I N S T A N D e. V. Gesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien e. V. (vormals Hämometerprüfstelle)

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1 I N S T A N D e. V. Gesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien e. V. (vormals Hämometerprüfstelle) WHO Collaborating Centre for Quality Assurance and Standardization in Laboratory Medicine in cooperation with Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten (DVV) Gesellschaft für Virologie (GfV) Deutsche Gesellschaft für Hygiene und Mikrobiologie (DGHM) EQAS Adviser: Assistant EQAS Adviser: Prof. Dr. Heinz Zeichhardt Dr. Hans-Peter Grunert CharitéCentrum für diagnostische und präventive Labormedizin Institut für Biotechnologische Diagnostik in der GBD Institut für Virologie, Campus Benjamin Franklin Potsdamer Chaussee 80, Berlin Hindenburgdamm 27, Berlin Tel.: +49-(0) ; Fax: +49-(0) Tel.: +49-(0) /23; Fax: +49-(0) HPGrunert@gmx.de Heinz.Zeichhardt@charite.de in cooperation with INSTAND-Geschäftsstelle Ubierstr Düsseldorf Telefon: +49 (0) Fax: +49 (0) instand@instand-ev.de Internet: Robert Koch-Institut Nationales Referenzzentrum für Influenza Dr. Brunhilde Schweiger Robert Koch-Institut Nordufer 20, Berlin Tel: , Fax: schweigerb@rki.de Friedrich-Loeffler-Institut Bundesforschungsinstitut für Tiergesundheit Nationales Referenzlabor für Aviäre Influenza PD Dr. Timm C. Harder Friedrich-Loeffler-Institut Bundesforschungsinstitut für Tiergesundheit Boddenblick 5a, Greifswald - Insel Riems Tel: , Fax: Timm.Harder@fli.bund.de August 2011 Final Report External Quality Assessment Scheme (EQAS) - March/April 2011 Virus Detection (Genome/Antigen) - Influenza A and B Viruses (Group 370) incl. Pandemic Influenza A(H1N1) 2009 Virus and Avian Influenza A(H5N1) Virus INSTAND-Target Value Laboratories: Charité - Universitätsmedizin Berlin, Campus Mitte, Institut für Medizinische Virologie, Nationales Konsiliarlaboratorium für Hantaviren: Prof. Dr. D. H. Krüger, PD Dr. J. Hofmann Klinikum der Johann Wolfgang Goethe-Universität, Institut für Medizinische Virologie, Frankfurt/Main: Prof. Dr. H. W. Doerr, Prof. Dr. H. Rabenau, PD Dr. A. Berger, Dr. R. Allwinn Labor Enders, Institut für Virologie, Infektiologie und Epidemiologie, Stuttgart: Prof. Dr. Gisela Enders & Partner Niedersächsisches Landesgesundheitsamt, Fachbereich Virologie, Hannover: Dr. A. Baillot Paul-Ehrlich-Institut, Bundesinstitut für Impfstoffe und biomedizinische Arzneimittel, WHO Collaborating Centre for Quality Assurance of Blood Products and in vitro Diagnostic Devices, Abt. Virologie, FG Molekulare Virologie, Langen: PD Dr. M. Nübling, Dr. M. Chudy Philipps Universität Marburg, Institut für Virologie, Nationales Konsiliarlaboratorium für Filoviren: Prof. Dr. S. Becker, Dr. M. Eickmann Uniklinik Köln, Institut für Virologie, Nationales Referenzzentrum für Papillom- und Polyomaviren: Prof. Dr. H. Pfister, Prof. Dr. U. Wieland, Dr. R. Kaiser Universität Duisburg-Essen, Universitätsklinikum Essen, Institut für Virologie, Nationales Referenzzentrum für Hepatitis-C-Viren, Nationales Konsiliarlaboratorium für Tollwut: Prof. Dr. M. Roggendorf, Prof. Dr. S. Ross Universitätsklinikum des Saarlandes, Institut für Virologie, Homburg/Saar; Prof. Dr. S. Smola, Prof. Dr. N. Müller-Lantzsch Universitätsklinikum Jena, Institut für Virologie und Antivirale Therapie, Nationales Konsiliarlaboratorium für HSV und VZV: Prof. Dr. A. Sauerbrei, Prof. Dr. P. Wutzler 370 Influenza March April 2011 Letter doc 1

2 Dear colleagues, Attached please find the final report for the EQA scheme of March/April 2011 for virus detection (genome/ antigen) - influenza A and B viruses incl. pandemic influenza A(H1N1) 2009 virus and avian influenza A(H5N1) virus. You receive your statement of participation and statement of your individual and - in case you succeeded in the EQA scheme - your certificate of successful participation (validity period: 12 months). The report for this EQA scheme is also available as PDF file on the INSTAND-Homepage under EQAS / Reports / Year and Category (Virus genome detection) in English language ( ) and German language ( ). Please note that the number of samples was reduced to 6 samples due to the current situation after the pandemic with influenza A(H1N1) 2009 virus. Summary In total 171 laboratories participated in this influenza EQA scheme which was performed after the pandemic with influenza A(H1N1) 2009 virus. Please see for details on the postpandemic phase given in the WHO declaration of 10 August For details on the current epidemiological situation in Germany please see "Arbeitsgemeinschaft für Influenza (AGI) am Robert Koch-Institut" Detection of pandemic influenza A(H1N1) 2009 virus One sample with pandemic influenza A(H1N1) 2009 virus was analyzed in this EQA scheme: - sample , vaccine strain A/California/7/2009 (H1N1), 1:80 diluted. All applied qualitative genome detection tests for pandemic influenza A(H1N1) 2009 virus revealed success rates of 100 each for this sample for the following test categories: - qualitative genome detection of influenza A viruses (w/o differentiation) (test category 10) - combined qualitative genome detection of influenza A and B viruses (test category 25) - qualitative genome detection of pandemic influenza A(H1N1) 2009 virus (with primers/probes specific for H1N1 or H1) (test category 13) - subtyping of influenza A viruses (test category 11) In contrast, the of rapid tests for antigen detection of influenza A (test category 30) showed a reduced success rate of 73.5 for this sample. Please note that reporting of the "positive" and "borderline" was accepted as result for test category 30. Considering also the result "borderline" ensured that this sample positive for pandemic influenza A(H1N1) 2009 virus was not misinterpreted as negative. The reduced success rate reflects the reduced analytical sensitivity of rapid tests for antigen detection in comparison to genome detection tests. Detection of avian influenza A(H5N1) virus One sample with avian influenza A(H5N1) virus was analyzed in this EQA scheme: - sample , strain A/chicken/Germany/R3294/2007 (H5N1), 1:100 diluted, chemically inactivated. All applied qualitative genome detection tests for the detection of this virus revealed success rates between 90.3 and 94.7 for the test categories 10, 25, 12 and 11. The of rapid tests for antigen detection of influenza A (test category 30) showed satisfactory success rates of Please note that reporting of the "positive" and "borderline" was accepted as result for test category 30. Considering also the result "borderline" ensured that this sample positive for pandemic influenza A(H1N1) 2009 virus was not misinterpreted as negative. Detection of seasonal influenza A(H3N2) and influenza A(H1N1) viruses Two samples with different seasonal influenza A virus s were analyzed in this EQA scheme: - sample , vaccine strain A/Brisbane/59/2007 (H1N1), 1:160 diluted, - sample , vaccine strain A/Perth/16/2009 (H3N2), 1:240 diluted. Applying qualitative genome detection tests for influenza A virus revealed success rates between 99.3 and 100 for these two samples for test categories 10, 25 and 11. The of rapid tests for antigen detection of influenza A (test category 30) showed satisfactory success rates of 100 each for both samples. Please note that only "positive" was accepted as result for both samples. Detection of seasonal influenza B virus One samples with seasonal influenza B virus were analyzed in this EQA scheme: - sample , vaccine strain B/Brisbane/60/2008, 1:5 diluted. Applying qualitative genome detection tests for influenza B virus (test categories 20 and 25) as well as rapid tests for antigen detection of influenza B (test category 40) revealed success rates of 100 each. Sample negative for influenza A and B viruses The success rates obtained with all applied test categories for the negative sample were between 98.6 and 100. Please see section "references" at the end of this report for further updated information. 370 Influenza March April 2011 Letter doc 2

3 1. Sample properties Sample No. Influenza virus Type Subtype Strain Dilution Origin pandemic influenza A H1N vaccine strain A/California/7/2009 1:80 infected cells (lysate) negative for influenza A and B non-infected cells (lysate) seasonal influenza A H1N1 vaccine strain A/Brisbane/59/2007 1:160 infected cells (lysate) seasonal influenza B vaccine strain B/Brisbane/60/2008 1:5 infected cells (lysate) avian influenza A H5N1 A/chicken/Germany/R3294/2007 1:100 allantoic fluid (chem. inactivated) seasonal influenza A H3N2 vaccine strain A/Perth/16/2009 1:240 infected cells (lysate) Please note: The samples for the influenza EQASs are not suitable for virus cultivation (e.g. shell vial technique) or antigen detection by immune fluorescence tests. The samples are either cell homogenates or allantoic fluids of embryonated eggs chemically inactivated. 2. Test categories and evaluations test categories Genome detection of influenza A and B viruses Qualitative genome detection of influenza A viruses (w/o diff.) (10) Qualitative genome detection of influenza B viruses (20) Combined qualitative genome detection of influenza A and B viruses (25) Qualitative genome detection of pandemic influenza A(H1N1) 2009 virus (with primers/probes specific for H1N1 or H1) (13) Qualitative genome detection of avian influenza A(H5N1) virus (with primers/probes specific for H5N1 or H5) (12) Subtyping of influenza A viruses (11) Quantitative genome detection of influenza A viruses (5) Quantitative genome detection of influenza B viruses (15) Antigen detection of influenza A and B viruses Qualitative antigen detection of influenza A viruses (30) Qualitative antigen detection of influenza A viruses (40) criteria for receiving a certificate (min. no. of ly determined samples) at least 6 of 6 samples at least 6 of 6 samples at least 6 of 6 samples at least 6 of 6 samples at least 6 of 6 samples at least 6 of 6 samples without evaluation without evaluation at least 6 of 6 samples at least 6 of 6 samples A result was not considered for evaluation in case you had specified that this result should only be taken as additional information and ignored as valid result. For receiving a certificate of successful participation it is required that you analyzed all 6 samples of the sample set ly with the same method in the corresponding test categories (100 ). The evaluation criteria for the of EQA schemes in virology follow the new Guideline of the German Medical Association (Bundesärztekammer/ RiliBÄK = Richtlinie der Bundesärztekammer zur Qualitätssicherung laboratoriumsmedizinischer Untersuchungen). Please see section B2 of the RiliBÄK "qualitative determinations in laboratory medicine = Qualitative laboratoriumsmedizinische Untersuchungen" (effective since with a transition period of two years; Deutsches Ärzteblatt, Jg. 108, Heft 30, , Seite A ). Section B3 of the RiliBÄK "direct detection and characterization of infectious diseases pathogens = Direkter Nachweis und Charakterisierung von Infektionserregern" is in progress. 3. Results The following table 1 comprises a summary of "ly" evaluated and gives details on the number of analyses as well as the success rates for the qualitative genome detection tests and rapid tests for antigen detection for each sample. Please note that different color codes have been used to discern seasonal influenza A and B, pandemic influenza A(H1N1) 2009 and avian influenza A(H5N1) types/s. Additionally a success rate for all 6 samples of the sample set is given for each of the corresponding test categories. These success rates refer to the number of participating laboratories. Laboratories having reported obtained by several methods are recorded only once in the corresponding test category. 370 Influenza March April 2011 Letter doc 3

4 Table 1: Qualitative detection of influenza A and B viruses Detected virus Test categories Sample Sample Sample Sample Sample Sample Sample pandemic influenza A virus (H1N1) 2009 (1:80 diluted) negative for influenza A and B viruses seasonal influenza A virus (H1N1) (1:160 diluted) seasonal influenza B virus (1:5 diluted) considered as "" avian influenza A virus (H5N1) (1:100 diluted) seasonal influenza A virus (H3N2) (1:240 diluted) Success rates for all 6 samples of the sample set & number of analyses with /total number of analyses (success rate in ) Influenza A virus positive negative positive negative positive positive Genome detection (10) 144/144 (100) 142/144 (98.6) 143/144 (99.3) 144/144 (100) 130/144 (90.3) 144/144 (100) Influenza B virus negative negative negative positive negative negative Genome detection (20) 125/125 (100) 125/125 (100) 125/125 (100) 125/125 (100) 123/125 (98.4) 124/125 (99.2) Influenza A/B v. combined positive negative positive positive positive positive Genome detection (25) 25/25 (100) 25/25 (100) 25/25 (100) 25/25 (100) 23/25 (92.0) 25/25 (100) Pandemic influenza A(H1N1) 2009 virus Genome detection with primers/probes specific for H1N1 or H1 (13) 89.4 (126/141) 98.4 (120/122) 92.0 (23/25) /143 (100) 143/143 (100) 143/143 (100) 143/143 (100) 142/143 (99.3) 143/143 (100) (136/137) Genome detection with primers/probes specific for 44/44 (100) 44/44 (100) 44/44 (100) 44/44 (100) 41/44 (93.2) 44/44 (100) 93.2 (41/44) Avian influenza A(H5N1) virus nd # nd # nd # nd # H5N1 / H5 nd # H5N1 or H5 (12) Influenza A H1N1 2009/H nd # H1N1 / H1 nd # H5N1 / H5 H3N2 / H3 Genome detection (11) 19/19 (100) 19/19 (100) 19/19 (100) 19/19 (100) 18/19 (94.7) 19/19 (100) Influenza A virus positive/borderline negative positive negative positive positive Antigen detection (30) 50/68 (73.5) 68/68 (100) 68/68 (100) 68/68 (100) 67/68 (98.5) 68/68 (100) Influenza B virus negative negative negative positive negative negative Antigen detection (40) 68/68 (100) 68/68 (100) 68/68 (100) 68/68 (100) 68/68 (100) 68/68 (100) # & nd = For samples and the reporting of "borderline" was accepted as additional result for tests for antigen detection of influenza A virus (in general rapid tests). Considering also the result "borderline" ensured that these samples positive for pandemic influenza A(H1N1) 2009 virus was not misinterpreted as negative. The success rates for all 6 samples of the sample set refer to the number of participating laboratories. Laboratories having reported obtained by several methods are recorded only once in the corresponding test category (18/19) 73.1 (49/67) 100 (67/67) 370 Influenza March April 2011 Letter doc 4

5 3.1. Qualitative detection of influenza A viruses Detection of pandemic influenza A(H1N1) 2009 virus The for sample positive for pandemic influenza A(H1N1) 2009 virus (vaccine strain A/California/7/2009) is differentiated according to the applied test categories (figure 1). Correct (positive ) General genome detection infl. A (10) Combined genome detection infl. A/B (25) Spec. genome detection pand. infl. A(H1N1) 2009 (13) Genome detection for subtyping infl. A (11) Antigen detection infl. A (30) No. of analyses N negative borderline positive; sample (1:80 dil.) Figure 1: Qualitative detection of pandemic influenza A(H1N1) 2009 virus Genome detection of influenza A viruses (without differentiation of s) (10) and combined genome detection of influenza A and B viruses (25) The of test category (10), screening PCRs for general genome detection of influenza A viruses (without differentiation), and test category (25), combined genome detection of influenza A and B viruses (without type differentiation), are summarized in table 1 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/section 4.1). For sample (1:80 diluted) 84/144 analyses for screening PCRs for general genome detection of influenza A viruses (without differentiation) (10) were performed with commercial test systems. The were as follows (figure 1): Sample (1:80 diluted) 144/144 (100) All applied screening PCRs for combined genome detection of influenza A and B viruses (without type differentiation) (25) were commercial tests (25/25 analyses each). Applying these tests led to the following (figure 1): Sample (1:80 diluted) 25/25 (100) 370 Influenza March April 2011 Letter doc 5

6 Genome detection of pandemic influenza A(H1N1) 2009 virus (with primers/probes specific for H1N1 or H1) (13) The of test category (13), specific genome detection exclusively for pandemic influenza A(H1N1) 2009 virus, are summarized in table 1 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/section 4.2). For sample (1:80 diluted) 90/143 analyses were performed with commercial test systems for the specific genome detection of pandemic influenza A(H1N1) 2009 virus (13). The were as follows (figure 1): Sample (1:80 diluted) 143/143 (100) In addition these tests showed a high reliability for the differentiation of pandemic influenza A(H1N1) 2009 virus from seasonal influenza A(H1N1) virus (sample ), seasonal influenza A(H3N2) virus (sample ) and avian influenza A(H5N1) virus (sample ) analyzed in this EQA scheme. Sample was falsely detected as pandemic influenza A(H1N1) 2009 virus in only 1/143 analyses (0.7) when a test for specific genome detection exclusively for pandemic influenza A(H1N1) 2009 virus was applied(see table 1) Genome detection for subtyping of influenza A viruses (11) The reported are assigned to test category (11), genome detection for subtyping of influenza A viruses, if a laboratory analyzed each sample on s of influenza A virus. These are summarized in table 1 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.4). For sample (1:80 diluted) the majority of specific RT-PCRs applied for subtyping of influenza A positive samples were in house tests (18/19 analyses each). Sequencing were not reported. The were as follows (figure 1): Sample (1:80 diluted) 19/19 (100) Antigen detection of influenza A viruses (30) The of test category (30), antigen detection of influenza A viruses, are summarized in table 1 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.5). For sample (1:80 diluted) the reporting of "borderline" was accepted as additional result for tests for antigen detection of influenza A virus (in general rapid tests). Considering also the result "borderline" ensured that this sample positive for pandemic influenza A(H1N1) 2009 virus was not misinterpreted as negative. For this sample all in test category (30) were achieved with commercial rapid tests (68/68 analyses). The summarized were as follows (figure 1): Sample (1:80 diluted) 50/68 (73.5) The for sample (1:80 diluted) thus led to 26.5 (18/68 analyses) false negative. These demonstrate the lower sensitivity of the applied rapid tests for antigen detection in comparison to the virus genome detection tests (figure 1). Please note that the commercial rapid tests revealed different sensitivities with the differently diluted samples (see tables with detailed according to test formats, manufacturers and names of test kits/section 4.5). 370 Influenza March April 2011 Letter doc 6

7 Detection of avian influenza A(H5N1) virus The for sample positive for avian influenza A(H5N1) virus (strain A/chicken/Germany/R3294/ 2007(H5N1) virus, 1:100 diluted, chemical inactivated) are differentiated according to the applied test categories (figure 2). Correct (positive ) General genome detection infl. A (10) Combined genome detection infl. A/B (25) Spec. genome detection avian infl. A(H5N1) (12) Genome detection for subtyping infl. A (11) Antigen detection infl. A (30) No. of analyses N negative borderline positive; sample (1:100 dil.) Figure 2: Qualitative detection of avian influenza A(H5N1) virus Genome detection of influenza A viruses (without differentiation of s) (10) and combined genome detection of influenza A and B viruses (25) The of test category (10), screening PCRs for general genome detection of influenza A viruses (without differentiation), and test category (25), combined genome detection of influenza A and B viruses (without type differentiation), are summarized in table 1 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/section 4.1). For sample (1:100 diluted) 84/144 analyses were performed with commercial test systems for screening PCRs for the general genome detection of influenza A viruses (without differentiation) (10). The were as follows (figure 2): Sample (1:100 diluted) 130/144 (90.3) All applied screening PCRs for combined genome detection of influenza A and B viruses (without type differentiation) (25) were commercial tests (25/25 analyses). Applying these tests led to the following (figure 2): Sample (1:100 diluted) 23/25 (92.0) Genome detection of avian influenza A(H5N1) virus (with primers/probes specific for H5N1 or H5) (12) The of test category (12), specific genome detection exclusively for avian influenza A(H5N1) virus, are summarized in table 1 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/section 4.3). For sample (1:100 diluted) 28/44 analyses for the specific genome detection of avian influenza A(H5N1) virus were performed with in house test systems and 16/44 analyses were performed with commercial test systems. The were as follows (figure 2): Sample (1:100 diluted) 41/44 (93.2) 370 Influenza March April 2011 Letter doc 7

8 In addition these tests showed a high reliability for the differentiation of avian influenza A(H5N1) virus from seasonal influenza A(H1N1) virus (sample ), seasonal influenza A(H3N2) virus (sample ) and pandemic influenza A(H1N1) 2009 virus (sample ) analyzed in this EQA scheme. No false positive were obtained for the detection of heterologous influenza A and B viruses (see table 1) Genome detection for subtyping of influenza A viruses (11) The reported are assigned to test category (11), genome detection for subtyping of influenza A viruses, if a laboratory analyzed each sample on s of influenza A virus. These are summarized in table 1 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.4). For sample (1:100 diluted) the majority of specific RT-PCRs applied for subtyping of influenza A positive samples were in house tests (18/19 analyses). Sequencing were not reported. The for this sample were as follows (figure 2): Sample (1:100 diluted) 18/19 (94.7) Antigen detection of influenza A viruses (30) The of test category (30), antigen detection of influenza A viruses, are summarized in table 1 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.5). For sample (1:100 diluted) the reporting of "borderline" was accepted as additional result for tests for antigen detection of influenza A virus (in general rapid tests). Considering also the result "borderline" ensured that this sample positive for avian influenza A(H5N1) virus was not misinterpreted as negative. For sample (1:100 diluted) all in test category (30) were achieved with commercial rapid tests (68/68 analyses). The were as follows (figure 2): Sample (1:100 diluted) 67/68 (98.5) Detection of seasonal influenza A viruses The for samples and positive for seasonal influenza A viruses (sample , vaccine strain A/Brisbane/59/2007(H1N1), 1:160 diluted; sample , vaccine strain A/Perth/16/2009(H3N2), 1:240 diluted) are differentiated according to the applied test categories (figure 3) Genome detection of influenza A viruses (without differentiation of s) (10) and combined genome detection of influenza A and B viruses (25) The of test category (10), screening PCRs for general genome detection of influenza A viruses (without differentiation), and test category (25), combined genome detection of influenza A and B viruses (without type differentiation), are summarized in table 1 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/section 4.1). In total 84/144 analyses were performed for both samples with commercial test systems for screening PCRs for general genome detection of influenza A viruses (without differentiation) (10). The for each of the samples were as follows (figure 3): Sample (A(H1N1) virus, 1:160 diluted) 143/144 (99.3) Sample (A(H3N2) virus, 1:240 diluted) 144/144 (100) All applied screening PCRs for combined genome detection of influenza A and B viruses (without type differentiation) (25) were commercial tests (25/25 analyses each). Applying these tests led to the following (figure 3): Sample (A(H1N1) virus, 1:160 diluted) 25/25 (100) Sample (A(H3N2) virus, 1:240 diluted) 25/25 (100) 370 Influenza March April 2011 Letter doc 8

9 Correct (positive ) General genome detection infl. A (10) Combined genome detection infl. A/B (25) Genome detection for subtyping infl. A (11) Antigen detection infl. A (30) N No. of analyses negative borderline positive; sample (1:160 diluted), A(H1N1) positive; sample (1:240 diluted), A(H3N2) Figure 3: Qualitative detection of seasonal influenza A viruses Genome detection for subtyping of influenza A viruses (11) The reported are assigned to test category (11), genome detection for subtyping of influenza A viruses, if a laboratory analyzed each sample on s of influenza A virus. These are summarized in table 1 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.4). For samples (H1N1, 1:160 diluted) and (H3N2, 1:240 diluted) the majority of specific RT-PCRs applied for subtyping of influenza A positive samples were in house tests (18/19 analyses each). Sequencing were not reported. The for each of the samples were as follows (figure 3): Sample (A(H1N1) virus, 1:160 diluted) 19/19 (100) Sample (A(H3N2) virus, 1:240 diluted) 19/19 (100) Antigen detection of influenza A viruses (30) The of test category (30), antigen detection of influenza A viruses, are summarized in table 1 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.5). For samples (H1N1, 1:160 diluted) and (H3N2, 1:240 diluted) all in test category (30) were achieved with commercial rapid tests (68/68 analyses each). The summarized for each of the samples were as follows (figure 3): Sample (A(H1N1) virus, 1:160 diluted) 68/68 (100) Sample (A(H3N2) virus, 1:240 diluted) 68/68 (100) 370 Influenza March April 2011 Letter doc 9

10 3.2. Qualitative detection of influenza B virus Detection of seasonal influenza B virus The for sample are differentiated according to the applied test categories (figure 4). Correct (positive ) General genome detection infl. B (20) Combined genome detection infl. A/B (25) Antigen detection infl. B (40) No. of analyses N negative borderline positive; sample (1:5 dil.) Figure 4: Qualitative detection of seasonal influenza B viruses Genome detection of influenza B viruses (20) and combined genome detection of influenza A and B viruses (25) The of test category (20), PCRs for genome detection of influenza B viruses, and test category (25), combined genome detection of influenza A and B viruses, are summarized in table 1 (please see also the tables with detailed according to test formats and manufacturers/section 4.1). In total 66/125 analyses for the genome detection of influenza B viruses (20) were performed with commercial test systems. The were as follows (figure 4): Sample (1:5 diluted) 125/125 (100) All applied screening PCRs for combined genome detection of influenza A and B viruses (without type differentiation) (25) were commercial tests (25/25 analyses). Applying these tests led to the following (figure 4): Sample (1:5 diluted) 25/25 (100) Antigen detection of influenza B viruses (40) All in test category (40), antigen detection of influenza B virus, were achieved with commercial rapid tests (68/68 analyses). These are summarized in table 1 (please see also the tables with detailed according to test formats and manufacturers/section 4.5). The were as follows (figure 4): Sample (1:5 diluted) 68/68 (100) 370 Influenza March April 2011 Letter doc 10

11 3.3. Qualitative detection of influenza A and B viruses - specificity Analysis of sample negative for influenza A and B viruses The for sample negative for influenza A and B viruses showed success rates between 98.6 and 100 negative. These are summarized in table 1 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/sections ). This reflects that all applied tests of the different test categories were reliably in respect to specificity Quantitative detection of influenza A and B viruses In total six participants reported quantitative for the genome detection of influenza A and B viruses. These are summarized in the following table 2 and are not evaluated (without any disadvantage for the certificate) due to the low number of analyses and the high level of variance. Table 2: Quantitative genome detection - without evaluation Testformat / manufacturer - name of test kit Participating laboratories (TN-No.) TaqMan / in house Sample Sample Sample Sample Sample Sample pandemic influenza A virus (H1N1) 2009 (1:80 dil.) copies/ml negative for influenza A and B viruses copies/ml seasonal influenza A virus (H1N1) (1:160 dil.) copies/ml seasonal influenza B virus (1:5 dil.) copies/ml avian influenza A virus (H5N1) (1:100 dil.) copies/ml seasonal influenza A virus (H3N2) (1:240 dil.) copies/ml TN TN TN TN 4297 ND ND ND ND ND LightCycler / TIB MolBiol - LightMix Kit Influenza A M2 TN LightCycler / in house TN ND = A detailed description of the for all samples including a differentiation according to the test formats and manufacturers is given in the tables attached to this report and structured as follows: 4. Tables including differentiation according to test formats and manufacturers 4.1. Qualitative genome detection of influenza A and B viruses (categories 10, 20 and 25) 4.2. Qualitative genome detection of pandemic influenza A(H1N1) 2009 virus (with primers/probes specific for H1N1 or H1) (category 13) 4.3. Qualitative genome detection of avian influenza A(H5N1) virus (with primers/probes specific for H5N1 or H5) (category 12) 4.4. Subtyping of influenza A viruses (category 11) 4.5. Qualitative antigen detection of influenza A and B viruses (categories 30 and 40) We gratefully acknowledge the good cooperation with Dr. Brunhilde Schweiger and her team (National Reference Center for Influenza at Robert Koch-Institute, Berlin, Germany) as well as with the INSTAND- Target Value Laboratories. Prof. Dr. H. Zeichhardt 370 Influenza March April 2011 Letter doc 11

12 References: CDC, 2011, Centers for Disease Control and Prevention (CDC), "Clinical Description & Lab Diagnosis of Influenza", DGPI, 2009, Deutsche Gesellschaft für pädiatrische Infektiologie (DGPI), " Aktualisierte Empfehlung der DGPI zur Diagnostik, Therapie und Prophylaxe der Infektion mit dem Neuen Influenza A/H1N1v-Virus bei Kindern und Jugendlichen", (Stand: ). ECDC, European Center for Disease Prevention and Control, Health Topics, Influenza Gesellschaft für Virologie, "Influenza aktuell - Stellungnahme der Gesellschaft für Virologie (GfV): Fragen und Antworten zur aktuellen Influenzasituation (echte Grippe) und zur Influenzaimpfung in Deutschland" (Stand ) OIE, World Organisation for Animal Health, Web portal on Avian Influenza RKI, Arbeitsgemeinschaft Influenza (gibt während der Influenza-Saison aktuelle Wochen- und Saisonberichte sowie eine Übersicht zu zirkulierenden Viren), RKI, RKI-Ratgeber Infektionskrankheiten Merkblätter für Ärzte, Influenza (Saisonale Influenza, Influenza A(H1N1) 2009, Aviäre Influenza), Mbl Influenza.ht ml (Stand: ) RKI, "Nachweis von H5N1-Viren (aviäre Influenza)", H5N1.html (Stand: ) WHO, 2010, "WHO recommendations for the post-pandemic period, (Stand: ) WHO, Global Alert and Response (GAR), Avian Influenza WHO, Global Alert and Response (GAR), Avian Influenza, Recommendations and laboratory procedures for detection of avian influenza A(H5N1) virus in specimens from suspected human cases, revised August 2007, Influenza March April 2011 Letter doc 12

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14 INSTAND e. V., Gesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien e. V. (vormals Hämometerprüfstellen) - in Zusammenarbeit mit Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten (DVV) Gesellschaft für Virologie (GfV) Deutsche Gesellschaft für Hygiene und Mikrobiologie (DGHM) 4.1 Qualitativer Genomnachweis von Influenza A- und B-Viren - Genomnachweis von Influenza A-Viren (ohne Subtyp-Differenzierung) (10) - Genomnachweis von Influenza B-Viren (20) - Kombinierter Genomnachweis von Influenza A- und B-Viren (25) 4.1 Qualitative genome detection of influenza A and B viruses - Genome detection of influenza A viruses (without differentiation of s) (10) - Genome detection of influenza B viruses (20) - Combined genome detection of influenza A and B viruses (25) 370 Influenza 10_20_25 Qual Genomnachweis Influenza A- und B-Viren Deckblatt doc

15 Gesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien e. V. (INSTAND) in Zusammenarbeit mit Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten e. V. (DVV) Gesellschaft für Virologie e. V. (GfV) Deutsche Gesellschaft für Hygiene und Mikrobiologie e. V. (DGHM) Ringversuch Virologie März 2011 PCR-/NAT u. AG Influenza A/B (370) Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza A-Virus : positiv gesamter Test Gesamt Positiv Grenzw. Negativ Quote PCR (11) eigene Herstellung nested PCR (12) eigene Herstellung Taq Man (13) eigene Herstellung Light Cycler (40) Roche - RealTime ready Influenza A/H1N eigene Herstellung TIB MolBiol - LightMix Kit Influenza A M astra Diagnostics - Influenza Screen&Type PIIM - AmpliGnost Influenza-Virus A/B TIB MolBiol - LightMix Kit InfA M2 & H1 sw Mikrogen - dia Human influenza A/B Argene - Flu A/B r-gene Multiplex PCR (70) Fast-track - FTD respiratory pathogens eigene Herstellung GenID - CAP Vir GenID - Influenza-typing Multipl. Taq Man (75) astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU CvirG / LgesQ Rv= :18h Bl. 1

16 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza B-Virus : negativ gesamter Test Gesamt Positiv Grenzw. Negativ Quote PCR (11) eigene Herstellung nested PCR (12) eigene Herstellung Taq Man (13) eigene Herstellung Light Cycler (40) eigene Herstellung Roche - RealTime ready Influenza B TIB MolBiol - LightMix Kit Influenza B astra Diagnostics - Influenza Screen&Type PIIM - AmpliGnost Influenza-Virus A/B Mikrogen - dia Human influenza A/B Argene - Flu A/B r-gene Multiplex PCR (70) eigene Herstellung sonstiger Hersteller Fast-track - FTD respiratory pathogens GenID - Influenza-screening GenID - CAP Vir Multipl. Taq Man (75) astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU Qualitative Ergebnisse für Probe Kombinierter qualitativer Genomnachweis von Influenza A und B : positiv gesamter Test Gesamt Positiv Grenzw. Negativ Quote Taq Man (13) CvirG / LgesQ Rv= :18h Bl. 2

17 Qualitative Ergebnisse für Probe Kombinierter qualitativer Genomnachweis von Influenza A und B Grp. 370 Light Cycler (40) Qiagen - artus Infl./H1 LC/RG RT-PCR Kit astra Diagnostics - Influenza Screen&Type PIIM - AmpliGnost Influenza-Virus A+B Qiagen - artus Influenza LC RT-PCR Kit Qiagen - artus Influenza/H5 LC RT-PCR Kit andere PCR (98) Qiagen - artus Infl./H1 LC/RG RT-PCR Kit CvirG / LgesQ Rv= :18h Bl. 3

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19 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza A-Virus : negativ gesamter Test Gesamt Positiv Grenzw. Negativ Quote PCR (11) eigene Herstellung nested PCR (12) eigene Herstellung Taq Man (13) eigene Herstellung Light Cycler (40) Roche - RealTime ready Influenza A/H1N eigene Herstellung TIB MolBiol - LightMix Kit Influenza A M astra Diagnostics - Influenza Screen&Type PIIM - AmpliGnost Influenza-Virus A/B TIB MolBiol - LightMix Kit InfA M2 & H1 sw Mikrogen - dia Human influenza A/B Argene - Flu A/B r-gene Multiplex PCR (70) eigene Herstellung Fast-track - FTD respiratory pathogens GenID - CAP Vir sonstiger Hersteller GenID - Influenza-typing Multipl. Taq Man (75) astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU CvirG / LgesQ Rv= :18h Bl. 4

20 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza B-Virus : negativ gesamter Test Gesamt Positiv Grenzw. Negativ Quote PCR (11) eigene Herstellung nested PCR (12) eigene Herstellung Taq Man (13) eigene Herstellung Light Cycler (40) eigene Herstellung Roche - RealTime ready Influenza B TIB MolBiol - LightMix Kit Influenza B astra Diagnostics - Influenza Screen&Type PIIM - AmpliGnost Influenza-Virus A/B Mikrogen - dia Human influenza A/B Argene - Flu A/B r-gene Multiplex PCR (70) sonstiger Hersteller Fast-track - FTD respiratory pathogens eigene Herstellung GenID - CAP Vir GenID - Influenza-screening Multipl. Taq Man (75) astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU Qualitative Ergebnisse für Probe Kombinierter qualitativer Genomnachweis von Influenza A und B : negativ gesamter Test Gesamt Positiv Grenzw. Negativ Quote Taq Man (13) CvirG / LgesQ Rv= :18h Bl. 5

21 Qualitative Ergebnisse für Probe Kombinierter qualitativer Genomnachweis von Influenza A und B Grp. 370 Light Cycler (40) Qiagen - artus Infl./H1 LC/RG RT-PCR Kit astra Diagnostics - Influenza Screen&Type PIIM - AmpliGnost Influenza-Virus A+B Qiagen - artus Influenza LC RT-PCR Kit Qiagen - artus Influenza/H5 LC RT-PCR Kit andere PCR (98) Qiagen - artus Infl./H1 LC/RG RT-PCR Kit CvirG / LgesQ Rv= :18h Bl. 6

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23 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza A-Virus : positiv gesamter Test Gesamt Positiv Grenzw. Negativ Quote PCR (11) eigene Herstellung nested PCR (12) eigene Herstellung Taq Man (13) eigene Herstellung Light Cycler (40) Roche - RealTime ready Influenza A/H1N eigene Herstellung TIB MolBiol - LightMix Kit Influenza A M astra Diagnostics - Influenza Screen&Type PIIM - AmpliGnost Influenza-Virus A/B TIB MolBiol - LightMix Kit InfA M2 & H1 sw Mikrogen - dia Human influenza A/B Argene - Flu A/B r-gene Multiplex PCR (70) eigene Herstellung Fast-track - FTD respiratory pathogens GenID - CAP Vir GenID - Influenza-typing Multipl. Taq Man (75) astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU CvirG / LgesQ Rv= :18h Bl. 7

24 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza B-Virus : negativ gesamter Test Gesamt Positiv Grenzw. Negativ Quote PCR (11) eigene Herstellung nested PCR (12) eigene Herstellung Taq Man (13) eigene Herstellung Light Cycler (40) eigene Herstellung Roche - RealTime ready Influenza B TIB MolBiol - LightMix Kit Influenza B astra Diagnostics - Influenza Screen&Type PIIM - AmpliGnost Influenza-Virus A/B Argene - Flu A/B r-gene Mikrogen - dia Human influenza A/B Multiplex PCR (70) Fast-track - FTD respiratory pathogens sonstiger Hersteller eigene Herstellung GenID - CAP Vir GenID - Influenza-screening Multipl. Taq Man (75) astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU Qualitative Ergebnisse für Probe Kombinierter qualitativer Genomnachweis von Influenza A und B : positiv gesamter Test Gesamt Positiv Grenzw. Negativ Quote Taq Man (13) CvirG / LgesQ Rv= :18h Bl. 8

25 Qualitative Ergebnisse für Probe Kombinierter qualitativer Genomnachweis von Influenza A und B Grp. 370 Light Cycler (40) Qiagen - artus Infl./H1 LC/RG RT-PCR Kit astra Diagnostics - Influenza Screen&Type Qiagen - artus Influenza LC RT-PCR Kit PIIM - AmpliGnost Influenza-Virus A+B Qiagen - artus Influenza/H5 LC RT-PCR Kit andere PCR (98) Qiagen - artus Infl./H1 LC/RG RT-PCR Kit CvirG / LgesQ Rv= :18h Bl. 9

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27 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza A-Virus : negativ gesamter Test Gesamt Positiv Grenzw. Negativ Quote PCR (11) eigene Herstellung nested PCR (12) eigene Herstellung Taq Man (13) eigene Herstellung Light Cycler (40) Roche - RealTime ready Influenza A/H1N eigene Herstellung TIB MolBiol - LightMix Kit Influenza A M astra Diagnostics - Influenza Screen&Type PIIM - AmpliGnost Influenza-Virus A/B Argene - Flu A/B r-gene TIB MolBiol - LightMix Kit InfA M2 & H1 sw Mikrogen - dia Human influenza A/B Multiplex PCR (70) eigene Herstellung Fast-track - FTD respiratory pathogens GenID - CAP Vir GenID - Influenza-typing Multipl. Taq Man (75) astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU CvirG / LgesQ Rv= :18h Bl. 10

28 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza B-Virus : positiv gesamter Test Gesamt Positiv Grenzw. Negativ Quote PCR (11) eigene Herstellung nested PCR (12) eigene Herstellung Taq Man (13) eigene Herstellung Light Cycler (40) eigene Herstellung Roche - RealTime ready Influenza B TIB MolBiol - LightMix Kit Influenza B astra Diagnostics - Influenza Screen&Type PIIM - AmpliGnost Influenza-Virus A/B Argene - Flu A/B r-gene Mikrogen - dia Human influenza A/B Multiplex PCR (70) Fast-track - FTD respiratory pathogens sonstiger Hersteller eigene Herstellung GenID - CAP Vir GenID - Influenza-screening Multipl. Taq Man (75) astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU Qualitative Ergebnisse für Probe Kombinierter qualitativer Genomnachweis von Influenza A und B : positiv gesamter Test Gesamt Positiv Grenzw. Negativ Quote Taq Man (13) CvirG / LgesQ Rv= :18h Bl. 11

29 Qualitative Ergebnisse für Probe Kombinierter qualitativer Genomnachweis von Influenza A und B Grp. 370 Light Cycler (40) Qiagen - artus Infl./H1 LC/RG RT-PCR Kit astra Diagnostics - Influenza Screen&Type Qiagen - artus Influenza LC RT-PCR Kit PIIM - AmpliGnost Influenza-Virus A+B Qiagen - artus Influenza/H5 LC RT-PCR Kit andere PCR (98) Qiagen - artus Infl./H1 LC/RG RT-PCR Kit CvirG / LgesQ Rv= :18h Bl. 12

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31 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza A-Virus : positiv gesamter Test Gesamt Positiv Grenzw. Negativ Quote PCR (11) eigene Herstellung nested PCR (12) eigene Herstellung Taq Man (13) eigene Herstellung Light Cycler (40) Roche - RealTime ready Influenza A/H1N eigene Herstellung TIB MolBiol - LightMix Kit Influenza A M astra Diagnostics - Influenza Screen&Type PIIM - AmpliGnost Influenza-Virus A/B TIB MolBiol - LightMix Kit InfA M2 & H1 sw Mikrogen - dia Human influenza A/B Argene - Flu A/B r-gene Multiplex PCR (70) Fast-track - FTD respiratory pathogens eigene Herstellung GenID - CAP Vir sonstiger Hersteller GenID - Influenza-typing Multipl. Taq Man (75) astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay astra Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU sonstiger Hersteller CvirG / LgesQ Rv= :18h Bl. 13