Combined use of Paracoccidioides brasiliensis recombinant 27-kilodalton and purified 87-kilodalton antigens in an enzyme-linked immunosorbent assay for serodiagnosis of paracoccidioidomycosis

Abstract

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.

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.

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; ×, antibody reactivity against combined purified 27- (0.06 μg/well) and 87-kDa (0.125 μg/well) proteins.

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; ×, antibody reactivity against combined purified 27- (0.06 μg/well) and 87-kDa (0.125 μg/well) proteins.

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; ×, antibody reactivity against combined purified 27- (0.06 μg/well) and 87-kDa (0.125 μg/well) proteins.