The effect of geo-climatic determinants on the distribution of cutaneous leishmaniasis in a recently emerging focus in eastern Iran.

BACKGROUND: Cutaneous leishmaniasis (CL) has been reported in recent years in South Khorasan Province, a desert region of eastern Iran, where the main species is Leishmania tropica. Little is known of the influence of geography and climate on its distribution, and so this study was conducted to determine geo-climatic factors by using geographic information system. METHODS: The home addresses of patients with CL patients who were diagnosed and notified from 2009 to 2017 were retrieved from the provincial health center and registered on the village/town/city point layer. The effects of mean annual rainfall (MAR) and mean annual humidity (MAH), mean annual temperature (MAT), maximum annual temperature (MaxMAT), minimum annual temperature (MinMAT), mean annual number of high-velocity wind days (MAWD), mean annual frosty days (MAFD) and snowy days (MASD), elevation, soil type and land cover on CL distribution were examined. The geographical analysis was done using ArcMap software, and univariate and multivariate binary logistic regression were applied to determine the factors associated with CL. RESULTS: A total of 332 CL patients were identified: 197 (59.3%) male and 135 (40.7%) female. Their mean age was 29.3 ± 2.1 years, with age ranging from 10 months to 98 years. CL patients came from a total of 86 villages/towns/cities. By multivariate analysis, the independent factors associated with increased CL were urban setting (OR = 52.102), agricultural land cover (OR = 3.048), and MAWD (OR = 1.004). Elevation was a protective factor only in the univariate analysis (OR = 0.999). Soil type, MAH, MAT, MinMAT, MaxMAT, and MAFD did not influence CL distribution in eastern Iran. CONCLUSIONS: The major risk zones for CL in eastern Iran were urban and agricultural areas with a higher number of windy days at lower altitudes. Control strategies to reduce human vector contact should be focused in these settings.

A novel diagnostic and prognostic approach for unresponsive patients with anthroponotic cutaneous leishmaniasis using artificial neural networks.

Cutaneous leishmaniasis (CL) imposes a major health burden throughout the tropical and subtropical regions of the globe. Unresponsive cases are common phenomena occurred upon exposure to the standard drugs. Therefore, rapid detection, prognosis and classification of the disease are crucial for selecting the proper treatment modality. Using machine learning (ML) techniques, this study aimed to detect unresponsive cases of ACL, caused by Leishmania tropica, which will consequently be used for a more effective treatment modality. This study was conducted as a case-control setting. Patients were selected in a major ACL focus from both unresponsive and responsive cases. Nine unique and relevant features of patients with ACL were selected. To categorize the patients, different classifier models such as k-nearest neighbors (KNN), support vector machines (SVM), multilayer perceptron (MLP), learning vector quantization (LVQ) and multipass LVQ were applied and compared for this supervised learning task. Comparison of the receiver operating characteristic graphs (ROC) and confusion plots for the above models represented that MLP was a fairly accurate prediction model to solve this problem. The overall accuracy in terms of sensitivity, specificity and area under ROC curve (AUC) of MLP classifier were 87.8%, 90.3%, 86% and 0.88%, respectively. Moreover, the duration of the skin lesion was the most influential feature in MLP classifier, while gender was the least. The present investigation demonstrated that MLP model could be utilized for rapid detection, accurate prognosis and effective treatment of unresponsive patients with ACL. The results showed that the major feature affecting the responsiveness to treatments is the duration of the lesion. This novel approach is unique and can be beneficial in developing diagnostic, prophylactic and therapeutic measures against the disease. This attempt could be a preliminary step towards the expansion of ML application in future directions.

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.

Quantitative proteomic analysis to determine differentially expressed proteins in axenic amastigotes of Leishmania tropica and Leishmania major.

Cutaneous leishmaniasis is commonly caused by Leishmania major and Leishmania tropica. In the present study, the differential expression of proteins was identified in the amastigote-like forms of L. tropica and L. major in Iranian isolates. Initially, the samples were cultured and identified using PCR-RFLP technique. The Leishmania isolates were then grown in host-free (axenic) culture and prepared to amastigote-like forms, followed by the extraction of their proteins. To identify significant differentially expressed proteins (DEPs) of two types of Leishmania, the label-free quantitative proteomic technique was used based on sequential window acquisition of all theoretical fragment ion spectra mass spectrometry. A total of 51 up/down-DEPs (fold change >2 and p-value <.05) were identified between the axenic amastigote forms of L. major and L. tropica. Of these, 34 and 17 proteins were up-regulated in L. major and L. tropica, respectively. Several enriched GO terms were identified via biological process analyses for DEPs; furthermore, the metabolic process and translation were disclosed as top category in the up-regulated proteins of both L. major and L. tropica species. Also, the KEGG analysis revealed carbon metabolism and metabolic pathways term as the top pathways in the proteins up-regulated in L. major and L. tropica, respectively. Taken together, the numerous novel DEPs identified between the studied species could help fully understand the molecular mechanisms of pathogenesis and provide potential drug targets and vaccine candidates.

Visualization of Leishmania tropica Infection in BALB/c Mice by Bioluminescence Imaging

Background: Leishmania tropica is the cause of more than one form of leishmaniasis and lacks a known reservoir animal. This study compares the potential infectivity of recombinant and wild-type L. tropica in BALB/c mice. Methods: The potential infectivity of recombinant L. tropicaEGFP or L. tropicaEGFP-LUC by two different, the subcutaneous and intradermal, routes was compared using a range of classical detection methods and bioluminescence imaging (BLI). Results: In addition to the results obtained from classical diagnostic approaches, the BLI signals were detected in footpads and ears of L. tropica-infected animals. The BLI revealed that a bioluminescence signal can be observed at the inoculation site. The stability of the BLI remained constant in the footpad, but the signal was detectable for only three months in the pinna due to the decline in infection over time. Conclusion: The presented data are a precise verification of the assumption that BALB/c mice could be used as an experimental model for L. tropica infectivity.

Failure of an Innovative Low-Cost, Noninvasive Thermotherapy Device for Treating Cutaneous Leishmaniasis Caused by Leishmania tropica in Pakistan.

Cutaneous leishmaniasis (CL), a neglected parasitic skin disease, is endemic in Pakistan, where Leishmania tropica and Leishmania major are the causative protozoan species. Standard treatment with antimonial injections is long, painful, and costly; has toxic side effects; and is not always available in public hospitals. Small pilot studies have previously evaluated a low-cost and noninvasive hand-held exothermic crystallization thermotherapy for cutaneous leishmaniasis (HECT-CL) device. We aimed to further establish the effectiveness, safety, and feasibility of HECT-CL in L. tropica. In a prospective observational study, patients with parasitological confirmation of CL were treated using the HECT-CL heat pack for 3 minutes with an initial temperature of 52-53°C for 7 consecutive days. Dried blood spot samples were taken for species identification by polymerase chain reaction (PCR). Effectiveness was assessed by using medical photographs and measurements of the lesion size at baseline and subsequent follow-up visits, for up to 180 days. We intended to enroll 317 patients. The HECT-CL treatment was easy to apply and well tolerated. Species identification demonstrated the presence of L. tropica. Interim analysis of 56 patients showed a failure rate of 91% at follow-up (median 45 days after treatment, interquartile range 30-60 days). Enrollment of patients was prematurely suspended because of futility. This study showed a high failure rate for HECT-CL thermotherapy in this setting. Leishmania tropica is known to be less sensitive to antileishmanial drugs, more temperature-resistant, and spontaneous healing is slower than that in L. major. More research is needed to identify low-cost, effective, and more patient-friendly treatment for L. tropica.

A single-group trial of end-stage patients with anthroponotic cutaneous leishmaniasis: Levamisole in combination with Glucantime in field and laboratory models.

Currently, there is no satisfactory treatment modality available for cutaneous leishmaniasis (CL). The major objective of the present study was to explore the effect of immunomodulator-levamisole in combination with Glucantime in end-stage unresponsive patients with anthroponotic CL (ACL). Twenty end-stage unresponsive patients with ACL were identified for participation in this single-group trial study. Simultaneously, each patient was received a combination of levamisole pills along with Glucantime during the remedy course. Several in vitro complementary experiments were performed to evaluate the mode of action of levamisole and Glucantime alone and in combination using a macrophage model, in vitro MTT assay, flow cytometry and quantitative real time PCR (qPCR). Overall, 75% of the patients showed complete clinical cure, 10% partially improved and the remaining (15%) had underlying chronic diseases demonstrated no response to the treatment regimen. In in vitro studies, there was no cytotoxic effect associated with these drugs in the range of our experiments. The findings by the flow cytometric analysis represented that the highest apoptotic values corresponded to the drugs combination (32.23%) at 200 µg/ml concentration. Finally, the gene expression level of IL-12 p40, iNOS and TNF-α promoted while the level of IL-10 and TGF-ß genes reduced as anticipated. The findings clearly indicated that the combination of levamisole and Glucantime should be considered in end-stage unresponsive patients with ACL who have not responded to basic treatments. The immunomodulatory role of levamisole in mounting immune system as documented by the in vitro experiments and further substantiated by this single-group trail study was highlighted.

Leishmania tropica: suggestive evidences for the effect of infectious dose on pathogenicity and immunogenicity in an experimental model.

Leishmania (L.) tropica is a causative agent of cutaneous and occasionally visceral or viscerotropic leishmaniasis in humans. The dose of parasites influences the course and outcome of disease in some Leishmania species. The effect of parasite dose on L. tropica infection in an experimental model was studied in the current paper. High and low doses of L. tropica were used for ear infection of BALB/c mice and lesion development, parasite load, and cytokine responses were assessed. L. major infection was used for comparison. Pre-infected mice were challenged in the footpad by a fixed high dose of L. tropica, and immune response and protection level were evaluated. High dose L. tropica infection in comparison to low dose results in higher lesion diameters, higher load of parasite in draining lymph node, higher levels of interferon-γ and interleukin-10, dissemination of parasite to spleen, and induction of protection against further L. tropica challenge. Comparison of L. tropica with L. major showed that L. tropica results in lower lesion diameters, more potential for growth in lymph nodes at early phases of infection, parasite dissemination to spleen, lower levels of IL-10, and a permanent lower cytokine response against low parasite dose in comparison to high dose. Our findings suggest that for L. tropica infection, only the high dose results in visceralization of the parasite and protection against further challenge of L. tropica. Therefore, the parasite dose may be an important factor in pathogenesis and immunity in L. tropica infection.

Risk factors for the expansion of cutaneous leishmaniasis by Leishmania tropica: Possible implications for control programmes.

Cutaneous leishmaniasis (CL) caused by Leishmania tropica is emerging in new areas, initially as outbreaks and then establishing endemic foci. There is little evidence of the risk factors and effectiveness of existing control measures, what limits our ability to generalize in different epidemiological settings. The disease is described as anthroponotic; however, zoonotic outbreaks have been reported in some countries. Our aim was to identify risk factors in a recently reported endemic focus in Morocco in order to design more effective control programmes. A case-control study was conducted from September 2014 to October 2015 for epidemiological data collection from families with and without CL cases. Sandflies were captured and L. tropica infection determined. The presence of potential animal reservoirs was evaluated. 71 CL cases (44 diagnosed between 2013 and 2015) and 137 healthy people were surveyed. The average age of the new cases was 33.1 ± 22.3 years, and 69.0% were women. Phlebotomus sergenti was the most abundant species with a density of 4.27 sandflies/trap/night and differences between houses with and without CL cases were detected (p-value = 0.014). Overall, 2.7% female P. sergenti and 3.0% dogs were positive for L. tropica. Human, cat, rabbit and bird blood was detected in blood-fed P. sergenti females. 45% people used preventive measures that were not translated into a reduction in the individual risk of acquiring CL. Exposure to P. sergenti was the only risk factor found, and the reduction in its density could be achieved through the improvement of water wells management, organic fertilizers' disposal and dogs control. The lack of effectiveness of indoor residual spraying and treated nets are attributable to poor compliance and misuse of them. In addition, result optimization of the awareness campaigns on the public is possible by involving patients with CL to explain their own experience.

Molecular epidemiology of human cutaneous leishmaniasis in Jericho and its vicinity in Palestine from 1994 to 2015.

Cutaneous leishmaniases (CL) are vector-borne parasitic diseases endemic in many countries of the Middle East including Palestine. Between 1994 and 2015, 2160 clinically suspected human cases of CL from the Jericho District were examined. Stained skin tissue smears and aspirates were checked by microscopy and cultured for promastigotes, respectively. For leishmanial species identification, amplification products from a PCR-ITS1 followed by RFLP analysis using Hae III. Data were analyzed using Epi Info free-software. The overall infection rate was 41.4% (895/2160), 56.3% (504/895) of the cases were male, 43.7% (391/895) female, 60.5% (514/849) children under age 14, 41.3% (259/627) of the cases were caused by Leishmaniamajor and 57.3% (359/627) by Leishmaniatropica. The case numbers peaked in 1995, 2001, 2004, and 2012. Statistically-significant clusters of cases caused by L. major were restricted to the Jericho District; those caused by L. tropica were from the districts of Jericho, Bethlehem, Nablus and Tubas. CL is seasonal and trails the sand fly season. Distribution of cases was parabolic with fewest in July. The monthly total number of cases of CL and just those caused by L. major correlated significantly with temperature, rainfall, relative humidity, evaporation, wind speed and sunshine (P<0.05, r2=0.7-0.9 and P<0.05, r2=0.5-0.8, respectively). Cases caused by L. tropica, significantly, had a single lesion compared to cases caused by L. major (P=0.0001), which, significantly, had multiple lesions (P=0.0001). This and previous studies showed that CL is present in all Palestinian districts. The surveillance of CL has increased public awareness and molecular biological methodology for leishmanial species identification is an essential addition to classical diagnosis. The overall results are discussed, correlated to climatic and environmental changes and large-scale human activities.

Comparing acute and chronic human cutaneous leishmaniasis caused by Leishmania major and Leishmania tropica focusing on arginase activity.

BACKGROUND: Cutaneous leishmaniasis (CL) in Iran is mainly caused by Leishmania major (L. major) and L. tropica. Arginase mediated L-arginine metabolism is an important issue in Leishmania parasite propagation. Arginase activity in human CL due to L. major and L. tropica have not been studied up to now. OBJECTIVES: We aimed to compare the clinical and laboratory aspects of acute and chronic CL, focussing on arginase activity. METHODS: In this case-control study, 30 patients with acute CL (duration ≤ 1 year), 13 patients with chronic CL (duration ≥ 2 year) and 11 healthy controls were recruited. Arginase activity was measured in skin biopsies of lesions, peripheral blood polymorphonuclear cells (PMNs), peripheral blood mononuclear cells (PBMCs) and plasma by standard methods. RESULTS: The median of arginase activity in the acute lesions was higher than in chronic samples and significantly higher than in healthy controls (P = 0.008). PMNs of both acute and chronic patients showed higher levels of arginase activity as compared to the levels in PBMCs and plasma. The median of arginase activity in the PMNs of patients with chronic CL was higher than that of patients with acute CL and significantly higher than that of the healthy controls (P = 0.010). CONCLUSION: The level of arginase activity in lesions of patients with acute and chronic CL was higher than the skin of healthy controls. The highest level of arginase activity was observed in PMNs from patients with chronic CL. This suggests that the high level of arginase activity in PMNs of patients with chronic CL may contribute to the chronicity.

Comparative study of viscerotropic pathogenicity of Leishmania major amastigotes and promastigotes based on identification of mitochondrial and nucleus sequences.

Experimental viscerotropic leishmaniasis is regularly caused by Leishmania tropica promastigotes. In the current investigation, the viscerotropic pathogenicities of Leishmania major amastigotes and promastigotes were compared and evaluated based on their heterogeneity traits and number of inoculated parasites in experimental mammalian hosts. Serous exudate from 50 patients was infected, 44 with L. major and 6 with L. tropica; only BALB/c mice inoculated with 1-2 × 10(4-6) L. major amastigotes manifested cutaneous lesions at the base of their tails. Five out of the 44 BALB/c mice inoculated with L. major died of sequela of viscerotropic adverse effect, while 2 × 10(6) L. major promastigotes showed viscerotropic signs in four BALB/c mice. The sequencing of the Cyt b gene showed a strain of L. major (GenBank accession number KM393221: haplotype diversity 0.9) containing two codon mutations, 86 and 126 in dead mice, whereas no significant mutant was observed in internal transcribed spacer (ITS)-ribosomal DNA (rDNA) sequences (haplotype diversity 0). Findings show that a lower dose of L. major amastigotes than promastigotes has more potential viscerotropic intensity in susceptible hosts. It illustrates that testing Cyt b as an evolutionary mitognome marker because of having its semi-conserved structure and low copy number is able to be utilized in the discrimination of new mutants.

Cutaneous leishmaniasis in North Africa: a review.

In North African countries, cutaneous leishmaniasis transmission has been increasing since the 1980s, with a significant increase in the incidence of cases and a spread of the geographical distribution. The disease currently represents a major public health problem with a productivity gap and an impediment for development, which results in dramatic socioeconomic and psycho-sanitary impacts. The incidence is more than thousands of cases every year in Algeria, Libya, Morocco, and Tunisia. In Egypt, only a few dozen cases per year are reported, mainly in the Sinai Peninsula. Three Leishmania species, associated with distinct eco-epidemiological and clinical patterns, are involved, namely Leishmania infantum, L. major, and L. tropica. However, L. major is by far the most frequent in Algeria, Libya, and Tunisia, with more than 90% of the registered cases. It is mainly encountered in rural areas under semi-arid, arid and Saharan climates. Leishmania tropica is more prevalent in Morocco, reaching 30-40% of isolates in some districts. Much data is still missing concerning the risk factors of the infection and the lesion development, as well as vector and reservoir ecology and behavior. The knowledge of such parameters, following multidisciplinary and integrated approaches, is crucial for better management and control of the disease, that also faces a lack of resources and efficient control measures.

The route of Leishmania tropica infection determines disease outcome and protection against Leishmania major in BALB/c mice.

Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.

Genetics of host response to Leishmania tropica in mice - different control of skin pathology, chemokine reaction, and invasion into spleen and liver.

BACKGROUND: Leishmaniasis is a disease caused by protozoan parasites of genus Leishmania. The frequent involvement of Leishmania tropica in human leishmaniasis has been recognized only recently. Similarly as L. major, L. tropica causes cutaneous leishmaniasis in humans, but can also visceralize and cause systemic illness. The relationship between the host genotype and disease manifestations is poorly understood because there were no suitable animal models. METHODS: We studied susceptibility to L. tropica, using BALB/c-c-STS/A (CcS/Dem) recombinant congenic (RC) strains, which differ greatly in susceptibility to L. major. Mice were infected with L. tropica and skin lesions, cytokine and chemokine levels in serum, and parasite numbers in organs were measured. PRINCIPAL FINDINGS: Females of BALB/c and several RC strains developed skin lesions. In some strains parasites visceralized and were detected in spleen and liver. Importantly, the strain distribution pattern of symptoms caused by L. tropica was different from that observed after L. major infection. Moreover, sex differently influenced infection with L. tropica and L. major. L. major-infected males exhibited either higher or similar skin pathology as females, whereas L. tropica-infected females were more susceptible than males. The majority of L. tropica-infected strains exhibited increased levels of chemokines CCL2, CCL3 and CCL5. CcS-16 females, which developed the largest lesions, exhibited a unique systemic chemokine reaction, characterized by additional transient early peaks of CCL3 and CCL5, which were not present in CcS-16 males nor in any other strain. CONCLUSION: Comparison of L. tropica and L. major infections indicates that the strain patterns of response are species-specific, with different sex effects and largely different host susceptibility genes.

Natural infection of North African gundi (Ctenodactylus gundi) by Leishmania tropica in the focus of cutaneous leishmaniasis, Southeast Tunisia.

North African gundis (Ctenodactylus gundi) were trapped in the Leishmania (L.) tropica focus of cutaneous leishmaniasis, situated in southeast Tunisia and evaluated for Leishmania infection by real-time kinetoplast DNA polymerase chain reaction (PCR). Species identification was performed by internal transcribed spacer one (ITS1)-PCR-restriction fragment length polymorphism (RFLP) and high-resolution melting (HRM) analysis of the 7SL RNA gene. Real-time PCR on blood was positive in 6 of 13 (46.2%) tested gundis. Leishmania tropica was identified in five infected gundis and Leishmania major in one specimen. Alignments of the ITS-1 DNA sequences and 7S-HRM curves analysis indicated that similar genotypes were present in humans, a sandfly, and gundis from the same region suggesting a potential role of this rodent as reservoir host of L. tropica in southeast Tunisia.

Intraspecific variability of natural populations of Phlebotomus sergenti, the main vector of Leishmania tropica.

An intraspecific study of Phlebotomus sergenti was performed on populations from Turkey, Syria, Israel, and Uzbekistan by four different approaches: geometric morphometrics, RAPD analysis, internal transcribed spacer 2 (ITS2) sequencing (nuclear marker), and cytochrome B sequencing (mitochondrial marker). In RAPD analysis, distinct clades were formed in accordance with the geographical origin of the specimens. There was no distinct grouping according to place of origin within the Turkish samples from various localities in south-eastern Anatolia, which suggests a gene flow between populations separated spatially by the Amanos mountains, a mountain range of a considerable altitude. The results of ITS2 rDNA sequencing complied with the previously published intraspecific division of P. sergenti into two branches, northeastern and southwestern. However, mtDNA haplotypes formed three lineages with specimens from Turkey and Israel, sharing a common clade. A previously postulated hypothesis about a complex of sibling species within P. sergenti is therefore questionable. Cytochrome B seems to be a more discriminative marker for intraspecific variability assessment.

Comparative in vivo expression of amastigote up regulated Leishmania genes in three different forms of Leishmaniasis.

In Old World Leishmania infections in India, Leishmania donovani is responsible for visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) while L. tropica is responsible for cutaneous leishmaniasis (CL) in humans. The molecular differences between the two species of Leishmania and within the same species causing distinct pathologies that govern the outcome of infection and pathogenesis in the human host are unknown. Quantitative expression of selected genes was evaluated directly in lesion tissues of VL, PKDL and CL patients. Assessment of in vivo mRNA level highlighted substantial differences in gene expression patterns, providing an indication of the genes involved in pathogenesis in the three different forms of Leishmaniasis.

Soluble CD26 and CD30 levels in patients with anthroponotic cutaneous leishmaniasis.

OBJECTIVE: Leishmania tropica is the causative agent of anthroponotic cutaneous leishmaniasis (CL) in Iran. The disease often heals within a year; however, the non-healing forms of disease are also known. The aim of the present study was the determination of the levels of soluble (s) CD26 and CD30 co-stimulatory molecules in sera of L. tropica-infected individuals. The correlations of sCD26 and sCD30 levels with clinical presentation of the disease were assessed. METHODS: The levels of sCD26 and sCD30 were determined by a sandwich enzyme-linked immunosorbent assay in sera from patients with acute and non-healing presentation of disease. RESULTS: The serum level of sCD26 was significantly higher in non-healing patients than in cases with acute CL (P<0.001). There was no significant difference in sCD26 level between patients with acute CL and healthy controls. However, the levels of sCD30 in sera from all L. tropica-infected individuals were higher than controls (P<0.001). A significant difference was also found in sCD30 level between non-healing cases and patients with acute CL (P<0.001). CONCLUSION: These findings suggest sCD30 is more relevant to clinical manifestation of cutaneous leishmaniasis than sCD26. The high sCD26 and sCD30 levels in non-healing patients reflect the presence of mixed Th1- and Th2-type responses in these patients.

Leishmania major and Leishmania tropica: II. Effect of an immunomodulator, S(2) complex on the enzymes of the parasites.

S(2) complex has been reported to have a direct antileishmanial effect. The possibility that the direct antileishmanial effect may be due to inhibition of key enzymes involved in glucose metabolism and/ or enzymes associated with virulence was investigated. Cell pellets were prepared from cultures of both axenic amastigotes and promastigotes of Leishmania major (MHOM/IQ/93/MRC6) and L. tropica (MHOM/IQ/93/MRC2). S(2) complex, at various concentrations, was added to the enzyme extracts prepared from the pellets. Results show that in the Embden-Meyerhof pathway, both hexokinase and glucose-phosphate isomerase but not fructophosphokinase were dose dependently inhibited. In the hexose-monophosphate shunt both glucose-6-phosphate dehydrogenase and ribose-5-phosphate isomerase were dose dependently inhibited. Malic dehydrogenase and malic enzyme from the citric-acid cycle were both dose dependently inhibited but succinate dehydrogenase from the same pathway was not inhibited. Both enzymes associated with virulence (protease and acid phosphatase), showed activation rather than inhibition at higher doses of S(2) complex. Thus, the direct antileishmanial effect of S(2) complex may result, partially or entirely, from the inhibition of enzymes that are necessary for the parasites' carbohydrate metabolism.