Kidist Bobosha

Introduction: To develop an in-house reference control DNA standard targeting the RLEP region of Mycobacterium leprae for use in quantitative polymerase chain reaction (qPCR) diagnostics, aiming to reduce dependence on animal models and external sources. Method: Previously characterized DNA samples from patients with leprosy were used to amplify and sequence the 450-base pair RLEP region. A specific 131-base pair segment within this region was targeted for qPCR assay development. The amplified products were purified; sequenced using Illumina next-generation sequencing technology; and validated through bioinformatics analyses, including Basic Local Alignment Search Tool (BLAST; National Institutes of Health) alignment and copy number assessment. Results: The consensus sequence aligned perfectly with the known RLEP region. The BLAST analysis revealed 37 copies of the target sequence throughout the genome, confirming the sequence’s utility as a sensitive diagnostic marker. The qPCR assay successfully detected the target in both reference and clinical samples, with melting curve analysis indicating high specificity. Discussion: An in-house M leprae RLEP control standard was successfully developed that provides a reliable, ethical, and resource-efficient alternative to traditional animal-derived controls for leprosy diagnosis using qPCR.

Background: Cutaneous leishmaniasis (CL) is a major public health concern, particularly in Ethiopia, where about 40 000 new cases occur annually, predominantly caused by Leishmania aethiopica. Clinical phenotypes include localized CL (LCL), mucocutaneous leishmaniasis (MCL) and diffuse CL (DCL). Despite the high disease burden, treatment options and high-quality data on treatment outcomes are limited. Objectives: To evaluate the effectiveness of standard treatments in Ethiopia to inform future clinical trials. Methods: We conducted an observational cohort study of patients with parasitologically confirmed cutaneous leishmaniasis at two specialized dermatology referral hospitals. Clinical- and patient-reported outcomes were assessed at baseline and at standardized times during follow-up. The primary clinical outcome measure (‘cure’) was complete re-epithelialization or flattening of the index lesion at day 90. Patient-reported outcomes were assessed using skin-specific and general quality-of-life scores. Results: We enrolled 666 participants from April 2022 to October 2023. Median patient age was 20 years (interquartile range 14–35) and 405 patients were male (60.8%). More than half (n = 372; 55.9%) had previously received traditional treatment for CL. Most participants had LCL (n = 390; 58.6%) or MCL (n = 261; 39.1%). Intramuscular sodium stibogluconate 20 mg kg–1 daily was the most frequently used systemic therapy, either alone or in combination with lesion-directed therapy (cryotherapy or intralesional sodium stibogluconate). At day 90, 28.3% (n = 83/293) of participants with LCL, 23.5% (v = 47/200) with MCL and 8% (n = 1/12) with DCL were deemed cured. By day 90, all patient-reported outcomes of skin health improved for those with LCL or MCL, but skin and CL-related quality-of-life scores did not improve for those with DCL. Most participants (n = 522/641; 81.4%) experienced at least one clinical adverse event and 7.2% (n = 46/641) had abnormal laboratory findings during treatment. Conclusions: Current treatment strategies have low cure rates in Ethiopia. Well-designed randomized controlled trials are urgently needed to improve management of CL caused by L. aethiopica.

Background: Tuberculosis remains the leading cause of death by a single infectious agent globally, with Ethiopia among the highest tuberculosis- and human immunodeficiency virus/tuberculosis–burden countries. Diagnostic gaps—particularly among household contacts (HHCs) unable to expectorate—hinder early case detection. Computer-aided detection software for chest radiography and nonrespiratory molecular assays, such as stool-based Xpert MTB/RIF testing, represent promising strategies for scalable screening. Methods: We conducted a prospective diagnostic accuracy study at St Luke Catholic Hospital, Oromia, Ethiopia, enrolling 478 participants (152 tuberculosis index patients and 326 HHCs). All HHCs ≥4 years underwent digital chest radiographic screening, with or without CAD4TB (Delft Imaging) software assistance, and provided stool and sputum samples for Xpert MTB/RIF testing. The accuracy of CAD4TB and stool Xpert testing was evaluated against sputum Xpert testing as the reference. Results: CAD4TB showed strong diagnostic performance, with a sensitivity of 0.77 (95% confidence interval, .70–.83) and specificity of 0.93 (.90–.96). Performance was higher among adults (sensitivity and specificity, 0.79 and 0.94) than in children (0.64 and 0.92). Stool and sputum Xpert testing demonstrated high concordance (Cohen’s κ = 0.76), with a sensitivity of 0.77 (95% confidence interval, .70–.84) and specificity of 0.97 (.93–.99). During the study, 10.6% of HHCs (34 of 321) were newly diagnosed microbiologically with tuberculosis. Conclusions: The combined use of CAD4TB and stool Xpert testing significantly improves tuberculosis detection, particularly among HHCs in high-burden, low-resource settings. This strategy is especially valuable in children and adults unable to produce sputum and where radiological expertise is limited.

No currently licensed vaccine reliably prevents pulmonary tuberculosis (TB), a leading cause of infectious disease mortality. Developing effective new vaccines requires identifying which Mycobacterium tuberculosis ( Mtb ) proteins are presented on major histocompatibility complex class II (MHC-II) by infected human phagocytes (target cells) and defining their capacity for recognition by CD4 ⁺ T cells. Vaccine designs must elicit T cell responses recognizing the same peptide-MHC complexes presented by infected cells. Although many human CD4 ⁺ T cell Mtb epitopes have been described, presentation on MHC-II by infected cells in most cases has not been directly evaluated. Using mass spectrometry (MS), we demonstrated that Mtb type VII secretion system (T7SS) substrates are enriched in the MHC-II repertoire of Mtb -infected human monocyte-derived phagocytes and that many of these antigens are immunogenic in people with prior evidence of Mtb infection. We next used MS to guide TB messenger RNA (mRNA) vaccine design, increasing the presentation of target MHC-II epitopes by orders of magnitude by incorporating design features that mirror aspects of antigen presentation dynamics in infected phagocytes. Our results provide a strategy for TB vaccine design that is guided by bottom-up unbiased discovery. Our approach combines targeted evaluation of antigen presentation in human cells paired with rapid iterative testing of mRNA vaccine designs to optimize antigen presentation before animal studies or human clinical trials.

Background: High-resolution metabolomics has shown promise for identifying blood-based biomarkers of tuberculosis (TB). We sought to discover a metabolic signature to detect pulmonary TB disease and monitor treatment response. Methods: Plasma from Ethiopian persons with pulmonary TB at diagnosis (n = 82) was compared to household contacts with TB symptoms (n = 104) and 2, 6, and 12 months after treatment initiation. Participants were divided into training and test sets for model building, with additional validation using independent cohorts from the countries of Georgia (n = 89) and South Africa (n = 85). Signatures were further evaluated in nonhuman primates infected with Mycobacterium tuberculosis (Mtb). Results: Among the metabolites that most significantly differed in concentration, tryptophan (Trp) and retinol were significantly decreased in persons with TB disease (45.2 vs 62.5 μM and 4.1 vs 8.2 μM, respectively), while kynurenine (Kyn) was significantly increased (2.1 vs 1.6 μM; q < .0001 for all). A signature that included the Kyn/Trp ratio and retinol showed excellent classification for TB disease (area under the curve [AUC] = 0.97). The signature had an AUC of 0.97 in HIV-positive and 0.95 in HIV-negative persons with TB disease from South Africa and 0.93 in TB patients from Georgia. In Ethiopian participants, signature scores decreased after 2 months (0.85 to 0.42) and 6 months of TB treatment (0.42 to 0.18; P < .0001 for both) to similar levels as controls. Plasma retinol also declined in nonhuman primates infected with Mtb 15–16 weeks after infection (5.9 vs 3.6 μM; P < .001). Conclusions: The plasma Kyn/Trp ratio and retinol represents a promising metabolic signature that could advance TB diagnostics.

Background: Human migration significantly contributes to the global spread of infectious diseases. In recent years, Ethiopia has experienced an increased influx of returnees from the Kingdom of Saudi Arabia. These migrants were often held in densely populated and inadequately ventilated detention centers and faced a heightened risk of tuberculosis transmission. This study investigates the genotypic drug resistance and transmission clusters of M. tuberculosis among returnees from various detention centers in the Kingdom of Saudi Arabia. Whole genome sequencing (WGS) is employed to characterize the genotypic drug resistance patterns and transmission clusters of M. tuberculosis isolates in presumptive TB cases. Methodology: Following symptom screening for presumptive TB, sputum samples were collected from 161 Xpert MTB/RIF-confirmed returnees between August and December 2022. The samples were further cultured on Lowenstein-Jensen media, with 66.5% (107/161) yielding positive results. The M. tuberculosis strains were classified, and genotypic drug susceptibility was predicted. Transmission clusters were identified using a distance threshold of 12 single nucleotide polymorphisms (SNPs). Results: Among the 88 isolates, Lineage 4 was the most prevalent, representing 65.9% of M. tuberculosis cases among returnees. Sub-lineage 4.2.2.2 was the most dominant within this group, comprising 33% (29/88) of the isolates. Among the isolates, 40 gene mutations conferring resistance to first- and second-line anti-TB drugs were identified. Sixteen transmission clusters were identified, suggesting possible transmission events and a likely origin within the detention centers of the Kingdom of Saudi Arabia (KSA). The proportion of MDR-TB among newly diagnosed cases was 2.3% (2/88). Conclusion: The clustering patterns of M. tuberculosis strain among KSA returnees possibly suggest increased transmission rates in congregate settings. Most importantly, the identified prevalence of MDR-TB, particularly among newly diagnosed TB cases, underscores the need to strengthen robust screening practices for returning migrants before they reintegrate into the community to curb TB/DR-TB transmission.

Aims: Mycobacterium tuberculosis (Mtb) remains a major global health challenge, particularly due to increasing drug resistance. Beyond the well-characterized mutations, the mechanisms involved in driving resistance appear to be more complex. This study investigated the differential gene expression of Ethiopian drug-resistant Mtb sub-lineage 4.2.2.2 clinical isolates through an integrated approach combining phenotypic, transcriptomic, and genomic analyses. Method and results: RNA sequencing was performed by isolating RNA from six Mtb strains (three drug-sensitive and three drug-resistant) during mid-logarithmic phase growth. Drug resistance was assessed through whole-genome analysis and phenotypic testing using the BACTEC Mycobacteria growth indicator tube (MGIT)™ 960 system. RNA profiling revealed significantly reduced expression of six genes: Rv0096, Rv2780, Rv3136, Rv3136A, Rv3137, and Rv3230c in drug-resistant isolates. These genes are not associated with known drug targets nor resistance mechanisms. Additionally, a discrepancy was noted between phenotypic resistance profiles and whole genome-based predictions, with the latter suggesting broader resistance. For instance, the missense mutation in rpoB p.Ser450Leu and katG p.Ser315Thr were identified with no change in phenotypic drug sensitivity to rifampicin and isoniazid, respectively. Conclusion: Identification of these differentially expressed genes and their networks could be useful in unraveling the complexities of Mtb drug resistance and in understanding the impact that drug resistance conferring mutations have on the physiology of drug-resistant Mtb.

No currently licensed vaccine reliably prevents pulmonary tuberculosis (TB), a leading cause of infectious disease mortality. Developing effective new vaccines will require identifying which of the roughly 4000 proteins in the Mycobacterium tuberculosis ( Mtb ) proteome are presented on MHC class II (MHC-II) by infected human phagocytes and can be recognized by CD4+ T cells to mediate protective immunity. Vaccines must also elicit T cell responses recognizing the same peptide-MHC complexes presented by infected cells, and successful presentation of target human MHC-II peptides is currently challenging to evaluate and optimize. Here, we define antigenic targets for TB vaccine development by using mass spectrometry (MS) for proteome-wide discovery of Mtb epitopes presented on MHC-II by infected human cells. We next iteratively design and evaluate candidate mRNA vaccine immunogens, revealing design principles that enhance presentation of target MHC-II peptides. Our results will inform the development of new TB vaccine candidates.

Background. Prevalence surveys in Ethiopia indicate smear negative pulmonary tuberculosis (SNPTB) taking the major share of the overall TB burden. It has also been a diagnostic dilemma worldwide leading to diagnostic delays and difficulty in monitoring treatment outcomes. This study determines and compares the clinical and imaging findings in SNPTB and smear positive PTB (SPPTB). Methodology. A case-control study was conducted on 313 PTB (173 SNPTB) patients. Data and sputum samples were collected from consented patients. Smear microscopy, GeneXpert, and culture analyses were performed on sputum samples. Data were analyzed using Stata version 17; a value < 0.05 was considered statistically significant. Results. Of the 173 SNPTB patients, 42% were culture positive with discordances between test results reported by health facilities and Armauer Hansen Research Institute laboratory using concentrated smear microscopy. A previous history of TB and fewer cavitary lesions were significantly associated with SNPTB. Conclusions. Though overall clinical presentations of SNPTB patients resemble those seen in SPPTB patients, a prior history of TB was strongly associated with SNPTB. Subject to further investigations, the relatively higher discrepancies seen in TB diagnoses reflect the posed diagnostic challenges in SNPTB patients, as a higher proportion of these patients are also seen in Ethiopia.

Background Detection delay is defined as the period between onset of first signs and symptoms of leprosy and the time of diagnosis, comprising of a ‘patient delay’ and a ‘health-system delay’ and reliability refers to the consistency of a measure. Three types of consistency are considered: over time (test-retest reliability), across items (internal consistency), and across different researchers (interrater reliability). For the case detection delay (CDD) two are applicable: test-retest reliability and interrater reliability. Methods The study was conducted in Ethiopia, Mozambique and Tanzania. The CDD questionnaire was administered to 79 leprosy patients. One month later, another researcher re-administered the CDD questionnaire with these same patients. Interrater reliability was assessed using the intra class correlation coefficient (ICC). The test-retest reliability was assessed among 69 leprosy patients by determining the CDD at one month difference, both times using the same rater, and then we determined the reliability by looking at the Pearson correlation between the two sets of CDD data. Results Results from 79 leprosy patients show that, 3 (3.8%) were children under 15 years of age and 25 (31.6%) were women. Interrater reliability: the first interviews led to a mean CDD of 24.0 months (95% CI =17.1 - 30.9). The second interviews, also led to a mean CDD of 24.0 months (95% CI =17.7 - 30.3). For the test-retest reliability, the mean CDD of the first and second interviews were 16.5 (95% CI =13.6 - 19.5) and 16.9 (95% CI =13.8 - 20.1) respectively: the interrater reliability measured with ICC was 0.89 (95% CI of 0.84 - 0.93). Test-retest repeatability coefficient was 0.90 (p = 0.01). Conclusion The PEP4LEP CDD tool to determine the case detection delay of newly diagnosed leprosy patients, was validated in the three African countries showing that both test-retest and interrater reliability measurements demonstrated good reliability of the instrument.