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Dr A. ASSAL

Second WHO consultation January 27 & 28 January, 2009 Standardization of PCR for Trypanosoma cruzi detection. Dr A. ASSAL. French Blood Services (EFS) PARIS. FRANCE. Usefulness of T. cruzi PCR.

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Dr A. ASSAL

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  1. Second WHO consultation January 27 & 28 January, 2009 Standardization of PCR for Trypanosoma cruzi detection Dr A. ASSAL French Blood Services (EFS) PARIS. FRANCE

  2. Usefulness of T. cruzi PCR • Parasitological tests (Strout, hemoculture or xenodiagnosis), have proven to be highly specific for T. cruzi detection but lack sensitivity. • Many studies show the superiority of PCR in comparison of traditional parsitological tests.

  3. Usefulness of T. cruzi PCR (2) • Acute and chronic chagasic patients • congenital transmission • Treatment efficacy • Disease reactivation after transplantation • Inconclusive serological results • Epidemiological studies

  4. Requirements for a reliable PCR • Sensitive > sensitivity of parasite detection. • Specific: detecting only T cruzi (but all lineages). • No carryover • Standardized => reproducible • Automated • Not expensive • Dozens of in-house PCRs with claimed high sensitivity and specificity, but with variable performance

  5. WHO/PAHO network for standardization of PCR for Chagas’ disease • Co-ordinator: INGEBI-CONICET, Buenos Aires, Argentina (Alejandro Schijman ) • Funding: WHO/TDR and Pan American Health Organization • Methods: • 26 participating laboratories (America's and Europe) • 3 panels A, B and C; tested blindly in duplicate • PCR workshop in Buenos Aires, November 2008, on final evaluation of selected PCRs • Output: "Practical laboratory guidelines for the use of PCR to detect T. cruzi in human peripheral blood for use in clinical research settings".

  6. Panel A 10-fold serial dilutions of T. cruzi DNA

  7. Panel B 10-fold serial dilutions of parasites in human blood

  8. Panel C • 45 blood Guanidine-EDTA samples from seropositive and seronegative individuals from endemic regions of Argentina, Brazil, Bolivia and Paraguay • Tested for extraction and amplification, in duplicate by each participating lab.

  9. DNA target 1- Kinetoplast DNA (kDNA)

  10. DNA target 2- Satellite DNA (kDNA

  11. Method 1: SAT- DNA, TaqMan RT-PCR Method 2: K- DNA, TaqMan RT-PCR RT-PCR used in our lab DNA target , primers and probes

  12. RT-PCR used in our lab (2) • DNA extraction: • High Pure PCR Template Preparation Kit (Roche Diagnostics), 200 µl of sample • Amplification kit: • LightCycler 480 Probes Master. • 5 µl extract in 20 µl final volume • Instrument: • LightCycler 480 (Roche Diagnostics)

  13. RESULTS on INGEBI panels

  14. RESULTS on INGEBI panels (2)

  15. Workshop standardization experiments • 18 PCR operators retested the 4 selected methods • Testing in one laboratory • Same extraction procedures • Same reagents including Taq Polymerase • Same thermocycler and cycling program

  16. Workshop PCR standardization Samples • 6 coded Blood samples, tested blindly: • M1 and M2: 2 seronegative samples • M3, M4 and M5, seropositive samples from chronic Chagas disease patients with increasing parasitic load • M6: seropositive sample from Chronic Chagas disease patient detected as positive by all laboratories that participated in the PCR network.

  17. Workshop standardization Experiment design Conventional PCR on kinetoplast DNA Conventional PCR on satellite DNA Real time PCR on satellite DNA (Sybergreen and TaqMan probes

  18. Specificity and SensitivityF. phenol , C QiAmp, k kDNA, S, Sat

  19. Results • Outcomes of different combinations statistically analyzed • Interpretation of the results still pending • Extraction: • Better sensitivity with phenol chloroform (ref) • Better specificity with silica membrane column • Amplification/detection: • DNA target: similar performance of kDNA and Sat-DNA • Better results with Real-Time PCR

  20. Workshop PCR Guide

  21. Workshop PCR Guide (2)

  22. Which PCR in Blood Transfusion ? • Is there a need for a PCR ? • Serology allows donation qualification. • PCR can be negative because parasitemia is low or absent or intermittent • Positivity of PCR in seropositive blood donors or patients is variable: • Barcelona: 20 % (Maria Piron personal data) • Madrid: 60 % (Maria Flores, personal data) • D. Leiby : 63 % (J Infect Dis, 2008)

  23. Which PCR in Blood Transfusion ? • Which PCR is the best ? • Satellite DNA or k DNA are both usable and give similar results • Extraction: • Sample volume (mixed with GE) • Phenol chloroform: • not usable in a blood bank setting (toxic and carryover risk) • Silica Column more convenient

  24. DNA Target Fluorescence Cycle numbers Fluorescence Crossing Point (Cycles) Cycle numbers log (nombre de copie) DNA dilution series Standard curve Quantitative PCR

  25. Normalization of parasite loads according to an internal standard and parasite satellite sequence group1 • The efficiency of the DNA extraction procedure measured by the amplification of the IS • A correction factor according to the representativity of satellite sequences in each parasite lineage group using melting temperatures 1Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in Chagas disease patients. T. Duffy, A.G. Schijman et al. Submitted

  26. Potential reference material for PCR QC • Different materials may be proposed • Quantified DNA of different strains from different lineages. • GEB spiked with known concentrations of parasites. Indefinite storage at + 4°C • Qualitative monitoring of PCR • Quantitative determination of parasite or DNA LOD using probit analysis

  27. Conclusions • Workshop approach to improve T. cruzi detection by PCR • Standardization of the different steps, from sample preparation to amplification and detection • PCR robust despite different panel shipment and storage • Promising preliminary step to reference material for PCR QC

  28. Acknowledgments • Alejandro Schijman (Argentina) • All the team of INGEBI • Maria Piron (Spain) • Frederic Auger (France)

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