Concentrations Of Fluconazole Research Articles

Distribution and Fluconazole Susceptibility Pattern of Fungi Isolated from Saloon Equipment in Rivers State, Nigeria

The distribution and susceptibility pattern of fungal isolates from barbing equipment to Fluconazlole in barber shops distributed in three major cities; Bori, Omoku, and Port Harcourt in Rivers State, Nigeria were investigated. The fungal load including dermatophytes on the equipment (brush, clipper and comb) were determined using swab plate technique in which Sabouraud dextrose agar and dermatophyte test medium were used as the culture medium. Inoculated plates were incubated at 25°C. Antifungal Susceptibility testing was carried in which fluconazole was used as the antifungal agent at concentrations of 100mg/ml, 50mg/ml, 25mg/ml, and 12.5mg/ml concentration using two-fold dilution. The mean range of the fungal counts for clipper, brush and comb were: 34.34x102 to 49.0x102 CFU/cm2, 66.95 to 93.26 CFU/cm2 and 40.09 to 74.61 x102 CFU/cm2, respectively. Brush had the highest fungal counts followed by the comb while the clipper had the least fungal counts. The fungal counts recorded in brushes were significantly higher (p≥0.05) than those recorded for the clippers. Total of forty-four fungal isolates belonging to six genera and their percentage occurrence include; Aspergillus flavus (11.36%) Aspergillus terrus (11.36%), Fusarium solani (22.72%), Mucor indicus (15.91%), Rhizopus nigricans (15.91%), Trichophyton rubrum (4.54%) and Penicillium italicum (18.2%). The susceptibility pattern showed that except for Trichophyton rubrum, all fungal isolates were completely inhibited at 100mg/ml concentration of fluconazole. Also, except for Mucor indicus, the Minimal inhibitory Concentration of fluconazole was recorded at 12.5mg/ml. The occurrence of these organisms in barbing equipment calls for concern and the need for regulation of the barbing industry to prevent disease outbreaks is recommended.

Development and validation of a sensitive LC-MS/MS method for determination of intracellular concentration of fluconazole in Candida albicans.

Systemic candidiasis is the fourth leading cause of healthcare-associated infections worldwide. The combination therapy based on existing antifungal agents is well-established to overcome drug resistance and restore antifungal efficacy against drug-resistant strains. In this study, a simple and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to quantify the intracellular fluconazole (FLC) content in the opportunistic human fungal pathogen Candida albicans. The cell lysates were prepared by lysing C. albicans cells with Precellys homogenizers and FLC was extracted with methylene chloride. The entire extraction approach was simple, precise and reliable. The extracts were separated on a Zorbax SB-C18 column using a mobile phase of acetonitrile (solvent A) and deionized water plus 0.1% formic acid. FLC and ketoconazole (KCZ, internal standard) were monitored in positive mode using electrospray ionization source. The multiple reaction monitoring transitions (precursor to product) were monitored for FLC m/z 307.1 → 238.2 and for the internal standard KCZ m/z 531.2 → 489.1. The linear for this method were in the range from 5.0 to 1000.0 ng/mL. The precision and accuracy of the samples were relative standard deviations (RSD) < 1.0% for intra-day and RSD < 0.51% for inter-day. The overall recovery of FLC from samples was higher than 77.61%. Furthermore, this method was successfully applied and validated in 36 clinical isolated strains. Taken together, we established a highly accurate, efficient, and reproducible method for quantifying the intracellular content of FLC in C. albicans.

P309 Clonal outbreak of Trichophyton tonsurans causing tinea capitis among wrestlers in Beijing, China

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectives Trichophyton tonsurans, an anthropophilic dermatophyte, mostly causes tinea capitis and tinea corporis and is often associated with outbreaks among athletes involved in combat sports, such as wrestlers and judo athletes. Here, we report an outbreak of tinea capitis caused by Trichophyton tonsurans among five juvenile athletes aged ten to fourteen in a wrestling team in Beijing, China.MethodsA total of 5 wrestlers aged 10-14 years presented with alopecia, erythema, scales, and pustule on the scalp (Fig. 1a). Scrapings from the lesions were performed by direct microscopic examination using 10% potassium hydroxide (KOH) with calcofluor white and fungal culture. The fungal pathogens were identified by morphology and sequencing of the internal transcribed spacer (ITS) regions. Multilocus genotyping analysis was performed by sequencing of the five gene loci including the ITS and non-transcribed spacer (NTS) of the ribosomal RNA (rRNA) locus, alkalineprotease-1 (ALP1), metalloprotease-5 (MEP5), carboxypeptidases Y (CarbY), which show intraspecies diversity and can be applied to epidemiological investigations and determining the route of infection transmission. Antifungal susceptibility of terbinafine (TBF), itraconazole (ITC), fluconazole (FLC), ketoconazole (KTC), and amphotericin B (AMB) against the causative fungal isolates was determined by broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) M38-A3 document.ResultsThe five patients were diagnosed with tinea capitis because of the scalp lesions, spores, and hyphae observed by direct microscopic examination (Fig. 1b), and positive fungal culture. The causative isolates were all identified as T. tonsurans by macroscopic and microscopic characteristics (Fig. 2) and sequencing of ITS regions. The sequences of ITS, NTS, ALP1, MEP5, and CarbY were identical among the five isolates, revealing a single genotype. The abovementioned sequences have been deposited to Genbank. The minimum inhibitory concentrations (MICs) of TBF, ITC, FLC, KTC, and AMB against five T. tonsurans isolates were 0.015 μg/ml, 0.015 μg/ml, 8 μg/ml, 0.06 μg/ml, and 0.5 μg/ml, respectively.ConclusionWe report an outbreak of tinea capitis caused by T. tonsurans among wrestlers in Beijing, China. Multilocus genotyping analysis revealed that all isolates consisted of a single genotype, suggesting the outbreak may be caused by a single strain of T. tonsurans. The isolates were all susceptible to the common antifungal drugs treating tinea capitis.

P018 Susceptibility profile of Candida albicans causing bloodstream infections against azole antifungals over 5 years: A prospective observational study

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesTo study the drug susceptibility profile of Candida albicans isolated from bloodstream infection (BSI) against azole anti-fungal agents (fluconazole, itraconazole, voriconazole, and posaconazole) in a tertiary care hospital over 5 years.MethodsAll blood culture bottles from patients attending our tertiary care hospital between January 2017 to December 2021 and growing Candida albicans were included in the analysis. All blood culture bottles were continuously monitored automatically inside BACTEC 9240 (BD Becton Dickinson, USA). Beep-positive bottles were further processed by preparation of Gram stain smears, sub-culture onto 5% sheep blood agar and MacConkey agar and spot inoculation onto Sabouraud's Dextrose agar (SDA). Isolates were identified as C. albicans using MALDI-TOF MS (Bruker Daltoniks, Bremen, Germany). Broth microdilution (BMD) was performed to determine minimum inhibitory concentration (MIC) of fluconazole, itraconazole, voriconazole, and posaconazole, in accordance with CLSI document M27-A3 and CLSI M27-S4. Representative isolates were further processed to determine the genetic basis of azole resistance (results will be shared at the conference).ResultsA total of 889 C. albicans were isolated from BSI over 5 years. The rates of resistant were 0.9%, 4.3%, and 1.4%; and that of susceptible dose dependent were 0.8%, 28.5%, and 0.4% for fluconazole, itraconazole, and voriconazole respectively (Fig. 1). The MIC90 of the isolates to fluconazole, itraconazole, voriconazole, and posaconazole were 2 μg/ml, 0.25 μg/ml, 0.5 μg/ml and 0.25 μg/ml, respectively (Fig. 2).ConclusionAzole antifungals continue to retain favorable MIC90 against C. albicans BSI isolates. Flucanozole continues to be the favored drug of choice due to low side-effects, cost-effectiveness, and least resistant rates.

S8.5d Cryptococcus neoformans and Cryptococcus gattii clinical isolates from Colombia develop heteroresistance to fluconazole at high concentrations

S8.5 Genotyping of Cryptococcus neoformans and C. gattii, September 23, 2022, 3:00 PM - 4:30 PMIntroductionCryptococcosis is a worldwide mycosis caused by Cryptococcus neoformans and Cryptococcus gattii. Although resistance to antifungals is infrequent, isolates with decreased susceptibility to fluconazole have been reported globally, including Colombia, which may be due to 1) heteroresistance, defined as the ability to adapt to increasing concentrations of this azolic antifungal, and 2) point mutations in the EGR11 gene encoding the fluconazole target enzyme, lanosterol 14-α-demethylase.ObjectiveTo determine the development of heteroresistance to fluconazole in C. neoformans and C. gattii clinical isolates from Colombia and to amplify and sequence the ERG11 gene of the isolates to seek for mutations that might characterize resistant or heteroresistant phenotypes.MethodsThe minimum inhibitory concentration (MIC) to fluconazole was determined in 31 and 24 isolates of C. neoformans and C. gattii, respectively, using broth microdilution. Heteroresistance was evaluated by plating each isolate on YPD agar that contained fluconazole at concentrations equal to the MIC of each isolate. Heteroresistant colonies were then replated at increasing concentrations of fluconazole, as high as 128 μg/ml.ResultsAll isolates were susceptible to fluconazole with MICs of 1 μg/ml (n = 3), 2 μg/ml (n = 6), 4 μg/ml (n = 17), 8 μg/ml (n = 23), 16 μg/ml (n = 5), and 32 μg/ml (n = 1). However, all isolates developed heteroresistant colonies, with increments in the MIC from 2 to 6 dilutions. Notably, 5 (16.1%) isolates of C. neoformans and 8 (33.3%) of C. gattii, grew up to 64 μg/ml of fluconazole, which is the MIC that defines resistance to this azole, and 1 (3,2%) isolate of C. neoformans and 4 (16.7%) of C. gattii grew up to 128 μg/ml of fluconazole. Currently, the ERG11 gene is being amplified for further sequencing. Conclusion: clinical isolates of C. neoformans and C. gattii that develop heteroresistance to fluconazole in high concentrations circulate in Colombia, which is important since this characteristic contributes to the relapse of cryptococcosis during therapy with this triazole.

P015 A study to demonstrate heteroresistance and tolerance to azoles in Candida tropicalis

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objective Candida tropicalis rarely exhibits a trailing growth phenomenon in in vitro antifungal susceptibility testing (AFST) of azoles. This phenomenon is considered as ‘sensitive’ as per current CLSI guidelines. This phenomenon has been linked to treatment failure and such isolates could either be heteroresistant (HR) or tolerant, which is known with bacteria. HR subtype (<1%) of an isogenic susceptible cell population grows at drug concentrations at least 8 times higher than the minimum inhibitory concentration (MIC). Tolerant cells (5%-90% of the population) endure antimicrobial treatment several times above MIC. Characterizing these isolates could help relate their clinical susceptibility profiles to the treatment outcome. The aim of the study was to differentiate heteroresistance and tolerance from true azole resistance in C. tropicalis clinical isolates.MethodsA total of 247 clinical isolates of C. tropicalis collected over a period of 7 months (September 2021 through March 2022) from PGIMER, Chandigarh were included in the study. Identification was done by MALDI-TOF MS and antifungal susceptibility testing to fluconazole was performed by CLSI broth microdilution method (M27-A3). Isolates exhibiting trailing growth phenomena were considered for further characterization. Cells from the trailing growth of each isolate were sub-cultured onto yeast extract peptone dextrose (YPD) agar to observe for phenotypic variations with and without fluconazole. The area under the population analysis (PAP) curve (AUC) was performed to determine the degree of heteroresistance. Yeast cells within the range of 102-105 cells/ml were spot inoculated in six replicates each onto a gradient of fluconazole (0.125-256 μg/ml) in YPD agar and incubated at 35°C for 5 days to determine the viable colony forming units (CFU).ResultsOut of the 225 fluconazole susceptible C. tropicalis isolates, 10 (4.4%) were found to exhibit a trailing growth phenomenon. A dose-response relationship showed a multimodal population distribution in these isolates with varying degrees of heteroresistance as demonstrated by area under curve that ranged from 171-1331. The heterogeneous subpopulations sampled from the growth at the highest fluconazole concentration (64 μg/ml) exhibited a similar MIC (± one 2-fold difference) as that of the parent isolate when tested individually. The heterogeneity range that determines a fold difference breakpoint of the isolates varied from 16 to >256.ConclusionAn isogenic population of cells under the effect of fluconazole could give rise to phenotypically different subpopulations. With repeated exposure to the azole drugs, these seemingly susceptible isolates can emerge as fully resistant population. Clinically, the implications include relapse, treatment failure, and persistent chronic infection, owing to which this phenomenon needs attention. Current CLSI guidelines do not provide any criteria to separately classify these isolates from the susceptible and resistant varieties. Hence a definitive cut-off is warranted to identify the HR and tolerant subtypes. The AUC-PAP method could be refined further for discriminating heteroresistance from true resistance.

A post-mortem analysis of tenofovir, lamivudine, efavirenz and fluconazole penetration in female genital tissues.

Optimal penetration of anti-infectives in the female genital tract (FGT) is paramount in the treatment and prevention of infectious diseases. While exposure of anti-infectives in lower FGT tissues (e.g. cervix, vagina) has been described, little data exist on upper genital tissues (e.g. ovary, uterus). Autopsies were performed and post-mortem tissues were collected within 24 h of death for female participants with advanced HIV in Uganda (n = 27). Tenofovir, lamivudine, efavirenz and fluconazole concentrations were measured using LC-MS/MS in plasma, ovarian, uterine, cervical and vaginal tissues. Tissue penetration was calculated as tissue-to-plasma concentration ratios (TPRs). TPRs of tenofovir, lamivudine and fluconazole were highest in vaginal tissue (medians 1.86, 1.83 and 0.94, respectively), while the TPR of efavirenz was highest in ovarian tissue (median 0.65). With cervix as a reference compartment, vaginal TPRs were significantly higher than cervical for all four drugs; TPRs of efavirenz in uterine and ovarian compartments were also significantly higher than cervical. Most of the post-mortem FGT samples had a TPR of greater than 1 for tenofovir and lamivudine, while less than 50% had a TPR of greater than 1 for both efavirenz and fluconazole. Penetration of anti-infectives was not homogeneous among the FGT compartments. Approximately 70% of FGT tissues had a TPR of greater than 1 for tenofovir and lamivudine, favouring the prevention of local HIV replication and transmission in the FGT.

Pharmacokinetics of fluconazole after oral administration to healthy beagle dogs

Fluconazole can be effective in the treatment of superficial mycoses in dogs. However, the pharmacokinetics of fluconazole have not yet been evaluated to determine its optimal dosing regimen. This study aimed to determine the plasma concentration of fluconazole after single and multiple administrations at two different dosages in dogs. Eight healthy beagle dogs were divided into two groups, and each group received either 5 or 10mg/kg of fluconazole per os. The pharmacokinetics of fluconazole was determined following single and multiple administrations p.o. Single- and multiple-dose treatment periods were separated by a washout period of seven days. Plasma concentrations of fluconazole were determined by established high-performance liquid chromatography coupled with tandem mass spectrometry system. In the 5mg/kg group, the mean maximum concentrations (Cmax ) and the area under the plasma concentrations (AUC0-24h ) were 4.84 μg/mL and 85.56 μg*h/mL, respectively, after single administration and 6.58 μg/mL and 119.52 μg*h/mL, respectively, after multiple administrations. In the 10mg/kg group, the Cmax and AUC0-24h were 5.67 μg/mL and 109.19 μg*h/mL, respectively, after single administration and 15.10μg/mL and 291.51 μg*h/mL, respectively, after multiple administrations. The Cmax (p< 0.001) and AUC0-24h (p< 0.001) were significantly lower in the 5mg/kg group than those in the 10mg/kg group at multiple administrations. Fluconazole accumulates in plasma and exhibits dose-proportional pharmacokinetics after multiple doses, and was safe and well tolerated at these doses for short-term administration.

A computer vision chemometric-assisted approach to access pH and glucose influence on susceptibility of Candida pathogenic strains.

Microorganisms adapt to environmental conditions as a survival strategy for different interactions with the environment. The adaptive capacity of fungi allows them to cause disease at various sites of infection in humans. In this study, we propose digital images as responses of a complete factorial 23. Furthermore, we compared two experimental approaches: the experimental design (3D) and the checkerboard assay (2D) to know the influence of pH, glucose, and fluconazole concentration on different strains of the genus Candida. The digital images obtained from the factorial 23 were used as input in the PCA-ANOVA to analyze the results of this experimental design. pH modification in the culture medium modifies the susceptibility in some species less adapted to this type of modification. For the first time, to the best of our knowledge, digital images were used as input to PCA-ANOVA to obtain information on Candida spp.. Therefore, a higher concentration of antifungals is needed to inhibit the same strain at a lower pH. In short, we present an alternative with less use of reagents and time. In addition, the use of digital images allows obtaining information about fungal susceptibility with three or more factors.

Molecular Typing of Dermatophytes Isolated from Pupils and Staff Members of Some Selected Schools in Anambra State, Nigeria

Dermatophytes are the fungal pathogens of human and animals infecting the keratinized tissues of the body namely skin, hair and nails. They include species of Trichophyton, Microsporum and Epidermophyton. This study was aimed at molecular typing of dermatophytes isolated from school pupils and staff members of some selected Schools in Anambra East and Ayamelum L.G.As of Anambra State in Nigeria. One thousand (1000) samples (scalp/hair, nail, feet, glabrous skin and groin/perianal) were collected from pupils and staff members of both gender and age bracket of (1 to 10, 11 to 20, 21 to 30 etc ) years that showed visible signs of skin infection located in these two Local Government Areas. Standard procedures were employed in processing of test samples and inoculation on dermatophyte test medium. The plates were incubated at room temperature (25 – 270C) for 7 – 10days for observation of fungal growth. Colonial, morphology, and molecular studies and sequencing were used for identification. Sensitivity was performed using sterilized discs (6mm) prepared from whatman No. 1 filter paper, impregnanted with different concentrations (25mg, 50mg, 100mg and 200mg) of terbinafine, itraconazole, ketoconazole, fluconazole and griseofulvin dissolved in 2% dimethylsuphuroxide (DMSO). Molecular studies were used as confirmatory tests on dermatophytic isolates using Sanger sequencing method. The results show that the dermatophytic isolates includes: Trichophyton tonsurans, Microsporum audounii, Trichophyton rubrum, Microsporum canis, Trichophyton violaceum, Trichophyton verrucosum, Trichophyton mentagrophytes, and Epidermorphyton flocossum. Results also revealed the nucleotide sequences of the dermatophytes and genetic relationships between isolated dermatophytes from different pupils and schools. PCR-RFLP was used as gold standard for the diagnosis and Confirmation of Source of infection of dermatophytes and can aid in initiating prompt and appropriate antifungal therapy. Phylogenetic tree was drawn to show the relationships between the isolates.

Genomic Diversity across Candida auris Clinical Isolates Shapes Rapid Development of Antifungal Resistance In Vitro and In Vivo.

ABSTRACTAntifungal drug resistance and tolerance pose a serious threat to global public health. In the human fungal pathogen, Candida auris, resistance to triazole, polyene, and echinocandin antifungals is rising, resulting in multidrug resistant isolates. Here, we use genome analysis and in vitro evolution of 17 new clinical isolates of C. auris from clades I and IV to determine how quickly resistance mutations arise, the stability of resistance in the absence of drug, and the impact of genetic background on evolutionary trajectories. We evolved each isolate in the absence of drug as well as in low and high concentrations of fluconazole. In just three passages, we observed genomic and phenotypic changes including karyotype alterations, aneuploidy, acquisition of point mutations, and increases in MIC values within the populations. Fluconazole resistance was stable in the absence of drug, indicating little to no fitness cost associated with resistance. Importantly, two isolates substantially increased resistance to ≥256 μg/mL fluconazole. Multiple evolutionary pathways and mutations associated with increased fluconazole resistance occurred simultaneously within the same population. Strikingly, the subtelomeric regions of C. auris were highly dynamic as deletion of multiple genes near the subtelomeres occurred during the three passages in several populations. Finally, we discovered a mutator phenotype in a clinical isolate of C. auris. This isolate had elevated mutation rates compared to other isolates and acquired substantial resistance during evolution in vitro and in vivo supporting that the genetic background of clinical isolates can have a significant effect on evolutionary potential.

Total bodyweight and sex both drive pharmacokinetic variability of fluconazole in obese adults.

Fluconazole is commonly used to treat or prevent fungal infections. It is typically used orally but in critical situations, IV administration is needed. Obesity may influence the pharmacokinetics and therapeutic efficacy of a drug. In this study, we aim to assess the impact of obesity on fluconazole pharmacokinetics given orally or IV to guide dose adjustments for the obese population. We performed a prospective pharmacokinetic study with intensive sampling in obese subjects undergoing bariatric surgery (n = 17, BMI ≥ 35 kg/m2) and non-obese healthy controls (n = 8, 18.5 ≤ BMI < 30.0 kg/m2). Participants received a semi-simultaneous oral dose of 400 mg fluconazole capsules, followed after 2 h by 400 mg IV. Population pharmacokinetic modelling and simulation were performed using NONMEM 7.3. A total of 421 fluconazole concentrations in 25 participants (total bodyweight 61.0-174 kg) until 48 h after dosing were obtained. An estimated bioavailability of 87.5% was found for both obese and non-obese subjects, with a 95% distribution interval of 43.9%-98.4%. With increasing total bodyweight, both higher CL and Vd were found. Sex also significantly impacted Vd, being 27% larger in male compared with female participants. In our population of obese but otherwise healthy individuals, obesity clearly alters the pharmacokinetics of fluconazole, which puts severely obese adults, particularly if male, at risk of suboptimal exposure, for which adjusted doses are proposed.

Lack of Association between Fluconazole Susceptibility and ERG11 Nucleotide Polymorphisms in Cryptococcus neoformans Clinical Isolates from Uganda.

Fluconazole is the drug of choice for cryptococcal meningitis (CM) monoprophylaxis in resource-limited settings such as Uganda. Emerging fluconazole resistance linked to mutations in the Cryptococcus neoformans ERG11 gene (CYP51) has been observed in clinical isolates. Currently, the single nucleotide polymorphisms [SNPs] in the Cryptococcus spp. ERG11 gene that could be responsible for fluconazole resistance are poorly characterized within Ugandan C. neoformans clinical isolates. If available, this information would be useful in the management of cryptococcosis among HIV patients. This cross-sectional study investigates the SNPs present in the coding region of the C. neoformans ERG11 gene to determine the relationship between the SNPs identified and fluconazole susceptibility of the clinical isolates. 310 C. neoformans isolates recovered from the Cerebrospinal Fluid (CSF) of patients with HIV and cryptococcal meningitis were examined. The fluconazole half-maximal inhibitory concentrations (IC50 range: 0.25–32 μg/mL) was determined using the microbroth dilution method. A total of 56.1% of the isolates had low IC50 values of <8 μg/mL while 43.9% had high IC50 values ≥ 8 μg/mL. We amplified and sequenced 600 bp of the ERG11 coding sequence from 40 of the clinical isolates. Novel synonymous and 2 missense mutations, S460T and A457V, were identified in the ERG11 gene. The identified SNPs were not associated with differences in fluconazole IC50 values in vitro (p = 0.179).

Inhibition of Hsp90 regulates fluconazole resistance in the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast, found in the lungs of immunocompromised hosts. In the human host, Cryptococcusis subject to multiple forms of environmental stress, including heat stress. Heat shock protein 90 (Hsp90) is an essential protein that has been shown to protect fungal cells against extreme temperature in the human host and to assist in proper cell division. Hsp90 has also been implicated in resistance to the anti‐fungal drug fluconazole. To better characterize the role of Hsp90 in fluconazole resistance in Cryptococcuswe addressed two questions: 1. Does concomitant partial inhibition of Hsp90 and treatment with fluconazole lead to increased sensitivity to fluconazole and 2. Does prolonged partial inhibition of Hsp90 prior to treatment with fluconazole lead to subsequent increase in development of fluconazole resistance. Using growth assays, disk assays, and a standardized e‐test to determine the Minimum Inhibitory Concentration (MIC) of fluconazole, we found that indeed, pharmacological partial inhibition of Hsp90 with radicicol, concomitant with fluconazole treatment leads to increased sensitivity to fluconazole. However, initial pre‐treatment of Cryptococcuswith sub‐inhibitory concentration of radicicol followed by exposure to fluconazole leads to development of fluconazole resistant Cryptococcuscolonies. We characterized the resistant colonies, which show a lower MIC to fluconazole compared to cells that were never exposed to radicicol and fluconazole. Cells derived from the resistant colonies exhibit slower growth on drug‐free media, and lose resistance to the drug after passaging in drug‐free media.Based on the fact that resistance to fluconazole is lost when fluconazole is removed from the environment, we hypothesize that compromising Hsp90 function during the pre‐treatment with radicicol triggers aneuploidy in Cryptococcus neoformans. Furthermore, we hypothesize that the resistant colonies are aneuploids that possess an altered chromosome number to maintain drug resistance in response to an environmental insult, such as presence of fluconazole.

Enhancing effect of chitosan on methylene blue-mediated photodynamic therapy against C. albicans: A study in planktonic growth, biofilms, and persister cells

Chitosan (CS) is a natural polymer extracted from the exoskeleton of crustaceans. Due to its cationic structure, CS has been studied as a possible enhancer of antimicrobial photodynamic therapy (aPDT). The objective was to evaluate the association of CS with methylene blue (MB)-mediated aPDT on Candida albicans, investigating its effects on planktonic growth, biofilms, and cells persistent to fluconazole. The ability of CS to interfere with MB absorption by Candida cells was also evaluated. For the assays, planktonic cells of C. albicans were cultivated for 24 h, and the biofilms were formed for 48 h. For the induction of persister cells, C. albicans was cultivated with high concentration of fluconazole for 48 h. Treatments were performed with MB, CS or MB+CS, followed by irradiation with LED (660 nm ). As results, aPDT with MB (300 µm) reduced the planktonic cells by 1.6 log10 CFU, while the MB+CS association led to a reduction of 4.8 log10 CFU. For aPDT in biofilms, there was a microbial reduction of 2.9 log10 CFU for the treatment with MB (600 µm) and 5.3 log10 CFU for MB+CS. In relation to persister cells, the fungal reductions were 0.4 log10 CFU for MB and 1.5 log10 CFU for MB+CS. In the absorption assays, the penetration of MB into Candida cells was increased in the presence of CS. It was concluded that CS enhanced the antimicrobial activity of aPDT in planktonic growth, biofilms, and persister cells of C. albicans, probably by facilitating the penetration of MB into fungal cells.

Investigation of fluconazole heteroresistance in clinical and environmental isolates of Cryptococcus neoformans complex and Cryptococcus gattii complex in the state of Amazonas, Brazil

Cryptococcosis affects many people and is caused by fungi of the Cryptococcus neoformans/Cryptococcus gattii complexes. These agents appear to become more resistant to antifungals when exposed to increasing concentrations of antifungals due to a phenomenon called heteroresistance.

Use of Modeling and Simulation to Predict the Influence of Triazole Antifungal Agents on the Pharmacokinetics of Crizotinib

Crizotinib is used for the treatment of c-ros oncogene 1-positive advanced non-small-cell lung cancer. Triazole antifungal agents are widely used for invasive fungal infections in clinical practice. To predict the potential influence of different triazoles (voriconazole, fluconazole, and itraconazole) on the pharmacokinetics of crizotinib by modeling and simulation the physiologically based pharmacokinetic models were established and validated in virtual cancer subjects through Simcyp software based on the essential physicochemical properties and pharmacokinetic data collected. The validated physiologically based pharmacokinetic models were applied to predict the drug-drug interactions between crizotinib and different triazoles (voriconazole, fluconazole, or itraconazole) in patients with cancer. Crizotinib and triazole antifungal agents were administered orally. The predicted plasma concentration vs time profiles of crizotinib, voriconazole, fluconazole, and itraconazole showed good agreement with observed, respectively. The geometric mean area under the plasma concentration-time curve (AUC) of crizotinib was increased by 84%, 58%, and 79% when coadministered with voriconazole, fluconazole, or itraconazole at multiple doses, respectively. The drug-drug interaction results showed increased pharmacokinetic exposure (maximum plasma concentration and area under the plasma concentration-time curve) of crizotinib when coadministrated with different triazoles (voriconazole>itraconazole>fluconazole). Among the 3 triazoles, voriconazole exhibited the most significant influence on the pharmacokinetic exposure of crizotinib. In clinic, adverse drug reactions and toxicity related to crizotinib should be carefully monitored, and therapeutic drug monitoring for crizotinib is recommended to guide dosing and optimize treatment when coadministered with voriconazole, fluconazole, or itraconazole.

Environmentally relevant concentrations of fluconazole alter the embryonic development, oxidative status, and gene expression of NRF1, NRF2, WNT3A, WNT8A, NRD1, and NRD2 of Danio rerio embryos

Up to date, there is little information published concerning fluconazole (FCZ) toxicity at environmentally relevant concentrations. Bearing in mind the above background of FCZ, we aimed to evaluate the embryotoxic effects environmentally relevant concentrations of FCZ (800-1000 ng/L) may induce in Danio rerio. Moreover, we also wanted to prove whether these FCZ concentrations could generate oxidative stress and alter the expression of several genes related to the antioxidant mechanisms, sterol and retinol biosynthesis, and embryogenesis. Our findings demonstrate that FCZ, at all concentrations, induced pericardial edema, yolk sac deformation, scoliosis, and tail malformation in embryos. Moreover, we also demonstrated this drug altered the redox equilibrium of fish, promoting the production of lipoperoxidation level, hydroperoxide content, and protein carbonyl content in a concentration-dependent manner. Concerning gene expression, FCZ downregulated wingless-type MMTV integration site family member 3a (WNT3A), wingless-type MMTV integration site family member 8a (WNT8A), N-arginine dibasic convertase 1 (NRD1), and N-arginine dibasic convertase 2 (NRD2) and upregulated cytochrome P450 family 26 subfamily a member 1 (CYP26A1), cytochrome P450 family 26 subfamily a member 1B (CYP261B), nuclear respiratory factor 1 (NRF1), and nuclear respiratory factor 2 (NRF2) in D. rerio larvae. Collectively, our results point out that FCZ, at low concentrations, may alter the embryogenesis, oxidative status, and expression of several genes in D. rerio embryos via an impairment in sterol and retinol biosynthesis. Thus, our results provide some of the first evidence that FCZ, even at environmentally relevant concentrations, is harmful to aquatic species.

Prevalence of Antifungal Resistance, Genetic Basis of Acquired Azole and Echinocandin Resistance, and Genotyping of Candida krusei Recovered from an International Collection.

This study was designed to evaluate the prevalence of antifungal resistance, genetic mechanisms associated with in vitro induction of azole and echinocandin resistance and genotyping of Candida krusei, which is intrinsically resistant to fluconazole and is recovered from clinical and nonclinical sources from different countries. Our results indicated that all the isolates were susceptible or had the wild phenotype (WT) to azoles, amphotericin B, and only 1.27% showed non-WT for flucytosine. Although 70.88% of the isolates were resistant to caspofungin, none of them were categorized as echinocandin-resistant as all were susceptible to micafungin and no FKS1 hot spot 1 (HS1) or HS2 mutations were detected. In vitro induction of azole and echinocandin resistance confirmed the rapid development of resistance at low concentrations of fluconazole (4 μg/ml), voriconazole (0.06 μg/ml), and micafungin (0.03 μg/ml), with no difference between clinical and nonclinical isolates in the resistance development. Overexpression of ABC1 gene and FKS1 HS1 mutations were the major mechanisms responsible for azole and echinocandin resistance, respectively. Genotyping of our 79 isolates coupled with 217 other isolates from different sources and geography confirmed that the isolates belong to two main subpopulations, with isolates from human clinical material and Asia being more predominant in cluster 1, and environmental and animals isolates and those from Europe in cluster 2. Our results are of critical concern, since realizing that the C. krusei resistance mechanisms and their genotyping are crucial for guiding specific therapy and for exploring the potential infection source.

Effect of Allium cepa loaded polyacrylonitrile and polyvinyl pyrrolidone nanofibers on Candida albicans growth and the expression of CDR1 and CDR2 genes.

Background and Purpose: This study aimed to assess the effect of Allium cepa ethanolic extract (ACE) loaded polyacrylonitrile (PAN) and polyvinyl pyrrolidone (PVP) nanofibers on Candida albicans (C. albicans) CDR1 and CDR2 genes expression.Materials and Methods: The minimum inhibitory concentrations (MICs) of ACE against C. albicans ATCC 10231 and clinical fluconazole (FLC)-resistant C. albicans PFCC 93-902 were determined using the Clinical and Laboratory Standards Institute (CLSI) protocol (M27-Ed4) at a concentration range of 45.3-5800 µg/ml. The nanofibers containing ACE (60 wt%) were fabricated using the electrospinning technique. The expression of the CDR1 and CDR2 genes was studied in the fungus exposed to ACE-loaded nanofibers and 0.5×MIC concentration of FLC using the real-time polymerase chain reaction.Results: MIC50 and MIC90 of ACE against FLC-resistant C. albicans were 725 and 1450 μg/mL, respectively. The expression of CDR1 (4.5-fold) and CDR2 (6.3-fold) were down-regulated after the exposure of FLC-resistant C. albicans to ACE-loaded nanofibers (P<0.05). Furthermore, the expression of CDR1 (2.8-fold) and CDR2 (3.2-fold) were up-regulated in FLC-treated C. albicans (P<0.05).Conclusion: The results revealed that nanofibers containing ACE interact with drug-resistant genes expressed in C. albicans. Further studies are recommended to investigate the mode of action and other biological activities of ACE-loaded nanofibers against C. albicans and other pathogenic fungi.