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Diagnosis of Pythiosis

The early diagnosis of pythiosis is of importance for its successful treatment. Common mistakes in diagnosis of P. insidiosum occurs frequently because it mimics other infections, and most healthcare professionals will treat with antibiotics, which does not cure this disease. Several methodologies have been used for its diagnosis: wet mount preparations, culture, histopathology, and serology.

Wet Mounts The collected tissue from the infected areas is sent to the laboratory in sterile distilled water at room temperature. Pieces of the tissue is placed with 10% KOH. The finding of sparsely septate hyphae may be indicative of P. insidiosum, or other fungal pathogen (zygomycetes).

Culture To isolate this organism is important to remember that P. insidiosum is highly inhibited by low temperatures. Those, transportation of the biopsied tissue in ice will decrease the chance to isolate this pathogen in culture. Samples, therefore, should be send to the laboratory in water and at room temperature. Small pieces of the biopsied tissue should be placed onto Sabouraud dextrose agar plates and incubated at 37°C. This temperature is ideal for P. insidiosum primary isolation. Incubations at room temperature delay its growth rate. After 24 to 48 hours small colonies should observed around the tissue. Pythium insidiosum growth rapidly at 37°C, thus in 5 days the plate would be cover by a submerged filamentous fungi-like colony.

Microscopically, hyphae without sporulation is observed. To identify P. insidiosum the formation of zoospores has to be induced in water cultures with, grass leaves, and some ions. Sporangia containing zoospores (asexual stage) will be observed at edges of the grass. The production of oogonium (sexual stage) is difficult and rarely found in the media commonly used in clinical labs.

Histopathology Tissue sections of the biopsied samples in H&E shows the typical eosinophilic inflammatory reaction discussed in animal and human disease. The hyphae of P. insidiosum, however, is difficult to observed with this stain. Silver stain and Periodic Acid-Schiff (PAS) are suggested for the proper identification of the hyphal elements of P. insidiosum in tissue. A differential identification of P. insidiosum and fungi developing filamentous structures, especially the zygomycetes, has to be performed.

Serology Several serologic tests have been developed to diagnose pythiosis in humans and animals. They are: Complement fixation, immunodiffusion, Enzyme-Linked Immunosorbent  Assay, immunoperoxidase assay, fluorescent antibodies, and western blot.

Complement fixation Complement fixation was developed in Australia .for the diagnosis of equine pythiosis. It is a sensitive test but it is not specific. This test is no longer in use in laboratories dealing with P. insidiosum.

Immunodiffusion ID This is the most popular serological test to diagnose pythiosis in humans and animals. The test is entirely specific, but it is highly sensitivity. The finding that the sera from some humans and dogs with proven pythiosis gave negative results in this test indicated that the diagnosis using ID has to be confirmed with a more sensitive test. Still, the ID test is recommended as an screen test for the putative diagnose of pythiosis in humans and animals.

Immunoperoxidasel assay This assay was developed to detect specifically the hyphae of P. insidiosum in the biopsied tissue from humans and animals. The technique use polyclonal antibodies against P. insidiosum and the Ag-Ab reaction is detected with a  peroxidase assay. The technique is as specific as the culture.

Enzyme-Linked Immunosorbent Assay ELISA This assay was introduced to be overcome the drawbacks of the ID test. The ELISA for P. insidiosum is specific and sensitive. This assay detected all proven cases of pythiosis in humans and dogs negative by the ID. It also showed to be helpful in detecting cases of cat, cattle, and equines with the disease. Our laboratory offers this ELISA test service to the national and international community. In addition, Pan American Veterinary Labs (www.pavlab.com, Tel: 800-856-9655) a Texan company offers ELISA, culture, fluorescent antibodies in fixed tissues, and molecular tools, such as PCR, to diagnose pythiosis.Fluorescent antibiodies This technique was developed to diagnosis pythiosis from fixed tissue samples and to identify P. insidiosum from culture. The technique specifically detected P. insidiosum hyphae and gave negative results when tested against Entomophthorales and Mucorales zygomycetes. Our laboratory offers this service to the national and international community. For details go to www.pavlab.com, Tel: 800-856-9655. 

Western blot This test have been mainly used for research purposes. It detects different antigenic proteins of P. insidiosum with sera from patients with Pythiosis. It is sensitive and specific.