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3.
Environ Sci Technol ; 56(18): 13245-13253, 2022 09 20.
Article in English | MEDLINE | ID: covidwho-2016515

ABSTRACT

Wastewater-based surveillance of the COVID-19 pandemic holds great promise; however, a point-of-use detection method for SARS-CoV-2 in wastewater is lacking. Here, a portable paper device based on CRISPR/Cas12a and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with excellent sensitivity and specificity was developed for SARS-CoV-2 detection in wastewater. Three primer sets of RT-LAMP and guide RNAs (gRNAs) that could lead Cas12a to recognize target genes via base pairing were used to perform the high-fidelity RT-LAMP to detect the N, E, and S genes of SARS-CoV-2. Due to the trans-cleavage activity of CRISPR/Cas12a after high-fidelity amplicon recognition, carboxyfluorescein-ssDNA-Black Hole Quencher-1 and carboxyfluorescein-ssDNA-biotin probes were adopted to realize different visualization pathways via a fluorescence or lateral flow analysis, respectively. The reactions were integrated into a paper device for simultaneously detecting the N, E, and S genes with limits of detection (LODs) of 25, 310, and 10 copies/mL, respectively. The device achieved a semiquantitative analysis from 0 to 310 copies/mL due to the different LODs of the three genes. Blind experiments demonstrated that the device was suitable for wastewater analysis with 97.7% sensitivity and 82% semiquantitative accuracy. This is the first semiquantitative endpoint detection of SARS-CoV-2 in wastewater via different LODs, demonstrating a promising point-of-use method for wastewater-based surveillance.


Subject(s)
SARS-CoV-2 , Waste Water , Biotin/genetics , CRISPR-Cas Systems , Fluoresceins , Nucleic Acid Amplification Techniques , Pandemics , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Waste Water/virology
4.
Nature ; 609(7925): 101-108, 2022 09.
Article in English | MEDLINE | ID: covidwho-1921636

ABSTRACT

As SARS-CoV-2 continues to spread and evolve, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing and/or sequencing capacity, which can also introduce biases1-3. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing4,5. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We developed and deployed improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detected emerging variants of concern up to 14 days earlier in wastewater samples, and identified multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.


Subject(s)
COVID-19 , SARS-CoV-2 , Waste Water , Wastewater-Based Epidemiological Monitoring , COVID-19/epidemiology , COVID-19/transmission , COVID-19/virology , Humans , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/classification , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sequence Analysis, RNA , Waste Water/virology
5.
Int J Environ Res Public Health ; 19(13)2022 06 24.
Article in English | MEDLINE | ID: covidwho-1911357

ABSTRACT

The SARS-CoV-2 virus, which is driving the current COVID-19 epidemic, has been detected in wastewater and is being utilized as a surveillance tool to establish an early warning system to aid in the management and prevention of future pandemics. qPCR is the method usually used to detect SARS-CoV-2 in wastewater. There has been no study using an immunoassay that is less laboratory-intensive than qPCR with a shorter turnaround time. Therefore, we aimed to evaluate the performance of an automated chemiluminescence enzyme immunoassay (CLEIA) for SARS-CoV-2 antigen in wastewater. The CLEIA assay achieved 100% sensitivity and 66.7% specificity in a field-captured wastewater sample compared to the gold standard RT-qPCR. Our early findings suggest that the SARS-CoV-2 antigen can be identified in wastewater samples using an automated CLEIA, reducing the turnaround time and improving the performance of SARS-CoV-2 wastewater monitoring during the pandemic.


Subject(s)
COVID-19 , Immunoenzyme Techniques , SARS-CoV-2 , Waste Water , Antigens, Viral/immunology , Antigens, Viral/isolation & purification , COVID-19/epidemiology , COVID-19/prevention & control , Humans , Immunoenzyme Techniques/methods , Luminescent Measurements , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Waste Water/virology , Wastewater-Based Epidemiological Monitoring
6.
Sci Rep ; 12(1): 2659, 2022 02 17.
Article in English | MEDLINE | ID: covidwho-1900621

ABSTRACT

The COVID-19 pandemic represents an unprecedented global crisis necessitating novel approaches for, amongst others, early detection of emerging variants relating to the evolution and spread of the virus. Recently, the detection of SARS-CoV-2 RNA in wastewater has emerged as a useful tool to monitor the prevalence of the virus in the community. Here, we propose a novel methodology, called lineagespot, for the monitoring of mutations and the detection of SARS-CoV-2 lineages in wastewater samples using next-generation sequencing (NGS). Our proposed method was tested and evaluated using NGS data produced by the sequencing of 14 wastewater samples from the municipality of Thessaloniki, Greece, covering a 6-month period. The results showed the presence of SARS-CoV-2 variants in wastewater data. lineagespot was able to record the evolution and rapid domination of the Alpha variant (B.1.1.7) in the community, and allowed the correlation between the mutations evident through our approach and the mutations observed in patients from the same area and time periods. lineagespot is an open-source tool, implemented in R, and is freely available on GitHub and registered on bio.tools.


Subject(s)
Mutation , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Software , Waste Water/virology , Humans
8.
Sci Rep ; 12(1): 3487, 2022 03 03.
Article in English | MEDLINE | ID: covidwho-1730315

ABSTRACT

Monitoring the progression of SARS-CoV-2 outbreaks requires accurate estimation of the unobservable fraction of the population infected over time in addition to the observed numbers of COVID-19 cases, as the latter present a distorted view of the pandemic due to changes in test frequency and coverage over time. The objective of this report is to describe and illustrate an approach that produces representative estimates of the unobservable cumulative incidence of infection by scaling the daily concentrations of SARS-CoV-2 RNA in wastewater from the consistent population contribution of fecal material to the sewage collection system.


Subject(s)
COVID-19/epidemiology , SARS-CoV-2/isolation & purification , Waste Water/virology , COVID-19/virology , Humans , Incidence
9.
PLoS One ; 16(11): e0258263, 2021.
Article in English | MEDLINE | ID: covidwho-1700786

ABSTRACT

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.


Subject(s)
COVID-19/virology , Ribonuclease P/genetics , SARS-CoV-2/genetics , Waste Water/virology , DNA Primers/genetics , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Specimen Handling/methods , Wastewater-Based Epidemiological Monitoring
10.
Environ Health Perspect ; 129(4): 45002, 2021 04.
Article in English | MEDLINE | ID: covidwho-1673983

ABSTRACT

BACKGROUND: Wastewater testing offers a cost-effective strategy for measuring population disease prevalence and health behaviors. For COVID-19, wastewater surveillance addresses testing gaps and provides an early warning for outbreaks. As U.S. federal agencies build a National Wastewater Surveillance System around the pandemic, thinking through ways to develop flexible frameworks for wastewater sampling, testing, and reporting can avoid unnecessary system overhauls for future infectious disease, chronic disease, and drug epidemics. OBJECTIVES: We discuss ways to transform a historically academic exercise into a tool for epidemic response. We generalize lessons learned by a global network of wastewater researchers around validation and implementation for COVID-19 and opioids while also drawing on our experience with wastewater-based epidemiology in the United States. DISCUSSION: Sustainable wastewater surveillance requires coordination between health and safety officials, utilities, labs, and researchers. Adapting sampling frequency, type, and location to threat level, community vulnerability, biomarker properties, and decisions that wastewater data will inform can increase the practical value of the data. Marketplace instabilities, coupled with a fragmented testing landscape due to specialization, may require officials to engage multiple labs to test for known and unknown threats. Government funding can stabilize the market, balancing commercial pressures with public good, and incentivize data sharing. When reporting results, standardizing metrics and contextualizing wastewater data with health resource data can provide insights into a community's vulnerability and identify strategies to prevent health care systems from being overwhelmed. If wastewater data will inform policy decisions for an entire community, comparing characteristics of the wastewater treatment plant's service population to those of the larger community can help determine whether the wastewater data are generalizable. Ethical protocols may be needed to protect privacy and avoid stigmatization. With data-driven approaches to sample collection, analysis, and interpretation, officials can use wastewater surveillance for adaptive resource allocation, pandemic management, and program evaluation. https://doi.org/10.1289/EHP8572.


Subject(s)
COVID-19 , Epidemiological Monitoring , SARS-CoV-2/isolation & purification , Waste Water/virology , Humans , Pandemics , United States
11.
Nat Commun ; 13(1): 635, 2022 02 03.
Article in English | MEDLINE | ID: covidwho-1671556

ABSTRACT

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID's EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir.


Subject(s)
SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Waste Water/virology , Water Microbiology , Adult , Aged , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/virology , Female , Genetic Variation , High-Throughput Nucleotide Sequencing , Humans , Male , Mice , Middle Aged , Mutation , New York City , Protein Binding , Rats , Spike Glycoprotein, Coronavirus/immunology , Young Adult
12.
Epidemiol Infect ; 150: e21, 2022 Jan 07.
Article in English | MEDLINE | ID: covidwho-1655367

ABSTRACT

Since the start of the coronavirus disease-2019 (COVID-19) pandemic, there has been interest in using wastewater monitoring as an approach for disease surveillance. A significant uncertainty that would improve the interpretation of wastewater monitoring data is the intensity and timing with which individuals shed RNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into wastewater. By combining wastewater and case surveillance data sets from a university campus during a period of heightened surveillance, we inferred that individual shedding of RNA into wastewater peaks on average 6 days (50% uncertainty interval (UI): 6-7; 95% UI: 4-8) following infection, and that wastewater measurements are highly overdispersed [negative binomial dispersion parameter, k = 0.39 (95% credible interval: 0.32-0.48)]. This limits the utility of wastewater surveillance as a leading indicator of secular trends in SARS-CoV-2 transmission during an epidemic, and implies that it could be most useful as an early warning of rising transmission in areas where transmission is low or clinical testing is delayed or of limited capacity.


Subject(s)
COVID-19/transmission , RNA, Viral/analysis , SARS-CoV-2/isolation & purification , Virus Shedding , Waste Water/virology , Time Factors
15.
Viruses ; 14(1)2022 01 13.
Article in English | MEDLINE | ID: covidwho-1625824

ABSTRACT

Infection with enterovirus D68 (EV-D68) has been linked with severe neurological disease such as acute flaccid myelitis (AFM) in recent years. However, active surveillance for EV-D68 is lacking, which makes full assessment of this association difficult. Although a high number of EV-D68 infections were expected in 2020 based on the EV-D68's known biannual circulation patterns, no apparent increase in EV-D68 detections or AFM cases was observed during 2020. We describe an upsurge of EV-D68 detections in wastewater samples from the United Kingdom between July and November 2021 mirroring the recently reported rise in EV-D68 detections in clinical samples from various European countries. We provide the first publicly available 2021 EV-D68 sequences showing co-circulation of EV-D68 strains from genetic clade D and sub-clade B3 as in previous years. Our results show the value of environmental surveillance (ES) for the early detection of circulating and clinically relevant human viruses. The use of a next-generation sequencing (NGS) approach helped us to estimate the prevalence of EV-D68 viruses among EV strains from other EV serotypes and to detect EV-D68 minor variants. The utility of ES at reducing gaps in virus surveillance for EV-D68 and the possible impact of nonpharmaceutical interventions introduced to control the COVID-19 pandemic on EV-D68 transmission dynamics are discussed.


Subject(s)
Enterovirus D, Human/isolation & purification , Waste Water/virology , COVID-19/epidemiology , COVID-19/prevention & control , Capsid Proteins/genetics , Enterovirus D, Human/classification , Enterovirus D, Human/genetics , Humans , Phylogeny , RNA, Viral/genetics , SARS-CoV-2 , Sequence Analysis, DNA , United Kingdom/epidemiology , Wastewater-Based Epidemiological Monitoring , Water Microbiology
16.
Yakugaku Zasshi ; 142(1): 11-15, 2022.
Article in Japanese | MEDLINE | ID: covidwho-1609123

ABSTRACT

The polio eradication program, launched in 1988, has successfully decreased the number of poliomyelitis patients worldwide. However, in areas with immunization gaps where oral polio vaccine coverage has dropped, outbreaks of more virulent vaccine-derived polioviruses (VDPVs) have become a threat to public health. In Japan, inactivated polio vaccine replaced oral polio vaccine as the routine immunization in 2012. Polio environmental surveillance (ES) has been conducted nationwide since 2013 to efficiently monitor the wild type poliovirus or VDPV, which may be imported from overseas. ES may also be utilized to detect other viruses in stool samples. We propose a method of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection based on the polio ES network, and establish a procedure to detect fragments of SARS-CoV-2 genome in wastewater solids. Our findings suggest that polio ES can be used to simultaneously monitor SARS-CoV-2 RNA fragments in sewage waters.


Subject(s)
Environmental Monitoring/methods , Poliovirus/isolation & purification , SARS-CoV-2/isolation & purification , Sewage/virology , Waste Water/virology , Disease Eradication , Humans , Japan , Poliovirus Vaccine, Inactivated , RNA, Viral/isolation & purification , SARS-CoV-2/genetics
17.
Viruses ; 13(12)2021 11 24.
Article in English | MEDLINE | ID: covidwho-1551630

ABSTRACT

During the four pandemic waves, a total of 560,504 cases and 10,178 deaths due to COVID-19 were reported in Croatia. The Alpha variant, dominant from March 2021 (>50% of positive samples), was rapidly replaced by Delta variants (>90%) by August 2021. Several seroprevalence studies were conducted in different populations (general population, children/adolescents, professional athletes, healthcare workers, veterinarians) and in immunocompromised patients (hemodialysis patients, liver/kidney transplant recipients). After the first pandemic wave, seroprevalence rates of neutralizing (NT) antibodies were reported to be 0.2-5.5%. Significantly higher seropositivity was detected during/after the second wave, 2.6-18.7%. Two studies conducted in pet animals (February-June 2020/July-December 2020) reported SARS-CoV-2 NT antibodies in 0.76% of cats and 0.31-14.69% of dogs, respectively. SARS-CoV-2 NT antibodies were not detected in wildlife. Environmental samples taken in the households of COVID-19 patients showed high-touch personal objects as most frequently contaminated (17.3%), followed by surfaces in patients' rooms (14.6%), kitchens (13.3%) and bathrooms (8.3%). SARS-CoV-2 RNA was also detected in 96.8% affluent water samples, while all effluent water samples tested negative. Detection of SARS-CoV-2 in humans, animals and the environment suggests that the 'One Health' approach is critical to controlling COVID-19 and future pandemics.


Subject(s)
COVID-19/epidemiology , COVID-19/virology , One Health , Pandemics , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/transmission , COVID-19/veterinary , Cats , Croatia/epidemiology , Dogs , Genetic Variation , Health Personnel , Humans , Pets , Prevalence , RNA, Viral , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Seroepidemiologic Studies , Waste Water/virology
18.
Microbiol Spectr ; 9(2): e0079221, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1526452

ABSTRACT

A wastewater surveillance program targeting a university residence hall was implemented during the spring semester 2021 as a proactive measure to avoid an outbreak of COVID-19 on campus. Over a period of 7 weeks from early February through late March 2021, wastewater originating from the residence hall was collected as grab samples 3 times per week. During this time, there was no detection of SARS-CoV-2 by reverse transcriptase quantitative PCR (RT-qPCR) in the residence hall wastewater stream. Aiming to obtain a sample more representative of the residence hall community, a decision was made to use passive samplers beginning in late March onwards. Adopting a Moore swab approach, SARS-CoV-2 was detected in wastewater samples just 2 days after passive samplers were deployed. These samples also tested positive for the B.1.1.7 (Alpha) variant of concern (VOC) using RT-qPCR. The positive result triggered a public health case-finding response, including a mobile testing unit deployed to the residence hall the following day, with testing of nearly 200 students and staff, which identified two laboratory-confirmed cases of Alpha variant COVID-19. These individuals were relocated to a separate quarantine facility, averting an outbreak on campus. Aggregating wastewater and clinical data, the campus wastewater surveillance program has yielded the first estimates of fecal shedding rates of the Alpha VOC of SARS-CoV-2 in individuals from a nonclinical setting. IMPORTANCE Among early adopters of wastewater monitoring for SARS-CoV-2 have been colleges and universities throughout North America, many of whom are using this approach to monitor congregate living facilities for early evidence of COVID-19 infection as an integral component of campus screening programs. Yet, while there have been numerous examples where wastewater monitoring on a university campus has detected evidence for infection among community members, there are few examples where this monitoring triggered a public health response that may have averted an actual outbreak. This report details a wastewater-testing program targeting a residence hall on a university campus during spring 2021, when there was mounting concern globally over the emergence of SARS-CoV-2 variants of concern, reported to be more transmissible than the wild-type Wuhan strain. In this communication, we present a clear example of how wastewater monitoring resulted in actionable responses by university administration and public health, which averted an outbreak of COVID-19 on a university campus.


Subject(s)
COVID-19/epidemiology , Disease Outbreaks , SARS-CoV-2/isolation & purification , Universities , Waste Water/virology , Wastewater-Based Epidemiological Monitoring , COVID-19/transmission , COVID-19/virology , Humans , Mass Screening , Ontario , Public Health , SARS-CoV-2/classification , SARS-CoV-2/genetics
19.
Sci Total Environ ; 810: 151283, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1510283

ABSTRACT

SARS-CoV-2 variants of concern (VoC) have been increasingly detected in clinical surveillance in Canada and internationally. These VoC are associated with higher transmissibility rates and in some cases, increased mortality. In this work we present a national wastewater survey of the distribution of three SARS-CoV-2 mutations found in the B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma) VoC, namely the S-gene 69-70 deletion, N501Y mutation, and N-gene D3L. RT-qPCR allelic discrimination assays were sufficiently sensitive and specific for detection and relative quantitation of SARS-CoV-2 variants in wastewater to allow for rapid population-level screening and surveillance. We tested 261 samples collected from 5 Canadian cities (Vancouver, Edmonton, Toronto, Montreal, and Halifax) and 6 communities in the Northwest Territories from February 16th to March 28th, 2021. VoC were not detected in the Territorial communities, suggesting the absence of VoC SARS-CoV-2 cases in those communities. Percentage of variant remained low throughout the study period in the majority of the sites tested, however the Toronto sites showed a marked increase from ~25% to ~75% over the study period. The results of this study highlight the utility of population level molecular surveillance of SARS-CoV-2 VoC using wastewater. Wastewater monitoring for VoC can be a powerful tool in informing public health responses, including monitoring trends independent of clinical surveillance and providing early warning to communities.


Subject(s)
SARS-CoV-2 , Waste Water/virology , COVID-19 , Canada , Humans , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification
20.
Sci Rep ; 11(1): 21368, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1493221

ABSTRACT

There is a need for wastewater based epidemiological (WBE) methods that integrate multiple, variously sized surveillance sites across geographic areas. We developed a novel indexing method, Melvin's Index, that provides a normalized and standardized metric of wastewater pathogen load for qPCR assays that is resilient to surveillance site variation. To demonstrate the utility of Melvin's Index, we used qRT-PCR to measure SARS-CoV-2 genomic RNA levels in influent wastewater from 19 municipal wastewater treatment facilities (WWTF's) of varying sizes and served populations across the state of Minnesota during the Summer of 2020. SARS-CoV-2 RNA was detected at each WWTF during the 20-week sampling period at a mean concentration of 8.5 × 104 genome copies/L (range 3.2 × 102-1.2 × 109 genome copies/L). Lag analysis of trends in Melvin's Index values and clinical COVID-19 cases showed that increases in indexed wastewater SARS-CoV-2 levels precede new clinical cases by 15-17 days at the statewide level and by up to 25 days at the regional/county level. Melvin's Index is a reliable WBE method and can be applied to both WWTFs that serve a wide range of population sizes and to large regions that are served by multiple WWTFs.


Subject(s)
COVID-19/epidemiology , SARS-CoV-2/genetics , Suburban Population , Urban Population , Waste Disposal Facilities , Waste Water/virology , Wastewater-Based Epidemiological Monitoring , Water Purification , COVID-19/virology , Genome, Viral , Humans , Minnesota/epidemiology , Prevalence , Prognosis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Risk Factors
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