Negative immunodiffusion test results obtained with sera of paracoccidioidomycosis patients may be related to low-avidity immunoglobulin G2 antibodies directed against carbohydrate epitopes

Abstract

Immunodiffusion (ID) is the serologic test most frequently used for the diagnosis and posttherapy follow-up of patients with paracoccidioidomycosis (PCM). The ID test is highly specific (100%), but its sensitivity is relatively low (90%), leading to false-negative results. The aim of this study was to determine the profiles of antibodies in sera from patients with proven PCM and with negative results in the ID test (IDneg) versus positive results in the ID test (IDpos). We analyzed 46 sera from patients with active PCM for total immunoglobulin G (IgG) and IgG subclass responses to Paracoccidioides brasiliensis gp43 antigen (treated or not treated with sodium metaperiodate) by enzyme-linked immunosorbent assay and immunoblotting. Immunoblotting showed that both IDneg and IDpos sera recognized predominantly the gp43 fraction of the P. brasiliensis antigen used in the ID test. IDneg sera contain low-avidity antibodies, low levels of specific IgG (total) and IgG1, and high levels of IgG2 compared with IDpos sera. The antibodies present in IDneg sera were predominantly directed against carbohydrate epitopes, since treatment with sodium metaperiodate resulted in a significant decrease in antibody reactivity. These data suggest that the lack of reactivity of sera from PCM patients in the ID test may be related to the production of low-avidity IgG2 antibodies directed against carbohydrate epitopes.

FIG. 1.

Metaperiodate-treated (MT) or untreated (UT) anti-gp43 IgG in ID^neg and ID^pos sera. Results are expressed as the absorbance at a 1:800 dilution. The horizontal bars represent the median, and the dotted line represents the cutoff (0.320). For the Wilcoxon test of UT ID^neg versus MT ID^neg sera, the P value was <0.0001; for UT ID^pos versus MT ID^pos sera, the P value was <0.0001.

FIG. 2.

Levels of anti-P. brasiliensis gp43 IgG1, IgG2, IgG3, and IgG4 in sera from ID^neg patients and ID^pos patients (AF and JF of PCM). Results are expressed as the AI. The horizontal bars in the boxes represent the median, and the error bars represent the standard deviations.

FIG. 3.

Antibody response to a P. brasiliensis crude antigen or CFA. (a) Lane 1, SDS-PAGE of P. brasiliensis CFA (silver stain); lanes 2 and 3, IgG immunoblot of representative ID^neg and ID^pos sera. (b) Top: IgG subclass immunoblots of a representative serum sample from one patient in each group: 1, AF PCM; 2, JF PCM; and 3, ID^neg. Bottom: respective ELISA results expressed as the AI.

FIG. 4.

IgG avidity of ID^neg and ID^pos sera. The horizontal bars represent the median. In a comparison of ID^neg versus ID^pos sera, the P value was 0.0018.