**3. Species identification**

Real time PCR (or quantitative PCR-qPCR), a molecular technique which has revolutionized the pathogen diagnosis, is considered to be the future reference method for molecular diagnosis. In recent years, qPCR assays based either on SYBR Green or TaqMan chemistries have been developed and evaluated for the detection, quantification and even species differ‐ entiation of *Leishmania spp* in a variety of clinical samples showing high sensitivity and reproducibility [45,46]. qPCR is considered to be a helpful tool for *Leishmania* diagnosis, monitoring during therapy, development of new drugs and diagnostic tools, comparison of drug efficacy or prophylactic schemes, and for epidemiological studies. Regarding diagnosis of leishmaniasis, the kinetic study of parasitemia in the immunocompromised hosts, the diagnosis of relapses and the quantification of the low parasitic load in asymptomatic patients

qPCR is highly sensitive especially at the lower parasite loads [48,49], specific and reproducible offering the ability to monitor therapy and to prevent relapses. The applications mentioned above make qPCR an attractive alternative to conventional PCR in routine diagnosis [47,49]. Some of the studies carried out so far and their findings regarding the detection threshold,

**threshold Sensitivity % Specificity % References**

**Detection**

kDNA BM, WB 0.001 p/ r [50] kDNA WB 0.07 p/ r 100 83.33 [51] kDNA BM, WB, LN, CS, S, L, LU,K, BC 0.03 p/ r [52] kDNA WB 0.004 p/ r [53] TRYP BS 98.7 59.8 [54] ITS1 WB, SB, S 0.25 p/s [55]

BM: Bone marrow, WB: whole blood, SB: Skin biopsy, CS: Conjunctival swab, LN: Lymph node, S: Spleen, L: Liver, LU: Lung, K: Kidney, BC: Buffy coat, BS: Biopsy specimen, p/r:parasites/reaction, p/s: parasite/sample TRYP: tryparedoxin

Given that PCR is restricted to well equiped laboratory settings, and that there is a need for simplification of the PCR assay and a demand for standardization and optimization [56], the described tools below may represent a good alternative for rapid and simple diagnosis of

Quantitative nucleic acid sequence-based amplification (QT-NASBA) has proven to be a very sensitive and specific assay in diagnostic microbiology which is based on the amplification of single-stranded RNA sequences. In fact, this technique detects RNA in a background of DNA [13]. Several QT-NASBA assays have been developed for the detection of *Leishmania* parasites including QT-NASBA combined with electro-chemiluminescence (ECL) [57,58] and QT-NASBA combined with oligochromatographic technology (OC) [12,59] for the detection of NASBA products. The QT-NASBA assays developed, are commonly based on amplification

**Table 2.** Detection threshold, sensitivity and specificity of qPCR using various targets in different tissues

leishmaniasis in endemic areas and epidemiological studies [12,57].

are of great interest [47].

**Target Tissue tested**

peroxidase gene

sensitivity and specificity are summarized in Table 2.

164 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

The species identification is useful in areas with various sympatric *Leishmania* species such as the southern Mediterranean Basin where CL is caused by *L. major*, *L. tropica* or *L.infantum* and South America where CL may be caused by *L.mexicana* and *L.amazonensis* as well as the species of the subgenus *L.(Viannia).* Regarding the areas where only one species is considered to be responsible for the disease, the species identification is an important tool for the differentiation between *Leishmania* species and lower trypanosomatids related to the monoxenous parasites of insects of the genera *Leptomonas* or *Herpetomonas* which are also considered to cause VL in Southern Europe, South America and in the Indian subcontinent. As far as it concerns the nonendemic areas, they seem to be at risk for parasite importation due to the increasing interna‐ tional travel and population migration [74].

In recent years, there has been great scientific interest in the development of molecular tools, based on PCR or other amplification techniques, for *Leishmania* parasites identification at species and even strain level. The molecular tools used, range from amplification and subse‐ quent RFLP or DNA sequence analysis of multicopy targets or multigene families, including coding and non-coding regions, and PCR-fingerprinting techniques to the recently developed MLST and MLMT with different discriminatory power, sensitivity and specificity while also each one has its specific advantages and drawbacks [74]. Additionally, in most cases, the level of polymorphism found with coding or repeated non-coding PCR-amplified sequences is not refined enough to distinguish between closely related strains while application of MLST and MLMT approaches may reveal important strain polymorphisms.

PCR assays amplifying the conserved region of kinetoplast minicircle DNA or SSU rDNA have been shown to be the most sensitive, but they are able to identify *Leishmania* parasites only to the generic and/or subgeneric level [34,35,41,62]. However, the kDNA PCR-RFLP assay has been used as a molecular marker for *Leishmania* identification at strain level and found to be discriminative between closely related organisms such as *L.infantum* MON-1. In this case, PCR-RFLP of whole minicircle DNA, a highly polymorphic assay, has been applied for differentia‐ tion between recrudescence and re-infection [75,76] and for *L.infantum* strain typing [77]. However, the interpretation of the RFLP patterns is difficult as well as the comparison of the results obtained between laboratories [74,77].

The targets used for species identification include the ribosomal internal transcribed spacer (ITS) [34,78,79]; the mini-exon gene [38,39]; repetitive nuclear DNA sequences [80]; the glucose-6-phosphate dehydrogenase gene [81]; gp63 genes [82]; hsp70 genes[83,84]; cyto‐ chrome b gene [85], 7SL RNA gene sequences [86].

Other PCR-based approaches used for *Leishmania* parasites identification at strain level include the sequences of cysteine protease B (cpb) gene [87–90], the gp63 [87,91], the ITS1 [33,92–94], the mini-exon [95] and the kinetoplast minicircles [76,96–99].

The digestion of ITS1 PCR product with the restriction enzyme HaeIII can distinguish all medically relevant *Leishmania* species. However, almost identical RFLP patterns arise for the representatives of the *L. donovani* complex *(L. donovani* and *L.infantum)* or *L. braziliensis* complex (*L. braziliensis, L. guyanensis, L. panamensis, L. peruviana* etc.) with a great variety of restriction enzymes [34]. According to Schönian et al, in such a case, the sequencing of the ITS1 PCR product will allow the species differentiation [74]. Nasereddin et al developed a simple reverse line blot hybridization (RLB) assay based on ITS1 sequences, which could distinguish all Old World *Leishmania* species, even *L. donovani* from *L.infantum*. This approach was found to be highly sensitive, approximately 10- to100-fold more sensitive than ITS1 PCR while the results obtained were comparable to those found by kDNA PCR [79]. Moreover, Talmi-Frank et al, described a new application of high resolution melt (HRM) analysis of a real time PCR product from the ITS1 region in samples from human, reservoir hosts and sand flies for rapid detection, quantification and speciation of Old World *Leishmania* species. In this assay, different charac‐ teristic high resolution melt analysis patterns were exhibited by *L.major*, *L.tropica*, *L. aethiopi‐ ca*, and *L.infantum* making this approach able to distinguish all Old World *Leishmania* species causing human disease, except *L. donovani* from *L.infantum* [55]. Recently, an alternative technique, PCR-fluorescent fragment length analysis (PCR-FFL), has been developed by Tomás-Pérez et al, for use in *Leishmania* while its use has been reported previously for species identification in Trypanosoma [100,101]. In this study the fluorescently tagged primers used, were designed in the rRNA fragment ITS1 and 7SL region. The amplified fragments were digested and their sizes were determined by an automated DNA sequencer. PCR-FFL was found to be accurate and more sensitive than PCR-RFLP analysis [101].

of the subgenus *L.(Viannia).* Regarding the areas where only one species is considered to be responsible for the disease, the species identification is an important tool for the differentiation between *Leishmania* species and lower trypanosomatids related to the monoxenous parasites of insects of the genera *Leptomonas* or *Herpetomonas* which are also considered to cause VL in Southern Europe, South America and in the Indian subcontinent. As far as it concerns the nonendemic areas, they seem to be at risk for parasite importation due to the increasing interna‐

In recent years, there has been great scientific interest in the development of molecular tools, based on PCR or other amplification techniques, for *Leishmania* parasites identification at species and even strain level. The molecular tools used, range from amplification and subse‐ quent RFLP or DNA sequence analysis of multicopy targets or multigene families, including coding and non-coding regions, and PCR-fingerprinting techniques to the recently developed MLST and MLMT with different discriminatory power, sensitivity and specificity while also each one has its specific advantages and drawbacks [74]. Additionally, in most cases, the level of polymorphism found with coding or repeated non-coding PCR-amplified sequences is not refined enough to distinguish between closely related strains while application of MLST and

PCR assays amplifying the conserved region of kinetoplast minicircle DNA or SSU rDNA have been shown to be the most sensitive, but they are able to identify *Leishmania* parasites only to the generic and/or subgeneric level [34,35,41,62]. However, the kDNA PCR-RFLP assay has been used as a molecular marker for *Leishmania* identification at strain level and found to be discriminative between closely related organisms such as *L.infantum* MON-1. In this case, PCR-RFLP of whole minicircle DNA, a highly polymorphic assay, has been applied for differentia‐ tion between recrudescence and re-infection [75,76] and for *L.infantum* strain typing [77]. However, the interpretation of the RFLP patterns is difficult as well as the comparison of the

The targets used for species identification include the ribosomal internal transcribed spacer (ITS) [34,78,79]; the mini-exon gene [38,39]; repetitive nuclear DNA sequences [80]; the glucose-6-phosphate dehydrogenase gene [81]; gp63 genes [82]; hsp70 genes[83,84]; cyto‐

Other PCR-based approaches used for *Leishmania* parasites identification at strain level include the sequences of cysteine protease B (cpb) gene [87–90], the gp63 [87,91], the ITS1 [33,92–94],

The digestion of ITS1 PCR product with the restriction enzyme HaeIII can distinguish all medically relevant *Leishmania* species. However, almost identical RFLP patterns arise for the representatives of the *L. donovani* complex *(L. donovani* and *L.infantum)* or *L. braziliensis* complex (*L. braziliensis, L. guyanensis, L. panamensis, L. peruviana* etc.) with a great variety of restriction enzymes [34]. According to Schönian et al, in such a case, the sequencing of the ITS1 PCR product will allow the species differentiation [74]. Nasereddin et al developed a simple reverse line blot hybridization (RLB) assay based on ITS1 sequences, which could distinguish all Old World *Leishmania* species, even *L. donovani* from *L.infantum*. This approach was found to be

tional travel and population migration [74].

166 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

results obtained between laboratories [74,77].

chrome b gene [85], 7SL RNA gene sequences [86].

the mini-exon [95] and the kinetoplast minicircles [76,96–99].

MLMT approaches may reveal important strain polymorphisms.

Regarding the hsp70 PCR-RFLP approach, it is considered to be useful for the *L.(Viannia)* species discrimination while its sensitivity is poor for *L.(Leishmania)* species. Diagnostic RFLP patterns for the *L.guyanensis* species complex as well as for *L. lainsoni* and *L. shawi* are produced after restriction with the enzyme HaeIII [84,102]. However, this assay was not able to discrim‐ inate between *L. braziliensis* and *L. peruviana* as well as *L. naiffi*, requiring a second restriction enzyme for the differentiation [102] while also *L. guyanensis* and *L. panamensis* both belonging to the *L. guyanensis* complex share identical RFLP pattern [83]. The discrimination of the species mentioned above is of great significance due to the fact that even if *L. braziliensis* is considered to be the main causative agent of MCL [103] other *L.(Viannia)* species are also suspected of causing MCL. Additionally, a differential response to antimonial treatment has been docu‐ mented [104–106]. This assay was suggested to be applicable on clinical samples [107,108].

Montalvo et al, extended the use of the hsp70 PCR-RFLP for identification of Old World and additional New World species and improved resolution within New World species complexes [108]. Recently, they developed an adequate and flexible toolbox which consists of one improved and three new PCR approaches based on hsp70 target amplification and subsequent RFLP, able to diagnose and identify the most medically relevant New and Old World *Leishmania* species. The new PCR variants were highly sensitive and specific and they pre‐ sented improved amplification efficiency in clinical samples compared to hsp70 PCR described previously by Garcia et al [84]. The choice of the most suitable PCR among the four described, depends on factors like the origin of infection, the sympatry of species, the imported versus endemic pathology, the clinical presentation and the clinical sample [109].

Fernandes et al first developed a PCR approach based on mini-exon gene [36] which was later adapted by Mauricio et al. In this study the mini-exon PCR-RFLP was compared with ITS1 PCR-RFLP. Both targets were shown to be able to identify the strains studied but mini-exon was found to be more polymorphic than ITS1 whereas neither ITS1 nor mini -exon produced as many robust groups as gp63 based restriction analyses published before [91,95]. Marfurt et al also developed a mini-exon PCR-RFLP assay [39]. The pair of primers deriving from the conserved region was able to amplify DNA from Old and New World *Leishmania* species while the diversity detected in the non transcribed spacers represented an informative phylogenetic marker. The digestion of the PCR products with one or two different restriction enzymes resulted in species-specific patterns allowing the species differentiation. Thus, they designed a mini-exon PCR-RFLP genotyping scheme, using different restriction enzymes. However, a single EaeI digest was informative enough for the speciation needed in clinical setting [39]. Furthermore, the repetitive character of this template made it highly sensitive even when applied to clinical samples [38]. On the other hand, when Bensoussan et al compared three PCR assays (kDNA,ITS1 and mini-exon used as targets) found that mini-exon presented the lowest sensitivity (53.8%) and suggested that this discrepancy may be attributed to the examination of stored clinical samples collected on filter papers instead of fresh samples, the extraction or the purification technique [110]. Rocha et al also adapted the PCR approach of Fernades et al and compared four PCR assays (kDNA and mini-exon used as targets) for the evaluation of New World *Leishmania* strains typing. Species belonging to the subgenus *Leishmania* were not amplified with the mini-exon target and the author suggested that this difference probably resulted from intraspecific variation [111]. Recently, in another study, ITS1 and mini-exon targets were compared with 18SrRNA in terms of sensitivity and discriminatory power in clinical samples, under routine laboratory settings. A new pair of primers for miniexon target was designed due to the inability of the previous published primers to amplify the target in all clinical samples while also the protocol was slightly modified in order to achieve better diagnostic sensitivity. However, ITS1 was found to be more sensitive and practical than mini-exon. In contrast, mini-exon was again more polymorphic and revealed a great discrim‐ inatory power in *L.(Viannia)* subgenus [32].

The *L.donovani* complex is the causative agent of visceral leishmaniasis, the most severe form of the disease. The discrimination between the representatives of *L.donovani* complex, *L.infantum* and *L.donovani*, is important as they are morphologically indistinguishable while also they are associated with different epidemiology, ecology and pathology as *L.donovani* is anthroponotic and *L.infantum* is anthropozoonotic. Moreover, there are not discriminative markers to identify certain strains which status is questioned. Thus, the development of molecular tools capable of identifying diagnostic markers and allowing a better understanding of phylogenetic relationships is of great importance. In a study a PCR assay based on cysteine proteinase B (cpb) was developed which was able to differentiate between the two species. The cathepsin-1 proteases CPB which belong to the papain-like superfamily, clan CA and family C1, play an important role in the host protein destruction and evasion of the host immune response [88,112]. CPB enzymes are encoded by a tandem array located in a single locus. Mundodi et al, have compared a *L. donovani* strain and a *L.chagasi*(syn *L.infantum*) strain and revealed at least five tandemly arranged genes [113]. Hide and Banuls, used the last repeats of the cluster (cpbE for *L.infantum* and cpbF for *L.donovani*) and designed a PCR assay able to differentiate the two species by their fragment length as *L.donovani* strains were characterized by a 741-bp product and *L.infantum* strains by a 702-bp product. This PCR assay did not generate amplification for other *Leishmania* species nor trypanosomatids. Although sensitive and specific in cultured parasites, the assay is not sensitive enough for diagnosis on clinical samples [88]. The fact that the species discrimination is based on 39 bp difference in PCR product may cause problems in species identification when using normal agarose gel electro‐ phoresis and where both species are not available for comparison. Thus, another cpb PCR assay was developed with subsequent digestion with DraIII which cuts the 741-bp amplicon of *L.donovani* into 400 and 341 bp and a PCR using a species-specific primer pair capable of amplifying a 317 bp of *L. donovani* whereas it did not amplify *L.infantum* [89]. Two cpb PCR-RFLP and one fluorogenic PCR assay for the molecular typing of *L.donovani* complex have been also developed and it was reported that the assays described were valid and informative for *Leishmania* typing in clinical samples [90,114]. Furthermore, a multilocus approach, using new and previously reported targets including cpb genes, was applied to neotropical isolates (*L.braziliensis, L.peruviana, L.guyanensis, L.lainsoni and L. amazonensis*) and was shown to be a highly robust method of distinguishing different strains [87].

the diversity detected in the non transcribed spacers represented an informative phylogenetic marker. The digestion of the PCR products with one or two different restriction enzymes resulted in species-specific patterns allowing the species differentiation. Thus, they designed a mini-exon PCR-RFLP genotyping scheme, using different restriction enzymes. However, a single EaeI digest was informative enough for the speciation needed in clinical setting [39]. Furthermore, the repetitive character of this template made it highly sensitive even when applied to clinical samples [38]. On the other hand, when Bensoussan et al compared three PCR assays (kDNA,ITS1 and mini-exon used as targets) found that mini-exon presented the lowest sensitivity (53.8%) and suggested that this discrepancy may be attributed to the examination of stored clinical samples collected on filter papers instead of fresh samples, the extraction or the purification technique [110]. Rocha et al also adapted the PCR approach of Fernades et al and compared four PCR assays (kDNA and mini-exon used as targets) for the evaluation of New World *Leishmania* strains typing. Species belonging to the subgenus *Leishmania* were not amplified with the mini-exon target and the author suggested that this difference probably resulted from intraspecific variation [111]. Recently, in another study, ITS1 and mini-exon targets were compared with 18SrRNA in terms of sensitivity and discriminatory power in clinical samples, under routine laboratory settings. A new pair of primers for miniexon target was designed due to the inability of the previous published primers to amplify the target in all clinical samples while also the protocol was slightly modified in order to achieve better diagnostic sensitivity. However, ITS1 was found to be more sensitive and practical than mini-exon. In contrast, mini-exon was again more polymorphic and revealed a great discrim‐

The *L.donovani* complex is the causative agent of visceral leishmaniasis, the most severe form of the disease. The discrimination between the representatives of *L.donovani* complex, *L.infantum* and *L.donovani*, is important as they are morphologically indistinguishable while also they are associated with different epidemiology, ecology and pathology as *L.donovani* is anthroponotic and *L.infantum* is anthropozoonotic. Moreover, there are not discriminative markers to identify certain strains which status is questioned. Thus, the development of molecular tools capable of identifying diagnostic markers and allowing a better understanding of phylogenetic relationships is of great importance. In a study a PCR assay based on cysteine proteinase B (cpb) was developed which was able to differentiate between the two species. The cathepsin-1 proteases CPB which belong to the papain-like superfamily, clan CA and family C1, play an important role in the host protein destruction and evasion of the host immune response [88,112]. CPB enzymes are encoded by a tandem array located in a single locus. Mundodi et al, have compared a *L. donovani* strain and a *L.chagasi*(syn *L.infantum*) strain and revealed at least five tandemly arranged genes [113]. Hide and Banuls, used the last repeats of the cluster (cpbE for *L.infantum* and cpbF for *L.donovani*) and designed a PCR assay able to differentiate the two species by their fragment length as *L.donovani* strains were characterized by a 741-bp product and *L.infantum* strains by a 702-bp product. This PCR assay did not generate amplification for other *Leishmania* species nor trypanosomatids. Although sensitive and specific in cultured parasites, the assay is not sensitive enough for diagnosis on clinical samples [88]. The fact that the species discrimination is based on 39 bp difference in PCR product may cause problems in species identification when using normal agarose gel electro‐

inatory power in *L.(Viannia)* subgenus [32].

168 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

Real-time PCR is considered to be a useful, sensitive, accurate and rapid tool for detection, quantification and even genetic characterization of *Leishmania* parasites. A LightCycler RT-PCR assay based on fluorescence melting curve analysis of PCR products generated from the minicircles of kDNA was developed. This assay was able to detect and differentiate four Old World *Leishmania* species ( *L. major* was differentiated from *L. donovani* and from *L.tropica* and *L.infantum*) [45]. In another study, a qPCR based on glucosephosphate isomerase (GPI) gene was able to discriminate between subgenus *Viannia* and the complexes *L.mexicana, L.donovani/ infantum* and *L.major* [115]. A qPCR based on glucose-6-phosphate dehydrogenase (g6pd) using either SYBR-Green or TaqMan probes has also been described. This assay was able to differentiate *L.braziliensis* from other *L. (Viannia)* species and from those of *L.(Leishmania*) [116]. Weirather et al used a set of primers and probes for serial qPCR assays based on kDNA which was able to detect and differentiate *Leishmania* species in clinical samples due to different melt temperature of the amplicon or by observing the presence or absence of some amplicons [117]. Recently, tryparedoxine peroxidase gene was used as amplification target in a qPCR assay able to identify Old-World *Leishmania* species causing CL [54]. An alternative 18S rDNA based qPCR using fluorescence resonance energy transfer probes (FRET) was able to discriminate the *L.donovani* complex, the *L.brasiliensis* complex, and species other than these based on the distinct melting temperature obtained [46]. Finally, a new qPCR assay based on FRET tech‐ nology and melting curve analysis was designed based on mannose phosphate isomerase (MPI) and 6-phosphogluconate dehydrogenase (6PGD) genes which found to be highly sensitive and discriminative for the five species of *Leishmania* being evaluated (*L.braziliensis, L.panamensis, L.guyanensis, L.peruviana* and *L.lainsoni*) [118].

MLEE, the technique which is regarded as the 'gold standard' for the identification of *Leishma‐ nia* parasites to species and subspecific levels and for genetic diversity studies, has been widely usedsinceits introduction[119].MLEEdetectsdifferentallelesofhousekeepinggenes indirectly by scoring the electrophoretic mobility of the enzymes they encode. The nucleotide differen‐ ces in the genes encoding the enzymes are reflected by their mobility differences. Thus, the parasites are identified by their enzymatic profile and are grouped in taxonomic units termed zymodemes, each one of whom consists of all the strains showing exactly the same profiles for all the enzymatic systems under study. Distinct combinations of isoenzyme mobilities for up to 15 enzymes have been assigned zymodeme numbers (MON-1–MON-274) [120].

However, this molecular method presents several disadvantages including the need for mass culture of *Leishmania* parasites and large amount of protein, it is timeconsuming, labourintensive, costly and technically demanding. It is also worth mentioning that the MLEE methods used in Europe and in South America are based on different enzyme panels and cannot be compared directly [74,93,119]. As far as it concerns its discriminatory power, it is considered to be poor due to its inability to detect nucleotide substitutions that do not change the amino acid composition and changes in the amino acid composition that does not modify the electrophoretic mobility. The discriminatory power of MLEE for classifications below species level is limited. For instance, the *L.infantum* zymodeme MON-1, the causative agent of the majority of visceral leishmaniasis cases around the Mediterranean basin and South America, has been shown to be genetically heterogeneous and polyphyletic with molecular markers presenting higher resolution level [121,122]. Moreover, other molecular studies do not always agree with the classification of *Leishmania* parasites by MLEE. For instance, the differentiation between the representatives of *L.donovani* complex, *L.donovani* and *L.infantum*, is based on only one enzymatic system (glutamate–oxaloacetate transaminase-GOT) making the species distinction poor. In fact, the zymodeme MON-30 which was regarded as *L.infan‐ tum*, has recently shown to be *L.donovani* [123,124]. Furthermore, the existence of *L.archibaldi* as a distinct species belonging to *L.donovani* complex was supported by MLEE but it was not in agreement with the results of many different molecular markers [125] while also *L.killicki* was not confirmed to be a separate species [94,126] and *L.donovani* zymodeme MON-37 was assigned to strains of different genetic background [74,120,127]. However, the codominant character of this molecular tool is advantageous as it is able to identify heterozygous profiles and thus potential hybrids while also if the proteins are highly polymeric, the distinction can be made between a heterozygous profile and a mixed infection [120].

RandomlyAmplifiedPolymorphicDNA(RAPD),asimpleprocess,distinctfromthePCR,based in the amplification of genomic DNA with short oligonucleotides of arbitrary nucleotide sequence used as primers, has been also applied for *Leishmania spp*. The primers are designed and used for the detection of polymorphisms without relying on prior knowledge of the DNA sequence tobeamplified[128].FromtheadventofRAPDtechnique [128,129]numerous studies, onlyafewofthemcanbecitedhere,havebeenpublishedreportingtheuseofRAPDasamolecular tool for *Leishmania* species identification and strain characterization. RAPD has been used for the investigationofthegenomicdiversityof*L.braziliensis* strains [130,131],*L.major*isolates [132], *L.donovani* complex [124,133,134] and *L.infantum* [77,121,135]. Regarding the use of RAPD in species identification, it has been applied for the differentiation between the species *L.brazilien‐ sis, L.mexicana, L.infantum, L.tropica, L.chagasi, L.amazonensis and L.major* [136], the identification anddifferentiationofOldWorldspecies at complex level[137] andrecentlyforthe characteriza‐ tion of clinical isolates responsible for kala-azar in India [138]. The main disadvantages of this technique are the need for parasite culture due to the use of non *Leishmania* specific primers and thepoorreproducibilityoftheassay.Moreover,thebandsof equal electrophoreticmobilitymay not be homologous and it is impossible to distinguish homozygous from heterozygous geno‐ types at specific loci because it is difficult to recognize allelic variants of randomly amplified polymorphic DNA markers in the absence of crossing data [74,120]

PCR hybridization is one of the first molecular methods for species identification and geno‐ typing. DNA probes have been designed for *Leishmania* species identification. The most

common target used for *Leishmania spp* identification is kDNA. DNA probes targeting kDNA have been applied for *L.major* [139], *L.infantum* [140], *L.aethiopica* [141], *L.mexicana* and *L.braziliensis* [142], and *L.mexicana*, *L.donovani* and *L.braziliensis* complexes [143]. Other specific probes developed include a cDNA probe, designed from a repetitive degenerate sequence isolated from *L.donovani*, which specifically hybridized only with isolates of the *L.donovani* complex [144] and two probes, the pDK10 and the pDK20, which were able to differentiate between the Old World *Leishmania* species belonging to *L.donovani* complex and between all Old World *Leishmania* species respectively [145,146]. DNA probes generated from mini-exon genes have also been developed [147]. Other probes developed so far include a *L.braziliensis* specific probe [148] and *L.guyanensis* specific one [149].

intensive, costly and technically demanding. It is also worth mentioning that the MLEE methods used in Europe and in South America are based on different enzyme panels and cannot be compared directly [74,93,119]. As far as it concerns its discriminatory power, it is considered to be poor due to its inability to detect nucleotide substitutions that do not change the amino acid composition and changes in the amino acid composition that does not modify the electrophoretic mobility. The discriminatory power of MLEE for classifications below species level is limited. For instance, the *L.infantum* zymodeme MON-1, the causative agent of the majority of visceral leishmaniasis cases around the Mediterranean basin and South America, has been shown to be genetically heterogeneous and polyphyletic with molecular markers presenting higher resolution level [121,122]. Moreover, other molecular studies do not always agree with the classification of *Leishmania* parasites by MLEE. For instance, the differentiation between the representatives of *L.donovani* complex, *L.donovani* and *L.infantum*, is based on only one enzymatic system (glutamate–oxaloacetate transaminase-GOT) making the species distinction poor. In fact, the zymodeme MON-30 which was regarded as *L.infan‐ tum*, has recently shown to be *L.donovani* [123,124]. Furthermore, the existence of *L.archibaldi* as a distinct species belonging to *L.donovani* complex was supported by MLEE but it was not in agreement with the results of many different molecular markers [125] while also *L.killicki* was not confirmed to be a separate species [94,126] and *L.donovani* zymodeme MON-37 was assigned to strains of different genetic background [74,120,127]. However, the codominant character of this molecular tool is advantageous as it is able to identify heterozygous profiles and thus potential hybrids while also if the proteins are highly polymeric, the distinction can

170 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

be made between a heterozygous profile and a mixed infection [120].

polymorphic DNA markers in the absence of crossing data [74,120]

RandomlyAmplifiedPolymorphicDNA(RAPD),asimpleprocess,distinctfromthePCR,based in the amplification of genomic DNA with short oligonucleotides of arbitrary nucleotide sequence used as primers, has been also applied for *Leishmania spp*. The primers are designed and used for the detection of polymorphisms without relying on prior knowledge of the DNA sequence tobeamplified[128].FromtheadventofRAPDtechnique [128,129]numerous studies, onlyafewofthemcanbecitedhere,havebeenpublishedreportingtheuseofRAPDasamolecular tool for *Leishmania* species identification and strain characterization. RAPD has been used for the investigationofthegenomicdiversityof*L.braziliensis* strains [130,131],*L.major*isolates [132], *L.donovani* complex [124,133,134] and *L.infantum* [77,121,135]. Regarding the use of RAPD in species identification, it has been applied for the differentiation between the species *L.brazilien‐ sis, L.mexicana, L.infantum, L.tropica, L.chagasi, L.amazonensis and L.major* [136], the identification anddifferentiationofOldWorldspecies at complex level[137] andrecentlyforthe characteriza‐ tion of clinical isolates responsible for kala-azar in India [138]. The main disadvantages of this technique are the need for parasite culture due to the use of non *Leishmania* specific primers and thepoorreproducibilityoftheassay.Moreover,thebandsof equal electrophoreticmobilitymay not be homologous and it is impossible to distinguish homozygous from heterozygous geno‐ types at specific loci because it is difficult to recognize allelic variants of randomly amplified

PCR hybridization is one of the first molecular methods for species identification and geno‐ typing. DNA probes have been designed for *Leishmania* species identification. The most MLEE has been recently modified in a direct sequencing allele detection method at each locus, called MLST. Partial sequences of approximately 700 bp in size, belonging to a defined set of housekeeping genes, are directly compared; the alleles are scored as identical or not and the same allele combinations are referred as sequence types. Alternatively, data analysis by sequencing of the alleles may be implemented. This technique was first used for bacterial pathogens whereas in *Leishmania*, steps have been taken to develop a MLST system [150]. The *L. donovani* complex has been studied by 2 sets of 5 loci for genes coding for enzymes used in MLEE [151,152]. These 10 targets in combination should be a complete MLST system for application in *L. donovani* complex. These studies showed that results from MLST are in agreement with results from MLEE whereas some discrepancies were found and MLST presented higher resolution level such as a silent Single Nucleotide Polymorphism (SNP) in gpi that distinguishes between strains of *L.infantum* [151]. Moreover, SNPs resulting in amino acid changes were also found in genes coding for enzymes giving indistinguishable electro‐ phoretic profiles such as in nh2, which has the same protein band size for all *L.donovani* complex strains. These authors reported that MLST could be applied directly to clinical samples or to small-volume cultures. Furthermore, it can be used to detect recombination indirectly and for population genetics studies [151]. Tsukayama et al investigated the intraspecific and interspe‐ cific variation in the coding sequences of four enzymes (gpi, mdh, mpi and 6pgd), used in the MLEE typing method, in order to identify SNPs able to discriminate among closely related species. The assay was applied to clinical samples and successfully identified the species of *Leishmania* responsible for the clinical disease [153]. However, the analysis did not include sufficient diversity of strains for each species [74]. Recently, in another study a combination of the previous published enzyme-coding genes (fh, g6pdh, icd, mpi and pgd) was used so as to differentiate the Chinese *Leishmania* isolates and to investigate their phylogenetic relationships [154]. MLST is likely to become the gold standard basis for taxonomy and identification of *Leishmania*.

MLMT is based on the amplification of microsatellites sequences, tandem repeats of a simple nucleotide motif, 1-6 nucleotides, which are distributed abundantly in the eukaryotic and prokaryotic genomes and may reveal important strain polymorphisms. These markers are very useful for studying genetic variation between closely related organisms. Length polymor‐ phisms in microsatellites sequences result from gain and loss of single repeat units which can be detected after amplification with specific to their flanking regions primers. MLMT ap‐ proaches developed so far for *Leishmania spp*, make use of sets of 14–20 unlinked microsatellite loci. Microsatellite loci with high discriminatory power and being suitable for characterizing closely related strains have been published for the *L.donovani* complex [155–158], *L.donovani* strains [127] *L.major* [159], *L.tropica* [126,160] and for species of the subgenus *L. (Viannia)* [161]. Moreover, as the genetic diversity of *L.infantum* strains has been the subject of intense interest, several studies used MLMT approaches for the evaluation of the genomic variation in *L.infantum* strains [122,135]. It is worth mentioning that when MLMT was compared with other molecular markers for strain typing of *L.infantum*, the results obtained with kDNA PCR-RFLP were comparable to MLMT. kDNA and MLMT presented the highest discriminatory power especially for the MON-1 strains discrimination and appeared to be the most adequate for strain fingerprinting. However, MLMT is advantageous over kDNA PCR-RFLP because of its better reproducibility and feasibility of inter-lab comparisons and the co-dominant character of the markers used, making MLMT suitable for population genetic studies [77]. MLMT is suitable for high-throughput analysis and the data obtained are reproducible and exchange‐ able between laboratories. Moreover, accurate, quality controlled microsatellite profiles can be stored in databases and compared between different laboratories. In contrast to MLEE, selection does not seem to act on polymorphisms in microsatellite length while also the codominant nature of these markers permits the detection of the allelic variants. MLMT can be used directly on biological samples without prior culture of the parasite. DNA extracted from specimens spotted on filter paper or glass slides or from old Giemsa stained microscope slides was successfully applied in MLMT approaches [155]. It is recommended to use a panel of 10–20 unlinked microsatellite markers in all studies for nearly every species because microsatellite sequences are prone to homoplasy. Additionally, polymorphic repeats are not conserved between different species of *Leishmania* [74,122,157].
