**2. Materials and methods**

Our study is a retrospective one that includes all CL diagnosed in the laboratory of Parasitology of the Farhat Hached teaching hospital, Sousse, Tunisia, between 1st January 2000 and 31th December 2012. Most of the patients were addressed to the laboratory for suspected CL by the department of Dermatology of the same hospital. Patients' data including age, sex, geographical origin, place of contamination, clinical presentation, treatment and outcome were collected.

The diagnosis of CL was achieved by the demonstration of amastigotes in Giemsa-stained cutaneous smears. Patients with lesions very evocative of CL but found negative on direct examination were further submitted to PCR, known to be more sensitive. Culture on NNN or coagulated rabbit serum (CRS) medium was mainly carried out for the purpose of typing strains either by isoenzymes' electrophoresis or molecular techniques.

#### **3. Results**

#### **1.** Cutaneous leishmaniasis cases

Over the 13 year study period, 2128 cases of CL were diagnosed in the laboratory, the majority of them by the demonstration of *Leishmania* amastigotes in dermal smears. Ninety five per cent of the affected patients originate from one of the following Central Tunisia Governorates (Districts), known to be endemic or highly endemic for ZCL [1, 2]: Sidi Bouzid, Kairouan, Mahdia and Sousse. The 5% remaining patients originate from Northern or Southern areas or from neighboring countries (Lybia, Algeria). Out of the 2128 cases, 2059 were confirmed (by typing strains) or very likely ZCL cases (according to the geographi‐ cal origin or place of contamination, seasonal occurrence, large amastigotes on stained smears, outcome, additional cases in families and neighbors). The 96 remaining cases were mostly *L. infantum* SCL, and few *L. killicki* CCL. The age of patients ranged from 1 month to 91 years (median 26.8 years). One thousand two hundred thirty four (1234) patients were females and 894 males (sex ratio F/M: 1.38).

Most of the patients presented with single or more often multiple ulcerated lesions located on the limbs, the face and to a lesser extent on the trunk. According to patients' declarations, the lesions appeared 10 days to > one year prior to first examination.

Patients with a few lesions (< 5) were treated with intralesional meglumine antimoniate (Glucantime®) and/or cryotherapy. Those with more lesions were treated with intramuscular Glucantime® for 14 to 20 days.

In most treated patients, scarring was obtained in 2 to 8 weeks. In a few of them, however, lesions persisted for a longer period and additional cures were delivered.

**2.** Cases with sporotrichoid nodules:

Out of the 2059 patients with ZCL, 34 (1.7%) developed SN. Fifteen of them were from Mahdia Governorate, 9 from Kairouan Governorate, 6 from Sousse Governorate and 4 from Sidi Bouzid Governorate. It is worth mentioning that Sidi Bouzid region, known to include the most active foci of ZCL in Central Tunisia, was less represented then the 3 remaining Governorates. Inside Governorates, the contamination of all 34 patients took place in areas known to be highly endemic for ZCL.

**2. Materials and methods**

218 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

treatment and outcome were collected.

**1.** Cutaneous leishmaniasis cases

females and 894 males (sex ratio F/M: 1.38).

Glucantime® for 14 to 20 days.

**2.** Cases with sporotrichoid nodules:

lesions appeared 10 days to > one year prior to first examination.

lesions persisted for a longer period and additional cures were delivered.

**3. Results**

Our study is a retrospective one that includes all CL diagnosed in the laboratory of Parasitology of the Farhat Hached teaching hospital, Sousse, Tunisia, between 1st January 2000 and 31th December 2012. Most of the patients were addressed to the laboratory for suspected CL by the department of Dermatology of the same hospital. Patients' data including age, sex, geographical origin, place of contamination, clinical presentation,

The diagnosis of CL was achieved by the demonstration of amastigotes in Giemsa-stained cutaneous smears. Patients with lesions very evocative of CL but found negative on direct examination were further submitted to PCR, known to be more sensitive. Culture on NNN or coagulated rabbit serum (CRS) medium was mainly carried out for the purpose of typing

Over the 13 year study period, 2128 cases of CL were diagnosed in the laboratory, the majority of them by the demonstration of *Leishmania* amastigotes in dermal smears. Ninety five per cent of the affected patients originate from one of the following Central Tunisia Governorates (Districts), known to be endemic or highly endemic for ZCL [1, 2]: Sidi Bouzid, Kairouan, Mahdia and Sousse. The 5% remaining patients originate from Northern or Southern areas or from neighboring countries (Lybia, Algeria). Out of the 2128 cases, 2059 were confirmed (by typing strains) or very likely ZCL cases (according to the geographi‐ cal origin or place of contamination, seasonal occurrence, large amastigotes on stained smears, outcome, additional cases in families and neighbors). The 96 remaining cases were mostly *L. infantum* SCL, and few *L. killicki* CCL. The age of patients ranged from 1 month to 91 years (median 26.8 years). One thousand two hundred thirty four (1234) patients were

Most of the patients presented with single or more often multiple ulcerated lesions located on the limbs, the face and to a lesser extent on the trunk. According to patients' declarations, the

Patients with a few lesions (< 5) were treated with intralesional meglumine antimoniate (Glucantime®) and/or cryotherapy. Those with more lesions were treated with intramuscular

In most treated patients, scarring was obtained in 2 to 8 weeks. In a few of them, however,

strains either by isoenzymes' electrophoresis or molecular techniques.

According to gender, females were more represented then males (23 *vs*. 11) and F/M sex ratio was higher as compared to common ZCL cases (2.1 *vs*. 1.4).

The age of patients with sporotrichoid form ranged from 2.5 years to 81 years. Median (47.8 years) was higher as compared to cases without SN (26.8 years).

In 28 (82.4%) patients, the nodules appeared spontaneously a few days to 3 weeks after the onset of the primary lesions. In 5 (4.7%) patients, the SN developed after intralesional anti‐ monial treatment and in one patient after a 3 day cure of intramuscular Glucantime®.

In the 34 patients, primary ulcers appeared 2 weeks to 5 months prior to the date of diagnosis (median 2.2 months). Their number ranged from 1 to 12 (median: 3.5). They measured 0.5 cm to 6 cm; ninety per cent were between 0.5 cm and 3 cm. They were mainly located on upper limbs: 27.4% on forearms, 26.2% on hands and fingers, 11.9% on arms, 9.5% on wrists and 4.8% on elbows. Lower limbs and other sites (nose, neck, eyelids, cheeks, back) were much less involved. Both sides of the body were equally represented.

The number of SN per patient ranged from 2 to > 10. Most of patients (76.5%) had 2 to 4 nodules; 5 had > 10 nodules.

The size of nodules ranged between 0.5 cm to 3 cm; the majority of them (90.7%) measured 0.5% cm to 1 cm.

Nodules mainly developed on forearms (59.5%) and arms (23.8%). Lower limbs and other sites were much less affected. The left side of the body was slightly more represented than the right one (56.1% *vs*. 43.9%).

In 3 out of the 34 patients, biopsy of nodules was performed. In none of them amastigotes could be demonstrated.

*Leishmania* strains isolated from 10 out the 34 patients were typed either by enzymes' electro‐ phoresis or by PCR according to Tordini et al. [7]. Nine strains were found to be *L. major* and one *L. killicki*.

Whenever SN were observed in patients initially treated with intralesional infiltrations of antimonials, the treatment was switched to the intramuscular form. Thirty three patients responded well to treatment and SN together with primary lesions scared in 2 to 8 weeks. In the last patient, the primary lesions and the nodules persisted up to 13 months. This patient was suffering of scleroderma and submitted to a long-term corticotherapy. In addition, she developed symptoms of antimonial toxicity while receiving intramuscular Glucantime®.
