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- Pettigrew, Melinda M.
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- National Science Foundation
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Swanson, Michele S. (Ed.)ABSTRACT Wastewater surveillance (WS), when coupled with advanced molecular techniques, offers near real-time monitoring of community-wide transmission of SARS-CoV-2 and allows assessing and mitigating COVID-19 outbreaks, by evaluating the total microbial assemblage in a community. Composite wastewater samples (24 h) were collected weekly from a manhole between December 2020 and November 2021 in Maryland, USA. RT-qPCR results showed concentrations of SARS-CoV-2 RNA recovered from wastewater samples reflected incidence of COVID-19 cases. When a drastic increase in COVID-19 was detected in February 2021, samples were selected for microbiome analysis (DNA metagenomics, RNA metatranscriptomics, and targeted SARS-CoV-2 sequencing). Targeted SARS-CoV-2 sequencing allowed for detection of important genetic mutations, such as spike: K417N, D614G, P681H, T716I, S982A, and D1118H, commonly associated with increased cell entry and reinfection. Microbiome analysis (DNA and RNA) provided important insight with respect to human health-related factors, including detection of pathogens and their virulence/antibiotic resistance genes. Specific microbial species comprising the wastewater microbiome correlated with incidence of SARS-CoV-2 RNA, suggesting potential association with SARS-CoV-2 infection. Climatic conditions, namely, temperature, were related to incidence of COVID-19 and detection of SARS-CoV-2 in wastewater, having been monitored as part of an environmental risk score assessment carried out in this study. In summary, the wastewater microbiome provides useful public health information, and hence, a valuable tool to proactively detect and characterize pathogenic agents circulating in a community. In effect, metagenomics of wastewater can serve as an early warning system for communicable diseases, by providing a larger source of information for health departments and public officials. IMPORTANCE Traditionally, testing for COVID-19 is done by detecting SARS-CoV-2 in samples collected from nasal swabs and/or saliva. However, SARS-CoV-2 can also be detected in feces of infected individuals. Therefore, wastewater samples can be used to test all individuals of a community contributing to the sewage collection system, i.e., the infrastructure, such as gravity pipes, manholes, tanks, lift stations, control structures, and force mains, that collects used water from residential and commercial sources and conveys the flow to a wastewater treatment plant. Here, we profile community wastewater collected from a manhole, detect presence of SARS-CoV-2, identify genetic mutations of SARS-CoV-2, and perform COVID-19 risk score assessment of the study area. Using metagenomics analysis, we also detect other microorganisms (bacteria, fungi, protists, and viruses) present in the samples. Results show that by analyzing all microorganisms present in wastewater, pathogens circulating in a community can provide an early warning for contagious diseases.more » « less
Abd El-Aty, A. M. (Ed.)Background Higher viral loads in SARS-CoV-2 infections may be linked to more rapid spread of emerging variants of concern (VOC). Rapid detection and isolation of cases with highest viral loads, even in pre- or asymptomatic individuals, is essential for the mitigation of community outbreaks. Methods and findings In this study, we analyze Ct values from 1297 SARS-CoV-2 positive patient saliva samples collected at the Clemson University testing lab in upstate South Carolina. Samples were identified as positive using RT-qPCR, and clade information was determined via whole genome sequencing at nearby commercial labs. We also obtained patient-reported information on symptoms and exposures at the time of testing. The lowest Ct values were observed among those infected with Delta (median: 22.61, IQR: 16.72–28.51), followed by Alpha (23.93, 18.36–28.49), Gamma (24.74, 18.84–30.64), and the more historic clade 20G (25.21, 20.50–29.916). There was a statistically significant difference in Ct value between Delta and all other clades (all p.adj<0.01), as well as between Alpha and 20G (p.adj<0.05). Additionally, pre- or asymptomatic patients (n = 1093) showed the same statistical differences between Delta and all other clades (all p.adj<0.01); however, symptomatic patients (n = 167) did not show any significant differences between clades. Our weekly testing strategy ensures that cases are caught earlier in the infection cycle, often before symptoms are present, reducing this sample size in our population. Conclusions COVID-19 variants Alpha and Delta have substantially higher viral loads in saliva compared to more historic clades. This trend is especially observed in individuals who are pre- or asymptomatic, which provides evidence supporting higher transmissibility and more rapid spread of emerging variants. Understanding the viral load of variants spreading within a community can inform public policy and clinical decision making.more » « less
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant has caused a dramatic resurgence in infections in the United Sates, raising questions regarding potential transmissibility among vaccinated individuals.
Between October 2020 and July 2021, we sequenced 4439 SARS-CoV-2 full genomes, 23% of all known infections in Alachua County, Florida, including 109 vaccine breakthrough cases. Univariate and multivariate regression analyses were conducted to evaluate associations between viral RNA burden and patient characteristics. Contact tracing and phylogenetic analysis were used to investigate direct transmissions involving vaccinated individuals.
The majority of breakthrough sequences with lineage assignment were classified as Delta variants (74.6%) and occurred, on average, about 3 months (104 ± 57.5 days) after full vaccination, at the same time (June-July 2021) of Delta variant exponential spread within the county. Six Delta variant transmission pairs between fully vaccinated individuals were identified through contact tracing, 3 of which were confirmed by phylogenetic analysis. Delta breakthroughs exhibited broad viral RNA copy number values during acute infection (interquartile range, 1.2-8.64 Log copies/mL), on average 38% lower than matched unvaccinated patients (3.29-10.81 Log copies/mL, P < .00001). Nevertheless, 49% to 50% of all breakthroughs, and 56% to 60% of Delta-infected breakthroughs exhibited viral RNA levels above the transmissibility threshold (4 Log copies/mL) irrespective of time after vaccination.
Delta infection transmissibility and general viral RNA quantification patterns in vaccinated individuals suggest limited levels of sterilizing immunity that need to be considered by public health policies. In particular, ongoing evaluation of vaccine boosters should specifically address whether extra vaccine doses curb breakthrough contribution to epidemic spread.
Yeager, Meredith (Ed.)Abstract Global sequencing of genomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to reveal new genetic variants that are the key to unraveling its early evolutionary history and tracking its global spread over time. Here we present the heretofore cryptic mutational history and spatiotemporal dynamics of SARS-CoV-2 from an analysis of thousands of high-quality genomes. We report the likely most recent common ancestor of SARS-CoV-2, reconstructed through a novel application and advancement of computational methods initially developed to infer the mutational history of tumor cells in a patient. This progenitor genome differs from genomes of the first coronaviruses sampled in China by three variants, implying that none of the earliest patients represent the index case or gave rise to all the human infections. However, multiple coronavirus infections in China and the United States harbored the progenitor genetic fingerprint in January 2020 and later, suggesting that the progenitor was spreading worldwide months before and after the first reported cases of COVID-19 in China. Mutations of the progenitor and its offshoots have produced many dominant coronavirus strains that have spread episodically over time. Fingerprinting based on common mutations reveals that the same coronavirus lineage has dominated North America for most of the pandemic in 2020. There have been multiple replacements of predominant coronavirus strains in Europe and Asia as well as continued presence of multiple high-frequency strains in Asia and North America. We have developed a continually updating dashboard of global evolution and spatiotemporal trends of SARS-CoV-2 spread (http://sars2evo.datamonkey.org/).more » « less
null (Ed.)Abstract Background Wastewater-based epidemiology (WBE) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be an important source of information for coronavirus disease 2019 (COVID-19) management during and after the pandemic. Currently, governments and transportation industries around the world are developing strategies to minimize SARS-CoV-2 transmission associated with resuming activity. This study investigated the possible use of SARS-CoV-2 RNA wastewater surveillance from airline and cruise ship sanitation systems and its potential use as a COVID-19 public health management tool. Methods Aircraft and cruise ship wastewater samples (n = 21) were tested for SARS-CoV-2 using two virus concentration methods, adsorption–extraction by electronegative membrane (n = 13) and ultrafiltration by Amicon (n = 8), and five assays using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR). Representative qPCR amplicons from positive samples were sequenced to confirm assay specificity. Results SARS-CoV-2 RNA was detected in samples from both aircraft and cruise ship wastewater; however concentrations were near the assay limit of detection. The analysis of multiple replicate samples and use of multiple RT-qPCR and/or RT-ddPCR assays increased detection sensitivity and minimized false-negative results. Representative qPCR amplicons were confirmed for the correct PCR product by sequencing. However, differences in sensitivity were observed among molecular assays and concentration methods. Conclusions The study indicates that surveillance of wastewater from large transport vessels with their own sanitation systems has potential as a complementary data source to prioritize clinical testing and contact tracing among disembarking passengers. Importantly, sampling methods and molecular assays must be further optimized to maximize detection sensitivity. The potential for false negatives by both wastewater testing and clinical swab testing suggests that the two strategies could be employed together to maximize the probability of detecting SARS-CoV-2 infections amongst passengers.more » « less