Journal Pre-proof J o u r n a l P r e -p r o o f 2 ABSTRACT Infection with SARS-CoV-2, the etiologic agent of the ongoing COVID-19 pandemic, is accompanied by the shedding of the virus in stool. Therefore, the quantification of SARS-CoV-2 in wastewater affords the ability to monitor the prevalence of infections amongst the population via wastewater-based epidemiology (WBE). In the current work, SARS-CoV-2 RNA was concentrated from wastewater in a catchment in Australia and viral RNA copies were enumerated using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) resulting in two positive detections within a six day period from the same wastewater treatment plant (WWTP). The estimated RNA copy numbers observed in the wastewater were then used to estimate the number of infected individuals in the catchment via Monte Carlo simulation. Given the uncertainty and variation in the input parameters, the model estimated a median range of 171 to 1,090 infected persons in the catchment, which is in reasonable agreement with clinical observations. This work highlights the viability of WBE for monitoring infectious diseases, such as COVID-19, in communities. The work also draws attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater.Journal Pre-proof de Roda Husman, 2020;Medema et al., 2020;Wu et al., 2020). Here, we report the first evidence for the presence of SARS-CoV-2 RNA in wastewater in Australia. Our preliminary findings demonstrate the applicability of WBE for COVID-19 surveillance as a potential tool for public health monitoring at the community level. Journal Pre-proof conditions ranging from 3 × 20 s at 8,000 rpm at a 10 s interval. From here on RNA was extracted using RNeasy Power Microbiome kit as per manufacturer's instruction. Method B began with centrifugation of wastewater samples (100-200 mL) at 4,750 g for 30 mins. Supernatant was then removed carefully without disturbing the pellet and Journal Pre-proof 4 /reaction) of Oncorhynchus keta (O. keta) was added in the DNAse and RNAse free water and the Cq value obtained acted as a reference point. If the Cq value of a wastewater sample increases compared to the reference Cq value, the sample is considered to have PCR inhibitors. Wastewater samples with a 2-Cq (quantification cycle) delay was considered to have RT-qPCR inhibition (Staley et al., 2012). All RNA samples were Journal Pre-proof average quality of 15 (SLIDINGWINDOW:4:15). Reads were cropped to 120bp (CROP:120), with any less than 120bp in length discarded (MINLEN:120). Overlapping forward and reverse reads were merged using bbmerge from the BBMap suite (ver. 38.41, https://sourceforge.net/projects/bbmap/). Quality-controlled, merged reads were then mapped Journal Pre-proof describing it, through the model 10,000 times. For each estimate of infected persons, the corresponding prevalence was calculated by dividing the number of persons infected by the number of persons in the catchment. Sensitivity of the estimated number o...

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We investigated the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater samples in southern Louisiana, USA. Untreated and treated wastewater samples were collected on five occasions over a four-month period from January to April 2020. The wastewater samples were concentrated via ultrafiltration (Method A), and an adsorption–elution method using electronegative membranes (Method B). SARS-CoV-2 RNA was detected in 2 out of 15 wastewater samples using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays (CDC N1 and N2). None of the secondary treated and final effluent samples tested positive for SARS-CoV-2 RNA. To our knowledge, this is the first study reporting the detection of SARS-CoV-2 RNA in wastewater in North America, including the USA. However, concentration methods and RT-qPCR assays need to be refined and validated to increase the sensitivity of SARS-CoV-2 RNA detection in wastewater.

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