Pohl-Albertyn, Carolina H.Albertyn, J.Musoke, JollyMosia, B.Moncho, Masego MaryJane2025-01-032025-01-032024http://hdl.handle.net/11660/12901Thesis (Ph.D.(Medical Microbiology))--University of the Free State, 2024𝗜𝗻𝘁𝗿𝗼𝗱𝘂𝗰𝘁𝗶𝗼𝗻: Invasive fungal infections contribute to a rise in morbidity and mortality, extended stay in hospital and higher health care cost. Due to the increased risk factors among the neonates, this population continues to bear the brunt as the morbidity and mortality due to 𝘊𝘢𝘯𝘥𝘪𝘥𝘢 𝘱𝘢𝘳𝘢𝘱𝘴𝘪𝘭𝘰𝘴𝘪𝘴 remains high. This is further complicated by the rising predominance of azole-resistant strains. 𝗔𝗶𝗺: This study aimed to identify the specific strains of C. 𝘱𝘢𝘳𝘢𝘱𝘴𝘪𝘭𝘰𝘴𝘪𝘴 𝘴𝘦𝘯𝘴𝘶 𝘭𝘢𝘵𝘰 cultured from neonatal and paediatric patients at Universitas Academic Hospital and to determine their virulence potential. The clinical outcome data of the patients were correlated with the strains’ data. 𝗠𝗲𝘁𝗵𝗼𝗱𝗼𝗹𝗼𝗴𝘆: The study was conducted at the Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein. This was a retrospective cross-sectional laboratory-based study. 𝘊𝘢𝘯𝘥𝘪𝘥𝘢 𝘱𝘢𝘳𝘢𝘱𝘴𝘪𝘭𝘰𝘴𝘪𝘴 as identified on Vitek®2 (bioMérieux Inc, Marcy l’Etoile, France), from invasive clinical samples sent for routine laboratory diagnosis to the Medical Microbiology laboratory from Universitas Hospital’s neonatal and paediatric wards, were used. A sample size of approximately 30 was estimated based on the numbers from the previous year’s however, only 21 strains were obtained, with one of the patients having two isolates. Therefore, 20 patients’ strains were obtained. The 𝘊𝘢𝘯𝘥𝘪𝘥𝘢 𝘱𝘢𝘳𝘢𝘱𝘴𝘪𝘭𝘰𝘴𝘪𝘴 strains used for the study were those already stored in the laboratory at -80°C from the year 2018 to 2020. Data obtained from Vitek®2 (bioMérieux Inc, Marcy l’Etoile, France) as well as patients’ demographic data and clinical information from patient clinical files were recorded on an Excel sheet for analysis. No patient names were recorded. Only study numbers were used. D1/D2 sequencing was performed for species differentiation. Clinical records were reviewed, time to positivity, species identification, antifungal susceptibility testing results and patients’ demographics were also retrieved from TrackCare. The QualiClean (QualiPharm, New Germany) used in the hospital setting at 1000 ppm for surface disinfection was tested against all the 20 strains to determine their susceptibility to the disinfectant. A crystal violet assay was conducted to determine biomasses of respective biofilms. For hydrolytic enzymes secretion, tributyrin agar was used for lipase activity, yeast carbon base bovine serum albumin agar for protease activity and the sabauraud dextrose agar supplemented with 8% egg yolk for phospholipase activity. Prostaglandin E2 production was evaluated by using the enzyme-linked immunosorbent assay (ELISA; Cayman Chemicals, Ann Arbour, USA) according to the manufacturer’s instructions. The biological method described by Brenner (Brenner, 1974) was followed for 𝘪𝘯 𝘷𝘪𝘷𝘰 relative virulence in 𝘊𝘢𝘦𝘯𝘰𝘳𝘩𝘢𝘣𝘥𝘪𝘵𝘪𝘴 𝘦𝘭𝘦𝘨𝘢𝘯𝘴 In addition, whole genome sequencing was conducted to study the ploidy of the study strains, genetic relatedness as well as the common antifungal resistance genes. 𝗥𝗲𝘀𝘂𝗹𝘁𝘀: The strains were all 𝘊𝘢𝘯𝘥𝘪𝘥𝘢 𝘱𝘢𝘳𝘢𝘱𝘴𝘪𝘭𝘰𝘴𝘪𝘴 𝘴𝘦𝘯𝘴𝘶 𝘴𝘵𝘳𝘪𝘤𝘵𝘰. Ninety percent of blood cultures had a time to positivity of 48 hours with 80% of the strains showing resistance to fluconazole and the intermediate resistance to voriconazole. For 70% of the strains, resistance to fluconazole and intermediate resistance to voriconazole was observed. In addition, the QualiClean disinfectant did not effectively inhibit any of the strains. Fifty-five percent of the patients were from neonatal intensive care unit followed by 35% from neonatal high care and 10% from paediatric wards. Eighty percent of the patients were males. Forty-one percent of the patients were categorized as very low birth weight and 55% delivered via caesarian section. The study population had multiple risk factors including the presence of invasive devices, total parenteral nutrition, gastrointestinal pathology, and administration of broad-spectrum antibiotics. Deaths were recorded for 47% of the patients. The strains produced biofilms, proteases, phospholipases, and prostaglandin E₂ in varying quantities. The three C. 𝘱𝘢𝘳𝘢𝘱𝘴𝘪𝘭𝘰𝘴𝘪𝘴 strains tested for their virulence in C. 𝘦𝘭𝘦𝘨𝘢𝘯𝘴, killed the nematodes rapidly when compared to the C. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 ATCC SC5314 strain. All strains were haploid and were found to group into four related Clades, with majority of the strains in Clade 4. Clade 4 also housed 94% of the fluconazoleresistant strains. The 𝘍𝘒𝘚1, 𝘔𝘙𝘙1 and 𝘌𝘙𝘎11 genes were highly conserved between strains with none of the mutations previously associated with resistance patterns. 𝗖𝗼𝗻𝗰𝗹𝘂𝘀𝗶𝗼𝗻: 𝘊𝘢𝘯𝘥𝘪𝘥𝘢 𝘱𝘢𝘳𝘢𝘱𝘴𝘪𝘭𝘰𝘴𝘪𝘴 𝘴𝘦𝘯𝘴𝘶 𝘴𝘵𝘳𝘪𝘤𝘵𝘰 is circulating with majority of strains resistant to fluconazole, suggesting that fluconazole should be avoided as the empiric therapy among this population. All strains were virulent and resisted the action of the disinfectant. The contact time will need to be increased or a different disinfectant used. Multiple infection prevention and control interventions including hand hygiene are also to be intensified. Further studies are required to identify the resistance mutations or novel mutations prevalent at the study site, including those outside the common regions.enCandidemia𝘊𝘢𝘯𝘥𝘪𝘥𝘢 𝘱𝘢𝘳𝘢𝘱𝘴𝘪𝘭𝘰𝘴𝘪𝘴Virulence factorsHydrolytic enzymesProstaglandin E₂Biofilm formationChlorine based disinfectant𝘊𝘢𝘦𝘯𝘰𝘳𝘩𝘢𝘣𝘥𝘪𝘵𝘪𝘴 𝘦𝘭𝘦𝘨𝘢𝘯𝘴Whole genome sequencingPloidyThesisUniversity of the Free State