**3. Materials and methods**

The laboratory of parasitology of Sousse, the third major Tunisian city which is located in the central, coastal part of Tunisia, was first created in 1986. It can be considered as a central laboratory ensuring the diagnosis of parasitic diseases' cases including leishmaniasis, sent by generalists, dermatologists, pediatricians or hematologists either from the same hospital or other hospitals mainly from governorates of central Tunisia like Kairouan, Sidi Bouzid and Mahdia. Results are usually sent back to the referring doctors so the patients can be managed as appropriate. Since its creation, the laboratory has been involved in the diagnosis of all forms of leishmaniasis.

For purposes of this retrospective study, VL and CL patients' data, including age, sex, geographical origin and the likely place of contamination, clinical presentation and evolution without or after treatment were collected.

#### **3.1 Diagnosis of cutaneous leishmaniasis**

The diagnosis of CL is usually achieved by the demonstration of amastigotes in Giemsastained smears from the fluid obtained by scraping the edges of the cutaneous lesion with a sterile lancet. In addition, the size of the identified *Leishmania* is carefully evaluated, because small amastigotes (< 3) are evocative of *L. infantum* and *L. killicki* rather than *L. major* whose amastigotes are larger. Over the last decade, lesions evocative of CL but found negative in direct examination were further submitted to PCR technique, known to be more sensitive.
