Combined Use of Paracoccidioides brasiliensis Recombinant 27 ...

JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2003, p. 1536–1542 0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.4.1536–1542.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 41, No. 4

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. Go ´mez,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, England1; Corporacio ń para Investigaciones Biolo ´gicas, Medellín, Colombia2; and Queens University Belfast, Whitla Medical Building, Belfast, Northern Ireland3 Received 16 October 2002/Returned for modification 21 November 2002/Accepted 14 December 2002

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, wellcharacterized antigens appears preferable and may yield optimum results. Accordingly an indirect enzymelinked 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. 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

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

* 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: [email protected]. 1536

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VOL. 41, 2003 TABLE 1. Sources of serum specimens tested by ELISA Source

No. of samplesa

PCM patients...................................................................................... 37 Histoplasmosis patients ..................................................................... 10 Aspergillosis patients ......................................................................... 10 Cryptococcosis patients ..................................................................... 10 Tuberculosis patients ......................................................................... 10 Healthy subjects ................................................................................. 50 a

All samples were taken at the moment of diagnosis.

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

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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. MATERIALS AND METHODS 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

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 ␮g/well). (b) Antibody reactivity against purified 87-kDa hsp (0.125 ␮g/well). (c) Antibody reactivity against combined purified 27- (0.125 ␮g/well) and 87-kDa (0.06 ␮g/well) proteins. (d) Antibody reactivity against combined purified 27(0.06 ␮g/well) and 87-kDa (0.125 ␮g/well) proteins. The dotted lines represent the ELISA cutoff points. O.D., optical density.

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TABLE 2. Sensitivities, specificities, and predictive values of ELISAs using individual and combined antigens Value obtained with antigen(s): Parametera

Sensitivity Specificity vs NHS Specificity vs other disease Predictive value of positive result (vs NHS) Predictive value of positive result (vs other diseases) Predictive value of negative result (vs NHS) Predictive value of negative result (vs other diseases) a

p27 (0.125 ␮g/well)

p87 (0.125 ␮g/well)

p27 (0.125 ␮g/well) ⫹ p87 (0.06 ␮g/well)

p27 (0.06 ␮g/well) ⫹ p87 (0.125 ␮g/well)

49 86 90 72 82 69 65

81 74 82.5 70 81 84 82.5

92 88 90 85 90 94 92

92 86 88 83 87 93 92

NHS, normal human serum. All values are percentages.

clinical presentations (13). Samples were collected between January 1988 and December 1997 at the Mycology Laboratory, Corporacio ń para Investigaciones Biolo ´gicas, 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. 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 ⫻ g for 10 min. The pellet was washed twice in phosphatebuffered 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 ⫻ 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; BioRad) 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 ⫻ 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 Corporacio ń para Investigaciones Biolo ´gicas. 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 ⫻ 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 ⫻ 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, SDSPAGE (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 ␮g/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 ␮l of 1% (wt/vol) bovine serum albumin in PBST/well for 2 h at 37°C. After three more washes, 100 ␮l 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 ␮l 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 (ANOVAType 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).

RESULTS 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. 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 ␮g/well) plus p87 (0.06 ␮g/well) and p27 (0.06 ␮g/well) plus p87 (0.125 ␮g/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. 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

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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 ␮g/well); ■, antibody reactivity against purified 87-kDa hsp (0.125 ␮g/well); Œ, antibody reactivity against combined purified 27(0.125 ␮g/well) and 87-kDa (0.06 ␮g/well) proteins; ⴛ, antibody reactivity against combined purified 27- (0.06 ␮g/well) and 87-kDa (0.125 ␮g/well) proteins.

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 87kDa 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. 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 ␮g/well); ■, antibody reactivity against purified 87-kDa hsp (0.125 ␮g/well); Œ, antibody reactivity against combined purified 27- (0.125 ␮g/well) and 87-kDa (0.06 ␮g/well) proteins; ⫻, antibody reactivity against combined purified 27- (0.06 ␮g/well) and 87-kDa (0.125 ␮g/ well) proteins.

DISCUSSION 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

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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 ␮g/well); ■, antibody reactivity against purified 87-kDa hsp (0.125 ␮g/well); Œ, antibody reactivity against combined purified 27- (0.125 ␮g/well) and 87-kDa (0.06 ␮g/well) proteins; ⫻, antibody reactivity against combined purified 27- (0.06 ␮g/well) and 87-kDa (0.125 ␮g/well) proteins.

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

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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. ACKNOWLEDGMENTS We thank the Overseas Research Scheme (ORS), United Kingdom, and the Wellcome Trust, United Kingdom, for their financial support. REFERENCES 1. Brummer, E., E. Castan ˜ eda, and A. Restrepo. 1993. Paracoccidioidomycosis: an update. Clin. Microbiol. Rev. 6:89–117. 2. Camargo, Z. P., J. L. Guesdon, E. Drouhet, and L. Improvisi. 1984. Enzyme linked immunosorbent assay (ELISA) in paraccocidioidomycosis. Comparison with counterimmunoelectrophoresis and erythroimmunoassay. Mycopathologia 88:31–37. 3. Cano, L. E., and A. Restrepo. 1987. Predictive value of serologic tests in the serodiagnosis and follow up of patients with paracoccidioidomycosis. Rev. Inst. Med. Trop. Sao Paulo 29:276–283. 4. Cisalpino, P. S., R. Puccia, L. M. Yamauchi, M. I. N. Cano, J. F. da Silveira, and L. R. Travassos. 1996. Cloning, characterization, and epitope expression of the major diagnostic antigen of Paracoccidioides brasiliensis. J. Biol. Chem. 271:4553–4560. 5. Cunha, D. A., R. M. Zancope´-Oliveira, M. Sueli, S. Felipe, S. M. SalemIzacc, G. S. Deepe, Jr., and C. M. Soares. 2002. Heterologous expression, purification, and immunological reactivity of a recombinant HSP60 from Paracoccidioides brasiliensis. Clin. Diagn. Lab. Immunol. 9:374–377. 6. Díez, S., B. L. Go ´mez., A. Restrepo, R. J. Hay, and A. Hamilton. 2002. Paraccocidioides brasiliensis 87-kilodalton antigen, a heat shock protein useful in diagnosis: characterization, purification and detection via immunohistochemistry. J. Clin. Microbiol. 40:359–365. 7. Do Valle, A. C., R. L. Costa, P. C. Fialho Monteiro, J. Von Helder, M. M. Muniz, and R. M. Zancope´-Oliveira. 2001. Interpretation and clinical correlation of serological tests in paracoccidioidomycosis. Med. Mycol. 39:373– 377. 8. Feige, U., and B. S. Polla. 1994. Heat shock proteins: the hsp70 family. Experientia 50:979–986. 9. Fifis, T., C. Costopoulos, L. A. Corner, and P. R. Wood. 1992. Serological reactivity to Mycobacterium bovis protein antigens in cattle. Vet. Microbiol. 30:343–354. 10. Figueroa, J., A. J. Hamilton, M. A. Bartholomew, L. E. Fenelon, and R. J. Hay. 1990. Preparation of species specific murine monoclonal antibodies against the yeast phase of Paracoccidioides brasiliensis. J. Clin. Microbiol. 28:1766–1769. 11. Figueroa, J., A. J. Hamilton, M. H. Allen, and R. J. Hay. 1994. Immunohistochemical detection of a novel 22- to 25-kilodalton glycoprotein of Paracoccidioides brasiliensis in biopsy material and partial characterization by using species specific monoclonal antibodies. J. Clin. Microbiol. 32:1566– 1574. 12. Figueroa, J., A. J. Hamilton, M. H. Allen, and R. J. Hay. 1995. Isolation and partial characterization of a Paracoccidioides brasiliensis 58kDa extracellular glycoprotein which is recognized by human immune sera. Trans. R. Soc. Trop. Med. Hyg. 89:566–572. 13. Franco, M., M. R. Montenegro, R. P. Mendes, S. A. Marquez, N. L. Dillon, and N. G. S. Mota. 1987. Paracoccidioidomycosis: a recently proposed classification of its clinical forms. Rev. Soc. Bras. Med. Trop. 20:129–132. 14. Gallen, R. S., and S. R. Gambino. 1975. Beyond normality. The predicted value and efficiency of medical diagnosis. John Wiley & Sons, Inc., New York, N.Y. 15. Go ´mez, B. L., J. I. Figueroa, A. J. Hamilton, B. Ortiz, M. A. Robledo, R. J. Hay, and A. Restrepo. 1997. Use of monoclonal antibodies in diagnosis of paracoccidioidomycosis: new strategies for detection of circulating antigens. J. Clin. Microbiol. 35:3278–3283. 16. Go ´mez, B. L., J. I. Figueroa, A. J. Hamilton, S. Díez, M. Rojas, A. M. Tobo ń, R. J. Hay, and A. Restrepo. 1998. Antigenemia in patients with paracoccidioidomycosis: detection of the 87-kilodalton determinant during and after antifungal therapy. J. Clin. Microbiol. 36:3309–3316. 17. Hamilton, A. J. 1998. Serodiagnosis of histoplasmosis, paracoccidioidomycosis and penicilliosis marneffei; current status and future trends. Med. Mycol. 36:351–364. 18. Hamilton, A. J., M. A. Bartholomew, L. E. Fenelon, J. I. Figueroa, and R. J. Hay. 1990. A murine monoclonal antibody exhibiting high species specificity for Histoplasma capsulatum var. capsulatum. J. Gen. Microbiol. 136:331–335. 19. Hiller, R., S. Laffer, C. Harwanegg, M. Huber, W. M. Schmidt, A. Twardosz,

20.

21.

22. 23. 24. 25.

26. 27. 28.

29.

30.

31.

32.

33. 34.

35. 36. 37. 38. 39. 40. 41.

B. Barletta, W. M. Becker, K. Blaser, H. Breiteneder, M. Chapman, R. Crameri, M. Duchene, F. Ferreira, H. Fiebig, K. Hoffmann-Sommergruber, T. P. King, T. Kleber-Janke, V. P. Kurup, S. B. Lehrer, J. Lidholm, U. Muller, C. Pini, G. Reese, O. Scheiner, A. Scheynius, H. D. Shen, S. Spitzauer, R. Suck, I. Swoboda, W. Thomas, R. Tinghino, M. Van HageHamsten, T. Virtanen, D. Kraft, M. W. Muller, and R. Valenta. 2002. Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment. FASEB J. 16:414–416. Izacc, S. M., F. J. Go ´mez, R. S. Jesuino, C. A. Fonseca, M. S. Felipe, G. S. Deepe, and C. M. Soares. 2001. Molecular cloning, characterization and expression of the heat shock protein 60 gene from the human pathogenic fungus Paracoccidioides brasiliensis. Med. Mycol. 39:445–455. Juvenale, M., G. M. B. Del Negro, A. J. S. Duarte, and G. Bernard. 2001. Antibody isotypes to a Paracoccidioides brasiliensis somatic antigen in subacute and chronic form paracoccidioidomycosis. J. Med. Microbiol. 50:127– 134. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. Lyashchenko, K. P., J. M. Pollock, R. Colangeli, and M. L. Gennaro. 1998. Diversity of antigen recognition by serum antibodies in experimental bovine tuberculosis. Infect. Immun. 66:5344–5349. Maresca, B., and G. S. Kobayashi. 1995. Hsps in dimorphic fungi, p. 119– 130. In R. Matthews and J. P. Burnie (ed.), Heat shock proteins in fungal infections. R. G. Landes Company, Austin, Tex. Martins, R., S. Marquez, M. Alves, D. Fecchio, and M. Franco. 1997. Serological follow-up of patients with paracoccidioidomycosis treated with itraconazole using dot-blot, ELISA and Western-blot. Rev. Inst. Med. Trop. Sao Paulo 39:261–269. Matthews, R. 1995. Hsps in candidiasis, p. 1–92. In R. Matthews and J. P. Burnie (ed.), Heat shock proteins in fungal infections. R. G. Landes Company, Austin, Tex. Matthews, R. C. 1992. Candida albicans HSP 90: link between protective and auto immunity. J. Med. Microbiol. 36:367–370. Matthews, R. C., B. Maresca, J. P. Burnie, A. Cardona, L. Carratu, S. Conti, G. S. Deepe, A. M. Florez, S. Franceschelli, E. Garcia, L. S. Gargano, G. S. Kobayashi, J. G. McEwen, B. L. Ortiz, A. M. Oviedo, L. Polonelli, J. Ponton, A. Restrepo, and A. Storlazzi. 1998. Stress proteins in fungal diseases. Med. Mycol. 36:45–51. McEwen, J. G., B. L. Ortiz., A. M. Garcia., A. M. Florez, S. Botero, and A. Restrepo. 1996. Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa protein from Paracoccidioides brasiliensis. Fungal Genet. Biol. 20:125–131. Mendes-Giannini, M. J., J. P. Bueno, M. A. Shikanai-Yasuda, A. W. Ferreira, and A. Masuda. 1989. Detection of the 43,000-molecular-weight glycoprotein in sera of patients with paracoccidioidomycosis. J. Clin. Microbiol. 27:2842–2845. Mendes-Giannini, M. J. S., J. P. Bueno, M. A. Shikanai-Yasuda, A. M. S. Stolf, A. Masuda, V. Amato Neto, and A. W. Ferreira. 1990. Antibody response to the 43 kDa glycoprotein in Paracoccidioides brasiliensis as a marker for the evaluation of patients under treatment. Am. J. Trop. Med. Hyg. 43:200–206. Mendes-Giannini, M. J. S., G. B. Del Negro, and A. M. Siqueira. 1994. Serodiagnosis, p. 345–363. In M. Franco, C. Da Silva Lacaz, A. Restrepo, and G. del Negro (ed.), Paracoccidioidomycosis. CRC Press, Inc., Boca Raton Fla. Ortiz, B. L., A. M. Garcia, A. Restrepo, and J. G. McEwen. 1996. Immunological characterization of the recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis. Clin. Diagn. Lab. Immunol. 3:239–241. Ortiz, B. L., S. Díez, M. E. Ura ń, J. M. Rivas, M. Romero, V. Caicedo, A. Restrepo, and J. G. McEwen. 1997. Use of the 27-kilodalton recombinant protein from Paracoccidioides brasiliensis in serodiagnosis of paracoccidioidomycosis. Clin. Diagn. Lab. Immunol. 5:826–830. Restrepo, A., and B. E. Jimenez. 1980. Growth of Paracoccidioides brasiliensis yeast phase in a chemically defined culture medium. J. Clin. Microbiol. 12:279–281. Restrepo, A. 2000. Paracoccidioides brasiliensis, p. 2768–2772. In G. L. Mandell, J. E. Bennett, and R. Dolin (ed.), Principles and practice of infectious diseases, 5th ed. Churchill Livingstone, Philadelphia, Pa. Taba, M. R. M., J. F. Da Silveira, L. R. Travassos, and S. Schenkman. 1989. Expression in Escherichia coli of a gene coding for epitopes of a diagnostic antigen of Paracoccidioides brasiliensis. Exp. Mycol. 13:223–230. Taborda, C. P., and Z. P. Camargo. 1994. Diagnosis of paracoccidioidomycosis by dot blot immunobinding assay for antibody detection using the purified and specific antigen gp43. J. Clin. Microbiol. 32:554–556. Voller, A., D. E. Bidwell, and A. Bartlett. 1980. Enzyme-linked immunosorbent assay, p. 359–371. In N. R. Rose and H. Friedman (ed.), Manual of clinical immunology. American Society for Microbiology, Washington, D.C. Walker, J. M. 1994. Basic protein and peptide protocols, vol. 32, p. 9–34. Humana Press Inc., Totowa, N.J. Zu ¨gel, U., and S. H. E. Kaufmann. 1999. Role of heat shock proteins in protection from and pathogenesis of infectious diseases. Clin. Microbiol. Rev. 12:19–39.