Genetic Diversity of Leishmania major Isolated from Different Dermal Lesions Using ITS2 Region.

BACKGROUND AND OBJECTIVE: Cutaneous leishmaniasis (CL) is still considered to be an uncontrolled endemic disease that spreads in many countries. The current study aimed to determine intra-species relationships of L. major using ITS2 sequencing. METHODS: The study was conducted from the beginning of March to the end of November 2022. All medical information regarding CL was collected from patients of Thi-Qar province who attended the Dermatology Department of Al-Hussein Teaching Hospital in Nasiriyah city. Seventy-three samples were selected for the molecular identification after confirming microscopy with Giemsa stain. In this study, the primers were designed using NCBI GenBank sequence database and Primer 3 plus primer design online software. RESULTS: The results recorded 21 (28.77%) positive samples of L. major using the internal transcribed spacer 2 region (ITS2) in ribosomal RNA gene. The local L. major IQN.1-IQN.10 were submitted to NCBI GenBank database with accession numbers OM069357.1-OM069366.1, respectively. The phylogenetic analysis revealed that local isolates of L. major showed a close relationship with NCBI-BLAST L. major Iran isolate (KU680848.1). CONCLUSION: ITS2-PCR is suitable for identifying Leishmania spp. and determining genetic diversity. A phylogenetic data analysis may provide an idea on the genetic homogeneity of local isolates and knowing the genetic origin of the dermal lesion. However, the local isolates showed genetic proximity to the KU680848.1 isolate. This signifies the possibility of infection prevalence from Iranian areas. In general, genetic variation of L. major isolates may give several clinical manifestations of the cutaneous lesion. Therefore, determination of the heterogeneity is important for detecting the infection origin, epidemiology, therapy, and control strategies.

Genome Analysis of Endotrypanum and Porcisia spp., Closest Phylogenetic Relatives of Leishmania, Highlights the Role of Amastins in Shaping Pathogenicity.

While numerous genomes of Leishmania spp. have been sequenced and analyzed, an understanding of the evolutionary history of these organisms remains limited due to the unavailability of the sequence data for their closest known relatives, Endotrypanum and Porcisia spp., infecting sloths and porcupines. We have sequenced and analyzed genomes of three members of this clade in order to fill this gap. Their comparative analyses revealed only minute differences from Leishmaniamajor genome in terms of metabolic capacities. We also documented that the number of genes under positive selection on the Endotrypanum/Porcisia branch is rather small, with the flagellum-related group of genes being over-represented. Most significantly, the analysis of gene family evolution revealed a substantially reduced repertoire of surface proteins, such as amastins and biopterin transporters BT1 in the Endotrypanum/Porcisia species when compared to amastigote-dwelling Leishmania. This reduction was especially pronounced for δ-amastins, a subfamily of cell surface proteins crucial in the propagation of Leishmania amastigotes inside vertebrate macrophages and, apparently, dispensable for Endotrypanum/Porcisia, which do not infect such cells.

Orientation of FtsH protease homologs in Trypanosoma brucei inner mitochondrial membrane and its evolutionary implications.

Trypanosoma brucei is an important human pathogen. In this study, we have focused on the characterization of FtsH protease, ATP-dependent membrane-bound mitochondrial enzyme important for regulation of protein abundance. We have determined localization and orientation of all six putative T.brucei FtsH homologs in the inner mitochondrial membrane by in silico analyses, by immunofluorescence, and with protease assay. The evolutionary origin of these homologs has been tested by comparative phylogenetic analysis. Surprisingly, some kinetoplastid FtsH proteins display inverted orientation in the mitochondrial membrane compared to related proteins of other examined eukaryotes. Moreover, our data strongly suggest that during evolution the orientation of FtsH protease in T. brucei varied due to both loss and acquisition of the transmembrane domain.

Genotyping of Cutaneous Leishmaniasis Cases Detected Before and After Migration with Real-Time Polymerase Chain Reaction in Hatay

Objective: Leishmania tropica (L. tropica) and Leishmania infantum (L. infantum) are the species causing cutaneous Leishmaniasis (CL) in Turkey. There was a wave of immigration due civil war in Syria in 2011. Migration from Syria, where CL is endemic, to other countries is thought to affect the number of CL cases and species diversity. The aim of the study was to typify the samples of CL positive, pre-migration and post-migration Turkish patients and importe (Syrian) patients whose smears were found in the archive and to reveal the difference of CL species before and after migration in Hatay. Methods: Smears of a total of 150 patients (50 Turkish patients before migration, 50 Turkish patients after migration and 50 Syrian patients) which had been prepared with dermal scraping, stained with Giemsa and determined as CL positive by microscope examination were included in the study. DNA isolation of selected preparations was performed and GZ-PZR analysis with ITS-1probe was performed for species determination. Results: L. infantum/donovani was detected in 40 (80%), L. tropica in 8 (16%), and L. major in 2 (4%) of the samples belonging to pre-immigration Turkish patients. L. infantum/donovani was detected in 28 (56%), L. major in 3 (6%) and L. tropica in 19 (%38) of the samples belonging to post-immigration Turkish patients. L. infantum/donovani was detected in 2 (4%), L. major in 1 (2%) and L. tropica in 47 (94%) of the samples belonging to Syrian patients. Conclusions: It was observed that in local cases in Hatay before immigration, L. infantum/donovani was the common species that caused CL and that after immigration L. tropica began to raise and that L. major was more encountered than before. It was concluded that Syrians coming to Hatay may have caused diversity in the Leishmania species which were the causative agents of CL, and that further research was needed on the subject.

Whole genome experimental maps of DNA G-quadruplexes in multiple species.

Genomic maps of DNA G-quadruplexes (G4s) can help elucidate the roles that these secondary structures play in various organisms. Herein, we employ an improved version of a G-quadruplex sequencing method (G4-seq) to generate whole genome G4 maps for 12 species that include widely studied model organisms and also pathogens of clinical relevance. We identify G4 structures that form under physiological K+ conditions and also G4s that are stabilized by the G4-targeting small molecule pyridostatin (PDS). We discuss the various structural features of the experimentally observed G-quadruplexes (OQs), highlighting differences in their prevalence and enrichment across species. Our study describes diversity in sequence composition and genomic location for the OQs in the different species and reveals that the enrichment of OQs in gene promoters is particular to mammals such as mouse and human, among the species studied. The multi-species maps have been made publicly available as a resource to the research community. The maps can serve as blueprints for biological experiments in those model organisms, where G4 structures may play a role.

Identification of Leishmania species using N-acetylglucosamine-1-phosphate transferase gene in a zoonotic cutaneous leishmaniasis focus of Iran.

BACKGROUND & OBJECTIVES: Ilam province is one of the oldest known endemic foci of zoonotic cutaneous leishmani- asis (CL) in Iran; and the recent studies have shown an increasing trend in the number of cases from the region. This study was aimed to investigate the parasite species and genetic diversity of isolates obtained from CL patients based on the N-acetylglucosamine-1-phosphate transferase (nagt) gene. METHODS: Exudate materials were collected from the swollen margin of the skin lesions of the patients suspected with CL who were referred to health centers laboratory of Mehran, Dehloran, Ilam and Malekshahi cities in the Ilam province. Demographic data were collected through a questionnaire. Smears were stained and examined microscopically. In total, 62 parasitologically positive samples were subjected to PCR-RFLP of nagt gene for identification of Leishmania species, in addition to genetic diversity investigation. RESULTS: Nearly, half of the positive cases were referred from Mehran followed by Dehloran City (40.4%). These included people from different age groups (1 to 73 yr), with majority being male (66.1%). The common site of lesions was hand (48.4%). Half of the patients had multiple lesions; most of them were wet ulcerative type. A 1450-60 bp band of the nagt gene was amplified from all the samples. Digestion patterns of acetyl-coenzyme A carboxylase 1 (ACC1) enzyme were similar to what expected for Leishmania major. No difference was observed at the nucleotide acid level or resulting amino acid in nine sequenced samples on the basis of phylogenetic analyses. However, intra- species differences (0.0015) were observed amongst the L. major isolates of present study and the other parts of Iran. INTERPRETATION & CONCLUSION: The findings of this study demonstrated that the main causative agent of CL in Ilam Province is L. major, and there is no considerable heterogeneity among the L. major isolates. Moreover, nagt gene proved to be an efficient marker for differentiating Leishmania species. Further studies with more samples need to be carried out to achieve a more comprehensive result on the genetic variation of L. major isolates.

Paraechinus aethiopicus (Ehrenberg 1832) and Atelerix algirus (Lereboullet 1842) hedgehogs: Possible reservoirs of endemic leishmaniases in Tunisia.

Rodents and dogs are the confirmed leishmaniases reservoir hosts in Tunisia. Recently, we described hedgehog Leishmania (L.) major and L. infantum infection in an L. infantum endemic area in the North-West. In order to assess if the observation could extend to other endemic areas and to highlight the potential role of hedgehogs as reservoir host, we aimed here at investigating their Leishmania infection in different foci in Tunisia located along a North-South transect, during and outside different transmission seasons. Based on morphological criteria, 2 hedgehogs' species, Atelerix algirus and Paraechinus aethiopicus were identified. Cytologic analysis showed presence of amastigotes in 9/22 samples corresponding to 4 Atelerix algirus specimens. Also, by combining 3 PCR tests targeting repeated DNA fragments using 13A/13B, Lei70R/Lei70L and nested T2/B4-L1/L4 specific primers, all hedgehogs (N = 12) showed a Leishmania infection. The infection rates were very high on spleen (91.66%), kidney (91.66%), blood (90.90%), liver (83.33%) and eye swabs (100%). Parasites were also detected in peritoneum. Three hedgehogs were found infected with L. infantum and the only Paraechinus aethiopicus specimen with L. major. A mixed L. major and L. infantum infection was identified in 8 animals, while the last one also had an L. tropica infection. Interestingly, 2 animals had skin lesions infected with L. major while all others appeared asymptomatic. There was a correlation between infected status and epidemiological profiles of the localities. Sequences and phylogeny indicated micro-heterogeneity and lack of correlation with sampling, season, or localities. We confirmed natural infection of Atelerix algirus and originally of Paraechinus aethiopicus in Tunisia. High rate of asymptomatic infection, parasitemia, proximity to transmission cycles, epidemiological patterns of infection together with hedgehogs' abundance, lifespan and lifestyle corroborate the hypothesis they constitute reservoir hosts.

Immunodetection and molecular determination of visceral and cutaneous Leishmania infection using patients' urine.

The diagnosis of leishmaniasis relies mainly on the use of invasive processes, to collect the biological material for detecting Leishmania parasites. Body fluids, which can be collected by non-invasive process, would greatly facilitate the leishmaniasis diagnosis. In the present study, we investigated the potency of urine immunoblotting to diagnose cutaneous and visceral leishmaniasis and we compared with routine molecular methods. A total of 80 samples, including 40 sera and their 40 corresponding urine samples were collected from 37 suspected patients with cutaneous and visceral leishmaniasis, and 3 healthy individuals (as control), in Ilam and Ardabil provinces of Iran. All sera and urine samples were analyzed, using immunoblotting. The confirmation of leishmaniasis infection was performed, using conventional and quantitative PCRs as well as by sequencing the amplicons. Among 37 suspected patients, 23 patients presented cutaneous lesions (CL) and 14 exhibited clinical symptoms reminiscent of visceral leishmaniasis (L. infantum). Among cutaneous patients, 15 were positive for zoonotic cutaneous leishmaniasis (L. major), and eight for anthroponotic cutaneous leishmaniasis (L. tropica). Molecular quantification of Leishmania parasites was performed on sera, urines and cutaneous biopsies of CL and VL patients, demonstrating that parasite load is lower in urines, compared to sera or biopsy. DNA can be detected in 20 out of 23 (86.9%) CL urine samples and in 13 out of 14 (92.8%) VL urine samples. Immunodetection analysis demonstrates that 22 out of 23 (95.6%) sera from CL patients and all patients suspected with VL are positive. For urine samples, 18 out of 23 (78.2%) urine of CL patients and 13 out of 14 (92.8%) urine of VL patients were positive, using Western blot. Therefore, immunodetection and molecular analysis using urine samples can be used as a diagnostic tool for surveying cutaneous and visceral leishmaniasis.

An outbreak of Leishmania major from an endemic to a non-endemic region posed a public health threat in Iraq from 2014-2017: Epidemiological, molecular and phylogenetic studies.

BACKGROUND: Cutaneous leishmaniasis (CL) is a neglected worldwide, zoonotic, vector-borne, tropical disease that is a threat to public health. This threat may spread from endemic to non-endemic areas. Current research has exploited epidemiological, molecular and phylogenetical studies to determine the danger of an outbreak of CL in the borderline area between northern and central Iraq from 2014-2017. METHODOLOGY/PRINCIPAL FINDINGS: For the first time, using sequence analysis of the cytochrome b gene, the occurrence of CL in the borderline area between northern and central Iraq was confirmed to be due to Leishmania major. The phylogenetic analysis indicated that it was closely related to the L. major MRHO/IR/75/ER strain in Iran. CONCLUSIONS AND SIGNIFICANCE: In conclusion, the genotype confirmation of the L. major strain will improve our understanding of the epidemiology of the disease. This is important for facilitating control programs to prevent the further spread of CL. Furthermore, this area could be considered as a model for further research on the risk of global CL epidemics in other non-endemic countries where both reservoir hosts and sandfly vectors are present.

Leishmania major cutaneous leishmaniasis outbreak in the Jordanian side of the Northern Jordan Valley.

Cutaneous leishmaniasis (CL) is endemic in many foci of Jordan and the Jordanian Mid Jordan Valley (JMJV) is the most affected and the incidence is quite high. The situation in the northern part of the Jordanian side of the Jordan Valley (NJJV) was different; before 2008, CL has rarely been reported from this area. From April 2008 to May 2009, passive detection followed by active detection was used to trace cases of CL from the NJJV. DNA was extracted from seven clinical isolates of Leishmania promastigotes and lesion scrapings spotted on filter papers obtained from 51 suspected CL patients living in the NJJV. The identity of the causative species of CL in the NJJV was investigated using ITS1-PCR followed by RFLP. In 2008/2009, 183 cases were clinically diagnosed of having CL in the NJJV. The parasites in five of the isolates and in 48 PCR-positive scrapings were classified as Leishmania major. In two isolates and in one PCR-positive scraping Leishmania tropica was identified. Investigations on the origin of CL cases revealed that the L. tropica cases were residents of two towns outside the NJJV. Herein, we report the clinical features, parasitological diagnosis, etiology, and the geographical distribution of CL cases from NJJV with the aim of documenting, for the first time, an outbreak in this area.

Molecular prevalence and estimated risk of cutaneous leishmaniasis in Libya.

BACKGROUND/PURPOSE: Cutanoeus leishmaniasis (CL) is an endemic disease in the Mediterranean area including Libya. The aim of the present study is to detect the prevalent Leishmania species obtained from smeared cutaneous lesions in addition to studying the diverse sociodemographic risk factors of the reported cases from different provinces of Libya. METHODS: A total of 250 archived microscopic slides from clinically suspected cases of CL attending the leishmaniasis clinic in the Dermatology Department, Tripoli Central Hospital, Tripoli, Libya, were microscopically examined. Leishmania-DNA was amplified using combined polymerase chain reaction (PCR) targeting kinetoplast-DNA (kDNA) and ribosomal internal transcribed spacer 1 (ITS1)-DNA with restriction fragment length polymorphism analysis for direct Leishmania species identification. RESULTS: Using kDNA and ITS1-PCR, 22.5% and 20% of cases were positive, respectively. Only 14.4% of cases were positive using microscopy. Nominating ITS1-PCR as the reference standard, kDNA-PCR assay was highly sensitive while microscopy was 100% specific but of limited sensitivity (72%) with a substantial agreement and an overall accuracy of 94.4%. Leishmania major and Leishmania tropica were the predominant species reported from the north-western provinces including Tripoli, Zintan, and Gharyan with their related subprovinces; Asabaa, Mizdan, Alkawasem, and Alorban. CL prevailed more among men and residents of rural areas. House wives and students were the most affected professions. Children were the least affected, while the middle-aged were the most affected age group. CONCLUSION: L. major and L. tropica are the predominant species in the north-western regions of Libya. ITS1-PCR-restriction fragment length polymorphism assay offered a sensitive, specific, and faster diagnostic method especially with negative parasitologic examination.

Comparative genomics of Tunisian Leishmania major isolates causing human cutaneous leishmaniasis with contrasting clinical severity.

Zoonotic cutaneous leishmaniasis caused by Leishmania (L.) major parasites affects urban and suburban areas in the center and south of Tunisia where the disease is endemo-epidemic. Several cases were reported in human patients for which infection due to L. major induced lesions with a broad range of severity. However, very little is known about the mechanisms underlying this diversity. Our hypothesis is that parasite genomic variability could, in addition to the host immunological background, contribute to the intra-species clinical variability observed in patients and explain the lesion size differences observed in the experimental model. Based on several epidemiological, in vivo and in vitro experiments, we focused on two clinical isolates showing contrasted severity in patients and BALB/c experimental mice model. We used DNA-seq as a high-throughput technology to facilitate the identification of genetic variants with discriminating potential between both isolates. Our results demonstrate that various levels of heterogeneity could be found between both L. major isolates in terms of chromosome or gene copy number variation (CNV), and that the intra-species divergence could surprisingly be related to single nucleotide polymorphisms (SNPs) and Insertion/Deletion (InDels) events. Interestingly, we particularly focused here on genes affected by both types of variants and correlated them with the observed gene CNV. Whether these differences are sufficient to explain the severity in patients is obviously still open to debate, but we do believe that additional layers of -omic information is needed to complement the genomic screen in order to draw a more complete map of severity determinants.

Risk Mapping and Situational Analysis of Cutaneous Leishmaniasis in an Endemic Area of Central Iran: A GIS-Based Survey.

INTRODUCTION: Cutaneous leishmaniasis (CL) is among the top 10 infectious disease priorities in the world, and the leading cause of morbidity in Iran. The present study was conducted to assess the risk of CL, and to determine some epidemiological features of the disease in endemic areas of Qom Province in Central Iran during 2009 to 2013. METHODS: Data regarding human cases of the disease were obtained from the Qom Province Health Center, prepared and stored in a spatial database created in ArcGIS10.3. A total of 9 out of 212 Leishmania spp. positive slides taken in 2013 from patients residing in Qom city were examined using molecular methods and the species of Leishmania was identified by PCR-RFLP. Those 9 patients had no history of travel outside the city. Spatial analysis and clustering methods were applied to find major hot spots and susceptible areas for the establishment of novel foci of the disease. Transmission patterns were examined for spatial autocorrelation using the Moran's I statistical application, and for the clustering of high or low values using the Getis-Ord Gi* statistics. RESULTS: During the period of study, a total of 1767 CL cases were passively reported in the area, out of which were 65% males and 35% females. The highest and lowest numbers of cases were reported in 2010 and 2013, respectively. Importantly, 979 cases were reported from urban areas, while the remainder came from rural areas. Leishmania major was detected as the causative agent of CL in the city of Qom. Remarkably, most patients recorded in Qom city were associated with a history of travel to the endemic areas of CL within the province, or to other endemic areas of the disease in Iran. Spatial distribution of CL cases revealed northeastern and southwestern quarters of the city were the major hot spots of the disease (P<0.05). Hot spot and CL transmission risk analysis across the province indicated that more than 40 villages were located in high and very high risk areas of CL transmission. CONCLUSIONS: Due to the existence of hot spots (P<0.05) of CL in successive years in some quarters of Qom city, along with detection of L. major from the patients without a history of travel, there may be potential of local transmission of the disease within the city. Therefore, it is necessary to conduct a comprehensive study concerning the hot spots of CL in Qom city for curtailing the incidence of the disease in the city. The methodology and the results of this study is essential in serving as a yardstick for subsequent similar studies that will be carried out in other endemic areas of CL in Iran and providing an adequate tool for the establishment of a national database of cutaneous leishmaniasis.

Genetic micro-heterogeneity of Leishmania major in emerging foci of zoonotic cutaneous leishmaniasis in Tunisia.

Tunisia is endemic for zoonotic cutaneous leishmaniasis (ZCL), a parasitic disease caused by Leishmania (L.) major. ZCL displays a wide clinical polymorphism, with severe forms present more frequently in emerging foci where naive populations are dominant. In this study, we applied the multi-locus microsatellite typing (MLMT) using ten highly informative and discriminative markers to investigate the genetic structure of 35 Tunisian Leishmania (L.) major isolates collected from patients living in five different foci of Central Tunisia (two old and three emerging foci). Phylogenetic reconstructions based on genetic distances showed that nine of the ten tested loci were homogeneous in all isolates with homozygous alleles, whereas one locus (71AT) had a 58/64-bp bi-allelic profile with an allele linked to emerging foci. Promastigote-stage parasites with the 58-bp allele tend to be more resistant to in vitro complement lysis. These results, which stress the geographical dependence of the genetic micro-heterogeneity, may improve our understanding of the ZCL epidemiology and clinical outcome.

First report of naturally infected Sergentomyia minuta with Leishmania major in Tunisia.

BACKGROUND: Many sand fly species are implicated in the transmission cycle of Leishmania parasites around the world. Incriminating new sand flies species, as vectors of Leishmania is crucial to understanding the parasite-vector transmission cycle in different areas in Tunisia and surrounding countries. FINDINGS: Seventy-four unfed females belonging to the genera Sergentomyia and Phlebotomus were collected in South Tunisia between June and November 2014, using sticky papers. PCR-RFLP (Restriction Fragment Length Polymorphism) analysis of the internal transcribed spacer 1 (ITS1) was used for Leishmania parasites detection and identification. Leishmania (L.) major (Yakimoff & Shokkor, 1914) was identified within two Sergentomyia (S.) minuta (Rondani, 1843) and one Phlebotomus papatasi (Scopoli, 1786). CONCLUSION: This is the first report of L. major identified from S. minuta in Tunisia. This novel finding enhances the understanding of the transmission cycle of L. major parasites of cutaneous leishmaniasis in an endemic area in South Tunisia.

Spatio-temporal Genetic Structuring of Leishmania major in Tunisia by Microsatellite Analysis.

In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic (Gafsa, Kairouan and Sidi Bouzid governorates) and collected during two periods: 1991-1992 and 2008-2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991-1992 and 2008-2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008-2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations.

Geographical sequence variation in the Leishmania major virulence factor P46.

Cutaneous leishmaniasis as caused by Leishmania major is a zoonotic infection with wide epidemiological impact. The L. major P46 virulence gene was shown to boost the parasite's virulence and extends its range of experimental hosts. Here we show that P46 is subject to significant geographical sequence variations that may reflect the adaption to different reservoir hosts. This view is supported by the results of passage experiments using P46 variants in different experimental hosts. Conversely, loss of P46 expression leads to attenuation both in vitro and in BALB/c mice. Although part of the L. major exosomal protein payload, P46 is not required for exosome-mediated immune modulation.

Comparison of conventional, molecular, and immunohistochemical methods in diagnosis of typical and atypical cutaneous leishmaniasis.

CONTEXT: Localized cutaneous leishmaniasis (CL) typically presents as papules, crusted nodules, plaques, or noduloulcerative lesions. Atypical CL does not show these features or mimic malignant lesion. In atypical forms, CL may be overlooked because of its similarity to other dermal diseases. OBJECTIVE: To compare conventional, molecular, and immunohistochemical methods in the diagnosis of typical and atypical CL. DESIGN: The kinetoplast DNA, nested, polymerase chain reaction assay and immunohistochemical methods were compared and validated against conventional methods, including cytology and pathology, using 100 specimens of typical and atypical lesions of suspected CL. RESULTS: Compared with other methods, polymerase chain reaction of the kinetoplast DNA showed the highest sensitivity (typical positive, 100%, 67 of 67; atypical positive, 94%, 31 of 33) and specificity (100%), followed by immunohistochemistry (typical positive, 97%, 65 of 67, with 100% specificity; atypical positives, 94%, 31 of 33, with 100% specificity), and cytology (typical positive, 79%, 53 of 67, with 100% specificity; atypical positive, 58%, 19 of 33, with 100% specificity), followed by pathology (typical positive, 70%, 47 of 67, with 100% specificity; atypical positive, 42%, 14 of 33, with 100% specificity). In addition, polymerase chain reaction enabled identification of 98% (98 of 100) of the positive samples that included strains of Leishmania major (99% [99 of 100] cases) and Leishmania tropica (1% [1 of 100] cases). CONCLUSIONS: Because cytology is cheap and easy to perform with high sensitivity, it is the preferred, primary approach for typical CL, but cytology and pathology do not have sufficient sensitivity for diagnosis of atypical CL cases. Nested polymerase chain reaction and immunohistochemistry are sensitive tests for diagnosis of both typical and atypical CL and are recommended as complementary tests in suspected CL with negative conventional microscopy results.

Kinetoplastid-specific histone variant functions are conserved in Leishmania major.

Regions of transcription initiation and termination in kinetoplastid protists lack known eukaryotic promoter and terminator elements, although epigenetic marks such as histone variants and the modified DNA base J have been localized to these regions in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Phenotypes of base J mutants vary significantly across trypanosomatids, implying divergence in the epigenetic networks governing transcription during evolution. Here, we demonstrate that the histone variants H2A.Z and H2B.V are essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast, H3.V is not essential for viability or normal growth in Leishmania. Steady-state transcript levels and the efficiency of transcription termination at convergent strand switch regions (SSRs) in H3V-null parasites were comparable to WT parasites. Our genetic tests show a conservation of histone variant phenotypes between L. major and T. brucei, unlike the diversity of phenotypes associated with genetic manipulation of the DNA base J modification.