Development of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis

Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay''s specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.     

Mycobacterium tuberculosis Genotypes Determined by Spoligotyping to Be Circulating in Colombia between 1999 and 2012 and Their Possible Associations with Transmission and Susceptibility to First-Line Drugs

Introduction

Tuberculosis (TB) remains a primary public health problem worldwide. The number of multidrug-resistant tuberculosis (MDR TB) cases has increased in recent years in Colombia. Knowledge of M. tuberculosis genotypes defined by spoligotyping can help determine the circulation of genotypes that must be controlled to prevent the spread of TB.

Objective

To describe the genotypes of M. tuberculosis using spoligotyping in resistant and drug-sensitive isolates and their possible associations with susceptibility to first-line drugs.

Methods

An analytical observational study was conducted that included 741 isolates of M. tuberculosis from patients. The isolates originated from 31 departments and were obtained by systematic surveillance between 1999 and 2012.

Results

In total 61.94% of the isolates were resistant to 1 or more drugs, and 147 isolates were MDR. In total, 170 genotypes were found in the population structure of Colombian M. tuberculosis isolates. The isolates were mainly represented by four families: LAM (39.9%), Haarlem (19%), Orphan (17%) and T (9%). The SIT42 (LAM 9) was the most common genotype and contained 24.7% of the isolates, followed by the genotypes SIT62 (Haarlem1), SIT53 (T1), and SIT50 (H3). A high clustering of isolates was evident with 79.8% of the isolates classified into 32 groups. The Beijing family was associated with resistant isolates, whereas the Haarlem and T families were associated with sensitive isolates. The Haarlem family was also associated with grouped isolates (p = 0.031).

Conclusions

A high proportion (approximately 80%) of isolates was found in clusters; these clusters were not associated with resistance to first-line drugs. The Beijing family was associated with drug resistance, whereas the T and Haarlem families were associated with susceptibility in the Colombian isolates studied.     

Broad diversity of Mycobacterium tuberculosis complex strains isolated from humans and cattle in Northern Algeria suggests a zoonotic transmission cycle

Mycobacterium tuberculosis complex (MTBC) comprises closely related species responsible for human and animal tuberculosis (TB). Efficient species determination is useful for epidemiological purposes, especially for the elucidation of the zoonotic contribution. In Algeria, data on MTBC genotypes are largely unknown. In this study, we aimed to investigate the occurrence and diversity of MTBC genotypes causing human and bovine TB in Northern Algeria. During a two-year sampling period (2017–2019) in two regions of Northern Algeria, we observed an overall prevalence of 6.5% of tuberculosis (TB) among slaughtered cattle, which is higher than previous Algerian data yet comparable to neighboring countries. A total of 296 Mycobacterium tuberculosis complex (MTBC) isolates were genotyped by spoligotyping: 181 from tissues with TB-like lesions collected from 181 cattle carcasses and 115 from TB patients. In human isolates, we identified 107 M. tuberculosis, seven M. bovis and one “M. pinnipedii-like”, while for bovine samples, 174 isolates were identified as M. bovis, three as M. caprae, three as “M. pinnipedii-like” and one as “M. microti-like”. The majority of isolates (89.2%) belonged to 72 different known Shared International Types (SIT) or M. bovis spoligotypes (SB), while we also identified seven new SB profiles (SB2695 to SB2701). Twenty-eight of the SB profiles were new to Algeria. Our data suggest zoonotic transmission in Sétif, where significantly more TB was observed among cattle (20%) compared to the slaughterhouses from the three other regions (5.4%–7.3%) (p < 0.0001), with the isolation of the same M. bovis genotypes from TB patients. The present study showed a high genetic diversity of MTBC isolated from human and cattle in Northern Algeria. Even though relatively small in terms of numbers, our data suggest the zoonotic transmission of TB from cattle to humans, suggesting the need for stronger eradication strategies for bovine TB.     

Comparative Genomics of Field Isolates of Mycobacterium bovis and M. caprae Provides Evidence for Possible Correlates with Bacterial Viability and Virulence

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly affect humans and animals worldwide. The life cycle of mycobacteria is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Recently, comparative genomics analyses have provided new insights into the evolution and adaptation of the MTBC to survive inside the host. However, most of this information has been obtained using M. tuberculosis but not other members of the MTBC such as M. bovis and M. caprae. In this study, the genome of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different lesion score, prevalence and host distribution phenotypes were sequenced. Genome sequence information was used for whole-genome and protein-targeted comparative genomics analysis with the aim of finding correlates with phenotypic variation with potential implications for tuberculosis (TB) disease risk assessment and control. At the whole-genome level the results of the first comparative genomics study of field isolates of M. bovis including M. caprae showed that as previously reported for M. tuberculosis, sequential chromosomal nucleotide substitutions were the main driver of the M. bovis genome evolution. The phylogenetic analysis provided a strong support for the M. bovis/M. caprae clade, but supported M. caprae as a separate species. The comparison of the MB1 and MB4 isolates revealed differences in genome sequence, including gene families that are important for bacterial infection and transmission, thus highlighting differences with functional implications between isolates otherwise classified with the same spoligotype. Strategic protein-targeted analysis using the ESX or type VII secretion system, proteins linking stress response with lipid metabolism, host T cell epitopes of mycobacteria, antigens and peptidoglycan assembly protein identified new genetic markers and candidate vaccine antigens that warrant further study to develop tools to evaluate risks for TB disease caused by M. bovis/M.caprae and for TB control in humans and animals.     

Identification of drug resistance mutations among Mycobacterium bovis lineages in the Americas

Identifying the Mycobacterium tuberculosis resistance mutation patterns is of the utmost importance to assure proper patient’s management and devising of control programs aimed to limit spread of disease. Zoonotic Mycobacterium bovis infection still represents a threat to human health, particularly in dairy production regions. Routinary, molecular characterization of M. bovis is performed primarily by spoligotyping and mycobacterial interspersed repetitive units (MIRU) while next generation sequencing (NGS) approaches are often performed by reference laboratories. However, spoligotyping and MIRU methodologies lack the resolution required for the fine characterization of tuberculosis isolates, particularly in outbreak settings. In conjunction with sophisticated bioinformatic algorithms, whole genome sequencing (WGS) analysis is becoming the method of choice for advanced genetic characterization of tuberculosis isolates. WGS provides valuable information on drug resistance and compensatory mutations that other technologies cannot assess. Here, we performed an analysis of the most frequently identified mutations associated with tuberculosis drug resistance and their genetic relationship among 2,074 Mycobacterium bovis WGS recovered primarily from non-human hosts. Full-length gene sequences harboring drug resistant associated mutations and their phylogenetic relationships were analyzed. The results showed that M. bovis isolates harbor mutations conferring resistance to both first- and second-line antibiotics. Mutations conferring resistance for isoniazid, fluoroquinolones, streptomycin, and aminoglycosides were identified among animal strains. Our findings highlight the importance of molecular surveillance to monitor the emergence of mutations associated with multi and extensive drug resistance in livestock and other non-human mammals.     

Evolution of Extensively Drug-Resistant Tuberculosis over Four Decades: Whole Genome Sequencing and Dating Analysis of Mycobacterium tuberculosis Isolates from KwaZulu-Natal

BackgroundThe continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents.ConclusionsIn the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic era. Subsequent accumulation of stepwise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts but also those that date back 50 y. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance.     

Phylogeography and transmission of M. tuberculosis in Moldova: A prospective genomic analysis

BackgroundThe incidence of multidrug-resistant tuberculosis (MDR-TB) remains critically high in countries of the former Soviet Union, where >20% of new cases and >50% of previously treated cases have resistance to rifampin and isoniazid. Transmission of resistant strains, as opposed to resistance selected through inadequate treatment of drug-susceptible tuberculosis (TB), is the main driver of incident MDR-TB in these countries.Methods and findingsWe conducted a prospective, genomic analysis of all culture-positive TB cases diagnosed in 2018 and 2019 in the Republic of Moldova. We used phylogenetic methods to identify putative transmission clusters; spatial and demographic data were analyzed to further describe local transmission of Mycobacterium tuberculosis. Of 2,236 participants, 779 (36%) had MDR-TB, of whom 386 (50%) had never been treated previously for TB. Moreover, 92% of multidrug-resistant M. tuberculosis strains belonged to putative transmission clusters. Phylogenetic reconstruction identified 3 large clades that were comprised nearly uniformly of MDR-TB: 2 of these clades were of Beijing lineage, and 1 of Ural lineage, and each had additional distinct clade-specific second-line drug resistance mutations and geographic distributions. Spatial and temporal proximity between pairs of cases within a cluster was associated with greater genomic similarity. Our study lasted for only 2 years, a relatively short duration compared with the natural history of TB, and, thus, the ability to infer the full extent of transmission is limited.ConclusionsThe MDR-TB epidemic in Moldova is associated with the local transmission of multiple M. tuberculosis strains, including distinct clades of highly drug-resistant M. tuberculosis with varying geographic distributions and drug resistance profiles. This study demonstrates the role of comprehensive genomic surveillance for understanding the transmission of M. tuberculosis and highlights the urgency of interventions to interrupt transmission of highly drug-resistant M. tuberculosis.

In a prospective genome surveillance study, Chongguang Yang and colleagues investigate the dynamics of multidrug-resistant tuberculosis transmission in Moldova.     

Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis

Genomic dissection of antibiotic resistance in bacterial pathogens has largely focused on genetic changes conferring growth above a single critical concentration of drug. However, reduced susceptibility to antibiotics—even below this breakpoint—is associated with poor treatment outcomes in the clinic, including in tuberculosis. Clinical strains of Mycobacterium tuberculosis exhibit extensive quantitative variation in antibiotic susceptibility but the genetic basis behind this spectrum of drug susceptibility remains ill-defined. Through a genome wide association study, we show that non-synonymous mutations in dnaA, which encodes an essential and highly conserved regulator of DNA replication, are associated with drug resistance in clinical M. tuberculosis strains. We demonstrate that these dnaA mutations specifically enhance M. tuberculosis survival during isoniazid treatment via reduced expression of katG, the activator of isoniazid. To identify DnaA interactors relevant to this phenotype, we perform the first genome-wide biochemical mapping of DnaA binding sites in mycobacteria which reveals a DnaA interaction site that is the target of recurrent mutation in clinical strains. Reconstructing clinically prevalent mutations in this DnaA interaction site reproduces the phenotypes of dnaA mutants, suggesting that clinical strains of M. tuberculosis have evolved mutations in a previously uncharacterized DnaA pathway that quantitatively increases resistance to the key first-line antibiotic isoniazid. Discovering genetic mechanisms that reduce drug susceptibility and support the evolution of high-level drug resistance will guide development of biomarkers capable of prospectively identifying patients at risk of treatment failure in the clinic.     

Pattern of Drug Resistance and Risk Factors Associated with Development of Drug Resistant Mycobacterium tuberculosis in Pakistan

Background

Drug resistant tuberculosis (DR-TB) is a major public health problem in developing countries such as Pakistan.

Objective

The current study was conducted to assess the frequency of drug resistant tuberculosis including multi drug resistance (MDR- TB) as well as risk factors for development of DR-TB, in Punjab, Pakistan.

Methodology

Drug susceptibility testing (DST) was performed, using proportion method, for 2367 culture positive Mycobacterium tuberculosis (MTB) cases that were enrolled from January 2012 to December 2013 in the province of Punjab, Pakistan, against first-line anti-tuberculosis drugs. The data was analyzed using statistical software; SPSS version 18.

Results

Out of 2367 isolates, 273 (11.5%) were resistant to at least one anti-TB drug, while 221 (9.3%) showed MDR- TB. Risk factors for development of MDR-TB were early age (ranges between 10–25 years) and previously treated TB patients.

Conclusion

DR-TB is a considerable problem in Pakistan. Major risk factors are previous history of TB treatment and younger age group. It emphasizes the need for effective TB control Program in the country.     

Microbiome-immune interactions in tuberculosis

Tuberculosis (TB) remains an infectious disease of global significance and a leading cause of death in low- and middle-income countries. Significant effort has been directed towards understanding Mycobacterium tuberculosis genomics, virulence, and pathophysiology within the framework of Koch postulates. More recently, the advent of “-omics” approaches has broadened our appreciation of how “commensal” microbes have coevolved with their host and have a central role in shaping health and susceptibility to disease. It is now clear that there is a diverse repertoire of interactions between the microbiota and host immune responses that can either sustain or disrupt homeostasis. In the context of the global efforts to combatting TB, such findings and knowledge have raised important questions: Does microbiome composition indicate or determine susceptibility or resistance to M. tuberculosis infection? Is the development of active disease or latent infection upon M. tuberculosis exposure influenced by the microbiome? Does microbiome composition influence TB therapy outcome and risk of reinfection with M. tuberculosis? Can the microbiome be actively managed to reduce risk of M. tuberculosis infection or recurrence of TB? Here, we explore these questions with a particular focus on microbiome-immune interactions that may affect TB susceptibility, manifestation and progression, the long-term implications of anti-TB therapy, as well as the potential of the host microbiome as target for clinical manipulation.     

Effect of Active Case Finding on Prevalence and Transmission of Pulmonary Tuberculosis in Dhaka Central Jail,Bangladesh

BackgroundUnderstanding tuberculosis (TB) transmission dynamics is essential for establishing effective TB control strategies in settings where the burden and risk of transmission are high. The objectives of this study were to evaluate the effect of active screening on controlling TB transmission and also to characterize Mycobacterium tuberculosis strains for investigating transmission dynamics in a correctional setting.MethodsThe study was carried out in Dhaka Central Jail (DCJ), from October 2005 to February 2010. An active case finding strategy for pulmonary TB was established both at the entry point to the prison and inside the prison. Three sputum specimens were collected from all pulmonary TB suspects and subjected to smear microscopy, culture, and drug susceptibility testing as well as genotyping which included deletion analysis, spoligotyping and analysis of mycobacterial interspersed repetitive units (MIRU).ResultsA total of 60,585 inmates were screened during the study period. We found 466 inmates with pulmonary TB of whom 357 (77%) had positive smear microscopy results and 109 (23%) had negative smear microscopy results but had positive results on culture. The number of pulmonary TB cases declined significantly, from 49 cases during the first quarter to 8 cases in the final quarter of the study period (p=0.001). Deletion analysis identified all isolates as M. tuberculosis and further identified 229 (70%) strains as ‘modern’ and 100 (30%) strains as ‘ancestral’. Analysis of MIRU showed that 347 strains (85%) exhibited unique patterns, whereas 61 strains (15%) clustered into 22 groups. The largest cluster comprised eight strains of the Beijing M. tuberculosis type. The rate of recent transmission was estimated to be 9.6%.ConclusionsImplementation of active screening for TB was associated with a decline in TB cases in DCJ. Implementation of active screening in prison settings might substantially reduce the national burden of TB in Bangladesh.     

Comparison of Gene Expression Profiles Between Pansensitive and Multidrug-Resistant Strains of Mycobacterium tuberculosis

Mycobacterium tuberculosis has developed resistance to anti-tuberculosis first-line drugs. Multidrug-resistant strains complicate the control of tuberculosis and have converted it into a worldwide public health problem. Mutational studies of target genes have tried to envisage the resistance in clinical isolates; however, detection of these mutations in some cases is not sufficient to identify drug resistance, suggesting that other mechanisms are involved. Therefore, the identification of new markers of susceptibility or resistance to first-line drugs could contribute (1) to specifically diagnose the type of M. tuberculosis strain and prescribe an appropriate therapy, and (2) to elucidate the mechanisms of resistance in multidrug-resistant strains. In order to identify specific genes related to resistance in M. tuberculosis, we compared the gene expression profiles between the pansensitive H37Rv strain and a clinical CIBIN:UMF:15:99 multidrug-resistant isolate using microarray analysis. Quantitative real-time PCR confirmed that in the clinical multidrug-resistant isolate, the esxG, esxH, rpsA, esxI, and rpmI genes were upregulated, while the lipF, groES, and narG genes were downregulated. The modified genes could be involved in the mechanisms of resistance to first-line drugs in M. tuberculosis and could contribute to increased efficiency in molecular diagnosis approaches of infections with drug-resistant strains.     

TLR2-Modulating Lipoproteins of the Mycobacterium tuberculosis Complex Enhance the HIV Infectivity of CD4+ T Cells

Co-infection with Mycobacterium tuberculosis accelerates progression from HIV to AIDS. Our previous studies showed that M. tuberculosis complex, unlike M. smegmatis, enhances TLR2-dependent susceptibility of CD4+ T cells to HIV. The M. tuberculosis complex produces multiple TLR2-stimulating lipoproteins, which are absent in M. smegmatis. M. tuberculosis production of mature lipoproteins and TLR2 stimulation is dependent on cleavage by lipoprotein signal peptidase A (LspA). In order to determine the role of potential TLR2-stimulating lipoproteins on mycobacterial-mediated HIV infectivity of CD4+ T cells, we generated M. smegmatis recombinant strains overexpressing genes encoding various M. bovis BCG lipoproteins, as well as a Mycobacterium bovis BCG strain deficient in LspA (ΔlspA). Exposure of human peripheral blood mononuclear cells (PBMC) to M. smegmatis strains overexpressing the BCG lipoproteins, LprF (p<0.01), LprH (p<0.05), LprI (p<0.05), LprP (p<0.001), LprQ (p<0.005), MPT83 (p<0.005), or PhoS1 (p<0.05), resulted in increased HIV infectivity of CD4+ T cells isolated from these PBMC. Conversely, infection of PBMC with ΔlspA reduced HIV infectivity of CD4+ T cells by 40% relative to BCG-infected cells (p<0.05). These results may have important implications for TB vaccination programs in areas with high mother-to-child HIV transmission.     

Molecular epidemiology of bovine tuberculosis in Northern Ghana identifies several uncharacterized bovine spoligotypes and suggests possible zoonotic transmission

ObjectiveWe conducted an abattoir-based cross-sectional study in the five administrative regions of Northern Ghana to determine the distribution of bovine tuberculosis (BTB) among slaughtered carcasses and identify the possibility of zoonotic transmission.MethodsDirect smear microscopy was done on 438 tuberculosis-like lesions from selected cattle organs and cultured on Lowenstein-Jensen media. Acid-fast bacilli (AFB) isolates were confirmed as members of the Mycobacterium tuberculosis complex (MTBC) by PCR amplification of IS6110 and rpoß. Characterization and assignment into MTBC lineage and sub-lineage were done by spoligotyping, with the aid of the SITVIT2, miruvntrplus and mbovis.org databases. Spoligotype data was compared to that of clinical M. bovis isolates from the same regions to identify similarities.ResultsA total of 319/438 (72.8%) lesion homogenates were smear positive out of which, 84.6% (270/319) had microscopic grade of at least 1+ for AFB. Two hundred and sixty-five samples (265/438; 60.5%) were culture positive, of which 212 (80.0%) were MTBC. Approximately 16.7% (34/203) of the isolates with correctly defined spoligotypes were negative for IS6110 PCR but were confirmed by rpoß. Spoligotyping characterized 203 isolates as M. bovis (198, 97.5%), M. caprae (3, 1.5%), M. tuberculosis (Mtbss) lineage (L) 4 Cameroon sub-lineage, (1, 0.5%), and M. africanum (Maf) L6 (1, 0.5%). A total of 53 unique spoligotype patterns were identified across the five administrative regions (33 and 28 were identified as orphan respectively by the SITVIT2 and mbovis.org databases), with the most dominant spoligotype being SIT1037/ SB0944 (77/203, 37.93%). Analysis of the bovine and human M. bovis isolates showed 75% (3/4) human M. bovis isolates sharing the same spoligotype pattern with the bovine isolates.ConclusionOur study identified that approximately 29% of M. bovis strains causing BTB in Northern Ghana are caused by uncharacterized spoligotypes. Our findings suggest possible zoonotic transmission and highlight the need for BTB disease control in Northern Ghana.     

Direct Detection by the Xpert MTB/RIF Assay and Characterization of Multi and Poly Drug-Resistant Tuberculosis in Guinea-Bissau,West Africa

BackgroundThis study aimed to evaluate the usefulness of the Xpert MTB/RIF assay for the rapid direct detection of M. tuberculosis complex (MTBC) strains and rifampicin resistance associated mutations in a resource-limited setting such as Guinea-Bissau and its implications in the management of tuberculosis (TB) and drug resistant tuberculosis, complementing the scarce information on resistance and genotypic diversity of MTBC strains in this West African country.ConclusionsThe Xpert MTB/RIF assay was reliable for the detection of rifampicin resistant MTBC strains directly from sputum samples of patients undergoing first-line treatment for two months, being more trustworthy than the simple presence of acid-fast bacilli in the smear. Its implementation is technically simple, does not require specialized laboratory infrastructures and is suitable for resource-limited settings when a regular source of electricity and maintenance is available as well as financial and operation sustainability is guaranteed by the health authorities. A high prevalence of MDR-TB among patients at risk of MDR-TB after two months of first-line treatment was found, in support of the WHO recommendations for its use in the management of this risk group.     

Transmission pattern of drug-resistant tuberculosis and its implication for tuberculosis control in eastern rural China

Objective

Transmission patterns of drug-resistant Mycobacterium tuberculosis (MTB) may be influenced by differences in socio-demographics, local tuberculosis (TB) endemicity and efficaciousness of TB control programs. This study aimed to investigate the impact of DOTS on the transmission of drug-resistant TB in eastern rural China.

Methods

We conducted a cross-sectional study of all patients diagnosed with drug-resistant TB over a one-year period in two rural Chinese counties with varying lengths of DOTS implementation. Counties included Deqing, with over 11 years'' DOTS implementation and Guanyun, where DOTS was introduced 1 year prior to start of this study. We combined demographic, clinical and epidemiologic information with IS6110-based restricted fragment length polymorphism (RFLP) and Spoligotyping analysis of MTB isolates. In addition, we conducted DNA sequencing of resistance determining regions to first-line anti-tuberculosis agents.

Results

Of the 223 drug-resistant isolates, 73(32.7%) isolates were identified with clustered IS6110RFLP patterns. The clustering proportion among total drug-resistant TB was higher in Guanyun than Deqing (26/101.vs.47/122; p,0.04), but not significantly different among the 53 multidrug-resistant isolates (10/18.vs.24/35; p,0.35). Patients with cavitary had increased risk of clustering in both counties. In Guanyun, patients with positive smear test or previous treatment history had a higher clustering proportion. Beijing genotype and isolates resistant to isoniazid and/or rifampicin were more likely to be clustered. Of the 73 patients with clustered drug-resistant isolates, 71.2% lived in the same or neighboring villages. Epidemiological link (household and social contact) was confirmed in 12.3% of the clustered isolates.

Conclusion

Transmission of drug-resistant TB in eastern rural China is characterized by small clusters and limited geographic spread. Our observations highlight the need for supplementing DOTS with additional strategies, including active case finding at the village level, effective treatment for patients with cavities and drug susceptibility testing for patients at increased risk for drug-resistance.     

Epidemiology and Clinical Characteristics of Pediatric Drug-Resistant Tuberculosis in Chongqing,China

To gain insight into the epidemiology of childhood drug resistant tuberculosis (DR-TB) in China that has the second largest burden of TB and the largest number of multidrug resistant (MDR) TB cases in the world, we performed the cross-sectional study to investigate drug resistance of four first-line anti-TB drugs (isoniazid, rifampicin, streptomycin and ethambutol) using Mycobacterium tuberculosis isolates from 196 culture-confirmed pediatric TB cases diagnosed in the Children’s Hospital of Chongqing Medical University, China during 2008–2013. Univariate and multivariate logistic regression analyses were performed to assess the associations between patient demographic and clinical characteristics and DR-and MDR-TB, respectively. Twenty-eight percent (56/196) of the study patients exhibited resistance to at least one of the four first-line anti-TB drugs tested. MDR was found in 4.6% (9/196) of the study patients. More than half (5/9, 55.6%) of the MDR cases were from a single county of Chongqing. A significant association was found between being acid-fast bacilli-smear negative and DR-TB (adjusted OR, 2.33; 95% CI, 1.13–4.80) and between having concurrent thoracic-extrathoracic involvement and MDR-TB (adjusted OR, 9.49; 95% CI, 1.05–85.92), respectively. The findings of this study indicate that the rate of DR is high among pediatric TB patients in Chongqing and suggest an urgent need for studies to identify MDR transmission hotspots in Chongqing, thereby contributing to the control DR- and MDR-TB epidemics in China. The study also generates new insight into the pathogenesis of DR and MDR M. tuberculosis strains and highlights the importance of studying childhood TB to the goal of global TB control.     

Multidrug-resistant and extensively drug-resistant tuberculosis in multi-ethnic region, Xinjiang Uygur Autonomous Region, China

Background

The multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has emerged as a global threat. Xinjiang is a multi-ethnic region and suffered second highest incidence of TB in China. However, epidemiological information on MDR and XDR TB is scarcely investigated.

Methodology/Principal Findings

A prospective study was conducted to analyze the prevalence of MDR and XDR TB and the differences of drug resistance TB between Chinese Han and other nationalities population at Chest Hospital of Xinjiang Uygur Autonomous Region, China. We performed in vitro drug susceptibility testing of Mycobacterium tuberculosis to first- and second-line anti-tuberculosis drugs for all 1893 culture confirmed positive TB cases that were diagnosed between June 2009 and June 2011. Totally 1117 (59.0%, 95% CI, 56.8%–61.2%) clinical isolates were resistant to ≥1 first-line drugs; the prevalence of MDR TB was 13.2% (95% CI, 11.7%–14.7%), of which, 77 (30.8%; 95% CI, 25.0%–36.6%) and 31 (12.8%; 95% CI, 8.6%–17.0%) isolates were pre-XDR and XDR TB respectively. Among the MDR/XDR TB, Chinese Han patients were significantly less likely to be younger with an odds ratio 0.42 for age 20–29 years and 0.52 for age 40–49 years; P _trend = 0.004), and Chinese Han patients has a lower prevalence of XDR TB (9.6%) than all the other nationality (14.9%).

Conclusions/Significance

The burden of drug resistance TB cases is sizeable, which highlights an urgent need to reinforce the control, detection and treatment strategies for drug resistance TB. However, the difference of MDR and XDR TB between Chinese Han and other nationalities was not observed.     

Tuberculosis caused by Mycobacterium africanum: Knowns and unknowns

Tuberculosis (TB), one of the deadliest threats to human health, is mainly caused by 2 highly related and human-adapted bacteria broadly known as Mycobacterium tuberculosis and Mycobacterium africanum. Whereas M. tuberculosis is widely spread, M. africanum is restricted to West Africa, where it remains a significant cause of tuberculosis. Although several differences have been identified between these 2 pathogens, M. africanum remains a lot less studied than M. tuberculosis. Here, we discuss the genetic, phenotypic, and clinical similarities and differences between strains of M. tuberculosis and M. africanum. We also discuss our current knowledge on the immune response to M. africanum and how it possibly articulates with distinct disease progression and with the geographical restriction attributed to this pathogen. Understanding the functional impact of the diversity existing in TB-causing bacteria, as well as incorporating this diversity in TB research, will contribute to the development of better, more specific approaches to tackle TB.     

Prevalence of bovine tuberculosis and characterization of the members of the Mycobacterium tuberculosis complex from slaughtered cattle in Rwanda

BackgroundBovine tuberculosis (bTB) is an endemic disease in Rwanda, but little is known about its prevalence and causative mycobacterial species. The disease causes tremendous losses in livestock and wildlife and remains a significant threat to public health.Materials and methodsA cross-sectional study employing a systematic random sampling of cattle (n = 300) with the collection of retropharyngeal lymph nodes and tonsils (n = 300) irrespective of granulomatous lesions was carried out in six abattoirs to investigate the prevalence and identify mycobacterial species using culture, acid-fast bacteria staining, polymerase chain reaction, and GeneXpert assay. Individual risk factors and the origin of samples were analysed for association with the prevalence.FindingsOf the 300 sample pools, six were collected with visible TB-like lesions. Our findings demonstrated the presence of Mycobacterium tuberculosis complex (MTBC) in 1.7% (5/300) of sampled slaughtered cattle. Mycobacterium bovis was isolated from 1.3% (4/300) animals while one case was caused by a rifampicin-resistant (RR) M. tuberculosis. Non-tuberculous mycobacteria were identified in 12.0% (36/300) of the sampled cattle. There were no significant associations between the prevalence and abattoir category, age, sex, and breeds of slaughtered cattle.ConclusionsThis study is the first in Rwanda to isolate both M. bovis and RR M. tuberculosis in slaughtered cattle indicating that bTB is present in Rwanda with a low prevalence. The isolation of RR M. tuberculosis from cattle indicates possible zooanthroponotic transmission of M. tuberculosis and close human-cattle contact. To protect humans against occupational zoonotic diseases, it is essential to control bTB in cattle and raise the awareness among all occupational groups as well as reinforce biosafety at the farm level and in the abattoirs.