• Parasitologic diagnosis
• Immunologic diagnosis
• Molecular methods (amplification of parasite DNA)

This is the simplest and most commonly performed procedure. The diagnosis is based on cytologic or histopathologic observation of amastigotes in giemsa stained smears of bone marrow or lymph node aspirates. Immunoperoxidase staining improves the detection of amastigotes in the infected tissues. This method is highly specific and cheap, but it has a low sensitivity.

Canine Bone Marrow cytology-Leishmania organisms concentrated within macrophages

Canine Integuement histopathology- immunoperoxidase stained amastigotes

This is the detection of antibodies (mainly IgG and especially IgG1) against Leishmania parasites or a specific cell-mediated response. The four main serological tests performed are IFAT, ELISA, DAT, and Western Blot. IFAT is considered the gold standard of tests, it has a high specificity and high sensitivity. IFAT uses the whole organism, which gives more repeatable and reliable results, rather than those using soluble Ag, such as complement fixation. ELISA is more sensitive, but less precise than IFAT. ELISA also cross reacts with Trypanosoma cruzi and Babesia.

PCR uses primers from the rRNA gene to identify parasites from a variety of samples, including canine and human bone marrow, lymph nodes, skin biopsies, and heparinized whole blood. PCR is useful in the diagnosis of Leishmania, for follow-up patients pre and post treatment, and for the identification of parasitic species. PCR is a very sensitive test, but is not always available.