Experimental Evaluation of Second-Line Oral Treatments of Visceral Leishmaniasis Caused by Leishmania infantum

In a murine model of Leishmania infantum visceral leishmaniasis, metronidazole, ketoconazole, fluconazole, itraconazole, and terbinafine were less effective than antimonial agents in reducing hepatic parasite load. Ketoconazole potentiated the effect of meglumine antimoniate reference therapy through its marked activity against spleen infection.     

Mechanism of Amphotericin B Resistance in Leishmania donovani Promastigotes

Amphotericin B (AmB)-resistant Leishmania donovani promastigotes were selected by increasing drug pressure, and their biological features were compared with those of the wild-type parent strain. The 50% inhibitory concentration for resistant cells was 20 times higher than that for the wild-type. Resistance was stable after more than 40 passages in drug-free medium, and resistant promastigotes were infective to macrophages in vitro but lost their virulence in vivo. They had 2.5 times longer generation time, decreased AmB uptake, and increased AmB efflux in comparison to the wild type. Fluorescence measurement with a specific plasma membrane probe, 1-[4-(trimethylammonio)-1,6-diphenylhexa]-1,3,5-triene, showed increased membrane fluidity in drug-resistant promastigotes. Analysis of lipid composition showed that in resistant cells saturated fatty acids were prevalent, with stearic acid as the major fatty acid, and the major sterol was an ergosterol precursor, the cholesta-5, 7, 24-trien-3β-ol and not ergosterol as in the AmB-sensitive strain.     

Efficacies of KY62 against Leishmania amazonensis and Leishmania donovani in Experimental Murine Cutaneous Leishmaniasis and Visceral Leishmaniasis

Current therapy for leishmaniasis is unsatisfactory because parenteral antimonial salts and pentamidine are associated with significant toxicity and failure rates. We examined the efficacy of KY62, a new, water-soluble, polyene antifungal, against cutaneous infection with Leishmania amazonensis and against visceral infection with Leishmania donovani in susceptible BALB/c mice. Mice were infected with L. amazonensis promastigotes in the ear pinna and in the tail and were treated with KY62 or amphotericin B. The cutaneous lesions showed a remarkable response to therapy with KY62 at a dose of 30 mg per kg of body weight per day. At this dose, the efficacy of KY62 was equivalent to or better than that of amphotericin B at 1 to 5 mg/kg/day. Mice infected intravenously with 10⁷ L. donovani promastigotes and treated with KY62 showed a 4-log reduction in the parasite burden in the liver and spleen compared to untreated mice. These studies indicate potent activity of KY62 against experimental cutaneous leishmaniasis caused by L. amazoniensis and against experimental visceral leishmaniasis caused by L. donovani.     

In Vitro Synergistic Effect of Amphotericin B and Allicin on Leishmania donovani and L. infantum

Current monotherapy against visceral leishmaniasis has serious side effects, and resistant Leishmania strains have been identified. Amphotericin B (AmB) has shown an extraordinary antileishmanial efficacy without emergence of resistance; however, toxicity has limited its general use. Results obtained showed, using a fixed-ratio analysis, that the combination of diallyl thiosulfinate (allicin) and AmB ranged from moderately synergic to synergic at low concentrations (0.07 μM AmB plus 35.45 μM allicin induced 95% growth inhibition). None of the treatments, alone or in combination, had noticeable adverse effects on macrophages (Mϕ) in the concentration range examined (allicin, 0.5, 1, 5 and 10 μM; AmB, 0.05, 0.075, and 0.1 μM). Allicin, AmB, or the combination did not affect the infection rate (percentage of infected Mϕ) of Leishmania. Allicin enhanced the activity of AmB on intracellular amastigotes of Leishmania donovani and L. infantum (ca. 45% reduction of amastigote burden with 0.05 μM AmB plus 10 μM allicin); this represented nearly a 2-fold reduction in the 50% inhibitory concentration (IC₅₀) of the antibiotic added alone. Results point toward the possible utility of testing this combination in vivo to reduce the toxicity associated with monotherapy with AmB.     

Sequential Chemoimmunotherapy of Experimental Visceral Leishmaniasis Using a Single Low Dose of Liposomal Amphotericin B and a Novel DNA Vaccine Candidate

Combination therapies for leishmaniasis, including drugs and immunomodulators, are one approach to shorten treatment courses and to improve the treatment of complex manifestations of the disease. We evaluated a novel T-cell-epitope-enriched DNA vaccine candidate (LEISHDNAVAX) as host-directed immunotherapy in combination with a standard antileishmanial drug in experimental visceral leishmaniasis. Here we show that the DNA vaccine candidate can boost the efficacy of a single suboptimal dose of liposomal amphotericin B in C57BL/6 mice.     

Serological studies on rK39 negative Visceral Leishmaniasis in an endemic focus of Leishmania donovani induced Cutaneous Leishmaniasis

Sri Lanka reports a focus of L. donovani induced cutaneous leishmaniasis (CL). Our more recent parasite and clinical studies and historical evidence point towards long term existence of Leishmania in the country, indicating a possible evolution leading to antigenic heterogenicity as well. In-house enzyme-linked immunosorbent assay (ELISA) that was developed during phase 1 study indicated >80% sero-positivity in local CL, while visceral leishmaniasis (VL) remained very rare with majority being negative when tested with rK39 assay. A novel serological tool was developed and sero-positivity of VL was assessed for the first time. The assay showed 100.0% sensitivity and 98.3% specificity for detection of VL. Samples were showed less positivity with established direct agglutination test (DAT) and rK39 strip test. The assay was less expensive than that of established rapid diagnostic tests (RDTs), culture and PCR assays. This assay may be useful in diagnosing clinical VL infections, detection of light microscopy (LM) negative patients, tracking post treatment stages, field screening of asymptomatic cases and in further serological studies.     

Development of Resistance to Amphotericin B in Candida lusitaniae Infecting a Human

Candida lusitaniae associated with infection in a patient with acute myelogenous leukemia developed resistance to amphotericin B during systemic treatment of the patient. The organism, when isolated initially, was inhibited by 0.31 mug of amphotericin B per ml in yeast nitrogen base agar, but when isolated (20 days later) just antemortem and postmortem, required 100 and 50 mug/ml, respectively, for complete inhibition at 48 h.     

Combination Therapy with Tamoxifen and Amphotericin B in Experimental Cutaneous Leishmaniasis

Leishmaniasis chemotherapy remains very challenging. The high cost of active drugs, along with the severity of their side effects and the increasing failure rate of the current therapeutic schemes, calls for the discovery of new active drugs and schemes of treatment. The use of combination therapy has gained much attention in recent years as a possible strategy for overcoming the various shortcomings in the present arsenal. We recently described the effectiveness of tamoxifen in murine models of leishmaniasis, and here, we investigated the interactions between tamoxifen and amphotericin B, one of the most potent drugs used in leishmaniasis treatment. The in vitro interactions were indifferent for the association of tamoxifen and amphotericin B. The association was also assayed in vivo in Leishmania amazonensis-infected BALB/c mice and was found to yield at least additive effects at low doses of both drugs.     

Identification of Novel Leishmania donovani Antigens that Help Define Correlates of Vaccine-Mediated Protection in Visceral Leishmaniasis

Visceral leishmaniasis (VL), caused by the intracellular parasite Leishmania donovani is a major public health problem in the developing world. But there is no effective and safe vaccine approved for clinical use against any form of leishmaniasis. Through reactivity with kala-azar patient and cured sera, polypeptides ranging from 91 to 31-kDa from L. donovani promastigotes were previously identified as potential protective vaccine candidates. In this study four polypeptides 91(LD91), 72 (LD72), 51(LD51) and 31 (LD31)-kDa were purified using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by electroelution. We compared the vaccine efficacy of these antigens encapsulated in cationic liposomes in BALB/c mice against challenge infection with L. donovani. Our results demonstrated that liposomal LD31 (74%–77%) and LD51 (72%–75%) vaccination reduced parasite burden to the greatest degree followed by liposomal LD72 (65%–67%) and LD91 (46%–49%). Analysis of the cytokine responses in immunized mice revealed that all the vaccinated groups produced prechallenge interferon-γ, interleukin-12 and interleukin-4. Interestingly, the degree of reduction in parasite load could be predicted by the magnitude of the cytokine responses which correlated inversely with the parasite burden both in liver and spleen. The 31, 51 and 72-kDa bands were identified as ATP synthase α chain, β-tubulin and heat shock 70-related protein 1 precursor of L. major, respectively using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF/TOF) mass spectrometry. These three leishmanial antigens have not been described before as successful vaccine candidates examined against in vivo VL model. Thus, these antigens can be potential components of future antileishmaniasis vaccines.     

Visceral Leishmaniasis in the BALB/c Mouse: A Comparison of the Efficacy of a Nonionic Surfactant Formulation of Sodium Stibogluconate with Those of Three Proprietary Formulations of Amphotericin B

In this study, treatment efficacies of a nonionic surfactant vesicle formulation of sodium stibogluconate (SSG-NIV) and of several formulations of amphotericin B were compared in a murine model of visceral leishmaniasis. Treatment with multiple doses of AmBisome, Abelcet, and Amphocil (total dose, 12.5 mg of amphotericin B/kg of body weight) resulted in a significant suppression of parasite burdens in liver (P < 0.0005) and spleen (P < 0.0005) compared with those of controls, with Abelcet having the lowest activity. Only AmBisome and Amphocil gave significant suppression of parasites in bone marrow (compared to control values, P < 0.005). In the acute-infection model, single-dose treatments of SSG-NIV (296 mg of Sb^V/kg), SSG solution (296 mg of Sb^V/kg), or AmBisome (8 mg of amphotericin B/kg) were equally effective against liver parasites (compared to control values, P < 0.0005). SSG-NIV and AmBisome treatment also significantly suppressed parasites in bone marrow and spleen (P < 0.005), with SSG-NIV treatment being more suppressive (>98% suppression in all three sites). Free-SSG treatment failed to suppress spleen or bone marrow parasites. Infection status influenced treatment outcome. In the chronic-infection model, the AmBisome single-dose treatment was less effective in all three infection sites and the SSG-NIV single-dose treatment was less effective in the spleen. The results of this study suggest that the antileishmanial efficacy of SSG-NIV compares favorably with those of the novel amphotericin B formulations.     

Efficacious Topical Treatment for Murine Cutaneous Leishmaniasis with Ethanolic Formulations of Amphotericin B

The goal of the present study was to evaluate the antileishmanial effects of topically applied lipid-based formulations containing amphotericin B (AmB) in CBA mice as a model for human cutaneous leishmaniasis. Such treatment, if efficacious, is expected to be superior to systemic treatments since, by acting in a localized manner, it will require lower, and therefore less toxic, drug dosages. Three preparations of AmB complexed to polar lipids were tested: Fungizone (mixed micelles composed of AmB and deoxycholate), Amphocil (AmB and cholesteryl sulfate complex), and ABPLC (AmB and phospholipid complex). All these formulations killed parasites in vitro with similar efficacies but were ineffective when they were applied topically. However, Amphocil and ABPLC, but not Fungizone, when dispersed in an aqueous solution containing 5 to 25% ethanol, induced a statistically significant improvement in lesion size from week 2 or 3 onward (a total of 15 mg of AmB per kg of body weight was applied over 3 weeks). AmB biodistribution measurements following topical application of Amphocil, determined by high-pressure liquid chromatography, showed that AmB was detectable in the skin but not in the internal organs. Application of at least 10 times more drug was necessary to obtain detectable levels of AmB in the internal organs. After application of therapeutic doses of ABPLC, very low levels of AmB were detected in the internal organs. These experiments show for the first time that AmB administered topically as a complex either with cholesteryl sulfate or with phospholipids and in the presence of ethanol can penetrate the skin and kill sensitive organisms in a localized manner by using very low total drug concentrations.     

Activity of a Heat-Induced Reformulation of Amphotericin B Deoxycholate (Fungizone) against Leishmania donovani

The heat treatment of amphotericin B deoxycholate (Fungizone), which was previously shown to induce superaggregation and decrease the toxicity of the drug to mammalian cells, increased its activity against Leishmania donovani in BALB/c mice, whereas it reduced its toxicity. Heat treatment preserved the activity of Fungizone against L. donovani HU3-infected mouse peritoneal macrophages.     

Detection of Resistance to Amphotericin B in Candida Isolates by Using Iso-Sensitest Broth

A major limitation of the National Committee for Clinical Laboratory Standards M27-A methodology is reliable detection of amphotericin B (AMB) resistance. The results obtained by using Iso-Sensitest, a synthetic medium, to detect AMB resistance were analyzed and compared with those obtained with RPMI and antibiotic medium 3 (AM3). The ability to detect AMB resistance with RPMI is not enhanced by using a higher inoculum, glucose supplementation at a final concentration of 20 g/liter, spectrophotometric reading, or 24 h of incubation time. Testing using AM3 and an inoculum of 10(3) CFU/ml detects resistance. Identification of resistant isolates is not improved by glucose supplementation, changes in reading method, or changes in incubation time. However, the use of AM3 as assay medium and an inoculum of 10(5) CFU/ml did not allow detection of AMB resistance. Testing using Iso-Sensitest medium appears to be similar to AM3 in detecting resistance. The most pronounced discrimination is achieved by testing in Iso-Sensitest supplemented with glucose and spectrophotometric reading after 24 h of incubation. The reproducibility of MIC testing was greatest for Iso-Sensitest-based procedures. Use of Iso-Sensitest produces both highly reproducible MICs and reliable identification of AMB-resistant Candida isolates.     

The LABCG2 Transporter from the Protozoan Parasite Leishmania Is Involved in Antimony Resistance

Treatment for leishmaniasis, which is caused by Leishmania protozoan parasites, currently relies on a reduced arsenal of drugs. However, the significant increase in the incidence of drug therapeutic failure and the growing resistance to first-line drugs like antimonials in some areas of Northern India and Nepal limit the control of this parasitic disease. Understanding the molecular mechanisms of resistance in Leishmania is now a matter of urgency to optimize drugs used and to identify novel drug targets to block or reverse resistant mechanisms. Some members of the family of ATP-binding cassette (ABC) transporters in Leishmania have been associated with drug resistance. In this study, we have focused our interest to characterize LABCG2''s involvement in drug resistance in Leishmania. Leishmania major parasites overexpressing the ABC protein transporter LABCG2 were generated in order to assess how LABCG2 is involved in drug resistance. Assays of susceptibility to different leishmanicidal agents were carried out. Analysis of the drug resistance profile revealed that Leishmania parasites overexpressing LABCG2 were resistant to antimony, as they demonstrated a reduced accumulation of Sb^III due to an increase in drug efflux. Additionally, LABCG2 was able to transport thiols in the presence of Sb^III. Biotinylation assays using parasites expressing LABCG2 fused with an N-terminal green fluorescent protein tag revealed that LABCG2 is partially localized in the plasma membrane; this supports data from previous studies which suggested that LABCG2 is localized in intracellular vesicles that fuse with the plasma membrane during exocytosis. In conclusion, Leishmania LABCG2 probably confers antimony resistance by sequestering metal-thiol conjugates within vesicles and through further exocytosis by means of the parasite''s flagellar pocket.     

Comparative Susceptibility of Candida albicans to Amphotericin B and Amphotericin B Methyl Ester

The in vitro antifungal activities of amphotericin B (AMB) and amphotericin B methyl ester (AME) were compared against 465 clinical isolates of Candida albicans. AMB and AME possessed comparable activity against half of the strains, but against the remainder of the strains the activity of AME was slightly lower than that of AMB. Rarely did AME show superior antifungal activity to AMB.     

Visceral Leishmaniasis in HIV Infection and AIDS: Clinical Features and Response to Therapy

The development of visceral leishmaniasis with atypical featuresin an AIDS patient, and the recent flurry of reports of visceralleishmaniasis in HIV-infected individuals prompted the reviewof its manifestations in the 47 reported cases. Splenomegaly,which is almost always a feature of visceral leishmaniasis inthe immunocompetent host, was absent in eight. Antibodies toLeishmania donovani, which are present in approximately 95 percent of immunocompetent patients with visceral leishmaniasis,were absent in 29 of 45 (66 per cent) of HIV-infected patientstested. Nine HIV-positive patients with visceral leishmaniasisdid not exhibit a primary clinical response to therapy withantimonials and of those who did show a response, relapse occurredin 13, at a mean 4.5 months after stopping therapy. Seventeenpatients are known to have died often in association with respiratorydisease; Leishmania was seen in one bronchial lavage specimenand in lung tissue in one post-mortem performed. In order toimprove the prognosis of visceral leishmaniasis in HIV-infectedpatients diagnosis will have to be made earlier, taking accountof the atypical features, and treatment will need to be improved,both initially and perhaps also by the use of long-term maintenancetherapy.     

Resveratrol Is Active against Leishmania amazonensis: In Vitro Effect of Its Association with Amphotericin B

Resveratrol is a polyphenol found in black grapes and red wine and has many biological activities. In this study, we evaluated the effect of resveratrol alone and in association with amphotericin B (AMB) against Leishmania amazonensis. Our results demonstrate that resveratrol possesses both antipromastigote and antiamastigote effects, with 50% inhibitory concentrations (IC₅₀s) of 27 and 42 μM, respectively. The association of resveratrol with AMB showed synergy for L. amazonensis amastigotes, as demonstrated by the mean sums of fractional inhibitory index concentration (mean ΣFIC) of 0.483, although for promastigotes, this association was indifferent. Treatment with resveratrol increased the percentage of promastigotes in the sub-G₀/G₁ phase of the cell cycle, reduced the mitochondrial potential, and showed an elevated choline peak and CH₂-to-CH₃ ratio in the nuclear magnetic resonance (NMR) spectroscopy analysis; all these features indicate parasite death. Resveratrol also decreased the activity of the enzyme arginase in uninfected and infected macrophages with and without stimulation with interleukin-4 (IL-4), also implicating arginase inhibition in parasite death. The anti-Leishmania effect of resveratrol and its potential synergistic association with AMB indicate that these compounds should be subjected to further studies of drug association therapy in vivo.