Mycobacterium Tuberculosis Cluster Research Articles

Sustained transmission over two decades of a previously unrecognised MPT64 negative Mycobacterium tuberculosis strain in Queensland, Australia: a whole genome sequencing study

MPT64 is a key protein used for Mycobacterium tuberculosis (MTB) complex strain identification. We describe protracted transmission of an MPT64 negative MTB strain in Queensland, Australia, and explore genomic factors related to its successful spread. All MPT64 negative strains identified between 2002 and 2022 by the Queensland Mycobacteria Reference Laboratory, and an additional 2 isolates from New South Wales (NSW), were whole genome sequenced. Bayesian modelling and phylogeographical analyses were used to assess their evolutionary history and transmission dynamics. Protein structural modelling to understand the putative functional effects of the mutated gene coding for MPT64 protein was performed. Forty-three MPT64 negative isolates were sequenced, belonging to a single MTB cluster of Lineage 4.1.1.1 strains. Combined with a UK dataset of the same lineage, molecular dating estimated 1990 (95% HPD 1987-1993) as the likely time of strain introduction into Australia. Although the strain has spread over a wide geographic area and new cases linked to the cluster continue to arise, phylodynamic analysis suggest the outbreak peaked around 2003. All MPT64 negative strains had a frame shift mutation (delAT, p.Val216fs) within the MPT64 gene, which confers two major structural rearrangements at the C-terminus of the protein. This study uncovered the origins of an MPT64 negative MTB outbreak in Australia, providing a richer understanding of its biology and transmission dynamics, as well as guidance for clinical diagnosis and public health action. The potential spread of MPT64 negative strains undermines the diagnostic utility of the MPT64 immunochromatographic test. This study was funded from an operational budget provided to the Queensland Mycobacterium Reference Laboratory by Pathology Queensland, Queensland Department of Health.

Characteristic SNPs defining the major multidrug-resistant Mycobacterium tuberculosis clusters identified by EuSeqMyTB to support routine surveillance, EU/EEA, 2017 to 2019.

BackgroundThe EUSeqMyTB project, conducted in 2020, used whole genome sequencing (WGS) for surveillance of drug-resistant Mycobacterium tuberculosis in the European Union/European Economic Area (EU/EEA) and identified 56 internationally clustered multidrug-resistant (MDR) tuberculosis (TB) clones.AimWe aimed to define and establish a rapid and computationally simple screening method to identify probable members of the main cross-border MDR-TB clusters in WGS data to facilitate their identification and track their future spread.MethodsWe screened 34 of the larger cross-border clusters identified in the EuSeqMyTB pilot study (2017-19) for characteristic single nucleotide polymorphism (SNP) signatures that could identify and define members of each cluster. We also linked this analysis with published clusters identified in previous studies and identified more distant genetic relationships between some of the current clusters.ResultsA panel of 30 characteristic SNPs is presented that can be used as an initial (routine) screen for members of each cluster. For four of the clusters, no unique defining SNP could be identified; three of these are closely related (within approximately 20 SNPs) to one or more other clusters and likely represent a single established MDR-TB clade composed of multiple recent subclusters derived from the previously described ECDC0002 cluster.ConclusionThe identified SNP signatures can be integrated into routine pipelines and contribute to the more effective monitoring, rapid and widespread screening for TB. This SNP panel will also support accurate communication between laboratories about previously identified internationally transmitted MDR-TB genotypes.

Erratum for Acosta et al., "Insights into the Complexity of a Dormant Mycobacterium tuberculosis Cluster Once Transmission Is Resumed".

Erratum for Acosta et al., "Insights into the Complexity of a Dormant Mycobacterium tuberculosis Cluster Once Transmission Is Resumed".

Insights into the Complexity of a Dormant Mycobacterium tuberculosis Cluster Once Transmission Is Resumed.

ABSTRACTGenotyping tools help identify the complexity in Mycobacterium tuberculosis transmission clusters. We carried out a thorough analysis of the epidemiological and bacteriological complexity of a cluster in Almería, Spain. The cluster, initially associated with Moroccan migrants and with no secondary cases identified in 4 years, then reappeared in Spanish-born individuals. In one case, two Mycobacterium tuberculosis clonal variants were identified. We reanalyzed the cluster, supported by the characterization of multiple cultured isolates and respiratory specimens, whole-genome sequencing, and epidemiological case interviews. Our findings showed that the cluster, which was initially thought to have restarted activity with just a single case harboring a small degree of within-host diversity, was in fact currently growing due to coincidental reactivation of past exposures, with clonal diversity transmitted throughout the cluster. In one case, within-host diversity was amplified, probably due to prolonged diagnostic delay.IMPORTANCE The precise study of the dynamics of tuberculosis transmission in socio-epidemiologically complex scenarios may require more thorough analysis than the standard molecular epidemiology strategies. Our study illustrates the epidemiological and bacteriological complexity present in a transmission cluster in a challenging epidemiological setting with a high proportion of migrant cases. The combination of whole-genome sequencing, refined and refocused epidemiological interviews, and in-depth analysis of the bacterial composition of sputa and cultured isolates was crucial in order to correctly reinterpret the true nature of this cluster. Our global approach allowed us to reinterpret correctly the unnoticed epidemiological and bacteriological complexity involved in the Mycobacterium tuberculosis transmission event under study, which had been overlooked by the usual molecular epidemiology approaches.

An optimized genomic VCF workflow for precise identification of Mycobacterium tuberculosis cluster from cross-platform whole genome sequencing data

Whole-genome sequencing (WGS) data allow for an inference of Mycobacterium tuberculosis (Mtb) clusters by using a pairwise genetic distance of ≤12 single nucleotide polymorphisms (SNPs) as a threshold. However, a problem of discrepancies in numbers of SNPs and genetic distance measurement is a great concern when combining WGS data from different next generation sequencing (NGS) platforms. We performed SNP variant calling on WGS data of 9 multidrug-resistant (MDR-TB), 3 extensively drug-resistant tuberculosis (XDR-TB) and a standard M. tuberculosis strain H37Rv from an Illumina/NextSeq500 and an Ion Torrent PGM. Variant calls were obtained using four different common variant calling tools, including Genome Analysis Toolkit (GATK) HaplotypeCaller (GATK-VCF workflow), GATK HaplotypeCaller and GenotypeGVCFs (GATK-GVCF workflow), SAMtools, and VarScan 2. Cross-platform pairwise SNP differences, minimum spanning networks and average nucleotide identity (ANI) were analysed to measure performance of the variant calling tools. Minimum pairwise SNP differences ranged from 2 to 14 SNPs when using GVCF workflow while maximum pairwise SNP differences ranged from 7 to 158 SNPs when using VarScan 2. ANI comparison between SNPs data from NextSeq500 and PGM of MDR-TB and XDR-TB showed maximum ANI of 99.7% and 99.0%, respectively, with GVCF workflow while the other SNP calling results showed lower ANI in a range of 98.6% to 95.1%. In this study, we suggest that the GVCF workflow showed the best performing variant caller to avoid cross-platform pairwise SNP differences.

The role of entry-screening procedures in the identification of multidrug-resistant Mycobacterium tuberculosis cluster cases amongst patients arriving in Europe from the horn of Africa, 2016-17

The role of entry-screening procedures in the identification of multidrug-resistant Mycobacterium tuberculosis cluster cases amongst patients arriving in Europe from the horn of Africa, 2016-17

The influence of mutation gene rpoB of Mycobacterium tuberculosis cluster I (507-534) on the elimination 25-desacetyl rifampicin in urine of tuberculosis subjects

The study aimed to evaluate the influence of mutation gene rpoB of Mycobacterium (M.) tuberculosis cluster I (507-534) on the elimination of 25-Desacetyl Rifampicin (25-DR) in the urine of tuberculosis (TB) subjects. Early morning sputum took from patient TB before treatment. Urine collected after 2 hours taken Fixed-Dose Combination (FDC) at days 7th treatment. All sputum were sequencing at Macrogen Korea Laboratory. Urine was analyzed by high-Performance Liquid Chromatography (HPLC) using the method of Lily et al. Mean (standard deviation) for mutation and non-mutation of rpoB M. tuberculosis group were 7.6147 (4.4478) and 4.5772 (1.7532) μg/ml, respectively. Shapiro-Wilk test showed normally distributed data, with significance 0.3. Independent t-test performed p-value 0.167 and confidence interval (CI) from -1.648 to 7.723. The mutation gene rpoB of M. tuberculosis cluster, I (507-534) in this study, did not affect elimination 25-DR in theurine of TB subjects statistically and clinically.

Evaluation of 24-locus MIRU-VNTR genotyping in Mycobacterium tuberculosis cluster investigations in four jurisdictions in the United States, 2006–2010

The U.S. Centers for Disease Control and Prevention (CDC) uses a combination of spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) analyses as part of the National TB Genotyping Service (NTGS). The NTGS expansion from 12-locus MIRU-VNTR (MIRU12) to 24-locus MIRU-VNTR (MIRU24) in 2009 enhanced the ability to discriminate Mycobacterium tuberculosis strains. In the current study, we investigated the MIRU24 concordance among epidemiologic-linked tuberculosis (TB) patients in four U.S. health jurisdictions. We also evaluated the programmatic benefits of combining MIRU24 and spoligotyping with epidemiologic evidence in identifying potential recent TB transmission. We examined 342 TB patients in 42 spoligotype/MIRU12 (PCRType) clusters (equivalent to 46 spoligotype/MIRU24 [GENType] clusters) to identify epidemiologic links among cases. GENType clusters, when compared to PCRType clusters, had 12 times higher odds of epidemiologic links being identified if patients were younger than 25 years and 3 times higher odds if patients resided in the same zip code, or had HIV infection. Sixty (18%) fewer PCRType-clustered patients would need investigations if clusters are defined using GENType instead of PCRType. An important advantage of defining clusters by MIRU24 is resource savings related to the reduced number of clustered cases needing investigation.

Whole Genome Sequencing Demonstrates Limited Transmission within Identified Mycobacterium tuberculosis Clusters in New South Wales, Australia.

Australia has a low tuberculosis incidence rate with most cases occurring among recent immigrants. Given suboptimal cluster resolution achieved with 24-locus mycobacterium interspersed repetitive unit (MIRU-24) genotyping, the added value of whole genome sequencing was explored. MIRU-24 profiles of all Mycobacterium tuberculosis culture-confirmed tuberculosis cases diagnosed between 2009 and 2013 in New South Wales (NSW), Australia, were examined and clusters identified. The relatedness of cases within the largest MIRU-24 clusters was assessed using whole genome sequencing and phylogenetic analyses. Of 1841 culture-confirmed TB cases, 91.9% (1692/1841) had complete demographic and genotyping data. East-African Indian (474; 28.0%) and Beijing (470; 27.8%) lineage strains predominated. The overall rate of MIRU-24 clustering was 20.1% (340/1692) and was highest among Beijing lineage strains (35.7%; 168/470). One Beijing and three East-African Indian (EAI) clonal complexes were responsible for the majority of observed clusters. Whole genome sequencing of the 4 largest clusters (30 isolates) demonstrated diverse single nucleotide polymorphisms (SNPs) within identified clusters. All sequenced EAI strains and 70% of Beijing lineage strains clustered by MIRU-24 typing demonstrated distinct SNP profiles. The superior resolution provided by whole genome sequencing demonstrated limited M. tuberculosis transmission within NSW, even within identified MIRU-24 clusters. Routine whole genome sequencing could provide valuable public health guidance in low burden settings.

Identifying Likely Transmission Pathways within a 10-Year Community Outbreak of Tuberculosis by High-Depth Whole Genome Sequencing.

BackgroundImproved tuberculosis control and the need to contain the spread of drug-resistant strains provide a strong rationale for exploring tuberculosis transmission dynamics at the population level. Whole-genome sequencing provides optimal strain resolution, facilitating detailed mapping of potential transmission pathways.MethodsWe sequenced 22 isolates from a Mycobacterium tuberculosis cluster in New South Wales, Australia, identified during routine 24-locus mycobacterial interspersed repetitive unit typing. Following high-depth paired-end sequencing using the Illumina HiSeq 2000 platform, two independent pipelines were employed for analysis, both employing read mapping onto reference genomes as well as de novo assembly, to control biases in variant detection. In addition to single-nucleotide polymorphisms, the analyses also sought to identify insertions, deletions and structural variants.ResultsIsolates were highly similar, with a distance of 13 variants between the most distant members of the cluster. The most sensitive analysis classified the 22 isolates into 18 groups. Four of the isolates did not appear to share a recent common ancestor with the largest clade; another four isolates had an uncertain ancestral relationship with the largest clade.ConclusionWhole genome sequencing, with analysis of single-nucleotide polymorphisms, insertions, deletions, structural variants and subpopulations, enabled the highest possible level of discrimination between cluster members, clarifying likely transmission pathways and exposing the complexity of strain origin. The analysis provides a basis for targeted public health intervention and enhanced classification of future isolates linked to the cluster.

Molecular Epidemiology of Mycobacterium Tuberculosis Strains in the North-West and West of Iran.

Background:Identifying Mycobacterium tuberculosis (MTB) transmission type is a key step in the control of this disease.Aim:This study aimed to determine the path and transmission type of MTB and the insertion sequence IS6110 band number and verify their relationship to demographic and clinical risk factors.Subjects and Methods:In this cross-sectional study, 64 MTB patients from three border provinces of Iran were selected after full clinical history and physical evaluation design. The drug susceptibility testing was carried out using the standard proportion technique on sputum samples. Isolates tested with restriction fragment length polymorphism technique used IS6110.Results:Recent transmission of disease was 33/50 (66%) based on clustering rate. The IS6110 band number had a significant relationship with drug resistance detected in proportion method tested by univariate linear regression (P < 0.01). Furthermore, the IS6110 band number had association with Bacillus Calmette–Guérin vaccination history (P = 0.02), sex (P < 0.01), and purified protein derivative (PPD) reaction size (P < 0.01) tested by multiple analysis. The risk of recent transmission inferred from the clustering rate was significantly higher in patients from Western provinces compared to those from the North-West province (P = 0.048). However, age (P = 0.39), gender (P = 0.16), vaccination history (P = 0.57), drug susceptibility, and PPD (P < 0.6) were independent of clustering. The largest cluster of up to six subjects was found in the Western provinces.Conclusion:Recent MTB transmission was much more common in the West compared to the North-West of Iran. Large MTB clusters with strong epidemiological links may be reflective of a disease outbreak. Correlation noted between the IS6110 band number and vaccination history; PPD size and female gender necessitates further studies.

Evaluation of large genotypic Mycobacterium tuberculosis clusters: contributions from remote and recent transmission

Tuberculosis genotypic clustering is used as a proxy for recent transmission. The association between clustering and recent transmission becomes problematic when the genotyping method lacks specificity in defining a cluster, as well as for clusters with extensive jurisdictional histories and/or common genotypes.We investigated the four largest spoligotype/12 loci MIRU-VNTR-defined clusters in Harris County, Texas from 2006–2012 to determine their historical contribution to tuberculosis morbidity, estimate the contributions from recent and remote transmission, and determine the impact of secondary genotyping on cluster definition. The clusters contained 189, 64, 51 and 38 cases. Each cluster was linked to cluster(s) previously identified by Houston Tuberculosis Initiative; 3 since 1995 and the fourth in 2002. Among cases for which timing of Mycobacterium tuberculosis transmission relative to tuberculosis disease could be ascertained, nearly equal proportions were associated with recent and remote transmission. The extent to which genotyping with an additional 12 MIRU-VNTR loci modified the cluster definition varied from little or no impact for the two smaller clusters to moderate impact for the larger clusters.Tuberculosis control measures to reduce morbidity associated with large clusters must involve strategies to identify and treat individuals who recently acquired infection, as well as persons infected for years.

Dynamic and complex Mycobacterium tuberculosis microevolution unrevealed by standard genotyping

We performed an in-depth analysis of a large Mycobacterium tuberculosis cluster involving nine isolates with identical or highly similar RFLP types. The strain involved in this transmission cluster microevolved and accumulated up to five differential IS6110 transposition events, several of them not revealed by standard genotyping approaches. Some of these events probably generated extended deletions. In two cases of tuberculosis within the cluster, we observed the simultaneous presence of both the reference and microevolved variants. A longitudinal genotypical survey of M. tuberculosis in a real clinical evolutionary scenario can reveal dynamic and complex microevolution events.

Conspicuous multidrug-resistant Mycobacterium tuberculosis cluster strains do not trespass country borders in Latin America and Spain

Multidrug-resistant Mycobacterium tuberculosis strain diversity in Ibero-America was examined by comparing extant genotype collections in national or state tuberculosis networks. To this end, genotypes from over 1000 patients with multidrug-resistant tuberculosis diagnosed from 2004 through 2008 in Argentina, Brazil, Chile, Colombia, Venezuela and Spain were compared in a database constructed ad hoc. Most of the 116 clusters identified by IS6110 restriction fragment length polymorphism were small and restricted to individual countries. The three largest clusters, of 116, 49 and 25 patients, were found in Argentina and corresponded to previously documented locally-epidemic strains. Only 13 small clusters involved more than one country, altogether accounting for 41 patients, of whom 13 were, in turn, immigrants from Latin American countries different from those participating in the study (Peru, Ecuador and Bolivia). Most of these international clusters belonged either to the emerging RDRio LAM lineage or to the Haarlem family of M. tuberculosis and four were further split by country when analyzed with spoligotyping and rifampin resistance-conferring mutations, suggesting that they did not represent ongoing transnational transmission events. The Beijing genotype accounted for 1.3% and 10.2% of patients with multidrug-resistant tuberculosis in Latin America and Spain, respectively, including one international cluster of two cases. In brief, Euro-American genotypes were widely predominant among multidrug-resistant M. tuberculosis strains in Ibero-America, reflecting closely their predominance in the general M. tuberculosis population in the region, and no evidence was found of acknowledged outbreak strains trespassing country borders.

Use of cluster-graphs from spoligotyping data to study genotype similarities and a comparison of three indices to quantify recent tuberculosis transmission among culture positive cases in French Guiana during a eight year period

BackgroundFrench Guiana has the highest tuberculosis (TB) burden among all French departments, with a strong increase in the TB incidence over the last few years. It is now uncertain how best to explain this incidence. The objective of this study was to compare three different methods evaluating the extent of recent TB transmission in French Guiana.MethodsWe conducted a population-based molecular epidemiology study of tuberculosis in French Guiana based on culture-positive TB strains (1996 to 2003, n = 344) to define molecular relatedness between isolates, i.e. potential transmission events. Phylogenetic relationships were inferred by comparing two methods: a "cluster-graph" method based on spoligotyping results, and a minimum spanning tree method based on both spoligotyping and variable number of tandem DNA repeats (VNTR). Furthermore, three indices attempting to reflect the extent of recent TB transmission (RTIn, RTIn-1 and TMI) were compared.ResultsMolecular analyses showed a total amount of 120 different spoligotyping patterns and 273 clinical isolates (79.4%) that were grouped in 49 clusters. The comparison of spoligotypes from French Guiana with an international spoligotype database (SpolDB4) showed that the majority of isolates belonged to major clades of M. tuberculosis (Haarlem, 22.6%; Latin American-Mediterranean, 23.3%; and T, 32.6%). Indices designed to quantify transmission of tuberculosis gave the following values: RTIn = 0.794, RTIn-1 = 0.651, and TMI = 0.146.ConclusionOur data showed a high number of Mycobacterium tuberculosis clusters, suggesting a high level of recent TB transmission, nonetheless an estimation of transmission rate taking into account cluster size and mutation rate of genetic markers showed a low ongoing transmission rate (14.6%). Our results indicate an endemic mode of TB transmission in French Guiana, with both resurgence of old spatially restricted genotypes, and a significant importation of new TB genotypes by migration of TB infected persons from neighgouring high-incidence countries.

Which Patients' Factors Predict the Rate of Growth of Mycobacterium tuberculosis Clusters in an Urban Community?

Factors influencing tuberculosis cluster growth are poorly understood. The authors examined clusters of two or more culture-confirmed Mycobacterium tuberculosis cases between January 1, 2001, and December 31, 2003, that had insertion sequence 6110 (IS6110) restriction fragment length polymorphism and spoligotype patterns identical to those of another study case. Genotypes first seen in New York, New York, before or during 1993 were considered historical; recent strains were those first seen after 1993. The authors examined the effect of the combined characteristics of infectiousness of the first two cases in a cluster on the rate of cluster growth. Genotyping was performed for 2,408 (91.8%) of the 2,623 tuberculosis cases diagnosed; 873 cases were in 212 clusters. Thirty-one clusters had historical strains, 153 were recent, and 28 were of unknown period. Patients' infectiousness was not associated with the rate of cluster growth among historical strain clusters. Among recent strain clusters, infectiousness of both of the initial cases was associated with a higher rate of cluster growth compared with clusters in which neither initial case was infectious, upon adjustment for male sex (rate ratio = 2.62, 95% confidence interval: 1.19, 5.78). The rate of genotype cluster growth should be monitored regardless of how long the strain has been present in the community. However, infectiousness in the first two cases may be useful to prioritize genotype cluster investigations.

Identification and evolution of an IS6110 low-copy-number Mycobacterium tuberculosis cluster.

A cohort of 56 patients infected with related strains of Mycobacterium tuberculosis, the S75 group, was identified in a New Jersey population-based study of all isolates with a low number of copies of the insertion element IS6110. Genotyping was combined with surveillance data to identify the S75 group and to elucidate its recent evolution. The S75 group had similar demographic and geographic characteristics. Seventeen persons (30%) were linked epidemiologically. The S75 group was segregated from other low-copy-number isolates on the basis of several independent molecular methods. This group included 3 IS6110 genotype variants: BE, H6, and C28, containing 1, 2, and 3 IS6110 insertions, respectively. IS6110 insertion site mapping and comparative sequence analysis strongly suggest a stepwise acquisition of IS6110 elements from BE to H6 to C28. S75 represents a locally produced strain cluster that has recently evolved. The combination of multiple molecular tools with traditional epidemiology provides novel insights into dissemination, local transmission, and evolution of M. tuberculosis.