SNP-IT Tool for Identifying Subspecies and Associated Lineages of Mycobacterium tuberculosis Complex.
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ABSTRACT:
SUBMITTER: Lipworth S
PROVIDER: S-EPMC6390766 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.
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Project description:Uganda is among the 22 countries in the world with a high burden of tuberculosis. The southwestern region of the country has consistently registered a high TB/HIV incidence rate. This study is aimed at characterizing the Mycobacterium tuberculosis complex (MTBC) genotypic diversity in southwestern Uganda. A total of 283 sputum samples from patients with pulmonary tuberculosis were genotyped using specific single nucleotide polymorphism markers for lineages 3 and 4. Most of the patients were males with a mean age of 34. The lineage 4 Ugandan family was found to be the most dominant strains accounting for 59.7% of all cases followed by lineage 3 at 15.2%. The lineage 4 non-Ugandan family accounted for 14.5% of all cases while 4.2% showed amplification for both lineage 4 and lineage 3. Eighteen samples (6.4%) of the strains remained unclassified since they could not be matched to any lineage based on the genotyping technique used. This study demonstrates that a wide diversity of strains is causing pulmonary tuberculosis in this region with those belonging to the lineage 4 Ugandan family being more predominant. However, to confirm this, further studies using more discriminative genotyping methods are necessary.
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Project description:Strain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ~92 k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ~7 k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineages than current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type.
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Project description:The polymerase chain reaction (PCR)-based detection of Mycobacterium tuberculosis (M. tuberculosis) complex (MTC) in clinical samples is a first-line approach by which to diagnose tuberculosis in clinical microbiology laboratories. In this study, the genome-wide profiling of 3,156 mycobacterial genomes using Roary determined the CRISPR-csm4 gene as specific for MTB. Real time (RT)-PCR and the PCR-sequencing of CRISPR-csm4, tested on a collection of 20 MTC and 5 nontuberculous mycobacteria, confirmed the 20 MTC isolates, whereas the 5 nontuberculous isolates were not detected. Further, 65 of the leftover clinical samples, including 25 GeneXpert-positive and 40 GeneXpert-negative samples, that were used to evaluate the CRISPR-csm4-MTB assay in the clinical microbiology laboratory setting yielded expected results in every case, further allowing for the identification of the M. tuberculosis Beijing lineage. RT-PCR and the PCR-sequencing of CRISPR-csm4 could be implanted in the clinical microbiology laboratory to complement the currently used assays, with the potential of increasing the specification of the MTC pathogens responsible for tuberculosis. IMPORTANCE The whole-genome sequence comparison of the Mycobacterium tuberculosis complex (MTC) genomic sequences that are available in the NCBI database identified a unique, specific gene to be used directly on clinical diagnostic samples to detect MTC against all species of mycobacteria and to differentiate between MTC species, lineages, and sublineages.
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Project description:Mycobacterium tuberculosis complex, Mycobacterium avium, and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial (n = 38) and nonmycobacterial (n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species (n = 57), archival clinical samples (n = 233), and strains isolated from various hosts (n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis. When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.
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Project description:This study was performed to investigate the role of dysglycemia on the genetic diversity of Mycobacterium tuberculosis (MTB) among pulmonary tuberculosis (TB) patients to build scientific evidence about the possible mechanisms of TB transmission. MTB isolates obtained of patients affected by pulmonary tuberculosis from health care facilities of North Lima-Peru, were analyzed using whole genome sequencing and 24-locus mycobacterial interspersed repetitive-unit -variable-number tandem repeats (MIRU-VNTR). Subsequently, clinical and epidemiological characteristics were associated with clustering, lineages and comorbid conditions. The analysis carried out 112 pulmonary TB patients from various health centers in North Lima, 17 (15%) had diabetes mellitus (DM) and 33 (29%) had pre-diabetes (PDM). Latin American-Mediterranean, Haarlem and Beijing were the most frequent MTB lineages found in those patients. Previous TB (adjusted odds ratio [aOR] = 3.65; 95%CI: 1.32-17.81), age (aOR = 1.12; 95%CI: 1.03-1.45) and Beijing lineage (aOR = 3.53; 95%CI: 1.08-13.2) were associated with TB-DM comorbidity. Alcoholism (aOR = 2.92; 95%CI: 1.10-8.28), age (aOR = 1.05; 95%CI: 1.03-1.12) and Haarlem lineage (aOR = 2.54; 95%CI: 1.04-6.51) were associated with TB-PDM comorbidity. Beijing and Haarlem lineages were independently associated with TB-DM and TB-PDM comorbidities, respectively. Although these findings may be surprising, we must be cautious to suggest that dysglycemia could be associated with a highly clustering and predisposition of MTB lineages related to a serious impact on the severity of TB disease, which requires further research.
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Project description:In this study we assessed first-line anti-tuberculosis drug resistance and the genotypic distribution of Mycobacterium tuberculosis complex (MTBC) isolates that had been collected from consecutive new tuberculosis patients enrolled in two clinical trials conducted in Guinea between 2005 and 2010. Among the total 359 MTBC strains that were analyzed in this study, 22.8% were resistant to at least one of the first line anti-tuberculosis drugs, including 2.5% multidrug resistance and 17.5% isoniazid resistance, with or without other drugs. In addition, further characterization of isolates from a subset of the two trials (n = 184) revealed a total of 80 different spoligotype patterns, 29 "orphan" and 51 shared patterns. We identified the six major MTBC lineages of human relevance, with predominance of the Euro-American lineage. In total, 132 (71.7%) of the strains were genotypically clustered, and further analysis (using the DESTUS model) suggesting significantly faster spread of LAM10_CAM family (p = 0.00016). In conclusion, our findings provide a first insight into drug resistance and the population structure of the MTBC in Guinea, with relevance for public health scientists in tuberculosis control programs.
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Project description:Bovine tuberculosis (bTB), traditionally associated with Mycobacterium bovis, presents significant public health and economic challenges worldwide. This study investigated the causative agents of bTB in slaughtered cattle and buffalo in Lahore, Pakistan. Of the 3,581 animals screened, 34 were identified with gross TB-like lesions. The lesions were processed for culture, PCR, and Sanger sequencing to identify the causative agents of the disease. The results identified 10 Mycobacterium orygis and 8 Mycobacterium tuberculosis sensu stricto isolates. Whole-genome sequencing was performed on two M. orygis isolates, and the sequences were phylogenetically compared to 93 publicly available M. orygis sequences. The results also demonstrated that the JB21 and JB22 primers, which have been previously commonly applied to detect M. bovis in Pakistan, are unable to distinguish between M. tuberculosis complex subspecies. The identification of M. orygis and M. tuberculosis as causative agents of bTB in this slaughterhouse in Punjab may have important implications in identifying cases of zoonotic TB in humans and applying appropriate molecular tools to identify the prevalence of the disease. The data from this study align with recent findings suggesting M. orygis is the predominant cause of bTB in South Asia.IMPORTANCEThe study findings hold significant relevance to the Journal of Clinical Microbiology, as they directly impact the field. The first-time identification of Mycobacterium orygis and Mycobacterium tuberculosis as the predominant causative agents of bovine tuberculosis in Lahore, Pakistan underscores the urgent need for enhanced diagnostic methods. The study emphasizes the importance of improved assays for the accurate detection and differentiation of Mycobacterium subspecies. Additionally, the research addresses zoonotic risk assessment and public health implications, advocating for a multidisciplinary approach that integrates clinical microbiology with veterinary and human health sectors. These insights contribute to clinical microbiology knowledge, shaping effective strategies for disease prevention, surveillance, and control. The study's potential to advance the field makes it well suited for publication in the Microbiology Spectrum journal.
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Project description:The development of whole-genome sequencing technologies is gradually leading to a more detailed description of the population structure of the Mycobacterium tuberculosis complex (MTBC). In this study, we correlated previously published classifications on a collection of more than 10,000 genomes and proposed a new, comprehensive nomenclature that unifies the existing ones. In total, we identified 169 lineages and sublineages of M. tuberculosis/M. africanum and 9 animal-adapted species. For the purpose of organizing these genotypes in a more streamlined manner, we stratified them into five hierarchical levels. To represent the classification and compare it with the reference, we compiled a confirmatory data set of 670 high-quality isolates, which includes all genotypes and species of MTBC, and this confirmatory data set can serve as a basis for further studies. We proposed a set of 213 robust barcoding single-nucleotide polymorphisms and a suitable workflow for reliable differentiation of genotypes and species within the complex. This work integrates the results of all the major systematized studies to date to provide an understanding of the global diversity of the MTBC population structure. The results of this work may ultimately help to reliably determine the pathogen genotype and associate it with traits that reflect its prevalence, virulence, vaccination, and treatment efficiency, as well as to reliably find natural features revealed during its spread. IMPORTANCE Through years of research into the Mycobacterium tuberculosis complex (MTBC), a number of ambiguous phylogenetic classifications have emerged, which often overlap with one another. In the present study, we have combined all major studies on MTBC classification and inferred a unified, most complete to date classification and accompanying SNP barcodes.
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