Ann C. Grimm

Development and Evaluation of EPA Method 1615 for Detection of Enterovirus and Norovirus in Water

ABSTRACT The U.S. EPA developed a sample concentration and preparation assay in conjunction with the total culturable virus assay for concentrating and measuring culturable viruses in source and drinking waters as part of the Information Collection Rule (ICR) promulgated in 1996. In an effort to improve upon this method, the U.S. EPA recently developed Method 1615: Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Method 1615 uses a culturable virus assay with reduced equipment and labor costs compared to the costs associated with the ICR virus method and introduces a new molecular assay for the detection of enteroviruses and noroviruses by reverse transcription-quantitative PCR. In this study, we describe the optimization of several new components of the molecular assay and examine virus recovery from ground, reagent-grade, and surface water samples seeded with poliovirus type 3 and murine norovirus. For the culturable virus and molecular assays, mean poliovirus recovery using the complete method was 58% and 20% in groundwater samples, 122% and 39% using low-titer spikes in reagent-grade water, 42% and 48% using high-titer spikes in reagent-grade water, and 11% and 10% in surface water with high turbidity, respectively. Murine norovirus recovery by the molecular assay was 30% in groundwater samples, less than 8% in both low- and high-titer spikes in reagent-grade water, and 6% in surface water with high turbidity. This study demonstrates the effectiveness of Method 1615 for use with groundwater samples and highlights the need for further research into its effectiveness with surface water.

Evaluation of the celite secondary concentration procedure and an alternate elution buffer for the recovery of enteric adenoviruses 40 and 41.

The effective recovery of adenovirus from water is a critical first step in developing a virus occurrence method able to provide accurate data for risk assessments and other applications. During virus concentration, electropositive filters are typically eluted with beef extract, undergo secondary concentration using either an organic flocculation or polyethylene glycol (PEG) precipitation technique and are ultimately resuspended in sodium phosphate buffer. In this study, an alternative secondary concentration procedure using celite was optimized by identifying the optimal celite and elution buffer to use. Two elution buffers, sodium phosphate and 1× PBS, were evaluated for their impact on real-time PCR. Sodium phosphate produced high levels of PCR inhibition compared to 1× PBS and so 1× PBS was used in subsequent experiments. The two secondary concentration techniques that were tested with adenovirus 40 and 41 gave recoveries of 69% and 65% for the optimized celite method and 75% and 109% for the organic flocculation method, respectively. Fine particle, calcinated celites in combination with 1× PBS elution buffer were shown to be effective at concentrating adenovirus 40 and 41 during secondary concentration and their subsequent detection using PCR. Heat extraction efficiencies were compared to samples processed using a DNA extraction kit to address possible virus aggregation issues. Samples processed through DNA extraction were found to produce realistic adenovirus recoveries compared to exaggerated recoveries using heat extraction.

Development of a molecular method to identify hepatitis E virus in water

Hepatitis E virus (HEV) causes an infectious form of hepatitis associated with contaminated water. By analyzing the sequence of several HEV isolates, a reverse transcription-polymerase chain reaction method was developed and optimized that should be able to identify all of the known HEV strains. When tested under laboratory conditions, this method was able to detect low levels of five diverse HEV variants. In addition, internal controls were constructed so that any PCR inhibition could be detected. Finally, virus-spiked environmental water samples were analyzed successfully with these assays.

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

Statistical approaches to developing a multiplex immunoassay for determining human exposure to environmental pathogens.

There are numerous pathogens that can be transmitted through water. Identifying and understanding the routes and magnitude of exposure or infection to these microbial contaminants are critical to assessing and mitigating risk. Conventional approaches of studying immunological responses to exposure or infection such as Enzyme-Linked Immunosorbent Assays (ELISAs) and other monoplex antibody-based immunoassays can be very costly, laborious, and consume large quantities of patient sample. A major limitation of these approaches is that they can only be used to measure one analyte at a time. Multiplex immunoassays provide the ability to study multiple pathogens simultaneously in microliter volumes of samples. However, there are several challenges that must be addressed when developing these multiplex immunoassays such as selection of specific antigens and antibodies, cross-reactivity, calibration, protein-reagent interferences, and the need for rigorous optimization of protein concentrations. In this study, a Design of Experiments (DOE) approach was used to optimize reagent concentrations for coupling selected antigens to Luminex™ xMAP microspheres for use in an indirect capture, multiplex immunoassay to detect human exposure or infection from pathogens that are potentially transmitted through water. Results from Helicobacter pylori, Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella typhimurium singleplexes were used to determine the mean concentrations that would be applied to the multiplex assay. Cut-offs to differentiate between exposed and non-exposed individuals were determined using finite mixed modeling (FMM). The statistical approaches developed facilitated the detection of Immunoglobulin G (IgG) antibodies to H. pylori, C. jejuni, Toxoplasma gondii, hepatitis A virus, rotavirus and noroviruses (VA387 and Norwalk strains) in fifty-four diagnostically characterized plasma samples. Of the characterized samples, the detection rate was 87.5% for H. pylori, and 100% for T. gondii assays and 89% for HAV. Further, the optimized multiplex assay revealed exposure/infection to several other environmental pathogens previously uncharacterized in the samples.

A Spike Cocktail Approach to Improve Microbial Performance Monitoring for Water Reuse.

  Water reuse, via either centralized treatment of traditional wastewater or decentralized treatment and on-site reuse, is becoming an increasingly important element of sustainable water management. Despite advances in waterborne pathogen detection methods, low and highly variable pathogen levels limit their utility for routine evaluation of health risks in water reuse systems. Therefore, there is a need to improve our understanding of the linkage between pathogens and more readily measured process indicators during treatment. This paper describes an approach for constructing spiking experiments to relate the behavior of viral, bacterial, and protozoan pathogens with relevant process indicators. General issues are reviewed, and the spiking protocol is applied as a case study example to improve microbial performance monitoring and health risk evaluation in a water reuse system. This approach provides a foundation for the development of novel approaches to improve real or near-real time performance monitoring of water recycling systems.

Immunoprevalence to Six Waterborne Pathogens in Beachgoers at Boquerón Beach, Puerto Rico: Application of a Microsphere-Based Salivary Antibody Multiplex Immunoassay

Waterborne infectious diseases are a major public health concern worldwide. Few methods have been established that are capable of measuring human exposure to multiple waterborne pathogens simultaneously using non-invasive samples such as saliva. Most current methods measure exposure to only one pathogen at a time, require large volumes of individual samples collected using invasive procedures, and are very labor intensive. In this article, we applied a multiplex bead-based immunoassay capable of measuring IgG antibody responses to six waterborne pathogens simultaneously in human saliva to estimate immunoprevalence in beachgoers at Boquerón Beach, Puerto Rico. Further, we present approaches for determining cutoff points to assess immunoprevalence to the pathogens in the assay. For the six pathogens studied, our results show that IgG antibodies against antigens from noroviruses GI.I and GII.4 were more prevalent (60 and 51.6%, respectively) than Helicobacter pylori (21.4%), hepatitis A virus (20.2%), Campylobacter jejuni (8.7%), and Toxoplasma gondii (8%) in the saliva of the study participants. The salivary antibody multiplex immunoassay can be used to examine immunoprevalence of specific pathogens in human populations.

The influence of incubation time on adenovirus quantitation in A549 cells by most probable number

Cell culture based assays used to detect waterborne viruses typically call for incubating the sample for at least two weeks in order to ensure that all the culturable virus present is detected. Historically, this estimate was based, at least in part, on the length of time used for detecting poliovirus. In this study, we have examined A549 cells infected with human adenovirus type 2, and have found that a three week incubation of virus infected cells results in a higher number of detected viruses by quantal assay than what is seen after two weeks of incubation, with an average 955% increase in Most Probable Number (MPN) from 2 weeks to 3 weeks. This increase suggests that the extended incubation time is essential for accurately estimating viral titer, particularly for slow-growing viruses, UV treated samples, or samples with low titers of virus. In addition, we found that for some UV-treated samples, there was no detectable MPN at 2 weeks, but after 3 weeks, MPN values were obtained. For UV-treated samples, the average increase in MPN from 2 weeks to 3 weeks was 1401%, while untreated samples averaged a change in MPN of 674%, leading us to believe that the UV-damaged viral DNA may be able to be repaired such that viral replication then occurs.

Optimization and evaluation of a method to detect adenoviruses in river water.

Adenoviruses are often implicated in recreational water disease outbreaks but existing methods for their detection perform poorly within these matrices. In this study, small volume (100mL) concentration was used to identify processes that promoted recovery of adenovirus from river water. Several alternative secondary concentration techniques were investigated and compared to the baseline method consisting of primary concentration via filtration, followed by celite mediated secondary concentration. The alternative secondary concentrations included multiple filter elutions, soaking the filter for 15 min prior to elution and concentration using pre-treated celite (river water, 1.5% and 3% milk) instead of a filter. Modifications of the viral nucleic acid extraction technique were also evaluated. Concentration using pre-treated celite and a modified extraction technique (10 min boil and a 1h ProK incubation at 37 °C) recovered significantly higher levels of adenovirus (P=0.001) than other methods tested. This optimized method increased recovery of spiked adenovirus (57 ± 27%) compared to baseline method performance (4 ± 3%) indicating that use of pre-treated celite as opposed to filtration significantly improves recovery. Application of the optimized concentration method to larger volume (1L) of river water resulted in similar recoveries (42 ± 19%) underlying the utility of this method to detect adenovirus from environmental samples.