Comparing Recovery Methods for Wastewater Surveillance of Arthropod-Borne and Enveloped Viruses

Abstract

The potential public health utility of arthropod-borne virus (arbovirus) wastewater surveillance (WWS) warrants the effort of establishing an effective sample preparation workflow. Here, we investigated different sample clarification, concentration, and extraction methods to determine the best workflow for recovery of spiked arboviral and murine hepatitis virus RNA signals in wastewater. We determined that the sample clarification method with the least loss is filtration through a 0.22 μm filter (9.06 ± 3.68%) when compared with centrifugation. Out of four different concentration methods (centrifugal ultrafiltration, polyethylene glycol precipitation, charged membrane capture and elution, and hollow-fiber ultrafiltration), centrifugal ultrafiltration, specifically using Macrosep Advance column (30 kDa), has the highest mean recovery of 84.76 ± 17.45%. With this method, we can reliably recover seeded arboviruses at concentrations corresponding to 10 infections in 400 to 180,000 people, depending on the shedding rate. As arboviruses are expected to be on the rise due to the expansion of their vectors’ geographic distribution, this research will lay the foundation for managing and reducing the impact of arboviral outbreaks through WWS-informed public health measures.

Co-incidence of BA.1 and BA.2 at the start of Singapore's Omicron wave revealed by Community and University Campus wastewater surveillance

“…Campus residential samples were processed by ultrafiltration using protein concentrators excluding a three-week period from 11 October to 2 November 2021 where the PEG method of viral particle concentration was used due to a global supply chain shortage of concentrators. Efficiencies of both methods for recovering SARS-CoV-2 were comparable (not statistically different) during internal evaluation (unpublished data; Chandra et al, 2023). Wastewater samples were first centrifuged at 4000g at 4 °C for 30 min to remove large debris.…”

Wastewater detection of emerging arbovirus infections: Case study of Dengue in the United States

“…Serotyping is based on PCR-confirmation. State-and county-aggregated incident case data were digitized from the weekly reports for the study period (18 June 2023 -30 September 2023; MMWR weeks [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]. Data on the specific DENV serotype responsible for each incident case are available for only state-aggregated, travel-associated cases, and county-aggregated locally-acquired cases.…”

“…25,26 Laboratory spike-in experiments have demonstrated that DENV can persist in wastewaters for days to weeks. 27–29…”

“…25,26 Laboratory spike-in experiments have demonstrated that DENV can persist in wastewaters for days to weeks. [27][28][29] Given the likely feasibility and potential for public health use of wastewater monitoring for dengue, we applied RT-PCR assays for DENV serotypes 1,2,3, and 4 to test wastewater solids from three wastewater treatment plants during summer 2023. These sites, located in a nonendemic area of the United States commonly experiencing both travel-associated and locally-acquired DENV infections, provide a valuable opportunity to assess the feasibility of our approach to identify DENV RNA in wastewater in an area with emerging dengue risk.…”

Wastewater Detection of Emerging Arbovirus Infections: Case Study of Dengue in the United States

“…Wastewater monitoring has been previously proposed as a promising tool for population-level monitoring of DENV, but limited studies testing natural wastewater have not detected the virus. , DENV is an enveloped virus in the Flaviviridae family with a single stranded RNA genome, and is shed in the urine and saliva of infected individuals and thus likely to be excreted into wastewater. − Quantitative data are limited, but shedding in urine has been estimated to be up to around 10 3 RNA copies/mL (or 10 6 RNA copies per day) at the peak of shedding, approximately 8–10 days after symptom onset. , Peak shedding occurs during days 0–7 in saliva. DENV shedding in human stool is currently unknown; however, Zika virus, another mosquito-borne virus of the same genus as DENV, has been detected in rectal swabs of infected humans, suggesting DENV might be shed in human stool as well. , Laboratory spike-in experiments have demonstrated that DENV can persist in wastewaters for days to weeks. − …”