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Candida blankii is a recently reported yeast causing rare cases of fungemia. This species presents high minimum inhibitory concentrations (MICs) to fluconazole and echinocandins. We report an atypical C. blankii metacarpophalangeal osteo-articular infection in a patient with Chronic Granulomatous Disease. The patient was initially treated by a combination of voriconazole and 5-fluorocytosine (5-FC). However, the treatment was subsequently changed to posaconazole and then to a combination of caspofungin and 5-FC.2012 Elsevier Ltd. All rights reserved.

ABSTRACT Mucormycosis is a severe and emerging invasive fungal infection associated with high mortality rates. Early diagnosis is crucial for initiating specific antifungal treatment, with molecular tools currently representing the most efficient diagnostic approach. Presently, a standardized in-house real-time PCR method is widely employed for diagnosing mucormycosis. Our study aimed to evaluate the agreement for the Mucorales DNA detection between two commercial real-time PCR assays—the Fungiplex Mucorales Real-Time PCR Kit and the MycoGENIE Aspergillus-Mucorales spp. Real-Time PCR Kit—in comparison with the in-house PCR. We retrospectively analyzed 58 samples previously identified as positive for Mucorales using the in-house PCR. These samples, obtained from 22 patients with proven or probable mucormycosis, were tested with both commercial kits. Additionally, samples from 40 patients without mucormycosis served as negative controls. Our findings revealed that the MycoGENIE Kit demonstrated superior performance in detecting Mucorales DNA in samples identified as positive by the in-house PCR. Notably, we observed minimal variability in cycle threshold (CT) values when comparing the results of the MycoGENIE Kit with those of the in-house PCR, with an average difference of 1.8 cycles. In contrast, the Fungiplex Kit exhibited a larger discrepancy in CT values compared to the in-house PCR, with an average difference of 4.1 cycles. The MycoGENIE Kit exhibited very good agreement (kappa of 0.82) with the in-house PCR for detecting Mucorales DNA across various sample types. These findings are important for the choice of kits that could be used to diagnose mucormycosis in clinical microbiology laboratories.IMPORTANCEEarly diagnosis of mucormycosis is crucial for initiating effective treatment. The detection of Mucorales DNA by PCR in serum has revolutionized the diagnosis of this infection. However, the use of in-house methods can be time consuming. The availability of a commercial kit eliminates the need for in-house assay development, reducing laboratory workload and ensuring consistent performance across different healthcare settings. Currently, there are several commercial assays available, but many have limited evaluation. In this study, we compared two commercial kits and found that the MycoGENIE Kit offers a promising alternative to the in-house method.

Transmission of dermatophytes, especially Trichophyton mentagrophytes genotype VII, during sexual intercourse has been recently reported. We report 13 such cases in France. All patients were male; 12 were men who have sex with men. Our findings suggest sexual transmission of this pathogen within a specific population, men who have sex with men.

Invasive fungal infections have recently been recognized by the WHO as a major health, epidemiological, and economic issue. Their high mortality rates and the emergence of drug resistance have driven the development of new molecules, including olorofim, an antifungal belonging to a new family of compounds, the orotomides. A review was conducted on the PubMed database and the ClinicalTrials.gov website to summarize the microbiological profile of olorofim and its role in the treatment of filamentous fungal infections. Twenty-four articles were included from the search and divided into two groups: an “in vitro” group focusing on minimum inhibitory concentration (MIC) results for various fungi and an “in vivo” group evaluating the pharmacokinetics (PK), pharmacodynamics (PD), efficacy, and tolerability of olorofim in animal models of fungal infection and in humans. Olorofim demonstrated in vitro and in vivo activity against numerous filamentous fungi, including azole-resistant Aspergillus fumigatus, various dermatophytes, and endemic and dimorphic fungi. in vitro results showed higher MICs for certain Fusarium species and dematiaceous fungi Alternaria alternata and Exophiala dermatitidis; further in vivo studies are needed. Published PK-PD data in humans are limited. The results of the ongoing phase III clinical trial are eagerly awaited to evaluate olorofim’s clinical impact.

(1) Background: Aspergillus flavus is a cosmopolitan mold with medical, veterinary, and agronomic concerns. Its morphological similarity to other cryptic species of the Flavi section requires molecular identification techniques that are not routinely performed. For clinical isolates of Aspergillus section Flavi, we present the molecular identification, susceptibility to six antifungal agents, and clinical context of source patients. (2) Methods: One hundred forty fungal clinical isolates were included in the study. These isolates, recovered over a 15-year period (2001–2015), were identified based on their morphological characteristics as belonging to section Flavi. After the subculture, sequencing of a part of the β-tubulin and calmodulin genes was performed, and resistance to azole antifungals was screened on agar plates containing itraconazole and voriconazole. Minimum inhibitory concentrations were determined for 120 isolates by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. (3) Results: Partial β-tubulin and calmodulin sequences analysis showed that 138/140 isolates were A. flavus sensu stricto, 1 isolate was A. parasiticus/sojae, and 1 was A. nomiae. Many of the isolates came from samples collected in the context of respiratory tract colonization. Among probable or proven aspergillosis, respiratory infections were the most frequent, followed by ENT infections. Antifungal susceptibility testing was available for isolates (n = 120, all A. flavus ss) from one hospital. The MIC range (geometric mean MIC) in mg/L was 0.5–8 (0.77), 0.5–8 (1.03), 0.125–2 (0.25), 0.03–2 (0.22), 0.25–8 (1.91), and 0.03–0.125 (0.061) for voriconazole, isavuconazole, itraconazole, posaconazole, amphotericin B, and caspofungin, respectively. Two (1.67%) isolates showed resistance to isavuconazole according to current EUCAST breakpoints with MICs at 8 mg/L for isavuconazole and voriconazole. One of these two isolates was also resistant to itraconazole with MIC at 2 mg/L. (4) Conclusions: The present characterization of a large collection of Aspergillus belonging to the Flavi section confirmed that A. flavus ss is the predominant species. It is mainly implicated in respiratory and ENT infections. The emergence of resistance highlights the need to perform susceptibility tests on section Flavi isolates.

Extensive dermatophytosis caused by terbinafine-resistant Trichophyton indotineae harboring Phe397Leu and Leu393Ser substitutions in the squalene epoxidase enzyme was diagnosed in France. Analysis of internal transcribed spacer sequences revealed the wide spread of this species in Asia and Europe. Detection of T. indotineae in animals suggests their possible role as reservoirs.

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.

The Antifungal Susceptibility Testing method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST-AFST) is a reference technique for the determination of the Minimum Inhibitory Concentration (MIC) of antifungals for Aspergillus fumigatus. However, it is time-consuming and requires expertise. Micronaut-AM (M-AM) is a fast, simple, time-saving, and ready-to-use new colorimetric method using an indicator (resazurin) to facilitate the visual reading. The aim of this retrospective study was to evaluate the performance of the M-AM system and compare it with the EUCAST broth microdilution reference method to determine the susceptibility of 77 A. fumigatus clinical strains to amphotericin B, itraconazole, voriconazole, and posaconazole. Overall, the essential agreements within ±2 dilutions were 100%, 62%, 58%, and 30% and the categorical agreements were 100%, 97%, 91%, and 87% for amphotericin B, itraconazole, voriconazole, and posaconazole, respectively. No categorical discrepancy was found for amphotericin B, but several categorical discordances were observed with azole antifungals. However, only 2 of the 16 azole-resistant strains confirmed by the cyp51A sequencing would have been misclassified by M-AM. The use of M-AM is probably suitable for the determination of the MICs of amphotericin B, but further evaluations are needed to confirm its usefulness for the determination of the MICs of azoles for A. fumigatus.

Abstract The in vitro interactions of isavuconazole with colistin were evaluated against 15 clinical Candida auris isolates by a microdilution checkerboard technique based on the EUCAST reference method for antifungal susceptibility testing and by agar diffusion using isavuconazole gradient concentration strips with or without colistin incorporated RPMI agar. Interpretation of the checkerboard results was done by the fractional inhibitory concentration index and by response surface analysis based on the Bliss model. By checkerboard, combination was synergistic for 93% of the isolates when interpretation of the data was done by fractional inhibitory concentration index, and for 80% of the isolates by response surface analysis interpretation. By agar diffusion test, although all MICs in combination decreased compared to isavuconazole alone, only 13% of the isolates met the definition of synergy. Essential agreement of EUCAST and gradient concentration strip MICs at +/− 2 log2 dilutions was 93.3%. Antagonistic interactions were never observed for any technique or interpretation model used.

Interactions of isavuconazole and colistin were evaluated against 57 common Candida strains belonging to the species Candida albicans (n = 10), Candida glabrata (n = 10), Candida kefyr (n = 8), Candida krusei (n = 10), Candida parapsilosis (n = 9), and Candida tropicalis (n = 10) by a broth microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference methodology for antifungal susceptibility testing. Results were analyzed with the fractional inhibitory concentration index and by the response surface analysis. Interpretation by the fractional inhibitory concentration index showed synergy for 50%, 80%, 90%, and 90% of the C. kefyr, C. krusei, C. glabrata, and C. tropicalis strains, respectively. Combination of isavuconazole with colistin against C. albicans and C. parapsilosis exhibited only indifference for 100% and 90% of the strains, respectively. The results were confirmed by response surface analysis for all species except for C. glabrata, for which an indifferent interaction was found for the majority of strains. Antagonistic interaction was never seen regardless of the interpretation model was used.

Introduction: Candida parapsilosis (C. parapsilosis) is a common non-albicans Candida species ranked as the second common cause of bloodstream infections. Azole resistance and elevated echinocandin MICs have been reported for these fungi. This study was conducted to determine the interactions between azoles and echinocandins against C. parapsilosis species complex. Materials and methods: Fifteen fluconazole-resistant clinical isolates of C. parapsilosis complex were included: C. parapsilosis sensu stricto (n = 7), C. orthopsilosis (n = 5) and C. metapsilosis (n = 3). The activity of azoles (fluconazole, itraconazole) and echinocandins (anidulafungin, micafungin) alone and in combination was determined using checkerboard broth microdilution. The results were determined based on the fractional inhibitory concentration index (FICI). Results: In vitro combination of fluconazole with anidulafungin was found to be synergistic (FICI 0.07–0.37) and decreased the MIC range from 4–64 μg/mL to 0.5–16 μg/mL for fluconazole and from 2–8 μg/mL to 0.125–1 μg/mL for anidulafungin. Similarly, interactions of fluconazole with micafungin (FICI 0.25–0.5), itraconazole with anidulafungin (FICI 0.15–0.37) and itraconazole with micafungin (FICI 0.09–0.37) were synergistic. Conclusion: The combination of fluconazole and itraconazole with either anidulafungin or micafungin demonstrated synergistic interactions against C. parapsilosis species complex, especially against isolates with elevated MIC values. However, the use of these combinations in clinical practice and the clinical relevance of in vitro combination results remain unclear.

Background and Purpose: Emergence and development of antifungal drug resistance in Candida species constitute a serious concern. Candida auris as an emerging multidrug-resistant fungus is the most important public health threat with high levels of mortality and morbidity. Almost all C. auris isolates are resistant to fluconazole, and there have been reports of elevated minimum inhibitory concentrations (MICs) to amphotericin B and echinocandins. To overcome the growing challenge of antifungal resistance, a valuable alternative option would be the use of drug combination. Materials and Methods: The present study evaluated the in vitro combination of nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, diclofenac and aspirin with fluconazole against fluconazole-resistant C. auris in comparison to other fluconazole-resistant Candida species, including C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei originating from patients with candidiasis. Results: The MIC ranges of fluconazole-ibuprofen and fluconazole-diclofenac decreased from 32-256 to 32-128 and 16-256 μg/ml, respectively and remained the same for fluconazole-aspirin against C. auris. However, the combination of fluconazole with ibuprofen resulted in a synergistic effect for 5 strains, including C. albicans (n=2), C. tropicalis (n=1), C. glabrata (n=1), and C. krusei (n=1), by decreasing the MIC of fluconazole by 2-3 log2 dilutions. Conclusion: Although the interaction of NSAIDs with fluconazole was not synergistic against fluconazole-resistant C. auris isolates, no antagonism was observed for any combinations. Therefore, combination with newer azole agents needs to be conducted.

Paecilomyces spp. are emerging fungal pathogens, where Paecilomyces lilacinus and Paecilomyces variotii are the most reported species. Taxonomic and phylogenetic revisions in this genus have shown that P. variotii represents a species complex, whereas P. lilacinus is related to another genus called Purpureocillium. The aims of this study were to identify clinical isolates of Paecilomyces spp. at the species level, and to determine their antifungal susceptibility profiles. 70 clinical Paecilomyces spp. isolates were identified by MALDI-TOF Mass Spectrometry (MS) and by multilocus rDNA genes sequencing including ITS and the D1/D2 genes. Among the 70 Paecilomyces spp. isolates, 28 were identified as P. lilacinum, 26 as P. variotii stricto sensu, and 16 as P. maximus. For antifungal susceptibility testing, Minimal Inhibitory Concentrations (MICs) or Minimal Effective Concentrations (MECs) were determined for 8 antifungals. All P. lilacinum isolates had high MICs and MECs of amphotericin B and echinocandins, respectively, unlike P. variotii and P. maximus. For azole drugs, MICs were molecule- and species- dependent. The differences in in vitro susceptibility to antifungals underline the importance of accurate species identification. The MALDI–TOF MS can be a good alternative in routine laboratory to ensure fast identification of Paecilomyces spp. and P. lilacinum.

Abstract Background Liver transplant recipients are at high risk of developing invasive aspergillosis and in particular by Aspergillus fumigatus which is the most commonly encountered species in this population. Other non-fumigatus Aspergillus species with reduced susceptibility to antifungal drugs can also be involved. Accurate identification associated to antifungal susceptibility testing is essential for therapy adjustment. We report a case of invasive pulmonary aspergillosis due to Aspergillus pseudodeflectus in a liver transplant recipient. To our knowledge, this is the first reported case of invasive aspergillosis due to this species with a reduced susceptibility to azoles. Case presentation A 64 year-old woman with drug-induced fulminant hepatitis underwent liver transplantation. Prophylactic treatment with caspofungin was introduced due to aspergillosis risk factors consisting in hemodialysis and fulminant hepatitis. Six weeks after transplantation, CT scan showed a right pulmonary opacity associated with an increase of galactomannan (index 5.4). Culture of BAL grew with several colonies of Aspergillus sp. The diagnosis of invasive aspergillosis was probable according to the EORTC criteria. The antifungal susceptibility tests (Etest®) revealed low MICs to echinocandins and amphotericin B) but high MICs to azoles. After these results, voriconazole was switched to liposomal amphotericin B. The patient died one month after diagnosis from a refractory septic shock with multiple organ failure. A molecular identification of isolate, based on partial β-tubulin and calmodulin genes, was performed and identified A. pseudodeflectus. Conclusions Our case raises the question of pathogenicity of this species, which belongs to Aspergillus section Usti and is genetically and morphologically very close to Aspergillus calidoustus that was previously reported in human transplant recipients.

In vitro interactions of broad-spectrum azole isavuconazole with flavonoid isoquercitrin were evaluated by a broth microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference methodology for antifungal susceptibility testing against 60 Candida strains belonging to the species Candida albicans (n = 10), Candida glabrata (n = 30), Candida kefyr (n = 6), Candida krusei (n = 5), Candida parapsilosis (n = 4), and Candida tropicalis (n = 5). The results were analyzed with the fractional inhibitory concentration index and by response surface analysis based on the Bliss model. Synergy was found for all C. glabrata strains, when the results were interpreted by the fractional inhibitory concentration index, and for 60% of the strains when response surface analysis was used. Interaction for all other species was indifferent for all strains tested, whatever interpretation model used. Importantly, antagonistic interaction was never observed.

Mucormycosis is a potentially fatal infection that presents in different clinical forms and occurs in patients with various risk factors. Recently, the COVID-19 epidemic has been responsible for an increase in the incidence of mucormycosis, particularly in India. As with other invasive filamentous fungal infections, there are no specific clinical or radiological signs, and we have fewer diagnostic tools available than for other invasive fungal infections. Therefore, the diagnosis of Mucormycosis remains difficult. Nevertheless, for optimal management, early and accurate diagnosis is important. According to the latest recommendations, diagnosis is based on direct examination of clinical specimens, and/or histopathology, and culture. There are also molecular tools for direct detection from clinical specimens, but these techniques are moderately recommended. The main problems with these molecular techniques are that, until now, they were not very well standardized; there was a great heterogeneity of DNA targets and methods, which resulted in variable sensitivity. It is in this field that most advances have been made in the last two years. Indeed, recent studies have evaluated the performance and kinetics of Mucorales qPCR in serum and have shown good sensitivity and specificity. Large inter-laboratory evaluations of qPCR in serum have also been performed and have demonstrated good qualitative and quantitative reproducibility. These new results suggest the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis. One way to achieve better reproducibility could be to use commercial methods. Currently, there are at least three commercial qPCRs for Mucorales (MucorGenius from PathoNostics, MycoGenie from Ademtech, and Fungiplex from Bruker) that can be used to test serum, respiratory samples, or biopsies. However, to date, there has been little evaluation of these methods. Overall, Mucorales PCR in tissue samples, in respiratory samples, and in serum is promising and its addition as a diagnostic tool in the definitions of invasive mucormycosis should be discussed.

Introduction: The complexity of biofilms constitutes a therapeutic challenge and the antimicrobial susceptibility of fungal-bacterial biofilms remains poorly studied. The filamentous fungus Aspergillus fumigatus (Af) and the Gram-negative bacillus Stenotrophomonas maltophilia (Sm) can form biofilms and can be co-isolated from the airways of cystic fibrosis (CF) patients. We previously developed an in vitro biofilm model which highlighted the antibiosis effect of Sm on Af, which was dependent on the bacterial fitness. The aim of the present study was to investigate the in vitro susceptibility of Af and Sm in mono- or polymicrobial biofilms to five antimicrobial agents alone and in two-drug combinations.Methods: Af and Sm clinical reference strains and two strains from CF sputa were tested through a planktonic and biofilm approaches. Af, Sm, or Af-Sm susceptibilities to amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), levofloxacin (LVX), and rifampicin (RFN) were evaluated by conventional planktonic techniques, crystal violet, XTT, qPCR, and viable plate count.Results: Af planktonic cells and biofilms in formation were more susceptible to AMB, ITC, and VRC than Af mature biofilms. Af mature biofilms were susceptible to AMB, but not to ITC and VRC. Based on viable plate count, a lower concentration of LVX and RFN was required to reduce Sm cell numbers on biofilms in formation compared with mature biofilms. The antibiosis effect of Sm on Af growth was more pronounced for the association of CF strains that exhibited a higher fitness than the reference strains. In Af-Sm biofilms, the fungal susceptibility to AMB was increased compared with Af biofilms. In contrast, the bacterial susceptibility to LVX decreased in Af-Sm biofilms and was fungal biomass-dependent. The combination of AMB (64 μg/mL) with LVX or RFN (4 μg/mL) was efficient to impair Af and Sm growth in the polymicrobial biofilm.Conclusion: Sm increased the Af susceptibility to AMB, whereas Af protected Sm from LVX. Interactions between Af and Sm within biofilms modulate susceptibility to antimicrobial agents, opening the way to new antimicrobial strategies in CF patients.

When Candida albicans and Candida dubliniensis isolates were tested for susceptibility to fluconazole and echinocandins using either EUCAST or Etest methods, differential patterns of growth were observed, independently of the methods used. For C. albicans, a trailing phenomenon (incomplete growth inhibition at supra-MICs) was observed with fluconazole in 90% and 93.3% for EUCAST and Etest, respectively, but not with echinocandins (50% for EUCAST and >86% for Etest). This suggests that the pathways involved in the trailing effect might be different between these two related species. Furthermore, clinical microbiologists must be aware of these species-specific patterns for a reliable MIC determination.

In recent years, we have moved from the sporadic description of terbinafine-resistant (TerR) Trichophyton spp. isolates to the Indian outbreak due to T. indotineae. Population flows have spread TerR worldwide, altering local epidemiology. We conducted a prospective multicentric study to determine the relative frequency of TerR isolates in France (Paris area) and of the newly introduced T. indotineae species. TerR isolates were screened by the terbinafine-containing-agar-medium (TCAM) method and confirmed by EUCAST. Sequencing methods were used to identify isolates to the species/genotype level and to analyze substitutions in the squalene epoxidase gene (SQLE). In total, 3 isolates out of 580 (T. rubrumn = 1; T. interdigitalen = 1; T. indotineaen = 1) grew on TCAM, showed terbinafine resistance by EUCAST and harbored the Phe397Leu (n = 2) or Leu393Ser (n = 1) substitution in the SQLE. ITS-sequencing of isolates of the T. mentagrophytes/interdigitale complex (n = 125) revealed a relative frequency of 4.8% for T. indotineae and the presence of T. mentagrophytes genotype VII. Despite the detection of terbinafine resistance, isolates from this complex remained susceptible to itraconazole, voriconazole and amorolfine. Terbinafine resistance is present in France and the dermatophyte epidemiology is changing. Efficient systems must be implemented to survey the evolution of newly introduced species and to identify TerR isolates.

The in vitro interaction of amphotericin B in combination with colistin was evaluated against a total of 86 strains comprising of 47 Candida species (10 Candida albicans, 15 Candida auris, five Candida glabrata, three Candida kefyr, five Candida krusei, four Candida parapsilosis and five Candida tropicalis), 29 Aspergillus species (five Aspergillus flavus, 10 Aspergillus fumigatus, four Aspergillus nidulans, five Aspergillus niger, and five Aspergillus terreus), and 10 Rhizopus species (seven Rhizopus arrhizus, one Rhizopus delemar and two Rhizopus microsporus) strains. For the determination of the interaction, a microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference method for antifungal susceptibility testing was used. Results of the checkerboard technique were evaluated by the fractional inhibitory concentration index (FICI) based on the Loewe additivity model for all isolates. Different inhibition endpoints were used to capture both the interaction at MIC and sub-MIC levels. Additionally, checkerboard technique results for Candida species were evaluated by response surface analysis based on the Bliss independence model. Against common Candida species, the combination was synergistic for 75% of the strains by FICI and for 66% of the strains by response surface analysis. For C. tropicalis, the interaction was antagonistic for three isolates by FICI, but antagonism was not confirmed by response surface analysis. Interestingly, synergistic and antagonistic FICIs were simultaneously present on checkboard microplates of all three strains. Against C. auris the combination was synergistic for 73% of the strains by response surface analysis and for 33% of the strains by FICI. This discrepancy could be related to the insensitivity of the FICI to detect weak interactions. Interaction for all other strains was indifferent. For Aspergillus and Rhizopus species combination exhibited only indifferent interactions against all tested strains.

Aspergillus fumigatus is an environmental filamentous fungus responsible for life-threatening infections in humans and animals. Azoles are the first-line treatment for aspergillosis, but in recent years, the emergence of azole resistance in A. fumigatus has changed treatment recommendations. The objective of this study was to evaluate the efficacy of voriconazole (VRZ) in a Galleria mellonella model of invasive infection due to azole-susceptible or azole-resistant A. fumigatus isolates. We also sought to describe the pharmacokinetics of VRZ in the G. mellonella model. G. mellonella larvae were infected with conidial suspensions of azole-susceptible and azole-resistant isolates of A. fumigatus. Mortality curves were used to calculate the lethal dose. Assessment of the efficacy of VRZ or amphotericin B (AMB) treatment was based on mortality in the lethal model and histopathologic lesions. The pharmacokinetics of VRZ were determined in larval hemolymph. Invasive fungal infection was obtained after conidial inoculation. A dose-dependent reduction in mortality was observed after antifungal treatment with AMB and VRZ. VRZ was more effective at treating larvae inoculated with azole-susceptible A. fumigatus isolates than larvae inoculated with azole-resistant isolates. The concentration of VRZ was maximal at the beginning of treatment and gradually decreased in the hemolymph to reach a Cmin (24 h) between 0.11 and 11.30 mg/L, depending on the dose. In conclusion, G. mellonella is a suitable model for testing the efficacy of antifungal agents against A. fumigatus.

Fungal ball (FB) rhinosinusitis (RS) is the main type of non-invasive fungal RS. Despite positive direct examination (DE) of biopsies, culture remains negative in more than 60% of cases. The aim of the study was to evaluate the performance/efficacy of targeted metagenomics (TM) to analyze microbiota and mycobiota in FB and find microbial associations. Forty-five sinus biopsies from patients who underwent surgery for chronic RS were included. After DE and culture, DNA was extracted, then fungal ITS1–ITS2 and bacterial V3–V4 16S rDNA loci were sequenced (MiSeqTM Illumina). Operational taxonomic units (OTUs) were defined via QIIME and assigned to SILVA (16S) and UNITE (ITS) databases. Statistical analyses were performed using SHAMAN. Thirty-eight patients had FB and seven had non-fungal rhinosinusitis (NFRS). DE and culture of FB were positive for fungi in 97.3 and 31.6% of patients, respectively. TM analysis of the 38 FB yielded more than one fungal genus in 100% of cases, with Aspergillus in 89.5% (34/38). Haemophilus was over-represented in FB with >1000 reads/sample in 47.3% (18/38) compared to NFRS (p < 0.001). TM allowed fungal identification in biopsies with negative culture. Haemophilus was associated with FB. Pathogenesis could result from fungi–bacteria interactions in a mixed biofilm-like structure.

Many thanks to all contributors to the Special Issue on “Antifungal Agents Recently Approved or Under Development (Current Knowledge and Future Perspectives)” [...]

Systemic fungal infections are associated with high mortality rates despite adequate treatment. Moreover, acquired resistance to antifungals is increasing, which further complicates the therapeutic management. One strategy to overcome antifungal resistance is to use antifungal combinations. In vitro, several techniques are used to assess drug interactions, such as the broth microdilution checkerboard, agar-diffusion methods, and time-kill curves. Currently, the most widely used technique is the checkerboard method. The aim of all these techniques is to determine if the interaction between antifungal agents is synergistic, indifferent, or antagonistic. However, the interpretation of the results remains difficult. Several methods of analysis can be used, based on different theories. The most commonly used method is the calculation of the fractional inhibitory concentration index. Determination of the usefulness of combination treatments in patients needs well-conducted clinical trials, which are difficult. It is therefore important to study antifungal combinations in vivo, in experimental animal models of fungal infections. Although mammalian models have mostly been used, new alternative animal models in invertebrates look promising. To evaluate the antifungal efficacy, the most commonly used criteria are the mortality rate and the fungal load in the target organs.

Invasive candidiasis remains one of the most prevalent systemic mycoses, and several studies have documented the presence of mixed yeast (MY) infections. Here, we describe the epidemiology, clinical, and microbiological characteristics of MY infections causing invasive candidiasis in a multicenter prospective study. Thirty-four centers from 14 countries participated. Samples were collected in each center between April to September 2018, and they were sent to a reference center to confirm identification by sequencing methods and to perform antifungal susceptibility testing, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A total of 6895 yeast cultures were identified and MY occurred in 150 cases (2.2%). Europe accounted for the highest number of centers, with an overall MY rate of 4.2% (118 out of 2840 yeast cultures). Of 122 MY cases, the most frequent combinations were Candida albicans/C. glabrata (42, 34.4%), C. albicans/C. parapsilosis (17, 14%), and C. glabrata/C. tropicalis (8, 6.5%). All Candida isolates were susceptible to amphotericin B, 6.4% were fluconazole-resistant, and two isolates (1.6%) were echinocandin-resistant. Accurate identification of the species involved in MY infections is essential to guide treatment decisions.

The in vitro interactions of isavuconazole in combination with colistin were evaluated against 55 clinical Aspergillus species isolates belonging to the five most important species (Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus) responsible for human aspergillosis by a microdilution checkerboard technique based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference method for antifungal susceptibility testing. Selected isolates (A. nidulans, n = 10; A. niger, n = 15) were additionally evaluated by an agar diffusion assay using isavuconazole gradient concentration strips with or without colistin incorporated Roswell Parc Memorial Institute (RPMI) agar. Interpretation of the checkerboard results was done by the fractional inhibitory concentration index. Using the checkerboard method, combination isavuconazole–colistin was synergistic for 100% of the 15 A. nidulans isolates and for 60% of the 20 A. niger isolates. No interactions were found for any of the other isolates. By agar diffusion assay, minimal inhibitory concentrations (MICs) in combination decreased compared to isavuconazole alone for 92% of the isolates. No interactions were found for any A. nidulans isolates, but synergy was observed for 40% of the A. niger isolates. A poor essential agreement of EUCAST and gradient concentration strip MICs at ± 2 log2 dilutions with 0% was obtained. Antagonistic interactions were never observed regardless of the technique used.

The interaction of isavuconazole with immunosuppressors (tacrolimus, cyclosporin A, or sirolimus) against 30 Aspergillus isolates belonging to the most common species responsible for invasive aspergillosis in humans (Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus) was evaluated in vitro by a microdilution checkerboard technique based on the EUCAST reference method for antifungal susceptibility testing. The interpretation of the results was performed based on the fractional inhibitory concentration index. The combination of isavuconazole with tacrolimus, cyclosporin A, or sirolimus, was synergistic for 56, 20, or 10% of the isolates, respectively. Interestingly synergy of the combination of isavuconazole with tacrolimus was also achieved for the majority of azole-resistant isolates of A. fumigatus, and for all A. niger isolates with isavuconazole minimal inhibitory concentrations ≥ 8 µg/mL. Antagonistic interactions were never observed for any combination tested.

The treatment of invasive fungal infections remains challenging and the emergence of new fungal pathogens as well as the development of resistance to the main antifungal drugs highlight the need for novel therapeutic strategies. Although in vitro antifungal susceptibility testing has come of age, the proper evaluation of therapeutic efficacy of current or new antifungals is dependent on the use of animal models. Mammalian models, particularly using rodents, are the cornerstone for evaluation of antifungal efficacy, but are limited by increased costs and ethical considerations. To circumvent these limitations, alternative invertebrate models, such as Galleria mellonella, have been developed. Larvae of G. mellonella have been widely used for testing virulence of fungi and more recently have proven useful for evaluation of antifungal efficacy. This model is suitable for infection by different fungal pathogens including yeasts (Candida, Cryptococcus, Trichosporon) and filamentous fungi (Aspergillus, Mucorales). Antifungal efficacy may be easily estimated by fungal burden or mortality rate in infected and treated larvae. The aim of the present review is to summarize the actual data about the use of G. mellonella for testing the in vivo efficacy of licensed antifungal drugs, new drugs, and combination therapies.

Introduction:Aspergillus fumigatus (Af) and Stenotrophomonas maltophilia (Sm) are pathogenic microorganisms, which coexist in the respiratory tract of cystic fibrosis (CF) patients. We recently developed an in vitro model of mixed biofilm associating Af ATCC 13073-GFP (Af13073) and Sm ATCC 13637 (Sm13637) and described an antibiosis effect. The present study aim was to assess the antibiosis of Sm on Af using different strains and to analyze the potential synergistic virulence of these strains in an in vivo Galleria mellonella model.Methods: The effect of Sm13637 was evaluated on eight Af strains and the effect of nine Sm strains was evaluated on Af13073. The strains originated from clinical cases (human and animal) and from environment. Fungal and bacterial inocula were simultaneously inoculated to initiate mixed biofilm formation. Fungal growth inhibition was analyzed by qPCR and CLSM and the fungal cell wall modifications by TEM analysis. The virulence of different Sm strains was assessed in association with Af in G. mellonella larvae.Results: All strains of Af and Sm were able to produce single and mixed biofilms. The antibiosis effect of Sm13637 was similar whatever the Af strain tested. On the other hand, the antibiosis effect of Sm strains was bacterial-fitness and strain dependent. One strain (1/9) originated from animal clinical case was never able to induce an antibiosis, even with high bacterial concentration. In the G. mellonella model, co-inoculation with Sm13637 and Af13073 showed synergism since the mortality was 50%, i.e., more than the summed virulence of both.Conclusion: Human clinical strains of Sm yielded in higher antibiosis effect on Af and in a thinner mixed biofilm, probably due to an adaptive effect of these strains. Further research covering Af increased wall thickness in the presence of Sm strains, and its correlation with modified antifungal susceptibility is encouraged in patients with chronic respiratory infections by these 2 microorganisms.

Mucormycosis is a life-threatening infection, occurring mainly in immunocompromised patients, but also in immunocompetent patients after traumatic injuries [...]

Antifungal susceptibility testing is an important tool for managing patients with invasive fungal infections, as well as for epidemiological surveillance of emerging resistance. For routine testing in clinical microbiology laboratories, ready-to-use commercial methods are more practical than homemade reference techniques. Among commercially available methods, the concentration gradient Etest strip technique is widely used. It combines an agar-based diffusion method with a dilution method that determinates a minimal inhibitory concentration (MIC) in µg/mL. Many studies have evaluated the agreement between the gradient strip method and the reference methods for both yeasts and filamentous fungi. This agreement has been variable depending on the antifungal, the species, and the incubation time. It has also been shown that the gradient strip method could be a valuable alternative for detection of emerging resistance (non-wild-type isolates) as Etest epidemiological cutoff values have been recently defined for several drug-species combinations. Furthermore, the Etest could be useful for direct antifungal susceptibility testing on blood samples and basic research studies (e.g., the evaluation of the in vitro activity of antifungal combinations). This review summarizes the available data on the performance and potential use of the gradient strip method.

More than 20 different species of Mucorales can be responsible for human mucormycosis. Accurate identification to the species level is important. The morphological identification of Mucorales is not reliable, and the currently recommended identification standard is the molecular technique of sequencing the internal transcribed spacer regions. Nevertheless, matrix-assisted laser desorption ionization time-of-flight mass spectrometry has been shown to be an accurate alternative for the identification of bacteria, yeasts, and even filamentous fungi. Therefore, 38 Mucorales isolates, belonging to 12 different species or varieties, mainly from international collections, including 10 type or neo-type strains previously identified by molecular methods, were used to evaluate the usefulness of matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the identification of human pathogenic Mucorales to the species level. One to three reference strains for each species were used to create a database of main spectrum profiles, and the remaining isolates were used as test isolates. A minimum of 10 spectra was used to build the main spectrum profile of each database strain. Interspecies discrimination for all the isolates, including species belonging to the same genus, was possible. Twenty isolates belonging to five species were used to test the database accuracy, and were correctly identified to the species level with a log-score >2. In summary, matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a reliable and rapid method for the identification of most of the human pathogenic Mucorales to the species level.

Biofilms are communal structures of microorganisms that have long been associated with a variety of persistent infections poorly responding to conventional antibiotic or antifungal therapy. Aspergillus fumigatus fungus and Stenotrophomonas maltophilia bacteria are examples of the microorganisms that can coexist to form a biofilm especially in the respiratory tract of immunocompromised patients or cystic fibrosis patients. The aim of the present study was to develop and assess an in vitro model of a mixed biofilm associating S. maltophilia and A. fumigatus by using analytical and quantitative approaches.An A. fumigatus strain (ATCC 13073) expressing a Green Fluorescent Protein (GFP) and an S. maltophilia strain (ATCC 13637) were used. Fungal and bacterial inocula (105 conidia/mL and 106 cells/mL, respectively) were simultaneously deposited to initiate the development of an in vitro mixed biofilm on polystyrene supports at 37°C for 24 h. The structure of the biofilm was analysed via qualitative microscopic techniques like scanning electron and transmission electron microscopy, and fluorescence microscopy, and by quantitative techniques including qPCR and crystal violet staining.Analytic methods revealed typical structures of biofilm with production of an extracellular matrix (ECM) enclosing fungal hyphae and bacteria. Quantitative methods showed a decrease of A. fumigatus growth and ECM production in the mixed biofilm with antibiosis effect of the bacteria on the fungi seen as abortive hyphae, limited hyphal growth, fewer conidia, and thicker fungal cell walls.For the first time, a mixed A. fumigatus-S. maltophilia biofilm was validated by various analytical and quantitative approaches and the bacterial antibiosis effect on the fungus was demonstrated. The mixed biofilm model is an interesting experimentation field to evaluate efficiency of antimicrobial agents and to analyse the interactions between the biofilm and the airways epithelium.

We report 20 episodes of infection caused by acquired echinocandin-resistant Candida spp. harboring diverse and new Fksp mutations. For 12 patients, initial isolates (low MIC, wild-type Fksp sequence) and subsequent isolates (after caspofungin treatment, high MIC, mutated Fksp) were genetically related.

We analyzed hospital records to provide a population-based estimate of zygomycosis incidence and trends over a 10-year period at a national level in France. Data showed an increasing incidence from 0.7/million in 1997 to 1.2/million in 2006 (p