Competitive binding inhibition enzyme-linked immunosorbent assay that uses the secreted aspartyl proteinase of Candida albicans as an antigenic marker for diagnosis of disseminated candidiasis

Abstract

The secreted aspartyl proteinases (Saps) of Candida albicans have been implicated as virulence factors associated with adherence and tissue invasion. The potential use of proteinases as markers of invasive candidiasis led us to develop a competitive binding inhibition enzyme-linked immunosorbent assay (ELISA) to detect Sap in clinical specimens. Daily serum and urine specimens were collected from rabbits that had been immunosuppressed with cyclophosphamide and cortisone acetate and infected intravenously with 10(7) C. albicans blastoconidia. Disseminated infection was confirmed by organ culture and histopathology. Although ELISA inhibition was observed when serum specimens from these rabbits were used, more significant inhibition, which correlated with disease progression, occurred when urine specimens were used. Urine collected as early as 1 day after infection resulted in significant ELISA inhibition (mean inhibition +/- standard error [SE] compared with preinfection control urine, 15.7% +/- 2.7% [P < 0.01]), and inhibition increased on days 2 through 5 (29.4% +/- 4.8% to 44.5% +/- 3.5% [P < 0.001]). Urine specimens from immunosuppressed rabbits infected intravenously with Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Aspergillus fumigatus, or Staphylococcus aureus were negative in the assay despite culture-proven dissemination. Nonimmunosuppressed rabbits receiving oral tetracycline and gentamicin treatment were given 2 x 10(8) C. albicans blastoconidia orally or intraurethrally to establish colonization of the gastrointestinal tract or bladder, respectively, without systemic dissemination; urine specimens from these rabbits also gave negative ELISA results. Dissemination to the kidney and spleen occurred in one rabbit challenged by intragastric inoculation, and urine from this rabbit demonstrated significant inhibition in the ELISA (mean inhibition +/- SE by day 3 after infection, 32.9% +/- 2.7% [P < 0.001]). The overall test sensitivity was 83%, the specificity was 92%, the positive predictive value was 84%, the negative predictive value was 91%, and the efficiency was 89% (166 urine samples from 33 rabbits tested). The specificity, positive predictive value, and efficiency could be increased to 97, 95, and 92%, respectively, if at least two positive test results were required for a true positive designation. The ELISA was sensitive and specific for the detection of Sap in urine specimens from rabbits with disseminated C. albicans infection, discriminated between colonization and invasive disease, reflected disease progression and severity, and has the potential to be a noninvasive means to diagnose disseminated candidiasis.

FIG. 1.

Time course for inhibition ELISA with C. albicans strain 36B-infected rabbits. Rabbits were infected on day 0, and urine was collected daily and tested in the inhibition ELISA. The number of rabbits tested for each day is shown in parentheses. *, P < 0.01 compared to day 0 inhibition; **, P < 0.001 compared to day 0 inhibition. Cross-hatched bars, mean percent ELISA inhibition values; open bars, SEs from the mean percent ELISA inhibition values.

FIG. 2.

Histopathological section of kidney tissue from a C. albicans-infected rabbit, showing tissue invasion with C. albicans (darkly staining material). Gormori silver stain was used. Approximate magnification, ×540.

FIG. 3.

Pepstatin A-agarose column chromatographic profiles for urine from a C. albicans-infected rabbit. Urine was applied to a pepstatin A-agarose column or a sham column (agarose support only, no pepstatin A), and effluent and eluate fractions were collected as described in Materials and Methods. Solid line, pepstatin A-agarose elution profile; dashed line, sham column elution profile; dotted and dashed line, pH elution gradient.

FIG. 4.

Western blot analysis of effluent and eluate fractions of urine from a C. albicans-infected rabbit passaged over pepstatin A-agarose or sham (agarose beads only, no pepstatin A) columns. Blots were tested with horseradish peroxidase-labeled polyclonal anti-Sap antibodies as described in Materials and Methods. Lane 1, sham column effluent (two bands noted); lane 2, pepstatin A column effluent (depleted, barely visible bands noted); lane 3, empty; lanes 4 and 5, duplicates of pepstatin A-agarose column eluate (two bands noted); lane 6, empty; and lane 7, sham column eluate (no bands noted). Molecular mass markers, BRL prestained high-molecular-mass standards (Bethesda Research Laboratories, Gaithersburg, Md.).