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Wkly Epidemiol Rec. WHO external quality assessment project for detecting influenza virus subtype A by polymerase chain reaction ? summary analysis, 2009

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  • Wkly Epidemiol Rec. WHO external quality assessment project for detecting influenza virus subtype A by polymerase chain reaction ? summary analysis, 2009

    WHO external quality assessment project for detecting influenza virus subtype A by polymerase chain reaction ? summary analysis, 2009 (Wkly Epidemiol Rec., edited)

    [Source Full Free Document: LINK. EDITED.]

    Weekly epidemiological record - Relev? ?pid?miologique hebdomadaire

    27 NOVEMBER 2009, 84th YEAR / 27 NOVEMBRE 2009, 84e ANN?E
    No. 48, 2009, 84, 493?504 - http://www.who.int/wer


    WHO external quality assessment project for detecting influenza virus subtype A by polymerase chain reaction ? summary analysis, 2009


    Introduction

    National Influenza Centres (NICs) have been the backbone of WHO?s Global Influenza Surveillance Network for more than 50 years. The centres collect specimens, conduct preliminary analyses and send representative virus isolates in a timely manner to WHO collaborating centres to support annual recommendations for composition of influenza vaccine for the next season. The NICs also have a key role in detecting viruses with pandemic potential, thus facilitating responses to outbreaks and preparedness for pandemics.

    During the past decade, polymerase chain reaction (PCR) has become the principal laboratory test for detecting emerging viruses, such as avian influenza A (H5N1) in 2004 and the pandemic influenza A (H1N1) 2009 virus.

    To monitor the quality and comparability of laboratories? performance for diagnosing both seasonal influenza and avian influenza A (H5N1), a WHO external quality assessment project was launched in 2007.

    The performance of laboratories that participated in the fi rst 4 influenza test panels dispatched during a 2-year period (2007?2008) have already been summarized and published.1

    In 2009, the project continued under the coordination of WHO?s Global Influenza Programme, with implementation by the H5 Reference Laboratory and National Influenza Centre at the Centre for Health Protection, China, Hong Kong Special Administrative Region (SAR), and with support from collaborating centres on influenza, other H5 reference laboratories and regional offices.

    Since the pandemic (H1N1) 2009 virus emerged in Mexico in late March 2009, the scope of the quality assessment project was expanded so that the pandemic virus was included in panel 6 in addition to the influenza viruses dispatched between 2007 and early 2009 in the first 5 panels; the other influenza viruses included were A (H1N1), A (H3N2) and A (H5N1)

    To distinguish from seasonal infl uenza A (H1N1) viruses, the pandemic virus is referred to as pandemic (H1N1) 2009 virus.

    This report summarizes the results for panels 5 and 6 of the quality assessment project; these were dispatched during 2009, between January?February and June?August, respectively.


    Preparation of panels

    Panels sent to participating laboratories consisted of vacuum-dried RNA specimens extracted from influenza A (H1N1), A (H3N2), A (H5N1) and pandemic (H1N1) 2009 viruses. Preparation was performed as previously described.1


    Composition of panels

    As with panels 1?4, panels 5 and 6 consisted of coded samples containing different concentrations of RNA from 5 viruses representing different genetic clusters of clade 1 and clade 2 H5N1, H1N1 and H3N2 viruses. Samples that contained pandemic (H1N1) 2009 virus were first included in panel 6. Details of the composition of each panel are shown in Table 1.

    Participants were instructed to reconstitute each sample with the reconstitution buffer, which was provided, prior to testing. Additionally, a questionnaire was included to obtain information on detection methods used, target genes tested and primer and probe sequences used.


    Distribution of panels and response of participants

    NICs and other registered national influenza laboratories were invited to participate before the panels were dispatched. Panel 5 was dispatched between January 2009 and February 2009; panel 6 was dispatched between June 2009 and August 2009. All panels were dispatched from the H5 Reference Laboratory and National Influenza Centre at the Centre for Health Protection in Hong Kong SAR at ambient temperature by courier service to participating laboratories in all 6 WHO regions. Participating laboratories were requested to notify the centre immediately after receiving the panel, either by fax or e-mail, and to return their results within 1 month. A preliminary report, which included the correct results, was sent to participants shortly after the closing date for each panel.

    Results for panel 5 were reported by 114 laboratories from 90 countries, areas or territories; results for panel 6 were reported by 129 laboratories from 102 countries, areas or territories. The results found by one laboratory participating in panel 6 were excluded from analysis owing to prolonged shipment time. Details of participating laboratories are shown in Table 2. Most participants received their panels within 1 week (panel 5, 93/114 [82%]; panel 6, 110/128 [86%]). For both panel 5 and panel 6, 96/242 (40%) participating laboratories reporting results included in the analysis were in WHO?s European Region; 45/242 (19%) were in the Region of the Americas; 36/242 (15%) were in the African Region; 37/242 (15%) in the Western Pacific Region; 16 (7%) were in the Eastern Mediterranean Region; and 12 (5%) were in the South-East Asia Region.


    Results

    The assessment criteria were the same as for panels 1?4,1 except the following criteria were used to assess results for samples of pandemic (H1N1) 2009 virus:

    (i) failure to detect pandemic (H1N1) 2009 virus samples or reporting the results as non pandemic (H1N1) 2009 virus subtype, or both, was recorded as an incorrect response;

    (ii) failure to report non-H1 and non-H3 test results for pandemic (H1N1) 2009 virus samples if pandemic (H1N1) 2009 virus subtyping was not performed was recorded as an incorrect response;
    and

    (iii) reporting ?pandemic (H1N1) 2009 virus and H5? for H5 samples was recorded as a correct response, if the United States Centers for Disease Control and Prevention (CDC) real-time reverse transcriptase PCR (RT-PCR) swine H1 protocol2 was used (see
    Discussion section for additional details).


    Laboratories? performance

    Panel 5

    A total of 87 participants returned correct results for all 10 samples in Panel 5 (Table 3). An additional 12 participants returned correct results for 9 samples, and 14 participants had correct results for 5?8 samples. One participating laboratory returned correct results for <5 samples.

    Only 1 participating laboratory reported a positive result for the 1 negative sample (sample 2009-03); thus, the false-positive rate was 1% (Table 1).

    For the 2 samples with higher concentrations of H5, 111 (97%) laboratories correctly reported sample 2009-07; 110 (96%) correctly reported sample 2009-08. For the 2 samples with lower concentrations of H5, 106 (93%) laboratories correctly reported sample 2009-02; 102 (89%) correctly reported sample 2009-06. For the 2 samples with similar concentrations of H5, 107 (94%) laboratories correctly reported sample 2009-04; 106 (93%) correctly reported sample 2009-09. For the 2 samples with similar concentrations of H1, sample 2009-10 was correctly reported by 105 (92%) and sample 2009-01 was correctly reported by 108 (95%).

    The 1 H3 sample (2009-05) was correctly reported by 107 (94%) participating laboratories.


    Panel 6

    A total of 101 participants returned correct results for all 10 samples (Table 3). An additional 12 participants returned correct results for 9 samples, and 13 participants had correct results for 5?8 samples. Two participants returned correct results for <5 samples.

    There were 4 samples of H5: 125 (98%) laboratories correctly reported sample 2009-15; 117 (91%) correctly reported 2009-11; 119 (93%) correctly reported 2009-19; and 114 (89%) correctly reported 2009-17. Overall performance on the 256 tests of samples with higher concentrations of H5 (2009-15, 2009-11) was 242/256 reported correctly (95%); for samples with lower concentrations of H5 (2009-19, 2009-17), 233/256 (91%) were reported correctly. The H5 sample for the clade 2.2 strain with a higher concentration (2009-20) was correctly reported by 122 (95%); the clade 2.2 strain with a lower concentration (2009-14) was correctly reported by 119 (93%).

    The 1 sample of H1 (2009-13) was correctly reported by 127 (99%) laboratories; the 1 sample of H3 (2009-18) was correctly reported by 120 (94%).

    There were 2 samples of pandemic (H1N1) 2009 virus: 2009-12 was reported correctly by 126 (98%) and 2009-16 was correctly reported by 128 (100%). The overall performance on the pandemic (H1N1) 2009 samples was 254/256 correct (99%).

    The false-positive rate could not be obtained since no negative sample was included.


    Methods of detection

    There were considerable variations in the PCR protocols used by laboratories to subtype H1, H3 and H5 viruses; this was seen previously.1 There was not much difference in test results despite the use of different PCR protocols. To detect swine influenza A viruses, 23 (18%) laboratories reported using primers targeting the nucleoprotein gene. For specific detection of pandemic (H1N1) 2009 virus, at least 22 different references were cited; 91/128 (71%) participants used the CDC protocol. The details on target genes, detection methods and sources of the primers and probes were included in the summary that was distributed to all participants with the results of the analysis.


    Comparison of laboratories? performance for all panels

    During 2007?2009, the number of laboratories participating in external quality assessment and reporting results that could be included in analysis increased, from 64 to 128 (Fig. 1). The proportion of laboratories with entirely correct results also increased, from 67% to 79% (panel 1, 43/64 [67%]; panel 2, 54/83 [65%]; panel 3, 70/95 [74%]; panel 4, 84/109 [77%]; panel 5, 87/114 [76%]; panel 6, 101/128 [79%]). The proportion of laboratories that correctly subtyped H5 virus ranged from 77% to 81% (panel 1, 49/64 [77%]; panel 2, 66/83 [80%]; panel 3, 78/95 [82%]; panel 4, 96/109 [88%]; panel 5, 93/114 [82%]; panel 6, 105/128 [82%]). The proportion of laboratories that correctly assessed the first samples of pandemic (H1N1) 2009 virus in panel 6 was 124/128 (97%).


    Factors affecting performance

    For panels 5 and 6, analyses consistently showed that laboratories using real-time RT-PCR for >1 test to detect the H5 gene had significantly better overall performance (P<0.05) than laboratories using only conventional PCR. However, the use of commercial kits did not have a significant (P>0.05) effect on laboratories? overall performance.


    Discussion

    This external quality assessment programme allowed participants to tailor their testing methods to detect circulating influenza viruses, including H1N1, H3N2 and the highly pathogenic avian H5N1 viruses. Improvements in diagnostic performance for detecting seasonal H1N1 and H3N2 viruses were shown by the higher proportion of H1 and H3 samples correctly identified in panels 5 and 6 (92?99%) when compared with results from the previous 4 panels (85?89%). The diagnostic performance in detecting H5N1 viruses correlated well with the viral load in the panels? samples. For the 3 clades of H5 dispatched in panels 5 and 6 with 2 different concentrations of viral RNA, the samples with higher concentrations were correctly identifi ed by 93?98% of laboratories, while samples with lower concentrations were correctly identifi ed by 89?93%.

    The pandemic (H1N1) 2009 virus was detected previously by using a combination of positive results for influenza A virus with negative results for influenza subtypes H1N1, H3N2 and H5N1. Accurate diagnosis is critical in guiding public health actions. On 28 April 2009, the first full genome sequences of pandemic (H1N1) 2009 virus strain California/04/2009 became available on the United States? National Institutes for Health?s GenBank sequence database; additionally, a protocol using real-time RT-PCR to detect and characterize pandemic (H1N1) 2009 virus was provided by the United States CDC and made available on WHO?s web site. This protocol provides 3 targets for detecting and characterizing swine influenza: (i) the matrix M gene for universal detection of type-A influenza viruses; (ii) the nucleoprotein A/NP pandemic gene for specific detection of swine-origin influenza A viruses, including pandemic (H1N1) 2009 virus; and (iii) the haemagglutinin A/H1 pandemic gene for specific detection of pandemic (H1N1) 2009 virus.

    Results from panel 6 revealed that the A/NP pandemic gene may detect both pandemic (H1N1) 2009 virus and H5 samples.

    Because cross-reactivity had not been anticipated, any participants reporting H5 samples as ?pandemic (H1N1) 2009 virus and H5? were categorized as providing the correct response.

    Although pandemic (H1N1) 2009 virus was first included in panel 6, 124/128 (97%) laboratories correctly detected the pandemic virus in samples. The possible explanations for this might be that there is (i) high sequence identity within each gene segment of the pandemic (H1N1) 2009 virus, 2 (ii) most RT-PCR assays were developed and evaluated using the first publicly released sequences, and (iii) most laboratories used the CDC?s protocols.

    The results of panels 5 and 6 provided a follow-up assessment of laboratories? proficiency in diagnosing influenza A and showed that an increased proportion of laboratories is capable of correctly reporting all samples. NICs and national influenza laboratories in countries without NICs are encouraged to participate in the external quality assessment project to benefit from continual monitoring of laboratory performance. It is expected that this project will help achieve and maintain high-quality influenza diagnostic capacity globally.


    Editorial note.

    The full report for each panel contains more information than is presented in this summary. All data from the project will be used by the Global Influenza Surveillance Network to assess needs and plans for action.

    WHO would like to thank all NICs and other influenza laboratories for participating in this project, for the time they spent completing the questionnaires, and for their willingness to share information for this analysis.

    For more information, please contact WHO?s Global Influenza Programme (e-mail: GISN@who.int).
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    1 See No. 45, 2008, pp. 401?412.
    2 Garten RJ et al. Antigenic and genetic characteristics of swine-origin 2009 A (H1N1) influenza viruses circulating in humans. Science, 2009, 325:197?201.

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