Cytologic, histopathologic confirmation remains mainstay of diagnosis

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.

Antigen detection for systemic mycoses

Antigen testing methods have been used in the diagnosis of cryptococcosis and aspergillosis. A method employing detection of capsular antigen has been used to find the organism in the body fluids of people. Antigen detection allows for monitoring the level of infection during treatment as quantitative results are available through the ELISA method. Measurement of antigens in urine shows cross reactivity with other causes of disseminated mycosis. Further studies in animals are needed.

Soluble capsular polysaccharide antigens are detected in body fluids of dogs or cats with cryptococcosis. This probably is the most common fungal serologic test used in veterinary practice. Latex-agglutination methods are the most widely used for this procedure. The test can be performed on serum or CSF and can detect the organism in very low concentrations and with high specificity. Proteolytic treatment of the serum prior to performing the test improves its accuracy.

Such measurements are used for diagnostic confirmation and as an index of the effectiveness of treatment. The result of the test is expressed as a titer based on the highest dilution of sera that gives reactivity.

Although the organism can be found as a commensal on mucous membranes, it does not enter the circulation where it can be measured until it proliferates and a granulomatous response develops in the tissues. Antigen enters the circulation and can be measured in the blood and CSF of infected animals.

Methods have been developed to detect Aspergillus antigens in body fluids and usually involve detection of the carbohydrate galactomannan. Inaccurate results have been obtained with the various methods although those with greater sensitivity have been more accurate. Unfortunately, reactions to other micro-organisms, even those causing urinary infections, have given false-positive test results.

Antigen testing has been used widely in diagnosis of human candidiasis. Several commercially available test systems are being used. Most systems detect a metabolite, D-arabinitol; however, its increase may be seen under other conditions. As disseminated infections of Candida have been rarely reported in dogs and cats, there is little information on the use of such procedures.

Molecular detection for systemic mycoses

PCR has been used to a limited degree in various fungal infections of dogs and cats. Generally, this method has been used to detect organisms isolated by culture. Multiplex PCR has been employed to detect a number of yeasts and filamentous fungi in clinical specimens. Reference laboratories use these methods to make finite testing on isolates. PCR has been used for specific identification of filamentous organisms that are often morphologically hard to distinguish in tissue specimens. Molecular methods must be interpreted in light of the clinical parameters and microscopic lesions in the tissues.

Real-time PCR has been used on peripheral blood to detect disseminated infections with Aspergillus or Candida in people. PCR studies on systemic mycoses such as blastomycosis and histoplasmosis have been limited. It has been used in situ in a case of canine histoplasmosis to confirm infection. PCR was also used to compare strains found in the soil and in corresponding human and canine infections.

PCR is used infrequently in the diagnosis of cryptococcosis because antigen detection is so sensitive, specific and widely available as a test kit. PCR has been used to detect the organism in serum, urine, CSF and tissues of cats. PCR has been used most to detect disseminated infections in people with aspergillosis and candidiasis. Samples tested include whole blood, urine, CSF or lavage fluids. Such results have been compared to those measuring serum galactomannan.

Real-time PCR has been used to diagnose disseminated infections in people and to monitor levels of infection following institution of therapy. There is no information of the use of this procedure for disseminated infection in dogs and cats. PCR has been used for rapid detection of urinary candidiasis in dogs and greatly shortened the interval for a confirmed determination of the infecting organism.

PCR has been used only for confirmation of lagenidiosis and pythiosis with testing of tissue specimens; however, there are no data concerning their use on sera or body fluids.

Toxoplasmosis

The cat is the definitive host of this disease caused by Toxoplasma gondii, and so the only difference is that oocysts may be detected in the cat. Otherwise diagnostic tests are aimed at the systemic manifestations of illness from spread of the infection into various body tissues.

Cats become infected most commonly through ingestion of bradyzoites in infected tissues. They shed oocysts only after ingestion of infected meat for a maximum period of two weeks, although the parasite remains encysted in their muscle for life. Re-shedding of oocysts does not occur under most circumstances. Oocysts are difficult to locate and distinguish from other Coccidia in fecal preparations, so this is not used as a definitive means.

Clinical signs in the acute disease are varied, depending on the tissue of encystment. Fever, lymphadenomegaly, hyperesthesia and uveitis have been seen. Therefore, specimens for diagnosis may be chosen depending on the system of involvement. Neonates or immunosuppressed cats develop systemic infection.

In older cats with disseminated encystation, infection of the lungs, CNS or muscle, hepatic, cardiac and ocular tissues are most common. In older or chronically infected animals, the clinical signs may be more insidious. CNS or ocular signs are most common in chronic reactivated infections associated with stress or immunosuppression.

Clinical signs in dogs also can involve the respiratory, neuromuscular or GI systems. Reactivated infections may be more chronic while disseminated disease involving parenchymal organs, such as the lung and liver, can be more rapid and fatal. Myocardial involvement may be more insidious and lead to arrhythmias or eventual myocardial failure. Chronic polymyositis is a syndrome observed in dogs with reactivated infections. Neurologic dysfunction is seen also with chronic infections and the signs observed depend on the location of the inflammation within the CNS.

Hematologic and serum chemistry profile abnormalities usually are nonspecific in acute illness and reflect inflammatory changes in many organs. Hypoalbuminemia may be observed in the acute infections and hyperglobulinemia in more chronic infections. Increased hepatic transaminases or creatine kinase activities are found in animals with acute hepatic or muscle necrosis.

Tachyzoites can rarely be detected in tissues and body fluids during acute illness. Radiographic findings may indicate an alveolar to interstitial infiltrate in the lung fields. Intestines or mesenteric nodes may be enlarged.

Detection of oocysts in the feces of cats is an inaccurate means of confirming a diagnosis. These oocysts are very small approaching the size of erythrocytes. The prevalence of oocysts is low and the excretion interval is 14 days or less. Only some cats develop diarrhea during the period of oocyst shedding and so indications for submitting specimens for fecal examination are absent.

Serologic testing

A number of serologic test methods have been used and no method is in itself definitive. Methods allowing for distinguishing between IgG and IgM antibodies have been valuable for indicating multisystemic infection in cats and for determining their public-health risk. Once infected, animals harbor Toxoplasma cysts for life.

Young kittens acquire maternal antibody in colostrum that can persist for up to 12 weeks. Serologic testing shows exposure to Toxoplasma is prevalent worldwide and increases with the age of the animal. A number of serologic assays are available; however, none by itself is definitive. Agglutination, IFA and ELISA assays are most commonly used.

Documentation of a positive agglutination or IgM titer generally indicates active or recent infection and may correlate with the period of oocyst shedding in cats. A change in IgG titer with paired sera can be used also. A single increased IgG titer can be associated with chronic or reactivated infections. A positive IgG titer in a cat actually confirms its acute infection period has passed. In other words, there is little risk of the cat shedding oocysts. For ocular or CNS infections, measurement of ratios of serum to aqueous humor or CSF levels has been used.

Organism detection

Organism detection is definitive for infection by Toxoplasma; however, finding the organism does not always confirm its role in clinical disease. Encysted forms may be quiescent while circulating tachyzoites often indicate acute systemic spread of infection. Cytologic identification can be helpful, but very insensitive. Toxoplasma antigens have been shown to be released from encysted muscle during chronic infection and this release is exacerbated by immunosuppression. PCR has been used to verify the presence of T. gondii in biologic specimens. Unfortunately, it is so sensitive that false-positive results may be caused by quiescent-encysted organisms in tissues. Therefore, antibody methods should still be considered the mainstay of diagnosis for this infection.

Dr. Hoskins is owner of DocuTech Services. He is a diplomate of the American College of Veterinary Internal Medicine with specialities in small animal pediatrics. He can be reached at (225) 955-3252, fax: (214) 242-2200 or e-mail: jdhoskins@mindspring.com

J ohnny D. Hoskins