Italian authorities and researchers from the Department of Food Security Coordination-Istituto Zooprofilattico Sperimentale, Department of Food Safety, Nutrition and Veterinary Public Health-Istituto Superiore di Sanità and the Department of Environment and Health- Istituto Superiore di Sanità have in a new study alarmingly found the prevalence of active SARS-CoV-2 virus in seafoods including bivalve mollusks in the harvesting areas around the coasts in France and Spain.
Bivalve shellfish are readily contaminated by human pathogens present in waters impacted by municipal sewage, and the detection of SARS-CoV-2 in feces of infected patients and in wastewater has drawn attention to the possible presence of the virus in bivalves.
The aim of this study was to collect data on SARS-CoV-2 prevalence in bivalve mollusks from harvesting areas of Campania region. A total of 179 samples were collected between September 2019 and April 2021 and were tested using droplet digital RT-PCR (dd RT-PCR) and real-time RT-PCR. Combining results obtained with different assays, SARS-CoV-2 presence was detected in 27/179 (15.1%) of samples.
A median viral concentration of 1.1 × 102 and 1.4 × 102 g.c./g was obtained using either Orf1b nsp14 or RdRp/gene E, respectively. Positive results were unevenly distributed among harvesting areas and over time, positive samples being more frequent after January 2021.
Partial sequencing of the spike region was achieved for five samples, one of which displaying mutations characteristic of the Alpha variant (lineage B.1.1.7). This study confirms that bivalve mollusks may bioaccumulate SARS-CoV-2 to detectable levels and that they may represent a valuable approach to track SARS-CoV-2 in water bodies and to monitor outbreak trends and viral diversity.
Table 3. Detection of SARS-CoV-2 in bivalve mollusk samples according to sampling site.
|Sampling Site||Position in the Coastline||SARS-CoV-2 Detection|
|N° of Positive Samples||% of Positive in the Site||% of Positive in the Total|
|Monte di Procida||North||1/14||7||0.6|
|Rada Santa Lucia||Center||4/16||25||2.2|
|Torre del Greco||South||2/32||6||1.1|
|Castellammare di Stabia||South||1/14||7||0.6|
The aim of the present study was the investigation of SARS-CoV-2 presence in bivalve shellfish of Campania region, in order to ascertain virus prevalence and concentration and investigate the possible use of shellfish for monitoring the spread of SARS-CoV-2. The coastal environment is subjected to contamination by a large variety of human viruses from sewage, which can be bio-accumulated by filter-feeding shellfish species.
Possible contamination by SARS-CoV-2 of coastal waters and other environmental compartments, such as estuaries and marine habitats, has been hypothesized by some authors, as well as the possibility to use shellfish as sentinels of their environment [12,17]. Although monitoring of SARS-CoV-2 in sewage discharges should be considered as the first choice for biocontrol, bivalves could be used as a surveillance tool, especially in case of non-point sources, direct wastewater discharges in small estuaries, and difficulties in sampling .
In addition, the continuous filter-feeding activity of bivalve mollusks, modeling their ability to bioaccumulate chemical and microbial contaminants, may help overcome the limitations of wastewater analysis deriving from low concentration of the viral target and limited analytical sensitivity. Some species such as mussels (Mytilus spp.) are already widely used as bioindicators for coastal water pollution monitoring . For these reasons, bivalves have good features to be considered sentinels for the detection of SARS-CoV-2 in the coastal environment.
It should be noted that the existence of RNA molecules binding with biofilm matrices provides a prolonged persistence of RNA in the environment (eRNA) and contributes to the conservation of eRNA in the marine environment much longer than expected [33,34], supporting the use of environmental analysis to trace viral spread and genetic variability.
However, in a risk assessment perspective, it should be considered that while SARS-CoV-2 RNA may be stable in river and sea waters, the presence of RNA alone does not correlate infectious virus [14,35,36]. A previous study on SARS-CoV-2 in bivalve shellfish from Polo et al. (2021)  using PCR viability assays, showed that real-time detection of SARS-CoV-2 RNA in clams and sediments did not correspond to intact capsids and, therefore, to infectious viral particles, and concluded that the risk to public health associated to SARS-CoV-2 in bivalve shellfish is extremely low, so that shellfish should not be inappropriately perceived as a risk or a vector of SARS-CoV-2.
Moreover, although the presence of SARS-CoV-2 RNA in wastewater influents has been confirmed, viral infectivity of positive samples in cell cultures has not been proven so far . Respiratory droplets and aerosols may contain high titers of viral particles [38,39,40] and SARS-CoV-2 infectivity is retained for over 3 h in experimentally produced aerosols .
On the contrary, occurrence of infectious virus in feces and urine has been questioned: detection of SARS-CoV-2 in human feces (reviewed in Foladori et al. 2020 ) is indeed a common feature in infected subjects, but isolation of infectious virus from this clinical specimen has been achieved in a limited number of cases [43,44,45,46], while other studies failed to do so [47,48,49].
Therefore, it may be hypothesized that feces and urine probably contain either low levels or no infectious SARS-CoV-2 particles and that, based on low predicted abundances and limited environmental survival, the likelihood of SARS-CoV-2 transmission though sewage-contaminated water or bivalve shellfish is extremely low or negligible .
In this study we chose to apply the current recommended method ISO15216-1:2019 for the detection of Norovirus and hepatitis A in mollusks, adapting it to SARS-CoV-2, given the existing evidence for a higher concentration of this virus in the digestive tissue of bivalve shellfish .
In the absence of a reference method for SARS-CoV-2 testing in foods, different analytical approaches (dd RT-PCR and real-time RT-PCR) and protocols based on different genetic targets (Orf1b nsp14 region, RdRp, E gene, and N gene) were included in the study, to provide a better estimate of SARS-CoV-2 prevalence. Further to this, to unambiguously confirm detection of SARS-CoV-2 and perform molecular characterization, all positive results obtained by dd RT-PCR or real-time RT-PCR were subjected to conventional nested RT-PCR followed by sequencing.
Overall, presence of SARS-CoV-2 was shown in 27/179 (15.1%) of samples, with a median viral concentration in positive samples of ~102 g.c./g, regardless of the applied assay. These results confirm, though with a significantly lower prevalence, the study by Polo et al. (2021)  on SARS-CoV-2 occurrence in clams and sediments collected from estuarine areas in Galicia (Spain) between May and July 2020, in which SARS-CoV-2 RNA was detected in 9/12 clam and 3/12 sediment samples. On the contrary, a similar survey conducted on oysters taken between April and August 2020 from several areas of the French coasts reported no detection of SARS-CoV-2 in over 180 samples .
Results from these studies, however, should be considered taking into account the concentration of the viral target in the shellfish matrix. Viral concentration in bivalve shellfish may be significantly affected by the viral load in the growing waters, which is a function of both viral input through wastewater discharge and virus dispersion in seawaters due to dilution effect and water circulation.
The two studies undertaken in France and Spain were conducted on samples collected in spring and summer 2020, in correspondence to the so-called first wave of the COVID-19 pandemic. Our study encompassed a longer observation period (September 2019 to April 2021) and highlights that, even though SARS-CoV-2 was detectable since February 2020 (i.e., at the beginning of the COVID-19 pandemic in Italy) and remained stable during the entirety of 2020, the number of positive samples increased since January 2021, following the so-called second wave of the epidemic.
This result probably reflects an increase of viral discharge in wastewaters, and consequently in seawaters, associated to the increase of infection cases in the population of the region Campania, as indicated by the trends reported by the national surveillance system for COVID-19.
Sewage discharge, however, may impact bivalve shellfish harvesting areas to a different level, based on viral particle dispersion in seawater (distance from the contamination source, dilution effects, water streams, tide effects, etc.). In our study, production areas along the whole coastline of region Campania (90 km) were included and a significant variability in SARS-CoV-2 detection was seen among relatively close areas of the northern part of the coastline, where virus was detected in 41% of samples from the site ‘Varcaturo’ (the closest site to the major wastewater treatment plants of the region), in 15% of those taken in ‘Bacoli’, and never detected in the sites ‘Pozzuoli’ and ‘Nisida’. SARS-CoV-2 was also detected in a significant proportion of samples collected in the site ‘Rada Santa Lucia’, the closest to the urban center of Naples.
Indeed, the sampling sites where SARS-CoV-2 was detected in bivalve mollusks represent the meeting points of multiple sewer systems. The positivity revealed on “Rada Santa Lucia”, for example, may be attributed to the contribute of a wide area with a high population density. In this site the treated wastewater coming from two sewer systems contributes together to generate the exposure on mussels.
On the other hand, virus was detected in a minority of samples collected in the three sites in the southern part of the coastline. These results highlight that high variability may be expected for SARS-CoV-2 detection depending on the characteristics of the sampling sites and may shed light on the differences in SARS-CoV-2 prevalence and concentration in our study, performed mostly on mussels (Mytilus galloprovincialis) grown in areas located approximately within 3 km from the coast, compared to the work by Polo et al. (2021) , which tested clams (Ruditapes philippinarum) grown in banks located in small estuaries influenced by tides and relatively close to wastewater treatment plants or sewage pump stations.
Further to the variables associated to SARS-CoV-2 concentration in growing waters, analytical issues may affect SARS-CoV-2 detection in complex food matrices such as bivalve shellfish. In our study, viral concentration was, with few exceptions, below 1 g.c./µL of tested RNA, hence in a concentration range close to the detection limit of molecular tests, a factor that—due to statistical probability—may affect target detection.
This effect, together with the intrinsic variability of amplification efficiency of different PCR assays, partially explain the inconsistency of the results obtained with the different protocols and the fact that only a minority of samples (5 of 19 considering dd RT-PCR results) were positive by two or more assays.
Interestingly, despite the low target concentration, amplification by RT-nested-PCR and molecular characterization by partial sequencing of the spike gene was achieved in five samples. In all such cases, amplification was obtained using PCR assays for short fragments (PCR IDs 973 and 975, ~320 bps) but not with the assay designed to amplify ~1600 bps of the spike gene (PCR ID 980).
This result is not surprising, as amplification of long fragments in complex matrices as foods or environmental samples is often challenging, due to low target concentration and fragmentation of nucleic acids. Significantly, the obtained amplifications showed 100% sequence identity to SARS-CoV-2 prototype strain (Wuhan sequence) in four samples collected between January and April 2021, but also displayed two mutations (N501Y and A570D) typical of the so-called ‘UK variant’, later renamed ‘Alpha variant’ according to WHO nomenclature (https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/, accessed on 5 September 2021), in one sample collected in March 2021, i.e., less than three months after the first isolation, on December 2020, of this Variant of Concern (VOC) in Italy (https://press.regione.puglia.it/-/covid-l-istituto-zooprofilattico-ha-isolato-il-virus-della-cosiddetta-variante-inglese, accessed on 5 September 2021).
reference link :https://www.mdpi.com/1660-4601/19/2/943/htm