Evaluation of a western blot test in an outbreak of acute pulmonary histoplasmosis

Abstract

A western blot (WB) test was evaluated for detection of antibodies against native glycosylated and chemically deglycosylated M and H antigens of Histoplasma capsulatum in serum obtained from patients during the acute phase of pulmonary histoplasmosis that occurred during an outbreak. Of 275 serum samples tested by immunodiffusion and complement fixation (CF) samples from 40 patients affected during this outbreak and from 37 negative controls were tested by WB test. A group of patients whose sera were negative for CF antibodies and precipitins early in the acute stage of histoplasmosis but who all seroconverted during convalescence 6 weeks later were tested with the WB test. Antibodies against untreated H and M antigens were detected at a 1:100 dilution by WB test in 45% of the 20 acute-phase serum samples and in all 20 of the convalescent-phase specimens. The WB test's sensitivity for acute-phase specimens increased to 90% (18 of 20 specimens) when H and M antigens were treated by periodate oxidation to inactivate susceptible carbohydrate epitopes. When native glycosylated antigens were used in the WB test, positive reactions were observed in negative control serum specimens (3 of 37 specimens; 8%) and in serum specimens obtained from asymptomatic persons screened as part of the outbreak investigation (13 of 20 specimens; 65%). These positive reactions were also attributed to glycosidic epitopes since the specificity of the WB test increased from 78 to 100% when periodate-treated H and M antigens were used. WB test with deglycosylated H and M antigens of histoplasmin provides a rapid, sensitive, and specific test to diagnose acute pulmonary histoplasmosis before precipitins can be detected.

FIG. 1

Representative WB reactions obtained with untreated (bottom) and periodate-treated (top) H and M antigens (∼120 and 94 kDa, respectively) probed with serum samples obtained from patients during an outbreak of acute pulmonary histoplasmosis. Lanes 1 to 20, acute-phase serum specimens; lanes 1A to 20A, serum samples from the same patients obtained 4 weeks later, during convalescence. MnAb, monoclonal antibodies specific for H and M antigens (see Materials and Methods section); C⁺, positive control (rabbit antiserum); C⁻, negative control (normal human serum). Numbers in left margin correspond to molecular-mass-size marker proteins.