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BackgroundCombatting the tuberculosis (TB) epidemic caused byMycobacterium tuberculosis(Mtb) necessitates a better understanding of the factors contributing to patient clinical outcomes and transmission. While host and environmental factors have been evaluated, the impact ofMtbgenetic background and phenotypic diversity is underexplored. Previous work has made associations betweenMtbgenetic lineages and some clinical and epidemiological features, but the bacterial traits underlying these connections are largely unknown.MethodsWe developed a high-throughput functional genomics platform for defining genotype-phenotype relationships across a panel ofMtbclinical isolates. These phenotypic fitness profiles function as intermediate traits which can be linked toMtbgenetic variants and associated with clinical and epidemiological outcomes. We applied this approach to a collection of 158Mtbstrains from a study ofMtbtransmission in Ho Chi Minh City, Vietnam.Mtbstrains were genetically tagged in multiplicate, which allowed us to pool the strains and assessin vitrocompetitive fitness using deep sequencing across a set of 14 host-relevant antibiotic and metabolic conditions. Phylogenetic and monogenic associations with these intermediate traits were identified and then associated with clinical outcomes.FindingsMtbclinical strains have a broad range of growth and drug response dynamics that can be clustered by their phylogenetic relationships. We identified novel monogenic associations withMtbfitness in various metabolic and antibiotic conditions. Among these, we find that mutations inRv1339, a phosphodiesterase, which were identified through their association with slow growth in glycerol, are further associated with treatment failure. We also identify a previously uncharacterized subclade of Lineage 1 strains (L1.1.1.1) that is phenotypically distinguished by slow growth under most antibiotic and metabolic stress conditionsin vitro. This clade is associated with cavitary disease, treatment failure, and demonstrates increased transmission potential.InterpretationHigh-throughput phenogenotyping of Mtb clinical strains enabled bacterial intermediate trait identification that can provide a mechanistic link betweenMtbgenetic variation and patient clinical outcomes.Mtbstrains associated with cavitary disease, treatment failure, and transmission potential display intermediate phenotypes distinguished by slow growth under various antibiotic and metabolic conditions. These data suggest that Mtb growth regulation is an adaptive advantage for host bacterial success in human populations, in at least some circumstances. These data further suggest markers for the underlying bacterial processes that govern these clinical outcomes.FundingNational Institutes of Allergy and Infectious Diseases: P01 AI132130 (SS, SMF); P01 AI143575 (XW, SMF); U19 AI142793 (QL, SMF); 5T32AI132120-03 (SS); 5T32AI132120-04 (SS); 5T32AI049928-17 (SS) Wellcome Trust Fellowship in Public Health and Tropical Medicine: 097124/Z/11/Z (NTTT) National Health and Medical Research Council (NHMRC)/A*STAR joint call: APP1056689 (SJD) The funding sources had no involvement in study methodology, data collection, analysis, and interpretation nor in the writing or submission of the manuscript.Research in contextEvidence before this studyWe used different combinations of the words mycobacterium tuberculosis, tuberculosis, clinical strains, intermediate phenotypes, genetic barcoding, phenogenomics, cavitary disease, treatment failure, and transmission to search the PubMed database for all studies published up until January 20th, 2022. We only considered English language publications, which biases our search. Previous work linkingMtbdeterminants to clinical or epidemiological data has made associations between bacterial lineage, or less frequently, genetic polymorphisms toin vitroorin vivomodels of pathogenesis, transmission, and clinical outcomes such as cavitary disease, treatment failure, delayed culture conversion, and severity. Many of these studies focus on the global pandemic Lineage 2 and Lineage 4Mtbstrains due in part to a deletion in a polyketide synthase implicated in host-pathogen interactions. There are a number ofMtbGWAS studies that have led to novel genetic determinants ofin vitrodrug resistance and tolerance. PreviousMtbGWAS analyses with clinical outcomes did not experimentally test any predicted phenotypes of the clinical strains. Published laboratory-based studies ofMtbclinical strains involve relatively small numbers of strains, do not identify the genetic basis of relevant phenotypes, or link findings to the corresponding clinical outcomes. There are two recent studies of other pathogens that describe phenogenomic analyses. One study of 331M. abscessusclinical strains performed one-by-one phenotyping to identify bacterial features associated with clearance of infection and another details a competition experiment utilizing three barcodedPlasmodium falciparumclinical isolates to assay antimalarial fitness and resistance.Added value of this studyWe developed a functional genomics platform to perform high-throughput phenotyping ofMtbclinical strains. We then used these phenotypes as intermediate traits to identify novel bacterial genetic features associated with clinical outcomes. We leveraged this platform with a sample of 158Mtbclinical strains from a cross sectional study ofMtbtransmission in Ho Chi Minh City, Vietnam. To enable high-throughput phenotyping of large numbers ofMtbclinical isolates, we applied a DNA barcoding approach that has not been previously utilized for the high-throughput analysis ofMtbclinical strains. This approach allowed us to perform pooled competitive fitness assays, tracking strain fitness using deep sequencing. We measured the replicative fitness of the clinical strains in multiplicate under 14 metabolic and antibiotic stress condition. To our knowledge, this is the largest phenotypic screen ofMtbclinical isolates to date. We performed bacterial GWAS to delineate theMtbgenetic variants associated with each fitness phenotype, identifying monogenic associations with several conditions. We then definedMtbphenotypic and genetic features associated with clinical outcomes. We find that a subclade ofMtbstrains, defined by variants largely involved in fatty acid metabolic pathways, share a universal slow growth phenotype that is associated with cavitary disease, treatment failure and increased transmission potential in Vietnam. We also find that mutations inRv1339, a poorly characterized phosphodiesterase, also associate with slow growthin vitroand with treatment failure in patients.Implications of all the available evidencePhenogenomic profiling demonstrates thatMtbstrains exhibit distinct growth characteristics under metabolic and antibiotic stress conditions. These fitness profiles can serve as intermediate traits for GWAS and association with clinical outcomes. Intermediate phenotyping allows us to examine potential processes by which bacterial strain differences contribute to clinical outcomes. Our study identifies clinical strains with slow growth phenotypes underin vitromodels of antibiotic and host-like metabolic conditions that are associated with adverse clinical outcomes. It is possible that the bacterial intermediate phenotypes we identified are directly related to the mechanisms of these outcomes, or they may serve as markers for the causal yet unidentified bacterial determinants. Via the intermediate phenotyping, we also discovered a surprising diversity inMtbresponses to the new anti-mycobacterial drugs that target central metabolic processes, which will be important in considering roll-out of these new agents. Our study and others that have identifiedMtbdeterminants of TB clinical and epidemiological phenotypes should inform efforts to improve diagnostics and drug regimen design.

Whole genome sequencing (WGS) and phenotypic drug susceptibility testing was performed on a collection of 2,542Mycobacterium tuberculosis (Mtb)isolates from tuberculosis (TB) patients recruited in Ho Chi Minh City (HCMC), Vietnam, to investigateMtbdiversity, the prevalence and phylodynamics of drug resistance, andin silicoresistance prediction with sequencing data. Amongst isolates tested phenotypically against first-line drugs, we observed high rates of streptomycin [STR, 37.7% (N=573/1,520)] and isoniazid resistance [INH, 25.7% (N=459/1,786)], and lower rates of resistance to rifampicin [RIF, 4.9% (N=87/1,786)] and ethambutol [EMB, 4.2% (N=75/1,785)]. Resistance to STR and INH was predicted moderately well when applying the TB-Profiler algorithm to WGS data (sensitivities of 0.81 and 0.87 respectively), while resistance to RIF and EMB was predicted relatively poorly (sensitivities of 0.70 and 0.44 respectively). Rates of multidrug-resistance [(MDR, 3.9% (N=69/1,786)], and resistance to a number of second-line drugs [Para-aminosalicylic acid (29.6% N=79/267), Amikacin (15.4% N=41/267) and Moxifloxacin (21.3%), N=57/267], were found to be high within a global context. Comparing rates of drug resistance among lineages, and exploring the dynamics of resistance acquisition through time, suggest the Beijing lineage (lineage 2.2) acquiresde novoresistance mutations at higher rates and suffers no apparent fitness cost acting to impede the transmission of resistance. We infer resistance to INH and STR to have arisen earlier, on average, than resistance to RIF, and to be more widespread across the phylogeny. The high prevalence of ‘background’ INH resistance, combined with high rates of RIF mono-resistance (20.7%, N=18/87) suggests that rapid assays for INH resistance will be valuable in this setting. These tests will allow the detection of INH mono-resistance, and will allow MDR isolates to be distinguished from isolates with RIF mono-resistance.

Background Meta-analysis of patients with isoniazid-resistant tuberculosis (TB) given standard first-line anti-TB treatment indicated an increased risk of multidrug-resistant TB (MDR-TB) emerging (8%), compared to drug-sensitive TB (0.3%). Here we use whole genome sequencing (WGS) to investigate whether treatment of patients with preexisting isoniazid-resistant disease with first-line anti-TB therapy risks selecting for rifampicin resistance, and hence MDR-TB. Methods Patients with isoniazid-resistant pulmonary TB were recruited and followed up for 24 months. Drug susceptibility testing was performed by microscopic observation drug susceptibility assay, mycobacterial growth indicator tube, and by WGS on isolates at first presentation and in the case of re-presentation. Where MDR-TB was diagnosed, WGS was used to determine the genomic relatedness between initial and subsequent isolates. De novo emergence of MDR-TB was assumed where the genomic distance was 5 or fewer single-nucleotide polymorphisms (SNPs), whereas reinfection with a different MDR-TB strain was assumed where the distance was 10 or more SNPs. Results Two hundred thirty-nine patients with isoniazid-resistant pulmonary TB were recruited. Fourteen (14/239 [5.9%]) patients were diagnosed with a second episode of TB that was multidrug resistant. Six (6/239 [2.5%]) were identified as having evolved MDR-TB de novo and 6 as having been reinfected with a different strain. In 2 cases, the genomic distance was between 5 and 10 SNPs and therefore indeterminate. Conclusions In isoniazid-resistant TB, de novo emergence and reinfection of MDR-TB strains equally contributed to MDR development. Early diagnosis and optimal treatment of isoniazid-resistant TB are urgently needed to avert the de novo emergence of MDR-TB during treatment., We investigated the sources of multidrug-resistant (MDR) tuberculosis (TB) in patients with isoniazid-resistant TB treated with first-line anti-TB therapy and show that reinfection with a new MDR-TB strain was just as common as the emergence of rifampicin resistance among these patients.

To examine the transmission dynamics of Mycobacterium tuberculosis (Mtb) isolated from tuberculosis patients in Ho Chi Minh City, Vietnam, we sequenced the whole genomes of 1,635 isolates and compared these with 3,144 isolates from elsewhere. The data identify an underlying burden of disease caused by the endemic Mtb lineage 1 associated with the activation of long-term latent infection, and a threefold higher burden associated with the more recently introduced Beijing lineage and lineage 4 Mtb strains. We find that Beijing lineage Mtb is frequently transferred between Vietnam and other countries, and detect higher levels of transmission of Beijing lineage strains within this host population than the endemic lineage 1 Mtb. Screening for parallel evolution of Beijing lineage-associated SNPs in other Mtb lineages as a signal of positive selection, we identify an alteration in the ESX-5 type VII-secreted protein EsxW, which could potentially contribute to the enhanced transmission of Beijing lineage Mtb in Vietnamese and other host populations.

Repeated emergence, not international dissemination, is behind the rise of multidrug-resistant lineage 4 tuberculosis., On the basis of population genomic and phylogeographic analyses of 1669 Mycobacterium tuberculosis lineage 4 (L4) genomes, we find that dispersal of L4 has been completely dominated by historical migrations out of Europe. We demonstrate an intimate temporal relationship between European colonial expansion into Africa and the Americas and the spread of L4 tuberculosis (TB). Markedly, in the age of antibiotics, mutations conferring antimicrobial resistance overwhelmingly emerged locally (at the level of nations), with minimal cross-border transmission of resistance. The latter finding was found to reflect the relatively recent emergence of these mutations, as a similar degree of local restriction was observed for susceptible variants emerging on comparable time scales. The restricted international transmission of drug-resistant TB suggests that containment efforts at the level of individual countries could be successful.

Tuberculosis (TB) is a leading cause of death from infectious disease and the global burden is now higher than at any point in history. Despite coordinated efforts to control TB transmission, the factors contributing to its successful spread remain poorly understood. Vietnam is identified as one of 30 high burden countries for TB and MDR-TB with an incidence of 137 TB cases per 100,000 individuals in 2015. Recent phylogenomic analyses of the causative agent Mycobacterium tuberculosis (Mtb) in other high-prevalence regions have provided insights into the complex processes underlying TB transmission. Here we examine the transmission dynamics of Mtb isolated from TB patients in Ho Chi Minh City (HCMC), Vietnam via whole genome analysis of 1,635 isolates and comparison with 3,085 isolates from other locations. The genomic data reveal an underlying burden of disease caused by endemic Mtb Lineage 1 associated with activation of long-term latent infection, on top of which is overlaid a three-fold higher burden associated with introduction of exotic Lineage 2 and 4 Mtb strains. We identify frequent transfer of Beijing lineage Mtb into the country, which are associated with higher levels of transmission in this host population than endemic Lineage 1 Mtb. We identify a mutation in the secreted protein EsxW in Beijing strains that also appears to be under positive selection in other Mtb lineages, which could potentially contribute to the enhanced transmission of the Beijing lineage in Vietnamese and other host populations.

Background: The optimal time to initiate antiretroviral therapy (ART) in human immunodeficiency virus (HIV)–associated tuberculous meningitis is unknown. Methods: We conducted a randomized, double-blind, placebo-controlled trial of immediate versus deferred ART in patients with HIV-associated tuberculous meningitis to determine whether immediate ART reduced the risk of death. Antiretroviral drugs (zidovudine, lamivudine, and efavirenz) were started either at study entry or 2 months after randomization. All patients were treated with standard antituberculosis treatment, adjunctive dexamethasone, and prophylactic co-trimoxazole and were followed up for 12 months. We conducted intention-to-treat, per-protocol, and prespecified subgroup analyses. Results: A total of 253 patients were randomized, 127 in the immediate ART group and 126 in the deferred ART group; 76 and 70 patients died within 9 months in the immediate and deferred ART groups, respectively. Immediate ART was not significantly associated with 9-month mortality (hazard ratio [HR], 1.12; 95% confidence interval [CI], .81–1.55; P = .50) or the time to new AIDS events or death (HR, 1.16; 95% CI, .87–1.55; P = .31). The percentage of patients with severe (grade 3 or 4) adverse events was high in both arms (90% in the immediate ART group and 89% in the deferred ART group; P = .84), but there were significantly more grade 4 adverse events in the immediate ART arm (102 in the immediate ART group vs 87 in the deferred ART group; P = .04). Conclusions:Immediate ART initiation does not improve outcome in patients presenting with HIV-associated tuberculous meningitis. There were significantly more grade 4 adverse events in the immediate ART arm, supporting delayed initiation of ART in HIV-associated tuberculous meningitis. Clinical Trials Registration: ISRCTN63659091.

There were 8.6 million cases and 1.3 million deaths from tuberculosis (TB) globally in 2012. Among the major challenges to global TB control and the ultimate goal of TB elimination is the increasing prevalence of drug resistant strains of Mycobacterium tuberculosis worldwide, coupled with an extremely limited pipeline of novel drug development. In 2012 there were an estimated 450,000 cases of muti- drug resistant TB (resistant to at least rifampicin and isoniazid) and 170,000 deaths due to multi-drug resistant (MDR) TB worldwide. The prevalence of resistance to isoniazid is extrememly high In some regions of the world, including Vietnam, where 25% of new smear positive patients and 54% of re treatment patients are infected with strains resistant to isoniazid. Treatment outcomes are known to be worse for patients with undiagnosed isoniazid resistant (INHR) TB treated with standard regimens but the majority of patients have successful outcomes. This thesis investigated risk factors for treatment failure among patients with isoniazid resistant TB in Ho Chi Minh City, Vietnam. \ud \ud Chapter one provides an introduction to tuberculosis and isoniazid resistant TB and chapter two describes the methodology of the studies decribed in the thesis. In chapter three, I investigate the treatment outcomes among a cohort of patients with isoniazid resistant tuberculosis treated according to National TB guidelines. The data show that unfavourable treatment outcomes are unacceptably high, at 19% among patients with INHR TB.\ud \ud In chapter four, I investigate three bacterial factors associated with unfavourable treatment outcomes among patients with isoniazid resistant TB, bacterial lineage, MIC to isoniazid and mutations responsible for isoniazid resistance. I show that the Beijing genotype is associated with young age, isoniazid resistance and multi-drug\ud resistance, and unfavourable outcome.\ud \ud In chapter five, I determine the prevalence of viral hepatitis co-infection among patients with INHR TB. Infection with hepatitis B is 9.8% and hepatitis C 4.6% among patients with INHR TB and is a risk factor for the development of anti TB drug-induced hepatitis (ATDIH) but not for unfavourable outcome.\ud \ud In chapter six, I determine the distribution of acetylator types for isoniazid among Vietnamese TB patients and show that acetylator status is not a risk factor for unfavourable outcome in patients with INHR TB.\ud \ud Overall, these studies show that INHR TB is a serious challenge to TB control efforts in Vietnam, with unacceptably high rates of treatment failure. In the final chapter, I discuss the implication of my findings and propose priorities for further research to address these issues.