Molecular epidemiology of norovirus infections and associated factors among patients with diarrheal diseases in the Amhara National Regional State, Ethiopia

Abstract

Background: Diarrheal disease is the second leading cause of morbidity, next to pneumonia, and the 10th leading cause of mortality globally, with an increasing burden in developing countries, including Ethiopia. Viruses are one of the most important etiologies of diarrhea and noroviruses (NoVs) are the leading of all. Globally, they cause gastroenteritis and diarrhea among all age groups, in both epidemic and sporadic cases. Noroviruses are genetically diverse groups with increased variability in different settings. Moreover, these viruses do not affect everyone equally due to different viral, sociodemographic and host factors. Understanding the magnitude and host susceptibility to NoV infections is important for controlling the infection. However, findings conducted on the molecular epidemiology of NoV infection, especially the association between NoV infection and blood groups, are lacking, and the available studies are inconsistent. Objectives: This project aimed to assess the prevalence, associted factors, genetic diversity and ABO blood association of norovirus infections among patients with diarrheal disease in selected health faclities in the Amara national regiolal state, Ethiopia. Methods: This multicenter health facility-based cross-sectional study was conducted from May 2021 to November 2021. The study was conducted in four areas, namely Bahir Dar, Gondar, Debre Tabor, and Debre Markos, each with three health facilities. The study participants were patients with gastroenteritis who demonstrated at least diarrhea. A total of 550 study participants of all age groups were included and proportionally distributed to each area and health facility based on the flow of diarrheal cases from the past six months of the previous year. A systematic random sampling technique was used to select study participants. Sociodemographic data and other factors were collected using pre-tested questionnaire. Fecal samples were collected from each study participant for molecular detection and characterization. Nuclease-free water was used to prepare a 10% stool suspension and loaded into an automatic extraction tool to extract RNA. Real-time reverse transcription PCR (rRTPCR) was employed for molecular detection and a set of primer pairs and probes that target the viral protein one (VP1)-encoding gene was used. We selected all rRT-PCR positive fecal samples for further genomic sequence. Conventional reverse transcription PCR was used to amplify the partial VP1 region and run in to the gel. Finally, the purified DNA amplicons were sequenced using Sanger sequence. Phylogenetic analysis was performed using MEGA7 software. In addition, blood samples were collected and analyzed on-site to assess the ABO blood groups using the hemagglutination technique. Data were entered and analyzed using SPSS version 23 software. A descriptive analysis was performed. Multiple logistic regression analyses were done to assess the associations between the dependent and independent variables. Variables with a P value < 0.05 at an adjusted odds ratio (AOR) in a 95% confidence interval (CI) did not cross one were considered statistically significant. Results: Five hundred nineteen out of 550 samples were analyzed (94.4% response rate). 46 out of 519 were positive for NoV, 8.9% (95% CI: 6.6–11.6). Norovirus infection was higher among the elderly (33.3%) and under-5 children (12.5%). Norovirus infection was more prevalent among patients from Debre Tabor (AOR = 3.8, 95% CI: 1.1–13) and Bahir Dar areas (AOR = 3.5, 95% CI: 1.03–12). Elderly (AOR = 7, 95% CI: 1.9–27) and under-5 children (AOR = 3.4, 95% CI: 2.7–13) were also more affected than adults. Norovirus infections were significantly higher among individuals with a history of diarrhea (AOR = 3.4, 95% CI: 1.4–8), family contact history (AOR = 5.3, 95% CI: 1.9–14.7), high frequency of diarrhea (AOR = 16.3, 95% CI: 6.6– 40), vomiting (AOR = 3.2, 95% CI: 1.5–7), and acute diarrhea (AOR = 13, 95% CI: 5–32.5), but participants with good hand washing practice were less likely infected (AOR = 0.7; 95% CI: 0.50.85). Both genogroup I (GI) and genogroup II (GII) were detected, with the predominance of NoV-GII (38/46; 82.6%). The sequence analysis showed that five distinct GII genotypes (GII.3, GII.6, GII.10, GII.17, and GII.21) and two GI genotypes (GI.3 and GI.5) were detected. Among all of the identified NoV genotypes, GII.3 was the predominant (13/29; 44.8%), followed by GII.21 (6/29; 20.7%), GII.17 (4/29; 13.8%), and GI.5 (3/29; 10.3%). Norovirus GII.21 was reported for the first time in Ethiopia. The GII.3 genotype was detected across all age groups, whereas GII.17 was common among the elderly. The genetic diversity and distribution of NoVs were significantly different across the study sites and age groups (P < 0.05). The phylogenetic analysis revealed a close relationship between the current strains and most strains reported from Ethiopia and elsewhere. Nearly half (249/519; 48%) of the study participants had blood group O. Of all 46 NoV-positive individuals, the majority (34/46; 74%) had blood group O, followed by blood group A (9/46; 19.6%). The risk of NoV infection was higher among patients with blood group O than with blood group B (AOR = 1.5, 95% CI = 2–15, P = 0.01), but there was no association for A and AB blood groups. At least one NoV-GII was identified in each of the blood groups, whereas NoV-GI affected individuals with blood groups O and A. Moreover, the GII.3 and GII.21 genotypes were common among blood group O individuals, whereas most (3/4; 75%) GII. 17 infections were found among blood group A individuals. Conclusions and recommendations: The prevalence of NoV was considerably high, with a predominance of NoV-GII. The positivity rate was higher among the extreme age groups and varied across the study areas. The genetic diversity of NoV was also considerably high as compared to the previous studies. The first-ever identification of the GII.21 genotype and predominance of GII.3 are the new addition to the growing evidence of the genetic diversity of NoVs in Ethiopia. In this study, the positivity rate of NoV was significantly increased among blood group O individuals. Norovirus-GII infected all blood groups, whereas NoV-GI selectively affected groups A and O. Hence, to obtain a comprehensive understanding of the virus`s epidemiology, a nationwide study is warranted. Moreover, to determine the relevance of our observation regarding the association between NoV infection and other host factors like the secrator antigens, future cohort studies are needed. In addition to this, all inclusive health education need to be given especially on the specific transmission modalities as well as on the prevention and control aspects of the virus.