Wastewater-based epidemiology (WBE) is an environmental approach to monitor community health through the analysis of sewage. The COVID-19 pandemic catalyzed scientists and public health professionals to revisit WBE as a tool to optimize resource allocation to mitigate disease spread and prevent outbreaks. Some studies have highlighted the value of WBE programs that coordinate with public health professionals; however, the details necessary for implementation are not well-characterized. To respond to this knowledge gap, this article documents the framework of a successful WBE program in Arizona, titled Wastewater Analysis for Tactical Epidemiological Response Systems (WATERS), detailing the developed structure and methods of communication that enabled public health preparedness and response actions. This communication illustrates how program operations were employed to reduce outbreak severity. The structure outlined here is customizable and may guide other programs in the implementation of WBE as a public health tool.

Digital polymerase chain reaction (dPCR) has emerged as a groundbreaking technology in molecular biology and diagnostics, offering exceptional precision and sensitivity in nucleic acid detection and quantification. This review highlights the core principles and transformative potential of dPCR, particularly in infectious disease diagnostics and environmental surveillance. Emphasizing its evolution from traditional PCR, dPCR provides accurate absolute quantification of target nucleic acids through advanced partitioning techniques. The review addresses the significant impact of dPCR in sepsis diagnosis and management, showcasing its superior sensitivity and specificity in early pathogen detection and identification of drug-resistant genes. Despite its advantages, challenges such as optimization of experimental conditions, standardization of data analysis workflows, and high costs are discussed. Furthermore, we compare various commercially available dPCR platforms, detailing their features and applications in clinical and research settings. Additionally, the review explores dPCR\'s role in water microbiology, particularly in wastewater surveillance and monitoring of waterborne pathogens, underscoring its importance in public health protection. In conclusion, future prospects of dPCR, including methodological optimization, integration with innovative technologies, and expansion into new sectors like metagenomics, are explored.

Escherichia coli, both commensal and pathogenic, can colonize plants and persist in various environments. It indicates fecal contamination in water and food and serves as a marker of antimicrobial resistance. In this context, 61 extended-spectrum β-lactamase (ESBL)-producing E. coli from irrigation water and fresh produce from previous studies were characterized using whole genome sequencing (Illumina MiSeq). The Center for Genomic Epidemiology and Galaxy platforms were used to determine antimicrobial resistance genes, virulence genes, plasmid typing, mobile genetic elements, multilocus sequence typing (MLST), and pathogenicity prediction. In total, 19 known MLST groups were detected among the 61 isolates. Phylogroup B1 (ST58) and Phylogroup E (ST9583) were the most common sequence types. The six ST10 (serotype O101:H9) isolates carried the most resistance genes, spanning eight antibiotic classes. Overall, 95.1% of the isolates carried resistance genes from three or more classes. The blaCTX-M-1, blaCTX-M-14, and blaCTX-M-15 ESBL genes were associated with mobile genetic elements, and all of the E. coli isolates showed a >90% predicted probability of being a human pathogen. This study provided novel genomic information on environmental multidrug-resistant ESBL-producing E. coli from fresh produce and irrigation water, highlighting the environment as a reservoir for multidrug-resistant strains and emphasizing the need for ongoing pathogen surveillance within a One Health context.

In regions without adequate centralized wastewater treatment plants, sample collection from rivers and sewers can be an alternative sampling strategy for wastewater surveillance. This study aimed to assess the feasibility of alternative sampling strategies by testing samples collected from rivers (n = 246) and sewers (n = 244) in the Kathmandu Valley between March 2021 and February 2022. All samples were concentrated using the skimmed-milk flocculation method and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was quantified using the nucleocapsid (N) and envelope (E) genes qPCR assays. Of the total, 75 % (371/490) of the samples tested positive using at least one qPCR assay, with concentrations ranging from 3.0 to 8.3 log10 gene copies/L. No significant correlation between concentrations of SARS-CoV-2 from both sewers and river with the number of confirmed coronavirus disease 2019 (COVID-19) cases in the Kathmandu valley was observed (p > 0.05). Despite the high concentration of SARS-CoV-2 in rivers and sewers, we hypothesize this finding to be a result of inaccurate number of clinical cases possibly due to inadequate clinical testing. This longitudinal study further supports the statement to consider sampling strategies from sewers and rivers for WBS in Nepal and other low and middle-income countries.

Precise and rapid methods are needed to improve monitoring approaches of L. pneumophila (Lp) in cooling towers (CTs) to allow timely operational adjustments and prevent outbreaks. The performance of liquid culture (ASTM D8429-21) and an online qPCR device were first compared to conventional filter plate culture (ISO 11731-2017), qPCR and semi-automated qPCR at three spiked concentrations of Lp (serogroup 1) validated by flow cytometry (total/viable cell count). The most accurate was qPCR, followed by liquid culture, online and semi-automated qPCR, and lastly, by a significant margin, filter plate culture. An industrial CT system was monitored using liquid and direct plate culture by the facility, qPCR and online qPCR. Direct plate and liquid culture results agreed at regulatory sampling point, supporting the use of the faster liquid culture for monitoring culturable Lp. During initial operation, qPCR and online qPCR results were within one log of culture at the primary pump before deviating after first cleaning. Other points revealed high spatial variability of Lp. The secondary pumps and chiller had the most positivity and highest concentrations by both qPCR and liquid culture compared to the basin and infeed tank. Altogether, this suggests that results from monthly compliance sampling at a single location with plate culture are not representative of Lp risks in this CT due to the high temporal and spatial variability. The primary pump, rather than the CT basin, should be designated for sampling, as it is representative of the health risk. An annual multi point survey of the system should be conducted to identify and target Lp hot spots. Generally, a combination of liquid culture for compliance and frequent qPCR for process control provides a more agile and robust monitoring scheme than plate culture alone, enabling early treatment adjustments, due to lower limit of detection (LOD) and turnover time.

After the first phase of the COVID-19 pandemic in Europe, a new highly pathogenic variant of echovirus 11 (E11) was detected. The aim of this study was to analyze the genetic diversity of Polish E11 environmental and clinical strains circulating between 2017 and 2023 as well as compare them with E11 strains isolated from severe neonatal sepsis cases reported in Europe between 2022 and 2023. Additionally, the study explores the effectiveness of environmental monitoring in tracking the spread of new variants. For this purpose, the complete sequences of the VP1 capsid protein gene were determined for 266 E11 strains isolated in Poland from 2017 to 2023, and phylogenetic analysis was performed. In the years 2017-2023, a significant increase in the detection of E11 strains was observed in both environmental and clinical samples in Poland. The Polish E11 strains represented three different genotypes, C3, D5 and E, and were characterized by a high diversity. In Poland, the intensive circulation of the new variant E11, responsible for severe neonatal infections with a high mortality in Europe, was detected in the years 2022-2023. This investigation demonstrates the important role of environmental surveillance in the tracking of enteroviruses circulation, especially in settings with limited clinical surveillance.

Wastewater monitoring provides essential information about water quality and the degree of contamination. Monitoring these waters helps identify and manage risks to public health, prevent the spread of disease, and protect the environment. Standardizing the appropriate and most accurate methods for the isolation and identification of viruses in wastewater is necessary. This review aims to present the major classes of viruses in wastewater, as well as the methods of concentration, isolation, and identification of viruses in wastewater to assess public health risks and implement corrective measures to prevent and control viral infections. Last but not least, we propose to evaluate the current strategies in wastewater treatment as well as new alternative methods of water disinfection.

Wastewater is a complex, but an ideal, matrix for disease monitoring and surveillance as it represents the entire load of enteric pathogens from a local catchment area. It captures both clinical and community disease burdens. Global interest in wastewater surveillance has been growing rapidly for infectious diseases monitoring and for providing an early warning of potential outbreaks. Although molecular detection methods show high sensitivity and specificity in pathogen monitoring from wastewater, they are strongly limited by challenges, including expensive laboratory settings and prolonged sample processing and analysis. Alternatively, biosensors exhibit a wide range of practical utility in real-time monitoring of biological and chemical markers. However, field deployment of biosensors is primarily challenged by prolonged sample processing and pathogen concentration steps due to complex wastewater matrices. This review summarizes the role of wastewater surveillance and provides an overview of infectious viral and bacterial pathogens with cutting-edge technologies for their detection. It emphasizes the practical utility of biosensors in pathogen monitoring and the major bottlenecks for wastewater surveillance of pathogens, and overcoming approaches to field deployment of biosensors for real-time pathogen detection. Furthermore, the promising potential of novel machine learning algorithms to resolve uncertainties in wastewater data is discussed.

Enteric infections due to viral pathogens are a major public health concern. Detecting the risk areas requires a strong surveillance system for pathogenic viruses in sources such as wastewater. Towards building an environmental surveillance system in Zambia, we aimed to identify group A rotavirus (RVA) and human adenovirus (HAdV) in wastewater. Convenient sampling was conducted at four study sites every Tuesday for five consecutive weeks. The research team focused on three different methods of viral concentration to determine the suitability in terms of cost and applicability for a regular surveillance system: the bag-mediated filtration system (BMFS), polyethylene glycol-based (PEG) precipitation, and skimmed milk (SM) flocculation. We screened 20 wastewater samples for HAdV and RVA using quantitative polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR). Of the 20 samples tested using qPCR, 18/20 (90%) tested positive for HAdV and 14/20 (70%) tested positive for RVA. For the genetic sequencing, qPCR positives were subjected to cPCR, of which 12 positives were successfully amplified. The human adenovirus was identified with a nucleotide identity range of 98.48% to 99.53% compared with the reference genome from GenBank. The BMFS and SM flocculation were the most consistent viral concentration methods for HAdV and RVA, respectively. A statistical analysis of the positives showed that viral positivity differed by site (p < 0.001). SM and PEG may be the most appropriate options in resource-limited settings such as Zambia due to the lower costs associated with these concentration methods. The demonstration of HAdV and RVA detection in wastewater suggests the presence of the pathogens in the communities under study and the need to establish a routine wastewater surveillance system for the identification of pathogens.

BACKGROUND: Rotavirus-induced viral gastroenteritis outbreaks result in over two million hospitalizations globally yearly. Wastewater-based epidemiology (WBE) has emerged as a crucial tool for detecting and monitoring viral outbreaks. The adoption of WBE has been instrumental in the early detection and surveillance of such viral outbreaks, providing a non-invasive method to assess public health.
OBJECTIVE: This study aims to utilize droplet digital polymerase chain reaction (ddPCR) technology to detect and quantify Rotavirus in wastewater samples collected from the Bhopal region of India, thereby contributing to the understanding and management of viral gastroenteritis outbreaks through environmental surveillance.
METHODS: In this study, we used ddPCR to detect and quantify Rotavirus in wastewater samples collected from the Bhopal region of India. We monitored its viral presence in municipal sewage treatment plants bi-weekly using an advanced ddPCR assay. Targeting the rotavirus non-structural protein 3 (NSP-3) region with custom primers and TaqMan probes, we detected virus concentration employing polyethylene glycol (PEG). Following RNA isolation, complementary DNA (cDNA) synthesis, and ddPCR analysis, our novel method eliminated standard curve dependence, propelling virus research and treatment forward.
RESULTS: Out of the 42 samples collected, a 16.60% positivity rate was observed, indicating a moderate presence of Rotavirus in Bhopal. The wastewater treatment plants (WWTP) attached to a hospital exhibited a 42.85% positivity rate, indicating the need for targeted monitoring. Leveraging ddPCR, precise quantification of rotavirus concentrations (ranging from 0.75 to 28.9 copies/µL) facilitated understanding and supported effective remediation.
CONCLUSIONS:  This study emphasizes the importance of vigilant wastewater surveillance, especially in WWTPs with higher rotavirus prevalence. The significance of ddPCR in comparison to conventional and real-time PCR lies in its superior sensitivity and specificity in detecting and quantifying positive samples. Furthermore, it can identify positive samples even in the smallest quantities without the need for a standard curve to evaluate. This makes ddPCR a valuable tool for accurate and precise detection and quantification of samples.