Candida auris - Comparison of Sensititre YeastOne and Vitek 2 AST systems for antifungal susceptibility testing - A single centre experience.

INTRODUCTION: Candida auris is emerging as an important cause of candidemia and deep seated candidal infection. We compared the susceptibility results of bloodstream Candida auris isolates by Vitek 2 with Sensititre YeastOne (SYO) method. METHODS: Forty-seven C.auris blood stream isolates were simultaneously tested for AFST by Vitek 2 and SYO. RESULTS: All strains were resistant to Fluconazole. 25.5% isolates showed pan-azole resistance. In comparison with SYO, lower MICs for voriconazole were noted with Vitek 2 (VME rate 76.1%). All strains were sensitive to anidulafungin and micafungin by SYO. For micafungin, Vitek 2 demonstrated higher MICs and an ME rate of 23.5%. Susceptibility interpretation of caspofungin by SYO was challenged by development of 'Eagle effect' resulting in sensitivity of 28.2%. We studied the evolution of caspofungin 'Eagle effect' with SYO by serial hourly MIC readings and noted that paradoxical growth commenced at 21 hours of incubation. Compared to SYO, Vitek 2 showed higher resistance rate to Amphotericin B with ME rate of 25.6%. CONCLUSION: Laboratories using commercial AFST systems for Candida auris need to be aware of the possibility of ME and VME for amphotericin B and voriconazole respectively with Vitek 2 and 'Eagle effect' for caspofungin with SYO.

Carbon substrates promotes stress resistance and drug tolerance in clinical isolates of Candida tropicalis.

Candida tropicalis is a human pathogen and one of the most prevalent non-Candida albicans Candida (NCAC) species causing invasive infections. Azole antifungal resistance in C. tropicalis is also gradually increasing with the increasing incidence of infections. The pathogenic success of C. tropicalis depends on its effective response in the host microenvironment. To become a successful pathogen, cellular metabolism, and physiological status determine the ability of the pathogen to counter diverse stresses inside the host. However, to date, limited knowledge is available on the impact of carbon substrate metabolism on stress adaptation and azole resistance in C. tropicalis. In this study, we determined the impact of glucose, fructose, and sucrose as the sole carbon source on the fluconazole resistance and osmotic (NaCl), oxidative (H2O2) stress adaptation in C. tropicalis clinical isolates. We confirmed that the abundance of carbon substrates influences or increases drug resistance and osmotic and oxidative stress tolerance in C. tropicalis. Additionally, both azole-resistant and susceptible isolates showed similar stress adaptation phenotypes, confirming the equal efficiency of becoming successful pathogens irrespective of drug susceptibility profile. To the best of our knowledge, our study is the first on C. tropicalis to demonstrate the direct relation between carbon substrate metabolism and stress tolerance or drug resistance.

Bullous erythema multiforme secondary to fluconazole intake: A unique case report managed with photobiomodulation therapy.

While fluconazole use is generally considered safe and well-tolerated, there has been an increasing number of reports regarding several adverse events. Therefore, the present study aimed to present a unique case in which photobiomodulation therapy (PBMT) was employed to manage bullous erythema multiforme lesions secondary to fluconazole intake. A 32-year-old female patient sought emergency dental care due to painful orofacial lesions that had developed two days after oral fluconazole use for recurrent vulvovaginal candidiasis. Given the acute clinical features, a diagnosis of bullous erythema multiforme secondary to fluconazole was established. Prednisone 20 mg was then prescribed for five days, and fluconazole intake was immediately discontinued. As the initial treatment strategies failed to show improvement in the clinical condition, three PBMT sessions were proposed every other day. Within seven days, almost complete wound healing was observed, and any pain complaints were no longer present. The resolution of orofacial lesions within a short period suggests that PBMT could be a promising tool for managing drug-induced bullous erythema multiforme. However, more studies are needed to confirm this statement.

Limonene synergistically augments fluconazole susceptibility in clinical Candida isolates from cleft lip and palate patients.

Background: Cleft lip and palate (CLP) patients are prone to Candida infections (oral thrush) mainly due to poor oral hygiene, repetitive surgeries, and orthodontic procedures. Aim: This study was undertaken to evaluate the antifungal efficacy of limonene against clinical Candida isolates from CLP patients. Materials and Methods: The antifungal efficacy of limonene was studied alone and in combination with fluconazole (FLC) against six standards, twenty nine FLC sensitive, and three FLC resistant clinical strains using broth dilution, checkerboard microdilution, agar disk diffusion, growth curves, and spot assays. Results: This nontoxic monoterpene gave low minimum inhibitory concentration (MIC) values of 300-375 µg/mL and 500-520 µg/mL for FLC susceptible and FLC resistant strains, respectively. It showed synergistic interaction with FLC in all clinical and standard Candida strains (fractional inhibitory concentration (FIC) index ≤0.5). Conclusion: Significant chemosensitization of FLC was observed even against resistant clinical isolates. Complete suppression of fungal growth was observed when using combinations. Negligible toxicity, easy availability, and potent antifungal properties suggest that limonene and FLC combinations in appropriate doses can make excellent antifungal mouthwashes during CLP treatment pre and post surgery. Impending in vivo studies are needed to validate the present data.

Uptake and translocation of pharmaceutically active compounds by olive tree (Olea europaea L.) irrigated with treated municipal wastewater.

Introduction: The use of treated municipal wastewater (TWW) represents a relevant opportunity for irrigation of agricultural crops in semi-arid regions to counter the increasing water scarcity. Pharmaceutically active compounds (PhACs) are often detected in treated wastewater, posing a risk to humans and the environment. PhACs can accumulate in soils and translocate into different plant tissues, reaching, in some cases, edible organs and entering the food chain. Methods: This study evaluated the uptake and translocation processes of 10 PhACs by olive trees irrigated with TWW, investigating their accumulation in different plant organs. The experiment was conducted in southern Italy, in 2-year-old plants irrigated with three different types of water: freshwater (FW), TWW spiked with 10 PhACs at a concentration of 200 µg L-1 (1× TWW), and at a triple dose (3× TWW), from July to October 2021. The concentration of PhACs in soil and plant organs was assessed, collecting samples of root, stem, shoot, leaf, fruit, and kernel at 0 (T0), 50 (T1), and 107 (T2) days of irrigation. PhACs extraction from soil and plant organs was carried out using the QuEChERS method, and their concentrations were determined by high-resolution mass spectrometry coupled with liquid chromatography. Results: Results of uptake factors (UF) showed a different behavior between compounds according to their physicochemical properties, highlighting PhACs accumulation and translocation in different plant organs (also edible part) in 1× TWW and 3× TWW compared to FW. Two PhACs, carbamazepine and fluconazole, showed interactions with the soil-plant system, translocating also in the aerial part of the plant, with a translocation factor (TF) greater than 1, which indicates high root-to-leaf translocation. Discussion: Findings highlight that only few PhACs among the selected compounds can be uptaken by woody plants and accumulated in edible parts at low concentration. No effects of PhACs exposure on plant growth have been detected. Despite the attention to be paid to the few compounds that translocate into edible organs, these results are promising for adapting wastewater irrigation in crops. Increasing knowledge about PhACs behavior in woody plants can be important for developing optimized wastewater irrigation and soil management strategies to reduce PhACs accumulation and translocation in plants.

Thermosensitive and mucoadhesive gels containing solid lipid nanoparticles loaded with fluconazole and niosomes loaded with clindamycin for the treatment of periodontal diseases: a laboratory experiment.

BACKGROUND: Periodontal diseases may benefit more from topical treatments with nanoparticles rather than systemic treatments due to advantages such as higher stability and controlled release profile. This study investigated the preparation and characterization of thermosensitive gel formulations containing clindamycin-loaded niosomes and solid lipid nanoparticles (SLNs) loaded with fluconazole (FLZ), as well as their in vitro antibacterial and antifungal effects in the treatment of common microorganisms that cause periodontal diseases. METHODS: This study loaded niosomes and SLNs with clindamycin and FLZ, respectively, and assessed their loading efficiency, particle size, and zeta potential. The particles were characterized using a variety of methods such as differential scanning calorimetry (DSC), dynamic light scattering (DLS), and Transmission Electron Microscopy (TEM). Thermosensitive gels were formulated by combining these particles and their viscosity, gelation temperature, in-vitro release profile, as well as antibacterial and antifungal effects were evaluated. RESULTS: Both types of these nanoparticles were found to be spherical (TEM) with a mean particle size of 243.03 nm in niosomes and 171.97 nm in SLNs (DLS), and respective zeta potentials of -23.3 and -15. The loading rate was 98% in niosomes and 51% in SLNs. The release profiles of niosomal formulations were slower than those of the SLNs. Both formulations allowed the release of the drug by first-order kinetic. Additionally, the gel formulation presented a slower release of both drugs compared to niosomes and SLNs suspensions. CONCLUSION: Thermosensitive gels containing clindamycin-loaded niosomes and/or FLZ-SLNs were found to effectively fight the periodontitis-causing bacteria and fungi.

Novel aza-bicyclic 2-isoxazoline acylhydrazone hybrids and their synergistic potential with fluconazole against a drug-resistant Candida albicans strain.

As the prevalence of drug-resistant Candida isolates continues to rise, the imperative for identifying novel compounds to enhance the arsenal of antifungal drugs becomes increasingly critical. Consequently, exploring new treatment strategies, including synthesizing molecular hybrids and applying combination therapy, is essential. For this reason, this study evaluated the efficacy of ten molecular hybrids of aza-bicyclic 2-isoxazoline-acylhydrazone belonging to two series 90 and 91 as possible anti-Candida agents. In addition, we also investigated the interaction between the hybrids and fluconazole, a commonly used antifungal drug. We evaluated the antifungal effect of aza-bicyclic 2-isoxazoline-acylhydrazone hybrid compounds against six Candida spp. strains that target planktonic cells. However, none of these new molecules were inhibitory active at the tested concentrations (2 to 1,024 µg/mL). Moreover, we analyzed the interaction between the ten new hybrid molecules and fluconazole using the checkerboard assay, employing two different methodologies for reading the plate. For this, one isolate fluconazole-resistant was selected. We observed that only one combination, 6-(4-tert-butylbenzoil)-4,5,6,6a-tetrahydro-3a-H-pirrole[3,2-d]isoxazole-3-carboxylic(furan-2-metilidene)-hydrazide (91e) and fluconazole, exhibited a synergistic interaction (FICI range 0.0781 to 0.4739). The combination successfully inhibited the growth of C. albicans CA2 fluconazole-resistant, and no interaction was observed in an isolate susceptible to fluconazole. Additionally, these results emphasize the continued need for research into new compounds and the importance of using combined approaches to increase their activity.

The efficacy of fluconazole for anti-fungal prophylaxis in peritoneal dialysis patients: A systematic review and meta-analysis.

INTRODUCTION AND OBJECTIVES: The efficacy of fluconazole as a prophylactic strategy in patients with chronic kidney disease (CKD) on peritoneal dialysis (PD) with prior antibiotic exposure is controversial in the current literature. This study aimed to compare a strategy of fluconazole prophylaxis versus no-prophylaxis for patients in PD on antibiotics for previous episodes of peritonitis. MATERIALS AND METHODS: We performed a systematic review and meta-analysis of observational studies and randomized controlled trials (RCTs) comparing fluconazole prophylaxis with no prophylaxis for PD-related peritonitis. The search was conducted on PubMed, EMBASE, and Cochrane Central in January 23, 2023. The outcome of interest was the occurrence of fungal peritonitis (FP). RESULTS: We included six studies (1 RCT, 5 observational) with 4515 occurrences of peritonitis, of which 1098 (24.8%) received fluconazole prophylaxis in variable doses, whereas 3417 (75.6%) did not receive prophylaxis during peritonitis episodes. Overall, fluconazole prophylaxis was associated with a lower incidence of FP (OR 0.22; 95% CI 0.12-0.41; p<0.001; I2=0%). Subgroup analysis of studies that administered daily doses of fluconazole also demonstrated a reduced incidence of FP in patients who received antifungal prophylaxis (OR 0.31; CI 0.14-0.69; p=0.004; I2=0%). CONCLUSIONS: In this meta-analysis of 4515 episodes of PD-related peritonitis, prophylaxis with fluconazole significantly reduced episodes of FP as compared with no antifungal prophylaxis.

Incorporation of Inclusion Complexes in the Dissolvable Microneedle Ocular Patch System for the Efficiency of Fluconazole in the Therapy of Fungal Keratitis.

Fluconazole (FNL) is one of the first-line treatments for fungal keratitis as it is an effective broad-spectrum antimicrobial commonly administered orally or topically. However, FNL has a very low water solubility, limiting its drug formulation, therapeutic application, and bioavailability through tissues. To overcome these limitations, this study aimed to develop FNL inclusion complexes (FNL-IC) with cyclodextrin (α-cyclodextrin, sulfobutylether-ß-cyclodextrin, and hydroxypropyl-γ cyclodextrin) and incorporate it into a dissolvable microneedle (DMN) system to improve solubility and drug penetration. FNL-IC was evaluated for saturation solubility, Fourier transform infrared spectroscopy, differential scanning calorimetry, in vitro release, minimum inhibitory concentration, minimum fungicidal concentration, and time-killing assay. DMN-FNL-IC was evaluated for mechanical and insertion properties, surface pH, moisture absorption ability, water vapor transmission, and drug content recovery. Moreover, ocular kinetic, ex vivo antimicrobial, in vivo antifungal, and chorioallantoic membrane (HET-CAM) assays were conducted to assess the overall performance of the formulation. Mechanical strength and insertion properties revealed that DMN-FNL-IC has great mechanical and insertion properties. The in vitro release of FNL-IC was significantly improved, exhibiting a 9-fold increase compared to pure FNL. The ex vivo antifungal activity showed significant inhibition of Candida albicans from 6.54 to 0.73 log cfu/mL or 100-0.94%. In vivo numbers of colonies of 0.87 ± 0.13 log cfu/mL (F2), 4.76 ± 0.26 log cfu/mL (FNL eye drops), 3.89 ± 0.24 log cfu/mL (FNL ointments), and 8.04 ± 0.58 log cfu/mL (control) showed the effectiveness of DMN preparations against other standard commercial preparations. The HET-CAM assay showed that DMN-FNL-IC (F2) did not show any vascular damage. Finally, a combination of FNL-IC and DMN was developed appropriately for ocular delivery of FNL, which was safe and increased the effectiveness of treatments for fungal keratitis.

Again and Again-Survival of Candida albicans in Urine Containing Antifungals.

BACKGROUND: Relapse of Candida albicans urinary tract infection (UTI) is frequent despite appropriate treatment, as commonly used antifungals such fluconazole and flucytosine are only fungistatics. To improve treatment of Candida UTI and decrease relapses, understanding the long-term metabolic activity and survival of C. albicans in urine containing antifungals at minimal inhibitory concentration (MIC) is needed. METHODS: we monitored the survival, metabolic activity and consumption of glucose and proteins by C. albicans using conventional methods and isothermal microcalorimetry (IMC). We also investigated the influence of dead Candida cells on the growth of their living counterparts. RESULTS: For 33 days, weak activity was observed in samples containing antifungals in which C. albicans growth rate was reduced by 48%, 60% and 88%, and the lag increased to 172 h, 168 h and 6 h for amphotericin, flucytosine and fluconazole, respectively. The metabolic activity peaks corresponded to the plate counts but were delayed compared to the exhaustion of resources. The presence of dead cells promoted growth in artificial urine, increasing growth rate and reducing lag in similar proportions. CONCLUSIONS: Even with antifungal treatment, C. albicans relapses are possible. The low metabolic activity of surviving cells leading to regrowth and chlamydospore formation possibly supported by autophagy are likely important factors in relapses.

Spectrum of activity and mechanisms of azole-bisphosphonate synergy in pathogenic Candida.

Candidiasis places a significant burden on human health and can range from common superficial vulvovaginal and oral infections to invasive diseases with high mortality. The most common Candida species implicated in human disease is Candida albicans, but other species like Candida glabrata are emerging. The use of azole antifungals for treatment is limited by increasing rates of resistance. This study explores repositioning bisphosphonates, which are traditionally used for osteoporosis, as antifungal synergists that can improve and revitalize the use of azoles. Risedronate, alendronate, and zoledronate (ZOL) were tested against isolates from six different species of Candida, and ZOL produced moderate antifungal activity and strong synergy with azoles like fluconazole (FLC), particularly in C. glabrata. FLC:ZOL combinations had increased fungicidal and antibiofilm activity compared to either drug alone, and the combination prevented the development of antifungal resistance. Mechanistic investigations demonstrated that the synergy was mediated by the depletion of squalene, resulting in the inhibition of ergosterol biosynthesis and a compromised membrane structure. In C. glabrata, synergy compromised the function of membrane-bound multidrug transporters and caused an accumulation of reactive oxygen species, which may account for its acute sensitivity to FLC:ZOL. The efficacy of FLC:ZOL in vivo was confirmed in a Galleria mellonella infection model, where combinations improved the survival of larvae infected with C. albicans and C. glabrata to a greater extent than monotherapy with FLC or ZOL, and at reduced dosages. These findings demonstrate that bisphosphonates and azoles are a promising new combination therapy for the treatment of topical candidiasis. IMPORTANCE: Candida is a common and often very serious opportunistic fungal pathogen. Invasive candidiasis is a prevalent cause of nosocomial infections with a high mortality rate, and mucocutaneous infections significantly impact the quality of life of millions of patients a year. These infections pose substantial clinical challenges, particularly as the currently available antifungal treatment options are limited in efficacy and often toxic. Azoles are a mainstay of antifungal therapy and work by targeting the biosynthesis of ergosterol. However, there are rising rates of acquired azole resistance in various Candida species, and some species are considered intrinsically resistant to most azoles. Our research demonstrates the promising therapeutic potential of synergistically enhancing azoles with non-toxic, FDA-approved bisphosphonates. Repurposing bisphosphonates as antifungal synergists can bypass much of the drug development pipeline and accelerate the translation of azole-bisphosphonate combination therapy.

N-Butylphthalide Potentiates the Effect of Fluconazole Against Drug-Resistant Candida glabrata and Candida tropicalis. Evidence for Its Mechanism of Action.

Objective: To define the antifungal activity of n-butylphthalide alone or in combination with fluconazole in Candida glabrata and Candida tropicalis. Methods: The antifungal activity of n-butylphthalide alone and in combination with fluconazole was investigated by the classical broth microdilution method and the time-killing curve method. The QRT-PCR method was used to determine gene expressions changes of mitochondrial respiratory chain enzymes, drug efflux pumps and drug target enzymes in Candida glabrata and Candida tropicalis after n-butylphthalide treatment. Results: The MIC values of n-butylphthalide against Candida glabrata and Candida tropicalis ranged from 16 to 64 µg·mL-1. The FICI value of the combination of n-butylphthalide and fluconazole against drug-resistant Candida glabrata and Candida tropicalis ranged from 0.5001 to 0.5315 with partial synergism. Time-killing curves showed that 256 µg·mL-1 n-butylphthalide significantly inhibited the growth of drug-resistant colonies of Candida glabrata and Candida tropicalis, and 128 µg·mL-1 n-butylphthalide combined with 1 µg·mL-1 fluconazole had an additive effect. N-butylphthalide could alter the expression of mitochondrial respiratory chain enzymes COX1, COX2, COX3, and CYTB genes in Candida glabrata and Candida tropicalis (P< 0.05) and downregulate the expression of the drug efflux pump genes CDR1 and CDR2 in drug-resistant Candida glabrata to 3.36% and 3.65%, respectively (P<0.001), but did not affect the drug target enzyme ERG11 in drug-resistant Candida tropicalis. Conclusion: N-butylphthalide had antifungal activity against Candida glabrata and Candida tropicalis. N-butylphthalide improved the activity of fluconazole against drug-resistant Candida glabrata by affecting the expression of mitochondrial respiratory chain enzyme genes and reversing the high expression of drug efflux pump genes CDR1 and CDR2.

Fabrication and Characterization of Pectin Films Containing Solid Lipid Nanoparticles for Buccal Delivery of Fluconazole.

Fluconazole (FZ) is a potential antifungal compound for treating superficial and systemic candidiasis. However, the use of conventional oral drug products has some limitations. The development of buccal film may be a potential alternative to oral formulations for FZ delivery. The present study involved the development of novel FZ-loaded solid lipid nanoparticles (FZ-SLNs) in pectin solutions and the investigation of their particle characteristics. The particle sizes of the obtained FZ-SLNs were in the nanoscale range. To produce pectin films with FZ-SLNs, four formulations were selected based on the small particle size of FZ-SLNs and their suitable polydispersity index. The mean particle sizes of all chosen FZ-SLNs formulations did not exceed 131.7 nm, and the mean polydispersity index of each formulation was less than 0.5. The properties of films containing FZ-SLNs were then assessed. The preparation of all FZ-SLN-loaded pectin films provided the mucoadhesive matrices. The evaluation of mechanical properties unveiled the influence of particle size variation in FZ-SLNs on the integrity of the film. The Fourier-transform infrared spectra indicated that hydrogen bonds could potentially form between the pectin-based matrix and the constituents of FZ-SLNs. The differential scanning calorimetry thermogram of each pectin film with FZ-SLNs revealed that the formulation was thermally stable and behaved in a solid state at 37 °C. According to a drug release study, a sustained drug release pattern with a burst in the initial stage for all films may be advantageous for reducing the lag period of drug release. All prepared films with FZ-SLNs provided a sustained release of FZ over 6 h. The films containing FZ-SLNs with a small particle size provided good permeability across the porcine mucosa. All film samples demonstrated antifungal properties. These results suggest the potential utility of pectin films incorporating FZ-SLNs for buccal administration.

A ketogenic diet enhances fluconazole efficacy in murine models of systemic fungal infection.

Invasive fungal infections are a significant public health concern, with mortality rates ranging from 20% to 85% despite current treatments. Therefore, we examined whether a ketogenic diet could serve as a successful treatment intervention in murine models of Cryptococcus neoformans and Candida albicans infection in combination with fluconazole-a low-cost, readily available antifungal therapy. The ketogenic diet is a high-fat, low-carbohydrate diet that promotes fatty acid oxidation as an alternative to glycolysis through the production of ketone bodies. In this series of experiments, mice fed a ketogenic diet prior to infection with C. neoformans and treated with fluconazole had a significant decrease in fungal burden in both the brain (mean 2.66 ± 0.289 log10 reduction) and lung (mean 1.72 ± 0.399 log10 reduction) compared to fluconazole treatment on a conventional diet. During C. albicans infection, kidney fungal burden of mice in the keto-fluconazole combination group was significantly decreased compared to fluconazole alone (2.37 ± 0.770 log10-reduction). Along with higher concentrations of fluconazole in the plasma and brain tissue, fluconazole efficacy was maximized at a significantly lower concentration on a keto diet compared to a conventional diet, indicating a dramatic effect on fluconazole pharmacodynamics. Our findings indicate that a ketogenic diet potentiates the effect of fluconazole at multiple body sites during both C. neoformans and C. albicans infection and could have practical and promising treatment implications.IMPORTANCEInvasive fungal infections cause over 2.5 million deaths per year around the world. Treatments for fungal infections are limited, and there is a significant need to develop strategies to enhance antifungal efficacy, combat antifungal resistance, and mitigate treatment side effects. We determined that a high-fat, low-carbohydrate ketogenic diet significantly potentiated the therapeutic effect of fluconazole, which resulted in a substantial decrease in tissue fungal burden of both C. neoformans and C. albicans in experimental animal models. We believe this work is the first of its kind to demonstrate that diet can dramatically influence the treatment of fungal infections. These results highlight a novel strategy of antifungal drug enhancement and emphasize the need for future investigation into dietary effects on antifungal drug activity.

Photoactivated riboflavin inhibits planktonic and biofilm growth of Candida albicans and non-albicans Candida species.

Fungal infections caused by Candida species pose a serious threat to humankind. Antibiotics abuse and the ability of Candida species to form biofilm have escalated the emergence of drug resistance in clinical settings and hence, rendered it more difficult to treat Candida-related diseases. Lethal effects of Candida infection are often due to inefficacy of antimicrobial treatments and failure of host immune response to clear infections. Previous studies have shown that a combination of riboflavin with UVA (riboflavin/UVA) light demonstrate candidacidal activity albeit its mechanism of actions remain elusive. Thus, this study sought to investigate antifungal and antibiofilm properties by combining riboflavin with UVA against Candida albicans and non-albicans Candida species. The MIC20 for the fluconazole and riboflavin/UVA against the Candida species tested was within the range of 0.125-2 µg/mL while the SMIC50 was 32 µg/mL. Present findings indicate that the inhibitory activities exerted by riboflavin/UVA towards planktonic cells are slightly less effective as compared to controls. However, the efficacy of the combination towards Candida species biofilms showed otherwise. Inhibitory effects exerted by riboflavin/UVA towards most of the tested Candida species biofilms points towards a variation in mode of action that could make it an ideal alternative therapeutic for biofilm-related infections.

Clinico-mycological and therapeutic updates on cutaneous dermatophytic infections in the era of Trichophyton indotineae.

Trichophyton indotineae has emerged as a novel dermatophyte species resulting in treatment recalcitrant skin infections. While the earliest reports came from India, T. indotineae has now spread to many parts of the world and is rapidly becoming a global health concern. Accurate identification of T. indotineae requires elaborate mycological investigations which is beyond the domain of routine microbiology testing. Extensive, non-inflammatory and atypical presentations are commonly seen with this novel species. T. indotineae shows an alarmingly high rate of mutations in the squalene epoxidase gene leading to lowered in vitro susceptibility to terbinafine. This has also translated into a lowered clinical response and requirement of a higher dose and much longer durations of treatment with the drug. Although the species remains largely susceptible to itraconazole, prolonged treatment durations are required to achieve cure with itraconazole. Fluconazole and griseofulvin do not have satisfactory in vitro or clinical activity. Apart from requirement of prolonged treatment durations, relapse postsuccessful treatment is a distressing and yet unexplained consequence of this "species-shift." Use of third generation azoles and combinations of systemic antifungals is unwarranted as both have not demonstrated clear superiority over itraconazole given alone, and the former is an important class of drugs for invasive mycoses.

Coadministration of fluconazole to boost subtherapeutic sirolimus concentrations: A case report.

Individual sirolimus whole blood concentrations are highly variable, critically influenced by the concomitant use of cytochrome P450 (CYP) 3A inducers or inhibitors, and also modulated by food. Therapeutic drug monitoring is therefore recommended, especially at treatment start or in circumstances that can influence sirolimus exposure. In this case report, we highlight the challenge of achieving therapeutic sirolimus concentrations and present pragmatic solutions with regimen adaptions, pharmacokinetic enhancement (use of a drug­drug interaction), concentration monitoring, and subsequent modeling of population pharmacokinetics to support treatment decisions. In a 69-year-old female patient with allogeneic hematopoietic stem cell transplantation, sirolimus concentrations were stable until she developed cerebral toxoplasmosis with tonic­clonic seizures. During treatment of this acute infection, sirolimus concentrations dropped to subtherapeutic levels and remained largely unaffected by dose increases. [Correction added on 4 May 2024, after first online publication: The word "tacrolimus concentrations" has been changed to "sirolimus concentrations" in the preceding sentence.] Only the simultaneous administration of the CYP3A4 inhibitor fluconazole and a shortening of the sirolimus dosing intervals to a (non-approved) twice-daily administration led to successful control of the concentrations, which ultimately even made a dose reduction possible. This intervention resulted in an increase of sirolimus mean trough concentration to 5.85 ng/mL, i.e., into the desired target range. Additionally, a higher ratio of sirolimus trough levels/daily dose from 26.9 to 109 ng/mL/mg/kg/day was achieved with the initiation of fluconazole. Thus, this case report describes the use of clinical pharmacological concepts and pharmacokinetic modeling to optimize treatment strategies in an individual patient. This strategy could be generalized to other CYP inhibitors and other treatment regimens.

Optimizing fluconazole-embedded transfersomal gel for enhanced antifungal activity and compatibility studies.

Fungal infections are of major concern all over the globe, and fluconazole is the most prevalently used drug to treat it. The goal of this research work was to formulate a fluconazole-embedded transfersomal gel for the treatment of fungal infections. A compatibility study between fluconazole and soya lecithin was performed by differential scanning calorimetry (DSC). Transfersomes were formulated by a thin-film hydration technique using soya lecithin and Span 80. A central composite design was adopted to prepare different formulations. Soya lecithin and Span 80 were chosen as independent variables, and the effect of these variables was studied on in vitro drug diffusion. Formulations were evaluated for entrapment efficiency and in vitro drug diffusion. The results of in vitro drug diffusion were analyzed using the analysis of variance (ANOVA) test. Optimized formulation was prepared based on the overlay plot and evaluated by scanning electron microscopy, DSC, vesicle size, polydispersity index (PDI), zeta potential, and in vitro drug diffusion studies. An optimized formulation was loaded into xanthan gum gel base and evaluated for pH, viscosity, in vitro and ex vivo drug diffusion, and antifungal activity. DSC studies revealed compatibility between fluconazole and soya lecithin. Entrapment efficiency and in vitro drug diffusion of various formulations ranged between 89.92% ± 0.20% to 97.28% ± 0.42% and 64% ± 1.56% to 85% ± 2.05%, respectively. A positive correlation was observed between in vitro drug diffusion and Span 80; conversely, a negative correlation was noted with soya lecithin. Entrapment efficiency, particle size, zeta potential, PDI, and drug diffusion of optimized formulation were 95.0% ± 2.2%, 397 ± 2 nm, -38 ± 5 mV, 0.43%, and 81 % ± 2%, respectively. SEM images showed well-distributed spherical-shaped transfersomes. In vitro, ex vivo drug diffusion and antifungal studies were conclusive of better diffusion and enhanced antifungal potential fluconazole in transfersomal formulation.

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.

Adhesion and biofilm formation by two clinical isolates of Trichosporon Cutaneum in various environmental conditions.

Trichosporon spp. is an emerging opportunistic pathogen and a common cause of both superficial and invasive infections. Although Trichosporon asahii is the most frequently isolated species, Trichosporon cutaneum is also widely observed, as it is the predominant agent in cases of white Piedra and onychomycosis. Trichosporon spp. is a known to produce biofilms, which serve as one of its virulence mechanisms, however, there is limited data available on biofilms formed by T. cutaneum. Thus, the aim of this study was to assess the adhesion and biofilm formation of two clinical isolates of T. cutaneum under various environmental conditions (including temperature, nutrient availability, and carbon source), as well as their tolerance to fluconazole. Adhesion was tested on common abiotic substrates (such as silicone, glass, and stainless steel), revealing that T. cutaneum readily adhered to all surfaces tested. CV staining was applied for the evaluation of the environment influence on biofilm efficiency and it was proved that the nutrient availability has a major impact. Additionaly, fluorescent staining was employed to visualize the morphology of T. cutaneum biofilm and its survival in the presence of fluconazole. Hyphae production was shown to play a role in elevated biofilm production in minimal medium and increased tolerance to fluconazole.