Date of Award

2026-05-01

Degree Name

Doctor of Philosophy

Department

Chemistry

Advisor(s)

Chuan Xiao

Abstract

Public health surveillance increasingly relies on wastewater-based epidemiology (WBE) as a non-invasive, community-level early warning system for infectious diseases like COVID-19 and influenza. However, detecting minute viral concentrations in complex wastewater matrices remains a significant analytical challenge. Traditional concentrating methods, such as ultrafiltration, are limited by high performance variability and significant viral signal loss due to necessary pretreatment steps, such as pre-clarification, which inadvertently discard solid-associated viral fractions. To address these limitations, this dissertation introduces and evaluates freeze-drying as a novel, pretreatment-free concentrating method for wastewater viral surveillance. By sublimating water, freeze-drying uniquely retains both liquid and solid-phase viruses within the sample matrix, while simultaneously preventing RNA degradation under a cryogenic environment. A systematic comparison with conventional ultrafiltration was conducted using influent wastewater samples from four Wastewater Treatment Plants at El Paso, Texas, collected during the early onset and waning phases of the COVID-19 pandemic. Freeze-drying achieved a significantly higher positive detection rate of SARS-CoV-2 than ultrafiltration. Furthermore, the method demonstrated greater recovery efficiency and a markedly lower method-specific detection limit. Beyond enhanced sensitivity, freeze-drying provided substantial operational advantages, including reduced consumable costs, improved biosafety through a sealed passive system, and greater scheduling flexibility due to stable frozen storage. Building upon the success of SARS-CoV-2 detection, the freeze-drying workflow was expanded to establish a comprehensive multiplex RT-qPCR surveillance system capable of simultaneously detecting SARS-CoV-2, Influenza A, and Influenza B. Controlled spiking experiments confirmed the method's robust sensitivity across a broad dynamic range, establishing a practical limit of detection for both influenza viruses. Finally, this work outlines future trajectories to refine WBE methodologies, proposing strategies to leverage freeze-drying for improved population biomarker normalization and addressing downstream PCR inhibition caused by ferric-based coagulation in wastewater treatment plants. Ultimately, this research establishes freeze-drying as a highly sensitive, sustainable, and scalable approach to environmental virology, offering a powerful tool to enhance global preparedness and community-level surveillance against emerging respiratory pathogens.

Language

en

Provenance

Received from ProQuest

File Size

251 p.

File Format

application/pdf

Rights Holder

Rui Dong

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Included in

Chemistry Commons

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