APOBEC3G protects the genome of human cultured cells and mice from radiation-induced damage.

Cytosine deaminases AID/APOBEC proteins act as potent nucleic acid editors, playing important roles in innate and adaptive immunity. However, the mutagenic effects of some of these proteins compromise genomic integrity and may promote tumorigenesis. Here, we demonstrate that human APOBEC3G (A3G), in addition to its role in innate immunity, promotes repair of double-strand breaks (DSBs) in vitro and in vivo. Transgenic mice expressing A3G successfully survived lethal irradiation, whereas wild-type controls quickly succumbed to radiation syndrome. Mass spectrometric analyses identified the differential upregulation of a plethora of proteins involved in DSB repair pathways in A3G-expressing cells early following irradiation to facilitate repair. Importantly, we find that A3G not only accelerates DSB repair but also promotes deamination-dependent error-free rejoining. These findings have two implications: (a) strategies aimed at inhibiting A3G may improve the efficacy of genotoxic therapies used to cure malignant tumours; and (b) enhancing A3G activity may reduce acute radiation syndrome in individuals exposed to ionizing radiation.

Failure to Detect Leishmania in the Blood of Patients with Old-World Cutaneous Leishmaniasis: Implications for Blood Donation.

Cutaneous leishmaniasis (CL) is endemic in Israel, caused mainly by Leishmania major (L. major) and L. tropica. In addition, returning travelers import another leishmanial species such as L. braziliensis. Although we are dealing with a skin disease, the blood bank in Israel does not accept blood donations from people infected with CL in cases of multiple lesions due to the possibility of transfusion. Our purpose was to investigate the prevalence of Leishmania in the blood of patients with active or previous CL. This pilot study screened patients with active or previous CL for parasites in their blood. All patients were infected in Israel or were returning travelers with leishmaniasis acquired in Latin America. Patients were seen at the Sheba Medical Center. In addition, patients were seen at their homes in L. tropica and L. major endemic regions in Israel. Blood samples were taken from each patient for culture and polymerase chain reaction (PCR). Altogether 62 blood samples were examined (L. tropica = 26, L. major = 33, and L. braziliensis = 3). Twenty-seven patients had an active disease and 35 were recovered. All blood cultures and PCR were negative for parasites except one blood sample that was PCR positive for L. braziliensis. The findings of our study, although a small sample, suggest that people with active or recent CL caused by L. major and L. tropica, do not harbor parasites in their blood. Thus, their exclusion from blood donation should be revisited. Further studies are needed with larger sample size and highly sensitive tests.

Synthesis and Antiplasmodial Activity of Bisindolylcyclobutenediones.

Malaria is one of the most dangerous infectious diseases. Because the causative Plasmodium parasites have developed resistances against virtually all established antimalarial drugs, novel antiplasmodial agents are required. In order to target plasmodial kinases, novel N-unsubstituted bisindolylcyclobutenediones were designed as analogs to the kinase inhibitory bisindolylmaleimides. Molecular docking experiments produced favorable poses of the unsubstituted bisindolylcyclobutenedione in the ATP binding pocket of various plasmodial protein kinases. The synthesis of the title compounds was accomplished by sequential Friedel-Crafts acylation procedures. In vitro screening of the new compounds against transgenic NF54-luc P. falciparum parasites revealed a set of derivatives with submicromolar activity, of which some displayed a reasonable selectivity profile against a human cell line. Although the molecular docking studies suggested the plasmodial protein kinase PfGSK-3 as the putative biological target, the title compounds failed to inhibit the isolated enzyme in vitro. As selective submicromolar antiplasmodial agents, the N-unsubstituted bisindolylcyclobutenediones are promising starting structures in the search for antimalarial drugs, albeit for a rational development, the biological target addressed by these compounds has yet to be identified.

Structure-activity relationships in a series of antiplasmodial thieno[2,3-b]pyridines.

BACKGROUND: Malaria is one of the most prevalent tropical infectious diseases. Since recently cases of artemisinin resistance were reported, novel anti-malarial drugs are required which differ from artemisinins in structure and biological target. The plasmodial glycogen synthase kinase-3 (PfGSK-3) was suggested as a new anti-malarial drug target. 4-Phenylthieno[2,3-b]pyridines were previously identified as selective PfGSK-3 inhibitors with antiplasmodial activity. The present study aims at identifying a molecular position on this scaffold for the attachment of side chains in order to improve solubility and antiplasmodial activity. Furthermore, the role of axial chirality in the compound class for antiplasmodial activity and PfGSK-3 inhibition was investigated. METHODS: 4-Phenylthieno[2,3-b]pyridines with substituents in 4-position of the phenyl ring were docked into the ATP binding site of PfGSK-3. The compounds were synthesized employing a Thorpe reaction as final step. The enantiomers of one congener were separated by chiral HPLC. All derivatives were tested for inhibition of asexual erythrocytic stages of transgenic NF54-luc Plasmodium falciparum. Selected compounds with promising antiplasmodial activity were further evaluated for inhibition of HEK293 cells as well as inhibition of isolated PfGSK-3 and HsGSK-3. The kinetic aqueous solubility was assessed by laser nephelometry. RESULTS: The para position at the 4-phenyl ring of the title compounds was identified as a suitable point for the attachment of side chains. While alkoxy substituents in this position led to decreased antiplasmodial activity, alkylamino groups retained antiparasitic potency. The most promising of these congeners (4h) was investigated in detail. This compound is a selective PfGSK-3 inhibitor (versus the human GSK-3 orthologue), and exhibits improved antiplasmodial activity in vitro as well as better solubility in aqueous media than its unsubstituted parent structure. The derivative 4b was separated into the atropisomers, and it was shown that the (+)-enantiomer acts as eutomer. CONCLUSIONS: The attachment of alkylamino side chains leads to the improvement of antiplasmodial activity and aqueous solubility of selective PfGSK-inhibitors belonging to the class of 4-phenylthieno[2,3-b]pyridines. These molecules show axial chirality, a feature of high impact for biological activity. The findings can be exploited for the development of improved selective PfGSK-3 inhibitors.

Amphotericin B-loaded nanoparticles for local treatment of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is an infectious, parasitic disease caused by the protozoan Leishmania. Amphotericin B (AMB) is a macrolide polyene antibiotic presenting potent antifungal and antileishmanial activity, but due to poor water solubility at physiological pH, side effects, and toxicity, its therapeutic efficiency is limited. In the present study, poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with AMB were generated to reduce drug toxicity and facilitate localized delivery over a prolonged time. AMB NPs were characterized for particle size, zeta potential, polydispersity index, and degree of aggregation. In vitro assessments demonstrated its sustained activity against Leishmania major promastigotes and parasite-infected macrophages. A single intralesional administration to infected BALB/c mice revealed that AMB NPs were more effective than AMB deoxycholate in terms of reducing lesion area. Taken together, these findings suggest that AMB NPs improve AMB delivery and can be used for local treatment of CL.

West Nile Virus: Seroprevalence in Animals in Palestine and Israel.

West Nile virus (WNV) epidemiological situation in Israel and Palestine, due to their unique location, draws attention following to the global spread of West Nile fever (WNF). Although much information is available from Israel on clinical cases and prevalence of WNV, clinical cases are rarely reported in Palestine, and prevalence is not known. The objectives of this study were to determine WNV seroprevalence in various domestic animals in Palestine and to reevaluate current seroprevalence, force of infection, and risk factors for WNV exposure in horses in Israel. Sera samples were collected from 717 animals from Palestine and Israel (460 horses, 124 donkeys, 3 mules, 50 goats, 45 sheep, and 35 camels). Two hundred and ten horses were sampled twice. The level of WNV antibodies was determined using commercial Enzyme-linked Immunosorbent Assay (ELISA) Kit. Seroprevalence in equids was 73%. Seroprevalence in Israel (84.6%) was significantly higher than in Palestine (48.6%). Seroprevalence in horses (82.6%) was significantly higher than in donkeys and mules (39.3%). Multivariable statistical analysis showed that geographical area, landscape features (altitude), environmental factors (land surface temperature during the day [LSTD]), species, and age significantly influenced WNV seroprevalence. Fourteen of 95 (14.7%) sheep and goats and 14/35 camels (40%) sampled in Palestine were seropositive for WNV. Of the horses that were sampled twice, 82.8% were seropositive for WNV at the first sampling, and all remained seropositive. Three of the seronegative horses, all from Palestine, converted to positive when resampled (8.5%). The results indicate that domestic animals in Palestine were infected with WNV in the past, and the seroconversion indicates that WNV was circulating in Palestine in the summer of 2014. Control measures to prevent human infection should be implemented in Palestine. Anti WNV antibodies in domestic animals suggest that those species can be used as sentinels for WNV activity in areas where most horses are either seropositive or vaccinated.

Antiplasmodial dihetarylthioethers target the coenzyme A synthesis pathway in Plasmodium falciparum erythrocytic stages.

BACKGROUND: Malaria is a widespread infectious disease that threatens a large proportion of the population in tropical and subtropical areas. Given the emerging resistance against the current standard anti-malaria chemotherapeutics, the development of alternative drugs is urgently needed. New anti-malarials representing chemotypes unrelated to currently used drugs have an increased potential for displaying novel mechanisms of action and thus exhibit low risk of cross-resistance against established drugs. RESULTS: Phenotypic screening of a small library (32 kinase-inhibitor analogs) against Plasmodium falciparum NF54-luc asexual erythrocytic stage parasites identified a diarylthioether structurally unrelated to registered drugs. Hit expansion led to a series in which the most potent congener displayed nanomolar antiparasitic activity (IC50 = 39 nM, 3D7 strain). Structure-activity relationship analysis revealed a thieno[2,3-d]pyrimidine on one side of the thioether linkage as a prerequisite for antiplasmodial activity. Within the series, the oxazole derivative KuWei173 showed high potency (IC50 = 75 nM; 3D7 strain), good solubility in aqueous solvents (1.33 mM), and >100-fold selectivity toward human cell lines. Rescue experiments identified inhibition of the plasmodial coenzyme A synthesis as a possible mode of action for this compound class. CONCLUSIONS: The class of antiplasmodial bishetarylthioethers reported here has been shown to interfere with plasmodial coenzyme A synthesis, a mechanism of action not yet exploited for registered anti-malarial drugs. The oxazole congener KuWei173 displays double-digit nanomolar antiplasmodial activity, selectivity against human cell lines, high drug likeness, and thus represents a promising chemical starting point for further drug development.

Epidemiological analysis of Leishmania tropica strains and giemsa-stained smears from Syrian and Turkish leishmaniasis patients using multilocus microsatellite typing (MLMT).

Turkey is located in an important geographical location, in terms of the epidemiology of vector-borne diseases, linking Asia and Europe. Cutaneous leishmaniasis (CL) is one of the endemic diseases in a Turkey and according to the Ministry Health of Turkey, 45% of CL patients originate from Sanliurfa province located in southeastern Turkey. Herein, the epidemiological status of CL, caused by L. tropica, in Turkey was examined using multilocus microsatellite typing (MLMT) of strains obtained from Turkish and Syrian patients. A total of 38 cryopreserved strains and 20 Giemsa-stained smears were included in the present study. MLMT was performed using 12 highly specific microsatellite markers. Delta K (ΔK) calculation and Bayesian statistics were used to determine the population structure. Three main populations (POP A, B and C) were identified and further examination revealed the presence of three subpopulations for POP B and C. Combined analysis was performed using the data of previously typed L. tropica strains and Mediterranean and Sanliurfa populations were identified. This finding suggests that the epidemiological status of L. tropica is more complicated than expected when compared to previous studies. A new population, comprised of Syrian L. tropica samples, was reported for the first time in Turkey, and the data presented here will provide new epidemiological information for further studies.

Controlled release of artemisone for the treatment of experimental cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is a leading cause of malarial mortality resulting from infection by Plasmodium falciparum. Treatment commonly involves adjunctive care and injections or transfusion of artemisinins. All artemisinins that are in current use are metabolized to dihydroxyartemisinin (DHA), to which there is already some parasite resistance. We used artemisone, a derivative that does not convert to DHA, has improved pharmacokinetics and anti-plasmodial activity and is also anti-inflammatory (an advantage given the immunopathological nature of CM). METHODS: We examined controlled artemisone release from biodegradable polymers in a mouse CM model. This would improve treatment by exposing the parasites for a longer period to a non-toxic drug concentration, high enough to eliminate the pathogen and prevent CM. The preparations were inserted into mice as prophylaxis, early or late treatment in the disease course. RESULTS: The most efficient formulation was a rigid polymer, containing 80 mg/kg artemisone, which cured all of the mice when used as early treatment and 60% of the mice when used as a very late treatment (at which stage all control mice would die of CM within 24 h). In those mice that were not completely cured, relapse followed a latent period of more than seven days. Prophylactic treatment four days prior to the infection prevented CM. We also measured the amount of artemisone released from the rigid polymers using a bioassay with cultured P. falciparum. Significant amounts of artemisone were released throughout at least ten days, in line with the in vivo prophylactic results. CONCLUSIONS: Overall, we demonstrate, as a proof-of-concept, a controlled-sustained release system of artemisone for treatment of CM. Mice were cured or if treated at a very late stage of the disease, depicted a delay of a week before death. This delay would enable a considerable time window for exact diagnosis and appropriate additional treatment. Identical methods could be used for other parasites that are sensitive to artemisinins (e.g. Toxoplasma gondii and Neospora caninum).

Novel 2-Phenoxyanilide Congeners Derived from a Hit Structure of the TCAMS: Synthesis and Evaluation of Their in Vitro Activity against Plasmodium falciparum.

The Tres Cantos Antimalarial Compound Set (TCAMS) is a publicly available compound library which contains 13533 hit structures with confirmed activity against Plasmodium falciparum, the infective agent responsible for malaria tropica. The TCAMS provides a variety of starting points for the investigation of new antiplasmodial drug leads. One of the promising compounds is TCMDC-137332, which seemed to be a good starting point due to its antiplasmodial potency and its predicted physicochemical properties. Several new analogues based on a 2-phenoxyanilide scaffold were synthesized by standard amide coupling reactions and were fully characterized regarding their identity and purity by spectroscopic and chromatographic methods. Furthermore, the results of the biological evaluation of all congeners against Plasmodium falciparum NF54 strains are presented. The findings of our in vitro screening could not confirm the presumed nanomolar antiplasmodial activity of TCMDC-137332 and its derivatives.

Use of peptide nucleic acids to manipulate gene expression in the malaria parasite Plasmodium falciparum.

One of the major concerns in treating malaria by conventional small drug molecules is the rapid emergence of drug resistance. Specific silencing of essential genes by antisense oliogomers has been proposed as an alternative approach that may result in antimalarial activity which is not associated with drug resistance. In addition, such an approach could be an important biological tool for studying many genes' function by reverse genetics. Here we present a novel methodology of using peptide nucleic acids (PNAs) as a useful tool for gene silencing in Plasmodium falciparum. PNAs, designed as specific antisense molecules, were conjugated to a cell penetrating peptide (CPP); namely, octa-D-lysine via the C-terminus, to allow facile delivery through cell membranes. PNAs added to P. falciparum cultures were found exclusively in infected erythrocytes and were eventually localized in nuclei of the parasites at all stages of intra erythrocytic development. We show that these PNAs specifically down regulated both a stably expressed transgene as well as an endogenous essential gene, which significantly reduced parasites' viability. This study paves the way for a simple approach to silence a variety of P. falciparum genes as means of deciphering their function and potentially to develop highly specific and potent antimalarial agents.

Molecular detection of Rickettsia africae, Rickettsia aeschlimannii, and Rickettsia sibirica mongolitimonae in camels and Hyalomma spp. ticks from Israel.

In this study, we aimed to identify and genetically characterize spotted fever group (SFG) rickettsiae in ticks, domestic one-humped camels, and horses from farms and Bedouin communities in southern Israel. A total of 618 ixodid ticks (Hyalomma dromedarii, Hyalomma turanicum, Hyalomma excavatum, and Hyalomma impeltatum) collected from camels and horses, as well as 152 blood samples from 148 camels and four horses were included in the study. Initial screening for rickettsiae was carried out by targeting the gltA gene. Positive samples were further analyzed for rickettsial ompA, 17kDa, ompB, and 16S rRNA genes. Rickettsia aeschlimannii DNA was detected in the blood of three camels and 14 ticks (H. dromedarii, H. turanicum, and H. excavatum). Rickettsia africae was found in six ticks (H. turanicum, H. impeltatum, H. dromedarii, and H. excavatum). In addition, Rickettsia sibirica mongolitimonae was detected in one H. turanicum tick. These findings represent the first autochthonous detection of R. africae in Israel. Previous detections of R. africae in Asia were reported from the Sinai Peninsula (Egypt) and Istanbul, only. Furthermore, we report for the first time the finding of R. aeschlimannii in H. turanicum and H. excavatum ticks, as well as the first identification of R. sibirica mongolitimonae in H. turanicum ticks. The tick species identified to harbor R. africae and other SFG rickettsiae have been reported to occasionally feed on people, and, therefore, physicians should be aware of the possible exposure of local communities and travelers, especially those in contact with camels, to these tick-borne rickettsial pathogens.

PfSec13 is an unusual chromatin-associated nucleoporin of Plasmodium falciparum that is essential for parasite proliferation in human erythrocytes.

In Plasmodium falciparum, the deadliest form of human malaria, the nuclear periphery has drawn much attention due to its role as a sub-nuclear compartment involved in virulence gene expression. Recent data have implicated components of the nuclear envelope in regulating gene expression in several eukaryotes. Special attention has been given to nucleoporins that compose the nuclear pore complex (NPC). However, very little is known about components of the nuclear envelope in Plasmodium parasites. Here we characterize PfSec13, an unusual nucleoporin of P. falciparum, which shows unique structural similarities suggesting that it is a fusion between Sec13 and Nup145C of yeast. Using super resolution fluorescence microscopy (3D-SIM) and in vivo imaging, we show that the dynamic localization of PfSec13 during parasites' intra-erythrocytic development corresponds with that of the NPCs and that these dynamics are associated with microtubules rather than with F-actin. In addition, PfSec13 does not co-localize with the heterochormatin markers HP1 and H3K9me3, suggesting euchromatic location of the NPCs. The proteins associated with PfSec13 indicate that this unusual Nup is involved in several cellular processes. Indeed, ultrastructural and chromatin immunoprecipitation analyses revealed that, in addition to the NPCs, PfSec13 is found in the nucleoplasm where it is associated with chromatin. Finally, we used peptide nucleic acids (PNA) to downregulate PfSec13 and show that it is essential for parasite proliferation in human erythrocytes.

Prevalence of Trypanosoma evansi in horses in Israel evaluated by serology and reverse dot blot.

Trypanosoma evansi is the cause of surra in horses, camels and other domestic animals. Following the first outbreak of surra in horses and camels in Israel in 2006, a survey of the prevalence of the parasite in the Israeli horse population was conducted using serology, PCR followed by the reverse dot blot (RDB) technique and blood smear microscopy. In total, 614 horses from 7 regions were sampled. The CATT/T. evansi kit was used for serology for all the horses. Horses from the Arava and Dead Sea region, where the first outbreak occurred, were sampled again one year later and both samples were subjected to serology and the RDB technique. The country wide seroprevalence was 4.6% (28/614). The seroprevalence in the Arava and Dead Sea region was 6.5% (9/139) in the first sampling compared with 4.1% (5/122) in the second, whereas the prevalence of RDB-positivity was 18.7% (26/139) in the first sampling and only 0.8% (1/122) in the second. All horses were asymptomatic except for one horse from the Arava and Dead Sea region that demonstrated clinical signs of surra combined with positive serology and RDB. The results of this study indicated that surra is prevalent in most regions of the country and thus should be considered an important differential diagnosis in horses and other domestic animals in Israel with chronic weight loss, edema or neurological signs.

Leishmania tropica in northern Israel: a clinical overview of an emerging focus.

BACKGROUND: In Israel, most cutaneous leishmaniasis (CL) is caused by Leishmania major. Recently a new focus of CL caused by Leishmania tropica has been described in Tiberias and the surrounding area of northern Israel. OBJECTIVE: The aim of this study was to evaluate clinical (size, number, location, and type of lesion) and laboratory (culture and polymerase chain reaction [PCR] analysis) parameters at diagnosis, response to treatment, and outcome of patients with CL due to L tropica. METHODS: Between September 2002 and March 2004, patients with direct smear-confirmed CL were evaluated; clinical records were reviewed and a telephone survey was performed. RESULTS: Forty nine patients, 34 (69%) male and 15 (31%) female, were studied. Mean age was 31.1 years (median 26 years, range 1-70); 76% of patients live in Tiberias and the surrounding area. The mean number of lesions was 2.6 (median 2, range 1-10). Lesions were commonly located on the face (61%) and upper limbs (57%). PCR analysis was performed in 27 patients and was positive for L tropica in 26. Fifty percent of patients studied received multiple therapeutic regimens because of incomplete response or treatment failure. Topical paromomycin was used in 44 patients (90%), with a complete response reported in only 17 (39%); of the 9 patients treated with intralesional sodium stibogluconate, a complete response was reported in 6 (67%); of the 5 patients treated with intravenous sodium stibogluconate, 4 (80%) were cured. LIMITATIONS: The relatively small number of patients studied combined with the fact that some were assessed retrospectively limit our conclusions. In addition, 50% of the patients studied received multiple therapeutic regimens because of failure of, or incomplete responses to, their initial therapy, thereby making comparisons difficult. CONCLUSIONS: The cure rate in those completing a course of antimony therapy, either 10 or more days of intravenous therapy or therapy administered intralesionally, was 75% (95% confidence interval [CI], 50.5-99.5%) as compared with 45% (95% CI, 28.9-60.5%) among those completing at least 10 days of topical paromomycin. To date, no standardized, simple, safe, and highly effective regimen for treating L tropica exists. Large, controlled clinical trials to evaluate current treatment regimens as well as new medications for CL, and especially CL attributed to L tropica, are urgently needed.

The effect of amphotericin b derivatives on Leishmania and immune functions.

The effects of a water-soluble amphotericin B (AmB)-arabinogalactan (AG) conjugate on several immune functions were investigated. The experiments measured the effects of AmB-AG on (1) release of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO), and interferon-gamma (IFN-gamma) from phagocytic cells and (2) cell-mediated immune responses. AmB-AG increased TNF-alpha release from mouse peritoneal macrophages and human monocytes but had no effect on IFN-gamma and NO release. A commercial preparation of nonconjugated AmB (Fungizone) also increased TNF-alpha production, but to a lesser extent than AmB-AG. AG alone had no effect on TNF-alpha production, proving that AmB caused the increased TNF-alpha production. AmB-AG and Fungizone were also tested for their effect on B- and T-cell proliferation. Neither compound altered T-lymphocyte responses to concanavalin A, but both inhibited the stimulation of B lymphocytes by lipopolysaccharides. However, Fungizone showed a stronger inhibitory effect on B cells. Allocytotoxicity was also inhibited by AmB-AG and more strongly by Fungizone. The increased production of TNF-alpha by cells treated with AmB-AG and the lower inhibitory effect of AmB-AG on lymphocyte stimulation and allocytotoxicity, as compared with Fungizone, explain the better therapeutic efficacy of the AmB-polysaccharide conjugate. AmB is active because of its preferential binding to ergosterol rather than cholesterol, the former sterol preferentially present in parasite surface membranes. This is also valid for the axenic amastigotes, which were sensitive to the AmB-AG. Overall, our results suggest that the antileishmanial activity of AmB-AG is mediated both directly and via modulation of immune functions.

Identification and differentiation of Leishmania species in clinical samples by PCR amplification of the miniexon sequence and subsequent restriction fragment length polymorphism analysis.

We recently developed a new PCR-restriction fragment length polymorphism (RFLP)-based assay using the miniexon sequence from the genus Leishmania. Here we report the application of this new genotyping method to naturally infected clinical samples for the differentiation of New and Old World Leishmania species. Of the newly developed assay and four currently applied diagnostic tests (i.e., in vitro cultivation, serology, and two other molecular assays using either the small subunit-internal transcribed spacer sequence or a repetitive genomic sequence), the miniexon assay showed the highest sensitivity, 89.7%, compared to 70.6, 57.1, 51.7, and 79.3%, respectively. Species differentiation was robust and reliable compared with that by two other Leishmania genotyping techniques. The assay provides a valuable tool for the identification of Leishmania directly from clinical samples and enables determination of the infecting species by a facile technique with high discrimination power. Since Leishmania causes a broad spectrum of diseases distinguished by different parasite and host factors, detection and characterization of the infecting species is crucial for the confirmation of a diagnosis as well as the establishment of the clinical prognosis and the initiation of an adequate therapeutic approach. The miniexon PCR-RFLP assay will facilitate such determination and might improve diagnosis and treatment of leishmaniasis.