GWAS for quantitative resistance phenotypes in Mycobacterium tuberculosis reveals resistance genes and regulatory regions.

Drug resistance diagnostics that rely on the detection of resistance-related mutations could expedite patient care and TB eradication. We perform minimum inhibitory concentration testing for 12 anti-TB drugs together with Illumina whole-genome sequencing on 1452 clinical Mycobacterium tuberculosis (MTB) isolates. We evaluate genome-wide associations between mutations in MTB genes or non-coding regions and resistance, followed by validation in an independent data set of 792 patient isolates. We confirm associations at 13 non-canonical loci, with two involving non-coding regions. Promoter mutations are measured to have smaller average effects on resistance than gene body mutations. We estimate the heritability of the resistance phenotype to 11 anti-TB drugs and identify a lower than expected contribution from known resistance genes. This study highlights the complexity of the genomic mechanisms associated with the MTB resistance phenotype, including the relatively large number of potentially causal loci, and emphasizes the contribution of the non-coding portion of the genome.

Performance of the G4 Xpert® MTB/RIF assay for the detection of Mycobacterium tuberculosis and rifampin resistance: a retrospective case-control study of analytical and clinical samples from high- and low-tuberculosis prevalence settings.

BACKGROUND: The Xpert® MTB/RIF (Xpert) assay is a rapid PCR-based assay for the detection of Mycobacterium tuberculosis complex DNA (MTBc) and mutations associated with rifampin resistance (RIF). An updated version introduced in 2011, the G4 Xpert, included modifications to probe B and updated analytic software. METHODS: An analytical study was performed to assess Xpert detection of mutations associated with rifampin resistance in rifampin-susceptible and -resistant isolates. A clinical study was performed in which specimens from US and non-US persons suspected of tuberculosis (TB) were tested to determine Xpert performance characteristics. All specimens underwent smear microscopy, mycobacterial culture, conventional drug-susceptibility testing and Xpert testing; DNA from isolates with discordant rifampin resistance results was sequenced. RESULTS: Among 191 laboratory-prepared isolates in the analytical study, Xpert sensitivity for detection of rifampin resistance associated mutations was 97.7% and specificity was 90.8%, which increased to 99.0% after DNA sequencing analysis of the discordant samples. Of the 1,096 subjects in the four clinical studies, 49% were from the US. Overall, Xpert detected MTBc in 439 of 468 culture-positive specimens for a sensitivity of 93.8% (95% confidence interval [CI]: 91.2%-95.7%) and did not detect MTBc in 620 of 628 culture-negative specimens for a specificity of 98.7% (95% CI: 97.5%-99.4%). Sensitivity was 99.7% among smear-positive cases, and 76.1% among smear-negative cases. Non-determinate MTBc detection and false-positive RIF resistance results were low (1.2 and 0.9%, respectively). CONCLUSIONS: The updated Xpert assay retained the high sensitivity and specificity of the previous assay versions and demonstrated low rates of non-determinate and RIF resistance false positive results.

Gyrase Mutations Are Associated with Variable Levels of Fluoroquinolone Resistance in Mycobacterium tuberculosis.

Molecular diagnostics that rapidly and accurately predict resistance to fluoroquinolone drugs and especially later-generation agents promise to improve treatment outcomes for patients with multidrug-resistant tuberculosis and prevent the spread of disease. Mutations in the gyr genes are known to confer most fluoroquinolone resistance, but knowledge about the effects of gyr mutations on susceptibility to early- versus later-generation fluoroquinolones and about the role of mutation-mutation interactions is limited. Here, we sequenced the full gyrA and gyrB open reading frames in 240 multidrug-resistant and extensively drug-resistant tuberculosis strains and quantified their ofloxacin and moxifloxacin MIC by testing growth at six concentrations for each drug. We constructed a multivariate regression model to assess both the individual mutation effects and interactions on the drug MICs. We found that gyrB mutations contribute to fluoroquinolone resistance both individually and through interactions with gyrA mutations. These effects were statistically significant. In these clinical isolates, several gyrA and gyrB mutations conferred different levels of resistance to ofloxacin and moxifloxacin. Consideration of gyr mutation combinations during the interpretation of molecular test results may improve the accuracy of predicting the fluoroquinolone resistance phenotype. Further, the differential effects of gyr mutations on the activity of early- and later-generation fluoroquinolones requires further investigation and could inform the selection of a fluoroquinolone for treatment.

Markerless tracking of small lung tumors for stereotactic radiotherapy.

PURPOSE: (1) To validate retrospective markerless tracking software for small lung tumors by comparing tracked motion in 4-dimensional planning computed tomography (4DCT) derived kV projection images and known tumor motion in the same 4DCT. (2) To evaluate variability of tumor motion using kV projection images from cone-beam computed tomography (CBCT) scans acquired on different days. METHODS: Nonclinical tumor tracking software (TTS) used a normalized cross correlation algorithm to track the tumor on enhanced kV projection images (e.g., from a CBCT scan). The reference dataset consisted of digitally reconstructed radiographs (DRRs) from one phase of a planning 4DCT. TTS matches two in-plane coordinates and obtains the out-of-plane coordinate by triangulating with match results from other projections. (1) To validate TTS, tracking results were compared with known 4DCT tumor motion for two patients (A and B). Projection images (1 image/1°) were digitally reconstructed for each 4DCT phase. From these, kV projection series were composed simulating full breathing cycles every 20° of gantry rotation [breathing period = 20°/(6°/s) = 3.33 s]. Reference templates were 360 "tumor enhanced" DRRs from the 4DCT expiration phase. TTS-derived tumor motion was compared to known tumor motion on 4DCT. (2) For five patients, TTS-assessed motion during clinical CBCT acquisition was compared with motion on the planning 4DCT, and the motion component in the Y (cranio-caudal)-direction was compared with the motion of an external marker box (RPM, real-time position management). RESULTS: (1) Validation results: TTS for case A (tumor 6.2 cm(3), 32 mm axial diameter) over 360° showed mean motion X (medial-lateral) = 3.4, Y = 11.5, and Z (ventral-dorsal) = 4.9 mm (1 SD < 1.0 mm). Corresponding 4DCT motion was X = 3.1, Y = 11.3, and Z = 5.1 mm. Correlation coefficients between TTS tumor motion and displacement of the tumor's center of mass (CoM) on 4DCT were 0.64, 0.96, and 0.82 (X, Y, and Z, respectively). For case B (4.1 cm(3), 20 mm diameter), due to temporarily decreased tumor visibility preventing TTS from resolving the tumor, robust tracking data were only available between angles 300°-40° and 120°-220°. Mean motion according to TTS was X = 2.0, Y = 7.7, and Z = 8.2 mm (1 SD < 0.9 mm). Tumor motion on 4DCT was X = 1.8, Y = 7.6, and Z = 9.5 mm and correlation coefficients between TTS motion and CoM displacement were 0.59, 0.95, and 0.93 (X, Y, and Z, respectively). (2) CLINICAL RESULTS: TTS revealed a mean intrafraction variation in tumor motion in Y-direction of >2.0 mm (1 SD) in four of five patients. In addition, clinical tumor motion amplitude differed from that seen on planning 4DCT. Internal and external structures that create abrupt density change (e.g., table-top edge, interface between lung/mediastinum and lung/heart) were observed to prevent 360° tracking of the tumor. Correlation coefficients between TTS motion in the Y-direction and the RPM signal (22 observations) ranged from 0.78 to 0.96. In 2D, 241 TTS matches at end-inspiration and end-expiration were visually validated: mean difference was 0.8 mm (SD = 0.7) for both. CONCLUSIONS: TTS can track small lung tumors if these are visible in kV projections. A 4DCT dataset can be used to validate kV tracking of moving targets. TTS and 4DCT displacement agreed to within 2 mm. TTS and RPM motion were closely associated but tumor motion during CBCT can vary from the planning 4DCT.

Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis.

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution--the independent fixation of mutations in the same nucleotide position 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 components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. 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.

First demonstration of combined kV/MV image-guided real-time dynamic multileaf-collimator target tracking.

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-dimensional projections, the tracking software triangulated the three-dimensional 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 approximately 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, and the dosimetric error was negligible. This system is a promising method for intrafraction motion management.

Dominant role of the sst1 locus in pathogenesis of necrotizing lung granulomas during chronic tuberculosis infection and reactivation in genetically resistant hosts.

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.

Evaluation of the effect of treatment of latent tuberculosis infection on QuantiFERON-TB gold assay results.

To evaluate the utility of the QuantiFERON-TB Gold assay for monitoring latent tuberculosis treatment efficacy, the assay was performed serially for healthcare workers receiving isoniazid therapy. After 9 months of isoniazid therapy, all of these healthcare workers remained QuantiFERON-TB Gold positive, and cellular proliferation assays revealed persistently strong purified protein derivative responses. These results do not support the use of the QuantiFERON-TB Gold assay to monitor therapy.

Discordant QuantiFERON-TB Gold test results among US healthcare workers with increased risk of latent tuberculosis infection: a problem or solution?

OBJECTIVE: In late 2006, our hospital implemented use of the QuantiFERON-TB Gold (QFT-G) assay, a whole-blood interferon-gamma release assay, for detection of tuberculosis infection. All newly hired healthcare workers (HCWs) with positive Mantoux tuberculin skin test (TST) results were routinely tested with the QFT-G assay, to take advantage of its higher specificity. We then undertook a quality assurance review to evaluate the QFT-G test results in HCWs with multiple risk factors for latent tuberculosis infection (LTBI). METHODS: The clinical records for TST-positive HCWs tested with the QFT-G assay were reviewed. HCWs with 2 or more risk factors commonly associated with LTBI were classified as "increased risk" (IR). IR HCWs who had negative QFT-G test results underwent repeat QFT-G testing and were offered testing with a different interferon-gamma release assay (T-SPOT.TB) and with extended T cell stimulation assays. RESULTS: Of 143 TST-positive HCWs tested with the QFT-G assay, 26 (18%) had positive results, 115 (81%) had negative results, and 2 (1%) had indeterminate results. Of 82 IR HCWs, 23 (28%) had positive QFT-G test results, and 57 (70%) had negative results. Of the 57 IR HCWs with negative results, 43 underwent repeat QFT-G testing: 41 had negative results again, and 2 had positive results. These 43 HCWs were also offered additional testing with the T-SPOT.TB diagnostic, and 36 consented: 31/36 tested negative, and 5/36 tested positive. Extended assays using the antigens ESAT-6 and CFP-10 confirmed the positive results detected by the overnight assays and yielded positive results for an additional 7/36 (19%) of individuals; strikingly, all 36 HCWs had strongly positive test results with assays using purified protein derivative. CONCLUSIONS: The extreme discordance between the results of our clinical diagnostic algorithm and the results of QFT-G testing raises concern about the sensitivity of the QFT-G assay for detection of LTBI in our HCWs. Results of extended stimulation assays suggest that many of our IR HCWs have indeed been sensitized to Mycobacterium tuberculosis. It is possible that the QFT-G assay identifies those at higher reactivation risk rather than all previously infected, but, in the absence of long-term follow-up data, we should interpret negative QFT-G results with some caution.

Specimen dilution increases the diagnostic utility of the gen-probe mycobacterium tuberculosis direct test.

The Mycobacterium Tuberculosis Direct (MTD) Test is widely used for detection of Mycobacterium tuberculosis complex (MTBC) in respiratory specimens. Based on high specificity, it is accepted that a positive MTD result, in proper clinical context, "rules in" a diagnosis of tuberculosis. Test utility for "ruling out" tuberculosis has been limited by lower sensitivity and negative predictive value (NPV), based in part on poorly defined reaction inhibitors in clinical specimens. After discovering that specimen dilution could decrease concentrations of reaction inhibitors without reaching the MTBC detection limit, the Massachusetts State Laboratory Institute in 2003 began routinely testing paired undiluted and diluted samples in parallel. We retrospectively analyzed results from 105 respiratory specimens (87 smear-positive, 18 smear-negative) tested consecutively over 23 months using this dilution protocol. MTD results for each sample pair were merged into a sum result (positive or negative) and compared with culture outcome as the "gold standard." We found that the MTD sum result always matched the culture outcome, even for smear-negative specimens. Accordingly, the MTD using the dilution protocol had sensitivity, specificity, positive predictive value, and NPV of 100% vs culture.