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Audit and improve! Evaluation of a real-time probe-based PCR assay with internal control for the direct detection of Mycobacterium tuberculosis complex

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Abstract

We retrospectively audited the performance of the commercial kit in use in our laboratory for the detection of Mycobacterium tuberculosis complex (MTBC) and found the sensitivity to be unacceptably low at 69% (52/75). We developed an in-house end-point polymerase chain reaction (PCR) detecting IS6110, an IS-like element of MTBC, and achieved a sensitivity of 90% (66/73) with the same DNA samples, re-emphasising the poor performance of the commercial kit. In order to avoid specificity issues surrounding gel-based PCR, we developed a probe-based real-time PCR assay with an internal control and achieved a sensitivity of 84%, specificity of 97% and diagnostic odds ratio (DOR) of 207. The evaluation was performed on clinically requested samples, so we expect the performance of the assay in real life to match the data from this evaluation. Centers for Disease Control and Prevention (CDC) guidelines recommending nucleic acid tests for the investigation of possible cases of tuberculosis are expected to promote the use of molecular assays. It is important that clinical laboratories do not assume that assays, in-house or commercial, will perform well or that they will continue to perform well. Audit at regular intervals is necessary to maintain confidence and to demonstrate that the assay works to specification in the real test population.

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Abbreviations

PCR:

Polymerase chain reaction

MTBC:

Mycobacterium tuberculosis complex

NAA:

Nucleic acid amplification

TB:

Tuberculosis

NTM:

Non-tuberculous mycobacteria

BSC:

Biosafety cabinet

PPV:

Positive predictive value

NPV:

Negative predictive value

DOR:

Diagnostic odds ratio

CI:

Confidence interval

6FAM:

6-carboxyfluorescein

BHQ:

Non-fluorescent “black hole” quencher

AFB:

Acid-fast bacilli

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Acknowledgements

The work at the Tan Tock Seng Hospital (TTSH) was supported and funded by the Molecular Biology Laboratory, TTSH, and in part by the Ministry of Health’s Health Service Development Program and the National Medical Research Council, Singapore. The work at the Institute of Molecular and Cell Biology (IMCB) was, in part, supported and funded by the Biomedical Research Council, which is part of Singapore’s Agency for Science, Technology, and Research (A*STAR). We thank Yon Hui Yi from the IMCB for the expert technical assistance.

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Authors and Affiliations

  1. Experimental Therapeutics Center, Agency for Science, Technology & Research, 31 Biopolis Way, #03-01 Nanos, Singapore, 138669, Singapore

    M. Inoue & S. Y. Wee

  2. Department of Laboratory Medicine, Tan Tock Seng Hospital (TTSH), 11 Jalan Tan Tock Seng, Singapore, 308433, Singapore

    W. Y. Tang & T. Barkham

Authors
  1. M. Inoue
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  2. W. Y. Tang
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  3. S. Y. Wee
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  4. T. Barkham
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Correspondence to T. Barkham.

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Inoue, M., Tang, W.Y., Wee, S.Y. et al. Audit and improve! Evaluation of a real-time probe-based PCR assay with internal control for the direct detection of Mycobacterium tuberculosis complex. Eur J Clin Microbiol Infect Dis 30, 131–135 (2011). https://doi.org/10.1007/s10096-010-1059-z

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  • DOI: https://doi.org/10.1007/s10096-010-1059-z

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