Implications of Race Adjustment in Lung-Function Equations.

BACKGROUND: Adjustment for race is discouraged in lung-function testing, but the implications of adopting race-neutral equations have not been comprehensively quantified. METHODS: We obtained longitudinal data from 369,077 participants in the National Health and Nutrition Examination Survey, U.K. Biobank, the Multi-Ethnic Study of Atherosclerosis, and the Organ Procurement and Transplantation Network. Using these data, we compared the race-based 2012 Global Lung Function Initiative (GLI-2012) equations with race-neutral equations introduced in 2022 (GLI-Global). Evaluated outcomes included national projections of clinical, occupational, and financial reclassifications; individual lung-allocation scores for transplantation priority; and concordance statistics (C statistics) for clinical prediction tasks. RESULTS: Among the 249 million persons in the United States between 6 and 79 years of age who are able to produce high-quality spirometric results, the use of GLI-Global equations may reclassify ventilatory impairment for 12.5 million persons, medical impairment ratings for 8.16 million, occupational eligibility for 2.28 million, grading of chronic obstructive pulmonary disease for 2.05 million, and military disability compensation for 413,000. These potential changes differed according to race; for example, classifications of nonobstructive ventilatory impairment may change dramatically, increasing 141% (95% confidence interval [CI], 113 to 169) among Black persons and decreasing 69% (95% CI, 63 to 74) among White persons. Annual disability payments may increase by more than $1 billion among Black veterans and decrease by $0.5 billion among White veterans. GLI-2012 and GLI-Global equations had similar discriminative accuracy with regard to respiratory symptoms, health care utilization, new-onset disease, death from any cause, death related to respiratory disease, and death among persons on a transplant waiting list, with differences in C statistics ranging from -0.008 to 0.011. CONCLUSIONS: The use of race-based and race-neutral equations generated similarly accurate predictions of respiratory outcomes but assigned different disease classifications, occupational eligibility, and disability compensation for millions of persons, with effects diverging according to race. (Funded by the National Heart Lung and Blood Institute and the National Institute of Environmental Health Sciences.).

Mutation rates and adaptive variation among the clinically dominant clusters of Mycobacterium abscessus.

Mycobacterium abscessus (Mab) is a multidrug-resistant pathogen increasingly responsible for severe pulmonary infections. Analysis of whole-genome sequences (WGS) of Mab demonstrates dense genetic clustering of clinical isolates collected from disparate geographic locations. This has been interpreted as supporting patient-to-patient transmission, but epidemiological studies have contradicted this interpretation. Here, we present evidence for a slowing of the Mab molecular clock rate coincident with the emergence of phylogenetic clusters. We performed phylogenetic inference using publicly available WGS from 483 Mab patient isolates. We implement a subsampling approach in combination with coalescent analysis to estimate the molecular clock rate along the long internal branches of the tree, indicating a faster long-term molecular clock rate compared to branches within phylogenetic clusters. We used ancestry simulation to predict the effects of clock rate variation on phylogenetic clustering and found that the degree of clustering in the observed phylogeny is more easily explained by a clock rate slowdown than by transmission. We also find that phylogenetic clusters are enriched in mutations affecting DNA repair machinery and report that clustered isolates have lower spontaneous mutation rates in vitro. We propose that Mab adaptation to the host environment through variation in DNA repair genes affects the organism's mutation rate and that this manifests as phylogenetic clustering. These results challenge the model that phylogenetic clustering in Mab is explained by person-to-person transmission and inform our understanding of transmission inference in emerging, facultative pathogens.

Phase variation as a major mechanism of adaptation in Mycobacterium tuberculosis complex.

Phase variation induced by insertions and deletions (INDELs) in genomic homopolymeric tracts (HT) can silence and regulate genes in pathogenic bacteria, but this process is not characterized in MTBC (Mycobacterium tuberculosis complex) adaptation. We leverage 31,428 diverse clinical isolates to identify genomic regions including phase-variants under positive selection. Of 87,651 INDEL events that emerge repeatedly across the phylogeny, 12.4% are phase-variants within HTs (0.02% of the genome by length). We estimated the in-vitro frameshift rate in a neutral HT at 100× the neutral substitution rate at [Formula: see text] frameshifts/HT/year. Using neutral evolution simulations, we identified 4,098 substitutions and 45 phase-variants to be putatively adaptive to MTBC (P < 0.002). We experimentally confirm that a putatively adaptive phase-variant alters the expression of espA, a critical mediator of ESX-1-dependent virulence. Our evidence supports the hypothesis that phase variation in the ESX-1 system of MTBC can act as a toggle between antigenicity and survival in the host.

Exploring gene content with pangene graphs.

MOTIVATION: The gene content regulates the biology of an organism. It varies between species and between individuals of the same species. Although tools have been developed to identify gene content changes in bacterial genomes, none is applicable to collections of large eukaryotic genomes such as the human pangenome. RESULTS: We developed pangene, a computational tool to identify gene orientation, gene order and gene copy-number changes in a collection of genomes. Pangene aligns a set of input protein sequences to the genomes, resolves redundancies between protein sequences and constructs a gene graph with each genome represented as a walk in the graph. It additionally finds subgraphs, which we call bibubbles, that capture gene content changes. Applied to the human pangenome, pangene identifies known gene-level variations and reveals complex haplotypes that are not well studied before. Pangene also works with high-quality bacterial pangenome and reports similar numbers of core and accessory genes in comparison to existing tools. AVAILABILITY AND IMPLEMENTATION: Source code at https://github.com/lh3/pangene; pre-built pangene graphs can be downloaded from https://zenodo.org/records/8118576 and visualized at https://pangene.bioinweb.org.

Drug resistance and epidemiological success of modern Mycobacterium tuberculosis lineages in western India.

BACKGROUND: Transmission is contributing to the slow decline of tuberculosis (TB) incidence globally. Drivers of TB transmission in India, the country estimated to carry a quarter of the World's burden, are not well studied. We conducted a genomic epidemiology study to compare epidemiological success, host factors and drug resistance (DR) among the four major Mycobacterium tuberculosis (Mtb) lineages (L1-4) circulating in Pune, India. METHODS: We performed whole-genome sequencing (WGS) of Mtb sputum culture-positive isolates from participants in two prospective cohort studies and predicted genotypic susceptibility using a validated random forest model. We used maximum likelihood estimation to build phylogenies. We compared lineage specific phylogenetic and time-scaled metrics to assess epidemiological success. RESULTS: Of the 642 isolates that underwent WGS, 612 met sequence quality criteria. Most isolates belonged to L3 (44.6%). The majority (61.1%) of multidrug-resistant isolates belonged to L2 (P < 0.001). In molecular dating, L2 demonstrated a higher rate and more recent resistance acquisition. We measured higher clustering, and time-scaled haplotypic density (THD) for L4 and L2 compared to L3 and/or L1 suggesting higher epidemiological success. L4 demonstrated higher THD and clustering (OR 5.1 (95% CI 2.3-12.3) in multivariate models controlling for host factors and DR. CONCLUSION: L2 shows a higher frequency of DR and both L2 and L4 demonstrate evidence of higher epidemiological success than L3 or L1 in the study setting. Our findings highlight the need for contact tracing around TB cases, and heightened surveillance of TB DR in India.

For Tuberculosis, Not "To Screen or Not to Screen?" but "Who?" and "How?"

Active case finding leveraging new molecular diagnostics and chest X-rays with automated interpretation algorithms is increasingly being developed for high-risk populations to drive down tuberculosis incidence. We consider why such an approach did not deliver a decline in tuberculosis prevalence in Brazilian prison populations and what to consider next.

Genotype-Phenotype Characterization of Serial Mycobacterium tuberculosis Isolates in Bedaquiline-Resistant Tuberculosis.

BACKGROUND: Emerging resistance to bedaquiline (BDQ) threatens to undermine advances in the treatment of drug-resistant tuberculosis (DRTB). Characterizing serial Mycobacterium tuberculosis (Mtb) isolates collected during BDQ-based treatment can provide insights into the etiologies of BDQ resistance in this important group of DRTB patients. METHODS: We measured mycobacteria growth indicator tube (MGIT)-based BDQ minimum inhibitory concentrations (MICs) of Mtb isolates collected from 195 individuals with no prior BDQ exposure who were receiving BDQ-based treatment for DRTB. We conducted whole-genome sequencing on serial Mtb isolates from all participants who had any isolate with a BDQ MIC >1 collected before or after starting treatment (95 total Mtb isolates from 24 participants). RESULTS: Sixteen of 24 participants had BDQ-resistant TB (MGIT MIC ≥4 µg/mL) and 8 had BDQ-intermediate infections (MGIT MIC = 2 µg/mL). Participants with pre-existing resistance outnumbered those with resistance acquired during treatment, and 8 of 24 participants had polyclonal infections. BDQ resistance was observed across multiple Mtb strain types and involved a diverse catalog of mmpR5 (Rv0678) mutations, but no mutations in atpE or pepQ. Nine pairs of participants shared genetically similar isolates separated by <5 single nucleotide polymorphisms, concerning for potential transmitted BDQ resistance. CONCLUSIONS: BDQ-resistant TB can arise via multiple, overlapping processes, including transmission of strains with pre-existing resistance. Capturing the within-host diversity of these infections could potentially improve clinical diagnosis, population-level surveillance, and molecular diagnostic test development.

Analysis of Genome-Wide Mutational Dependence in Naturally Evolving Mycobacterium tuberculosis Populations.

Pathogenic microorganisms are in a perpetual struggle for survival in changing host environments, where host pressures necessitate changes in pathogen virulence, antibiotic resistance, or transmissibility. The genetic basis of phenotypic adaptation by pathogens is difficult to study in vivo. In this work, we develop a phylogenetic method to detect genetic dependencies that promote pathogen adaptation using 31,428 in vivo sampled Mycobacterium tuberculosis genomes, a globally prevalent bacterial pathogen with increasing levels of antibiotic resistance. We find that dependencies between mutations are enriched in antigenic and antibiotic resistance functions and discover 23 mutations that potentiate the development of antibiotic resistance. Between 11% and 92% of resistant strains harbor a dependent mutation acquired after a resistance-conferring variant. We demonstrate the pervasiveness of genetic dependency in adaptation of naturally evolving populations and the utility of the proposed computational approach.

Transcriptomic responses to antibiotic exposure in Mycobacterium tuberculosis.

Transcriptional responses in bacteria following antibiotic exposure offer insights into antibiotic mechanism of action, bacterial responses, and characterization of antimicrobial resistance. We aimed to define the transcriptional antibiotic response (TAR) in Mycobacterium tuberculosis (Mtb) isolates for clinically relevant drugs by pooling and analyzing Mtb microarray and RNA-seq data sets. We generated 99 antibiotic transcription profiles across 17 antibiotics, with 76% of profiles generated using 3-24 hours of antibiotic exposure and 49% within one doubling of the WHO antibiotic critical concentration. TAR genes were time-dependent, and largely specific to the antibiotic mechanism of action. TAR signatures performed well at predicting antibiotic exposure, with the area under the receiver operating curve (AUC) ranging from 0.84-1.00 (TAR <6 hours of antibiotic exposure) and 0.76-1.00 (>6 hours of antibiotic exposure) for upregulated genes and 0.57-0.90 and 0.87-1.00, respectfully, for downregulated genes. This work desmonstrates that transcriptomics allows for the assessment of antibiotic activity in Mtb within 6 hours of exposure.

A Genome-wide Association Study Reveals a Novel Susceptibility Locus for Pancreas Divisum at 3q29.

INTRODUCTION: Pancreas divisum (PD) is a common congenital anomaly of the pancreas, but its genetic basis remains unknown. The purpose of this genome-wide association study was to identify genetic loci associated with PD. METHODS: Using the Mass General Brigham Biobank, patients diagnosed with PD were identified. Quality control and imputation were performed using standard approaches. Single nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) ≥ 5% were tested for association with PD using mixed linear model-based association analysis. The significance threshold was set at 5 × 10-8. RESULTS: A total of 13,940 subjects were included, of which 251 (1.8%) were diagnosed with PD. A genetic locus in chromosome 3q29 was found to be associated with PD (lead SNP rs3850646, MAFPD = 34.6% vs. MAFcontrols = 26.4%, beta = 0.0106, P = 1.47 × 10-8). The identified locus is located in the phosphatidylinositol glycan anchor biosynthesis class Xand p21 activated kinase 2genes. The heritability of PD was estimated at 27.5%. (Expression quantitative trait loci) and chromatin interaction analysis found 12 genes whose expression may be regulated by SNPs in this genomic locus. CONCLUSIONS: The results of this study suggest that a genetic locus at 3q29 is associated with PD. This locus is in the phosphatidylinositol glycan anchor biosynthesis class X and p21 activated kinase 2 genes. Twelve candidate genes were identified whose expression may be regulated by this locus. These findings may help us understand both normal and aberrant pancreatic development and may aid in clinical evaluation and genetic counseling of patients with PD and associated diseases, such as acute pancreatitis.

Benchmarking the empirical accuracy of short-read sequencing across the M. tuberculosis genome.

MOTIVATION: Short-read whole-genome sequencing (WGS) is a vital tool for clinical applications and basic research. Genetic divergence from the reference genome, repetitive sequences and sequencing bias reduces the performance of variant calling using short-read alignment, but the loss in recall and specificity has not been adequately characterized. To benchmark short-read variant calling, we used 36 diverse clinical Mycobacterium tuberculosis (Mtb) isolates dually sequenced with Illumina short-reads and PacBio long-reads. We systematically studied the short-read variant calling accuracy and the influence of sequence uniqueness, reference bias and GC content. RESULTS: Reference-based Illumina variant calling demonstrated a maximum recall of 89.0% and minimum precision of 98.5% across parameters evaluated. The approach that maximized variant recall while still maintaining high precision (<99%) was tuning the mapping quality filtering threshold, i.e. confidence of the read mapping (recall = 85.8%, precision = 99.1%, MQ ≥ 40). Additional masking of repetitive sequence content is an alternative conservative approach to variant calling that increases precision at cost to recall (recall = 70.2%, precision = 99.6%, MQ ≥ 40). Of the genomic positions typically excluded for Mtb, 68% are accurately called using Illumina WGS including 52/168 PE/PPE genes (34.5%). From these results, we present a refined list of low confidence regions across the Mtb genome, which we found to frequently overlap with regions with structural variation, low sequence uniqueness and low sequencing coverage. Our benchmarking results have broad implications for the use of WGS in the study of Mtb biology, inference of transmission in public health surveillance systems and more generally for WGS applications in other organisms. AVAILABILITY AND IMPLEMENTATION: All relevant code is available at https://github.com/farhat-lab/mtb-illumina-wgs-evaluation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

COVID-19 bacteremic co-infection is a major risk factor for mortality, ICU admission, and mechanical ventilation.

BACKGROUND: Recent single-center reports have suggested that community-acquired bacteremic co-infection in the context of Coronavirus disease 2019 (COVID-19) may be an important driver of mortality; however, these reports have not been validated with a multicenter, demographically diverse, cohort study with data spanning the pandemic. METHODS: In this multicenter, retrospective cohort study, inpatient encounters were assessed for COVID-19 with community-acquired bacteremic co-infection using 48-h post-admission blood cultures and grouped by: (1) confirmed co-infection [recovery of bacterial pathogen], (2) suspected co-infection [negative culture with ≥ 2 antimicrobials administered], and (3) no evidence of co-infection [no culture]. The primary outcomes were in-hospital mortality, ICU admission, and mechanical ventilation. COVID-19 bacterial co-infection risk factors and impact on primary outcomes were determined using multivariate logistic regressions and expressed as adjusted odds ratios with 95% confidence intervals (Cohort, OR 95% CI, Wald test p value). RESULTS: The studied cohorts included 13,781 COVID-19 inpatient encounters from 2020 to 2022 in the University of Alabama at Birmingham (UAB, n = 4075) and Ochsner Louisiana State University Health-Shreveport (OLHS, n = 9706) cohorts with confirmed (2.5%), suspected (46%), or no community-acquired bacterial co-infection (51.5%) and a comparison cohort consisting of 99,170 inpatient encounters from 2010 to 2019 (UAB pre-COVID-19 pandemic cohort). Significantly increased likelihood of COVID-19 bacterial co-infection was observed in patients with elevated ≥ 15 neutrophil-to-lymphocyte ratio (UAB: 1.95 [1.21-3.07]; OLHS: 3.65 [2.66-5.05], p < 0.001 for both) within 48-h of hospital admission. Bacterial co-infection was found to confer the greatest increased risk for in-hospital mortality (UAB: 3.07 [2.42-5.46]; OLHS: 4.05 [2.29-6.97], p < 0.001 for both), ICU admission (UAB: 4.47 [2.87-7.09], OLHS: 2.65 [2.00-3.48], p < 0.001 for both), and mechanical ventilation (UAB: 3.84 [2.21-6.12]; OLHS: 2.75 [1.87-3.92], p < 0.001 for both) across both cohorts, as compared to other risk factors for severe disease. Observed mortality in COVID-19 bacterial co-infection (24%) dramatically exceeds the mortality rate associated with community-acquired bacteremia in pre-COVID-19 pandemic inpatients (5.9%) and was consistent across alpha, delta, and omicron SARS-CoV-2 variants. CONCLUSIONS: Elevated neutrophil-to-lymphocyte ratio is a prognostic indicator of COVID-19 bacterial co-infection within 48-h of admission. Community-acquired bacterial co-infection, as defined by blood culture-positive results, confers greater increased risk of in-hospital mortality, ICU admission, and mechanical ventilation than previously described risk factors (advanced age, select comorbidities, male sex) for COVID-19 mortality, and is independent of SARS-CoV-2 variant.

Tuberculosis Pathways to Care and Transmission of Multidrug Resistance in India.

Rationale: India is experiencing a regional increase in cases of multidrug-resistant tuberculosis (MDR-TB). Objectives: Given the complexity of MDR-TB diagnosis and care, we sought to address key knowledge gaps in MDR risk factors, care delays, and drivers of delay to help guide disease control. Methods: From January 2018 to September 2019, we conducted interviews with adults registered with the National TB Elimination Program for MDR (n = 128) and non-MDR-TB (n = 269) treatment to quantitatively and qualitatively study care pathways. We collected treatment records and GeneXpert-TB/RIF diagnostic reports. Measurements and Main Results: MDR-TB was associated with young age and crowded residence. GeneXpert rifampicin resistance diversity was measured at 72.5% Probe E. Median time from symptom onset to diagnosis of MDR was 90 days versus 60 days for non-MDR, Wilcoxon P < 0.01. Delay decreased by a median of 30 days among non-MDR patients with wider access to GeneXpert, Wilcoxon P = 0.02. Pathways to care were complex, with a median (interquartile range) of 4 (3-5) and 3 (2-4) encounters for MDR and non-MDR, respectively. Of patients with MDR-TB, 68% had their first encounter in the private sector, and this was associated with a larger number of subsequent healthcare encounters and catastrophic expenditure. Conclusions: The association of MDR with young age, crowding, and low genotypic diversity raises concerns of ongoing MDR transmission fueled by long delays in care. Delays are decreasing with GeneXpert use, suggesting the need for routine use in presumptive TB. Qualitatively, we identify the need to improve patient retention in the National TB Elimination Program and highlight patients' trust relationship with private providers.

Whole Genome Sequencing Assessing Impact of Diabetes Mellitus on Tuberculosis Mutations and Type of Recurrence in India.

BACKGROUND: Evidence describing the impact of diabetes mellitus (DM) on the recurrence and mutation rate of Mycobacterium tuberculosis (Mtb) is limited. METHODS: This study was nested in 3 cohort studies of tuberculosis (TB) patients with and without DM in India. Paired Mtb isolates recovered at baseline and treatment failure/recurrence underwent whole genome sequencing. We compared acquisition of single-nucleotide polymorphisms (SNPs), TB drug resistance mutations, and type of recurrence (endogenous reactivation [<8 SNPs] or exogenous reinfection [≥8 SNPs]) by DM status. RESULTS: Of 1633 enrolled in the 3 parent cohorts, 236 (14.5%) had microbiologically confirmed TB treatment failure/recurrence; 76 Mtb isolate pairs were available for sequencing (22 in TB-DM and 54 in TB-only). The SNP acquisition rate was overall was 0.43 (95% confidence interval [CI], .25-.64) per 1 person-year (PY); 0.77 (95% CI, .40-1.35) per 1 PY, and 0.44 (95% CI, .19-.86) per 1 PY at treatment failure and recurrence, respectively. Significant difference in SNP rates by DM status was seen at recurrence (0.21 [95% CI, .04-.61]) per 1 PY for TB-only vs 1.28 (95% CI, .41-2.98) per 1 PY for TB-DM; P = .02). No significant difference in SNP rates by DM status was observed at treatment failure. Acquired TB drug resistance was seen in 4 of 18 (22%) in TB-DM vs 4 of 45 (9%) in TB-only (P = .21). Thirteen (17%) participants had exogenous reinfection; the reinfection rate at recurrence was 25% (3/12) for TB-DM vs 17% (4/24) in TB-only (P = .66). CONCLUSIONS: Considerable intrahost Mtb mutation rates were present at recurrence among patients with DM in India. One-fourth of patients with DM had exogenous reinfection at recurrence.

Role of Epistasis in Amikacin, Kanamycin, Bedaquiline, and Clofazimine Resistance in Mycobacterium tuberculosis Complex.

Antibiotic resistance among bacterial pathogens poses a major global health threat. Mycobacterium tuberculosis complex (MTBC) is estimated to have the highest resistance rates of any pathogen globally. Given the low growth rate and the need for a biosafety level 3 laboratory, the only realistic avenue to scale up drug susceptibility testing (DST) for this pathogen is to rely on genotypic techniques. This raises the fundamental question of whether a mutation is a reliable surrogate for phenotypic resistance or whether the presence of a second mutation can completely counteract its effect, resulting in major diagnostic errors (i.e., systematic false resistance results). To date, such epistatic interactions have only been reported for streptomycin that is now rarely used. By analyzing more than 31,000 MTBC genomes, we demonstrated that the eis C-14T promoter mutation, which is interrogated by several genotypic DST assays endorsed by the World Health Organization, cannot confer resistance to amikacin and kanamycin if it coincides with loss-of-function (LoF) mutations in the coding region of eis. To our knowledge, this represents the first definitive example of antibiotic reversion in MTBC. Moreover, we raise the possibility that mmpR (Rv0678) mutations are not valid markers of resistance to bedaquiline and clofazimine if these coincide with an LoF mutation in the efflux pump encoded by mmpS5 (Rv0677c) and mmpL5 (Rv0676c).

Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing.

BACKGROUND: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear. METHODS: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide 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 profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance. RESULTS: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted. CONCLUSIONS: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.).

Systematic review of mutations associated with resistance to the new and repurposed Mycobacterium tuberculosis drugs bedaquiline, clofazimine, linezolid, delamanid and pretomanid.

BACKGROUND: Improved genetic understanding of Mycobacterium tuberculosis (MTB) resistance to novel and repurposed anti-tubercular agents can aid the development of rapid molecular diagnostics. METHODS: Adhering to PRISMA guidelines, in March 2018, we performed a systematic review of studies implicating mutations in resistance through sequencing and phenotyping before and/or after spontaneous resistance evolution, as well as allelic exchange experiments. We focused on the novel drugs bedaquiline, delamanid, pretomanid and the repurposed drugs clofazimine and linezolid. A database of 1373 diverse control MTB whole genomes, isolated from patients not exposed to these drugs, was used to further assess genotype-phenotype associations. RESULTS: Of 2112 papers, 54 met the inclusion criteria. These studies characterized 277 mutations in the genes atpE, mmpR, pepQ, Rv1979c, fgd1, fbiABC and ddn and their association with resistance to one or more of the five drugs. The most frequent mutations for bedaquiline, clofazimine, linezolid, delamanid and pretomanid resistance were atpE A63P, mmpR frameshifts at nucleotides 192-198, rplC C154R, ddn W88* and ddn S11*, respectively. Frameshifts in the mmpR homopolymer region nucleotides 192-198 were identified in 52/1373 (4%) of the control isolates without prior exposure to bedaquiline or clofazimine. Of isolates resistant to one or more of the five drugs, 59/519 (11%) lacked a mutation explaining phenotypic resistance. CONCLUSIONS: This systematic review supports the use of molecular methods for linezolid resistance detection. Resistance mechanisms involving non-essential genes show a diversity of mutations that will challenge molecular diagnosis of bedaquiline and nitroimidazole resistance. Combined phenotypic and genotypic surveillance is needed for these drugs in the short term.

Rifampicin and rifabutin resistance in 1003 Mycobacterium tuberculosis clinical isolates.

OBJECTIVES: Drug-resistant TB remains a public health challenge. Rifamycins are among the most potent anti-TB drugs. They are known to target the RpoB subunit of RNA polymerase; however, our understanding of how rifamycin resistance is genetically encoded remains incomplete. Here we investigated rpoB genetic diversity and cross-resistance between the two rifamycin drugs rifampicin and rifabutin. METHODS: We performed WGS of 1003 Mycobacterium tuberculosis clinical isolates and determined MICs of both rifamycin agents on 7H10 agar using the indirect proportion method. We generated rpoB mutants in a laboratory strain and measured their antibiotic susceptibility using the alamarBlue reduction assay. RESULTS: Of the 1003 isolates, 766 were rifampicin resistant and 210 (27%) of these were rifabutin susceptible; 102/210 isolates had the rpoB mutation D435V (Escherichia coli D516V). Isolates with discordant resistance were 17.2 times more likely to harbour a D435V mutation than those resistant to both agents (OR 17.2, 95% CI 10.5-27.9, P value <10-40). Compared with WT, the D435V in vitro mutant had an increased IC50 of both rifamycins; however, in both cases to a lesser degree than the S450L (E. coli S531L) mutation. CONCLUSIONS: The observation that the rpoB D435V mutation produces an increase in the IC50 of both drugs contrasts with findings from previous smaller studies that suggested that isolates with the D435V mutation remain rifabutin susceptible despite being rifampicin resistant. Our finding thus suggests that the recommended critical testing concentration for rifabutin should be revised.