Leishmaniases is a group of diseases caused by more than 20 species of the protozoan genus Leishmania in 98 countries and regions with 350 million people living at risk. The main forms of human disease are visceral (VL), cutaneous (CL) and mucocutaneous (MCL) leishmaniasis [1], [2].

Turkey is of special epidemiological interest because it lies at the crossroad between Asia and Europe and it comprises seven geographical regions with environmental and ecological differences. Human leishmaniasis, both visceral (>40 cases yearly) and cutaneous (>2000 cases yearly) have been reported for centuries in Turkey. Two species of Leishmania are prevalent in Turkey, causing anthroponotic CL (Leishmania tropica), and zoonotic VL (Leishmania infantum) [3], [4]. There is also a single report about the occurrence of L. major variants in specific regions of the country [5].

Human VL and CanL are endemic throughout Mediterranean, Ege, Marmara and Black Sea Regions of western Turkey while sporadic in other regions with higher infection rates in dog populations than human cases. There were two zymodemes of L. infantum (MON1 and MON-98) in dog isolates while all human VL isolates were identified as L. infantum MON-1 by isoenzyme analyses [6]–[8].

Anthroponotic CL caused by L. tropica is highly endemic in the Southeastern Anatolia, East Mediterranean and Ege Regions of Turkey [3], [6]–[9]. In the South Anatolia, L. infantum in addition to L. tropica has also been reported as a causative agent for human CL [6], [10], [11]. Leishmania major is known to be endemic in the countries bordering Turkey to the south: Syria, Iraq and Iran [6]. Parasites isolated from CL patients in Sanliurfa province located in Southeastern Region were all identified as L. tropica MON304 while in Aydin province located in western part, Ege region, were identified as L. tropica MON303 (87%) and L. tropica MON304 (13%) [unpublished data].

A universal PCR method targeting the internal transcribed spacer 1 (ITS1) region between the SSU and 5.8S rRNA genes were described for the direct diagnosis of different clinical manifestations of leishmaniasis and parasite identification. This method is applicable where several more than one parasite species are aetiologically relevant. It is highly specific and sensitive detecting approximately 0.2 parasites per sample [12]. Most of the medically important Leishmania species are then readily distinguished by DNA sequencing or restriction enzyme analysis of the PCR product. ITS1 PCR restriction fragment length polymorphism (RFLP) are used for direct species identification in patient tissues, blood or other samples without prior parasite culturing, microscopic analysis or other technique [6], [12]–[14].

In endemic areas, the presence of multiple Leishmania species with overlapping clinical features and geographical distribution requires the development of sensitive laboratory tests with Leishmania species identification in order to evaluate the prognosis of human and canine leishmaniasis and to choose appropriate therapies. Species identification will also contribute to better understanding the epidemiology of leishmaniases in Turkey [15], [16]. Species identification of the agents of leishmaniasis in Turkey is crucial, since the country comprises seven geographical regions with environmental and ecological differences. ITS1 PCR RFLP was successfully performed for clinical samples collected from human leishmaniasis and CanL cases from Turkey [6].

The main aim of the present study was to design a real time PCR method based on internal transcribed spacer 1 (ITS1) region in the diagnosis of all clinical forms of leishmaniasis and identifying of parasite directly from clinical samples (human and canine) or Leishmania isolates. We further sequenced the PCR products for confirmation and assigning the inter- and intraspecies heterogeneity.

The various leishmaniases are caused by different species of Leishmania, some of which co-exist in the same region like several endemic areas in Turkey; therefore it is crucial to distinguish the species for diagnosis, treatment and epidemiological purposes. Microscopical examination of stained tissue preparations and culture of tissue aspirates for diagnosis, and multilocus enzyme electrophoresis (MLEE) for species identification are accepted as gold standards. However, all the conventional methods applied in the diagnosis of leishmaniasis have medium to low sensitivity and the amplification of DNA by PCR, using different genomic and kDNA targets which is shown to be more sensitive is gradually replacing the traditional methods for the diagnosis of leishmaniasis. Besides, molecular phylogeny studies of the parasite have increasingly suggested new approaches regarding treatment, prognosis of the disease, the distribution of Leishmania species in human and animal hosts, as well as in insect vectors for designing appropriate control measures [12], [20]–[22].

Different PCR/PCR-RFLP based methods targeting kinetoplastic DNA, telomeric sequences, gp63, miniexons, β-tubulin, or ribosomal RNA encoding genes (particularly the internal transcribed spacers, ITS) and recently microsatellites, have been proposed for species identification in Leishmania parasites using isolates and clinical samples [23], [24]. The ITS1 of the ribosomal DNA repeat unit (rDNA-ITS1) has previously been exploited for Old World Leishmania species discrimination using RFLP [12]–[14], reverse hybridization assays [25], and sequencing [26]. There are an estimated 20 to 200 identical copies in the Leishmania genome, making it a good target for analyzing low parasite quantities [13], [14], [27]. A new technique applying PCR-SSCP (single-strand conformational polymorphism) was published recently by Chargui et al. The authors notified that SSCP provides more resolution especially when PCR product has weak band on agarose gel and is less expensive than RFLP method [24].

In our study, we present a real-time ITS1-PCR method that can diagnose three Old World Leishmania species L. donovani complex, L. tropica and L. major, using newly designed probes to diagnose and simultaneously differentiate between Turkish species in clinical samples. The real time ITS1 PCR assay clearly identified the leishmanial species in 81.58% of all clinical samples. L. infantum was identified in 80.43% of human and dog visceral samples while L. tropica was detected in 6.52%. In five isolates (C41, C56, C087, C39, C40) from 3 VL patient and 2 dogs, the agent was diagnosed as L. tropica. This finding was also supported by isoenzyme typing of the strain C87, resulting as L. tropica MON-315 in Montpellier leishmaniasis reference center. This is the first report notifying L. tropica as a causative agent of human and canine VL in Turkey although, L. tropica has been reported to be isolated from human and canine VL cases in different countries [28]–[34]. Several studies and case reports published from neighbouring country, Iran, showed that L. tropica can rarely cause visceral leishmaniasis. Alborzi et al [35] identified only one L. tropica among 64 bone marrow/spleen samples while another group also found only one L. tropica out of 11 isolates obtained from dogs in Iran [36].

L. infantum is reported as a rare causative agent of CL most notably in the Mediterranean Basin countries such as Tunisia, Algeria, Morocco, Spain, Italy, Portugal, Greece and France [2], [37]–[40]. Although L. tropica is the main causative agent of CL in Turkey, L. infantum has dominancy in the South Anatolia of Turkey, mainly in Hatay and Adana provinces according to identification of Leishmania strains by molecular techniques using miniexon gene PCR-RFLP [10], [11], kDNA-PCR [41] and ITS1 PCR-RFLP [6] assays. In our study, it is confirmed that both Leishmania species can be found in clinical samples from CL patients in this area. L. infantum is also isolated from proven vector sand fly species, P. tobbi by Svobodova et al. in the region [11].

So far only little is known about the population structures of the two Leishmania species in Turkey and the correlation with geographical origin, biogeographical parameters, clinical outcome, involved animal reservoir (dogs) and the transmitting sand fly species. Several pioneer studies were performed that used PCR-RFLP and/or sequence analysis of the ITS rDNA, minicircle kinetoplast DNA, miniexon, NAGT [5], [6], [10], [42] mainly for species identification. In Turkey, nine zymodemes of L. tropica and four of L. infantum were described so far [43, unpublished data]. The high degree of heterogeneity in L. tropica species has been reported [13], [14], [44] and shown by MLEE [30]. This variation was reported in different level in Old World Leishmania species by ITS PCR-RFLP analysis from highest to lowest in order of L. tropica>L. aethiopica>L. major>L. donovani [44]. A study using PCR-RFLP/sequencing based on ITS1 region was performed in Iran using clinical samples from CL patients and they showed six different genotype groups of L. tropica [45]. In our study, the heterogeneity of L. tropica was also observed in phylogenetic analysis. L. infantum isolates constituted a single group while strains determined as L. tropica showed several different groups.

The main difficulty in the present study was to observe two peaks in some samples. It is probably due to genetic variety in ITS1 region. Genetic polymorphism in ITS region of different strains of same Leishmania species and possibility of heterogeneity in individual copies was described by El-Tai et al [17]. In a recent study, after digestion of the amplification product with the HaeIII, the ITS1 PCR assay clearly identified the leishmanial species in CL samples in only 72.3% and L. tropica was found to be the most dominant [20]. In our study, the failing of species identification in 18.42% of the samples could be either due to the minuteness of the DNA, possibility of a mix infection, hybridization of different species or intraspecies variations in Turkish Leishmania parasites as also commented by Kifaya et al. [20]. We also found that there are 6 copies of our probe region containing variable part in ITS1 sequence (data not shown). Therefore, we evaluated that if all copies are identical in the DNA sample one peak was obtained; if not, several copies have one or two bases difference, two peaks were obtained but always one peak is higher. In the case of detection of two peaks, we decided to take higher one for species identification with the support of sequence analysis and it can be acceptable that the identification in species level was done in all clinical samples. Hereby, we can speculate that our assay could also propose intragenomic heterogeneity of particular isolates and/or Leishmania DNA samples obtained from clinical materials. The statistical analyses of melting temperatures in the samples having one and two peaks was also performed and standart deviation was found very low in one peak samples than two peak samples. After we compared these results with sequencing results, we also found highest variability in L. tropica group. Gelanew et al. performed PCR-RFLP/direct sequencing assay using L. donovani strains and the direct sequencing of both strands of ITS1 DNA showed the presence of multiple peaks in the chromatograms, which could possibly have resulted from: (i) the presence of multiple strains or clones of Leishmania; (ii) the presence of a hybrid genotype; (iii) intragenomic variation in the multicopy ITS1; or a combination of these [46]. This should be studied furtherly by multiple gene targets and innovative methods like Multilocus Sequence Typing (MLST) of the genes encoding the proteins used for MLEE and Multilocus Microsatellite Typing (MLMT) as well as experimental animal infection studies using Turkish isolates.

Two aims were achieved through the analysis of real time ITS1 PCR products by sequencing of the reference and local strains as designing new probes and proving the sensitivity of the technique at the genus and species identification level. The results of isolates in the optimization step also showed that real time ITS1 PCR results are highly concordant with MLEE analysis (96.07%). We can also propose that the method can identify inter- and intraspecies variability based on ITS1 region but cannot differentiate L. donovani complex species within each other like other molecular assays using ITS1 region [26]. In this point, we would like to emphasize that the studies addressed to L. donovani complex species (L. donovani and L. infantum) identification should be planned.

In conclusion, the proposed method presents a sufficient sensitivity for fast and correct diagnosis of leishmaniasis in all type of clinical samples but due to the samples giving two peaks the ability of the method for species identification is limited and needs further analyses. However, the higher peak was always very well matched with the results of sequence analysis. Genotypic variations based on ITS1 region of Leishmania parasites in Turkey within species and intraspecies were determined. The findings in this study were showed that L. tropica is one of the causative agents of human and canine visceral leishmaniasis in Turkey.