Resistance to the first-line anti-tuberculosis (TB) drug, isoniazid (INH), is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a minimum inhibitory concentration of ≥0.25 μg/mL and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a superior method for detection of INH-R MTBC compared to current targeted molecular testing methods and could provide earlier diagnosis of drug-resistant TB.

BACKGROUND: Nucleic acid amplification (NAA) tests rapidly detect Mycobacterium tuberculosis complex directly from clinical specimens, providing valuable results for those evaluated for tuberculosis. METHODS: We analyzed characteristics of cases with NAA testing performed, compared cases with positive and negative NAA test results, and calculated turnaround time and time to treatment for all verified cases reported to the National Tuberculosis Surveillance System in the United States during 2011-2017. RESULTS: Among 67082 verified tuberculosis cases with NAA testing information, 30820 (45.9%) were reported as not having an NAA test performed; the proportion without NAA testing declined annually, from 60.5% in 2011 to 33.6% in 2017. Of 67082 verified cases, 27912 (41.6%) had positive, 8215 (12.2%) had negative, and 135 (0.2%) had indeterminate NAA test results. Among the 33937 cases with an acid-fast bacilli (AFB) smear-positive result, 24093 (70.9%) had an NAA test performed; 11490 of the 30244 (38.0%) with an AFB smear-negative result had an NAA test performed. Although sputum was the most common specimen type tested, 79.8% (7023/8804) of nonsputum specimen types had a positive NAA test result. Overall, 63.7% of cases with laboratory testing had NAA test results reported <6 days following specimen collection; for 13891 cases not yet on treatment, median time to treatment after the laboratory report date was 2 days. CONCLUSIONS: Our analyses demonstrate increased NAA test utilization between 2011 and 2017. However, a large proportion of cases did not have an NAA test performed, reflecting challenges in broader uptake, suggesting an opportunity to expand use of this diagnostic methodology.

Fluoroquinolones (FQ) are crucial components of multidrug-resistant tuberculosis (MDR TB) treatment. Varying levels of resistance are associated with specific mutations within the Quinolone Resistance Determining Region (QRDR) of gyrA We sequenced the QRDR from serial isolates of MDR TB patients in the Preserving Effective TB Treatment Study (PETTS) with baseline (FQ(R)) or acquired FQ resistance (FQ(ACQR)) using the IonTorrent™ Personal Genome Machine to a depth of 10,000x and reported single nucleotide polymorphisms in ≥1% of reads. FQ(R) isolates harbored 15 distinct alleles with 1.3 (max=6) on average per isolate. Eighteen alleles were identified in FQ(ACQR) isolates with an average of 1.6 (max=9) per isolate. Isolates from 78% of FQ(ACQR) individuals had mutant alleles identified within 6 months of treatment initiation. Asp94Gly followed by Ala90Val were the predominant alleles in initial FQ-resistant isolates. Seventy-seven percent (36/47) of FQ(ACQR) group patients had isolates with FQ resistance alleles prior to changes to the FQ component of their treatment. Unlike individuals treated initially with other FQs, none of the 21 individuals treated initially with levofloxacin developed genotypic or phenotypic FQ resistance, although, country of residence is likely a contributing factor since 69% of these individuals were from a single country. Initial detection of phenotypic and genotypic resistance occurred simultaneously for most; however, phenotypic resistance occurred earlier in isolates harboring mixtures of very low-abundance (<1% of reads) alleles while genotypic resistance often occurred earlier for low-level resistance-associated alleles. Understanding factors influencing acquisition and evolution of FQ resistance could reveal strategies for improved treatment success.

BACKGROUND: Mutations in the genes inhA, katG and rpoB confer resistance to anti-tuberculosis (TB) drugs isoniazid and rifampin. We questioned whether specific mutations in these genes were associated with different clinical and microbiological characteristics. METHODS: In a multi-country prospective cohort study of MDR-TB, we identified inhA, katG and rpoB mutations in sputum isolates using the Hain MTBDRplus line probe assay. For specific mutations, we performed bivariate analysis to determine relative risk of baseline or acquired resistance to other TB drugs. We compared time-to-sputum-culture-conversion (TSCC) using Kaplan-Meier curves and stratified Cox regression. RESULTS: In total, 447 participants enrolled January 2005-December 2008 from seven countries were included. Relative to rpoB S531L, isolates with rpoB D516V had less cross-resistance to rifabutin, increased baseline resistance to other drugs, and increased acquired fluoroquinolone resistance.Relative to mutation of katG only, mutation of inhA promoter and katG was associated with increased acquired fluoroquinolone resistance and slower TSCC (125.5 vs. 89.0 days). CONCLUSIONS: Specific mutations in inhA and katG are associated with differences in resistance to other drugs and TSCC. Molecular testing may make it possible to tailor treatment and assess additional drug resistance risk according to specific mutation profile.

Background: The American Thoracic Society, U.S. Centers for Disease Control and Prevention, European Respiratory Society, and Infectious Diseases Society of America jointly sponsored this new practice guideline on the treatment of drug-resistant tuberculosis (DR-TB). The document includes recommendations on the treatment of multidrug-resistant TB (MDR-TB) as well as isoniazid-resistant but rifampin-susceptible TB.Methods: Published systematic reviews, meta-analyses, and a new individual patient data meta-analysis from 12,030 patients, in 50 studies, across 25 countries with confirmed pulmonary rifampin-resistant TB were used for this guideline. Meta-analytic approaches included propensity score matching to reduce confounding. Each recommendation was discussed by an expert committee, screened for conflicts of interest, according to the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) methodology.Results: Twenty-one Population, Intervention, Comparator, and Outcomes questions were addressed, generating 25 GRADE-based recommendations. Certainty in the evidence was judged to be very low, because the data came from observational studies with significant loss to follow-up and imbalance in background regimens between comparator groups. Good practices in the management of MDR-TB are described. On the basis of the evidence review, a clinical strategy tool for building a treatment regimen for MDR-TB is also provided.Conclusions: New recommendations are made for the choice and number of drugs in a regimen, the duration of intensive and continuation phases, and the role of injectable drugs for MDR-TB. On the basis of these recommendations, an effective all-oral regimen for MDR-TB can be assembled. Recommendations are also provided on the role of surgery in treatment of MDR-TB and for treatment of contacts exposed to MDR-TB and treatment of isoniazid-resistant TB.

Resistance to the first-line anti-tuberculosis (TB) drug, isoniazid (INH), is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a minimum inhibitory concentration of >/=0.25 mug/mL and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a useful tool for detecting uncommon INH resistance mutations that would otherwise be missed by current targeted molecular testing methods, and suggest that its use (or use of expanded conventional or NGS-based targeted sequencing) may provide earlier diagnosis of INH-R TB.

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.