Multicentric Analysis of the Species Distribution and Antifungal Susceptibility of Clinical Isolates from Aspergillus Section Circumdati.

The clinical involvement and antifungal susceptibility of Aspergillus section Circumdati are poorly known. We analyzed 52 isolates, including 48 clinical isolates, belonging to 9 species inside the section Circumdati. The whole section exhibited, by the EUCAST reference method, a poor susceptibility to amphotericin B, but species/series-specific patterns were observed for azole drugs. This underlines the interest in getting an accurate identification inside the section Circumdati to guide the choice of antifungal treatment in clinical practice.

Etest ECVs/ECOFFs for Detection of Resistance in Prevalent and Three Nonprevalent Candida spp. to Triazoles and Amphotericin B and Aspergillus spp. to Caspofungin: Further Assessment of Modal Variability.

Susceptibility testing is an important tool in the clinical setting; its utility is based on the availability of categorical endpoints, breakpoints (BPs), or epidemiological cutoff values (ECVs/ECOFFs). CLSI and EUCAST have developed antifungal susceptibility testing, BPs, and ECVs for some fungal species. Although the concentration gradient strip bioMérieux Etest is useful for routine testing in the clinical laboratory, ECVs are not available for all agent/species; the lack of clinical data precludes development of BPs. We reevaluated and consolidated Etest data points from three previous studies and included new data. We defined ECOFFinder Etest ECVs for three sets of species-agent combinations: fluconazole, posaconazole, and voriconazole and 9 Candida spp.; amphotericin B and 3 nonprevalent Candida spp.; and caspofungin and 4 Aspergillus spp. The total of Etest MICs from 23 laboratories (Europe, the Americas, and South Africa) included (antifungal agent dependent): 17,242 Candida albicans, 244 C. dubliniensis, 5,129 C. glabrata species complex (SC), 275 C. guilliermondii (Meyerozyma guilliermondii), 1,133 C. krusei (Pichia kudriavzevii), 933 C. kefyr (Kluyveromyces marxianus), 519 C. lusitaniae (Clavispora lusitaniae), 2,947 C. parapsilosis SC, 2,214 C. tropicalis, 3,212 Aspergillus fumigatus, 232 A. flavus, 181 A. niger, and 267 A. terreus SC isolates. Triazole MICs for 66 confirmed non-wild-type (non-WT) Candida isolates were available (ERG11 point mutations). Distributions fulfilling CLSI ECV criteria were pooled, and ECOFFinder Etest ECVs were established for triazoles (9 Candida spp.), amphotericin B (3 less-prevalent Candida spp.), and caspofungin (4 Aspergillus spp.). Etest fluconazole ECVs could be good detectors of Candida non-WT isolates (59/61 non-WT, 4 of 6 species).

Comparison of the MICs Obtained by Gradient Concentration Strip and EUCAST Methods for Four Azole Drugs and Amphotericin B against Azole-Susceptible and -Resistant Aspergillus Section Fumigati Clinical Isolates.

Reference methods used to assess the drug susceptibilities of Aspergillus fumigatus isolates consisted of EUCAST and CLSI standardized broth microdilution techniques. Considering the increasing rate and the potential impact on the clinical outcome of azole resistance in A. fumigatus, more suitable techniques for routine testing are needed. The gradient concentration strip (GCS) method has been favorably evaluated for yeast testing. The aim of this study was to compare the CGS test with EUCAST broth microdilution for amphotericin B (AMB), posaconazole (PCZ), itraconazole (ITZ), voriconazole (VRZ), and isavuconazole (ISA). A total of 121 Aspergillus section Fumigati strains were collected, including 24 A. fumigatus sensu stricto strains that were resistant to at least one azole drug. MICs were determined using GCS and EUCAST methods. Essential agreement between the 2 methods was considered when MICs fell within ±1 dilution or ±2 dilutions of the 2-fold dilution scale. Categorical agreement was defined as the percentage of strains classified in the same category (susceptible, intermediate, or resistant) with both methods. Essential agreements with ±1 dilution and ±2 dilutions were 96.7, 93.4, 90.0, 89.3, and 95% and 100, 99.2, 100, 97.5, and 100% for AMB, PCZ, ITZ, VRZ, and ISA, respectively. Categorical agreements were 94.3, 86.1, 89.3, and 88.5% for AMB, PCZ, ITZ, and VRZ, respectively. Detection of resistance was missed with the GCS for one strain (4.1%) for PCZ and for 2 strains (8.3%) for ISA. Determination of ITZ MICs using the GCS allowed the detection of 91.7% of azole-resistant strains. The GCS test appears to be a valuable method for screening azole-resistant A. fumigatus clinical isolates.

Multicentric Analysis of the Species Distribution and Antifungal Susceptibility of Cryptic Isolates from Aspergillus Section Fumigati.

The antifungal susceptibility of Aspergillus cryptic species is poorly known. We assessed 51 isolates, belonging to seven Fumigati cryptic species, by the EUCAST reference method and the concentration gradient strip (CGS) method. Species-specific patterns were observed, with high MICs for azole drugs, except for Aspergillus hiratsukae and Aspergillus tsurutae, and high MICs for amphotericin B for Aspergillus lentulus and Aspergillus udagawae Essential and categorical agreements between EUCAST and CGS results were between 53.3 and 93.3%.

Species Distribution and Comparison between EUCAST and Gradient Concentration Strips Methods for Antifungal Susceptibility Testing of 112 Aspergillus Section Nigri Isolates.

Aspergillus niger, the third species responsible for invasive aspergillosis, has been considered as a homogeneous species until DNA-based identification uncovered many cryptic species. These species have been recently reclassified into the Aspergillus section Nigri However, little is yet known among the section Nigri about the species distribution and the antifungal susceptibility pattern of each cryptic species. A total of 112 clinical isolates collected from 5 teaching hospitals in France and phenotypically identified as A. niger were analyzed. Identification to the species level was carried out by nucleotide sequence analysis. The MICs of itraconazole, voriconazole, posaconazole, isavuconazole, and amphotericin B were determined by both the EUCAST and gradient concentration strip methods. Aspergillus tubingensis (n = 51, 45.5%) and Aspergillus welwitschiae (n = 50, 44.6%) were the most common species while A. niger accounted for only 6.3% (n = 7). The MICs of azole drugs were higher for A. tubingensis than for A. welwitschiae The MIC of amphotericin B was 2 mg/liter or less for all isolates. Importantly, MICs determined by EUCAST showed no correlation with those determined by the gradient concentration strip method, with the latter being lower than the former (Spearman's rank correlation tests ranging from 0.01 to 0.25 depending on the antifungal agent; P > 0.4). In conclusion, A. niger should be considered as a minority species in the section Nigri The differences in MICs between species for different azoles underline the importance of accurate identification. Significant divergences in the determination of MIC between EUCAST and the gradient concentration strip methods require further investigation.

Species Identification and In Vitro Antifungal Susceptibility of Aspergillus terreus Species Complex Clinical Isolates from a French Multicenter Study.

Aspergillus section Terrei is a species complex currently comprised of 14 cryptic species whose prevalence in clinical samples as well as antifungal susceptibility are poorly known. The aims of this study were to investigate A. Terrei clinical isolates at the species level and to perform antifungal susceptibility analyses by reference and commercial methods. Eighty-two clinical A. Terrei isolates were collected from 8 French university hospitals. Molecular identification was performed by sequencing parts of beta-tubulin and calmodulin genes. MICs or minimum effective concentrations (MECs) were determined for 8 antifungal drugs using both EUCAST broth microdilution (BMD) methods and concentration gradient strips (CGS). Among the 79 A. Terrei isolates, A. terreus stricto sensu (n = 61), A. citrinoterreus (n = 13), A. hortai (n = 3), and A. alabamensis (n = 2) were identified. All strains had MICs of ≥1 mg/liter for amphotericin B, except for two isolates (both A. hortai) that had MICs of 0.25 mg/liter. Four A. terreus isolates were resistant to at least one azole drug, including one with pan-azole resistance, yet no mutation in the CYP51A gene was found. All strains had low MECs for the three echinocandins. The essential agreements (EAs) between BMD and CGS were >90%, except for those of amphotericin B (79.7%) and itraconazole (73.4%). Isolates belonging to the A section Terrei identified in clinical samples show wider species diversity beyond the known A. terreus sensu stricto Azole resistance inside the section Terrei is uncommon and is not related to CYP51A mutations here. Finally, CGS is an interesting alternative for routine antifungal susceptibility testing.

Unique case report of a chromomycosis and Listeria in soft tissue and cerebellar abscesses after kidney transplantation.

BACKGROUND: Chromomycosis is a rare mycotic infection encountered in tropical and subtropical regions. The disease presents as a slowly-evolving nodule that can become infected with bacteria. Here, we describe a unique association of abscesses caused by a chromomycosis and Listeria monocytogenes in a kidney transplant recipient, and didactically expose how the appropriate diagnosis was reached. CASE PRESENTATION: A 49-year old male originating from the Caribbean presented a scalp lesion which was surgically removed in his hometown where it was misdiagnosed as a sporotrichosis on histology, 3 years after he received a kidney transplant. He received no additional treatment and the scalp lesion healed. One year later, an abscess of each thigh due to both F. pedrosoi and L. monocytogenes was diagnosed in our institution. A contemporary asymptomatic cerebellar abscess was also found by systematic MRI. An association of amoxicillin and posaconazole allowed a complete cure of the patient without recurring to surgery. Histological slides from the scalp lesion were re-examined in our institution and we retrospectively concluded to a first localisation of the chromomycosis. We discuss the possible pathophysiology of this very unusual association. CONCLUSION: In this case of disseminated listeriosis and chromomycosis, complete cure of the patients could be reached with oral anti-infectious treatment only.

Multicenter Comparison of the Etest and EUCAST Methods for Antifungal Susceptibility Testing of Candida Isolates to Micafungin.

In vitro susceptibility of 933 Candida isolates, from 16 French hospitals, to micafungin was determined using the Etest in each center. All isolates were then sent to a single center for determination of MICs by the EUCAST reference method. Overall essential agreement between the two tests was 98.5% at ±2 log2 dilutions and 90.2% at ±1 log2 dilutions. Categorical agreement was 98.2%. The Etest is a valuable alternative to EUCAST for the routine determination of micafungin MICs in medical mycology laboratories.

In vitro combination of voriconazole and miltefosine against clinically relevant molds.

Invasive infections caused by filamentous fungi are a major threat for immunocompromised patients. Innate/acquired resistance to antifungal drugs might necessitate combination therapies. We assessed the potential combination of voriconazole with miltefosine, an original drug with antifungal activity against 33 clinically relevant mold isolates, including both azole-susceptible and -resistant Aspergillus. Using complete inhibition as an endpoint, interactions were indifferent for 32/33 isolates. An alternative 50% inhibition endpoint showed synergistic interactions for 14/33 isolates. Antagonism was absent.

PCR-based detection of Toxoplasma gondii DNA in blood and ocular samples for diagnosis of ocular toxoplasmosis.

PCR detection of Toxoplasma gondii in blood has been suggested as a possibly efficient method for the diagnosis of ocular toxoplasmosis (OT) and furthermore for genotyping the strain involved in the disease. To assess this hypothesis, we performed PCR with 121 peripheral blood samples from 104 patients showing clinical and/or biological evidence of ocular toxoplasmosis and from 284 (258 patients) controls. We tested 2 different extraction protocols, using either 200 µl (small volume) or 2 ml (large volume) of whole blood. Sensitivity was poor, i.e., 4.1% and 25% for the small- and large-volume extractions, respectively. In comparison, PCR with ocular samples yielded 35.9% sensitivity, while immunoblotting and calculation of the Goldmann-Witmer coefficient yielded 47.6% and 72.3% sensitivities, respectively. Performing these three methods together provided 89.4% sensitivity. Whatever the origin of the sample (ocular or blood), PCR provided higher sensitivity for immunocompromised patients than for their immunocompetent counterparts. Consequently, PCR detection of Toxoplasma gondii in blood samples cannot currently be considered a sufficient tool for the diagnosis of OT, and ocular sampling remains necessary for the biological diagnosis of OT.

Aspergillus fumigatus harbouring the sole Y121F mutation shows decreased susceptibility to voriconazole but maintained susceptibility to itraconazole and posaconazole.

OBJECTIVES: Voriconazole, itraconazole and posaconazole are members of the azole family and widely used for the treatment of aspergillosis. They act by inhibiting the activity of the fungal Cyp51A enzyme. The emergence of environmental azole-resistant Aspergillus fumigatus strains raises major concerns for human health. METHODS: Recently, a new cyp51A-mediated resistance mechanism (namely TR46/Y121F/T289A) was described in clinical samples and patient-frequented environmental sites. In an azole-naive patient, we isolated an A. fumigatus strain that was not susceptible to voriconazole but was susceptible to itraconazole and posaconazole. RESULTS: A molecular analysis indicated a single Y121F substitution without the TR46 or T289A alterations, which to our knowledge has never been reported. Structure modelling and molecular dynamics offered an explanation for the resistance profile consistent with the structural differences between the three azoles. CONCLUSIONS: Taken together, these observations suggest an original mechanism conferring resistance to azoles mediated by cyp51A of environmental origin. This uncommon susceptibility pattern might represent a 'missing link' between the wild-type A. fumigatus and the fully azole-resistant strain harbouring the TR46/Y121F/T289A mutations.

Emergence of echinocandin-resistant Candida spp. in a hospital setting: a consequence of 10 years of increasing use of antifungal therapy?

Since their introduction in the 2000s, echinocandin drugs have become widely used for the treatment and prophylaxis of invasive fungal infections and, notably, invasive candidiasis. Although cases of breakthrough candidiasis in patients receiving echinocandins have been reported, clinical failure during echinocandin treatment due to the acquisition of resistance by a normally susceptible Candida spp. isolate is considered rare. To date, no publications have been published correlating the use of echinocandins and the emergence of echinocandin resistance among Candida species. So, our goal is to report an initial analysis of echinocandin use in relation to the emergence of resistant Candida isolates. We report here a single-centre experience of the emergence of eight resistant isolates belonging to normally susceptible Candida species in six patients receiving echinocandins. We describe the context and analyse the use of echinocandins over the previous decade. For seven of these isolates, we identified FKS gene mutations involved in decreased susceptibility. Seven isolates were obtained in 2011, on the heels of a ten-fold increase in caspofungin use over the preceding decade. In contrast, in 2012, the use of echinocandins decreased in our institution by 19.5 % and, in that year, only one Candida-resistant isolate was detected, despite the stable global epidemiology of invasive candidaemia. This work underlines the necessity of improving the prescription of antifungal drugs. Improvement in the monitoring of strain susceptibility should also be considered in order to better detect the emergence of resistant or non-susceptible yeast strains.

Rapid emergence of echinocandin resistance during Candida kefyr fungemia treatment with caspofungin.

Echinocandin drugs are widely used for the treatment of candidemia. Resistance is considered rare, and only a few cases of breakthrough candidiasis in patients receiving echinocandin have been reported worldwide. We report here for the first time a Candida kefyr isolate that acquired echinocandin resistance very rapidly after the initiation of caspofungin treatment for candidemia. We characterized the FKS gene mutation responsible for the resistance via the comparison of isolates sampled before and during treatment.

Secretome of human bronchial epithelial cells in response to the fungal pathogen Aspergillus fumigatus analyzed by differential in-gel electrophoresis.

BACKGROUND: For years, the analysis of innate responses to the major mold pathogen Aspergillus fumigatus has been restricted to specialized cells, such as professional phagocytes. More recently, the contribution of the airway epithelial barrier has been assessed and studies have shown that it was able to sense and react to the Aspergillus infection, for example, by producing cytokines. METHODS: To further explore the reaction of the respiratory epithelium to the fungus, we analyzed the proteome response of a human bronchial epithelial cell line to Aspergillus infection using difference gel electrophoresis. We studied the protein pattern of BEAS-2B cell culture supernatant after interaction of the cells with Aspergillus during a 15-hour coculture. RESULTS: We found formerly unknown aspects of bronchial cell behavior during Aspergillus infection: bronchial cells are able to develop both cellular defense mechanisms (ie, thioredoxin system activation) and immune reactions (ie, lysosomal degranulation and cathepsin activation) in response to the fungal aggression. CONCLUSIONS: Bronchial epithelial cells appear to be a more important effector of antifungal defense than expected. Degranulation of lysosomal enzymes that might be responsible for both fungal growth inhibition and host cell damage suggests that inductors/inhibitors of these pathways may be potential targets of therapeutic intervention.

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.

Direct genotyping of Toxoplasma gondii in ocular fluid samples from 20 patients with ocular toxoplasmosis: predominance of type II in France.

We report the direct genotyping analysis of Toxoplasma gondii in ocular samples collected from 20 patients, as well as associated clinical and epidemiological data. This work was aimed at better understanding the impact of genotypes of Toxoplasma gondii strains on toxoplasmic retinochoroiditis. For this purpose, we studied the aqueous humor (AH) or vitreous humor (VH) of 20 patients presenting with ocular toxoplasmosis (OT) in 2 hospitals in France. Genetic characterization was obtained with microsatellite markers in a multiplex PCR assay. In contrast to the results of previous studies, we found no association between atypical Toxoplasma gondii genotypes and the occurrence of OT. Considering the local epidemiological data, our OT patients seemed to be infected more frequently by ordinary type II strains found in the environment. In conclusion, direct genotyping of Toxoplasma gondii strains from aqueous or vitreous humor showed a predominance of the type II genotype in ocular toxoplasmosis; this may be due to a high exposure rate of this genotype in humans.

Discrepancies between a new highly sensitive Toxoplasma gondii ELISA assay and other reagents: interest of Toxo IgG Western blot.

Immunodiagnostic assays are commonly used to screen for maternal toxoplasmic seroconversion during pregnancy. The introduction to the market of a new highly sensitive IgG assay, the Elecsys Toxo IgG test, has resulted in discrepancy issues with other immunoassays because of a lack of standardisation. Western blot appears to be a good alternative gold standard to the dye test, as the latter is not routinely available. For the present prospective study, we compared the analytical performances of two immunoassays, Elecsys Toxo IgG (Roche Diagnostics) and Platelia Toxo IgG (Bio-Rad, Marnes la Coquette, France), to Toxo II IgG Western blot (LDBio, Lyon, France) using 231 consecutive sera with low or equivocal IgG titres. Of these 231 sera, 213 presented discrepancies, which showed the importance of a confirmation test. Of the Elecsys Toxo IgG-positive results, 100% were confirmed by the Western blot with a positive threshold of 30 IU/ml for Elecsys; in the equivocal area (1-30 IU/ml), Western blot is negative in 54% of cases. Our results suggest that the lower diagnostic cut-off of Platelia Toxo IgG should be further reduced. Our study indirectly confirms that monitoring, especially for pregnant women, must be done in the same laboratory using the same technique. The ability to diagnose very early seroconversion using Western blot merits further study.

Pneumocystosis: a network survey in the Paris area 2003-2008.

The aims of this network group were to collect epidemiological data of PcP cases in 14 hospitals in the Paris area and to determine the Di-Hydro Pteroate Synthase (DHPS) genotypes, genetic markers for possible sulfamide resistance. From January 1, 2003 to December 31, 2008, 993 (mean 166/year) PcP cases have been reported. Sixty-five percent of patients were HIV-positive. The median count of CD4 lymphocytes was 32/mm(3) (30 in HIV-positive patients, 152 in HIV-negative patients). In HIV-positive patients, PcP revealed the HIV infection in 39%. Among 304 PcP occurring in HIV known infected patients, no prophylaxis was prescribed for 64%; cotrimoxazole prophylaxis had been prescribed to 47 patients but only one of them had the right compliance. In HIV-negative patients (264), corticosteroids were prescribed in 59% and cytotoxic chemotherapies in 34%; 78% did not receive prophylaxis. One hundred sixty nine tumoral pathologies and 116 transplantations were notified. The mortality rate was 16% at day 14 (13% in HIV-positive patients, 26% in HIV-negative patients). Mutations in DHPS genes were detected in 18.5% of samples; 12.5% of patients were infected with several strains. The total annual number of cases has been stable for five years but the proportion of HIV-negative patients increased from 25% to 43%.

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.