DOI

10.17077/etd.f70k-5ykh

Document Type

Dissertation

Date of Degree

Spring 2019

Access Restrictions

Access restricted until 07/29/2020

Degree Name

PhD (Doctor of Philosophy)

Degree In

Occupational and Environmental Health

First Advisor

Nonnenmann, Matthew

First Committee Member

Peters, Thomas

Second Committee Member

Brown, Grant

Third Committee Member

Ford, Bradley

Fourth Committee Member

Diekema, Daniel

Fifth Committee Member

Field, R. William

Abstract

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.

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.

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.

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.

Keywords

Bioaerosols, Industrial Hygiene, Norovirus, Toilet Aerosol

Pages

xiii, 107 pages

Bibliography

Includes bibliographical references (pages 91-104).

Copyright

Copyright © 2019 Corey Lee Boles

Available for download on Wednesday, July 29, 2020

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