Trends in antifungal resistance in Candida from a multicenter study conducted in Madrid (CANDIMAD study): fluconazole-resistant C. parapsilosis spreading has gained traction in 2022.

We previously conducted a multicenter surveillance study on Candida epidemiology and antifungal resistance in Madrid (CANDIMAD study; 2019-2021), detecting an increase in fluconazole-resistant Candida parapsilosis. We here present data on isolates collected in 2022. Furthermore, we report the epidemiology and antifungal resistance trends during the entire period, including an analysis per ward of admission. Candida spp. incident isolates from blood cultures and intra-abdominal samples from patients cared for at 16 hospitals in Madrid, Spain, were tested with the EUCAST E.Def 7.3.2 method against amphotericin B, azoles, micafungin, anidulafungin, and ibrexafungerp and were molecularly characterized. In 2022, we collected 766 Candida sp. isolates (686 patients; blood cultures, 48.8%). Candida albicans was the most common species found, and Candida auris was undetected. No resistance to amphotericin B was found. Overall, resistance to echinocandins was low (0.7%), whereas fluconazole resistance was 12.0%, being higher in blood cultures (16.0%) mainly due to fluconazole-resistant C. parapsilosis clones harboring the Y132F-R398I ERG11p substitutions. Ibrexafungerp showed in vitro activity against the isolates tested. Whereas C. albicans was the dominant species in most hospital wards, we observed increasing C. parapsilosis proportions in blood. During the entire period, echinocandin resistance rates remained steadily low, while fluconazole resistance increased in blood from 6.8% (2019) to 16% (2022), mainly due to fluconazole-resistant C. parapsilosis (2.6% in 2019 to 36.6% in 2022). Up to 7 out of 16 hospitals were affected by fluconazole-resistant C. parapsilosis. In conclusion, rampant clonal spreading of C. parapsilosis fluconazole-resistant genotypes is taking place in Madrid.

Antifungal resistance in Candida spp within the intra-abdominal cavity: study of resistance acquisition in patients with serial isolates.

OBJECTIVES: Antifungal susceptibility testing is mostly conducted on blood-cultured Candida spp isolates. Because the intra-abdominal cavity has been highlighted as a hidden echinocandin-resistant C. glabrata reservoir, we assessed whether testing sequential isolates from a given patient might increase the chances of detecting antifungal resistance. METHODS: Intra-abdominal initial and sequential isolates from the same species from patients included in the CANDIdaemia in MADrid study (January 2019 to June 2022) were studied. We assessed antifungal susceptibility to amphotericin B, azoles, anidulafungin, micafungin, and ibrexafungerp using European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology and molecularly characterized resistant isolates. RESULTS: We collected 308 isolates (C. albicans [n = 179/308; 58.1%], C. glabrata [n = 101/308; 32.8%], C. tropicalis [n = 17/308; 5.5%], and C. parapsilosis [n = 11/308; 3.6%]) from 112 patients distributed as incident (n = 125/308) and sequential (n = 183/308). Per patient resistance rates of fluconazole (13.4% [15/112] vs. 8% [9/112]); 5.4% proportions difference (95% CI, -2.7% to 13.5%, p 0.09) and echinocandins (8.9% [10/112] vs. 1.8% [2/112]); 7.1% proportions difference (95% CI; 1.2-12.9%; p 0.01) were higher when considering all available isolates than only incident isolates. Resistance was detected in 18 of 112 patients and would have been overlooked in 11 of 18 (61.1%) patients if only incident isolates had been studied. Of the patients who harboured fluconazole or echinocandin-resistant isolates, 14 of 15 and 8 of 10 had received or were receiving fluconazole or echinocandins, respectively. DISCUSSION: Testing sequential Candida isolates from intra-abdominal samples is required to detect antifungal resistance, particularly to echinocandins, in patients whose incident isolates turned out to be susceptible. Furthermore, patients with echinocandin-resistant infections had frequently used echinocandins and had common secondary resistance acquisition.

Yeasts from blood cultures in the wake of the COVID-19 pandemic in a tertiary care hospital: Shift in species epidemiology, steady low antifungal resistance and full in vitro ibrexafungerp activity.

Several institutions reported a rise not only in fungemia incidence but also in the number of cases caused by Candida auris or fluconazole-resistant C. parapsilosis during the COVID-19 pandemic. Since the pandemic broke out in early 2020, we studied its impact on fungemia incidence, species epidemiology, potential patient-to-patient transmission, and antifungal resistance in 166 incident yeast isolates collected from January 2020 to December 2022. Isolates were molecularly identified, and their antifungal susceptibilities to amphotericin B, azoles, micafungin, anidulafungin, and ibrexafungerp were studied following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) method, and genotyped. The fungemia incidence (episodes per 1000 admissions) tended to decrease over time (2020 = 1.60, 2021 = 1.36, 2022 = 1.16); P > .05). Species distribution was C. albicans (50.6%, n = 84), C. parapsilosis (18.7%, n = 31), C. glabrata (12.0%, n = 20), C. tropicalis (11.4%, n = 19), C. krusei (3.0%, n = 5), other Candida spp. (1.2%, n = 2), and non-Candida yeasts (3.0%, n = 5). The highest and lowest proportions of C. albicans and C. parapsilosis were detected in 2020. The proportion of isolates between 2020 and 2022 decreased in C. albicans (60.3% vs. 36.7%) and increased in C. parapsilosis (10.3% vs. 28.6%; P < .05) and C. tropicalis (8.8% vs. 16.3%; P > .05). Only three C. albicans intra-ward clusters involving two patients each were detected, and the percentages of patients involved in intra-ward clusters reached 9.8% and 8.0% in 2020 and 2021, respectively, suggesting that clonal spreading was not uncontrolled. Fluconazole resistance (5%) exhibited a decreasing trend (P > .05) over time (2020 = 7.6%; 2021 = 4.2%; and 2022 = 2.1%). Ibrexafungerp showed high in vitro activity.

Fungemia incidence increased during the COVID-19 pandemic in our hospital, however, clonal spreading was not uncontrolled. The proportion of C. parapsilosis and C. tropicalis cases constantly increased. Antifungal resistance remained very low, and fluconazole-resistant C. parapsilosis was undetected.

Correction: Díaz-García et al. Candida Genotyping of Blood Culture Isolates from Patients Admitted to 16 Hospitals in Madrid: Genotype Spreading during the COVID-19 Pandemic Driven by Fluconazole-Resistant C. parapsilosis. J. Fungi 2022, 8, 1228.

In the original publication [...].

Candida Genotyping of Blood Culture Isolates from Patients Admitted to 16 Hospitals in Madrid: Genotype Spreading during the COVID-19 Pandemic Driven by Fluconazole-Resistant C. parapsilosis.

BACKGROUND: Candidaemia and invasive candidiasis are typically hospital-acquired. Genotyping isolates from patients admitted to different hospitals may be helpful in tracking clones spreading across hospitals, especially those showing antifungal resistance. METHODS: We characterized Candida clusters by studying Candida isolates (C. albicans, n = 1041; C. parapsilosis, n = 354, and C. tropicalis, n = 125) from blood cultures (53.8%) and intra-abdominal samples (46.2%) collected as part of the CANDIMAD (Candida in Madrid) study in Madrid (2019-2021). Species-specific microsatellite markers were used to define the genotypes of Candida spp. found in a single patient (singleton) or several patients (cluster) from a single hospital (intra-hospital cluster) or different hospitals (widespread cluster). RESULTS: We found 83 clusters, of which 20 were intra-hospital, 49 were widespread, and 14 were intra-hospital and widespread. Some intra-hospital clusters were first detected before the onset of the COVID-19 pandemic, but the number of clusters increased during the pandemic, especially for C. parapsilosis. The proportion of widespread clusters was significantly higher for genotypes found in both compartments than those exclusively found in either the blood cultures or intra-abdominal samples. Most C. albicans- and C. tropicalis-resistant genotypes were singleton and presented exclusively in either blood cultures or intra-abdominal samples. Fluconazole-resistant C. parapsilosis isolates belonged to intra-hospital clusters harboring either the Y132F or G458S ERG11p substitutions; the dominant genotype was also widespread. CONCLUSIONS: the number of clusters-and patients involved-increased during the COVID-19 pandemic mainly due to the emergence of fluconazole-resistant C. parapsilosis genotypes.

Blood and intra-abdominal Candida spp. from a multicentre study conducted in Madrid using EUCAST: emergence of fluconazole resistance in Candida parapsilosis, low echinocandin resistance and absence of Candida auris.

OBJECTIVES: We prospectively monitored the epidemiology and antifungal susceptibility of Candida spp. from blood cultures and intra-abdominal samples in patients admitted to hospitals in the Madrid area. METHODS: Between 2019 and 2021, we prospectively collected incident isolates [one per species, patient and compartment (blood cultures versus intra-abdominal samples)] from patients admitted to any of 16 hospitals located in Madrid. We studied the antifungal susceptibilities to amphotericin B, triazoles, micafungin, anidulafungin and ibrexafungerp following the EUCAST E.Def 7.3.2 procedure. RESULTS: A total of 2107 Candida spp. isolates (1895 patients) from blood cultures (51.7%) and intra-abdominal samples were collected. Candida albicans, the Candida glabrata complex, the Candida parapsilosis complex, Candida tropicalis and Candida krusei accounted for 96.9% of the isolates; in contrast, Candida auris was undetected. Fluconazole resistance in Candida spp. was higher in blood cultures than in intra-abdominal samples (9.1% versus 8.2%; P > 0.05), especially for the C. parapsilosis complex (16.6% versus 3.6%, P < 0.05), whereas echinocandin resistance tended to be lower in blood cultures (0.5% versus 1.0%; P > 0.05). Resistance rates have risen, particularly for fluconazole in blood culture isolates, which increased sharply in 2021. Ibrexafungerp showed in vitro activity against most isolates. Species distributions and resistance rates varied among hospitals. CONCLUSIONS: Whereas no C. auris isolates were detected, fluconazole-resistant C. parapsilosis isolates have been spreading across the region and this has pulled up the rate of fluconazole resistance. In contrast, the rate of echinocandin resistance continues to be low.

Evidence of Fluconazole-Resistant Candida parapsilosis Genotypes Spreading across Hospitals Located in Madrid, Spain and Harboring the Y132F ERG11p Substitution.

We have been monitoring the antifungal resistance in Candida parapsilosis isolates collected from inpatients at Madrid metropolitan area hospitals for the last 3 years. The study aimed to elucidate the presence of fluconazole-resistant C. parapsilosis genotypes in Madrid. From January 2019 to December 2021, a total of 354 C. parapsilosis isolates (n = 346 patients) from blood (76.6%) or intraabdominal samples were collected and genotyped using species-specific microsatellite markers. Antifungal susceptibilities to amphotericin B, the triazoles, micafungin, anidulafungin, and ibrexafungerp were performed according to EUCAST E.Def 7.3.2; the ERG11 gene was sequenced in fluconazole-resistant isolates. A total of 13.6% (n = 48/354) isolates (one per patient) were found to be resistant to fluconazole and non-wild-type to voriconazole but fully susceptible to ibrexafungerp. Resistant isolates were mostly sourced from blood (n = 45/48, 93.8%) and were detected in five hospitals. Two hospitals accounted for a high proportion of resistant isolates (n = 41/48). Resistant isolates harbored either the Y132F ERG11p amino acid substitution (n = 43) or the G458S substitution (n = 5). Isolates harboring the Y132F substitution clustered into a clonal complex involving three genotypes (one genotype accounted for n = 39/43 isolates) that were found in four hospitals. Isolates harboring the G458S substitution clustered into another genotype found in a fifth hospital. C. parapsilosis genotypes demonstrating resistance to fluconazole have been spreading across hospitals in Madrid, Spain. Over the last 3 years, the frequency of isolation of such isolates and the number of hospitals affected is on the rise.

Impact of the COVID-19 Pandemic on the Clinical Profile of Candidemia and the Incidence of Fungemia Due to Fluconazole-Resistant Candida parapsilosis.

Severely ill COVID-19 patients are at high risk of nosocomial infections. The aim of the study was to describe the characteristics of candidemia during the pre-pandemic period (January 2019−February 2020) compared to the pandemic period (March 2020−September 2021). Antifungal susceptibilities were assessed using the EUCAST E.Def 7.3.2 broth dilution method. Fluconazole-resistant C. parapsilosis isolates (FRCP) were studied for sequencing of the ERG11 gene. The incidence of candidemia and C. parapsilosis bloodstream infection increased significantly in the pandemic period (p = 0.021). ICU admission, mechanical ventilation, parenteral nutrition and corticosteroids administration were more frequent in patients with candidemia who had been admitted due to COVID-19. Fifteen cases of FRCP fungemia were detected. The first case was recorded 10 months before the pandemic in a patient transferred from another hospital. The incidence of FRCP in patients admitted for COVID-19 was 1.34 and 0.16 in all other patients (p < 0.001). ICU admission, previous Candida spp. colonization, arterial catheter use, parenteral nutrition and renal function replacement therapy were more frequent in patients with candidemia due to FRCP. All FRCP isolates showed the Y132F mutation. In conclusion, the incidence of candidemia experienced an increase during the COVID-19 pandemic and FRCP fungemia was more frequent in patients admitted due to COVID-19.

ΔF659 and F659S substitutions at the HS1 of FKS2 gene, along with E655A and W715L upstream and downstream substitutions, correlate with high ibrexafungerp MICs against Candidaglabrata.

OBJECTIVES: Ibrexafungerp is a new inhibitor of Candida spp glucan synthase. We previously set the ibrexafungerp wild-type upper limit (wtUL) against Candida glabrata. We here assessed which FKS2 gene substitutions confer an ibrexafungerp non-wild-type phenotype in C. glabrata isolates. METHODS: We studied a set of C. glabrata (n = 34) isolates showing resistance to micafungin and anidulafungin (n = 28) or only to anidulafungin (n = 6) and harbouring 10 different FKS2 gene substitutions. Antifungal susceptibility to ibrexafungerp was tested according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) E.Def 7.3.2 procedure and isolates were considered ibrexafungerp non-wild type according to the statistical wtUL (minimum inhibitory concentration [MIC] ≥2) or visual wtUL (MIC ≥4). RESULTS: Ibrexafungerp MICs against the isolates ranged from 0.06 to 4 mg/L. Four FKS2 gene substitutions (ΔF659, F659S, E655A, and W715L) were exclusively found in isolates showing an ibrexafungerp MIC above the statistical wtUL (≥2 mg/L) whereas isolates harbouring other substitutions were found to be ibrexafungerp wild type. The use of the visual wtUL (MIC ≥4 mg/L) bisected the population of isolates harbouring such substitutions. DISCUSSION: C. glabrata isolates showing an ibrexafungerp MIC ≥2 mg/L may be considered non-wild type and are prone to harbour ΔF659, F659S, E655A, and W715L substitutions at the FKS2 gene. It is worth noting that substitutions ΔF659 and F659S were located at the beginning of the HS1 of FKS2 gene of C. glabrata. The role of other substitutions on conferring a non-wild-type phenotype to ibrexafungerp is not well elucidated.

In vitro activity of ibrexafungerp against Candida species isolated from blood cultures. Determination of wild-type populations using the EUCAST method.

OBJECTIVES: Ibrexafungerp is a new oral glucan synthase inhibitor with in vivo and in vitro activity against Candida spp., including echinocandin- and azole-resistant isolates. We studied the in vitro activity of ibrexafungerp against Candida species isolated from blood cultures and assessed wild-type upper limits against the five Candida species most frequently associated to candidaemia. METHODS: Isolates (n = 958) causing incident episodes of candidaemia in patients admitted to Gregorio Marañón hospital (Madrid, Spain) between January 2007 and April 2021 were studied. Antifungal susceptibility to ibrexafungerp, fluconazole, micafungin and anidulafungin was tested (EUCAST E.Def 7.3.2) and wild-type upper limits determined against C. albicans (n = 462), C. glabrata (n = 120), C. parapsilosis (n = 249), C. tropicalis (n = 73) and C. krusei (n = 24). fksgene sequencing was carried out in non-wild-type isolates. RESULTS: Ibrexafungerp showed antifungal in vitro activity against the studied isolates. Wild-type upper limits for ibrexafungerp were >0.25 mg/L against C. albicans, >1 mg/L against C. parapsilosis, C. glabrata, and C. tropicalis, and >2 mg/L against C. krusei. Percentages of ibrexafungerp non-wild-type isolates were low (C. parapsilosis and C. krusei, 0%; C. albicans, 0.22% (1/462); C. glabrata, 0.83% (1/120); and C. tropicalis, 1.37% (1/73)). Ibrexafungerp proved in vitro activity against fluconazole- or echinocandin-resistant isolates. DISCUSSION: We show in vitro activity of ibrexafungerp against the tested Candida species. Furthermore, we provide ibrexafungerp wild-type upper limits, which allows defining the wild-type populations of the five most relevant Candida species.

Antifungal Susceptibility Testing Identifies the Abdominal Cavity as a Source of Candida glabrata-Resistant Isolates.

To identify unrecognized niches of resistant Candida isolates and compartmentalization, we retrospectively studied the antifungal susceptibility of 1,103 Candida spp. isolates from blood cultures, nonblood sterile samples, and nonsterile samples. Antifungal susceptibility was assessed by EUCAST E.Def 7.3.2; sequencing and genotyping of the fks1-2 and erg11 genes were carried out for non-wild-type isolates. Resistance compartmentalization (presence of resistant and susceptible isogenic isolates in different anatomical sites of a given patient) was studied. Clinical charts of patients carrying non-wild-type isolates were reviewed. Most isolates (63%) were Candida albicans, regardless the clinical source; Candida glabrata (27%) was the second most frequently found species in abdominal cavity samples. Fluconazole and echinocandin resistance rates were 1.5 and 1.3%, respectively, and were highest in C. glabrata. We found 22 genotypes among non-wild-type isolates, none of them widespread across the hospital. Fluconazole/echinocandin resistance rates of isolates from the abdominal cavity (3.2%/3.2%) tended to be higher than those from blood cultures (0.7%/1.3%). Overall, 15 patients with different forms of candidiasis were infected by resistant isolates, 80% of whom had received antifungals before or at the time of isolate collection; resistance compartmentalization was found in six patients, mainly due to C. glabrata. The highest antifungal resistance rate was detected in isolates from the abdominal cavity, mostly C. glabrata. Resistance was not caused by the spread of resistant clones but because of antifungal treatment. Resistance compartmentalization illustrates how resistance might be overlooked if susceptibility testing is restricted to bloodstream isolates.

Lack of relationship between genotype and virulence in Candida species / Ausencia de relación entre el genotipo y la virulencia en especies de Candida

BACKGROUND: The virulence of isolates among different Candida species causing candidemia may play a role in the prognosis of the patients. Furthermore, the potential relationship between genotype and virulence is still unclear and need to be further studied. AIMS: We aim to assess the relationship between genotype and virulence in Candida species using a Galleria mellonella larvae infection model. METHODS: One hundred and ninety-four isolates from 68 clusters (Candida albicans, 114/41; Candida parapsilosis, 74/24; Candida tropicalis, 6/3) were compared against the same number of each species singleton genotypes in terms of survival of G. mellonella larvae. RESULTS: The median of survival and the IQR ranges of clusters and singleton were as follows: C. albicans (2 days, IQR 1.5-2 vs. 2 days, IQR 1-2.25), C. parapsilosis (2 days, IQR 1.5-2.6 vs. 2 days, IQR 2-3.3), and C. tropicalis (1 day, IQR 1-3.5 vs. 2 days, IQR 2-3.5; p < 0.05). High intra-cluster variability in terms of median of survival was found regardless the species. CONCLUSIONS: No relationship between genotype and virulence in Candida was observed with the G. mellonella model

ANTECEDENTES: La virulencia de cepas de diferentes especies de Candida causantes de candidemia puede jugar un papel en el pronóstico de los pacientes, y su estudio en el modelo de infección en Galleria mellonella puede ser útil para entender su contribución general a la infección. Además, la potencial relación entre genotipo y virulencia requiere de más estudios. OBJETIVOS: Se evaluó la relación entre genotipo y virulencia en especies de Candida mediante el modelo de infección de larvas de G. mellonella. MÉTODOS: Se estudió la supervivencia de las larvas infectadas con 194 aislados incluidos en 68 clusters (Candida albicans, 114/41; Candida parapsilosis, 74/24; Candida tropicalis, 6/3) y con el mismo número de aislados con genotipos únicos por especie. RESULTADOS: La mediana de supervivencia y los rangos intercuartílicos (IQR) de clusters y genotipos únicos se muestra a continuación: C. albicans (2 días, IQR: 1,5-2 vs. 2 días, IQR: 1-2,25), C. parapsilosis (2 días, IQR: 1,5-2,6 vs. 2 días, IQR: 2-3,3), y C. tropicalis (un día, IQR: 1-3,5 vs. 2 días, IQR: 2-3,5; p < 0,05). Encontramos una importante variabilidad en la mediana de supervivencia entre cepas del mismo cluster, independientemente de la especie analizada. CONCLUSIONES: No se encontró relación entre el genotipo y la virulencia entre los aislados de Candida evaluados mediante el modelo de infección de G. mellonella

Monitoring the Epidemiology and Antifungal Resistance of Yeasts Causing Fungemia in a Tertiary Care Hospital in Madrid, Spain: Any Relevant Changes in the Last 13 Years?

We conducted an updated analysis on yeast isolates causing fungemia in patients admitted to a tertiary hospital in Madrid, Spain, over a 13-year period. We studied 896 isolates associated with 872 episodes of fungemia in 857 hospitalized patients between January 2007 and December 2019. Antifungal susceptibility was assessed by EUCAST EDef 7.3.2. Mutations conferring azole and echinocandin resistance were further studied, and genotyping of resistant clones was performed with species-specific microsatellite markers. Candida albicans (45.8%) was the most frequently identified species, followed by the Candida parapsilosis complex (26.4%), Candida glabrata (12.3%), Candida tropicalis (7.3%), Candida krusei (2.3%), other Candida spp. (3.1%), and non-Candida yeasts (2.8%). The rate of fluconazole resistance in Candida spp. was 4.7%, ranging from 0% (C. parapsilosis) to 9.1% (C. glabrata). The overall rate of echinocandin resistance was 3.1%. Resistance was highly influenced by the presence of intrinsically resistant species. Although the number of isolates between 2007 and 2013 was almost 2-fold higher than that in the period from 2014 to 2019 (566 versus 330), fluconazole resistance in Candida spp. was greater in the second period (3.5% versus 6.8%; P < 0.05), while overall resistance to echinocandins remained stable (3.5% versus 2.4%; P > 0.05). Resistant clones were collected from different wards and/or time points, suggesting that there were no epidemiological links. The number of fungemia episodes has been decreasing over the last 13 years, with a slight increase in the rate of fluconazole resistance and stable echinocandin resistance. Antifungal resistance is not the cause of the spread of resistant clones.

Lack of relationship between genotype and virulence in Candida species.

BACKGROUND: The virulence of isolates among different Candida species causing candidemia may play a role in the prognosis of the patients. Furthermore, the potential relationship between genotype and virulence is still unclear and need to be further studied. AIMS: We aim to assess the relationship between genotype and virulence in Candida species using a Galleria mellonella larvae infection model. METHODS: One hundred and ninety-four isolates from 68 clusters (Candida albicans, 114/41; Candida parapsilosis, 74/24; Candida tropicalis, 6/3) were compared against the same number of each species singleton genotypes in terms of survival of G. mellonella larvae. RESULTS: The median of survival and the IQR ranges of clusters and singleton were as follows: C. albicans (2 days, IQR 1.5-2 vs. 2 days, IQR 1-2.25), C. parapsilosis (2 days, IQR 1.5-2.6 vs. 2 days, IQR 2-3.3), and C. tropicalis (1 day, IQR 1-3.5 vs. 2 days, IQR 2-3.5; p<0.05). High intra-cluster variability in terms of median of survival was found regardless the species. CONCLUSIONS: No relationship between genotype and virulence in Candida was observed with the G. mellonella model.

Azole resistance survey on clinical Aspergillus fumigatus isolates in Spain.

OBJECTIVES: We aimed to assess the percentage of azole resistance in Aspergillus fumigatus in Spain. METHODS: Thirty participating Spanish hospitals stored all morphologically identified A. fumigatus sensu lato clinical isolates-regardless their clinical significance-from 15 February to 14 May 2019. Isolates showing azole resistance according to the EUCAST 9.3.2 methodology were molecularly identified and the cyp51A gene was studied in A. fumigatus sensu stricto isolates. RESULTS: Eight hundred and forty-seven isolates from 725 patients were collected in 29 hospitals (A. fumigatus sensu stricto (n = 828) and cryptic species (n = 19)). Isolates were mostly from the lower respiratory tract (94.0%; 797/847). Only cryptic species were amphotericin B resistant. Sixty-three (7.4%) out of the 847 isolates were resistant to ≥1 azole(s). Azole resistance was higher in cryptic species than in A. fumigatus sensu stricto (95%, 18/19 vs. 5.5%, 45/828); isavuconazole was associated to the lowest number of non-wild type isolates. The dominant mechanism of resistance was the presence of TR34-L98H substitutions (n = 24 out of 63). Out of the 725 patients, 48 (6.6%) carried either cryptic species (n = 14) or A. fumigatus sensu stricto (n = 34; 4.7%) resistant isolates. Aspergillus fumigatus sensu stricto harbouring either the TR34-L98H (n = 19) or TR46/Y121F/T289A (n = 1) mutations were detected in patients in hospitals located at 7/24 studied cities. DISCUSSION: Of the patients, 6.6% carry azole-resistant A. fumigatus sensu lato isolates in Spain. TR34-L98H is the dominant cyp51A gene substitutions, although its presence is not widespread.

Candidemia Candida albicans clusters have higher tendency to form biofilms than singleton genotypes†.

The capacity of Candida spp. to form biofilms allows them to attach either to living or inert surfaces, promoting their persistence in hospital environments. In a previous study, we reported strain-to-strain variations in Candida spp. biofilm development, suggesting that some genotypes may be greater biofilm formers than others. In this study, we hypothesize that isolates pertaining to clusters may be found more frequently in the environment due to their ability to form biofilms compared to singleton genotypes. Two hundred and thirty-nine Candida spp. isolates (78 clusters) from candidemia patients admitted to 16 hospitals located in different cities and countries-and the same number of singleton genotypes used as controls-were tested in terms of biofilm formation using the crystal violet and the XTT reduction assays. Candida albicans clusters showed higher biofilm formation in comparison to singleton genotypes (P < .01). The biofilms formed by intra-hospital C. albicans clusters showed higher metabolic activity (P < .05). Furthermore, marked variability was found among species and type of cluster. We observed that the higher the number of isolates, the higher the variability of biofilm production by isolates within the cluster, suggesting that the production of biofilm by isolates of the same genotype is quite diverse and does not depend on the type of cluster studied. In conclusion, candidemia Candida spp. clusters-particularly in the case of C. albicans-show significantly more biomass production and metabolic activity than singleton genotypes.

Susceptibility of uncommon Candida species to systemic antifungals by the EUCAST methodology.

The incidence of infections by uncommon Candida species has increased in recent years, however, in vitro susceptibility data are scarce. Here we assess the susceptibility of C. krusei, C. dubliniensis, C. lusitaniae, and C. guilliermondii complex isolates (n = 120) to antifungal agents by the EUCAST methodology. C. dubliniensis proved to be the most susceptible species, similar to that of C. albicans (P < .05), whereas C. guilliermondii was the least susceptible. Two C. krusei isolates were echinocandin-resistant and harbored a point mutation (L701M) in the FKS1. Some isolates were either fluconazole-resistant (C. lusitaniae, n = 2) or fluconazole non-wild type (C. guilliermondii, n = 3).

Does the composition of polystyrene trays affect Candida spp. biofilm formation?

The biofilm formation ability of Candida species seems to have a role in the prognosis of patients with candidemia. Biofilm formation is usually tested using 96 well flat bottom polystyrene microtiter plates, although the type of plastic used is not commonly reported. This study compares biofilm formation by Candida spp. on six types of plates from three brands (three non-tissue-treated and three tissue-treated). Thirty isolates of each of the following species were selected: C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, as well as 15 isolates of C. krusei (n = 135 isolates) from patients with candidemia. Biofilm production was evaluated by measuring biomass production and metabolic activity. Our results show higher biomass production and metabolic activity of biofilms formed on non-tissue-treated plates in comparison to those formed on tissue-treated plates (P < .001). We only found significant differences in metabolic activity of biofilms formed on non-tissue-treated plates (P < .003). All comparisons including biofilm formation and metabolic activity among plates of the same brand yielded higher biofilm formation on non-treated plates compared to treated plates (P < .001). Significant difference in biomass production by C. parapsilosis was only seen when comparing between the various tissue-treated plastics (P < .03). In contrast, comparisons of different non-tissue-treated tray brands yielded significant metabolic activity differences for all species except for C. parapsilosis (P < .05). Biofilm formation and metabolic activity is significantly affected by the plastic composition of non-tissue-treated trays leading to increased biofilm formation.

Low and constant micafungin concentrations may be sufficient to lead to resistance mutations in FKS2 gene of Candida glabrata.

We studied the ability of five echinocandin-susceptible C. glabrata isolates to acquire in vitro resistance to anidulafungin and micafungin. All isolates became phenotypically resistant after 2-4 days of exposure to low and constant micafungin concentrations (P < .05). Mutations in the HS1 region of the FKS2 gene were found in all isolates. The acquisition of resistance was not related to the previous use of antifungal treatment in the patients or the presence of mutations at MSH2 gene. We found differences (P < .0001) in the median survival of Galleria mellonella larvae infected with FKS2 mutant isolates (5 days) and wild-type isolates (3 days).