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

Dr A. ASSAL

French Blood Services (EFS) PARIS. FRANCE

Second WHO consultation

January 27 & 28 January, 2009

Standardization of PCR for

Trypanosoma cruzi detection

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.

•Acute and chronic chagasic patients

• congenital transmission

• Treatment efficacy

• Disease reactivation after transplantation

• Inconclusive serological results

• Epidemiological studies

Usefulness of T. cruzi PCR (2)

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 B10-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 RT-PCR

Method 2: K- DNA,

TaqMan RT-PCR

• 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)

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 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

• 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

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

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

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)