The performance and costs of XTT, resazurin, MTS and luciferin as viability dyes in in vitro susceptibility testing of Madurella mycetomatis.

BACKGROUND: in vitro susceptibility testing for the non-sporulating fungus Madurella mycetomatis is performed with a hyphal suspension as starting inoculum and a viability dye for endpoint reading. Here we compared the performance of four different viability dyes for their use in in vitro susceptibility testing of M. mycetomatis. METHODS: To compare the reproducibility and the agreement between the viability dyes 2,3-bis-(2-methoxy-4-nitro-5-sulfphenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), resazurin, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) and luciferin, the in vitro susceptibilities of 14 genetically diverse M. mycetomatis isolates were determined for itraconazole and amphotericin B. The reproducibility, agreement, price and ease of use were compared. RESULTS: Each of the four dyes gave highly reproducible results with >85.7% reproducibility. Percentage agreement ranged between 78.9% and 92.9%. Resazurin was the most economical to use (0.0009 €/minimal inhibitory concentration [MIC]) and could be followed in real time. Luciferin omitted the need to transfer the supernatant to a new 96-well plate, but cost 6.07 €/MIC. CONCLUSION: All four viability dyes were suitable to determine the in vitro susceptibility of M. mycetomatis against itraconazole and amphotericin B. Based on the high reproducibility, high percentage agreement, price and possibility to monitor in real time, resazurin was the most suited for routine in vitro susceptibility testing in the diagnostic laboratory in mycetoma-endemic countries. Because luminescence could be measured directly without the need to transfer the supernatant to a new 96-well plate, luciferin is suitable for drug-screening campaigns. LAY SUMMARY: To determine the in vitro susceptibility testing in the non-sporulating fungus Madurella mycetomatis, a viability dye is needed for endpoint reading. In this study we tested the viability dyes XTT, resazurin, MTS and luciferin for their use in in vitro susceptibility testing. It appeared that they all could be used but there were differences in time to result and costs associated with them.

Two promising Bacillus-derived antifungal lipopeptide leads AF4 and AF5 and their combined effect with fluconazole on the in vitro Candida glabrata biofilms.

Introduction: Candida species are endowed with the ability to produce biofilms, which is one of the causes of pathogenicity, as biofilms protect yeasts from antifungal drugs. Candida glabrata (Nakaseomyces glabrata) is one of the most prevalent pathogenic yeasts in humans and a biofilm producer. Methods: The study was aimed at evaluating the combined effects of two highly promising antifungal biomolecules (AF4 and AF5) lipopeptide in nature, chromatographically purified to homogeneity from Bacillus subtilis (B. subtilis) and the standard antifungal fluconazole (at different concentrations) to demonstrate C. glabrata biofilm formation inhibition. Biofilm production and inhibition were evaluated by quantification of the biofilm biomass and metabolic activity using crystal violet (CV) staining and XTT reduction assays, respectively. Microscopic techniques such as confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM) were employed to visualize biofilm formation and inhibition. Results and Discussion: Compared to untreated and fluconazole-treated biofilms, an enhanced in vitro anti-biofilm effect of the antifungal lipopeptides AF4/AF5 alone and their combinations with fluconazole was established. The lipopeptides AF4/AF5 alone at 8 and 16 µg/mL exhibited significant biomass and metabolic activity reductions. SEM and CSLM images provided evidence that the lipopeptide exposure results in architectural alterations and a significant reduction of C. glabrata biofilms, whereas (2', 7'-dichlorofluorescin diacetate (DCFDA) and propidium iodide (PI) analyses showed reactive oxygen species (ROS) generation along with membrane permeabilization. The estimation of exopolysaccharides (EPS) in AF4/AF5-treated biofilms indicated EPS reduction. The combinations of fluconazole (64/128 µg/mL) and AF4/AF5 lipopeptide (16 µg/mL) were found to significantly disrupt the mature (24 h) biofilms as revealed by CSLM and SEM studies. The CSLM images of biofilms were validated using COMSTAT. The FTIR-analyses indicate the antibiofilm effects of both lipopeptides on 24 h biofilms to support CSLM and SEM observations. The combinations of fluconazole (64/128 µg/mL) and AF4/AF5 lipopeptide were found to disrupt the mature biofilms; the study also showed that the lipopeptides alone have the potentials to combat C. glabrata biofilms. Taken together, it may be suggested that these lipopeptide leads can be optimized to potentially apply on various surfaces to either reduce or nearly eradicate yeast biofilms.

Betanin Attenuates Epigenetic Mechanisms and UV-Induced DNA Fragmentation in HaCaT Cells: Implications for Skin Cancer Chemoprevention.

Dermal photoaging refers to the skin's response to prolonged and excessive ultraviolet (UV) exposure, resulting in inflammation, changes to the tissue, redness, swelling, and discomfort. Betanin is the primary betacyanin in red beetroot (Beta vulgaris) and has excellent antioxidant properties. Yet, the specific molecular mechanisms of betanin in HaCaT cells have not been fully clarified. The objective of this study was to investigate the activity of betanin and the underlying mechanisms in HaCaT cells; furthermore, in this study, we explored the protective effect of various concentrations of betanin against UVB irradiation on HaCaT cells. Additionally, we assessed its influence on the transcription of various epigenetic effectors, including members of the DNA methyltransferase (DNMT) and histone deacetylase (HDAC) families. Our findings demonstrate a notable downregulation of genes in HaCaT cells, exhibiting diverse patterns upon betanin intake. We considered the involvement of DNMT and HDAC genes in distinct stages of carcinogenesis and the limited exploration of the effects of daily exposure dosages. Our results indicate that betanin may protect the skin from damage caused by UV exposure. Further investigation is essential to explore these potential associations.

Detection and Differentiation of Xanthomonas translucens Pathovars translucens and undulosa from Wheat and Barley by Duplex Quantitative PCR.

Two probe-based quantitative PCR (qPCR) systems, namely P-Xtt and P-Xtu, were developed to diagnose cereal bacterial leaf streak pathogens Xanthomonas translucens pv. translucens and pv. undulosa, respectively. P-Xtt is specific to pv. translucens, and P-Xtu is specific to pv. undulosa, pv. cerealis, pv. secalis, and pv. pistaciae. P-Xtt and P-Xtu worked on all accessible strains of pv. translucens and pv. undulosa, respectively. Both systems could detect 100 copies of the target gBlock DNA. The two systems could be used in both singleplex qPCR and duplex qPCR with similar efficiencies. On genomic DNA from strains of various X. translucens pathovars, both singleplex and duplex qPCR could specifically detect and differentiate pv. translucens and pv. undulosa. The duplex qPCR could detect pv. translucens and pv. undulosa from genomic DNA of 1,000 bacterial cells. On infected barley and wheat grain samples and on one infected wheat leaf sample, the duplex qPCR showed similar efficiency compared to a previously published qPCR system but with the additional capability of pathovar differentiation. The duplex qPCR system developed in this study will be useful in studies on bacterial leaf streak and detection/differentiation of the pathogens.

Electrochemical assay of mammalian cell viability.

We present the first use of amperometric detection to assess the viability of mammalian cells in continuous mode, directly in the cell culture medium. Vero or HeLa cells were injected into electrochemical sensors equipped with a 3-electrode system and containing DCIP 50 µM used as the redox mediator. DCIP was reduced by the viable cells and the reduced form was detected amperometrically at 300 mV vs silver pseudo-reference. The continuous regeneration of the oxidized form of the mediator ensured a stable redox state of the cell environment, allowing the cells to survive during the measurement time. The electrochemical response was related to cell metabolism (no response with dead cells or lysed cells) and depended on both mediator concentration and cell density. The protocol was applied to both cells in suspension and adhered cells. It was also adapted to detect trans-plasma membrane electron transfer (tPMET) by replacing DCIP by ferricyanide 500 µM and using linear scan voltammetry (2 mV/s). The pioneering results described here pave the way to the development of routine electrochemical assays for cell viability and for designing a cell-based analytical platform.

Testing a Method for Evaluation of the Viability of Biofilm-Forming Bacteria after Exposure to Disinfectants.

A method for determining the viability of opportunistic pathogenic bacteria at the stage of biofilm formation after exposure to disinfectants with different active components was tested. The method is based on oxidation of tetrazolium salts by metabolically active cells with the formation of colored formazan derivatives and their quantitative spectrophotometry. The cell viability in the biofilm decreased after exposure to quaternary ammonium compounds and chlorine-containing disinfectants, but their effect was reversible. Dissemination of cells that had retained viability from the biofilm occurred after 24 h. The algorithm of testing, necessary controls, counting, and data interpretation are specified. The method can be recommended for use in laboratory diagnostics and clinical practice.

In Vitro and In Silico Evaluations of the Antileishmanial Activities of New Benzimidazole-Triazole Derivatives.

Benzimidazole and triazole rings are important pharmacophores, known to exhibit various pharmacological activities in drug discovery. In this study, it was purposed to synthesize new benzimidazole-triazole derivatives and evaluate their antileishmanial activities. The targeted compounds (5a-5h) were obtained after five chemical reaction steps. The structures of the compounds were confirmed by spectral data. The possible in vitro antileishmanial activities of the synthesized compounds were evaluated against the Leishmania tropica strain. Further, molecular docking and dynamics were performed to identify the probable mechanism of activity of the test compounds. The findings revealed that compounds 5a, 5d, 5e, 5f, and 5h inhibited the growth of Leishmania tropica to various extents and had significant anti-leishmanial activities, even if some orders were higher than the reference drug Amphotericin B. On the other hand, compounds 5b, 5c, and 5g were found to be ineffective. Additionally, the results of in silico studies have presented the existence of some interactions between the compounds and the active site of sterol 14-alpha-demethylase, a biosynthetic enzyme that plays a critical role in the growth of the parasite. Therefore, it can be suggested that if the results obtained from this study are confirmed with in vivo findings, it may be possible to obtain some new anti-leishmanial drug candidates.

Antimicrobial Synergistic Effects of Linezolid and Vancomycin with a Small Synthesized 2-Mercaptobenzothiazole Derivative: A Challenge for MRSA Solving.

Methicillin-resistant Staphylococcus aureus (MRSA) emerged as one of the leading causes of persistent human infections and makes it difficult to treat bacteremia, especially with biofilm formation. In this work, we investigated the in vitro synergism between Linezolid (LNZ) and Vancomycin (VAN) with a 2-mercaptobenzothiazole derivative, resulting in a new small-molecule antibacterial compound that we named BTZ2e, on several clinical MRSA, MRSE (methicillin-resistant Staphylococcus epidermidis) and control (ATCC Collection) strains in their planktonic and biofilms cultures. The broth microdilution method evaluated the susceptibility of planktonic cells to each investigated antibiotic combined with BTZ2e. The biofilm's metabolic activity was studied with the XTT reduction assay. As a result, in this study, biofilm formation was significantly suppressed by the BTZ2e treatment. In terms of minimal biofilm inhibitory concentration (MBIC), BTZ2e revealed an MBIC50 value of 32 µg/mL against methicillin-susceptible S. aureus (MSSA) and 16 µg/mL against methicillin-resistant S. aureus ATCC 43300 biofilms. An inhibition range of 32 µg/mL and 256 µg/mL was registered for the clinical isolates. Interestingly, a synergistic effect (FICI ≤ 0.5) was encountered for the combination of BTZ2e with LNZ and VAN on several planktonic and sessile strains. In particular, the best result against planktonic cells emerged as a result of the synergistic association between LNZ and BTZ2e, while against sessile cells, the best synergistic association resulted from VAN and BTZ2e. The consistent results indicate BTZ2e as a promising adjuvant against multi-resistant strains such as MRSA and MRSE.

Isoniazid Loaded PCL-PEG Copolymer Nanoparticles for Sustained Release Application.

BACKGROUND: Tuberculosis (TB) is still a major cause of death worldwide, despite possibly curable therapies. Neurotoxicity, optic neuritis, and severe liver damage are side effects of isoniazid, a powerful first-line anti-TB drug. OBJECTIVE: We investigated the use of PCL-PEG copolymer to sustain the release of isoniazid to reduce its adverse effects. METHODS: In the present work, PCL-PEG copolymer was synthesized and characterized. Isoniazid-loaded nanoparticles (Inp) were prepared using a PCL-PEG copolymer. Furthermore, a 23 half factorial design was employed for the optimization of drug and emulsifier concentration in Inp. Full characterization of the nanoparticles was performed in terms of drug loading, entrapment efficiency, particle size, zeta potential, and in vitro drug release. The morphology, FTIR, DSC, and PXRD evaluation of the optimized Batch Inp F13 were studied. Stability was evaluated by storing the freeze-dried Inp F13 at various temperatures. RESULTS: The entrapment efficiency and drug loading of nanoparticles prepared by double emulsion solvent evaporation were found to be the highest. The release study revealed that all batches of nanoparticles exhibited sustained drug release (60.26 - 88.59%) for 5 days. The cytotoxicity study conducted on Mycobacterium tuberculosis revealed a gradual release of isoniazid from Inp, reaching the maximum (on the 15th day) compared to plain isoniazid (on the 4th day). At 0.8 µg/mL concentration, the inhibitory activity of Inp F13 was maintained for 15 days, indicating sustained release of isoniazid. CONCLUSION: The nanoparticles having PCL:PEG in a 95:5 ratio, with 0.5% PVA and initial drug loading of 3 mg, produced the optimum batch. Isoniazid-loaded PCL-PEG nanoparticles allowed controlled (sustained) release of isoniazid.

In Vitro Effectiveness of Soft Contact Lens Solutions Available on the Dutch Market against Acanthamoeba Species.

Acanthamoeba keratitis is almost universally associated with contact lens (CL) use. Until today, however, CL solution manufacturing protocols lack testing of anti-amoebic activity. This study investigates the effectiveness of CL solutions available on the Dutch market against trophozoites and cysts of Acanthamoeba castellanii and Acanthamoeba polyphaga. Sixteen CL solutions were tested: 13 multiple purpose solutions (MPS), 2 hydrogen peroxidase solutions (HPS) and 1 povidone-iodine-based solution (PIS). The Spearman-Karber (SK) log reduction method and an XTT colorimetric assay were used to evaluate the effectiveness at the manufacturer's minimum recommended disinfection time (MMRDT) and after eight hours. At the MMRDT, one MPS showed an SK mean log reduction (MLR) of >3.0 against A. castellanii trophozoites. Two additional MPS and both HPS reached this threshold after eight hours. The SK MLR values for A. polyphaga trophozoites were between 1 and 3 at all time points. Using the XTT colorimetric assay, only HPS 1 showed >99.9% reduction (equivalent to 3 log reduction) in metabolic activity of A. castellanii trophozoites after eight hours. For A. polyphaga, both HPS and PIS showed a metabolic reduction of >99.9% after eight hours. Cysts were resistant against all solutions. We conclude that following the manufacturer's guidelines, few solutions provide sufficient effectiveness against Acanthamoeba trophozoites and none against cysts. The results underline the importance of adequate hygiene when handling CLs.

Antioxidant, Cytotoxicity, Apoptotic Properties of Extracts of Andricus sternlichti galls and Their Phenolic Characterisation by HPLC.

Quercus L. galls have been used in Western and Eastern cultures for various diseases in traditional medicine. Galls are also used in the East for many purposes, including consumption as food, commercial inks, leather tanning. In the current study, Andricus sternlichti Bellido, Pujade-Villar & Melika, 2003 galls were extracted in different solvents. The possible antioxidant effects of gall extracts were determined using 7 different methods (ß-carotene-linoleic acid assay, Phosphomolybdenum assay, DPPH and ABTS radical scavenging activity, CUPRAC and FRAP assay, Metal Chelating activity) to support each other. Total phenolic, flavonoid and tannin amounts of extracts are calculated by using standard curves. In addition, HPLC method used to characterize the phenolic component with 15 different standards. The MIA PaCa-2 cell lines was preferred to identify possible cytotoxic activities of galls. Expression of some genes (Bax, Bcl-2, FAS, BID, caspase-3, caspase-8, caspase-9, caspase-10, FADD, TRADD) role in the apoptosis was determined to investigate apoptotic effects of extracts. According the results, the gall extracts of A. sternlichti may be considered as a potential source of biological agents for their antioxidant capacity and rich bioactive compounds. The gall extracts exhibit antiproliferative activity via regulating expressions of apoptotic genes.

Evaluation of Cytotoxicity of Calcium Silicate-based Mineral Trioxide Aggregate Sealers: A Systematic Review of In Vitro Studies.

AIM: This review aimed to evaluate the in vitro studies done with regard to the cytotoxicity associated with mineral trioxide aggregate (MTA)-based root canal sealers. BACKGROUND: Root canal sealers are used during endodontic treatment as fillers to seal the gaps between the canal gutta-percha cone and canal walls. It is necessary to understand the cytotoxicity of these materials on human-derived cells as these materials interact with human cells periapically. REVIEW RESULTS: Six in vitro studies were chosen for review. In these selected studies, along with MTA-based root canal sealers, other sealers were tested for cytotoxicity on human periodontal ligament (PDL) stem cells, human PDL fibroblasts, and human osteoblast cells. Regarding cytotoxicity, the studies were diverse, and most were based on 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide) (MTT) assay. In general, the studies suggested that root canal sealers cause mild to severe cytotoxic effects and that several factors influence this effect, such as material setting time, concentration, and duration of exposure. CONCLUSION: All studies in the review indicated that MTA. Fillapex must be used cautiously as it exhibited the highest cytotoxic effect compared to other MTA-based and non-MTA-based sealers. CLINICAL SIGNIFICANCE: Endodontic sealers do serve the purpose of bridging the gaps between the gutta-percha cone and the canal wall but knowing its biocompatibility becomes important as the material is extruded beyond the apical foramen where it comes in contact with the surrounding tissues. The effect of sealers on the surrounding tissues affects the healing and prognosis of the treatment.

Observation of α-Synuclein Preformed Fibrils Interacting with SH-SY5Y Neuroblastoma Cell Membranes Using Scanning Ion Conductance Microscopy.

Parkinson's disease (PD) is the second-most prevalent neurodegenerative disorder in the U.S. α-Synuclein (α-Syn) preformed fibrils (PFFs) have been shown to propagate PD pathology in neuronal populations. However, little work has directly characterized the morphological changes on membranes associated with α-Syn PFFs at a cellular level. Scanning ion conductance microscopy (SICM) is a noninvasive in situ cell imaging technique and therefore uniquely advantageous to investigate PFF-induced membrane changes in neuroblastoma cells. The present work used SICM to monitor cytoplasmic membrane changes of SH-SY5Y neuroblastoma cells after incubation with varying concentrations of α-Syn PFFs. Cell membrane roughness significantly increased as the concentration of α-Syn PFFs increased. Noticeable protrusions that assumed a more crystalline appearance at higher α-Syn PFF concentrations were also observed. Cell viability was only slightly reduced, though statistically significantly, to about 80% but independent of the dose. These observations indicate that within the 48 h treatment period, PFFs continue to accumulate on the cell membranes, leading to membrane roughness increase without causing prominent cell death. Since PFFs did not induce major cell death, these data suggest that early interventions targeting fibrils before further aggregation may prevent the progression of neuron loss in Parkinson's disease.

Comparison of cell viability assessment and visualization of Aspergillus niger biofilm with two fluorescent probe staining methods.

Filamentous fungi are ubiquitous and frequent components of biofilms. A means to visualize them and quantify their viability is essential for understanding their development and disruption. However, quantifying filamentous fungal biofilms poses challenges because, unlike yeasts and bacteria, they are not composed of discrete cells of similar size. This research focused on filamentous fungal biofilms that are representative of those in the built environment. The objective of this study was to develop a rapid method to examine biofilm structure and quantify live (metabolically active/ membrane undamaged) and dead (inactive/ membrane damaged) cells in Aspergillus niger biofilms utilizing a fluorescent probe staining method and confocal laser scanning microscopy (CLSM). For this, we compared two commercially available probe staining kits that have been developed for bacterial and yeast systems. One method utilized the classic cell stain FUN 1 that exhibits orange-red fluorescent intravacuolar structures in metabolically active cells, while dead cells are fluoresced green. The second method utilized a combination of SYTO9 and propidium iodide (PI), and stains cells based on their membrane morphology. SYTO9 is a green fluorescent stain with the capacity to penetrate the living cell walls, and PI is a red fluorescent stain that can only penetrate dead or dying cells with damaged cell membranes. Following staining, the biofilms were imaged using CLSM and biofilm volumes and thickness were quantified using COMSTAT, a computer program that measures biofilm accumulation from digital image stacks. The results were compared to independent measurements of live-dead cell density, as well as a classic cell viability assay-XTT. The data showed that the combination of SYTO9 and PI is optimal for staining filamentous fungal biofilms.

Addressing artifacts of colorimetric anticancer assays for plant-based drug development.

Cancer has become the silent killer in less-developed countries and the most significant cause of morbidity worldwide. The accessible and frequently used treatments include surgery, radiotherapy, chemotherapy, and immunotherapy. Chemotherapeutic drugs traditionally involve using plant-based medications either in the form of isolated compounds or as scaffolds for synthetic drugs. To launch a drug in the market, it has to pass through several intricate steps. The multidrug resistance in cancers calls for novel drug discovery and development. Every year anticancer potential of several plant-based compounds and extracts is reported but only a few advances to clinical trials. The false-positive or negative results impact the progress of the cell-based anticancer assays. There are several cell-based assays but the widely used include MTT, MTS, and XTT. In this article, we have discussed various pitfalls and workable solutions.

A Simple Method for Growth of Candida albicans Biofilms Under Continuous Media Flow and for Recovery of Biofilm Dispersed Cells.

Majority of nosocomial infections are associated with biofilms growing on indwelling medical devices. These biofilms are nourished by a continuous flow of body fluids and subjected to shear stress forces. Cells dispersed from C. albicans biofilms are highly virulent and developmentally distinct from their parent biofilms. To study biofilm dispersed cells, it becomes imperative to isolate newly dispersed cells using a flow biofilm model. In this chapter, we detail the methods underlying assembly and workings of a simple flow biofilm model using materials commonly available in most microbiological laboratories. Biofilms developed using this system are robust and particularly suitable for studies requiring large amounts of biofilm (dispersed) cells for downstream analyses. Importantly, this apparatus mimics in vivo flow conditions, thereby making it a physiologically relevant model.

A Simple 96-Well Plate-Based Method for Development of Candida Biofilms Under Static Conditions.

We describe a rapid and simple in vitro method for development of Candida biofilms under static growth conditions. This 96-well microtiter-based method measures metabolic activity of sessile cells and can also be easily adapted for antifungal susceptibility testing. The entire procedure takes 2-3 days to complete, reliably quantifies biofilms, and provides reproducible results that are imperative toward the standardization of antifungal susceptibility testing of biofilms.

Cell Viability Assays for Candida auris.

Cell viability assays are useful for assessing the efficacy of antifungal therapeutics and disinfection strategies in vitro. In recent years these assays have been fundamental for the testing of conventional and novel therapies against the nosocomial fungal pathogen Candida auris. Here we provide detailed descriptions of methods for assessing cellular viability of Candida auris in vitro, such as metabolic assays (XTT and resazurin), colony-forming unit counting, live/dead quantitative PCR, and fluorescent staining for microscopic analyses.

Detection of Xanthomonas translucens pv. undulosa, pv. translucens, and pv. secalis by Quantitative PCR.

A probe-based quantitative PCR (qPCR) protocol was developed for detection and evaluation of the wheat bacterial leaf streak pathogen Xanthomonas translucens pathovar (pv.) undulosa. The protocol can also detect X. translucens pv. translucens and X. translucens pv. secalis but can't differentiate the three pathovars. When tested on nontarget DNA (i.e., from plant; bacteria other than X. translucens pv. undulosa, X. translucens pv. translucens, and X. translucens pv. secalis; and culture of microorganisms from wheat grains), the qPCR showed a high specificity. On purified X. translucens pv. undulosa DNA, the qPCR was more sensitive than a loop-mediated isothermal amplification assay. When DNA samples from a set of serial dilutions of X. translucens pv. undulosa cells were tested, the qPCR method could repeatedly generate quantification cycle (Cq) values from the dilutions containing ≥1,000 cells. Since 2 µl of the total 50 µl of DNA was used in one reaction, one qPCR reaction could detect the presence of the bacteria in samples containing as few as 40 bacterial cells. The qPCR could detect the bacteria from both infected grain and leaf tissues. For seed testing, a protocol for template preparation was standardized, which allowed one qPCR reaction to test DNA from the surface of one wheat grain. Thus, the qPCR system could detect X. translucens pv. undulosa, X. translucens pv. translucens, and/or X. translucens pv. secalis in samples where the bacteria had an average concentration of ≥40 cells per grain.

Fenticonazole nitrate loaded trans-novasomes for effective management of tinea corporis: design characterization, in silico study, and exploratory clinical appraisal.

The current investigation aimed for loading fenticonazole nitrate (FTN), an antifungal agent with low aqueous solubility, into trans-novasomes (TNs) for management of tinea corporis topically. TNs contain Brij® as an edge activator besides the components of novasomes (cholesterol, Span 60, and oleic acid) owing to augment the topical delivery of FTN. TNs were fabricated applying ethanol injection method based on D-optimal experiment. TNs were evaluated with regard to entrapment efficiency percent (EE%), particle size (PS), polydispersity index (PDI), and zeta potential (ZP). Further explorations were conducted on the optimum formulation (F7). F7 showed spherical appearance with EE%, PS, PDI, and ZP of 100.00 ± 1.10%, 358.60 ± 10.76 nm, 0.51 ± 0.004, and -30.00 ± 0.80 mV, respectively. The in silico study revealed the ability of the FTN-cholesterol complex to maintain favorable interactions throughout the molecular dynamics simulation (MDS) study. Moreover, Trichophyton mentagrophytes growth was inhibited effectively by F7 than by FTN suspension applying 2,3-bis(2-methyloxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. Furthermore, a clinical appraisal on patients with tinea corporis fungal lesions confirmed the superiority of F7 compared to Miconaz® cream in the magnitude of clinical cure of tinea corporis. Thereby, TNs could be considered as promising vesicles for enhancing the antifungal potential of FTN for the topical management of tinea corporis.