Antigen detection in the diagnosis of Invasive Aspergillosis

Aspergillus species are known to release exo-antigens during growth in vitro and in vivo. Low levels of antigens may be present in body fluids of patients with invasive Aspergillus infection such as serum, urine and CSF. A number of methods have been developed and evaluated which employ antibodies directed against antigens produced by Aspergillus. These methods include latex agglutination (1), radioimmunoassay (2), ELISA inhibition (3) and sandwich ELISA (4). The detection limits for antigen of these assays are 15, 10, 4 to 5, and 1 ng/ml respectively (4). Two antigen detection kits are commercially available and have been evaluated in several institutes.

The latex agglutination (LA) test (Pastorex Aspergillus, Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) employs the rat monoclonal antibody EB-A2 to detect Aspergillus galactomannan (5). This assay has been evaluated with serum mainly from patients receiving treatment for hematological malignancies and although the specificity of the LA-test was high, substantial variation in sensitivity was observed (Table 1) (1,6-12).

Table 1. Performance of the Pastorex Aspergillus latex antigen agglutination (LA) test and the Platelia^® Aspergillus sandwich ELISA with serum from patients with proven or probable invasive aspergillosis and controls.

Furthermore, in most patients galactomannan was detected in the serum only during advanced stages of disease (10-12). The LA-test was found positive in only 12% of serum samples from patients with hematological malignancies on the day the diagnosis of invasive pulmonary aspergillosis was made (20). However, in 15 of 18 patients with pulmonary infection galactomannan was detected in bronchoalveolar lavage (BAL) fluid which appeared to be a good indicator of disease (20). Nevertheless, the LA-test may contribute in the diagnosis of invasive aspergillosis especially if cultures remain negative, serial samples are obtained (10,12) or when performed with BAL fluid specimens (20).

Recently, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed (Platelia® Aspergillus, Sanofi Diagnostics Pasteur) which employs the same monoclonal antibody as the latex agglutination test (4). The detection limit of the sandwich ELISA was lowered 10-fold by employing the monoclonal antibody as a captor and detector (4), thus allowing 0.5 to 1.0 ng/ml of galactomannan to be detected. Several investigations have shown that the sensitivity of the sandwich ELISA was higher that that of the LA-test (Table 1) (4,7,12,15-19,21).

BMT, bone marrow transplantation; AIDS, acquired immunodeficiency syndrome; ND, not done; LA = latex agglutination test (Pastorex); ELISA = Platelia® sandwich enzyme linked immunosorbent assay.

Furthermore, galactomannan could be detected in the serum at an earlier stage of infection than could be achieved by the LA-test (7,12). With the sandwich ELISA galactomannan could be detected in the serum at an early stage of infection, often before clinical signs and symptoms or radiological abnormalities suggestive of invasive aspergillosis became apparent (Table 2)(21,22).

Table 2. Temporal relationships between antigenemia and clinical and radiological signs and symptoms (23).

Galactomannan was also detected by sandwich ELISA in BAL fluid specimens from patients with invasive aspergillosis and an excellent correlation was found between serum and BAL fluid ELISA results (23). However, serum samples obtained from patients with ELISA positive BAL fluid were found to be positive up to 30 days before the bronchoscopy was performed (23), which supported previous observations that in some patients antigenemia may proceed the development of clinical signs and symptoms. In animal models the concentration of galactomannan in the serum was shown to correspond with the extent of tissue burden with Aspergillus (4), and there is some evidence that the course of the antigen titer corresponds with the clinical outcome of disease (15,24). Indeed, circulating galactomannan reversed in six of ten pediatric patients during antifungal treatment (15) and in a patient with invasive aspergillosis following BMT who responded to antifungal treatment (24). Furthermore, correlation between antigenemia and survival has also been observed among 31 patients with proven or probable invasive aspergillosis by using an in-house inhibition ELISA (25). However, the value of antigen detection for monitoring the response to antifungal treatment remains to be established in prospective trials.

False positive reactivity of the ELISA was found in up to 8% of serum samples and occurred especially with samples obtained within 30 days of BMT or cytotoxic chemotherapy (7,26,27), and in premature infants (28). False positive reactivity in a single serum sample can be overcome by performing ELISA with additional serum samples (12) and most investigators now define positive as two separate positive samples (21). If positive, other tests and procedures are required to localise and confirm the presence of infection.

Until now the sandwich ELISA has been evaluated predominately with serum samples from patients receiving treatment for hematological malignancies. The presence of a well-defined period of increased risk for invasive aspergillosis, e.g. during the neutropenic episode, enables the sandwich ELISA to be used as a screening test to identify patients with an increased risk for invasive aspergillosis (29). This approach may allow those with invasive aspergillosis to be identified at an early stage of infection and to benefit from early antifungal treatment. The feasibility of such an approach is presently under investigation including a cost-benefit analysis. The performance of the sandwich ELISA and the optimal schedule for obtaining samples from other patient groups at high risk for invasive aspergillosis, such as solid organ transplant recipients, those treated with corticosteroids, and patients with AIDS remains to be investigated.

P.E. Verweij
Consultant microbiologist, University Hospital Nijmegen
p.verweij@mmb.azn.nl

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