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Journal of Clinical Microbiology, April 2003, p. 1536-1542, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1536-1542.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Combined Use of Paracoccidioides brasiliensis Recombinant 27-Kilodalton and Purified 87-Kilodalton Antigens in an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Paracoccidioidomycosis

Soraya Díez,^1,2* Beatriz L. Gómez,^1,2 Juan G. McEwen,² Angela Restrepo,² Rod J. Hay,^1,3 and Andrew J. Hamilton¹

St. John's Institute of Dermatology, Guy's Hospital, London, England,¹ Corporación para Investigaciones Biológicas, Medellín, Colombia,² Queens University Belfast, Whitla Medical Building, Belfast, Northern Ireland³

Received 16 October 2002/ Returned for modification 21 November 2002/ Accepted 14 December 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The diagnosis of paracoccidioidomycosis (PCM) has relied on the identification of the host's humoral response by using a variety of immunological methods, such as complement fixation and immunodiffusion. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of undefined antigens. The use of combinations of purified, well-characterized antigens appears preferable and may yield optimum results. Accordingly an indirect enzyme-linked immunosorbent assay (ELISA) using combinations of the previously described 27-kDa recombinant antigen and the 87-kDa heat shock protein were used for diagnosis and follow-up of patients with PCM. A total of 37 patients classified according to their clinical presentations (7 with the acute or subacute form of the disease, 22 with the chronic form of the disease, and 8 with the chronic unifocal form) were studied. Eighteen of these patients were also evaluated at every follow-up appointment. Forty serum samples from patients with other diseases and 50 serum samples from healthy individuals were also studied. Detection of anti-27-kDa and anti-87-kDa antibodies in sera of patients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 92% with a specificity of 88% in comparison with normal human sera and 90% in comparison with the heterologous sera. These results demonstrated a significant increase in sensitivity and specificity compared to results when the antigens were used separately. Thus, the use of combinations of well-defined antigens appears to offer clear advantages over the use of single antigens when diagnosing PCM.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Paracoccidioidomycosis (PCM), one of the most prevalent systemic fungal mycoses in Latin America, is caused by Paracoccidioides brasiliensis, a thermally dimorphic fungus (1, 36). Two forms of the disease are recognized, the chronic form (adult type) and the acute or subacute form (juvenile type) (13). The disease varies in severity and primarily involves the lungs, with subsequent dissemination to other organs and systems; secondary lesions may occur in the mucous membranes, the skin, the lymph nodes, and the adrenal glands (1, 36). Prompt and accurate diagnosis is of the utmost importance, since it allows initiation of specific therapy.

The definitive diagnosis of PCM is typically dependent on laboratory-based procedures, principally the visualization of the fungus in biopsy and clinical specimens and/or its isolation by culture; however, the former is insensitive, and the latter is time-consuming (1). Consequently, serological testing is an important tool not only for disease diagnosis but also for monitoring patients' responses to treatment (7, 25, 32). Antibodies to P. brasiliensis can be detected in patients' sera by serological techniques, such as complement fixation, immunodiffusion, and immunoenzymatic assays (2, 3, 38). Historically, the use of complex mixtures of undefined antigens has imposed important limitations on such tests; cross-reactivity has been a problem, as has the absence of antigen standardization (17). Variation in antigen production can arise from differences in the strains used, in the fungus growth phase chosen, in the incubation time, and in the culture media employed (32).

As a consequence, efforts have been directed toward the purification and characterization of defined, serodiagnostically useful P. brasiliensis antigens. These include a 22- to 25-kDa protein (11), a 58-kDa glycoprotein (12), and an 87-kDa protein that has been purified and subsequently characterized as a member of the HSP70 family (6). Initially, this glycoprotein was detected in the sera of patients with PCM by an inhibition enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody (15, 16). This inhibition ELISA proved useful for both early diagnosis and follow-up observations of PCM patients (16).

Simultaneously, progress has also been made in the application of recombinant proteins to the serodiagnosis of PCM. An example is the recombinant 43-kDa glycoprotein used in various serodiagnostic tests (4, 30, 37), while a recombinant 27-kDa antigen has also been used to detect immune responses by ELISA (29, 33, 34). Recently, an hsp60 antigen was cloned and used as a serodiagnostic marker (5, 20).

In all these cases, the antigens were used individually. Considering the large number of antigenic epitopes expressed by P. brasiliensis (21), it is probable that patients respond to several of them simultaneously. Consequently, superior diagnostic results may be achieved by using combinations of either purified or recombinant antigens to improve the specificity and sensitivity of any given test. Accordingly, in this paper, we describe the first application of a mixture of defined P. brasiliensis antigens to the diagnosis of PCM. The antigens chosen for study were the previously purified 87-kDa hsp (6) and the recombinant 27-kDa protein (34) used previously in an indirect ELISA for the detection of antibodies in the sera of PCM patients.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patients and serum samples. A total of 37 serum samples taken at the moment of diagnosis were obtained from patients with mycologically confirmed (by direct examination, isolation by culture, and positive serological test) PCM. Eighteen of these patients were also evaluated at every follow-up appointment; six of them had the acute or subacute form of the disease, eight had the chronic multifocal form, and four had the chronic unifocal form according to their respective clinical presentations (13). Samples were collected between January 1988 and December 1997 at the Mycology Laboratory, Corporación para Investigaciones Biológicas, Medellín, Colombia. Forty serum samples from patients with other mycoses (confirmed by culture and serology) were also evaluated (Table 1). Negative controls consisting of 50 serum samples from healthy individuals from the area of endemicity were included in the study.

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TABLE 1. Sources of serum specimens tested by ELISA

Preparation and purification of the 27-kDa antigen. Escherichiacoli DH5 expressing the recombinant 27-kDa antigen (29, 34) was grown on slants of brain heart infusion medium (Oxoid, Basingstoke, Hampshire, England) containing 50 mg of ampicillin (Sigma, Dorset, England)/liter and incubated at 37°C for 24 h. The cultures were then transferred to a 500-ml flask containing 200 ml of liquid brain heart infusion medium plus ampicillin, which was then placed in a gyratory shaker incubator at 125 rpm and 37°C for 24 h. The cells were then harvested by centrifugation at 3,000 x g for 10 min. The pellet was washed twice in phosphate-buffered saline (PBS) (0.01 M; pH 7.2) and frozen using liquid nitrogen; it was then disrupted by mechanical maceration. This homogenate was centrifuged at 10,000 x g for 20 min at 4°C. The supernatant was recovered, and the total protein concentration was measured by the Bradford technique (Bio-Rad, Hercules, Calif.) (40). Subsequent purification of the 27-kDa antigen was performed by preparative gel electrophoresis, using the Prep-Cell system (model 491; Bio-Rad) as described previously (34). Briefly, a resolving gel of 12% (vol/vol) acrylamide (with 0.1% [wt/vol] sodium dodecyl sulfate [SDS]) was prepared and loaded with 30 mg of the recombinant bacterium antigen, which was then run at 12 W for 10 h with 2.5-ml fractions collected every minute. The fractions were precipitated with chilled acetone and left at -20°C for 2 h; samples were then centrifuged at 12,000 x g for 15 min at 4°C. The resultant pellets were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), enzyme-linked immunosorbent assay (ELISA), and immunoenzyme development to test the purity of the protein (see below).

Fungal strain: preparation and purification of the 87-kDa antigen. P. brasiliensis CIB 339 was obtained from the culture collection of the Corporación para Investigaciones Biológicas. P. brasiliensis cytoplasmic yeast antigen (CYA) was prepared as previously described (6). Briefly, cells were subcultured on slants of synthetic McVeigh-Morton medium (35) and incubated at 36°C for 3 days. The cultures were transferred to 500-ml flasks containing 200 ml of synthetic McVeigh-Morton liquid medium, which were then placed in a gyratory shaker incubator at 125 rpm and 36°C. Seven days later, the cells were killed with thimerosal (0.02% [wt/vol]; Sigma, Poole, United Kingdom) and harvested by centrifugation at 3,000 x g for 15 min. The pellet was washed twice in PBS, pH 7.2, and treated with a mixture of protease inhibitors (6). The pellet was frozen using liquid nitrogen and disrupted by mechanical maceration, and the homogenate was then centrifuged at 10,000 x g for 30 min at 4°C. The supernatant was recovered, and the total protein concentration was measured by the Bradford technique (40). The purification of the 87-kDa protein from CYA was done as described previously (6) using a 7.5% resolving gel on a Prep-Cell with 30 mg of P. brasiliensis CYA loaded on each run. The fractions were collected, and pellets were obtained and analyzed as described above.

SDS-PAGE, ELISA, and immunoenzyme development of Western blots. To evaluate the fractions obtained during the purification of both proteins, SDS-PAGE (using 10 and 12% [vol/vol] gels), ELISA, and Western immunoblotting using polyvinylidene difluoride membranes (Immobilon P; Millipore Corp., Watford, United Kingdom) were used as previously described (10, 18, 22). Pooled PCM patients' sera were used to detect the 27-kDa protein, and monoclonal antibody P1B was used to identify the 87-kDa protein (15). The proteins were also visualized by Coomassie blue staining (0.1% [wt/vol]) and/or silver staining (Bio-Rad).

Antibody detection by indirect ELISA. Microtiter plates (Maxisorp; Nunc A/S) were coated with the purified 27- and 87-kDa proteins (either individually or in combination at concentrations varying between 0.032 and 4.0 µg/well) diluted in 0.06 M carbonate buffer, pH 9.6 (39), and left for 30 min at room temperature and then incubated overnight at 4°C. The plates were washed three times with PBS plus Tween 20 (0.05% [vol/vol]), pH 7.2 (PBST), and blocked with 200 µl of 1% (wt/vol) bovine serum albumin in PBST/well for 2 h at 37°C. After three more washes, 100 µl of human sera (at dilutions varying from 1:1,000 to 1:8,000 in the blocking solution) was added to each plate, and the plates were incubated at 37°C for 1 h. After the plates were washed as described previously, 100 µl of peroxidase-labeled goat anti-human immunoglobulin G (Jackson, West Grove, Pa.) was added to each well, and the plates were incubated for another hour at 37°C. After another cycle of washing, the reaction was visualized with a solution of o-phenylenediamine (0.2 mg/ml; Sigma) and 0.05% (vol/vol) H₂O₂. The reaction was stopped with 4 N H₂SO₄ after 5 min of incubation in the dark. The optical density was measured at 492 nm using an automated ELISA plate reader (microplate reader; Bio-Rad, Richmond, Calif.). All tests were run in triplicate.

Statistical analysis. Data were analyzed by analysis of variance (ANOVA-Type III) of square sum. Comparisons within the factors were performed by a multiple-range test using the least-significant-difference test. Sensitivity, specificity, and predictive values were determined by the statistical method of Gallen and Gambino (14).

Top Abstract Introduction Materials and Methods Results Discussion References

 
According to statistical procedures, the ELISA cutoff point was estimated to be 1.4 times the mean point of the negative control readings (34). Consequently, samples with a higher optical density were considered positive.

When used individually, both the 27- and the 87-kDa antigens were recognized by a large number of PCM patients' sera (Fig. 1a and b). However, the reactivity to the 27-kDa antigen was inferior to the reaction to the 87-kDa antigen—thus, only 18 of the 37 PCM serum samples recognized the 27-kDa antigen, whereas 30 (81%) recognized the purified 87-kDa hsp.

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FIG. 1. Indirect ELISA for the detection of anti-27- and anti-87-kDa antibodies in sera from patients with PCM or other diseases and healthy controls from areas of endemicity. The groups studied are indicated by numbers as follows: 1, PCM, pretreatment sera (n = 37); 2, normal human serum (n = 50); 3, histoplasmosis (n = 10); 4, aspergillosis (n = 10); 5, cryptococcosis (n = 10); 6, tuberculosis (n = 10). (a) Antibody reactivity against recombinant purified 27-kDa antigen (0.125 µg/well). (b) Antibody reactivity against purified 87-kDa hsp (0.125 µg/well). (c) Antibody reactivity against combined purified 27- (0.125 µg/well) and 87-kDa (0.06 µg/well) proteins. (d) Antibody reactivity against combined purified 27- (0.06 µg/well) and 87-kDa (0.125 µg/well) proteins. The dotted lines represent the ELISA cutoff points. O.D., optical density.

A number of different combinations of the 27- and 87-kDa antigen concentrations were tested (data not shown), and the two most effective, p27 (0.125 µg/well) plus p87 (0.06 µg/well) and p27 (0.06 µg/well) plus p87 (0.125 µg/well) are shown in Fig. 1c and d. A total of 34 PCM sera were reactive when either of these antigen combinations was used. This gave an overall sensitivity for the combined-antigen ELISA of 92% (Table 2), which was superior to the sensitivity of either antigen used alone. Positive and negative predictive values are also included in Table 2; these also demonstrated the superiority of the combined-antigen approach.

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TABLE 2. Sensitivities, specificities, and predictive values of ELISAs using individual and combined antigens

In terms of specificity in the ELISA, when used individually, the 27-kDa antigen was greatly superior to the 87-kDa antigen (Fig. 1a and b). In reference to sera from patients with other diseases or from healthy subjects, the 27-kDa antigen-87-kDa antigen combination proved to be at least as specific in the ELISA as the 27-kDa antigen used alone (Fig. 1c and d and Table 2). The best results were obtained when the 27-kDa antigen was used at a higher concentration than the 87-kDa hsp, with 88 and 90% specificities against sera from patients with other diseases and normal sera, respectively (Table 2).

Eighteen patients with different clinical forms of PCM, as indicated previously, were also evaluated at every follow-up appointment. In the ELISA, patients with the acute form showed a greater reactivity against the combined 27- and 87-kDa antigens than against the individual antigens (Fig. 2). Whichever combination of the 27- and 87-kDa antigen concentrations (i.e., 27-kDa antigen concentration higher than 87-kDa antigen concentration or vice versa) was used, the results were always superior to the equivalent figures with the single antigens. Similar results were observed for patients with the chronic unifocal (Fig. 3) and the multifocal (Fig. 4) forms of the disease.

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FIG. 2. Findings at follow-up for six patients with the acute form of PCM by indirect ELISA. , antibody reactivity against purified 27-kDa antigen (0.125 µg/well); , antibody reactivity against purified 87-kDa hsp (0.125 µg/well); , antibody reactivity against combined purified 27- (0.125 µg/well) and 87-kDa (0.06 µg/well) proteins; x, antibody reactivity against combined purified 27- (0.06 µg/well) and 87-kDa (0.125 µg/well) proteins.

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FIG. 3. Findings at follow-up for four patients with the chronic unifocal form of PCM by indirect ELISA. , antibody reactivity against purified 27-kDa antigen (0.125 µg/well); , antibody reactivity against purified 87-kDa hsp (0.125 µg/well); , antibody reactivity against combined purified 27- (0.125 µg/well) and 87-kDa (0.06 µg/well) proteins; x, antibody reactivity against combined purified 27- (0.06 µg/well) and 87-kDa (0.125 µg/well) proteins.

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FIG. 4. Findings at follow-up for eight patients with the chronic multifocal form of PCM by indirect ELISA. , antibody reactivity against purified 27-kDa antigen (0.125 µg/well); , antibody reactivity against purified 87-kDa hsp (0.125 µg/well); , antibody reactivity against combined purified 27- (0.125 µg/well) and 87-kDa (0.06 µg/well) proteins; x, antibody reactivity against combined purified 27- (0.06 µg/well) and 87-kDa (0.125 µg/well) proteins.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The detection of antibody by serological methods plays an important role in the diagnosis of PCM (2, 3, 17, 32, 36). However, the complexity of the antigens traditionally used meant that regardless of the methodology employed, it almost invariably resulted in problems associated with a lack of antigen standardization and specificity (17, 36). Recent work using protein purification methods and recombinant protein technology has identified a series of candidate antigens (4, 5, 6, 11, 12, 29, 31, 34, 37) that may be used in place of complex antigenic extracts, thus offering a solution to such problems. However, the use of individual purified or recombinant antigens in diagnostic tests can itself be problematic when, for example, particular antigens are recognized by a relatively low percentage of the patients tested, resulting in low sensitivity. Accordingly, the use of combinations of characterized antigens may be advantageous.

In this report, we describe for the first time the use of a multiantigen approach for the diagnosis of PCM, using a standard ELISA format. Although multiple antigens have been used in the diagnosis of allergic reactions (19) and bovine tuberculosis (9, 23), this appears to be the first application of such methodology to the diagnosis of any human mycosis.

To study this question, we chose to use two well-characterized proteins that had previously been studied in our respective laboratories (6, 33, 34). The 27-kDa protein was originally cloned and used as an immunological marker in 1996 (29); subsequently, Ortiz and collaborators elaborated on its use in antibody detection (34). The P. brasiliensis 87-kDa protein was previously detected in sera of patients with PCM (15, 16) and was subsequently purified and characterized as an hsp (6). There is, of course, no reason why this multiantigen approach could not be used with other diagnostic P. brasiliensis antigens, such as the 43- and 60-kDa antigens (4, 5, 11, 12, 37). Indeed, combinations of three or even four antigens could be used in genuine "antigen cocktails" if desired.

By using a cocktail comprised of the 27- and 87-kDa antigens in the ELISA, we have apparently combined the best diagnostic features of each component; thus, the high specificity of the 27-kDa antigen has been merged with the high sensitivity of the 87-kDa hsp. Ortiz et al. (34) previously reported a sensitivity of 78% when using the 27-kDa protein in an indirect ELISA. However, the present study (which employed different parameters from those described by Ortiz et al.) suggested that when used by itself under the conditions described here, the 27-kDa antigen has an unacceptably low sensitivity of 49%. In contrast, the 87-kDa antigen showed high sensitivity (81%) when used in isolation but suffered from low specificity. The latter is almost certainly related to the fact that the 87-kDa antigen is an hsp; such proteins are shared by many different fungal pathogens (8, 24, 28, 41) and are known to be highly homologous.

Hsps have been previously shown to be important markers of disease when used in isolation in the diagnosis of several fungal infections, including candidosis (6, 26, 27, 28). The work described in this communication has shown that the combination of an hsp with another defined protein is a useful tool in the diagnosis of PCM. As such, hsp-protein combinations may also be broadly applicable in the diagnosis of other fungal pathogens, such as Candida albicans

The multiantigen approach was not only useful for direct diagnosis, it also demonstrated greater sensitivity during the follow-up of patients. However, antigen detection is probably superior to antibody detection when following up patients during and after treatment (16), as antibody is a lagging indicator. Future studies will focus on combining the methodology described here with that previously reported for antigen detection (15, 16) in order to develop the best all-round strategy for diagnosis and management of PCM.

 
We thank the Overseas Research Scheme (ORS), United Kingdom, and the Wellcome Trust, United Kingdom, for their financial support.

 
* Corresponding author. Mailing address: Dermatology Laboratory, 5th Floor, Thomas Guy House, Guy's Hospital, London SE1 9RT, United Kingdom. Phone: 44 (0) 207 955 4663. Fax: 44 (0) 207 955 2103. E-mail: soraya.diez{at}kcl.ac.uk.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, April 2003, p. 1536-1542, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1536-1542.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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