Rapid identification of multidrug-resistant Mycobacterium tuberculosis isolates by rpoB gene scanning using high-resolution melting curve PCR analysis

Ariane Tatjana Pietzka, Alexander Indra, Anna Stöger, Josef Zeinzinger, Miriam Konrad, Petra Hasenberger, Franz Allerberger, Werner Ruppitsch
Austrian Agency for Health and Food Safety, Vienna, Austria


Background: Multidrug-resistant (MDR) Mycobacterium tuberculosis poses a serious threat to the control of tuberculosis and constitutes an increasing public health problem. The availability of rapid in vitro susceptibility tests is a prerequisite for optimal patient treatment. Rifampicin resistance caused by diverse mutations in the rpoB gene is an established and widely used surrogate marker for MDR-TB. We used a high-resolution melting (HRM) curve analysis approach to scan for mutations in the rpoB gene. Methods: A total of 49 MDR-TB and 19 fully susceptible non-MDR-TB isolates, as determined by conventional drug susceptibility testing using the BACTEC-MGIT960 system, were used to evaluate the suitability of HRM curve analysis as a rapid and accurate screening system for rifampicin resistance. Results: HRM analysis of the rpoB cluster I site allowed the correct allocation of 44 of the 49 MDR-TB isolates and all of the non-MDR-TB isolates. Three of five MDR-TB isolates (60%) falsely identified as non-MDR-TB harboured the V176F mutation that could be specifically detected by an additional HRM assay. The combined HRM analysis of all strains and isolates exhibited 95.9% sensitivity and 100% specificity. Conclusions: With a positive predictive value of 100% and a negative predictive value of at least 99.9% this combined HRM curve analysis is an ideal screening method for the tuberculosis laboratory, with minimal requirements of cost and time. The method is a closed-tube assay that can be performed in an interchangeable 96- or 384-well microplate format enabling a rapid, reliable, simple and cost-effective handling of even large sample numbers.

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