The epidemiology and antifungal sensitivity of clinical Cryptococcus neoformans and Cryptococcus gatti isolates from Bloemfontein, South Africa

English: In this dissertation, an attempt was made to study the epidemiology of cryptococcosis by first estimating the incidence rates over a two-year period, 2011 and 2012. The major findings from this part of the study included establishing that: 1) cases were more prevalent among Blacks (Africans, Coloureds and Indians), and this is in line with the assertion by WHO that diseases such as cryptococcosis are more poverty-related, 2) the distribution pattern of cryptococcosis across different age groups mirrored that of HIV-infected persons, and 3) the number of cryptococcosis cases were quite low for the study period (representing less than 0.1 % of the Bloemfontein population), this was surprising and unexpected given the huge HIV positive population in South Africa, and by extension in Bloemfontein, that is at risk of acquiring this AIDS-defining illness. It is documented in literature that the currently employed methods for the routine diagnosis of cryptococcosis often yield inconsistent test results, thereby; influencing the number of reported cases, which are important for health officials. But more importantly, these inconsistencies have far reaching consequences as they may negatively influence patient outcomes. Therefore, we sought to investigate the usefulness of molecular methods in identifying the etiological agents of cryptococcosis viz. Cr. neoformans and Cr. gattii. Here, the ITS, including the 5.8 gene, intra-specific variation between the tested strains allowed for their delineation into three traditional varieties of Cr. neoformans. To be specific, we identified: 1) 51 strains of Cr. neoformans var. grubii, 2) 13 strains of Cr. neoformans var. neoformans, and 3) 6 strains of Cr. neoformans var. gattii. Given the geographical distribution of Cr. gattii, thought to be limited to the tropics, we sought to confirm the six positive cases obtained from the molecular identification study by cultivating all 70 strains on CGB media. Here, only the six strains of Cr. gattii (constituting Cr. neoformans var. gattii) were able to turn the media blue via hydrolyzing glycine whereas all 64 Cr. neoformans (constituted by Cr. neoformans var. neoformans and Cr. neoformans var. grubii) strains we unable to do so. Thus confirming the molecular test results. Perhaps, the important finding from the molecular study, is the uncovering of a restriction site for the enzyme SspI, which is present only in the distinct species, Cr. neoformans but absent in the distinct species, Cr. gattii. This is important as this eliminates sequencing from the identification process, thus shortening the time required to obtain test results and simultaneously cuts down the operational costs. In addition, it also makes it easier to optimise the protocol, as laboratory technicians will require no specialised training. Although a patient’s outcome is dependent on the timely release of an accurate diagnosis, treatment is also crucial. Today, the widespread usage of antifungals has led to increased resistance. Therefore, there is a constant need to find alternative drugs in order to improve patient outcomes. In this part of the study, we considered antimitochondrial drugs i.e. aspirin and oligomycin, as possible candidate drugs for controlling the growth of Cr. neoformans and Cr. gattii. In vitro susceptibility results, based on a direct comparative experiment, revealed that aspirin was more inhibitory than oligomycin, with 1 mM aspirin yielding at least 70 % growth reduction. Meanwhile, the checkerboard assay revealed that aspirin was not synergistic with fluconazole, however; it was indifferent, which is a frequent outcome in combined therapy. In future, it will be prudent to directly compare aspirin with fluconazole. Nonetheless, in this study, aspirin was proven to be useful as an antifungal agent with the highest concentration tested, 1 mM aspirin, being well within the recommended doses in the blood.