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.

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.

A smooth tubercle bacillus from Ethiopia phylogenetically close to the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) includes several human- and animal-adapted pathogens. It is thought to have originated in East Africa from a recombinogenic Mycobacterium canettii-like ancestral pool. Here, we describe the discovery of a clinical tuberculosis strain isolated in Ethiopia that shares archetypal phenotypic and genomic features of M. canettii strains, but represents a phylogenetic branch much closer to the MTBC clade than to the M. canettii strains. Analysis of genomic traces of horizontal gene transfer in this isolate and previously identified M. canettii strains indicates a persistent albeit decreased recombinogenic lifestyle near the emergence of the MTBC. Our findings support that the MTBC emergence from its putative free-living M. canettii-like progenitor is evolutionarily very recent, and suggest the existence of a continuum of further extant derivatives from ancestral stages, close to the root of the MTBC, along the Great Rift Valley.

Pulmonary tuberculosis case notification and burden of drug resistance among children under 15 years of age in Ethiopia: sub-analysis from third-round drug resistance tuberculosis survey.

INTRODUCTION: Data on the burden of bacteriologically confirmed childhood Tuberculosis (PTB) and drug-resistant TB in Ethiopia is limited due to difficulties related to its diagnosis in this population. Therefore, this study aimed to assess bacteriologically confirmed childhood PTB Case Notification Rates (CNRs) and the burden of Drug Resistant-Tuberculosis among children in Ethiopia. METHOD: Retrospective secondary clinical and laboratory data were obtained from 3rd round national DR-TB survey which was conducted between August 2017 and January 2019. We used IBM SPSS 24 for sub-analysis of 3rd round Drug Resistant-Tuberculosis data. Descriptive statistics were used in computing the association between the sociodemographic characteristics and PTB CNRs, and the strength of the associations was determined using binary logistic regression with Odds ratios (OR) with a 95% confidence interval (CI). RESULT: Overall, 102 bacteriologically confirmed childhood PTB cases were identified with a median age of 12 (range 1-14) years. Of these, 54 (52.9%) were females and 81 (79.4%) lived in rural areas. HIV-TB co-infection cases were 5/102 (4.3%) and the majority (98%) of cases were newly diagnosed children. Nationally, the incidence of bacteriologically confirmed childhood PTB was calculated to be 5.1 per 100,000 children. The burden of Drug Resistant-Tuberculosis to at least one of the five first-line anti-TB drugs tested was five (6.5%) cases and one (1.3%) was found to be a Multi-drug resistant tuberculosis case. Drug-resistant tuberculosis was significantly associated with the age group 10-14 years (P = 0.002; [AOR] 29.76; [95% CI, 3.51-252.64]) and children living in urban areas (P = 0.027; [AOR] 5.76; 95% CI, 1.22-27.09). CONCLUSION: Bacteriologically confirmed childhood PTB cases increased as the age of the children increased. Most of the bacteriologically confirmed childhood PTB and the identified drug Resistant-Tuberculosis cases were new cases. Also, rural children were more affected by TB than their urban, counterparts Drug Resistant-Tuberculosis was higher in urban resident children.

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.

Optimization of the Simple One-Step Stool Processing Method to Diagnose Tuberculosis: Evaluation of Robustness and Stool Transport Conditions for Global Implementation.

Stool is recommended as an alternative specimen for the diagnosis of tuberculosis (TB) in young children, as they cannot easily produce sputum. The Simple One-Step (SOS) stool processing method is a new and simple stool processing method for the detection of Mycobacterium tuberculosis (MTB) using Xpert MTB/RIF Ultra (Xpert-Ultra). We determined the robustness of the SOS stool processing method and stool specimen transport conditions in participants with confirmed TB. We processed stool using the standard protocol after simulated "transport," varying time, and temperature, and experimented with slightly modified processing steps. We included 2,963 Xpert-Ultra test results from 132 stool specimens of 47 TB participants, including 11 children aged <10 years. We compared Xpert-Ultra processing errors and MTB positivity rates between standard and modified procedures. Minor deviations from the standard SOS protocol did not significantly impact the Xpert-Ultra test outcomes. The rate of Xpert-Ultra processing errors significantly increased with noncold-chain transport, exposure of stool to sample reagent at room temperature or beyond 12 h, and adding >0.8 g of stool. We found that almost all steps in the current SOS stool processing method provide optimal Xpert-Ultra results but recommend an adjustment to use a wider range of stool amounts (0.3 to 0.8 g) than advised previously (0.8 g). With this adaptation, stool-based diagnosis of TB using the SOS stool processing method can be scaled-up. IMPORTANCE The manuscript will support the global implementation and scale-up of the SOS stool method in routine settings. It also provides important insights on the optimal stool transport conditions and robustness of the SOS method, which can be used for bacteriological diagnosis of TB in children at the lowest levels of the healthcare system, avoiding lengthy healthcare-seeking pathways and additional costs.

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.

The Simple One-step stool processing method for detection of Pulmonary tuberculosis: A study protocol to assess the robustness, stool storage conditions and sampling strategy for global implementation and scale-up.

BACKGROUND: The Xpert MTB/RIF Ultra (Xpert-Ultra) assay provides timely results with good sensitivity and acceptable specificity with stool specimens in children for bacteriological confirmation of tuberculosis (TB). This study aims to optimize the Simple One-Step (SOS) stool processing method for testing stool specimens using the Xpert-Ultra in children and adults in selected health facilities in Addis Ababa, Ethiopia. The study is designed to assess the robustness of the SOS stool method, to help fine-tune the practical aspects of performing the test and to provide insights in stool storage conditions and sampling strategies before the method can be implemented and scaled in routine settings in Ethiopia as well as globally. METHODS AND DESIGN: The project "painless optimized diagnosis of TB in Ethiopian children" (PODTEC) will be a cross sectional study where three key experiments will be carried out focusing on 1) sampling strategy to investigate if the Xpert-Ultra M. tuberculosis (MTB) -positivity rate depends on stool consistency, and if sensitivity can be increased by taking more than one stool specimen from the same participant, or doing multiple tests from the same stool specimen, 2) storage conditions to determine how long and at what temperature stool can be stored without losing sensitivity, and 3) optimization of sensitivity and robustness of the SOS stool processing method by varying stool processing steps, stool volume, and storage time and conditions of the stool-sample reagent mixture. Stool specimens will be collected from participants (children and adults) who are either sputum or naso-gastric aspiration (NGA) and/or stool Xpert-Ultra MTB positive depending on the experiment. Stool specimens from these participants, recruited from 22 sites for an ongoing related study, will be utilized for the PODTEC experiments. The sample size is estimated to be 50 participants. We will use EpiData for data entry and Stata for data analysis purposes. The main analyses will include computing the loss or gain in the Xpert-Ultra MTB positivity rate and rates of non-determinate Xpert-Ultra test results per experiment compared to the Xpert-Ultra MTB result of stool processed according to the published standard operating procedures for SOS stool processing. The differences in the MTB positivity rate by regarding testing more than one sample per child, and using different storage, and processing conditions, will be also compared to the baseline (on-site) Xpert-Ultra result.

Detection of Mycobacterium tuberculosis and rifampicin resistance by Xpert® MTB/RIF assay among presumptive tuberculosis patients in Addis Ababa, Ethiopia from 2014 to 2021.

Objective: This study aimed to determine the frequencies and trends of Mycobacterium tuberculosis and rifampicin resistance among presumptive tuberculosis patients in Ethiopia, who were tested using the Xpert MTB/RIF assay between 2014 and 2021. Methods: Data were collected retrospectively from patient registries. Laboratory-based data were extracted from the national tuberculosis (TB) referral laboratory database. All patients referred to the National Tuberculosis Reference Laboratory (NTRL) for TB diagnosis from all over the country between March 1, 2014 and September 30, 2021, and tested using the Xpert MTB/RIF assay, were included. The extracted data were entered into a Microsoft Excel sheet and analyzed by Statistical Package for Social Sciences (SPSS) version 23. Results: Among a total of 13 772 individuals tested using the Xpert MTB/RIF assay, the majority (8223; 59.7%) were males, and 48.5% (6678) of the individuals were aged between 15 and 39 years. Mycobacterium tuberculosis (MTB) was detected in 17.0% (2347) of the examined individuals. Of the detected MTB cases, nearly 9.9% (233) were rifampicin resistant (RR-TB), while 24 (1.0%) were RR-intermediate. Among all RR-TB cases, more than half (125; 53.6%) were detected in males, and 105 were new TB cases. Extrapulmonary (EPTB) patients had a greater rate of rifampicin resistance (11.0%) than pulmonary (PTB) patients (9.6%). Conclusion: The frequency of TB and RR-TB remains high in the study setting. RR-TB was found to have a statistically significant association with previous anti-TB medication treatment. As a result, improving treatment adherence in recognized instances could assist in preventing MTB and RR-TB cases.

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.

Effect of sputum quality and role of Xpert® MTB/ RIF assay for detection of smear-negative pulmonary tuberculosis in same-day diagnosis strategy in Addis Ababa, Ethiopia.

Background: There is limited information on the performance of the Xpert® MTB/RIF test for diagnosis of smear-negative pulmonary tuberculosis (SNPT) and rifampicin resistance (RR) in the same-day diagnosis approach. The effects of sputum quality and other factors affecting the Xpert performance are also under-investigated. Objective: This study aimed to determine the performance of the Xpert® MTB/RIF test for detection of SNPT and RR in the same-day diagnosis strategy and the effect of sputum quality and other factors on its performance. Methods: A cross-sectional study was conducted from August 2017 to January 2018 across 16 health facilities in Addis Ababa, Ethiopia. Two spot sputum samples were collected from 418 presumptive SNPT patients, tested with Xpert® MTB/RIF, then compared to tuberculosis culture. Additionally, culture isolates were tested for RR by BACTEC MGIT™ 960 drug susceptibility testing (DST) and MTBDRplus version 2. Results: The Xpert® MTB/RIF test detected 24 (5.7%) SNPT cases, with a sensitivity of 92.3% (75.9% - 97.9%) and specificity of 99.2% (97.8% - 99.7%) compared with tuberculosis culture. Xpert® MTB/RIF also detected three (11.58%) RR strains with 100.0% concordance with BACTEC MGIT™ 960 DST and MTBDRplus results. Three blood-stained SNPT samples were positive by Xpert (30.0%), which was 6.9 times higher compared to salivary sputum (odds ratio: 6.9, 95% confidence interval: 1.36-34.96, p = 0.020). Conclusion: The performance of the Xpert® MTB/RIF to detect SNPT and RR in same-day diagnosis is high. However, SNPT positivity varies among sputum qualities, and good sample collection is necessary for better test performance.

Pre-extensively drug-resistant tuberculosis among multidrug-resistant tuberculosis patients in Ethiopia: a laboratory-based surveillance study.

Background: The rise of drug-resistant tuberculosis (DR-TB) has presented a substantial challenge to the national tuberculosis (TB) control program. Understanding the epidemiology of pre-extensively drug-resistant tuberculosis (pre-XDR-TB) could help clinicians to adapt MDR-TB treatment regimens at an earlier stage. This study aimed to assess second-line anti-TB drug resistance among MDR-TB patients in Ethiopia using routine laboratory-based data. Methods: Laboratory-based cross-sectional data were collected from the national TB reference laboratory and seven regional tuberculosis culture laboratories in Ethiopia from July 2019 to March 2022. The required data, such as drug-susceptibility testing (DST) results and sociodemographics, were collected on a structured checklist from laboratory registration books and electronic databases. Data were entered into a Microsoft Excel spreadsheet and analyzed using SPSS version 23. Descriptive statistics were performed to show the distribution and magnitude of drug resistance. Results: Second-line drugs (SLDs) susceptibility testing was performed for 644 MDR isolates, of which 19 (3%) were found to be pre-XDR-TB cases. Of the total MDR-TB isolates, 19 (3%) were resistant to at least one fluoroquinolone drug, while 11 (1.7%) were resistant to at least one injectable second-line drug. Of the 644 MDR-TB isolates, 1.9% (5/261) pre-XDR were from new MDR-TB cases, while 3.7% (14/383) were from previously treated MDR-TB patients. The most frequently identified mutations, based on MTBDRsl results, were in codon A90V of the gyrA gene (77.3%) and A1401G of the rrs gene (45.5%). Conclusion: The overall prevalence of pre-XDR-TB in Ethiopia is considerable. The majority of SLD resistance mutations were in the gyrA gene at position A90V. Modern, rapid DST is necessary to enable identification of pre-XDR-TB and XDR-TB in supporting proper regimen administration for patients.

Evaluation of modified bleach technique for the detection of acid fast bacilli in lymph node aspirate at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia.

Background: Tuberculous Lymphadenitis (TBLN) is the commonest form of extrapulmonary tuberculosis. Definitive diagnosis of TBLN is difficult due to the low efficacy of the routine diagnostic techniques as compared to culture. Objective: To determine whether prior bleach concentration can improve the detection of acid-fast bacilli when compared with conventional Ziehl-Neelsen (ZN) staining by using culture as the gold standard. Methods: Cross-sectional study was conducted from March 01, 2015 to May 30, 2015. The study subjects were clinically suspected TBLN patients. Fine needle aspiration (FNA) was performed. Two different smears were prepared directly from the first needle pass for cytology and ZN staining. The remaining aspirate was used for the modified bleach concentration. The second needle pass aspirate was transferred into a sterile container containing sterile normal saline. The sediment was inoculated on the Lowenstein Jensen (LJ) medium. Measurement parameters for reliability and validity were used to analyze the results. Result: A total of 93 participants were included in the study. Fifty-six out of the 93 (60.2 %) cases were positive for mycobacterium tuberculosis complex on LJ culture. The detection rates of direct ZN staining and modified bleach method were 20.4 % and 44.1 %, respectively. 73.1 % of the cases showed cytomorphological features consistent with TBLN by cytologic examination. The sensitivities of direct ZN staining and modified bleach method and cytomorphology were 32.0 %, 67.8 %, and 92.8 %, respectively. Conclusion: Implementation of bleach concentration increases the detection rate of AFB over the direct ZN method. The bleach method can also be easily performed and provide a safe working environment by reducing infections.

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.

Utility of line probe assay in detecting drug resistance and the associated mutations in patients with extrapulmonary tuberculosis in Addis Ababa, Ethiopia.

Introduction: Molecular tests allow rapid detection of Mycobacterium tuberculosis and drug resistance in a few days. Identifying the mutations in genes associated with drug resistance may contribute to the development of appropriate interventions to improve tuberculosis control. So far, there is little information in Ethiopia about the diagnostic performance of line probe assay (LPA) and the M. tuberculosis common gene mutations associated with drug resistance in extrapulmonary tuberculosis. Thus, this study aimed to assess the frequency of drug resistance-associated mutations in patients with extrapulmonary tuberculosis (EPTB) and to compare the agreement and determine the utility of the genotypic in the detection of drug resistance in Addis Ababa, Ethiopia. Methods: A cross-sectional study was conducted on stored M. tuberculosis isolates. The genotypic and phenotypic drug susceptibility tests were performed using LPA and BACTEC-MGIT-960, respectively. The common mutations were noted, and the agreement and the utility of the LPA were determined using the BACTEC-MGIT-960 as a gold standard. Results: Of the 151 isolates, the sensitivity and specificity of MTBDRplus in detecting isoniazid resistance were 90.9% and 100%, respectively. While for rifampicin, it was 100% and 99.3% for sensitivity and specificity, respectively. The katG S315Tl was the most common mutation observed in 85.7% of the isoniazid-resistant isolates. In the case of rifampicin, the most common mutation (61.9%) was observed at position rpoB S531L. Mutations in the gyrA promoter region were strongly associated with Levofloxacin and Moxifloxacin resistance. Conclusion: Line probe assay has high test performance in detecting resistance to anti-TB drugs in EPTB isolates. The MTBDRplus test was slightly less sensitive for the detection of isoniazid resistance as compared to the detection of rifampicin. The most prevalent mutations associated with isoniazid and rifampicin resistance were observed at katG S315Tl and rpoB S531L respectively. Besides, all the fluoroquinolone-resistant cases were associated with gyrA gene. Finally, a validation study with DNA sequencing is recommended.

Diagnostic accuracy of Truenat Tuberculosis and Rifampicin-Resistance assays in Addis Ababa, Ethiopia.

BACKGROUND: Rapid and sensitive Tuberculosis (TB) diagnosis closer to patients is a key global TB control priority. Truenat assays (MTB, MTB Plus, and MTB-RIF Dx) are new TB molecular diagnostic tools for the detection of TB and Rifampicin (RIF)-resistance from sputum samples. The diagnostic accuracy of the assays is needed prior to implementation in clinical use in Ethiopia. This study aimed to determine the sensitivity and specificity of Truenat assays; and aimed to compare the assays to the Xpert MTB/RIF assay. METHODS: A prospective evaluation study was conducted among 200 presumptive TB patients in microscopy centers in Addis Ababa, Ethiopia from May 2019 to December 2020. Culture (Solid and Liquid methods) and phenotypic (liquid method) drug susceptibility testing (DST) were used as a reference standard. RESULTS: Of 200 adult participants, culture confirmed TB cases were 25 (12.5%), and only one isolate was resistant to RIF by phenotypic DST. The sensitivity of Truenat MTB was 88.0% [95% CI 70.1, 95.8], while 91.7 [95% CI 74.2, 97.7] for Truenat MTB Plus at the microscopy centers. The specificity of Truenat MTB was 97.2% [95% CI 93.1, 98.9], while for Truenat MTB Plus was 97.2% [95% CI 93.0, 99.0]. The sensitivity of Truenat MTB was 90.5% while for MTB Plus, 100% compared to the Xpert MTB/RIF assay. CONCLUSION: Truenat assays were found to have high diagnostic accuracy. The assays have the potential to be used as a point of care (POC) TB diagnostic tests.

Mycobacterial Lineages Associated with Drug Resistance in Patients with Extrapulmonary Tuberculosis in Addis Ababa, Ethiopia.

BACKGROUND: In Ethiopia, tuberculosis (TB) is one of the most common causes of illness and death. However, there is limited information available on lineages associated with drug resistance among extrapulmonary tuberculosis patients in Ethiopia. In this study, researchers looked into Mycobacterium tuberculosis lineages linked to drug resistance in patients with extrapulmonary tuberculosis in Addis Ababa, Ethiopia. METHODS: On 151 Mycobacterium tuberculosis isolates, a cross-sectional analysis was performed. Spoligotyping was used to characterize mycobacterial lineages, while a phenotypic drug susceptibility test was performed to determine the drug resistance pattern. Data were analyzed using SPSS version 23. RESULTS: Among 151 Mycobacterium tuberculosis complex (MTBC) genotyped isolates, four lineages (L1-L4), and Mycobacterium bovis were identified. The predominantly identified lineage was Euro-American (73.5%) followed by East-African-Indian (19.2%). Any drug resistance (RR) and multidrug-resistant (MDR) tuberculosis was identified among 16.2% and 7.2% of the Euro-American lineage, respectively, while it was 30.8% and 15.4% among the East-African-Indian lineages. Among all three preextensively drug-resistance (pre-XDR) cases identified, two isolates belong to T3-ETH, and the other one strain was not defined by the database. There was no statistically significant association between any type of drug resistance and either lineage or sublineages of Mycobacterium tuberculosis. CONCLUSION: A higher proportion of any type of drug resistance and MDR was detected among the East-African-Indian lineage compared to others. However, there was no statistically significant association between any type of drug resistance and either lineages or sublineages. Thus, the authors recommend a large-scale study.

Molecular characterization and drug resistance patterns of Mycobacterium tuberculosis complex in extrapulmonary tuberculosis patients in Addis Ababa, Ethiopia.

BACKGROUND: Molecular characterization of Mycobacterium tuberculosis (MTB) is important to understand the pathogenesis, diagnosis, treatment, and prevention of tuberculosis (TB). However, there is limited information on molecular characteristics and drug-resistant patterns of MTB in patients with extra-pulmonary tuberculosis (EPTB) in Ethiopia. Thus, this study aimed to determine the molecular characteristics and drug resistance patterns of MTB in patients with EPTB in Addis Ababa, Ethiopia. METHODS: This study was conducted on frozen stored isolates of EPTB survey conducted in Addis Ababa, Ethiopia. A drug susceptibility test was performed using BACTEC-MGIT 960. Species and strain identification were performed using the Geno-Type MTBC and spoligotyping technique, respectively. Data were entered into the MIRU-VNTRplus database to assess the spoligotype patterns of MTB. Analysis was performed using SPSS version 23, and participants' characteristics were presented by numbers and proportions. RESULTS: Of 151 MTB isolates, 29 (19.2%) were resistant to at least one drug. The highest proportion of isolates was resistant to Isoniazid (14.6%) and Pyrazinamide (14.6%). Nine percent of isolates had multidrug-resistant TB (MDR-TB), and 21.4% of them had pre-extensively drug-resistant TB (pre-XDR-TB). Among the 151 MTB isolates characterized by spoligotyping, 142 (94.6%) had known patterns, while 9 (6.0%) isolates were not matched with the MIRU-VNTRplus spoligotype database. Of the isolates which had known patterns, 2% was M.bovis while 98% M. tuberculosis. Forty-one different spoligotype patterns were identified. The most frequently identified SpolDB4 (SIT) wereSIT149 (21.2%), SIT53 (14.6%) and SIT26 (9.6%). The predominant genotypes identified were T (53.6%), Central Asia Strain (19.2%) and Haarlem (9.9%). CONCLUSION: The present study showed a high proportion of MDR-TB and pre-XDR-TB among EPTB patients. The strains were mostly grouped into SIT149, SIT53, and SIT26. The T family lineage was the most prevalent genotype. MDR-TB and pre-XDR-TB prevention is required to combat these strains in EPTB. A large scale study is required to describe the molecular characteristics and drug resistance patterns of MTB isolates in EPTB patients.

Low Prevalence of Mycobacterium bovis in Tuberculosis Patients, Ethiopia.

An estimated 17% of all tuberculosis cases in Ethiopia are caused by Mycobacterium bovis. We used M. tuberculosis complex isolates to identify the prevalence of M. bovis as the cause of pulmonary tuberculosis. Our findings indicate that the proportion of pulmonary tuberculosis due to M. bovis is small (0.12%).

Prevalence of tuberculosis, multidrug resistant tuberculosis and associated risk factors among smear negative presumptive pulmonary tuberculosis patients in Addis Ababa, Ethiopia.

BACKGROUND: The diagnoses of active smear negative PTB, remains difficult. As a result, treatment is often carried out empirically relaying on clinical criteria. The distribution and magnitude of smear negative PTB, smear negative MDR-TB and associated factors in the same day diagnosis strategy are not clearly known in the study area. Therefore, this study aimed to determine the prevalence of TB, MDR-TB and associated risk factors among presumptive smear negative pulmonary tuberculosis patients in Addis Ababa, Ethiopia. METHODS: Analytic cross sectional study design was used. A total of 418 smear negative presumptive pulmonary TB patients were enrolled from selected health facilities since August 01, 2017 to January 5, 2018. Sputum samples were examined by Ziehl Neelsen microscopy, Xpert MTB/RIF assay and Culture. Drug susceptibility testing was performed by line probe assay and BACTEC MGIT 960 system. These laboratory tests were performed in Ethiopian Public Health Institute, National TB Reference Laboratory. Data was analyzed by SPSS Ver.20. RESULTS: From the total of 418 enrolled patients, 27 (6.5%) were Xpert MTB/ RIF and 26 (6.4%) were culture confirmed smear negative PTB patients. The positivity rate among male and female was 10.2 and 3.5% (p = 0.005) respectively. From 26 culture positive isolates 3 (11.54%) were MDR TB; from MDR-TB confirmed isolates 2/23 (8.7%) were among new and 1/3 (33.3%) was among retreatment smear negative presumptive pulmonary TB patients. All Rifampicin resistant smear negative pulmonary TB isolates by Xpert MTB/ RIF assay were found to be MDR TB and 7/26 (26.9%) isolates were INH mono resistant. History of migration found to be a potential factor for developing smear negative pulmonary TB. CONCLUSION: In this study a significant proportion of smear negative pulmonary TB was diagnosed. Furthermore, a high smear negative multi drug resistant (MDR) TB and other mono drug resistant TB prevalence was confirmed. Due to the limitations of smear microscopy which is used as a primary diagnostic tool, these TB strains are missed to be diagnosed and transmission continues in the community.