HLA class II locus and susceptibility to podoconiosis.

BACKGROUND: Podoconiosis is a tropical lymphedema resulting from long-term barefoot exposure to red-clay soil derived from volcanic rock. The World Health Organization recently designated it as a neglected tropical disease. Podoconiosis develops in only a subgroup of exposed people, and studies have shown familial clustering with high heritability (63%). METHODS: We conducted a genomewide association study of 194 case patients and 203 controls from southern Ethiopia. Findings were validated by means of family-based association testing in 202 family trios and HLA typing in 94 case patients and 94 controls. RESULTS: We found a genomewide significant association of podoconiosis with the single-nucleotide polymorphism (SNP) rs17612858, located 5.8 kb from the HLA-DQA1 locus (in the allelic model: odds ratio, 2.44; 95% confidence interval [CI], 1.82 to 3.26; P=1.42×10(-9); and in the additive model: odds ratio, 2.19; 95% CI, 1.66 to 2.90; P=3.44×10(-8)), and suggestive associations (P<1.0×10(-5)) with seven other SNPs in or near HLA-DQB1, HLA-DQA1, and HLA-DRB1. We confirmed these associations using family-based association testing. HLA typing showed the alleles HLA-DRB1*0701 (odds ratio, 2.00), DQA1*0201 (odds ratio, 1.91), and DQB1*0202 (odds ratio, 1.79) and the HLA-DRB1*0701-DQB1*0202 haplotype (odds ratio, 1.92) were risk variants for podoconiosis. CONCLUSIONS: Association between variants in HLA class II loci with podoconiosis (a noncommunicable disease) suggests that the condition may be a T-cell-mediated inflammatory disease and is a model for gene-environment interactions that may be relevant to other complex genetic disorders. (Funded by the Wellcome Trust and others.).

Clinical and pharmacogenomic implications of genetic variation in a Southern Ethiopian population.

Africa is home to genetically diverse human populations. We compared the genetic structure of the Wolaita ethnic population from Southern Ethiopia (WETH, n=120) with HapMap populations using genome-wide variants. We investigated allele frequencies of 443 clinically and pharmacogenomically relevant genetic variants in WETH compared with HapMap populations. We found that WETH were genetically most similar to the Kenya Maasai and least similar to the Japanese in HapMap. Variant alleles associated with increased risk of adverse reactions to drugs used for treating tuberculosis (rs1799929 and rs1495741 in NAT2), thromboembolism (rs7294, rs9923231 and rs9934438 in VKORC1), and HIV/AIDS and solid tumors (rs2242046 in SLC28A1) had significantly higher frequencies in WETH compared with African ancestry HapMap populations. Our results illustrate that clinically relevant pharmacogenomic loci display allele frequency differences among African populations. We conclude that drug dosage guidelines for important global health diseases should be validated in genetically diverse African populations.

Investigation of the high rates of extrapulmonary tuberculosis in Ethiopia reveals no single driving factor and minimal evidence for zoonotic transmission of Mycobacterium bovis infection.

BACKGROUND: Ethiopia, a high tuberculosis (TB) burden country, reports one of the highest incidence rates of extra-pulmonary TB dominated by cervical lymphadenitis (TBLN). Infection with Mycobacterium bovis has previously been excluded as the main reason for the high rate of extrapulmonary TB in Ethiopia. METHODS: Here we examined demographic and clinical characteristics of 953 pulmonary (PTB) and 1198 TBLN patients visiting 11 health facilities in distinct geographic areas of Ethiopia. Clinical characteristics were also correlated with genotypes of the causative agent, Mycobacterium tuberculosis. RESULTS: No major patient or bacterial strain factor could be identified as being responsible for the high rate of TBLN, and there was no association with HIV infection. However, analysis of the demographic data of involved patients showed that having regular and direct contact with live animals was more associated with TBLN than with PTB, although no M. bovis was isolated from patients with TBLN. Among PTB patients, those infected with Lineage 4 reported "contact with other TB patient" more often than patients infected with Lineage 3 did (OR = 1.6, CI 95% 1.0-2.7; p = 0.064). High fever, in contrast to low and moderate fever, was significantly associated with Lineage 4 (OR = 2.3; p = 0.024). On the other hand, TBLN cases infected with Lineage 4 tended to get milder symptoms overall for the constitutional symptoms than those infected with Lineage 3. CONCLUSIONS: The study suggests a complex role for multiple interacting factors in the epidemiology of extrapulmonary TB in Ethiopia, including factors that can only be derived from population-based studies, which may prove to be significant for TB control in Ethiopia.

Molecular typing and drug sensitivity testing of Mycobacterium tuberculosis isolated by a community-based survey in Ethiopia.

BACKGROUND: The identification of circulating TB strains in the community and drug sensitivity patterns is essential for the tuberculosis control program. This study was undertaken to identify M. tuberculosis strains circulating in selected communities in Ethiopia as well as to evaluate the drug sensitivity pattern of these strains. METHOD: This study was a continuation of the Ethiopian National TB Prevalence Survey that was conducted between 2010 and 2011. Culture-positive isolates of M. tuberculosis from previous study were typed using region of difference (RD) 9-based polymerase chain reaction (PCR) and spoligotyping. Drug sensitivity testing was conducted using the indirect proportion method on Lowenstein-Jensen media. RESULT: All 92 isolates were confirmed as M. tuberculosis by RD9-based PCR and spoligotyping of 91 of these isolates leds to the identification of 41 spoligotype patterns. Spoligotype revealed higher diversity (45 %) and among this 65.8 % (27/41) were not previously reported. The strains were grouped into 14 clusters consisting of 2-15 isolates. The dominant strains were SIT53, SIT149 and SIT37 consisting of 15, 11, and 9 isolates, respectively. Our study reveals 70 % (64/91) clustered strains and only 39.1 % (25/64) occurred within the same Kebele. Further assignment of the strains to the lineages showed that 74.7 % (68/91) belonged to Euro-American lineage, 18.6 % (17/91) to East Africa Indian lineage and the remaining 6.5 % (6/91) belonged to Indo-oceanic lineage. Valid drug susceptibility test results were available for 90 of the 92 isolates. Mono-resistance was observed in 27.7 % (25/90) and poly-resistance in 5.5 % (5/90) of the isolates. Moreover, multi-drug resistance (MDR-TB) was detected in 4.4 % of the isolates whilst the rest (60/90) were susceptible to all drugs. The highest level of mono-resistance, 26.6 % (24/90), was observed for streptomycin with majority (91.1 %) of streptomycin mono-resistant strains belonging to the Euro-American lineage. CONCLUSION: In this study, the strains of M. tuberculosis circulating in selected sites of Ethiopia were identified along with the drug sensitivity patterns. Thus, these findings are useful for the TB Control Program of the country.

Mycobacterial lineages causing pulmonary and extrapulmonary tuberculosis, Ethiopia.

Molecular typing of 964 specimens from patients in Ethiopia with lymph node or pulmonary tuberculosis showed a similar distribution of Mycobacterium tuberculosis strains between the 2 disease manifestations and a minimal role for M. bovis. We report a novel phylogenetic lineage of M. tuberculosis strongly associated with the Horn of Africa.

Population-based prevalence survey of tuberculosis in the Tigray region of Ethiopia.

BACKGROUND: Population based prevalence survey is an important epidemiological index to measure the burden of tuberculosis (TB) disease and monitor progress towards TB control in high burden countries like Ethiopia. This study was aimed to estimate the prevalence of bacteriologically confirmed pulmonary tuberculosis (PTB) in the Tigray region of Ethiopia. METHODS: Sixteen rural and urban villages were randomly selected in a stratified multistage cluster sampling. Individuals aged 15 years and older were screened by symptom inquiry for PTB. Those individuals who were symptomatic of PTB provided two sputum samples for smear microscopy, culture and molecular typing. RESULTS: The study covering 4,765 households screened a total of 12,175 individuals aged 15 years and above. The overall weighted prevalence of bacteriologically confirmed PTB in the Tigray region of Ethiopia was found to be 216/100,000 (95% CI: 202.08, 230.76) while the weighted prevalence of smear-positive PTB was 169/100,000 (95% CI: 155.53, 181.60). The prevalence of bacteriologically confirmed TB was higher amongst males (352/100 000; 95% CI: 339.05, 364.52) than females (162/100 000; 95% CI: 153.60, 171.17) and among rural (222/100,000; 95% CI: 212.77-231.53) as compared to urban residents (193/100,000; 95% CI: 183.39-203.59). CONCLUSIONS: This study found a relatively higher prevalence smear-positive PTB in the region than in a same period nationwide survey and identified a significant number of undetected PTB cases. The urgency for improved TB case detection and intensified community awareness is emphasized.

Prevalence study on bovine tuberculosis and molecular characterization of its causative agents in cattle slaughtered at Addis Ababa municipal abattoir, Central Ethiopia.

A cross-sectional study was conducted on 500 cattle slaughtered at Addis Ababa abattoir to determine the prevalence of bovine tuberculosis (BTB) and characterize its causative agents. Postmortem examination, mycobacteriological culturing, region of difference-4 (RD4)-based PCR and spoligotyping were applied. The prevalence of BTB was 5 % on the basis of postmortem inspection alone but 1.2 % based on molecular confirmation. Factors including age, sex, and breed showed statistically significant association with BTB (p < 0.05). Gross lesions were observed most frequently (68 %) in the lungs and lung-associated lymph nodes compared to other organs and lymph nodes. Of the 25 grossly suspicious TB lesions processed and cultured, only six (24 %) were culture-positive, yielding Mycobacterium bovis confirmed by RD4 deletion typing. Further characterization of the six M. bovis isolates at the strain level by using spoligotyping revealed that one did not belong to any previously known type, while the others belonged to types SB1176 (two), SB1477 (two), and SB0133 (one). The new strain was submitted to the international M. Bovis.org database for international code designation. The study confirms the considerable prevalence of bovine tuberculosis in cattle slaughtered at Addis Ababa abattoir and highlights the need for control of bovine tuberculosis in the country.

Lack of methoxy-mycolates characterizes the geographically restricted lineage 7 of Mycobacterium tuberculosis complex.

Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of 'ancient' lineage 1 and prior to the Eurasian dispersal of 'modern' lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages - such as constitutive expression of nitrate reductase - as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host-pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe.

Drug Resistance in Tuberculous Lymphadenitis: Molecular Characterization.

Background: Drug-resistant tuberculosis (TB) epidemic in high-TB-incidence countries, particularly Ethiopia, remains a significant challenge. As a result, we investigated the drug resistance, common gene mutation, and molecular characterization of mycobacterial isolates from patients with suspected tuberculous lymphadenitis (TBLN). Methodology. A cross-sectional study of 218 FNA samples from TBLN patients inoculated on Lowenstein-Jensen media was carried out. The culture isolates were identified as MTB by polymerase chain reaction (PCR) and the difference-9 (RD9) test region. In addition, the GenoType MTBDRplus assay tested the first and second-line MTB drugs, and the spoligotyping strain-dependent polymorphism test was determined. Results: Among the 50 culture-positive isolates, 14% (7/50) had drug resistance caused by a gene mutation. Out of these, 4 (8%) isolates were mono-resistant to isoniazid drug, which is caused by a gene mutation in katG in the region of interrogated at codon 315 in the amino acid sequence of S315T1, and 3 (6%) isolates were resistant to both rifampicin and isoniazid drugs. The mutation was observed for katG (at codon 315 with a change in the sequence of amino acid S315T) and rpoB (at codon 530-533 with a change in the sequence of amino acid S531L (S450L)) genes. The most prevalent spoligotypes were orphan and SIT53 strains. Conclusion: The predominance of INH mono-resistance poses a critical risk for the potential development of MDR-TB, as INH mono-resistance is a typical pathway to the occurrence of MDR-TB. The orphan and SIT53 (T) strains were the most common in the study area, and a drug-resistant strain caused by a common gene mutation could indicate the transmission of clonal-resistant strains in the community.

African 2, a clonal complex of Mycobacterium bovis epidemiologically important in East Africa.

We have identified a clonal complex of Mycobacterium bovis isolated at high frequency from cattle in Uganda, Burundi, Tanzania, and Ethiopia. We have named this related group of M. bovis strains the African 2 (Af2) clonal complex of M. bovis. Af2 strains are defined by a specific chromosomal deletion (RDAf2) and can be identified by the absence of spacers 3 to 7 in their spoligotype patterns. Deletion analysis of M. bovis isolates from Algeria, Mali, Chad, Nigeria, Cameroon, South Africa, and Mozambique did not identify any strains of the Af2 clonal complex, suggesting that this clonal complex of M. bovis is localized in East Africa. The specific spoligotype pattern of the Af2 clonal complex was rarely identified among isolates from outside Africa, and the few isolates that were found and tested were intact at the RDAf2 locus. We conclude that the Af2 clonal complex is localized to cattle in East Africa. We found that strains of the Af2 clonal complex of M. bovis have, in general, four or more copies of the insertion sequence IS6110, in contrast to the majority of M. bovis strains isolated from cattle, which are thought to carry only one or a few copies.

Bovine tuberculosis at a cattle-small ruminant-human interface in Meskan, Gurage region, Central Ethiopia.

BACKGROUND: Bovine tuberculosis (BTB) is endemic in Ethiopian cattle. The aim of this study was to assess BTB prevalence at an intensive contact interface in Meskan Woreda (district) in cattle, small ruminants and suspected TB-lymphadenitis (TBLN) human patients. METHODS: The comparative intradermal test (CIDT) was carried out for all animals involved in the cross-sectional study and results interpreted using a > 4 mm and a > 2 mm cut-off. One PPD positive goat was slaughtered and lymph nodes subjected to culture and molecular typing. In the same villages, people with lymphadenitis were subjected to clinical examination. Fine needle aspirates (FNA) were taken from suspected TBLN and analyzed by smear microscopy and molecular typing. RESULTS: A total of 1214 cattle and 406 small ruminants were tested for BTB. In cattle, overall individual prevalence (> 2 mm cut-off) was 6.8% (CI: 5.4-8.5%) with 100% herd prevalence. Only three small ruminants (2 sheep and 1 goat) were reactors. The overall individual prevalence in small ruminants (> 2 mm cut-off) was 0.4% (CI: 0.03-5.1%) with 25% herd prevalence. Cattle from owners with PPD positive small ruminants were all PPD negative. 83% of the owners kept their sheep and goats inside their house at night and 5% drank regularly goat milk.FNAs were taken from 33 TBLN suspected cases out of a total of 127 screened individuals with lymph node swellings. Based on cytology results, 12 were confirmed TBLN cases. Nine out of 33 cultures were AFB positive. Culture positive samples were subjected to molecular typing and they all yielded M. tuberculosis. M. tuberculosis was also isolated from the goat that was slaughtered. CONCLUSIONS: This study highlighted a low BTB prevalence in sheep and goats despite intensive contact with cattle reactors. TBLN in humans was caused entirely by M. tuberculosis, the human pathogen. M. tuberculosis seems to circulate also in livestock but their role at the interface is unknown.

Molecular Epidemiology of Mycobacterium tuberculosis strains isolated from pulmonary tuberculosis patients in south Ethiopia.

INTRODUCTION: Understanding the epidemiology of tuberculosis is limited by lack of genotyping data. We sought to characterize the drug susceptibility testing patterns and genetic diversity of M. tuberculosis isolates in southern Ethiopia. METHODOLOGY: A cross-sectional study was conducted among newly diagnosed sputum smear positive patients with tuberculosis visiting nine health facilities in southern Ethiopia from June 2015 to May 2016. Three consecutive sputum samples (spot-morning-spot) per patient were examined using acid-fast bacilli smear microscopy with all smear positive specimens having acid-fast bacilli cultures performed. M. tuberculosis isolates had drug susceptibility testing performed using indirect proportion method and were genotyped with RD9 deletion analysis and spoligotyping. Mapping of strain was made using geographic information system. RESULTS: Among 250 newly diagnosed patients with tuberculosis, 4% were HIV co-infected. All 230 isolates tested were M. tuberculosis strains belonging to three lineages: Euro-American, 187 (81%), East-African-Indian, 31 (14%), and Lineage 7 (Ethiopian lineage), 8 (4%); categorized into 63 different spoligotype patterns, of which 85% fell into 28 clusters. M. tuberculosis strains were clustered by geographic localities. The dominant spoligotypes were SIT149 (21%) and SIT53 (19%). Drug susceptibility testing found that 14% of isolates tested were resistant to > 1 first line anti- tuberculosis drugs and 11% to INH. SIT 149 was dominant among drug resistant isolates. CONCLUSIONS: The study revealed several clusters and drug resistant strains of M. tuberculosis in the study area, suggesting recent transmission including of drug resistant tuberculosis. Wider monitoring of drug susceptibility testing and geospatial analysis of transmission trends is required to control tuberculosis in southern Ethiopia.

Epidemiology of Mycobacterium tuberculosis lineages and strain clustering within urban and peri-urban settings in Ethiopia.

BACKGROUND: Previous work has shown differential predominance of certain Mycobacterium tuberculosis (M. tb) lineages and sub-lineages among different human populations in diverse geographic regions of Ethiopia. Nevertheless, how strain diversity is evolving under the ongoing rapid socio-economic and environmental changes is poorly understood. The present study investigated factors associated with M. tb lineage predominance and rate of strain clustering within urban and peri-urban settings in Ethiopia. METHODS: Pulmonary Tuberculosis (PTB) and Cervical tuberculous lymphadenitis (TBLN) patients who visited selected health facilities were recruited in the years of 2016 and 2017. A total of 258 M. tb isolates identified from 163 sputa and 95 fine-needle aspirates (FNA) were characterized by spoligotyping and compared with international M.tb spoligotyping patterns registered at the SITVIT2 databases. The molecular data were linked with clinical and demographic data of the patients for further statistical analysis. RESULTS: From a total of 258 M. tb isolates, 84 distinct spoligotype patterns that included 58 known Shared International Type (SIT) patterns and 26 new or orphan patterns were identified. The majority of strains belonged to two major M. tb lineages, L3 (35.7%) and L4 (61.6%). The observed high percentage of isolates with shared patterns (n = 200/258) suggested a substantial rate of overall clustering (77.5%). After adjusting for the effect of geographical variations, clustering rate was significantly lower among individuals co-infected with HIV and other concomitant chronic disease. Compared to L4, the adjusted odds ratio and 95% confidence interval (AOR; 95% CI) indicated that infections with L3 M. tb strains were more likely to be associated with TBLN [3.47 (1.45, 8.29)] and TB-HIV co-infection [2.84 (1.61, 5.55)]. CONCLUSION: Despite the observed difference in strain diversity and geographical distribution of M. tb lineages, compared to earlier studies in Ethiopia, the overall rate of strain clustering suggests higher transmission and warrant more detailed investigations into the molecular epidemiology of TB and related factors.

Identification of drug susceptibility pattern and mycobacterial species in sputum smear positive pulmonary tuberculosis patients with and without HIV co-infection in north west Ethiopia.

BACKGROUND: Ethiopia is among the high-burden countries of tuberculosis (TB) in the world Since mycobacterial culture and susceptibility testing are not routinely performed in Ethiopia, recent data on susceptibility patterns and the mycobacterial species cultured from sputum smear positive patients are limited. OBJECTIVES: The aim was to determine first line anti-TB drug susceptibility of Mycobacterium tuberculosis isolates obtained from consecutive newly diagnosed smear positive pulmonary TB patients in north west Ethiopia. METHODOLOGY: A retrospective cross sectional study was conducted using previously collected sputum samples (n=180) kept at the referral hospital of the University of Gondar at -20 degrees C. Sputum samples were cultured on Lowenstein Jensen (LJ) medium. Conventional Polymerase Chain Reaction (PCR) using RD4 primers to identify the M. tuberculosis complex was performed on cultured isolates. Ninety eight (84.4%) of the 116 isolates identified as M. tuberculosis were tested for their drug susceptibility pattern using the proportion method Clinical baseline data including body mass index, body temperature, clinical symptoms and erythrocyte sedimentation rate were obtained. RESULTS: The culture retrieval rate of previously frozen sputum samples was 64.4% (116/180). All the isolated mycobacterial species (n=116) were confirmed as belonging to the M. tuberculosis complex by PCR. Of 98 isolates for which the drug susceptibility test was done, 15.3% (15/98) were found to be resistant to one or more antimycobacterial drugs, and resistance to isoniazid and streptomycin was most common with 8.2% (8/98) and 6.1% (6/98) respectively. TB patients co infected with HIV had increased erythrocyte sedimentation rate, higher age and lower sputum smear grade than HIV negative TB patients. CONCLUSIONS: No mycobacteria other than M. tuberculosis were detected in sputum smear positive TB-patients. Although no multi drug resistant strain was observed, relatively high rates of INH resistance were found in this region. Culture facilities are urgently needed in regional centers to increase diagnostic sensitivity and monitor developing trends of drug resistance in Ethiopia.

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

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

An African origin for Mycobacterium bovis.

BACKGROUND AND OBJECTIVES: Mycobacterium bovis and Mycobacterium caprae are two of the most important agents of tuberculosis in livestock and the most important causes of zoonotic tuberculosis in humans. However, little is known about the global population structure, phylogeography and evolutionary history of these pathogens. METHODOLOGY: We compiled a global collection of 3364 whole-genome sequences from M.bovis and M.caprae originating from 35 countries and inferred their phylogenetic relationships, geographic origins and age. RESULTS: Our results resolved the phylogenetic relationship among the four previously defined clonal complexes of M.bovis, and another eight newly described here. Our phylogeographic analysis showed that M.bovis likely originated in East Africa. While some groups remained restricted to East and West Africa, others have subsequently dispersed to different parts of the world. CONCLUSIONS AND IMPLICATIONS: Our results allow a better understanding of the global population structure of M.bovis and its evolutionary history. This knowledge can be used to define better molecular markers for epidemiological investigations of M.bovis in settings where whole-genome sequencing cannot easily be implemented. LAY SUMMARY: During the last few years, analyses of large globally representative collections of whole-genome sequences (WGS) from the human-adapted Mycobacterium tuberculosis complex (MTBC) lineages have enhanced our understanding of the global population structure, phylogeography and evolutionary history of these pathogens. In contrast, little corresponding data exists for M. bovis, the most important agent of tuberculosis in livestock. Using whole-genome sequences of globally distributed M. bovis isolates, we inferred the genetic relationships among different M. bovis genotypes distributed around the world. The most likely origin of M. bovis is East Africa according to our inferences. While some M. bovis groups remained restricted to East and West Africa, others have subsequently dispersed to different parts of the world driven by cattle movements.

Line-probe assay and molecular typing reveal a potential drug resistant clone of Mycobacterium tuberculosis in Ethiopia.

BACKGROUND: Antimicrobial resistance is a global concern of increasing significance. Multidrug resistant tuberculosis (MDR-TB) is spreading worldwide. It is important to monitor trends of antimycobacterial resistance. This is particularly true for high TB burden countries such as Ethiopia where disproportionally less drug sensitivity data are reported from. METHODS: The prevalence of drug resistance was assessed with the line probe assay GenoType MTBDRplus in a set of 161 M. tuberculosis strains that were selected from four common lineages and sub-lineages previously identified in Ethiopia. Most of the tested M. tuberculosis isolates had been genotyped by established Spoligotyping and MIRU-VNTR typing methods. RESULTS: The proportion of MDR-TB among the isolates was 3.1%. Mono-resistance was 1.2% to rifampicin and 4.3% to isoniazid, and resistance to either of the two first line drugs was 8.7%. Strains of Lineage 4 had the highest resistance rate (13.6%) followed by Lineage 3 (4.9%). None of the isolates representing Lineages 1 and Lineage 7 were drug resistant. Multidrug resistance among pulmonary TB and TB lymphadenitis clinical isolates was 2.8 and 3.7%, respectively. Drug resistance of strains carrying the most prevalent spoligotype in Ethiopia - SIT149 - was further explored. Stratification by MIRU-VNTR identified one genotype with a high rate of drug resistance against Rifampicin and Isoniazid and circulation of a potential MDR-TB clone is proposed. CONCLUSION: Although the strain selection was not fully randomized, the overall M. tuberculosis drug resistance rate in this strain set was 8.7% while the rate of MDR was 3.1%. In parallel, we identified a sub-lineage that showed a high rate of resistance to both rifampicin and isoniazid. These resistant strains may belong to a clone of M. tuberculosis that is circulating in the highlands of Ethiopia.

Molecular identification of Mycobacterium tuberculosis causing Pulmonary Tuberculosis in Sudan.

BACKGROUND: Tuberculosis (TB) is a major public health problem in Sudan. However, the strains responsible for the epidemic of TB have been poorly characterized. The aim of this study was to characterize the causative agent of TB isolates circulating in Kassala and Gezira States during 2011-2012. METHOD: Ninety two Mycobacterium tuberculosis complex (MTBC) strains were isolated from sputum samples of pulmonary TB patients, attending the Teaching Hospitals in Kassala and Gezira states. Molecular identification was performed using region of difference 9 (RD9) deletion and spoligotyping methods. Spoligotype patterns were compared with those listed in the international SITVIT2 spoligotyping database. RESULTS: The results determined the circulation of Euro-American, Central Asian, and Indo-Oceanic Lineages. They were clustered in the following clades: Manu2 (64.8%), CAS (22.7%), EAI (4.5), LAM2 (2.3%), H3 (1.1%), T (1.1%), T3-ETH (1.1%), T5-RUS1 (1.1%). Comparison with the international multi-marker database SITVIT2, 76.1% of isolates matched the database, while 23.9% of the isolates were not represented in SITVIT2 (orphans). SIT54 (Manu2) was the most common strain circulating in the two states. CONCLUSION: The study showed that a large variety of Mycobacterium tuberculosis (MTB) genotypes were circulating in the two states. Further studies on identification and molecular characterization of mycobacterial are needed to understand the biodiversity, rate of transmission and the associated risk factors of MTB in Sudan.

Identification and Characterization of Mycobacterium Tuberculosis Isolates from Cattle Owners in North Western and North Eastern Parts of Rural Ethiopia.

Tuberculosis (TB) is a major global public health problem resulting in a considerable morbidity and mortality worldwide. Ethiopia ranks 8th among the 22 high TB burden countries. Establishing an appropriate and improved intervention strategy to prevent and control tuberculosis requires the presence of evidence based data on the genetic diversity of its causative agent. The current research work, therefore, was to differentiate strains of Mycobacterium tuberculosis isolated from pulmonary tuberculosis patients who own cattle in North Eastern and North Western parts of Ethiopia using molecular techniques. Sputum samples were collected from Acid Fast Bacilli (AFB) positive pulmonary tuberculosis patients and cultured on Lowenstein-Jensen (LJ) medium containing glycerol and sodium pyruvate. Deoxyribonucleic acid (DNA) was extracted from each positive culture, spoligotyping and single nucleotide polymorphisms were performed to further differentiate strains of M. tuberculosis, after deletion typing PCR confirmed that all the isolates were Mycobacterium tuberculosis. The mean age of study participants was 35.7 years (18-63 years) + 13.24. The majority (55.7%) were from North Gondar zone. Spoligotyping revealed that (47/50) 94% had interpretable patterns and 3 lineages namely; East-Africa-Indian (57.4%), Euro-American-African (EAA lineage-Lineage 4) 38.3% and Ethiopian (lineage-7) 2/50 (4.3%). Lineage 7 was registered in North Wollo zone only. In this study 8 clusters (with cluster size ranging from 2-8), 8 unique and 10 new patters were recorded. Spoligotype International Types (SIT) (21, 25, 26, 35, 53, 109, 149 and 289), were found as clusters and of these SIT 25 (7) and SIT 289 (8) were the predominant ones. Our study proved that 3 Mycobacterium tuberculosis lineages, namely; the ancient, intermediate between the modern lineages as well as modern were identified. Besides, considerable clustering was seen, which indicates the presence of current TB transmission in the study areas.