Multisite community-scale monitoring of respiratory and enteric viruses in the effluent of a nursing home and in the inlet of the local wastewater treatment plant

ABSTRACT

The aim of this study was to evaluate whether community-level monitoring of respiratory and enteric viruses in wastewater can provide a comprehensive picture of local virus circulation. Wastewater samples were collected weekly at the wastewater treatment plant (WWTP) inlet and at the outlet of a nearby nursing home (NH) in Burgundy, France, during the winter period of 2022/2023. We searched for the pepper mild mottle virus as an indicator of fecal content as well as for the main respiratory viruses [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza, and respiratory syncytial virus] and enteric viruses (rotavirus, sapovirus, norovirus, astrovirus, and adenovirus). Samples were analyzed using real-time reverse transcription PCR-based methods. SARS-CoV-2 was the most frequently detected respiratory virus, with 66.7% of positive samples from the WWTP and 28.6% from the NH. Peaks of SARS-CoV-2 were consistent with the chronological incidence of infections recorded in the sentinel surveillance and the nearby hospital databases. The number of positive samples was lower in the NH than in WWTP for the three respiratory viruses. Enteric viruses were frequently detected, most often sapovirus and norovirus genogroup II, accounting both for 77.8% of positive samples in the WWTP and 57.1% and 37%, respectively, in the NH. The large circulation of sapovirus was unexpected in particular in the NH. Combined wastewater surveillance using simple optimized methods can be a valuable tool for monitoring viral circulation and may serve as a suitable early warning system for identifying both local outbreaks and the onset of epidemics. These results encourage the application of wastewater-based surveillance (WBS) to SARS-CoV2, norovirus, and sapovirus.

IMPORTANCE

WBS provides valuable information on the spread of epidemic viruses in the environment using appropriate and sensitive detection methods. By monitoring the circulation of viruses using reverse transcription PCR methods in wastewater from the inlet of a wastewater treatment plant and the outlet of a nearby retirement home (connected to the same collective sewer network), we aimed to demonstrate that implementing combined WBS at key community sites allows effective detection of the occurrence of respiratory (influenza, respiratory syncytial virus, and SARS-CoV-2) and enteric (norovirus, rotavirus, and sapovirus) virus infections within a given population. This analysis on a localized scale provided new information on the viral circulation in the two different sites. Implementing WBS to monitor the circulation or the emergence of infectious diseases is an important means of alerting the authorities and improving public health management. WBS could participate actively to the health of humans, animals, and the environment.

INTRODUCTION

Wastewater-based surveillance (WBS) aims to search for or quantify potential pathogens in wastewater, especially upstream of wastewater treatment plants (WWTPs), and to monitor the emergence and spread of infectious diseases in populations (1–3). The coronavirus disease 2019 (COVID-19) pandemic sparked a particular interest in wastewater safety, resulting in numerous WBS studies reporting on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surveillance in WWTPs (4). This approach was deployed worldwide during the COVID-19 pandemic, which modeled the dynamics of the coronavirus (5–9). Monitoring epidemics in human populations is particularly difficult for infections that are not systematically diagnosed or when the number of asymptomatic or pre-symptomatic carriers is significant, as for SARS-CoV-2 (10, 11). Moreover, while there are sentinel surveillance networks for certain diseases (e.g., Sentiweb in France), the data are often not complete and based on a clinical approach that does not confirm the cause of the infection. The WBS approach is a non-invasive means of monitoring the health status of a population. It can provide early warnings and epidemic surveillance, with a cost:benefit ratio that is much lower than for mass screening campaigns (12).

WBS provides valuable information about the circulation of viruses, especially as it can be adapted to any pathogen that is excreted in sufficient quantities in feces and urine (13, 14). Infected individuals usually shed enteric viruses at high concentrations, up to 10¹³ copies/g of feces in children, which is why these viruses are commonly found in wastewater (15–18). In contrast to enteric pathogens, the excretion of respiratory viruses in stools of infected individuals is rather low because it stems mainly from the ingestion of infected respiratory mucus (19). Real-time PCR assays were chosen for virus detection over more sophisticated methods like metagenomics as they can be processed by local laboratories with experience in PCR. These techniques are adapted to territories, and they can be used to provide an adapted local health response in real time, as opposed to the longer and more cumbersome individual approach to the detection of contamination. WBS has been shown to be an effective tool for monitoring viral spread and transmission, especially SARS-CoV-2 and enteric viruses, and a few studies have investigated other major respiratory pathogens like influenza virus (IV) and respiratory syncytial virus (RSV) (20–23).

Moreover, the few studies reporting on WBS in nursing homes (NHs) have focused only on SARS-CoV-2 (24–27). We therefore chose to analyze samples collected at a WWTP and an NH to determine if there is any difference in the pathogens excreted by a larger population and those from the residents of a single nursing home community living in a relatively enclosed space. WBS from WWTPs reveals the extent of viral circulation present in a large population, while those obtained from NHs can provide real-time information on the infectious status of their residents. Tracking the major infectious pathogens in wastewater can greatly benefit public health within the community.

MATERIALS AND METHODS

Study settings and sampling sites

This WBS consisted of a weekly collection of wastewater samples for 29 weeks (W), from 14 September 2022 (W37/2022) to 30 March 2023 (W13/2023), to detect respiratory and enteric viruses. The WBS involved both the WWTP and the NH of Précy-sous-Thil, a village situated in Burgundy, France, with a population of around 709 inhabitants according to the most recent census from the Institut National de la Statistique et des Études Économiques (Insee) (28). The two sites belong to the same collective sewer network and are 1,800 m apart (Fig. 1). The WWTP collects effluents with a flow rate of 126 m³/day (i.e., approximately 5,250 L/hour) from the entire population of the village, while the collector of the NH receives wastewater from 50 elderly residents and 34 caregivers and managers. Temperature and rainfall data were obtained from the database of the local station of the Météo-France network (29). Data for temperature and rainfall were collected at noon every day. The monthly meteorological data for both weather variables were combined to provide a monthly average.

Fig 1.

Collective sewer network map of Précy-sous-Thil, Côte d’Or, Bourgogne. The maps are from www.sesam21.fr, reproduced with permission.

The wastewater sample collection, the extraction, and the respiratory virus analyses were conducted by the Laboratoire Départemental de Côte d’Or (LDCO) in Dijon, France, which is a routine field laboratory for local public health. Enteric virus detection was performed by the French National Reference Centre for Gastroenteritis Viruses, Dijon, France.

Epidemiological data

Epidemiological data on SARS-CoV-2, RSV, and IV detections were obtained from the Dijon Bourgogne University Hospital (DBUH), which is the hospital linked to Précy-sous-Thil and located approximately 70 km east. The number of SARS-CoV-2-positive cases was also based on data recorded in the Précy-sous-Thil area through the French government’s contact tracing application called “TousAntiCovid.” The sum of the data for the 7 days prior to the sampling day corresponded to the weekly incidence. IV cases in Burgundy were based on data recorded by the DBUH, and national clinical influenza cases were recorded by the French public health services [Santé Publique France (SpF)]. Information on patients in the nursing home with symptoms of SARS-CoV-2 was kindly provided by the administrators of the nursing home. Epidemiological data on clinical acute gastroenteritis cases in Burgundy were obtain from the Sentinelles network, which is a French primary care health research and surveillance network that collects large national data in general medicine and pediatrics for health monitoring and research purposes (www.sentiweb.fr/) (Data S1).

Wastewater sampling

The raw effluent arriving at the WWTP passes through a pre-filtration grid, which retains the largest debris before the effluent is discharged into a first passive collection tank. This reservoir has a volume of 110 m³ and a very regular brewing. Sampling was carried out under satisfactory safety conditions. As soon as the level rises, a partial emptying of this tank is automatically triggered, the swirl generated by the pump mixing the entire tank. During emptying, the eddy created by the pumps mixes the entire tank and what remains “waiting” is representative of the contents of the previous emptyings. The first tank, which was an open reservoir, was sampled using a 1-L liquid sampling dipper inserted in a bottle holder, the sampler taking care to vary the depth of stirring locally. The samples were taken around 10:30 a.m. to account for the morning peak in toilet use.

Sampling was performed at the NH outlet with an AS950 isothermal automatic sampler (Hydreka) where the facility’s general collector flows. A small pipe of 15 cm was connected to the general pipe and remained in place for 6 months. Each week, a new 17-L collector tank was installed for the automatic sampling of 100 mL every 10 minutes for a total of 14.5 L sampled in 24 hours. The next day, the container was shaken vigorously before 1 L of the homogenized mixture was collected in a disposable 1-L bottle. Collection and filtration were performed in the field.

Both NH and WWTP samples were then processed in the same manner. After further stirring, a volume of 120–150 mL was sampled into a container on which a Whatman grade 2V pleated filter (particle retention = 8 µm) was placed, allowing the liquid to flow by gravity. Finally, 20 mL of liquid was withdrawn using a 20-mL disposable plastic syringe with a 5-µm filter attached. Approximately 10 mL was collected for transfer to the laboratory. The filter was changed in the middle of the operation due to clogging caused by the particle load. Each week, the identified wastewater sample was returned to the LDCO in isothermal boxes at 4°C. Nucleic acids were extracted on the day of collection.

Nucleic acid extraction and reverse transcription PCRs

Nucleic acids from 300 µL of each wastewater sample were extracted in duplicate at the LDCO using a KingFisher 96 extraction platform with BioExtract Superball (BioSellal, Dardilly, France) to obtain a volume of 60 µL of eluate. To check the extraction quality and the absence of reverse transcription PCR (RT-PCR) inhibitors, 5 µL of internal positive control from the Environmental Covid & Flu and the Environmental SARS-CoV-2 Bio-T kits was added to each sample prior to extraction. In addition, negative control samples consisting of RNase- free water were extracted along each extraction series following the same protocol.

SARS-CoV-2 and influenza A and B genome detections were performed using the Environmental SARS-CoV-2 (ref. # BIOTK137) and Environmental Covid & Flu (ref. # BIOTK133) Bio-T kits (BioSellal), respectively, according to the manufacturer’s recommendations. For human RSV A and B detection, the Premium DX Coldplex kit (BioSellal) was used first, according to the manufacturer’s recommendations. However, due to its very low sensitivity (data not shown), it was replaced by the prototype version of the Environmental HRSV Bio-T kit (BioSellal) on the remaining volumes of eluates. All respiratory RT-PCRs were performed on an ABI PRISM 7500 FAST Real-Time PCR System (Life Technology, Thermo Fisher Scientific, Waltham, MA, USA) at the LDCO. It is worth noting that the Environmental SARS-CoV-2 Bio-T kit uses a pepper mild mottle virus (PMMoV) gene to help normalize viral loads by using the human fecal matter content present in the wastewater sample. PMMoV is an RNA virus that infects pepper plants, which is present in wastewater, and is highly associated with human fecal content (30).

Enteric virus genome detection assays were performed in parallel by the French National Reference Centre for gastroenteritis viruses. Norovirus genogroup I (NoV GI) and norovirus genogroup II (NoV GII), rotavirus A (RVA), sapovirus (SaV) genogroups I, II, IV, and V, and human astrovirus (hAstV) were detected using specific semi-quantitative in-house RT-PCR assays (NF EN ISO 15189 certified), as previously performed (31–36); for primer and probes sequences, see Table S2. Adenoviruses (AdVs) were detected using the Adenovirus R-gene PCR kit from bioMérieux (Marcilly l’Étoile, France), according to the manufacturer’s instructions. Internal quality controls were used to validate the assays. All enteric RT-PCRs were performed on a QuantStudio 5 Real-Time PCR System (Life Technology, ThermoFisher Scientific). A standard curve was generated using a quantified plasmid for PPMoV and SARS-CoV (E and N genes) that allows quantification ( Data S3). For the other viruses, plasmids were not available and analyses were qualitative.

Statistical analysis

All analyses were performed with STATA software. Data for PPMoV and SARS-CoV-2 are presented as means ± standard errors of the means. Comparisons between the virus positivity in the two groups, WWTP and NH, were done using the χ² test and the Fisher exact test. A P value of less than 0.05 was considered statistically significant.

RESULTS

Our study was based on sewage samples collected at the outlet of an NH and at the inlet of a WWTP in the same village (Fig. 1). Weekly sampling was performed for 29 consecutive weeks, except weeks 3 and 12 in 2023. During the sample period, from mid-September 2022 to the end of March 2023, the weekly mean ambient temperature ranged from −2.3°C to 16.7°C, and the weekly cumulative rainfall ranged from 0 to 88 mm (Data S4).

Virus positivity

Sample results are presented in Table 1. The means of viral load and Ct for PPMoV in the WWTP (4 × 10⁵ ± 0.6 × 10⁵ copies/mL, Ct 30.31 ± 0.25) and the NH (5.1 × 10⁵ ± 1 × 10⁵ copies/mL, Ct 30.48 ± 0.22) indicated a similar fecal content, making it possible to compare each viral circulation in the WWTP and in the NH. Ct values can be used as a proxy for viral load.

TABLE 1.

Rates of virus positivity in the WWTP and the nursing home

Virus	WWTP		NH		P valueb
	n/total of samplesa	%	n/total of samplesa	%
SARS-CoV-2	18/27	66.7	8/28	28.6	<0.01
Influenza A and B	5/20	25.0	1/21	4.8
RSV A and B	9/22	40.9	3/23	13.0	<0.05
Norovirus GI	19/26	73.1	7/27	25.9	<0.01
Norovirus GII	21/27	77.8	10/27	37.0	<0.01
Sapovirus	21/27	77.8	16/28	57.1
Rotavirus	8/27	29.6	2/27	7.4	<0.05
Astrovirus	0/27	0	0/27	0
Adenovirus	1/27	3.7	0/27	0
Pepper mild mottle virus	27/27	100.0	28/28	100.0

^a Total number of samples is <28 when unsuccessful preliminary tests were done.

^b P values <0.05 and <0.01 are indicated.

Regarding respiratory viruses, most samples from the WWTP were positive for SARS-CoV-2 (66.7%), while the positivity rate was lower in the NH (28.6%) (P = 0.007) (Fig. 2). At the most, W44 and W51, the SARS-CoV-2 load values were around 8.9 × 10⁴ copies/mL in the WWTP reservoir, while in the NH, the mean SARS-CoV-2 load values were equal to 1.95 × 10⁴ copies/mL during 3 weeks at the beginning of October, W39–41. Similarly, the positive rates were higher in the WWTP than in the NH for RSV (40.9% vs 13.0%, respectively) and IV (25.0% vs 4.8%, respectively).

Fig 2.

SARS-CoV-2 virus wastewater detection in Précy-sous-Thil during the study period, from mid-September to end-March 2023. (A) SARS-CoV-2 RT-PCR detection of E gene (dark colors) and N gene (light colors) in the nursing home (orange shades) and the WWTP (blue shades). Missing data are indicated with gray crosses. The references are the weekly flu-like illness (ILI) incidence per 100,000 inhabitants in the administrative area of Précy-sous-Thil (light green histogram) and the weekly number of SARS-CoV-2 cases detected in the regional Dijon Hospital (light gray histogram). (B) SARS-CoV-2 viral load in the nursing home (orange) and in the WWTP (blue). Data are expressed in copies per milliliter, ×10³.

Regarding enteric viruses, caliciviruses [i.e., noroviruses (NoVs) and SaV] were the most frequently detected gastroenteritis viruses throughout the study period. The positive rates were higher in the WWTP than in the NH for NoV GI (73.1% vs 25.9%, respectively; P = 0.001) and NoV GII (77.8% vs 37.0%, respectively; P = 0.003). In contrast, the positive rates for SaV in the WWTP and the NH were not significantly different (77.8% vs 57.1%, respectively; P = 0.152). RSV (WWTP, 40.9%; NH, 13%), IV (WWTP, 25.0%; NH, 4.8%), and RVA (WWTP, 29.6%; NH, 7.4%) presented low frequencies in the nursing home. Milder positive rates were found for RVA (29.6% vs 7.4%, respectively), while AdV (3.7% vs 0%) was barely detected and hAstV was not. Overall, the number of positive samples was lower in the NH than in the WWTP except for SARS-CoV-2 and SaV.

Temporal positivity

The temporal profiles of positivity in the WWTP and NH samples were similar, although in the NH there was a break in the detection of the most common viruses (SARS-CoV-2, NoV GI and GII, and SaV) from weeks 43 to 48.

The detection of SARS-CoV-2 started in September (Fig. 2), on W38/2022 in the WWTP. It remained stable and then decreased in W45/2022 before increasing again from W46/2022 to W52/2022 (i.e., mid-November to late December 2022). From the beginning of 2023 until the end of March 2023, there was almost no detection. The profiles of SARS-CoV-2 detection in the WWTP and the NH were in line with the dynamics of the SARS-CoV-2 epidemics, which was represented by its incidence in the Côte-d’Or department and its detection rate at the DBUH (Data S1). It is worth noting that SARS-CoV-2 was detected in wastewater in mid-September, while the peak of incidence was in early October. As soon as viruses were detected, both the mayor of the village and the director of the NH could have been be notified, showing that data from wastewater samples can provide real-time information without requiring invasive individual testing.

RSV was detected in the wastewater samples during the peak of the epidemic in the DBUH (Fig. 3; Supplemental Data S1), from W43/2022 to W2/2023, in the WWTP and occasionally in the NH. In 2023, from January to the end of March, RSV was almost not detected. Influenza virus was mostly detected from W44/2022, during the pre-epidemic period, as reported by SpF, to W48/2022 in the WWTP, and at one point during W6/2023. However, there was no similarity with the peak of the epidemics reported in the DBUH and SpF databases, which was from W50/2022 to W2/2023 (Fig. 3; Supplemental Data S1). The detection of IV in the WWTP may be the result of a local, early, and brief outbreak in Précy-sous-Thil, although this could not be certified. In the NH, IV was detected only on W9/2023, while all NH residents were vaccinated against IV in the fall of 2022 (Fig. 3).

Fig 3.

Respiratory viruses in wastewater of Précy-sous-Thil: (A) influenza A and B viruses (IV) and (B) respiratory syncytial virus (RSV). RT-PCR detection of IV (A) and RSV (B) in the nursing home (blue curves) and in the WWTP (orange curves). The references are the weekly number of IV and RSV cases, respectively, in the Dijon Burgundy Hospital; histogram (light gray).

NoVs and SaVs were frequently detected in the WWTP and in the NH, except in the NH in November, where there was almost no circulation of enteric viruses (W44–49) (Fig. 4). NoV circulation was expected during the study period, but the high circulation of SaV was rather surprising. SaV mostly causes sporadic cases or epidemics in infants and children, but its prevalence in France is not fully known. In contrast, RVA was only sporadically detected (Fig. 4). Rotavirus is usually responsible for winter epidemics in children, peaking between January and April, but also regularly affects the elderly.

Fig 4.

RT-PCR detection of enteric viruses in the two sites. Norovirus genogroups I (A) and II (B), rotavirus (C), and sapovirus (D) in the WWTP and in the nursing home. Astrovirus and adenovirus were not detected. The reference is the clinical acute gastroenteritis incidence per 100,000 inhabitants in Burgundy [acute gastroenteritis (AGE) incidence, light green].

Finally, no respiratory viruses, including SARS-Cov-2, were detected in the WWTP from W1/2023 to W13/2023, while NoVs and SaV were actively circulating. Our study provides information on relative viral circulation in the two sites, depending on the virus involved. Indeed, concerning the viruses mainly found, we showed that SARS-CoV-2 circulated at the same period in the two sites but that an increased signal in the WWTP wastewater was observed before an increase in NH, suggesting possible viral pressure from outside on the NH. This has implications for wastewater surveillance, emphasizing the importance of WWTP surveillance for SARS-CoV-2. However, the relative circulation in these two sites of NoVs and SaV seems less time-lagged. This implies the need to monitor both sites. The monitoring would help put in place direct interventions via early warning.

DISCUSSION

The purpose of this study was to demonstrate the effectiveness of WBS in a rural area using simple methods for sampling and extraction, combined with RT-PCRs suited to environmental samples for respiratory and enteric virus tracking. This approach enabled us to perform population-based epidemic biomonitoring of a small village through its WWTP and, at the same time, to track viral infections among residents in an NH, which is a relatively enclosed space. Viral circulations in the WWTP and the nearby NH were compared. Wastewater-based epidemiological studies are usually conducted at the inlet of WWTPs, which cover multiple municipalities, in order to collect data on a large scale. Our study compared the local circulation of viruses in the village and the NH based on the results obtained from samples from the two selected sites. The positivity rate for SARS-CoV-2 was considerably higher than those for IV and RSV. NoVs and SaV were the most frequently detected enteric viruses. All viruses found in the WWTP were found in the NH samples; however, we showed that SaV had an unexpected high level of circulation in the NH. Although less than in the initial years of the pandemic, SARS-CoV-2 continued to circulate during fall and winter of 2022–2023, consistent with the drop in temperatures (37, 38). Soon after SARS-CoV-2 was first identified, researchers suggested that the virus could infect enterocytes, which may explain the gastrointestinal symptoms commonly observed in COVID-19 patients (39). However, the presence of SARS-CoV-2 in stool could disrupt the intestinal microbiota and generate inflammation, resulting in diarrhea (40, 41).

Additionally, studies have revealed that SARS-CoV-2 RNA can be detected in stool samples from 50% to 100% of patients who have tested positive for SARS-CoV-2 (42–45). Some viral excretion has also been reported in urine (42, 44), which explains its high detection in wastewater. In the NH, all residents were inoculated with four vaccine doses, and only one resident experienced mild clinical symptoms. COVID-19 vaccines significantly reduce disease severity and mortality, but vaccination does not stop viral shedding (46, 47). The SARS-CoV-2 vaccine has demonstrated efficacy in preventing severe forms of COVID-19, and the spread of the virus in the NH seems to originate from asymptomatic or nearly asymptomatic individuals (48). In accord with these findings, we found rather high SARS-CoV-2 load values in the NH samples during weeks 39–41 despite the vaccination of personnel and residents, which also suggests that, though it does provide clinical protection, the vaccine does not effectively stop viral shedding. Therefore, despite being clinically protected, these individuals are capable of transmitting SARS-CoV-2 to others.

Considering that the annual seasonal patterns of outbreaks can vary, WBS offers a tool for the early identification of local circulation of viruses. In France, in 2022–2023, the epidemic waves of both RSV and IV started earlier than usual and last longer, presumably because of the low circulation of RSV and IV during the COVID-19 pandemic, which may have caused a lack of immune stimulation and subsequently increased susceptibility to these pathogens (49, 50). SARS-CoV-2 circulation may have been influenced by other infection dynamics through viral interference (50, 51). Moreover, the non‐pharmaceutical interventions have variable efficacy on the different viruses, including RSV and IV (52, 53), and SARS-CoV-2 (54, 55).

In our study, RSV, but not IV, matched with the epidemiological references. In the NH, where all residents were vaccinated, IV was scarcely detected. We did not observe robust evidence of IV and RSV circulation in WWTP and NH. First, this could be linked to vaccination, particularly in the NH, for flu, but more generally to low shedding of respiratory viruses in feces, except SARS-CoV-2. A recent review reported only 36% of IV-positive and 14% of RSV-positive stools from patients with confirmed viral infections (14). Respiratory viruses in feces may result from swallowing of mucus rather than from gastrointestinal infection (19), explaining their low abundance in wastewater. Some virus excretion has been reported in the urine of IV-positive patients, but none was found for RSV (14). Therefore, viral surveillance in sewer systems is indicative of infections but is less sensitive for RSV and IV. Even if some time points were missing, we show that RSV and influenza can be detected with our method.

Overall, enteric viruses were highly present in wastewater samples from both sites. NoVs and SaVs were frequently detected, as expected. NoVs affect people of all age groups with a global prevalence of 18% (56). GI NoVs are usually involved in food- and waterborne outbreaks throughout the year, whereas GII NoVs are mostly spread person-to-person during the winter season. Here, the two genogroups were detected similarly throughout the study period in both the WWTP and the NH. This shows a high circulation of NoVs among the two populations. In particular, GI NoVs were detected as frequently as GII NoVs, even though cases of gastroenteritis involving NoVs are usually due to GII NoVs during the winter outbreak season, especially GII.4, and less frequently to GI NoVs. Surprisingly, SaV was detected as frequently as NoVs in the WWTP despite a much lower reported prevalence of around 3.4% (56–58). The positivity rate for SaV was also high in the NH. Although SaV infections are relatively uncommon and more often linked to children (58), they have been reported at higher positivity rates as an occasional cause of outbreaks in hospitals and other health care facilities (58, 59). In contrast, RVA was rarely detected in either site, although there were a few periods of circulation in the WWTP. While outbreaks can occasionally occur among NH residents and more generally among the elderly, the limited detection of RVA is consistent with the fact that immunity to this virus is acquired in infancy and generally persists throughout life. The low RVA detection in the WWTP effluent may thus be explained by the small number of young children living in the village, who account for only 15.8% of children under 14 years old according to the Insee census (28). It should be noted that the use of diapers by certain bedridden residents likely to be infected poses a potential limitation to virus detection in healthcare facilities. Feces from these individuals are not delivered to the sewer systems, which reduces the sensitivity of monitoring. Nonetheless, WBS can certainly play a key role in controlling the spread of viruses, notably among the elderly, and serve as an early warning system for the implementation of protective measures. Its primary advantage is that it avoids invasive sampling in patients, which is particularly useful for NHs seeing as regular or too frequent nasopharyngeal testing would be less acceptable in asymptomatic frail residents. An increase in positivity rate or viral load in wastewater can indicate the need for reinforced hygiene measures during both pre-epidemic and epidemic phases without invasive sampling.

Virus detection, as reported here, is mostly qualitative and appropriate for WBS. However, although PCRs efficiently detect viral genomes, this does not necessarily indicate virus infectivity. Furthermore, correlating a viral load in wastewater with the number of infected patients is quite challenging due to the variable sum of symptomatic and asymptomatic patients, who often present different viral loads (60). The Ct value and the resulting load depend on various factors including the virus type, the chronology of the infection, the intensity of symptoms, patient age, and overall health status (immunodepression, prematurity, chronic illness, etc.) (61, 62).

The BioSellal environmental reagents utilized in our tests did not require extensive sample pre-treatment prior to PCR testing, despite the various factors affecting virus concentration suggested for SARS-CoV-2 (63). Neither did we include any of the numerous methods or additional steps that have been proposed to enhance virus determination (e.g., pre-treatment using glycine buffer to help virion recovery from organic matter, polyethylene glycol (PEG) precipitation, adsorption-precipitation with aluminum hydroxide, supplemental MgCl₂, ultrafiltration-concentration with Centricon filter, or the use of an Innovalep concentrating pipette) (20, 64–66). While the described pre-concentration steps can somewhat improve recovery, they all add additional costs and complicate the pre-treatment process. During preliminary tests, two pre-treatment strategies were selected: concentration with Centricon and ultracentrifugation. We did not proceed with these pre-treatments because of the low improvement of recovery (at the most, 3.6 Ct) associated with either a higher cost (Centricon) or a cumbersome process (ultracentrifugation). However, we found that both the rapid treatment of water samples and the design of the PCRs were key factors for detection. Some recent studies suggested that viruses may partition more favorably into the solid fraction of wastewater matrices than the liquid fraction. Thus, pre-treatments that contribute to removing solid-associated viruses could enhance virus recovery (35, 65, 67–69). Although the sewer system design may affect genome detection and final virus concentrations in samples due to potential degradation of viral RNAs during transit within the network (70), our study reports similar results between the two sampling sites. This demonstrates that our approach can be easily implemented in many places to enable local monitoring of respiratory and enteric viruses.

Finally, to the best of our knowledge, this report is one of the first involving WBS for a variety of viruses in an NH. The implementation of WBS in both WWTP and NH provided precise monitoring of virus spread in the environment. Our study demonstrates that wastewater-based epidemiological studies on a community level can effectively detect the occurrence of respiratory and enteric virus infections. While this study was limited to weekly sampling and focused on a limited period covering the winter epidemic peak of respiratory and enteric viruses in a temperate region, continuous, year-round WBS combined with increased sampling frequency during sensitive periods may provide informative and detailed data on circulating human pathogens and help to identify the onset of epidemics. Our data underline the importance of implementing WBS at key sites to report on the circulation or the emergence of infectious diseases. The simplicity of our method makes it well suited for fast deployment and use in rural areas. Additional uses for this method could include sampling in lakes and ponds to look for emergent or animal viruses. Further studies detecting specific strains or variants could significantly assist in providing valuable information to safeguard public health.

Conclusion

Our study was designed to assess the potential of community-scale effluent monitoring in a rural area as a means of rapid and efficient detection of pathogens within the served community. We provide evidence of significant detection of respiratory and enteric viruses in both the WWTP and NH. Moreover, this method could be included in local, regional, national, and global WBS networks looking for human and animal pathogens. To achieve optimal health outcomes, the interconnection between people, animals, plants, and their shared environment must be considered, as proposed by the One Health concept.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.01158-24.

File S1. aem.01158-24-s0001.xlsx.

Epidemiological data.

File S3. aem.01158-24-s0002.xlsx.

Standard curve SARS-CoV-2.

File S4. aem.01158-24-s0003.xlsx.

Meteorological data.

File S5. aem.01158-24-s0004.docx.

Virus positivity statistics.

Table SI. aem.01158-24-s0005.docx.

Primers for detection of gastroenteritis viruses.

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