Wastewater surveillance for SARS-CoV-2: Lessons learnt from recent studies to define future applications

Abstract

Wastewater-based epidemiology (WBE) is successful in the detection of the spread of SARS-CoV-2. This review examines the methods used and results of recent studies on the quantification of SARS-CoV-2 in wastewater. WBE becomes essential, especially with virus transmission path uncertainty, limitations on the number of clinical tests that could be conducted, and a relatively long period for infected people to show symptoms. Wastewater surveillance was used to show the effect of lockdown on the virus spread. A WBE framework tailored for SARS-CoV-2 that incorporates lessons learnt from the reviewed studies was developed. Results of the review helped outline challenges facing the detection of SARS-CoV-2 in wastewater samples. A comparison between the various studies with regards to sample concentration and virus quantification was conducted. Five different primers sets were used for qPCR quantification; however, due to limited data availability, there is no consensus on the most sensitive primer. Correlating the slope of the relationship between the number of gene copies vs. the cumulative number of infections normalized to the total population served with the average new cases, suggests that qPCR results could help estimating the number of new infections. The correlation is improved when a lag period was introduced to account for asymptomatic infections. Based on lessons learnt from recent studies, it is recommended that future applications should consider the following: 1) ensuring occupational safety in managing sewage collection and processing, 2) evaluating the effectiveness of greywater disinfection, 3) measuring viral RNA decay due to biological and chemical activities during collection and treatment, 4) assessing the effectiveness of digital PCR, and 5) conducting large scale international studies that follow standardized protocols.

Keywords: COVID-19; SARS-CoV-2; Virus concentration; Wastewater virus surveillance; Wastewater-based epidemiology.

Graphical abstract

Fig. 1

Inactivation rate coefficient of viruses and virus surrogates in sewage (a) and pasteurized sewage (b) based on reported log inactivation values. When multiple log inactivation values were reported, an average inactivation rate coefficient value was determined. SARS: Severe acute respiratory syndrome coronavirus (SARS-CoV), 229E: Human coronavirus 229E, FIPV: Feline infectious peritonitis virus, PV1: Poliovirus 1, MHV: Mouse hepatitis virus, TGEV: transmissible gastroenteritis virus, Phi6: Pseudomonas phage Phi6, MS2: Enterobacteria phage MS2. Rectangles refer to non-enveloped viruses (PV1 and MS2), while the rest are for enveloped viruses or enveloped virus surrogates. Note that the value for SARS in (b) is for sterilized water. [1] (Duan et al., 2003), [2] (Wang et al., 2005), [3] (Gundy et al., 2008), [4] (Casanova et al., 2009), [5] (Casanova and Weaver, 2015), [6] (Ye et al., 2016), [7] (Aquino de Carvalho et al., 2017).

Fig. 2

Framework for a wastewater-based epidemiology system.

Fig. 3

Schematic representation of SARS-CoV-2 genome and virus particle. (A) SARS-CoV-2 full-length genome, location of all the genes and proteolytic processing of the polyproteins encoded by the ORF1a and ORF1b genes. The genome is followed by schematic of the full-length genomic RNA (capped and polyadenylated, poly A), and location of the primers and probes being currently used for real time qPCR assays for virus detection. (B) Graphic representation of the virus particle along with its major structural proteins.

Fig. 4

Schematic representation of SARS-CoV-2 life cycle. See text for details. ACE2, angiotensin-converting enzyme-2; TMPRSS2, transmembrane serine protease 2; RTC, replication-transcription complex; ER, endoplasmic reticulum; ERGIC, endoplasmic reticulum–Golgi intermediate compartment. The blue arrow denotes (−) strand RNA transcribed as an intermediate in the genome replication process. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Schematic illustration of the general strategy being employed currently to collect wastewater or sludge samples, process for virus inactivation, virus concentration, and RNA isolation, followed by viral RNA detection using RT qPCR. Each step is shown systematically with key information provided next to the arrows.

Fig. 6

Timeline of published articles on the detection of SARS-CoV-2in wastewater against the date of first reported case of SARS-CoV-2 in patients.

Fig. 7

Reported wastewater sampling locations in the reviewed articles. 1) Sewer network, 2) WWTP influent, 3) WWTP sludge, 4) WWTP treated, and 5) WWTP effluent in addition to surface water.

Fig. 8

Common wastewater sampling locations and sample type in recently published studies on the detection and quantification of SARS-CoV-2 in wastewater systems.

Fig. 9

Average number of samples analyzed in recently published studies on the detection and quantification of SARS-CoV-2 in wastewater systems.

Fig. 10

Virus concentration methods used for SARS-CoV-2 in literature. a) Different virus concentration methods applied in the reviewed studies (P-F = precipitation-flocculation, UC = ultracentrifugation, UF = ultrafiltration, CMF = charged membrane filtration, A-E = adsorption-elution) b) Gene copies/ mL of wastewater vs. Ct values at different locations. Locations correspond to the following studies: Montana (Nemudryi et al., 2020), Spain (Randazzo et al., 2020a), Australia (Ahmed et al., 2020a), Turkey (Kocamemi et al., 2020a). (Note: the figure repeats the count of a study that uses different methods to the same sample location, or the same method for different sample locations.)

Fig. 11

Comparative analysis of the Ct values obtained relative to viral copy numbers observed for different real time PCR assays used in SARS-CoV-2 RNA surveillance WBE. The Ct values are normalized to copies of SARS-CoV-2 RNA detected per milliliter of wastewater tested in each study.

Fig. 12

Relationship between normalized cumulative infection per 100,000 people and gene copies /mL of wastewater measured plotted per primer at each location studied for the SARS-CoV-2 in wastewater. References of the locations in the figure are indicated in Table 2.