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.

Keywords: disease surveillance; enteric viruses; environmental monitoring; environmental surveillance; poliovirus; sludge.

Figure 1

(a) Method 1 and (b) Method 2 experimental designs for preliminary investigations. PV1 is poliovirus type 1. DI is deionized. SM is skimmed milk flocculation.

Figure 2

Percentage contribution of each elution to total mean recovery (48.4%) of poliovirus type 1 (PV1) from samples processed by Method 1 during the elution optimization and extraction optimization. Total contribution adds to 100%.

Figure 3

Recovery of poliovirus type 1 (PV1) from samples processed by Method 1 during preliminary (Prelim.) and elution optimization (Opt.) experiments. Box and whisker plot: lower, middle, and upper box lines show the first, second, and third quartiles, respectively; whiskers show the minimum and maximum data points; markers ‘×’ show the mean; and circles show the outliers.

Figure 4

Recovery of poliovirus type 1 (PV1) from samples processed by Method 1 during preliminary (Prelim.) and extraction optimization (Opt.) experiments. Box and whisker plot: lower, middle, and upper box lines show the first, second, and third quartiles, respectively; whiskers show the minimum and maximum data points; and markers ‘×’ show the mean.

Figure 5

Mean recovery of poliovirus type 1 (PV1) from samples processed with or without acid adsorption.

Figure 6

A flow diagram showing the processes for the optimized method for enteric virus recovery from primary sewage sludge.