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

[摘要] English: In this dissertation, an attempt was made to study the epidemiology of cryptococcosisby first estimating the incidence rates over a two-year period, 2011 and 2012. The majorfindings from this part of the study included establishing that: 1) cases were moreprevalent among Blacks (Africans, Coloureds and Indians), and this is in line with theassertion by WHO that diseases such as cryptococcosis are more poverty-related, 2)the distribution pattern of cryptococcosis across different age groups mirrored that ofHIV-infected persons, and 3) the number of cryptococcosis cases were quite low for thestudy period (representing less than 0.1 % of the Bloemfontein population), this wassurprising and unexpected given the huge HIV positive population in South Africa, andby extension in Bloemfontein, that is at risk of acquiring this AIDS-defining illness. It isdocumented in literature that the currently employed methods for the routine diagnosisof cryptococcosis often yield inconsistent test results, thereby; influencing the number ofreported cases, which are important for health officials. But more importantly, theseinconsistencies have far reaching consequences as they may negatively influencepatient outcomes. Therefore, we sought to investigate the usefulness of molecularmethods in identifying the etiological agents of cryptococcosis viz. Cr. neoformans andCr. gattii. Here, the ITS, including the 5.8 gene, intra-specific variation between thetested 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 thetropics, we sought to confirm the six positive cases obtained from the molecularidentification study by cultivating all 70 strains on CGB media. Here, only the six strainsof Cr. gattii (constituting Cr. neoformans var. gattii) were able to turn the media blue viahydrolyzing glycine whereas all 64 Cr. neoformans (constituted by Cr. neoformans var.neoformans and Cr. neoformans var. grubii) strains we unable to do so. Thus confirmingthe molecular test results. Perhaps, the important finding from the molecular study, isthe uncovering of a restriction site for the enzyme SspI, which is present only in thedistinct species, Cr. neoformans but absent in the distinct species, Cr. gattii. This isimportant as this eliminates sequencing from the identification process, thus shorteningthe time required to obtain test results and simultaneously cuts down the operationalcosts. In addition, it also makes it easier to optimise the protocol, as laboratorytechnicians will require no specialised training. Although a patient's outcome isdependent 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 andoligomycin, as possible candidate drugs for controlling the growth of Cr. neoformansand 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 atleast 70 % growth reduction. Meanwhile, the checkerboard assay revealed that aspirinwas not synergistic with fluconazole, however; it was indifferent, which is a frequentoutcome in combined therapy. In future, it will be prudent to directly compare aspirinwith fluconazole. Nonetheless, in this study, aspirin was proven to be useful as anantifungal agent with the highest concentration tested, 1 mM aspirin, being well withinthe recommended doses in the blood.

[发布机构] University of the Free State