Method Development for Enteric Virus Recovery from Primary Sludge

Abstract

Enteric viruses, such as poliovirus, are a leading cause of gastroenteritis, which causes 2–3 million deaths annually. Environmental surveillance of wastewater supplements clinical surveillance for monitoring enteric virus circulation. However, while many environmental surveillance methods require liquid samples, some at-risk locations utilize pit latrines with waste characterized by high solids content. This study’s objective was to develop and evaluate enteric virus concentration protocols for high solids content samples. Two existing protocols were modified and tested using poliovirus type 1 (PV1) seeded into primary sludge. Method 1 (M1) utilized acid adsorption, followed by 2 or 3 elutions (glycine/sodium chloride and/or threonine/sodium chloride), and skimmed milk flocculation. Method 2 (M2) began with centrifugation. The liquid fraction was filtered through a ViroCap filter and eluted (beef extract/glycine). The solid fraction was eluted (beef extract/disodium hydrogen phosphate/citric acid) and concentrated by skimmed milk flocculation. Recovery was enumerated by plaque assay. M1 yielded higher PV1 recovery than M2, though this result was not statistically significant (26.1% and 15.9%, respectively). M1 was further optimized, resulting in significantly greater PV1 recovery when compared to the original protocol (p < 0.05). This method can be used to improve understanding of enteric virus presence in communities without liquid waste streams.