Summary: The fungal cell wall, a conserved and highly dynamic structure, is essential for virulence and viability of fungal pathogens and is an important antifungal drug target. The cell wall integrity (CWI) signalling pathway regulates shape and biosynthesis of the cell wall. In this work we identified, localized and functionally characterized four putative CWI stress sensors of Aspergillus fumigatus, an airborne opportunistic human pathogen and the cause of invasive aspergillosis. We show that Wsc1 is specifically required for resistance to echinocandin antifungals. MidA is specifically required for elevated temperature tolerance and resistance to the cell wall perturbing agents congo red and calcofluor white. Wsc1, Wsc3 and MidA additionally have overlapping functions and are redundantly required for radial growth and conidiation. We have also analysed the roles of three Rho GTPases that have been implicated in CWI signalling in other fungi. We show that Rho1 is essential and that conditional downregulation of rho1 or deletion of rho2 or rho4 results in severely impaired CWI. Our data indicate significant functional differences between the CWI stress sensors of yeasts and moulds.

Magnusiomyces clavatus and Magnusiomyces capitatus are emerging yeasts with intrinsic resistance to many commonly used antifungal agents. Identification is difficult, and determination of susceptibility patterns with commercial and reference methods is equally challenging. For this reason, few data on invasive infections by Magnusiomyces spp. are available.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized diagnostics in culture-based microbiology. Commonly used MALDI-TOF MS systems in clinical microbiology laboratories are MALDI Biotyper (Bruker Daltonics) and Vitek MS (bioMérieux), but recently the new EXS2600 (Zybio) has been launched. This study aimed to evaluate the performance of the three devices by comparing the results to 16S rRNA gene sequencing. A set of 356 previously collected difficult-to-identify bacteria was tested in parallel with the three systems. Only the direct smear method and simple formic acid extraction were applied. Valid results were achieved for 98.6%, 94.4%, and 93.3% of all isolates by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Of all valid results, agreement with sequencing data was achieved in 98.9%, 98.5%, and 99.7% by MALDI Biotyper, EXS2600, and Vitek MS, respectively. Considering only the isolates with valid measurements at the single-species level, misidentification rates were 0%, 2.6%, and 1.1% for MALDI Biotyper, EXS2600, and Vitek MS, respectively. Apart from minor performance differences, our data demonstrate that the three systems provide comparable results and are suitable for use in medical diagnostic laboratories.

Background: The incidence of Aspergillus and Candida CNS infection, which are characterised by high mortality rates, is underestimated. This underdiagnosis presumably results from the limitations of available diagnostic tools and the need for invasive sampling. Little is known about the role of serologic biomarkers in the setting of CNS aspergillosis and candidiasis.Patients, materials and methods: Serum and cerebrospinal fluid (CSF; 10) samples of 19 patients, whose CNS specimens yielded growth of Aspergillus or Candida, were analysed for different biomarkers for fungal infection, that is galactomannan (GM), galactomannoprotein (GP), mannan, anti‐mannan‐antibodies and β‐1,3‐D‐glucan (BDG). Serum and CSF specimens of time‐matched patients (two each for every case of fungal CNS infection) were included as controls.Results: Galactomannan, GP and BDG seropositivity was found in one, two and three of five cases of CNS aspergillosis. BDG and mannan/anti‐mannan‐antibody sensitivity in proven CNS candidiasis was 40% and 20%, respectively. Applying the serum cut‐off, sensitivity in CSF testing was 100% for GM and BDG and 50% for mannans. While serum specificity for all assays ranged from 89 to 97%, specificity for CSF BDG was only 70%. No false‐positive GM results from CSF were obtained.Conclusion: Sensitivity for diagnosing CNS aspergillosis and CNS candidiasis from serum is mediocre for all serological biomarkers. GM testing in CSF proved excellent performance. With a sensitivity of 100% but a specificity of only 70%, CSF BDG might be most useful when used in patients with a high pre‐test probability.

Mycobacterium abscessus is a major respiratory pathogen in cystic fibrosis (CF) patients. Recently it has been shown that dominant global clonal complexes (DCCs) have spread worldwide among CF patients.

The early diagnosis of invasive aspergillosis (IA) relies mainly on computed tomography imaging and testing for fungal biomarkers such as galactomannan (GM). We compared an established ELISA for the detection of GM with a turbidimetric assay for detection of the panfungal biomarker β-D-glucan (BDG) for early diagnosis of IA. A total of 226 serum specimens from 47 proven and seven probable IA cases were analysed. Sensitivity was calculated for samples obtained closest to the day of IA-diagnosis (d0). Additional analyses were performed by including samples obtained during the presumed course of disease. Most IA cases involved the respiratory system (63%), and Aspergillus fumigatus was the most frequently isolated species (59%). For proven cases, sensitivity of BDG/GM analysis was 57%/40%. Including all samples dating from –6 to +1 weeks from d0 increased sensitivities to 74%/51%. Sensitivity of BDG testing was as high as or higher than GM testing for all subgroups and time intervals analysed. BDG testing was less specific (90–93%) than GM testing (99–100%). Combining BDG and GM testing resulted in sensitivity/specificity of 70%/91%. Often, BDG testing was positive before GM testing. Our study backs the use of BDG for diagnosis of suspected IA. We suggest combining BDG and GM to improve the overall sensitivity.