https://doi.org/10.1016/j.watres.2022.118535

Making waves: Wastewater surveillance of SARS-CoV-2 in an endemic future

During the COVID-19 pandemic, genomics and bioinformatics have emerged as essential public health tools. The genomic data acquired using these methods have supported the global health response, facilitated the development of testing methods and allowed the timely tracking of novel SARS-CoV-2 variants. Yet the virtually unlimited potential for rapid generation and analysis of genomic data is also coupled with unique technical, scientific and organizational challenges. Here, we discuss the application of genomic and computational methods for efficient data-driven COVID-19 response, the advantages of the democratization of viral sequencing around the world and the challenges associated with viral genome data collection and processing. 

/pdf/unlocking-capacities-of-genomics-for-the-covid-19-response-11dktkz4.pdf

Unlocking capacities of genomics for the COVID-19 response and future pandemics

https://doi.org/10.1016/j.scitotenv.2022.161196

Normalisation of SARS-CoV-2 concentrations in wastewater: The use of flow, electrical conductivity and crAssphage

The PCR technique has been known to the general public since the pandemic outbreak of COVID-19. This technique has progressed through three stages: from simple PCR to real-time fluorescence PCR to digital PCR. Among them, the microfluidic-based droplet digital PCR technique has attracted much attention and has been widely applied due to its advantages of high throughput, high sensitivity, low reagent consumption, low cross-contamination, and absolute quantification ability. In this review, we introduce various designs of microfluidic-based ddPCR developed within the last decade. The microfluidic-based droplet generation methods, thermal cycle strategies, and signal counting approaches are described, and the applications in the fields of single-cell analysis, disease diagnosis, and pathogen detection are introduced. Further, the challenges and prospects of microfluidic-based ddPCR are discussed. We hope that this review can contribute to the further development of the microfluidic-based ddPCR technique.

/pdf/advances-in-droplet-digital-polymerase-chain-reaction-on-2ep9i1d8.pdf

Advances in droplet digital polymerase chain reaction on microfluidic chips.

Wastewater-based epidemiology has been used to measure SARS-CoV-2 prevalence in cities worldwide as an indicator of community health, however, few longitudinal studies have followed SARS-CoV-2 in wastewater in small communities from the start of the pandemic or evaluated the influence of tourism on viral loads. Therefore the objective of this study was to use measurements of SARS-CoV-2 in wastewater to monitor viral trends and variants in a small island community over a twelve-month period beginning May 1, 2020, before the community re-opened to tourists. Wastewater samples were collected weekly and analyzed to detect and quantify SARS-CoV-2 genome copies. Sanger sequencing was used to determine genome sequences from total RNA extracted from wastewater samples positive for SARS-CoV-2. Visitor data was collected from the local Chamber of Commerce. We performed Poisson and linear regression to determine if visitors to the Cedar Key Chamber of Commerce were positively associated with SARS-CoV-2-positive wastewater samples and the concentration of SARS-CoV-2 RNA. Results indicated that weekly wastewater samples were negative for SARS-CoV-2 until mid-July when positive samples were recorded in four of five consecutive weeks. Additional positive results were recorded in November and December 2020, as well as January, March, and April 2021. Tourism data revealed that the SARS-CoV-2 RNA concentration in wastewater increased by 1.06 Log10 genomic copies/L per 100 tourists weekly. Sequencing from six positive wastewater samples yielded two complete sequences of SARS-CoV-2, two overlapping sequences, and two low yield sequences. They show arrival of a new variant SARS-CoV-2 in January 2021. Our results demonstrate the utility of wastewater surveillance for SARS-CoV-2 in a small community. Wastewater surveillance and viral genome sequencing suggest that population mobility likely plays an important role in the introduction and circulation of SARS-CoV-2 variants among communities experiencing high tourism and who have a small population size. 

Wastewater surveillance for SARS-CoV-2 in a small coastal community: Effects of tourism on viral presence and variant identification among low prevalence populations

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the coronavirus disease 2019 (COVID-19) pandemic to track disease burden through analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the health care system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven wastewater treatment plants (WTPs), collected from August 2020 to January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single-nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater, and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically important SARS-CoV-2 variants along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. IMPORTANCE Wastewater-based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the coronavirus disease 2019 (COVID-19) pandemic to track the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) without adding burden to health care systems. In this study, we used metatranscriptomics and reverse transcription-droplet digital PCR (RT-ddPCR) to assay RNA viruses across Southern California wastewater from August 2020 to January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single-nucleotide variants through sequencing. Likewise, wastewater treatment plants (WTPs) harbored different viromes, and we detected other human pathogens, such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

RNA viromics of Southern California wastewater and detection of SARS-CoV-2 single nucleotide variants.

Recent research has elucidated many factors which play a role in the development and composition of human microbiomes. In this study we briefly examine the microbiomes of saliva and fecal samples from 71 indigenous individuals, and chicha samples from 28 single family households in a remote community in the Ecuadorian Amazon. Fecal and saliva samples were collected at two separate time points whereas chicha samples were collected at four time points, once each day of the fermentation process. In total 324 samples were collected: 113 saliva, 108 chicha, and 103 fecal. Microbial composition and diversity were assessed using shotgun metagenome sequence data. Chicha samples were found to be nearly entirely composed of the order Lactobacillales, accounting for 90.1% of the relative abundance. Saliva samples also contained a high relative abundance of Lactobacillales (31.9%) as well as being composed of Neisseriales (12.8%), Actinomycineae (8.7%), Bacteroidales (7.0%), Clostridiales (6.8%), Micrococcineae (6.5%), and Pasteurellales (6.0%). Fecal samples were largely composed of the three orders Clostridiales (33.7%), Bacteroidales (21.9%), and Bifidobacteriales (16.5%). Comparison of α-diversity, as calculated by Shannon9s Diversity Index, in mothers and their offspring showed no significant difference between the two groups in either fecal or saliva samples. Comparison of β-diversity in fecal and saliva samples, as calculated by the Bray-Curtis Dissimilarity measure, within household units and between differing households showed that members of the same household were significantly less dissimilar to each other than to members of other households in the community. Average microbiome composition for individuals within fecal and saliva samples was assessed to determine the impact of an individual9s household on the composition of their microbiome. Household was determined to have a significant impact on both fecal and oral microbiome compositions.

https://biorxiv.org/content/10.1101/2020.10.02.323097v1.full.pdf

Comparing Fecal, Saliva and Chicha Microbiomes Between Mothers and Children in an Indigenous Ecuadorian Cohort

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early-warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the COVID-19 pandemic to track disease burden through analyses of SARS-CoV-2 RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the healthcare system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven treatment plants (WTPs), collected August 2020 – January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically-important SARS-CoV-2 variants, along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. Importance Wastewater based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the COVID-19 pandemic to track the spread of SARS-CoV-2 without adding burden to healthcare systems. In this study, we used metatranscriptomics and RT-ddPCR to assay RNA viruses across Southern California wastewater from August 2020 – January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single nucleotide variants through sequencing. Likewise, WTPs harbored different viromes, and we detected other human pathogens such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

/pdf/rna-viromics-of-southern-california-wastewater-and-detection-2vo9w7hzt3.pdf

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RNA viromics of Southern California wastewater and detection of SARS-CoV-2 single nucleotide variants.

Comparing Fecal, Saliva and Chicha Microbiomes Between Mothers and Children in an Indigenous Ecuadorian Cohort

RNA viromics of Southern California wastewater and detection of SARS-CoV-2 single nucleotide variants.