Masters Degrees (Medical Virology)

Permanent URI for this collection

https://scholar.sun.ac.za/handle/10019.1/3155

Browse

Browsing Masters Degrees (Medical Virology) by Subject "Antibiotics -- Therapeutic use"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    The role of underdiagnosed viruses in neonatal sepsis
    (Stellenbosch : Stellenbosch University, 2022-12) Mahlangu, Tshegofatso Perseverance; Van Zyl, Gert Uves; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.
    ENGLISH ABSTRACT: Background Neonatal sepsis is a clinical syndrome that is difficult to diagnose due to the unspecific symptoms that patients present with. Several different diagnostic techniques are used to diagnose neonatal sepsis; but currently, blood cultures are regarded as the gold standard for diagnosis of bacterial sepsis. Broad spectrum antibiotics are administered to neonates with suspected sepsis and the therapy is adjusted accordingly after diagnosis. This is, however, inappropriate therapy if sepsis is caused by viral and not bacterial agents. Numerous studies have found certain viruses to cause sepsis or a sepsis-like syndrome, but because viruses require virus specific assays, they may be underdiagnosed. Although the incidence of viral neonatal sepsis has been low, the effect of its underdiagnosis has detrimental effects. Therefore, in this study, a rapid detection multiplex polymerase chain reaction (PCR) was developed to potentially assist with the diagnosis of viral neonatal sepsis. A third-generation sequencing (TGS) technology was used for species specific identification of the agents detected by the multiplex PCR. TGS technology is increasingly utilised in medical research and appears promising for future rapid infectious agent diagnosis due to its real-time and long read sequencing. Methods Various sample types were included: residual patient laboratory samples with known positive diagnoses (none of which were from neonatal sepsis patients), external quality assurance panel samples and culture isolates from known agents. These samples were used to first optimise a singleplex assay followed by the multiplex assay. The assay was developed to have two rounds of PCR to ensure increased sensitivity. Two different extraction methods were compared to determine the method best suited for the assay. In addition, a simple sensitivity test was conducted using serially diluted samples for both the singleplex, and multiplex assays. As a result of the presence of an unknown fragment in the second-round multiplex PCR non-template controls (NTCs), several troubleshooting experiments were performed. Products with the incorrect band size or present in NTCs, which were observed during assay optimisation, were initially investigated with Sanger sequencing. Thereafter Oxford Nanopore Technology (ONT) sequencing was used to confirm the PCR amplified products. In addition, ONT sequencing was used to attempt to identify the unknown fragment in the NTCs. Results The singleplex assay was successfully optimised, however, during the multiplex optimisation an unknown fragment was discovered in the second round PCR NTCs. Troubleshooting excluded all other sources and we concluded that the “contaminant” was possibly an impurity of bacterial origin carried over from recombinant technology used in the production of the enzyme system. A new enzyme system was introduced to continue the multiplex optimisation, but the sensitivity was lower than with the previous enzyme system. As a result, the previous enzyme system was utilised for all targets except bacterial targets, for which a separate reaction including the 16s rRNA primers were used. Thereafter, the multiplex assay was further optimised. ONT sequencing confirmed that the correct agents were amplified, rendering the entire workflow successful. The unknown fragment of bacterial origin discovered in the NTCs was sequenced with ONT as well as Sanger sequencing. However, both platforms failed to confirm the specific bacterial species present. Conclusion The multiplex assay developed in this study can successfully amplify the intended agents but only at high concentrations. The comparison of sensitivity of the singleplex versus the multiplex versions of the assay revealed that the multiplex assay was significantly less sensitive. ONT sequencing could rapidly confirm all the amplified agents, and this is beneficial for diagnostic purposes. Currently, this study serves as a proof of concept because the multiplex assay, in its current form, does not appear to be suitable for primary (uncultured) diagnostic samples due to inadequate sensitivity for a low target concentration input.