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Trans-renal DNA for infectious disease diagnosis

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Trans-renal DNA for infectious disease diagnosis Powered By Docstoc
					        Trans-renal DNA for
         infectious disease
             diagnosis
Clare Green, Jim Huggett, Alimuddin Zumla

Centre for Infectious Diseases and International Health

University College London
                                     clare.green@ucl.ac.uk
          Centre for Infectious Diseases & International Health (CIDIH)




Outline

1. Trans-renal DNA: a brief history and current
   knowledge

2. Using qPCR to define urinary DNA

3. Putting these lessons into practice: TB trDNA

4. Summary
            Centre for Infectious Diseases & International Health (CIDIH)




What is trans-renal DNA?




                                                                                    treatment




                                                                                                                DNAse &
                                                                                                DNAse




                                                                                                                RNAse
                                                                                                        RNAse
                                                                         B




                                                                                    No
                                        A




                                      A: Botezatu, I et al. (2000) Clinical Chemistry, 46:8; 1078-1084
                                      B: Umansky, S., Xenomics Ltd, USA




 • Cell free nucleic acids             • Small molecular weight
                                         molecules
             Centre for Infectious Diseases & International Health (CIDIH)




The advantages of a trDNA approach for
infectious disease diagnosis

 • Non-invasive sample

 • Abundant sample

 • Ease of collection, outside health care facility by the
   patient

 • Diagnosis irrespective of infection site
                    Centre for Infectious Diseases & International Health (CIDIH)




TrDNA timeline
     1997                                 2002
     CNAPs                                Routine foetal
     discovered                           RHD genotyping                       Infectious
                                                                               disease
                                                                               diagnosis
                                 2000
                                 TrDNA
                                 defined                                     2008
  Lo (1997) Lancet : 350: 485-87                                             Ultrashort
  Botezatu (2000) ,Clinical Chemistry :46:8; 1078-84
  Finning (2002), Transfusion :42: 1079-85
  Melkonyan, (2008), Ann. N.Y. Acad. Sci. :137: 83-81
                                                                             fragments
                     Centre for Infectious Diseases & International Health (CIDIH)




TrDNA for infectious disease diagnosis
Nucleic acids in urine used to diagnose infections outside the genito-urinary tract

         1996 Legionella spp?                                 2006
                                                              Plasmodium
                     1998 Wucheria                            falciparum
                          bancrofti?


                                                                 2007
                                                                 Leishmania infantum
                                                                 Dengue virus
                           1999
                           Mycobacterium tuberculosis
            Centre for Infectious Diseases & International Health (CIDIH)




The advantages of a trDNA approach for
tuberculosis diagnosis

 • 9 million people develop TB annually

 • 1.7 million people die from TB per year

 • Up to 70 % of TB patients are HIV positive in sub-
   Saharan Africa
 • Current diagnostics insensitive & case detection
   rates low especially in sub-Saharan Africa
 • Smear microscopy based on method developed in
   1882
                    Centre for Infectious Diseases & International Health (CIDIH)




TB trDNA
EU framework 6 project involving partners in Europe and Africa

     Centre for Infectious
         Diseases and                                                Foundation for
     International Health                                            Innovative New
       (CIDIH), UCL, UK                                            Diagnostics (FIND),
                                                                   Geneva, Switzerland
       Ludwig Maximilan
       University, (LMU)                                                Instituto Nazionale
       Munich, Germany                                                     per le Malattie
                                                                          Infettive (INMI),
                                                                            Rome, Italy



        University of Zambia                                    Mbeya Medical Research
        (UNZA) & University                                      Programme (MMRP),
         Teaching Hospital                                         Mbeya, Tanzania
       (UTH), Lusaka, Zambia
                    Centre for Infectious Diseases & International Health (CIDIH)




TrDNA for TB diagnosis
Detection of Mycobacterium tuberculosis nucleic acids in urine in patients with pulmonary
tuberculosis
                                          2002 Kafwabulula (n=63)

                                                     2005 Torrea (n=247)




                                                                           2008
                                                                           Cannas (n=43)
                          1999                               2006
                          Aceti (n=13)                       Rebollo (n=27)
                          Centre for Infectious Diseases & International Health (CIDIH)




TrDNA for TB diagnosis
Detection of Mycobacterium tuberculosis nucleic acids in urine in patients with pulmonary
tuberculosis
   100 %                                    Detection sensitivity


     50 %




   600 bp                                        Amplicon size


   300 bp




               Aceti 99        Kafwabaluala 02             Torrea 05   Rebollo 06    Cannas 08
            Centre for Infectious Diseases & International Health (CIDIH)




Inconsistencies and unknowns

1. Extraction method
                                                             Sample

                                               Extract DNA
2. Storage of urine prior
                                                               DNA
  to extraction
                                          Measure DNA by
                                               PCR


3. Target amplified                                          Result
             Centre for Infectious Diseases & International Health (CIDIH)




Addressing the inconsistencies
Using qPCR
1. Extraction method                              Human DNA assay
                                                  targeting ALU J SINEs
2. Storage of urine prior to
   extraction                                     Inhibition measured by
                                                  SPUD assay

3. Target amplified
   Mycobacterium tuberculosis specific targets
   - IS6110
   - IS1081
                Centre for Infectious Diseases & International Health (CIDIH)




Performance of the qPCR assays
  Assay    Use              Size   Efficiency   R2         Linear dynamic          Cq
                            (bp)                           range                   NTC
  ALU J    Human DNA        94     ~1.00        ~0.998     5 x 106 to 5 x 103      26.87
           quantification

  IS6110   MTB DNA          41     ~0.96        ~0.997     8 x 106   to 8          >40
           quantification

  IS1081   MTB DNA          39     ~0.98        ~0.998     25 x 105 to 25          >40
           quantification
                                                                     PCR efficiency = 10 -1/slope -1


   ALU J                                                 IS6110              IS1081
             Centre for Infectious Diseases & International Health (CIDIH)




Extraction techniques
Extraction technique optimised for DNA recovery & inhibition

              DNA recovery
                     Centre for Infectious Diseases & International Health (CIDIH)




Extraction techniques
Assessed on patient urines
  Undiluted extract                                        Country




  Extracts diluted 1:5                                      Sex




 Nolan (2006) Analytical Biochemistry 351: 308-10
 Huggett (2008) BMC Research Notes 1:70
Summary of urine extraction and detection


 • Q sepharose method chosen for extraction of DNA
   from urine

 • In extractions from larger volumes of urine, a 1:5
   dilution is required to remove inhibition


 • The amount of human DNA in urine from Italy,
   Zambia and Tanzania are comparable at baseline
          Centre for Infectious Diseases & International Health (CIDIH)




Stability of DNA in stored urine
Italy
          Centre for Infectious Diseases & International Health (CIDIH)




Stability of DNA in stored urine
Zambia
            Centre for Infectious Diseases & International Health (CIDIH)




Summary of stability of DNA in stored urine


 • Different stability of DNA in the urine of healthy
   individuals in Italy and Zambia


 • To avoid any potential degradation, extract DNA
   from urine as soon as possible after collection
                     Centre for Infectious Diseases & International Health (CIDIH)




MTB specific assays: target choice
Multi-copy targets
   1. IS6110
      • Present in 0 to 25 copies
        in the MTB complex
        • Regions of the insertion sequence have
          homology with other Mycobacterium species

    1. IS1081
      • Present at 6 copies in M. tuberculosis, M. bovis,
         M. avium & M. xenopi

  Van Embden (1993) Journal of Clinical Microbiology; 31:2: 406-9
              Centre for Infectious Diseases & International Health (CIDIH)




Target choice
Ultra-short amplification
   • Q sepharose extraction methods
   enabled detection of ultrashort
   fragments
   • Shorter targets improved MTB
   detection sensitivity in one individual


   • Our assays employ SYBR green
   and rely on good primer design for
   MTB specificity

                                            Meklonyan (2008) Ann. N.Y. Acad. Sci. 1:137: 83-81
               Centre for Infectious Diseases & International Health (CIDIH)




Putting these lessons into practice
Optimising TB trDNA detection




          IS1081   IS6110   IS6110



          Q sepharose       Wizard
            Centre for Infectious Diseases & International Health (CIDIH)




Summary
• Urine is a very variable sample

• ALU J can be used to determine human DNA content
  of sample prior to disease specific analysis

• Wizard extraction allows a larger volume of
  extracted DNA to be analysed in one qPCR assay
  (no qPCR inhibition by the extract)
             Centre for Infectious Diseases & International Health (CIDIH)




The future

• Characterise the trDNA phenomenon with respect to
  disease progression


• Using the qPCR assays outlined, examine MTB
  trDNA in well defined patient groups and DNA in
  urine further – work in progress
               Centre for Infectious Diseases & International Health (CIDIH)




Thank you
Centre for Infectious Diseases &                University of Zambia (UNZA)
International Health (CIDIH), UCL               Glendah Kalunga
Jim Huggett      Alimuddin Zumla                Peter Mwaba
Gillian Rodger    Tanya Novak                   Patrick Katemangwe
Nina Witt         Mike Taylor

Ludwig Maximilan University           &     Mbeya Medical Research Programme
(LMU)                                       (MMRP)
Michael Hoelscher                           Gasper Nyika
Klaus Reither                               Jutta Jung


                     Instituto Nazionale per le Malattie Infettive (INMI)
                     Angela Cannas       Enrico Giradi     Ludovica Calvo

                     Foundation for Innovative New Diagnostics (FIND)
                     Elizabeth Talbot   Mark Perkins