Cytologic, histopathologic confirmation remains mainstay of diagnosis

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May 01, 2007

Q Please review laboratory diagnosis of systemic mycoses and toxoplasmosis.

A In addition to tickborne disease, veterinarians need to know more about diagnosing systemic fungal diseases and toxoplasmosis.

Dr. Craig E. Greene at the 2006 American College of Veterinary Internal Medicine Forum in Louisville, Ky., gave a lecture titled "Laboratory Diagnosis of Systemic Mycoses and Toxoplasmosis." Some relevant points in Dr. Greene's lecture are reviewed here:

Systemic mycoses

Initial diagnosis of systemic fungal infections generally involves microscopic examination, depending on the fungal infection involved. Culture of many of these agents is avoided in many cases. Where the organism cannot be identified, serologic methods have been used as supportive tools; however, the specificity of these tests is variable among the different agents.

Culture for systemic mycoses

Fungal culture is not generally used for deep mycoses because of the risk to public health. Airborne spread of propagated fungal elements can be extremely hazardous. Culture of fungal agents must be correlated with other methods because microscopic confirmation of their presence is needed.

In some cases, mucosal isolation of such organisms may indicate normal colonization. The definitive diagnosis is made by demonstration of yeasts in specimens from the patient.

Cytologic and histopathologic confirmation is the mainstay of diagnosis.

Many stains can be used to detect the yeasts in exudates or aspirates. For histopathologic examination, special stains such as periodic acid-Schiff, Gomori's methenamine silver, or Grocott's stains are used. Depending on the fungus, organisms can be identified intracellularly or extracellularly. Staining can be achieved with other immunochemical stains and light microscopy. Genetic detection methods such as PCR or in-situ hybridization can be used also.

Antibody titers may be unreliable for many infections as environmental exposure may cause an increase. Antibody measurement might be considered if searching for organisms is unrewarding but with microscopic evidence of inflammation compatible with fungal cause.

Antibody detection for systemic mycoses

For blastomycosis, the agar gel immunodiffusion test (AGID) is the most commonly employed serologic test. The results are qualitative, as precipitin bands are visually detected and finding a positive result may support the diagnosis (but is nonspecific).

The test may be positive in dogs that were exposed to the agent but have contained or eliminated the infection. False-negative results may be found early in the infection.

Few infected cats have been evaluated with this method. Other test methods such as ELISA have been developed; however, the accuracy and sensitivity of this method has not been extensively evaluated.

In cases of suspected histoplasmosis, serologic testing usually involves AGID or complement fixation; however, false-nega-tive results have been found in animals with immunosuppression associated with disseminated infections. Cross-reaction occurs in some animals with blastomycosis. There is no accurate means of diagnosing histoplasmosis in dogs and cats using antibody detection.

Antibody detection methods are used most commonly in coccidioidomycosis to confirm infection where organisms cannot be demonstrated by cytologic or histopathologic methods.

The standard tests have been the tube precipitin and complement fixation methods. The former detects predominantly IgM and the latter detects IgG. AGID and ELISA methods have also been employed to detect these specific antibody classes. Positive results with tests detecting IgM are typical or early or active infection, or with reactivation of infection associated with immunosuppression. IgG titers are increased in chronic or past infections.

In some cases with superficial or localized hilar node infections, antibody test results are negative in infected dogs. With more limited studies, these serologic tests also appear to be useful in the diagnosis of infection in cats.

Many methods have been employed to detect Aspergillus antibody in diseased dogs and cats. For nasal and disseminated infections, results have been unpredictable despite the methods employed. False-positive and false-negative results have been obtained.

Testing for specific antibodies in serum has been a valuable tool in the diagnosis of pythiosis and lagenidiosis. Initially immunoblot analysis was used; however, the current assay uses an ELISA method that allows for quantitation of antibody levels. Results using this test seem to be sensitive and specific, and levels can be used to monitor the response to infection.