**4. Results**

#### **4.1 Cutaneous leishmaniasis**

Over the 25 year period study, 4329 patients were investigated for CL. Most of them were referred to our laboratory by the service of Dermatology of Farhat Hached hospital, Sousse. *Leishmania* parasites were demonstrated in 2087 cases (48.2%). In addition, out of 86 PCR performed on samples found negative in direct examination of dermal Giemsa stained smears, 17 were positive. So, the total number of CL cases diagnosed during the study period was 2104. Most of them were diagnosed during the last decade.

Out of the 2104 diagnosed cases 50 were confirmed or very likely SCL form. Fourteen came from areas known to be endemic for SCL (Le Kef, Jendouba, Siliana, Zaghouan and Bizerte governorates). The remaining 36 cases were from areas located in central Tunisian governorates where SCL has never been described: 13 were from Monastir, 12 from Sousse, 6 from Mahdia and 5 from Kairouan. Out of the 36 cases, 13 originated from areas known to be endemic for ZCL, in Kairouan, Mahdia and Sousse governorates. The age of patients ranged from to 5.5 to 63 years (median = 28.5 years). Twenty six were males and 24 were females (sex ratio M/F = 1.08).

In three patients, the isolate proved to be *L. killicki*. The first patient was a 5 year old child from Meknassi in Sidi Bouzid governorate where CCL was unknown. The second was a 30 year old woman from Ghomrassen, known to be endemic for CCL. The third patient was a 21 year old woman who came from Nasrallah, one of the most active ZCL foci in Kairouan governorate and from where no CCL cases were reported before.

All the remaining 2051 patients were suffering from ZCL. 1182 were females and 869 were males (sex ratio F/M = 1.36). Their age ranged from 1 month to 90 years (median = 28 years). The annual distribution of ZCL cases diagnosed over the 25 year period is shown in figure 9. The number of cases ranged from 5 in 1997 to 443 in 2004.

The place of contamination could be ascertained in 1873 out of the 2051 patients. In the 178 remaining cases, the geographical origin of contamination could not be determined with certainty because of multiple displacements of patients across two, three or more endemic areas. In addition, some patients were originating from Libya and Algeria and were not included in the analysis of the spatio-temporal distribution of ZCL cases.

The distribution of the 1873 ZCL cases according to the area where the contamination took place is given in table 3. Most of the patients came from Sidi Bouzid (610 = 32.6%), Mahdia (494 = 26.4%), Kairouan (369 = 19.7%), and Sousse (306 = 16.3%) governorates.

The distribution of cases according to delegations inside the four governorates mentioned above is shown in figure 10, and the annual distribution of diagnosed cases in figure 11. According to seasonal distribution 1745 (85.1 %) cases were diagnosed between October and February. All confirmed CL patients were treated with local or parenteral Nmethylglucamine antimoniate (glucantime) together with cryotherapy in those with few lesions. Most of treated patients responded well to antimonial treatment, and scarring of

Later, additional strains isolated from CL patients were typed by a novel Multiplex PCR (Saadi

In addition, specimens from patients with lesions highly evocative of CL but found negative in parasitological examination were addressed for PCR to the laboratory of parasitology of the Faculty of Pharmacy, Monastir, where a molecular biology unit has just been set up.

Over the 25 year period study, 4329 patients were investigated for CL. Most of them were referred to our laboratory by the service of Dermatology of Farhat Hached hospital, Sousse. *Leishmania* parasites were demonstrated in 2087 cases (48.2%). In addition, out of 86 PCR performed on samples found negative in direct examination of dermal Giemsa stained smears, 17 were positive. So, the total number of CL cases diagnosed during the study

Out of the 2104 diagnosed cases 50 were confirmed or very likely SCL form. Fourteen came from areas known to be endemic for SCL (Le Kef, Jendouba, Siliana, Zaghouan and Bizerte governorates). The remaining 36 cases were from areas located in central Tunisian governorates where SCL has never been described: 13 were from Monastir, 12 from Sousse, 6 from Mahdia and 5 from Kairouan. Out of the 36 cases, 13 originated from areas known to be endemic for ZCL, in Kairouan, Mahdia and Sousse governorates. The age of patients ranged from to 5.5 to 63 years (median = 28.5 years). Twenty six were males and 24 were

In three patients, the isolate proved to be *L. killicki*. The first patient was a 5 year old child from Meknassi in Sidi Bouzid governorate where CCL was unknown. The second was a 30 year old woman from Ghomrassen, known to be endemic for CCL. The third patient was a 21 year old woman who came from Nasrallah, one of the most active ZCL foci in Kairouan

All the remaining 2051 patients were suffering from ZCL. 1182 were females and 869 were males (sex ratio F/M = 1.36). Their age ranged from 1 month to 90 years (median = 28 years). The annual distribution of ZCL cases diagnosed over the 25 year period is shown in

The place of contamination could be ascertained in 1873 out of the 2051 patients. In the 178 remaining cases, the geographical origin of contamination could not be determined with certainty because of multiple displacements of patients across two, three or more endemic areas. In addition, some patients were originating from Libya and Algeria and were not

The distribution of the 1873 ZCL cases according to the area where the contamination took place is given in table 3. Most of the patients came from Sidi Bouzid (610 = 32.6%), Mahdia

The distribution of cases according to delegations inside the four governorates mentioned above is shown in figure 10, and the annual distribution of diagnosed cases in figure 11. According to seasonal distribution 1745 (85.1 %) cases were diagnosed between October and February. All confirmed CL patients were treated with local or parenteral Nmethylglucamine antimoniate (glucantime) together with cryotherapy in those with few lesions. Most of treated patients responded well to antimonial treatment, and scarring of

et al., in preparation) as part of the IAEA project that is still ongoing.

period was 2104. Most of them were diagnosed during the last decade.

governorate and from where no CCL cases were reported before.

figure 9. The number of cases ranged from 5 in 1997 to 443 in 2004.

included in the analysis of the spatio-temporal distribution of ZCL cases.

(494 = 26.4%), Kairouan (369 = 19.7%), and Sousse (306 = 16.3%) governorates.

PCR was performed according to Chargui et al., 2005.

**4. Results** 

**4.1 Cutaneous leishmaniasis** 

females (sex ratio M/F = 1.08).

Fig. 9. Annual distribution of 2051 zoonotic cutaneous leishmaniasis cases (1986-2010).


Table 3. Distribution of 1873 zoonotic cutaneous leishmaniasis cases according to governorate and delegation.

Retrospective Analysis of Leishmaniasis in

**4.2 Visceral leishmaniasis** 

Tunisia (Mahdia, Monastir, Kasserine).

tests were negative.

Central Tunisia: An Update on Emerging Epidemiological Trends 241

Fig. 11. Annual distribution of 1779 zoonotic cutaneous leishmaniasis cases in Sidi Bouzid

Up to 2010, 2784 serum samples were addressed to our laboratory for suspected VL cases. Anti-*Leishmania* antibodies were demonstrated in 944 sera. FAT and rK39 DST were positive in 944 (99.2%) and 822 (87.1%) cases respectively. In all positive patients, VL was confirmed by the demonstration of amastigotes in bone marrow and/or cytocentrifuged blood and/or favourable outcome under antimonial treatment. In 8 confirmed VL cases, both serological

Three hundred and eight blood samples were investigated by the cytoconcentration technique. Among them, 201 were from confirmed VL patients, and amastigotes were demonstrated in 88 (43.7%) of them. In six cases, bone marrow examination was also negative. Apart from the confirmed VL FAT negative cases, all the remaining negative sera were from patients suffering from diseases other than VL, mainly leukaemia and various haematological disorders. Nine hundred and thirty four of the VL patients were children, aged 2 months to 14 years (median: 26.8 months). Eighteen were adults aged 19 to 39 years (median: 25.9 years); one of them was a HIV+ woman. The number of annually diagnosed cases ranged from 14 cases in 2004 to 83 cases in 2005. The highest proportion of VL cases (60%) was from Kairouan governorate, known to include the most active foci of VL in Tunisia, followed by Sousse governorate and, to a lesser extent from other areas in central

All confirmed VL patients were treated with glucantime, usually a single 21 to 28 day course. The outcome was favourable in the majority of them. Death occurred in 18 (1.9%) patients. All were children. In 4 of them, no response to antimonials was observed despite an adequate treatment regimen. They were assumed to be resistant to antimonials even

(A), Mahdia (B), Kairouan (C) and Sousse (D) governorates (1986 to 2010).

Thus, the total number of VL cases diagnosed over the 25 year period is 952.

Fig. 10. Distribution of 1779 zoonotic cutaneous leishmaniasis cases diagnosed over the 25 year period originating from Sidi Bouzid, Mahdia, Kairouan and Sousse. The dots represent the number of cases. The limits of the governorates are shown in bold lines. The administrative district subdivisions are illustrated by different colours in each governorate.

lesions was obtained in a few weeks (one to three) after the treatment was initiated. However, in some patients, the outcome was unexpectedly atypical in that the lesions took much more delay to heal as demonstrated by the persistence of *Leishmania* in direct examination. In some adequately treated patients, the lesions persisted longer than one year and up to 4.5 years in one of them. In another patient, nearly 100 glucantime injections were needed before the lesions resolved. In some additional cases, new lesions appeared while the patient was under specific treatment for previous ulcers. On the other hand, in many patients treated with *in situ* antimonial infiltrations, sporotrichoid nodules developed a few days or weeks later, next to the treated lesion.

Fig. 11. Annual distribution of 1779 zoonotic cutaneous leishmaniasis cases in Sidi Bouzid (A), Mahdia (B), Kairouan (C) and Sousse (D) governorates (1986 to 2010).

### **4.2 Visceral leishmaniasis**

240 Current Topics in Tropical Medicine

Fig. 10. Distribution of 1779 zoonotic cutaneous leishmaniasis cases diagnosed over the 25

represent the number of cases. The limits of the governorates are shown in bold lines. The

lesions was obtained in a few weeks (one to three) after the treatment was initiated. However, in some patients, the outcome was unexpectedly atypical in that the lesions took much more delay to heal as demonstrated by the persistence of *Leishmania* in direct examination. In some adequately treated patients, the lesions persisted longer than one year and up to 4.5 years in one of them. In another patient, nearly 100 glucantime injections were needed before the lesions resolved. In some additional cases, new lesions appeared while the patient was under specific treatment for previous ulcers. On the other hand, in many patients treated with *in situ* antimonial infiltrations, sporotrichoid nodules developed

year period originating from Sidi Bouzid, Mahdia, Kairouan and Sousse. The dots

administrative district subdivisions are illustrated by different colours in each

a few days or weeks later, next to the treated lesion.

governorate.

Up to 2010, 2784 serum samples were addressed to our laboratory for suspected VL cases. Anti-*Leishmania* antibodies were demonstrated in 944 sera. FAT and rK39 DST were positive in 944 (99.2%) and 822 (87.1%) cases respectively. In all positive patients, VL was confirmed by the demonstration of amastigotes in bone marrow and/or cytocentrifuged blood and/or favourable outcome under antimonial treatment. In 8 confirmed VL cases, both serological tests were negative.

Thus, the total number of VL cases diagnosed over the 25 year period is 952.

Three hundred and eight blood samples were investigated by the cytoconcentration technique. Among them, 201 were from confirmed VL patients, and amastigotes were demonstrated in 88 (43.7%) of them. In six cases, bone marrow examination was also negative. Apart from the confirmed VL FAT negative cases, all the remaining negative sera were from patients suffering from diseases other than VL, mainly leukaemia and various haematological disorders. Nine hundred and thirty four of the VL patients were children, aged 2 months to 14 years (median: 26.8 months). Eighteen were adults aged 19 to 39 years (median: 25.9 years); one of them was a HIV+ woman. The number of annually diagnosed cases ranged from 14 cases in 2004 to 83 cases in 2005. The highest proportion of VL cases (60%) was from Kairouan governorate, known to include the most active foci of VL in Tunisia, followed by Sousse governorate and, to a lesser extent from other areas in central Tunisia (Mahdia, Monastir, Kasserine).

All confirmed VL patients were treated with glucantime, usually a single 21 to 28 day course. The outcome was favourable in the majority of them. Death occurred in 18 (1.9%) patients. All were children. In 4 of them, no response to antimonials was observed despite an adequate treatment regimen. They were assumed to be resistant to antimonials even

Retrospective Analysis of Leishmaniasis in

(Aoun et al., 2008; Haouas et al., 2005).

et al., 2001; Kallel et al., 2008b).

Central Tunisia: An Update on Emerging Epidemiological Trends 243

CCL, known to be confined to limited areas in the presaharian region of Tataouine, has itself shown spread towards areas far from its original endemic foci, up to Kairouan governorate

The described changes are supposed to mainly originate in the agricultural development and the ecological transformations that occurred in the concerned areas and led to a marked increase in irrigated surfaces that in turn helped the reservoirs (mainly rodents) and vectors to abundantly proliferate, and created biotopes very suitable for the *Leishmania* cycles to establish and amplify (Aoun et al., 2008; Ben Abda et al., 2009; Ben Salah et al., 2007). In addition, urbanization of previously rural areas made the populations' access to medical facilities easier, and consequently more and more leishmaniasis cases could be detected and diagnosed. The impact of climate change and variability on leishmaniasis (due to el Niño) was shown in Colombia and is associated to shifts in insect and animal distributions (Cardenas et al., 2008). This phenomenon was also shown to impact on the incidence of VL in Brazil and models were established to predict high risk years for VL (Franke et al., 2002). In Tunisia, the relationship between climate variability and leishmaniasis is not well studied. However, incidence rate of VL was shown to be positively correlated with mean yearly rainfall and continentality index; a rainy year is followed 2 years later by an increase in VL cases likely modulated by the intensity of transmission to dogs and by the influence on sandfly abundance (Ben-Ahmed et al., 2009). Distribution of sandflies classically associated to VL was also shown to be dependent on bioclimate (Zhioua et al., 2007), likewise for the distribution of *Phlebotomus papatasi*, the vector of ZCL (Chelbi et al., 2009). Knowledge on the epidemiological patterns and trends in leishmaniasis has much increased over the last 3 decades mainly because of a better identification of the *Leishmania* species involved in the natural cycles of the parasite. This knowledge greatly benefited of the availability of techniques used in typing isolates obtained from humans or from reservoirs and sandflies. In this respect, IEE or molecular techniques allowed a more precise identification of the *Leishmania* at the species, zymodeme and genomic (schizodeme) level (Ben Hammouda et al., 2000; Ben Ismail et al., 1992; Ben Said et al., 2006; Guerbouj et al., 2007; Guizani et al., 1993, 1994a, 1994b; Guizani et al., 2002; Hanafi

As far as zymodemes are concerned, it was shown that *L. major* was much more homogeneous than *L. infantum*. Indeed, all *L. major* strains obtained from humans, rodents and sandflies proved to be identical to the *L. major* MON 25 reference strain (Aoun et al., 2008 ; Ben Ismail et al., 1986; Haouas et al., 2007 ; Kallel et al., 2005). In contrast, at least three *L*. *infantum* zymodemes occur in Tunisia : i) the MON 1, mainly causing VL and to a lesser extent SCL ; ii) the MON 24, the most common agent of SCL, that also causes VL ; iii) the MON 80, which is very rarely isolated (Aoun et al., 1999, 2008; Bel Hadj et al., 2000, 2002; Gramiccia et al., 1991; Haouas et al., 2007). These findings show that a single zymodeme may cause quite different diseases; and raise the as yet poorly documented question of pathogenesis and virulence of strains in cause. In addition, it was shown that the distribution of both MON 1 and MON 24 *L. infantum* zymodemes was different according to the geographical areas the patients originated from. Indeed, *L. infantum* MON 24 zymodeme was more frequently reported in VL cases from Kairouan governorate as compared to northern VL foci where MON 1 zymodeme is predominant. This was attributed to the emergent character of Kairouan foci which may favour and select atypical or rare variants (Aoun et al., 2008; Ben Abda et al., 2009). Involvement of different

though the resistance could not be assessed by *in vitro* testing of the strain. In an additional child, initially treated by three successive courses of glucantime, healing was ultimately obtained with ambisome (liposomal amphotericin B).

#### **4.3 Typing of** *Leishmania* **strains**

Over the 25 year study period 103 *Leishmania* strains were typed by either IEE or PCR or both. More than 150 isolates were *in vitro* cultured. Fifteen of them were from bone-marrow aspirates in VL cases. All were typed by IEE and found to be *L. infantum*. Most cultured isolates were from CL patients. Eighty one of them could be maintained in culture media, cryopreserved and typed by IEE. The remaining isolates were either lost after repeated subculture or contaminated during sampling or subculturing. It should be mentioned that strains addressed to the laboratory of Monastir, were typed and identified at the zymodeme level, whereas those typed in the LEEP were only identified at the species level.

Sixty four isolates were typed by molecular techniques: 4 were typed by K-DNA-PCR and PCR-RFLP, and 60 by multiplex PCR.

Overall, 88 CL strains were typed by either IEE or PCR technique: 7 were *L. infantum*, 3 were *L. killicki* and 78 were *L. major*. All *L. major* strains that were typed at the zymodeme level were found to be *L. major* MON 25.
