Evaluation of the Vitek 2 system for antifungal susceptibility testing of Candida auris using a representative international panel of clinical isolates: overestimation of amphotericin B resistance and underestimation of fluconazole resistance.

Although the Vitek 2 system is broadly used for antifungal susceptibility testing of Candida spp., its performance against Candida auris has been assessed using limited number of isolates recovered from restricted geographic areas. We therefore compared Vitek 2 system with the reference Clinical and Laboratory Standards Institute (CLSI) broth microdilution method using an international collection of 100 C. auris isolates belonging to different clades. The agreement ±1 twofold dilution between the two methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention's (CDC's) tentative resistance breakpoints and Vitek 2-specific wild-type upper limit values (WT-ULVs) were determined. The CLSI-Vitek 2 agreement was poor for 5-flucytosine (0%), fluconazole (16%), and amphotericin B (29%), and moderate for voriconazole (61%), micafungin (67%), and caspofungin (81%). Significant interpretation errors were recorded using the CDC breakpoints for amphotericin B (31% CA, 69% major errors; MaEs) and fluconazole (69% CA, 31% very major errors; VmEs), but not for echinocandins (99% CA, 1% MaEs for both micafungin and caspofungin) for which the Vitek 2 allowed correct categorization of echinocandin-resistant FKS1 mutant isolates. Discrepancies were reduced when the Vitek 2 WT-ULV of 16 mg/L for amphotericin B (98% CA, 2% MaEs) and of 4 mg/L for fluconazole (96% CA, 1% MaEs, 3% VmEs) were used. In conclusion, the Vitek 2 system performed well for echinocandin susceptibility testing of C .auris. Resistance to fluconazole was underestimated whereas resistance to amphotericin B was overestimated using the CDC breakpoints of ≥32 and ≥2 mg/L, respectively. Vitek 2 minimun inhibitory concentrations (MICs) >4 mg/L indicated resistance to fluconazole and Vitek 2 MICs ≤16 mg/L indicated non-resistance to amphotericin B.

Development of an agar-based screening method for terbinafine, itraconazole, and amorolfine susceptibility testing of Trichophyton spp.

Resistance in dermatophytes is an emerging global public health issue. We, therefore, developed an agar-based method for screening Trichophyton spp. susceptibility to terbinafine (TRB), itraconazole (ITC), and amorolfine (AMF) and validated it using molecularly characterized isolates. Α total of 40 Trichophyton spp. isolates, 28 TRB wild type (WT) (13 T. rubrum, 10 T. mentagrophytes, 5 T. interdigitale) and 12 TRB non-WT (7 T. rubrum, 5 T. indotineae) with different alterations in the squalene epoxidase (SQLE) gene, were used. The optimal test conditions (inoculum and drug concentrations, incubation time, and temperature) and stability over time were evaluated. The method was then applied for 86 WT Trichophyton spp. clinical isolates (68 T. rubrum, 7 T. interdigitale, 6 T. tonsurans, 5 T. mentagrophytes) and 4 non-WT T. indotineae. Optimal growth of drug-free controls was observed using an inoculum of 20 µL 0.5 McFarland after 5-7 days of incubation at 30°C. The optimal concentrations that prevented the growth of WT isolates were 0.016 mg/L of TRB, 1 mg/L of ITC, and 0.25 mg/L of AMF, whereas 0.125 mg/L of TRB was used for the detection of Trichophyton strong SQLE mutants (MIC ≥0.25 mg/L). The agar plates were stable up to 4 months. Inter-observer and inter-experimental agreement were 100%, and the method successfully detected TRB non-WT Trichophyton spp. strains showing 100% agreement with the reference EUCAST methodology. An agar-based method was developed for screening Trichophyton spp. in order to detect TRB non-WT weak and strong mutant isolates facilitating their detection in non-expert routine diagnostic laboratories.

Optimization of the EUCAST reference broth microdilution method for echinocandin susceptibility testing of Aspergillus fumigatus.

BACKGROUND: Because of the high inoculum (105 cfu/mL) used in the EUCAST susceptibility testing of Aspergillus spp., determination of the minimal effective concentration (MEC) of echinocandins is challenging as the morphological differences are subtle. METHODS: The MECs of 10 WT and 4 non-WT Aspergillus fumigatus isolates were determined with the EUCAST E.Def 9.4. Plates were inoculated with increasing inocula (102-105 cfu/mL) and after 24 and 48 h of incubation, MECs were determined macroscopically (magnifying mirror) and microscopically (inverted microscope) by two observers, spectrophotometrically (OD at 405 nm) and colorimetrically (absorbance at 450/630 nm after 2 h incubation with 400 mg/L XTT/6.25 µM menadione). The interobserver (between observers)/intermethod (compared with the microscopic method) essential agreement (EA, ±1 2-fold dilution) and categorical agreement (CA) were determined for each inoculum. RESULTS: Echinocandin-induced microscopic hyphal alterations or macroscopic changes in turbidity were subtle with a 105 cfu/mL inoculum compared with the lower inocula of 103 and 102 cfu/mL, where more distinct changes in turbidity and formation of characteristic rosettes were obvious at the MEC after 48 h. A 105 cfu/mL inoculum resulted in wider MEC distributions (3-6 dilutions) and lower interobserver EA (69%), macroscopic-microscopic EA (26%) and CA (71%) compared with a 103 cfu/mL inoculum (2-3 dilutions, 100%, 100% and 100%, respectively). Spectrophotometric readings using a 103 cfu/mL inoculum showed good EA (57-93%) and excellent CA (86%-100%), while the XTT assay demonstrated excellent EA (93%) and CA (100%). CONCLUSIONS: A 48 h incubation using a 103 cfu/mL inoculum improved echinocandin MEC determination for A. fumigatus with the EUCAST method, while the colorimetric assay could allow automation.

Changing Epidemiology of Tinea Capitis in Athens, Greece: The Impact of Immigration and Review of Literature.

Mass population movements have altered the epidemiology of tinea capitis (TC) in countries receiving refugees. Periodic monitoring of the local pathogen profiles may serve as a basis for both the selection of appropriate empirical antifungal therapy and the implementation of preventive actions. Therefore, we investigated the impact of an unprecedented immigration wave occurring in Greece since 2015 on the epidemiological trends of TC. All microbiologically confirmed TC cases diagnosed during the period 2012-2019 in a referral academic hospital for dermatological disorders in Athens, Greece, were retrospectively reviewed. A total of 583 patients were recorded, where 348 (60%) were male, 547 (94%) were children and 160 (27%) were immigrants from Balkan, Middle Eastern, Asian as well as African countries. The overall annual incidence of TC was 0.49, with a significant increase over the years (p = 0.007). M. canis was the predominant causative agent (74%), followed by T. violaceum (12%), T. tonsurans (7%) and other rare dermatophyte species (7%). M. canis prevalence decreased from 2014 to 2019 (84% to 67%, p = 0.021) in parallel with a three-fold increase in T. violaceum plus T. tonsurans rates (10% to 32%, p = 0.002). An increasing incidence of TC with a shift towards anthropophilic Trichophyton spp. in Greece could be linked to the immigration flows from different socioeconomic backgrounds.

Overestimation of Amphotericin B Resistance in Candida auris with Sensititre YeastOne Antifungal Susceptibility Testing: a Need for Adjustment for Correct Interpretation.

Significant variation in minimal inhibitory concentrations (MIC) has been reported for amphotericin B (AMB) and C. auris, depending on the antifungal susceptibility testing (AFST) method. Although the Sensititre YeastOne (SYO) is widely used in routine laboratory testing, data regarding its performance for the AFST of C. auris are scarce. We tested AMB against 65 C. auris clinical isolates with the SYO and the reference methodology by the Clinical and Laboratory Standards Institute (CLSI). The essential agreement (EA, ±1 dilution) between the two methods and the categorical agreement (CA) based on the Centers for Disease Control and Prevention (CDC)'s tentative breakpoint of MIC ≥ 2 mg/L were determined. The SYO wild type upper limit value (WT-UL) was determined using the ECOFFinder. The modal (range) CLSI growth inhibitory MIC was lower than the SYO colorimetric MIC [1(0.25-1) versus 2(1-8) mg/L, respectively]). The CLSI-colorimetric SYO EA was 29% and the CA was 11% (89% major errors; MaE). MaE were reduced when the SYO WT-UL of 8 mg/L was used (0% MaE). Alternatively, the use of SYO growth inhibition endpoints of MIC-1 (75% growth inhibition) or MIC-2 (50% growth inhibition) resulted in 88% CA with 12% MaE and 97% CA with 3% MaE, respectively. In conclusion, SYO overestimated AMB resistance in C. auris isolates when colorimetric MICs, as per SYO instructions and the CDC breakpoint of 2 mg/L, were used. This can be improved either by using the method-specific WT-UL MIC of 8 mg/L for colorimetric MICs or by determining growth inhibition MIC endpoints, regardless of the color. IMPORTANCE Candida auris is an emerging and frequently multidrug-resistant fungal pathogen that accounts for life-threatening invasive infections and nosocomial outbreaks worldwide. Reliable AF is important for the choice of the optimal treatment. Commercial methods are frequently used without prior vigorous assessment. Resistance to AMB was over-reported with the commercial colorimetric method Sensititre YeastOne (SYO). SYO produced MICs that were 1 to 2 twofold dilutions higher than those of the reference CLSI method, resulting in 89% MaE. MaE were reduced using a SYO-specific colorimetric wild type upper limit MIC value of 8 mg/L (0%) or a 50% growth inhibition endpoint (3%).

Can bronchial secretion cultures identify the etiologic agent of COVID-19-associated pulmonary aspergillosis in ICU patients? Comparison with a species-specific Aspergillus PCR in serum.

Aspergillus spp. isolated from non-BAL cultures of coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) patients may reflect colonization rather than infection. Sera (n = 181) from 49 adult ICU CAPA patients (24 probable and 25 possible CAPA) with bronchial secretions (BS) culture positive for Aspergillus spp. were collected and tested for Aspergillus DNA detection by species-specific real-time PCR. Overall, 30/49 (61%) patients were PCR positive. BS culture/serum PCR agreement was moderate (21/30; 70%). Based on serum PCR positive patients, all CAPAs were due to A. fumigatus (80%), A. flavus (10%), and A. terreus (10%). No A. niger/A. nidulans or mixed infections were found despite positive BS cultures.

Discordant results were observed between bronchial secretion cultures and species-specific serum PCR (30%) with A. fumigatus being by far the most common etiological agent of CAPA (80%). No A. niger/A. nidulans or mixed infections were found despite positive cultures.

Filamentous Fungal Keratitis in Greece: A 16-Year Nationwide Multicenter Survey.

In a multicenter, prospective study of filamentous fungal keratitis in Greece, predisposing factors, etiology, treatment practices, and outcome, were determined. Corneal scrapings were collected from patients with clinical suspicion of fungal keratitis, and demographic and clinical data were recorded. Fungal identification was based on morphology, molecular methods, and matrix assisted laser desorption ionization time-of-flight mass-spectrometry. A total of 35 cases were identified in a 16-year study period. Female to male ratio was 1:1.7 and median age 48 years. Corneal injury by plant material, and soft contact lens use were the main risk factors (42.8% and 31.4%, respectively). Trauma was the leading risk factor for men (68.1%), contact lens use (61.5%) for women. Fusarium species were isolated more frequently (n = 21, 61.8%). F. solani was mostly associated with trauma, F. verticillioides and F. proliferatum with soft contact lens use. Other fungi were: Purpureocillium lilacinum (14.7%), Alternaria (11.8%), Aspergillus (8.8%), and Phoma foliaceiphila, Beauveria bassiana and Curvularia spicifera, one case each. Amphotericin B and voriconazole MIC50s against Fusarium were 2 mg/L and 4 mg/L respectively. Antifungal therapy consisted mainly of voriconazole locally or both locally and systemically, alone or in combination with liposomal AmB. Cure/improvement rate with antifungal therapy alone was 52%, keratoplasty was required in 40% of cases, and enucleation in 8%. In conclusion, filamentous fungal keratitis in Greece is rare, but with considerable morbidity. A large proportion of cases resulted in keratoplasty despite appropriate antifungal treatment.

Pan-Echinocandin Resistant C. parapsilosis Harboring an F652S Fks1 Alteration in a Patient with Prolonged Echinocandin Therapy.

The isolation of a pan-echinocandin-resistant Candida parapsilosis strain (anidulafungin, caspofungin, micafungin and rezafungin EUCAST MICs > 8 mg/L) from urine of a patient following prolonged exposure to echinocandins (38 days of micafungin followed by 16 days of anidulafungin) is described. The isolate harbored the novel alteration F652S in the hotspot 1 region of fks1. Isogenic C. parapsilosis bloodstream isolates collected up to 1.5 months earlier from the same patient were susceptible to echinocandins (anidulafungin, caspofungin and micafungin EUCAST MICs 1−2, 1 and 1 mg/L, respectively) and contained wild-type FKS1 sequences. This is the first report of pan-echinocandin resistance in C. parapsilosis associated with an aminoacid change in hotspot 1 region of fks1.

Breakthrough Acute Necrotizing Invasive Fungal Rhinosinusitis by Alternariaalternata in a Patient with Acute Lymphoblastic Leukemia on Anidulafungin Therapy and Case-Based Literature Review.

Alternaria spp. have emerged as opportunistic pathogens particularly in immunosuppressed patients. A case of a breakthrough acute invasive fungal rhinosinusitis (AIFRS), caused by Alternaria alternata, is reported in a patient with acute lymphoblastic leukemia (ALL) on anidulafungin therapy, who was successfully treated with liposomal amphotericin B and surgical intervention. To date, 20 cases of AIFRS due to Alternaria spp. have been described, 19 in the USA and 1 in Chile, making this case report the first case of AIFRS due to Alternaria in Europe. The patients had median (range) age 25 (2-56) years (65% female), almost all of them (19/20) with hematological diseases and severe neutropenia (8-41 days pre-infection). Amphotericin B was the most frequently used antifungal agent, either alone or in combination. In all of the cases, systemic antifungal therapy was combined with surgery. Despite stabilization or improvement of the AIFRS, mortality was 38% (5 days to 8 months post-surgical debridement) due to their underlying disease or other infections without sign of AIFRS at autopsy.

A fatal neonatal case of fungemia due to Exophiala dermatitidis-case report and literature review.

BACKGROUND: Systemic infections caused by the black yeast-like fungus Exophiala dermatitidis are rare, but are associated with high mortality especially in immunocompromised patients. We report the first case of E. dermatitidis fungemia in a premature extremely low birth weight (ELBW) neonate who succumbed despite antifungal therapy with liposomal amphotericin (AMB) and fluconazole. A systematic review of all fungemia cases due to E. dermatitidis was also conducted aiming for a better understanding of the risk factors, treatment strategies and outcomes. CASE PRESENTATION: A male, ELBW premature neonate, soon after his birth, developed bradycardia, apnoea and ultimately necrotizing enterocolitis with intestinal perforation requiring surgical intervention. Meanwhile, he had also multiple risk factors for developing bloodstream infection, such as intubation, mechanical ventilation, central venous catheter (CVC), parenteral nutrition, empirical and prolonged antibiotic use. His blood cultures were positive, firstly for Acinetobacter junii and then for Klebsiella pneumoniae together with E. dermatitidis while on fluconazole prophylaxis and antibiotic empiric therapy. Despite the treatment with broad spectrum antibiotics, liposomal AMB and fluconazole, the newborn succumbed. A literature review identified another 12 E. dermatitidis bloodstream infections, mainly in patients with hematologic malignancies and solid organ transplant recipients (61%), with overall mortality 38% despite CVC removal and antifungal therapy. CONCLUSIONS: Due to the rarity of E. dermatitidis infections, little is known about the characteristics of this yeast, the identification methods and the optimal therapy. Identification by common biochemical tests was problematic requiring molecular identification. Resolution of neonatal fungemia is difficult despite proper antifungal therapy especially in cases with multiple and severe risk factors like the present one. Therapeutic intervention may include CVC removal and treatment for at least 3 weeks with an azole (itraconazole or fluconazole after susceptibility testing) or AMB monotherapy but not echinocandins or AMB plus azole combination therapy.

Comparative Evaluation of Vitek 2 and Etest versus Broth Microdilution for Ceftazidime/Avibactam and Ceftolozane/Tazobactam Susceptibility Testing of Enterobacterales and Pseudomonas aeruginosa.

Ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T) are novel antibiotics with activity against multidrug-resistant Gram-negative pathogens. Nevertheless, resistance to both agents has been reported emphasizing the need for accurate and widely accessible susceptibility testing. In the present study, Vitek 2 and Etest CAZ and C/T MIC results for 100 non-repetitive clinical isolates (83 Enterobacterales and 17 P. aeruginosa, whereof 69 challenge isolates) were compared to the standard broth microdilution (BMD) method. EUCAST breakpoints were used for assessing the categorical (CA) and essential (EA) agreement between the methods along with the corresponding error rates. The Vitek 2 performance was comparable to that of BMD for testing both antimicrobial agents exceeding the ISO requirements (CA 98-99%, EA 96-100%, major errors (MEs) 0-1%, very major error (VMEs) 1%). Likewise, the Etest provided accurate results for CZA and C/T testing against Enterobacterales and P. aeruginosa, respectively (CA 100%, EA 97-100%, MEs 0%, VMEs 0%). On the contrary, EA of 85% and 6% VME rate were found for CZA Etest and P. aeruginosa. Overall, Vitek 2 measurements of CZA and C/T susceptibility correlated closely with the reference BMD, indicating that it can represent a suitable alternative to BMD for susceptibility testing of Enterobacterales and P. aeruginosa. The Etest did not fulfill the ISO performance criteria of EA and VME for CZA and P. aeruginosa. Further studies are needed to assess whether the Etest allows a reliable assessment of CZA and C/T EUCAST MICs.

Field Evaluation of the New Rapid NG-Test® SARS-CoV-2 Ag for Diagnosis of COVID-19 in the Emergency Department of an Academic Referral Hospital.

Background: As the COVID-19 pandemic resurges affecting large numbers of patients, rapid, and accurate diagnosis using point-of-care tests is very important. Objectives: To evaluate the NG-Test® SARS-CoV-2 Ag (NG-Test) immunoassay for qualitative detection of SARS-CoV-2 antigen in nasopharyngeal (NP) and oropharyngeal (OP) samples compared with RT-PCR, in patients attending the Emergencies of an academic referral hospital. Methods: All adult ambulatory patients presenting to the Emergencies of "Attikon" University hospital (Athens, Greece) within three consecutive hours per day between December 2020 and March 2021 and for whom SARS-CoV-2 PCR testing was requested were included. Two NP and one OP samples obtained from each participant were analyzed to determine the diagnostic performance [sensitivity, specificity, positive/negative predictive values (PPV/NPV)] of the NG-Test (NP/OP swabs) in comparison to the reference RT-PCR (NP swab). Results: Overall, 134/263 (51%) patients tested were RT-PCR positive, whereof 108 (overall sensitivity 81%, 95% CI 73-87%) were NP NG-Test positive (PPV 99%, NPV 83%) and 68 (overall sensitivity 51%, 95% CI 42-59%) were OP NG-Test positive (PPV 100%, NPV 66%). The test's specificity (95% CI) was 99% (95-100%) and 100% (96-100%) for NP and OP swabs, respectively. The assay's sensitivity (95% CI) for high viral load (Ct ≤25) was 99% (92-100%) and 71% (60-81%) for NP and OP swabs, respectively. Conclusions: NG-Test using NP swabs detected almost all patients with high viral loads, showing satisfactory performance as a point-of-care test for NP samples obtained from patients with acute infection.

Evaluation of the Dynamiker® Fungus (1-3)-ß-D-Glucan Assay for the Diagnosis of Invasive Aspergillosis in High-Risk Patients with Hematologic Malignancies.

INTRODUCTION: The Dynamiker® Fungus (1-3)-ß-D-glucan assay (DFA) allows the testing of samples in smaller batches compared to the well-established Fungitell® assay (FA) making the assay cost-effective in centers with small numbers of samples. Evaluations of its performance for the diagnosis of invasive aspergillosis (IA) are limited. Therefore, we compared the two assays and evaluated their clinical performance in diagnosing IA. METHODS: A total of 60 adult hematology patients were screened for IA, 13 with probable IA, 19 with possible IA, and 28 with no IA. Serum specimens (n = 166) were collected twice-weekly and tested for (1-3)-ß-D-glucan (BDG) using FA and DFA which were compared quantitatively with Spearman rank correlation analysis and qualitatively with the Chi-square test. Agreement and error rates were determined using FA as the reference method. Sensitivity, specificity, and positive predictive and negative predictive values in diagnosing IA were calculated. RESULTS: The performance of the DFA was highly consistent with that of the FA, both quantitatively (rs = 0.913) and qualitatively (kappa = 0.725). The agreement was 85% with 8% minor, no major, and 7% very major errors (FA+/DFA-). Using a cut-off value of 20 pg/mL for DFA, very major errors were reduced to 1%, although 5% major errors were detected. BDG levels were lower with DFA than FA (slope 0.653 ± 0.031). Sensitivity, specificity, positive predictive value, and negative predictive value (NPV) was 67%, 53%, 44%, and 74% for FA, and 53%, 67%, 49%, and 71% for DFA, respectively. The optimal BDG positivity threshold calculated did not lead to significant test quality improvement for either assay. However, a higher % of patients with probable IA (62%) had ≥ 2 consecutive positive specimens compared to patients with no IA (FA-BDG 26%, p = 0.10, and DFA-BDG 10%, p = 0.01) leading to improved sensitivity and NPV (71% and 85% for DFA, and 95% and 96% for FA, respectively). CONCLUSION: DFA could be a valuable alternative to the FA, particularly in laboratories with small numbers of samples. The results of the BDG testing should be carefully interpreted in the high-risk setting of patients with hematologic malignancies. Higher NPV was found using as criterion ≥ 2 consecutive positive samples for diagnosing IA.

Fungemia due to Moesziomyces aphidis (Pseudozyma aphidis) in a premature neonate. Challenges in species identification and antifungal susceptibility testing of rare yeasts.

Premature neonates are at particularly increased risk to develop invasive infections with excessive case fatality due to their low birth weight, enteral malabsorbtion, insufficient microbial defenses and underdeveloped anatomic barriers. We present a case of Moesziomyces aphidis (syn. Pseudozyma aphidis) fungemia in a newborn with severe morbidity and prolonged hospital stay. Phenotypic tests failed to identify the isolate whereas commercial antifungal susceptibility tests failed to detect resistance to fluconazole. To the best of our knowledge, this is the first case of M. aphidis fungemia in a premature neonate in whom complete clinical resolution occurred after liposomal amphotericin B administration. Our case is the third Pseudozyma spp. infection described in Europe. Twenty-one cases have been described globally. Common risk factors were central venus catheter (80%), previous antibiotic treatment (80%), hematologic malignancies (27%) and solid tumors (20%) with 3 cases reported in neonates. The most commonly used antifungal therapy was amphotericin B followed by oral voriconazole or itraconazole. Our report highlights the clinical importance of rare yeasts species in neonates, emphasizes the roles of prematurity and lower birth weight as major risk factors for invasive infections with high morbidity. Reliable identification and susceptibility testing of these rare yeasts is a key issue for an adequate therapy and better outcome.

Increasing Incidence and Shifting Epidemiology of Candidemia in Greece: Results from the First Nationwide 10-Year Survey.

Globally, candidemia displays geographical variety in terms of epidemiology and incidence. In that respect, a nationwide Greek study was conducted, reporting the epidemiology of Candida bloodstream infections and susceptibility of isolates to antifungal agents providing evidence for empirical treatment. All microbiologically confirmed candidemia cases in patients hospitalized in 28 Greek centres during the period 2009-2018 were recorded. The study evaluated the incidence of infection/100,000 inhabitants, species distribution, and antifungal susceptibilities of isolated strains. Overall, 6057 candidemic episodes occurred during the study period, with 3% of them being mixed candidemias. The average annual incidence was 5.56/100,000 inhabitants, with significant increase over the years (p = 0.0002). C. parapsilosis species complex (SC) was the predominant causative agent (41%), followed by C. albicans (37%), C. glabrata SC (10%), C. tropicalis (7%), C. krusei (1%), and other rare Candida spp. (4%). C. albicans rates decreased from 2009 to 2018 (48% to 31%) in parallel with a doubling incidence of C. parapsilosis SC rates (28% to 49%, p < 0.0001). Resistance to amphotericin B and flucytosine was not observed. Resistance to fluconazole was detected in 20% of C. parapsilosis SC isolates, with a 4% of them being pan-azole-resistant. A considerable rising rate of resistance to this agent was observed over the study period (p < 0.0001). Echinocandin resistance was found in 3% of C. glabrata SC isolates, with 70% of them being pan-echinocandin-resistant. Resistance rate to this agent was stable over the study period. This is the first multicentre nationwide study demonstrating an increasing incidence of candidemia in Greece with a species shift toward C. parapsilosis SC. Although the overall antifungal resistance rates remain relatively low, fluconazole-resistant C. parapsilosis SC raises concern.

Epidemiology and Incidence of COVID-19-Associated Pulmonary Aspergillosis (CAPA) in a Greek Tertiary Care Academic Reference Hospital.

INTRODUCTION: Invasive pulmonary aspergillosis is an emerging complication among intensive care unit (ICU) patients with COVID-19 (CAPA). In the present study, all CAPA cases during the first year of the pandemic were reviewed in critically ill patients at a 650-bed tertiary Greek COVID-19 reference hospital. METHODS: Data regarding patients admitted to the ICU of Attikon Hospital in Athens, Greece, between 22 March 2020 and 28 February 2021 with a positive PCR for SARS-CoV-2 infection were reviewed. Clinical and microbiological records were analysed including demographic, clinical, laboratory and radiological features, treatment and outcomes. CAPA was determined according to the recent 2020 ECMM/ISHAM definitions. RESULTS: A total of 179 patients were admitted in the ICU and 6 (3.3%) patients were diagnosed with CAPA (4 probable and 2 possible CAPA) with 5/6 with co-infection with multidrug-resistant (MDR) gram-negative pathogens. No patient had a history of immunosuppression. All suffered from acute respiratory distress syndrome. The median (range) time from intubation to diagnosis was 6 (1-14) days. Five patients had positive Aspergillus cultures in bronchial secretions (1 A. fumigatus, 1 A. flavus, 1 A. fumigatus + A. flavus, 1 A. fumigatus + A. terreus and 1 A. terreus) while culture was negative in one patient. All isolates were susceptible to antifungal drugs. Serum galactomannan (GM), pan-Aspergillus PCR and (1,3)-ß-D-glucan (BDG) were positive in 4/6 (67%), 5/6 (83%, 3/5 in two consecutive samples) and 4/6 (67%, in consecutive samples) patients, respectively. GM and PCR positive bronchial secretions had GM indices > 9.95 and PCR Ct < 34. All were treated with antifungal drugs with 5 out of 6 receiving isavuconazole. Mortality was 67% (4/6) with 1/4 attributed to CAPA (two died as a result of bacterial septic shock and one as a result of multiorgan failure). CONCLUSION: The incidence of CAPA in ICU patients was 3.3% and it was associated with approximately a 17% attributable mortality in the setting of MDR gram-negative pathogen co-infections.

A Corneal Perforation Related to Beauveria Bassiana and Post-Penetrating Keratoplasty Management Discussion.

Fungal keratitis is an infection that is insidious and frequently misdiagnosed. Those with chronic eye surface conditions, contact lenses, systemic immunosuppression, and diabetes have been the most frequently affected with fungal keratitis. An 84-year-old male patient with a history of bilateral penetrating keratoplasty (PK) for keratoconus presented with pain and decreased visual acuity on his left eye. A corneal perforation was found, which was treated immediately with a full-thickness corneal transplant. The specimen was sent for bacterial and fungal cultures. Topical corticosteroids were prescribed postoperatively. Beauveria bassiana was isolated from the corneal scrapings. The postoperative treatment was modified by reducing the dose of corticosteroid and adding topical natamycin together with systemic posaconazole. No recurrence occurred in the transplant four months postoperatively under topical dexamethasone 0.1% b.i.d.  This is the first case of keratitis and perforation in a previously transplanted cornea. Due to the rarity of the infection, there are no clear guidelines for postoperative prophylaxis in B. bassiana infection. Either the continuation of corticosteroids or the switch to another immunosuppressive therapy and selecting the appropriate antifungal regimen posed a significant therapeutic dilemma.

Molecular Epidemiology and Antifungal Susceptibility of Trichophyton Isolates in Greece: Emergence of Terbinafine-Resistant Trichophytonmentagrophytes Type VIII Locally and Globally.

Trichophyton isolates with reduced susceptibility to antifungals are now increasingly reported worldwide. We therefore studied the molecular epidemiology and the in vitro antifungal susceptibility patterns of Greek Trichophyton isolates over the last 10 years with the newly released EUCAST reference method for dermatophytes. Literature was reviewed to assess the global burden of antifungal resistance in Trichophyton spp. The in vitro susceptibility of 112 Trichophyton spp. molecularly identified clinical isolates (70 T. rubrum, 24 T. mentagrophytes, 12 T. interdigitale and 6 T. tonsurans) was tested against terbinafine, itraconazole, voriconazole and amorolfine (EUCAST E.DEF 11.0). Isolates were genotyped based on the internal transcribed spacer (ITS) sequences and the target gene squalene epoxidase (SQLE) was sequenced for isolates with reduced susceptibility to terbinafine. All T. rubrum, T. interdigitale and T. tonsurans isolates were classified as wild-type (WT) to all antifungals, whereas 9/24 (37.5%) T. mentagrophytes strains displayed elevated terbinafine MICs (0.25-8 mg/L) but not to azoles and amorolfine. All T. interdigitale isolates belonged to ITS Type II, while T. mentagrophytes isolates belonged to ITS Type III* (n = 11), VIII (n = 9) and VII (n = 4). All non-WT T. mentagrophytes isolates belonged to Indian Genotype VIII and harbored Leu393Ser (n = 5) and Phe397Leu (n = 4) SQLE mutations. Terbinafine resistance rates ranged globally from 0-44% for T. rubrum and 0-76% for T. interdigitale/T. mentagrophytes with strong endemicity. High incidence (37.5%) of terbinafine non-WT T. mentagrophytes isolates (all belonging to ITS Type VIII) without cross-resistance to other antifungals was found for the first time in Greece. This finding must alarm for susceptibility testing of dermatophytes at a local scale particularly in non-responding dermatophytoses.

Performance, Correlation and Kinetic Profile of Circulating Serum Fungal Biomarkers of Invasive Aspergillosis in High-Risk Patients with Hematologic Malignancies.

As conventional microbiological documentation of invasive aspergillosis (IA) is difficult to obtain, serum fungal biomarkers are important adjunctive diagnostic tools. Positivity rates and the kinetic profiles of galactomannan (GM), 1,3-ß-D-glucan (BDG) and Aspergillus DNA (PCR) were studied in high-risk patients with hematologic malignancies. GM, BDG and PCR data from serial serum specimens (n = 240) from 93 adult hematology patients with probable (n = 8), possible (n = 25) and no (n = 60) IA were retrospectively analyzed. Positivity rates and sensitivity/specificity/positive/negative predictive values (NPV) of each fungal biomarker alone and in combination were estimated. The three markers were compared head-to-head and correlated with various biochemical, demographic and patient characteristics. The positivity rates for patients with probable/possible/no IA were 88%/8%/0% for GM (X2 = 55, p < 0.001), 62%/46%/35% for BDG (X2 = 2.5, p = 0.29), 62%/33%/27% for PCR (X2 = 3.9, p = 0.15), 50%/4%/0% for GM + BDG and GM + PCR (X2 = 31, p < 0.001), 50%/8%/22% for BDG + PCR (X2 = 6.5, p = 0.038) and 38%/4%/0% for GM + BDG + PCR (X2 = 21, p < 0.001). Higher agreement (76%) and negative correlation (rs = -0.47, p = 0.0017) was found between GM index and PCR Ct values. The sensitivity and NPV was 45-55% and 90-92% when biomarkers assessed alone and increased to 75-90% and 93-97%, respectively when combined. Weak significant correlations were found between GM, PCR and BDG results with renal/liver function markers (r = 0.11-0.57) with most GM+ and PCR+ samples found in the first and second week of clinical assessment, respectively and BDG later on. Different positivity rates, time profiles and performances were found for the three biomarkers advocating the combination of GM with PCR for the early diagnosis of IA, whereas the high NPV of combined biomarkerscould help excluding IA.

Comparative Pharmacodynamics of Echinocandins against Aspergillus fumigatus Using an In Vitro Pharmacokinetic/Pharmacodynamic Model That Correlates with Clinical Response to Caspofungin Therapy: Is There a Place for Dose Optimization?

Echinocandins have been used as primary therapy of invasive aspergillosis (IA), with suboptimal results at standard dosing. Here, we explored the efficacy of dose escalation in a validated in vitro pharmacokinetic/pharmacodynamic (PK/PD) model. Six echinocandin wild-type (WT) and three non-WT A. fumigatus isolates were tested in an in vitro PK/PD model simulating anidulafungin, caspofungin, and micafungin exposures with a free drug maximum concentration (fCmax) of 0.01 to 16 mg/liter and a half-life (t1/2) of 8 to 22 h. The relationship between the area under the dosing interval time-free drug concentration curve (fAUC0-24)/minimum effective concentration (MEC) and % aberrant mycelium formation was analyzed. PK/PD indices associated with 50 to 99.99% maximal activity (EI50 to EI99.99) were correlated with the clinical outcome of a 50-mg/day standard dose of caspofungin. The probability of target attainment (PTA) was calculated for different dosing regimens of each echinocandin via Monte Carlo analysis. A sigmoidal PK/PD relationship was found for WT isolates with EI99 values of 766, 8.8, and 115 fAUC0-24/CLSI MEC for anidulafungin, caspofungin, and micafungin, respectively. No aberrant mycelia were observed for non-WT isolates, irrespective of their MEC and drug exposure. The EI99, EI99.9, and EI99.99 values corresponded to 2-, 3-, and 4-log10 formation of aberrant mycelia and correlated with survival, favorable, and complete response rates to caspofungin primary therapy in patients with IA. A very low PTA (<13%) was found for the standard doses of all echinocandins, whereas a PTA of ≥90% was found with 100 and 150 mg/day of caspofungin and 1,400 mg/day micafungin against WT isolates. For anidulafungin, the PTA for 1,500 mg/day was 10%. Among the three echinocandins, only caspofungin at 2 or 3 times the licensed dosing was associated with a high PTA. Caspofungin dose escalation might deserve clinical validation.