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Contribution of the Leishmania P-glycoprotein-related gene ltpgpA to oxyanion resistance.

Oxyanions in the form of pentavalent antimony compounds are currently the drug of choice for treating leishmaniasis. Leishmania mutants resistant to high concentrations of the oxyanion arsenite were obtained in a stepwise selection procedure. Amplification of the H locus P-glycoprotein-related gene ltpgpA, as part of extrachromosomal circles, is a frequent event in arsenite-resistant cells, but was observed only in cells resistant to high concentrations of the metalloid salts. Revertants grown in the absence of the drug lost their ltpgpA-containing amplicon and part of their resistance. The results of previous transfection experiments in Leishmania had suggested that ltpgpA is only involved in low level resistance to arsenite and antimonite. The results of this study using transfection of ltpgpA alleles isolated from arsenite-resistant mutants or of whole circular amplicons containing ltpgpA demonstrate clearly that this P-glycoprotein-related gene is involved in low level resistance to oxyanions, including the pentavalent antimony-containing drug Pentostam. By site-directed mutagenesis, the LtpgpA protein was shown to require an intact nucleotide-binding site to confer arsenite resistance. Under the experimental conditions used, decreased accumulation of the 73AsO2- drug was not observed in the ltpgpA transfectants. One possibility is that LtpgpA-mediated arsenite resistance could result from sequestration of the toxic anion in an intracellular compartment. The results indicate that despite the fact that ltpgpA amplification is a frequent event in oxyanion-resistant mutants, it contributes only slightly to the overall resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Double blind, randomized controlled trial, to evaluate the effectiveness of a controlled nitric oxide releasing patch versus meglumine antimoniate in the treatment of cutaneous leishmaniasis [NCT00317629].

BACKGROUND: Cutaneous Leishmaniasis is a worldwide disease, endemic in 88 countries, that has shown an increasing incidence over the last two decades. So far, pentavalent antimony compounds have been considered the treatment of choice, with a percentage of cure of about 85%. However, the high efficacy of these drugs is counteracted by their many disadvantages and adverse events. Previous studies have shown nitric oxide to be a potential alternative treatment when administered topically with no serious adverse events. However, due to the unstable nitric oxide release, the topical donors needed to be applied frequently, making the adherence to the treatment difficult. The electrospinning technique has allowed the production of a multilayer transdermal patch that produces a continuous and stable nitric oxide release. The main objective of this study is to evaluate this novel nitric oxide topical donor for the treatment of cutaneous leishmaniasis. METHODS AND DESIGN: A double-blind, randomized, double-masked, placebo-controlled clinical trial, including 620 patients from endemic areas for Leishmaniasis in Colombia was designed to investigate whether this patch is as effective as meglumine antimoniate for the treatment of cutaneous leishmaniasis but with less adverse events. Subjects with ulcers characteristic of cutaneous leishmaniasis will be medically evaluated and laboratory tests and parasitological confirmation performed. After checking the inclusion/exclusion criteria, the patients will be randomly assigned to one of two groups. During 20 days Group 1 will receive simultaneously meglumine antimoniate and placebo of nitric oxide patches while Group 2 will receive placebo of meglumine antimoniate and active nitric oxide patches. During the treatment visits, the medications will be daily administered and the presence of adverse events assessed. During the follow-up, the research group will visit the patients at days 21, 45, 90 and 180. The healing process of the ulcer, the health of the participants, recidivisms and/or reinfection will also be assessed. The evolution of the ulcers will be photographically registered. In case that the effectiveness of the patches is demonstrated, a novel and safe therapeutic alternative for one of the most important public health problems in many countries will be available to patients.

Journal Article↗

Role of the ABC transporter MRPA (PGPA) in antimony resistance in Leishmania infantum axenic and intracellular amastigotes.

Antimonial compounds are the mainstay for the treatment of infections with the protozoan parasite Leishmania. We present our studies on Leishmania infantum amastigote parasites selected for resistance to potassium antimonyl tartrate [Sb(III)]. Inside macrophages, the Sb(III)-selected cells are cross-resistant to sodium stibogluconate (Pentostam), the main drug used against Leishmania. Putative alterations in the level of expression of more than 40 genes were compared between susceptible and resistant axenic amastigotes using customized DNA microarrays. The expression of three genes coding for the ABC transporter MRPA (PGPA), S-adenosylhomocysteine hydrolase, and folylpolyglutamate synthase was found to be consistently increased. The levels of cysteine were found to be increased in the mutant. Transfection of the MRPA gene was shown to confer sodium stibogluconate resistance in intracellular parasites. This MRPA-mediated resistance could be reverted by using the glutathione biosynthesis-specific inhibitor buthionine sulfoximine. These results highlight for the first time the role of MRPA in antimony resistance in the amastigote stage of the parasite and suggest a strategy for reversing resistance.

ATP-Binding Cassette Transporters↗

[American cutaneous leishmaniasis: special features in diagnosis and therapy].

HISTORY AND CLINICAL FINDINGS: Three weeks after returning from a trip to Guatemala, a 33-year-old man developed two ulcers with indurated edges on his right leg and painful lymph nodes in the right groin. His general condition was not impaired. EXAMINATIONS: Histological examination revealed cellular infiltrates of the corium by lymphocytes and plasma cells, always accompanied by epithelial cells and multinuclear giant cells. Special stainings were unable to detect pathogens but Leishmania brasiliensis was identified using PCR. The Leishmania culture remained negative. THERAPY AND COURSE: After 7-day intravenous therapy with 20 mg/kg/d pentostam (pentavalent antimonial compound), the patient developed gastrointestinal complaints, coupled with a marked elevation of transaminases. Therapy was discontinued until the transaminase values normalized, then continued in reduced dosage (12 mg/kg body weight) for 23 days. The ulcers and lymphadenitis healed under this therapy. CONCLUSION: The diagnosis of American cutaneous Leishmaniasis may be complicated by the relative lack of pathogens in the lesions. PCR diagnosis are very helpful here. The therapy must be systemic owing to the danger of progression to mucocutaneous Leishmaniasis. The standard therapeutic pentostam has, however, a high rate of side effects and administration is exclusively intravenous.

Adult↗

HIV-1 replication is stimulated by sodium stibogluconate, the therapeutic mainstay in the treatment of leishmaniasis.

Leishmaniasis is an important opportunistic disease among patients infected with human immunodeficiency virus (HIV)-1. The pentavalent antimony compound sodium stibogluconate is a drug of choice for the treatment of leishmaniasis. Because sodium stibogluconate acts as an inhibitor of phosphotyrosyl phosphatases and such inhibitors can promote HIV-1 replication, we tested the effect of this compound on virus gene expression. Using pseudotyped reporter viruses and fully infectious laboratory-adapted and clinical strains of HIV-1, we report that sodium stibogluconate induces an increase in HIV-1 transcription and virus replication in primary CD4(+) T cells and in thymic histocultures. This activation is a slow process and appears to involve the transcription factors nuclear factor- kappa B and activator protein 1, as well as the Syk, Jun, and mitogen-activated protein kinase/extracellular signal-related kinase signal-transduction pathways. In addition, the effect seems to be partly mediated by a soluble factor. Altogether, these findings might reveal clinical implications for the treatment of leishmaniasis in HIV-1-infected patients.

Antimony Sodium Gluconate↗

Multidentate aryloxide and oxo-aryloxide complexes of antimony: synthesis and structural characterization of [eta4-N(o-C6H4O)3]Sb(OSMe2), {{[eta3-N(o-C6H4OH)- (o-C6H4O)2]Sb}2(mu2-O)}2 and {[eta3-PhN(o-C6H4O)2]Sb}4(mu3-O)2.

Antimony compounds that feature multidentate aryloxide ligands, namely [eta4-N(o-C6H4O)3]Sb(OSMe2), {{[eta3-N(o-C6H4OH)(o-C6H4O)2]Sb}2(mu2-O)}2, and {[eta3-PhN(o-C6H4O)2]Sb}4(mu3-O)2 have been synthesized from N(o-C6H4OH)3 and PhN(o-C6H4OH)2 and structurally characterized by X-ray diffraction. While [eta4-N(o-C6H4O)3]Sb(OSMe2) exists as a discrete mononuclear species, the oxo complexes {{[eta3-N(o-C6H4OH)(o-C6H4O)2]Sb}2(mu2-O)}2 and {[eta3-PhN(o-C6H4O)2]Sb}4(micro3-O)2 are multinuclear. Specifically, the dinuclear fragment {[eta3-N(o-C6H4OH)(o-C6H4O)2]Sb}2(mu2-O)} exists in a dimeric form due to the bridging oxo ligand participating in an intermolecular hydrogen bonding interaction, while the dinuclear fragment {[eta3-PhN(o-C6H4O)2]Sb}2(mu-O) exists in a dimeric form due to the bridging oxo ligand serving as a donor to the antimony of a second fragment. The structures of {{[eta3-N(o-C6H4OH)(o-C6H4O)2]Sb}2(mu2-O)}2 and {[eta3-PhN(o-C6H4O)2]Sb}4(mu3-O)(2), therefore, indicate that an oxo ligand bridging two Sb(III) centers is sufficiently electron rich to serve as both an effective hydrogen bond acceptor and as a ligand for an additional Sb(III) center.

Journal Article↗

Sodium stibogluconate (Pentostam) overdose during treatment of American cutaneous leishmaniasis.

A 27-year-old woman who acquired cutaneous leishmaniasis in Central America was inadvertently treated with 10 times the intended daily dose of the pentavalent antimonial compound sodium stibogluconate (Pentostam): 8500 mg (143 mg/kg) instead of 850 mg. The patient felt "wiped out" during the 4-h infusion of the drug. After the mistake in dosing was discovered, she was vigorously hydrated and carefully monitored in an intensive care unit for greater than 48 h. Her vital signs were stable, and no arrhythmias were noted. Her alanine aminotransferase level rose briefly to 2.4 times the upper limit of normal, and her white blood cell count briefly fell 43% to a low of 3700/microliter. Her skin lesions subsequently healed without further therapy. Although sodium stibogluconate has been associated with a variety of side effects, in this case, a single high dose of the drug was tolerated without serious toxicity.

Adult↗

Interaction of the catalytic and the membrane subunits of an oxyanion-translocating ATPase.

Resistance to arsenical and antimonial compounds in Escherichia coli is due to active extrusion of these compounds from cells expressing the ars operon. The arsenical pump is an ion-translocating ATPase which consists of two polypeptide components, the ArsA and ArsB proteins. The ArsB protein, the inner membrane component of the pump, has been shown to function as the membrane anchor for the catalytic subunit, the ArsA protein. The properties and nature of interaction between these two components of the pump were investigated using an in vitro binding assay. Purified ArsA protein bound to the membrane in a saturable manner. In the absence of arsenite or antimonite an apparent positive cooperativity in the binding of the ArsA protein to membrane vesicles containing the ArsB protein was observed. In the presence of arsenite or antimonite binding became hyperbolic, with a 10-fold decrease in the concentration of ArsA protein required for half-maximal binding, without any change in the stoichiometry of the complex. Addition of ATP had little affect on membrane binding of the ArsA ATPase subunit. In the presence or absence of the anionic substrates binding was maximal in a pH range 7.5-8.5.

Adenosine Triphosphatases↗

Binding of ArsR, the repressor of the Staphylococcus xylosus (pSX267) arsenic resistance operon to a sequence with dyad symmetry within the ars promoter.

arsR, the first gene of the Staphylococcus xylosus (pSX267) arsenic/antimonite resistance (ars) operon encodes a negative regulatory protein, ArsR, which mediates inducibility of the resistances by arsenic and antimony compounds. ArsR, which has no obvious DNA-binding motif in its primary structure, was purified from an ArsR-overproducing Escherichia coli strain and identified as a DNA-binding protein by its behaviour in gel mobility shift assays. ArsR had a specific affinity for a 312 bp DNA restriction fragment carrying the ars promoter; the minimum sequence complexed by ArsR was a 75 bp polymerase chain reaction (PCR) fragment, which mainly comprised the -35 and -10 regions of the promoter. The effect of inducers on the DNA-binding activity of ArsR was examined by in vitro induction assays; only arsenite inhibited DNA-binding of the repressor. DNase I footprinting revealed two protected regions within the promoter region, spanning 23 and 9 nucleotides, respectively. Furthermore, a new cleavage site for DNase I between the protected regions was made accessible by binding of the repressor. The footprints cover a region of three inverted repeats located between the -35 and -10 motifs of the ars promoter. By high resolution footprinting with the hydroxy radical, five sites of close contact between the protein and DNA were identified.

Antimony↗

Aminosidine and its combination with sodium stibogluconate in the treatment of diffuse cutaneous leishmaniasis caused by Leishmania aethiopica.

Treatment of diffuse cutaneous leishmaniasis (DCL) caused by Leishmania aethiopica remains unsatisfactory as the parasite is relatively insensitive to antimonial compounds. Reports of the clinical effectiveness of aminosidine sulphate, especially in combination with sodium stibogluconate, in visceral leishmaniasis and the finding that this antibiotic is potent against L. aethiopica in vitro, prompted us to evaluate its usefulness in DCL. Two patients with long-standing, active DCL were treated for 60 d with aminosidine sulphate, 14 mg/kg/d parenterally. The skin lesions resolved completely in both patients although they relapsed subsequently. Synergism between aminosidine and stibogluconate was demonstrated in vitro against parasites isolated from the patients. This led us to administer combined therapy, aminosidine sulphate 14 mg/kg/d and sodium stibogluconate 10 mg/kg/d, to the 2 patients in relapse and to another, third patient. Treatment was continued for 2 months beyond parasitological cure. Side effects were minimal. Following treatment, a return of specific cell-mediated immunity occurred, as expressed by a moderate infiltration of lymphocytes into the lesions and by lymphocyte proliferation in vitro in the presence of live Leishmania antigen, with synthesis of interleukin-2 and interferon gamma with one patient and interleukin 4 with the other. During follow-up periods of 2 to 21 months after treatment, no sign of relapse was seen.

Animals↗

Epidemic visceral leishmaniasis in southern Sudan: treatment of severely debilitated patients under wartime conditions and with limited resources.

OBJECTIVES: 1) To determine the proportions of patients with visceral leishmaniasis who had various treatment outcomes when cared for under wartime conditions and with limited resources and 2) to identify patient characteristics associated with the outcomes. DESIGN: Cohort study. SETTING: Médecins sans Frontières-Holland's treatment center in Duar, Western Upper Nile Province, an area in southern Sudan that has been severely affected by Sudan's civil war and a massive epidemic of visceral leishmaniasis. PATIENTS: 3076 consecutive patients who had visceral leishmaniasis, were admitted to the treatment center the first year the center was operational (August 1990 to July 1991), and were treated with the pentavalent antimonial compound sodium stibogluconate. MEASUREMENTS: Patient characteristics on admission and four mutually exclusive treatment outcomes (default during first admission, death during first admission, discharge and readmission for retreatment [relapse], and discharge and no readmission for retreatment [successful treatment]). RESULTS: The patients had a median age of 15 years and were notably anemic (median hemoglobin level, 77g/L) and malnourished (median body mass index of adults [> or = 18 years of age], 15.2 kg/m2); most (91.0%) had been sick less than 5 months. Although patients could not be monitored after treatment to document cure, most (2562 [83.3%]) were successfully treated; 336 (10.9%) died during their first admission, and 79 are known to have relapsed (3.0% of those discharged alive [that is, those whose final treatment outcome was successful treatment or relapse]). In univariable analysis, young and older age (<5 or > or = 45 years of age), long duration of illness (> or = 5 months), markedly low hemoglobin level or body mass index, large spleen, high parasite density, and vomiting at least once during the treatment course were associated with death. In multiple logistic regression analysis of data for a subgroup of 1207 adults (those who did not default or relapse and for whom data were recorded on age, sex, duration of illness, hemoglobin level, body mass index, and spleen size), the approximate risk ratios for death were 2.2 (95% Cl, 1.4 to 3.6) for those with a long duration of illness, 3.6 (Cl, 2.1 to 5.9) for those 45 years of age or older, 4.6 (Cl, 2.2 to 9.4) for those with a hemoglobin level less than 60 g/L, and 12.2 (Cl, 3.2 to 47.2) for those with a body mass index less than 12.2 kg/m2. CONCLUSION; Despite the severe debility of the patients and the exceptionally difficult circumstances under which they were treated, most fared remarkably well.

Adolescent↗

Drug resistance in Indian visceral leishmaniasis.

Throughout the world, pentavalent antimonial compounds (Sb(v)) have been the mainstay of antileishmanial therapy for more than 50 years. Sb(v) has been highly effective in the treatment of Indian visceral leishmaniasis (VL: kala-azar) at a low dose (10 mg/kg) for short durations (6-10 days). But in the early 1980s reports of its ineffectiveness emerged, and the dose of Sb(v) was eventually raised to 20 mg/kg for 30-40 days. This regimen cures most patients with VL except in India, where the proportion of patients unresponsive to Sb(v) has steadily increased. In hyperendemic districts of north Bihar, 50-65% patients fail treatment with Sb(v). Important reasons are rampant use of subtherapeutic doses, incomplete duration of treatment and substandard drugs. In vitro experiments have established emergence of Sb(v) resistant strains of Leishmania donovani, as isolates from unresponsive patients require 3-5 times more Sb(v) to reach similarly effectiveness against the parasite as in Sb(v) responders. Anthroponotic transmission in India has been an important factor in rapid increase in the Sb(v) refractoriness. Pentamidine was the first drug to be used and cured 99% of these refractory patients, but over time even with double the amount of initial doses, it cures only 69-78% patients now and its use has largely been abandoned in India. Despite several disadvantages, amphotericin B is the only drug available for use in these areas and should be used as first-line drug instead of Sb(v). The new oral antileishmanial drug miltefosine is likely to be the first-line drug in future. Unfortunately, development of newer antileishmanial drugs is rare; two promising drugs, aminosidine and sitamaquine, may be developed for use in the treatment of VL. Lipid associated amphotericin B has an excellent safety and efficacy profile, but remains out of reach for most patients because of its high cost.

Aminoquinolines↗

Chemotherapeutic approaches to protozoa: kinetoplastida--current level of knowledge and outlook.

The possibilities for treating haemoflagellate infections (African trypanosomiasis) are very limited (Table 1; Mehlhorn and Schrevel 1995; Croft 1997; Hunter 1997; Wang 1997; Trouiller and Olliaro 1998). All the available drugs have severe side-effects in humans and animals. Vaccination is not really an option, in view of the wide antigen variability. At present, there are several drug combinations in clinical trials: suramin/eflornithine, suramin/metronidazole, suramin/pentamidine, melarsoprol/pentamidine, melarsoprol/nifurtimox and nifurtimox/eflornithine. Some of these combinations were successful in treating resistant Trypanosoma brucei rhodesiense and/or T. b. gambiense infections (Keiser et al. 2001). In leishmaniasis, the tendency is still to resort to the old antimony compounds, with their severe side effects. At present, miltefosine is in clinical phase and is the first oral drug against visceral leishmaniasis (Jha et al. 1999). Two drugs are currently used against Chagas' disease, although these do not cure chronic effects. There is no prospect of novel drugs in this indication either (Pecoul et al. 1999; Morel 2000).

Animals↗

[Active antihelminitic alkaloids: active in vitro against Leishmanic Tropica the protozoa involved in leishmaniasis].

Leishmaniasis caused by protozoan Leishmania ssp., is an endemic parasitic disease in Central and South America. The chemotherapeutic agents against Leishmania ssp. (pentavalent antimony compounds, pentamidine and amphothericine B) are toxic and expensive products. Basing on the Bolivian folk medicine, we tried to find new active principles. Fourteen isoquinoline alkaloids, especially bisbenzylisoquinoline alkaloids extracted from Annonaceae, Berberidaceae, Hernandiaceae and Menispermaceae, demonstrate highly effective activity against this protozoan. Among them gyrocarpine, daphnandrine and obaberine seem to be of particular interest. The therapeutic effect was studied by biological assays on culture forms in vitro three strains of Leishmania, L. donovani, L. braziliensis (cutaneous and mucocutaneous leishmaniasis), L. mexicana amazonensis (cutaneous) and L. donovani (visceral leishmaniasis).

Alkaloids↗

Disseminated cutaneous leishmaniasis after visceral disease in a patient with AIDS.

Leishmaniasis is emerging as a common and serious opportunistic disease for patients with HIV infection. Almost all cases of HIV-Leishmania coinfection have been described in Mediterranean countries and they occur with various clinical presentations, ranging from typical visceral forms to asymptomatic or atypical cases, including cutaneous and mucocutaneous leishmaniasis. Pentavalent antimony compounds have been the mainstays of antileishmanial therapy for half a century and new lipid formulations of amphotericin B seem reliable, but the most effective treatment remains unknown. We describe a patient who was HIV infected and an intravenous drug user, with an unusual disseminated cutaneous leishmaniasis, after an initial visceral disease and after a 13-month maintenance treatment with liposomal amphotericin. The severe concurrent immunosuppression probably played an essential role in leading to this atypical cutaneous form, characterized by diffuse, nonulcerated, nonscabby maculopapular lesions.

AIDS-Related Opportunistic Infections↗

Leishmaniasis in Sudan. Mucosal leishmaniasis.

Sudanese mucosal leishmaniasis is a chronic infection of the upper respiratory tract and/or oral mucosa caused mainly by Leishmania donovani. The disease occurs in areas of the country endemic for visceral leishmaniasis, particularly among Masalit and other closely related tribes in western Sudan. The condition may develop during or after an attack of visceral leishmaniasis, but in most cases it is a primary mucosal disease. Unlike South American mucocutaneous leishmaniasis, mucosal leishmaniasis in Sudan is not preceded or accompanied by a cutaneous lesion. Pathologically, the lesions show a mixture of macrophages, plasma cells and lymphocytes. An epithelioid granuloma may also be found. Parasites are scanty. Diagnosis is established by demonstration of parasites in smears or biopsies, by culture or animal inoculation, or with the aid of the polymerase chain reaction. Most patients give positive results in the direct agglutination test and leishmanin skin test. Patients respond well to treatment with pentavalent antimony compounds.

Antiprotozoal Agents↗

[Short course treatment for visceral leishmaniasis with liposomal amphotericin B in immunocompetent patients].

INTRODUCTION: Visceral leishmaniasis is endemic in southern Europe. Traditional treatment consists of pentavalent antimonial compounds. However, treatment failures, the treatment's long duration, and toxicity have led to the introduction of new therapies, such as liposomal amphotericin B (LAB). In this study we evaluate the safety and efficacy of LAB at a maximum dose of 4 mg/kg/day on days 1, 2, 3, 4, 5, and 10. PATIENTS AND METHODS: A prospective, observational, open study was conducted in 13 Spanish centers. The diagnosis of visceral leishmaniasis was based on visualization of Leishmanias sp. in bone marrow aspirate or culture or positive serology together with compatible clinical symptoms. RESULTS: Thirty-two immunocompetent children aged from 7 months to 7 years were treated. All the children had rapid clinical response and bone marrow aspirate performed on day 21 was normal in the 24 patients (100 %) who underwent this procedure. In the remaining eight children efficacy was assessed by clinical response. Two relapses were observed. Cure was achieved in 18 patients (90.0 %) and in 87.5 % of the patients with microbiological confirmation of the disease. No adverse events were detected. CONCLUSIONS: A total dosage of 24 mg/kg of liposomal amphotericin B administered in 6 doses within 10 days is safe and effective for the treatment of visceral leishmaniasis and reduces the length of hospital stay.

Amphotericin B↗

Alternate energy coupling of ArsB, the membrane subunit of the Ars anion-translocating ATPase.

The arsenical resistance (ars) operon of the conjugative R-factor R773 confers resistance to arsenical and antimonial compounds in Escherichia coli, where resistance results from active extrusion of arsenite catalyzed by the products of the arsA and arsB genes. Previous in vivo studies on the energetics of arsenite extrusion showed that expression of both genes produced an ATP-coupled arsenite extrusion system that was independent of the electrochemical proton gradient. In contrast, in cells expressing only the arsB gene, arsenite extrusion was coupled to electrochemical energy and independent of ATP, suggesting that the Ars transport system exhibits a dual mode of energy coupling depending on the subunit composition. In vitro the ArsA-ArsB complex has been shown to catalyze ATP-coupled uptake of 73AsO2(-1) in everted membrane vesicles. However, transport catalyzed by ArsB alone has not previously been observed in vitro. In this study we demonstrate everted membrane vesicles prepared from cells expressing only arsB exhibit uptake of 73AsO2(-1) coupled to electrochemical energy.

Adenosine Triphosphatases↗