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Concept: Flucytosine

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Invasive aspergillosis remains a major cause of death among the immunocompromised population and those receiving long-term immunosuppressive therapy. In light of increased azole resistance, variable outcomes with existing echinocandin mono and combination therapy, and persistent high mortality rates, new antifungal agents for the treatment of invasive aspergillosis are clearly needed.SCY-078 is the first in class triterpenoid antifungal, a novel class of glucan synthase inhibitors, with broadin vitroandin vivoactivity against a broad spectrum ofCandidaandAspergillusIn vitrotesting of clinical strains ofAspergillus fumigatusand non-fumigatusstrains showed potent fungistatic activity of SCY-078 (minimum effective concentration, MEC90= 0.125 μg/ml) as compared with amphotericin B (MIC90= 8 μg/ml) and voriconazole (MIC90= 2 μg/ml). Combination testing of SCY-078 with isavuconazole or voriconazole demonstrated synergistic activity against the majority of the azole-susceptible strains tested, and SCY-078 in combination with amphotericin B was synergistic against the azole-susceptible strains, as well as one known resistantcyp51Amutant. SCY-078 may be an important additional antifungal for first-line or salvage mono or combination treatment of invasive aspergillosis.

Concepts: Mortality rate, Opportunistic infection, Aspergillosis, Antifungals, Flucytosine, Candidiasis, Ergosterol, Echinocandin

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Background Cryptococcal meningitis accounts for more than 100,000 human immunodeficiency virus (HIV)-related deaths per year. We tested two treatment strategies that could be more sustainable in Africa than the standard of 2 weeks of amphotericin B plus flucytosine and more effective than the widely used fluconazole monotherapy. Methods We randomly assigned HIV-infected adults with cryptococcal meningitis to receive an oral regimen (fluconazole [1200 mg per day] plus flucytosine [100 mg per kilogram of body weight per day] for 2 weeks), 1 week of amphotericin B (1 mg per kilogram per day), or 2 weeks of amphotericin B (1 mg per kilogram per day). Each patient assigned to receive amphotericin B was also randomly assigned to receive fluconazole or flucytosine as a partner drug. After induction treatment, all the patients received fluconazole consolidation therapy and were followed to 10 weeks. Results A total of 721 patients underwent randomization. Mortality in the oral-regimen, 1-week amphotericin B, and 2-week amphotericin B groups was 18.2% (41 of 225), 21.9% (49 of 224), and 21.4% (49 of 229), respectively, at 2 weeks and was 35.1% (79 of 225), 36.2% (81 of 224), and 39.7% (91 of 229), respectively, at 10 weeks. The upper limit of the one-sided 97.5% confidence interval for the difference in 2-week mortality was 4.2 percentage points for the oral-regimen group versus the 2-week amphotericin B groups and 8.1 percentage points for the 1-week amphotericin B groups versus the 2-week amphotericin B groups, both of which were below the predefined 10-percentage-point noninferiority margin. As a partner drug with amphotericin B, flucytosine was superior to fluconazole (71 deaths [31.1%] vs. 101 deaths [45.0%]; hazard ratio for death at 10 weeks, 0.62; 95% confidence interval [CI], 0.45 to 0.84; P=0.002). One week of amphotericin B plus flucytosine was associated with the lowest 10-week mortality (24.2%; 95% CI, 16.2 to 32.1). Side effects, such as severe anemia, were more frequent with 2 weeks than with 1 week of amphotericin B or with the oral regimen. Conclusions One week of amphotericin B plus flucytosine and 2 weeks of fluconazole plus flucytosine were effective as induction therapy for cryptococcal meningitis in resource-limited settings. (ACTA Current Controlled Trials number, ISRCTN45035509 .).

Concepts: AIDS, Africa, Tuberculosis, Amphotericin B, Antifungals, Flucytosine, Antifungal drug, World Health Organization essential medicines

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The fungal pathogen Cryptococcus neoformans poses a major threat to immunocompromised patients and is a leading killer of human immunodeficiency virus (HIV)-infected patients worldwide. Cryptococci are known to manipulate host macrophages and can either remain latent or proliferate intracellularly within the host phagocyte, a favourable niche that also renders them relatively insensitive to antifungal agents. Here we report an attempt to address this limitation by using a fluorescence-based drug screening method to identify potential inhibitors of intracellular proliferation of C. neoformans. The Prestwick Chemical Library(®) of FDA-approved small molecules was screened for compounds that limit the intracellular replication of a fluorescently-tagged C. neoformans reference strain (H99-GFP) in macrophages. Preliminary screening revealed 19 of 1200 compounds that could significantly reduce intracellular growth of the pathogen. Secondary screening and host cell cytotoxicity assays highlighted fendiline hydrochloride as a potential drug candidate for the development of future anticryptococcal therapies. Live cell imaging demonstrated that this Ca(2+) channel blocker strongly enhanced phagosome maturation in macrophages leading to improved fungal killing and reduced intracellular replication. Whilst the relatively high dose of fendiline hydrochloride required renders it unfit for clinical deployment against cryptococcosis, this study highlights a novel approach for identifying new lead compounds and unravels a pharmacologically promising scaffold towards the development of novel antifungal therapies for this neglected disease.

Concepts: HIV, AIDS, Immune system, Cryptococcus neoformans, Enzyme inhibitor, Flucytosine, Basidiomycota, Fluconazole

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Rhodotorula species are emergent fungal pathogens capable of causing invasive infections, primarily fungemia. They are particularly problematic in immunosuppressed patients when using a central venous catheter. In this study, we evaluated the species distribution of 51 clinical and 8 environmental Rhodotorula species isolates using the ID32C system and internal transcribed spacer (ITS) sequencing. Antifungal susceptibility testing and biofilm formation capability using a crystal violet staining assay were performed. Using ITS sequencing as the gold standard, the clinical isolates were identified as follows: 44 R. mucilaginosa isolates, 2 R. glutinis isolates, 2 R. minuta isolates, 2 R. dairenensis isolates, and 1 Rhodosporidium fluviale isolate. The environmental isolates included 7 R. mucilaginosa isolates and 1 R. slooffiae isolate. Using the ID32C system, along with a nitrate assimilation test, only 90.3% of the isolates tested were correctly identified. In the biofilm formation assay, R. mucilaginosa and R. minuta exhibited greater biofilm formation ability compared to the other Rhodotorula species; the clinical isolates of R. mucilaginosa showed greater biofilm formation compared to the environmental isolates (P = 0.04). Amphotericin B showed good in vitro activity (MIC ≤ 1 μg/ml) against planktonic cells, whereas voriconazole and posaconazole showed poor activity (MIC(50)/MIC(90), 2/4 μg/ml). Caspofungin and fluconazole MICs were consistently high for all isolates tested (≥64 μg/ml and ≥ 4 μg/ml, respectively). In this study, we emphasized the importance of molecular methods to correctly identify Rhodotorula species isolates and non-R. mucilaginosa species in particular. The antifungal susceptibility profile reinforces amphotericin B as the antifungal drug of choice for the treatment of Rhodotorula infections. To our knowledge, this is the first study evaluating putative differences in the ability of biofilm formation among different Rhodotorula species.

Concepts: Molecular biology, Biofilm, Catheter, Antifungals, Flucytosine, Candidiasis, Ergosterol, Antifungal drug

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The high-affinity reductive iron uptake system that includes a ferroxidase (Cfo1) and an iron permease (Cft1) is critical for the pathogenesis of Cryptococcus neoformans. In addition, a mutant lacking CFO1 or CFT1 not only has reduced iron uptake but also displays a markedly increased susceptibility to azole antifungal drugs. Altered antifungal susceptibility of the mutants was of particular interest because the iron uptake system has been proposed as an alternative target for antifungal treatment. In this study, we used transcriptome analysis to begin exploring the molecular mechanisms of altered antifungal susceptibility in a cfo1 mutant. The wild-type strain and the cfo1 mutant were cultured with or without the azole antifungal drug fluconazole and their transcriptomes were compared following sequencing with Illumina Genome Analyzer IIx (GAIIx) technology. As expected, treatment of both strains with fluconazole caused elevated expression of genes in the ergosterol biosynthetic pathway that includes the target enzyme Erg11. Additionally, genes differentially expressed in the cfo1 mutant were involved in iron uptake and homeostasis, mitochondrial functions and respiration. The cfo1 mutant also displayed phenotypes consistent with these changes including a reduced ratio of NAD(+)/NADH and down-regulation of Fe-S cluster synthesis. Moreover, combination treatment of the wild-type strain with fluconazole and the respiration inhibitor diphenyleneiodonium dramatically increased susceptibility to fluconazole. This result supports the hypothesis that down-regulation of genes required for respiration contributed to the altered fluconazole susceptibility of the cfo1 mutant. Overall, our data suggest that iron uptake and homeostasis play a key role in antifungal susceptibility and could be used as novel targets for combination treatment of cryptococcosis. Indeed, we found that iron chelation in combination with fluconazole treatment synergistically inhibited the growth of C. neoformans.

Concepts: AIDS, Gene, Gene expression, Cryptococcus neoformans, Flucytosine, Antifungal drug, Fluconazole, Cryptococcosis

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Cryptococcus gattii is responsible for an expanding epidemic of serious infections in Western Canada and the Northwestern United States (NW), some patients with these infections respond poorly to azole antifungals, and high azole MICs have been reported in NW C. gattii. In this study, 25 NW C. gattii had higher MICs for multiple azoles (but not amphotericin B) than did 34 non-NW C. gattii or 20 Cryptococcus neoformans strains. We therefore examined the roles in azole resistance of overexpression of or mutations in the gene (ERG11) encoding the azole target enzyme. ERG11/ACT1 mRNA ratios were higher in C. gattii than in C. neoformans, but these ratios did not differ in NW and non-NW C. gattii strains, nor did they correlate with fluconazole MICs within any group. Three NW C. gattii strains with low azole MICs and 2 with high azole MICs had deduced Erg11p sequences that differed at one or more position from that of the fully-sequenced NW C. gattii strain R265. However, the azole MICs of conditional S. cerevisiae erg11 mutants expressing the 5 variant ERG11s were within 2-fold of the azole MICs of S. cerevisiae expressing the ERG11s from C. gattii R265, non-NW C. gattii strain WM276, or C. neoformans strains H99 or JEC21. We conclude that neither ERG11 overexpression nor variations in ERG11 coding sequences was responsible for the high azole MICs observed in the NW C. gattii strains we studied.

Concepts: Fungus, Yeast, Cryptococcus neoformans, Flucytosine, Basidiomycota, Cryptococcus, Yeasts, Cryptococcosis

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Molecular epidemiology studies on cryptococcemia are limited. This study aimed to describe the clinical features of patients with bloodstream infections by Cryptococcus sp. in a public tertiary hospital in Mato Grosso do Sul, as well as identify the fungus' molecular type and determine its antifungal susceptibility. Molecular typing was performed using URA5 restriction fragment length polymorphism PCR, and antifungal susceptibility was determined by microdilution method standardized by the Clinical and Laboratory Standards Institute. Over 14 years, 48 patients were diagnosed with cryptococcemia. The majority (72.9 %) was male with a median age of 40 years; 81.3 % of the patients had HIV/AIDS and 72.9 % died. Cryptococcus neoformans was the most commonly isolated species (97.9 %). Molecular analysis identified the genotypes C. neoformans VNI (93.7 %), C. neoformans VNII (4.2 %), and Cryptococcus gattii VGII (2.1 %). In vitro, these fungi were not resistant to fluconazole, itraconazole, voriconazole, and amphotericin B. This is the first description of the molecular types of cryptococcemia agents in central-west Brazil. Its high lethality, especially in HIV-negative patients, suggests that early diagnosis and prompt antifungal therapy are crucial for a good clinical outcome.

Concepts: Fungus, Cryptococcus neoformans, Antifungals, Flucytosine, Basidiomycota, Cryptococcus, Fluconazole, Cryptococcosis

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Abstract Objective: The conventional liposomal amphotericin B causes many unwanted side effects like blood disorder, nephrotoxicity, dose-dependent side effects, highly variable oral absorption and formulation-related instability. The objective of the present investigation was to develop cost-effective nanoemulsion as nanocarreir for enhanced and sustained delivery of amphotericin B into the skin. Methods and characterizations: Different oil-in-water nanoemulsions were developed by varying the composition of hydrophilic (Tween® 80) surfactants and co-surfactant by the spontaneous titration method. The developed formulation were characterized, optimized, evaluated and compared for the skin permeation with commercial formulation (fungisome 0.01% w/w). Optimized formulations loaded with amphotericin B were screened using varied concentrations of surfactants and co-surfactants as decided by the ternary phase diagram. Results and discussion: The maximum % transmittance obtained were 96.9 ± 1.0%, 95.9 ± 3.0% and 93.7 ± 1.2% for the optimized formulations F-I, F-III and F-VI, respectively. These optimized nanoemulsions were subjected to thermodynamic stability study to get the most stable nanoemulsions (F-I). The results of the particle size and zeta potential value were found to be 67.32 ± 0.8 nm and -3.7 ± 1.2 mV for the final optimized nanoemulsion F-I supporting transparency and stable nanoemulsion for better skin permeation. The steady state transdermal flux for the formulations was observed between 5.89 ± 2.06 and 18.02 ± 4.3 µg/cm(2)/h whereas the maximum enhancement ratio were found 1.85- and 3.0-fold higher than fungisome and drug solution, respectively, for F-I. The results of the skin deposition study suggests that 231.37 ± 3.6 µg/cm(2) drug deposited from optimized nanoemulsion F-I and 2.11-fold higher enhancement ratio as compared to fungisome. Optimized surfactants and co-surfactant combination-mediated transport of the drug through the skin was also tried and the results were shown to have facilitated drug permeation and skin perturbation (SEM). Conclusion: The combined results suggested that amphotericin B nanoemulsion could be a better option for localized topical drug delivery and have greater potential as an effective, efficient and safe approach.

Concepts: Thermodynamics, Topical, Amphotericin B, Gilead Sciences, Antifungals, Flucytosine, Ergosterol, Nephrotoxicity

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Cryptococcus neoformans and C. gattii are the etiologic agents of cryptococcosis, a life-threatening disease in both immunocompromised and immunocompetent hosts. Antifungal resistance has been evaluated using different methods, breakpoints, and sizes of test populations and it is an emerging as a significant issue worldwide. A total of 176 (95 clinical and 81 environmental) C. neoformans and eight clinical C. gattii isolates were evaluated to determine the minimal inhibitory concentration (MIC) according to the Clinical and Laboratory Standards Institute method. A total of 10.5% of the C. neoformans clinical isolates were resistant to amphotericin B (AMB), and 6.2% of the environmental isolates were resistant to fluconazole (FLZ). Environmental and clinical isolates presented epidemiologic cut-off values (ECVs) of 64 and 16 to FLZ and 1 and 2 to AMB, respectively. All of the C. gattii isolates showed high susceptibility to most drugs evaluated. Clinical isolates had lower susceptibility than environmental isolates to AMB and itraconazole whereas environmental isolates had lower susceptibility than the clinical isolates to FLZ, voriconazole, and ketoconazole. However, no difference was found in the susceptibility of the two species. The MICs and ECVs to antifungals can help to select the best therapeutic option for tracking epidemiological resistance among clinical and environmental isolates of Cryptococcus spp. around the world.

Concepts: AIDS, Cryptococcus neoformans, Antifungals, Flucytosine, Cryptococcus, Fluconazole, Minas Gerais, Cryptococcosis

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Fungal infections create a significant risk to pediatric lung transplant recipients. However, no international consensus guidelines exist for fungal infection prevention strategies. It was the aim to describe the current strategies of antifungal prophylaxis in pediatric lung transplant centers. A self-administered, web-based survey on current practices to prevent fungal infection was circulated to centers within the IPLTC. Twenty-one (88%) IPLTC centers participated, predominantly from Europe and the US. More than 50% of respondents perform adult and pediatric lung transplant operations. Twenty-four percent use universal prophylaxis, 28% give prophylaxis to all patients but stratify the antifungal coverage based on pretransplant risk, and 48% target prophylaxis to only the children with CF or pretransplantation fungal colonization. Commonly, centers aim to target Aspergillus and Candida infection. Monotherapy with either voriconazole or inhaled amphotericin B is used in the majority of centers. Institutions utilize prophylactic therapy for variable time periods (40% 3-6 months; 30% ≥12 months). Alternative drugs were prescribed for lack of tolerance, toxicity, or positive surveillance culture. TDM (itraconazole/voriconazole) was used in 86% of centers. The survey revealed a wide range of antifungal prophylaxis strategies as current international practice in pediatric lung transplant recipients.

Concepts: Medicine, Infectious disease, Pleural effusion, Organ transplant, Antifungals, Flucytosine, Candidiasis, Ergosterol