Detection of histoplasma antigen by a quantitative enzyme immunoassay

Abstract

The second-generation Histoplasma antigen immunoassay is semiquantitative, expressing results as a comparison to a negative control, which requires repeat testing of the prior specimen with the current specimen to accurately determine a change in antigen. Reporting results in this manner often is confusing to the ordering physician and laboratory. Development of a quantitative assay could improve accuracy, reduce interassay variability, and eliminate the need to test the prior sample with the current sample in the same assay. Calibrators with known concentrations of Histoplasma antigen were used to quantitate antigen in specimens from patients with histoplasmosis and from controls. Samples from cases of disseminated histoplasmosis or other mycoses and controls were tested to evaluate the performance characteristics of the quantitative assay. Paired specimens were evaluated to determine if quantitation eliminated the need to test the current and prior specimens in the same assay to assess a change in antigen. The sensitivity in samples from patients with AIDS and disseminated histoplasmosis was 100% in urine and 92.3% in serum. Cross-reactions occurred in 70% of other endemic mycoses, but not in aspergillosis. Specificity was 99% in controls with community-acquired pneumonia, medical conditions in which histoplasmosis was excluded, or healthy subjects. A change in antigen level categorized as an increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in nanograms/milliliter determined from testing current and prior specimens in different assays. Sensitivity, specificity, and interassay precision are excellent in the new third-generation quantitative Histoplasma antigen immunoassay.

FIG. 1.

ROC curve depicting assay sensitivity and specificity, based upon testing serum and urine specimens from 65 patients with AIDS and disseminated histoplasmosis and 100 controls with mycoplasma pneumonia, other conditions for which histoplasmosis was excluded, and healthy subjects. With a cutoff OD of 0.065 (three times the mean normal), the sensitivity is 95.4% and the specificity is 99.0%.

FIG. 2.

Sensitivity in disseminated histoplasmosis in samples from patients with AIDS and specificity in samples from controls. The antigen concentration, in nanograms/milliliter, is shown on the vertical axis. The sensitivity in urine was 100%, and in serum it was 92.3%; the specificity was 99.0% in controls without fungal infections.

FIG. 3.

Interassay agreement of changes in nanograms/milliliter in histoplasmosis cases with paired current and prior specimens, comparing results determined in the same assay (same day) with those determined in different assays (different days). Data below the 0 value represent pairs in which the concentration decreased in the current specimen compared to that of the prior specimen, and data above 0 represent pairs in which the concentration increases in the current specimen compared to that of the prior specimen.

FIG. 4.

Changes in antigen concentration between a current and prior specimen first categorized according to the change in antigen EU in the same assay and then plotted with the change in antigen concentration calculated quantitatively as nanograms/milliliter from separate days' assays. The number/total (percent) shown on the column designates agreement between the category of change in nanograms/milliliter as determined from different days' assays with change in antigen EU determined from the same assay. For the determination of agreement, a significant change in nanograms/milliliter was defined as >3 ng/ml when the concentration in the prior specimen was <20 ng/ml (designated by closed circles on the graph) and >15% when the concentration in the prior specimen was ≥20 ng/ml (open circles).