Sampling strategies for wastewater surveillance: Evaluating the variability of SARS-COV-2 RNA concentration in composite and grab samples.
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ABSTRACT:
SUBMITTER: Augusto MR
PROVIDER: S-EPMC8882035 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We monitored the concentration of indicator viruses crAssphage and pepper mild mottle virus (PMMoV) and human pathogen adenovirus (HAdV) in influent from a wastewater treatment plant in Brisbane, Australia in 1-h and 24-h composite samples. Over three days of sampling, the mean concentration of crAssphage gene copies (GC)/mL in 24-h composite samples did not differ significantly (p = 0.72-0.92), while for PMMoV GC/mL (p value range: 0.0002-0.0321) and HAdV GC/mL (p value range: 0.0028-0.0068) significant differences in concentrations were observed on one day of sampling compared to the other two. For all three viruses, the variation observed in 1-h composite samples was greater than the variation observed in 24-h composite samples. For crAssphage, in 54.1% of 1-h composite samples, the concentration was less than that observed in 24-h composite samples; whereas for PMMoV and HAdV the concentration was less in 79.2 and 70.9% of 1-h composite samples, respectively, compared to the relevant 24-h composite samples. Similarly, the concentration of crAssphage in 1-h compared to 24-h composite samples did not differ (p = 0.1082) while the concentrations of PMMoV (p < 0.0001) and HAdV (p < 0.0001) in 1-h composite samples were significantly different from 24-h composite samples. These results suggest that 24-h composite samples offer increased analytical sensitivity and decreased variability compared to 1-h composite samples when monitoring wastewater, especially for pathogenic viruses with low infection rates within a community. Thus, for wastewater-based epidemiology applications, 24-h composite samples are less likely to produce false negative results and erroneous public health information.
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Project description:The presence of antibiotics in the environment and their subsequent impact on resistance development has raised concerns globally. Hospitals are a major source of antibiotics released into the environment. To reduce these residues, research to improve knowledge of the dynamics of antibiotic release from hospitals is essential. Therefore, we undertook a study to estimate seasonal and temporal variation in antibiotic release from two hospitals in India over a period of two years. For this, 6 sampling sessions of 24 hours each were conducted in the three prominent seasons of India, at all wastewater outlets of the two hospitals, using continuous and grab sampling methods. An in-house wastewater sampler was designed for continuous sampling. Eight antibiotics from four major antibiotic groups were selected for the study. To understand the temporal pattern of antibiotic release, each of the 24-hour sessions were divided in three sub-sampling sessions of 8 hours each. Solid phase extraction followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used to determine the antibiotic residues. Six of the eight antibiotics studied were detected in the wastewater samples. Both continuous and grab sampling methods indicated that the highest quantities of fluoroquinolones were released in winter followed by the rainy season and the summer. No temporal pattern in antibiotic release was detected. In general, in a common timeframe, continuous sampling showed less concentration of antibiotics in wastewater as compared to grab sampling. It is suggested that continuous sampling should be the method of choice as grab sampling gives erroneous results, it being indicative of the quantities of antibiotics present in wastewater only at the time of sampling. Based on our studies, calculations indicate that from hospitals in India, an estimated 89, 1 and 25 ng/L/day of fluroquinolones, metronidazole and sulfamethoxazole respectively, might be getting released into the environment per 100 hospital beds.
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Project description:The shedding of pathogens by infected humans enables the use of sewage monitoring to conduct wastewater-based epidemiology (WBE). Although most WBE studies use data from large sewage treatment plants, timely data from smaller catchments are needed for targeted public health action. Traditional sampling methods, like autosamplers or grab sampling, are not conducive to quick ad hoc deployments and high-resolution monitoring at these smaller scales. This study develops and validates a cheap and easily deployable passive sampler unit, made from readily available consumables, with relevance to the COVID-19 pandemic but with broader use for WBE. We provide the first evidence that passive samplers can be used to detect SARS-CoV-2 in wastewater from populations with low prevalence of active COVID-19 infections (0.034 to 0.34 per 10,000), demonstrating their ability for early detection of infections at three different scales (lot, suburb, and city). A side by side evaluation of passive samplers (n = 245) and traditionally collected wastewater samples (n = 183) verified that the passive samplers were sensitive at detecting SARS-CoV-2 in wastewater. On all 33 days where we directly compared traditional and passive sampling techniques, at least one passive sampler was positive when the average SARS-CoV-2 concentration in the wastewater equaled or exceeded the quantification limit of 1.8 gene copies per mL (n = 7). Moreover, on 13 occasions where wastewater SARS-CoV-2 concentrations were less than 1.8 gene copies per mL, one or more passive samplers were positive. Finally, there was a statistically significant (p < 0.001) positive relationship between the concentrations of SARS-CoV-2 in wastewater and the levels found on the passive samplers, indicating that with further evaluation, these devices could yield semi-quantitative results in the future. Passive samplers have the potential for wide use in WBE with attractive feasibility attributes of cost, ease of deployment at small-scale locations, and continuous sampling of the wastewater. Further research will focus on the optimization of laboratory methods including elution and extraction and continued parallel deployment and evaluations in a variety of settings to inform optimal use in wastewater surveillance.
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Project description:Use of environmental DNA (eDNA) to assess distributions of aquatic and semi-aquatic macroorganisms is promising, but sampling schemes may need to be tailored to specific objectives. Given the potentially high variance in aquatic eDNA among replicate grab samples, compositing smaller water volumes collected over a period of time may be more effective for some applications. In this study, we compared eDNA profiles from composite water samples aggregated over three hours with grab water samples. Both sampling patterns were performed with identical autosamplers paired at two different sites in a headwater stream environment, augmented with exogenous fish eDNA from an upstream rearing facility. Samples were filtered through 0.8 μm cellulose nitrate filters and DNA was extracted with a cetyl trimethylammonium bromide procedure. Eukaryotic and bacterial community profiles were derived by amplicon sequencing of 12S ribosomal, 16S ribosomal, and cytochrome oxidase I loci. Operational taxa were assigned to genus with a lowest common ancestor approach for eukaryotes and to family with the RDP Classifier software for prokaryotes. Eukaryotic community profiles were more consistent with composite sampling than grab sampling. Downstream, rarefaction curves suggested faster taxon accumulation for composite samples, and estimated richness was higher for composite samples as a set than for grab samples. Upstream, composite sampling produced lower estimated richness than grab samples, but with overlapping standard errors. Furthermore, a bimodal pattern of richness as a function of sequence counts suggested the impact of clumped particles on upstream samples. Bacterial profiles were insensitive to sample method, consistent with the more even dispersion expected for bacteria compared with eukaryotic eDNA. Overall, samples composited over 3 h performed equal to or better than triplicate grab sampling for quantitative community metrics, despite the higher total sequencing effort provided to grab replicates. On the other hand, taxon-specific detection rates did not differ appreciably and the two methods gave similar estimates of the ratio of the common fish genera Salmo and Coregonus at each site. Unexpectedly, Salmo eDNA dropped out substantially faster than Coregonus eDNA between the two sites regardless of sampling method, suggesting that differential settling affects the estimation of relative abundance. We identified bacterial patterns that were associated with eukaryotic diversity, suggesting potential roles as biomarkers of sample representativeness.
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Project description:Restoring all facility operations after the 2001 Amerithrax attacks took years to complete, highlighting the need to reduce remediation time. Some of the most time intensive tasks were environmental sampling and sample analyses. Composite sampling allows disparate samples to be combined, with only a single analysis needed, making it a promising method to reduce response times. We developed a statistical experimental design to test three different composite sampling methods: 1) single medium single pass composite (SM-SPC): a single cellulose sponge samples multiple coupons with a single pass across each coupon; 2) single medium multi-pass composite: a single cellulose sponge samples multiple coupons with multiple passes across each coupon (SM-MPC); and 3) multi-medium post-sample composite (MM-MPC): a single cellulose sponge samples a single surface, and then multiple sponges are combined during sample extraction. Five spore concentrations of Bacillus atrophaeus Nakamura spores were tested; concentrations ranged from 5 to 100 CFU/coupon (0.00775 to 0.155 CFU/cm2). Study variables included four clean surface materials (stainless steel, vinyl tile, ceramic tile, and painted dry wallboard) and three grime coated/dirty materials (stainless steel, vinyl tile, and ceramic tile). Analysis of variance for the clean study showed two significant factors: composite method (p< 0.0001) and coupon material (p = 0.0006). Recovery efficiency (RE) was higher overall using the MM-MPC method compared to the SM-SPC and SM-MPC methods. RE with the MM-MPC method for concentrations tested (10 to 100 CFU/coupon) was similar for ceramic tile, dry wall, and stainless steel for clean materials. RE was lowest for vinyl tile with both composite methods. Statistical tests for the dirty study showed RE was significantly higher for vinyl and stainless steel materials, but lower for ceramic tile. These results suggest post-sample compositing can be used to reduce sample analysis time when responding to a Bacillus anthracis contamination event of clean or dirty surfaces.
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Project description:Wastewaters can be analyzed to generate population-level data for public health surveillance, such as antibiotic resistance monitoring. To provide representative data for the contributing population, bacterial isolates collected from wastewater should originate from different individuals and not be distorted by a selection pressure in the wastewater. Here we use Escherichia coli diversity as a proxy for representativeness when comparing grab and composite sampling at a major municipal wastewater treatment plant influent and an untreated hospital effluent in Gothenburg, Sweden. All municipal samples showed high E. coli diversity irrespective of the sampling method. In contrast, a marked increase in diversity was seen for composite compared to grab samples from the hospital effluent. Virtual resampling also showed the value of collecting fewer isolates on multiple occasions rather than many isolates from a single sample. Time-kill tests where individual E. coli strains were exposed to sterile-filtered hospital wastewater showed rapid killing of antibiotic-susceptible strains and significant selection of multi-resistant strains when incubated at 20 °C, an effect which could be avoided at 4 °C. In conclusion, depending on the wastewater collection site, both sampling method and collection/storage temperature could significantly impact the representativeness of the wastewater sample.
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Project description:Wastewater1 surveillance of SARS-CoV-2 may be a useful supplement to clinical surveillance as it is shed in feces, there are many asymptomatic cases, and diagnostic testing can have capacity limitations and extended time to results. Although numerous studies have utilized wastewater surveillance for SARS-CoV-2, the methods used were developed and/or standardized for other pathogens. This study evaluates multiple methods for concentration and recovery of SARS-CoV-2 and seeded human coronavirus OC43 from municipal primary wastewater and/or sludge from the Greater Seattle Area (March-July 2020). Methods evaluated include the bag-mediated filtration system (BMFS), with and without Vertrel™ extraction, skimmed milk flocculation, with and without Vertrel™ extraction, polyethylene glycol (PEG) precipitation, ultrafiltration, and sludge extraction. Total RNA was extracted from wastewater concentrates and analyzed for SARS-CoV-2 and OC43 with RT-qPCR. Skimmed milk flocculation without Vertrel™ extraction performed consistently over time and between treatment plants in Seattle-area wastewater with the lowest average OC43 Cq value and smallest variability (24.3; 95% CI: 23.8-24.9), most frequent SARS-CoV-2 detection (48.8% of sampling events), and highest average OC43 percent recovery (9.1%; 95% CI: 6.2-11.9%). Skimmed milk flocculation is also beneficial because it is feasible in low-resource settings. While the BMFS had the highest average volume assayed of 11.9 mL (95% CI: 10.7-13.1 mL), the average OC43 percent recovery was low (0.7%; 95% CI: 0.4-1.0%). Ultrafiltration and PEG precipitation had low average OC43 percent recoveries of 1.0% (95% CI: 0.5-1.6%) and 3.2% (95% CI: 1.3-5.1%), respectively. The slopes and efficiency for the SARS-CoV-2 standard curves were not consistent over time, confirming the need to include a standard curve each run rather than using a single curve for multiple plates. Results suggest that the concentration and detection methods used must be validated for the specific water matrix using a recovery control to assess performance over time.
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Project description:In search of practical and affordable tools for wastewater-based surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), three independent field experiments were conducted using three passive sampler sorbents (electronegative membrane, cotton bud, and gauze) in Guelph, Ontario, Canada. Total daily cases during this study ranged from 2 to 17/100,000 people and 43/54 traditionally collected wastewater samples were positive for SARS-CoV-2 with mean detectable concentrations ranging from 8.4 to 1780 copies/ml. Viral levels on the passive samplers were assessed after 4, 8, 24, 48, 72, and 96 hrs of deployment in the wastewater and 43/54 membrane, 42/54 gauze, and 27/54 cotton bud samples were positive. A linear accumulation rate of SARS-CoV-2 on the membranes was observed up to 48 hours, suggesting the passive sampler could adequately reflect wastewater levels for up to two days of deployment. Due the variability in accumulation observed for the cotton buds and gauzes, and the pre-processing steps required for the gauzes, we recommend membrane filters as a simple cost-effective option for wastewater-based surveillance of SARS-CoV-2.
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Project description:Wastewater-based epidemiology is now widely used in many countries for the routine monitoring of SARS-CoV-2 and other viruses at a community level. However, efficient sample processing technologies are still under investigation. In this study, we compared the performance of the novel Nanotrap® Microbiome Particles (NMP) concentration method to the commonly used polyethylene glycol (PEG) precipitation method for concentrating viruses from wastewater and their subsequent quantification and sequencing. For this, we first spiked wastewater with SARS-CoV-2, influenza and measles viruses and norovirus and found that the NMP method recovered 0.4%-21% of them depending on virus type, providing consistent and reproducible results. Using the NMP and PEG methods, we monitored SARS-CoV-2, influenza A and B viruses, RSV, enteroviruses and norovirus GI and GII and crAssphage in wastewater using quantitative PCR (qPCR)-based methods and next-generation sequencing. Good viral recoveries were observed for highly abundant viruses using both methods; however, PEG precipitation was more successful in the recovery of low-abundance viruses present in wastewater. Furthermore, samples processed with PEG precipitation were more successfully sequenced for SARS-CoV-2 than those processed with the NMP method. Virus recoveries were enhanced by high sample volumes when PEG precipitation was applied. Overall, our results suggest that the NMP concentration method is a rapid and easy virus concentration method for viral targets that are abundant in wastewater, whereas PEG precipitation may be more suited to the recovery and analysis of low-abundance viruses and for next generation sequencing.
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Project description:Much of what is known and theorized concerning passive sampling techniques has been developed considering chemical analytes. Yet, historically, biological analytes, such as Salmonella typhi, have been collected from wastewater via passive sampling with Moore swabs. In response to the COVID-19 pandemic, passive sampling is re-emerging as a promising technique to monitor SARS-CoV-2 RNA in wastewater. Method comparisons and disease surveillance using composite, grab, and passive sampling for SARS-CoV-2 RNA detection have found passive sampling with a variety of materials routinely produced qualitative results superior to grab samples and useful for sub-sewershed surveillance of COVID-19. Among individual studies, SARS-CoV-2 RNA concentrations derived from passive samplers demonstrated heterogeneous correlation with concentrations from paired composite samples ranging from weak (R2 = 0.27, 0.31) to moderate (R2 = 0.59) to strong (R2 = 0.76). Among passive sampler materials, electronegative membranes have shown great promise with linear uptake of SARS-CoV-2 RNA observed for exposure durations of 24 to 48 h and in several cases RNA positivity on par with composite samples. Continuing development of passive sampling methods for the surveillance of infectious diseases via diverse forms of fecal waste should focus on optimizing sampler materials for the efficient uptake and recovery of biological analytes, kit-free extraction, and resource-efficient testing methods capable of rapidly producing qualitative or quantitative data. With such refinements passive sampling could prove to be a fundamental tool for scaling wastewater surveillance of infectious disease, especially among the 1.8 billion persons living in low-resource settings served by non-traditional wastewater collection infrastructure.
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