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.

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.

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.

Population Genomics of Mycobacterium tuberculosis in Ethiopia Contradicts the Virgin Soil Hypothesis for Human Tuberculosis in Sub-Saharan Africa.

Colonial medical reports claimed that tuberculosis (TB) was largely unknown in Africa prior to European contact, providing a "virgin soil" for spread of TB in highly susceptible populations previously unexposed to the disease [1, 2]. This is in direct contrast to recent phylogenetic models which support an African origin for TB [3-6]. To address this apparent contradiction, we performed a broad genomic sampling of Mycobacterium tuberculosis in Ethiopia. All members of the M. tuberculosis complex (MTBC) arose from clonal expansion of a single common ancestor [7] with a proposed origin in East Africa [3, 4, 8]. Consistent with this proposal, MTBC lineage 7 is almost exclusively found in that region [9-11]. Although a detailed medical history of Ethiopia supports the view that TB was rare until the 20(th) century [12], over the last century Ethiopia has become a high-burden TB country [13]. Our results provide further support for an African origin for TB, with some genotypes already present on the continent well before European contact. Phylogenetic analyses reveal a pattern of serial introductions of multiple genotypes into Ethiopia in association with human migration and trade. In place of a "virgin soil" fostering the spread of TB in a previously naive population, we propose that increased TB mortality in Africa was driven by the introduction of European strains of M. tuberculosis alongside expansion of selected indigenous strains having biological characteristics that carry a fitness benefit in the urbanized settings of post-colonial Africa.

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.

Admixture mapping of tuberculosis and pigmentation-related traits in an African-European hybrid cattle population.

Admixture mapping affords a powerful approach to genetic mapping of complex traits and may be particularly suited to investigation in cattle where many breeds and populations are hybrids of the two divergent ancestral genomes, derived from Bos taurus and Bos indicus. Here we design a minimal genome wide SNP panel for tracking ancestry in recent hybrids of Holstein-Friesian and local Arsi zebu in a field sample from a region of high bovine tuberculosis (BTB) endemicity in the central Ethiopian highlands. We first demonstrate the utility of this approach by mapping the red coat color phenotype, uncovering a highly significant peak over the MC1R gene and a second peak with no previously known candidate gene. Secondly, we exploit the described differential susceptibility to BTB between the ancestral strains to identify a region in which Bos taurus ancestry associates, at suggestive significance, with skin test positivity. Interestingly, this association peak contains the toll-like receptor gene cluster on chromosome 6. With this work we have shown the potential of admixture mapping in hybrid domestic animals with divergent ancestral genomes, a recurring condition in domesticated species.

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.

Genotyping of Pulmonary Mycobacterium tuberculosis Isolates from Sudan Using Spoligotyping.

Tuberculosis (TB) remains a major public health problem worldwide due to its high risk of person-to-person transmission, morbidity and mortality [1]. Sudan has a high burden of tuberculosis. Spoligotyping (spacer oligonucleotide typing) a rapid method for genotyping of Mycobacterium tuberculosis using the principle of reverse hybridization. The ecology of the prevalent mycobacteria strain can vary depending on country and region. The aim of this study was to determine the genotyping of Mycobacterium tuberculosis isolated from Sudan using spoligotyping SPOLDB4. A total of 75 Mycobacterium tuberculosis sputum samples were collected from pulmonary Tuberculosis patients attending references Laboratories and diagnostic centers in Khartoum and Eastern Sudan in (2011-2013). The mycobacteria were genotyped using Spoligotyping technique and data obtained were analyzed and compared to the SPOLDB4 database. Among the 75 isolate analyzed, 57(76%) were identified by SPOLDB4 and 18 (24%) could not be matched to any lineages. The most prevalent genotype cluster was MANU2 38 (50.7%) followed by CASI Delhi 8 (10.7%). In the study SIT54 was the most common pattern 37 (49.3%) followed by SIT25 6(8%).

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.

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.

Novel genomic signals of recent selection in an Ethiopian population.

The recent feasibility of genome-wide studies of adaptation in human populations has provided novel insights into biological pathways that have been affected by adaptive pressures. However, only a few African populations have been investigated using these genome-wide approaches. Here, we performed a genome-wide analysis for evidence of recent positive selection in a sample of 120 individuals of Wolaita ethnicity belonging to Omotic-speaking people who have inhabited the mid- and high-land areas of southern Ethiopia for millennia. Using the 11 HapMap populations as the comparison group, we found Wolaita-specific signals of recent positive selection in several human leukocyte antigen (HLA) loci. Notably, the selected loci overlapped with HLA regions that we previously reported to be associated with podoconiosis-a geochemical lymphedema of the lower legs common in the Wolaita area. We found selection signals in PPARA, a gene involved in energy metabolism during prolonged food deficiency. This finding is consistent with the dietary use of enset, a crop with high-carbohydrate and low-fat and -protein contents domesticated in Ethiopia subsequent to food deprivation 10 000 years ago, and with metabolic adaptation to high-altitude hypoxia. We observed novel selection signals in CDKAL1 and NEGR1, well-known diabetes and obesity susceptibility genes. Finally, the SLC24A5 gene locus known to be associated with skin pigmentation was in the top selection signals in the Wolaita, and the alleles of single-nucleotide polymorphisms rs1426654 and rs1834640 (SLC24A5) associated with light skin pigmentation in Eurasian populations were of high frequency (47.9%) in this Omotic-speaking indigenous Ethiopian population.

A 24-well plate assay for simultaneous testing of first and second line drugs against Mycobacterium tuberculosis in a high endemic setting.

BACKGROUND: Early detection of drug resistance is one of the priorities of tuberculosis (TB) control programs as drug resistance is increasing. New molecular assays are only accessible for a minority of the second line drugs and their availability in high endemic settings is also hampered by high cost and logistic challenges. Therefore, we evaluated a previously developed method for drug susceptibility testing (DST) including both first- and second line anti-TB drugs for use in high endemic areas. RESULTS: Baseline mycobacterial isolates from 78 consecutive pulmonary TB patients from Addis Ababa, Ethiopia who were culture positive for Mycobacterium tuberculosis at the end of a two-month directly observed treatment short course (DOTS) were included. The isolates were simultaneously tested for isoniazid, rifampicin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide and para-aminosalicylic acid susceptibility using the indirect proportion method adopted for 24-well agar plates containing Middlebrook 7H10 medium. Applying the 24-well plate assay, 43 (55.1%) isolates were resistant to one or more of the first line drugs tested (isoniazid, rifampicin and ethambutol). MDR-TB was identified in 20.5% of this selected group and there was a perfect correlation for rifampicin resistance with the results from the genotype MTBDRplus assay. All isolates were susceptible to aminoglycosides and fluoroquinolones in agreement with the genotype MTBDRsl assay. The only tested second line drug associated to resistance was ethionamide (14.1% resistant). The method was reproducible with stable results for internal controls (one multi-drug resistant (MDR) and one pan-susceptible strain (H37Rv) and DST results could be reported at two weeks. CONCLUSIONS: The 24-well plate method for simultaneous DST for first- and second line drugs was found to be reproducible and correlated well to molecular drug susceptibility tests. It is likely to be useful in high-endemic areas for surveillance as well as for the detection of second line drug resistance in targeted groups such as in those who fail empirical MDR treatment.

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.

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.