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Background Candida parapsilosis is the second or third most frequently isolated Candida species related to nosocomial infections, even overtaking Candida albicans in some hospitals. C. parapsilosis constitutes a complex of closely related species: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis. Accurate detection of these species is of importance, as the incidence of C. orthopsilosis has been reported to surpass that of Candida krusei. Objective To evaluate the diagnostic utility of two PCR-RFLP methods targeting the SADH and FKS1 genes and to determine the prevalence of cryptic species in 96 bloodstream isolates of C. parapsilosis from 93 patients. Methods Restriction patterns of the SADH and FKS1 genes were analysed, and sequencing of the D1/D2 regions of the ribosomal RNA was used to evaluate the reliability of both PCR-RFLP methods. Results In our study, 77 C. parapsilosis sensu stricto, 13 C. orthopsilosis and five C. metapsilosis were identified by sequencing. Both PCR-RFLP methods demonstrated strong agreement with D1/D2 sequencing in the identification of C. parapsilosis and C. orthopsilosis, while both methods were unable to identify the C. metapsilosis isolates. Moreover, unexpected restriction patterns were observed for two isolates on SADH PCR-RFLP and for four isolates on FKS1 PCR-RFLP. Mixed bloodstream infections of C. parapsilosis sensu stricto and C. orthopsilosis were detected for three patients, for which differential growth characteristics were observed. Conclusion The molecular method chosen for identification could have an impact on determination of the real prevalence of C. metapsilosis in candidaemia, and mixed fungaemias can remain undetected.

International audience; Susceptibility testing is an important tool in the clinical setting; its utility is based on the availability of categorical endpoints, breakpoints (BPs) or epidemiological cutoff values (ECVs/ECOFFs). CLSI and EUCAST have developed antifungal susceptibility testing, BPs and ECVs for some fungal species. Although the Concentration Gradient Strip BioMerieux Etest is useful for routine testing in the clinical laboratory, ECVs are not available for all agent/species; the lack of clinical data precludes development of BPs. We re-evaluated and consolidated Etest data points from three previous studies, and included new data. We defined ECOFFinder Etest ECVs for three sets of species/agent combinations: fluconazole, posaconazole and voriconazole and 8 Candida spp.; amphotericin B and 3 non-prevalent Candida spp.; and caspofungin and 5 Aspergillus spp. The total of Etest MICs from 23 laboratories (Europe, the Americas, South Africa) included (antifungal agent/dependent): 17,242 Candida albicans, 244 C. dubliniensis, 5,129 C. glabrata species complex (SC), 275 C. guilliermondii (Meyerozyma guilliermondii), 1,133 C. krusei (Pichia kudriavzevii), 933 C. kefyr (Kluyveromyces marxianus), 519 C. lusitaniae (Clavispora lusitaniae), 2,947 C. parapsilosis SC, 2,214 C. tropicalis, 3,212 Aspergillus fumigatus, 232 A. flavus, 181 A. niger, and 267 A. terreus SC isolates. Triazole MICs for 66 confirmed non-wild-type (non-WT) Candida isolates were available (ERG11 point mutations). Distributions fulfilling CLSI ECV criteria were pooled and ECOFFinder Etest ECVs were established for triazoles (9 Candida spp.); amphotericin B (3 less-prevalent Candida spp.) and caspofungin (4 Aspergillus spp.). Etest fluconazole ECVs could be good detectors of Candida non-WT isolates (59/61 Non-WT: 4 of 6 species).

Background: The epidemiology of candidemia has changed over the last decades and varies widely among geographic areas. Aims: We examined in children (aged 0-14) with candidemia the trends in the incidence rate of this infection, as well as the clinical characteristics of the patients, in order to optimize the prognosis and the control measures of this serious disease. Methods: A retrospective cohort study of candidemia in the period 2011-2018 in the neonatal intensive care unit (NICU), pediatric ICU ( PICU) and pediatric wards of a tertiary hospital, was conducted. The clinical course, Candida species isolated, antifungal susceptibility, outcome and incidence rates were analyzed and compared. Results: We diagnosed 68 episodes of candidemia in 62 children, 48% occurred in the NICU, 31% in the PICU and 21% in pediatric wards. Candida albicans was the most frequent species isolated in NICU infants (53%), and Candida parapsilosis predominated among PICU patients (59%) and pediatric wards (50%). One third of NICU infants had invasive candidiasis (IC), most of them having extremely low birth weight (ELBW) (35%). All isolates were susceptible to the antifungal administered. Over time, the incidence of candidemia decreased in the PICU (from 2.2 to 0.3 episodes/1000 patient-days, OR = 0.6; 95%CI 0.5-0.8), whereas in the NICU and in the wards remained stable. Mortality occurred mostly in NICU patients (26%), predominated in ELBW infants and did not change over time. Conclusions: The higher incidence and mortality of candidemia and IC observed in preterm infants requires a continuous evaluation of practices and diagnostic methods which will allow improving the prognosis of this most vulnerable population. (C) 2020 Asociacion Espanola de Micologia. Published by Elsevier Espana, S.L.U. All rights reserved.

Resumen Antecedentes Las estrategias terapeuticas actuales poseen una limitada eficacia para erradicar biopeliculas de Candida formadas en la superficie de los dispositivos biomedicos. Pocos estudios han evaluado la eficacia de los antifungicos sobre biopeliculas de Candida tropicalis. Objetivos Evaluar la actividad de la anfotericina B (AMB) y la anidulafungina (AND), solas y combinadas, sobre biopeliculas de C. tropicalis desarrolladas en superficies de politetrafluoroetileno (teflon - PTFE) y titanio mediante ensayos de letalidad-tiempo. Metodos Los ensayos se realizaron en un CDC Biofilm Reactor sobre biopeliculas de 24 h de maduracion formadas en discos de PTFE y titanio. Las concentraciones ensayadas fueron 40 mg/l para AMB y 8 mg/l para AND, tanto para su uso por separado como combinadas. Tras 24, 48 y 72 h de exposicion a los antifungicos se determinaron las ufc/cm2 mediante agitacion vorticial y cultivo cuantificado previa sonicacion. Resultados AMB redujo las celulas viables adheridas a PTFE y titanio en mas de un 99%, y AND lo hizo en un 89,3% en PTFE y 96,8% en titanio. La combinacion AMB + AND fue menos activa que la AMB sola tanto en PTFE (descenso en ufc/cm2 de 3,09 Log10 vs. 1,08 en la combinacion) como en titanio (4,51 vs. 1,53 en la combinacion), siendo la interaccion indiferente en ambas superficies. Conclusiones AMB es mas activa que AND sobre biopeliculas de C. tropicalis. La eficacia sobre las biopeliculas es mayor en el titanio. La combinacion AMB + AND es menos eficaz que AMB sola en ambas superficies.

Background Candida auris is an emerging multidrug-resistant yeast that can cause invasive infections and is associated with high mortality. It is typically resistant to fluconazole and voriconazole and, some cases, also to echinocandins and amphotericin B. This species, phylogenetically related to Candida haemulonii, is frequently misidentified by commercial identification techniques in clinical laboratories; therefore, the real prevalence of C. auris infections may be underestimated. Aims To describe the clinical and microbiological features of the first four cases of C. auris fungemia episodes observed in the European continent. Methods The four patients were hospitalized in the adult surgical intensive care unit. A total of 8 isolates (two per patient) from blood and catheter tip were analyzed. Results All isolates were misidentified as Saccharomyces cerevisiae by AuxaColor 2, and as Candida sake by API ID20C. VITEK MS technology misidentified one isolate as Candida lusitaniae, another as C. haemulonii and could not identify the other six. C. auris identification was confirmed by ITS rDNA sequencing. All isolates were fluconazole (MIC >256 mg/l) and voriconazole (MIC 2 mg/l) resistant and susceptible to posaconazole, itraconazole, echinocandins and amphotericin B. Conclusions C. auris should be regarded as an emerging pathogen, which requires molecular methods for definitive identification. Our isolates were highly resistant to fluconazole and resistant to voriconazole, but susceptible to the other antifungals tested, which emphasizes the importance of accurately identifying this species to avoid therapeutic failures.

We determined the in vitro activity of fenticonazole against 318 vaginitis isolates of Candida and bacterial species and selected 28 isolates for time-kill studies. At concentrations equal to 4× MIC, fenticonazole reached the 99.9% killing endpoint by ∼10 h for Staphylococcus aureus , Streptococcus agalactiae , and Escherichia coli and by ∼17 h for Candida albicans and Candida parapsilosis ; and at concentrations equal to 8× MIC, by ∼19 and ∼20 h for Candida glabrata and Candida tropicalis , respectively.

Although the Sensititre Yeast-One (SYO) and Etest methods are widely utilized, interpretive criteria are not available for triazole susceptibility testing of Candida or Aspergillus species. We collected fluconazole, itraconazole, posaconazole, and voriconazole SYO and Etest MICs from 39 laboratories representing all continents for (method/agent-dependent) 11,171 Candida albicans, 215 C. dubliniensis, 4,418 C. glabrata species complex, 157 C. guilliermondii (Meyerozyma guilliermondii), 676 C. krusei (Pichia kudriavzevii), 298 C. lusitaniae (Clavispora lusitaniae), 911 C. parapsilosis sensu stricto, 3,691 C. parapsilosis species complex, 36 C. metapsilosis, 110 C. orthopsilosis, 1,854 C. tropicalis, 244 Saccharomyces cerevisiae, 1,409 Aspergillus fumigatus, 389 A. flavus, 130 A. nidulans, 233 A. Niger, and 302 A. terreus complex isolates. SYO/Etest MICs for 282 confirmed non-wild-type (non-WT) isolates were included: ERG11 (C. albicans), ERG11 and MRR1 (C. parapsilosis), cyp51A (A. fumigatus), and CDR2 and CDR1 overexpression (C. albicans and C. glabrata, respectively). Interlaboratory modal agreement was superior by SYO for yeast species and by the Etest for Aspergillus spp. Distributions fulfilling CLSI criteria for epidemiological cutoff value (ECV) definition were pooled, and we proposed SYO ECVs for S. cerevisiae and 9 yeast and 3 Aspergillus species and Etest ECVs for 5 yeast and 4 Aspergillus species. The posaconazole SYO ECV of 0.06 g/ml for C. albicans and the Etest itraconazole ECV of 2 g/ml for A. fumigatus were the best predictors of non-WT isolates. These findings support the need for method-dependent ECVs, as, overall, the SYO appears to perform better for susceptibility testing of yeast species and the Etest appears to perform better for susceptibility testing of Aspergillus spp. Further evaluations should be conducted with more Candida mutants. Fil: Espinel-Ingroff, A.. Virginia Commonwealth University Medical Center; Fil: Turnidge, J.. University of Adelaide; Australia Fil: Alastruey-Izquierdo, A.. Centro Nacional de Microbiologia; Fil: Botterel, F.. Mycologie; Fil: Canton, Eduardo. Instituto de Investigación Sanitaria la Fe; Fil: Castro, C.. Hospital Universitario de Valme; Fil: Chen, Y.-C.. National Taiwan University Hospital; Fil: Chen, Y.. Public Health Ontario; Fil: Chryssanthou, E.. Karolinska University Hospital; Fil: Dannaoui, E.. Universite Paris Descartes; Fil: Garcia, Guillermo Manuel. Universidad Nacional del Litoral; Argentina Fil: Gonzalez, G.M.. Universidad Autónoma de Nuevo León; Fil: Govender, N.P.. University of the Witwatersrand; Sudáfrica Fil: Guinea, J.. Hospital General Universitario Gregorio Marañon; Fil: Kidd, S.. National Mycology Reference Centre; Fil: Lackner, M.. Medizinische Universitat Innsbruck; Fil: Lass-Flörl, C.. Medizinische Universitat Innsbruck; Fil: Linares-Sicilia, M.J.. Universidad de Cordoba, Facultad de Medicina; Fil: López-Soria, L.. Hospital Universitario Cruces; Fil: Magobo, R.. Universite Paris Descartes; Fil: Pelaez, T.. Hospital Universitario Central de Asturias; Fil: Quindós, G.. Universidad del Pais Vasco - Euskal Herriko Unibertsitatea, Campus Bizkaia; Fil: Rodriguez-Iglesia, M.A.. Universidad de Cádiz; España Fil: Ruiz, M.A.. Instituto de Investigación Sanitaria Aragón; Fil: Sánchez-Reus, F.. Hospital de la Santa Creu I Sant Pau; Fil: Sanguinetti, M.. Università Cattolica del Sacro Cuore, Rome; Fil: Shields, R.. University of Pittsburgh at Johnstown; Estados Unidos. University of Pittsburgh; Estados Unidos Fil: Szweda, P.. Gdan´ Sk University Of Technology; Fil: Tortorano, A.. Università degli Studi di Milano; Italia Fil: Wengenack, N.L.. Mayo Clinic In Rochester, Minnesota; Fil: Bramati, S.. Azienda Ospedaliera San Gerardo Monza; Fil: Cavanna, C.. Fondazione Irccs Policlinico San Matteo; Fil: DeLuca, C.. Humanitas Research Hospital; Fil: Gelmi, M.. Spedali Civili Di Brescia; Fil: Grancini, A.. Ospedale Maggiore Policlinico Milano; Fil: Lombardi, G.. Ospedale Niguarda, Milan; Fil: Meletiadis, J.. University Of Athens Medical School; Fil: Negri, C.E.. Universidade Federal de Sao Paulo; Fil: Passera, M.. Microbiology Institute; Fil: Peman, J.. Hospital Universitari I Politècnic la Fe; Fil: Prigitano, A.. Università degli Studi di Milano; Italia Fil: Sala, E.. Asst Lariana; Fil: Tejada, M.. Irccs Policlinico San Donato

Candida auris is an emerging pathogen causing candidaemia outbreaks in several countries for which azole, amphotericin B (AmB) and echinocandin resistance has been reported. In this study, the antifungal susceptibilities of 73 Spanish C. auris isolates (56 bloodstream and 17 urine) to eight antifungal agents were determined using three methods. Isolates were identified by internal transcribed spacer (ITS) sequencing, and minimum inhibitory concentrations (MICs) of fluconazole, isavuconazole, itraconazole, posaconazole, voriconazole, anidulafungin, micafungin and AmB were determined by EUCAST methodology and Sensititre (R) YeastOne (R) (SYO) (bloodstream isolates) and Liofilchem (R) MIC Test Strip (all isolates). Agreement between the methods was analysed and the MICs (ours and published data) were categorised using recently proposed epidemiological cut-offvalues (ECVs). Fluconazole MICs were >64 mg/L, whilst >60% of voriconazole MICs were >1 mg/L by the three methods. Posaconazole was the most active azole (EUCAST geometric mean MIC, 0.053 mg/L), followed by isavuconazole (0.066 mg/L) and itraconazole (0.157 mg/L). Echinocandins MICs were 93% for the eight antifungals. For this new C. auris clade, all isolates were resistant to fluconazole, and MICs for anidulafungin, micafungin and AmB were 93%) was observed between EUCAST and SYO. (C) 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

The number of biomedical devices (intravascular catheters, heart valves, joint replacements, etc.) that are implanted in our hospitals has increased exponentially in recent years. Candida species are pathogens which are becoming more significant in these kinds of infections. Candida has two forms of development: planktonic and in biofilms. A biofilm is a community of microorganisms which adhere to a surface and are enclosed by an extracellular matrix. This form of development confers a high resistance to the antimicrobial agents. This is the reason why antibiotic treatments usually fail and biomedical devices may have to be removed in most cases. Unspecific adhesion mechanisms, the adhesion-receptor systems, and an intercellular communication system called quorum sensing play an essential role in the development of Candida biofilms. In general, the azoles have poor activity against Candida biofilms, while echinocandins and polyenes show a greater activity. New therapeutic strategies need to be developed due to the high morbidity and mortality and high economic costs associated with these infections. Most studies to date have focused on bacterial biofilms. The knowledge of the formation of Candida biofilms and their composition is essential to develop new preventive and therapeutic strategies.

Background: Candida parapsilosis, Candida metapsilosis and Candida orthopsilosis are emerging as relevant causes of candidemia. Moreover, they show differences in their antifungal susceptibility and virulence. The echinocandins are different in terms of in vitro antifungal activity against Candida. Time-kill (TK) curves represent an excellent approach to evaluate the fungicidal activity of antifungal drugs. Aims: To compare the fungicidal activities of anidulafungin, caspofungin and micafungin against C. parapsilosis species complex by TK curves. Methods: Antifungal activities of three echinocandins against C. parapsilosis, C. metapsilosis and C. orthopsilosis were studied by TK curves. Drug concentrations assayed were 0.25, 2 and 8 mu g/ml. CFU/ml were determined at 0, 2, 4, 6, 24 and 48 h. Results: Killing activities of echinocandins were species-, isolates- and concentration-dependent. Anidulafungin reached the fungicidad endpoint for 6 out of 7 isolates (86%); it required between 13.34 and 29.67 h to reach this endpoint for the three species studied, but more than 48 h were needed against one isolate of C orthopsilosis (8 mu g/ml). Caspofungin fungicidal endpoint was only achieved with 8 mu g/ml against one isolate of C. metapsilosis after 30.12 h (1 out of 7 isolates; 14%). Micafungin fungicidal endpoint was reached in 12.74-28.38 h (8 mu g/ml) against one isolate each of C parapsilosis and C orthopsilosis, and against both C. metapsilosis isolates (4 out of 7 isolates; 57%). Conclusions: C. metapsilosis was the most susceptible species to echinocandins, followed by C. orthopsilosis and C. parapsilosis. Anidulafungin was the most active echinocandin against C parapsilosis complex. (C) 2019 Asociacion Espanola de Micologia. Published by Elsevier Espana, S.L.U. All rights reserved.

Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.

Contains fulltext : 196638.pdf (Publisher’s version ) (Open Access) Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.

Anidulafungin, caspofungin, and micafungin killing activities against Candida glabrata , Candida bracarensis , and Candida nivariensis were evaluated by the time-kill methodology. The concentrations assayed were 0.06, 0.125, and 0.5 μg/ml, which are achieved in serum. Anidulafungin and micafungin required between 13 and 26 h to reach the fungicidal endpoint (99.9% killing) against C. glabrata and C. bracarensis . All echinocandins were less active against C. nivariensis .

We evaluated the activity of (1) amphotericin-B (AMB), combined with rifampicin (RIF), clarithromycin (CLA), N-acetylcysteine (NAC), ethylenediaminetetraacetic acid (EDTA), and farnesol (FAR) (1000, 1000, 1000, 4000, and 30,000 mg/L, and 300 microM, respectively), against Candida tropicalis biofilms formed on polytetrafluoroethylene (PTFE) and (2) anidulafungin (ANF) combined with the same compounds at 8, 10, 5, 40, and 30 mg/L, and 30 microM, respectively, against biofilms formed on titanium. Biofilm growth kinetics were performed in a CDC Biofilm Reactor (CBR). PTFE or titanium disks were removed from the CBR at 24, 48, 72, and 96 h to determine the Log10CFU/cm(2). Killing kinetics were performed by adding the drugs to 24-h-mature biofilms (time 0). Disks were removed after 24, 48, and 72 h of drug exposure to determine Log10CFU/cm(2). Viable cells in biofilms were 4.73 and 4.29 Log10CFU/cm(2) on PTFE and titanium, respectively. Maximum Log10 decreases in CFU/cm(2) depend on the combination and were: 3.53 (AMB + EDTA), 2.65 (AMB + RIF), 3.07 (AMB + NAC), 2.52 (AMB + CLA), 1.49 (AMB + FAR), 2.26 (ANF + EDTA), 2.45 (ANF + RIF), 2.47 (ANF + NAC), 1.52 (ANF + CLA), and 0.44 (ANF + FAR). In conclusion, EDTA, NAC, RIF, and CLA improve the activity of AMB and ANF against biofilms developed on both surfaces, which could be an effective strategy against C. tropicalis biofilm-related infections.

Method-dependent Etest epidemiological cutoff values (ECVs) are not available for susceptibility testing of either Candida or Aspergillus species with amphotericin B or echinocandins. In addition, reference caspofungin MICs for Candida spp. are unreliable. Candida and Aspergillus species wild-type (WT) Etest MIC distributions (microorganisms in a species-drug combination with no detectable phenotypic resistance) were established for 4,341 Candida albicans , 113 C. dubliniensis , 1,683 C. glabrata species complex (SC), 709 C. krusei , 767 C. parapsilosis SC, 796 C. tropicalis , 1,637 Aspergillus fumigatus SC, 238 A. flavus SC, 321 A. niger SC, and 247 A. terreus SC isolates. Etest MICs from 15 laboratories (in Argentina, Europe, Mexico, South Africa, and the United States) were pooled to establish Etest ECVs. Anidulafungin, caspofungin, micafungin, and amphotericin B ECVs (in micrograms per milliliter) encompassing ≥97.5% of the statistically modeled population were 0.016, 0.5, 0.03, and 1 for C. albicans ; 0.03, 1, 0.03, and 2 for C. glabrata SC; 0.06, 1, 0.25, and 4 for C. krusei ; 8, 4, 2, and 2 for C. parapsilosis SC; and 0.03, 1, 0.12, and 2 for C. tropicalis . The amphotericin B ECV was 0.25 μg/ml for C. dubliniensis and 2, 8, 2, and 16 μg/ml for the complexes of A. fumigatus , A. flavus , A. niger , and A. terreus , respectively. While anidulafungin Etest ECVs classified 92% of the Candida fks mutants evaluated as non-WT, the performance was lower for caspofungin (75%) and micafungin (84%) cutoffs. Finally, although anidulafungin (as an echinocandin surrogate susceptibility marker) and amphotericin B ECVs should identify Candida and Aspergillus isolates with reduced susceptibility to these agents using the Etest, these ECVs will not categorize a fungal isolate as susceptible or resistant, as breakpoints do.

Invasive fungal infections (IFIs) are difficult to diagnose and cause a high mortality to an expanding spectrum of patients. Culture of clinical samples has limitations for the diagnosis of IFI and alternative procedures have been developed. Among them, serum determination of galactomannan or beta-1,3-d-glucan, and antimicelium and antimannan antibodies are relevant. The use of molecular procedures and mass spectrometry (MALDI-TOF) are encouraging tools for the optimization of the diagnosis and management of patients with IFI. The proposal of species-specific breakpoint to classify the isolates as resistant or susceptible to antifungal agents and the necessity of monitor the azole serum levels deserve greater attention.

Treatment of disseminated Trichosporon infections still remains difficult. Amphotericin B frequently displays inadequate fungicidal activity and echinocandins have no meaningful antifungal effect against this genus. Triazoles are currently the drugs of choice for the treatment of Trichosporon infections. This study evaluates the inhibitory and fungicidal activities of five triazoles against 90 clinical isolates of Trichosporon asahii . MICs (μg/ml) were determined according to Clinical and Laboratory Standards Institute microdilution method M27-A3 at 24 and 48 h using two endpoints, MIC-2 and MIC-0 (the lowest concentrations that inhibited ∼50 and 100% of growth, respectively). Minimum fungicidal concentrations (MFCs; μg/ml) were determined by seeding 100 μl of all clear MIC wells (using an inoculum of 10 4 CFU/ml) onto Sabouraud dextrose agar. Time-kill curves were assayed against four clinical T. asahii isolates and the T. asahii ATCC 201110 strain. The MIC-2 (∼50% reduction in turbidity compared to the growth control well)/MIC-0 (complete inhibition of growth)/MFC values that inhibited 90% of isolates at 48 h were, respectively, 8/32/64 μg/ml for fluconazole, 1/2/8 μg/ml for itraconazole, 0.12/0.5/2 μg/ml for voriconazole, 0.5/2/4 μg/ml for posaconazole, and 0.25/1/4 μg/ml for isavuconazole. The MIC-0 endpoints yielded more consistent MIC results, which remained mostly unchanged when extending the incubation to 48 h (98 to 100% agreement with 24-h values) and are easier to interpret. Based on the time-kill experiments, none of the drugs reached the fungicidal endpoint (99.9% killing), killing activity being shown but at concentrations not reached in serum. Statistical analysis revealed that killing rates are dose and antifungal dependent. The lowest concentration at which killing activity begins was for voriconazole, and the highest was for fluconazole. These results suggest that azoles display fungistatic activity and lack fungicidal effect against T. asahii . By rank order, the most active triazole is voriconazole, followed by itraconazole ∼ posaconazole ∼ isavuconazole > fluconazole.

Resumen Introduccion Existen pocos estudios epidemiologicos sobre candidemia en poblacion pediatrica en Espana. Nuestro objetivo es conocer la epidemiologia de candidemia en estos pacientes. Metodos Estudio prospectivo, observacional, multicentrico en 44 hospitales espanoles sobre episodios de candidemia en poblacion pediatrica desde 0 a 15 anos de edad ocurridos entre enero de 2009 y febrero de 2010. Resultados Se estudiaron 197 episodios con 200 aislamientos de levaduras. La especie mas frecuentemente aislada fue Candida parapsilosis sensu stricto (43%) seguida de C. albicans (36%), C. tropicalis (6%), C. orthopsilosis y C. glabrata (4% cada una). C. albicans predomino en neonatos, mientras que C. parapsilosis predomino en el resto de las edades. Por comunidades autonomas, en Baleares, Cataluna y Canarias se aislo mas frecuentemente C. albicans , y en Andalucia, Castilla y Leon, Galicia, Madrid y Valencia fue mas prevalente C. parapsilosis . El porcentaje de cepas resistentes al fluconazol fue del 1,5% (4,1% con los nuevos criterios especie especificos del CLSI). La resistencia al fluconazol fue menor en neonatos que en el resto de los grupos. Neonatologia fue el area donde mas frecuentemente se detectaron estos episodios (31,5%). La presencia de cateter y la prematuridad se asociaron, de manera independiente, con candidemia por C. albicans con un riesgo casi 6 y 2 veces mayor, respectivamente, en el analisis multivariante. Conclusiones La epidemiologia de candidemia pediatrica varia entre las comunidades autonomas, pero globalmente C. parapsilosis y C. albicans son por este orden las especies mas frecuentes y muestran unas tasas de resistencia al fluconazol inferiores al 5%.

The U.S. Clinical and Laboratory Standards Institute (CLSI) and the European Committee of Antimicrobial Susceptibility Testing (AFST-EUCAST) have developed broth microdilution methodologies for testing yeasts and filamentous fungi (molds). The mission of these methodologies is to identify in vitro antifungal resistance, which is accomplished by the use of either clinical breakpoints (CBPs), or to a lesser degree, epidemiologic cutoff values (ECVs). The newly adjusted and species-specific CLSI CBPs for Candida spp. versus fluconazole and voriconazole have ameliorated some of the differences between the two methodologies. In the absence of CBPs for mold testing, CLSI ECVs are available for six Aspergillus species versus the triazoles, caspofungin and amphotericin B. Recently, breakpoints were developed by the EUCAST for certain Aspergillus spp. versus amphotercin B, itraconazole and posaconazole, which to some extent are comparable to ECVs. We summarize these latest accomplishments, which have made possible the harmonization of some susceptibility cutoffs, if not methodologies for some agent/species combinations.

Current 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.To 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.Assays 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/cmAMB 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/cmAMB 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.

Objectives: To evaluate the in vitro activity of anidulafungin combined with amphotericin B or voriconazole against Candida spp. biofilms. Methods: Four Candida albicans, four Candida tropicalis, four Candida glabrata, two Candida parapsilosis and two Candida orthopsilosis blood isolates were tested by the microdilution chequerboard method combined with the XTT metabolic assay. Biofilm MIC was defined as the lowest concentration producing 50% metabolic inhibition with respect to control (BMIC50). Concentrations in the combinations ranged from 1/8xBMIC(50) to 4xBMIC(50) found for each antifungal tested alone. Results: Anidulafungin plus amphotericin B acted synergistically against C. albicans and C. glabrata biofilms [fractional inhibitory concentration index (FICI): 0.082-0.387], but showed no interaction against C. tropicalis, C. parapsilosis and C. orthopsilosis (FICI: 0.516-2.099). The combination of these antifungals failed to completely remove biofilms of C. albicans and C. glabrata, decreasing the metabolic activity of the biofilms up to 80% and 95%, respectively, which did not occur when each antifungal was used alone. Anidulafungin plus voriconazole showed no interaction against all isolates. Using a less stringent criterion previously proposed to define synergism (FICI, 1) and antagonism (FICI. 1.25), antagonistic interactions were found against some isolates. Conclusions: Anidulafungin with amphotericin B results in a synergistic effect against C. albicans and C. glabrata biofilms at serum concentrations of the drugs, but showed no interaction against C. tropicalis and C. parapsilosis complex. Anidulafungin plus voriconazole showed no interaction against the five Candida species assayed. Biofilms of C. tropicalis were found to be the most resistant towards the combinations assayed. The results presented may be of potential interest in the clinical setting.

Objectives: Candida lusitaniae fungaemia, although infrequent (1%), is more common in immunocompromised patients than Candida albicans. Although infections produced by Candida spp. are therapeutic targets for treatment with echinocandins, little information is available regarding their killing kinetics against C. lusitaniae. The objectives of this study were to determine the killing kinetics of anidulafungin, micafungin and caspofungin against four blood isolates of C. lusitaniae by time –kill methodology. Methods: Time–kill studies were performed in RMPI 1640 medium (5 mL, inoculum � 10 5 cfu/mL). The number of cfu/mL was determined at 0, 2, 4, 6 and 24 h. The anidulafungin concentrations assayed were 0.03, 0.12, 0.5, 2 and 8 mg/L, while micafungin and caspofungin concentrations were 0.25, 1, 4, 16 and 32 mg/L. Results: MIC ranges were 0.03–1 mg/L (anidulafungin), 0.016–0.06 mg/L (micafungin) and 0.03–1 mg/L (caspofungin). The mean maximum log decrease in cfu/mL was reached with 2 mg/L anidulafungin (1.85+0.4 log), 32 mg/L caspofungin (5.5+0.2 log) and 32 mg/L micafungin (2.65+1.9 log). Only caspofungin and micafungin reached the fungicidal endpoint (99.9% growth reduction or a 3 log decrease) with 32 mg/L at 22.8 h (caspofungin) and 26.5 h (micafungin). Analysis of variance showed significant differences in killing activity among isolates, but not among concentrations reached in serum or echinocandins. Conclusions: Anidulafungin and micafungin exhibit greater killing rates than caspofungin. Caspofungin was the only echinocandin that reached the fungicidal endpoint before 24 h, but at drug concentrations (≥16 mg/L) not usually reached in serum. The echinocandin killing rate was isolate dependent and concentration independent.

Objectives: The aims of this work were to study the epidemiological profiles, differences in echinocandin susceptibilities and clinical relevance of the Candida parapsilosis sensu lato species isolated from proven fungaemia cases at La Fe University Hospital of Valencia (Spain) from 1995 to 2007. Results: The prevalence of these species was: C. parapsilosis sensu stricto, 74.4%; Candida orthopsilosis, 23.54%; and Candida metapsilosis, 2.05%. The incidence of the species complex as agents of fungaemia remained stationary until 2005 and doubled in 2006. The incidence of C. orthopsilosis showed an increasing trend during the study period, while C. parapsilosis sensu stricto incidence diminished. Also, an important epidemiological change was observed starting in 2004, when 86.5% of the C. parapsilosis sensu lato strains were found in adult patients, while before that year only 13.5% of the isolates were found in this population. Conclusions: Echinocandin drug susceptibility testing using the CLSI M27-A3 document showed a wide range of MIC values (0.015–4 mg/L), with micafungin being the most potent in vitro inhibitor followed by anidulafungin and caspofungin (MIC geometric mean of 0.68, 0.74 and 0.87 mg/L, respectively). C. metapsilosis was the most susceptible species of the complex to anidulafungin and micafungin in vitro (MIC50 for anidulafungin and micafungin: 0.06 mg/L), while there were no differences between C. parapsilosis sensu lato species when caspofungin MIC50s were compared (MIC50 1.00 mg/L). Differences in caspofungin in vitro susceptibility were observed between the different clinical service departments of La Fe Hospital.

To determine the ability of voriconazole to inhibit the formation of biofilms.A total of 38 blood isolates of Candida spp. (8 Candida albicans, 10 Candida tropicalis, 10 Candida glabrata, 7 Candida parapsilosis sensu stricto and 3 Candida orthopsilosis) and C. albicans ATCC 90028 and ATCC 64548 were assessed. Biofilm formation was quantified using XTT reduction assays. The inhibition of biofilm formation was determined (i) in the presence of 0.06 and 0.25 mg/L voriconazole, and (ii) on surfaces previously coated with 0.06, 0.25, 1, 4 and 16 mg/L voriconazole.Voriconazole reduced biofilm formation under both conditions, the extent depending on the species, isolate and drug concentration. In the presence of 0.25 mg/L, the highest reduction was found for C. parapsilosis (79% ± 8.6%), followed by C. albicans (64.5% ± 6.3%), C. tropicalis (53.3% ± 13.1%) and C. glabrata (23.8% ± 11.2%). This reduction was significant (P 0.05) for all isolates tested. After coating the wells with voriconazole, biofilm formation was reduced in all Candida spp. examined, C. albicans being the species with the highest reduction (68.8% with 16 mg/L) and C. parapsilosis complex and C. glabrata the lowest.As voriconazole reduces biofilm formation it may be a good candidate for the prevention of Candida biofilm-related infections although further studies using voriconazole-impregnated catheter tubing or prostheses are required to confirm these results.

Methods developed for testing filamentous fungi (molds) include standardized broth microdilution (Clinical and Laboratory Standards Institute [CLSI] and European Committee for Antimicrobial Susceptibility Testing [AFST-EUCAST]) methods and disk diffusion (CLSI) methods. Quality control limits also are available from CLSI for MIC (minimal inhibitory concentration), MEC (minimal effective concentration), and zone diameters. Although clinical breakpoints based on correlations of in vitro results with clinical outcome have not been established, epidemiologic cutoff values have been defined for six Aspergillus species and the triazoles, caspofungin, and amphotericin B. The link between resistance molecular mechanisms, elevated MICs, and clinical treatment failure has also been documented, especially for Aspergillus and the triazoles. Other insights into the potential clinical value of high MICs have also been reported. Various commercial methods (e.g., YeastOne, Etest, and Neo-Sensitabs) have been evaluated in comparison with reference methods. This review summarizes and discusses these developments.

Data on fungemia epidemiology and antifungal susceptibility of isolates from children are scarce, leading frequently to pediatric empirical treatment based on available adult data. The present study was designed to update the epidemiological, mycological, and in vitro susceptibility data on fungal isolates from children with fungemia in Spain. All fungemia episodes were identified prospectively by blood culture over 13 months at 30 hospitals. Tests of susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, and micafungin were performed at participant institutions by a microdilution colorimetric method. New species-specific clinical breakpoints for fluconazole, voriconazole, and echinocandins were also applied. A total of 203 episodes of fungemia in 200 children were identified. A higher proportion of fungal isolates was from general wards than intensive care units (ICU). Candida parapsilosis (46.8%), Candida albicans (36.5%), Candida tropicalis (5.9%), Candida glabrata (3.9%), and Candida guilliermondii (2.5%) were the leading species. C. parapsilosis was the predominant species except in neonates. C. albicans was the most frequent in neonatal ICU settings (51.9%). Intravascular catheter (79.3%), surgery (35%), prematurity (30%), and neutropenia (11%) were the most frequent predisposing factors. Most Candida isolates (95.1%) were susceptible to all antifungals. When the new species-specific clinical breakpoints were applied, all C. parapsilosis isolates were susceptible to echinocandins except one, which was micafungin resistant. This is the largest published series of fungemia episodes in the pediatric setting. C. parapsilosis is the most prevalent species in Spain, followed by C. albicans and C. tropicalis . Resistance to azole and echinocandin agents is extremely rare among Candida species. The fluconazole resistance rate in Spain has decreased in the last 10 years.

A 13-month prospective multicenter study including 44 hospitals was carried out to evaluate the epidemiology of Candida parapsilosis complex candidemia in Spain. Susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, anidulafungin, caspofungin, and micafungin was tested by the microdilution colorimetric method. A total of 364 C. parapsilosis complex isolates were identified by molecular methods: C. parapsilosis (90.7%), Candida orthopsilosis (8.2%), and Candida metapsilosis (1.1%). Most candidemias ( C. parapsilosis , 76.4%; C. orthopsilosis , 70.0%; C. metapsilosis , 100%) were observed in adults. No C. orthopsilosis or C. metapsilosis candidemias occurred in neonates. C. parapsilosis was most frequent in adult intensive care unit (28.8%), surgery (20.9%), and internal medicine (19.7%) departments; and C. orthopsilosis was most frequent in hematology (28.6%), pediatrics (12.0%), and neonatology (11.5%) departments. The geographic distribution of C. orthopsilosis and C. metapsilosis was not uniform. According to CLSI clinical breakpoints, all C. orthopsilosis and C. metapsilosis isolates were susceptible to the nine agents tested. Resistance (MICs > 1 mg/liter) was observed only in C. parapsilosis : amphotericin B, posaconazole, itraconazole, and caspofungin (0.3% each), anidulafungin (1.9%), and micafungin (2.5%). Applying the new species-specific fluconazole and echinocandin breakpoints, the rates of resistance to fluconazole for C. parapsilosis and C. orthopsilosis increased to 4.8% and 0.3%, respectively; conversely, for C. parapsilosis they shifted from 1.9 to 0.6% (anidulafungin) and from 2.5 to 0.6% (micafungin). Our study confirms the different prevalence of C. parapsilosis complex candidemia among age groups: neither C. orthopsilosis nor C. metapsilosis was isolated from neonates; interestingly, C. metapsilosis was isolated only from adults and the elderly. The disparity in antifungal susceptibility among species could be important for therapy.

Micafungin Etest and Clinical and Laboratory Standards Institute (CLSI) MICs were compared for 337 Candida spp. isolates. The performance of Etest for testing the susceptibilities of Candida spp. to micafungin was evaluated by the assessment of both categorical (CA) and essential (EA) agreements. The CA was evaluated 2 ways: (i) by the ability of Etest to separate resistant (nontreatable) from susceptible (treatable) isolates by using the newly adjusted species-specific micafungin clinical breakpoints (CBPs) that are available for most of the common species tested and (ii) by the ability to separate wild type (WT) from non-WT isolates or those harboring FKS mutations (with reduced echinocandin susceptibility) by using micafungin epidemiologic cutoff values (ECVs). Etest and CLSI MICs were in EA when the MICs were within 2 log(2) dilutions. Based on agreement percentages, our data indicated that Etest is suitable to test micafungin for most of the Candida species evaluated (overall EA 94.7%; overall CA according to CBPs 97.2% and according to ECVs 97.3%). However, the number of resistant isolates was small, so further evaluations are needed with a higher number of such isolates including more resistant or those with known mechanisms of resistance (non-WT).

Resumen Antecedentes Recientemente se ha observado un incremento de las fungemias causadas por especies diferentes de Candida albicans y una disminucion de la sensibilidad de los microorganismos responsables al fluconazol. Objetivos Evaluar la epidemiologia y la sensibilidad al fluconazol de los casos de fungemia en Espana en 2009, comparando los resultados con los obtenidos entre los anos 1997-1999 (Peman J, et al. Eur J Clin Microbiol Infect Dis. 2005). Metodos Estudio prospectivo multicentrico con 44 centros participantes realizado desde enero de 2009 a febrero de 2010. Los aislamientos fungicos procedentes de hemocultivo fueron recogidos en cada centro, donde se realizo el estudio de sensibilidad antifungica mediante microdilucion colorimetrica (Sensititre Yeast One). Resultados Desde enero de 2009 a febrero de 2010 se recogieron 1.377 aislamientos en hemocultivos, correspondientes a 1.357 episodios de fungemia. Las fungemias se observaron principalmente en mayores de 64 anos (46,7%) y el 8,6% en menores de 1 ano. C. albicans (44,7%), Candida parapsilosis (29,1%), Candida glabrata (11,5%), Candida tropicalis (8,2%) y Candida krusei (1,9%) fueron las especies mas frecuentes, pero su distribucion no fue geograficamente homogenea. En los ultimos 10 anos la incidencia de C. albicans ha aumentado significativamente en Cataluna (39,1 vs. 54,7%, P = 0,03) y reducido en la Comunidad Valenciana (49,1 vs. 34,6%, P = 0,01). C. parapsilosis ha disminuido en Cataluna (29 vs. 12,4%, P = 0,002) y Extremadura (58,3 vs. 20%, P = 0,01). La sensibilidad a fluconazol fue similar en toda Espana pero en los aislamientos de C. albicans la resistencia fue diez veces superior en mayores de 64 anos. Sin embargo, la tasa de resistencia (CMI > 32 mg/L) global ha disminuido con respecto a la obtenida hace 10 anos (3,7 vs. 2,5% actual), sobre todo en C. albicans (3 vs. 1,6%). Conclusiones En los ultimos 10 anos la distribucion de las especies causantes de fungemia en Espana y la sensibilidad al fluconazol no han variado significativamente, aunque se observa una menor tasa de resistencia. La distribucion de las especies varia segun la unidad de hospitalizacion, hospital y Comunidad Autonoma.

The echinocandins anidulafungin, caspofungin, and micafungin have a broad and similar spectrum of in vitro and in vivo activity against most Candida spp. Minimal inhibitory concentrations (MICs) for Candida spp. are usually below 1 μg/mL for most isolates. The exceptions are Candida parapsilosis and C. guilliermondii. Species-specific clinical breakpoints (CBPs) and epidemiologic cutoff values (ECVs) have been proposed by the Clinical and Laboratory Standards Institute (CLSI) for the eight most common Candida spp. versus each echinocandin; these values are useful to detect in vitro antifungal resistance (CBPs) and to identify isolates harboring fks mutations or having reduced susceptibility (ECVs). This paper presents a review of the literature (2006–2010) regarding the in vitro activity similarities or differences among the three echinocandins against Candida spp.; different parameters or measurements of in vitro potency were evaluated. The focus of the review is the non-Candida albicans species.

We compared the susceptibilities of 302 isolates (209 Candida spp., 89 Cryptococcus neoformans and four Trichosporon asahii) against amphotericin B (AMB), flucytosine (5FC), fluconazole (FLC) and voriconazole (VRC) obtained with an automated commercial system (VITEK 2, bioMérieux, Spain) and the Clinical and Laboratory Standards Institute (CLSI M27-A3) reference broth microdilution method (BMD). Reference BMD MIC endpoints were determined visually after 24-72 h of incubation, depending on the species, and VITEK 2 system MIC endpoints were determined spectrophotometrically by automated components of this equipment. For Candida spp. and T. asahii, the overall MIC agreement between of the results of the VITEK 2 system and the 24/48-h BMD was: 34/62% for AMB; 96.3% at 24/48-h for 5FC; 87.8/87.3% for FLC and 95.3/92% for VRC, respectively. The overall categorical agreement between both methods was: 98.5/97.6% for AMB at 24/48-h; 95.3% for 5FC at 24/48-h; 85.4/84.4% at 24/48-h for FLC; and 97.6/92.95% at 24/48-h for VRC. For C. neoformans, essential agreement was good for FLC (91%) and 5FC (84.2%) but not so good for AMB (69%). Excellent categorical agreement was obtained for all antifungal agents tested except for 5FC (69.7%). This new system could play an important role in the clinical laboratory, but more studies are necessary to verify its ability to identify resistant isolates.

Failure to respond to antifungal therapy could be due to in vitro resistance (intrinsic or developed during therapy) or clinical resistance. In vitro resistance is mostly due to genetic mutations (resistance mechanisms), and it is associated with high minimal inhibitory concentrations (MICs), minimal effective concentrations (MECs), and/or clinical failure. Clinical breakpoints (CBPs) and/or epidemiologic cutoff values (ECVs) are useful to detect the in vitro antifungal resistance when isolates are tested by standardized methods. ECVs are available from the Clinical and Laboratory Standards Institute (CLSI) for Candida spp. versus echinocandins (anidulafungin, caspofungin, and micafungin) and triazoles (fluconazole, posaconazole, and voriconazole). Lately, the CLSI has adjusted to species-specific CBPs for Candida spp. versus fluconazole, similar to those of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and versus echinocandins. However, the available voriconazole EUCAST and CLSI CBPs differ. In the absence of CBPs, EUCAST and CLSI assigned ECVs for various Aspergillus spp. and triazoles. This article reviews emerging resistance, laboratory detection, and clinical relevance as reported in the literature in the past 3 to 4 years.

The Clinical and Laboratory Standards Institute (CLSI) has standardized broth microdilution and disk diffusion methodology for testing filamentous fungi (molds) that cause invasive disease. Quality control MIC (minimal inhibitory concentration) and MEC (minimal effective concentration; echinocandins only) limits are also available in the recently published CLSI M38-A2 document. Although breakpoints based on correlations of in vitro results and clinical outcome have not been established, MIC or MEC and zone diameter categories for five antifungal agents and various mold species, as well as epidemiologic cutoffs for Aspergillus fumigatus versus the triazoles, have been recently documented. Some insights of the potential clinical value of reference methods also have been reported. During the past few years, the potential utility of various commercial methods has been evaluated by comparing them with reference methodology. This review summarizes and discusses the advantages and disadvantages of these developments.

We evaluated the in vitro activities of anidulafungin, micafungin, and caspofungin againstCandida kruseiby determining MIC and minimum fungicidal concentration (MFC) measurements and by the time-kill method. The geometric mean (GM)-MIC/GM-MFC values were 0.1/0.34, 0.25/0.44, and 1/2.29, respectively. The mean times to reach 99.9% growth reduction were 19.1 ± 18.2 h (mean ± standard deviation) for 2 mg/liter anidulafungin, 37.4 ± 8.8 h for 2 mg/liter caspofungin, and 30.7 ± 12.2 h for 1 mg/liter micafungin. Anidulafungin exhibited the highest time-kill rate, followed by micafungin. The three echinocandins showed fungicidal activity at concentrations reached in serum.

Background: Micafungin is a new and very useful pharmacological tool for the treatment of invasive mycoses with a wide antifungal spectrum for the most common pathogenic fungi. Micafungin is especially active against the genera Candida and Aspergillus. Its antifungal mechanism is based on the inhibition of the β-1,3D-glucan synthesis, an essential molecule for the cell wall architecture, with different con sequences for Candida and Aspergillus, being micafungin fungicide for the former and fungistatic for the latter. Aim: To describe the in vitro antifungal spectrum of micafungin based in the scientific and medical lite rature of recent years. Methods: We have done a bibliographic retrieval using the scientific terms, “micafungin”, “activity”

The high morbidity, mortality, and health care costs associated with invasive fungal infections, especially in the critical care setting and immunocompromised host, have made it an excellent target for prophylactic, empiric, and preemptive therapy interventions principally based on early identification of risk factors. Early diagnosis and treatment are associated with a better prognosis. In the last years there have been important developments in antifungal pharmacotherapy. An approach to the new diagnosis tools in the clinical mycology laboratory and an analysis of the use new antifungal agents and its application in different clinical situations has been made. Furthermore, an attempt of developing a state of the art in each clinical scenario (critically ill, hematological, and solid organ transplant patients) has been performed, trying to choose the best strategy for each clinical situation (prophylaxis, pre-emptive, empirical, or targeted therapy). The high mortality rates in these settings make mandatory the application of early de-escalation therapy in critically ill patients with fungal infection. In addition, the possibility of antifungal combination therapy might be considered in solid organ transplant and hematological patients.

Currently, no standardized method to study the in vitro activity of antifungal agents on biofilms is available, thus, the comparison among different authors is difficult. The studies discussed in this review use the XTT reduction to measure the activity of antifungals on biofilms of 24 h of maturation. To date, biofilm anidulafungin MICs of 47 isolates of Candida spp. (25 Candida albicans, 16 Candida tropicalis, 5 Candida dubliniensis and 1 Candida parapsilosis) have been published. The geometric mean MIC of anidulafungin on biofilms of Candida spp. is of 1.18 microg/ml. Against isolates of species with great capacity of biofilm formation, the geometric mean MIC is 0.325 (C. albicans), 2 (C. parapsilosis) and 0.5 microg/ml (C. dubliniensis). No echinocandin has activity on C. tropicalis biofilms. In addition, anidulafungin can be used for lock therapy of catheters since it is the echinocandin with the least in vitro paradoxical effect.

Resumen Las infecciones invasoras producidas por Candida spp. han aumentando considerablemente y se estan convirtiendo en una causa importante de morbilidad y mortalidad en pacientes inmunocomprometidos. Un gran numero de manifestaciones de las candidiasis estan relacionadas con la formacion de biopeliculas sobre dispositivos y materiales medicos. Las biopeliculas de Candida spp. son refractarias al tratamiento antifungico convencional. El objetivo de este estudio es doble: 1) determinar la prevalencia de aislamientos productores de biopelicula a partir de cepas procedentes de cateter (16 C. albicans ) y de hemocultivo (dos C. albicans y 30 C. tropicalis ) y, 2) determinar la actividad de la anfotericina B y la anidulafungina sobre biopeliculas de C. albicans y C. tropicalis de 24 y 48 h de maduracion. La produccion de biopelicula y la actividad de los antifungicos sobre las biopeliculas se determino mediante el ensayo de reduccion de una sal de tetrazolio (XTT). Un 62,5% de los aislamientos de cateter y un 56,25% de los procedentes de hemocultivo produjeron biopelicula. Por especies, un 68,42% de los aislamientos de C. albicans y un 53,33% de los de C. tropicalis formaron biopelicula. Las biopeliculas fueron mas resistentes a la anfotericina y la anidulafungina que sus homologas planctonicas. La eliminacion completa de la biopelicula no se consiguio ni siquiera a las concentraciones mas altas de antifungicos ensayadas. La anidulafungina fue mas activa que la anfotericina sobre biopeliculas de C. albicans de 24 h de maduracion (MG-CMIB 2 0,354 frente a 0,686 μg/ml), pero no fue activa sobre biopeliculas de C. tropicalis . Sin embargo, la anfotericina B fue mas activa sobre las biopeliculas de 48 h de maduracion de ambas especies.

Objectives To evaluate the suitability of disc diffusion (DD) assay for testing posaconazole activity and to corroborate its activity against recently isolated yeasts by the CLSI reference microdilution M27-A2 method. Methods A total of 224 yeast isolates (7 species with 52 to 11 isolates each, and 15 species with 1 to 6 isolates) were evaluated, 125 were recent bloodstream isolates, 30 isolates from other sources and six ATCC isolates that included amphotericin B-resistant Candida albicans ATCC 200955, Candida lusitaniae (ATCC 200950, 200951, 200952 and 200953) and amphotericin B- and itraconazole-resistant Candida tropicalis ATCC 200956. MICs were determined at 24 and 48 h by following the CLSI guidelines, document M27-A2. DD testing was performed by following CLSI M44-A document with 5 microg posaconazole discs. Inhibition zone diameters were measured at the transition point at which growth decreased at both 24 and 48 h. Results DD showed very good reproducibility, with coefficient of variability median value 4.56. Posaconazole demonstrated good in vitro activity against all clinical isolates, including the emerging species and amphotericin B-resistant ATCC isolates except for C. tropicalis ATCC 200956 (posaconazole MIC >or= 16 mg/L). Only 1.5% and 4.1% of isolates were inhibited by >2 mg/L posaconazole at 24 and 48 h. Good correlation was obtained between methods (R = 0.763 at 24 h and 0.602 at 48 h). DD detected posaconazole-resistant isolates (MIC > 2 mg/L). Conclusions DD could be an alternative to the microdilution reference method, as no major discrepancies were detected.

Resumen Voriconazol se ha desarrollado para afrontar la necesidad creciente de nuevos y eficaces antifungicos para tratar las micosis invasoras. Esta revision describe el espectro de actividad in vitro de voriconazol basandose en los datos obtenidos en estudios publicados en los ultimos tres anos. Esta valoracion demuestra que voriconazol tiene un espectro antifungico muy amplio que engloba a los patogenos fungicos mas comunes frente a los que desarrolla una accion fungistatica (Candida) y fungicida contra Aspergillus y otros hongos filamentosos. De forma global, mas del 95% de todos los aislamientos de Candida son sensibles a voriconazol y menos del 3% son resistentes. Tasas de actividades parecidas o mejores se han descrito para Aspergillus, Cryptococcus y el resto de levaduras y mohos de importancia medica. Tambien discutimos las limitaciones planteadas por la aparicion de resistencias cruzadas a los azoles en algunos aislamientos de Candida glabrata y la reducida actividad de voriconazol contra Scedosporium prolificans, su actividad sobre las biopeliculas fungicas y de la gran utilidad potencial de la combinacion de voriconazol con otros antifungicos.

Native valve endocarditis caused by Aspergillus spp. is an uncommon disease with a high mortality rate. Generally, Aspergillus is isolated from affected valve in post-mortem or biopsy specimens. However, its isolation from blood cultures is exceedingly rare. We report a case of fungal endocarditis in a native mitral valve with the isolation of Aspergillus fumigatus both in valve vegetation and in blood culture bottles. The patient underwent valve replacement and antifungal treatment with voriconazole and caspofungin, but he died on post-operative day 45 with disseminated aspergillosis confirmed by necropsy. Paradoxically, galactomannan antigen detection in serum was negative. This is the third case of Aspergillus endocarditis with positive blood culture reported in the literature.

We compared the Neo-Sensitabs tablet assay to both reference M27-A2 broth microdilution and M44-A disk diffusion methods for testing susceptibilities of 110 isolates of Candida spp. and Cryptococcus neoformans to amphotericin B, caspofungin, fluconazole, itraconazole, and voriconazole. Neo-Sensitabs assay inhibition zone diameters in millimeters on three agars (Mueller-Hinton agar supplemented with 2% dextrose and 0.5 μg/ml methylene blue [MGM], Shadomy [SHA], and RPMI 1640 [RPMI, 2% dextrose]) were obtained at 24 to 72 h. The correlation coefficient of Neo-Sensitabs results with MICs was similar to that of the disk method for most of the five agents on MGM ( R , 0.80 to 0.89 versus 0.76 to 0.89, respectively). Overall, superior correlation was observed at 24 h for most agents. The exception was amphotericin B ( R values of 0.68 and 0.5 for disk and tablet, respectively, at 48 h versus 0.68 and 0.48, respectively, at 24 h). In general, Neo-Sensitabs results were less consistent on SHA and RPMI agars. Although agreement by breakpoint category of Neo-Sensitabs and disk results with CLSI method M27-A2 was also similar on MGM (92.7 to 98.2% versus 95.5 to 100%, respectively), the Neo-Sensitabs method failed to identify two of the six isolates with high amphotericin B MICs. These data suggest the potential value of the Neo-Sensitabs assay for testing at least four of the five agents against yeasts evaluated in the clinical laboratory.

Major burn patients have characteristics that make them especially susceptible to candidemia, but few studies focused on this have been published. The objectives were to evaluate the epidemiological, microbiological and clinical aspects of candidemia in major burn patients, determining factors associated with a poorer prognosis and mortality. We conducted a retrospective observational study of candidemia between 1996 and 2012 in major burn patients admitted to the La Fe University Hospital, Valencia, Spain. The study included 36 episodes of candidemia in the same number of patients, 55.6% men, mean age 37.33 years and low associated comorbidity. The incidence of candidemia varied between 0.26 and 6.09 episodes/1000 days stay in the different years studied. Candida albicans was the most common species (61.1%) followed by Candida parapsilosis (27.8%). Candidemia by C. krusei, C. glabrata or C. tropicalis were all identified after 2004. Central vascular catheter (CVC) was established as a potential source of candidemia in 36.1%, followed by skin and soft tissues of thermal injury (22.2%) and urinary tract (8.3%). Fluconazole was used in 19 patients (52.7%) and its in vitro resistance rate was 13.9%. The overall mortality was 47.2%, and mortality related to candidemia was 30.6%. Factors associated with increased mortality were those related to severe infection and shock. CVC was the most usual focus of candidemia. Fluconazole was the most common antifungal drug administered. The management of candidemia in major burn patients is still a challenge.

Neither breakpoints (BPs) nor epidemiological cutoff values (ECVs) have been established for Candida spp. with anidulafungin, caspofungin, and micafungin when using the Sensititre YeastOne (SYO) broth dilution colorimetric method. In addition, reference caspofungin MICs have so far proven to be unreliable. Candida species wild-type (WT) MIC distributions (for microorganisms in a species/drug combination with no detectable phenotypic resistance) were established for 6,007 Candida albicans , 186 C. dubliniensis , 3,188 C. glabrata complex, 119 C. guilliermondii , 493 C. krusei , 205 C. lusitaniae , 3,136 C. parapsilosis complex, and 1,016 C. tropicalis isolates. SYO MIC data gathered from 38 laboratories in Australia, Canada, Europe, Mexico, New Zealand, South Africa, and the United States were pooled to statistically define SYO ECVs. ECVs for anidulafungin, caspofungin, and micafungin encompassing ≥97.5% of the statistically modeled population were, respectively, 0.12, 0.25, and 0.06 μg/ml for C. albicans , 0.12, 0.25, and 0.03 μg/ml for C. glabrata complex, 4, 2, and 4 μg/ml for C. parapsilosis complex, 0.5, 0.25, and 0.06 μg/ml for C. tropicalis , 0.25, 1, and 0.25 μg/ml for C. krusei , 0.25, 1, and 0.12 μg/ml for C. lusitaniae , 4, 2, and 2 μg/ml for C. guilliermondii , and 0.25, 0.25, and 0.12 μg/ml for C. dubliniensis . Species-specific SYO ECVs for anidulafungin, caspofungin, and micafungin correctly classified 72 (88.9%), 74 (91.4%), 76 (93.8%), respectively, of 81 Candida isolates with identified fks mutations. SYO ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin, micafungin, and especially caspofungin, since testing the susceptibilities of Candida spp. to caspofungin by reference methodologies is not recommended.

Voriconazole is a new triazole developed for the treatment of life-threatening fungal infections. The drug is available for both oral and intravenous administration; the oral formulation has excellent bioavailability. The side-effect profile of voriconazole presents an acceptable safety and tolerability spectrum: transient visual disturbances, liver enzyme abnormalities, and skin rashes are the most frequently reported side effects but rarely lead to discontinuation. The potential for drug–drug interactions is high, because of its extensive hepatic metabolism. Careful attention to dosage is required, and serum levels and the effects of interacting drugs should be monitored. Review of 25 470 isolates of yeasts and 3216 isolates of filamentous fungi showed voriconazole to have broad-spectrum activity against pathogenic yeasts including intrinsically fluconazole-resistant isolates such as Candida krusei, dimorphic fungi, and opportunistic moulds like Aspergillus spp, amphotericin-B-resistant Aspergillus terreus, Fusarium spp, and Scedosporium apiospermum. It displays excellent clinical efficacy in patients with fluconazole-resistant and -susceptible Candida infections, invasive bone and central nervous system aspergillosis, and various refractory fungal infections. Voriconazole has been approved by the US Food and Drug Administration and by the European Medicines Agency for the treatment of invasive aspergillosis, serious infections caused by Fusarium and S. apiospermum, fluconazole-resistant invasive Candida infections, and candidemia in nonneutropenic patients.