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.

The Simple One-Step (SOS) Stool Processing Method for Use with the Xpert MTB/RIF Assay for a Child-Friendly Diagnosis of Tuberculosis Closer to the Point of Care.

Young children cannot easily produce sputum for diagnosis of pulmonary tuberculosis (TB). Alternatively, Mycobacterium tuberculosis complex bacilli can be detected in stool by using the Xpert MTB/RIF (Ultra) assay (Xpert). Published stool processing methods contain somewhat complex procedures and require additional supplies. The aim of this study was to develop a simple one-step (SOS) stool processing method based on gravity sedimentation only, similar to Xpert testing of sputum samples, for the detection of M. tuberculosis in stool samples. We first assessed whether the SOS stool method could provide valid Xpert results without the need for bead-beating, dilution, and filtration steps. We concluded that this was the case, and we then validated the SOS stool method by testing spiked stool samples. By using the SOS stool method, 27 of the 29 spiked samples gave valid Xpert results, and M. tuberculosis was recovered from all 27 samples. The proof of principle of the SOS stool method was demonstrated in routine settings in Addis Ababa, Ethiopia. Nine of 123 children with presumptive TB had M. tuberculosis-positive results for nasogastric aspiration (NGA) samples, and 7 (77.8%) of those children also had M. tuberculosis-positive Xpert results for stool samples. Additionally, M. tuberculosis was detected in the stool samples but not the NGA samples from 2 children. The SOS stool processing method makes use of the standard Xpert assay kit, without the need for additional supplies or equipment. The method can potentially be rolled out to any Xpert site, bringing a bacteriologically confirmed diagnosis of TB in children closer to the point of care.

Phenotypic and genotypic drug sensitivity profiles of Mycobacterium tuberculosis infection and associated factors in northeastern Ethiopia.

BACKGROUND: Tuberculosis is a devastating and a deadly disease despite the novel advances in its diagnostic tools and drug therapy. Drug resistant Mycobacterium contributes a great share to tuberculosis mortality. Status of drug resistance and patients' awareness toward the disease is unknown in northeastern Ethiopia. Thus, the aim of this study was to determine the phenotypic and genotypic drug sensitivity patterns and associated factors in Oromia Special Zone and Dessie Town, northeastern Ethiopia. METHODS: In a cross-sectional study, 384 smear positive tuberculosis cases were recruited and Löwenstein-Jensen culture was done. The performance of GenoTypic MTBDRplus assay using the conventional BACTEC MGIT 960 as a "gold standard" was determined. Drug resistant strains were identified using spoligotyping. Pearson Chi-square test was used to determine the association of drug sensitivity test and tuberculosis type, lineages, dominant strains and clustering of the isolates. RESULTS: The 384 smear positive Mycobacterium samples were cultured on LJ media of which 29.2% (112/384) as culture positive. A fair agreement was found between MTBDRplus assay and the conventional MGIT test in detecting the Mycobacterium tuberculosis with sensitivity, specificity, positive and negative predictive value of 94.2, 30.2, 68.4 and 76.5%, respectively. Among LJ culture positive samples 95 of them gave valid result for MTBDRplus assay and 16.8% (16/95) as drug resistant. Similarly, MGIT subculture was made for the 112 isolates and 69 of them gave positive result with 15.9% (11/69) as drug resistant. Cohen's kappa value showed almost a perfect agreement between the two testing methods in detecting rifampicin (sensitivity 100% and specificity 98.3%) and multi-drug resistance (sensitivity 83.3% and specificity 100%). Spoligotyping identified 76.5% (13/17) of the drug resistant isolates as Euro-American and family 33 as the predominant family. Significant association was observed between drug resistant isolates and the dominant strains (χ2: 34.861; p = 0.040) of the Mycobacterium. CONCLUSION: Higher magnitude of drug resistance was found in the study area. The GenoTypic MDRTBplus assay had an acceptable drug sensitivity testing performance.

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%).

Does Xpert® MTB/RIF assay give rifampicin resistance results without identified mutation? Review of cases from Addis Ababa, Ethiopia.

BACKGROUND: Xpert® MTB/RIF assay is currently used in Ethiopia for the rapid diagnosis of Mycobacterium tuberculosis (MTB) and mutations that confer Rifampicin resistance. Rifampicin resistance is determined based on any mutation in the 81 bp of rpoB gene using five overlapping probes represented as Probe A (codons 507-511), Probe B (codons 512-518), Probe C (codons 518-523), Probe D (codons 523-529) and Probe E (codons 529-533). In this review, we assessed the frequency of missed probe types for Rifampicin Resistance results. METHODS: Data were reviewed from specimens received and tested using Xpert® MTB/RIF assay at Ethiopian National Tuberculosis Reference Laboratory, in Addis Ababa from 15 July 2016 to 31 December 2018 retrospectively. All archived data were reviewed carefully to describe missed probe types and the quantity of DNA in the sample. RESULTS: A total of 100 specimens were reported as MTB Detected Rifampicin Resistance Detected by Xpert® MTB/RIF assay. More than half (55%) of these results were reported from male patients. The median age was 28.0 years (5 months to 88 years). Majorities (62%) of the cases were detected from sputum. Among the total of 38 extrapulmonary samples, lymph node aspirates were accounted for 50% (19/38). The most common mutations (81.0%) were found in the Probe E region followed by Probe D (10.0%), and Probe B (3.0%). Mutations in Probe A and Probe C regions were not observed. However, six (6.0%) Rifampicin resistance cases were found without any missed probe type. The delta Ct max is ≥4.3. No specimen yielded Rifampicin resistance associated with more than one probe failure or mutation combinations. CONCLUSION: Mutations associated with Probe E (codons 529-533) region were identified as the commonest rpoB gene mutations. The Rifampicin resistance results found without any identified missing probe needs further study. The lower DNA amount was observed in extrapulmonary specimens compared with sputum.

Intestinal parasites co-infection and associated factors among active pulmonary tuberculosis patients in selected health centers, Addis Ababa, Ethiopia: unmatched case control study.

BACKGROUND: In co-endemic areas, rate of intestinal parasites and tuberculosis (TB) co-infection thought to be high. However, there are limited studies on the epidemiology of this co-infection in Ethiopia. Therefore, the present study aimed to generate evidence on intestinal parasites co-infection rate and associated factors among pulmonary tuberculosis patients (PTB) and their household contacts in Addis Ababa, Ethiopia. METHODS: Unmatched case-control study was conducted. Data were collected from 91 PTB patients (cases) and 89 household contacts (controls). Socio-demographic characteristics and associated factors were collected using structured questionnaire. Sputum, stool and blood specimens were collected, processed and examined for PTB, intestinal parasites and Human Immunodeficiency virus anti-body test, respectively. Data were entered and analyzed by Statistical Packages for Social Sciences (SPSS) Version 20. Descriptive statistics, Fisher's exact test, binary logistic regression, and odds ratio were used. P-value of < 0.05 was considered as statistically significant. RESULTS: The infection rate of intestinal parasites based on one stool samples in PTB patients and controls was 22 and 9%, respectively. The difference was statistically significant (COR = 2.85;95% CI = 1.18-6.87). The most prevalent intestinal parasite in PTB patients was Gardia lamblia (8.8%, 8), followed equally by Ascaris lumbricoides, Haymenolopsis nana and Entamoeba histolytica/dispar (4.4%, 4). Co-infection in PTB patients was associated with body mass index (BMI) < 18.5 (AOR = 6.71;95% CI = 1.65-27.25) and dirty material in finger nails (AOR = 8.99;95% CI = 2.46-32.78). There was no variable associated with parasitic infections in controls in our analysis, which might be due to the low prevalence of intestinal parasites'. CONCLUSIONS: There was a statistical significant difference in the infection rate of intestinal parasites in PTB patients compared to healthy household contacts. The consequence of co-infection on developing an active disease, disease severity and treatment efficacy needs to be investigated in future.

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.

Evaluation of genotype MTBDRplus VER 2.0 line probe assay for the detection of MDR-TB in smear positive and negative sputum samples.

BACKGROUND: Multi drug resistant tuberculosis (MDR-TB) poses formidable challenges to TB control due to its complex diagnostic and treatment challenges and often associated with a high rate of mortality. Accurate and rapid detection of MDR-TB is critical for timely initiation of treatment. Line Probe Assay (LPA) is a qualitative in vitro diagnostic test based on DNA-STRIP technology for the identification of the M. tuberculosis complex and its resistance to rifampicin (RMP) and/or isoniazid (INH). Hain Lifescience, GmbH, Germany has improved the sensitivity of Genotype MTBDRplus VER 2.0 LPA for the detection of MDR-TB; with the possibility of applying the tool in smear negative sputum samples. METHOD: A cross sectional study was conducted on 274 presumptive MDR-TB patients referred to the National TB Reference Laboratory (NTRL), Ethiopian Public Health Institute (EPHI) who submitted sputum samples for laboratory diagnosis of drug resistant-TB testing. Seventy-two smear and culture positive samples processed in smear positive direct LPA category and 197 smear negative sputum samples were processed for direct LPA. Among the smear negative samples 145 (73.6%) were culture negative and 26 (13.2%) were culture positive. All specimens were processed using NALC-NaOH method and ZN smear microscopy done from sediments. Genotype MTBDRplus VER 2.0 done from processed sputum sediments and the result was compared against the reference, BACTEC MGIT 960 culture and DST. Sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 assay was determined and P-value <0.05 was considered as statistically significant. RESULTS: The sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 LPA were 96.4, 100, 100 and 96.9%, respectively for the detection of MDR-TB from direct smear positive sputum samples. The sensitivity, specificity, PPV and NPV of Genotype MTBDR plus VER 2.0 LPA were 77.8, 97.2, 82.4 and 97.2%, respectively, for the detection of M. tuberculosis from direct smear negative sputum samples. Fourteen (53.8%) samples had valid results with LPA among the 26 smear negative culture positive samples. The remaining 8 (30.8%) and 4 (15.4%) were invalid and negative with LPA, respectively. The sensitivity and specificity of Genotype MTBDRplus VER 2.0 LPA were 100% for the detection of MDR-TB among 14 direct smear negative and culture positive sputum samples. The most common mutations associated with RMP and INH resistance were S531L and S315TL, respectively. A single rare mutation (C15T/A16G) was detected for INH resistance. CONCLUSION: The diagnostic performance of Genotype MTBDRplus VER 2.0 LPA in direct smear positive sputum sample was highly sensitive and specific for early detection of MDR-TB. However, the diagnostic performance of this molecular assay in direct smear negative sputum sample was low and showed a high level of invalid results for detection of M. tuberculosis and its resistance to RMP and/or INH so it is unlikely to implement Genotype MTBDRplus VER 2.0 for the detection of MDR-TB in direct smear negative sample in our routine settings. The sensitivity of the assay should be improved for detection of MDR-TB in direct smear negative sputum specimens.

Genotypic and phenotypic drug resistance patterns of Mycobacterium tuberculosis isolated from presumptive pulmonary tuberculosis patients in Ethiopia: A multicenter study.

BACKGROUND: The emergence of drug-resistant tuberculosis (DR-TB) has been a major obstacle to global tuberculosis control programs, especially in developing countries, including Ethiopia. This study investigated drug resistance patterns and associated mutations of Mycobacterium tuberculosis Complex (MTBC) isolates from the Amhara, Gambella, and Benishangul-Gumuz regions of Ethiopia. METHODS: A cross-sectional study was conducted using 128 MTBC isolates obtained from patients with presumptive tuberculosis (TB). Phenotypic (BACTEC MGIT 960) and genotypic (MTBDRplus and MTBDRsl assays) methods were used for drug susceptibility testing. Data were entered into Epi-info and analyzed using SPSS version 25. Frequencies and proportions were determined to describe drug resistance levels and associated mutations. RESULTS: Of the 127 isolates recovered, 100 (78.7%) were susceptible to four first-line anti-TB drugs. Any drug resistance, polydrug resistance, and multi-drug resistance (MDR) were detected in 21.3% (27), 15.7% (20), and 15% (19) of the isolates, respectively, by phenotypic and/or genotypic methods. Mono-resistance was observed for Isoniazid (INH) (2, 1.6%) and Streptomycin (STR) (2, 1.6%). There were two genotypically discordant RIF-resistant cases and one INH-resistant case. One case of pre-extensively drug-resistant TB (pre-XDR-TB) and one case of extensively drug-resistant TB (XDR-TB) were identified. The most frequent gene mutations associated with INH and rifampicin (RIF) resistance were observed in the katG MUT1 (S315T1) (20, 76.9%) and rpoB (S531L) (10, 52.6%) genes, respectively. Two MDR-TB isolates were resistant to second-line drugs; one had a mutation in the gyrA MUT1 gene, and the other had missing gyrA WT1, gyrA WT3, and rrs WT1 genes without any mutation. CONCLUSIONS: The detection of a significant proportion of DR-TB cases in this study suggests that DR-TB is a major public health problem in Ethiopia. Thus, we recommend the early detection and treatment of DR-TB and universal full first-line drug-susceptibility testing in routine system.

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.

Acquired bedaquiline and fluoroquinolones resistance during treatment follow-up in Oromia Region, North Shewa, Ethiopia.

Background: Bedaquiline (BDQ) is an effective drug currently used for multidrug-resistant or rifampicin-resistant TB (MDR/RR-TB) and pre-extensively drug-resistant TB (pre-XDR-TB) treatment. However, resistance to this new drug is emerging. We discussed the characteristics of the first patient in Ethiopia who acquired BDQ and fluoroquinolones (FQs) resistance during treatment follow-up. Case report: In this case report, we present the case of a 28-year-old male pulmonary TB patient diagnosed with MDR-TB who is a resident of the Oromia Region of North Shewa, Mulona Sululta Woreda, Ethiopia. Sputum specimen was collected initially and for treatment monitoring using culture and for phenotypic drug susceptibility testing (DST) to first-line and second-line TB drugs. Initially, the patient was infected with a mycobacterial strain resistant to the first-line anti-TB drugs Rifampicin (RIF), Isoniazid (INH), and Pyrazinamide (PZA). Later, during treatment, he acquired additional drug resistance to ethambutol (EMB), ofloxacin (OFX), levofloxacin (LFX), moxifloxacin (MFX), and BDQ. The patient was tested with MTBDRplus and MTBDRsl to confirm the presence of resistance-conferring mutation and mutation was detected in rpoB, katG, and gyrA genes. Finally, the patient was registered as having extensively drug-resistant tuberculosis (XDR-TB) and immediately started an individualized treatment regimen. Conclusion: This case report data has revealed the evolution of BDQ resistance during treatment with a BDQ-containing regimen in Ethiopia. Therefore, there is a need for DST to new second-line drugs to monitor and prevent the spread of DR-TB.

Diagnostic accuracy, feasibility and acceptability of stool-based testing for childhood tuberculosis.

Background: Childhood tuberculosis (TB) diagnosis remains challenging, partly because children cannot provide sputum. This study evaluated the diagnostic accuracy of the Simple One-Step (SOS) stool method with Xpert MTB/RIF Ultra (Xpert-Ultra) for childhood TB compared to culture and Xpert-Ultra on a respiratory sample (RS) and clinical diagnosis. It also assessed the feasibility and acceptability of stool testing according to laboratory staff, and caregivers' sample preference. Methods: We enrolled children (≤10 years) with presumptive pulmonary tuberculosis in Ethiopia. RS was tested using Xpert-Ultra and culture; stool samples were tested using the SOS stool method with Xpert-Ultra. Laboratory staff and caregivers' opinions were assessed using standardised questionnaires. Results: Of the 898 children enrolled, 792, 832 and 794 were included for assessing the diagnostic accuracy of SOS stool with Xpert-Ultra against culture, RS Xpert-Ultra and clinical diagnosis, respectively, yielding sensitivity estimates for SOS stool with Xpert-Ultra of 69.1% (95% confidence interval (CI) 56.0-79.7%), 76.8% (95% CI 64.2-85.9%) and 59.0% (95% CI 47.9-69.2%), respectively. The specificity was ≥98.8% for all comparisons. The rate of non-determinate test results was 2.8% after one repeat test. According to laboratory staff, stool collection was feasible and acceptable and the SOS stool method was easy to perform. Most caregivers (75%) preferred stool for TB diagnosis over RS. Conclusion: This study shows that SOS stool Xpert-Ultra testing offers a good alternative to RS testing for TB in children who cannot spontaneously produce a sputum sample and would otherwise need to undergo invasive procedures to obtain RS for diagnosis.

Epidemiology of extensively drug-resistant tuberculosis among patients with multidrug-resistant tuberculosis: A systematic review and meta-analysis.

OBJECTIVES: To estimate the pooled proportion of extensively drug-resistant tuberculosis (XDR-TB) and pre-extensively drug-resistant tuberculosis (pre-XDR-TB) in patients with multidrug-resistant TB (MDR-TB). METHODS: We systematically searched articles from electronic databases: MEDLINE (PubMed), ScienceDirect, and Google Scholar. We also searched gray literature from the different literature sources main outcome of the review was either XDR-TB or pre-XDR-TB in patients with MDR-TB. We used the random-effects model, considering the substantial heterogeneity among studies. Heterogeneity was assessed by subgroup analyses. STATA version 14 was used for analysis. RESULTS: A total of 64 studies that reported on 12,711 patients with MDR-TB from 22 countries were retrieved. The pooled proportion of pre-XDR-TB was 26% (95% confidence interval [CI]: 22-31%), whereas XDR-TB in MDR-TB cases was 9% (95% CI: 7-11%) in patients treated for MDR-TB. The pooled proportion of resistance to fluoroquinolones was 27% (95% CI: 22-33%) and second-line injectable drugs was 11% (95% CI: 9-13%). Whereas the pooled resistance proportions to bedaquiline, clofazimine, delamanid, and linezolid were 5% (95% CI: 1-8%), 4% (95% CI: 0-10%), 5% (95% CI; 2-8%), and 4% (95% CI: 2-10%), respectively. CONCLUSION: The burden of pre-XDR-TB and XDR-TB in MDR-TB were considerable. The high burdens of pre-XDR-TB and XDR-TB in patients treated for MDR-TB suggests the need to strengthen TB programs and drug resistance surveillance.

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.

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.

Bacteriologically confirmed extrapulmonary tuberculosis and the associated risk factors among extrapulmonary tuberculosis suspected patients in Ethiopia: A systematic review and meta-analysis.

BACKGROUND: The actual burden of bacteriologically confirmed extrapulmonary tuberculosis (EPTB) and risk factors in Ethiopia is not well known due to the lack of a strong surveillance system in Ethiopia. Thus, this study was conducted to estimate the pooled prevalence of bacteriologically confirmed EPTB and the associated risk factors among persons suspected to have non-respiratory tuberculosis in Ethiopia. METHODS: A systematic review and meta-analysis of published studies reporting the prevalence of EPTB from searched electronic databases; Science Direct, PubMed, and Google Scholar was estimated spread across the research periods, nationally, and in different areas, using a fixed-effects model. We used I2 to analyze heterogeneity in the reported prevalence of bacteriologically confirmed extrapulmonary tuberculosis. RESULTS: After reviewing 938 research articles, 20 studies (19 cross-sectional and 1 retrospective) from 2003 to 2021 were included in the final analyses. The pooled prevalence of bacteriologically confirmed EPTB was 43% (95%CI; 0.34-0.52, I2 = 98.45%). The asymmetry of the funnel plot revealed the presence of publication bias. Specifically the pooled prevalence of bacteriologically confirmed EPTB based on smear microscopy, Xpert MTB/RIF assay, and culture were 22% (95%CI; 0.13-0.30, I2 = 98.56%), 39% (95%CI; 0.23-0.54, I2 = 98.73%) and 49% (95%CI; 0.41-0.57, I2 = 96.43%) respectively. In this study, a history of pulmonary tuberculosis (PTB) contact with PTB patients, contact with live animals, consumption of raw milk, HIV-positive, male, and lower monthly income, were found to be independently associated with bacteriologically confirmed EPTB. CONCLUSION: Ethiopia has a high rate of bacteriologically confirmed EPTB. A history of previous PTB, being HIV-positive and having contact with PTB patients were the most reported risk factors for EPTB in the majority of studies. Strengthening laboratory services for EPTB diagnosis should be given priority to diagnose EPTB cases as early as possible.

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.

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.

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.