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1. Detection of SARS-CoV-2 RNA in commercial passenger aircraft and cruise ship wastewater: a surveillance tool for assessing the presence of COVID-19 infected travellers

   https://doi.org/10.1093/jtm/taaa116  

   Ahmed, Warish; Bertsch, Paul M; Angel, Nicola; Bibby, Kyle; Bivins, Aaron; Dierens, Leanne; Edson, Janette; Ehret, John; Gyawali, Pradip; Hamilton, Kerry A; et al (July 2020, Journal of Travel Medicine)

   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. 

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2. Wastewater surveillance for bacterial targets: current challenges and future goals

   https://doi.org/10.1128/aem.01428-23  

   Philo, Sarah E; De_León, Kara B; Noble, Rachel T; Zhou, Nicolette A; Alghafri, Rashed; Bar-Or, Itay; Darling, Amanda; D'Souza, Nishita; Hachimi, Oumaima; Kaya, Devrim; et al (January 2024, Applied and Environmental Microbiology)

   Elkins, Christopher A (Ed.)

   ABSTRACT Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods. IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs. 

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3. Building-level wastewater surveillance using tampon swabs and RT-LAMP for rapid SARS-CoV-2 RNA detection

   https://doi.org/10.1039/D1EW00496D  

   Bivins, Aaron; Lott, Megan; Shaffer, Marlee; Wu, Zhenyu; North, Devin; Lipp, Erin K.; Bibby, Kyle (December 2021, Environmental Science: Water Research & Technology)

   Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has demonstrated useful correlation with both coronavirus disease 2019 (COVID-19) cases and clinical testing positivity at the community level. Wastewater surveillance on college campuses has also demonstrated promising predictive capacity for the presence and absence of COVID-19 cases. However, to date, such monitoring has most frequently relied upon composite samplers and reverse transcription quantitative PCR (RT-qPCR) techniques, which limits the accessibility and scalability of wastewater surveillance, particularly in low-resource settings. In this study, we trialed the use of tampons as passive swabs for sample collection and reverse transcription loop-mediated isothermal amplification (RT-LAMP), which does not require sophisticated thermal cycling equipment, to detect SARS-CoV-2 RNA in wastewater. Results for the workflow were available within three hours of sample collection. The RT-LAMP assay is approximately 20 times less analytically sensitive than RT-droplet digital PCR. Nonetheless, during a building-level wastewater surveillance campaign concurrent with independent weekly clinical testing of all students, the method demonstrated a three-day positive predictive value (PPV) of 75% (excluding convalescent cases) and same-day negative predictive value (NPV) of 80% for incident COVID-19 cases. These predictive values are comparable to that reported by wastewater monitoring using RT-qPCR. These observations suggest that even with lower analytical sensitivity the tampon swab and RT-LAMP workflow offers a cost-effective and rapid approach that could be leveraged for scalable building-level wastewater surveillance for COVID-19 potentially even in low-resource settings. 

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4. Wastewater Infrastructure as a Public Health Tool: Agent-Based Modeling of Surveillance Strategies in a COVID-19 Context

   https://doi.org/10.3390/systems13121093  

   Xiang, Lin; Keck, James W; Gallimore, James; Sakhaei, Amirmohammad; Loh, Elizabeth; Berry, Scott M (December 2025, Systems)

   Wastewater-based epidemiology (WBE) played a vital role during the COVID-19 pandemic by providing early warnings of outbreaks through SARS-CoV-2 RNA detection in sewage. Many rural communities did not benefit from WBE because limited centralized sewer infrastructure challenged conventional WBE surveillance strategies. We present a multi-agent computer model simulating COVID-19 spread in a U.S. county with both sewered and non-sewered zones to assess the performance of WBE in this setting. We evaluate how the sewage service status of the first SARS-CoV-2 carrier, cross-zone community mobility, and WBE detection thresholds influence outbreak detection timing at the county’s wastewater treatment plant under basic reproduction numbers (R0) of 4 and 8. Our key findings include that (1) a detection threshold of 10 gc/mL can identify outbreaks up to six days earlier than a threshold of 50 gc/mL; (2) outbreaks originating in non-sewered zones are detected 1–2 days later, compared with outbreaks in sewered zones; and (3) cross-zone community mobility impacts detection timing only when outbreaks begin in non-sewered zones. Furthermore, once detected, disease prevalence can increase by five- to eleven-fold within the following week. These results underscore the importance of WBE sensitivity and tailored surveillance strategies in both sewered and non-sewered zones of a community. Strengthening WBE capabilities at local treatment facilities can improve early outbreak detection, thereby supporting timely public health interventions. 

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5. Detecting and supporting COVID-19 outbreak response in a small-scale nursing home through passive wastewater surveillance of SARS-CoV-2

   https://doi.org/10.1128/aem.00757-26  

   Jeon, Min Ki; Di, Doris_Yoong Wen; Steadmon, Maria; Remigio, Stella Maris; O'Brien, Pamela; Strange, Daniel P; Chanthasouvanh, Vila; Ornellas, Dayna; Desmond, Edward; Yan, Tao (July 2026, Applied and Environmental Microbiology)

   Elkins, Christopher A (Ed.)

   ABSTRACT Passive wastewater sampling has emerged as a cost-effective approach for pathogen monitoring, but its application in very small populations has not been well studied. This study reports the use of tampons as passive samplers in a small-scale nursing home of 24 residents to detect respiratory viruses, including SARS-CoV-2, influenza A, influenza B, and respiratory syncytial virus (RSV). Weekly wastewater surveillance by reverse transcription digital PCR (RT-dPCR) for a 1-year period resulted in a cluster of consecutive SARS-CoV-2 detections over 4 weeks. When wastewater SARS-CoV-2 concentrations reached higher levels in the third and fourth weeks, whole-genome sequencing achieved >98% genome coverage, identifying lineage LB.1.5, while lower-concentration samples produced only partial genomes. Clinical testing was initiated in the fourth week when clinical symptoms also emerged, and confirmed SARS-CoV-2 infection in 15 of 24 residents and staff. Sequencing of the clinical samples confirmed LB.1.5 in 11 cases, corroborating the wastewater findings. Influenza A subtypes H3N2 and H1N1 were additionally detected in wastewater by targeted enrichment sequencing, and RSV was detected once by RT-dPCR, neither of which was associated with confirmed clinical cases. These results demonstrate that wastewater surveillance can precede clinical symptoms for early detection, and passive sampling using tampons is a practical and sensitive strategy for outbreak surveillance in small-scale facilities. The study also highlights the need for clear response protocols that include rapid clinical testing and cohorting measures when wastewater viral concentrations exceed defined thresholds.IMPORTANCENursing homes are among the highest-risk settings for infectious disease outbreaks, yet early detection remains challenging due to the high prevalence of asymptomatic infections and limited resources for routine clinical surveillance. This study demonstrates that inexpensive tampon-based passive wastewater samplers can detect SARS-CoV-2 up to 3 weeks before clinical symptoms emerge in a nursing home with only 24 residents, which is the smallest population in which this method has been validated. Wastewater-derived genomes matched clinical sequencing quality, enabling identification of a novel SARS-CoV-2 lineage new to Hawai'i. These findings support passive wastewater surveillance as a practical early warning tool for small congregate care facilities, provided that clear institutional response protocols are in place. 

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