Genetic diversity of Leishmania donovani that causes cutaneous leishmaniasis in Sri Lanka: a cross sectional study with regional comparisons
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Leishmania donovani is the etiological agent of visceral leishmaniasis (VL) in the Indian subcontinent. However, it is also known to cause cutaneous leishmaniasis (CL) in Sri Lanka. Sri Lankan L. donovani differs from other L. donovani strains, both at the molecular and biochemical level. To investigate the different species or strain-specific differences of L. donovani in Sri Lanka we evaluated sequence variation of the kinetoplastid DNA (kDNA).
Parasites isolated from skin lesions of 34 CL patients and bone marrow aspirates from 4 VL patients were genotyped using the kDNA minicircle PCR analysis. A total of 301 minicircle sequences that included sequences from Sri Lanka, India, Nepal and six reference species of Leishmania were analyzed.
Haplotype diversity of Sri Lankan isolates were high (H d = 0.757) with strong inter-geographical genetic differentiation (F ST > 0.25). In this study, L. donovani isolates clustered according to their geographic origin, while Sri Lankan isolates formed a separate cluster and were clearly distinct from other Leishmania species. Within the Sri Lankan group, there were three distinct sub-clusters formed, from CL patients who responded to standard antimony therapy, CL patients who responded poorly to antimony therapy and from VL patients. There was no specific clustering of sequences based on geographical origin within Sri Lanka.
This study reveals high levels of haplotype diversity of L. donovani in Sri Lanka with a distinct genetic association with clinically relevant phenotypic characteristics. The use of genetic tools to identify clinically relevant features of Leishmania parasites has important therapeutic implications for leishmaniasis.
KeywordsSkin lesions Antimony resistance Visceral leishmaniasis kDNA minicircle sequences Haplotype diversity Geographical clustering Phenotypic characteristics Nepal India
Fetal bovine serum
Pair wise fixation index
Intra-lesional sodium stibogluconate
Internal transcribed spacer
Multiple sequence alignment
Polymerase Chain Reaction
Post kala-azar dermal leishmaniasis
Cutaneous leishmaniasis that showed poor response to antimony therapy in Sri Lanka
Uncomplicated cutaneous leishmaniasis in Sri Lanka
Visceral disease in Sri Lanka
Leishmaniases, caused by the Leishmania species, are a group of vector-borne parasitic diseases with a high disease burden in the Indian sub-continent [1, 2]. The main clinical forms are cutaneous leishmaniasis (CL), muco-cutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL), which are caused by different species and subspecies of Leishmania [1, 3]. Leishmania donovani is widely recognized to cause VL in the old world; while in the new world VL is caused by a closely related species, L. infantum [3, 4, 5, 6]. The causative agent of CL in Sri Lanka is L. donovani zymodeme MON-37 [7, 8], which is closely related to the common VL causing zymodeme MON-2 in India .
CL is now considered as an endemic disease in Sri Lanka, with a steady increase in the number of cases detected [10, 11]. Since 2001, more than 6500 cases of CL  have been reported along with a few MCL and VL cases [13, 14, 15]. The steady increase of CL in Sri Lanka is complicated by the appearance and spread of CL that does not respond to the standard antimonial drugs routinely used in clinical practice . Initial studies in Sri Lanka have indicated that both CL and VL are caused by L. donovani- MON-37 [7, 15]. However, the existence and contribution of intra-species variation that influences the clinical manifestations of leishmaniasis and the treatment response to antimonial drugs in Sri Lanka is unknown.
Leishmania donovani virulence factors involved in pathogenesis, visceral organ invasion and parasite persistence, have been extensively studied over several decades [17, 18, 19]. The A2 protein production [18, 20, 21], lipophosphoglycan (LPG) activity [22, 23, 24], glycoprotein 63 (gP63) gene expression [25, 26], acid phosphatase activity [18, 27] and variations in the mini-exon genes of chromosome 36  are known factors that influence the virulence of Leishmania parasites. Further analysis of Sri Lankan L. donovani isolates is thus warranted in order to gain a better understanding of differences in virulence and pathogenicity.
The genetic characterization of Leishmania parasites can be achieved via the analysis of several genetic markers such as mini-exons, microsatellites, ribosomal internal transcribed spacer (ITS) regions and minicircles of kinetoplast DNA [28, 29, 30, 31]. The kinetoplast DNA (kDNA) network in Leishmania is composed of two types of DNA rings; maxicircles and minicircles. The Leishmania parasite minicircle kDNA is approximately 1 kb in size, containing a conserved region that is ~200 bp in length and a variable region of ~600 bp. Leishmania minicircle kDNA provides an ideal target for the genotyping of Leishmania parasite as the sequence differences allow for accurate discrimination between species [32, 33, 34].
Previous studies using the minicircle kDNA footprint assay have enabled rapid identification of known or unknown species with a high level of sensitivity . This method is considered to be better suited as compared to other frequently used speciation methods, such as Restriction Fragment Length Polymorphism (RFLP) and Southern blot which depend on low-copy PCR targets (non–minicircle DNA regions) and have limitations in Leishmania with regard to species identification [35, 36, 37].
The aim of this study was to use the kDNA footprint assay to study L. donovani strain specific sequence diversity and determine the association between sequence variations and distinct clinical characteristics in individuals with leishmaniasis in Sri Lanka.
Study setting and study population
Nepal and India
Parasite DNA extracts collected via archived samples of L. donovani parasites (from skin lesions of post kala-azar dermal leishmaniasis (PKDL) patients and bone marrow of VL patients) maintained at collaborative centers in India (n = 27) and Nepal (n = 26), were used for comparative experiments. The kDNA sequences from clinical isolates stored in public databases (GenBank) were also included in the analyses.
Laboratory confirmation of leishmaniasis
Prior to inclusion in the study, diagnosis of infection was confirmed in all study subjects by the demonstration of Leishmania parasites by oil immersion (X1000) microscopy of Giemsa-stained smears of lesion aspirates or slit-skin scrapings of CL patients and bone marrow aspirates of VL patients, either directly or following in-vitro culture. Parasites were grown in M199 medium (Gibco, Invitrogen, USA) supplemented with 15% heat inactivated fetal bovine serum (FBS) (Gibco, Invitrogen, USA) with Penicillin-Streptomycin (Sigma-Aldrich, USA) and the cultures were maintained at 26 °C. Cultured parasites were used for DNA extraction with the use of QIAamp DNA minikit (Qiagen, USA). Laboratory-confirmed patients were referred to the local dermatologist (for CL) and/or consultant physicians (for VL) for treatment according to Sri Lankan Ministry of Health guidelines. IL-SSG poor-responders in the CL group were those re-referred to the department for repeat investigations due to delay or absence of clinical cure.
DNA preparation and sequencing
Extracted DNA was subjected to a fluorescence-based PCR followed by cloning and sequencing of the resulting DNA fragment according to established protocols . The PCR reaction for kDNA amplification was performed using primers from conserved regions across kinetoplast minicircles of Leishmania spp. (primer JW11–5’CCTATTTTACACCAACCCCCAGT3’ and primer JW12–5’GGGTAGGGGCGTTCTGCGAAA3’) which generate an approximately 120 bp amplicon [34, 41, 42, 43]. Subsequently the amplified products were ligated in the pGEM-T vector and the resulting ligation products were transformed in to E. coli TOP 10 competent cells and plated for blue/white screening on plates containing ampicillin (100 μg/ml), X-gal (20 mg/ml) and iso-propyl-thio-galactoside (10 μl/ml of 100 mM). The resulting white colonies indicated transformed cells and blue colonies represented non-transformed ones. Individual white transformed colonies were used for plasmid DNA preparation followed by restriction digestion of 1 μg of plasmids using EcoRI enzyme at 37 °C (Promega, USA), according to the manufacturer’s recommendations. The total reaction volume was subjected to electrophoresis, in 2% agarose gel to confirm the presence of an insert. The inserts from the individual clones were sequenced with M13 forward primer.
Minicircle sequence analysis of parasite isolates
A total of 301 L. donovani sequences of parasite isolates from Sri Lanka (n = 38), India (n = 27) and Nepal (n = 26) were analyzed, along with minicircle sequences of 6 Leishmania reference species from different countries . Multiple sequence alignment (MSA) was done, via CLUSTAL-X MSA Program, version 2.0, with a gap opening penalty of 10.00; gap extension penalty of 0.05; DNA transition weight of 0.50 [44, 45]. The DnaSP software was utilized to measure haplotype diversity (H d ), Nucleotide diversity (N d) and pair wise fixation index (F ST ) for each group . Furthermore, Tajima’s D Test  and Fu’s Fs test  were also performed for detection of population growth (neutrality test for selection). The Haplotype diversity is defined as H d = [n/(n − 1)] [1 − Σ P i 2 ], with n being the number of sequences analyzed and p i is the relative frequency of haplotype i. The nucleotide diversity is defined as, N d = [n/(n − 1)] [1 − Σ x ij 2 ], where x ij is the relative frequency of nucleotide variant j (j = 1, 2, 3 and 4 correspond to A, C, G and T) at site i. H d > 0.5 and N d > 0.1 reflect high haplotype and nucleotide diversity, respectively within the study population [46, 49].
Tajima’s D Test is a measure of selection through the patterns of genetic diversity within populations, whether they are consistent with neutral expectations. Positive D value suggests a recent population bottleneck or balancing selection, while negative D value suggests population expansion or purifying selection . Fu’s Fs test is another measure of selection, which is based on the infinite sites model of mutation, and tests the number of alleles in a population and indicates whether they are consistent with neutral expectations. Fu’s statistic is thought to be a more sensitive indicator of population expansion and genetic hitchhiking than Tajimas D. F ST measures genetic differentiation among subpopulations. According to established standards, F ST values from 0.0–0.05 indicate little genetic differentiation, whereas values that range from 0.05–0.15, 0.15–0.25 and >0.25 are considered to be representing moderate, great and strong genetic differentiation, respectively [51, 52]. The ‘isolation by distance analysis’ was performed using IBDWS . Three populations were defined for this analysis viz., Sri Lanka, India and Nepal.
Phylogenetic analysis was done using all sequences (n = 301) generated as described above. Previously published minicircle sequences of L. donovani (DD8), L. infantum (TK), L.infantum (SP), L. major (Al), L. braziliensis (P), L.tropica (TK) and L. amazonensis from the minicircle sequence database of GenBank, were also used in the phylogenetic analysis as reference sequences. The phylogenetic trees were constructed using the neighbor joining method, implemented in MEGA7.0.7 . The evolutionary distances were computed using the Maximum Composite Likelihood method and depicted in units of number of base substitutions per site [54, 55, 56]. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The resulting Guide Trees, showed cluster relationships among the sequences and allowed the assignment of test sequences to one of the reference species.
PCR amplification of Leishmania kDNA sequence segments from Sri Lanka showed a single approximately 120-bp band on 2% agarose gel, which was consistent with the product size of L. donovani as shown previously . The multiple sequence alignment clearly showed a high level of genetic variation within the variable regions, whereas the conserved regions showed 83% nucleotide identity on average, in all sequences (Additional file 1).
Genetic diversity parameters of Leishmania donovani isolates from Sri Lanka, India and Nepal
Haplotype diversity (H d )
Nucleotide diversity (N d )
Number of sequences used (n)
Number of polymorphic sites(S)
Number of Haplotypes (h)
Tajima’s D (P-value)
Fu’s Fs tests(P-value)
kDNA based genetic distances (F ST ) between populations of Leishmania parasites from selected geographical areas
This study was aimed to examine genetic diversity, population structure, phylogenetic relationships as well as the association between genotypic and phenotypic characteristics of L. donovani in Sri Lanka with the use of a minicircle-based strain specific sequence diversity assay. Attempts were also made to investigate Sri Lankan L. donovani sequences in relation to those in the neighboring countries of India and Nepal as well as the rest of the world. Distinct genetic clustering of Sri Lankan parasites according to their phenotypic properties, coupled with an admixed nature of the population was observed for the first time in Sri Lanka.
In the multiple sequence alignment, unique conserved regions could be clearly identified with minor gaps and nucleotide mismatches, since such regions remain conserved across many species and between classes of the same species. The existence of minicircle sequence haplotype diversity among different species and within the same species of Leishmania is fairly well established [41, 57, 58, 59, 60, 61]. The haplotype diversity value based on minicircle sequences revealed the degree of diversity within the Sri Lankan isolates with different phenotypic properties. Furthermore, lack of clear genotypic pattern in relation to geography within the country was revealed, indicating a population mix or presence of gene flow between parasite populations that may be still in the process of expansion. High levels of haplotype and nucleotide diversity were observed in Sri Lankan, Nepalese and Indian sequences. However, the highest haplotype diversity and nucleotide diversity were observed for the Indian parasite sequences indicating the highly diverse nature of the Indian isolates when compared to the Sri Lankan and Nepalese counterparts.
Indeed, a recent population expansion or an existence of an excess number of alleles often observed in genetic hitch-hiking was evident from Fu’s Fs test for Sri Lankan sequences, where a negative Tajima’s D test implies purifying selection. In contrast, positive Fs value observed in both Nepal and Indian sequences are an indication of deficient alleles, suggestive of a recent population bottleneck, and a positive Tajima’s D points towards a balancing selection.
The degree of variability within Leishmania species has been linked to the number of vectors and/or animal reservoir(s) involved in the transmission of these parasites [62, 63]. Therefore, the high variability found in Leishmania isolates in Sri Lanka, could be due to its transmission associated with several sub-populations of sand flies that belong to the genus Phlebotomus reported from various parts of Sri Lanka [64, 65].
This study confirms that, sequences of the Sri Lankan, Indian and Nepalese (VL and PKDL) parasites, clustered within the L. donovani complex, while CL causing parasites obtained from skin lesions of Indian patients, formed a sister cluster with L. tropica. None of the Sri Lankan CL and VL parasites were found to be associated with L. infantum, a closely related subspecies within the L. donovani complex [5, 61]. However, within the L. donovani group of sequences, the Sri Lankan parasites formed a distinct cluster, in agreement with previous observations made using different genotyping tools . This may indicate the parasite’s existence over a prolonged period within the country, though the clinical cases were recognized only recently . Interestingly, the Sri Lankan parasites belonged to a group, which was well differentiated from other dermotropic Leishmania parasites viz., L. tropica, L. major, L. amazonensis and L. braziliensis indicating potential population isolation.
There were three distinct sub-clusters seen within the Sri Lankan group, comprising sequences from visceral leishmaniasis (VL) patients, cutaneous leishmaniasis patients (CL-S), and CL cases that showed poor response to the standard drug SSG (CL-DR). However, no specific clustering was observed in relation to their geographical origins within Sri Lanka. The phylogenetic differentiation within CL-S, CL-DR and VL may be due to the genetic mutations/variations associated with these three clinical groups (viz. VL, uncomplicated CL and CL with poor response to antimonial drugs, which is currently used as the first line of treatment in Sri Lanka) .
It is possible that multiple genetic strains of L. donovani exist within Sri Lanka, as reported from other geographic regions in the world [19, 31, 35]. Occasional reports on atypical cutaneous leishmaniasis reported in literature, indicate that more than one strain of L. donovani is likely to cause cutaneous disease [5, 6, 29]. L. donovani strains of the same genetic and/or geographical group are also known to result in different clinical outcomes . This study with Sri Lankan isolates further affirms that there is sub-genetic diversity in L. donovani in the Indian subcontinent, which warrants further investigation on how that diversity translates into pathogenesis and its implications on patient management and treatment outcome.
Sri Lankan L. donovani parasites are likely to have evolved more recently as a novel group compared to other members of the genus, since the sequences from these isolates were found in the most peripheral branches of the phylogram. Given Sri Lanka’s geographic position as an island close to India, the parasites may have been in circulation within the country long enough to adopt such independent genetic characteristics. However, it is unlikely that these parasites were introduced within the last 2–3 decades, when the disease became apparent. As suggested by past genetic studies, the L. donovani complex has originated 3–4 million years ago [31, 35]. Initial cases reported in Sri Lanka about 30 years ago were all imported cases from the Middle East and Africa [67, 68]. Leishmania parasites are known to find new ecologies along with the immigration of people [55, 68, 69]. It is possible that the parasites may have been introduced to Sri Lanka by locals returning after employment overseas or by foreign immigrants [5, 68]. Sri Lankan Leishmania parasites may have co-evolved with the ecological settings along with its vector and possible reservoir hosts. However, this remains mere speculation with the precise origin of the Sri Lankan L. donovani parasites being debatable and not addressed through the present study.
Use of the kDNA minicircle region can be used as a surrogate genotyping tool for the identification of species-specific sequence footprints among different species of Leishmania and for the identification of new Leishmania species or strains with relative ease and reasonable accuracy. Such tools can be cost-effective in resource limited settings and provide for rapid identification of parasite species. However, such data obtained are unlikely to provide accurate information on evolutionary history of the parasite, which in turn may require high resolution evaluation of the whole genome and sequence analysis of markers that are representative of the entire parasite genome.
For the first time this study reveals the extent of genetic diversity of L. donovani in Sri Lanka and a clear clustering nature of local parasites according to their antimonial sensitivity and tissue localization. Furthermore, the genetic differentiation between CL-DR and CL-S suggests a likely genetic basis for poor responsiveness to antimonial drugs and possible drug resistance. Overall, genetic variations associated with specific functional characteristics are likely to influence the disease phenotype, which is of clinical relevance and significance. Studies are underway to use whole genome sequence information with more detailed and higher resolution mapping, to investigate the parasite genetic variations associated with distinct clinical manifestations.
We acknowledge the Dermatologists in all study areas for assistance with patient care, Mr. Sudath Weerasinghe and Ms. Nirosha Pathirana at Faculty of Medicine, Colombo, Ms. Ginni Arora at IMM for technical assistance and Dr. G. Sreenivas and Dr. Ranadhir Dey for valuable comments on the manuscript. Dr. John Hobson at Food and Drug Administration, USA is also acknowledged for language editing.
Research reported in this publication was supported by University of Colombo grant AP/3/2011/CG/01 and the National Institute of Allergy And Infectious Diseases of the National Institutes of Health, USA under Award Number R01AI099602 and U01AI136033. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Availability of data and materials
The datasets generated and/or analyzed during the current study are available in the [GenBank] repository (GenBank reference numbers KU201288-KU201312, KU 220245-KU220268) or are available from the corresponding author on request.
Conceived and designed the experiments-UK, NDK, HN, AS, SR, NRB, YS. Performed the experiments-UK, KA, THW, KR, MAB. Analyzed the data-UK, AS, HUP, DZ, GZ. Wrote the paper- UK, NDK, Read and approved the manuscript- all authors.
Ethics approval and consent to participate
Written informed consent was obtained from all individuals. Ethical approval for the study was granted by the Ethics Review Committee, Faculty of Medicine, University of Colombo (ERC-12-068).
Consent for publication
The authors declare that they have no competing interests. None of the authors of this paper has a financial or personnel relationship with people or organizations that could inappropriately influence or bias the content of the paper.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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