WATER NEWS: a field approach for sustainable detection of pathogens in wastewater

Abstract

Wastewater surveillance is a critical tool in responding to the COVID-19 pandemic, yet traditional centralized methods are unsustainable due to their high costs and complex implementation requirements. Here, we introduce Wastewater Analysis and Tracking for Epidemiological Recognition: Necessary Early Warning System (WATER NEWS)-a field clustered regularly interspaced short palindromic repeats-diagnostic approach designed for robust and cost-effective pathogen detection in wastewater treatment plants (WWTPs). WATER NEWS combined a one-pot assay, an optimized reporter probe, on-site nucleic acid extraction, an improved freeze-drying process and a user-friendly, low-cost, battery-operated device, thereby overcoming the limitations of field-deployable wastewater surveillance. In evaluations across 25 WWTPs in 10 Chinese cities, WATER NEWS achieved sensitivities exceeding 90% and specificities of 100% in SARS-CoV-2 detection from the influent and effluent samples of WWTPs. Notably, the system consistently detected SARS-CoV-2 presence within 20 days at a local WWTP. Economic analysis reveals that our approach achieved a 6.5-fold cost reduction compared with clinical testing and costs nearly half that of traditional wastewater surveillance methods, making it a viable and sustainable option for wastewater surveillance.

Keywords: CRISPR–diagnostic; field; pathogen detection; wastewater surveillance; wastewater treatment plants.

Figure 1.

WATER NEWS for field wastewater surveillance. Enabling the concentration of pathogens, purification of nucleic acids, rapid one-pot detection and lyophilized reagents with the deployment of compact, battery-powered diagnostic microdevices. The schematics shown were created by figdraw.com.

Figure 2.

Design of one-pot assay for mpox detection. (a and b) Schematic illustrations of both canonical and suboptimal PAM-mediated cis cleavages in one-pot assay. (c) One canonical PAM (TTTA) and two suboptimal PAMs (TGTC, GTTA) were designed for the f3l and b6r genes. (d and e) The discrepancy performances in the one-pot test between canonical and suboptimal PAMs. Three crRNAs targeting one canonical PAM (TTTA) and two suboptimal PAMs were designed for the (d) f3l and (e) b6r genes, respectively. (f) Schematic illustrations of Probe-1- and Probe 2-mediated trans cleavages in one-pot assay. (g, h) Comparison of the cleavage rates of suboptimal PAM-mediated one-pot assays for f3l and b6r genes with two probes. (i and j) LoD of one-pot assays for the (i) f3l and (j) b6r genes using suboptimal PAM (GTTA) and Probe-2. The fluorescence values at 20 min of the one-pot reaction were compared after subtracting the initial fluorescence values. Mean ± SD of n = 3 technical replicates and NC represents reactions without substrate for (d and e) and (i and j). Statistical significance was analysed by using a two-tailed t-test: ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3.

Optimizing lyophilization strategies for one-pot reactions. (a) Schematic of the benefits of lyophilizing one-pot reagents. (b and c) One-pot fluorescence on pseudoviruses (f3l–3.9 × 10² and b6r–1.2 × 10³ cp/μl) before and after lyophilization by using the different freezing methods. ‘Before lyophilization’ refers to testing reagents that have not been freeze-dried. ‘LN’ denotes freeze-drying after initial freezing in liquid nitrogen, while ‘–20°C’ and ‘–80°C’ represent freeze-drying after being frozen at the respective temperatures. ‘–20°C/LN’ and ‘–80°C/LN’ indicate freezing for 2 h at –20°C or –80°C, respectively, followed by placement in liquid nitrogen for further freezing and lyophilization. Fluorescence was measured after 20 min. (d and e) One-pot fluorescence using lyophilized (–80°C/LN) reagents that were kept at 25°C, 4°C and –20°C. (f and g) Using hydrated reagents to determine the LoD of one-pot reaction with different concentrations of mpox DNA (f, f3l gene; g, b6r gene) based on chlorimetric readout. (h) Diagram of the portable microdevice. (i) Rapid detection of mpox with portable handheld reader. Virus loads: f3l: 0–3.9 × 10² cp/μL, b6r: 0–1.2 × 10³ cp/μL. The schematics shown in (a) were created by figdraw.com.

Figure 4.

Mpox detection in municipal wastewater and natural water samples by WATER NEWS. (a–c) Wastewater samples were collected from the three WWTPs by adopting different treatment processes. (d) Natural water samples were collected from Yexi Lake and Chaohu Lake. (e and f) WATER NEWS assays of wastewater samples and contrived samples (e, f3l gene; f, b6r gene). The results were comparable to those of qPCR analysis. (g and h) WATER NEWS assays of the natural water samples and contrived samples (g, f3l gene; h, b6r gene). The results were comparable to those of qPCR analysis. Virus loads: f3l–3.9 × 10² and b6r–1.2 × 10³ cp/μL.

Figure 5.

SARS-COV-2 detection in human saliva and urban sewage by WATER NEWS. (a) Schematic diagram illustrating the detection of SARS-COV-2 in saliva and sewage. (b) Comparison of the detection efficiency of WATER NEWS for N gene with 1, 10 or 100 fM at 37°C and 42°C. (c and d) Comparison of the detection limit of N gene by using RT–qPCR and WATER NEWS. The fluorescence values at 20 min of the one-pot reaction were compared after subtracting the initial fluorescence values. (e) Using WATER NEWS to detect both positive and negative samples from human saliva. (f and g) Using WATER NEWS to detect the influent and effluent samples from 25 WWTPs in 10 cities of China. Sensitivity: the proportion of true positives correctly identified by the test. Specificity: the proportion of true negatives correctly identified by the test. The threshold was set as the mean of three negative experiments plus three times the standard deviation. If the fluorescence value exceeded this threshold, then it was defined as positive; otherwise, it was defined as negative. Statistical significance was analysed by using a two-tailed t-test: ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The schematics shown in (a, e, g) were created by figdraw.com.

Figure 6.

Real-time and field early warning of COVID-19 in Hefei City by using WATER NEWS. (a) Schematic diagram illustrating the field deployment for diagnosing SARS-CoV-2 in wastewater. (b) Comparison of the efficiency of magnetic bead adsorption and column membrane method for capturing SARS-CoV-2 nucleic acids in influent and excess sludge. (c) Real-time and field detection of SARS-CoV-2 in influent and excess sludge from WWTPs with on-site images. (d and f) Re-examination of the field diagnostic results by using WATER NEWS in the laboratory. (e and g) Confirmation of WATER NEWS results by using RT–qPCR. (h) Monthly costs of clinical testing, standard wastewater surveillance paradigm and WATER NEWS. (i) Comparison of the costs and benefits using the ROI. (j) Monthly cost savings (benefits) of WATER NEWS based on different monitored population sizes. Statistical significance was analysed by using a two-tailed t-test: ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.