Enteric viruses are the leading cause of foodborne disease, with human norovirus (HuNoVs) the most prevalent, and hepatitis A virus (HAV) the more severe. Fresh and frozen berry fruits are a recognized vehicle for transmission, gaining increased international attention. The detection of these viruses is complicated because: (i) they cannot be cultivated routinely in vitro; (ii) their concentrations in foods are frequently low; (iii) and sample matrices are complex. ISO- standardized methods, released in the last decade, are widely used, but there remain complexities in their applications, interpretations, and risk-based decision making based on results. This paper describes deliberations of an International Expert Panel asked to address the following: (i) methods most often used to detect viruses in fresh and frozen berries; (ii) role of sampling in test reliability; (iii) means by which testing results are interpreted; (iv) typical uses of testing by various stakeholder sectors; (v) role/use of confirmatory testing; (vi) how testing results are used by various stakeholder sectors; and (vii) the overall value of testing. Critical unanswered questions are discussed, such as the relationship between RT-qPCR positive results and infection risk (virus infectivity) and the role of testing in risk management. Perhaps the most comprehensive work of its kind, this paper highlights the unique challenges posed by emerging molecular-based detection methods applied to non-cultivable foodborne pathogens and sets a stage for the questions that beg answers as these methods become more widely and routinely used.

Enterovirus (EV) infections are common and often mild or asymptomatic but can occasionally cause serious illness in children. Routine clinical surveillance typically underestimates the full spectrum of circulating EVs, whereas wastewater-based epidemiology captures a broader viral diversity and may serve as an early warning tool. We conducted a 5-year surveillance (2020-2024) in Barcelona, Spain, combining biweekly wastewater sampling with clinical EV reporting during the SARS-CoV-2 pandemic and subsequent relaxation of restrictions. We quantified EV genome copies in sewage by RT-qPCR and performed partial VP1 sequencing on wastewater concentrates and on clinical specimens. Over the study period, wastewater EV loads broadly paralleled reported case trends, with good correlations over the years except in 2022. A monotonic relationship was observed for certain EV type trends, including CVA6, CVB2, CVB3, E11, and EV-D68, but there was low robustness in their predictive capacity for clinical cases. Overall, deep sequencing revealed 122 distinct EV types in wastewater versus 92 types in clinical samples; 66 types were found in both sources, whereas 56 types appeared only in sewage. Sequences from wastewater and clinical sources showed high genetic similarity. Dominant EV types shifted over time highlighting the virus dynamics. These findings demonstrate that integrating WS with clinical surveillance yields a more comprehensive picture of EV circulation, uncovering hidden viral diversity and enabling early detection of emerging public health threats.

Hepatitis A virus (HAV) egresses from hepatocytes cloaked in exosomes (eHAV). However, the traffic network used for its release from polarized hepatocytes is not completely understood. We propose that eHAV biogenesis may follow not only an ESCRT-mediated pathway but also the syndecan-syntenin-ALIX pathway. The Bro1 and the V domains of ALIX bind to the pX extension of VP1 and the VP2-late domains of the unmature capsid, respectively. A Serine-to-Glycine replacement at position 134 of VP2, closely located with the first late domain, facilitates the interaction with ALIX promoting the syndecan-syntenin-ALIX pathway and improving the basolateral egress, preferentially using RAB35. This replacement is conserved in hepatoviruses infecting a wide range of mammalian species, but not in hepatoviruses infecting chimpanzees and humans. An inefficient basolateral egress could be a strategy to escape the antiviral cellular response in apes.

In 2024, circulating vaccine-derived poliovirus type 2 (cVDPV2) was detected in wastewater samples in Finland, Germany, Poland, Spain and the United Kingdom (UK). All strains were genetically linked, but sequence analysis showed high genetic diversity among the strains identified within individual wastewater sites and countries and an unexpected high genetic proximity among isolates from different countries. Taken together these results, with sequential samples having tested positive in various sites, a broader geographic distribution beyond positive sampling sites must be considered.

Rotaviruses (RVs) represent the principal causative agents of severe gastroenteritis leading to high mortality rates, especially in children < 5 years old in both developed and developing countries. Although the first generation of live attenuated RV vaccines such as RotaTeq and Rotarix achieved partial success in reducing the number of RV deaths worldwide, several concerns, such as low efficacy especially in developing countries, safety, and cost, imply a dire need to develop these vaccines. Also, sensitive methods to estimate the immunogenicity of the candidate recombinant subunit VP6 vaccines in vitro are of great need. In the present study, 1232 bp of the most frequent full-length VP6 in clinical and environmental isolates in Egypt with 98% nucleotide identity and 98% amino acid identity in comparison to human RoV Wa reference strain was expressed in E. coli. Examination of the sensitivity of antibodies produced in the male rabbits which were immunized intramuscularly with 20 mu g of the purified VP6 protein indicated a sensitivity up to 1/24000 dilution of antibodies against the expressed protein using ELISA. Introducing antibodies into the MA104 cell line was performed using electroporation to neutralize the human rotavirus Wa strain VP6 when exposed after viral uncoating. The efficiency of the antibodies which act intracellularly to neutralize the infectious human rotavirus Wa strain used in challenge was 1.4, 1.6, and 1.8 log10 for high, moderate, and low viral titres at 1/10 dilution of the antibodies and the efficiency decreased gradually by increasing the dilution.

Background/Objectives: Hepatitis A virus (HAV) yearly causes over 150 million new infections and around 40,000 deaths. Current vaccines are based on strains that grow poorly in cell culture, leading to high production costs and limited availability. This study aimed to compare the immunogenic properties of a novel HAV vaccine candidate based on the fast-growing HM175-HP strain with those of the parental slow-growing HM175-L0 strain, which derives from the cytopathic HM175 strain, like the prototype strain used in certain existing vaccines. Methods: The humoral and cellular immune response elicited by either HM175-HP or HM175-L0 vaccines was assessed in female BALB/c mice. Results: Both HM175-HP and HM175-L0 vaccines induced comparable levels of anti-HAV IgG, as well as similar numbers of antibody-secreting cells and cellular proliferation rates in immunized mice. Importantly, anti-HAV antibodies developed by HM175-HP-immunized mice were able to neutralize the HM175-L0 strain. In addition, both vaccines induced anti-HAV IgG1 antibodies, which are associated with Th2 immune response, but the HM175-HP vaccine showed a tendency to produce a greater IgG2a response, suggesting that it might elicit a higher Th1 response, which is of utmost importance for host defense against viruses. Conclusions: Our findings indicated that the fast-growing HM175-HP strain has similar immunogenic properties to the vaccine prototype-like HM175-L0, making it a promising candidate to reduce the elevated costs and time-consuming procedures of producing the current HAV vaccines. The novel HM175-HP-based vaccine would therefore facilitate mass vaccination programs and prevent vaccine shortages.

Rotaviruses (RVs) represent the principal causative agent of severe gastroenteritis leading to high mortality rates, especially in children < 5 years in both developed and developing countries. Although, the first-generation of live attenuated RV vaccines such as RotaTeq and Rotarix achieved partial success in reducing the number of RV deaths worldwide, several concerns, such as low efficacy especially in developing countries, safety, and cost imply a dire need to develop these vaccines. Also, sensitive methods to estimate the immunogenicity of the candidate recombinant subunit VP6 vaccines in vitro are of great need. In the present study, 1232 bp of the most frequent full length VP6 in clinical and environmental isolates in Egypt with 98% nucleotides identity and 98% amino acid identity in comparison to human RoV Wa reference strain was expressed in E.coli . The examination of the sensitivity of the antibodies produced in the male rabbits which were immunized intramuscularly with 20 µg of the purified VP6 proteins, indicated a sensitivity up to 1/24000 dilution of antibodies against the expressed protein using ELISA. Introduce antibodies into MA104 cell line was performed using electroporation to neutralize the human rotavirus Wa strain VP6 when exposed after viral uncoating. Higher sensitivity of neutralization in relation to immunoperoxidase was observed for the estimation of the antibodies which act intracellularly against high and low infectious units of human rotavirus Wa strain in vitro . Promising sensitivity of the produced antibodies against the infectious human RV Wa strain was observed.

Abstract Wastewater analysis serves as a complementary tool to clinical data in monitoring both the quantity and diversity of circulating enteroviruses among the population. While enterovirus infections typically result in asymptomatic or mild disease, certain types can lead to severe symptoms, highlighting the importance of tracking their prevalence and distribution. Here we present both a RTqPCR assay targeting a conserved region in the 5’ end of the enterovirus genome to detect and quantify its presence in sewage along with a sequencing pipeline based in MinION platform (Oxford Nanopore Technologies) to characterize circulating enterovirus types in samples with sufficient virus load (Cq≤33). Two sequencing approaches are possible: one targeting all enterovirus types (1), and the other specifically focusing on Cluster C enterovirus (2), including poliovirus. The obtained data are processed using a designed pipeline to quantify the proportions of different enterovirus types, providing valuable insights into the spread of potentially severe types.

Enteroviruses (EV) are a highly diverse group of viruses multiplying primarily in the gastrointestinal tract and/or the upper respiratory tract, initially distributed in two separate genera: Enterovirus and Rhinovirus, respectively. According to the similarities in genome organization and particle structure, rhinovirus species were later reclassified as also belonging to genus Enterovirus. Human EV infections are usually asymptomatic or causing mild clinical manifestations. Nevertheless, some EV infections may derive in severe neural complications, including acute flaccid paralysis (AFP) such as poliomyelitis, whose etiological agent is poliovirus, a member of the Enterovirus C species. The inactivated polio vaccine (IPV) and particularly the oral attenuated polio vaccine (OPV) have contributed to the virtual eradication of the disease. However, sustained global circulation of vaccine-derived poliovirus 2 (cVDPV2), originated from the genetic instability of OPV strain 2 and intertypic recombination between Sabin OPV strains and members of the Enterovirus C species, still causes outbreaks of AFP worldwide. In addition, humanitarian crises, in particular armed conflicts, hamper polio vaccination campaigns and facilitate the occurrence of cases. Additionally, besides poliovirus, other EV may also cause AFP, among them EV A71 or EV D68, and it is highly advisable to implement wastewater surveillance to elucidate the occurrence of not only polioviruses, but also of other EV susceptible to derive in serious neural complications, since the screening of viral RNA in cerebrospinal fluid samples in patients suffering from AFP is not a reliable diagnostic tool.

Wastewater-based surveillance can be a valuable tool to monitor viral circulation and serve as an early warning system. For respiratory viruses that share similar clinical symptoms, namely SARS-CoV-2, influenza, and respiratory syncytial virus (RSV), identification in wastewater may allow differentiation between seasonal outbreaks and COVID-19 peaks. In this study, to monitor these viruses as well as standard indicators of fecal contamination, a weekly sampling campaign was carried out for 15 months (from September 2021 to November 2022) in two wastewater treatment plants that serve the entire population of Barcelona (Spain). Samples were concentrated by the aluminum hydroxide adsorption-precipitation method and then analyzed by RNA extraction and RT-qPCR. All samples were positive for SARS-CoV-2, while the positivity rates for influenza virus and RSV were significantly lower (10.65 % for influenza A (IAV), 0.82 % for influenza B (IBV), 37.70 % for RSV-A and 34.43 % for RSV-B). Gene copy concentrations of SARS-CoV-2 were often approximately 1 to 2 logarithmic units higher compared to the other respiratory viruses. Clear peaks of IAV H3:N2 in February and March 2022 and RSV in winter 2021 were observed, which matched the chronological incidence of infections recorded in the Catalan Government clinical database. In conclusion, the data obtained from wastewater surveillance provided new information on the abundance of respiratory viruses in the Barcelona area and correlated favorably with clinical data.

Here we analyze SARS-CoV-2 genome copies in Catalonia's wastewater during the Omicron peak and develop a mathematical model to estimate the number of infections and the temporal relationship between reported and unreported cases. 1-liter samples from 16 wastewater treatment plants were collected and used in a compartmental epidemiological model. The average correlation between genome copies and reported cases was 0.85, with an average delay of 8.8 days. The model estimated that 53% of the population was infected, compared to the 19% reported cases. The under-reporting was highest in November and December 2021. The maximum genome copies shed in feces by an infected individual was estimated to range from 1.4×108 gc/g to 4.4×108 gc/g. Our framework demonstrates the potential of wastewater data as a leading indicator for daily new infections, particularly in contexts with low detection rates. It also serves as a complementary tool for prevalence estimation and offers a general approach for integrating wastewater data into compartmental models.

During the evolution of the SARS-CoV-2 pandemic, new variants of the virus have emerged and spread worldwide. The increased transmissibility and proclivity of some of these variants to cause more serious disease threatens public health responses against the virus, and they are classified as variants of concern (Variants of Concern, VOCs). While Next-Generation-Sequencing (NGS) is the gold standard to identify VOC, it cannot always be rapidly implemented in some settings to provide information as an early warning tool. Duplex quantitative real time RTqPCR assays offer a sensitive and easy-to-use tool to detect, discriminate, and estimate relative proportions of SARS-CoV-2 variants containing VOC-specific signature mutations from variants lacking it, using allelic discrimination probes. We developed three multiplexed RTqPCR assays that can detect Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) VOCs by targeting 22281_22289DelCTTTACTTG, 28262_28263InsAACA and 22029_22034DelAGTTCA deletions/insertions in their genomes, respectively. Beta and Delta deletions are mapped to the S gene (residues 241/243 and 157/158, respectively), while Gamma insertion is located between the end of ORF8 and the beginning of N gene. Our specific duplex RTqPCR assays have been adapted from a previously designed duplex RTqPCR assay used to estimate the relative proportion of genomes containing 21765-21770DelTACATG mutation affecting residues HV69/70, a signature mutation of Alpha VOC (Carcereny et al., 2021). All duplex RTqPCR assays targeting signature mutations of VOCs may be used as a complementary tool to NGS for rapid variant tracking and surveillance in wastewater-based epidemiology.

Enteric viruses are the major cause of gastroenteritis and enteric hepatitis worldwide, but in some areas like Saudi Arabia, little is known about their presence in water sources. The available information from clinical samples is not enough to figure out their actual prevalence. The aim of this study was to gather information for the first time in Saudi Arabia on the presence of the Norovirus (NoV) genogroup GI and GII, hepatitis A virus (HAV), and hepatitis E virus (HEV) in water. For this purpose, thirteen monthly samples were collected from Lake Wadi Hanifa and surrounding wells from December 2014 to November 2015. Viruses were detected and quantified using real-time RT-qPCR. Despite HEV findings being anecdotic, our results highlight interesting behaviors of the other viruses. There was a higher prevalence of noroviruses in Wadi Hanifa samples than in well water samples (46.43% vs. 12.5% of NoV GI; 66.67% vs. 8.33% of NoV GII). On the contrary, similar levels of HAV positivity were observed (40.48% in surface water vs. 43.06% in well water). Also, a strong influence of flooding events on HAV and NoV GI occurrence was observed in both surface and well water samples, with NoV GII apparently not affected.

Abstract Background Among the most prevalent enteric viruses associated with gastroenteritis are norovirus (NoV), rotavirus (RV), astrovirus (AstV), sapovirus (SaV) and adenovirus (AdV). The diagnosis of infections associated with gastroenteritis viruses is mainly based on the detection of the genome of these viruses by molecular methods, mainly qPCR and RTqPCR reactions. The aim of this study was to validate the diagnostic performance of 7 qPCR diagnostic kits of the “VIASURE Real Time PCR Detection Kits” series with respect to commercial reference kits used for the diagnosis of these pathogens. Methods A total of 318 clinical stool samples were used, including: 213 stool samples from children from sporadic cases of gastroenteritis collected between 2016–2018; 94 samples from outbreaks of gastroenteritis caused by NoV between 2017–2019; and 11 samples from healthy children with asymptomatic enteric virus infections collected between 2017–2018. Seven VIASURE kits were used for the specific detection of enteric viruses. At the same time, samples were also tested with the following commercial kits used as reference methods: RIDA®GENE Viral Stool Panel II (rotavirus, adenovirus 40/41 and astrovirus) and RIDA®GENE Sapovirus, RIDA®GENE Norovirus I & II. In the case of AdV, the positivity of the samples was confirmed using the qPCR protocol published by Heim et al. (2003). Results The results obtained are summarized in Table 1. Conclusion The results obtained show a high sensitivity and specificity of the VIASURE kits for the detection of rotavirus, adenovirus, sapovirus, astrovirus and norovirus GI and GII (in the latter case using both the monoplex and multiplex product). In addition, it should be noted that all these kits share the same thermal protocol, thus allowing their joint use for the simultaneous diagnosis of these 5 enteric viruses.

Besides nasopharyngeal swabs, monkeypox virus (MPXV) DNA has been detected in a variety of samples such as saliva, semen, urine and fecal samples. Using the environmental surveillance network previously developed in Spain for the routine wastewater surveillance of SARS-CoV-2 (VATar COVID-19), we have analyzed the presence of MPXV DNA in wastewater from different areas of Spain. Samples (n = 312) from 24 different wastewater treatment plants were obtained between May 9 (week 19 of 2022) and August 4 (week 31 of 2022). Following concentration of viral particles by a validated aluminum adsorption-precipitation method, a qPCR procedure allowed us to detect MPXV DNA in 56 wastewater samples collected from May 16 to August 4, 2022, with values ranging between 2.2 x 103 to 8.7 x 104 genome copies (gc)/L. This study shows that MPXV DNA can be reproducibly detected by qPCR in longitudinal samples collected from different Spanish wastewater treatment plants. According to data from the National Epidemiological Surveillance Network (RENAVE) in Spain a total of 6,119 cases have been confirmed as of August 19, 2022. However, and based on the wastewater data, the re-ported clinical cases seem to be underestimated and asymptomatic infections may be more frequent than expected.

The pathogenic mechanisms determining the diverse clinical outcomes of HEV infection (e.g., self-limiting versus chronic or symptomatic versus asymptomatic) are not yet understood. Because specific microRNA signatures during viral infection inform the cellular processes involved in virus replication and pathogenesis, we investigated plasma microRNA profiles in 44 subjects, including patients with symptomatic acute (AHE, n = 7) and chronic (CHE, n = 6) hepatitis E, blood donors with asymptomatic infection (HEV BDs, n = 9), and anti-HEV IgG(+) IgM(-) (exposed BDs, n = 10) and anti-HEV IgG(-) IgM(-) (naive BDs, n = 12) healthy blood donors. By measuring the abundance of 179 microRNAs in AHE patients and naive BDs by reverse transcription-quantitative PCR (RT-qPCR), we identified 51 potential HEV-regulated microRNAs (P value adjusted for multiple testing by the Benjamini-Hochberg correction [P-BH] < 0.05). Further analysis showed that HEV genotype 3 infection is associated with miR-122, miR-194, miR-885, and miR-30a upregulation and miR-221, miR-223, and miR-27a downregulation. AHE patients showed significantly higher levels of miR-122 and miR-194 and lower levels of miR-221, miR-27a, and miR-335 than HEV BDs. This specific microRNA signature in AHE could promote virus replication and reduce antiviral immune responses, contributing to the development of clinical symptoms. We found that miR-194, miR-335, and miR-221 can discriminate between asymptomatic HEV infections and those developing acute symptoms, whereas miR-335 correctly classifies AHE and CHE patients. Our data suggest that diverse outcomes of HEV infection result from different HEV-induced microRNA dysregulations. The specific microRNA signatures described offer novel information that may serve to develop biomarkers of HEV infection outcomes and improve our understanding of HEV pathogenesis, which may facilitate the identification of antiviral targets.IMPORTANCE There is increasing evidence that viruses dysregulate the expression and/or secretion of microRNAs to promote viral replication, immune evasion, and pathogenesis. In this study, we evaluated the change in microRNA abundance in patients with acute or chronic HEV infection and asymptomatic HEV-infected blood donors. Our results suggest that different HEV-induced microRNA dysregulations may contribute to the diverse clinical manifestations of HEV infection. The specific microRNA signatures identified in this study hold potential as predictive markers of HEV infection outcomes, which would improve the clinical management of hepatitis E patients, particularly of those developing severe symptoms or chronic infections. Furthermore, this study provides new insights into HEV pathogenesis that may serve to identify antiviral targets, which would have a major impact because no effective treatments are yet available. There is increasing evidence that viruses dysregulate the expression and/or secretion of microRNAs to promote viral replication, immune evasion, and pathogenesis. In this study, we evaluated the change in microRNA abundance in patients with acute or chronic HEV infection and asymptomatic HEV-infected blood donors.

Abstract Background Acute Gastroenteritis (AGE) are the second most common infectious diseases, only overtaken by Respiratory Tract Infections. Sign and symptoms of AGE illness include nausea, diarrhoea, vomits, fever and abdominal pain. In infants and children, gastroenteritis is one of the most important causes of morbidity and mortality all around the Globe. Since their identification in the 1970s, enteric viruses are considered a leading cause of gastroenteritis worldwide. Among these, rotaviruses (RV) are considered to be the main cause of AGE in young children, whereas noroviruses (NoV) affect people of all ages. In addition, sapoviruses (SaV), human astroviruses (hAstV), human adenoviruses (AdV) are also relevant. In the recent years, molecular methods, such as real-time PCR, have been widely used for detection of viral genome of enteric viruses due to their high sensitivity and specificity, becoming the gold standard. The aim of this study is the comparison of different commercial real-time PCR assays for the detection of human enteric viruses. Material and methods Three hundred and eighteen clinical stool samples were selected from different groups collected between 2016 and 2019, including specimens from symptomatic adults and symptomatic and asymptomatic children. Samples were stored at −20°C. The collection included a minimum of 25 samples positive for RV, 55 positives for AdV, 16 positives for AstV, 22 positives for SaV and 65 positives for NoV (31 for GI and 34 for GII). All specimens were extracted using the Maxwell®RSC Blood DNA Kit on the Maxwell RSC equipment (Promega). Samples were analysed using the following assays: RIDA®GENE Viral Stool Panel II, RIDA®GENE Sapovirus and RIDA®GENE Norovirus I & II (R-Biopharm), Vitassay qPCR kits for the detection of Rotavirus, Astrovirus, Norovirus GI, Norovirus GII, and Sapovirus. Results obtained with Vitassay were compared to R-Biopharm assays. Sensitivity and specificity were calculated using the software MetaDisc 1.4. Discrepant samples were tested in triplicate. Results After assessing the discrepant results, 29/114 samples were positive for RV in both RIDA®GENE and Vitassay kit. This latter reported 2 false negative (FN) and 4 false positive (FP) results when compared to RIDA®GENE. Sensitivity was 93.5% (78.6–99.2) and specificity was 95.2% (88.1–98.7). For AstV, 27/138 were positive in both kits. Vitassay qPCR reported 1 FN and 2 FP, obtaining sensitivity and specificity values of 96.4% (81.7–99.9) and 98.6% (94.9–99.8), respectively. Regarding SaV, Vitassay reported 100% (88.8%-100) of sensitivity and 97.8% (88.5–99.9) of specificity, as 1 FP was obtained. 30/89 samples tested for Norovirus GI were detected by Vitassay qPCR Norovirus GI and 25/89 RIDA®GENE Norovirus I & II (R-Biopharm). On the other hand, Vitassay qPCR Norovirus GII reported 31/81 positive samples for NoV I, whereas RIDA®GENE obtained 30 positive results. In both cases, sensitivity was of 100%, whereas specificity was over 92%. Conclusions Vitassay real-time PCR assays for the detection of different enteric viruses present high specificity and sensitivity, which is essential for a proper diagnosis and epidemiology assessment. In addition, Vitassay qPCR kit presents the advantage of being a ready-to-use lyophilised kit.

Shortly after the beginning of the SARS-CoV-2 pandemic, many countries implemented sewage sentinel systems to monitor the circulation of the virus in the population. A fundamental part of these surveillance programs is the variant tracking through sequencing approaches to monitor and identify new variants or mutations that may be of importance. Two of the main sequencing platforms are Illumina and Oxford Nanopore Technologies. Here, we compare the performance of MiSeq (Illumina) and MinION (Oxford Nanopore Technologies), as well as two different data processing pipelines, to determine the effect they may have on the results. MiSeq showed higher sequencing coverage, lower error rate, and better capacity to detect and accurately estimate variant abundances than MinION R9.4.1 flow cell data. The use of different variant callers (LoFreq and iVar) and approaches to calculate the variant proportions had a remarkable impact on the results generated from wastewater samples. Freyja, coupled with iVar, may be more sensitive and accurate than LoFreq, especially with MinION data, but it comes at the cost of having a higher error rate. The analysis of MinION R10.4.1 flow cell data using Freyja combined with iVar narrows the gap with MiSeq performance in terms of read quality, accuracy, sensitivity, and number of detected mutations. Although MiSeq should still be considered as the standard method for SARS-CoV-2 variant tracking, MinION’s versatility and rapid turnaround time may represent a clear advantage during the ongoing pandemic.

Background SARS-CoV-2, the virus responsible for the COVID-19 pandemic, can be detected in stool samples and subsequently shed in the sewage system. The field of Wastewater-based epidemiology (WBE) aims to use this valuable source of data for epidemiological surveillance, as it has the potential to identify unreported infections and to anticipate the need for diagnostic tests. Objectives The objectives of this study were to analyze the absolute concentration of genome copies of SARS-CoV-2 shed in Catalonia’s wastewater during the Omicron peak in January 2022, and to develop a mathematical model capable of using wastewater data to estimate the actual number of infections and the temporal relationship between reported and unreported infections. Methods We collected twenty-four-hour composite 1-liter samples of wastewater from 16 wastewater treatment plants (WWTPs) in Catalonia on a weekly basis. We incorporated this data into a compartmental epidemiological model that distinguishes between reported and unreported infections and uses a convolution process to estimate the genome copies shed in sewage. Results The 16 WWTPs showed an average correlation of 0.88 ± 0.08 (ranging from 0.96 to 0.71) and an average delay of 8.7 ± 5.4 days (ranging from 0 to 20 days). Our model estimates that about 53% of the population in our study had been infected during the period under investigation, compared to the 19% of cases that were detected. This under-reporting was especially high between November and December 2021, with values up to 10. Our model also allowed us to estimate the maximum quantity of genome copies shed in a gram of feces by an infected individual, which ranged from 4.15 × 107 gc/g to 1.33 × 108 gc/g . Discussion Although wastewater data can be affected by uncertainties and may be subject to fluctuations, it can provide useful insights into the current trend of an epidemic. As a complementary tool, WBE can help account for unreported infections and anticipate the need for diagnostic tests, particularly when testing rates are affected by human behavior-related biases. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was partially funded by the Catalan Agency for Water (ACA), the Catalan Public Health Agency (ASPCAT) from the Department of Health, and the Health Innovation Program from the General Research Directorate (DGRIS) of the Generalitat de Catalunya. The authors would like to thank the EU and The Spanish State Research Agency for funding project PCI2021-121928, in the frame of the collaborative international consortium SARA financed under the ERA-NET AquaticPollutants Joint Transnational Call (GA n 869178). We kindly acknowledge Promega for the assignment of a Maxwell AS4500 nucleic acids extraction System. This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 945413 and from the Universitat Rovira i Virgili (URV). Disclaimer: This work reflects only the author's view and the Agency is not responsible for any use that may be made of the information it contains. AA acknowledges the Joint Appointment Program at Pacific Northwest National Laboratory (PNNL). PNNL is a multi-program national laboratory operated for the U.S. Department of Energy (DOE) by Battelle Memorial Institute under Contract No. DE-AC05-76RL01830, the European Union's Horizon Europe Programme under the CREXDATA project, grant agreement no. 101092749, support by Ministerio de Econom\'ia y Competitividad (Grants No.\ PID2021-128005NB-C21), Generalitat de Catalunya (Grant No.\ 2017SGR-896) and Universitat Rovira i Virgili (Grant No.\ 2019PFR-URV-B2-41). A.A.\ also acknowledges ICREA Academia 2023, and the James S.\ McDonnell Foundation (Grant No.\ 220020325). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data analyzed are available online

Wastewater-based epidemiology has shown to be an efficient tool to track the circulation of SARS-CoV-2 in communities assisted by wastewater treatment plants (WWTPs). The challenge comes when this approach is employed to help Health authorities in their decision-making. Here, we describe the roadmap for the design and deployment of SARSAIGUA, the Catalan Surveillance Network of SARS-CoV-2 in Sewage. The network monitors, weekly or biweekly, 56 WWTPs evenly distributed across the territory and serving 6 M inhabitants (80% of the Catalan population). Each week, samples from 45 WWTPs are collected, analyzed, results reported to Health authorities, and finally published within less than 72 h in an online dashboard ( https://sarsaigua.icra.cat ). After 20 months of monitoring (July 20–March 22), the standardized viral load (gene copies/day) in all the WWTPs monitored fairly matched the cumulative number of COVID-19 cases along the successive pandemic waves, showing a good fit with the diagnosed cases in the served municipalities (Spearman Rho = 0.69). Here we describe the roadmap of the design and deployment of SARSAIGUA while providing several open-access tools for the management and visualization of the surveillance data.