Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters

Abstract

dPathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size. Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. Here, we compared the efficiency of virus recovery for three rapid methods of concentrating two microbial source tracking (MST) viral markers human adenoviruses (HAdVs) and polyomaviruses (HPyVs) from one liter tap water and river water samples on HA membranes (90 mm in diameter). Samples were spiked with raw sewage, and viral adsorption to membranes was promoted by acidification (method A) or addition of MgCl 2 (methods B and C). Viral nucleic acid was extracted directly from membranes (method A), or viruses were eluted with NaOH and concentrated by centrifugal ultrafiltration (methods B and C). No inhibition of qPCR was observed for samples processed by method A, but inhibition occurred in river samples processed by B and C. Recovery efficiencies of HAdVs and HPyVs were ϳ10-fold greater for method A (31 to 78%) than for methods B and C (2.4 to 12%). Further analysis of membranes from method B revealed that the majority of viruses were not eluted from the membrane, resulting in poor recovery. The modification of the originally published method A to include a larger diameter membrane and a nucleic acid extraction kit that could accommodate the membrane resulted in a rapid virus concentration method with good recovery and lack of inhibitory compounds. The frequently used strategy of viral absorption with added cations (Mg 2؉) and elution with acid were inefficient and more prone to inhibition, and will result in underestimation of the prevalence and concentrations of HAdVs and HPyVs markers in environmental waters. Discharges from sewage treatment plants (STPs), storm water drains, improperly designed septic systems, and fecal contamination from livestock and wildlife are known to degrade environmental water quality in terms of elevating fecal indicator bacteria and possibly pathogen concentrations (1-5). Fecal indicator bacteria such as Escherichia coli and Enterococcus spp. have been widely used as an indirect measure of microbial risk associated with environmental waters. However, identifying the health risks associated with enteric viruses and protozoa by monitoring fecal indicator bacteria has been questioned (5-8).The risk of infectious disease associated with recreational water use may well be of viral etiology (9). The transmission of these viruses occurs via the fecal-oral route, nasal mucosa or the conjunctiva and the infected individual may shed up to 10 11 viral particles/gram of feces (10). Some of these viruses are more res...

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