Invasive fungal infection has become a major cause of morbidity and mortality in immunocompromised patients. Rapid identification of pathogenic fungi to species level is critical for disease treatment. A real-time LightCycler assay aiming at rapid detection and species identification of pathogenic fungi from clinical isolates was developed. Template DNAs of different species were amplified and detected in real time by employing SYBR Green fluorescent dye. The target sequences for species-level detection were located between the 18S and 28S rDNA. Seven fungal species encountered frequently in the clinical setting, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida guilliermondii and Cryptococcus neoformans, could be discriminated by species-specific primers and confirmed by melting-curve analyses. The range of linearity was from 1 ng to 1 pg (ìl À1 water) and the sensitivity was 1 pg fungal DNA ìl À1 . Identification by this real-time PCR method matched biochemical identification for all 58 clinical strains. Therefore, the method is simple, rapid and sensitive enough for detection and identification of several fungal species.
INTRODUCTIONInvasive fungal infections have become major causes of morbidity and mortality among immunocompromised patients (Dasbach et al., 2000;Ellis et al., 2001), such as some neutropenic patients with haematological malignancies and recipients of allogenic bone marrow transplants (Denning, 1998) as well as individuals with AIDS (Mitchell & Perfect, 1995). Candida species are now the fourth most frequent cause of nosocomial blood-stream infections in critically ill patients in the United States. At a teaching hospital in Taiwan, yeast infection was identified as the leading cause of nosocomial infection (Chen et al., 1997). The increasing prevalence of yeast infections highlights the need for simple and rapid methods to identify clinically important fungi in a microbiological laboratory.Each Candida species has a different degree of susceptibility to common antifungal agents. For instance, Candida krusei is innately resistant and Candida glabrata, Candida guilliermondii and Candida dubliniensis are less susceptible to fluconazole than other Candida species (Orozco et al., 1998;Piemonte et al., 1996). Emergence of secondary resistance in Candida lusitaniae to amphotericin B has also been observed and monitored closely (Pfaller et al., 2003). Current recommendations suggest that invasive fungal infections, such as candidiasis and aspergillosis, should be treated empirically, because the current diagnoses are difficult and time-consuming (Rex et al., 2000;Stevens et al., 2000). However, there is great concern that such practice would result in the emergence of resistant fungal pathogens.As more and more alternative antifungal agents with various spectra of activities are developed and become available, treatment according to accurate diagnosis has become even more important. Therefore, rapid species identification will be more critical for ...