Re: Indonesia, Tangerang - Man, 18, Suspected H5N1 Bird Flu Died - Declared negative
Re: Indonesia, Tangerang - Man, 18, Suspected H5N1 Bird Flu Died - Declared negative
[Source: World Health Organization, full PDF document: (
LINK). Extract, edited.]
Weekly epidemiological record, Relev? ?pid?miologique hebdomadaire
20 january 2012, 87th year / 20 janvier 2012, 87e ann?e - No. 3, 2012, 87, 29?36 -
http://www.who.int/wer
Detection of influenza virus subtype A by polymerase chain reaction: WHO external quality assessment project summary analysis, 2011
Introduction
Global virological surveillance of influenza has been conducted through the WHO Global Influenza Surveillance and Response System (GISRS), formerly known as the Global Influenza Surveillance Network (GISN), for >50 years.(1) Currently 136 institutions in 106 countries are recognized by WHO as National Influenza Centres (NICs) which form the backbone of GISRS. The laboratory network also comprises 6 WHO Collaborating Centres, 4 essential regulatory laboratories and ad hoc groups established to address specific emerging issues.
GISRS monitors the evolution of influenza viruses and provides information about recommendations in areas including laboratory diagnosis, composition of vaccines, antiviral susceptibility and risk assessment. The GISRS laboratory network also serves as a global alert mechanism for the emergence of influenza viruses with pandemic potential.
During the past decade, an increasing number of NICs have adopted the polymerase chain reaction (PCR) as the principal method for both routine laboratory diagnosis and surveillance of seasonal influenza viruses and other emerging viruses, such as avian influenza A (H5N1) and the pandemic influenza A (H1N1) 2009 virus [hereafter A(H1N1)pdm09, to differentiate it from seasonal influenza A (H1N1) virus(2)]. WHO initiated an external quality assessment project in 2007 to monitor the quality and comparability of the performance of the participating laboratories.
Summaries of the performance of participating laboratories in response to the first 8 virus panels sent for analysis (2007?2010) have been reported in the Weekly Epidemiological Record.(3, 4, 5)
In 2011, the project continued under the coordination of the WHO Global Influenza Programme and was implemented by the H5 Reference Laboratory and National Influenza Centre at the Centre for Health Protection, China, Hong Kong Special Administration Region (SAR); it also received support from WHO Collaborating Centres for Reference and Research on Influenza, other H5 reference laboratories and WHO regional offices. This report summarizes the results of the assessments of virus panels 9 and 10, which were dispatched to the participating laboratories in 2011.
Preparation of virus panels
Vacuum-dried RNA samples of influenza A and influenza B, and γ-ray inactivated influenza A viruses, were dispatched to participating laboratories. RNA samples were prepared as described previously. (3, 4) Viruses were grown in Madin?Darby canine kidney (MDCK) cells, the culture fluid inactivated by γ-ray irradiation and then prepared as for the vacuum-dried RNA samples.
Composition of panels
Panel 9 and panel 10 consisted of 10 coded samples containing different concentrations of RNA from different genetic clades of A(H5N1), A(H1N1), A(H3N2), A(H1N1)pdm09 and influenza B viruses and 2 γ-ray inactivated A(H3N2) or A(H1N1) viruses. Samples that did not contain virus were included in both panels. Details of the composition of the panels are shown in Table 1. Participants were instructed to reconstitute each sample with the provided buffer prior to testing. A questionnaire on methods of detection and gene targets used by the laboratories was also included.
Distribution of panels and response of participants
NICs and other national influenza laboratories were invited to participate before the panels were dispatched. In 2011, panel 9 was dispatched during January?March, and panel 10 was dispatched during June?July. Both panels were dispatched from the H5 Reference Laboratory and National Influenza Centre at the Centre of Health Protection in Hong Kong SAR at ambient temperature by courier service to participating laboratories in each of the 6 WHO regions as previously described.(3, 4, 5)
(...)
Results for panel 9 were reported by 158 laboratories from 123 countries, areas and territories, and results for panel 10 were reported by 159 laboratories from 126 countries, areas and territories. The majority of participants received the panels within 1 week of dispatch (panel 9, 129/158 [81.6%]; panel 10, 130/159 [81.8%]).
Results
The criteria used to assess the performance of the participating laboratories in panels 9 and 10 were the same as those used for panels 1?8.(3, 4, 5)
Performance of laboratories
Panel 9
Of the 158 laboratories, 122 (77.2%) returned correct results for all of the 12 samples. An additional 7 (4.4%) participants returned 1 incorrect result and 29 (18.4%) returned >1 incorrect results (Table 2). Eight participating laboratories reported a positive result for negative sample(s) (sample 2011-02 or 2011-03) (Table 1); the false-positive rate was 5.1%. For the paired A(H5N1) RNA samples from genetic clade 2.3.2 (sample 2011-01 and 2011-07) with similar concentrations, 143 (90.5%) laboratories reported correct results. For the other paired samples containing different concentrations of clade 2.2 H5 virus (sample 2011-06 and 2011-10), 150 (94.9%) laboratories reported a correct test result for sample 2011-06, and 151 (95.6%) reported a correct test result for sample 2011-10.
For the paired A(H1N1)pdm09 RNA samples (sample 2011-04 and 2011-05) containing similar concentrations, 145 (94.3%) laboratories reported correct results for both samples.
For the A(H3N2) RNA samples (sample 2011-09) and influenza B RNA samples (sample 2011-08), 152 (96.2%) participants reported correct results.
For the γ-ray inactivated A(H1N1) (V01-2011) and A(H3N2) (V02-2011) samples, 146 (92.4%) laboratories reported correct results for both samples.
Panel 10
Of the 159 laboratories, 124 (78.0%) returned correct results for all the 12 samples. An additional 12 (7.5%) participants returned 1 incorrect result and 23 (14.5%) returned >1 incorrect results (Table 2).
(...)
Three participating laboratories reported a positive result for the negative sample (2011-19) (Table 1); the false-positive rate was 1.9%.
For the paired A(H5N1) RNA samples from genetic clade 2.3.2 (samples 2011-12 and 2011-18) containing similar concentrations, 147 (92.5%) laboratories reported correct results. For the other paired samples containing different concentrations of clade 2.3.4 H5 virus (sample 2011-14 and 2011-16), 147 (92.5%) laboratories reported a correct test result for sample 2011-14, and 150 (94.3%) reported a correct result for sample 2011-16.
For the paired A(H1N1)pdm09 RNA samples (sample 2011-17 and 2011-20) containing similar concentrations, 156 (98.1%) laboratories reported correct results for both samples.
For the A(H3N2) RNA sample (sample 2011-15), 155 (97.5%) participants reported correct results, while for the 2 lineage of influenza B RNA samples (sample 2011-11 and 2011-13), 155 (97.5%) participants reported correct results for both samples.
For the paired γ-ray inactivated A(H3N2) (V03-2011 and V04-2011) samples, 140 (88.1%) laboratories reported correct results for both samples.
Methods of detection
As seen previously, there was considerable variation in the testing strategies and PCR protocols used by participating laboratories to screen for influenza type A and type B viruses and subtype H1, H3 and H5 viruses.(3, 4, 5)
More than half of the participants used the protocols from the United States Centers for Disease Control and Prevention. There was little difference in test results despite the use of different PCR protocols.
Details on target genes, detection methods and source of primers/probes and enzymes used were included in the summary report of performance that was distributed to all participants.
Comparison of laboratory performance for all panels
From 2007 through 2011, the number of laboratories participating in external quality assessment, and which reported results in time to be included in the analysis, increased steadily from 64 in panel 1 to 158 in panel 8 and remained stable in panel 9 to panel 10 (ranged from 158 to 160). Although the percentage of laboratories with all correct results showed a small decrease in panel 9 (77%) and panel 10 (78%) as compared to panel 8 (86%), for H5 virus detection, the percentage of laboratories that reported all correct results increased from 77% in panel 1 to 91% in panel 8 and remained stable in performance in panel 9 (90%) and panel 10 (89%).
Factors affecting performance
Analysis of panel 9 and panel 10 results showed that:
i) the use of real-time PCR in ≥1 test for the H5 gene detection,
ii) the use of commercial diagnostic kits and
iii) the use of a second H5 assay did not have a significant effect on the laboratory performance for H5 gene detection.
Discussion
In the past 5 years, the number of laboratories participating in external quality assessment remained stable since the PCR examination of panel 8 viruses. Improvement was observed in the performance for A(H5N1) detection. While A(H5N1) viruses from different clades (2.2, 2.3.2, 2.3.4) were included in panel 9 and 10, the percentage of laboratories reporting all correct results for samples containing A(H5N1) virus remained high, at 90% in panel 9 and 89% in panel 10 (Figure 1). However, with the introduction of γ-ray inactivated virus samples that required an additional step of RNA extraction in panel 9 and panel 10, the number of participating laboratories which returned all correct results decreased from 86% in panel 8 to 77% and 78%, respectively.
The scope of EQAP has been expanded between 2007 and 2011. Panels 1 to 5 contained RNA from A(H1N1), A(H3N2) and A(H5N1) viruses. With the emergence of A(H1N1)pdm09virus in March 2009, the scope of EQAP was expanded to include the A (H1N1)pdm09 virus in panel 6. In 2010, to meet the demand of NICs, samples containing influenza B virus were included in panel 7 for the first time. In 2011, in order to assess the performance of RNA extraction, γ-ray inactivated virus samples were first included in panel 9. It is planned to replace RNA samples with γ-ray inactivated virus samples in panel 11. NICs, and designated national influenza laboratories in countries without NICs, are encouraged to continue their participation in the external quality assessment project, in order to benefit from monitoring of their performance, optimizing protocols and exchanging information. It is expected that this project will continue to strengthen the global capacity for rapid identification of influenza viruses.
(1) See
http://www.who.int/influenza/gisrs_laboratory/en/
(2) See
http://www.who.int/influenza/gisrs_laboratory/terminology_ah1n1pdm09/en/
(3) See No. 45, 2008, pp. 401?412.
(4) See No. 48, 2009, pp. 493?504.
(5) See No. 3, 2011, pp. 17?24.
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