1. WHO EQAP for the detection of influenza A virus subtype by PCR Centre for Health Protection Hong Kong SAR, China

2. Aims/objectives To monitor quality and standards of performance To monitor quality and standards of performance To facilitate information exchange To facilitate information exchange To identify problems with assays To identify problems with assays To provide mechanisms to remedy any deficiencies revealed To provide mechanisms to remedy any deficiencies revealed

3. Benefits to participants Able to Able to compare performancecompare performance provide evidence of qualityprovide evidence of quality minimize errorsminimize errors identify training if neededidentify training if needed

4. Methodology Targets Targets mostly NICs and some non-NICsmostly NICs and some non-NICs Materials Materials dried RNA of H5, H1, H3, H1v virusesdried RNA of H5, H1, H3, H1v viruses Schedule Schedule twice a yeartwice a year Data analysis Data analysis questionnaires onquestionnaires on testing strategy testing strategy test methodology test methodology good laboratory practice (GLP) good laboratory practice (GLP)

5. Distribution of panels and response of participants WHORegion No. of laboratories Invited Received samples Reported results P 1 P 2 P 3 P 4 P 5 P 1 P 2 P 3 P 4 P 5 P 1 P 2 P 3 P 4 P 5 AFR101112191968916176681317 AMR2628282727616192224514162123 EMR89910102567824667 EUR505153525735404345483439434545 SEAR9910883456634556 WPR192120212115171719181416171916 Total122129132137142679099115121648395109114

6. AFRO, AMRO, EMRO, EURO SEARO, WPRO Response of participants No. invited No. receivedNo. reported

7. Reasons for laboratories not receiving panels Problem No. (%) of laboratories Panel 1Panel 2Panel 3Panel 4Panel 5 (N=122)(N=129)(N=132)(N=137)(N=142) No response38 (31)26 (20)20 (15)11 (8)9 (6) Unwilling to participate5 (4)7 (5) 10 (7)9 (6) Import permit or logistical problems12 (10)6 (5) 1 (1)3 (2)

8. Panel Contents No. of samples in the panel Panel 1Panel 2Panel 3Panel 4Panel 5Panel 6 2007 2008 2009 Feb-MarAug-OctJan-FebJun-JulJan-FebJun-Aug H5 sample: - clade 12112 - clade 2.1 2 2 1 1  - clade 2.2 2 2 1 1  - clade 2.3.2222 - clade 2.3.422 1 1 2 H1 sample11112 H3 sample11111 H1v sample Negative sample24221 Total101410 Composition of panels

9. Conventional Real-time 20 40 6080 Panel 2 Panel 3 Panel 4 Panel 5 H5 PCR 20 40 60 80 Panel 2 Panel 3 Panel 4 Panel 5 H1 PCR 20 40 60 80 Panel 2 Panel 3 Panel 4 Panel 5 H3 PCR Method of detection (% of participants)

10. Nucleic acid amplification tests Most were developed in-house Most were developed in-house the primes/probes:the primes/probes: most commonly adapted from other researchers most commonly adapted from other researchers minority were own designed minority were own designed Minority were commercial kits Minority were commercial kits

11. Assessment criteria The performance of individual laboratories was assessed by adding up the number of correct results. The performance of individual laboratories was assessed by adding up the number of correct results. Incorrect responses: Incorrect responses: failing to detect H5 samples and/or reporting the results as non-H5 subtypefailing to detect H5 samples and/or reporting the results as non-H5 subtype failing to detect H1 samples and/or reporting the results as non-H1 subtypefailing to detect H1 samples and/or reporting the results as non-H1 subtype failing to detect H3 samples and/or reporting the results as non-H3 subtypefailing to detect H3 samples and/or reporting the results as non-H3 subtype failing to report correct influenza A test results for H1/H3 samples if H1/H3 subtyping was not performedfailing to report correct influenza A test results for H1/H3 samples if H1/H3 subtyping was not performed reporting positive results for a sample that did not contain any viral RNAreporting positive results for a sample that did not contain any viral RNA

12. Performance of laboratories Performance No. (%) of laboratories Panel 1Panel 2Panel 3Panel 4Panel 5 (N=64)(N=83)(N=95)(N=109)(N=114) All samples correct43 (67)54 (65)70 (74)84 (77) 87 (76) All but 1 sample correct6 (9)14 (17)10 (11)11 (10) 12 (11) 50 – 89% of samples correct 9 (14)12 (14)11 (12)10 (9) 14 (12) <50% of samples correct6 (9)3 (4)4 (4) 1 (1)

13. Testing errors made by laboratories Comparison factors No. (%) of laboratories Panel 1 Panel 2 Panel 3 Panel 4Panel 5 (N=64)(N=83)(N=95)(N=109)(N=114) Incorrect H5 results15(23) 17(20) 17(18)13(12)21(18) False-positive results9(14) 5(6) 2(2)6(6)1(1)

14. Overall performance 10 20 30 40 50 60 70 80 90100AFRAMREMREURSEARWPRTotal Panel 1 Panel 2 Panel 3 Panel 4 Panel 5

15. H5 performance 10 20 30 40 50 60 70 80 90 100 AFRAMREMREURSEARWPRTotal Panel 1 Panel 2 Panel 3 Panel 4 Panel 5

16. AFRO, AMRO, EMRO, EURO SEARO, WPRO Performance % of all correct % of H5 all correct

17. Problems identified in EQAP Positive control not used appropriately Positive control not used appropriately Lab contamination Lab contamination Misinterpretation of results Misinterpretation of results Primers and probes mismatch Primers and probes mismatch

18. The sequences of the H5 primers/probes were obtained from PubMed and WHO website. The positions of the oligonucleotides are based on the HA gene of A/HongKong/156/1997(H5N1), GenBank accession number AF046088. Dir, direction; F, forward; R, reverse The primers/probes sequences of the H5 gene used by participants

19. The most widely adopted H5 PCR primers/probes Panel No. of laboratories reportedresultsperformed H5 subtyping adopted CDC primers/probes with incorrect H5 results a NN %b%b%b%bN %c%c%c%cN %d%d%d%d Panel 2 8381981721318 Panel 3 9594992931414 Panel 4 10910899444125 Panel 5 1141141004741511 a Incorrect results were due to either false-positive, false negative or any non-H5 results reported in H5 samples. b Percentages are based on the number of laboratories reported results. c Percentages are based on the number of laboratories performed H5 subtyping. d Percentages are based on the number of laboratories adopted CDC primers/probes.

20. Factors affecting H5 performance Panel Technical factor a Real-time assay Commercial kit TAT > 28 days Panel 2 0.0020.678 Not applicable Panel 3 0.0060.6370.002 Panel 4 0.5650.1880.945 Panel 5 0.0060.3480.022 a P values were calculated by Yates-corrected chi-square test.

21. H1v performance in Panel 6 AFRO AMRO EMRO EURO SEARO WPRO % of H1v all correct One participant each in AFRO, AMRO and EMRO reported incorrect H1v subtyping results.

22. SourceNo. * % Centres for Disease Control and Prevention7178 Institut Pasteur, Paris, France55 Robert Koch-Institute44 Health Protection Agency, United Kingdom33 Other 19 sources191 H1v primers/probes adopted by 91 participants in Panel 6 * More than one set of primers/probes were used by 10 participants, the total number is larger than 91.

23. GLP Survey 2007 survey composed of 73 questions 2007 survey composed of 73 questions 2008 survey composed of 25 questions 2008 survey composed of 25 questions Both surveys composed questions on the following seven categories: Both surveys composed questions on the following seven categories: personnelpersonnel quality managementsquality managements design, equipment and consumablesdesign, equipment and consumables pre-analytical procedurespre-analytical procedures analytical proceduresanalytical procedures post-analytical procedurespost-analytical procedures reporting and record keepingreporting and record keeping safetysafety

24. Parameter GLP in 20072008 Nn%Nn% Quality management - Laboratory has a continuous improvement programme 846881928592 - Laboratory is accredited by national/international laboratory accreditation organizations 855362922932 Facility design - Laboratory has separate rooms for: - sample preparation 846780949399 - reagent preparation 848095949298 - amplification and product detection 848399949298 - Laboratory has documented policy requiring a unidirectional workflow 847083946468 Quality management and facility design

25. Parameter GLP in 20072008 Nn%Nn% Examination procedures - Laboratory has SOP for the following individual testing procedure: - viral RNA extraction 848095929098 - preparation of in-house controls 774964924863 - Controls included when performing molecular diagnostic tests: - positive control 827895939198 - negative control 797494 9399 Post-examination procedures - Laboratory has worksheet to record: - the date of testing 85849991 100 - the reagent expiry dates 845970915965 - Laboratory has another staff to countercheck the test results 855969946771 Examination procedures and post- examination procedures

26. Factors affecting performance based on GLP analysis Lab did not return all correct results tends to meet less quality parameters; significantly more likely (p < 0.05) not having Lab did not return all correct results tends to meet less quality parameters; significantly more likely (p < 0.05) not having - audits of personnel - separate rooms for all steps involving PCR - programme to monitor equipment - programme to monitor equipment - 100 samples in recent representative month - SOP on preparation of in-house controls - established test turn-around-time

27. Way forward Regularly review results of EQAP Regularly review results of EQAP Identify problems related to test protocols Identify problems related to test protocols Enhance performance through training Enhance performance through training

28. Thank You