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The validation of genome-wide association signals for tuberculosis (TB) susceptibility and the development of type 2 diabetes (T2D) across diverse populations remain problematic. The ancestry-specific variants (coding and non-coding) that contribute to previously identified differentially expressed genes (DEG) in patients with TB, T2D and comorbid TB-T2D, remain unknown. Identifying ancestry-specific expression quantitative trait loci (eQTLs) can aid in distinguishing the most probable disease-causing variants for population-specific therapeutic interventions. Therefore, this study conductedcis-eQTL mapping in TB, T2D and TB-T2D patients to identify variants associated with DEG. Both genotyping (Infinium H3A array with ∼2.3 M markers) and RNA sequencing data of 96 complex multi-way admixed South Africans were used for this purpose. Importantly, both global-and local ancestry adjustment were included in statistical analysis to account for complex admixture. Unique gene-variant pairs were associated with TB-T2D on chromosome 7p22 whilst adjusting for Bantu-speaking African ancestry (PRKAR1B:rs4464850; P=7.68e-07) and Khoe-San ancestry (PRKAR1B:rs117842122; P=3.66e-07). In addition,IFITM3(a biomarker for the development of TB) was associated with three SNPs (rs11025530, rs3808990, and rs10896664) on chromosome 11p15 while adjusting for Khoe-San ancestry. Our results also indicated that the upregulation of theNLRP6inflammasome is strongly associated with people with TB-T2D while adjusting for Khoe-San ancestry. Three African-specific eGenes (NLRP6, IFITM3andPRKAR1B) would have been missed if local ancestry adjustment was not conducted. This study determined a list of ancestry-specific eQTLs in TB-T2D patients that could potentially guide the search for new therapeutic targets for TB-T2D in African populations.Author SummaryThe limitation of genome-wide association study (GWAS) is that the particular biological pathway impacted by a variant might not be evident. eQTL mapping can be conducted to determine the impact that a genetic variant might have on the expression of a specific gene in a biological pathway. In this study the use ofcis-eQTL mapping was explored to elucidate the underlying genetic variants that regulate gene expression between TB-T2D and T2D patients, and between TB patients and healthy controls with multi-way genetic admixture from South Africa. Using RNA sequencing data and newly genotyped dataset of 96 individuals (Illumina Infinium H3Africa array with ∼2.5 M markers), we were able to identify ancestry-specific eQTLs. eQTLs of indigenous Khoe-San ancestral origin were identified in genetic regions previously implicated in TB and T2D in African populations. If local ancestry was not incorporated in thecis-eQTL mapping analysis these important African-specific eQTLs would have been missed. Our results provide a list of possible ancestry-specific causal variants associated with TB-T2 comorbidity that could guide the search for new therapeutic targets for African-specific populations. Including populations with complex ancestry and admixture in genetic studies is necessary to improve the quality of genetic research in sub-Saharan African groups.

In South Africa, mycobacterial culture is regarded as the gold standard for the detection of Mycobacterium tuberculosis complex (MTBC) infection in wildlife even though it is regarded as “imperfect.” We compared a novel decontamination and mycobacterial culture technique (TiKa) to the conventional mycobacterium growth indicator tube (MGIT) system using known amounts of bacilli and clinical samples from MTBC-infected African buffaloes ( Syncerus caffer), white rhinoceros ( Ceratotherium simum), and African elephants ( Loxodonta africana). Use of the TiKa-KiC decontamination agent on samples spiked with 10,000 to 10 colony forming units (cfu) of M. bovis (SB0121) and M. tuberculosis (H37Rv) had no effect on isolate recovery in culture. In contrast, decontamination with MGIT MycoPrep resulted in no growth of M. bovis samples at concentrations

Diagnosis of bovine tuberculosis (bTB) may be confounded by immunological cross-reactivity to Mycobacterium bovis antigens when animals are sensitised by certain nontuberculous mycobacteria (NTMs). Therefore, this study aimed to investigate NTM species diversity in African buffalo (Syncerus caffer) respiratory secretions and tissue samples, using a combination of novel molecular tools. Oronasal swabs were collected opportunistically from 120 immobilised buffaloes in historically bTB-free herds. In addition, bronchoalveolar lavage fluid (BALF; n=10) and tissue samples (n=19) were obtained during post-mortem examination. Mycobacterial species were identified directly from oronasal swab samples using the Xpert MTB/RIF Ultra qPCR (14/120 positive) and GenoType CMdirect (104/120 positive). In addition, all samples underwent mycobacterial culture, and PCRs targeting hsp65 and rpoB were performed. Overall, 55 NTM species were identified in 36 mycobacterial culture-positive swab samples with presence of esat-6 or cfp-10 detected in 20 of 36 isolates. The predominant species were M. avium complex and M. komanii. Nontuberculous mycobacteria were also isolated from 6 of 10 culture-positive BALF and 4 of 19 culture-positive tissue samples. Our findings demonstrate that there is a high diversity of NTMs present in buffaloes, and further investigation should determine their role in confounding bTB diagnosis in this species.

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in wildlife. Confirmation of M. bovis infection relies on mycobacterial culture, which is time-consuming. Collection and transportation of infectious material also pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to effectively inactivate infectious organisms, making it a safe method for handling infectious samples. This study investigated an in-field sampling technique for rapid, safe detection of M. bovis in buffalo tissues. Potentially infected tissues from bTB test-positive buffaloes were swabbed at post-mortem examination and stored in PrimeStore MTM at ambient temperature until Xpert MTB/RIF Ultra testing was performed. Additionally, tissue samples were frozen and transported before homogenisation for culture and Ultra testing. Oral swabs were collected from M. bovis-unexposed buffaloes as a negative control cohort. Mycobacterium tuberculosis complex (MTBC) DNA was detected by Ultra in 13/16 tissue swabs and 9/16 matched tissue homogenates from culture-confirmed M. bovis-positive buffalo tissues. MTBC DNA was not detected in swabs from M. bovis-unexposed animals, showing the potentially high specificity of Ultra with PrimeStore swabs. PrimeStore MTM sample processing, in combination with the Ultra assay, has the potential to provide a safe, rapid post-mortem screening test for M. bovis in buffaloes.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, is a multi-host disease which negatively affects the wildlife industry, with adverse consequences for conservation, ecotourism, and game/wildlife sales. Although interspecies transmission has been reported between some wildlife hosts, the risk of spread in complex ecosystems is largely unknown. As a controlled disease, tools for accurate detection of M. bovis infection are crucial for effective surveillance and management, especially in wildlife populations. There are, however, limited species-specific diagnostic tests available for wildlife. Hippopotamuses are rarely tested for M. bovis infection, and infection has not previously been confirmed in these species. In this study, blood and tissue samples collected from common hippopotamus (Hippopotamus amphibius) residing in a bTB-endemic area, the Greater Kruger Protected area (GKPA), were retrospectively screened to determine whether there was evidence for interspecies transmission of M. bovis, and identify tools for M. bovis detection in this species. Using the multi-species DPP® VetTB serological assay, a bTB seroprevalence of 8% was found in hippopotamus from GKPA. In addition, the first confirmed case of M. bovis infection in a free-ranging common hippopotamus is reported, based on the isolation in mycobacterial culture, genetic speciation and detection of DNA in tissue samples. Importantly, the M. bovis spoligotype (SB0121) isolated from this common hippopotamus is shared with other M. bovis-infected hosts in GKPA, suggesting interspecies transmission. These results support the hypothesis that M. bovis infection may be under recognized in hippopotamus. Further investigation is needed to determine the risk of interspecies transmission of M. bovis to common hippopotamus in bTB-endemic ecosystems and evaluate serological and other diagnostic tools in this species.

Background Diabetes mellitus (DM) patients are three times more likely to develop tuberculosis (TB) than the general population. Active TB screening in people with DM is part of a bidirectional approach. The aim of this study was to conduct pragmatic active TB screening among DM patients in four countries to inform policy. Methods DM patients were recruited in Indonesia (n=809), Peru (n=600), Romania (n=603) and South Africa (n=51). TB cases were diagnosed using an algorithm including clinical symptoms and chest X-ray. Presumptive TB patients were examined with sputum smear and culture. Results A total of 171 (8.3%) individuals reported ever having had TB (South Africa, 26%; Indonesia, 12%; Peru, 7%; Romania, 4%), 15 of whom were already on TB treatment. Overall, 14 (0.73% [95% confidence interval 0.40 to 1.23]) TB cases were identified from screening. Poor glucose control, smoking, lower body mass index, education and socio-economic status were associated with newly diagnosed/current TB. Thirteen of the 14 TB cases diagnosed from this screening would have been found using a symptom-based approach. Conclusions These data support the World Health Organization recommendation for routine symptom-based screening for TB in known DM patients in high TB-burden countries. DM patients with any symptoms consistent with TB should be investigated and diagnostic tools should be easily accessible.

Elephant endotheliotropic herpesvirus (EEHV) infection can cause acute, often fatal, EEHV hemorrhagic disease in free-ranging and human-managed Asian elephants (Elephas maximus) and human-managed African elephants (Loxodonta africana). However, significant knowledge gaps exist pertaining to the presence of EEHV in free-ranging African elephant populations. We retrospectively screened 142 opportunistically collected samples (blood, n=98; bronchoalveolar lavage (BAL) fluid, n=21; trunk wash (TW) fluid, n=23) obtained between 2010 and 2020 from 98 free-ranging African elephants in the Kruger National Park, South Africa, for the presence of different EEHVs, as well as determining the real-time quantitative PCR positivity rate in this population. With the use of validated, previously published DNA extraction and real-time quantitative PCR protocols provided by the National Elephant Herpesvirus Laboratory (Washington, DC, USA), EEHV was detected in nine male African elephants from samples collected in 2011 (n=1), 2013 (n=1), 2018 (n=2), 2019 (n=4), and 2020 (n=1). Viral detection was more common in respiratory compared with blood samples. Six elephants tested positive for EEHV2 subtype (blood, n=2; BAL, n=3; TW, n=2), including one individual that tested positive on matched respiratory samples (BAL and TW). Four elephants tested positive for EEHV3-4-7 (blood, n=1; BAL, n=2; TW, n=1), whereas EEHV6 was not detected in any of the study animals. One elephant tested positive for both EEHV2 and EEHV3-4-7 in the same BAL sample. Even though the levels of viremia varied between 158 and 1,292 viral genome equivalents/mL blood and viral shedding of EEHV2 and EEHV3-4-7 was detected in respiratory samples, no clinical signs were observed in these apparently healthy elephants. These findings are consistent with reports of asymptomatic EEHV infection in human-managed African elephants.

BackgroundPeople with diabetes are more likely to develop tuberculosis (TB) and to have poor TB treatment outcomes than those without. We previously showed that blood transcriptomes in people with TB-diabetes (TB-DM) co-morbidity have excessive inflammatory and reduced interferon responses at TB diagnosis. It is unknown whether this persists through treatment, potentially underlying adverse outcomes.MethodsPulmonary TB patients were recruited in South Africa, Indonesia and Romania, and classified as having TB-DM, TB with prediabetes, TB-related hyperglycaemia or uncomplicated TB, based on glycated haemoglobin (HbA1c) concentration at TB diagnosis and after 6 months of TB treatment. Gene expression in blood samples collected at diagnosis and at regular intervals throughout treatment was measured by unbiased RNA-Seq and targeted Multiplex Ligation-dependent Probe Amplification.ResultsGene expression was modulated by TB treatment in all groups but to different extents, such that differences remained in people with TB-DM relative to TB-only throughout, including genes involved in innate responses, anti-microbial immunity and the inflammasome. People with prediabetes or with TB-related hyperglycaemia had gene expression more similar to people with TB-DM than TB-only throughout treatment. The overall pattern of change was similar across clinical groups irrespective of glycaemic index, permitting models predictive of TB treatment to be developed.ConclusionsThe exacerbated transcriptome changes seen in TB-DM take longer to resolve during TB treatment, indicating that prolonged treatment or host-directed therapy may be needed to improve TB treatment outcomes. Development of transcriptome-based biomarker signatures of TB-treatment response should include people with diabetes to be useful across populations.Key PointsHost blood transcriptomes are altered in tuberculosis, and further altered with diabetes co-morbidity. We have shown that there is similar resolution of transcriptomes through treatment, but with differing magnitude and kinetics in TB patients with or without diabetes.

Mycobacterium bovis infection in wildlife species occurs worldwide. However, few cases of M. bovis infection in captive elephants have been reported. We describe 2 incidental cases of bovine tuberculosis in free-ranging African elephants (Loxodonta africana) from a tuberculosis-endemic national park in South Africa and the epidemiologic implications of these infections.

In South Africa, mycobacterial culture is regarded as the gold standard for the detection of Mycobacterium tuberculosis complex (MTBC) infection in wildlife even though it is regarded as “imperfect.” We compared a novel decontamination and mycobacterial culture technique (TiKa) to the conventional mycobacterium growth indicator tube (MGIT) system using known amounts of bacilli and clinical samples from MTBC-infected African buffaloes (Syncerus caffer), white rhinoceros (Ceratotherium simum), and African elephants (Loxodonta africana). Use of the TiKa-KiC decontamination agent on samples spiked with 10,000 to 10 colony forming units (cfu) of M. bovis (SB0121) and M. tuberculosis (H37Rv) had no effect on isolate recovery in culture. In contrast, decontamination with MGIT MycoPrep resulted in no growth of M. bovis samples at concentrations

Coxiellosis, or Query (Q) fever, a disease caused by the intracellular bacteria Coxiella burnetii, was recently described in a managed breeding herd of white rhinoceros (Ceratotherium simum) in the southeastern United States. Clinical disease often results in abortion and could represent a conservation challenge for this species. In addition to the reproductive and herd management consequences, coxiellosis is also a zoonotic disease. Infection or clinical disease in any free-ranging rhinoceros species in a national park setting has not been previously described. In this study, evidence of prior infection was measured by immunofluorescent antibody titers in 89 serum samples collected from white rhinoceros within private reserves and a national park in South Africa. Total seropositivity was 48/89 (53.9% [95% CI, 43.6-63.9%]). Animals on private reserves had a seropositivity of 21/51 (41.1% [95% CI, 27.1-55.2%]), and national park rhinoceros had a higher rate of seropositivity at 71.0% [95% CI, 55.9-86.2%] (27/38; P= 0.004). Adults had a higher seropositivity compared with subadults (P= 0.03). There was no difference in seropositivity between sexes (P > 0.05). Results demonstrate that South African white rhinoceros populations are exposed to Coxiella, which could result in underrecognized reproductive consequences. Further studies should investigate potential implications for public health and conservation management of this species.

Mycobacterium bovis has the largest host range of the Mycobacterium tuberculosis complex and infects domestic animal species, wildlife, and humans. The presence of global wildlife maintenance hosts complicates bovine tuberculosis (bTB) control efforts and further threatens livestock and wildlife-related industries. Thus, it is imperative that early and accurate detection of M. bovis in all affected animal species is achieved. Further, an improved understanding of the complex species-specific host immune responses to M. bovis could enable the development of diagnostic tests that not only identify infected animals but distinguish between infection and active disease. The primary bTB screening standard worldwide remains the tuberculin skin test (TST) that presents several test performance and logistical limitations. Hence additional tests are used, most commonly an interferon-gamma (IFN-γ) release assay (IGRA) that, similar to the TST, measures a cell-mediated immune (CMI) response to M. bovis. There are various cytokines and chemokines, in addition to IFN-γ, involved in the CMI component of host adaptive immunity. Due to the dominance of CMI-based responses to mycobacterial infection, cytokine and chemokine biomarkers have become a focus for diagnostic tests in livestock and wildlife. Therefore, this review describes the current understanding of host immune responses to M. bovis as it pertains to the development of diagnostic tools using CMI-based biomarkers in both gene expression and protein release assays, and their limitations. Although the study of CMI biomarkers has advanced fundamental understanding of the complex host-M. bovis interplay and bTB progression, resulting in development of several promising diagnostic assays, most of this research remains limited to cattle. Considering differences in host susceptibility, transmission and immune responses, and the wide variety of M. bovis-affected animal species, knowledge gaps continue to pose some of the biggest challenges to the improvement of M. bovis and bTB diagnosis.

Coxiellosis, or Query (Q) fever, a disease caused by the intracellular bacteria

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in wildlife. Confirmation of M. bovis infection relies on mycobacterial culture, which is time-consuming. Collection and transportation of infectious material also pose a human health risk. PrimeStore® Molecular Transport Medium (MTM) has been shown to effectively inactivate infectious organisms, making it a safe method for handling infectious samples. This study investigated an in-field sampling technique for rapid, safe detection of M. bovis in buffalo tissues. Potentially infected tissues from bTB test-positive buffaloes were swabbed at post-mortem examination and stored in PrimeStore® MTM at ambient temperature until Xpert® MTB/RIF Ultra testing was performed. Additionally, tissue samples were frozen and transported before homogenisation for culture and Ultra testing. Oral swabs were collected from M. bovis-unexposed buffaloes as a negative control cohort. Mycobacterium tuberculosis complex (MTBC) DNA was detected by Ultra in 13/16 tissue swabs and 9/16 matched tissue homogenates from culture-confirmed M. bovis-positive buffalo tissues. MTBC DNA was not detected in swabs from M. bovis-unexposed animals, showing the potentially high specificity of Ultra with PrimeStore® swabs. PrimeStore® MTM sample processing, in combination with the Ultra assay, has the potential to provide a safe, rapid post-mortem screening test for M. bovis in buffaloes.

Background Inequality is rife throughout South Africa. The first wave of COVID-19 may have affected people in lower socioeconomic groups worse than the affluent. The SARS-CoV-2 seroprevalence and the specificity of anti-SARS-CoV-2 antibody tests in South Africa is not known. Methods We tested 405 volunteers representing all socioeconomic strata from the workforce of a popular shopping and tourist complex in central Cape Town with the Abbott SARS-CoV-2 IgG assay. We assessed the association between antibody positivity and COVID-19 symptom status, medical history, and sociodemographic variables. We tested 137 serum samples from healthy controls collected in Cape Town prior to the COVID-19 pandemic, to confirm the specificity of the assay in the local population. Results Of the 405 volunteers tested one month after the first peak of the epidemic in Cape Town, 96(23.7%) were SARS-CoV-2 IgG positive. Of those who tested positive, 46(47.9%) reported no symptoms of COVID-19 in the previous 6 months. Seropositivity was significantly associated with living in informal housing, residing in a subdistrict with low income-per household, and having a low-earning occupation. The specificity of the assay was 98.54%(95%CI 94.82%-99.82%) in the pre-COVID controls. Conclusions There is a high background seroprevalence in Cape Town, particularly in people of lower socioeconomic status. Almost half of cases are asymptomatic, and therefore undiagnosed by local testing strategies. These results cannot be explained by low assay specificity.

Several types of T cells have suppressive activity, like the interleukin (IL)-10 induced and producing Tr1 cells, transforming growth factor (TGF)-β producing Th3 cells, natural killer T (NKT) cells, the CD8+FoxP3- T cell population, γ/δ TCR+ cells and natural FoxP3+ regulatory T cells (Tregs) [1–3]. CD4+CD25+ Tregs expressing the transcription factor forkhead box P3 (FoxP3) are considered ‘professional’ Tregs as they already have suppressive functions prior to encountering antigens and retain their suppressive function after clonal expansion while continuing to express FoxP3 [4]. Suppressor T cells constitute approximately 10% of CD4+ T cells, and co-express CD25.

Immunological assays are the basis for many diagnostic tests for infectious diseases in animals and humans. Application in wildlife species, including the African elephant (Loxodonta africana), is limited however due to lack of information on immune responses. Since many immunoassays require both identified biomarkers of immune activation as well as species-specific reagents, it is crucial to have knowledge of basic immunological responses in the species of interest. Cytokine gene expression assays (GEAs) used to measure specific immune responses in wildlife have frequently shown that targeted biomarkers are often species-specific. Therefore, the aim of this study was to identify elephant-specific cytokine biomarkers to detect immune activation and to develop a GEA, using pokeweed mitogen stimulated whole blood from African elephants. This assay will provide the foundation for the development of future cytokine GEAs that can be used to detect antigen specific immune responses and potentially lead to various diagnostic tests for this species.

Pulmonary tuberculosis (PTB) is characterized by lung granulomas, inflammation and tissue destruction. Here we used within-subject peripheral blood gene expression over time to correlate with the within-subject lung metabolic activity, as measured by positron emission tomography (PET) to identify biological processes and pathways underlying overall resolution of lung inflammation. We used next-generation RNA sequencing and [18F]FDG PET-CT data, collected at diagnosis, week 4, and week 24, from 75 successfully cured PTB patients, with the [18F]FDG activity as a surrogate for lung inflammation. Our linear mixed-effects models required that for each individual the slope of the line of [18F]FDG data in the outcome and the slope of the peripheral blood transcript expression data correlate, i.e., the slopes of the outcome and explanatory variables had to be similar. Of 10,295 genes that changed as a function of time, we identified 639 genes whose expression profiles correlated with decreasing [18F]FDG uptake levels in the lungs. Gene enrichment over-representation analysis revealed that numerous biological processes were significantly enriched in the 639 genes, including several well known in TB transcriptomics such as platelet degranulation and response to interferon gamma, thus validating our novel approach. Others not previously associated with TB pathobiology included smooth muscle contraction, a set of pathways related to mitochondrial function and cell death, as well as a set of pathways connecting transcription, translation and vesicle formation. We observed up-regulation in genes associated with B cells, and down-regulation in genes associated with platelet activation. We found 254 transcription factor binding sites to be enriched among the 639 gene promoters. In conclusion, we demonstrated that of the 10,295 gene expression changes in peripheral blood, only a subset of 639 genes correlated with inflammation in the lungs, and the enriched pathways provide a description of the biology of resolution of lung inflammation as detectable in peripheral blood. Surprisingly, resolution of PTB inflammation is positively correlated with smooth muscle contraction and, extending our previous observation on mitochondrial genes, shows the presence of mitochondrial stress. We focused on pathway analysis which can enable therapeutic target discovery and potential modulation of the host response to TB.

Effective screening methods are critical for preventing the spread of bovine tuberculosis (bTB) among livestock and wildlife species. The tuberculin skin test (TST) remains the primary test for bTB globally, although performance is suboptimal. African buffaloes (Syncerus caffer) are a maintenance host of Mycobacterium bovis in South Africa, tested using the single intradermal tuberculin test (SITT) or comparative test (SICTT). The interpretation of these tests has been based on cattle thresholds due to the lack of species-specific cut-off values for African buffaloes. Therefore, the aims of this study were to calculate buffalo-specific thresholds for different TST criteria (SITT, SICTT, and SICTT72h that calculates the differential change at 72 h only) and compare performance using these cut-off values. The results confirm that 3 mm best discriminates M. bovis-infected from unexposed control buffaloes with sensitivities of 69 % (95 % CI 60-78; SITT and SICTT) and 76 % (95 % CI 65-83; SICTT72h), and specificities of 86 % (95 % CI 80-90; SITT), 96 % (95 % CI 92-98; SICTT72h) and 97 % (95 % CI 93-99; SICTT), respectively. A comparison between TST criteria using buffalo-specific thresholds demonstrates that the comparative TST performs better than the SITT, although sensitivity remains suboptimal. Therefore, further research and the addition of ancillary tests, such as cytokine release assays, are necessary to improve M. bovis detection in African buffaloes.

Wildlife tuberculosis is a major economic and conservation concern globally. Bovine tuberculosis (bTB), caused byMycobacterium bovis(M. bovis), is the most common form of wildlife tuberculosis. In South Africa, to date,M. bovisinfection has been detected in 24 mammalian wildlife species. The identification ofM. bovisinfection in wildlife species is essential to limit the spread and to control the disease in these populations, sympatric wildlife species and neighboring livestock. The detection ofM. bovis-infected individuals is challenging as only severely diseased animals show clinical disease manifestations and diagnostic tools to identify infection are limited. The emergence of novel reagents and technologies to identifyM. bovisinfection in wildlife species are instrumental in improving the diagnosis and control of bTB. This review provides an update on the diagnostic tools to detectM. bovisinfection in South African wildlife but may be a useful guide for other wildlife species.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, is a multi-host disease which negatively affects the wildlife industry, with adverse consequences for conservation, ecotourism, and game/wildlife sales. Although interspecies transmission has been reported between some wildlife hosts, the risk of spread in complex ecosystems is largely unknown. As a controlled disease, tools for accurate detection of M. bovis infection is crucial for effective surveillance and management, especially in wildlife populations. There are however, limited species-specific diagnostic tests available for wildlife. Hippopotamuses are rarely tested for M. bovis infection and infection has not previously been confirmed in these species. In this study, blood and tissue samples collected from common hippopotamus (Hippopotamus amphibius) residing in a bTB endemic area, the Greater Kruger Protected area (GKPA), were retrospectively screened to determine whether there was evidence for interspecies transmission of M. bovis, and to identify tools for M. bovis detection in this species. Using the multi-species DPP® VetTB serological assay, a bTB seroprevelance of 8% was found in hippopotamus from GKPA. In addition, the first confirmed case of M. bovis infection in a free-ranging common hippopotamus is reported, based on the isolation in mycobacterial culture, genetic speciation, and detection of DNA in tissue samples. Importantly, the M. bovis spoligotype (SB0121) isolated from this common hippopotamus is shared with other M. bovis-infected hosts in GKPA, suggesting interspecies transmission. These results support the hypothesis that M. bovis infection may be under recognized in hippopotamus. Further investigation is needed to determine the risk of interspecies transmission of M. bovis to common hippopotamus in bTB endemic ecosystems and evaluate serological and other diagnostic tools in this species.

Oxidized cholesterols have emerged as important signaling molecules of immune function, but little is known about the role of these oxysterols during mycobacterial infections. We found that expression of the oxysterol-receptor GPR183 was reduced in blood from patients with tuberculosis (TB) and type 2 diabetes (T2D) compared to TB patients without T2D and was associated with TB disease severity on chest x-ray. GPR183 activation by 7α,25-hydroxycholesterol (7α,25-OHC) reduced growth of Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis BCG in primary human monocytes, an effect abrogated by the GPR183 antagonist GSK682753. Growth inhibition was associated with reduced IFN-β and IL-10 expression and enhanced autophagy. Mice lacking GPR183 had significantly increased lung Mtb burden and dysregulated IFNs during early infection. Together, our data demonstrate that GPR183 is an important regulator of intracellular mycobacterial growth and interferons during mycobacterial infection.Graphical Abstract

As a recognized Mycobacterium bovis maintenance host, the African buffalo (Syncerus caffer) poses transmission risks to livestock, humans and other wildlife. Early detection of M. bovis infection is critical for limiting its spread. Currently, tests detecting cell-mediated immune responses are used for diagnosis in buffaloes. However, these may have suboptimal sensitivity or specificity, depending on the blood stimulation method. Recent evidence suggests that assays using combinations of host cytokine biomarkers may increase diagnostic performance. Therefore, this study aimed to investigate the application of a MILLIPLEX

BackgroundDiabetes mellitus (DM) increases active tuberculosis (TB) risk and worsens TB outcomes, jeopardizing TB control especially in TB-endemic countries with rising DM prevalence rates. We assessed DM status and clinical correlates in TB patients across settings in Indonesia, Peru, Romania, and South Africa.MethodsAge-adjusted DM prevalence was estimated using laboratory glycated hemoglobin (HbA1c) or fasting plasma glucose in TB patients. Detailed and standardized sociodemographic, anthropometric, and clinical measurements were made. Characteristics of TB patients with or without DM were compared using multilevel mixed-effect regression models with robust standard errors.ResultsOf 2185 TB patients (median age 36.6 years, 61.2% male, 3.8% human immunodeficiency virus–infected), 12.5% (267/2128) had DM, one third of whom were newly diagnosed. Age-standardized DM prevalence ranged from 10.9% (South Africa) to 19.7% (Indonesia). Median HbA1c in TB–DM patients ranged from 7.4% (Romania) to 11.3% (Indonesia). Compared to those without DM, TB–DM patients were older and had a higher body mass index (BMI) (P value < .05). Compared to those with newly diagnosed DM, TB patients with diagnosed DM had higher BMI and HbA1c, less severe TB, and more frequent comorbidities, DM complications, and hypertension (P value < .05).ConclusionsWe show that DM prevalence and clinical characteristics of TB–DM vary across settings. Diabetes is primarily known but untreated, hyperglycemia is often severe, and many patients with TB–DM have significant cardiovascular disease risk and severe TB. This underlines the need to improve strategies for better clinical management of combined TB and DM.

Type 2 diabetes (T2D) is a prevalent risk factor for tuberculosis (TB), but most studies on TB-T2D have focused on TB patients, been limited to one community, and shown a variable impact of T2D on TB risk or treatment outcomes. We conducted a cross-sectional assessment of sociodemographic and metabolic factors in adult TB contacts with T2D (versus no T2D), from the Texas-Mexico border to study Hispanics, and in Cape Town to study South African Coloured ethnicities. The prevalence of T2D was 30.2% in Texas-Mexico and 17.4% in South Africa, with new diagnosis in 34.4% and 43.9%, respectively. Contacts with T2D differed between ethnicities, with higher smoking, hormonal contraceptive use and cholesterol levels in South Africa, and higher obesity in Texas-Mexico (p < 0.05). PCA analysis revealed striking differences between ethnicities in the relationships between factors defining T2D and dyslipidemias. Our findings suggest that screening for new T2D in adult TB contacts is effective to identify new T2D patients at risk for TB. Furthermore, studies aimed at predicting individual TB risk in T2D patients, should take into account the heterogeneity in dyslipidemias that are likely to modify the estimates of TB risk or adverse treatment outcomes that are generally attributed to T2D alone.

The development of an improved vaccine to stimulate an effective response against Mycobacterium tuberculosis (MTB) infection and disease will be a major breakthrough in the fight against TB. A lack of tools to adequately track the progression or resolution of events in TB pathogenesis that occur at bacterial loads below the threshold for culture in human samples seriously hampers vaccine development and evaluation. In this review we discuss recent studies that use new imaging applications, modalities and analysis techniques to provide insight into the dynamic processes of MTB infection and disease that are challenging to monitor. These include early infection, the spectrum of latency and subclinical disease, the paucibacillary state induced by treatment, and events leading to recurrence, including relapse.

The study describes the novel use of the Xpert MTB/RIF Ultra assay for detection of Mycobacterium tuberculosis complex (MTBC) DNA in samples from white rhinoceros (Ceratotherium simum) and African elephants (Loxodonta africana). Culture negative respiratory sample matrices were spiked to determine if the Ultra could detect MTBC DNA in rhinoceros and elephant samples. Rhinoceros bronchial alveolar lavage fluid (BALF) was found to have an inhibitory effect on the Ultra. In this study, the limit of detection (LOD) of M. tuberculosis H37Rv in all spiked animal samples were 2 CFU/ml compared to 15.6 CFU/ml for humans, while the LOD for M. bovis SB0121 was 30 CFU/ml compared to 143.4 CFU/ml for M. bovis BCG in humans. Screening was performed on stored tissue and respiratory samples from known MTBC-infected animals and MTBC DNA was detected in 92% of samples collected from six rhinoceros and two elephants. Conversely, 83% of culture-negative tissue and respiratory samples from uninfected animals tested negative on the Ultra. In conclusion, the Ultra assay appears to be a sensitive and rapid diagnostic test for the detection of MTBC DNA from tissue and respiratory samples collected from African elephants and rhinoceros. Furthermore, the Ultra assay could provide a new tool for the detection of MTBC in various sample types from other wildlife species.

Biomarkers for TB treatment response and outcome are needed. This study characterize changes in immune profiles during TB treatment, define biosignatures associated with treatment outcomes, and explore the feasibility of predictive models for relapse. Seventy-two markers were measured by multiplex cytokine array in serum samples from 78 cured, 12 relapsed and 15 failed treatment patients from South Africa before and during therapy for pulmonary TB. Promising biosignatures were evaluated in a second cohort from Uganda/Brazil consisting of 17 relapse and 23 cured patients. Thirty markers changed significantly with different response patterns during TB treatment in cured patients. The serum biosignature distinguished cured from relapse patients and a combination of two clinical (time to positivity in liquid culture and BMI) and four immunological parameters (TNF-β, sIL-6R, IL-12p40 and IP-10) at diagnosis predicted relapse with a 75% sensitivity (95%CI 0.38-1) and 85% specificity (95%CI 0.75-0.93). This biosignature was validated in an independent Uganda/Brazil cohort correctly classifying relapse patients with 83% (95%CI 0.58-1) sensitivity and 61% (95%CI 0.39-0.83) specificity. A characteristic biosignature with value as predictor of TB relapse was identified. The repeatability and robustness of these biomarkers require further validation in well-characterized cohorts.

Although currently available model organisms such as Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) have significantly contributed to our understanding of tuberculosis (TB) biology, these models have limitations such as differences in genome size, growth rates and virulence. However, attenuated Mycobacterium tuberculosis strains may provide more representative, safer models to study M. tuberculosis biology. For example, the M. tuberculosis ΔleuDΔpanCD double auxotroph, has undergone rigorous in vitro and in vivo safety testing. Like other auxotrophic strains, this has subsequently been approved for use in biosafety level (BSL) 2 facilities. Auxotrophic strains have been assessed as models for drug-resistant M. tuberculosis and for studying latent TB. These offer the potential as safe and useful models, but it is important to understand how well these recapitulate salient features of non-attenuated M. tuberculosis. We therefore performed a comprehensive comparison of M. tuberculosis H37Rv and M. tuberculosisΔleuDΔpanCD. These strains demonstrated similar in vitro and intra-macrophage replication rates, similar responses to anti-TB agents and whole genome sequence conservation. Shotgun proteomics analysis suggested that M. tuberculosisΔleuDΔpanCD has a heightened stress response that leads to reduced bacterial replication during exposure to acid stress, which has been verified using a dual-fluorescent replication reporter assay. Importantly, infection of human peripheral blood mononuclear cells with the 2 strains elicited comparable cytokine production, demonstrating the suitability of M. tuberculosisΔleuDΔpanCD for immunological assays. We provide comprehensive evidence to support the judicious use of M. tuberculosisΔleuDΔpanCD as a safe and suitable model organism for M. tuberculosis research, without the need for a BSL3 facility.