Mycobacterium tuberculosis is successfully evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including the genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly and 7 previously sequenced M. tuberculosis genomes, we identified genomewide signatures of positive selection specific to the 47 resistant genomes. By searching for convergent evolution, the independent fixation of mutations at the same nucleotide site or gene, we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode pathways of cell wall biosynthesis, transcriptional regulation and DNA repair. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

BackgroundThe World Health Organization recommends universal drug susceptibility testing for Mycobacterium tuberculosis complex to guide treatment decisions and improve outcomes. We assessed whether DNA sequencing can accurately predict antibiotic susceptibility profiles for first-line anti-tuberculosis drugs. MethodsWhole-genome sequences and associated phenotypes to isoniazid, rifampicin, ethambutol and pyrazinamide were obtained for isolates from 16 countries across six continents. For each isolate, mutations associated with drug-resistance and drug-susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These were predicted to be pan-susceptible if predicted susceptible to isoniazid and to other drugs, or contained mutations of unknown association in genes affecting these other drugs. We simulated how negative predictive value changed with drug-resistance prevalence.Results10,209 isolates were analysed. The greatest proportion of phenotypes were predicted for rifampicin (9,660/10,130; (95.4%)) and the lowest for ethambutol (8,794/9,794; (89.8%)). Isoniazid, rifampicin, ethambutol and pyrazinamide resistance was correctly predicted with 97.1%, 97.5% 94.6% and 91.3% sensitivity, and susceptibility with 99.0%, 98.8%, 93.6% and 96.8% specificity, respectively. 5,250 (89.5%) drug profiles were correctly predicted for 5,865/7,516 (78.0%) isolates with complete phenotypic profiles. Among these, 3,952/4,037 (97.9%) predictions of pan-susceptibility were correct. The negative predictive value for 97.5% of simulated drug profiles exceeded 95% where the prevalence of drug-resistance was below 47.0%. ConclusionsPhenotypic testing for first-line drugs can be phased down in favour of DNA sequencing to guide anti- tuberculosis drug therapy.

Purpose-For intrafraction motion management, a real-time tracking system was developed by combining fiducial marker-based tracking via simultaneous kilovoltage (kV) and megavoltage (MV) imaging and a dynamic multileaf-collimator (DMLC) beam-tracking system.Methods and Materials-The integrated tracking system employed a Varian Trilogy system equipped with kV/MV imaging systems and a Millennium 120-leaf MLC. A gold marker in elliptical motion (2-cm superior-inferior, 1-cm left-right, 10 cycles/min) was simultaneously imaged by the kV and MV imagers at 6.7 Hz, and segmented in real time. With these two 2D projections, the tracking software triangulated the 3D marker position and repositioned the MLC leaves to follow the motion. Phantom studies were performed to evaluate time delay from image acquisition to MLC adjustment, tracking error, and dosimetric impact of target motion with and without tracking.Results-The time delay of the integrated tracking system was ~450 ms. The tracking error using a prediction algorithm was 0.9±0.5 mm for the elliptical motion. The dose distribution with tracking showed better target coverage and less dose to surrounding region over no tracking. The failure rate of the gamma test (3%/3-mm criteria) was 22.5% without tracking , but was reduced to 0.2% with tracking.Conclusion-For the first time, a complete tracking system combining kV/MV image-guided target tracking and DMLC beam tracking was demonstrated. The average geometric error was less than 1 mm, while the dosimetric error was negligible. This system is a promising method for intrafraction motion management.

Significant host heterogeneity in susceptibility to tuberculosis exists both between and within mammalian species. Using a mouse model of infection with virulent Mycobacterium tuberculosis (Mtb), we identified the genetic locus sst1 that controls the progression of pulmonary tuberculosis in immunocompetent hosts. In this study, we demonstrate that within the complex, multigenic architecture of tuberculosis susceptibility, sst1 functions to control necrosis within tuberculosis lesions in the lungs; this lung-specific sst1 effect is independent of both the route of infection and genetic background of the host. Moreover, sst1-dependent necrosis was observed at low bacterial loads in the lungs during reactivation of the disease after termination of anti-tuberculosis drug therapy. We demonstrate that in sst1-susceptible hosts, nonlinked host resistance loci control both lung inflammation and production of inflammatory mediators by Mtb-infected macrophages. Although interactions of the sst1-susceptible allele with genetic modifiers determine the type of the pulmonary disease progression, other resistance loci do not abolish lung necrosis, which is, therefore, the core sst1-dependent phenotype. Sst1-susceptible mice from tuberculosis-resistant and -susceptible genetic backgrounds reproduce a clinical spectrum of pulmonary tuberculosis and may be used to more accurately predict the efficacy of anti-tuberculosis interventions in genetically heterogeneous human populations.