Development and evaluation of a rapid latex agglutination test using a monoclonal antibody to identify Candida dubliniensis colonies

Abstract

Cell components of the dimorphic pathogenic fungus Candida dubliniensis were used to prepare monoclonal antibodies (MAbs). One MAb, designated 12F7-F2, was shown by indirect immunofluorescence to be specific for a surface antigen of Candida dubliniensis yeast cells. No reactivity was observed with other fungal genera or with other Candida species, including Candida albicans, that share many phenotypic features with C. dubliniensis. The use of different chemical and physical treatments for cell component extraction suggested that the specific epitope probably resides on a protein moiety absent from C. albicans. However, we failed to identify the target protein by Western blotting, owing to its sensitivity to heat and sodium dodecyl sulfate. MAb 12F7-F2 was further used to develop a commercial latex agglutination test to identify C. dubliniensis colonies (Bichro-dubli Fumouze test; Fumouze Diagnostics). The test was validated on yeast strains previously identified by PCR and on fresh clinical isolates; these included 46 C. dubliniensis isolates, 45 C. albicans isolates, and other yeast species. The test had 100% sensitivity and specificity for C. dubliniensis isolated on Sabouraud dextrose, CHROMagar Candida, and CandiSelect media and 97.8% sensitivity for C. dubliniensis grown on Candida ID medium. The test is rapid (5 min) and easy to use and may be recommended for routine use in clinical microbiology laboratories and for epidemiological investigations.

FIG. 1.

Coomassie blue staining of 5 to 15% SDS-PAGE gels loaded with crude Zymolyase extracts (lanes A1 and A2) and 1.7 M (lanes B1 and B2), 1.6 M (lanes B3 and B4), and 1.5 M (lanes B5 and B6) HIC fractions of C. albicans (lanes A1, B1, B3, and B5) and C. dubliniensis (lanes A2, B2, B4, and B6) yeast cells grown at 22°C for 48 h on SDA. The molecular masses of standard proteins are listed on the left of the gels. Relevant components are indicated by arrowheads.

FIG. 2.

Immunofluorescence (A, C, and E) and phase-contrast (B, D, and F) photomicrographs of the same microscopic fields of C. dubliniensis strain ATCC MYA-646 stained with immune serum from a mouse immunized with the HIC 1.7 to 1.6 M ammonium sulfate fraction (A and B) or stained with MAb 12F7-F2 (C and E), after growth on SDA for 24 h at 22°C (A to D) or in medium 199 for 3 h at 37°C (E and F). Note the heterogeneous fluorescent labeling of yeast cells (arrows) and the lack of germ tube labeling. Bars, 10 μm.

FIG. 3.

MAb 12F7-F2 reactivity of chemical and enzymatic extracts of C. dubliniensis MYA-646 and C. albicans 66396 grown for 48 h at 37°C. Results are the means of triplicate determinations ± standard deviations for two independent experiments. 2ME, 2-mercaptoethanol.