EXPERTISE, SERVICE PROVISION AND CUSTOMER RELATIONS QUALITY OF MEDICAL LABORATORIES CLINICAL BIOLOGY COMMISSION COMMITTEE OF EXPERTS EXTERNAL QUALITY ASSESSMENT IN CLINICAL BIOLOGY DEFINITIVE GLOBAL ANNUAL REPORT Molecular Biology Microbiology 2015 IPH/Molecular Biology/Microbiology/8 Expertise, service provision and customer relations Quality of medical laboratories J. Wytsmanstreet, 14 1050 Brussels Belgium www.wiv-isp.be
ISSN: 2294-3471 COMMITTEE OF EXPERTS IPH (secretariat) TEL: 026425321 026425322 FAX: 026425645 Survey coordinator: Bernard China Substitute survey coordinator: Kris Vernelen Experts: TEL: e-mail: TEL: e-mail: 026425385 bernard.china@wiv-isp.be 026425529 kvernelen@wiv-isp.be Name Institution Email Dr. Hans de Beenhouwer O.L.V. Ziekenhuis, Aalst hans.de.beenhouwer@olvz-aalst.be Dr. Nicolas Hougardy Clinique Sud Luxembourg, Arlon nhougardy@clinsudlux.be Dr. Greet Ieven UZA, Antwerpen Greet.Ieven@uza.be Dr. Koen Magerman Virga Jesse Ziekenhuis, Hasselt koen.magerman@virgajesse.be Dr. Marjan Van Esbroeck ITM, Antwerpen mvesbroeck@itg.be Dr. Marijke Reynders AZ sint Jan, Brugge marijke.reynders@azsintjan.be Dr. Ina Benoy Labo Lokeren Ina.benoy@riatol.be Committee of experts: 24/06/2016 Authorisation to release report: By Bernard China, Survey coordinator, 28/06/2016. 2/36 FORM 43/125/E V6
TABLE OF CONTENTS INTRODUCTION... 5 I. THE BACTERIA... 6 1. CHLAMYDIA TRACHOMATIS AND NEISSERIA GONORRHEAE... 6 1.1. THE SAMPLES... 6 1.2. CHLAMYDIA TRACHOMATIS... 7 1.2.1. Proficiency per sample... 7 1.2.2. Summary of the results... 7 1.2.3. scoring of the laboratories... 8 1.2.4. Comparison of the used methods... 8 1.3. DETECTION OF NEISSERIA GONORRHOEAE... 10 1.3.1. Proficiency per sample... 10 1.3.2. Summary of the results.... 10 1.3.3. Scoring of the laboratories... 10 1.3.4. Proficiency per method... 12 2. MYCOBACTERIUM TUBERCULOSIS... 13 2.1. The samples... 13 2.2. Proficiency per sample... 13 2.3. Scores of the laboratories.... 14 2.4. Proficiency per method... 14 II. THE VIRUSES... 15 1. VARICELLA-ZOSTER VIRUS... 15 1.1. The samples... 15 1.2. Proficiency per sample... 15 1.3. Scores of the laboratories... 15 1.4. Proficiency per method... 16 2.ENTEROVIRUSES... 17 2.1. The samples... 17 2.2. Proficiency per sample... 17 2.3. Scores of the laboratories... 17 2.4. Proficiency per method... 18 3. HERPES SIMPLEX VIRUS... 19 3.1. The samples... 19 3.2. Proficiency per sample... 19 FORM 43/125/E V6 3/36
3.3. Scores of the laboratories... 19 3.4. Proficiency per method... 20 4. HEPATITIS B VIRUS... 21 4.1. The samples... 21 4.2. Proficiency per sample : qualitative results... 21 4.3. Score per laboratory... 21 4.4. Methods... 21 4.5. Quantitative results... 22 5. HEPATITIS C VIRUS... 24 5.1. The samples... 24 5.2. Proficiency per sample... 24 5.3. Score per laboratory... 24 5.4. Methods... 24 5.5. Quantitative results... 25 6. HCV GENOTYPING... 27 6.1. The samples... 27 6.2. Proficiency per sample... 28 6.3. Methods... 29 7. HIGH RISK PAPILLOMAVIRUS... 30 7.1. The samples... 30 7.2. Results per sample... 31 7.3. Scores of the labs... 31 7.4. Proficiency per method... 32 III. PARASITES... 34 1. TOXOPLASMA GONDII IN AMNIOTIC FLUID.... 34 1.1. The samples... 34 1.2. Proficiency per sample... 34 1.3. Score per laboratory... 35 1.4. Proficiency per method... 35 CONCLUSIONS... 36 FORM 43/125/E V6 4/36
INTRODUCTION In 2015, in the molecular microbiology domain the same surveys as previous years were organized. The survey corresponded to the parameters of article 24 (N. gonorrhoeae, qualitative HCV, M. tuberculosis, Chlamydia trachomatis) and 24 bis (quantitative HCV, HCV genotyping, HBV, enterovirus, HSV, VZV, Toxoplasma gondii) of the INAMI/RIZIV nomenclature. In 2015, the samples for the Chlamydia trachomatis, Neisseiria gonorrhoeae and Toxoplasma gondii were produced by the IPH. The other samples were provided by QCMD (www.qcmd.org) performing also the data collection and the data treatment. For each parameter a panel of samples was sent. The panels include negative, strong positive or frequently detected, positive or detected, weak positive or infrequently detected samples (table Int. 1). Table Int.1. Sample type and associated score Sample type Scoring Negative Strong positive/frequently detected (>95%) Positive/detected (65<X<95%) Weak positive/ infrequently detected (<65%) 0 if success, 3 if failing 0 if success, 3 if failing 0 if success, 2 if failing 0 if success, 1 if failing Each result is associated with a score. In case of correct answer, the score was 0. In case of wrong answer the score depended of the sample type. For a negative sample or for a strong positive sample, a score of 3 was attributed to a wrong result. For a positive sample a score of 2 was attributed to a wrong result. Finally for a weak positive sample a score of 1 was attributed to a wrong result. Therefore, the lower the score of a lab the better it is. Moreover, QCMD attributed a score of 3 for a non-determined result. This appears to us questionable since very often the laboratory has not the opportunity to repeat the assay in case of a technical problem. Therefore, the IPH decided to attribute no penalty for a not determined result. In 2015, 6 surveys were organized: Survey Datum parameters 2015.01 10/02/2015 Neisseria gonorrhoeae and Chlamydia trachomatis 2015.02 10/03/2015 Toxoplasma gondii. 2015.03 09/06/2015 Mycobacterium tuberculosis 2015.04 22/06/2015 VZV-HSV-EV 2015.05 27/07/2015 HCV-HBV-HCV genotyping 2015.06 07/09/2015 High risk HPV FORM 43/125/E V6 5/36
I. THE BACTERIA 1. CHLAMYDIA TRACHOMATIS AND NEISSERIA GONORRHEAE 1.1. THE SAMPLES In 2015, a panel was constituted by IPH for the detection of C. trachomatis and N. gonorroheae in urine and swabs. C. trachomatis LG2 was produced by St Pierre Hospital Brussels and validated by the national reference centre (ITM, Antwerpen). N. Gonorrheae was produced by IPH and validated by the reference centre (ITM, Antwerpen). Table I.1. The samples sample Matrix* C. trachomatis N. gonorrheae BMM 205-1 urine Negative Negative BMM2015-2 urine Negative Frequently detected BMM2015-3 urine Frequently detected Frequently detected BMM2015-4 Urine Frequently detected Negative BMM2015-5 Urine Frequently detected Frequently detected BMM2015-6 Swabs Frequently detected Negative BMM2015-7 Swabs Frequently detected Frequently detected BMM 2015-8 Swabs Frequently detected Frequently detected BMM2015-9 Swabs Detected Frequently detected BMM2015-10 Swabs Negative Negative *: For urine the bacteria were diluted in filtrated human urine (singe donor) and for swabs,the bacteria were diluted in m4rt transport medium. The number of participants was 59 among them 41 were accredited. FORM 43/125/E V6 6/36
1.2. CHLAMYDIA TRACHOMATIS 1.2.1. PROFICIENCY PER SAMPLE The results of the proficiency per sample for the detection of C. trachomatis are shown on table I.2. Table I.2. Proficiency per sample sample Matrix Status Negative Positive ND comment BMM2015-1 urine Negative 58 0 1 1 ND BMM2015-2 urine Negative 53 5 0 5 false positive BMM2015-3 urine Frequently detected 0 59 0 OK BMM2015-4 Urine Frequently detected 1 58 0 1 false negative BMM2015-5 Urine Frequently detected 1 58 0 1 false negative BMM2015-6 swabs Frequently detected 0 59 0 OK BMM2015-7 swabs Frequently detected 0 59 0 OK BMM 2015-8 swabs Frequently detected 0 59 0 OK BMM2015-9 swabs Detected 15 42 2 15 false negative 2 ND BMM2015-10 swabs Negative 56 0 3 3 ND The results are summarized in table I.3. 1.2.2. SUMMARY OF THE RESULTS Table I.3. Summary of the results. Result N (%) Total number 590 (100%) Correct answer 562 (95.3%) Wrong answer 28 (4.7%) False negative 17 (60.7%) False positive 5 (17.9%) Not determined 6 (21.4%) 59 series of results and 10 samples per series representing 590 encoded results. The number of correct answers was 562 out of 590 (95.3%). 17 false negative results were encoded especially for sample 2015-9 (15/17) which was a very low contaminated sample (<10 copies/ml). 5 false positive results and 6 not determined samples were encoded. FORM 43/125/E V6 7/36
1.2.3. SCORING OF THE LABORATORIES The minimum score was 0 in case of 100% of good results and the maximum score was 29 in case of 100% of wrong answers. 40 laboratories (67.8%) have a score of 0. 13 laboratories (22.0%) have a score of 2. 4 laboratories (6.8%) have a score of 3 1 laboratory (1.7%) has a score of 5 1 laboratory (1.7%) has a score of 8. 1.2.4. COMPARISON OF THE USED METHODS Table I.4. Proficiency comparison of the methods used for the detection of C. trachomatis in urine Method N total Good Results % FP FN ND Ranking 131.Bio-Rad Dx CT/NG/MG assay 2 20 20 100 0 0 0 1 171. CT/NG Presto 1 10 10 100 0 0 0 1 119. Artus CT/NG 1 10 9 90 0 0 1 1 101. Abbott RealTime CT 1 10 10 100 0 0 0 1 102. Abbott RealTime CT/NG 17 170 165 97.1 2 2 1 2 201. Roche cobas 4800 CT/NG Test 11 110 106 96.4 0 4 0 3 141. GenXpert CT/NG 7 70 67 95.7 0 0 3 4 111. Artus C. trachomatis Plus PCR Kit 2 20 19 95 0 1 0 5 998. Home made Realtime 4 40 38 95 0 2 0 5 159. Diagenode CT/NG Real time PCR 2 20 19 95 0 0 1 5 202. COBAS TaqMan CT Test v2.0 8 80 75 93.8 2 3 0 6 203. Rohe Amplicor CT/NG 1 10 9 90 0 1 0 7 121. BD Probetec ET CT/GC/AC Amplified DNA Assay 2 20 15 75 1 4 0 8 Total 59 590 562 95.3 5 17 6 FN : false negative, FP : false positive FORM 43/125/E V6 8/36
Table I.5. proficiency per sample, per method and per laboratory Method 2015-1 2015-2 2015-3 2015-4 2015-5 2015-6 2015-7 2015-8 2015-9 2015-10 Negative Negative FD FD FD FD FD FD D Negative 101 102 102 102 102 102 102 102 102 102 0 0 0 0 ND 102 102 102 102 102 0 0 0 2 0 102 0 0 0 2 0 102 0 3 0 0 0 102 0 3 0 0 0 111 0 0 0 2 0 111 121 0 3 0 2 0 121 0 0 0 3 3 0 0 0 2 0 131 131 141 141 ND 0 0 ND 0 141 141 141 141 0 0 0 0 ND 141 159 0 0 0 0 ND 159 171 201 201 201 201 201 201 201 0 0 0 2 0 201 0 0 0 2 0 201 0 0 0 2 0 201 201 0 0 0 2 0 202 202 202 202 0 0 0 2 0 202 0 0 0 2 0 202 0 0 0 2 0 202 0 3 0 0 0 202 0 3 0 0 0 203 0 0 0 2 0 998 998 998 0 0 0 2 0 998 0 0 0 2 0 119 0 0 0 ND 0 FD: freqeduently detected, D: detect, ND: not determined FORM 43/125/E V6 9/36
1.3. DETECTION OF NEISSERIA GONORRHOEAE 57 Belgian laboratories encoded results. 1.3.1. PROFICIENCY PER SAMPLE Table I.6. Proficiency per sample. Sample Status Positive Negative ND Comment BMM2015-1 Negative 0 56 1 1 wrong result BMM2015-2 Frequently Detected 56 1 0 1 wrong result BMM2015-3 Frequently Detected 56 1 0 1 wrong result BMM2015-4 Negative 2 55 0 2 wrong results BMM2105-5 Frequently Detected 54 2 1 3 wrong results BMM2015-6 Negative 0 57 0 OK BMM2015-7 Frequently Detected 56 0 1 1 wrong result BMM2015-8 Frequently Detected 56 0 1 1 wrong result BMM2015-9 Frequently Detected 55 0 2 2 wrong results BMM2015-10 Negative 1 54 2 3 wrong results 1.3.2. SUMMARY OF THE RESULTS. 57 laboratories and 10 samples representing 570 results. Table I.7. Summary of the results Results N (%) Correct answer 555 (97.4%) Wrong answer 15 ( 2.6%) False negative 4 (26.7%) False positive 3 (20.0%) Not determined 8 (53.3%) The number of correct results was 555 out of 570 (97.4%). 4 false negative results were encoded representing 1% of the expected positive results. 3 false positive results were encoded. 1.3.3. SCORING OF THE LABORATORIES The minimum score was 0 and the maximum score was 30. 52 laboratories (91.2%) obtained a ideal score of 0 4 laboratories (7.0%) obtained a score of 3 1 laboratory (1.8%) obtained a score of 9. FORM 43/125/E V6 10/36
Table I.8. proficiency per method Method N total Good Results % FP FN ND Ranking 203. Roche Amplicor CT/NG 1 10 10 100 0 0 0 1 171. CT/NG Presto 1 10 10 100 0 0 0 1 201. Roche cobas 4800 CT/NG Test 11 110 110 100 0 0 0 1 121.BD Probetec ET CT/GC/AC Amplified DNA Assay 2 20 20 100 0 0 0 1 119. Artus CT/NG 2 20 20 100 0 0 0 1 151. Diagenode NG Real time PCR 5 50 50 100 0 0 0 1 998. Home made Realtime 7 70 69 98.6 1 0 0 2 102. Abbott RealTime CT/NG 17 170 167 98.2 1 0 2 3 Bio-Rad Dx CT/NG/MG assay 2 20 19 95 0 1 0 4 141.Xpert CT/NG 7 70 66 94.3 1 0 3 5 211.TIB MolBiol N. gonorrhoeae 2 20 14 70 0 3 3 6 Total 57 570 555 97.4 3 4 8 N: number of participants, FP: false positive, FN: false negative, ND: not determined. FORM 43/125/E V6 11/36
1.3.4. PROFICIENCY PER METHOD Table I.9. Proficiency per method, per sample and per laboratory Method 2015-1 2015-2 2015-3 2015-4 2015-5 2015-6 2015-7 2015-8 2015-9 2015-10 Neg FD FD Neg FD Neg FD FD FD Neg Score 102 ND 102 0 102 0 102 0 102 0 0 0 3 0 3 102 0 102 0 102 0 102 0 102 0 102 0 102 0 0 0 0 ND 0 102 0 102 0 102 0 102 0 102 0 119 0 121 0 121 0 131 0 131 0 0 0 0 3 3 141 0 0 0 3 0 3 141 ND 0 0 ND 0 0 141 0 141 0 141 0 141 0 0 0 0 ND 0 141 0 151 0 151 0 151 0 151 0 171 0 201 0 201 0 201 0 201 0 201 0 201 0 201 0 201 0 201 0 201 0 209 0 211 0 3 3 0 3 0 ND ND ND 0 9 211 0 998 0 998 0 998 0 998 0 0 0 0 3 3 998 0 998 0 998 0 999 0 999 0 203 0 FORM 43/125/E V6 12/36
2. MYCOBACTERIUM TUBERCULOSIS The sample series included 5 samples, 3 cerebrospinal fluid samples and 2 sputum samples. 24 laboratories have encoded results. 2.1. THE SAMPLES Table I.10. Characteristics of the samples. Sample Matrix content Status MTB15-01 Synthetic CSF M. tuberculosis complex Frequently Detected MTB15-02 Synthetic CSF M. tuberculosis complex Detected MTB15-03 Synthetic sputum M. tuberculosis complex Frequently Detected MTB15-04 Synthetic sputum M. Xenopi Negative MTB15-05 Synthetic CSF No Mycobacterium Negative 2.2. PROFICIENCY PER SAMPLE Table I.11. Proficiency per sample. Sample Status Positive Negative ND Comment MTB15-01 Frequently Detected 24 0 0 OK MTB15-02 Detected 22 2 0 2 wrong results MTB15-03 Frequently Detected 24 0 0 OK MTB15-04 Negative 0 21 3 3 wrong results MTB15-05 Negative 0 24 0 OK 24 participants and 5 samples representing 120 results. 115 correct results were encoded (95.8%). FORM 43/125/E V6 13/36
2.3. SCORES OF THE LABORATORIES. The minimum score was 0 and the maximum score was 14. 22 Belgian laboratories obtained the ideal score of 0 and 2 laboratories obtained a score of 2. 2.4. PROFICIENCY PER METHOD Table I.12. proficiency per method, per sample and per laboratory. METHOD N MTB15-01 MTB15-02 MTB15-03 MTB15-04 MTB15-05 FD Detected FD Negative Negative BD Molecular Diagnostics 1 Cepheid Xpert kit 12 0 2 0 0 0 0 0 0 ND 0 Conventional In-House PCR 1 PathoFinder Real Time PCR 1 Real-time In-House PCR 4 Roche Cobas Taqman 0 0 0 ND 0 4 0 0 0 ND 0 TIB-MolBiol LightMix 1 0 2 0 0 0 FORM 43/125/E V6 14/36
II. THE VIRUSES 1. VARICELLA-ZOSTER VIRUS 18 Belgian laboratories encoded results. 1.1. THE SAMPLES Table II.1. Characteristic of the samples. Sample Matrix content Status VZV-15-01 Transport Medium VZV strain 9/84 Frequently detected VZV-15-02 Transport Medium VZV strain 9/84 Frequently detected VZV-15-03 Transport Medium VZV strain Ellen Detected VZV-15-04 Transport Medium VZV strain OKA Frequently detected VZV-15-05 Transport Medium VZV negative Negative 1.2. PROFICIENCY PER SAMPLE Table II.2. Proficiency per sample Sample Status Positive Negative Comment VZV-15-01 Frequently detected 18 0 0 VZV-15-02 Frequently detected 18 0 0 VZV-15-03 Detected 17 1 1 wrong result VZV-15-04 Frequently detected 18 0 0 VZV-15-05 Negative 0 18 0 18 participants and 5 samples representing 90 results. The number of correct answers was 89 out of 90 (98.9%). 1 false negative result representing 1.4% of the expected positive results. 1.3. SCORES OF THE LABORATORIES The score can ranged from 0 to 14. 17 laboratories (94.4%) obtained the ideal score of 0. 1 laboratory (5.6%) obtained a score of 2 FORM 43/125/E V6 15/36
1.4. PROFICIENCY PER METHOD Table II.3a. Proficiency per method. N N results Good Results % FN FP Ranking Qiagen Artus VZV RG PCR 2 10 10 100.0 0 0 1 Biomérieux HSV1 HSV2 VZV R-gene kit 7 35 35 100.0 0 0 1 Diagenode VZVirus Real-Time PCR 1 5 5 100 0 0 1 Realtime in house 8 40 39 97.5 1 0 2 18 90 89 98.9% 1 0 N : number of laboratories Table II.3b. Proficiency per method, per sample and per laboratory Method N VZV15-01 VZV15-02 BVZV15-03 VZV15-04 VZV15-05 FD FD Detected FD Negative biomerieux R-gene Quant Kit 7 Diagenode Real Time kit 1 Qiagen Artus Real Time 2 Real-time In-House PCR 8 0 0 2 0 0 FORM 43/125/E V6 16/36
2.ENTEROVIRUSES 25 Belgian laboratories encoded results. 2.1. THE SAMPLES Table II.4. Characteristics of the samples. Sample Matrix Content Status EV15-01 Transport Medium Coxsackievirus B3 Frequently detected EV15-02 Transport Medium Coxsackievirus B4 Frequently detected EV15-03 Transport Medium Enterovirus 68 Frequently detected EV15-04 Transport Medium Enterovirus 71 Frequently detected EV15-05 Transport Medium EV negative Negative 2.2. PROFICIENCY PER SAMPLE Table II.5. Proficiency per sample. Sample Status Negative Positive Comment EV15-01 Frequently detected 0 25 OK EV15-02 Frequently detected 0 25 OK EV15-03 Frequently detected 0 25 OK EV15-04 Frequently detected 0 25 OK EV15-05 Negative 25 0 OK 25 participants and 5 samples representing 125 results. The number of correct answers was 125 out of 125 (100 %). The cumulative score can range from 0 to 15. All the laboratories obtained an ideal score of 0 2.3. SCORES OF THE LABORATORIES FORM 43/125/E V6 17/36
2.4. PROFICIENCY PER METHOD The proficiency per method is listed in tables II.8 Table II.6. Proficiency per method KitType N N results N Good results % FN FP ranking Cepheid Xpert EV 14 70 70 100 0 0 1 Biomerieux Enterovirus R-gene 2 10 10 100 0 0 1 Realtime in house 9 45 45 100 0 0 1 Total 25 125 125 100 0 0 N: number of participants ; 3 : false positive ; 1 : false negative. FORM 43/125/E V6 18/36
3. HERPES SIMPLEX VIRUS 22 Belgian laboratories encoded results. 3.1. THE SAMPLES Table II.7. Characteristics of the samples. Sample Matrix Content Status HSV15-1 Transport Medium HSV1 HSV15-2 Transport Medium HSV1 HSV15-3 Transport Medium HSV2 HSV15-4 Transport Medium HSV2 HSV15-5 Transport Medium HSV negative Frequently detected Frequently Detected Detected Frequently Detected Negative 3.2. PROFICIENCY PER SAMPLE Table II.8. Proficiency per sample. Sample Status Negative Positive Comment HSV15-1 Frequently detected 0 22 OK HSV15-2 Frequently Detected 0 22 OK HSV15-3 Detected 1 21 1 wrong result HSV15-4 Frequently Detected 0 22 0 HSV15-5 Negative 22 0 0 22 participants and 5 samples representing 110 results. The number of correct answers was 109 out of 110 (99.1%). 1 false negative result representing 1.1% of the expected positive results was encoded. 3.3. SCORES OF THE LABORATORIES The cumulative score can range from 0 to 14. 21 laboratories (95.4%) obtained an ideal score of 0 1 laboratory ( 4.6%) obtained a score of 2 FORM 43/125/E V6 19/36
3.4. PROFICIENCY PER METHOD The proficiency of the methods IS illustrated in tables II.9a and II.9b. Table II.9a. proficiency per method. KitType N N results N good results % FN FP Ranking Qiagen HSV-1/2 PCR Kit (RG) 3 15 15 100 0 0 1 Realtime In house 12 60 60 100 0 0 1 Abbott HSV 1/2 PCR Kit 1 5 5 100 0 0 1 Biomérieux HSV1 HSV2 VZV R-gene kit 6 30 29 96.7 1 0 2 Total 22 110 109 99.1% 1 0 N : number of participant Table II.9b. Proficiency per method, per sample and per participant Method N HSV15-01 HSV15-02 HSV15-03 HSV15-04 HSV15-05 FD FD Detected FD Negative Abbott Realtime 1 biomerieux R-gene Quant Kit 6 0 0 2 0 0 Qiagen Artus Real Time 3 Real-time In-House PCR 12 FORM 43/125/E V6 20/36
4. HEPATITIS B VIRUS 20 laboratories encoded results. Table II.10. Characteristics of the samples. 4.1. THE SAMPLES Samples Matrix Content status HBV15-01 Plasma HBV type A Frequently Detected HBV15-02 Plasma HBV type A Frequently Detected HBV15-03 Plasma HBV type D Frequently detected HBV15-04 Plasma No HBV Negative 4.2. PROFICIENCY PER SAMPLE : QUALITATIVE RESULTS Table II.11. Proficiency per sample Sample Status Negative Positive Comment HBV15-01 Frequently Detected 0 20 Ok HBV15-02 Frequently Detected 0 20 Ok HBV15-03 Frequently detected 0 20 Ok HBV15-04 Negative 20 0 Ok 20 participants and 4 samples representing 80 results. All the encoded results were qualitatively correct. 4.3. SCORE PER LABORATORY The cumulative score could range from 0 to 22. 17 laboratories (100%) obtained an ideal score of 0. Table II.12. Used methods 4.4. METHODS KitType N Abbott RealTime HBV test 8 Altona-RealStar HBV PCR Kit 1.0 1 Qiagen artus HBV PCR Kit (RG) 3 Roche COBAS TaqMan HBV Test 5 RealTime in house PCR 2 Conventional in House PCR 1 FORM 43/125/E V6 21/36
4.5. QUANTITATIVE RESULTS 4.5.1. results distributions The laboratories have also encoded quantitative results (Log 10 IU /ml) (Figure II.1). 15-1 15-2 15-3 Figure II.1. boxplot distribution of the quantitative results (Log10 IU/mL) per sample. 4.5.2. Z Scores The target value was the median of the values. A z-score was calculated according to the statistical method used in IPH (https://www.wivisp.be/qml/activities/external_quality/_down/_fr/methodes_statistiques_appliquees_a_eeq.pdf ). Z= Median-Value /SD SD=(P75-P25)/1,349 A Z score>3 is considered as a wrong result. Table II.13. Repartition of z score per sample. Sample Z <1 1<Z<2 2<Z<3 Z>3 Comment HBV15-01 14 4 2 OK HBV15-02 13 5 2 OK HBV15-03 15 2 2 1 1 wrong result Z scores per method FORM 43/125/E V6 22/36
Table II.14. Z scores per sample and per participant Kit HBV15-01 HBV15-02 HBV15-03 HBV15-04 Target value Log10 IU/mL) 2.90 2.87 3.67 No target SD 0.18 0.20 0.18 ND Method Z score Z score Z score Abbott Realtime 0.25-2.94-0.31 NEGATIVE Abbott Realtime -0.55-0.37-0.44 NEGATIVE Abbott Realtime -0.93-0.37-0.72 NEGATIVE Abbott Realtime -0.27-0.23-0.07 NEGATIVE Abbott Realtime -1.08-0.73-0.99 NEGATIVE Abbott Realtime -0.11-0.69-0.81 NEGATIVE Abbott Realtime -1.93-1.93-3.57 NEGATIVE Abbott Realtime 2.19 2.01 2.40 NEGATIVE Altona Diagnostics RealStar 0.98 1.31 0.26 NEGATIVE Conventional In House PCR 1.27 1.41 0.66 NEGATIVE Qiagen Artus Real Time 0.83 0.76 0.19 NEGATIVE Qiagen Artus Real Time 1.13 0.47 0.90 NEGATIVE Qiagen Artus Real Time 0.75 0.80 1.35 NEGATIVE Real-time In-House PCR -0.15-0.32-0.18 NEGATIVE Real-time In-House PCR -2.55-1.76-2.61 NEGATIVE Roche Cobas Taqman 0.11 0.23 0.82 NEGATIVE Roche Cobas Taqman -0.67-0.54 0.01 NEGATIVE Roche Cobas Taqman 0.22 1.16 1.36 NEGATIVE Roche Cobas Taqman -0.14 0.44-0.01 NEGATIVE Roche Cobas Taqman 0.64 0.61 0.98 NEGATIVE Table II. 15. Proficiency per method Method N Spl Good % Z<1 1<Z<2 2<Z<3 Z>3 ranking RocheCOBAS TaqMan HBV Test 5 15 15 100 13 2 0 0 1 RealTime in House PCR 2 6 6 100 3 1 2 0 1 Conventional in house PCR 1 3 3 100 1 2 0 0 1 Qiagen artus HBV PCR Kit (RG) 3 9 9 100 7 2 0 0 1 Altona-RealStar HBV PCR Kit 1.0 1 3 3 100 1 1 1 0 1 Abbott RealTime HBV test 8 24 23 95.8 16 3 4 1 2 total 20 60 59 98.3 41 11 7 1 FORM 43/125/E V6 23/36
27 Belgian laboratories encoded results. 5. HEPATITIS C VIRUS 5.1. THE SAMPLES Table II.16. Characterization of the samples. Samples Matrix Content Status HCV15-01 Plasma HCV type 1b Frequently detected HCV15-02 Plasma HCV type 1b Frequently detected HCV15-03 Plasma HCV type 3a Frequently detected HCV15-04 Plasma No HCV Negative 5.2. PROFICIENCY PER SAMPLE Table II.17. Proficiency per sample. Sample Status Negative Positive Comment HCV15-01 Frequently detected 0 27 Ok HCV15-02 Frequently detected 0 27 Ok HCV15-03 Frequently detected 0 27 Ok HCV15-04 Negative 27 0 Ok 27 participants and 4 samples representing 108 results. All the laboratories encoded correct answers. 27 laboratories obtained an ideal score of 0. 5.3. SCORE PER LABORATORY Table II. 18. Used methods 5.4. METHODS Method N Abbott real time PCR 12 Altona diagnostics RealStar 1 In house PCR 2 Qiagen Artus RT 2 Roche Cobas Taqman 10 Total 27 FORM 43/125/E V6 24/36
5.5. QUANTITATIVE RESULTS 24 laboratories encoded quantitative results. 5.5.1. Distribution of the results. Log10 IU/ml 15-01 15-02 15-03 Figure II.2. boxplots distribution of the results (Log10 IU/mL) per sample. 5.5.2. Z scores The target value was the median of the values. A z-score was calculated according to the statistical method used in IPH (https://www.wivisp.be/qml/activities/external_quality/_down/_fr/methodes_statistiques_appliquees_a_eeq.pdf ). Z= Median-Value /SD SD=(P75-P25)/1,349 A Z score>3 is considered as a wrong result FORM 43/125/E V6 25/36
Table II.19. Distribution of the Z scores per sample Sample N Z <1 1<Z<2 2<Z<3 Z>3 Comment HCV15-01 26 18 6 2 0 Ok HCV15-02 26 21 5 0 0 Ok HCV15-03 26 19 7 0 0 Ok Total 78 58 18 2 0 Ok % 100 74.4 23.1 2.5 0 5.5.3. Z scores per method Table II.20. Z scores per method BE-IPH_HCV15-01 BE-IPH_HCV15-02 BE-IPH_HCV15-03 Status Frequently detected Frequently detected Frequently detected Target value (Log10 IU/mL) 4.13 4.15 2.81 SD 0.37 0.33 0.38 Method N Zscore Zsocre Z score -0.84-0.80-0.71-0.25-0.29 0.01 0.00-0.56-1.48-0.98-0.91 0.72 0.95 0.54 0.76 Abbott Realtime 12-0.17 0.17-0.36-1.02-1.02-0.45-0.23-0.20-0.23-0.56-0.51-0.47-1.03-0.85-0.43-0.32-0.23-0.11-0.65 0.12-0.70 Altona Diagnostics RealStar 1 1.35 0.77 1.68 Qiagen Artus Real Time 2 1.04 1.01 1.21 0.25 0.04 1.01 In-House PCR 2-2.30-1.90-0.66 1.48 1.09 1.72 0.14 0.08 0.95 0.17-0.04 1.16 0.85 0.78 0.50 0.83 0.35-0.50 Roche Cobas Taqman 9 0.03-0.12 1.20-0.28-0.43-0.02 2.07 1.33 0.58 0.60 0.45-0.14 1.05 0.74 0.08 FORM 43/125/E V6 26/36
6. HCV GENOTYPING 15 Laboratories encoded results. 6.1. THE SAMPLES Table II.21. Characteristic of the samples. Samples Matrix Content HCV15-01 Plasma HCV type 1a HCV15-02 Plasma HCV type 1b HCV15-03 Plasma HCV type 1b HCV15-04 Plasma HCV type 3a HCV15-05 Plasma HCV type 4a HCV15-06 Plasma HCV type 5a HCV15-07 Plasma HCV type 5a HCV15-08 Plasma No HCV FORM 43/125/E V6 27/36
6.2. PROFICIENCY PER SAMPLE Table II.22. Proficiency per sample. Samples Expected result Introduced Results Comment HCV15-01 HCV type 1a 15 type 1a Good answer HCV15-02 HCV type 1b 12 type 1b Good answer 3 type 1 wrong answer HCV15-03 HCV type 1b 12 type 1b 3 type 1 HCV15-04 HCV type 3a 9 type 3a 5 type 3 1 type 4a HCV15-05 HCV type 4a 2 type 4a 13 type 4 Good answer wrong answer Good answer Good answer Wrong answer Good answer Good answer HCV15-06 HCV type 5a 10 type 5a 5 type 5 Good answer Good answer HCV15-07 HCV type 5a 9 type 5a 5 type 5 1 type 6 Good answer Good answer Wrong answer HCV15-08 No HCV 15 not detected Good answer 15 laboratories and 8 samples representing 120 results. The number of correct answers was 112 out of 120 (93.3%). The fact to fail in the detection of genotype 1a or 1b has been considered by the experts as wrong. FORM 43/125/E V6 28/36
6.3. METHODS Table II.23. Proficiency per method per sample and per participant. Method N GT15-01 GT15-02 GT15-03 GT15-04 GT15-05 GT15-06 GT15-07 GT15-08 1a 1b 1b 3a 4a 5a 5a Negative 1a 1 1 3 4 5 5 Negative 1a 1 1 3 4 5 5 Negative Abbott Realtime 5 1a 1b 1b 3 4 5 5 Negative 1a 1 1 3 4 5 5 Negative 1a 1b 1b 3 4 5 5 Negative In-House PCR 2 1a 1b 1b 4a 4a 5a 5a Negative 1a 1b 1b 3a 4a 5a 5a Negative 1a 1b 1b 3a 4 5a 5a Negative 1a 1b 1b 3a 4 5a 5a Negative 1a 1b 1b 3a 4 5a 5a Negative Siemens Versant 8 1a 1b 1b 3a 4 5a 5a Negative 1a 1b 1b 3a 4 5a 5a Negative 1a 1b 1b 3a 4 5a 6 Negative 1a 1b 1b 3a 4 5a 5a Negative 1a 1b 1b 3a 4 5a 5a Negative FORM 43/125/E V6 29/36
7. HIGH RISK PAPILLOMAVIRUS 40 Belgian laboratories encoded results 7.1. THE SAMPLES Table II.24. Characteristics of the samples Sample Matrix Content Status HPV15-01 PreservCyt+ Caski Cells HPV16 Frequently detected HPV15-02 PreservCyt+ Caski Cells HPV16 Frequently detected HPV15-03 PreservCyt+ Hela Cells HPV18 Frequently detected HPV15-04 PreservCyt+ Hela Cells HPV18 Frequently detected HPV15-05 PreservCyt+ CC10b Cells HPV45 Detected HPV15-06 PreservCyt+ Caski/Hela Cells HPV16/18 Frequently detected HPV15-07 PreservCyt+ SiHa Cells HPV16 Infrequently detected HPV15-08 PreservCyt No HPV Negative HPV15-09 PreservCyt+ BSM Cells HPV45/56 Detected HPV15-10 PreservCyt+ Caski/CC10b Cells HPV16/45 Frequently detected FORM 43/125/E V6 30/36
7.2. RESULTS PER SAMPLE Table II. 25. Proficiency per sample Sample Status Positive Negative ND comment HPV15-01 Frequently detected 39 0 1 1 wrong result HPV15-02 Frequently detected 39 0 1 1 wrong result HPV15-03 Frequently detected 39 0 1 1 wrong result HPV15-04 Frequently detected 39 0 1 1 wrong result HPV15-05 Detected 35 4 1 5 wrong results HPV15-06 Frequently detected 39 0 1 1 wrong result HPV15-07 Infrequently detected 20 18 2 20 wrong results HPV15-08 Negative 0 38 2 2 wrong results HPV15-09 Detected 28 11 1 12 wrong results HPV15-10 Frequently detected 38 1 1 2 wrong results Table II. 26. Summary of the results Characteristics N % Total results 400 100 Correct answers 354 88.5 Wrong answers 46 11.5 False negative 34 73.9 Not determined 12 22.1 7.3. SCORES OF THE LABS The score can range from 0 to 26. 1 laboratory encoded only not determined results and was not evaluated. 12 laboratories obtained the ideal score of 0 14 laboratories obtained the score of 1 7 laboratories obtained the score of 2 3 laboratories obtained the score of 3 3 laboratories obtained the score of 5 FORM 43/125/E V6 31/36
7.4. PROFICIENCY PER METHOD Table II. 27. Proficiency per method and per sample Method N Abbott Realtime 15 Cepheid Xpert kit Greiner bio-one 1 1 Hologic Aptima 2 Hologic Cervista NucliSENS EasyQ 1 1 Qiagen Digene HC 5 Real-time In-House PCR 5 Roche Cobas 7 Roche Linear Assay Sacace Real TM 1 1 HPV15-01 HPV15-02 HPV15-03 HPV15-04 HPV15-05 HPV15-06 HPV15-07 HPV15-08 HPV15-09 HPV15-10 FD FD FD FD Detected FD IFD Negative detected FD Score 0 0 0 ND ND 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 0 1 0 1 0 0 0 1 0 1 0 0 0 1 0 1 0 0 0 1 0 1 0 0 0 1 0 1 0 0 0 1 0 0 0 0 2 2 0 0 0 2 0 2 0 0 0 2 3 5 0 0 0 0 2 2 0 0 0 2 0 2 0 0 0 2 0 2 0 1 0 2 0 3 0 0 0 2 0 2 ND ND ND ND ND ND ND ND ND ND ND 0 1 0 0 0 1 0 1 0 0 0 1 0 1 0 0 0 1 0 1 0 0 0 1 0 0 0 2 0 2 0 1 0 2 0 3 0 1 0 2 0 3 0 0 0 0 2 0 1 0 2 0 5 0 0 0 0 2 0 1 0 2 0 5 0 0 0 0 0 1 0 0 0 1 0 0 1 0 0 0 1 0 0 1 0 0 0 1 FORM 43/125/E V6 32/36
Table II.28 Proficiency per method Method N N answers N good answers % FP FN ND Ranking Roche Linear Assay 1 10 10 100 0 0 0 1 Roche Cobas 7 70 68 97.1 0 2 0 2 Abbott Realtime 15 150 140 90.3 0 8 2 3 Greiner bio-one 1 10 9 90 0 1 0 4 Hologic Aptima 2 20 18 90 0 2 0 4 NucliSENS EasyQ 1 10 9 90 0 1 0 4 Sacace Real TM 1 10 9 90 0 1 0 4 Cepheid Xpert kit 1 10 8 80 0 2 0 5 Hologic Cervista 1 10 8 80 0 2 0 5 Real-time In-House PCR 5 50 39 78 0 11 0 6 Qiagen Digene HC 5 50 36 72 0 4 10 7 Total 40 400 354 88.5 0 34 12 FORM 43/125/E V6 33/36
III. PARASITES The panel included 4 samples for the detection of T. gondii in amniotic fluid and 4 samples for the detection of T. gondii in plasma. 14 participants have encoded results. 1. TOXOPLASMA GONDII IN AMNIOTIC FLUID. 1.1. THE SAMPLES Table III.1. Characteristic of the samples. Sample Matrix Content Status BMM2015/11 Plasma Toxoplasma gondii Infrequently detected BMM2015/12 Plasma Toxoplasma gondii Frequently Detected BMM2015/13 Plasma No T. gondii Negative BMM2015/14 plasma Toxoplasma gondii Frequently detected BMM2015/15 Buffer Toxoplasma gondii Negative BMM2015/16 Buffer Toxoplasma gondii Frequently Detected BMM2015/17 Buffer No T. gondii Negative BMM2015/18 Buffer Toxoplasma gondii Frequently detected 1.2. PROFICIENCY PER SAMPLE Table III.2. Proficiency per sample Sample Status Positive Negative ND Comment BMM2015/11 Infrequently detected 3 10 1 11 wrong results BMM2015/12 Frequently Detected 14 0 0 Ok BMM2015/13 Negative 0 13 1 1 wrong result BMM2015/14 Frequently detected 13 1 0 1 wrong result BMM2015/15 Frequently detected 14 0 0 Ok BMM2015/16 Frequently Detected 14 0 0 Ok BMM2015/17 Negative 0 14 0 Ok BMM2015/18 Frequently detected 13 1 0 1 wrong result 14 laboratories and 8 samples, representing 112 results. The number of correct results was 98 out of 112 (87.5%). 12 false negative results were encoded representing 14.3% of the expected positive samples. Most of these false negative results come from the sample 2015/11 which is a very low concentrated sample (<10 tachyzoites per ml). Finally, 2 not determined results were encoded due to inhibition of the PCR reaction. FORM 43/125/E V6 34/36
The score can ranged from 0 to 22. 4 laboratories obtained an ideal score of 0. 8 laboratories obtained a score of 1. 2 laboratories obtained a score of 4. 1.3. SCORE PER LABORATORY 1.4. PROFICIENCY PER METHOD 13 laboratories used an in house real time method and 1 laboratory used a commercial kit based on LAMP technique (Diasorin). Table III.3. Proficiency per method, per sample and per participant Method 2015/11 2015/12 2015/13 2015/14 2015/15 2015/16 2015/17 2015/18 score IFD FD Negative FD FD FD Negative FD Diasorin Iam Toxo 0 0 0 0 1 0 0 1 1 0 0 1 1 0 0 1 1 0 3 4 1 0 0 1 1 0 0 3 0 0 0 0 4 0 0 0 0 Realtime In house 0 0 0 0 1 0 0 1 1 0 0 1 ND 0 ND 0 1 0 0 1 1 0 0 1 IFD: Infrequently detected, FD: frequently detected. FORM 43/125/E V6 35/36
CONCLUSIONS The different surveys were classified in function of their percentage of correct answers (table IV.1). Table IV.I. Classification of the surveys in function of their proficiency. Survey N Res Good Results % Wrong results FP FN ND SP SE # HBV 20 80 80 100.0% 0 0 0 0 100.0% 100.0% 1 HCV 27 108 108 100.0% 0 0 0 0 100.0% 100.0% 1 EV 25 125 125 100.0% 0 0 0 0 100.0% 100.0% 1 HSV 22 110 109 99.1% 1 0 1 0 100.0% 98.9% 2 VZV 18 90 89 98.9% 1 0 1 0 100.0% 98.6% 3 NG 57 570 555 97.4% 15 3 4 8 98.7% 98.8% 4 MTB 24 120 115 95.8% 5 0 2 3 100.0% 97.2% 5 CT 59 590 562 95.3% 28 5 17 6 97.2% 95.9% 6 HCV GT 15 120 112 93.3% 8 0 NA 8 100% NA 7 HPV 40 400 354 88.5% 46 0 34 12 100% 90.6% 8 TG 14 112 98 87.5% 14 0 12 2 100.0% 85.7% 9 Total 321 2425 2307 95.1% 118 8 71 39 98.7% 96.1% CT: Chlamydia trachomatis; MTB Mycobacterium tuberculosis; NG: Neisseria gonorrhoeae; TG: Toxoplasma gondii ; N : number of participants; Res; umber of encoded results, FP: false positive; FN: false negative; ND: not determined; SE: sensitivity; SP: specificity; #: ranking. The results indicated that the percentage of correct results ranged from 87.5% (TG) to 100% (HBV, HCV, EV). The general percentage of correct answers was 95.1%. The specificity ranged from 97.2 to 100%. The sensitivity ranged from 85.7 to 100%. The comparison of the surveys is presented in an indicative way. Indeed, the composition of the different panels can be different from survey to survey. END Scientific Institute of Public Health, Brussels 2016. This report may not be reproduced, published or distributed without the consent of the WIV-ISP. FORM 43/125/E V6 36/36