Evaluation of Secondary Concentration Methods for Poliovirus Detection in Wastewater

Abstract

Effective surveillance of human enteric viruses is critical to estimate disease prevalence within a community and can be a vital supplement to clinical surveillance. This study sought to evaluate simple, effective, and inexpensive secondary concentration methods for use with ViroCap™ filter eluate for environmental surveillance of poliovirus. Wastewater was primary concentrated using cartridge ViroCap filters, seeded with poliovirus type 1 (PV1), and then concentrated using five secondary concentration methods (beef extract-Celite, ViroCap flat disc filter, InnovaPrep® Concentrating Pipette, polyethylene glycol [PEG]/sodium chloride [NaCl] precipitation, and skimmed-milk flocculation). PV1 was enumerated in secondary concentrates by plaque assay on BGMK cells. Of the five tested methods, PEG/NaCl precipitation and skimmed-milk flocculation resulted in the highest PV1 recoveries. Optimization of the skimmed-milk flocculation method resulted in a greater PV1 recovery (106 ± 24.8%) when compared to PEG/NaCl precipitation (59.5 ± 19.4%) (p = 0.004, t-test). The high PV1 recovery, short processing time, low reagent cost, no required refrigeration, and requirement for only standard laboratory equipment suggest that the skimmed-milk flocculation method would be a good candidate to be field-validated for secondary concentration of environmental ViroCap filter samples containing poliovirus.

Keywords: Enteric viruses; Environmental surveillance; Poliovirus; Skimmed-milk flocculation; Wastewater.

Fig. 1

Preliminary secondary concentration PV1 percent recoveries from wastewater primary concentrates. Beef extract-Celite (n = 10); ViroCap flat disc filter (n = 3); concentrating pipette (n = 4); PEG/NaCl precipitation (n = 5); skimmed-milk flocculation (4 h shaking at room temperature) (n = 6). Box-and-whisker plot: lower and upper box lines show the first and third quartiles, respectively; middle box lines show the median; whiskers show the minimum and maximum; ‘×’ markers show the mean; circles show the outliers. PV1 poliovirus type 1, PEG polyethylene glycol, NaCl sodium chloride

Fig. 2

PV1 recovery from competitive secondary concentration methods: optimized skimmed-milk flocculation (n = 8) and PEG/NaCl precipitation (n = 8). Error bars represent 95% confidence intervals. PV1 poliovirus type 1, PEG polyethylene glycol, NaCl sodium chloride. Optimized skimmed-milk flocculation includes 5% w/v skimmed-milk solution, 2 h shaking at room temperature (20–25 °C), and centrifugation at 4500×g