Aspergillus Testing

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 (original/bulk antigen) Discontinued

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.

Antibody (new/Afmp1p antigen)

• Afmp1p is a main cell wall antigen present in Aspergillus fumigatus with similar structures in other fungal species. 
  
• It has been shown to be a target of antibody responses in humans, animal models, and in falcons with confirmed infection.
  
• In early validation results (in-lab), significantly higher reactivity was observed in penguins with confirmed and suspected infection. Low and negative values could be obtained from normal penguins.  In a limited number of cases with repeated measures, levels decreased with treatment.
  
• Albeit with a low sample size, it appears this ELISA has applicability to other avian species.
  
• Results are expressed as an optical density reading with results < 1.0 negative; results > 1.0 positive; results >1.5 strongly positive.

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.

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.

Gliotoxin

Gliotoxin is made during the replication of fungal hyphae and is a known virulence factor of aspergillosis. A positive result should be considered confirmatory of fungal infection – be aware that some other fungal species can make this toxin. A negative result does not rule out infection.

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. Gliotoxin testing should be considered to have the highest specificity but the sensitivity is currently not known. Afmp1p antibody testing shows promise in penguins but more work is needed for full validation in other species.  Please share case information when available.

Antibody (original/bulk antigen) Discontinued 

• Previously, we have shown that in contrast to psittacine species, penguin, waterfowl (and other avian species in zoological institutions and aquaria), and raptor species often have moderate to high detectable levels of reactivity on the ELISA for Aspergillus antibody. High antibody levels had also previously been described by others.
• Recent work with samples from Aspergillus infected dolphins has demonstrated a cross reactivity with Coccidiomyces and Cryptococcus antibody in the same ELISA. It is likely that there are other common fungal elements which also account for the high antibody reactivity in penguins in addition to actual antibody against Aspergillus sp. Thus, at face value, the presence of antibody alone (as part of a panel approach of testing) is not diagnostic in penguin species. Our current study (3) found no significant difference in antibody levels in clinically normal and Aspergillus infected penguins.
• The antibody ELISA performed at the University of Miami Avian & Wildlife Laboratory has remained the same over several years. Data collected since 06/05 shows a mean of 1.8 and SD of 0.4 for African penguins. In review of over 2000 samples without information regarding clinical status – 11% negative (<1.4), 17% weak positive (1.4-1.6), 34% moderate positive (1.7-1.9), 38% strong positive (>2.0).
• There is an appreciation of facility differences for antibody reactivity. This may reflect inside/outside housing or other husbandry related issues that may promote or reduce exposure to Aspergillus and cross reactive antigens. In addition, there is an appreciation of differences by age – young birds clearly show lower reactivity. In these situations, perhaps obtaining a baseline antibody index may be helpful in the differential diagnosis of aspergillosis at a later point. For example, a mean value of samples from one newly constructed facility with younger penguins is 1.5; many penguin facilities which are older range 1.7-2.1.
• In a similar line of support, a previous study on captive and wild Humboldt penguins (8) found a higher antibody index in captive penguins (2.08 mean vs 1.69 mean). In addition, antibody levels were examined at 3 different institutions (means 2.08, 2.54, 1.69). It may be proposed that facilities could perform a review of long term housed penguins to determine mean antibody index (perhaps also by age). Animals with higher antibody index values may reflect additional levels of exposure or possible current/previous infection.

Antibody (new/Afmp1p antigen)

• Afmp1p is a main cell wall antigen present in Aspergillus fumigatus with similar structures in other fungal species. 
  
• It has been shown to be a target of antibody responses in humans, animal models, and in falcons with confirmed infection.
  
• In early validation results (in-lab), significantly higher reactivity was observed in penguins with confirmed and suspected infection. Low and negative values could be obtained from normal penguins.  In a limited number of cases with repeated measures, levels decreased with treatment.
  
• Albeit with a low sample size, it appears this ELISA will also have applicability in other avian species.
  
• Results are expressed as an optical density reading with results < 1.0 negative; results > 1.0 positive; results >1.5 strongly positive.

Galactomannan

• Previously, we have shown a significant increase in galactomannan levels in birds infected with Aspergillus sp. This study involved a group of known positive cases in several different species. It was inclusive of some Aspergillus positive penguin cases. When examined as a group as in the current study, there was no significant difference in mean galactomannan levels in clinically normal and Aspergillus infected penguins. Anecdotally, we appreciate that galactomannan levels can appear increased near the endpoint of disease and in penguins (or avian species in general) with no to low levels of antibody. We hypothesize the low levels of antibody (versus high) allow for higher levels of circulating galactomannan that then can be detected in the assay.
• The galactomannan ELISA performed at the University of Miami Avian & Wildlife Laboratory has remained the same over several years. In review of nearly 2000 samples without clinical data, only 10% of the samples were positive for galactomannan.

Electrophoresis

• Across a multi species study, we have previously shown that beta and beta/gamma globulin increases are the more common electrophoretic abnormality observed in Aspergillus infected birds.
• In the study, the A/G ratio was found to be significantly lower. This was due to a significant decrease in prealbumin and alpha 1 globulins (p<0.005). The alpha 2, beta, and gamma globulins were all significantly increased (p<0.005). Notably, the Aspergillus diseased cases were similar to the non-_Aspergillus_ inflammatory controls except for alpha 2 globulins (mean 1.13g/dL vs. 0.86g/dL). This is a novel finding which has not been previously appreciated.
• The Avian & Wildlife Laboratory does have specific reference intervals for several penguin species. When you submit for protein electrophoresis, please indicate the species of penguin – not just “penguin” – for the best interpretation.
• Hemolysis of penguin plasma samples can and often does results in artifactual increases in the beta fraction. The gamma fraction is also often involved.

3-hydroxybutyrate

• In the current study, 3-hydroxybutyrate (3HB) was found to be significantly increased in Aspergillus-diseased penguins (p=0.002) vs. clinically normal controls as well as non-aspergillosis inflammatory disease controls (3). This assay is now available for sample submission in the Avian & Wildlife Laboratory. Approximately 0.1ml on non-hemolyzed serum or plasma is required.
• Normal penguins had a mean value of 0.94 mmol/L. Concentrations of >1.90 mmol/L were observed in birds with apparent peak clinical signs. A high value of 6.00 mmol/L was reported in a penguin near death. 3HB returns to normal in convalescence. Overall, the specificity was 78.6%.

Electrophoresis + 3HB combination

Using a cutoff of 0.94 mmol/L for 3HB and an increase in alpha 2 globulins (>1.05g/dL), the specificity was 98.7%. The negative predictive value as 77.8% and the positive predictive value was 79.9%. The combination of elevated beta globulins and 3HB resulted in a NPV of 81.3% and PPV of 60%. Thus, the combined tests have a high probability of indicating a patient is not infected with Aspergillus.

Gliotoxin

Gliotoxin is made during the replication of fungal hyphae and is a known virulence factor of aspergillosis. A positive result should be considered confirmatory of fungal infection – be aware that some other fungal species can make this toxin. A negative result does not rule out infection

Summary

• Antibody and galactomannan measures can have value but should be used judiciously based on the clinical signs of the animal and desire to obtain a baseline on a clinically normal animal.
• Electrophoresis is an excellent primary assessment to utilize. Changes in alpha 2 globulins appear to be unique to aspergillosis diseased penguins vs. clinically abnormal/inflammatory (non-aspergillosis) controls.
• 3-hydroxyutyrate also provides a good specificity for the detection of aspergillosis. While additional studies need to be undertaken to address the effects of age and anorexia/diet, this tool can be used in conjunction with the other panel tests.
• Gliotoxin should be considered confirmatory of infection. The utility of this test as a screening test in apparently normal penguins is not known.
• Afmp1p antibody reactivity is significantly increased in penguins with confirmed and suspected infection. Early data suggests utility in other avian species.  Feedback on submissions is greatly appreciated.
• Other test options – including other biomarkers – are currently under development at the University of Miami Avian & Wildlife Laboratory.

The new Dolphin Aspergillus ELISA features cytoplasmic antigens from Aspergillus fumigatus and has been used extensively to diagnose acute and chronic infection in humans. Preliminary work with dolphin samples indicates a substantial improvement in specificity over the Aspergillus Antibody (Dolphin) assay and a moderate significant correlation with the Aspergillus Immunoblot.

It is intended for the detection of antibody to A. fumigatus. Lower reactivity is presumed to be present with infection of some other Aspergillus species. The assay will be performed, at minimum, one time per week. Approximately 0.1ml serum or plasma is needed. The results are expressed as AU/ml.

Antibody

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

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

Galactomannan

C. Cray, D. Reavill, A. Romagnano, F. Van Sant, D. Champagne, R. Stevenson, V. Rolfe, C. Griffin, S. Clubb. Galactomannan assay and plasma protein electrophoresis findings in psittacine birds with aspergillosis. Journal of Avian Medicine & Surgery 2009;23:125-135.

C. Cray, T. Watson, M. Rodriguez, K.L. Arheart. Application of galactomannan analysis and protein electrophoresis in the diagnosis of aspergillosis in avian species. Journal of Zoo & Wildlife Medicine 2009;40:64-70.

Electrophoresis

A. Valdivia, K. Ortega, S. Bhattacharya, and C. Cray. Capillary electrophoresis assessment of plasma protein changes in an African penguin (Spheniscus demersus) with aspergillosis. ACS Omega dx.doi.org/10.1021/acsomega.0c04983, 2020.

G. Desoubeaux, M. Rodriguez, E. Bronson, G. Sirpenski, C. Cray. Application of 3-hydroxybutyrate measurement and plasma protein electrophoresis in the diagnosis of aspergillosis in African penguins (Spheniscus demersus), Journal of Zoo & Wildlife Medicine, 49(3)696-703, 2018.

C. Cray, D. Reavill, A. Romagnano, F. Van Sant, D. Champagne, R. Stevenson, V. Rolfe, C. Griffin, S. Clubb. Galactomannan assay and plasma protein electrophoresis findings in psittacine birds with aspergillosis. Journal of Avian Medicine & Surgery 2009;23:125-135.

C. Cray, T. Watson, M. Rodriguez, K.L. Arheart. Application of galactomannan analysis and protein electrophoresis in the diagnosis of aspergillosis in avian species. Journal of Zoo & Wildlife Medicine 2009;40:64-70.

Biomarkers

A. Valdivia, K. Ortega, S. Bhattacharya, and C. Cray. Capillary electrophoresis assessment of plasma protein changes in an African penguin (Spheniscus demersus) with aspergillosis. ACS Omega dx.doi.org/10.1021/acsomega.0c04983, 2020.

G. Desoubeaux, D. Chauvin, M. Piqueras Soria, E. Bronson, S. Bhattacharya, G. Sirpenski, E. Bailly, and C. Cray. Translational proteomic study to address host protein changes during aspergillosis. PLoS ONE 13(7): e0200843. https://doi.org/10.1371/journal.pone.0200843, 2018.

G. Desoubeaux, M. Rodriguez, E. Bronson, G. Sirpenski, C. Cray. Application of 3-hydroxybutyrate measurement and plasma protein electrophoresis in the diagnosis of aspergillosis in African penguins (Spheniscus demersus), Journal of Zoo & Wildlife Medicine, 49(3)696-703, 2018.

Dolphins

G. Desoubeaux, M. Piqueras, C. Le-Bert, V. Fravel, T. Clauss, A.J. Delaune, R. Daniels, E.D. Jensen, J.E. Flower, G.D. Bossart, S.K. Bhattacharya, and C. Cray. Labeled quantitative mass spectrometry to study the host response during aspergillosis in the common bottlenose dolphin (Tursiops truncatus). Veterinary Microbiology, 232: 42-49, 2019.

G Desoubeaux, C. Le-Bert, V. Fravel, T. Clauss, A.J. Delaune, J. Soto, E.D. Jensen, J.E. Flower, R. Wells, G.D. Bossart, C. Cray. Evaluation of commercial Aspergillus Western blot IgG kit and a genus-specific ELISA for the diagnosis of aspergillosis in common bottlenose dolphins (Tursiops truncatus). Medical Mycology, 56(7):847-856, 2018.

General

R. Hauck, C. Cray, and M. Franca. Spotlight on avian pathology: aspergillosis. Avian Pathology, 49(2):115-118, 2020.
G. Desoubeaux and C. Cray. Animal models of aspergillosis. Comparative Medicine, 68(2):109-123, 2018.

G. Desoubeaux, D. Chauvin, M. Piqueras Soria, E. Bronson, S. Bhattacharya, G. Sirpenski, E. Bailly, and C. Cray. Translational proteomic study to address host protein changes during aspergillosis. PLoS ONE 13(7): e0200843. https://doi.org/10.1371/journal.pone.0200843, 2018.

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

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