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Meeting of the WHO expert working group on surveillance of influenza antiviral susceptibility, Geneva, July 2013 (WHO, WER, November 1 2013, edited)
[Source: World Health Organization, Weekly Epidemiological Record, full PDF document: (LINK). Extract.]
Weekly epidemiological record / Relev? ?pid?miologique hebdomadaire, 1st NOVEMBER 2013, 88th year / No. 44-45, 2013, 88, 477?488, http://www.who.int/wer
Meeting of the WHO expert working group on surveillance of influenza antiviral susceptibility, Geneva, July 2013
The WHO expert working group on surveillance of influenza antiviral susceptibility (AVWG) was formed in 2011 to support the WHO Global Influenza Surveillance and Response System (GISRS). This report provides a summary of the deliberations during the 3rd meeting of the AVWG, held on 4?5 July 2013 in Geneva.
The working group includes representatives from the WHO Collaborating Centres for Reference and Research on Influenza (WHO CCs), National Influenza Centres (NICs) and public health institutes.(1) An expert in clinical virology was also invited to attend this meeting.
The GISRS generates timely virological data for monitoring of and response to seasonal influenza, and risk assessment of influenza viruses with pandemic potential. Vigilance and robust surveillance of antiviral susceptibility is essential to guide clinical management and national/regional antiviral stockpile policies as part of the pandemic preparedness strategy. The AVWG develops practical approaches for NICs of GISRS through review of methodologies available for antiviral susceptibility surveillance, advises on practical antiviral susceptibility surveillance strategy for GISRS, and provides guidance on the interpretation of laboratory antiviral susceptibility surveillance data. The work of the AVWG provided the basis for the development of WHO guidelines, available on the WHO website dedicated to influenza antiviral susceptibility monitoring.(2)
The 3rd meeting of the AVWG continued and further developed the work carried out or planned during the previous meetings.
Antiviral susceptibility testing capabilities
Phenotypic methods, measuring the neuraminidase inhibitor (NAI) concentration required to cause a 50% inhibition (IC50) of a standard amount of neuraminidase activity, are methods of choice of WHO CCs, backed up by full neuraminidase (NA) gene sequencing of those viruses that have IC50 values requiring further investigation.
For M2 blockers, sequencing of the gene encoding M2 is used to assess the presence of well-defined markers of resistance. Similarly, many laboratories have developed genotypic assays, which can be applied to clinical specimens, based on Sanger sequencing, pyrosequencing or single nucleotide polymorphism (SNP) analysis to look for well-defined markers of reduced inhibition by NAIs.
NICs of GISRS use a wide variety of techniques dependent on available resources. The laboratories of all AVWG members use phenotypic methods to test for susceptibility to oseltamivir and zanamivir and 3 WHO CCs also test for susceptibility to laninamivir and peramivir.
The AVWG agreed to investigate other sources for antivirals and expensive reagents like MUNANA(3) substrate for the fluorescence-based neuraminidase inhibition (NAI) assay. However, any change of reagent source requires validation for which panels of reference viruses with known antiviral susceptibility profiles are available.(2) Reference viruses remain important controls in phenotypic and genotypic assays. Cost reduction based on using a reduced number of drug concentrations in the NAI assay as an initial screen for viruses with reduced inhibition is practised at the WHO CC in London and the NIC in China, Hong Kong, SAR. The AVWG will further explore implementation of these methods as less expensive alternative screening methods. However, laboratories establishing phenotypic testing for the first time are recommended to gain experience with the original methods first.
Antiviral susceptibility overview
Since the meeting of the AVWG in 2012, all influenza A(H1N1)pdm09 and A(H3N2) viruses screened for M2 blocker susceptibility encoded the S31N substitution in M2 associated with resistance and there has been no change observed in the proportions of viruses showing (highly) reduced inhibition by NAIs. Among the A(H1N1)pdm09 viruses tested the proportion with highly reduced oseltamivir inhibition ranged from 0% in the WHO South-East Asia Region up to 2.4% in Japan, with the majority being associated with the NA-H275Y amino acid substitution. Among A(H3N2) and B viruses less than 1% of viruses tested showed (highly) reduced NAI inhibition. Of these A(H3N2) viruses a small number were associated with the NA-R292K or NA-E119V amino acid substitutions.
Initial reports on A(H7N9) virus showed discrepancy between detection of NA-R292K (according to the numbering of N2 in the H3N2 virus) and low IC50. Purified NA-R292K variant virus showed highly reduced oseltamivir inhibition. A wild-type/NAR292K variant mixture in the initial virus isolate and a different pH optimum for neuraminidase activity was the cause of the low IC50,(4) underlining the importance of combining phenotypic and genotypic methods and critical evaluation of the combined results.
Amino acid substitutions associated with reduced inhibition
The list of NA amino acid substitutions associated with (highly) reduced inhibition on the website was reviewed. The I221V substitution in influenza B viruses associated with reduced oseltamivir inhibition has now been added. In addition, the AVWG established clear criteria for the inclusion of substitutions in the table: these are substitutions that occur naturally or are induced by antiviral treatment of patients, but not those that have been artificially induced by reverse genetics experiments, in animal experiments, or selected in cell culture with or without antiviral drug pressure (i.e. where the substitution is not present in the virus from the clinical specimen). Alerts to, and specifications of, (highly) reduced inhibition substitutions that might have been induced by virus isolation in cell or embryonated egg culture will be included in the accompanying text. The AVWG recommends sequencing of clinical specimens directly if such substitutions are detected in cultured viruses.
Use of the inhibition category criteria
The AVWG evaluated the use of the criteria established during its second meeting for reporting of the levels of susceptibility, graded according to fold-change in IC50 compared to susceptible seasonal or reference virus baseline IC50 levels as normal, reduced and highly reduced inhibition.(1)
Several studies using this new terminology have been published.
Implementation of the terminology for both phenotypic and genotypic data reporting by European Union countries to the European CDC antiviral susceptibility database has further advocated the use of this terminology.
The AVWG recognises that under certain circumstances some viruses carrying amino acid substitutions previously associated with a mild level of reduced inhibition fall just below the threshold for reduced inhibition and are considered to show normal inhibition by strict application of the criteria.
This also holds for a substitution that by itself does not qualify for the reduced inhibition category but instead enhances the inhibition level of other substitutions conferring (highly) reduced inhibition. Therefore, a clear interpretative comment should accompany reporting of such substitutions and their associated category of inhibition, especially if it involves patient treatment.
To set normal inhibition baselines for calculation of fold change, the AVWG further specified that the median IC50 (of previous season data or ?floating? current season median) after removal of obvious outliers should be used when data for at least 15 viruses are available. During the emergence of a novel virus infecting humans, a recent wild type specific control virus should be used to determine the normal inhibition baseline. For clarity and uniformity the AVWG decided to translate substitution interpretation into similar terminology, i.e. amino acid substitution previously associated with normal, reduced or highly reduced inhibition for a given neuraminidase inhibitor.
FluNet developments
Presently, the perceived highest risk of clinical resistance emerging is the NA-H275Y substitution in A(H1N1)pdm09 viruses and GISRS capacity is currently weighted towards molecular methodology for detection of this substitution. FluNet has been developed to include monitoring for this substitution and aggregated collection of data by week of specimen collection will include details on patient setting (hospital/community/unknown) and oseltamivir exposure; this is planned to be in practice before the end of 2013.
Pilot inclusion of NA-H275Y A(H1N1)pdm09 virus in the 12th WHO EQAP panel
The AVWG evaluated the pilot inclusion of an A(H1N1)pdm09 virus with NA-H275Y substitution in the 12th WHO External Quality Assessment Project (EQAP) panel for the detection of influenza virus type A by PCR.
Participating laboratories were asked to apply their available molecular method(s) to detect the NA-H275Y substitution. About a quarter (n=44) of all laboratories participating in the EQAP (n=158) performed molecular detection of the substitution. Approximately 60% used SNP real-time RT-PCR, 40% Sanger sequencing and 10% pyrosequencing, with some laboratories applying more than one technique. Of the 44 laboratories, 43 (98%) correctly identified NA-H275Y. The AVWG encouraged the EQAP team to keep NA-H275Y in the panel and to consider inclusion of a mixture of wild-type and NA-H275Y variant to simulate clinical specimens, often collected during an oseltamivir treatment course.
Practical guidance document
Draft chapters of the Practical guidance for National Influenza Centres establishing/implementing neuraminidase inhibitor susceptibility surveillance were reviewed. The final version will be ready for distribution before the end of 2013. This guidance is complementary to the AVWG web pages and the laboratory protocols provided therein.
Review of regional activities identified effective practical training as a common need. Although one-on-one training, sometimes within a trainee?s institute, is provided by the WHO CCs and other laboratories in GISRS, effective implementation requires the availability of reagents, equipment, and software for immediate application and dissemination of what has been taught. Regular follow-up with trainees to address unforeseen difficulties and practicalities was considered a necessary part of the training programme. A step-wise plan to establish and implement effective antiviral susceptibility monitoring will be part of the practical guidance.
Although the WHO CCs receive a wealth of viruses and specimens for testing, primarily to assess virus antigenic properties and inform vaccine recommendations, representativeness by country based on population size is not evenly distributed. In addition, a review of specimen referral forms used by the WHO CCs showed that the virological, epidemiological and clinical information requested with the shared specimens varies widely in the number and type of parameters collected. With the increased use of NAIs, data such as antiviral treatment status, health care setting (community or hospital), and immune status (compromised or competent) are important for monitoring antiviral susceptibility.
The AVWG decided to explore standardization of the specimen referral forms.
Different criteria for the selection of specimens to be tested and which viruses to refer to a WHO CC are required to address the questions of antigenicity and antiviral susceptibility. Although the current WHO guidance does include antiviral susceptibility monitoring,(5) these recommendations do not take into sufficient account the testing capability and capacity in countries.
In the USA a right-size surveillance approach has been developed and is under evaluation. In Europe recommendations for specimen selection and specimen referral stratified according to NIC capability and capacity are followed. The AVWG considered that in the practical guidance document these aspects of specimen selection and virus referral should be addressed in more detail for the global network.
Future objectives
The AVWG will continue to review its recommendations to ensure they remain appropriate. The main focus of the coming year will be to finalise the guidance document, and the implementation and evaluation of new FluNet antiviral susceptibility reporting criteria. Further developments of the database for antiviral data will be considered in the future. The AVWG will explore with the WHO EQAP team the inclusion of further specimens for antiviral susceptibility evaluation in the next WHO EQAP panel in 2014.
Despite successful implementation of the criteria for levels of drug inhibition, constant appraisal of these criteria is needed to ensure they remain applicable as circulating seasonal influenza viruses evolve or novel influenza viruses cause zoonoses.
Similarly, issues concerning training and implementation of antiviral susceptibility testing methods will be the subject of future AVWG meetings.
Finally, the AVWG agreed to work towards an annual global update on antiviral susceptibility for seasonal influenza viruses, and possibly zoonotic influenza viruses, but not other animal influenza viruses.
The role of the AVWG will be strengthened to support the GISRS in ensuring effective antiviral susceptibility surveillance through providing guidance and recommendations on relevant and cost-effective methods, correct data interpretation and balanced coverage.
_________
(1) See No. 39, 2012, 87, 369?374.
(2) Laboratory methodologies for testing the antiviral susceptibility of influenza viruses. Geneva, World Health Organization, 2012 (http://www.who.int/influenza/gisrs_laboratory/antiviral_susceptibility/en/index.html accessed August 2013)
(3) 2?-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid.
(4) Sleeman K et al. R292K substitution and susceptibility of influenza A(H7N9) viruses to neuraminidase inhibitors. Emerging Infectious Diseases, 2013 Sep. doi:10.3201/eid1909.130724. [Epub ahead of print]
(5) Selection of clinical specimens for virus isolation and of viruses for shipment from National Influenza Centres to WHO Collaborating Centres. Geneva, World Health Organization, 2010 (http://www.who.int/influenza/gisrs_laboratory/national_influenza_centres/20101206_specimens_selected_for_virus_isolation_an d_shipment.pdf, accessed August 2013)
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Weekly epidemiological record / Relev? ?pid?miologique hebdomadaire, 1st NOVEMBER 2013, 88th year / No. 44-45, 2013, 88, 477?488, http://www.who.int/wer
Meeting of the WHO expert working group on surveillance of influenza antiviral susceptibility, Geneva, July 2013
The WHO expert working group on surveillance of influenza antiviral susceptibility (AVWG) was formed in 2011 to support the WHO Global Influenza Surveillance and Response System (GISRS). This report provides a summary of the deliberations during the 3rd meeting of the AVWG, held on 4?5 July 2013 in Geneva.
The working group includes representatives from the WHO Collaborating Centres for Reference and Research on Influenza (WHO CCs), National Influenza Centres (NICs) and public health institutes.(1) An expert in clinical virology was also invited to attend this meeting.
The GISRS generates timely virological data for monitoring of and response to seasonal influenza, and risk assessment of influenza viruses with pandemic potential. Vigilance and robust surveillance of antiviral susceptibility is essential to guide clinical management and national/regional antiviral stockpile policies as part of the pandemic preparedness strategy. The AVWG develops practical approaches for NICs of GISRS through review of methodologies available for antiviral susceptibility surveillance, advises on practical antiviral susceptibility surveillance strategy for GISRS, and provides guidance on the interpretation of laboratory antiviral susceptibility surveillance data. The work of the AVWG provided the basis for the development of WHO guidelines, available on the WHO website dedicated to influenza antiviral susceptibility monitoring.(2)
The 3rd meeting of the AVWG continued and further developed the work carried out or planned during the previous meetings.
Antiviral susceptibility testing capabilities
Phenotypic methods, measuring the neuraminidase inhibitor (NAI) concentration required to cause a 50% inhibition (IC50) of a standard amount of neuraminidase activity, are methods of choice of WHO CCs, backed up by full neuraminidase (NA) gene sequencing of those viruses that have IC50 values requiring further investigation.
For M2 blockers, sequencing of the gene encoding M2 is used to assess the presence of well-defined markers of resistance. Similarly, many laboratories have developed genotypic assays, which can be applied to clinical specimens, based on Sanger sequencing, pyrosequencing or single nucleotide polymorphism (SNP) analysis to look for well-defined markers of reduced inhibition by NAIs.
NICs of GISRS use a wide variety of techniques dependent on available resources. The laboratories of all AVWG members use phenotypic methods to test for susceptibility to oseltamivir and zanamivir and 3 WHO CCs also test for susceptibility to laninamivir and peramivir.
The AVWG agreed to investigate other sources for antivirals and expensive reagents like MUNANA(3) substrate for the fluorescence-based neuraminidase inhibition (NAI) assay. However, any change of reagent source requires validation for which panels of reference viruses with known antiviral susceptibility profiles are available.(2) Reference viruses remain important controls in phenotypic and genotypic assays. Cost reduction based on using a reduced number of drug concentrations in the NAI assay as an initial screen for viruses with reduced inhibition is practised at the WHO CC in London and the NIC in China, Hong Kong, SAR. The AVWG will further explore implementation of these methods as less expensive alternative screening methods. However, laboratories establishing phenotypic testing for the first time are recommended to gain experience with the original methods first.
Antiviral susceptibility overview
Since the meeting of the AVWG in 2012, all influenza A(H1N1)pdm09 and A(H3N2) viruses screened for M2 blocker susceptibility encoded the S31N substitution in M2 associated with resistance and there has been no change observed in the proportions of viruses showing (highly) reduced inhibition by NAIs. Among the A(H1N1)pdm09 viruses tested the proportion with highly reduced oseltamivir inhibition ranged from 0% in the WHO South-East Asia Region up to 2.4% in Japan, with the majority being associated with the NA-H275Y amino acid substitution. Among A(H3N2) and B viruses less than 1% of viruses tested showed (highly) reduced NAI inhibition. Of these A(H3N2) viruses a small number were associated with the NA-R292K or NA-E119V amino acid substitutions.
Initial reports on A(H7N9) virus showed discrepancy between detection of NA-R292K (according to the numbering of N2 in the H3N2 virus) and low IC50. Purified NA-R292K variant virus showed highly reduced oseltamivir inhibition. A wild-type/NAR292K variant mixture in the initial virus isolate and a different pH optimum for neuraminidase activity was the cause of the low IC50,(4) underlining the importance of combining phenotypic and genotypic methods and critical evaluation of the combined results.
Amino acid substitutions associated with reduced inhibition
The list of NA amino acid substitutions associated with (highly) reduced inhibition on the website was reviewed. The I221V substitution in influenza B viruses associated with reduced oseltamivir inhibition has now been added. In addition, the AVWG established clear criteria for the inclusion of substitutions in the table: these are substitutions that occur naturally or are induced by antiviral treatment of patients, but not those that have been artificially induced by reverse genetics experiments, in animal experiments, or selected in cell culture with or without antiviral drug pressure (i.e. where the substitution is not present in the virus from the clinical specimen). Alerts to, and specifications of, (highly) reduced inhibition substitutions that might have been induced by virus isolation in cell or embryonated egg culture will be included in the accompanying text. The AVWG recommends sequencing of clinical specimens directly if such substitutions are detected in cultured viruses.
Use of the inhibition category criteria
The AVWG evaluated the use of the criteria established during its second meeting for reporting of the levels of susceptibility, graded according to fold-change in IC50 compared to susceptible seasonal or reference virus baseline IC50 levels as normal, reduced and highly reduced inhibition.(1)
Several studies using this new terminology have been published.
Implementation of the terminology for both phenotypic and genotypic data reporting by European Union countries to the European CDC antiviral susceptibility database has further advocated the use of this terminology.
The AVWG recognises that under certain circumstances some viruses carrying amino acid substitutions previously associated with a mild level of reduced inhibition fall just below the threshold for reduced inhibition and are considered to show normal inhibition by strict application of the criteria.
This also holds for a substitution that by itself does not qualify for the reduced inhibition category but instead enhances the inhibition level of other substitutions conferring (highly) reduced inhibition. Therefore, a clear interpretative comment should accompany reporting of such substitutions and their associated category of inhibition, especially if it involves patient treatment.
To set normal inhibition baselines for calculation of fold change, the AVWG further specified that the median IC50 (of previous season data or ?floating? current season median) after removal of obvious outliers should be used when data for at least 15 viruses are available. During the emergence of a novel virus infecting humans, a recent wild type specific control virus should be used to determine the normal inhibition baseline. For clarity and uniformity the AVWG decided to translate substitution interpretation into similar terminology, i.e. amino acid substitution previously associated with normal, reduced or highly reduced inhibition for a given neuraminidase inhibitor.
FluNet developments
Presently, the perceived highest risk of clinical resistance emerging is the NA-H275Y substitution in A(H1N1)pdm09 viruses and GISRS capacity is currently weighted towards molecular methodology for detection of this substitution. FluNet has been developed to include monitoring for this substitution and aggregated collection of data by week of specimen collection will include details on patient setting (hospital/community/unknown) and oseltamivir exposure; this is planned to be in practice before the end of 2013.
Pilot inclusion of NA-H275Y A(H1N1)pdm09 virus in the 12th WHO EQAP panel
The AVWG evaluated the pilot inclusion of an A(H1N1)pdm09 virus with NA-H275Y substitution in the 12th WHO External Quality Assessment Project (EQAP) panel for the detection of influenza virus type A by PCR.
Participating laboratories were asked to apply their available molecular method(s) to detect the NA-H275Y substitution. About a quarter (n=44) of all laboratories participating in the EQAP (n=158) performed molecular detection of the substitution. Approximately 60% used SNP real-time RT-PCR, 40% Sanger sequencing and 10% pyrosequencing, with some laboratories applying more than one technique. Of the 44 laboratories, 43 (98%) correctly identified NA-H275Y. The AVWG encouraged the EQAP team to keep NA-H275Y in the panel and to consider inclusion of a mixture of wild-type and NA-H275Y variant to simulate clinical specimens, often collected during an oseltamivir treatment course.
Practical guidance document
Draft chapters of the Practical guidance for National Influenza Centres establishing/implementing neuraminidase inhibitor susceptibility surveillance were reviewed. The final version will be ready for distribution before the end of 2013. This guidance is complementary to the AVWG web pages and the laboratory protocols provided therein.
Review of regional activities identified effective practical training as a common need. Although one-on-one training, sometimes within a trainee?s institute, is provided by the WHO CCs and other laboratories in GISRS, effective implementation requires the availability of reagents, equipment, and software for immediate application and dissemination of what has been taught. Regular follow-up with trainees to address unforeseen difficulties and practicalities was considered a necessary part of the training programme. A step-wise plan to establish and implement effective antiviral susceptibility monitoring will be part of the practical guidance.
Although the WHO CCs receive a wealth of viruses and specimens for testing, primarily to assess virus antigenic properties and inform vaccine recommendations, representativeness by country based on population size is not evenly distributed. In addition, a review of specimen referral forms used by the WHO CCs showed that the virological, epidemiological and clinical information requested with the shared specimens varies widely in the number and type of parameters collected. With the increased use of NAIs, data such as antiviral treatment status, health care setting (community or hospital), and immune status (compromised or competent) are important for monitoring antiviral susceptibility.
The AVWG decided to explore standardization of the specimen referral forms.
Different criteria for the selection of specimens to be tested and which viruses to refer to a WHO CC are required to address the questions of antigenicity and antiviral susceptibility. Although the current WHO guidance does include antiviral susceptibility monitoring,(5) these recommendations do not take into sufficient account the testing capability and capacity in countries.
In the USA a right-size surveillance approach has been developed and is under evaluation. In Europe recommendations for specimen selection and specimen referral stratified according to NIC capability and capacity are followed. The AVWG considered that in the practical guidance document these aspects of specimen selection and virus referral should be addressed in more detail for the global network.
Future objectives
The AVWG will continue to review its recommendations to ensure they remain appropriate. The main focus of the coming year will be to finalise the guidance document, and the implementation and evaluation of new FluNet antiviral susceptibility reporting criteria. Further developments of the database for antiviral data will be considered in the future. The AVWG will explore with the WHO EQAP team the inclusion of further specimens for antiviral susceptibility evaluation in the next WHO EQAP panel in 2014.
Despite successful implementation of the criteria for levels of drug inhibition, constant appraisal of these criteria is needed to ensure they remain applicable as circulating seasonal influenza viruses evolve or novel influenza viruses cause zoonoses.
Similarly, issues concerning training and implementation of antiviral susceptibility testing methods will be the subject of future AVWG meetings.
Finally, the AVWG agreed to work towards an annual global update on antiviral susceptibility for seasonal influenza viruses, and possibly zoonotic influenza viruses, but not other animal influenza viruses.
The role of the AVWG will be strengthened to support the GISRS in ensuring effective antiviral susceptibility surveillance through providing guidance and recommendations on relevant and cost-effective methods, correct data interpretation and balanced coverage.
_________
(1) See No. 39, 2012, 87, 369?374.
(2) Laboratory methodologies for testing the antiviral susceptibility of influenza viruses. Geneva, World Health Organization, 2012 (http://www.who.int/influenza/gisrs_laboratory/antiviral_susceptibility/en/index.html accessed August 2013)
(3) 2?-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid.
(4) Sleeman K et al. R292K substitution and susceptibility of influenza A(H7N9) viruses to neuraminidase inhibitors. Emerging Infectious Diseases, 2013 Sep. doi:10.3201/eid1909.130724. [Epub ahead of print]
(5) Selection of clinical specimens for virus isolation and of viruses for shipment from National Influenza Centres to WHO Collaborating Centres. Geneva, World Health Organization, 2010 (http://www.who.int/influenza/gisrs_laboratory/national_influenza_centres/20101206_specimens_selected_for_virus_isolation_an d_shipment.pdf, accessed August 2013)
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