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13 pages, 1176 KiB

Open AccessArticle

Use of Capsid Integrity-qPCR for Detecting Viral Capsid Integrity in Wastewater

by Jessica L. Kevill, Kata Farkas, Nicola Ridding, Nicholas Woodhall, Shelagh K. Malham and Davey L. Jones

Viruses 2024, 16(1), 40; https://doi.org/10.3390/v16010040 - 26 Dec 2023

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Abstract

Quantifying viruses in wastewater via RT-qPCR provides total genomic data but does not indicate the virus capsid integrity or the potential risk for human infection. Assessing virus capsid integrity in sewage is important for wastewater-based surveillance, since discharged effluent may pose a public [...] Read more.

Quantifying viruses in wastewater via RT-qPCR provides total genomic data but does not indicate the virus capsid integrity or the potential risk for human infection. Assessing virus capsid integrity in sewage is important for wastewater-based surveillance, since discharged effluent may pose a public health hazard. While integrity assays using cell cultures can provide this information, they require specialised laboratories and expertise. One solution to overcome this limitation is the use of photo-reactive monoazide dyes (e.g., propidium monoazide [PMAxx]) in a capsid integrity-RT-qPCR assay (ci-RT-qPCR). In this study, we tested the efficiency of PMAxx dye at 50 μM and 100 μM concentrations on live and heat-inactivated model viruses commonly detected in wastewater, including adenovirus (AdV), hepatitis A (HAV), influenza A virus (IAV), and norovirus GI (NoV GI). The 100 μM PMAxx dye concentration effectively differentiated live from heat-inactivated viruses for all targets in buffer solution. This method was then applied to wastewater samples (n = 19) for the detection of encapsulated AdV, enterovirus (EV), HAV, IAV, influenza B virus (IBV), NoV GI, NoV GII, and SARS-CoV-2. Samples were negative for AdV, HAV, IAV, and IBV but positive for EV, NoV GI, NoV GII, and SARS-CoV-2. In the PMAxx-treated samples, EV, NoV GI, and NoV GII showed −0.52–1.15, 0.9–1.51, and 0.31–1.69 log reductions in capsid integrity, indicating a high degree of potentially infectious virus in wastewater. In contrast, SARS-CoV-2 was only detected using RT-qPCR but not after PMAxx treatment, indicating the absence of encapsulated and potentially infectious virus. In conclusion, this study demonstrates the utility of PMAxx dyes to evaluate capsid integrity across a diverse range of viruses commonly monitored in wastewater. Full article

(This article belongs to the Special Issue Wastewater-Based Epidemiology (WBE) in COVID-19 Pandemics)

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13 pages, 2193 KiB

Open AccessArticle

SARS-CoV-2 RNA in Wastewater and Bivalve Mollusk Samples of Campania, Southern Italy

by Annalisa Lombardi, Antonia Voli, Andrea Mancusi, Santa Girardi, Yolande Thérèse Rose Proroga, Biancamaria Pierri, Renato Olivares, Luigi Cossentino, Elisabetta Suffredini, Giuseppina La Rosa, Giovanna Fusco, Antonio Pizzolante, Amalia Porta, Pietro Campiglia, Ida Torre, Francesca Pennino and Alessandra Tosco

Viruses 2023, 15(8), 1777; https://doi.org/10.3390/v15081777 - 21 Aug 2023

Cited by 2 | Viewed by 1169

Abstract

SARS-CoV-2 can be detected in the feces of infected people, consequently in wastewater, and in bivalve mollusks, that are able to accumulate viruses due to their ability to filter large amounts of water. This study aimed to monitor SARS-CoV-2 RNA presence in 168 [...] Read more.

SARS-CoV-2 can be detected in the feces of infected people, consequently in wastewater, and in bivalve mollusks, that are able to accumulate viruses due to their ability to filter large amounts of water. This study aimed to monitor SARS-CoV-2 RNA presence in 168 raw wastewater samples collected from six wastewater treatment plants (WWTPs) and 57 mollusk samples obtained from eight harvesting sites in Campania, Italy. The monitoring period spanned from October 2021 to April 2022, and the results were compared and correlated with the epidemiological situation. In sewage, the ORF1b region of SARS-CoV-2 was detected using RT-qPCR, while in mollusks, three targets—RdRp, ORF1b, and E—were identified via RT-dPCR. Results showed a 92.3% rate of positive wastewater samples with increased genomic copies (g.c.)/(day*inhabitant) in December–January and March–April 2022. In the entire observation period, 54.4% of mollusks tested positive for at least one SARS-CoV-2 target, and the rate of positive samples showed a trend similar to that of the wastewater samples. The lower SARS-CoV-2 positivity rate in bivalve mollusks compared to sewages is a direct consequence of the seawater dilution effect. Our data confirm that both sample types can be used as sentinels to detect SARS-CoV-2 in the environment and suggest their potential use in obtaining complementary information on SARS-CoV-2. Full article

(This article belongs to the Special Issue Wastewater-Based Epidemiology (WBE) in COVID-19 Pandemics)

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14 pages, 3229 KiB

Open AccessArticle

SARS-CoV-2 Variants Detection Strategies in Wastewater Samples Collected in the Bangkok Metropolitan Region

by Ratanaporn Tangwangvivat, Supaporn Wacharapluesadee, Papassorn Pinyopornpanish, Sininat Petcharat, Suthida Muangnoicharoen Hearn, Nattakarn Thippamom, Chadaporn Phiancharoen, Piyapha Hirunpatrawong, Phattra Duangkaewkart, Ananporn Supataragul, Chadaporn Chaiden, Wiriyachayon Wechsirisan, Nantaporn Wandee, Krongkan Srimuang, Leilani Paitoonpong, Rome Buathong, Chonticha Klungthong, Vichan Pawun, Soawapak Hinjoy, Opass Putcharoen and Sopon Iamsirithaworn Show full author list Hide full author list

Viruses 2023, 15(4), 876; https://doi.org/10.3390/v15040876 - 29 Mar 2023

Cited by 2 | Viewed by 1642

Abstract

Wastewater surveillance is considered a promising approach for COVID-19 surveillance in communities. In this study, we collected wastewater samples between November 2020 and February 2022 from twenty-three sites in the Bangkok Metropolitan Region to detect the presence of SARS-CoV-2 and its variants for [...] Read more.

Wastewater surveillance is considered a promising approach for COVID-19 surveillance in communities. In this study, we collected wastewater samples between November 2020 and February 2022 from twenty-three sites in the Bangkok Metropolitan Region to detect the presence of SARS-CoV-2 and its variants for comparison to standard clinical sampling. A total of 215 wastewater samples were collected and tested for SARS-CoV-2 RNA by real-time PCR with three targeted genes (N, E, and ORF1ab); 102 samples were positive (42.5%). The SARS-CoV-2 variants were determined by a multiplex PCR MassARRAY assay to distinguish four SARS-CoV-2 variants, including Alpha, Beta, Delta, and Omicron. Multiple variants of Alpha–Delta and Delta–Omicron were detected in the wastewater samples in July 2021 and January 2022, respectively. These wastewater variant results mirrored the country data from clinical specimens deposited in GISAID. Our results demonstrated that wastewater surveillance using multiple signature mutation sites for SARS-CoV-2 variant detection is an appropriate strategy to monitor the presence of SARS-CoV-2 variants in the community at a low cost and with rapid turn-around time. However, it is essential to note that sequencing surveillance of wastewater samples should be considered complementary to whole genome sequencing of clinical samples to detect novel variants. Full article

(This article belongs to the Special Issue Wastewater-Based Epidemiology (WBE) in COVID-19 Pandemics)

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13 pages, 2204 KiB

Open AccessArticle

Simple Wastewater Preparation Protocol Applied to Monitor the Emergence of the Omicron 21L/BA.2 Variant by Genome Sequencing

by Nathalie Wurtz, Maelle Boussier, Louis Souville, Gwilherm Penant, Alexandre Lacoste, Philippe Colson, Bernard La Scola and Sarah Aherfi

Viruses 2023, 15(2), 268; https://doi.org/10.3390/v15020268 - 17 Jan 2023

Cited by 2 | Viewed by 1455

Abstract

Detecting and monitoring viruses in wastewater samples have been reported as useful ways of tracking SARS-CoV-2 epidemic trends. However, there is currently no unanimously recognised method of processing samples to identify and quantify SARS-CoV-2 variants in wastewater. We aimed to implement a method [...] Read more.

Detecting and monitoring viruses in wastewater samples have been reported as useful ways of tracking SARS-CoV-2 epidemic trends. However, there is currently no unanimously recognised method of processing samples to identify and quantify SARS-CoV-2 variants in wastewater. We aimed to implement a method that was as simple as possible in order to be used universally. In a study performed between January 2022 and June 2022 in the city of Marseille, France, we first evaluated the impact of the sample preservation strategy. We then compared ultracentrifugation to ultrafiltration and several steps of filtration to determine the optimal approach for virus concentration. As a proof-of-concept, the definitive protocol was applied to next-generation sequencing of SARS-CoV-2 in wastewater to monitor the emergence of the Omicron variant in the city. For sewage water to be processed in the week following the sampling, storage at +4 °C is sufficient, with less than 1 Ct loss. Filtration with a 5 µm syringe filter, then with a 0.8 µm filtration unit, followed by ultrafiltration was the optimal protocol, leading to an average increase of 3.24 Ct when the starting Ct was on average 38 in the wastewater. This made it possible to observe the emergence of the Omicron 21L/BA.2 variant after Omicron 21K/BA.1 by genome sequencing over a period ranging from 20 February to 10 April 2022 in agreement with observations based on patient data. To conclude, by using a simple method requiring only basic filters and a centrifuge as equipment, it is possible to accurately track the relative incidence rates and the emergence of SARS-CoV-2 variants based on sewage samples. Full article

(This article belongs to the Special Issue Wastewater-Based Epidemiology (WBE) in COVID-19 Pandemics)

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12 pages, 2210 KiB

Open AccessArticle

Expanding the Pathogen Panel in Wastewater Epidemiology to Influenza and Norovirus

by Rudolf Markt, Fabian Stillebacher, Fabiana Nägele, Anna Kammerer, Nico Peer, Maria Payr, Christoph Scheffknecht, Silvina Dria, Simon Draxl-Weiskopf, Markus Mayr, Wolfgang Rauch, Norbert Kreuzinger, Lukas Rainer, Florian Bachner, Martin Zuba, Herwig Ostermann, Nina Lackner, Heribert Insam and Andreas Otto Wagner

Viruses 2023, 15(2), 263; https://doi.org/10.3390/v15020263 - 17 Jan 2023

Cited by 10 | Viewed by 3295

Abstract

Since the start of the 2019 pandemic, wastewater-based epidemiology (WBE) has proven to be a valuable tool for monitoring the prevalence of SARS-CoV-2. With methods and infrastructure being settled, it is time to expand the potential of this tool to a wider range [...] Read more.

Since the start of the 2019 pandemic, wastewater-based epidemiology (WBE) has proven to be a valuable tool for monitoring the prevalence of SARS-CoV-2. With methods and infrastructure being settled, it is time to expand the potential of this tool to a wider range of pathogens. We used over 500 archived RNA extracts from a WBE program for SARS-CoV-2 surveillance to monitor wastewater from 11 treatment plants for the presence of influenza and norovirus twice a week during the winter season of 2021/2022. Extracts were analyzed via digital PCR for influenza A, influenza B, norovirus GI, and norovirus GII. Resulting viral loads were normalized on the basis of NH₄-N. Our results show a good applicability of ammonia-normalization to compare different wastewater treatment plants. Extracts originally prepared for SARS-CoV-2 surveillance contained sufficient genomic material to monitor influenza A, norovirus GI, and GII. Viral loads of influenza A and norovirus GII in wastewater correlated with numbers from infected inpatients. Further, SARS-CoV-2 related non-pharmaceutical interventions affected subsequent changes in viral loads of both pathogens. In conclusion, the expansion of existing WBE surveillance programs to include additional pathogens besides SARS-CoV-2 offers a valuable and cost-efficient possibility to gain public health information. Full article

(This article belongs to the Special Issue Wastewater-Based Epidemiology (WBE) in COVID-19 Pandemics)

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19 pages, 2068 KiB

Open AccessArticle

New RT-PCR Assay for the Detection of Current and Future SARS-CoV-2 Variants

by Antonio Marchini, Mauro Petrillo, Amy Parrish, Gerhard Buttinger, Simona Tavazzi, Maddalena Querci, Fay Betsou, Goffe Elsinga, Gertjan Medema, Tamir Abdelrahman, Bernd Gawlik and Philippe Corbisier

Viruses 2023, 15(1), 206; https://doi.org/10.3390/v15010206 - 11 Jan 2023

Cited by 8 | Viewed by 3629

Abstract

Multiple lineages of SARS-CoV-2 have been identified featuring distinct sets of genetic changes that confer to the virus higher transmissibility and ability to evade existing immunity. The continuous evolution of SARS-CoV-2 may pose challenges for current treatment options and diagnostic tools. In this [...] Read more.

Multiple lineages of SARS-CoV-2 have been identified featuring distinct sets of genetic changes that confer to the virus higher transmissibility and ability to evade existing immunity. The continuous evolution of SARS-CoV-2 may pose challenges for current treatment options and diagnostic tools. In this study, we have first evaluated the performance of the 14 WHO-recommended real-time reverse transcription (RT)-PCR assays currently in use for the detection of SARS-CoV-2 and found that only one assay has reduced performance against Omicron. We then developed a new duplex real-time RT-PCR assay based on the amplification of two ultra-conserved elements present within the SARS-CoV-2 genome. The new duplex assay successfully detects all of the tested SARS-CoV-2 variants of concern (including Omicron sub-lineages BA.4 and BA.5) from both clinical and wastewater samples with high sensitivity and specificity. The assay also functions as a one-step droplet digital RT-PCR assay. This new assay, in addition to clinical testing, could be adopted in surveillance programs for the routine monitoring of SARS-CoV-2’s presence in a population in wastewater samples. Positive results with our assay in conjunction with negative results from an Omicron-specific assay may provide timely indication of the emergence of a novel SARS-CoV-2 variant in a certain community and thereby aid public health interventions. Full article

(This article belongs to the Special Issue Wastewater-Based Epidemiology (WBE) in COVID-19 Pandemics)

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10 pages, 1199 KiB

Open AccessArticle

Using Wastewater Surveillance to Compare COVID-19 Outbreaks during the Easter Holidays over a 2-Year Period in Cape Town, South Africa

by Nomfundo Mahlangeni, Renée Street, Suranie Horn, Angela Mathee, Noluxabiso Mangwana, Stephanie Dias, Jyoti Rajan Sharma, Pritika Ramharack, Johan Louw, Tarylee Reddy, Swastika Surujlal-Naicker, Sizwe Nkambule, Candice Webster, Mongezi Mdhluli, Glenda Gray, Christo Muller and Rabia Johnson

Viruses 2023, 15(1), 162; https://doi.org/10.3390/v15010162 - 05 Jan 2023

Cited by 1 | Viewed by 1909

Abstract

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown to be an important approach to determine early outbreaks of infections. Wastewater-based epidemiology (WBE) is regarded as a complementary tool for monitoring SARS-CoV-2 trends in communities. In this study, the changes [...] Read more.

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown to be an important approach to determine early outbreaks of infections. Wastewater-based epidemiology (WBE) is regarded as a complementary tool for monitoring SARS-CoV-2 trends in communities. In this study, the changes in the SARS-CoV-2 RNA levels in wastewater during Easter holidays in 2021 and 2022 in the City of Cape Town were monitored over nine weeks. Our findings showed a statistically significant difference in the SARS-CoV-2 RNA viral load between the study weeks over the Easter period in 2021 and 2022, except for study week 1 and 4. During the Easter week, 52% of the wastewater treatment plants moved from the lower (low viral RNA) category in 2021 to the higher (medium to very high viral RNA) categories in 2022. As a result, the median SARS-CoV-2 viral loads where higher during the Easter week in 2022 than Easter week in 2021 (p = 0.0052). Mixed-effects model showed an association between the SARS-CoV-2 RNA viral loads and Easter week over the Easter period in 2021 only (p < 0.01). The study highlights the potential of WBE to track outbreaks during the holiday period. Full article

(This article belongs to the Special Issue Wastewater-Based Epidemiology (WBE) in COVID-19 Pandemics)

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11 pages, 1017 KiB

Open AccessArticle

Wastewater-Based Epidemiology Mitigates COVID-19 Outbreaks at a Food Processing Facility near the Mexico-U.S. Border—November 2020–March 2022

by Gabriel K. Innes, Bradley W. Schmitz, Paul E. Brierley, Juan Guzman, Sarah M. Prasek, Martha Ruedas, Ana Sanchez, Subhadeep Bhattacharjee and Stephanie Slinski

Viruses 2022, 14(12), 2684; https://doi.org/10.3390/v14122684 - 30 Nov 2022

Cited by 2 | Viewed by 1550

Abstract

Background: Wastewater-based epidemiology (WBE) has the potential to inform activities to contain infectious disease outbreaks in both the public and private sectors. Although WBE for SARS-CoV-2 has shown promise over short time intervals, no other groups have evaluated how a public-private partnership could [...] Read more.

Background: Wastewater-based epidemiology (WBE) has the potential to inform activities to contain infectious disease outbreaks in both the public and private sectors. Although WBE for SARS-CoV-2 has shown promise over short time intervals, no other groups have evaluated how a public-private partnership could influence disease spread through public health action over time. The aim of this study was to characterize and assess the application of WBE to inform public health response and contain COVID-19 infections in a food processing facility. Methods: Over the period November 2020–March 2022, wastewater in an Arizona food processing facility was monitored for the presence of SARS-CoV-2 using Real-Time Quantitative PCR. Upon positive detection, partners discussed public health intervention strategies, including infection control reinforcement, antigen testing, and vaccination. Results: SARS-CoV-2 RNA was detected on 18 of 205 days in which wastewater was sampled and analyzed (8.8%): seven during Wild-type predominance and 11 during Omicron-variant predominance. All detections triggered the reinforcement of infection control guidelines. In five of the 18 events, active antigen testing identified asymptomatic workers. Conclusions: These steps heightened awareness to refine infection control protocols and averted possible transmission events during periods where detection occurred. This public-private partnership has potentially decreased human illness and economic loss during the COVID-19 pandemic. Full article

(This article belongs to the Special Issue Wastewater-Based Epidemiology (WBE) in COVID-19 Pandemics)

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16 pages, 22957 KiB

Open AccessArticle

SARS-CoV-2 Monitoring in Wastewater Reveals Novel Variants and Biomarkers of Infection

by Jenna McGowan, Monica Borucki, Hicham Omairi, Merina Varghese, Shahnaz Vellani, Sukanya Chakravarty, Shumin Fan, Srestha Chattopadhyay, Mashuk Siddiquee, James B. Thissen, Nisha Mulakken, Joseph Moon, Jeffrey Kimbrel, Amit K. Tiwari, Roger Travis Taylor, Dae-Wook Kang, Crystal Jaing, Ritu Chakravarti and Saurabh Chattopadhyay

Viruses 2022, 14(9), 2032; https://doi.org/10.3390/v14092032 - 13 Sep 2022

Cited by 3 | Viewed by 2664

Abstract

Wastewater-based epidemiology (WBE) is a popular tool for the early indication of community spread of infectious diseases. WBE emerged as an effective tool during the COVID-19 pandemic and has provided meaningful information to minimize the spread of infection. Here, we present a combination [...] Read more.

Wastewater-based epidemiology (WBE) is a popular tool for the early indication of community spread of infectious diseases. WBE emerged as an effective tool during the COVID-19 pandemic and has provided meaningful information to minimize the spread of infection. Here, we present a combination of analyses using the correlation of viral gene copies with clinical cases, sequencing of wastewater-derived RNA for the viral mutants, and correlative analyses of the viral gene copies with the bacterial biomarkers. Our study provides a unique platform for potentially using the WBE-derived results to predict the spread of COVID-19 and the emergence of new variants of concern. Further, we observed a strong correlation between the presence of SARS-CoV-2 and changes in the microbial community of wastewater, particularly the significant changes in bacterial genera belonging to the families of Lachnospiraceae and Actinomycetaceae. Our study shows that microbial biomarkers could be utilized as prediction tools for future infectious disease surveillance and outbreak responses. Overall, our comprehensive analyses of viral spread, variants, and novel bacterial biomarkers will add significantly to the growing body of literature on WBE and COVID-19. Full article

(This article belongs to the Special Issue Wastewater-Based Epidemiology (WBE) in COVID-19 Pandemics)

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9 pages, 2768 KiB

Open AccessBrief Report

The Rise and Fall of Omicron BA.1 Variant as Seen in Wastewater Supports Epidemiological Model Predictions

by Michal Liddor Naim, Yu Fu, Marilou Shagan, Itay Bar-Or, Robert Marks, Qun Sun, Rony Granek and Ariel Kushmaro

Viruses 2023, 15(9), 1862; https://doi.org/10.3390/v15091862 - 31 Aug 2023

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Abstract

The COVID-19 pandemic caused by the SARS-CoV-2 virus has inflicted significant mortality and morbidity worldwide. Continuous virus mutations have led to the emergence of new variants. The Omicron BA.1 sub-lineage prevailed as the dominant variant globally at the beginning of 2022 but was [...] Read more.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has inflicted significant mortality and morbidity worldwide. Continuous virus mutations have led to the emergence of new variants. The Omicron BA.1 sub-lineage prevailed as the dominant variant globally at the beginning of 2022 but was subsequently replaced by BA.2 in numerous countries. Wastewater-based epidemiology (WBE) offers an efficient tool for capturing viral shedding from infected individuals, enabling early detection of potential pandemic outbreaks without relying solely on community cooperation and clinical testing resources. This study integrated RT-qPCR assays for detecting general SARS-CoV-2 and its variants levels in wastewater into a modified triple susceptible-infected-recovered-susceptible (SIRS) model. The emergence of the Omicron BA.1 variant was observed, replacing the presence of its predecessor, the Delta variant. Comparative analysis between the wastewater data and the modified SIRS model effectively described the BA.1 and subsequent BA.2 waves, with the decline of the Delta variant aligning with its diminished presence below the detection threshold in wastewater. This study demonstrates the potential of WBE as a valuable tool for future pandemics. Furthermore, by analyzing the sensitivity of different variants to model parameters, we are able to deduce real-life values of cross-variant immunity probabilities, emphasizing the asymmetry in their strength. Full article

(This article belongs to the Special Issue Wastewater-Based Epidemiology (WBE) in COVID-19 Pandemics)

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