Combination Of Amphotericin B Research Articles

Clinical Characteristics, Prognosis Factors and Metagenomic Next-Generation Sequencing Diagnosis of Mucormycosis in Patients with Hematologic Diseases: A Single-Center Retrospective Study

Mucormycosis is a serious disorder with severe inflammatory response and tissue damage caused by the infection of Mucorales. Mucormycosis usually occurs in immunocompromised population, high mortality of mucormycosis is one of the challenges facing in patients with hematologic diseases. New diagnostic methods and antifungal strategies may improve prognosis of mucormycosis. The aim of our study was to describe the diagnostic value of metagenomic next⁃generation sequencing (mNGS) and identify the prognostic factors of mucormycosis. We conducted a retrospective study of hematologic patients suffered from mucormycosis and treated with monotherapy [amphotericin B (AmB) or posaconazole] or combination therapy (AmB and posaconazole). All the patients were diagnosed as mucormycosis by the 2019 EORTC/MSG criteria from March 2012 to September 2022. The primary outcome was 84-day all-cause mortality after diagnosis. Multivariate cox regression analysis was performed. We described the prognostic factors of mucormycosis in multivariate survival analysis, and investigated the application of different diagnostic methods. Ninety-five patients were included, with “proven” ( n=27), “probable” ( n=16) mucormycosis confirmed by traditional diagnostic methods, and “possible” ( n=52) mucormycosis with positive mNGS results. The median age was 44 (1-68) years. The most common primary diseases was acute leukemia (69.5%, n=66), and 32.6% of patients ( n=31) received hematopoietic stem cell transplantation. The most common pathogen species was Rhizopus spp (34.7%, n=33). The most common infection type was pulmonary infection (84.2%, n=80), followed by disseminated infection (8.4%, n=8) and rhino-orbito-cerebral infection (4.2%, n=4). Breakthrough infection occurred in 47 patients (49.5%). There were 13 patients (13.7%) receiving amphotericin B (AmB), 22 patients (23.2%) receiving posaconazole (Pos), and 60 patients (63.2%) receiving AmB plus Pos. A total of 9 patients (9.5%) underwent surgical treatment. With a median follow up of 79 (1-1015) days, the overall mortality rate at 42 days and 84 days was 33.7% and 44.2%, respectively. Possible patients and probable patients had similar diagnosis processes, overall survival rates (44.2% vs 50.0%, p=0.685) and overall response rates to effective drugs (44.0% vs 37.5%, p=0.647). Furthermore, the median diagnostic time was shorter in possible patients than proven and probable patients (14 vs 26 days, p<0.001). Combination therapy was associated with better survival compared to monotherapy at six weeks after treatment (78.8% vs 53.1%, p=0.0075). Multivariate analysis showed that combination therapy was the protective factor (HR=0.338, 95% CI: 0.162-0.703, p=0.004), though diabetes (HR=3.864, 95% CI: 1.897-7.874, p<0.001) and hypoxemia (HR=3.536, 95% CI: 1.874-6.673, p<0.001) were risk factors for mortality. Mucormycosis is a life-threatening infection in patients with hematologic diseases. Early management of diabetes and hypoxemia may improve the prognosis. Moreover, mNGS may be valuable evidence for microbiologic diagnosis, and combination of AmB and posaconazole is an effective treatment.

Amphotericin B May Decrease the Serum Level of Voriconazole: A Case Report and Brief Review of Literature

BackgroundCombination therapy with amphotericin B (AmB) and Voriconazole are sometimes used due to reducing AmB-related adverse reactions and improving outcomes. However, there was no report whether AmB would affect the serum level of Voriconazole.Case PresentationA patient was in presumption of invasive fungal infection. Voriconazole and amphotericin B was combined for administration. The results showed that serum levels of Voriconazole were dramatically reduced when combined AmB, whereas went up when receiving Voriconazole alone.ConclusionAmB might decrease the level of Voriconazole. Such a combination of AmB and Voriconazole cannot be considered appropriate until more data are available.

Preparation of amphotericin B-loaded hybrid liposomes and the integration of chitin-binding proteins for enhanced antifungal activity

Amphotericin B (AMB) is a gold standard antifungal drug because of its broad-spectrum activity toward pathogenic yeasts and molds. Because of its low solubility in water and toxicity toward humans, several lipid-based formulations that either increase the aqueous solubility or decrease the side effects have been employed in practical use. In our previous research, we found that the combination of AMB with an artificial palmitoylated chitin-binding domain from Pteris ryukyuensis chitinase (LysM-Pal) resulted in synergistic antifungal action against Trichoderma viride. Herein, we prepared hybrid liposomal formulations by combining a commercially available AMB formulation and liposomes with different surface charges to explore key factors in the antifungal activity. The characterization of AMB-loaded liposomal formulations (AMB-LFs), including particle size distribution and zeta potential, showed that anionic and neutral AMB-LFs could stably encapsulate AMB. The combination of either anionic or neutral AMB-LFs with unmodified LysM decreased the minimum inhibitory concentration of AMB. The combination of neutral AMB-LF with LysM-Pal resulted in a further decrease in the MIC, up to 15-fold compared with that of the neutral AMB-LF alone. Our results demonstrate the potential utility of lipid-based liposomal formulations of AMB combined with lipid-modified proteinaceous binders to tackle fungal infections.

Amphotericin B and Curcumin Co-Loaded Porous Microparticles as a Sustained Release System against Candida albicans.

Amphotericin B (AMB) is an antifungal drug used for serious fungal infections. However, AMB has adverse reactions such as nephrotoxicity, which limit the clinical application of AMB alone or in combination with other antifungal drugs. Nano or micro drug delivery systems (DDS) have been proven to be effective in reducing the toxic and side effects of drugs. Further, the combination of AMB with other compounds with antifungal activity, such as curcumin (CM), may enhance the synergistic effects. Herein, AMB and CM were co-loaded into porous poly (lactic-co-glycolic acid) (PLGA) microparticles (MPs) to prepare AMB/CM-PLGA MPs. The AMB/CM-PLGA MPs showed a remarkably reduced hemolysis (62.2 ± 0.6%) compared to AMB (80.9 ± 1.1%). The nephrotoxicity of AMB/CM-PLGA MPs is significantly lower than that of AMB. In vitro, AMB/CM-PLGA MPs had better inhibitory effects on the adhesion and biofilm formation of Candida albicans compared with AMB. Experiments on mice infected with C. albicans showed that AMB/CM-PLGA MPs have a better therapeutic effect than AMB in vivo. In summary, AMB/CM-PLGA MPs may be a novel and promising therapeutic candidate for fungal infection.

Enhancement of the Antifungal Activity of Chitinase by Palmitoylation and the Synergy of Palmitoylated Chitinase with Amphotericin B.

Combinations of antifungal drugs can have synergistic antifungal activity, achieving high therapeutic efficacy while minimizing the side effects. Amphotericin B (AMB) has been used as a standard antifungal drug for fungal infections; however, because of its high toxicity, new strategies to minimize the required dose are desirable. Chitinases have recently received attention as alternative safe antifungal agents. Herein, we report the combination of palmitoylated chitinase domains with AMB to enhance the antifungal activity. The chitin-binding domain (LysM) from Pteris ryukyuensis chitinase was site-specifically palmitoylated by conjugation reaction catalyzed by microbial transglutaminase. The palmitoylated LysM (LysM-Pal) exhibited strong antifungal activity against Trichoderma viride, inhibiting the growth completely at a concentration of 2 μM. This antifungal effect of LysM-Pal was mainly due to the effect of anchoring of palmitic acid motif to the plasma membrane of fungi. A combination of AMB with LysM-Pal resulted in synergistic enhancement of the antifungal activity. Intriguingly, LysM-Pal exhibited higher level of antifungal activity enhancement than palmitoylated catalytic domain (CatD) and fusion of LysM and CatD. Addition of 0.5 μM LysM-Pal to AMB reduced the minimal inhibition concentration of AMB to 0.31 μM (2.5 μM without LysM-Pal). The possible mechanism of the synergistic effect of AMB and LysM-Pal is destabilization of the plasma membrane by anchoring of palmitic acid and ergosterol extraction by AMB and destabilization of the chitin layer by LysM binding. The combination of LysM-Pal with AMB can drastically reduce the dose of AMB and may be a useful strategy to treat fungal infections.

Silver(I) and Copper(II) Complexes of 1,10-Phenanthroline-5,6-Dione Against Phialophora verrucosa: A Focus on the Interaction With Human Macrophages and Galleria mellonella Larvae.

Phialophora verrucosa is a dematiaceous fungus that causes mainly chromoblastomycosis, but also disseminated infections such as phaeohyphomycosis and mycetoma. These diseases are extremely hard to treat and often refractory to current antifungal therapies. In this work, we have evaluated the effect of 1,10-phenanthroline-5,6-dione (phendione) and its metal-based complexes, [Ag (phendione)2]ClO4 and [Cu(phendione)3](ClO4)2.4H2O, against P. verrucosa, focusing on (i) conidial viability when combined with amphotericin B (AmB); (ii) biofilm formation and disarticulation events; (iii) in vitro interaction with human macrophages; and (iv) in vivo infection of Galleria mellonella larvae. The combination of AmB with each of the test compounds promoted the additive inhibition of P. verrucosa growth, as judged by the checkerboard assay. During the biofilm formation process over polystyrene surface, sub-minimum inhibitory concentrations (MIC) of phendione and its silver(I) and copper(II) complexes were able to reduce biomass and extracellular matrix production. Moreover, a mature biofilm treated with high concentrations of the test compounds diminished biofilm viability in a concentration-dependent manner. Pre-treatment of conidial cells with the test compounds did not alter the percentage of infected THP-1 macrophages; however, [Ag(phendione)2]ClO4 caused a significant reduction in the number of intracellular fungal cells compared to the untreated system. In addition, the killing process was significantly enhanced by post-treatment of infected macrophages with the test compounds. P. verrucosa induced a typically cell density-dependent effect on G. mellonella larvae death after 7 days of infection. Interestingly, exposure to the silver(I) complex protected the larvae from P. verrucosa infection. Collectively, the results corroborate the promising therapeutic potential of phendione-based drugs against fungal infections, including those caused by P. verrucosa.

Fingolimod Potentiates the Antifungal Activity of Amphotericin B.

Candida albicans (C. albicans) is an opportunistic human fungal pathogen that can cause severe infection in clinic. Its incidence and mortality rate has been increasing rapidly. Amphotericin B (AMB), the clinical golden standard antifungal agent, has severe side effects that limit its clinical application. Thus, lowering the concentration and increasing the efficacy of AMB in a combinatorial antifungal therapy have been pursued by both industry and academia. Here we identify that fingolimod (FTY720), an immunomodulatory drug used for oral treatment of relapsing-remitting multiple sclerosis, can potentiate the efficacy of AMB against C. albicans growth synergistically. Furthermore, we observe an antifungal efficacy of FTY720 in combination with AMB against diverse fungal pathogens. Intriguingly, cells treated with both drugs are hypersensitive to endothelial endocytosis and macrophage killing. This is later found to be due to the hyperaccumulation of reactive oxygen species and the corresponding increase in activities of superoxide dismutase and catalase in the cells that received combinatorial treatment. Therefore, the combination of AMB and FTY720 provides a promising antifungal strategy.

A Novel Cryptococcal Meningitis Therapy: The Combination of Amphotericin B and Posaconazole Promotes the Distribution of Amphotericin B in the Brain Tissue.

The deficient brain tissue distribution of amphotericin B (AMPB) seriously restricts its treatment for the clinical efficacy of cryptococcus neoformans meningitis (CNM). We strive to develop a tactic to increase its concentration in brain tissue. We aimed to investigate whether the combination of AMPB and posaconazole (POS) could be more effective in the treatment of CNM and to elucidate its potential mechanisms. HPLC analysis was used to analyze the concentration of AMPB in mouse serum, brain tissue, and BCECs cells. Schrodinger molecular docking, in vitro plasma balance dialysis, and ultrafiltration analysis were performed to evaluate the combinative effect of AMPB and POS with serum albumin and POS on AMPB plasma protein binding. H&E staining and colonization culture experiment of CN were employed to assess the effect of POS on the efficacy of AMPB. POS + AMPB significantly reduced the concentration of plasma total AMPB and increased its concentration in the brain tissue. However, the P-gp inhibitor zosuquidar, BCRP inhibitor Ko143, and a common inhibitor of both, elacridar, had no significant effect on its concentration. Molecular docking, balance dialysis, and ultrafiltration analysis showed that AMPB and POS had potential binding properties to serum albumin. Meanwhile, 4 and 8 μg/mL POS could significantly increase the concentration of free AMPB in plasma. POS and three inhibitors all had no significant effect on the uptake of AMPB by BCECs, but serum albumin had. The therapeutic effect of CNM in mice was confirmed that AMPB and AMPB+POS could restrain the infiltration of neutrophils and lymphocytes in cortical neurons and improve the bleeding and markedly inhibit the proliferation of CN. Collectively, we propose that POS competitively binds to the plasma protein sites of AMPB, thereby increasing its level in the brain tissue. Meanwhile, POS could enhance the efficacy of AMPB in the treatment of CNM, which may be independent of P-gp and BCRP proteins.

Modulated Response of Aspergillus fumigatus and Stenotrophomonas maltophilia to Antimicrobial Agents in Polymicrobial Biofilm.

Introduction: The complexity of biofilms constitutes a therapeutic challenge and the antimicrobial susceptibility of fungal-bacterial biofilms remains poorly studied. The filamentous fungus Aspergillus fumigatus (Af) and the Gram-negative bacillus Stenotrophomonas maltophilia (Sm) can form biofilms and can be co-isolated from the airways of cystic fibrosis (CF) patients. We previously developed an in vitro biofilm model which highlighted the antibiosis effect of Sm on Af, which was dependent on the bacterial fitness. The aim of the present study was to investigate the in vitro susceptibility of Af and Sm in mono- or polymicrobial biofilms to five antimicrobial agents alone and in two-drug combinations.Methods: Af and Sm clinical reference strains and two strains from CF sputa were tested through a planktonic and biofilm approaches. Af, Sm, or Af-Sm susceptibilities to amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), levofloxacin (LVX), and rifampicin (RFN) were evaluated by conventional planktonic techniques, crystal violet, XTT, qPCR, and viable plate count.Results: Af planktonic cells and biofilms in formation were more susceptible to AMB, ITC, and VRC than Af mature biofilms. Af mature biofilms were susceptible to AMB, but not to ITC and VRC. Based on viable plate count, a lower concentration of LVX and RFN was required to reduce Sm cell numbers on biofilms in formation compared with mature biofilms. The antibiosis effect of Sm on Af growth was more pronounced for the association of CF strains that exhibited a higher fitness than the reference strains. In Af-Sm biofilms, the fungal susceptibility to AMB was increased compared with Af biofilms. In contrast, the bacterial susceptibility to LVX decreased in Af-Sm biofilms and was fungal biomass-dependent. The combination of AMB (64 μg/mL) with LVX or RFN (4 μg/mL) was efficient to impair Af and Sm growth in the polymicrobial biofilm.Conclusion: Sm increased the Af susceptibility to AMB, whereas Af protected Sm from LVX. Interactions between Af and Sm within biofilms modulate susceptibility to antimicrobial agents, opening the way to new antimicrobial strategies in CF patients.

Combination therapy with amphotericin B and doxorubicin encapsulated in mannosylated nanomicelles for visceral leishmaniasis

Drugs used to treat visceral leishmaniasis (VL) have severe side effects and face increasing resistance. We have created a mannose-functionalized nanomicelle carrying two drugs amphotericin B (AmB) and doxorubicin (DOX). The nanomicelles AmB&DOX@MCC preferentially target macrophages where Leishmania parasites reside and replicate, by the interaction between mannose residues on the micelle and mannose receptors (MRs) on the macrophage membrane. The combination of AmB and DOX leads to enhanced anti-leishmanial therapeutic efficacy through a synergistic effect. The mannose-fabricated nanomicelle is a promising formulation for leishmaniasis treatment.

Time-kill assays of amphotericin B plus anidulafungin against Candida tropicalis biofilms formed on two different biomaterials.

To determine the fungicidal activity by time-killing assays of amphotericin B (AMB) combined with anidulafungin (ANF) against biofilms of 2 clinical isolates of Candida tropicalis and the reference strain ATCC® 750, developed on polytetrafluoroethylene (PTFE) and titanium, using the CDC Biofilm Reactor (CBR) as an in vitro model. Biofilms were developed for 24 hours on the disk surfaces and then exposed to AMB (40 mg/L), ANF (8 mg/L), alone and combined. At predetermined time points after drug exposure, biofilms were removed from the disk surface by vortexing-sonication to quantify viable biofilm cells. Drug activity was dependent on strain and time. After exposure to AMB, the mean decrease in viable cells attached to PTFE was 2.23 ± 0.89 Log10 cfu/cm2 (range 0.6-3.56 Log10), and on titanium 2.91 ± 1.04 (range 1.49-4.51 Log10). The reduction with ANF was 0.78 ± 0.5 (0.03-1.58 Log10) on PTFE and 0.8 ± 2.26 (0.42-1.16 Log10) on titanium. The reduction obtained with the combination of AMB + ANF was 1.8 ± 1.07 (0.22-3.54 Log10) on PTFE and 1.97 ± 0.49 (1.36-2.84 Log10) on titanium. The interaction was classified as indifferent with a tendency to antagonism. The activity of antifungal agents depends on the biomaterial surfaces the biofilm forming capacity of the isolate. AMB + ANF is less effective than AMB alone on both surfaces. Thus, the combination of these antifungals does not seem to add additional benefits to the treatment of C. tropicalis biofilm-related infections.

Actividad de la anfotericina B y la anidulafungina, solas y combinadas, frente a biopelículas de Candida tropicalis formadas sobre Teflon® y titanio

BackgroundCurrent therapeutic strategies have a limited efficacy against Candida biofilms that form on the surfaces of biomedical devices. Few studies have evaluated the activity of antifungal agents against Candida tropicalis biofilms. ObjectivesTo evaluate the activity of amphotericin B (AMB) and anidulafungin (AND), alone and in combination, against C. tropicalis biofilms developed on polytetrafluoroethylene (teflon -PTFE) and titanium surfaces using time-kill assays. MethodsAssays were performed using the CDC Biofilm Reactor equipped with PTFE and titanium disks with C. tropicalis biofilms after 24h of maturation. The concentrations assayed were 40mg/l for AMB and 8mg/l for AND, both alone and combined. After 24, 48 and 72h of exposure to the antifungals, the cfu/cm2 was determined by a vortexing-sonication procedure. ResultsAMB reduced biofilm viable cells attached to PTFE and titanium by ≥99% and AND by 89.3% on PTFE and 96.8% on titanium. The AMB+AND combination was less active than AMB alone, both on PTFE (decrease of cfu/cm2 3.09 Log10vs. 1.08 when combined) and titanium (4.51 vs. 1.53 when combined), being the interaction irrelevant on both surfaces. ConclusionsAMB is more active than AND against C. tropicalis biofilms. Yeast killing rates are higher on titanium than on PTFE surfaces. The combination of AMB plus AND is less effective than AMB alone on both surfaces.

Lysine enhances the effect of amphotericin B against <italic>Candida albicans in vitro</italic>

Amphotericin B (AmB) is a polyene antibiotic produced by Streptomyces nodosus and has been used for >50 years in the treatment of acute systemic fungal infections. In the present study, we demonstrated that lysine, an essential amino acid, could enhance the effect of AmB against Candida albicans in vitro, although lysine itself did not exert a fungicidal effect. In addition, the combination of AmB with lysine could provide an enhanced action against Candida parapsilosis and Cryptococcus neoformans compared with AmB alone. Lysine could also enhance the antifungal effect of caspofungin or nystatin. An enhanced effect of the combination of lysine with AmB was observed for the prevention of biofilm and hypha formation. Furthermore, our results demonstrated that lysine-mediated oxidative damage, such as the generation of endogenous reactive oxygen species, may be the mechanism underlying the enhancing effect of lysine on AmB. Our results also showed that CaMCA1 gene plays an important role in increasing the sensitivity of C. albicans cells upon AmB treatment. Using AmB together with lysine may be a promising strategy for the therapy of disseminated candidiasis.

Amphotericin B-silver hybrid nanoparticles: synthesis, properties and antifungal activity

High antifungal activity is reported, in comparison with commercially available products, of a novel hybrid system based on silver nanoparticles synthesized using a popular antifungal macrocyclic polyene amphotericin B (AmB) acting both as a reducing and stabilizing/capping agent. The synthesis reaction proceeds in an alkaline environment which prevents aggregation of AmB itself and promotes nanoparticle formation. The innovative approach produces monodisperse (PDI=0.05), AmB-coated silver nanoparticles (AmB-AgNPs) with the diameter ~7nm. The products were characterized using imaging (electron microscopy) and spectroscopic (UV–vis and infrared absorption, dynamic light scattering and Raman scattering) methods. The nanoparticles were tested against Candida albicans, Aspergillus niger and Fusarium culmorum species. For cytotoxicity studies CCD-841CoTr and THP-1 cell lines were used. Particularly high antifungal activity of AmB-AgNPs is interpreted as the result of synergy between the antifungal activity of amphotericin B and silver antimicrobial properties (Ag+ ions release). From the Clinical EditorAmphotericin B (AmB) is a common agent used for the treatment against severe fungal infections. In this article, the authors described a new approach in using a combination of AmB and silver nanoparticles, in which the silver nanoparticles were synthesized and stabilized by AmB. Experimental data confirmed synergistic antifungal effects between amphotericin B and silver. This novel synthesis process could potentially be important in future drug development and fabrication.

Amphotericin B Resistance in Aspergillus terreus Is Overpowered by Coapplication of Pro-oxidants.

Invasive fungal infections have significantly increased over the past decades in immunocompromised individuals and high-risk patients. Amphotericin B (AmB) exerts a powerful and broad activity against a vast array of fungi and has a remarkably low rate of microbial resistance. However, most isolates of Aspergillus terreus developed an intrinsic resistance against AmB, and during this study, we characterized the mode of action of this polyene antifungal drug in more detail in resistant (ATR) and rare susceptible (ATS) clinical isolates of A. terreus. We illustrate that AmB treatment changes cellular redox status and promotes the generation of high levels of reactive oxygen species (ROS) in ATS. In contrast, ATR isolates were able to cope better with AmB-induced oxidative stress. Most importantly, we demonstrate in this study that coapplication of anti- and pro-oxidants significantly affects AmB efficacy in an antithetic manner--antioxidants and ROS-scavenging agents increase AmB tolerance in susceptible strains, while pro-oxidants render formerly resistant isolates considerably susceptible to the antifungal drug also in vivo in a Galleria animal model. Thereby, our study provides novel therapeutic options to treat formerly resistant fungal strains by a combination of AmB and pro-oxidant compounds.

Enhancement of the antibiofilm activity of amphotericin B by polyamine biosynthesis inhibitors

The aim of this study was to investigate the effect of polyamine biosynthesis inhibitors on the activity of amphotericin B (AmB) against Candida albicans biofilms and to clarify the underlying mechanisms. The antibiofilm activity of AmB was significantly enhanced when used in combination with the polyamine biosynthesis inhibitors 1,4-diamino-2-butanone (DAB) and α-difluoromethylornithine (DFMO). Further study showed that DAB and DFMO also enhanced the antibiofilm activity of several other antifungal agents. Moreover, the combination of AmB and polyamine biosynthesis inhibitors resulted in an increase in intracellular levels of reactive oxygen species. In addition, caspase activity and transcription of the caspase-encoding gene CaMCA1 were greatly increased upon combined treatment with polyamine biosynthesis inhibitors and AmB. Consistently, the biofilm formed by a Δcamca1 mutant exhibited greater viability and lower caspase activity than that of the wild-type strain upon combined treatment. These data provide useful information for the development of new strategies to enhance the antibiofilm activities of antifungal agents.

Postantifungal effect of the combination of caspofungin with voriconazole and amphotericin B against clinicalCandida kruseiisolates

We evaluated the postantifungal effects (PAFEs) of caspofungin (CAS), voriconazole (VOR), amphotericin B (AmB), and the combinations of CAS + VOR and CAS + AmB against 30 clinical Candida krusei isolates at 0.25, 1 and 4 times the MIC of each individually and in the indicated combinations. Antifungals were removed after 1 hour and colony counts were performed at 0, 2, 6, 24, and 48 h. VOR did not display any measurable PAFE regardless of antifungal concentrations, while AmB and CAS exhibited dose-dependent PAFE. The most effective agent producing a prolonged PAFE in this study was CAS. Although the combination of CAS with VOR generated longer PAFEs at 0.25 and 1 times their respective MICs in comparison with CAS alone, this combination was indifferent rather than synergistic. However, the combination of CAS with AmB at 4 times their MICs exhibited the best performance, reducing the colony counts during the 48 h after removal of drugs and resulted in synergic interaction in respect to 20 (67%) isolates. Consequently, CAS has a prolonged PAFE in vitro against C. krusei isolates, and the combination of AmB + CAS may increase significantly the efficacy of CAS. Our data may be useful in optimizing dosing regimens for these agents and their combinations, although further studies are needed to explore the clinical usefulness of our results.

Dependence of vacuole disruption and independence of potassium ion efflux in fungicidal activity induced by combination of amphotericin B and allicin against Saccharomyces cerevisiae

Allicin selectively enhances the fungicidal activity of amphotericin B (AmB). It also accelerates AmB-induced vacuole disruption but does not affect AmB-induced potassium ion efflux in Saccharomyces cerevisiae and Candida albicans. The fungicidal activity of AmB alone or combined with allicin was further evaluated based on the relationship among cell viability, vacuole disruption and potassium ion efflux in S. cerevisiae. Lethality and vacuole disruption caused by AmB alone were completely restricted when K(+) and Mg(2+) were added to the growth medium. On the other hand, in identical conditions, the combination of AmB and allicin induced both lethality and vacuole disruption. S. cerevisiae Δerg6 cells, which lack ergosterol in plasma membrane, were mostly resistant to AmB as well as the combination of AmB and allicin against both lethality and vacuole disruption. The incorporation of AmB into the cytoplasm of Δerg6 cells was significantly reduced in comparison with that in parent cells, regardless of the presence of allicin. Our results suggest that the fungicidal activity of AmB combined with allicin is involved in vacuole disruption but not in potassium ion efflux, and that the expression of allicin-mediated activity of AmB requires the presence of ergosterol in the plasma membrane.

Allicin enhances the oxidative damage effect of amphotericin B against Candida albicans

Amphotericin B (AmB) is the gold standard of antifungal treatment for the most severe invasive mycoses. In addition to the interaction of AmB with ergosterol in the fungi cell membrane, several studies have demonstrated oxidative damage involved in the fungicidal activity of AmB. In this study, allicin, an allyl sulphur compound from garlic, was shown to enhance significantly the effect of AmB against Candida albicans in vitro and in vivo, although allicin did not exert a fungicidal effect. Further study first demonstrated that allicin-mediated oxidative damage, such as phospholipid peroxidation in the plasma membrane, via influencing the defence of C. albicans against oxidative damage may be the cause of the synergistic interaction between allicin and AmB. We envision that a combination of AmB with allicin may prove to be a promising strategy for the therapy of disseminated candidiasis.

Efficacy of recombinant gamma interferon for treatment of systemic cryptococcosis in SCID mice.

We have previously shown that gamma interferon (IFN-gamma) is a useful adjunct to therapy of experimental systemic cryptococcosis in normal mice. To better emulate AIDS patients, SCID mice were infected intravenously with Cryptococcus neoformans. Mice received no therapy, 3 mg of amphotericin B (AmB) per kg of body weight, or 10(5) U of IFN-gamma alone (prophylactically and therapeutically or only therapeutically) or with AmB. In the first experiment, >75% of the mice survived. Therapy with AmB alone was efficacious compared to no therapy in all organs. Both regimens of IFN-gamma alone were efficacious in the brain and lungs, and the combination of AmB and IFN-gamma showed significant synergy in the kidneys. AmB alone cured 40% of mice of infection, whereas the combination regimens cured >50% of the mice and 90% of the brain infections. In a second study, IFN-gamma again proved efficacious alone, and when given with AmB its efficacy was improved. Therapeutic IFN-gamma alone was effective only in the liver compared to no therapy, and the combination regimen, although highly effective, showed no significant synergy. In a third experiment, AmB alone or in combination with IFN-gamma prolonged survival compared to no therapy or IFN-gamma alone. The combination regimen showed significant synergy over AmB alone in the brain, liver, kidneys, and lungs. AmB alone cured no mice of infections in more than two organs, whereas AmB in combination with IFN-gamma cured 55% of infections in three or more organs. These results indicate that IFN-gamma has therapeutic efficacy in severely immunodeficient animals, especially in combination with AmB. Significant synergistic activity was noted in all organs except the spleen. Overall, IFN-gamma has utility as an adjunctive therapy against systemic cryptococcosis in the severely immunocompromised host.