Aspergillus Testing: Antibody, EPH, Galactomannan

About the Assays

The antibody test is performed in the ELISA format and results are currently reported quantitatively using an index. This result is the magnitude greater than the negative controls run within the assay.  That is, an index of 2.0 means the test sample is 2 fold more reactive on the assay.  The galactomannan assay is a commercial assay for a specific dominant Aspergillus antigen.  A pretreatment of the sample is performed to eliminate the problem of antigen complexed with antibody. The result is also a calculated index. Electrophoresis is a standardized technique to quantitate the plasma proteins.

Sample Collection and Submission

Fresh non-hemolyzed plasma samples (<5 days old) are desirable.  Green-top (lithium heparin) tubes with separator gel are preferred.  Longer term refrigeration of samples or non-sterile preparation may result in exogenous Aspergillus contamination and false positive results.

Interpretation of Results

Interpretation of the results should always be performed in conjunction with clinical presentation and other diagnostic information.  Be aware that a significant number of confirmed Aspergillus cases have been found with weak antibody or galactomannan results as well as abnormal EPH (see summary).  In addition, normal birds may have weakly positive results.

Antibody Results
Weak positive results may indicate one of the following: an old titer from previous infection, active antibody production in the initial stages of infection, poor production of antibody by the patient.  Positive or strong positive results represent seroconversion by the patient and can indicate previous or active infection or repeated exposure in older birds.

Galactomannan Results
Confirmed cases have shown a range of negative and positive results. The timing of the sample draw and site of infection will have impact on the results. In human medicine, suspect cases are often subjected to sequential sampling to increase the chance of finding a positive result.  That said, the following data will greatly aid in interpretation.

Protein Electrophoresis Results
Protein electrophoresis (EPH) is used as a gauge of the stimulation of inflammatory processes and humoral immunity – both of which may be present in birds with infectious disease such as aspergillosis.  In suspect and confirmed cases of infection, only 25-40% of the cases had normal EPH when tested as part of the Aspergillus Panel. Of the abnormal EPH results, beta and/or gamma globulin increases were most common. Alpha globulin changes (alone) were seen in less than 25% of the cases.

Summary

Current data from confirmed and suspect cases indicates the use of the combination of antibody, galactomannan and EPH testing to have greater diagnostic accuracy than any one test alone.  There is no doubt from the study that normal birds can have positive results on the tests. What is notable is that value of tests is not necessarily as single tests but as a panel. The more positives (or abnormals) that are recorded on the panel, the higher the likelihood that the bird is a true positive. That is, the specificity of the panel increases with positive/abnormal results on the 3 tests. As expected, a trade off is present with decreasing sensitivity.

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