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JAC Advance Access originally published online on April 15, 2009
Journal of Antimicrobial Chemotherapy 2009 63(6):1121-1127; doi:10.1093/jac/dkp124
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© The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

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

Ariane T. Pietzka{dagger}, Alexander Indra{dagger}, Anna Stöger, Josef Zeinzinger, Miriam Konrad, Petra Hasenberger, Franz Allerberger and Werner Ruppitsch*

Austrian Agency for Health and Food Safety, Spargelfeldstrasse 191, 1220 Vienna, Austria

Received 4 September 2008; returned 25 January 2009; revised 20 February 2009; accepted 12 March 2009

* Corresponding author. Tel: +43-50555-32204; Fax: +43-50555-32219; E-mail: werner.ruppitsch{at}ages.at

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.

Keywords: HRM , rifampicin resistance , MDR-TB , SNP analysis , mutation detection

{dagger} Both authors contributed equally to this work.


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