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.

Single-cell eQTL models reveal dynamic T cell state dependence of disease loci.

Non-coding genetic variants may cause disease by modulating gene expression. However, identifying these expression quantitative trait loci (eQTLs) is complicated by differences in gene regulation across fluid functional cell states within cell types. These states-for example, neurotransmitter-driven programs in astrocytes or perivascular fibroblast differentiation-are obscured in eQTL studies that aggregate cells1,2. Here we modelled eQTLs at single-cell resolution in one complex cell type: memory T cells. Using more than 500,000 unstimulated memory T cells from 259 Peruvian individuals, we show that around one-third of 6,511 cis-eQTLs had effects that were mediated by continuous multimodally defined cell states, such as cytotoxicity and regulatory capacity. In some loci, independent eQTL variants had opposing cell-state relationships. Autoimmune variants were enriched in cell-state-dependent eQTLs, including risk variants for rheumatoid arthritis near ORMDL3 and CTLA4; this indicates that cell-state context is crucial to understanding potential eQTL pathogenicity. Moreover, continuous cell states explained more variation in eQTLs than did conventional discrete categories, such as CD4+ versus CD8+, suggesting that modelling eQTLs and cell states at single-cell resolution can expand insight into gene regulation in functionally heterogeneous cell types.

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.

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.

COVID-19 vaccination strategies in Africa: A scoping review of the use of mathematical models to inform policy.

OBJECTIVE: Mathematical models are vital tools to understand transmission dynamics and assess the impact of interventions to mitigate COVID-19. However, historically, their use in Africa has been limited. In this scoping review, we assess how mathematical models were used to study COVID-19 vaccination to potentially inform pandemic planning and response in Africa. METHODS: We searched six electronic databases: MEDLINE, Embase, Web of Science, Global Health, MathSciNet and Africa-Wide NiPAD, using keywords to identify articles focused on the use of mathematical modelling studies of COVID-19 vaccination in Africa that were published as of October 2022. We extracted the details on the country, author affiliation, characteristics of models, policy intent and heterogeneity factors. We assessed quality using 21-point scale criteria on model characteristics and content of the studies. RESULTS: The literature search yielded 462 articles, of which 32 were included based on the eligibility criteria. Nineteen (59%) studies had a first author affiliated with an African country. Of the 32 included studies, 30 (94%) were compartmental models. By country, most studies were about or included South Africa (n = 12, 37%), followed by Morocco (n = 6, 19%) and Ethiopia (n = 5, 16%). Most studies (n = 19, 59%) assessed the impact of increasing vaccination coverage on COVID-19 burden. Half (n = 16, 50%) had policy intent: prioritising or selecting interventions, pandemic planning and response, vaccine distribution and optimisation strategies and understanding transmission dynamics of COVID-19. Fourteen studies (44%) were of medium quality and eight (25%) were of high quality. CONCLUSIONS: While decision-makers could draw vital insights from the evidence generated from mathematical modelling to inform policy, we found that there was limited use of such models exploring vaccination impacts for COVID-19 in Africa. The disparity can be addressed by scaling up mathematical modelling training, increasing collaborative opportunities between modellers and policymakers, and increasing access to funding.

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.

The Impact of Vitamin A Deficiency on Tuberculosis Progression.

BACKGROUND: Although previous studies have shown that vitamin A deficiency is associated with incident tuberculosis (TB) disease, the direction of the association has not been established. We investigated the impact of vitamin A deficiency on TB disease progression. METHODS: We conducted a longitudinal cohort study nested within a randomized clinical trial among HIV-infected patients in Haiti. We compared serial vitamin A levels in individuals who developed TB disease to controls matched on age, gender, follow-up time, and time to antiretroviral therapy initiation. We also evaluated histopathology, bacterial load, and immune outcomes in TB infection in a guinea pig model of dietary vitamin A deficiency. RESULTS: Among 773 participants, 96 developed incident TB during follow-up, 62.5% (60) of whom had stored serum samples obtained 90-365 days before TB diagnosis. In age- and sex- adjusted and multivariate analyses, respectively, incident TB cases were 3.99 times (95% confidence interval [CI], 2.41 to 6.60) and 3.59 times (95% CI, 2.05 to 6.29) more likely to have been vitamin A deficient than matched controls. Vitamin A-deficient guinea pigs manifested more extensive pulmonary pathology, atypical granuloma morphology, and increased bacterial growth after experimental TB infection. Reintroduction of dietary vitamin A to deficient guinea pigs after established TB disease successfully abrogated severe disease manifestations and altered cellular immune profiles. CONCLUSIONS: Human and animal studies support the role of baseline vitamin A deficiency as a determinant of future TB disease progression.

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.

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.

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.

Children as sentinels of tuberculosis transmission: disease mapping of programmatic data.

BACKGROUND: Identifying hotspots of tuberculosis transmission can inform spatially targeted active case-finding interventions. While national tuberculosis programs maintain notification registers which represent a potential source of data to investigate transmission patterns, high local tuberculosis incidence may not provide a reliable signal for transmission because the population distribution of covariates affecting susceptibility and disease progression may confound the relationship between tuberculosis incidence and transmission. Child cases of tuberculosis and other endemic infectious disease have been observed to provide a signal of their transmission intensity. We assessed whether local overrepresentation of child cases in tuberculosis notification data corresponds to areas where recent transmission events are concentrated. METHODS: We visualized spatial clustering of children < 5 years old notified to Peru's National Tuberculosis Program from two districts of Lima, Peru, from 2005 to 2007 using a log-Gaussian Cox process to model the intensity of the point-referenced child cases. To identify where clustering of child cases was more extreme than expected by chance alone, we mapped all cases from the notification data onto a grid and used a hierarchical Bayesian spatial model to identify grid cells where the proportion of cases among children < 5 years old is greater than expected. Modeling the proportion of child cases allowed us to use the spatial distribution of adult cases to control for unobserved factors that may explain the spatial variability in the distribution of child cases. We compare where young children are overrepresented in case notification data to areas identified as transmission hotspots using molecular epidemiological methods during a prospective study of tuberculosis transmission conducted from 2009 to 2012 in the same setting. RESULTS: Areas in which childhood tuberculosis cases are overrepresented align with areas of spatial concentration of transmission revealed by molecular epidemiologic methods. CONCLUSIONS: Age-disaggregated notification data can be used to identify hotspots of tuberculosis transmission and suggest local force of infection, providing an easily accessible source of data to target active case-finding intervention.

Vitamin D status and risk of incident tuberculosis disease: A nested case-control study, systematic review, and individual-participant data meta-analysis.

BACKGROUND: Few studies have evaluated the association between preexisting vitamin D deficiency and incident tuberculosis (TB). We assessed the impact of baseline vitamins D levels on TB disease risk. METHODS AND FINDINGS: We assessed the association between baseline vitamin D and incident TB in a prospective cohort of 6,751 HIV-negative household contacts of TB patients enrolled between September 1, 2009, and August 29, 2012, in Lima, Peru. We screened for TB disease at 2, 6, and 12 months after enrollment. We defined cases as household contacts who developed TB disease at least 15 days after enrollment of the index patient. For each case, we randomly selected four controls from among contacts who did not develop TB disease, matching on gender and year of age. We also conducted a one-stage individual-participant data (IPD) meta-analysis searching PubMed and Embase to identify prospective studies of vitamin D and TB disease until June 8, 2019. We included studies that assessed vitamin D before TB diagnosis. In the primary analysis, we defined vitamin D deficiency as 25-(OH)D < 50 nmol/L, insufficiency as 50-75 nmol/L, and sufficiency as >75nmol/L. We estimated the association between baseline vitamin D status and incident TB using conditional logistic regression in the Lima cohort and generalized linear mixed models in the meta-analysis. We further defined severe vitamin D deficiency as 25-(OH)D < 25 nmol/L and performed stratified analyses by HIV status in the IPD meta-analysis. In the Lima cohort, we analyzed 180 cases and 709 matched controls. The adjusted odds ratio (aOR) for TB risk among participants with baseline vitamin D deficiency compared to sufficient vitamin D was 1.63 (95% CI 0.75-3.52; p = 0.22). We included seven published studies in the meta-analysis and analyzed 3,544 participants. In the pooled analysis, the aOR was 1.48 (95% CI 1.04-2.10; p = 0.03). The aOR for severe vitamin D deficiency was 2.05 (95% CI 0.87-4.87; p trend for decreasing 25-(OH)D levels from sufficient vitamin D to severe deficiency = 0.02). Among 1,576 HIV-positive patients, vitamin D deficiency conferred a 2-fold (aOR 2.18, 95% CI 1.22-3.90; p = 0.01) increased risk of TB, and the aOR for severe vitamin D deficiency compared to sufficient vitamin D was 4.28 (95% CI 0.85-21.45; p = 0.08). Our Lima cohort study is limited by the short duration of follow-up, and the IPD meta-analysis is limited by the number of possible confounding covariates available across all studies. CONCLUSION: Our findings suggest vitamin D predicts TB disease risk in a dose-dependent manner and that the risk of TB disease is highest among HIV-positive individuals with severe vitamin D deficiency. Randomized control trials are needed to evaluate the possible role of vitamin D supplementation on reducing TB disease risk.

Correction to: Detection of Mycobacterium tuberculosis in pediatric stool samples using TruTip technology.

Following publication of the original article [1]. The authors reported that there is a mistake in Fig. 1: the number of patients in the control group its 449 patients, instead of 455.

Detection of Mycobacterium tuberculosis in pediatric stool samples using TruTip technology.

BACKGROUND: Rapid and accurate diagnosis of childhood tuberculosis (TB) is challenging because children are often unable to produce the sputum sample required for conventional tests. Stool is an alternative sample type that is easy to collect from children, and studies investigating the use of stool for molecular detection of Mycobacterium tuberculosis (Mtb) have led to promising results. Our objective was to evaluate stool as an alternative specimen to sputum for Mtb detection in children. We did so using the TruTip workstation (Akonni Biosystems), a novel automated lysis and extraction platform. METHODS: We tested stool samples from 259 children aged 0-14 years old, in Lima, Peru who presented with TB symptoms. Following extraction with TruTip, we detected the presence of Mtb DNA by IS6110 real-time PCR. We calculated assay sensitivity in two groups: (1) children with culture confirmed TB (N = 22); and (2) children with clinically-diagnosed unconfirmed TB (N = 84). We calculated specificity among children in whom TB was ruled out (N = 153). Among children who were diagnosed with TB, we examined factors associated with a positive stool test. RESULTS: Assay sensitivity was 59% (95% confidence interval [CI]: 39-80%) and 1.2% (95% CI: 0.0-6.5%) in children with culture-confirmed and clinically-diagnosed unconfirmed TB, respectively, and specificity was 97% (95% CI: 93-99%). The assay detected Mtb in stool of 7/7 children with smear-positive TB (100% sensitivity; 95% CI: 59-100%), and in 6/15 of children with smear-negative, culture-confirmed TB (40% sensitivity; 95% CI: 16-68%). Older age, smear positivity, culture positivity, ability to produce sputum and cavitary disease were associated with a positive stool result. CONCLUSION: Testing of stool samples with the TruTip workstation and IS6110 amplification yielded sensitivity and specificity estimates comparable to other tests such as Xpert. Future work should include detection of resistance using the TruTip closed amplification system and assay optimization to improve sensitivity in children with low bacillary loads.

Using routinely collected laboratory data to identify high rifampicin-resistant tuberculosis burden communities in the Western Cape Province, South Africa: A retrospective spatiotemporal analysis.

BACKGROUND: South Africa has the highest tuberculosis incidence globally (781/100,000), with an estimated 4.3% of cases being rifampicin resistant (RR). Control and elimination strategies will require detailed spatial information to understand where drug-resistant tuberculosis exists and why it persists in those communities. We demonstrate a method to enable drug-resistant tuberculosis monitoring by identifying high-burden communities in the Western Cape Province using routinely collected laboratory data. METHODS AND FINDINGS: We retrospectively identified cases of microbiologically confirmed tuberculosis and RR-tuberculosis from all biological samples submitted for tuberculosis testing (n = 2,219,891) to the Western Cape National Health Laboratory Services (NHLS) between January 1, 2008, and June 30, 2013. Because the NHLS database lacks unique patient identifiers, we performed a series of record-linking processes to match specimen records to individual patients. We counted an individual as having a single disease episode if their positive samples came from within two years of each other. Cases were aggregated by clinic location (n = 302) to estimate the percentage of tuberculosis cases with rifampicin resistance per clinic. We used inverse distance weighting (IDW) to produce heatmaps of the RR-tuberculosis percentage across the province. Regression was used to estimate annual changes in the RR-tuberculosis percentage by clinic, and estimated average size and direction of change was mapped. We identified 799,779 individuals who had specimens submitted from mappable clinics for testing, of whom 222,735 (27.8%) had microbiologically confirmed tuberculosis. The study population was 43% female, the median age was 36 years (IQR 27-44), and 10,255 (4.6%, 95% CI: 4.6-4.7) cases had documented rifampicin resistance. Among individuals with microbiologically confirmed tuberculosis, 8,947 (4.0%) had more than one disease episode during the study period. The percentage of tuberculosis cases with rifampicin resistance documented among these individuals was 11.4% (95% CI: 10.7-12.0). Overall, the percentage of tuberculosis cases that were RR-tuberculosis was spatially heterogeneous, ranging from 0% to 25% across the province. Our maps reveal significant yearly fluctuations in RR-tuberculosis percentages at several locations. Additionally, the directions of change over time in RR-tuberculosis percentage were not uniform. The main limitation of this study is the lack of unique patient identifiers in the NHLS database, rendering findings to be estimates reliant on the accuracy of the person-matching algorithm. CONCLUSIONS: Our maps reveal striking spatial and temporal heterogeneity in RR-tuberculosis percentages across this province. We demonstrate the potential to monitor RR-tuberculosis spatially and temporally with routinely collected laboratory data, enabling improved resource targeting and more rapid locally appropriate interventions.

Genotyping Multidrug-Resistant Mycobacterium tuberculosis from Primary Sputum and Decontaminated Sediment with an Integrated Microfluidic Amplification Microarray Test.

There is a growing awareness that molecular diagnostics for detect-to-treat applications will soon need a highly multiplexed mutation detection and identification capability. In this study, we converted an open-amplicon microarray hybridization test for multidrug-resistant (MDR) Mycobacterium tuberculosis into an entirely closed-amplicon consumable (an amplification microarray) and evaluated its performance with matched sputum and sediment extracts. Reproducible genotyping (the limit of detection) was achieved with ∼25 M. tuberculosis genomes (100 fg of M. tuberculosis DNA) per reaction; the estimated shelf life of the test was at least 18 months when it was stored at 4°C. The test detected M. tuberculosis in 99.1% of sputum extracts and 100% of sediment extracts and showed 100% concordance with the results of real-time PCR. The levels of concordance between M. tuberculosis and resistance-associated gene detection were 99.1% and 98.4% for sputum and sediment extracts, respectively. Genotyping results were 100% concordant between sputum and sediment extracts. Relative to the results of culture-based drug susceptibility testing, the test was 97.1% specific and 75.0% sensitive for the detection of rifampin resistance in both sputum and sediment extracts. The specificity for the detection of isoniazid (INH) resistance was 98.4% and 96.8% for sputum and sediment extracts, respectively, and the sensitivity for the detection of INH resistance was 63.6%. The amplification microarray reported the correct genotype for all discordant phenotype/genotype results. On the basis of these data, primary sputum may be considered a preferred specimen for the test. The amplification microarray design, shelf life, and analytical performance metrics are well aligned with consensus product profiles for next-generation drug-resistant M. tuberculosis diagnostics and represent a significant ease-of-use advantage over other hybridization-based tests for diagnosing MDR tuberculosis.