Highlights
•Low-cost and rapid paper microfluidic device for on-site wastewater surveillance
•Sensing platform for pandemic preparedness and public health
•Field testing of SARS-CoV-2 in wastewater from quarantine hotels
Summary
Tracking genomic sequences as microbial biomarkers in wastewater has been used to determine community prevalence of infectious diseases, contributing to public health surveillance programs worldwide. Here, we report upon a low-cost, rapid, and user-friendly paper microfluidic platform for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza detection, using loop-mediated isothermal amplification, with signal read using a mobile phone camera. Sample-to-answer results were collected in less than 1.5 h, providing rapid multiplexed detection of viruses in wastewater, with a detection limit of <20 copies mL−1. The device was subsequently used for on-site testing of SARS-CoV-2 in wastewater samples from four quarantine hotels at London Heathrow Airport, showing comparable results to those obtained using polymerase chain reaction. This sensing platform, which enables rapid and localized testing without requiring samples to be sent to centralized laboratories, provides a potentially important public health tool for pandemic preparedness, with a variety of future wastewater surveillance applications in community settings.
Introduction
Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), brought about significant public health and socioeconomic challenges across the globe.1 While the healthcare outcomes of the disease in severe cases are well documented, the Centers for Disease Control and Prevention (CDC) noted that in mild cases, the symptoms of the disease are similar to a number of other respiratory infections, and they emphasized the importance of distinguishing between infections caused by SARS-CoV-2 and other respiratory viruses, particularly those causing influenza.2
For many infectious diseases,3 including both influenza and COVID-19,4 during an active infection, microbial materials are excreted in feces and survive within sewage for several days. Wastewater surveillance has previously been shown to be capable of detecting a wide range of such diseases within community settings, acting as an early warning tool through the analysis of readily available aggregated samples.5 However, limitations in the technologies remain, preventing their more widespread use, including the local availability of diagnostic centers, with appropriately trained staff.
Currently, wastewater surveillance samples are sent to centralized analytical laboratories, with analysis involving using either polymerase chain reaction (PCR) assay,6,7 as a gold standard analytical method, and/or genome sequencing for the identification of new variants with associated lengthy times for results to be reported (typically 24–48 h). Notwithstanding such delays, in one example, wastewater-based epidemiology (WBE) was successfully performed across 45 sewage sites in the UK, demonstrating the emergence of SARS-CoV-2, 4–5 days in advance of the formal diagnosis of individual clinical cases.8 In a second example, deep sequencing of 94 urban water catchments was used to infer the spatiotemporal abundance of predefined variants of SARS-CoV-2 from a large number of wastewater samples.9
In addition to challenges around the length of the analysis time (during which period, nucleic acid degradation may occur during sample storage and transport), the presence of PCR inhibitors in sewage may also lead to inconsistent results. Point-of-need in situ testing with rapid target amplification is needed to overcome these limitations, enabling the sensitive detection of low viral loads (often associated with sample dilution by rainwater runoff in the wastewater).
As an alternative to laboratory-based PCR, isothermal amplification methods such as loop-mediated isothermal amplification (LAMP) have attracted increasing attention for the detection of a range of pathogens,10 providing a less complex assay workflow by negating the demand for complex thermocycling equipment. Using such isothermal amplification protocols, RNA viral biomarkers can be easily integrated in the form of reverse-transcription loop-mediated isothermal amplification (RT-LAMP), allowing detection of SARS-CoV-211 as well as influenza A and influenza B.12 However, many of these LAMP-based detection methods still rely upon centralized laboratories, preventing their wider application in point-of-care diagnostics.
Paper-based microdevices have been proposed as a method of providing point-of-need diagnostics that are affordable, sensitive, specific, user-friendly, rapid, robust, equipment-free, and deliverable to end users (ASSURED criteria).13 When such assays include real-time connectivity and ease of specimen collection, tests have come to be known as RE-ASSURED.14 Proof-of-concept methods have been developed to integrate nucleic acid extraction, purification, elution, amplification, and detection into affordable and easy-to-use tests.15 The whole analysis process can be completed by “origami” folding of the paper device, without the need for fixed power supplies.
With advances in microfluidic technology, LAMP-based assays have now been implemented on paper-based tests, enabling the detection of a variety of pathogens.16 For example, the paper-origami technique was used to enable DNA extraction, amplification, and array-based fluorescence detection of malaria species,17 and it was applied for the multiplexed detection of bovine infectious reproductive diseases in rural India.18 Such devices have been further developed to perform multiplexed lateral flow test (LFT) detection for malaria field testing in Uganda, Africa,19,20 demonstrating that paper-based devices can be readily integrated into field-ready modules and provide excellent detection capabilities comparable to laboratory-based instruments. In addition, RT-LAMP has been integrated with LFT for the clinical detection of RNA viruses, including hepatitis C.21
Most recently, the combination of paper microfluidic devices and WBE has provided new insights into SARS-CoV-2 detection using a paper device based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a and RT-LAMP for the detection of N, E, and S genes in the laboratory using wastewater spiked with genomic sequences and/or viruses.22 The concept of the potential use of SARS-CoV-2 testing in sewage has also been described, to trace the source of COVID-19 and to determine the presence of SARS-CoV-2 carriers in a community.23
Early warning sensing systems based upon WBE and paper tests are now expected to act as sentinels to highlight the emergence of new disease outbreaks and to help monitor prevalence and control future outbreaks.7 In order to demonstrate the power of community sensing, at the point of need, we now describe a device using RT-LAMP assay for rapid detection of SARS-CoV-2 and influenza A/B in community wastewater. The method comprised a simple hand-held syringe-based sample preparation system to enrich pathogen samples from wastewater, enabling the extraction, purification, amplification, and in situ detection.
The paper microfluidic device was first optimized using a model surrogate virus porcine reproductive and respiratory syndrome virus (PRRSV) in order to define channel geometries and sensing area sizes. To assess the sensitivity and specificity of the test for pathogen detection, transcribed RNA at different concentrations was spiked into water to mimic real samples in a laboratory setting. Subsequently, to optimize the platform in real samples, we tested 20 wastewater samples collected by Anglian Water (using a laboratory-based RT-qPCR assay as a gold standard reference).
Finally, we implemented the test for the local analysis of wastewater within a government-designated quarantine hotel near London Heathrow Airport, showing its potential for rapid and on-site wastewater surveillance. The technique not only shows the value of the methodology for localized community testing in well-resourced environments, but it also illustrates the potential application for the paper microfluidic device for pandemic preparedness in the resource-limited settings, where there is an increasing need to develop rapid, portable, low-cost, and user-friendly platforms to detect pathogens.
Results and discussion
Optimization of a paper microfluidic device for virus detection
During the COVID-19 outbreak between 2020 and 2023, the spread of the pandemic was monitored and mitigated by designing detection platforms to distinguish viruses in wastewater.24 In general, these systems only provided infection trends, with the effectiveness of the methods being limited by samples having to be transported to centralized laboratories for analysis (and data generated was retrospective and not “real time”). We chose to overcome these limitations by developing a paper-based LFT for wastewater epidemiology, measuring nucleic acid biomarkers in situ, at the point of sampling.
We first optimized the paper microfluidic device based upon a general testing framework (Figure 1). Initial device design was studied using PRRSV as a surrogate virus (Figure S1). The cycle threshold (Ct) is the number of cycles at which the intensity of the fluorescent signal exceeds the threshold signal for positivity.25 The optimal pore size, channel width, and channel length of the paper device were concluded to be 4.0 mm, 1.5 mm, and 2.5 mm, respectively. We subsequently confirmed the validity of the developed device for virus detection in wastewater, using spiked genomic samples, samples from a wastewater company, and finally in a community setting within four quarantine hotels during pandemic.
