Wastewater surveillance of SARS-CoV-2 RNA is increasingly being incorporated into public health efforts to respond to the COVID-19 pandemic. In order to obtain the maximum benefit from these efforts, approaches to wastewater monitoring need to be rapid, sensitive, and relatable to relevant epidemiological parameters. In this study, we present an ultracentrifugation-based method for the concentration of SARS-CoV-2 wastewater RNA and use crAssphage, a bacteriophage specific to the human gut, to help account for RNA loss during transit in the wastewater system and sample processing. With these methods, we were able to detect, and sometimes quantify, SARS-CoV-2 RNA from 20 mL wastewater samples within as little as 4.5 hours. Using known concentrations of bovine coronavirus RNA and deactivated SARS-CoV-2, we estimate recovery rates of approximately 7-12% of viral RNA using our method. Results from 24 sewersheds across Upstate New York during the spring and summer of 2020 suggested that stronger signals of SARS-CoV-2 RNA from wastewater may be indicative of greater COVID-19 incidence in the represented service area approximately one week in advance. SARS-CoV-2 wastewater RNA was quantifiable in some service areas with daily positives tests of less than 1 per 10,000 people or when weekly positive test rates within a sewershed were as low as 1.7%. crAssphage DNA concentrations were significantly lower during periods of high flow in almost all areas studied. After accounting for flow rate and population served, crAssphage levels per capita were estimated to be about 1.35 × 10 11 and 2.42 × 10 8 genome copies per day for DNA and RNA, respectively. A negative relationship between per capita crAssphage RNA and service area size was also observed likely reflecting degradation of RNA over long transit times. Our results reinforce the potential for wastewater surveillance to be used as a tool to supplement understanding of infectious disease transmission obtained by traditional testing and highlight the potential for crAssphage co-detection to improve interpretations of wastewater surveillance data.
Wastewater surveillance of SARS-CoV-2 has become an attractive tool for combating the spread of COVID-19 by assessing the presence or levels of the virus shed in a population. However, the methods to quantify viral RNA and to link those quantities to the level of infection within the community vary. In this study, we sought to identify and optimize scalable methods for recovery of viral nucleic acids from wastewater and attempted to use a constitutive member of the gut virome, human-specific crAssphage, to help account for unknown levels of SARS-CoV-2 decay and dilution in the wastewater infrastructure. Results suggest that ultracentrifugation of a small volume of wastewater through a 50% sucrose cushion followed by total nucleic acid extraction yielded quantifiable virus in an area with a modest number of COVID-19 cases. Further, the ratio of log10(SARS-CoV-2):log10(crAssphage) appears to be associated with the cumulative incidence of COVID-19 in the Syracuse, NY area. In areas where ultracentrifuges are available, these methods may be used to link SARS-CoV-2 quantities in wastewater to levels of transmission within communities with sewer service.
Bog turtles (Glyptemys muhlenbergii) are listed as Species of Greatest Conservation Need (SGCN) for wildlife action plans in every state it occurs and multi-state efforts are underway to better characterize extant populations and prioritize restoration efforts. However, traditional sampling methods can be ineffective due to the turtle’s wetland habitat, small size, and burrowing nature. Molecular methods, such as qPCR, provide the ability to overcome this challenge by effectively quantifying minute amounts of turtle DNA left behind in its environment (eDNA). Developing such methods for bog turtles has proved difficult partly because of the high sequence similarity between bog turtles and closely-related, cohabitating species, most often wood turtles (Glyptemys insculpta). Additionally, substrates containing bog turtle eDNA are often rich in organics or other substances that frequently inhibit both DNA extraction and qPCR amplification. Here, we describe the development and validation of a qPCR assay, BT3, targeting the mitochondrial cytochrome oxidase I gene that correctly identifies bog turtles with 100% specificity and sensitivity when tested on 201 blood samples collected from six species over a wide geographic range. We also developed a full-process internal control employing a genetically modified strain of Caenorhabditis elegans to improve DNA extraction methods, limit false negative results due to qPCR inhibition, and measure total DNA recovery from each sample. Using the internal control, we found that DNA recovery varied by over an order of magnitude between samples and likely explains the lack of bog turtle detection in some cases. Methods presented herein are highly-specific and may offer a more cost effective, non-invasive tool to supplement bog turtle population assessments in the Eastern United States. Poor or differential DNA recovery, which remains unmeasured in the vast majority of eDNA studies, significantly reduced the ability to detect bog turtle in their natural environment.
Wastewater entering sewer networks represents a unique source of pooled epidemiological information. In this study, we coupled online solid-phase extraction with liquid chromatography-high resolution mass spectrometry to achieve high-throughput analysis...
Infectious disease surveillance is vitally important to maintaining health security, but these efforts are challenged by the pace at which new pathogens emerge. Wastewater surveillance can rapidly obtain population-level estimates of disease transmission, and we leverage freedom from disease principles to make use of nondetection of SARS-CoV-2 in wastewater to estimate the probability that a community is free from SARS-CoV-2 transmission. From wastewater surveillance of 24 treatment plants across upstate New York from May through December of 2020, trends in the intensity of SARS-CoV-2 in wastewater correlate with trends in COVID-19 incidence and test positivity (⍴ > 0.5), with the greatest correlation observed for active cases and a 3-day lead time between wastewater sample date and clinical test date. No COVID-19 cases were reported 35% of the time the week of a nondetection of SARS-CoV-2 in wastewater. Compared to the United States Centers for Disease Control and Prevention levels of transmission risk, transmission risk was low (no community spared) 50% of the time following nondetection, and transmission risk was moderate or lower (low community spread) 92% of the time following nondetection. Wastewater surveillance can demonstrate the geographic extent of the transmission of emerging pathogens, confirming that transmission risk is either absent or low and alerting of an increase in transmission. If a statewide wastewater surveillance platform had been in place prior to the onset of the COVID-19 pandemic, policymakers would have been able to complement the representative nature of wastewater samples to individual testing, likely resulting in more precise public health interventions and policies.
Estimating population size and species distribution is essential for fisheries management and conservation. Traditionally, estimates rely on live-capture and visual surveys; however, these approaches are challenging for low density or elusive species and sensitive habitats. Environmental DNA (eDNA) has shown potential to improve fisheries management, offering a sensitive tool for species detection while reducing some of the unintended harm, uncertainties, and cost of traditional approaches. For eDNA to be incorporated into quantitative population estimates, variability in factors such as DNA shedding and decay must be understood. We assess shedding and decay rates for three developmental stages of muskellunge (Esox masquinongy), a recreational species of social and economic importance and an apex predator of conservation priority in the St. Lawrence River. By housing fish at different biomass levels, we attempt to assess how crowding, and underlying behavioral and/or metabolic responses, affects shedding and decay. Additionally, we collected water from spawning bays and compared eDNA detections to live-capture data. Total eDNA shedding rates for muskellunge were similar to values reported in previous studies of freshwater fishes and ranged from 9.92 × 10 3 copies/h/fish for larvae to 1.32 × 10 6 copies/h/fish for juveniles. Adjusting shedding rates for fish mass revealed no significant difference between larvae and juveniles. eDNA decay rates varied between life stages and experimental aquaria, with coefficients ranging from 0.064 to 0.259. We were unable to detect eDNA from hardened embryos even at high density. Muskellunge DNA was quantified in over 27% of samples from spawning bays, including bays where muskellunge were not captured with traditional approaches. The recovery of muskellunge eDNA in quantifiable levels, often in the absence of live capture, highlights the potential for eDNA approaches to supplement muskellunge monitoring and management. eDNA shedding and decay rates estimated here may also aid in interpretation of future data.
Aquatic fecal contamination poses human health risks by introducing pathogens in water that may be used for recreation, consumption, or agriculture. Identifying fecal contaminant sources, as well as the factors that affect their transport, storage, and decay, is essential for protecting human health. However, identifying these factors is often difficult when using fecal indicator bacteria (FIB) because FIB levels in surface water are often the product of multiple contaminant sources. In contrast, microbial source-tracking (MST) techniques allow not only the identification of predominant contaminant sources but also the quantification of factors affecting the transport, storage, and decay of fecal contaminants from specific hosts. We visited 68 streams in the Finger Lakes region of Upstate New York, United States, between April and October 2018 and collected water quality data (i.e., Escherichia coli, MST markers, and physical–chemical parameters) and weather and land-use data, as well as data on other stream features (e.g., stream bed composition), to identify factors that were associated with fecal contamination at a regional scale. We then applied both generalized linear mixed models and conditional inference trees to identify factors and combinations of factors that were significantly associated with human and ruminant fecal contamination. We found that human contaminants were more likely to be identified when the developed area within the 60 m stream buffer exceeded 3.4%, the total developed area in the watershed exceeded 41%, or if stormwater outfalls were present immediately upstream of the sampling site. When these features were not present, human MST markers were more likely to be found when rainfall during the preceding day exceeded 1.5 cm. The presence of upstream campgrounds was also significantly associated with human MST marker detection. In addition to rainfall and water quality parameters associated with rainfall (e.g., turbidity), the minimum distance to upstream cattle operations, the proportion of the 60 m buffer used for cropland, and the presence of submerged aquatic vegetation at the sampling site were all associated based on univariable regression with elevated levels of ruminant markers. The identification of specific features associated with host-specific fecal contaminants may support the development of broader recommendations or policies aimed at reducing levels of aquatic fecal contamination.
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