Wastewater surveillance for bacterial targets: current challenges and future goals.

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs.

Extracellular Calcium Receptor as a Target for Glutathione and Its Derivatives.

Extracellular glutathione (GSH) and oxidized glutathione (GSSG) can modulate the function of the extracellular calcium sensing receptor (CaSR). The CaSR has a binding pocket in the extracellular domain of CaSR large enough to bind either GSH or GSSG, as well as the naturally occurring oxidized derivative L-cysteine glutathione disulfide (CySSG) and the compound cysteinyl glutathione (CysGSH). Modeling the binding energies (ΔG) of CySSG and CysGSH to CaSR reveals that both cysteine derivatives may have greater affinities for CaSR than either GSH or GSSG. GSH, CySSG, and GSSG are found in circulation in mammals and, among the three, CySSG is more affected by HIV/AIDs and aging than either GSH or GSSG. The beta-carbon linkage of cysteine in CysGSH may model a new class of calcimimetics, exemplified by etelcalcetide. Circulating glutathionergic compounds, particularly CySSG, may mediate calcium-regulatory responses via receptor-binding to CaSR in a variety of organs, including parathyroids, kidneys, and bones. Receptor-mediated actions of glutathionergics may thus complement their roles in redox regulation and detoxification. The glutathionergic binding site(s) on CaSR are suggested to be a target for development of drugs that can be used in treating kidney and other diseases whose mechanisms involve CaSR dysregulation.

Water mite assemblages reveal diverse genera, novel DNA barcodes and transitional periods of intermediate disturbance.

Water mites are important constituents of aquatic ecosystems, but their biodiversity is poorly understood. The goal of this study was to improve knowledge of water mite assemblages in the Detroit River through combined use of morphological and cytochrome oxidase I (COI) DNA barcode data and to elucidate seasonal water mite diversity. The diversity of water mites collected from Blue Heron Lagoon at Belle Isle, an island in the Detroit River, is described. Novel DNA barcodes for Albia, Hydrochoreutes, Madawaska, and Axonopsis are reported with a species level barcode for Lebertia. Novel DNA barcodes may represent the presence of previously undescribed variants or new species of several genera. The prevalence of water mites is higher in the summer, but a different pattern is observed for diversity. The diversity of water mites, by several measures, varies seasonally with lower diversity in summer and winter months and higher diversity during seasonal transitions. For these organisms, we interpret seasonal change as an intermediate disturbance resulting in increased biodiversity.

Nutritional Correlates of Human Oral Microbiome.

BACKGROUND: Despite many potential effects of the oral microbiome on oral and systemic health, scant information is available regarding the associations between diet and the oral microbiome. METHODS: Oral rinse DNA samples from 182 participants in a population-based case-control study for colorectal cancer were used to amplify a V3-V4 region of bacterial 16S rRNA gene. The amplicons were sequenced using Illumina MiSeq paired end chemistry on 2 runs, yielding approximately 33 million filtered reads that were assigned to bacterial classes. Relative abundances of each class and family as well microbial diversity/richness indices were correlated with selected dietary intakes from a food frequency questionnaire. RESULTS: Saturated fatty acids (SFAs) and vitamin C intakes were consistently correlated with alpha (within-subjects) diversity indexes in both richness and diversity. SFA intake was positively correlated with relative abundance of betaproteobacteria and fusobacteria. Vitamin C and other vitamins with correlated intakes-for example, the B vitamins and vitamin E-exhibited positive correlations with fusobacteria class, its family Leptotrichiaceae and a clostridia family Lachnospiraceae. In addition, glycemic load was positively correlated with Lactobacillaceae abundance. CONCLUSION: The observed associations in this study were modest. However, the results suggest that the effects of diets are likely to be habitat specific, and observations from the gut microbiome are not transferrable to the oral microbiome. Further studies are warranted, incorporating a range of host biomarkers, such as cytohistological, molecular, or biochemical measurements, in order to address biological consequences of these dietary intakes in human oral health.

Early detection monitoring for aquatic non-indigenous species: Optimizing surveillance, incorporating advanced technologies, and identifying research needs.

Following decades of ecologic and economic impacts from a growing list of nonindigenous and invasive species, government and management entities are committing to systematic early- detection monitoring (EDM). This has reinvigorated investment in the science underpinning such monitoring, as well as the need to convey that science in practical terms to those tasked with EDM implementation. Using the context of nonindigenous species in the North American Great Lakes, this article summarizes the current scientific tools and knowledge - including limitations, research needs, and likely future developments - relevant to various aspects of planning and conducting comprehensive EDM. We begin with the scope of the effort, contrasting target-species with broad-spectrum monitoring, reviewing information to support prioritization based on species and locations, and exploring the challenge of moving beyond individual surveys towards a coordinated monitoring network. Next, we discuss survey design, including effort to expend and its allocation over space and time. A section on sample collection and analysis overviews the merits of collecting actual organisms versus shed DNA, reviews the capabilities and limitations of identification by morphology, DNA target markers, or DNA barcoding, and examines best practices for sample handling and data verification. We end with a section addressing the analysis of monitoring data, including methods to evaluate survey performance and characterize and communicate uncertainty. Although the body of science supporting EDM implementation is already substantial, research and information needs (many already actively being addressed) include: better data to support risk assessments that guide choice of taxa and locations to monitor; improved understanding of spatiotemporal scales for sample collection; further development of DNA target markers, reference barcodes, genomic workflows, and synergies between DNA-based and morphology-based taxonomy; and tools and information management systems for better evaluating and communicating survey outcomes and uncertainty.

Eurytemora carolleeae in the Laurentian Great Lakes revealed by phylogenetic and morphological analysis.

In the Laurentian Great Lakes, specimens of Eurytemora have been reported as E. affinis since its invasion in the late 1950s. During an intensive collection of aquatic invertebrates for morphological and molecular identification in Western Lake Erie in 2012-2013, several specimens of Eurytemora were collected. Analysis of these specimens identified them as the recently described species E. carolleeae Alekseev and Souissi 2011. This result led us to assess E. carolleeae's identifying features, geographic distribution and historical presence in the Laurentian Great Lakes in view of its recent description in 2011. Cytochrome oxidase I (COI) DNA sequences of Eurytemora specimens were identified as closer (2 - 4% different) to recently described E. carolleeae than to most Eurytemora affinis sequences (14% different). Eurytemora from other areas of the Great Lakes and from North American rivers as far west as South Dakota (Missouri River) and east to Delaware (Christina River) also keyed to E. carolleeae. Morphological analysis of archival specimens from 1962 and from all the Great Lakes were identified as E. carolleeae. Additionally, Eurytemora drawings in previous publications from studies in the Holarctic region were reassessed to determine if these specimens were E. carolleeae. Additional morphological characters that may distinguish the North American E. carolleeae from other taxa are also described. We conclude that E. carolleeae is the correct name for the species of Eurytemora that has inhabited the Great Lakes since its invasion, as established by both morphological and COI sequence comparisons to reference keys and sequence databases in present and archival specimens.

Invertebrates as model organisms for research on aging biology.

Invertebrate model systems, such as nematodes and fruit flies, have provided valuable information about the genetics and cellular biology involved in aging. However, limitations of these simple, genetically tractable organisms suggest the need for other model systems, some of them invertebrate, to facilitate further advances in the understanding of mechanisms of aging and longevity in mammals, including humans. This paper introduces 10 review articles about the use of invertebrate model systems for the study of aging by authors who participated in an 'NIA-NIH symposium on aging in invertebrate model systems' at the 2013 International Congress for Invertebrate Reproduction and Development. In contrast to the highly derived characteristics of nematodes and fruit flies as members of the superphylum Ecdysozoa, cnidarians, such as Hydra, are more 'basal' organisms that have a greater number of genetic orthologs in common with humans. Moreover, some other new model systems, such as the urochordate Botryllus schlosseri, the tunicate Ciona, and the sea urchins (Echinodermata) are members of the Deuterostomia, the same superphylum that includes all vertebrates, and thus have mechanisms that are likely to be more closely related to those occurring in humans. Additional characteristics of these new model systems, such as the recent development of new molecular and genetic tools and a more similar pattern to humans of regeneration and stem cell function suggest that these new model systems may have unique advantages for the study of mechanisms of aging and longevity.

Glutathione-Based Photoaffinity Probe Identifies Caffeine as a Positive Allosteric Modulator of the Calcium-Sensing Receptor.

The calcium-sensing receptor (CaSR), abundantly expressed in the parathyroid gland and kidney, plays a central role in calcium homeostasis. In addition, CaSR exerts multimodal roles, including inflammation, muscle contraction, and bone remodeling, in other organs and tissues. The diverse functions of CaSR are mediated by many endogenous and exogenous ligands, including calcium, amino acids, glutathione, cinacalcet, and etelcalcetide, that have distinct binding sites in CaSR. However, strategies to evaluate ligand interactions with CaSR remain limited. Here, we developed a glutathione-based photoaffinity probe, DAZ-G, that analyzes ligand binding to CaSR. We showed that DAZ-G binds to the amino acid binding site in CaSR and acts as a positive allosteric modulator of CaSR. Oxidized and reduced glutathione and phenylalanine effectively compete with DAZ-G conjugation to CaSR, while calcium, cinacalcet, and etelcalcetide have cooperative effects. An unexpected finding was that caffeine effectively competes with DAZ-G's conjugation to CaSR and acts as a positive allosteric modulator of CaSR. The effective concentration of caffeine for CaSR activation (<10 µM) is easily attainable in plasma by ordinary caffeine consumption. Our report demonstrates the utility of a new chemical probe for CaSR and discovers a new protein target of caffeine, suggesting that caffeine consumption can modulate the diverse functions of CaSR.

Improved passive sampling methods for wastewater to enable more sensitive detection of SARS-CoV-2 and its variants.

Wastewater-based epidemiology (WBE) can be used as a part of a long-term strategy for detecting and responding rapidly to new outbreaks of infectious disease in the community. However, wastewater collected by grab samples may miss marker presence, and composite auto-sampling throughout a day is technically challenging and costly. Tampon swabs can be used as passive collectors of wastewater markers over hours, but recovery of the captured markers is a challenge. Our goal was to improve tampon elution methods for virus detection and variant analysis to increase the likelihood of detection near the Limit of Detection (LOD) and to potentially detect new or rare variants in a new outbreak. Counts of SARS-CoV-2 N1 and N2 markers in grab samples were compared to markers eluted from tampons that had been immersed in 3 sewersheds for 4-6 h during June to December 2023. We compared tampon elution methods that used different elution volumes, pressure, and amounts of Tween 20, evaluated after automated magnetic bead purification and RT-ddPCR of SARS-CoV-2 markers. Overall, method "SwabM2" in which tampons were eluted by high pressure squeeze in a 50 mL syringe after adding 2 mL of 0.5 X TE + 0.075 % Tween-20 yielded a median four-fold higher concentration of final purified SARS-CoV-2 markers than paired grab samples and significantly more than other tested tampon elution methods (p < 0.0001). Method SwabM2 was more likely to yield enough extracted nucleic acids for sequencing and also gave higher quality variant sequences than two other tampon elution methods. Variant analysis captured the Fall 2023 transition of variants from XBB to JN and "H" lineages. In summary, we demonstrated a tampon-based wastewater collecting and elution method that yielded higher counts, more detections near the LOD, and higher quality variant sequences compared to both grab samples and other tampon-based passive-collecting wastewater methods.

New species and a fascinating diversity of Chironomidae (Diptera, Insecta) in and around an overlooked urban vernal pool.

In this study, the biodiversity of Chironomidae was investigated in Palmer Park Pond A, an urban vernal pond in Detroit, Michigan, USA. This study is developed as part of our ongoing Public Environmental Outreach Program at the Detroit Exploration and Nature Center in Palmer Park. Twenty-one Chironomidae species were discovered in and on the adjacent riparian vegetation of this pond using molecular and morphological methods. Three species Bryophaenocladiuspalmerparcum Namayandeh & Hudson sp. nov., Limnophyesstagnum Namayandeh, Guerra & Ram sp. nov., and Rheocricotopus (s. s.) angustus Namayandeh & Hudson sp. nov. are new to science. Bryophaenocladiuspalmerparcum sp. nov. and L.stagnum sp. nov. are unusual Orthoclads, with B.palmerparcum sp. nov. possessing a setose, short, and wide anal point and L.stagnum sp. nov. lacking lanceolate setae on both sexes. Based on the shape of superior volsella, R.angustus sp. nov., belongs to the effusus group, which was also confirmed by DNA barcoding molecular analysis. In this study, a new faunistic record was also found for the Nearctic as well as four new faunistic records for the state of Michigan. Ephemeral aquatic habitats such as vernal pools are often overlooked or destroyed by urbanization activities, controlling vector species, creating groomed fields, and/or residential development. Therefore, finding these new species demonstrates the biodiversity value of vernal ponds as important habitats, further motivating us to preserve them.

Rapid Micromolding of Sub-100 µm Microfluidic Channels Using an 8K Stereolithographic Resin 3D Printer.

Engineering microfluidic devices relies on the ability to manufacture sub-100 micrometer fluidic channels. Conventional lithographic methods provide high resolution but require costly exposure tools and outsourcing of masks, which extends the turnaround time to several days. The desire to accelerate design/test cycles has motivated the rapid prototyping of microfluidic channels; however, many of these methods (e.g., laser cutters, craft cutters, fused deposition modeling) have feature sizes of several hundred microns or more. In this paper, we describe a 1-day process for fabricating sub-100 µm channels, leveraging a low-cost (USD 600) 8K digital light projection (DLP) 3D resin printer. The soft lithography process includes mold printing, post-treatment, and casting polydimethylsiloxane (PDMS) elastomer. The process can produce microchannels with 44 µm lateral resolution and 25 µm height, posts as small as 400 µm, aspect ratio up to 7, structures with varying z-height, integrated reservoirs for fluidic connections, and a built-in tray for casting. We discuss strategies to obtain reliable structures, prevent mold warpage, facilitate curing and removal of PDMS during molding, and recycle the solvents used in the process. To our knowledge, this is the first low-cost 3D printer that prints extruded structures that can mold sub-100 µm channels, providing a balance between resolution, turnaround time, and cost (~USD 5 for a 2 × 5 × 0.5 cm3 chip) that will be attractive for many microfluidics labs.

Alaskacladius gen. nov., (Diptera: Chironomidae), a unique new orthoclad from Alaska.

We describe a new genus Alaskacladius gen. nov., based on the adult stages collected from Alaska, USA, and British Columbia, Canada. Molecular and morphological assessment of adult specimens supports the presence of a new genus. Alaskacladius gen. nov., is related to the genera Doithrix Sther & Sublette, 1983; Georthocladius Strenzke, 1941; Parachaetocladius Wlker, 1959; and Pseudorthocladius Goetghebuer, 1932. Based on the molecular analysis result and intergeneric K2P distance obtained from Cytochrome Oxidase I (COI) genes, Alaskacladius is closest and forms a sister group with Doithrix.

Wastewater Monitoring for Infectious Disease: Intentional Relationships between Academia, the Private Sector, and Local Health Departments for Public Health Preparedness.

The public health emergency caused by the COVID-19 pandemic stimulated stakeholders from diverse disciplines and institutions to establish new collaborations to produce informed public health responses to the disease. Wastewater-based epidemiology for COVID-19 grew quickly during the pandemic and required the rapid implementation of such collaborations. The objective of this article is to describe the challenges and results of new relationships developed in Detroit, MI, USA among a medical school and an engineering college at an academic institution (Wayne State University), the local health department (Detroit Health Department), and an environmental services company (LimnoTech) to utilize markers of the COVID-19 virus, SARS-CoV-2, in wastewater for the goal of managing COVID-19 outbreaks. Our collaborative team resolved questions related to sewershed selection, communication of results, and public health responses and addressed technical challenges that included ground-truthing the sewer maps, overcoming supply chain issues, improving the speed and sensitivity of measurements, and training new personnel to deal with a new disease under pandemic conditions. Recognition of our complementary roles and clear communication among the partners enabled city-wide wastewater data to inform public health responses within a few months of the availability of funding in 2020, and to make improvements in sensitivity and understanding to be made as the pandemic progressed and evolved. As a result, the outbreaks of COVID-19 in Detroit in fall and winter 2021-2022 (corresponding to Delta and Omicron variant outbreaks) were tracked in 20 sewersheds. Data comparing community- and hospital-associated sewersheds indicate a one- to two-week advance warning in the community of subsequent peaks in viral markers in hospital sewersheds. The new institutional relationships impelled by the pandemic provide a good basis for continuing collaborations to utilize wastewater-based human and pathogen data for improving the public health in the future.

Passive swab versus grab sampling for detection of SARS-CoV-2 markers in wastewater.

Early detection of the COVID-19 virus, SARS-CoV-2, is key to mitigating the spread of new outbreaks. Data from individual testing is increasingly difficult to obtain as people conduct non-reported home tests, defer tests due to logistics or attitudes, or ignore testing altogether. Wastewater based epidemiology is an alternative method for surveilling a community while maintaining individual anonymity; however, a problem is that SARS-CoV-2 markers in wastewater vary throughout the day. Collecting grab samples at a single time may miss marker presence, while autosampling throughout a day is technically challenging and expensive. This study investigates a passive sampling method that would be expected to accumulate greater amounts of viral material from sewers over a period of time. Tampons were tested as passive swab sampling devices from which viral markers could be eluted with a Tween-20 surfactant wash. Six sewersheds in Detroit were sampled 16-22 times by paired swab (4 h immersion before retrieval) and grab methods over a five-month period and enumerated for N1 and N2 SARS-CoV-2 markers using ddPCR. Swabs detected SARS-CoV-2 markers significantly more frequently (P < 0.001) than grab samples, averaging two to three-fold more copies of SARS-CoV-2 markers than their paired grab samples (p < 0.0001) in the assayed volume (10 mL) of wastewater or swab eluate. No significant difference was observed in the recovery of a spiked-in control (Phi6), indicating that the improved sensitivity is not due to improvements in nucleic acid recovery or reduction of PCR inhibition. The outcomes of swab-based sampling varied significantly between sites, with swab samples providing the greatest improvements in counts for smaller sewersheds that otherwise tend to have greater variation in grab sample counts. Swab-sampling with tampons provides significant advantages in detection of SARS-CoV-2 wastewater markers and are expected to provide earlier detection of new outbreaks than grab samples, with consequent public health benefits.

Sensitive detection of SARS-CoV-2 molecular markers in urban community sewersheds using automated viral RNA purification and digital droplet PCR.

Wastewater based epidemiology (WBE) has emerged as a strategy to identify, locate, and manage outbreaks of COVID-19, and thereby possibly prevent surges in cases, which overwhelm local to global health care networks. The WBE process is based on assaying municipal wastewater for molecular markers of the SARS-CoV-2 virus. Standard processes for purifying viral RNA from municipal wastewater are often time-consuming and require the handling of large quantities of wastewater, negatively affecting throughput, timely reporting, and safety. We demonstrate here an automated, faster system to purify viral RNA from smaller volumes of wastewater but with increased sensitivity for detection of SARS-CoV-2 markers. We document the effectiveness of this new approach by way of comparison to the PEG/NaCl/Qiagen method prescribed by the State of Michigan for SARS-CoV-2 wastewater monitoring and show its application to several Detroit sewersheds. Specifically, compared to the PEG/NaCl/Qiagen method, viral RNA purification using the PerkinElmer Chemagic™ 360 lowered handling time, decreased the amount of wastewater required by ten-fold, increased the amount of RNA isolated per µl of final elution product by approximately five-fold, and effectively removed ddPCR inhibitors from most sewershed samples. For detection of markers on the borderline of viral detectability, we found that use of the Chemagic™ 360 enabled the measurement of viral markers in a significant number of samples for which the result with the PEG/NaCl/Qiagen method was below the level of detectability. The improvement in detectability of the viral markers might be particularly important for early warning to public health authorities at the beginning of an outbreak. Applied to sewersheds in Detroit, the technique enabled more sensitive detection of SARS-CoV-2 markers with good correlation between wastewater signals and COVID-19 cases in the sewersheds. We also discuss advantages and disadvantages of several automated RNA purification systems, made by Promega, PerkinElmer, and ThermoFisher.

Variation in the diversity of bacterial communities and potential sources of fecal contamination of beaches in the Huron to Erie corridor.

Understanding the diversity of bacteria and E.coli levels at beaches is important for managing health risks. This study compared temporal changes of the bacterial communities of Belle Isle Beach (Detroit, MI) and Sand Point Beach (Windsor, ONT), both located near the Lake St. Clair origin of the Detroit River. Water samples collected 4 days/week for 12 weeks in summer, were subjected to 16S rRNA analysis of amplicon sequencing and E. coli enumeration. Bacterial communities changed over time, as determined by cluster dendrogram analysis, exhibiting different communities in July and August than in June and different communities at the two beaches. After June, alpha diversity decreased and relative abundance of Enterobacter (Gammaproteobacteria) increased at Sand Point; whereas, Belle Isle maintained its alpha diversity and dominance by Betaproteobacteria and Actinobacteria. Contamination at both beaches is dominated by birds (23% to 50% of samples), while only ∼10% had evidence of human-associated bacteria. High E. coli at both beaches was often associated with precipitation. Nearshore sampling counts were higher than waist-deep sampling counts. Despite the dynamic changes in bacterial communities between the two beaches, this analysis based on 16S rRNA amplicon sequencing is able to provide information about bacterial types associated with high E. coli levels and to use bacterial sequences to more precisely determine sources and health relevance of contaminants.

Molecular diet studies of water mites reveal prey biodiversity.

Water mites are diverse aquatic invertebrates that provide potentially important ecosystem and economic services as bioindicators and mosquito biocontrol; however, little is known about water mite digestive physiology, including their diet in nature. Water mites, much like their spider relatives, liquefy their prey upon consumption. This results in the absence of morphologically identifiable prey in water mite mid-gut. Previous studies have reported associations in the field of water mites with presumed prey and laboratory observations of water mites feeding on specific organisms offered for ingestion; however, the present work aims to determine what water mites have ingested in nature based on molecular studies of gut contents from freshly collected organisms from the field. To elucidate water mite prey, we used next-generation sequencing to detect diverse cytochrome oxidase I DNA barcode sequences of putative prey in the guts of 54 specimens comprising two species of Lebertia and a few specimens of Arrenurus (2) and Limnesia (1). To our knowledge this is the first molecular study of the diets of water mites as they feed in nature. While the presence of chironomid DNA confirmed previous observations of midge larvae as part of the diets of Lebertia, we also found the DNA of diverse organisms in all four species of water mites, including the DNA of mosquitoes in 6 specimens of Lebertia and a large number of previously unknown prey, especially from oligochaete worms. These studies thereby reveal a greater diversity of prey and a potentially broader significance than previously appreciated for water mites in aquatic food webs. Molecular studies like this can detect water mite predators of mosquito larvae and add knowledge of water mite predatory contributions to freshwater food webs.

Wastewater surveillance for SARS-CoV-2 on college campuses: Initial efforts, lessons learned and research needs.

Background: Wastewater surveillance for SARS-CoV-2 is an emerging approach to help identify the risk of a COVID-19 outbreak. This tool can contribute to public health surveillance at both community (wastewater treatment system) and institutional (e.g., colleges, prisons, nursing homes) scales. Objectives: This research aims to understand the successes, challenges, and lessons learned from initial wastewater surveillance efforts at colleges and university systems to inform future research, development and implementation. Methods: This paper presents the experiences of 25 college and university systems in the United States that monitored campus wastewater for SARS-CoV-2 during the fall 2020 academic period. We describe the broad range of approaches, findings, resource needs, and lessons learned from these initial efforts. These institutions range in size, social and political geographies, and include both public and private institutions. Discussion: Our analysis suggests that wastewater monitoring at colleges requires consideration of information needs, local sewage infrastructure, resources for sampling and analysis, college and community dynamics, approaches to interpretation and communication of results, and follow-up actions. Most colleges reported that a learning process of experimentation, evaluation, and adaptation was key to progress. This process requires ongoing collaboration among diverse stakeholders including decision-makers, researchers, faculty, facilities staff, students, and community members.

Wastewater Surveillance for SARS-CoV-2 on College Campuses: Initial Efforts, Lessons Learned, and Research Needs.

Wastewater surveillance for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging approach to help identify the risk of a coronavirus disease (COVID-19) outbreak. This tool can contribute to public health surveillance at both community (wastewater treatment system) and institutional (e.g., colleges, prisons, and nursing homes) scales. This paper explores the successes, challenges, and lessons learned from initial wastewater surveillance efforts at colleges and university systems to inform future research, development and implementation. We present the experiences of 25 college and university systems in the United States that monitored campus wastewater for SARS-CoV-2 during the fall 2020 academic period. We describe the broad range of approaches, findings, resources, and impacts from these initial efforts. These institutions range in size, social and political geographies, and include both public and private institutions. Our analysis suggests that wastewater monitoring at colleges requires consideration of local information needs, sewage infrastructure, resources for sampling and analysis, college and community dynamics, approaches to interpretation and communication of results, and follow-up actions. Most colleges reported that a learning process of experimentation, evaluation, and adaptation was key to progress. This process requires ongoing collaboration among diverse stakeholders including decision-makers, researchers, faculty, facilities staff, students, and community members.

Smoking and other personal characteristics as potential predictors for fecal bacteria populations in humans.

BACKGROUND: Intestinal microbes have been postulated to play an important role in the development of colorectal cancer. Recently developed methods for preserving and delivering fecal samples at ambient temperature to the laboratory for molecular analysis of bacterial constituents were used to test associations of bacterial populations with epidemiologic risk factors for colorectal cancer. MATERIAL/METHODS: Real-time PCR targeting 16S rRNA gene sequences was used to quantify three intestinal bacterial groups relative to total DNA in stool samples preserved with RNAlater from 62 subjects. Subjects' medical and family history, race, diet, weight, height, and personal habits including smoking were obtained through structured questionnaires. RESULTS: Bacteroides DNA proportions were relatively stable among individuals and relatively independent of dietary intake or other personal factors. Clostridium (coccoides group) DNA was positively associated with total fat and vitamin C intake. Desulfovibrio DNA amount tended to be higher in African Americans than in other races. Furthermore, Desulfovibrio DNA increased progressively with pack-years of cigarette smoking. The relative DNA quantity (%) was more than 17 times higher in the subjects who smoked at least 15 pack-years compared with never-smokers (P-value for a linear trend =0.001). In addition, Desulfovibrio DNA (%) decreased with increased calcium, vitamin E, and dietary fiber intake. However, only smoking remained significant in multivariable analysis. CONCLUSIONS: Although the study was limited by its sample size, these results suggest that smoking (or possibly unmeasured dietary confounders) may exert modulatory effects on the bacterial populations of the gastrointestinal tract. The study also demonstrates collection, preservation, and sample delivery procedures suitable for large epidemiological studies.