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Network of expertise on animal influenza

OIE - World Organization for Animal HealthFAO - Food and Agriculture Organization of the United Nations

WHO PCR Meeting, Geneva, 14-15 th June, 2011 - Meeting of the Technical Group on PCR Diagnosis of Influenza Virus Infections

Day 1 

W. Zhang, GISRS, WHO – Greetings and opening Introduction. Review of history of the technical group since the 1997s H5N1 cases in Hong Kong when WHO H5 reference labs were founded. 

T. Besellaar, GISRS, WHO -Review of last year's meetings and update on the outcomes. Lab manual was finalized and posted on the WHO website along with an updated CDC pdm2009 protocol. Goals and outcomes for this year's meeting were outlined. It was also stressed that virus isolation is still required and that NICs submit un-typeables to CCs.

Updates from participating CCs
W. Dayan (China) – pdmH1N1 detected in all regions of China, Jan 1-June 5th 2011 there have been over 8500 cases with 243 being severe and 70 fatal. No new cases of H5N1 or H9N2 since June 2010. Have an in-house developed PCR assay to detect Influenza B subtypes Vic and Yam. 

Stephen Lindstrom – CDC, Diagnostic Development Team. Update of pdmH1N1 assays from CDC; assay is now posted on the WHO website as a link. Kits available from the CDC (5 target kit:FluA/B,hH1/H3,RP,avianH5; 3 other kits more specific detection kits containing portions of the 5 target kit). Seeing reduced sensitivity to H5 clade 2.3.4, new reverse primer addresses this issue. Have provisionally devised an H9 assay due to prevalence of H9s in Bangladesh, but not deemed necessary at this time so validation is "on hold". Other kits include "H9 Asian", "1918 HA", "H7 Euro or N.Am HA", "Sw N.Am trp. reassort".

John Franks, St. Jude, Memphis (Webster/Webby lab): Avian/swine surveillance, up to 6000 samples annually. Have detected a number of H9 samples from birds in Bangladesh in BYFs, poultry and have 1 human isolate. Starting a collaboration with Colombians to do surveillance on avian influenza. 

Leo Poon, Hong Kong: Surveillance of pigs  slaughtered in abattoir (500/month) using qPCR assay for all 8 pH1N1 gene segments (Clin Chem 2010). Phylogenetic analysis published for Hong Kong SIV (Nature 2011, Science 2010) and re-introduction of pH1N1 in swine (EID 2011). Starting to see mismatches in H5N1 primers recommended by the WHO; especially wrt clade 2.2 and to a lesser extent 2.3.2. Find it very useful to use universal RT-PCR primers for sequencing genes (HA=Phipps, NA=Castillo-Alvarez, Polymerases=Li, others=Hoffman).

P. Reading, VIDRL (Melbourne): finding an increase in number of participating centres submitting samples vs. isolates. Increased H3N2 activity (covered in current vaccine) but appears to have difficulty in isolation of co-circulation of H1 and H3 viruses. Influenza B seems to be out-competing influenza A wrt Ct values. Rod Daniel (UK WHO CC) indicates the co-infections are confounding results in the UK as well; is this due to live vaccination ?

A. Gaynor (NAMRU-3; Egypt): Post-revolution has resulted in isolates needing to be shipped to CDC Atlanta for isolation. H9s being detected through migratory bird surveillance, publication is in progress. All H5 deaths to date have been due to contact with sick birds.  

W. Ampofo (NIC, Ghanan): reporting difficulties with import permits as they pertain to migratory/wild bird samples for testing; wondering if OFFLU can suggest/help with easing this issue. Active swine surveillance program, samples being sent to Egypt for analysis. 

Outcome of day 1

H9 surveillance is required at the human/animal interface due to the increasing numbers of H9 human infections in S.E. Asia. CDC has kits to assay for this but will only provide to centres seeing increased H9 infections. Also hoping for more swine surveillance with an interest in gene reassortments in swine-looking to OFFLU to take lead on this issue.

Strong interest in WHO CCs and H5 Reference Centres participating in avian influenza RT-PCR proficiency panels. Due to increasing evolution of H5s and mutations in WHO primer sites, very interested in acquiring  the latest H5N1s isolated from Indonesia for testing their current assays. Not all labs have requisite H5 isolates to test/validate PCR assays. 

Looking into using the CDC China influenza B protocol to assist screen due to inability to grow H3 viruses. Will compare to the protocols being used in Germany/Norway. 

Day 2

Peter Cheng, Hongkong (SAR): Reviews ring trial program by WHO. Samples included seasonal strains H1, H3, B, pdmH1N1 and different clades of HPAIV H5N1. Rounds of proficiency testing twice a year, now have 160 laboratories participating. For first time panel participants had to extract their own RNA from samples, previous panels had lyophilized RNA. On average favourable outcomes (up to 90% correct results), outcomes dropped this year compared to previous due to contaminants upon extraction (2 labs) and mutations in clade 2.3.2 that affect probe site. EQAP was accredited in accordance with ISO 17043 in 2010. Temperature logger included in select panel packages. A 32 question GLP survey was completed by labs. Many labs found the questions vague and lengthy, going forward will make survey clearer and more concise. Survey found labs have good compliance with SOPs, equipment maintenance and designated work areas/equipment; areas needing improvement involve inability to perform evaluation of reagents/assays and lack of auditing programs.  

Stephen Lindstrom (CDC Atlanta):  CDC rolling out a performance evaluation program for molecular detection of influenza; panel  sent out simulating clinical cases (patient history provided). Requested information from labs wrt protocol and reagents used. All results to be sent back in an electronically locked form (fields have limited fill-in options), easier to analyze. Set up in accordance to US CLIA requirements. September is the roll-out date for the CDC website on “Laboratory Support for Influenza Surveillance”. 

R. Fasce (Chile): Review of PCR protocols and gaps. Identified a number of overlapping protocols, recommended that this be trimmed down. Don’t have a good FluB Yamagata/Victoria protocol (will look at China’s conventional RT-PCR). Current gaps are: only one H7 protocol due to genetic variability may not capture all H7s; no current WHO endorsed H9 protocol.

Discussion of value of PCR in diagnosis: 

Participants felt still required for high-throughput and novel strain detection, aids in pandemic plans (control/detection). There remains the issue of virus adaptation to tissue culture resulting in mutations. Workshops  will go forward and also focus on multiplexing, deployment of reagents and strains to test assays with. PCR has lowered number of un-typeables 

Discussion on role of sequencing: 

SOP not recommended due to differing platforms/chemistry….would be difficult to validate for the number of labs performing. Found to be beneficial for resistance determination; preferred over SNPs as this method has too much variation around target region to validate. WHO held a sequencing workshop in Singapore. The EU document will be modified and added to WHO website, will contain general guidelines and genes/sites of importance. Quality of material on Genbank is poor, most are now using GISAID; St. Jude’s is obligated to use own database. No guidance yet for next generation sequencing, still too new and generates so much data to analyze.

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