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.

Gender-specific expression of GIBBERELLIC ACID INSENSITIVE is critical for unisexual organ initiation in dioecious Spinacia oleracea.

While unisexual flowers have evolved repeatedly throughout angiosperm families, the actual identification of sex-determining genes has been elusive, and their regulation within populations remains largely undefined. Here, we tested the mechanism of the feminization pathway in cultivated spinach (Spinacia oleracea), and investigated how this pathway may regulate alternative sexual development. We tested the effect of gibberellic acid (GA) on sex determination through exogenous applications of GA and inhibitors of GA synthesis and proteasome activity. GA concentrations in multiple tissues were estimated by enzyme-linked immunosorbent assay analysis. Gene function was investigated and pathway analysis was performed through virus-induced gene silencing. Relative gene expression levels were estimated by quantitative reverse transcription-polymerase chain reaction. Inhibition of GA production and proteasome activity feminized male flowers. However, there was no difference in GA content in tissues between males and females. We characterized a single DELLA family transcription factor gene (GIBBERELLIC ACID INSENSITIVE (SpGAI)) and observed inflorescence expression in females two-fold higher than in males. Reduction of SpGAI expression in females to male levels phenocopied exogenous GA application with respect to flower development. These results implicate SpGAI as the feminizing factor in spinach, and suggest that the feminizing pathway is epistatic to the masculinizing pathway. We present a unified model for alternative sexual development and discuss the implications for established theory.

Hormonal interactions and gene regulation can link monoecy and environmental plasticity to the evolution of dioecy in plants.

Most models for dioecy in flowering plants assume that dioecy arises directly from hermaphroditism through a series of independent feminizing and masculinizing mutations that become chromosomally linked. However, dioecy appears to evolve most frequently through monoecious grades. The major genetic models do not explain the evolution of unisexual flowers in monoecious and submonoecious populations, nor do they account for environmentally induced sexual plasticity. In this review, we explore the roles of environmental stress and hormones on sex determination, and propose a model that can explain the evolution of dioecy through monoecy, and the mechanisms of environmental sex determination. Environmental stresses elicit hormones that allow plants to mediate the negative effects of the stresses. Many of these same hormones are involved in the regulation of floral developmental genes. Recent studies have elucidated the mechanisms whereby these hormones interact and can act as switchpoints in regulatory pathways. Consequently, differential concentrations of plant hormones can regulate whole developmental pathways, providing a mechanism for differential development within isogenic individuals such as seen in monoecious plants. Sex-determining genes in such systems will evolve to generate clusters of coexpressed suites. Coexpression rather than coinheritance of gender-specific genes will define the sexual developmental fate. Therefore, selection for gender type will drive evolution of the regulatory sequences of such genes rather than their synteny. Subsequent mutations to hyper- or hyposensitive alleles within the hormone response pathway can result in segregating dioecious populations. Simultaneously, such developmental systems will remain sensitive to external stimuli that modify hormone responses.