Azole resistance screening in Aspergillus fumigatus sensu stricto using the azole-containing agar method (EUCAST E.Def 10.2): conidial suspension filtration and inoculum adjustment before inoculum preparation may not be needed.

Azole resistance screening in Aspergillus fumigatus sensu stricto can be routinely carried out by using azole-containing agar plates (E.Def 10.2 procedure); however, conidial suspension filtering and inoculum adjustment before inoculum preparation are time-consuming. We evaluated whether skipping the filtration and inoculum adjustment steps negatively influenced the performance of the E.Def 10.2 procedure. A. fumigatus sensu stricto isolates (n = 98), previously classified as azole susceptible or azole resistant (E.Def 9.4 method), were studied. Azole-resistant isolates had either the wild-type cyp51A gene sequence (n = 1) or the following cyp51A gene substitutions: TR34-L98H (n = 41), G54R (n = 5), TR46-Y121F-T289A (n = 1), or G448S (n = 1). In-house azole-containing agar plates were prepared according to the EUCAST E.Def 10.2 procedure. Conidial suspensions obtained by adding distilled water (Tween 20 0.1%) were either filtered and the inocula adjusted to 0.5 McFarland or left unfiltered and unadjusted. Agreements between the agar screening methods using inocula prepared by each procedure were high for itraconazole (99%), voriconazole (100%), and posaconazole (94.9%). Sensitivity and specificity (considering the susceptibility category as per the microdilution E.Def 9.4 method as the gold standard) of E.Def 10.2 were 100% to rule in or rule out resistance when unfiltered and unadjusted suspensions were used; the resistance phenotype of isolates harboring the TR34-L98H, G54R, or TR46-Y121F-T289A substitutions was correctly detected. Unfiltered and unadjusted conidial suspensions do not negatively influence the performance of the E.Def 10.2 method when screening for azole resistance in A. fumigatus sensu stricto. IMPORTANCE: Azole resistance screening in Aspergillus fumigatus sensu stricto can be routinely carried out by using azole-containing plates (E.Def 10.2 procedure); however, conidial suspension filtering and inoculum adjustment before inoculation of plates are time-consuming. We, here, showed that unfiltered and unadjusted conidial suspensions do not negatively influence the performance of the E.Def 10.2 method when screening for azole resistance in A. fumigatus sensu stricto.

Clonal spread of fluconazole-resistant C. parapsilosis in patients admitted to a referral hospital located in Burgos, Spain, during the COVID-19 pandemic.

BACKGROUND: Fluconazole-resistant Candida parapsilosis (FRCP) is a matter of concern in Spain. OBJECTIVES: We here report a FRCP spread across a 777-bed referral hospital located in Burgos, Spain, during the COVID-19 pandemic. PATIENTS/METHODS: In April 2021, an FRCP isolate (MIC = 64 mg/L, E-test®) from a hospitalised patient was detected. Up to June 2022, all C. parapsilosis isolates (n = 35) from hospitalised patients (n = 32) were stored and genotyped using microsatellite markers, and their antifungal susceptibilities were studied (EUCAST); FRCP isolates were molecularly characterised. RESULTS: We detected 26 FRCP isolates collected between 2021 (n = 8) and 2022 (n = 18); isolates were susceptible to amphotericin B, echinocandins and ibrexafungerp. FRCP isolates were grouped into three genotypes: CP-707 and CP-708 involved isolates harbouring the Y132F + R398I ERG11p substitutions (n = 24) and were clonally related; the remaining CP-675 genotype involved isolates harbouring the G458S ERG11p substitution (n = 2). FRCP genotypes were genetically related to the FRCP genotypes found in Madrid and were unrelated to fluconazole-susceptible ones. Patients harbouring FRCP were mainly (n = 22/23) admitted to intensive care units. Most patients had received broad-spectrum antibiotics (n = 22/23), and/or antifungal therapy with azoles (n = 14/23) within the 30 days prior to FRCP isolation. Thirteen patients were colonised, 10 of whom were infected and presented candidaemia (n = 8/10), endovascular infection (n = 1/10) or complicated urinary infection (n = 1/10). Overall nonattributable 30-day mortality was 17% (n = 4/23). CONCLUSIONS: We report an outbreak caused by FRCP affecting patients admitted to the ICU of a referral hospital located in Burgos. Patients harbouring FRCP had a higher fluconazole use than those carrying susceptible isolates.

Fluconazole-resistant Candida parapsilosis genotypes from hospitals located in five Spanish cities and one in Italy: Description of azole-resistance profiles associated with the Y132F ERG11p substitution.

BACKGROUND: Fluconazole-resistant Candida parapsilosis is a matter of concern. OBJECTIVES: To describe fluconazole-resistant C. parapsilosis genotypes circulating across hospitals in Spain and Rome and to study their azole-resistance profile associated with ERG11p substitutions. PATIENTS/METHODS: We selected fluconazole-resistant C. parapsilosis isolates (n = 528 from 2019 to 2023; MIC ≥8 mg/L according to EUCAST) from patients admitted to 13 hospitals located in five Spanish cities and Rome. Additionally, we tested voriconazole, posaconazole, isavuconazole, amphotericin B, micafungin, anidulafungin and ibrexafungerp susceptibility. RESULTS: Of the 53 genotypes found, 49 harboured the Y132F substitution, five of which were dominating city-specific genotypes involving almost half the isolates. Another genotype involved isolates harbouring the G458S substitution. Finally, we found two genotypes with the wild-type ERG11 gene sequence and one with the R398I substitution. All isolates were fully susceptible/wild-type to amphotericin B, anidulafungin, micafungin and ibrexafungerp. The azole-resistance patterns found were: voriconazole-resistant (74.1%) or voriconazole-intermediate (25.2%), posaconazole-resistant (10%) and isavuconazole non-wild-type (47.5%). Fluconazole-resistant and voriconazole non-wild-type isolates were likely to harbour substitution Y132F if posaconazole was wild type; however, if posaconazole was non-wild type, substitution G458S was indicated if isavuconazole MIC was >0.125 mg/L or substitution Y132F if isavuconazole MIC was ≤0.125 mg/L. CONCLUSIONS: We detected a recent clonal spread of fluconazole-resistant C. parapsilosis across some cities in Spain, mostly driven by dominating city-specific genotypes, which involved a large number of isolates harbouring the Y132F ERG11p substitution. Isolates harbouring substitution Y132F can be suspected because they are non-susceptible to voriconazole and rarely posaconazole-resistant.

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.

Amphotericin B, itraconazole, posaconazole, and isavuconazole MICs against clinical Mucorales isolates obtained by visual inspection and spectrophotometric reading according to the EUCAST 9.4 procedure.

We compared the antifungal susceptibility of 92 Mucorales isolates obtained by visual inspection and spectrophotometric readings following EUCAST (European Committee on Antimicrobial Susceptibility Testing) testing. Amphotericin B minimum inhibitory concentrations (MICs) were up to 1 mg/l against most isolates and variable among species, except for Cunninghamella bertholletiae. Posaconazole MICs against most isolates were up to 1 mg/l and high against Mucor circinelloides, some Rhizopus arrhizus, and Rhizopus microsporus. Isavuconazole MICs ranged between 1 and 8 mg/l but were invariably >8 mg/l against M. circinelloides and C. bertholletiae. The agreement between MICs obtained by visual endpoint or spectrophotometric readings was moderate and higher when using the ≥90% fungal growth inhibition endpoint.

The agreement between minimum inhibitory concentration (MIC) values obtained by visual inspection or spectrophotometric readings was moderate and higher when the ≥90% fungal growth inhibition endpoint was chosen. Isavuconazole presented higher MICs than posaconazole, regardless of the inhibition endpoint used.

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.

Gradient diffusion strips for detecting azole resistance in Aspergillus fumigatus sensu lato.

BACKGROUND: Studies comparing gradient diffusion strips (GDSs) and the EUCAST E.Def 9.4 microdilution method are scarce, thwarted by a low number of isolates, and restricted to selected antifungal agents. OBJECTIVES: We evaluated the performance of GDSs to detect azole resistance in A. fumigatus, including cryptic species. PATIENTS/METHODS: A. fumigatus sensu stricto (n = 89) and cryptic species (n = 52) were classified as susceptible or resistant to itraconazole, voriconazole, posaconazole and isavuconazole (EUCAST E.Def 9.4; clinical breakpoints v10). A. fumigatus sensu stricto azole-resistant isolates had the following cyp51A gene mutations: TR34 -L98H (n = 24), G54R (n = 5), TR46 -Y121F-T289A (n = 1), F46Y-M172V-N248T-D255E-E427K (n = 1), F165L (n = 1) and cyp51A gene wild type (n = 3). GDSs (ETEST®, bioMèrieux, Marcy-l'Etoile, France and Liofilchem®, Roseto degli Abruzzi, Italy) MICs were obtained by following the manufacturer's guidelines. RESULTS: For A. fumigatus sensu stricto, itraconazole MICs >1.5 mg/L, voriconazole >0.38 mg/L, posaconazole >0.75 mg/L, and isavuconazole >0.5 mg/L correctly separated resistant from susceptible isolates with two exceptions. Considering the aforementioned cut-off MICs, sensitivity/specificity values of GDSs to detect azole resistance were: itraconazole (97%/100%), voriconazole (97%/100%), posaconazole (97%/100%) and isavuconazole (93.3%/100%). For cryptic species isolates, voriconazole MICs >1 mg/L and isavuconazole >0.75 mg/L separated resistant isolates from susceptible isolates with 15 and 27 exceptions, respectively. Considering the aforementioned cut-off MICs, sensitivity/specificity values were as follows: voriconazole (68.1%/100%) and isavuconazole (25%/100%). For itraconazole and posaconazole, it was not possible to establish cut-off values. CONCLUSIONS: We set tentative cut-off MIC values to correctly spot resistant Aspergillus fumigatus sensu stricto isolates using GDSs. The performance against cryptic species was poor.

EUCAST-Obtained Olorofim MICs against Aspergillus and Scedosporium Species and Lomentospora prolificans Showed High Agreements between Visual Inspection and Spectrophotometric Readings.

Previous studies show high agreement between MIC spectrophotometric readings and visual inspection of azoles and amphotericin B against Aspergillus fumigatus isolates. Here, we tested and compared the in vitro activity of a novel antifungal, olorofim, against Aspergillus spp., Scedosporium spp., and Lomentospora prolificans by visual inspection and spectrophotometric readings. Clinical isolates of Aspergillus (n = 686) and Scedosporium (n = 36) spp. and L. prolificans (n = 13) were tested. Olorofim MICs were evaluated-following the EUCAST E.Def 9.4 procedure-by visual inspection or spectrophotometric readings (combinations of either ≥90% or ≥95% fungal growth inhibition endpoints compared to drug-free control endpoints and different wavelengths [405 nm, 450 nm, 492 nm, 540 nm, and 620 nm]). We observed high in vitro activity of olorofim against all tested Aspergillus spp. (MICs up to 0.06 mg/L), except for A. calidoustus, and against L. prolificans and Scedosporium spp. (MICs up to 0.125 mg/L). The combination of ≥90% fungal growth inhibition endpoints at wavelengths of ≥492 nm resulted in high essential agreements with A. fumigatus and lesser agreement with non-fumigatus Aspergillus, Scedosporium spp., and L. prolificans, although the number of isolates studied was low. This single-center study shows high agreement among olorofim MICs against A. fumigatus by visual inspection and spectrophotometric readings (≥90% fungal growth inhibition endpoints and wavelengths of ≥492 nm) and encouraging results against non-fumigatus Aspergillus spp., Scedosporium spp., and L. prolificans.

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.

Multicentre validation of a modified EUCAST MIC testing method and development of associated epidemiologic cut-off (ECOFF) values for rezafungin.

OBJECTIVES: Rezafungin EUCAST MIC testing has been associated with notable inter-laboratory variation, which prevented ECOFF setting for C. albicans. We assessed in vitro susceptibility and reproducibility for a modified EUCAST methodology and established associated wild-type upper limits (WT-ULs). METHODS: MICs against 150 clinical Candida isolates (six species), molecularly characterized fks mutants (n = 13), and QC strains (n = 6) were determined at six laboratories according to E.Def 7.3 but using Tween 20 supplemented medium. WT-ULs were determined using the derivatization method, the ECOFFinder programme and visual inspection. Consensus WT-ULs were determined. RESULTS: The laboratory- and species-specific MIC distributions were Gaussian with >99.5% MICs within four 2-fold dilutions except for C. parapsilosis (92.8%). The following consensus WT-UL were determined: C. albicans 0.008 mg/L; C. dubliniensis and C. glabrata 0.016 mg/L; C. krusei and C. tropicalis 0.03 mg/L; and C. parapsilosis 4 mg/L. Adopting these WT-UL, six clinical isolates were non-wild-type, five of which harboured Fks alterations. For 11/13 mutants, all 670 MICs were categorized as non-wild-type whereas MICs for C. glabrata Fks2 D666Y and C. tropicalis Fks1 R656R/G overlapped with the corresponding wild-type distributions. Repeat testing of six reference strains yielded 98.3%-100% of MICs within three 2-fold dilutions except for C. albicans CNM-CL-F8555 (96%) and C. parapsilosis ATCC 22019 (93.3%). CONCLUSIONS: The modified EUCAST method significantly improved inter-laboratory variation, identified wild-type populations and allowed perfect separation of wild-type and fks mutants except for two isolates harbouring weak mutations. These consensus WT-UL have been accepted as ECOFFs and will be used for rezafungin breakpoint setting.

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.

Gastrointestinal tract Candida spp colonization shows mostly a monoclonal pattern: an intra-patient pilot study.

Gastrointestinal tract Candida genotypes may associate with isolates later causing infections. We genotyped Candida spp isolates (n = 200 individual colonies) from rectal swabs to assess whether gastrointestinal gut colonization is caused by a single genotype (monoclonal pattern) or a combination of them (polyclonal pattern). C. glabrata showed a sheer monoclonal pattern. C. parapsilosis and C. tropicalis showed a monoclonal pattern involving the presence of either exclusively identical genotypes or a combination of clonally-related genotypes; in the latter case, a dominant genotype was always found. C. albicans showed mostly a polyclonal pattern involving a combination of dominant clonally-related genotypes and unrelated genotypes. LAY SUMMARY: We genotyped C. albicans, C. parapsilosis, C. tropicalis, and C. glabrata isolates prospectively from rectal swabs to study the gastrointestinal colonization pattern in the patients. Gastrointestinal tract colonization is mostly monoclonal and commonly dominated by one genotype.

Evaluation of the Vitek Ms system for the identification of filamentous fungi.

The implementation of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of fungal isolates remains challenging and has been limited to experienced laboratories in sample preparation and in-house libraries construction. However, the development of commercial kits for standardized fungal sample preparation and updated reference libraries can fill this gap. This study aimed to evaluate the performance of the commercial VITEK MS Mould Kit (bioMérieux, Marcy l'Etoile, France) and the VITEK MS system (bioMérieux) for identification using a panel of fungal species of clinical interest. Overall, 200 isolates belonging to 13 genera and 43 fungal species were analyzed with the VITEK MS system equipped with the v3.2 IVD database. Overall, 89.0% of the isolates were correctly identified, 41.5 and 43.5% at species and complex level, respectively. For an additional 4.0% of the identifications, correlation at the genus level was reported. The remaining 21 isolates (10.5%) could not be identified among which 85.0% (18/21) were species not claimed in the database. One Syncephalastrum isolate was misidentified as Rhizopus microsporus complex. Specifically, 100% of the Scedosporium/Lomentospora, 97.1% of the Fusarium, 65.7% of the Mucorales and 86.4% of the Aspergillus isolates were correctly identified at the species and complex level. The methodology described allows for an easy implementation of MALDI-TOF MS for routine identification of fungal species in a fast and reliable manner. Although further improvement in the databases is still required, an important number of fungal species can be correctly identified at the species level using this method. LAY SUMMARY: The use of MALDI-TOF for fungal identification remains a challenge. In this study, using a commercial protein extraction kit and updated database, VITEK MS system was able to identify up to 89.0% of a diverse collection of 200 filamentous fungi representing 43 fungal species.

Screening of azole resistance in Aspergillus fumigatus using the EUCAST E.Def 10.2 azole-containing agar method: a single study suggests that filtration of conidial suspensions prior to inoculum preparation may not be needed.

BACKGROUND: Azole resistance screening in A. fumigatus isolates can be routinely carried out by using azole-containing plates (E.Def 10.2 method), that requires filtering conidial suspensions prior inoculum adjustment. OBJECTIVES: We evaluated whether skipping the filtration step of conidial suspensions negatively influences the performance of the E.Def 10.2. Patients/Methods A. fumigatus sensu stricto isolates (n=92), classified as azole-susceptible or azole-resistant according to the EUCAST microdilution E.Def 9.4 method, were studied. Azole-resistant isolates had either wild type cyp51A gene sequence (n = 3) or the TR34 -L98H (n = 26), G54R (n = 5), TR46 -Y121F-T289A (n = 1), F46Y-M172V-N248T-D255E-E427K (n = 1), F165L (n=1), or G448S (n=1) cyp51A gene substitutions. In-house azole-containing agar plates were prepared according to the EUCAST E.Def 10.2 procedure. Conidial suspensions were obtained by adding distilled water (Tween 20 0.1%). Subsequently, the suspensions were either filtered or left unfiltered prior to inoculum adjustment to 0.5 McFarland. Using microdilution as the gold standard, agreement, sensitivity, and specificity of the agar plates inoculated with two inoculums were assessed. RESULTS: Agreements for the agar screening method with either unfiltered or filtered conidial suspensions were high for itraconazole (100%), voriconazole (100%), and posaconazole (97.8%). Sensitivity (100%) and specificity (98.2%) of the procedure to rule in or out resistance when unfiltered suspensions were used were also high. Isolates harbouring the TR34 -L98H, G54R, and TR46 -Y121F-T289A substitutions were detected with the modified method. CONCLUSIONS: Unfiltered conidial suspensions does not negatively influence the performance of the E.Def 10.2 method when screening for A. fumigatus sensu stricto.

Aspergillus endocarditis in the recent years, report of cases of a multicentric national cohort and literature review.

OBJECTIVES: (1) To describe the incidence, clinical characteristics, treatment and outcome of Aspergillus Endocarditis (AE) in a nationwide multicentric cohort (GAMES). (2) To compare the AE cases of the GAMES cohort, with the AE cases reported in the literature since 2010. (3) To identify variables related to mortality. METHODS: We recruited 10 AE cases included in the GAMES cohort (January 2008-December 2018) and 51 cases from the literature published from January 2010 to July 2019. RESULTS: 4528 patients with infectious endocarditis (IE) were included in the GAMES cohort, of them 10 (0.2%) were AE. After comparing our 10 cases with the 51 of the literature, no differences were found. Analysing the 61 AE cases together, 55.7% were male, median age 45 years. Their main underlying conditions were as follows: prosthetic valve surgery (34.4%) and solid organ transplant (SOT) (19.7%). Mainly affecting mitral (36.1%) and aortic valve (29.5%). Main isolated species were as follows: Aspergillus fumigatus (47.5%) and Aspergillus flavus (24.6%). Embolisms occurred in 54%. Patients were treated with antifungals (90.2%), heart surgery (85.2%) or both (78.7%). Overall, 52.5% died. A greater mortality was observed in immunosuppressed patients (59.4% vs. 24.1%, OR = 4.09, 95%CI = 1.26-13.19, p = .02), and lower mortality was associated with undergoing cardiac surgery plus azole therapy (28.1% vs. 65.5%, OR = 0.22, 95%CI = 0.07-0.72, p = .01). CONCLUSIONS: AE accounts for 0.2% of all IE episodes of a national multicentric cohort, mainly affecting patients with previous valvular surgery or SOT recipients. Mortality remains high especially in immunosuppressed hosts and azole-based treatment combined with surgical resection are related to a better outcome.

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.

Azole-Resistant Aspergillus fumigatus Clinical Isolate Screening in Azole-Containing Agar Plates (EUCAST E.Def 10.1): Low Impact of Plastic Trays Used and Poor Performance in Cryptic Species.

Azole-containing agar is used in routine Aspergillus fumigatus azole resistance screening. We evaluated the impact of the type of plastic used to prepare in-house agar plates on the procedure's performance against A. fumigatus sensu stricto and cryptic species. A. fumigatus sensu stricto (n = 91) and cryptic species (n = 52) were classified as susceptible or resistant (EUCAST E.Def 9.3.2; clinical breakpoints v10). In-house azole-containing agar plates were prepared following EUCAST E.Def 10.1 on three types of multidish plates. We assessed the sensitivity, specificity, and agreement values of the agar plates to screen for azole resistance. Overall, sensitivity and specificity values of the agar screening method were 100% and 93.3%, respectively. The type of tray used did not affect these values. All isolates harboring TR34-L98H substitutions were classified as resistant to itraconazole and voriconazole by the agar method; however, false susceptibility (very major error) to posaconazole was not uncommon and happened in isolates with posaconazole MICs of 0.25 mg/liter. Isolates harboring G54R and TR46-Y121F-T289A substitutions were correctly classified by the agar method as itraconazole/posaconazole resistant and voriconazole resistant, respectively. False resistance (major error) occurred in isolates showing tiny fungal growth. Finally, agreements between both procedures against cryptic species were much lower. Azole-containing agar plates are a convenient and reliable tool to screen for resistance in A. fumigatus sensu stricto; the type of plastic tray used minimally affects the method. On the contrary, the performance against cryptic species is rather poor.

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.

Spectrophotometric azole and amphotericin B MIC readings against Aspergillus fumigatus sensu lato using the EUCAST 9.3.2 methodology. Are ≥90 and ≥95% fungal growth inhibition endpoints equally suitable?

We recently reported high essential (97.1%) and categorical (99.6%) agreements between azole and amphotericin B MICs against Aspergillus fumigatus sensu lato obtained by visual and spectrophotometric readings using a ≥ 95% fungal growth endpoint and following the EUCAST methodology (doi: 10.1128/AAC.01693-20). Here, we compared the aforementioned MICs against spectrophotometric MIC readings obtained using a ≥ 90% inhibition endpoint. Spectrophotometric readings using either ≥ 90% or ≥ 95% fungal growth inhibition resulted in high categorical (>99.9%) agreements with visual MIC readings against A. fumigatus sensu stricto. In contrast, agreements with visual MICs against cryptic species were higher with the use of a ≥ 95% fungal growth inhibition endpoint. LAY SUMMARY: Spectrophotometrically obtained MIC readings using either ≥ 90% or ≥ 95% fungal growth inhibition endpoints and following the EUCAST methodology are suitable against A. fumigatus sensu stricto. However, the ≥ 95% fungal growth inhibition endpoint is preferred against cryptic species.

Whole genome sequencing confirms Candida albicans and Candida parapsilosis microsatellite sporadic and persistent clones causing outbreaks of candidemia in neonates.

Whole genome sequencing has been extensively used to describe infection outbreaks, although with limited application on Candida albicans and Candida parapsilosis.We retrospectively studied all patients admitted to the neonatal care unit diagnosed with candidemia caused by C. albicans (n = 46) or C. parapsilosis (n = 31) between 2007 and 2010 (Period 1) and 2011 and 2014 (Period 2). All isolates were genotyped by microsatellite markers. A cluster was defined as a group of ≥ 2 patients infected by strains with identical genotypes. For the validation of microsatellite markers and outbreak investigation, phylogenetic analyses and whole genome pairwise strain comparisons were performed.The number of episodes was significantly higher in Period 1 than in Period 2 (51 vs 32; P = 0.003); the reduction in the number of cases coincided with the educational campaign for catheter care implementation in 2011. Overall, eight genotypes were clusters involving 29 patients. All C. albicans (n = 5) and C. parapsilosis (n = 3) clusters were found during Period 1 before the educational campaign. No statistically significant differences were found between the percentage of C. albicans and C. parapsilosis clusters, but the percentage of patients associated to the clusters was significantly higher for C. parapsilosis clusters in comparison to C. albicans clusters (52 vs 28.2%; P = 0.03). Whole genome sequencing confirmed microsatellite-defined clusters with high bootstrap values.Whole genome sequences confirmed microsatellite-defined clusters, corroborating the presence of outbreaks. Persistent or sporadic Candida clusters causing candidemia in neonates disappeared after the implementation of catheter care educational campaigns. LAY SUMMARY: We retrospectively studied all patients admitted to the neonatal care unit diagnosed with candidemia caused by C. albicans or C. parapsilosis. Reliable whole genome sequences confirmed microsatellite-defined clusters, corroborating the presence of outbreaks before educational campaigns for catheter care.