Giuseppe
Emeritus
Meeting of the WHO working group on polymerase chain reaction protocols for detecting subtype influenza A viruses ? Geneva, June 2010 (WHO WER, edited)
[Source: World Health Organization, Weekly Epidemiological Record, full PDF document (LINK). Extract, edited.]
Weekly epidemiological record
Relev? ?pid?miologique hebdomadaire
12 november 2010, 85th year / 12 novembre 2010, 85e ann?e
No. 46, 2010, 85, 453?460
http://www.who.int/wer
Meeting of the WHO working group on polymerase chain reaction protocols for detecting subtype influenza A viruses ? Geneva, June 2010
The WHO working group on polymerase chain reaction (PCR) protocols for detecting subtype influenza A viruses held its third meeting in Geneva in June 2010.
Participants included representatives from WHO Collaborating Centres for Reference and Research on Influenza, WHO H5 Reference Laboratories, Essential Regulatory Laboratories, National Influenza Centres and the World Organisation for Animal Health?United Nations Food and Agriculture Organisation Network of Expertise on Animal Influenza.
The first and second meetings of the working group were held in September 2007 and April 2009. The objectives of the meeting in June 2010 were to discuss the updating of protocols, the availability of new PCR test kits and the use of new diagnostic techniques, as well as surveillance activities, the external quality assessment of laboratories, antiviral resistance and human serological studies to influenza A viruses. A brief account of the outcomes of the working group?s discussions is presented in this article.
Updated protocols, primer and probe kits, and diagnostic techniques
An updated PCR protocol is being used at the WHO Collaborating Centre at the United States Centers for Disease Control and Prevention (CDC), United States. Kits are available to detect and characterize influenza viruses using real-time reversetranscriptase PCR (RT-PCR); the kits have been tested for quality control. The 5-tar-get kit contains 7 primer?probe sets: universal influenza A, influenza B, human seasonal (A)H1, human (A)H3, Asian avian (A)H5 (2 kits) and human RNase P gene; the positive control kit consists of a human seasonal influenza control and an avian (A)H5N1 control. Since being introduced, the assay has been adapted to improve its sensitivity and specificity for pandemic A(H1N1) 2009 viruses, and the current panel has >96% sensitivity and >96% specificity for upper respiratory specimens. Because testing procedures differ depending on the type of equipment available, the 5-target kit has been split into component kits to allow flexibility in testing: there is an influenza universal A/B kit, an influenza A subtyping kit (seasonal H1 and H3), and an avian (A)H5N1 kit.
During 2010?2011, the CDC will continue to donate RT?PCR kits and updated control reagents to National Influenza Centres which are members of WHO?s Global Influenza Surveillance Network. Several other institutes will provide control reagents for the molecular detection of influenza viruses. The CDC protocol has been adapted for use by many influenza laboratories. WHO collaborating centres and H5 reference laboratories are encouraged to evaluate new reagents and kits developed by other laboratories.
Participants agreed that the PCR methods described in the current WHO document on the laboratory diagnosis of pandemic A(H1N1) 2009 virus in humans(1) should be updated and simplified, and links should be provided to the institution that contributed each protocol. Because many publications refer to the 2009 protocol, it should be archived on the WHO web site for reference.
Participants from H5 reference laboratories reported on new protocols that have been developed to detect reassortant influenza viruses.
Most laboratories in the Global Influenza Surveillance Network conduct molecular testing first and then virus isolation. It was agreed that positive samples with a cycle threshold value <30 should be cultured, and a representative number of samples that test negative by RT?PCR (approximately 5%) should be cultured also.
National Influenza Centres should be able to perform confirmatory testing for diagnosis through culture and should be reminded to send samples for which the virus cannot be subtyped to WHO collaborating centres immediately.
The need for virus isolation was highlighted because antigenic characterization of virus isolates is essential for monitoring antigenic drift and updating the composition of influenza vaccines to ensure a close match with circulating viruses. Issues of safety in virus isolation should be addressed explicitly, and each country should conduct its own risk assessment, using WHO recommendations.(2)
Training in RT?PCR techniques for influenza diagnosis is available at WHO collaborating centres, H5 reference laboratories and National Influenza Centres with this capacity. It was agreed that such training should be coordinated through WHO to avoid duplication of efforts.
The working group also agreed that training is more effective when conducted in the laboratory where the training will be used because the techniques can be applied immediately. In addition, training in good specimen collection is particularly important.
Surveillance
Surveillance activities at a number of institutions were described; these institutions included St Jude Children?s Research Hospital, Memphis, Tennessee, United States; the University of Hong Kong, Hong Kong Special Administrative Region, China; the Pasteur Institute, Paris, France; and the State Research Centre of Virology and Biotechnology, Novosibirsk, Russian Federation.
Assays for detecting (A)H5N1, porcine influenza and other animal influenza viruses A representative from the Network of Expertise on Animal Influenza described their PCR-related activities.
Progress has been made in detecting porcine influenza, and a list of validated methods, with an algorithm for laboratory testing(3) is available.
The working group agreed that an inventory of virus types and sequences would be useful since individual laboratories in the Global Influenza Surveillance Network currently keep their own reference viruses. In view of the variability of the (A)H5 viral sequences, the 2010 Egyptian (A)H5N1 viruses should be considered for use as references to validate and update the WHO (A)H5 RT?PCR detection protocols. Multiplex assays are being used for detection, but they are not recommended in view of their lower sensitivity and specificity.
External quality assessment project
The value of and the need to continue WHO?s external quality assessment project for PCR detection of influenza A viruses was discussed and affirmed by the working group. The quality assessment project has considerably improved the quality of results from global influenza laboratories and confidence in their diagnostic capacity. The quality assessment project sends panels of dried RNA to participating laboratories twice a year, and once a year a survey is sent to assess good laboratory practice. Test results are reported within 1 month, and a summary of the analysis is published annually.
The working group recommended that quality assessment panels should include, if possible, materials that allow assessment of nucleic acid extraction. The quality assessment project should consider changing the dates for receipt of the panels to April and November to avoid the peak of seasonal influenza activity.
Testing for antiviral resistance
The working group reviewed methods for detecting antiviral resistance to drugs that target influenza neuraminidase and M2, emphasizing that the type of assay affects interpretation. Genotypic testing identifies alteration of the viral genome; phenotypic testing requires virus isolation. The gold standard for identifying resistance in influenza viruses is the phenotypic assay, which can detect resistance or reduced susceptibility that genotypic assays fail to detect. Genotypic assays, including PCR-based methods, which most National Influenza Centres have the capacity to conduct, may be used as a screening method for surveillance for antiviral drug susceptibility.
The working group agreed that laboratories that use only genotypic assays should refer potentially resistant specimens to WHO collaborating centres, which will assess the results using phenotypic assays. The working group also agreed that guidelines are needed to help laboratories in the Global Influenza Surveillance Network interpret test results, taking into consideration the limitations of the assays and the need for clinical interpretation of the results. The working group recommended that, under coordination by WHO, the Global Influenza Surveillance Network should report data on antiviral drug susceptibility as part of the National Influenza Centres? reporting to FluNet.
Several hands-on training courses on testing for antiviral resistance have been conducted, and some institutions are planning courses in 2010 and 2011.
Assays for serological responses to influenza (A)H1 viruses
Data from human serological studies on pandemic A(H1N1) 2009 virus infection and laboratory methods used by the Health Protection Agency, London, England, and the CDC to detect antibodies to the virus were exchanged at the meeting. Participants discussed the results of the various studies, and the advantages and disadvantages of the different serological methods used. The WHO Expert Committee on Biological Standardization will decide the international unit for the proposed standard antibody.
Practical training sessions in serological techniques are being run at the CDC and at the Health Protection Agency, London, England. The need for serological training in National Influenza Centres is unclear, but a modest need could be accommodated by offering individual or small-group training at WHO collaborating centres and other competent laboratories on a case-bycase basis. Training requests should be made to WHO to allow coordination of these activities.
As WHO?s manual for laboratory diagnosis and virological surveillance of influenza is nearing completion and will be published shortly on the WHO web site, the working group agreed that the manual should be updated electronically with the most recent versions of the laboratory protocols as they become available
(1) WHO information for laboratory diagnosis of pandemic (H1N1) 2009 virus in humans ? revised. Geneva, World Health Organization, 2009 ((LINK), accessed October 2010).
(2) Laboratory biorisk management for laboratories handling pandemic influenza A (H1N1) 2009 virus. Geneva, World Health Organization, 2010 ((LINK), accessed 20 October 2010).
(3) OFFLU interim guidance on detecting pandemic H1N12009 in pigs: laboratory testing algorithm. Paris, World Organisation for Animal Health?United Nations Food and Agriculture Organization Network of Expertise on Animal Influenza, 2009 ((LINK), accessed 20 October 2010).
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[Source: World Health Organization, Weekly Epidemiological Record, full PDF document (LINK). Extract, edited.]
Weekly epidemiological record
Relev? ?pid?miologique hebdomadaire
12 november 2010, 85th year / 12 novembre 2010, 85e ann?e
No. 46, 2010, 85, 453?460
http://www.who.int/wer
Meeting of the WHO working group on polymerase chain reaction protocols for detecting subtype influenza A viruses ? Geneva, June 2010
The WHO working group on polymerase chain reaction (PCR) protocols for detecting subtype influenza A viruses held its third meeting in Geneva in June 2010.
Participants included representatives from WHO Collaborating Centres for Reference and Research on Influenza, WHO H5 Reference Laboratories, Essential Regulatory Laboratories, National Influenza Centres and the World Organisation for Animal Health?United Nations Food and Agriculture Organisation Network of Expertise on Animal Influenza.
The first and second meetings of the working group were held in September 2007 and April 2009. The objectives of the meeting in June 2010 were to discuss the updating of protocols, the availability of new PCR test kits and the use of new diagnostic techniques, as well as surveillance activities, the external quality assessment of laboratories, antiviral resistance and human serological studies to influenza A viruses. A brief account of the outcomes of the working group?s discussions is presented in this article.
Updated protocols, primer and probe kits, and diagnostic techniques
An updated PCR protocol is being used at the WHO Collaborating Centre at the United States Centers for Disease Control and Prevention (CDC), United States. Kits are available to detect and characterize influenza viruses using real-time reversetranscriptase PCR (RT-PCR); the kits have been tested for quality control. The 5-tar-get kit contains 7 primer?probe sets: universal influenza A, influenza B, human seasonal (A)H1, human (A)H3, Asian avian (A)H5 (2 kits) and human RNase P gene; the positive control kit consists of a human seasonal influenza control and an avian (A)H5N1 control. Since being introduced, the assay has been adapted to improve its sensitivity and specificity for pandemic A(H1N1) 2009 viruses, and the current panel has >96% sensitivity and >96% specificity for upper respiratory specimens. Because testing procedures differ depending on the type of equipment available, the 5-target kit has been split into component kits to allow flexibility in testing: there is an influenza universal A/B kit, an influenza A subtyping kit (seasonal H1 and H3), and an avian (A)H5N1 kit.
During 2010?2011, the CDC will continue to donate RT?PCR kits and updated control reagents to National Influenza Centres which are members of WHO?s Global Influenza Surveillance Network. Several other institutes will provide control reagents for the molecular detection of influenza viruses. The CDC protocol has been adapted for use by many influenza laboratories. WHO collaborating centres and H5 reference laboratories are encouraged to evaluate new reagents and kits developed by other laboratories.
Participants agreed that the PCR methods described in the current WHO document on the laboratory diagnosis of pandemic A(H1N1) 2009 virus in humans(1) should be updated and simplified, and links should be provided to the institution that contributed each protocol. Because many publications refer to the 2009 protocol, it should be archived on the WHO web site for reference.
Participants from H5 reference laboratories reported on new protocols that have been developed to detect reassortant influenza viruses.
Most laboratories in the Global Influenza Surveillance Network conduct molecular testing first and then virus isolation. It was agreed that positive samples with a cycle threshold value <30 should be cultured, and a representative number of samples that test negative by RT?PCR (approximately 5%) should be cultured also.
National Influenza Centres should be able to perform confirmatory testing for diagnosis through culture and should be reminded to send samples for which the virus cannot be subtyped to WHO collaborating centres immediately.
The need for virus isolation was highlighted because antigenic characterization of virus isolates is essential for monitoring antigenic drift and updating the composition of influenza vaccines to ensure a close match with circulating viruses. Issues of safety in virus isolation should be addressed explicitly, and each country should conduct its own risk assessment, using WHO recommendations.(2)
Training in RT?PCR techniques for influenza diagnosis is available at WHO collaborating centres, H5 reference laboratories and National Influenza Centres with this capacity. It was agreed that such training should be coordinated through WHO to avoid duplication of efforts.
The working group also agreed that training is more effective when conducted in the laboratory where the training will be used because the techniques can be applied immediately. In addition, training in good specimen collection is particularly important.
Surveillance
Surveillance activities at a number of institutions were described; these institutions included St Jude Children?s Research Hospital, Memphis, Tennessee, United States; the University of Hong Kong, Hong Kong Special Administrative Region, China; the Pasteur Institute, Paris, France; and the State Research Centre of Virology and Biotechnology, Novosibirsk, Russian Federation.
Assays for detecting (A)H5N1, porcine influenza and other animal influenza viruses A representative from the Network of Expertise on Animal Influenza described their PCR-related activities.
Progress has been made in detecting porcine influenza, and a list of validated methods, with an algorithm for laboratory testing(3) is available.
The working group agreed that an inventory of virus types and sequences would be useful since individual laboratories in the Global Influenza Surveillance Network currently keep their own reference viruses. In view of the variability of the (A)H5 viral sequences, the 2010 Egyptian (A)H5N1 viruses should be considered for use as references to validate and update the WHO (A)H5 RT?PCR detection protocols. Multiplex assays are being used for detection, but they are not recommended in view of their lower sensitivity and specificity.
External quality assessment project
The value of and the need to continue WHO?s external quality assessment project for PCR detection of influenza A viruses was discussed and affirmed by the working group. The quality assessment project has considerably improved the quality of results from global influenza laboratories and confidence in their diagnostic capacity. The quality assessment project sends panels of dried RNA to participating laboratories twice a year, and once a year a survey is sent to assess good laboratory practice. Test results are reported within 1 month, and a summary of the analysis is published annually.
The working group recommended that quality assessment panels should include, if possible, materials that allow assessment of nucleic acid extraction. The quality assessment project should consider changing the dates for receipt of the panels to April and November to avoid the peak of seasonal influenza activity.
Testing for antiviral resistance
The working group reviewed methods for detecting antiviral resistance to drugs that target influenza neuraminidase and M2, emphasizing that the type of assay affects interpretation. Genotypic testing identifies alteration of the viral genome; phenotypic testing requires virus isolation. The gold standard for identifying resistance in influenza viruses is the phenotypic assay, which can detect resistance or reduced susceptibility that genotypic assays fail to detect. Genotypic assays, including PCR-based methods, which most National Influenza Centres have the capacity to conduct, may be used as a screening method for surveillance for antiviral drug susceptibility.
The working group agreed that laboratories that use only genotypic assays should refer potentially resistant specimens to WHO collaborating centres, which will assess the results using phenotypic assays. The working group also agreed that guidelines are needed to help laboratories in the Global Influenza Surveillance Network interpret test results, taking into consideration the limitations of the assays and the need for clinical interpretation of the results. The working group recommended that, under coordination by WHO, the Global Influenza Surveillance Network should report data on antiviral drug susceptibility as part of the National Influenza Centres? reporting to FluNet.
Several hands-on training courses on testing for antiviral resistance have been conducted, and some institutions are planning courses in 2010 and 2011.
Assays for serological responses to influenza (A)H1 viruses
Data from human serological studies on pandemic A(H1N1) 2009 virus infection and laboratory methods used by the Health Protection Agency, London, England, and the CDC to detect antibodies to the virus were exchanged at the meeting. Participants discussed the results of the various studies, and the advantages and disadvantages of the different serological methods used. The WHO Expert Committee on Biological Standardization will decide the international unit for the proposed standard antibody.
Practical training sessions in serological techniques are being run at the CDC and at the Health Protection Agency, London, England. The need for serological training in National Influenza Centres is unclear, but a modest need could be accommodated by offering individual or small-group training at WHO collaborating centres and other competent laboratories on a case-bycase basis. Training requests should be made to WHO to allow coordination of these activities.
As WHO?s manual for laboratory diagnosis and virological surveillance of influenza is nearing completion and will be published shortly on the WHO web site, the working group agreed that the manual should be updated electronically with the most recent versions of the laboratory protocols as they become available
(1) WHO information for laboratory diagnosis of pandemic (H1N1) 2009 virus in humans ? revised. Geneva, World Health Organization, 2009 ((LINK), accessed October 2010).
(2) Laboratory biorisk management for laboratories handling pandemic influenza A (H1N1) 2009 virus. Geneva, World Health Organization, 2010 ((LINK), accessed 20 October 2010).
(3) OFFLU interim guidance on detecting pandemic H1N12009 in pigs: laboratory testing algorithm. Paris, World Organisation for Animal Health?United Nations Food and Agriculture Organization Network of Expertise on Animal Influenza, 2009 ((LINK), accessed 20 October 2010).
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