A Prevalence Risk Analysis of Waterborne Transmission of SARS-CoV-2

We statistically analyzed 31 published studies comprising 113 water samples collected from 17 countries for SARS-CoV-2 positivity. The pooled estimated prevalence of viral RNA in the tested samples was 64.1% [95% CI:51.6%, 74.9%] with considerable heterogeneity (I2: 90.1%, P<0.001). Notably, wastewater, sewage, hospital septic-tank, biological sludge, and effluent demonstrated statistical significance (P<0.05) for RNA positivity. The country-wise pooled estimated prevalence for Germany, India, Turkey, Spain, the Netherlands, Italy, the USA, and Japan were 88% (76%, 94%), 85% (33%, 98%), 83% (43%, 97%), 78% (54%, 92%), 60% (41%, 77%), 53% (36%, 70%), 53% (27%, 77%), and 25% (13%,43%), respectively. Further subgroup analyses showed that the prevalence of SARS-CoV-2 among the tested water samples was significantly higher in middle-income countries compared to high-income groups. Our data, therefore, suggests wastewater-based epidemiological surveillance as an important tool for community-wide monitoring of SARS-CoV-2.

The recently emerged SARS-CoV-2, which has caused the devastating CoV-2 disease-19  pandemic, is the seventh and third most pathogenic CoV after SARS-CoV-1 and MERS-CoV [6,7]. Similar to SARS-CoV-1 and MERS-CoV, transmission of SARS-CoV-2 from 'asymptomatic' individuals during the 'pre-symptomatic' state has also been observed [8,9]. Notably, a proportion of SARS-CoV-2-infected patients have also shown gastrointestinal and hepatobiliary manifestations, including fecal shedding of high-titer infectious particles [10][11][12][13][14][15][16][17][18]. In the last two years of the COVID-19 pandemic, there have been a growing number of reports on the worldwide detection of SARS-CoV-2 in wastewater, raw sewage, hospital septic tanks, biological sludge and effluent, lakes, and rivers [19]. In view of the highlighted fecal contamination of water, a potential risk of waterborne transmission of SARS-CoV-2 in countries with poor sanitation and inadequate wastewater management has been envisaged. Here, we have statistically analyzed the global prevalence of SARS-CoV-2 in different water sources based on published reports, and accessed the risk of waterborne spread of COVID-19.

Literature Search Strategy
A structured online search for peer-reviewed articles published in English (2020-2021) was conducted on PubMed, Europe PMC, MEDLINE, EMBASE, and Google Scholar portals, including the Cochrane Library, using phrases: enteric or diarrheal coronaviruses or SARS-CoV-2, gastrointestinal or fecal shedding of SARS-CoV-2, Waterborne or fecaloral transmission of COVID-19, detection of SARS-CoV-2 in wastewater or water samples, etc. The present study followed the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-analysis, or PRISMA [20], and Meta-analysis of Observational Studies in Epidemiology, or MOOSE [21]. The quality of the study was appraised using the Newcastle-Ottawa quality scale [22].

Inclusion Criteria
The eligibility of each published study followed the inclusion criteria: (i) original research or observational studies; and (ii) study samples of wastewater-based epidemiological surveillance for SARS-CoV-2. The exclusion criteria consisted of articles published in other languages or as conference abstracts, especially reporting on biological or excretory fluid specimens from COVID-19 patients to avoid inter-study variance. Study eligibility was independently assessed by the authors, and any disagreements were resolved by mutual discussion and consent.

Process of Systematic Review and Data Retrieval
All retrieved full articles were first screened for their titles and abstracts to determine their eligibility before systematic review and meta-analysis. Further, standardized data on the first author's name, year of publication, country of origin, study design, sample size, method of water concentration, and diagnostic technique of SARS-CoV-2 detection were collected for the analysis.

Measurements and Statistical Analysis
The prevalence of SARS-CoV-2 in water samples in each region subjected to statistical analysis was expressed in percentage (%). All data were presented as mean±standard deviation (SD) or event rate with a 95% confidence interval, and P<0.05 was considered statistically significant. All statistical analyses were performed using comprehensive metaanalysis software. Data were also assessed for Higgins I2 statistics, which quantify heterogeneity levels as minimal (1-40%), moderate (30-60%), substantial (50-90%), and considerable (90-100%).

Quantitative Detection of Viral Load in Water Samples
Since the first report on the detection of SARS-CoV-2 in the fecal sample of clinically confirmed COVID-19 patients [23], its plausible waterborne transmission through contaminated water has become an important water-based epidemiological issue [19]. Subsequently, ample data on wastewater or sewage surveillance for SARS-CoV-2 has emerged from across the world [24]. Of these, several studies have reported the detection of SARS-CoV-2 RNA in water samples collected from different sources (Table 1). Our analysis of the published data from different countries and water sources on the occurrences of SARS-CoV-2 confirmed by detectable viral RNA was in accordance with the set eligibility criteria. Of these, most studies employed the molecular diagnostic test (RT-PCR) to detect the viral RNA rather than quantifying RNA (RT-qPCR) expressed as genome copy (gc) number. Notably, higher titers of SARS-CoV-2 were reported in wastewater samples as compared to clinical specimens [17].
Owing to the samples' origins in different water sources and geographic regions of variable endemicity or socioeconomic status, variable occurrences of SARS-CoV-2 were reported. The use of different sample volumes, methods of virus filtration or concentration, and RNA quantifications in different units therefore greatly challenged our comparative analysis between studies. Nonetheless, the overall detection rate of SARS-CoV-2 in raw sewage or wastewater samples ranged between 13.0% and 100%, with optimal viral RNA over 106 gc/L. Notably, the first published report on SARS-CoV-2 detection in Dutch untreated sewage samples used the ultrafiltration method and RT-qPCR for RNA quantification in the range of 2.6×103 to 2.2×106 gc/L (Table 1). In contrast, while polyethylene glycol precipitation and ultracentrifugation of American raw sewage samples had SARS-CoV-2 RNA loads ranging 103−105 gc/L [17], viral RNA concentrations in French wastewater samples ranged between 5×104 and 3×106 gc/L (Table 1). Further examples include the use of aluminum flocculation-based concentration methods and the quantification of SARS-CoV-2 RNA as 2.5 ×105 gc/L in Spanish wastewater [25], which corroborated the German data based on ultracentrifugation and ultrafiltration of viral RNA [26]. Interestingly, a comparatively lower level of SARS-CoV-2 RNA (2.5×103 copies/L) was reported in secondary-treated wastewater samples in Japan, suggesting the importance of water treatment in reducing the viral contamination (Table 1).

Country-Wise Prevalence Risk of Waterborne Spread of SARS-CoV-2
In the country-wise analysis, seven studies from the USA demonstrated a prevalence of 53% [27%, 77%], followed by four studies from India with a prevalence of 85% [95% CI: 33%, 98%] with substantial heterogeneity (Table 3). Of these, while the highest prevalence of waterborne SARS-CoV2 RNA was observed in France, the UK, Germany, and Ecuador, the least prevalence was observed in Israel, followed by Japan (Figure 3).

Conclusion
The fecal shedding of high-titer SARS-CoV-2 in COVID-19 patients has been recently corroborated with several reports on the detection of SARS-CoV-2 in wastewater, raw sewage, hospital septic tanks, biological sludge and effluent, lakes, and rivers worldwide. In view of this, our meta-analysis of pooled samples showed data about a 64% prevalence risk of waterborne transmission of SARS-CoV-2 with considerable heterogeneity. Of the various water sources, wastewater, raw sewage, hospital septic tanks, biological sludge, and effluent demonstrated statistically significant contamination with SARS-CoV-2. The pooled estimation of country-wise prevalence of SARS-CoV-2 was substantially high in Germany, India, Turkey, and Spain, moderate in the Netherlands, Italy, and the USA, and minimal in Japan. In addition, the prevalence of SARS-CoV-2 among water samples was significantly higher in middle-income countries compared to high-income countries. This is very likely due to the lack of focused water surveillance on enteric coronaviruses in general and the knowledge gaps in their circulation, persistence, and post-treatment inactivation. Because costly and time-consuming diagnostics are not feasible in such a pandemic situation, wastewater-based epidemiological surveillance should be considered an important tool for community-wide monitoring of COVID-19. However, tracking the source of SARS-CoV-2 contamination and spread, as well as its genetic variants, in near realtime would be most challenging.

Author Contributions
Conceptualization, M.K.P.; resources, M.A.P. and A.R.A.; data curation, A.R.A. and M.A.P.; writing-original draft preparation, A.R.A. and M.K.P.; writing-review and editing, M.K.P. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement
The data presented in this study are available in the article.

Funding
The authors received no financial support for the research, authorship, and/or publication of this article.

Institutional Review Board Statement
Not applicable.

Informed Consent Statement
Not applicable.