Aspergillosis

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. No significant differences between the mean antibody indices of the normal vs. confirmed infection birds were observed. However, when the data was examined as percent positive cases (index > 1.4), 69% of the normal birds were negative versus only 42% of the confirmed cases. Thus, infected birds were more likely to be seropositive but the magnitude of positive index value was not significant among the clinical groups. Penguins, raptors, and common zoological collection species are often seropositive whereas psittacine species are rarely seropositive.

Galactomannan Results

Confirmed cases have shown a range of negative and positive results but significant differences have been observed in normal vs. confirmed cases. In other research, the magnitude of the index has been associated with the degree of the burden of infection.

Galactomannan Mean +/- SE
Negative Case 0.64 +/- 0.18
Suspect Case 1.12 +/- 0.11
Confirmed Case 1.68 +/- 0.33

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 our recent large study of confirmed cases, whereas only 30% of the normal group had abnormal EPH results, 72% of the confirmed group had abnormal EPH results. Increases in beta and beta/gamma globulins were most common.

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.

References

Franca M, Cray C, Shivaprasad HL. Serological testing for aspergillosis in broiler chickens and commercial turkeys. Avian Diseases, 56(1):160-164, 2012.

Cray C. New testing options for the diagnosis of avian aspergillosis. ExoticDVM, 12(1): 32- 34, 2010.

Cray C. New testing options for the diagnosis of aspergillosis. Part II: Clinical interpretation of aspergillosis test results. ExoticDVM, 12(2):29-31, 2010.

Cray C, Reavill DR, Romagnano A, et al. Galactomannan assay and protein electrophoresis findings in psittacine birds with aspergillosis. J Av Med Surg 2009;23(2):125-135.

Cray C, Watson T, Arheart KL. Serosurvey and diagnostic application of antibody titers to Aspergillus in avian species. Avian Diseases, 2009;53:491-494.

Cray C, Watson T, Rodriguez M, Arheart K. Application of galactomannan analysis and protein electrophoresis in the diagnosis of aspergillosis in avian species. J Zoo Wild Med, 40(1):64-70, 2009.