Identification and molecular characterization of Mycobacterium bovis DNA in GeneXpert® MTB/RIF ultra-positive, culture-negative sputum from a rural community in South Africa.

This study investigated the presence of Mycobacterium bovis (M. bovis) DNA in archived human sputum samples previously collected from residents who reside adjacent to the M. bovis-endemic Hluhluwe-iMfolozi wildlife park, South Africa (SA). Sixty-eight sputum samples were GeneXpert MTB/RIF Ultra-positive for M. tuberculosis complex (MTBC) DNA but culture negative for M. tuberculosis. Amplification and Sanger sequencing of hsp65 and rpoB genes from DNA extracted from stored heat-inactivated sputum samples confirmed the presence of detectable amounts of MTBC from 20 out of the 68 sputum samples. Region of difference PCR, spoligotyping and gyrB long-read amplicon deep sequencing identified M. bovis (n = 10) and M. tuberculosis (n = 7). Notably, M. bovis spoligotypes SB0130 and SB1474 were identified in 4 samples, with SB0130 previously identified in local cattle and wildlife and SB1474 exclusively in African buffaloes in the adjacent park. M. bovis DNA in sputum, from people living near the park, underscores zoonotic transmission potential in SA. Identification of spoligotypes specifically associated with wildlife only and spoligotypes found in livestock as well as wildlife, highlights the complexity of TB epidemiology at wildlife-livestock-human interfaces. These findings support the need for integrated surveillance and control strategies to curb potential spillover and for the consideration of human M. bovis infection in SA patients with positive Ultra results.

The met and unmet health needs for HIV, hypertension, and diabetes in rural KwaZulu-Natal, South Africa: analysis of a cross-sectional multimorbidity survey.

BACKGROUND: The convergence of infectious diseases and non-communicable diseases in South Africa is challenging to health systems. In this analysis, we assessed the multimorbidity health needs of individuals and communities in rural KwaZulu-Natal and established a framework to quantify met and unmet health needs for individuals living with infectious and non-communicable diseases. METHODS: We analysed data collected between May 25, 2018, and March 13, 2020, from participants of a large, community-based, cross-sectional multimorbidity survey (Vukuzazi) that offered community-based HIV, hypertension, and diabetes screening to all residents aged 15 years or older in a surveillance area in the uMkhanyakude district in KwaZulu-Natal, South Africa. Data from the Vukuzazi survey were linked with data from demographic and health surveillance surveys with a unique identifier common to both studies. Questionnaires were used to assess the diagnosed health conditions, treatment history, general health, and sociodemographic characteristics of an individual. For each condition (ie, HIV, hypertension, and diabetes), individuals were defined as having no health needs (absence of condition), met health needs (condition that is well controlled), or one or more unmet health needs (including diagnosis, engagement in care, or treatment optimisation). We analysed met and unmet health needs for individual and combined conditions and investigated their geospatial distribution. FINDINGS: Of 18 041 participants who completed the survey (12 229 [67·8%] were female and 5812 [32·2%] were male), 9898 (54·9%) had at least one of the three chronic diseases measured. 4942 (49·9%) of these 9898 individuals had at least one unmet health need (1802 [18·2%] of 9898 needed treatment optimisation, 1282 [13·0%] needed engagement in care, and 1858 [18·8%] needed a diagnosis). Unmet health needs varied by disease; 1617 (93·1%) of 1737 people who screened positive for diabetes, 2681 (58·2%) of 4603 people who screened positive for hypertension, and 1321 (21·7%) of 6096 people who screened positive for HIV had unmet health needs. Geospatially, met health needs for HIV were widely distributed and unmet health needs for all three conditions had specific sites of concentration; all three conditions had an overlapping geographical pattern for the need for diagnosis. INTERPRETATION: Although people living with HIV predominantly have a well controlled condition, there is a high burden of unmet health needs for people living with hypertension and diabetes. In South Africa, adapting current, widely available HIV care services to integrate non-communicable disease care is of high priority. FUNDING: Fogarty International Center and the National Institutes of Health, the Bill & Melinda Gates Foundation, the South African Department of Science and Innovation, the South African Medical Research Council, the South African Population Research Infrastructure Network, and the Wellcome Trust. TRANSLATION: For the isiZulu translation of the abstract see Supplementary Materials section.

Prevalence of Mycobacterium tuberculosis in Sputum and Reported Symptoms Among Clinic Attendees Compared With a Community Survey in Rural South Africa.

BACKGROUND: Tuberculosis (TB) case finding efforts typically target symptomatic people attending health facilities. We compared the prevalence of Mycobacterium tuberculosis (Mtb) sputum culture-positivity among adult clinic attendees in rural South Africa with a concurrent, community-based estimate from the surrounding demographic surveillance area (DSA). METHODS: Clinic: Randomly selected adults (≥18 years) attending 2 primary healthcare clinics were interviewed and requested to give sputum for mycobacterial culture. Human immunodeficiency virus (HIV) and antiretroviral therapy (ART) status were based on self-report and record review. Community: All adult (≥15 years) DSA residents were invited to a mobile clinic for health screening, including serological HIV testing; those with ≥1 TB symptom (cough, weight loss, night sweats, fever) or abnormal chest radiograph were asked for sputum. RESULTS: Clinic: 2055 patients were enrolled (76.9% female; median age, 36 years); 1479 (72.0%) were classified HIV-positive (98.9% on ART) and 131 (6.4%) reported ≥1 TB symptom. Of 20/2055 (1.0% [95% CI, .6-1.5]) with Mtb culture-positive sputum, 14 (70%) reported no symptoms. Community: 10 320 residents were enrolled (68.3% female; median age, 38 years); 3105 (30.3%) tested HIV-positive (87.4% on ART) and 1091 (10.6%) reported ≥1 TB symptom. Of 58/10 320 (0.6% [95% CI, .4-.7]) with Mtb culture-positive sputum, 45 (77.6%) reported no symptoms. In both surveys, sputum culture positivity was associated with male sex and reporting >1 TB symptom. CONCLUSIONS: In both clinic and community settings, most participants with Mtb culture-positive sputum were asymptomatic. TB screening based only on symptoms will miss many people with active disease in both settings.

Computer-aided interpretation of chest radiography reveals the spectrum of tuberculosis in rural South Africa.

Computer-aided digital chest radiograph interpretation (CAD) can facilitate high-throughput screening for tuberculosis (TB), but its use in population-based active case-finding programs has been limited. In an HIV-endemic area in rural South Africa, we used a CAD algorithm (CAD4TBv5) to interpret digital chest x-rays (CXR) as part of a mobile health screening effort. Participants with TB symptoms or CAD4TBv5 score above the triaging threshold were referred for microbiological sputum assessment. During an initial pilot phase, a low CAD4TBv5 triaging threshold of 25 was selected to maximize TB case finding. We report the performance of CAD4TBv5 in screening 9,914 participants, 99 (1.0%) of whom were found to have microbiologically proven TB. CAD4TBv5 was able to identify TB cases at the same sensitivity but lower specificity as a blinded radiologist, whereas the next generation of the algorithm (CAD4TBv6) achieved comparable sensitivity and specificity to the radiologist. The CXRs of people with microbiologically confirmed TB spanned a range of lung field abnormality, including 19 (19.2%) cases deemed normal by the radiologist. HIV serostatus did not impact CAD4TB's performance. Notably, 78.8% of the TB cases identified during this population-based survey were asymptomatic and therefore triaged for sputum collection on the basis of CAD4TBv5 score alone. While CAD4TBv6 has the potential to replace radiologists for triaging CXRs in TB prevalence surveys, population-specific piloting is necessary to set the appropriate triaging thresholds. Further work on image analysis strategies is needed to identify radiologically subtle active TB.

Convergence of infectious and non-communicable disease epidemics in rural South Africa: a cross-sectional, population-based multimorbidity study.

BACKGROUND: There has been remarkable progress in the treatment of HIV throughout sub-Saharan Africa, but there are few data on the prevalence and overlap of other significant causes of disease in HIV endemic populations. Our aim was to identify the prevalence and overlap of infectious and non-communicable diseases in such a population in rural South Africa. METHODS: We did a cross-sectional study of eligible adolescents and adults from the Africa Health Research Institute demographic surveillance area in the uMkhanyakude district of KwaZulu-Natal, South Africa. The participants, who were 15 years or older, were invited to participate at a mobile health camp. Medical history for HIV, tuberculosis, hypertension, and diabetes was established through a questionnaire. Blood pressure measurements, chest x-rays, and tests of blood and sputum were taken to estimate the population prevalence and geospatial distribution of HIV, active and lifetime tuberculosis, elevated blood glucose, elevated blood pressure, and combinations of these. FINDINGS: 17 118 adolescents and adults were recruited from May 25, 2018, to Nov 28, 2019, and assessed. Overall, 52·1% (95% CI 51·3-52·9) had at least one active disease. 34·2% (33·5-34·9) had HIV, 1·4% (1·2-1·6) had active tuberculosis, 21·8% (21·2-22·4) had lifetime tuberculosis, 8·5% (8·1-8·9) had elevated blood glucose, and 23·0% (22·4-23·6) had elevated blood pressure. Appropriate treatment and optimal disease control was highest for HIV (78·1%), and lower for elevated blood pressure (42·5%), active tuberculosis (29·6%), and elevated blood glucose (7·1%). Disease prevalence differed notably by sex, across age groups, and geospatially: men had a higher prevalence of active and lifetime tuberculosis, whereas women had a substantially high prevalence of HIV at 30-49 years and an increasing prevalence of multiple and poorly controlled non-communicable diseases when older than 50 years. INTERPRETATION: We found a convergence of infectious and non-communicable disease epidemics in a rural South African population, with HIV well treated relative to all other diseases, but tuberculosis, elevated blood glucose, and elevated blood pressure poorly diagnosed and treated. A public health response that expands the successes of the HIV testing and treatment programme to provide multidisease care targeted to specific populations is required to optimise health in such settings in sub-Saharan Africa. FUNDING: Wellcome Trust, Bill & Melinda Gates Foundation, the South African Department of Science and Innovation, South African Medical Research Council, and South African Population Research Infrastructure Network. TRANSLATION: For the isiZulu translation of the abstract see Supplementary Materials section.

Increased Neutrophil Count and Decreased Neutrophil CD15 Expression Correlate With TB Disease Severity and Treatment Response Irrespective of HIV Co-infection.

Tuberculosis remains a leading cause of death globally despite curative treatment, partly due to the difficulty of identifying patients who will not respond to therapy. Simple host biomarkers that correlate with response to drug treatment would facilitate improvement in outcomes and the evaluation of novel therapies. In a prospective longitudinal cohort study, we evaluated neutrophil count and phenotype at baseline, as well as during TB treatment in 79 patients [50 (63%) HIV-positive] with microbiologically confirmed drug susceptible TB undergoing standard treatment. At time of diagnosis, blood neutrophils were highly expanded and surface expression of the neutrophil marker CD15 greatly reduced compared to controls. Both measures changed rapidly with the commencement of drug treatment and returned to levels seen in healthy control by treatment completion. Additionally, at the time of diagnosis, high neutrophil count, and low CD15 expression was associated with higher sputum bacterial load and more severe lung damage on chest x-ray, two clinically relevant markers of disease severity. Furthermore, CD15 expression level at diagnosis was associated with TB culture conversion after 2 months of therapy (OR: 0.14, 95% CI: 0.02, 0.89), a standard measure of early TB treatment success. Importantly, our data was not significantly impacted by HIV co-infection. These data suggest that blood neutrophil metrics could potentially be exploited to develop a simple and rapid test to help determine TB disease severity, monitor drug treatment response, and identify subjects at diagnosis who may respond poorly to treatment.

Population-level emergence of bedaquiline and clofazimine resistance-associated variants among patients with drug-resistant tuberculosis in southern Africa: a phenotypic and phylogenetic analysis.

BACKGROUND: Bedaquiline and clofazimine are important drugs in the treatment of drug-resistant tuberculosis and are commonly used across southern Africa, although drug susceptibility testing is not routinely performed. In this study, we did a genotypic and phenotypic analysis of drug-resistant Mycobacterium tuberculosis isolates from cohort studies in hospitals in KwaZulu-Natal, South Africa, to identify resistance-associated variants (RAVs) and assess the extent of clofazimine and bedaquiline cross-resistance. We also used a comprehensive dataset of whole-genome sequences to investigate the phylogenetic and geographical distribution of bedaquiline and clofazimine RAVs in southern Africa. METHODS: In this study, we included M tuberculosis isolates reported from the PRAXIS study of patients with drug-resistant tuberculosis treated with bedaquiline (King Dinuzulu Hospital, Durban) and three other cohort studies of drug-resistant tuberculosis in other KwaZulu-Natal hospitals, and sequential isolates from six persistently culture-positive patients with extensively drug-resistant tuberculosis at the KwaZulu-Natal provincial referral laboratory. Samples were collected between 2013 and 2019. Microbiological cultures were done as part of all parent studies. We sequenced whole genomes of included isolates and measured bedaquiline and clofazimine minimum inhibitory concentrations (MICs) for isolates identified as carrying any Rv0678 variant or previously published atpE, pepQ, and Rv1979c RAVs, which were the subject of the phenotypic study. We combined all whole-genome sequences of M tuberculosis obtained in this study with publicly available sequence data from other tuberculosis studies in southern Africa (defined as the countries of the Southern African Development Community), including isolates with Rv0678 variants identified by screening public genomic databases. We used this extended dataset to reconstruct phylogenetic relationships across lineage 2 and 4 M tuberculosis isolates. FINDINGS: We sequenced the whole genome of 648 isolates from 385 patients with drug-resistant tuberculosis recruited into cohort studies in KwaZulu-Natal, and 28 isolates from six patients from the KwaZulu-Natal referral laboratory. We identified 30 isolates with Rv0678 RAVs from 16 (4%) of 391 patients. We did not identify any atpE, pepQ, or Rv1979c RAVs. MICs were measured for 21 isolates with Rv0678 RAVs. MICs were above the critical concentration for bedaquiline resistance in nine (43%) of 21 isolates, in the intermediate category in nine (43%) isolates, and within the wild-type range in three (14%) isolates. Clofazimine MICs in genetically wild-type isolates ranged from 0·12-0·5 µg/mL, and in isolates with RAVs from 0·25-4·0 µg/mL. Phylogenetic analysis of the extended dataset including M tuberculosis isolates from southern Africa resolved multiple emergences of Rv0678 variants in lineages 2 and 4, documented two likely nosocomial transmission events, and identified the spread of a possibly bedaquiline and clofazimine cross-resistant clone in eSwatini. We also identified four patients with pepQ frameshift mutations that may confer resistance. INTERPRETATION: Bedaquiline and clofazimine cross-resistance in southern Africa is emerging repeatedly, with evidence of onward transmission largely due to Rv0678 mutations in M tuberculosis. Roll-out of bedaquiline and clofazimine treatment in the setting of limited drug susceptibility testing could allow further spread of resistance. Designing strong regimens would help reduce the emergence of resistance. Drug susceptibility testing is required to identify where resistance does emerge. FUNDING: Wellcome Trust, National Institute of Allergy and Infectious Diseases and National Center for Advancing Translational Sciences of the National Institutes of Health.

Clofazimine has delayed antimicrobial activity against Mycobacterium tuberculosis both in vitro and in vivo.

OBJECTIVES: The anti-leprosy drug clofazimine has been shown to have antimicrobial activity against Mycobacterium tuberculosis and has been associated with treatment-shortening activity in both clinical and preclinical studies of TB chemotherapy. However, a reported lack of early bactericidal activity (EBA) in TB patients has raised questions regarding the usefulness of clofazimine as an anti-TB drug. Our objective was to systematically evaluate the EBA of clofazimine in vitro and in vivo to provide insight into how and when this drug exerts its antimicrobial activity against M. tuberculosis. METHODS: We evaluated the 14 day EBA of clofazimine (i) in vitro at concentrations ranging from 4 times below to 4 times above the MIC for M. tuberculosis and (ii) in vivo in infected BALB/c mice at doses ranging from 1.5 to 100 mg/kg/day, and serum clofazimine levels were measured. In both experiments, isoniazid was used as the positive control. RESULTS: In vitro, clofazimine, at any concentration tested, did not exhibit bactericidal activity during the first week of exposure; however, in the second week, it exhibited concentration-dependent antimicrobial activity. In vivo, clofazimine, at any dose administered, did not exhibit bactericidal activity during the first week, and limited antimicrobial activity was observed during the second week of administration. While serum clofazimine levels were clearly dose dependent, the antimicrobial activity was not significantly related to the dose administered. CONCLUSIONS: Our data suggest that clofazimine's delayed antimicrobial activity may be due more to its mechanism of action rather than to host-related factors.

Clofazimine Contributes Sustained Antimicrobial Activity after Treatment Cessation in a Mouse Model of Tuberculosis Chemotherapy.

Experimental and clinical studies have indicated that the antileprosy drug clofazimine may contribute treatment-shortening activity when included in tuberculosis treatment regimens. Clofazimine accumulates to high levels in tissues, has a long half-life, and remains in the body for months after administration is stopped. We hypothesized that in tuberculosis treatment, accumulated clofazimine may contribute sustained antimicrobial activity after treatment cessation, and we used the BALB/c mouse model of chronic tuberculosis chemotherapy to address this hypothesis. Mycobacterium tuberculosis-infected mice were treated for 4 weeks or 8 weeks with either isoniazid alone, clofazimine alone, the first-line regimen rifampin-isoniazid-pyrazinamide-ethambutol, or a first-line regimen where clofazimine was administered in place of ethambutol. To evaluate posttreatment antimicrobial activity, bacterial regrowth in the lungs and spleens was assessed at the day of treatment cessation and 2, 4, 6, and 8 weeks after treatment was stopped. Bacterial regrowth was delayed in all mice receiving clofazimine, either alone or in combination, compared to the mice that did not receive clofazimine. This effect was especially evident in mice receiving multidrug therapy. In mice not receiving clofazimine, bacterial regrowth began almost immediately after treatment was stopped, while in mice receiving clofazimine, bacterial regrowth was delayed for up to 6 weeks, with the duration of sustained antimicrobial activity being positively associated with the time that serum clofazimine levels remained at or above the 0.25-µg/ml MIC for M. tuberculosis Thus, sustained activity of clofazimine may be important in the treatment-shortening effect associated with this drug.

Pharmacokinetics and pharmacodynamics of clofazimine in a mouse model of tuberculosis.

The antileprosy drug clofazimine has shown potential for shortening tuberculosis treatment; however, the current dosing of the drug is not evidence based, and the optimal dosing is unknown. Our objective was to conduct a preclinical evaluation of the pharmacokinetics and pharmacodynamics of clofazimine in the mouse model of tuberculosis, with the goal of providing useful information on dosing for future studies. Pharmacokinetic parameters were evaluated in infected and uninfected BALB/c mice. Pharmacodynamic parameters were evaluated in Mycobacterium tuberculosis-infected mice that were treated for 12 weeks with one of six different clofazimine dosing regimens, i.e., doses of 6.25, 12.5, and 25 mg/kg of body weight/day and 3 regimens with loading doses. Clofazimine progressively accumulated in the lungs, livers, and spleens of the mice, reaching levels of greater than 50 µg/g in all tissues by 4 weeks of administration, while serum drug levels remained low at 1 to 2 µg/ml. Elimination of clofazimine was extremely slow, and the half-life was dependent on the duration of drug administration. Clofazimine exhibited dose-dependent tissue and serum concentrations. At any dose, clofazimine did not have bactericidal activity during the first 2 weeks of administration but subsequently demonstrated potent, dose-independent bactericidal activity. The antituberculosis activity of clofazimine was dependent on neither the dose administered nor the drug concentrations in the tissues, suggesting that much lower doses could be effectively used for tuberculosis treatment.