Molecular Methods for Pathogenic Bacteria Detection and Recent Advances in Wastewater Analysis

With increasing concerns about public health and the development of molecular techniques, new detection tools and the combination of existing approaches have increased the abilities of pathogenic bacteria monitoring by exploring new biomarkers, increasing the sensitivity and accuracy of detection, quantification, and analyzing various genes such as functional genes and antimicrobial resistance genes (ARG). Molecular methods are gradually emerging as the most popular detection approach for pathogens, in addition to the conventional culture-based plate enumeration methods. The analysis of pathogens in wastewater and the back-estimation of infections in the community, also known as wastewater-based epidemiology (WBE), is an emerging methodology and has a great potential to supplement current surveillance systems for the monitoring of infectious diseases and the early warning of outbreaks. However, as a complex matrix, wastewater largely challenges the analytical performance of molecular methods. This review synthesized the literature of typical pathogenic bacteria in wastewater, types of biomarkers, molecular methods for bacterial analysis, and their recent advances in wastewater analysis. The advantages and limitation of these molecular methods were evaluated, and their prospects in WBE were discussed to provide insight for future development.

Reliability of Wastewater Analysis for Monitoring COVID-19 Incidence Revealed by a Long-Term Follow-Up Study

Wastewater-based epidemiology has been used for monitoring human activities and waterborne pathogens. Although wastewaters can also be used for tracking SARS-CoV-2 at the population level, the reliability of this approach remains to be established, especially for early warning of outbreaks. We collected 377 samples from different treatment plants processing wastewaters of >1 million inhabitants in Valencia, Spain, between April 2020 and March 2021. Samples were cleaned, concentrated, and subjected to RT-qPCR to determine SARS-CoV-2 concentrations. These data were compared with cumulative disease notification rates over 7 and 14 day periods. We amplified SARS-CoV-2 RNA in 75% of the RT-qPCRs, with an estimated detection limit of 100 viral genome copies per liter (gc/L). SARS-CoV-2 RNA concentration correlated strongly with disease notification rates over 14-day periods (Pearson r = 0.962, P < 0.001). A concentration >1000 gc/L showed >95% sensitivity and specificity as an indicator of more than 25 new cases per 100,000 inhabitants. Albeit with slightly higher uncertainty, these figures were reproduced using a 7-day period. Time series were similar for wastewaters data and declared cases, but wastewater RNA concentrations exhibited transient peaks that were not observed in declared cases and preceded major outbreaks by several weeks. In conclusion, wastewater analysis provides a reliable tool for monitoring COVID-19, particularly at low incidence values, and is not biased by asymptomatic cases. Moreover, this approach might reveal previously unrecognized features of COVID-19 transmission.

CoWWAn: Model-based assessment of COVID-19 epidemic dynamics by wastewater analysis

We present COVID-19 Wastewater Analyser (CoWWAn) to reconstruct the epidemic dynamics from SARS-CoV-2 viral load in wastewater. As demonstrated for various regions and sampling protocols, this mechanistic model-based approach quantifies the case numbers, provides epidemic indicators and accurately infers future epidemic trends. In situations of reduced testing capacity, analysing wastewater data with CoWWAn is a robust and cost-effective alternative for real-time surveillance of local COVID-19 dynamics.

Assessment of Environmental Pollution and Human Exposure to Pesticides by Wastewater Analysis in a Seven-Year Study in Athens, Greece

Pesticides have been used in large amounts around the world for decades and are responsible for environmental pollution and various adverse effects on human health. Analysis of untreated wastewater can deliver useful information on pesticides’ use in a particular area and allow the assessment of human exposure to certain substances. A wide-scope screening method, based on liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry, was applied, using both target and suspect screening methodologies. Daily composite influent wastewater samples were collected for seven or eight consecutive days in Athens between 2014 and 2020 and analyzed for 756 pesticides, their environmental transformation products and their human metabolites. Forty pesticides were quantified at mean concentrations up to 4.9 µg/L (tralkoxydim). The most abundant class was fungicides followed by herbicides, insect repellents, insecticides and plant growth regulators. In addition, pesticide transformation products and/or metabolites were detected with high frequency, indicating that research should be focused on them. Human exposure was evaluated using the wastewater-based epidemiology (WBE) approach and 3-ethyl-carbamoyl benzoic acid and cis-1,2,3,6-tetrahydrophthalimide were proposed as potential WBE biomarkers. Wastewater analysis revealed the presence of unapproved pesticides and indicated that there is an urgent need to include more transformation products in target databases.