Rotavirus infection among children and calves: Genotype diversity, zoonotic potential and evaluation of EpiTuub® fecal rotavirus antigen rapid test kit in Amhara National Regional State, Ethiopia

Abstract

Background: Rotavirus is the leading cause of severe gastroenteritis in children and neonatal calves worldwide. It is estimated that more than 80% of all rotavirus-related deaths among children occur in resource-limited countries in South Asia and sub-Saharan Africa. Though four groups (A, B, C and H) are known to cause acute gastroenteritis in humans, nearly all the commonly detected rotavirus that affect human beings belong to rotavirus A (RVA). In Ethiopia little is known about the epidemiology of rotavirus in human and cattle population. Moreover, there are no routine diagnostic platforms for rotavirus infection in Ethiopian healthcare settings. Therefore, this study aimed to assessing the magnitude, genotype diversity, zoonotic potential, and evaluation of the diagnostic performance of EpiTuub® Fecal Rotavirus Antigen rapid test kit among under-five children with acute gastroenteritis and calves in Amhara National Regional State, Ethiopia. Methods: A multi-center hospital and farm based cross-sectional study design was employed in Amhara National Regional State, Ethiopia from February 2021 to December 2022. Socio demographic and clinical data were collected using a structured questionnaire by trained nurses. Stool samples and fecal samples were collected from under-five children and calves with acute gastroenteritis, respectively. Samples were stored in a -80 oC freezer until the laboratory work was done. Viral RNA extraction was done using viral RNA extraction kit. The samples were tested using one-step RT-PCR and EpiTuub® Fecal Rotavirus Antigen Rapid Test Kit in parallel. Sanger sequencing technology was used for sequencing VP7 and VP4 genes of RVA. Data were entered and analyzed by SPSS version 29 software. Descriptive analysis was employed to determine the frequencies while Pearson’s Chi-squared test was employed to investigate associations between outcome and explanatory variables. The sensitivity, specificity, and positive and negative predictive values of the EpiTuub® Fecal Rotavirus Antigen Rapid Test Kit were determined using one step RT-PCR as a gold standard. Moreover, the agreement of the rapid test kit with one step RT-PCR was determined by kappa statistics and receiver operators’ curve (ROC) analysis was done to assess the overall diagnostic accuracy of the rapid test kits. Phylogenetic analysis with 1000 bootstrap replicates was performed using Molecular Evolutionary Genetics Analysis (MEGA) version 11 software. Protein modeling and comparative analysis of antigenic epitopes of VP7 and VP4 sequences were used to evaluate the presence of antigenic mismatches between circulating and vaccine virus strains. Statistical significance was set at p < 0.05 for all analyses. Results: The prevalence of RVA infection among diarrheic children was 94/537 (17.5%, 95% CI = 14.3–21%). The most prevalent G-types identified were G3 (37%), G12 (28%), and G1 (20%). The predominant P-type was P[8] (51%); followed by P[6] (29%) and P[4] (14%). The three major G/P combinations observed were G3P[8] (32.8%), G12P[6] (28.4%), and G1P[8] (19.4%). Phylogenetic analysis revealed the clustering of Ethiopian strains with the globally reported strains. Many strains exhibited amino acid differences in the VP4 (VP8* domain) and VP7 proteins compared to vaccine strains, potentially affecting virus neutralization (Paper I). The prevalence of RVA among calves was 41/266 (15.4%, 95% CI=11.3%-19.5%) while the herd-level prevalence was 24/94 (25.53%). The circulating G-types in calves were G6, G8, and G10. On the other hand, the circulating P-types were P[1], P[4], P[8], P[11], P[13] and P[14]. In this study, G10 (41%) and P[11] (38%) genotypes were the most dominant G and P-types, respectively. The G10P[11] (37.5%), G6P[14] (18.8%), and G6P[1] (12.5%) were the dominant G/P combinations circulating among calves in the study area. The circulating bovine RVA strains in the present study were closely related to globally reported bovine and human RVA strains (Paper II). The Fecal Rotavirus Antigen Rapid Test Kit has shown a sensitivity of 75.5% and specificity of 98.2% against one-step RT-PCR. The kit was also found to have 89.9% and 95.0% positive and negative predictive values, respectively. The Fecal Rotavirus Antigen Rapid Test Kit has shown a substantial agreement (78.7%, p=0.0001) with one-step RT-PCR. The overall accuracy of the Fecal Rotavirus Antigen Rapid Test Kit was excellent with the area under the ROC curve of 86.9% (95% CI=81.6, 92.1%) (p=.0001) (Paper III). Conclusions: Despite the high RVA vaccination rate, the prevalence of RVA infection remains high among diarrheic children. There is an observable shift in circulating RVA genotypes from G1 to G3, alongside the emergence of unusual G/P genotype combinations such as G9P[4]. Many of these circulating RVA strains have shown amino acid substitutions that may allow for neutralization escape. Rotavirus A infection among calves was high with zoonotic potential. Fecal Rotavirus Antigen Rapid Test kit has a substantial agreement with RT-PCR and can be an alternative diagnostic approach to be used as a point-of-care test in Ethiopian healthcare settings VIII | P a g e IX | P a g e where resources are limited precluding the use of one step RT-PCR for the diagnosis of RVA infection. Furthermore, the kit could be used in the evaluation and monitoring of rotavirus vaccine effectiveness in resource limited settings. Further studies are warranted to comprehend the emergence of these unusual RVA strains and the diverse factors influencing the vaccine's diminished effectiveness in developing countries