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Respiratory viruses in wastewater compared to clinical samples, Leuven, Belgium – Volume 30, Issue 1 – January 2024 – Journal of Emerging Infectious Diseases

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Author affiliation: KU Leuven Rega Institute, Leuven, Belgium (A. Rector, M. Bloemen, M. Thijssen, M. Van Ranst, E. Wolants); KU Leuven Academic Center for General Practice, Leuven (B. Pussig); Leuven University Hospital (K. Beuselinck, M. Van Ranst).

Since the beginning of the COVID-19 pandemic, wastewater-based surveillance has been used to track circulating levels of SARS-CoV-2 (1,2). To this end, we started collecting samples from a regional wastewater treatment plant in Leuven, Belgium, in December 2020. We found that sewage-based surveillance is an objective indicator of SARS-CoV-2 community circulation. This can be of great value if testing is required. limited (3).

Many patients with acute respiratory infections (ARI) do not seek medical attention, allowing these infections to go undetected. Obtaining detailed information on the circulation of respiratory viruses in communities is key to understanding the societal burden of respiratory viruses. This knowledge could enable better prediction and management of outbreaks and guide doctors’ diagnoses. Current approaches are typically based on limited reporting by sentinel physicians and laboratories, which can lead to significant data bias. We investigated whether wastewater sampling could provide an alternative method for monitoring the circulation of respiratory pathogens at the population level.

We used an in-house developed multiplex quantitative PCR respiratory panel to evaluate 112 wastewater samples collected weekly over a 2-year period at a large regional treatment plant in Leuven for the presence of respiratory pathogens. Screened (Appendix) (Four). We investigated whether the respiratory viruses found in the wastewater matched those detected in samples from patients with respiratory infections at the University Hospital Leuven (UZL).Five). At UZL, patient samples were only tested with a respiratory panel in case of severe lower respiratory tract infections in immunocompromised or critically ill patients. Therefore, these clinical samples are not completely representative of locally circulating respiratory pathogens, especially if those pathogens primarily cause mild infections. Where possible, clinical sample data were supplemented with epidemiological data from surveillance laboratories across Belgium reported by Sciensano, but these were only available for a limited number of viruses (6). Nonpharmacological interventions during the coronavirus disease (COVID-19) pandemic influenced the timing and levels of viral circulation.

Figure 1

Respiratory viruses measured in wastewater and positive clinical samples, Leuven, Belgium, January 2021 to December 2022: A) Influenza viruses.  B) respiratory syncytial virus.  C) HMPV; D) PIV-1; E) PIV-2; F) PIV-3; G) PIV-4; H) AdV. , dots represent individual measurements) and the weekly number of PCR-positive tests detected in UZL (bar graph) shows the evolution of viruses detected in wastewater. Plots were created using R version 4.1.1 (The R Foundation for Statistical Computing, and the ggplot2 package version 3.3.5 ( generated. A larger version of this figure is available at  AdV, adenovirus;  Ct, cycle threshold.  HMPV, human metapneumovirus.  PIV, parainfluenza virus;  Respiratory syncytial virus, respiratory syncytial virus.  UZL, University Hospital Leuven.

Figure 1. Respiratory viruses and positive clinical samples measured in wastewater, Leuven, Belgium, January 2021 to December 2022: A) Influenza virus. B) respiratory syncytial virus. C) HMPV; D) PIV-1; E) PIV-2; F) PIV-3; G) PIV-4; is)…

Influenza A was repeatedly identified from mid-February to mid-May 2022 (Figure 1, panel A). This pattern is consistent with positive clinical samples at UZL, where the peak of influenza A was from March to May 2022, with few cases occurring outside of that period. This is also consistent with data from Ciensano, which showed a mild influenza season in 2020-21 and a late season (end of February to end of April) in 2021-22.6), caused almost exclusively by influenza A (7). The beginning of the 2022-2023 influenza epidemic, with simultaneous influenza A and B strains, was reflected in positive reactions in wastewater samples as of mid-December.

The off-season peak of respiratory syncytial virus (RSV) in spring 2021, seen in clinical samples from UZL and data reported by Sciensano, was reflected in positive wastewater samples from March to July 2021 (Figure 1, panel B). From mid-October 2021 to the end of July 2022, RSV was detected in almost all wastewater samples. Ciensano’s data also showed that in the 2021-22 season he had a low continued presence of RSV (6). Since August 2022, RSV has re-emerged in wastewater. Levels increased in November and December 2022. The number of positive clinical specimens at UZL and sentinel laboratories remained low until the end of October 2022, followed by a strong RSV outbreak in November and December 2022.

From late September to December 2021, human metapneumovirus was almost continuously detected in wastewater, consistent with a large number of positive samples at UZL (Figure 1, Panel C). Human metapneumovirus reappeared in wastewater in late October 2022, followed by an increase in positive samples at UZL in November and December.

Parainfluenza virus (PIV) type 1 was mainly detected in wastewater samples in autumn 2022, coinciding with the increase in positive cases in UZL. PIV-2 has been sporadically detected in wastewater since autumn 2021, corresponding to a low number of positive cases in UZL from November 2021 to November 2022. PIV-3 was almost always detected in wastewater samples. A clear peak occurred from February to May 2021, coinciding with the number of positive samples at UZL. PIV-4 was detectable from August to December 2021 and from September to December 2022, with sporadic outbreaks during that time. This data also shows an association with the number of positive samples in UZL (Figure 1, panels D-G).

Figure 2

Number of respiratory viruses and positive clinical specimens measured in wastewater, Leuven, Belgium, January 2021 to December 2022: A) HBoV.  B) EV/RV and EV-D68.  C) HPeV; D) SARS-CoV-1; SARS-CoV-2; and MERS-CoV.  E) HCoV-NL63; F) HCoV-229E; G) HCoV-OC43; H) HCoV-HKU-1. The graph shows the weekly number of PCR-positive tests detected in the in-house developed multiplex quantitative PCR respiratory panel (line graph, dots represent individual measurements) and UZL (bar graph) in wastewater. It shows the evolution of the detected viruses. Plots were created using R version 4.1.1 (The R Foundation for Statistical Computing, and the ggplot2 package version 3.3.5 ( generated. A larger version of this figure is available at  Ct, cycle threshold.  EV, enterovirus.  HBoV, bocavirus; HCoV, human coronavirus.  HPeV, parechovirus.  MERS-CoV, Middle East Respiratory Syndrome Coronavirus.  RV, rhinovirus;  UZL, University Hospital Leuven.

Figure 2. Number of respiratory viruses and positive clinical specimens measured in wastewater, Leuven, Belgium, January 2021 to December 2022: A) HBoV. B) EV/RV and EV-D68. C) HPeV; D) SARS-CoV-1; SARS-CoV-2; and MERS-CoV. workman)…

Adenovirus and human bocavirus (HBoV) were consistently detected at high concentrations in all wastewater samples (Figure 1, panel H; Figure 2, panel A). This finding is consistent with the continuous circulation of adenovirus in 2020-2021 and 2021-22 reported by Sciensano and previous studies on ARI samples in which adenovirus infections were detected throughout the year. Masu(8). The continued high level detection of adenovirus and HBoV in wastewater is inconsistent with the low number of positive samples found in her ARI patients at UZL. Of the four HBoV genotypes, HBoV1 is mainly associated with respiratory symptoms in children with his ARI, and HBoV2 is associated with gastroenteritis (9,Ten). All HBoV genotypes are known to be present in stool and are frequently detected in wastewater samples (11). Adenovirus infections can cause gastrointestinal symptoms even when the primary site of infection is the respiratory tract (12). The presence of HBoV and adenovirus in wastewater samples may be associated with intestinal infections rather than respiratory infections.

Enteroviruses/rhinoviruses were continuously detected in wastewater, but enterovirus D68 (EV-D68) was only present from early September to December 2021. The highest concentrations were detected in October 2021 (Figure 2, Panel B). These findings, consistent with the increase in EV-D68 infections in Europe in September 2021, suggest a regional outbreak of EV-D68 infections in autumn 2021 (13). In UZL, 33 EV-D68 positive samples were detected during the study period, most of them between October 2021 and January 2022. In mid-September 2022, EV-D68 reappeared in wastewater. Only a small number of positive samples were reported in UZL. In the same region, detection of EV-D68 in wastewater preceded positive cases, indicating that wastewater monitoring can be used as a sensitive early warning signal for the spread of EV-D68.

Human parechovirus (HPeV) infections are common in children. Illnesses range from gastroenteritis and respiratory infections to neurological disorders, especially in newborns (14). His HPeV was consistently detected in almost all wastewater samples throughout the study, but in few positive clinical samples (Fig. 2, panel C). The presence of HPeV in wastewater may be associated with enteric infections or subclinical respiratory infections, although hospital spillover is limited.

SARS-CoV assays in respiratory panels did not detect SARS-CoV-2 until late September 2021 (Figure 2, Panel D). This assay targets a conserved region of the open reading frame 1ab polyprotein gene to enable detection of SARS-CoV-1 and SARS-CoV-2, and therefore has low sensitivity. A decrease in sensitivity was observed in validation experiments on clinical samples, but did not negatively impact accuracy in routine clinical practice (Four). However, this analysis is not sensitive enough for environmental monitoring. Among the four endemic seasonal coronaviruses that infect humans, human coronavirus (HCoV) NL63 was mainly detected in sewage during the fall and winter of 2022, while HCoV-229E and HCoV-OC43 was present in most samples throughout. HCoV-HKU-1 was mainly detected between winter 2021 and summer 2022. All positive clinical samples were also reported during this period (Figure 2, panels E to H). A small number of HCoV-positive clinical specimens were detected at UZL, especially for HCoV-NL63 and HCoV-229E, but this is probably not due to lack of circulation, but rather due to the mild nature of endemic coronavirus infection (usually hospitalization This is thought to be because it does not require

By testing two years of wastewater sample collection using an in-house respiratory panel, we effectively detected the presence and seasonal variation of most respiratory viruses tested. These findings demonstrate the potential of wastewater sampling for population-level pathogen monitoring and early detection of outbreaks, and address limitations associated with limited sentinel testing data. Our study highlights the role of sewage-based epidemiology in complementing clinical surveillance of respiratory viruses, improving our understanding of community viral circulation, and supporting public health efforts.

Dr. Rector is a senior researcher at the KU Leuven Rega Institute for Medical Research in Leuven, Belgium. Her main research interests are molecular evolutionary virology and epidemiology of respiratory viruses, with a focus on respiratory syncytial viruses.


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