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First international proficiency study on West Nile virus molecular detection.

West Nile. ROBERT KOCH INSTITUT. First international proficiency study on West Nile virus molecular detection. Source: H. Gelderblom, RKI. Matthias Niedrig , Sonja Linke Christian Drosten Hervé Zeller . WN. WN. WN. WN. WN. WN. WN.

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First international proficiency study on West Nile virus molecular detection.

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  1. West Nile ROBERT KOCH INSTITUT First international proficiency study on West Nile virus molecular detection. Source: H. Gelderblom, RKI Matthias Niedrig, Sonja Linke Christian Drosten Hervé Zeller ENIVD 06/06 15:10

  2. WN WN WN WN WN WN WN Distribution of disease vectors and WN virus in Europe Aedes albopictus Ochlerotatus atropalpus Ochlerotatus japonicus source: Snow & Ramsdale, Biologist (2002) 49 (2) Hubalek & Halauzka; EID, (1999) 5 (5) ENIVD 05/06 15:10

  3. Import of suspected or confirmed WN cases into Europe eight cases in total ENIVD 05/06 15:10

  4. Preparation of PCR samples for the External Quality Assurance (EQA) • cell culture supernatants of infected cells with the respective viruses • heat treatment for 1h at 56°C and gamma irradiation of 30 kGy • check for infectivity by cell cultivation • dilute with human plasma, aliquots of 100 µl are frozen at –70°C before freeze-drying for 12 hours • samples were distributed by normal mail service ENIVD 05/06 15:10

  5. Phylogenetic tree of some tested WNV samples and reference strains Source: Alex Platonov ENIVD 05/06 15:10 based on the UPGMA method by using 216 nt sequence of 5'-UTR/C gene fragment

  6. Evaluation of the WN-PCR EQA ENIVD 05/06 15:10

  7. Evaluation of WN PCR diagnostic by EQA (1) ENIVD 05/06 15:10

  8. Evaluation of the WNV-PCR diagnostic by EQA (2) ENIVD 05/06 15:10

  9. Probit analysis of the fractions of laboratories achieving positive results (y-axis) in relation to the virus RNA concentration in a given positive sample (x-axis). Data points represent individual samples in the proficiency test panel. The thick line is the regression line calculated on the basis of a probit model (dose-response curve), the thin lines are 95% confidence lineage 2 Fraction of laboratories Log RNA copies / ml Evaluation of WNV PCR diagnostic by EQA (3) lineage 1 ENIVD 05/06 15:10

  10. Results WNV PCR EQA • Does the PCR method used has an influence on the quality of the results?n= 28; 15 real time PCR, 11 RT-PCR, 2 commercial • Do other factors influence the PCR results ?genome preparation: n= 28, 19 Qiagen, 5 Roche, 4 other There is no significant correlation between the PCR method and the quality of the results. There is no significant correlation between the genome preparation method and the quality of the results. ENIVD 05/06 15:10

  11. - Conclusions - WNV PCR EQA • using cell culture supernatants, diluting samples with human plasma and freeze-drying before shipping is useful for sample preparation- gamma-irradiation is a suitable method for inactivation of samples • 7 laboratories showed excellent results (sensitivity of 100%), whereas 10 laboratories that were less sensitive need to improve their methods • PCR methods for detection of WNV should also detect lineage 2 which is now present in Europe. • 4 laboratories create false positive results ENIVD 05/06 15:10

  12. EQA studies performed by the ENIVD between 1999 and 2005 ENIVD 05/06 15:10

  13. What‘s coming next ENIVD 05/06 15:10

  14. Acknowledgement: H. Bin, Central Virol. Laboratory, Sheba Medical Center, Tel Hashomer, Israel D. J. Gubler, CDC, Fort Collins, USA A. Platonov, Central Research Institute of Epidemiology, Moscow, Russia H. Zeller, Jean Mérieux-INSERM P4 laboratory, Institut Pasteur, Lyon, France C. Drosten, Bernhard Nocht Institut, Hamburg, Germany Stephan W. Aberle, Franz Heinz, Medical University of Vienna, Austria Parts of this project were supported by theEuropean Network for diagnostics of "Imported" viral Diseases (ENIVD) presently funded by DG-SANCO of the European Community under the program „AIDS and other communicable diseases“ thanks for your attention ENIVD 05/06 15:10

  15. European Network for diagnostics of "Imported" viral Diseases (ENIVD) for further information: www.enivd.de www.enivd.net www.enivd.org ENIVD 06/06 15:10

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