1. WHO EQAP for the detection of influenza A virus subtype by PCR Wilina Lim 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 help develop testing strategies To help develop testing strategies To provide mechanisms to remedy any deficiencies revealed To provide mechanisms to remedy any deficiencies revealed Requirement of lab accreditation in accordance with international standard such as ISO 15189 Requirement of lab accreditation in accordance with international standard such as ISO 15189

3. Benefits identified by NICs To monitor feasibility to ship samples to countries laboratory capability timeliness of reporting

4. 1A. Invitation 2. Preparation of Panels 4. Data Collection 3. Panel Distribution 5. Preliminary report 6. Data Analysis 7. Final report QAP Process 1B. New Participants The EQAP has been accredited in accordance With ISO 17043

5. Preparation of panels Include different subtypes/clades Include different subtypes/clades Dried RNA of influenza AH5, H1, H3, H1v and influenza B viruses Gamma-ray inactivated seasonal influenza samples Verify sample content Verify sample content Verify sufficient homogeneity Verify sufficient homogeneity Samples in final packaged form selected and tested Assure sufficient stability Assure sufficient stability Test over a range of storage conditions prior to distribution Samples were tested after 7 days of storage at 37 o C using both conventional and real-time PCR assays

6. Temperature survey Monitor the temperature change during shipment Monitor the temperature change during shipment Target participants Target participants With temperature record higher than 37 ℃ during sample dispatch periodWith temperature record higher than 37 ℃ during sample dispatch period Panel 9: Jan-Mar 2011Panel 9: Jan-Mar 2011 Region No. of logger sent returned AFRO51 AMRO32 EMRO22 WPRO32 Total137

7. Temperature survey on EQAP panel 9 shipment Longest duration with temperature higher than 37 ℃ was 6 hours

8. Temperature above 37 ℃ were recorded during the transit in daytime

9. Panel contents No. of samples in the panel Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 Panel 6 Panel 7 Panel 8 Panel 9 200720072008200820092009201020102011 Feb-MarAug-OctJan-FebJun-JulJan-FebJun-AugJan-MarJun-AugJan-Mar RNA sample H5 sample: - clade 1 - clade 1 2112 2 - clade 2.1 - clade 2.12211 2 - clade 2.2 - clade 2.22211 22 2 - clade 2.3.2 - clade 2.3.2 222 22 - clade 2.3.4 - clade 2.3.42211222 H1 sample 11112111 H3 sample 111111111 H1pdm sample 2222 Influenza B sample 111 Negative sample 24221 112 Inactivated virus sample H1 sample 1 H3 sample 1 Total101410101010101012 Composition of panels

10. WHO region No. of laboratories participated and reported results Panel 1Panel 2Panel 3Panel 4Panel 5Panel 6Panel 7Panel 8Panel 9 2007 2008 2009 2010 2011 Feb-MarAug-OctJan-FebJun-JulJan-FebJun-AugJan-MarJun-AugJan-Mar AFR668131719212224 AMR51416212322263332 EMR246679111314 EUR3439434545515259 SEAR345566688 WPR14161719162123 All648395109114128139158160 Response of participants

11. Problems encountered No PCR capacity No PCR capacity No reagents No reagents Delay in obtaining import permit Delay in obtaining import permit Varying requirements at the customs Varying requirements at the customs Shipment detained at customs for prolonged period Shipment detained at customs for prolonged period

12. Reasons for laboratories not receiving panels among total invited

13. Performance of participants

14. Panel No. of participants invitedrespondedparticipatedreceivedreportedall correct Panel 1944332 Panel 2955442 Panel 31076554 Panel 4876654 Panel 5886665 Panel 6886664 Panel 7876665 Panel 8988887 Panel 9988886 Performance of SEAR participants % of all correct

15. Panel No. of participants invitedrespondedparticipatedreceivedreportedall correct Panel 119161615148 Panel 2211717171610 Panel 3201919171713 Panel 4212119191916 Panel 5212020181614 Panel 622 2116 Panel 723 19 Panel 824 2322 Panel 924 2321 Performance of WPR participants % of all correct

16. Performance in Panel 8

17. Performance in Panel 9

18. Problems identified in EQAP Inconsistent technical performance Inconsistent technical performance 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 Transcriptional error Transcriptional error

19. False negative results due to probe mis-matches An example of a H5 real-time PCR primer/probe set An example of a H5 real-time PCR primer/probe set Forward primer: 1 bp mis-match Forward primer: 1 bp mis-match Probe: 2 bp mis-matches Probe: 2 bp mis-matches Reverse primer: none Reverse primer: none

20. 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 2010 survey composed of 32 questions 2010 survey composed of 32 questions Questions on the following seven categories: 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

21. Molecular diagnosis (PCR) & Good laboratory practice (GLP) Laboratories returning completed GLP survey forms 2007 2007 64 ( 96%) 2008 200894(82%) 2010 2010 142 (89%)

22. Good laboratory practices More than 80% of laboratories Separate work room for molecular diagnosis (99%) Separate set of equipment and consumables in each working area (94%) Equipment maintenance programme (87%) Control materials for molecular diagnosis (100%) Standard operating procedures (94%) Separate work room for molecular diagnosis (99%) Separate set of equipment and consumables in each working area (94%) Equipment maintenance programme (87%) Control materials for molecular diagnosis (100%) Standard operating procedures (94%) Less than 80% of laboratories Evaluation of the reagents used for molecular tests (67%) Evaluation of the sensitivity/specificity of the molecular tests (59%) Internal audit programme (54%) Accredited by international/national scheme (34%) Countercheck results (78%) Evaluation of the reagents used for molecular tests (67%) Evaluation of the sensitivity/specificity of the molecular tests (59%) Internal audit programme (54%) Accredited by international/national scheme (34%) Countercheck results (78%) Data from GLP survey 2010

23. GLP and EQAP performance Compare GLP with EQAP results Group A (laboratories returned correct answers for all 10 samples) Group B (laboratories returned less than 10 corrects answers) Laboratories with less good performance (Group B) 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 - more samples in recent representative month - SOP on preparation of in-house controls - established test turn-around-time

24. Reagents/tests evaluation, internal audit and accreditation in laboratories of different WHO regions Data from GLP survey 2010

25. The way forward Review Review - materials for simulated specimens/scope - type/subtype/clade to include in the panel - frequency of shipment Enhance performance through training Enhance performance through training Accreditation by recognized authority Accreditation by recognized authority

26. http://www.wpro.who.int/sites/htl/documents/Laboratory+Quality+Standards+and+Implementation.htm

27. Thank You