Ethambutol (EMB) is used as a part of drug regimens for treatment of tuberculosis (TB). Susceptibility of Mycobacterium tuberculosis complex (MTBC) isolates to EMB can be discerned by DNA sequencing to detect mutations in the embB gene associated with resistance. US Public Health Laboratories (PHL) primarily use growth-based drug susceptibility test (DST) methods to determine EMB resistance. The Centers for Disease Control and Prevention (CDC) provides a service for molecular detection of drug resistance (MDDR) by DNA sequencing and concurrent growth-based DST using agar proportion. PHL and CDC test results were compared for 211 MTBC samples submitted to CDC from September 2009 through February 2011. Concordance between growth-based DST results from PHL and CDC was 88.2%. A growth-based comparison of 39 samples, where an embB mutation associated with EMB resistance was detected, revealed a higher percentage of EMB resistance by CDC (84.6%) than by PHL (59.0%) which was significant (P value = 0.002). Discordance between all growth-based test results from PHL and CDC was also significant (P value = 0.003). Most discordance was linked to false susceptibility using the BACTEC MGIT 960 (MGIT) growth-based system. Our analysis supports coalescing growth-based and molecular results for an informed interpretation of potential EMB resistance.

Newer generation fluoroquinolones moxifloxacin (MXF) and levofloxacin (LVX) are becoming more common components of TB treatment regimens. However, the critical concentrations for testing susceptibility of Mycobacterium tuberculosis to MXF and LVX are not yet well established. Additionally, the degree of cross-resistance between ofloxacin (OFX) and these newer fluoroquinolones has not been thoroughly investigated. In this study, minimal inhibitory concentrations (MICs) for MXF and LVX and susceptibility to the critical concentration of OFX were determined using the agar proportion method for 133 isolates of M. tuberculosis. Most isolates resistant to OFX had LVX MICs >1 mug/mL and MXF MICs >0.5mug/mL. The presence of mutations within the GyrA QRDR correlated well with increased MICs, and the level of LVX and MXF resistance was dependent on the specific GyrA mutation present. Substitutions Ala90Val, Asp94Ala and Asp94Tyr resulted in low-level MXF resistance (MICs >0.5 but ≤2 mug/mL) while other mutations led to MXF MIC >2mug/mL. Based on these results, a critical concentration of 1 mug/mL is suggested for LVX and 0.5 mug/ml for MXF drug susceptibility testing by agar proportion with reflex testing for MXF at 2 mug/mL.

Multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis complex (MTBC) are defined by resistance to at least rifampin (RMP) and isoniazid (INH). Rapid and accurate detection of multidrug resistance is essential for effective treatment and interruption of disease transmission of tuberculosis (TB). Over-diagnosis of MDR TB may result in treatment with second-line drugs that are more costly, less effective, and more poorly tolerated than first-line drugs. CDC offers rapid confirmation of MDR TB by molecular detection of mutations associated with resistance (MDDR) to RMP and INH along with analysis for resistance to other first-line and second-line drugs. Simultaneously, CDC does growth-based phenotypic drug susceptibility testing (DST) by the indirect agar proportion method for a panel of first-line and second-line antituberculous drugs. We reviewed discordance between molecular and phenotypic DST for INH and RMP for 285 isolates submitted as MTBC to CDC September 2009-February 2011. We compared CDC's results with those from the submitting public health laboratories (PHL). Concordance between molecular and phenotypic testing at CDC was 97.4% for RMP and 92.5% for INH resistance. Concordance between CDC's molecular testing and PHL DST results was 93.9% for RMP and 90.0% for INH. Overall concordance between CDC molecular and PHL DST results was 91.7% for RMP and INH collectively. Discordance was primarily attributable to absence of known INH-resistance mutations in isolates INH resistant by DST and detection of mutations associated with low-level RMP resistance in isolates that were RMP susceptible by phenotypic DST. Both molecular and phenotypic test results should be considered for diagnosis of MDR TB.

Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis.

The emergence of multi and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure patients receive effective treatment and become non-infectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs, isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB), and the second-line drugs, amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin, (CIP) and ofloxacin (OFX). Nine loci were sequenced: rpoB for resistance to RIF, katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical M. tuberculosis complex isolates, representing a variety of antibiotic resistance patterns, genotypes and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values (as percentages) for the first-line drug loci were rpoB (97.1, 93.6), katG (85.4, 100), inhA (16.5, 100), katG and inhA together (90.6, 100) pncA (84.6, 85.8), and embB (78.6, 93.1). The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates, support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis (Mtb). The Rifampicin Resistance Determinant Region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampicin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTBC) strains from Nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High resolution melting (HRM) analysis following real-time PCR was used to identify Mtb strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific SNP transversion mutations. This method was used to screen 252 Mtb clinical isolates including 154 RIF resistant strains and 174 INH resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated a sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance, and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in Mtb strains and offers several advantages over current molecular and culture-based techniques.