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Articles by Shannon M. Griffin in JoVE
Other articles by Shannon M. Griffin on PubMed
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Development and Evaluation of EPA Method 1615 for Detection of Enterovirus and Norovirus in Water
Applied and Environmental Microbiology.
Jan, 2013 |
Pubmed ID: 23087037 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.
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Application of Salivary Antibody Immunoassays for the Detection of Incident Infections with Norwalk Virus in a Group of Volunteers
Journal of Immunological Methods.
Sep, 2015 |
Pubmed ID: 25985985 Norovirus infection is the most common cause of acute gastroenteritis in developed countries. Developing an assay based on a non-invasive biomarker for detecting incident norovirus infections could improve disease surveillance and epidemiological investigations. This project involved analysis of IgA and IgG norovirus-specific antibody responses in saliva samples from a Norwalk virus (Genogroup I, genotype 1 norovirus) challenge study involving infected and symptomatic, and non-infected asymptomatic individuals. Saliva was collected at the challenge, and two weeks and 40 days post-challenge. Samples were analyzed using the Luminex fluorometric and Meso Scale Discovery (MSD) electrochemiluminescence immunoassays. Recombinant P domains of Norwalk virus capsid protein, as well as similar recombinant proteins of two genogroup II noroviruses (VA387 and VA207) were used as antigens. Immunoconversions were defined as >4-fold increase in antibody responses to the norovirus antigens. Various sample pre-treatment options, buffers, saliva dilution ratios, and data adjustment approaches to control for sample-to-sample variability in saliva composition were compared using the Luminex assay. The results suggest that adjusting responses to the norovirus antigens for responses to the protein purification tag, glutathione-S-transferase (GST), significantly improved the odds of producing a correct immunoconversion test result. IgG-based tests were more accurate compared to IgA-based tests. At optimal conditions, both Luminex and MSD assays for Norwalk-specific IgG antibodies correctly identified all infected and non-infected individuals. There was no evidence of cross-reactivity of anti-Norwalk virus antibodies with genogroup II noroviruses. These results suggest that salivary antibody responses can be used for the detection of incident infections with Norwalk virus in prospective surveys.
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Statistical Approaches to Developing a Multiplex Immunoassay for Determining Human Exposure to Environmental Pathogens
Journal of Immunological Methods.
Oct, 2015 |
Pubmed ID: 26070441 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.
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