Wastewater Surveillance Can Uncover Circulation of RSV, Influenza, Other Viruses

Evaluation of wastewater for respiratory viruses is effective, can yield results within 24 hours, and can help to guide public health programs and clinical decision-making.

Wastewater surveillance performed during the COVID-19 pandemic yielded useful information on respiratory viruses circulating in the community, including RSV, influenza, rhinovirus, and seasonal coronaviruses. These are among study findings recently published in The Lancet Microbe.

Wastewater-based epidemiology was used in monitoring infection rates for SARS-CoV-2, influenza A, and respiratory syncytial virus (RSV) at a community level. For the current study, investigators retrospectively analyzed wastewater surveillance data gathered during the pandemic to gain broader understanding of the circulation and occurrence of viral respiratory infections.

The current analysis involved 216 samples obtained at San José-Santa Clara Regional Wastewater Facility in California from February 1, 2021, to June 21, 2022. The researchers developed and validated hydrolysis probe-based reverse transcription-polymerase chain reaction (RT-PCR) assays that target respiratory viral genomes and applied the assays to wastewater solids obtained 3 times per week at that wastewater treatment plant over a 17-month period during the COVID-19 pandemic.

The RT-PCR assays were used to measure concentrations of respiratory syncytial virus (RSV) A and RSV B, influenza A (IAV) and influenza B (IBV), human metapneumovirus (HMPV), human parainfluenza (HPIV; 1-4), seasonal human coronaviruses (HCoVs 229E, OC43, NL63, and HKU-1), and human rhinovirus (HRV) RNA. Viral RNA concentrations were then compared with positivity rates for viral infections from clinical specimens submitted to the California Sentinel Clinical Laboratories to assess concordance in the data.

Wastewater-based epidemiology can be used to obtain information on circulation of respiratory viruses at a localised, community level without the need to test many individuals because a single sample of wastewater represents the entire contributing community.

RNA for all viruses was detected in the wastewater samples, though in small amounts for IAV, IBV and RSV A. The median (interquartile range [IQR]) concentrations in units of copies per gram in order of the highest to lowest (with zero representing nondetection) were:

  • 35,000 (17,000-56,000) for HCoV;
  • 4300 (0-9500) for HRV;
  • 3500 (1400-6300) for HPIV;
  • 1700 (0-5400) for RSV B;
  • 760 (0-2000) for HMPV;
  • 0 (0-660) for IAV;
  • 0 (0-0) for IBV; and
  • 0 (0-0) for RSV A.

Notably, viral RNA from IAV was detected in 60 (28%) samples, in 12 (6%) samples for IBV, and in 30 (14%) samples for RSV A. The median concentration for SARS-CoV-2 was 48,000 (25,000-130,000) copies per gram.

Concentrations were generally lowest in February 2021 and increased until January, 2022, when RNA concentrations for all viruses had a steep decrease. Afterward, viral RNA concentrations began to increase until the end of the study except for RSV B.

With respect to HCoV, concentrations of OC43, HKU-1, 229E, and NL63 RNA in 18 samples were measured to assess which HCoV were circulating during the study period. OC43 RNA accounted for most of the total HCoV RNA in 15 of 18 samples.

The investigators also evaluated whether state-aggregated positivity rates for respiratory viral infections were associated with wastewater concentrations of viral RNA, and the associations were significant and positive for all viruses with the exception of IBV. The Kendall’s tau values between positivity rates and wastewater concentration were 0.47 (P <10–7) for HRV, 0.45 (P <10–5) for IAV, 0.52 (P <10–9) for HPIV, 0.57 (P <10–10) for RSV (with use of RSV B wastewater concentrations as RSV A was mostly nondetected), 0.32 (P =.0002) for HMPV, 0.32 (P =.0001) for HCoV, and -.010 (P =.92) for IBV. Wastewater concentrations and positivity rates combined among viruses were positively correlated (tau =0.43; P <10–15).

The state-aggregated positivity rates for individual HCoV infections were comparable to those of the relative occurrence of their RNA in wastewater solids.

The researchers noted that their analysis between wastewater data and clinical data was limited by the use of passive surveillance data and the lack of local data matching the sewershed on respiratory disease occurrence.

“Wastewater-based epidemiology can be used to obtain information on circulation of respiratory viruses at a localised, community level without the need to test many individuals because a single sample of wastewater represents the entire contributing community,” said the study authors. “Results from wastewater can be available within 24 h of sample collection, generating real time information to inform public health responses, clinical decision making, and individual behaviour modifications.”

Disclosure: Some of the study authors declared affiliations with a research organization. Please see the original reference for a full list of authors’ disclosures.


Boehm AB, Hughes B, Duong D, et al. Wastewater concentrations of human influenza, metapneumovirus, parainfluenza, respiratory syncytial virus, rhinovirus, and seasonal coronavirus nucleic-acids during the COVID-19 pandemic: a surveillance study. Lancet Microbe. Published online March 22, 2023. doi: 10.1016/S2666-5247(22)00386-X