Table 12.

Practical guide for evaluating skin lesions that develop after accidental exposures to Leishmaniaspp.a

General comments
 To increase sensitivity, use several techniques and obtain multiple specimens per technique. Even under optimal circumstances, the maximum overall sensitivity of this approach, using conventional parasitologic methods, may be only ∼70–75% and is even lower with chronic lesions and mucosal disease.
 After cleansing the skin with 70% ethanol, inject anesthetic (i.e., 1% lidocaine with epinephrine 1:100,000) through intact skin into the dermis under the area to be sampled. High concentrations of anesthetic could inhibit parasite growth in culture, as could residual iodine if iodine is used to cleanse the skin. Before obtaining dermal scrapings and biopsy specimens, debride eschar from the relevant portions of the lesions and apply pressure with sterile gauze to achieve hemostasis and to avoid making bloody smears.
Obtain needle aspirates for leishmanial culture.
 Obtain 3–5 aspirates from different lesions or different portions of a lesion. Draw up ∼0.1 ml of preservative-free sterile 0.9% saline into a 1.0–3.0-ml syringe. For ulcerative lesions, insert the needle through intact skin into the dermis of the active border. Use a 23- to 27-gauge needle; use small-gauge needles for facial lesions. Repeatedly move the needle back and forth under the skin, tangentially to the ulcer, simultaneously rotating the syringe and applying gentle suction, until pink-tinged tissue fluid is noted in the hub of the needle. If no aspirate is obtained, inject 0.05–0.1 ml of saline under the skin and resume suction.
 Discharge each aspirate into a separate tube of culture medium (e.g., Novy-MacNeal-Nicolle medium). Thin smears of aspirates typically are suboptimal unless a cytospin preparation is used.
Obtain biopsy specimens for cultures and histopathology.
 Obtain one or two full-thickness punch biopsy specimens at the active border of one or more lesions, with some of the specimen from nonulcerated tissue.
 Divide the specimen into three portions, or obtain multiple biopsy specimens:
  Use one portion for leishmanial culture and, if appropriate, for bacterial, mycobacterial, and fungal cultures.
  Use one portion for impression smears (i.e., touch preparations; see below).
  Use one portion for histologic examination of tissue stained with hematoxylin and eosin; Giemsa; and, if appropriate, special stains to exclude mycobacterial, fungal, and other infectious etiologies. Although histopathology generally is the least sensitive technique for diagnosing cutaneous leishmaniasis (sensitivity, <20% in some studies), it is useful for excluding other diagnoses. Amastigotes are more easily recognizable in touch preparations and in thin smears of tissue scrapings (see below).
  PCR, monoclonal antibody analyses, and animal inoculation can also be done.
Make tissue impression smears.
 Grasp the biopsy specimen with forceps. Gently blot the cut surface onto a clean paper towel or gauze to remove excess blood. Gently press the blotted surface, with a rolling or circular motion, onto a glass slide. Repeat in a parallel row down the slide. Air dry the slide, fix in methanol, and stain with Giemsa.
Obtain dermal scrapings for thin smears.
 Obtain 3–5 dermal scrapings from different lesions or different portions of a lesion (e.g., beneath the necrotic lip of the lesion). If aspirates and biopsy specimens for culture will also be obtained from these lesions, obtain the dermal scrapings last to minimize the risk of contaminating the sites. Some practitioners use the slit-skin smear technique and first make an incision before obtaining dermal scrapings. For this technique, pinch the skin to exclude blood and use a scalpel blade to incise a slit, several millimeters long and deep, through intact skin into the dermis. For ulcerative lesions, start the incision in the active border and proceed radially out across several millimeters of intact skin.
 Obtain tissue fluid and flecks of tissue by scraping the dermis (e.g., beneath the necrotic lip of the lesion or along the walls of the incision) with a sharp instrument (e.g., a scalpel blade or stainless steel spatula). After obtaining as much tissue as possible, make as thin a smear as possible. Air dry the slide, fix in methanol, and stain with Giemsa. Although dermal scrapings can also be cultured, the risk for contamination is high.
Examine slides by light microscopy.
 Slides should be examined under oil immersion for amastigotes, the tissue stage of the leishmanial parasite. Amastigotes are obligate intracellular organisms. However, on slides (e.g., thin smears of dermal scrapings), amastigotes may also be found extracellularly. Amastigotes are round-to-oval structures, 2–4 μm long, with 2 prominent internal organelles (i.e., a nucleus and a kinetoplast, which is a rod-shaped, specialized mitochondrial structure with extranuclear DNA). When stained with Giemsa, the cytoplasm of the amastigote typically is pale blue and the nucleus and kinetoplast are pinkish red or violet blue.
  • a This table is adapted from reference80 with permission from the publisher. For questions about the diagnosis and treatment of leishmaniasis, call the CDC Division of Parasitic Diseases at (770) 488-7775 or (770) 488-7760. CDC can provide culture medium and perform isoenzyme analysis to identify the causative species.