Monkeypox viral nucleic acids detected using both DNA and RNA extraction workflows.

Molecular methods have been used to detect human pathogens in wastewater with sampling typically performed at wastewater treatment plants (WWTP) and upstream locations within the sewer system. A wastewater-based surveillance (WBS) program was established at the University of Miami (UM) in 2020, which included measurements of SARS-CoV-2 levels in wastewater from its hospital and within the regional WWTP. In addition to the development of a SARS-CoV-2 quantitative PCR (qPCR) assay, qPCR assays to detect other human pathogens of interest were also developed at UM. Here we report on the use of a modified set of reagents published by the CDC to detect nucleic acids of Monkeypox virus (MPXV) which emerged during May of 2022 to become a concern worldwide. Samples collected from the University hospital and from the regional WWTP were processed through DNA and RNA workflows and analyzed by qPCR to detect a segment of the MPXV CrmB gene. Results show positive detections of MPXV nucleic acids in the hospital and wastewater treatment plant wastewater which coincided with clinical cases in the community and mirrored the overall trend of nationwide MPXV cases reported to the CDC. We recommend the expansion of current WBS programs' methods to detect a broader range of pathogens of concern in wastewater and present evidence that viral RNA in human cells infected by a DNA virus can be detected in wastewater.

Degradation rates influence the ability of composite samples to represent 24-hourly means of SARS-CoV-2 and other microbiological target measures in wastewater.

The utility of using severe-acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA for assessing the prevalence of COVID-19 within communities begins with the design of the sample collection program. The objective of this study was to assess the utility of 24-hour composites as representative samples for measuring multiple microbiological targets in wastewater, and whether normalization of SARS-CoV-2 by endogenous targets can be used to decrease hour to hour variability at different watershed scales. Two sets of experiments were conducted, in tandem with the same wastewater, with samples collected at the building, cluster, and community sewershed scales. The first set of experiments focused on evaluating degradation of microbiological targets: SARS-CoV-2, Simian Immunodeficiency Virus (SIV) - a surrogate spiked into the wastewater, plus human waste indicators of Pepper Mild Mottle Virus (PMMoV), Beta-2 microglobulin (B2M), and fecal coliform bacteria (FC). The second focused on the variability of these targets from samples, collected each hour on the hour. Results show that SARS-CoV-2, PMMoV, and B2M were relatively stable, with minimal degradation over 24-h. SIV, which was spiked-in prior to analysis, degraded significantly and FC increased significantly over the course of 24 h, emphasizing the possibility for decay and growth within wastewater. Hour-to-hour variability of the source wastewater was large between each hour of sampling relative to the variability of the SARS-CoV-2 levels calculated between sewershed scales; thus, differences in SARS-CoV-2 hourly variability were not statistically significant between sewershed scales. Results further provided that the quantified representativeness of 24-h composite samples (i.e., statistical equivalency compared against hourly collected grabs) was dependent upon the molecular target measured. Overall, improvements made by normalization were minimal within this study. Degradation and multiplication for other targets should be evaluated when deciding upon whether to collect composite or grab samples in future studies.

Comparison of Electronegative Filtration to Magnetic Bead-Based Concentration and V2G-qPCR to RT-qPCR for Quantifying Viral SARS-CoV-2 RNA from Wastewater

Methods of wastewater concentration (electro-negative filtration (ENF) versus magnetic bead-based concen-tration (MBC)) were compared for the analysis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), beta-2 micro-globulin, and human coronavirus OC43. Using ENF as the concentration method, two quantitative polymerase chain reaction (qPCR) analytical methods were also compared: volcano second generation (V2G)-qPCR and reverse transcriptase (RT)-qPCR measuring three different targets of the virus responsible for the COVID-19 illness (N1, modified N3, and ORF1ab). Correlations between concentration methods were strong and statistically significant for SARS-CoV-2 (r = 0.77, p < 0.001) and B2M (r = 0.77, p < 0.001). Comparison of qPCR analytical methods indicate that, on average, each method provided equivalent results with average ratios of 0.96, 0.96, and 1.02 for N3 to N1, N3 to ORF1ab, and N1 to ORF1ab and were supported by significant (p < 0.001) correlation coefficients (r = 0.67 for V2G (N3) to RT (N1), r = 0.74 for V2G (N3) to RT (ORF1ab), r = 0.81 for RT (N1) to RT (ORF1ab)). Overall results suggest that the two concentration methods and qPCR methods provide equivalent results, although variability is observed for individual measurements. Given the equivalency of results, additional advantages and disadvantages, as described in the discussion, are to be considered when choosing an method.

Relationships between SARS-CoV-2 in Wastewater and COVID-19 Clinical Cases and Hospitalizations, with and without Normalization against Indicators of Human Waste.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in wastewater has been used to track community infections of coronavirus disease-2019 (COVID-19), providing critical information for public health interventions. Since levels in wastewater are dependent upon human inputs, we hypothesize that tracking infections can be improved by normalizing wastewater concentrations against indicators of human waste [Pepper Mild Mottle Virus (PMMoV), ß-2 Microglobulin (B2M), and fecal coliform]. In this study, we analyzed SARS-CoV-2 and indicators of human waste in wastewater from two sewersheds of different scales: a University campus and a wastewater treatment plant. Wastewater data were combined with complementary COVID-19 case tracking to evaluate the efficiency of wastewater surveillance for forecasting new COVID-19 cases and, for the larger scale, hospitalizations. Results show that the normalization of SARS-CoV-2 levels by PMMoV and B2M resulted in improved correlations with COVID-19 cases for campus data using volcano second generation (V2G)-qPCR chemistry (r s = 0.69 without normalization, r s = 0.73 with normalization). Mixed results were obtained for normalization by PMMoV for samples collected at the community scale. Overall benefits from normalizing with measures of human waste depend upon qPCR chemistry and improves with smaller sewershed scale. We recommend further studies that evaluate the efficacy of additional normalization targets.