Development of Assays Using Hexokinase and Phosphoglucomutase Gene Sequences That Distinguish Strains of Leishmania tropica from Different Zymodemes and Microsatellite Clusters and Their Application to Palestinian Foci of Cutaneous Leishmaniasis

Background/Objectives Palestinian strains of L.tropica characterized by multilocus enzyme electrophoresis (MLEE) fall into two zymodemes, either MON-137 or MON-307. Methodology/Principle Findings Assays employing PCR and subsequent RFLP were applied to sequences found in the Hexokinase (HK) gene, an enzyme that is not used in MLEE, and the Phosphoglucomutase (PGM) gene, an enzyme that is used for MLEE, to see if they would facilitate consigning local strains of L.tropica to either zymodeme MON-137 or zymodeme MON-307. Following amplification and subsequent double digestion with the restriction endonucleases MboI and HaeIII, variation in the restriction patterns of the sequence from the HK gene distinguished strains of L.tropica, L.major and L.infantum and also exposed two genotypes (G) among the strains of L.tropica: HK-LtG1, associated with strains of L.tropica of the zymodemes MON-137 and MON-265, and HK-LtG2, associated with strains of L.tropica of the zymodemes MON-307, MON-288, MON-275 and MON-54. Following amplification and subsequent digestion by the restriction endonuclease MboI, variation in the sequence from the PGM gene also exposed two genotypes among the strains of L.tropica: PGM-G1, associated only with strains of L.tropica of the zymodeme MON-137; and PGM-G2, associated with strains of L.tropica of the zymodemes MON-265, MON-307, MON-288, MON-275 and MON-54, and, also, with six strains of L.major, five of L.infantum and one of L.donovani. The use of the HK and PGM gene sequences enabled distinction the L.tropica strains of the zymodeme MON-137 from those of the zymodeme MON-265. This genotyping system ‘correctly’ identified reference strains of L.tropica of known zymodemal affiliation and also from clinical samples, with a level of sensitivity down to <1 fg in the case of the former and to 1 pg of DNA in the case of the latter. Conclusions/Significance Both assays proved useful for identifying leishmanial parasites in clinical samples without resource to culture and MLEE.


Introduction
Many methods employing various techniques and targets have been used for the diagnosis of leishmaniases and characterization and identification of their causative agents. Nuclear DNA [1][2][3], miniexon genes [4] kinetoplast DNA [5] and the gp63 gene [6] are among the targets. Multilocus enzyme electrophoresis (MLEE) [7,8] is the standard accepted method for identifying and distinguishing leishmanial parasites at the species level and is based on variation in the electrophoretic mobility of enzymes isolated from leishmanial organisms. This is performed in a few specialized reference laboratories, which presents some limitations and is expensive and time consuming, requiring large quantities of cultured leishmanial promastigotes. At the end of the procedure, strains are consigned to various zymodemes. is not available in Palestinian and Israeli diagnostic clinics so other methods, i. e., nDNA and kDNA analysis [1,[9][10][11] and excreted factor (EF) serotyping [12] have been used. Though these tend to parallel characterization by MLEE, they cannot be linked directly to the genetics underlying the enzymes examined by MLEE.
Here, sequences from the genes for Hexokinase (HK) and Phosphoglucomutase (PGM) have been tested as targets for methods incorporating, consecutively, a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) to identify strains of the three Old World leishmanial species L. major, L. tropica and L. infantum and attempt to differentiate among strains of L. tropica and indicate to which zymodemes they probably belong, and also apply the two techniques to the diagnosis of leishmaniases. Genotyping results derived through the PCR RFLP system were firstly validated using reference strains and then applied to clinical samples from cases of CL.

Isolation of parasites, clinical samples and DNA extraction
The 95 strains of L. tropica used were from different geographical areas (Table 1). Thirteen of which represented five different zymodemes [8,13,14]. In addition, Drs. Christophe Ravel and Gert de Auwera, Université Montpellier 1, Centre Hospitalier Universitaire de Montpellier, Laboratoire de Parasitologie and Centre National de Reference des Leishmania, Montpellier, France, provided DNA samples from 17 strains. Forty-five strains of L. tropica of unknown zymodemal affiliation were analyzed by the HK and PGM PCR RFLP assays developed at Al-Quds University. Ten of the Palestinian strains of L. tropica analysed had been characterized by MLEE (Table 1) and [14]. Six strains of L. Sixty-one cases of CL diagnosed on clinical grounds only and registered as such by the Palestinian Ministry of Health (PMOH) were later confirmed, using their tissue aspirates that had been spotted onto filter papers. This included identification of the parasites. Genomic DNA was extracted from pellets of promas-tigotes and from amastigotes in skin tissue aspirates collected on filter papers. A high purification template PCR kit (Roche Diagnostics GmbH, Mannheim, Germany) was used and the DNA was eluted in 40 ml Tris EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The use of patient samples was approved by the Research Ethics Committee of Al-Quds University, Jerusalem. All the reference strains (Table 1) and only the positive clinical samples that had been confirmed previously and had had their parasites identified by an ITS1PCR and consecutive RFLP analysis [1] were analyzed here. To evaluate the possibility of employing the HK and PGM PCR RFLP assays established here without isolating the parasites, 61 tissue aspirates from positive cases caused by L. tropica spotted onto filter paper were assessed.

Selection of oligonucleotides
A combined analytical approach was used to differentiate between the species L. tropica, L. major and, L. infantum and, in particular, to separate Palestinian strains of L. tropica of different zymodemal affiliation. This was done by carrying out an HK PCR RFLP assay and a PGM PCR RFLP assay.
GenBank listed sequences of PGM for L. major and L. infantum but none had been published for L. tropica. In this study, the sequences of PGM from two strains of L. This generated a 1700 bp amplicon. These sequences were used as references for this study. Samples were purified using a PCR product purification kit (Roche Diagnostics GmbH, Mannheim, Germany). The amplicons were aligned against the sequences of L. major and L. infantum and the nucleotide differences among strains of L. tropica were exposed, using the CLUSTAL program (.http://multalin. toulouse.inra.fr/multalin/) The primers PGMLtF:59-TCCGTGA GAAGGACGGTATC-39 and PGMLtR:59-AGGGTCCGTG-TAGCTGAAGT-39 were used in a PCR RFLP assay to reveal the sequence that differentiates strains of L. tropica of the zymodeme MON-137 from those of the other zymodemes explored here.

Author Summary
The species L. major, L. tropica and L. infantum exist in Palestine and Israel where the first two cause CL and the third usually causes VL although cases of CL without visible signs of VL have been reported from Palestine. This means that diagnosis of locally acquired leishmaniases requires identification of their causative agents for further management of cases. Two molecular biological methods based on sequences from the genes of the enzymes HK and PGM and employing PCRs and consecutive RFLPs were developed and used together to distinguish among strains of the three species and between the two subtypes of L. tropica found in Palestinian foci that coincide with zymodemes MON-137 and MON-307. They were applied to, both, isolated parasites grown as promastigotes and to amastigotes in tissue preparations from cases and were able to identify strains and indicate their zymodemal affiliations.

PCR amplification conditions for HK and PGM sequences
HK and PGM PCR reactions were done in a volume of 25 ml that contained 1 ml of DNA template and PCR-Ready Supreme mix (Syntezza Bioscience, Jerusalem, Israel) in a Gene Amp PCRsystem 9700 thermal cycler (Applied Biosystems, CA, USA), using the following conditions: initial denaturation for 5 min. at 98uC, followed by 38 cycles at 94uC for 45 s, 55uC for 45 s, and 72uC for 30 s and final elongation at 72uC for 7 min. For each reaction, DNA from strains of L. major and L. infantum, and strains of L. tropica belonging to the various zymodemes mentioned above were used as positive controls and distilled water was used as a negative control. Ten ml of each PCR product were run in 2.5% agarose gels. For all the species of Leishmania, the amplified products of the HK sequence were 197 bp and those of the PGM sequence were 278 bp.

Selection of the HK and PGM PCR restriction endonucleases
To select appropriate endonucleases for distinguishing between strains of L. infantum, L. major, and L. tropica, reference sequences of HK from GenBank were used. The NEBcutter V2.0 program available at the website http://tools.neb.com/NEBcutter2/index. php was able to differentiate these species. Theoretically, a double digestion of the PCR product with the endonucleases HaeIII and MboI should give three different digestion patterns, each corresponding to the three species and, as digestion with just MboI proved insufficient to separate L. tropica and L. infantum, double digestion with MboI and HaeIII was carried out, in the same tube at the same time.
The PGM gene sequences of all the strains of L. tropica were mapped for restriction site polymorphisms. The restriction enzyme MboI was selected to produce fragment sizes able to separate strains of L. tropica of the zymodeme MON-137 from all the other types of strain examined, which included strains of L. tropica that were not of the zymodeme MON-137 and strains of L. major and L. infantum.
For, both, the HK PCR RFLP and the PGM PCR RFLP, 15 ml of PCR product were incubated with 10 U endonuclease (MBI Fermentas), i. e., MboI and HaeIII, together in the same tube at the same time in the former and, only MboI for the latter, for 2 hr at 37uC, according to the manufacturer's instructions. Digestion products were electrophoresed in a 3% agarose gel (FMC BioProducts, Rockland, ME) in TAE buffer. Digestion patterns were stained with ethidium bromide and visualized by UV light. A DNA 50 bp molecular weight ladder (Promega, Madison, WI) was added to gels to indicate the molecular size of the digestion fragments.

Sensitivity and specificity of the HK PCR and PGM PCR
To determine the minimal amount of leishmanial DNA needed for amplification of the HK and PGM gene sequences by their respective PCRs for their RFLP analyses, each of a series of tenfold dilutions of leishmanial DNA extracted from the promastigotes of the strains LRC-L890 and -L758 was amplified. Concentrations of DNA were measured by a Nanodrop ND-1000 UV-Vis spectrophotometer (Thermo Fisher Scientific Inc, Waltham MA, USA). A search for the similarity in genetic sequences in GenBank revealed 100% specificity of the primers used for strains of Leishmania.

Sequencing of Hexokinase
The PCR amplification products from the strains L. tropica ISER/IL/1998/LRC-L758 and L. tropica MHOM/PS/2002/ LRC-L890, representing the zymodemes MON-137 and MON-307, respectively, were purified with a PCR product purification kit (Roche Diagnostics GmbH, Mannheim, Germany) and sequenced (Applied biosystem, genetic analyzer, Foster City, CA). The sequences were processed and aligned, using the CLUSTAL software package, and nucleotide differences were detected with the programs BioEdit and Clustal X (Fig. 1).

Ethical statement
A total of sixty-one tissue aspirates spotted onto filter papers were collected from CL patients. All tested samples were anonymized. The study protocol was approved by the Research Ethics Committee of Al-Quds University.

PCR RFLP of the HK gene
RFLP analysis of the 197 bp amplicon from the HK gene of the reference strains of L. major, L. infantum, L. donovani and the two zymodemal types of L. tropica gave their expected patterns, and different restriction fragment patterns were generated for the three different species and between the two zymodemal types of L. tropica (Figs. 2a and 2b).
The digested amplicons of the strains of L. tropica gave two different restriction patterns: those, whose profiles displayed four bands of 81, 49, 37, 30 bp, of which the latter two appear as a single broad band, that were assigned to the genotype HK-LtG1 and, co-incidentally, belonged to either zymodeme MON-137 or zymodeme MON-265; and those, whose profile displayed the same four bands of 81, 49, 37, 30 bp plus an extra one of approximately 130 bp that were assigned to the genotype HK-LtG2 and, co-incidentally, belonged to the other zymodemes mentioned above (Fig. 2a, Table 1). This was confirmed by sequence analysis of the PCR products of the two strains of L. tropica, ISER/IL/1998/LRC-L758 and MHOM/PS/2002/   Table 1 and Fig. 2c).

Sensitivity and specificity of the HK PCR and PGM PCR
For the PGM sequence of the strains LRC-L890 and -L758, a 278 bp PCR product was clearly detected at a dilution of the  genomic DNA that contained an estimated 1 pg of DNA, which is considered to be equivalent to 10 leishmanial parasites. The minimal amount of amplified DNA detectable by the HK PCR was less than 1 fg of DNA, which is considered to be equivalent to 0.01 leishmanial parasites [15] (Figs. 3a and b). Other cutaneous infections that could be confused clinically with CL are bacterial and fungal infections, for example, staphylococcal and streptococcal infections. However, regarding the clinical specificity and utility of this leishmanial parasite-specific PCR-RFLP diagnostic assay, a BLAST search gave perfect matches with 100% query coverage identity for the HK sequence of leishmanial species amplified with the primers used in the HK PCR. It also gave 100% coverage identity with the registered sequence XM 001682945.1 from L. major and the three registered sequences XM 001564729.1, XM 001465338.1, AM 50223.91 from L. infantum. The BLAST search also gave perfect matches with 100% query coverage identity for the PGM sequence of leishmanial species amplified with the primers used in the PGM PCR. It also gave 100% coverage identity with the registered sequence from L. donovani FR799608.1, from L. major FR796455.1 and L. major XM001682945.1, from L. infantum FR796455.1, and from L. mexicana FR799574.1. Sequences from Trypanosoma cruzi, African trypanosomes, Crithidia luciliae, Crithidia fasiculata, and Leptomonas collosoma were not found in the GenBank Megablast and could not be compared. No amplification products were observed when either the DNA of Trypanosoma cruzi or human DNA was used as templates in this PCR approach (data not shown).

RFLP analysis of the HK and PGM gene sequences from local and foreign strains of Leishmania
The restriction profiles of 73 out of 99 DNA samples amplified by the HK PCR and doubly digested with MboI and HaeIII were identical to that of the reference strain of L. tropica ISER/IL/ 1998/LRC-L758, representing the zymodeme MON-137, and were genotype HK-LtG1; 26 were identical to that of the reference strain of L. tropica MHOM/PS/2002/52JnM18 ( = LRC-L890), representing the zymodeme MON-307, and were genotype HK-LtG2. Ten of the 55 Palestinian strains of L. tropica had been sizes were compared with a 50 bp molecular weight ladder (MW), nd = not done; c) of the PCR products from the Phosphoglucomutase (PGM) genes of the leishmanial reference strain L. tropica LRC-L863 and local Palestinian strains after single digestion with the endonuclease MboI. Only the heavier differentiating band is shown. Uncut PCR product from L. tropica MHOM/PS/02/79JnF20 ( = LRC-L885) was used as a control and molecular sizes were compared with a 50 bp molecular weight ladder (MW). doi:10.1371/journal.pntd.0002464.g002 Interestingly, 19 strains of L. tropica of known zymodemal type and four whose zymodemal type was not known, all from geographical regions other than Palestine and Israel, displayed PGM PCR RFLP profiles, indicating they were genotype PGM-G2 and HK PCR RFLP profiles, indicating they were genotype HK-LtG2 (Table 1).Whereas, the strains belonging to the zymodemes MON-119 and 71 from Kenya and Yemen, respectively, and the two Namibian strains of unknown zymodemal type, were genotype PGM-G2 but genotype HK-LtG1 (Table 1).

Application of the HK and PGM PCR RFLP assays to clinical samples
Of the 61 positive aspirates amplified by the HK and PGM PCRs, 56 gave a product of 197 bp and 43 gave a product of 278 bp, respectively. All the PCR positive samples were subjected to HK RFLP analysis of their amplicons in comparison to the reference strains and consigned to their species. Forty-six of the 56 samples were L. tropica. The restriction profile and corresponding genotype, HK-LtG1, were like those of the reference strains of the zymodemes MON-137 and MON-265. Thirty-eight of the 46 samples that were positive by the HK PCR were also identified by PGM RFLP analysis and were genotype PGM-G1 like the reference strains of the zymodeme MON-137 (data not shown).

Discussion
Identification of leishmanial species is an integral part of the diagnosis of leishmaniases. Many methods have been used for this and together they have revealed considerable micro-heterogeneity within each different species. For example, strains of L. tropica show considerable variation in their microsatellite profiles [16] and in their enzyme profiles that have consigned them to 35 different zymodemes to date [7,14,17,18]. The strains of L. tropica causing CL in Palestine display two types of enzyme profile, affirming the existence of two zymodemes, MON-137 and MON-307 [14], that coincide with genetic clusters separated by multilocus microsatellite typing (MLMT) [14]. Strains of the zymodeme MON-137 have also been isolated in Israel as have strains of the four zymodemes MON-288, MON-275, MON-265 and MON-54. MLEE showed that most of the Palestinian strains of L. tropica considered here belonged to the zymodeme MON-137, strains of which in addition to occurring in Israel also occur in Jordan and Egypt [7,11,19,20]. Strains of L. tropica of the zymodeme MON-307 have not been isolated in these countries or any other countries where leishmaniases occur.
Of the two enzymes used here in PCR RFLP analyses, one, PGM, is included among the 15 enzymes used to generate enzyme profiles according to the system applied at the 'Centre National de référence des Leishmania' in Montpellier, the other, HK, is not. The ability to distinguish the species, L. major, L. tropica and L. infantum from one another by employing one DNA sequence from the gene for HK compared favourably with the separation of these species by cellulose acetate enzyme analysis (CAE) when using only the HK enzyme where Kreutzer [21,22] did not manage to separate these three species. However, Mebrahtu [23], applying the same CAE system, did manage to distinguish the species, L. major, L. tropica and L. infantum from one another using the HK enzyme. Also, employing this one DNA sequence from the gene for HK enabled the separation of strains of L. tropica into two genotypes associated with different groups of zymodemes (Table 1).
RFLP analysis before and, even more so, after the introduction of the polymerase chain reaction has proved very useful in identifying pathogens like leishmanial parasites as isolates from clinical samples, vectors and animal hosts and in situ in infected tissues [24]. The HK PCR RFLP developed here was, in addition, able to indicate genetic sub-types of the species L. tropica that appear to be directly related to zymodemal sub-types [1], which might be of epidemiological significance. However, for routine diagnostic use, it is difficult to achieve the required level of sensitivity and discrimination in a single step. Umasankar et al. [25], have shown that the HK gene displays sufficient polymorphism to be able to use sequencing of hexokinase loci to distinguish the species, L. major, L. tropica and L. infantum, which are the species circulating in Palestine. A sequence from the HK gene was selected here, which, in addition, also separated strains of L. tropica into two previously exposed genotypes: HK-LtG1, associated with the strains of the zymodemes MON-137 and MON-265; and HK-LtG2, associated with the strains of the zymodemes MON-307, MON-288, MON-275 and MON-54 [14].
This method easily distinguished between the Palestinian strains of the zymodemes MON-137 and MON-307, the only two zymodemes shown to occur in the Jenin District of Palestine [26] and one wonders if all the Palestinian strains examined here that were either genotype HK-LtG1 or genotype HK-LtG2 would prove to be, respectively, strains of the zymodemes MON-137 (or, possibly, MON-265, not found in Palestine so far) and MON-307 (or, possibly, MON-288, MON-275, MON-54, also not found in Palestine so far) if their enzyme profiles were determined.
At this time, the clinical and epidemiological relevance of being able to distinguish between strains of L. tropica belonging to zymodemes MON 137 and MON 265 is uncertain but could prove significant with continued epidemiological and chemotherapeutic studies. Presently, it is of biological interest and useful for population genetics. There seems to be no precise correlation between clinical form and species of Leishmania. A single species and even strains within a single zymodeme can cause lesions of different style and the same type of lesion can be produced by strains of different leishmanial species [7]. The lack of correlation between clinical presentation and enzyme polymorphism of L. infantum has been noted Pratlong [27]. Also, at this time, chemotherapy would be the same irrespective of the leishmanial species. Regarding epidemiology, the sand fly vector(s) transmitting the two sub-types of the Palestinian strains of L. tropica described here have not been identified. It would be well to know if they are transmitted by the same vector or each sub-type of L. tropica has a different species of vector. For example, in Israel, Phlebotomus (Adlerius) arabicus was shown to be sand fly vector of L. tropica of the zymodeme MON-265 in a focus just north of the Sea of Galilee [28][29][30] whereas Phlebotomus (Paraphlebotomus) sergenti was shown to be sand fly vector of L. tropica of the zymodeme MON-137 [11].
The RFLP profiles of the HK sequences of these two zymodemal types of strain of L. tropica, generated after digesting their PCR amplicons were congruent with the DNA sequencing (Fig. 2). The PGM PCR RFLP assay was not suitable for distinguishing the leishmanial species mentioned above, but it did separate strains identified as strains of L. Since the genotype HK-LtG1 encompasses strains of the zymodemes MON-137 and MON-265 but the genotype PGM-G1 encompasses only strains of the zymodeme MON-137, an assay, using both underlying sequences, was employed to separate cases caused by strains of L. tropica of the zymodeme MON-137 from cases caused by strains of L. tropica of the other zymodemes mentioned. However, this assay could not be used to distinguish cases caused by strains of L. tropica of the zymodeme MON-307 from cases caused by strains of L. tropica of the other zymodemes mentioned. While this indicates a limitation to its general use compared with MLEE done using many enzyme systems, it is much simpler and less expensive to apply. Since Palestinian strains of L. tropica have been of either the zymodeme MON-137 or the zymodeme MON-307, one could assume that strains not shown to be of the zymodeme MON-137 are of the zymodeme MON-307. The results of this method do increase our knowledge of the genetic diversity of the species L. tropica and does enable the differentiation of the two sub-populations of L. tropica circulating in the Palestinian West Bank region.
The clustering together of Palestinian and Israeli strains of L. tropica of the zymodemes MON-137 and MON-265 within the genotype HK-LtG1 but their separation by the genotypes of their PGM corresponds with their interrelationship based on other genetic (microsatellite and kinetoplast DNA profiles), antigenic (excreted factor (EF) serotypes), and biochemical criteria (enzyme profiles) [14,16]. Microsatellite analysis of the Palestinian and Israeli strains of L. tropica belonging to the zymodemes MON-137 and MON-265 showed that they formed a single main population Figure 4. Scheme of the interrelationship between the genotypes derived from the PCR RFLP profiles of the chosen hexokinase gene sequence after double digestion of the PCR product with MboI and HaeIII and the phosphoglucomutase gene sequence after single digestion of the PCR product with just MboI, and their correlation to the zymodemes and microsatellite clusters encompassing Palestinian and Israeli strains of L. tropica: genotype HK-LtG1 = Hexokinase L. tropica group 1, genotype HK-LtG2 = Hexokinase L. tropica group 2, genotype PGM-G1 = Phosphoglucomutase group 1, genotype PGM-G2 = Phosphoglucomutase group 2. Zymodemal designations were taken from, Rioux [8] and Pratlong [7], and microsatellite clusters from Schwenkenbecher [16]. doi:10.1371/journal.pntd.0002464.g004