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In vitro interaction of anidulafungin with voriconazole was tested by a microdilution broth checkerboard technique and an agar diffusion method against 30 Aspergillus clinical isolates belonging to five different species. By using a complete inhibition endpoint, indifferent interactions were observed for 97% of the isolates by the checkerboard technique (FIC index from 0.5 to 2) and for 100% of the isolates by the agar diffusion method (variation of -2 to +1 log(2) dilutions).

Unlabelled: Cryptococcosis is a multifaceted fungal infection with variable clinical presentation and outcome. As in many infectious diseases, this variability is commonly assigned to host factors. To investigate whether the diversity of Cryptococcus neoformans clinical (ClinCn) isolates influences the interaction with host cells and the clinical outcome, we developed and validated new quantitative assays using flow cytometry and J774 macrophages. The phenotype of ClinCn-macrophage interactions was determined for 54 ClinCn isolates recovered from cerebrospinal fluids (CSF) from 54 unrelated patients, based on phagocytic index (PI) and 2-h and 48-h intracellular proliferation indexes (IPH2 and IPH48, respectively). Their phenotypes were highly variable. Isolates harboring low PI/low IPH2 and high PI/high IPH2 values were associated with nonsterilization of CSF at week 2 and death at month 3, respectively. A subset of 9 ClinCn isolates with different phenotypes exhibited variable virulence in mice and displayed intramacrophagic expression levels of the LAC1, APP1, VAD1, IPC1, PLB1, and COX1 genes that were highly variable among the isolates and correlated with IPH48. Variation in the expression of virulence factors is thus shown here to depend on not only experimental conditions but also fungal background. These results suggest that, in addition to host factors, the patient's outcome can be related to fungal determinants. Deciphering the molecular events involved in C. neoformans fate inside host cells is crucial for our understanding of cryptococcosis pathogenesis., Importance: Cryptococcus neoformans is a life-threatening human fungal pathogen that is responsible for an estimated 1 million cases of meningitis/year, predominantly in HIV-infected patients. The diversity of infecting isolates is well established, as is the importance of the host factors. Interaction with macrophages is a major step in cryptococcosis pathogenesis. How the diversity of clinical isolates influences macrophages' interactions and impacts cryptococcosis outcome in humans remains to be elucidated. Using new assays, we uncovered how yeast-macrophage interactions were highly variable among clinical isolates and found an association between specific behaviors and cryptococcosis outcome. In addition, gene expression of some virulence factors and intracellular proliferation were correlated. While many studies have established that virulence factors can be differentially expressed as a function of experimental conditions, our study demonstrates that, under the same experimental conditions, clinical isolates behaved differently, a diversity that could participate in the variable outcome of infection in humans.

Objectives: An increase in invasive aspergillosis (IA) due to azole-resistant Aspergillus fumigatus isolates has been reported for 10 years. Our study aimed to estimate the prevalence of azole resistance in isolates prospectively collected in patients with haematological diseases., Methods: One hundred and eighteen isolates were collected from 89 consecutive patients over 4 years. Fifty-one patients had proven or probable IA. Species identification was ascertained based on β-tubulin gene sequencing. The MICs of azole drugs were determined using Etest(®), and the cyp51A gene and its promoter were sequenced to detect mutations., Results: All isolates were identified as A. fumigatus and all of them but one had itraconazole and voriconazole MICs of ≤ 2 mg/L and posaconazole MICs of ≤ 0.25 mg/L. An isolate for which the itraconazole MIC was high (itraconazole MIC = 16 mg/L; voriconazole MIC = 0.38 mg/L; and posaconazole MIC = 0.25 mg/L) was recovered from a patient naive to azole treatment and had a new G432S substitution. To establish whether this mutation existed in other isolates, the 1426-2025 bp cyp51A locus was sequenced for all. G432S was not found., Conclusions: In A. fumigatus, the prevalence of azole resistance is currently low in the haematological population in the Paris area. Surveillance programmes for azole resistance to adapt antifungal treatments are warranted for clinical isolates of A. fumigatus.

We report a rare case of dermatophytosis due to Microsporum praecox in a 28-year-old female horse rider. The skin lesion was located on the right external malleolus. Microscopic examination of skin scrapings revealed a dermatophyte which was also isolated in culture. The identification of M. praecox was confirmed by molecular biology (sequence analysis of PCR products amplified from internal transcribed spacer regions with universal primers). Combined antifungal therapy with oral terbinafine and topical cyclopiroxolamide resulted in complete remission of the fungal lesion within 1 month. Since 1944, only 29 cases of human M. praecox infection have been reported in the literature. The clinical features and treatment of these cases are reviewed. The prevalence of M. praecox infection is probably underestimated, and systematic molecular identification could improve our understanding of the epidemiology of this fungal dermatosis.

Delays in the identification of microorganisms are a barrier to the establishment of adequate empirical antibiotic therapy of bacteremia. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) allows the identification of microorganisms directly from colonies within minutes. In this study, we have adapted and tested this technology for use with blood culture broths, thus allowing identification in less than 30 min once the blood culture is detected as positive. Our method is based on the selective recovery of bacteria by adding a detergent that solubilizes blood cells but not microbial membranes. Microorganisms are then extracted by centrifugation and analyzed by MALDI-TOF-MS. This strategy was first tested by inoculating various bacterial and fungal species into negative blood culture bottles. We then tested positive patient blood or fluid samples grown in blood culture bottles, and the results obtained by MALDI-TOF-MS were compared with those obtained using conventional strategies. Three hundred twelve spiked bottles and 434 positive cultures from patients were analyzed. Among monomicrobial fluids, MALDI-TOF-MS allowed a reliable identification at the species, group, and genus/family level in 91%, 5%, and 2% of cases, respectively, in 20 min. In only 2% of these samples, MALDI-TOF MS did not yield any result. When blood cultures were multibacterial, identification was improved by using specific databases based on the Gram staining results. MALDI-TOF-MS is currently the fastest technique to accurately identify microorganisms grown in positive blood culture broths.

MALDI-TOF MS can be used for the identification of microorganism species. We have extended its application to a novel assay of Candida albicans susceptibility to fluconazole, based on monitoring modifications of the proteome of yeast cells grown in the presence of varying drug concentrations. The method was accurate, and reliable, and showed full agreement with the Clinical Laboratory Standards Institute's reference method. This proof-of-concept demonstration highlights the potential for this approach to test other pathogens.

Trichoderma longibrachiatum, a filamentous fungus, was recently described as an emerging pathogen in immunocompromised patients. Here, we report the first case, to our knowledge, of isolated invasive lung infection by T. longibrachiatum in a patient with hematologic malignancy. The infection mimicked invasive pulmonary aspergillosis and was successfully treated with a combination of voriconazole and caspofungin.