The First Isoenzymatic Characterizations of the Leishmania Strains Responsible for Cutaneous Leishmaniasis in the Area of Annaba (Eastern Algeria)

: The epidemiological situation of cutaneous leishmaniasis in the region of Annaba (North Eastern Algeria) is explored for the first time. During a clinical survey carried out in the Annaba Hospital between 2004 and 2008, the parasitological study of lesions has revealed 259 positive cases (43.31%). Isoenzymatic identification of 16 strains isolated showed the presence of three Leishmania species and four zymodemes. The hypothesis of the presence of L. infantum is confirmed with two zymodemes, MON-1 and MON-24. The unexpected presence of L. major MON-25 certifies the sub-stantial extension of this zoonotic form to the North. The isolation of L.killicki , whose presence was unsuspected, reiter-ated the interest of eco-epidemiological analysis of households affected by the disease. Moreover, it is a new zymodeme never isolated so far, the MON-306.


INTRODUCTION
The leishmaniases are known from Algeria for more than a century. Their annual incidence in the country is estimated around 30,000 reported cases in the recent years, of which 29,500 are cutaneous leishmaniasis (CL) cases [1].
ZCL recently outspread from its usual foci and is becoming more frequent in Northern Algeria, coexisting with the visceral form, which raises questions of epidemiology. To answer these questions, epidemiological studies are required, particularly the isolation and identification of all strains circulating among humans, reservoir hosts and vectors . Recently, a third species responsible for CL, Leishmania killicki, has been identified in the focus of Ghardaia, Southwestern Algeria [13].
*Address correspondence to this author at the Service de Parasitologie-Mycologie , Hôpital IBN SINA, CHU Annaba, Algérie; Tel/Fax: 00213 38865686; E-mail: r.mansouri@facmed-annaba.com Furthermore, foci of L.killicki in Tunisia [14][15][16] and L.tropica in Morocco [17][18][19] had been previously reported but not in Algeria, although the bio-climatic conditions and ecological epidemiology are similar. This shows the interest of establishing structures which are able to isolate all strains in endemic foci.
In spite leishmaniases were previously reported from the Annaba region, no Leishmania strains have been isolated and typed from this region as yet. The aim of this work is to provide a better understanding of the epidemiology of leishmaniasis in the region of Annaba, by isolation and identification of strains circulating in the area.
For each patient, an information form was carefully filled up, collecting the administrative, epidemiologic and clinical information. The samples studied were lesion scrapings col-lected under sterile conditions. These samples were examined after Giemsa staining and cultured on NNN medium at 25°C. Several subcultures, during one to five weeks, were often required to obtain a positive culture. The isolates obtained were sent to the Service de Biologie Parasitaire, Institut Pasteur d'Algérie for mass culture and cryopreservation in liquid nitrogen.
The strains were characterised by isoenzyme analysis based on starch gel electrophoresis according to Rioux et al.(1990)  Isoelectrofocusing was used as a complementary technique to provide greater resolving power.

RESULTS
The parasitological diagnostic of the samples revealed 259 positive cases (43.31%). Cultures allowed us to isolate 22 strains of Leishmania, of which 16 were typed by isoenzyme electrophoresis . All patients were infected in their wilaya of origin and did not recall any notion of travel outside, so their cases should be considered as autochthonous. Positive cases were mainly from the wilaya of Annaba, with 68 cases (26.26%), followed by the province of Guelma with 63 cases (24.33%).
The isoenzymatic identification of these 16 strains showed the presence in our sampling of three Leishmania species and four zymodemes : 8 were L. major zymodeme MON-25, 7 were L. infantum, of which two zymodemes were identified (MON-1, 2 strains, and MON-24, 5 strains) and a single strain corresponded to a new zymodeme of L. killicki ( Table 1). The enzymatic profile of this strain was a new variant enzyme which the CNRL of Montpellier has assigned the code MON-306, which differs by ME from the zymodeme MON-301 isolated from Ghardaia [13]( Table 2).
The geographical distribution of zymodemes identified is illustrated in Fig. (1), and fully documented in Table 1. The species isolated in the wilaya of Annaba was L.killicki. In the wilaya of El Tarf, L. infantum MON-1 was isolated from a cutaneous lesion in a patient who had been infected in the   The species L. infantum occurred in infected patients in the wilaya of Tebessa, which seems surprising considering the semi-arid character of this Sub-Saharan location. Indeed, among the three cutaneous strains identified, there is L. infantum. Moreover, it is the zymodeme MON-1 with a usual viscerotropic tropism ( Table 1).

DISCUSSION
Strain identification is a basic investigation useful for the knowledge of Leishmania foci and the understanding of the clinical outcome of leishmaniasis. The ecoepidemiological studies of leishmaniasis have made real progress thanks to the isoenzyme identification [20][21][22][23][24]. Identical clinical lesions may occur due to different species of Leishmania, which stresses the high interest of isoenzyme identification of the strains occurring in the area of Annaba [25]. In the present study, the region of Annaba was explored for the first time. Identification of isolates from patients infected in this area allowed us to type by isoenzyme electrophoresis 16 strains of Leishmania isolated from cutaneous forms.
As in other countries of North Africa, we were able to detect in the Annaba region the presence of three Leishmania species, namely L. infantum. L.major and L.killicki. In Algeria, the authors identified in previous work the two complexes L.major and L. infantum, but L. killicki remained unknown until its recent identification in the focus of Ghardaia (Southwestern Algeria) [13]. During a study on CL in Tunisia, Kallel et al. (2005) had also identified three species, L. infantum (MON-1 and MON-24), L.major (MON-25) and L.killicki .
Looking closely at the strains isolated from cutaneous forms, we deduced that the two CL clinical forms, namely the SCL and ZCL, are prevalent in the area of Annaba. In fact, L.major responsible for the ZCL has been identified in the wilaya of Skikda, Guelma, Tebessa and Souk Ahras at the same time with L. infantum, responsible for SCL. Regarding El Tarf, one strain was identified, L. infantum responsible for the SCL. At Annaba, a new species, L.killicki zymodeme MON-306, was identified, responsible for the chronic cutaneous leishmaniasis (CCL).
SCL has been described in many Mediterranean countries [27][28][29][30][31][32]. It coexists geographically with canine leishmaniasis and the Mediterranean infantile visceral leishmaniasis, which are also due to zymodemes of the same taxon. In Algeria, this SCL form has also been called Northern cutaneous form, as it was first described by [33] and called by them "Clou de Mila" to distinguish it from the ZCL called "Clou de Biskra", and described in 1860 by Hamel. At that time, the causative agent, the vector and the reservoir were unknown. The answer to the first question relating to the causative agent was provided by Belazzoug et al., in 1985, who isolated for the first time L. infantum MON-24. This classical agent of SCL remains the most common dermotropic variant zymodeme. Harrat et al. (1996) during a study at the Institute Pasteur of Algiers, have isolated 14 strains of L. infantum MON-24 from patients with SCL, compared to a single strain of L. infantum MON-1.
Zymodeme MON-24 was reported not only in the North African countries, Northern Tunisia [30,34] and Morocco [35], but also in some South European countries, South of France [25,36], Italy [37], Spain [29] and Greece [31]. In Tunisia, Kallel et al. (2005)  L. infantum MON-1 is the principal agent of Mediterranean VL [38]. However, it was isolated in cases of SCL in Algeria [3,32,39], in Tunisia [40], in France [25],in Spain [29], in Greece [31], in Italy [23] and in Portugal [41]. It has been described in Tunisia first in SCL by Aoun et al. (2000), with two strains against six of L. infantum MON-24. L. infantum MON-1 was also found in rare cutaneous forms in Biskra [3,42] isolated it from dogs in Kabylie and Biskra. Identify [43] 91 strains of L. infantum MON-24 and 7 strains of L. infantum MON-1 from SCL lesions. Concerning the unexpected presence in Skikda (locality of Oued el Aneb) of the L.major species, typically responsible for ZCL of arid areas, it could be explained by a more important extension towards North of this form, in relation to the presence of the vector and rodent hosts and a change in eco-epidemiological conditions related to climate warming. This species we are reporting was isolated in 2006, but the patient contamination occurred in 2005, a year during which outbreaks of CL, including L.major ZCL, occurred in many regions of Algeria [1,6].
In our study, L. infantum and L.major were identified in Guelma and Souk Ahras wilayas which are of humid and sub-humid climates, favorable to L. infantum but currently not to L.major, which is usually prevailing in arid and semiarid biotopes. In Tébessa also, where both species coexist, the presence of two distinct areas, the highlands to the North and the Sub-Saharan area to the South, could explain this coexistence. However, the assumption of the extension of the two species, from North to South for L. infantum and from South to North for L. major, is likely. In Tunisia, Kallel et al. (2005), also reported the presence of L. infantum MON-1 and MON-24 in Beja governorate bordering the Wilaya of El Tarf, in Jendouba governorate bordering the wilaya of Souk Ahras and in El Kef Governorate neighboring the two wilayas of Souk Ahras and Tebessa.
Concerning ZCL, it was formerly restricted to arid and semi-arid areas but an extension to the Highlands of Algeria has occurred. The species responsible, L.major MON-25, has spread from the old foci of Southern Algeria, namely Biskra in the East and Abadla in the West, to the neighboring regions. This species prevails in an endemic state in Northern Saharan areas and is responsible for epidemics. The only zymodeme isolated from this complex in the whole Maghreb, and particularly in Algeria, is MON-25.
In Tunisia, ZCL was also limited, for a long time, to the arid and semi-arid areas. It progressed initially towards the areas of the Tunisian Center [44] then in Béja and Jendouba (NorthTunisia) governorates bordering the two wilayas El Tarf and Souk Ahras. The authors link this increase to a migration of the rodents Meriones [26].
Concerning L.killicki species, responsible for CCL, it was isolated from a cutaneous lesion in a patient originating from the wilaya of Annaba. It is about a patient of male sex, 17 years old.The lesion localized to the eyelid was 1 cm in diameter. The strain identified proved to be L.killicki with a different profile from that of the Tunisian isolates which correspond to the MON-8.
L.killicki was discovered during an epidemic of cutaneous leishmaniasis in the South-East of Tunisia by Rioux et al., 1986. This species was initially included within the L.tropica complex. It was specified as a phylogenetic complex distinct from L.tropica [45].
CCL due to L.killicki MON-8 is distributed in the Sub-Saharan arid areas of South Tunisia [14] and was found by Kallel et al., 2005 in Gafsa (Center of Tunisia) probably reflecting a geographical spread. Haouas et al.,2005 andAoun et al., 2008 reported the presence of L.killicki outside its original foci (South-East) in areas of the ZCL and visceral leishmaniasis.

CONCLUSION
The coexistence of the L. infantum, L.tropica, L.killicki and L.major complexes in neighboring countries, Morocco and Tunisia and the recent identification of L.killicki in the foci of Ghardaia (Southwestern Algeria) and its present identification in the area of Annaba prove the need for further isolation and identification of strains for a better understanding of eco-epidemiological aspects in the areas affected by the disease.