**2.4 Visceral leishmanasis (VL)**

234 Current Topics in Tropical Medicine

delegation which is the main transmission focus in Sousse governorate, and led to an

The ongoing outbreak that started in 1982 near the Sidi-Saad dam may be explained by the interruption, as a result of the construction of the dam, in the flooding that frequently occurred in the area and used to decimate a high proportion of rodents every year. In addition, the enrichment of the area's ground water helped the chenopodiaceae to grow abundantly, thereby increasing the food source of *Psammomys*. On the other hand, *Atriplex*, a plant grown in large quantities as a sheep fodder is much appreciated by *Psammomys.*

Fig. 6. Spread of zoonotic cutaneous leishmaniasis during the period 1982- 1986 (Ben Ismail

**1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010**

Fig. 7. Annual number of reported zoonotic cutaneous leishmaniasis cases at the national

Furthemore, humidity created by the dam is highly suitable for the sandflies.

obvious decrease of incidence in both foci.

R., unpublished).

**0**

level from 1982 up to 2010.

**2000**

**4000**

**6000**

**8000**

**Number of cases** 

**10000**

**12000**

**14000**

**16000**

VL has been known to sporadically occur in Tunisia, since 1903 where the first case of mediterranean VL was described in a child living in the suburbs of Tunis. The disease is caused by *Leishmania infantum*, mostly zymodeme MON 1 and to a lesser extent zymodemes MON 24 and MON 80 (Aoun et al., 2001, 2008; Bel Hadj et al., 1996, 2000, 2002; Ben Ismail et al., 1986; Haouas et al., 2005; Kallel et al., 2008b). It is transmitted by *Phlebotomus perniciosus* sandfly, and the dog is the exclusive reservoir, with an infection prevalence rate ranging from 5% to 26 % (Ben Ismail et al., 1986; Ben Rachid et al., 1992; Bouratbine et al., 1998).


Since 1903, 2449 VL cases were reported in Tunisia (Table 2).

Table 2. Number of visceral leishmaniasis cases reported in Tunisia (1903-2010)

Up to 1981, incidence was low to moderate and nearly all cases were reported from Zaghouan, North-West (Le Kef, Béja, Jendouba, Siliana), Tunis and Sousse governorates, located in the humid, sub-humid and semi-arid zones (Anderson et al., 1934, 1938; Ben Ismail et al., 1986; Ben Rachid et al., 1983; Chadli et al., 1968; Khaldi et al., 1991; Nicolle, 1912; Vermeil, 1956). From the early 1980s, the incidence markedly increased and the disease progressed towards the south, mainly to Kairouan governorate and to a lesser extent to Sfax, Sidi Bouzid, Kasserine and Tozeur governorates, together with an increase in canine leishmaniasis (Ayadi et al., 1991; Bel Hadj et al., 1996; Ben Salah et al., 2000; Besbès et al., 1994; Bouratbine et al., 1998; Chargui et al., 2007; Pousse et al., 1995). Indeed, from the 1980s, the region of Kairouan emerged as a highly active VL focus, and by 1991-1992 it was recognized as the most active one, with 30 to 55 % of reported cases (Anonymous).

The distribution of VL is given in figure 8. Highest number of cases was reported in 1922 (n = 130), 1993 and 2006 (n = 122), and 2005 (n = 120). It is worth mentioning that VL has nearly disappeared between 1974 and 1980, as a result of the anti-malaria campaign which included extensive insecticide spraying (Ben Rachid et al., 1983). The disease sporadically occurs in rural and to a lesser extent in suburban areas and mainly affects children under five years, cases in immunocompetent adults being less than 5 % (Ben Ismail & Ben Rachid, 1989; Ben Rachid et al., 1983; Besbès et al., 1994; Bouratbine et al, 1998; Hammoud et al.,

Retrospective Analysis of Leishmaniasis in

**3.2 Diagnosis of visceral leishmaniasis** 

**3.2.2 Serodiagnosis of leishmaniasis** 

Monastir, where an IEE unit was set up in 2001.

Saghrouni et al., 2009.

**3.4 Molecular biology** 

**3.3 Culture of** *Leishmania*

examination are later sent to us together with sera specimens.

aspiration is no more needed and the diagnosis of VL is established.

Sera showing fluorescence at 1/200 dilution are considered positive.

though isolates from VL bone-marrow aspirates were occasionally cultured.

**3.2.1 Parasitological diagnosis** 

Central Tunisia: An Update on Emerging Epidemiological Trends 237

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.

Diagnosis of VL is usually based on the demonstration of amastigotes in Giemsa stained bone-marrow aspirates. However, in our laboratory, bone-marrow smears are mainly carried out and read in doubtful cases, because they usually are performed and interpreted by the haematologists and sometimes the pediatricians themselves and the results of the

For the purpose of parasitological diagnosis of VL, since 1996, we have been using the cytoconcentration of peripheral blood according to the technique of Petithory et al, 1997. Cytoconcentration is usually practiced first, and when positive the painful bone marrow

All sera from suspected or confirmed VL cases are sent to our laboratory for serodiagnosis. Our reference technique is the fluorescent antibody test (FAT). It is carried out by using *Leishmania*-spot IF slides (bioMerieux, France) on sera serially diluted starting from 1/100.

In addition to FAT, we have been using, since 2003, the rK39 dipstick test (DST). The first one we used was from Inbios International, USA (kalazar detect). Then, since 2007 and up to date, we have been using the Dia.Med IT Leish (Dia Med, Switzerland), according to

Our laboratory has mainly been involved in the culture of dermotropic *Leishmania*, even

Culture was mainly carried out for the purpose of typing strains by isoenzyme electrophoresis (IEE) technique. Media we have been using are the NNN (Nicolle Novy Mc Neal), the BHI (brain heart infusion agar) and the CRS (coagulated rabbit serum), the last being best for cutaneotropic *L. infantum* strains. *Leishmania* isolates were typed either in the IEE unit of the LEEP or in the laboratory of parasitology of the Faculty of Pharmacy,

Up to 2008, our laboratory was not equipped for molecular biology techniques. In 2007, as part of a collaborative project on "molecular tools for accurate diagnosis and better assessment of leishmaniases", codirected by the LEEP and our own laboratory and financed by the IAEA, a molecular biology unit was set up in our department and has just started working. In the meanwhile we have been collaborating with the molecular biology unit of the LEEP, mainly for identifying *Leishmania* strains. The first study, conducted in 2005, aimed at identifying strains isolated from patients suspected for having SCL, by using K-DNA-PCR according to Smyth et al., 1992 and PCR-RFLP according to Guerbouj et al., 2001 (Ben Said et al., 2006).

2004; Pousse et al. 1995). In HIV Tunisian patients, VL is infrequent; it is best diagnosed by PCR (Kallel et al., 2007).

Fig. 8. Actual distribution of visceral leishmaniasis in Tunisia.
