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

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

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

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)

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 AFR AMR EMR EUR SEAR WPR Total

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

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)

Panel Contents No. of samples in the panel Panel 1Panel 2Panel 3Panel 4Panel 5Panel Feb-MarAug-OctJan-FebJun-JulJan-FebJun-Aug H5 sample: - clade clade  - clade  - clade clade  H1 sample11112 H3 sample11111 H1v sample Negative sample24221 Total Composition of panels

Conventional Real-time Panel 2 Panel 3 Panel 4 Panel 5 H5 PCR Panel 2 Panel 3 Panel 4 Panel 5 H1 PCR Panel 2 Panel 3 Panel 4 Panel 5 H3 PCR Method of detection (% of participants)

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

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

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)

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)

Overall performance AFRAMREMREURSEARWPRTotal Panel 1 Panel 2 Panel 3 Panel 4 Panel 5

H5 performance AFRAMREMREURSEARWPRTotal Panel 1 Panel 2 Panel 3 Panel 4 Panel 5

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

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

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 AF Dir, direction; F, forward; R, reverse The primers/probes sequences of the H5 gene used by participants

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 Panel Panel Panel 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.

Factors affecting H5 performance Panel Technical factor a Real-time assay Commercial kit TAT > 28 days Panel Not applicable Panel Panel Panel a P values were calculated by Yates-corrected chi-square test.

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.

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.

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

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

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

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 samples in recent representative month - SOP on preparation of in-house controls - established test turn-around-time

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

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