Methods for Antifungal Susceptibility Testing of the Cryptococcus neoformans/C. gattii Complex: Strengths and Limitations.

When method-dependent categorical endpoints are available, namely either BPs or ECVs, MICs could aid in selecting the best treatment agent(s). BPs can categorize an isolate as either susceptible or resistant while the ECVs/ECOFFs can distinguish the wild type (WT, no known resistance mechanisms) from the Non-WT (NWT, harboring resistant mechanisms). Our literature review focused on the Cryptococcus species complex (SC) and the available methods and categorization endpoints. We also covered the incidence of these infections as well as the numerous Cryptococcus neoformans SC and C. gattii SC genotypes. The most important agents to treat cryptococcal infections are fluconazole (widely used), amphotericin B, and flucytosine. We provide data from the collaborative study that defined CLSI fluconazole ECVs for the most common cryptococcal species or genotypes and modes. EUCAST ECVs/ECOFFs are not yet available for fluconazole. We have summarized the incidence of cryptococccal infections (2000-2015) where fluconazole MICs were obtained by reference and commercial antifungal susceptibility tests. This occurrence is documented all over the world and those fluconazole MICs are mostly categorized by available CLSI ECVs/BPs as "resistant" instead of non-susceptible strains, including those by the commercial methods. As expected, the agreement between the CLSI and commercial methods is variable because SYO and Etest data could yield low/variable agreement (<90%) versus the CLSI method. Therefore, since BPs/ECVs are species and method dependent, why not gather sufficient MICs by commercial methods and define the required ECVs for these species?

In Vitro Antifungal Activity of Ibrexafungerp (SCY-078) Against Contemporary Blood Isolates From Medically Relevant Species of Candida: A European Study.

Background: Ibrexafungerp (SCY-078) is the newest oral and intravenous antifungal drug with broad activity, currently undergoing clinical trials for invasive candidiasis. Objective: The aim of this study was to assess the in vitro activity of ibrexafungerp and comparators against a collection of 434 European blood isolates of Candida. Methods: Ibrexafungerp, caspofungin, fluconazole, and micafungin minimum inhibitory concentrations (MICs) were collected from 12 European laboratories for 434 blood isolates, including 163 Candida albicans, 108 Candida parapsilosis, 60 Candida glabrata, 40 Candida tropicalis, 29 Candida krusei, 20 Candida orthopsilosis, 6 Candida guilliermondii, 2 Candida famata, 2 Candida lusitaniae, and 1 isolate each of Candida bracarensis, Candida catenulata, Candida dubliniensis, and Candida kefyr. MICs were determined by the EUCAST broth microdilution method, and isolates were classified according to recommended clinical breakpoints and epidemiological cutoffs. Additionally, 22 Candida auris from different clinical specimens were evaluated. Results: Ibrexafungerp MICs ranged from 0.016 to ≥8 mg/L. The lowest ibrexafungerp MICs were observed for C. albicans (geometric MIC 0.062 mg/L, MIC range 0.016-0.5 mg/L) and the highest ibrexafungerp MICs were observed for C. tropicalis (geometric MIC 0.517 mg/L, MIC range 0.06-≥8 mg/L). Modal MICs/MIC50s (mg/L) against Candida spp. were 0.125/0.06 for C. albicans, 0.5/0.5 for C. parapsilosis, 0.25/0.25 for C. glabrata, 0.5/0.5 for C. tropicalis, 1/1 for C. krusei, 4/2 for C. orthopsilosis, and 0.5/0.5 for C. auris. Ibrexafungerp showed activity against fluconazole- and echinocandin-resistant isolates. If adopting wild-type upper limits, a non-wild-type phenotype for ibrexafungerp was only observed for 16/434 (3.7%) isolates: 11 (4.6%) C. parapsilosis, 4 (5%) C. glabrata, and 1 (2.5%) C. tropicalis. Conclusion: Ibrexafungerp showed a potent in vitro activity against Candida.

Antifungal Resistance among Less Prevalent Candida Non-albicans and Other Yeasts versus Established and under Development Agents: A Literature Review.

Fungal diseases and antifungal resistance continue to increase, including those caused by rare or emerging species. However, the majority of the published in vitro susceptibility data are for the most common fungal species. We reviewed the literature in order to pool reference minimal inhibitory concentration (MIC) data (Clinical and Laboratory Standards Institute-CLSI and European Committee on Antimicrobial Susceptibility-EUCAST) for rare/non-prevalent Candida and other yeast species. MIC results were compared with those for Candida albicans, C. glabrata, and C. krusei. Data were listed for twenty rare and emerging Candida spp., including C. auris, as well as two Cryptococcus spp., two Trichosporon spp., Saccharomyces cerevisiae and five Malassezia spp. The best detectors of antimicrobial resistance are the breakpoints, which are not available for the less common Candida species. However, epidemiological cutoff values (ECVs/ECOFFs) have been calculated using merely in vitro data for both reference methods for various non-prevalent yeasts and recently the CLSI has established ECVs for other Candida species. The ECV could identify the non-wild type (NWT or mutants) isolates with known resistance mechanisms. Utilizing these ECVs, we were able to report additional percentages of NWT, especially for non-prevalent species, by analyzing the MIC distributions in the literature. In addition, since several antifungal drugs are under development, we are listing MIC data for some of these agents.

Recent changes in candidemia trends in a tertiary hospital (2011-2018).

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.

Recent changes in candidemia trends in a tertiary hospital (2011-2018) / Cambios recientes en la evolución de la candidemia en un hospital terciario (2011-2018)

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

ANTECEDENTES: La epidemiología de la candidemia varía con el tiempo y entre las áreas geográficas. OBJETIVOS: Se ha estudiado en niños (0-14 años) con candidemia la evolución de la tasa de incidencia y las características clínicas de los pacientes para optimizar el pronóstico y las medidas de control de esta grave enfermedad. MÉTODOS: Se llevó a cabo un estudio de cohorte retrospectivo de los casos de candidemia en la unidad de cuidados intensivos neonatales (UCIN), UCI pediátrica (UCIP) y salas pediátricas de un hospital terciario, entre los años 2011 y 2018. Se compara el curso clínico, las especies de Candida, la sensibilidad antifúngica y las tasas de incidencia. RESULTADOS: Se diagnosticaron 68 episodios de candidemia en 62 niños; el 48% de ellos tuvieron lugar en UCIN, el 31% en UCIP y el 21% en salas pediátricas. Candida albicans fue la especie más frecuente en UCIN (53%), y Candida parapsilosis predominó en UCIN (59%) y salas pediátricas (50%). Un tercio de los bebés de la UCIN tenía candidiasis invasora (CI) y la mayoría presentaba extremado bajo peso al nacimiento (EBPN) (35%). Con el tiempo, la incidencia de candidemia disminuyó en la UCIP (de 2,2 a 0,3 episodios/1.000 días/paciente, OR: 0,6; IC 95%: 0,5-0,8), mientras que en la UCIN y en las salas permaneció estable. La mortalidad se produjo principalmente en pacientes de UCIN (26%), predominó en lactantes EBPN y no cambió con el tiempo. CONCLUSIONES: La mayor incidencia y mortalidad de la candidemia y CI observadas en lactantes prematuros requiere una evaluación continua de prácticas y métodos de diagnóstico que permitan mejorar el pronóstico de esta población más vulnerable

Genotyping Reveals High Clonal Diversity and Widespread Genotypes of Candida Causing Candidemia at Distant Geographical Areas.

The objectives of this study were to gain further insight on Candida genotype distribution and percentage of clustered isolates between hospitals and to identify potential clusters involving different hospitals and cities. We aim to genotype Candida spp. isolates causing candidemia in patients admitted to 16 hospitals in Spain, Italy, Denmark, and Brazil. Eight hundred and eighty-four isolates (Candida albicans, n = 534; C. parapsilosis, n = 282; and C. tropicalis, n = 68) were genotyped using species-specific microsatellite markers. CDC3, EF3, HIS3, CAI, CAIII, and CAVI were used for C. albicans, Ctrm1, Ctrm10, Ctrm12, Ctrm21, Ctrm24, and Ctrm28 for C. tropicalis, and CP1, CP4a, CP6, and B for C. parapsilosis. Genotypes were classified as singletons (genotype only found once) or clusters (same genotype infecting two or more patients). Clusters were defined as intra-hospital (involving patients admitted to a single hospital), intra-ward (involving patients admitted to the same hospital ward) or widespread (involving patients admitted to different hospitals). The percentage of clusters and the proportion of patients involved in clusters among species, genotypic diversity and distribution of genetic diversity were assessed. Seven hundred and twenty-three genotypes were detected, 78 (11%) being clusters, most of which (57.7%; n = 45/78) were intra-hospital clusters including intra-ward ones (42.2%; n = 19/45). The proportion of clusters was not statistically different between species, but the percentage of patients in clusters varied among hospitals. A number of genotypes (7.2%; 52/723) were widespread (found at different hospitals), comprising 66.7% (52/78) of clusters, and involved patients at hospitals in the same city (n = 21) or in different cities (n = 31). Only one C. parapsilosis cluster was a widespread genotype found in all four countries. Around 11% of C. albicans and C. parapsilosis isolates causing candidemia are clusters that may result from patient-to-patient transmission, widespread genotypes commonly found in unrelated patients, or insufficient microsatellite typing genetic discrimination.

Utility of two PCR-RFLP-based techniques for identification of Candida parapsilosis complex blood isolates.

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.

In Vitro Activity of Fenticonazole against Candida and Bacterial Vaginitis Isolates Determined by Mono- or Dual-Species Testing Assays.

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. At concentrations equal to 2× MIC, fenticonazole required ∼20 h to reach the above endpoint against C. albicans in mixed culture with S. aureus, S. agalactiae, or E. coli versus ∼17 h against C. albicans in pure culture. Supra-MICs are achievable in topically treated patients' vaginal surfaces.

Killing kinetics of anidulafungin, caspofungin and micafungin against Candida parapsilosis species complex: Evaluation of the fungicidal activity.

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 8µg/ml. CFU/ml were determined at 0, 2, 4, 6, 24 and 48h. 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 29.67h to reach this endpoint for the three species studied, but more than 48h were needed against one isolate of C. orthopsilosis (8µg/ml). Caspofungin fungicidal endpoint was only achieved with 8µg/ml against one isolate of C. metapsilosis after 30.12h (1 out of 7 isolates; 14%). Micafungin fungicidal endpoint was reached in 12.74-28.38h (8µg/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.

Killing kinetics of anidulafungin, caspofungin and micafungin against Candida parapsilosis species complex: Evaluation of the fungicidal activity / Cinéticas de letalidad de la anidulafungina, la caspofungina y la micafungina frente a las especies del complejo Candida parapsilosis: evaluación de la actividad fungicida

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 μg/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 29.67h to reach this endpoint for the three species studied, but more than 48h were needed against one isolate of C. orthopsilosis (8 μg/ml). Caspofungin fungicidal endpoint was only achieved with 8μg/ml against one isolate of C. metapsilosis after 30.12 h (1 out of 7 isolates; 14%). Micafungin fungicidal endpoint was reached in 12.74-28.38h (8μg/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

Antecedentes: Candida parapsilosis, Candida metapsilosis y Candida orthopsilosis son causas relevantes de candidemia. Además, muestran diferencias en la sensibilidad a los fármacos antifúngicos. Las equinocandinas muestran diferente actividad antifúngica in vitro frente a Candida. Las curvas de tiempo-letalidad (TK) representan una excelente aproximación para evaluar la actividad fungicida de los fármacos antifúngicos. Objetivos: Comparar la actividad fungicida de la anidulafungina, la caspofungina y la micafungina frente al complejo C. parapsilosis mediante las curvas de TK. Métodos: Se estudió la actividad de tres equinocandinas frente a C. parapsilosis, C. metapsilosis y C. orthopsilosis mediante las curvas de TK. Las concentraciones ensayadas fueron 0,25, 2 y 8 μg/ml. Se determinaron las UFC/ml a las 0, 2, 4, 6, 24 y 48 h. Resultados: La actividad de las equinocandinas fue especie-, aislamiento- y concentración-dependiente. La anidulafungina alcanzó el límite fungicida frente a 6 de 7 aislamientos (86%), y necesitó 13,34-29,67h para alcanzar este límite en las tres especies estudiadas; para un aislamiento de C. orthopsilosis, requirió más de 48h (8μg/ml). El límite fungicida de la caspofungina solo se alcanzó con 8 μg/ml frente a un aislamiento de C. metapsilosis después de 30,12h (1 de 7 aislamientos; 14%). La micafungina alcanzó este límite en 12,74-28,38 h (8 μg/ml) frente a un aislamiento de C. parapsilosis y C. orthopsilosis y frente a ambos aislamientos de C. metapsilosis (4 de 7 aislamientos; 57%). Conclusiones: C. metapsilosis fue la especie más sensible a las equinocandinas, seguida de C. orthopsilosis y C. parapsilosis. La anidulafungina fue la equinocandina más activa frente al complejo C. parapsilosis

Outbreak of Candida auris in Spain: A comparison of antifungal activity by three methods with published data.

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® YeastOne® (SYO) (bloodstream isolates) and Liofilchem® 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-off values (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 ≤0.5 mg/L by SYO and EUCAST. The overall lowest AmB MICs (≤0.25 mg/L) were obtained by EUCAST. Essential agreement (±2 dilutions) between EUCAST and SYO was >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 ≤1 mg/L using dilution methods. Voriconazole MICs were method-dependent. The number of non-wild-type (non-WT) isolates depended on the ECV applied; by the 97.5% ECV all isolates were WT except for isavuconazole (1.8% non-WT). Good essential agreement (>93%) was observed between EUCAST and SYO.

Methodologies for in vitro and in vivo evaluation of efficacy of antifungal and antibiofilm agents and surface coatings against fungal biofilms.

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.

Time-kill assays of amphotericin B plus anidulafungin against Candida tropicalis biofilms formed on two different biomaterials.

PURPOSE: To determine the fungicidal activity by time-killing assays of amphotericin B (AMB) combined with anidulafungin (ANF) against biofilms of 2 clinical isolates of Candida tropicalis and the reference strain ATCC® 750, developed on polytetrafluoroethylene (PTFE) and titanium, using the CDC Biofilm Reactor (CBR) as an in vitro model. METHODS: Biofilms were developed for 24 hours on the disk surfaces and then exposed to AMB (40 mg/L), ANF (8 mg/L), alone and combined. At predetermined time points after drug exposure, biofilms were removed from the disk surface by vortexing-sonication to quantify viable biofilm cells. RESULTS: Drug activity was dependent on strain and time. After exposure to AMB, the mean decrease in viable cells attached to PTFE was 2.23 ± 0.89 Log10 cfu/cm2 (range 0.6-3.56 Log10), and on titanium 2.91 ± 1.04 (range 1.49-4.51 Log10). The reduction with ANF was 0.78 ± 0.5 (0.03-1.58 Log10) on PTFE and 0.8 ± 2.26 (0.42-1.16 Log10) on titanium. The reduction obtained with the combination of AMB + ANF was 1.8 ± 1.07 (0.22-3.54 Log10) on PTFE and 1.97 ± 0.49 (1.36-2.84 Log10) on titanium. The interaction was classified as indifferent with a tendency to antagonism. CONCLUSIONS: The activity of antifungal agents depends on the biomaterial surfaces the biofilm forming capacity of the isolate. AMB + ANF is less effective than AMB alone on both surfaces. Thus, the combination of these antifungals does not seem to add additional benefits to the treatment of C. tropicalis biofilm-related infections.

Actividad de la anfotericina B y la anidulafungina, solas y combinadas, frente a biopelículas de Candida tropicalis formadas sobre Teflon(R) y titanio / Activity of amphotericin B and anidulafungin, alone and combined, against Candida tropicalis biofilms developed on Teflon(R) and titanium

Antecedentes. 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. Objetivos. 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. Métodos. Los ensayos se realizaron en un CDC Biofilm Reactor sobre biopelículas de 24h de maduración formadas en discos de PTFE y titanio. Las concentraciones ensayadas fueron 40mg/l para AMB y 8mg/l para AND, tanto para su uso por separado como combinadas. Tras 24, 48 y 72h de exposición a los antifúngicos se determinaron las ufc/cm2 mediante agitación vorticial y cultivo cuantificado previa sonicación. Resultados. 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 Log10vs. 1,08 en la combinación) como en titanio (4,51 vs. 1,53 en la combinación), siendo la interacción indiferente en ambas superficies. Conclusiones. 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 (AU)

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 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/cm2 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/cm2 3.09 Log10vs. 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 (AU)

[Activity of amphotericin B and anidulafungin, alone and combined, against Candida tropicalis biofilms developed on Teflon® and titanium]. / Actividad de la anfotericina B y la anidulafungina, solas y combinadas, frente a biopelículas de Candida tropicalis formadas sobre Teflon® y titanio.

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 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/cm2 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/cm2 3.09 Log10vs. 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.

Nosocomial fungemia by Candida auris: first four reported cases in continental Europe / Fungemia nosocomial por Candida auris: primeros cuatro casos en Europa continental

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 >256mg/l) and voriconazole (MIC 2mg/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 (AU)

Antecedentes. Candida auris es una levadura multirresistente de reciente aparición que puede causar infecciones invasivas asociadas con una elevada mortalidad. Habitualmente, C. auris es resistente al fluconazol y el voriconazol, y en algunos casos, también a las equinocandinas y la anfotericina B. Esta especie, relacionada filogenéticamente con Candida haemulonii, no se identifica por las técnicas comerciales habitualmente disponibles en los laboratorios clínicos, por lo que la prevalencia real de las infecciones causadas por C. auris puede estar subestimada. Objetivos. Describir las características clínicas y microbiológicas de los cuatro primeros casos de fungemia por C. auris observados en el continente europeo. Métodos. Los cuatro pacientes eran adultos y estaban en la unidad de cuidados intensivos quirúrgicos. Se analizaron un total de 8 aislamientos (dos por paciente), obtenidos a partir de un hemocultivo y de punta de catéter. Resultados. Todos los aislamientos se identificaron erróneamente como Saccharomyces cerevisiae por AuxaColor 2 y como Candida sake por API ID20C. El sistema VITEK MS identificó erróneamente un aislamiento como Candida lusitaniae, otro como C. haemulonii y no pudo identificar los seis aislamientos restantes. La identificación de C. auris se confirmó mediante secuenciación de la región ITS del ADNr. Todos los aislamientos fueron resistentes al fluconazol (CMI>256mg/l) y el voriconazol (CMI 2mg/l) y sensibles al posaconazol, el itraconazol, las equinocandinas y la anfotericina B. Conclusiones. C. auris es un agente patógeno de reciente aparición que actualmente solo puede ser identificado mediante secuenciación molecular. Nuestros aislamientos fueron muy resistentes al fluconazol y resistentes al voriconazol, pero sensibles a los otros antifúngicos ensayados, lo cual destaca la importancia de identificar correctamente esta especie en la práctica asistencial para evitar fracasos terapéuticos (AU)

Nosocomial fungemia by Candida auris: First four reported cases in continental Europe.

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 >256mg/l) and voriconazole (MIC 2mg/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.

Activity of Amphotericin B and Anidulafungin Combined with Rifampicin, Clarithromycin, Ethylenediaminetetraacetic Acid, N-Acetylcysteine, and Farnesol against Candida tropicalis Biofilms.

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.

Candidiasis asociada a biopelículas / Candida biofilm-related infections

El número de dispositivos biomédicos (catéteres intravasculares, válvulas cardíacas, prótesis articulares, etc.) que se colocan en nuestros hospitales ha aumentado de manera exponencial en los últimos años. Las especies de Candida son organismos patógenos que cada vez tienen mayor relevancia en este tipo de infecciones. Candida tiene dos formas de desarrollo: planctónica y en biopelícula. Una biopelícula es una comunidad de microorganismos adheridos a una superficie y englobados por una matriz extracelular. Esta forma de desarrollo confiere a los microorganismos una gran resistencia a los agentes antimicrobianos. Esto es la causa de que los tratamientos antibióticos fracasen habitualmente y haya que retirar los dispositivos en la mayor parte de las ocasiones. Los mecanismos de adherencia inespecíficos, los sistemas de adhesina-receptor, y el sistema de comunicación intercelular denominado quorum sensing juegan un papel esencial en el desarrollo de las biopelículas de Candida. En general, los azoles tienen poca actividad frente a las biopelículas de Candida, mientras que las equinocandinas y los polienos muestran una mayor actividad. Es necesario desarrollar nuevas estrategias terapéuticas debido a la elevada morbimortalidad y el gran gasto económico asociado a este tipo de infecciones. La mayor parte de los estudios realizados hasta la fecha se han centrado en las biopelículas bacterianas. El conocimiento del proceso de formación de las biopelículas de Candida, así como su composición, es fundamental para poder desarrollar nuevas estrategias preventivas o terapéuticas (AU)

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 (AU)

[Candida biofilm-related infections]. / Candidiasis asociada a biopelículas.

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.