Simple and rapid detection of Candida albicans DNA in serum by PCR for diagnosis of invasive candidiasis

Abstract

A rapid and sensitive PCR assay for the detection of Candida albicans DNA in serum was established. DNA from human serum samples was purified using the QIAamp blood kit, which proved to be a fast and simple method for isolating minute amounts of Candida DNA from clinical specimens for diagnosis of invasive candidiasis. Universal primer sequences used in the PCR assay are derived from the internal transcribed spacer rRNA gene of fungi, whereas the biotinylated hybridization probe used in a DNA enzyme immunoassay (DEIA) binds specifically to C. albicans DNA. The sensitivity of this PCR-DEIA method is very high; the detection limit for genomic Candida DNA is one C. albicans genome per assay. Blood from uninfected and infected persons, ranging from healthy volunteers, patients with mucocutaneous infections, and patients at risk to develop a systemic Candida infection to patients with an established systemic candidiasis, was analyzed for the presence of C. albicans to diagnose fungal infection. Candida DNA could not be detected in sera of 16 culture-negative controls and from 11 nonsystemic candidal infections by PCR or DEIA. Blood cultures from patients at risk were all negative for Candida, whereas all blood cultures from systemic candidiasis patients were positive. However, Candida DNA could be detected by PCR and DEIA in the serum from three out of nine patients who were at risk for a systemic infection and in the serum of all seven patients who had already developed an invasive Candida infection. PCR is more sensitive than blood culture, since some of the patients at risk for invasive yeast infection, whose blood cultures were all negative for Candida, tested positive in the PCR amplification. These results indicate the potential value of PCR for detecting C. albicans in serum samples and for identifying patients at risk for invasive candidiasis.

FIG. 1

Ethidium bromide-stained polyacrylamide gel of PCR products obtained with primers ITS3 and ITS4 and DNAs from different Candida species. Lanes: 1, molecular weight marker (100-bp ladder); 2, C. albicans; 3, C. parapsilosis; 4, C. glabrata; 5, C. krusei; 6, C. pelliculosa; 7, C. tropicalis; 8, C. rugosa; 9, C. guilliermondii; 10, molecular weight marker (100-bp ladder).

FIG. 2

Ethidium bromide-stained polyacrylamide gel of PCR products obtained with DNA extracted from serum samples of systemic candidiasis patients (group 4). Lanes: 1, molecular weight marker (100-bp ladder); 2, C. albicans DNA; 3, patient 37; 4, patient 38; 5, patient 39a (before therapy); 6, patient 39b (after therapy); 7, patient 40; 8, negative control. The size of the C. albicans PCR product is indicated by an arrow.