In vitro activity of isavuconazole against clinically isolated yeasts from Chile.

Isavuconazole is the last antifungal agent approved by the FDA and available for treatment of fungal infections. In the present study, the in vitro activity of isavuconazole against several yeasts was investigated. Two hundred forty-six isolates were included: 64 Candida albicans, 53 Candida parapsilosis sensu stricto, 48 Cryptococcus neoformans species complex, 27 C. glabrata sensu stricto, 17 C. lusitaniae, 17 C. tropicalis, 5 C. orthopsilosis, 4 C. krusei, 3 C. guilliermondii sensu stricto, 3 C. pelliculosa, 2 C. dubliniensis, 1 C. auris, 1 C. fermentati and 1 Trichosporon asahii. All isolates were recovered from clinical isolates from Chile, being 221 from hemoculture, 22 from cerebrospinal fluid, 1 pleural fluid, and 1 from tissue culture. The minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs) of isavuconazole were determined. Isavuconazole demonstrated good in vitro activity against all species tested. MIC90 values and MFC ranges of isavuconazole for Candida albicans were 0.03 mg/L and 0.03- > 16 mg/L respectively. Non-Candida albicans species isolates were inhibited by ≤ 1 mg/L, with MFC ranges from < 0.03- > 16 mg/L. Also, isavuconazole was active against the non-Candida yeasts, being inhibited with MIC values ≤ 0.06 mg/L. Isavuconazole has exhibited potent in vitro activity against clinical isolates of Candida spp., Cryptococcus neoformans species complex, and other clinical yeast in Chile. Despite the results obtained in the present work, additional clinical studies are necessary to verify the level of efficacy of this azole.

[Outbreak of fungemias by Sarocladium kiliense in eight public hospitals per intrinsic contamination of ondansetron intravenous]. / Brote de fungemias por Sarocladium kiliense en ocho hospitales públicos por contaminación intrínseca de ondansetrón intravenoso.

Sarocladium kiliense is a saprophyte fungus that can cause opportunistic infections associated to invasive procedures. We report a multi-hospital nosocomial outbreak of fungemias due to this agent. Patients with positive blood culture to this agent were studied after six bloodstream infections identified in three Chilean hospitals in July 2013 were reported to Ministry of Health National Infection and Prevention Control Program. In general, there were mild clinical manifestations, without deaths attributable to the infection. Epidemiological and micro-biological study identified 65 cases in 8 hospitals, mostly pediatric patients in chemotherapy. Initial studies of 94 different drugs and medical devices had negative results, until a second analysis of specific blisters and their pharmaceutical matrix selected by epidemiological criteria identified an intrinsic contamination of ondansetron blisters from a specific producer used in all the patients. A recall of contaminated ondansetron blisters was performed in all the country, after which the outbreak was contained. Surveillance and response of local and national infection prevention and control programs and laboratory support were key to control of a national multi-hospital common source outbreak due to contamination of a drug by an unusual fungus.

Brote de fungemias por Sarocladium kiliense en ocho hospitales públicos por contaminación intrínseca de ondansetrón intravenoso / Outbreak of fungemias by Sarocladium kiliense in eight public hospitals per intrinsic contamination of ondansetron intravenous

Resumen Introducción: Sarocladium kiliense es un hongo saprófito que puede generar infecciones oportunistas asociadas a procedimientos invasores. Se informa un brote multicéntrico nosocomial de fungemias de fuente común por este agente. Luego del reporte de cinco casos en pacientes en tres hospitales al Programa de Control de Infecciones del Ministerio de Salud de Chile en julio de 2013, se estudiaron a nivel nacional todos los pacientes con hemocultivo positivo para este agente. Se trató de cuadros clínicos leves a moderados, sin muertes atribuibles. El estudio identificó 65 casos en 8 hospitales, en su mayoría pacientes pediátricos en quimioterapia. Estudios iniciales de 94 muestras de cuatro fármacos y dispositivos usados en todos los casos resultaron negativas hasta que, en un segundo análisis de lotes seleccionados por criterios epidemiológicos y su matriz farmacéutica, se identificó la contaminación intrínseca de ampollas de ondansetrón de un productor específico, que se usó en todos los casos. Se realizó un retiro nacional de las ampollas de los tres lotes contaminados del fármaco, después de lo cual se contuvo el brote. La vigilancia de infecciones en los hospitales y el programa nacional coordinado con los laboratorios de microbiología fueron claves para identificar un brote multicéntrico de fuente común por contaminación de un fármaco por un hongo inusual.

Sarocladium kiliense is a saprophyte fungus that can cause opportunistic infections associated to invasive procedures. We report a multi-hospital nosocomial outbreak of fungemias due to this agent. Patients with positive blood culture to this agent were studied after six bloodstream infections identified in three Chilean hospitals in July 2013 were reported to Ministry of Health National Infection and Prevention Control Program. In general, there were mild clinical manifestations, without deaths attributable to the infection. Epidemiological and micro-biological study identified 65 cases in 8 hospitals, mostly pediatric patients in chemotherapy. Initial studies of 94 different drugs and medical devices had negative results, until a second analysis of specific blisters and their pharmaceutical matrix selected by epidemiological criteria identified an intrinsic contamination of ondansetron blisters from a specific producer used in all the patients. A recall of contaminated ondansetron blisters was performed in all the country, after which the outbreak was contained. Surveillance and response of local and national infection prevention and control programs and laboratory support were key to control of a national multi-hospital common source outbreak due to contamination of a drug by an unusual fungus.

Whole-Genome Sequencing to Determine Origin of Multinational Outbreak of Sarocladium kiliense Bloodstream Infections.

We used whole-genome sequence typing (WGST) to investigate an outbreak of Sarocladium kiliense bloodstream infections (BSI) associated with receipt of contaminated antinausea medication among oncology patients in Colombia and Chile during 2013-2014. Twenty-five outbreak isolates (18 from patients and 7 from medication vials) and 11 control isolates unrelated to this outbreak were subjected to WGST to elucidate a source of infection. All outbreak isolates were nearly indistinguishable (<5 single-nucleotide polymorphisms), and >21,000 single-nucleotide polymorphisms were identified from unrelated control isolates, suggesting a point source for this outbreak. S. kiliense has been previously implicated in healthcare-related infections; however, the lack of available typing methods has precluded the ability to substantiate point sources. WGST for outbreak investigation caused by eukaryotic pathogens without reference genomes or existing genotyping methods enables accurate source identification to guide implementation of appropriate control and prevention measures.

The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor.

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca(2+), but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca(2+). This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca(2+), absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized anti-phospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site ((645)R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q(660)) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.

Ca2+/Calmodulin and Apo-Calmodulin Both Bind to and Enhance the Tyrosine Kinase Activity of c-Src.

Src family non-receptor tyrosine kinases play a prominent role in multiple cellular processes, including: cell proliferation, differentiation, cell survival, stress response, and cell adhesion and migration, among others. And when deregulated by mutations, overexpression, and/or the arrival of faulty incoming signals, its hyperactivity contributes to the development of hematological and solid tumors. c-Src is a prototypical member of this family of kinases, which is highly regulated by a set of phosphorylation events. Other factor contributing to the regulation of Src activity appears to be mediated by the Ca2+ signal generated in cells by different effectors, where the Ca2+-receptor protein calmodulin (CaM) plays a key role. In this report we demonstrate that CaM directly interacts with Src in both Ca2+-dependent and Ca2+-independent manners in vitro and in living cells, and that the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibits the activation of this kinase induced by the upstream activation of the epidermal growth factor receptor (EGFR), in human carcinoma epidermoide A431 cells, and by hydrogen peroxide-induced oxidative stress, in both A431 cells and human breast adenocarcinoma SK-BR-3 cells. Furthermore, we show that the Ca2+/CaM complex strongly activates the auto-phosphorylation and tyrosine kinase activity of c-Src toward exogenous substrates, but most relevantly and for the first time, we demonstrate that Ca2+-free CaM (apo-CaM) exerts a far higher activatory action on Src auto-phosphorylation and kinase activity toward exogenous substrates than the one exerted by the Ca2+/CaM complex. This suggests that a transient increase in the cytosolic concentration of free Ca2+ is not an absolute requirement for CaM-mediated activation of Src in living cells, and that a direct regulation of Src by apo-CaM could be inferred.

Characterization of phospho-(tyrosine)-mimetic calmodulin mutants.

Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro.

Evaluation of the correlation of caspofungin MICs and treatment outcome in murine infections by wild type strains of Candida parapsilosis.

We have evaluated the in vitro activity of caspofungin against 36 wild-type strains of Candida parapsilosis sensu stricto using 3 techniques: broth microdilution, disk diffusion, and the determination of minimal fungicidal concentration (MFC). The first 2 methods showed a good in vitro activity of caspofungin, but the MFCs were ≥2 dilutions above their corresponding MICs. In a murine model of disseminated infection, we evaluated the efficacy of caspofungin at 5 mg/kg against 8 strains of C. parapsilosis representing different degrees of in vitro susceptibility (0.12-1 µg/mL). All the isolates responded to treatment and (1→3)-ß-D-glucan levels were reduced in all the cases; however, the study revealed differences among isolates, since caspofungin reduced the tissue burden of mice infected with isolates with MICs ≤0.5 µg/mL but was less effective against those with MICs of 1 µg/mL.

Evaluation of the in vitro activity of voriconazole as predictive of in vivo outcome in a murine Aspergillus fumigatus infection model.

We have evaluated the in vitro activity of voriconazole against 61 strains of Aspergillus fumigatus by using broth microdilution, disk diffusion, and minimal fungicidal concentration procedures. We observed an excellent correlation between the results obtained with the three methods. Five percent of the strains showed MICs greater than or equal to the epidemiological cutoff value (ECV; 1 µg/ml). To assess whether MICs were predictive of in vivo outcome, we tested the efficacy of voriconazole at 25 mg/kg of body weight daily in an immunosuppressed murine model of disseminated infection using 10 strains representing various patterns of susceptibility to the drug as determined by the in vitro study. Voriconazole prolonged survival and reduced fungal load in the kidneys and brain in those mice infected with strains with MICs of ≤0.25 µg/ml, while in mice infected with strains with MICs of 0.5 to 2 µg/ml, the efficacy was varied and strain dependent and in mice infected with the strain with a MIC of 4 µg/ml, the antifungal did not show efficacy. Voriconazole reduced galactomannan antigenemia against practically all strains with a MIC of <4 µg/ml. Our results demonstrate that some relationship exists between voriconazole MICs and in vivo efficacy; however, further studies testing additional strains are needed to better ascertain which MIC values can predict clinical outcome.

MIC values of voriconazole are predictive of treatment results in murine infections by Aspergillus terreus species complex.

We evaluated the efficacy of voriconazole against nine strains of Aspergillus terreus with different MICs (0.12 to 4 µg/ml) by using a murine model. Markers of efficacy included survival, tissue burden, galactomannan antigenemia, and drug serum levels. Voriconazole was especially effective in prolonging survival and reducing the fungal load in infections by strains that showed MICs that were less than or equal to the epidemiological cutoff value (1 µg/ml). In vitro data might be useful for predicting the outcome of A. terreus infections.

Are epidemiologic cut-off values predictors of the in vivo efficacy of azoles in experimental aspergillosis?

We tried to correlate the in vitro activity and the in vivo efficacy of voriconazole (VRC) and posaconazole (POS) against Aspergillus flavus and Aspergillus niger. The in vitro susceptibility of a large set of isolates was determined by broth microdilution and disk diffusion methods, while the in vivo efficacy was assessed in a murine model of disseminated infection. For A. flavus, VRC showed efficacy even for strains with MICs above the epidemiologic cutoff value (ECV) (1 µg/mL). For A. niger, VRC was ineffective against those strains for which the MIC was 1 dilution below the corresponding ECV (2 µg/mL). POS showed efficacy against all the strains of both species included in the study, although isolates with MICs > ECV were not tested.

Efficacy of posaconazole in a murine model of disseminated infection caused by Apophysomyces variabilis.

OBJECTIVES: We evaluated the in vitro activity of posaconazole and amphotericin B against several clinical strains of the mucoralean fungus Apophysomyces variabilis, and their efficacy in a murine model of disseminated infection caused by that fungus. METHODS: The in vitro susceptibility of seven strains of A. variabilis to posaconazole and amphotericin B was determined by using a broth microdilution method. The in vivo efficacy of both drugs, posaconazole at 20 mg/kg twice daily orally by gavage and amphotericin B at 0.8 mg/kg once daily intravenously, was evaluated against six of the strains previously tested in vitro using immunocompetent mice. RESULTS: In general, MICs of both drugs were within the range of susceptibility or intermediate susceptibility. Posaconazole and amphotericin B were able to significantly reduce the percentages of positive cultures in the affected tissues. However, in general, posaconazole significantly improved survival (median, 23 days; range, 7-30 days) compared with untreated controls (median, 6 days; range, 4-7 days) and, in some cases, with respect to the animals treated with amphotericin B (median, 15 days; range, 5-30 days). CONCLUSIONS: Our results demonstrate the efficacy of posaconazole in the treatment of a disseminated murine infection caused by A. variabilis. However, further clinical studies are required to ascertain the potential use in human infections caused by this fungus.

Experimental murine model of disseminated infection by Saksenaea vasiformis: successful treatment with posaconazole.

We have determined the in vitro activity of amphotericin B (AMB) and posaconazole (PSC) against Saksenaea vasiformis using broth microdilution and disk diffusion methods and determined the minimal fungicidal concentration (MFC). PSC was found to have the greatest in vitro activity in all cases and was the most efficacious in prolonging survival and reducing the fungal load in an immunocompetent murine model of disseminated infection caused by four strains of the fungus.

In vitro and in vivo activities of posaconazole and amphotericin B in a murine invasive infection by Mucor circinelloides: poor efficacy of posaconazole.

The in vitro susceptibility of 17 strains of Mucor circinelloides to amphotericin B and posaconazole was ascertained by using broth microdilution and disk diffusion methods and by determining the minimal fungicidal concentration (MFC). We evaluated the efficacy of posaconazole at 40 mg/kg of body weight/day and amphotericin B at 0.8 mg/kg/day in a neutropenic murine model of disseminated infection by M. circinelloides by using 6 different strains tested previously in vitro. In general, most of the posaconazole MICs were within the range of susceptibility or intermediate susceptibility, while the small inhibition zone diameters (IZDs) were indicative of nonsusceptibility for all isolates tested. The MFCs were ≥ 3 dilutions higher than the corresponding MICs. In contrast, amphotericin B showed good activity against all of the strains tested regardless of the method used. The in vivo studies demonstrated that amphotericin B was effective in prolonging survival and reducing the fungal load. Posaconazole showed poor in vivo efficacy with no correlation with the MIC values. The results suggested that posaconazole should be used with caution in the treatment of infections caused by Mucor circinelloides or by strains of Mucor not identified to the species level.

Histopathology and antifungal treatment of experimental murine chromoblastomycosis caused by Cladophialophora carrionii.

OBJECTIVES: A murine model of chromoblastomycosis caused by Cladophialophora carrionii was used to compare the efficacy of posaconazole and voriconazole with that of terbinafine and itraconazole, the currently used drugs in the management of chromoblastomycosis. METHODS: Athymic nude mice were infected with 2 × 10(7) cfu of a clinical isolate of C. carrionii. When typical lesions were established, treatments with posaconazole at 20 mg/kg/day, voriconazole at 20 mg/kg/day, itraconazole at 50 mg/kg/day or terbinafine at 250 mg/kg/day were initiated. Treatment efficacy was evaluated for 4 months by measuring the size of the lesions, observing any histopathological changes and culturing the excised tissue. RESULTS: Posaconazole was the only drug that reduced the initial lesion size, while voriconazole and terbinafine reduced growth relative to controls. CONCLUSIONS: This study suggests that the newer triazoles have potential in the treatment of chromoblastomycosis caused by C. carrionii.

Combined therapy of voriconazole and anidulafungin in murine infections by Aspergillus flavus.

The efficacy of the combination of anidulafungin (AFG) at 1 mg/kg plus voriconazole (VRC) at 25 mg/kg was evaluated in a murine model of disseminated infection by Aspergillus flavus using three isolates previously tested in vitro. All the combinations showed indifferent in vitro interaction with the exception of one, which showed synergy. In general, the combined treatment prolonged the survival and reduced the fungal load in comparison with AFG alone, and only in a few cases, it improved the results of the VRC monotherapy. The combination of the two drugs and VRC alone reduced the galactomannan levels in serum in comparison with the control group.

Anidulafungin in treatment of experimental invasive infection by Candida parapsilosis: in vitro activity, (1-->3)-beta-D-glucan and mannan serum levels, histopathological findings, and in vivo efficacy.

We have evaluated the in vitro activity of anidulafungin (AFG) against 31 strains of Candida parapsilosis sensu stricto by using broth microdilution, disk diffusion, and minimal fungicidal concentration (MFC) determination procedures. The two first methods showed a high level of activity of the drug, while MFCs were 1 to 5 dilutions higher than their corresponding MICs. To assess if MICs were predictive of in vivo outcomes, six strains representing different AFG MICs (0.12 to 2 µg/ml) were tested in a murine model of disseminated infection treated with different doses of the drug (1, 5, or 10 mg/kg of body weight). AFG was able to prolong the survival of mice infected with all the strains tested but was able to reduce the tissue burden of those mice infected only with the strains that showed the lowest MIC (0.12 µg/ml).

In vitro activity and in vivo efficacy of posaconazole in treatment of murine infections by different isolates of the Aspergillus terreus complex.

Posaconazole (PSC) is an antifungal drug recommended as an alternative for the treatment of invasive aspergillosis in patients who are refractory or intolerant to primary antifungal therapy. We have evaluated the in vitro activity of PSC against 21 strains of the Aspergillus terreus complex using both broth microdilution and disk diffusion (Neo Sensitabs) methods. PSC showed the same high level of activity against all the strains with the two in vitro methods used. We developed a murine model of disseminated infection to evaluate the efficacy of PSC at 5, 10, or 20 mg/kg of body weight twice a day by using 6 different strains chosen randomly. PSC showed good efficacy, especially at 20 mg/kg, as measured by prolonged survival, tissue burden reduction, histopathology, and lowered galactomannan levels. The PSC levels in serum on the fourth day of treatment were higher than the MICs for the strains tested.

In vitro activity and in vivo efficacy of anidulafungin in murine infections by Aspergillus flavus.

Anidulafungin (AFG) showed high activity against 27 strains of Aspergillus flavus by use of broth microdilution and disk diffusion methods. This drug was effective in vivo in a murine model of disseminated infection with five isolates tested. AFG was able to prolong survival and reduce tissue burden of infected mice but not able to reduce galactomannan serum concentrations. The AFG serum levels were above the corresponding minimum effective concentrations (MEC) for all of the strains tested.

Two new species of Mucor from clinical samples.

Two new species in the order Mucorales, Mucor velutinosus and Mucor ellipsoideus, isolated from human clinical specimens in the USA, are described and illustrated. The former species is similar to Mucor ramosissimus, from which it can be differentiated by its ability to grow at 37°C and produce verrucose sporangiospores. Mucor ellipsoideus is also able to grow and sporulate at 37°C like M. indicus, the nearest phylogenetic species in this study, however, the former has narrow ellipsoidal sporangiospores in contrast to the subglobose to ellipsoidal sporangiospores of M. indicus. Analysis of the sequences of the ITS and the D1-D2 regions of the rRNA genes confirmed the novelty of these species. The in vitro antifungal susceptibility of the new species showed that amphotericin B was active against all isolates and posaconazole and itraconazole showed low activity.