key: cord-0764255-571hf9gi authors: Hughes, B.; Duong, D.; White, B. J.; Wigginton, K.; Chan, E.; Wolfe, M.; Boehm, A. title: Respiratory Syncytial Virus (RSV) RNA in wastewater settled solids reflects RSV clinical positivity rates date: 2021-12-05 journal: nan DOI: 10.1101/2021.12.01.21267014 sha: 0647a5b9d398eab63d701de6963c959fedd7952b doc_id: 764255 cord_uid: 571hf9gi Wastewater based epidemiology (WBE) uses concentrations of infectious agent targets in wastewater to infer infection trends in the contributing community. To date, WBE has been used to gain insight into infection trends of gastrointestinal diseases, but its application to respiratory diseases has been limited to COVID-19. Here we report Respiratory Syncytial Virus (RSV) genomic RNA can be detected in wastewater settled solids at two publicly owned treatment works (POTWs). We further show that its concentration in settled solids is strongly associated with clinical positivity rates for RSV at sentinel laboratories across the state in 2021, a year with anomalous seasonal trends in RSV disease. Given that RSV infections have similar clinical presentations to COVID-19, can be life threatening for some, and immunoprophylaxis distribution for vulnerable people is based on outbreak identification, WBE represents an important tool to augment current RSV surveillance and public health response efforts. Respiratory syncytial virus (RSV) is a single-stranded, -sense RNA, enveloped virus that infects the 45 respiratory tract of humans. RSV infections are usually self-limiting in healthy individuals but can 46 become severe, particularly for infants and immunocompromised or elderly individuals. Symptoms 47 include congestion, dry cough, fever, and trouble breathing in severe cases. Up to 1-2% of infants under 48 6 months with RSV are hospitalized, and RSV is responsible for up to a third of hospitalizations for acute 49 respiratory infections (ARI) in children <5 in the United States 1 . RSV is reportable to public health 50 officials in some locations in the United States, but many RSV infections are not identified because 51 people do not require or seek care and testing is not always done when patients encounter the 52 healthcare system. Given the similarities in presentation between COVID-19 and RSV, the challenge in 53 obtaining data on RSV in communities, and the high risk associated with infection in infants, there is a 54 need to better understand RSV incidence among US populations. 55 56 Wastewater-based epidemiology (WBE) is a population health monitoring approach that uses 57 concentrations of pathogenic agents in wastewater to gain insight into infection rates in the contributing 58 community 2 . Using wastewater to understand community infection rates is a passive method that has 59 the benefit of capturing inputs from an entire community without a participation burden. WBE has been 60 used to gain insight into community infection rates of non-enveloped viruses including poliovirus 3 , 61 norovirus 4 , and hepatitis A 5 , which are excreted in stool of infected individuals and transmitted via the 62 fecal-oral route. Widespread implementation of WBE since the beginning of the COVID-19 pandemic 63 shows strong associations between SARS-CoV-2 RNA in wastewater influent 6-8 and settled solids 9-11 and 64 laboratory-confirmed COVID-19 incidence rates. Although COVID-19 is primarily a respiratory disease, 65 SARS-CoV-2 RNA is excreted in stool of those infected 12-14 . length, GC content, and melt temperatures) are provided in Table S1 . 107 108 Primers and probe sequences were screened for specificity in silico using NCBI Blast, and then tested in specificity testing was between 10 3 and 10 4 copies per well; each target from the respiratory panel was 118 run in a single well, and the SARS-CoV-2 gRNA was tested in 8 wells. 119 120 Wastewater samples. Two publicly owned treatment works (POTWs) that serve populations of Santa 121 Clara County, California, USA (San José-Santa Clara Regional Wastewater Facility, RWF) and San Mateo 122 County, California, USA (Silicon Valley Clean Water, SVCW) were included in the study. RWF and SVCW 123 serve approximately 1,500,000 and 220,000 people, respectively; further descriptions of these POTWs 124 can be found in Wolfe et al. 22 . Samples of approximately 50 mL of settled solids were collected by POTW staff using sterile technique in 127 clean, labeled bottles. Grab samples were taken at SVCW. At RWF, POTW staff manually collected a 24 h 128 composite sample 21 . Samples were immediately stored at 4°C, transported to the lab, and processed 129 within 6 hours of collection. Samples were collected daily for a larger COVID-19 wastewater surveillance effort starting in November 132 2020 22 , and a subset of these samples are used in the present study and were chosen to span the period 133 prior to and including increasing RSV incidence in the state 23 . Although RSV incidence usually peaks in 134 the winter, the 2020-2021 was an anomalous year with limited to no RSV cases and increasing cases 135 beginning in the summer (Figure 1 ). One sample was analyzed every other week during January, 136 February, and March; one sample was analyzed per week in April and May, and then three samples per 137 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. gRNA. The ddRT-PCR methods applied to wastewater solids to measure the SARS-CoV-2 N gene, PMMoV, and 165 BCoV are provided in detail elsewhere, including details relating to EMMI 31 guidelines and are not 166 repeated here 22 . Here, we provide methods for the multiplexed SARS-CoV-2 N and RSV N gene assays 167 which have not been reported previously. For this multiplexed assay, ddRT-PCR was performed on 20 µl 168 samples from a 22 µl reaction volume, prepared using 5.5 µl template, mixed with 5.5 µl of One-Step RT- Droplets were generated using the AutoDG Automated Droplet Generator (Bio-Rad, Hercules, CA). PCR 176 was performed using Mastercycler Pro (Eppendforf, Enfield, CT) with with the following cycling 177 conditions: reverse transcription at 50°C for 60 minutes, enzyme activation at 95°C for 5 minutes, 40 178 cycles of denaturation at 95°C for 30 seconds and annealing and extension at 61°C for 30 seconds, 179 enzyme deactivation at 98°C for 10 minutes then an indefinite hold at 4°C. The ramp rate for 180 temperature changes were set to 2°C/second and the final hold at 4°C was performed for a minimum of 181 30 minutes to allow the droplets to stabilize. Droplets were analyzed using the QX200 Droplet Reader 182 (Bio-Rad). A well had to have over 10,000 droplets for inclusion in the analysis. All liquid transfers were 183 performed using the Agilent Bravo (Agilent Technologies, Santa Clara, CA). 184 185 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. PMMoV were calculated for each week of 2021 through 12 Nov 2021 to match the frequency of RSV 217 positivity rate reporting; the weekly average was calculated using all available data (between 1 and 3 218 data points). We tested the null hypothesis that weekly average wastewater concentrations were not 219 associated with weekly positivity rates from sentinel laboratories using Kendall's tau. We used linear 220 regression to determine the rate of change of RSV positivity with RSV wastewater concentrations. 221 Measurements below the detection limit were replaced with 500 copies/g (the approximate detection 222 limit). All statistical analyses were implemented in R studio (version 1.4.1106). Results and Discussion 225 RSV Assay specificity. In silico analysis indicated no cross reactivity between the RSV assay and 226 deposited sequences in NCBI. When challenged against the respiratory virus panel and SARS-CoV-2 227 gRNA no cross reactivity was observed; and the assay amplified gRNA from both RSV A and RSV B. 228 Positive controls and NTCs run on the sample plate were positive and negative. These results suggest 229 that the RSV ddRT-PCR assay is specific to both RSV subtypes. 230 231 RSV RNA concentrations in wastewater solids. All positive and negative controls were positive and 232 negative respectively, indicating assays performed well and without contamination. BCoV recoveries 233 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. ; https://doi.org/10.1101/2021.12.01.21267014 doi: medRxiv preprint were higher than 10% and PMMoV concentrations within the expected range for the POTW suggesting 234 an efficient and acceptable recovery of RNA during RNA extraction ( Figure S1 ). Concentrations of the RSV N gene in wastewater solids ranged from non-detect to 6.2x10 4 copies/g dry 237 weight. At both POTWs RSV levels were non-detect until late June at which time concentrations began 238 to increase through the last sample collected in mid-November (Figure 1 ). Concentrations of RSV RNA 239 were positively and significantly correlated at the two POTW (Kendall's tau = 0.52, p<10 -12 ). Paired, log10- RNA in wastewater solids when laboratory-confirmed COVID-19 incidence rates are between 1/100,000 257 and 10/100,000 22 . The positivity rates used herein were calculated using data from sentinel laboratories 258 through the state rather than from the sewershed areas, and thus do not provide direct information on 259 population incidence rates. Additional work to compile RSV incidence data in the sewersheds may allow 260 for estimates of disease burden associated with RSV in wastewater beyond outbreak identification and 261 trends. Understanding how RSV RNA in wastewater relates to incidence rates will also require a better 262 understanding of RSV shedding from infected individuals. In particular, there is no quantitative data on 263 RSV RNA concentrations in stool of infected patients and the potential for sputum or other bodily 264 secretions to contribute RSV RNA to wastewater should be considered. The Acknowledgements. This work was funded by a gift from the CDC Foundation. We acknowledge the 280 following individuals for assistance with wastewater solids collection: Payal Sarkar (RWF), Noel Enoki 281 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted December 5, 2021. Tables S1 and S2 show primer design criteria and 286 systematic review process, and Figures S1 and S2 show PMMoV results and RSV normalized by PMMoV, 287 respectively. 288 289 290 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted December 5, 2021. ; https://doi.org/10.1101/2021.12.01.21267014 doi: medRxiv preprint 429 Figure 2 . Weekly state-wide RSV positivity rate at sentinel laboratories versus weekly average RSV 430 concentrations in wastewater settled solids at two POTW. Linear curve fit between log10-transformed 431 concentrations and positivity rate is shown for both POTWs. Samples with RSV that are non-detect are 432 shown with a value of 500 cp/g. 433 434 . CC-BY-NC 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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