Molecular epidemiology and drug sensitivity of Mycobacterium tuberculosis in homeless individuals in the Addis Ababa city, Ethiopia.

Although homeless segment of the society could be the hotspots for tuberculosis (TB) transmission, there is little data on TB in homeless individuals in Ethiopia. The objective of this study was to investigate the molecular epidemiology and drug sensitivity of Mycobacterium tuberculosis (M. tuberculosis) isolated from homeless individuals in Addis Ababa, Ethiopia. The study was conducted on 59 M. tuberculosis isolates, which were recovered by the clinical screening of 5600 homeless individuals and bacteriological examination of 641 individuals with symptoms of pulmonary tuberculosis (PTB). Region of difference-9 (RD9) based polymerase-chain reaction (PCR), Spoligotyping and 24-loci Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing were used for genotyping of the isolates. In addition, drug sensitivity test was performed on the isolates using BD Bactec Mycobacterial Growth Inhibition Tube (MGIT) 960. Fifty-eight of the 59 isolates were positive by spoligotyping and spoligotyping International type (SIT) 53, SIT 37, and SIT 149 were the dominant spoligotypes; each consisting of 19%, 15.5%, and10.3% of the isolates, respectively. The majority of the isolates (89.7%) were members of the Euro-American (EA) major lineage. MIRU-VNTR identified Ethiopia_3, Delhi/CAS, Ethiopia_2, TUR, X-type, Ethiopia_H37Rv-like strain, Haarlem and Latin-American Mediterranean (LAM) sub lineages. The proportion of clustering was 77.6% (45/58) in spoligotyping while it was 39.7% (23/58) in 24-loci MIRU-VNTR typing. Furthermore, the proportion of clustering was significantly lowered to 10.3% (6/58) when a combination of spoligotyping and 24-loci MIRU-VNTRplus was used. The recent transmission index (RTI) recorded by spoligotyping, 24-loci MIRU-VNTR typing, and a combination of the two genotyping methods were 58.6%, 27.6% and 5.2%, respectively. Young age and living in groups were significantly associated with strain clustering (P < 0.05). The drug sensitivity test (DST) result showed 8.9% (4/58) of the isolates were resistant to one or more first line ant-TB drugs; but multidrug resistant isolate was not detected. Clustering and RTI could suggest the transmission of TB in the homeless individuals, which could suggest a similar pattern of transmission between homeless individuals and the general population. Hence, the TB control program should consider homeless individuals during the implementation of TB control program.

Prevalence of paratuberculosis in cattle based on gross and microscopic lesions in Ethiopia.

BACKGROUND: Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic progressive granulomatous enteritis mainly affecting domestic and wild ruminants worldwide. Although paratuberculosis could be prevail in Ethiopia, there is a scarcity of epidemiological data on paratuberculosis in the country. Thus, this study was conducted to estimate the prevalence of paratuberculosis based on gross and microscopic lesions in cattle slaughtered at ELFORA Abattoir, central Ethiopia. Small intestines and associated lymph nodes of 400 apparently healthy cattle which were slaughtered at ELFORA export abattoir were examined for gross and microscopic lesions of paratuberculosis. The microscopic lesions were classified into four grades (I-IV) based on the type and number of cells infiltrated into the lesion. The prevalence of paratuberculosis was estimated on the basis of gross as well as microscopic lesion of paratuberculosis. RESULTS: The prevalence of paratuberculosis was 11.25% (95% Confidence interval, CI = 0.083-0.148) on the basis of gross lesion. However, relatively lower prevalence (2.0%, 95% CI = 0.01, 0.039) was recorded based on microscopic lesion. The gross lesions were characterized by intestinal thickening, mucosal corrugations and enlargement of associated mesenteric lymph nodes. On the other hand, the microscopic lesions were characterized by granuloma of different grades ranging from grade I to grade III lesions. CONCLUSIONS: The present study indicated the occurrence of paratuberculosis in cattle of Ethiopia based on the detection of gross and microscopic lesions consistent with the lesion of paratuberculosis. The result of this study could be used as baseline information for future studies on the epidemiology and economic significance of paratuberculosis.

Equine Histoplasmosis in Ethiopia: Phylogenetic Analysis by Sequencing of the Internal Transcribed Spacer Region of rRNA Genes.

Equine histoplasmosis commonly known as epizootic lymphangitis (EL) is a neglected granulomatous disease of equine that is endemic to Ethiopia. It is caused by Histoplasma capsulatum variety farciminosum, a dimorphic fungus that is closely related to H. capsulatum variety capsulatum. The objective of this study was to undertake a phylogenetic analysis of H. capsulatum isolated from EL cases of horses in central Ethiopia and evaluate their relationship with H. capsulatum isolates in other countries and/or clades using the internal transcribed spacer (ITS) region of rRNA genes. Clinical and mycological examinations, DNA extraction, polymerase chain reaction (PCR), Sanger sequencing, and phylogenetic analysis were used for undertaking this study. Additionally, sequence data of Histoplasma isolates were retrieved from GenBank and included for a comprehensive phylogenetic analysis. A total of 390 horses were screened for EL and 97 were positive clinically while H. capsulatum was isolated from 60 horses and further confirmed with PCR, of which 54 were sequenced. BLAST analysis of these 54 isolates identified 29 H. capsulatum isolates and 14 isolates from other fungal genera while the remaining 11 samples were deemed insufficient for further downstream analysis. The phylogenetic analysis identified five clades, namely, African, Eurasian, North American 1 and 2, and Latin American A and B. The Ethiopian isolates were closely aggregated with isolates of the Latin American A and Eurasian clades, whereas being distantly related to isolates from North American 1 and 2 clades as well as Latin American B clade. This study highlights the possible origins and transmission routes of Histoplasmosis in Ethiopia.

Spoligotyping of Clinical Isolates of Mycobacterium tuberculosis Complex Species in the Oromia Region of Ethiopia.

Background: Tuberculosis (TB) is a leading cause of morbidity and mortality in Ethiopia. Investigation of the Mycobacterium tuberculosis complex (MTBC) species circulating in the Ethiopian population would contribute to the efforts made to control TB in the country. Therefore, this study was conducted to investigate the MTBC species and spoligo patterns in the Oromia region (central) of Ethiopia. Methods: A cross-sectional study design was used to recruit 450 smear positive pulmonary TB (PTB) cases from the Oromia region between September 2017 and August 2018. Mycobacteria were isolated from sputum samples on the Lowenstein Jensen (LJ) medium. Molecular identification of the isolates was performed by spoligotyping. The results of spoligotyping were transferred into a query box in the SITVIT2 database and Run TB-Lineage in the TB Insight website for the identification of spoligo international type (SIT) number and linages of the isolates, respectively. Statistical Product and Service Solutions (SPSS) 20 was applied for statistical analysis. Results: Three hundred and fifteen isolates were grouped under 181 different spoligotype patterns. The most dominantly isolated spoligotype pattern was SIT149 and it consisted of 23 isolates. The majority of the isolates were grouped under Euro-American (EA), East-African-Indian (EAI), and Indo-Oceanic (IO) lineages. These lineages consisted of 79.4, 9.8, and 9.8% of the isolates, respectively. One hundred and sixty-five of the isolates were classified under 31 clustered spoligotypes whereas the remaining 150 were singleton types. Furthermore, 91.1% of the total isolates were classified as orphan types. Clustering of spoligotypes was associated (p < 0.001) with EAI lineage. Conclusion: SIT149 and EA lineage were predominantly isolated from the Oromia region substantiating the findings of the similar studies conducted in other regions of Ethiopia. The observation of significant number of singleton and orphan spoligotypes warrants for additional genetic typing of the isolates using method(s) with a better discriminatory power than spoligotyping.

Seroprevalence and Associated Risk Factors of Ovine Brucellosis in South Omo Zone, Southern Ethiopia.

BACKGROUND: Brucellosis is a zoonotic disease with economic and public health significance in developing countries that rely on livestock production including Ethiopia. This study intended to establish the seroprevalence and associated risk factors of ovine brucellosis. METHODS: A cross-sectional study was carried out on seroepidemiology of ovine brucellosis from January 2017 to June 2020 G.C in five districts of South Omo zone, Southern Ethiopia. A total of 1536 sera samples were collected from sheep and serially tested using modified Rose Bengal plate test, competitive enzyme-linked immunosorbent assay, and complement fixation test to detect antibodies against natural infection by Brucella species. A structured questionnaire was used to collect data from individual animals, and flocks for the analysis of the association between expounding and outcome variables. Data were analyzed using STATA version 14.0 and potential risk factors for seropositivity of brucellosis were analyzed using logistic regression. RESULTS: The study discovered an overall 5.40% (95% CI: 6.34, 14.25) and 39.74% (95% CI: 6.50, 6.97) seroprevalence of ovine brucellosis at individual and flock level, respectively, by a confirmatory test. Age groups, sex, flock size, district, history of abortion, and body condition were significantly associated risk factors with Brucella seropositivity (p < 0.05). CONCLUSION: To conclude, the prevalence of ovine brucellosis in the South Omo Zone was relatively high which needs integrated intervention approaches in place to curb the spread of the disease.

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.

Retrospective study on bovine clinical mastitis and associated milk loss during the month of its peak occurrence at the National Dairy Farm in the Emirate of Abu Dhabi, United Arab Emirates.

Background: Commercial dairy establishments are relatively young in the United Arab Emirates (UAE), and as a result, there is lack of epidemiological data on mastitis in dairy farms. Methods: A retrospective data of seven years (2015-2021) were used to estimate the cumulative average monthly incidence rate of bovine clinical mastitis and evaluate associated milk loss at the National Dairy Farm. Data were extracted from the records of lactating dairy cows (n = 1300-1450) and analyzed using repeated measure and one-way ANOVA, non-parametric Spearman correlation, paired and unpaired t tests. Results: The highest average cumulative monthly incidence rate was 49 cases per 1000 cows-year that was recorded in 2019 while the lowest was 19 cases per 1000 cows-year in 2021. The cumulative average monthly incidence rate of clinical mastitis significantly (p < 0.001) varied among the seven years. The cumulative average monthly incidence rate was associated with average monthly humidity (p < 0.01) and average monthly rainfall (p < 0.05); however, it was not associated with the average monthly temperature (p > 0.05). The average daily milk yield of cows with clinical mastitis (Mean ± SEM; 18.6 ± 0.54 kg) was significantly (p < 0.001) lower than the average daily milk yield of clinical mastitis free cows (40.5 ± 0.29 kg). The largest average monthly milk loss due to clinical mastitis was 5% of the average total monthly milk production in 2019 while the lowest was 2% of the average total monthly milk production in 2021. Conclusion: The result of the study indicated the direct influence of weather conditions such as increased rainfall and humidity, which caused an upsurge in the incidence rate of clinical mastitis, leading to an increased loss in milk and hence the economy of the dairy farm. Proactive preventive measures along with good dairy farm practices that help mitigate the impacts of harsh weather conditions are recommended.

Field evaluation of specific mycobacterial protein-based skin test for the differentiation of Mycobacterium bovis-infected and Bacillus Calmette Guerin-vaccinated crossbred cattle in Ethiopia.

Bovine tuberculosis (bTB) challenges intensive dairy production in Ethiopia and implementation of the test and slaughter control strategy is not economically acceptable in the country. Vaccination of cattle with Bacillus Calmette-Guerin (BCG) could be an important adjunct to control, which would require a diagnostic test to differentiate Mycobacterium bovis (M. bovis)-infected and BCG-vaccinated animals (DIVA role). This study describes an evaluation of a DIVA skin test (DST) that is based on a cocktail (DSTc) or fusion (DSTf) of specific (ESAT-6, CFP-10 and Rv3615c) M. bovis proteins in Zebu-Holstein-Friesians crossbred cattle in Ethiopia. The study animals used were 74 calves (35 BCG vaccinated and 39 unvaccinated) aged less than 3 weeks at the start of experiment and 68 naturally infected 'TB reactor' cows. Six weeks after vaccination, the 74 calves were tested with the DSTc and the single intradermal cervical comparative tuberculin (SICCT) test. The TB reactor cows were tested with the DSTc and the SICCT test. Reactions to the DSTc were not observed in BCG-vaccinated and unvaccinated calves, while SICCT test reactions were detected in vaccinated calves. DSTc reactions were detected in 95.6% of the TB reactor cows and single intradermal tuberculin positive reactions were found in 98.2% (95% confidence interval, CI, 92.1-100%). The sensitivity of the DSTc was 95.6% (95% CI, 87.6-99.1%), and significantly (p < .001) higher than the sensitivity (75%, 95% CI, 63.0-84.7%) of the SICCT test at 4 mm cut-off. DSTf and DSTc reactions were correlated (r = 0.75; 95% CI = 0.53-0.88). In conclusion, the DSTc could differentiate M. bovis-infected from BCG-vaccinated cattle in Ethiopia. DST had higher sensitivity than the SICCT test. Hence, the DSTc could be used as a diagnostic tool for bTB if BCG vaccination is implemented for the control of bTB in Ethiopia and other countries.

Pathology of Bovine Tuberculosis in Three Breeds of Dairy Cattle and Spoligotyping of the Causative Mycobacteria in Ethiopia.

Different breeds of cattle were observed to have a variable degree of susceptibility to bovine tuberculosis (bTB). The screening of bTB was conducted on 720 dairy cattle consisting of three breeds using the single intradermal cervical comparative tuberculin (SICCT) test. Besides this, 43 SICCT test-positive cattle were used to compare the severity of the pathology of bTB among the three breeds and to identify the causative mycobacteria using spoligotyping. The overall SICCT test positivity was 17.92% (129/720) by pooling all animals in the three farms. There was a significant difference in SICCT test positivity among the three breeds (χ2 = 71.06; p < 0.001); the highest (25.34%) was recorded in the crossbreed followed by the Boran breed (10.08%), while the least (3.14%) was recorded in the Jersey breed. On other hand, the highest median pathology score (10.0, interquartile range, IQR = 6.0-17.0) was recorded in Boran followed by cross (5.0, IQR = 3.5-7.5), while the least (3.0, IQR = 2.25-3.0) was recorded in Jersey. Thus, the difference in the median pathology scores was significant [Kruskal Wallis χ ( 2 ) 2 = 18.78, p < 0.001] among the three breeds. Furthermore, multivariate analysis using ordinal logistic regression by considering age, sex, breed, reproductive status, and location of the farms also showed a significant [ χ ( 2 ) 2 = 11.97, p < 0.01] difference in pathology scores among the three breeds of cattle. Even at a single-herd level at Holeta, the difference in severity of pathology between the Boran and crossbreeds was significant (U = 33.5; p < 0.01). Culture positivity was 39% in 108 suspicious tissues. Fourteen Mycobacterium bovis (M. bovis) and two Mycobacterium tuberculosis (M. tuberculosis) were isolated from the lesions. All the 14 M. bovis isolates belonged to SB0912, while the two M. tuberculosis belonged to SIT54. In conclusion, although the frequency of the SICCT test positivity was high in the crossbreed, a more severe pathology was observed on the Boran (zebu) breed. In addition M. tuberculosis was isolated from TB lesions of dairy cattle, demonstrating the role of M. tuberculosis in causing TB in cattle.

Evaluation of the Efficacy of BCG in Protecting Against Contact Challenge With Bovine Tuberculosis in Holstein-Friesian and Zebu Crossbred Calves in Ethiopia.

Bovine tuberculosis (bTB) is prevalent in intensive dairy farms in Ethiopia. Vaccination could be an alternative control approach given the socio-economic challenges of a test-and-slaughter control strategy. The efficacy of the BCG was evaluated on 40 Holstein-Friesian (HF) and zebu crossbred calves recruited from single intradermal cervical comparative tuberculin (SICCT) test negative herds and randomly allocated into two groups. Twenty-two calves were vaccinated within 2 weeks of age, and 18 were kept as a control. Six weeks post-vaccination, the two groups were exposed and kept mixed with known SICCT test positive cows for 1 year. Immune responses were monitored by interferon gamma (IFN-γ) release assay (IGRA), SICCT test, and antibody assay. Vaccinated calves developed strong responses to the SICCT test at the sixth week post-vaccination, but did not respond to ESAT-6/CFP-10 peptide antigen-based IGRA. During the exposure, IFN-γ response to the specific peptide cocktail [F (2.44, 92.67) = 26.96; p < 0.001] and skin reaction to the specific proteins cocktail [F (1.7, 64.3); p < 0.001] increased progressively in both groups while their antibody responses were low. The prevalence of bTB was 88.9% (95% CI: 65.3-98.6) and 63.6% (95% CI: 40.7-83.8) in the control and vaccinated calves, respectively, based on Mycobacterium bovis isolation, giving a direct protective efficacy estimate of 28.4% (95% CI: -2.7 to 50.1). The proportion of vaccinated calves with lesion was 7.0% (34/484) against 11.4% (45/396) in control calves, representing a 38% (95% CI: 5.8-59.4) reduction of lesion prevalence. Besides, the severity of pathology was significantly lower (Mann-Whitney U-test, p < 0.05) in vaccinated (median score = 2.0, IQR = 0-4.75) than in control (median score = 5, IQR = 3.0-6.25) calves. Moreover, survival from M. bovis infection in vaccinated calves was significantly (log-rank test: χ2 = 6.749, p < 0.01) higher than that of the control calves. In conclusion, the efficacy of BCG was low, but the reduced frequency and severity of lesion in vaccinated calves could suggest its potential role in containing onward transmission.

Protein and Microbial Biomarkers in Sputum Discern Acute and Latent Tuberculosis in Investigation of Pastoral Ethiopian Cohort.

Differential diagnosis of tuberculosis (TB) and latent TB infection (LTBI) remains a public health priority in high TB burden countries. Pulmonary TB is diagnosed by sputum smear microscopy, chest X-rays, and PCR tests for distinct Mycobacterium tuberculosis (Mtb) genes. Clinical tests to diagnose LTBI rely on immune cell stimulation in blood plasma with TB-specific antigens followed by measurements of interferon-γ concentrations. The latter is an important cytokine for cellular immune responses against Mtb in infected lung tissues. Sputum smear microscopy and chest X-rays are not sufficiently sensitive while both PCR and interferon-γ release assays are expensive. Alternative biomarkers for the development of diagnostic tests to discern TB disease states are desirable. This study's objective was to discover sputum diagnostic biomarker candidates from the analysis of samples from 161 human subjects including TB patients, individuals with LTBI, negative community controls (NCC) from the province South Omo, a pastoral region in Ethiopia. We analyzed 16S rRNA gene-based bacterial taxonomies and proteomic profiles. The sputum microbiota did not reveal statistically significant differences in α-diversity comparing the cohorts. The genus Mycobacterium, representing Mtb, was only identified for the TB group which also featured reduced abundance of the genus Rothia in comparison with the LTBI and NCC groups. Rothia is a respiratory tract commensal and may be sensitive to the inflammatory milieu generated by infection with Mtb. Proteomic data supported innate immune responses against the pathogen in subjects with pulmonary TB. Ferritin, an iron storage protein released by damaged host cells, was markedly increased in abundance in TB sputum compared to the LTBI and NCC groups, along with the α-1-acid glycoproteins ORM1 and ORM2. These proteins are acute phase reactants and inhibit excessive neutrophil activation. Proteomic data highlight the effector roles of neutrophils in the anti-Mtb response which was not observed for LTBI cases. Less abundant in the sputum of the LTBI group, compared to the NCC group, were two immunomodulatory proteins, mitochondrial TSPO and the extracellular ribonuclease T2. If validated, these proteins are of interest as new biomarkers for diagnosis of LTBI.

Detection of Mycobacterium tuberculosis complex DNA in CD34-positive peripheral blood mononuclear cells of asymptomatic tuberculosis contacts: an observational study.

BACKGROUND: Haematopoietic stem cells expressing the CD34 surface marker have been posited as a niche for Mycobacterium tuberculosis complex bacilli during latent tuberculosis infection. Our aim was to determine whether M tuberculosis complex DNA is detectable in CD34-positive peripheral blood mononuclear cells (PBMCs) isolated from asymptomatic adults living in a setting with a high tuberculosis burden. METHODS: We did a cross-sectional study in Ethiopia between Nov 22, 2017, and Jan 10, 2019. Digital PCR (dPCR) was used to determine whether M tuberculosis complex DNA was detectable in PBMCs isolated from 100 mL blood taken from asymptomatic adults with HIV infection or a history of recent household or occupational exposure to an index case of human or bovine tuberculosis. Participants were recruited from HIV clinics, tuberculosis clinics, and cattle farms in and around Addis Ababa. A nested prospective study was done in a subset of HIV-infected individuals to evaluate whether administration of isoniazid preventive therapy was effective in clearing M tuberculosis complex DNA from PBMCs. Follow-up was done between July 20, 2018, and Feb 13, 2019. QuantiFERON-TB Gold assays were also done on all baseline and follow-up samples. FINDINGS: Valid dPCR data (ie, droplet counts >10 000 per well) were available for paired CD34-positive and CD34-negative PBMC fractions from 197 (70%) of 284 participants who contributed data to cross-sectional analyses. M tuberculosis complex DNA was detected in PBMCs of 156 of 197 participants with valid dPCR data (79%, 95% CI 74-85). It was more commonly present in CD34-positive than in CD34-negative fractions (154 [73%] of 197 vs 46 [23%] of 197; p<0·0001). Prevalence of dPCR-detected M tuberculosis complex DNA did not differ between QuantiFERON-negative and QuantiFERON-positive participants (77 [78%] of 99 vs 79 [81%] of 98; p=0·73), but it was higher in HIV-infected than in HIV-uninfected participants (67 [89%] of 75 vs 89 [73%] of 122, p=0·0065). By contrast, the proportion of QuantiFERON-positive participants was lower in HIV-infected than in HIV-uninfected participants (25 [33%] of 75 vs 73 [60%] of 122; p<0·0001). Administration of isoniazid preventive therapy reduced the prevalence of dPCR-detected M tuberculosis complex DNA from 41 (95%) of 43 HIV-infected individuals at baseline to 23 (53%) of 43 after treatment (p<0·0001), but it did not affect the prevalence of QuantiFERON positivity (17 [40%] of 43 at baseline vs 13 [30%] of 43 after treatment; p=0·13). INTERPRETATION: We report a novel molecular microbiological biomarker of latent tuberculosis infection with properties that are distinct from those of a commercial interferon-γ release assay. Our findings implicate the bone marrow as a niche for M tuberculosis in latently infected individuals. Detection of M tuberculosis complex DNA in PBMCs has potential applications in the diagnosis of latent tuberculosis infection, in monitoring response to preventive therapy, and as an outcome measure in clinical trials of interventions to prevent or treat latent tuberculosis infection. FUNDING: UK Medical Research Council.

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.

Differences in plasma proteomes for active tuberculosis, latent tuberculosis and non-tuberculosis mycobacterial lung disease patients with and without ESAT-6/CFP10 stimulation.

BACKGROUND: Tuberculosis (TB) is one of the world's most problematic infectious diseases. The pathogen Mycobacterium tuberculosis (Mtb) is contained by the immune system in people with latent TB infection (LTBI). No overt disease symptoms occur. The environmental and internal triggers leading to reactivation of TB are not well understood. Non-tuberculosis Mycobacteria (NTM) can also cause TB-like lung disease. Comparative analysis of blood plasma proteomes from subjects afflicted by these pathologies in an endemic setting may yield new differentiating biomarkers and insights into inflammatory and immunological responses to Mtb and NTM. METHODS: Blood samples from 40 human subjects in a pastoral region of Ethiopia were treated with the ESAT-6/CFP-10 antigen cocktail to stimulate anti-Mtb and anti-NTM immune responses. In addition to those of active TB, LTBI, and NTM cohorts, samples from matched healthy control (HC) subjects were available. Following the generation of sample pools, proteomes were analyzed via LC-MS/MS. These experiments were also performed without antigen stimulation steps. Statistically significant differences using the Z-score method were determined and interpreted in the context of the proteins' functions and their contributions to biological pathways. RESULTS: More than 200 proteins were identified from unstimulated and stimulated plasma samples (UPSs and SPSs, respectively). Thirty-four and 64 proteins were differentially abundant with statistical significance (P < 0.05; Benjamini-Hochberg correction with an FDR < 0.05) comparing UPS and SPS proteomic data of four groups, respectively. Bioinformatics analysis of such proteins via the Gene Ontology Resource was indicative of changes in cellular and metabolic processes, responses to stimuli, and biological regulations. The m7GpppN-mRNA hydrolase was increased in abundance in the LTBI group compared to HC subjects. Charged multivesicular body protein 4a and platelet factor-4 were increased in abundance in NTM as compared to HC and decreased in abundance in NTM as compared to active TB. C-reactive protein, α-1-acid glycoprotein 1, sialic acid-binding Ig-like lectin 16, and vitamin K-dependent protein S were also increased (P < 0.05; fold changes≥2) in SPSs and UPSs comparing active TB with LTBI and NTM cases. These three proteins, connected in a STRING functional network, contribute to the acute phase response and influence blood coagulation. CONCLUSION: Plasma proteomes are different comparing LTBI, TB, NTM and HC cohorts. The changes are augmented following prior blood immune cell stimulation with the ESAT-6/CFP-10 antigen cocktail. The results encourage larger-cohort studies to identify specific biomarkers to diagnose NTM infection, LTBI, and to predict the risk of TB reactivation.

Establishment of COVID-19 testing laboratory in resource-limited settings: challenges and prospects reported from Ethiopia.

The Coronavirus pandemic is recording unprecedented deaths worldwide. The temporal distribution and burden of the disease varies from setting to setting based on economic status, demography and geographic location. A rapid increase in the number of COVID-19 cases is being reported in Africa as of June 2020. Ethiopia reported the first COVID-19 case on 13 March 2020. Limited molecular laboratory capacity in resource constrained settings is a challenge in the diagnosis of the ever-increasing cases and the overall management of the disease. In this article, the Ethiopian Public Health Institute (EPHI) shares the experience, challenges and prospects in the rapid establishment of one of its COVID-19 testing laboratories from available resources. The first steps in establishing the COVID-19 molecular testing laboratory were i) identifying a suitable space ii) renovating it and iii) mobilizing materials including consumables, mainly from the Malaria and Neglected Tropical Diseases (NTDs) research team at the EPHI. A chain of experimental design was set up with distinct laboratories to standardize the extraction of samples, preparation of the master mix and detection. At the commencement of sample reception and testing, laboratory contamination was among the primary challenges faced. The source of the contamination was identified in the master mix room and resolved. In summary, the established COVID-19 testing lab has tested more than 40,000 samples (August 2020) and is the preferred setting for research and training. The lessons learned may benefit the further establishment of emergency testing laboratories for COVID-19 and/or other epidemic/pandemic diseases in resource-limited settings.

Molecular epidemiology of clinical Mycobacterium tuberculosis complex isolates in South Omo, Southern Ethiopia.

BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis complex (MTBC). Mapping the genetic diversity of MTBC in high TB burden country like Ethiopia is important to understand principles of the disease transmission and to strengthen the regional TB control program. The aim of this study was to investigate the genetic diversity of Mycobacterium tuberculosis complex (MTBC) isolates circulating in the South Omo, southern Ethiopia. METHODS: MTBC isolates (N = 156) were genetically analyzed using spacer oligotyping (spoligotyping) and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing. Major lineages and lineages were identified using MTBC databases. Logistic regression was used to correlate patient characteristics with strain clustering. RESULTS: The study identified Euro-American (EA), East-African-Indian (EAI), Indo-Oceanic (IO), Lineage_7/Aethiops vertus, Mycobacterium bovis and Mycobacterium africanum major lineages in proportions of 67.3% (105/156), 22.4% (35/156), 6.4% (10/156), 1.9% (3/156), 1.3% (2/156) and 0.6% (1/156), respectively. Lineages identified were Delhi/CAS 23.9% (37/155), Ethiopia_2 20.6% (32/155), Haarlem 14.2% (22/155), URAL 14.2%(22/155), Ethiopia_3 8.4% (13/155), TUR 6.5% (10/155), Lineage_7/Aethiops vertus 1.9% (3/155), Bovis 1.3% (2/155), LAM 1.3% (2/155), EAI 0.6% (1/155), X 0.6% (1/155) and Ethiopia H37Rv-like strain 0.6% (1/155). Of the genotyped isolates 5.8% (9/155) remained unassigned. The recent transmission index (RTI) was 3.9%. Orphan strains compared to shared types (AOR: 0.09, 95% CI: 0.04-0.25) were associated with reduced odds of clustering. The dominant TB lineage in pastoral areas was EAI and in non-pastoral areas was EA. CONCLUSION: The epidemiological data, highly diverse MTBC strains and a low RTI in South Omo, provide information contributing to the TB Control Program of the country.

Cellular and Cytokine Responses in the Granulomas of Asymptomatic Cattle Naturally Infected with Mycobacterium bovis in Ethiopia.

Cell (CD3+ T cell and CD68+ macrophages), cytokine (interferon gamma-positive [IFN-γ+] and tumor necrosis factor alpha-positive [TNF-α+]), and effector molecule (inducible nitric oxide synthase-positive [iNOS+]) responses were evaluated in the lymph nodes and tissues of cattle naturally infected with Mycobacterium bovis Detailed postmortem and immunohistochemical examinations of lesions were performed on 16 cows that were positive by the single intradermal cervical comparative tuberculin (SICCT) test and that were identified from dairy farms located around the city of Addis Ababa, Ethiopia. The severity of the gross lesion was significantly higher (P = 0.003) in M. bovis culture-positive cows (n = 12) than in culture-negative cows (n = 4). Immunohistochemical techniques showed that in culture-positive cows, the mean immunolabeling fraction of CD3+ T cells decreased as the stage of granuloma increased from stage I to stage IV (P < 0.001). In contrast, the CD68+ macrophage, IFN-γ+, TNF-α+, and iNOS+ immunolabeling fractions increased from stage I to stage IV (P < 0.001). In the early stages, culture-negative cows showed a significantly higher fraction of CD68+ macrophage (P = 0.03) and iNOS+ (P = 0.007) immunolabeling fractions than culture-positive cows. Similarly, at advanced granuloma stages, culture-negative cows demonstrated significantly higher mean proportions of CD3+ T cells (P < 0.001) than culture-positive cows. Thus, this study demonstrates that, following natural infection of cows with M. bovis, as the stage of granuloma increases from stage I to stage IV, the immunolabeling fraction of CD3+ cells decreases, while the CD68+ macrophage, IFN-γ+, TNF-α+, and iNOS+ immunolabeling fractions increases.

Prevalence of latent tuberculosis infection and associated risk factors in prison in East Wollega Zone of western Ethiopia.

BACKGROUND: Latent tuberculosis infection (LTBI) is the major source of active TB and is an obstacle to the strategy of World Health Organization to end TB by 2035. In Ethiopia, there are hundreds of prisons and they are conducive settings for the transmission of TB and could serve as the sources of infection to the general public. However, there is little data on the epidemiology of TB in prisons in Ethiopia. The objective of the present study was to estimate the prevalence of LTBI and evaluate associated risk factors in prisons in East Wollega Zone in western Ethiopia. METHODS: A cross-sectional design and systematic sampling technique were used to select 352 prisoners from a total of 2620 prisoners during the two months (May and June, 2019). The selected inmates were consented for their willingness to participate in the study. Thereafter, they were interviewed and 2ml of blood sample was collected from each prisoner and screened for LTBI using interferon-gamma release assay (IGRA). The data were analyzed using SPSS version 25 and logistic regression was used to model the likelihood of LTBI occurrence and to identify risk factors associated with LTBI. RESULTS: The prevalence of LTBI was 51.2% (95% CI: 46.45-57%) and higher prevalence was recorded in males (53%) than in females (43.5%) although the difference was not significant. Prisoners whose age ≥45 years (AOR = 2.48, 95%CI, 1.04-5.9), who chewed khat (AOR = 2.27; 95% CI, 1.27-4.19), who were prisoned over a year (AOR = 1.81, 95%CI, 1.04-3.18) and who were in overcrowded pens (AOR = 1.91, 95% CI, 1.002-3.65) were at higher risk of LTBI. CONCLUSIONS: The prevalence of LTBI in prisoners in West Wollega Zone of western Ethiopia was high and could serve as sources of infection to the public. Hence optimum handling of prisoners, and regular follow up and treatment of TB cases in prisons were recommended to minimize the burden of TB in the Zone.

Tuberculosis at Farmer-Cattle Interface in the Rural Villages of South Gondar Zone of Northwest Ethiopia.

BACKGROUND: Tuberculosis (TB) has been an important public health concern in Ethiopia, particularly at areas of human-animal intersection. However, limited epidemiological information is available in this respect in the country. Therefore, the present study was conducted to investigate the transmission of TB at human-cattle interface, associated risk factors and public awareness about the disease at South Gondar Zone, northwest Ethiopia. METHODS: A cross sectional study was conducted between March 2015 and April 2018 on 186 farmers and 476 cattle in South Gondar Zone, northwest Ethiopia. Bacteriological examination, region of difference (RD) 9-based polymerase chain reaction (PCR), single intradermal comparative tuberculin test (SICTT), and questionnaire were used for undertaking this study. RESULTS: Culture positivity in farmers was 59.7% (111/186) and all the culture positive isolates were M. tuberculosis. About 68% (74/111) of culture positive respondents did not know about the transmission of TB from cattle to human or vice versa. The animal and herd prevalence of bovine TB were 1.5% (7/476) and 7.4% (7/95), respectively. Although the result was not statistically significant, the odds of bovine TB in cattle owned by TB positive households was slightly higher than those owned by TB free households (adjusted odds ratio, AOR = 1.39; 95% CI: 0.31-7.10; p = 0.76). CONCLUSION: Although SIDCTT reactivity was slightly higher in cattle owned by TB positive households, all the human isolates were M. tuberculosis and no M. bovis was isolated from farmers, which could be due to the low prevalence of bovine TB in the area.

Evaluation of Mycobacterium tuberculosis lipoarabinomannan antigen assay and rapid serology blood test for the diagnosis of bovine tuberculosis in Ethiopia.

BACKGROUND: Bovine tuberculosis (bTB) is prevalent in dairy cattle in Ethiopia. Currently used diagnostic tools such as the single intradermal comparative tuberculin test (SICTT) are time consuming and labor intensive. A rapid, easy-to-use and cost-effective diagnostic test would greatly contribute to the control of bTB in developing countries like Ethiopia. In the present study, two point-of-care diagnostic tests were evaluated for the detection of bTB: LIONEX® Animal TB Rapid test, a membrane-based test for the detection of antibodies to Mycobacterium bovis in blood and ALERE® Determine TB Lipoarabinomannan (LAM) Ag, an immunoassay for the detection of lipoarabinomannan (LAM) antigen (Ag) of mycobacteria in urine. A combination of the SICTT and gamma interferon (IFN-γ) test was used as the gold standard for the validation of these point-of-care tests, as it was not feasible to slaughter the study animals to carry out the historical gold standard of mycobacterial culture. A total of 175 heads of cattle having three different bTB infection categories (positive SICTT, negative SICTT, and unknown SICTT status) were used for this study. RESULT: The sensitivity and specificity of TB LAM Ag were 72.2% (95% CI = 62.2, 80.4) and 98.8% (95% CI = 93.6, 99.7), respectively, while the sensitivity and specificity of the LIONEX Animal TB rapid test assay were 54% (95% CI = 44.1 64.3) and 98.8% (95% CI = 93.6, 99.7) respectively. The agreement between TB LAM Ag and SICTT was higher (κ = 0.85; 95% CI = 0.65-0.94) than between TB LAM Ag and IFN-γ (κ = 0.67; 95% CI = 0.52-0.81). The agreement between LIONEX Animals TB Rapid blood test and SICTT was substantial, (κ = 0.63; 95% CI = 0.49-0.77) while the agreement between LIONEX Animal TB rapid blood test and IFN-γ test was moderate (κ = 0.53; 95% CI = 0.40-0.67). Analysis of receiver operating curve (ROC) indicated that the area under the ROC curve (AUC) for TB LAM Ag was 0.85 (95% CI = 0.79-0.91) while it was 0.76 (95% CI; =0.69-0.83) for LIONEX Animal TB rapid test assay. CONCLUSION: This study showed that TB LAM Ag had a better diagnostic performance and could potentially be used as ancillary either to SICTT or IFN-γ test for diagnosis of bTB.