Population-level monitoring of SARS-CoV-2 by wastewater testing using an integrated microfluidic chip

Introduction: : Surveillance of emerging infectious diseases is essential for implementing preventive and control strategies during outbreaks; this surveillance is often performed using clinical testing of individuals. However, large-scale clinical testing to track infectious diseases within populations is resource-intensive and expensive. Wastewater-based epidemiology (WBE) tracks the prevalence of pathogens in a community via regular wastewater testing. WBE is a powerful public health tool that can act as an early warning system and inform health officials about the spread of infectious diseases in a community. WBE minimizes the cost of disease monitoring at a population level compared to traditional clinical testing. However, widespread adoption of WBE in low-resource settings is hindered by the lack of infrastructure, limited technical capacity, and the need for trained personnel. Those limitations are seen at almost every step of the WBE workflow.
A point-of-use wastewater testing device can revolutionize WBE, in the same way point-of-care (POC) testing has revolutionized clinical testing. In this work, we have utilized immiscible filtration assisted by surface tension (IFAST) to concentrate and extract nucleic acids from wastewater. IFAST uses the immiscibility of oil in water as a barrier between the sample and other buffers, allowing the nucleic acid bound-magnetic beads to move easily between buffers using a simple magnet. IFAST technique is coupled with loop-mediated isothermal amplification (LAMP), resulting in an integrated, easy-to-use, and inexpensive concentration-extraction-identification device (CEID) for semi-quantitative identification of the target pathogens in wastewater samples.

Materials and

Methods: : The chip was fabricated out of 1/8 in thick PMMA sheets (McMaster-Carr, 8589K41) using a CO2 laser cutter (Universal Laser Systems, VLS3.50) and enclosed with pressure-sensitive adhesive films (ThermoFisher Scientific, AB-1170). While the wells were cut out of the PMMA sheet, the connection channels were fabricated using the engraving option of the laser cutter (Figure 1A).
For sample processing, 2 drops of silicone oil were added to the oil chambers, and 160 and 80 uL of nuclease-free water were added to the wash and elution wells, respectively. Next, 2 mL of wastewater sample was transferred to a tube and mixed with 3 mL of lysis buffer and 120 uL of paramagnetic particles (PMPs). The mixture of sample, lysis buffer, and PMPs was then loaded to the sample well. An external magnet collected all the PMPs inside the sample well. The PMPs were then pulled through the series of wells by a linear manual movement of the magnet (Figure 1B). Once in the elution well, the PMPs were mixed by pipetting up and down. The CEID was then placed on a hot plate at 65°C for 5 minutes to ensure elution of nucleic acids. The SARS-CoV-2 colorimetric RT-LAMP assay targets the nucleocapsid gene. The extracted nucleic acid was mixed with the WarmStart Colorimetric LAMP 2X Master Mix with UDG (NEB, M1804L), and 2.5 uL of 10X primer mix in the amplification wells, and incubated at 65°C for 30 min. A color change from pink to yellow indicates a positive sample.

Results, Conclusions, and Discussions:: After optimizing CEID, we evaluated its performance for the extraction of SARS-CoV-2 from wastewater. As shown in Figure 2A, the on-chip extraction resulted in higher SARS-CoV-2 copies per elution compared to other methods at all different concentrations. In the next step, the sensitivity of colorimetric RT-LAMP in the detection of SARS-CoV-2 was measured using serial dilutions of spiked samples. The amplification curves are shown in Figure 2B using the ratio of absorbance at 560 and 480 nm. This ratio can quantify the amount of color change from pink to yellow. RT-LAMP was able to amplify SARS-CoV-2 down to 5 copies per reaction. At high concentrations of the target, the RT-LAMP Ct correlated with the number of copies per reaction. However, at concentrations lower than 100 copies per reaction, a similar correlation did not hold (Figure 2C). This suggested that RT-LAMP can be used as a qualitative method for concentrations lower than 100 copies per reaction.
Finally, the CEID was used to monitor the presence of SARS-CoV-2 over 6 weeks in the wastewater samples from two different locations, a wastewater treatment plant, and a hospital sewage manhole. The results were then reported in copies per mL of wastewater on the left Y-axis in Figures 3A-D. For the integrated CEID both the RT-LAMP replicate positivity (out of 3 replicates) and RT-LAMP Ct (in minutes) were reported. As shown in Figures 3A and 3B, the trend of SARS-CoV-2 concentration was also seen in the replicate positivity. The RT-LAMP Ct was also highly correlated with the SARS-CoV-2 concentrations as measured by RT-qPCR. CEID measured SARS-CoV-2 in wastewater in a semi-quantitative manner and indicated SARS-CoV-2 concentration trends with replicate positivity, as well as RT-LAMP time to positivity.
The total per sample cost is about $11 for the CEID. Moreover, the CEID only requires a hot plate at 65 °C, which can be reached using a battery-powered PTC heating element. The CEID takes about 40 minutes, of which only 5 minutes is hands-on time. CEID has the potential to expand to other infectious diseases by implementing RT-LAMP assays for other pathogens such as influenza or RSV.

Acknowledgements and/or References (Optional):: The work was supported by the National Institutes of Health (NIH) grants 1U01DA053903-01 and P30 ES026529, the Centers for Disease Control and Prevention (CDC) contract BAA 75D301-20-R-68024, and National Science Foundation (NSF) grant 2154934 and 2412446. M.D.B. thanks the Lighthouse Beacon Foundation for providing a graduate fellowship.