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...
Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates ( k ). The average T 90 (time required for 1-log 10 reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T 90 for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58–43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.
The effects of the short-term resuspension of a contaminated anoxic estuarine sediment on solid-phase metal speciation have been studied. Preliminary experiments investigated the oxidation rates of model metal sulfide phases to provide mechanistic information for interpreting the observations on the natural sediment. FeS and MnS model phases were particularly labile and oxidized rapidly in aerated waters. In contrast, CdS, CuS, PbS, and ZnS model phases were kinetically stable over periods of several hours. The oxidation rate of free sulfide (HS -) was significantly slower than that of FeS and MnS. Upon sediment resuspension, the rapid decrease in acid volatile sulfide (AVS) could be accounted for by the oxidation of iron monosulfide phases. Over prolonged resuspension periods (>300 min), AVS decreased to values lower than the simultaneously extracted metals [SEM ) ∑Cd, Cu, Ni, Pb, Zn (1 M HCl extraction, 30 min)] concentration, indicating that a significant fraction of trace metal sulfide phases may be oxidized during resuspension events. During 8-h sediment resuspension experiments, SEM(Cu) increased from 0.1 to ca. 2 µmol/g while the SEM measured for the other metals remained constant. The increase in SEM(Cu) was shown to be an artifact of the AVS/SEM analytical procedure. Fe(OH) 3 , formed through the oxidation of FeS, dissolved upon acidification to produce Fe 3+ (aq), which subsequently oxidized acid-insoluble copper sulfide mineral phases in the sediment. The implications of these observations for natural systems and for the assessment of metal toxicity using AVS/SEM procedures are discussed.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus which causes COVID-19, has spread rapidly across the globe infecting millions of people and causing significant health and economic impacts. Authorities are exploring complimentary approaches to monitor this infectious disease at the community level. Wastewater-based Epidemiological (WBE) approaches to detect SARS-CoV-2 RNA in municipal wastewater are being developed worldwide as an environmental surveillance approach to inform health authority decision-making. Owing to the extended excretion of SARS-CoV-2 RNA in stool, WBE can surveil large populated areas with a longer detection window providing unique information on the presence of pre-symptomatic and asymptomatic cases that are unlikely to be screened by clinical testing. Herein, we analysed SARS-CoV-2 RNA in 24-h composite wastewater samples ( n = 63) from three wastewater treatment plants (WWTPs) in Brisbane, Queensland, Australia from 24 th of February to 1 st of May 2020. A total of 21 samples were positive for SARS-CoV-2, ranging from 135 to 11,992 gene copies (GC)/100 mL of wastewater. Detections were made in a Brisbane South WWTP in late February 2020, up to three weeks before the first cases were reported there. Wastewater samples were generally positive during the period with highest caseload data. The positive SARS-CoV-2 RNA detection in wastewater while there were limited clinical reported cases demonstrates the potential of WBE as an early warning system. When integrated into disease surveillance and monitoring systems, wastewater monitoring data may assist management efforts to identify hotspots and target localised public health responses, such as increased individual testing and the provision of health warnings.
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