Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria.
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.
The University of Texas Health Science Center Houston, School of Public Health at Brownsville, Brownsville, TX 78520, USA.
Diana I
DI
Gomez
Susan P
SP
Fisher-Hoch
Andrea S
AS
Bordt
Teresa N
TN
Quitugua
Jaime
J
Robledo
Nataly
N
Alvarez
Nidia
N
Correa
Joseph B
JB
McCormick
Blanca I
BI
Restrepo
D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D003105:Colombia; D012321:DNA-Directed RNA Polymerases; D006801:Humans; D008800:Mexico; D008826:Microbial Sensitivity Tests / Q000379:methods; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D013781:Texas; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis* / Q000382:microbiology
D000995:Antitubercular Agents; D001426:Bacterial Proteins; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin