Precise genome editing underlines the distinct contributions of mutations in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in Candida parapsilosis.

Candida parapsilosis has recently emerged as a major threat due to the worldwide emergence of fluconazole-resistant strains causing clonal outbreaks in hospitals and poses a therapeutic challenge due to the limited antifungal armamentarium. Here, we used precise genome editing using CRISPR-Cas9 to gain further insights into the contribution of mutations in ERG11, ERG3, MRR1, and TAC1 genes and the influence of allelic dosage to antifungal resistance in C. parapsilosis. Seven of the most common amino acid substitutions previously reported in fluconazole-resistant clinical isolates (including Y132F in ERG11) were engineered in two fluconazole-susceptible C. parapsilosis lineages (ATCC 22019 and STZ5). Each mutant was then challenged in vitro against a large array of antifungals, with a focus on azoles. Any possible change in virulence was also assessed in a Galleria mellonella model. We successfully generated a total of 19 different mutants, using CRISPR-Cas9. Except for R398I (ERG11), all remaining amino acid substitutions conferred reduced susceptibility to fluconazole. However, the impact on fluconazole in vitro susceptibility varied greatly according to the engineered mutation, the stronger impact being noted for G583R acting as a gain-of-function mutation in MRR1. Cross-resistance with newer azoles, non-medical azoles, but also non-azole antifungals such as flucytosine, was occasionally noted. Posaconazole and isavuconazole remained the most active in vitro. Except for G583R, no fitness cost was associated with the acquisition of fluconazole resistance. We highlight the distinct contributions of amino acid substitutions in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in C. parapsilosis.

Chemical composition, antifungal, antioxidant and hemolytic activities of Moroccan Thymus capitatus essential oil.

This study aimed to define the chemical composition of Moroccan Thymus capitatus essential oil, and to investigate its in vitro antioxidant and antifungal activities against human pathogenic fungi. Chemical analysis using GC-FID and GC-MS system revealed 28 constituents, representing 99% of total compounds. Oxygenated monoterpenes represented the highest proportion (79.79%), among which carvacrol (75.73%) was the predominant compound, followed by linalol (2.26%). Monoterpene hydrocarbons represented the second major fraction (16.29%): within them, the predominant constituents were γ-terpinene (5,55%), ρ-cymene (5,50%), and ß-caryophyllene (2.73%). Antioxidant activity was performed by DPPH scavenging, ß-carotene bleaching inhibition, and ferric reducing power. T. capitatus revealed pronounced DPPH radical scavenging activity (IC50=110.53µg.mL-1), strong ferric reducing ability (EC50=644.4µg.mL-1), and a remarkable degree of protection against lipid peroxidation during ß-carotene bleaching inhibition (IC50=251.76µg.mL-1). Antifungal activity was carried out against Candida, Aspergillus, and Rhizopus species by microdilution method.  T. capitatus   exhibited potent anticandidal activity (MIC=125-500µg.mL-1) and strong inhibition against filamentous fungi (MIC=250-500µg.mL-1). Its hemolytic activity against human erythrocytes had a low toxic effect at concentrations lower than 1250µg.mL-1. The useful antioxidant properties and broad antifungal effect of T. capitatus EO confirm its considerable potential for the food industry and for phytopharmaceutical production.

Highly Conserved gsc1 Gene of Pneumocystis jirovecii in Patients with or without Prior Exposure to Echinocandins.

Echinocandins are noncompetitive inhibitors of the GSC1 subunit of the enzymatic complex involved in synthesis of 1,3-beta-d-glucan, a cell wall component of most fungi, including Pneumocystis spp. Echinocandins are widely used for treating systemic candidiasis and rarely used for treating Pneumocystis pneumonia. Consequently, data on P. jirovecii gsc1 gene diversity are still scarce compared to that for the homologous fks1 gene of Candida spp. In this study, we analyzed P. jirovecii gsc1 gene diversity and the putative selection pressure of echinocandins on P. jirovecii. gsc1 gene sequences of P. jirovecii specimens from two patient groups were compared. One group of 27 patients had prior exposure to echinocandins, whereas the second group of 24 patients did not, at the time of P. jirovecii infection diagnoses. Two portions of the P. jirovecii gsc1 gene, HS1 and HS2, homologous to hot spots described in Candida spp., were sequenced. Three single-nucleotide polymorphisms (SNPs) at positions 2204, 2243, and 2303 close to the HS1 region and another SNP at position 4540 more distant from the HS2 region were identified. These SNPs represent synonymous mutations. Three gsc1 HS1 alleles, A, B, and C, and two gsc1 HS2 alleles, a and b, and four haplotypes, Ca, Cb, Aa, and Ba, were defined, without significant difference in haplotype distribution in both patient groups (P = 0.57). Considering the identical diversity of P. jirovecii gsc1 gene and the detection of synonymous mutations in both patient groups, no selection pressure of echinocandins among P. jirovecii microorganisms can be pointed out so far.

Genotypic, proteomic, and phenotypic approaches to decipher the response to caspofungin and calcineurin inhibitors in clinical isolates of echinocandin-resistant Candida glabrata.

BACKGROUND: Echinocandin resistance represents a great concern, as these drugs are recommended as first-line therapy for invasive candidiasis. Echinocandin resistance is conferred by mutations in FKS genes. Nevertheless, pathways are crucial for enabling tolerance, evolution, and maintenance of resistance. Therefore, understanding the biological processes and proteins involved in the response to caspofungin may provide clues indicating new therapeutic targets. OBJECTIVES: We determined the resistance mechanism and assessed the proteome response to caspofungin exposure. We then evaluated the phenotypic impact of calcineurin inhibition by FK506 and cephalosporine A (CsA) on caspofungin-resistant Candida glabrata isolates. METHODS: Twenty-five genes associated with caspofungin resistance were analysed by NGS, followed by studies of the quantitative proteomic response to caspofungin exposure. Then, susceptibility testing of caspofungin in presence of FK506 and CsA was performed. The effects of calcineurin inhibitor/caspofungin combinations on heat stress (40°C), oxidative stress (0.2 and 0.4 mM menadione) and on biofilm formation (polyurethane catheter) were analysed. Finally, a Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of the combinations on larval survival. RESULTS: F659-del was found in the FKS2 gene of resistant strains. Proteomics data showed some up-regulated proteins are involved in cell-wall biosynthesis, response to stress and pathogenesis, some of them being members of calmodulin-calcineurin pathway. Therefore, the impact of calmodulin inhibition was explored. Calmodulin inhibition restored caspofungin susceptibility, decreased capacity to respond to stress conditions, and reduced biofilm formation and in vivo pathogenicity. CONCLUSIONS: Our findings confirm that calmodulin-calcineurin-Crz1 could provide a relevant target in life-threatening invasive candidiasis.

Collateral consequences of agricultural fungicides on pathogenic yeasts: A One Health perspective to tackle azole resistance.

Candida and Cryptococcus affect millions of people yearly, being responsible for a wide array of clinical presentations, including life-threatening diseases. Interestingly, most human pathogenic yeasts are not restricted to the clinical setting, as they are also ubiquitous in the environment. Recent studies raise concern regarding the potential impact of agricultural use of azoles on resistance to medical antifungals in yeasts, as previously outlined with Aspergillus fumigatus. Thus, we undertook a narrative review of the literature and provide lines of evidence suggesting that an alternative, environmental route of azole resistance, may develop in pathogenic yeasts, in addition to patient route. However, it warrants sound evidence to support that pathogenic yeasts cross border between plants, animals and humans and that environmental reservoirs may contribute to azole resistance in Candida or other yeasts for humans. As these possibilities could concern public health, we propose a road map for future studies under the One Health perspective.

Invasive Aspergillosis Due to Aspergillus Section Usti: A Multicenter Retrospective Study.

BACKGROUND: Aspergillus spp. of section Usti (A. ustus) represent a rare cause of invasive aspergillosis (IA). This multicenter study describes the epidemiology and outcome of A. ustus infections. METHODS: Patients with A. ustus isolated from any clinical specimen were retrospectively identified in 22 hospitals from 8 countries. When available, isolates were sent for species identification (BenA/CaM sequencing) and antifungal susceptibility testing. Additional cases were identified by review of the literature. Cases were classified as proven/probable IA or no infection, according to standard international criteria. RESULTS: Clinical report forms were obtained for 90 patients, of whom 27 had proven/probable IA. An additional 45 cases were identified from literature review for a total of 72 cases of proven/probable IA. Hematopoietic cell and solid-organ transplant recipients accounted for 47% and 33% cases, respectively. Only 8% patients were neutropenic at time of diagnosis. Ongoing antimold prophylaxis was present in 47% of cases. Pulmonary IA represented 67% of cases. Primary or secondary extrapulmonary sites of infection were observed in 46% of cases, with skin being affected in 28% of cases. Multiple antifungal drugs were used (consecutively or in combination) in 67% of cases. The 24-week mortality rate was 58%. A. calidoustus was the most frequent causal agent. Minimal inhibitory concentrations encompassing 90% isolates (MIC90) were 1, 8, >16, and 4 µg/mL for amphotericin B, voriconazole, posaconazole, and isavuconazole, respectively. CONCLUSIONS: Aspergillus ustus IA mainly occurred in nonneutropenic transplant patients and was frequently associated with extrapulmonary sites of infection. Mortality rate was high and optimal antifungal therapy remains to be defined.

The Extent of Aspergillosis in Critically Ill Patients With Severe Influenza Pneumonia: A Multicenter Cohort Study.

OBJECTIVES: To determine the frequency and prognosis of invasive pulmonary aspergillosis in critically ill patients with severe influenza pneumonia. DESIGN: Retrospective multicenter cohort study. SETTING: Five French ICUs. PATIENTS: Patients with influenza admitted to ICU between 2009 and 2018. MEASUREMENTS AND MAIN RESULTS: Of the 524 patients admitted for severe influenza diagnosed with a positive airway reverse-transcriptase polymerase chain reaction test, 450 (86%) required mechanical ventilation. A lower respiratory tract sample yielded with Aspergillus (Asp+) in 28 patients (5.3%). Ten patients (1.9%) were diagnosed with putative or proven invasive pulmonary aspergillosis, based on the validated AspICU algorithm. A multivariate model was built to identify independent risk factors for Aspergillus-positive pulmonary culture. Factors independently associated with Aspergillus-positive culture were liver cirrhosis (odds ratio = 6.7 [2.1-19.4]; p < 0.01), hematologic malignancy (odds ratio = 3.3 [1.2-8.5]; p = 0.02), Influenza A(H1N1)pdm09 subtype (odds ratio = 3.9 [1.6-9.1]; p < 0.01), and vasopressor requirement (odds ratio = 4.1 [1.6-12.7]; p < 0.01). In-hospital mortality of Asp+ patients was 36% versus 21% in patients without Aspergillus-positive pulmonary culture (p = 0.09). CONCLUSIONS: In this large retrospective multicenter cohort of critically ill patients, putative invasive pulmonary aspergillosis according to AspICU algorithm was a relatively rare complication of influenza. Patients at higher risk of Aspergillus pulmonary colonization included those with liver cirrhosis, hematologic malignancy, H1N1pdm09 influenza A virus, and requiring vasopressors. Our results provide additional data on the controversial association between severe influenza and invasive pulmonary aspergillosis. Reaching a consensual definition of invasive pulmonary aspergillosis becomes mandatory and confers further prospective research.

Anti-protozoal and anti-fungal evaluation of 3,5-disubstituted 1,2-dioxolanes.

Endoperoxides are a class of compounds, which is well-known for their antimalarial properties, but few reports exist about 3,5-disubstituted 1,2-dioxolanes. After having designed a new synthetic route for the preparation of these substances, they were evaluated against 4 different agents of infectious diseases, protozoa (Plasmodium and Leishmania) and Fungi (Candida and Aspergillus). Whereas moderate antifungal activity was found for our products, potent antimalarial and antileishmanial activities were observed for a few compounds. The nature of the substituents linked to the endoperoxide ring seems to play an important role in the bioactivities.

Impact of calmodulin inhibition by fluphenazine on susceptibility, biofilm formation and pathogenicity of caspofungin-resistant Candida glabrata.

BACKGROUND: In recent decades, Candida glabrata has emerged as a frequent cause of life-threatening fungal infection. In C. glabrata, echinocandin resistance is associated with mutations in FKS1/FKS2 (ß-1,3-glucan synthase). The calmodulin/calcineurin pathway is implicated in response to antifungal stress and calcineurin gene disruption specifically reverses Fks2-mediated resistance of clinical isolates. OBJECTIVES: We evaluated the impact of calmodulin inhibition by fluphenazine in two caspofungin-resistant C. glabrata isolates. METHODS: C. glabrata isolates were identified by ITS1/ITS4 (where ITS stands for internal transcribed spacer) sequencing and the echinocandin target FKS1/FKS2 genes were sequenced. Susceptibility testing of caspofungin in the presence of fluphenazine was performed by a modified CLSI microbroth dilution method. The effect of the fluphenazine/caspofungin combination on heat stress (37°C or 40°C), oxidative stress (0.2 and 0.4 mM menadione) and biofilm formation (polyurethane catheter) was analysed. A Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of this combination on larval survival. RESULTS: F659del was found in the FKS2 gene of both resistant strains. In these clinical isolates, fluphenazine increased susceptibility to caspofungin and reduced their thermotolerance. Furthermore, the fluphenazine/caspofungin combination significantly impaired biofilm formation in an in vitro polyurethane catheter model. All these features participated in the increasing survival of infected G. mellonella after combination treatment in comparison with caspofungin alone. CONCLUSIONS: In a repurposing strategy, our findings confirm that calmodulin could provide a relevant target in life-threatening fungal infectious diseases.

Biological exploration of a novel 1,2,4-triazole-indole hybrid molecule as antifungal agent.

(2-(2,4-Dichlorophenyl)-3-(1H-indol-1-yl)-1-(1,2,4-1H-triazol-1-yl)propan-2-ol (8 g), a new 1,2,4-triazole-indole hybrid molecule, showed a broad-spectrum activity against Candida, particularly against low fluconazole-susceptible species. Its activity was higher than fluconazole and similar to voriconazole on C. glabrata (MIC90 = 0.25, 64 and 1 µg/mL, respectively), C. krusei (MIC90 = 0.125, 64 and 0.125 µg/mL, respectively) and C. albicans (MIC90 = 0.5, 8 and 0.25 µg/mL, respectively). The action mechanisms of 8 g were also identified as inhibition of ergosterol biosynthesis and phospholipase A2-like activity. At concentration as low as 4 ng/mL, 8g inhibited ergosterol production by 82% and induced production of 14a-methyl sterols, that is comparable to the results obtained with fluconazole at higher concentration. 8 g demonstrated moderate inhibitory effect on phospholipase A2-like activity being a putative virulence factor. Due to a low MRC5 cytotoxicity, this compound presents a high therapeutic index. These results pointed out that 8 g is a new lead antifungal candidate with potent ergosterol biosynthesis inhibition.

Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in Candida orthopsilosis.

BACKGROUND: Azoles are one of the main antifungal classes for the treatment of candidiasis. In the current context of emerging drug resistance, most studies have focused on Candida albicans, Candida glabrata or Candida auris but, so far, less is known about the underlying mechanisms of resistance in other species, including Candida orthopsilosis. OBJECTIVES: We investigated azole resistance in a C. orthopsilosis clinical isolate recovered from a patient with haematological malignancy receiving fluconazole prophylaxis. METHODS: Antifungal susceptibility to fluconazole was determined in vitro (CLSI M27-A3) and in vivo (in a Galleria mellonella model of invasive candidiasis). The CoERG11 gene was then sequenced and amino acid substitutions identified were mapped on the predicted 3D structure of CoErg11p. A clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) genome-editing strategy was used to introduce relevant mutations into a fluconazole-susceptible C. orthopsilosis isolate. RESULTS: Compared with unrelated C. orthopsilosis isolates, the clinical isolate exhibited both in vitro and in vivo fluconazole resistance. Sequencing of the CoERG11 gene identified several amino acid substitutions, including two possibly involved in fluconazole resistance (L376I and G458S). Both mutations mapped close to the active site of CoErg11p. Engineering these mutations in a different genetic background using CRISPR-Cas9 demonstrated that G458S, but not L376I, confers resistance to fluconazole and voriconazole. CONCLUSIONS: Our data show that the G458S amino acid substitution in CoERG11p, but not L376I, contributes to azole resistance in C. orthopsilosis. In addition to highlighting the potential of CRISPR-Cas9 technology for precise genome editing in the field of antifungal resistance, we discuss some points that are critical to improving its efficiency.

Comparison between MALDI-TOF MS and MicroScan in the identification of emerging and multidrug resistant yeasts in a fourth-level hospital in Bogotá, Colombia.

BACKGROUND: The introduction of MALDI-TOF MS in the clinical microbiology laboratory has modified the approaches for the identification of fungi. Thanks to this tool, it is possible to identify cryptic species, which possess critical susceptibility patterns. Clinical strains were identified using the MicroScan and MALDI-TOF MS systems. Discrepant results from both methods were investigated using ITS rDNA barcoding. Finally, these isolates were also tested for in vitro susceptibility. RESULTS: The percentage of agreement between both methods to 498 yeast isolates was of 93.6% (32 discrepant isolates). The concordance of ITS sequencing with MALDI-TOF MS was higher (99%) than that of MicroScan (94%). Several of these discordant yeasts displayed high MICs for antifungal agents. CONCLUSIONS: Our study highlights the need of the MS and molecular approaches such as MALDI-TOF MS and ITS rDNA barcoding for the correct identification of emerging or cryptic yeast species; besides, some of these could be multidrug resistant. This work was the first experience in the implementation of the MALDI-TOF MS technology in Colombia. We found the first uncommon yeasts including Candida auris and we could identify Trichosporon faecalis. Our work highlights a clear necessity of an accurate yeast identification as a much more pertinent technique than the susceptibility profiles, because the most unusual yeasts exhibit resistance profiles to the few available antifungals.

Multivalent Fucosides with Nanomolar Affinity for the Aspergillus fumigatus Lectin FleA Prevent Spore Adhesion to Pneumocytes.

FleA (or AFL), a fucose lectin, was recently identified in the opportunistic mold Aspergillus fumigatus, which causes fatal lung infections in immunocompromised patients. We designed di-, hexa- and octavalent fucosides with various spacer arm lengths to block the hexameric FleA through chelation. Microcalorimetry measurements showed that the ethylene glycol (EG) spacer arm length has a strong influence on the binding affinity of the divalent fucosides. The relationship between the EG length and chelate binding efficiency to FleA was explored according to polymer theory. Hexa- and octavalent compounds based on cyclodextrin and octameric silsesquioxane scaffolds were nanomolar FleA inhibitors, surpassing their monovalent fucose analogue by more than three orders of magnitude. Importantly, some of the fucosides were highly efficient in preventing fungal spore adhesion to bronchoepithelial cells, with half maximal inhibitory concentration values in the micromolar range. We propose that the synergistic antiadhesive effect observed can be ascribed to chelate binding to FleA and to the formation of conidium aggregates, as observed by optical microscopy. These fucosides are promising tools that can be used to better understand the role of FleA in conidia pathogenicity and host defenses against invasive aspergillosis.

Benzofuro[3,2-d]pyrimidines inspired from cercosporamide CaPkc1 inhibitor: Synthesis and evaluation of fluconazole susceptibility restoration.

In a context of growing resistance to classical antifungal therapy, the design of new drugs targeting alternative pathways is highly expected. Benzofuro[3,2-d]pyrimidine derivatives, derived from (-)-cercosporamide, were synthesized and evaluated as potential Candida albicans PKC inhibitors in the aim of restoring susceptibility to azole treatment. Co-administration assay of benzofuropyrimidinedione 23 and fluconazole highlighted a synergistic effect on inhibition of cell growth of a Candida albicans resistant strain.

PCR-based detection of Aspergillus fumigatus and absence of azole resistance due to TR34 /L98H in a french multicenter cohort of 137 patients with fungal rhinosinusitis.

Fungal rhinosinusitis (FRS) has a worldwide distribution, comprises distinct clinical entities but is mostly due to Aspergillus among which Aspergillus fumigatus plays a major role in European countries. Although, there is accumulating evidence for the emergence of environmentally acquired-azole resistance in A. fumigatus (such as TR34 /L98H) in various clinical settings, there is few data for patients with FRS. In this study, we aimed to investigate the prevalence of A. fumigatus azole resistance due to TR34 /L98H in a multicentre cohort of patients with FRS. One hundred and thirty-seven patients with FRS admitted between 2002 and 2016 at four French medical centres were retrospectively enrolled. Clinical and mycological findings were collected. Aspergillus fumigatus and the TR34 /L98H alteration conferring azole resistance were investigated directly from clinical samples using the commercial CE-IVD marked MycoGENIE® A. fumigatus real-time PCR assay. Fungal ball was the more frequent clinical form (n = 118). Despite the presence of fungal hyphae at direct microscopic examination, mycological cultures remained negative for 83 out of the 137 patients (60.6%). The PCR assay proved to be useful allowing the identification of A. fumigatus and etiological diagnosis in 106 patients (77.4%) compared with 44 patients (32.1%) when using culture as the reference method. Importantly, neither TR34 nor L98H alterations were evidenced.

Specific Human and Candida Cellular Interactions Lead to Controlled or Persistent Infection Outcomes during Granuloma-Like Formation.

A delayed type of multicellular process could be crucial during chronic candidiasis in determining the course of infection. This reaction, consisting of organized immune cells surrounding the pathogen, initiates an inflammatory response to avoid fungal dissemination. The goal of the present study was to examine, at an in vitro cellular scale, Candida and human immune cell interaction dynamics during a long-term period. By challenging human peripheral blood immune cells from 10 healthy donors with 32 Candida albicans and non-albicans (C. glabrata, C. tropicalis, C. parapsilosis, C. dubliniensis, C. lusitaniae, C. krusei, and C. kefyr) clinical isolates, we showed that Candida spp. induced the formation of granuloma-like structures within 6 days after challenge, but their sizes and the respective fungal burdens differed according to the Candida species. These two parameters are positively correlated. Phenotypic characteristics, such as hypha formation and higher axenic growth rate, seem to contribute to yeast persistence within granuloma-like structures. We showed an interindividual variability of the human response against Candida spp. Higher proportions of neutrophils and elevated CD4+/CD8+ T cell ratios during the first days after challenge were correlated with early production of gamma interferon (IFN-γ) and associated with controlled infection. In contrast, the persistence of Candida could result from upregulation of proinflammatory cytokines such as interleukin-6 (IL-6), IFN-γ, and tumor necrosis factor alpha (TNF-α) and a poor anti-inflammatory negative feedback (IL-10). Importantly, regulatory subsets of NK cells and CD4lo CD8hi doubly positive (DP) lymphocytes at late stage infiltrate granuloma-like structures and could correlate with the IL-10 and TNF-α production. These data offer a base frame to explain cellular events that guide infection control or fungal persistence.

Home Environment as a Source of Life-Threatening Azole-Resistant Aspergillus fumigatus in Immunocompromised Patients.

A case of fatal aspergillosis due to a TR46/Y121F/T289A azole-resistant Aspergillus fumigatus is reported. Environmental investigations at the patient's residence led to the recovery of TR46/Y121F/T289A isolates, genotypically indistinguishable from the clinical isolate, supporting for the first time the direct role of household as potential source of azole-resistant invasive aspergillosis.

Prevalence of the reversed halo sign in neutropenic patients compared with non-neutropenic patients: Data from a single-centre study involving 27 patients with pulmonary mucormycosis (2003-2016).

Pulmonary mucormycosis (PM) is a life-threatening infection and the diagnosis can be challenging. The objective was to retrospectively explore the value of the RHS in our cohort of 27 patients with mucormycosis and its relation to neutropenia. This was a retrospective study including all patients with a diagnosis of probable or proven invasive PM according to the 2008 EORTC/MSG criteria between September 2003 to April 2016. Fisher's exact test and Mann-Whitney test, with a P-value statistically significant under .05 (P<.05), were used to compare neutropenic and non-neutropenic groups. 27 patients were eligible. The RHS could be identified in 78% of cases in the neutropenic group, and was less common in the non-neutropenic group (31%) (P<.05). Reticulations inside ground-glass opacity in case of RHS were present in 13 out of 15 patients (87%). Mucorales DNA detection by PCR on serum provided, a median time to the first PCR-positive sample of 3 days (-33 to +60 days) before diagnosis was confirmed. Six patients had IPA co-infection. In conclusion, RHS is more frequent in case of PM in neutropenic patients compare to non-neutropenic patients. Its presence in immunocompromised patients should be sufficient to promptly start Mucorales-active antifungal treatment, while its absence especially in non-neutropenic cases should not be sufficient to exclude the diagnosis.

The amino acid substitution N136Y in Candida albicans sterol 14alpha-demethylase is involved in fluconazole resistance.

Resistance to fluconazole antifungal is an ongoing impediment to a successful treatment of Candida albicans infections. One of the most prevalent mechanisms leading to azole resistance is genetic alterations of the 14α-demethylase, the target of azole antifungals, through point mutations. Site-directed mutagenesis and molecular modeling of 14α-demethylase rationalize biological data about the role of protein substitutions in the azole treatment failure. In this work, we investigated the role of N136Y substitution by site-directed mutagenesis into Pichia pastoris guided by structural analysis. Single amino acid substitutions were created by site-directed mutagenesis into P. pastoris with C. albicans ERG11 gene as template. In vitro susceptibility of P. pastoris transformants expressing wild-type and mutants to azole compounds was determined by CLSI M27-A2 and spot agar methods. The fluconazole effect on ergosterol biosynthesis was analyzed by gas chromatography-mass spectrometry. By microdilution and spot tests, N136Y transformants showed a reduced in vitro susceptibility to fluconazole compared to wild-type controls. As expected, ergosterol/lanosterol ratios were higher in N136Y transformants compared to the wild-type controls after treatment with fluconazole. Molecular modeling suggests that residue Asn136 located within the first mutation hot spot, could play a role during heme and azole binding. These results provide new insights into the structural basis for 14α-demethylase-azole interaction and could guide the design of novel azole antifungals.