Alternate: COVID-19 outbreak monitoring through SARS-CoV-2 wastewater surveillance: a case-study for medium outbreaks in small urban environments

Environmental surveillance through wastewater monitoring has been implemented for a long time for poliovirus and antimicrobial resistance. Different principles for sampling and sample processing have been used in the past and sufficient comparative data have not been published to unequivocally prove that there is an advantage of any one principle over the others for detection of low-level poliovirus circulation in human populations. Most studies published to date on the use of environmental surveillance for SARS-CoV-2 have been from high-resource settings. However, approaches are needed that can be applied in lower-resource settings, where a greater proportion of the population is not connected to sewers and instead uses pit toilets or septic tanks. Possibilities include testing surface water contaminated by sewage. Several scientific teams around the world have begun to investigate the course of the virus in the population through epidemiology of sewage. The Netherlands, Australia and France were among the first to suggest that. According to these studies, the SARS-CoV-2 genome was detected in untreated wastewater at concentrations over 106 copies per liter, while in the corresponding study that carried out in France, in treated wastewater samples, the corresponding concentration of the viral genome was up to nearly 105 copies per liter (Wurtzer et al., 2020). However, more research is needed on the use of computational models to estimate the viral particles each person secretes at a time and how they are diluted in the drains to match the actual number of infected individuals. Here, we describe the implementation of the environmental surveillance for COVID-19 during the 2nd pandemic wave and seek to refine and streamline a protocol that could be applied in medium-sized urban settings during medium-sized outbreaks. 2. Materials & Methods 2.1. Sampling Network and Procedure The wastewater treatment plant was the reference sampling point for the whole city. Moreover, and in collaboration with the Water and Sanitation Department of Ioannina City (DEYAI), we identified sampling spots on the urban matrix that corresponded to specific well-defined urban regions of varying population density and covering the entire city. Finally, we also identified transmission and morbidity hotspots as follows: hospitals, nursing homes, hospices, and refugee camps. A surveillance map was constructed. For the WW treatment plant, WW was subsampled from 24-hour composites of total plant influent collected by WW treatment plant personnel. Subsamples consisted of 1lt drawn from daily 24-hour total plant intake composites using a WaterSam WS Porti 1, Portable Water Sampler. Samples were routinely collected once per week from April 2020 to November 2020 corresponding to the period before and during the COVID-19 city outbreak related to the second wave of the pandemic. For the sentinels, early-morning (7am-11am) grab 1-lt sampling was performed once weekly. We opted for early morning sampling since defecation in the general population is most frequent in the early morning (Heaton KW et al, 1992) and thus higher viral shedding was more probable. The samples were placed in an isothermal container with ice, transported to the laboratory into approximately 2 hours and stored at 4oC until further analysis. 2.2. Sample Pre-treatment & Viral Precipitation Before the treatment of the samples, the physicochemical parameters, mainly pH, conductivity, and UV254nm absorption were measured. The samples were pre-treated with the Skimmed Milk Flocculation method before isolating the viral genome to increase the chances of detecting SARS-CoV-2 RNA in wastewater (Calgua B. Et al. 2008). The Skimmed Milk Flocculation method is based on the absorption of the viruses to the flocks of skimmed milk. After sample analysis and pre-treatment, the viral RNA and DNA isolation kit (NucleoSpin Microbial DNA, Macherey-Nagel) was used according to the manufacturer's instructions. Following extraction of the viral genome, the extracted samples were measured in NanoDrop 2000 / 2000c Spectrophotometers (Thermo Scientific) to record their purity as well as the concentration of the viral nucleic acids. 2.3. RNA Extraction & RT-qPCR analysis The molecular detection of SARS-CoV-2 was performed using a commercially available RT-qPCR assay (Viasure SARS-CoV-2 Real Time PCR Detection Kit). The assay is based on the amplification of a conserved region of ORF1ab and N genes for SARS-CoV-2 using specific primers and a fluorescent-labeled probe the sequences of which have not been published by the company. A total of 5μl RNA extract was transferred into reaction tubes containing 15 μl PCR reagents. RT was performed at 450C for 15 min and amplification was performed for 1 cycle of 950C for 2 min and 45 cycles of 950C for 10 sec, 600C for 50 sec followed by a final cooling step at 40C. A CFX-96 instrument (Bio Rad Laboratories) was used for all amplification reactions. Both the efficiency of the RNA extraction and RT-qPCR were evaluated in all samples using the internal control (IC) RNA. 2.4. Recovery of SARS-CoV-2 from wastewater and limit of detection To determine the efficiency of recovering SARS-CoV-2 from wastewater using our precipitation and RNA purification methods, we first spiked synthetic RNA (Viasure SARS-CoV-2 Real Time PCR Detection Kit, CerTest Biotec) and added it in the resuspended precipitate at the final step of the extraction procedure. This approach was used to monitor successful handling of each sample in every step of the method. Assays showed recovery efficiencies of greater than 90% when input concentrations were log(6) and log(4) genome copies/ L. Efficiency declined rapidly when input was log(3) genome copies/ L and below. The limit of detection for SARS-CoV-2 virus extracted from wastewater samples was tested using serial dilutions of 105 to 10 copies / L of non-replicative recombinant virus particles with sequences from SARS-CoV-2 genome (Accuplex SARS-CoV-2 Verification panel, Seracare) into sterilized wastewater. Spiked wastewater samples were extracted and assayed using the protocols described above. Greater than 95% of qPCR reactions using the CDC N1 and N2 primers/ hydrolysis probes produced a positive detection when SARS-CoV-2 was diluted to as little as 100 copies/ mL (N = 20 for each dilution). The spiking experiment was performed in triplicates. 2.5. Statistical analysis All statistical analysis was performed using STATA 14.0. 3. Results 3.1. Sampling grid and sample characteristics The sampling grid consisted of 12 sampling spots. 151 wastewater samples were collected over a period of 8 months. Twelve sampling points were included. Six points were coming from 'hotspots' of interest such as nursing homes (n=3), tertiary hospital and 2 refugee camps. 3.2. Limit of detection In all cases, only the viral N gene could be detected by RT-qPCR and only in the concentration of 105 RNA copies/L (Figure 1). This concentration was therefore considered to be the limit of detection of our method and the criterion according to which our samples would be characterized as positive or negative for the detection of SARS-CoV-2 in wastewater. 3.3.RNA concentration and extraction Nanodrop spectrophotometer measurements showed that all samples had similar concentrations of nucleic acids (data not shown). Four samples (2,65%) showed inhibition of the internal control used in the RT-PCR reaction and were excluded from further analysis. Whenever, inhibition of the internal control was observed new samples from the same spot were collected. For all samples, the physico-chemical analysis for pH, conductivity, absorption at UV 254 nm and the results showed no statistically significant differences. 3.4. SARS-CoV-2 detection in wastewater samples Based on that, we were able to confirm the virus presence in 32 wastewater samples by detecting the Orf1ab genomic region and/or the N gene. Samples with low viral load showed N gene amplification only. 3.5. Wastewater SARS-CoV-2 detection and correlation with clinical cases Across the entire network, we detected the first positive sample in our system in week 21 of the surveillance and continued to detect positive samples up until week 34 of the surveillance following closely the outbreak course (Table 1) and without interruption for 13 weeks. A total of 32 wastewater samples were found positive in several spots of sampling ranging from 1 to 7 positive samples per week (median 2.5 positive samples per week). Of particular note, the peak of positive wastewater samples appeared by end-October, slightly preceding the apparent increase in new clinical cases. The number of human cases increased considerably by the beginning of November and remained high by late-November (Figure 2).