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Visceral Leishmania 1. Microscopy Parasites may be found in a splenic aspirate, liver biopsy or bone
marrow biopsy. These techniques, especially splenic aspirate and liver biopsy, can be hazardous and require previous expertise in the procedure. Amastigotes may be scanty and therefore difficult to see Smears.
a. Air dry smears. b. Fix in methanol for 1 minute c. Stain with Giemsa 1 in 10 in buffered distilled water pH 6.8 for 30 minutes (or use the rapid Field's stain)
d. Wash the slide in buffered water and drain dry
Amastigotes of leishmania should be seen in positive smears. They are approximately 2-4 µm in size, oval and are frequently seen within the cytoplasm of the macrophage. The amastigotes possess a nucleus
and a rod - shaped kinetoplast within the cytoplasm. In many samples a very small number of parasites are present. Extensive searching of the film is necessary. 2. Culture The
aspirates can be cultured in Novy-Nicolle-MacNeal (NNN) or Schneider's Drosophila medium. In culture the amastigote stage converts to the promastigote stage. However, this is not a rapid technique, as the parasites may
take anything from 10 - 21 days to grow. 3. Serodiagnosis VL produces large amounts of specific IgG which can be used for diagnosis. Currently the most used sero diagnostic tests are
Indirect-immuno Fluorescent Antibody Test (IFAT), Enzyme Linked Immunosorbent Assay (ELISA) and Direct Agglutination Test (DAT). Cutaineous Leishmaniasis 1. Slit skin smear. The margin of the lesion contains amastigotes whereas the centre contains debris and dead skin material. This the margin of the lesion is
aseptically punctured with a hypodermic needle and syringe containing a small amount of saline. The aspirate which is drawn up into the needle is examined microscopically and/or cultured using the method
described in visceral leishmaniasis. 2. Polymerase chain reaction Gene amplification techniques are powerful and sensitive methods and are useful in diagnosis of cutaneous leishmaniasis
particularly when organisms cannot be detected microscopically. It is also very useful for the speciation of Leishmania parasites thus the correct treatment can be administered. Mucocutaineous Leishmaniasis 1. Slit skin smear.
The margin of the lesion contains amastigotes whereas the centre contains debris and dead skin material. This the margin of the
lesion is aseptically punctured with a hypodermic needle and syringe containing a small amount of saline. The aspirate which is drawn up into the needle is examined microscopically and/or cultured using the
method described in visceral leishmaniasis. 2. Polymerase chain reaction Gene amplification techniques are powerful and sensitive methods and are useful in diagnosis of cutaneous leishmaniasis
particularly when organisms cannot be detected microscopically. It is also very useful for the speciation of Leishmania parasites thus the correct treatment can be administered. |
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| [UKNEQAS Parasitology] [Teaching Information] [Intended results] [UKNEQAS Study Day] |