PE/PPE mutations in the transmission of Mycobacterium tuberculosis in China revealed by whole genome sequencing.

OBJECTIVE: This study aims to examine the impact of PE/PPE gene mutations on the transmission of Mycobacterium tuberculosis (M. tuberculosis) in China. METHODS: We collected the whole genome sequencing (WGS) data of 3202 M. tuberculosis isolates in China from 2007 to 2018 and investigated the clustering of strains from different lineages. To evaluate the potential role of PE/PPE gene mutations in the dissemination of the pathogen, we employed homoplastic analysis to detect homoplastic single nucleotide polymorphisms (SNPs) within these gene regions. Subsequently, logistic regression analysis was conducted to analyze the statistical association. RESULTS: Based on nationwide M. tuberculosis WGS data, it has been observed that the majority of the M. tuberculosis burden in China is caused by lineage 2 strains, followed by lineage 4. Lineage 2 exhibited a higher number of transmission clusters, totaling 446 clusters, of which 77 were cross-regional clusters. Conversely, there were only 52 transmission clusters in lineage 4, of which 9 were cross-regional clusters. In the analysis of lineage 2 isolates, regression results showed that 4 specific gene mutations, PE4 (position 190,394; c.46G > A), PE_PGRS10 (839,194; c.744 A > G), PE16 (1,607,005; c.620T > G) and PE_PGRS44 (2,921,883; c.333 C > A), were significantly associated with the transmission of M. tuberculosis. Mutations of PE_PGRS10 (839,334; c.884 A > G), PE_PGRS11 (847,613; c.1455G > C), PE_PGRS47 (3,054,724; c.811 A > G) and PPE66 (4,189,930; c.303G > C) exhibited significant associations with the cross-regional clusters. A total of 13 mutation positions showed a positive correlation with clustering size, indicating a positive association. For lineage 4 strains, no mutations were found to enhance transmission, but 2 mutation sites were identified as risk factors for cross-regional clusters. These included PE_PGRS4 (338,100; c.974 A > G) and PPE13 (976,897; c.1307 A > C). CONCLUSION: Our results indicate that some PE/PPE gene mutations can increase the risk of M. tuberculosis transmission, which might provide a basis for controlling the spread of tuberculosis.

Unraveling tuberculosis patient cluster transmission chains: integrating WGS-based network with clinical and epidemiological insights.

Background: Tuberculosis remains a global health threat, and the World Health Organization reports a limited reduction in disease incidence rates, including both new and relapse cases. Therefore, studies targeting tuberculosis transmission chains and recurrent episodes are crucial for developing the most effective control measures. Herein, multiple tuberculosis clusters were retrospectively investigated by integrating patients' epidemiological and clinical information with median-joining networks recreated based on whole genome sequencing (WGS) data of Mycobacterium tuberculosis isolates. Methods: Epidemiologically linked tuberculosis patient clusters were identified during the source case investigation for pediatric tuberculosis patients. Only M. tuberculosis isolate DNA samples with previously determined spoligotypes identical within clusters were subjected to WGS and further median-joining network recreation. Relevant clinical and epidemiological data were obtained from patient medical records. Results: We investigated 18 clusters comprising 100 active tuberculosis patients 29 of whom were children at the time of diagnosis; nine patients experienced recurrent episodes. M. tuberculosis isolates of studied clusters belonged to Lineages 2 (sub-lineage 2.2.1) and 4 (sub-lineages 4.3.3, 4.1.2.1, 4.8, and 4.2.1), while sub-lineage 4.3.3 (LAM) was the most abundant. Isolates of six clusters were drug-resistant. Within clusters, the maximum genetic distance between closely related isolates was only 5-11 single nucleotide variants (SNVs). Recreated median-joining networks, integrated with patients' diagnoses, specimen collection dates, sputum smear microscopy, and epidemiological investigation results indicated transmission directions within clusters and long periods of latent infection. It also facilitated the identification of potential infection sources for pediatric patients and recurrent active tuberculosis episodes refuting the reactivation possibility despite the small genetic distance of ≤5 SNVs between isolates. However, unidentified active tuberculosis cases within the cluster, the variable mycobacterial mutation rate in dormant and active states, and low M. tuberculosis genetic variability inferred precise transmission chain delineation. In some cases, heterozygous SNVs with an allelic frequency of 10-73% proved valuable in identifying direct transmission events. Conclusion: The complex approach of integrating tuberculosis cluster WGS-data-based median-joining networks with relevant epidemiological and clinical data proved valuable in delineating epidemiologically linked patient transmission chains and deciphering causes of recurrent tuberculosis episodes within clusters.

Surveillance of close contacts of patients with infectious tuberculosis: a prospective cohort study.

BACKGROUND: A long-term follow-up of close contacts to monitor their infection status is essential to formulate a promising screening strategy. The study aimed to assess the dynamics of tuberculosis (TB) infection using Interferon-γ release assay (IGRA) and determine risk factors associated with TB infection. METHODS: Definite TB patients were interviewed and their household contacts were screened for TB infection by IGRA during 12-month longitudinal investigation. RESULTS: We included in our analyses 184 household contacts of 92 index TB patients. 87 individuals (47.3%) in contact group progressed to TB infection, of whom 86 developed into IGRA positive within 24 weeks. Close contacts with a higher age and comorbidities are easier to exhibit TB infection. Analysis showed that risk factors for becoming IGRA-positive individuals included residence, older age, comorbidities, BCG scar and high bacterial load. Contacts with BCG scar had a lower IGRA-positive rate. CONCLUSION: IGRA conversion generally occurs within 24 weeks after exposure. The TB transmission happens since subclinical TB stage and the presence of BCG scar is an independent protective factor reducing risk of TB infection among close contacts. Repeated IGRA tests are sensible to conducted among close contacts at 24 weeks after exposure to identify the IGRA-positive individuals.

The role of whole genome sequencing in identifying occupational tuberculosis among healthcare workers: two case reports.

In this case report, we present case reports for two nurses, both working in departments of respiratory medicine, who developed tuberculosis (TB). For each individual case, whole genome sequencing (WGS) revealed only one specific match within a genomic distance of <6 single-nucleotide polymorphisms. The subsequent epidemiological investigations confirmed that both nurses had relevant exposures to their corresponding match 1139 and 1704 days before presenting with TB symptoms, respectively. Twenty-two studies were identified that reported using genotyping to identify occupational transmission of Mycobacterium tuberculosis to healthcare workers. Only two studies applied WGS, both conducted in resource-rich countries, comparable to the present Danish investigation. When comparing the two WGS studies to the other studies that used older genotyping techniques, WGS provided a higher resolution and much more detailed information. Consequently, the epidemiological investigations were more straightforward. In conclusion, WGS is a powerful tool for determining whether M. tuberculosis transmission is occupational as demonstrated for the two cases in this study.

Confirmation of the invasive American mink (Neogale vison) as carrier of Mycobacterium bovis in southern Chile.

Bovine tuberculosis (bTB) is a chronic infectious-contagious disease with worldwide distribution, caused by the zoonotic pathogen Mycobacterium bovis. It is believed that the existence of wild cycles may hamper the success of bTB control strategies worldwide, where wildlife species could be reservoirs of this bacterial agent across their native (e.g., European badgers, wild boars) or non-indigenous (e.g., brushtail possum in New Zealand) ranges. However, further studies are required to understand the potential risk posed by non-native wildlife in becoming carriers of M. bovis in other neglected latitudes, such as the Southern Cone of South America. In this study, we performed a specific M. bovis-RD4 real-time PCR (qPCR) assay to detect bacterial DNA in tissues from the invasive American mink (Neogale vison) in Los Ríos region, Chile. We detected M. bovis DNA in blood samples collected from 13 out of 186 (7 %) minks with known sex and age. We did not find any significant differences in bacterial DNA detection according to mink sex and age. We found that 92 % (12/13) of specimens were positive in lung, 39 % (5/13) in mediastinal lymph node, and 15 % (2/13) in mesenteric lymph node, which suggest that both respiratory and digestive pathways as possible routes of transmission between infected hosts and minks. Our study is the first report on M. bovis molecular detection in invasive minks in an area where the largest cattle population in the country is located. Furthermore, this area is characterized by a low within-herd prevalence of M. bovis infection in cattle, with a relatively low number of infected herds, and so far, no attempts at eradicating the disease have been successful.

Population structure and history of Mycobacterium bovis European 3 clonal complex reveal transmission across ecological corridors of unrecognized importance in Portugal.

Mycobacterium bovis causes animal tuberculosis in livestock and wildlife, with an impact on animal health and production, wildlife management, and public health. In this work, we sampled a multi-host tuberculosis community from the official hotspot risk area of Portugal over 16 years, generating the largest available data set in the country. Using phylogenetic and ecological modeling, we aimed to reconstruct the history of circulating lineages across the livestock-wildlife interface to inform intervention and the implementation of genomic surveillance within the official eradication plan. We find evidence for the co-circulation of M. bovis European 1 (Eu1), Eu2, and Eu3 clonal complexes, with Eu3 providing sufficient temporal signal for further phylogenetic investigation. The Eu3 most recent common ancestor (bovine) was dated in the 1990s, subsequently transitioning to wildlife (red deer and wild boar). Isolate clustering based on sample metadata was used to inform phylogenetic inference, unravelng frequent transmission between two clusters that represent an ecological corridor of previously unrecognized importance in Portugal. The latter was associated with transmission at the livestock-wildlife interface toward locations with higher temperature and precipitation, lower agriculture and road density, and lower host densities. This is the first analysis of M. bovis Eu3 complex in Iberia, shedding light on background ecological factors underlying long-term transmission and informing where efforts could be focused within the larger hotspot risk area of Portugal. IMPORTANCE: Efforts to strengthen surveillance and control of animal tuberculosis (TB) are ongoing worlwide. Here, we developed an eco-phylodynamic framework based on discrete phylogenetic approaches informed by M. bovis whole-genome sequence data representing a multi-host transmission system at the livestock-wildlife interface, within a rich ecological landscape in Portugal, to understand transmission processes and translate this knowledge into disease management benefits. We find evidence for the co-circulation of several M. bovis clades, with frequent transmission of the Eu3 lineage among cattle and wildlife populations. Most transition events between different ecological settings took place toward host, climate and land use gradients, underscoring animal TB expansion and a potential corridor of unrecognized importance for M. bovis maintenance. Results stress that animal TB is an established wildlife disease without ecological barriers, showing that control measures in place are insufficient to prevent long-distance transmission and spillover across multi-host communities, demanding new interventions targeting livestock-wildlife interactions.

Genomic and spatial analysis reveal the transmission dynamics of tuberculosis in areas with high incidence of Zhejiang, China: A prospective cohort study.

In the mountainous, rural regions of eastern China, tuberculosis (TB) remains a formidable challenge; however, the long-term molecular epidemiological surveillance in these regions is limited. This study aimed to investigate molecular and spatial epidemiology of TB in two mountainous, rural counties of Zhejiang Province, China, from 2015 to 2021, to elucidate the recent transmission and drug-resistance profiles. The predominant Lineage 2 (L2) Beijing family accounted for 80.1% of total 532 sequenced Mycobacterium tuberculosis (Mtb) strains, showing consistent prevalence over seven years. Gene mutations associated with drug resistance were identified in 19.4% (103/532) of strains, including 47 rifampicin or isoniazid-resistant strains, eight multi-drug-resistant (MDR) strains, and five pre-extensively drug-resistant (pre-XDR) strains. Genomic clustering revealed 53 distinct clusters with an overall transmission clustering rate of 23.9% (127/532). Patients with a history of retreatment and those infected with L2 strains had a higher risk of recent transmission. Spatial and epidemiological analysis unveiled significant transmission hotspots, especially in densely populated urban areas, involving various public places such as medical institutions, farmlands, markets, and cardrooms. The study emphasizes the pivotal role of Beijing strains and urban-based TB transmission in the western mountainous regions in Zhejiang, highlighting the urgent requirement for specific interventions to mitigate the impact of TB in these unique communities.

First time whole genome sequencing of Mycobacterium bovis from the environment supports transmission at the animal-environment interface.

Spreading of Mycobacterium bovis causing animal tuberculosis (TB) at livestock-wildlife-environment interfaces remains a significant problem. Recently, we provided evidence of widespread environmental contamination of an endemic animal TB setting with viable and dormant M. bovis cells able to recover metabolic activity, making indirect transmission via environmental contamination plausible. We now report the first whole genome sequences of M. bovis recovered from the environment. We establish epidemiological links at the environment-animal interface by phylogenomic comparison of these M. bovis genomes with those isolated from livestock and wild ungulates from the same area. Environmental and animal genomes are highly intertwined and distribute similarly into the same M. bovis lineages, supporting several instances of environmental contamination. This study provides compelling evidence of M. bovis excretion into the environment and viability maintenance, supporting the environment as a potential source of new infection. These insights have clear implications for policy formulation, advocating environmental surveillance and an ecosystem perspective in TB control programs. ENVIRONMENTAL IMPLICATION: We report the first whole genome sequences of M. bovis from the environment and establish epidemiological links at the environment-animal interface, demonstrating close phylogenomic relatedness of animal and environmental M. bovis. Definitive evidence of M. bovis excretion into the environment with viability maintenance is provided, supporting the environment as a potential source of new infection. Implications of this work include methodological innovations offering a tool to resolve indirect transmission chains and support customized biosecurity measures. Policy formulation aiming at the control of animal tuberculosis and cost mitigation should consider these findings, encouraging environmental surveillance in official eradication programmes.

HIV co-infection is associated with reduced Mycobacterium tuberculosis transmissibility in sub-Saharan Africa.

Persons living with HIV are known to be at increased risk of developing tuberculosis (TB) disease upon infection with Mycobacterium tuberculosis (Mtb). However, it has remained unclear how HIV co-infection affects subsequent Mtb transmission from these patients. Here, we customized a Bayesian phylodynamic framework to estimate the effects of HIV co-infection on the Mtb transmission dynamics from sequence data. We applied our model to four Mtb genomic datasets collected in sub-Saharan African countries with a generalized HIV epidemic. Our results confirm that HIV co-infection is a strong risk factor for developing active TB. Additionally, we demonstrate that HIV co-infection is associated with a reduced effective reproductive number for TB. Stratifying the population by CD4+ T-cell count yielded similar results, suggesting that, in this context, CD4+ T-cell count is not a better predictor of Mtb transmissibility than HIV infection status alone. Together, our genome-based analyses complement observational household contact studies, and more firmly establish the negative association between HIV co-infection and Mtb transmissibility.

Mycobacterium tuberculosis Infection in School Contacts of Tuberculosis Cases: A Systematic Review and Meta-Analysis.

Substantial tuberculosis transmission occurs outside of households, and tuberculosis surveillance in schools has recently been proposed. However, the yield of tuberculosis outcomes from school contacts is not well characterized. We assessed the prevalence of Mycobacterium tuberculosis infection among close school contacts by performing a systematic review. We searched PubMed, Elsevier, China National Knowledge Infrastructure, and Wanfang databases. Studies reporting the number of children who were tested overall and who tested positive were included. Subgroup analyses were performed by study location, index case bacteriological status, type of school, and other relevant factors. In total, 28 studies including 54,707 school contacts screened for M. tuberculosis infection were eligible and included in the analysis. Overall, the prevalence of M. tuberculosis infection determined by the QuantiFERON Gold in-tube test was 33.2% (95% CI, 0.0-73.0%). The prevalences of M. tuberculosis infection based on the tuberculin skin test (TST) using 5 mm, 10 mm, and 15 mm as cutoffs were 27.2% (95% CI, 15.1-39.3%), 24.3% (95% CI, 15.3-33.4%), and 12.7% (95% CI, 6.3-19.0%), respectively. The pooled prevalence of M. tuberculosis infection (using a TST ≥5-mm cutoff) was lower in studies from China (22.8%; 95% CI, 16.8-28.8%) than other regions (36.7%; 95% CI, 18.1-55.2%). The pooled prevalence of M. tuberculosis infection was higher when the index was bacteriologically positive (43.6% [95% CI, 16.5-70.8%] versus 23.8% [95% CI, 16.2-31.4%]). These results suggest that contact investigation and general surveillance in schools from high-burden settings merit consideration as means to improve early case detection in children.

Community Voices and Whole-Genome Sequencing for Tuberculosis: Storytelling and the Importance of Listening.

One of the primary public health functions of a tuberculosis (TB) program is to arrest the spread of infection. Traditionally, TB programs have relied on epidemiological information, gathered through contact tracing, to infer that transmission has occurred between people. The ability of drawing such inferences is extensively context dependent. Where epidemiological information has been strong, such as 2 cases of TB occurring sequentially within a single household, confidence in such inferences is high; conversely, public health authorities have been less certain about the significance of TB cases merely occurring in the same wider social group or geographic area. Many current laboratory tests for TB used globally may be sufficient to confirm a diagnosis and guide appropriate therapy but still be insufficiently precise for distinguishing two strains reliably. In short, drawing inferences regarding a chain of transmissions has always been as much art as science.

Management of newborns and healthcare workers exposed to isoniazid-resistant congenital tuberculosis in the neonatal intensive care unit.

BACKGROUND: Management of newborns and healthcare workers (HCWs) exposed to congenital tuberculosis (TB) in the neonatal intensive care unit (NICU) has been reported rarely. AIM: To outline a contact investigation process for individuals exposed to congenital TB in the NICU and investigate nosocomial transmission. Additionally, to assess the efficacy and safety of window prophylaxis in exposed newborns. METHODS: A baby, born at a gestational age of 28 + 1 weeks, was diagnosed with isoniazid-resistant congenital TB on the 39th day of admission to the level IV NICU. Newborns and HCWs exposed cumulatively for ≥8 h underwent contact investigation and follow-up for a year. FINDINGS: Eighty-two newborns underwent contact investigation. All newborns displayed normal chest X-rays, and 42 hospitalized newborns tested negative for acid-fast bacilli stain and Xpert® MTB/RIF assay in their endotracheal sputum or gastric juices. Eighty received window prophylaxis: six of 75 on rifampin experienced mild adverse events, and none of the five on levofloxacin. After 12 weeks, five (6.1%) had a positive tuberculin skin test, all of whom had already received the Bacillus Calmette-Guérin vaccine and tested negative on TB interferon-gamma releasing assay. Of 119 exposed HCWs, three (2.5%) were diagnosed with latent TB infection and completed a four-month rifampin therapy. There was no active TB disease among exposed newborns and HCWs during a one-year follow-up. CONCLUSION: Timely diagnosis of congenital TB is crucial for minimizing transmission among exposed neonates and HCWs in the NICU setting. In cases of isoniazid-resistant index patients, even premature newborns may consider the use of rifampin or levofloxacin for window prophylaxis.

Community-Wide Universal HIV Test and Treat Intervention Reduces Tuberculosis Transmission in Rural Uganda: A Cluster-Randomized Trial.

BACKGROUND: Human immunodeficiency virus (HIV) treatment reduces tuberculosis (TB) disease and mortality; however, the population-level impact of universal HIV-test-and-treat interventions on TB infection and transmission remain unclear. METHODS: In a sub-study nested in the SEARCH trial, a community cluster-randomized trial (NCT01864603), we assessed whether a universal HIV-test-and-treat intervention reduced population-level incident TB infection in rural Uganda. Intervention communities received annual, population-level HIV testing and patient-centered linkage. Control communities received population-level HIV testing at baseline and endline. We compared estimated incident TB infection by arms, defined by tuberculin skin test conversion in a cohort of persons aged 5 and older, adjusting for participation and predictors of infection, and accounting for clustering. RESULTS: Of the 32 trial communities, 9 were included, comprising 90 801 participants (43 127 intervention and 47 674 control). One-year cumulative incidence of TB infection was 16% in the intervention and 22% in the control; SEARCH reduced the population-level risk of incident TB infection by 27% (adjusted risk ratio = 0.73; 95% confidence interval [CI]: .57-.92, P = .005). In pre-specified analyses, the effect was largest among children aged 5-11 years and males. CONCLUSIONS: A universal HIV-test-and-treat intervention reduced incident TB infection, a marker of population-level TB transmission. Investments in community-level HIV interventions have broader population-level benefits, including TB reductions.

Genomic epidemiology sheds light on the emergence and spread of Mycobacterium bovis Eu2 Clonal Complex in Portugal.

ABSTRACTAnimal tuberculosis (TB) remains a serious concern for animal and human health. Mycobacterium bovis circulates in multi-host systems, dominated by the European 2 clonal complex (Eu2) in Iberia. In this work, we use genomic epidemiology to infer the emergence, spread, and spatiotemporal patterns of Eu2 in the official epidemiological risk area of animal TB in Portugal. Phylogenetic analysis of 144 M. bovis whole-genome sequences from cattle, wild boar, and red deer, representing the 2002-2021 period, distinguished three Eu2 clades that evolved independently. The major Eu2 clade underwent phylodynamic inferences to estimate the time and location of outbreaks, host transitions, and spatial diffusion as well. The origin of this Eu2 clade was attributed to the red deer population in the Castelo Branco district, near the border with Spain. Most host transitions were intraspecific (80%), while interspecific transmissions between wildlife species (wild boar-red deer), and between wild boar and cattle, were highly supported. Phylogeographic reconstruction evidenced that most transitions (82%) occur within municipalities, highlighting local transmission corridors.Our study indicates that M. bovis continues to spread at the cattle-wildlife interface within the animal TB hotspot area, possibly driven by the foraging behaviour of wild boar near agricultural lands. Red deer seems to be an important driver of TB within wildlife hosts, while the wild boar links the multi-host wildlife community and livestock. This work highlights the value of combining genomic epidemiology with phylodynamic inference to resolve host jumps and spatial patterns of M. bovis, providing real-time clues about points of intervention.

Comparing Mycobacterium tuberculosis transmission reconstruction models from whole genome sequence data.

Genomic epidemiology is routinely used worldwide to interrogate infectious disease dynamics. Multiple computational tools exist that reconstruct transmission networks by coupling genomic data with epidemiological models. Resulting inferences can improve our understanding of pathogen transmission dynamics, and yet the performance of these tools has not been evaluated for tuberculosis (TB), a disease process with complex epidemiology including variable latency and within-host heterogeneity. Here, we performed a systematic comparison of six publicly available transmission reconstruction models, evaluating their accuracy when predicting transmission events in simulated and real-world Mycobacterium tuberculosis outbreaks. We observed variability in the number of transmission links that were predicted with high probability (P ≥ 0.5) and low accuracy of these predictions against known transmission in simulated outbreaks. We also found a low proportion of epidemiologically supported case-contact pairs were identified in our real-world TB clusters. The specificity of all models was high, and a relatively high proportion of the total transmission events predicted by some models were true links, notably with TransPhylo, Outbreaker2, and Phybreak. Our findings may inform the choice of tools in TB transmission analyses and underscore the need for caution when interpreting transmission networks produced using probabilistic approaches.

Guia para Controle de Tuberculose em Instituições de Acolhimento para População em Situação de Rua / Guide to Tuberculosis Control in Reception Institutions for Population in Street Situation

Este guia se destina a profissionais que atuam, principalmente, nas Instituições de Acolhimento destinadas à População em Situação de Rua (PSR). Entretanto, vários conceitos e informações que serão apresentados aqui podem ser usados em outros espaços de acolhimento e de oferta de cuidados a esta população, como os de grupos informais e de organizações públicas, governamentais ou não-governamentais.

Perguntas e respostas sobre tuberculose para educadores comunitários - Elaboração participativa de material educativo / Questions and answers about tuberculosis for community educators - Participatory development of educational material

Esta cartilha é resultado de um processo de construção participativa de material educativo entre educadores e lideranças comunitárias do Fórum TB/RJ e profissionais de saúde.

Characterisation of drug-resistant Mycobacterium tuberculosis mutations and transmission in Pakistan.

Tuberculosis, caused by Mycobacterium tuberculosis, is a high-burden disease in Pakistan, with multi-drug (MDR) and extensive-drug (XDR) resistance, complicating infection control. Whole genome sequencing (WGS) of M. tuberculosis is being used to infer lineages (strain-types), drug resistance mutations, and transmission patterns-all informing infection control and clinical decision making. Here we analyse WGS data on 535 M. tuberculosis isolates sourced across Pakistan between years 2003 and 2020, to understand the circulating strain-types and mutations related to 12 anti-TB drugs, as well as identify transmission clusters. Most isolates belonged to lineage 3 (n = 397; 74.2%) strain-types, and were MDR (n = 328; 61.3%) and (pre-)XDR (n = 113; 21.1%). By inferring close genomic relatedness between isolates (< 10-SNPs difference), there was evidence of M. tuberculosis transmission, with 55 clusters formed consisting of a total of 169 isolates. Three clusters consist of M. tuberculosis that are similar to isolates found outside of Pakistan. A genome-wide association analysis comparing 'transmitted' and 'non-transmitted' isolate groups, revealed the nusG gene as most significantly associated with a potential transmissible phenotype (P = 5.8 × 10-10). Overall, our study provides important insights into M. tuberculosis genetic diversity and transmission in Pakistan, including providing information on circulating drug resistance mutations for monitoring activities and clinical decision making.