Diversity of enteroviruses detected in sewage samples and stool specimens from Gauteng province, South Africa

Abstract

Enteroviruses (EVs) are a genus of enteric viruses belonging to the family Picornaviridae. Enteroviruses infect humans as well as other mammals causing a wide range of disease symptoms from asymptomatic infection, mild febrile illnesses and acute gastroenteritis (AGE) to some life-threatening clinical manifestations like acute flaccid paralysis and meningitis. Replication of enteroviruses takes place within the intestinal tract leading to shedding of infectious viral particles in the stool of infected patients. Therefore, stool and wastewater remain valuable sample types for the screening of enteroviruses as the analysis of stool specimens allows for diagnoses of specific enteroviruses in a symptomatic patient, while the investigation of wastewater is a valuable tool for the assessment of community spread and asymptomatic infections. A study by Janse van Rensburg showed a steep decline in EV detections in the cerebrospinal fluid (CSF) specimens, a common infection site for EVs, of patients with aseptic meningitis and encephalitis after the introduction of coronavirus disease 2019 (COVID-19) lockdown measures. To assess whether EVs were still present during a similar period (2019-2022) the following study was conducted in two parts. In the first part of the study stool specimens submitted to the National Health Laboratory Service Tshwane Academic Division (NHLS-TAD) laboratory from public hospitals, were collected. These specimens were obtained from hospitalised patients with AGE and were collected between 2019 and 2021. The specimens were screened for the presence of EVs using a real-time polymerase chain reaction assay (RT-PCR) after which all EV positive specimens were genotyped using Sanger sequencing and basic local alignment search tool (BLAST) analysis. IV

Phylogenetic analysis was performed to determine the evolutionary relationships present. The second part of the study involved the screening of wastewater samples, taken from four sites in the Gauteng province of South Africa between March 2021 and April 2022, for the presence of EVs. These samples’ nucleic acids were extracted using an automated extraction method and were screened using the same RT-PCR as in the first part of the study. All EV positive samples then underwent next-generation sequencing (NGS) to determine the identity of all EVs in each sample after which phylogenetic analysis was again performed as in part one. In total 138 stool specimens were screened, 26 specimens from 2019, 64 specimens from 2020 and 48 specimens from 2021. Overall, 21/138 (15.2%) of the stool specimens tested positive for the presence of enterovirus. The genotypes detected were Enterovirus C99 in 28% (6/21), Poliovirus 3 (Vaccine strain) in 28% (6/21) and Poliovirus 1 (Vaccine strain) in 24% (5/21). There were single detections of Coxsackievirus A2, A5, A9, Echovirus E25 and E30. Two co-infections of two EV strains were detected: 1 case of both Poliovirus 1 and 3 and 1 case of Enterovirus C99 and Coxsackievirus A9. In the second part of the study, 151 wastewater samples were screened and EVs were detected in 22% (33/151) of these samples. Breaking it down by site, site 1 had a positivity ratio of 23% (9/39), site 2 had a positivity rate of 40% (16/40), site 3 had a positivity ratio of 18% (6/33) and site 4 had a positivity ratio of 5% (2/39). Of the 33 positive samples, nucleic acids from 31 samples were successfully sequenced utilising next generation sequencing technology. Sequencing of EV strains from these samples revealed 29 different genotypes with the most dominantly detected genotype being EV-A119, which was detected in 87% (27/31) of samples. This was followed by coxsackievirus A9, which was detected in 48% (15/31) of samples, enterovirus A76, which was detected in 39% (12/31), and enterovirus C99 detected in 35% (11/31) of samples. The results of this study indicate that EVs continued to circulate in the community during the COVID-19 pandemic and its associated lockdown measures. It also indicated a large diversity of EVs present in the community and provided a good starting point for future research.