Browsing by Subject "laboratory sciences"

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    Open Access
    Evaluation of two SARS-CoV-2 immunoassays
    (2022) Naidoo, Michelle; Hardie, Diana
    Aim: The purpose of this study is to verify the performance of the Roche Elecsys ® antinucleocapsid (qualitative) and anti-spike (quantitative) SARS-CoV-2 immunoassays to determine whether the performance of the assays is acceptable for diagnostic use in the Groote Schuur Hospital virology/chemistry laboratory, as well as other National Health Laboratory Service (NHLS) laboratories in South Africa. Methods: We performed a verification study using de-identified remnant serum or plasma samples. Standard verification experiments including sensitivity, specificity and precision were performed. Pre-pandemic samples were used to assess specificity. Samples with a linked positive SARS-CoV-2 polymerase chain reaction (PCR) result on a respiratory sample >10 days before the serum/plasma collection date were used to assess sensitivity. Additionally, postvaccine humoral response and other parameters was assessed in a cohort of laboratory staff. Results: For the anti-nucleocapsid antibody assay, specificity was 99.7% based on 316 samples and sensitivity 91.3% based on 404 samples. For the anti-spike antibody assay, the specificity based on 194 samples was 100%, and the sensitivity based on 384 samples was 93.8%. Both assays demonstrated acceptable precision. Furthermore, the anti-spike antibody assay sensitivity was >92% during the first three waves in South Africa, dominated by different SARS-CoV-2 variants. Post-vaccine seroconversion in 115 staff with no evidence of prior natural infection was 99% and hybrid immunity produced higher anti-spike antibody titres compared to vaccine-only participants. Conclusion: Both immunoassays met our acceptance criteria. Both assays can be used for seroprevalence studies. The anti-nucleocapsid immunoassay assay is valuable in confirming past natural infection in patients with previous asymptomatic infection, previous symptomatic infection where no PCR was done or PCR-negative patients who present to hospital with COVID-19 during the second week of illness or later. Most importantly, the antispike immunoassay can be used as a reliable, cheap, and easily accessible surrogate marker of post-vaccine humoral immune response and we recommend using this to confirm and monitor humoral immune response in patients with risk factors for non-seroconversion following vaccination and increased risk for morbidity and mortality following infection with SARS-CoV-2.
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    Open Access
    Exploring the phylodynamics, genetic reassortment and RNA secondary structure formation patterns of orthomyxoviruses by comparative sequence analysis
    (2019) Nindo, Fredrick Nzabanyi; Martin, Darren
    RNA viruses are among the most virulent microorganisms that threaten the health of humans and livestock. Among the most socio-economically important of the known RNA viruses are those found in the family Orthomyxovirus. In this era of rapid low-cost genome sequencing and advancements in computational biology techniques, many previously difficult research questions relating to the molecular epidemiology and evolutionary dynamics of these viruses can now be answered with ease. Using sequence data together with associated meta-data, in chapter two of this dissertation I tested the hypothesis that the Influenza A/H1N1 2009 pandemic virus was introduced multiple times into Africa, and subsequently dispersed heterogeneously across the continent. I further tested to what degree factors such as road distances and air travel distances impacted the observed pattern of spread of this virus in Africa using a generalised linear modelbased approach. The results suggested that their were multiple simultaneous introductions of 2009 pandemic A/H1N1 into Africa, and geographical distance and human mobility through air travel played an important role towards dissemination. In chapter three, I set out to test two hypotheses: (1) that there is no difference in the frequency of reassortments among the segments that constitute influenza virus genomes; and (2) that there is epochal temporal reassortment among influenza viruses and that all geographical regions are equally likely sources of epidemiologically important influenza virus reassortant lineages. The findings suggested that surface segments are more frequently exchanges than internal genes and that North America/Asia, Oceania, and Asia could be the most likely source locations for reassortant Influenza A, B and C virus lineages respectively. In chapter four of this thesis, I explored the formation of RNA secondary structures within the genomes of orthomyxoviruses belonging to five genera: Influenza A, B and C, Infectious Salmon Anaemia Virus and Thogotovirus using in silico RNA folding predictions and additional molecular evolution and phylogenetic tests to show that structured regions may be biologically functional. The presence of some conserved structures across the five genera is likely a reflection of the biological importance of these structures, warranting further investigation regarding their role in the evolution and possible development of antiviral resistance. The studies herein demonstrate that pathogen genomics-based analytical approaches are useful both for understanding the mechanisms that drive the evolution and spread of rapidly evolving viral pathogens such as orthomyxoviruses, and for illuminating how these approaches could be leveraged to improve the management of these pathogens.
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    Open Access
    Heterologous production of recombinant peptidylglycine α-amidating monooxygenase for the production of biosimilar α-amidated peptides
    (2022) Morrison, David Graham; Sturrock, Edward; Steenkamp L S
    A biological method for peptide synthesis provides increased production capacity of inexpensive peptide pharmaceuticals with environmentally safe procedures relative to current chemical peptide synthesis. Most precursor peptides are readily produced from yeast and bacterial systems using recombinant DNA technologies but require C-terminal amidation for maximum biological activity. Peptidylglycine α-amidating monooxygenase (PAM) is the only enzyme that catalyses the C-terminal amidation of peptides in vivo through its two catalytic cores, peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidylglycine αamidating lyase (PAL). The cost and limited quantities of the commercial PAM variants available have necessitated research into low cost, scalable quantities of PAM and peptide amidation to enable inexpensive biological peptide production. In the present study, an assay for measuring the product of PAM activity, glyoxylate, was developed based on a 2-aminobenzaldehyde-glycine-glyoxylate (AGG) absorbance assay. The AGG chromophore synthesised was identified with ultra-performance liquid chromatography mass-spectroscopy (UPLC-MS). PAM activity was measurable with glyoxylate between 25 µM and 1600 µM with the AGG assay. Furthermore, the activity of PHM alone was measured by the inclusion of an alkaline hydrolysis step to lyse glyoxylate as a substitute for PAL catalytic activity. Multiple candidate proteins and DNA sequences for PAM were identified by genetic sequence searches and a novel fungal PHM modelled in silico. Fourteen PAM, PHM, PAL and truncated constructs were expressed in the non-conventional yeast host, Yarrowia lipolytica. The novel fungal PHM's nutrient, temperature, and pH conditions were optimised to maximise protein expression. Enzyme purification was optimised with scalable industrial appropriate methods to purify milligram amounts of fungal PHM. The AGG assay was validated with a commercially obtained PAM, demonstrating a simple medium-throughput method to measure PAM activity. The novel fungal PHM was characterised with a pH optimum of 4.0 and maximum enzymatic activity at 45°C. Deglycosylation of fungal PHM enhanced enzyme activity by 1.83 fold, but lowered the temperature optimum to 37°C. The novel PHM and alkaline hydrolysis catalysed the conversion of the peptide pharmaceutical precursor for exenatide into its final bioactive form.