The assessment of sewage treatment efficacy through the detection and characterisation of enteroviruses

Abstract

Enteroviruses (EVs) are small RNA viruses that have been responsible for outbreaks of hand, foot and mouth disease, viral meningitis, encephalitis and paralytic poliomyelitis. Water may act as a reservoir for waterborne EVs that are faecally shed by infected populations and subsequently introduced to water sources. The inefficient removal of EVs with wastewater treatment and its discharge into natural water sources may pose as a potential health concern if utilised by the public for domestic, agricultural or recreational purposes. An environmental surveillance (ES) complements the symptomatic surveillance of EVs since EV infections are generally asymptomatic and can, therefore, be detected in sewage as a representative of the infected population. By combining direct reverse transcription – polymerase chain reaction (RT-PCR) and integrated cell culture-RT-PCR (ICC-RT-PCR) detection and characterisation it provides a broader epidemiological overview of the current EV diversity in South Africa and the survival of specific EVs after wastewater treatment. The aim of this project was to determine the viral removal efficiency of six selected wastewater treatment plants by detecting and characterising EVs in wastewater, wastewater discharge and surface water, with molecular and viral isolation methods. Between April 2015 and March 2016, 156 water samples were collected and viruses were recoverd. The recovered viral suspensions were inoculated onto Buffalo green monkey kidney cells (BGM), L20B murine cells and Primary liver carcinoma cells (PLC/PRF/5). Enteroviruses were detected in 69.2% of the recovered viral suspensions from the water samples with direct RT-nested PCR (RT-nPCR), of which 45.4%, 43.5% and 11.1% were from wastewater, wastewater discharge and surface water, respectively. With ICC-real-time RT-PCR, 52.6% of the harvested cell culture extracts were positive for EV, of which 56.1%, 32.9% and 11% were from wastewater, wastewater discharge and surface water, respectively. The most diverse species were the EV-B species that predominated in cell culture, with echovirus 6, coxsackievirus (CV)-B3 and CV-A13 being most predominantly identified in wastewater discharge after viral amplification in cell culture. The most prevalent species were the EV-C species and predominated in the recovered viral suspensions, with EV-C99, CV-A20 and CV-A22 being most predominantly identified in the wastewater discharge with direct RT-nPCR characterisation. The majority of EVs and EV types were isolated from the PLC/PRF/5 cell line. A combination of viral isolation and molecular detection of EVs enhanced the detection of EVs in water sources and provided a wider epidemiological overview of EVs that are shown to be more resilient to treatment and environmental exposure. To conclude, it is evident that wastewater treatment systems do not efficiently remove EVs, specifically potentially viable EVs, which are subsequently discharged into natural water sources. If natural water sources become a reservoir for a wide diversity of EVs that may be potentially infectious to humans, it might raise a public health concern.