Browsing by Author "Abegaz Woldaregay"

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    Extended-Spectrum β-Lactamase and Carbapenemase Producing Escherichia Coli O157:H7 among Diarrheic Patients in Shashemene, west Arsi, Ethiopia
    (Addis Ababa University, 2023-06) Ayalneh Shimelis ; Abegaz Woldaregay ; Gebreselassie Solomon ; Teferi Mekonnen; Beshah Biruk
    Background: Escherichia coli (E. coli) O157:H7 has been found in various sources across the globe, and until recently, it was uncommon for this pathogen to produce Extended Spectrum β-Lactamase (ESBL) and Carbapenemase. However, recent reports from different regions have shown that ESBL-producing E. coli O157:H7is becoming more prevalent. In Ethiopia, while there is sufficient knowledge about the epidemiology of E. coli O157:H7 in the country's different food supply chains, there is a lack of information regarding the extent of disease caused by this pathogen and its production of ESBL and Carbapenemase. Objectives: To isolate E. coli O157:H7, determine its antimicrobial susceptibility profiles, characterize its ESBL andCarbapenemase production from stool specimens collected among diarrheic patients and explore the association of E.coli O157:H7 infection with demographic and clinical features of diarrheal patients in Shashemene, west Arsi, Ethiopia. Methods: A total of 423 study participants were included from July 1, 2022, to November 25, 2022, in this prospective healthcare facility-based cross-sectional study among all patients with diarrhea. The bacterial pathogen was isolated and identified by colony characteristics, Gram stain, and standard biochemical tests using API 20E as well as utilization of sorbitol and serotyping by antisera for O157 antigen. Each identified isolate was screened and tested for ESBL and Carbapenemase production phenotypical and further characterized at the molecular level for 2 Carbapenemase (blaNDM, blaKPC) and 3 ESBL coding gens (blaCTX-M, blaTEM, and blaSHV). The data were entered into SPSS version 26.0 software for analysis. Bi-variant and multi-variant analyses Were employed using a logistic regression model for further analysis and were interpreted based on the odds ratio and level of statisticasignificance at a p-value <0.05. Result: Upon laboratory investigation, E. coli O157:H7 strain was found in 38/423 (9%) study participants from this majority of the participants [262 (61.9%)] were males with a 1.6:1 male: female ratio and 81(19.1%) of the participants were less than five years old and 14 (3.3%) of patients were elders aged above 55 years. Living in urban area, having domestic animal and having ≥5 family size were significantly associated with E. coli O157:H7 cases. High antimicrobial resistance was observed on Ampicillin [38, (100%)] followed by Amoxicillin with clavulanic acid [34,(89.5%)]. However, all isolates were sensitive to ciprofloxacin. Twenty-seven (71.1%) and 12 (31.6%) isolates were phenotypically confirmed to be ESBL and carbapenemase producers, respectively. The genotypic testing revealed that the most abundantly found ESBL genes were blaTEM group 15 (79%) followed by blaCTX-M group 12 (63%) and blaSHV group 2(10%). Additionally, from the 12 carbapenemase-positive isolates, 8 (66.6%) were confirmed to have the blaKPC group gene and none of the isolates were positive for blaNDM group. Conclusion: The E. coli O157:H7 isolates from this study exhibited a high level of resistance to some of the antimicrobials tested. The magnitude of ESBL and Carbapenemase production among these isolates was found to be high. High resistance of Ampicillin and Amoxicillin/Clavulanic acid was observed among ESBL, and carbapenemase-producing isolates and Ciprofloxacin was found to be the most effective drug against both ESBL producers and non-producers. blaTEM group gene was the most abundant ESBL coding gene found and blaKPC group gene was the only gene found in our isolates that code for Carbapenamese.
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    Genomic Characterization of HIV-1 Isolates from Ethiopian Patients: Baseline Studies on Antiretroviral Drug Resistance and Sub-type Variations
    (Addis Ababa University, 2011-05) Abegaz Woldaregay ; Mengistu Yohannes
    Background: Several studies were conducted in the past two decades in Ethiopia to understand the genotypic characteristics of prevalent HIV subtypes in the country. However, the majority of the information gathered relied on sequencing of the envelope and a certain portion of the gag gene fragments of the HIV genome. Considering the facts that relying only on sequencing of the env and gag regions has inherent inadequacy in characterizing HIV subtypes, that long time has elapsed since genomic characterization of HIV isolates in Ethiopia, that non-C subtypes have expanded in the neighboring countries, and that a new selective pressure coming from the recently initiated ARV treatment has been introduce into current HIV isolates circulating in the country as a consequence of which new HIV subtypes might be currently circulating in the country, this PhD study was, therefore, undertaken to understand the genomic characteristics of HIV isolates from ARV drug-naïve and drug-experienced persons by sequencing the protease (PR) and reverse transcriptase (RT) genes using standard RT-PCR/PCR amplification and genome sequencing protocols. In addition, two test evaluation studies were carried out: one on use of In house brewed genotyping system, and the other on use of DBS as source of specimen for genotypic HIV drug resistance monitoring. Materials and Methods: Different set of criteria were used to select eligible HIV-infected people to take part in three study groups: recently infected drug-naïve antenatal care (ANC) attendee pregnant women (recent drug-naïve), chronically infected drug-naïve but who were eligible to start ART (chronic drug-naïve), and chronically infected heavily treated patients (chronic drug-experienced). For drug resistance assays, list of mutations from three algorithms were utilized. For evaluations of the performance of In-house genotyping system and DBS as source sample, the commercial genotyping system ViroSeq TM and plasma samples, respectively, were used as standards. Mean nucleotide similarities of paired sequences generated from Inhouse/ViroSeq TM genotyping system and DBS/plasma were determined on pairwise alignment sequences using ClustalW multiple alignment program in BioEdit Version 7.0.9.0 software. For other purposes, FASTA-formatted nucleotide and amino acid sequences were aligned and analyzed using various on-line and off-line software. Descriptive, χ2 test, and Student t test statistical data analyses were used as appropriate, with significant level of 0.05. Results: Among chronically infected participant groups, 74% of drug-naïve and 84% of drug-experienced participants were in the advanced WHO clinical stages III and IV at baseline. Nearly 92% of chronic drug-naïve patients had CD4+ count of <200 cells/µL, and 70% had over 100,000 copies/ml of plasma viral RNA. Mean baseline CD4+ T cell count for chronic drug-experienced patients was ~114 cells/µL. Moreover, mean plasma viral RNA and mean CD4+ T cell counts for these patients at the time of genotyping were 289,128 copies/ml and 238 cells/µL, respectively, after median treatment duration of 42 months. The performance of the In–house genotyping system relative to the performance of the commercial ViroSeq TM genotyping system both in amplifying and genotyping the specimens in this study was 100%. It had also mean concordance value of 98.72% at the nucleotide level. Similarly, the performance of DBS as specimen for drug resistance genotyping relative to the performance of plasma specimens was 100% for both amplification and genotyping. The mean nucleotide concordance rate between paired DBS/plasma sequences was 98.82%. The prevalence of transmitted drug resistance mutations was 4.9% among recent drug-naïve persons. Among chronically infected drug-naïve patients, 22 major resistance mutations were detected: eight against PIs, eight against NRTIs, and 6 against NNRTIs. Virological treatment failure among treatment experienced patients was 31%; 70% of successfully genotyped viremic specimens harbored at least one confirmed major drug resistance mutation from any class. Drug class-wide mutations were 3% against PIs, 62% against NRTIs, 68% against NNRTIs, 61% against any NRTI and NNRTI double class mutations, and 3% against all the three classes of drugs in use. Nearly 37% of D4T- and 20% of AZT containing initial regimens were responsible for selection of at least one NRTI resistance mutations. Of the initial drug regimens, ~27% of treated patients with NVP-containing regimens or ~55% of NVP exposed patients with genotyped viral isolates harbored at least one major NNRTI resistance mutation. Likewise, ~29% of all EFV-treated patients or 96% of EFV-exposed patients with genotyped viral isolates had at least one NNRTI resistance mutation. With regards to subtype distribution among the study participants, ~96% of the sequences tested were of subtype C both at PR and RT genes. Sequences from two individuals (1%) were identified as subtype B, while the remaining 3% were subtyped, at least by one genotyping algorithm, as mosaic forms at the PR and RT genes; including four (2%) BCs, one (0.5%) CRF_02AG (or BG? or ABG?), and one (0.5%) A1D. Analysis of genome diversity among the three groups of study participants showed that while certain non resistance mutations had high prevalence in all the three groups, others showed differential occurrences. Synonymous/non-synonymous substitution analysis and test of Shannon Entropy have also shown genome diversity differences among the three groups with distinct positional patterns, diversity at certain patches in the RT region being most prominent. Discussions and Conclusions: Among chronically infected patients, the high baseline viral RNA load, low CD4+ T cell count, and more persons in clinical stages III and IV during treatment initiation show that treatment was started late after the patients’ virological, immunological and clinical conditions have already been deteriorated. These poor baseline conditions were probably reflected by the high viral load values and low CD4+ T cell counts observed after treatment period of over three years among heavily treated patients. The transmitted drug resistance mutation rate documented in this study was in the WHO’s ‘low prevalence category’. However, more surveillance works of greater scope are required in terms of both geographic coverage and study population in order to understand the current prevalence of transmitted drug resistance. The detection of 22 drug resistance mutations among drug-naïve patients starting ART indicates the need for undertaking resistance testing before initiating therapy whenever settings allow. Among drug experienced patients, the most important drugs which were associated with NRTI and NNRTI resistance mutations were D4T and EFV, respectively. Suboptimal therapy could be the source of resistance mutations against these two drugs. It could also be due to impaired absorption of the two drugs, as the pharmacokinetics of ART drugs have not been investigated under the settings of Ethiopian patients. This therefore calls for the need to conduct therapeutic drug monitoring (TDM) at least in research settings on Ethiopian patients taking the various drugs. A lot of so-called non-resistance mutations occurred at positions known to confer resistance. Since most resistance/non-resistance mutation identification was done based on subtype B genetic background, and since virus variants behave differently under different settings, it would be of great benefit if phenotypic drug resistance profiles of isolates from Ethiopia with these non-resistance mutations are investigated. With regards to subtype distribution, the overwhelming majority of the isolates belonged to subtype C at the pol region investigated in this study, showing that still this subtype has not been replaced by any other in Ethiopia. However, variants with mosaic genomic regions have been detected, showing the possibility of finding subtypes other than C, particularly recombinant forms, if further sequencing is made from genomic region larger than the PR and RT regions. Because of discrepancies between various genotyping algorithms in assigning subtypes, and because none of these mosaic forms had exact similarity with those previously known subtype identity, the mosaic variants identified in this study might be considered as Unique Recombinant Forms (URFs). Worth noting, however, is the population sequencing method used in this study might have masked presence of minority populations both in terms of variants with drug resistance mutations and those with different subtype profiles. In the latter case, use of only the short pol region might have contributed to the ambiguity of subtype identification in the non-C isolates detected in this study.
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    Isolation and Antimicrobial Resistance Determination of Escherichia Coli O157:H7 from Raw Meat in Selected Abattoirs and Butcher Shops, Addis Ababa, Ethiopia.
    (Addis Ababa University, 2021-08) Misker Nahom; Abegaz Woldaregay ; Lakew Matios
    Background: Foodborne illness and death due to foodborne diseases caused by highly dangerous pathogens is common in the world; especially it is widespread in developing nations. In Ethiopia, the consumption of meat as a raw is well practiced among the people, which is a potential cause of foodborne illness. Cattle are considered as reservoir of pathogens like the highly virulent Escherichia coli O157:H7 (E. coli O157:H7). Low standard of meat handling and safety practices, lack of protecting personal hygiene among workers of meat supply chain and lack of awareness about E. coli O157:H7-caused infection are major problems in Ethiopia. Furthermore, there is paucity of information regarding the epidemiology of the pathogen along the beef carcass supply chain at Addis Ababa Abattoir Enterprise (AAAE). Therefore, the purpose of this study was to isolate E. coli O157: H7 and determine the site of contamination of raw beef meat at AAAE and its carcass supply chain of butcher shops in Yeka sub-city, Addis Ababa, Ethiopia Methodology: A total of 210 samples from carcass swab, raw minced beef meat and pooled environmental sources were collected along the supply chain. Collected samples were cultured to isolate E. coli O157: H7 and identified using biochemical tests and Biolog bacterial identification system. Antimicrobial susceptibility tests were performed on E. coli O157: H7 isolates using the Kirby-Bauer disk diffusion method on Muller-Hinton agar. Observation was used to assess the overall hygiene and sanitation of the beef-selling environment, and a questionnaire was used to assess the workers' knowledge, attitude, and practice. Following data entry into an Excel spreadsheet, the data was exported to and analyzed using statistical software (SPSS version 26.0). Descriptive statistics and the Chi-square (X2 ) test were used, and a p-value (p< 0.05) was considered statistically significant. Result: The overall prevalence of E. coli O157:H7 was 2/210 (0.95%) and the two isolates were detected from knife swab sample at slaughter house and sample from minced raw beef meat at butcher shop. In the case of the antimicrobial susceptibility test, both isolates were susceptible to ciprofloxacin, gentamicin, meropenem, chloramphenicol, amoxicillin clavulanate and cefotaxime, but resistant to sulfamethoxazole + trimethoprim, amoxicillin, streptomycin and ampicillin. Conclusion: Overall, although the prevalence of E. coli O157:H7 was low, the results indicated that the contamination detected from raw beef meat from butcher shop and the utensil in the abattoir is a matter of concern that calls for an effort to improve the hygienic status. Hence, to ensure the hygienic quality of meat, everyone involved in carcass slaughter, distribution and serving to the customers in the chain should be trained for sanitary and hygienic practices of these meat supply services including providing basic trainings to the personnel involved in the industry. Key words: AAAE, Antimicrobial susceptibility, Butcher shop, E. coli O157:H7.
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    Sero-Prevanence of Hepatitis-B and C Infection and the knowledge,Attitude and Practice among Healthcare Workers and Traditional Healers at Selected Sites of Kolfe Keranio Sub-city, Addis Ababa,Ethiopia.
    (Addis Ababa University, 2019-06) Negerie Michael ; Abegaz Woldaregay
    Background: Hepatitis B and C viruses are the leading causes for global morbidity and mortality of viral hepatitis. According to the world health organization report, globally an estimated 257 million and 150 million people are living with chronic HBV and HCV respectively. The risk of these infectious blood borne pathogens are highest on the health care settings and the health care workers or people who work in the field of medicine are at highest risk for these infections. In Ethiopia, although reports showed that more than 60% of chronic liver disease and up to 80% of hepato-cellular cancers have occurred due to hepatitis B and C viral infections, still there is limited surveillance data regarding the impacts of these infectious pathogens among the health care workers and the impact of hepatitis was totally neglected among the traditional healers. So far in Ethiopia, very limited studies were conducted to show the prevalence and risk factors of hepatitis among allopathic health care workers. Yet, no studies have been conducted to show the burdens of hepatitis and risk factors among traditional healers. Therefore, further studies should be conducted to show the impact of hepatitis among the healers alongside the aliphatic health care workers. Objective: the aim of this study was to assess the sero-prevalence and associated risk factors of hepatitis B and C among the health care workers and the traditional healers at selected study sites of Kolfe-Keraniyo sub city Addis Ababa Ethiopia. Method: An institution based cross-sectional study was conducted from the study period of November, 2017toJanuary; 2019.Study participants were selected using a multi-stage sampling technique. A multi-item standardized questioner was used to collect data on the demographic information and potential risk factors for hepatitis B and C. Five to ten milliliters of blood was collected from each study participants for sero-prevalence study. The serum from each study participants was screened for hepatitis B surface antigen and anti-hepatitis C antibody by using rapid screening test kits. All positive samples for hepatitis B surface antigen had confirmed for the presence of hepatitis B envelop antigen (HBeAg) and hepatitis B nucleic acid (DNA)by laboratory test method of enzyme linked immunosorbant assay (ELISA).In addition, all hepatitis C positive samples had confirmed for the presence of hepatitis C nucleic acid (HCV-RNA) by enzyme linked immunosorbant assay test method. Finally, collected data were entered into Epi-Data 3.1” software and analyzed by statistical software program SPSS version 20.0 (SPSS, Chicago, IL, USA). Results: in the study period, 248 (95.4% response rate) study participants had administered the questioner properly and gave blood for sero-prevalence study. The overall prevalence rate for of HBV was 2.8 % (CI= 0.7-5.4%) and for HCV was 0.8 % (CI=0.3-1.9%). Most (82.8%) study participants had good knowledge of hepatitis B and C transmission and preventive strategies. However, only 64% of participants had positive risk perception (attitude) and only 43% of the study participants had good hepatitis preventive practice. The overall prevalence rate for occupational was 56% among participants with in the past three years. Exposure for blood, body fluids, needle stick and sharp injuries were 79%, 56%, 27% and 63% respectively. Among all, only 155(62.5%) study participants had a history of anti hepatitis B vaccination and only 65 (25% of the total) was fully vaccinated. Generally, we find the risk factor analysis for hepatitis C was very difficult since its’ sero-prevalence was too small. However, lengths of working time per week (COR= 9.1 p<0.043), non-compliance to safety measures (COR=9.55 P< 0.038), needle stick injuries (COR= 7.22, P<0.02) and absence of HBV vaccination history (COR= 10.62, P< 0.03) were found to be significantly associated with hepatitis B sero-positivity on univariate analysis. Finally, absence of history of vaccination remains the only significant (AOR= 9.2, P<0.02) predictor risk factor for hepatitis B infection after removing cofounding risk factors on multivariate logistic regression analysis. Conclusion and recommendation: despite having good knowledge of hepatitis transmission and preventive measures, hepatitis risk perception (attitude) of the study participants and their hepatitis preventive practices were not good. Frequencies of occupational exposures were high yet the immunization coverage and the infection prevention training coverage were poor. Our study revealed like the allopathic health workers the risk of hepatitis is high. These all shortcoming indicate the need for regular infection prevention trainings, enhanced immunization coverage and inclusive health policy targeting the traditional healers.
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    The Comparison of Xpert® Xpress with Cobas® SARS-CoV-2 Assay on Individual and Pooled Nasophary Neal Swabs and Whole Genome Sequencing of SARS-CoV-2 Samples Collected in Selected Hospitals of Addis Ababa, Ethiopia.
    (Addis Ababa University, 2023-07) Zerihun Betselot; Abegaz Woldaregay ; Gebre Selassie; Kebede Habtamu ; Alemu Ayinalem
    Background: Xpert® Xpress and Cobas® are assays offering rapid test for the detection of SARS-CoV-2. Pooled testing allows for continued testing even when supplies are relatively scarce. Whole genome sequencing of SARS-CoV-2 refers to the process of determining the complete genetic sequence of the virus, providing a detailed and comprehensive analysis of its entire genome. The agreement between assays for SARS-CoV-2 detection from individual and pooled samples is uncertain in Ethiopia. Moreover, there is limited availability of whole genome sequencing data for SARS-CoV-2, leading to insufficient information regarding the circulating variants, lineages, and mutations. Objective: The study aimed to compare the performance of Xpert® Xpress with Cobas® SARS CoV-2 assays for SARS-CoV-2 detection from individual and pooled nasopharyngeal swabs and whole genome sequencing of SARS-CoV-2 to identify lineages, sub-lineages and mutations for nucleocapsid and envelop gene, in Ethiopia. Methods: A cross-sectional study design was utilized on individuals suspected of having COVID-19 in seven selected hospitals in Addis Ababa, Ethiopia, between June 25, and July 20, 2022. The samples were tested for SARS-CoV-2 using the GeneXpert® and Cobas 8800®. A total of fifty samples that tested positive with the Xpert® were chosen and divided into four groups according to their CT-value and then pooled with negative samples and subjected to testing using both assays. Positive samples with a CT-value of ≤30 were selected for sequencing. The CovidSeq Assay was used to prepare sequencing libraries, which were then sequenced on the Illumina Miseq. Data were entered into Epi data version 3.1 and exported to SPSS version 28 for statistical analysis. Analysis was performed by using descriptive statistics, pearson correlation, and cohen’s kappa. Nextflow work flow manager and different bioinformatics tools were used for the analysis of variants, lineages, and mutations. Results: In this study, a total of 440 nasopharyngeal swabs were collected, and the overall rate of positivity for SARS-CoV-2 was found to be 100 (22.73%). The overall agreement between the assays from individual samples was 91.73(95% CI, 89.99% to 92.66 %, κ=0.715) indicating a substantial level of agreement. The Xpert® assay showed a high positive percent agreement (98.25%) and negative percent agreement (90.71%). The overall agreement between the assays from pooled samples was 89%, (95% CI, 86.31% to 91.52% κ=0.552). All 16 sequenced samples were identified as the Omicron variant, with ten samples assigned to clade 22A. The consensus XII sequence length ranged from 29,578 to 29,903, with a GC% ranging from 33.1% to 39.0%. The average length of amino acids in the sequences was 9519. The pairwise identity between the samples and the reference genome ranged from 98.6% to 99.9%. Several amino acid changes were observed in the sequenced samples, including P13L, P151S, N E31del, N R32del, N S33del, N P151S, N R203K, N G204R, and N S413R on the N genes. Likewise, there was one common mutation at position 9 in the E gene, E31del. Conclusion: The Xpert® performed better than the Cobas® assay in detecting positive results due to its ability to detect low viral load cases, utilizing the more sensitive nucleocapsid gene instead of the ORF1a gene used in the Cobas® assay. Confirming presumptive positive results by different methods and using multiple molecular assays targeting different genes is important to ensure high sensitivity in detecting RNA. Pooling up to six samples was possible regardless of viral load and pool size for Xpert®. But viral load effect was significant in all pool size of Cobas® assay. The agreement on individual samples indicated that both assays can be used interchangeably for diagnostic purposes. Furthermore, there were variations in amino acid length, the presence of both unique and existing mutations, and variations in mutation frequencies among the analyzed sequences. Key words: Agreement, Cobas® SARS-CoV-2 assay, COVID-19, Xpert® Xpress SARS-CoV-2 assay, Pooling, SARS-COV-2, WGS