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Isolation
of Leishmania Organisms
The diagnosis
of leishmaniasis
is made by microscopic identification of the nonmotile, intracellular
form (amastigote) in stained sections from lesions, and by culture of
the motile, extracellular form (promastigote) on suitable media.
Slides should be fixed with methanol and Giemsa stained before they are
sent unless reagents are not available. Serologic tests are also
available to detect for anti-leishmanial antibodies; however, these tests
are often not sensitive, particularly for diagnosing cutaneous leishmaniasis,
and do not provide a definitive diagnosis. Examination of scrapings,
impression smears, and biopsy smears can be performed at CDC, with prior
notification of the appropriate state health laboratory.
Leishmanial culture is also available on needle aspirates or punch biopsy lesions. CDC can provide culture medium (typically Novy-MacNeal-Nicolle [NNN] medium). Keep this refrigerated until it is used (stable for 2-4 weeks) and bring it to room temperature right before inoculation. Once inoculated, keep the culture at room temperature and mail as soon as possible by overnight mail. If culture medium is needed, call 770-488-4475.
The needle aspirate should be obtained by first drawing approximately 0.1 ml of preservative-free sterile 0.9% NaCl into a 1.0 to 3.0ml syringe. Insert the needle (23-27 gauge) through intact skin into the dermis of the active border. Move the needle back and forth repeatedly under the skin simultaneously rotating the syringe and applying gentle suction until pink-tinged tissue juice is noted in the hub of the syringe. After the aspirate is obtained, discharge into the leishmanial culture medium. For biopsy specimens, obtain 1 or 2 full thickness punch specimens at the active border of the lesion. Use 1 specimen for culture and the other for impression smears. For culture, place the punch biopsy into the culture medium. The culture tubes should be kept at room temperature prior to and during shipment. The cultures will be kept 4 weeks at CDC and if the culture becomes positive and grows, isoenzyme studies will be performed for species identification.
For more information on Leishmania testing, contact the Division of Parasitic Diseases at 770-488-4475.
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