Cerebral aspergillosis in the era of new antifungals: The CEREALS national cohort study Nationwide CEREbral Aspergillosis Lesional study (CEREALS).

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 ß-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.

Antifungal susceptibility testing practices in mycology laboratories in France, 2018.

A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation.

Multicentre study to determine the Etest epidemiological cut-off values of antifungal drugs in Candida spp. and Aspergillus fumigatus species complex.

OBJECTIVES: To determine the Etest-based epidemiological cut-off values (ECVs) for antifungal agents against the most frequent yeast and Aspergillus fumigatus species isolated in 12 French hospitals. METHODS: For each antifungal agent, the Etest MICs in yeast and A. fumigatus isolates from 12 French laboratories were retrospectively collected from 2004 to 2018. The ECVs were then calculated using the iterative statistical method with a 97.5% cut-off. RESULTS: Forty-eight Etest ECVs were determined for amphotericin B, caspofungin, micafungin, anidulafungin, fluconazole, voriconazole, posaconazole and itraconazole, after pooling and analysing the MICs of 9654 Candida albicans, 2939 Candida glabrata SC, 1458 Candida parapsilosis SC, 1148 Candida tropicalis, 575 Candida krusei, 518 Candida kefyr, 241 Candida lusitaniae, 131 Candida guilliermondii and 1526 Aspergillus fumigatus species complex isolates. These ECVs were 100% concordant (identical or within one two-fold dilution) with the previously reported Etest-based ECVs (when available), and they were concordant in 76.1% of cases with the Clinical and Laboratory Standards Institute ECVs and in 81.6% of cases with the European Committee on Antimicrobial Susceptibility Testing ECVs. CONCLUSIONS: On the basis of these and other previous results, we recommend the determination of method-dependent ECVs. Etest ECVs should not be used instead of breakpoints, but may be useful to identify non-wild-type isolates with potential resistance to antifungal agents, and to indicate that an isolate may not respond as expected to the standard treatment.

Comparison of matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) systems for the identification of moulds in the routine microbiology laboratory.

OBJECTIVES: The purpose of this study was to compare the efficiency of mould identification of two matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) systems - Vitek MS (VMS) and Microflex LT (MLT) - and the MSI application. METHODS: Moulds were collected retrospectively and prospectively to display epidemiological diversity of a microbiology laboratory. All of them were identified via sequencing. Strains were then identified using the VMS v3.0, the MLT, and the MSI software applied on MLT spectra. Rates of correct identifications to the species, to the complex, and to the genus level were compared with the molecular reference standard. RESULTS: A total of 102 isolates were collected. The rate of correct identification to the species level with the MLT was 42.2% (43/102) with a threshold of 1.7 (vs. 16.7% (17/102) with a threshold of 2.0, p < 0.05). The VMS performed better than the MLT with a threshold of 1.7 for species (49.0% (50/102), p 0.33) and complex level identifications (71.6% (73/102) vs. 54.9% (56/102), p < 0.05). However the highest performances were observed when the MLT spectra were analysed via the Mass Spectrometry Identification (MSI) software reaching 90.2% (92/102) of correct identification to the species, 92.2% (94/102) to the species complex and 94.1% (96/102) to the genus level. CONCLUSIONS: The VMS performed better than the MLT for mould identification. However, it remains of utmost importance to expand commercial databases, as performances of the MLT highly improved when using the MSI software and its extended database, reaching far above the VMS system. Thus the VMS could benefit from the use of this online tool.

Aspergillus antibody detection: diagnostic strategy and technical considerations from the Société Française de Mycologie Médicale (French Society for Medical Mycology) expert committee.

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.

Prospective Evaluation of a New Aspergillus IgG Enzyme Immunoassay Kit for Diagnosis of Chronic and Allergic Pulmonary Aspergillosis.

Anti-Aspergillus IgG antibodies are important biomarkers for the diagnosis of chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis (ABPA). We compared the performance of a new commercial enzyme immunoassay (EIA) (Bordier Affinity Products) with that of the Bio-Rad and Virion\Serion EIAs. This assay is novel in its association of two recombinant antigens with somatic and metabolic antigens of Aspergillus fumigatus In a prospective multicenter study, 436 serum samples from 147 patients diagnosed with CPA (136 samples/104 patients) or ABPA (94 samples/43 patients) and from 205 controls (206 samples) were tested. We obtained sensitivities of 97%, 91.7%, and 86.1%, and specificities of 90.3%, 91.3%, and 81.5% for the Bordier, Bio-Rad, and Virion\Serion tests, respectively. The Bordier kit was more sensitive than the Bio-Rad kit (P < 0.01), which was itself more sensitive than the Virion\Serion kit (P = 0.04). The Bordier and Bio-Rad kits had similar specificity (P = 0.8), both higher than that of the Virion\Serion kit (P = 0.02). The area under the receiver operating characteristic (ROC) curves confirmed the superiority of the Bordier kit over the Bio-Rad and the Virion\Serion kits (0.977, 0.951, and 0.897, respectively; P < 0.01 for each comparison). In a subset analysis of 279 serum samples tested with the Bordier and Bio-Rad kits and an in-house immunoprecipitin assay (IPD), the Bordier kit had the highest sensitivity (97.7%), but the IPD tended to be more specific (71.2 and 84.7%, respectively; P = 0.10). The use of recombinant, somatic, and metabolic antigens in a single EIA improved the balance of sensitivity and specificity, resulting in an assay highly suitable for use in the diagnosis of chronic and allergic aspergillosis.

[Internal and external quality controls for Elisa techniques of aspergillosis serodiagnosis: proposals of the group "sérodiagnostic fongique" of the Société française de mycologie médicale]. / Contrôles internes et externes de qualité pour les techniques Elisa de sérodiagnostic aspergillaire: propositions du groupe «sérodiagnostic fongique¼ de la Société française de mycologie médicale.

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.

[Aspergillus serology, from yesterday to today for tomorrow]. / Sérologie aspergillaire, d'hier à aujourd'hui pour demain.

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.

Invasive aspergillosis: an important risk factor on the short- and long-term survival of acute myeloid leukemia (AML) patients.

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.

Toxoplasma gondii: comparison of human CD34+ and monocyte-derived dendritic cells after parasite infection.

Human dendritic cells (DC) obtained in vitro from CD34(+) progenitors (CD34-DC) or blood monocytes (mo-DC) are different DC which may be used in a model of T. gondii infection. We compared the survival, infection rate and cell surface receptor expression of both DC types after living T. gondii tachyzoite infection. CD34-DC appeared less resistant to the parasite than mo-DC. At 48h post-infection, chemokine receptors responsible for DC homing and migration were absent in mo-DC, while down regulation of CCR6 and up regulation of CCR7 was observed in CD34-DC. This result, suggesting migration ability of CD34-DC, was confirmed by in vitro migration experiments against different chemokines. Tachyzoite supernatant, used as chemokine, attracted immature CD34-DC as observed by MIP3alpha, while MIP3beta, as expected, attracted mature CD34-DC. Under similar conditions, no significant difference was noticed between mature or immature mo-DC. These data indicated that CD34-DC represent an alternative model that allows migration assay of infected DC by T. gondii.

Toxoplasma gondii regulates recruitment and migration of human dendritic cells via different soluble secreted factors.

We investigated in vitro the properties of soluble factors produced by Toxoplasma gondii on the recruitment, maturation and migration of human dendritic cells (DC) derived from CD34+ progenitor cells. We used soluble factors including excreted secreted antigens (ESA) produced under various conditions by the virulent type I RH strain (ESA-RH) and the less virulent PRU type II strain (ESA-PRU). Soluble factors of both T. gondii strains appeared to possess a chemokine-like activity that attracted immature DC. This recruitment activity required the presence of functional CCR5 molecules on the cell membrane. Incubation of DC for 24 h with ESA triggered the migration of a large percentage of these cells towards the chemokine MIP-3beta; ESA-PRU was more efficient than ESA-RH. ESA produced in absence of exogenous protein and crude extract did not induce DC migration but retained recruitment activity. These data indicate that recruitment activity and migration-inducing activity are not governed by the same factors. Moreover, incubation of DC for 48 h with ESA did not modify the expression of costimulation or maturation markers (CD83, CD40, CD80, CD86 or HLA-DR), but induced a decrease in CCR6 expression associated with an increased expression of CCR7. Taken together, these results suggest that T. gondii controls recruitment and migration of immature DC by different soluble factors and may induce a dysfunction in the host-specific immune response.

Candida species distribution in bloodstream cultures in Lyon, France, 1998-2001.

In order to determine the types of Candida spp. isolated from bloodstream cultures in Lyon, France, a retrospective study of isolates collected at five different bacteriology laboratories from 1998 to 2001 was conducted. During this period Candida spp. were isolated from 190 patients hospitalized in the internal medicine (32%), hematology (23%) and surgery (23%) wards, and in intensive care units (22%). C. albicans was the leading cause of Candida infection (49.5%), followed by C. glabrata (12.6%) and C. parapsilosis (12.1%). Among the onco-hematology patients, the major cause of candidemia was C. krusei (34%), followed by C. albicans (19%), while these two species were identified in 4% and 59% of patients in the other wards, respectively. In the single onco-hematology ward that was specialized in treating acute myeloid leukemia, 14 C. krusei isolates were identified in this study, which contrasts with the single C. krusei isolate recorded between 1992 and 1996. Since C. krusei has inherent resistance to the antifungal agent fluconazole, prophylactic use of fluconazole in these patients was investigated, but no relationship between these two parameters was found.

Binding of live conidia of Aspergillus fumigatus activates in vitro-generated human Langerhans cells via a lectin of galactomannan specificity.

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.

Evaluation of different commercial ELISA methods for the serodiagnosis of systemic candidosis.

Different commercial enzyme-linked immunsorbent assays (ELISA) were evaluated in a preliminary study for diagnosis of systemic candidosis: Biomerica and Virotech GmbH, which allowed immunoglobulin G detection, and Platelia, which associated total antibody to antigen detection. They were tested with a home-made ELISA and compared with the routine techniques used in the hospital laboratory: indirect immunofluorescence and counter-immunoelectrophoresis. Sera were obtained from patients with probable or proven systemic candidosis (groups 3 and 4, n=8 and n=14, respectively) and from patients without systemic candidosis who were divided into controls (n=10), those hospitalized without Candida isolation (group 1, n=10) and those hospitalized with Candida isolation in a peripheral site (group 2, n=18). The immunoglobulin G ELISAs showed a higher sensitivity associated with lower specificity compared to the indirect immunofluorescence, counter-immunoelectrophoresis and total immunoglobulin ELISAs. Mannan antigen detection showed the highest specificity (78.9%). Its association with the detection of total anti-Candida immunoglobulins was more sensitive than the association of indirect immunofluorescence with counter-immunoelectrophoresis (95.4% versus 59%, respectively) with a specificity of 52.6% (versus 55.2%). Interest in the use of commercial ELISAs, more particularly the Platelia tests, has to be confirmed in a prospective study with follow-up of the patients.

Sterol composition of itraconazole-resistant and itraconazole-susceptible isolates of Aspergillus fumigatus.

Sterol composition of four clinical isolates of Aspergillus fumigatus resistant to itraconazole was determined by gas chromatography--mass spectrometry and compared with that of four susceptible strains. For all strains, the major sterol was ergosterol. Sterol compositions were qualitatively and quantitatively similar for the resistant and susceptible strains. These results suggest that itraconazole resistance is not related, for the strains studied, to alterations in the ergosterol synthesis pathway.