RAPID DIAGNOSTIC TEST FOR HUMAN FUNGAL PATHOGENS

LOCALIZED AND SYSTEMIC MYCOSES CAUSED BY PATHOGENIC FUNGI (E.G., THE YEASTS CANDIDA ALBICANS AND CRYPTOCOCCUS NEOFORMANS) REPRESENT A SERIOUS HEALTH THREAT, ESPECIALLY TOIMMUNOCOMPROMISED PATIENTS AMONG WHOM INVASIVE CANDIDIASIS HAS HIGH MORBIDITY AND MORTALITY. CURRENTLY, IDENTIFICATIONOF FUNGAL PATHOGENS REQUIRES ISOLATION OF PURE CULTURES, FOLLOWED BY TESTING FOR SUBSTRATE ASSIMILATION AND FERMENTATION PATTERNS. THE LONG-TERM OBJECTIVE IS DEVELOPMENT OF A SERIES OF INNOVATIVE DIAGNOSTIC TESTS FOR SPECIFIC HUMAN FUNGAL PATHOGENS IN CLINICAL SAMPLES USING RAPID NUCLEIC ACID HYBRIDIZATION METHODOLOGY. THE IMMEDIATEGOAL IS TO FOCUS ON DEVELOPMENT OF A DIAGNOSTIC TEST FOR CANDIDA ALBICANS BASED ON HYBRIDIZATION OF A SPECIFIC DNA PROBE WITH HIGHLY AMPLIFIED RIBOSOMAL RNA (RRNA) SEQUENCES IN DISRUPTED YEAST CELLS. IN PHASE I, CANDIDA ALBICANS RRNAGENE FRAGMENTS WILL BE CLONED USING PURIFIED, IN VITRO RADIOLABELED 18S RRNA AS A PROBE. SUBCLONES FOUND TO MAP INA PUTATIVE UNIQUE REGION CORRESPONDING TO NUCLEOTIDES 642 TO702 OF PREVIOUSLY DESCRIBED EUKARYOTIC 18S RRNAS WILL BE DNASEQUENCED. DNA PROBES HOMOLOGOUS TO THIS AND OTHER REGIONS WILL BE ISOLATED OR SYNTHESIZED. THEY WILL THEN BE TESTED FOR SPECIFIC HYBRIDIZATION TO CANDIDA ALBICANS RNA IN PURE AND MIXED CULTURES. IN PHASE II, TEST FORMATS AND PROCEDURES WILL BE OPTIMIZED FOR USE WITH CLINICAL SAMPLES.