dr a. assal french blood services (efs) paris. france second who consultation january 27 & 28...
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Dr A. ASSAL
French Blood Services (EFS) PARIS. FRANCE
Second WHO consultation
January 27 & 28 January, 2009
Standardization of PCR for
Trypanosoma cruzi detection
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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.
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•Acute and chronic chagasic patients
• congenital transmission
• Treatment efficacy
• Disease reactivation after transplantation
• Inconclusive serological results
• Epidemiological studies
Usefulness of T. cruzi PCR (2)
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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
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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".
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Panel A
10-fold serial dilutions of T. cruzi DNA
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Panel B10-fold serial dilutions of parasites in
human blood
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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.
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DNA target
1- Kinetoplast DNA (kDNA)
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DNA target
2- Satellite DNA (kDNA
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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
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• 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)
RT-PCR used in our lab (2)
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RESULTS on INGEBI panels
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RESULTS on INGEBI panels (2)
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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
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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.
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Workshop standardization
Experiment design
Conventional PCR on kinetoplast DNA
Conventional PCR on satellite DNA
Real time PCR on satellite DNA (Sybergreen and TaqMan probes
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Specificity and Sensitivity F. phenol , C QiAmp, k kDNA, S, Sat
Especificidad por metodo para M1 y M2 246 esp_FS_ esp_CS_ Muestra esp_FK esp_FS RT esp_Ck esp_CS RT 1 0.61111 0.77778 0.83333 0.94444 0.94444 0.83333 2 0.77778 0.88889 0.83333 0.94444 0.88889 0.83333 Sensibilidad por metodo para M3 - M6 sen_FS_ sen_CS_ Muestra sen_FK sen_FS RT sen_Ck sen_CS RT 3 0.50000 0.44444 0.44444 0.27778 0.11111 0.16667 4 0.77778 0.94444 0.88889 0.44444 0.50000 0.44444 5 0.77778 0.94444 0.88889 0.55556 0.66667 0.50000 6 0.83333 0.88889 0.94444 0.94444 0.94444 1.00000
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• 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
Results
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Workshop PCR Guide
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Workshop PCR Guide (2)
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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)
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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
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Quantitative PCR
Cycle numbers
Flu
ore
scence
DNA Target
Cro
ssin
g P
oin
t (C
ycl
es)
log (nombre de copie)
Flu
ore
scence
Cycle numbers
DNA dilution series Standard curve
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Normalization of parasite loads according to an internal standard and parasite satellite
sequence group 1
1) The efficiency of the DNA extraction procedure measured by the amplification of the IS
2) A correction factor according to the representativity of satellite sequences in each parasite lineage group using melting temperatures
1 Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in Chagas disease patients. T. Duffy, A.G. Schijman et al. Submitted
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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
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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
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Acknowledgments
• Alejandro Schijman (Argentina)
• All the team of INGEBI
• Maria Piron (Spain)
• Frederic Auger (France)