The first insight into Mycobacterium tuberculosis complex isolates in the lower northern region in Thailand.

BACKGROUND: Tuberculosis (TB) remains an important infectious disease and different genotypes have been reported. This study aimed to investigate the genetic diversity and molecular epidemiology of TB in the lower northern region of Thailand, where genotyping data are limited. METHODS: A total of 159 Mycobacterium tuberculosis complex (MTBC) isolates from this region were genotyped by spoligotyping and the major spoligotypes were further subdivided by the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method. RESULTS: Spoligotyping identified 34 types and classified them into 14 clusters. East African-Indian (EAI) groups were the most frequent (44.7%), followed by Beijing (36.5%), with a higher prevalence of drug resistance. By 15-loci MIRU-VNTR typing, the major groups of the Beijing and EAI2_NTB were further differentiated into 44 and 21 subtypes forming 9 and 5 subclusters with cluster rates of 0.26 and 0.44, respectively. The Hunter-Gaston Discriminatory Index among the Beijing and EAI2_NTB groups were 0.987 and 0.931, respectively, indicating high diversity. CONCLUSIONS: This is the first look at the MTBC genotypes in the lower northern region of Thailand, which could aid in understanding the distribution and potential spread of MTBC and Mycobacterium bovis in the target region to support TB control in Thailand.

The Eradication of M. Caprae Tuberculosis in Wild Boar (Sus Scrofa) in the Bieszczady Mountains, Southern Poland - An Administrative Perspective.

Introduction: Animal tuberculosis (TB) is a zoonotic disease caused by acid-fast bacteria belonging to the Mycobacterium tuberculosis complex (MTBC). Both animals and humans are susceptible to infection by the MTBC. Interspecies transmission is also possible, including to livestock and humans. In the years 1997-2013, many tuberculosis cases were recorded in European bison in the Bieszczady Mountains; more alarmingly, TB was also recorded in wild boar in the years 2013-2020. Material and Methods: In the years 2013-2020, 104 wild boar from the Bieszczady Mountains were tested for TB through necropsy, mycobacterial culture, strain identification and spoligotyping. Results: The microbiological examination confirmed TB in 46 wild boar; these infections were identified as M. caprae, spoligotype SB2391. Conclusion: Free-living European bison are at risk of TB infection from wild boar carrying M. caprae. This situation also poses a risk to local cattle. There is a need for further activities aimed at monitoring the disease, preventing further transmission, and minimising the risk to public health.

Mycobacterium bovis in Egyptian mongoose, Spain.

Tuberculosis caused by Mycobacterium bovis and other related mycobacteria has been reported in a wide range of mammals worldwide. In the case of the Herpestidae family, Mycobacterium mungi and M. bovis, both belonging to the Mycobacterium tuberculosis Complex, have been reported in banded mongooses (Mungos mungo) in Africa and in Egyptian mongooses (Herpestes ichneumon) in Portugal, respectively. Thus, we hypothesized that Tuberculosis may occur in Egyptian mongooses from Spain. Twenty-five found dead Egyptian mongooses were necropsied in order to detect macroscopic TB-compatible lesions and mandibular lymph nodes and lungs were cultured onto mycobacteria-specific growth media. We isolated M. bovis in 3/25 Egyptian mongooses (12.00%, IC95: 4.17-29.96%) and identified spoligotypes SB0121 (2/3) and SB0134 (1). No macroscopic TB-compatible lesions were observed. To the best of our knowledge, this is the first report of M. bovis in Egyptian mongoose in Spain, as well as the only study that includes spolygotyping in this species. Although the absence of visible lesions suggests a minor role of the Egyptian mongoose in Tuberculosis epidemiology, further research thereon is encouraged.

The Emergence of Novel Spoligotypes of Highly Drug-Resistant Mycobacterium tuberculosis Isolates in Fujian, China.

Background: Here, we aimed to determine the population structure of Mycobacterium tuberculosis (MTB) genotypes in Fujian and explore risk factors associated with infection with the Beijing genotype. We also explored the association between Beijing genotype and drug resistance. Methods: Representative MTB isolates obtained during provincial-level routine anti-tuberculosis drug resistance surveillance conducted since 2013 in 11 Fujian counties were analyzed using McSpoligotyping. In vitro drug susceptibilities to anti-tuberculosis drugs were determined using the standard Löwenstein-Jensen proportion method. Results: Overall, 477 MTB isolates were included in the study, of which 245 isolates belonged to the Beijing genotype family and 232 possessed non-Beijing genotypes. Ultimately, a total of 204 spoligotypes were identified that included 58 spoligotype international types (SITs) from the SITVITWEB database and 146 novel spoligotypes. As compared to patients <25 years of age (control group), elderly patients (≥65 years of age) were more likely to be infected with non-Beijing genotypes [aOR 18.69, 95% CI (5.80-60.26)], with risk of infection with non-Beijing genotypes increasing with age [aOR 3.73, 95% CI (1.67-8.30) for patients 45-64 years of age]. Additionally, as compared to isolates with Beijing and other non-Beijing genotypes, significantly greater proportions of isolates with novel spoligotypes exhibited PTO- and PAS-resistance. Moreover, a markedly higher proportion of isolates with novel spoligotypes exhibited OFX-resistance as compared to isolates with other non-Beijing genotypes. Conclusion: Our data demonstrated that the Beijing genotype is the predominant MTB sublineage in Fujian and that the prevalence rate of infection with this MTB genotype decreases with advancing patient age. Notably, the prevalence rate of this genotype in Fujian TB patients is relatively lower than in other regions of China. In addition, the emergence of novel spoligotypes of highly drug-resistant MTB isolates highlights the urgent need for ongoing molecular genotyping surveillance in Fujian.

Spoligotype-based population structure and isoniazid-resistance gene mutation of Mycobacterium tuberculosis isolates from Thailand.

OBJECTIVES: The present study aims to investigate the population structure of Thai Mycobacterium tuberculosis (MTB) isolates and anti-tuberculosis (TB) drug resistance and to determine the most frequent genetic mutations conferring isoniazid (INH) resistance. METHODS: Genomic DNA from 287 MTB clinical isolates were extracted and used for spoligotyping, amplification and sequencing analysis of the region of different (RD) 105, and of the INH resistance (IR) associated genes, inhA, katG and oxyR-ahpC genes. RESULTS: Eighty-one clinical isolates were resistant to at least one first-line drug; 53 of these were resistant to INH. All strains were classified into three lineages based on their spoligotypes: East Asia (EA)/Beijing, Indo-Oceanic (IO), and Euro-American (EuA). EA and IO lineages revealed a strong association with anti-TB drug resistance (P = 0.005 and 0.013, respectively). A total of 33 mutations were found among IR isolates, which for 28 (84.8%), 3 (9.1%), and 2 (6.1%) occurred in katG, inhA, and oxyR-ahpC genes, respectively. Moreover, the most common mutations found were 54.7% of IR presented Ser315Thr at katG (54.7%) and C-15T at inhA (15.1%) presented. This result suggests the involvement of other genetic markers or other mechanisms of resistance. CONCLUSION: This study provides information about strains diversity, drug resistance profiles, and their possible association. EA and IO lineages were predominant in Thailand, and they were highly associated with anti-TB drug resistance. Testing two mutations including katGSer315Thr and inhA-15C→T could detect 68% of the IR strains.

A Comprehensive Evaluation of GeneLEAD VIII DNA Platform Combined to Deeplex Myc-TB® Assay to Detect in 8 Days Drug Resistance to 13 Antituberculous Drugs and Transmission of Mycobacterium tuberculosis Complex Directly From Clinical Samples.

The GeneLEAD VIII (Diagenode, Belgium) is a new, fully automated, sample-to-result precision instrument for the extraction of DNA and PCR detection of Mycobacterium tuberculosis complex (MTBC) directly from clinical samples. The Deeplex Myc-TB® assay (Genoscreen, France) is a diagnostic kit based on the deep sequencing of a 24-plexed amplicon mix allowing simultaneously the detection of resistance to 13 antituberculous (antiTB) drugs and the determination of spoligotype. We evaluated the performance of a strategy combining the both mentioned tools to detect directly from clinical samples, in 8 days, MTBC and its resistance to 13 antiTB drugs, and identify potential transmission of strains from patient-to-patient. Using this approach, we screened 112 clinical samples (65 smear-negative) and 94 MTBC cultured strains. The sensitivity and the specificity of the GeneLEAD/Deeplex Myc-TB approach for MTBC detection were 79.3% and 100%, respectively. One hundred forty successful Deeplex Myc-TB results were obtained for 46 clinical samples and 94 strains, a total of 85.4% of which had a Deeplex Myc-TB susceptibility and resistance prediction consistent with phenotypic drug susceptibility testing (DST). Importantly, the Deeplex Myc-TB assay was able to detect 100% of the multidrug-resistant (MDR) MTBC tested. The lowest concordance rates were for pyrazinamide, ethambutol, streptomycin, and ethionamide (84.5%, 81.5%, 73%, and 55%, respectively) for which the determination of susceptibility or resistance is generally difficult with current tools. One of the main difficulties of Deeplex Myc-TB is to interpret the non-synonymous uncharacterized variants that can represent up to 30% of the detected single nucleotide variants. We observed a good level of concordance between Deeplex Myc-TB-spoligotyping and MIRU-VNTR despite a lower discriminatory power for spoligotyping. The median time to obtain complete results from clinical samples was 8 days (IQR 7-13) provided a high-throughput NGS sequencing platform was available. Our results highlight that the GeneLEAD/Deeplex Myc-TB approach could be a breakthrough in rapid diagnosis of MDR TB in routine practice.

Characterization of Mutations Associated with Streptomycin Resistance in Multidrug-Resistant Mycobacterium tuberculosis in Zambia.

Streptomycin (STR) is recommended for the management of multidrug-resistant tuberculosis (MDR-TB). Streptomycin resistance-conferring mutation types and frequency are shown to be influenced by genotypes of circulating strains in a population. This study aimed to characterize the mutations in MDR-TB isolates and examine their relationship with the genotypes in Zambia. A total of 138 MDR-TB isolates stored at the University Teaching Hospital Tuberculosis Reference Laboratory in Zambia were analyzed using spoligotyping and sequencing of STR resistance-associated genes. Streptomycin resistance was observed in 65.9% (91/138) of MDR-TB isolates. Mutations in rpsL, rrs, and gidB accounted for 33%, 12.1%, and 49.5%, respectively. Amino acid substitution K43R in rpsL was strongly associated with the CAS1_Kili genotype (p < 0.0001). The combination of three genes could predict 91.2% of STR resistance. Clustering of isolates based on resistance-conferring mutations and spoligotyping was observed. The clustering of isolates suggests that the increase in STR-resistant MDR-TB in Zambia is largely due to the spread of resistant strains from inadequate treatment. Therefore, rapid detection of STR resistance genetically is recommended before its use in MDR-TB treatment in Zambia.

Diversidad genética de Mycobacterium tuberculosis circulando en la jurisdicción V de Jalapa, Veracruz, México / Genetic diversity of Mycobacterium tuberculosis circulating in the jurisdiction V from Jalapa, Veracruz, Mexico

ANTECEDENTES: El estado de Veracruz se ubica en el sureste de México y presenta una alta prevalencia de tuberculosis (TBC) y drogo resistencia. Sin embargo, la composición de los genotipos circulantes es poco conocida. OBJETIVO: Caracterizar la diversidad genética de la TBC en la jurisdicción sanitaria V del estado de Veracruz. MÉTODOS: Estudio transversal realizado en aislados clínicos de pacientes con TBC residentes de la jurisdicción V. Se determinó la sensibilidad a medicamentos de primera línea. La genotipificación se realizó mediante espoligotipificación y MIRU-VNTR 15 loci. RESULTADOS: Entre los 74 aislados analizados se observó resistencia a un fármaco en 44 (59%) aislados. Linaje L4 (EuroAmericano) se presentó en 73 aislados. Se identificaron cinco sublinajes; H (40%), T (22%), LAM (16%), X (13%) y U (7%). El 32% de los aislados se agrupó mediante su espoligotipo y 40% en 10 complejos clonales. CONCLUSIONES: Es la primera descripción sobre la estructura genética de TBC en la región central de Veracruz. La diversidad de genotipos podría contribuir a su dispersión en la región. Esta información será útil para el desarrollo de intervenciones y reducir el impacto de TBC en la población.

BACKGROUND: The state of Veracruz is placed in southeastern Mexico and has a high prevalence of tuberculosis (TB) and drug resistance. Nevertheless, the composition of circulating genotypes in the central region of the state is partially known. AIM: To characterize the genetic diversity of TB in the sanitary jurisdiction V of the state of Veracruz. METHODS: A cross-sectional study was conducted among clinical isolates from patients with TB living in the jurisdiction V, in Jalapa Ver., Mexico. Sensitivity to first-line drugs was determined, and genotyping was performed by spoligotyping and MIRU-VNTR 15 loci. RESULTS: Among the 74 isolates analyzed, resistance to one drug was observed in 44 isolates. L4 (EuroAmerican) was the major lineage identified. Five sublineages were the most abundant; H (40%), T (22%), LAM (16%), X (13%) and U (7%). Only 32% of the isolates were clustered by spoligotype and 40% were placed in ten clonal complexes. CONCLUSIONS: This is the first description of the genetic structure of TB in the central region of Veracruz. The diversity of genotypes could contribute to its dispersion. This information will be useful for the development of interventions to reduce the impact of TB in the population.

Spoligotype-specific risk of finding lesions in tissues from cattle infected by Mycobacterium bovis.

BACKGROUND: Although the pathogenic effect of members of the Mycobacterium tuberculosis complex in susceptible hosts is well known, differences in clinical signs and pathological findings observed in infected animals have been reported, likely due to a combination of host and pathogen-related factors. Here, we investigated whether Mycobacterium bovis strains belonging to different spoligotypes were associated with a higher risk of occurrence of visible/more severe lesions in target organs (lungs and/or lymph nodes) from infected animals. A large collection of 8889 samples belonging to cattle were classified depending on the presence/absence of tuberculosis-like lesions and its degree of severity. All samples were subjected to culture irrespective of the presence of lesions, and isolates retrieved were identified and subjected to spoligotyping. The association between the presence/severity of the lesions and the isolation of strains from a given spoligotype was assessed using non-parametric tests and Bayesian mixed multivariable logistic regression models that accounted for origin (region and herd) effects. RESULTS: Results suggested a difference in severity in lesioned samples depending on the strain's spoligotype. An association between specific spoligotypes and presence of lesions was observed, with a higher risk of finding lesions in animals infected with strains with spoligotypes SB0120, SB0295 and SB1142 compared with SB0121, and in those coming from certain regions in Spain. CONCLUSIONS: Our results suggest that strains belonging to certain spoligotypes may be associated with a higher probability in the occurrence of gross/macroscopic lesions in infected cattle, although these observational findings should be confirmed in further studies that allow accounting for the effect of other possible confounders not considered here, and ultimately through experimental studies.

Genetic Diversity and Potential Paths of Transmission of Mycobacterium bovis in the Amazon: The Discovery of M. bovis Lineage Lb1 Circulating in South America.

Bovine tuberculosis (bTB) has yet to be eradicated in Brazil. Herds of cattle and buffalo are important sources of revenue to people living in the banks of the Amazon River basin. A better understanding of Mycobacterium bovis (M. bovis) populational structure and transmission dynamics affecting these animals can significantly contribute in efforts to improve their sanitary status. Herein, we sequenced the whole genome of 22 M. bovis isolates (15 from buffalo and 7 from cattle) from 10 municipalities in the region of the Lower Amazon River Basin in Brazil and performed phylogenomic analysis and Single Nucleotide Polymorphism (SNP)-based transmission inference to evaluate population structure and transmission networks. Additionally, we compared these genomes to others obtained in unrelated studies in the Marajó Island (n = 15) and worldwide (n = 128) to understand strain diversity in the Amazon and to infer M. bovis lineages. Our results show a higher genomic diversity of M. bovis genomes obtained in the Lower Amazon River region when compared to the Marajó Island, while no significant difference was observed between M. bovis genomes obtained from cattle and buffalo (p ≥ 0.05). This high genetic diversity is reflected by the weak phylogenetic clustering of M. bovis from the Lower Amazon River region based on geographic proximity and in the detection of only two putative transmission clusters in the region. One of these clusters is the first description of inter-species transmission between cattle and buffalo in the Amazon, bringing implications to the bTB control program. Surprisingly, two M. bovis lineages were detected in our dataset, namely Lb1 and Lb3, constituting the first description of Lb1 in South America. Most of the strains of this study (13/22) and all 15 strains of the Marajó Island carried no clonal complex marker, suggesting that the recent lineage classification better describe the diversity of M. bovis in the Amazon.

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.

Single-nucleotide polymorphism-based epidemiological analysis of Korean Mycobacterium bovis isolates.

BACKGROUND: Bovine tuberculosis (TB) is caused by Mycobacterium bovis, a well-known cause of zoonotic tuberculosis in cattle and deer, and has been investigated in many physiological and molecular studies. However, detailed genome-level studies of M. bovis have not been performed in Korea. OBJECTIVES: To survey whole genome-wide single-nucleotide polymorphism (SNP) variants in Korean M. bovis field isolates and to define M. bovis groups in Korea by comparing SNP typing with spoligotyping and variable number tandem repeat typing. METHODS: A total of 46 M. bovis field isolates, isolated from laryngopharyngeal lymph nodes and lungs of Korean cattle, wild boar, and Korean water deer, were used to identify SNPs by performing whole-genome sequencing. SNP sites were confirmed via polymerase chain reaction using 87 primer pairs. RESULTS: We identified 34 SNP sites with different frequencies across M. bovis isolates, and performed SNP typing and epidemiological analysis, which divided the 46 field isolates into 16 subtypes. CONCLUSIONS: Through SNP analysis, detailed differences in samples with identical spoligotypes could be detected. SNP analysis is, therefore, a useful epidemiological tracing tool that could enable better management of bovine TB, thus preventing further outbreaks and reducing the impact of this disease.

Genotype Diversity of Mycobacterium bovis and Pathology of Bovine Tuberculosis in Selected Emerging Dairy Regions of Ethiopia.

Bovine tuberculosis (bTB) is endemic in Ethiopia with higher prevalence in cattle, particularly in the central parts. Spread of Mycobacterium bovis (M. bovis) to wider regions is inevitable in uncontrolled conditions. This study was conducted to explore the pathology, characterize M. bovis strains, and describe genotypic diversity to demonstrate possible epidemiological links in emerging dairy areas of Ethiopia, namely, Mekelle and Gondar. Twenty-seven bTB positive cattle identified by the Single Intradermal Comparative Cervical Tuberculin (SICCT) test were subjected to post-mortem inspection to determine lesion distribution and pathological score. Samples from tissues with visible tuberculous or suspected non-visible lesions were processed and cultured following a standard protocol. Isolates identified as M. bovis by Region of Difference (RD)-based Polymerase Chain Reaction (PCR) were also spoligotyped to determine their spoligotype patterns. Post-mortem inspection of visceral organs indicated bTB suggestive lesions in 41% of the animals, with 25% being in the lungs. Lymph nodes from 77% of the animals had lesions. Fifty-five isolates identified from 24 of the slaughtered animals were confirmed as M. bovis. No other mycobacterial species were isolated. Spoligotyping classified strains from 21 of these animals into seven spoligotype patterns: SB0133, SB0134, SB1176, SB2233, SB2290, SB2467, and SB2520. More than one spoligotype were identified from five of these animals, and none of the last four spoligotypes had been reported in Ethiopia before. SB0134 was the most predominant type (47%) followed by SB0133 (25.5%). SB0133, SB2290, SB2467, and SB1176 are spoligotypes lacking spacers 3-7, characteristics of M. bovis strains of the African 2 (Af2) clonal complex, while SB0134, SB2233, and SB2520 do not belong to any of the established clonal complexes and likely to have a different evolutionary history. Despite a small sample size, the present study showed strain diversity with multiple genotypes identified in a single herd and even within a single animal, and the genotypes showed no sign of geographical localization, which could be a consequence of significant movement of bTB diseased cattle around the country, spreading the disease. Therefore, any future control programme of bTB in Ethiopia needs to address the risks of cattle movement.

Frequency of mutations associated with resistance to first- and second-line drugs in multidrug-resistant Mycobacterium tuberculosis isolates.

INTRODUCTION: Tuberculosis is considered one of the most fatal diseases worldwide, with an estimation of 10.1 million cases. In this study, whole-genome sequencing was used to determine the genomic characterisation of 40 Mycobacterium tuberculosis isolates from patients with different nationalities hospitalised in the Czech Republic. MATERIALS AND METHODS: Susceptibility testing for first-line drugs was performed. DNA was sequenced using the Illumina MiSeq platform. Spoligotype single-nucleotide polymorphisms and mutations in antibiotic-resistant genes were detected, and phylogenetic analysis was performed. RESULTS: Samples showing phenotypic resistance to at least one drug were 12 to streptomycin, 11 to isoniazid, 7 to rifampicin, 6 to ethambutol and 5 to pyrazinamide. Phenotypic and genotypic profiles did not match in all cases, suggesting the presence of a novel mutation in some cases and a low expression of resistant genes in others. The presented phylogeny enables the correct assignation of M. tuberculosis lineages and sublineages. Our results suggest that the most dominant lineage in our samples was lineage 4 (33/40). CONCLUSION: To our knowledge, this is the first study using this approach to be done in the Czech Republic. Lineage 4 was the predominant lineage identified among our samples. Nevertheless, the dominance of Lineage 4 along with other lineages suggests that infections can originate from different sources.

Comparative analysis of genomic variability for drug-resistant strains of Mycobacterium tuberculosis: The special case of Belarus.

Mycobacterium tuberculosis (M.tb) is the leading cause of death from an infectious disease. Drug resistant tuberculosis (DR-TB) threatens to exacerbate challenges in diagnostics and treatment. It is important to monitor strains circulating in countries with heavy burden of DR-TB, to make informed decisions about treatment, and because in these countries there is an elevated probability that DR-TB may advance to the totally drug resistant form. The TB Portals Program (TBPP, https://TBPortals.niaid.nih.gov) formed a global network of participating institutions and hospitals collecting and analyzing de-identified clinical, imaging and socioeconomic data, augmenting these with genomic sequencing results. TB Portals database includes complete M.tb genomes, with the information about spoligotypes, strains, and genomic variants related to drug resistance. Within the framework of TB Portals, we created Data Exploration Portal (DEPOT), to facilitate visualization and statistical analysis of user-defined cohorts from the entire TB Portals database. A continuing TB Portals research objective is to actively monitor and examine genomic variability that may account for observed differences in DR-TB incident rates and/or difficulties with diagnosis and treatment. Our analysis identified that several genomic variants implicated in drug resistance or improved fitness of the pathogen, were significantly more frequent in M.tb strains circulating in Belarus in comparison with other countries. Further studies are necessary to reveal whether the corresponding genomic variants may explain unusually high burden of drug-resistant M.tb in Belarus and suggest improvements for diagnostic and drug therapies.

A review of published spoligotype data indicates the diversity of Mycobacterium tuberculosis from India is under-represented in global databases.

BACKGROUND: Mycobacterium tuberculosis (MTBC) lineages differ in clinical presentation, virulence, transmission, drug resistance and immunological responses. Despite having the largest burden of tuberculosis (TB) in the world, strains from India are underrepresented in international databases. We reviewed published spoligotype data to determine the distribution and diversity of MTBC lineages in India. METHODS: A Pubmed/MEDLINE search identified 34 M. tuberculosis spoligotyping studies from India. Spoligotype patterns were extracted and the Spoligotype International Type (SIT) number, sub-lineage and lineage determined. Minimum Spanning Trees were used to determine relationships between patterns. RESULTS: We identified 1528 spoligotype patterns distributed across 8300 isolates; 6733 isolates belonged to 472 SITs, with 53% of all isolates belonging to 12 SITs with at least 100 isolates each. Lineage 1 and Lineage 3 made up 67% of all isolates, although a lineage could not be assigned for 16% of isolates. Lineage 1 isolates were most common in Southern, Western and Eastern India, and Lineage 3 was most common in Northern and Central India. The RULE, CBN and KBBN lineage prediction algorithms from the TB-lineage tools performed variably, with the correct lineage predicted correctly for only 64% of patterns with known lineage. Using a consensus definition, 64% of the 1359 isolates with unknown lineage were assigned to Lineage 1, and 14% each were assigned to Lineages 3 and 4. With these lineage assignments, 80% of all isolates belonged to either Lineage 1 or Lineage 3. CONCLUSION: Our findings indicate significant M. tuberculosis diversity in India. The documentation of 1056 orphan and unreported patterns indicate that this diversity is under-represented in global databases.

Genetically Related Mycobacterium bovis Strains Displayed Differential Intracellular Growth in Bovine Macrophages.

Molecular typing of bacterial isolates provides a powerful approach for distinguishing Mycobacterium bovis (M. bovis) genotypes. It is known that M. bovis strain virulence plays a role in prevalence and spread of the disease, suggesting that strain virulence and prevailing genotypes are associated. However, it is not well understood whether strain virulence correlates with particular genotypes. In this study, we assessed the in vitro intracellular growth of 18 M. bovis isolates in bovine macrophages as an indicator of bacterial virulence and sought a relationship with the genotype identified by spoligotyping. We found 14 different spoligotypes-11 were already known and three spoligotypes had never been reported before. We identified 2 clusters that were phylogenetically related, containing 10 and 6 strains, respectively, and 2 orphan strains. Intracellular growth and phagocytic rates of 18 M. bovis strains were heterogeneous. Our results suggest that M. bovis intracellular growth and phagocytosis are independent of the bacterial lineage identified by spoligotyping.

Insight into genetic diversity of Mycobacterium tuberculosis in Kandy, Sri Lanka reveals predominance of the Euro-American lineage.

OBJECTIVE: Sri Lanka is a country where the molecular epidemiology of Mycobacterium tuberculosis (MTB) is poorly explored. Therefore, this study was performed to identify circulating lineages/sub-lineages of MTB and their transmission patterns. METHODS: DNA was extracted from 89 isolates of MTB collected during 2012 and 2013 from new pulmonary tuberculosis patients in Kandy, Sri Lanka and analyzed by spoligotyping, large sequence polymorphism (LSP), mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing, and drug resistance-associated gene sequencing. RESULTS: The predominant lineage was lineage 4 (Euro-American, 45.9%), followed by lineage 1 (Indo-Oceanic, 29.4%), lineage 2 (East-Asian, 23.5%), and lineage 3 (Central-Asian, 1.2%). Among 26 spoligotype patterns, eight were undesignated or new types and seven of these belonged to lineage 4. Undesignated lineage 4/SIT124 (n=2/8) and SIT3234 (n=8/8) clustered together based on 24-locus MIRU-VNTR typing. The dominant sub-lineage was Beijing/SIT1 (n=19), with the isoniazid resistance katG G944C mutation (Ser315Thr) detected in two of them. CONCLUSIONS: The population structure of MTB in Kandy, Sri Lanka was different from that in the South Asian region. The clonal expansion of locally evolved lineage 4/SIT3234 and detection of the pre-multidrug resistant Beijing isolates from new tuberculosis patients is alarming and will require continuous monitoring.

Exportation of MDR TB to Europe from Setting with Actively Transmitted Persistent Strains in Peru.

We performed a cross-border molecular epidemiology analysis of multidrug-resistant tuberculosis in Peru, Spain, and Italy. This analysis revealed frequent transmission in Peru and exportation of a strain that recreated similar levels of transmission in Europe during 2007-2017. Transnational efforts are needed to control transmission of multidrug-resistant tuberculosis globally.

The most common spoligotype of Mycobacterium bovis isolated in the world and the recommended loci for VNTR typing; A systematic review.

Mycobacterium bovis is a neglected zoonotic organism that epidemiological studies are of crucial importance in identifying its source, control it and prevent it from spreading. The aim of this study was to investigate the most common spoligotypes of Mycobacterium bovis circulating around the world and introduce the most and least strong determine powers of loci for VNTR. We have used different databases such as ISC, science direct, Embase (Elsevier), Web of Science, Scopus and Medline via PubMed. Searches were performed by key words including: Mycobacterium bovis, MIRU -VNTR, spoligotyping and discrimination power. Finally, thirty-one articles were selected after filtering out some titles, abstracts and full texts. Spoligotype SB0120 was the most common circulating type on several continents while SB0121 existed in Europe, Africa and America. SB0140 was also detected in Asia, Europe and America. QUB3232 and QUB11b were more appropriate loci among the loci with high discriminatory power. MIRU 10 and MIRU4 were among the loci with poor discriminatory power. Taking the published data into consideration, SB0120 and SB0121 are predominant spoligotypes of M. bovis circulating among animals around the world. Determining the most common spoligotype of M. bovis is the key to find source of infection, control and prevent the disease.