Characterisation of microbial communities and quantification of antibiotic resistance genes in Italian wastewater treatment plants using 16S rRNA sequencing and digital PCR.

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in humans, animals and environment is a growing threat to public health. Wastewater treatment plants (WWTPs) are crucial in mitigating the risk of environmental contamination by effectively removing contaminants before discharge. However, the persistence of ARB and ARGs even after treatment is a challenge for the management of water system. To comprehensively assess antimicrobial resistance dynamics, we conducted a one-year monitoring study in three WWTPs in central Italy, both influents and effluents. We used seasonal sampling to analyze microbial communities by 16S rRNA, as well as to determine the prevalence and behaviour of major ARGs (sul1, tetA, blaTEM, blaOXA-48, blaCTX-M-1 group, blaKPC) and the class 1 Integron (int1). Predominant genera included in order: Arcobacter, Acinetobacter, Flavobacterium, Pseudarcobacter, Bacteroides, Aeromonas, Trichococcus, Cloacibacterium, Pseudomonas and Streptococcus. A higher diversity of bacterial communities was observed in the effluents compared to the influents. Within these communities, we also identified bacteria that may be associated with antibiotic resistance and pose a significant threat to human health. The mean concentrations (in gene copies per liter, gc/L) of ARGs and int1 in untreated wastewater (absolute abundance) were as follows: sul1 (4.1 × 109), tetA (5.2 × 108), blaTEM (1.1 × 108), blaOXA-48 (2.1 × 107), blaCTX-M-1 group (1.1 × 107), blaKPC (9.4 × 105), and int1 (5.5 × 109). The mean values in treated effluents showed reductions ranging from one to three log. However, after normalizing to the 16S rRNA gene (relative abundance), it was observed that in 37.5 % (42/112) of measurements, the relative abundance of ARGs increased in effluents compared to influents. Furthermore, correlations were identified between ARGs and bacterial genera including priority pathogens. This study improves our understanding of the dynamics of ARGs and provides insights to develop more effective strategies to reduce their spread, protecting public health and preserving the future efficacy of antibiotics.

Assessing the Prevalence of Astroviruses in Water Environments: A Systematic Review and Meta-analysis.

Astroviruses (AstVs) are a major cause of gastroenteritis, especially in children. They can be transmitted through various pathways, including environmental contamination via water matrices. This study aimed to investigate the prevalence of AstV in different types of water, such as untreated and treated wastewater, surface water (e.g., rivers, lakes, and seawater), groundwater, drinking water, and other water matrices (e.g., irrigation water, gray water, reservoir water, floodwater, and pig slaughterhouse effluents). The meta-analysis included 80 articles, and the overall prevalence of AstV in water matrices was 36.6% [95% confidence interval (CI) of 29.6-44.0]. The highest prevalence was found in untreated wastewater at 56.8% (95% CI of 41.5-71.5), followed by treated wastewater at 48.5% (95% CI of 30.6-66.5), surface water at 28.6% (95% CI of 21.1-36.7), other matrices at 9.8% (95% CI of 0.7-25.3), drinking water at 3.3% (95% CI of 0.2-8.7), and groundwater at 0.5% (95% CI of 0.0-3.4). The most frequent AstVs detected in water environments were human AstVs, but canine and feline AstVs were also detected. Our findings highlight the importance of water as a potential route for AstV transmission, even in high-income countries. Effective water surveillance and treatment measures are necessary to minimize AstV environmental circulation and human infection through water.

Characterization of SARS-CoV-2 Variants in Military and Civilian Personnel of an Air Force Airport during Three Pandemic Waves in Italy.

We investigated SARS-CoV-2 variants circulating, from November 2020 to March 2022, among military and civilian personnel at an Air Force airport in Italy in order to classify viral isolates in a potential hotspot for virus spread. Positive samples were subjected to Next-Generation Sequencing (NGS) of the whole viral genome and Sanger sequencing of the spike coding region. Phylogenetic analysis classified viral isolates and traced their evolutionary relationships. Clusters were identified using 70% cut-off. Sequencing methods yielded comparable results in terms of variant classification. In 2020 and 2021, we identified several variants, including B.1.258 (4/67), B.1.177 (9/67), Alpha (B.1.1.7, 9/67), Gamma (P.1.1, 4/67), and Delta (4/67). In 2022, only Omicron and its sub-lineage variants were observed (37/67). SARS-CoV-2 isolates were screened to detect naturally occurring resistance in genomic regions, the target of new therapies, comparing them to the Wuhan Hu-1 reference strain. Interestingly, 2/30 non-Omicron isolates carried the G15S 3CLpro substitution responsible for reduced susceptibility to protease inhibitors. On the other hand, Omicron isolates carried unusual substitutions A1803V, D1809N, and A949T on PLpro, and the D216N on 3CLpro. Finally, the P323L substitution on RdRp coding regions was not associated with the mutational pattern related to polymerase inhibitor resistance. This study highlights the importance of continuous genomic surveillance to monitor SARS-CoV-2 evolution in the general population, as well as in restricted communities.

Surveillance for rat hepatitis E in wastewater networks, Italy.

IMPORTANCE: Hepatitis E virus (HEV) infection constitutes a significant health problem worldwide. In recent years, in addition to the zoonotic HEV3 and HEV4, emerging highly divergent hepevirus of rat origin (rat HEV [RHEV]) has been associated with human acute and chronic hepatitis. As environmental surveillance can be a complementary tool to explore emerging viruses of human and rodent origin, we investigated the epidemiology and the genetic variability of RHEV targeting 14 wastewater treatment plants in an Italian geographic area considered a hot spot for HEV infection in humans. Our results revealed that RHEV is a significant component of the wastewater microbiota with viral RNA detected in 43.9% of the specimens tested, adding further evidence to the need to investigate more in depth the real burden of RHEV infections in humans.

Wastewater-based Epidemiology and SARS-CoV-2: Variant Trends in the Apulia Region (Southern Italy) and Effect of Some Environmental Parameters.

During the COVID-19 pandemic, wastewater monitoring has been used to monitor the levels of SARS-CoV-2 RNA entering the sewerage system. In Italy, the Istituto Superiore di Sanità coordinated the SARI project (Sorveglianza Ambientale Reflue in Italia) to detect SARS-CoV-2 and its variants. In this study, the concentration of SARS-CoV-2 and its variants in raw wastewater against COVID-19 cases was evaluated together with the effect of temperature and precipitation on virus spread. We validated a predictive model, proposed by De Giglio et al., 2021, to establish the number of COVID-19 cases/100,000 inhabitants. A receiver operating characteristic curve model was applied to predict the number of COVID-19 cases and Poisson regression was applied to study the effect of temperature and rainfall on viral load. In Apulia, from October 2021 to December 2022, we analyzed 1041 samples, of which 985 (94.6%) tested positive for SARS-CoV-2. Median atmospheric temperature was inversely proportional to viral load in wastewater; no correlation was found with precipitation. The predictive model confirmed that at least 11 cases/100,000 inhabitants would occur in the 15 days following the detection of the virus in wastewater. Environmental surveillance of SARS-CoV-2 can be used to map the virus and its variants.

SARS-CoV-2 RNA in Wastewater and Bivalve Mollusk Samples of Campania, Southern Italy.

SARS-CoV-2 can be detected in the feces of infected people, consequently in wastewater, and in bivalve mollusks, that are able to accumulate viruses due to their ability to filter large amounts of water. This study aimed to monitor SARS-CoV-2 RNA presence in 168 raw wastewater samples collected from six wastewater treatment plants (WWTPs) and 57 mollusk samples obtained from eight harvesting sites in Campania, Italy. The monitoring period spanned from October 2021 to April 2022, and the results were compared and correlated with the epidemiological situation. In sewage, the ORF1b region of SARS-CoV-2 was detected using RT-qPCR, while in mollusks, three targets-RdRp, ORF1b, and E-were identified via RT-dPCR. Results showed a 92.3% rate of positive wastewater samples with increased genomic copies (g.c.)/(day*inhabitant) in December-January and March-April 2022. In the entire observation period, 54.4% of mollusks tested positive for at least one SARS-CoV-2 target, and the rate of positive samples showed a trend similar to that of the wastewater samples. The lower SARS-CoV-2 positivity rate in bivalve mollusks compared to sewages is a direct consequence of the seawater dilution effect. Our data confirm that both sample types can be used as sentinels to detect SARS-CoV-2 in the environment and suggest their potential use in obtaining complementary information on SARS-CoV-2.

Wastewater-Based Epidemiology as a Tool to Detect SARS-CoV-2 Circulation at the Community Level: Findings from a One-Year Wastewater Investigation Conducted in Sicily, Italy.

Wastewater-based epidemiology is a well-established tool for detecting and monitoring the spread of enteric pathogens and the use of illegal drugs in communities in real time. Since only a few studies in Italy have investigated the correlation between SARS-CoV-2 in wastewater and the prevalence of COVID-19 cases from clinical testing, we conducted a one-year wastewater surveillance study in Sicily to correlate the load of SARS-CoV-2 RNA in wastewater and the reported cumulative prevalence of COVID-19 in 14 cities from October 2021 to September 2022. Furthermore, we investigated the role of SARS-CoV-2 variants and subvariants in the increase in the number of SARS-CoV-2 infections. Our findings showed a significant correlation between SARS-CoV-2 RNA load in wastewater and the number of active cases reported by syndromic surveillance in the population. Moreover, the correlation between SARS-CoV-2 in wastewater and the active cases remained high when a lag of 7 or 14 days was considered. Finally, we attributed the epidemic waves observed to the rapid emergence of the Omicron variant and the BA.4 and BA.5 subvariants. We confirmed the effectiveness of wastewater monitoring as a powerful epidemiological proxy for viral variant spread and an efficient complementary method for surveillance.

Exploring the Astrovirome of Shellfish Matrices Using Nanopore Sequencing.

Astroviruses are important human enteric pathogens transmissible with contaminated food and water. Astroviruses have also been identified in mammals, birds, lower vertebrates and invertebrates. The genetic diversity of human and animal astroviruses poses a challenge for diagnostics and taxonomy. As a proof of concept, we used a panastrovirus consensus primer set, able to amplify in a nested RT-PCR protocol a 400-nt-long fragment of the RNA-dependent RNA polymerase of most members of the Astroviridae family, in conjunction with a nanopore sequencing platform, to generate information on the astrovirome in filter-feeding mollusks. Amplicons generated from bivalve samples were used to generate libraries for deep sequencing. In three samples, only one unique RdRp sequence type was obtained. However, in seven samples and in three barcodes with eleven pooled samples, we identified a variety of known and unknown RdRp sequence types, in most cases distantly related to astrovirus sequences available in the databases. In total, 37 different sequence contigs were generated. Avian-origin astrovirus sequences were predominant, likely due to contamination of shellfish harvesting waters by marine birds. Astroviruses of the aquatic eco-system were also identified, whereas human astroviruses were not detected.

Occurrence of Hepatitis A Virus in Water Matrices: A Systematic Review and Meta-Analysis.

Hepatitis A is a common form of viral hepatitis. It is usually transmitted through the ingestion of contaminated food and water. This systematic review was carried out to summarise the overall prevalence of Hepatitis A virus (HAV) in different water matrices: untreated and treated wastewater, surface water, groundwater, drinking water, and others (e.g., irrigation water and floodwater). The literature search was performed in four databases: PubMed, Web of Science, Global Index Medicus, and Excerpta Medica Database. Heterogeneity (I2) was assessed using the χ2 test on the Cochran Q statistic and H parameters. A total of 200 prevalence data from 144 articles were included in this meta-analysis. The overall prevalence of HAV in water matrices was 16.7% (95% CI: 13.4−20.3). The prevalence for individual matrix was as follows: 31.4% (95% CI: 23.0−40.4) untreated wastewater, 18.0% (95% CI: 9.5−28.2) treated wastewater, 15.0% (95% CI: 10.1−20.5) surface water, 2.3% (95% CI: 0.1−6.0) in groundwater, 0.3% (95% CI: 0.0−1.7) in drinking water, and 8.5% (95% CI: 3.1−15.6) in other matrices. The prevalence was higher in low-income economies (29.0%). Africa and Eastern Mediterranean were the regions with higher HAV prevalence values. This study showed a high heterogeneity (I2 > 75%) with a significant publication bias (p value Egger test < 0.001). The results of this review suggest that water matrices could be an important route of HAV transmission even in industrialized countries, despite the lower prevalence compared to less industrialized countries, and the availability of advanced water management systems. More effective water/wastewater treatment strategies are needed in developing countries to limit the environmental circulation of HAV.

Detection of Monkeypox Virus DNA in Airport Wastewater, Rome, Italy.

Environmental surveillance can be a complementary tool for detecting pathogens circulating in communities. We detected monkeypox virus DNA in wastewater from Italy's largest airport by using real-time PCR assays targeting the G2R region and F3L and N3R genes and sequencing. Wastewater surveillance can be quickly adapted to investigate emerging threats.

Calibration of Methods for SARS-CoV-2 Environmental Surveillance: A Case Study from Northwest Tuscany.

The current pandemic has provided an opportunity to test wastewater-based epidemiology (WBE) as a complementary method to SARS-CoV-2 monitoring in the community. However, WBE infection estimates can be affected by uncertainty factors, such as heterogeneity in analytical procedure, wastewater volume, and population size. In this paper, raw sewage SARS-CoV-2 samples were collected from four wastewater treatment plants (WWTPs) in Tuscany (Northwest Italy) between February and December 2021. During the surveillance period, viral concentration was based on polyethylene glycol (PEG), but its precipitation method was modified from biphasic separation to centrifugation. Therefore, in parallel, the recovery efficiency of each method was evaluated at lab-scale, using two spiking viruses (human coronavirus 229E and mengovirus vMC0). SARS-CoV-2 genome was found in 80 (46.5%) of the 172 examined samples. Lab-scale experiments revealed that PEG precipitation using centrifugation had the best recovery efficiency (up to 30%). Viral SARS-CoV-2 load obtained from sewage data, adjusted by analytical method and normalized by population of each WWTP, showed a good association with the clinical data in the study area. This study highlights that environmental surveillance data need to be carefully analyzed before their use in the WBE, also considering the sensibility of the analytical methods.

SARS-CoV-2 in Atmospheric Particulate Matter: An Experimental Survey in the Province of Venice in Northern Italy.

Analysis of atmospheric particulate matter (PM) has been proposed for the environmental surveillance of SARS-CoV-2. The aim of this study was to increase the current knowledge about the occurrence of SARS-CoV-2 in atmospheric PM, introduce a dedicated sampling method, and perform a simultaneous assessment of human seasonal coronavirus 229E. Thirty-two PM samples were collected on quartz fiber filters and six on Teflon using a low- and high-volumetric rate sampler, respectively, adopting a novel procedure for optimized virus detection. Sampling was performed at different sites in the Venice area (Italy) between 21 February and 8 March 2020 (n = 16) and between 27 October and 25 November 2020 (n = 22). A total of 14 samples were positive for Coronavirus 229E, 11 of which were collected in October-November 2020 (11/22; positivity rate 50%) and 3 in February-March 2020 (3/16 samples, 19%). A total of 24 samples (63%) were positive for SARS-CoV-2. Most of the positive filters were collected in October-November 2020 (19/22; positivity rate, 86%), whereas the remaining five were collected in February-March 2020 at two distinct sites (5/16, 31%). These findings suggest that outdoor PM analysis could be a promising tool for environmental surveillance. The results report a low concentration of SARS-CoV-2 in outdoor air, supporting a scarce contribution to the spread of infection.

The Geological Characteristics of the Vadose Zone Influence the Impact of Treated Wastewater on the Groundwater Quality (SCA.Re.S. Project 2019-2020).

This study evaluated whether some chemical and microbial contaminants in treated sewage effluents from two wastewater treatment plants (WWTP) reached the groundwater when they drained through a fractured karst vadose zone (WWTP-K) and a porous vadose zone (WWTP-P). Forty-five samples of sewage water (SW), treated water (TW), and monitoring well (MW), collected from WWTP-P (24) and WWTP-K (21), were analyzed for a range of microbiological and chemical properties. The E. coli and Salmonella counts were below the limits outlined in the Legislative Decree 152/06 in effluents from both types of WWTP. Enteric viruses were found in 37.5% and 12.5% of the SW and TW from WWTP-P, respectively. The percentages of Pepper mild mottle virus isolated were higher in TW (62.5% in WWTP-P, 85.7% in WWTP-K) than in SW and MW. The residual concentrations of contaminants of emerging concern (CEC) of each drug category were higher in the MW downstream of WWTP-K than of WWTP-P. Our results showed that the porous vadose zone was more effective at reducing the contaminant loads than the fractured karst one, especially the CEC, in the effluent. The legislation should include other parameters to minimize the risks from treated effluent that is discharged to soil.

An Italian survey on the microbiological safety of toys containing aqueous media.

AIMS: The purpose of the present investigation is to fill the current gap in information regarding the microbiological quality of toys containing aqueous media and the related risks for users. METHODS: Over 18 years, a total of 491 sealed toys containing aqueous media were analysed using conventional microbial culture methods. In addition, molecular methods (PCR/nested RT-PCR, followed by Sanger sequencing) were employed to test for enteric viruses (enteroviruses and adenoviruses) in a subset of toys; subsequently, the infectivity of the positive samples was tested on cell cultures. RESULTS: Of the examined toys, 23.8% were noncompliant with the limits of the European guideline. The most frequently exceeded limits were those for Aerobic bacteria (84.6%), and Pseudomonas aeruginosa (29.9%). Other opportunistic bacterial species that were frequently detected were Stenotrophomonas maltophilia, Pseudomonas fluorescens, Burkholderia cepacia, Sphingomonas paucimobilis and Comamonas acidovorans. In a subset of 28 samples, adenovirus (25%) and enterovirus (11%) genome was also found to be present, although the samples with viral positivity did not show infectivity after inoculation on appropriate cell monolayers. CONCLUSIONS: The results indicate a condition of microbial exposure related to the use of toys containing aqueous media. SIGNIFICANCE AND IMPACT OF STUDY: The investigation highlights the need for more stringent monitoring during the production, packaging and storage of toys containing aqueous matrices in order to safeguard children's health.

The wave of the SARS-CoV-2 Omicron variant resulted in a rapid spike and decline as highlighted by municipal wastewater surveillance.

This paper highlights the extraordinarily rapid spread of SARS-CoV-2 loads in wastewater that during the Omicron wave in December 2021-February 2022, compared with the profiles acquired in 2020-21 with 410 samples from two wastewater treatment plants (Trento+suburbs, 132,500 inhabitants). Monitoring of SARS-CoV-2 in wastewater focused on: (i) 3 samplings/week and analysis, (ii) normalization to calculate genomic units (GU) inh-1 d-1; (iii) calculation of a 7-day moving average to smooth daily fluctuations; (iv) comparison with the 'current active cases'/100,000 inh progressively affected by the mass vaccination. The time profiles of SARS-CoV-2 in wastewater matched the waves of active cases. In February-April 2021, a viral load of 1.0E＋07 GU inh-1 d - 1 corresponded to 700 active cases/100,000 inh. In July-September 2021, although the low current active cases, sewage revealed an appreciable SARS-CoV-2 circulation (in this period 2.2E＋07 GU inh-1 d-1 corresponded to 90 active cases/100,000 inh). Omicron was not detected in wastewater until mid-December 2021. The Omicron spread caused a 5-6 fold increase of the viral load in two weeks, reaching the highest peak (2.0-2.2E＋08 GU inh-1 d-1 and 4500 active cases/100,000 inh) during the pandemic. In this period, wastewater surveillance anticipated epidemiological data by about 6 days. In winter 2021-22, despite the 4-7 times higher viral loads in wastewater, hospitalizations were 4 times lower than in winter 2020-21 due to the vaccination coverage >80%. The Omicron wave demonstrated that SARS-CoV-2 monitoring of wastewater anticipated epidemiological data, confirming its importance in long-term surveillance.

Novel subtypes and unexpected heterogeneity of hepatitis E viral strains in wild boar captured in a small area in Central Italy.

Wild boar is the main sylvatic reservoir of the genotype 3 of hepatitis E virus (HEV). The occurrence of HEV-3 human cases has been linked to the consumption of raw or undercooked pig and wild boar meat and liver. The zoonotic transmission of HEV-3 has been confirmed by sequencing identical or strictly related viral strains in humans, wild boar and derived food. The HEV sequences classified within the HEV-3 genotype are highly variable, and although only one serotype has been identified so far, the observed differences allow for the further classification of the HEV-3 genotype into subtypes, named in alphabetical order. Compared to human and pig strains, an even higher heterogeneity is observed among strains infecting wild boar. In the present study, the genetic variability of eight HEV-3 strains detected in wild boars sampled in a small geographical area in Central Italy (Lazio and Umbria regions) was investigated by full genome sequencing and phylogenetic analysis. The strains were classified within the HEV-3a, HEV-3c, HEV-3f subtypes and within two new recently proposed subtypes. Results demonstrate - despite the relatively small geographic area of origin - an unexpected divergence within HEV-3 strains hosted by the investigated wild boar population and highlights the need for extensive sequencing of HEV in reservoirs to fully understand diversity, geographical distribution and evolution of this group of viruses.

Wastewater-based epidemiology for early warning of SARS-COV-2 circulation: A pilot study conducted in Sicily, Italy.

There is increasing evidence of the use of wastewater-based epidemiology to integrate conventional monitoring assessing disease symptoms and signs of viruses in a specific territory. We present the results of SARS-CoV-2 environmental surveillance activity in wastewater samples collected between September 2020 and July 2021 in 9 wastewater treatment plants (WTPs) located in central and western Sicily, serving over 570,000 residents. The presence of SARS-CoV-2, determined in 206 wastewater samples using RT-qPCR assays, was correlated with the notified and geo-referenced cases on the areas served by the WTPs in the same study period. Overall, 51% of wastewater samples were positive. Samples were correlated with 33,807 SARS-CoV-2 cases, reported in 4 epidemic waves, with a cumulative prevalence of 5.9% among Sicilian residents. The results suggest that the daily prevalence of SARS-CoV-2 active cases was statistically significant and higher in areas with SARS-CoV-2 positive wastewater samples. According to these findings, the proposed method achieves a good sensitivity profile (78.3%) in areas with moderate or high viral circulation (≥133 cases/100,000 residents) and may represent a useful tool in the management of epidemics based on an environmental approach, although it is necessary to improve the accuracy of the process.

Global prevalence and case fatality rate of Enterovirus D68 infections, a systematic review and meta-analysis.

A substantial amount of epidemiological data has been reported on Enterovirus D68 (EV-D68) infections after the 2014 outbreak. Our goal was to map the case fatality rate (CFR) and prevalence of current and past EV-D68 infections. We conducted a systematic review (PROSPERO, CRD42021229255) with published articles on EV-68 infections in PubMed, Embase, Web of Science and Global Index Medicus up to January 2021. We determined prevalences using a model random effect. Of the 4,329 articles retrieved from the databases, 89 studies that met the inclusion criteria were from 39 different countries with apparently healthy individuals and patients with acute respiratory infections, acute flaccid myelitis and asthma-related diseases. The CFR estimate revealed occasional deaths (7/1353) related to EV-D68 infections in patients with severe acute respiratory infections. Analyses showed that the combined prevalence of current and past EV-D68 infections was 4% (95% CI = 3.1-5.0) and 66.3% (95% CI = 40.0-88.2), respectively. The highest prevalences were in hospital outbreaks, developed countries, children under 5, after 2014, and in patients with acute flaccid myelitis and asthma-related diseases. The present study shows sporadic deaths linked to severe respiratory EV-D68 infections. The study also highlights a low prevalence of current EV-D68 infections as opposed to the existence of EV-D68 antibodies in almost all participants of the included studies. These findings therefore highlight the need to implement and/or strengthen continuous surveillance of EV-D68 infections in hospitals and in the community for the anticipation of the response to future epidemics.

Detection of SARS-CoV-2 RNA in Bivalve Mollusks by Droplet Digital RT-PCR (dd RT-PCR).

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.

SARS-CoV-2 detection in nasopharyngeal swabs: Performance characteristics of a real-time RT-qPCR and a droplet digital RT-PCR assay based on the exonuclease region (ORF1b, nsp 14).

The emergence and spread of SARS-CoV-2 has led to a compelling request for accurate diagnostic tests. The aim of this study was assessing the performance of a real-time RT-qPCR (rt RT-qPCR) assay and of a droplet digital RT-PCR (dd RT-PCR) targeting the nsp14 genome region for the detection of SARS-CoV-2 in nasopharyngeal swabs. A total of 258 nasopharyngeal swabs were analyzed with the nsp14 assays and, for comparison, with a reference assay targeting the RdRp and E genes. Conflicting results were further investigated by two additional protocols, the Centers for Disease Control and Prevention (CDC) real-time targeting N1/N2, and a nested RT-PCR for the spike region. Agreement of results was achieved on 226 samples (156 positive and 70 negative), 8 samples were positive in the reference assay and in the nsp14 rt RT-qPCR but negative with the dd RT-PCR, and 24 samples provided different combinations of results with the three assays. Sensitivity, specificity and accuracy (95 %C.I.) of the nsp14 assays were: 100.0 % (97.4-100.0), 98.7 % (92.1-100.0), and 99.6 % (97.5-100.0) for the rt RT-qPCR; 92.4 % (87.4-95.6), 100.0 % (94.2-100.0), and 94.7 % (91.1-97.0) for the dd RT-PCR. The results of the study support the use of the nsp14 real-time RT-qPCR and ddPCR for the detection of SARS-CoV-2 in nasopharyngeal swabs.