Evaluating the transmission risk of SARS-CoV-2 from sewage pollution.

The presence of SARS-CoV-2 in untreated sewage has been confirmed in many countries but its incidence and infection risk in contaminated waters is poorly understood. The River Thames in the UK receives untreated sewage from 57 Combined Sewer Overflows (CSOs), with many discharging dozens of times per year. This study investigated if such discharges provide a pathway for environmental transmission of SARS-CoV-2. Samples of wastewater, surface water, and sediment collected close to six CSOs on the River Thames were assayed over eight months for SARS-CoV-2 RNA and infectious virus. Bivalves were also sampled as an indicator species of viral bioaccumulation. Sediment and water samples from the Danube and Sava rivers in Serbia, where raw sewage is also discharged in high volumes, were assayed as a positive control. No evidence of SARS-CoV-2 RNA or infectious virus was found in UK samples, in contrast to RNA positive samples from Serbia. Furthermore, this study shows that infectious SARS-CoV-2 inoculum is stable in Thames water and sediment for <3 days, while SARS-CoV-2 RNA is detectable for at least seven days. This indicates that dilution of wastewater likely limits environmental transmission, and that detection of viral RNA alone is not an indication of pathogen spillover.

Surveillance of SARS-CoV-2 RNA in open-water sewage canals contaminated with untreated wastewater in resource-constrained regions.

Sewage-based surveillance for COVID-19 has been described in multiple countries and multiple settings. However, nearly all are based on testing sewage treatment plant inflows and outflows using structured sewage networks and treatment systems. Many resource-limited countries worldwide have open canals, lakes and other such waste-contaminated water bodies that act as a means of sewage effluent discharge. These could serve as hyperlocal testing points for detecting COVID-19 incidence using the effluents from nearby communities. However, a sensitive, robust and economical method of SARS-CoV-2 RNA detection from open waste contaminated water bodies in resource-constrained regions is currently lacking. A protocol employed in Bangalore, India, where SARS-CoV-2 RNA levels were evaluated using two open canal systems during the first and second waves in the present study. SARS-CoV-2 RNA was measured using two strategies: a modified TrueNATTM microchip-based rapid method and traditional real-time reverse transcription-PCR (rRT-PCR), which were compared for analytical sensitivity, cost and relative ease of use. SARS-CoV-2 RNA levels were detected at lower levels during the earlier half compared to the later half of the first wave in 2020. The opposite trend was seen in the second wave in 2021. Interestingly, the change in RNA levels corresponded with the community burden of COVID-19 at both sites. The modified TrueNATTM method yielded concordant results to traditional rRT-PCR in sensitivity and specificity and cost. It provides a simple, cost-effective method for detecting and estimating SARS-CoV-2 viral RNA from open-water sewage canals contaminated with human excreta and industrial waste that can be adopted in regions or countries that lack structured sewage systems.

A review on detection of SARS-CoV-2 RNA in wastewater in light of the current knowledge of treatment process for removal of viral fragments.

The entire globe is affected by the novel disease of coronavirus 2019 (COVID-19 or 2019-nCoV), which is formally recognised as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The World Health Organisation (WHO) announced this disease as a global pandemic. The presence of SARS-CoV-2 RNA in unprocessed wastewater has become a cause of worry due to these emerging pathogens in the process of wastewater treatment, as reported in the present study. This analysis intends to interpret the fate, environmental factors and route of transmission of SARS-CoV-2, along with its eradication by treating the wastewater for controlling and preventing its further spread. Different recovery estimations of the virus have been depicted by the detection of SARS-CoV-2 RNA in wastewater through the viral concentration techniques. Most frequently used viral concentration techniques include polyethylene glycol (PEG) precipitation, ultrafiltration, electronegative membrane, and ultracentrifugation, after which the detection and quantification of SARS-CoV-2 RNA are done in wastewater samples through quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The wastewater treatment plant (WWTP) holds the key responsibility of eliminating pathogens prior to the discharge of wastewater into surface water bodies. The removal of SARS-CoV-2 RNA at the treatment stage is dependent on the operations of wastewater treatment systems during the outbreak of the virus; particularly, in the urban and extensively populated regions. Efficient primary, secondary and tertiary methods of wastewater treatment and disinfection can reduce or inactivate SARS-CoV-2 RNA before being drained out. Nonetheless, further studies regarding COVID-19-related disinfectants, environment conditions and viral concentrations in each treatment procedure, implications on the environment and regular monitoring of transmission need to be done urgently. Hence, monitoring the SARS-CoV-2 RNA in samples of wastewater under the procedure of wastewater-based epidemiology (WBE) supplement the real-time data pertaining to the investigation of the COVID-19 pandemic in the community, regional and national levels.

Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater.

There is currently a clear benefit for many countries to utilize wastewater-based epidemiology (WBE) as part of ongoing measures to manage the coronavirus disease 2019 (COVID-19) global pandemic. Since most wastewater virus concentration methods were developed and validated for nonenveloped viruses, it is imperative to determine the efficiency of the most commonly used methods for the enveloped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Municipal wastewater seeded with a human coronavirus (CoV) surrogate, murine hepatitis virus (MHV), was used to test the efficiency of seven wastewater virus concentration methods: (A-C) adsorption-extraction with three different pre-treatment options, (D-E) centrifugal filter device methods with two different devices, (F) polyethylene glycol (PEG 8000) precipitation, and (G) ultracentrifugation. MHV was quantified by reverse-transcription quantitative polymerase chain reaction and the recovery efficiency was calculated for each method. The mean MHV recoveries ranged from 26.7 to 65.7%. The most efficient methods were adsorption-extraction methods with MgCl2 pre-treatment (Method C), and without pre-treatment (Method B). The third most efficient method used the Amicon® Ultra-15 centrifugal filter device (Method D) and its recovery efficiency was not statistically different from the most efficient methods. The methods with the worst recovery efficiency included the adsorption-extraction method with acidification (A), followed by PEG precipitation (F). Our results suggest that absorption-extraction methods with minimal or without pre-treatment can provide suitably rapid, cost-effective and relatively straightforward recovery of enveloped viruses in wastewater. The MHV is a promising process control for SARS-CoV-2 surveillance and can be used as a quality control measure to support community-level epidemic mitigation and risk assessment.