Epidemiological trends show a dramatic increase in the prevalence of fungal infections, and in the isolation of multidrug-resistant species, such as Candida auris. CHROMagarTM Candida (CC; CHROMagar, Paris, France) and other chromogenic media, which are widely used in the clinical laboratory because they allow a rapid identification of most Candida species. Recently, CHROMagarTM Candida Plus (CC-Plus; CHROMagar, Paris, France) was developed to detect and differentiate C. auris in addition to other major clinical Candida species, such as C. albicans, C. tropicalis, C. glabrata, or C. krusei. C. auris colonies display a differential light blue color with a blue halo. A multicentric study was designed to evaluate the performance of the CC-Plus medium in the detection of Candida species in patients' surveillance and environmental samples from three Spanish hospitals with active C. auris outbreaks. A total of 364 patients' surveillance samples and 212 environmental samples were tested. Samples were inoculated in CC and CC-Plus in parallel, and the plates were read at 24 and 48 h. All recovered colonies were presumptively identified according to colony color described by manufacturer, and the definitive identification was performed by mass spectrometry at 48 h. A total of 134 C. auris isolates were obtained (101 from patients' surveillance samples, and 33 from environmental samples). Sensitivity, specificity, and predictive positive and negative values were 99.5%, 100%, 100%, and 99.1%, respectively, for the main clinical Candida species, showing that CC-Plus is comparable to CC, with the advantage of being able to differentiate C. auris from C. parapsilosis. Furthermore, CC-Plus was able to detect one C. albicans, one C. glabrata, and eight C. auris that did not grow in CC. Additionally, the yeast colonies were generally larger, suggesting that this novel medium could be a richer medium, and suitable for surveillance and environmental cultures of C. auris and other clinically relevant Candida species.

BACKGROUND:Microbiological contamination is one of the main risks that must be controlled in tissue banking practices. For this reason, strict donor selection criteria are applied, disinfection protocols are used, and microbiological monitoring is performed at various stages. AIM:To detect Candida auris in arterial allografts and assess its origin. METHODS:Data on two multi-tissue donations with positive microbiological cultures for C. auris were analysed. Risk factors for microbiological contamination were assessed at procurement, processing and post storage. FINDINGS:C. auris was only isolated in cultures from arteries, and was not detected in cultures from cornea, musculoskeletal tissue or skin (even in the axillary-rectal sample taken from one donor). CONCLUSION:The donor's own skin was identified as the most likely source to explain the contamination of arteries by C. auris. Due to the pathogenicity of this fungus and difficulties associated with its correct identification, the implementation of measures for its detection in tissue donations is recommended.

BACKGROUND:Scedosporium species and Lomentospora prolificans (Sc/Lp) are emerging molds that cause invasive disease associated with a high mortality rate. After Aspergillus, these molds are the second filamentous fungi recovered in lung transplant (LT) recipients. AIMS:Our objective was to evaluate the incidence, risk factors and outcome of Sc/Lp infections in LT recipients at a tertiary care hospital with a national reference LT program. METHODS:A nine-year retrospective study was conducted. RESULTS:During this period, 395 LT were performed. Positive cultures for Sc/Lp were obtained from twenty-one LT recipients. Twelve patients (incidence 3.04%) developed invasive scedosporiosis (IS). In 66.7% of the patients with IS the invasive infection was defined as a breakthrough one. The main sites of infection were lungs and paranasal sinuses. Most of the patients received combination antifungal therapy. The IS crude mortality rate after 30 days was 16.7%, and 33.3% after a year. CONCLUSIONS:Our study highlights improved survival rates associated with combination antifungal therapy in LT recipients and underlines the risk of breakthrough infections in patients with allograft dysfunction on nebulized lipidic amphotericin B prophylaxis. In addition to pretransplant colonization, acute or chronic organ dysfunctions seem to be the main risk factors for IS.

Background: 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.Objectives: 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.Methods: Assays were performed using the CDC Biofilm Reactor equipped with PTFE and titanium disks with C. tropicalis biofilms after 24 h of maturation. The concentrations assayed were 40 mg/l for AMB and 8 mg/l for AND, both alone and combined. After 24, 48 and 72 h of exposure to the antifungals, the cfu/cm(2) was determined by a vortexing-sonication procedure.Results: AMB 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/cm(2) 3.09 Log(10) vs. 1.08 when combined) and titanium (4.51 vs. 1.53 when combined), being the interaction irrelevant on both surfaces.Conclusions: AMB 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. (C) 2017 Asociacion Espanola de Micologia. Published by Elsevier Espana, S.L.U. All rights reserved.

Las estrategias terapéuticas actuales poseen una limitada eficacia para erradicar biopelículas de Candida formadas en la superficie de los dispositivos biomédicos. Pocos estudios han evaluado la eficacia de los antifúngicos sobre biopelículas de Candida tropicalis. Evaluar la actividad de la anfotericina B (AMB) y la anidulafungina (AND), solas y combinadas, sobre biopelículas de C. tropicalis desarrolladas en superficies de politetrafluoroetileno (teflón - PTFE) y titanio mediante ensayos de letalidad-tiempo. Los ensayos se realizaron en un CDC Biofilm Reactor sobre biopelículas de 24 h de maduración 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 exposición a los antifúngicos se determinaron las ufc/cm2 mediante agitación vorticial y cultivo cuantificado previa sonicación. AMB redujo las células viables adheridas a PTFE y titanio en más de un 99%, y AND lo hizo en un 89,3% en PTFE y 96,8% en titanio. La combinación 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 combinación) como en titanio (4,51 vs. 1,53 en la combinación), siendo la interacción indiferente en ambas superficies. AMB es más activa que AND sobre biopelículas de C. tropicalis. La eficacia sobre las biopelículas es mayor en el titanio. La combinación AMB + AND es menos eficaz que AMB sola en ambas superficies. 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 24 h of maturation. The concentrations assayed were 40 mg/l for AMB and 8 mg/l for AND, both alone and combined. After 24, 48 and 72 h of exposure to the antifungals, the cfu/cm2 was determined by a vortexing-sonication procedure. AMB reduced biofilm viable cells attached to PTFE and titanium by ≥99% and AND by 89.3% on PTFE and 96.8% on titanium. The AMB + AND combination was less active than AMB alone, both on PTFE (decrease of cfu/cm2 3.09 Log10 vs. 1.08 when combined) and titanium (4.51 vs. 1.53 when combined), being the interaction irrelevant on both surfaces. AMB 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.

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 µM, 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 µM, 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/cm2. 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/cm2. Viable cells in biofilms were 4.73 and 4.29 Log10CFU/cm2 on PTFE and titanium, respectively. Maximum Log10 decreases in CFU/cm2 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.

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 1/8 × BMIC50 to 4 × BMIC50 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.

The Streptococcus bovis group (SBG) is a well-known cause of endocarditis, but its role in osteoarticular infections (OAIs) has not been well described.We analyzed all patients with OAIs by SBG diagnosed in our hospital (1988–2014). We selected those cases with septic arthritis and osteomyelitis, as defined according to clinical, microbiological, and imaging studies. Identification of the strains was performed by using the API 20 Strep and the GP card of the Vitek 2 system, and confirmed the identification by molecular methods. In addition, we reviewed the literature to select all cases of OAI by SBG during the period 1980–2015.From the 83 cases of OAI included in the analysis (21 from our center and 62 from the literature review), 59 were osteomyelitis (57 of them spondylodiscitis) and 24 were arthritis (2 with associated spondylodiscitis). The mean age was 66.9 years, and 79.2% of the patients were men. Endocarditis (IE) was associated with 59% of the cases and this association was greater for osteomyelitis than for arthritis (78.9% vs. 13.6%; P = 0.001). OAI was a presenting symptom in 63% of the cases of IE. Colonoscopy was performed in 64 cases, which detected colorectal neoplasm (CRN) in 46 patients (71.8%), almost all asymptomatic. Some 69.5% of these neoplasm were carcinomas or advanced adenomas. The blood cultures were positive in 78.3% cases. In 45 cases, the S. bovis species was identified; in 82.2% of the cases the cause was Streptococcus gallolyticus subsp. gallolyticus. The mortality was 7.2%, which in no case was attributable to the OAI.OAIs are frequently the initial manifestation of IE caused by SBG. S. gallolyticus causes most of these infections. Echocardiogram and colonoscopy are therefore mandatory, given the species’ close association with IE and CRN.

Dissemination of multidrug-resistant Klebsiella pneumoniae in the hospital environment represents a primary target of resistance containment and stewardship programs. At present, polymyxins, mostly in combination, exemplify a last-resort alternative. Colistin-resistant K. pneumoniae isolates harboring OXA-48 plus CTX-M-15 (n = 21) with the simultaneous colistin-susceptible counterparts (n = 9) were recovered from 14 hospitalized patients (January 2014–January 2015) admitted in different wards. In most cases, patients had not previously received colistin. Genetic relatedness experiments demonstrated that 93% (28/30) of isolates belonged to the ST11 high-risk clone. Heteroresistance and the fitness cost of colistin resistance were addressed in susceptible and resistant isolates as well as in in vitro–obtained stable mutants, and results appeared to be strain dependent. Whole genome sequencing demonstrated molecular changes in pmrA, pmrB, and mgrB genes. Plasmid-mediated colistin resistance genes were not found. Colistin resistance in multidrug-resistant K. pneumoniae isolates should be continuously monitored to detect its potential emergence, even in patients not previously exposed to colistin.

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 (798.6), followed by C. albicans (64.56.3), C. tropicalis (53.313.1) and C. glabrata (23.811.2). This reduction was significant (P0.05) 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.

The in vitro activity of posaconazole and voriconazole was evaluated by MIC, minimum fungicidal concentration (MFC), and time-kill methods. MFCs were determined for 15 Candida krusei and time-killing curves for 5 of these isolates. MFCs were obtained transferring 100 microL from clear MIC wells onto Sabouraud dextrose agar. Time-kill studies were performed in RPMI 1640 medium (5 mL, inoculum approximately 10(5) colony-forming unit [CFU]/mL). Geometric mean (GM) MIC and GM-MFC were 0.2 and 0.72 mg/L for posaconazole and 0.4 and 2.64 mg/L for voriconazole. The killing rate was isolate and concentration dependent; reductions in CFUs start at >or=0.12 mg/L (posaconazole) and >or= 0.5 mg/L (voriconazole). The mean time to reach a 90% growth reduction was 41.5+/-14.2 h (8 mg/L posaconazole) and 48.7+/-61.3 h (32 mg/L voriconazole). By time kill, a 99% killing rate was not reached by either agent. Both methods demonstrated that posaconazole (more active and greater killing rate) and voriconazole have fungicidal activity against C. krusei.

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.

Invasive infections caused by Candida spp. are increasing worldwide and are becoming an important cause of morbidity and mortality in immunocompromised patients. A large number of manifestations of candidiasis are associated with the formation of biofilms on inert or biological surfaces. Candida spp. biofilms are recalcitrant to treatment with conventional antifungal therapies. The aim of this study was dual 1) to determine the prevalence of biofilm producers among clinical isolates from catheter (16 C. albicans ) and blood culture (2 C. albicans and 30 C. tropicalis), and 2) to determine the activity of amphotericin B and anidulafungin against C. albicans and C. tropicalis biofilms of 24 and 48 hours of maturation. Biofilms were developed using a 96-well microtitre plate model and production and activity of antifungal agents against biofilms were determined by the tetrazolium (XTT) reduction assay. Of catheter and blood isolates, 62.5 and 56.25%, respectively, produced biofilms. By species, 68.42% of C. albicans and 53.33% of C. tropicalis were biofilm producers. C. albicans biofilms showed more resistance to amphotericin B and anidulafungin than their planktonic counterparts. Complete killing of biofilms was never achieved, even at the highest concentrations of the drugs tested. Anidulafungin displayed more activity than amphotericin B against C. albicans biofilms of 24 hours of maturation (GM MIC 0.354 vs. 0.686 microg/ml), but against C. tropicalis biofilms amphotericin B was more active (GM MIC 11.285 vs. 0.476 microg/ml). In contrast, against biofilms with 48 hours maturation, amphotericin B was more active against both species.

The effect of culture medium and incubation time on the fungicidal activity of caspofungin and on the frequency of paradoxical effect has been studied in 144 blood culture isolates (42, Candida tropicalis, 32 Candida glabrata, 21 Candida krusei, 20 Candida parapsilosis, 11 Candida guilliermondii, 4 Candida famata, 3 Candida lusitaniae, 3 Blastoschizomyces capitatus, 2 Saccharomyces cerevisiae, 2 Yarrowia lipolytica, 1 Candida inconspicua, 1 Candida lambica, 1 Candida sake y 1 Pichia ohmeri). The media assayed were standard RPMI 1640, Antibiotic Medium #3 (AM#3) and AM#3 with 2% of dextrose (AM#3-G). MIC(2) (>or= 50% growth inhibition) and MIC(0) (100% inhibition) at 24 and 48h and the minimum fungicidal concentration (MFC) were determined. The greatest activity of caspofungin was obtained in AM#3 medium followed by AM#3-G and RPMI. Geometric means (GM) MIC(2) at 24/48 h incubation were 0.06/0.12, 0.1/0.2 and 0.65/0.89 mg/l, respectively. GMMIC(0) was 0.1/0.17 (AM#3), 0.16/0.25 (AM#3-G) and 1.06/1.7 mg/l (RPMI). Caspofungin showed fungicidal activity in the three media (GM-MFC, 0.28, 0.38 and 3 mg/l in AM#3, AM#3-G and RPMI, respectively). The percentage of caspofungin tolerant isolates (MFC >or=8 x MIC(0)) was 8.09% (AM#3), 8.3% (AM#3-G) and 13.9% (RPMI). RPMI was the medium less affected by the incubation time and AM#3 the most affected (93.7 and 78.2% of isolates the MIC(2) increased or=2 mg/l (AM#3) and >or=4 mg/l (AM#3-G). The highest frequency was obtained in RPMI medium and C. tropicalis species and the lowest frequency for C. krusei y C. glabrata.

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 mu g/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 mu g/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.