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J Antimicrob Chemother. 2009 Jun;63(6):1121-7. doi: 10.1093/jac/dkp124. Epub 2009 Apr 15.

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

Author information

1
Austrian Agency for Health and Food Safety, Vienna, Austria.

Erratum in

  • J Antimicrob Chemother. 2009 Aug;64(2):436.

Abstract

BACKGROUND:

Multidrug-resistant (MDR) Mycobacterium tuberculosis poses a serious threat to the control of tuberculosis (TB) 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 non-MDR-TB isolates. Three of the 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 TB 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.

PMID:
19369271
DOI:
10.1093/jac/dkp124
[Indexed for MEDLINE]

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