Dissertation
Optimization of sampling and quantification methods for aerosolized norovirus
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2019
DOI: 10.17077/etd.f70k-5ykh
Abstract
<p>Norovirus is the most common pathogen to cause acute gastroenteritis in the world. Symptoms of acute gastroenteritis include vomiting and/or diarrhea, along with fever, abdominal pain, and malaise. Annually, norovirus causes 685 million cases of acute gastroenteritis and 200,000 deaths, worldwide. Among the 685 million cases occurring every year, 19-21 million occur in the United States. Norovirus can spread through direct or indirect contact (e.g., contaminated food or water). In addition, recent evidence has suggested that norovirus can also be spread via aerosolization. However, no study has determined an indoor generation source for aerosolized norovirus. Therefore, the goals of this study were to optimize sampling and quantification methods for the collection of aerosolized norovirus. Upon optimization, the last was to investigate a potential indoor generation source (i.e., toilet flushing) of aerosolized norovirus. To achieve this goal we devised three studies.</p>
<p>In the first study, we optimized a sampling method for the collection of aerosolized norovirus using murine norovirus (MNV) as a surrogate. Optimization of the sampling method was performed using two bioaerosol samplers (SKC BioSampler and the National Institute for Occupational Safety and Health [NIOSH] Bioaerosol Cyclone Sampler 251) and two sampling media (Hanks Balanced Salt Solution [HBSS] and Phosphate Buffered Saline [PBS]). Murine norovirus was aerosolized in a bioaerosol chamber and later collected using the optimized sampler/media combination. After collection, viral RNA was extracted from MNV collected samples and quantified using quantitative polymerase chain reaction (qPCR). Intact capsids of MNV were assessed using propidium monoazide dye in combination with qPCR and confirmed with transmission electron microscopy. There were a total of 10 trials conducted, with each trial lasting for 30 minutes. The SKC BioSampler collected a significantly higher concentration of MNV than the NIOSH-251 sampler did (p-value < 0.0001). However, there were no significant differences in the relative percent of MNV that remained viable between both samplers (p-value = 0.2215). The use of HBSS sampling media yielded a higher concentration of MNV than PBS media (p-value = 0.0125). However, PBS media maintained viability at a significantly higher percentage than HBSS media (p-value < 0.0001). The results support the optimization of a sampling method for the collection of aerosolized MNV and possibly norovirus in different sampling environments.</p>
<p>In the second study, we optimized the quantification method for MNV. A relatively new quantification system, droplet digital polymerase chain reaction (ddPCR), was evaluated using the same extracted samples collected in the first study to determine if the same overall outcome could be achieved. In addition, a MNV standard was directly compared between the qPCR and ddPCR. When comparing the same standard, the mean observed concentrations were similar to the nominal concentration. The limit of detection for both instruments was 5 copies per reaction. The coefficient of variation was lower across all ddPCR results than the qPCR results. The range of the R2 was larger for ddPCR compared to qPCR. As for the analysis of bioaerosol samples collected from the first study, the SKC BioSampler collected a significantly higher concentration of MNV compared to the NIOSH-251 sampler (p-value = 0.0002). However, there were no significant differences in the relative percent of MNV that remained viable in both samplers (p-value = 0.6734). The use of HBSS sampling media yielded a higher concentration of MNV than PBS media (p-value = 0.0190). However, PBS media maintained viability at a significantly higher percentage than HBSS media (p-value = 0.0004). The use of ddPCR allows for a simpler workflow and fewer samples and resources. These results support that both PCR systems yield similar results and overall outcomes, thus presenting an optimized quantification method for MNV.</p>
<p>In the third study, we used the optimized sampling and quantification methods to conduct a field trial investigation of a potential indoor aerosolization source for norovirus (toilet flushing). To inform bioaerosol sampler placement, two optical particle counters monitored particle size and number distribution of aerosol produced from flushing a toilet across three variables (height, position, and side). The location with the highest mean particle concentration, and where bioaerosol sampling occurred, was behind the toilet and 0.15 m above the toilet bowl rim. A flushometer type toilet was seeded with 105 and 106 PFU/mL of MNV and then flushed. Upon flushing, a SKC BioSampler and Coriolis µ sampler were activated to collect aerosolized MNV. Samples were extracted and then quantified using RT-ddPCR, and viability was quantified using PMA: RT-ddPCR. The concentration of MNV collected after seeding the toilet water ranged from 2.18 x 105 – 9.65 x 106 total copies of MNV. Positive samples of airborne MNV were detected using the Coriolis µ sampler with collected concentrations ranging from 383 – 684 RNA copies/m3 of air. Sample viability for bioaerosol samples were unable to be quantified. The relative percent of MNV virions that remained intact in seeded toilet water was 37-79%. This study provides the first evidence that MNV, a NV surrogate, can be aerosolized when a toilet is flushed.</p>
Details
- Title: Subtitle
- Optimization of sampling and quantification methods for aerosolized norovirus
- Creators
- Corey Lee Boles - University of Iowa
- Contributors
- Matthew Nonnenmann (Advisor)Thomas Peters (Committee Member)Grant Brown (Committee Member)Bradley Ford (Committee Member)Daniel Diekema (Committee Member)R. William Field (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Occupational and Environmental Health
- Date degree season
- Spring 2019
- DOI
- 10.17077/etd.f70k-5ykh
- Publisher
- University of Iowa
- Number of pages
- xiii, 107 pages
- Copyright
- Copyright © 2019 Corey Lee Boles
- Language
- English
- Date submitted
- 10/31/2019
- Description illustrations
- illustrations
- Description bibliographic
- Includes bibliographical references (pages 91-104).
- Public Abstract (ETD)
Every year in the United States norovirus causes 19-21 million cases of acute gastroenteritis, 1.7-1.9 million outpatient visits, 400,000 emergency department visits, 56,000 – 71,000 hospitalization, and 570 – 800 death. Norovirus is also a global concern with 685 million cases of acute gastroenteritis and 200,000 deaths occurring globally every year. Therefore, it is important to understand how norovirus is transmitted and implement approaches to limit human exposure. Recent evidence has suggested that norovirus can become aerosolized; however, an indoor aerosolization source has yet to be determined. A leading hypothesis in the scientific literature suggests that norovirus can become aerosolized via the flushing of a toilet. However, this hypothesis has not been directly investigated. Furthermore, sampling and quantification methods for aerosolized norovirus can vary greatly between studies. Therefore, an evaluated sampling and quantification method is required to maintain consistency across studies.
This study focuses on optimizing sampling and quantification methods for the collection of aerosolized norovirus. The optimized sampling method can be used in occupational, residential, and environmental settings for the collection of aerosolized norovirus. Lastly, these optimized sampling and quantification methods can be used to determine an indoor aerosolization source for norovirus. This study has three aims: (1) optimization of the bioaerosol sampling method for NV, (2) optimization of molecular quantification methods for NV, and (3) determine if flushing a toilet aerosolizes norovirus by using optimized sampling and quantification methods. Upon complete ion, this work allows for the optimization of sampling and quantification methods for the collection of aerosolized norovirus, as well as, determining an indoor generation for aerosolized norovirus. Furthermore, the findings of this research will allow for broader investigation of environmental contamination and worker exposure to NV.
- Academic Unit
- Occupational and Environmental Health
- Record Identifier
- 9983776788202771
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