Population structure and spatial distribution of Mycobacterium tuberculosis in Ethiopia.

Ethiopia is one of the countries with a high tuberculosis (TB) burden, yet little is known about the spatial distribution of Mycobacterium tuberculosis (Mtb) lineages. This study identifies the spoligotyping of 1735 archived Mtb isolates from the National Drug Resistance Survey, collected between November 2011 and June 2013, to investigate Mtb population structure and spatial distribution. Spoligotype International Types (SITs) and lineages were retrieved from online databases. The distribution of lineages was evaluated using Fisher's exact test and logistic regression models. The Global Moran's Index and Getis-Ord Gi statistic were utilized to identify hotspot areas. Our results showed that spoligotypes could be interpreted and led to 4 lineages and 283 spoligotype patterns in 91% of the isolates, including 4% of those with multidrug/rifampicin resistance (MDR/RR) TB. The identified Mtb lineages were lineage 1 (1.8%), lineage 3 (25.9%), lineage 4 (70.6%) and lineage 7 (1.6%). The proportion of lineages 3 and 4 varied by regions, with lineage 3 being significantly greater than lineage 4 in reports from Gambella (AOR = 4.37, P < 0.001) and Tigray (AOR = 3.44, P = 0.001) and lineage 4 being significantly higher in Southern Nations Nationalities and Peoples Region (AOR = 1.97, P = 0.026) than lineage 3. Hotspots for lineage 1 were located in eastern Ethiopia, while a lineage 7 hotspot was identified in northern and western Ethiopia. The five prevalent spoligotypes, which were SIT149, SIT53, SIT25, SIT37 and SIT26 account for 42.8% of all isolates under investigation, while SIT149, SIT53 and SIT21 account for 52-57.8% of drug-resistant TB cases. TB and drug resistant TB are mainly caused by lineages 3 and 4, and significant proportions of the prevalent spoligotypes also influence drug-resistant TB and the total TB burden. Regional variations in lineages may result from both local and cross-border spread.

BCG vaccination reduces bovine tuberculosis transmission, improving prospects for elimination.

Bacillus Calmette-Guérin (BCG) is a routinely used vaccine for protecting children against Mycobacterium tuberculosis that comprises attenuated Mycobacterium bovis. BCG can also be used to protect livestock against M. bovis; however, its effectiveness has not been quantified for this use. We performed a natural transmission experiment to directly estimate the rate of transmission to and from vaccinated and unvaccinated calves over a 1-year exposure period. The results show a higher indirect efficacy of BCG to reduce transmission from vaccinated animals that subsequently become infected [74%; 95% credible interval (CrI): 46 to 98%] compared with direct protection against infection (58%; 95% CrI: 34 to 73%) and an estimated total efficacy of 89% (95% CrI: 74 to 96%). A mechanistic transmission model of bovine tuberculosis (bTB) spread within the Ethiopian dairy sector was developed and showed how the prospects for elimination may be enabled by routine BCG vaccination of cattle.

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.

Genomic transmission clusters and circulating lineages of Mycobacterium tuberculosis among refugees residing in refugee camps in Ethiopia.

BACKGROUND: Understanding the transmission dynamics of Mycobacterium tuberculosis (Mtb) could benefit the design of tuberculosis (TB) prevention and control strategies for refugee populations. Whole Genome Sequencing (WGS) has not yet been used to document the Mtb transmission dynamics among refugees in Ethiopia. We applied WGS to accurately identify transmission clusters and Mtb lineages among TB cases in refugee camps in Ethiopia. METHOD AND DESIGN: We conducted a cross-sectional study of 610 refugees in refugee camps in Ethiopia presenting with symptoms of TB. WGS data of 67 isolates was analyzed using the Maximum Accessible Genome for Mtb Analysis (MAGMA) pipeline; iTol and FigTree were used to visualize phylogenetic trees, lineages, and the presence of transmission clusters. RESULTS: Mtb culture-positive refugees originated from South Sudan (52/67, 77.6%), Somalia (9/67, 13.4%). Eritrea (4/67, 6%), and Sudan (2/67, 3%). The majority (52, 77.6%) of the isolates belonged to Mtb lineage (L) 3, and one L9 was identified from a Somalian refugee. The vast majority (82%) of the isolates were pan-susceptible Mtb, and none were multi-drug-resistant (MDR)-TB. Based on the 5-single nucleotide polymorphisms cutoff, we identified eight potential transmission clusters containing 23.9% of the isolates. Contact investigation confirmed epidemiological links with either family or social interaction within the refugee camps or with neighboring refugee camps. CONCLUSION: Four lineages (L1, L3, L4, and L9) were identified, with the majority of strains being L3, reflecting the Mtb L3 dominance in South Sudan, where the majority of refugees originated from. Recent transmission among refugees was relatively low (24%), likely due to the short study period. The improved understanding of the Mtb transmission dynamics using WGS in refugee camps could assist in designing effective TB control programs for refugees.

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.

Prevalence of tuberculosis and associated factors among presumptive TB refugees residing in refugee camps in Ethiopia.

BACKGROUND: Tuberculosis (TB) causes significant morbidity and mortality in refugee populations. Although Ethiopia is the third largest refugee-hosting country in Africa, there is limited published data on the prevalence and associated factors of TB in refugees. The objective of this study was to estimate the prevalence of bacteriologically confirmed pulmonary TB (PTB) and explore associated factors in presumptive TB refugees residing in refugee camps in Ethiopia. METHODS: A facility-based cross-sectional study was conducted between February and August 2021 in refugee camps in Ethiopia. Data were collected consecutively from 610 presumptive TB refugees who attended for TB diagnosis in selected refugee camp clinics in Ethiopia. A pre-tested questionnaire was used to collect data, and sputum samples were collected from eligible study participants. The Xpert Mycobacterium tuberculosis (MTB)/Rifampicin (RIF) assay was performed on direct spot sputum samples, whereas morning sputum samples were processed and inoculated for bacteriological culture using Mycobacterium Growth Indicator Tube (MGIT) and Lowsteen Jensen (LJ) methods. The statistical software package (STATA version 14) was used for statistical analysis. A logistic regression model was used for the evaluation of the association between bacteriologically confirmed TB cases and the associated factors. Descriptive statistics were used for the expression of the results, and statistical significance was assumed at p < 0.05. RESULTS: Out of 610 study participants, more than half were female (54.9%), and the mean age was 37.9 years (SD, 16.64). The prevalence of bacteriologically confirmed PTB cases among refugees residing in refugee camps in Ethiopia was 13.3% (95% CI, 10.7-16.2%) using the Xpert MTB/RIF assay and/or culture. MTB was detected in 12.8% (95% CI, 10.2-15.7%) of the individuals using the Xpert MTB/RIF assay, while culture positivity was observed in 11.6% (95% CI, 9.2-14.5%). The multivariable logistic regression model showed South Sudan origins (adjusted odds ratio, AOR = 7.74; 95% CI, 3.05-19.64), age group, 19-38 years old (AOR = 5.66; 95% CI, 1.86-17.28), and male sex (AOR = 2.69; 95% CI, 1.58-4.56) were significantly associated with the bacteriologically confirmed TB among refugees residing in refugee camps in Ethiopia. CONCLUSION: The prevalence of bacteriologically confirmed PTB among presumptive TB refugees residing in refugee camps in Ethiopia was high. The national TB program should strengthen TB prevention and control activities in the refugee camps of Ethiopia. Moreover, an active TB survey program should be implemented in refugee camps in Ethiopia.

Molecular Characteristics of Bovine Viral Diarrhea Virus Strains Isolated from Persistently Infected Cattle.

In this study, we reported the isolation, identification, and molecular characteristics of nine BVDV strains that were isolated from the serum of persistently infected cattle. The new strains were designated as BVDV TJ2101, TJ2102, TJ2103, TJ2104, TJ2105, TJ2106, TJ2107, TJ2108 and TJ2109. The TJ2102 and TJ2104 strains were found to be cytopathic BVDV, and the other strains were non-cytopathic BVDV. An alignment and phylogenetic analysis showed that the new isolates share 92.2-96.3% homology with the CP7 strain and, thus, were classified as the BVDV-1b subgenotype. A recombination analysis of the genome sequences showed that the new strains could be recombined by the major parent BVDV-1a NADL strain and the minor parent BVDV-1m SD-15 strain. Some genome variations or unique amino acid mutations were found in 5'-UTR, E0 and E2 of these new isolates. In addition, a potential linear B cell epitopes prediction showed that the potential linear B cell epitope at positions 56-61 is highly variable in BVDV-1b. In conclusion, the present study has identified nine strains of BVDV from persistently infected cattle in China. Further studies on the virulence and pathogenesis of these new strains are recommended.

Genetic diversity and drug sensitivity profile of Mycobacterium tuberculosis among children in Ethiopia.

BACKGROUND: Worldwide, tuberculosis (TB) affects about one million children every year. The burden of the disease is higher in developing countries. However, there is limited information on the lineages and drug sensitivity patterns of Mycobacterium tuberculosis (M. tuberculosis) infecting children in these countries, including Ethiopia. Thus, this study aimed to characterize the different lineages of the M. tuberculosis complex causing childhood pulmonary tuberculosis and evaluate the drug-sensitivity patterns to the first-line anti-TB drugs. METHOD: A total of 54 stored cultures were used in this study. The region of difference 9 (RD9) based polymerase chain reaction (PCR) and spoligotyping were employed for the identification of the isolates at the species and lineages level respectively. Lineage identification was done by using the pre-existing database. Identification of clustering of the spoligotype patterns was by using the SPOLIDB3-based model. The result was retrieved by the most probable family format. Furthermore, the phenotypic, and genotypic drug-sensitivity test (DST) was performed using Mycobacterium Growth Indicator Tube (MGIT™ 960) and GenoTypeMTBDRplus assay respectively. Data analysis was done using SPSS version 27 software. RESULT: Spoligotyping produced 39 interpretable results for M. tuberculosis. The majority (74.4%) of them were clustered into 7 groups, while the rest (25.6%) were single. The Euro-American (EA) lineage was the predominant lineage (64.1%) followed by the East-African Indian (EAI) (30.8%) and M. Africanum (5.1%) lineages. The most predominant subtypes were SIT37 (15.4%), SIT149 (12.8%), SIT25 (7.7%), and SIT53 (7.7%). Furthermore, of the identified SITs, T1 and CAS families consisted of 38.5% and 28.2% of the lineages respectively. Drug susceptibility was 91.9% by phenotypic method and 97.4% by molecular assay. The overall prevalence of any resistance was 7.8% and there was a single MDR-TB. CONCLUSION: Many of the isolates belong to the modern lineages (Euro American) representing the most common circulating strains in the country. More importantly, despites the tiny isolates tested, drug resistance is low. To fully describe the molecular epidemiology of MTBC lineages in children, we recommend a prospective large-scale study.

Whole genome sequencing of Ethiopian Brucella abortus isolates expands the known diversity of an early branching sub-Saharan African lineage.

Brucellosis remains one of the most significant zoonotic diseases globally, responsible for both considerable human morbidity and economic losses due to its impacts on livestock productivity. Despite this, there remain significant evidence gaps in many low- and middle-income countries, including those of sub-Saharan Africa. Here we report the first molecular characterisation of Brucella sp. from Ethiopia. Fifteen Brucella sp. isolates from an outbreak in cattle from a herd in central Ethiopia were identified as Brucella abortus, using bacterial culture and molecular methods. Sequencing of the Ethiopian B. abortus isolates allowed their phylogenetic comparison with 411 B. abortus strains of diverse geographical origins, using whole genome single nucleotide polymorphisms (wgSNP). The Ethiopian isolates belonged to an early-branching lineage (Lineage A) previously only represented by data from two strains, both of sub-Saharan African origin (Kenya and Mozambique). A second B. abortus lineage (Lineage B), also comprised solely of strains originating from sub-Saharan Africa, was identified. The majority of strains belonged to one of two lineages of strains originating from a much broader geographical range. Further analyses based on multi-locus sequence typing (MLST) and multi-locus variable-number tandem repeat analysis (MLVA) expanded the number of B. abortus strains available for comparison with the Ethiopian isolates and were consistent with the findings from wgSNP analysis. MLST profiles of the Ethiopian isolates expanded the sequence type (ST) diversity of the early branching lineage of B. abortus, equivalent to wgSNP Lineage A. A more diverse cluster of STs, equivalent to wgSNP Lineage B, was comprised solely of strains originating from sub-Saharan Africa. Similarly, analysis of B. abortus MLVA profiles (n = 1891) confirmed that the Ethiopian isolates formed a unique cluster, similar to only two existing strains, and distinct from the majority of other strains of sub-Saharan African origin. These findings expand the known diversity of an under-represented lineage of B. abortus and suggest a potential evolutionary origin for the species in East Africa. In addition to providing information concerning Brucella species extant within Ethiopia this work serves as the basis for further studies on the global population structure and evolutionary history of a major zoonotic pathogen.

Prevalence of pulmonary tuberculosis in homeless individuals in the Addis Ababa City, Ethiopia.

Background: Homeless individuals are at a high risk of infection with Mycobacterium tuberculosis (M. tuberculosis) as compared to the general population. The number of homeless individuals has been increasing in Addis Ababa City during the last three decades due to the migration of rural inhabitants to the City for better living conditions. The objective of this study was to estimate the prevalence of pulmonary tuberculosis (PTB) and evaluate associated risk factors in homeless individuals in Addis Ababa City. Methods: A total of 5,600 homeless individuals were screened for PTB symptoms using WHO guideline between February 2019 and December 2020. Sputum samples were cultured from individuals with symptoms of PTB for mycobacterial isolation. Logistic regression analysis was used to identify factors associated with PTB. Results: The prevalence of bacteriologically confirmed cases was 1.1% (59/5,600) or 10.54 per 1000 population. Multinomial logistic regression analysis showed that being homeless for more than 5 years, body mass index (BMI) < 18.5, smoking cigarette, living in a group of more than five individuals, close contact with chronic coughers, imprisonment and HIV infection were significantly associated with the prevalence of PTB in homeless individuals (P < 0.05). Conclusion: In conclusion, the result of this study indicated that the prevalence of PTB in homeless individuals was higher than the prevalence of PTB in the general population of Addis Ababa City requiring for the inclusion of the homeless individuals in the TB control program.

Molecular typing and drug sensitivity profiles of M. Tuberculosis isolated from refugees residing in Ethiopia.

Background: Refugees in developing countries have poor access to Tuberculosis (TB) care and control services. The understanding of genetic diversity and drug sensitivity patterns of M. tuberculosis (MTB) is important for the TB control program. However, there is no evidence that shows the drug sensitivity profiles and genetic diversity of MTB circulating among refugees residing in Ethiopia. This study aimed to investigate the genetic diversity of MTB strains and lineages, and to identify the drug sensitivity profiles of MTB isolated from refugees residing in Ethiopia. Methods: A cross-sectional study was conducted among 68 MTB positive cases isolated from presumptive TB refugees from February to August 2021. Data and samples were collected in the refugee camp clinics and both rapid TB Ag detection and region of difference (RD)-9 deletion typing were used to confirm the MTBs. Drug susceptibility test (DST) and molecular typing were done using Mycobacterium Growth Indicator Tube (MGIT) method and spoligotyping respectively. Results: DST and spoligotyping results were available for all 68 isolates. The isolates were grouped into 25 spoligotype patterns, which consisted of 1-31 isolates with 36.8% strain diversity. The international shared type (SIT)25 was predominant spoligotype pattern consisting of 31 (45.6%) isolates, followed by SIT24 comprising 5 (7.4%) isolates. Further investigation showed that 64.7% (44/68) of the isolates were belonged to CAS1-Delhi family and 75% (51/68) of the isolates were belonged to lineage(L)-3. Multi-drug resistance (MDR)-TB was observed only in one isolate (1.5%) for first-line anti-TB drugs and the highest level of mono-resistance, 5.9% (4/68), was observed for PZA(Pyrazinamide). Mono-resistance was observed in 2.9 % (2/68) and while 97.0% (66/68) of the MTB positive cases were susceptible to the second-line anti-TB drugs. Conclusion: The findings are useful evidence for the TB screening, treatment and control in refugee populations and surrounding communities in Ethiopia.

Molecular epidemiology of tuberculosis in the Somali region, eastern Ethiopia.

Background: Tuberculosis (TB) is one of the leading causes of morbidity and mortality in low-income countries like Ethiopia. However, because of the limited laboratory infrastructure there is a shortage of comprehensive data on the genotypes of clinical isolates of Mycobacterium tuberculosis (M. tuberculosis) complex (MTBC) in peripheral regions of Ethiopia. The objective of this study was to characterize MTBC isolates in the Somali region of eastern Ethiopia. Methods: A cross-sectional study was conducted in three health institutions between October 2018 and December 2019 in the capital of Somali region. A total of 323 MTBC isolates (249 from pulmonary TB and 74 from extrapulmonary TB) were analyzed using regions of difference 9 (RD 9)-based polymerase chain reaction (PCR) and spoligotyping. Results: Of the 323 MTBC isolates, 99.7% (95% CI: 99.1-100%) were M. tuberculosis while the remaining one isolate was M. bovis based on RD 9-based PCR. Spoligotyping identified 71 spoligotype patterns; 61 shared types and 10 orphans. A majority of the isolates were grouped in shared types while the remaining grouped in orphans. The M. tuberculosis lineages identified in this study were lineage 1, 2, 3, 4, and 7 with the percentages of 7.4, 2.2, 28.2, 60.4, and 0.6%, respectively. Most (87.9%) of the isolates were classified in clustered spoligotypes while the remaining 12.1% isolates were singletons. The predominant clustered spoligotypes identified were SIT 149, SIT 21, SIT 26, SIT 53, and SIT 52, each consisting of 17.6, 13.3, 8.4, 7.4, and 5%, respectively. Lineage 3 and lineage 4, as well as the age group (15-24), were associated significantly with clustering. Conclusion: The MTBC isolated from TB patients in Somali region were highly diverse, with considerable spoligotype clustering which suggests active TB transmission. In addition, the Beijing spoligotype was isolated in relatively higher frequency than the frequencies of its isolation from the other regions of Ethiopia warranting the attention of the TB Control Program of the Somali region.

Drug sensitivity of clinical isolates of Mycobacterium tuberculosis and its association with bacterial genotype in the Somali region, Eastern Ethiopia.

Background: Drug resistance is becoming a major bottleneck for tuberculosis (TB) control programs in countries with high TB burdens. Although several studies were conducted on the drug sensitivity of Mycobacterium tuberculosis (M. tuberculosis) in central Ethiopia, there is a lack of data on the drug sensitivity of M. tuberculosis in the peripheral regions of the country including in the Somali region. Therefore, the objective of this study was to evaluate the drug sensitivity of M. tuberculosis and its association with bacterial genotype and evaluate the performance of Xpert MTB/RIF (Xpert) in detecting resistance to rifampicin (RIF). Methods: A total of 302 M. tuberculosis were tested using the BD BACTEC-Mycobacteria Growth Indicator Tube 960 (MGIT 960) system for their drug sensitivity to the first-line anti-TB drugs. Besides, the drug sensitivity of 10 multidrug-resistant (MDR) M. tuberculosis isolates was evaluated for the second-line anti-TB drugs. Additionally, 177 of the 302 isolates were tested for genotypic drug resistance using Xpert. Chi-square and Fisher's exact tests were used for the evaluation of the association between variables and drug sensitivity. Results: The overall prevalence of resistance to at least one drug was 11.6% (95% CI: 7.9-15.2%), while the prevalence of MDR was 3.3% (95% CI: 1.3-5.3%). Two of the 10 MDR isolates were resistant to capreomycin. The spoligotype Shared International Type (SIT) 149 was significantly associated with either monoresistance or MDR (p < 0.05). Of the 177 isolates tested by Xpert, 6.2% (11/177) were RIF-resistant. Discordant between Xpert and MGIT 960 was observed in one isolate and linked with probe-binding delay (ΔCT max = 5.8). The sensitivity and specificity of the Xpert assay were 100 and 99.4%, respectively, while its positive and negative predictive values were 90.9 and 100%, respectively. Conclusion: The magnitude of MDR M. tuberculosis in the Somali region of Ethiopia was higher than the national prevalence of MDR-TB warranting the strengthening of the TB control program in the Somali region. Besides, drug resistance was associated with SIT 149 spoligotype (genotype). The Xpert assay was observed to have high sensitivity and specificity in detecting RIF-resistant M. tuberculosis, which is encouraging for its application widely.

Minimum inhibitory concentrations of rifampin and isoniazid among multidrug and isoniazid resistant Mycobacterium tuberculosis in Ethiopia.

INTRODUCTION: Traditionally, single critical concentrations of drugs are utilized for Mycobacterium tuberculosis (Mtb) drug susceptibility testing (DST); however, the level of drug resistance can impact treatment choices and outcomes. Mutations at the katG gene are the major genetic mutations in multidrug resistant (MDR) Mtb and usually associated with high level resistance. We assessed the minimum inhibitory concentrations (MICs) of MDR or rifampin resistant (RR) and isoniazid (INH) resistant Mtb isolates to determine the quantification of drug resistance among key anti-tuberculosis drugs. METHODS: The study was conducted on stored Mtb isolates collected as part of a national drug resistance survey in Ethiopia. MIC values were determined using Sensititre™ MYCOTB plates. A line probe assay (MTBDRplus) was also performed to identify genetic determinants of resistance for all isolates. RESULTS: MIC testing was performed on 74 Mtb isolates including 46 MDR, 2 RR and 26 INH phenotypically resistant isolates as determined by the Löwenstein Jensen (LJ) method. Four (15%) INH resistant Mtb isolates were detected as borderline rifampin resistance (MIC = 1 µg/ml) using MYCOTB MIC plates and no rifampin resistance mutations were detected by LPA. Among the 48 MDR/RR TB cases, 9 (19%) were rifabutin susceptible (MIC was between ≤0.25 and 0.5µg/ml). Additionally, the MIC for isoniazid was between 2-4 µg/ml (moderate resistance) for 58% of MDR TB isolates and 95.6% (n = 25) of the isolates had mutations at the katG gene. CONCLUSION: Our findings suggest a role for rifabutin treatment in a subset of RR TB patients, thus potentially preserving an important drug class. The high proportion of moderate level INH resistant among MDR Mtb isolates indicates the potential benefit of high dose isoniazid treatment in a high proportion of katG gene harboring MDR Mtb isolates.

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.

Genotypic and phenotypic drug-resistance detection and prevalence of heteroresistance in patients with isoniazid- and multidrug-resistant tuberculosis in Ethiopia.

Objective: To assess the agreement between genotypic and phenotypic methods for detecting drug resistance, and examine the prevalence of heteroresistance among isoniazid (INH)- and multidrug/rifampicin-resistant (MDR/RR) TB. Method: In total, 127 Mycobacterium tuberculosis (Mtb) isolates, including 65 MDR/RR and 62 INH resistant, were used. First-line drug susceptibility testing (DST) was performed using the LJ method to determine the percentage of resistant bacteria. All drug-resistant isolates underwent testing with LPA. Heteroresistance was defined as simultaneous detection of wild-type and resistance-conferring mutations using LPA. Result: The sensitivity of LPA (compared with LJ DST) was 96% for any INH-resistant TB and 94% for any RR TB. The prevalence of heteroresistance among the 123. Mtb isolates was 9.8%. The percentage of resistant bacteria ranged from 1% to 10% for heteroresistant TB. Rifampicin heteroresistance was detected in 1.6% of MDR TB patients. INH heteroresistance was detected in 1.6% and 16.7% of MDR and INH-resistant TB patients, respectively. The proportion of INH heteroresistance was significantly higher (p = 0.030) in persons living with HIV. Conclusion: Some phenotypic drug resistances were not captured by LPA. The prevalence and percentage of resistant bacteria among heteroresistant TB highlight the importance of LPA for early detection of heteroresistant TB.

Traditional medicinal plants used in the treatment of tuberculosis in Ethiopia: A systematic review.

Background: Majority of people in Ethiopia heavily rely on traditional medicinal plants to treat a number of diseases including tuberculosis (TB). However, there has been lack of comprehensive evidences on taxonomic distribution of medicinal plant species, methods of preparation of remedies from these plants and how the remedies are administered. This systematic review is designed to examine and synthesize available evidences focusing on medicinal plants that have been used for TB treatment in Ethiopia. Methods: Research findings related to ethno-botanical and pharmacological approaches of TB remedies were retrieved from databases. Electronic libraries of Ethiopian Universities and relevant church-based religious books were also reviewed as additional sources. Evidences are searched and organized in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. Result: From a total of 68 research documents that reported use of plants for treatment of TB 98 plants species belonging to 82 genera and 49 families were identified. The most frequently reported plant species belonged to family Lamiaceae (n = 8), Euphorbiaceae (n = 7), Cucurbitaceae (n = 6) and Fabaceae (n = 6). Croton macrostachyus, Allium sativum, and Myrsine Africana were the most often mentioned anti-TB medicinal plants. Shrubs (35.7%) and trees (29.6%) were reported as dominant growth forms while plant roots (31.6%) and leaves (28.6%) were frequently used plant parts for the preparations of the treatment. The most favored administration route was oral (59.1%). About 87% of the preparations were made from fresh plant materials. No experimental/clinical evidence was presented for 79.6%(78/98) of the reported plants to support their anti-mycobacterial activities. Conclusion: In Ethiopia, the number of herbal remedies is enormous and their use for TB treatment is a common practice. However, majority of them are not yet backed up by evidence generated through scientific experimentation and this warrants further experimental and clinical validations. Moreover, the efficacy, toxicity and safety tests should be initiated and this would help in the rapid identification of new anti-TB regimens, and possibly it would lead to developing more effective new plant-based drugs. This systematic review will serve as a reference for the selection of plants for developing new anti-TB regimens.

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.

Utility of the Intradermal Skin Test in a Test-and-Cull Approach to Control Bovine Tuberculosis: A Pilot Study in Ethiopia.

Bovine tuberculosis (bTB) is one of the top three, high-priority, livestock diseases in Ethiopia and hence, the need for evaluation of potential control strategies is critical. Here, we applied the test-and-segregate followed by cull strategy for the control of bTB in the intensive Alage dairy farm in Ethiopia. All cattle reared on this farm were repeatedly skin tested using the Comparative Cervical Tuberculin (CCT) test for a total of five times between 2015 and 2021. During the first (October 2015) and second (March 2017) rounds of testing, all reactor animals (>4 mm) were culled, while those that were deemed as inconclusive (1-4 mm) were segregated and retested. At retest, animals with CCT >2 mm were removed from the herd. In the third (December 2017) and fourth (June 2018) rounds of tuberculin testing, a more stringent approach was taken wherein all reactors per the severe mode of CCT test interpretation (>2 mm) were culled. A final herd status check was performed in May 2021. In summary, the number of CCT positives (>4 mm) in the farm dropped from 23.1% (31/134) in October 2015 to 0% in December 2017 and remained 0% until May 2021. In contrast, the number of Single Cervical Tuberculin (SCT) test positives (≥4 mm) increased from 1.8 to 9.5% (from 2017 to 2021), indicating that CCT test might not be sufficient to effectively clear the herd of bTB. However, a more stringent approach would result in a drastic increase in the number of false positives. The total cost of the bTB control effort in this farm holding 134-200 cattle at any given time was conservatively estimated to be ~US$48,000. This, together with the need for culling an unacceptably high number of animals based on skin test status, makes the test-and-cull strategy impractical for nationwide implementation in Ethiopia and other low- and middle-income countries (LMICs) where the infection is endemic. Hence, there is an increased emphasis on the need to explore alternate, affordable measures such as vaccination alongside accurate diagnostics to help control bTB in endemic settings.