Molecular detection of Enteropathogens from diarrheic stool of HIV positive patients in Gondar, Ethiopia

Seid et al. BMC Infectious Diseases (2018) 18:354 https://doi.org/10.1186/s12879-018-3265-8 RESEARCH ARTICLE Open Access Molecular detection of Enteropathogens from diarrheic stool of HIV positive patients in Gondar, Ethiopia Lubaba Seid1, William Stokes2, Abebe Genetu Bayih1, Sisay Getie1, Aberham Abere1, Habtie Tesfa1 and Dylan R. Pillai1,2,3,4* Abstract Background: Infectious diarrhea is a common problem in the developing world, especially among people living with HIV/AIDS. Traditional diagnostic methods such as stool culture and microscopic examination are limited by resources and poor sensitivity. The use of molecular diagnostics for enteropathogen detection in this region of sub-Saharan Africa has not been fully explored. We sought to identify risk factors and characterize enteropathogens from diarrheic stools of HIV-positive patients in Gondar, Ethiopia using multiplex molecular panels targeting key infectious agents. Methods: A cross-sectional study of 100 stool samples was performed. Samples were collected consecutively from HIV- positive patients presenting with diarrhea at University of Gondar Hospital clinic, a major center in NW Ethiopia. Genomic DNA was extracted from stool and processed using a multiplex molecular panel Allplex™ [Seegene, Canada]. Correlations between patient characteristics, symptoms, public health risk factors, and enteropathogen type (s) were studied. Eighty-six samples were successfully analyzed by molecular methods. Results: The mean age was 35 with 43% male. Eighty percent lived in an urban area, 18% had access to well water only, and 81% practiced proper hand hygiene. The majority of patients (72%) were receiving HAART with a median CD4 cell count of 362/μL. Multiple pathogens were detected in 94% of specimens, with an average of 5 enteropathogens per sample. Common bacteria, viruses, and parasites detected were Shigella spp./enteroinvasive E. coli (80%), enterotoxigenic E. coli (73%), Norovirus (16%) and B. hominis (62%). CD4 cell count < 500/ μL was associated with the presence of viruses (p = 0.004) and the absence of STEC (p = 0.010). The use of HAART or CD4 levels was not associated with the number of enteropathogens detected. Conclusions: Diarrheic stool from HIV-positive outpatients in Gondar, Ethiopia had on average 5 enteropathogens present in their stool. Shigellaspp./enteroinvasive E. coli and enterotoxigenic E. coli are the major pathogens, not dissimilar to immunocompetent individuals in low income countries. Keywords: HIV, Enteropathogens, Molecular, Diarrhea, Diagnostics * Correspondence: 1 Department of Medical Parasitology; School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia 2 Department of Medicine, Cumming School of Medicine, University of Calgary, Calgary, Canada Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Seid et al. BMC Infectious Diseases (2018) 18:354 Background Diarrheal illness contributes to significant mortality and morbidity within the HIV-positive population [1]. It accounts for 1 in 9 deaths for children worldwide and the death rate increases 11 -fold in children with HIV [2]. Furthermore, diarrhea is associated with reduced quality of life and can cause psychological and social burden on afflicted patients [3, 4]. This is most notable in low-resource settings such as Ethiopia in which HIV/AIDS occurs in 1.5% of adults [5]. The variety of organisms known to cause diarrhea presents an inherent challenge in treatment to the clinician, and, in diagnosis, to the laboratory due to shortage of reagents, laboratory set up and skilled manpower in developing countries. Clinical laboratories currently utilize an array of different methodologies to test for bacterial, parasitic, and viral causes of diarrhea among HIV/AIDS patients, a strategy that suffers from poor sensitivity, potentially long turnaround times, and complicated ordering practices and workflows [6–14]. Additionally, there are limited or no testing methods routinely available for most diarrheagenic Escherichia coli strains [15] and certain enteric viruses. Molecular techniques for enteropathogen detection provides a comprehensive, rapid, and streamlined alternative to conventional methods for the microbiological diagnosis of diarrhea in the laboratory setting. The potential advantages include improved performance parameters, a more extensive menu of pathogens, and a turnaround time as short as 1 h [15]. Diagnostic yields in terms of pathogens detected are also approximately 30% higher and multiple enteropathogens are more commonly detected per sample, occurring in up to 35% of positive samples [16]. Prevalence of enteropathogens is regionally dependent, with stark differences between developed and developing countries. A study conducted in the United States on 1556 diarrheal stools using the Biofire FilmArray® platform detected a wide variety of enteropathogens in approximately half (832) of the stool samples collected. In the study, EPEC (41.8%), C. difficile (24.5%), EAEC (13.1%) and norovirus GI/GII (8.4%) were the most prevalent organisms detected in positive samples and 73% had multiple enteropathogens [15]. In contrast, a study on 91 diarrheal stool of symptomatic Peruvians demonstrated higher prevalence of diarrheagenic E. coli strains and mixed enteropathogens. The most common organisms detected in their study was Shigella spp./ enteroinvasive E. coli (EIEC) at 93%, G. lamblia (70%), enteroaggregative E. coli (EAEC) (60%), and enterotoxigenic E. coli (ETEC) (48%). In addition, 100% of their samples contained mixed enteropathogens [17]. Our study’s aim was to identify epidemiological risk factors and characterize enteropathogens from diarrheic stools Page 2 of 7 of HIV-positive patients in Gondar, Ethiopia using multiplex molecular panels targeting key infectious agents. Methods Patient population Diarrheic stool was collected consecutively from all pediatric and adult patients with HIV who attended an antiretroviral therapy (ART) clinic at University of Gondar Hospital, Ethiopia from January 2016, to May 2016. All patients who had diarrhea, defined as 3 or more loose or watery stools during a 24-h period, were included in the study. Acute and chronic diarrhea was defined as diarrhea lasting less than 2 weeks and greater than 4 w (...truncated)