Doctoral Degrees (Medical Microbiology)
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Browsing Doctoral Degrees (Medical Microbiology) by Author "Goedhals, Dominique"
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Item Open Access Drug resistance mutations in newly diagnosed HIV-infected infants and in children and adolescents with virological failure on ART(University of the Free State, 2020-12) Barakzai, Iqra; Goedhals, DominiqueIntroduction and aim: South Africa has never experienced an epidemic of the same extent and magnitude as the one with human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS).The roll out of antiretroviral therapy (ART) and other interventions, such as prevention of mother to child transmission (PMTCT), has greatly reduced the incidence of new HIV infections among children. However, early initiation of ART among children has increased their overall exposure to antiretrovirals (ARVs), resulting in an increased prevalence of acquired drug resistance (ADR) on treatment. Presence of drug resistant virus further complicates clinical management of HIV infection, resulting in fewer present and future treatment options. Therefore, drug resistance testing has been recommended in selecting appropriate treatment regimens among children and adolescents diagnosed with virological failure. The aim of the current study was to determine and characterise drug resistance mutations in newly diagnosed HIV-infected children and in children and adolescents with virological failure on ART. Methods: To detect drug resistance mutations, the present study developed and validated a Sanger sequencing assay using dried blood spot (DBS) samples. The Sanger assay was used to perform HIV drug resistance testing on DBS samples testing positive on the early infant diagnosis (EID) platform. Lastly, a retrospective analysis was performed of resistance data from samples of children and adolescents submitted to the routine diagnostic laboratory. Results: A high level of concordance was found among major mutations detected using the Sanger DBS and plasma assays. Since all the discordant mutations were either minor or existed as mixtures, the interpretation of resistance profiles was found to be identical. The DBS assay was then used to detect pre-treatment drug resistance (PDR) among samples from children on the EID platform. Overall, PDR was detected among 73% of the children, with the majority of the children showing resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) (72%). Dual class resistance was most prevalent to NNRTIs and NRTIs (11% [33/308]), followed by NNRTIs + protease inhibitors (PIs) (0.6% [2/308]). Triple class resistance (NNRTI + NRTI + PI) was very rare and was only detected in two children. Protease resistance was very rare among the children and none of the children displayed high-level resistance against any PI. Results from children (0 to 9 years) and adolescents (10 to 19 years) with virological failure while on ART indicated that most of the children were resistant to NRTIs (86% [79/92]), followed by NNRTIs (75% [69/92]) and PIs (28% [26/92]). Among adolescents, prevalence of resistance to NNRTIs (87% [146/167]) was the highest, followed by NRTIs (66% [111/167]) and PIs (30% [51/167]). Dual class resistance to NRTIs + NNRTIs was the most common among both children (44% [41/92]) and adolescents (38%, [63/167]). Triple class resistance (NNRTI+NRTI+PI) was detected in 18% of children (17/92) and 19% of adolescents (32/167). Conclusion: The prevalence of infants, children and adolescents experiencing treatment failure due to the presence of transmitted, acquired and PDR is increasing. While the implementation of virological monitoring and genotypic testing play an essential role in identifying and managing drug resistance among children and adolescents experiencing treatment failure, these services are limited in many developing countries. The current study supports the implementation of HIV drug resistance testing for national surveys and optimal clinical management of HIV-infected children and adolescents.Item Open Access Immune responses to Crimean-Congo haemorrhagic fever virus and molecular characterization of viral isolates(University of the Free State, 2014-02) Goedhals, Dominique; Burt, F. J.; Paweska, J.Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus belonging to the family Bunyaviridae, genus Nairovirus. The distribution of the virus correlates with that of the principal vector, ticks belonging to the genus Hyalomma. This includes areas in Africa, Asia, Eastern Europe and the Middle East, with recent emergence in Turkey, Greece and India. CCHFV is associated with haemorrhagic fever in humans, with a case fatality rate of up to 30%. Current patient management relies on supportive therapy and administration of ribavirin, but the efficacy of this antiviral drug is controversial. Although an inactivated vaccine has been used in Eastern Europe and the former Soviet Union, it has not been accepted for widespread use. An understanding of immune correlates is therefore needed to guide further development of therapeutic and preventative interventions. This study aimed to investigate immune responses in survivors of CCHF in South Africa, focusing on the presence of detectable memory T lymphocyte responses and the identification of epitopic regions within the nucleoprotein and glycoproteins. In order to ensure applicability of identified epitopes to geographically distinct isolates, viral sequence diversity was also investigated by means of next generation sequencing and phylogenetic studies. A synthetic overlapping peptide library was used to screen for interferon gamma production by peripheral blood mononuclear cells from survivors of CCHFV infection in ELISPOT assays. Ten potential epitopic regions were identified, the majority of which were located on the nucleoprotein with only two regions identified on the glycoprotein GC in a single patient. Long-lived memory CD8+ T cell responses were detected in survivors of CCHF up to 13 years after infection. These findings indicate the presence of effective long term cellular immune responses which could be modulated through vaccination and gives an indication of epitopic regions that should be considered in candidate vaccines and testing vaccine immunogenicity. The presence of detectable memory responses in the absence of reexposure or chronic infection will allow future studies to fully characterize T cell responses in survivors. With an expanding area of CCHFV endemicity, safe, sensitive and specific serological assays are required for diagnostic and serosurveillance purposes. As the biosafety level 4 facilities required to culture the virus are lacking in many endemic areas, alternative means of producing reagents for diagnostic assays are needed which will not pose a safety risk to laboratory workers. The use of synthetic peptides in serological assays is one such alternative approach. In addition, identification of immunodominant epitopic regions may have application in vaccine development if they induce protective immunity. The peptide library was used to screen for antibodies recognizing human defined linear B cell epitopic regions in survivors of CCHFV infection by means of an enzyme-linked immunosorbent assay (ELISA). Two potential epitopic regions were identified on the GC glycoprotein with reactivity in 13 – 14 of 15 patients tested. Further investigation will be required to determine whether these epitopic regions also correlate with immune protection and to identify non-contiguous B cell epitopes which are likely to play an important role in antibody induction during natural infection with CCHFV. With new foci of CCHFV infections emerging in recent years, it is important to ascertain whether genomic variation will influence applicability of vaccine candidates and diagnostic assays in distinct geographic areas. Next generation sequencing techniques were used to obtain complete genome sequences for ten southern African CCHFV isolates. This is the first application of next generation sequencing technology to CCHFV isolates and proved to be a rapid and cost effective alternative to standard Sanger sequencing which can be effectively applied to the approximately 20kb CCHFV genome. The phylogenetic results confirmed that although there is extensive variability among geographically distinct CCHFV isolates at a genomic level, conserved areas are present which could be targeted for vaccine development and diagnostic purposes. The genetic variability seen results from point mutations and segment reassortment, which was shown to occur commonly in southern African CCHFV isolates. Despite the extensive variation in primary sequence, at a protein level, the motifs involved in protein function are well conserved. Prediction software analysis confirmed the presence of conserved OTU-like cysteine protease and RNA dependent RNA polymerase (RdRp) domains in the L segment of diverse southern African CCHV isolates. The RdRp is essential for viral replication while the OTU-like protease likely plays a role in immune evasion and therefore affects viral pathogenicity. Analysis of the M segment showed conservation of the basic protein coding strategy, with two structural and three non-structural glycoproteins. However, amino acid variation was notable across all predicted proteins but particularly in the variable mucin-like domain which is thought to play a role in viral pathogenicity. This study identifies targets for further investigation of viral pathogenicity which may include in vivo studies in animal models and mutagenicity assays.