key: cord-0771319-mgegxka0
authors: Gonçalves, José; Koritnik, Tom; Mioč, Verica; Trkov, Marija; Bolješič, Maja; Berginc, Nataša; Prosenc, Katarina; Kotar, Tadeja; Paragi, Metka
title: Detection of SARS-CoV-2 RNA in hospital wastewater from a low COVID-19 disease prevalence area
date: 2020-10-28
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.143226
sha: cf95e05cf8e855512ed570534f453f9d1bc1bf86
doc_id: 771319
cord_uid: mgegxka0

Previous studies on SARS-CoV and MERS-CoV reported the detection of viral RNA in the stool of both symptomatic and asymptomatic individuals. These clinical observations suggest that municipal and hospital wastewater from affected communities may contain SARS-CoV-2 RNA. Recent studies have also reported the presence of SARS-CoV-2 RNA in human feces. Wastewater-based epidemiology (WBE) is a promising approach to understand the prevalence of viruses in a given catchment population, as wastewater contains viruses from symptomatic and asymptomatic individuals. The current study reports the first detection of SARS-CoV-2 RNA in untreated wastewater in Slovenia. Two sizes of centrifugal filters were tested: 30 kDa and 10 kDA AMICON® Ultra-15 Centrifugal Filters, where 10 kDA resulted in a higher concentration factor and higher recovery efficiency. The results in hospital wastewater show that WBE can be used for monitoring COVID -19 and could be applied in municipal wastewater treatment plants as a potential complementary tool for public health monitoring at population level.

Coronavirus Disease 2019 is an ongoing global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Symptoms of COVID-19 patients include dry cough, myalgia, fatigue, fever, shortness of breath, diarrhea, anosmia and ageusia Guan et al., 2020; Huang et al., 2020; Sharifian-Dorche et al., 2020; . SARS-CoV-2 has spread to almost all countries and territories of the world with more than 30 million confirmed cases and 950 000 deaths (WHO, 2020) .

Previous studies on SARS-CoV and MERS-CoV reported the detection of viral RNA in the stool of both symptomatic and asymptomatic individuals (Corman et al., 2016; Leung et al., 2003) . Recent studies also reported the presence of SARS-CoV-2 RNA in human stool (Cai et al., 2020; Gao et al., 2020; Tang et al., n.d.) . These clinical observations suggest that municipal and hospital wastewater from affected communities may contain SARS-CoV-2 viral particles and/or RNA (Ahmed et al., 2020a) . Wastewater-based epidemiology (WBE) is a promising approach to understand the prevalence of viruses in a given catchment population, as wastewater contains viruses from symptomatic and asymptomatic individuals.

WBE is particularly important for early warning of disease outbreaks and information on the effectiveness of public health interventions, as previously demonstrated for enteric viruses such as norovirus, rotavirus, hepatitis A virus and poliovirus (Asghar et al., 2014; Gonçalves et al., 2018; Hellmér et al., 2014) . When it was first proposed to track SARS-CoV-2 RNA, the prevailing scientific opinion was that the virus can be released into wastewater at insufficiently high rates and that both the viral particles and its RNA may be too unstable to A concentration step is usually employed as a preceding preparative step prior to molecular detection of viral DNA or RNA from wastewaters. SARS-CoV-2 RNA has been concentrated from wastewater by PEG precipitation , electronegative filters, ultracentrifugal membrane filters (Medema et al., 2020; Sherchan et al., 2020) , ultracentrifugation (Wurtzer et al., 2020) , Al(OH) 3 adsorption-precipitation (Randazzo et al., 2020) and by the adaptation of the standard WHO protocol for Poliovirus surveillance (WHO, 2003) .

The current study reports the first detection of SARS-CoV-2 RNA in untreated hospital wastewater in Slovenia. The results show that WBE is a potential tool that could be used as an early warning for COVID-19 and could be applied in municipal wastewater treatment plants as a potential complementary tool for public health monitoring at population level.

Anonymous positive nasopharyngeal swab specimens for SARS-CoV-2 were collected from patients diagnosed with COVID-19 in the Department for Public Health Microbiology Ljubljana, National Laboratory for Health, Environment and Food. These native samples were used to spike the wastewater samples to test the performance of the concentration step and serially diluted 10-fold to 1 x 10 5 followed by RT -qPCR detection in triplicates. A standard curve was obtained by plotting the Cq values for each dilution against the Log 10 dilution for RdRP and E genes and used to calculate the recovery efficiency and concentration factor for each gene, and relative quantification of the water samples tested.

One liter of untreated hospital wastewater samples per day were collected from June 1 to 15, 2020 (15 wastewater samples) from a pumping station representing the Department of J o u r n a l P r e -p r o o f Infectious Diseases, University Medical Center Ljubljana catchment area, starting when no positive COVID-19 patients were admitted to the hospital. As part of the main hospital complex in Slovenia, the Department of Infectious Diseases has 10 beds in the intensive care unit, 68 beds for adult patients on 3 wards and 51 beds for children and accompanying adults.

The sampling personnel wore standard personal protective face equipment during wastewater sampling, including long trousers, steel-capped boots, hard hats, face mask, safety goggles and gloves. Samples were collected using a refrigerated automatic sampler (Avalanche, Teledyne ISco, USA), resulting in a 24-hour cumulative sample (set to sample 70 ml every 10 minutes). The samples were transported to the laboratory on ice and stored at -70 ºC until further analysis.

Wastewater samples were mixed and 100 ml of each sample was used for the concentration with 30 kDa and 10 kDa Amicon® Ultra 15 Centrifugal Filters (Merck KGaA, Germany), as described by Qiu et al., 2016 . The samples were pre-filtered with a glass fiber filter membrane with 0.7 µm pore size (Sartorius AG, Germany). The centrifugal devices had a sample volume of 20 ml, therefore, the 100 ml sample was divided evenly and centrifuged five times at 4000

x g for 20 minutes. A fraction was collected before the concentration step (BC), the sample that passed through the filter (FC), and the concentrated sample that remained on the filter (E). Each fraction was stored at -70°C until further analysis. A Negative Control of Isolation (NCI) was used with each viral RNA extraction set and prepared in the same manner as a sample, except that nuclease-free water was added in place of the sample. The NCI was used to monitor possible cross-contamination during viral RNA extraction.

SARS-CoV-2 RNA was detected by targeting the genes E and RdRP, as described by Corman The cycle conditions for RT-qPCR assays were: 55 °C for 5 min for reverse transcription, followed by 95 °C for 5 min and 45 cycles of 95 °C for 5 s, 60 °C for 15 s and 72 °C for 15 s.

All experiments were performed in three repetitions. A Negative Control (NC) and a Positive Control (PC) were added to each RT -qPCR to monitor the performance of the RT-qPCR. The quantification cycle (Cq) for each individual amplification was determined using the software 2.6. Calculation of RdRP and E genes recovery efficiency and concentration factor 1 mL of anonymized positive nasopharyngeal swab specimens were spiked into 1 mL of a wastewater sample and incubated at room temperature at a rate of 500 rpm for 15 minutes.

Total viral RNA was extracted from the spiked sample. The extracted RNA was 10-fold serially diluted in nuclease-free water from neat 1 x 10 5 and assayed for RdRP and E genes with RT-qPCR in triplicate. A standard curve was obtained by plotting the Cq values for each dilution against the Log 10 dilution for each target (Figure 1 ). Using the equation for the standard curve, the recovery efficiencies and concentration factors for RdRP and E gene were calculated as follows: Where E stands for the eluted sample remaining on the filter and BC for the sample before J o u r n a l P r e -p r o o f concentration.

Based on the properties of SARS-CoV-2, especially the fact that it is a spherical particle with a diameter between 60 and 140 nm ( replicates and a clear amplification was observed in at least one of the two targets.

As summarized in In total, 13.4% (2/15) of the samples were positive before any concentration step, 26.7% In previous studies (Sherchan et al., 2020) Centricon® Plus-70 was used, which allows the concentration of viruses from larger water volumes in one step. However, its use requires a centrifuge that can hold a volume of 70 ml, which is not generally available in clinical microbiology laboratories. The AMICON® Ultra-15 was able to hold a volume of up to 20 ml in a standard-sized centrifuge, making it easier to use in other laboratories with similar J o u r n a l P r e -p r o o f settings (Medema et al., 2020; Wu et al., 2020) .

30 kDa and 10 kDA AMICON® Ultra-15 Centrifugal Filters successfully concentrated and recovered SARS-CoV-2 RNA from wastewater as shown in TABLE 2, where 10 kDA leads to a higher concentration factor and higher recovery efficiency, but also to higher variability.

Ultrafiltration based on centrifugal filters seems to be an efficient method to recover SARS-CoV-2 RNA. These methods were previously used for the concentration of viruses from wastewater and environmental waters (Ikner et al., 2012; Rosario et al., 2009) laboratory equipment is required for the isolation of SARS-CoV-2 in cell cultures. In the current study, the Cqs for both targets ranged from 29.65 to 38.12. The observed Cqs were slightly lower than in previous studies, where the Cqs ranged from 34 to 40 (Medema et al., 2020; Randazzo et al., 2020; Wu et al., 2020) .

During the sampling period, the number of hospitalized patients with COVID-19 was low and ranged from 0 to 4. As shown in Figure 2 , SARS-CoV-2 RNA was detected in hospital wastewater when only one patient was hospitalized. The hypothetical number of copies were calculated according to the strandard curve obtained from the spiked wastewater sample (section 2.6.). The standard curve obtained is a limitation in the current study, as it may not mimic the potential association of SARS-CoV-2 RNA with particles. In addition, the variation in the hypothetical number of copies may be due to the composition of the wastewater, particularly turbidity, with the strong precipitation and/or differences in viral shedding rate of the patients. Current results show that surveillance of wastewater for SARS-CoV-2 RNA provides a useful epidemiological approach that can help to monitor the ongoing pandemic and support public health measures. The present study complements the increasing number of studies that are establishing an important link between wastewater surveillance and epidemiology COVID-19.

The present study is the first to report the detection of SARS-CoV-2 RNA in wastewater in Slovenia using RT-qPCR and it shows that viral concentration methods are an essential step to accurately detect SARS-CoV-2 RNA in wastewaters. The results from this study show that 10 kDA centrifugal filters can be a successful method to concentrate SARS-CoV-2 RNA from J o u r n a l P r e -p r o o f Copies

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TABLES TABLE 1. Primers and probes used to detect SARS-CoV-2 RNA and their sequences