Mycobacterial Interspersed Repetitive Units-variable Number Of Tandem Repeats Research Articles

24 loci MIRU-VNTR analysis and pattern of drug resistance in pre-extensively drug resistant pulmonary tuberculosis in Bangladesh.

Phylogenetic diversity and distinct phylogeographic distribution of Mycobacterium tuberculosis (MTB) contribute to regional differences in drug resistance. The emergence of pre-extensively drug resistant tuberculosis (Pre-XDR-TB) becomes obstacles to achieve End TB strategy in Bangladesh. This cross-sectional study was conducted to identify the strains of different lineages of MTB, their variations of distribution among Pre-XDR-TB cases and to observe the linkage of particular strains of MTB with drug resistance. A total of 33 Pre-XDR-TB isolates were enrolled in this study. All isolates were confirmed as MTB by MPT 64 antigen detection and genotyped by 24 loci Mycobacterial Interspersed Repetitive Unit-Variable Number of Tandem Repeats (MIRU-VNTR) analysis. Drug resistance was detected by second line Line probe assay (LPA). Beijing was the predominant strain 16 (48.48%), followed by Delhi/CAS 5(15.15%), LAM 4 (12.12%) and Harlem 3(9.10%), EAI 2(6.06%), Cameroon 2(6.06%) and NEW-1 1(3.03%). There were 31 different genotypes consisting of 2 clusters and 29 singletons. All the clustered strains were belonged to Beijing lineage. Recent transmission occurred manly by Beijing strains, showed low transmission rate (12.1%). Of 33 isolates 30(90.90%) were Fluoroquinolones resistant, the mutations involved was Asp94Gly in gyr A MUT 3C gene 13(39.39%) in quinolone resistance determining region (QRDR) followed by 11 (33.33%) in gyr A MUT 1. Three (9.10%) isolates showed resistant to injectable 2nd line drugs and all mutation occurs in G1484T of rrs MUT 2. Beijing lineage was predominant in treatment failure and relapse cases. Levofloxacin was resistant to all Pre-XDR-TB cases, but moxifloxacin showed low level resistance. QUB 26 was the most discriminatory locus (0.85) among 24 loci whereas MIRU 2 was the least (0.03). 24 loci MIRU-VNTR analysis shows high discriminatory index (0.71), found to be powerful tool for genotyping of Pre-XDR-TB, which is the first study in Bangladesh that enhanced the current TB control policy.

Mycobacteriosis in Various Pet and Wild Birds from Germany: Pathological Findings, Coinfections, and Characterization of Causative Mycobacteria.

ABSTRACTA total of 50 birds diagnosed with mycobacteriosis were examined for pathomorphological lesions, coinfections, and causative agents. Mycobacterial species were identified and isolates differentiated using multilocus sequence typing (MLST) and mycobacterial interspersed repetitive-unit variable-number of tandem-repeat (MIRU-VNTR) analysis. Possible associations between mycobacterial species, pathomorphological findings, coinfections, bird orders, and husbandry conditions were evaluated statistically. Mycobacteria were isolated from 34 birds (13 of 22 Psittaciformes, 12 of 18 Passeriformes, five of six Columbiformes, and four other orders) belonging to 26 species in total. Mycobacterium genavense (Mg) was cultured from 15 birds, Mycobacterium avium subsp. avium (Maa) from 20 birds, and Mycobacterium avium subsp. hominissuis (Mah) from three birds; hence, four birds had mixed infections. About equal numbers of psittacines and passerines were infected with Ma and Mg. The genetic diversity differed; Mg isolates belonged to one MLST type, Maa to six, and Mah to three combined genotypes. Several coinfections were detected; viruses and/or endoparasites affected 44%, fungi 38%, and bacteria 29% of the birds. Pathological findings and mycobacteriosis-affected organs were independent of coinfections. Overall, gross pathological findings were more often seen in mycobacteriosis caused by Ma (95%) compared with Mg (66%). Organ distribution of mycobacteriosis was independent of the mycobacterial species. Pathomorphological changes were seen in the small intestine of 71% and the lung of 65% of the birds, suggesting oral or pulmonal ingestion of mycobacteria. There were no associations between mycobacterial species and bird orders or bird husbandry conditions. Not only Mg, but also Maa and Mah, were clearly identified as primary cause of mycobacteriosis in pet birds.IMPORTANCE In this study, the causative agents and confounding factors of mycobacteriosis in a set of pet and some wild birds from Germany were examined. Not only Mycobacterium genavense, but also M. avium subsp. avium and M. avium subsp. hominissuis, contributed to mycobacteriosis in these birds. Various coinfections did not affect the manifestation of mycobacteriosis. Due to different gross necropsy findings, however, a different pathogenicity of the two species was assumed. New strains of M. avium subsp. hominissuis originating from birds were identified and characterized, which is important for epidemiological studies and for understanding the zoonotic role of this pathogen, as the subsp. hominissuis represents an increasing public health concern. The study provides some evidence of correlation between M. avium subsp. avium genotypes and virulence which will have to be confirmed by broader studies.

Country-wide genotyping of Mycobacterium tuberculosis complex in Singapore, 2011–2017

ObjectiveTo describe the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) and factors associated with its transmission in Singapore. MethodsSpoligotyping, 24-loci mycobacterial interspersed repetitive units – variable number of tandem repeats (MIRU-VNTR) typing and demographic data from the national TB notification registry of MTBC culture-positive cases notified from January 2011 to December 2017 were analysed. ResultsOf the 12,046 culture-positive cases notified, complete spoligotyping and MIRU-VNTR typing results were available for 8690 (72.1%) belonging to 4950 (57.0%) local-born and 3740 (43.0%) foreign-born persons. From these, 4810 (55.3%) were identified in 883 clusters. The proportion of recent transmission was 45.2%. The East-Asian Lineage 2 accounted for 4045 (47.1%) of isolates, and disproportionately accounted for large clusters. Clustered cases were more likely to be older than 50 years, male, Malay, local-born, Singapore citizens or Permanent Residents, of lower socioeconomic status, imprisoned; to harbour East-Asian Lineage 2 strain; to have cavitary pulmonary TB, positive sputum smear or be recalcitrant treatment defaulters. They were less likely to have multidrug-resistant, or isoniazid or rifampicin mono-resistant TB. ConclusionWe demonstrated the diversity of MTBC strains and, notwithstanding the likely over-estimation of clustering using these genotyping methods, elucidated factors associated with TB transmission in Singapore.

Genetic Diversity and Transmission of Multidrug-Resistant Mycobacterium tuberculosis strains in Lusaka, Zambia

Zambia is among the 30 high tuberculosis burden countries in the world. Despite increasing reports of multidrug-resistant tuberculosis (MDR-TB) in routine surveillance, information on the transmission of MDR Mycobacterium tuberculosis strains is largely unknown. This study elucidated the genetic diversity and transmission of MDR M. tuberculosis strains in Lusaka, Zambia. Eighty-five MDR M. tuberculosis samples collected from 2013 to 2017 at the University Teaching Hospital were used. Drug-resistance associated gene sequencing, spoligotyping, 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR), and multiplex PCR for RD-Rio sub-lineage identification were applied. The identified clades were LAM (48%), CAS (29%), T (14%), X (6%) and Harlem (2%). Strains belonging to SITs 21/CAS1-Kili and 20/LAM1 formed the largest clonal complexes. Combined spoligotyping and 24 loci-MIRU-VNTR revealed 47 genotypic patterns with a clustering rate of 63%. Ninety-five percent of LAM strains belonged to the RD-Rio sub-lineage. The high clustering rate suggested that a large proportion of MDR-TB was due to recent transmission rather than the independent acquisition of MDR. This spread was attributed to clonal expansion of SIT21/CAS1-Kili and SIT20/LAM1 strains. Therefore, TB control programs recommending genotyping coupled with conventional epidemiological methods can guide measures for stopping the spread of MDR-TB.

Mycobacterium tuberculosis typing using Allele-specific oligonucleotide multiplex PCR (ASO-PCR) method.

Mycobacterium tuberculosis (M. tuberculosis) genotyping provides valuable information related to the origin and the evolution of the isolates. This study aimed to evaluate the applicability of single-nucleotide polymorphisms (SNPs) technique for lineages identification of M. tuberculosis and compare it with mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) method. The lineages of 162 clinically isolates were evaluated using six pair primers by Multiplex-PCR based on SNPs. Among 162 isolates, 70 (43.2%) isolates were lineage 4, following that 62 (38.3%) and 22 (13.6%) isolates were lineage 3 and 2, respectively. The method could not type 8 (4.9%) isolates. Moreover, we could identify 71 out of 79 unknown isolates resulted from the MIRU-VNTR method. The results showed that the SNP typing method has the potential to determine the lineages of M. tuberculosis as a rapid laboratory screening test.

Genetic diversity and primary drug resistance transmission in Mycobacterium tuberculosis in southern Mexico

Tuberculosis is a global human health threat, especially in developing countries. The present study aimed to describe the genetic diversity of Mycobacterium tuberculosis and to measure the transmission rates of primary and acquired resistance. A total of 755 M. tuberculosis isolates from a cohort study of patients with culture-confirmed pulmonary tuberculosis in Orizaba, Veracruz, performed between 1995 and 2010 were genotyped by the 24-locus mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) method. Drug susceptibility was determined. Logistic regression models were constructed to identify the variables associated with resistance and clusters. The recent transmission index (RTI), the Hunter-Gaston discrimination index (HGDI) for the MIRU-VNTR test and allelic diversity (h) were calculated. The Haarlem and LAM lineages were the most common in the population. A total of 519 isolates were grouped into 128 clusters. The overall drug resistance rate was 19%, isoniazid monoresistance (10%) was the most common, and 3.4% of the isolates were multidrug resistant. Among the 116 isolates resistant to at least one drug, the primary and acquired resistance rates were 81.9% and 18.1%, respectively. Primary resistance was associated with belonging to a cluster (aOR 4.05, 95% CI 1.5-11.2, p = 0.007). Previous treatment history (aOR 9.05, 95% CI 3.6-22.5, p < 0.001) and LAM lineage (aOR 4.25, 95% CI 1.4-12.7, p = 0.010) were associated with multidrug-resistant tuberculosis (MDR-TB). The RTI was 51.7%, and the 24-locus MIRU-VNTR HGDI was 0.98. The alleles with the greatest diversity were 4056-QUB26 (h = 0.84), 2163b-QUB11b (h = 0.79), and 424-Mtub04 (h = 0.72). Primary resistance transmission, high LAM lineage prevalence and its association with MDR-TB represent public health problems. The implementation of molecular tools is needed to improve the existing control surveillance tuberculosis program.

Genetic diversity of Mycobacterium bovis evaluated by spoligotyping and MIRU‐VNTR in an intensive dairy cattle breeding area in Mexico

Bovine tuberculosis (bTB) is mainly caused by Mycobacterium bovis. In Mexico, dairy cattle play an important role in the persistence and spread of the bacillus. In order to describe M.bovis genetic diversity, we genotyped a total of 132 strains isolated from slaughtered cattle with bTB suggestive lesions between 2009 and 2010 in Hidalgo, Mexico, using a panel of 9-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) and spoligotyping. We found 21 spoligotypes, and 124 isolates were grouped in 13 clusters. The most frequent spoligotypes were SB0121 (49, 37.1%) and SB0673 (27, 20.5%); three new spoligotypes were reported SB02703, SB02704 and SB02705. We observed 37 MIRU-VNTR patterns, 107 isolates were grouped in 12 clusters and 25 isolates were unique. Spoligotypes SB0121, SB0673, SB0140, SB0145 and SB0120 showed marked subdivision applying MIRU-VNTR method; meanwhile, spoligotypes SB0971 and SB0327 showed single MIRU-VNTR profiles. The Hunter-Gaston discriminatory index (HGDI) was 0.88, 0.78 and 0.90 for 9-loci MIRU-VNTR, spoligotyping and both methods, respectively. Additionally, allelic diversity (h) analysis showed high diversity for QUB3232, QUB26 and QUB11b with h=0.79, 0.66 and 0.63, respectively. Overall, high genetic variability was observed among M.bovis isolates. Thus, the use of 9-loci MIRU-VNTR panel is enough to describe genetic diversity, evolution and distribution of M.bovis. This study supports the use of these tools for subsequent epidemiological studies in high incidence areas.

Integrative transnational analysis to dissect tuberculosis transmission events along the migratory route from Africa to Europe.

Growing international migration has increased the complexity of tuberculosis transmission patterns. Italy's decision to close its borders in 2018 made of Spain the new European porte entrée for migration from the Horn of Africa (HA). In one of the first rescues of migrants from this region at the end of 2018, tuberculosis was diagnosed in eight subjects, mainly unaccompanied minors. Mycobacterium tuberculosis isolates from these recently arrived migrants were analysed by Mycobacterial Interspersed Repetitive-Unit/Variable-Number of Tandem Repeat (MIRU-VNTR) and subsequent whole genome sequencing (WGS) analysis. Data were compared with those from collections from other European countries receiving migrants from the HA and a strain-specific PCR was applied for a fast searching of common strains. Infections in a cellular model were performed to assess strain virulence. MIRU-VNTR analysis allowed identifying an epidemiological cluster involving three of the eight cases from Somalia (0 single-nucleotide polymorphisms between isolates, HA cluster). Following detailed interviews revealed that two of these cases had shared the same migratory route in most of the trip and had spent a long time at a detention camp in Libya. To confirm potential en route transmission for the three cases, we searched the same strain in collections from other European countries receiving migrants from the HA. MIRU-VNTR, WGS and a strain-specific PCR for the HA strain were applied. The same strain was identified in 12 cases from Eritrea diagnosed soon after their arrival in 2018 to the Netherlands, Belgium and Italy. Intracellular replication rate of the strain did not reveal abnormal virulence. Our study suggests a potential en route transmission of a pan-susceptible strain, which caused at least 15 tuberculosis cases in Somalian and Eritrean migrants diagnosed in four different European countries.

Molecular epidemiology of Mycobacterium tuberculosis in adult Filipino TB-HIV co-infected patients.

BACKGROUND: TB is the leading cause of death from a single infectious disease, particularly among people living with HIV (PLHIV). Molecular epidemiology provides information on prevalent genotypes of Mycobacterium tuberculosis and disease transmission dynamics, which aid in TB control. Identification of mutations that confer drug resistance is essential for the rapid diagnosis of drug-resistant TB, especially in high TB burden settings, like the Philippines.METHODS: This study aimed to determine mutations in M. tuberculosis drug resistance-conferring genes and circulating genotypes in PLHIV. MIRU-VNTR (mycobacterial interspersed repetitive unit-variable number of tandem repeats) typing using a set of 24-loci and sequencing of drug resistance-conferring genes were performed in 22 M. tuberculosis isolates from TB-HIV co-infected patients.RESULTS: The prevalence of resistance to any drug was 31.8%, 18.2% for isoniazid monoresistance, 4.5% for streptomycin monoresistance and 9.1% for multidrug resistance. The identified mutations in the katG, rpoB, pncA, rpsL and gyrA genes have been reported in the literature; none was found in the inhA and embB genes. All isolates belonged to the EAI2-Manila family and were grouped into four clusters based on their phenotypic drug resistance and mutation profiles.CONCLUSION: The use of 24-loci set may be used as a more discriminatory MIRU-VNTR typing in settings where the East African-Indian lineage is predominant, like the Philippines.

Overlapping prison/community tuberculosis outbreaks in Costa Rica revealed by alternative analysis of suboptimal material.

Costa Rica has a low incidence of tuberculosis. Thus, identifying transmission hotspots is key to implement interventions. A tuberculosis outbreak was suspected in a prison in Costa Rica. Given the suboptimal quality of the samples received in our laboratory in Madrid, we applied alternative schemes for their analysis. In the first scheme, we bypassed the standard approach of applying systematic mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) and used a strain-specific polymerase chain reaction (PCR) that allowed identifying a cluster involving six cases (C1). The second scheme followed the canonical MIRU-VNTR path coupled with a whole-genomic amplification step, by which a second unsuspected overlapping cluster (C2), was detected in the same prison. These findings justified the implementation of a surveillance programme adapted to local resources based on a tailored multiplex allele-specific oligonucleotide (ASO)-PCR targeting C1 and C2. Presence of the C2 strain at a different prison was determined. ASO-PCR was applied extensively and alerted to the active circulation of one of the strains within and beyond prisons. Our study shows that alternative methodological strategies may provide useful data in settings with lack of resources for performing systematic standard molecular epidemiology programmes and/or with suboptimal material for analysis.

Molecular Epidemiologic Analysis of Mycobacterium tuberculosis among Prison Inmates in Selected Prisons in the Philippines

Tuberculosis (TB) remains an important public health problem in developing countries like the Philippines. The success of the National TB Control Program depends on a clear understanding of the dynamics of transmission and spread of TB in high-risk populations in the community. We conducted a molecular epidemiologic analysis of M tuberculosis isolates collected from inmates with pulmonary TB in selected prisons in the Philippines. A total of 25 isolates were characterized and genotyped using Spoligotyping and 15-loci MIRU-VNTR (mycobacterial interspersed repetitive units–variable number of tandem repeats) typing. The majority of the patients were male (84%) and aged 30–49 yr old (68%). Eighteen (72%) of the culture-positive patients had severe pulmonary TB, 13 (52%) were smear-positive, and seven (28%) were classified as having a high bacillary load. Twenty isolates (80%) were susceptible to all the first-line drugs. Two (8%) were multidrug- resistant (MDR) and isolated from patients in the same prison, one of which was resistant to all first-line drugs. Three isolates (12%) were streptomycin-monoresistant. There were nine identified Spoligo-International Types (SITs), with SIT19 as the predominant (40%). One isolate (4%) did not match any SIT in the SpoIDB4 database, while three were not assessed due to inadequate DNA for analysis. The distribution of strains according to major M tuberculosis clades were as follows: EAI2_Manilla (48%) &gt; LAM2 (20%) &gt; LAM6 (8%) = LAM9 (8%). Spoligotyping identified two clusters and 13 genotypes (four unique strains) with a Hunter-Gaston discriminatory index (HGDI) of 0.83. MIRU-VNTR typing identified two clusters and 23 genotypes (HGDI = 0.993). Combined Spoligotyping and MIRU-VNTR typing also identified two clusters and 23 genotypes (HGDI = 0.993). There were no significant associations shown among host demographic factors, severity of the disease, drug resistance, and M tuberculosis strain. We conclude that our patient population was infected predominantly by M tuberculosis belonging to the EAI2_Manila clade

Circulating strains of Mycobacterium tuberculosis: 24 loci MIRU-VNTR analysis in Bangladesh.

Bangladesh is among the high burden countries for tuberculosis (TB) and multidrug resistant TB (MDR-TB). As the genetic diversity and distinct phylogeographic distribution of Mycobacterium tuberculosis are responsible for regional differences in drug resistance, this cross sectional study was conducted to identify the circulating M. tuberculosis strains belonging to different lineages among pulmonary tuberculosis and, to investigate the contribution of distinct M. tuberculosis lineages to rifampicin resistant (RR) and rifampicin sensitive (RS) TB. A total of 40 RR and 20 RS isolates were enrolled in this study, all of which confirmed as M. tuberculosis by MPT 64 antigen detection. Furthermore, all isolates were genotyped by 24 loci Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR), thus comprising the first study to employ this approach in Bangladesh. Beijing was the predominant lineage (26.8%) followed by EAI (23.2%), Delhi/CAS (16.1%), H37Rv (8.9%), Haarlem (7.1%), LAM (5.4%), Cameroon (3.6%) and a NEW-1 (1.8%). Four (7.1%) isolates remained as unidentified. Beijing strains were the significantly predominant (36.8%; p=0.0135) among the RR isolates in comparison with other strains whereas EAI was the predominant (38.8%) lineage among RS isolates. Also, approximately 13% RR isolates showed genotypic resistance against fluoroquinolones by LPA and, hence, classed as pre-XDR TB albeit no specific lineage was found associated with these latter strains. A low transmission rate (10.5%) and high genetic diversity was detected in this setting with all the clustered strains herein identified belonging to the Beijing lineage. This study highlights 24 loci MIRU-VNTR analysis as a powerful tool for genotyping of Mycobacterium tuberculosis in this setting as it shows a high discriminatory index (0.81).

Molecular epidemiology of clinical Mycobacterium tuberculosis complex isolates in South Omo, Southern Ethiopia

BackgroundTuberculosis (TB) is caused by Mycobacterium tuberculosis complex (MTBC). Mapping the genetic diversity of MTBC in high TB burden country like Ethiopia is important to understand principles of the disease transmission and to strengthen the regional TB control program. The aim of this study was to investigate the genetic diversity of Mycobacterium tuberculosis complex (MTBC) isolates circulating in the South Omo, southern Ethiopia.MethodsMTBC isolates (N = 156) were genetically analyzed using spacer oligotyping (spoligotyping) and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing. Major lineages and lineages were identified using MTBC databases. Logistic regression was used to correlate patient characteristics with strain clustering.ResultsThe study identified Euro-American (EA), East-African-Indian (EAI), Indo-Oceanic (IO), Lineage_7/Aethiops vertus, Mycobacterium bovis and Mycobacterium africanum major lineages in proportions of 67.3% (105/156), 22.4% (35/156), 6.4% (10/156), 1.9% (3/156), 1.3% (2/156) and 0.6% (1/156), respectively. Lineages identified were Delhi/CAS 23.9% (37/155), Ethiopia_2 20.6% (32/155), Haarlem 14.2% (22/155), URAL 14.2%(22/155), Ethiopia_3 8.4% (13/155), TUR 6.5% (10/155), Lineage_7/Aethiops vertus 1.9% (3/155), Bovis 1.3% (2/155), LAM 1.3% (2/155), EAI 0.6% (1/155), X 0.6% (1/155) and Ethiopia H37Rv-like strain 0.6% (1/155). Of the genotyped isolates 5.8% (9/155) remained unassigned. The recent transmission index (RTI) was 3.9%. Orphan strains compared to shared types (AOR: 0.09, 95% CI: 0.04–0.25) were associated with reduced odds of clustering. The dominant TB lineage in pastoral areas was EAI and in non-pastoral areas was EA.ConclusionThe epidemiological data, highly diverse MTBC strains and a low RTI in South Omo, provide information contributing to the TB Control Program of the country.

Mycobaterial interspersed repetitive units-variable number of tandem repeats analysis and pattern of drug resistance in extended drug resistant (XDR) TB isolates from pulmonary tuberculosis patients in Bangladesh

Background: To investigate the spread of specific genotypes in a defined geographical area and to determine any relationship of these genotypes with drug resistance the most essential method is molecular typing. It allows a rapid and precise species differentiation.&#x0D; Objective: This study was intended to observe the genotypes of XDR mycobacterium tuberculosis by determining 24 loci MIRU-VNTR analysis.&#x0D; Methods: To gain an insight about molecular typing of MTB and drug resistance-associated mutations in XDR-TB isolates a total of 98 multi drug resistant tuberculosis (MDR-TB) isolates collected through Xpert MTB/RIF assay. They were subjected to 2nd line (Fluoroquinolones, kanamycin, capreomycin and amikacin) drug susceptibility testing through line probe assay (LPA) in a view to detect extensively drug resistant tuberculosis (XDR-TB). Genotyping was done for XDR-TB isolates using 24 loci Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) using the online tool at http://www.MIRU-VNTRplus.org.. Out of 98 MDR-TB isolates 11(11.23%) XDR-TB isolates were typed and analysed.&#x0D; Results: Twenty four loci MIRU-VNTR genotyping involving similarity searching and phylogenetic tree analysis revealed that six (54.60%) XDR-TB isolates belonged to Beijing strain, Other MTB strain also detected were Delhi/CAS two(18.20%), Haarlem two(18.20%) and New-1, one (9.10%) in number. Minimum spanning tree analysis showed two strain of Beijing family form a clonal complex. Beijing strains were more common among younger age group and within urban population. Beijing strains were also predominant in treatment failure patient. Only one new case of XDR-TB belongs to Delhi/CAS family. Second line mycobacterial drug resistance (MTBDRsl) detected by LPA showed the most prevalent mutations involved in Fluoroquinolones drug resistance (FQ) was Asp94Gly in gyrA gene (54.55%) in quinolone resistance determining region (QRDR) and for Injectable 2nd line Drug resistance (ISL) was A1401G, C1402T in 16S rrs gene (100%).. All XDR-TB isolates showed resistance to Levofloxacin in 2nd line LPA but Moxifloxacin showed low level resistance to some cases.&#x0D; Conclusion: Molecular typing of XDR- TB isolates and pattern of drug resistance associated mutations in XDR-TB isolates in Bangladesh have not been reported previously. The result of this study highlights the need to reinforce the TB policy in Bangladesh with regard to control the spread and transmission as well as detection and treatment strategies regarding XDR-TB.&#x0D; Bangladesh Med Res Counc Bull 2020; 46(1): 22-28

Local adaptive evolution of two distinct clades of Beijing and T families of Mycobacterium tuberculosis in Chongqing: a Bayesian population structure and phylogenetic study

BackgroundBeijing sub-pedigree 2 (BSP2) and T sub-lineage 6 (TSL6) are two clades belonging to Beijing and T family of Mycobacterium tuberculosis (MTB), respectively, defined by Bayesian population structure analysis based on 24-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR). Globally, over 99% of BSP2 and 89% of TSL6 isolates were distributed in Chongqing, suggesting their possible local adaptive evolution. The objective of this paper is to explore whether BSP2 and TSL6 originated by their local adaptive evolution from the specific isolates of Beijing and T families in Chongqing.MethodsThe genotyping data of 16 090 MTB isolates were collected from laboratory collection, published literatures and SITVIT database before subjected to Bayesian population structure analysis based on 24-loci MIRU-VNTR. Spacer Oligonucleotide Forest (Spoligoforest) and 24-loci MIRU-VNTR-based minimum spanning tree (MST) were used to explore their phylogenetic pathways, with Bayesian demographic analysis for exploring the recent demographic change of TSL6.ResultsPhylogenetic analysis suggested that BSP2 and TSL6 in Chongqing may evolve from BSP4 and TSL5, respectively, which were locally predominant in Tibet and Jiangsu, respectively. Spoligoforest showed that Beijing and T families were genetically distant, while the convergence of the MIRU-VNTR pattern of BSP2 and TSL6 was revealed by WebLogo. The demographic analysis concluded that the recent demographic change of TSL6 might take 111.25 years.ConclusionsBSP2 and TSL6 clades might originate from BSP4 and TSL5, respectively, by their local adaptive evolution in Chongqing. Our study suggests MIRU-VNTR be combined with other robust markers for a more comprehensive genotyping approach, especially for families of clades with the same MIRU-VNTR pattern.

Genetic diversity and drug resistance pattern of Mycobacterium tuberculosis strains isolated from pulmonary tuberculosis patients in the Benishangul Gumuz region and its surroundings, Northwest Ethiopia.

Tuberculosis (TB) remains a major global public health problem and is the leading cause of death from a single bacterium, Mycobacterium tuberculosis (MTB) complex. The emergence and spread of drug-resistant strains aggravate the problem, especially in tuberculosis high burden countries such as Ethiopia. The supposedly high initial cost of laboratory diagnosis coupled with scarce financial resources has limited collection of information about drug resistance patterns and circulating strains in peripheral and emerging regions of Ethiopia. Here, we investigated drug susceptibility and genetic diversity of mycobacterial isolates among pulmonary tuberculosis patients in the Benishangul Gumuz region and its surroundings in northwest Ethiopia. In a cross-sectional study, 107 consecutive sputum smear-positive pulmonary tuberculosis (PTB) patients diagnosed at two hospitals and seven health centers were enrolled between October 2013 and June 2014. Sputum samples were cultured at Armauer Hansen Research Institute (AHRI) TB laboratory, and drug susceptibility testing (DST) was performed against Isoniazid, Rifampicin, Ethambutol, and Streptomycin using the indirect proportion method. Isolates were characterized using polymerase chain reaction (PCR)based Region of Difference 9 (RD9) testing and spoligotyping. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) for Windows version 24.0. Of 107 acid-fast-bacilli (AFB) smear-positive sputum samples collected, 81.3% (87/107) were culture positive. A PCR based RD9 testing revealed that all the 87 isolates were M. tuberculosis. Of these isolates, 16.1% (14/87) resistance to one or more drugs was observed. Isoniazid monoresistance occurred in 6.9% (6/87). Multidrug resistance (MDR) was observed in two isolates (2.3%), one of which was resistant to all the four drugs tested. Spoligotyping revealed that the majority, 61.3% (46/75) of strains could be grouped into ten spoligotype patterns containing two to 11 isolates each while the remaining 38.7% (29/75) were unique. SIT289 (11 isolates) and SIT53 (nine isolates) constituted 43.5% (20/46) among clustered isolates while 29.3% (22/75) were ''New" to the database. The dominant families were T, 37% (28/75), CAS, 16.0% (12/75), and H, 8% (6/75), adding up to 51.3% (46/75) of all isolates identified. The current study indicates a moderate prevalence of MDR TB. However, the observed high monoresistance to Isoniazid, one of the two proxy drugs for MDR-TB, reveals the hidden potential threat fora sudden increase in MDR-TB if resistance to Rifampicin would increase. Clustered spoligotype patterns suggest ongoing active tuberculosis transmission in the area. The results underscore the need for enhanced monitoring of TB drug resistance and epidemiological studies in this and other peripheral regions of the country using robust molecular tools with high discriminatory power such as the Mycobacterial Interspersed Repetitive Units -Variable Number of Tandem Repeats (MIRU-VNTR) typing and whole-genome sequencing (WGS).

Tracing cross species transmission of Mycobacterium bovis at the wildlife/livestock interface in South Africa

BackgroundBovine tuberculosis (bTB) affects cattle and wildlife in South Africa with the African buffalo (Syncerus caffer) as the principal maintenance host. The presence of a wildlife maintenance host at the wildlife/livestock interface acting as spill-over host makes it much more challenging to control and eradicate bTB in cattle. Spoligotyping and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) genotyping methods were performed to investigate the genetic diversity of Mycobacterium bovis (M. bovis) isolates from cattle and wildlife, their distribution and transmission at the wildlife/livestock interface in northern Kwa-Zulu Natal (KZN), South Africa.ResultsSB0130 was identified as the dominant spoligotype pattern at this wildlife/livestock interface, while VNTR typing revealed a total of 29 VNTR profiles (strains) in the KZN province signifying high genetic variability. The detection of 5 VNTR profiles shared between cattle and buffalo suggests M. bovis transmission between species. MIRU-VNTR confirmed co-infection in one cow with three strains of M. bovis that differed at a single locus, with 2 being shared with buffalo, implying pathogen introduction from most probably unrelated wildlife sources.ConclusionOur findings highlight inter and intra species transmission of bTB at the wildlife/livestock interface and the need for the implementation of adequate bTB control measures to mitigate the spread of the pathogen responsible for economic losses and a public health threat.

Polyclonal infection as a new scenario in Mycobacterium caprae epidemiology

Portugal is one of the European Union countries with an ongoing eradication program for bovine tuberculosis (TB), which does not include systematic goat testing. However, surveillance in small ruminants is increasingly important, since goat and sheep can harbour Mycobacterium caprae and be an infection source to cattle with impact in the success of bovine TB control. Furthermore, the information regarding the epidemiology and biology of M. caprae is quite limited comparing to the cognate bovine-adapted ecotype, M. bovis.In this work, we applied spoligotyping and MIRU-VNTR (Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats) to M. caprae obtained between 2003 and 2014 from 55 animal hosts, including goat (n = 29), cattle (n = 21), sheep (n = 1) and wild boar (n = 4) from Portugal. The molecular analysis revealed a unique spoligotyping pattern (SB0157) and 24 MIRU types. Genotyping of serial M. caprae from herds with recurrent outbreaks enabled further discrimination of epidemiologically related isolates, supporting a clonal structure in Portugal and denoting the emergence of clonal diversity at the herd level, more apparent for MIRU4. Results suggest a founder effect and adaptive genotypic divergence, paving the way for sympatric speciation.Double allele findings at MIRU4 in over 20 % of infected animals indicates that co-infection and in vivo microevolution may be frequent in the goat-adapted ecotype. While polyclonal infection appears common in M. caprae epidemiology, the functional significance of subtle genotypic variations remains to be disclosed, namely at the interface with the host, to expand knowledge on the epidemiology and biology of this neglected ecotype.

Population structure of drug-resistant Mycobacterium tuberculosis in Central Asia

BackgroundDrug-resistant tuberculosis (TB) is a major public health concern threathing the success of TB control efforts, and this is particularily problematic in Central Asia. Here, we present the first analysis of the population structure of Mycobacterium tuberculosis complex isolates in the Central Asian republics Uzbekistan, Tajikistan, and Kyrgyzstan.MethodsThe study set consisted of 607 isolates with 235 from Uzbekistan, 206 from Tajikistan, and 166 from Kyrgyzstan. 24-loci MIRU-VNTR (Mycobacterial Interspersed Repetitive Units - Variable Number of Tandem Repeats) typing and spoligotyping were combined for genotyping. In addition, phenotypic drug suceptibility was performed.ResultsThe population structure mainly comprises strains of the Beijing lineage (411/607). 349 of the 411 Beijing isolates formed clusters, compared to only 33 of the 196 isolates from other clades. Beijing 94–32 (n = 145) and 100–32 (n = 70) formed the largest clusters. Beijing isolates were more frequently multidrug-resistant, pre-extensively resistant (pre-XDR)- or XDR-TB than other genotypes.ConclusionsBeijing clusters 94–32 and 100–32 are the dominant MTB genotypes in Central Asia. The relative size of 100–32 compared to previous studies in Kazakhstan and its unequal geographic distribution support the hypothesis of its more recent emergence in Central Asia. The data also demonstrate that clonal spread of resistant TB strains, particularly of the Beijing lineage, is a root of the so far uncontroled MDR-TB epidemic in Central Asia.

Genetic Variation of the Mycobacterium tuberculosis in North of Iran; the Golestan Province

Background: Genotyping of Mycobacterium tuberculosis (M.tub) is an essential step for several purposes, including the epidemiological studies and the tuberculosis (TB) control programs. Golestan province in the Southeast of the Caspian Sea is the second Iranian high burden TB province. Objectives: This study aimed to determine the genetic diversity of M.tub isolates in Golestan province located in the north of Iran. Methods: In this cross-sectional study, all non-repetitive confirmed M.tub isolates, which were collected from patients with TB in Golestan province in 2016, were used for genotyping. After DNA extraction, PCR was done for 15 loci of mycobacterial interspersed repetitive unit-variable-number of tandem repeats (MIRU-VNTRs) for the 162 M.tub isolates. Then genetic diversity and genetic relationships between them were assessed by best match analysis using tools on MIRU-VNTRplus database. Antibiotic susceptibility patterns of M.tub isolates was determined by the proportional method. Statistical analysis was done by SPSS and R software. Results: Out of 162 M.tub isolates, 156 genotype patterns were obtained, which 150 of which were unique. Nine of 15 loci were highly discriminative. The clustering rates were 3.7%. The prevalence of polyclonal infection was 2.46%. Also, HGDI and AHGDI were 0.999 and 0.979, respectively. The New-1 (22.2%) and Beijing (13.6%) sub-lineages had the highest prevalence in this region. Although there was no significant correlation between demographic criteria and M.tub genotypes, still Beijing isolates showed more treatment failure (18.2% vs. 0.7%) and resistance to streptomycin (40.9% vs. 7.8%) compared to others (P < 0.05). The assessment of the minimum spanning tree showed that the members of the clonal complex were limited except for members of Beijing. The PCA analysis showed that the combination of Qub11b and Mtub30 loci was diagnostic for Beijing sub-lineage. Conclusions: Owing to the high genotypic diversity of M.tub isolates in this region, relying on tuberculosis control program just based on close contact treatment is not sufficient, and we require to apply another approach.