Competitive Binding Inhibition Enzyme-Linked Immunosorbent Assay That Uses the Secreted Aspartyl Proteinase ofCandida albicansas 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 ؋ 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.

Rapid Differentiation of Aspergillus Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay

“…Rabbit models of disseminated candidiasis and IA, previously described by our laboratory (26,49), were used as the source of infected tissues. DNA extracted from frozen, homogenized kidney tissue was PCR amplified using primers ITS3 and ITS4.…”

“…Rabbit models of IA and candidiasis developed previously (26,49) were used to detect fungal DNA in tissues from infected animals. Rabbits were euthanized and necropsied on day 3 after infection, and tissue samples (5 by 5 by 1 mm) were placed immediately after removal onto aluminum foil, wrapped tightly, and submerged into liquid nitrogen for 1 min.…”

Aspartic Protease Inhibitors as Potential Anti-Candida albicans Drugs: Impacts on Fungal Biology, Virulence and Pathogenesis

“…For example, high titers of anti-Sap antibodies were observed in the sera of patients with candidiasis [123], and Sap antigens were detected in biopsies of oral epithelial lesions collected from HIVinfected patients [105]. Regarding the Saps immunogenic properties, a competitive binding inhibition enzyme-linked immunosorbent assay (ELISA) was described as sensitive and specific for the detection of Sap antigens in urine specimens from rabbits with disseminated C. albicans infection, being a potential antigenic marker to be used in a noninvasive means in order to diagnose disseminated candidiasis [124].…”

Dissecting Candida albicans Infection from the Perspective of C. albicans Virulence and Omics Approaches on Host–Pathogen Interaction: A Review

“…Of all Candida species, C. albicans remains the most common species of bloodstream infections in hospitals. C. albicans is responsible for 59% of nosocomial candidemia and for 55% of bloodstream infections [9,10,11]. …”