       ously contemplated. Clofazimine (CFZ)
              sas experiências de esquemas alternativos.        used to treat hanseniasis, amikacin (AM)
              Associava-se de uma só vez todas as drogas        and, more recently, the new quinolones
              programáticas, cirurgias de ressecção pulmo-      were tested. Cure or even the greater sur-
              nares e uso de antigos fármacos não norma-        vival seen in those who progressed without
              tizados pelo PNCT, como a canamicina              alternative treatment was the basis for as-
              (KM), o ácido para-aminossalicílico (PAS),        sessing the regimens’ efficacy11-13.
              tiossemicarbazona (TSCZ) e outros. Foram          In 1995, the Centro de Referência Professor
              testadas a clofazenina (CFZ) usada no trata-      Hélio Fraga do MS (CRPHF/MS)/Rio de
              mento da hanseníase, a amicacina (AM) e,          Janeiro organised a national protocol with a
              mais recentemente, as novas quinolonas. A         regimen associating AM, levofloxacin
              efetividade dos esquemas tomava como base         (LFX), terizidone (TRZ), CFZ and E (if
              a cura ou mesmo a sobrevida maior encon-          still sensitive), tested in three reference cen-
              trada entre os que evoluíam sem tratamento        tres, from 1995 to 1997, with fair efficacy
              alternativo11,12,13.                              rates (n = 187– 56/48) in comparison with
              Em 1995, o Centro de Referência Profes-           international studies14.
              sor Hélio Fraga, do MS (CRPHF/MS)/                In 2000, the CRPHF/MS initiated an
              Rio de Janeiro, organiza um protocolo na-         MDR-TB epidemiological surveillance pro-
              cional com esquema associando AM, levo-           gramme and, in 2004, had an agreement
              floxacino (LFX), terizidona (TRZ), CFZ            with a non-profit making Brazilian civil as-
              e E (se ainda sensível), testado em três          sociation “Project MSH” (Management
              centros de referências entre 1995 a 1997,         Sciences for Health) financed by the USAID
              com taxas de eficácia/efetividade razoáveis       (United States Agency for International De-
              (n=187-56/48), quando confrontados                velopment), guaranteeing the financing of
              com estudos internacionais14.                     the alternative drugs. A guide to MDR-TB
              Em 2000, inicia no CRPHF/MS o Progra-             epidemiological surveillance was drawn up,
              ma de Vigilância Epidemiológica de TBMR           summarising the country’s collected know-
              e em 2004 um convénio com a associação            ledge, establishing diagnostic, treatment,
              civil brasileira sem fins lucrativos, “Projeto    prevention and biosafety norms, epidemio-
              MSH” (Management Sciences for Health), e          logical surveillance guidelines, human re-
              com recursos financiados pela USAID (Uni-         sources training and financial support for
              ted States Agency for International Develop-      carrying out the programme. A nationwide
              ment), garante o financiamento das drogas         notification system specifically for MDR-
              alternativas. Um guia de vigilância epide-        TB was instituted15.
              miológica de TBMR foi elaborado conden-           Subsequent studies and routine review of
              sando o conhecimento acumulado no país,           notifications showed much smaller numbers
              estabelecendo normas para o diagnóstico,          than predicted, with regional variations dif-

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tratamento, prevenção e biossegurança,                     ferentiated by local socioeconomic situa-
orientações para a vigilância epidemiológi-                tions, the quality of the local program and
ca, formação de recursos humanos e provi-                  by a probable under-notification due to the
são de recursos materiais para execução do                 meagre amount of culture and sensitivity
programa. Foi instituído um sistema de no-                 tests available15 (Fig. 3).
tificação específico para a TBMR, aplicado                 The current alternative regimen for MDR-
nacionalmente15.                                           TB patients used in Brazil within the pro-
Estudos posteriores e revisão de notificados               gramme is AM (or S if sensitive) for 12
na rotina acabaram por apresentar números                  months; OFX, TRZ and E (if sensitive) for
bem menores do que os estimados, com va-                   18 months and Z (if sensitive) for six
riações regionais diferenciadas pela situação              months. Some Units use metronidazol
sócioeconômica, pela qualidade do progra-                  (MTZ) instead of Z for 18 months. Cure
ma local e por uma provável subnotificação                 rates range from 62% to 85%, the rate of
devido à baixa oferta da cultura e testes de               abandonment from 5-7%, failure 10-15%
sensibilidade15 (Fig. 3).                                  and the mortality rate dropped from 33%
O esquema alternativo atual para os porta-                 to 11%, depending on the greater or lesser
dores de TBMR usado no país dentro desse                   degree of organisation and quality of treat-
programa é: AM (ou S se sensível) por 12                   ment15,16.
meses; OFX, TRZ e E (se sensível) por 18                   The MDR-TB cases were mostly post-pri-
meses e Z (se sensível) por seis meses. Al-                mary (74-80%), with the primaries around
guns serviços usam, ao invés da Z, o metro-                6-8%, especially between contacts and risk
nidazol (MTZ), por 18 meses. As taxas de                   groups (conscripts, the homeless, health
cura variam entre 62 a 85%, com o abando-                  care professionals and others with high ex-

Fig. 3 – Casos de TBMR no Brasil (1994-2006) (n = 2632 casos – incidência anual média: 75 000 casos/ano)

Fig. 3 – MDR-TB cases in Brazil (1994-2006) (n=2632 cases, mean annual rate 75 000 cases/year)

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                                                                                      Fernando Augusto Fiuza de Melo

              no entre 5 e 7%, a falência entre 10 e 15%          posure) and 11-20% undetermined (lack
              e o óbito decresceu de 33 para 11%, depen-          of information capable of establishing a
              dendo do maior ou menor grau de organiza-           group). There is a low incidence of MDR-
              ção e qualidade do atendimento15,16.                TB in patients co-infected with HIV in
              Os casos de TBMR são maioritariamente pós-          Brazil, between 1.6% and 3%. We cannot
              -primários de 74 a 80%, sendo os primários          state if there is a trend to maintain this
              em torno de 6 a 8%, especialmente entre con-        number or if it will increase, or even if
              tatos e grupos de risco (conscritos, sem-abrigo     there was under-notification15-17. The vast
              e profissionais de saúde e outros com alta ex-      majority of cases, around 80%, present bi-
              posição) e, 11 a 20%, indeterminados (ausên-        lateral pulmonary lesions, which rules out
              cia de informações capazes de estabelecer o         routine surgical procedures, be it associat-
              grupo). A ocorrência de TBMR entre co-              ed to treatment or hygiene (?) to prevent
              infectados pelo VIH no país é baixa, entre 1,6      recurrences, as some Brazilian surgeons
              e 3%. Não se pode afirmar se há uma tendên-         propose18.
              cia a manter esse número ou se deve aumentar        Cases of extensively drug-resistant tubercu-
              a ocorrência, ou mesmo se não existe uma            losis (XDR-TB) with resistance to two usual
              subnotificação15,16,17. A grande maioria dos ca-    and three alternative drugs, seen after 2000,
              sos apresenta lesões pulmonares bilaterais, cer-    have been observed in Brazil. The scarcity of
              ca de 80%, o que inviabiliza condutas cirúrgi-      drug sensitivity testing, in particular to al-
              cas de rotina, seja associado ao tratamento ou      ternative drugs, makes it hard to select for
              higiénicas (?), para prevenir recidiva, como        these forms of disease. In the Instituto Cle-
              propõem alguns cirurgiões no país18.                mente Ferreira, which has automated sus-
              Casos de TB superresistentes (TB-XDR),              ceptibility testing and agreements with units
              com resistência a duas drogas usuais e três al-     which evaluate resistance using minimal in-
              ternativas, observados a partir de 2000, vêm        hibitory concentration (MIC) in liquid me-
              sendo encontrados no Brasil. As baixas ofer-        dium and reading using the Alamar-Blue
              tas de testes de sensibilidade, em especial para    technique, 34 quinolone-resistant cases
              drogas alternativas, dificultam a seleção des-      were recently documented with 16 resistant
              sas formas da doença. No Instituto Clemente         to AM and 18 to S. Treatment with the al-
              Ferreira, que conta com TS automatizados e          ternative regimen indicated for MDR-TB
              tem convénios com serviços que avaliam a            resulted in nine cures and 25 failures, with
              resistência pelo MIC em meio líquido e lei-         17 deaths among the latter. A cause for
              tura pela técnica de Alamar-Blue, um levan-         alarm was finding three cases of primary
              tamento recente documentou 34 casos resis-          XDR-TB, indicating that super-resistant
              tentes a quinolonas, sendo 16 resistentes a         bacilli could be in our midst and not only
              AM e 18 a S. Tratados com o esquema alter-          occasioned by treatment errors19,20.
              nativo indicado para TBMR, o resultado foi
              nove curas e 25 falências, entre estes 17 óbi-      Note: Brazil, based on a review of the prog-
              tos. Um dado alarmante foi o encontro de            ress of primary and post-primary resistance,
              três casos de TB-XDR primários, um indício          decided to change the initial E-1 with asso-
              de que bacilos superresistentes podem estar         ciation of E as the fourth drug in the attack

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circulando no nosso meio, e não apenas pro-                stage, phased in from 2010 on. The in-
duzidos por erros terapêuticos19,20.                       creased rates of resistance to H, MDR-TB
                                                           in patients co-infected with TB-HIV/AIDS
Nota: O Brasil, com base em revisão da evo-                and longevity of the population with a stock
lução da resistência primária e pós-primária,              of latent TB selected in the 30 years of use
decidiu e está alterando o E-1 inicial com                 of R+H were considered. This change is in
associação do E como quarta droga na fase                  the use of the association of drugs in tablet
de ataque, com implantação gradativa no                    form with fixed combined dosage (4 in 1)
início de 2010. Foram considerados os au-                  and also changes the reserve E-3 now for
mentos da taxa de resistência a H da TB-                   MDR-TB patients and considers those who
-MDR em coinfetados TB-HIV/AIDS e da                       cannot find a cure within the system as
longevidade populacional com estoque de                    XDR-TB cases21.
TB-latente selecionado nos 30 anos de uso
de R+H. Essa mudança faz-se com o uso de
associação dos fármacos na forma de com-
primidos com dose fixa combinada (4 em
1), altera também o E-3 de reserva agora
para doentes portadores de TB-MDR e pas-
sa a considerar os que não conseguem a cura
dentro do sistema como TB-XDR21.

1. Melo FAF. Evolução dos conhecimentos, o controle e      6. Melo FAF, Afiune JB, Ribeiro LHG, Almeida, EA,
algumas questões pendentes na tuberculose pulmonar         Castelo A. Resistência primária do M. tuberculosis num
multirresistente, Pneumologia: atualização e reciclagem.   serviço ambulatorial de referência em São Paulo:
Sociedade Paulista de Pneumologia e Tisiologia 2003;       evolução por três décadas e comparação com outros es-
5:285-292.                                                 tudos nacionais. J Pneumol 1996; 22:3-8.
2. CDC/USA. National action plan to combat of              7. Ministério da Saúde/Fundação Nacional de Saúde/
multidrug-resistant tuberculosis. Meting the challenge     Centro de Referência Prof. Hélio Fraga-Rio de Janeiro
of multidrug-resistant tuberculosis: summary of a con-     Documento Básico da Reunião de Avaliação operacional
ference. Management of persons exposed to multidrug-       e epidemiológica do PNCT na década de 80. Bol Pneu-
-resistant tuberculosis. MMWR 1992; 41(RR-11).             mol Sanit 1993, número especial.
3. Snider DE Jr, Roper WL. The news tuberculosis           8. Ministério da Saúde/Secretaria de Vigilância em
(Eds.). N Engl J Med 1992; 267:703-705.                    Saúde/Centro de Referência Prof. Hélio Fraga-Rio de
4. Gerhardt G, Teixeira GM, Hijjar MA, Feitosa JVP,        Janeiro. Análise da situação da tuberculose no Brasil nos
Penna MLF. Resultados iniciales del tratamiento de         anos 90 e início da década atual. Bol Pneumol Sanit
corta duración en condiciones de rutina en los servicios   2005; 13:133-179.
de salud del Brasil. Bol UICT 1982; 57:87-92.              9. Campos HS, Melo FAF. Efetividade do esquema 3
5. Ministério da Saúde/Fundação Nacional de Saúde/         (3sSZEEt/9EEt) no retratamento da tuberculose na rotina
Centro de Referência Prof. Hélio Fraga-Rio de Janeiro      das unidades de saúde. Bol Pneumol Sanit 2000; 8:7-14.
– Sociedade Brasileira de Pneumologia e Tisiologia.        10. Melo FAF, Ide Neto J, Seiscento M, Pinto JA, Afiune
Controle da tuberculose: uma proposta de integração        JB. Tuberculose multirresistente. J Pneumol 1993; 19:73-
ensino-serviço. 5.ª ed. 2000.                              -82.

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                        a experiência brasileira de controlo da multi-droga-resistência
                                                                                                   Fernando Augusto Fiuza de Melo

              11. Picon PD, Pereira AN, Dias CA. Tuberculose pul-          16. Ministério da Saúde/Secretaria de Vigilância em
              monar: análise terapêutica de 62 casos crônicos. Rev         Saúde/Centro de Referência Prof. Hélio Fraga-Rio de
              AMERIGS 1980; 24:36-8.                                       Janeiro. Dados coletados no Sistema de Informação da
              12 Comissão de terceira linha do Hospital Sanatório          Tuberculose Multirresistente (Sistema TBMR), me-
              Partenon: Eficácia terapêutica do esquema de terceira        diante uso de senha. 2008.
              linha ofloxacina–amicacina–tiacetazona-hidrazida para        17. Melo FAF, Afiune JB, Ide Neto J, Almeida EA, Spa-
              tuberculose multirresistente. J Pneumol 1995; 21:215-        da DTA, Antel ANL, Cruz ML. Aspectos epidemiológi-
              -224.                                                        cos da tuberculose multirresistente em serviço de refe-
              13. Seiscento M, Fiuza de Melo FA, Ide Neto J, No-           rência na cidade de São Paulo. Rev da Soc Brasil Med
              ronha AML, Afiune JB, Inomata T, Cruz ML. Tubercu-           Trop 2003; 36:733-740.
              lose multirresistente (TBMR): aspectos clínico-laborato-     18. Leite LPS, Costa ALP, Andrade RNS, Galvão T.
              riais, epidemiológicos e terapêuticos. J Pneumol 1997;       Tratamento cirúrgico adjuvante de tuberculose pulmonar
              23:237-244.                                                  multirresistente. Jornal de Pneumologia 1997; 23:11-14.
              14. Dalcolmo MP, Fortes A, Melo FAF, Motta R, Ide            19 Emergence of Mycobacterium tuberculosis with exten-
              Neto J, Cardoso N, Andrade M, Barreto AW, Gerhardt           sive resistance to second line drugs worldwide 2000 –
              G. Estudo de efetividade de esquemas alternativos para       2004. MMWR 2006; 55(11).
              o tratamento da tuberculose multirresistente no Brasil. J    20. Savioli MTG, Melo FAF, Morrone N e Rodrigues
              Pneumol 1999; 25:70-77.                                      DS. Tuberculosis with extensive resistance to drugs in a
              15. Ministério da Saúde/ Secretaria de Vigilância em         TB reference center in Sao Paulo, Brazil. Poster accepted
              Saúde/Centro de Referência Prof. Hélio Fraga-Rio de          CHEST, San Diego, California 2009.
              Janeiro. Tuberculose multirresistente: guia de vigilância    21. III Diretrizes para a tuberculose da Sociedade Bra-
              epidemiológica; Rio de Janeiro, 2007.                        sileira de Pneumologia e Tisiologia. J Bras Pneumol
                                                                           2009; 35:1018-1048.

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Sebastien Gagneux1                                     Forças evolutivas do Mycobacterium tuberculosis

                                                       Evolutionary forces in Mycobacterium tuberculosis

A análise da diversidade genética de patogé-                         Analyzing the genetic diversity of bacterial
nios bacterianos ajuda a compreender me-                             pathogens helps to better understand the
lhor a epidemiologia e a evolução destes mi-                         epidemiology and evolution of these mi-
cróbios. A compreensão da evolução de                                crobes. Understanding the evolution of
patogénios é particularmente importante                              pathogens is particularly important in to-
nos dias de hoje, com a crescente resistência                        day’s era of increasing antimicrobial resis-
antimicrobiana1. Porém, o estudo da diversi-                         tance1. However, studying the genetic di-
dade genética do complexo Mycobacterium                              versity of the Mycobacterium tuberculosis
tuberculosis (CMTB) é um desafio, porque a                           complex (MTBC) is challenging because
variação da sequência de ADN nestes orga-                            the DNA sequence variation in these or-
nismos é baixa2 e os métodos-padrão de ge-                           ganisms is low2, and standard genotyping
notipagem, como a tipagem de sequência                               tools such as multilocus sequence typing
multiloco (TSML), já bem estabelecida nou-                           (MLST), which have been well established
tras bactérias3, proporcionam pouca infor-                           in other bacteria3, provide little informa-
mação4. Além disso, deveriam usar-se dife-                           tion4. Moreover, different genotyping
rentes métodos de genotipagem quando se                              tools should be used when addressing dif-
investigam questões diferentes5. A investiga-                        ferent research questions5. Classical mo-
ção epidemiológica molecular clássica da                             lecular epidemiological investigation of
transmissão de doenças e a diferenciação en-                         disease transmission and differentiating
tre recidiva e reinfeção exógena requer mar-                         between relapse and exogenous re-infec-
cadores de genotipagem com grande força                              tion requires genotyping markers with a
discriminatória6, enquanto as análises evolu-                        high discriminatory power6, whereas evo-
tivas deveriam apoiar-se em marcadores filo-                         lutionary analyses should rely on phyloge-
geneticamente robustos7. Infelizmente,                               netically robust markers7. Unfortunately,
ocorre frequentemente trade-off entre essas                          there is often a trade-off between these

1 Division of Mycobacterial Research, MRC National Institute for Medical Research, London, UK


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                                         Forças evolutivas do Mycobacterium tuberculosis
                                                                                              Sebastien Gagneux

              propriedades nos marcadores individuais            properties in individual markers used for
              usados na genotipagem, particularmente             genotyping, and this is particularly true
              quando se estudam bactérias geneticamente          when studying genetically monomorphic
              monomórficas, como CMTB8,9. Com o                  bacteria such as MTBC8,9. In MTBC, spo-
              CMTB, a spoligotipagem e a tipagem por             ligotyping and MIRU-VNTR typing have
              MIRU-VNTR têm sido usadas com grande               been used very successfully for fine typing
              sucesso para tipagem e investigação epide-         and molecular epidemiological investiga-
              miológica molecular, mas essas técnicas são        tions, but these techniques are limited
              limitadas quando se inferem relações filoge-       when inferring phylogenetic relationships
              néticas entre estirpes10. Isto deve-se ao facto    between strains10. This is because spoligo-
              de a spoligotipagem e a tipagem por MIRU-          typing and MIRU-VNTR typing rely on
              -VNTR dependerem de marcadores mole-               repetitive molecular markers that change
              culares repetitivos que se alteram rapidamen-      rapidly, and as a consequence these mar-
              te e, por consequência, esses marcadores têm       kers exhibit a high rate of convergent evo-
              um elevado índice de evolução convergen-           lution10. By contrast, genomic deletions,
              te10. Em contraste, as deleções genómicas ou       also known as large sequence polymor-
              polimorfismos de sequência (LSP) e os poli-        phisms (LSPs), and single nucleotide poly-
              morfismos de um nucleótido único (SNP)             morphisms (SNPs) accumulate relatively
              aglomeram-se de modo relativamente lento           slowly in the MTBC genome and can thus
              no genoma de CMTB, podendo assim se-               be used to define phylogenetically robust
              rem usados para definir linhagens filogeneti-      strain lineages11.
              camente robustas11.                                We have previously shown that LSPs de-
              Já mostrámos que os LSP definem seis li-           fine six main strain lineages within the
              nhagens de estirpes principais no CMTB             human MTBC12. These include the two
              humano12, que incluem as duas linhagens            lineages generally referred to as M. africa-
              geralmente referidas como M. africanum             num West-Africa 1 and 2. We have also
              África Ocidental 1 e 2. Mostrámos também           shown that these lineages were congruent
              que estas linhagens eram correspondentes a         with groupings found based on SNP-typ-
              agrupamentos observados na tipagem de              ing11. However, because of the inherent
              SNP11. No entanto, devido a limitações ine-        limitations of those previous studies, the
              rentes a esses estudos anteriores, a real dis-     actual genetic distance within and be-
              tância genética entre linhagens e nelas pró-       tween strain lineages remained unknown.
              prias é ainda desconhecida. Para definir           To define more quantitatively the genetic
              mais quantitativamente a diversidade gené-         diversity in MTBC, we performed a large-
              tica em CMTB, realizámos uma análise de            scale multilocus sequence analysis (MLSA)
              sequências multilocos em larga escala              of 108 globally representative strains13.
              (MLSA) de 108 estirpes globalmente repre-          These included members of the animal-
              sentativas13, que incluíram membros de             adapted MTBC like M. bovis, M. microti,
              CMTB adaptado a animais, como M. bovis,            M. caprae, and M. pinnipedii. For all of
              M. microti, M. caprae e M. pinnipedii. Para        these 108 strains, we generated the com-
              todas estas 108 estirpes, gerámos a sequên-        plete DNA sequence of 89 genes, which

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Forças evolutivas do Mycobacterium tuberculosis
Sebastien Gagneux

cia de ADN completa de 89 genes, que             comprised housekeeping genes, virulence
compreenderam genes de housekeeping, ge-         genes, and antigenic genes. We used the
nes de virulência e genes antigénicos. Usá-      complete gene concatenates to define a
mos os genes concatenatos completos para         new DNA sequence-based phylogeny of
definir uma nova filogenia do CMTB com           MTBC13. This phylogeny is the most
base na sequência do ADN13. Esta é a mais        comprehensive and most robust phyloge-
abrangente e robusta filogenia do CMTB           ny of MTBC to date14, and is also highly
até à data14 e é também altamente congruen-      congruent with our previous LSP-based
te com a nossa anterior classificação do         classification of MTBC into six main li-
CMTB, baseada na análise de LSP, em seis         neages15. The quantitative nature of the
linhagens principais15. A natureza quantita-     new DNA sequence data offered some
tiva dos novos dados das sequências de ADN       new insights. In particular, while all ani-
proporcionou um novo discernimento. Em           mal-adapted forms of MTBC clustered
particular, enquanto todas as formas de          together, they represented only a sub-set
CMTB adaptadas de animais se agrupavam,          of all the genetic diversity observed across
representavam apenas um subgrupo da di-          the whole phylogeny, suggesting that the
versidade genética observada em toda a filo-     genetic diversity of human MTBC is more
genia, sugerindo que a diversidade genética      pronounced than previously thought13.
do CMTB humano é mais pronunciada do             Furthermore, the two M. africanum li-
que anteriormente se pensava13. Além disso,      neages, which are almost exclusively ob-
as duas linhagens M. africanum, quase ex-        served in West Africa or the most ances-
clusivamente observadas na África Ociden-        tral lineages. Together with the fact that
tal, são as mais ancestrais. Conjuntamente       Africa is the only continent that harbours
com o facto de ser a África o único conti-       all six main lineages of human MTBC,
nente onde se encontram as seis mais im-         these findings support to view that MTBC
portantes linhagens do CMTB humano,              originated in Africa12.
estes achados apoiam a teoria de que o           Our new MLSA data allowed us to study
CMTB terá sido originado em África12.            the evolutionary history of human MTBC
Os dados do nosso novo estudo por análise de     in more detail. When we compared the ge-
sequências multilocos permitiram-nos estudar     netic diversity data to the geographic dis-
a história evolutiva do CMTB humano em           tances separating the places of origin of the
maior detalhe. Quando comparámos os dados        strains included in the study, we found sta-
da diversidade genética com as distâncias geo-   tistically significant correlations between
gráficas que separam os locais de origem das     these two measures13. Our results supported
estirpes incluídas no estudo, encontrámos cor-   an ‘Out-of-and-back-to-Africa’ scenario for
relações estatisticamente significativas entre   the evolution of human MTBC13. Accord-
estas duas medidas13. Os presentes resultados    ing to this scenario, MTBC originated in
apoiaram um cenário ‘Fora-de-e-de-volta-a-       Africa and spread originally out of Africa ac-
-África’ para a evolução do CMTB humano13.       companying ancient human migrations
De acordo com este cenário, o CMTB origi-        which occurred approximately 50 000 years
nou-se na África e espalhou-se originalmente     ago. Through these ancient migrations,

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                                        Forças evolutivas do Mycobacterium tuberculosis
                                                                                             Sebastien Gagneux

              para fora desse continente, acompanhando          three evolutionarily “modern” lineages of
              antigas migrações humanas que ocorreram há        MTBC seeded areas in Europe, India, and
              aproximadamente 50 000 anos. Através dessas       China, respectively. These regions expe-
              antigas migrações, três ‘modernas’ linhagens      rienced strong human population increase
              evolutivas de CMTB espalharam-se por áreas        during the last few hundred years, leading
              da Europa, da Índia e da China, respectiva-       to an expansion of these MTBC lineages.
              mente. Essas regiões passaram por grande au-      Concomitantly, these modern lineages star-
              mento da população humana durante as últi-        ted to spread globally and back to Africa,
              mas centenas de anos, o que levou a uma           through recent waves of colonization, trade,
              expansão destas linhagens de CMTB. Conco-         and conquest.
              mitantemente, estas linhagens modernas co-        Another interesting observation coming
              meçaram a disseminar-se globalmente e re-         out of our MLSA work was that the ratio
              gressaram a África, através de recentes ondas     of nonsynonymous SNPs to synonymous
              de colonização, comércio e conquista.             SNPs (a measure known as dN/dS) is
              Outra observação interessante que decorreu        much higher in MTBC than in most other
              da nossa análise de sequências multilocos foi     bacteria13. A high dN/dS in bacterial
              que o rácio SNP não sinónimos: SNP sinóni-        pathogens has generally been associated
              mos (uma medida conhecida como dN/dS) é           with recent ancestry16. In other words,
              muito maior no MTBC do que na maioria             there has not been enough time of purify-
              das outras bactérias13. Um dN/dS elevado em       ing selection to remove nonsynonymous
              patogénios bacterianos tem normalmente            SNPs, the majority of which tend to be
              sido associado à ancestralidade recente16. Por    slightly deleterious to bacterial fitness.
              outras palavras, ainda não houve tempo sufi-      Because synonymous SNPs accumulate
              ciente para a purificação da seleção eliminar     over time without being removed by na-
              SNP não sinónimos, a maioria dos quais ten-       tural selection a high dN/dS can be in-
              dem a ser ligeiramente nocivos à competência      dicative of recent emergence. However,
              bacteriana. Porque os SNP sinónimos se acu-       our analyses supported the view that the
              mulam ao longo do tempo sem serem elimi-          reason dN/dS is high in MTBC is because
              nados pela seleção natural, um elevado dN/dS      purifying selection in this organism is re-
              pode ser indicador de ocorrência recente. Po-     duced, most likely as consequence of in-
              rém, as nossas análises apoiam a noção de que     creased random genetic drift associated
              a razão dN/dS é elevada no CMTB porque a          with the serial population bottlenecks
              purificação da seleção deste organismo é redu-    during patient-to-patient transmission13.
              zida, provavelmente pela consequência do au-      This suggest that ‘chance’, and not just
              mento aleatório da deslocação genética asso-      natural selection has been driving the evo-
              ciada às acumulações de populações durante a      lution of MTBC.
              transmissão doente a doente13. Isto sugere que    In summary, our MLSA work confirmed
              o ‘acaso’, e não apenas a seleção natural, tem    that the genetic population structure of
              conduzido a evolução do CMTB.                     human MTBC consists of six main strain
              Em resumo, o presente trabalho de análise de      lineages. Our findings also show that the
              sequências multilocos confirmou que a estru-      human MTBC lineages are more geneti-

S 24   R e v i s t a      P o r t u g u e s a                  d e    P n e u m o l o g i a
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Forças evolutivas do Mycobacterium tuberculosis
Sebastien Gagneux

tura genética da população humana de                         cally diverse than previously thought.
CMTB consiste em seis linhagens de estirpes                  There is mounting evidence that MTBC
principais. Os achados mostram também que                    lineage can affect the outcome of tuber-
as estirpes do CMTB humano são mais gene-                    culosis infection and disease17,18,19,20. Our
ticamente diversas do que se pensava anterior-               sequencing work identified many phylo-
mente. Há evidência crescente de que a linha-                genetically informative SNPs, which will
gem de CMTB pode afetar a evolução da                        allow researchers to rapidly classify
tuberculose infeção e doença17-20. O presente                strains into robust groupings10, and to
trabalho de sequenciação identificou muitos                  further explore lineage-specific diffe-
SNPS filogeneticamente informativos, o que                   rences in experimental and clinical con-
permitirá aos investigadores classificar rapida-             texts. Our population genetic analyses
mente estirpes em agrupamentos robustos10 e                  revealed that the genetic diversity in
continuar a explorar diferenças específicas das              MTBC has been shaped by both ancient
linhagens nos contextos experimental e clíni-                and more recent human migrations, and
co. As nossas análises de genética de popula-                that random genetic drift might be an
ções revelaram que a diversidade genética do                 important driving force in the evolution
CMTB foi moldada por migrações humanas                       of MTBC. As DNA sequencing costs
antigas e mais recentes e que a deslocação ge-               continue to decrease, full genome-se-
nética aleatória pode ser uma importante for-                quencing has the potential to become
ça condutora na evolução do CMTB. À me-                      the “ultimate” genotyping tools for bac-
dida que os custos da sequenciação de ADN                    teria21. Comparative genome sequencing
continuam a baixar, a sequenciação do geno-                  of large strain collections of MTBC will
ma completo tem potencial para se tornar o                   improve our understanding of the evolu-
instrumento ‘final’ para genotipagem de bac-                 tionary forces shaping the genetic diver-
térias21. A sequenciação genómica comparati-                 sity in this important pathogen 15.
va de grandes coleções de estirpes de CMTB
melhorará a nossa compreensão das forças
evolutivas que moldam a diversidade genética
deste importante patogénio15.

1. Borrell S, Gagneux S. Infectiousness, reproductive fit-   4. Baker L, Brown T, Maiden MC, Drobniewski F. Si-
ness and evolution of drug-resistant Mycobacterium tu-       lent nucleotide polymorphisms and a phylogeny for My-
berculosis. Int J Tuberc Lung Dis 2009; 13:1456-1466.        cobacterium tuberculosis. Emerg Infect Dis 2004; 10:
2. Sreevatsan S, Pan X, Stockbauer KE, Connell ND,           1568-1577.
Kreiswirth BN, et al. Restricted structural gene poly-       5. Feil EJ. Small change: keeping pace with microevolu-
morphism in the Mycobacterium tuberculosis complex           tion. Nat Rev Microbiol 2004; 2: 483-495.
indicates evolutionarily recent global dissemination.        6. Mathema B, Kurepina NE, Bifani PJ, Kreiswirth
Proc Natl Acad Sci USA 1997; 94: 9869-9874.                  BN. Molecular epidemiology of tuberculosis: cur-
3. Maiden MC. Multilocus sequence typing of bacteria.        rent insights. Clin Microbiol Rev 2006; 19: 658-
Annu Rev Microbiol 2006; 60: 561-588.                        -685.

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                                             Vol XVI Suplemento 1 A Janeiro 2010
                                              Forças evolutivas do Mycobacterium tuberculosis
                                                                                                                Sebastien Gagneux

              7. Achtman M, Wagner M. Microbial diversity and the        evolution of Mycobacterium tuberculosis. Nat Rev
              genetic nature of microbial species. Nat Rev Microbiol     Microbiol 2009; 7: 537-544.
              2008; 6: 431-440.                                          15. Comas I, Gagneux S. The past and future of tuber-
              8. Achtman M. Evolution, population structure, and         culosis research. PLoS Pathog 2009; 5: e1000600.
              phylogeography of genetically monomorphic bacterial        16. Rocha EP, Smith JM, Hurst LD, Holden MT,
              pathogens. Annu Rev Microbiol 2008; 62: 53-70.             Cooper JE, et al. Comparisons of dN/dS are time de-
              9. Pearson T, Okinaka RT, Foster JT, Keim P. Phyloge-      pendent for closely related bacterial genomes. J Theor
              netic understanding of clonal populations in an era of     Biol 2006; 239: 226-235.
              whole genome sequencing. Infect Genet Evol 2009; 9:        17. Caws M, Thwaites G, Dunstan S, Hawn TR, Thi Ngoc
              1010-1019.                                                 Lan N, et al. The influence of host and bacterial genotype
              10. Comas I, Homolka S, Niemann S, Gagneux S. Geno-        on the development of disseminated disease with Mycobac-
              typing of genetically monomorphic bacteria: ADNse-         terium tuberculosis. PLoS Pathog 2008; 4: e1000034.
              quencing in Mycobacterium tuberculosis highlights the      18. de Jong BC, Hill PC, Aiken A, Awine T, Antonio M,
              limitations of current methodologies. PLoS One 2009;       et al. Progression to active tuberculosis, but not trans-
              4: e7815.                                                  mission, varies by Mycobacterium tuberculosis lineage in
              11. Gagneux S, Small PM. Global phylogeography of          the Gambia. J Infect Dis 2008; 198: 1037-1043.
              Mycobacterium tuberculosis and implications for tuber-     19. de Jong BC, Hill PC, Brookes RH, Gagneux S, Jef-
              culosis product development. Lancet Infect Dis 2007; 7:    fries DJ, et al. Mycobacterium africanum elicits an attenua-
              328-337.                                                   ted T cell response to early secreted antigenic target, 6
              12. Gagneux S, Deriemer K, Van T, Kato-Maeda M, de         kDa, in patients with tuberculosis and their household
              Jong BC, et al. Variable host-pathogen compatibility in    contacts. J Infect Dis 2006; 193: 1279-1286.
              Mycobacterium tuberculosis. Proc Natl Acad Sci USA         20. Thwaites G, Caws M, Chau TT, D’Sa A, Lan NT, et
              2006; 103: 2869-2873.                                      al. The relationship between Mycobacterium tuberculo-
              13. Hershberg R, Lipatov M, Small PM, Sheffer H, Nie-      sis genotype and the clinical phenotype of pulmonary
              mann S, et al. High functional diversity in Mycobacte-     and meningeal tuberculosis. J Clin Microbiol 2008; 46:
              rium tuberculosis driven by genetic drift and human de-    1363-1368.
              mography. PLoS Biol 2008; 6:e311.                          21. Medini D, Serruto D, Parkhill J, Relman DA, Do-
              14. Smith NH, Hewinson RG, Kremer K, Brosch R,             nati C, et al. Microbiology in the post-genomic era. Nat
              Gordon SV. Myths and misconceptions: the origin and        Rev Microbiol 2008; 6: 419-430.

S 26   R e v i s t a        P o r t u g u e s a                         d e       P n e u m o l o g i a
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Joseph O Falkinham III1                                    Epidemiologia e ecologia de micobactérias não

                                                           Epidemiology and ecology of nontuberculous

As micobactérias não tuberculosas (MNT) são                        Nontuberculous mycobacteria (NTM) are
patogénios oportunistas dos humanos e animais                      opportunistic human and animal environ-
encontrados no meio ambiente1,2. Nos Estados                       mental pathogens1,2. In the United States,
Unidos, a prevalência de doenças por MNT                           the prevalence of NTM disease is increasing
vem aumentando 8% ao ano, atingindo atual-                         by 8% per year and now stands at almost 35
mente quase 35 casos por cada 100 0003. Em                         cases per 100 0003. In Ontario, Canada the
Ontário, Canadá, a prevalência de doença pul-                      prevalence of NTM pulmonary disease in-
monar por MNT aumentou de 1,5 para 9,0                             creased from 1.5 to 9.0 per 100 000 (6-fold
por 100 000 (6 vezes) no período 1997-20034.                       increase) over the period 1997-20034.
A evidência de que o ambiente é a fonte das                        Evidence that the environment was the
doenças por MNT em seres humanos foi                               source of NTM disease in humans was gained
conseguida a partir do ADN de isolados de                          from the identity of DNA fingerprints of
MNT de doentes com SIDA, de água de be-                            NTM isolates from AIDS patients and drink-
ber5 e de doentes imunocompetentes e de                            ing water5 and from immunocompetent pa-
isolados de MNT de terra de vasos6 ou de um                        tients and NTM isolates from potting soils6
chuveiro doméstico7. Os seres humanos estão                        or a home shower7. Humans are continually
continuamente expostos a MNT, uma vez                              exposed to nontuberculous mycobacteria
que estes vivem e crescem normalmente nos                          (NTM) as they are normal inhabitants and
sistemas de distribuição da água de beber8, e                      grow in drinking water distribution systems8
20% das amostras recolhidas em biofilmes                           and 20 % of showerhead biofilms (swab sam-
nas cabeças dos chuveiros domésticos nos Es-                       ples) collected from households in the United
tados Unidos tinham M. avium9.                                     States had M. avium9.

1   Department of Biological Sciences, Virginia Tech,
    Blacksburg, Virginia 24061-0406
    Phone 1-540-231-5931
    Fax 1-540-231-9307

        R e v i s t a                     P o r t u g u e s a                   d e      P n e u m o l o g i a     S 27
                                                        Vol XVI Suplemento 1 A Janeiro 2010
                   Epidemiologia e ecologia de micobactérias não tuberculosas (MNT)
                                                                                            Joseph O Falkinham III

              Os fatores de risco para doenças por MNT            Risk factors for NTM disease include: re-
              incluem: imunocompetência diminuída devi-           duced immune competence as a result of
              do a infeção por VIH, cancro, quimioterapia,        HIV infection, cancer, chemotherapy, or
              ou imunossupressão associada a transplanta-         immunosuppression associated with trans-
              ção, doença pulmonar preexistente, como             plantation, preexisting lung disease such as
              pneumoconiose, silicose e doença do pulmão          pneumoconiosis, silicosis, and black lung
              negro, alterações na arquitetura normal do          disease, altered normal chest architecture,
              tronco, alcoolismo e hábitos tabágicos10. Re-       alcoholism, and smoking10. Recently, mu-
              centemente, mutações no regulador da con-           tations in the cystic fibrosis transmem-
              dutância transmembranária na fibrose quísti-        brane conductance regulator (CFTR) and
              ca (RCTFQ) e genes de α-1-antitripsina têm          α-1-antitrypsin genes have been associated
              sido associados ao risco aumentado de doença        with increased risk of NTM pulmonary
              pulmonar por MNT11. É preocupante o facto           disease11. Quite alarming is the fact that
              de a doença pulmonar por MNT ter aumen-             pulmonary NTM disease has increased
              tado drasticamente entre homens e mulheres          dramatically amongst elderly slender men
              idosos magros que, não tendo os fatores de          and women, who lack the classic risk fac-
              risco clássicos para doença micobacteriana,         tors for mycobacterial disease, yet appear
              parecem ser substancialmente mais suscetíveis       to be substantially more susceptible to
              a doenças por MNT12. Com o envelhecimen-            NTM disease12. As the human population
              to das populações, é expectável que o número        ages, it would be expected that the number
              de idosos com doença pulmonar por MNT               of elderly with NTM pulmonary disease
              aumente, com o consequente peso para os             will increase, placing further demands on
              prestadores de cuidados de saúde. A terapêu-        healthcare providers. Recommended the-
              tica recomendada para infeções por MNT              rapy for NTM infections include multiple
              inclui múltiplos antibióticos (e.g., claritromi-    antibiotics (e.g., clarithromycin, ethambu-
              cina, etambutol e rifampin) por períodos de         tol, and rifampin) for periods as long as 24
              24 meses13.                                         months13.
              As micobactérias não tuberculosas sobrevi-          NTM survive, grow, and persist in a num-
              vem, crescem e persistem em habitats parti-         ber of habitats that are shared with humans
              lhados por seres humanos e animais. São oli-        and animals. NTM are oligotrophs; able to
              gotróficos, podendo crescer em água com             grow in water containing greater than 50
              mais de 50 μg de carbono orgânico assimilá-         μg assimilable organic carbon (AOC/)L14.
              vel (COA/)L14. A presença de grandes quan-          The presence of high numbers of NTM in
              tidades de MNT em pântanos de águas escu-           coastal acidic, brown water swamps15 and
              ras e ácidas15 e no solo de pinhais16 deve-se,      pine forest soils16 is due, in part, to the
              em parte, ao crescimento estimulado pela            growth stimulation by soil organic matter
              matéria orgânica do solo raramente metabo-          material rarely metabolized by other mi-
              lizada por outros microrganismos, nomeada-          croorganisms; namely humic and fulvic
              mente ácidos húmico e fúlvico17.                    acids17.
              As micobactérias têm uma verdadeira mem-            Mycobacteria have a true outer mem-
              brana externa18, cujos ácidos micólicos de          brane18, whose long chain mycolic acids

S 28   R e v i s t a      P o r t u g u e s a                    d e    P n e u m o l o g i a
                                   Vol XVI Suplemento 1 A Janeiro 2010
Epidemiologia e ecologia de micobactérias não tuberculosas (MNT)
Joseph O Falkinham III

cadeia longa contribuem para a hidrofobici-                 contribute to the hydrophobicity, imper-
dade, impermeabilidade e crescimento lento                  meability, and slow growth of NTM cells19.
das células de MNT19. A hidrofobicidade da                  The surface hydrophobicity of NTM cells
superfície das células de MNT é a mais eleva-               is the highest amongst bacteria and is a
da de todas as bactérias e é uma importante                 major determinant of phagocytosis by
determinante da fagocitose dos macrófagos20.                macrophages20. Although the hydrophobic
Embora a parede hidrofóbica reduza o índice                 wall reduces the rate of transfer of hydro-
de transferência de nutrientes hidrofílicos,                philic nutrients, it contributes to disinfec-
contribui para a resistência a desinfetantes                tant- (e.g., chlorine and biocides) and anti-
(e.g., cloro e biócidos) e a antibióticos21,22.             biotic-resistance21,22.
As MNT penetram nos sistemas de trata-                      NTM enter a water treatment system on
mento de águas aderentes a partículas, sobre-               particulates, survive disinfection, and
vivem à desinfeção e crescem em biofilmes na                grow in biofilms in the absence of com-
ausência de competidores mortos pela disin-                 petitors killed by disinfection8. Biofilm
feção8. A formação de biofilme também au-                   formation also increases NTM resistance
menta a resistência das MNT a desinfetan-                   to disinfectants23 and antibiotics24. Hydro-
tes23 e a antibióticos24. A hidrofobicidade                 phobicity also promotes the aerosolization
também promove a aerossolização de mico-                    of mycobacteria from water to air in envi-
bactérias da água para o ar em ambientes                    ronments such as showers and hot tubs in
como chuveiros e banheiras domésticos e nas                 the home and occupations where aerosols
profissões onde se manuseiam aerossóis25.                   are generated25.

1. Wallace RJ, Brown BA, Griffith DE. Nosocomial            7. Falkinham JO III, et al. Mycobacterium avium in a
outbreaks pseudo-outbreaks caused by nontuberculous         shower linked to pulmonary disease. J Water Health
mycobacteria. Annu Rev Microbiol 1998; 52;453-490.          2008; 6;209-213.
2. Falkinham JO III. Surrounded by mycobacteria: non-       8. Falkinham JO III, Norton CD, LeChevallier MW.
tuberculous mycobacteria in the human environment. J        Factors influencing numbers of Mycobacterium avium,
Appl Microbiol 2009; 107;356-367.                           Mycobacterium intracellulare, and other Mycobacteria
3. Iseman MD, Marras TK. The importance of nontu-           in drinking water distribution systems. Appl Environ
berculous mycobacterial lung disease. Am J Respir Crit      Microbiol 2001; 67;1225-1231.
Care Med 2008; 178:999-1000.                                9. Feazel LM, et al. Opportunistic pathogens enriched
4. Marras TK, et al. Isolation prevalence of pulmonary      in showerhead biofilms. Proc Natl Acad Sci USA
non-tuberculous mycobacteria in Ontario, 1997-2003.         2009;106;16393-16399.
Thorax 2007; 62;661-666.                                    10. Marras TK, Daley CL. Epidemiology of human pul-
5. von Reyn CF, et al. Persistent colonisation of potable   monary infection with nontuberculous mycobacteria.
water as a source of Mycobacterium avium infection in       Clin Chest Med 2002; 23;553-567.
AIDS. Lancet 1994;343;1137-1141.                            11. Kim JS, et al. Nontuberculous mycobacterial infec-
6. De Groote MA, et al. Relationships between Myco-         tion: CT scan findings, genotype, and treatment res-
bacterium isolates from patients with pulmonary myco-       ponsiveness. Chest 2005; 128;3863-3869.
bacterial infection and potting soils. 2006; Appl Envi-     12. Kennedy TP, Weber DJ. Nontuberculous mycobac-
ron Microbiol 2006;72;7602-7606.                            teria – an underappreciated cause of geriatric lung di-

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                                            Vol XVI Suplemento 1 A Janeiro 2010
                    Epidemiologia e ecologia de micobactérias não tuberculosas (MNT)
                                                                                                               Joseph O Falkinham III

              sease. Am Rev Respir Crit Care Med 1994;149;1654-              19. Brennan PJ, Nikaido H. The envelope of mycobac-
              -1658.                                                         teria. Annu Rev Biochem 1995; 64;29-63.
              13. Griffith DE, et al. An official ATS/IDSA statement:        20. van Oss CJ, Gillman CF, Neumann AW. Phagocytic
              Diagnosis, treatment, and prevention of nontubercu-            engulfment and cell adhesiveness. 1975; Marcel Dekker,
              lous mycobacterial diseases. Am J Respir Crit Care Med         New York.
              2007; 175;367-416.                                             21. Rastogi N, et al. Multiple drug resistance in Myco-
              14. Norton CD, LeChevallier MW, Falkinham JO III.              bacterium avium – Is the wall architecture responsible
              Survival of Mycobacterium avium in a model distribu-           for the exclusion of antimicrobial agents. Antimicrob
              tion system. Water Res 2005; 38;1457-1466.                     Agents Chemother 1981; 20;666-677.
              15. Kirschner RA Jr, Parker BC, Falkinham JO III. Epi-         22. Taylor R, et al. Chlorine, chloramine, chlorine diox-
              demiology of infection by nontuberculous mycobacteria.         ide, and ozone susceptibility of Mycobacterium avium.
              Mycobacterium avium, Mycobacterium intracellulare, and         Appl Environ Microbiol 2000; 66;1702-1705.
              Mycobacterium scrofulaceum in acid, brown-water swamps         23. Steed KA Falkinham JO III. Effect of growth in bio-
              of the southeastern United States and their association        films on chlorine susceptibility of Mycobacterium avium
              with environmental variables. Am Rev Respir Dis 1992;          and Mycobacterium intracellulare. Appl Environ Micro-
              145;271-275.                                                   biol 2006; 72;4007-4011.
              16. Iivanainen EK, et al. Mycobacteria in boreal coniferous    24. Falkinham, JO III. Growth in catheter biofilms and
              forest soils. FEMS Microbiol Ecol 1997;23;325-332.             antibiotic resistance of Mycobacterium avium. J Med
              17. Kirschner RA, Parker BC, Falkinham JO III. Hu-             Microbiol 2007; 56;250-254.
              mic and fulvic acids stimulate the growth of Mycobacte-        25. Parker BC, George KL, Falkinham JO III. Epidemio-
              rium avium. FEMS Microbiol Ecol 1995; 30;327-332.              logy of infection by nontuberculous mycobacteria. IV.
              18. Hoffmann C, et al. Disclosure of the mycobacterial         Preferential aerosolization of Mycobacterium intracellu-
              outer membrane: Cryo-electron tomography and vitre-            lare from natural waters. Am Rev Respir Dis 1984;
              ous sections reveal the lipid bilayer structure. Proc Natl     123;652-656.
              Acad Sci USA 2008; 105;3963-3967.

S 30   R e v i s t a         P o r t u g u e s a                            d e      P n e u m o l o g i a
                                        Vol XVI Suplemento 1 A Janeiro 2010
Jean-Pierre Zellweger1                            O uso da análise de libertação de gama interferão como
                                                  auxiliar no controlo da tuberculose

                                                  The use of interferon gamma release assays as an aid in
                                                  the control of tuberculosis

As análises de libertação de gama interferão              Interferon Gamma Release Assays (IGRAs)
(IGRA) são testes in vitro que detetam a                  are in vitro tests detecting the presence of
presença de infeção por tuberculose latente               latent tuberculosis infection (LTBI) in as-
(ITBL) em pessoas assintomáticas que po-                  ymptomatic persons who may have been
dem ter sido infetadas por Mycobacterium                  infected by Mycobacterium tuberculosis in a
tuberculosis num passado recente ou remoto                recent or remote past and who may benefit
e que podem beneficiar dum tratamento                     from a preventive treatment to decrease the
preventivo para diminuir o risco de reativa-              risk of later reactivation of tuberculosis1, 2.
ção posterior da tuberculose1, 2.                         Contact investigation is the search for se-
A investigação dos contactos é a busca de casos           condary cases of tuberculosis among the
secundários de tuberculose entre os contactos             contacts of a primary (index) case and the
dum caso primário, e a busca de contactos com             search for contacts with a latent tuberculosis
uma infeção latente de tuberculose que possam             infection who may benefit from a preven-
beneficiar dum tratamento preventivo de                   tive treatment in order to avoid the future
modo a evitar a futura progressão para a doen-            progression to active disease. This is the se-
ça ativa. Esta é a segunda prioridade no contro-          cond priority for the control of tuberculosis,
lo da tuberculose, depois da deteção e trata-             after the detection and treatment of active
mento dos casos ativos. Se feito de modo                  cases. If conducted in a systematic way and
sistemático, e em países com entidades bem                in countries with a well-organized TB ma-
organizadas no controlo da TB, esta atividade             nagement team, this activity may be very
pode ser eficaz em termos de custo.                       cost-effective.
A deteção de casos secundários assenta essen-             The detection of secondary cases relies
cialmente nos exames clínico, radiológico e               mainly on the clinical, radiological and bac-
bacteriológico dos contactos com queixas. A               teriological examination of contacts with

1 Swiss Lung Association, Berne, Switzerland


     R e v i s t a                    P o r t u g u e s a              d e       P n e u m o l o g i a      S 31
                                               Vol XVI Suplemento 1 A Janeiro 2010
                    O uso da análise de libertação de gama interferão como auxiliar
                                                        no controlo da tuberculose
                                                                                              Jean-Pierre Zellweger

              deteção de contactos com infeção latente de        complaints. The detection of contacts with
              tuberculose, por definição assintomática,          a latent tuberculosis infection, by definition
              apoia-se no uso de testes indiretos capazes de     asymptomatic, relies on the use of indirect
              detetar a sensibilização anterior de linfócitos    tests able to detect the prior sensitization of
              T por antigénios micobacterianos. O tradi-         T lymphocytes by mycobacterial antigens.
              cional teste cutâneo de tuberculina (TCT),         The time-honoured tuberculin skin test
              com que se faz a maioria dos estudos epide-        (TST), with which most of the epidemio-
              miológicos, enferma de graves deficiências,        logical studies are performed, suffers from
              entre as quais a baixa especificidade. As novas    severe deficiencies, among which a low
              análises de libertação de gama interferão,         specificity. The new Interferon-Gamma Re-
              com a sua alta especificidade, são muito mais      lease Assays, with their high specificity, are
              promissoras e permitem uma melhor seleção          much more promizing and allow a better
              dos contactos que podem beneficiar duma            targeting of the contacts who may benefit
              terapêutica preventiva, evitando a prescrição      from a preventive therapy, avoiding the
              dum tratamento desnecessário para contac-          prescription of an unnecessary treatment to
              tos não infetados que podem ter um TCT             uninfected contacts who may have a false-
              falso positivo, devido, por exemplo, ao BCG,       positive TST, for instance due to BCG,
              ao efeito booster ou à sensibilização com mi-      booster effect or sensitization with non-tu-
              cobactérias não tuberculosas3.                     berculous mycobacteria3.
              Basicamente, os testes IGRA assentam no            Basically, the IGRA tests rely on the same
              mesmo fenómeno imunológico dos testes              immunological phenomenon as the tuber-
              cutâneos de tuberculina, mas duma maneira          culin skin tests, but they do it in a much
              muito mais específica, porque os testes não        more specific way, because the tests are not
              são influenciados por uma anterior vacina-         influenced by a prior vaccination with BGC
              ção com BGC ou por uma infeção com a               or by an infection with most of the non-tu-
              maioria das micobactérias não tuberculosas         berculous mycobacteria present in the envi-
              presentes no ambiente. Assim, as indicações        ronment. Therefore, the indications and the
              e o uso de testes IGRA são fundamental-            use of the IGRA tests are fundamentally the
              mente as mesmas dos testes cutâneos de tu-         same as for the tuberculin skin tests4:
                                                                 1. Detection of LTBI in persons in contact
              1. Deteção de ITBL em pessoas em con-                 with an index case of tuberculosis;
                 tacto com um caso primário de tuber-            2. Detection of LTBI in persons with a
                 culose;                                            high risk of tuberculosis, if infected
              2. Deteção de ITBL em pessoas com alto                (immunosuppressed patients, patients
                 risco de tuberculose, se infetadas (doen-          receiveing or due to receive immuno-
                 tes imunossuprimidos, doentes a rece-              suppressive therapy, small children);
                 ber ou programados para receber terapia         3. Surveillance of exposed health care
                 imunossupressora, crianças);                       workers (as the test can be repeated
              3. Vigilância de trabalhadores dos serviços           without risk of inducing a booster ef-
                 de saúde expostos (uma vez que o teste             fect);

S 32   R e v i s t a      P o r t u g u e s a                   d e     P n e u m o l o g i a
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O uso da análise de libertação de gama interferão como auxiliar
no controlo da tuberculose
Jean-Pierre Zellweger

   pode ser repetido sem risco de indução        4. Aid to the diagnosis of tuberculosis in
   do efeito booster);                              cases where a bacteriological examina-
4. Auxiliar no diagnóstico de tuberculose           tion is not feasible or not reliable (severe
   em casos em que o exame bacteriológi-            extrapulmonary TB, TB in children).
   co não é viável ou fiável (TB extra-
   pulmonar grave, TB em crianças).              The level of IGRA response is proportional
                                                 with the intensity of exposure to tuberculo-
O nível de resposta ao IGRA é proporcional       sis, and may change under preventive or cu-
à intensidade da exposição à tuberculose e       rative treatment, possibly indicating that
pode mudar sob tratamento preventivo ou          the number of living mycobacteria has also
curativo, possivelmente indicando que o nú-      decreased. It is therefore possible that the
mero de micobactérias vivas também dimi-         change in the level of IGRA reflects the ef-
nuiu. Assim, é possível que a alteração no       fect of treatment, but there is unfortunately
nível de IGRA reflita o efeito do tratamento,    up to now no solid proof that a decrease in
mas infelizmente, até agora, não foi possível    the level is clearly correlated with the eradi-
provar que a diminuição do nível esteja clara-   cation of mycobacteria and could be used as
mente relacionada com a erradicação de mi-       a documentation of cure5. Furthermore,
cobactérias e possa ser usada para documen-      there is no proof that a positive test result
tar a cura5. Além disso, não está provado que    always documents the persistence of living
um teste positivo evidencie sempre a persis-     mycobacteria. It may be that the test only
tência de micobactérias vivas. É possível que    detects a lasting immune response to a prior
o teste detete apenas uma resposta imune a       contact with mycobacteria that have disap-
um anterior contacto com micobactérias que,      peared in between 6
entretanto, tenham desaparecido6                 IGRAs may contribute to the diagnosis
Os testes IGRA podem contribuir para o           of difficult TB cases. In smear-negative
diagnóstico de casos difíceis de TB. Na          pulmonary tuberculosis and in extrapul-
tuberculose pulmonar com um esfregaço            monary tuberculosis, it may be very dif-
negativo, e na tuberculose extrapulmonar         ficult to obtain the bacteriological docu-
pode ser muito difícil obter evidência bac-      mentation of the presence of mycobacteria.
teriológica da presença de micobactérias.        The release of interferon-gamma from
A libertação de gama interferão de linfó-        lymphocytes isolated from the organs po-
citos isolados a partir de órgãos potencial-     tentially involved in cases of suspect tu-
mente envolvidos em casos suspeitos de           berculosis (BAL in smear-negative pul-
tuberculose (lavagem broncoalveolar na           monary tuberculosis, CSF in tuberculous
tuberculose pulmonar com um esfregaço            meningitis, peritoneal fluid in abdominal
negativo, líquido cerebrospinal na menin-        tuberculosis) can be measured and is ele-
gite tuberculosa, líquido peritoneal na tu-      vated in cases with a final diagnosis of
berculose abdominal) pode ser medida e           tuberculosis. In such cases, the determi-
está elevada em casos com um diagnóstico         nation of the level of Interferon-Gamma
final de tuberculose. Nesses casos, a deter-     release can be used as an aid to the diag-
minação do nível de libertação de gama           nosis of tuberculosis7.

    R e v i s t a          P o r t u g u e s a                d e       P n e u m o l o g i a      S 33
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                     O uso da análise de libertação de gama interferão como auxiliar
                                                         no controlo da tuberculose
                                                                                                   Jean-Pierre Zellweger

              interferão pode ajudar no diagnóstico da               IGRAs may predict the future development
              tuberculose7.                                          of TB disease. Not all cases in contact with
              Os testes IGRA podem indicar o futuro de-              a case of tuberculosis will be infected and
              senvolvimento da doença. Nem todas as pes-             not all infected contacts will develop tuber-
              soas em contacto com um caso de tuberculo-             culosis. The risk of reactivation of tubercu-
              se são infetadas e nem todos os contactos              losis is estimated to be 10% for contacts
              infetados virão a desenvolver tuberculose.             with a positive tuberculin skin test. Recent
              Estima-se o risco de reativação da tuberculo-          studies have demonstrated that the risk of
              se como sendo de 10% para contactos com                future reactivation is higher in contacts
              um TCT positivo. Estudos recentes demons-              with a positive IGRA test than in negative
              traram que o risco de futura reativação é              contacts, independently from the result of
              maior em contactos com um IGRA positivo                the tuberculin skin test8. Therefore, a posi-
              do que em contactos negativos, independen-             tive IGRA test result may have a higher
              temente do resultado do TCT8. Por isso, um             predictive value for the risk of future reacti-
              IGRA positivo pode ter maior valor preditivo           vation than the tuberculin skin test. As the
              para risco de reativação futura do que o TCT.          proportion of contacts with a positive
              Como a proporção de contactos com um                   IGRA test is lower than the proportion of
              IGRA positivo é menor do que a dos contac-             contacts with a positive tuberculin skin test,
              tos com um teste cutâneo de tuberculina po-            the number of contacts considered as in-
              sitivo, o número de contactos considerados             fected and who may benefit from a preven-
              infetados e que podem beneficiar de um tra-            tive treatment is lower if IGRA are used as
              tamento preventivo é menor se os testes                a definition. Using IGRA for the detection
              IGRA forem usados como definição. A utili-             of infected contacts and selection of con-
              zação de testes IGRA na deteção de contactos           tacts in need of a preventive treatment has
              infetados e na seleção de contactos que neces-         therefore a sparing effect9.
              sitam de tratamento preventivo tem, assim,             In spite of their superiority, the IGRAs are
              um efeito moderador9.                                  not totally devoid of problems in practice
              Apesar da sua superioridade, os testes IGRA            and the best use of them is still a matter of
              não são totalmente isentos de problemas na             debate. The intra-observer and inter-ob-
              prática e a sua melhor utilização ainda vem sen-       server variability are low, but the level of re-
              do debatida. A variabilidade inter e intra-            sponse may vary if the test is repeated and
              observador é baixa, mas o nível de resposta pode       there are spontaneous conversions and re-
              variar se o teste for repetido, verificando-se con-    versions in the absence of exposure or treat-
              versões e reversões espontâneas na ausência de         ment10-12. More of the changes are observed
              exposição ou de tratamento10-12. Observam-se           for cases with borderline responses. There-
              ainda mais alterações nos casos com respostas          fore, proposals have been made for an in-
              borderline. Assim, têm sido propostos aumentos         crease in the cut-off for positivity and for
              no cut-off da positividade e na definição da con-      the definition of conversion in repeated
              versão na repetição dos testes13.                      testing13.
              Algumas linhas de orientação recomendam                Some Guidelines recommend their use only
              o seu uso apenas para confirmação dum                  for the confirmation of positive TST among

S 34   R e v i s t a       P o r t u g u e s a                      d e     P n e u m o l o g i a
                                    Vol XVI Suplemento 1 A Janeiro 2010
O uso da análise de libertação de gama interferão como auxiliar
no controlo da tuberculose
Jean-Pierre Zellweger

TCT positivo entre contactos (o chamado                       contacts (the so-called two-step testing pro-
teste de dois passos), enquanto outras reco-                  cedure) whereas others recommend the rou-
mendam a substituição do TCT de rotina                        tine replacement of the TST by IGRAs. Per-
por IGRA. Fazer um só teste é mais fácil e                    forming only one test is easier, and avoids a
evita a possível influência de um TCT ante-                   possible influence of a prior TST on the
rior na resposta do IGRA.                                     IGRA response.

1. Menzies D, Pai M, Comstock G. Meta-analysis: new           8. Diel R, Loddenkemper R, Meywald-Walter K, Nie-
tests for the diagnosis of latent tuberculosis infection:     mann S, Nienhaus A. Predictive value of a whole-blood
areas of uncertainty and recommendations for research.        IFN-{gamma} assay for the development of active TB
Ann Intern Med 2007; 146(5):340-354.                          disease. Am J Respir Crit Care Med 2008; 177:1164-
2. Pai M, Zwerling A, Menzies D. Systematic review:           -1170.
T-cell-based assays for the diagnosis of latent tuberculo-    9. Diel R, Nienhaus A, Lange C, Schaberg T. Cost opti-
sis infection: an update. Ann Intern Med 2008;                mization of screening for latent tuberculosis in close
149(3):177-184.                                               contacts. Eur Respir J 2006; 28:35-44.
3. Diel R, Loddenkemper R, Meywald-Walter K,                  10. Pai M, Joshi R, Dogra S, et al. T-cell assay conver-
Gottschalk R, Nienhaus A. Comparative performance             sions and reversions among household contacts of tu-
of tuberculin skin test, QuantiFERON-TB-Gold in               berculosis patients in rural India. Int J Tuberc Lung Dis
tube assay, and T-Spot.TB test in contact investigations      2009; 13(1):84-92.
for tuberculosis. Chest 2009; 135(4):1010-1018.               11. van Zyl-Smit RN, Pai M, Peprah K, et al. Within-
4. Zellweger JP. Latent tuberculosis: which test in which     -subject variability and boosting of T-cell interferon-
situation? Swiss Med Wkly 2008; 138(3-4):31-37.               -gamma responses after tuberculin skin testing. Am J
5. Pai M, Menzies D. Interferon-gamma release assays:         Respir Crit Care Med 2009; 180(1):49-58.
what is their role in the diagnosis of active tuberculosis?   12. Detjen AK, Loebenberg L, Grewal HM et al. Short-
Clin Infect Dis 2007; 44(1):74-77.                            -term reproducibility of a commercial interferon-gamma
6. Mack U, Migliori GB, Sester M, et al. LTBI: latent         release assay. Clin Vaccine Immunol 2009; 16:1170-
tuberculosis infection or lasting immune responses to         -1175
M. tuberculosis? A TBNET consensus statement. Eur             13. Pai M, Dendukuri N, Wang L, Joshi R, Kalantri S,
Respir J 2009; 33(5):956-973.                                 Rieder HL. Improving the estimation of tuberculosis
7. Jafari C, Ernst M, Strassburg A, et al. Local immuno-      infection prevalence using T-cell-based assay and mix-
diagnosis of pulmonary tuberculosis by enzyme-linked          ture models. Int J Tuberc Lung Dis 2008;12(8):895-
immunospot. Eur Respir J 2008; 31(2):261-265.                 -902.

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                                              Vol XVI Suplemento 1 A Janeiro 2010
Lee W Riley1                                              Regulação da composição lipídica da parede celular do
                                                          Mycobacterium tuberculosis e o seu efeito na persistência
                                                          bacteriana in vitro

                                                          Regulation of Mycobacterium tuberculosis cell wall lipid
                                                          composition and its effects on in vitro bacterial

Introdução                                                          Introduction
A marca da infeção humana por Mycobacte-                            The hallmark of human infection by My-
rium tuberculosis é a sua capacidade de per-                        cobacterium tuberculosis is its ability to re-
manecer latente durante vários anos, com                            main latent for many years, only to reacti-
posterior reativação para doença ativa (tu-                         vate to cause active disease (tuberculosis).
berculose). Pouco se sabe sobre como este                           Very little is known about how this orga-
organismo permanece em latência e o que                             nism remains latent and what triggers it to
desencadeia a sua reativação. Frequente-                            reactivate. Much attention is often paid to
mente atribui-se aos lípidos do M. tubercu-                         the lipids of M. tuberculosis as playing a
losis um papel na patogénese, porque grande                         role in pathogenesis. This is because a large
parte do genoma do M. tuberculosis é dedi-                          proportionof the genome of M. tuberculo-
cado à biossíntese e à degradação dos lípi-                         sis is dedicated to the biosynthesis and
dos1. Só no metabolismo dos ácidos gordos                           degradation of lipids1. Nearly 250 genes
quase 250 genes estão envolvidos, compara-                          are involved in metabolism of fatty acids
tivamente com apenas cerca de 50 em orga-                           alone, compared to only about 50 in or-
nismos como a Escherichia coli. Mais de                             ganisms such as Escherichia coli. More than
50% do peso seco da parede celular do M.                            50% of the dry weight of the cell wall of
tuberculosis é composto por lípidos1-3 e, além                      M. tuberculosis is composed of lipids1-3,
disso, o organismo utiliza os lípidos como                          and furthermore, the organism utilizes
fonte de energia durante a sua persistência                         lipids as energy source during its persis-
num mamífero hospedeiro4. Esta despro-                              tence in a mammalian host4. This dispro-
porcionada atenção dispensada ao metabo-                            portionate attention paid to lipid metabo-
lismo lipídico é muito provavelmente uma                            lism is very likely a major strategy this
estratégia adotada por este organismo como                          organism has adopted to defend against

1 MD, School of Public Health, University of California, Berkeley


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                                                     Vol XVI Suplemento 1 A Janeiro 2010
Regulação da composição lipídica da parede celular do Mycobacterium tuberculosis
                                    e o seu efeito na persistência bacteriana in vitro
                                                                                                      Lee W Riley

               defesa contra o ambiente hostil do seu ni-         the hostile environment of its natural
               cho natural – os granulomas humanos.               niche – the human granulomas.
               Os ácidos micólicos são componentes lipídi-        Mycolic acids are the major lipid consti-
               cos importantes e os mais abundantes ácidos        tutent and the most abundant fatty acid
               gordos na parede celular do M. tuberculosis5,6.    found in the cell wall of M. tuberculosis5,6.
               O envelope celular do M. tuberculosis contém       The M. tuberculosis cell envelope contains 3
               três classes de micolatos – ácidos alfa, ceto e    classes of mycolates-alpha, keto- and me-
               metoximicólicos6. Os micolatos estão nor-          thoxy-mycolic acid6. Mycolates are normally
               malmente ligados à camada arabinogalactana         attached to the arabinogalactan layer and
               e ao carboidrato de trealose6,7. As alterações     carbohydrate trehalose6,7. Structural altera-
               estruturais dos ácidos micólicos afetam a viru-    tions of mycolic acids affect the virulence
               lência do M. tuberculosis. A ausência de ácido     property of M. tuberculosis. Absence of oxy-
               micólico oxigenado está associada à alteração      genated mycolic acid is associated with at-
               do M. tuberculosis num modelo de rato8. A          tenuation of M. tuberculosis in a mouse
               cisciclopropanação de ácidos micólicos me-         model8. Cis-cyclopropanation of mycolic
               diados por ciclopropano sintetase é necessária     acids mediated by cyclopropane synthase is
               à formação do fator corda e à virulência em        needed for cord formation as well as for
               ratos9,10. Por outro lado, a transciclopropana-    virulence in mice9,10. On the other hand,
               ção de ácidos micólicos oxigenados é impor-        transcyclopropanation of oxygenated my-
               tante para a virulência na direção oposta. Um      colic acids is important for virulence in the
               mutante do M. tuberculosis sem anéis transci-      opposite direction. An M. tuberculosis mu-
               clopropanos nestes ácidos micólicos é hipervi-     tant lacking transcyclopropane rings in
               rulenta no modelo de rato11.                       these mycolic acids is hypervirulent in the
               O conjunto destas observações sugere um            mouse model11.
               papel importante dos lípidos e, em particu-        Taken together, these observations suggest
               lar, dos ácidos micólicos, na modificação da       an important role for lipids, and in particu-
               resposta imune do hospedeiro e no poste-           lar, mycolic acids, in modifying host im-
               rior resultado clínico. Os autores vêm estu-       mune response and the subsequent clinical
               dando a regulação dos lípidos do M. tuber-         outcome. We have been studying the regu-
               culosis, com particular atenção a um               lation of M. tuberculosis lipids focusing on a
               conjunto de operões que parecem servir             set of operons that appear to serve as lipid
               como sistemas transportadores de lípidos na        transporter systems in the cell wall – mce
               parede celular – mce operões.                      operons.

               Papel dos mce operões na                           Role of the mce operons in cell
               modificação da estrutura da                        wall structural modification
               parede celular                                     M. tuberculosis contains 4 homologous co-
               O Mycobacterium tuberculosis contém qua-           pies of an operon designated mce1-41. We
               tro cópias homólogas de um operão desig-           showed that M. tuberculosis disrupted in the
               nado mce1-41. Já demonstrámos que o M.             mce1 operon is hypervirulent in mice12. The

S 38   R e v i s t a       P o r t u g u e s a                   d e     P n e u m o l o g i a
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Regulação da composição lipídica da parede celular do Mycobacterium tuberculosis
e o seu efeito na persistência bacteriana in vitro
Lee W Riley

tuberculosis danificado no operão mce1 é hi-   mutant cannot elicit a strong Th1-type im-
pervirulento em ratos12. O mutante não         mune response and causes aberrant migra-
provoca uma forte resposta imune do tipo       tion of pro-inflammatory cells, resulting in
Th1 e causa migração aberrante de células      poorly organized granulomas in the mouse
pró-inflamatórias, resultando em granulo-      lungs12. The lack of adequate Th-1 type res-
mas mal organizados nos pulmões dos ra-        ponse and organized granuloma formation
tos12. A falta de resposta imune do tipo Th1   precludes control of bacterial proliferation
adequada e de formação granulomatosa or-       in mice, which leads to early death in this
ganizada impede o controlo da proliferação     animal. This observation was made in im-
bacteriana no rato, o que leva à morte pre-    munocompetent BALB/c as well as in
matura neste animal. Esta observação foi       C57BL/6 mice, both of which exhibit dif-
obtida em ratos BALB/c e C57BL/6 imu-          ferential Th1 type response to wild type M.
nocompetentes, exibindo ambos resposta         tuberculosis infection12,13.
diferencial tipo Th1 à infeção por M. tuber-   The mce1 operon is negatively regulated
culosis do tipo selvagem12,13.                 intracellularly by mce1R, located imme-
O mce1 operão é regulado negativamente         diately upstream14. Mce1R belongs to the
no meio intracelular por mce1R, localizado     FadR subfamily of GntR transcriptional
imediatamente acima14. O mce1R pertence à      regulators1,14. A mutant disrupted in mce1R
subfamília FadR de reguladores transcricio-    gene is also hypervirulent in mice but for
nais GntR1,14. Um mutante do gene mce1R        an opposite reason. It causes accelerated
também é hipervirulento em ratos, mas pela     immunopathologic response with rapid
razão oposta, pois causa uma resposta imu-     progression to death of the animal follow-
nopatológica acelerada, com rápida progres-    ing massive granuloma formation in their
são para a morte do animal após maciça for-    lungs15. The animal dies, not from bacte-
mação granulomatosa nos pulmões15. A           rial proliferation but from the hyper-proin-
morte do animal deve-se, não à proliferação    flammatory response. Therefore, we dis-
bacteriana, mas à resposta hiperpró-           covered that the absence of the mce1 operon
-inflamatória. Descobrimos, assim, que a       expression (as with the mce1 operon mu-
ausência de expressão do operão mce1 (como     tant) is associated with poorly organized
com o operão mce1 mutante) está associada      granulomas and aberrant pro-inflammato-
aos granulomas mal organizados e migração      ry cell migration, while its constitutive ex-
aberrante de células pró-inflamatórias, en-    pression (as with the mce1R mutant) causes
quanto a sua expressão constitutiva (como      massive granuloma formation in mouse
com o mutante mce1R) causa maciça forma-       lungs. Both of these outcomes result in ad-
ção granulomatosa nos pulmões dos ratos.       verse clinical outcomes in mice. We there-
Ambos os efeitos resultam em consequên-        fore reasoned that the mce1 operon serves
cias clínicas adversas nestes animais, pelo    to homeostatically regulate the host im-
que achámos que o mce1 operão serve para       mune response to maintain granuloma
regular homeostaticamente a resposta imu-      structures that allow not only the host to
ne do hospedeiro para manter as estruturas     survive but also for M. tuberculosis to re-
granulomatosas que permitem, não só a so-      main persistent.

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                                   e o seu efeito na peresistência bacteriana in vitro
                                                                                                       Lee W Riley

               brevivência do hospedeiro, mas também a              A phylogenomic analysis of the M. tubercu-
               persistência do M. tuberculosis.                     losis mce operons showed that related ope-
               A análise filogenómica dos operões mce do M.         rons are found among most members of
               tuberculosis mostrou que operões relacionados        Actinomycetales and that they encode a fam-
               se encontram entre a maioria dos membros de          ily of ABC lipid uptake transporters16. One
               Actinomycetales e que estes codificam uma fa-        of the gene products of the mce1 operon is
               mília de transportadores ABC de captação de          FadD5, which is similar in sequence (43%)
               lípidos16. Um dos produtos do operão mce1 é          to the E. coli fatty-acyl-CoA synthetase
               o FadD5, semelhante na sequência (43%) em            FadD17. In addition, the M. tuberculosis Mc-
               E. coli da acil-CoA sintetase FadD17. Além           e1R protein is 33% identical to the FadR
               disso, a proteína Mce1R do M. tuberculosis é         transcriptional repressor protein found in E.
               33% idêntica à proteína repressora transcri-         coli, which binds fatty-acyl CoA and indu-
               cional FadR encontrada em E. coli, que se liga       ces the expression of genes involved in fatty
               à acyl CoA-gorda e induz a expressão dos ge-         acid degradation and transport18.
               nes envolvidos na degradação e transporte dos        M. tuberculosis disrupted in fadD5 is atte-
               ácidos gordos18.                                     nuated in mice and diminished in growth in
               O M. tuberculosis danificado em fadD5 é ate-         vitro in minimal medium supplied with my-
               nuado em ratos e tem crescimento reduzido            colic acid as the sole carbon source19. Thus,
               in vitro em meio mínimo, com ácido micóli-           FadD5 may serve to recycle mycolic acids
               co como única fonte de carbono19. Assim,             that may be released from dying M. tubercu-
               FadD5 pode servir para reciclar ácidos micó-         losis during a natural course of infection.
               licos que possam ser libertados na morte do          Such a recycling mechanism may contribute
               M. tuberculosis durante o desenvolvimento            to the long-term survival of live population
               natural da infeção. Este mecanismo de reci-          of M. tuberculosis in a granuloma environ-
               clagem pode contribuir para a sobrevivência          ment. Thus, the mce1 operon may comprise
               a longo prazo da população viva de M. tuber-         a mycolic acid import system.
               culosis num ambiente granulomatoso. Deste            Interestingly, another group proposed that
               modo, o operão mce1 pode incluir um siste-           the mce4 operon may serve as a cholesterol
               ma de importação de ácidos micólicos.                import system20. At this time, the function
               Curiosamente, outro grupo propôs que o               of mce2 and mce3 operons as lipid trans-
               operão mce4 pode ser um sistema de impor-            porters is not established.
               tação de colesterol20. Atualmente, a função          Nevertheless, these observations suggest
               dos operões mce2 e mce3 como transporta-             that during different stages of infection,
               dores de lípidos ainda não está estabelecida.        M. tuberculosis utilizes a variety of carbon
               No entanto, estas observações sugerem que,           sources for its long-term survival using
               durante diferentes estádios da infeção, o M.         several distinct lipid assimilation systems.
               tuberculosis utiliza uma variedade de fontes de      These carbon sources may become diffe-
               carbono para a sua sobrevivência a longo pra-        rentially available to M. tuberculosis du-
               zo, utilizando vários sistemas distintos de assi-    ring the turnover of granuloma cells.
               milação de lípidos. Estas fontes de carbono          Some of the lipids may be released from
               podem tornar-se diferencialmente disponíveis         these cells turning over, while others may

S 40   R e v i s t a       P o r t u g u e s a                     d e    P n e u m o l o g i a
                                     Vol XVI Suplemento 1 A Janeiro 2010
Regulação da composição lipídica da parede celular do Mycobacterium tuberculosis
e o seu efeito na persistência bacteriana in vitro
Lee W Riley

ao M. tuberculosis durante o turnover das célu-     come from a subset of M. tuberculosis cells
las granulomatosas, quando alguns dos lípidos       that are killed by effector molecules pro-
podem ser libertados por estas células, enquan-     duced by cells that comprise the granulo-
to outros podem advir de um subgrupo de             mas. In all of these situations, M. tubercu-
células de M. tuberculosis mortas pelas molécu-     losis appears to be assured of continued
las efetoras produzidas por células que consti-     supply of energy for a long period of time.
tuem os granulomas. Em todas estas situações,       Thus, any disruption of this balance be-
o M. tuberculosis parece ter o fornecimento         tween bacterial population and granulo-
contínuo de energia assegurado por um longo         ma cell turnover could lead to either com-
período de tempo. Assim, qualquer interrup-         plete elimination of the bacteria (e.g., by
ção deste equilíbrio entre a população bacte-       treatment with anti-tuberculosis drugs)
riana e o turnover das células granulomatosas       or active disease production (e.g., from
poderia levar à completa eliminação das bacté-      immunosuppression, old age, high initial
rias (e.g., por tratamento com fármacos anti-       infectious inoculum, and certain strain-
tuberculose) ou à produção da doença ativa          related factors).
(e.g., por imunossupressão, idade avançada,
inóculo infeccioso inicial elevado e certos fato-
res relacionados com a estirpe).                    Conclusions
                                                    The highly conserved mce-operon-like
                                                    ABC transporters across members of the
Conclusões                                          Actinomycetales, most of which are soil sa-
Os transportadores ABC semelhantes ao operão        prophytes, support the idea that M. tuber-
mce conservados nos membros dos Actinomyce-         culosis itself descended from some soil my-
tales, a maioria dos quais saprófitas de solo,      cobacteria. In the soil reservoir, saprophytes
apoiam a ideia de que o próprio M. tuberculosis     derive their carbon nutrients as energy
descende de alguma micobactéria de solo. No         source from dead organic matter. M. tuber-
reservatório do solo, as saprófitas obtêm os seus   culosis has as its natural reservoir the human
nutrientes e carbono como fonte de energia a        host, in particular, the granuloma structure
partir de matéria orgânica morta. O M. tubercu-     in human organs. It appears that M. tuber-
losis tem como seu reservatório natural o hospe-    culosis has simply re-adapted its ancestral
deiro humano, particularmente a estrutura gra-      carbon-sequestration function its ancestors
nulomatosa dos órgãos humanos. Parece que o         used in soil to derive carbon released from
M. tuberculosis simplesmente readaptou a fun-       dead granuloma cells and bacteria in the
ção ancestral de sequestração de carbono usada      human reservoir. Unfortunately, for hu-
pelos seus antepassados ao retirar o carbono li-    mans, this metabolic readaptation has led
bertado pelas células granulomatosas mortas e       M. tuberculosis to become one of the most
bactérias no reservatório humano. Infelizmente,     common infectious causes of death in
para os humanos, esta readaptação metabólica        adults worldwide.
levou a que o M. tuberculosis se tornasse uma das
mais frequentes causas de morte por infeção no
adulto em todo o mundo.

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Regulação da composição lipídica da parede celular do Mycobacterium tuberculosis
                                    e o seu efeito na persistência bacteriana in vitro
                                                                                                                            Lee W Riley

               1. Cole ST BR, Parkhill J, Garnier T, Churcher C, Har-        10. Glickman MS, Jacobs WR. Microbial pathogenesis
               ris D, Gordon SV, Eiglmeier KGS, Barry CE 3rd, Tekaia         of Mycobacterium tuberculosis: Dawn of a discipline. Cell
               F, Badcock K, Basham D, Brown D, Chillingworth T,             2001; 104:477-485.
               CR, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin         11. Rao V, Gao F, Chen B, Jacobs WR Jr, Glickman,
               N, Holroyd SHT, Jagels K, Krogh A, McLean J, Moule            MS. Trans-cyclopropanation of mycolic acids on treha-
               S, Murphy L, Oliver K,, Osborne JQM, Rajandream               lose dimycolate suppresses Mycobacterium tuberculosis
               MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares          -induced inflammation and virulence. J Clin Invest
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               2000; 406:735-738.                                            in the mce1 operon negative transcriptional regulator.
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S 42   R e v i s t a          P o r t u g u e s a                           d e       P n e u m o l o g i a
                                         Vol XVI Suplemento 1 A Janeiro 2010
Jesus Gonzalo Asensio1                                    Uma nova vacina viva contra a tuberculose com base na
Ainhoa Arbues1                                            inativação do phoP
Dessi Marinova1
Carlos Martín1
                                                          A new live tuberculosis vaccine based on phoP inactivation

A BCG viva atenuada é a atual vacina con-                        The attenuated live BCG is the current vac-
tra a tuberculose (TB). Embora venha sen-                        cine against tuberculosis (TB). It has been
do usada há mais de 80 anos, esta vacina                         used for more than 80 years but is ineffec-
não é eficaz na proteção contra a TB pulmo-                      tive at providing protection against adult
nar no adulto, sendo necessárias novas vaci-                     pulmonary TB. New tuberculosis vaccine
nas e novas estratégias de vacinação que ofe-                    candidates and TB vaccination strategies
reçam melhor proteção contra esta doença                         conferring better protection against pulmo-
do que a vacina BCG existente. As vacinas                        nary tuberculosis than the current vaccine
vivas atenuadas estão entre as mais eficazes                     BCG are needed. Live attenuated vaccines
contra doenças infecciosas humanas devido                        are among the most effective vaccines
à resposta imune alargada e de longa dura-                       against human infectious disease due to the
ção por elas induzida.                                           broad and long-lived immune response they
Na última década verificou-se um ressurgi-                       induce.
mento global de tuberculose, e as estratégias                    In the recent decade, a global pipeline of
pioneiras para o desenvolvimento de vaci-                        novel TB candidates has emerged. Pioneer-
nas mais eficazes levaram à descoberta de                        ing strategies for the development of more
subunidades de vacinas que provaram não                          effective vaccines today have lead to the dis-
oferecer melhor proteção do que a BCG em                         covery of subunit vaccines, which have
vários modelos animais, mas melhoravam a                         proved ineffective at providing better pro-
eficácia da BCG quando usadas numa estra-                        tection than BCG in various animal mo
tégia de vacinação prime-boost. Formas dife-                     dels, but could improve the efficacy of BCG
rentes de BCG prime e boost com estratégias                      used in a prime-boost strategy. Different
de subunidades estão a ser utilizadas em en-                     heterologous prime BCG and boost with
saios clínicos, com o objetivo de melhorar a                     subunit strategies are in clinical trials with

1   Department of Microbiology, University of Zaragoza. Spain

        R e v i s t a                    P o r t u g u e s a                  d e      P n e u m o l o g i a      S 43
                                                     Vol XVI Suplemento 1 A Janeiro 2010
         Uma nova vacina viva contra a tuberculose com base na inativação do phoP
                                                      Jesus Gonzalo Asensio, Ainhoa Arbues, Dessi Marinova, Carlos Martín

              eficácia da BCG. Mais recentemente,                the aim to improve efficacy of BCG. More
              iniciaram-se ensaios clínicos com vacinas          recently, clinical trials with recombinant
              BCG recombinantes, com o intuito de en-            BCG vaccines have started with the aim to
              contrar candidatas para serem usadas como          find candidates to be used as prime, preven-
              vacinas prime preventivas. Outra estratégia        tive vaccines. Another innovative strategy,
              inovadora, as vacinas vivas baseadas no My-        live vaccines based on rationally attenuated
              cobacterium tuberculosis racionalmente ate-        Mycobacterium tuberculosis are promising
              nuado, são candidatas promissoras (algumas         candidates (some of which in late preclini-
              delas no fim da investigação pré-clínica),         cal investigation), is a promising new BCG-
              um novo instrumento substituto da BCG,             replacement tool with better protective ef-
              com melhor eficácia protetora contra as for-       ficacy against pulmonary forms of TB.
              mas pulmonares de TB.                              Based upon the observation that the phoP
              Com base na observação de que a codifica-          gene coding for the transcription factor
              ção pelo gene phoP do fator de transcrição         phoP is essential for M. tuberculosis virulence
              PhoP é essencial para a virulência do M. tu-       (Perez et al., 2001), we rationally attenuated
              berculosis (Perez et al., 2001), atenuámos ra-     the tubercle bacillus by inactivating phoP.
              cionalmente o bacilo da TB por inativação          The mutant demonstrated strong attenua-
              do phoP. O mutante demonstrou forte ate-           tion in cellular and animal models and re-
              nuação em modelos celular e animal e resul-        sulted more attenuated than BCG in im-
              tou mais atenuado do que a BCG em rati-            munocompromised SCID mice (Martin et
              nhos SCID imunocomprometidos (Martin               al., 2006). The vaccine candidate protected
              et al., 2006). A vacina candidata protegeu         guinea pigs and non-human primates
              porquinhos-da-índia e primatas não huma-           against tuberculosis infection (Martin et al.,
              nos contra a infeção tuberculosa (Martin et        2006, Verreck et al., 2009).
              al., 2006, Verreck et al., 2009).                  Recent molecular studies (Gonzalo Asensio
              Estudos moleculares recentes (Gonzalo Asen-        et al., 2008) have demonstrated that phoP
              sio et al., 2008) demonstraram que o fator de      transcription factor controls the expression
              transcrição phoP controla a expressão de apro-     of approximately 80 genes (many of which
              ximadamente 80 genes (muitos dos quais im-         implicated in M. tuberculosis virulence), ac-
              plicados na virulência do M. tuberculosis),        counting for about 2% of the open reading
              responsável por cerca de 2% da open reading        frame (ORF) in M. tuberculosis genome.
              frame (ORF) no genoma do M. tuberculosis.          Based on these studies it could be deduced
              Com base nestes estudos, pode pensar-se que        that the attenuated phenotype and the pro-
              o fenótipo atenuado e a imunidade protetora        tective immunity conferred by the phoP
              conferida pelo mutante phoP podem ser ex-          mutant can be explained by the altered
              plicados pelo mecanismo de ação alterado do        mechanism of action of the transcription
              fator de transcrição phoP, conforme descrito e     factor phoP, as described below and summa-
              resumido na Fig. 1.                                rized in Fig. 1.
              O mutante phoP diminui a regulação da              The phoP mutant decreases byosinthesis
              biossíntese dos complexos lípidos da parede        regulation of complex mycobacterial cell-
              celular micobacteriana, incluindo a diacil-        wall lipids, including diacyltrehalose (DAT)

S 44   R e v i s t a      P o r t u g u e s a                  d e       P n e u m o l o g i a
                                  Vol XVI Suplemento 1 A Janeiro 2010
Uma nova vacina viva contra a tuberculose com base na inativação do phoP
Jesus Gonzalo Asensio, Ainhoa Arbues, Dessi Marinova, Carlos Martín

 ~2% ORF no genoma do M. tuberculosis sob controlo do phoP / ~2% ORFs in the M. tuberculosis genome under phoP control

                               RESPIRAÇÃO AERÓBICA E ANAERÓBICA/                            RESPOSTAS AO STRESS
                                AEROBIC & ANAEROBIC RESPIRATION                         E AO CHOQUE TÉRMICO/STRESS
                                                                                          & HEAT-SHOCK RESPONSES

        SL, DAT, PAT.../                                                                                    REGIÃO RD1 E SECREÇÃO DE ESAT-6/
      LIPID METABOLISM                                                                                       RD1 REGION & ESAT-6 SECRETION
         SL, DAT, PAT...

                                                                 Mutante phoP/
                                                                 PhoP mutant

       Membrana plasmática/

        Plasma membrane

                                        RESPOSTAS HIPÓXICAS PRECOCES

 Gonzalo Asensio et al. JBC 2006        E DURADOURAS/EARLY & ENDURING                                             Cell
                                              HYPOXIC RESPONSES                           PERSISTÊNCIA/          lysate

                                                                                                                 Frigui et al. PLoS Path 2008

Fig. 1 – Estudos moleculares de vacina baseada em phoP (Gonzalo-Asensio, et al., 2008)

Fig. 1 – Molecular studies of phoP-based vaccine (Gonzalo-Asensio et al., 2008)

trealose (DAT) e poliaciltrealose (PAT) im-                   and polyacyltrehalose (PAT) implicated in
plicadas na imunomodulação do sistema                         immunomodulation of the host immune
imunológico hospedeiro (Saavedra R et al.                     system (Saavedra R et al 2005), which are
2005), não expressos pelo mutante phoP                        not expressed by the phoP mutant (Gonzalo
(Gonzalo Asensio et al. 2006).                                Asensio et al 2006).
Também se observou desregulação dos genes                     Down regulation of genes within the region
na região da diferença 1 (RD1) (Gonzalo                       of difference 1 (RD1) (Gonzalo-Asensio et
Asensio et al., 2008) necessária à virulência e à             al., 2008) required for virulence and ESAT-
secreção de ESAT-6 no mutante phoP quando                     6 secretion were also observed in the phoP
comparado com a estirpe do tipo selvagem                      mutant when compared to wild-type strain
(Friguie et al., 2008). A RD1 restringe-se às                 (Friguie et al 2008). RD1 is restricted to
estirpes virulentas do complexo M. tuberculosis               virulent M. tuberculosis complex (MTBC)
(CMTB) e perde-se por todas as subestirpes de                 strains and is lost by all BCG substrains
BCG (Behr et al., 1999, Pym et al., 2003).                    (Behr et al., 1999, Pym et al., 2003).

     R e v i s t a                 P o r t u g u e s a                            d e     P n e u m o l o g i a                                                             S 45
                                             Vol XVI Suplemento 1 A Janeiro 2010
         Uma nova vacina viva contra a tuberculose com base na inativação do phoP
                                                         Jesus Gonzalo Asensio, Ainhoa Arbues, Dessi Marinova, Carlos Martín

              O mutante phoP evidenciou regulação altera-           The phoP mutant showed altered regula-
              da e controlo de outras funções-chave neces-          tion and control of other key functions re-
              sárias à sobrevivência dos microrganismos na          quired for successful survival of the micro-
              célula hospedeira. Como se mostra na Fig. 1,          organism within the host cell. As illustrated
              estas funções incluem respostas hipóxicas             in Figure 1, these functions include early
              precoces e duradouras, respiração aeróbica e          and enduring hypoxic responses, aerobic
              anaeróbica, respostas ao stress e ao choque tér-      and anaerobic respiration, stress and heat-
              mico, metabolismo lipídico (normalmente               shock responses, lipid metabolism (normal-
              desregulado na estirpe avirulenta H37Ra) e            ly down regulated in the avirulent strain
              expressão do fator de persistência ICL, im-           H37Ra), and expression of the persistence
              portante para a persistência intracelular do          factor ICL, important for intracellular per-
              M. tuberculosis durante a infeção (Gonzalo            sistence of M. tuberculosis during infection
              Asensio et al., 2008). O ICL é regulado posi-         (Gonzalo-Asensio et al 2008). ICL is posi-
              tivamente no mutante phoP e esta caracterís-          tively regulated in the phoP mutant and
              tica poderia explicar o adequado estado de            this characteristic could explain the ade-
              persistência, importante para a apresentação          quate persistence state important for im-
              melhorada do antigénio pelo mutante duran-            proved antigen presentation by the mutant
              te a vacinação.                                       during vaccination.
              Estas observações foram usadas para construir         These observations were used to construct a
              uma nova geração duma vacina viva baseada na          new generation of a live vaccine based on
              inativação do phoP com uma segunda mutação            phoP inactivation carrying a second addi-
              adicional que afecta a síntese duma nova famí-        tional mutation which affects the synthesis
              lia de lípidos associada à virulência da M. tuber-    of a new family of lipids associated with M.
              culosis depois do consenso de Genebra sobre os        tuberculosis virulence following the Geneva
              passos essenciais para o desenvolvimento clíni-       consensus on essential steps towards clinical
              co de novas vacinas micobacterianas vivas ate-        development of new live attenuated
              nuadas (Kamath et al., 2005). O elemento fi-          mycobacterial vaccines (Kamath et al.,
              nal é a primeira candidata a vacina viva              2005). The final construct is the first live
              atenuada desenvolvida de acordo com o con-            attenuated candidate vaccine developed
              senso e recomendações de Genebra para vaci-           according with and fulfilling the Geneva
              nas micobacterianas vivas, com o objectivo de         consensus       requirements      for     live
              a apresentar ao Millennium Development Goal           mycobacterial vaccines with the aim to
              no combate à TB. Ensaios pré-clínicos rigoro-         deliver on the Millennium Development
              sos feitos até à data com o protótipo da vacina       Goal to combat TB. Rigorous preclinical
              viva baseada no phoP demonstraram a sua ade-          studies to date with the live phoP-based
              quada atenuação, segurança, imunogenicidade           vaccine prototype have demonstrated proof
              e eficácia na proteção contra as formas respira-      of principle for adequate attenuation, safety,
              tórias de TB em modelos animais (Perez et al.,        immunogenicity, and protective efficacy
              2001, Williams et al., 2005, Martin et al.,           against respiratory forms of TB in stringent
              2006, Aguilar et al., 2007, Cardona et al.,           animal models (Perez et al., 2001, Williams
              2009, Verreck et al., 2009).                          et al., 2005, Martin et al., 2006, Aguilar et

S 46   R e v i s t a       P o r t u g u e s a                     d e      P n e u m o l o g i a
                                    Vol XVI Suplemento 1 A Janeiro 2010
Uma nova vacina viva contra a tuberculose com base na inativação do phoP
Jesus Gonzalo Asensio, Ainhoa Arbues, Dessi Marinova, Carlos Martín

Os estudos moleculares com o mutante                       al., 2007, Cardona et al., 2009, Verreck et
phoP ajudaram na compreensão do comple-                    al., 2009).
xo puzzle por detrás do mecanismo de ação                  The molecular studies with the phoP mutant
do fator de transcrição do phoP necessário à               have helped begin to understand the intricate
virulência, persistência e sobrevivência in-               puzzle behind the mechanism of action of the
tracelular da estirpe do tipo selvagem du-                 PhoP transcription factor required for
rante a infeção, e cuja expressão alterada é               virulence, persistence and intracellular survival
provavelmente responsável pela adequada                    of the wild-type strain during infection and
atenuação do fenótipo do protótipo da vaci-                whose altered expression probably accounts
na. Além disso, a eliminação dos lípidos                   for the adequate attenuation phenotype of the
imunomoduladores extraíveis da parede ce-                  prototype vaccine. Moreover, elimination of
lular (DAT e PAT) resultante da inativação                 the extractable immunomodulatory cell-wall
do gene phoP e a deficiente síntese da com-                lipids (DAT and PAT) as a result of the targeted
plexa determinante da virulência e dos lípi-               inactivation of the phoP gene, and the impaired
dos da parede celular PDIM (Camacho et                     synthesis of the complex virulence determinant
al., 1999, Cox et al., 1999), devido à segun-              and cell-wall lipid PDIM (Camacho et al.,
da mutação geneticamente conseguida no                     1999, Cox et al., 1999) due to the second
protótipo da vacina baseada no phoP, tam-                  genetically engineered mutation in the
bém contribuem para o fenótipo atenuado                    prototype phoP-based vaccine further
desta nova candidata.                                      contribute to the attenuated phenotype of this
Investigação atual de qualidade proporciona                novel candidate.
forte apoio científico a esta vacina de nova               Strong to date research provides robust
geração em franco progresso e em vias de ser               scientific support adducing this new
clinicamente avaliada quanto à segurança e                 generation vaccine well–advanced to progress
eficácia em seres humanos. Tendo em conta                  from research to development and be
a falta de correlativos imunológicos de pro-               clinically evaluated for vaccine safety and
teção, é indispensável realizar ensaios clíni-             efficacy for the first time in human. Moreover,
cos de fase 3 para conhecermos o valor real                considering the lack of immunological
de qualquer nova vacina usada profilatica-                 correlates of protection, Phase 3 clinical trials
mente contra a TB.                                         are indispensible to tell us the real value of
A parceria Stop TB estima que pelo menos                   any new vaccine used as a prophylactic tool
20 vacinas candidatas deveriam iniciar os                  against TB.
ensaios de segurança de fase I antes de 2015,              Stop TB partnership estimate that at least
com o propósito de conseguir o licencia-                   20 vaccine candidates should enter phase I
mento para uma vacina eficaz contra a TB                   safety trials before 2015 with the goal to
(Young, Dye, 2006). A descoberta e utiliza-                reach an effective licensed vaccine against
ção de uma nova vacina contra a TB que                     TB (Young and Dye, 2006). The discovery
ofereça melhor proteção contra as formas                   and use of a new TB vaccine that confers
pulmonares da doença do que a atual BCG                    improved protection against pulmonary
é crucial para alcançar o objetivo de erradi-              forms of TB than the current BCG is key to
car a tuberculose até 2050.                                reach the 2050 objective of TB eradication.

    R e v i s t a                P o r t u g u e s a                     d e       P n e u m o l o g i a       S 47
                                           Vol XVI Suplemento 1 A Janeiro 2010
         Uma nova vacina viva contra a tuberculose com base na inativação do phoP
                                                                 Jesus Gonzalo Asensio, Ainhoa Arbues, Dessi Marinova, Carlos Martín

              Aguilar D, Infante E, Martin C, Gormley E, Gicquel B,          Kamath AT, Fruth U, Brennan MJ, Dobbelaer R, Hu-
              Hernandez Pando R. Immunological responses and pro-            brechts P, Ho MM, Mayner RE, Thole J, Walker KB,
              tective immunity against tuberculosis conferred by vac-        Liu M, Lambert PH. New live mycobacterial vaccines:
              cination of Balb/C mice with the attenuated Mycobacte-         the Geneva consensus on essential steps towards clinical
              rium tuberculosis (phoP) SO2 strain. Clin Exp Immunol          development. Vaccine 2005; 23: 3753-3761.
              2007; 147: 330-338.                                            Martin C, Williams A, Hernandez-Pando R, Cardona PJ,
              Asensio JA, Arbues A, Perez E, Gicquel B, Martin C.            Gormley E, Bordat Y, Soto CY, Clark SO, Hatch GJ, Agui-
              Live tuberculosis vaccines based on phoP mutants: a            lar D, Ausina V, Gicquel B. The live Mycobacterium tuber-
              step towards clinical trials. Expert Opin Biol Ther 2008;      culosis phoP mutant strain is more attenuated than BCG
              8:201-211.                                                     and confers protective immunity against tuberculosis in
              Camacho LR, Ensergueix D, Perez E, Gicquel B, Guil-            mice and guinea pigs. Vaccine 2006; 24: 3408-3419.
              hot C. Identification of a virulence gene cluster of Myco-     Perez E, Samper S, Bordas Y, Guilhot C, Gicquel B, Mar-
              bacterium tuberculosis by signature-tagged transposon          tin C. An essential role for phoP in Mycobacterium tuber-
              mutagenesis. Mol Microbiol 1999; 34: 257-267.                  culosis virulence. Mol Microbiol 2001; 41: 179-187.
              Cardona PJ, Asensio JG, Arbues A, Otal I, Lafoz C, Gil         Saavedra R, Segura E, Tenorio EP, López-Marín LM.
              O, Caceres N, Ausina V, Gicquel B, Martin C. Exten-            Mycobacterial trehalos-containing glycolipid with im-
              ded safety studies of the attenuated live tuberculosis vac-    munomodulatory activity on human CD4+ and CD8+
              cine SO2 based on phoP mutant. Vaccine 2009; 27:               T-cells. Micr Inf 2006; 8:533-540.
              2499-2505.                                                     Verreck FA, Vervenne RA, Kondova I, van Kralingen
              Cox JS, Chen B, McNeil M, Jacobs WR Jr. Complex                KW, Remarque EJ, Braskamp G, van der Werff NM,
              lipid determines tissue-specific replication of Mycobacte-     Kersbergen A, Ottenhoff TH, Heidt PJ, Gilbert SC,
              rium tuberculosis in mice. Nature 1999; 402: 79-83.            Gicquel B, Hill AV, Martin C, McShane H, Thomas
              Frigui W, Bottai D, Majlessi L, Monot M, Josselin E,           AW. MVA.85A boosting of BCG and an attenuated,
              Brodin P, Garnier T, Gicquel B, Martin C, Leclerc C,           phoP deficient M. tuberculosis vaccine both show pro-
              Cole ST, Brosch R. Control of M. tuberculosis ESAT-6           tective efficacy against tuberculosis in rhesus macaques.
              secretion and specific T cell recognition by PhoP. PLoS        PLoS One 2009; 4: e5264.
              Pathog 2008; 4: e33.                                           Williams A, Hatch GJ, Clark SO, Gooch KE, Hatch
              Gonzalo Asensio J, Maia C, Ferrer NL, Barilone N,              KA, Hall GA, Huygen K, Ottenhoff TH, Franken KL,
              Laval F, Soto CY, Winter N, Daffe M, Gicquel B, Mar-           Andersen P, Doherty TM, Kaufmann SH, Grode L,
              tin C, Jackson M. The virulence-associated two-                Seiler P, Martin C, Gicquel B, Cole ST, Brodin P, Pym
              -component PhoP-PhoR system controls the biosynthe-            AS, Dalemans W, Cohen J, Lobet Y, Goonetilleke N,
              sis of polyketide-derived lipids in Mycobacterium              McShane H, Hill A, Parish T, Smith D, Stoker NG,
              tuberculosis. J Biol Chem 2006; 281: 1313-1316.                Lowrie DB, Kallenius G, Svenson S, Pawlowski A, Blake
              Gonzalo-Asensio J, Mostowy S, Harders-Westerveen J,            K, Marsh PD. Evaluation of vaccines in the EU TB vac-
              Huygen K, Hernandez-Pando R, Thole J, Behr M, Gic-             cine cluster using a guinea pig aerosol infection model
              quel B, Martin C. PhoP: a missing piece in the intricate       of tuberculosis. Tuberculosis (Edinb) 2005; 85: 29-38.
              puzzle of Mycobacterium tuberculosis virulence. PLoS           Young D, Dye C. The development and impact of tu-
              ONE 2008; 3: e3496.                                            berculosis vaccines. Cell 2006; 124: 683-687.

S 48   R e v i s t a         P o r t u g u e s a                            d e      P n e u m o l o g i a
                                        Vol XVI Suplemento 1 A Janeiro 2010
Ruth McNerney1                                           Simpósio: Testes rápidos para o rastreio preliminar da

                                                         Symposium: Point-of-care tests for tuberculosis

Pensa-se que há mais casos de tuberculose                        It is believed there are more cases of tu-
no mundo hoje do que em qualquer outra                           berculosis in the world today than at any
altura através da história. As estimativas da                    previous time in history. WHO estimates
Organização Mundial de Saúde (OMS) para                          for 2007 suggested a prevalence of 13.7
2007 sugeriam uma prevalência de 13,7 mi-                        million, with 9.3 million incident cases
lhões, com 9,3 milhões de novos casos e 1,8                      and 1.8 million deaths. It is primarily a
milhões de mortes. Esta é essencialmente                         disease of poverty, with Asia and Africa
uma doença de pobreza, contando a Ásia e a                       between them sharing over ninety percent
África juntas mais de noventa por cento dos                      of the global TB burden. In the absence of
casos mundiais. Na ausência duma vacina                          an effective vaccine control depends on
eficaz, o controlo depende da prontidão do                       prompt treatment to reduce the viable
tratamento para reduzir a carga bacilar viá-                     bacillary load, thus reducing the infec-
vel, diminuindo assim a contagiosidade de                        tiousness of individuals with open pul-
indivíduos com formas pulmonares da doen-                        monary forms of the disease. Through the
ça. Através dos esforços de iniciativas de                       efforts of international health initiatives
saúde internacionais, como as do Global                          such as the Global Fund to fight HIV/
Fund to fight HIV/AIDS, Tuberculosis and                         AIDS, Tuberculosis and Malaria treat-
Malaria, o tratamento da TB está agora dis-                      ment for TB is now widely available and
ponível e de forma gratuita para a maioria                       free for the majority patients. However,
das pessoas. Porém, o acesso ao tratamento                       access to treatment is dependent on diag-
está dependente do diagnóstico e os atrasos                      nosis and delays in detection may result
na deteção podem resultar na deterioração                        in deterioration of the patient and in-
do doente e aumentar as oportunidades de                         creased opportunity for transmission.

1   Department of Infectious and Tropical Diseases,
    London School of Hygiene & Tropical Medicine
    Keppel Street, London, WC1E 7HT, UK

        R e v i s t a                    P o r t u g u e s a                  d e     P n e u m o l o g i a    S 49
                                                      Vol XVI Suplemento 1 A Janeiro 2010
                               Testes rápidos para o rastreio preliminar da tuberculose
                                                                                                Ruth McNerney

              transmissão. Infelizmente, o diagnóstico         Unfortunately diagnosis is not straight-
              nem sempre é fácil, uma vez que os sinto-        forward as symptoms are not specific,
              mas não são específicos, particularmente em      particularly in those with an underlying
              pessoas com problemas de saúde concomi-          health problem such as HIV related im-
              tantes, como a imunossupressão relacionada       munosupression. The majority of TB cases
              com o VIH.                                       reside in countries with weak and poorly
              A maioria dos casos de TB residem em paí-        resourced health care systems where labo-
              ses com sistemas de saúde fracos e com pou-      ratory facilities for diagnosis are limited
              cos recursos, onde os laboratórios que reali-    in number and distribution. Access may
              zam exames de diagnóstico são limitados          involve long journeys and considerable
              em número e distribuição geográfica, e cujo      expense, a situation compounded by the
              acesso pode envolver longas viagens e despe-     insensitivity of the available tests and need
              sa considerável. Esta situação é agravada        for multiple visits. Delays also arise from
              pela insensibilidade dos testes disponíveis e    health seeking behaviour, where indivi-
              pela necessidade de múltiplas consultas. Os      duals do not seek medical assistance. Re-
              atrasos também se devem ao comportamen-          luctance to seek a diagnosis reflects the
              to dos doentes, quando estes não procuram        chronic nature of the disease, a lack of
              uma consulta médica. A relutância em obter       awareness and, in some settings a fear of
              um diagnóstico reflete a natureza crónica da     stigmatization and social exclusion. WHO
              doença, a sua não perceção e, nalguns casos,     estimates suggest that during 2006 four
              o receio de estigmatização e exclusão social.    million cases of TB remained undiag-
              As estimativas da OMS sugerem que, du-           nosed. Unlike conditions such as malaria
              rante 2006, quatro milhões de casos de TB        or HIV there are no sensitive rapid tests
              não foram diagnosticados. Ao contrário do        and no reliable point-of-care (POC) de-
              que acontece com patologias como a malá-         vices that can be used by non specialist
              ria ou o VIH, não há testes rápidos nem dis-     personnel within the community. To ad-
              positivos (POC) suficientemente fiáveis para     dress these issues a panel of international
              uso de pessoal não especializado na comuni-      experts were invited to speak in a sympo-
              dade.                                            sium on the topic of point-of-care tests
              Um painel de especialistas internacionais        for the diagnosis of tuberculosis disease.
              foi convidado para um simpósio onde seria        The event was organized by the POC sub-
              abordado o tópico dos testes rápidos para o      group of the STOP TB working group on
              diagnóstico da tuberculose. O evento foi         New Diagnostics in collaboration with
              organizado pelo subgrupo POC do grupo            the European Society of Mycobacteriolo-
              de trabalho STOP TB sobre novos diagnós-         gy. The intention of the symposium was
              ticos, em colaboração com a Sociedade Eu-        to increase awareness of the need for im-
              ropeia de Micobacteriologia. A intenção do       proved access to TB diagnosis, to update
              simpósio foi aumentar a perceção da neces-       diagnostic practitioners and the research
              sidade de melhorar o acesso ao diagnóstico       community on the current status of POC
              da TB, atualizar os técnicos de diagnóstico      test development and to debate research
              e os investigadores sobre o desenvolvimen-       needs and priorities.

S 50   R e v i s t a     P o r t u g u e s a                  d e    P n e u m o l o g i a
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Testes rápidos para o rastreio preliminar da tuberculose
Ruth McNerney

to e o estado atual dos testes POC, e ainda     The potential of POC tests to aid TB
debater as necessidades e prioridades da in-    control was discussed by Catharina Boe-
vestigação. O potencial dos testes POC na       hme (Foundation for Innovative New Dia-
ajuda ao controlo da TB foi discutido por       gnostics). The ideal test would not require
Catharina Boehme (Foundation for Innova-        specialist skills or a laboratory infrastruc-
tive New Diagnostics). O teste ideal não de-    ture, would yield a result within one visit,
veria necessitar de técnicos especializados     would be highly specific and have a sensi-
nem de infraestruturas laboratoriais, facul-    tivity superior to that of smear microsco-
taria o resultado no próprio dia da consul-     py. Anyone found positive would be im-
ta, teria uma elevada especificidade e sensi-   mediately registered and referred for
bilidade superior à da microscopia de           treatment. Tests that are not more sensi-
esfregaço. Os indivíduos com um resultado       tive than laboratory based tests would
positivo seriam imediatamente registados e      have a role if they could be used to reduce
referenciados para tratamento. Os testes        diagnostic delay. Alternatively, a screen-
com sensibilidade inferior à dos testes com     ing test that had high sensitivity and a
base no laboratório teriam um papel se pu-      reasonable specificity could be used to
dessem ser usados para reduzir os atrasos no    screen high risk populations, where a po-
diagnóstico. Alternativamente, um teste de      sitive result triggered referral for confir-
avaliação com elevada sensibilidade e espe-     mation prior to initiation of treatment.
cificidade razoável poderia ser usado no ras-   The current difficulties in accessing treat-
treio de populações de alto risco, em que,      ment were illustrated through the testa-
com um resultado positivo, o doente seria       ment of a former TB patient from Zam-
referenciado para confirmação do diagnós-       bia, a country hard hit by both TB and
tico antes do início do tratamento.             HIV/AIDS. Carol Nyirenda (National
As atuais dificuldades no acesso ao trata-      Co-ordinator, Community Initiative for
mento foram ilustradas pelo testemunho de       Tuberculosis, HIV/AIDS and Malaria) had
uma doente com TB natural da Zâmbia,            presented at her local health clinic in
país severamente atingido por TB e VIH/         2003 with a cough of over two months
/SIDA. Em 2003, Carol Nyirenda (co-             duration. Unable to produce sputum for
ordenadora nacional da Community Initiati-      testing she was sent home with suspected
ve for Tuberculosis, HIV/AIDS and Malaria)      chronic bronchitis. Over the following six
dirigiu-se ao seu centro de saúde, queixando-   months she had a total of four chest x-
-se de tosse que durava havia mais de dois      rays but was not offered an HIV test dur-
meses. Como a doente não conseguia pro-         ing that time. She was finally offered
duzir expetoração para análise, foi enviada     treatment for her TB more than six
para casa com suspeita de bronquite cróni-      months after reporting symptoms. Suc-
ca. Durante os seis meses seguintes foi sub-    cessfully treated she now campaigns on
metida a um total de quatro raios-X, mas        behalf of people living with HIV and TB.
não lhe foi proposto um teste VIH nesse pe-     She proposed that a test be developed that
ríodo. Foi-lhe, finalmente, proporcionado o     could be used by community volunteers,
tratamento para a TB mais de seis meses de-     a test that could be used in rural settings

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                                                                                                 Ruth McNerney

              pois de ter referido os sintomas. Tratada         where there is no electricity and a test that
              com sucesso, Carol Nyirenda agora defende         was affordable by people living in com-
              ativamente os interesses de doentes com           munities affected by TB and HIV.
              VIH e TB, e propôs que seja desenvolvido          Countries where TB is endemic frequently
              um teste que possa ser usado por voluntá-         lack the legistrative framework to regulate
              rios comunitários, um teste que possa tam-        diagnostic test devices. In such an environ-
              bém ser usado em áreas rurais onde não há         ment test kits may be marketed without
              eletricidade e que possa ser disponibilizado      evidence of their benefit and in some cir-
              a pessoas que residem em comunidades afe-         cumstances have been accompanied by
              tadas por TB e VIH.                               misleading claims regarding their perfor-
              Os países onde a TB é endémica frequente-         mance. Rosanna Peeling (London School of
              mente não dispõem da moldura legislativa          Hygiene & Tropical Medicine) presented
              para regular dispositivos e testes de diagnós-    data from a study where the performance
              tico. Nessas regiões, os kits-teste podem ser     of 19 commercially available rapid diag-
              comercializados sem evidência de benefício        nostic tests for tuberculosis was assessed
              e, em algumas circunstâncias, foram acom-         using a panel of 355 sera collected from
              panhadas de alegações enganadoras quanto          eight geographically diverse sites. Unfortu-
              ao desempenho. Rosanna Peeling (London            nately none of the tests were found to per-
              School of Hygiene & Tropical Medicine) apre-      form well enough to replace the current
              sentou dados dum estudo em que o desem-           test of smear microscopy. When compared
              penho de 19 testes rápidos de diagnóstico         to a combined reference standard of mi-
              da tuberculose disponíveis comercialmente         croscopy, culture, radiography and clinical
              foi avaliado usando um painel de 355 amos-        follow-up the test with the highest sensi-
              tras séricas recolhidas em diversas regiões       tivity of 59.71% had a specificity of just
              geográficas. Infelizmente, nenhum dos tes-        57.72%. The two tests with the highest
              tes demonstrou um desempenho suficiente-          specificity (98.66%) had sensitivities of
              mente bom para substituir os atuais testes        0.97 and 2.43% respectively. A full copy of
              de microscopia de esfregaço. Quando com-          the report may be downloaded from the
              parado com a combinação de referência-            Special Programme for Research and Train-
              -padrão (microscopia, cultura, radiografia e      ing in Tropical Diseases (TDR) website.
              follow-up clínico), o teste com a maior sen-
              sibilidade (59,71%) tinha uma especificida-       tdr-research-publications/diagnostics-eva-
              de de apenas 57,72%. Os dois testes com           luation-2. The failure of current know-
              maior especificidade (98,66%) tinham sen-         ledge to provide a rapid test for TB has led
              sibilidades de 0,97 e 2,43%, respectivamen-       to recognition of the need for novel ap-
              te. É possível obter uma cópia deste relató-      proaches. Gerd Michel (Foundation for In-
              rio no website do Special Programme for           novative New Diagnostics) described the
              Research and Training in Tropical Diseases        application of genome, proteome and me-
              (TDR):          tabolomic based technologies to biomarker
              cations/tdr-research-publicationsdiagnos-         discovery. The search for suitable detection
              tics -evaluation-2.                               targets has expanded from traditional pro-

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Ruth McNerney

O insucesso do conhecimento atual em for-        tein chemistry to a broader range of struc-
necer um teste rápido para TB levou ao re-       tural components and metabolites. Tests
conhecimento da necessidade de novas             based on detection of lipoarabinomannan
abordagens. Gerd Michel (Foundation for          (LAM) a lipopolysaccharide component of
Innovative New Diagnostics) descreveu téc-       the cell wall have not so far achieved suffi-
nicas baseadas na aplicação de tecnologias       cient sensitivity but intriguingly in one
do conhecimento do genoma, proteoma e            study appeared to function better in HIV
metabolómica para a descoberta de biomar-        positive individuals than those that were
cadores. A busca de alvos adequados foi          not co infected. New strategies are also be-
além da química de proteínas tradicional         ing investigated for nucleic acid based
para um âmbito mais alargado de compo-           analysis with the development of assays for
nentes estruturais e metabólitos. Os testes      fragmented transrenal DNA in urine. POC
baseados na deteção de lipoarabinomanano         tests need to be both rapid and easy to per-
(LAM), um componente lipossacarídeo da           form. One of the simplest and fastest ways
parede celular, ainda não conseguiram sen-       to identify substances is by their aroma and
sibilidade suficiente, mas, curiosamente,        volatile organic compound (VOC) analysis
num estudo, pareceram funcionar melhor           is emerging as field of interest. Proof of
em indivíduos positivos para VIH do que          concept for TB diagnosis has been provi-
nos que não estavam coinfectados.                ded through the training of African pouch
Com o desenvolvimento de ensaios de ADN          rats to smell TB in sputum samples. Al-
transrenal fragmentado na urina, novas estra-    though not as sensitive as smear microsco-
tégias estão também a ser investigadas para      py the animals are able to screen a sample
análises com base em ácidos nucleicos. Os        in just a few seconds. Tools for detecting
testes POC precisam de ser rápidos e fáceis de   VOC at low concentrations are being in-
fazer. Uma das maneiras mais simples e rápi-     vestigated, including the adaptation of
das de identificar substâncias é pela análise    novel instrumentation developed for mili-
do seu aroma e a identificação de compostos      tary use and bioterror agent detection.
orgânicos voláteis (COV), e vem emergindo        However, early optimism regarding the ap-
como um campo de interesse. A prova de           plication of electronic nose technology has
conceito do diagnóstico da TB foi consegui-      dissipated following the realisation that
da com o treino de ratos pouch africanos para    they lack the robustness required for a dia-
cheirarem TB em amostras de expectoração.        gnostic test. A more analytical approach is
Embora não tão sensível como a microscopia       now being pursued and Ruth McNerney
de esfregaço, os animais conseguem avaliar       (London School of Hygiene & Tropical Medi-
uma amostra em apenas alguns segundos. Os        cine) outlined some of the challenges fa-
utensílios para detectar baixas concentrações    cing those searching for VOC biomarkers
de COV estão a ser investigados, incluindo a     that are predictive of TB disease. Small
adaptação de novos instrumentos desenvolvi-      volatile molecules lack the distinctive cha-
dos para uso militar e para deteção de agentes   racter of macromolecules traditionally used
de bioterrorismo. No entanto, o optimismo        to predict infection and they are more of-
precoce no que respeita à aplicação da tecno-    ten found in nature. To complicate matters

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                                                                                                 Ruth McNerney

              logia do nariz electrónico dissipou-se depois     metabolites produced by Mycobacterium
              da constatação de que lhe falta a robustez ne-    tuberculosis vary depending on the avai-
              cessária a um teste de diagnóstico. Uma abor-     lability of nutrients and other environmen-
              dagem mais analítica está agora em curso e        tal factors, thus VOC emitted during in-
              Ruth McNerney (London School of Hygiene           fection may differ with the site of infection
              & Tropical Medicine) referiu alguns dos desa-     and stage of disease. Similarly, VOC emit-
              fios que se apresentam a quem investiga bio-      ted by the host in response to infection
              marcadores em COV preditivos de TB. As            may alter with disease progression. The
              pequenas moléculas voláteis não têm o cará-       complexity and dynamic nature of VOC
              ter distinto das macromoléculas tradicional-      emission suggest that to attain high sensi-
              mente usadas para indicar a presença de in-       tivity and specificity a multivariate analyti-
              fecção e encontram-se mais frequentemente         cal approach will be required.
              na natureza. Para complicar, os metabólitos       There are many steps along the path of
              produzidos por Mycobacterium tuberculosis         test development and Amy P Wong (X
              variam, dependendo da disponibilidade de          PRIZE Foundation) discussed the route
              nutrientes e outros fatores ambientais, pelo      and identified potential barriers to the de-
              que os COV emitidos durante uma infeção           livery of a successful new diagnostic. Once
              podem diferir com o local da infecção e o es-     biomarkers have been discovered and tes-
              tádio da doença. Do mesmo modo, os COV            ted with prototype devices the capacity to
              emitidos pelo hospedeiro em resposta à infe-      manufacture must be developed and a
              ção podem sofrer alterações com a progressão      route to market established. New tech-
              da doença. A complexidade e a natureza di-        nologies may require innovative manufac-
              nâmica da emissão de COV sugere que para          turing practices to ensure delivery of ro-
              atingir elevada sensibilidade e especificidade    bust devices at a competitive price.
              será necessária uma abordagem analítica mul-      Improved guidance on optimum test
              tivariada.                                        specification is badly needed. Uncertainty
              Há muitos passos ao longo do caminho do           regarding the market for POC TB tests
              desenvolvimento de testes e Amy P. Wong           also acts as a disincentive to potential in-
              (X PRIZE Foundation) discutiu a rota e            vestors. Innovative mechanisms for re-
              identificou as potenciais barreiras ao apare-     warding test development are required to
              cimento de um novo diagnóstico de suces-          stimulate participation and facilitate the
              so. Quando os biomarcadores tiverem sido          development of devices that can be used
              descobertos e testados com protótipos de          to detect TB wherever in the world there
              dispositivos, a capacidade de fabrico será        is a need. In the words of an ex TB patient
              desenvolvida e estabelecer-se-á uma rota de       “TB infection and disease is not just about
              mercado. As novas tecnologias podem re-           numbers, for some of us it is a reality…
              querer métodos de fabrico inovadores que          we sit and watch TB reversing all the ef-
              assegurem a robustez dos dispositivos a pre-      forts and gains of the HIV fight, as we die
              ços competitivos. É necessária melhor             one by one from TB. It is not about num-
              orientação das especificações de novos tes-       bers, there are real people at the end of
              tes. A incerteza quanto ao mercado para           the chain we have run out of time”.

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Testes rápidos para o rastreio preliminar da tuberculose
Ruth McNerney

testes POC de TB também funciona como
desincentivo para potenciais investidores.
São necessários mecanismos inovadores que
compensem o desenvolvimento de testes,
de modo a estimular a participação e a faci-
litar o desenvolvimento de dispositivos que
possam ser usados para detectar a TB em
qualquer parte do mundo. Nas palavras de
um ex-doente de TB, “a infeção por TB e a
própria doença não são apenas números,
para alguns de nós é uma realidade… va-
mos assistindo à inversão, pela TB, de todos
os esforços e ganhos na luta do VIH, con-
forme vamos morrendo, um a um, de TB.
Não se trata de números, somos pessoas no
extremo da cadeia e o nosso tempo está a

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Thomas M Shinnick1                                           Criar capacidade laboratorial para a tuberculose:
                                                             Necessidades e estratégias

                                                             Building tuberculosis laboratory capacity: Needs and

Palavras-chave                                                             Key-words
Planos estratégicos nacionais, abordagem de                                National strategic plans, systems approach,
sistemas, rede de laboratórios de saúde pública.                           public health laboratory networks.

Fundamentos                                                                Background
No relatório “TB Global 2009”1, a Organi-                                  In the 2009 Global TB report1, the World
zação Mundial de Saúde (OMS) utilizou                                      Health Organization used information from
informação de modelos epidemiológicos e                                    epidemiologic models and program data to
dados de programas para estimar que, em                                    estimate that in 2007 there were 9.27 mil-
2007, havia 9,27 milhões de novos casos de                                 lion new cases on TB; 1.37 million (15%)
tuberculose (TB); 1,37 milhões (15%) de                                    cases among persons living with HIV;
casos entre pessoas infetadas com VIH;                                     511 000 new cases of multidrug-resistant
511 000 novos casos de TB multirresistente                                 TB (MDR TB); and 50 000 new cases of
(MDR TB); e 50 000 novos casos de TB ex-                                   extensively drug-resistant TB (XDR TB).
tensamente resistente a fármacos (XDR                                      These are only estimates, in part, because
TB). Estas são apenas estimativas, em parte                                there are large gaps in the availability of TB
devido a falhas na disponibilidade de servi-                               laboratory services in many regions of the
ços laboratoriais de TB em muitas regiões                                  world1,2. Only about 60% of new TB cases
do mundo1.2. Apenas cerca de 60% dos no-                                   are laboratory confirmed and only about
vos casos de TB são confirmados pelo labo-                                 5% of MDR TB cases are actually identified
ratório e só aproximadamente 5% dos casos                                  and reported.

1   PhD, Associate Director for Global Laboratory Activities, Division of TB Elimination,

Centers for Disease Control e Prevention
1600 Clifton Road, MS-G35, Atlanta Georgia 30333 USA

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                                                                                                 Thomas M Shinnick

              de MDR TB são realmente identificados e             The inadequate laboratory capacity hinders
              reportados.                                         diagnosis, case management, and disease
              A inadequada capacidade laboratorial preju-         surveillance. This is particularly important
              dica o diagnóstico, a gestão dos casos e a vi-      for patients with drug-resistant TB because
              gilância da doença. Isto é particularmente          effective care often does not begin until re-
              importante para os doentes com TB resis-            sults of drug-susceptibility tests are avai-
              tente a fármacos porque, frequentemente, os         lable. Indeed, the emergence of MDR TB
              cuidados eficazes só se iniciam depois dos          and XDR TB has led to the recognition that
              resultados dos testes de suscetibilidade a fár-     the lack of TB laboratory capacity is a global
              macos. O aparecimento da MDR TB e da                crisis. Factors that have contributed to the
              XDR TB levou ao reconhecimento de que a             gaps in TB laboratory services include: 1) a
              falta de capacidade laboratorial representa         lack of recognition of the importance of the
              uma crise global. Os fatores que têm contri-        laboratory in TB treatment and control; 2)
              buído para as falhas nos serviços laborato-         poor communication between National TB
              riais de TB incluem: 1) falta de reconheci-         Programs and those providing TB labora-
              mento da importância do laboratório no              tory services; 3) inadequate human and fi-
              tratamento e no controlo da doença; 2) má           nancial resources for TB laboratories; 4)
              comunicação entre os programas nacionais            lack of infrastructure and physical facilities;
              de TB e as entidades que fornecem os servi-         and 5) biosafety concerns4.
              ços laboratoriais de TB; 3) recursos humanos        Recognizing the important role of the labo-
              e financeiros inadequados nesses laborató-          ratory, the 2007 World Health Assembly5
              rios; 4) falta de infraestruturas e de instala-     endorsed the call of the Global Plan to Stop
              ções; e 5) preocupações com biossegurança4.         TB2 for universal access to culture and drug-
              Ao reconhecer o importante papel do labora-         susceptibility testing. Universal access will
              tório, a 2007 World Health Assembly5 endos-         require the capacity perform 120 million
              sou o apelo do Global Plan to Stop TB2 para         microscopy investigations, 60 million cul-
              acesso universal a testes de cultura e de susce-    ture investigations, and 6 million drug-sus-
              tibilidade a fármacos. O acesso universal re-       ceptibility investigations per year3,6. Meet-
              presenta capacidade para fazer anualmente           ing this goal will require establishing 5000
              120 milhões de exames microscópicos, 60             new microscopy centers and training 9000
              milhões de testes de cultura e 6 milhões de         new microscopy technicians as well as estab-
              testes de suscetibilidade a fármacos3,6. Cum-       lishing 2000 new culture and drug-suscep-
              prir com este objetivo representará criar 5000      tibility testing laboratories and training
              novos centros de microscopia, treinar 9000          23 000 new technicians. At least US$ 1 bil-
              novos técnicos de microscopia e criar ainda         lion will be needed annually for building
              2000 novos laboratório para testes de cultura       TB laboratory infrastructure and recurring
              e testes de suscetibilidade a fármacos e treinar    costs. However, the benefit to cost ratio of
              23 000 novos técnicos. Serão necessários            such investments is estimated to be 9:1 in
              anualmente pelo menos mil milhões de dóla-          populations with a high prevalence of HIV
              res americanos para construir e manter infra-       infection7. Meeting the universal access
              estruturas laboratoriais de TB. No entanto,         goals could save countries in sub-Saharan

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Thomas M Shinnick

estima-se que o rácio custo-benefício de tais     Africa alone as much as US$ 52 billion an-
investimentos seja de 9:1 em populações com       nually.
elevada prevalência de infeção por VIH7. Se
se conseguir o objetivo de acesso universal, os
países na África subsariana poderão poupar        National laboratory strategic
cerca de 52 mil milhões de dólares america-       plans
nos anualmente.                                   creating and sustaining the needed labora-
                                                  tory services will require considerable re-
                                                  sources, efforts, and political will. The 2008
Planeamento estratégico para                      Maputo Declaration on Strengthening La-
laboratórios nacionais                            boratory Systems8 calls upon national go-
Para criar e manter os serviços laboratoriais     vernments to take ownership of their labo-
em falta serão necessários recursos conside-      ratory systems and develop national strategic
ráveis, dedicação e vontade política. A De-       laboratory plans that integrate laboratory
claração de Maputo de 2008 sobre Fortale-         support for the major diseases of public
cimento dos Sistemas de Laboratórios8             health importance. National governments
apelou para os governos tomarem posse das         should establish a department of laboratory
redes de serviços laboratoriais e implemen-       systems within the Ministry of Health to
tarem estratégias nacionais de reforço e          provide leadership and should set as a prio-
apoio a esses serviços e, consequentemente,       rity developing a laboratory policy within
às principais doenças que ameaçam a saúde         the health development plan that will guide
pública. Os governos deveriam criar um de-        the implementation of a strategic laboratory
partamento de sistemas laboratoriais, no          plan.
âmbito do Ministério da Saúde, capaz de           Strategic planning efforts should include all
gerir essa rede de sistemas e assumir como        disease programs, clinical services, and other
prioridade elaborar uma política laborato-        stakeholders and strive to create a laboratory
rial inserida no plano nacional de saúde.         system based on quality laboratory manage-
Os esforços de planeamento estratégico de-        ment principles. To implement and sustain
veriam incluir programas para todas as doen-      the strategic plans, national governments
ças, serviços clínicos e outras estruturas e a    should: 1) acknowledge the importance of
padronização dos serviços laboratoriais as-       laboratory services in disease control; 2) en-
sente na qualidade. Para implementar e            sure that health sector strengthening plans
manter o planeamento estratégico, os gover-       include adequately budgeted components
nos deveriam: 1) reconhecer a importância         for laboratory capacity development; 3) co-
dos serviços laboratoriais no controlo das        ordinate efforts of all departments, disease-
doenças; 2) assegurar que o fortalecimento        specific programs, donors, and technical
dos planos do sector da saúde inclui compo-       partners responsible for laboratory services;
nentes adequadamente orçamentados para            4) commit adequate human and financial
o desenvolvimento da capacidade laborato-         resources for laboratory services; 5) develop
rial; 3) coordenar os esforços de todos os        and implement a human resource policy to
departamentos, programas específicos para         create a qualified laboratory workforce and

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                                                                                              Thomas M Shinnick

              cada doença, dadores e parcerias técnicas         identify and remove barriers to laboratory
              responsáveis pelos serviços de laboratório;       staff career development, remuneration, re-
              4) consignar recursos humanos e financeiros       tention, and sustainability of technical com-
              adequados para esses serviços; 5) desenvol-       petency, and 6) engage all laboratory service
              ver e implementar uma política de recursos        providers (e.g., private, academic, and pub-
              humanos, a fim de se obterem profissionais        lic health laboratories) to improve access to
              qualificados para os laboratórios, e identifi-    quality-assured TB diagnostic testing.
              car e afastar barreiras ao desenvolvimento
              das suas carreiras e remunerações, bem como
              a sustentação da sua competência técnica; e       Building laboratory networks
              6) contratar fornecedores de serviços de la-      An essential component of efforts to com-
              boratório (e.g., privados, académicos e labo-     bat infectious diseases is a network of labo-
              ratórios de saúde pública) para melhorar o        ratories that can provide reliable laboratory
              acesso aos testes de qualidade controlada         testing for diagnosis, treatment, and moni-
              para o diagnósticos da TB.                        toring of therapy. Such a network should
                                                                strive to be integrated across the diseases of
                                                                public health importance, especially HIV
              Construção de redes de                            and TB. A systems approach to creating
              laboratórios                                      such a network is necessary to optimize la-
              Uma componente essencial no esforço de            boratory testing and information exchange
              combate a doenças infeciosas é uma rede de        and to ensure that appropriate services are
              laboratórios que forneça testes de diagnóstico    available in every program. Comprehensive
              fiáveis, tratamento e monitorização da tera-      laboratory strengthening efforts involve as-
              pêutica. Tal rede deveria ser posta ao serviço    sessment and understanding of the struc-
              de patologias com particular importância na       ture, performance, and cost of the labora-
              saúde pública, especialmente o VIH e a TB.        tory network; development of a referral and
              Uma abordagem de sistemas na criação dessa        information network to ensure prompt flow
              rede é fundamental para otimizar os testes de     of specimens and information; and use of
              laboratório e a troca de informações necessá-     quality-improvement principles to evaluate
              ria para assegurar que os serviços apropriados    and improve the performance of the labora-
              estarão disponíveis em cada programa. A           tory network9.
              abrangência desse esforço envolve avaliação e     A challenge to an integrated approach is
              compreensão da estrutura, do desempenho e         that at the global level programs and fund-
              do custo da rede de laboratórios; desenvolvi-     ing are directed to specific diseases. Such
              mento duma rede de referência e informação        vertical global programs are often mirrored
              que assegure o rápido fluxo de amostras e de      by vertical national programs and laborato-
              resultados; e adoção de princípios de melho-      ries. For a variety of reasons including fund-
              ramento da qualidade para avaliar e melhorar      ing sources, technical requirements, and
              o desempenho da rede de laboratórios9.            biosafety concerns, the national reference
              Um desafio na abordagem integrada é o facto       laboratory for a disease in many countries is
              do nível global de programas e de subsídios       a specialized, free-standing institution sepa-

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Thomas M Shinnick

serem dirigidos a doenças específicas. Esses      rate from the general public health labora-
programas verticais globais refletem frequen-     tory system and other national reference
temente programas e laboratórios verticais        laboratories. Also, national reference labora-
nacionais. Por uma variedade de razões, in-       tories often concentrate on support for epi-
cluindo origens de recursos, necessidades téc-    demiologic, surveillance, or research activi-
nicas e preocupações com a biossegurança, o       ties and may have a limited, direct role in
laboratório de referência nacional para uma       patient care. Such an emphasis may limit
doença em muitos países é uma instituição         the perceived value of integrating the ser-
individual especializada, separada do sistema     vices of national reference laboratories across
geral de laboratórios de saúde pública e de       disease programs.
outros laboratórios nacionais de referência.      On the other hand, national reference la-
Além disso, os laboratórios nacionais de refe-    boratories often have responsibility for de-
rência concentram-se frequentemente no            veloping, leading, and monitoring a net-
apoio a actividades epidemiológicas, de vigi-     work of public health laboratories that
lância ou de investigação, e podem ter um         provide services for diagnosis and patient
papel direto limitado no cuidado de doentes,      care. Such network laboratories may pro-
o que pode ser uma limitação no que respeita      vide services for more than one disease-
ao valor da integração de serviços de labora-     specific program and represent opportuni-
tórios nacionais de referência nos programas      ties to build linkages between programs
dirigidos a doenças específicas.                  and harmonize or integrate services. In-
Por outro lado, os laboratórios nacionais de      deed, at the most peripheral level of the
referência têm frequentemente a responsabi-       health system, laboratory services are often
lidade de desenvolver, dirigir e monitorizar as   fully integrated with one technician doing
redes de laboratórios de saúde pública que        testing for all diseases.
fornecem serviços de diagnóstico e de cuida-      Building laboratory capacity is by itself
dos a doentes. Essas redes podem fornecer         an opportunity to build linkages between
serviços para mais de um programa de doen-        programs and integrate services. That is,
ças específicas, representando oportunidades      although training of bench-level techni-
de associação de programas e harmonização         cians relies on disease-specific expertise,
ou integração de serviços. Na verdade, ao ní-     laboratory capacity building relies more
vel mais periférico do sistema de saúde, os       on cross-cutting expertise in infrastruc-
serviços de laboratório estão muitas vezes to-    ture, biosafety, facilities, human resource
talmente integrados, com um técnico que           development, specimen referral, supply
executa os testes para todas as doenças.          chain management, logistics, equipment,
Conceber a capacidade do laboratório é, por       maintenance, quality assurance pro-
si só, uma oportunidade para construir liga-      grams, management principles, informa-
ções entre programas e para integrar servi-       tion systems, data management, and ac-
ços, isto é, embora o treinamento de técnicos     creditation processes. A potential role
assente na experiência específica de determi-     for a department of laboratory systems
nadas doenças, a capacidade de construir la-      within the Ministry of Health of a coun-
boratórios tem mais que ver com perícia           try would be to oversee programs that

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                                                                                              Thomas M Shinnick

              transversal em infraestruturas, biosseguran-       address these issues in coordinated, inte-
              ça, instalações, recursos humanos, referência      grated manner.
              de espécimes, gestão da cadeia logística, lo-      Generally, it should be the responsibility of
              gística, equipamento, manutenção, progra-          the national TB reference laboratory, in col-
              mas de garantia de qualidade, princípios de        laboration and coordination with the na-
              boa gestão, sistemas de informação, gestão         tional TB program, other disease programs,
              de dados e acreditação de processos. Um pa-        and MOH departments, to develop, imple-
              pel potencial para um departamento de sis-         ment, and monitor a laboratory network
              temas de laboratórios, sob a alçada do minis-      that assures access to quality TB testing and
              tério da saúde de um país, seria o de              complete, timely reporting. In many coun-
              inspecionar os programas que tratam destes         tries, there exists a network of AFB-smear
              temas de modo coordenado e integrado.              microscopy laboratories overseen by the na-
              Geralmente, deveria ser responsabilidade do        tional TB reference laboratory, which could
              laboratório de TB nacional de referência, em       serve as a starting point for developing the
              colaboração e coordenação com o programa           laboratory capacity needed to address drug-
              nacional de TB, programas de outras doenças        resistant TB and HIV-associated TB. How-
              e departamentos do ministério da saúde, para       ever, care must be taken to avoid creating an
              desenvolver, implementar e monitorizar uma         isolated, vertical TB laboratory network.
              rede de laboratórios que assegure o acesso a       Where possible, TB laboratory services
              testes de TB com qualidade. Em muitos paí-         should be an integral part of primary health
              ses, existem redes de laboratórios de micros-      care and a national public health laboratory
              copia de esfregaços para bacilos ácido-álcool      network.
              resistentes (BAAR) inspecionados pelo labo-
              ratório de TB de referência nacional que po-
              deriam servir de ponto de partida para o de-       Resources and assistance
              senvolvimento da capacidade laboratorial           Resources are becoming available to assist
              necessária ao processamento de testes de TB        countries with TB laboratory capacity build-
              resistente a fármacos e TB associada a VIH.        ing. The Global Laboratory Initiative (GLI)
              Porém, há que ter o cuidado de evitar criar        of the Stop TB Partnership (http://www.
              uma rede de laboratórios de TB isolada e ver-; 6) is working to develop
              tical. Sempre que possível, os serviços dos la-    1) a roadmap for ensuring quality TB diag-
              boratórios TB devem ser uma parte integral         nostics services within national laboratory
              dos cuidados de saúde primários e duma rede        strategic plans, 2) guidance on norms and
              nacional de laboratórios de saúde pública.         standards for TB laboratory testing, equip-
                                                                 ment, and biosafety, 3) global policy gui-
                                                                 dance, 4) training materials, and 5) human
              Recursos e assistência                             resource strategies.
              Começam a surgir recursos de apoio a países        Laboratory consultants are needed to assist
              com capacidade para construir laboratórios         countries with assessment, strategic plan-
              para testar TB. A Global Laboratory Initiative     ning, implementation, and evaluation. Cur-
              (GLI) da Stop TB Partnership (http://www.          rently expert laboratory diagnostic advice

S 62   R e v i s t a      P o r t u g u e s a                   d e    P n e u m o l o g i a
                                   Vol XVI Suplemento 1 A Janeiro 2010
Criar capacidade laboratorial para a tuberculose: necessidades e estratégias
Thomas M Shinnick está a trabalhar para de-     and expertise has been provided by industria-
senvolver: 1) um roteiro para assegurar servi-    lized-world laboratory experts, often on an
ços de diagnóstico de TB com qualidade,           ad hoc basis with little or no coordination
integrados no planeamento estratégico de          with global partners. The WHO Global
laboratórios; 2) orientação nas normas e pa-      Plan 2006-20152 indicated that the current
drões para laboratórios de testes de TB, equi-    system of short workshops and consultancy
pamento e biossegurança; 3) orientação na         visits are insufficient to bridge the training
política global; 4) materiais de formação; e      gap of laboratory personnel in high burden
5) estratégias de recursos humanos.               countries and to build sustainable laborato-
São necessários consultores de serviços de la-    ry systems. The GLI is working to develop a
boratório para ajudar os países na avaliação,     consensus strategy for laboratory capacity
planeamento estratégico e implementação.          building, courses to train consultants in the
Presentemente, aconselhamento e experiên-         implementation of the consensus strategy,
cia têm sido fornecidos por peritos de labo-      and a mechanism to ensure coordination of
ratórios de países industrializados, frequen-     capacity building efforts within a country
temente numa base ad hoc, com pouca ou            and among partners. The critical aspects in
nenhuma coordenação com parceiros glo-            the consulting process are to develop a con-
bais. O Plano Global 2006-2015 da OMS2            sistent approach to laboratory capacity
indicava que o actual sistema de curtas           building; to promote the use of laboratory
workshops e visitas de aconselhamento são         systems and tests appropriate for resource-
insuficientes para suprir a falta de pessoal de   limited, high-burden countries; and to en-
laboratório em países com estas carências e       sure that the expertise needed to develop,
para criar sistemas laboratoriais sustentáveis.   implement, and evaluate a strategic plan for
A GLI está a trabalhar no desenvolvimento         laboratory capacity building will be avai-
duma estratégia de consenso para criar capa-      lable to countries. Note that there is greater
cidade laboratorial, cursos para consultores      need for expertise in laboratory capacity
de implementação da estratégia de consenso        building than there is for expertise in di-
e um mecanismo que assegure a coordena-           sease-specific laboratory techniques. A goal
ção de esforços em cada país e entre parcei-      is to have cadre of consultants who can be
ros. Os aspetos críticos no processo de con-      stationed in country for extended periods of
sultadoria são: desenvolver uma abordagem         time that would be able to assist countries
consistente para criar a capacidade laborato-     in building an integrated laboratory net-
rial; promover o uso de sistemas e testes de      work capable of providing the laboratory
laboratório apropriados para países com re-       services needed to combat TB, HIV, and
cursos limitados; e assegurar que a experiên-     malaria.
cia necessária para desenvolver, implementar
e avaliar o planeamento estratégico referido
seja disponibilizada. De notar que há uma         Conclusion
maior necessidade de experiência para criar       An effective response to the call for univer-
capacidade laboratorial do que para as técni-     sal access to TB diagnostics requires a mas-
cas específicas de laboratório. Um dos obje-      sive scale-up of TB laboratory services and

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                                                                                                            Thomas M Shinnick

              tivos é conseguir quadros de consultores que               correspondingly large commitment of re-
              possam ser colocados nos países durante lon-               sources. To maximize the impact and sus-
              gos períodos de tempo, a fim de ajudarem na                tainability of the investments in TB labora-
              criação duma rede integrada de laboratórios                tory capacity building, a broad health
              capaz de fornecer os serviços necessários ao               systems strengthening approach is needed.
              combate de tuberculose, VIH e malária.                     Harmonized efforts using a systems ap-
                                                                         proach to build to a strong laboratory net-
                                                                         work that addresses the needs of all diseases
              Conclusão                                                  of public health importance is a benefit to
              Uma resposta eficaz ao apelo para acesso                   all disease programs and contributes to ge-
              universal ao diagnóstico da TB exige um au-                neral health sector strengthening and quali-
              mento maciço dos serviços laboratoriais e                  ty patient care.
              correspondente atribuição de recursos. Para
              maximizar o impacto e a sustentabilidade
              destes investimentos é imprescindível uma
              abordagem alargada dos sistemas de saúde.
              A harmonização de esforços na criação
              duma sólida rede de sistemas laboratoriais
              capaz de responder às necessidades de todas
              as patologias de importância para a saúde
              pública é um benefício para todos os pro-
              gramas de saúde e contribui para o fortaleci-
              mento do setor da saúde em general e para a
              qualidade dos cuidados aos doentes.

              1. World Health Organization. Global tuberculosis          Geneva: World Health Organization; 2009. Available
              control: epidemiology, strategy, financing: WHO re-        online at
              port 2009 (WHO/HTM/TB/2009.411). Geneva:                   tlenecks/bottlenecks_chapter5.pdf. Accessed Nov. 1,
              World Health Organization; 2009. Available online at       2009.
              4. Abdel Aziz M, Ryszewska K, Laszlo A, Blanc L. Stra-
              en. Accessed Nov. 1, 2009.                                 tegic approach for the strengthening of laboratory
              2. Stop TB Partnership e World Health Organiza-            services for tuberculosis control, 2006-2009 (WHO/
              tion. Global Plan to Stop TB 2006–2015 (WHO/               HTM/TB/2006.364). Geneva: World Health Organi-
              HTM/STB/2006.35). Geneva: World Health Or-                 zation; 2006. Available online at: http://whqlibdoc.
              ganization; 2006. Available online at http://www.
                 pdf. Accessed on Nov. 1, 2009.
              PlanFinal.pdf. Accessed Nov. 1, 2009.                      5. World Health Organization. 60th World Health As-
              3. Report of a Ministerial Meeting of High M/XDR-          sembly. Resolutions and Decisions. World Health As-
              -TB Burden Countries, Beijing China, April 1-3,            sembly Resolution WHA 60.19: Tuberculosis control:
              2009. Key bottlenecks in M/XDR-TB control and pa-          progress and long-term planning. Geneva: World Health
              tient care. 5. Responding to the laboratory bottleneck.    Organization; 2007. Available online at http://apps.

S 64   R e v i s t a        P o r t u g u e s a                         d e      P n e u m o l o g i a
                                      Vol XVI Suplemento 1 A Janeiro 2010
Criar capacidade laboratorial para a tuberculose: necessidades e estratégias
Thomas M Shinnick -en.               WDSP/IB/2007/08/01/000158349_20070801103922
pdf. Accessed Nov. 1, 2009.                                 /Rendered/PDF/wps4295.pdf. Accessed Nov. 1, 2009.
6. Global Laboratory Initiative. Moving tuberculosis (TB)   8. World Health Organization. Maputo Declaration on
laboratory capacity strengthening forward: A Global La-     Strengthening Laboratory Sistems. Presented at: Con-
boratory Initiative. Supporting document for 15th Annual    sensus Meeting on Clinical Laboratory Testing Harmo-
Meeting of the Stop TB Partnership Coordinating Board,      nization and Standardization; January 22-24, 2008;
Bagamoyo Tanzania, November 28-29, 2008. Geneva:            Maputo, Mozambique. Available at: http://www.who.
Stop Tb Partnership; 2009. Available online at http://      int/diagnostics_laboratory/Maputo-Declaration_2008.               pdf. Accessed Nov. 1, 2009.
Tanzania/assets/documents/2.08-11.1%20GLI%20Syn             9. Centers for Disease Control and Prevention (Shin-
opsis%20FINAL.pdf. Accessed Nov. 1, 2009.                   nick TM, Iademarco MF, Ridderhof JC). National plan
7. Laxminarayan R, Klein E, Dye C, Floyd K, Darley S,       for reliable tuberculosis laboratory services using a sis-
Adeyi O. Economic Benefit of Tuberculosis Control           tems approach: recommendations from CDC and the
(August 1, 2007). World Bank Policy Research Working        Association of Public Health Laboratories Task Force on
Paper Series, No. 4295, 2007. Available at http://www.      Tuberculosis Laboratory Services. MMWR (RR-6)                 2005; 54:1-12.

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Afranio Kritski1                                         Experiência da Rede Brasileira de Pesquisa em
                                                         Tuberculose no desenvolvimento e avaliação de novos
                                                         métodos de diagnóstico em tuberculose

                                                         The experience of the Brazilian Tuberculosis Research
                                                         Network in the development and evaluation of new
                                                         methods of diagnosing tuberculosis

Introdução                                                            Introduction
De acordo com estimativa da Organização                               Brazil ranks 18th out of the 22 countries
Mundial de Saúde (OMS) de 2009, o Brasil                              that bear 80% of the worldwide tuberculo-
está em 18.º lugar entre os 22 países que cons-                       sis (TB) burden, according to the World
tituem 80% da carga global da tuberculose                             Health Organization (WHO) in 2009. TB
(TB). A TB continua a ser a principal causa de                        is the leading cause of mortality in HIV in-
morte em indivíduos infetados com VIH,                                fected persons, despite all HIV infected per-
mesmo nos submetidos a HAART (terapia                                 sons having received highly active antiretro-
antirretroviral potente) oferecida gratuitamen-                       viral therapy (HAART) free of charge since
te a todas as pessoas infetadas com VIH, desde                        1997.
1997. Tal como acontece noutros países em                             As in other developing nations, there is a
desenvolvimento, há alguma dificuldade em                             significant gap in communication and un-
harmonizar a comunicação e o entendimento                             derstanding between TB programme ex-
entre especialistas programáticos em TB, aca-                         perts, academics, the community, and non-
démicos e comunidade, e as organizações não                           governmental organisations. In 2001, there
governamentais. Em 2001, a investigação da                            was no TB research policy in place regard-
TB não estava sujeita a quaisquer linhas de                           ing: a) the development of new products, b)
orientação quanto a: a) desenvolvimento de                            technological innovation and c) the incor-
novos produtos; b) inovação tecnológica; e c)                         poration of new tools in public and private
incorporação de novos instrumentos nos seto-                          sectors.
res público e privado. A Rede Nacional de In-                         In recognition of this gap, a National TB
vestigação da Tuberculose (REDE-TB) foi                               Research Network was established in 2002
criada, em 2002, para aproximar as diferentes                         to bring together different constituencies to

1   Coordinator, Diagnostic Area, Brazilian TB Research Network (Rede TB). Academic Tuberculosis Programme, Medical School of the Federal University
    of Rio de Janeiro, Brazil

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       Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento
                       e avaliação de novos métodos de diagnóstico em tuberculose
                                                                                                   Afranio Kritski

              sensibilidades e promover uma estratégia inte-    promote an integrated, multi-disciplinary
              grada, multidisciplinar e multi-institucional     and multi-institutional strategy to TB con-
              para controlo e investigação da TB no Brasil.     trol and research in Brazil (
              (Rede-TB – Nesta apresen-        This article describes the Network’s work in
              tação, serão comentadas as atividades da área     the area of diagnosis.
              diagnóstica da Rede TB.

              Desenvolvimento, avaliação                        The development, evaluation
              e incorporação de testes                          and incorporation of diagnostic
              diagnósticos no Brasil                            tests in Brazil
              A avaliação da incorporação de novas tecno-       Evaluating the incorporation of new TB
              logias diagnósticas em TB tem sido também         diagnostic techniques was prioritised by the
              priorizada pela Organização Mundial de            WHO following the 2006 launch of the
              Saúde após o lançamento do Plano Global           Global Plan Against TB – WHO/STOP
              de TB da OMS/STOP, em 2006 (http://               (
             strategy/en/). Since 2002, we have learned
              en/). A partir de 2002, apercebemo-nos de         that the diagnostics development and
              que o desenvolvimento diagnóstico e o pro-        evaluation process proposed by WHO has
              cesso de avaliação proposto pela OMS rece-        received different attention and funding by
              beram das partes interessadas atenção e           the stakeholders. A lot of interest and avai-
              apoios diferentes. Verificou-se um enorme         lable funding has been allocated to the fol-
              interesse e disponibilidade de fundos relati-     lowing stages: a) pre-clinical: discovery and
              vamente às seguintes fases: a) pré-clínica:       research, development, b) phase 1: evalua-
              descoberta e investigação, desenvolvimento;       tion – proof of principle; c) phase 2: labora-
              b) fase 1: avaliação – prova de princípio; c)     tory evaluations, and d) phase 3: evaluation
              fase 2: avaliações laboratoriais; e d) fase 3:    of field trials for performance analysis. There
              avaliações de ensaios de campo para análise       has been a scarcity of interest and available
              de desempenho. Mas a fase 4 despertou me-         funding allocated, however, to phase 4: cost,
              nos interesse e obteve menos fundos: custo,       impact studies and policy transfer, prequali-
              estudos de impacto e, para a transferência        fication, bulk procurement, negotiated pri-
              de políticas, pré-qualificação, aprovisiona-      cing and access analysis.
              mento, preços negociados, análise de acesso.      In 2007 there was an evaluation of the trends
              Em 2007, procedeu-se à avaliação das ten-         in scientific articles on TB in Brazil pub-
              dências dos artigos científicos sobre tuber-      lished between 1986 and 2006. Of 1054 TB
              culose no Brasil publicados entre 1986 e          publications assessed containing the word
              2006. Entre os 1054 trabalhos avaliados,          ‘tuberculosis’ with the authors affiliated to
              que continham a palavra tuberculose e cujos       Brazilian Institutions, 486 (46.1%) articles
              autores estavam ligados a instituições brasi-     were related to descriptive, review and case
              leiras, 486 (46,1%) artigos estavam relacio-      series reports. Only 70 (6.7%) articles des-
              nados com descrição e revisão de casos e sé-      cribed operational/effectiveness studies or
              ries. Apenas 70 (6,7%) artigos eram estudos       clinical trials (Kritski AL et al., 2007).

S 68   R e v i s t a      P o r t u g u e s a                  d e     P n e u m o l o g i a
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Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento
e avaliação de novos métodos de diagnóstico em tuberculose
Afranio Kritski

operacionais/de eficácia ou ensaios clínicos.    In 2002, we were approached by a company
(Kritski AL, et al., 2007).                      interested in having its serological tests regis-
Em 2002, fomos abordados por uma empre-          tered at the Brazilian regulatory agency (AN-
sa que manifestou interesse em ter os seus       VISA) for future commercial use. The evalua-
testes serológicos registados numa agência       tion of these serological tests showed a high
reguladora brasileira (ANVISA) para futura       specificity (95%) in healthy control subjects,
comercialização. A avaliação desses testes se-   but a low specificity in TB suspects (46%)
rológicos demonstrou uma elevada especifi-       (Gounder C et al., 2002). Fortunately, they
cidade (95%) entre os controlos saudáveis,       withdrew from the Brazilian market. These
mas baixa especificidade (46%) entre os in-      results are similar to those described in the
divíduos suspeitos de serem portadores de        systematic review published in 2007 (Stein-
TB (Gounder C, et al., 2002). Felizmente, a      gart et al., 2007). The authors concluded
empresa deixou o mercado brasileiro. Esses       that there is currently no indication for com-
resultados são semelhantes aos descritos na      mercial serologic testing kits or in-house
revisão sistemática publicada em 2007            tests in the diagnosis of TB in the world.
(Steingart et al., 2007). Os autores concluí-    Healthcare professionals should be warned
ram que no momento não há indicação de           not to use these techniques in clinical prac-
kits sorológicos comercializados ou testes in    tice. Unfortunately, these tests are still sold
house no diagnóstico da TB no mundo. Os          in developing nations and no strict recom-
profissionais de saúde deveriam ser alertados    mendations on them has been issued by in-
para não utilizarem tais tecnologias na sua      ternational organisations. In 2005 contact
prática clínica. Lamentavelmente, esses tes-     was made with the management section of
tes ainda estão à venda em países em desen-      the in vitro diagnostic products section of
volvimento, não tendo sido publicitadas          the National Regulatory Agency (ANVISA)
quaisquer recomendações sobre este facto         and an evaluation of TB diagnostic products
pelas organizações internacionais. Em 2005,      tested registered at ANVISA in the period
iniciou-se uma interação com a “Gerência         2000 to 2004 was carried out: 48 had been
de produtos para diagnóstico de uso in vitro”    registered since 2000; 33 had no valid regis-
da Agência Reguladora Nacional (ANVISA)          tration, but still remained on the market and
e efetuou-se uma avaliação dos testes diag-      14 (30%) were immunoserological tests.
nósticos de tuberculose registados na ANVISA     These tests had been registered without prior
no período de 2000 a 2004: 48 foram regis-       validation in different clinical settings in
tados desde 2000, 33 não tinham registo vá-      Brazil. These results highlighted the gap be-
lido mas continuavam no mercado e 14             tween the regulatory agency, academics and
(30%) eram testes imunosserológicos. Estes       the policy makers who deal with the incor-
testes foram registados em diferentes cená-      poration of new technologies in Brazil.
rios clínicos no Brasil, sem validação prévia.   Over the last few years, Brazil, along with
Estes resultados acentuam a dificuldade de       other developing countries, has seen new
harmonização entre a agência reguladora, os      molecular technique kits for diagnosing TB
académicos e os decisores que tratam da in-      or resistant TB enter the market, both poly-
corporação de novas tecnologias no Brasil.       merase reaction (PCR) and phenotype

     R e v i s t a         P o r t u g u e s a                 d e       P n e u m o l o g i a       S 69
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       Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento
                       e avaliação de novos métodos de diagnóstico em tuberculose
                                                                                                  Afranio Kritski

              Nos últimos anos, como ocorre noutros paí-       (MGIT960) kits. These technologies were
              ses em desenvolvimento, no Brasil foram          made available almost exclusively in pri-
              comercializadas novas tecnologias molecula-      vately owned laboratories. They were even-
              res (kits de PCR) ou fenotípicas (MGIT960)       tually incorporated into healthcare units of
              no diagnóstico de TB e TB resistente. Entre-     the National Health System, but were dis-
              tanto, tais tecnologias têm sido disponibili-    continued as they were not considered
              zadas praticamente na rede privada de labo-      Ministry of Health priorities.
              ratórios. Eventualmente foram incluídos em
              unidades de saúde do sistema público de          Phenotype techniques for
              saúde, mas sem continuidade, por não terem       diagnosing resistant TB
              sido incorporados na lista de prioridades do     A study performed in three TB Research
              ministério.                                      Network mycobacteriology laboratories
                                                               showed a strong agreement between
                                                               MGIT960 performance and the three gold
              Testes fenotípicos para o                        standard methods for diagnosing resistant
              diagnóstico de TB resistente                     TB; the proportion method, Bactec 460
              Por meio de estudo realizado em três labo-       and the resistance ratio method (Giampa-
              ratórios de micobacteriologia participantes      glia et al., 2007). Another study, performed
              da Rede-TB, observou-se elevada concor-          by researchers in the Honduras and at the
              dância entre a performance do MGIT960 e          John Hopkins University, evaluated the per-
              os três métodos considerados padrão-ouro         formance of the microscopic-observation
              para o diagnóstico de TB resistente: a) Mé-      drug-susceptibility (MODS) assay in 854
              todo de proporções; b) Bactec 460; e c) Ra-      patients with suspected pulmonary TB.
              zão da resistência (Giampaglia et al., 2007).    MODS sensitivity and specificity was
              Em outro estudo, em colaboração com pes-         96.5% and 92.6%, respectively, and time to
              quisadores nas Honduras e na Universidade        diagnosis was quicker for MODS (6 days; 5
              John Hokpins, avaliamos a performance do         – 7 interquartile), than Lowenstein Jensen
              MODS em 854 doentes suspeitos de TB              (LJ) medium (21 days; 17 – 25 inter-
              pulmonar. A sensibilidade e a especificida-      quartile), (Arias M et al., 2007).
              de do MODS foi respectivamente de 96,5%          Studies on the MODS assay suggest that
              e de 92,6%, o tempo do diagnóstico infe-         MODS may be of use in diagnosing drug-
              rior para MODS (6 dias; interquartil de 5 a      susceptible TB as in addition to its sensiti-
              7), em comparação com LJ (21 dias; inter-        vity and specificity – similar to that of stan-
              quartil 17 a 25 dias) (Arias M et al., 2007)     dardised culture media – it offers a far
              Os estudos sobre a técnica MODS sugerem          quicker diagnosis. It does, however, require
              que ela pode ser útil no diagnóstico da TB       laboratory techniciens with a high degree of
              sensível às drogas, pois, além de apresentar     proficiency and biosafety as it uses a liquid
              sensibilidade e especificidade similares aos     medium in Petri dishes. Furthermore, we
              meios de cultura padronizados, o diagnósti-      evaluated the MODS performance for drug
              co é bem mais rápido. Entretanto, requer         resistant TB (DR-TB). In 351 DR-TB sus-
              técnicos de laboratório com elevado grau         pects, MODS had sensitivity and specificity

S 70   R e v i s t a     P o r t u g u e s a                  d e     P n e u m o l o g i a
                                  Vol XVI Suplemento 1 A Janeiro 2010
Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento
e avaliação de novos métodos de diagnóstico em tuberculose
Afranio Kritski

de proficiência e de biossegurança, em ra-    for isoniazid and rifampicin respectively of
zão do uso de meio líquido em placas de       97.2%, 79%, and of 96.4%, 86.5% (Mello
Petri. Avaliámos, ainda, o desempenho do      FCQ, et al., 2007). These results suggest
MODS com a TB resistente (DR-TB). En-         that MODS might be used as screening
tre 351 indivíduos suspeitos de DR-TB, o      technique for DR-TB diagnosis.
MODS teve sensibilidade e especificidade
para isoniazida e rifampicina de 97,2%,
79%, 96,4% e 86,5%, respetivamente            Molecular biology techniques for
(Mello FCQ, et al., 2007). Estes resultados   diagnosing TB and resistant TB
sugerem que o MODS poderá ser usado           There is a great variation in the accuracy of
para triagem no diagnóstico da DR-TB.         commercial molecular tests for the diagno-
                                              sis of active TB, mainly in immunosup-
                                              pressed patients, with lesser sensitivity than
Tecnologias de biologia molecular             specificity (Palomino, 2009, Ling 2008,
para diagnóstico de TB e TB                   Barnard, 2008). Further, performance re-
resistente                                    sults from studies have led to some molecu-
Há grande variabilidade da precisão dos       lar techniques being approved by the regu-
testes moleculares comercializados no diag-   latory bodies of industrialised countries and
nóstico da TB ativa, principalmente em        sold for use in respiratory samples, that is to
doentes imunossuprimidos, com menores         investigate pulmonary TB in adult patients
valores de sensibilidade em relação a espe-   with no prior history of TB treatment. The
cificidade (Palomino, 2009, Ling 2008,        molecular tests currently sold should not be
Barnard, 2008). Além disso, em razão dos      used to diagnose other forms of TB or mo-
resultados obtidos em estudos de perfor-      nitor treatment and do not replace a myco-
mance, alguns testes moleculares foram        bacterial culture exam.
aprovados nos órgãos regulatórios de países   In developing nations, positive direct mi-
industrializados e comercializados para uso   croscopy results have a high positive predic-
em amostras respiratórias, ou seja, para a    tive value for tuberculosis diagnosis. In
investigação de TB pulmonar, em doentes       those regions, molecular tests may provide a
adultos, sem história prévia de tratamento    higher impact for diagnosis of smear nega-
antiTB. Portanto, os testes moleculares co-   tive TB cases (SNTB), especially among
mercializados no momento não deveriam         HIV seropositive cases. However, little data
ser utilizados para o diagnóstico de outras   are available on the cost effectiveness and
formas de TB, monitoramento do trata-         clinical utility of PCR in SNTB, in a setting
mento, e não substituem o exame de cultu-     with a high burden of TB/HIV co-infection
ra para micobactérias.                        (van Cleef, 2005). We evaluated the perfor-
Nos países desenvolvidos, os resultados po-   mance of the PCR dot-blot (developed by
sitivos da microscopia direta têm um eleva-   Brazilian scientists) in parallel with pretest
do valor indicativo positivo no diagnóstico   probability (clinical suspicion) in patients
da tuberculose. Nestas regiões, os testes     suspected of having SNTB, in a prospective
moleculares podem ter maior impacto no        study of 213 individuals with clinical and

     R e v i s t a       P o r t u g u e s a               d e       P n e u m o l o g i a      S 71
                                 Vol XVI Suplemento 1 A Janeiro 2010
       Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento
                       e avaliação de novos métodos de diagnóstico em tuberculose
                                                                                                  Afranio Kritski

              diagnóstico de casos de TB com esfregaço         radiological suspicion of SNTB in a TB/
              negativo TB (ENTB), especialmente entre          HIV referral hospital in southern Brazil.
              seropositivos para VIH. Porém, há poucos         There was no difference in the sensitivity of
              estudos sobre a relação custo-benefício e        PCR in relation to HIV status (Scherer LC
              utilidade clínica da PCR na ENTB, em re-         et al., 2007).
              giões com elevada incidência de coinfeção        In the same study group we compared two
              TB/VIH (van Cleef, 2005). Avaliámos o            strategies: use of acid fast bacilli smear mi-
              desempenho da PCR dot-blot (desenvolvida         croscopy by Ziehl-Neelsen staining (AFB
              por cientistas brasileiros) paralelamente à      smear) plus culture and AFB smear plus
              probabilidade pré-teste (suspeita clínica)       colorimetric test (PCR dot-blot) and per-
              em doentes suspeitos de ENTB, num estu-          formed a cost analysis that included health
              do prospectivo de 213 indivíduos com sus-        services and patient costs. The total screen-
              peita clínica e radiológica de ENTB num          ing costs were 3.7 times higher for AFB
              hospital de referência de TB/VIH, no Sul         smear plus culture than for AFB smear plus
              do Brasil. Não se observou diferença na          PCR dot-blot costs (USD 5,651.560 versus
              sensibilidade da PCR relativamente ao esta-      USD 1,513. 760). AFB smear plus PCR
              tuto de VIH(Scherer LC, et al., 2007). No        dot-blot was more cost-effective than AFB
              mesmo grupo de estudo, comparámos duas           smear plus culture, when the cost of treating
              estratégias: o uso de microscopia de esfrega-    all correctly diagnosed cases was considered
              ço com bacilos acid fast por coloração Ziehl-    (Scherer LC et al., 2009). These results show
              -Neelsen (esfregaço AFB) e cultura; e esfre-     that molecular tests, even in developing na-
              gaço AFB e teste colorimétrico (PCR              tions, are welcome and play an important
              dot-blot), e efectuámos uma análise de cus-      role in SNTB diagnosis, cutting diagnostic
              tos que incluiu serviços de saúde e custos       delay, morbid-mortality and M. tuberculosis
              com os doentes. Os custos totais de scree-       transmission to the community, even in re-
              ning foram 3,7 vezes para o esfregaço AFB        gions with a high rate of HIV infection.
              e cultura versus os custos para esfregaço
              AFB e PCR dot-blot (US$ 5 651 560 versus
              US$ 1 513 ,760). O esfregaço AFB e PCR           Molecular tests for diagnosing
              dot-blot mostrou melhor relação custo-           multi-drug resistant TB (MDR-TB)
              -benefício do que o esfregaço AFB e cultura      Of the molecular tests sold worldwide for a
              quando se considerou o custo de tratar to-       swift diagnosis of resistant TB, the following
              dos os casos corretamente diagnosticados         are accurate: INNO-LIPA Rif.TB kit (Inno-
              (Scherer LC et al., 2009), Estes resultados      genetics, Zwijndrecht, Belgium), GenoType®
              mostraram que os testes moleculares, mes-        MDRTB and GenoType®MDRTB plus as-
              mo nos países desenvolvidos, são bem acei-       says (Hain Lifescience, GMBH, Germany),
              tes e desempenham um papel importante            TB Research Network researchers under-
              no diagnóstico da ENTB, diminuindo não           took a study into the accuracy of selected
              só o tempo necessário para o diagnóstico,        samples using the INNO-Lipa Rif.TB kit
              mas ainda a morbilidade/mortalidade e a          and found a high level of agreement with
              transmissão do M. tuberculosis à comunida-       the reference tests in selected samples (Oli-

S 72   R e v i s t a     P o r t u g u e s a                  d e     P n e u m o l o g i a
                                  Vol XVI Suplemento 1 A Janeiro 2010
Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento
e avaliação de novos métodos de diagnóstico em tuberculose
Afranio Kritski

de, mesmo em regiões com grande incidên-         veira M, 2005). That said, there are as yet
cia de infecção por VIH.                         no published studies in the literature into
                                                 the performance of these molecular tests
                                                 under routine conditions in developing
Testes moleculares para o                        countries.
diagnóstico de TB multirresistente               We recently evaluated the performance of
Entre os testes moleculares comercializados, a   DNA sequencing in 38 (26%) patients (99
nível mundial, para o diagnóstico rápido de      clinical samples) with discordant results be-
TB resistente que mostraram boa acurácia: o      tween MODS and the proportion method.
kit INNO-LIPA Rif.TB (Innogenetics, Zwi-         Surprisingly, we found high rate of hetero-
jndrecht, Bélgica), o ensaio de GenoType®        resistance to RIF and/or INH (21.7%) and
MDRTB e GenoType® MDRTBplus (Hain                of superinfection (28.9%) using the spolig-
Lifescience, GMBH, Alemanha), pesquisa-          otyping technique, with the majority Lam,
dores da Rede TB realizaram estudo de acurá-     Haarlem, and T1 (Andrade et al., 2009).
cia em amostras selecionadas ao utilizarem o     Similar results were seen in South Africa
kit INNO-Lipa Rif.TB e observaram elevada        and in Uzbekistan. Van Rie et al. (Van Rie
concordância com testes de referência em         2005) evaluated 186 TB patients, finding
amostras selecionadas. [Oliveira M, 2005).       superinfection in 23% (14/62) retreatment
Entretanto, até ao momento, não há estudos       cases and in 17% (21/1254) of cases. Hof-
publicados na literatura sobre a performance     mann-Thiel et al. (Hofmann-Thiel S et al.
de tais testes moleculares em condições de ro-   2009), evaluating 35 TB cases, found super-
tina, em países em desenvolvimento. Recen-       infection in 8.6%.
temente, avaliamos o desempenho da sequen-       In both studies the majority of superinfec-
ciação de ADN em 38 (26%) doentes (99            tion was related to the M.tb Beijing family.
amostras clínicas) com resultados discordan-     More recently, Hillemann et al. (Hillemann
tes entre MODS e métodos proporcionais.          D, 2009), compared the accuracy of the
Surpreendentemente, verificámos um elevado       new MTBDRsl assay for extensively drug-
índice de heterorresistência a RIF e/ou INH      resistant TB (XDR-TB) which included de-
(21,7%) e de superinfeção (28,9%), usando a      tection of fluoroquinolone, amikacin-capreo-
técnica spoligotyping, sendo a maioria Lam,      mycin and ethambutol resistance testing.
Haarlem, e T1 [Andrade et al., 2009]. Resul-     Among 106 selected clinical isolates, resis-
tados semelhantes foram observados na África     tance to rifampicin and isoniazid was found
do Sul e no Usbequistão. Van Rie et al. (2005)   in 63 (59.4%) cases. Ofloxacin resistance
avaliaram 186 doentes com TB, encontrando        was found in 32 (30.2%) cases and hete-
superinfeção em 23% (14/62) de casos relata-     roresistance was observed in 21.9% (7/32).
dos e em 17% (21/1254) dos casos. Hofmann-       The high rates of heteroresistance and su-
-Thiel et al. (Hofmann-Thiel S, et al., 2009)    perinfection identified in those studies in
avaliaram 35 casos de TB, encontrando super-     clinical samples collected from DR-TB sus-
infeção em 8,6%. Em ambos estudos, a maio-       pects highlight the need to evaluate the im-
ria das superinfeções esteve relacionada com a   pact of the use of line probe assays in DR-
família M.tb Beijing. Mais recentemente,         TB suspects before its incorporation into

     R e v i s t a         P o r t u g u e s a                d e      P n e u m o l o g i a     S 73
                                    Vol XVI Suplemento 1 A Janeiro 2010
       Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento
                       e avaliação de novos métodos de diagnóstico em tuberculose
                                                                                                 Afranio Kritski

              Hillemann et al. (Hillemann D, 2009), com-         clinical practice, especially in developing
              pararam a precisão do novo ensaio MTBDR-           nations.
              sl para XDR-TB, que incluiu a deteção da           More recently, the TB Research Network
              resistência aos testes com fluoroquinolona,        along with researchers from the Interna-
              amikacin-capreomicina e etambutol. Entre           tional Union Against Tuberculosis of the
              106 isolados clínicos selecionados, observa-       Management Science for Health of the
              ram resistência a rifampicina e a isoniazida em    Centro de Referencia Prof Helio Fraga da
              63 (59,4%) dos casos. Observaram resistência       Fiocruz and technicians from the National
              à ofloxacina em 32 (30,2%) e heteror-              TB programme and the Ministry of Health’s
              resistência em 21,9% (7/32). Os elevados ín-       Department of Science Technology drew
              dices de heterorresistência e de superinfeção      up feasibility and economic impact research
              identificados nesses estudos em amostras clí-      protocols agreements. These are to be car-
              nicas colhidas de indivíduos suspeitos de DR-      ried out in different regions of Brazil and
              -TB acentuam a necessidade de avaliar o im-        include use of the Xpert™ MTB / Rif (Cep-
              pacto do uso de ensaios line probe nestes          heid, Sunnyvale, CA, USA) test for diag-
              suspeitos, antes da sua incorporação na práti-     nosing TB and resistant TB and use of the
              ca clínica, especialmente nos países em desen-     GenoType® MDRTB plus test and MTB-
              volvimento.                                        DRsl assay (Hain Lifescience, GMBH,
              Mais recentemente, a Rede TB, juntamente           Germany) for the management of patients
              com pesquisadores da Union International           with suspected MDR-TB and XDR-TB,
              Contra Tuberculosis, do Management Science         respectively.
              for Health, do Centro de Referência Prof. Hé-
              lio Fraga, da Fiocruz, e técnicos do Programa
              Nacional de TB e do Departamento de Ciên-
              cia e Tecnologia do Ministério da Saúde, ela-
              boraram uma plataforma de protocolos de
              pesquisa de viabilidade e impacto econômico
              a serem realizados em diferentes regiões do
              país, que inclui o uso do teste Xpert™ MTB/
              Rif (Cepheid, Sunnyvale, CA, EUA) para o
              diagnóstico de TB e TB resistente e o uso do
              teste GenoType® MDRTBplus e MTBDRsl
              assay (Hain Lifescience, GMBH, Alemanha)
              para o manuseio do doentes suspeitos de TB-
              -MDR e TB-XDR, respetivamente

S 74   R e v i s t a      P o r t u g u e s a                   d e    P n e u m o l o g i a
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Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento
e avaliação de novos métodos de diagnóstico em tuberculose
Afranio Kritski

Andrade MK, Dalcolmo M, Marsico AG, Mello FCQ,                mann H. Mechanisms of heteroresistance to isoniazid
Dorman S, Rossetti ML, Fonseca LS, de Oliveira MM,            and rifampin of Mycobacterium tuberculosis in Tashkent,
Kritski A. 40th World Conference on Lung Health.              Uzbekistan. Eur Respir J 2009;33(2):368-374. Epub
Cancun, México 3-7 December, 2009 (www.worldlung-             2008 Oct 1.                                                  Hillemann D, Rüsch-Gerdes S, Richter E. Feasibility of
Arias M, Mello FC, Pavon A, Marsico AG, Alvarado-             the GenoType MTBDRsl assay for fluoroquinolone,
-Galvez C, Rosales S, Pessoa CL, Perez, Andrade MK,           amikacin-capreomycin, and ethambutol resistance tes-
Kritski AL, Fonseca LS, Chaisson RE, Kimerling ME,            ting of Mycobacterium tuberculosis strains and clinical
Dorman SE. The microscopic observation drug suscep-           specimens. J Clin Microbiol 2009; 47(6):1767-1772.
tibility (MODS) assay for detection of tuberculosis and       Epub 2009 Apr 22.
tuberculosis drug resistance: results from a multi-center     Kritski AL, Villa TS, Trajman A, Lapa e Silva, JR Me-
study. Clin Infect Dis 2007;44(5):674-80. Epub 2007           dronho RA, Ruffino-Netto A. Two decades of research
Jan 22.                                                       on tuberculosis in Brazil: state of the art of scientific
Barnard M, Albert H, Coetzee G, O’Brien R, Bosman             publications. Rev Saude Publica 2007;41(Supl):9-14.
ME. Rapid molecular screening for multidrug-resistant         Ling DI, Flores LL, Riley LW, Pai M. Commercial
tuberculosis in a high-volume public health laboratory        nucleic-acid amplification tests for diagnosis of pulmo-
in South Africa. Am J Resp Crit Care 2008, 177: 787-          nary tuberculosis in respiratory specimens: meta-analysis
-792.                                                         and meta-regression. PLoS One 2008; 2:e1536.
de Oliveira MM, da Silva Rocha A, Cardoso Oele-               Mello FCQ, Arias M, Pavón A, Marsico AG, Alvarado-
mann M, Gomes HM, Fonseca L, Werneck-Barreto                  -Gálvez C, Rosales |S, Pessoa CEC, Pérez M, Andrade
AM, Valim AM, Rossetti ML, Rossau R, Mijs W, Van-             MK, Kritski AL, Fonseca LS, Chaisson RE, Kimerling
derborght B, Suffys P. Rapid detection of resistance          M, Dorman, SE. Clinical evaluation of the micros-
against rifampicin in isolates of Mycobacterium tuber-        copic observation drug susceptibility (MODS) assay
culosis from Brazilian patients using a reverse-phase         for detection of Mycobacterium tuberculosis resis-
hybridization assay. J Microbiol Methods 2003;                tance to isoniazid or rifampin. J Clin Microbiol 2007;
53(3):335-342.                                                45(10):3387-3389.
Flores LL, PaiM, Colford JM, Riley LW. In-house nucleic       Palomino JC. Molecular detection, identification and
acid amplification tests for the detection of Mycobacterium   drug resistance detection in Mycobacterium tuberculosis.
tuberculosis in sputum specimens: meta-analysis and me-       Minireview. FEMS Immunol Med Microbiol 2009; 1-9.
taregression. BMC Microbiol 2005; 5: 55.                      Rede Brasileira de Pesquisa em Tuberculose – Rede Tb
Giampaglia MS, Martins MC, Vieira GBO, Vinhas SA,             (
da Silva Telles MA, Palaci M, Marsico AG, Hadad DJ,           Scherer LC, Sperhacke RD, Mello FCQ, C Jarczewski,
Mello FCQ, Kritski A, Siddiqi S, Fonseca LS. Multi-           Cafrune P, Minghelli S, Osorio M, Rossetti ML, Kritski
-center Evaluation of Automated Bactec MGIT 960               AL. PCR colorimetric dot-blot assay and clinical pretest
System for testing susceptibility of M. tuberculosis as       probability for diagnosis of pulmonary tuberculosis in
compared with BACTEC 460TB, proportion and re-                smear-negative patients BMC Public Health 2007;
sistance ratio methods in Southeast of Brazil. Int J Tu-      7(1):356.
berc Lung Dis 2007; 11(9):986-991.                            Scherer LC, Sperhacke RD, Ruffino-Netto A, Rossetti,
Global Plan against TB – WHO/STOP em 2006                     MLR, Kritski AL. Cost-effectiveness analysis of the
(        PCR associated with Ziehl Neelsen smear examination
Gounder C, Mello FCQ, Conde MB; Kritski A, Chais-             (ZN) for the rapid diagnosis of pulmonary tuberculosis
son RE, Dorman S. Field evaluation of a rapid immuno-         in subjects with and without HIV in a hospital setting.
chromatographic test for tuberculosis. J Clin Microbiol       BMC Public Health 2009 (in press).
2002; 40(6):1477-1451.                                        Steingart KR, Henry M, Laal S, Hopewell PC, Ramsay
Hofmann-Thiel S, van Ingen J, Feldmann K, Turaev L,           A, Menzies D, Cunningham J, Weldingh K, Pai M.
Uzakova GT, Murmusaeva G, van Soolingen D, Hoff-              Commercial serological antibody detection tests for the

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                                              Vol XVI Suplemento 1 A Janeiro 2010
       Experiência da Rede Brasileira de Pesquisa em Tuberculose no desenvolvimento
                       e avaliação de novos métodos de diagnóstico em tuberculose
                                                                                                                  Afranio Kritski

              diagnosis of pulmonary tuberculosis: a systematic re-       losis drug-resistance patterns. Am J Respir Crit Care
              view. PLoS Med 2007 Jun; 4(6):e202. Erratum in: PLoS        Med 2005; 172(5):636-642. Epub 2005 Jun 9.
              Med 2007; 4(8):e254.                                        WHO – 2007 recomendations – use of liquid médium
              Van Cleeff M, Kivihya-Ndugga L, Githui W, Ng’ang’a          for TB and drug resistant TB diagnosis http://www.
              L, Kibuga D, Odhiambo J, Klatser P. Cost-effectiveness
              of polymerase chain reaction versus Ziehl-Neelsen smear     WHO – 2008 recommendations – use of line molecular
              microscopy for diagnosis of tuberculosis in Kenya. Int J    probe assays for drug resistant TB diagnosis (http://
              Tuberc Lung Dis 2005; 9(8):877-883.               
              Van Rie A, Victor TC, Richardson M, Johnson R, van          en/index.html\).
              der Spuy GD, Murray EJ, Beyers N, Gey van Pittius           [WHO] World Health Organization 2009. Global Tu-
              NC, van Helden PD, Warren RM. Reinfection and               berculosis Control. WHO Report. Geneva, Switzer-
              mixed infection cause changing Mycobacterium tubercu-       land.

S 76   R e v i s t a        P o r t u g u e s a                          d e      P n e u m o l o g i a
                                       Vol XVI Suplemento 1 A Janeiro 2010
Moisés Palaci1                                          Ensaios clínicos de novas drogas e testes diagnósticos
                                                        em tuberculose: Desafios micobacteriológicos

                                                        Clinical trials of new tuberculosis drugs and diagnostic
                                                        tests: Mycobacteriological challenges

Desde que a Organização Mundial de Saúde                              Since the World Health Organization’s 1993
declarou a da tuberculose (TB) como emer-                             declaration that tuberculosis (TB) was a
gência global em 1993, esta doença, histori-                          worldwide emergency, this historically im-
camente importante e igualmente negligen-                             portant and equally neglected disease has
ciada, vem recebendo mais atenção por parte                           received more attention from agencies dedi-
das agências dedicadas ao seu controlo e ao                           cated to its controlling and financing re-
financiamento de pesquisas. Como conse-                               search. As a consequence of this and of re-
quência deste facto e do desenvolvimento                              cent scientific and technological advances,
científico e tecnológico alcançado nos últi-                          new diagnostic tests and compounds with
mos anos, novos testes diagnósticos e com-                            therapeutic potential have emerged. These
postos com potencial terapêuticos têm surgi-                          have obliged manufacturers and clinical re-
do e obrigado os fabricantes e sites de                               search sites to evaluate and register them in
pesquisa clínica a avaliá-los para serem regis-                       regulatory bodies. The main methodology
tados em agências regulatórias. Em ensaios                            used in clinical trials to evaluate new drugs
clínicos para avaliação de novas drogas, a                            is early bactericidal activity (EBA), described
principal metodologia utilizada é a atividade                         by Mitchison1, which consists of quantify-
bactericida precoce (early bactericidal activity                      ing the colony forming unit (CFU) in spu-
– EBA) descrita por Mitchson1, que consiste                           tum samples collected over a 12-h period
em quantificar a carga bacilar (CFU) presen-                          for the first 7 days of treatment.
te em amostras de escarro recolhidas por um                           This methodology is based on the reduction
período de 12 horas durante os primeiros 7                            in the number of CFU over the first two days
dias de tratamento. Tal metodologia é basea-                          being statistically related to the efficacy of the
da no facto de a redução do número de CFU                             treatment regimens instituted, making it a

1Núcleo de Doenças Infecciosas da Universidade Federal do Espírito Santo, Brasil/Infectious Diseases Unit, Universidade Federal do Espírito Santo, Brazil

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                                                       desafios micobacteriológicos
                                                                                                       Moisés Palaci

              durante os primeiros dois dias estar estatisti-    clinical parameter of reduced infectiousness2,3.
              camente relacionada com a eficácia dos es-         EBA studies are performed to compare the
              quemas terapêuticos empregues e representa         activity of various doses of a drug, different
              um parâmetro clínico de redução da infec-          drugs of the same class and different classes of
              ciosidade2,3. Os estudos de EBA são realiza-       drugs4. This type of clinical trial implies heavy
              dos para comparar a atividade de várias doses      financial investment, professionals qualified
              de uma droga, diferentes drogas dentro da          in good clinical and laboratory practices and a
              mesma classe e diferentes classes de drogas4.      serious commitment to and understanding of
              A realização deste tipo de ensaio clínico re-      patients. Accordingly, the mycobacteriology
              quer elevado investimento financeiro, profis-      laboratory faces heavy responsibility and a se-
              sionais qualificados em boas práticas clínicas     ries of challenges to be able to play a success-
              e laboratoriais e um sério comprometimento         ful role in clinical trials. Some of the obstacles
              e compreensão dos doentes. Diante destes           encountered and studies undertaken to over-
              factos, o laboratório de micobacteriologia as-     come them are summarised below.
              sume grande responsabilidade e enfrenta
              muitos desafios para cumprir com êxito o seu
              papel nos ensaios clínicos. A seguir são des-      Sample collection over 12 and 16 hrs
              critos resumidamente alguns dos obstáculos e       Collecting a pool of sputum under these condi-
              estudos realizados para superá-los.                tions requires the patient being admitted to the
                                                                 hospital, which involves separation of the pa-
                                                                 tient from his/her family, the costs of hospita-
              Recolha de amostras 12 a 16                        lization stay, an increased risk of culture con-
              horas                                              tamination and increased length of patient
              Um pool de escarro recolhido nestas condi-         monitoring. To see if a pool of sputum collected
              ções requer internação do doente, o seu dis-       over 5-h in the morning contained similar CFU
              tanciamento da família, custo financeiro de        to a pool collected over a 12-h period, Nasci-
              internação, maior risco de contaminação da         mento et al. (study currently in final stage) when
              cultura e longo período de tempo para mo-          comparing CFU in the sputum of patients with
              nitoramento. Com o objetivo de verificar se        pulmonary TB collected over 5-h and 12-h pe-
              o pool de escarro recolhido durante 5 horas        riods, did not find any statistically significant
              pela manhã poderia conter uma carga bacilar        differences in the two groups evaluated (6.88
              semelhante ao pool recolhido num período           log10 CFU/ml vs. 6.95 log10 CFU/ml, respec-
              de 12 horas, Nascimento et al (estudo em           tively) and, thus, showed that sputum collected
              fase final), ao comparar a carga bacilar em        over 5-h could be used in future clinical trials.
              amostras de escarro de doentes com tubercu-
              lose pulmonar, recolhidas por períodos de 5
              e 12 horas, não observaram diferenças esta-        New markers of bacteriological
              tísticas significativas entre os dois grupos       clearance in response to anti-
              avaliados (6,88 log10 CFU/ml, e 6,95 log10         tuberculosis therapy
              CFU/ml, respectivamente) e demonstraram            Microbiological parameters can easily be as-
              assim que uma recolha monitorizada de es-          sessed in spontaneously expectorated spu-

S 78   R e v i s t a      P o r t u g u e s a                   d e     P n e u m o l o g i a
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Ensaios clínicos de novas drogas e testes diagnósticos em tuberculose:
desafios micobacteriológicos
Moisés Palaci

carro durante 5 horas poderá ser utilizada       tum, but quantitative culture is time con-
em futuros ensaios clínicos.                     suming and labour intensive. The ideal
                                                 marker would measure events early during
                                                 treatment and be accurate regardless of the
Novos marcadores de depuração                    drug action or regimen being tested. Re-
bacteriológica em resposta à                     cently, Liwen et al.5 assessed and reported
terapia antituberculose                          mRNA levels by quantitative RT-PCR in
Os parâmetros microbiológicos podem ser          sputum specimens from TB patients receiv-
facilmente avaliados na expectoração espon-      ing monotherapy in an early bactericidal
tânea, mas a cultura quantitativa é demorada     activity study of fluoroquinolones and in
e trabalhosa. O marcador ideal mediria even-     those receiving a standard rifampin-based
tos no decurso do início do tratamento e se-     regimen in an IL-2 trial. These authors
ria rigoroso, independentemente da ação ou       demonstrated that messenger RNA for the
do regime a ser testado. Recentemente,           glyoxylate cycle enzyme isocitrate lyase de-
Liwen et al.5 avaliaram e reportaram níveis      clined at similar rates in patients receiving
de mRNA por RT-PCR quantitativa em               isoniazid, gatifloxacin, levofloxacin, and
amostras recolhidas de doentes com TB sob        moxifloxacin monotherapy. Isocitrate lyase
monoterapia, num anterior estudo de ativi-       (icl) mRNA correlated highly with CFU in
dade bactericida em fluoroquinolonas, e nos      sputum prior to therapy and during 7 days
que estavam sob um regime-padrão com             of monotherapy in all treatment arms. icl
base em rifampina num ensaio de IL-2. Os         mRNA was detectable in sputum of culture-
autores demonstraram que o mensageiro            positive TB patients receiving a rifampin-
RNA para a isocitrato liase da enzima do ci-     based regimen for 2 months. In addition,
clo glioxilato teve taxas de declínio seme-      they also demonstrated that fibronectin-
lhantes em doentes a receberem monoterapia       binding protein (fbpB) mRNA decreased
com isoniazida, gatifloxicina, levofloxacina e   0.47 log10 molecules/ml/d from baseline to
moxifloxacina. A isocitrato liase (icl) mRNA     day 2, the only mRNA correlating with the
esteve altamente relacionada com as unida-       early bactericidal activity of isoniazid mono-
des que formavam colónias na expectoração        therapy. In conclusion, icl and fbpB mRNAs
antes da terapia e durante 7 dias de monote-     are reliable markers of M. tuberculosis viabil-
rapia em todos os grupos de tratamento.          ity, however, both of these uses will require
Detectou-se icl mRNA na expectoração de          larger longitudinal studies to validate the re-
doentes com cultura positiva de TB em regi-      liability of icl mRNA as a surrogate marker
me de tratamento com base em rifampina           of response to drug therapy for TB.
durante 2 meses. Além disso, também de-
monstraram que a proteína de ligação à fi-
bronectina (fbpB) mRNA diminuiu 0,47             Lower rates of contamination
log10 moléculas/ml/d desde o início até ao       of mycobacterial culture
segundo dia, o único mRNA que se relacio-        In clinical trials to evaluate treatment re-
nou com a atividade bactericida precoce da       gimes that require patient follow-up for up
monoterapia com isoniazida. Em conclusão,        to 2 years after cure, and in studies to eva-

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                                                       desafios micobacteriológicos
                                                                                                      Moisés Palaci

              icl e fbpB mRNAs são marcadores fiáveis de          luate new diagnostic tests, contaminated
              viabilidade do M. tuberculosis; no entanto,         sputum cultures could result in high costs,
              estas duas utilizações carecem de estudos           elimination of data from the statistical ana-
              longitudinais mais extensos que validem a           lysis (time points where contamination oc-
              fiabilidade de icl mRNA como marcador               curred) or even exclusion of the patient
              substituto de resposta à terapia farmacológi-       from the study. In the face of these difficul-
              ca para TB.                                         ties, Peres et al. (article submitted for publi-
                                                                  cation) performed a pragmatic clinical trial
                                                                  to evaluate the efficacy of methods of intra-
              Diminuição das taxas de                             oral antisepsis in reducing the contamina-
              contaminação de culturas                            tion rate of cultures from patients suspected
              de micobactérias                                    of TB. The authors found that of the three
              Em ensaios clínicos para avaliação de regimes       intra-oral hygiene procedures employed
              terapêuticos que exigem o monitoramento do          separately (water only; chlorhexidine diglu-
              doente em até dois anos após a sua cura, e em       conate and cetylpyridinium chloride), only
              estudos para avaliação de novos testes diagnós-     the one using chlorhexidine digluconate re-
              ticos, culturas de escarro contaminadas podem       sulted in a significant reduction of the cul-
              causar prejuízos económicos, eliminação de          ture contamination rate, particularly in
              dados da análise estatística (time points onde      MGIT liquid medium (Becton Dickinson).
              ocorreram contaminação) ou até a exclusão do        They also found that using a higher concen-
              doente do estudo. Diante destas situações pro-      tration of PANTA antimicrobial solution
              blemáticas, Peres et al. (artigo submetido a pu-    (PANTA 2x) in patients’ control samples,
              blicação) realizaram um ensaio clínico prag-        significantly reduced the population of con-
              mático para avaliar a eficácia de métodos de        taminating organisms in the MGIT liquid
              antissepsia intrabucal na redução da taxa de        medium, without, however, lowering the
              contaminação de culturas de doentes suspeitos       rate of M. tuberculosis detection.
              de tuberculose. Os autores constataram que,
              dentre os três procedimentos de higienização
              bucais utilizados isoladamente (somente água,       Sputum sample division
              digluconato de clorexidina e cloreto de cetilpi-    Performing clinical trials to evaluate new di-
              ridínio), apenas o realizado como digluconato       agnostic methods and treatments often re-
              de clorexidina permitiu uma redução signifi-        quires sputum sample division for compara-
              cativa da taxa de contaminação das culturas,        tive analysis. In this case, the samples must
              sobretudo no meio líquido MGIT (Becton              be divided equally, maintaining a similar
              Dickinson). Verificaram ainda que o uso de          CFU in each part. The use of a mucolitic
              uma concentração maior do antimicrobiano            agent is recommended for this, but there are
              PANTA (PANTA 2x) nas amostras-controlo              no studies proving its efficacy or that of
              dos doentes reduziu significativamente a po-        other procedures for sample division. Mo-
              pulação de organismos contaminantes no              rais et al. (unpublished data) used a quanti-
              meio MGIT, sem, contudo, reduzir a taxa de          tative culture technique to verify if CFU
              deteção do M. tuberculosis.                         was similar in divided samples after the di-

S 80   R e v i s t a      P o r t u g u e s a                    d e     P n e u m o l o g i a
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Ensaios clínicos de novas drogas e testes diagnósticos em tuberculose:
desafios micobacteriológicos
Moisés Palaci

Divisão em amostras de escarro                        gestion of sputum samples by chemical pro-
A realização de ensaios clínicos para avaliação       cedure (N-Acetyl-L-Cysteine 50mg/ml-
de novos métodos diagnósticos e terapêuticos          10% of the sample volume for 15 minutes),
frequentemente requer a divisão da amostra de         or mechanically (agitation with glass beads).
escarro para análise comparativa. Neste caso é        No statistically significant differences were
necessário que a divisão da amostra ocorra de         seen when the results of the groups were
maneira equitativa, mantendo a carga bacilar          compared. In the first group (NALC), ali-
semelhante entre as partes. Recomenda-se o            quot I had mean 5.66 (±0.92) log10 and ali-
uso de um agente mucolítico para esta finalida-       quot II mean 5.65 (±0.94) log10 CFU/ml. In
de; contudo, por não se dispor de estudos que         the second group (glass beads) the means
comprovem a eficiência deste ou de outros             were 5.53 (±0.91) and 5.49 (±0.94) log10C-
procedimentos de divisão de amostras, Morais          FU/ml for each aliquot, respectively, thus
et al (dados ainda não publicados), utilizando        showing that both chemical and physical
técnica de cultura quantitativa, propuseram-se        procedures can be used with equal efficacy
verificar se após a digestão de amostras escarro      for division of sputum samples.
por procedimento químico (N-acetil-L-cisteina         As mentioned above, clinical treatment trials
50 mg/ml- 10% do volume da amostra duran-             for TB are complex and expensive. Few sites
te 15 minutos), ou mecânico (agitação com             worldwide have the necessary requirements
pérolas de vidro), a carga bacilar seria seme-        and teams to perform them. If we consider
lhante nas amostras divididas. Ao comparar os         the modest investment made in this area,
resultados dos grupos entre si não observaram         important advances have been seen in re-
diferenças estatísticas significativas. Para o pri-   cent years. There are, however, many chal-
meiro grupo que fez uso imediato do NALC              lenges which have yet to be met. One such
foi observada na aliquota I uma média de 5,66         challenge is the validation of the results de-
(±0,92) log10, enquanto na aliquota II 5,65           scribed above and the development of sim-
(±0,94) log10 UFC/ml. Para o segundo grupo,           ple tests able to determine sensitivity to
processado com pérolas de vidro, foi observado        drugs for latent bacteria and bacterial viabil-
uma média de 5,53 (±0,91) e 5,49 (±0,94)              ity over the long course of TB treatment.
log10 UFC/ml em cada aliquota, respetivamen-
te, demonstrando assim que tanto o procedi-
mento químico quanto o físico podem ser uti-
lizados com igual eficiência para divisão de
amostras de escarro.
Como destacado anteriormente, os ensaios
clínicos terapêuticos em tuberculose são
complexos e onerosos. Poucos sites no mun-
do possuem os requisitos e equipas necessá-
rias à sua realização. Se considerarmos o mo-
desto investimento feito nesta área,
importantes avanços ocorreram nos últimos
anos. Entretanto, muitos outros desafios

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              Ensaios clínicos de novas drogas e testes diagnósticos em tuberculose:
                                                       desafios micobacteriológicos
                                                                                                                          Moisés Palaci

              ainda necessitam de ser superados. Dentre os
              quais a validação dos resultados descritos e o
              desenvolvimento de testes simples e capazes
              de determinar a sensibilidade a drogas de
              bactérias em fase de latência e a viabilidade
              bacteriana durante o longo período de trata-
              mento da tuberculose.

              1. Mitchison DA. Assessment of new sterilizing drugs           4. Johnson JL, Hadad DJ, Boom WH, Daley CL, Pelo-
              for treating pulmonary tuberculosis by culture at 2            quin CA, Eisenach KD, Jankus DD, Debanne SM,
              months. Am Rev Respir Dis 1993; 147: 1062-1063.                Charlebois ED, Maciel E, Palaci M, Dietze R. Early and
              2. Jindani A, Aber VR, Edwards EA, Mitchison DA.               extended early bactericidal activity of levofloxacin, gati-
              The early bactericidal activity of drugs in patients with      floxacin and moxifloxacin in pulmonary tuberculosis.
              pulmonary tuberculosis. Am Rev Respir Dis 1980;                Int J Tuberc Lung Dis 2006; 10:605-612.
              121:939-949.                                                   5. Li L, Mahan CS, Palaci M, Horter L, Loeffelholz L,
              3. Kennedy N, Fox R, Kisyombe GM, Saruni AOS,                  Johnson JL, Dietze R, Debanne SM, Joloba ML, Okw-
              Uiso LO, Ramsay ARC, Ngowi FI, Gillespie S. Early              era A, Boom WH, Eisenach KD. Sputum Mycobacte-
              bactericidal and sterilizing activities of ciprofloxacin in    rium tuberculosis mRNA as a marker of bacteriologic
              pulmonary tuberculosis. Am Rev Respir Dis 1993;                clearance in response to anti-tuberculosis therapy. J Clin
              148:1547-1551.                                                 Microbiol 2009; 18 [Epub ahead of print].

S 82   R e v i s t a         P o r t u g u e s a                            d e       P n e u m o l o g i a
                                        Vol XVI Suplemento 1 A Janeiro 2010
Karina de Prince1                                    Avaliação das moléculas com atividade antiTB das
Fernando R Pavan1                                    plantas do cerrado brasileiro
Daisy N Sato2
Wagner Villegas5
Sergio RA Leite5                                     Screening of molecules with anti-TB activity from the
Clarice QF Leite1*                                   brazilian cerrado plants

Introdução                                                        Introduction
A tuberculose (TB) ainda é, em todo o mun-                        Across the world, tuberculosis (TB) remains
do, a doença infeciosa mais frequente e im-                       the most frequent and important infectious
portante, causando morbilidade e morte.                           disease causing morbidity and death. A
Um terço da população mundial está infeta-                        third of the world’s population is infected
da com Mycobacterium tuberculosis (MTB) e                         with Mycobacterium tuberculosis (MTB),
aproximadamente dois milhões de mortes                            and approximately two million deaths are
são atribuídas à TB anualmente1. Entre to-                        attributable to TB annually1. Among all the
dos os países do continente americano, o                          countries in the Americas, Brazil reports the
Brasil tem o segundo maior índice de morbi-                       second-highest TB mortality and morbidity,
lidade e de morte por TB, com uma preva-                          with a prevalence of 62 in /100 0002. The
lência de 62/100 0002. O ressurgimento glo-                       global resurgence of TB and the rapid emer-
bal da TB e o rápido aparecimento da                              gence of MDR-TB, underscore the impor-
tuberculose multirresistente (MDR-TB) su-                         tance of the development of new antituber-
blinha a importância do desenvolvimento de                        culous drugs3.
novos fármacos antituberculose3.                                  Concerning the search for new compounds
Na busca de novos compostos a partir de                           from Plants, trees have provided many drugs
plantas, as árvores forneceram muitos fárma-                      in the past, and remain a rich source of no-
cos no passado e permanecem uma importan-                         vel compounds. Natural products have re-
te fonte de novos compostos. Recentemente                         cently received a lot of attention as potential
os produtos naturais têm sido alvo de muita                       anti-TB agents4-6. In Brazil, there is a tradi-
atenção como potenciais agentes antiTB4-6.                        tional knowledge of how to use native plants

1 Faculdade de Ciências Farmacêuticas, UNESP, CEP 14801-902, Araraquara (SP), Brasil
2 Instituto Adolfo Lutz, Ribeirão Preto, CEP 14085-410, Ribeirão Preto (SP), Brasil
3 Departamento de Química, Universidade Federal de São Carlos, CP 676, CEP 13565-905, São Carlos (SP), Brasil

4 Instituto de Química de São Carlos, Universidade de São Paulo, CP 780, 13560-970, São Carlos – SP, Brasil

5 Instituto de Química de Araraquara, UNESP, CEP 14801-902, Araraquara – SP, Brasil.


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       Avaliação das moléculas com atividade antiTB das plantas do cerrado brasileiro
                               Karina de Prince, Fernando R Pavan, Daisy N Sato, Wagner Villegas, Sergio RA Leite, Clarice QF Leite

                 No Brasil, existe um conhecimento tradicio-             to treat several diseases because many com-
                 nal de como utilizar plantas nativas no trata-          munities don’t have access to synthetic me-
                 mento de várias doenças, porque muitas co-              dicines and use those plants in treatments7.
                 munidades não têm acesso a medicamentos e               Although several hundred natural plant
                 usam essas plantas para se tratarem7.                   products with antimycobacterial activity
                 Embora tenham sido encontradas várias                   have been found, none of them have
                 centenas de produtos naturais de plantas                moved towards drug development, be-
                 com atividade antimicobacteriana, nenhu-                cause of difficulties, such as very low com-
                 ma delas atingiu a fase de desenvolvimento              pound availability, high structural com-
                 para fármaco devido a dificuldades, como a              plexity and lack of follow-up studies of
                 escassez de compostos, a elevada complexi-              promising leads
                 dade estrutural e a falta de estudos de acom-
                 panhamento de indícios promissores.
                                                                         In this context, we started the project in-
                 Objetivo                                                tegrating chemical analysis and anti-TB
                 Neste contexto, iniciámos o projeto inte-               activity tests of plants, especially species
                 grando análises químicas e testes de activi-            that compose the “Brazilian Cerrado”, a
                 dade antiTB de plantas, especialmente de                savannah like biome that predominates in
                 espécies que compõem o cerrado brasileiro,              the center-west region of the country. It
                 bioma do tipo savana que predomina no                   includes more than several thousands na-
                 planalto central brasileiro e que inclui mi-            tive vascular plant species, grouped in
                 lhares de espécies vasculares nativas agrupa-           hundreds of families. Many of these plants
                 das em centenas de famílias. Muitas destas              are commonly used as natural remedies
                 plantas são habitualmente usadas como re-               by people living in this area to treat va-
                 médios naturais pela população local para               rious illnesses.
                 tratar doenças várias.

                 Material e métodos                                      Material and methods
                 Os especímenes de plantas do cerrado foram              The cerrado plant specimens were collected
                 colhidas nos estados de Tocantins (aproxima-            in the states of Tocantins (at nearly 11° S by
                 damente 11° S, 48° O) e Mato Grosso do Sul              48° W) and Mato Grosso do Sul (approxi-
                 (aproximadamente 21° S, 56° O). Para a sepa-            mately 21° S by 56° W). To perform the
                 ração fitoquímica, usámos técnicas cromato-             phytochemical separation, we used chro-
                 gráficas, essencialmente adequadas para sepa-           matographic techniques, mainly suitable for
                 ração de substâncias polares (GPC, XAD2,                polar substances (GPC, XAD2, DCCC,
                 DCCC, HSCC, HPLC, etc.). Para determi-                  HSCC, HPLC, etc). To determine the
                 nar a estrutura dos compostos isolados usámos           structure of the isolated compounds we
                 principalmente métodos espectrofotométri-               used mainly spectrophotometric methods
                 cos, como NMR, IR, UV e MS. Avaliámos a                 such as NMR, IR, UV and MS. To evaluate
                 atividade dos extratos, frações enriquecidas e          the activity of the extracts, enriched frac-

S 84     R e v i s t a       P o r t u g u e s a                       d e        P n e u m o l o g i a
                                      Vol XVI Suplemento 1 A Janeiro 2010
Avaliação das moléculas com atividade antiTB das plantas do cerrado brasileiro
Karina de Prince, Fernando R Pavan, Daisy N Sato, Wagner Villegas, Sergio RA Leite, Clarice QF Leite

substâncias puras contra o M. tuberculosis por               tions and pure substances against M. tuber-
resazurin microtiter assay (REMA), de acordo                 culosis we used the Resazurin Microtiter As-
com Palomino et al. (2002) e M. tuberculosis                 say (REMA) according Palomino et al.
H37Rv ATCC 272948.                                           (2002) and the M. tuberculosis H37Rv ATCC
                                                             27294 strain8.

Resultados e discussão
Sessenta e cinco extratos de 37 plantas distri-              Results and discussion
buídas por 18 famílias foram testadas contra                 sixty five extracts from 37 plants, distribu-
M. tuberculosis. Vinte e seis por cento dos ex-              ted in 18 families have been tested against
tratos avaliados demonstraram actividade                     M. tuberculosis. Out of all the extracts as-
promissora, nomeadamente concentração                        sayed, 26% showed promising activity,
inibitória mínima (CIM) ≤ 125 μg/m, 13                       namely MICs below or equal to 125 μg/mL,
(76%) deles extratos de clorofórmio e 4                      13 (76%) of these coming from chloroform
(24%) de metanol. Esses extratos foram sele-                 extract and 4 (24%) from methanol extract.
cionados para fracionamento guiado por                       These extracts were selected for activity
atividade e avaliação detalhada das proprie-                 guided-fractionation and detailed evalua-
dades antituberculosas, sendo a sua compo-                   tion of the anti-tuberculosis properties, and
sição química analisada (Quadro I).                          their chemical composition was analysed
Do extrato de clorofórmio de B. fagifolia, a                 and showed in Table I.
mistura de lupeol, α-amirina e β-amirina re-                 From the B. fagifolia chloroform extract, the
velaram CIM mais baixa (31,25 μg/mL) do                      mixture of lupeol, α-amyrin and β-amyrin
que lupeol, α-amirina ou β-amirina isola-                    displayed a lower MIC (31.25 μg/mL) than
dos, cujos valores CIM foram ≥ 62,5 μg/mL.                   isolated lupeol, α-amyrin or β-amyrin
A mistura de lupeol e acetatos de α-amirina                  whose MIC value were higher than or equal
e β-amirina mostraram o mesmo valor CIM                      to 62.5 μg/mL. The mixture containing lu-
(31,25 μg/mL), sugerindo que a acetilação                    peol and α-amyrin and β-amyrin acetates
de α-amirina e β-amirina não influencia a                    showed the same MIC value of 31.25 μg/
sua atividade9. Observou-se a mesma situa-                   mL, suggesting that the acetylation of
ção nas frações enriquecidas de extrato de                   α-amyrin and β-amyrin does not influence
clorofórmio de B. crassa. As CIM de 31,25                    their activity9. The same situation was found
para a mistura de α-e β-amirina e de 62,5                    in the enriched fractions from the B. crassa
μg/mL, o dobro do valor para o acetato puro                  chloroform extract. MICs of 31.25 for the
de α-amirina, confirma o efeito sinergístico                 mixture of α-and β-amyrin and 62.5 μg/
entre os componentes destas misturas contra                  mL, double the value for the pure acetate of
M. tuberculosi10.                                            α-amyrin, confirms the synergistic effect
Para C. adamantium, a 5,7-dihidroxi-6,8-di                   among the components of these mixture
-C-metilflavanona (A) isolada mostrou uma                    against M. tuberculosis10.
CIM superior a 250 e 2’, 4’-dihidroxi-3’,5’-                 For C. adamantium, the isolated 5,7-dihy-
dimetil-6’-metoxicalcona (B) uma CIM de                      droxy-6,8-di-C-methylflavanone (A) showed
62,5 μg/mL, enquanto as suas misturas                        higher MIC of 250 and 2’,4’-dihydroxy-3’,5’-

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       Avaliação das moléculas com atividade antiTB das plantas do cerrado brasileiro
                                    Karina de Prince, Fernando R Pavan, Daisy N Sato, Wagner Villegas, Sergio RA Leite, Clarice QF Leite

                 Quadro I – Valores CIM das frações enriquecidas e compostos testados contra M. tuberculosis
                  Compostos                                                                                            CIM (μg/mL)
                  Fração enriquecida / compostos de clorofórmio extrato de B. fagifolia
                  Mistura de lupeol, α-e β-amirina                                                                         31,25
                  Mistura de lupeol, acetatos de α- e β-amirina                                                            31,25
                  α-amirina                                                                                                 62,5
                  α-amirina acetato                                                                                         62,5
                  Dotriacontano                                                                                             62,5
                  Ácido básico                                                                                              2,5
                  Fração enriquecida / compostos de extrato de clorofórmio de B. crassa
                  Mistura de α-e β- amirina                                                                                31,25
                  Mistura de acetato de α-e β- amirina                                                                     31,25
                  Mistura de ácidos ursólico e oleanólico                                                                  62,5
                  Compostos de extrato de clorofórmio de C. adamantium
                  5,7-dihidroxi-6,8-di-C-metilflavanona (A)                                                                  250
                  2’,4’-dihidroxi-3’,5’-dimetil-6’-metoxicalcona (B)                                                       62,5
                  Mistura A + B (2:8)                                                                                        7,8
                  Mistura A + B (3:7)                                                                                       15,6
                  Mistura A + B (7:3)                                                                                      31,25
                  Mistura A + B (8:2)                                                                                       62,5
                  Compostos de extrato de clorofórmio de Qualea parviflora
                  Lupeol                                                                                                    62,5
                  Lupenona                                                                                                  125
                  Ácido betulínico                                                                                         31,25
                  Ácido 3 epibetulínico                                                                                    62,5
                  Friedelina                                                                                                125
                  β sitosterol                                                                                              125

                 Table I – MIC values of enriched fractions and compounds tested against M. tuberculosis
                  Compounds                                                                                           MIC (μg/mL)
                  Enriched fraction/compounds from chloroform extract of B. fagifolia
                  Mixture of lupeol, α-and β-amyrin                                                                       31.25
                  Mixture of lupeol, acetates of α- and β-amyrin                                                          31.25
                  α-amyrin                                                                                                 62.5
                  α-amyrin acetate                                                                                         62.5
                  Dotriacontane                                                                                            62.5
                  Bassic acid                                                                                               2.5
                  Enriched fraction/compounds from chloroform extract of B. crassa
                  Mixture of α-and β-amyrin                                                                               31.25
                  Mixture of α-and β-amyrin acetate                                                                       31.25
                  Mixture of ursolic and oleanolic acid                                                                   62.5
                  Compounds from chloroform extract of C. adamantium
                  5,7-dihydroxy-6,8-di-C-methylflavanone (A)                                                                250
                  2’,4’-dihydroxy-3’,5’-dimethyl-6’-methoxychalcone (B)                                                   62.5
                  Mixture A + B (2:8)                                                                                       7.8
                  Mixture A + B (3:7)                                                                                      15.6
                  Mixture A + B (7:3)                                                                                     31.25
                  Mixture A + B (8:2)                                                                                      62.5
                  Compounds from chloroform extract of Qualea parviflora
                  Lupeol                                                                                                   62.5
                  Lupenona                                                                                                 125
                  Betulinic acid                                                                                          31.25
                  3 epi betulinic acid                                                                                     62.5
                  Friedelin                                                                                                125
                  β sitosterol                                                                                             125

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Avaliação das moléculas com atividade antiTB das plantas do cerrado brasileiro
Karina de Prince, Fernando R Pavan, Daisy N Sato, Wagner Villegas, Sergio RA Leite, Clarice QF Leite

(A+B), em vários rácios, mostrou vários                      dimethyl-6’-methoxychalcone (B) a MIC of
CIM, entre 62,5 μg/mL para o rácio 8:2                       62.5 μg/mL, while their mixtures (A+B), in
(A+B) e a atividade muito melhor de 7,8                      several ratios, showed various MICs, ranging
μg/mL para o rácio 2:8. Há aqui claro siner-                 from 62.5 μg/mL for the ratio 8:2 (A+B)
gismo entre os compostos A e B quando                        down to the much higher activity of 7.8 μg/
misturados, e este sinergismo depende for-                   mL, for the ratio 2:8. Here there is a clear syn-
temente do seu rácio de concentração5. Nos                   ergism between compounds A and B when
compostos isolados de Qualea parviflora,                     mixed and this synergism depends strongly
apesar da grande semelhança estrutural de                    on their concentration ratio5. For compounds
lupeol e lupenona, observou-se uma redu-                     isolated from Qualea parviflora, despite the
ção duas vezes maior da atividade antiTB de                  great structural similarity of lupeol and lupe-
lupeol, que se deveu à substituição de                       none a two-fold reduction of anti-TB activity
β-hidroxilato C3 no lupeol para cetona na                    was observed with lupeol. This was due to the
lupenona. Do mesmo modo, também se                           substitution of β hydroxylat C3 in lupeol to
observou uma redução duas vezes maior da                     the ketone on lupenone. Similarly a two fold
atividade antiTB entre os ácidos betulínico                  reduction of anti-TB activity was observed
(CIM 31,2 μg/mL) e epibetulínico (62,5μg/                    between betulinic (MIC 31,2 μg/mL) and
mL), devido à epimerização no ácido epibe-                   epi-betulinic acid (62,5μg/mL), due to the
tulínico de β-hidroxil C3. Esses resultados                  epimerization in epi-betulinic acid of the β
corroboram os do trabalho de Cantrells et                    hydroxyl C3. Those results corroborate the
al. (2001), em que nos triterpenos, β-hidroxil               report of Cantrells et al. (2001) in that within
em C3 é importante para a atividade anti-                    triterpenes, the β hydroxyl on C3 is impor-
TB4. A melhor MIC observada foi no triter-                   tant for anti-TB activity4. The best MIC
peno do ácido básico de B. fagifolia, que                    found was for the triterpene bassic acid from
mostrou forte atividade antitubercular, com                  B. fagifolia, this showed strong antitubercular
valores CIM de 2,5 μg/mL9. Este valor de                     activity, with MIC values of 2.5 μg/mL9. This
concentração inibitória é comparável aos                     inhibitory concentration value is comparable
dos fármacos antiTB de primeira linha,                       to those of first-line tuberculosis drugs, such
como etambutol (1-5 μg/mL) e estreptomi-                     as ethambutol (1-5 μg/mL) and streptomycin
cina (2-8 μg/mL), e melhor do que a pirazi-                  (2-8 μg/mL), and better than pyrazinamide
namida (20-100μg/mL)11.                                      (20-100μg/mL)11.

Conclusão                                                    Conclusion
Os resultados indicam que as plantas do                      The results indicated that plants of the “cer-
cerrado podem proporcionar frações e com-                    rado” can provide fractions and compounds
postos com atividade antituberculose pro-                    with promising anti – tuberculosis activity.

    R e v i s t a                 P o r t u g u e s a                        d e         P n e u m o l o g i a   S 87
                                            Vol XVI Suplemento 1 A Janeiro 2010
       Avaliação das moléculas com atividade antiTB das plantas do cerrado brasileiro
                                 Karina de Prince, Fernando R Pavan, Daisy N Sato, Wagner Villegas, Sergio RA Leite, Clarice QF Leite

                 1. Global Alliance for TB Drug Development, www.          6. Honda NK, Pavan FR, Coelho RG, Leite SRA,
       , accessed June 1, 2009.                    Micheletti AC, Lopes TIB, Misutsi MY, Beatriz A, Brum
                 2. Malaspina AC, Cavalcanti HR, Leite CQF, Machado        RL, Leite CQF. Phytomedicine (2009) in press.
                 SM, Viana BH, Silva RM, Hage EF, Figueiredo WM,           7. Almeida SP, Proença CEB, Sano SM, Ribeiro Cerrado
                 Marques E, Ferrazoli L, Arbex M, Lessi M, Fonseca LS,     JF. Espécies vegetais úteis. In: Sano SM, Almeida SP
                 Rigouts L, Saad MH. Jpn J Infect Dis (2008); 231-         (Eds.). Planaltina, Distrito Federal, Brazil, 38-39.
                 -233.                                                     8. Palomino JC, Martin A, Camacho M, Guerra H,
                 3. Lourenço MCS, Ferreira ML, Souza MV, Peralta MA,       Swings J, Portaels F. Antimicrob Agents Chemoter
                 Vasconcelos TR, Henriques MGO. Eur J Med Chem             2002; 2720-2722.
                 2008; 43:1344-1347.                                       9. Higuchi CT, Sannomiya M, Pavan FR, Leite SRA,
                 4. Cantrell CL, Franzblau SG, Fischer NH. Planta Med      Sato DN, Franzblau SG, Sacramento LVS, Vilegas W,
                 2001; 67:1-10.                                            Leite CQF. eCAM (2008) doi:10.1093/ecam/nen077.
                 5. Pavan FR, Leite CQF, Coelho RG, Coutinho ID,           10. Higuchi CT, Pavan FR, Leite CQF, Sannomiya M,
                 Honda NK, Cardoso CAL, Vilegas W, Leite SRA, Sato         Vilegas W, Leite SRA, Sacramento LVS, Sato DN.
                 DN. Quim Nova 2009; 32:1222-1226.                         Quim Nova 2008; 31:1719-1721.
                                                                           11. Collins L, Franzblau SG. Antimicrob Agent Che-
                                                                           mother 1997; 1004-1009.

S 88     R e v i s t a         P o r t u g u e s a                       d e        P n e u m o l o g i a
                                        Vol XVI Suplemento 1 A Janeiro 2010
Caroline Allix-Béguec3                        Novas ferramentas de fácil utilização para genotipagem
Christine Hubans3                             padronizada e com qualidade controlada de estirpes do
Stéphanie Ferreira3
Philip Supply1,2,3                            complexo Mycobacterium tuberculosis

                                              New, easy-to-use tools for standardised and quality-
                                              controlled genotyping of Mycobacterium tuberculosis
                                              complex strains

A tipagem harmonizada e fiável de bactérias           Harmonized and reliable typing of patho-
patogénicas permite a fácil identificação de          genic bacteria permits easy identification
clones em circulação local ou internacional-          of locally or internationally circulating
mente, o que é essencial para bons serviços de        clones, which is essential for optimal epi-
vigilância epidemiológica e de controlo de            demiological surveillance and disease con-
doenças. Isto aplica-se particularmente a             trol. This is especially true for diseases such
doenças como a tuberculose (TB), com inci-            as tuberculosis (TB), with worldwide dis-
dência mundial e emergência global de estir-          tribution and global emergence of drug-
pes resistentes a fármacos. Além do mais, a           resistant strains. In addition, typing can
tipagem pode ajudar nas decisões terapêuti-           guide therapeutic decisions, for instance in
cas, por exemplo, em casos de suspeição de            case of suspected laboratory errors or con-
erros ou contaminações laboratoriais. A tipa-         taminations. Mycobacterial interspersed
gem pela metodologia MIRU-VNTR (myco-                 repetitive unit-variable number of tandem
bacterial interspersed repetitive unit-variable       repeat (MIRU-VNTR) typing has become
number of tandem repeat) tornou-se impor-             a major method for fast and high-resolu-
tante para a genotipagem rápida e de alta re-         tion genotyping of Mycobacterium tubercu-
solução dos isolados do complexo Mycobacte-           losis complex isolates. A system based on
rium tuberculosis. Um consórcio internacional         24 loci has been proposed for international
que inclui 10 laboratórios europeus e ameri-          standardization by an international con-
canos1 propôs um sistema baseado em 24 loci           sortium including 10 European and Ame-
para padronização internacional. Estudos de           rican laboratories1. In population-based
população mostraram que a tipagem MIRU-               studies, standard MIRU-VNTR typing
-VNTR padrão tem um valor de previsão                 was shown to have an equal to slightly bet-

2 Institut Pasteur de Lille, Lille
3 Genoscreen, Lille, France


     R e v i s t a                    P o r t u g u e s a          d e       P n e u m o l o g i a      S 89
                                           Vol XVI Suplemento 1 A Janeiro 2010
Novas ferramentas de fácil utilização para genotipagem padronizada e com qualidade
                   controlada de estirpes do complexo Mycobacterium tuberculosis
                                                     Caroline Allix-Béguec, Christine Hubans, Stéphanie Ferreira, Philip Supply

                igual a ligeiramente melhor do que o anterior       ter predictive value than the previous gold
                padrão-ouro, IS6110 RFLP, no estudo da              standard IS6110 RFLP for the study of TB
                transmissão de TB, em ambientes com carac-          transmission, in settings with epidemio-
                terísticas epidemiológicas representativas das      logical characteristics representative of
                de muitos países desenvolvidos. A interroga-        those of many developed countries. PCR-
                ção baseada na PCR de até 24 marcadores             based interrogation of up to 24 indepen-
                independentes e bem calibrados facilita a rá-       dent and well-calibrated markers facilitates
                pida e fiável elucidação dos mecanismos mo-         prompt and reliable molecular-guided elu-
                leculares de situações complexas que envol-         cidation of complex situations involving
                vam potenciais surtos, infeções mistas ou           potential outbreak cases, mixed infections
                reinfeções2-6. Como resultado, este método          or re-infections2-6. As a result, this method
                vem sendo adoptado internacionalmente,              is being internationally adopted, often in
                frequentemente em combinação com spoligo-           combination with spoligotyping, as the
                typing, como o novo método de referência            new reference method for TB molecular
                para epidemiologia molecular da TB, por             epidemiology, e.g. by the US CDC, large
                exemplo, pelo Center for Disease Control            European research and epidemiological
                (CDC) nos Estados Unidos, grandes consór-           surveillance consortiums and National or
                cios europeus de investigação e de vigilância       Regional reference Centers.
                epidemiológica e por centros de referência          New, easy-to-use tools and services have re-
                nacionais ou regionais.                             cently become available, which facilitate
                Novos equipamentos e serviços de uso fácil          quality-controlled use of this technique, as
                que ficaram recentemente disponíveis vie-           well as interpretation of the results obtained.
                ram facilitar o controlo de qualidade da uti-       MIRU-VNTR typing services are proposed
                lização desta técnica, bem como a interpreta-       and already used by international Reference
                ção dos resultados obtidos. Os serviços de          Centers and laboratories, for outsourcing
                tipagem MIRU-VNTR são propostos e usa-              their genotyping (including of M. bovis
                dos já por centros de referência e laborató-        strains) and/or for QA/QC evaluation.
                rios internacionais para realização de genoti-      Quality-controlled MIRU-VNTR Calibra-
                pagem (incluindo de estirpes de M. bovis) e/        tion, Validation and Typing kits, as well as
                /ou para avaliação do sistema de garantia de        dedicated data management software and
                qualidade QA/QC. Os kits de calibração,             on-site trainings greatly facilitate standar-
                validação e tipagem MIRU-VNTR de qua-               dized set up and efficient use of MIRU-
                lidade controlada, bem como o software para         VNTR typing in user’s laboratory.
                gestão de dados e formação on-site, facilitam       With the MIRU-VNTR Typing Kit, 24
                enormemente a padronização e a utilização           markers are amplified from purified DNA
                eficiente da tipagem MIRU-VNTR no labo-             or crude DNA extract from mycobacterial
                ratório do utilizador.                              colonies or cell pellets from liquid cul-
                Com o kit de tipagem MIRU-VNTR, 24                  tures, using 8 triplex PCR and fluorescent
                marcadores são ampliados a partir de ADN            primers specific for the flanking regions of
                purificado ou de extrato bruto de ADN de            the targeted loci. Amplified fragment are
                colónias micobacterianas ou de grânulos de          separated by capillary electrophoresis on

S 90    R e v i s t a       P o r t u g u e s a                  d e         P n e u m o l o g i a
                                    Vol XVI Suplemento 1 A Janeiro 2010
Novas ferramentas de fácil utilização para genotipagem padronizada e com qualidade
controlada de estirpes do complexo Mycobacterium tuberculosis
Caroline Allix-Béguec, Christine Hubans, Stéphanie Ferreira, Philip Supply

células de culturas líquidas, usando 8 triplex                 ABI platforms to determine the PCR
PCR e primers fluorescentes específicos para                   product sizes. As the length of the repeat
os flancos dos loci alvo. Os fragmentos am-                    units is known, these sizes reflect the num-
pliados são separados por eletroforese capi-                   bers of repeated sequences in the ampli-
lar em plataformas ABI para determinação                       fied loci. Analysis is done using ABI Gene-
das dimensões dos produtos da PCR. Con-                        Mapper® software customized with
forme se vão sabendo os tamanhos das uni-                      optimized modules provided in GENO-
dades repetidas, estes refletem os números                     SCREEN MIRU-VNTR Typing Calibra-
das sequências dos loci ampliados. A análise                   tion Kit. The final result is a portable nu-
é feita usando o software ABI GeneMapper®,                     merical genotype, corresponding to the
equipado com módulos optimizados forne-                        repeat number in each locus. Strain geno-
cida no GENOSCREEN MIRU-VNTR ty-                               types can then be analyzed and compared
ping calibration kit. O resultado final é um                   to reference strain genotypes using local
genótipo numérico portátil, que correspon-                     databases or, a
de ao número repetido em cada locus. As                        multi-functional identification database
estirpes dos genótipos podem então ser ana-                    freely accessible via the Internet.
lisadas e comparadas com as estirpes de ge-                    The MIRU-VNTR Calibration Kit is de-
nótipos de referência, recorrendo a bancos                     signed for standardised implementation and
de dados locais ou a www.miru-vntrplus.                        validation of MIRU-VNTR technique in
org, um banco de dados de identificação                        user’s laboratory. Relative migration be-
multifuncional de livre acesso na Internet.                    tween the size standard and the PCR pro-
O kit de calibragem MIRU-VNTR foi de-                          ducts depends on the MIRU-VNTR locus
senhado para implementação e validação                         and alleles, and differs upon polymer used
padronizada da técnica MIRU-VNTR no                            for capillary electrophoresis and between
laboratório do utilizador. A migração relati-                  instruments4. Therefore, instrument-speci-
va entre o padrão de tamanho e os produtos                     fic GeneMapper bin sets are created in order
do PCR depende dos locus e alelos do                           to calibrate for these effects. Each bin de-
MIRU-VNTR e difere segundo o polímero                          fines the observed size range for a given re-
usado para eletroforese capilar e entre ins-                   peat number (allele) determined from 4 dif-
trumentos4. Assim, os bin sets GeneMapper                      ferent runs. These bin sets are specifically
específicos dos instrumentos são criados                       created using the Bin Set Creator software
com o fim de calibrar para estes efeitos.                      and 24 marker-specific Allelic Ladders, in
Cada bin define o âmbito de tamanhos ob-                       accordance with the Allelic Ladder sizes
servados para um dado número repetido                          obtained on user’s DNA Analyzer. To vali-
(alelo) determinado a partir de 4 sequências                   date the process, a panel of 12 reference
diferentes. Estes bin sets são criados usando                  samples (including one negative control)
o software Bin Set Creator e 24 marcado-                       with known allelic profiles is provided. Af-
res allelic ladders, de acordo com os tama-                    ter PCR amplification and electrophoretic
nhos dos allelic ladder obtidos no analisa-                    separation, allelic profiles are determined
dor de ADN do utilizador. Para validar o                       using the created bin sets and checked for
processo, é fornecido um painel de 12                          validation.

     R e v i s t a                 P o r t u g u e s a                       d e     P n e u m o l o g i a     S 91
                                              Vol XVI Suplemento 1 A Janeiro 2010
Novas ferramentas de fácil utilização para genotipagem padronizada e com qualidade
                   controlada de estirpes do complexo Mycobacterium tuberculosis
                                                              Caroline Allix-Béguec, Christine Hubans, Stéphanie Ferreira, Philip Supply

                Fig. 1 – Tipagem MIRU-VNTR. A curva, as caixas e as setas à direita representam um cromossoma da estirpe do complexo
                M. tuberculosis, unidades repetidas dos loci MIRU-VNTR e os primers usados para ampliação PCR, respectivamente.
                Vinte e quatro loci MIRU-VNTR são analisados via 8 PCR multiplex. O resultado final é um genótipo numérico, que corres-
                ponde ao número de unidades repetidas de cada um dos 24 marcadores

                Fig. 1 – MIRU-VNTR typing. Close curve, orange boxes and arrows on the right represent a M. tuberculosis complex strain
                chromosome, repeat units of MIRU-VNTR loci, and labeled primers used for PCR amplification, respectively. Twenty-four
                MIRU-VNTR loci are analysed via 8 multiplex PCRs. The final result is a numerical genotype, corresponding to the repeat
                unit number of each of the 24 markers

                amostras de referência (incluindo um con-                    The MIRU-VNTR Data Manager software
                trolo negativo) com perfis alélicos conheci-                 is designed to facilitate data management
                dos. Após ampliação por PCR e separação                      after GeneMapper analysis. Features include
                eletroforética, determinam-se os perfis aléli-               quality control operations (verification of
                cos usando os bin sets criados e validados.                  controls, etc), data management (missing
                O software do gestor de dados MIRU-                          data and double alleles, creation of new run
                -VNTR foi desenhado para facilitar a gestão                  spreadsheets, merging and cross-control of
                de dados após análise do GeneMapper. As                      GeneMapper® project results), and final for-
                características incluem operações de contro-                 matting for compatibility with MIRU-VN-
                lo de qualidade (verificação dos controlos,                  TRPlus database. MIRU-VNTR training is
                etc.), gestão dos dados (dados em falta e ale-               proposed in user’s laboratory, and includes
                los duplos, criação de novas folhas de cálcu-                calibration and validation of user’s platform,
                lo, intercalação e controlo cruzado dos re-                  as well as training on technical and scien-
                sultados do projecto GeneMapper®) e                          tific interpretation. Dedicated MIRU-VN-
                formatação final para compatibilidade com                    TR Support is provided to kit users via spe-
                o banco de dados MIRU-VNTRPlus. A for-                       cific e-mailbox and telephone.

S 92    R e v i s t a          P o r t u g u e s a                         d e        P n e u m o l o g i a
                                         Vol XVI Suplemento 1 A Janeiro 2010
Novas ferramentas de fácil utilização para genotipagem padronizada e com qualidade
controlada de estirpes do complexo Mycobacterium tuberculosis
Caroline Allix-Béguec, Christine Hubans, Stéphanie Ferreira, Philip Supply

mação MIRU-VNTR, que se propõe tenha                           It is hoped that the availability of these tools
lugar no laboratório do utilizador, inclui ca-                 for easier and more efficient real-time geno-
libragem e validação da plataforma do utili-                   typing will contribute to improved molecu-
zador, bem como formação quanto à inter-                       lar-guided TB control and surveillance.
pretação técnica e científica. Aos utilizadores
dos kits MIRU-VNTR é oferecido apoio
específico via e-mail e telefone.
Espera-se que a disponibilidade destes apa-
relhos para genotipagem em tempo real
mais fácil e eficiente contribua para melho-
rar o controlo e a vigilância molecular da

1. Supply P, et al. Proposal for standardization of opti-      4. Allix C, Supply P, Fauville-Dufaux M. Utility of fast
mized mycobacterial interspersed repetitive unit-variable-     mycobacterial interspersed repetitive unit-variable
-number tandem repeat typing of Mycobacterium tuber-           number tandem repeat genotyping in clinical mycobac-
culosis. J Clin Microbiol 2006 44(12):4498-510.                teriological analysis. Clin Infect Dis 2004; 39(6):783-
2. Alonso-Rodriguez N, et al. Evaluation of the new ad-        -789.
vanced 15-loci MIRU-VNTR genotyping tool in Myco-              5. Allix-Béguec C, Fauville-Dufaux M, Supply P. Three-
bacterium tuberculosis molecular epidemiology studies.         -year population-based evaluation of standardized my-
BMC Microbiol 2008; 8:34.                                      cobacterial interspersed repetitive-unit-variable-number
3. Oelemann MC, et al. Assessment of an optimized              tandem-repeat typing of Mycobacterium tuberculosis. J
mycobacterial interspersed repetitive-unit-variable-           Clin Microbiol 2008; 46(4):1398-1406.
-number tandem-repeat typing system combined with              6. Shamputa IC, et al. Mixed infection and clonal repre-
spoligotyping for population-based molecular epidemi-          sentativeness of a single sputum sample in tuberculosis
ology studies of tuberculosis. J Clin Microbiol 2007;          patients from a penitentiary hospital in Georgia. Respir
45(3):691-697.                                                 Res 2006; 7:99.

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Elvira Richter1                      Simpósio: Avaliação externa da qualidade

                                     Symposium: External quality assurance

Os objetivos deste simpósio foram:            The aims of this symposium were:
• avaliar a situação actual                   • to estimate the current situation
• explorar os progressos na AEQ               • to explore advances in EQA
• desenvolver redes                           • to develop networks
• promover melhor colaboração                 • to promote better collaboration

Três oradores informaram quanto aos dife-     Three speakers provided information to dif-
rentes aspectos destes objetivos.             ferent aspects of these aims.

A AEQ num país com baixa                      EQA in a low incidence, high
incidência e elevados                         income country (Germany)
rendimentos (Alemanha)                        The TB situation in Germany is character-
Há anos que a situação da tuberculose (TB)    ized since years by a decrease of the number
na Alemanha se caracteriza por uma dimi-      of patients with an incidence of 5.5 per
nuição do número de doentes, com uma          100,000 inhabitants in the year 2008. 43%
incidência de 5,5 por 100 000 habitantes      of patients with culture confirmed pulmo-
em 2008. Quarenta e três por cento dos        nary TB were smear negative. 20% of all
doentes com TB pulmonar confirmada por        patients present with exclusively extrapul-
cultura tiveram esfregaços negativos. Vinte   monary TB. The rate of resistant strains
por cento de todos os doentes apresenta-      reaches 11% for any resistance and 2% for
vam exclusivamente TB extrapulmonar. O        MDR. XDR strains are reported. Infections
índice de estirpes resistentes atinge 11%     with non tuberculous mycobacteria (NTM)
para qualquer resistência e 2% para a TB      comprise lymphadenitis in children, adults

1   NRL, Borstel, Germany

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                                                                                                    Elvira Richter

              multirresistente (MDR). Estão descritas es-       with underlying diseases (e.g. COPD, bron-
              tirpes extensivamente resistentes (XDR).          chiectasis), or others (like swimming pool
              As infecções com micobactérias não tuber-         granuloma).
              culosas (MNT) incluem a linfadenite em            This situation is reflected by the specific
              crianças, adultos com doenças subjacentes         kind of EQA aiming to the evaluation of:
              [e.g., doença pulmonar obstrutiva crónica
              (DPOC), bronquiectasia), ou outras (como          1) specificity and sensitivity of microscopy
              o granuloma de piscina).                             (positive and negative slides prepared for
              Esta situação reflete-se no tipo específico de       staining with the routine technique of
              AEQ com o objetivo de avaliar:                       the laboratory)
                                                                2) sensitivity of culture techniques (speci-
              1) A especificidade e sensibilidade da mi-           mens with or without mycobacteria [TB
                 croscopia (lâminas positivas e negativas          or NTM] are prepared for primary isola-
                 preparadas para coloração com a técnica           tion and identification of TB or NTM)
                 de rotina do laboratório);                     3) sensitivity and specificity of nucleic acid
              2) A sensibilidade das técnicas de cultura           amplification techniques (sputum speci-
                 [amostras com ou sem micobactérias                mens are prepared for detection of spe-
                 (TB ou MNT), preparados para isola-               cific nucleic acids of TB)
                 mento primário e identificação de TB ou        4) reliability and rapidity of susceptibility
                 MNT];                                             testing (INH, RMP, EMB, SM, and
              3) A sensibilidade e especificidade das téc-         PZA)
                 nicas de ampliação de ácidos nucleicos         5) the accuracy of identification of TB bac-
                 (as amostras de expetoração são prepara-          teria and NTM (different mycobacterial
                 dos para deteção de ácidos nucleicos es-          species are prepared for identification to
                 pecíficos de TB);                                 species level)
              4) A fiabilidade e rapidez dos testes de sus-
                 cetibilidade (INH, RMP, EMB, SM, e             Before sending EQA specimens to the labo-
                 PZA);                                          ratories, regulations (e.g transportation, or
              5) O rigor da identificação das bactérias da      the permission for handling with TB bacte-
                 TB e das MNT (diferentes espécies mi-          ria) have to be clarified.
                 cobacterianas são preparadas para identi-      Some important causes for false results can
                 ficação a nível de espécie).                   be found by analysis of the data and addi-
                                                                tional reanalysis in cooperation with the
              Antes do envio das amostras para AEQ nos          laboratory that reported false results. Rea-
              laboratórios, é necessário esclarecer os regu-    sons for false positive microscopic results
              lamentos (e.g., transporte, ou autorização        were found to follow from staining in cu-
              para o manuseamento de bactérias de TB).          vettes, not as single slides, or misidentifica-
              Algumas causas importantes para os falsos         tion of staining artefacts. False positive cul-
              resultados podem ser detectadas através da        ture results derive from laboratory cross
              análise dos dados e reanálise adicional desses    contamination. Incorrect susceptibility re-
              dados com a colaboração do laboratório que        sults are most often found when testing SM,

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Elvira Richter

forneceu os falsos resultados. Observaram-      and in part PZA. Performing species identi-
-se falsos positivos de microscopia após co-    fication, sometimes significant errors occur.
loração em cuvettes, e não em lâminas indi-     The main reason for this is the insufficiency
viduais, ou identificação errada dos produtos   of biochemical analyses alone.
de coloração. Os resultados falsos positivos    Some challenges have to be addressed in the
de culturas resultam de contaminação cru-       preparation of samples for EQA.
zada no laboratório. Encontram-se com al-
guma frequência resultados com suscetibili-     – The question of a real estimation of sen-
dade incorreta ao testar SM e, em parte,          sitivity (with regard to microscopy, nu-
PZA. Ao fazer a identificação de especis,         cleic acid amplification, and primary iso-
ocorrem por vezes erros significativos, sendo     lation). The preparation of samples with
a principal razão a insuficiência da utiliza-     low amount of bacteria is difficult since it
ção exclusiva de análises bioquímicas.            can result in single samples without my-
Na preparação de amostras para AEQ há             cobacteria.
que abordar alguns desafios.                    – To avoid the use of MDR strains, iso-
                                                  lates with uncommon resistance patterns
– A questão da verdadeira avaliação da sen-       are selected. This does not reflect a ‚real
  sibilidade (no que respeita a microscopia,      life situation‘.
  ampliação dos ácidos nucleicos e isola-       – There is a contrast between scientific vs.
  mento primário). A preparação de amos-          diagnostic needs as we see in the over-
  tras com pequenas quantidades de bacté-         whelming number of NTM-species with
  rias é difícil, porque pode resultar em         sequences delivered in the Nucleotide
  amostras individuais sem micobactérias.         Databases, but only few different species
– Para evitar o uso de estirpes MDR, são          isolated in the routine laboratory.
  selecionados isolados com padrões de re-
  sistência pouco comuns. Isto não reflete
  uma “situação da vida real”.                  Organization and EQA in the
– Há um contraste entre as necessidades         Latvian TB laboratory network
  científicas e as do diagnóstico, como se      The TB incidence in Latvia was characte-
  observa no espantoso número de espé-          rized by a continuous decrease until approx.
  cies de MNT com sequências que cons-          1990, when a sharp increase was noted,
  tam das bases de dados de nucleótidos,        which peaked around 2000. Since then, the
  com apenas algumas espécies diferentes        incidence again decreased as to 40.3 per
  isoladas em laboratório.                      100,000 inhabitants for the year 2008.
                                                The Latvian TB-Laboratory Network is
                                                composed of the National Reference Labo-
Organização e AEQ na Rede de                    ratory in Riga, of 5 regional culture and mi-
Laboratórios de TB na Letónia                   croscopy laboratories, and of several labora-
A incidência de TB na Letónia foi caracteri-    tories (at district hospitals, policlinics,
zada por uma contínua diminuição até            private labs) facilitating AFB microscopy at
aproximadamente 1990, quando se verifi-         PHC level in each of 26 districts.

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              cou um brusco aumento, com pico cerca do            All microscopy centers are audited and cer-
              ano 2000. Desde então, a incidência voltou          tified based on international standards for
              a descer, atingindo 40,3 por 100 000 habi-          testing and medical laboratories (ISO
              tantes em 2008.                                     17025, ISO 15189). Internal Quality Con-
              A Rede de Laboratórios de TB na Letónia é           trol is performed by staining and microsco-
              composta pelo Laboratório Nacional de Refe-         py of known positive and negative smears.
              rência (LNR), em Riga, 5 laboratórios regio-        The culture laboratories perform decon-
              nais para trabalho de cultura e microscopia e       tamination of specimens and culture. Cul-
              ainda vários outros laboratórios (em hospitais      tures will be sent to NRL for identification
              distritais, policlínicas, laboratórios privados)    and drug susceptibility tests (DST). The
              que proporcionam microscopia de esfregaço           National Reference Laboratory performs
              para pesquisa de bacilos ácido-álcool resisten-     smear microscopy, culture of specimens,
              tes (BAAR) a nível PHC em cada um dos 26            DST for 1st line drugs (+ Kanamycin), and
              distritos.                                          2nd line DST for MDR-TB strains.
              Todos os centros de microscopia são audita-         Furthermore, the NRL provides the external
              dos e certificados com base em padrões inter-       quality assurance for all laboratories by a
              nacionais (ISO 17025, ISO 15189). O con-            panel of 5 fixed smears twice a year. For this,
              trolo de qualidade interno é feito por coloração    5 slides prepared from patient sputum, com-
              e microscopia de esfregaços positivos e negati-     prising negative, scanty, and positive speci-
              vos conhecidos. Os laboratórios de cultura          mens, are provided to the laboratories. These
              fazem descontaminação de amostras e de cul-         stain and read the slides, record the results
              turas. As culturas são enviadas para o LNR          and send back results and slides to NRL. Fi-
              para identificação e testes de suscetibilidade a    nally, the NRL rereads the slides, assesses
              fármacos. O laboratório nacional de referên-        staining and results, and issues reports on
              cia realiza microscopia de esfregaços, cultura      performance. Furthermore, the NRL moni-
              de amostras, testes de susceptibilidade para        tors the quality of sputum smear microscopy
              fármacos de 1.ª linha (+ Kanamicina) e TSF          procedure during supervisory visits.
              de 2ª linha para estirpes de MDR-TB.                For EQA of the performance of the culture,
              Além disso, o LNR efectua o controlo da             2 specimens are sent to each lab twice a year.
              qualidade externo de todos os laboratórios,         In detail, each specimen is divided into two
              duas vezes por ano, utilizando um painel de         parts, one part is decontaminated and incu-
              5 esfregaços fixados. Com este fim, 5 lâmi-         bated at NRL, and the other is sent to the
              nas preparadas a partir de expetoração de           culture laboratory. The culture laboratory
              doentes, contendo amostras negativas e po-          performs decontamination, inoculation, in-
              sitivas, são fornecidas aos laboratórios, que       cubation, record of results and sends the re-
              processam e interpretam as lâminas, regis-          sults back to the NRL. Beside this, the NRL
              tam os resultados e enviam-nos ao LNR               monitors the quality of culture procedures
              juntamente com as respectivas lâminas. Fi-          during supervisory visits.
              nalmente, o LNR reexamina as lâminas,               The NRL itself yearly participated in an ex-
              avalia a coloração e os resultados e emite re-      ternal proficiency programme for 1st line
              latórios sobre o desempenho. Este LNR               DST provided by in Swedish Institute for

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também controla a qualidade do procedi-           Infectious Diseases Control. Since 2008 the
mento da microscopia dos esfregaços de ex-        NRL itself became Supranational Reference
petoração durante visitas de inspecção.           Laboratory and thus taking part in the year-
Para a AEQ do desempenho da cultura, 2            ly EQA rounds for 1st and 2nd line DST.
amostras são enviadas a cada laboratório
duas vezes por ano. Cada amostra é dividida
em duas partes, sendo uma parte desconta-         The implementation a of quality
minada e incubada no LNR e a outra envia-         assurance as prerequisite for the
da para o laboratório de cultura. Este proce-     introduction of a new diagnostic
de à descontaminação, inoculação, incubação       test.
e registo dos resultados, que envia ao LNR.       The integration of public health laborato-
Além disto, o LNR também controla a qua-          ries is one of the main targets in fighting
lidade dos procedimentos de cultura durante       emerging infectious diseases. Quality assu-
visitas de inspecção.                             rance of the test procedures is a prerequisite
O próprio LNR participou anualmente               to obtain reliable and useful results. Quality
num programa externo de proficiência para         assurance is composed of internal quality
TSF de 1.ª linha disponibilizado pelo Insti-      control and external quality assessment. For
tuto Sueco para o Controlo de Doenças In-         the integration of new molecular techniques
fecciosas. A partir de 2008, o LNR passou a       into a TB laboratory, internal quality con-
ser o Laboratório Supranacional de Referên-       trol comprises several aspects, like elaborate
cia, tomando, assim, parte nas atividades         infrastructure, equipment that is well main-
AEQ anuais para TSF de 1.ª e 2.ª linha.           tained and operated and procedures guided
                                                  by efficient SOPs. Test reagents need to be
                                                  controlled with every LOT, its integrity
A implementação do controlo da                    needs to be checked for every shipment and
qualidade como requisito para a                   proper transport and storage has to be con-
introdução de um novo teste de                    trolled. The test procedures itself require
diagnóstico                                       several quality control steps, like contami-
A integração de laboratórios de saúde pública     nation controls, negative controls at differ-
é um dos principais alvos na luta contra as do-   ent levels of the test process, the correct in-
enças infeciosas. O controlo da qualidade do      terpretation of adequate and inadequate
procedimento dos testes é um requisito essen-     runs, and the implementation of certain
cial para a obtenção de resultados úteis e fiá-   quality indicators. Furthermore, personnel
veis. O controlo da qualidade compreende o        need to be trained and re-trained.
controlo interno da qualidade e a avaliação ex-   External proficiency testing can be conduc-
terna da qualidade. Para a integração de novas    ted by analysis of duplicate specimens from
técnicas moleculares num laboratório de TB,       one patient. As example, proficiency testing
o controlo interno da qualidade compreende        for line probe assays was performed in four
vários aspectos, como uma infraestrutura ela-     laboratories in India as prerequisite for the
borada, utilização e manutenção adequadas         implementation of the new test. The advan-
do equipamento e orientação nos procedi-          tage of panel testing for external quality

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               mentos por SOP eficientes. Os reagentes têm        control is the revision of the entire proce-
               de ser controlados com cada LOT, a sua inte-       dure, the directed use of selected strains
               gridade verificada em cada remessa e o trans-      with certain resistances, with common and
               porte e armazenagem devidamente fiscaliza-         uncommon mutations, as well as the use of
               dos. O processamento dos testes envolve vários     NTM species. However, costs of panel tests
               passos de controlo da qualidade, como con-         (including preparation and shipment) have
               trolo de contaminação, controlos negativos         to be taken into account. Cheaper alterna-
               em diferentes níveis do processo, interpretação    tives for EQA of line probe assays may be
               correta das sequências adequadas e inadequa-       achieved by sending of digitized results for
               das e implementação de certos indicadores de       interpretation as well as for re-checking.
               qualidade. Há ainda que treinar o pessoal e        The best solution has to be found for diffe-
               proporcionar a sua atualização contínua.           rent situations.
               Os testes externos de proficiência podem ser       The speakers were: Elvira Richter, NRL,
               efetuados pela análise de duplicados de            Borstel, Germany; Girts Skenders, State
               amostras de um doente. Como exemplo, os            Agency of TB and Lung Disease, Latvia;
               testes de proficiência para ensaios line probe     Akos Somoskövy, FIND, Geneva, Switzer-
               foram realizados em quatro laboratórios, na        land.
               Índia, um requisito essencial para a imple-        The symposium was sponsored by IN-
               mentação dos novos testes. A vantagem dos          STAND e.V., Society for Research Promo-
               testes de painel para controlo externo da          tion of Quality Assurance in Medical Labo-
               qualidade consiste na revisão de todo o pro-       ratories, WHO Collaborating Centre,
               cedimento, no uso direcionado de estirpes          Düsseldorf, Germany.
               selecionadas com determinadas resistências,
               com mutações comuns e incomuns, bem
               como na utilização de espécies de MNT.
               Há, no entanto, que ter em conta o custo
               dos testes de painel (incluindo preparação e
               expedição). O envio de resultados digitali-
               zados para interpretação, bem como para
               reverificação, pode constituir uma alternati-
               va menos dispendiosa para AEQ dos ensaios
               line probe, devendo a melhor solução ser
               apreciada para cada situação.
               Foram oradores: Elvira Richter, NRL, Bors-
               tel, Alemanha; Girts Skenders, State Agency
               of TB and Lung Disease, Letónia; Akos So-
               moskövy, FIND, Geneva, Suissa.
               O simpósio foi apoiado por INSTAND e.V.
               Society for Research Promotion of Quality As-
               surance in Medical Laboratories, WHO Colla-
               borating Centre, Düsseldorf, Alemanha.

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Normas de Publicação
Instructions for Authors

                                        Normas de publicação na
                                        Revista Portuguesa de Pneumologia

                                        Portuguese Journal of Pulmonology
                                        Instructions for authors

A Revista Portuguesa de Pneumologia considera para          Revista Portuguesa de Pneumologia / The Portuguese
publicação trabalhos (artigos originais, de revisão, de     Journal of Pulmonology publishes papers (original ar-
actualização, casos clínicos, cartas ao editor, resumos     ticles, revised articles, updated articles, case reports,
críticos a livros, etc.) relacionados directa ou indirec-   letters to the editor, book reviews, etc) which are di-
tamente com o Aparelho Respiratório.                        rectly or indirectly related to the respiratory system.
As opiniões expressas são da exclusiva responsabilida-      The opinions expressed are the exclusive responsibili-
de dos autores.                                             ty of the authors.

Os artigos publicados ficarão propriedade da Re-            The articles published are the intellectual property
vista Portuguesa de Pneumologia, não podendo ser            of Revista Portuguesa de Pneumologia/ The Portu-
reproduzidos, no todo ou em parte, sem autoriza-            guese Journal of Pulmonology and may not be re-
ção do editor.                                              produced, in part or in whole, without permission
                                                            from the editor.
A aceitação dos originais enviados para publicação é
condicionada à avaliação pelo Conselho Científico           All submissions are subject to a screening process by
da Revista. Nesta avaliação os artigos poderão ser:         the Journal’s Scientific Board. There are three asses-
                                                            sments possible:
a) aceites sem alterações;                                  a) accepted for publication as is;
b) aceites após as modificações propostas e aceites pe-     b) accepted for publication after the proposed altera-
   los autores;                                                tions, accepted by the authors;
c) recusados.                                               c) rejected.
Apresentação dos trabalhos – Os textos devem ser            Manuscript instructions – The manuscripts submit-
escritos em português, dactilografados, com margens         ted should be in Portuguese, typed, doubled spaced
largas (25 mm), a dois espaços, numa só face do papel       with ample (25mm) margins, and on one side of A4
e em três exemplares com as páginas numeradas no            sized paper. Three copies should be submitted, with
canto superior direito.                                     the pages numbered in the upper right corner.
Solicita-se a todos os autores que enviem artigos para      All articles submitted for publication must be sent
publicação que o façam acompanhados do respecti-            in addition in a computerised format. The compu-

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                                                  NORMAS DE PUBLICAÇÃO/INSTRUCTIONS FOR AUTHORS

vo suporte magnético, que indiquem o programa              ter program used must be clearly stated, and particu-
de computador em que foram executados e que te-            lar attention must be paid to the reproduction of
nham em atenção à reprodução das imagens (que              images, which should ideally be in JPG or TIFF for-
deverá ser feita, idealmente, em suporte JPG ou            mat, to give a clear printed quality.
TIFF) de modo a que fiquem nítidas na sua impres-
são tipográfica.                                           If an image, figure or graph has been previously pu-
                                                           blished, written permission from the editor in ques-
Chama-se a atenção que a transcrição de imagens, qua-      tion must be submitted to safeguard the author’s in-
dros ou gráficos de outras publicações deverá ter a pré-   tellectual property rights.
via autorização dos respectivos editores para dar cum-
primento às normas que regem os direitos de autor.         Articles may also be submitted in English. In this
Poder-se-ão considerar para publicação artigos redigi-     case, the summary, the title and key words should
dos em inglês. Neste caso, deve incluir-se o resumo, o     also be submitted in Portuguese.
título e as palavras-chave, também em português.
                                                           The authores should categorise their submissions as
Deverão ser referenciados, pelos próprios autores,         original articles, revised articles, case studies, letters
como artigos originais, de revisão, cartas ao editor, ou   to the editor, technical notes, etc.
                                                           All original articles shall be also printed in English,
Todos os artigos originais serão também publicados         after being translated by the Revista Portuguesa de
em inglês, após retroversão para esta língua, pela(s)      Pneumologia / The Portuguese Journal of Pulmonology´s
tradutora(s) da Revista Portuguesa de Pneumologia.         translators. Authors may submit their articles already
Caso os autores assim o entendam, poderão enviar os        translated, if they so wish.
artigos já traduzidos.
                                                           Form – As far as possible, the convention will be ob-
Estrutura – Sempre que possível será adoptado o es-        served of beginning each new part of the paper on a
quema convencional em que se iniciará cada parte do        new page and in the following order:
trabalho numa nova página pela seguinte ordem:
                                                           a) Title page:
a) Na primeira página:                                         – the full title of the paper in Portuguese and
   – título do trabalho em português e inglês                    English;
b) Na segunda página:                                      b) Second page:
   – o nome dos autores com os respectivos títulos             – the full names of the authors and their academic
     académicos e/ou profissionais;                              and /or professional titles;
   – os serviços onde foi realizado, nome dos seus di-         – the full name and address of the institution(s) at
     rectores e os respectivos endereços.                        which the work was carried out and the full
c) Na(s) página(s) seguinte(s):                                  name of the institution(s) director(s).
   – o resumo em português que não deverá ultrapas-        c) Following page(s):
     sar 250 palavras para os trabalhos originais e de         – a summary in Portuguese, not exceeding 250
     150 para os casos clínicos;                                 words for original articles and 150 for case reports;

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      – os resumos em inglês com características idênti-            – a summary in English, with identical characte-
        cas ao do inicial em português;                               ristics to that in Portuguese;
      – as palavras-chave, em português e inglês (3 a               – between three and 10 key words in Portuguese
        10), que servirão de base à indexação do artigo,              and English, which will be used to index the ar-
        de acordo com a terminologia do Index Medi-                   ticle, using terms from the Medical Subject Hea-
        cus “Medical Subject Headings”.                               dings list of the Index Medicus.
 d)   O texto que, no caso dos artigos originais, terá em      d)   Original articles shall include, as a general rule,
      geral: Introdução, Material e Métodos, Resulta-               the following: Introduction, Subjects and Metho-
      dos, Discussão e Conclusões                                   ds, Results, Discussion and Conclusions.
 e)   O texto, também em inglês, tratando-se de um artigo      e)   Original articles may be written in English if the
      original, e caso o(s) autor(es) assim o entendam fazer        authors so wish.
 f)   Agradecimentos                                           f)   Acknowledgments.
 g)   Bibliografia                                             g)   References.
 h)   Quadros e Figuras.                                       h)   Tables and Figures.

 Bibliografia – As referências bibliográficas devem ser        References – The bibliographical references should be
 numeradas por ordem consecutiva da sua primeira               numbered consecutively, in the order in which they are
 citação no texto. Devem ser identificadas no texto            cited in the text and should be identified in the text with
 com números árabes. As referências devem conter, no           Arabic numerals. Each reference to a journal article
 caso das revistas, o nome do primeiro autor (apelido          should contain the surname and initial of the first author
 e nome), seguido dos restantes, do título do artigo,          followed by the rest. These should be followed by the ti-
 do nome da publicação e da sua identificação (ano,            tle of the article, the title of the publication and its iden-
 volume e páginas).                                            tifying years, volume number and the page numbers.

 Quadros e figuras – Os quadros e figuras devem ser            Tables and figures – Each table and figure should be
 apresentados em páginas separadas, em condições de            on a separate page, and in such conditions as to be re-
 reprodução. Devem ser acompanhados da respectiva le-          produced. Each table and figure should have its own
 genda em página à parte, mencionando no verso a lápis         brief description on a separate page and bear its sequen-
 o número de ordem. Todos os gráficos deverão ser apre-        ce number on its back, written in pencil. Each table and
 sentados através de fotografia do respectivo original.        figure should be represented via a copy of the original.

 Modificações e revisões – No caso da aceitação do ar-         Alterations and changes – Should an article be ac-
 tigo ser condicionada a modificações, estas devem ser         cepted for publication subject to alteration, these must
 realizadas pelos autores no prazo máximo de vinte dias.       be made by the authors within a twenty day period.

 As provas tipográficas serão da responsabilidade da           Publishing proofs – These are the editorial board’s
 Redacção, se os autores não indicarem o contrário.            responsibility, unless the authors state otherwise.
 Neste caso elas deverão ser feitas no prazo determina-        Should this latter be the case, the proofs should be
 do pela Redacção, em função das necessidades edito-           concluded within a deadline set by the editorial bo-
 riais da Revista.                                             ard, in line with the editorial norms of the Journal.

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                                     Vol XVI Suplemento 1 A Janeiro 2010
                                                 NORMAS DE PUBLICAÇÃO/INSTRUCTIONS FOR AUTHORS

Cartas ao editor – Devem constituir um comentário         Letters to the editor – These should contain a criti-
crítico a um artigo da Revista ou uma pequena nota        cal appraisal of a Journal article or a small comment
sobre um tema ou caso clínico. Não devem exceder as       on a theme or a case study. They should not exceed
500 palavras, nem conter mais de um quadro ou figu-       500 words, contain more than one table or figure and
ra e um máximo de 6 referências bibliográficas. As        have a maximum of 6 bibliographical references. Au-
respostas do(s) autor(es) devem obedecer às mesmas        thors’ replies should observe these same norms.
                                                          Submissions for publication – Papers should be sent to
Pedido de publicação – Os trabalhos deverão ser en-       the editorial board, addressed to the editor: Sociedade
viados à Redacção, em nome do editor, para a sede da      Portuguesa Pneumologia office: SPP – Rua Ivone Silva,
SPP, Rua Ivone Silva, n.º 6 – 6.º Esq., Edifício Arcis,   n.º 6 – 6.º Esq., Edifício Arcis, 1069-130 Lisboa, Portu-
1069-130 Lisboa, Portugal, acompanhados de uma            gal. Submissions should be accompanied by a letter re-
carta com pedido de publicação, subscrito por todos       questing that the work be submitted for publication,
os autores, indicação da cedência do copyright e que      signed by all of the authors, stating that they waive their
não foram publicados ou enviados para publicação          intellectual property rights and that they work has not be
em outra revista nacional ou estrangeira. Não serão       published or submitted for publication in any other Por-
aceites trabalhos já publicados ou enviados simul-        tuguese of international journal. Work already publi-
taneamente a outras revistas.                             shed or already sent to other journals will be rejected.

Deverão ser acompanhados pelo endereço electró-           Submissions must bear the e-mail address of the
nico do autor principal para eventuais pedidos de         corresponding author in order to facilitate contact
esclarecimentos por parte da Redacção.                    with the editorial team should any clarification be
Os trabalhos também poderão ser enviados por via
electrónica (e-mail:      Papers may should be sent via electronic mail to:
Nota final – Para um mais completo esclarecimento
sobre este assunto aconselha-se a leitura dos requisi-    Final note – For a fuller clarification of this matter, a
tos do International Commitee of Medical Journal Edi-     reading of the requirements of the International Com-
tors, publicados na integra no N Engl J Med 1991;         mittee of Medical Journal Editors, published in full in
324:424-428.                                              the N Engl J Med 1991; 324:424-428, is advised.

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