Leishmaniasis Species Research Articles

Molecular Identification of Cutaneous Leishmaniasis Species in the Northcentral Iranian Province of Alborz: Is There a New Focus on Cutaneous Leishmaniasis in the Province?

Cutaneous leishmaniasis (CL) is an endemic infection in the Middle East, including Iran that is also spreading to new foci. We aimed to determine the leishmaniasis species causing CL in Alborz province. Overall, out of 55-suspected CL patients referred to health centers in Alborz Province, north central Iran in 2019, 40 patients had positive smear for CL based on optical microscopy. The internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA (rDNA) was amplified by PCR. Leishmania species were identified by PCR-restriction fragment length polymorphism (PCR-RFLP) using BshF I (Hae III) enzyme. Out of the 40 positive patients with CL, 34 cases (85%) had been caused by Leishmania (L) major and six (15%) by L. tropica. Fifteen patients had no history of traveling to the disease endemic areas, of which nine were Iranians. Skin lesions and scars caused by CL were mostly observed on the hands and face. Moreover, more than two skin lesions were observed in 22 cases (55%), all of which were infected with L. major. A single skin ulcer was seen in 18 (45%) of the CL patients. Climate change, reduced rainfall, and demographic changes such as migration into Alborz Province and the increasing marginalization of the population and their entry to settle in new areas might have caused natural transmission of both L. tropica and L. major in this province.

Identification of Leishmania species and frequency distribution of LRV1 and LRV2 viruses on cutaneous leishmaniasis patients in Isfahan Province, Iran

PurposeLeishmaniasis is one of the most serious health problems in developing countries. Iran is one of the endemic regions of cutaneous leishmaniasis. Leishmania RNA virus (LRV) is a dsRNA virus member of the Totiviridae family, which was first detected in the promastigotes of Leishmania braziliensis guyanensis. Our study aimed to investigate possible changes in the predominant and causative strains of CL and screening the LRV1 and LRV2 species genome from Leishmania species isolated from the lesions of patients. Materials and methodsDirect smear samples obtained from 62 patients with leishmaniasis referring to the Skin Diseases and Leishmaniasis Research Center in Isfahan province during 2021–2022 were examined. Total DNA extraction procedures and conservation of site-specific multiplex PCR and nested PCR were performed for detecting Leishmania species. The molecular identification of LRV1 and LRV2 viruses, samples were used for total RNA extraction and real-time (RT)-PCR analysis, followed by conducting a restriction enzyme assay to confirm the PCR products. ResultsOf the total Leishmania isolates, 54 and 8 isolates were identified as L. major and L. tropica, respectively. LRV2 was identified in 18 samples affected by L. major, while LRV1 was only detected in one of the samples with L. tropica. No LRV2 was found in any samples with L. tropica. The results showed that there was a significant relationship between LRV1 and the type of leishmaniasis (Sig. ​= ​0.009, P ​≤ ​0.05), while this relationship was not observed between LRV2 and the type of leishmaniasis. ConclusionsThe presence of a significant number of LRV2 in isolated samples, as well as the recognition of LRV1 in one of the Old World leishmaniasis species, which is a new result, could pave the way for investigating further aspects of this disease and successful treatment strategies in future studies.

Similarity but not equivalence: Ecological niche comparison between sandflies from the Pleistocene and future scenarios in Central and South America.

Sandfly species (Diptera: Psychodidae) are suspected or proven vectors of Leishmania spp. in the American region. Understanding niche conservatism (NC) in insect vectors allows an understanding of constraints on adaptive responses, and thus implications for disease ecology. Therefore, in this study, the authors evaluated NC in three vector species of leishmaniasis (Lutzomyia gomezi, Psathyromyia shannoni and Pintomyia ovallesi) in Central and South America. For this, the authors performed niche identity and similarity testing through paired comparisons in ENMTools and niche overlap in Niche Analyst. The authors found that species niches were more similar to each other than if the points had been randomly extracted, and they also found extensive similarity between Pa. shannoni and Lu. gomezi niches and in Pa. shannoni niches over different timescales. The authors suggest Pa. shannoni as a priority species due to fundamental niche similarity with phylogenetically related species and also its extensive evolutionary history and ecological plasticity that could affect the emergence and resurgence of leishmaniasis in areas endemic by this vector.

Molecular Study of Cutaneous Leishmaniasis Species among Soldiers with Dermal Ulcers in Zahedan, Iran

Molecular Study of Cutaneous Leishmaniasis Species among Soldiers with Dermal Ulcers in Zahedan, Iran

Molecular identification of cutaneous leishmaniasis species using kinetoplast DNA (kDNA) gene in southeast of Iran

Cutaneous leishmaniasis (CL) is one of the most common parasitical diseases in many regions of Iran and plays a significant role in the inhibition of socioeconomic growth of this deprived province. The present study aimed to molecular identification of cutaneous leishmaniasis species in patients from Chabahar and Konark district, in Sistan and Baluchestan province, Iran. This descriptive cross-sectional study was carried out on 250 patients suspected of cutaneous leishmaniasis (CL). Samples of the skin lesions and ulcers suspected to CL were collected from patients and diagnosed by parasitological test and molecular methods, respectively. The microscopic test detected 80 out of 250 (32%) suspected patients to CL, while 80 out of 80 (100%) were positive for Leishmania kDNA. Two species L. major and L. tropica were responsible for 9 and 69 cases, respectively. Our results were consistent with the previous reports in Iran, revealing L. tropica as the most causative agent of CL Further investigation are demanded to identify the hosts, reservoirs, disease species and vectors in this part of the world.

In-depth comparison of cell-based methodological approaches to determine drug susceptibility of visceral Leishmania isolates.

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity.

Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle.

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the Leishmania genus. In Australia, leishmaniasis is an imported disease that is presenting itself at increased rates because of international travel, the influx of immigrants, and deployment of military operations to endemic regions. Although Leishmania species are morphologically indistinguishable, there is a strong correlation between some causative species of leishmaniasis and the subsequent response to the treatments available and patient outcome. Consequently, identification of the infective species is imperative as misidentification can result in the administering of an ineffective drug. The aim of this study was to develop a simple diagnostic tool with high sensitivity and specificity, which is capable of detecting the presence of the parasite and accurately differentiating the causative species in question. Using the advantageous properties of the maxi-circle kinetoplast DNA, a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) targeting the ND7 gene was developed for the analysis of imported cases of human leishmaniasis in Australia. Designed as a dual analysis, concurrent PCR of Leishmania maxi-circle DNA and digestion with two separate enzymes (NlaIII and HpyCH4IV), this study provides an appraisal on 24 imported cases of leishmaniasis between 2008 and 2017. Five Leishmania species were reported, with members of the Viannia subgenus being the most common. The implementation of novel diagnostic procedures for leishmaniasis such as the one reported here is needed to establish a gold standard practice for the diagnosis and treatment of leishmaniasis.

Molecular Diagnosis and Phylogenetic Analysis of 5.8s rDNA Gene of Cutaneous Leishmaniasis Species in Holy Karbala/Iraq

Molecular Diagnosis and Phylogenetic Analysis of 5.8s rDNA Gene of Cutaneous Leishmaniasis Species in Holy Karbala/Iraq

Pediatric Old World cutaneous leishmaniasis treated with oral fluconazole: A case series.

Cutaneous leishmaniasis has an incidence of 0.7-1.2million cases per year and represents a growing concern in the outpatient dermatologic practice in Europe because of imported cases due to increased travel to risk areas and to immigration phenomena. When dealing with children, the treatment can be challenging because of side effects and pain of classic antimonial therapy leading to poor rates of course completion and requirement of sedation for several children. We retrospectively studied three cases of cutaneous leishmaniasis in pediatric patients, between the ages of 3 and 6years of age, treated with oral fluconazole. We examined the efficacy, the tolerability, the safety profile and the cosmetic result of fluconazole at a dose of 6mg/kg/daily for 6weeks. The patients had a complete resolution of their lesions with minimal scarring. No adverse effect was reported. The leishmaniasis species identified were L.major or L.tropica. Considering sides effects and the parents' and the clinician's concern for systemic treatment in the pediatric population, fluconazole represents a valid, safe and easily manageable option for Old World cutaneous leishmaniasis in pediatric outpatients caused by L.major or L.tropica.

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.

ROS regulate differentiation of visceralizing Leishmania species into the virulent amastigote form.

Leishmania virulence and disease development critically depends on the ability of Leishmania promastigotes to infect, differentiate into amastigote forms and replicate inside mammalian host macrophages. Understanding changes associated with amastigote differentiation in axenic culture conditions is key to identifying virulence factors. Here we compared efficiency of the conventional pH-temperature-dependent shift method to induce amastigote differentiation with the recently identified trigger for differentiation mediated by mitochondrial reactive oxygen species (ROS). Using two different visceral leishmaniasis species, L. infantum and. L. donovani, we show that ROS-generating methods such as iron deprivation or exposure to sub-lethal concentrations of H2O2 or menadione are significantly more effective in promoting promastigoteamastigote differentiation than the low pH-high temperature shift, leading to higher survival rates, morphological changes and gene expression patterns characteristic of the amastigote stage. Notably, both H2O2 and menadione-mediated differentiation did not require up-regulation of the mitochondrial electron transport chain (ETC)-associated protein p27, suggesting that treatment with oxidants bypasses the necessity to upregulate mitochondrial activity, a precondition for mROS generation. Our findings confirm that ROS-induced differentiation occurs in multiple Leishmania species, including the medically important visceralizing species, and provide mechanistic rationale for earlier reports demonstrating markedly increased virulence of L. infantum promastigotes pre-treated with oxidative reagents.

Interventions for Old World cutaneous leishmaniasis.

Interventions for Old World cutaneous leishmaniasis.

Identification of Leishmania Species for Cutaneous leishmaniasis in Gonabad, Bardaskan and Kashmar, Central Khorasan, 2015

Background: About 90% of cutaneous leishmaniasis (CL) cases are reported from 7 countries including Iran. In this study, the cutaneous leishmaniasis species causing CL in Gonabad, Bardaskan, Kashmar cities in central Khorasan (Iran) were identified by kDNA-PCR. Methods: During the study, 93 suspected patients with CL, who were referred to the dermatology research center in these cities, were evaluated based on age, clinical forms, and place of residence. Direct microscopy was employed for parasitological diagnosis and PCR from skin ulcers performed using specific kDNA primers. Data were analyzed using the SPSS software. Results: Among 93 individuals with skin ulcers suspected to CL, the results of 81 direct smears were positive. PCR bands were observed in 84 examined samples, of which 68 (81%) samples were identified for Leishmania tropica and 16 (19%) samples for L. major. 12 patients were from Bardaskan with 10 cases of L. tropica, 23 patients from Gonabad with 20 cases of L. tropica, and 49 patients from Kashmar with 38 cases of L. tropica. Most of the lesions were located on hands (37%), the most clinical feature was papule (75%), and most of patients were 21 - 30 years old. Conclusions: Previous epidemiologic studies have indicated that anthroponotic cutaneous leishmaniasis is the only dominant CL in the center and south of Khorasan. However, this study introduced new foci of zoonotic cutaneous leishmaniasis in these areas. Kashmar city was introduced as a new focus of zoonotic cutaneous leishmaniasis.

Real-time PCR using FRET technology for Old World cutaneous leishmaniasis species differentiation.

BackgroundRecently, there has been a re-emergence of cutaneous leishmaniasis in endemic countries and an increase in imported cases in non-endemic countries by travelers, workers, expatriates, immigrants, and military force personnel. Old World cutaneous leishmaniasis is caused primarily by Leishmania major, L. tropica and L. aethiopica. Despite their low sensitivity, diagnosis traditionally includes microscopic and histopathological examinations, and in vitro cultivation. Several conventional PCR techniques have been developed for species identification, which are time-consuming and labour-intensive. Real-time PCR using SYBR green dye, although provides rapid detection, may generate false positive signals. Therefore, a rapid and easy method such as a FRET-based real-time PCR would improve not only the turn-around time of diagnosing Old World cutaneous Leishmania species but will also increase its specificity and sensitivity.MethodsA FRET-based real-time PCR assay which amplifies the cathepsin L-like cysteine protease B gene encoding a major Leishmania antigen was developed to differentiate L. major, L. tropica, and L. aethiopica in one single step using one set of primers and probes. Assay performance was tested on cutaneous and visceral strains of Leishmania parasite cultures and isolates of other protozoan parasites as well as human biopsy specimen.ResultsThe assay readily differentiates between the three Old World cutaneous leishmaniasis species based on their melting curve characteristics. A single Tm at 55.2 ± 0.5 °C for L. aethiopica strains was distinguished from a single Tm at 57.4 ± 0.2 °C for L. major strains. A double curve with melting peaks at 66.6 ± 0.1 °C and 48.1 ± 0.5 °C or 55.8 ± 0.6 °C was observed for all L. tropica strains. The assay was further tested on biopsy specimens, which showed 100 % agreement with results obtained from isoenzyme electrophoresis and Sanger sequencing.ConclusionCurrently, there are no published data on real-time PCR using FRET technology to differentiate between Old World cutaneous Leishmania species. In summary, our assay based on specific hybridization addresses the limitations of previous PCR technology and provides a single step, reliable method of species identification and rapid diagnostic applications.

Isolation and identification of cutaneous leishmaniasis species by PCR-RFLP in Ilam province, the west of Iran.

Cutaneous leishmaniasis (CL) is one of the most common parasitic diseases and public health problems in Iran. CL is endemic in most parts of Ilam province, in the west of Iran. The distance from the center of country, the great number of divers rural areas, and lack of specialists and laboratory facilities have been the major causes of Leishmania species remaining unknown in this region. Polymerase chain reaction followed by restriction fragment length polymorphism was performed to identify the Leishmania species in 61 patients with cutaneous lesions. Eventually L. major was confirmed as the cause of cutaneous leishmaniasis in Ilam province, the west of Iran.

DNA barcoding for identification of sand flies (Diptera: Psychodidae) in India

About 50 species of sand flies have been reported to be prevalent in India. We explored the utility of the DNA barcode approach towards species identification of these medically important insects. A total of 62 specimens belonging to seven morphologically identified species of two genera, Phlebotomus and Sergentomyia, collected from Puducherry Union Territory, Maharashtra and Rajasthan states of India were subjected to the analysis. Neighbor-joining (NJ) analysis of DNA barcode sequences identified the individuals of seven morphological species into eight distinct species, as presented in the designed NJ tree. This methodology delineated morphologically identified species, S.bailyi, into two genetically isolated groups. Also, this study characterizes DNA barcodes of P.argentipes and P.papatasi, the vector species of leishmaniasis in India, for the first time.

Genotyping of isolated cutaneous Leishmaniasis species by polymerase chain reaction- restriction fragment length polymorphism (PCR- RFLP) analysis in endemic foci of Isfahan, Iran

Genotyping Cutaneous Leishmaniasis (CL) species is important for selecting the appropriate method to control and prevent disease, determine the health effects of drugs, prepare and evaluate the vaccine and finally to determine disease vectors and reservoirs. In order to identify Leishmania species, endemic foci of Iran center (Isfahan) was used in this study using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) method. For this purpose, 305 clinical samples were collected from patients referred to health centers in the city of Isfahan. Two samples were collected from each patient for direct slide and culture. Nucleic acid of samples was separated using deoxyribonucleic acid (DNA) extraction kit and kept in 20°C until the test time. Amplify of ITS1 area using L5.8s and LITSr primers showed 350 bp in a number of isolated cases and 450 bp in some others. Enzyme digestion of bands was compared using the enzyme HaeIII with the digested areas of reference strains. The resulted product of a number of isolates was analyzed after determining the sequence. The results showed that 193 isolated cases had 350 bp bonds and after the enzyme digestion revealed 220 and 140 bp bands that were related to the Leishmania major and 7 isolated cases with 350 bp bands that revealed bands 200 and 60 bp after enzymatic digestion that was related to Leishmania tropica and 55 isolated cased had 450 bp bond and after enzyme digestion, revealed 300 and 150 bp parts and after determining the sequence with Crithidia fasciculata and Crithidia luciliae they were matched to about 96%. It seems that by planning new studies and completing current studies, more conclusive results can be accessed and therefore it seems that control, prevention and treatment of the disease in the future may be associated with changed strategies.

Localized Leishmanial Lymphadenopathy: An Unusual Manifestation of Leishmaniasis in a Traveler in Southern Europe

A 38-year-old male patient presented with enlarged nuchal and sinistral supraclavicular lymph nodes of about 1-month history. He had no further signs or symptoms of disease and was not immunocompromised. His travel history included India, South America, and Africa but only southern Europe during more recent years. Leishmania parasites were histologically detected in a lymph node and Leishmania (Leishmania) infantum, the causative species of leishmaniasis in the European Mediterranean area, was identified by PCR and sequencing. Leishmanial infection should be included in the differential diagnosis of localized lymph node enlargement in returnees from endemic countries including southern Europe.

Leishmania spp. identification by polymerase chain reaction–restriction fragment length polymorphism analysis and its applications in French Guiana

Leishmania (Viannia) guyanensis was for many years the only species commonly identified in French Guiana, but precise species identifications were quite rare. We describe a new restriction fragment length polymorphism–polymerase chain reaction technique using a 615-bp fragment of the RNA polymerase II gene and 2 restriction enzymes, TspRI and HgaI. Seven reference strains ( Leishmania (Leishmania) amazonensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) braziliensis, L. (V.) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Leishmania) major, Leishmania (Leishmania) infantum) and 112 clinical samples from positive lesions were used for the development of the technique. The rates of positive species identification were 85.7% for punch skin biopsy specimens, 93.1% for positive Giemsa-stained smears, and 100% for positive culture supernatants. In the framework of cutaneous leishmaniasis species surveillance for the 2006 to 2008 period, parasite identification was carried out for 199 samples from different patients. The prevalence of the various Leishmania spp. was 84.4% for L. (V.) guyanensis, 8.0% for L. (V.) braziliensis, 5.0% for L. (L.) amazonensis, and 2.6% for L. (V.) lainsoni. L. (V.) braziliensis seems to be locally an emerging pathogen.

A new focus of zoonotic cutaneous leishmaniasis in Shiraz, Iran

Cutaneous leishmaniasis (CL) continues to be an increasing public health problem in Iran. Shiraz, a city in Fars Province in southwestern Iran, is one of the endemic foci of CL. However, there is no formal report of endemicity of CL in rural areas of Shiraz. To our knowledge, this is the first report of an epidemic of zoonotic CL in this area. The purpose of this study was to examine the epidemiological features of leishmaniasis in three villages in rural areas of Shiraz and to identify the causative species of leishmaniasis using nested PCR with clinical direct samples. The prevalence of infection among 1000 inhabitants of the three villages was 23.2%. Prevalences of ulcers and scars were 7 and 16.2%, respectively. The most infected age group was 0-9 years, with a rate of 14.2%. Children are more than twice as susceptible to the infection. In this study, the Leishmania major strain was identified in the majority of cases. The results of this epidemiological study indicate a high prevalence of leishmaniasis in rural areas of Shiraz.