MIRU-VNTR Loci Research Articles

A Genome-Focused Investigation Reveals the Emergence of a Mycobacterium tuberculosis Strain Related to Multidrug-Resistant Tuberculosis in the Amazon Region of Brazil.

A previous study in Pará, Northern Brazil, described a strain of Mycobacterium tuberculosis with a unique genotype (SIT2517/T1) associated with multidrug-resistant tuberculosis (MDR-TB). To improve our understanding of MDR-TB transmission dynamics of these strains within this region, we performed phenotypic and genotypic drug susceptibility testing (pDST/gDST), 24-loci mycobacterial interspersed repetitive units (MIRU-VNTR) genotyping, whole-genome sequencing (WGS) and geo-epidemiology analysis. Of the 28 SIT2517/T1 isolates, 19 (67.9%) could be genotyped by 24-loci MIRU-VNTR and 15 by WGS. All belonged to sublineage 4.1.1.3, distinct from other representative Lineage 4 isolates identified in Brazil. The MDR phenotype determined by pDST was confirmed by gDST, the latter also demonstrating the presence of additional mutations conferring pre-extensively drug-resistance (pre-XDR). Discrepancies between gDST and pDST were observed for pyrazinamide and fluoroquinolones. Thirteen out of 15 isolates analyzed by WGS were clustered when applying a 12 single nucleotide polymorphisms (SNPs) cutoff. The SIT2517/T1 isolates were distributed across the metropolitan regions of Belém and Collares municipalities, showing no geographic clustering. WGS-transmission network analysis revealed a high likelihood of direct transmission and the formation of two closely linked transmission chains. This study highlights the need to implement TB genomic surveillance in the Brazilian Amazon region.

Prevalence, drug resistance, and genotypic diversity of the RDRio subfamily of Mycobacterium tuberculosis in Ecuador: a retrospective analysis for years 2012-2016.

A major sublineage within the Mycobacterium tuberculosis (MTB) LAM family characterized by a new in-frame fusion gene Rv3346c/55c was discovered in Rio de Janeiro (Brazil) in 2007, called RDRio, associated to drug resistance. The few studies about prevalence of MTB RDRio strains in Latin America reported values ranging from 3% in Chile to 69.8% in Venezuela, although no information is available for countries like Ecuador. A total of 814 MTB isolates from years 2012 to 2016 were screened by multiplex PCR for RDRio identification, followed by 24-loci MIRU-VNTR and spoligotyping. A total number of 17 MTB RDRio strains were identified, representing an overall prevalence of 2.09% among MTB strains in Ecuador. While 10.9% of the MTB isolates included in the study were multidrug resistance (MDR), 29.4% (5/17) of the RDRio strains were MDR. This is the first report of the prevalence of MTB RDRio in Ecuador, where a strong association with MDR was found, but also a very low prevalence compared to other countries in Latin America. It is important to improve molecular epidemiology tools as a part of MTB surveillance programs in Latin America to track the transmission of potentially dangerous MTB stains associated to MDR TB like MTB RDRio.

Epidemiological Analysis of Human and Animal Originated Mycobacterium bovis Strains by Spoligotyping and MIRU-VNTR Methods

This study aims to investigate the genotypic similarities between human and animal-originated isolates by spoligotyping and 24 loci MIRU-VNTR for molecular epidemiological analysis of M. bovis isolates. In this study, isolates were obtained between 2019-2022 from 58 humans and 50 bovines. All isolates were initially identified with the GenoType MTBC kit and genotyped with spoligotyping and 24 loci MIRU-VNTR methods and their lineage relationships were shown in the dendrogram. When the human and animal-originated isolates were tested by the spoligotyping method, eight different clusters and 29 different genotypes were observed. Among these genotypes, the most common ones were found to be SIT1118/SB0989 (19.23%), SIT482/SB0120 (16.35%), SIT685/SB0288 (12.5%) and they were detected in isolates of both human and animal-originated. SB1593 (12.5%) was detected only in isolates of animal-originated. Other genotypes were found as SIT3529/SB0920, SIT1185/SB0897, SIT3710/SB1595, SIT688/SB0129, SIT3687/SB1625, SB0419, SB2466, SB1231, and SB2510. Nine different clusters and 55 different genotypes were obtained with MIRU-VNTR. ETR-C, QUB2163b, QUB26, and Mtub04 had the most allelic diversity. It was observed that MIRU02, MIRU20, MIRU24, MIRU27, and MIRU39 did not indicate allelic diversity. In conclusion, it was seen that the spoligotyping was easier to implement and evaluate compared to MIRU-VNTR. It was concluded that both tests can be used safely. Similar genotypes in humans and animals indicate the importance of zoonotic transmission of bovine tuberculosis.

A first insight into tuberculosis transmission at the border of Ecuador and Colombia: a retrospective study of the population structure of Mycobacterium tuberculosis in Esmeraldas province.

Tuberculosis (TB) is a major public health concern in Ecuador and Colombia, considering that both countries are high-burden TB settings. Molecular epidemiology is crucial to understand the transmission dynamics of Mycobacterium tuberculosis complex (MTBC) and to identify active transmission clusters of regional importance. We studied the potential transmission of TB between Colombia and Ecuador through the analysis of the population structure of MTBC lineages circulating in the Ecuadorian province of Esmeraldas at the border with Colombia. A total of 105 MTBC strains were characterized by 24-loci MIRU-VNTR and spoligotyping. MTBC lineage 4 is only present in Esmeraldas; no MTBC strains belonging to Lineage 2-sublineage Beijing were found despite its presence in other provinces of Ecuador and, in Colombia. Genotyping results revealed a high degree of diversity for MTBC in Esmeraldas: Neither active transmission clusters within this province nor including MTBC strains from Colombia or other provinces of Ecuador were found. Our data suggest that tuberculosis dynamics in this rural and isolated area may be not related to highly transmitted strains but could be influenced by other health determinants that favor TB relapse such as poverty and poor health system access. Further studies including a larger number of MTBC strains from Esmeraldas are necessary to test this hypothesis.

Prevalence, Transmission and Genetic Diversity of Pyrazinamide Resistance Among Multidrug-Resistant Mycobacterium tuberculosis Isolates in Hunan, China.

China is a country with a burden of high rates of both TB and multidrug-resistant TB (MDR-TB). However, published data on pyrazinamide (PZA) resistance are still limited in Hunan province, China. This study investigated the prevalence, transmission, and genetic diversity of PZA resistance among multidrug-resistant Mycobacterium tuberculosis isolates in Hunan province. Drug susceptibility testing (DST) with the Bactec MGIT 960 PZA kit and pyrazinamidase (PZase) testing were conducted on all 298 MDR clinical isolates. Moreover, 24-locus MIRU-VNTR and DNA sequencing of pncA, rpsA, and panD genes were conducted on 180 PZA-resistant (PZA-R) isolates. The prevalence of PZA resistance among MDR-TB strains reached 60.4%. Newly diagnosed PZA-R TB patients and clustered isolates with identical pncA, rpsA, and panD mutations showed that transmission of PZA-R isolates played a significant role in the formation of PZA-R TB. Ninety-eight mutation patterns were observed in the pncA among 180 PZA-R isolates, and seventy-one (72.4%) were point mutations. Twenty-four of these mutations are new, including 2 base substitutions (V93G and T153S) and 22 nucleotide deletions or insertions. The W119C was found in PZA-S isolates, on the other hand, F94L and V155A mutations were found in both PZA resistant and susceptible isolates with positive PZase activity, indicating that they were not associated with PZA resistance. This is not entirely in line with the WHO catalogue. Ten novel rpsA mutations were found in 10 PZA-R isolates, which all combined with mutations in pncA. Thus, it is unpredictable whether these mutations in rpsA can impact PZA resistance. No panD mutation was found in all PZA-R isolates. DNA sequencing of pncA and PZase activity testing have great potential in predicting PZA resistance.

Mycobacterium tuberculosis genotypes in an ethnically diverse area with millions of pilgrims and thousands of immigrants

BackgroundImmigration is considered as a risk factor of tuberculosis (TB). Qom province receives millions of pilgrims and significant numbers of immigrants each year. Most of the immigrants to Qom, arrive from neighboring TB-endemic countries. This study aimed to identify the current circulating Mycobacterium tuberculosis genotypes in Qom province using 24-locus MIRU-VNTR genotyping.MethodsEighty six M. tuberculosis isolates were collected during 2018–2022 from patients referring to Qom TB reference laboratory. The DNA of isolates was extracted and followed by 24 loci MIRU-VNTR genotyping which performed using the web tools available on MIRU-VNTRplus.ResultsOf 86 isolates, 39 (45.3%) were of Delhi/CAS genotype, 24 (27.9%) of NEW-1, 6 (7%) of LAM, 6 (7%) of Beijing, 2 (2.3%) of UgandaII, 2 (2.3%) of EAI, 1 of S (1.2%) and 6 (7%) did not match with profiles present in MIRUVNTRplus database.ConclusionsAbout half of the isolates belong to Afghan immigrants; which warns health policy makers about the future situation of TB in Qom. Also, the similarity of Afghan and Iranian genotypes provides evidence that immigrants partake in the circulation of M. tuberculosis. This study underpin the studies about the circulating M. tuberculosis genotypes, their geographical distribution, the association of TB risk factors with these genotypes and the impact of immigration on the situation of TB in Qom province.

Molecular Epidemiology of Clinical Mycobacterium tuberculosis Isolates from Southern Xinjiang, China Using Spoligotyping and 15-Locus MIRU-VNTR Typing.

In the last decades, the molecular epidemiological investigation of Mycobacterium tuberculosis has significantly increased our understanding of tuberculosis epidemiology. However, few such studies have been done in southern Xinjiang, China. We aimed to clarify the molecular epidemic characteristics and their association with drug resistance in the M. tuberculosis isolates circulating in this area. A total of 347 isolates obtained from southern Xinjiang, China between Sep, 2017 and Sep, 2019 were included to characterize using a 15-locus MIRU-VNTR (VNTR-15China) typing and spoligotyping, and test for drug susceptibility profiles. Then the lineages and clustering of the isolates were analyzed, as well as their association with drug resistance. Spoligotyping results showed that 60 spoligotype international types (SITs) containing 35 predefined SITs and 25 Orphan or New patterns, and 12 definite genotypes were found, and the top three prevalent genotypes were Beijing genotype (207, 59.7%), followed by CAS1-Delhi (46, 13.6%), and Ural-2 (30, 8.6%). The prevalence of Beijing genotype infection in the younger age group (≤30) was more frequent than the two older groups (30~59 and ≥60 years old, both P values <0.05). The Beijing genotype showed significantly higher prevalence of resistance to isoniazid, rifampicin, ethambutol, multi-drug or at least one drug than the non-Beijing genotype (All P values ≤0.05). The estimated proportion of tuberculosis cases due to transmission was 18.4% according to the cluster rate acquired by VNTR-15China typing, and the Beijing genotype was the risk factor for the clustering (OR 9.15, 95% CI: 4.18-20.05). Our data demonstrated that the Beijing genotype is the dominant lineage, associated with drug resistance, and was more likely to infect young people and contributed to tuberculosis transmission in southern Xinjiang, China. These findings will contribute to a better understanding of tuberculosis epidemiology in this area.

Genomic epidemiology of Mycobacterium avium subsp. paratuberculosis isolates from Canadian dairy herds provides evidence for multiple infection events.

Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen responsible for paratuberculosis or Johne's Disease (JD) in ruminants, which is responsible for substantial economic losses worldwide. MAP transmission primarily occurs through the fecal-oral route, and the introduction of an MAP infected animal into a herd is an important transmission route. In the current study, we characterized MAP isolates from 67 cows identified in 20 herds from the provinces of Quebec and Ontario, Canada. Whole genome sequencing (WGS) was performed and an average genome coverage (relative to K-10) of ∼14.9 fold was achieved. The total number of SNPs present in each isolate varied from 51 to 132 and differed significantly between herds. Isolates with the highest genetic variability were generally present in herds from Quebec. The isolates were broadly separated into two main clades and this distinction was not influenced by the province from which they originated. Analysis of 8 MIRU-VNTR loci and 11 SSR loci was performed on the 67 isolates from the 20 dairy herds and publicly available references, notably major genetic lineages and six isolates from the province of Newfoundland and Labrador. All 67 field isolates were phylogenetically classified as Type II (C-type) and according to MIRU-VNTR, the predominant type was INMV 2 (76.1%) among four distinct patterns. Multilocus SSR typing identified 49 distinct INMV SSR patterns. The discriminatory index of the multilocus SSR typing was 0.9846, which was much higher than MIRU-VNTR typing (0.3740). Although multilocus SSR analysis provides good discriminatory power, the resolution was not informative enough to determine inter-herd transmission. In select cases, SNP-based analysis was the only approach able to document disease transmission between herds, further validated by animal movement data. The presence of SNPs in several virulence genes, notably for PE, PPE, mce and mmpL, is expected to explain differential antigenic or pathogenetic host responses. SNP-based studies will provide insight into how MAP genetic variation may impact host-pathogen interactions. Our study highlights the informative power of WGS which is now recommended for epidemiological studies and to document mixed genotypes infections.

More insights about genomic population structure of Mycobacterium avium subspecies paratuberculosis (Map) from multiple hosts in west and central provinces of Iran using a boosted genotyping approach

To investigate the population genetic of Mycobacterium avium subsp. paratuberculosis (Map) in Iran, Mycobacterial Interspersed Repetitive Units (MIRUs) and Multi Locus Short Sequence Repeat (MLSSR) system were employed. Numerous genotypes by MIRU (N = 11) and MLSSR (N = 9) methods bearing discriminatory indices of 0.90 and 0.79 respectively, were obtained. Browsing the INRA–Nouzilly list (http://mac-inmv.tours.inra.fr/) detected 3 of the found patterns as new types. Some loci either MIRU-VNTR or SSR proved more polymorphic and therefore are recommended to be applied in priority for strain typing in the Iranian environment. While identical MIRU-VNTR or MLSSR patterns were detected among different conspecifics and geographical locations, dissimilar types were also observed at the same farms an indication of coexistence of Map strains within one herd. We suggest extension of the genotyping work described here to include more endogenous isolates in order to better analysis of transmission and virulence in epidemiology and control of paratuberculosis.

Fingerprinting of Mycobacterium tuberculosis isolates by MIRU-VNTR genotyping and detection of isoniazid resistance by real-time PCR.

Introduction. Tuberculosis (TB) is a great public health problem in developing countries such as Egypt. Genotyping of Mycobacterium tuberculosis isolates has a prominent role in the field of TB prevention.Aim. This study aimed to evaluate real-time PCR using Minor Groove Binder (MGB) probes and to identify circulating lineages/sub-lineages of M. tuberculosis and their transmission patterns.Hypothesis. We hypothesize that MIRU-VNTR technique is efficient in identifying circulating M. tuberculosis lineages in Egypt.Methodology. Fifty sputum specimens positive for acid-fast bacilli were included. Isoniazid (INH) resistance was detected using the 1 % proportion method. Real-time PCR using MGB-probes was used for simultaneous detection of TB infection and INH resistance. Partial sequencing of the katG gene was used to confirm INH resistance results. A standard 15 Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (15-MIRU-VNTR) approach was used for genotyping through the MIRU-VNTRplus online platform.Results. Only seven specimens showed phenotypic resistance to INH. M. tuberculosis was detected in all samples, while a mutation in the katG gene codon 315 was detected only in five samples, which were also phenotypically INH-resistant. Sequencing of the katG gene showed codon 315 mutation genotypically and phenotypically in the five INH-resistant isolates. Molecular genotyping of M. tuberculosis isolates revealed that the majority of isolates (26/50, 52 %) belonged to the S family of lineage_4. A low clustering rate (2 %) was observed among our isolates. According to the Hunter-Gaston Discriminatory Index (HGDI), 11 MIRU-VNTR loci were highly or moderately discriminative, while four loci were less polymorphic.Conclusion. MIRU-VNTR genotyping revealed a low clustering rate with a low recent transmission rate of M. tuberculosis strains in Alexandria, Egypt.

A comparative analysis of molecular genotypes of Mycobacterium tuberculosis isolates from HIV-positive and HIV-negative patients.

SettingTuberculosis Research Laboratory, Division of Clinical Microbiology and Molecular Medicine, Department of Laboratory Medicine, All India Institute of Medical Sciences, and the National Institute of Tuberculosis and Respiratory Diseases (NITRD), both situated in New Delhi.ObjectivesWe aimed to identify the distribution of various genotypes of M. tuberculosis among HIV-positive and HIV-negative patients suspected of having Tuberculosis, seen at the National Institute of Tuberculosis and Respiratory Diseases, New Delhi, which is a tertiary care dedicated TB hospital.Patients and methodsGenotyping by Spoligotyping and 24 loci MIRU-VNTR was performed and analyzed using SITVITWEB and MIRU-VNTRplus. Drug susceptibility patterns were also analyzed.ResultsA total of 503 subjects who were PTB/EPTB suspected were recruited and 287 were culture positive. Among them, 276 had growth of Mycobacterium tuberculosis (MTB) and in 11 patients non-tuberculous mycobacteria (NTM) were grown. The isolation rate of NTM was predominantly from HIV positive [10 of 130 (7.6%)] patients. Of the total isolates of MTB, 156 (56.5%) were from HIV negative patients and 120 (43.5%) were from HIV positive patients. All 276 M. tuberculosis isolates were genotyped and tested for drug susceptibility patterns. The CAS genotype was most predominant [153 (55.4%)], followed by Beijing lineage [44 (15.9%)], East African India [25 (9.1%)] and others [54 (19.6%)]. Beijing genotype was significantly more common in HIV positive patients (22.5%) than in HIV negative patients (10.9%). In MIRU-VNTR analysis, clustering was found to be more frequent in CAS strains irrespective of HIV status. In the HIV positive group, spoligotyping could differentiate various genotypes in 90% of isolates and MIRU-VNTR analysis in 84.2% of isolates. The clustering of various MTB strains was more associated with drug resistance.ConclusionThe Beijing lineage was predominant in HIV-TB coinfected cases, even though the Central Asian Strain (CAS) was overall more predominant in the region.

Prevalence and transmission of pyrazinamide-resistant Mycobacterium tuberculosis in Hunan Province,China

Objective: To provide a scientific reference for the prevention and treatment of pyrazinamide-resistant tuberculosis (PZA-R TB), we analyzed the prevalence and risk factors of pyrazinamide-resistant tuberculosis in Hunan province and described the genotyping and clustering characteristics of the pyrazinamide-resistant Mycobacterium tuberculosis (PZA-R MTB) isolates. Methods: The drug susceptibility test results of first-line anti-tuberculosis drugs including isoniazid (INH), rifampicin (RFP), streptomycin (SM), ethambutol (EMB) and pyrazinamide (PZA), and the characteristics of patients were collected from 3 862 tuberculosis patients in Hunan Chest Hospital (Institute of Tuberculosis Control and Prevention) from January 2016 to December 2018. The prevalence of PZA-R TB was calculated and risk factors were analyzed by univariate and multivariable logistic regression analysis. Two hundred and twelve Mycobacterium tuberculosis isolates selected from June 2017 to June 2018 were genotyped using the 24-loci MIRU-VNTR system. The genetic difference value (h), and the Hunter-Gaston index (HGI) were used to evaluate the resolution and variation for the 24 loci. MIRU-VNTR results were analyzed using BioNumerics 5.0 software to conduct cluster analysis. Clustered isolates were further analyzed by pncA gene sequencing. Results: The rate of PZA-R TB among tuberculosis patients and MDR patients was 14.7%(566/3 862) and 60.5%(511/844), respectively. Multivariable logistic regression analysis showed that patients who were INH mono-resistance and MDR had a higher risk of developing PZA resistance, compared with TB patients who were pan-sensitive to anti-TB drugs (INH, RFP, SM, and EMB). The adjusted OR value (95%CI) was 13.08(5.67-30.18), 298.41(164.88-540.08), respectively, and P values were all less than 0.01. Clustering analysis showed that 65 strains formed 19 clusters, the clustering rate was 30.7%(65/212). Of 19 clusters, eight clusters had at least two isolates with identical pncA mutation types within each cluster. In eight clusters, cluster 4, 6, 16 had four, three, and two patients who lived in the same county, respectively, thus providing probable epidemiological links for the recent transmission of PZA-R Mycobacterium tuberculosis. At least 47.6%(101/212) of PZA drug-resistant TB patients were suggestive of primary drug resistance caused by transmission. Conclusions: The prevalence of PZA-R TB was severe in Hunan province. PZA susceptibility testing should be performed for isolates resistant to any first-line anti-tuberculosis drugs, especially for MDR-MTB isolates. Nearly half of tuberculosis patients were suggestive of primary drug resistance caused by transmission. The prevention and treatment strategy of PZA-R TB should focus on the standardized treatment and management of patients as well as control of the source of infection.

A retrospective cohort study on the treatment outcomes and genotyping of isoniazid-resistant tuberculosis patients in Eastern China

Isoniazid resistance might be overlooked because of the priority of detection of rifampicin-resistant tuberculosis. It was urgent to reveal the current situation of isoniazid-resistant tuberculosis (HR-TB), including unfavorable outcomes and bacterial factors. A retrospective cohort study was undertaken including 120 patients with HR-TB and 193 patients with drug-sensitive tuberculosis (DS-TB). 24-loci MIRU-VNTR and Spoligotyping were adopted for genotyping. We found 106 cases (88.3%) of HR-TB and 165 cases (85.5%) of DS-TB were treated with the first-line drugs. Meanwhile, 12 (10.0%) patients of the HR-TB group and 7 (3.63%) patients of the DS-TB group involved adverse treatment outcomes (χ2=5.271, P=0.028). Seventy-eight DNA from HR Mycobacterium tuberculosis and 114 DNA from DS M. tuberculosis were available for MIRU-VNTR and Spoligotyping. The clustering rate was 17.9% (14/78) for HR-TB and 16.7% (19/114) for DS-TB, and reached no significant difference (χ2=0.05, P=0.8171). The Beijing family strains accounted for 83.7% (65/78) of HR-TB and 80.0% (91/114) of DS-TB (χ2=0.37, P=0.5407). The adverse treatment outcomes for HR-TB all occurred in patients infected with Beijing family strains (13.8%), but no difference was found between Beijing and non-Beijing genotypes (P=0.342). Adverse outcomes were significantly more frequent in patients with HR-TB than in those with DS-TB, and most of the patients with HR-TB were receiving a standard first-line regimen. Although the clustering rate and Beijing genotype distribution amongst HR-TB and DS-TB showed no significant difference, the Beijing genotype was the dominant genotype and its proportion was slightly higher amongst HR-TB than amongst DS-TB.

Association between Mycobacterium tuberculosis genotype and diabetes mellitus/hypertension: a molecular study

BackgroundA paucity of studies focused on the genetic association that tuberculosis (TB) patients with non-communicable diseases (NCDs) are more likely to be infected with Mycobacterium tuberculosis (MTB) with more potent virulence on anti-TB drug resistance than those without NCDs. The study aimed to document the predominant genotype, determine the association between MTB genotypes and NCD status and drug resistance.MethodsWe conducted a molecular study in 105 TB patients based on a cross-sectional study focused on the comorbid relationship between chronic conditions and TB among 1773 subjects from September 1, 2019 to August 30, 2020 in Guizhou, China. The participants were investigated through face-to-face interviews, followed by NCDs screening. The DNA of MTB isolates was extracted prior to genotyping using 24 loci MIRU-VNTR. The subsequent evaluations were performed by phylogenetic trees, combined with tests of statistical power, Chi-square or Fisher and multivariate logistic regression analysis.ResultsThe Beijing family of Lineage 2 (East Asia) was the predominant genotype accounting for 43.8% (46/105), followed by Lineage 4 (Euro-America) strains, including Uganda I (34.3%, 36/105), and the NEW-1 (9.5%, 10/105). The proportion of Beijing strain in patients with and without NCDS was 28.6% (8/28) and 49.4% (38/77), respectively, with a statistical power test value of 24.3%. No significant association was detected between MTB genotype and NCD status. A low clustering rate (2.9%) was identified, consisting of two clusters. The rates of global, mono-, poly- and multi-drug resistance were 16.2% (17/105), 14.3% (15/105), 1.0% (1/105) and 4.8% (5/105), respectively. The drug-resistant rates of rifampicin, isoniazid, and streptomycin, were 6.7% (7/105), 11.4% (12/105) and 5.7% (6/105), respectively. Isoniazid resistance was significantly associated with the Beijing genotype of Lineage 2 (19.6% versus 5.1%).ConclusionsThe Lineage 2 East Asia/Beijing genotype is the dominant genotype of the local MTB with endogenous infection preponderating. Not enough evidence is detected to support the association between the MTB genotype and diabetes/hypertension. Isoniazid resistance is associated with the Lineage 2 East Asia/Beijing strain.

Diagnostic and public health investigation of Mycobacterium tuberculosis infection in a dog in Ontario, Canada

A 4-y-old, female mixed-breed dog was presented to the Ontario Veterinary College for further evaluation of multiple pulmonary and hepatic masses, intrathoracic lymphadenitis, and recent development of a pyogranulomatous pleural effusion. Along with other comprehensive tests, a thoracic lymph node biopsy was performed, and Mycobacterium tuberculosis complex infection was confirmed by real-time PCR. The dog’s condition declined post-operatively, and euthanasia was elected. Postmortem examination confirmed severe granulomatous pneumonia, hepatitis, intrathoracic and intraabdominal lymphadenitis, omentitis, and nephritis. Line-probe assays performed on samples collected postmortem confirmed the species as M. tuberculosis. 24-loci MIRU-VNTR genotyping, spoligotyping, and whole-genome sequencing revealed relations to known human isolates, but no epidemiologic link to these cases was investigated. Given the concern for potential human exposure during this animal’s disease course, a public health investigation was initiated; 45 individuals were tested for M. tuberculosis exposure, and no subsequent human infections related to this animal were identified. Our case highlights the need for more readily available, minimally invasive testing for the diagnosis of canine mycobacteriosis, and highlights the ability of canid species to act as potential contributors to the epidemiology of M. tuberculosis infections.

Molecular characterization of Mycobacterium bovis strains isolated from cattle and humans by spoligotyping and 24-locus MIRU-VNTR, and prevalence of positive IGRA in slaughterhouse workers in Southern Turkey.

Mycobacterium bovis is a zoonotic member of the Mycobacterium tuberculosis complex with a wide range of hosts, mainly cattle. Molecular epidemiological studies should be conducted to determine the transmission route, zoonotic risk factors, and phylogenetic relationships of M. bovis strains. Aims: This study aimed to characterize bovine and human M. bovis isolates by molecular methods. Molecular characterization and clonal relationship of strains isolated from tissue and organ samples of 76 cattle with positive tuberculin tests were collected from a slaughterhouse, and four M. bovis strains isolated from clinical materials of patients with suspected pulmonary TB isolates were analyzed using 24-locus MIRU-VNTR and spoligotyping methods. QuantiFERON-TB Gold Plus (QFT-Plus; Qiagen) was used to determine the prevalence of latent TB infection among 21 slaughterhouse personnel including 7 veterinarians, 12 butchers, 1 caretaker, and 1 veterinary technician. SB0288/SIT685 type was detected in both cattle and humans by the spoligotyping method. When evaluating MIRU-VNTR, the presence of a 100% compatible pattern between human and bovine isolates was not detected, but some human samples were found to be 91.6% similar to a bovine sample. In addition, 21 slaughterhouse workers were screened with the interferon gamma-released assay (IGRA) and a 23.8% positivity was detected. Clonal similarity was determined between the bovine and human isolates using the MIRU-VNTR and spoligotyping methods and IGRA positivity in the occupational group suggested that M. bovis might be associated with pulmonary tuberculosis in humans.

Genotypic Characterization of Mycobacterium bovis Isolates From Dairy Cattle Diagnosed With Clinical Tuberculosis.

Molecular diagnosis of bovine tuberculosis plays an essential role in the epidemiological knowledge of the disease. Bovine tuberculosis caused by Mycobacterium bovis represents a risk to human health. This study aimed to perform the genotypic characterization of M. bovis isolated from bovines diagnosed as tuberculosis from dairy herds in the state of Pernambuco, Brazil. Granulomas from 30 bovines were sent for microbiological culture, and colonies compatible with Mycobacterium spp. were obtained in at least one culture from 17/30 granulomas. All isolates were confirmed to be M. bovis by spoligotyping and 24loci MIRU-VNTR typing. While spoligotyping characterized the isolates as SB0121, SB0295, SB0852, SB0120, and an unclassified genotype, 24loci MIRU-VNTR rendered two clusters of two isolates each and 13 unique profiles. Loci ETR-A showed higher discriminatory power, and loci (ETR-B, ETR-C, MIRU16, MIRU27, and QUB26) showed moderate allelic diversity. This is the first study on the genetic variability of the infectious agent cause of bovine TB in Pernambuco and demonstrates variability of strains in the state. Thus, it corroborates the importance of this microorganism as agent of bovine tuberculosis and its zoonotic potential, this epidemiological tool being a determinant in the rigor of the sanitary practices of disease control in dairy herds.

Genotypic diversity of multi- and pre-extremely drug-resistant Mycobacterium tuberculosis isolates from Morocco.

In Morocco, the prevalence of multidrug resistant tuberculosis (MDR-TB) continues to increase especially within previously treated cases; these MDR cases may evolve to extensively drug resistant tuberculosis (XDR-TB) raising major concern to TB control programs. From an epidemiological window, scarce informations are available about the genetic diversity of Mycobacterium tuberculosis (MTB) strains fueling these forms of resistance. The aim of this study was to assess to genetic diversity of MDR-MTB strains. Hence, this prospective study was conducted on patients diagnosed with MDR-TB at Pasteur Institute of Casablanca from 2010 to 2013. A total of 70 MDR-MTB isolates were genotyped by spoligotyping and 15-loci MIRU-VNTR methods. Spoligotyping generated four orphan patterns, five unique profiles whereas 61 strains were grouped in nine clusters (2 to 25 strains per cluster), the clustering rates being 87.1%. Subtyping by 15 loci MIRU-VNTR splitted all clusters already established by spoligotyping and generated 70 unique profiles not recognized in SITVIT2 database; clustering rate was equal to zero. HGDI analysis of 15 loci MIRU demonstrated that eight out of 15 loci were highly discriminant. Of note, all pre-XDR strains belongs to many clades, meaning that there no association between gyrA mutants and particular clade. Overall, the data generated by this study (i) describe the population structure of MDR MTBC in Morocco which is highly homogenous, (ii) confirm that TB in Morocco is almost exclusively transmitted by modern and evolutionary lineages with high level of biodiversity seen by MIRU, and (iii) validate the use of optimized 15-loci MIRU-VNTR format for future investigations in Morocco.

Molecular diversity of Mycobacterium tuberculosis complex in Sikkim, India and prediction of dominant spoligotypes using artificial intelligence

In India, tuberculosis is an enormous public health problem. This study provides the first description of molecular diversity of the Mycobacterium tuberculosis complex (MTBC) from Sikkim, India. A total of 399 Acid Fast Bacilli sputum positive samples were cultured on Lőwenstein–Jensen media and genetic characterisation was done by spoligotyping and 24-loci MIRU-VNTR typing. Spoligotyping revealed the occurrence of 58 different spoligotypes. Beijing spoligotype was the most dominant type constituting 62.41% of the total isolates and was associated with Multiple Drug Resistance. Minimum Spanning tree analysis of 249 Beijing strains based on 24-loci MIRU-VNTR analysis identified 12 clonal complexes (Single Locus Variants). The principal component analysis was used to visualise possible grouping of MTBC isolates from Sikkim belonging to major spoligotypes using 24-MIRU VNTR profiles. Artificial intelligence-based machine learning (ML) methods such as Random Forests (RF), Support Vector Machines (SVM) and Artificial Neural Networks (ANN) were used to predict dominant spoligotypes of MTBC using MIRU-VNTR data. K-fold cross-validation and validation using unseen testing data set revealed high accuracy of ANN, RF, and SVM for predicting Beijing, CAS1_Delhi, and T1 Spoligotypes (93–99%). However, prediction using the external new validation data set revealed that the RF model was more accurate than SVM and ANN.

Genetic diversity of Mycobacterium tuberculosis using 24-locus MIRU-VNTR typing and Spoligotyping in Upper Myanmar

MIRU-VNTR typing and Spoligotyping are the useful molecular tools for TB epidemiology study. Information regarding genetic diversity and tuberculosis (TB) transmission in Upper Myanmar only is scares. We determined the genetic diversity of Mycobacterium tuberculosis (Mtb) and TB transmission from Upper Myanmar TB Reference Laboratory, Mandalay Region, including Mandalay (72), Shan (22), Magway (15), Sagaing (13), Nay Pyi Taw (8), Kachin (7), Chin (2) and Kayah (1). One hundred and forty Mtb isolates were genotyped using 24-locus MIRU-VNTR typing and spoligotyping. Lineage classification and TB transmission analysis were performed. 24-locus MIRU-VNTR typing identified 135 unique profiles and two clusters compared to 35 spoligotyping profiles which contained 12 clusters and 23 unique isolates, Beijing (n=100, 71.4%) was found to be prominent lineage by combine two methods. The expected proportion attributable to recent transmission based on clustering rate was 2.1%. One cluster case was more likely to be in MDR patient. Our findings showed Beijing genotypes were dominant in Upper Myanmar. The usage and analysis of 24-locus MIRU-VNTR typing might prove useful for our broader understanding of TB outbreaks and epidemiology than spoligotyping. The genotypic pattern of this combined method suggests that the lower transmission rate may be due to a higher possibility of reactivation cases in Upper Myanmar.