scholarly journals Sewage surveillance for the presence of SARS-CoV-2 genome as a useful wastewater based epidemiology (WBE) tracking tool in India

2020 ◽  
Author(s):  
Sudipti Arora ◽  
Aditi Nag ◽  
Jasmine Sethi ◽  
Jayana Rajvanshi ◽  
Sonika Saxena ◽  
...  
Keyword(s):  
Public Health ◽  
Viral Genome ◽  
Local Population ◽  
Health Data ◽  
Health Concern ◽  
N Gene ◽  
List Type ◽  
Hospital Wastewater ◽  
Rt Pcr ◽  
Wastewater Samples

AbstractThe infection with SARS-CoV-2 is reported to be accompanied by the shedding of the virus in stool samples of infected patients. Earlier reports have suggested that COVID-19 agents can be present in the fecal and sewage samples and thus it can be a good indication of the pandemic extent in a community. However, no such studies have been reported in the Indian context so far. Since, several factors like local population physiology, the climatic conditions, sewage composition, and processing of samples could possibly affect the detection of the viral genome, it becomes absolutely necessary to check for the presence of the SARS-CoV-2 in the wastewater samples from wastewater treatment plants (WWTPs) serving different localities of Jaipur city, which has been under red zone (pandemic hotspots) since early April 2020. Samples from different local municipal WWTPs and hospital wastewater samples were collected and wastewater based epidemiology (WBE) studies for the presence of SARS-CoV-2 were carried out using the RT-PCR technique to confirm the presence of different COVID-19 target genes namely S gene, E gene, ORF1ab gene, RdRp gene and N gene in the viral load of wastewater samples. In the present study, the untreated wastewater samples from the municipal WWTPs and hospital wastewater samples showed the presence of SARS-CoV-2 viral genome, which was correlated with the increased number of COVID-19 positive patients from the concerned areas, as per reported in the publically available health data. This is the first study that investigated the presence of SARS-CoV-2 viral genome in wastewater, at higher ambient temperature (above 40°C), further validating WBE as a potential tool in predicting and mitigating outbreaks.HighlightsThe study reports detection of SARS-CoV-2 in sewage in India.The presence of SARS-CoV-2 was confirmed by RT-PCR.The presence of viral genome was detected at high ambient temperatures of 40-45° C.Corroborates trends in the WWTPs showing viral genome with public health data.Treated effluent from WWTPs appears safe for reuse with low public health concern.Graphical Abstract

scholarly journals Sewage surveillance for the presence of SARS-CoV-2 genome as a useful wastewater based epidemiology (WBE) tracking tool in India

2020 ◽  
Vol 82 (12) ◽  
pp. 2823-2836
Author(s):  
Sudipti Arora ◽  
Aditi Nag ◽  
Jasmine Sethi ◽  
Jayana Rajvanshi ◽  
Sonika Saxena ◽  
...  
Keyword(s):  
Viral Genome ◽  
Target Genes ◽  
Wastewater Treatment Plants ◽  
Health Data ◽  
N Gene ◽  
Rt Pcr ◽  
Untreated Wastewater ◽  
Indian Context ◽  
Wastewater Samples ◽  
Potential Tool

Abstract The infection with SARS-CoV-2 is reported to be accompanied by the shedding of the virus in fecal samples of infected patients. Earlier reports have suggested that COVID-19 agents can be present in the sewage samples and thus it can be a good indication of the pandemic extent in a community. However, no such studies have been reported in the Indian context. Hence, it becomes absolutely necessary to detect the presence of the SARS-CoV-2 in the wastewater samples from wastewater treatment plants (WWTPs) serving different localities of Jaipur city. Samples from different WWTPs and hospital wastewater samples were collected and wastewater based epidemiology (WBE) studies were carried out using the RT-PCR to confirm the presence of different COVID-19 target genes namely S gene, E gene, ORF1ab gene, RdRp gene and N gene. The results revealed that the untreated wastewater samples showed the presence of SARS-CoV-2 viral genome, which was correlated with the increased number of COVID-19 positive patients from the concerned areas, as reported in the publically available health data. This is the first study that investigated the presence of SARS-CoV-2 viral genome in wastewater, at higher ambient temperature (45 °C), further validating WBE as potential tool in predicting and mitigating outbreaks.

scholarly journals Epidemiology of COVID-19 and effect of public health interventions, Chennai, India, March - October 2020

2021 ◽  
Author(s):  
M Jagadeesan ◽  
Parasuraman Ganeshkumar ◽  
Prabhdeep Kaur ◽  
Hemalatha Masanam Sriramulu ◽  
Manikandanesan Sakthivel ◽  
...  
Keyword(s):  
Public Health ◽  
Case Fatality ◽  
Contact Tracing ◽  
Severe Illness ◽  
List Type ◽  
Rt Pcr ◽  
Case Fatality Ratio ◽  
Metropolitan City ◽  
Lessons Learnt ◽  
Public Health Strategies

AbstractObjectivesTo describe the public health strategies and their effect in controlling the COVID-19 pandemic from March to October 2020 in Chennai, India.SettingChennai, a densely populated metropolitan city in Southern India, was one of the five cities which contributed to more than half of the COVID-19 cases in India.ParticipantsWe collected the de-identified line list of all the 192,450 COVID-19 case-patients reported from 17 March to 31 October 2020 in Chennai and their contacts for the analysis. We defined a COVID-19 case-patient based on the RT-PCR positive test in one of the Government approved labs.Outcome measuresThe primary outcomes of interest were incidence of COVID-19 per million population, case fatality ratio, deaths per million and the effective reproduction number (Rt). We also analysed the indicators for surveillance, testing, contact tracing and isolation.ResultsOf the 192,450 RT-PCR confirmed COVID-19 case-patients reported in Chennai from 17 March-31 October 2020, 114,889 (60%) were males. The highest incidence was 41,064 per million population among the 61-80 years. The incidence peaked during June 2020 at 5239 per million and declined to 3,627 per million in October 2020. The city reported 3,543 deaths, with a case fatality ratio (CFR) of 1.8% and the crude death rate was 431 per million. When lockdown began, Rt was high (4.2) in March and fluctuated from April to June 2020. The Rt dropped below one by the first week of July and remained so until October 2020, even with the relaxation of restrictionsConclusionThe combination of public health strategies controlled the COVID-19 epidemic in a large, densely populated city in India. We recommend continuing the interventions to prevent resurgence, even as vaccination is being rolled out.StrengthsWe did a comprehensive analysis of COVID-19 strategies and outcome in a large, densely populated metropolitan city in India.We documented that the community-centric public health strategies were feasible and effective in controlling the COVID-19 outbreak even in a large, thickly populated cityThe lessons learnt are relevant to similar settings in low-and middle-income countries. Given the ongoing multiple waves of COVID-19 and the difficulty in controlling the transmission, our experience and lessons learnt will be valuable for policymakers and scientific advisors globallyLimitationsWe analysed the data available from the GCC database and not from the hospitals where patients with moderate to severe illness were admitted. Hence, we could not report the severity of illness among admitted patients.Second, the COVID-19 incidence might have been underestimated while testing was low during the early phase of the epidemic

scholarly journals Failed detection of the full-length genome of SARS-CoV-2 by ultra-deep sequencing from the recovered and discharged patients retested viral PCR positive

2020 ◽  
Author(s):  
Fengyu Hu ◽  
Fengjuan Chen ◽  
Yaping Wang ◽  
Teng Xu ◽  
Xiaoping Tang ◽  
...  
Keyword(s):  
Public Health ◽  
Deep Sequencing ◽  
Viral Genome ◽  
Full Length ◽  
Health Concern ◽  
Public Health Concern ◽  
Low Concentration ◽  
Discharged Patients ◽  
Full Length Genome

AbstractOver 10 percent of recovered and discharged patients retested positive for SARS-CoV-2, raising a public health concern whether they could be potential origins of infection. In this study, we found that detectable viral genome in discharged patients might only mean the presence of viral fragments, and could hardly form an infection origin for its extremely low concentration.

scholarly journals Mink SARS-CoV-2 infection in Poland – short communication

2021 ◽  
Vol 65 (1) ◽  
pp. 1-5
Author(s):  
Katarzyna Domańska-Blicharz ◽  
Anna Orłowska ◽  
Marcin Smreczak ◽  
Krzysztof Niemczuk ◽  
Ewelina Iwan ◽  
...  
Keyword(s):  
Public Health ◽  
Short Communication ◽  
Genetic Relatedness ◽  
Farm Workers ◽  
N Gene ◽  
Whole Genome ◽  
Rt Pcr ◽  
Genome Sequences ◽  
Blood Samples ◽  
Epidemiological Situation

AbstractIntroductionSince April 2020, when the first SARS-CoV-2 infection was reported in mink and subsequently in mink farm workers in the Netherlands, it has been confirmed that human-to-mink and mink-to-human transmission can occur. Later, SARS-CoV-2 infections in mink were reported in many European and North American countries.Material and methodsSamples from 590 mink from a total of 28 farms were tested by real-time RT-PCR. Whole genome sequences from one positive farm were generated and genetic relatedness was established.ResultsSARS-CoV-2 RNA was detected on a breeder farm with stock of 5,850 mink. Active viraemia was confirmed in individually tested samples with Ct values respectively between 19.4 and 29.6 for E and N gene fragments. Further testing of samples from culled animals revealed 70% positivity in throat swabs and 30% seropositivity in blood samples. Phylogenetic analysis of full-length nucleotide sequences of two SARS-CoV-2 isolates revealed that they belong to the 20B Nextstrain clade. Several nucleotide mutations were found in analysed samples compared to the reference Wuhan HU-1 strain and some of them were nonsynonymous.ConclusionWe report the infection of mink with SARS-CoV-2 on one farm in Poland and the results of subsequent analysis of virus sequences from two isolates. These data can be useful for assessment of the epidemiological situation of SARS-CoV-2 in Poland and how it endangers public health.

Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y, and del156-157/R158G) in settled solids using digital RT-PCR v2

2021 ◽  
Author(s):  
Bridgette Hughes ◽  
Bradley J. White ◽  
Marlene K. Wolfe ◽  
Krista Wigginton ◽  
Alexandria B Boehm
Keyword(s):  
Quantitative Analysis ◽  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Population Level ◽  
Digital Pcr ◽  
N Gene ◽  
Rt Pcr ◽  
Pcr Inhibitor ◽  
Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one for HV69-70, one for E484K/N501Y, and one for del156-157/R158G) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y,del156-157/R158G, and Del143-145) in settled solids using digital RT-PCR v2

2021 ◽  
Author(s):  
Bridgette Hughes ◽  
Bradley J. White ◽  
Marlene K. Wolfe ◽  
Krista Wigginton ◽  
Alexandria B Boehm
Keyword(s):  
Quantitative Analysis ◽  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Population Level ◽  
Digital Pcr ◽  
N Gene ◽  
Rt Pcr ◽  
Pcr Inhibitor ◽  
Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one each for HV69-70, E484K/N501Y, del156-157/R158G, and Del143-145) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

scholarly journals A Case of SARS-CoV-2 Clinical Relapse after 4 Negative RT-PCR Tests in Greece: Recurrence or Reinfection?

2021 ◽  
Vol 14 ◽  
pp. 117954762110098
Author(s):  
Fotis Konstantinou ◽  
Ioanna Skrapari ◽  
Eleni Bareta ◽  
Nikolaos Bakogiannis ◽  
Anna-Maria Papadopoulou ◽  
...  
Keyword(s):  
Public Health ◽  
Clinical Syndrome ◽  
Health Concern ◽  
Clinical Relapse ◽  
Test Results ◽  
Rt Pcr ◽  
Public Health Concern ◽  
Pcr Test

SARS-CoV-2 pandemic is the greatest public health concern of the year 2020. There are several worldwide reports of patients who have managed to recover from SARS-CoV-2 infection with negative PCR test results, that for unknown reasons convert back to positive PCR. We report a case of a patient in our hospital who developed positive PCR test results for SARS-CoV-2, after 4 consecutive results that were negative, along with a full-blown clinical syndrome of SARS-CoV-2 infection.

High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR v2

2021 ◽  
Author(s):  
Aaron Topol (Verily Life Sciences) ◽  
marlene.wolfe not provided ◽  
Brad White (Verily Life Sciences) ◽  
Krista Wigginton ◽  
Alexandria B Boehm
Keyword(s):  
Quantitative Analysis ◽  
Nucleic Acid ◽  
High Throughput ◽  
Rna Extraction ◽  
Digital Pcr ◽  
N Gene ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Pcr Inhibitor ◽  
Wastewater Samples

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.

scholarly journals RT-qPCR detection of SARS-CoV-2 mutations S 69-70 del, S N501Y and N D3L associated with variants of concern in Canadian wastewater samples

2021 ◽  
Author(s):  
Shelley W Peterson ◽  
Ravinder Lidder ◽  
Jade Daigle ◽  
Quinn Wonitowy ◽  
Audra Nagasawa ◽  
...  
Keyword(s):  
Public Health ◽  
Early Warning ◽  
Population Level ◽  
N Gene ◽  
Allelic Discrimination ◽  
Molecular Surveillance ◽  
Screening And Surveillance ◽  
Relative Quantitation ◽  
Clinical Surveillance ◽  
Wastewater Samples

SARS-CoV-2 variants of concern (VoC) have been increasingly detected in clinical surveillance in Canada and internationally. These VoC are associated with higher transmissibility rates and in some cases, increased mortality. In this work we present a national wastewater survey of the distribution of three SARS-CoV-2 mutations found in the B.1.1.7, B.1.351, and P.1 VoC, namely the S-gene 69-70 deletion, N501Y mutation, and N-gene D3L. RT-qPCR allelic discrimination assays were sufficiently sensitive and specific for detection and relative quantitation of SARS-CoV-2 variants in wastewater to allow for rapid population-level screening and surveillance. We tested 261 samples collected from 5 Canadian cities (Vancouver, Edmonton, Toronto, Montreal, and Halifax) and 6 communities in the Northwest Territories from February 16th to March 28th, 2021. VoC were not detected in the Territorial communities, suggesting the absence of VoC SARS-CoV-2 cases in those communities. Percentage of variant remained low throughout the study period in the majority of the sites tested, however the Toronto sites showed a marked increase from ~25% to ~75% over the study period. The results of this study highlight the utility of population level molecular surveillance of SARS-CoV-2 VoC using wastewater. Wastewater monitoring for VoC can be a powerful tool in informing public health responses, including monitoring trends independent of clinical surveillance and providing early warning to communities.

scholarly journals Development of genus-specific universal primers for the detection of flaviviruses

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Tomo Daidoji ◽  
Ronald Enrique Morales Vargas ◽  
Katsuro Hagiwara ◽  
Yasuha Arai ◽  
Yohei Watanabe ◽  
...  
Keyword(s):  
Public Health ◽  
Viral Genome ◽  
Health And Safety ◽  
High Sensitivity ◽  
Infected Mosquito ◽  
Health Concern ◽  
Universal Primers ◽  
The Novel ◽  
Universal Primer ◽  
Mosquito Cells

Abstract Background Flaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods. Methods We developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates. Results The novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates. Conclusion This PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology.

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