Analysis of the cyp51 genes contribution to azole resistance in Aspergillus section Nigri with the CRISPR-Cas9 technique.

Cyp51 contribution to azole resistance has been broadly studied and characterized in Aspergillus fumigatus, whereas it remains poorly investigated in other clinically relevant species of the genus, such as those of section Nigri In this work, we aimed to analyze the impact of cyp51 genes (cyp51A and cyp51B) on the voriconazole (VRC) response and resistance of Aspergillus niger and Aspergillus tubingensis We generated CRISPR-Cas9 cyp51A and cyp51B knock-out mutants from strains with different genetic backgrounds and diverse patterns of azole susceptibility. Single gene deletions of cyp51 genes resulted in 2 to 16-fold decrease of the VRC Minimum Inhibitory Concentration (MIC) values, which were below the VRC Epidemiological Cutoff Value (ECV) established by the Clinical and Laboratory Standards Institute (CLSI) irrespective of their parental strains susceptibilities. Gene expression studies in the tested species confirmed that cyp51A participates more actively than cyp51B in the transcriptional response of azole stress. However, ergosterol quantification revealed that both enzymes comparably impact the total ergosterol content within the cell, as basal and VRC-induced changes to ergosterol content was similar in all cases. These data contribute to our understanding on Aspergillus azole resistance, especially in non-fumigatus species.

A revision of malbranchea-like fungi from clinical specimens in the United States of America reveals unexpected novelty.

The fungi of the order Onygenales can cause important human infections; however, their taxonomy and worldwide occurrence is still little known. We have studied and identified a representative number of clinical fungi belonging to that order from a reference laboratory in the USA. A total of 22 strains isolated from respiratory tract (40%) and human skin and nails (27.2%) showed a malbranchea-like morphology. Six genera were phenotypically and molecularly identified, i.e. Auxarthron/Malbranchea (68.2%), Arachnomyces (9.1%), Spiromastigoides (9.1%), and Currahmyces (4.5%), and two newly proposed genera (4.5% each). Based on the results of the phylogenetic study, we synonymized Auxarthron with Malbranchea, and erected two new genera: Pseudoarthropsis and Pseudomalbranchea. New species proposed are: Arachnomyces bostrychodes, A. graciliformis, Currahmyces sparsispora, Malbranchea gymnoascoides, M. multiseptata, M. stricta, Pseudoarthropsis crassispora, Pseudomalbranchea gemmata, and Spiromastigoides geomycoides, along with a new combination for Malbranchea gypsea. The echinocandins showed the highest in vitro antifungal activity against the studied isolates, followed by terbinafine and posaconazole; in contrast, amphotericin B, fluconazole, itraconazole and 5-fluorocytosine were less active or lacked in vitro activity against these fungi.

Sarocladium and Acremonium infections: New faces of an old opportunistic fungus.

The genera Acremonium and Sarocladium comprise a high diversity of morphologically and genetically related fungi generally found in the environment, although a few species, mainly Sarocladium kiliense and Acremonium egyptiacum, can also be involved in many human infections. Clinical management of opportunistic infections caused by these fungi is very complex, since their correct identification is unreliable, and they generally show poor antifungal response. More than 300 clinical cases involving a broad range of Acremonium/Sarocladium infections have so far been published, and with this review we aim to compile and provide a detailed overview of the current knowledge on Acremonium/Sarocladium human infections in terms of presentation, diagnosis, treatments and prognoses. We also aim to summarise and discuss the data currently available on their antifungal susceptibility, emphasising the promising results obtained with voriconazole as well as their impact in terms of animal infections.

Improvement of the pharmacokinetic/pharmacodynamic relationship in the treatment of invasive aspergillosis with voriconazole. Reduced drug toxicity through novel rapid release formulations.

Voriconazole (VCZ) is currently the first-line treatment for invasive aspergillosis, although the doses are limited by its poor solubility and high hepatic toxicity. The aim of this study was to develop a solid self-dispersing micellar system of VCZ to improve the pharmacokinetic/pharmacodynamic (PK/PD) relationship and reduce hepatotoxicity. In this work, solid micellar systems of VCZ are formulated with different polysorbate 80 ratios using mannitol as a hydrophilic carrier. The novel micellar systems were characterized by scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC) and dissolution studies. Self-dispersing micellar systems reduced VCZ crystallinity, leading to an improvement in its dissolution rate. The in vitro susceptibility test also revealed that the most common microorganisms in invasive aspergillosis exhibited low minimum inhibitory concentration (MIC) values for micellar systems. Pharmacokinetic studies indicated an improvement in bioavailability for MS-1:3:0.05, and changes in its biodistribution to different organs. MS-1:3:0.05 showed an increased concentration in lungs and a significant decrease in VCZ accumulated in the liver.

Azole resistance mechanisms in Aspergillus: update and recent advances.

Aspergillus fumigatus is the main causal agent of invasive aspergillosis (IA), however other species of the genus can also cause IA, such as Aspergillus flavus, Aspergillus terreus, Aspergillus niger and related cryptic species. This infectious disease mainly affects immunosuppressed patients and is linked to elevated mortality rates. As voriconazole is the treatment of choice for this condition, the relevant increase in the number of azole-resistant isolates in recent years has gathered alarming attention, as it also translates into an increase in clinical failures. In this review, we summarise and discuss the azole resistance molecular data described to date in the most clinically prevalent sections of Aspergillus, including mechanisms involving the target proteins Cyp51 and ATP-binding cassette (ABC) or major facilitator superfamily (MFS) efflux pumps. Other resistance mechanisms proposed but not yet fully characterised are also discussed.

Current knowledge on the etiology and epidemiology of Scopulariopsis infections.

Scopulariopsis is a common fungus in the environment, characterized by its intrinsic resistance to the available antifungal drugs. Around 70 cases of infection by this fungus have been described in the literature. Pulmonary and disseminated infections are the most common and their treatment is difficult; therefore, very diverse approaches have been taken, with varied results. A successful outcome has been reported in only a few cases, generally attributed to a multitreatment strategy combining medical and surgical procedures that ultimately led to the resection of the infected tissue if possible, identification of the mould, and an aggressive long-term antifungal therapy. Although most of the infections are caused by Scopulariopsis brevicaulis, a few other species have also been linked to these cases, although molecular evidence has not been proven for all of them. On this basis, more knowledge on the epidemiology, presentation, diagnosis, treatment, and prognosis of these unusual infections would improve their management. This review aims to compile the current data on Scopulariopsis infections.

Increased Efficacy of Oral Fixed-Dose Combination of Amphotericin B and AHCC® Natural Adjuvant against Aspergillosis.

Invasive pulmonary aspergillosis represents one of the most serious fungal infections among immunocompromised patients. In this study, we aimed to analyze the in vivo efficacy of prophylactic oral amphotericin B (AMB) encapsulated in modified chitosan-nanoparticles (Nanomerics' Molecular Envelope Technology (MET)) supplemented with a standardized extract of cultured Lentinula edodes mycelia (AHCC®) in a murine model of pulmonary aspergillosis. We determined fungal burden and survival of mice and additionally, we carried out a cytokine analysis in an attempt to understand the immunomodulation of the extract. Our results evidenced equivalent efficacy between orally administered AMB-MET and the intravenous liposomal AMB marketed formulation. Addition of the AHCC® supplement significantly improved efficacy in terms of burden reduction and survival increase of both oral and intravenous AMB therapies compared to the untreated control group. Moreover, a protective effect of the extract was observed in terms of weight loss. Regarding the cytokine profiles, the Th1 immune response was stimulated in treated animals when compared to the control group. This response was marked by an enhancement in the MCP-1, GM-CSF, VEGF, RANTES and IL-17 levels and a decrease in the IL-6, a biomarker related to the severity of the infection.

New Insights into the Cyp51 Contribution to Azole Resistance in Aspergillus Section Nigri.

Invasive aspergillosis (IA) is a severe condition mainly caused by Aspergillus fumigatus, although other species of the genus, such as section Nigri members, can also be involved. Voriconazole (VRC) is the recommended treatment for IA; however, the prevalence of azole-resistant Aspergillus isolates has alarmingly increased in recent years, and the underlying resistance mechanisms in non-fumigatus species remain unclear. We have determined the in vitro susceptibility of 36 strains from section Nigri to VRC, posaconazole (POS), and itraconazole (ITC), and we have explored the role of Cyp51A and Cyp51B, both targets of azoles, in azole resistance. The three drugs were highly active; POS displayed the best in vitro activity, while ITC and VRC showed MICs above the established epidemiological cutoff values in 9 and 16% of the strains, respectively. Furthermore, expression studies of cyp51A and cyp51B in control condition and after VRC exposure were performed in 14 strains with different VRC susceptibility. We found higher transcription of cyp51A, which was upregulated upon VRC exposure, but no correlation between MICs and cyp51 transcription levels was observed. In addition, cyp51A sequence analyses revealed nonsynonymous mutations present in both, wild-type and non-wild-type strains of A. niger and A. tubingensis Nevertheless, a few mutations were exclusively present in non-wild-type A. tubingensis strains. Altogether, our results suggest that azole resistance in section Nigri is not clearly explained by Cyp51A protein alteration or by cyp51 gene upregulation, which indicates that other mechanisms might be involved.

Antifungal susceptibility of Saccharomyces cerevisiae and therapy in a murine model of disseminated infection / Sensibilidad antifúngica de Saccharomyces cerevisiae en un modelo murino de infección diseminada

Background: The incidence of systemic infections by Saccharomyces cerevisiae has increased in recent years, especially among immunocompromised patients. Amphotericin B, voriconazole or echinocandins have been used with favorable outcome against systemic infections by this fungus. However, clinical experience is limited and no in vivo studies have been conducted. Aims: We evaluated the in vitro activity of nine antifungal compounds against S.cerevisiae and the in vivo efficacy of those three antifungals showing the highest in vitro activity by using a murine model of systemic infection. Methods: Minimal inhibitory concentrations (MICs) were determined by the microdilution method against three strains of S. cerevisiae. After intravenous infection with 5 × 107 CFUs, animals received liposomal amphotericin B (5 mg/kg), voriconazole (25 mg/kg) or anidulafungin (5 mg/kg). Treatment efficacy was assessed by determining of CFUs/g in liver, kidney, brain, lung and spleen. Results: 5-Fluorocytosine was the most in vitro active compound followed by amphotericin B, voriconazole and anidulafungin. The in vivo study showed that liposomal amphotericin B was the most effective drug driving highest fungal clearance. Conclusions: All treatments reduced the fungal load in comparison to the control group, being liposomal amphotericin B the most effective drug followed by anidulafungin and finally voriconazole

Antecedentes: La incidencia de infecciones sistémicas causadas por Saccharomyces cerevisiae ha aumentado en los últimos años, especialmente entre pacientes inmunodeprimidos. A pesar de que la anfotericina B, el voriconazol o las equinocandinas han dado buen resultado en infecciones sistémicas por este hongo, no se han establecido recomendaciones terapéuticas sólidas. Objetivos: Se evaluó la actividad in vitro de nueve antifúngicos frente a S. cerevisiae y la eficacia in vivo de los tres fármacos con mayor actividad in vitro mediante un modelo murino de infección sistémica. Métodos: Se determinaron las concentraciones mínimas inhibitorias (CMIs) frente a tres cepas de S. cerevisiae por el método de microdilución. Después de la inoculación intravenosa con 5 × 107UFC, los ratones fueron tratados con anfotericina B liposomal (5 mg/kg), voriconazol (25 mg/kg) o anidulafungina (5 mg/kg). La eficacia de los tratamientos se estableció basándose en la determinación de UFC/g en hígado, riñón, cerebro, pulmón y bazo. Resultados: La 5-fluorocitosina fue el compuesto más activo in vitro, seguido por la anfotericina B liposomal, el voriconazol y la anidulafungina. En el estudio in vivo, la anfotericina B liposomal fue el fármaco más eficaz en términos de reducción de la carga fúngica y esterilización de los órganos estudiados. Conclusiones: Todos los tratamientos redujeron la carga fúngica en comparación con el grupo control, y la anfotericina B liposomal fue el antifúngico más efectivo, seguido de la anidulafungina y el voriconazol

Antifungal susceptibility of Saccharomyces cerevisiae and therapy in a murine model of disseminated infection.

BACKGROUND: The incidence of systemic infections by Saccharomyces cerevisiae has increased in recent years, especially among immunocompromised patients. Amphotericin B, voriconazole or echinocandins have been used with favorable outcome against systemic infections by this fungus. However, clinical experience is limited and no in vivo studies have been conducted. AIMS: We evaluated the in vitro activity of nine antifungal compounds against S.cerevisiae and the in vivo efficacy of those three antifungals showing the highest in vitro activity by using a murine model of systemic infection. METHODS: Minimal inhibitory concentrations (MICs) were determined by the microdilution method against three strains of S. cerevisiae. After intravenous infection with 5×107 CFUs, animals received liposomal amphotericin B (5mg/kg), voriconazole (25mg/kg) or anidulafungin (5mg/kg). Treatment efficacy was assessed by determining of CFUs/g in liver, kidney, brain, lung and spleen. RESULTS: 5-Fluorocytosine was the most in vitro active compound followed by amphotericin B, voriconazole and anidulafungin. The in vivo study showed that liposomal amphotericin B was the most effective drug driving highest fungal clearance. CONCLUSIONS: All treatments reduced the fungal load in comparison to the control group, being liposomal amphotericin B the most effective drug followed by anidulafungin and finally voriconazole.

Efficacy, Biodistribution, and Nephrotoxicity of Experimental Amphotericin B-Deoxycholate Formulations for Pulmonary Aspergillosis.

An experimental micellar formulation of 1:1.5 amphotericin B-sodium deoxycholate (AMB:DCH 1:1.5) was obtained and characterized to determine its aggregation state and particle size. The biodistribution, nephrotoxicity, and efficacy against pulmonary aspergillosis in a murine model were studied and compared to the liposomal commercial formulation of amphotericin B after intravenous administration. The administration of 5 mg/kg AMB:DCH 1:1.5 presented 2.8-fold-higher lung concentrations (18.125 ± 3.985 µg/g after 6 daily doses) and lower kidney exposure (0.391 ± 0.167 µg/g) than liposomal commercial amphotericin B (6.567 ± 1.536 and 5.374 ± 1.157 µg/g in lungs and kidneys, respectively). The different biodistribution of AMB:DCH micelle systems compared to liposomal commercial amphotericin B was attributed to their different morphologies and particle sizes. The efficacy study has shown that both drugs administered at 5 mg/kg produced similar survival percentages and reductions of fungal burden. A slightly lower nephrotoxicity, associated with amphotericin B, was observed with AMB:DCH 1:1.5 than the one induced by the liposomal commercial formulation. However, AMB:DCH 1:1.5 reached higher AMB concentrations in lungs, which could represent a therapeutic advantage over liposomal commercial amphotericin B-based treatment of pulmonary aspergillosis. These results are encouraging to explore the usefulness of AMB:DCH 1:1.5 against this disease.