Masters Degrees (Medical Virology)
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Browsing Masters Degrees (Medical Virology) by browse.metadata.advisor "Nkosi, Nokwazi"
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- ItemA real-time reverse transcription polymerase chain reaction for the sensitive detection of hepatitis a virus in various clinical and environmental samples(Stellenbosch : Stellenbosch University, 2023-03) Mpazi, Nothukela; Nkosi, Nokwazi; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.ENGLISH SUMMARY: Hepatitis A virus (HAV) is a growing public health concern worldwide due to its contribution to acute viral hepatitis. In South Africa, HAV data has been accounted for in clinical and environmental studies separately. Simultaneous detection of the HAV in clinical and environmental samples in the Western Cape province has not yet been explored. The current serological method of HAV detection has been linked to cross-reactivity between antibodies resulting in false positive results. Supplemental use of real-time reverse transcription (RT) polymerase chain reaction (PCR) may have the benefit of limiting cross-reactivity and serving as an early detection method in outbreak settings as it detects HAV ribonucleic acid (RNA) two weeks prior to seroconversion. The aim was to assess the presence of HAV (RNA) in various clinical and environmental wastewater samples using an in-house established real-time RT-PCR assay and to describe circulating HAV genotype(s). The objectives were to identify serologically tested HAV residual serum, plasma and stool samples referred to the National Health Laboratory Service (NHLS) Medical Virology Laboratories at Tygerberg Hospital (TBH), Groote Schuur Hospitals (GSH); and Western Cape Blood Service (WCBS); to screen untreated wastewater environmental samples for HAV presence from the South African Medical Research Council (SAMRC); to analyse data by age, sex, liver function enzyme parameters and location of retrieval using Microsoft Excel and to assess circulating genotype (s) through Sanger sequencing. Two different primers and probes were evaluated for HAV RNA detection in 353 samples comprising of 179 clinical (serum, stool, and plasma) and 174 environmental (untreated wastewater RNA eluates) samples obtained through convenience sampling from TBH; GSH NHLS serology laboratories; WCBS and SAMRC (from five Stellenbosch University residences and two Cape Town areas). BioEdit software; Genome detectives Krisp genotyping tool version 2.43 and Mega 11 software were used to assess HAV genotype(s) on selected clinical samples only. HAV RNA was positive in 43.6% (78/179) clinical samples and 32.7% (57/174) environmental samples. In the ages younger 15 years HAV RNA positive samples were higher in males in compared to females. In contrast age groups >16 years HAV RNA positive samples were higher in females compared to males. The City of Cape Town areas had a higher detection of HAV RNA positive environmental samples (>40%), while a 28% detection was recorded from the Stellenbosch University student residence samples. The identified circulating HAV genotype was HAV IB. Using the two primer and probe sets this assay successfully detected HAV RNA in various clinical and environmental untreated wastewater samples offering a 92% and 100% sensitivity for the first and second primer set, respectively. Specificity was 100% and 75% respectively. Performance characteristics of second primer set evaluated limited by sample number evaluated. Using assays with 100% specificity might be used in diagnostic settings as a supplemental confirmatory technique. This study was semi-qualitative in nature, future studies may assess the quantitative aspect and cost related to the implementation of the assay in diagnostic settings.