The early
diagnosis of pythiosis is important for its successful treatment. 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 are 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 it is important to
remember that P. insidiosum is severely inhibited by low temperatures.
Thus, transportation of the biopsy tissue in ice will decrease the chance
to isolate this pathogen in culture. Samples, therefore, should be sent
to the laboratory in water and at room temperature. Small pieces of
the biopsy tissue should be placed onto Sabouraud dextrose agar plates
and incubated at 37C, the ideal temperature for P. insidiosum
primary isolation. Pythium insidiosum grows rapidly at 37C,
but incubation at room temperature delays its growth rate. Small colonies
should be observed around the tissue sample after 24 to 48 hours at
37C. At 5 days the plate will be filled with submerged filamentous fungi-like
growth.
Microscopically, hyphae without sporulation are 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 rare.
Histopathology: Tissue sections of the biopsy samples
in H&E stains show a typical eosinophilic inflammatory reaction.
The hyphae of P. insidiosum, however, are difficult to observe
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.
Serology: Several serologic tests have been developed
to diagnose pythiosis in humans and animals. They are: Complement fixation,
immunodiffusion, Enzyme-Linked Immunosorbent Assay (ELISA), immunoperoxidase
assay, fluorescent antibodies, and western blot.
Complement fixation for the diagnosis of equine pythiosis was developed
in Australia. It is a sensitive test but it is not specific. This test
is no longer in use in the laboratories dealing with P. insidiosum.
Immunodiffusion (ID) has been the most widely employed serological test
to diagnose pythiosis in humans and animals. The test is very specific
but it has demonstrated a low level of sensitivity. The finding that
the sera from some humans and dogs with proven pythiosis gave negative
results indicated that the diagnosis using ID has to be confirmed with
a more sensitive test.
6/16/04 |