Chlamydia trachomatis

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1 Chlamydia trachomatis 2012 EQA Programme A Final Report QAB (CTDNA12A) Dr Anton M van Loon Scientific Experts on behalf of QCMD Report authorised by the QCMD Executive in May 2012 A UKAS accredited proficiency testing provider (no. 4385) Not to be reproduced or quoted without permission of QCMD. Any queries about this report should be addressed to the QCMD Neutral Office. Unit 5, Technology Terrace, Todd Campus, West of Scotland Science Park, Glasgow, G20 0XA, Scotland Tel: +44 (0) , Fax: +44 (0) , info@qcmd.org, Web:

2 Percentage of datasets 2012 Chlamydia trachomatis A 1. Programme aims To determine the qualitative performance of participants testing for Chlamydia trachomatis (C. trachomatis) using molecular technologies. To determine the incidence of false positive results and to compare performance with previous distributions. Participants are encouraged to read the QCMD Participants' Manual, which can be downloaded from the QCMD website. Any queries about this report should be addressed to the QCMD Neutral Office (neutraloffice@qcmd.org). 2. Programme details CTDNA12A Date of panel distribution 06/02/2012 Number of respondents 236 (97%) Number of participants 244 Number of datasets submitted 263 Number of countries 33 Number of qualitative datasets submitted 260 (99%) Number of qualitative and quantitative datasets submitted Eight participants did not return results. Three of these withdrew officially, citing 'assay not offered' (n=2) and 'technical issues' (n=1). 3 (1%) 3. Panel composition content * matrix conc.** Copies/vial status type CTA12-05 C. trachomatis Urine 4,000 1,000 Frequently detected Core CTA12-03 C. trachomatis Urine 1, Detected Core CTA12-04 C. trachomatis Urine Detected CTA12-01 C. trachomatis Swedish Variant Urine 2, Detected CTA12-02 Ct. negative urine Urine Negative Core CTA12-07 C. trachomatis Sim. Swab 6,250 1,563 Frequently detected Core CTA12-06 C. trachomatis Sim. Swab 1, Frequently detected Core CTA12-08 C. trachomatis Sim. Swab Detected Core CTA12-10 C. trachomatis Sim. Swab Detected CTA12-09 Ct. negative swab Sim. Swab Negative Core : QCMD panel sample codes for the samples distributed to participants. content: bacterial content of the panel samples. matrix: material used as a matrix in preparation of the panel samples. conc.: consensus values calculated from all of the data returned by participants, once outliers had been removed. The values are not technology specific and should not be used by participants for method comparison or as a target for individual laboratory assessment. status: the sample status assigned to each panel sample. Please see Appendix A for more information. type: panel samples classified as core proficiency samples. *Urine: Chlamydia trachomatis-negative urine collected from healthy adult volunteers. Sim. Swab: containing 1/10 dilution of TE buffer designed to simulate a clinical swab sample when reconstituted with sample transport medium. ** The EQA panel consisted of five lyophilized urine samples that participants were instructed to reconstitute with 4.0 ml of water, and 5 lyophilized simulated swab samples that participants were instructed to reconstitute with 4.0ml of their CT transport medium for swabs. 4. Programme results 4.1. Qualitative performance on the core proficiency samples 100.0% 90.0% 80.0% 70.0% 60.0% 50.0% 40.0% 30.0% 20.0% 10.0% 0.0% 87.5% 6.5% 4.9% 0.4% 0.8% 0.0% 0.0% 0.0% 7/7 6/7 5/7 4/7 3/7 2/7 1/7 0/7 Number of core samples correct The QCMD EQA panels contain a range of samples, designed to look at different aspects of assay performance. Panel members are designated core proficiency samples on the basis of scientific information, clinical relevance and clinical experience (published literature and professional clinical guidelines) and, where available and appropriate, established target performance limits taken from previous QCMD EQA distributions. Laboratories are expected to correctly analyse and report the core proficiency samples in order to show acceptable proficiency. 2 of 7

3 4.2. Qualitative analysis of the EQA data for all panel samples The number (percentage) of correct qualitative results are presented below. Qualitative data were returned by participants as 'positive', 'negative' or 'not determined'. Not determined results were counted as incorrect for all panel samples (positive or negative). QCMD organises datasets according to commercial and in-house technology groups, which are Conventional PCR, Real time PCR, NASBA, SDA, TMA and bdna. Where datasets were reported as other for a technology or kit method this was reviewed by the QCMD Neutral Office and assigned to an appropriate group where possible. Table i: Number of correct qualitative results per panel member and technology type conc. content datasets n=263 n=16 n=5 n % n % n % n % n % n % n % CTA12-05 C. trachomatis 1, CTA12-03 C. trachomatis CTA12-04 C. trachomatis CTA12-01 C. trachomatis Swedish Variant CTA12-02 Ct. negative urine CTA12-07 C. trachomatis 1, CTA12-06 C. trachomatis CTA12-08 C. trachomatis CTA12-10 C. trachomatis CTA12-09 Ct. negative swab PCR Total Conventional Real time SDA f TMA g Commercial a In-house b Commercial c In-house d n=169 n=24 n=24 n=25 Table ii: Qualitative performance scores per technology type Total PCR All technologies Conventional Real time SDA f TMA g status Commercial a In-house b Commercial c In-house d n=263 n=16 n=5 n=169 n=24 n=24 n= CTA12-05 Frequently detected CTA12-03 Detected CTA12-04 Detected CTA12-01 Detected CTA12-02 Negative CTA12-07 Frequently detected CTA12-06 Frequently detected CTA12-08 Detected CTA12-10 Detected CTA12-09 Negative Key to Table i and ii : QCMD panel sample codes for the samples distributed to participants. content: bacterial content of the panel samples. conc.: consensus values calculated from all of the data returned by participants, once outliers had been removed. The values are not technology specific and should not be used by participants for method comparison or as a target for individual laboratory assessment. Total datasets: number and percentage of datasets reporting the correct qualitative result for each panel sample. status: the sample status assigned to each panel sample. Please see Appendix A for more information. Total. All technologies: number of datasets awarded each score (0 to 3). A breakdown of the results for all datasets is also provided based on technology type. a: Hain Lifescience GenoQuick CT (n=1), Roche Amplicor CT/NG (n=4), Roche COBAS Amplicor CT/NG (n=8), Sacace Chlamydia trachomatis 330/740 IC (n=1), Seeplex STI Master ACE Detection (n=2). b: Details not presented. c: Abbott RealTime CT (n=5), Abbott RealTime CT/NG (n=40), ABI Chlamydia trachomatis RG detect (n=1), BD ProbeTec CT Qx Amplified DNA Assay (n=1), Bio-Rad Dx CT/NG/MG Assay (n=1), Diagenode Dia-CT/NG-050 (n=9), GeneProof Chlamydia Trachomatis PCR Kit (n=2), Goffin Presto CT-NG assay (n=4), IAB C. trachomatis RG detect (n=3), Minerva Biolabs Onar Ct qpcr Kit (n=1), Nanogen Chlamydia tr. Q-PCR Alert Kit (n=5), QIAGEN artus C. trachomatis PCR Kit (TM) (n=2), QIAGEN artus C. trachomatis Plus PCR Kit (LC) (n=5), QIAGEN artus C. trachomatis Plus PCR Kit (RG) (n=7), Roche cobas 4800 CT/NG Test (n=37), Roche COBAS TaqMan CT Test v2.0 (n=34), Sacace N. gonorrhoeae/c. trachomatis/t. vaginalis/m. genitalium Real-TM (n=1), Seegene Anyplex CT/NG Real-time Detection (n=1), Seegene Anyplex II STI-7 Detection Assay (n=1), Seegene Seeplex STD6 ACE Detection (n=4), Siemens Versant CT/GC DNA 1.0 assay (n=2), TIB MOLBIOL LightMix Kit (n=3). d: Details not presented. f: BD Diagnostics BD ProbeTec ET System (n=7), BD Diagnostics Viper ProbeTec ET (n=2), BD Probetec ET CT/GC Amplified DNA Assay (n=6), BD Probetec ET CT/GC/AC Amplified DNA Assay (n=9). g: Gen-Probe APTIMA Combo 2 Assay (n=19), Gen-Probe APTIMA CT Assay (n=6). 3 of 7

4 4.3. Qualitative analysis of the EQA data by assay target gene Participants were asked to specify the target gene of their assay when submitting their results. Out of the 263 datasets received by QCMD 255 (97%) contained information on the target gene. conc. content Total datasets Cryptic plasmid Cryptic plasmid + other target n=263 n=127 n=94 n % n % n % n % n % n % n % CTA12-05 C. trachomatis 1, CTA12-03 C. trachomatis CTA12-04 C. trachomatis CTA12-01 C. trachomatis Swedish Variant CTA12-02 Ct. negative urine CTA12-07 C. trachomatis 1, CTA12-06 C. trachomatis CTA12-08 C. trachomatis CTA12-10 C. trachomatis CTA12-09 Ct. negative swab n=26 n=5 Other* n=3 Not reported : QCMD panel sample codes for the samples distributed to participants. content: bacterial content of the panel samples. conc.: predefined specifications for QCMD internal purposes only. Values should not be used by participants for method comparison or as a target for individual laboratory performance assessment. Total datasets: number and percentage of datasets reporting the correct qualitative result for each panel sample. A breakdown of the results for all datasets is also provided based on target gene. * 16S and ompa (n=1), inca AF (n=1), pmph Gene (n=1). rrna ompa n= Quantitative results per laboratory Three participants provided quantitative results; these are shown below content conc. (copies/ml) Summary statistics (log 10 ) Mean SD %CV Lab a Lab a Lab b ii dataset 1 i copies/ml dataset 2 i copies/ml copies/ml CTA12-05 C. trachomatis 1, ,900 CTA12-03 C. trachomatis ,000 CTA12-04 C. trachomatis CTA12-01 C. trachomatis Swedish Variant CTA12-07 C. trachomatis 1, ,500 CTA12-06 C. trachomatis ,500 CTA12-08 C. trachomatis CTA12-10 C. trachomatis : QCMD panel sample codes for the samples distributed to participants. content: bacterial content of the panel samples. conc.: predefined specifications for QCMD internal purposes only. Values should not be used by participants for method comparison or as a target for individual laboratory performance assessment. Summary statistics (log10): the mean, standard deviation and coefficient of variation expressed as a percentage are presented. These values were calculated from the log concentration of participants results. The following assays were used. i: QIAGEN artus C. trachomatis PCR Kit (TM) ii: Real time in-house PCR on the ABI PRISM 7900 HT. 4 of 7

5 5. Comments General observations The number of participants in the 2012 QCMD Chlamydia trachomatis (CT) A EQA programme increased by 9.4% from 223 in 2011A to 244 in this EQA programme. The majority of datasets were generated using commercial kits, with only 11.0% (n=29) of datasets utilising inhouse assays. PCR was the dominant technology in this EQA programme, with 81.4% datasets returned using this technique. The majority of PCR datasets were generated using real time PCR assays (90.2%; n=193). Of the real time PCR datasets 87.6% were generated using commercially developed kits. Qualitative performance All core samples were detected by 87.5% of participants. This is an increase on the 80.5% of the datasets that detected all core samples in the 2011 CT A EQA programme. The 2012 CT A EQA panel contained dilution series containing Chlamydia trachomatis in both urine and simulated swab. Most participants (over 90%) were able to detect all but the lowest concentration samples in both dilution series. Little difference in performance was observed based on technology group, although both in-house and commercial real time PCR assays look to have performed slightly better than other technology groups particularly on the lowest concentration samples in both dilution series. Four not determined and two false positive results (0.7%) were reported on the negative urine panel sample CTA One not determined and two false positive results (0.7%) were also reported on the negative simulated swab panel sample CTA This gives an overall false positivity of (0.7%) which compares favourably to the percentage of false positive results reported in the 2011A C. trachomatis EQA programme (1.1%). Results for the Swedish variant Chlamydia trachomatis Panel sample CTA12-01 contained the Swedish variant (Ripa et al: 2007) of C. trachomatis at lower concentration. For most technology groups the percent range of correct positive results was between 60% and 90%. For Conventional Commercial PCR technologies it was much lower at only 31.3%, however this does represent a continued improvement on previous EQA programmes. Results by assay target gene The most commonly used target gene in datasets received by QCMD was the cryptic plasmid (n=127; 49.8%) only, or in combination with another target gene (n=88; 36.7%). This represents an increase in the proportion of datasets returned using another target gene as well as the cryptic plasmid. No major differences in performance were observed between assay targets genes used. Analysis of the quantitative EQA data Only three quantitative datasets were returned to QCMD which did not allow full quantitative analysis. Individual quantitative results returned to QCMD are presented in section 4.5. Although the numbers of quantitative results remains low (2010: n=3) this area may develop in the future. The variability in the quantitative results returned indicates that the availability of EQA and reference materials will be of value in the establishment of quantitative assays. References Ripa T, Nilsson PA. A Chlamydia trachomatis strain with a 377-bp deletion in the cryptic plasmid causing false-negative nucleic acid amplification tests. Sexually Transmitted Diseases, 2007; 34(5): Acknowledgements Data analysis and report generation were performed by the QCMD Neutral Office. QCMD The QCMD EQA programme samples, associated reports and data generated during this programme are intended for External Quality Assessment (EQA) and Proficiency Testing (PT) purposes only. QCMD operates according to a strict Code of Practice which is in line with ISO/IEC and associated standards. Data reported in QCMD programmes is representative of a laboratory s standard diagnostic testing protocols irrespective of the technology they use. The data provided in the reports are based on technical information provided by the individual laboratories as part of the assessment process, as such it does not constitute a formal technology method comparison. All text and images produced by QCMD are the property of QCMD unless otherwise stated. The reproduction and use of these materials is not permitted without the express written consent of QCMD. The use of the information provided in QCMD reports for commercial purposes is strictly prohibited. 5 of 7

6 Appendix A Assigning the sample status status is assigned by peer-group consensus, based on the qualitative results returned by all participants. It is not a measure of the 'strength' of a positive sample nor is it technology-dependent, and is used solely for the scoring of the EQA data. The rationale for the sample status is: Frequently detected: More than 95% of datasets recorded the correct positive result. Detected: Between 65% and 95% of datasets recorded the correct positive result. Infrequently detected: Less than 65% of datasets recorded the correct positive result. Negative: A panel sample that does not contain the target and produces an unequivocal negative result. Scoring system for qualitative EQA data The scores awarded for qualitative EQA data were based on the sample status. The scoring system is represented in the following table, where 0 is 'highly satisfactory' and 3 is 'highly unsatisfactory'. Colour has been included as an extra visual aid. Scoring system based on the assigned sample status status Participant's result Negative Not determined Positive Frequently detected Detected Infrequently detected Negative of 7

7 Appendix B: Manufacturers' Section All commercial manufacturers of molecular diagnostic assays are encouraged to independently test the EQA panel and present the results of their own analysis of the EQA panel in this section of the report. The information and data are provided independent of QCMD and at the sole discretion and liability of the assay manufacturer. The "manufacturers' section" is intended to provide EQA users of a particular commercial manufacturer's assay with additional information and is based on data and information supplied independent of the QCMD EQA process. Whilst every effort has been made to ensure that the content of this section of the report is both reliable and accurate, QCMD advises that the information should not be relied upon in any way as representing a comprehensive description of the commercial manufacturer's assay and that users should discuss this detail directly with the manufacturer concerned. QCMD also advises that it cannot guarantee, and assumes no legal liability or responsibility for, the accuracy, reliability or completeness of the information contained within this section of the report. B.1.1. Panel results provided by Roche Molecular Systems Roche Molecular Systems, Pleasanton, CA tested the QCMD CTDNA12A EQA panels and a summary of their qualitative results is provided in Table B1. The results from all EQA participant laboratories are presented for comparison. B.1.2. Qualitative analysis of the EQA data The number (percentage) of correct qualitative results is presented in Table B1. Qualitative data were returned as 'positive', 'negative' or 'not determined'. Not determined results were counted as incorrect for all panel samples (positive or negative). Table B1: Number of correct qualitative results per panel member and technology type Total content matrix conc. datasets type n=263 Total Roche datasets Roche technologies Cobas 4800 a Cobas TaqMan b Cobas Amplicor c results d n=83 n=37 n=34 n=12 n=1 n % n % n % n % n % n % CTA12-05 C. trachomatis Urine 1,000 Core CTA12-03 C. trachomatis Urine 250 Core CTA12-04 C. trachomatis Urine CTA12-01 C. trachomatis Sv Urine CTA12-02 Ct. negativ e urine Urine Core CTA12-07 C. trachomatis Sim. Swab 1,563 Core CTA12-06 C. trachomatis Sim. Swab 313 Core CTA12-08 C. trachomatis Sim. Swab 63 Core CTA12-10 C. trachomatis Sim. Swab CTA12-09 Ct. negativ e swab Sim. Swab Core Roche Key to Table B1 : QCMD panel sample codes for the samples distributed to participants. content: bacterial content of the panel samples. matrix: material used as a matrix in preparation of the panel samples. conc.: predefined specifications for QCMD internal purposes only. Values should not be used by participants for method comparison or as a target for individual laboratory performance assessment. type: panel samples classified as core proficiency samples. Total datasets: number and percentage of datasets reporting the correct qualitative result for each panel sample. Roche technologies: number and percentage of datasets Roche Molecular Systems technologies reporting the correct qualitative result for each panel sample a: Qualitative results returned by EQA participants using the Roche cobas 4800 CT/NG Test. b: Qualitative results returned by EQA participants using the Roche COBAS TaqMan CT Test v2.0. c: Qualitative results returned by EQA participants using the Roche COBAS Amplicor CT/NG Test. d: Qualitative results returned from Roche Molecular Systems using Roche cobas 4800 CT/NG Test. 7 of 7