Leishmaniasis is a parasitic infectious disease comprised from a variety of syndromes, very different the one with the other in their epidemiology, pathogenesis and clinical picture. According to the World Health Organization (WHO), leishmaniasis is considered as one of the most important parasitic diseases (WHO, 1990). It is endemic in 88 countries in five continents with 12 million people afflicted world wide from it and another 350 million living at risk of infection. About 1-1.5 million new cases of cutaneous leishmaniasis (CL) and 500,000 of visceral leishmaniasis (VL), rise per year (Desjeux, 1996).
The disease is attributed to Leishmania, haemoflagellate protozoan parasites of the Trypanosomatidae family. In many localities more than one Leishmania species co-exist with overlapping animal hosts and vectors. It is a zoonotic disease but it is still not known to what extent that affects the epidemiology, transmission and planning of control measures.
During the recent years parallel to the development of techniques based on the genomic information of organisms, there is an increased effort to apply this knowledge to the applied fields of disease diagnostics. In the course of not many years now, some methods were developed for the diagnosis of leishmaniasis based on the genomic information of the parasite. Some of them could detect the parasite very successfully, some not and some are still standing under question. The aim of this study was the evaluation of PCR methods for detection, species identification and determination of genetic variation in L. infantum.
RFLP (restriction fragment length polymorphism) of the ITS (internally transribed spacer), and fingerprinting with single arbitrary primers deriving from the core sequence of the phage M13, from the intergenic tRNA spacers (T3B) and the simple repeat sequences (GTG) 5 and (GACA) 4 (Schönian et al., 1996; Schönian et al., 2000), were applied. Those methods have two general goals: first to discriminate if possible between closely related species and complexes and second to establish a degree of evolutionary relationships. This leads to the construction of molecular evolutionary trees that provide a logical framework to view the evolutionary process in these organisms. 36 well characterised Leishmania strains, 21 canine isolates from Northern Greece and 9 human isolates went through both those methods.
Those isolates that were identified as L. infantum, coming from the whole Mediterranean basin, were processed through a PCR-SSCP (polymerase chain reaction-single stranded conformation polymorphism) with codominant markers, to search levels of genetic variation and heterogeneity among strains of L. infantum. For this 8 sets of codominant primer pairs were used and 42 Leishmania donovani complex strains (38 L. infantum, 2 L. chagasi and 2 L. donovani).
The 42 Leishmania donovani complex strains and 24 clinical canine samples went also through a diagnostic PCR that amplified a 560 bp variable fragment of the ssu rRNA gene (Meredith et al., 1993; Osman et al., 1998a), in order to evaluate detection efficiencies also in canine samples and clinical speciments.
The results of all procedures combined together exploit and establish even more the situation of whether the L. infantum population of the Mediterranean area is a clonal or a recombinant one. Previous studies based on non-molecular approaches and techniques have suggested a clonal population (Jimenez et al, 1997) of the same dominant clone (MON1).
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