Development and Application of Methods for Sensitive and Specific Detection of SARS-CoV-2 and Variants in Clinical and Environmental Samples
Creators: Kumblathan, Teresa
Publisher: name: University of Alberta Library
Year: 2024

<p>The COVID-19 pandemic, caused by SARS-CoV-2, had far-reaching global health consequences, with the virus spreading rapidly via respiratory droplets and aerosols. Accurate and timely diagnostic testing became critical for guiding public health policies and preventing the spread of infections. The standard clinical testing of SARS-CoV-2 RNA involves nasopharyngeal swabs (NPS) sample collection and reverse transcription quantitative polymerase chain reaction (RT-qPCR) detection. Clinical testing for public health surveillance quickly became overwhelmed due to constraints on available diagnostic supplies and human resources. Therefore, testing became restricted to specific patients. To complement standard clinical testing and alleviate some of these testing constraints, I designed and developed alternative approaches. <br>
SARS-CoV-2 is present in oral fluids, but viral detection has proven challenging due to the heterogenous and viscous matrix of these fluids, which hinders subsequent analysis, and the limited resources pose obstacles for large-scale community biomonitoring. This led to my first proposed project with my colleagues as part of the pandemic preparedness team to develop new sampling and testing methods for monitoring SARS-CoV-2 in oral fluids. To achieve sensitive and reliable analysis of viral RNA in oral fluids, I integrated viral inactivation, RNA release and preservation, and subsequent direct detection of SARS-CoV-2 on magnetic beads. The unique formulation of the viral inactivation and RNA preservation (VIP) buffer enabled patients to self-collect samples, minimizing the need for healthcare professionals and transmission of infection. The VIP buffer also enabled sample stability for at least 3 weeks. This method offered a limit of detection of 25 RNA copies per 200 μL of sample and 9-111x higher sensitivity than the Centers for Disease Control and Prevention recommended kit. This new integrated method successfully analyzed more than 200 patient samples and was also used for pooled sample analysis.<br>
Due to the overwhelming strain on clinical testing resources, wastewater surveillance (WS) was promoted as an alternative for community biomonitoring of SARS-CoV-2 because viral particles were proven to enter wastewater via stools of infected patients. My comprehensive literature evaluation on SARS-CoV-2 WS highlighted challenges such as complex matrices, lack of standardized procedures, and poor and irreproducible recoveries. To overcome these challenges and address my second objective of implementing WS to complement clinical testing, I developed a robust method for highly sensitive wastewater detection of SARS-CoV-2. Viral particles and free RNA were captured from both phases of wastewater using an electronegative membrane (EM), followed by incubation in the VIP-Mag buffers, and direct RT-qPCR detection. My method&#39;s capability of detecting trace and diverse concentrations of SARS-CoV-2 in wastewater is attributed to the enhanced recovery (80%) and efficient removal of PCR inhibitors. I analyzed 120 wastewater samples and consistently detected higher levels of RNA than the provincial reference lab.<br>
To expand the capacity of detecting the rapidly evolving SARS-CoV-2 variants, I proposed my third objective to develop multiplex RT-qPCR assays for the early detection VOCs in wastewater. To address this objective, I developed three multiplex assays using naturally selected mutations capable of differentiating Alpha, Beta, Delta, and Omicron sub-variants. These assays have excellent efficiencies (90–104%) for all targets and LODs of 4-28 RNA copies per reaction. Analysis of 294 wastewater samples revealed that the trends of Alpha, Beta, Gamma, Delta, and Omicron sub-variants aligned with clinical trends, and suggested early wastewater detection of certain variants prior to reported clinical cases. <br>
Finally, to establish a universal platform for the detection of various co-circulating viruses, my fourth objective was to demonstrate that my WS protocol can be easily adapted for other viruses. To address this objective, I used the enveloped Omicron and the non-enveloped Norovirus (NoV) as examples of structurally different, but clinically significant viruses. My WS protocol successfully quantified NoV (genotypes I and II) and Omicron subvariants in the same sets of 94 wastewater samples with high recovery (72% and 80%, respectively). The results showed seasonal trends of NoV and Omicron variants in the same wastewater systems which matched clinical trends and revealed an inverse relationship between the presence of these viruses. <br>
Overall, my methods and strategies highlight the importance of robust platforms for clinical testing and community surveillance of enveloped and non-enveloped viruses. These techniques demonstrate the adaptability of platforms for future biomonitoring of community infections such as COVID-19, and beyond.</p>