1. Introduction
Coronavirus disease 2019 (COVID-19) has become a pandemic worldwide, resulting in
nearly 6.7 million deaths (World Health Organization, 2022). The SARS-CoV-2 viral particles or associated genetic fragments are excreted in
the stool and body fluids of infected individuals (Tran et al., 2021). Therefore, wastewater surveillance for the SARS-CoV-2 pathogen is an effective
way to track the health of entire communities. Wastewater surveillance serves as a
sensitive indicator to determine the magnitude of SARS-CoV-2 circulation within the
population and if its transmission is on the rise or decline. This global approach
for addressing COVID-19 emphasizes the potential of wastewater data to complement
existing established epidemic control measures. SARS-CoV-2, has already been detected
in many wastewater treatment plants (Medema et al., 2020; Randazzo et al., 2020) during the early stage of the pandemic (Ahmed, Angel et al., 2020; Fernández-de-Mera et al., 2021; Haramoto et al., 2020; La Rosa et al., 2020; Medema et al., 2020; Sherchan et al., 2020). A robust population-scale testing strategy for SARS-CoV-2 based on rapid, reliable,
decentralized, and inexpensive diagnostic testing is a high priority for clinical
testing and wastewater monitoring. Consistent with mask-wearing, frequent hand washing,
and social distancing, this testing approach could be sufficient to prevent and contain
major outbreaks while COVID-19 immunization programs are underway. Therefore, quantifying
SARS-CoV-2 in wastewater treatment plant allows for monitoring the infection among
the community via wastewater-based epidemiology (WBE) (Ahmed, Bivins et al., 2020). However, wastewater surveillance is beneficial for early warning and monitoring
of disease outbreaks and to inform the effectiveness of public health interventions
against enteric viruses such as previously demonstrated norovirus, hepatitis A virus,
and poliovirus (Asghar et al., 2014; Hellmer et al., 2014).
Wastewater surveillance for COVID-19 provides many benefits and is a cost-effective
way to investigate the transmission dynamics of an entire community (Larsen and Wigginton, 2020).
Particularly in regions lacking access to clinical testing or facing unavailability,
as well as in areas with a high volume of patients, wastewater-based surveillance
offers an alternative solution to quantify disease trends at the population level
(Beattie et al., 2022). At present wastewater surveillance of SARS-CoV-2 RNA has been used in at least
55 countries to monitor the presence and support management of COVID-19 in many Communities
(Ahmed, Angel et al., 2020; Bertrand et al., 2021; Carrillo-Reyes et al., 2021; Gibas et al., 2021; Kumar et al., 2020; Medema et al., 2020; Naughton et al., 2021; Navarro et al., 2021; Prado et al., 2020; Randazzo et al., 2020; Rimoldi et al., 2020; Westhaus et al., 2021). For the monitoring of COVID-19 through wastewater surveillance, a set of intricate
environmental microbiology methods are employed. These procedures encompassed wastewater
sampling techniques, isolation of genetic fragments from complex wastewater matrices
leading to the identification and quantification of viral RNA. This primary method
employed for this purpose involved utilizing polymerase chain reaction (PCR)-based
assays (Ahmed, Angel et al., 2020; Ahmed, Simpson et al., 2022; Pecson et al., 2021). However, wastewater samples often contain inhibitors, such as pharmaceuticals,
personal care products, household detergents, industrial effluents, and metals which,
may affect PCR amplification (Cao et al., 2012; Schrader et al., 2012). PCR inhibition can be minimized using digital PCR (dPCR) (Ahmed, Smith et al., 2022, Tiwari et al., 2022). However, sample analyzing using dPCR is expensive and often not high throughput.
Besides this, it requires trained personnel to perform and interpret results. The
availability of microfluidic technologies is a critical barrier, and many reagents
and equipment are unavailable in underdeveloped countries where they are more vulnerable
to viral infections (Kojabad et al., 2021).
This review paper particularly focused on the rapid and alternative methods which
are needed for SARS-CoV-2 RNA detection in wastewater for routine wastewater monitoring
and the social implementation of diseases surveillance. The developed methods described
in this study are efficient and applied virus detection systems with comparable reliable
sensitivity. This paper provides an overview of current available methods used for
virus concentration in wastewater and the sensitivity analysis for the specific recovery
of SARS-CoV-2 in sewage.
2. Sampling Strategy, Handling and Storage
Wastewater sampling for the pathogen is used to evaluate the trends in infection within
the community contributing water to the sewer system. According to the centers for
disease control and prevention (CDC, NWSS), there are two primary sample collection
methods for wastewater surveillance, grab and composite samples.
Collecting a grab sample is straightforward and does not require expensive auto sampler.
The grab sample provides a snapshot of wastewater at the time of sample collection
and could be less representative.
Composite samples are collected by putting multiple grab samples at a specified frequency
over time, either by continuous sampling or mixing discrete samples. Collection of
composite samples can be possible manually or by using automated samplers. A composite
sample represents the average wastewater characteristics during the compositing period.
Composite samples are more representative of fecal community contributions than grab
samples, and 24-hour composite sample is a more reliable daily average of viral concentration
(Sherchan et al., 2020). The suggested sampling depth for surface water samples should be 6 - 12 inches
below the water surface.
Wastewater samples containing SARS-CoV-2 must be managed in accordance with guidelines.
During transit to the laboratory, water samples should be either iced or refrigerated
at a temperature below 10°C. It is crucial to prevent samples from freezing, and the
use of insulated containers is recommended to maintain the storage temperature effectively.
Additionally, ensure that sample bottles are tightly closed and remain above the water
level during transportation. The experiment should be done as soon as possible after
the collection of samples. Also, sample storage and pre-treatment steps including
temperature, time, and handling may impact the concentration of virus recovered (Ahmed, Smith et al., 2022, Islam et al., 2022). Therefore, many research for WBE of pathogens has focused on developing the best
practices for viral concentration, extraction, and quantification (Ciesielski et al., 2021; LaTurner et al., 2021; Perez-Cataluna et al., 2021) however, a better understanding of sample storage and pre-processing steps is necessary
to ensure effective detection and recovery regardless of the methods used.
3. Concentration Methods
Throughout the Coronavirus 2 (SARS-CoV-2) pandemic, a range of strategies has been
implemented to detect the virus’s spread in the population. Wastewater-based epidemiology
(WBE) has emerged as an excellent tool for assessing viral circulation in communities.
To ensure reliable results, (Salvo et al., 2021) assessed three low-cost virus enrichment methods: polyethylene glycol (PEG) precipitation,
skim milk flocculation (SM), and aluminum polychloride flocculation (PAC). They utilized
Pseudomonas aeruginosa bacteriophage PP7 as a surrogate for non-enveloped viruses and Bovine Coronavirus
(BCoV) as a surrogate for enveloped viruses, with a specific focus on SARS-CoV-2.
The research findings indicate that PEG precipitation is a suitable approach for virus
concentration, proving effective for both enveloped and non-enveloped viruses in wastewater.
It demonstrates greater sensitivity compared to SM flocculation and PAC flocculation.
Moreover, a literature review reveals that many other countries have also adopted
PEG precipitation methods to concentrate SARS-CoV-2 nucleic acids (Table 1). This methodology can be applied in WBE studies to monitor the dynamics of the SARS-CoV-2
pandemic, especially in developing countries with limited economic resources.
Table 1. Concentration methods used to detect SARS-CoV-2 nucleic acids in different countries wastewater treatment plants
|
Country
|
Sampling site
|
Sample volume (ml)
|
Concentration method
|
References
|
|
China
|
Sewage
|
100
|
Subjected to polyethylene glycol precipitation
|
Zhang, Ling et al., 2020 |
|
Japan
|
Sewage
|
200-5000
|
Electronegative membrane-vortex (EMV) and membrane adsorption
|
Haramoto et al., 2020 |
|
Australia
|
Sewage
|
100-200
|
Electronegative membrane filter Ultrafiltration
|
Ahmed, Angel et al., 2020 |
|
USA
|
Sewage
|
40
|
Polyethylene glycol (PEG) precipitation
|
Wu et al., 2020 |
|
Brazil
|
Sewage
|
40
|
Ultracentrifugation
|
Prado et al., 2020 |
|
Spain
|
Sewage
|
200
|
Aluminum flocculation (beef extract precipitation)
|
Randazzo et al., 2020 |
|
France
|
Sewage
|
11
|
Ultracentrifugation
|
Wurtzer et al. 2020 |
|
Italy
|
Sewage
|
250
|
Polyethylene glycol (PEG) precipitation/dextran
|
La Rosa et al., 2020 |
|
Germany
|
Sewage
|
45
|
Ultrafiltration
|
Westhaus et al., 2020
|
|
Netherlands
|
Sewage
|
250
|
(PEG) precipitation
|
Medema et al., 2020 |
|
India
|
Sewage
|
50
|
PEG precipitation
|
Kumar et al., 2020 |
|
Turkey
|
Sewage
|
250
|
Ultrafiltration and PEG precipitation
|
Kocamemi et al., 2020 |
|
Israel
|
Sewage
|
250–1000
|
PEG/alum precipitation
|
Bar Or et al., 2020 |
3.1 PEG precipitation method
Polyethylene glycol (PEG) precipitation is one of the most conventional methods for
virus concentration (Haramoto et al., 2018; Lewis and Metcalf, 1988; Torii et al., 2022). As PEG is an inert and biocompatible polymer, PEG is preferentially applied for
trap solvents and acts as an “inert solvent sponge” (Atha and Ingham, 1981). When the concentration exceeds the saturation solubility (Atha and Ingham, 1981; Lewis and Metcalf, 1988), PEG methods are frequently applied for the concentration and precipitation of proteins
where sequestrating water molecules from the solvation layer around the proteins of
the viral capsid, enhancing the virus-virus interactions and resulting in the precipitation
(Torii et al., 2022). The advantages of PEG precipitation are that it can be performed using essential
laboratory equipment (Ahmed, Bivins et al., 2020) with relatively low running costs compared to other methods (e.g., ultrafiltration).
Other studies have also reported the applicability for the detection of SARS-CoV-2
RNA in wastewater (Hata et al., 2021; Kumar et al., 2020; Torii et al., 2021; Wu et al., 2020) and resulted in high efficiency in the recovery of RNA viruses (Amdiouni et al. 2012). The PEG method is beneficial for concentrating viruses from wastewater samples,
given the presence of multiple DNA/RNA viruses in such samples (Adriaenssens et al., 2018; Ng et al., 2012). Also, the procedures of PEG precipitation methods are primarily dependent on executors,
like several analytes as supernatant or filtrate of raw wastewater and non-pretreated
raw wastewater were added with a different concentration of salt and PEG and the incubation
time for the precipitation varied from 0 h to overnight incubation (Ahmed, Angel et al., 2020; Alexander et al., 2020; Barril et al., 2021; Chavarria-Miró et al., 2021; D’Aoust et al., 2021; Gerrity et al., 2021; Graham et al., 2021; LaTurner et al., 2021; Pecson et al., 2021; Pérez-Cataluña et al., 2021; Philo et al., 2021; Sapula et al., 2021; Torii et al., 2021).
In their 2021 report, Pecson et al. highlighted varied process recovery efficiencies
(ranging from 0.03% to 78%) for human coronavirus OC43 using PEG precipitation methods.
Interestingly, these discrepancies were observed even when employing identical wastewater
samples. A drawback of this method is that PEG induces the precipitation of diverse
proteins, including enzymes. This precipitation may interfere with or inhibit subsequent
viral genome detection through PCR amplification methods, leading to non-selective
precipitation (Masclaux et al., 2013; Shieh et al., 1995).
3.2 Skim milk and Aluminum polychloride flocculation
Skim milk flocculation, initially developed (Calgua et al. 2008) as the primary concentration method for adenovirus recovery from seawater, is also
employed for retrieving viruses from wastewater samples. The process involves three
key physical steps: i) the virus adsorbs to pre-aggregated skim milk proteins, ii)
flocs containing the adsorbed virus precipitate, and iii) the precipitate dissolves
in a phosphate buffer solution. In a previous study, a successful combination of elution
with glycine buffer and skim milk flocculation was employed to recover HAdV, JCPyV,
and NoVGII from raw municipal sewage samples (Calgua et al., 2013; Salvo et al., 2021). Their study shows that PEG precipitation and skim milk flocculation have a similar
percentage of recovery for enveloped and non-enveloped viruses using PP7 and BCoV
as surrogates of each one. Another study shows skim milk flocculation for HAdV and
RoV recovery from WWTP wastewater samples (Assis et al., 2018). They also revealed that higher recoveries of HAdV and RoV were obtained by eliminating
the initial centrifugation step and doubling the concentration of skim milk. The centrifugation
step was eliminated because the treated effluent contained less solids. The advantages
of this concentration method are that a large number of samples can be concentrated
because no special equipment is required, and the number of processing steps is reduced
(Calgua et al., 2008).
The aluminum polychloride (PAC) flocculation concentration technique exhibited high
efficiency in the recovery of feline calicivirus (FCV) from wastewater. To mitigate
the risk of handling SARS-CoV-2, FCV was utilized as a process control for this concentration
technique. Among eleven concentration methods, two protocols, one based on PEG precipitation
and the other on PAC flocculation, demonstrated notable effectiveness in FCV recovery
from wastewater (62.2% and 45.0%, respectively). Subsequently, both methods were tested
for the specific recovery of SARS-CoV-2. The PAC flocculation technique exhibited
a lower limit of detection (4.3 × 102 GC/mL) compared to PEG precipitation (4.3 × 103 GC/mL) (Barril et al., 2021). However, the study revealed that while this method recovered PP7 with a low percentage
of efficiency, it did not successfully recover BCoV. Consequently, aluminum polychloride
flocculation exhibited lower recovery efficiency and success in viral concentration
compared to PEG and SM flocculation methods (Salvo et al., 2021).
4. Extraction Methods
All viruses possess genome materials that are either RNA or DNA (Artika et al., 2020). The viral genomic material can be classified as either single-stranded or double-stranded,
with nucleic acid strands having positive (+) or negative (-) polarities. The structure
of the viral genome may be linear or circular, and viruses can have either segmented
or complete genomes (Guttman, 2013; Murphy, 1988; O’Carroll and Rein, 2016). In most PCR-based amplification processes, the template is DNA; however, in the
case of RNA viruses, the RNA is reverse-transcribed into complementary DNA (cDNA).
The quality and purity of these bio-macromolecules significantly affect the efficiency
of amplification and quantification methods. The isolation and purification of DNA/RNA
involve dissolution, purification, and recovery steps. DNA extraction methods encompass
boiling, column methods, magnetic beads, and FTA cards (Barbosa et al., 2016).
Studies focusing on virus detection in wastewater samples often rely on commercially
available DNA and RNA kits. The most common DNA extraction kits utilize columns with
silica-based membranes (Barbosa et al., 2016), categorized as solid phase-DNA extraction methods (Barbosa et al., 2016; Butler, 2010). Examples of silica-based membrane kits frequently used for extracting viral nucleic
acids from wastewater samples include those mentioned by Barbosa et al. (2016).
For RNA extraction, researchers commonly employ kits such as the RNeasy Power Microbiome
kit and RNeasy Water Kit (Ahmed, Bertsch et al., 2020; Ando et al., 2022). Automated extractors, as utilized by Ibrahim et al. (2017) and Di Bonito et al. (2017), facilitate the extraction of viral nucleic acids from influent and effluent wastewater
samples. Most automated extractors use magnetic beads that bind to nucleic acids,
leaving impurities in the solution. Elution is then performed to recover DNA bound
to the beads (Barbosa et al., 2016). The advantages of using an automated extractor include high throughput and low
variability of assay results (Dundas et al., 2008).
This review paper aims to provide guidelines for sensitive and cost-effective virus
detection, aiding in the development, optimization, and validation of the SARS-CoV-2
assay to achieve successful virus detection and consistent measurements in wastewater
samples. Immunoassays are employed when quantifying an unknown concentration of an
analyte within a sample. To ensure accurate determination, an immunoassay must be
developed based not only on standard assay development criteria but also on its ability
to accurately measure the value of a wastewater sample. Firstly, there is a need to
establish the critical success factors of the assay. Subsequently, the assay is developed
to establish proof of concept. During the optimization phase, the quantifiable range
of the immunoassay method is determined by calculating a precision profile in the
matrix in which the experimental wastewater samples will be measured. A spiked recovery
is then conducted by adding the analyte to the matrix and determining the percent
recovery of the analyte in the matrix. If the precision profile falls within the desired
working range, the immunoassay validation is completed by assaying spiked recovery
samples over several days. However, if the precision profile limits do not meet the
desired working range, further immunoassay optimization is necessary before validation
(Cox et al., 2019). Fig. 1 depicts the flowchart illustrating the development, optimization, and validation
processes for detection and quantification.
Fig. 1. Detection and quantification development, optimization, and validation flow chart.
5. Alternative Detection Methods
5.1 RT-LAMP
The standard for COVID-19 testing is RT-PCR to detect the genetic material of SARS-CoV-2
in nasopharyngeal (NP) samples. Although highly reliable, RT-PCR diagnostics are complex,
laborious, and expensive. Their global use needed more sample collection steps and
reagents for viral RNA extraction early in the pandemic (Amaral et al., 2021). On the other hand, Loop-mediated isothermal amplification (LAMP) is a DNA amplification
method that allows rapid and sensitive detection of specific genes (Nagamine et al., 2002; Notomi et al., 2000; Tomita et al., 2008). LAMP combined with reverse transcription (RT-LAMP) has been successfully used for
the detection of several respiratory RNA viruses (Ahn et al., 2019; Bhadra et al., 2015; Hong et al., 2004; Jayawardena et al., 2007; Lee et al., 2017) including SARS-CoV-2 (Thompson and Lei, 2020). RT-LAMP stands out as a reliable substitute for RT-PCR, characterized by its exceptional
specificity and sensitivity, cost-effectiveness, and rapid turnaround time, typically
within 30 minutes. Because RT-LAMP amplifies the genetic material of viruses at a
constant temperature and diagnostic tests based on RT-LAMP require only a heat block
or a water bath, set to a single temperature and they can be performed anywhere essential
resources are available. Reaction products can be analyzed via conventional DNA intercalation
dyes, agarose gel electrophoresis, UV illumination, or real-time fluorescence (Quyen et al., 2019). Alternatively, end-point colorimetric readouts are also possible through the detection
of reaction by-products, such as pyrophosphate and protons, which are released during
DNA polymerization after the incorporation of deoxynucleotide triphosphates. LAMP
colorimetric methods detect turbidity, triggered by the accumulation of magnesium
pyrophosphate (Nagamine et al., 2002), or color changes, occurring when complexometric indicators (Goto et al., 2009; Tomita et al., 2008), pH-sensitive dyes (Tanner et al., 2015) or even DNA-intercalating dyes (Fischbach et al., 2015; Lamb et al., 2020; Park et al., 2020) are incorporated into the reaction. The simple technical and instrumental requirements
of colorimetric RT-LAMP tests make them extremely attractive for point-of-care (POC)
use and implementation in low-resource settings (Fig. 2). Colorimetric RT-LAMP has been successfully used for the detection of SARS-CoV-2
in NP fluids from COVID-19 patients (Anahtar et al., 2020; Buck et al., 2020; Butler, 2020; Dao et al., 2020; Huang et al., 2020; Kellner et al., 2020; Park et al., 2020; Rabe and Cepko, 2020; Yu et al., 2020; Zhang, Odiwuor et al., 2020).
Fig. 2. Colorimetric RT-LAMP method.
Therefore, LAMP offers a practical and swift substitute for traditional PCR or qPCR
in the viral context. The amplification in LAMP doesn’t necessitate sophisticated
equipment, as the reaction is maintained at a constant temperature, typically around
65 °C (Tomita et al., 2000). Many amplification methods are susceptible to contamination,
often stemming from products of prior experiments transmitted through the environment,
researcher attire, or laboratory apparatus. Contaminant products may serve as templates
in new reactions, leading to false positives in certain instances (Dhama et al., 2014; Hsieh et al., 2014). In this regard, the LAMP process is notably vulnerable and responsive compared
to alternative detection methods. Studies demonstrate the potential application of
RT-LAMP for detecting SARS-CoV-2 in wastewater, offering a more cost-effective and
expeditious alternative to RT-qPCR or RT-ddPCR for the epidemiological monitoring
of COVID-19 and other viral infections (Amoah et al., 2021).
LAMP, developed by Notomi et al. in (2000), relies on the utilization of a minimum of four primers to initiate the polymerase-driven
extension of the gene sequence.The mechanism of RT-LAMP is based on automated cyclic
strand displacement DNA synthesis. In the LAMP reaction, polymerase gene amplification
proceeds by repeating two elongation reactions that occur through loop regions. Two
pairs of primers are used, inside and outside primer pairs. These primers are specifically
designed for the reaction. Each internal primer is complementary to one amplification
chain and has the same sequence as the internal region of the same chain. The elongation
reaction is sequentially repeated by DNA polymerase-mediated strand-displacement synthesis
with the stem mentioned above loop region as a step. This method works on the basic
principle of producing large quantities of DNA amplification products with complementary
sequences and alternating and repeating structures (Notomi et al., 2015).
However, a primer set to be used for detecting the SARS-CoV-2 virus using RT-LAMP
has been developed. This assay can detect the virus even with low sample concentrations.
The sample preparation for this can be carried out in just one tube within minutes.
Furthermore, only three buffers, a pulse-spin mini-centrifuge, and a 65°C heat block
are needed to apply this method at institutions.
RT-LAMP can achieve high specificity due to its targeting sequence. Unlike other techniques,
RT-LAMP uses six independent sequences initially and four independent sequences later
to recognize the target sequence. Primer recognition of the target genome results
in a robust colorimetric response, allowing detection without requiring highly specialized
or costly equipment. The primers designed for the target several key areas of coronavirus
genomes, including the ORF1ab gene, S gene, and N gene. ORF1ab is involved in the
replication of the viral genome, whereas the S gene is important for COVID-19 binding
to human ACE2 protein. The N gene is a nucleocapsid protein conserved in most coronaviruses.
A key improvement in the COVID-19 LAMP assay is the speed and ease at which it can
be carried out.
Furthermore, the color change associated with the presence of viral RNA, at levels
as low as 80 copies per mL sample, is visible by the eye, and therefore detection
equipment is not needed. This was achieved by using a pH indicator. Amplification
of nucleic acids causes the release of pyrophosphate and hydrogen ions, which lead
to decreases in pH, therefore making it possible to combine RT-LAMP with a visible
pH indicator to infer the presence of COVID-19. A similar method relies on the turbidity
of the sample, which increases with the amount of genetic material, to measure viral
content. Amplification and detection can also be performed by agarose gel analysis.
Therefore, RT-LAMP can be one of preferable technology for using COVID-19 detection
due to its accuracy and relatively simple equipment. This technology is possible to
applied in non-standard institutions, such as airports or rural hospitals, medical
centers, and wastewater treatment plants. Designing robust, field-based platforms
that can withstand variations in environmental conditions will broaden the utility
of RT-LAMP for on-site testing in both clinical and environmental surveillance scenarios.
Addressing the present challenges and embracing future perspectives will contribute
to the continued advancement and widespread adoption of the RT-LAMP method including
diagnostics, environmental monitoring, and point-of-care applications.
5.2 ELISA
Enzyme-linked immunosorbent assay (ELISA) is a method that detects the presence of
microbial antigens in various matrices which uses plates coated with viral proteins,
usually the N or S protein, to detect specific antibodies (Boonham et al., 2014; Lino et al., 2022). The principle of this method is antigen binding to its specific antibody and eliciting
a change in color or fluorescence due to the resultant enzyme activity. After adding
the sample, the binding of any antibodies to the viral proteins occurs. In the case
of a positive sample, the presence of the antibody–protein complex will be detected
by a color change or fluorescence after adding a marked antibody. The first step of
the process is binding an antigen at a specific antibody immobilized on a surface,
commonly in a set of 96-well microtiter plates. A second enzyme-linked antibody, specific
for the same antigen, forms an antibody-antigen-antibody sandwich. The enzyme-coupled
antibody reacts with a substrate that changes color when modified by the enzyme. The
change in color or fluorescence is correlated with the concentration of the probed
antigens in the sample (Gan and Patel, 2013). This method is faster than RT-qPCR and requires minimal equipment; However, there
is a risk of cross-reactivity to antibodies from other coronaviruses (Lv et al., 2020). Additionally, these tests are inconsistent during the first 15 days after infection.
Early detection is impossible because the human immune system takes several days to
create a detectable antibody response (Udugama et al., 2020).
Moreover, this diagnosis is usually based on detecting just one protein. These limitations
make these tests prone to inaccurate results, given the high mutation rate of the
virus. Although limited in practice for diagnosis, these tests help estimate the number
of individuals who have been in contact with SARS-CoV-2 and whether or not they develop
symptoms (Katsarou et al., 2019).
An ELISA test requires one or more antibodies with specificity for a particular antigen.
Samples containing an unknown antigen are non-specifically or immobilized explicitly
on solid support (Fig. 3). After the antigen is immobilized, a detection antibody is added to form a complex
with the antigen. The detection antibody may be covalently linked to the enzyme or
may itself be detected by a secondary antibody linked to the enzyme via bioconjugation.
The antibody incubation part of ELISA is similar to the western blot. The plate is
usually washed with a mild detergent solution between each step to remove specifically
unbound proteins or antibodies. After a final wash step, the plate is spread with
the addition of enzyme-substrate to generate a visual queue indicating the amount
of antigen in the sample.
Fig. 3. Schematic representation of the mechanism of ELISA.
5.3 Bio-Sensors
A biosensor is a device that combines a biological component that detects an analyte
and a transducer that detects a physicochemical reaction to produce a measurable signal.
A biosensor consists of three components: a bioreceptor, a transducer, and a signal
processor. A bioreceptor is a biological element, and the binding of an analyte to
a bioreceptor will cause the type of change to be detected by the transducer. This
change is converted into a measurable signal, and the signal processor is responsible
for displaying it to the electronics (Misra et al., 2021). Biosensors can be largely classified into electrochemical, thermal, optical, and
piezoelectric types according to the type of transducer. One of the techniques used
to increase the sensitivity of biosensors and lower the detection limit is the addition
of nanoparticles. Depending on the type of material, it can exhibit photoluminescence,
magnetic ability, low toxicity, high stability, or good biocompatibility and conductivity
(Ibrahim et al., 2021). Conversely, an additional benefit is their adaptability for chemical modification
to conjugate with nucleic acid probes, viral proteins, antibodies, or other ligands.
Various biosensors based on nanoparticles are currently under development for the
detection of COVID-19. Nevertheless, the advantages are the same. It is fast, cheap,
portable, user-friendly, highly sensitive, and specific. However, the use of nanoparticles
usually comes with a need to optimize these systems due to their very untapped potential.
Although several biosensors have already been developed or adapted to detect SARS-CoV-2,
their use is rare, as most are still in the process of optimization and validation
and general commercialization still needs to be improved (Lino et al., 2022).
A biosensor comprises two main components: a biological part, encompassing enzymes,
antibodies, etc., that primarily interact with analyte particles and induce a physical
change in these particles, and a transducer part that collects information from the
biological segment, converting, amplifying, and displaying it. To create a biosensor,
biological particles are immobilized on the transducer surface, serving as a point
of contact between the transducer and analyte. Biosensors are capable of detecting
biological substances, with bioreceptors derived from DNA, enzymes, antibodies, etc.
Transducers utilized in biosensors find applications in various fields, including
electrochemical, piezoelectric, optical, and thermal (Fig. 4). Biomarkers and biosensors enable the detection and tracing of bacteria and pathogens,
while biomarkers and biosensors also facilitate drug delivery to target tissues.
Fig. 4. Function of a biosensor (Kumar et al., 2018).
5.4 EPISENS-S
The Efficient and Practical virus Identification System with ENhanced Sensitivity
for Solids (EPISENS-S) method presents a practical approach for detecting SARS-CoV-2
RNA in wastewater, employing direct RNA extraction from wastewater pellets formed
through low-speed centrifugation. This technique involves two distinct steps: a first-step
RT-preamplifier before total RNA extraction and qPCR from the solid fraction of wastewater,
utilizing SARS-CoV-2 and Pepper Mild Mottle Virus (PMMoV)-specific reverse primers
for qPCR of targets with different concentrations in wastewater of RT-preamplifier
products, allowing for quantification.
To evaluate detection sensitivity, the method was tested using wastewater samples
injected with heat-inactivated SARS-CoV-2 at concentrations ranging from 2.11 × 103 to 2.11 × 106 copies/L. Results demonstrated that the EPISENS-S method exhibited a sensitivity
2-fold higher than the conventional method (general RT-qPCR after PEG precipitation;
PEG-QVR-qPCR) (Ando et al., 2022).
The limited sensitivity of existing methods for detecting SARS-CoV-2 RNA in wastewater
has hindered the widespread adoption of WBE in Japan. The development of a highly
sensitive method for detecting low-concentration SARS-CoV-2 RNA in wastewater is urgently
needed (Ando et al., 2022). Consequently, it has been suggested that the solid-phase wastewater assay may offer
greater sensitivity in SARS-CoV-2 RNA detection compared to the aqueous phase assay.Effective
social implementation of WBE demands a method that is simple, time-efficient, and
highly sensitive, as timely data collection is crucial for authorities to make informed
decisions to mitigate infections or promote socio-economic activity. Table 2 provides a comparative analysis of sensitive SARS-CoV-2 detection methods (Lino et al., 2022).
Table 2. Summary and comparison of the sensitive detection method of SARS-CoV-2 (Lino et al., 2022)
|
Methods
|
Principle
|
Positive
|
Negative
|
Cost
|
|
RT-LAMP
|
Converting COVID-19’s RNA to cDNA by transcriptase enzyme is performed and temperature
is between 60 and 65°C.
|
Fast, easy to perform high specificity and sensitivity, no expensive equipment required.
|
Difficulty in primer design, there are challenges to using LAMP for multiplex assays
in a single sample and in quantitation of target DNA.
|
Cost effective
|
|
ELISA
|
Antibody binding to coated COVID-19 Antigens on ELISA plates to form and detect complexes
with a labeled secondary antibody generated color or fluorescence.
|
Excellent sensitivity and specificity, faster and cheaper than RT-PCR.
|
Only detects 1 target, risk of cross-reactivity, needs a laboratory setting and technicians.
|
Moderate
|
|
Bio-Sensors
|
Depends on the type of sensor
|
Rapid, Fast, portable, continuous, cheap, high specificity and sensitivity.
|
Needs optimization, can be affected by environmental changes and contamination.
|
Expensive
|
|
EPISENS-S
|
Extraction of RNA from solid fraction and one step RT-Preamp prior to qPCR.
|
Highly sensitive and practically usable, effective for untreated and undiluted wastewater
samples.
|
Difficult to apply this method in secondary-treated wastewater or environmental water,
which contains only a small number of suspended solids
|
Cheap
|
|
GeneXpert
|
Cartridge based clinical test on a portable platform
|
Sensitive and rapid detection possible for SARS-CoV-2. Also, time consuming effective
method.
|
Detection limit is less than 50 copy (cp)/mL in a clinical setting
|
Moderate
|
Based on this research background, Ando et al. (2022) developed an advanced and efficient method for detecting SARS-CoV-2 RNA in wastewater.
EPISENS-S, was specifically designed for routine monitoring to facilitate the social
implementation of WBE (Fig. 5). The EPISENS-S method involves low-speed centrifugation of wastewater, direct RNA
extraction from the resulting pellet, RT pre-amplification, and qPCR using a commercial
kit. To enhance accuracy, the method also incorporates the quantification of the endemic
PMMoV, an RNA virus prevalent in wastewater (Kitajima et al., 2018), to prevent misinterpretation of SARS-CoV-2 results. The concentrations of RNA in
wastewater can be influenced by transient fecal intensity and precipitation-induced
dilution (Ando et al., 2022; Graham et al., 2021; Kim et al., 2022).
Fig. 5. Detection function of EPISENS-S method (Ando et al., 2022).
5.5 GeneXpert
GeneXpert is a molecular diagnostic platform commonly used for the detection of various
infectious diseases, including tuberculosis and COVID-19. The GeneXpert system is
a cartridge-based rapid molecular clinical test for SARS-CoV-2 on a portable platform
that can use wastewater as an input. GeneXpert demonstrated a detection limit of SARS-CoV-2
of 32 copies/mL in wastewater with a sample turnaround time of less than 1 hour (Daigle et al., 2022). An alternative possible option for rapid detection for wastewater sample testing
is the Cepheid GeneXpert system, which enables rapid, fully automated, cartridge-based
clinical testing. Recently, Cepheid launched the Xpert Xpress-SARS-CoV-2/Flu/RSV combination
test for the detection of SARS-CoV-2, Influenza A, and Influenza, a rapid diagnostic
multiplex test with a run time of 37 minutes (Johnson et al., 2021) and respiratory syncytial virus (RSV). This assay performs reverse transcription-quantitative
PCR (RT-qPCR) targeting the envelope (E) and nucleocapsid (N2) regions of the SARS-CoV-2
genome. Compared to other rapid diagnostic tests, GeneXpert has several characteristics
that make it an ideal candidate for detection of SARS-CoV-2 in wastewater.
The extraction phase of the assay uses a filtration system that separates and concentrates
viral particles while removing many of the inhibitors often present in wastewater.
Moreover, this assay is one of the most sensitive rapid tests reported with a detection
limit of less than 50 copy (cp)/mL in a clinical setting (Becker et al., 2020; Johnson et al., 2021; Wolters et al., 2020; Zhen et al., 2020). GeneXpert’s detection limit can be further improved by monitoring the endpoint
fluorescence of the assay, a method used to improve sensitivity in clinical settings
when performing high multiplex sample pooling. Finally, this test is quantitative
and provides cycle threshold (CT) values from which SARS-CoV-2 can be estimated
using a standard curve. At this observed level of sensitivity, GeneXpert can act as
an early detection system in remote communities in conjunction with a preprocessing
method for concentration (Daigle et al., 2023). Therefore, the summary and comparison
of the sensitive detection methods for SARS-CoV-2 have been presented in Table 2.
6. Conclusions
The worldwide pandemic caused by SARS-CoV-2 has emphasized the importance of effective
detection methods. Although several technologies are already developed, COVID-19 diagnosis
fundamentally relies on PCR techniques. To better track and anticipate COVID-19 disease
trends, there is a need for an easy to-use, sensitive, and rapid wastewater test for
SARS-CoV-2, particularly in remote communities or in resource-limited settings. Consequently,
this study aimed to explore the use several methods as solution for SARS-CoV-2 testing
in wastewater, which would allow for the decentralization of testing to sampling sites
and the capacity to generate near-real-time data to better guide public health actions.
However, the current research and development of sensitive and rapid technologies
are RT-LAMP, ELISA, Biosensors, GeneXpert allows a wide range of potential options
for SARS-CoV-2 detection and also for other viruses as well. Nonetheless, there are
parameters to consider before choosing the best test for each situation. The factors
that may limit testing costs are response time, availability of infrastructure, equipment,
and specialized personnel.
Additionally, the emergence of new virus strains poses a challenge, potentially impacting
the efficacy of currently commercialized detection methods. Hence, there is a crucial
need for ongoing genomic surveillance of the SARS-CoV-2 virus worldwide. This continuous
monitoring is essential to anticipate potential failures in COVID-19 tests and to
facilitate the timely replacement and update of affected testing methods. In conclusion,
the foremost challenge posed by the SARS-CoV-2 epidemic to human health necessitates
robust research aimed at developing rapid, cost-effective, sensitive, and portable
early diagnostic tools. The detection of SARS-CoV-2 in wastewater and sewage from
municipal treatment plants holds the potential to expedite mass COVID-19 diagnosis
even before clinical tests are universally accessible. Therefore, a persistent focus
on monitoring COVID-19 threats in sewage and wastewater, coupled with environmental
monitoring of public spaces and the advancement of more effective disinfection methods,
promises to mitigate the spread and impact of the global COVID-19 pandemic. It can
be confidently asserted that technological advancements in virus detection will empower
the scientific community and medical institutions to better prepare for future biological
threats and viral pandemics.
Wastewater-based surveillance is a powerful tool to provide an impartial measure of
the spread of COVID-19 in a community. This work describes wastewater rapid test for
SARS-CoV-2 based on a widely deployed technique. The advantages of easy-to-use wastewater
testing for SARS-CoV-2 are important, to deliver faster results that support surveillance
in remote communities, improve access to testing, and enable an immediate public health
response. The application of wastewater rapid testing in remote communities also demonstrated
the usefulness of rapid detection technology by facilitating the detection of COVID-19
clusters and triggering public health actions. Wastewater surveillance will become
increasingly important in post-vaccination pandemic settings as individuals with asymptomatic/mild
infection continue to transmit SARS-CoV-2 but are unlikely to be tested.