Evaluation of the Performance of the Dynamiker Fungus (1-3)-ß-D-Glucan and Fungitell Assay for Diagnosis of Candidemia: Need for New Cut-off Development and Test Validation.

(1-3)-Beta-D Glucan (BDG) detection has shown to be a highly effective tool to diagnose invasive fungal infections. Therefore, this study aimed to compare clinical characteristics of the Fungitell (FA) and Dynamiker Fungus (1-3)-ß-D-Glucan assay (DFA) for the diagnosis of candidemia. Using DFA and FA, the BDG levels of 57 serum samples from case and control groups were determined. The kappa coefficient (κ) and Spearman's rank correlation (rs) were used to examine the consistency of assays on a quantitative and qualitative level, respectively. The sensitivity, specificity, and accuracy were 94.6 %, 65.0 %, and 87.7% for DFA, and 94.6 %, 75.0 %, and 89.4 % for FA, respectively. The performance of the DFA for the diagnosis of candidemia was highly consistent with that of the FA, both quantitatively (rs: 0.9) and qualitatively (kappa = 0.78). Collectively, the DFA assay performed excellently in comparison to the FA for the diagnosis of candidemia.

Species distribution and antifungal susceptibility profiles of yeasts isolated from onychomycosis: a cross-sectional study with insights into emerging species.

Candida onychomycosis is a common fungal infection affecting the nails, primarily caused by Candida (C.) species. Regarding the increasing trend of Candida onychomycosis and the antifungal resistant phenomenon in recent years, this study aims to evaluate the epidemiological characteristics of Candida onychomycosis, the distribution of emerging species, and the antifungal susceptibility profiles of isolates. Onychomycosis caused by yeast species was confirmed through direct examination and culture of nail scraping among all individuals suspected to have onychomycosis and referred to a medical mycology laboratory between June 2019 and March 2022. Species of yeast isolates were identified using the multiplex PCR and PCR-RFLP methods. The antifungal susceptibility of isolates to common antifungal agents and imidazole drugs was evaluated according to the M-27-A3 CLSI protocol. Among 101 yeast strains isolated from onychomycosis, Candida parapsilosis complex (50.49%) was the most common species, followed by C. albicans (20.79%) and C. tropicalis (10.89%). Rare species of yeasts such as C. guilliermondii and Saccharomyces cerevisiae were also identified by molecular methods. Results obtained from antifungal susceptibility testing showed significant differences in MIC values of isoconazole, fenticonazole, and sertaconazole among different species. Overall, a fluconazole-resistant rate of 3% was found among Candida species. Moreover, there was a statistically significant difference in MICs of fenticonazole and clotrimazole between the two most prevalent causative species, C. parapsilosis complex and C. albicans. Correct identification of the causative agents of onychomycosis and performing susceptibility testing could be helpful in choosing the most appropriate antifungal therapy.

Direct molecular analysis of Malassezia species from the clinical samples of patients with pityriasis versicolor.

Background and Purpose: Species identification of Malassezia using culture-dependent methods is time-consuming due to their fastidious growth requirements. This study aimed to evaluate a rapid and accurate molecular method in order to diagnose the pityriasis versicolor (PV) and identify Malassezia species from direct clinical samples. Materials and Methods: Skin scraping or tape samples from patients with PV and healthy volunteers as the control group were collected. Diagnosis of PV was confirmed by direct microscopic examination. The DNA extraction was performed according to the steel-bullet beating method. Polymerase chain reaction-restriction fragment length polymorphism assay using HhaI restriction enzyme was applied for the identification and differentiation of Malassezia species. Results: The PCR method was able to detect Malassezia in 92.1% of specimens which were also confirmed with microscopic examination. Statistically, a significant association was observed between the results of the two assays (P < 0.001). Moderate agreement was identified between the two methods to diagnose the PV in both populations (Kappa: 0.55). Considering microscopic examination as the gold standard method for confirmation of PV, the sensitivity, specificity, positive predictive value, and negative predictive value values of the PCR assay for recognition of PV were 85%, 75%, 92%, and 60%, respectively. M. globosa and M. restricta were the most prevalent species isolated from patients. Conclusion: In this study, the two-step molecular method based on the amplification of the D1/D2 domain and digestion of the PCR product by one restriction enzyme was able to diagnose and identify Malassezia directly from clinical samples. Consequently, it can be said that the molecular-based method provides more facilities to identify fastidious species, such as M. restricta.

Species identification, antifungal susceptibility patterns, and vitamin D3 level in women with vaginal candidiasis: a case-control study in Iran.

Vulvovaginal candidiasis (VVC) is a fungal infection that is a global issue of women's health due to its association with morbidity, infertility, and economic costs. This study aimed to compare the vitamin D3 levels between women with VVC to healthy controls and determine the species distribution and susceptibility pattern of isolates. Species identification was performed using sequencing of the ITS-rDNA regions and amplification of the HWP1 gene. Antifungal susceptibility testing was determined by the disk diffusion method. Moreover, serum vitamin D3 levels were measured using a commercial ELISA (enzyme-linked immunosorbent assay) kit. Our results indicated that vitamin D3 level in women with VVC was lower than those of healthy women (p-value < .001). Candida albicans complex (62.8 percent) was the most common species, and most species were susceptible to fluconazole, itraconazole, ketoconazole, and nystatin. In conclusion, our study revealed a potential link between vitamin D3 deficiency and VVC in women. Although our findings showed significantly lower vitamin D3 levels in women with VVC, further research is needed to establish a definitive causative relationship between vitamin D3 deficiency and VVC. Nonetheless, our study highlights the potential importance of maintaining adequate levels of vitamin D3 and the need for further exploration in this area.

[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives as new therapeutic candidates against urease positive microorganisms: design, synthesis, pharmacological evaluations, and in silico studies.

Regarding the important role of the urease enzyme as a virulence factor in urease-positive microorganisms in this study, new series of [1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives were designed and synthesized. All compounds evaluated against urease enzyme exhibiting IC50 values of 0.87 ± 0.09 to 8.32 ± 1.21 µM as compared with thiourea as the positive control (IC50 = 22.54 ± 2.34 µM). The kinetic evaluations of 6a as the most potent derivative recorded a competitive type of inhibition. Molecular dynamic simulations of the 6a derivative were also conducted, showing that 6a occupied the active site with closed state. Antimicrobial activities of all derivatives were performed, and 6f (R = 3-Cl), 6g (R = 4-Cl), and 6h (R = 3,4-diCl) analogs demonstrated significant antifungal activities with MIC values of 1, 2, and 0.5 µg/mL compared with fluconazole with MIC = 2 µg/mL. Synthesized analogs also exhibited potent urease inhibitory activities against C. neoformans (IC50 = 83.7-118.7 µg/mL) and P. mirabilis (IC50 = 74.5-113.7 µg/mL), confirming their urease inhibitory potential. The results demonstrated that the designed scaffold could be considered a suitable pharmacophore to develop potent urease inhibitors.

New thioxothiazolidinyl-acetamides derivatives as potent urease inhibitors: design, synthesis, in vitro inhibition, and molecular dynamic simulation.

To identify potent urease inhibitors, in the current study, a series of thioxothiazolidinyl-acetamides were designed and synthesized. The prepared compounds were characterized by spectroscopic techniques, including FTIR, 1HNMR, 13CNMR, and elemental analysis. In the enzymatic assessments, it was demonstrated that all derivatives had significant urease inhibition with IC50 values in the range of 1.473-9.274 µM in comparison with the positive control hydroxyurea (IC50 = 100.21 ± 2.5 µM) and thiourea (IC50 = 23.62 ± 0.84 µM). Compound 6i (N-benzyl-3-butyl-4-oxo-2-thioxothiazolidine-5-carboxamide) was the most active agent with an IC50 value of 1.473 µM. Additionally, kinetic investigation and in silico assessments of 6i was carried out to understand the type of inhibition and behavior of the most potent derivative within the binding site of the enzyme. Noteworthy, the anti-urease assay against P. vulgaris revealed 6e and 6i as the most active agents with IC50 values of 15.27 ± 2.40 and 17.78 ± 3.75 µg/mL, respectively. Antimicrobial evaluations of all compounds reveal that compounds 6n and 6o were the most potent antimicrobial agents against the standard and resistant S. aureus. 6n and 6o also showed 37 and 27% inhibition in the development of biofilm by S. aureus at 512 µg/ml. Furthermore, the MTT test showed no toxicity up to 100 µM. Taken together, the study suggests that the synthesized thioxothiazolidinyl-acetamides bases derivatives may serve as potential hits as urease inhibitors.

Assessment of Risk Factors and Clinical Outcomes in Hospitalized COVID-19 Patients with Candida spp. Co-infections: Species Distribution and Antifungal Susceptibility Patterns of Isolates.

INTRODUCTION: Fungal co-infections are considered an important complication in hospitalized patients with SARS-CoV-2 that can be attributed to disease aggravation, increased mortality, and poor outcomes. This study was conducted to determine the species distribution and antifungal susceptibility patterns of Candida isolates from hospitalized COVID-19 patients in Shiraz, Iran, in addition to associated risk factors and outcomes of co-infections with Candida species. MATERIALS AND METHODS: In this single-center study, a total of 106 hospitalized COVID-19 patients were evaluated for clinical characteristics and outcomes. Species identification was performed by ITS1-5.8S-ITS2 gene sequencing. Antifungal susceptibility testing to fluconazole, itraconazole, voriconazole, posaconazole, caspofungin, amphotericin B, and nystatin was determined according to the M27-A3/S4 CLSI protocol. RESULTS: Candida species were recovered from 48% (51/106) of hospitalized COVID-19 patients. Statistical analysis showed that patients who had heart failure, bacterial co-infection, and were receiving empirical antifungal therapy had a higher risk of developing Candida co-infection. In total, 71 Candida isolates were recovered, of which C. albicans (69%) was the most prevalent isolate. The majority of the Candida isolates were susceptible to all classes of tested antifungal drugs. DISCUSSION: Our results elucidate a high rate of Candida co-infections among hospitalized COVID-19 patients. Comorbidities such as heart failure, HTN, COPD, bacterial infections as well as therapeutic interventions including catheterization, mechanical ventilation, and ICU admission increased the risk of Candida spp. isolation from the bloodstream, respiratory tract and urine samples, which led to a higher in-hospital mortality rate. Additionally, obtained data clarified that empirical antifungal therapy was not as successful as anticipated.

Quantitative analysis of in vitro biofilm formation by clinical isolates of dermatophyte and antibiofilm activity of common antifungal drugs.

BACKGROUND: The ability of dermatophytes to develop biofilm, as one of the virulence factors in fungal infections which contribute to antifungal resistance, is an outstanding aspect of dermatophytosis that has been noted recently. Because of the paucity of data about the biofilm formation by dermatophytes and their susceptibility to antifungal drugs, this study evaluated the biofilm formation by clinical isolates of dermatophytes and antibiofilm activity of common antifungals widely used to manage dermatophytosis. METHODS: The ribosomal DNA internal transcribed spacer (ITS) regions sequencing for species identification of 50 clinical dermatophyte isolates was performed. The ability of isolates to form biofilm and inhibitory activity of itraconazole, terbinafine, and griseofulvin against biofilm formation was assayed by the crystal violet staining method. Optical microscopy and scanning electron microscopy (SEM) were applied for the visualization of the biofilm structures. RESULTS: Trichophyton (T.) mentagrophytes (n: 14; 28%) and T. rubrum (n: 13;26%) were included in more than half of the dermatophyte isolates. Biofilm formation was observed in 37 out of 50 (74%) isolates that were classified as follows: nonproducers (n: 13; 26%), weak producers (n: 4; 8%), moderate producers (n: 16; 32%), and strong producers (n: 17; 34%) by comparison of the absorbance of biofilms produced by clinical strains with control. The mean IC50 values for terbinafine, griseofulvin, and itraconazole were 2.42, 3.18, and 3.78 µg/ml, respectively. CONCLUSIONS: The results demonstrated that most of the clinical dermatophyte isolates are capable to form biofilm in vitro with variable strength. Moreover, terbinafine can be suggested as the first-line choice for the treatment of biofilm-formed dermatophytosis.

Vitamin D3: A promising antifungal and antibiofilm agent against Candida species.

Background and Purpose: Candida species are opportunistic fungal pathogens that cause mild to life-threatening infections in both immunocompetent and immunocompromised populations. The increasing prevalence of drug-resistant Candida species has posed a significant challenge to the management of infections in clinical settings. Therefore, this study aimed to investigate the direct antifungal and antibiofilm effect of vitamin D3 against Candida species. Materials and Methods: The antifungal activity of vitamin D3 was evaluated by broth microdilution method based on the Clinical and Laboratory Standard Institute. Prevention of biofilm formation by Candida albicans was measured using the XTT assay following exposure to different concentrations of vitamin D3. Moreover, expression of Agglutinin-like sequence gene 1 (ALS1), hyphal wall protein gene (HWP1), secreted aspartyl proteinase 6 gene (SAP6), and morphogenesis pathway regulatory gene (EFG1) were analyzed by real-time polymerase chain reaction using the comparative Ct method (ΔΔ Ct) after exposure to vitamin D3. Results: Vitamin D3 showed antifungal activity against Candida species ranging from 1-128 µg/mL. Furthermore, vitamin D3 inhibited biofilm formation in a dose-dependent manner, with IC50 of 7.5 µg/mL. Treatment with vitamin D3 resulted in significant upregulation of the EFG1, ALS1, and SAP6 genes under hypha-inducing conditions to overcome environmental challenges. Conclusion: Results of the current study demonstrated that vitamin D3 has a significant inhibitory effect on Candida growth and biofilm formation. Considering its demonstrated antifungal and antibiofilm properties, vitamin D3 holds promise as a potential agent for medical applications.

Epidemiology, clinical features, therapeutic interventions and outcomes of mucormycosis in Shiraz: an 8-year retrospective case study with comparison between children and adults.

Mucormycosis is an invasive fungal infection with high morbidity and mortality rate despite the early diagnosis and proper therapeutic interventions. Given the importance of epidemiological data in reviewing the attitude toward infectious diseases in developing countries, the current retrospective case study aimed to compare the epidemiological aspects, risk factors, clinical characteristics, therapeutic interventions, and outcomes of mucormycosis between adults and children during eight years (2013-2021) in the main infectious disease referral centers in the southwest of Iran. The median age of 164 patients included in this study was 47 years (IQR 22-59). The median length of hospitalization was 33 days.The annual incidence of mucormycosis-related hospitalizations was estimated 1.76 per 10,000 admissions during the study period. Moreover, the incidence of infection was 2.4 times higher in males than females in children. Diabetes mellitus was the most frequent predisposing factor in adults (46.0%). The main risk factor in children was hematologic malignancy (52.6%), but a considerable proportion of them (28.9%) were immunocompetent.The most frequent antifungal agent used was liposomal amphotericin B (82.3%) as monotherapy. The combination therapy was used more in adults (15.8%) than children (7.9%). In addition, surgical intervention with antifungal therapy was considered the most effective therapeutic approach. The in-hospital mortality rate was 14.6% for adults, whereas it was zero for children. Our findings provide a recent epidemiologic analysis of mucormycosis among hospitalized patients in both children and adults. Mucormycosis mainly affects individuals with diabetes mellitus or hematological malignancies and presents as rhino-orbito-cerebral form. Proven diagnosis of mucormycosis according to clinical manifestations and histopathology observations accompanied by proper antifungal treatments may improve survival rates.

Evaluation of different DNA extraction methods based on steel-bullet beating for molecular diagnosis of onychomycosis.

BACKGROUND: Considering increased trends toward molecular methods for detection/identification of fungi causing onychomycosis, the aim of this study is comparison three DNA extraction methods based on steel-bullet beating to extract DNA from nail. METHODS: Ex -vivo onychomycosis model was developed using bovine hoof with Candida albicans and Aspergillus flavus. For two models, total DNA was extracted using the three different methods. In method 1, the extraction and purification were performed by steel-bullet beating and phenol chloroform protocol, respectively. In method 2, a freezing step were applied before beating. The purification step in method 3 was carried out using a commercial kit, although DNA extraction was done similarly to method 1 in that approach. To evaluate the efficacy of each method, the extracted genomic DNA was amplified with Polymerase Chain Reaction (PCR) using Internal Transcribed Spacer (ITS) regions. Moreover, 50 nail samples were evaluated for onychomycosis using direct microscopy examination as well as PCR in order to evaluate the diagnostic efficiency of the optimal DNA extraction method. RESULTS: Regarding the desirable quality of the extracted DNA, cost effectiveness, and simplicity, method 1 could be used to extract DNA effectively. Additionally, the obtained data showed that PCR had a higher detection rate of fungal agents in the nail samples than direct microscopic examination. CONCLUSIONS: This study demonstrated that the mechanical disruption of the cell wall by steel-bullet beating is a useful and practical method to improve the quantity and quality of fungal DNA thorough the extraction process.

Design and synthesis of novel nitrothiazolacetamide conjugated to different thioquinazolinone derivatives as anti-urease agents.

The present article describes the design, synthesis, in vitro urease inhibition, and in silico molecular docking studies of a novel series of nitrothiazolacetamide conjugated to different thioquinazolinones. Fourteen nitrothiazolacetamide bearing thioquinazolinones derivatives (8a-n) were synthesized through the reaction of isatoic anhydride with different amine, followed by reaction with carbon disulfide and KOH in ethanol. The intermediates were then converted into final products by treating them with 2-chloro-N-(5-nitrothiazol-2-yl)acetamide in DMF. All derivatives were then characterized through different spectroscopic techniques (1H, 13C-NMR, MS, and FTIR). In vitro screening of these molecules against urease demonstrated the potent urease inhibitory potential of derivatives with IC50 values ranging between 2.22 ± 0.09 and 8.43 ± 0.61 µM when compared with the standard thiourea (IC50 = 22.50 ± 0.44 µM). Compound 8h as the most potent derivative exhibited an uncompetitive inhibition pattern against urease in the kinetic study. The high anti-ureolytic activity of 8h was confirmed against two urease-positive microorganisms. According to molecular docking study, 8h exhibited several hydrophobic interactions with Lys10, Leu11, Met44, Ala47, Ala85, Phe87, and Pro88 residues plus two hydrogen bound interactions with Thr86. According to the in silico assessment, the ADME-Toxicity and drug-likeness profile of synthesized compounds were in the acceptable range.

Evaluation of electrospun poly (vinyl alcohol)-based nanofiber mats incorporated with Zataria multiflora essential oil as potential wound dressing.

Infections, especially those caused by multi-drug resistant pathogens, result in serious problems in wound healing process. In this study, Zataria multiflora (ZM) essential oil, as a strong natural antimicrobial agent, is incorporated into poly (vinyl alcohol)-based nanofiber mats to fabricate a novel wound dressing. Different amounts of ZM essential oil (0, 2, 5 and 10% (v/v)) were incorporated into chitosan/poly(vinyl alcohol)/gelatin (CS/PVA/Gel) solutions and then were successfully electrospun into beadless and uniform fibers with 95 ±â€¯14, 154 ±â€¯27, 187 ±â€¯40 and 218 ±â€¯58 nm in diameters, respectively. The produced nanofiber mats (CS/PVA/Gel/ZM) were chemically crosslinked by glutaraldehyde vapor. The chemical compositions of ZM essential oil and nanofiber mats were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) and Fourier Transform Infrared Spectroscopy (FTIR), respectively. The antimicrobial activity of the CS/PVA/Gel/ZM nanofiber mats was determined by the AATCC100 method. The nanofiber mat loaded with 10% of ZM essential oil completely inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans after 24 h of incubation. Swelling investigations showed that the produced nanofibers have a substantial ability to take up water, in the range of 400-900%. Mechanical properties of the nanofiber mats were studied by tensile testing. Furthermore, they were found to be non-toxic by biocompatibility assays on mouse fibroblast (L929) cells. The obtained results have demonstrated that CS/PVA/Gel nanofiber mats, loaded with ZM essential oil, are promising alternatives to conventional wound dressings.

Modified magnetic nanoparticles by PEG-400-immobilized Ag nanoparticles (Fe3O4@PEG-Ag) as a core/shell nanocomposite and evaluation of its antimicrobial activity.

BACKGROUND: Noble metal nanoparticles, due to their good physicochemical properties, have been exploited in biological applications. Among these metals, nanosilver has attracted great attention because of its optical properties and broad-spectrum antimicrobial activities with no drug tolerance. PURPOSE: The present study has attempted to conduct chemical synthesis of Fe3O4@PEG-Ag core/shell nanocomposites in aqueous solutions through co-precipitation of Fe3+ and Fe2+ ions, encapsulating the iron oxide core by poly (ethylene-glycol) (PEG) improve its hydrophilicity and biocompatibility, and immobilizing silver ions by application of NaBH4 as a reducing agent. PATIENTS AND METHODS: The synthesized structures were characterized by Fourier-transform infrared (FT-IR), field emission scanning electron microscopy, energy-dispersive X-ray spectrum, wavelength-dispersive X-ray, vibrating sample magnetometer, inductively coupled plasma-mass spectrometry and transmission electron microscopy methods. Antimicrobial activity of the nanostructures against Staphylococcus aureus, Escherichia coli and Candida albicans was evaluated by broth microdilution based on the methods suggested by Clinical Laboratory Standard Institute. Furthermore, the nanocomposite was tested for possible anti-parasitic effects against Leishmania major promastigotes by MTT assay. Also, its impacts on bacterial cell morphology were defined using atomic force microscopy. Moreover, toxicity of the nanostructure related to animal cell line was determined based on MTT assay. RESULTS: In general, the synthesized core/shell nanostructure can demonstrate noticeable activity against the evaluated representative microorganisms while its toxicity against animal cell line is not considerable. CONCLUSION: This nanostructure can be applied as a smart drug delivery system with the help of an external magnetic field or it can be used as a powerful antibiotic agent along with other antibiotics that can form a shell on its structure.