Reconsidering Mycobacterium bovis as a proxy for zoonotic tuberculosis: a molecular epidemiological surveillance study
Shannon C DuffySreenidhi SrinivasanMegan A. SchillingTod StuberSarah N DanchukJoy S. MichaelManigandan VenkatesanNitish BansalSushila MaanNaresh JindalDeepika ChaudharyPremanshu DandapatRobab KataniShubhada K. ChotheMaroudam VeerasamiSuelee Robbe‐AustermanNicholas JuleffVivek KapurMarcel A. Behr
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BackgroundZoonotic tuberculosis is defined as human infection with Mycobacterium bovis. Although globally, India has the largest number of human tuberculosis cases and the largest cattle population, in which bovine tuberculosis is endemic, the burden of zoonotic tuberculosis is unknown. The aim of this study was to obtain estimates of the human prevalence of animal-associated members of the Mycobacterium tuberculosis complex (MTBC) at a large referral hospital in India.MethodsWe did a molecular epidemiological surveillance study of 940 positive mycobacteria growth indicator tube (MGIT) cultures, collected from patients visiting the outpatient department at Christian Medical College (Vellore, India) with suspected tuberculosis between Oct 1, 2018, and March 31, 2019. A PCR-based approach was applied to subspeciate cultures. Isolates identified as MTBC other than M tuberculosis or as inconclusive on PCR were subject to whole-genome sequencing (WGS), and phylogenetically compared with publicly available MTBC sequences from south Asia. Sequences from WGS were deposited in the National Center for Biotechnology Information Sequence Read Archive, accession number SRP226525 (BioProject database number PRJNA575883).FindingsThe 940 MGIT cultures were from 548 pulmonary and 392 extrapulmonary samples. A conclusive identification was obtained for all 940 isolates; wild-type M bovis was not identified. The isolates consisted of M tuberculosis (913 [97·1%] isolates), Mycobacterium orygis (seven [0·7%]), M bovis BCG (five [0·5%]), and non-tuberculous mycobacteria (15 [1·6%]). Subspecies were assigned for 25 isolates by WGS, which were analysed against 715 MTBC sequences from south Asia. Among the 715 genomes, no M bovis was identified. Four isolates of cattle origin were dispersed among human sequences within M tuberculosis lineage 1, and the seven M orygis isolates from human MGIT cultures were dispersed among sequences from cattle.InterpretationM bovis prevalence in humans is an inadequate proxy of zoonotic tuberculosis. The recovery of M orygis from humans highlights the need to use a broadened definition, including MTBC subspecies such as M orygis, to investigate zoonotic tuberculosis. The identification of M tuberculosis in cattle also reinforces the need for One Health investigations in countries with endemic bovine tuberculosis.FundingBill & Melinda Gates Foundation, Canadian Institutes for Health Research.Keywords:
Mycobacterium tuberculosis complex
Molecular Epidemiology
ABSTRACT Background Studies on human intraocular tuberculosis (IOTB) are extremely challenging. For understanding the pathogenesis of IOTB, it is important to investigate the mycobacterial transcriptional changes in ocular environment. Methods Mice were challenged intravenously with Mycobacterium tuberculosis H37Rv and at 45 days post-infection, experimental IOTB was confirmed based on bacteriological and molecular assays. M. tuberculosis transcriptome was analyzed in the infected eyes using microarray technology. The identified M. tuberculosis signature genes were further validated and investigated in human IOTB samples using real-time polymerase chain reaction. Results Following intravenous challenge with M. tuberculosis, 45% (5/12) mice showed bacilli in the eyes with positivity for M. tuberculosis ribonucleic acid in 100% (12/12), thus confirming the paucibacillary nature of IOTB similar to human IOTB. M. tuberculosis transcriptome in these infected eyes showed significant upregulation of 12 M. tuberculosis genes and five of these transcripts (Rv0962c, Rv0984, Rv2612c, Rv0974c and Rv0971c) were also identified in human clinically confirmed cases of IOTB. Conclusions Differentially expressed mycobacterial genes identified in an intravenously challenged paucibacillary mouse IOTB model and presence of these transcripts in human IOTB samples highlight the possible role of these genes for survival of M. tuberculosis in the ocular environment, thus contributing to pathogenesis of IOTB.
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Objective To develop a new,rapid and easy method for species identification of Mycobacterium tuberculosis complex(MTBC).Methods Seven sets of primers(Mtbc1-7) for MTBC were used to amplify the genomic DNA from the reference strains and 96 clinical isolates.Results The specificities of 7 sets of primers were identified by PCR with 3 MTBC reference strains,9 non-mycobacterium reference strains and 27 non-tuberculosis mycobacterium(NTM) reference strains. The results showed that mycobacterium and non-mycobacterium were identified by Mtbc1;NTM and MTBC by Mtbc2;M.microti by Mtbc3;M.bovis and M.bovis BCG by Mtbc4;M.africanum subtypeI and M.tuberculosis by Mtbc5;M.bovis BCG by Mtbc4 and Mtbc6 together;M.canettii by Mtbc5 and Mtbc7 together.Of 96 clinical isolates,20 MOTT,3 M.microti,1 M.bovis,1 M.bovis BCG,3 M.africanum subtypeⅠ,1 M.caprae,3 M.canettii,64 M.africanum subtypeⅡ and M.tuberculosis were identified.Conclusion PCR is a rapid and effective method for the species identification of MTBC,and can be used for molecular epidemiology.
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ABSTRACT The species identification of members of the Mycobacterium tuberculosis complex is critical to the timely initiation of both appropriate antibiotic therapy and proper public health control measures. However, the current commercially available molecular assays identify mycobacteria only to the complex level and are unable to differentiate M. tuberculosis from the closely related M. bovis and M. bovis BCG. We describe here a rapid and robust two-step, multiplex, real-time PCR assay based on genomic deletions to definitively identify M. tuberculosis, M. bovis, M. bovis BCG, and other members of the complex. When tested against a panel of well-characterized mycobacterial reference strains, the assay was both sensitive and specific, correctly identifying all strains. We applied this assay to 60 clinical isolates previously identified as M. tuberculosis complex and found 57 M. tuberculosis isolates and 3 M. bovis BCG isolates from patients who had received intravesical BCG. Furthermore, analysis of 15 clinical specimens previously identified as M. bovis by spoligotyping revealed an isolate of M. tuberculosis that had been misidentified. We propose that this assay will allow the routine identification of M. tuberculosis complex members in the clinical laboratory.
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Mycobacterium tuberculosis (M. tuberculosis) causes an enormous burden of disease worldwide. As a central aspect of its pathogenesis, M. tuberculosis grows in macrophages, and host and microbe influence each other's metabolism. To define the metabolic impact of M. tuberculosis infection, we performed global metabolic profiling of M. tuberculosis–infected macrophages. M. tuberculosis induced metabolic hallmarks of inflammatory macrophages and a prominent signature of cholesterol metabolism. We found that infected macrophages accumulate cholestenone, a mycobacterial-derived, oxidized derivative of cholesterol. We demonstrated that the accumulation of cholestenone in infected macrophages depended on the M. tuberculosis enzyme 3β-hydroxysteroid dehydrogenase (3β-Hsd) and correlated with pathogen burden. Because cholestenone is not a substantial human metabolite, we hypothesized it might be diagnostic of M. tuberculosis infection in clinical samples. Indeed, in 2 geographically distinct cohorts, sputum cholestenone levels distinguished subjects with tuberculosis (TB) from TB-negative controls who presented with TB-like symptoms. We also found country-specific detection of cholestenone in plasma samples from M. tuberculosis–infected subjects. While cholestenone was previously thought to be an intermediate required for cholesterol degradation by M. tuberculosis, we found that M. tuberculosis can utilize cholesterol for growth without making cholestenone. Thus, the accumulation of cholestenone in clinical samples suggests it has an alternative role in pathogenesis and could be a clinically useful biomarker of TB infection.
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Bovine tuberculosis
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Tuberculosis is one of the most dangerous infectious diseases and has among the highest mortality rates of all infectious diseases. There are 9 million cases of active tuberculosis reported annually; however, an estimated one-third of the world's population is infected with Mycobacterium tuberculosis and remains asymptomatic. Despite the great progress in its diagnosis and treatment, tuberculosis is still a serious health and social problem. The contact between the immune system and Mycobacterium tuberculosis initiates cell-specific (Th1) and humoral-specific (Th2) responses. Many studies about the presence of antituberculotic antibodies in the serum have produced inconsistent results because of a high proportion of false-positive or false-negative results. The purpose of this study was to confirm whether circulating immune complexes (CIC) isolated from the serum of patients with tuberculosis are accompanied by antigenic proteins typical of Mycobacterium tuberculosis.We assayed serum samples from 42 patients with tuberculosis. The control group consisted of the sera samples taken from 45 healthy subjects. The immunochemical analysis of dissociated immune complexes using the dot blot method demonstrated positive reaction on the presence of Mycobacterium tuberculosis antigens in all patients with tuberculosis.All patients with tuberculosis demonstrated a high serum concentration of CIC protein. The mean serum concentration of CIC protein was significantly higher in patients than in controls: 0.081 g/l in the control group and 0.211 g/l in the tuberculosis patients.The analysis of CIC suggests that it may be a helpful test for patients with tuberculosis because of its quickness, simplicity of the idea, and limited invasiveness.
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Transcriptional Biomarkers of Differentially Detectable Mycobacterium tuberculosis in Patient Sputum
Certain populations of Mycobacterium tuberculosis go undetected by standard diagnostics but can be enumerated using limiting dilution assays. These differentially detectable M. tuberculosis (DD M. tuberculosis) populations may have relevance for persistence due to their drug tolerance. It is unclear how well DD M. tuberculosis from patients is modeled by a recently developed
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Extensively drug-resistant tuberculosis
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Introduction. Relapses of tuberculosis are fairly rare nowdays and they represent the onset of tuberculosis two, or more than two years after completion of previous treatment. Material and Methods. In the previous period, relapses of tuberculosis occurred in 141 patients (87 male and 54 female). Their mean age was 46.2 years. Results. Relapses of tuberculosis occurred after 11.3 years, on average. All patients presented with pulmonary tuberculosis, and two patients also had pulmonary and extrapulmonary tuberculosis(bones). Resistance was one of the statistically significant factors for relapse of tuberculosis. Resistance to one antituberculotic agent was most common - 8 patients, resistance to two drugs - 4 patients, resistance to three drugs - 4 patients, resistance to four drugs in 5 patients. Due to these findings on resistant strains of mycobacterium tuberculosis, a huge number of patients with relapses of tuberculosis had full recovery and completed the treatment. Conclusion. The importance of resistant strains of mycobacterium tuberculosis is really huge in our conditions. The findings of these resistant strains of mycobacterium tuberculosis and adequate medical treatment are obligatory nowadays. .
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There is considerable variability in the outcome of Mycobacterium tuberculosis infection. We hypothesized that Mycobacterium africanum was less likely than M. tuberculosis to transmit and progress to tuberculosis disease.In a cohort study of patients with tuberculosis and their household contacts in The Gambia, we categorized 1808 HIV-negative tuberculosis contacts according to exposure to M. tuberculosis or M. africanum. Positive skin test results indicated transmission, and development of tuberculosis during 2 years of follow-up indicated progression to disease.Transmission rates were similar, but rates of progression to disease were significantly lower in contacts exposed to M. africanum than in those exposed to M. tuberculosis (1.0% vs. 2.9%; hazard ratio [HR], 3.1 [95% confidence interval {CI}, 1.1-8.7]). Within M. tuberculosis sensu stricto, contacts exposed to a Beijing family strain were most likely to progress to disease (5.6%; HR relative to M. africanum, 6.7 [95% CI, 2.0-22]).M. africanum and M. tuberculosis transmit equally well to household contacts, but contacts exposed to M. africanum are less likely to progress to tuberculosis disease than those exposed to M. tuberculosis. The variable rate of progression by lineage suggests that tuberculosis variability matters in clinical settings and should be accounted for in studies evaluating tuberculosis vaccines and treatment regimens for latent tuberculosis infection.
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