This week in TB R&D – 7 July 2010

6 Jul 2010
by Working Group

Cepheid® MTB/RIF Assay cartridge

In our latest ‘Sanatorium Files’ post ‘Diagnosis Dilemma’, we commented on the current advances in TB diagnostics. We’re following up on this theme for the next couple of posts to share the scientific data supporting some of the most promising of these new diagnostic tools.

This year, two reports were published in the Journal of Clinical Microbiology on the sensitivity and specificity of the Xpert® MTB/RIF assay in identifying M. tb. The Xpert® MTB/RIF assay is a fully automated, single-step real-time PCR based DNA diagnostic tool. The assay uses a GeneXpert instrument and plastic microfluidic cartridges which contain all necessary buffers and reagents, including Bacillus globigii DNA (a positive control). The ‘necessary buffers and reagents’ contain heminested PCR primers for the amplification of a sequence in the rpoB gene that is specific for members of the M. tb complex and are able to probe for mutations in the 81 base pair rifampin resistance-determining region (RRDR) of the rpoB gene.

Sputum samples are mixed with a sample reagent supplied with the assay, incubated briefly at room temperature, loaded onto a cartridge and then into the GeneXpert machine. Based upon the Xpert® MTB/RIF assay’s defined algorithm, sputum samples can be identified as TB negative, TB drug sensitive, or TB probable drug-resistant in approximately 2hrs.

In the first report, Helb and colleagues determined the limit of detection (the number of bacilli that can be detected with 95% confidence) for M. tb DNA to be approximately 131 CFU/ml of sputum independent of whether fresh or frozen sputum samples are used. Evaluation of the proprietary sample reagent to reduce possible biohazards demonstrated its ability to significantly inactivate live M. tb in sputum samples. Moreover, the Xpert® MTB/RIF assay could correctly identify M. tb samples that were mixed with high numbers of different nontuberculous mycobacteria (NTM) suggesting that the presence of NTM will not produce a false positive M. tb signal.

Testings of clinical sputum samples from suspected TB patients from Vietnam and from culture-positive patients with a previous history of TB from Uganda were conducted to evaluate the ability of the Xpert® MTB/RIF assay to distinguish positive verses negative sputa and probable rifampin resistance, respectively. The results were then compared to standard solid and/or liquid medium culture. Results showed that the Xpert® MTB/RIF assay could correctly identify positive M. tb as well as distinguish drug resistant sputa.

In a second report, Blakemore and colleagues further extended the evaluation of the Xpert® MTB/RIF assay by investigating the dynamic range of the assay, and its sensitivity, specificity, and RIF-resistance detection in heterogeneous sputum samples. In addition, they examined whether contamination of sputum samples with the MTBDRplus amplicon (a line-probe assay) could affect the ability of the Xpert® MTB/RIF assay to accurately identify TB samples. Overall, results suggested that the Xpert® MTB/RIF assay has a wide dynamic range, is able to reproducibly identify various M. tb strains and rpoB mutations, and is not significantly affected by NTM (in agreement with Helb et al.) or deliberate contamination with the MTBDRplus amplicon.

In your opinion, Is the Xpert® MTB/RIF assay a validated, rapid TB diagnostic tool? The rapid detection of TB and drug-resistance in 2hrs is an advantage over standard culture practices; however what are the foreseeable disadvantages? Is this a point-of-care diagnostic? What impact will this have in the field and at what cost in low economic countries? What are your thoughts? Please share them below.

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