A nationwide survey of clinical laboratory methodologies for fungal infections

Goodwin, Shirley; Fiedler‐Kelly, Jill; Grasela, Thaddeus H.; Schell, Wiley A.; Perfect, John R.

doi:10.1080/02681219280000201

Cited by 13 publications

(11 citation statements)

References 17 publications

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“…Therefore, rapid species identification will be more critical for effective disease therapy and control (Polak, 2003). Conventional diagnostic tests, such as blood culture and biochemical tests, which lack sufficient sensitivity and specificity for early diagnosis of invasive fungal infections, may often require 2 or more days and may be inaccurate (Espinel-Ingroff et al, 1998;Goodwin et al, 1992;Hazen, 1995 In this paper, we describe a simple real-time PCR assay with the LightCycler system employing species-specific primers and SYBR Green fluorescent dye for detection and species identification of fungal strains. This assay offers the advantage that the conventional PCR can be easily adapted to realtime format without the need for complicated probe design.…”

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Species identification of medically important fungi by use of real-time LightCycler PCR

Hsu

Chen

et al. 2003

Journal of Medical Microbiology

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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 ...

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Species identification of medically important fungi by use of real-time LightCycler PCR

Hsu

Chen

et al. 2003

Journal of Medical Microbiology

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“…Since opportunistic mycoses are often grave, the early, rapid, and accurate identification of the pathogenic fungus is critical for timely, appropriate management. The conventional identification of pathogenic fungi in the clinical microbiology laboratory is based on morphological and physiological tests, often requires 3 or more days, and may be inaccurate (12,14).…”

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Rapid Identification of Pathogenic Fungi Directly from Cultures by Using Multiplex PCR

2002

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A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus. Another set of previously published ITS primers, CN4 and CN5, were used to identify Cryptococcus neoformans. Three sets of primers were used in one multiplex PCR to identify three different species. Six different species of pathogenic fungi can be identified with two multiplex PCRs. Furthermore, instead of using templates of purified genomic DNA, we performed the PCR directly from yeast colonies or cultures, which simplified the procedure and precluded contamination during the extraction of DNA. A total of 242 fungal isolates were tested, representing 13 species of yeasts, four species of Aspergillus, and three zygomycetes. The multiplex PCR was tested on isolated DNA or fungal colonies, and both provided 100% sensitivity and specificity. However, DNA from only about half the molds could be amplified directly from mycelial fragments, while DNA from every yeast colony was amplified. This multiplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify common clinical fungal isolates.

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“…However, in spite of the advances in biochemical commercial systems, so far they do not allow for the rapid diagnosis of invasive fungal infections (Goodwin et al 1992;Hazen 1995;Espinel-Ingroff et al 1998), leading to a late introduction of the specific antifungal therapy (Goodwin et al 1992;Hazen 1995;Espinel-Ingroff et al 1998;Hsu et al 2003;Huang et al 2006). In this sense, molecular biology techniques, such as PCR, might be more accurate and efficient, due to their reproducibility, high specificity, and sensitivity (Hidalgo et al 2000;Kanbe et al 2002Kanbe et al , 2003Hsu et al 2003), allowing same day identification.…”

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confidence: 99%