Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria

Diana I. Gomez, Susan P. Fisher-Hoch, Andrea S. Bordt, Teresa N. Quitugua, Jaime Robledo, Nataly Alvarez, Nidia Correa, Joseph B. McCormick, Blanca I. Restrepo

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. Rifampicin (RIF) resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes ≤2 days. However, qPCR identification of resistant isolates, particularly for isolates with mixed RIF-susceptible and RIF-resistant bacteria, is reader dependent and limits its clinical use. The aim of this study was to develop an objective, reader-independent method to define rpoB mutants using beacon qPCR. This would facilitate the transition from a research protocol to the clinical setting, where high-throughput methods with objective interpretation are required. For this, DNAs from 107 M. tuberculosis clinical isolates with known susceptibility to RIF by culture-based methods were obtained from 2 regions where isolates have not previously been subjected to evaluation using molecular beacon qPCR: the Texas-Mexico border and Colombia. Using coded DNA specimens, mutations within an 81-bp hot spot region of rpoB were established by qPCR with 5 beacons spanning this region. Visual and mathematical approaches were used to establish whether the qPCR cycle threshold of the experimental isolate was significantly higher (mutant) compared to a reference wild-type isolate. Visual classification of the beacon qPCR required reader training for strains with a mixture of RIF-susceptible and RIF-resistant bacteria. Only then had the visual interpretation by an experienced reader had 100% sensitivity and 94.6% specificity versus RIF resistance by culture phenotype and 98.1% sensitivity and 100% specificity versus mutations based on DNA sequence. The mathematical approach was 98% sensitive and 94.5% specific versus culture and 96.2% sensitive and 100% specific versus DNA sequence. Our findings indicate the mathematical approach has advantages over the visual reading, in that it uses a Microsoft Excel template to eliminate reader bias or inexperience, and allows objective interpretation from high-throughput analyses even in the presence of a mixture of RIF-resistant and RIF-susceptible isolates without the need for reader training.

Original languageEnglish
Pages (from-to)37-46
Number of pages10
JournalDiagnostic Microbiology and Infectious Disease
Volume67
Issue number1
DOIs
StatePublished - May 2010
Externally publishedYes

Bibliographical note

Funding Information:
This project was supported by grants NIH-R21 AI 064297-01A1 and by the Hispanic Health Research Center EXPORT grant NIHMHD P20 MD000170 020.

Keywords

  • Colombia
  • MDR-TB
  • Molecular beacon
  • Mycobacterium
  • Real-time polymerase chain reaction
  • Rifampicin resistance
  • Texas-Mexico border
  • Tuberculosis

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