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 17 July 2012 Final Report External Quality Assessment Scheme (EQAS) - March/April 2012 Virus Detection (Genome/Antigen) - Influenza A and B Viruses (Group 370) incl. Influenza A(H1N1) pdm09 Virus and Avian Influenza A(H5N1) Virus INSTAND-Target Value Laboratories: Robert Koch-Institut, Abt. Infektionskrankheiten, FG 12 Virale Infektionen, Nationales Referenzzentrum für Influenza, Berlin: Dr. B. Schweiger 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, PD 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 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. U. Dittmer, Prof. Dr. S. Ross, Prof. Dr. M. Roggendorf 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 2012 Letter a.doc 1

2 Dear colleagues, Below please find the final report of the INSTAND External Quality Assessment (EQA) scheme "Virus Detection (genome/antigen) - influenza A and B viruses incl. influenza A(H1N1) pdm09 virus* and avian influenza A(H5N1) virus" March/April You receive the following participation documents for this EQA scheme by mail: - certificate of successful participation, - statement of participation, - statement of individual. You have already been informed about changes as to the INSTAND EQA schemes in recent final reports and information letters. For details please see the INSTAND homepage under "Information on INSTAND External Quality Assessment (EQA) Schemes in Virus Diagnostics/as of 11 May 2012" (please use the download button) These changes concern: New RiliBÄK (Guideline of the German Medical Association) and validity period of certificates of INSTAND EQA schemes in virus diagnostics. Mailing of participation documents of a defined EQA scheme term in virus diagnostics (certificate of successful participation, statement of participation, statement of individual ). Electronic supply of pre-evaluations and final reports of the EQA schemes in virus diagnostics. Please note that INSTAND can only inform you about the electronic supply of pre-evaluations and final reports of the EQA schemes in virus diagnostics when you have already returned your address (see information letter as of 12 March 2012). Release of final reports of EQA schemes in virus diagnostics on the INSTAND homepage. Each final report of a defined EQA scheme will be released on the INSTAND homepage immediately after completion as PDF file under EQAS / Reports / Year and Category (Virus immunology / Virus genome detection) in English language ( and in German language ( 1 Test categories, statement of and evaluation criteria for this EQA scheme As already recently practiced, the certificate of successful participation of a defined EQA scheme in virus diagnostics is assigned to a defined test category. This EQA scheme comprises the following test categories (Table 1): Table 1: Test categories, statement of and evaluation criteria Test categories Genome detection of influenza A and B viruses (5) Quantitative genome detection of influenza A viruses - Quantitativer Genomnachweis von Influenza A-Viren (10) Qualitative genome detection of influenza A viruses (without differentiation) - Qualitativer Genomnachweis von Influenza A-Viren (ohne Subtyp-Differenzierung) (11) Subtyping of influenza A viruses - Subtypisierung von Influenza A-Viren Statement of (the following statements of were requested) genome equivalents/ml = copies/ml or below level of detection positive or below level of detection / negative or indeterminate Hx or HxNx or below level of detection / negative or Criteria for receiving a certificate (min. no. of ly determined samples) without evaluation 6 of 6 samples 6 of 6 samples The which were reported in copies/ml are without evaluation due to the low number of analyses and the high level of variance (without disadvantage for the certificate). For details please see Table 4. * Influenza A(H1N1) pdm09 virus is the new nomenclature of the formerly named pandemic influenza A(H1N1) 2009 virus. 370 Influenza March April 2012 Letter a.doc 2

3 Table 1: Test categories, statement of and evaluation criteria (continued) Test categories Genome detection of influenza A and B viruses (12) Qualitative genome detection of avian influenza A(H5N1) virus (with primers/probes specific for H5N1 or H5) - Qualitativer Genomnachweis von aviärem Influenza A(H5N1)-Virus (mit H5N1- oder H5-spezfischen Primern/Sonden) Test category 12 concerns only test systems for the exclusive detection of avian influenza A(H5N1) virus (13) Qualitative genome detection of influenza A(H1N1) pdm09 virus* (with primers/probes specific for H1N1 or H1) - Qualitativer Genomnachweis von Influenza A(H1N1) pdm09-virus (mit H1N1- oder H1-spezfischen Primern/Sonden) Test category 13 concerns only test systems for the exclusive detection of influenza A(H1N1) pdm09 virus (15) Quantitative genome detection of influenza B viruses - Quantitativer Genomnachweis von Influenza B-Viren (20) Qualitative genome detection of influenza B viruses - Qualitativer Genomnachweis von Influenza B-Viren (25) Combined qualitative genome detection of influenza A and B viruses - Kombinierter qualitativer Genomnachweis von Influenza A- und B-Viren Antigen detection of influenza A and B viruses (30) Qualitative antigen detection of influenza A viruses - Qualitativer Antigennachweis von Influenza A-Viren (40) Qualitative antigen detection of influenza A viruses - Qualitativer Antigennachweis von Influenza B-Viren Statement of (the following statements of were requested) H5 or H5N1 or below level of detection / negative or H1 pdm09 or H1N1 pdm09 or below level of detection / negative or genome equivalents/ml = copies/ml or below level of detection positive or below level of detection / negative or indeterminate positive or negative or borderline Criteria for receiving a certificate (min. no. of ly determined samples) 6 of 6 samples 6 of 6 samples without evaluation 6 of 6 samples 6 of 6 samples 6 of 6 samples 6 of 6 samples * Influenza A(H1N1) pdm09 virus is the new nomenclature of the formerly named pandemic influenza A(H1N1) 2009 virus. The which were reported in copies/ml are without evaluation due to the low number of analyses and the high level of variance (without disadvantage for the certificate). For details please see Table 4. These test categories are individually evaluated in the certificate of successful participation and listed in all participation and evaluation documents. The evaluation criteria for the of EQA schemes for the detection of virus specific antibodies follow the new Guideline of the German Medical Association, RiliBÄK section E 2 (specific requirements on EQA schemes for qualitative laboratory medical analyses = Spezielle Anforderungen an Ringversuche bei qualitativen laboratoriumsmedizinischen Untersuchungen). For receiving a certificate of successful participation for a defined EQA scheme it is required that you analyzed all samples of the sample set ly with the same method in the corresponding test categories (100 according to the target values). Please note: A corresponding proceeding is applied for tests for virus antigen and genome detection. Example - Program "Virus Detection (genome/ antigen) - influenza A and B viruses incl. influenza A(H1N1) pdm09 virus and avian influenza A(H5N1) virus" (370): All 6 samples of the sample set have to be tested ly with the same method in test category 10 "Qualitative genome detection of influenza A viruses (without differentiation) - Qualitativer Genomnachweis von Influenza A-Viren (ohne Subtyp-Differenzierung)". The same applies for test categories 5, 11, 12, 13, 15, 20, 25, 30 and 40 of this EQA scheme. 370 Influenza March April 2012 Letter a.doc 3

4 Please note for the quantitative evaluation that quantitative which were reported in copies/ml are without evaluation due to the low number of analyses and the high level of variance (without disadvantage for the certificate). For details please see Table 4. 2 Sample properties Please see Table 2 for details on the properties of the samples of this EQA scheme. Table 2: Sample properties Sample No. Influenza virus Type Subtype Strain Dilution Origin seasonal influenza A H3N2 vaccine strain A/Perth/16/2009 1:150 infected cells (lysate) avian influenza A H5N1 A/chicken/Germany/R3294/2007 1:90 allantoic fluid (chem. inactivated) influenza A (pandemic) H1N1 pdm09 vaccine strain A/California/7/2009 1:100 infected cells (lysate) negative for influenza A and B non-infected cells (lysate) seasonal influenza A H1N1 vaccine strain A/Brisbane/59/2007 1:60 infected cells (lysate) seasonal influenza B vaccine strain B/Brisbane/60/2008 1:5 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. For details on sample properties, and success rates please see Table 3. The simultaneous reporting of different for one and the same sample cannot be accepted and will be evaluated as a missing value, e.g. the simultaneous reporting of a "positive" and "indeterminate" result will not be accepted. 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. 370 Influenza March April 2012 Letter a.doc 4

5 3 Results Summary The EQA scheme influenza A and B viruses of March/April 2012 revealed the following : Detection of influenza A(H1N1) pdm09 virus One sample with influenza A(H1N1) pdm09 virus was analyzed in this EQA scheme: - sample , vaccine strain A/California/7/2009 (H1N1), 1:100 diluted. All applied qualitative genome detection tests for the detection of this virus in test categories 10, 11, 13 and 25 (see Table 1) revealed success rates between 95.7 and 100 (see Table 3). In contrast, the of rapid tests for antigen detection of influenza A (test category 30) showed a reduced success rate of 72.3 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 influenza A(H1N1) pdm09 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), 1:90 diluted, chemically inactivated. All applied qualitative genome detection tests for the detection of this virus in test categories 10, 11, 12 and 25 (see Table 1) revealed success rates between 87.0 and 92.6 (see Table 3). The of rapid tests for antigen detection of influenza A (test category 30) showed a success rate of 100. 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 influenza A(H1N1) pdm09 virus was not misinterpreted as negative. Detection of seasonal influenza A(H1N1) and influenza A(H3N2) viruses Two samples with different seasonal influenza A virus s were analyzed in this EQA scheme: - sample , vaccine strain A/Perth/16/2009 (H3N2), 1:150 diluted, - sample , vaccine strain A/Brisbane/59/2007 (H1N1), 1:60 diluted. Applying qualitative genome detection tests for influenza A virus revealed success rates between 95.7 and 100 for these two samples for test categories 10, 11 and 25 (see Table 3). The of rapid tests for antigen detection of influenza A (test category 30) showed for both samples a success rate of 100. Please note that "borderline" was also accepted as result for sample 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 in test categories 20 and 25 (see Table 1) revealed success rates of 100, respectively (see Table 3). The of rapid tests for antigen detection of influenza B in test category 40 showed a success rate of 100. Sample negative for influenza A and B viruses The success rates obtained with all applied test categories for the negative sample were between 99.3 and 100. For details on the present epidemiological situation please see homepages: - Arbeitsgemeinschaft für Influenza (AGI) am Robert Koch-Institut: - European Centre for Disease Prevention and Control (ECDC): - World Health Organization (WHO): References are given at the end of this report for further information. The following Table 3 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, influenza A(H1N1) pdm09 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 2012 Letter a.doc 5

6 Table 3: Summary of sample properties, and success rates - qualitative detection of influenza A and B viruses Detected virus (Test categories) Sample Sample Sample Sample Sample Sample Sample Success seasonal avian negative for seasonal seasonal rates for all influenza A virus influenza A virus influenza A virus influenza A and B influenza A virus influenza B virus 6 samples of (H3N2) (H5N1) (H1N1) pdm09 viruses (H1N1) the sample (1:150 diluted) (1:90 diluted) (1:100 diluted) (1:60 diluted) (1:5 diluted) set & considered as "" number of analyses with /total number of analyses (success rate in ) Influenza A virus positive positive positive negative positive negative Genome detection (10) 147/147 (100) 134/147 (91.2) 146/147 (99.3) 146/147 (99.3) 147/147 (100) 145/147 (98.6) Influenza B virus negative negative negative negative negative positive Genome detection (20) 139/139 (100) 139/139 (100) 139/139 (100) 139/139 (100) 139/139 (100) 139/139 (100) Influenza A/B v. combined positive positive positive negative positive positive Genome detection (25) 27/27 (100) 25/27 (92.6) 27/27 (100) 27/27 (100) 27/27 (100) 27/27 (100) 88.6 & (124/140) & 100 & (135/135) & 92.6 & (25/27) & Influenza A(H1N1) pdm09 virus negative / nd # negative / nd # H1N1 pdm09 / H1 pdm09 negative / nd # nd # negative / nd # 97.1 & Genome detection with primers/probes specific for H1N1 or H1 (13) 143/143 (100) 142/143 (99.3) 142/143 (99.3) 143/143 (100) 140/143 (97.9) 143/143 (100) (132/136) & Avian influenza A(H5N1) virus negative / nd # H5N1 / H5 negative / nd # negative / nd # negative / nd # negative / nd # 91.9 & Genome detection with primers/probes specific for H5N1 or H5 (12) 37/37 (100) 34/37 (91.9) 37/37 (100) 37/37 (100) 37/37 (100) 37/37 (100) Influenza A H3N2 / H3 H5N1 / H5 H1N1 pdm09 / H1 pdm09 negative / nd # H1N1 / H1 negative / nd # 82.6 & Genome detection (11) 23/23 (100) 20/23 (87.0) 22/23 (95.7) 23/23 (100) 22/23 (95.7) 23/23 (100) Influenza A virus (34/37) & (19/23) & positive / positive / positive / borderline borderline borderline negative positive negative 71.4 & Antigen detection (30) 65/65 (100) 65/65 (100) 47/65 (72.3) 65/65 (100) 65/65 (100) 65/65 (100) Influenza B virus negative negative negative negative negative positive Antigen detection (40) 65/65 (100) 65/65 (100) 65/65 (100) 65/65 (100) 65/65 (100) 65/65 (100) # & nd = The reporting of "borderline" was accepted as result in addition to the reporting of "positive" for samples , and (test category 30). Considering also the result "borderline" ensured that these positive samples were 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. (45/63) & 100 & (63/63) & 370 Influenza March April 2012 Letter a.doc 6

7 3.1 Qualitative detection of influenza A viruses Detection of influenza A(H1N1) pdm09 virus The for sample positive for influenza A(H1N1) pdm09 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 infl. A(H1N1) pdm09 (13) Genome detection for subtyping infl. A (11) Antigen detection infl. A (30) No. of analyses N negative borderline positive; sample (1:100 dil.) Figure 1: Qualitative detection of influenza A(H1N1) pdm09 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 3 (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) 96/147 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 (1:100 diluted) 146/147 (99.3) All applied screening PCRs for combined genome detection of influenza A and B viruses (without type differentiation) (25) were commercial tests (27/27 analyses). Applying these tests led to the following (Figure 1): Sample (1:100 diluted) 27/27 (100) 370 Influenza March April 2012 Letter a.doc 7

8 Genome detection of influenza A(H1N1) pdm09 virus (with primers/probes specific for H1N1 or H1) (13) The of test category (13), specific genome detection exclusively for influenza A(H1N1) pdm09 virus, are summarized in Table 3 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/section 4.2). For sample (1:100 diluted) 99/143 analyses were performed with commercial test systems for the specific genome detection of influenza A(H1N1) pdm09 virus (13). The were as follows (Figure 1): Sample (1:100 diluted) 142/143 (99.3) These tests showed a high reliability for the differentiation of influenza A(H1N1) pdm09 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. The for specific genome detection of influenza A(H1N1) pdm09 virus were false positive for the following samples (see Table 3). Sample : seasonal influenza A(H1N1) virus 3/143 false positive (2.1) Sample : avian influenza A(H5N1) virus 1/143 false positive (0.7) 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 3 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.4). For sample the majority of specific RT-PCRs applied for subtyping of influenza A positive samples were in house tests (20/23 analyses). Sequencing were not reported. The were as follows (Figure 1): Sample (1:100 diluted) 22/23 (95.7) Antigen detection of influenza A viruses (30) The of test category (30), antigen detection of influenza A viruses, are summarized in Table 3 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.5). For sample 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" as result ensured that this sample positive for influenza A(H1N1) pdm09 virus was not misinterpreted as negative. For this sample the majority of in test category (30) were achieved with commercial rapid tests (64/65 analyses). The summarized were as follows (Figure 1): Sample (1:100 diluted) 47/65 (72.3) The for sample thus led to 27.7 (18/65 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 2012 Letter a.doc 8

9 3.1.2 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, 1:90 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 (1:90 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 3 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/section 4.1). For sample (1:90 diluted) 96/147 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 (1:90 diluted) 134/147 (91.2) All applied screening PCRs for combined genome detection of influenza A and B viruses (without type differentiation) (25) were commercial tests (27/27 analyses). Applying these tests led to the following (Figure 2): Sample (1:90 diluted) 25/27 (92.6) 370 Influenza March April 2012 Letter a.doc 9

10 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 3 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/section 4.3). For sample (1:90 diluted) 29/37 analyses for the specific genome detection of avian influenza A(H5N1) virus were performed with in house test systems. The were as follows (Figure 2): Sample (1:90 diluted) 34/37 (91.9) 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 influenza A(H1N1) pdm09 virus (sample ) analyzed in this EQA scheme. No false positive were obtained for the detection of heterologous influenza A and B viruses (see Table 3) 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 3 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.4). For sample the majority of specific RT-PCRs applied for subtyping of influenza A positive samples were in house tests (20/23 analyses). Sequencing were not reported. The for this sample were as follows (Figure 2): Sample (1:90 diluted) 20/23 (87.0) Antigen detection of influenza A viruses (30) The of test category (30), antigen detection of influenza A viruses, are summarized in Table 3 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.5). For sample 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 (1:90 diluted) all in test category (30) were achieved with commercial rapid tests (64/65 analyses). The were as follows (Figure 2): Sample (1:90 diluted) 65/65 (100) Detection of seasonal influenza A viruses The for samples and positive for seasonal influenza A viruses (sample , vaccine strain A/Perth/16/2009(H3N2), 1:150 diluted; sample , vaccine strain A/Brisbane/59/2007(H1N1), 1:60 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 3 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/section 4.1). In total 96/147 analyses were performed for each of the two 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(H3N2) virus, 1:150 diluted) 147/147 (100) Sample (A(H1N1) virus, 1:60 diluted) 147/147 (100) 370 Influenza March April 2012 Letter a.doc 10

11 All applied screening PCRs for combined genome detection of influenza A and B viruses (without type differentiation) (25) were commercial tests (27/27 analyses each). Applying these tests led to the following (Figure 3): Sample (A(H3N2) virus, 1:150 diluted) 27/27 (100) Sample (A(H1N1) virus, 1:60 diluted) 27/27 (100) 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 (1:150 diluted), A(H3N2) positive; sample (1:60 diluted), A(H1N1) 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 3 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.4). For samples and the majority of specific RT-PCRs applied for subtyping of influenza A positive samples were in house tests (20/23 analyses each). Sequencing were not reported. The for each of the samples were as follows (Figure 3): Sample (A(H3N2) virus, 1:150 diluted) 23/23 (100) Sample (A(H1N1) virus, 1:60 diluted) 22/23 (95.7) Antigen detection of influenza A viruses (30) The of test category (30), antigen detection of influenza A viruses, are summarized in Table 3 (please also see the tables with detailed according to test formats, manufacturers and names of test kits/section 4.5). For samples and all in test category (30) were achieved with commercial rapid tests (64/65 analyses each). The summarized for each of the samples were as follows (Figure 3): Sample (A(H3N2) virus, 1:150 diluted) 65/65 (100) Sample (A(H1N1) virus, 1:60 diluted) 65/65 (100) For sample 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 seasonal influenza A(H3N2) virus was not misinterpreted as negative. 370 Influenza March April 2012 Letter a.doc 11

12 3.2 Qualitative detection of influenza B virus Detection of seasonal influenza B virus The for sample positive for seasonal influenza B viruses (vaccine strain B/Brisbane/60/2008, 1:5 diluted) 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 3 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/section 4.1). In total 85/139 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) 139/139 (100) All applied screening PCRs for combined genome detection of influenza A and B viruses (without type differentiation) (25) were commercial tests (27/27 analyses). Applying these tests led to the following (Figure 4): Sample (1:5 diluted) 27/27 (100) Antigen detection of influenza B viruses (40) The majority of in test category (40), antigen detection of influenza B virus, was achieved with commercial rapid tests (64/65 analyses). These are summarized in Table 3 (please see also the tables with detailed according to test formats, manufacturers and names of test kits /section 4.5). The were as follows (Figure 4): Sample (1:5 diluted) 65/65 (100) 370 Influenza March April 2012 Letter a.doc 12

13 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 99.3 and 100 negative. These are summarized in Table 3 (please see also the tables with detailed according to test formats, manufacturers and names of test kits/sections ). This reflects that in most cases the applied tests of the different test categories were reliable in respect to specificity. 3.4 Quantitative detection of influenza A and B viruses In total nine participants reported quantitative for the genome detection of influenza A and B viruses. These are summarized in the following Table 4 and are not evaluated (without any disadvantage for the certificate) due to the low number of analyses and the high level of variance. Testformat / manufacturer - name of test kit Participating laboratories (TN-No.) TaqMan / in house Table 4: Quantitative genome detection - without evaluation Sample Sample Sample Sample Sample Sample seasonal influenza A virus (H3N2) (1:150 diluted) copies/ml avian influenza A virus (H5N1) (1:90 diluted) copies/ml influenza A virus (H1N1) pdm09 (1:100 diluted) copies/ml negative for influenza A and B viruses copies/ml seasonal influenza A virus (H1N1) (1:60 diluted) copies/ml seasonal influenza B virus (1:5 diluted) copies/ml TN TN TN TN TN TN LightCycler / in house TN TN LightCycler / LightMix Kit Influenza A M2 (TibMolBiol) TN 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, manufacturers and names of test kits 4.1 Qualitative genome detection of influenza A and B viruses (categories 10, 20 and 25) 4.2 Qualitative genome detection of influenza A(H1N1) pdm09 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) Surplus samples of the current and previous EQA schemes in virus diagnostics are available for test assessment of your virus diagnostics. Please contact INSTAND for details. We gratefully acknowledge the good cooperation with Dr. 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 2012 Letter a.doc 13

14 References: CDC, 2012, 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, "Nachweis von H5N1-Viren (aviäre Influenza)", (Stand: ) WHO, 2010, "WHO recommendations for the post-pandemic period, (Stand: ) WHO, Global Alert and Response (GAR), Avian Influenza Influenza March April 2012 Letter a.doc 14

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16 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) (Testkategorie 10) Genomnachweis von Influenza B-Viren (Testkategorie 20) Kombinierter Genomnachweis von Influenza A- und B-Viren (Testkategorie 25) 4.1 Qualitative genome detection of influenza A and B viruses Genome detection of influenza A viruses (without differentiation of s) (Test category 10) Genome detection of influenza B viruses (Test category 20) Combined genome detection of influenza A and B viruses (Test category 25) 370 Influenza 10_20_25 Qual Genomnachweis Influenza A- und B-Viren Deckblatt doc

17 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 2012 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 Methode / Hersteller gesamt positiv grenzw. negativ Quote PCR (11) eigene Herstellung Taq Man (13) eigene Herstellung sonstiger Hersteller Light Cycler (40) eigene Herstellung Roche - RealTime ready Influenza A/H1N altona Diagnostics - Influenza Screen&Type TIB MolBiol - LightMix Kit Influenza A M PIIM - AmpliGnost Influenza-Virus A/B Diff R-Biopharm - RIDA GENE Flu Mikrogen - dia Human influenza A/B TIB MolBiol - LightMix Kit InfA M2 & H1 sw Multiplex PCR (70) sonstiger Hersteller Fast-track - FTD respiratory pathogens GenID - CAP Vir eigene Herstellung GenID - Influenza-typing Multipl. Taq Man (75) altona Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay altona Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU Mikrogen - dia Human influenza A/B Cepheid - SmartCycler FluA/B CvirG / LgesQ Rv= :20h Bl. 1

18 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza B-Virus : negativ gesamter Test Gesamt Positiv Grenzw. Negativ Quote Methode / Hersteller gesamt positiv grenzw. negativ Quote PCR (11) eigene Herstellung nested PCR (12) eigene Herstellung Taq Man (13) eigene Herstellung sonstiger Hersteller Light Cycler (40) eigene Herstellung Roche - RealTime ready Influenza B altona Diagnostics - Influenza Screen&Type TIB MolBiol - LightMix Kit Influenza B PIIM - AmpliGnost Influenza-Virus A/B Diff R-Biopharm - RIDA GENE Flu Mikrogen - dia Human influenza A/B Multiplex PCR (70) sonstiger Hersteller Fast-track - FTD respiratory pathogens GenID - CAP Vir eigene Herstellung GenID - Influenza-screening Multipl. Taq Man (75) altona Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay altona Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU Mikrogen - dia Human influenza A/B Cepheid - SmartCycler FluA/B CvirG / LgesQ Rv= :20h Bl. 2

19 Grp. 370 Qualitative Ergebnisse für Probe Kombinierter qualitativer Genomnachweis von Influenza A und B : positiv gesamter Test Gesamt Positiv Grenzw. Negativ Quote Methode / Hersteller gesamt positiv grenzw. negativ Quote Taq Man (13) Light Cycler (40) Qiagen - artus Infl./H1 LC/RG RT-PCR Kit altona 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 Cepheid - GeneXpert Flu Assay CvirG / LgesQ Rv= :20h Bl. 3

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21 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza A-Virus : positiv gesamter Test Gesamt Positiv Grenzw. Negativ Quote Methode / Hersteller gesamt positiv grenzw. negativ Quote PCR (11) eigene Herstellung Taq Man (13) eigene Herstellung sonstiger Hersteller Light Cycler (40) eigene Herstellung Roche - RealTime ready Influenza A/H1N altona Diagnostics - Influenza Screen&Type TIB MolBiol - LightMix Kit Influenza A M PIIM - AmpliGnost Influenza-Virus A/B Diff R-Biopharm - RIDA GENE Flu Mikrogen - dia Human influenza A/B TIB MolBiol - LightMix Kit InfA M2 & H1 sw Multiplex PCR (70) sonstiger Hersteller Fast-track - FTD respiratory pathogens GenID - CAP Vir eigene Herstellung GenID - Influenza-typing Multipl. Taq Man (75) altona Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay altona Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU Mikrogen - dia Human influenza A/B Cepheid - SmartCycler FluA/B CvirG / LgesQ Rv= :20h Bl. 4

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

23 Grp. 370 Qualitative Ergebnisse für Probe Kombinierter qualitativer Genomnachweis von Influenza A und B : positiv gesamter Test Gesamt Positiv Grenzw. Negativ Quote Methode / Hersteller gesamt positiv grenzw. negativ Quote Taq Man (13) Light Cycler (40) Qiagen - artus Infl./H1 LC/RG RT-PCR Kit altona 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 Cepheid - GeneXpert Flu Assay CvirG / LgesQ Rv= :20h Bl. 6

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25 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza A-Virus : positiv gesamter Test Gesamt Positiv Grenzw. Negativ Quote Methode / Hersteller gesamt positiv grenzw. negativ Quote PCR (11) eigene Herstellung Taq Man (13) eigene Herstellung sonstiger Hersteller Light Cycler (40) eigene Herstellung Roche - RealTime ready Influenza A/H1N altona Diagnostics - Influenza Screen&Type TIB MolBiol - LightMix Kit Influenza A M PIIM - AmpliGnost Influenza-Virus A/B Diff R-Biopharm - RIDA GENE Flu Mikrogen - dia Human influenza A/B TIB MolBiol - LightMix Kit InfA M2 & H1 sw Multiplex PCR (70) sonstiger Hersteller GenID - CAP Vir Fast-track - FTD respiratory pathogens GenID - Influenza-typing eigene Herstellung Multipl. Taq Man (75) altona Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay altona Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU Cepheid - SmartCycler FluA/B Mikrogen - dia Human influenza A/B CvirG / LgesQ Rv= :20h Bl. 7

26 Grp. 370 Qualitative Ergebnisse für Probe Qualitativer Genomnachweis von Influenza B-Virus : negativ gesamter Test Gesamt Positiv Grenzw. Negativ Quote Methode / Hersteller gesamt positiv grenzw. negativ Quote PCR (11) eigene Herstellung nested PCR (12) eigene Herstellung Taq Man (13) eigene Herstellung sonstiger Hersteller Light Cycler (40) eigene Herstellung Roche - RealTime ready Influenza B altona Diagnostics - Influenza Screen&Type TIB MolBiol - LightMix Kit Influenza B PIIM - AmpliGnost Influenza-Virus A/B Diff R-Biopharm - RIDA GENE Flu Mikrogen - dia Human influenza A/B Multiplex PCR (70) sonstiger Hersteller GenID - CAP Vir Fast-track - FTD respiratory pathogens eigene Herstellung GenID - Influenza-screening Multipl. Taq Man (75) altona Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD respiratory pathogens andere PCR (98) Cepheid - GeneXpert Flu Assay altona Diagnostics - Influenza Screen&Type eigene Herstellung Fast-track - FTD FLU Mikrogen - dia Human influenza A/B Cepheid - SmartCycler FluA/B CvirG / LgesQ Rv= :20h Bl. 8