Aspergillus Fumigatus antigen detection in sera from patients at risk for invasive aspergillosis

Abstract

We have developed an inhibition enzyme immunoassay (inhibition-EIA) to monitor for the occurrence of invasive aspergillosis (IA) in sera from 45 immunocompromised (IC) patients. The test uses rabbit polyclonal antibodies and a mixture of components from Aspergillus fumigatus, containing three predominant antigens with molecular weights of 18,000, 33,000, and 56,000. Circulating antigens were found in five of seven proven cases of IA due to A. fumigatus. In two of the five positive cases, antigenemia was detected with inhibition-EIA earlier than with X ray or other biological methods. No antigens were detected in the sera from two patients with proven IA due to Aspergillus flavus and Aspergillus terreus nor in the sera from four patients with probable IA. Circulating antigens were not detected in the control group, composed of 30 healthy adult blood donors. Four of the 32 at-risk patients examined, though they displayed no definite evidence of IA, gave a positive result in this test. The sensitivity, specificity, and positive predictive value of inhibition-EIA were 71.4, 94.4, and 71.2%, respectively. The data were compared with those obtained by a latex agglutination assay of galactomannan (GM) that was positive in only one patient with probable IA. The higher sensitivity obtained by inhibition-EIA may well be due to its ability to detect circulating antigens other than GM in the sera of IC patients with IA. Detecting these antigens may improve the diagnosis of IA, as they may serve as markers of this infection.

FIG. 1

Optimization of the parameters of inhibition-EIA. Three rabbit hyperimmune sera were tested: anti-CF27, anti-CF37, and anti-CF42 (1:1,000). In this case, the plate was coated overnight with CF37 antigen at 2 μg/ml, and peroxidase conjugate was used at 1:1,500. The highest % inh was obtained experimentally with anti-CF27.

FIG. 2

Detection of antigen in a patient (number 2) with proven IA (in trachea, vertebra, and brain) in terms of number of days after initial detection. Positive cultures were found first in trachea (first arrow), then in brain (second arrow), and finally in vertebra (third arrow). In this case, antigenemia detected (+) in heated sera was transient and repeated. It was positive at 4 of 13 time points and in all of the earlier stages (up to 60 days). The detection of circulating antigens in several samples may be necessary. Unheated serum samples gave negative results. The error bars represent standard deviations.