First Baseline of Circulating Genotypic Lineages of Mycobacterium tuberculosis in Patients from the Brazilian Borders with Argentina and Paraguay

Background

At the triple border Brazil/Paraguay/Argentina there is easy mobility from one city to another for economic and tourism activities. This constant and fast population mobility is mainly to visit Iguazu Falls, in the Iguazu River, on the border of the Brazilian state of Paraná and the Argentina. As the incidence of tuberculosis is high in this setting, our study aimed to establish a first baseline of circulating genotypic lineages of Mycobacterium tuberculosis.

Methodology/Principal Findings

This study included 120 patients from 10 cities in southwestern Paraná, Brazil with pulmonary symptoms, from July 2009 to July 2011. Information about sex, age, clinical features and address was collected by reviewing the national tuberculosis notification database. Of these, 96 (80%) isolates were identified as M. tuberculosis and 22 (22.9%) were drug resistant (20, 20.8% INH mono-resistant and 2, 2.1% multidrug-resistant). All isolates were subjected to genotyping by Spoligotyping and MIRU-VNTR typing. The distribution of the isolates analyzed by spoligotyping revealed 30 distinct patterns. The four mainly detected clades were Latin American and Mediterranean (LAM), ill-defined T, Haarlem (H) and S. The MIRU-VNTR showed 85 distinct patterns. Spoligotyping combined to MIRU-VNTR allowed 90 distinct patterns.

Conclusions/Significance

Our study demonstrated that there is significant molecular diversity in circulating M. tuberculosis, with predominance of the LAM and T clades in cities of southwestern Paraná, Brazil, bordering Argentina and Paraguay.     

Genetic diversity of Mycobacterium tuberculosis isolates from Tibetans in Tibet, China

Background

Tuberculosis (TB) is a serious health problem in Tibet where Tibetans are the major ethnic group. Although genotyping of Mycobacterium tuberculosis (M. tuberculosis) isolates is a valuable tool for TB control, our knowledge of population structure of M. tuberculosis circulating in Tibet is limited.

Methodology/Principal Findings

In our study, a total of 576 M. tuberculosis isolates from Tibetans in Tibet, China, were analyzed via spoligotyping and 24-locus MIRU-VNTR. The Beijing genotype was the most prevalent family (90.63%, n = 522). Shared-type (ST) 1 was the most dominant genotype (88.89%, n = 512). We found that there was no association between the Beijing genotype and sex, age and treatment status. In this sample collection, 7 of the 24 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index. An informative set of 12 loci had similar discriminatory power with 24 loci set.

Conclusions/Significance

The population structure of M. tuberculosis isolates in Tibetans is homogeneous and dominated by Beijing genotype. The analysis of 24-locus MIRU-VNTR data might be useful to select appropriate VNTR loci for the genotyping of M. tuberculosis.     

First Insights into the Phylogenetic Diversity of Mycobacterium tuberculosis in Nepal

Background

Tuberculosis (TB) is a major public health problem in Nepal. Strain variation in Mycobacterium tuberculosis may influence the outcome of TB infection and disease. To date, the phylogenetic diversity of M. tuberculosis in Nepal is unknown.

Methods and Findings

We analyzed 261 M. tuberculosis isolates recovered from pulmonary TB patients recruited between August 2009 and August 2010 in Nepal. M. tuberculosis lineages were determined by single nucleotide polymorphisms (SNP) typing and spoligotyping. Drug resistance was determined by sequencing the hot spot regions of the relevant target genes. Overall, 164 (62.8%) TB patients were new, and 97 (37.2%) were previously treated. Any drug resistance was detected in 50 (19.2%) isolates, and 16 (6.1%) were multidrug-resistant. The most frequent M. tuberculosis lineage was Lineage 3 (CAS/Delhi) with 106 isolates (40.6%), followed by Lineage 2 (East-Asian lineage, includes Beijing genotype) with 84 isolates (32.2%), Lineage 4 (Euro-American lineage) with 41 (15.7%) isolates, and Lineage 1 (Indo-Oceanic lineage) with 30 isolates (11.5%). Based on spoligotyping, we found 45 different spoligotyping patterns that were previously described. The Beijing (83 isolates, 31.8%) and CAS spoligotype (52, 19.9%) were the dominant spoligotypes. A total of 36 (13.8%) isolates could not be assigned to any known spoligotyping pattern. Lineage 2 was associated with female sex (adjusted odds ratio [aOR] 2.58, 95% confidence interval [95% CI] 1.42–4.67, p = 0.002), and any drug resistance (aOR 2.79; 95% CI 1.43–5.45; p = 0.002). We found no evidence for an association of Lineage 2 with age or BCG vaccination status.

Conclusions

We found a large genetic diversity of M. tuberculosis in Nepal with representation of all four major lineages. Lineages 3 and 2 were dominating. Lineage 2 was associated with clinical characteristics. This study fills an important gap on the map of the M. tuberculosis genetic diversity in the Asian region.     

The Genotypic Population Structure of Mycobacterium tuberculosis Complex from Moroccan Patients Reveals a Predominance of Euro-American Lineages

Background

Tuberculosis (TB) remains a major health problem in Morocco. Characterization of circulating Mycobacterium tuberculosis genotypic lineages, important to understand the dynamic of the disease, was hereby addressed for the first time at a national level.

Methodology/Principal Findings

Spoligotyping was performed on a panel of 592 M. tuberculosis complex strains covering a 2-year period (2004–2006). It identified 129 patterns: 105 (n = 568 strains) corresponded to a SIT number in the SITVIT2 database, while 24 patterns were labeled as orphan. A total of 523 (88.3%) strains were clustered vs. 69 or 11.7% unclustered. Classification of strains within 3 large phylogenetical groups was as follows: group 1– ancestral/TbD1+/PGG1 (EAI, Bovis, Africanum), group 2– modern/TbD1−/PGG1 group (Beijing, CAS), group 3– evolutionary recent/TbD1−/PGG2/3 (Haarlem, X, S, T, LAM; alternatively designated as the Euro-American lineage). As opposed to group 3 strains (namely LAM, Haarlem, and T) that predominated (86.5% of all isolates), 6 strains belonged to group 2 (Beijing n = 5, CAS n = 1), and 3 strains (BOV_1 n = 2, BOV_4-CAPRAE) belonged to ancestral group 1 (EAI and AFRI lineage strains were absent). 12-loci MIRU-VNTR typing of the Casablanca subgroup (n = 114 strains) identified 71 patterns: 48 MITs and 23 orphan patterns; it allowed to reduce the clustering rate from 72.8% to 29.8% and the recent transmission rate from 64% to 20.2%.

Conclusion

The M. tuberculosis population structure in Morocco is highly homogeneous, and is characterized by the predominance of the Euro-American lineages, namely LAM, Haarlem, and T, which belong to the “evolutionary recent” TbD1−/PGG2/3 phylogenetic group. The combination of spoligotyping and MIRUs decreased the clustering rate significantly, and should now be systematically applied in larger studies. The methods used in this study appear well suited to monitor the M. tuberculosis population structure for an enhanced TB management program in Morocco.     

Strain Classification of Mycobacterium tuberculosis Isolates in Brazil Based on Genotypes Obtained by Spoligotyping,Mycobacterial Interspersed Repetitive Unit Typing and the Presence of Large Sequence and Single Nucleotide Polymorphism

Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC¹⁰³, RD^Rio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as “LAM-like”, 79.3% of these presenting the LAM-specific SNP fbpC¹⁰³. The frequency of RD^Rio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RD^Rio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.     

Clonal population of Mycobacterium tuberculosis strains reside within multiple lung cavities

Background

Unsuccessful treatment outcomes among patients with multi-/extensively- drug resistant tuberculosis (TB) have hampered efforts involved in eradicating this disease. In order to better understand the etiology of this disease, we aimed to determine whether single or multiple strains of Mycobacterium tuberculosis (MTB) are localized within lung cavities of patients suffering from chronic progressive TB.

Methodology/Findings

Multiple cavity isolates from lung of 5 patients who had undergone pulmonary resection surgery were analyzed on the basis of their drug susceptibility profile, and genotyped by spoligotyping and 24-loci MIRU-VNTR. The patients past history including treatment was studied. Three of the 5 patients had extensive drug resistant TB. Heteroresistance was also reported within different cavity isolates of the lung. Both genotyping methods reported the presence of clonal population of MTB strain within different cavities of the each patient, even those reporting heteroresistance. Four of the 5 patients were infected with a population of the Beijing genotype. Post-surgery they were prescribed a drug regimen consisting of cycloserine, a fluoroquinolone and an injectable drug. A 6 month post-surgery follow-up reported only 2 patients with positive clinical outcome, showing sputum conversion.

Conclusion

Identical spoligotype patterns and MIRU-VNTR profiles between multiple cavities of each patient, characterize the presence of clonal population of MTB strains (and absence of multiple MTB infection).     

Tuberculous Lymphadenitis in Ethiopia Predominantly Caused by Strains Belonging to the Delhi/CAS Lineage and Newly Identified Ethiopian Clades of the Mycobacterium tuberculosis Complex

Background

Recently, newly defined clades of Mycobacterium tuberculosis complex (MTBC) strains, namely Ethiopia 1–3 and Ethiopia H37Rv-like strains, and other clades associated with pulmonary TB (PTB) were identified in Ethiopia. In this study, we investigated whether these new strain types exhibit an increased ability to cause TB lymphadenitis (TBLN) and raised the question, if particular MTBC strains derived from TBLN patients in northern Ethiopia are genetically adapted to their local hosts and/or to the TBLN.

Methods

Genotyping of 196 MTBC strains isolated from TBLN patients was performed by spoligotyping and 24-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) typing. A statistical analysis was carried out to see possible associations between patient characteristics and phylogenetic MTBC strain classification.

Results

Among 196 isolates, the majority of strains belonged to the Delhi/CAS (38.8%) lineage, followed by Ethiopia 1 (9.7%), Ethiopia 3 (8.7%), Ethiopia H37RV-like (8.2%), Ethiopia 2 and Haarlem (7.7% each), URAL (3.6%), Uganda l and LAM (2% each), S-type (1.5%), X-type (1%), and 0.5% isolates of TUR, EAI, and Beijing genotype, respectively. Overall, 15 strains (7.7%) could not be allocated to a previously described phylogenetic lineage. The distribution of MTBC lineages is similar to that found in studies of PTB samples. The cluster rate (35%) in this study is significantly lower (P = 0.035) compared to 45% in the study of PTB in northwestern Ethiopia.

Conclusion

In the studied area, lymph node samples are dominated by Dehli/CAS genotype strains and strains of largely not yet defined clades based on MIRU-VNTR 24-loci nomenclature. We found no indication that strains of particular genotypes are specifically associated with TBLN. However, a detailed analysis of specific genetic variants of the locally contained Ethiopian clades by whole genome sequencing may reveal new insights into the host-pathogen co-evolution and specific features that are related to the local host immune system.     

Molecular Typing of Mycobacterium Tuberculosis Complex by 24-Locus Based MIRU-VNTR Typing in Conjunction with Spoligotyping to Assess Genetic Diversity of Strains Circulating in Morocco

Background

Standard 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing allows to get an improved resolution power for tracing TB transmission and predicting different strain (sub) lineages in a community.

Methodology

During 2010–2012, a total of 168 Mycobacterium tuberculosis Complex (MTBC) isolates were collected by cluster sampling from 10 different Moroccan cities, and centralized by the National Reference Laboratory of Tuberculosis over the study period. All isolates were genotyped using spoligotyping, and a subset of 75 was genotyped using 24-locus based MIRU-VNTR typing, followed by first line drug susceptibility testing. Corresponding strain lineages were predicted using MIRU-VNTRplus database.

Principal Findings

Spoligotyping resulted in 137 isolates in 18 clusters (2–50 isolates per cluster: clustering rate of 81.54%) corresponding to a SIT number in the SITVIT database, while 31(18.45%) patterns were unique of which 10 were labelled as “unknown” according to the same database. The most prevalent spoligotype family was LAM; (n = 81 or 48.24% of isolates, dominated by SIT42, n = 49), followed by Haarlem (23.80%), T superfamily (15.47%), >Beijing (2.97%), > U clade (2.38%) and S clade (1.19%). Subsequent 24-Locus MIRU-VNTR typing identified 64 unique types and 11 isolates in 5 clusters (2 to 3isolates per cluster), substantially reducing clusters defined by spoligotyping only. The single cluster of three isolates corresponded to two previously treated MDR-TB cases and one new MDR-TB case known to be contact a same index case and belonging to a same family, albeit residing in 3 different administrative regions. MIRU-VNTR loci 4052, 802, 2996, 2163b, 3690, 1955, 424, 2531, 2401 and 960 were highly discriminative in our setting (HGDI >0.6).

Conclusions

24-locus MIRU-VNTR typing can substantially improve the resolution of large clusters initially defined by spoligotyping alone and predominating in Morocco, and could therefore be used to better study tuberculosis transmission in a population-based, multi-year sample context.     

Genotypic diversity and drug susceptibility patterns among M. tuberculosis complex isolates from South-Western Ghana

Objective

The aim of this study was to use spoligotyping and large sequence polymorphism (LSP) to study the population structure of M. tuberculosis complex (MTBC) isolates.

Methods

MTBC isolates were identified using standard biochemical procedures, IS6110 PCR, and large sequence polymorphisms. Isolates were further typed using spoligotyping, and the phenotypic drug susceptibility patterns were determined by the proportion method.

Result

One hundred and sixty-two isolates were characterised by LSP typing. Of these, 130 (80.25%) were identified as Mycobacterium tuberculosis sensu stricto (MTBss), with the Cameroon sub-lineage being dominant (N = 59/130, 45.38%). Thirty-two (19.75%) isolates were classified as Mycobacterium africanum type 1, and of these 26 (81.25%) were identified as West-Africa I, and 6 (18.75%) as West-Africa II. Spoligotyping sub-lineages identified among the MTBss included Haarlem (N = 15, 11.53%), Ghana (N = 22, 16.92%), Beijing (4, 3.08%), EAI (4, 3.08%), Uganda I (4, 3.08%), LAM (2, 1.54%), X (N = 1, 0.77%) and S (2, 1.54%). Nine isolates had SIT numbers with no identified sub-lineages while 17 had no SIT numbers. MTBss isolates were more likely to be resistant to streptomycin (p<0.008) and to any drug resistance (p<0.03) when compared to M. africanum.

Conclusion

This study demonstrated that overall 36.4% of TB in South-Western Ghana is caused by the Cameroon sub-lineage of MTBC and 20% by M. africanum type 1, including both the West-Africa 1 and West-Africa 2 lineages. The diversity of MTBC in Ghana should be considered when evaluating new TB vaccines.     

Multidrug Resistant Mycobacterium tuberculosis: A Retrospective katG and rpoB Mutation Profile Analysis in Isolates from a Reference Center in Brazil

Background

Multidrug resistance is a critical factor in tuberculosis control. To gain better understanding of multidrug resistant tuberculosis in Brazil, a retrospective study was performed to compare genotypic diversity and drug resistance associated mutations in Mycobacterium tuberculosis isolates from a national reference center.

Methods and Findings

Ninety-nine multidrug resistant isolates from 12 Brazilian states were studied. Drug-resistance patterns were determined and the rpoB and katG genes were screened for mutations. Genotypic diversity was investigated by IS6110-RFLP and Luminex 47 spoligotyping. Mutations in rpoB and katG were seen in 91% and 93% of the isolates, respectively. Codon 315 katG mutations occurred in 82.8% of the isolates with a predominance of the Ser315Thr substitution. Twenty-five isolates were clustered in 11 groups with identical IS6110-RFLP patterns while 74 showed unique patterns with no association between mutation frequencies or susceptibility profiles. The most prevalent spoligotyping lineages were LAM (47%), T (17%) and Haarlen (12%). The Haarlen lineage showed a higher frequency of codon 516 rpoB mutations while codon 531 mutations prevailed in the other isolates.

Conclusions

Our data suggest that there were no major multidrug resistant M. tuberculosis strains transmitted among patients referred to the reference center, indicating an independent acquisition of resistance. In addition, drug resistance associated mutation profiles were well established among the main spoligotyping lineages found in these Brazilian multidrug resistant isolates, providing useful data for patient management and treatment.     

Population Structure among Mycobacterium tuberculosis Isolates from Pulmonary Tuberculosis Patients in Colombia

Background

Phylogeographic composition of M. tuberculosis populations reveals associations between lineages and human populations that might have implications for the development of strategies to control the disease. In Latin America, lineage 4 or the Euro-American, is predominant with considerable variations among and within countries. In Colombia, although few studies from specific localities have revealed differences in M. tuberculosis populations, there are still areas of the country where this information is lacking, as is a comparison of Colombian isolates with those from the rest of the world.

Principal Findings

A total of 414 M. tuberculosis isolates from adult pulmonary tuberculosis cases from three Colombian states were studied. Isolates were genotyped using IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, and 24-locus Mycobacterial interspersed repetitive units variable number tandem repeats (MIRU-VNTRs). SIT42 (LAM9) and SIT62 (H1) represented 53.3% of isolates, followed by 8.21% SIT50 (H3), 5.07% SIT53 (T1), and 3.14% SIT727 (H1). Composite spoligotyping and 24-locus MIRU- VNTR minimum spanning tree analysis suggest a recent expansion of SIT42 and SIT62 evolved originally from SIT53 (T1). The proportion of Haarlem sublineage (44.3%) was significantly higher than that in neighboring countries. Associations were found between M. tuberculosis MDR and SIT45 (H1), as well as HIV-positive serology with SIT727 (H1) and SIT53 (T1).

Conclusions

This study showed the population structure of M. tuberculosis in several regions from Colombia with a dominance of the LAM and Haarlem sublineages, particularly in two major urban settings (Medellín and Cali). Dominant spoligotypes were LAM9 (SIT 42) and Haarlem (SIT62). The proportion of the Haarlem sublineage was higher in Colombia compared to that in neighboring countries, suggesting particular conditions of co-evolution with the corresponding human population that favor the success of this sublineage.     

High Prevalence of Shared International Type 53 among Mycobacterium tuberculosis Complex Strains in Retreated Patients from C?te d’Ivoire

Background

Genotyping methods are useful tools to provide information on tuberculosis epidemic. They can allow a better response from health authorities and the implementation of measures for tuberculosis control. This study aimed to identify the main lineages and clades of Mycobacterium tuberculosis complex strains circulating in Côte d’Ivoire.

Methods/Main Findings

Strains isolated from sputum samples of patients ongoing retreatment from all the country were characterized by spoligotyping and by MIRU-VNTR. Profiles obtained by spoligotyping were first compared to the SITVIT/SpolDB4 database for family assignment. Of 194 strains analysed, 146 (75.3%) belonged to the T lineage. The most predominant spoligotype was the shared international type 53 with 135 strains (69.6%). In contrast with neighbouring countries, LAM (11 strains, 5.7%) and H (9 strains 4.6%) lineages were slightly represented. Only 3 Beijing strains (1.5%) and 4 strains of Mycobacterium africanum (2%) were found. Analysis of the results obtained with MIRU-VNTR revealed also a high level of clustering.

Conclusion/Significance

The population of Mycobacterium tuberculosis complex strains among retreatment cases in Côte d’Ivoire exhibits a low diversity, allowing to assume recent transmission and locally based infection.     

Spoligotyping and drug resistance analysis of Mycobacterium tuberculosis strains from national survey in China

Background

Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), is one of the major causes of death in the world today. Although China has the second largest global case rate of tuberculosis, a systematic study of TB prevalence in China has not been completed. From 2006 to 2007, the base line surveillance of tuberculosis was carried out by Ministry of Health, and more than 4000 representative strains were selected from 31 provinces in China.

Methodology/Principal Findings

The aim of the present research was to survey the genotypes of representative Mycobacterium tuberculosis (M. tuberculosis) strains from China using spacer oligonucleotide typing (spoligotyping), and to analyze the relationship between genotype and drug resistance for the first time. A total of 4017 clinical isolates were collected from 2007 to 2008 throughout China. Among those M. tuberculosis isolates, 2500 (62.2%) isolates were Beijing genotypes. The percentage of Beijing genotypes in northern China was higher than in southern China (76.5% vs. 53.2%). Additionally, the frequencies of rifampin-resistant, ofloxacin-resistant and multidrug-resistant isolates were significantly higher in Beijing genotype strains than non-Beijing strains. Furthermore, a novel genotype named “China Southern genotype (CS)” was only isolated from Fujian and Guangdong provinces. Hence, it is very practical to uncover the reason for prevalence of the CS type in southern China.

Conclusions/Significance

In conclusion, Beijing family genotypes were still the predominant genotype throughout China, which exhibited a greater correlation with rifampin-resistance, ofloxacin-resistance and MDR phenotypes than other TB spoligotypes, and some regions of China showed several unique characters in the distribution of M. tuberculosis genotypes. Our research represents an important contribution for the TB control and research community, which completes broad pictures on drug resistance levels and distribution of M. tuberculosis strain types over China.     

Distribution of spoligotyping defined genotypic lineages among drug-resistant Mycobacterium tuberculosis complex clinical isolates in Ankara, Turkey

Background

Investigation of genetic heterogeneity and spoligotype-defined lineages of drug-resistant Mycobacterium tuberculosis clinical isolates collected during a three-year period in two university hospitals and National Tuberculosis Reference and Research Laboratory in Ankara, Turkey.

Methods and Findings

A total of 95 drug-resistant M. tuberculosis isolates collected from three different centers were included in this study. Susceptibility testing of the isolates to four major antituberculous drugs was performed using proportion method on Löwenstein–Jensen medium and BACTEC 460-TB system. All clinical isolates were typed by using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) methods. Seventy-three of the 95 (76.8%) drug resistant M. tuberculosis isolates were isoniazid-resistant, 45 (47.4%) were rifampicin-resistant, 32 (33.7%) were streptomycin-resistant and 31 (32.6%) were ethambutol-resistant. The proportion of multidrug-resistant isolates (MDR) was 42.1%. By using spoligotyping, 35 distinct patterns were observed; 75 clinical isolates were grouped in 15 clusters (clustering rate of 79%) and 20 isolates displayed unique patterns. Five of these 20 unique patterns corresponded to orphan patterns in the SITVIT2 database, while 4 shared types containing 8 isolates were newly created. The most prevalent M. tuberculosis lineages were: Haarlem (23/95, 24.2%), ill-defined T superfamily (22/95, 23.2%), the Turkey family (19/95, 20%; previously designated as LAM7-TUR), Beijing (6/95, 6.3%), and Latin-America & Mediterranean (LAM, 5/95 or 5.3%), followed by Manu (3/95, 3.2%) and S (1/95, 1%) lineages. Four of the six Beijing family isolates (66.7%) were MDR. A combination of IS6110-RFLP and spoligotyping reduced the clustering rate from 79% to 11.5% among the drug resistant isolates.

Conclusions

The results obtained showed that ill-defined T, Haarlem, the Turkey family (previously designated as LAM7-TUR family with high phylogeographical specifity for Turkey), Beijing and LAM were predominant lineages observed in almost 80% of the drug-Resistant M. tuberculosis complex clinical isolates in Ankara, Turkey.     

Mycobacterium tuberculosis Genotypes Determined by Spoligotyping to Be Circulating in Colombia between 1999 and 2012 and Their Possible Associations with Transmission and Susceptibility to First-Line Drugs

Introduction

Tuberculosis (TB) remains a primary public health problem worldwide. The number of multidrug-resistant tuberculosis (MDR TB) cases has increased in recent years in Colombia. Knowledge of M. tuberculosis genotypes defined by spoligotyping can help determine the circulation of genotypes that must be controlled to prevent the spread of TB.

Objective

To describe the genotypes of M. tuberculosis using spoligotyping in resistant and drug-sensitive isolates and their possible associations with susceptibility to first-line drugs.

Methods

An analytical observational study was conducted that included 741 isolates of M. tuberculosis from patients. The isolates originated from 31 departments and were obtained by systematic surveillance between 1999 and 2012.

Results

In total 61.94% of the isolates were resistant to 1 or more drugs, and 147 isolates were MDR. In total, 170 genotypes were found in the population structure of Colombian M. tuberculosis isolates. The isolates were mainly represented by four families: LAM (39.9%), Haarlem (19%), Orphan (17%) and T (9%). The SIT42 (LAM 9) was the most common genotype and contained 24.7% of the isolates, followed by the genotypes SIT62 (Haarlem1), SIT53 (T1), and SIT50 (H3). A high clustering of isolates was evident with 79.8% of the isolates classified into 32 groups. The Beijing family was associated with resistant isolates, whereas the Haarlem and T families were associated with sensitive isolates. The Haarlem family was also associated with grouped isolates (p = 0.031).

Conclusions

A high proportion (approximately 80%) of isolates was found in clusters; these clusters were not associated with resistance to first-line drugs. The Beijing family was associated with drug resistance, whereas the T and Haarlem families were associated with susceptibility in the Colombian isolates studied.     

Molecular Epidemiology and Clinical Characteristics of Drug-Resistant Mycobacterium tuberculosis in a Tuberculosis Referral Hospital in China

Background

Despite the large number of drug-resistant tuberculosis (TB) cases in China, few studies have comprehensively analyzed the drug resistance-associated gene mutations and genotypes in relation to the clinical characteristics of M. tuberculosis (Mtb) isolates.

Methodology/Principal Findings

We thus analyzed the phenotypic and genotypic drug resistance profiles of 115 Mtb clinical isolates recovered from a tuberculosis referral hospital in Beijing, China. We also performed genotyping by 28 loci MIRU-VNTR analysis. Socio-demographic and clinical data were retrieved from medical records and analyzed. In total, 78 types of mutations (including 42 previously reported and 36 newly identified ones) were identified in 115 Mtb clinical isolates. There was significant correlation between phenotypic and genotypic drug resistance rates for first-line anti-TB drugs (P<0.001). Genotyping revealed 101 MIRU-VNTR types, with 20 isolates (17.4%) being clustered and 95 isolates (82.6%) having unique genotypes. Higher proportion of re-treatment cases was observed among patients with clustered isolates than those with unique MIRU-VNTR genotypes (75.0% vs. 41.1%). Moreover, clinical epidemiological links were identified among patients infected by Mtb strains belonging to the same clusters, suggesting a potential of transmission among patients.

Conclusions/Significance

Our study provided information on novel potential drug resistance-associated mutations in Mtb. In addition, the genotyping data from our study suggested that enforcement of the implementation of genotyping in diagnostic routines would provide important information for better monitor and control of TB transmission.     

Extrapulmonary Tuberculosis: Mycobacterium tuberculosis Strains and Host Risk Factors in a Large Urban Setting in Brazil

Background

Factors related to the development of extrapulmonary forms of tuberculosis (EPTB) are still poorly understood, particularly in high-endemic countries like Brazil. The objective of the paper is to determine host and Mycobacterium tuberculosis (MTB) strain-related factors associated with the development of EPTB in Espírito Santo state, Brazil.

Methods and Findings

We conducted a retrospective laboratory-based surveillance study of new tuberculosis (TB) cases diagnosed in Espírito Santo state, Brazil between 1998 and 2007. We genotyped 612 isolates of MTB from 606 TB patients using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) typing and compared sociodemographic and clinical characteristics of patients with pulmonary TB (PTB) and EPTB. Among 606 patients, 464 (77%) had PTB, 79 (13%) had EPTB, 51 (8%) had both, and 12 (2%) had miliary TB. The IS6110 RFLP analysis demonstrated that 250 (41%) isolates belonged to clustered RFLP patterns, 27 (11%) of which were from EPTB. We identified 73 clusters including 35 (48%) composed of 2 isolates each. By spoligotyping, 506 (83%) MTB isolates fell into known patterns and 106 (17%) fell into patterns with no family assignment; 297 (48%) isolates belonged to the Latin-American Mediterranean family. Higher school level (4-7 years OR: 0.16 95% CI 0.34-0.73 and > 8 years of education, OR 0.06 95% CI 0.009-0.50) white ethnicity (OR: 2.54 95% CI 1.03-6.25) and HIV infection (OR: 16.83 95% CI 5.23-54.18) were associated with EPTB. No specific strain lineage or percentage of clustering was associated with EPTB.

Conclusions

These results demonstrate that risk factors for EPTB are related more to host than to MTB strain lineage characteristics.     

Reduced transmissibility of East African Indian strains of Mycobacterium tuberculosis

Background

Mycobacterium tuberculosis (MTB) has been classified into 4 main lineages. Some reports have associated certain lineages with particular clinical phenotypes, but there is still insufficient information regarding the clinical and epidemiologic implications of MTB lineage variation.

Methods

Using large sequence polymorphisms we classified MTB isolates from a population-based study in Montreal, Canada into the 4 major lineages, and identified the associated clinical and epidemiologic features. In addition, IS6110-RFLP and spoligotyping were used as indicators of recent TB transmission. The study population was divided into a derivation cohort, diagnosed between 2001 and 2007, and a separate validation cohort, diagnosed between 1996 and 2000.

Results

In the derivation cohort, when compared to the other MTB lineages, the East African-Indian (EAI) lineage was associated with lower rates of TB transmission, as measured by: positive TST among close contacts of pulmonary TB cases (adjusted odds ratio 0.6: [95% confidence interval 0.4–0.9]), and clustered TB cases (0.3: [<0.001–0.6]). Severe forms of TB were also less likely among the EAI group (0.4: [<0.001–0.8]). There were no significant differences when comparing patients with the other MTB lineages. In the validation cohort, the EAI lineage was associated with lower rates of positive TST among contacts (0.5: [0.3–0.9]) and a trend towards less clustered TB cases (0.5: [0.1–1.8]) when compared to the other lineages. Disease severity among the different groups was not significantly different in the validation cohort.

Conclusions

We conclude that in Montreal, EAI strains were associated with reduced transmission compared to other MTB lineages.     

Transmission of Mycobacterium tuberculosis between Farmers and Cattle in Central Ethiopia

Background

Transmission of Mycobacterium tuberculosis (M. tuberculosis) complex could be possible between farmers and their cattle in Ethiopia.

Methodology/Principal Findings

A study was conducted in mixed type multi-purposes cattle raising region of Ethiopia on 287 households (146 households with case of pulmonary tuberculosis (TB) and 141 free of TB) and 287 herds consisting of 2,033 cattle belonging to these households to evaluate transmission of TB between cattle and farmers. Interview, bacteriological examinations and molecular typing were used for human subjects while comparative intradermal tuberculin (CIDT) test, post mortem and bacteriological examinations, and molecular typing were used for animal studies. Herd prevalence of CIDT reactors was 9.4% and was higher (p<0.01) in herds owned by households with TB than in herds owned by TB free households. Animal prevalence was 1.8% and also higher (p<0.01) in cattle owned by households with TB case than in those owned by TB free households. All mycobacteria (141) isolated from farmers were M. tuberculosis, while only five of the 16 isolates from cattle were members of the M. tuberculosis complex (MTC) while the remaining 11 were members of non-tuberculosis mycobacteria (NTM). Further speciation of the five MTC isolates showed that three of the isolates were M. bovis (strain SB1176), while the remaining two were M. tuberculosis strains (SIT149 and SIT53). Pathology scoring method described by “Vordermeier et al. (2002)” was applied and the average severity of pathology in two cattle infected with M. bovis, in 11 infected with NTM and two infected with M. tuberculosis were 5.5, 2.1 and 0.5, respectively.

Conclusions/Significance

The results showed that transmission of TB from farmers to cattle by the airborne route sensitizes the cows but rarely leads to TB. Similarly, low transmission of M. bovis between farmers and their cattle was found, suggesting requirement of ingestion of contaminated milk from cows with tuberculous mastitis.     

Genotypic Diversity Analysis of Mycobacterium tuberculosis Strains Collected from Beijing in 2009, Using Spoligotyping and VNTR Typing

Background

Tuberculosis (TB) is a serious problem in China. While there have been some studies on the nationwide genotyping of Mycobacterium tuberculosis (M. tuberculosis), there has been little detailed research in Beijing, the capital of China, which has a huge population. Here, M. tuberculosis clinical strains collected in Beijing during 2009 were genotyped by classical methods.

Methodology/Principal Findings

Our aim was to analyze the genetic diversity of M. tuberculosis strains within the Beijing metropolitan area. We characterized these strains using two standard methods, spoligotyping (n = 1585) and variable number of tandem repeat (VNTR) typing (n = 1053). We found that the most prominent genotype was Beijing family genotype. Other genotypes included the MANU, T and H families etc. Spoligotyping resulted in 137 type patterns, included 101 unclustered strains and 1484 strains clustered into 36 clusters. In VNTR typing analysis, we selected 12-locus (QUB-11b, MIRU10, Mtub21, MIRU 23, MIRU39, MIRU16, MIRU40, MIRU31, Mtub24, Mtub04, MIRU20, and QUB-4156c) and named it 12-locus (BJ) VNTR. VNTR resulted in 869 type patterns, included 796 unclustered strains and 257 strains clustered into 73 clusters. It has almost equal discriminatory power to the 24-locus VNTR.

Conclusions/Significance

Our study provides a detailed characterization of the genotypic diversity of M. tuberculosis in Beijing. Combining spoligotyping and VNTR typing to study the genotyping of M. tuberculosis gave superior results than when these techniques were used separately. Our results indicated that Beijing family strains were still the most prevalent M. tuberculosis in Beijing. Moreover, VNTR typing analyzing of M. tuberculosis strains in Beijing was successfully accomplished using 12-locus (BJ) VNTR. This method used for strains genotyping from the Beijing metropolitan area was comparable. This study will not only provide TB researchers with valuable information for related studies, but also provides guidance for the prevention and control of TB in Beijing.