Molecular characterization of invasive yeast isolates with increased resistance to antifungals

Invazivne okužbe z glivami so v porastu zaradi izboljšanih metod zdravljenja populacije, ki ima večje tveganje za razvoj teh okužb. Mednje sodijo imunska oslabelost zaradi prejemanja bioloških zdravil, kemoterapevtikov in kortikosteroidov, nevtropenija ob zdravljenju s presaditvijo krvotvornih matičnih celic, parenteralna prehrana, hemodializa, umetna ventilacija, žilni katetri, ekstremne starostne skupine, nedonošenčki ter bolniki z rakavo boleznijo. Zaradi pozne diagnostike in slabo občutljivih metod, ki privedejo do diagnoze, je zdravljenje pogosto uvedeno pozno, kar je pomemben dejavnik, ki vpliva na prognozo bolezni. Invazivno kandidozo v našem geografskem področju najpogosteje povzročata vrsti Candida albicans in Candida glabrata. V raziskavi smo želeli opredeliti odpornost kliničnih izolatov C. albicans in C. glabrata, ki so z metodo antimikotičnega gradienta izkazali mejno občutljivost ali odpornost proti anidulafunginu in mikafunginu. Z referenčno mikrodilucijsko metodo po EUCAST smo izolate potrdili kot občutljive ali odporne. Sledila je molekularna opredelitev 26 izolatov C. glabrata, ki so bili opredeljeni kot odporni. Izolacija DNA-izolatov je potekala na napravi MagNA Pure Compact (Roche, Mannheim, Nemčija). Sledila je verižna reakcija s polimerazo na napravi Veriti (Applied Biosystems, Foster City, Združene države Amerike), ki ji je sledila agarozna gelska elektroforeza za namen določevanja FKS-mutacij, ki posredujejo odpornost proti ehinokandinom. V raziskavi smo ugotovili, da so bile mutacije FKS prisotne pri 69,2 % odpornih izolatov C. glabrata.The incidence of invasive fungal infections has increased. We assign this to improved methods of treating the population at risk for developing invasive fungal infections. Risk factors include immunodeficiency caused by treatments with biological drugs, chemotheraupetics, corticosteroids, neutropenia connected to hematopoietic stem cell transplantation, parentheral nutrition, hemodialysis, artificial ventilation, venous catheters, extremes of age, prematurely born babies and cancer patients. Late diagnosis and low sensitivity of methods for detecting fungal infections are the reasons for delayed start of treatment, which has major effect on the prognosis of the disease. Most of the cases of invasive candidiasis in our geographic region are caused by Candida albicans and Candida glabrata. Our goal was to determine susceptibility of clinical isolates C. albicans and C. glabrata, which were defined as intermediate or resistant to anidulafungin and micafungin with gradient diffusion method. We retested selected isolates with reference microdilution EUCAST method and they were determined as susceptible or resistant. Twenty-six resistant isolates of C. glabrata were selected for molecular characterization. The isolation of DNA was conducted on MagNA Pure Compact (Roche, Mannheim, Germany). The next step was polymerase chain reaction on Veriti (Applied Biosystems, Foster City, United States of America), followed by agarose gel electrophoresis for determination of FKS mutations, which confer resistance to echinocandins. We have concluded that FKS mutations were present in 69, 2 % resistant isolates C. glabrata