Repetitive Unit-variable Number Tandem Repeat Research Articles

Drug resistance and population structure of M.tuberculosis isolates from prisons and communities in Ethiopia

BackgroundThe population structure and drug resistance pattern of Mycobacterium tuberculosis complex (MTBC) isolates in Ethiopian prisons and some communities is still unknown.MethodsA comparative cross sectional study was conducted on 126 MTBC strains isolated from prisons and communities in southwestern, southern and eastern Ethiopia. Phenotypic drug susceptibility testing was performed with the MGIT960 system. Combined 24-loci Mycobacterium interspersed repetitive unit-variable number tandem repeat and spacer oligonucleotide typing methods were used to study the MTBC population structure. The obtained data from prisons and communities were compared using statistical tests and regression analysis.ResultsA diverse population structure with 11 different lineages and sub-lineages was identified. The predominant strains were the recently described Ethiopia_H37Rv like (27.52%) and Ethiopia_3 (16.51%) with equal lineage distribution between prisons and communities. 28.57% of prison strains and 31.82% of community strains shared the identical genotype with at least one other strain. The multidrug-resistance (MDR) prevalence of the community was 2.27% whereas that of prisons was 9.52%. The highest mono resistance was seen against streptomycin (15.89%).ConclusionTuberculosis in communities and prisons is caused by a variety of MTBC lineages with predominance of local Ethiopian lineages. The increasing prevalence of MDR MTBC strains is alarming. These findings suggest the need for new approaches for control of MDR tuberculosis in Ethiopia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-2041-x) contains supplementary material, which is available to authorized users.

Isolation of Mycobacterium bovis from Free-Ranging Wildlife in South Korea.

We demonstrate Mycobacterium bovis infection in wild boar ( Sus scrofa ) in South Korea. During 2012-15, we attempted to isolate M. bovis from 847 wild animals, mainly Korean water deer ( Hydropotes inermis argyropus), raccoon dogs ( Nyctereutes procyonoides ), and wild boar, from 11 regions in South Korea. We isolated M. bovis from three of 118 wild boar (2.5%) captured in Gyeonggi Province, where bovine tuberculosis (bTB) outbreaks have also occurred in livestock. Spoligotypes and mycobacterial interspersed repetitive units-variable number tandem repeats types of these M. bovis isolates (SB0140 and SB1040, 4-2-3-3-7-5-5-4-4-3-4-3 and 5-2-3-3-7-5-5-4-3-10-5-2; MIRU4, MIRU16, MIRU27, MIRU31, ETR-A, ETR-B, ETR-C, QUB11b, QUB26, QUB3336, VNTR2401, and VNTR3171) have also been identified from farmed livestock such as cattle ( Bos taurus coreanae), Formosan sika deer ( Cervus nippon taiouanus), and American elk ( Cervus canadensis ) in the country. In South Korea, bTB appears to be endemic in livestock, and there are numerous opportunities for contact between wild boar and livestock due to high population densities and broad activity ranges. Our results support the hypothesis that M. bovis is transmitted between domestic and wild animals.

English

Mycobacterium ulcerans (MU), the causative agent of Buruli ulcer (BU), skin disease, is considered to be an environmental pathogen. The pathogenic virulence of MU is being linked to the expression of toxin called Mycolactone. Genetic analyses have shown the high diversity with variable number tandem repeats (VNTR) and mycobacterial interspersed repetitive units (MIRU) in M. ulcerans and in mycolactone producing Mycobacteria (MPMs). The purpose of this study is the molecular characterization of potentially pathogenic environmental mycobacteria strain, apart from the M. ulcerans, from aquatic environments in Cote d’Ivoire. A total of 473 samples were collected comprising of 251 water and 222 sediment based on sampling sites. The sediments were the most contaminated by mycobacteria with 60% as against 43.3% in water samples from the hyper endemic areas. In hypo-endemic areas, water was the most contaminated with 53.57% against 43.24% in sediment. Microscopy by Ziehl-Neelsen-staining, and PCR diagnostics using IS2404 and KR were performed on strains. 20% fast growing isolated mycobacteria species including Mycobacterium mucogenicum, Mycobacterium peregrinum and Mycobacterium sp. was found carrying the IS2404 gene previously found in M. ulcerans. 9.23% of strains carry the ketoreductase (KR) genes, one of the synthesis of mycolactone enzymes. In terms of genetic analysis using the MIRU/VNTR, the MIRU 1 was the most amplified sequence, and LOCUS 6 less amplified; no known profile have been identified in this study. This study is the first step taken in order to understand different skin infections encountered in Cote d’Ivoire. Key words: Mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR; ketoreductase, LOCUS 6, IS2404, Mycobacteria, Buruli ulcer.

Genomic Diversity of Mycobacterium tuberculosis Complex Strains in Cantabria (Spain), a Moderate TB Incidence Setting.

BackgroundTuberculosis (TB) control strategies are focused mainly on prevention, early diagnosis, compliance to treatment and contact tracing. The objectives of this study were to explore the frequency and risk factors of recent transmission of clinical isolates of Mycobacterium tuberculosis complex (MTBC) in Cantabria in Northern Spain from 2012 through 2013 and to analyze their clonal complexity for better understanding of the transmission dynamics in a moderate TB incidence setting.MethodsDNA from 85 out of 87 isolates from bacteriologically confirmed cases of MTBC infection were extracted directly from frozen stocks and genotyped using the mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) method. The MIRU-VNTRplus database tool was used to identify clusters and lineages and to build a neighbor joining (NJ) phylogenetic tree. In addition, data were compared to the SITVIT2 database at the Pasteur Institute of Guadeloupe.ResultsThe rate of recent transmission was calculated to 24%. Clustering was associated with being Spanish-born. A high prevalence of isolates of the Euro-American lineage was found. In addition, MIRU-VNTR profiles of the studied isolates corresponded to previously found MIRU-VNTR types in other countries, including Spain, Belgium, Great Britain, USA, Croatia, South Africa and The Netherlands. Six of the strains analyzed represented clonal variants.ConclusionTransmission of MTBC is well controlled in Cantabria. The majority of TB patients were born in Spain. The population structure of MTBC in Cantabria has a low diversity of major clonal lineages with the Euro-American lineage predominating.

Molecular epidemiology of Mycobacterium tuberculosis in Gansu province of China.

Mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) and Beijing family typing based on detecting the deletion of RD105 sequence are two common genotyping methods used to study the molecular epidemiologic characteristics of Mycobacterium (M.) tuberculosis. We collected 218 strains of M. tuberculosis between 2004 and 2006 in the Linxia Hui Autonomous Prefecture of Gansu province in Northwest China. MIRU-VNTR analysis and Beijing family typing based on detecting the deletion of RD105 sequence were used to type the 218 strains, and their typing power was evaluated to look for practical and efficient genotyping methods suitable for the region. The MIRU typing yielded 115 distinct genotypes, including 98 unique isolates and 17 different clusters containing 120 isolates (55.05%); the cluster rate was 47.25%. By detecting the deletion of RD105 sequence, 188 of 218 (86.23%) isolates belonged to Beijing family. Combination of Beijing family typing and MIRU typing yielded 118 distinct patterns, including 101 unique isolates and 17 clusters containing 117 isolates (54.13%). The largest cluster contained 58 strains with MIRU genotype of 223325173533 which contained 50 strains belonging to Beijing family and 8 strains belonging to non-Beijing family. The Beijing family strains occupied a large proportion and the Beijing family MIRU genotype 223325173533 is a dominant strain in Linxia of Gansu. Combining detecting the deletion of RD105 and MIRU typing together provides a simple, fast, and effective method which is low in cost and might be practical and suitable for M. tuberculosis genotyping in China.

Outbreak of tuberculosis in a closed setting: views on transmission based on results from molecular and conventional methods

This study describes an outbreak of tuberculosis (TB) in a nursing home for men with mental disorders where residency is lengthy or permanent. This type of setting can provide a model of transmission as contact with the rest of society is extremely limited. To determine if cases of TB, diagnosed around the same time and in the same place, are linked based on results using molecular and conventional methods. The strains of Mycobacterium tuberculosis were analysed by drug resistance testing and mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTRV). Microbiological results were related to clinical history and time of diagnosis. Nine patients were diagnosed with TB, and strains were recovered from seven of these patients. Unexpectedly, the strains with the same genotype showed different patterns of resistance, and only two strains demonstrated identical patterns. MIRU-VNTR analysis demonstrated that one patient was infected with two different strains. Variation between the strains indicates that the outbreak may have arisen from several sources of infection. The variation in resistance indicates that rapid emergence of antimicrobial resistance is possible. As such, several questions are raised concerning source of infection, development of disease, resistance and mixed infections.

High prevalence of Mycobacterium tuberculosis mixed infection in the capital of moderate tuberculosis incidence country

ObjectiveRecent studies using molecular epidemiological techniques have demonstrated mixed infection with multiple strains of Mycobacterium tuberculosis especially in countries with high tuberculosis (TB) burden. We aimed to determine the prevalence of mixed infection among patients with TB in the capital of Iran as a country with moderate incidence rate. MethodsSamples were collected randomly from January 2011 to December 2013 in Tehran, capital of Iran. A total of 75 M. tuberculosis isolates were genotyped by 24 loci mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-VNTR) for screening the mixed infection. ResultsTwenty patients (20/75) were identified with mixed infection, and the estimated rate of mixed infection was 26.6%. Thirteen out of the 24 loci were able to detect the mixed infection in our study. ConclusionsMixed infections occur at high prevalence among studied Iranian TB patients. Further research is inevitable to evaluate the association of mixed infection and disease progression and treatment.

Phylogenomic exploration of the relationships between strains of Mycobacterium avium subspecies paratuberculosis.

BackgroundMycobacterium avium subspecies paratuberculosis (Map) is an infectious enteric pathogen that causes Johne’s disease in livestock. Determining genetic diversity is prerequisite to understanding the epidemiology and biology of Map. We performed the first whole genome sequencing (WGS) of 141 global Map isolates that encompass the main molecular strain types currently reported. We investigated the phylogeny of the Map strains, the diversity of the genome and the limitations of commonly used genotyping methods.ResultsSingle nucleotide polymorphism (SNP) and phylogenetic analyses confirmed two major lineages concordant with the former Type S and Type C designations. The Type I and Type III strain groups are subtypes of Type S, and Type B strains are a subtype of Type C and not restricted to Bison species.We found that the genome-wide SNPs detected provided greater resolution between isolates than currently employed genotyping methods. Furthermore, the SNP used for IS1311 typing is not informative, as it is likely to have occurred after Type S and C strains diverged and does not assign all strains to the correct lineage. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) differentiates Type S from Type C but provides limited resolution between isolates within these lineages and the polymorphisms detected do not necessarily accurately reflect the phylogenetic relationships between strains.WGS of passaged strains and coalescent analysis of the collection revealed a very high level of genetic stability, with the substitution rate estimated to be less than 0.5 SNPs per genome per year.ConclusionsThis study clarifies the phylogenetic relationships between the previously described Map strain groups, and highlights the limitations of current genotyping techniques. Map isolates exhibit restricted genetic diversity and a substitution rate consistent with a monomorphic pathogen. WGS provides the ultimate level of resolution for differentiation between strains. However, WGS alone will not be sufficient for tracing and tracking Map infections, yet importantly it can provide a phylogenetic context for affirming epidemiological connections.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2234-5) contains supplementary material, which is available to authorized users.

Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods.

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units–variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications.

A study on pre-XDR & XDR tuberculosis & their prevalent genotypes in clinical isolates of Mycobacterium tuberculosis in north India

Background & objectives:Pre-extensively drug resistant (pre-XDR) and extensively drug resistant tuberculosis (XDR-TB) have been areas of growing concern, and are posing threat to global efforts of TB control. The present study was planned to study the presence of pre-XDR and XDR Mycobacterium tuberculosis and their genotypes in clinical isolates obtained from previously treated cases of pulmonary TB.Methods:A total of 219 isolates obtained from previously treated cases of pulmonary TB were subjected to first-line (streptomycin, isoniazid, rifampicin and ethambutol) and second-line (ofloxacin, kanamycin, capreomycin and amikacin) drug susceptibility testing on solid Lowenstein-Jensen medium by proportion method. Genotyping was done for pre-XDR and XDR-TB isolates using 12 loci Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR).Results:Multi-drug resistance was observed in 39.7 per cent (87/219) isolates. Pre-XDR and XDR M. tuberculosis isolates amongst 87 multi-drug resistant (MDR) TB isolates were 43 (49.4%) and 10 (11.4%), respectively. Two most dominant genotypes among pre-XDR and XDR M. tuberculosis isolates were Beijing and Delhi/CAS types.Interpretation & conclusions:Resistance to second-line anti-tubercular drugs should be routinely assessed in areas endemic for TB. Similar genotype patterns were seen in pre-XDR and XDR-TB isolates. Beijing and Delhi/CAS were predominant genotypes.

Mycobacterium tuberculosis causing tuberculous lymphadenitis in Maputo, Mozambique.

BackgroundThe zoonosis bovine tuberculosis (TB) is known to be responsible for a considerable proportion of extrapulmonary TB. In Mozambique, bovine TB is a recognised problem in cattle, but little has been done to evaluate how Mycobacterium bovis has contributed to human TB. We here explore the public health risk for bovine TB in Maputo, by characterizing the isolates from tuberculous lymphadenitis (TBLN) cases, a common manifestation of bovine TB in humans, in the Pathology Service of Maputo Central Hospital, in Mozambique, during one year.ResultsAmong 110 patients suspected of having TBLN, 49 had a positive culture result. Of those, 48 (98 %) were positive for Mycobacterium tuberculosis complex and one for nontuberculous mycobacteria. Of the 45 isolates analysed by spoligotyping and Mycobacterial Interspersed Repetitive Unit – Variable Number Tandem Repeat (MIRU-VNTR), all were M. tuberculosis. No M. bovis was found.Cervical TBLN, corresponding to 39 (86.7 %) cases, was the main cause of TBLN and 66.7 % of those where from HIV positive patients.We found that TBLN in Maputo was caused by a variety of M. tuberculosis strains. The most prevalent lineage was the EAI (n = 19; 43.2 %). Particular common spoligotypes were SIT 48 (EAI1_SOM sublineage), SIT 42 (LAM 9), SIT 1 (Beijing) and SIT53 (T1), similar to findings among pulmonary cases.ConclusionsM. tuberculosis was the main etiological agent of TBLN in Maputo. M. tuberculosis genotypes were similar to the ones causing pulmonary TB, suggesting that in Maputo, cases of TBLN arise from the same source as pulmonary TB, rather than from an external zoonotic source. Further research is needed on other forms of extrapulmonary TB and in rural areas where there is high prevalence of bovine TB in cattle, to evaluate the risk of transmission of M. bovis from cattle to humans.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0603-5) contains supplementary material, which is available to authorized users.

First insight into the genetic population structure of Mycobacterium tuberculosis isolated from pulmonary tuberculosis patients in Egypt

The present study aimed to assess the population structure of Mycobacterium tuberculosis (MTB) isolates from Egypt. A total of 230 MTB isolates were analysed using spoligotyping, large sequence polymorphism (LSPs), mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and multi-locus sequence typing (MLST). The majority of isolates (93.0%) belonged to lineage 4, including 44.3, 13.4 and 10.8% of the ill-defined T clade, LAM and Haarlem families, respectively, and lineage 3 was identified in 7.0% of the isolates. MIRU-VNTRs typing allowed efficient discrimination of the spoligotype-defined clusters, including spoligo-international types (SIT) 53, 34, and 4, into 56 patterns, including 13 clusters and 43 unique patterns. A new SNP at position 311614 was identified in all six isolates to form the biggest MIRU-VNTR cluster, which suggested a recent clonal expansion. This SNP could possibly be used as a genetic marker for robust discriminations of Egyptian MTB isolates belonging to SIT53. The combination of spoligotyping, 12 MIRU-VNTRs loci and MLST provided insight into the genetic diversity and transmission dynamics of the Egyptian MTB genotypes and could be a key to implementation of effective control measures by public health authorities.

An investigation on the population structure of mixed infections of Mycobacterium tuberculosis in Inner Mongolia, China

Mixed infections of Mycobacterium tuberculosis strains have attracted more attention due to their increasing frequencies worldwide, especially in the areas of high tuberculosis (TB) prevalence. In this study, we accessed the rates of mixed infections in a setting with high TB prevalence in Inner Mongolia Autonomous Region of China. A total of 384 M.tuberculosis isolates from the local TB hospital were subjected to mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing method. The single clones of the strains with mixed infections were separated by subculturing them on the Löwenstein-Jensen medium. Of these 384 isolates, twelve strains (3.13%) were identified as mixed infections by MIRU-VNTR. Statistical analysis indicated that demographic characteristics and drug susceptibility profiles showed no statistically significant association with the mixed infections. We further subcultured the mixed infection strains and selected 30 clones from the subculture for each mixed infection. Genotyping data revealed that eight (8/12, 66.7%) strains with mixed infections had converted into single infection through subculture. The higher growth rate was associated with the increasing proportion of variant subpopulation through subculture. In conclusion, by using the MIRU-VNTR method, we demonstrate that the prevalence of mixed infections in Inner Mongolia is low. Additionally, our findings reveal that the subculture changes the population structures of mixed infections, and the subpopulation with higher growth rate show better fitness, which is associated with high proportion among the population structure after subculture. This study highlights that the use of clinical specimens, rather than subcultured isolates, is preferred to estimate the prevalence of mixed infections in the specific regions.

Genotypic characteristics of Mycobacterium tuberculosis circulating in Xinjiang, China

Background: Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis (MTB), poses a serious threat to human health. We investigated the genotypes of MTB in the high prevalence province Xinjiang, China. Methods: From March 2010 to May 2013, 381 MTB isolates from patients with pulmonary TB were analyzed by molecular typing of 24 mycobacterial interspersed repetitive unit-variable number tandem repeat loci and PCR detection of the deleted regions of difference of the Beijing/W lineage and its sublineages. Results: These isolates were shown to be highly polymorphic and to be composed of 345 unique genotypes, including 30 genotype clusters consisting of 2 or 3 strains and 315 individual genotypes. The genotype clustering rate was 17.32% and recent transmission index was low (9.45%). The Beijing/W lineage strains accounted for 57.48% of the isolates, and this predominant family strain was further subdivided into four sublineages: 181 (69.86%), 207 (14.61%), 105 (10.96%), and 150 (4.56%). Conclusions: The Beijing/W lineage (especially sublineage 181) strains were predominant and were associated with the transmissibility of TB in Xinjiang. Based on our data, we hypothesize that the circulating MTB strains in Xinjiang have significant genetic diversity and that the majority of the TB in Xinjiang may be explained by non-recent transmission emerging by endogenous reactivation. The possibility of outbreak is low, and current measures to control TB should first focus on standardized treatment of TB patients to prevent reactivation of latent infections.

Genetic diversity and molecular epidemiology of multidrug-resistant Mycobacterium tuberculosis in Minas Gerais State, Brazil.

BackgroundWe aimed to characterize the genetic diversity of drug-resistant Mycobacterium tuberculosis (MTb) clinical isolates and investigate the molecular epidemiology of multidrug-resistant (MDR) tuberculosis from Minas Gerais State, Brazil.MethodsOne hundred and four MTb clinical isolates were assessed by IS6110-RFLP, 24-locus mycobacterial interspersed repetitive units variable-number tandem repeats (MIRU-VNTR), TB-SPRINT (simultaneous spoligotyping and rifampicin-isoniazid drug-resistance mutation analysis) and 3R-SNP-typing (analysis of single-nucleotide polymorphisms in the genes involved in replication, recombination and repair functions).ResultsFifty-seven different IS6110-RFLP patterns were found, among which 50 had unique patterns and 17 were grouped into seven clusters. The discriminatory index (Hunter and Gaston, HGDI) for RFLP was 0.9937. Ninety-nine different MIRU-VNTR patterns were found, 95 of which had unique patterns and nine isolates were grouped into four clusters. The major allelic diversity index in the MIRU-VNTR loci ranged from 0.6568 to 0.7789. The global HGDI for MIRU-VNTR was 0.9991. Thirty-two different spoligotyping profiles were found: 16 unique patterns (n = 16) and 16 clustered profiles (n = 88). The HGDI for spoligotyping was 0.9009. The spoligotyped clinical isolates were phylogenetically classified into Latin-American Mediterranean (66.34 %), T (14.42 %), Haarlem (5.76 %), X (1.92 %), S (1.92 %) and U (unknown profile; 8.65 %). Among the U isolates, 77.8 % were classified further by 3R-SNP-typing as 44.5 % Haarlem and 33.3 % LAM, while the 22.2 % remaining were not classified. Among the 104 clinical isolates, 86 were identified by TB-SPRINT as MDR, 12 were resistant to rifampicin only, one was resistant to isoniazid only, three were susceptible to both drugs, and two were not successfully amplified by PCR. A total of 42, 28 and eight isolates had mutations in rpoB positions 531, 526 and 516, respectively. Correlating the cluster analysis with the patient data did not suggest recent transmission of MDR-TB.ConclusionsAlthough our results do not suggest strong transmission of MDR-TB in Minas Gerais (using a classical 100 % MDR-TB identical isolates cluster definition), use of a smoother cluster definition (>85 % similarity) does not allow us to fully eliminate this possibility; hence, around 20–30 % of the isolates we analyzed might be MDR-TB transmission cases.

Genotype heterogeneity of Mycobacterium tuberculosis within geospatial hotspots suggests foci of imported infection in Sydney, Australia

In recent years the State of New South Wales (NSW), Australia, has maintained a low tuberculosis incidence rate with little evidence of local transmission. Nearly 90% of notified tuberculosis cases occurred in people born in tuberculosis-endemic countries. We analyzed geographic, epidemiological and genotypic data of all culture-confirmed tuberculosis cases to identify the bacterial and demographic determinants of tuberculosis hotspot areas in NSW. Standard 24-loci mycobacterium interspersed repetitive unit-variable number tandem repeat (MIRU-24) typing was performed on all isolates recovered between 2009 and 2013. In total 1692/1841 (91.9%) cases with confirmed Mycobacterium tuberculosis infection had complete MIRU-24 and demographic data and were included in the study. Despite some year-to-year variability, spatio-temporal analysis identified four tuberculosis hotspots. The incidence rate and the relative risk of tuberculosis in these hotspots were 2- to 10-fold and 4- to 8-fold higher than the state average, respectively. MIRU-24 profiles of M. tuberculosis isolates associated with these hotspots revealed high levels of heterogeneity. This suggests that these spatio-temporal hotspots, within this low incidence setting, can represent areas of predominantly imported infection rather than clusters of cases due to local transmission. These findings provide important epidemiological insight and demonstrate the value of combining tuberculosis genotyping and spatiotemporal data to guide better-targeted public health interventions.

Whole genome sequence analysis of multidrug-resistant Mycobacterium tuberculosis Beijing isolates from an outbreak in Thailand

The Mycobacterium tuberculosis Beijing family is often associated with multidrug resistance and large outbreaks. Conventional genotyping study of a community outbreak of multidrug-resistant tuberculosis (MDR-TB) that occurred in Kanchanaburi Province, Thailand was carried out. The study revealed that the outbreak was clonal and the strain was identified as a member of Beijing family. Although, the outbreak isolates showed identical spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeats patterns, a discrepancy regarding ethambutol resistance was observed. In-depth characterization of the isolates through whole genome sequencing of the first and the last three isolates from our culture collection showed them to belong to principal genetic group 1, single nucleotide polymorphism (SNP) cluster group 2, sequence type 10. Compared with the M. tuberculosis H37Rv reference genome, 1242 SNPs were commonly found in all isolates. The genomes of these isolates were shown to be clonal and highly stable over a 5-year period and two or three unique SNPs were identified in each of the last three isolates. Genes known to be associated with drug resistance and their promoter regions, where applicable, were analyzed. The presence of low or no fitness cost mutations for drug resistance and an additional L731P SNP in the rpoB gene was observed in all isolates. These findings might account for the successful transmission of this MDR-TB strain.

Genetic diversity of Mycobacterium tuberculosis isolates obtained from three distinct population groups in the Central Province, Sri Lanka

ObjectiveTo characterize the Mycobacterium tuberculosis (M. tuberculosis) isolates by spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing to understand how M. tuberculosis strains transmit among the study population. MethodsSpoligotyping and MIRU-VNTR were used to genotype M. tuberculosis isolates obtained from three distinct population groups in Sri Lanka. General population suspected of having tuberculosis attending the Chest Clinic, Kandy (n = 78), patients having tuberculosis in Bogambara prison, Kandy (n = 22) and estate workers having tuberculosis in the Central Province, Sri Lanka (n = 50), from January 2012 to April 2014 were included in the study. ResultsAmong 150 isolates, a total of 19 distinct families were observed including 6 major spoligotyping-based families; East-African-Indian (39.33%), Haarlem (20%), Beijing (8.6%), Central European family T (6.5%), European family X (5.2%) and Central and Middle Eastern Asian (0.6%). Beijing strains were only identified among the general population. MANU strains were significant (36.36%) among the prisoners who had clustered with the MANU strains of the general population indicating contact cases and a possible transmission index within a particular geographical area. Haarlem 3 (34%) was the predominant strain among the estate workers. There was a close epidemiological relationship between the prisoners and the estate workers in the population. ConclusionsThe first insight of 15 loci MIRU-VNTR typing in conjunction with spoligotyping in a population in Sri Lanka demonstrated the feasibility and the applicability of these techniques to differentiate strains, their heterogeneity and the predominance of several worldwide distributed spoligotypes.

The impact of isoniazid resistant Mycobacterium tuberculosis on the epidemic of multi-drug resistant tuberculosis

Objective To investigate the impact of isoniazid (INH)-resistant Mycobacterium tuberculosis (Mtb) on the prevalence and dissemination of multi-drug resistant tuberculosis (MDR-TB). Methods A total of 251 patients diagnosed with tuberculosis in designated hospitals of Guanyun, Jiangsu and Deqing, Zhejiang from 2010 to 2011 were included in the study. The drug susceptibility tests (DST) were performed on all the Mtb isolates available from the sputum cultures. Mycobacteral interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) was conducted for genotyping for all available Mtb isolates. Chi-square test, Fisher exact test, ANOVA and non-conditional Logistic regression modelling were applied for data analysis. Results Among 251 patients with Mtb isolates and DST results available, 72(28.7%) were resistant to INH, including 13 were INH mono-drug resistant. Of the remaining 59 INH-resistant Mtb, 34(13.5%) were resistant to rifampin TB and 25 were resistant to streptomycin and/or ethambutol. The clustering analysis based on MIRU-VNTR genotyping revealed 29 clustered genotypes (including 105 isolates) and 146 unique genotypes (including 119 isolates). Twenty-five clusters contained drug resistant Mtb and 16 clusters of them comprised by 37 INH-resistant isolates and 20 MDR-TB isolates, which accounted for 51.4% of the INH-resistant isolates and 58.8% of the MDR-TB isolates. Single factor analysis showed that sex, age, previous tuberculosis treatment history and sputum smear results were all related to INH-resistant tuberculosis and MDR-TB (all P<0.05). Multiple factors analysis showed that previous tuberculosis treatment history was risk factor of MDR-TB (OR=8.40, 95%CI: 3.342-21.105), while the risk factors of INH-resistant tuberculosis were previous tuberculosis treatment history (OR=3.52, 95%CI: 1.570-7.910), pulmonary caviry (OR=2.27, 95%CI: 1.075-4.799) and sputum smear results (OR=0.50, 95%CI: 0.275-0.892). Conclusions That INH-resistant strain may evolve to the MDR-TB after recent transmission is a possible trend. Patients with previous treatment history and advanced age are at high risk of INH-resistant tuberculosis and MDR-TB. Key words: Mycobacterium tuberculosis; Isoniazid; Tuberculosis, multidrug-resistant

Mycobacterium tuberculosis lineage 7 strains are associated with prolonged patient delay in seeking treatment for pulmonary tuberculosis in Amhara Region, Ethiopia.

Recent genotyping studies of Mycobacterium tuberculosis in Ethiopia have reported the identification of a new phylogenetically distinct M. tuberculosis lineage, lineage 7. We therefore investigated the genetic diversity and association of specific M. tuberculosis lineages with sociodemographic and clinical parameters among pulmonary TB patients in the Amhara Region, Ethiopia. DNA was isolated from M. tuberculosis-positive sputum specimens (n=240) and analyzed by PCR and 24-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) analysis and spoligotyping. Bioinformatic analysis assigned the M. tuberculosis genotypes to global lineages, and associations between patient characteristics and genotype were evaluated using logistic regression analysis. The study revealed a high diversity of modern and premodern M. tuberculosis lineages, among which approximately 25% were not previously reported. Among the M. tuberculosis strains (n=138) assigned to seven subgroups, the largest cluster belonged to the lineage Central Asian (CAS) (n=60; 26.0%), the second largest to lineage 7 (n=36; 15.6%), and the third largest to the lineage Haarlem (n=35; 15.2%). Four sublineages were new in the MIRU-VNTRplus database, designated NW-ETH3, NW-ETH1, NW-ETH2, and NW-ETH4, which included 24 (10.4%), 18 (7.8%), 8 (3.5%), and 5 (2.2%) isolates, respectively. Notably, patient delay in seeking treatment was significantly longer among patients infected with lineage 7 strains (Mann-Whitney test, P<0.008) than in patients infected with CAS strains (adjusted odds ratio [AOR], 4.7; 95% confidence interval [CI], 1.6 to 13.5). Lineage 7 strains also grew more slowly than other M. tuberculosis strains. Cases of Haarlem (OR, 2.8; 95% CI, 1.2 to 6.6) and NW-ETH3 (OR, 2.8; 95% CI, 1.0 to 7.3) infection appeared in defined clusters. Intensified active case finding and contact tracing activities in the study region are needed to expedite diagnosis and treatment of TB.