Phenotypic and immunological methods:
Leishmania organisms have been classified as different species primarily on the basis of clinical, biological, geographical and epidemiological criteria. During the last decade a number of methods have become available for the differentiation of Leishmania isolates. For epidemiological purposes, the most useful taxonomic techniques has long been isoenzyme analysis (Miles et al., 1980; Evans et al., 1984;
WHO, 1990; Andresen et al., 1996). The technique still remains the ‘gold standard’ for Leishmania taxonomy and its principal advantage is to provide a stable marker for clusters of geographical isolates within each given species (Hommel, 1999). However, it is restricted in the way that, it assays the genotype indirectly, so that nucleotide substitutions that do not change the amino acid composition remain undetected, changes in the amino acid composition that do not change the electrophoretic mobility may also be not observed (Lewin, 2000). Further more isoenzyme analysis is slow, laborious, and expensive, requiring cultivation of the isolates and the estimation of the profiles of 10-20 different enzymes (Andresen et al., 1996; Noyes et al., 1996). Other techniques include monoclonal antibodies, which are currently being assessed to identify New World species but suffer from their relative lack of specificity for isolates from widely spread foci (McMahon-Pratt & David, 1981; Noyes et al., 1996).
1.1.9.2 Molecular biological methods:
Different DNA-based methods have been used for characterization of isolates of Leishmania at
(a) Genus level: by performing PCR using Leishmania common primers depending on direct identification of the parasite in clinical materials (Uliana et al., 1991; Hassan et al., 1993; Piarroux et al., 1993 & 1994; Andresen et al., 1997; Osman et al., 1997 &
1998).
(b) Species level: different methods could be applied, such as PCR which depends on species specific primers (Ibrahim et al., 1994), analysis of restriction fragment length polymorphism (RFLP), where the substrate can be whole genomic DNA, kDNA or PCR amplification products, analysis of kinetoplast and nuclear DNA including Southern blot hybridisation with specific DNA probes (Jackson et al., 1984; Beverley et al., 1987; Barker, 1989; Van Eys et al., 1989&1991), DNA fingerprinting using DNA probes complementary to repetitive DNA sequences (Macedo et al., 1992), PCR fingerprinting approaches using single arbitrary or non-specific primers (Williams et al., 1990; Tibayrenc et al.,1993; Pogue et al., 1995a&b; Schönian et al, 1996), molecular karyotyping (Lighthall and Giannini,1992) and sequencing of species specific genes e.g. sequence of the first part of ITS (located between the small subunit rRNA and the 5.8S rRNA genes), obtained at Microbiology laboratory, Institüt fur Microbiologie und Hygiene, Berlin, Germany, are found to be almost species specific.
(c) Strain level: these methods are capable to detect variation among isolates of Leishmania belonging to the same species such as sequencing, PCR-fingerprinting approach which uses single non specific primers and the restriction analysis of the amplified ITS region (Schönian et al., 2000; Schönian et al., 2001), single-stranded conformation polymorphisms, SSCP (Van Eyes et al., 1992; Lewin, 2000).
High levels of intra- and inter-species variation were observed in New World Leishmania species of the Viannia subgenus by amplifying and restricting the rapidly evolving internal transcribed spacers (ITS) in the ribosomal operons. Using this aproach it has been made possible to distinguish species and strains of New World Leishmania isolates based on their characteristic restriction patterns (Cupolillo et al., 1995). However, the disadvantage of this technique is that it detects either point mutations that affect the restriction site or deletions or insertions which change the size of the fragment (Orita et al., 1989). To obtain information about the part of the sequence not covered by this analysis, other techniques must be employed.
PCR fingerprinting methods do not depend upon previous knowledge or availability of the DNA sequence of the target (Welsh & McCllelland, 1990; Williams et al., 1990;
Tibayrenc et al., 1993). Using these PCR techniques, detection of genetic polymorphisms among species and strains of Leishmania has been described (Pogue et al., 1995a&b; Noyes et al., 1996; Schönian et al., 1996). However, these methods can only be used on cultured parasites. Contaminating host DNA would mask the signal from the parasite DNA (Noyes et al., 1996).
Analysis of kinetoplast sequences as well as of electrophoretic karyotyping have proven less reliable for identifying Leishmania isolates because extensive polymorphism has been observed by these methods among strains of the same species (Rogers & Wirth, 1987; Bishop & Akinsehinwa, 1989; Bastien et al., 1990;
Henriksson et al., 1996; Wincker et al., 1997).
Direct DNA sequencing of PCR products is another approach commonly used for development of typing methods, population genetic studies, designing specific primer and detection of sequence variation in a particular gene (Bevan et al., 1992; Reddy, 1995). Although it is possible to detect polymorphisms in a sequence, it is not possible to separate and hence define the number of sequence variants, if significant levels of size and sequence heterogeneity exist in a particular DNA e.g. (hypervariable region of DNA) then it may be difficult or even impossible to read a sequence (Gasser & Chilton, 1995). To overcome this problem PCR products are usually cloned into a plasmid vector. Once PCR product is cloned, it may be a tedious and time consuming (Gasser, 1997). Although nucleic acid sequencing is the most informative technique available for genotypic studies, it is labour intensive, difficult and expensive. Sequencing can be faster and easier if an automated sequencer is used.
PCR- based mutation scanning techniques provide powerful alternatives for studying genetic variation in populations (Cotton, 1993; Lessa & Applebaum, 1993). These methods, which rely on physical properties or the modification of DNA molecules of the same or very similar size, that differ by one or more nucleotides, their excellent capacity and potentiality to resolve sequence variability has not been exploited for the study of genetic variation in parasites (Gasser, 1997).
Single-stranded conformation polymorphisms (SSCP) analysis has been developed to scan genes for single base differences which could be useful as genetic markers (Orita et al., 1989; Gasser, 1997; Gasser & Zhu, 1999). This method relies mainly on the principle that the electrophoretic mobility of a single-stranded DNA molecule in a non-denaturing gel is dependant on its structure and sizes (length). The single stranded molecules have secondary and tertiary structures (conformations) as a result of base pairing between complementary nucleotides within each strand. SSCP showed species-specific patterns as well as intra-species variation in an entomological study (Hiss et al., 1994). So far, only a single report describes the use of isotopic SSCP as a tool for the identification of Leishmania at the complex level based on the observation of point mutations in the central part of the SSU rRNA genes (Van Eys et al., 1992).