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

Abstract

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.

Afrikaans: Met hierdie verhandeling is gepoog om die epidemiologie van cryptococcose te bestudeer, deur eerstens die voorkoms daarvan oor ‘n tydperk van twee jaar, 2011 en 2012, te skat. Die hoofbevindinge van hierdie deel van die studie sluit in: 1) gevalle was meer algemeen onder swart mense (Afrikane, Kleurlinge en Indieërs), en dit stem ooreen met die stelling deur die WGO dat siektes soos cryptococcose meer armoede verwant is, 2) die verspreidingspatroon van cryptococcose oor verskillende ouderdomsgroepe is dieselfde as die van persone met MIV en 3) die hoeveelheid cryptococcosegevalle was laag vir die studietydperk (verteenwoordig minder as 0.1 % van die bevolking van Bloemfontein), dit was verrassend en onverwags gegewe die groot MIV positiewe bevolking in Suid-Afrika, en dus in Bloemfontein, wat die risiko loop om hierdie VIGS-definiërende siekte op te doen. Uit die literatuur is dit bekend dat die metodes wat tans gebruik word vir die roetine diagnose van cryptococcose, dikwels teenstrydige resultate lewer, en sodoender die hoeveelheid gerapporteerde gevalle beïnvloed, wat belangrik is vir gesondheidsbeamptes. Belangriker nog, hierdie strydighede het verreikende gevolge aangesien dit die pasiënte se uitkomste negatief mag beïnvloed. Dus het ons gepoog om die bruikbaarheid van molekulêre metodes vir die identifikasie van die veroorsakende agente van cryptococcose, nl. Cr. neoformans en Cr. gattii te bepaal.. Hier het die intra-spesifieke variasie in die ITS, insluitend die 5.8 geen, tussen die stamme hulle afbakening in die drie tradisionele variëteite van Cr. neoformans toegelaat. Om spesifiek te wees, het ons die volgende geïdentifiseer: 1) 51 stamme van Cr. neoformans var. grubii, 2) 13 stamme van Cr. neoformans var. neoformans, en 3) 6 stamme van Cr. neoformans var. gattii. Gegewe die geografiese verspreding van Cr. gattii, wat vermoedelik beperk is tot die trope, het ons gepoog om die ses positiewe gevalle geïdentifiseer deur die molekulêre identifikasiestudie te bevestig deur al 70 stamme op CGB media te kweek. Slegs die ses Cr. gattii stamme (bestaande uit Cr. neoformans var. gatti) kon die media blou kleur via die hidrolise van glisien, terwyl al 64 Cr. neoformans stamme (bestaande uit Cr. neoformans var. neoformans en Cr. neoformans var. grubii) dit nie kon doen nie. Dus bevestig dit die molekulêre resultate. Die belangrikste bevinding van die molekulêre studie is moontlik die onthulling van ‘n beperkingsnypunt vir die ensiem SspI, wat slegs teenwoordig is in die spesie, Cr. neoformans maar afwesig is in die spesie, Cr. gattii. Dit is belangrik aangesien dit basispaaropeenvolgingbepaling uit die identifikasieproses haal, en dus die tyd wat nodig is om resultate te kry asook die operasionele koste veminder. Daar benewens vergemaklik dit die optimisering van die protokol, aangesien laboratoriumtegnici geen gespesialiseerde opleiding sal nodig hê nie. Alhoewel ‘n pasiënt se uitkoms afhang van die tydige beskikbaarstelling van ‘n akkurate diagnose, is behandeling ook noodsaaklik. Huidiglik het die algemene gebruik van antifungale middels gelei tot verhoogde weerstandbiedendheid. Dus is daar ‘n volgehoue noodsaaklikheid om alternatiewe middels te vind om pasiënte se uitkomste te verbeter. In hierdie deel van die studie is die anti-mitochondriale middels, aspirien en oligomisien, geëvalueer as moontlike kandidate vir die beheer van die groei van Cr. neoformans en Cr. gattii. In vitro vatbaarheidsresultate, gebaseer op ‘n direkte vergelykende eksperiment, het getoon dat aspirien meer inhiberend is as oligomisien, met 1 mM aspirien wat groei met ten minste 70 % verminder het. Intussen het die skaakbordtoets getoon dat aspirien nie sinergisties was saam met flukonazool nie, maar onverskillig, wat dikwels die uitkoms van kombinasieterapie is. In die toekoms sal dit wys wees om aspirien direk met flukonazool te vergelyk. Nietemin is aspirien in hierdie studie bewys as ‘n nuttige antifungale middel met die hoogste getoetste konsentrasie, 1 mM aspirien, wat binne die aanbevole dosis in die bloed is.