Multimodally profiling memory T cells from a tuberculosis cohort identifies cell state associations with demographics, environment and disease.

Multimodal T cell profiling can enable more precise characterization of elusive cell states underlying disease. Here, we integrated single-cell RNA and surface protein data from 500,089 memory T cells to define 31 cell states from 259 individuals in a Peruvian tuberculosis (TB) progression cohort. At immune steady state >4 years after infection and disease resolution, we found that, after accounting for significant effects of age, sex, season and genetic ancestry on T cell composition, a polyfunctional type 17 helper T (TH17) cell-like effector state was reduced in abundance and function in individuals who previously progressed from Mycobacterium tuberculosis (M.tb) infection to active TB disease. These cells are capable of responding to M.tb peptides. Deconvoluting this state-uniquely identifiable with multimodal analysis-from public data demonstrated that its depletion may precede and persist beyond active disease. Our study demonstrates the power of integrative multimodal single-cell profiling to define cell states relevant to disease and other traits.

T cell autoreactivity directed toward CD1c itself rather than toward carried self lipids.

The hallmark function of αß T cell antigen receptors (TCRs) involves the highly specific co-recognition of a major histocompatibility complex molecule and its carried peptide. However, the molecular basis of the interactions of TCRs with the lipid antigen-presenting molecule CD1c is unknown. We identified frequent staining of human T cells with CD1c tetramers across numerous subjects. Whereas TCRs typically show high specificity for antigen, both tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self lipids. Such extreme polyspecificity was attributable to binding of the TCR over the closed surface of CD1c, with the TCR covering the portal where lipids normally protrude. The TCR essentially failed to contact lipids because they were fully seated within CD1c. These data demonstrate the sequestration of lipids within CD1c as a mechanism of autoreactivity and point to small lipid size as a determinant of autoreactive T cell responses.

History of tuberculosis disease is associated with genetic regulatory variation in Peruvians.

A quarter of humanity is estimated to have been exposed to Mycobacterium tuberculosis (Mtb) with a 5-10% risk of developing tuberculosis (TB) disease. Variability in responses to Mtb infection could be due to host or pathogen heterogeneity. Here, we focused on host genetic variation in a Peruvian population and its associations with gene regulation in monocyte-derived macrophages and dendritic cells (DCs). We recruited former household contacts of TB patients who previously progressed to TB (cases, n = 63) or did not progress to TB (controls, n = 63). Transcriptomic profiling of monocyte-derived DCs and macrophages measured the impact of genetic variants on gene expression by identifying expression quantitative trait loci (eQTL). We identified 330 and 257 eQTL genes in DCs and macrophages (False Discovery Rate (FDR) < 0.05), respectively. Four genes in DCs showed interaction between eQTL variants and TB progression status. The top eQTL interaction for a protein-coding gene was with FAH, the gene encoding fumarylacetoacetate hydrolase, which mediates the last step in mammalian tyrosine catabolism. FAH expression was associated with genetic regulatory variation in cases but not controls. Using public transcriptomic and epigenomic data of Mtb-infected monocyte-derived dendritic cells, we found that Mtb infection results in FAH downregulation and DNA methylation changes in the locus. Overall, this study demonstrates effects of genetic variation on gene expression levels that are dependent on history of infectious disease and highlights a candidate pathogenic mechanism through pathogen-response genes. Furthermore, our results point to tyrosine metabolism and related candidate TB progression pathways for further investigation.

A positively selected FBN1 missense variant reduces height in Peruvian individuals.

On average, Peruvian individuals are among the shortest in the world1. Here we show that Native American ancestry is associated with reduced height in an ethnically diverse group of Peruvian individuals, and identify a population-specific, missense variant in the FBN1 gene (E1297G) that is significantly associated with lower height. Each copy of the minor allele (frequency of 4.7%) reduces height by 2.2 cm (4.4 cm in homozygous individuals). To our knowledge, this is the largest effect size known for a common height-associated variant. FBN1 encodes the extracellular matrix protein fibrillin 1, which is a major structural component of microfibrils. We observed less densely packed fibrillin-1-rich microfibrils with irregular edges in the skin of individuals who were homozygous for G1297 compared with individuals who were homozygous for E1297. Moreover, we show that the E1297G locus is under positive selection in non-African populations, and that the E1297 variant shows subtle evidence of positive selection specifically within the Peruvian population. This variant is also significantly more frequent in coastal Peruvian populations than in populations from the Andes or the Amazon, which suggests that short stature might be the result of adaptation to factors that are associated with the coastal environment in Peru.

Who Transmits Tuberculosis to Whom: A Cross-Sectional Analysis of a Cohort Study in Lima, Peru.

RATIONALE: The persistent burden of TB disease emphasizes the need to identify individuals with TB for treatment and those at a high risk of incident TB for prevention. Targeting interventions towards those at high risk of developing and transmitting tuberculosis is a public health priority. OBJECTIVES: We aimed to identify characteristics of individuals involved in tuberculosis transmission in a community setting, which may guide the prioritization of targeted interventions. METHODS: We collected clinical and socio-demographic data from a cohort of tuberculosis patients in Lima, Peru. We used whole-genome sequencing data to assess the genetic distance between all possible pairs of patients; we considered pairs to be the result of a direct transmission event if they differed by three or fewer SNPs and we assumed that the first diagnosed patient in a pair was the transmitter and the second to be the recipient. We used logistic regression to examine the association between host factors and the likelihood of direct tuberculosis transmission. MAIN RESULTS: Analyzing data from 2,518 tuberculosis index patients, we identified 1,447 direct transmission pairs. Regardless of recipient attributes, individuals less than 34 years old, males, and those with a history of incarceration had a higher likelihood of being transmitters in direct transmission pairs. Direct transmission was more likely when both patients were drinkers or smokers. CONCLUSIONS: This study identifies men, young adults, former prisoners, alcohol consumers, and smokers as priority groups for targeted interventions. Innovative strategies are needed to extend tuberculosis screening to social groups like young adults and prisoners with limited access to routine preventive care. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Spatial scale of tuberculosis transmission in Lima, Peru.

Spatially targeted interventions may be effective alternatives to individual or population-based prevention strategies against tuberculosis (TB). However, their efficacy may depend on the mechanisms that lead to geographically constrained hotspots. Local TB incidence may reflect high levels of local transmission; conversely, they may point to frequent travel of community members to high-risk areas. We used whole-genome sequencing to explore patterns of TB incidence and transmission in Lima, Peru. Between 2009 and 2012, we recruited incident pulmonary TB patients and their household contacts, whom we followed for the occurrence of TB disease. We used whole-genome sequences of 2,712 Mycobacterial tuberculosis isolates from 2,440 patients to estimate pariwise genomic distances and compared these to the spatial distance between patients' residences. Genomic distances increased rapidly as spatial distances increased and remained high beyond 2 km of separation. Next, we divided the study catchment area into 1 × 1 km grid-cell surface units and used household spatial coordinates to locate each TB patient to a specific cell. We estimated cell-specific transmission by calculating the proportion of patients in each cell with a pairwise genomic distance of 10 or fewer single-nucleotide polymorphisms. We found that cell-specific TB incidence and local transmission varied widely but that cell-specific TB incidence did not correlate closely with our estimates of local transmission (Cohen's k = 0.27). These findings indicate that an understanding of the spatial heterogeneity in the relative proportion of TB due to local transmission may help guide the implementation of spatially targeted interventions.

Chest Radiograph Screening for Detecting Subclinical Tuberculosis in Asymptomatic Household Contacts, Peru.

The World Health Organization's end TB strategy promotes the use of symptom and chest radiograph screening for tuberculosis (TB) disease. However, asymptomatic early states of TB beyond latent TB infection and active disease can go unrecognized using current screening criteria. We conducted a longitudinal cohort study enrolling household contacts initially free of TB disease and followed them for the occurrence of incident TB over 1 year. Among 1,747 screened contacts, 27 (52%) of the 52 persons in whom TB subsequently developed during follow-up had a baseline abnormal radiograph. Of contacts without TB symptoms, persons with an abnormal radiograph were at higher risk for subsequent TB than persons with an unremarkable radiograph (adjusted hazard ratio 15.62 [95% CI 7.74-31.54]). In young adults, we found a strong linear relationship between radiograph severity and time to TB diagnosis. Our findings suggest chest radiograph screening can extend to detecting early TB states, thereby enabling timely intervention.

Dual TCR-α Expression on Mucosal-Associated Invariant T Cells as a Potential Confounder of TCR Interpretation.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are highly abundant in human blood and tissues. Most MAIT cells have an invariant TCRα-chain that uses T cell receptor α-variable 1-2 (TRAV1-2) joined to TRAJ33/20/12 and recognizes metabolites from bacterial riboflavin synthesis bound to the Ag-presenting molecule MHC class I related (MR1). Our attempts to identify alternative MR1-presented Ags led to the discovery of rare MR1-restricted T cells with non-TRAV1-2 TCRs. Because altered Ag specificity likely alters affinity for the most potent known Ag, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), we performed bulk TCRα- and TCRß-chain sequencing and single-cell-based paired TCR sequencing on T cells that bound the MR1-5-OP-RU tetramer with differing intensities. Bulk sequencing showed that use of V genes other than TRAV1-2 was enriched among MR1-5-OP-RU tetramerlow cells. Although we initially interpreted these as diverse MR1-restricted TCRs, single-cell TCR sequencing revealed that cells expressing atypical TCRα-chains also coexpressed an invariant MAIT TCRα-chain. Transfection of each non-TRAV1-2 TCRα-chain with the TCRß-chain from the same cell demonstrated that the non-TRAV1-2 TCR did not bind the MR1-5-OP-RU tetramer. Thus, dual TCRα-chain expression in human T cells and competition for the endogenous ß-chain explains the existence of some MR1-5-OP-RU tetramerlow T cells. The discovery of simultaneous expression of canonical and noncanonical TCRs on the same T cell means that claims of roles for non-TRAV1-2 TCR in MR1 response must be validated by TCR transfer-based confirmation of Ag specificity.

Utility of Mycobacterium tuberculosis genome sequencing snapshots to assess transmission dynamics over time.

We explored the utility of brief Mycobacterium tuberculosis whole-genome sequencing (WGS) "snapshots" at a sentinel site within Lima, Peru for evaluating local transmission dynamics over time. Within a 17 km2 area, 15/70 (21%) isolates with WGS collected during 2011-2012 and 22/81 (27%) collected during 2020-2021 were clustered (p = 0.414), and additional isolates clustered with those from outside the area. Isolates from the later period were disproportionately related to large historic clusters in Lima from the earlier period. WGS snapshots at a sentinel site may not be useful for monitoring transmission, but monitoring the persistence of large transmission clusters might be.

Sensitivity of Various Case Detection Algorithms for Community-based Tuberculosis Screening.

Using data from 388 people diagnosed with tuberculosis through a community-based screening program in Lima, Peru, we estimated that cough screening followed by sputum smear microscopy would have detected only 23% of cases found using an algorithm of radiographic screening followed by rapid nucleic acid amplification testing and clinical evaluation.

Low detection rate of RT-PCR-confirmed COVID-19 using IgM/IgG rapid antibody tests in a large community sample in Lima, Peru.

BACKGROUND: Rapid IgM/IgG antibody tests were largely used in lieu of RT-PCR tests as part of COVID-19 public health response activities in Lima, Peru. To assess their utility, we explored the relationship between the time since onset of several COVID-19-related symptoms and the sensitivity of a rapid combined IgM/IgG antibody test. METHODS: We collected data from a community sample of individuals (n = 492) who received concurrent RT-PCR and rapid IgM/IgG antibody testing between May 2020 and March 2021. We estimated the sensitivity of the antibody test, against the RT-PCR test, by weeks since symptom onset via segmented regression analysis. RESULTS: The overall sensitivity of the rapid IgM/IgG antibody test was 46.7% (95% CI, 42.4-51.2%). Among 372 (75.6%) participants who reported COVID-19-related symptoms, sensitivity increased from 30.4% (95% CI, 24.7-36.6%) in week 1 after symptom onset to 83.3% (95% CI, 41.6-98.4%) in week 4. The test sensitivity increased by 31.9% (95% CI, 24.8-39.0%) per week until week 2 to 3, then decreased by - 6.0% (95% CI, - 25.7-13.7%) per week thereafter. CONCLUSION: Rapid antibody tests are a poor substitute for RT-PCR testing, regardless of presenting symptoms. This highlights the need for future pandemic planning to include timely and equitable access to gold-standard diagnostics, treatment, and vaccination.

The Contribution of Chest Radiography to the Clinical Management of Children Exposed to Tuberculosis.

Rationale: Although World Health Organization guidelines emphasize contact investigation for tuberculosis (TB)-exposed children, data that support chest radiography as a useful tool are lacking. Objectives: We evaluated the diagnostic and prognostic information of chest radiography in children exposed to TB and measured the efficacy of isoniazid preventive therapy (IPT) in those with relevant radiographic abnormalities. Methods: Between September 2009 and August 2012, we enrolled 4,468 TB-exposed children who were screened by tuberculin skin testing, symptom assessment, and chest radiography. Those negative for TB disease were followed for 1 year for the occurrence of new TB diagnoses. We assessed the protective efficacy of IPT in children with and without abnormal chest radiographs. Measurements and Main Results: Compared with asymptomatic children with normal chest films, asymptomatic children with abnormal radiographs were 25.1-fold more likely to have coprevalent TB (95% confidence interval [CI], 1.02-613.76) and 26.7-fold more likely to be diagnosed with incident TB disease during follow-up (95% CI, 10.44-68.30). Among the 29 symptom-negative and CXR-abnormal child contacts, 20% (3/15) of the isoniazid recipients developed incident TB, compared with 57% (8/14) of those who did not receive IPT (82% IPT efficacy). Conclusions: Our results strongly support the use of chest radiography as a routine screening tool for the evaluation of child TB contacts, which is readily available. Radiographic abnormalities not usually considered suggestive of TB may indicate incipient or subclinical disease, although TB preventive treatment is adequate in most cases.

Prevalence of Severe Acute Respiratory Syndrome Coronavirus 2 Antibodies Among Market and City Bus Depot Workers in Lima, Peru.

We report severe acute respiratory syndrome coronavirus 2 antibody positivity among market and city bus depot workers in Lima, Peru. Among 1285 vendors from 8 markets, prevalence ranged from 27% to 73%. Among 488 workers from 3 city bus depots, prevalence ranged from 11% to 47%. Self-reported symptoms were infrequent.

Smoking Cessation in Tuberculosis Patients and the Risk of Tuberculosis Infection in Child Household Contacts.

BACKGROUND: While previous studies have shown that cigarette smoking increases the infectiousness of tuberculosis patients, the impact of smoking cessation on tuberculosis transmissibility has not been evaluated. METHODS: Between 2009 and 2012, we enrolled 4500 tuberculosis patients and followed 14 044 household contacts in Lima, Peru. Tuberculosis patients were classified into 4 categories: never smoked, quit in the distant past (stopped smoking >2 months prior to time of diagnosis), recently quit (stopped smoking ≤2 months prior to time of diagnosis), and active smokers. We used a modified Poisson generalized estimating equation to assess the risk of tuberculosis infection of child contacts at enrollment and by 6 months of follow-up. RESULTS: In total, 1371 (76.8%) child contacts were exposed to patients who had never smoked, 211 (11.8%) were exposed to distant quitters, 155 (8.7%) were exposed to recent quitters, and 49 (2.7%) were exposed to active smokers. Compared with child contacts of index patients who had never smoked, child contacts of recent quitters had a similar risk of tuberculosis infection at enrollment (adjusted risk ratio, 95% confidence intervals [0.81, 0.50-1.32]) and by six months of follow-up (0.76, 0.51-1.13); and by 6 months of follow-up (aRR, 0.76; 95% CI, .51-1.13); child contacts of recent quitters had a significantly reduced risk of tuberculosis infection compared with contacts of active smokers (enrollment 0.45, 0.24-0.87; 6-month follow-up 0.48, 0.29-0.79). CONCLUSIONS: Our results show that the adverse effects of smoking on the transmissibility of tuberculosis are significantly reduced shortly after quitting smoking, reinforcing the importance of smoking cessation interventions in tuberculosis control.

Mycobacterium tuberculosis releases an antacid that remodels phagosomes.

Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.

A TCR ß-Chain Motif Biases toward Recognition of Human CD1 Proteins.

High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR ß-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1+ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1-encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.

Isoniazid Preventive Therapy in Contacts of Multidrug-Resistant Tuberculosis.

Rationale: The World Health Organization recommends the use of isoniazid (INH) alone or in combination with rifapentine to treat latent tuberculosis infections. The recent rise of drug-resistant tuberculosis has complicated the choice of treatment regimen for latent tuberculosis infection.Objectives: To evaluate the effects of INH preventive therapy on the contacts of patients with multidrug-resistant tuberculosis.Methods: In a prospective cohort study conducted between September 2009 and August 2012, we identified 4,500 index patients with tuberculosis and 14,044 tuberculosis-exposed household contacts who we followed for 1 year for the occurrence of incident tuberculosis disease. Although Peruvian national guidelines specify that INH preventive therapy should be provided to contacts aged 19 years old or younger, only half this group received INH preventive therapy.Measurements and Main Results: Among 4,216 contacts under 19 years of age, 2,106 contacts (50%) initiated INH preventive therapy at enrollment. The protective effect of INH was more extreme in contacts exposed to drug-sensitive tuberculosis (adjusted hazard ratio, 0.30; 95% confidence interval, 0.18-0.48) and to multidrug-resistant tuberculosis (adjusted hazard ratio, 0.19; 95% confidence interval, 0.05-0.66) compared with those exposed to mono-INH-resistant tuberculosis (adjusted hazard ratio, 0.80; 95% confidence interval, 0.23-2.80). In the second independent study, tuberculosis occurred in none of the 76 household contacts who received INH preventive therapy compared with 3% (8 of 273) of those who did not.Conclusions: Household contacts who received INH preventive therapy had a lower incidence of tuberculosis disease even when they had been exposed to an index patient with multidrug-resistant tuberculosis. INH may have a role in the management of latent multidrug-resistant tuberculosis infection.

Peripheral Blood Mucosal-Associated Invariant T Cells in Tuberculosis Patients and Healthy Mycobacterium tuberculosis-Exposed Controls.

BACKGROUND: In human blood, mucosal-associated invariant T (MAIT) cells are abundant T cells that recognize antigens presented on non-polymorphic major histocompatibility complex-related 1 (MR1) molecules. The MAIT cells are activated by mycobacteria, and prior human studies indicate that blood frequencies of MAIT cells, defined by cell surface markers, decline during tuberculosis (TB) disease, consistent with redistribution to the lungs. METHODS: We tested whether frequencies of blood MAIT cells were altered in patients with TB disease relative to healthy Mycobacterium tuberculosis-exposed controls from Peru and South Africa. We quantified their frequencies using MR1 tetramers loaded with 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil. RESULTS: Unlike findings from prior studies, frequencies of blood MAIT cells were similar among patients with TB disease and latent and uninfected controls. In both cohorts, frequencies of MAIT cells defined by MR1-tetramer staining and coexpression of CD161 and the T-cell receptor alpha variable gene TRAV1-2 were strongly correlated. Disease severity captured by body mass index or TB disease transcriptional signatures did not correlate with MAIT cell frequencies in patients with TB. CONCLUSIONS: Major histocompatibility complex (MHC)-related 1-restrictied MAIT cells are detected at similar levels with tetramers or surface markers. Unlike MHC-restricted T cells, blood frequencies of MAIT cells are poor correlates of TB disease but may play a role in pathophysiology.

Two Clinical Prediction Tools to Improve Tuberculosis Contact Investigation.

BACKGROUND: Efficient contact investigation strategies are needed for the early diagnosis of tuberculosis (TB) disease and treatment of latent TB infections. METHODS: Between September 2009 and August 2012, we conducted a prospective cohort study in Lima, Peru, in which we enrolled and followed 14 044 household contacts of adults with pulmonary TB. We used information from a subset of this cohort to derive 2 clinical prediction tools that identify contacts of TB patients at elevated risk of progressing to active disease by training multivariable models that predict (1) coprevalent TB among all household contacts and (2) 1-year incident TB among adult contacts. We validated the models in a geographically distinct subcohort and compared the relative utilities of clinical decisions based on these tools to existing strategies. RESULTS: In our cohort, 296 (2.1%) household contacts had coprevalent TB and 145 (1.9%) adult contacts developed incident TB within 1 year of index patient diagnosis. We predicted coprevalent disease using information that could be readily obtained at the time an index patient was diagnosed and predicted 1-year incident TB by including additional contact-specific characteristics. The area under the receiver operating characteristic curves for coprevalent TB and incident TB were 0.86 (95% confidence interval [CI], .83-.89]) and 0.72 (95% CI, .67-.77), respectively. These clinical tools give 5%-10% higher relative utilities than existing methods. CONCLUSIONS: We present 2 tools that identify household contacts at high risk for TB disease based on reportable information from patient and contacts alone. The performance of these tools is comparable to biomarkers that are both more costly and less feasible than this approach.

Mycobacterium tuberculosis Beijing Lineage and Risk for Tuberculosis in Child Household Contacts, Peru.

Few studies have prospectively compared the relative transmissibility and propensity to cause disease of Mycobacterium tuberculosis Beijing strains with other human-adapted strains of the M. tuberculosis complex. We assessed the effect of Beijing strains on the risk for M. tuberculosis infection and disease progression in 9,151 household contacts of 2,223 culture-positive pulmonary tuberculosis (TB) patients in Lima, Peru. Child contacts exposed to Beijing strains were more likely than child contacts exposed to non-Beijing strains to be infected at baseline, by 12 months of follow-up, and during follow-up. We noted an increased but nonsignificant tendency for child contacts to develop TB. Beijing strains were not associated with TB in adult contacts. These findings suggest that Beijing strains are more transmissible in children than are non-Beijing strains.