Platelet-derived biomaterial controls aspergillus fumigatus keratitis by decreasing fungal burden: an in vivo study.

Fungal keratitis is a severe corneal infection characterized by suppurative and ulcerative lesions. Aspergillus fumigatus is a common cause of fungal keratitis. Antifungal drugs, such as natamycin, are currently the first-line treatment for fungal keratitis, but their ineffectiveness leads to blindness and perforation. Additionally, the development of fungal resistance makes treating fungal keratitis significantly more challenging. The present study used platelet-derived biomaterial (PDB) to manage A. fumigatus keratitis in the animal model. Freezing and thawing processes were used to prepare PDB, and then A. fumigatus keratitis was induced in the mice. Topical administration of PDB, natamycin, and plasma was performed; quantitative real-time PCR (qPCR) and histopathologic examination (HE) were used to assess the inhibitory effect of the mentioned compounds against fungal keratitis. The qPCR results showed that PDB significantly decreased the count of A. fumigatus compared to the control group (P-value ≤ 5). Natamycin also remarkably reduced the count of fungi in comparison to the untreated animal, but its inhibitory effect was not better than PDB (P-value > 5). The findings of HE also demonstrated that treatment with PDB and natamycin decreased the fungal loads in the corneal tissue. However, plasma did not show a significant inhibitory effect against A. fumigatus. PDB is intrinsically safe and free of any infections or allergic responses; additionally, this compound has a potential role in decreasing the burden of A. fumigatus and treating fungal keratitis. Therefore, scientists should consider PDB an applicable approach to managing fungal keratitis and an alternative to conventional antifungal agents.

Breaking Down the Barriers of Drug Resistance and Corneal Permeability with Chitosan-Poly(ethylene glycol)-LK13 Peptide Conjugate to Combat Fungal Keratitis.

Fungal keratitis (FK) is a leading cause of preventable blindness and eye loss. The poor antifungal activity, increased drug resistance, limited corneal permeability, and unsatisfactory biosafety of conventional antifungal eye drops are among the majority of the challenges that need to be addressed for currently available antifungal drugs. Herein, this study proposes an effective strategy that employs chitosan-poly(ethylene glycol)-LK13 peptide conjugate (CPL) in the treatment of FK. Nanoassembly CPL can permeate the lipophilic corneal epithelium in the transcellular route, and its hydrophilicity surface is a feature to drive its permeability through hydrophilic stroma. When encountering fungal cell membrane, CPL dissembles and exposes the antimicrobial peptide (LK13) to destroy fungal cell membranes, the minimum inhibitory concentration values of CPL against Fusarium solani (F. solani) are always not to exceed 8 µg peptide/mL before and after drug resistance induction. In a rat model of Fusarium keratitis, CPL demonstrates superior therapeutic efficacy than commercially available natamycin ophthalmic suspension. This study provides more theoretical and experimental supports for the application of CPL in the treatment of FK.

Ergosterol promotes aggregation of natamycin in the yeast plasma membrane.

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability became compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and caused a reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogues of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. Importantly, binding and membrane aggregation of natamycin was paralleled by a decrease of the dipole potential in the PM, and this effect was enhanced in the presence of myriocin. We conclude that ergosterol promotes binding and aggregation of natamycin in the yeast PM, which can be synergistically enhanced by inhibitors of sphingolipid synthesis.

Enhancing the bioavailability and antioxidant activity of natamycin E235-ferulic acid loaded polyethylene glycol/carboxy methyl cellulose films as anti-microbial packaging for food application.

This study investigated the development of biodegradable films made from a combination of polyethylene glycol (PEG), carboxymethyl cellulose (CMC) and mixtures from natamycin and ferulic acid. The films were characterized for their surface microstructure, antioxidant activity, thermal stability, mechanical properties, permeability and antifungal/bacterial activity. The addition of natamycin and ferulic acid to the film matrix enhanced antioxidant activity, thermal stability, antimicrobial activity, reduced the water vapor permeability (WVP) to 1.083 × 10-10 g × m-1s-1Pa-1, imparted opaque color and increased opacity up to 3.131 A mm-1. The attendance of natamycin and ferulic acid inside films created a clear roughness shape with agglomerates on the surface of films and caused a clear inhibition zone for Aspergillus niger, E. coli and C. botulinum. The utilization of PG/CMC/N-F packaging material on Ras cheese had a noticeable effect, resulting in a slight decrease in moisture content from 34.23 to 29.17 %. Additionally, it helped maintain the titrable acidity within the range of 0.99 % to 1.11 % and the force required for puncture from 0.035 to 0.052 N with non-significant differences. Importantly, these changes did not significantly affect the sensory qualities of Ras cheese during the storage period.

Antifungal susceptibility profiles for fungal isolates from corneas and contact lenses in the United Kingdom.

OBJECTIVE: To report the identification and results of susceptibility testing for fungal isolates from the cornea or contact lens care systems. MATERIALS AND METHODS: In this retrospective epidemiological study, we searched the results of fungal cultures from cornea or contact lens systems referred for identification and susceptibility testing to the United Kingdom National Mycology Reference Laboratory between October 2016 and March 2022. For each fungal isolate, we recorded the genus and species of the fungus and the minimum inhibitory concentration (MIC) to six antifungal agents available to treat corneal infection (amphotericin, econazole, itraconazole, natamycin, posaconazole, and voriconazole). RESULTS: There were 600 isolates from 585 patients, comprising 374 (62%) from corneal samples and 226 from contact lenses and care systems, of which 414 (69%) isolates were moulds (filamentous fungi) and 186 (31%) were yeasts. The most frequent moulds isolated were Fusarium spp (234 isolates, 39%) and Aspergillus spp (62, 10%). The most frequent yeasts isolated were Candida spp (112, 19%), predominantly Candida parapsilosis (65, 11%) and Candida albicans (33, 6%), with 35 isolates (6%) of Meyerozyma guilliermondii. In vitro susceptibility was greatest for natamycin (347 moulds tested, mode 4 mg/L, range 0.25-64 mg/L; 98 yeasts tested, mode 4 mg/L, range 0.5-32 mg/L), with susceptibility for 94% for moulds and 99% yeasts. Of the 16 isolates interpreted as highly resistant to natamycin (MIC ≥16 mg/L), 13 were Aspergillus flavus complex. CONCLUSIONS: In vitro susceptibility supports the use of natamycin for the empiric treatment of fungal keratitis in the UK.

Efficacy and toxic action of the natural product natamycin against Sclerotinia sclerotiorum.

BACKGROUND: Sclerotinia stem rot caused by Sclerotinia sclerotiorum seriously endangers oilseed rape production worldwide, and the occurrence of fungicide-resistant mutants of S. sclerotiorum leads to control decline. Thus, it is critical to explore new green substitutes with different action mechanisms and high antifungal activity. Herein, the activity and the action mechanism of natamycin against S. sclerotiorum were evaluated. RESULTS: Natamycin showed potent inhibition on the mycelial growth of S. sclerotiorum, and half-maximal effective concentration (EC50 ) values against 103 S. sclerotiorum strains ranged from 0.53 to 4.04 µg/mL (mean 1.44 µg/mL). Natamycin also exhibited high efficacy against both carbendazim- and dimethachlone-resistant strains of S. sclerotiorum on detached oilseed rape leaves. No cross-resistance was detected between natamycin and carbendazim. Natamycin markedly disrupted hyphal form, sclerotia formation, integrity of the cell membrane, and reduced the content of oxalic acid and ergosterol, whereas it increased the reactive oxygen species (ROS) and malondialdehyde content. Interestingly, exogenous addition of ergosterol could reduce the inhibition of natamycin against S. sclerotiorum. Importantly, natamycin significantly inhibited expression of the Cyp51 gene, which is contrary to results for the triazole fungicide flusilazole, indicating a different action mechanism from triazole fungicides. CONCLUSION: Natamycin is a promising effective candidate for the resistance management of S. sclerotiorum. © 2023 Society of Chemical Industry.

Improved Topical Ophthalmic Natamycin Suspension for the Treatment of Fungal Keratitis.

Purpose: Natamycin (NT) is used as a first-line antifungal prescription in the treatment of fungal keratitis (FK) and is commercially available as a 5% w/v ophthalmic suspension. NT shows poor water solubility and light sensitivity. Thus, the present investigation is aimed to enhance the fraction of NT in solution in the commercial formulation by adding cyclodextrins (CDs), thereby improving the delivery of the drug into deeper ocular tissues. Methods: The solubility of NT in different CDs, the impact of ultraviolet (UV) light exposure, stability at 4°C and 25°C, in vitro release, and ex vivo transcorneal permeation studies were performed. Results: NT exhibited the highest solubility (66-fold) in randomly methylated-ß-cyclodextrin (RM-ßCD) with hydroxypropyl-ßCD (HP-ßCD) showing the next highest solubility (54-fold) increase in comparison to market formulation Natacyn® as control. The stability of NT-CD solutions was monitored for 2 months (last-time point) at both storage conditions. The degradation profile of NT in NT-RM-ßCD and NT-HP-ßCD solutions under UV-light exposure followed first-order kinetics exhibiting half-lives of 1.2 h and 1.4 h, respectively, an almost 3-fold increase over the control solutions. In vitro release/diffusion studies revealed that suspensions containing RM-ßCD and HP-ßCD increased transmembrane flux significantly (3.1-fold) compared to the control group. The transcorneal permeability of NT from NT-RM-ßCD suspension exhibited an 8.5-fold (P < 0.05) improvement compared to Natacyn eyedrops. Furthermore, the addition of RM-ßCD to NT suspension increases the solubilized fraction of NT and enhances transcorneal permeability. Conclusion: Therefore, NT-RM-ßCD formulations could potentially lead to a decreased frequency of administration and significantly improved therapeutic outcomes in FK treatment.

Study on the Antifungal Activity and Potential Mechanism of Natamycin against Colletotrichum fructicola.

In this investigation, the antifungal activity, its influence on the quality of apples, and the molecular mechanism of natamycin against Colletotrichum fructicola were systematically explored. Our findings indicated that natamycin showed significant inhibition against C. fructicola. Moreover, it efficaciously maintained the apple quality by modulating the physicochemical index. Research on the antifungal mechanism showed that natamycin altered the mycelial microstructure, disrupted the plasma membrane integrality, and decreased the ergosterol content of C. fructicola. Interestingly, the exogenous addition of ergosterol weakened the antifungal activity of natamycin. Importantly, natamycin markedly inhibited the expression of Cyp51A and Cyp51B genes in C. fructicola, which was contrary to the results obtained after treatment with triazole fungicide flusilazole. All these results exhibited sufficient proof that natamycin had enormous potential to be conducive as a promising biopreservative against C. fructicola on apples, and these findings will advance our knowledge on the mechanism of natamycin against pathogenic fungi.

The preparation and therapeutic effects of ß-glucan-specific nanobodies and nanobody-natamycin conjugates in fungal keratitis.

Fungal keratitis (FK) is a severe infectious corneal disease. Since traditional eye drops exhibit poor dissolution and high corneal toxicity, the efficacy of current treatments for FK remains limited. It is needed to develop new approaches to control the cornea damage from FK. In this study, a nanobody (Nb) specific to ß-glucan in the fungal cell wall was prepared. The conjugate of the Nb with natamycin (NAT), a traditional antifungal drug, was synthesized. Firstly, we found the Nb specific to ß-glucan inhibited fungal growth by disrupting cell wall and biofilm formation.. In addition, the content of ß-glucan in the fungal cell wall decreased after Nb treatment. The Nb also reduced the adhesion ability of fungal conidia to human corneal epithelial cells (HCECs). Further, we examined the difference between NAT and Nb-NAT in antifungal growth. Nb-NAT showed better antifungal effects than NAT which was caused by the interaction between Nb and ß-glucan. Moreover, Nb concentration below 0.5 mg/mL did not affect the viability of HCECs. Nb-NAT had less cytotoxicity and ocular surface irritation than NAT. Nb specific to ß-glucan attenuated Aspergillus fumigatus (A. fumigatus) virulence and relieved inflammatory responses in FK. Nb-NAT treatment of the cornea improved therapeutic effects compared with NAT. It decreased clinical scores and expression level of inflammatory factors. To our knowledge, this study is the first to report a Nb specific to ß-glucan and Nb-NAT for the treatment of FK. These unique functions of the Nb specific to ß-glucan and Nb-NAT would render it as an alternative molecule to control fungal infections including FK. STATEMENT OF SIGNIFICANCE: Fungal keratitis is a corneal disease with a high rate of blindness. Due to the poor dissolution and high corneal toxicity exhibited by traditional eye drops, the efficacy of current therapeutic treatments for fungal keratitis (FK) remains limited. To enhance the therapeutic effect of natamycin in treating fungal keratitis, this study developed an innovative approach by preparing a ß-glucan-specific nanobody and loading it with the antifungal drug natamycin. The ß-glucan-specific nanobody has the ability to control both fungal pathogen invasion and inflammation, which can cause damage to the cornea in FK. The conjugation with the ß-glucan-specific nanobody significantly increased the antifungal capacity of natamycin and reduced its toxicity. The further application of natamycin conjugated with the ß-glucan-specific nanobody could be expanded to other diseases caused by fungal pathogen infections.

Fabrication of carboxymethyl chitosan films for cheese packaging containing gliadin-carboxymethyl chitosan nanoparticles co-encapsulating natamycin and theaflavins.

In this study, gliadin-carboxymethyl chitosan composite nanoparticles (GC NPs) co-encapsulated natamycin (Nata) and theaflavins (TFs) were constructed and added as an antioxidant, antifungal, and structural enhancer to carboxymethyl chitosan (CMCS) films. The stabilized GC NPs with a particle size of 160.7 ± 2.8 nm, a zeta potential of -29.0 ± 0.9 mV, and a protein content in the supernatant of 96 ± 1 % could be fabricated. Tests of pH and salt ions showed that the stability of NPs dispersion was based on electrostatic repulsion. Co-encapsulation of TFs enhanced the photostability of Nata and the antioxidant activity of the NPs dispersion. The interactions between gliadin with Nata and TFs were studied by molecular simulations. As a functional additive, the addition of Nata/TFs-GC NPs could improve the optical properties, mechanical properties, water-blocking capability, and antifungal and antioxidant activities of the CMCS films. The in-vivo test showed that the functional film could be used to inhibit the growth of Aspergillus niger on cheese.

Natamycin Ocular Delivery: Challenges and Advancements in Ocular Therapeutics.

Fungal keratitis, an ocular fungal infection, is one of the leading causes of monocular blindness. Natamycin has long been considered the mainstay drug used for treating fungal keratitis and is the only US Food and Drug Administration (USFDA)-approved drug, commercially available as a topical 5% w/v suspension. Furthermore, ocular fungal infection treatment takes a few weeks to months to recover, and the available marketed antifungal suspensions are associated with poor residence time, limited bioavailability (< 5%) and high dosing frequency as well as minor irritation and discomfort. Despite these challenges, natamycin is still the preferred drug choice for treating fungal keratitis, as it has fewer side effects and less ocular toxicity and is more effective against Fusarium species than other antifungal agents. Several novel therapeutic approaches for the topical delivery of natamycin have been reported to overcome the challenges posed by the conventional dosage forms and to improve ocular bioavailability for the efficient management of fungal keratitis. Current progress in the delivery systems uses approaches aimed at improving the corneal residence time, bioavailability and antifungal potency, thereby reducing the dose and dosing frequency of natamycin. In this review, we discuss the various strategies explored to overcome the challenges present in ocular drug delivery of natamycin and improve its bioavailability for ocular therapeutics.

Sensitivity of Mucor piriformis to Natamycin and Efficacy of Natamycin Alone and with Salt and Heat Treatments Against Mucor Rot of Stored Mandarin Fruit.

Mucor rot caused by Mucor piriformis is an emerging postharvest disease of mandarin fruit in California. Natamycin is a newly registered biofungicide for postharvest use on citrus and some other fruits. In the study, baseline sensitivity to natamycin in 50 isolates of M. piriformis was determined in vitro. The mean EC50 (effective concentration to inhibit sporangiospore germination by 50%) and MIC (minimum inhibitory concentration to inhibit mycelial growth by 100%) values were 0.59 µg/ml and less than 1.0 µg/ml, respectively. Natamycin at the label rate of 920 µg/ml alone or in combination with 3% potassium sorbate (PS) or 3% sodium carbonate (SC) applied at 20 or 50°C was evaluated for control of Mucor rot on inoculated 'Tango' mandarin fruit. Natamycin alone reduced Mucor rot incidence on stored mandarin fruit from 100% among nontreated control fruit to approximately 30%, a reduction of more than 70% compared to the nontreated control, while 3% PS and 3% SC had no to little control. When applied at 50°C, natamycin and 3% PS reduced Mucor rot incidence by 65.0 and 31.2%, respectively; while natamycin in combination with 3% PS reduced disease incidence by 92.5% compared to the nontreated control after 2 weeks of storage at 5°C. This combined treatment remained effective even when the application of the treatment was delayed for 6 and 12 h after inoculation. However, the effectiveness of the treatments declined when storage was extended to 3 or 4 weeks. Natamycin can be an effective tool to control Mucor rot on mandarin fruit, and minimizing the period of extended storage could help maintain the control efficacy of natamycin.

Natamycin Has an Inhibitory Effect on Neofusicoccum parvum, the Pathogen of Chestnuts.

This research aimed to investigate natamycin's antifungal effect and its mechanism against the chestnut pathogen Neofusicoccum parvum. Natamycin's inhibitory effects on N. parvum were investigated using a drug-containing plate culture method and an in vivo assay in chestnuts and shell buckets. The antifungal mechanism of action of natamycin on N. parvum was investigated by conducting staining experiments of the fungal cell wall and cell membrane. Natamycin had a minimum inhibitory concentration (MIC) of 100 µg/mL and a minimum fungicidal concentration (MFC) of 200 µg/mL against N. parvum. At five times the MFC, natamycin had a strong antifungal effect on chestnuts in vivo, and it effectively reduced morbidity and extended the storage period. The cell membrane was the primary target of natamycin action against N. parvum. Natamycin inhibits ergosterol synthesis, disrupts cell membranes, and causes intracellular protein, nucleic acid, and other macromolecule leakages. Furthermore, natamycin can cause oxidative damage to the fungus, as evidenced by decreased superoxide dismutase and catalase enzyme activity. Natamycin exerts a strong antifungal effect on the pathogenic fungus N. parvum from chestnuts, mainly through the disruption of fungal cell membranes.

The therapeutic role and mechanism of 4-Methoxycinnamic acid in fungal keratitis.

Fungal keratitis is an infectious vision-threatening disease that has a poor prognosis, and the clinical therapeutic drugs have multiple limitations, such as epithelial toxicity and low bioavailability. Therefore, new antifungal treatment strategies must be developed. 4-Methoxycinnamic acid (MCA) is a widely occurring natural phenolic acid that has been proven to have multiple effects, such as antibacterial, antifungal, anti-inflammatory, neuroprotective, and inhibiting cancer. In this research, we explored the effects and underlying mechanisms of MCA on A. fumigatus keratitis and the antifungal effects of the combination of MCA and natamycin (NATA) on A. fumigatus. We found that MCA exerts antifungal effects by inhibiting the synthesis of the fungal cell wall, changing the permeability of fungal cell membranes. Moreover, the MCA-NATA combination exhibited synergy for A. fumigatus. In addition, MCA exerted an anti-inflammatory effect by downregulating the inflammatory factors (IL-1ß, TNF-α, IL-6, and iNOS) in C57BL/6 mice and RAW264.7 cells. The anti-inflammatory mechanism of MCA was associated with the Mincle signal pathway. In summary, MCA acts as a potential therapeutic drug for fungal keratitis and a potential antifungal sensitizer for natamycin. MCA inhibits fungal cell wall synthesis, destroys the permeability of fungal cell membranes, and mediates the anti-inflammatory, immune response of the host.

Development of natamycin-loaded zein-casein composite nanoparticles by a pH-driven method and application to postharvest fungal control on peach against Monilinia fructicola.

This work fabricated natamycin-loaded zein-casein nanoparticles (N-Z/C NPs) by a pH-driven approach and applied to control postharvest peach brown rot caused by Monilinia fructicola. When casein and phosphoric acid were used as a stabilizer and neutralizing acid, respectively, NPs with mean particle sizes < 100 nm and zeta-potentials < -30 mV could be obtained. The NPs could increase the aqueous dispersibility of natamycin and showed high stability against environmental changes, which could be attributed to both hydrophobic stacking and hydrogen bonds between natamycin and zein. Besides, the effects of N-Z/C NPs on the storage of peach were assessed in vitro and in vivo. Nanoencapsulation did not affect the antifungal activities of natamycin. The NPs with 20 and 80 µg/mL of natamycin could basically inhibit the spore germination and mycelial growth of M. fructicola, respectively. The N-Z/C NPs coatings exhibited better results than natamycin in controlling of peach brown rot.

Application of natamycin and farnesol as bioprotection agents to inhibit biofilm formation of yeasts and foodborne bacterial pathogens in apple juice processing lines.

Biofilms serve as a reservoir for pathogenic and spoilage microorganisms, and their removal from different surfaces is a recurring problem in the beverage industry. This study aimed to investigate the effect of a combination of natamycin (NAT, 0.01 mmol/l) and farnesol (FAR, 0.6 mmol/l) against biofilms on ultrafiltration (UF) membranes and stainless steel (SS) surfaces using apple juice as food matrix. The co-adhesion of Rhodotorula mucilaginosa, Candida tropicalis, C. krusei and C. kefyr (mixed-yeast) with Listeria monocytogenes, Salmonella enterica or Escherichia coli O157:H7 (multi-species) in presence of NAT + FAR was evaluated for 2, 24, 48 h. In biofilms treated with NAT + FAR were observed by cell quantification and microscopy, inhibition of the filamentous yeast forms, disruption of the tri-dimensional structure and a high detachment of yeast cells. NAT + FAR affected the biofilms independently of the surfaces used and the presence (or not) of bacteria. L. monocytogenes was the most susceptible (p < 0.001) in multi-species biofilms, followed by E. coli O157:H7 on both surfaces (p < 0.001), whereas the growth of S. enterica was reduced (p < 0.05) in SS but not in UF-membranes (p > 0.05). Since the combination NAT + FAR affected the structure and viability of yeast species and foodborne pathogens in multi-species biofilms developed on UF-membranes and SS surfaces, the combination proposed could be considered a promising control agent to prevent biofilms in apple juice processing lines.

Semisynthetic Amides of Polyene Antibiotic Natamycin.

Natamycin is a macrolide polyene antibiotic, characterized by a potent broad spectrum antifungal activity and low toxicity. However, it is not used for the treatment of systemic mycoses due to its low bioavailability and low solubility in aqueous solutions. In order to create new semisynthetic antifungal agents for treatment of mycoses, a series of water-soluble amides of natamycin were synthesized. Antifungal activities of natamycin derivatives were investigated against Candida spp., including a panel of Candida auris clinical isolates and filamentous fungi. Toxicity for mammalian cells was assayed by monitoring antiproliferative activity against human postnatal fibroblasts (HPF) and human embryonic kidney cells (HEK293). By comparing leakage of contents from ergosterol versus cholesterol containing vesicles, a ratio that characterizes the efficacy and safety of natamycin and its derivatives was determined (EI, efficiency index). Ability of all tested semisynthetic natamycines to prevent proliferation of the yeast Candida spp. cells was comparable or even slightly higher to those of parent antibiotic. Interestingly, amide 8 was more potent than natamycin (1) against all tested C. auris strains (MIC values 2 µg/mL vs 8 µg/mL, respectively). Among 7 derivatives, amide 10 with long lipophilic side chains showed the highest EI and strong antifungal activity in vitro but was more toxic against HPF. In vivo experiments with amide 8 showed in vivo efficacy on a mouse candidemia model with a larger LD50/ED50 ratio in comparison to amphotericin B.

Microbiology Profile of COVID-19-Associated Rhino-Orbital Mucormycosis Pathogens in South India.

This study describes the microbiological and histopathological features of patients with COVID-19-associated rhino-orbital mucormycosis (ROM) seen at the L V Prasad Eye Institute between May and August 2021. Diagnosed clinically and radiologically, 24 patients with ROM were included in the study. Deep nasal swabs or endoscopically collected nasal swabs or orbital tissues were submitted for microbiological evaluation and in vitro susceptibility testing by microbroth dilution for natamycin, amphotericin B, caspofungin, posaconazole, ketoconazole, and voriconazole. Cultures were processed by 28S ribosomal DNA polymerase chain reaction and molecular sequencing. A portion of orbital tissues was also sent for histopathological evaluation. The age of the patients ranged from 27 to 75 (mean 48.58 ± 14.09) years and the majority (79%) were male. Nineteen patients were known to be diabetic prior to developing ROM and 18 patients had recovered from active COVID-19 infection. Thirteen patients had a history of hospitalization during COVID-19 infection and eight received steroids. Of the 24 samples, microbiological evaluation identified Rhizopus arrhizus in 12, Rhizopus microsporus in 9, Lichtheimia ramosa in 2, and Rhizopus delemar in 1. Twelve isolates were tested for antifungal susceptibility and all were susceptible to natamycin and amphotericin B. The susceptibility to posaconazole was high, with minimum inhibitory concentration (MIC) < 2 µg/mL for 10/12 (84%) isolates, whereas the MIC of other drugs varied. Histopathological examination of tissues showed acute fulminant disease, granuloma formation, and vascular invasion by the fungal pathogens in these specimens. Rhizopus arrhizus was predominantly associated with ROM and most isolates were susceptible to amphotericin B and posaconazole. Further studies are needed to corroborate the findings and explain possible underlying links.

Evaluation of antifungal susceptibility and clinical characteristics in fungal keratitis in a tertiary care center in North India.

Purpose: To study the antifungal susceptibility of common corneal pathogenic fungi to antifungal agents in the North Indian population. Methods: Prospective study of the antifungal sensitivity testing (natamycin, amphotericin B, voriconazole, itraconazole, fluconazole, posaconazole, caspofungin, micafungin) of fungal isolates from 50 cases of culture positive fungal keratitis by using E test method. Details noted included demographic data, visual acuity, clinical details, grade of keratitis, healing time, and success in medical management. Results: Of 50 patients with fungal keratitis (mean age: 40.28 ± 16.77 years), 12 eyes healed within 3 weeks, 14 had a delayed healing response, and 24 had chronic keratitis. Among the 15 cases of Fusarium isolates, 93.3% were sensitive to natamycin, while 40% to amphotericin B; 66.6% to voriconazole, 13.4% to itraconazole and fluconazole each. 80% of Fusarium cases (n = 12) showed susceptibility to posaconazole. Among Aspergillus flavus isolates, 53.4% (n = 8) were sensitive to natamycin, with only 40% (n = 7) showing sensitivity to amphotericin B and good susceptibility to azoles. MIC against susceptible Fusarium spp. for natamycin was 3-16 µg/mL, amphotericin B: 1-8 µg/mL, voriconazole: 0.5-1.5 µg/mL, itraconazole: 0.5-12 µg/mL, posaconazole: 0.094-1.5 µg/mL. MIC against Aspergillus flavus was natamycin: 8-32 µg/mL, amphotericin B: 0.5-16 µg/mL, voriconazole: 0.025-4 µg/mL, itraconazole: 0.125-8 µg/mL, posaconazole: 0.047-0.25 µg/mL; against Aspergillus niger isolates, to natamycin was 6 µg/mL (n=1), amphotericin B 8-12 µg/mL (n = 3), voriconazole: 0.125-0.19 µg/mL (n = 3), itraconazole: 0.38-0.75 µg/mL, posaconazole: 0.064-0.19 µg/mL and against Aspergillus fumigatus (n = 1), was natamycin4 µg/mL, amphotericin B - 8 µg/mL, voriconazole 0.25 µg/mL, itraconazole 1 µg/mL, and posaconazole 0.19 µg/mL. MIC against susceptible Acremonium spp. for natamycin was 1.5-16 µg/mL, amphotericin B: 0.5-8 µg/mL, voriconazole: 0.19-3 µg/mL, itraconazole: 0.125 µg/mL, posaconazole: 0.125-0.5 µg/mL and against susceptible Curvularia was natamycin 0.75-4 µg/mL, amphotericin B 0.5-1 µg/mL, voriconazole 0.125-0.19 µg/mL, itraconazole 0.047-0.094 µg/mL, posaconazole 0.047-0.094 µg/mL. MIC against Mucor spp.+ Rhizopus spp. (n = 1) was natamycin: 8 µg/mL, amphotericin B: 0.75 µg/mL, posaconazole: 1.5 µg/mL. MIC against of Alternaria (n = 1) was voriconazole: 0.19 µg/mL, posaconazole: 0.094 µg/mL. MIC against Penicillium (n=1) was natamycin: 8 µg/mL, voriconazole: 0.25 µg/mL, itraconazole: 0.5 µg/mL, and Posaconazole: 0.125 µg/mL. Conclusion: Our observations highlight the variations in susceptibility to antifungal agents. Posaconazole seems to be effective with low MIC against common corneal pathogenic fungal isolates.

Drug-loaded mesoporous carbon with sustained drug release capacity and enhanced antifungal activity to treat fungal keratitis.

Fungal keratitis is a severe infectious corneal disease with a high rate of incidence and blindness. Since traditional treatments natamycin (NATA) eye drops, exhibit poor dissolution and bioavailability, and the efficacy of current therapeutic approaches remains limited. In this study, we innovatively utilized mesoporous carbon (Meso-C) and microporous carbon (Micro-C) as nanocarriers loaded with the antifungal drug NATA and silver nanoparticles (Ag-NPs). Porous carbon loaded with NATA and Ag-NPs has not previously been studied in fungal keratitis. Due to the mesoporous structure, high surface area and larger pore volume of Meso-C, it displayed greater superiority in sustained drug release and drug dispersity than Micro-C. Moreover, Meso-C could adsorb inflammatory cytokines during fungal infection. In vitro, Meso-C/NATA/Ag showed excellent antifungal effects. In vivo, compared with pure NATA treatment, Meso-C/NATA/Ag exhibited significantly improved therapeutic effects and reduced dosing frequency when treating fungal keratitis. Our study is the first to report the sustained drug release and improved drug dispersity of Meso-C/NATA and demonstrates that NATA and Ag-NPs-loaded Meso-C has therapeutic effects against fungal keratitis.