Poster W7 - Ultrafiltration-Based Method for Monitoring SARS-CoV-2 Resource Limited Settings

Introduction: Wastewater-based surveillance has found utility for COVID-19 tracking. Such surveillance is useful because people infected with SARS-CoV-2 (the virus that causes COVID-19) can shed the virus in their urine and feces, even if they don’t have symptoms. Wastewater surveillance can serve as an early warning that COVID-19 is spreading in a community before data at healthcare facilities are collected. For example, our prior research has unveiled a significant correlation between the concentration of the virus in local sanitary wastewater and subsequent increases in patient numbers. Specifically, a spike in viral concentration in wastewater can precede a surge in hospital admissions by 7 to 10 days. This insight is particularly crucial for areas remote from medical facilities, as it allows for the prediction of pandemic outbreaks and the preparatory mobilization of medical resources without the need of medical data. However, wastewater-based SARS-CoV-2 surveillance is heavily lab dependent and complex to operate, which hinders the practice in communities with limited infrastructure and geographic isolation. Thus, despite valuable predictive data from wastewater samples, their potential for surveillance has yet to be fully harnessed. We present an approach here that has streamlined the entire sample processing methodology for wastewater-based virus surveillance, rendering it more accessible, cost-effective, and suitable for non-specialists, thereby enabling the routine monitoring of wastewater viral concentrations as an early pandemic alert system at resource limited settings.

Materials and

Methods: To extract SARS-CoV-2 from wastewater for analysis using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), it's crucial to concentrate the viral particles and eliminate inhibitors that might interfere with virus quantification through RT-qPCR. The sample processing procedure consists of three main stages: (1) viral purification, (2) viral concentration and (3) RNA extraction (Figure 1). The conventional procedure relies on centrifugation to remove large particles and suspended solids (Stage 1). This is followed by ultrafiltration through a centrifugal filter unit to concentrate viral particles (Stage 2), and affinity chromatography to concentrate and purify the virus RNA (Stage 3). To simplify the instrument-dependent procedure, our approach utilizes a manual pump to pass wastewater spiked with SARS-CoV-2 through a series of polyvinylidene difluoride (PVDF) membrane filters with progressively finer pores, specifically arranged in a sequence of 30μm, 25μm, 5μm, 3μm, 0.45μm, and finally 0.22μm (Filter set A, Stage 1). Subsequently, the sample is passed through a 100 kDa cut-off Regenerated Cellulose membrane, which retains viral particles on the surface of the filter, effectively concentrating the virus for further analysis (Filter B, Stage 2). Following sample concentration, we utilized ultrasonic waves to elute the virus into water followed by lysing the viral particles using a solution of 0.075% Tween®20 + 25 mM Tris HCl . For RNA purification, we employed a glass fiber membrane dipstick to wash away inhibitors and elute purified viral RNA in the PCR mixture (Stage 3). The whole process requires minimal equipment.

Results, Conclusions, and Discussions: Our results show that Filter Set A is able to reduce the turbidity of wastewater samples ranging between 60-120 NTU (Nephelometric Turbidity Units) to 2.6-3.9 NTU which indicates that 97.2 % of the suspended solids have been removed (Figure 2A). When the wastewater sample after Filter Set A was further processed by the conventional approaches in the next two stages and the resulting RNA quantified by RT-qPCR, consistently lower CT values for Filter Set A was observed compared to ultracentrifugation across all concentrations, suggesting Filter Set A is efficient in removing large particles and suspended solids. Note that the CT value from RT-qPCR is inversely proportional to the viral RNA concentration, i.e. a lower CT value corresponds to a higher viral concentration in the sample.
Next Filter Set A and Filter B was combined to purify and concentrate SARS-CoV-2 virus from wastewater samples, followed with conventional RNA extraction and RT-qPCR. Consistently lower CT values for Filter Set A + Filter B suggest that Filter B is more efficient at retaining the virus compared to the traditional method (Figure 2C). To individually assess the performance of Filter B, an additional experiment was conducted using Filter set A as a preliminary stage. This experiment focused on comparing the performance of Filter B with the Centricon filter on samples processed by Filter set A. The CT values of the samples processed by Filter B were 4 to 5 units lower than those processed by the Centricon filter, indicating that the concentrations obtained using Filter B were more than 10 times higher than samples processed by the Centricon filter (Figure 2.D). Furthermore, the filtrate through Filter B contains comparable or less virus than the filtrate collected in the conventional method, indicating Filter B perform well in retaining the virus in the concentrate with minimal losses (Figure 2.B).
Lastly, we compared the performance of the dipstick membrane against a commercial RNA extraction kit (RNeasy PowerMicrobiome Kit by Qiagen) for RNA purification and elution (Figure 3).

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