Tropica Isolates Research Articles

Autochthonous Leishmaniasis Caused by Leishmania tropica, Identified by Using Whole-Genome Sequencing, Sri Lanka.

Cutaneous leishmaniasis is atypical in Sri Lanka because Leishmania donovani, which typically causes visceral disease, is the causative agent. The origins of recently described hybrids between L. donovani and other Leishmania spp. usually responsible for cutaneous leishmaniasis remain unknown. Other endemic dermotropic Leishmania spp. have not been reported in Sri Lanka. Genome analysis of 27 clinical isolates from Sri Lanka and 32 Old World Leishmania spp. strains found 8 patient isolates clustered with L. tropica and 19 with L. donovani. The L. tropica isolates from Sri Lanka shared markers with strain LtK26 reported decades ago in India, indicating they were not products of recent interspecies hybridization. Because L. tropica was isolated from patients with leishmaniasis in Sri Lanka, our findings indicate L. donovani is not the only cause of cutaneous leishmaniasis in Sri Lanka and potentially explains a haplotype that led to interspecies dermotropic L. donovani hybrids.

A comparative genomics approach reveals a local genetic signature of Leishmania tropica in Morocco.

In Morocco, cutaneous leishmaniasis (CL) caused by Leishmania (L.) tropica is an important health problem. Despite the high incidence of CL in the country, the genomic heterogeneity of these parasites is still incompletely understood. In this study, we sequenced the genomes of 14 Moroccan isolates of L. tropica collected from confirmed cases of CL to investigate their genomic heterogeneity. Comparative genomics analyses were conducted by applying the recently established Genome Instability Pipeline (GIP), which allowed us to conduct phylogenomic and principal components analyses (PCA), and to assess genomic variations at the levels of the karyotype, gene copy number, single nucleotide polymorphisms (SNPs) and small insertions/deletions (INDELs) variants. Read-depth analyses revealed a mostly disomic karyotype, with the exception of the stable tetrasomy of chromosome 31. In contrast, we identified important gene copy number variations across all isolates, which affect known virulence genes and thus were probably selected in the field. SNP-based cluster analysis of the 14 isolates revealed a core group of 12 strains that formed a tight cluster and shared 45.1 % (87 751) of SNPs, as well as two strains (M3015, Ltr_16) that clustered separately from each other and the core group, suggesting the circulation of genetically highly diverse strains in Morocco. Phylogenetic analysis, which compared our 14 L. tropica isolates against 40 published genomes of L. tropica from a diverse array of locations, confirmed the genetic difference of our Moroccan isolates from all other isolates examined. In conclusion, our results indicate potential regional variations in SNP profiles that may differentiate Moroccan L. tropica from other L. tropica strains circulating in endemic countries in the Middle East. Our report paves the way for future research with a larger number of strains that will allow correlation of diverse phenotypes (resistance to treatments, virulence) and origins (geography, host species, year of isolation) to defined genomic signals such as gene copy number variations or SNP profiles that may represent interesting biomarker candidates.

In Vitro Study on Four Types of Commercial Lectins on Leishmania infantum, L. major and L. tropica with Stage-Specific Binding and Leishmania Species Identification.

We aimed to verify the susceptibility of Leishmania infantum, L. major and L. tropica, to commercial lectins in order to identify the three Leishmania species. The degree of agglutination was determined both macroscopically and microscopically and was scored negative (-) to positive (from 1+- 4+) based on their percentage of agglutination. Jacalin and UEA-1 were capable of agglutination of L. infantum isolates in both logarithmic and stationary phases at a concentration of 1000 μg/ml (100%). L. tropica isolates showed agglutination with the lectin UEA-1 in both logarithmic and stationary phases (62.5% and 87.5%). L. major and L. tropica showed 75% agglutination with lectin Jacalin in both logarithmic and stationary phases. L. tropica isolates showed 25% agglutination with the lectin WGA in the logarithmic phase. L. infantum, L. major and L. tropica isolates showed 25, 12.5 and 37.5% agglutination in the stationary phase, however, did not show agglutination in logarithmic phases. L. major isolates showed 12.5% agglutination with the lectin PHA in the stationary phase, however, were incapable of agglutination with the L. tropica and L. infantum in both logarithmic and stationary phases. Despite the fact, that JCA and I-UEA lectins were not able to completely separate L. infantum, L. major and L. tropica. WGA lectin and PHA lectin can help in separating the species of Leishmania parasites.

Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis

Leishmania RNA virus was detected the first time in the New World Leishmania species. Recent studies were also showed the presence of Leishmania RNA virus 2 (LRV2) in Old Word Leishmania species including Turkish L. major and L. tropica isolates. This study aimed to increase the sensitivity of qPCR with a modification in the denaturation step of cDNA preparation protocol. In this study, LRV2+ three L. major, two L. tropica strains and L. major control strain (MHOM/SU/73/5-ASKH) were included. Total RNA isolation was done using different numbers of Leishmania promastigotes (108, 105 and 103). Before cDNA synthesis, samples were denatured at 95 °C for 2 min, as a modification of the kit procedure. qPCR was undertaken using 0.5 mM primers (LRV F-HR/LRV R-HR) diluted in SYBR Green Master mix. We observed lower Ct values in amplicons with the modified version than with the classical kit protocol for cDNA synthesis, in all of the strains used in the study. The addition of pre-denaturation step at 95 °C showed lower Ct values meaning the sensitivity increased. Different parasite dilutions showed similar results. It is important to increase the sensitivity especially with the aim for detecting LRV in clinical samples obtained from patients probably have less number of parasites. The presence and burden of the virus can help to understand the relationship between the clinical findings and the pathogenicity of the parasite which may lead to changes in the course of treatment.

Molecular-based detection of Leishmania tropica isolates among sensitive, resistant, and relapsed patients treated with Meglumine Antimoniate.

The appearance of resistance to pentavalent antimony, as the mainline of treatment for Cutaneous Leishmaniasis (CL) has been reported from Iran. According to the patients' laboratory and clinical history, 96 archived slides of patients infected with Leishmania tropica (L. tropica) treated with Meglumine Antimoniate (Glucantime®) were selected. After microscopic examination, Nested Polymerase Chain Reaction (Nested-PCR) and Restriction Fragment Length Polymorphism (RFLP) assays were done for each sample. In Nested-PCR, all positive samples were characterized as L. tropica. Additionally, some positive products of sensitive, resistant, and recidivans cases were selected to check their differentiations by sequencing software. In RFLP, various patterns of schizodemes were detected according to the reference patterns. Most sensitive cases of L. tropica (treated with Glucantime®) were categorized as schizodeme B, and most resistant cases were identified as schizodeme B and D. In recidivans cases, 91% of specimens categorized as schizodeme A and B. However, study on the type of L. tropica isolates that are resistant or sensitive to Glucantime® could be helpful before drug therapy.

High genome plasticity and frequent genetic exchange in Leishmania tropica isolates from Afghanistan, Iran and Syria.

The kinetoplastid protozoan Leishmania tropica mainly causes cutaneous leishmaniasis in humans in the Middle East, and relapse or treatment failure after treatment are common in this area. L. tropica's digenic life cycle includes distinct stages in the vector sandfly and the mammalian host. Sexual reproduction and genetic exchange appear to occur more frequently than in other Leishmania species. Understanding these processes is complicated by chromosome instability during cell division that yields aneuploidy, recombination and heterozygosity. This combination of rare recombination and aneuploid permits may reveal signs of hypothetical parasexual mating, where diploid cells fuse to form a transient tetraploid that undergoes chromosomal recombination and gradual chromosomal loss. The genome-wide SNP diversity from 22 L. tropica isolates showed chromosome-specific runs of patchy heterozygosity and extensive chromosome copy number variation. All these isolates were collected during 2007-2017 in Sweden from patients infected in the Middle East and included isolates from a patient possessing two genetically distinct leishmaniasis infections three years apart with no evidence of re-infection. We found differing ancestries on the same chromosome (chr36) across multiple samples: matching the reference genome with few derived alleles, followed by blocks of heterozygous SNPs, and then by clusters of homozygous SNPs with specific recombination breakpoints at an inferred origin of replication. Other chromosomes had similar marked changes in heterozygosity at strand-switch regions separating polycistronic transcriptional units. These large-scale intra- and inter-chromosomal changes in diversity driven by recombination and aneuploidy suggest multiple mechanisms of cell reproduction and diversification in L. tropica, including mitotic, meiotic and parasexual processes. It underpins the need for more genomic surveillance of Leishmania, to detect emerging hybrids that could spread more widely and to better understand the association between genetic variation and treatment outcome. Furthering our understanding of Leishmania genome evolution and ancestry will aid better diagnostics and treatment for cutaneous leishmaniasis caused by L.tropica in the Middle East.

Detection of Leishmania tropica Using Nested-PCR and Some of Their Virulence Factors in Thi-Qar Province, Iraq

Cutaneous leishmaniasis is one of endemic diseases in Iraq. It is considered as widely health problem and is an uncontrolled disease. The aim of the study is to identify of Leishmania species that cause skin lesions among patients in Thi-Qar Province, South of Iraq, also to detect some virulence factors of L. tropica. This study includes three local locations, Al-Hussein Teaching, Suq Al-Shyokh General and Al-Shatrah General Hospitals in Province for the period from the beginning of December 2018 to the end of September 2019. The samples were collected from 80 patients suffering from cutaneous leishmaniasis, both genders, different ages, various residence places and single and multiple lesions. Nested-PCR technique was used to amplify kinetoplast minicircle fragments DNA (kDNA). Conventional-PCR was performed for determination of some virulence factors (LPG1, GP63, CPA and PPG1 genes). The electrophoresis findings of kDNA gene showed two species of the parasite found in the study area, 65 samples were positive for cutaneous leishmaniasis, L. tropica at 750bp and L. major at 560bp. Generally, L. tropica (57.5%) was the most common specie and L. major (23.75%) appeared in a low level. There are no significant differences between the infections of males and females, while there are significant differences at the comparison between age groups. All virulence genes (LPG1, GP63, CPA and PPG1) appeared in all L. tropica isolates with high percentage (100%). L. tropica is the major specie which that caused CL in Thi-Qar province, while L. major appeared in low incidence. The virulence genes, which were reviewed, are necessary and important in pathogenesis of L. tropica.

A Nuclear Magnetic Resonance-Based Metabolomic Study to Identify Metabolite Differences between Iranian Isolates of Leishmania major and Leishmania tropica.

Background: Cutaneous leishmaniasis caused by Leishmania species (L. spp) is one of the most important parasitic diseases in humans. To gain information on the metabolite variations and biochemical pathways between L. spp, we used the comparative metabolome of metacyclic promastigotes in the Iranian isolates of L. major and L. tropica by proton nuclear magnetic resonance (1H-NMR).Methods: L. tropica and L. major were collected from three areas of Iran, namely Gonbad, Mashhad, and Bam, between 2017 and 2018, and were cultured. The metacyclic promastigote of each species was separated, and cell metabolites were extracted. 1H-NMR spectroscopy was applied, and the data were processed using ProMatab in MATLAB (version 7.8.0.347). Multivariate statistical analyses, including the principal component analysis and the orthogonal projections to latent structures discriminant analysis, were performed to identify the discriminative metabolites between the two L. spp. Metabolites with variable influences in projection values of more than one and a P value of less than 0.05 were marked as significant differences. Results: A set of metabolites were detected, and 24 significantly differentially expressed metabolites were found between the metacyclic forms of L. major and L. tropica isolates. The top differential metabolites were methionine, aspartate, betaine, and acetylcarnitine, which were increased more in L. tropica than L. major (P<0.005), whereas asparagine, 3-hydroxybutyrate, L-proline, and kynurenine were increased significantly in L. major (P<0.01). The significantly altered metabolites were involved in eight metabolic pathways. Conclusion: Metabolomics, as an invaluable technique, yielded significant metabolites, and their biochemical pathways related to the metacyclic promastigotes of L. major and L. tropica. The findings offer greater insights into parasite biology and how pathogens adapt to their hosts.

The potential therapeutic role of PTR1 gene in non-healing anthroponotic cutaneous leishmaniasis due to Leishmania tropica.

BackgroundDrug resistance is a common phenomenon frequently observed in countries where leishmaniasis is endemic. Due to the production of the pteridine reductase enzyme (PTR1), drugs lose their efficacy, and consequently, the patient becomes unresponsive to treatment. This study aimed to compare the in vitro effect of meglumine antimoniate (MA) on non‐ healing Leishmania tropica isolates and on MA transfected non‐healing one to PTR1.MethodsTwo non‐healing and one healing isolates of L. tropica were collected from patients who received two courses or one cycle of intralesional MA along with biweekly liquid nitrogen cryotherapy or systemic treatment alone, respectively. After confirmation of L. tropica isolates by polymerase chain reaction (PCR), the recombinant plasmid pcDNA‐rPTR (antisense) was transfected via electroporation and cultured on M199. Isolates in form of promastigotes were treated with different concentrations of MA and read using an enzyme‐linked immunosorbent assay (ELISA) reader and the half inhibitory concentration (IC50) value was calculated. The amastigotes were grown in mouse macrophages and were similarly treated with various concentrations of MA. The culture glass slides were stained, and the mean number of intramacrophage amastigotes and infected macrophages were assessed in triplicate for both stages.ResultsAll three transfected isolates displayed a reduction in optical density compared with the promastigotes in respective isolates, although there was no significant difference between non‐healing and healing isolates. In contrast, in the clinical form (amastigotes), there was a significant difference between non‐healing and healing isolates (p < 0.05).ConclusionThe results indicated that the PTR1 gene reduced the efficacy of the drug, and its inhibition by antisense and could improve the treatment of non‐healing cases. These findings have future implications in the prophylactic and therapeutic modality of non‐ healing Leishmania isolates to drug.

Molecular Typing and Phylogenetic Analysis of Cutaneous Leishmania from Iraqi Patients

This study aimed for molecular characterization of local isolates of L. tropica and L. major and theirrelatedness to reference isolated from neighboring countries. Exudates and dermal tissues were collectedfrom the cutaneous lesions of 50 newly diagnosed patients with CL. Leishmania DNA was extracted fromlesion exudate and 5.8S rRNA gene was amplified using two sets of specific primers through nested PCR. Atotal of 12 PCR products were directly sequenced and the resultant sequences were aligned with referencesequences in National Center for Biotechnology Information (NCBI). A phylogenetic tree was constructedfor local Leishmania isolates with the reference isolates using Mega 6 software. According to alignmentresults out of 12 sequences, 9 sequences were belonging to L. major, while 3 sequences were belonging toL. tropica. All local L. major isolates were very close to reference isolate KP773404.1 (an Iranian isolate),while all local L. tropica isolates were very close to reference isolate KY612611.1 (an Iranian isolate) withan identity of 98%. Phylogenetic trees confirmed that closest isolates for local Leishmania (whether L.tropica or L. major) was the Iranian isolate. These data indicate that local isolates of L. tropica and L. majorare closely related to Iranian isolates.

Expanded genome-wide comparisons give novel insights into population structure and genetic heterogeneity of Leishmania tropica complex.

Leishmania tropica is one of the main causative agents of cutaneous leishmaniasis (CL). Population structures of L. tropica appear to be genetically highly diverse. However, the relationship between L. tropica strains genomic diversity, protein coding gene evolution and biogeography are still poorly understood. In this study, we sequenced the genomes of three new clinical L. tropica isolates, two derived from a recent outbreak of CL in camps hosting Syrian refugees in Lebanon and one historical isolate from Azerbaijan to further refine comparative genome analyses. In silico multilocus microsatellite typing (MLMT) was performed to integrate the current diversity of genome sequence data in the wider available MLMT genetic population framework. Single nucleotide polymorphism (SNPs), gene copy number variations (CNVs) and chromosome ploidy were investigated across the available 18 L. tropica genomes with a main focus on protein coding genes. MLMT divided the strains in three populations that broadly correlated with their geographical distribution but not populations defined by SNPs. Unique SNPs profiles divided the 18 strains into five populations based on principal component analysis. Gene ontology enrichment analysis of the protein coding genes with population specific SNPs profiles revealed various biological processes, including iron acquisition, sterols synthesis and drug resistance. This study further highlights the complex links between L. tropica important genomic heterogeneity and the parasite broad geographic distribution. Unique sequence features in protein coding genes identified in distinct populations reveal potential novel markers that could be exploited for the development of more accurate typing schemes to further improve our knowledge of the evolution and epidemiology of the parasite as well as highlighting protein variants of potential functional importance underlying L. tropica specific biology.

Comparison of &lt;i&gt;in vitro&lt;/i&gt; Resistance of Wild &lt;i&gt;Leishmania&lt;/i&gt; İsolates, Which are Resistant to Pentavalent Antimonial Compounds, Against Drugs Used in the Treatment of Leishmaniasis

Meglumine antimoniate (Glucantime®) and Sodium stibogluconate (Pentostam®) are used for the treatment of cutaneous leismaniasis in Turkey. There is a reported resistance to these drugs in recent years. The aim of the present study was to compare the in vitro sensitivities of resistant Leishmania isolates against Amphotericin B, Miltefosine, Meglumine Antimoniate, Paromomycin and Sodium Stibogluconate. Five Leishmania isolates of patients with cutaneous leishmaniasis, who showed no clinical recovery despite two consecutive meglumine antimoniate treatments, which were stored in the Parasite Bank in Manisa Celal Bayar University Medical Faculty were selected. They were genotyped with Real-Time PCR using specific primers and probes to ITS1 region. Drug resistance levels of each Leishmania isolate were analysed for Amphotericin B, Miltefosine, Meglumine Antimoniate, Paromomycin, and Sodium Stibogluconate at concentrations of 500, 250, 125, 50, 25 μg/mL with XTT method and hemocytometer. It was observed that the resistant Leishmania tropica isolates showed no resistance to Amphotericin B, and were sensitive to Miltefosine, Sodium Stibogluconate, Paromomycin and Meglumin Antimonate, respectively. In addition, Leishmania tropica (MHOM/AZ/1974/SAF-K27) isolate of the control group could stay viable in none of the drug concentrations of five agents in the study. It was determined that none of the selected resistant L. tropica isolates showed resistance to Amphotericin B and that was also shown statistically (p<0.05). The results of this study are important in guiding clinicians and researchers who conduct studies on drugs and search for new drug molecules.

VULGARIS ACTIVITY AGAINST OF Leishmania tropica PROMASTIGOTES; IN VITRO.

Cutaneous leishmaniasis (CL) is a major public health problem and an endemic disease in Iraqi population, Leishmania tropica is one of the causes of leishmaniasis in Baghdad. Considering the inefficiency of current drugs and the fact that some varieties of Leishmania are resistant to these treatments, new drugs are being researched in order to find a more selective and effective therapy with fewer side effects. Therefore, our research group conducted studies on new therapeutic agents. This study is intended to investigate the effect ethanol extract of Chara vulgaris at concentrations (15.6-500) µg/mL in the growth rate and viability of leishmania tropica isolates and compared with pentostam (3.12-100) µg/mL In-vitro. (15.6, 31.25, 62.5, 125, 250, 500 µg/mL) in vitro by MTT assay [3-(4.5-dimethylthiazol-2-yl)- 2.5-diphenyl tetrazolium bromide)], to investigate its effect on the proliferation of promastigotes. Three incubation periods (24, 48, 72 hr.).

Intra-Species Diversity of Leishmania major and L. tropica from Clinical Isolates of Cutaneous Leishmaniasis in Southwest Iran Inferred by ITS1-rDNA

Cutaneous leishmaniasis (CL) as a public health concern is increasingly circulating by causative agents of Leishmania tropica and L. major in Iran. As regard to recent treatment failure and controlling problems, the accurate elucidation of heterogeneity traits and taxonomic status of Leishmania spp. should be broadly addressed by policymakers. This study was designed to determine the genetic variability and molecular characterization of L. major and L. tropica from Iranian CL patients. One hundred positive Giemsa-stained slides were taken from clinical isolates of CL at Pol-e-Dokhtar County, Southwest Iran, from May 2014 to Sep 2016. DNAs were directly extracted and amplified by targeting ribosomal internal transcribed spacer (ITS) gene following microscopic observation. To identify Leishmania spp. amplicons were digested by restriction enzyme HaeIII subsequent PCR-RFLP technique. To reconfirm, the isolates were directly sequenced to conduct diversity indices and phylogenetic analysis. Based upon the RFLP patterns, 84 and 16 isolates were explicitly identified to L. tropica and L. major respectively. No co-infection was found in clinical isolates. The high genetic diversity of L. tropica (Haplotype diversity 0.9) was characterized compared to L. major isolates (Hd 0.476). The intra-species diversity for L. tropica and L. major isolates corresponded to 3%-3.9% and 0%-0.4%, respectively. Findings indicate the L. tropica isolates with remarkable heterogeneity than L. major are predominantly circulating at Pol-e-Dokhtar County. Occurrence of high genetic variability of L. tropica may be noticed in probable treatment failure and/or emerging of new haplotypes; however, more studies are warranted from various geographic regions of Southwest Iran, using large sample size.

Molecular Identification of Agents of Human Cutaneous Leishmaniasis and Canine Visceral Leishmaniasis in Different Areas of Iran Using Internal Transcribed Spacer 1 PCR-RFLP

Leishmaniasis is a major medical health problem and distributes in nearly half of 31 provinces of Iran. We aimed to identify cutaneous and visceral Leishmania spp. isolated from infected humans and domestic dogs in various regions of Iran, 2010-2013. DNA was extracted from 108 lesion exudate samples of suspected patients to cutaneous leishmaniasis and nine liver and spleen aspirates of infected dogs cultured in RPMI-1640 and amplified using partial sequence of ITS1 gene. The PCR amplicons were digested using HaeIII endonuclease enzyme and used in restriction fragment length polymorphism (RFLP) assay. Then, 48 amplicons representing various hosts were sequenced and compared to sequences from GenBank databases using BLAST. PCR-RFLP analysis showed that 60 and 48 CL patients were infected by Leishmania tropica and L. major, respectively. From nine canine visceral leishmaniasis (CVL) isolates, eight isolates were identified as L. infantum and one as L. tropica. The greatest similarity of 95.7% in ITS1 region was seen between L. infantum and L. major. Furthermore, the lowest similarity with 65.7% was seen between L. tropica and L. major. Intra-species comparison of ITS1 region in L. infantum, L. major and L. tropica isolates were showed 100%, 98.2% and 72.4 % similarities, respectively. PCR-RFLP based on ITS1 region is an appropriate method to distinguish three Leishmania spp. of L. major, L. tropica, and L. infantum. In intra-species comparison of ITS1 region, genotypic variations showed that L. tropica isolates were more heterogeneous than L. major and L. infantum isolates.

Antimony susceptibility of Leishmania isolates collected over a 30-year period in Algeria

BackgroundIn Algeria, the treatment of visceral and cutaneous leishmanioses (VL and CL) has been and continues to be based on antimony-containing drugs. It is suspected that high drug selective pressure might favor the emergence of chemoresistant parasites. Although treatment failure is frequently reported during antimonial therapy of both CL and VL, antimonial resistance has never been thoroughly investigated in Algeria. Determining the level of antimonial susceptibility, amongst Leishmania transmitted in Algeria, is of great importance for the development of public health policies.Methodology/Principal findingsWithin the framework of the knowledge about the epidemiology of VL and CL amassed during the last 30 years, we sampled Leishmania isolates to determine their susceptibility to antimony. We analyzed a total of 106 isolates including 88 isolates collected between 1976 and 2013 in Algeria from humans, dogs, rodents, and phlebotomines and 18 collected from dogs in France. All the Algerian isolates were collected in 14 localities where leishmaniasis is endemic. The 50% inhibitory concentrations (IC50) of potassium antimony tartrate (the trivalent form of antimony, Sb(III)) and sodium stibogluconate (the pentavalent form of antimony, Sb(V)) were determined in promastigotes and intramacrophage amastigotes, respectively. The epidemiological cutoff (ECOFF) that allowed us to differentiate between Leishmania species causing cutaneous or visceral leishmaniases that were susceptible (S+) or insusceptible (S-) to the trivalent form of antimony was determined. The computed IC50 cutoff values were 23.83 μg/mL and 15.91 μg/mL for VL and CL, respectively. We report a trend of increasing antimony susceptibility in VL isolates during the 30-year period. In contrast, an increase in the frequency of S- phenotypes in isolates causing CL was observed during the same period. In our study, the emergence of S- phenotypes correlates with the inclusion of L. killicki (syn: L. tropica) isolates that cause cutaneous leishmaniasis and that have emerged in Algeria during the last decade.Conclusion/SignificanceOur results provide insight into the spatiotemporal dynamics of Leishmania antimony susceptibility in Algeria. We highlight the need for the future implementation of an effective methodology to determine the antimony susceptibility status of Leishmania isolates to detect the emergence of and prevent the dissemination of drug-resistant strains.

Geographical distribution and molecular characterization for cutaneous leishmaniasis species by sequencing and phylogenetic analyses of kDNA and ITS1 loci markers in south-eastern Iran

This study aimed to explore geographic distribution and molecular characterization of cutaneous leishmaniasis (CL) species by amplifying two popular markers in kinetoplast DNA and internal transcribed spacer 1 loci by nested-PCR, and characterized by sequencing and phylogenetic analyses. Findings demonstrated that two species co-existed in the province: L. tropica (88.5%) and L. major (11.5%). All gender and age groups were equally infected, although males, 21–30 years old, exhibited a significantly higher infection. Sequencing and phylogenetic analyses of 34 randomly selected samples showed that L. tropica isolates exhibited some degree of heterogeneity. Both anthroponotic CL and zoonotic CL are present in south-eastern Iran with predominance of L. tropica species. Some level of heterogeneity was observed in L. tropica isolates which possibly reflects different colonies in the area. Implementation of diagnostic tools directly from clinical samples could be an important strategic approach for exploration of spatial distribution, molecular characterization and phylogenetic analyses.

Variability of Cutaneous Leishmaniasis Lesions Is Not Associated with Genetic Diversity of Leishmania tropica in Khyber Pakhtunkhwa Province of Pakistan.

Leishmania tropica is the causative agent of cutaneous leishmaniasis in Pakistan. Here, intraspecific diversity of L. tropica from northern Pakistan was investigated using multilocus microsatellite typing. Fourteen polymorphic microsatellite markers were typed in 34 recently collected L. tropica isolates from Pakistan along with 158 archival strains of diverse Afro-Eurasian origins. Previously published profiles for 145 strains of L. tropica originating from different regions of Africa, Central Asia, Iran, and Middle East were included for comparison. Six consistently well-supported genetic groups were resolved: 1) Asia, 2) Morroco A, 3) Namibia and Kenya A, 4) Kenya B/Tunisia and Galilee, 5) Morocco B, and 6) Middle East. Strains from northern Pakistan were assigned to Asian cluster except for three that were placed in a geographically distant genetic group; Morocco A. Lesion variability among these Pakistani strains was not associated with specific L. tropica genetic profile. Pakistani strains showed little genetic differentiation from strains of Iraq, Afghanistan, and Syria (FST = 0.00-0.06); displayed evidence of modest genetic flow with India (FST = 0.14). Furthermore, genetic structuring within these isolates was not geographically defined. Pak-Afghan cluster was in significant linkage disequilibrium (IA = 1.43), had low genetic diversity, and displayed comparatively higher heterozygosity (FIS = -0.62). Patterns of genetic diversity observed suggest dominance of a minimally diverse clonal lineage within northern Pakistan. This is surprising as a wide clinical spectrum was observed in patients, suggesting the importance of host and other factors. Further genotyping studies of L. tropica isolates displaying different clinical phenotypes are required to validate this potentially important observation.

Expression analysis of activated protein kinase C gene (LACK1) in antimony sensitive and resistant Leishmania tropica clinical isolates using real-time RT-PCR.

Resistance to pentavalent antimonial drugs has become a serious problem in the treatment of cutaneous leishmaniasis in some endemic areas. Investigations on molecular markers involved in drug resistance are essential for monitoring of the disease. Leishmania-activated C kinase gene (LACK1) is involved in multiple central processes such as signal transduction. According to the probable role of the LACK1 gene in antimony resistance, we used real-time reverse transcription-polymerase chain reaction (PCR) to investigate the expression of this gene in clinical L. tropica strains, which were resistant or sensitive to meglumine antimoniate. We analyzed the expression level of LACK in 18 sensitive and 14 resistant L. tropica isolates collected from patients with anthroponotic cutaneous leishmaniasis. After cDNA synthesis, gene expression analysis was performed by quantitative real-time PCR using SYBR Green. In addition, the full length of the LACK gene from six reference strains was cloned and sequenced then deposited in the NCBI database to confirm our strains. Real-time reverse transcription-PCR revealed that the average RNA expression level of LACK in isolates from unresponsive and responsive patients were 0.479 and 4.583, respectively, and expression of LACK was significantly downregulated (9.56-fold) in resistant isolates compared to sensitive ones. Results of the present study suggest the probable role of the LACK gene in antimony resistance. Moreover, it can be considered as a potential marker for monitoring antimony resistance in clinical isolates. However, further studies are required to exploit the biological functions of it in antimony resistance.

Genetic diversity of Leishmania tropica strains isolated from clinical forms of cutaneous leishmaniasis in rural districts of Herat province, Western Afghanistan, based on ITS1-rDNA

Despite the high incidence of cutaneous leishmaniasis (CL) in Afghanistan, there is a little information concerning epidemiological status of the disease and phylogenetic relationship and population structure of causative agents. This study was conducted to determine the prevalence and distribution of CL cases and investigate the Leishmania tropica population structure in rural districts of Heart province in the West of Afghanistan in comparison to neighboring foci. Overall, 4189 clinically suspected CL cases from 177 villages (including 12 districts) in Herat province were enrolled in the referral laboratory of WHO sub-office in Herat city from January 2012 to December 2013. 3861 cases were confirmed as CL by microscopic examination of Giemsa-stained slides. ITS1 PCR–RFLP analysis showed dominance of L. tropica (more than 98%) among 127 randomly chosen samples. Analysis of the ITS1 sequences revealed 4 sequence types among the 21 L. tropica isolates. Comparison of sequence types from Herat rural districts with the representatives of L. tropica from Iran, India, and Herat city showed two main population groups (cluster A and B). All isolates from Herat province, India and Southeast, East, and Central Iran were found exclusively in cluster A. The close proximity of West Afghanistan focus and Birjand county as the capital of Southern Khorasan province in East Iran can explain relatively equal to the genetic composition of L. tropica in these two neighboring regions. In addition, two populations were found among L. tropica isolates from Herat rural districts. Main population showed more similarity to some isolates from Birjand county in East Iran while minor population probably originated from the Southeast and East Iranian L. tropica. Recent study provided valuable information concerning the population structure of L. tropica and epidemiology of ACL in the West of Afghanistan, which could be the basis for molecular epidemiology studies in other regions of Afghanistan.