1. Standardization of PCR for Trypanosoma cruzi detection
Second WHO consultation
January 27 & 28 January, 2009
Standardization of PCR for Trypanosoma cruzi detection d
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. d

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 d

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 d

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". d

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

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

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. d

9. 1- Kinetoplast DNA (kDNA)
DNA target
1- Kinetoplast DNA (kDNA) d

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
Method 1:
SAT- DNA, TaqMan RT-PCR
Method 2:
K- DNA, TaqMan RT-PCR d

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) d

13. RESULTS on INGEBI panelsd

14. RESULTS on INGEBI panels (2)d

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 d

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. d

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

18. Specificity and Sensitivity F. phenol , C QiAmp, k kDNA, S, Sat
The method makes the differencve
RT PCR better than conventionnal PCR d

19. Outcomes of different combinations statistically analyzed
Results
Outcomes of different combinations statistically analyzed
Interpretation of the results still pending
The method makes the differencve
RT PCR better than conventionnal PCR
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 d

20. Workshop PCR Guide d

21. Workshop PCR Guide (2) d

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) d

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 d

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

25. Normalization of parasite loads according to an internal standard and parasite satellite sequence group 1
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 d
1 Accurate 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)