Download Dr. Assal - World Health Organization

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
• 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.
Usefulness of T. cruzi PCR (2)
•Acute and chronic chagasic patients
• congenital transmission
• Treatment efficacy
• Disease reactivation after transplantation
• Inconclusive serological results
• Epidemiological studies
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
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".
Panel A
10-fold serial dilutions of T. cruzi DNA
Panel B
10-fold serial dilutions of parasites in
human blood
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.
DNA target
1- Kinetoplast DNA (kDNA)
DNA target
2- Satellite DNA (kDNA
RT-PCR used in our lab
DNA target , primers and probes
Method 1:
SAT- DNA,
TaqMan RTPCR
Method 2:
K- DNA,
TaqMan RTPCR
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)
RESULTS on INGEBI panels
RESULTS on INGEBI panels (2)
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
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.
Workshop standardization
Experiment design
Conventional PCR on kinetoplast DNA
Conventional PCR on satellite DNA
Real time PCR on satellite DNA (Sybergreen and TaqMan probes
Specificity and Sensitivity
F. phenol , C QiAmp, k kDNA, S, Sat
Especificidad por metodo para M1 y M2
246
Muestra
esp_FK
esp_FS
esp_FS_
RT
esp_Ck
esp_CS
esp_CS_
RT
1
2
0.61111
0.77778
0.77778
0.88889
0.83333
0.83333
0.94444
0.94444
0.94444
0.88889
0.83333
0.83333
Sensibilidad por metodo para M3 - M6
Muestra
sen_FK
sen_FS
sen_FS_
RT
sen_Ck
sen_CS
sen_CS_
RT
3
4
5
6
0.50000
0.77778
0.77778
0.83333
0.44444
0.94444
0.94444
0.88889
0.44444
0.88889
0.88889
0.94444
0.27778
0.44444
0.55556
0.94444
0.11111
0.50000
0.66667
0.94444
0.16667
0.44444
0.50000
1.00000
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
Workshop PCR Guide
Workshop PCR Guide (2)
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)
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
Quantitative PCR
Fluorescence
DNA Target
Crossing Point (Cycles)
Fluorescence
Cycle numbers
Cycle numbers
DNA dilution series
log (nombre de copie)
Standard curve
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
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
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
Acknowledgments
• Alejandro Schijman (Argentina)
• All the team of INGEBI
• Maria Piron (Spain)
• Frederic Auger (France)