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Yosef Gebretensaie,1,* Abay Atnafu,2,* Selfu Girma,2 Yonas Alemu,3 Kassu Desta1,* 1Department of Medical Laboratory Science, Addis Ababa University, Addis Ababa, Ethiopia; 2Mycobacterial Disease Research Directorate, Armauer Hansen Research Institute, Addis Ababa, Ethiopia; 3Department of Microbiology, Immunology, and Parasitology, College of Health Science, Addis Ababa University, Addis Ababa, Ethiopia*These authors contributed equally to this workCorrespondence: Yosef Gebretensaie, Tel +251913063450, Email yoseftakelove@gmail.comBackground: Urinary tract infections (UTIs) brought a significant and serious health-related problem that may lead to the subsequent development of serious indications with the challenge of the emergence of antibiotic resistance. Therefore, the choice of antibiotics depends on the accuracy of the diagnostic tool of UTIs to minimize false results that may subject patients to wrong treatments. This study aimed to determine the prevalence of bacteriuria, associated factors, and AMR pattern of UTI-suspected patients.Methods: A cross-sectional study was conducted from March to May 2022, at Arsho Advanced Medical Laboratory (AAML), Addis Ababa, Ethiopia. Species identification and isolation from bacterial colonies were characterized by gram stain and biochemical properties followed by antibiotic susceptibility testing using the Kirby-Bauer method on Muller-Hinton agar. Logistic regression analysis was carried out to determine the association between the independent variables and significant bacterial growth to identify factors that affect the prevalence of UTI. A test is considered statistically significant that has a P value less than 0.05.Results: Out of 141 (31.6%) which yielded significant bacteriuria, 16 different species of bacterial uropathogens were identified. A total of 105/446 (91 Gram-negative and 14 Gram-positive) of bacterial growth in the female gender and 36/446 (33 Gram-negative and 3 Gram positive) in male were observed with a P value of 0.03. The most predominant bacteria were E. coli followed by Klebsiella pneumoniae. Amoxicillin had shown the highest resistance rate (100%) followed by Ampicillin (98.9%). Females and participants with previous infections were shown to be associated with significant bacterial growth.Conclusion: Based on our study finding, the ordinarily used antibiotics seem to face emerging resistant strains, which needs considerable and due attention to the impact of UTI in developing countries including Ethiopia. History of previous UTIs and female gender were shown to be possible risk factors associated with UTIs.Keywords: prevalence, uropathogen, urinary tract infection, antimicrobial resistance pattern

Ashenafi Alemu,1 Selfu Girma,1 Solomon H Mariam1,2 1Armauer Hansen Research Institute, Addis Ababa, Ethiopia; 2Infectious Diseases Program, Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, EthiopiaCorrespondence: Solomon H Mariam, Email solomon.hm@aau.edu.etBackground: Infectious diseases caused by pathogenic members of the family Enterobacteriaceae cause mortality and morbidity in humans. These are mediated mainly via toxins or virulence factors in combination with multiple antimicrobial resistance (MAR) against antimicrobials intended to treat infections. Resistance can be transferred to other bacteria, possibly also in association with other resistance determinants and/or virulence properties. Food-borne bacterial infections are one of the major causes of infections in humans. The level of scientific information about foodborne bacterial infections in Ethiopia is very limited at best.Methods: Bacteria were isolated from commercial dairy foods. These were cultured in appropriate media for identification at the family level (Enterobacteriaceae) based on Gram-negative, catalase-positive, oxidase-negative, and urease-negative phenotypes, followed by testing for the presence of virulence factors and resistance determinants to various antimicrobial classes using phenotypic and molecular tests.Results: Twenty Gram-negative bacteria isolated from the foods were found to be resistant to almost all antimicrobials belonging to the phenicol, aminoglycoside, fluoroquinolone, monobactam, and β-lactam classes. All of them were multiple-drug-resistant. The resistance to the β-lactams was due to the production of β-lactamases and were also mostly resistant to some of the β-lactam/β-lactamase inhibitor combinations. Some isolates also contained toxins.Conclusion: This small-scale study demonstrated the presence, in the isolates, of high levels of virulence factors and resistance to major antimicrobials that are in clinical use. Most treatment being empirical, there can be not only a high degree of treatment failure but also the likelihood for further development and dissemination of antimicrobial resistance. Since dairy foods are animal products, there is an urgent need to control animal-food-human transmission mechanisms, restrict antimicrobial use in animal agriculture, and improve clinical treatment from the usual empirical treatment to more targeted and effective treatment.Keywords: dairy isolates, gram-negative bacteria, multiple antimicrobial resistance, virulence, β-lactamase inhibitor

Mycobacterium leprae is a slow-growing species of mycobacteria that cannot be cultured in axenic media. This presents a number of challenges for monitoring treatment efficacy and advancing new drugs and regimens for treating leprosy. We previously developed a molecular viability assay (MVA) which measures expression of hsp18 and esxA transcripts to determine viability of M. leprae directly from infected tissue. The objective of the current study was to determine the utility of the MVA for practical use on clinical specimens. Leprosy cases from the Philippines (N = 199), Ethiopia (N = 40), and Nepal (N = 200) were diagnosed by clinical examination, slit-skin smears (SSS) from index sites, and/or histopathology. Biopsy specimens for MVA were collected from an active lesion and stored in 70% ethanol. DNA and RNA were extracted from the tissue, and M. leprae were enumerated on the DNA fraction via RLEP qPCR. Based on this count, DNased RNA was normalized to the equivalent of 3x103M. leprae per reverse transcription reaction, and hsp18 and esxA transcripts were amplified by PCR on the resulting cDNA. There was a strong correlation between RLEP enumeration on the specific biopsy specimen for MVA and the average SSS bacterial index (BI) in all three cohorts (p < 0.001). The MVA could be performed on most biopsies with an average SSS BI ≥ 2 and showed a decrease in M. leprae viability with increasing duration of leprosy multidrug therapy (R2 = 0.81, p < 0.001). The MVA also detected viable M. leprae in relapse patients where it showed significant correlation with the mouse footpad assay (p = 0.018). The MVA is a M. leprae-specific, sensitive, and relatively quick test. Clinically, the MVA would likely be most useful to monitor treatment, confirm suspected relapse cases, and determine efficacy of new leprosy drugs in clinical trials.

Sosina Ayalew,1,2 Teklu Wegayehu,2 Hawult Taye,1 Liya Wassie,1 Selfu Girma,1 Stefan Berg,3 Adane Mihret1 1Armauer Hansen Research Institute, Addis Ababa, Ethiopia; 2Department of Biology, College of Natural Sciences, Arba Minch University (AMU), Arba Minch, Ethiopia; 3Bacteriology Department, Animal and Plant Health Agency, Weybridge, UKCorrespondence: Sosina Ayalew Tel +251 912166324Email absosina2011@gmail.comBackground: Tuberculosis lymphadenitis (TBLN) is a growing public health concern in Ethiopia. However, there is limited information available on gene mutations conferring drug resistance and genetic diversity of M. tuberculosis isolates from TBLN patients.Methods: Drug resistance and genetic diversity analysis were done on 91 M. tuberculosis isolates from culture positive TBLN patients collected between 2016 and 2017. Detection of mutations conferring resistance was carried out using GenoType MTBDRplus VER 2.0. Thereafter, isolates were typed using spoligotyping.Results: Out of the 91 strains, mutations conferring resistance to rifampicin (RIF) and isoniazid (INH) were observed in two (2.2%) and six (6.6%) isolates, respectively. The two RIF resistant isolates displayed a mutation at codon 531 in the rpoB gene with amino acid change of S531L. Among the six INH resistant strains, four isolates had shown mutation at the KatG gene at codon 315 with amino acid change of S315T, one isolate had a mutation at the inhA gene at codon 15 with amino acid change of C15T and one isolate had a mutation at the inhA gene with unknown amino acid change. All drug resistant isolates were from treatment naive TBLN patients. The dominantly identified Spoligo International Types (SITs) were SIT25, SIT149, and SIT53, respectively; these accounted for 43% of the total number of strains. The isolates were grouped into four main lineages; Lineage 1 (2, 2.2%), Lineage 3 (38, 41.7%), Lineage 4 (49, 53.8%) and Lineage 7 (2, 2.2%). Four out of six (66.7%) isolates with drug resistance conferring mutations belonged to clustered strains (strains with shared SIT).Conclusion: The detection of drug resistant conferring mutation in treatment naïve TBLN patients together with detection of drug resistant isolates among clustered strains might suggest resistant strains’ transmission in the community. This needs to be carefully considered to prevent the spread of drug resistant clones in the country.Keywords: drug resistant, genetic diversity, mutation, tuberculosis lymphadenitis

Background Tuberculosis lymphadenitis (TBLN) is a growing public health concern in Ethiopia. However, there is limited information available on gene mutations conferring drug resistance and genetic diversity of M. tuberculosis isolates from TBLN patients. Methods Drug resistance and genetic diversity analysis were done on 91 M. tuberculosis isolates from culture positive TBLN patients collected between 2016 and 2017. Detection of mutations conferring resistance was carried out using GenoType MTBDRplus VER 2.0. Thereafter, isolates were typed using spoligotyping. Results Out of the 91 strains, mutations conferring resistance to rifampicin (RIF) and isoniazid (INH) were observed in two (2.2%) and six (6.6%) isolates, respectively. The two RIF resistant isolates displayed a mutation at codon 531 in the rpoB gene with amino acid change of S531L. Among the six INH resistant strains, four isolates had shown mutation at the KatG gene at codon 315 with amino acid change of S315T, one isolate had a mutation at the inhA gene at codon 15 with amino acid change of C15T and one isolate had a mutation at the inhA gene with unknown amino acid change. All drug resistant isolates were from treatment naive TBLN patients. The dominantly identified Spoligo International Types (SITs) were SIT25, SIT149, and SIT53, respectively; these accounted for 43% of the total number of strains. The isolates were grouped into four main lineages; Lineage 1 (2, 2.2%), Lineage 3 (38, 41.7%), Lineage 4 (49, 53.8%) and Lineage 7 (2, 2.2%). Four out of six (66.7%) isolates with drug resistance conferring mutations belonged to clustered strains (strains with shared SIT). Conclusion The detection of drug resistant conferring mutation in treatment naïve TBLN patients together with detection of drug resistant isolates among clustered strains might suggest resistant strains' transmission in the community. This needs to be carefully considered to prevent the spread of drug resistant clones in the country.

Background The comparatively straightforward and cheaper light-emitting diode fluorescent microscope (LEDFM) was suggested by WHO to replace conventional microscope in tuberculosis (TB) laboratories. However, the comparable efficacy of each of those techniques differs from laboratory to laboratory. We investigated the efficacy of LEDFM for the diagnosis of tuberculous lymphadenitis (TBLN) patients. Methods A cross-sectional study was conducted on 211 samples from clinically suspected tuberculous lymphadenitis patients. Three smears were prepared from FNA on microscope slides for cytomorphology study, Auramine O (AO), and for Ziehl-Neelsen (ZN) staining. The left-over samples were inoculated onto Lowenstein-Jensen (LJ) media. Statistical analysis was done using STATA version 11. The sensitivity, specificity, positive and negative predictive values were calculated by considering the culture results as the gold standard using a 95% confidence interval. Results Among 211 samples 49.7% (105) were positive by cytomorphology, 32.7% (69) by LEDFM, 23.69% (50) by LJ culture, and 13.7% (29) by ZN. Compared to the gold standard sensitivity of ZN, LEDFM, and cytomorphology were 30% [95% CI: 17.9–44.6], 66% [95% CI: 51.2–78.8] 78% [95% CI: 64–88.5], respectively. The specificity of ZN, LEDFM, and cytomorphology was 91.3% [95% CI: 85.8–95.2], 77.6% [95% CI: 70.4–83.8], 58.8% [95% CI: 50.7–66.5], respectively. Conclusion LED fluorescence microscopy gives a legitimate option in contrast to conventional ZN techniques in terms of its higher sensitivity, a bit lower specificity, time-saving, and minimal effort.

Recent studies suggest an essential role of the innate immune effector cells neutrophils and monocytes in protection or disease progression in the early course of Leishmania infection. In areas endemic for cutaneous leishmaniasis in Ethiopia most individuals are exposed to bites of infected sandflies. Still only a minor ratio of the inhabitants develops symptomatic disease. Neutrophils, followed by monocytes, are the first cells to be recruited to the site of Leishmania infection, the initial response of neutrophils to parasites appears to be crucial for the protective response and disease outcome. Our working hypothesis is that neutrophils and/or monocytes in localized cutaneous leishmaniasis (LCL) patients may have defects in function of innate immune cell that contribute to failure to parasite clearance that lead to establishment of infection. The response of cells in Ethiopian LCL patients and healthy controls to Leishmania aethiopica and to the Toll like receptor (TLR) agonists lipopolysaccharide (LPS) and macrophage activating lipopeptide-2 (MALP-2) was investigated by assessing the cell surface expression of CD62L (on neutrophil and monocyte) and CD66b (only on neutrophil), as well as reactive oxygen species (ROS) production by using whole blood-based assays in vitro. No impaired response of neutrophils and monocytes to the microbial constituents LPS and MALP-2 was observed. Neutrophils and monocytes from LCL patients responded stronger to Leishmania aethiopica in the applied whole blood assays than cells from healthy individuals. These experimental findings do not support the hypothesis regarding a possible dysfunction of neutrophils and monocytes in cutaneous leishmaniasis. On the contrary, these cells react stronger in LCL patients as compared to healthy controls. The differential response to L. aethiopica observed between LCL patients and healthy controls have the potential to serve as biomarker to develop FACS based diagnostic/ prognostic techniques for LCL.

The clinical course and outcome of cutaneous leishmaniasis (CL) vary due to the infecting Leishmania species and host genetic makeup that result in different immune responses against the parasites. The host immune response to Leishmania aethiopica (L.aethiopica), the causative agent of CL in Ethiopia, is poorly understood. To contribute to the understanding of the protective immune response in CL due to L.aethiopica, we characterized the cytokine response to L. aethiopica in patients with the localized form of CL (LCL) and age-and sex-matched apparently healthy controls. By applying a whole blood based in vitro culture we found enhanced release of TNF, IL-6, MCP-1 or CCL2, IP-10 or CXCL10, MIP-1β or CCL4 and IL-8 or CXCL8- but not of IL-10CL patients in response to L. aethiopica compared to the controls. No difference was observed between LCL cases and controls in the secretion of these cytokines and chemokines in whole blood cultures treated with the TLR-ligands LPS, MALP-2 or polyI: C. The observed increased secretion of the pro-inflammatory cytokines/chemokines reflects an enhanced response against the parasites by LCL patients as compared to healthy controls rather than a generally enhanced ability of blood leukocytes from LCL patients to respond to microbial constituents. Our findings suggest that the enhanced production of pro-inflammatory cytokines/chemokines is associated with localized cutaneous leishmaniasis caused by L.aethiopica.

Background, Diagnosis of leprosy mainly relies on clinical examination due to the inconsistent sensitivity and poor reproducibility of the current laboratory tests. Utilisation of alternative methods to the standard Ziehl Neelsen (ZN), Fite-Faraco (FF) and Haematoxylin and Eosin (H&E) staining procedures may eventually improve leprosy diagnosis., Methodology/Principal findings, In this comparative study, the performance of the fluorescent Auramine O (AO) staining and polymerase chain reaction (PCR) was assessed with different skin samples using a combination of ZN, FF and H&E staining as the gold standard. AO, ZN, FF, H&E and PCR tests were performed on slit skin smears (SSS) and/or punch biopsies collected from 141 clinically confirmed leprosy cases and 28 non-leprosy skin samples. DNA was extracted from punch biopsies using two different methods with or without mechanical lysis., Sensitivities were 87.6%, 59.3% and 77% for H&E, ZN and FF, respectively, whereas it reached 65.5% and 77.9% for AO in SSS and tissue sections and 91.1% for PCR in tissue samples. Morover, samples with low bacillary index, sensitivity of AO staining (61.8%) was similar to FF (60%, p>0.05) and lower than PCR (86.6%, p, Conclusions/Significance, Our results showed that for diagnostic purposes, analysis of skin section is more sensitive than SSS, especially for samples with low bacillary load. AO staining on SSS and tissue sections was not significantly better than other routine diagnostic tests but considerably more user friendly. The sensitivity of PCR was higher than current standard methods and increased when combined with more efficient DNA extraction using mechanical and chemical lysis. Therefore, we recommend AO staining for the diagnosis of leprosy in lower health facilities such as health centres and district hospitals and PCR diagnosis at referral level and research centres.

Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients’ skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance., Leprosy is caused by the yet-uncultured pathogen Mycobacterium leprae. Here, Benjak et al. obtain M. leprae genome sequences from DNA extracted from patients' skin biopsies and, by analysing 154 genomes from 25 countries, provide insight into the pathogen’s evolution and antimicrobial resistance.

Background Leprosy reactions are a significant cause of morbidity in leprosy population. Erythema nodosum leprosum (ENL) is an immunological complication affecting approximately 50% of patients with lepromatous leprosy (LL) and 10% of borderline lepromatous (BL) leprosy. ENL is associated with clinical features such as skin lesions, neuritis, arthritis, dactylitis, eye inflammation, osteitis, orchitis, lymphadenitis and nephritis. ENL is treated mainly with corticosteroids and corticosteroids are often required for extended periods of time which may lead to serious adverse effects. High mortality rate and increased morbidity associated with corticosteroid treatment of ENL has been reported. For improved and evidence-based treatment of ENL, documenting the systems affected by ENL is important. We report here the clinical features of ENL in a cohort of patients with acute ENL who were recruited for a clinico-pathological study before and after prednisolone treatment. Materials and methods A case–control study was performed at ALERT hospital, Ethiopia. Forty-six LL patients with ENL and 31 non-reactional LL matched controls were enrolled to the study and followed for 28 weeks. Clinical features were systematically documented at three visits (before, during and after predinsolone treatment of ENL cases) using a specifically designed form. Skin biopsy samples were obtained from each patient before and after treatment and used for histopathological investigations to supplement the clinical data. Results Pain was the most common symptom reported (98%) by patients with ENL. Eighty percent of them had reported skin pain and more than 70% had nerve and joint pain at enrolment. About 40% of the patients developed chronic ENL. Most individuals 95.7% had nodular skin lesions. Over half of patients with ENL had old nerve function impairment (NFI) while 13% had new NFI at enrolment. Facial and limb oedema were present in 60% patients. Regarding pathological findings before treatment, dermal neutrophilic infiltration was noted in 58.8% of patients with ENL compared to 14.3% in LL controls. Only 14.7% patients with ENL had evidence of vasculitis at enrolment. Conclusion In our study, painful nodular skin lesions were present in all ENL patients. Only 58% patients had dermal polymorphonuclear cell infiltration showing that not all clinically confirmed ENL cases have neutrophilic infiltration in lesions. Very few patients had histological evidence of vasculitis. Many patients developed chronic ENL and these patients require inpatient corticosteroid treatment for extended periods which challenges the health service facility in resource poor settings, as well as the patient’s quality of life., Author summary Leprosy reactions (Type 1 and 2) are important causes of nerve damage and illness. Erythema Nodosum Leprosum (ENL) also called type 2 reactions is a severe multisystem immune-mediated complication of borderline and lepromatous leprosy. ENL causes high morbidity and mortality and usually requires urgent medical attention. ENL can occur before, during, or after completion of MDT. The diagnosis and treatment of ENL is largely based on clinical symptoms. However, the clinical symptoms are heterogeneous and may vary from patient to patient. Although thalidomide is an effective drug for ENL treatment, it is not available in many leprosy endemic countries including Ethiopia. In spite of its adverse effects, in many endemic countries corticosteroid is the only available drug for ENL treatment, usually being used for prolonged periods. Therefore, alternative and effective drugs are required to reduce the burden of ENL. To establish which drugs will be effective in the treatment of ENL it is necessary to have a clear picture of the clinical and histological features of the disease. We systematically documented these features of ENL and compared them with matched non-reactional LL controls. Thus, the findings will help to develop better ENL diagnosis and treatment options.