How well do routine molecular diagnostics detect rifampicin heteroresistance inMycobacterium tuberculosis?
Kamela C. S. NgPhilip SupplyFrank CobelensCyril GaudinJulián González-MartínBouke C. de JongLeen Rigouts
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ABSTRACT Rifampicin heteroresistance – where rifampicin-resistant and -susceptible tuberculosis (TB) bacilli co-exist – may result in failed standard TB treatment and potential spread of rifampicin-resistant strains. Detection of rifampicin heteroresistance in routine rapid diagnostic tests (RDTs) allows for patients to receive prompt and effective multidrug-resistant-TB treatment, and may improve rifampicin-resistant TB control. The limit of detection (LOD) of rifampicin heteroresistance for phenotypic drug susceptibility testing by the proportion method is 1%, yet is insufficiently documented for RDTs. We therefore aimed to determine, for the four RDTs (XpertMTB/RIF, XpertMTB/RIF Ultra, GenoTypeMTBDR plus v2.0, and GenoscholarNTM+MDRTBII), the LOD per probe and mutation, validated by colony-forming-unit-counting and targeted deep sequencing (Deeplex-MycTB). We selected one rifampicin-susceptible and four rifampicin-resistant strains, with mutation D435V, H445D, H445Y, and S450L respectively, mixed them in various proportions in triplicate, tested them with each RDT, and determined the LODs per mutation type. Deeplex-MycTB revealed concordant proportions of the minority resistant variants in the mixtures. The Deeplex-MycTB-validated-LODs ranged from 20-80% for XpertMTB/RIF, 20-70% for Xpert Ultra, 5-10% for GenoTypeMTBDR plus v2.0, and 1-10% for GenoscholarNTM+MTBII for the different mutations. Deeplex-MycTB, GenoTypeMTBDR plus v2.0, and GenoscholarNTM+MDRTBII, provide explicit information on rifampicin heteroresistance for the most frequently detected mutations. Classic Xpert and Ultra report rifampicin heteroresistance as rifampicin resistance, while Ultra may denote rifampicin heteroresistance through ‘mixed patterns’ of wild-type and mutant melt probe melt peak temperatures. Overall, our findings inform end-users that the threshold for reporting resistance in case of rifampicin heteroresistance is the highest for Classic Xpert and Ultra, to resolve phenotypic and genotypic discordant rifampicin-resistant TB results.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.
Pathogenesis
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The objective: to identify factors of a higher risk to develop tuberculosis in children exposed to tuberculosis cases resistant or sensitive to rifampicin. Subjects 161 children under 6 years old exposed to tuberculosis in their families were enrolled in the retrospective study. The children were divided into 2 groups: Group 1 ( n = 92) – children free of TB; Group 2 ( n = 69) – children ill with TB. In each group, two subgroups were identified: those with index cases susceptible to rifampicin (RS) 1A ( n = 40) and 2A ( n = 43) and those with index cases resistant to rifampicin (RR) 1Б ( n = 52) and 2Б ( n = 26). Results. Exposure to isoniazid resistant tuberculosis increases the risk of TB in the child by 12 times, on the opposite, drug resistance to rifampicin provides no impact on the risk to develop tuberculosis. If the child is exposed to 2 cases of tuberculosis and more, the risk to develop tuberculosis increases by 2-14 times. BCG vaccination of the child exposed to tuberculosis reduces the risk to develop tuberculosis by 2-13 times.
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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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Current theory in the molecular epidemiology of tuberculosis holds that tuberculosis cases harboring Mycobacterium tuberculosis strains with a common deoxyribonucleic acid (DNA) fingerprint are the result of recent M. tuberculosis transmission. Here we propose a mathematical approach independent of DNA fingerprinting to estimating the percentage of recent transmissions responsible for current tuberculosis incidence. The “short-term reproductive number” of tuberculosis is defined as the average number of tuberculosis cases developing within 1 year of infection. Multiplying the short-term reproductive number by the number of tuberculosis cases in each year and dividing by the subsequent year’s tuberculosis case burden equals the proportion of tuberculosis cases in the subsequent year that are due to recent transmission. We carried out separate calculations for human immunodeficiency virus (HIV)-negative and HIV-positive tuberculosis cases. We applied the model to pulmonary (infectious) tuberculosis cases diagnosed in New York City during 1989–1993, using tuberculosis and AIDS surveillance data. Model-based estimates of the proportion of tuberculosis due to recent transmission were lower than estimates based on DNA fingerprints. Reconciliation of these divergent estimates may require the re-estimation of model parameters from data collected de novo, additional model development, and further advances in DNA fingerprinting methods.
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ABSTRACT The long-term persistence of Mycobacterium tuberculosis in communities with high tuberculosis prevalence is a serious problem aggravated by the presence of drug-resistant tuberculosis strains. Drug resistance in an individual patient is often discovered only after a long delay, particularly if the diagnosis is based on current culture-based drug sensitivity testing methods. During such delays, the patient may transmit tuberculosis to his or her contacts. Rapid diagnosis of drug resistance would be expected to reduce this transmission and hence to decrease the prevalence of drug-resistant strains. To investigate this quantitatively, a mathematical model was constructed, assuming a homogeneous population structure typical of communities in South Africa where tuberculosis incidence is high. Computer simulations performed with this model showed that current control strategies will not halt the spread of multidrug-resistant tuberculosis in such communities. The simulations showed that the rapid diagnosis of drug resistance can be expected to reduce the incidence of drug-resistant cases provided the additional measure of screening within the community is implemented.
Extensively drug-resistant tuberculosis
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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
Tuberculosis diagnosis
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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. .
Mycobacterium tuberculosis complex
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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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