Assessment and validation of enrichment and target capture approaches to improve Mycobacterium tuberculosis WGS from direct patient samples.

Within-host Mycobacterium tuberculosis (Mtb) diversity may detect antibiotic resistance or predict tuberculosis treatment failure and is best captured through sequencing directly from sputum. Here, we compared three sample pre-processing steps for DNA decontamination and studied the yield of a new target enrichment protocol for optimal whole-genome sequencing (WGS) from direct patient samples. Mtb-positive NALC-NaOH-treated patient sputum sediments were pooled, and heat inactivated, split in replicates, and treated by either a wash, DNase I, or benzonase digestion. Levels of contaminating host DNA and target Mtb DNA were assessed by quantitative PCR (qPCR), followed by WGS with and without custom dsDNA target enrichment. The pre-treatment sample has a high host-to-target ratio of DNA (6,168 ± 1,638 host copies/ng to 212.3 ± 59.4 Mtb copies/ng) that significantly decreased with all three treatments. Benzonase treatment resulted in the highest enrichment of Mtb DNA at 100-fold compared with control (3,422 ± 2,162 host copies/ng to 11,721 ± 7,096 Mtb copies/ng). The custom dsDNA probe panel successfully enriched libraries from as little as 0.45 pg of Mtb DNA (100 genome copies). Applied to direct sputum the dsDNA target enrichment panel increased the percent of sequencing reads mapping to the Mtb target for all three pre-processing methods. Comparing the results of the benzonase sample sequenced both with and without enrichment, the percent of sequencing reads mapping to the Mtb increased to 90.95% from 1.18%. We demonstrate a low limit of detection for a new custom dsDNA Mtb target enrichment panel that has a favorable cost profile. The results also demonstrate that pre-processing to remove contaminating extracellular DNA prior to cell lysis and DNA extraction improves the host-to-Mtb DNA ratio but is not adequate to support average coverage WGS without target capture.

To Test or Not? Xpert MTB/RIF as an Alternative to Smear Microscopy to Guide Line Probe Assay Testing for Drug-Resistant Tuberculosis.

Xpert MTB/RIF (Xpert) revolutionized tuberculosis (TB) diagnosis. Laboratory decision making on whether widely-used reflex drug susceptibility assays (MTBDRplus, first-line resistance; MTBDRsl, second-line) are conducted is based on smear status, with smear-negative specimens often excluded. We performed receiver operator characteristic (ROC) curve analyses using bacterial load information (smear microscopy grade, Xpert-generated semi-quantitation categories and minimum cycle threshold [CTmin] values) from Xpert rifampicin-resistant sputum for the prediction of downstream line probe assay results as "likely non-actionable" (no resistance or susceptible results generated). We evaluated actionable-to-non-actionable result ratios and pay-offs with missed resistance versus LPAs done universally. Smear-negatives were more likely than smear-positive specimens to generate a non-actionable MTBDRplus (23% [133/559] versus 4% [15/381]) or MTBDRsl (39% [220/559] versus 12% [47/381]) result. However, excluding smear-negatives would result in missed rapid diagnoses (e.g., only 49% [264/537] of LPA-diagnosable isoniazid resistance would be detected if smear-negatives were omitted). Testing smear-negatives with a semi-quantitation category ≥ "medium" had a high ratio of actionable-to-non-actionable results (12.8 or a 4-fold improvement versus testing all using MTBDRplus, 4.5 or 3-fold improvement for MTBDRsl), which would still capture 64% (168/264) and 77% (34/44) of LPA-detectable smear-negative resistance, respectively. Use of CTmins permitted optimization of this ratio with higher specificity for non-actionable results but decreased resistance detected. Xpert quantitative information permits identification of a smear-negative subset in whom the payoffs of the ratio of actionable-to-non-actionable LPA results with missed resistance may prove acceptable to laboratories, depending on context. Our findings permit the rational expansion of direct DST to certain smear-negative sputum specimens.

Poikilocytosis of Angora goats is associated with erythrocyte density and reticulocytosis.

Angora goats in South Africa experience several syndromes that result in notable morbidity and mortality in juveniles and adults, but not kids. Insight into their causes is hampered by the lack of normal reference values for this breed, and the present study therefore aimed to characterise (1) differences in the haematology of healthy kids at birth and weaning, and (2) the haematology of apparently healthy yearlings. Selected variables were measured by blood smear analysis, and complete blood counts were performed using an ADVIA 2120i. Variables at 1, 11, and 20 weeks of age were compared using the Friedman test and associations between variables of yearlings were determined by correlation analysis. In kids, red blood cell count, mean corpuscular haemoglobin concentration (MCHC), and poikilocytosis increased over time, while mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV) decreased. Yearlings displayed a lower MCHC, and higher haemoglobin distribution width than previously reported for goats, and these were positively correlated with poikilocytosis, as were reticulocyte counts. White cell counts of yearlings exceeded normal values previously reported for goats, with some individuals displaying remarkably high mature neutrophil counts. Changes in haemoglobin variant expression or cation and water fluxes are possible explanations for the findings in kids, while in yearlings, the associations between MCHC, HDW, poikilocytosis, and reticulocytosis suggest alterations in red cell hydration in adulthood that are associated with increased red cell turnover. These findings may prove informative in the further investigation of various clinical syndromes in this population.

Smoked drug use in patients with TB is associated with higher bacterial burden.

BACKGROUND: Smoking of illicit drugs may lead to more rapid TB disease progression or late treatment presentation, yet research on this topic is scant. We examined the association between smoked drug use and bacterial burden among patients newly initiated on drug-susceptible TB (DS-TB) therapy.METHODS: Data from 303 participants initiating DS-TB treatment in the Western Cape Province, South Africa, were analyzed. Smoked drug use was defined as self-reported or biologically verified methamphetamine, methaqualone and/or cannabis use. Proportional hazard and logistic regression models (adjusted for age, sex, HIV status and tobacco use) examined associations between smoked drug use and mycobacterial time to culture positivity (TTP), acid-fast bacilli sputum smear positivity and lung cavitation.RESULTS: People who smoked drugs (PWSD) comprised 54.8% (n = 166) of the cohort. TTP was faster for PWSD (hazard ratio 1.48, 95% CI 1.10-1.97; P = 0.008). Smear positivity was higher among PWSD (OR 2.28, 95% CI 1.22-4.34; P = 0.011). Smoked drug use (OR 1.08, 95% CI 0.62-1.87; P = 0.799) was not associated with increased cavitation.CONCLUSIONS: PWSD had a higher bacterial burden at diagnosis than those who do not smoke drugs. Screening for TB among PWSD in the community may facilitate earlier linkage to TB treatment and reduce community transmission.

Emergence of Canonical and Noncanonical Genomic Variants following In Vitro Exposure of Clinical Mycobacterium tuberculosis Strains to Bedaquiline or Clofazimine.

In Mycobacterium tuberculosis, bedaquiline and clofazimine resistance occurs primarily through Rv0678 variants, a gene encoding a repressor protein that regulates mmpS5/mmpL5 efflux pump gene expression. Despite the shared effect of both drugs on efflux, little else is known about other pathways affected. We hypothesized that in vitro generation of bedaquiline- or clofazimine-resistant mutants could provide insight into additional mechanisms of action. We performed whole-genome sequencing and determined phenotypic MICs for both drugs on progenitor and mutant progenies. Mutants were induced through serial passage on increasing concentrations of bedaquiline or clofazimine. Rv0678 variants were identified in both clofazimine- and bedaquiline-resistant mutants, with concurrent atpE SNPs occurring in the latter. Of concern was the acquisition of variants in the F420 biosynthesis pathway in clofazimine-resistant mutants obtained from either a fully susceptible (fbiD: del555GCT) or rifampicin mono-resistant (fbiA: 283delTG and T862C) progenitor. The acquisition of these variants possibly implicates a shared pathway between clofazimine and nitroimidazoles. Pathways associated with drug tolerance and persistence, F420 biosynthesis, glycerol uptake and metabolism, efflux, and NADH homeostasis appear to be affected following exposure to these drugs. Shared genes affected by both drugs include Rv0678, glpK, nuoG, and uvrD1. Genes with variants in the bedaquiline resistant mutants included atpE, fadE28, truA, mmpL5, glnH, and pks8, while clofazimine-resistant mutants displayed ppsD, fbiA, fbiD, mutT3, fadE18, Rv0988, and Rv2082 variants. These results show the importance of epistatic mechanisms as a means of responding to drug pressure and highlight the complexity of resistance acquisition in M. tuberculosis.

Management of rifampicin-resistant TB: programme indicators and care cascade analysis in South Africa.

BACKGROUND: Xpert® MTB/RIF was expected to revolutionise the management of rifampicin-resistant TB (RR-TB) by enabling rapid and decentralised diagnosis of rifampicin (RIF) resistance.METHODS: We performed a care cascade analysis for a cohort of RR-TB patients managed under programmatic conditions. Cumulative incidences of time to completion of the RR-TB care cascade steps were estimated, reasons for delay or attrition from the cascade investigated and WHO programme indicators for monitoring of RR-TB programmes calculated.RESULTS: Of 502 patients diagnosed with RR-TB using Xpert, 64% initiated multidrug-resistant TB (MDR-TB) treatment immediately, 20% after some first-line treatment, 16% never initiated MDR-TB treatment, mainly because of death (44%) or loss to follow-up (26%) soon after diagnosis. A supplementary sputum sample was collected within 14 days of treatment in 58.8% of cases. Only 63% of RR-TB cases were assessed for isoniazid resistance, and only 65% of MDR-TB cases were evaluated for pre-XDR-TB (extensively drug-resistant TB). Treatment was individualised in 57% of pre-XDR and 68% of XDR-TB patients. Only 8% completed the entire RR-TB care cascade as intended.CONCLUSION: Fidelity to the RR-TB algorithm was poor, with substantial losses at each step of the cascade, highlighting the fact that implementation of novel technologies needs to be accompanied by health system strengthening to maximise impact.

Bacterial Genotyping of Central Nervous System Tuberculosis in South Africa: Heterogenic Mycobacterium tuberculosis Infection and Predominance of Lineage 4.

Tuberculous meningitis (TBM), the most severe extrapulmonary manifestation of tuberculosis, is caused by the pathogen Mycobacterium tuberculosis The M. tuberculosis complex includes seven lineages, all described to harbor a unique geographical dissemination pattern and clinical presentation. In this study, we set out to determine whether a certain M. tuberculosis lineage demonstrated tropism to cause TBM in patients from Cape Town, South Africa. DNA was extracted from formalin-fixed paraffin-embedded central nervous system (CNS) tissue from a unique neuropathological cohort of 83 TBM patients, collected between 1975 and 2012. M. tuberculosis lineages 1, 2, 3, and 4 were determined using an allele-specific PCR and Sanger sequencing. Of the 83 patient specimens tested, bacterial characterization could be performed on 46 specimens (55%). M. tuberculosis lineage 4 was present in 26 patient specimens (56%), and non-lineage 4 was identified in 10 cases (22%). Moreover, genomic heterogeneity was detected in the CNS specimens of 7 adults and 3 children. We could show that infection of the CNS is not restricted to a single M. tuberculosis lineage and that even young children with rapid progression of disease can harbor more than one M. tuberculosis lineage in the CNS.

Deciphering Within-Host Microevolution of Mycobacterium tuberculosis through Whole-Genome Sequencing: the Phenotypic Impact and Way Forward.

The Mycobacterium tuberculosis genome is more heterogenous and less genetically stable within the host than previously thought. Currently, only limited data exist on the within-host microevolution, diversity, and genetic stability of M. tuberculosis As a direct consequence, our ability to infer M. tuberculosis transmission chains and to understand the full complexity of drug resistance profiles in individual patients is limited. Furthermore, apart from the acquisition of certain drug resistance-conferring mutations, our knowledge on the function of genetic variants that emerge within a host and their phenotypic impact remains scarce. We performed a systematic literature review of whole-genome sequencing studies of serial and parallel isolates to summarize the knowledge on genetic diversity and within-host microevolution of M. tuberculosis We identified genomic loci of within-host emerged variants found across multiple studies and determined their functional relevance. We discuss important remaining knowledge gaps and finally make suggestions on the way forward.

Antigen-specific interferon-gamma release is decreased following the single intradermal comparative cervical skin test in African buffaloes (Syncerus caffer).

Effective disease management of wildlife relies on the strategic application of ante-mortem diagnostic tests for early identification and removal of M. bovis-infected animals. To improve diagnostic performance, interferon-gamma release assays (IGRAs) are often used in conjunction with the tuberculin skin test (TST). Since buffaloes are major maintenance hosts of M. bovis, optimal application of bovine TB diagnostic tests are especially important. We aimed to determine whether the timing of blood collection relative to the TST has an influence on IFN-γ production and diagnostic outcome in African buffaloes. Release of IFN-γ in response to bovine purified protein derivative (PPD), avian PPD and PC-HP® and PC-EC® peptides was measured by Bovigam® and an in-house IGRA in a group of Bovigam®-positive and - negative buffaloes at the time the TST was performed and three days later. There was significantly lower IFN-γ release in response to these antigens post-TST in Bovigam®-positive buffaloes, but no significant changes in Bovigam®-negative buffaloes. Also, a significantly greater proportion of buffaloes were Bovigam®-positive prior to the TST than three days later. We therefore recommend that blood samples for use in IGRAs be collected prior to or at the time the TST is performed to facilitate the correct identification of greater numbers of IGRA-positive buffaloes.

Detection of tuberculosis by automatic cough sound analysis.

OBJECTIVE: Globally, tuberculosis (TB) remains one of the most deadly diseases. Although several effective diagnosis methods exist, in lower income countries clinics may not be in a position to afford expensive equipment and employ the trained experts needed to interpret results. In these situations, symptoms including cough are commonly used to identify patients for testing. However, self-reported cough has suboptimal sensitivity and specificity, which may be improved by digital detection. APPROACH: This study investigates a simple and easily applied method for TB screening based on the automatic analysis of coughing sounds. A database of cough audio recordings was collected and used to develop statistical classifiers. MAIN RESULTS: These classifiers use short-term spectral information to automatically distinguish between the coughs of TB positive patients and healthy controls with an accuracy of 78% and an AUC of 0.95. When a set of five clinical measurements is available in addition to the audio, this accuracy improves to 82%. By choosing an appropriate decision threshold, the system can achieve a sensitivity of 95% at a specificity of approximately 72%. The experiments suggest that the classifiers are using some spectral information that is not perceivable by the human auditory system, and that certain frequencies are more useful for classification than others. SIGNIFICANCE: We conclude that automatic classification of coughing sounds may represent a viable low-cost and low-complexity screening method for TB.

Widespread use of incorrect PCR ramp rate negatively impacts multidrug-resistant tuberculosis diagnosis (MTBDRplus).

The scale-up of rapid drug resistance testing for TB is a global priority. MTBDRplus is a WHO-endorsed multidrug-resistant (MDR)-TB PCR assay with suboptimal sensitivities and high indeterminate rates on smear-negative specimens. We hypothesised that widespread use of incorrect thermocycler ramp rate (speed of temperature change between cycles) impacts performance. A global sample of 72 laboratories was surveyed. We tested 107 sputa from Xpert MTB/RIF-positive patients and, separately, dilution series of bacilli, both at the manufacturer-recommended ramp rate (2.2 °C/s) and the most frequently reported incorrect ramp rate (4.0 °C/s). Mycobacterium tuberculosis-complex DNA (TUB-band)-detection, indeterminate results, accuracy, and inter-reader variability (dilution series only) were compared. 32 respondents did a median (IQR) of 41 (20-150) assays monthly. 78% used an incorrect ramp rate. On smear-negative sputa, 2.2 °C/s vs. 4.0 °C/s improved TUB-band positivity (42/55 vs. 32/55; p = 0.042) and indeterminate rates (1/42 vs. 5/32; p = 0.039). The actionable results (not TUB-negative or indeterminate; 41/55 vs. 28/55) hence improved by 21% (95% CI: 9-35%). Widespread use of incorrect ramp rate contributes to suboptimal MTBDRplus performance on smear-negative specimens and hence limits clinical utility. The number of diagnoses (and thus the number of smear-negative patients in whom DST is possible) will improve substantially after ramp rate correction.

Exploring the potential of T7 bacteriophage protein Gp2 as a novel inhibitor of mycobacterial RNA polymerase.

Over the past six decades, there has been a decline in novel therapies to treat tuberculosis, while the causative agent of this disease has become increasingly resistant to current treatment regimens. Bacteriophages (phages) are able to kill bacterial cells and understanding this process could lead to novel insights for the treatment of mycobacterial infections. Phages inhibit bacterial gene transcription through phage-encoded proteins which bind to RNA polymerase (RNAP), thereby preventing bacterial transcription. Gp2, a T7 phage protein which binds to the beta prime (ß') subunit of RNAP in Escherichia coli, has been well characterized in this regard. Here, we aimed to determine whether Gp2 is able to inhibit RNAP in Mycobacterium tuberculosis as this may provide new possibilities for inhibiting the growth of this deadly pathogen. Results from an electrophoretic mobility shift assay and in vitro transcription assay revealed that Gp2 binds to mycobacterial RNAP and inhibits transcription; however to a much lesser degree than in E. coli. To further understand the molecular basis of these results, a series of in silico techniques were used to assess the interaction between mycobacterial RNAP and Gp2, providing valuable insight into the characteristics of this protein-protein interaction.

Development of a Gene Expression Assay for the Diagnosis of Mycobacterium bovis Infection in African Lions (Panthera leo).

Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON® -TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions.

Isolation and Potential for Transmission of Mycobacterium bovis at Human-livestock-wildlife Interface of the Serengeti Ecosystem, Northern Tanzania.

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is a multihost pathogen of public health and veterinary importance. We characterized the M. bovis isolated at the human-livestock-wildlife interface of the Serengeti ecosystem to determine the epidemiology and risk of cross-species transmission between interacting hosts species. DNA was extracted from mycobacterial cultures obtained from sputum samples of 472 tuberculosis (TB) suspected patients and tissue samples from 606 livestock and wild animal species. M. bovis isolates were characterized using spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Tandem Repeats (MIRU-VNTR) on 24 loci. Only 5 M. bovis were isolated from the cultured samples. Spoligotyping results revealed that three M. bovis isolates from two buffaloes (Syncerus caffer) and 1 African civet (Civettictis civetta) belonged to SB0133 spoligotype. The two novel strains (AR1 and AR2) assigned as spoligotype SB2290 and SB2289, respectively, were identified from indigenous cattle (Bos indicus). No M. bovis was detected from patients with clinical signs consistent with TB. Of the 606 animal tissue specimens and sputa of 472 TB-suspected patients 43 (7.09%) and 12 (2.9%), respectively, yielded non-tuberculous mycobacteria (NTM), of which 20 isolates were M. intracellulare. No M. avium was identified. M. bovis isolates from wildlife had 45.2% and 96.8% spoligotype pattern agreement with AR1 and AR2 strains, respectively. This finding indicates that bTB infections in wild animals and cattle were epidemiologically related. Of the 24 MIRU-VNTR loci, QUB 11b showed the highest discrimination among the M. bovis strains. The novel strains obtained in this study have not been previously reported in the area, but no clear evidence for recent cross-species transmission of M. bovis was found between human, livestock and wild animals.

Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates.

BACKGROUND: Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. METHODS: Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. RESULTS: Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30% to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. CONCLUSIONS: Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection. This suggests that the genome of M. tuberculosis is more dynamic than previously thought, suggesting preparedness to respond to a changing environment.

Molecular Epidemiological Interpretation of the Epidemic of Extensively Drug-Resistant Tuberculosis in South Africa.

We show that the interpretation of molecular epidemiological data for extensively drug-resistant tuberculosis (XDR-TB) is dependent on the number of different markers used to define transmission. Using spoligotyping, IS6110 DNA fingerprinting, and DNA sequence data, we show that XDR-TB in South Africa (2006 to 2008) was predominantly driven by the acquisition of second-line drug resistance.

Phylogeny to function: PE/PPE protein evolution and impact on Mycobacterium tuberculosis pathogenicity.

The pe/ppe genes represent one of the most intriguing aspects of the Mycobacterium tuberculosis genome. These genes are especially abundant in pathogenic mycobacteria, with more than 160 members in M. tuberculosis. Despite being discovered over 15 years ago, their function remains unclear, although various lines of evidence implicate selected family members in mycobacterial virulence. In this review, we use PE/PPE phylogeny as a framework within which we examine the diversity and putative functions of these proteins. We report on the evolution and diversity of the respective gene families, as well as the implications thereof for function and host immune recognition. We summarize recent findings on pe/ppe gene regulation, also placing this in the context of PE/PPE phylogeny. We collate data from several large proteomics datasets, providing an overview of PE/PPE localization, and discuss the implications this may have for host responses. Assessment of the current knowledge of PE/PPE diversity suggests that these proteins are not variable antigens as has been so widely speculated; however, they do clearly play important roles in virulence. Viewing the growing body of pe/ppe literature through the lens of phylogeny reveals trends in features and function that may be associated with the evolution of mycobacterial pathogenicity.

Phage-based detection of bacterial pathogens.

Bacterial pathogens cause significant morbidity and mortality annually to both humans and animals. With the rampant spread of drug resistance and the diminishing effectiveness of current antibiotics, there is a pressing need for effective diagnostics for detection of bacterial pathogens and their drug resistances. Bacteriophages offer several unique opportunities for bacterial detection. This review highlights the means by which bacteriophages have been utilized to achieve and facilitate specific bacterial detection.

Mutation rate and the emergence of drug resistance in Mycobacterium tuberculosis.

The emergence and spread of multidrug-resistant strains of Mycobacterium tuberculosis remains a major concern of tuberculosis control programmes worldwide, as treatment depends on low-efficacy, toxic compounds that often lead to poor outcomes. M. tuberculosis develops drug resistance exclusively through chromosomal mutations, in particular single-nucleotide polymorphisms. Moreover, in laboratory assays the organism exhibits a spontaneous mutation rate that is at the lower end of the bacterial spectrum. Despite this, whole-genome sequencing technology has identified unexpected genetic diversity among clinical M. tuberculosis populations. This suggests that the mycobacterial mutation rate may be modulated within the host and, in turn, implies a potential role for constitutive and/or transient mutator strains in adaptive evolution. It also raises the possibility that environmental factors might act as key mutagens during M. tuberculosis infection. Here we consider the elements that might influence the mycobacterial mutation rate in vivo and evaluate the potential roles of constitutive and transient mutator states in the generation of drug resistance mutations. In addition, we identify key research questions that will influence future efforts to develop novel therapeutic strategies for a disease that continues to impose a significant global health burden.