SARS-CoV-2 circulation in Croatian wastewaters and the absence of SARS-CoV-2 in bivalve molluscan shellfish

Abstract

The circulation of SARS-CoV-2 in the environment has been confirmed numerous times, whilst research on the bioaccumulation in bivalve molluscan shellfish (BMS) has been rather scarce. The present study aimed to fulfil the knowledge gap on SARS-CoV-2 circulation in wastewaters and surface waters in this region and to extend the current knowledge on potential presence of SARS-CoV-2 contamination in BMS. The study included 13 archive wastewater and surface water samples from the start of epidemic and 17 influents and effluents from nine wastewater treatment plants (WWTP) of different capacity and treatment stage, sampled during the second epidemic wave. From that period are the most of 77 collected BMS samples, represented by mussels, oysters and warty venus clams harvested along the Dalmatian coast. All samples were processed according to EN ISO 15216-1 2017 using Mengovirus as a whole process control. SARS-CoV-2 detection was performed by real-time and conventional RT-PCR assays targeting E, N and nsp14 protein genes complemented with nsp14 partial sequencing. Rotavirus A (RVA) real-time RT-PCR assay was implemented as an additional evaluation criterion of virus concentration techniques. The results revealed the circulation of SARS-CoV-2 in nine influents and two secondary treatment effluents from eight WWTPs, while all samples from the start of epidemic (wastewaters, surface waters) were negative which was influenced by sampling strategy. All tertiary effluents and BMS were SARS-CoV-2 negative. The results of RVA amplification were beneficial in evaluating virus concentration techniques and provided insights into RVA dynamics within the environment and community. In conclusion, the results of the present study confirm SARS-CoV-2 circulation in Croatian wastewaters during the second epidemic wave while extending the knowledge on wastewater treatment potential in SARS-CoV-2 removal. Our findings represent a significant contribution to the current state of knowledge that considers BMS of a very low food safety risk regarding SARS-CoV-2.

“pySewage”: a hybrid approach to predict the number of SARS-CoV-2-infected people from wastewater in Brazil

“…The prevalence of infection ranged from 0.05 to 0.38%, showing a positive correlation with clinical observations. Other studies detected SARS-CoV-2 viral RNA in wastewater in India (Arora et al 2020 ), Germany (Westhaus et al 2021 ), the USA (Wu et al 2021 ; Barua et al 2022 ), Croatia (Brnić et al 2022 ), and the United Arab Emirates (Hasan et al 2021 ).…”

SARS-CoV-2 Whole-Genome Sequencing Using Oxford Nanopore Technology for Variant Monitoring in Wastewaters

“…Mengovirus, a murine picornavirus, was used as a process control for nucleic acid (NA) extraction. MgV or other non-enveloped viruses were used as process control for SARS-CoV-2 WBE by other teams previously ( Barril et al, 2021 ; Alamin et al, 2022 ; Brnić et al, 2022 ). Here it was considered adequate as our concentration step relies on ultracentrifugation, which is efficient on both enveloped and non-enveloped virions, and early tests showed similar efficiencies using a porcine coronavirus (data not shown).…”

Very low likelihood that cultivated oysters are a vehicle for SARS-CoV-2: 2021–2022 seasonal survey at supermarkets in Kyoto, Japan

“…Oysters and other bivalves are known as ‘filter feeders’ because they filter organic matter, including viruses and bacteria, present in seawater through their midgut gland and concentrate them in their bodies ( Brnić et al, 2022 ; Desdouits et al, 2021 ; Le Guernic et al, 2022 ; Mancusi et al, 2022 ; Polo et al, 2021 ). Previous studies have elucidated a marked infection cycle that arises when norovirus from the feces of infected people seeps into the aquatic environment through sewage and accumulates in the bodies of oysters and other bivalves cultivated in coastal areas.…”

“…The latter are consumed by humans without sufficient heating needed to kill the virus ( Brnić et al, 2022 ; Desdouits et al, 2021 ; Le Guernic et al, 2022 ). Although several studies have reported a low risk of SARS-CoV-2 transmission through food ( Bondad-Reantaso et al, 2020 ; Godoy et al, 2021 ), concern remains that a similar infection cycle may arise with SARS-CoV-2, although no details of such a cycle are yet known.…”

“…Methods for detecting norovirus in oysters, other foods and fecal matter have been reported in Europe ( EFSA, 2019 ; ISO, 2017 ) and by government testing guidelines in Japan ( Imamura et al, 2021 ; MAFF, 2019 ; MHLW, 2003 ; NIHS, 2010 ). However, no method has been established for detecting SARS-CoV-2 in bivalves, with studies from European nations that reported detecting the virus having only modified the method used for norovirus ( Brnić et al, 2022 ; Desdouits et al, 2021 ; Le Guernic et al, 2022 ; Mancusi et al, 2022 ; Polo et al, 2021 ). Since both infectious viral particles and non-infectious fragmented RNAs accumulate in the midgut gland of oysters, methods used to detect RNA directly from oyster homogenates may overestimate the actual risk ( Imamura et al, 2021 , MAFF, 2019 , 2021 ; Polo et al, 2021 ).…”