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First international proficiency study on West Nile virus molecular detection.

Matthias Niedrig, Sonja Linke

Christian Drosten Hervé Zeller

Source: H. Gelderblom, RKI

ROBERT KOCH INSTITUT

West Nile

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Distribution of disease vectors and WN virus in Europe

source: Snow & Ramsdale, Biologist (2002) 49 (2)Hubalek & Halauzka;EID, (1999) 5 (5)

Aedes albopictus

Ochlerotatus atropalpus

Ochlerotatus japonicus

WN

WN WNWN WN

WNWN

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Import of suspected or confirmed WN cases into Europe

Period of time

Imported from

Imported to Viral agent

Number of

cases/deaths

Type of traveller Reference

Nov 1994 Ivory Coast Switzerland Ebola virus 1/0 Repatriation 7

Apr 1996 Brazil Switzerland Yellow Fever virus 1/1 Tourist 8

Sep 1997 Ghana Germany Suspected VHF1 1/1 Refugee 9

Nov 1998 The Gambia Belgium Suspected VHF2 1/1 Tourist 10

Aug 1999 Ivory Coast Germany Yellow Fever virus 1/1 Business 11

Jan 2000 i.a. Ivory Coast Germany Lassa virus 1/1 Tourist 12

Feb 2000 Sierra Leone UK Lassa virus 1/1 Repatriation 13

Mar 2000 Nigeria Germany Lassa virus 1/1 Medical transport 14

Jul 2000 Sierra Leone The Netherlands Lassa virus 1/1 Business 15

Dec 2000 Kenya Germany Suspected VHF3 1/1 Tourist 16

Mar 2001 Sierra Leone Germany Suspected VHF2 1/0 Repatriation 17

Mar 2001 i.a. Chile France Hantavirus 1/0 Tourist 18

Aug 2001 Bulgaria Germany CCHF virus 1/0 Tourist Pers. comm. a)

Sep 2001 Chad France RVF virus 2/0 Business 19

Nov 2001 The Gambia Belgium Yellow Fever virus 1/1 Tourist 20

Sep 2002 Nepal Spain Suspected VHF1 1/0 Tourist Pers. comm. b)

Oct 2002 Cameroon Rep.of Ireland Suspected VHF2 1/0 Business Pers. comm. c)

Feb 2003 Sierra Leone UK Lassa virus 1/0 Business 21

Feb 2003 i.a China different countries SARS-CoV 33/1 Tourist/Business 6

Mar 2003 USA France West Nile virus 1/0 Tourist Pers. comm. d)

Aug 2003 USA Germany West Nile virus 2/0 Tourist 22

Jun 2004 USA France Hantavirus 1/1 Tourist Pers. comm. d)

Sep 2004 USA Germany West Nile virus 1/0 Tourist 22

Oct 2004 USA France West Nile virus 3/0 Tourist Pers. comm. d)

Oct 2004 Tunisia France West Nile virus 1/0 Tourist Pers. comm. d)

Nov 2004 Senegal France CCHF virus 1/0 Repatriation Pers. comm. d)

eight cases in total

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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

Preparation of PCR samples for the External Quality Assurance (EQA)

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Phylogenetic tree of some tested WNV samples and reference strains

WN-HNY1999/AF202541/ WN-IS-98-STD-STORK/AF481864/ WN-NY99-F382-99/AF196835/ WN-MNY-2741/AF206518/ WN-NY99-eqhs/AF260967/ 12 ENIVD 9 ENIVD 2 ENIVD AY278441 Ast99-901 WN-NY2000-grouse3282/AF404755/ 4 ENIVD WN-Eg101/AF260968/ WN-ITALY98-EQUINE/AF404757/ AY490240 Chin strain WN-Volgograd-1999 AF317203 WN-Romania-1996M/AF260969/ AY262283 KN3829 C univittatus 5 ENIVD AY268132 PaAn001 AY268133 PaH001 Kunjin-MRM61C/D00246/ AY274505 Kunjin Pakun WN-Sarafend/AY688948/ WN-B956/AY532665/ 1 ENIVD WN-WNFCG/M12294/ 6 ENIVD AY277251 LEIV-Krnd88-190 D marginatus

0.01

based on the UPGMA method by using 216 nt sequence of 5'-UTR/C gene fragment

Source: Alex Platonov

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Evaluation of the WN-PCR EQA #2 #9 #12 #4 #5 #1 #6 #10 #11 #3 #7 #8

WN-NY WN-NY WN-NY WN-NY WN France WN Uganda Kunjin SLE / JEV YF-17D/TBE Dengue 1-4 neg. neg.

9,4E+05 9,4E+04 9,4E+03 9,4E+02 1,0E+06 1,0E+03 1,0E+03 1,0E+03 1,0E+03 1,0E+03

1 8,7x103 2,0x105 2,7x102 7,8x103 2,0x106 4,3x105 2,6x103 - - - - - 11 100,02 1,2x107 7,8x105 1,5x105 3,6x104 8x106 1,37x106 - - - - - - 11 100,05 WN 1 WN 1 WN 1 WN-1 WN 1 WN 2 WN - SLE - - DEN 3 - - 11 100,06 WN-NY WN-NY WN-NY WN-NY WN WN WN - SLE - YF - DEN 2 - - 11 100,07 + + + + + (+) + - - - - - 11 100,0

19 + + + + + + - - - - - - 11 100,04 + + + + + WN 2 WN-2 - - Flavi - DEN 2 - - 11 100,0

27* WN 1,2x106 WN 3,4x104 WN 3,4x103 WN 3,4x103 WN 1,4x106 WN 1,7x105 WN ~102 - WN ~102 (+) - DEN - - 10 90,913 + + + (-) + + + - - - - - 10 90,926 WN WN WN WN WN (-) WN WN (+) - - - - 10 90,921 1,5x106 1,5x105 1,5x103 1,5x103 1,5x104 (-) - - - - - - 10 90,918 3,8x106 5,6x105 7,4x104 5,1x103 7,7x106 (-) - - - - - - 10 90,922 + + + + + (-) - - - - - - 10 90,923 + + + + + (-) - - - - - - 10 90,915 + + + + + (-) - - - - - - 10 90,93 + + + + + (-) - - - - - - 10 90,9

31 + + + + + (-) - - - - - - 10 90,932 WN-NY WN-NY + (-) WN-France WN-Uganda + - - - DEN 2 - - 10 90,914 + + + + + (-) - - - - - +/- 9 81,833 + + + + + (-) + WN (+) - - - - 9 81,820 + + + (-) + (-) - - - - - - 9 81,816 WN1 4,5x104 WN1 8,5x103 WN1 4,2x102 1x102 (-) (-) WN-2 1,7x105 - - n.d. - - 8 72,78 + + (-) (-) + (-) - - - - DEN - - 8 72,7

10 + + (-) (-) + (-) - - - - - - 8 72,730 + + (-) (-) + (-) - - - - - - 8 72,729 + + (-) (-) + (-) - - - - - - 8 72,737 + (-) (-) (-) + + - - - WN (+) - - 7 63,624 + (-) (-) (-) + (-) - - - - - - 7 63,634 (-) (-) (-) (-) (-) (-) - - - - - - 5 45,5

labo

rato

ry n

°

Scorecorrect results

in %

samples n°

copy N° [ge/ml]

origin

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Evaluation of WN PCR diagnostic by EQA (1)

WNV-1 copy N° [ge / ml]

N°of laboratories %

100.000 28 96,6

10.000 26 89,7

1.000 22 75,9

100 19 65,5

WNV-2 10 34,5

YF, SLE, DEN 1 - 4, TBE, neg.

4 2,9

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Evaluation of the WNV-PCR diagnostic by EQA (2)

1,0E+02

1,0E+03

1,0E+04

1,0E+05

1,0E+06

1,0E+07

1,0E+08

1 2 3 4

sample N°

cop

y N

° [g

e/m

l]

Reihe1 Reihe2 Reihe3 Reihe4

Reihe5 Reihe6 Reihe7

control lab. 1 lab. 2 lab. 27

lab. 21 lab. 16 lab. 18

# 2 # 9 # 12 # 4

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Evaluation of WNV PCR diagnostic by EQA (3)

Log RNA copies / ml

Fra

ctio

n o

f la

bo

rato

ries

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

lineage 1

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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.

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- 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

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EQA studies performed by the ENIVD between 1999 and 2005

1999/2000* Hantavirus Serology 13 10 3/1 11 (85 % ) published2001/2002 Hantavirus Serology 18 14 14/6 13 (72 % ) published

1999* Dengue virus Serology 13 10 2/2 11 (85 % ) published2002 Dengue virus Serology 18 16 18/2 15 (83 % ) published

2002/2003 Dengue virus PCR 13 12 7/3 5 (38 % ) published2002/2003 Filovirus PCR 14 13 7/5** 7 (50 % ) published2002/2003 Lassa virus PCR 14 13 8/5** 8 (57 % ) published2002/2003 Orthopox virus PCR 23 15 13/5** 10 (43 % ) published

2004 SARS-CoV PCR 62 37 7/3 54 (87 % ) published2004 SARS-CoV Serology 30 19 5/6 13 (43 % ) published

2004/2005 Orthopox virus PCR 34 18 11/9 85% / 58% *** published2005 West Nile Virus PCR 28 20 6/5 17 (60% ) submitted2005 West Nile Virus Serology 28 20 4/6 20 (71% ) submitted2005 Tick borne Enc. PCR 23 16 9/3 9 (39% ) submitted2005 Tick borne Enc. Serology 42 25 8/5 25 (60% ) submitted2006 Yellow Fever PCR / Serology study planed

* Pre-evaluation panel tested before running the respective EQA to optimise sample preparation and shipping pro-cedures. ** The same negative samples were included in the three test panels for diagnostic of Filo-, Lassa- or Orthopox virus. *** consist out of two panels: one for sensitivity and specificity including inhibiting factors

No. of participants (countries)

No. of samples positive/negative

No. of labs with good overall proficiency

ReferencePeriod of

timeViral agent

Type of methods

No. of participants

(labs)

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What‘s coming next

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Acknowledgement:

Parts of this project were supported by the European 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“

H. Bin, Central Virol. Laboratory, Sheba Medical Center, Tel Hashomer, IsraelD. 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, GermanyStephan W. Aberle, Franz Heinz, Medical University of Vienna, Austria

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European Network for diagnostics of "Imported" viral Diseases (ENIVD)

for further information: www.enivd.de www.enivd.net www.enivd.org