Mycobacterium tuberculosis

Transcription

1 Mycobacterium tuberculosis 2012 EQA Programme Final Report QAB (MTBDNA12) Prof. Udo Reischl Scientific Expert on behalf of QCMD Report authorised by the QCMD Executive in September 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 1. Programme aims To assess the proficiency of laboratories in the molecular detection of Mycobacterium tuberculosis (M. tuberculosis) complex. 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 2. Programme details MTBDNA12 Date of panel distribution 30/07/2012 Number of respondents 181 (93%) Number of participants 195 Number of datasets submitted 199 Number of countries 32 Number of qualitative datasets submitted 193 (97%) Number of qualitative and quantitative datasets submitted Fourteen participants did not return results. Five of these withdrew officially, citing 'assay not offered' (n=2), 'technical issues' (n=1) and 'other' (n=2). 3. Panel composition Sample Sample content * Sample ** matrix Sample conc. Cells/sample Sample status 6 (3%) Sample type MTB12-05 M. tuberculosis complex PRB 10,000 Frequently detected Core MTB12-03 M. tuberculosis complex PRB 1,000 Frequently detected Core MTB12-01 M. tuberculosis complex PRB 500 Detected Core MTB12-02 M. tuberculosis complex PRB 100 Detected MTB12-04 Mtb negative PRB Negative Core MTB12-07 M. tuberculosis complex Sputum 10,000 Frequently detected Core MTB12-06 M. tuberculosis complex Sputum 500 Detected Core MTB12-09 M. tuberculosis complex Sputum 500 Detected Core MTB12-10 M. tuberculosis complex Sputum 100 Detected MTB12-08 Mtb negative Sputum Negative Core Sample: QCMD panel sample codes for the samples distributed to participants. Sample content: bacterial content of the panel samples. Sample matrix: material used as a matrix in preparation of the panel samples. Sample 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. Sample status: the sample status assigned to each panel sample. Please see Appendix A for more information. Sample type: panel samples classified as core proficiency samples. * Samples contained M. bovis BCG, which belongs to the M. tuberculosis complex and is considered to be nonpathogenic. ** PRB: Protein Rich Buffer (0.1% Tween 20, 0.5% Bovine serum albumin in phosphate buffered saline to simulate cerebrospinal fluid). Sputum: Pooled sputum negative for Mtb complex. Panel samples MTB12-06 and -09 were duplicate samples. 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% 74.4% 17.1% 3.0% 4.0% 1.0% 0.5% 0.0% 0.0% 0.0% 8/8 7/8 6/8 5/8 4/8 3/8 2/8 1/8 0/8 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 6

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 NASBA datasets In-house b PCR Sample Sample Sample Sample conc. Total Conventional Real time e SDA f TMA g content matrix Cells/sample Commercial a Commercial c In-house d n=199 n=19 n=6 n=116 n=46 n=4 n=4 n=4 n % n % n % n % n % n % n % n % MTB12-05 M. tuberculosis complex PRB 10, MTB12-03 M. tuberculosis complex PRB 1, MTB12-01 M. tuberculosis complex PRB MTB12-02 M. tuberculosis complex PRB MTB12-04 Mtb negative PRB MTB12-07 M. tuberculosis complex Sputum 10, MTB12-06 M. tuberculosis complex Sputum MTB12-09 M. tuberculosis complex Sputum MTB12-10 M. tuberculosis complex Sputum MTB12-08 Mtb negative Sputum Table ii: Qualitative performance scores per technology type Total All technologies Conventional Sample Sample status Commercial a In-house b PCR Real time NASBA e SDA f TMA g Commercial c In-house d n=199 n=19 n=6 n=116 n=46 n=4 n=4 n= MTB12-05 Frequently detected MTB12-03 Frequently detected MTB12-01 Detected MTB12-02 Detected MTB12-04 Negative MTB12-07 Frequently detected MTB12-06 Detected MTB12-09 Detected MTB12-10 Detected MTB12-08 Negative Key to Table i and ii Sample: QCMD panel sample codes for the samples distributed to participants. Sample content: bacterial content of the panel samples. Sample matrix: material used as a matrix in preparation of the panel samples. Sample 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. 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: Ecoli s.r.o Amplisens MBT-EPh PCR kit (n=1), Roche COBAS Amplicor MTB (n=12), Seegene MTB Nested ACE Detection (n=1), Seegene Seeplex MTB ACE detection (n=1), Seegene Seeplex MTB Nested ACE Detection (n=3), Seegene Seeplex MTB/NTM ACE detection (n=1). b: Details not presented. c: AnDiaTec Mycobacterium tuberculosis Complex real time PCR Kit (n=1), Bioneer AccuPower MTB & NTM Real-Time PCR Kit (n=1), Cepheid Xpert MTB/RIF (n=46), Diagenode DIA-MT-050 (n=4), GeneProof Mycobacterium tuberculosis PCR Kit (n=1), Hain Lifescience FluoroType MTB (n=1), Nanogen MTB Q-PCR Alert Kit (n=8), PathoFinder RealAccurate MTB kit (n=5), Progenie Molecular Realcycler MTBC (n=1), QIAGEN artus M. tuberculosis PCR Kit (LC) (n=2), QIAGEN artus M. tuberculosis PCR Kit (RG) (n=6), QIAGEN artus M. tuberculosis PCR Kit (TM) (n=1), QIAGEN artus Mycobac. diff. PCR Kit (LC) (n=3), Roche Cobas Taqman MTB test (n=25), Roche Lightcycler Mycobacterium Detection Kit (n=3), Sacace MTB Real-TM (n=1), Seegene Anyplex MTB/NTM (n=6), TIB MolBiol LightMix kit Mycobacterium tuberculosis (n=1). d: Details not presented. e: Hain Lifescience GenoType MTBDRplus (n=3), Vircell Speed-oligo Mycobacteria (n=1). f: Becton Dickinson ProbeTec ET (n=4). g: Gen-Probe Amplified MTD (n=4). 3 of 6

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 199 qualitative datasets received by QCMD 162 (81.4%) contained information on the target gene. Sample Sample Sample conc. content Cells/sample Total datasets IS S rdna rpob IS6110 & MPB64 n=199 n=72 n=36 n=33 n=6 n=15 n=37 n % n % n % n % n % n % n % MTB12-05 M. tuberculosis complex 10, MTB12-03 M. tuberculosis complex 1, MTB12-01 M. tuberculosis complex MTB12-02 M. tuberculosis complex MTB12-04 Mtb negative MTB12-07 M. tuberculosis complex 10, MTB12-06 M. tuberculosis complex MTB12-09 M. tuberculosis complex MTB12-10 M. tuberculosis complex MTB12-08 Mtb negative Other* Not reported Sample: QCMD panel sample codes for the samples distributed to participants. Sample content: bacterial content of the panel samples. Sample 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-12S rrna (n=1), gyrase (n=1), Insertion Sequence IS6110 and Intergenic Transcribed Spacers between 16S and 23S rrna gene (n=1), intergenic transcribed spacer (n=1), IS/ rpob/katg/inha (n=1), IS6110 & 16S-23S ITS (n=1), IS6110, MPB64 and16s rrna gene (n=1), M.tuberculosis complex DNA and rpob gene (n=2), MPB64 and MTBIS6110, rpob (n=1), MPB-70 protein gene (n=1), MTB target DNA, Rif target DNA-rpo B gene (n=1), Multi copy gene (n=1), rpob, katg (n=1), TB gene and rpo B gene (n=1). 4.4 Quantitative results per laboratory Three participants returned six datasets containing quantitative information; these are shown below. Sample Sample content Sample conc. (cells/sample) Summary statistics (log 10 ) Mean SD %CV dataset 1 i dataset 2 ii dataset 3 iii dataset 4 iv MTB12-05 M. tuberculosis complex 10, ,292 10,738 4,505 4,014 39,760 3,200 MTB12-03 M. tuberculosis complex 1, ,569 1,116 2,498 2, MTB12-01 M. tuberculosis complex , ,469 1, MTB12-02 M. tuberculosis complex , MTB12-07 M. tuberculosis complex 10, ,098 7,055 7,740 5,796 8, MTB12-06 M. tuberculosis complex , MTB12-09 M. tuberculosis complex , , MTB12-10 M. tuberculosis complex ,134 2,063 1, Lab a lab b v cells/vial lab c vi cells/vial Sample: QCMD panel sample codes for the samples distributed to participants. Sample content: bacterial content of the panel samples. Sample conc.: predefined specifications for QCMD internal purposes only. 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. Quantitative results were generated by testing the panel samples using the following. i: QIAGEN artus M. tuberculosis PCR Kit (RG); ii:qiagen artus M. tuberculosis PCR Kit (TM), iii: QIAGEN artus M. tuberculosis PCR Kit (LC); iv: QIAGEN artus Mycobac. diff. PCR Kit (LC); v: Real time in-house PCR; vi: QIAGEN artus M. tuberculosis PCR Kit (RG). 4 of 6

5 5. Comments General comments In the QCMD EQA programme 195 participants submitted a total of 199 datasets. PCR continues to be the dominant technology in this EQA programme, with 94% of datasets returned using this technique. The majority of PCR datasets were generated using real time PCR assays (86.6%; n=162). Of the real time PCR datasets 71.6% were generated using commercially developed kits. Qualitative analysis All core samples were detected by 74.4% of participants. This was similar to the 74.9% of datasets that detected all core samples in a similar EQA panel in the 2011 programme. The EQA panel contained two dilution series: one in simulated CSF (PRB) and the other in sputum. A comparison of the results reported by participants on samples in the two different matrices demonstrated better performance on the simulated CSF samples compared to the sputum samples particularly with the samples containing a low number of target organisms. This was also observed in previous programmes. It is well known that molecular MTB detection procedures are highly demanding with respect to an efficient lysis of the rigid cell walls of mycobacterial organisms during the sample preparation steps and target nucleic acid extraction procedures. So the overall performance of a given assay is not only dependent on the selection of a suitable target gene, primer design / sensitive and economic amplification & detection modules - but also on the type of lysis protocol implemented in the individual workflow. The spectrum of current lysis protocols runs from simple boiling to proteolytic enzyme digestions, sonication or alkaline lysis procedures - and the efficiencies of certain protocols with respect to lysis of the rigid mycobacterial cell walls has proven to be highly variable in routine practice (especially when applied to NALC-NaOH decontaminated respiratory specimens - the routine situation). This aspect was apparent and has to be considered in the assessment of the qualitative performance scores per technology type. Four false positive results and four not determined results were reported on the negative PRB panel sample (MTB12-04) and two false positive results and one not determined result were reported on the negative sputum panel sample (MTB12-08). This gave an overall false positivity of 1.5% that was comparable to the 1.8% false positivity in A total of 162 datasets provided information on the target genes used. The most commonly targeted gene was the insertion sequence IS6001 which was targeted either individually or in combination with another gene in 51.9% of datasets. Little difference was observed in the reporting of the correct results across the genes. Quantitative analysis Only six quantitative datasets were returned to QCMD which did not allow full quantitative analysis. Section 4.4 shows the individual quantitative results returned to QCMD Although the numbers of quantitative results remains low 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. 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 6

6 Appendix A Assigning the sample status Sample 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 Sample status Participant's result Negative Not determined Positive Frequently detected Detected Infrequently detected Negative of 6