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The aim was to develop an RT-qPCR targeting Aspergillus fumigatus and compare its performance to that of Aspergillus fumigatus qPCR for the diagnosis of invasive aspergillosis (IA). Samples from patients of the Lyon University hospitals for whom a suspicion of IA led to the realization of an Aspergillus fumigatus qPCR molecular diagnostic test over a 2-year period were included. The patients were classified according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC-MSGERC) criteria for suspected IA; RT-qPCR and qPCR assays were performed on all included samples. The sensitivities and specificities of RT-qPCR and qPCR were calculated and compared using the results of the EORTC-MSGERC classification as reference. The cycle threshold (Ct) results were compared according to IA classification and sample type. Among the 193 samples analyzed, 91 were classified as IA excluded, 46 as possible IA, 53 as probable IA, and 3 as proven IA. For all sample types, RT-qPCR was significantly more sensitive than qPCR for all IA classifications with an additional 17/102 samples detected ( P -value < 0.01). For plasma samples, sensitivity was significantly higher and specificity significantly lower using RT-qPCR for all IA classifications ( P -value < 0.001). The mean Ct obtained with RT-qPCR were significantly lower than those obtained with qPCR for all IA classifications and all sample types ( P -value < 0.001 and P -value < 0.0001, respectively). RT-qPCR presents a higher sensitivity than qPCR for the diagnosis of IA due to Aspergillus fumigatus , particularly in samples with an intrinsically low fungal load.IMPORTANCE Aspergillus fumigatus belongs to the critical priority group of the World Health Organization fungal priority pathogens list. Invasive aspergillosis (IA) is a life-threatening infection with poor prognosis and challenging diagnosis. PCR has been integrated into the 2020 European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions for IA diagnosis. However, due to frequent low fungal burdens, its sensitivity needs to be improved. This work presents an innovative method for detecting total nucleic acids, corresponding to both ribosomal RNA and DNA, that enables IA diagnosis with greater sensitivity than conventional techniques, especially in non-invasive samples such as blood, enhancing the monitoring of this infection in high-risk patients., Competing Interests: The authors declare no conflict of interest.

Background: Cerebral aspergillosis (CA) is a life-threatening disease for which diagnosis and management remain challenging. Detailed analyses from large cohorts are lacking., Methods: We included 119 cases of proven (n = 54) or probable (n = 65) CA diagnosed between 2006 and 2018 at 20 French hospitals. Data were collected at baseline and during follow-up. Cerebral imaging was reviewed centrally by two neuroradiologists., Results: The most frequent underlying conditions were hematological malignancy (40%) and solid organ transplantation (29%). Galactomannan was detected in the serum of 64% of patients. In 75% of cases, at least one of galactomannan, Aspergillus PCR, and β-d-glucan was positive in the cerebrospinal fluid. Six-week mortality was 45%. Two distinct patterns of disease were identified according to presumed route of dissemination. Presumed haematogenous dissemination (n = 88) was associated with a higher frequency of impaired consciousness (64%), shorter time to diagnosis, the presence of multiple abscesses (70%), microangiopathy (52%), detection of serum galactomannan (69%) and Aspergillus PCR (68%), and higher six-week mortality (54%). By contrast, contiguous dissemination from the paranasal sinuses (n = 31) was associated with a higher frequency of cranial nerve palsy (65%), evidence of meningitis on cerebral imaging (83%), macrovascular lesions (61%), delayed diagnosis, and lower six-week mortality (30%). In multivariate analysis and in a risk prediction model, haematogenous dissemination, hematological malignancy and the detection of serum galactomannan were associated with higher six-week mortality., Conclusion: Distinguishing between hematogenous and contiguous dissemination patterns appears to be critical in the workup for CA, as they are associated with significant differences in clinical presentation and outcome., Competing Interests: Conflict of interest disclosure Dr Serris, Dr J. Benzakoun, Dr R. Porcher, Dr G. Hekimian, Dr L. Lelièvre, Dr F. Bonneville, Dr M-E. Bougnoux, Dr S. Houze, Dr S. Bretagne, Dr S Kremer, Dr V Letscher-Bru, Dr V. Pourcher, Dr S. Poirée, Dr FA, Dr P. Tattevin have nothing to declare., Dr. Danion reports personal fees from Gilead, outside the submitted work., Dr F. Cotton declares personal fees from Bayer Schering, Biogen IDEC, Bracco imaging, Guerbet, Novartis outside the submitted work., Dr M. Wolff declares lectures fees from MSD and from Correvio outside the submitted work., Dr A. Fekkar declares personal fees from Gilead, MSD, Pfizer and Janssen, outside the submitted work., Dr. Gangneux reports personal fees from Pfizer, personal fees from Gilead, outside the submitted work., Dr. Cassaing reports non-financial support and other from Gilead Sciences, non-financial support and other from Pfizer France, non-financial support and other from MSD france, outside the submitted work., Dr. Kremer reports personal fees from biogen, personal fees from alexion, personal fees from roche, personal fees from bayer, outside the submitted work., Pr. Lanternier reports personal fees from Gilead, F2G outside the submitted work., (Copyright © 2021 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Until now, there has been no consensus on the best method for the detection of anti-Aspergillus antibodies, a key diagnostic tool for chronic aspergilloses. To better appreciate the usage of and confidence in these techniques, the Société Française de Mycologie Médicale (French Society for Medical Mycology; SFMM) performed a two-step survey of French experts. First, we administered an initial survey to French labs performing Aspergillus serology to depict usage of the different techniques available for Aspergillus serology. Second, an opinion poll was conducted of 40 experts via an online questionnaire. Each item was rated from 1 to 9 according to the level of agreement. The initial survey revealed that enzyme-linked immunosorbent assay (ELISA) (81%) and immunoelectrophoresis (IEP) (67%) were the most commonly used techniques for screening and confirmation, respectively. The distinction between screening and confirmation techniques was confirmed by the experts (median = 7) with a 44.2% variation coefficient. Only ELISA for screening and IEP and Western blot (WB) for confirmation were clearly considered valuable methods (median ≥8 with variation coefficients less than 30%). The use of a confirmation technique was recommended in the case of a positive result in a compatible clinical context (cystic fibrosis, for example) or during the patient's follow-up. In the case of discordant results between the screening and confirmation techniques, the experts recommended greater confidence in the results obtained with the confirmation technique. All experts emphasized the need to standardize Aspergillus serology techniques and to better define the place of each of them in the diagnosis of aspergillosis., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Graft-versus-host disease (GVHD) is a known risk factor for invasive aspergillosis (IA), but remains poorly studied in relation to Clostridium difficile infection (CDI). We report a case of a 58-years-old patient who developed an IA within a protected room, CDI and GVHD after allogeneic allogeneic peripheral blood stem cell transplantation (PBSCT). Factors associated with this complex condition in patients receiving allogeneic PBSCT need to be identified., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the end of May 2012, a meeting of the group "sérodiagnostic fongique" of the "Société française de mycologie médicale" had concerned quality controls to use, in particular, in the follow-up of Elisa techniques. A preliminary investigation showed that the internal quality controls (CIQ), according to the terms defined by the accreditation, were not systematically used. In June, was published the new guide of the COFRAC SH-GTA-06 on quality controls, this text being applicable on July 1st, 2012. It incited the working group to formulate proposals on the choice of the CIQ for antigen and antibody Elisa in the aspergillosis serodiagnosis. Informations on the external evaluations of the quality (EEQ) have also been given to better define for what we can expect from it. All these controls will allow every laboratory to better master the used techniques and their conditions of realization. A strengthened dialogue between the users and the manufacturers should incite these last actors to improve the supplied kits. It will drive later to an improvement of the reliability of the results obtained by these techniques and their interest in the aspergillosis diagnosis., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Invasive aspergillosis (IA) during induction chemotherapy of acute myeloid leukemia (AML) could worsen the prognosis. Our objective was to study how the development of IA during AML interferes with the therapeutic strategy and to evaluate its impact on the short- and long-term survival. Newly diagnosed AML patients between the years 2004 and 2007 were retrospectively analyzed. The outcome was death of the patient. A Cox proportional hazards model with the diagnosis of IA and post-induction response evaluation as the main exposure was fitted. Overall, 262 patients were analyzed and 58 IA were observed. The 2-year survival of patients having had remission of AML was 54% and, for patients with failure of chemotherapy, it was 5% (p < 0.001). The 2-year survival of patients having had IA was 14%, and without IA, it was 32% (p = 0.01). Multivariate analysis showed that IA was associated with a higher risk of death in case of remission compared to no IA (hazard ratio [HR] = 1.66 [1.05-2.65], p = 0.031) and also in case of failure (HR = 6.43, p < 0.001). IA was associated with an increased risk of death for patients if they were either in remission or in failure after induction chemotherapy.

Anti-Aspergillus antibody detection has been performed for over 50 years for the diagnosis of different chronic Aspergillus infections, starting with aspergilloma and later with chronic necrotizing pulmonary aspergillosis. It also enters into definition criteria for allergic broncho-pulmonary aspergillosis and contributes to the initial diagnosis of the aspergillosis, to the follow-up under treatment or to the detection of exacerbations. For the acute invasive aspergillosis, antibody detection has low interest compared to galactomannan antigen detection. Serology results have to be interpreted together with other clinical, radiological and biological, mycological criteria. This review describes the origins, the technical evolutions and the current place of Aspergillus serology in France. Finally, future improvements are discussed., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Aspergillus fumigatus is the most common aetiological fungus responsible for human pulmonary aspergilloses. This study investigated the primary contact between Langerhans cells (LC), corresponding to dendritic cells present in pulmonary mucosa and live conidia of A. fumigatus. LC play a key role in antigen presentation for initiation of the primary T cell response. In vitro-generated LC (iLC) were differentiated from cultured human cord blood CD34+ cells and incubated at 4 degrees C or 37 degrees C with fluorescein-isothiocyanate (FITC)-stained conidia or control latex beads. In vitro, conidia were shown by microscopy and cytometry to adhere to iLC in a dose- and time-dependent manner. This adhesion was not limited to iLC because interstitial dendritic and other cells also fluoresced in the presence of conidia-FITC. A lectin other than mannose receptor-type lectin was demonstrated to be responsible of conidial binding. Inhibition of binding was observed with heterologous galactomannan and EDTA, indicating a C-lectin-like receptor with galactomannan structure specificity. After binding only a few conidia were internalized in acidic vesicles, as indicated by the cessation of conidial fluorescence. Conidial binding was followed by activation and maturation of iLC, suggesting that LC present in the lung may play a role in cellular host defence against aspergilloses.

The in-vivo activity of amphotericin B and itraconazole against a clinical isolate of Aspergillus terreus was determined in a murine model of disseminated aspergillosis. MICs of amphotericin B and itraconazole for the strain, determined by an NCCLS-based technique, were 2 microg/ml and 1 microg/ml, respectively. Mice infected intravenously were treated with either itraconazole (50 or 100 mg/kg/day) or amphotericin B 4.5 mg/kg/day for 10 days. Treatment with both doses of itraconazole significantly prolonged the survival rates compared with those for untreated mice. In comparison, mortality rate and median survival time were identical for mice treated with amphotericin B and for mice given no therapy, indicating that the strain was highly resistant to amphotericin B in this model. Analysis of sterol composition showed that the major sterol was ergosterol. This suggests that amphotericin B resistance was not related to a modified sterol profile.

An animal model of disseminated aspergillosis was used to test the in-vivo activity of itraconazole against four isolates of Aspergillus fumigatus. Two reference isolates of A. fumigatus known to be resistant to itraconazole in vitro and in vivo were used as control isolates, and two new isolates were tested under the same conditions. For each isolate MICs for itraconazole and amphotericin B were determined by an NCCLS-based method. Mice infected intravenously were treated either with itraconazole 100 mg/ kg/day or amphotericin B 4.5 mg/kg/day for 10 days. Amphotericin B showed good in-vivo activity against all four isolates. For one strain, which had a low in-vitro MIC for itraconazole, in-vivo therapy with itraconazole prolonged the survival of mice and reduced fungal burdens in organs compared with untreated controls. In mice infected with a strain with a high MIC of >16 mg/L, itraconazole neither prolonged survival nor reduced fungal load in organs compared with controls. It is concluded that there is a relationship between MIC and treatment outcome in mice for A. fumigatus infection.

A new immunoelectrophoresis system, the Paragon system, was evaluated in three different hospital centres with the aim of improving standardization of the serodiagnosis of human aspergillosis. To select the most efficient antigen, various commercial and home-made antigens were first tested on 19 sera from 19 patients with highly probable aspergillosis. The value measured using the Paragon anti-Aspergillus antibody detection system was then compared with the results obtained by conventional serological diagnostic methods (conventional immunoelectrophoresis, enzyme linked-immunosorbent assay, indirect immunofluorescence): this step was performed using the first 19 sera as well as 16 other sera from 13 patients with suspected aspergillosis. Concordant results were obtained in 28 cases. The discrepancies observed with seven sera were probably related to differences in the nature of the antigens. Paragon immunoelectrophoresis proved to be a practicable technique requiring only a small amount of serum and giving results within a shorter time than competitive methods (24-48 h). Its major drawbacks compared with conventional immunoelectrophoresis are some difficulties in reading, fewer precipitin lines and the relatively high cost of routine analysis.