PCR-enzyme immunoassay of rDNA in the diagnosis of candidemia and comparison with amplicon detection by agarose gel electrophoresis

Abstract

We have developed a semi-nested PCR-enzyme immunoassay (snPCR-EIA) for the detection of Candida species in serum specimens, and the sensitivity of amplicon detection was compared with the detection of amplified product by agarose gel electrophoresis (AGE). The universal outer primers amplified the 3′ end of 5.8S and the 5′ end of 28S rDNA including the internally transcribed spacer 2 (ITS2) in PCR with genomic DNA as template from all the tested Candida species. The biotin-labeled species-specific primers derived from ITS2 from the four commonly encountered Candida species, viz. C. albicans, C. tropicalis, C. parapsilosis and C. glabrata, together with digoxigenin-labeled reverse primer amplified species-specific DNA in the reamplification step of the snPCR. The snPCR-EIA was positive for genomic DNA recovered from 0.06 Candida cells in culture and one organism/ml in spiked serum specimens. Evaluation of snPCR-EIA and snPCR-AGE for specific identification of Candida species with 26 clinical Candida isolates showed 100% concordant results with Vitek and ID32C yeast identification systems. Further evaluation of snPCR-EIA and snPCR-AGE for detection of Candida species in serum samples from culture proven ( n=6) and suspected ( n=10) patients showed concordance with the corresponding species isolated in culture. The serum samples from none of the healthy volunteers ( n=10) were positive for the presence of Candida DNA by snPCR-EIA or snPCR-AGE. Our results show that the snPCR-EIA has the same sensitivity as snPCR-AGE, however, it offers additional advantages of simultaneous testing of a large number of serum samples and avoids the use of ethidium bromide, a potent mutagen. The snPCR-EIA could, therefore, be a method of choice for the diagnosis of candidemia.