Semiquantitative polymerase chain reaction enzyme immunoassay for diagnosis of disseminated candidiasis

Abstract

A polymerase chain reaction enzyme immunoassay (PCR-EIA) was developed for the semiquantitative of circulating candidal DNA in disseminated candidiasis due to Candida albicans. Polymerase chain reaction was based on primers from the internal transcribed ribosomal region. Binding of the product to a streptavidin-coated microtitration plate was mediated by a biotinylated capture probe. The product was digoxigenylated during PCR; this was the tag to which antibody was bound in the subsequent EIA. The optical density (OD) endpoint was < 0.1 in 15 sera from patients with no evidence of candidal infection (group 1) and in 13 of the 16 sera from colonized patients (group 2); it was > 0.1 in the other three sera from group 2 blood culture-negative patients who required intravenous amphotericin B for cure. The OD was positive in 28 patients with disseminated candidiasis (group 3), defined as positive blood cultures and successful treatment with amphotericin B (n = 11), positive blood culture confirmed at autopsy (n = 11), or negative blood culture first proven at necropsy (n = 6). In patients from whom multiple samples were available, recovery correlated with an optical density of < 0.1 by day 4 in four patients and by day 13 in the rest. In the five patients with fatal outcome from whom multiple samples were available, the mean OD rose from 0.174 to 0.668. Samples seeded with Candida albicans blastoconidia demonstrated that on OD of 0.220 was equivalent to 10 cfu. Assay of the group 3 sera by a commercial antigen detection test gave a corresponding sensitivity of 60% which rose to 67.9% when an in-house reverse passive latex agglutination test was used.