Giuseppe
Emeritus
[Source: World Health Organization, Weekly Epidemiological Record, full PDF document: (LINK). Edited.]
Weekly epidemiological record / Relev? ?pid?miologique hebdomadaire
28 september 2012, 87th year / 28 septembre 2012, 87e ann?e, No. 39, 2012, 87, 369?38 - http://www.who.int/wer
Meetings of the WHO working group on surveillance of influenza antiviral susceptibility ? Geneva, November 2011 and June 2012
The WHO expert working group on surveillance of influenza antiviral susceptibility (AVWG) was formed in 2011 to meet the needs of the Global Influenza Surveillance and Response System (GISRS). The AVWG includes representatives from the WHO Collaborating Centres for Reference and Research on Influenza (WHOCCs), National Influenza Centres (NICs), public health institutes and research laboratories with expertise in influenza antiviral susceptibility surveillance. Experts from the broader scientific community will be included as needs arise. The overall role of the AVWG is: to develop practical approaches for NICs of the GISRS through review of methodologies available for antiviral susceptibility surveillance; to advise on the appropriate surveillance strategy; and to guide interpretation of laboratory surveillance data. The outputs of the AVWG are communicated to GISRS laboratories through GISRS EZcollab, a restricted online platform for GISRS communication, and the GISRS pages of the WHO web site.(1)
The GISRS has been monitoring the evolution of influenza viruses and providing timely virological data that is necessary for epidemic and pandemic risk assessment and response. During the past 10 years antiviral drugs have provided an important intervention for the treatment and prophylaxis of influenza virus infection.
In countries where antiviral drugs are licensed, national guidance on their use in clinical management is in place. The use and stockpiling of antiviral drugs are key components of the pandemic preparedness plans of many countries. Surveillance of antiviral susceptibility is therefore essential for public health.
Currently, 2 classes of anti-influenza drugs are available for the treatment or prophylaxis of influenza: adamantanes and neuraminidase inhibitors. The rapid global spread of adamantane-resistant A(H3N2) viruses in the mid-2000s, leading to recommendations to discontinue use of this category of antivirals, first demonstrated that antiviral-resistant influenza viruses represent a genuine public health risk.(2) Initial surveillance and research found that emergence of neuraminidase inhibitor (NI) resistant viruses was rare, and, in contrast to adamantane-resistant viruses, NI resistant viruses showed reduced replication and transmissibility.(3) This perspective changed with the emergence of oseltamivir-resistant former seasonal influenza A (H1N1) viruses. First detected in Norway in 2008, these viruses with a single neuraminidase (NA) amino acid substitution (H275Y) emerged in the absence of high drug use, were no more virulent than their sensitive progenitors, but transmitted readily.
By early 2009, oseltamivir-resistant A(H1N1) viruses had become predominant, circulating globally(4) until being replaced by the A(H1N1)pdm09 virus in 2009?2010. Prior to 2009, NI use was largely focused in Japan and the USA; however, the onset of the pandemic in 2009 brought with it a sharp increase in NI usage in the rest of the world. In patients exposed to oseltamivir through prophylaxis or treatment, A(H1N1)pdm09 viruses with NA H275Y substitution causing resistance were detected from relatively early in the pandemic period, although the risk of this variant becoming transmissible was unknown.(5) Several localised transmission events were detected in 2009?2010, but the identification of a community cluster of oseltamivir-resistant A(H1N1)pdm09 viruses in Australia in 2011 increased the possibility that such a virus could emerge and spread widely.(6) Continued vigilance and robust surveillance are critical to ensure that any signals of transmissible antiviral-resistant viruses are detected early. In order to meet the public health needs, WHO has convened two meetings of the AVWG to date, on November 11 2011 and June 28?29 2012. This report provides a summary of both meetings.
Achievements of year 1
The AVWG, in the first meeting, confirmed the role of the group, setting out the broad aims and objectives.
The focus is on reviewing the scope of antiviral surveillance performed by the GISRS, the distribution of antiviral testing capacity, the laboratory methodologies available and the level of necessary surveillance.
GISRS antiviral susceptibility surveillance capacity
The WHO GISRS antiviral testing and sequencing survey in 2010 revealed that antiviral surveillance capability is disproportionately distributed with significant gaps in the South American, Eastern Mediterranean, South-East Asian and African areas.(7) A considerable amount of antiviral susceptibility testing is carried out by the WHOCCs, and provides broad baseline susceptibility data each season. However, NICs are in a position to generate more timely antiviral sensitivity data than the WHOCCs, thereby potentially assisting with clinical management and the early detection of resistant strains in the community. Training and capacity development are required, focusing on those geographical areas lacking antiviral susceptibility surveillance capability, with particularly attention to regions of higher antiviral drug use.
GISRS antiviral susceptibility surveillance methodologies
The AVWG reviewed the current methodologies for antiviral susceptibility testing. A summary was prepared of the costs of reagents and equipment, level of training required and the advantages and disadvantages for the various genotypic and phenotypic assays. These summaries and standard operating procedures recommended by the AVWG will be available through the WHO GISRS web pages.
Adamantane resistance is associated with a well-defined genotype, thus molecular assays (e.g. M gene sequencing or pyrosequencing) are the method of choice for adamantane susceptibility surveillance. For NI susceptibility testing, a wide range of methodologies is in use across the GISRS network, both (i) genotypic, including full length NA sequencing, short range pyrosequencing and real time polymerase chain reaction (PCR) for specific substitutions, and (ii) phenotypic, including enzyme inhibition assays measuring IC50 values using either a fluorescent or chemiluminescent substrate against 1 or more of the NI drugs.
As the molecular mechanisms of NI resistance have not been fully characterised, phenotypic enzyme inhibition assays have been adopted as the gold standard for surveillance, supported by sequencing. Changes to multiple drug susceptibilities can be measured, whether due to known, novel or combinations of substitutions. Nevertheless, many laboratories have implemented molecular screening assays for NI resistance substitutions, by real time PCR, sequencing or pyrosequencing. These screening assays are advantageous as they can be performed with high sensitivity directly on the clinical specimen without the need for virus isolation. The rapid screens can be focused on detection of the substitutions most relevant for the clinical setting and surveillance, as such laboratories have predominantly implemented assays based on the most commonly detected NA H275Y substitution in A(H1N1)pdm09 viruses.
GISRS antiviral susceptibility surveillance strategy
As adamantanes are not recommended for use and all currently circulating influenza A subtypes are resistant to these drugs, the AVWG recommended that surveillance for adamantane resistance should not be a current priority for NICs. A review was carried out of the amino acid substitutions associated with reductions in NI susceptibility, their frequency of detection and the patient setting from which they arose, e.g. treated versus untreated, and community versus hospitalized. This allowed identification of substitutions detected at the highest frequency in a clinical setting. The AVWG agreed that while it was clear that several amino acid substitutions have been associated with reductions in NI susceptibility, the NA H275Y substitution in influenza A viruses with the N1 gene is the only one which occurs with sufficient frequency to warrant a specific screening test.
Achievements of year 2
Working towards the second meeting, the focus moved towards addressing the issues of interpretation of antiviral susceptibility data, reporting, practical guidance for laboratories seeking to implement antiviral surveillance, and the most appropriate means to monitor quality in laboratories.
Interpretation criteria to define phenotypic surveillance data
One of the major challenges for NI antiviral susceptibility surveillance is interpretation of the data produced. For phenotypic testing, data are reported as IC50 values: the concentration of drug required to inhibit enzyme activity by 50%. However, currently there are insufficient data to describe the clinical implications of minor changes in IC50 values and/or the associated amino acid substitutions. To ensure consistency in reporting of surveillance data for NI susceptibility, the AVWG has established a set of criteria to define the antiviral susceptibility of viruses based on the fold change of their IC50 value compared to reference IC50 values. For influenza A, use of normal (<10-fold increase), reduced (10?100-fold increase) and highly reduced (>100-fold increase) inhibition, and for influenza B the same criteria but using <5-fold, 5?50-fold and >50-fold increases, is recommended.
These criteria are intended to bring uniformity to the reporting and analysis of surveillance data from GISRS but not to change clinical or any other practice.
Reporting of antiviral data in FluNet of GISRS
Currently antiviral surveillance data are mainly reported via the WHOCCs or, in the case of Europe, the European Centre for Disease Prevention and Control surveillance system (TESSy) and the WHO/Europe influenza surveillance EuroFlu antiviral databases. Additions to FluNet are required to enable NICs outside Europe to report data electronically. FluNet collects aggregated data, whereas in the case of antiviral resistance reporting, a virus-based reporting strategy is preferable.
However, this level of reporting involves a high number of variables and complexity. As the highest present risk of resistance emerging is the NA H275Y substitution in A(H1N1)pdm09 viruses, GISRS capacity is weighted towards molecular methodology for detection of this mutation. In order to bring in the additions to FluNet gradually, the AVWG agreed minimal additions, namely collection of aggregated data on viruses tested for the H275Y mutation, detailing the patient setting (hospital/community) and the genotype (H275/Y275). The value of data on antiviral treatment history, along with other clinical and epidemiological factors, in interpreting resistance data was recognised, but these fields were not added to the template for FluNet at the present time.
External quality assessment of antiviral susceptibility testing
External quality assessment is an important tool to monitor capabilities of laboratories in the GISRS network and to identify training needs. As a greater number of laboratories incorporate antiviral susceptibility testing methods into their surveillance capacity, it is important to ensure high data quality. In line with the recommended surveillance strategy of molecular screening for NA H275Y in A(H1N1)pdm09 and the proposed FluNet-based reporting of this data, the AVWG recommended the inclusion of a H275Y variant virus in the WHO PCR External Quality Assessment Project (EQAP) panels. The next EQAP will be in 2013, and a sample will be included whereby laboratories can use the molecular-based assay of their choice (e.g. real time reverse transcription PCR, pyrosequencing or NA sequencing) for the detection of the H275Y substitution.
Laboratories will not be scored on this antiviral test, but the data will be used to assess the sample preparation and cost and analysis implications, with a view to preparing a wider panel in future, incorporating further samples with different amino acid substitutions, as appropriate.
Practical guidance for establishing antiviral susceptibility testing
The AVWG agreed the content and format of a guidance document, including necessary practical information for laboratories considering implementing laboratory testing for antiviral surveillance.
Future objectives
The AVWG will continue to review its recommendations on methodology and surveillance strategies for GISRS to ensure that they remain appropriate. The main focus of the coming year will be to undertake the preparatory work for the inclusion of an oseltamivir-resistant (H275Y) A(H1N1)pdm09 virus in the next WHO PCR EQAP panel in 2013. The results of the antiviral testing in the EQAP will be evaluated and ways forward to expand or continue antiviral EQAP will be the subject of discussion in future meetings. The basic additions to FluNet will be evaluated and further developments of the database for antiviral data will be considered in the future. There will need to be constant appraisal of the criteria for categorization of levels of drug inhibition, to ensure these remain applicable as circulating influenza viruses evolve.
Issues surrounding the training of laboratories in antiviral testing methods will be an important subject of future meetings of the AVWG. Training needs to be targeted at a reasonable level, to laboratories with the capability to implement long-term testing and with financial security to purchase and maintain equipment and reagent stocks. The continued role of the AVWG will be to support the GISRS to meet the specific public health need for appropriate antiviral surveillance, through advice and recommendations on surveillance strategies, relevant and cost effective methods, valid data interpretation and operational aspects of GISRS.
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Weekly epidemiological record / Relev? ?pid?miologique hebdomadaire
28 september 2012, 87th year / 28 septembre 2012, 87e ann?e, No. 39, 2012, 87, 369?38 - http://www.who.int/wer
Meetings of the WHO working group on surveillance of influenza antiviral susceptibility ? Geneva, November 2011 and June 2012
The WHO expert working group on surveillance of influenza antiviral susceptibility (AVWG) was formed in 2011 to meet the needs of the Global Influenza Surveillance and Response System (GISRS). The AVWG includes representatives from the WHO Collaborating Centres for Reference and Research on Influenza (WHOCCs), National Influenza Centres (NICs), public health institutes and research laboratories with expertise in influenza antiviral susceptibility surveillance. Experts from the broader scientific community will be included as needs arise. The overall role of the AVWG is: to develop practical approaches for NICs of the GISRS through review of methodologies available for antiviral susceptibility surveillance; to advise on the appropriate surveillance strategy; and to guide interpretation of laboratory surveillance data. The outputs of the AVWG are communicated to GISRS laboratories through GISRS EZcollab, a restricted online platform for GISRS communication, and the GISRS pages of the WHO web site.(1)
The GISRS has been monitoring the evolution of influenza viruses and providing timely virological data that is necessary for epidemic and pandemic risk assessment and response. During the past 10 years antiviral drugs have provided an important intervention for the treatment and prophylaxis of influenza virus infection.
In countries where antiviral drugs are licensed, national guidance on their use in clinical management is in place. The use and stockpiling of antiviral drugs are key components of the pandemic preparedness plans of many countries. Surveillance of antiviral susceptibility is therefore essential for public health.
Currently, 2 classes of anti-influenza drugs are available for the treatment or prophylaxis of influenza: adamantanes and neuraminidase inhibitors. The rapid global spread of adamantane-resistant A(H3N2) viruses in the mid-2000s, leading to recommendations to discontinue use of this category of antivirals, first demonstrated that antiviral-resistant influenza viruses represent a genuine public health risk.(2) Initial surveillance and research found that emergence of neuraminidase inhibitor (NI) resistant viruses was rare, and, in contrast to adamantane-resistant viruses, NI resistant viruses showed reduced replication and transmissibility.(3) This perspective changed with the emergence of oseltamivir-resistant former seasonal influenza A (H1N1) viruses. First detected in Norway in 2008, these viruses with a single neuraminidase (NA) amino acid substitution (H275Y) emerged in the absence of high drug use, were no more virulent than their sensitive progenitors, but transmitted readily.
By early 2009, oseltamivir-resistant A(H1N1) viruses had become predominant, circulating globally(4) until being replaced by the A(H1N1)pdm09 virus in 2009?2010. Prior to 2009, NI use was largely focused in Japan and the USA; however, the onset of the pandemic in 2009 brought with it a sharp increase in NI usage in the rest of the world. In patients exposed to oseltamivir through prophylaxis or treatment, A(H1N1)pdm09 viruses with NA H275Y substitution causing resistance were detected from relatively early in the pandemic period, although the risk of this variant becoming transmissible was unknown.(5) Several localised transmission events were detected in 2009?2010, but the identification of a community cluster of oseltamivir-resistant A(H1N1)pdm09 viruses in Australia in 2011 increased the possibility that such a virus could emerge and spread widely.(6) Continued vigilance and robust surveillance are critical to ensure that any signals of transmissible antiviral-resistant viruses are detected early. In order to meet the public health needs, WHO has convened two meetings of the AVWG to date, on November 11 2011 and June 28?29 2012. This report provides a summary of both meetings.
Achievements of year 1
The AVWG, in the first meeting, confirmed the role of the group, setting out the broad aims and objectives.
The focus is on reviewing the scope of antiviral surveillance performed by the GISRS, the distribution of antiviral testing capacity, the laboratory methodologies available and the level of necessary surveillance.
GISRS antiviral susceptibility surveillance capacity
The WHO GISRS antiviral testing and sequencing survey in 2010 revealed that antiviral surveillance capability is disproportionately distributed with significant gaps in the South American, Eastern Mediterranean, South-East Asian and African areas.(7) A considerable amount of antiviral susceptibility testing is carried out by the WHOCCs, and provides broad baseline susceptibility data each season. However, NICs are in a position to generate more timely antiviral sensitivity data than the WHOCCs, thereby potentially assisting with clinical management and the early detection of resistant strains in the community. Training and capacity development are required, focusing on those geographical areas lacking antiviral susceptibility surveillance capability, with particularly attention to regions of higher antiviral drug use.
GISRS antiviral susceptibility surveillance methodologies
The AVWG reviewed the current methodologies for antiviral susceptibility testing. A summary was prepared of the costs of reagents and equipment, level of training required and the advantages and disadvantages for the various genotypic and phenotypic assays. These summaries and standard operating procedures recommended by the AVWG will be available through the WHO GISRS web pages.
Adamantane resistance is associated with a well-defined genotype, thus molecular assays (e.g. M gene sequencing or pyrosequencing) are the method of choice for adamantane susceptibility surveillance. For NI susceptibility testing, a wide range of methodologies is in use across the GISRS network, both (i) genotypic, including full length NA sequencing, short range pyrosequencing and real time polymerase chain reaction (PCR) for specific substitutions, and (ii) phenotypic, including enzyme inhibition assays measuring IC50 values using either a fluorescent or chemiluminescent substrate against 1 or more of the NI drugs.
As the molecular mechanisms of NI resistance have not been fully characterised, phenotypic enzyme inhibition assays have been adopted as the gold standard for surveillance, supported by sequencing. Changes to multiple drug susceptibilities can be measured, whether due to known, novel or combinations of substitutions. Nevertheless, many laboratories have implemented molecular screening assays for NI resistance substitutions, by real time PCR, sequencing or pyrosequencing. These screening assays are advantageous as they can be performed with high sensitivity directly on the clinical specimen without the need for virus isolation. The rapid screens can be focused on detection of the substitutions most relevant for the clinical setting and surveillance, as such laboratories have predominantly implemented assays based on the most commonly detected NA H275Y substitution in A(H1N1)pdm09 viruses.
GISRS antiviral susceptibility surveillance strategy
As adamantanes are not recommended for use and all currently circulating influenza A subtypes are resistant to these drugs, the AVWG recommended that surveillance for adamantane resistance should not be a current priority for NICs. A review was carried out of the amino acid substitutions associated with reductions in NI susceptibility, their frequency of detection and the patient setting from which they arose, e.g. treated versus untreated, and community versus hospitalized. This allowed identification of substitutions detected at the highest frequency in a clinical setting. The AVWG agreed that while it was clear that several amino acid substitutions have been associated with reductions in NI susceptibility, the NA H275Y substitution in influenza A viruses with the N1 gene is the only one which occurs with sufficient frequency to warrant a specific screening test.
Achievements of year 2
Working towards the second meeting, the focus moved towards addressing the issues of interpretation of antiviral susceptibility data, reporting, practical guidance for laboratories seeking to implement antiviral surveillance, and the most appropriate means to monitor quality in laboratories.
Interpretation criteria to define phenotypic surveillance data
One of the major challenges for NI antiviral susceptibility surveillance is interpretation of the data produced. For phenotypic testing, data are reported as IC50 values: the concentration of drug required to inhibit enzyme activity by 50%. However, currently there are insufficient data to describe the clinical implications of minor changes in IC50 values and/or the associated amino acid substitutions. To ensure consistency in reporting of surveillance data for NI susceptibility, the AVWG has established a set of criteria to define the antiviral susceptibility of viruses based on the fold change of their IC50 value compared to reference IC50 values. For influenza A, use of normal (<10-fold increase), reduced (10?100-fold increase) and highly reduced (>100-fold increase) inhibition, and for influenza B the same criteria but using <5-fold, 5?50-fold and >50-fold increases, is recommended.
These criteria are intended to bring uniformity to the reporting and analysis of surveillance data from GISRS but not to change clinical or any other practice.
Reporting of antiviral data in FluNet of GISRS
Currently antiviral surveillance data are mainly reported via the WHOCCs or, in the case of Europe, the European Centre for Disease Prevention and Control surveillance system (TESSy) and the WHO/Europe influenza surveillance EuroFlu antiviral databases. Additions to FluNet are required to enable NICs outside Europe to report data electronically. FluNet collects aggregated data, whereas in the case of antiviral resistance reporting, a virus-based reporting strategy is preferable.
However, this level of reporting involves a high number of variables and complexity. As the highest present risk of resistance emerging is the NA H275Y substitution in A(H1N1)pdm09 viruses, GISRS capacity is weighted towards molecular methodology for detection of this mutation. In order to bring in the additions to FluNet gradually, the AVWG agreed minimal additions, namely collection of aggregated data on viruses tested for the H275Y mutation, detailing the patient setting (hospital/community) and the genotype (H275/Y275). The value of data on antiviral treatment history, along with other clinical and epidemiological factors, in interpreting resistance data was recognised, but these fields were not added to the template for FluNet at the present time.
External quality assessment of antiviral susceptibility testing
External quality assessment is an important tool to monitor capabilities of laboratories in the GISRS network and to identify training needs. As a greater number of laboratories incorporate antiviral susceptibility testing methods into their surveillance capacity, it is important to ensure high data quality. In line with the recommended surveillance strategy of molecular screening for NA H275Y in A(H1N1)pdm09 and the proposed FluNet-based reporting of this data, the AVWG recommended the inclusion of a H275Y variant virus in the WHO PCR External Quality Assessment Project (EQAP) panels. The next EQAP will be in 2013, and a sample will be included whereby laboratories can use the molecular-based assay of their choice (e.g. real time reverse transcription PCR, pyrosequencing or NA sequencing) for the detection of the H275Y substitution.
Laboratories will not be scored on this antiviral test, but the data will be used to assess the sample preparation and cost and analysis implications, with a view to preparing a wider panel in future, incorporating further samples with different amino acid substitutions, as appropriate.
Practical guidance for establishing antiviral susceptibility testing
The AVWG agreed the content and format of a guidance document, including necessary practical information for laboratories considering implementing laboratory testing for antiviral surveillance.
Future objectives
The AVWG will continue to review its recommendations on methodology and surveillance strategies for GISRS to ensure that they remain appropriate. The main focus of the coming year will be to undertake the preparatory work for the inclusion of an oseltamivir-resistant (H275Y) A(H1N1)pdm09 virus in the next WHO PCR EQAP panel in 2013. The results of the antiviral testing in the EQAP will be evaluated and ways forward to expand or continue antiviral EQAP will be the subject of discussion in future meetings. The basic additions to FluNet will be evaluated and further developments of the database for antiviral data will be considered in the future. There will need to be constant appraisal of the criteria for categorization of levels of drug inhibition, to ensure these remain applicable as circulating influenza viruses evolve.
Issues surrounding the training of laboratories in antiviral testing methods will be an important subject of future meetings of the AVWG. Training needs to be targeted at a reasonable level, to laboratories with the capability to implement long-term testing and with financial security to purchase and maintain equipment and reagent stocks. The continued role of the AVWG will be to support the GISRS to meet the specific public health need for appropriate antiviral surveillance, through advice and recommendations on surveillance strategies, relevant and cost effective methods, valid data interpretation and operational aspects of GISRS.
- Summary of influenza antiviral susceptibility surveillance findings, September 2010 - March 2011. Geneva, World Health Organization, 2011 (http://www.who.int/influenza/gisrs_laboratory/antiviral_susceptibility/en/index.html, accessed September 2012).
- Bright RA et al. Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern. Lancet, 2005, 366: 1175?1181.
- Gubareva LV, Fry AM. Current challenges in the risk assessment of neuraminidase inhibitor-resistant influenza viruses. Journal of Infectious Diseases, 2010, 201(5): 656?658.
- Influenza A(H1N1) virus resistance to oseltamivir. Geneva, World Health Organization, 2008 (http://www.who.int/influenza/patient_care/antivirals/oseltamivir_summary/en/, accessed September 2012).
- No. 44, 2009, 84, 453?459.
- Hurt AC et al. Community transmission of oseltamivir-resistant A(H1N1)pdm09 influenza. New England Journal of Medicine, 2011, 365: 2541-2542.
- No. 17, 2011, 86, 166?169.
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