Advancing research through collaboration is at the heart of the JHU TRAC mission. Research is organized around scientific programs and working groups that represent a cross-section of member strengths and disciplines. Learn more about key dates and information for past awards. Details for the 2025 funding cycle will be announced in December 2024.
Awarded Research
JHU TRAC FACULTY DEVELOPMENTAL AWARDEE 2024
Investigating the role of CCRL2 as an adjunctive host-directed therapeutic target in TB
Recipient: Theodoros Karantanos, MD, PhD
We discovered that C-C motif receptor-like 2 (CCRL2), a chemokine receptor-like protein, is upregulated in high-risk myelodysplastic syndrome and secondary acute myeloid leukemia. Its knockdown suppresses cancer cell growth and increases cancer cell sensitivity to the limited available therapies. Using novel antibody-drug conjugates (ADCs) as a targeted therapy to bind to antigens present on cell surfaces leading to cell death, we have been able to target and kill CCRL2-expressing cancer cells selectively. Since CCRL2 is expressed in dendritic cells and macrophages, and may be linked with Mycobacterium tuberculosis (Mtb) persistence via M2 macrophage polarization, dendritic cell maturation inhibition, and chemerin deprivation, we tested a novel CCRL2 ADC (conjugated with the cytotoxic drug SG3199) as a host-directed therapy for tuberculosis (TB). We hypothesized that the selective killing of Mtb-infected CCRL2-expressing macrophages and dendritic cells will result in Mtb release in the vicinity, enhancing the antigen recognition and facilitating clearance. We found that the CCRL2 ADC enhanced the adjunctive therapeutic response of the four-drug first-line TB regimen after 7 weeks of treatment in a chronic TB mouse model without associated toxicity. This proposal explores the role of CCRL2 in TB immunity and whether novel CCRL2-targeted approaches can shorten curative TB treatment in preclinical models. This study will help the PI obtain preliminary data to submit an R21 proposal focusing on understanding the immune mechanisms of the selective CCRL2-targeting host-directed therapy approaches. This project is in collaboration with Styliani Karanika, MD (Assistant Professor of Medicine, Division of Infectious Diseases)
JHU TRAC FACULTY DEVELOPMENTAL AWARDEE 2024
Characterizing the impact and strategies to optimize the effectiveness of multidrug-resistant tuberculosis preventive therapy among household contacts: a case study from Vietnam
Recipient: Parastu Kasaie, PhD
The initial findings from the V-QUIN trial (presented in Nov-2023) highlight the effectiveness of tuberculosis preventive therapy (TPT) for presumed rifampin-/multidrug-resistant-tuberculosis (RR/MDR-TB) among household contacts. However, the clinical trial results alone may be inadequate for supporting future implementation beyond research settings due to resource constraints. The long-term impact of RR/MDR-TPT extends beyond trial’s duration, offering benefits such as slowing disease progression and preventing secondary transmissions. To assess population-level impact and optimize implementation, we propose a dynamic agent-based simulation calibrated to individual-level data from the V-QUIN trial in Vietnam. Utilizing this model, we aim to project the long-term indirect impact of RR/MDR-TPT at the household- and population-level, and to evaluate future impact under enhancements in household contact interventions and community-wide RR/MDR-TB care strategies. Dr. Kasaie has led the development of this simulation, previously applied to explore the impact of RR/MDR-TPT in India. Currently Dr. Kasaie is extending her K01 award at no cost, focusing on HIV/STI coinfection dynamics. Leveraging Dr.Kasaie’s expertise in agent-based modeling and our collaborations with V-QUIN trial, we aim to gather preliminary data for expanding this project with an NIH R01 grant. The TRAC award will support Dr. Kasaie’s efforts and cover research expenses for proposed scientific aims.
JHU TRAC FACULTY DEVELOPMENTAL AWARDEE 2024
Brain-derived Extracellular Vesicles’ Participation in Tuberculous Meningitis Pathogenesis
Recipient: Elizabeth Tucker, MD
Central nervous system (CNS) tuberculosis (TB), most often TB meningitis (TBM), is the most severe form of TB and disproportionately affects young children. Current treatment is inadequate, with more than 75% of children left with life-long neurologic disability. Neuroinflammation driven by microglia is integral to pathogenesis, but the mechanisms remain poorly understood. Currently, adjunctive steroids are used to dampen neuroinflammation but they do not prevent neurological sequalae and only improve survival in some patients. Therefore, there is a critical need for a mechanistic understanding of the host inflammatory response to develop treatment that not only cures but also limits neurological injury. To investigate the host inflammatory response, we developed a young rabbit model of TBM that replicates key neuropathological features of human disease, including microglial activation and motor deficits. Extracellular vesicles (EVs) have emerged as important mediators of cellular communication, carrying RNAs, lipids and proteins that can modulate the behavior of recipient cells. Brain-derived EVs have a key role in neurodevelopment, neurodegenerative diseases and neuroinflammation where their secretion is increased. EVs have been investigated in pulmonary TB with differential effects based on the cell of origin; Mtb-derived EVs worsened infection but host-derived EVs (macrophage and neutrophils) promoted bacterial clearance. However, in TBM, neither immune cell-specific nor brain-derived EVs have been investigated, ignoring the unique immunopathology within the CNS.
In this TRAC proposal, we will investigate whether brain-derived EVs, especially microglia-derived EVs, contribute to the propagation of neuroinflammation in TBM. In Aim 1, EVs will be isolated from primary rabbit microglia cultures with and without Mtb infection to characterize its biochemical properties and immunogenicity. Aim 2 will use our in vivo pediatric rabbit TBM model to isolate brain-derived EVs and identify their cellular origin using Immuocapture of Cell-specific small Extracellular vesicles (ICE), an immunoaffinity-based method optimized by collaborator, Sam Das, that harnesses EV’s expression of cell surface markers from the cell of origin, which is an intrinsic property of EV-biogenesis. There are several areas of innovation that have the potential to revolutionize diagnosis and host-directed therapy in TBM. (1) Studying brain-derived EVs in TBM is completely unexplored. Microglia-derived EVs are associated with neuroinflammation and could elucidate new treatment targets. (2) Utilization of young TBM rabbit model allows investigation of the infection in a developing brain where microglia have unique roles in both immune defense and normal development. (3) Utilization of ICE is a novel approach to identify and capture cell-specific EVs, especially in rabbits.
These experiments will provide crucial preliminary data on the role of brain-derived EVs, especially microglia-derived EVs, in propagating neuroinflammation in TBM. They are designed to launch an entirely new direction of TBM research to unravel the mechanisms that lead to brain injury and death. This aligns perfectly with my long-term goals to better understand the host inflammatory response resulting in brain injury, to inform adjunctive therapeutic strategies, and to design diagnostic and therapeutic monitoring tools in order to improve outcomes in TBM. These experiments and other ongoing work will provide preliminary data to support new R01/R21 (ESI/NI) grant applications to explore the following topics: 1) Mechanisms by which brain-derived EVs propagate neuroinflammation; 2) EV-based diagnostic panel for TBM; 3) Targeting EVs to dampen neuroinflammation, such as altering EV-biogenesis; 4) Utilization of EVs as nanoparticles to deliver therapy. Additionally, the Catalyst Award will allow us to deepen collaborations already in place, including with Dr. Sam Das who is an Anesthesiology and Critical Care Medicine (ACCM) researcher with expertise in EV-based research. In summary, obtaining a TRAC Award will provide me the resources to transition and focus on this novel area of TBM research and would be transformative to my career trajectory and to the TBM field.
JHU TRAC POSTDOCTORAL DEVELOPMENTAL AWARDEE 2024
Evaluation of Risk Factors for Tuberculosis Infection Among Household Child and Adolescent Contacts in South Africa
Recipient: Evan Shirey, MD
TB remains a leading cause of child mortality in sub-Saharan Africa, and TB incidence in South Africa remains among the highest globally [1,2]. Although WHO guidelines now recommend provision of preventive treatment to older children and adolescents with household TB exposures, barriers to universal implementation remain and limited data exist regarding which contacts are at highest risk of infection.
Assessing Household Tuberculosis Transmission using detection of Differentially Culturable Tubercle Bacteria (HoTT-DCTB) is a prospective observational cohort study of patients with TB and their household contacts in South Africa (PI: Neil Martinson, 5R01AI147349). I propose a nested study within the HoTT-DCTB cohort to identify risk factors for incident TB infection among child and adolescent contacts.
We will add a tuberculin skin test (TST) to the demographic and TB exposure history already obtained. We will analyze these factors for association with TB infection and develop a risk score to identify those children and adolescents who are at highest risk of infection.
Improved understanding of transmission factors, particularly among older children and adolescents, will assist with developing targeted TB prevention interventions that may be effective and cost-effective for TB programs, which I plan to pilot within a future K23 application.
JHU TRAC FACULTY DEVELOPMENTAL AWARDEE 2023
Deciphering Mitochondrial Responses to Mycobacterium tuberculosis Infection of Macrophages
Recipient: Anne Hamacher-Brady, PhD
It is generally accepted that Mycobacterium tuberculosis (Mtb) modulates host cell death responses, bearing the potential for host-directed therapies. However, underlying mechanisms are incompletely understood, and a comprehensive characterization of contributing cell death modes is lacking. Mitochondria regulate programmed cell death, inflammatory and innate immune signaling. A recent study of our laboratory demonstrated that direct endolysosomal targeting of mitochondria is integral to mitochondrial outer membrane permeabilization, aka MOMP (Wang et al., Dev Cell, 2020). Based on our preliminary observation of Mtb infection-associated targeting of endolysosomes to mitochondria, albeit in apparent absence of functional MOMP, we propose to investigate the hypothesis that Mtb infection alters endolysosome-mitochondrial interaction-based regulation of mitochondrial cell death signaling.
Specific Aims
1) Targeted investigation of potential regulators of endolysosome-mitochondrial interactions during Mtb-induced macrophage cell death.
2) Proteomic mapping of Mtb infection-induced endolysosome-mitochondrial contact sites through TurboID-based proximity labeling.”
JHU TRAC FACULTY DEVELOPMENTAL AWARDEE 2023
Mycobacterium Tuberculosis Transmission Networks in Urban Uganda: Evidence from Spatial and Genomic Data
Recipient: Tess Ryckman, PhD
The proposed project seeks to characterize Mtb transmission in an urban Ugandan community in terms of (1) recency, (2) spatial distribution, and (3) known risk factors. To accomplish this objective, I plan three aims:
AIM 1: Identify transmission clusters. I will use phylogenetic techniques to identify transmission clusters: groups of individuals likely linked by transmission based on genomic distance. The resulting scale of clustering will provide evidence on the extent to which TB is driven by recent transmission within a densely populated urban community. I hypothesize that most isolates will be related to at least one other isolate (based on a standard genetic distance of 12 SNPs), given the small size of the study area (< 2 km2), intensity of screening, and my hypothesis that recent local transmission drives TB epidemics in high-burden countries like Uganda. Evidence of substantial recent localized transmission could imply that community-based efforts to find and treat people with undiagnosed TB could have major local-level impact in communities heavily affected by TB.
AIM 2: Describe the spatial distribution of transmission. I will combine pairwise genetic distances with GPS data on residential and screening locations to describe the association between genetic relatedness and space. I will also merge the transmission clusters from Aim 1 with GPS data to formally analyze the geographic scope of Mtb transmission, aiming to identify transmission “hotspots” within the study area and whether there is a spatial radius relevant to contact tracing efforts. I hypothesize that the probability of two individuals being in the same transmission cluster will be lower for people residing >1km vs <0.5 km apart. Evidence of spatial clustering could support efforts to augment spatially targeted case finding and prevention efforts.
AIM 3: Assess factors associated with the risk of transmission. I will make use of Bayesian phylogenetic techniques that enable inference regarding transmission directionality and then use statistical models to assess associations between hypothesized risk factors and the likelihood of transmitting Mtb. For clusters in which directionality is most uncertain, I will send a subset of isolates for deep sequencing, which can provide evidence on within-host diversity that can clarify transmission directionality. I hypothesize that key TB risk factors that affect contact patterns (e.g., incarceration history, young age) increase the risk of being a source of transmission, while others (e.g., HIV) may only increase the risk of acquiring TB. Findings can inform strategies for focused case detection (e.g., at release from correctional facilities or at venues frequented by young men).
The proposed research will provide unique insights on the scope of Mtb transmission networks in an urban African community – a starting point for evidence-based design of targeted interventions in these settings. Furthermore, this research will help me secure larger-scale funding by extending and bolstering the rationale for research I recently proposed in an initial K01 submission.
JHU TRAC POSTDOCTORAL DEVELOPMENTAL AWARDEE 2023
Deciphering the hormonal and genetic mechanisms mediating sex differences in tuberculosis: using a sst1-susceptible ‘Four Core Genotype’ mouse model
Recipient: Manish Gupta, PhD
TB afflicts more men than women with a global M/F morbidity and mortality ratio of ~1.7 but the underlying biologic basis for this sex bias is largely unknown. Previously we used the ‘Four Core Genotypes’ (FCG) mouse model in the standard C57BL/6 mouse background. FCG mice yield four genotypes that are XX or XY, each genotype with either testes or ovaries. Following Mtb infection we found that gonadal male mice (XX-males or XY-males) died significantly earlier than gonadal female mice (XX-females or XY-females. Of note, XY complemented females were more susceptible to Mtb infection than XX. This suggests that sex hormones have a major influence in the rapid the progression of pulmonary TB but that the sex chromosome complement also plays role with XX genotypes being protective. We further characterized these observations with other modalities (lung bacterial burden, histology, immunophenotyping, hormone/cytokine measurements, and bulk-RNAseq), and we are preparing a manuscript. However, the major limitation of our earlier work is that C57BL/6 mice do not develop human-like necrotic granulomas.
With TRAC funding, I propose to test the Mtb susceptibility of FCG mice in the B6sst1-susceptible (B6.Sst1S) background in which mice develop human-like, caseous, necrotic granulomas. By multiple backcross events, I have successfully generated a stable lineage of B6.Sst1S FCG mice and validated the superinduction of type I interferons in their BMDMs post TNF-α stimulation. Using these sst1-susceptible FCGs, I propose to perform low-dose Mtb infections and measure multiple outcome parameters: time-to-death, lung Mtb burden, CyTOF immunophenotyping, spatial transcriptomics, PET/CT imaging, hormone/cytokine measurements, etc. Further, we will correlate epigenetic profiles (ATAC-Seq) with bulk gene expression levels (RNA-Seq) in order to seek cellular evidence of escape from X chromosome inactivation in the BMDMs of these FCGs post Mtb infection. The results should enable me to discriminate the impact of genetics and sex steroids that might differentially modulate innate and adaptive immune responses in males and females, leading to sex differences in disease susceptibility. Research addressing the biological bases for the male bias is likely to provide novel insights into the pathogenesis of active TB disease and latent TB infection. I also hope to use these findings as preliminary data to apply for a K99-R00 and/or K22 award that will enable me to start an independent research career.
JHU TRAC POSTDOCTORAL DEVELOPMENTAL AWARDEE 2023
Characterization of immunometabolic drivers of tuberculosis infection
Recipient: Sadiya Parveen, PhD
Mtb is known to induce diverse metabolic changes in the host (Russell, Lamprecht, et al. 2019). However, the mechanism driving most of these metabolic changes remains understudied. We recently demonstrated that a novel glutamine metabolism antagonist JHU083, that was discovered by Drs Jonthan Powell and Barbara Slusher at JHU, substantially reduces lung bacillary burden and improves the survival of Mtb-infected mice. Additionally, we have shown that JHU083 modulates level of host metabolites in the whole lung tissue and boosts effector T-cell immunity in murine models of tuberculosis (Parveen et al.; under revision at Nat Comm; BioRxiv 2023). Single-cell RNA sequencing-based analysis revealed that JHU083 treatment affected metabolic pathways in a cell type-dependent fashion. The glutamine metabolism antagonist JHU083 downregulated transcripts associated with glutaminolysis in T-cells but upregulated it in macrophages. However, effect of JHU083 upon the metabolites in specific immune cell types remain to be deciphered.
Delineating the fundamental differences in how JHU083 affects the metabolome of macrophages vs. T-cells may lead to identifying host metabolic pathways crucial to the containment of TB. The present proposal aims to characterize these immune cell type-specific metabolic differences. We hypothesize that JHU083-mediated immunometabolic reprogramming is immune cell type dependent. To test this hypothesis, untargeted metabolomic analysis of macrophages and T-cells in vivo (isolated from lungs by magnetic-bead enrichment), with and without JHU083 treatment will be conducted. The targeted pathways will be glycolysis, glutaminolysis, beta-oxidation, amino acid utilization, Kreb’s cycle, and oxidative phosphorylation. These aims will lead to the identification of the host metabolic pathways that are critical for TB containment. We will also likely identify the fundamental differences in how JHU083 affects macrophages versus T-cells. This analysis will likely lead to identifying novel metabolic targets that will provide unique insights toward developing novel host-directed therapies for TB. Despite being largely descriptive, the results will provide preliminary data crucial for generating novel hypotheses for future R01 grants.
JHU TRAC DEVELOPMENTAL AWARDEE 2022
Development of a novel diagnostic assay for pediatric tuberculosis
Recipient: Subhra Chakraborty, PhD, MPH
Tuberculosis (TB) is a significant problem in both adults and children. Diagnosis of pediatric TB is challenging. Children fail to produce quality sputum, and therefore sensitivity and specificity of the current sputum-based assays are low. Since children swallow sputum, a stool specimen is a potential alternative for bacteriological confirmation of pulmonary TB. Previous studies with stool using Xpert had limitations, including low analytical and diagnostic sensitivity and a lack of a standard stool processing method. Previously, we innovated a low-resource-settings-adapted simple and sensitive stool processing method coupled with isothermal amplification assay named “RLDT” to detect enteric pathogens (JHTV C16784). We evaluated this assay in several endemic countries, and has attracted multiple grants, including NIH R21 and R01. We propose to develop RLDT platform-based “TB-RIF-RLDT,” a simple, rapid, and sensitive assay to detect TB and resistance to rifampin (RIF) from stool in children with the following specific aims: a) Develop TB-RIF-RLDT using spiked stool samples with M. tuberculosis bacteria. b) Determine performance specifications of TB-RIF-RLDT. c) Evaluate TB-RIF-RLDT at JHU with stool samples from TB-positive and negative children. With the preliminary data from this feasibility pilot study, we will apply for NIH grants to evaluate the TB-RIF RLDT in endemic countries.
JHU TRAC DEVELOPMENTAL AWARDEE 2022
A therapeutic mRNA vaccine targeting Mycobacterium tuberculosis persisters
Recipient: Styliani Karanika, MD
The key stringent response enzyme RelMtb is essential for prolonged Mycobacterium tuberculosis (Mtb) survival and antibiotic tolerance in vivo. We have demonstrated that DNA vaccination targeting RelMtb enhanced the activity of isoniazid in a murine tuberculosis (TB) model. After we fused relMtb to the gene encoding Macrophage Inflammatory Protein-3 alpha (MIP-3α), targeting the antigen to immature dendritic cells, we showed substantially enhanced adjunctive therapeutic efficacy of that vaccine resulting in robust Th1 pathway response. The recently demonstrated safety and efficacy of mRNA vaccines in the current pandemic raise the potential of using that vaccine platform, which is closer to the clinical application than are current DNA vaccine candidates due to the historical difficulties with their effective delivery into human cells. This proposal explores whether a similar mRNA vaccine delivered with biodegradable nanoparticles would show equivalent immunogenicity and therapeutic efficacy in TB preclinical models. This study will help the PI obtain preliminary data to submit an R21 proposal focusing on understanding the immune mechanisms of the MIP-3α/relMtb mRNA vaccine and whether it shortens the curative TB treatment in preclinical models. This project is in collaboration with Markham Lab (Bloomberg School of Public Health) and Green Lab (Biomedical Engineering, Johns Hopkins University).
JHU TRAC DEVELOPMENTAL AWARDEE 2022
Exploring Host Determinants of Mycobacterium tuberculosis Control
Recipient: Eileen Scully, MD, PhD
Mtb infection is associated with a range of clinical outcomes, from latent infection with few clinical indicators through severe, progressive infection. While there are several conditions associated with a heightened risk of transition between latent and active infection, there are fewer known mechanisms for why immune control is lost. Understanding the precise immunologic features associated with development of progressive infection will better inform host-directed therapeutics for TB disease. Two features of increased risk are HIV infection and pregnancy. This proposal will explore these two distinct, but often overlapping, risk conditions. In the first aim, studies will build on preliminary work that has already defined transcriptional and epigenetic shifts in the monocyte compartment within individuals with HIV infection before and after HIV viral suppression. We will use these same samples to define the capacity of these monocytes to control Mtb infection with a goal of linking the changes observed after HIV viral suppression to pathways of immune control. In the second aim, we will explore the impact of pregnancy on the development and differentiation of monocyte derived macrophages. These studies will model the first and third trimester of pregnancy with in vitro conditions and test the impact on Mtb control. For both HIV infection and pregnancy, the drivers of increased risk of progressive disease are unknown, and there is a clear need for optimization of treatment. The work from this proposal will be used to expand on previous, HIV-focused studies to support a new application for funding on the impact of HIV on TB innate immune control, and as data to support an R01 on the immunologic determinants of pregnancy related risk of active TB in women with and without HIV coinfection.
JHU TRAC DEVELOPMENTAL AWARDEE 2022
Assessing the role of preventive therapy in advancing goals of TB Free Nepal
Recipient: Sourya Shrestha, PhD
A national prevalence survey completed in 2019 estimated that the prevalence of tuberculosis (TB) in Nepal was 416 per 100,000. This was 1.8 times larger than previously estimated by the World Health Organization (WHO); and it revealed that the majority of incident cases (54%) are not notified. As in many high-burden countries, reducing the burden of TB has been a major challenge in Nepal, despite advances in TB diagnosis and treatment, and more intensive active case finding efforts. This prompted the Ministry of Health to announce a bold vision entitled “TB Free Nepal” in 2020, with a goal of reducing TB incidence by 25% in the next five years, and ultimately eliminating TB by 2050. Scaling up TB preventive therapy (TPT) is a major component of this vision. However, it is unclear (i) how impactful TPT will be in a high-transmission setting (where individuals may have high risk of re-exposure on one hand, whereas only treating diagnosed TB cases may be too little too late); (ii) whether prioritizing scale up of preventive therapy is cost-effective (given challenges associated with testing for latent TB, TPT uptake, and completion of TPT); and (iii) what implementation strategies for TPT scale up should be prioritized to make progress toward the vision of TB Free Nepal.
This pilot study, will augment an ongoing study (2021-2023; IMPACT 2 TB; PI: Dr. Maxine Caws; funded by Nick Simons Foundation) that assesses the feasibility of implementing 3HP (3 months of weekly isoniazid and rifapentine) in two districts in Nepal: a predominantly rural western district, Pyuthan, and densely populated district with large urban center, Chitwan. We propose to conduct two primary research activities: 1. Collection of site-specific data on implemention of preventive therapy from Pyuthan and Chitwan including (a) yield (numbers of people diagnosed with TB and LTBI, using TST); (b) prevalence of LTBI among household contacts (c) uptake of TPT; (d) TPT completion; and (e) costs of WHO screening algorithm. Additionally, we will recruit local study staff to collate programmatic data that are already collected as a part of the ongoing study and to collect additional data on household contacts. 2. Development of transmission models, calibrated to rural and urban settings, to project the epidemiological impact, and cost-effectiveness of TPT. Dr. Shrestha will train a junior researcher currently based at Birat Nepal Medical Trust (BNMT), Mr. Rajan Paudel, to develop these models under his supervision with input from the PI of ongoing IMPACT 2 TB study, Dr Maxine Caws, and mentor Dr David Dowdy.
JHU TRAC DEVELOPMENTAL AWARDEE 2022
Determination of the mid-treatment molecular clock for multidrug resistant tuberculosis
Recipient: Jeffrey Tornheim, MD, MPH
MDR-TB represents 3% of new and 18% of repeat TB infections, with 70% representing primary infection with resistant Mtb. Several molecular epidemiology programs use a well-established Mtb “molecular clock” to rule-out transmission between infected people by comparing acquisition of single nucleotide polymorphisms–SNPs–over time. Unfortunately, the Mtb molecular clock was originally defined among laboratory isolates or people with drug-susceptible TB without selective pressure from treatment. Recently, large mutation rate differences have been found by Mtb clade, undermining the generalizability of established thresholds. Similarly, selection pressure of multidrug MDR-TB treatment likely increases the rate of genetic polymorphisms identified before culture conversion, with direct implications for contact tracing and transmission inferences. To determine the molecular clock of MDR-TB during effective MDR-TB treatment, we will leverage a biorepository of well-characterized isolates collected through a completed longitudinal cohort study in which participants with MDR-TB completed baseline susceptibility testing, individualized treatment, and monthly sputum collection until symptom resolution. Of 204 study participants with baseline WGS results, 58 additional culture-positive mid-treatment samples are available from 41 participants. We will complete WGS from longitudinal samples to identify the time-normalized average pairwise SNP distances between paired MDR-TB isolates, with secondary stratification by phenotypic resistance and treatment regimen.