Mycobacterium Tuberculosis Lineages Research Articles

Effects of Mycobacterium tuberculosis lineages and regions of difference (RD) virulence gene variation on tuberculosis recurrence.

BackgroundChina ranks second in the incidence of tuberculosis (TB), and the virulence and infectivity of Mycobacterium tuberculosis (M.tb) in different lineages are different. The variation of virulence genes in the M.tb regions of difference (RD) may be the reason for differences in pathogenicity. Studying the relationship between virulence gene mutations in the RD region of clinical strains of M.tb and TB relapse can provide basic data for the study of TB prevention and control.MethodsA total of 155 M.tb clinical strains were collected in Kashgar Prefecture. Whole-genome sequencing (WGS) was conducted, and mutations in virulence genes in the M.tb RD region were analyzed. The maximum likelihood method was implemented using IQ-TREE software. Logistic regression was used to analyze the relationship between lineage, RD region virulence gene variation, and patient relapse.ResultsThe 155 strains of M.tb in Kashgar Prefecture belong to 3 M.tb lineages: L2 (45.80%), L3 (32.90%), and L4 (21.30%). In relapsed patients, L2 (70.83%, 17/24) was significantly higher than the other lineages (29.17%, 7/24; P<0.05). Relapse was significantly correlated with L2 [odds ratio (OR) =3.505; 95% confidence interval (CI): 1.341–9.158; P=0.011]. In the virulence genes of the RD region, g.4357804 (T→G, OR =4.278; 95% CI: 1.594–11.481; P=0.004), g.4359653 (C→T, OR =3.356; 95% CI: 1.303–8.644; P=0.012), and g.2627618 (C→A, OR =2.676; 95% CI: 1.101–6.502; P=0.030) mutations were significantly associated with patient relapse. The mutation frequencies of g.4357804, g.4359653, and g.2627618 in L2 were significantly higher than those in the non-L2 group (P<0.05).ConclusionsPatients infected with L2 are more prone to relapse, and RD region virulence gene variation is an important factor for the strong pathogenicity and easy relapse after infection associated with L2.

Effects of Increasing Concentrations of Rifampicin on Different Mycobacterium tuberculosis Lineages in a Whole-Blood Bactericidal Activity Assay.

High-dose rifampicin improved bactericidal activity and culture conversion in early-phase tuberculosis (TB) trials, done mainly in Africa. We performed a whole-blood bactericidal activity (WBA) study to determine whether the effects of high-dose rifampicin differ across globally relevant TB strains and whether effects are similar in dormant bacilli that will be required for enhancing cure. Whole blood from healthy volunteers was spiked with rifampicin (range, 0.63 to 60 mg/L) and incubated with one of four Mycobacterium tuberculosis clinical strains (Haarlem, Latin American-Mediterranean [LAM], East African-Indian [EAI], and Beijing lineages) or a dormant strain (streptomycin-starved 18b [ss18b]). Change in bacterial CFU was estimated after inoculation of WBA cultures in MGIT. WBA increased with higher concentrations of rifampicin in all strains. At rifampicin concentrations up to 5 mg/L, the rates of increase in WBA per unit increase in rifampicin concentration were similar in all 4 clinical strains (P > 0.51). Above 5 mg/L, EAI (P < 0.001) and Beijing (P = 0.007) strains showed greater increases in WBA than did LAM; Haarlem was similar to LAM. The dormant strain showed a lower rate of increase in WBA than clinical strains at rifampicin concentrations up to 5 mg/L; above 5 mg/L, the rate of increase was similar to those in the LAM, Beijing, and Haarlem strains. Increasing rifampicin concentration enhanced WBA in all strains; the greatest effects were seen in strains common in Asia, suggesting that early-phase trial findings may be generalizable beyond Africa. Similar effects of high concentrations of rifampicin on the dormant strain support the concept that this intervention may enhance sterilizing activity.

Revised nomenclature and SNP barcode for Mycobacterium tuberculosis lineage 2.

Mycobacterium tuberculosis (Mtb) lineage 2 (L2) strains are present globally, contributing to a widespread tuberculosis (TB) burden, particularly in Asia where both prevalence of TB and numbers of drug resistant TB are highest. The increasing availability of whole-genome sequencing (WGS) data worldwide provides an opportunity to improve our understanding of the global genetic diversity of Mtb L2 and its association with the disease epidemiology and pathogenesis. However, existing L2 sublineage classification schemes leave >20 % of the Modern Beijing isolates unclassified. Here, we present a revised SNP-based classification scheme of L2 in a genomic framework based on phylogenetic analysis of >4000 L2 isolates from 34 countries in Asia, Eastern Europe, Oceania and Africa. Our scheme consists of over 30 genotypes, many of which have not been described before. In particular, we propose six main genotypes of Modern Beijing strains, denoted L2.2.M1–L2.2.M6. We also provide SNP markers for genotyping L2 strains from WGS data. This fine-scale genotyping scheme, which can classify >98 % of the studied isolates, serves as a basis for more effective monitoring and reporting of transmission and outbreaks, as well as improving genotype-phenotype associations such as disease severity and drug resistance. This article contains data hosted by Microreact.

Interaction between M. tuberculosis Lineage and Human Genetic Variants Reveals Novel Pathway Associations with Severity of TB.

Tuberculosis (TB) remains a major public health threat globally, especially in sub-Saharan Africa. Both human and Mycobacterium tuberculosis (MTBC) genetic variation affect TB outcomes, but few studies have examined if and how the two genomes interact to affect disease. We hypothesize that long-term coexistence between human genomes and MTBC lineages modulates disease to affect its severity. We examined this hypothesis in our TB household contact study in Kampala, Uganda, in which we identified three MTBC lineages, of which one, L4.6-Uganda, is clearly derived and hence recent. We quantified TB severity using the Bandim TBscore and examined the interaction between MTBC lineage and human single-nucleotide polymorphisms (SNPs) genome-wide, in two independent cohorts of TB cases (n = 149 and n = 127). We found a significant interaction between an SNP in PPIAP2 and the Uganda lineage (combined p = 4 × 10−8). PPIAP2 is a pseudogene that is highly expressed in immune cells. Pathway and eQTL analyses indicated potential roles between coevolving SNPs and cellular replication and metabolism as well as platelet aggregation and coagulation. This finding provides further evidence that host–pathogen interactions affect clinical presentation differently than host and pathogen genetic variation independently, and that human–MTBC coevolution is likely to explain patterns of disease severity.

Mycobacterial Lineages Associated with Drug Resistance in Patients with Extrapulmonary Tuberculosis in Addis Ababa, Ethiopia.

Background In Ethiopia, tuberculosis (TB) is one of the most common causes of illness and death. However, there is limited information available on lineages associated with drug resistance among extrapulmonary tuberculosis patients in Ethiopia. In this study, researchers looked into Mycobacterium tuberculosis lineages linked to drug resistance in patients with extrapulmonary tuberculosis in Addis Ababa, Ethiopia. Methods On 151 Mycobacterium tuberculosis isolates, a cross-sectional analysis was performed. Spoligotyping was used to characterize mycobacterial lineages, while a phenotypic drug susceptibility test was performed to determine the drug resistance pattern. Data were analyzed using SPSS version 23. Results Among 151 Mycobacterium tuberculosis complex (MTBC) genotyped isolates, four lineages (L1–L4), and Mycobacterium bovis were identified. The predominantly identified lineage was Euro-American (73.5%) followed by East-African-Indian (19.2%). Any drug resistance (RR) and multidrug-resistant (MDR) tuberculosis was identified among 16.2% and 7.2% of the Euro-American lineage, respectively, while it was 30.8% and 15.4% among the East-African-Indian lineages. Among all three preextensively drug-resistance (pre-XDR) cases identified, two isolates belong to T3-ETH, and the other one strain was not defined by the database. There was no statistically significant association between any type of drug resistance and either lineage or sublineages of Mycobacterium tuberculosis. Conclusion A higher proportion of any type of drug resistance and MDR was detected among the East-African-Indian lineage compared to others. However, there was no statistically significant association between any type of drug resistance and either lineages or sublineages. Thus, the authors recommend a large-scale study.

BCG re-vaccination in Malawi: 30-year follow-up of a large, randomised, double-blind, placebo-controlled trial

SummaryBackgroundA large, double-blind, randomised, placebo-controlled trial of repeat BCG found 49% efficacy against leprosy but no protection against tuberculosis after 6–9 years’ follow-up in 1995. We report here additional follow-up, which resulted in greatly increased tuberculosis case numbers, and allowed subgroup analysis.MethodsNearly 47 000 individuals of all ages living in northern Malawi with a BCG vaccine scar were randomly assigned (1:1) between 1986 and 1989 to receive a second BCG or placebo. The investigators and project staff remained masked to all interventions. Enhanced passive surveillance ensured ascertainment of tuberculosis and leprosy to the end of 2018. Tuberculosis case definitions included rigorous microbiological or histological confirmation. Prespecified subgroup analyses were by tuberculosis type, age at vaccination, time since vaccination, previous tuberculin reactivity, HIV status and Mycobacterium tuberculosis lineage. The original trial is registered with ISRCTN registry, ISRCTN11311670.FindingsIn follow-up until Dec 31, 2018, 824 participants had developed tuberculosis, including 786 with pulmonary disease, of whom 383 (63%) of 607 with known HIV status were HIV positive. There was no effect of a second BCG overall (odds ratio [OR] 0·92; 95% CI 0·80–1·05), or for pulmonary (0·93; 0·81–1·07), or lymph node tuberculosis (0·60; 0·31–1·17). The OR was lower for those with known HIV-negative tuberculosis (0·77; 0·59–1·00), for those vaccinated as children (aged <5 years, 0·74; 0·41–1·35; aged 5–14 years, 0·77; 0·60–0·99), and for cases arising at least 20 years after vaccination (0·79; 0·63–1·01). There were no differences by tuberculin status at vaccination, or lineage. There was no evidence of protection against leprosy beyond 10 years after vaccination (although there have been only nine diagnostically certain cases since 1995).InterpretationThere was no evidence that repeat BCG vaccination provides appreciable protection against overall tuberculosis in this rural African population with a high prevalence of HIV. Subgroup effects should not be overinterpreted given the multiple analyses done. However, the evidence for modest protection against HIV-negative tuberculosis, and for a delayed benefit in those vaccinated as children, is consistent with other observations in the literature.FundingLEPRA, Wellcome Trust, Bill & Melinda Gates Foundation.

CXCL17 Is Dispensable during Hypervirulent Mycobacterium tuberculosis HN878 Infection in Mice.

CXCL17 is a novel mucosal chemokine that mediates myeloid cell recruitment and bactericidal activity and highly expressed in the respiratory tract. However, its role in tuberculosis (TB) immunopathogenesis or protection remains unknown. In this study, we evaluated the function of CXCL17 in a mouse model of aerosol infection with the clinical W-Beijing lineage Mycobacterium tuberculosis hypervirulent HN878 strain. Our results show that CXCL17 production increases in the lung of M. tuberculosis–infected mice during acute and chronic stages of infection. Moreover, in vitro M. tuberculosis infection of epithelial cells and myeloid cells induces production of CXCL17. In humans, lower serum CXCL17 levels are observed among active pulmonary TB patients when compared with subjects with latent TB infection and healthy controls, suggesting a protective role. However, mice treated with rCXCL17 show similar lung bacterial burden and inflammation compared with control animals, despite an increased lung myeloid cell accumulation. Finally, CXCL17−/− mice are not more susceptible to TB than wild-type animals. These findings suggest that CXCL17 is induced in both murine epithelial and myeloid cells upon M. tuberculosis infection and increased expression during human latent TB infection. However, CXCL17 may have a dispensable role during pulmonary TB.

Genetic composition and evolution of the prevalent Mycobacterium tuberculosis lineages 2 and 4 in the Chinese and Zhejiang Province populations

BackgroundThere are seven human-adaptation lineages of Mycobacterium tuberculosis (Mtb). Tuberculosis (TB) dissemination is strongly influenced by human movements and host genetics. The detailed lineage distribution evolution of Mtb in Zhejiang Province is unknown. We aim to determine how different sub-lineages are transmitted and distributed within China and Zhejiang Province.MethodsWe analysed whole-genome sequencing data for a worldwide collection of 1154 isolates and a provincial collection of 1296 isolates, constructed the best-scoring maximum likelihood phylogenetic tree. Bayesian evolutionary analysis was used to calculate the latest common ancestor of lineages 2 and 4. The antigenic diversity of human T cell epitopes was evaluated by calculating the pairwise dN/dS ratios.ResultsOf the Zhejiang isolates, 964 (74.38%) belonged to lineage 2 and 332 (25.62%) belonged to lineage 4. The distributions of the sub-lineages varied across the geographic regions of Zhejiang Province. L2.2 is the most ancient sub-lineage in Zhejiang, first appearing approximately 6897 years ago (95% highest posterior density interval (HDI): 6513–7298). L4.4 is the most modern sub-lineage, first appearing approximately 2217 years ago (95% HDI: 1864–2581). The dN/dS ratios showed that the epitope and non-epitope regions of lineage 2 strains were significantly (P < 0.001) more conserved than those of lineage 4.ConclusionsAn increase in the frequency of lineage 4 may reflect its successful transmission over the last 20 years. The recent common ancestors of the sub-lineages and their transmission routes are relevant to the entry of humans into China and Zhejiang Province. Diversity in T cell epitopes may prevent Mycobacterium tuberculosis from being recognized by the immune system.

Epidemiology of Mycobacterium tuberculosis lineages and strain clustering within urban and peri-urban settings in Ethiopia.

Previous work has shown differential predominance of certain Mycobacterium tuberculosis (M. tb) lineages and sub-lineages among different human populations in diverse geographic regions of Ethiopia. Nevertheless, how strain diversity is evolving under the ongoing rapid socio-economic and environmental changes is poorly understood. The present study investigated factors associated with M. tb lineage predominance and rate of strain clustering within urban and peri-urban settings in Ethiopia. Pulmonary Tuberculosis (PTB) and Cervical tuberculous lymphadenitis (TBLN) patients who visited selected health facilities were recruited in the years of 2016 and 2017. A total of 258 M. tb isolates identified from 163 sputa and 95 fine-needle aspirates (FNA) were characterized by spoligotyping and compared with international M.tb spoligotyping patterns registered at the SITVIT2 databases. The molecular data were linked with clinical and demographic data of the patients for further statistical analysis. From a total of 258 M. tb isolates, 84 distinct spoligotype patterns that included 58 known Shared International Type (SIT) patterns and 26 new or orphan patterns were identified. The majority of strains belonged to two major M. tb lineages, L3 (35.7%) and L4 (61.6%). The observed high percentage of isolates with shared patterns (n = 200/258) suggested a substantial rate of overall clustering (77.5%). After adjusting for the effect of geographical variations, clustering rate was significantly lower among individuals co-infected with HIV and other concomitant chronic disease. Compared to L4, the adjusted odds ratio and 95% confidence interval (AOR; 95% CI) indicated that infections with L3 M. tb strains were more likely to be associated with TBLN [3.47 (1.45, 8.29)] and TB-HIV co-infection [2.84 (1.61, 5.55)]. Despite the observed difference in strain diversity and geographical distribution of M. tb lineages, compared to earlier studies in Ethiopia, the overall rate of strain clustering suggests higher transmission and warrant more detailed investigations into the molecular epidemiology of TB and related factors.

Distribution of Mycobacterium tuberculosis lineages in South America

Molecular genotyping of Mycobacterium tuberculosis allows for the identification of circulating lineages and sublineages in the population and their relationship with migratory movements. The purpose of this review is to describe the phylogeography of Mycobacterium tuberculosis reported in South American countries that was analyzed using genotyping tools, analyze the Tuberculosis hotspots for the region and determine the impact of the COVID-19 pandemic on the Tuberculosis control program. The Latin American Mediterranean (LAM) sublineage belonging to the Euro-American lineage (Lineage 4) presents the highest prevalence in South America and is followed by the Beijing sublineage belonging to the East Asian lineage (Lineage 2). The Beijing sublineage is considered of worldwide interest because of its association with multidrug-resistant tuberculosis (MDR-TB), which is almost entirely distributed in South America, with Peru being the country with the highest prevalence for this sublineage. On the other hand, the Indo-Oceanic (Lineage 1), India-East Asia (Lineage 3) and West- African 2 (Lineage 6) sublineages have been reported with lower prevalence in South America. The molecular techniques used in the genotyping studies for Mycobacterium tuberculosis in South America were as follows: typing by complementary oligonucleotide spacer sequences (Spoligotyping), restriction-hybridization patterns (IS6110-RFLP, PGRS-RFLP), mycobacterial interspaced repeat units-variable number tandem repeats (MIRU-VNTR) and whole genome sequencing (WGS). At present, Brazil and Peru are the hotspots for tuberculosis and MDR-TB in South America, where the control of tuberculosis wholly affected by the COVID-19 pandemic. Thus, there have been significant impacts on containment programs and possible post-pandemic scenarios such that scientific contributions will need to be evaluated and implemented with new strategies for prevention, diagnosis, treatment and control of Tuberculosis.

Phenotypic and genotypic features of the Mycobacterium tuberculosis lineage 1 subgroup in central Vietnam

Mycobacterium tuberculosis (Mtb) has different features depending on different geographic areas. We collected Mtb strains from patients with smear-positive pulmonary tuberculosis in Da Nang, central Vietnam. Using a whole genome sequencing platform, including genome assembly complemented by long-read-sequencing data, genomic characteristics were studied. Of 181 Mtb isolates, predominant Vietnamese EAI4_VNM and EAI4-like spoligotypes (31.5%), ZERO strains (5.0%), and part of EAI5 (11.1%) were included in a lineage-1 (L1) sublineage, i.e., L1.1.1.1. These strains were found less often in younger people, and they genetically clustered less frequently than other modern strains. Patients infected with ZERO strains demonstrated less lung infiltration. A region in RD2bcg spanning six loci, i.e., PE_PGRS35, cfp21, Rv1985c, Rv1986, Rv1987, and erm(37), was deleted in EAI4_VNM, EAI4-like, and ZERO strains, whereas another 118 bp deletion in furA was specific only to ZERO strains. L1.1.1.1-sublineage-specific deletions in PE_PGRS4 and PE_PGRS22 were also identified. RD900, seen in ancestral lineages, was present in majority of the L1 members. All strains without IS6110 (5.0%) had the ZERO spoligo-pattern. Distinctive features of the ancestral L1 strains provide a basis for investigation of the modern versus ancestral Mtb lineages and allow consideration of countermeasures against this heterogeneous pathogen.

Characterisation of secretome-based immune responses of human leukocytes infected with various Mycobacterium tuberculosis lineages.

BackgroundDifferences in immune responses against different lineages of Mycobacterium tuberculosis (Mtb), and by different types of immune cell, are still poorly understood. We aimed to compare the secretome-based immune responses among three Mtb lineages and among immune-cell types. The immune responses were also investigated during infection and when the bacilli had been eliminated from the immune cells.MethodsHuman primary leukocytes were infected with strains representing three lineages of Mtb (East-Asian, Indo-Oceanic and Euro-American). Label-free GeLC MS/MS proteomic analysis of secretomes was performed. The response of each immune-cell type was compared with the appropriate interactome database for each.ResultsThe expression pattern of proteins secreted by Mtb-infected leukocytes differed among Mtb lineages. The ancestral lineage (IO lineage) had a greater ability to activate MMP14 (associated with leukocyte migration) than did the more recent lineages (EA and EuA). During infection, proteins secreted by macrophages, dendritic cells, neutrophils and B-cells were associated with cell proliferation. Following clearance of Mtb, proteins associated with interferon signaling were found in macrophages, dendritic cells and neutrophils: proteins associated with antigen processing were found in B-cells and regulatory T-cells. Expression of immune response-related proteins from many immune-cell types might be suppressed by Mtb infection. Our study has provided a better insight into the host-pathogen interaction and immune response against different Mtb lineages.

Era of TB elimination: Growing need to understand diversities of Mycobacterium tuberculosis lineages!

IntroductionThe mycobacterium tuberculosis complex (MTBC) has highly clonal population structure which made the organism spread globally mirroring human migration out of Africa and resulted in the formation of seven lineages. We conducted this study to determine the proportion of spoligotype lineages and drug susceptibility profile of Mycobacterium tuberculosis isolates among smear positive TB patients attending a tertiary care hospital in Mysore, Karnataka, India. MethodsIt is a descriptive study conducted at JSS Hospital a tertiary care centre at Mysore, India during 2018–19. The sputum smear positive samples were subjected to solid culture and drug susceptibility testing and spoligotyping for identification of lineages. ResultsOf the 100 samples which were culture positive, 94 isolates were clustered into five spoligotype international types with SIT-126 (EAI-5) being the largest cluster of 46 (46%) isolates, followed by SIT-62 (H1) with 24 (24%), SIT -26 (CAS 1-DELHI) with 20 (20%), SIT-53 (T1) with 03 (3%) and SIT-482 (BOV-1) with 01 (1%). Among the remaining six isolates, two had unique Cameroon spoligotypes and four were orphans ConclusionThe study finding reveals that a diverse pattern of genotypes is circulating in the region of which EAI-5, Harleem (H1) and CAS-DELHI pattern forms the majority (88%). It is evident that there is a wide range of MTB genetic lineages in circulation and further research is needed to understand the diversity across the country.

Estimation of the global burden of Mycobacterium tuberculosis lineage 1.

Tuberculosis is still problematic as it affects large numbers of people globally. Mycobacterium tuberculosis Lineage 1 (L1) or Indo Oceanic Lineage, one of widespread major lineages, has a specific geographic distribution and high mortality. It is highly diverse and endemic in several high burden countries. However, studies on the global burden of L1 and its sublineages remain limited. This may lead to the underestimation of the importance of its variance in developing and applying tuberculosis control measures. This study aimed to estimate the number of patients infected with M. tuberculosis L1 and its sublineages worldwide. The proportion of L1 among tuberculosis patients was searched in published reports from countries around the world and the number of patients was calculated based on a WHO report on country incidences and populations. The numbers of patients infected with the five major sublineages, namely L1.1.1, L1.1.2, L1.1.3, L1.2.1, and L1.2.2 were estimated where information was available. It was found that L1 accounted for 28% of global tuberculosis cases in 2012 and 2018. Over 80% of the L1 global burden was in India, the Philippines, Indonesia and Bangladesh, which are also among the countries with highest absolute numbers of tuberculosis patients in the world. Globally, the estimated number of patients infected with M. tuberculosis L1.2.1 and L1.1.2 was over 1.1 million and of patients infected with L1.1.1 was about 200,000. This study demonstrated that L1 contributes significantly to the global burden of tuberculosis. To achieve the End TB Strategy, more attention needs to be paid to the responses of M. tuberculosis L1 to various control measures.

Dysglycemia is associated with Mycobacterium tuberculosis lineages in tuberculosis patients of North Lima-Peru.

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.

Robust barcoding and identification of Mycobacterium tuberculosis lineages for epidemiological and clinical studies

BackgroundTuberculosis, caused by bacteria in the Mycobacterium tuberculosis complex (MTBC), is a major global public health burden. Strain-specific genomic diversity in the known lineages of MTBC is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of drug resistance. Fast and accurate tracking of MTBC strains is therefore crucial for infection control, and our previous work developed a 62-single nucleotide polymorphism (SNP) barcode to inform on the phylogenetic identity of 7 human lineages and 64 sub-lineages.MethodsTo update this barcode, we analysed whole genome sequencing data from 35,298 MTBC isolates (~ 1 million SNPs) covering 9 main lineages and 3 similar animal-related species (M. tuberculosis var. bovis, M. tuberculosis var. caprae and M. tuberculosis var. orygis). The data was partitioned into training (N = 17,903, 50.7%) and test (N = 17,395, 49.3%) sets and were analysed using an integrated phylogenetic tree and population differentiation (FST) statistical approach.ResultsBy constructing a phylogenetic tree on the training MTBC isolates, we characterised 90 lineages or sub-lineages or species, of which 30 are new, and identified 421 robust barcoding mutations, of which a minimal set of 90 was selected that included 20 markers from the 62-SNP barcode. The barcoding SNPs (90 and 421) discriminated perfectly the 86 MTBC isolate (sub-)lineages in the test set and could accurately reconstruct the clades across the combined 35k samples.ConclusionsThe validated 90 SNPs can be used for the rapid diagnosis and tracking of MTBC strains to assist public health surveillance and control. To facilitate this, the SNP markers have now been incorporated into the TB-Profiler informatics platform (https://github.com/jodyphelan/TBProfiler).

Higher genome mutation rates of Beijing lineage of Mycobacterium tuberculosis during human infection

Mycobacterium tuberculosis (Mtb) strains of Beijing lineage have caused great concern because of their rapid emergence of drug resistance and worldwide spread. DNA mutation rates that reflect evolutional adaptation to host responses and the appearance of drug resistance have not been elucidated in human-infected Beijing strains. We tracked and obtained an original Mtb isolate of Beijing lineage from the 1999 tuberculosis outbreak in Japan, as well as five other isolates that spread in humans, and two isolates from the patient caused recurrence. Three isolates were from patients who developed TB within one year after infection (rapid-progressor, RP), and the other three isolates were from those who developed TB more than one year after infection (slow-progressor, SP). We sequenced genomes of these isolates and analyzed the propensity and rate of genomic mutations. Generation time versus mutation rate curves were significantly higher for RP. The ratio of oxidative versus non-oxidation damages induced mutations was higher in SP than RP, suggesting that persistent Mtb are exposed to oxidative stress in the latent state. Our data thus demonstrates that higher mutation rates of Mtb Beijing strains during human infection is likely to account for the higher adaptability and an emergence ratio of drug resistance.

Geno-Spatial Distribution of Mycobacterium Tuberculosis and Drug Resistance Profiles in Myanmar-Thai Border Area.

Worldwide, studies investigating the relationship between the lineage of Mycobacterium tuberculosis (MTB) across geographic areas has empowered the “End TB” program and understand transmission across national boundaries. Genomic diversity of MTB varies with geographical locations and ethnicity. Genomic diversity can also affect the emergence of drug resistance. In Myanmar, we still have limited genetic information about geographical, ethnicity, and drug resistance linkage to MTB genetic information. This study aimed to describe the geno-spatial distribution of MTB and drug resistance profiles in Myanmar–Thailand border areas. A cross-sectional study was conducted with a total of 109 sequenced isolates. The lineages of MTB and the potential associated socio-demographic, geographic and clinical factors were analyzed using Fisher’s exact tests. p value of statistically significance was set at < 0.05. We found that 67% of the isolates were lineage 1 (L1)/East-African-Indian (EAI) (n = 73), followed by lineage 2 (L2)/Beijing (n = 26), lineage 4 (L4)/European American (n = 6) and lineage 3 (L3)/Delhi/Central Asian (n = 4). “Gender”, “type of TB patient”, “sputum smear grading” and “streptomycin resistance” were significantly different with the lineages of MTB. Sublineages of L1, which had never been reported elsewhere in Myanmar, were detected in this study area. Moreover, both ethnicity and lineage of MTB significantly differed in distribution by patient location. Diversity of the lineage of MTB and detection of new sublineages suggested that this small area had been resided by a heterogeneous population group who actively transmitted the disease. This information on distribution of lineage of MTB can be linked in the future with those on the other side of the border to evaluate cross-border transmission.

Comparison of hspX gene sequence in the Beijing and non-Beijing Mycobacterium tuberculosis

PurposeThe pathogenicity of various lineages of Mycobacterium tuberculosis (MTB) is different. This could be due to the difference in survival ability within the host macrophage. The alpha crystalline secretion protein, a product of the hspx gene, is one of the bacterial protection factors in these stressful situations. The Beijing family, part of the East Asian lineage, was reported to be more virulent. Regarding the importance of this protein in pathogenicity, this study was conducted to investigate the polymorphism of the hspx gene in Beijing family compared to non- Beijing strains. MethodDNA of 50 MTB isolates were extracted by boiling method. The existence of hspx gene was determined using PCR-specific primer and finally PCR product was sequenced to examine the polymorphism in both direct and reverse directions. Sequencing results were aligned by chromas software. ResultsThe hspx gene was detected in all of the Beijing and non-Beijing isolates. The polymorphism in the sequences of this gene were not observed in all of the MTB isolates. DiscussionThis study indicated that hspx gene is protected. Also it has showed that lineage type was not related to the sequence of hspX gene, but the expression of this protein may be different, which requires further studies.

Phylogeny magnitude of Mycobacterium tuberculosis based on genomic analysis

Mycobacterium tuberculosis (MTB) is mostly found in humans, and it can cause more than two million deaths each year with increasing morbidity. Although lineages of MTB show identical nucleotide relationships, they have different characteristics such as evolution, transmission, drug resistance, host interaction, latency, and vaccine effectiveness. It is necessary to have better understanding of MTB relationships based on similarities in genome sizes and phylogenetic analysis. This paper observes the relationships of MTB based on nucleotide through phylogenetic frameworks. The MTB species consist of six lineages, and each lineage has various size of genomes . This difference contributes to virulence of MTB affecting levels of severity, morbidity, and mortality of diseases. Genetic diversity of MTB can contribute to global threats in the world such as outbreak of tuberculosis, Multi Drug Resistant (MDR) and Extensively Drug Resistant (XDR) tuberculosis.