Spoligotype analysis of Mycobacterium bovis isolates from cattle and assessment of zoonotic TB transmission among individuals working in bovine TB-infected dairy farms in Ethiopia.

Bovine tuberculosis (bTB) is a disease with impact on dairy productivity, as well as having the potential for zoonotic transmission. Understanding the genetic diversity of the disease agent Mycobacterium bovis is important for identifying its routes of transmission. Here we investigated the level of genetic diversity of M. bovis isolates and assessed the zoonotic potential in risk groups of people working in bTB-infected dairy farms in central Ethiopia. M. bovis was isolated and spoligotyped from tissue lesions collected from slaughtered cattle as well as from raw milk collected from bTB positive cows in dairy farms from six urban areas of central Ethiopia. From consented dairy farm workers, knowledge and practices related to zoonotic TB transmission, together with demographic and clinical information, was collected through interviews. Sputum or Fine Needle Aspirate (FNA) samples were collected from suspected TB cases. Spoligotyping of 55 M. bovis isolates that originated either from cattle tissues with tuberculous lesion or from raw milk revealed seven spoligotype patterns where SB1176 was the most prevalent type (47.3%). Most isolates (89.1%) were of the M. bovis African 2 clonal complex. All sputum and FNA samples from 41 dairy farm workers with symptoms of TB were culture negative for any mycobacteria. Among the 41 TB suspected farm workers, 61% did not know about bTB in cattle and its zoonotic potential, and over two-third of these workers practiced raw milk consumption. Our spoligotype analysis suggests a wider transmission of a single spoligotype in the study area. The results reported here may be useful in guiding future work to identify the source and direction of bTB transmission and hence design of a control strategy. Isolation of M. bovis from milk, knowledge gap on zoonotic TB and practice of consumption of raw milk in the study population showed potential risk for zoonotic transmission.

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.

Global prevalence of Mycobacterium bovis infections among human tuberculosis cases: Systematic review and meta-analysis.

Tuberculosis (TB) is a chronic communicable bacterial disease caused by Mycobacterium tuberculosis complex (MTBC) species. M. tuberculosis is the main causative agent of human TB, and cattle are the primary host of Mycobacterium bovis; due to close interaction between cattle and humans, M. bovis poses a zoonotic risk. This review summarizes and estimates the prevalence of M. bovis infection among human cases. Studies reporting TB prevalence data that were published in English during 10 years from 20 April 2009 to 17 April 2019 were identified through search of PubMed and other sources. Quality of studies and risk of bias were assessed using standard tools for prevalence study reports. Characteristics of included studies and their main findings were summarized in tables and discussed with narrative syntheses. Meta-analysis was performed on 19 included studies, with a total of 7,185 MTBC isolates identified; 702 (9.7%) of them were characterized as of subspecies M. bovis, but there was a large prevalence difference between the studies, ranging from 0.4% to 76.7%. The genotyping-based studies reported significantly lower prevalence of zoonotic TB than did the studies based on older techniques. The overall pooled prevalence of M. bovis aggregated from all included studies was 12.1% of the total MTBC isolates, while the corresponding pooled figure from the 14 genotyping-based studies was only 1.4%. Generally, human M. bovis cases reported from different countries of the world suggest that the impact of zoonotic TB is still important in all regions. However, it was difficult to understand the true picture of the disease prevalence because of methodological differences. Future investigations on zoonotic TB should carefully consider these differences when evaluating prevalence results.

Population structure and transmission of Mycobacterium bovis in Ethiopia.

Bovine tuberculosis (bTB) is endemic in cattle in Ethiopia, a country that hosts the largest national cattle herd in Africa. The intensive dairy sector, most of which is peri-urban, has the highest prevalence of disease. Previous studies in Ethiopia have demonstrated that the main cause is Mycobacterium bovis, which has been investigated using conventional molecular tools including deletion typing, spoligotyping and Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). Here we use whole-genome sequencing to examine the population structure of M. bovis in Ethiopia. A total of 134 M. bovis isolates were sequenced including 128 genomes from 85 mainly dairy cattle and six genomes isolated from humans, originating from 12 study sites across Ethiopia. These genomes provided a good representation of the previously described population structure of M. bovis, based on spoligotyping and demonstrated that the population is dominated by the clonal complexes African 2 (Af2) and European 3 (Eu3). A range of within-host diversity was observed amongst the isolates and evidence was found for both short- and long-distance transmission. Detailed analysis of available genomes from the Eu3 clonal complex combined with previously published genomes revealed two distinct introductions of this clonal complex into Ethiopia between 1950 and 1987, likely from Europe. This work is important to help better understand bTB transmission in cattle in Ethiopia and can potentially inform national strategies for bTB control in Ethiopia and beyond.

Factors associated with localization of tuberculosis disease among patients in a high burden country: A health facility-based comparative study in Ethiopia.

INTRODUCTION: In contrast to most tuberculosis (TB) high burden countries, Ethiopia has for a long time reported a very high percentage of extra pulmonary TB (EPTB), which is also reflected in population based estimations reported by the World Health Organization (WHO). Particularly a steadily higher proportion of cervical tuberculous lymphadenitis (TBLN) has been described. Here we identify clinical and demographic factors associated with anatomic site of the TB disease. METHOD: A health facility based comparative study was conducted among TBLN and PTB patients who visited selected health facilities in Ethiopia during 2016 and 2017. Associated risk factors were identified through a multivariate logistic regression model using R-studio. RESULT: A total of 1,890 study participants, 427 TBLN and 1,463 PTB patients, were included. The mean age of TBLN patients (29 years ± 14.4 SD) was lower than that of PTB cases (36 years ± 15.0 SD). There were slightly more women diagnosed with TBLN (51.1%) while nearly 6 out of 10 male patients were diagnosed with PTB (58.9%). Most significantly, younger age groups (<15 Years) were more likely to develop cervical TBLN than older people (>56 years), with an AOR of 9.76 (95% CI: 4.87, 19.56). The odds of cervical TBLN among women [1.69 (1.30, 2.20)] was higher than that for men. In addition, adjusted estimates suggested that, compared with PTB, renal diseases [3.41 (1.29, 9.02)] and the presence of other concomitant chronic illness [1.61 (1.23, 2.09)] had a significant association with TBLN. CONCLUSION: Generally, the risk of developing a particular form of TB disease is usually associated with demographic and medical history of an infected individual. Hence, the current symptom based screening, which primarily rely on chronic cough in many countries, may lead to missing significant portions of TBLN cases.

Drug Resistance Conferring Mutation and Genetic Diversity of Mycobacterium tuberculosis Isolates in Tuberculosis Lymphadenitis Patients; Ethiopia.

BACKGROUND: Tuberculosis lymphadenitis (TBLN) is a growing public health concern in Ethiopia. However, there is limited information available on gene mutations conferring drug resistance and genetic diversity of M. tuberculosis isolates from TBLN patients. METHODS: Drug resistance and genetic diversity analysis were done on 91 M. tuberculosis isolates from culture positive TBLN patients collected between 2016 and 2017. Detection of mutations conferring resistance was carried out using GenoType MTBDRplus VER 2.0. Thereafter, isolates were typed using spoligotyping. RESULTS: Out of the 91 strains, mutations conferring resistance to rifampicin (RIF) and isoniazid (INH) were observed in two (2.2%) and six (6.6%) isolates, respectively. The two RIF resistant isolates displayed a mutation at codon 531 in the rpoB gene with amino acid change of S531L. Among the six INH resistant strains, four isolates had shown mutation at the KatG gene at codon 315 with amino acid change of S315T, one isolate had a mutation at the inhA gene at codon 15 with amino acid change of C15T and one isolate had a mutation at the inhA gene with unknown amino acid change. All drug resistant isolates were from treatment naive TBLN patients. The dominantly identified Spoligo International Types (SITs) were SIT25, SIT149, and SIT53, respectively; these accounted for 43% of the total number of strains. The isolates were grouped into four main lineages; Lineage 1 (2, 2.2%), Lineage 3 (38, 41.7%), Lineage 4 (49, 53.8%) and Lineage 7 (2, 2.2%). Four out of six (66.7%) isolates with drug resistance conferring mutations belonged to clustered strains (strains with shared SIT). CONCLUSION: The detection of drug resistant conferring mutation in treatment naïve TBLN patients together with detection of drug resistant isolates among clustered strains might suggest resistant strains' transmission in the community. This needs to be carefully considered to prevent the spread of drug resistant clones in the country.

A Randomized, Placebo-Controlled Trial Evaluating Safety and Immunogenicity of the Killed, Bivalent, Whole-Cell Oral Cholera Vaccine in Ethiopia.

Killed whole-cell oral cholera vaccine (OCV) has been a key component of a comprehensive package including water and sanitation measures for recent cholera epidemics. The vaccine, given in a two-dose regimen, has been evaluated in a large number of human volunteers in India, Vietnam, and Bangladesh, where it has demonstrated safety, immunogenicity, and clinical efficacy. We conducted a double-blind randomized placebo-controlled trial in Ethiopia, where we evaluated the safety and immunogenicity of the vaccine in 216 healthy adults and children. OCV was found to be safe and elicited a robust immunological response against Vibrio cholerae O1, with 81% adults and 77% children demonstrating seroconversion 14 days after the second dose of vaccine. This is the first study to evaluate safety and immunogenicity of the vaccine in a population outside Asia using a placebo-controlled, double-blind, randomized study design.