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Department of Medical Microbiology and Hospital Hygiene, University Hospital, D-18057 Rostock,1 Institute of Medical Microbiology and Hygiene at the Helios Medical Center, D-99089 Erfurt, Germany2
Received 18 November 2002/ Returned for modification 18 January 2003/ Accepted 3 April 2003
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ABSTRACT |
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Mannan is the main soluble immunodominant component of the outer cell wall layer of Candida species (6). The mannans are released from the cell walls of the yeasts during infection and circulate in the bloodstream (3, 4, 11). By using the monoclonal antibody EB-CA1 for the detection of 
-linked oligomannoside antigens of C. albicans, a latex agglutination assay was introduced in 1991 (1); subsequently, a more sensitive, one-stage sandwich microplate enzyme immunoassay (i.e., the PLATELIA CANDIDA Ag EIA; Bio-Rad, Munich, Germany) was developed in 1999 (11). The detection limit of this assay is 0.25 ng of mannan per ml of serum, and the clinical specificity and sensitivity are 98 and 40%, respectively (11).
In the present study, we evaluated the PLATELIA CANDIDA Ag EIA for cross-reactivities with the cell wall mannan antigens of 63 clinically relevant fungal species, including yeasts, molds, dermatophytes, and dimorphic fungi.
Fungal strains. A total of 130 fungal strains of 63 clinically relevant species were tested, including 96 strains of yeasts, 27 strains of molds, 5 strains of dermatophytes, and 2 strains of dimorphic fungi (Table 1).
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For the preparation of antigen extracts, four to five colonies of yeasts and dimorphic fungi were suspended in 2 ml of sterile phosphate-buffered saline (PBS; pH 7.2). Molds and dermatophytes were harvested and suspended in sterile PBS by using wet swabs, yielding a suspension of both conidia and hyphal fragments, as confirmed by microscopic examination. The fungal suspensions were vortexed and adjusted to a density of McFarland 5.0. Aliquots of the McFarland 5.0 standard suspensions (500 µl) in PBS were transferred into 1.5-ml safe-lock Eppendorf tubes (Eppendorf, Hamburg, Germany), kept in a boiling water bath for 5 min, and subsequently centrifuged at 10,000 x g for 10 min. Thirty microliters of the supernatants was diluted into 300 µl of 6% bovine serum albumin solution (BSA; DiaMed AG, Cressier sur Morat, Switzerland).
Control of antigen extracts. Successful antigen extractions were confirmed by testing the extracts of Aspergillus flavus, A. fumigatus, A. nidulans, A. niger, A. tamarii, A. terreus, Penicillium chrysogenum, P. marneffei, and Fusarium oxysporum in the Platelia Aspergillus EIA (Bio-Rad), because these molds are known to cross-react with the antibody of this assay (5). All antigen extracts tested positive. The antigen extracts of the four Cryptococcus neoformans strains tested positive by the Pastorex Crypto Plus latex agglutination test (Bio-Rad).
Detection of mannan antigens by PLATELIA CANDIDA Ag EIA. The EIA (Bio-Rad) was performed according to the instructions of the manufacturer by using 300 µl of fungal antigen extracts diluted in BSA in place of patient's serum.
In each experiment, we included a total of seven control samples: (i) one negative control serum sample, (ii-v) a series of four standard positive control samples of known mannan antigen concentrations (0.25, 0.5, 1.0, and 2.0 ng/ml, respectively), (vi) one positive control serum sample with an antigen concentration between 1.0 and 2.0 ng/ml, and (vii) one control of BSA without fungal supplement.
Briefly, 300 µl of the experimental samples and the controls was added to 100 µl of EDTA treatment solution in 1.5-ml safe-lock Eppendorf tubes. The tubes were kept in boiling water for 3 min and centrifuged at 10,000 x g for 10 min. Fifty microliters of the supernatants was transferred into EB-CA1-coated wells, which contained 50 µl of horseradish peroxidase-conjugated EB-CA1. The plates were covered with adhesive film and incubated at 37°C for 90 min. After incubation, the adhesive film was removed, and the plates were washed five times each with 370 µl of washing solution. Two hundred microliters of tetramethylbenzidine chromogen solution was added to each well, and the plates were incubated in darkness at room temperature for 30 min. The enzymatic reaction was stopped by adding 100 µl of stopping solution (1.5 N sulfuric acid) in each well, and the absorbance was read at 450 and 620 nm on a microplate reader (LP400; Bio-Rad). Reactions were performed in duplicate. The mannan concentrations were calculated from the calibration curve of standard controls.
Test medium for the fungal antigen extracts. As PBS was found to lead to nonspecific false-positive results in the assay, several media, including 0.85% sodium chloride, sterile distilled water, human serum, and a series of BSA solutions ranging in concentration from 0.1 to 6%, were examined. All the protein-free solutions and the low BSA concentrations up to 2.0% yielded results that varied between 0.1 and 0.8 ng/ml and thus could not safely be considered negative. The BSA solutions with concentrations of 4.0 and 6.0% and human serum were consistently negative (<0.25 ng/ml). The 6% BSA solution was used as the diluent for the fungal antigen extracts because it resembles the protein concentration in human serum.
Assay controls. The results of the standard controls consistently fell into the range of the values given by the manufacturer. The results from the additional control of plain 6% BSA solution without fungal antigen remained below the detection level in each experiment (<0.25 ng/ml).
Reproducibility. When aliquots from one antigen extract of C. albicans ATCC 24433 were tested in four separate experiments, the antigen concentrations were 0.7, 0.6, 1.2, and 0.9 ng/ml (mean, 0.85 ng/ml; standard deviation, 0.23 ng/ml). Four separate antigen extracts of C. albicans ATCC 76615 and of C. glabrata RV6/97, respectively, were prepared and tested in separate experiments. The antigen concentrations of the four extracts of C. albicans ATCC 76615 were 0.8, 0.6, 0.9, and 0.6 ng/ml (mean, 0.73 ng/ml; standard deviation, 0.13 ng/ml). The antigen concentrations of the four extracts of C. glabrata RV6/97 were 1.9, 1.5, 2.1, and 2.0 ng/ml (mean, 1.88 ng/ml; standard deviation, 0.23 ng/ml).
Cross-reactivity of the PLATELIA CANDIDA Ag EIA. Among 63 clinically relevant fungal species, isolates of the seven species C. albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. lusitaniae, and C. tropicalis led to positive results. G. candidum and F. verticillioides yielded borderline-positive to weakly positive reactions. This finding was confirmed by repeated testing of the strain HD4274/00 of F. verticillioides on five independent occasions. The identity of this strain was confirmed by the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. Antigen preparations from 54 other fungal species did not lead to detectable amounts of mannan (Table 1).
The PLATELIA CANDIDA Ag EIA was developed for the detection of circulating Candida mannan antigen in the bloodstream of patients suffering from invasive candidiasis. The test is based on the use of a monoclonal antibody, EB-CA1, which recognizes a mannopentose epitope of C. albicans. This epitope is present in large amounts on numerous mannoproteins in the cell wall of C. albicans. It has also been found in large amounts on mannoproteins of C. glabrata, C. tropicalis, and C. guilliermondii but has only been found to a lesser extent on C. krusei, C. kefyr, and C. parapsilosis (2, 12). As invasive candidiasis is caused more and more by nonalbicans species (7, 8), an ideal antigen test would cross-react with a wide range of different Candida species but would remain negative with the antigens from other clinically relevant fungi. Therefore, we checked the cross-reactivity of the PLATELIA CANDIDA Ag EIA by subjecting BSA solutions spiked with antigen extracts from a wide variety of fungi to this assay. The test proved to be acceptably reproducible in our hands—one individual antigen extract as well as separately prepared antigen extracts from one fungal strain showed very similar results upon separate, independent testing. We recorded positive results with preparations from seven different Candida species, including the most prevalent pathogenic species—C. albicans, C. glabrata, and C. tropicalis.
In contrast to Jacquinot et al., who reported weak cross-reactivities of EB-CA1 with C. parapsilosis and C. krusei when Western blot analysis was used (2), we were unable to measure antigens from the five strains of C. parapsilosis and the four strains of C. krusei that we tested. Two clinical studies (10, 13) support our finding of weak cross-reactions of these Candida species. They reported a sensitivity for the detection of mannanemia of 63 to 78% for infections caused by C. albicans, C. glabrata, and C. tropicalis, while that for C. parapsilosis and C. krusei was only 30 to 44%.
One strain of F. verticillioides and two strains of G. candidum repeatedly led to borderline-positive results. Antigen preparations from Fusarium species other than F. verticillioides did not cross-react. The reactivity of the PLATELIA CANDIDA Ag EIA with serum samples from patients with invasive fusariosis is unknown.
In conclusion, the PLATELIA CANDIDA Ag EIA may be used for the detection of circulating antigens of C. albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. lusitaniae, and C. tropicalis. We were unable to record a reactivity of the assay with antigens from C. parapsilosis and C. krusei. Weak cross-reactions appear to be possible with Geotrichum and Fusarium, which rarely cause invasive infections. No false-positive results were caused by Cryptococcus or Aspergillus, which represent other common invasive fungal pathogens.
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ACKNOWLEDGMENTS |
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We thank Gabriele Born, Anjana Gröger, and Barbara Schirlitz for excellent technical assistance, and we thank Andreas Podbielski for critical reading of the manuscript.
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FOOTNOTES |
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REFERENCES |
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