scholarly journals Wastewater sequencing uncovers early, cryptic SARS-CoV-2 variant transmission

2021 ◽  
Author(s):  
Smruthi Karthikeyan ◽  
Joshua I Levy ◽  
Peter De Hoff ◽  
Greg Humphrey ◽  
Amanda Birmingham ◽  
...  
Keyword(s):  
Sequence Data ◽  
Virus Concentration ◽  
Clinical Testing ◽  
University Campus ◽  
Infection Dynamics ◽  
Abundance Estimates ◽  
Two Factors ◽  
Critical Issues ◽  
Multiple Virus ◽  
Wastewater Samples

As SARS-CoV-2 becomes an endemic pathogen, detecting emerging variants early is critical for public health interventions. Inferring lineage prevalence by clinical testing is infeasible at scale, especially in areas with limited resources, participation, or testing/sequencing capacity, which can also introduce biases. SARS-CoV-2 RNA concentration in wastewater successfully tracks regional infection dynamics and provides less biased abundance estimates than clinical testing. Tracking virus genomic sequences in wastewater would improve community prevalence estimates and detect emerging variants. However, two factors limit wastewater-based genomic surveillance: low-quality sequence data and inability to estimate relative lineage abundance in mixed samples. Here, we resolve these critical issues to perform a high-resolution, 295-day wastewater and clinical sequencing effort, in the controlled environment of a large university campus and the broader context of the surrounding county. We develop and deploy improved virus concentration protocols and deconvolution software that fully resolve multiple virus strains from wastewater. We detect emerging variants of concern up to 14 days earlier in wastewater samples, and identify multiple instances of virus spread not captured by clinical genomic surveillance. Our study provides a scalable solution for wastewater genomic surveillance that allows early detection of SARS-CoV-2 variants and identification of cryptic transmission.

scholarly journals Detection of SARS-CoV-2 RNA Throughout Wastewater Treatment Plants and A Modeling Approach to Understand COVID-19 Infection Dynamics in Winnipeg, Canada

2021 ◽  
Author(s):  
Kadir Yanac ◽  
Adeola Adegoke ◽  
Liqun Wang ◽  
Qiuyan Yuan ◽  
Miguel Uyaguari
Keyword(s):  
Wastewater Treatment ◽  
Wastewater Treatment Plants ◽  
Water Cycle ◽  
Positive Control ◽  
Virus Concentration ◽  
Potential Contribution ◽  
Sample Collection ◽  
Infection Dynamics ◽  
Wastewater Samples ◽  
Concentration Methods

Although numerous studies have detected SARS-CoV-2 in wastewater and attempted to find correlations between the concentration of SARS-CoV-2 and the number of cases, no consensus has been reached on sample collection and processing, and data analysis. Moreover, the fate of SARS-CoV-2 in wastewater treatment plants is another issue, specifically regarding the discharge of the virus into environmental settings and the water cycle. The current study monitored SARS-CoV-2 in influent and effluent wastewater samples with three different concentration methods and sludge samples over six months (July to December 2020) to compare different virus concentration methods, assess the fate of SARS-CoV-2 in wastewater treatment plants, and describe the potential relationship between SARS-CoV-2 concentrations in influent and infection dynamics. Skimmed milk flocculation (SMF) resulted in higher recoveries of an internal positive control, Armored RNA, and higher positivity rate of SARS-CoV-2 in samples compared to ultrafiltration methods employing a prefiltration step to eliminate solids. Our results suggested that SARS-CoV-2 may predominate in solids and therefore, concentration methods focusing on both supernatant and solid fractions may result in better recovery. SARS-CoV-2 was detected in influent and primary sludge samples but not in secondary and final effluent samples, indicating a significant reduction during primary and secondary treatments. SARS-CoV-2 was first detected in influent on September 30th, 2020. A decay-rate formula was applied to estimate initial concentrations of late-processed samples with SMF. A model based on shedding rate and new cases was applied to estimate SARS-CoV-2 concentrations and the number of active shedders. Inferred sensitivity of observed and modeled concentrations to the fluctuations in new cases and test-positivity rates indicated a potential contribution of newly infected individuals to SARS-CoV-2 loads in wastewater.

scholarly journals Detection of SARs-CoV-2 in wastewater, using the existing environmental surveillance network: An epidemiological gateway to an early warning for COVID-19 in communities

2020 ◽  
Author(s):  
Salmaan Sharif ◽  
Aamer Ikram ◽  
Adnan Khurshid ◽  
Muhammad Salman ◽  
Nayab Mehmood ◽  
...  
Keyword(s):  
Early Warning ◽  
High Speed ◽  
Sequence Data ◽  
Rna Extraction ◽  
Study Finding ◽  
Warning Signs ◽  
Virus Concentration ◽  
Two Phase ◽  
Surveillance Network ◽  
Wastewater Samples

AbstractBackgroundThe ongoing COVID-19 pandemic caused by SARs-CoV-2 was transmitted person to person via droplet infections and fecal-oral transmission. This illustrates the probability of environmentally facilitated transmission, mainly the sewage.MethodWe used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value.ResultsA total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID patients and COVID 19 patient quarantine center drainage strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater.DiscussionThis study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as early warning system to monitor viral tracking and circulation in cities with lower COVID-19 disease burden or heavily populated areas where door-to-door tracing may not be possible. However, attention needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.

scholarly journals Detection of SARs-CoV-2 in wastewater using the existing environmental surveillance network: A potential supplementary system for monitoring COVID-19 transmission

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0249568
Author(s):  
Salmaan Sharif ◽  
Aamer Ikram ◽  
Adnan Khurshid ◽  
Muhammad Salman ◽  
Nayab Mehmood ◽  
...  
Keyword(s):  
High Speed ◽  
Sequence Data ◽  
Close Contact ◽  
Rna Extraction ◽  
Study Finding ◽  
Warning Signs ◽  
Virus Concentration ◽  
Two Phase ◽  
Surveillance Network ◽  
Wastewater Samples

The ongoing COVID-19 pandemic is caused by SARs-CoV-2. The virus is transmitted from person to person through droplet infections i.e. when infected person is in close contact with another person. In January 2020, first report of detection of SARS-CoV-2 in faeces, has made it clear that human wastewater might contain this virus. This may illustrate the probability of environmentally facilitated transmission, mainly the sewage, however, environmental conditions that could facilitate faecal oral transmission is not yet clear. We used existing Pakistan polio environment surveillance network to investigate presence of SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for surety and confirmatory of positivity. A Two-phase separation method is used for sample clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in Cq value. A total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV-2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV-2 RNA positive samples from areas with COVID 19 patients and quarantine center strengthen the findings and use of wastewater surveillance in future. Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in wastewater. This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an epidemiologic potential that can be used as supplementary system to monitor viral tracking and circulation in cities with lower COVID-19 testing capacity or heavily populated areas where door-to-door tracing may not be possible. However, attention is needed on virus concentration and detection assay to increase the sensitivity. Development of highly sensitive assay will be an indicator for virus monitoring and to provide early warning signs.

scholarly journals Specific Detection of Arcobacter and Campylobacter Strains in Water and Sewage by PCR and Fluorescent In Situ Hybridization

2003 ◽  
Vol 69 (2) ◽  
pp. 1181-1186 ◽  
Author(s):  
Yolanda Moreno ◽  
Salut Botella ◽  
José Luis Alonso ◽  
María A. Ferrús ◽  
Manuel Hernández ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Sequence Data ◽  
23S Rrna ◽  
Specific Detection ◽  
Specific Primers ◽  
Detection Range ◽  
Water And Wastewater ◽  
Wastewater Samples

ABSTRACT The aim of this study was to evaluate PCR and fluorescent in situ hybridization (FISH) techniques for detecting Arcobacter and Campylobacter strains in river water and wastewater samples. Both 16S and 23S rRNA sequence data were used to design specific primers and oligonucleotide probes for PCR and FISH analyses, respectively. In order to assess the suitability of the methods, the assays were performed on naturally and artificially contaminated samples and compared with the isolation of cells on selective media. The detection range of PCR and FISH assays varied between 1 cell/ml (after enrichment) to 103 cells/ml (without enrichment). According to our results, both rRNA-based techniques have the potential to be used as quick and sensitive methods for detection of campylobacters in environmental samples.

scholarly journals Rapid incidence estimation from SARS-CoV-2 genomes reveals decreased case detection in Europe during summer 2020

2021 ◽  
Author(s):  
Maureen Smith ◽  
Maria Trofimova ◽  
Ariane Weber ◽  
Yannick Duport ◽  
Denise Kühnert ◽  
...  
Keyword(s):  
In Silico ◽  
Sequence Data ◽  
Case Detection ◽  
Viral Genomes ◽  
Infection Dynamics ◽  
True Number ◽  
Incidence Estimation ◽  
Novel Method ◽  
National Testing ◽  
Testing Policies

Abstract By May 2021, over 160 million SARS-CoV-2 diagnoses have been reported worldwide. Yet, the true number of infections is unknown and believed to exceed the reported numbers by several fold. National testing policies, in particular, can strongly affect the proportion of undetected cases. Here, we propose a novel method (GInPipe) that reconstructs SARS-CoV-2 incidence profiles within minutes, solely from publicly available, time-stamped viral genomes. We validated GInPipe against in silico generated outbreak data and elaborate phylodynamic analyses. We apply the method to reconstruct incidence histories from sequence data for Denmark, Scotland, Switzerland, and Victoria (Australia). GInPipe reconstructs the different pandemic waves robustly and remarkably accurate. We demonstrate how the method can be used to investigate the effects of changing testing policies on the probability to diagnose and report infected individuals. Specifically, we find that under-reporting was highest in mid 2020 in parts of Europe, coinciding with changes towards more liberal testing policies at times of low testing capacities. Due to the increased use of real-time sequencing, it is envisaged that GInPipe can complement established surveillance tools to monitor the SARS-CoV-2 pandemic. We anticipate that the method is particularly useful in settings where diagnostic and reporting infrastructures are insufficient. In ‘post-pandemic’ times, when diagnostic efforts are decreased, GInPipe may facilitate the detection of hidden infection dynamics.

scholarly journals Rapid incidence estimation from SARS-CoV-2 genomes reveals decreased case detection in Europe during summer 2020

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maureen Rebecca Smith ◽  
Maria Trofimova ◽  
Ariane Weber ◽  
Yannick Duport ◽  
Denise Kühnert ◽  
...  
Keyword(s):  
Real Time ◽  
Sequence Data ◽  
Considerable Proportion ◽  
Case Detection ◽  
Viral Genomes ◽  
Case Ascertainment ◽  
Infection Dynamics ◽  
Incidence Estimation ◽  
Testing Policies

AbstractBy October 2021, 230 million SARS-CoV-2 diagnoses have been reported. Yet, a considerable proportion of cases remains undetected. Here, we propose GInPipe, a method that rapidly reconstructs SARS-CoV-2 incidence profiles solely from publicly available, time-stamped viral genomes. We validate GInPipe against simulated outbreaks and elaborate phylodynamic analyses. Using available sequence data, we reconstruct incidence histories for Denmark, Scotland, Switzerland, and Victoria (Australia) and demonstrate, how to use the method to investigate the effects of changing testing policies on case ascertainment. Specifically, we find that under-reporting was highest during summer 2020 in Europe, coinciding with more liberal testing policies at times of low testing capacities. Due to the increased use of real-time sequencing, it is envisaged that GInPipe can complement established surveillance tools to monitor the SARS-CoV-2 pandemic. In post-pandemic times, when diagnostic efforts are decreasing, GInPipe may facilitate the detection of hidden infection dynamics.

scholarly journals A Comparison of Four Commercially Available RNA Extraction Kits for Wastewater Surveillance of SARS-CoV-2 in a College Population

2021 ◽  
Author(s):  
Megan O'Brian ◽  
Zachary C Rundell ◽  
Michelle D Nemec ◽  
Laura M Langan ◽  
Jeffrey A Back ◽  
...  
Keyword(s):  
Public Health ◽  
New England ◽  
Rna Extraction ◽  
University Campus ◽  
Population Densities ◽  
Soil Microbe ◽  
Processing Times ◽  
College Population ◽  
Public Health Officials ◽  
Wastewater Samples

Localized wastewater surveillance has allowed for public health officials to gain a broader understanding of SARS-CoV-2 viral prevalence in the community allowing public health officials time to prepare for impending outbreaks. Given variable levels of virus in the population through public health interventions, proper concentration and extraction of viral RNA is a key step in ensuring accurate detections. With many commercial RNA extraction kits and methodologies available, the performance of 4 different kits were evaluated for SARS-CoV-2 RNA detection in wastewater, specifically focusing on their applicability to lower population densities such as those at university campus dorms. Raw wastewater samples were collected at 4 sites on a college campus over a 24 hour period as a composite sample. Included in these sites was an isolation site that housed students that tested positive for Covid-19 via nasopharyngeal swabs. These samples were analyzed using the following kits: Qiagen All Prep PowerViral DNA/RNA kit, New England BioLabs Monarch RNA MiniPrep Kit, and Zymo Quick RNA-Viral Kit, and the Zymo Quick-RNA Fecal/Soil Microbe MicroPrep Kit. All four sites were processed according to the manufacturers guidelines. Extractions were then quantified with RT-qPCR one-step reactions using an N2 primer and a linearized plasmid standard. While the Zymo Quick-RNA Fecal/Soil Microbe MicroPrep Kit (also known as the Zymo Environ Water RNA Kit) only recovered approximately 73% (+/- 38%) SARS-CoV-2 RNA compared to the Zymo Quick-RNA Viral kit, it was the most time efficient kit to yield comparable results. This extraction kit had a cumulative processing time of approximately five hours compared, while the other three kits had processing times between approximately 9 and 9.5 hours. Based on the current research, the most effective kits for smaller population densities are pellet based and include a homogenization, inhibitor removal, and RNA preservation step.

scholarly journals Genome Sequencing of SARS-CoV-2 Allows Monitoring of Variants of Concern through Wastewater

Water ◽  
2021 ◽  
Vol 13 (21) ◽  
pp. 3018
Author(s):  
Malte Herold ◽  
Aymeric Fouquier d′Hérouël ◽  
Patrick May ◽  
Francesco Delogu ◽  
Anke Wienecke-Baldacchino ◽  
...  
Keyword(s):  
Genetic Material ◽  
Population Based ◽  
Epidemiological Surveillance ◽  
Clinical Samples ◽  
Short Read ◽  
Regional Clusters ◽  
Subsequent Time ◽  
Infection Dynamics ◽  
Time Period ◽  
Wastewater Samples

Monitoring SARS-CoV-2 in wastewater has shown to be an effective tool for epidemiological surveillance. More specifically, RNA levels determined with RT-qPCR have been shown to track with the infection dynamics within the population. However, the surveillance of individual lineages circulating in the population based on genomic sequencing of wastewater samples is challenging, as the genetic material constitutes a mixture of different viral haplotypes. Here, we identify specific signature mutations from individual SARS-CoV-2 lineages in wastewater samples to estimate lineages circulating in Luxembourg. We compare circulating lineages and mutations to those detected in clinical samples amongst infected individuals. We show that especially for dominant lineages, the allele frequencies of signature mutations correspond to the occurrence of particular lineages in the population. In addition, we provide evidence that regional clusters can also be discerned. We focused on the time period between November 2020 and March 2021 in which several variants of concern emerged and specifically traced the lineage B.1.1.7, which became dominant in Luxembourg during that time. During the subsequent time points, we were able to reconstruct short haplotypes, highlighting the co-occurrence of several signature mutations. Our results highlight the potential of genomic surveillance in wastewater samples based on amplicon short-read data. By extension, our work provides the basis for the early detection of novel SARS-CoV-2 variants.

scholarly journals Co-exposure to multiple Ranavirus types enhances viral infectivity and replication in a larval amphibian system

2018 ◽  
Author(s):  
Joseph R. Mihaljevic ◽  
Jason T. Hoverman ◽  
Pieter T.J. Johnson
Keyword(s):  
Ambystoma Tigrinum ◽  
Viral Infectivity ◽  
Virus Species ◽  
Frog Virus ◽  
Larval Amphibians ◽  
Infection Dynamics ◽  
Animal Populations ◽  
Exposed Individual ◽  
Multiple Virus ◽  
Chorus Frog

AbstractMultiple pathogens commonly co-occur in animal populations, yet few studies demonstrate how co-exposure of individual hosts scales up to affect transmission. Although viruses in the genus Ranavirus are globally widespread and multiple virus species or strains likely co-occur in nature, no studies have examined how co-exposure affects infection dynamics in larval amphibians. We exposed individual Rana aurora (Northern red-legged frog) larvae to Ambystoma tigrinum virus (ATV), frog virus 3 (FV3), or an FV3-like strain isolated from a frog-culturing facility in Georgia, USA (RCV-Z2). We compared single-virus to pairwise co-exposures, while experimentally accounting for dosage. Co-exposure to ATV and FV3-like strains resulted in almost twice as many infected individuals compared to single-virus exposures, suggesting an effect of co-exposure on viral infectivity. The viral load in infected individuals exposed to ATV and FV3 was also higher than the single-dose FV3 treatment, suggesting an effect of co-exposure on viral replication. In a follow-up experiment, we examined how the co-occurrence of ATV and FV3 affected epizootics in mesocosm populations of larval Pseudacris triseriata (Western chorus frog). Although ATV did not generally establish within host populations (<4% prevalence), when ATV and FV3 were both present, this co-exposure resulted in a larger epizootic of FV3. Our results emphasize the importance of multi-pathogen interactions in epizootic dynamics and have management implications for natural and commercial amphibian populations.

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