Use of Preservative Agents and Antibiotics for Increased Poliovirus Survival on Positively Charged Filters

Abstract

Environmental surveillance of poliovirus (PV) and other non-enveloped viruses can help identify silent circulation and is necessary to certify eradication. The bag-mediated filtration system is an efficient method to filter large volumes of environmental waters at field sites for monitoring the presence of viruses. As filters may require long transit times to off-site laboratories for processing, viral inactivation or overgrowth of bacteria and fungi can interfere with virus detection and quantification (Miki and Jacquet in Aquatic Microb Ecol 51(2):195–208, 2008). To evaluate virus survival over time on ViroCap™ filters, the filters were seeded with PV type 1 (PV1) and/or MS2 and then dosed with preservatives or antibiotics prior to storage and elution. These filters were stored at various temperatures and time periods, and then eluted for PV1 and MS2 recovery quantification. Filters dosed with the preservative combination of 2% sodium benzoate and 0.2% calcium propionate had increased virus survival over time when stored at 25 °C, compared to samples stored at 25 °C with no preservatives. While elution within 24 h of filtration is recommended, if storage or shipping is required then this preservative mixture can help preserve sample integrity. Addition of an antibiotic cocktail containing cephapirin, gentamicin, and Proclin™ 300 increased recovery after storage at 4 and 25 °C, when compared to storage with no antibiotics. The antibiotic cocktail can aid sample preservation if access to appropriate antibiotics storage is available and sample cold chain is unreliable. This study demonstrated that the use of preservatives or antibiotics is a simple, cost-effective method to improve virus detection from ViroCap cartridge filters over time.

Feasibility of the Bag-Mediated Filtration System for Environmental Surveillance of Poliovirus in Kenya

“…MS2 was initially chosen as an internal control because it is non-pathogenic, found in the environment, and is similar to enteric viruses (Grabow 2001). In a previous study, with MS2 pre-seeded onto ViroCap filters, 10 L PV1-spiked Seattle influent wastewater filtered through these ViroCap filters, and preservatives added to the filters prior to elution, the average MS2 recovery (84.6%) was greater than the average PV1 recovery spiked into the 10 L influent wastewater (53.5%) (Fagnant et al 2017). The erratic recovery of MS2 seen in this pilot study may have been due to various factors including deaggregation, MS2 presence in the environment, complications with the double agar layer assay, and/or seeding at different levels than anticipated.…”

“…Technicians from the Kenya Medical Research Institute (KEMRI) Center for Virus Research in Nairobi, Kenya and the University of Pretoria in Pretoria, South Africa were trained in-person on the use of the BMFS field kit and laboratory protocols (Fagnant et al 2014(Fagnant et al , 2017(Fagnant et al , 2018. Trainees received detailed illustrated protocols, a one-page "quicksheet" reference protocol, and a training video describing the BMFS field sampling protocol.…”

“…For future BMFS studies, if filters are shipped for processing, the addition of preservatives is recommended, to stabilize virus and reduce bacterial and fungal growth in the case of cold-chain failure during shipment. The addition of preservatives has been found to increase virus survival in ViroCap filters held for up to 7 days at temperatures up to 25 °C (Fagnant et al 2017). Future research to determine the effect of these preservatives on unconcentrated water samples should be examined for potential use with other sampling methods.…”

“…BMFS enables collection of large sample volumes (3-6 L wastewater or wastewater-impacted waters) and the ability for in-field filtration through a charged cartridge filter. Prior to field sampling, bacteriophage MS2 can be pre-seeded onto the filters as an internal control (Fagnant et al 2017). After filtration, the samples are transported by reverse cold chain to the laboratory for further processing.…”

“…After filtration, the samples are transported by reverse cold chain to the laboratory for further processing. In the laboratory, a preservative mixture is added to improve the survival of PV over time on the filters followed by filter elution and secondary concentration (Fagnant et al 2017(Fagnant et al , 2018Zhou et al 2018). Processing large volumes allows for an increased effective volume assayed, while on-site filtration eliminates transport of large water samples and reduces required laboratory space.…”