Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay

Abstract

We describe a rapid and reproducible PCR assay for quantitation of the Candida albicans ribosomal DNA (rDNA) in clinical blood samples based on the TaqMan principle (Applied Biosystems), in which a signal is generated by cleavage of a template-specific probe during amplification. We used two fluorogenic probes based on universal, fungus-specific primers, one for the detection of C. albicans species DNA and one for the detection of all Candida genus DNA. C. albicans blastoconidia mixed with whole blood in a titration experiment yielded a linear PCR signal over a range of 3 orders of magnitude. The TaqMan-based PCR assay for C. albicans exhibited a low limit of detection (5 CFU/ml of blood) and an excellent reproducibility (96 to 99%). While the C. albicans species-specific probe had 100% specificity for C. albicans, all Candida genus-specific probes cross-reacted with other organisms likely to coinfect patients with C. albicans infections. On the basis of these data, we determined the C. albicans loads with a species-specific probe from 122 blood samples from 61 hematology or oncology patients with clinically proven or suspected systemic Candida infections. Eleven positive samples exhibited a wide range of C. albicans loads, extending from 5 to 100,475 CFU/ml of blood. The sensitivity and specificity of the present assay were 100 and 97%, respectively, compared with the results of blood culture. These data indicate that the TaqMan-based PCR assay for quantitation of C. albicans with a species-specific probe provides an attractive alternative for the identification and quantitation of C. albicans rDNA in pure cultures and blood samples.

FIG. 1.

Quantitation of C. albicans blastoconidia in seeded human blood specimens and comparative sensitivities of DNA detection by using the CA-FAM and All-CAN-TET probes. Serially diluted C. albicans blastoconidia were mixed with blood, and the extracted DNA was used in duplicate as the template in the TaqMan assay. (A) The relative fluorescence refers to the increase in the fluorescence emission of the reporter dye relative to that of the passive reference dye. The threshold fluorescence was determined by using the CA-FAM probe. No signal was obtained when double-distilled water was used as a negative control. (B) Log₁₀ concentration of blastoconidia plotted against the mean C_T values generated from the amplification plots in panel A (CA-FAM probe) and those generated with the All-CAN-TET probe.

FIG. 2.

Linear range for the TaqMan assay for C. albicans by using the CA-FAM and All-CAN-TET probes. Two identical dilution series of C. albicans DNA (1,000 to 0.05 pg DNA, corresponding to 10⁵ to 5 CFU) were used as external standards. Each dilution was tested in triplicate. Linear regression analysis was performed on the mean C_T values for each dilution series plotted against the log₁₀ concentration of standard DNA.