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=110.53 µg mL-1), strong ferric reducing ability (EC50=644.4 µg mL-1), and a remarkable degree of protection against lipid peroxidation during ß-carotene bleaching inhibition (IC50=251.76 µg mL-1). Antifungal activity was carried out against Candida, Aspergillus, and Rhizopus species by microdilution method. T. capitatus exhibited potent anticandidal activity (MIC=125-500 µg mL-1) and strong inhibition against filamentous fungi (MIC=250-500 µg mL-1). Its hemolytic activity against human erythrocytes had a low toxic effect at concentrations lower than 1250 µg 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.

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.

Is the C-Terminal Domain an Effective and Selective Target for the Design of Hsp90 Inhibitors against Candida Yeast?

Improving the armamentarium to treat invasive candidiasis has become necessary to overcome drug resistance and the lack of alternative therapy. In the pathogenic fungus Candida albicans, the 90-kDa Heat-Shock Protein (Hsp90) has been described as a major regulator of virulence and resistance, offering a promising target. Some human Hsp90 inhibitors have shown activity against Candida spp. in vitro, but host toxicity has limited their use as antifungal drugs. The conservation of Hsp90 across all species leads to selectivity issues. To assess the potential of Hsp90 as a druggable antifungal target, the activity of nine structurally unrelated Hsp90 inhibitors with different binding domains was evaluated against a panel of Candida clinical isolates. The Hsp90 sequences from human and yeast species were aligned. Despite the degree of similarity between human and yeast N-terminal domain residues, the in vitro activities measured for the inhibitors interacting with this domain were not reproducible against all Candida species. Moreover, the inhibitors binding to the C-terminal domain (CTD) did not show any antifungal activity, with the exception of one of them. Given the greater sequence divergence in this domain, the identification of selective CTD inhibitors of fungal Hsp90 could be a promising strategy for the development of innovative antifungal drugs.

Discovery of New Broad-Spectrum Anti-Infectives for Eukaryotic Pathogens Using Bioorganometallic Chemistry.

Drug resistance observed with many anti-infectives clearly highlights the need for new broad-spectrum agents to treat especially neglected tropical diseases (NTDs) caused by eukaryotic parasitic pathogens, including fungal infections. Herein, we show that the simple modification of one of the most well-known antifungal drugs, fluconazole, with organometallic moieties not only improves the activity of the parent drug but also broadens the scope of application of the new derivatives. These compounds were highly effective in vivo against pathogenic fungal infections and potent against parasitic worms such as Brugia, which causes lymphatic filariasis and Trichuris, one of the soil-transmitted helminths that infects millions of people globally. Notably, the identified molecular targets indicate a mechanism of action that differs greatly from that of the parental antifungal drug, including targets involved in biosynthetic pathways that are absent in humans, offering great potential to expand our armamentarium against drug-resistant fungal infections and neglected tropical diseases (NTDs) targeted for elimination by 2030.

Antifungal efficacy of imidazo[1,2-a]pyrazine-based thiosemicarbazones and thiazolidinediones against Sporothrix species.

Aim: To evaluate antifungal potential of 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine hybrids based on thiosemicarbazones and thiazolidinediones against pathogenic Sporothrix species. Methods: Antifungal activity of nine compounds were assessed by broth microdilution. Interactions between active compounds and itraconazole were evaluated by the checkerboard assay using non-wild-type isolates. Cytotoxicity of the compounds was determined. Results: Four C-3 substituted analogs showed antifungal activity, unrelated to thiosemicarbazone or thiazolidinedione functions. Synergistic interactions between the four compounds and itraconazole, and low toxicity on mouse fibroblast cells were observed. Activity of 5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine hybrids against Sporothrix depended on the substitution on the imidazopyrazine ring. Conclusion: Antifungal potential, overcoming itraconazole resistance and low toxicity indicate the possible use of that series of compounds in a therapeutic alternative for treatment of sporotrichosis.

The Mortality Attributable to Candidemia in C. auris Is Higher than That in Other Candida Species: Myth or Reality?

Candida auris has become a major health threat due to its transmissibility, multidrug resistance and severe outcomes. In a case-control design, 74 hospitalised patients with candidemia were enrolled. In total, 22 cases (29.7%) and 52 controls (C. albicans, 21.6%; C. parapsilosis, 21.6%; C. tropicalis, 21.6%; C. glabrata, 1.4%) were included and analysed in this study. Risk factors, clinical and microbiological characteristics and outcomes of patients with C. auris and non-auris Candida species (NACS) candidemia were compared. Previous fluconazole exposure was significantly higher in C. auris candidemia patients (OR 3.3; 1.15-9.5). Most C. auris isolates were resistant to fluconazole (86.3%) and amphotericin B (59%) whilst NACS isolates were generally susceptible. No isolates resistant to echinocandins were detected. The average time to start antifungal therapy was 3.6 days. Sixty-three (85.1%) patients received adequate antifungal therapy, without significant differences between the two groups. The crude mortality at 30 and 90 days of candidemia was up to 37.8% and 40.5%, respectively. However, there was no difference in mortality both at 30 and 90 days between the group with candidemia by C. auris (31.8%) and by NACS (42.3%) (OR 0.6; 95% IC 0.24-1.97) and 36.4% and 42.3% (0.77; 0.27-2.1), respectively. In this study, mortality due to candidemia between C. auris and NACS was similar. Appropriate antifungal therapy in both groups may have contributed to finding no differences in outcomes.

Acquired fluconazole resistance and genetic clustering in Diutina (Candida) catenulata from clinical samples.

OBJECTIVES: Diutina (Candida) catenulata is an ascomycetous yeast isolated from environmental sources and animals, occasionally infecting humans. The aim of this study is to shed light on the in vitro antifungal susceptibility and genetic diversity of this opportunistic yeast. METHODS: Forty-five D. catenulata strains isolated from various sources (including human and environmental sources) and originating from nine countries were included. Species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and confirmed via internal transcribed spacer ribosomal DNA barcoding. In vitro antifungal susceptibility was determined for seven systemic antifungals via the gradient strip method after 48 hours of incubation at 35°C using Etest® (Biomérieux) or Liofilchem® strips. Isolates exhibiting fluconazole minimal inhibitory concentrations (MICs) of ≥8 µg/mL were investigated for mutations in the ERG11 gene. A novel microsatellite genotyping scheme consisting of four markers was developed to assess genetic diversity. RESULTS: MIC ranges for amphotericin B, caspofungin, micafungin, isavuconazole, and posaconazole were 0.19-1 µg/mL, 0.094-0.5 µg/mL, 0.012-0.064 µg/mL, 0.003-0.047 µg/mL, and 0.006-0.032 µg/mL, respectively. By comparison, a broad range of MICs was noted for fluconazole (0.75 to >256 µg/mL) and voriconazole (0.012-0.38 mg/L), the higher values being observed among clinical strains. The Y132F amino acid substitution, associated with azole resistance in various Candida species (C. albicans, C. tropicalis, C. parapsilosis, and C. orthopsilosis), was the main substitution identified. Although microsatellite typing showed extensive genetic diversity, most strains with high fluconazole MICs clustered together, suggesting human-to-human transmission or a common source of contamination. DISCUSSION: The high rate of acquired fluconazole resistance among clinical isolates of D. catenulata is of concern. In this study, we highlight a link between the genetic diversity of D. catenulata and its antifungal resistance patterns, suggesting possible clonal transmission of resistant isolates.

The Novodiag® Stool parasites assay, an innovative high-plex technique for fast detection of protozoa, helminths and microsporidia in stool samples: a retrospective and prospective study.

OBJECTIVES: We provide the first evaluation of the CE-IVD marked Novodiag® stool parasites assay (NVD), allowing rapid and high-plex detection of 26 distinct targets, encompassing protozoans, helminths and microsporidia in stool samples. METHODS: A total of 254 samples (n = 205 patients) were prospectively processed by the NVD and our routine procedure (RP). Performances of the NVD were compared with RP. Samples only positive by the NVD assay were investigated by external PCR assays. Sensitivity and specificity (Se/Sp) and time from sample receipt to results were determined for each method. The NVD was also evaluated against 77 additional samples positive for a wide range of parasites. RESULTS: Overall positivity rate was 16.9% for RP compared with 34% using the NVD assay, and 164 samples (66%) were negative by both methods. Only 30 positive samples (12%) showed full concordance between RP and NVD. Fifty-three discordant samples were sent for external investigations. Except for Giardia intestinalis and Trichuris spp., higher Se was observed for the NVD assay for Blastocystis spp. (100% vs. 63%), Dientamoeba fragilis (100% vs. 0%), Schistosoma spp. (100% vs. 17%), and Enterobius vermicularis (100% vs. 67%) but roughly similar to RP for the remaining parasites tested. False-positive results were identified for Blastocystis spp., G. intestinalis, and Trichuris spp. using the NVD assay. The NVD mostly provides a diagnosis on the day of sample receipt compared with a mean of three days with RP. CONCLUSIONS: Besides some limitations, the NVD is a new diagnostic strategy allowing rapid and high-plex detection of gastrointestinal parasites from unpreserved stools.

Title: Le test Novodiag® Stool parasites, une technique high-plex innovante pour la détection rapide des protozoaires, helminthes et microsporidies dans les échantillons de selles : une étude rétrospective et prospective. Abstract: Objectifs : Nous présentons la première évaluation du kit Novodiag® Stool parasite (NVD) marqué CE-IVD, permettant la détection rapide de 26 cibles distinctes dans les selles (protozoaires, helminthes et microsporidies). Méthodes : Un total de 254 échantillons (n = 205 patients) a été traité prospectivement par le NVD et notre procédure de routine (PR). Les performances du NVD ont été comparées à celles de la PR. Seuls les échantillons positifs au test NVD ont été étudiés par des PCR externes. La sensibilité et la spécificité (Se/Sp) ainsi que le temps écoulé entre la réception de l'échantillon et les résultats ont été déterminés pour chaque méthode. Le NVD a également été évalué par rapport à 77 échantillons supplémentaires positifs pour un large éventail de parasites. Résultats : Le taux de positivité global était de 16,9 % pour la PR contre 34 % avec le NVD, et 164 échantillons (66 %) étaient négatifs par les deux méthodes. Seuls 30 échantillons positifs (12 %) ont montré une concordance complète entre la PR et le NVD. Cinquante-trois échantillons discordants ont été envoyés pour des investigations externes. À l'exception de Giardia intestinalis et de Trichuris spp., des Se plus élevées ont été observées pour le test NVD pour Blastocystis spp. (100 % contre 63 %), Dientamoeba fragilis (100 % contre 0 %), Schistosoma spp. (100 % contre 17 %), Enterobius vermicularis (100 % contre 67 %) mais étaient à peu près similaires à la PR pour les autres parasites testés. Des faux positifs ont été identifiés pour Blastocystis spp., G. intestinalis et Trichuris spp. en utilisant le NVD. Le NVD fournit le plus souvent un diagnostic le jour de la réception du prélèvement contre une moyenne de trois jours avec la PR. Conclusions : Malgré quelques limites, le test NVD est une nouvelle stratégie de diagnostic permettant une détection rapide et high-plex des parasites gastro-intestinaux à partir de selles non conservées.

Prototheca Infections and Ecology from a One Health Perspective.

Prototheca microalgae were only recognized as pathogens of both humans and animals in the 1960s; however, since then, these microbes have been drawing increasing interest in both human and veterinary medicine. The first human outbreak of protothecosis in a tertiary care chemotherapy ward in 2018 further highlighted the need to understand in more depth and detail their ecology, etiology, pathogenesis and routes of transmission between different hosts, environments and habitats from a One Health perspective. Protothecal infections have been reported in a growing number of cattle herds around the world in recent decades, and Prototheca has become an important bovine mastitis pathogen in certain countries and regions. The survival of Prototheca in the environment and its ability to spread in the herd pose a serious challenge to the management of infected dairy farms. Prevention of the disease is particularly important, as there is no effective and reliable treatment for it and the chances of self-healing are minimal. Therefore, the development of more effective drugs is needed for the treatment of human and animal protothecosis. The prudent use of antibiotics and their replacement by alternative or preventive measures, when possible, may further contribute to the control of protothecal infections.

CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata.

Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.