Invasive fungal infections (IFI) are a major clinical challenge, particularly in immunocompromised patients, and are associated with high morbidity and mortality. With the increasing prevalence of immunosuppressive conditions and ageing populations, the incidence of IFI is rising globally. This survey aims to evaluate the diagnostic and therapeutic capacities for IFI in Belgium, the Netherlands, and Luxembourg (Benelux), a region of high azole-resistance among Aspergillus fumigatus isolates. A survey evaluating the diagnostic and therapeutic capacity for IFI was conducted in the Benelux. Data were collected from specialists via an online case report form between March and September 2023. The survey addressed patient characteristics, access to microbiology labs, diagnostic methods (microscopy, culture, molecular diagnostics, etc.), IFI incidence, and the availability of antifungal drugs and therapeutic drug monitoring. In total, 32 hospitals responded to the questionnaire (12 [38%] from the Netherlands, 19 [59%] from Belgium and one [3%] from Luxembourg). Antifungal susceptibility tests were available in 29 institutions (91%), constituting 84% of the centres in Belgium and 100% for the Netherlands (p = 0.265). Aspergillus PCR testing was available in 12 centres in Belgium (63%) while in 11 centres in the Netherlands (92%, p = 0.108). Mucorales PCR testing was available in 56% of centres. Treatment with at least one amphotericin B formulation was only available in 84% of the responding centres. Therapeutic drug monitoring (TDM), although recommended, was possible for voriconazole in 26 centres (81%) while for posaconazole in 24 centres (75%). Significantly more testing (diagnostic tests and TDM) was outsourced in Belgium compared to the Netherlands (p < 0.001). Antifungal susceptibility testing is widely available in Belgium and the Netherlands, but implementation in areas with high azole resistance for Aspergillus fumigatus is not yet universal, and techniques vary. Tests for coinfections, like Mucorales PCR, were only available in half of the centres. More testing is outsourced in Belgium, likely due to differences in reference centre organisation, country size, transport, and reimbursement. Delays in diagnosis can impact patient outcomes, so awareness of test availability and transport times is crucial.

ABSTRACTIntroductionFungaemia due to non‐Candida and non‐Cryptococcus yeasts is uncommon but clinically significant, particularly in immunocompromised hosts. We aimed to describe the epidemiology, microbiology and outcomes of bloodstream infections (BSIs) caused by these organisms.MethodsWe identified all BSIs due to non‐Candida and non‐Cryptococcus yeasts over a 20‐year period using statewide laboratory and administrative health databases.ResultsSeventy‐five unique episodes were identified. The most frequent genera were Trichosporon (n = 31, 41.3%), Rhodotorula (n = 26 34.7%) and Saccharomyces (n = 10, 13.3%) species. Antifungal susceptibility testing performed in 33 (44%) episodes revealed high MICs (&gt; 16 mg/L) to echinocandins for Trichosporon and Rhodotorula species. Fluconazole MICs were universally elevated ( 32 mg/L) in Rhodotorula spp. but lower in Saccharomyces cerevisiae (2–4 mg/L). Voriconazole and posaconazole had good in vitro activity across all genera where tested. Thirty‐day mortality was 22.7%, with the highest rate observed in S. cerevisiae (50.0%). Mortality was associated with malignancy (aHR 4.71, 95% CI 1.00–22.25), heart failure (aHR 11.31, 95% CI 1.66–77.14) and intensive care unit (ICU) admission (aHR 7.05, 95% CI 0.99–50.36). The presence of a central line may be protective (aHR 0.17, 95% CI 0.03–1.04). Rhodotorula infection was associated with lower mortality on univariable analysis (HR 0.11, 95% CI 0.14–0.86) compared with Trichosporon species.ConclusionAlthough rare, fungaemia due to non‐Candida and non‐Cryptococcus yeasts is associated with significant mortality and antifungal resistance. Species identification and susceptibility testing are crucial to guide treatment. Increased awareness is essential in high‐risk patients, particularly those with malignancy, heart failure, or requiring ICU admission.

Candida (Candidozyma) auris has distinct genetic clades. Clade distinction is relevant for infection control and epidemiological purposes. State-of-the-art typing methodologies for clade distinction are based on genomic approaches, such as short tandem repeat (STR) analysis and whole-genome sequencing (WGS). However, they are time-consuming and expensive. Fourier transform infrared spectroscopy (FT-IR) is an alternative tool for strain typing based on their unique biochemical spectral profiles. To apply FT-IR to differentiate C. auris clades and evaluate epidemiological relationships based on biochemical data among isolates from a multicenter C. auris outbreak in the state of Pernambuco, northeastern Brazil. Sixty-nine C. auris strains from clades I, II, III, and IV were analysed. Fifty-nine were clade IV strains obtained from three outbreaks that took place in Pernambuco state, northeastern Brazil. An adjusted FT-IR spectroscopy protocol was applied to obtain carbohydrates and lipid fingerprints. Short Tandem Repeat (STR) analysis was used in order to validate the spectroscopy approach. The adjusted preparation protocol for FT-IR analysis improved the spectral quality by 31.42% compared to standard protocol. FT-IR allowed us to discriminate C. auris clades I to IV. Moreover, important similarities were observed in C. auris clade IV strains obtained from two of the three hospitals, based on polysaccharides (1300-800 cm-1) plus lipids (3000-2800 cm-1 and 1500-1400 cm-1) spectra. STR confirmed the similarity results obtained by FT-IR, clustering the strains from two different hospitals. The IR Biotyper is fast, easy-to-use, and a promising alternative for moderate-to-high-complexity laboratories to differentiate C. auris clades. Furthermore, this technique has the potential for isolate-level source tracking, which could be valuable for monitoring transmission routes in clinical settings.

The differential diagnosis of invasive pulmonary aspergillosis (IPA), pulmonary mucormycosis (PM), bacterial pneumonia (BP) and pulmonary tuberculosis (PTB) are challenging due to overlapping clinical and imaging features. Manual CT lesion segmentation is time-consuming, deep-learning (DL)-based segmentation models offer a promising solution, yet disease-specific models for these infections remain underexplored. We aimed to develop and validate dedicated CT segmentation models for IPA, PM, BP and PTB to enhance diagnostic accuracy. Methods：Retrospective multi-centre data (115 IPA, 53 PM, 130 BP, 125 PTB) were used for training/internal validation, with 21 IPA, 8PM, 30 BP and 31 PTB cases for external validation. Expert-annotated lesions served as ground truth. An improved 3D U-Net architecture was employed for segmentation, with preprocessing steps including normalisations, cropping and data augmentation. Performance was evaluated using Dice coefficients. Results：Internal validation achieved Dice scores of 78.83% (IPA), 93.38% (PM), 80.12% (BP) and 90.47% (PTB). External validation showed slightly reduced but robust performance: 75.09% (IPA), 77.53% (PM), 67.40% (BP) and 80.07% (PTB). The PM model demonstrated exceptional generalisability, scoring 83.41% on IPA data. Cross-validation revealed mutual applicability, with IPA/PTB models achieving > 75% Dice for each other's lesions. BP segmentation showed lower but clinically acceptable performance ( ＞72%), likely due to complex radiological patterns. Disease-specific DL segmentation models exhibited high accuracy, particularly for PM and PTB. While IPA and BP models require refinement, all demonstrated cross-disease utility, suggesting immediate clinical value for preliminary lesion annotation. Future efforts should enhance datasets and optimise models for intricate cases.

Identification of filamentous fungi still poses a major challenge to laboratories. Matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) is a promising tool, since it offers low-cost and fast results. Different MALDI-TOF MS systems are available for routine laboratories. This is a comprehensive head-to-head comparison of three devices and the respective reference spectrum databases. A set of 77 pre-characterised isolates of filamentous fungi was measured (in duplicates) parallelly with three MALDI-TOF MS systems after 24 h, 48 h and 72 h of incubation: (1) Biotyper smart ('BT', Bruker Daltonics),(2) EXS2600 ('EXS', Zybio) and (3) VITEK MS PRIME ('VITEK', bioMérieux). After three measurements, no valid results at the species level ('green category') were obtained for 18%, 21% and 14% of isolates by BT, EXS and VITEK. Depending on the MALDI-TOF MS system, validity rates ranged from 58%-82% for the different time points. Correct and valid results were obtained for 82%, 73% and 81% of isolates by BT, EXS and VITEK. BT was the system that required the most duplicate measurements. EXS displayed the highest rate of misidentification events. VITEK had the highest rate of unidentified isolates, which were featured within its database. Based on our experience, all three devices proved to be suitable for routine diagnostics. The timepoint of measurement had a major impact on the quality of analysis and should be considered by the user. Lowering the validity cut-off levels might increase the performance of the EXS system.

Due to the paucity of fungal elements in the tissue, the sensitivity of histopathological tests for the diagnosis of cutaneous sporotrichosis remains low, particularly for low-virulent Sporothrix species. We retrospectively analysed and re-evaluated the role of histopathological examination in the diagnosis of cutaneous sporotrichosis caused by Sporothrix globosa. Retrospective analysis and pathological reassessment were conducted for 69 sporotrichosis cases caused by S. globosa between 2013 and 2024. Statistical analysis using prevalence ratios was conducted to characterise the clinicopathological and epidemiological aspects. Histopathological examination revealed mixed inflammatory cell infiltration and granulomatous changes in all cases. Periodic acid-Schiff (PAS) staining was performed in 29 tissue specimens, with positivity observed in 4 cases (13.8%, 4/29). Following supplemental staining and re-evaluation of the slides, PAS staining revealed positivity in 50.7% (35/69) of cases, demonstrating yeast forms (91.4%, 32/35) with asteroid bodies (n = 2) and rare hyphae (n = 1). Currently, the role of histopathological examination in the diagnosis of sporotrichosis is limited. Our study showed that the frequency of detecting S. globosa based on histopathological examination is considerably high. Nevertheless, achieving high positive rates necessitates the collaborative efforts of experienced mycologists. The predominance of suppurative granulomas or neutrophils is related to the presence of the fungus in tissue sections from human patients.

The recent rise in the global incidence of fluconazole resistance in C. parapsilosis has become a significant public health concern. Epidemiological studies suggest that fluconazole resistance in C. parapsilosis spreads through endemic clones. We, therefore, investigated the molecular epidemiology of fluconazole-resistant C. parapsilosis in our centre. C. parapsilosis isolates from 2016 through 2022 were investigated for antifungal susceptibility. Fluconazole-resistant isolates were analysed for ERG11 mutation using Sanger sequencing. Gene expression profiles of ERG11, CDR1 and MDR1 were assessed by real-time qPCR. The epidemiological relationship of resistant and susceptible isolates of C. parapsilosis was investigated using short tandem repeat typing. Additionally, biofilm production and cell wall ergosterol contents were also quantified and compared. Among 572 C. parapsilosis isolates, 48 (8.4%) were resistant to fluconazole. Of 28 recoverable resistant isolates, 17.9% (5/28) were wild-type and 82.1% (23/28) harboured the following ERG11 mutations: Y132F (n = 3), K143R (n = 10) and K143R + R398I (10/28). Significant fold-changes were observed in ERG11 (p = 0.037) and MDR1 (p = 0.008) gene expressions in fluconazole resistant compared to susceptible isolates. Contrary to global reports, STR typing suggested a limited clonal transmission of resistant C. parapsilosis with multiple introductions of resistant isolates in our centre. On fluconazole exposure, ergosterol content significantly increased (p < 0.01) in resistant isolates, particularly in isolates harbouring ERG11K143R + R398I mutations. In contrast, fluconazole-susceptible isolates formed comparatively higher baseline biofilm (p < 0.05) than resistant isolates with ERG11K143R mutation. The current study underscores the need for continuous molecular surveillance and tailored therapeutic options for effective management of fluconazole resistance in C. parapsilosis.