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					                                           Activities in 2009
                                                Leptospirosis
                                                 Dr R.A. Hartskeerl
                          OIE Reference laboratory for Leptospirosis, KIT Biomedical Research
                               Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands
                                   Tel.: (+31-20) 566.5438, Fax: (+31-20) 697.1841
                                           E-mail address: r.hartskeerl@kit.nl




                     Summary of general activities related to the disease


1.   Test(s) in use/or available for the specified disease at your laboratory


                                                                                                       Diagnosis/
                                                                                    Surveillance
            Test                  For                Specificity                                       research in
                                                                               (diagnosis in humans)
                                                                                                         animals

           ELISA               Antibody                  Genus                          601                  0

       Microscopic             Antibody               Serogroup                         601                  69
     Agglutination Test
          (MAT)

           Culture              Antigen                 Serovar                         180                  0

       Real time PCR             DNA            Pathogenic Leptospira                   112                  2
                                                        spp.


     Animals. For the sero-diagnosis in animals, the centre received sera from 45 mice, 19 cows, 2 dogs, 2 sea lions and
     1 lion. Investigations of the sera from the mice concerned requests for a quality check. All sera were negative.
     PCR (negative) was performed on tissue samples from the lion and a brown rat. Except for the sea lions (USA), all
     animals were from the Netherlands.

     Humans. Human diagnosis forms the basis for the national surveillance task. In 2009, a total of 656 (compared to
     787 in 2008) human specimens were tested. In 24 cases (20 males, 4 females) the diagnosis leptospirosis was
     established. In 3 cases this was based on a positive real time PCR. To our knowledge 1 patient died (only positive
     in PCR). Five infections acquired in The Netherlands were due to occupational activities, with 2 potentially
     associated with contact to farm animals. For 1 additional infection acquired in The Netherlands the probable cause
     is unknown. In addition, 17 sera from the USA and 5 from Finland have been tested by MAT and IgM ELISA and
     53 serum samples from the outbreak in Sri Lanka have been tested by MAT. PCR on a set of 28 samples from
     patients without microbiological diagnosis obtained from a local academic hospital revealed 3 (10.7%) positive
     samples stressing that under-diagnosis also poses a major problem in The Netherlands


2.   Production and distribution of diagnostic reagents

     During the last decade, the reference centre has equipped more than 100 laboratories all over the world with
     various reference materials, including Leptospira reference strains, (panels of) rabbit anti-Leptospira reference
     sera and mAbs that enable these laboratories to quickly and easily identify most locally occurring strains. In 2009,
     mAbs, Leptospira strains, culture media, ELISA reagents, rabbit anti-Leptospira sera, DNA, primers and probes
     were sent to several institutions in Australia, Belgium, Brazil, Canada, China (Hong Kong), Costa Rica, Czech
     Republic, Croatia, Cuba, Denmark, Germany, Indonesia, Ireland, Italy, Mexico, Nigeria, Romania, Slovenia,




Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009                                           1
                                                      Leptospirosis




     South Korea, Spain, Sri Lanka, Switzerland, Thailand, The Netherlands, United Kingdom and United States of
     America. These are all OIE member countries.


                         Activities specifically related to the mandate
                                 of OIE Reference Laboratories

3.   International harmonisation and standardisation of methods for diagnostic testing or the
     production and testing of vaccines

     The centre has a CCKL accreditation (ISO 15189-based). This ISO 15189-based accreditation is most suitable for
     the centre that also functions as National and WHO/FAO reference centre and forms the diagnostic centre for
     human leptospirosis in The Netherlands. In a joint effort with the Leptospirosis Reference Unit, Department of
     Microbiology and Immunology, County Hospital, Hereford and the National Serology Reference Laboratory
     (NRL), Australia, Melbourne, the reference centre in Amsterdam performed the eight annual round of an
     international proficiency testing of the MAT. Late 2009, 93 panels were sent to participants, mostly veterinary
     institutions and (national) reference centres.


4.   Preparation and supply of international reference standards for diagnostic tests or vaccines

     The reference laboratory provides reference rabbit sera and mAbs for typing and checking of (diagnostic) strains
     and provides backstopping to laboratories by performing quality control and typing of problem strains.


5.   Research and development of new procedures for diagnosis and control

     Research focuses on new simple and rapid serological methods and molecular tests for early diagnosis. Since
     2004, a lateral flow test from Zephyr Biomedicals, India was included in a prospective evaluation. A preliminary
     report over the period 2004-2007 was drafted to aid decisions making during the leptospirosis outbreak in the
     Philippines. Further joint research within the framework of formal (externally financed) and informal
     collaborations was aimed at the validation (following the OIE scheme) of a real time PCR for the detection of
     Leptospira in human and animal specimens and of molecular typing tools, the unravelling of the pathogenesis of
     leptospirosis and the surveillance and epidemiology of (animal and human) leptospirosis in several regions in the
     world.


6.   Collection, analysis and dissemination of epizootiological data relevant to international disease
     control

     In 2009, the reference centre received or obtained 21 Leptospira isolates for typing as follows: 3 human isolates
     from Sri Lanka, 2 rodent isolates from the Azores islands, 9 rat isolates from Madagascar, 6 isolates from various
     origins from USA and 1 human isolate from The Netherlands. Two reference strains were received from Slovenia
     for an identity check.


7.   Provision of consultant expertise to OIE or to OIE Members

     The centre participated in requests and questionnaires from OIE and other animal health related investigations.

     In 2009, the centre (i) provided consultancy to WHO concerning the set-up of guidelines for diagnosis, treatment
     and surveillance in South East Asia and the evaluation of a leptospirosis epidemic in the Philippines and (ii)
     participates in the WHO Leptospirosis Burden Epidemiology Reference Group (LERG).


8.   Provision of scientific and technical training to personnel from other OIE Members

     In 2009, the centre did not contribute to an international leptospirosis course. The centre guided several MSc and
     PhD students and trainees from Turkey, Malaysia, Slovenia, Romania and The Netherlands.




2                                                 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
                                                          Leptospirosis




9.   Provision of diagnostic testing facilities to other OIE Members

     See point 2 and 4.

     Agent identification is carried out using rabbit sera against reference Leptospira strains and panels with
     monoclonal antibodies (mAbs) that agglutinate serovars in a characteristic way. The Cross Agglutinin Absorption
     Test (CAAT) is the gold standard for serovar determination. DNA determination is done by (species) specific
     PCRs and gene-sequencing. Total number of isolates identified in 2009: 7 isolates, i.e. 3 isolates from Sri Lanka,
     2 isolates from humans in The Netherlands and 2 isolates from rodents in Portugal.
     Additionally, checks were performed on 2 reference strains from Slovenia, 3 strains from Czech Republic and 6
     animal vaccine strains from The Netherlands.


10. Organisation of international scientific meetings on behalf of OIE or other international bodies

     Co-organization of the international scientific Meeting of the International Leptospirosis Society, "LeptoCON"
     2009, Cochin, India, 21-24 September 2009.


11. Participation in international scientific collaborative studies

     The centre performed in 2009 the following project: ECDC public tender OJ/2009/05/19-PROC/2009/018
     “Scientific input into the selection and processing of datasets for the European Environment and Epidemiology
     (E3) network” started in October 2009. The OIE Reference Laboratory participates in the consortium for the
     inclusion of leptospirosis as a typical water-borne disease.


12. Publication and dissemination of information relevant to the work of OIE (including list of
    scientific publications, internet publishing activities, presentations at international conferences)

         Presentations at international conferences and meetings

     Presentation. Hartskeerl RA, Invited speaker. Leptospirosis: a prototype Neglected Infectious Disease. 70th
     Anniversary Congress of Instituto Pedro Kouri, 2-6 June 2009, Havana, Cuba.

     Presentation. Hartskeerl RA. Invited speaker. Burden of leptospirosis. WHO-SEA meeting 16-18 September 2009,
     Chennai, India

     Presentation. Goris MGA, Wagenaar JFP, Hartskeerl RA, Gorp ECM van, Poll T van der, Veer C van 't. Innate
     immune response to pathogenic and non-pathogenic Leptospira is dependent of both TLR2 and TLR4 signaling in
     human whole blood. 6th International Leptospirosis Society Meeting "LeptoCON" 2009, Cochin, India, 21-24
     September 2009.

     Presentation. Hartskeerl RA, Invited speaker. Leptospirosis: Current status and future trends in human
     leptospirosis. Aravind Eye Hospital, Madurai, India, 26-27 September 2009.

     Presentation. Hartskeerl RA. Invited speaker. Human Leptospirosis at a global perspective.. ISC conference
     Emerging Zoonotic Diseases, 13-18 October 2009, Cairo, Egypt.

     Poster. Ahmed A, Engelberts MFM, Ahmed N, Hartskeerl RA. Development and validation of a real-time PCR for
     the early diagnosis of leptospirosis. 6th International Leptospirosis Society Meeting "LeptoCON" 2009, Cochin,
     India, 21-24 September 2009.

     Poster. Barragan VA, Mejia ME, Zapata S, Travez A, Haake DA, Hartskeerl RA, Trueba GA. Enviromental
     Bacteria Foster the growth of Saprophytic but not Pathogenic Leptospira. 6th International Leptospirosis Society
     Meeting "LeptoCON" 2009, Cochin, India, 21-24 September 2009.

     Poster. Carmona-Gasca CA, Hartskeerl R, Ahmed N, Lemuel A, Lara L, De la Pena-Moctezuma A. Evaluation of
     gspD in MLST scheme for the identification of Leptospira isolates. 6th International Leptospirosis Society
     Meeting "LeptoCON" 2009, Cochin, India, 21-24 September 2009.



Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009                                         3
                                                      Leptospirosis




    Poster. Chappel RJ, Goris MGA, Palmer MF, Hartskeerl RA. Lessons Learned from the Results of Proficiency
    Testing for the Leptospirosis Microscopic Agglutination Test. 6th International Leptospirosis Society Meeting
    "LeptoCON" 2009, Cochin, India, 21-24 September 2009.

    Poster. Galloway RL, Buchholz AE, Whelen AC, Zaanen van W, Hartskeerl RA. Characterization of Leptospira
    isolates from patients in Hawaii using molecular and serological techniques. 6th International Leptospirosis
    Society Meeting "LeptoCON" 2009, Cochin, India, 21-24 September 2009.

    Poster. Munoz-Zanzi, C, Neils L, Hrnickel L, Belongia E, Hartskeerl R, Lesina B. Estimation of sensitivity and
    specificity of screening tests for leptospirosis in traditionally low risk populations. 6th International Leptospirosis
    Society Meeting "LeptoCON" 2009, Cochin, India, 21-24 September 2009.

    Poster. Rebeiro G, Gouveia EL,Almeida SF, Flannery B, Ramos JG, Pitangueira Filho JC, De Carvalho AL,
    Villasboas Bisneto JC, Aires T, Queiroz A, Fraga DB, Santos AC, Salgado K, Reis RB, Hartskeerl R, Reis MG,
    Ko A. Cyclic rainfall - Associated epidemics of severe leptospirosis in the urban setting. 6th International
    Leptospirosis Society Meeting "LeptoCON" 2009, Cochin, India, 21-24 September 2009.

    Poster. Zeng L, Guo Z, Liu Y, Zeng Y, Ahmed N, Hartskeerl RA. Surveillance of leptospirosis at SiChuan
    Province in 2008. 6th International Leptospirosis Society Meeting "LeptoCON" 2009, Cochin, India, 21-24
    September 2009.

    Poster. Hopman J, Stelma FF, Ahmed A, Ven van der AJ, Brouwer AE, Keuter M, Hartskeerl RA, Becker PJ,
    Galama JM, Sturm PD, Sauerwein RW. Microbiological analysis of travelers with febrile illness returning to the
    Netherlands. 6th European Congress on Tropical Medicine and International Health, Verona, Italy, September 6-
    10, 2009.

       Scientific publications in peer-reviewed journals

    Houwers DJ, Wagenaar JA, Hartskeerl RA, Hautvast JLA, Stinis HPJ, Ruijs WLM, Lenaers SJMM. Leptospirose
    (ziekte van Weil) bij de hond: een risico voor de mens? Tijdschrift voor Diergeneeskunde, 2009, 134, 392-394.

    Wagenaar JFP, Goris MGA, Sakundarno A, Gasem H, Isbandrio B, Moalli F, Mantovani A, Boer KR, Hartskeerl
    R, Garlanda C, Gorp ECM van. Long pentraxin PTX3 is associated with mortality and disease severity in severe
    Leptospirosis. Journal of Infection Society, 2009, 58, 425-432.

    Ahmed A, Engelberts MFM, Boer RK, Ahmed N, Hartskeerl RA. Development and Validation of a Real-Time
    PCR for Detection of Pathogenic Leptospira Species in Clinical Materials. PLoS ONE, 2009, 9, 1-8
    (doi:10.1371/journal.pone.0007093).

    Wagenaar JF, Gasem MH, Goris MG, Leeflang M, Hartskeerl RA, van der Poll T, van 't Veer C, van Gorp EC.
    Soluble ST2 Levels Are Associated with Bleeding in Patients with Severe Leptospirosis. PLoS Neglected Tropical
    Diseases, 2009, 3, e453.

    Gasem MH, Wagenaar JF, Goris MG, Adi MS, Isbandrio BB, Hartskeerl RA, Rolain JM, Raoult D, van Gorp EC.
    Murine typhus and leptospirosis as causes of acute undifferentiated fever, Indonesia. Emerging Infectious
    Diseases, 2009, 15,975-7.

    Wagenaar JF, Goris MG, Partiningrum DL, Isbandrio B, Hartskeerl RA, Brandjes DP, Meijers JC, Gasem MH,
    van Gorp EC. Derangements of the coagulation system in patients with severe leptospirosis is associated with
    severe bleeding and mortality. Tropical Medicine and International Health, in press

    Gonçalves AT, Paiva C, Melo-Mota F, Vieira ML, Carreira T, Nunes MS, Mota-Vieira L, Ahmed A, Hartskeerl
    RA, Hyde K and Collares-Pereira M. First isolation of human Leptospira strains (Azores, Portugal). International
    Journal of Infectious Diseases, in press.

    Abela-Ridder B, Sikkema R, Hartskeerl RA. Estimating the burden of human leptospirosis. International Journal
    of Antimicrobial Agents, in press.




4                                                 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
                                                          Leptospirosis




13. Inscription of diagnostic kits on the OIE Register

     i)    Did you participate in expert panels for the validation of candidate kits for inscription on the
           OIE Register? If yes, for which kits?
           No

     ii)   Did you submit to the OIE candidate kits for inscription on the OIE Register? If yes, for
           which kits?
           No
                                                       _______________




Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009                             5

				
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