TY - JOUR
T1 - Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria
AU - Gomez, Diana I.
AU - Fisher-Hoch, Susan P.
AU - Bordt, Andrea S.
AU - Quitugua, Teresa N.
AU - Robledo, Jaime
AU - Alvarez, Nataly
AU - Correa, Nidia
AU - McCormick, Joseph B.
AU - Restrepo, Blanca I.
N1 - 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.
PY - 2010/5
Y1 - 2010/5
N2 - 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.
AB - 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.
KW - Colombia
KW - MDR-TB
KW - Molecular beacon
KW - Mycobacterium
KW - Real-time polymerase chain reaction
KW - Rifampicin resistance
KW - Texas-Mexico border
KW - Tuberculosis
UR - http://www.scopus.com/inward/record.url?scp=77951497849&partnerID=8YFLogxK
U2 - 10.1016/j.diagmicrobio.2009.12.007
DO - 10.1016/j.diagmicrobio.2009.12.007
M3 - Artículo en revista científica indexada
C2 - 20227226
AN - SCOPUS:77951497849
SN - 0732-8893
VL - 67
SP - 37
EP - 46
JO - Diagnostic Microbiology and Infectious Disease
JF - Diagnostic Microbiology and Infectious Disease
IS - 1
ER -