Microbial detection in and surveillance of various water sources: methodology development to data analysis
Date
2021
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Environmental water matrices are microbially, chemically, and physically complex with constantly changing physicochemical parameters and microbial populations. This situation creates a unique set of challenges in monitoring the sources for pathogen contamination, particularly viral pathogens. Enteric viruses, including human noroviruses and hepatitis A virus, have been of major concern in managing the food supply and maintaining public health. Methods for the recovery, detection, and quantification of enteric viruses from drinking water were previously established by the EPA and treatments are required to maintain a 3-log reduction of enteric viruses under the National Primary Drinking Water Regulations. ☐ However, when evaluating surface, reclaimed, and wastewater for viral contamination there are no standardized methods for recovery, detection, or quantification. These types of water, when compared to groundwater or typical drinking water sources, are highly variable and heterogenous. Each water source has a unique set of physicochemical properties and microbial communities that fluctuate over time. The development of a method that is ideal for all sources throughout the world during all times is unlikely. Viruses are also incredibly diverse morphologically ranging in size from 20 to 970 nm, occurring in icosahedral, rod, and oblong shapes, and containing a variety of surface structures such as spikes. Enteric viruses are typically icosahedral, non-enveloped, ranging between 20 and 90 nm in diameter. Some enteric viruses, like adenovirus, contain spikes similar to the devastating respiratory virus SARS-CoV-2 that aid in infection of the human host. Unlike enteric viruses, severe acute respiratory coronavirus 2 (SARS-CoV-2), is enveloped with an outer phospholipid membrane. This membrane is more easily damaged by disinfectants than the interior protein-based nucleocapsid. However, when attempting to recover SARS-CoV-2, the remaining outer membrane may hinder recovery through traditional enteric virus recovery methods. ☐ When utilizing wastewater-based epidemiology (WBE) methods for tracking viral infection within a population, the factors influencing recovery and detection of those viruses must be scrutinized to ensure sufficient and reliable data collection. Adaptation of enteric virus methods can accelerate the gain in knowledge needed to establish methods for recovery of SARS-CoV-2 from wastewater sources and perform surveillance of COVID-19. The capital and time investments placed towards battling the COVID-19 pandemic through research of SARS-CoV-2 presence in wastewater has developed an infrastructure which can serve to aid in future enteric virus research. ☐ An investigation of an effective recovery method for enteric viruses from non-traditional irrigation water sources, surface and reclaimed, was performed. Volumes of water (ten to forty liters) were collected, and viruses recovered using positively charged filtration. The viruses were eluted, and viruses concentrated using centrifugal ultra-filtration. Viruses were detected after extraction of nucleic acids using RT-qPCR. Primers and probes for each virus were selected based on their previous documented use of successful detection from environmental water sources. Norovirus GI and GII, hepatitis A virus, and Aichi virus presence were compared to the fecal indicator virus pepper mild mottle virus (PMMoV), atmospheric and physicochemical data including precipitation, temperature, cloud cover, turbidity, salinity, and pH. ☐ Recovery of viruses after recovery and treatment methodologies are employed is variable across method and virus type. Treatments used at wastewater treatment plants (WWTP) were generally designed to reduce the bacterial populations or cause inactivation of the pathogen through physical, biological, or chemical processes. These processes may include filtration, aeration, chlorination, and ultra-violet (UV) light disinfection. Similarly, treatments employed for agricultural water remediation can include physical, chemical, and biological methods, however filtration is widely used due to the ease of implementation and maintenance. Zero-valent iron can be combined with traditional sand filters to improve the efficacy of removing biological contaminants by causing a combination of physical and chemical reactions. Employing treatments to water sources can reduce the viral populations, though complications may arise in attempts to use those viruses as for indicators for surveillance purposes. ☐ Viruses are affected differently by treatments and recovery methods due to differences in structure. Therefore, when using viruses as indicators of fecal contamination, process controls, or surrogates for other pathogens or organisms, effort should be made to determine the disparity in recovery among the targets. Untreated or unprocessed water samples, from environmental or waste origins, must be evaluated and compared to the treated or processed samples. When utilizing methods typically employed for enteric virus recovery for detection of other organisms, namely SARS-CoV-2, assessment of the process efficacy must be performed. ☐ A centrifugal ultrafiltration method used for detection of enteric viruses in non-traditional irrigation water sources was adapted for recovery of SARS-CoV-2 from wastewater. Wastewater was composited over twenty-four-hour-periods at locations across New Castle County, Delaware, United States during the COVID-19 pandemic. Samples were filtered prior to concentration by centrifugal ultrafiltration using 10-kDa centrifugal ultrafiltration units. Viral concentrate was collected, nucleic acids were extracted, and detection was performed via real-time RT-qPCR molecular assay. SARS-CoV-2 was detected using primers and probes designed to target the N1 and N2 regions of the viral genome per the current CDC recommendations. Pepper mild mottle virus (PMMoV) and Tulane virus (TV) were detected using primers and probes documented as successfully detecting the targets from environmental water sources. Recovery of the viral targets was compared as alterations were made to the recovery method. Thermal processing and prolonged storage prior to wastewater filtration and filter units of varying pore size and membrane materials were evaluated. Variation in recovery across samples from different sources was also evaluated. The data collected provided insight into the adjustments needed during data analysis and additional data needed to obtain a representative data set. ☐ SARS-CoV-2 levels in wastewater, with the appropriate modifications, can be used alongside COVID-19 clinical case information to track the spread of the pandemic throughout a population. Wastewater was collected weekly and results compared to the new daily COVID-19 cases reported in New Castle County and subsets within the area. Increases in SARS-CoV-2 concentrations preceded reported rising of COVID-19 cases by thirteen to twenty-seven days for wastewater treatment plant sources. Additional investigation and possible alterations are needed to apply the statistical techniques to wastewater sources other than treatment plants, such as pump stations. ☐ The inclusion of negative results, preliminary findings, and acknowledgment of obstacles encountered has broadened the knowledge base surrounding viruses and their recovery from water sources. Implementation of new technologies in the laboratory and expectations for data sharing have created a platform for both current and future research in this area. Now that the foundation has been laid during the COVID-19 pandemic, it can be used to accelerate wastewater-based epidemiology for not only SARS-CoV-2 but all viral pathogens of concern.
Description
Keywords
Epidemiology, Food safety, Viruses, Wastewater, Water