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.

Twenty Years in EUCAST Anti-Fungal Susceptibility Testing: Progress & Remaining Challenges.

Since its inception in 2002, the EUCAST Antifungal Susceptibility Testing Subcommittee (AFST) has developed and refined susceptibility testing methods for yeast, moulds and dermatophytes, and established epidemiological cut-off values and breakpoints for antifungals. For yeast, three challenges have been addressed. Interpretation of trailing growth in fluconazole susceptibility testing, which has been proven without impact on efficacy if below the 50% endpoint. Variability in rezafungin MIC testing due to laboratory conditions, which has been solved by the addition of Tween 20 to the growth medium in E.Def 7.4. And third, interpretation of MICs for rare yeast with no breakpoints, where recommendations have been established for MIC-based clinical advice. For moulds, refinements include the validation of spectrophotometer reading for A. fumigatus to facilitate objective MIC determination, and for dermatophytes the establishment of a microdilution method with automated reading and a selective medium to minimise the risk of contaminations. Recent initiatives involve development and validation of agar-based screening assays for detection of potential azole and echinocandin resistance in A. fumigatus and Aspergillus species, respectively, and of terbinafine resistance in Trichophyton species. Moreover, the development of a EUCAST guidance document for molecular resistance testing represents an advancement, particularly for identifying target gene alterations associated with resistance. In summary, EUCAST AFST continues to play a pivotal role in standardizing AFST and facilitating accurate interpretation of susceptibility data for clinical decision-making. Adoption of EUCAST breakpoints for commercial test methods, however, requires thorough validation to ensure concordance with EUCAST reference testing species-specific MIC distributions.

Aspergillus granulosus femoral osteomyelitis in a cardiac transplant patient: first reported case and literature review.

Aspergillus osteomyelitis is a rare complication of extrapulmonary invasive aspergillosis, which usually presents as spondylodiscitis. The clinical picture is usually paucisymptomatic and of long evolution, which leads to diagnostic difficulties, especially in immunosuppressed patients presenting a delayed systemic host response. We report a case of femoral osteomyelitis caused by Aspergillus granulosus in a heart transplant recipient successfully treated with a combined surgical and antifungal approach. A 65-year-old heart transplant male presented with left knee pain lasting 3 months. X-ray and magnetic resonance imaging identified a lesion with aggressive characteristics at the distal third of the left femur, due to which the patient underwent excisional surgery. Aspergillus granulosus was cultured from the removed material and antifungal treatment with oral isavuconazole was started. Chest imaging excluded pulmonary aspergillosis, while the positron emission tomography/computed tomography (PET/CT) identified a remnant of a prosthetic vascular graft sewn to the proximal third of the right axillary artery, through which a catheter-based micro-axial left ventricular assist device was implanted previously as bridge to transplant therapy. The patient presented a rapid clinical improvement with complete functional recovery following the surgical treatment and the antifungal therapy and finally underwent surgical removal of the residual vascular graft. This is the first reported episode of long bone osteomyelitis due to A. granulosus that occurred in a heart transplant recipient without pulmonary infection and was successfully treated with isavuconazole. The PET/CT was useful in supporting the diagnostic process and follow-up. Cryptic fungal species can cause invasive infections, particularly in immunocompromised patients. Molecular methods are crucial in fungal identification.

Fluconazole-resistant Candida parapsilosis: fast detection of the Y132F ERG11p substitution, and a proposed microsatellite genotyping scheme.

OBJECTIVES: We propose fast and accurate molecular detection of the Y132F ERG11p substitution directly on pure-cultured Candida parapsilosis isolates. We also assessed a discriminative genotyping scheme to track circulating genotypes. METHODS: A total of 223 C. parapsilosis isolates (one patient each) from 20 hospitals, located in Spain and Italy were selected. Isolates were fluconazole-resistant (n = 94; harbouring the Y132F ERG11p substitution [n = 85], the G458S substitution [n = 6], the R398I substitution [n = 2], or the wild-type ERG11 gene sequence) or fluconazole-susceptible (n = 129). Two targeted-A395T-mutation PCR formats (conventional and real-time) were engineered and optimized on fluconazole-susceptible and fluconazole-resistant pure-cultured isolates, thus skipping DNA extraction. Two genotyping schemes were compared: Scheme 1 (CP1, CP4a, CP6, and B markers), and Scheme 2 (6A, 6B, 6C, CP1, CP4a, and CP6 markers). RESULTS: The screening performed using both PCR formats showed 100% specificity (fluconazole-susceptible isolates; n = 129/129) and sensitivity (Y132F isolates; n = 85/85) values; however, results were available in 3 and 1.5 hours with the conventional and real-time PCR formats, respectively. Overall, Scheme 1 showed higher genetic diversity than Scheme 2, as shown by the number of alleles detected (n = 98; mean 23, range 13-38), the significantly higher observed and expected heterozygosity, and the probability of identity index (2.5 × 10-6). Scheme 2 markers did not provide further genotypic discrimination of Y132F fluconazole-resistant genotypes. CONCLUSION: Both proposed PCR formats allow us to speed up the accurate detection of substitution Y132F ERG11p in C. parapsilosis isolates with 100% specificity and sensitivity. In addition, we recommend CP1, CP4a, CP6, and B microsatellite markers for genotyping fluconazole-resistant isolates.