Okoh, A.I.Msolo, L.Mayoyo, Okuhle2026-08-172026-08-172024Mayoyo, O. (2024) Incidence and diversity of SARS-CoV-2 from wastewater samples recovered from selected municipal wastewater treatment facilities in Buffalo City Metropolitan Municipality, Eastern Cape Province, South Africa. PhD (Microbiology) thesis. Alice: University of Fort Hare.http://hdl.handle.net/20.500.11837/4825PhD thesisWastewater surveillance has gained traction since the Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) induced global health crisis. SARS-CoV-2 is an enveloped Betacoronavirus that has evolved into numerous variants. The virus and its genomic variants contributed to COVID-19 transmission dynamics, disease severity, immune-response escape, and economic collapse in developing countries. This study elucidated the incidence and diversity of SARS-CoV-2 in municipal wastewaters in the Buffalo City Metropolitan Municipality (BCMM). Wastewater influent samples were aseptically collected monthly from ten wastewater treatment facilities in the municipality over a 12-month period from June 2021 to June 2022 to represent the four seasons of autumn, spring, summer and winter in South Africa. A total of 494 wastewater-influent samples were collected, and the total viral RNA was extracted from the samples following the standard operating protocols. The Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) was utilised to screen for the nucleocapsid phosphoprotein-inducing gene of SARS-CoV-2. Subsequently, the TaqMan SARS-CoV-2 mutation panel genotyping-molecular-specific RT-PCR assay, targeting the single nucleotide polymorphisms (SNPs) (delH69V70, E484K, K417N, L452R, N501Y, P681H and P681R), was used to rapidly detect SARS-CoV-2 variants with the panel related to the B.1617.2 and the B.1.1.529 lineages of SARS-CoV-2 variants. About 71% (n = 351) of the 494 samples were positive for the nucleocapsid gene, indicating SARS-CoV-2 presence. The total viral load retrieved from all study sites in winter (2021) was a total concentration of 22.08 × 104 GC/mL, spring (2021) 13.09 × 104 GC/mL, summer (2021-2022) 7.61 × 104 GC/mL and autumn (2022) 29.30 × 104 GC/mL. The circulation of variants of concern (VOC) was ascertained as 93% of SARS-CoV-2 samples (n = 45) with viral loads ≥ 1000 GC/mL were positive for the target SNPs. The predominant amino acid mutation was the N501Y (22%), shared between Alpha (B.1.17), Beta (B.1.33, B.1.351) and Omicron lineages. The geospatial analysis revealed the EB WWTP, a combined sewer network, as the diverse SARS-CoV-2 genome hotspot. Nevertheless, the spike protein mutations linked to VOCs were circulating in other areas. Overall, a direct correlation was observed between SARS-CoV-2 signals in wastewater, VOC-linked SNPs and clinical cases in the region. A significant spike in all three factors was observed in September 2021, during the third epidemic. VOC Delta (B.1.617.2) was the predominant variant during this period. Furthermore, a more significant peak was in SARS-CoV-2 with a total viral load exceeding 20 × 104 GC/mL, L452R, N501Y and P681H were detected more frequently during this epidemic driven by BA.4 & BA.5. This finding emphasises the significance of wastewater-based epidemiology as a non-invasive populationbased surveillance strategy to monitor circulating pathogens and diseases. Quantitative Microbial Risk Assessment (QMRA) model was carried out to evaluate the potential occupational health risks of exposure to SARS-CoV-2 amongst wastewater treatment plant workers at different exposure scenarios. As expected, the P(i) values for wastewater treatment plant (WWTP) operators working for 6 hours without personal protective equipment (PPE) and inadvertently exposed to up to 20 mL of contaminated bioaerosol, droplets and fomites were 35.9 × 10-2 (winter 2021), 5.1 × 10-2 (spring 2021), 6.8 × 10-2 (summer 2021-2022), and 18.2 × 10-2 (autumn 2022). There was no significant correlation between the seasons and the potential health risks (p ≥ 0.05). Therefore, the potential health risks associated with the incidence of the SARS-CoV-2 genome in wastewater across the epidemiological seasons in the study WWTPs are low at a significance level of 95%. Nevertheless, the controversies in the literature regarding the viability of SARS-CoV-2 in the wastewater milieu accentuate the coronavirus as a public health concern and demand stringent precautionary measures in occupational settings. Irrevocably, the incidence of the SARS-CoV-2 genome in the municipal wastewaters of the BCCM is an indicator of the circulation of the virus in the metropolitan. The implication of occupational and environmental safety protocols is crucial to curb the dissemination of the pathogen, and wastewater-based epidemiology coupled with QMRA are imperative approaches that could be continuously employed as complementary tools for clinical surveillance. This surveillance strategy for communicable diseases is imperative for reaching the 2030 Agenda for Sustainable Development.enCOVID-19 (Disease) -- South AfricaSingle nucleotide polymorphisms -- South africaSewageThesis