Fusarium Keratitis: A Systematic Review (1969 to 2023).

BACKGROUND: Mycotic keratitis (MK) represents a corneal infection, with Fusarium species identified as the leading cause. Fusarium is a genus of filamentous fungi commonly found in soil and plants. While many Fusarium species are harmless, some can cause serious infections in humans and animals, particularly Fusarium keratitis, that can lead to severe ocular infections, prevalent cause of monocular blindness in tropical and subtropical regions of the world. Due to its incidence and importance in ophthalmology, we conducted a systematic analysis of clinical cases to increase our understanding of Fusarium keratitis by gathering clinical and demographic data. METHODS: To conduct an analysis of Fusarium keratitis, we looked through the literature from the databases PubMed, Embase, Lilacs, and Google Scholar and found 99 papers that, between March 1969 and September 2023, corresponded to 163 cases of Fusarium keratitis. RESULTS: Our analysis revealed the Fusarium solani species complex as the predominant isolate, with females disproportionately affected by Fusarium keratitis. Notably, contact lens usage emerged as a significant risk factor, implicated in nearly half of cases. Diagnosis primarily relied on culture, while treatment predominantly involved topical natamycin, amphotericin B, and/or voriconazole. Surprisingly, our findings demonstrated a prevalence of cases originating from the United States, suggesting potential underreporting and underestimation of this mycosis in tropical regions. This shows the imperative for heightened vigilance, particularly in underdeveloped regions with substantial agricultural activity, where Fusarium infections may be more prevalent than currently reported. CONCLUSION: Our study sheds light on the clinical complexities of Fusarium keratitis and emphasizes the need for further research and surveillance to effectively tackle this vision-threatening condition. Furthermore, a timely identification and early initiation of antifungal treatment appear to be as important as the choice of initial treatment itself.

Favorable outcome of Lasiodiplodia theobromae keratomycosis : a clinical case and systematic review.

BACKROUD: Keratitis caused by Lasiodiplodia theobromae is rare and typically associated with a poor prognosis. Current literature lacks sufficient evidence on effective management of patients with this condition. CASE PRESENTATION: A 74-year-old former agricultural worker presented with a red right eye, discomfort, and decreased visual acuity, progressing over three days without treatment. Examination revealed type 2 diabetes and a non-perforating, spiculated corneal abscess with a hypopyon in the right eye. Initial treatment included a triple antibiotic therapy and supportive care. Direct mycological examination identified numerous septate mycelial filaments. Antifungal treatment with natamycin and voriconazole, both topically and orally, was initiated. Cultures confirmed Lasiodiplodia theobromae. The patient showed significant improvement. Treatment continued for eight weeks, with a final visual acuity of 20/50 due to a stromal scar. CONCLUSION: An extensive literature review conducted in November 2023, using databases such as PubMed and Google Scholar with the keywords "lasiodiplodia" and "keratitis" yielded no previous cases of this specific condition being managed solely with the combined use of natamycin and voriconazole. This antifungal combination is commonly included in most management protocols for fungal keratitis. Factors such as the use of corticosteroids and delayed diagnosis were noted to adversely affect the prognosis. This case and this systematic review underscores the potential for non-surgical management options in severe fungal keratitis.

Treatment Strategies for Filamentous Fungi Keratitis.

INTRODUCTION: This research aims to describe clinical findings, epidemiology and treatment outcomes in patients with filamentous fungi keratitis of a tertiary centre in Germany over a 7-year period and to compare the efficacy of different antifungal treatments and the effect of additive topical steroids. METHODS: This retrospective study included 25 eyes of 23 patients from October 2013 to December 2020 with cultural isolates of filamentous fungi and corresponding keratitis. Best-corrected visual acuity (BCVA), clinical signs, symptoms, risk factors and outcome were extracted from medical records. RESULTS: Improvement of BVCA was noted in 68% of eyes. Mean BCVA of the study population increased from 0.75 logMAR [median 0.40, standard deviation (SD) 0.82, range 0-2.3] to 0.48 logMAR (median 0.10, SD 0.88, range - 0.1 to 3). The most commonly used antifungal topical treatment was a combination of natamycin 5% and voriconazole 2% (44% of eyes), followed by voriconazole 2% in 36% of cases. An antiinflammatory topical steroid was applied in 52%. In 16% of the eyes, penetrating keratoplasty (pKP) was performed. CONCLUSION: Diagnosis of filamentous fungi keratitis is often difficult or delayed. Outcomes can be poor even with intensive treatment because of high resistance to common antifungals. Access to natamycin 5% seems to lead to favourable outcomes in filamentous fungi keratitis.

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.

Utilizing confocal microscopy and topical natamycin in metarhizium keratitis of Caribbean origin.

A 32-year-old contact lens-wearing man with recent travel history to the Caribbean was referred for a corneal infiltrate in the left eye that worsened following 1-week of steroid-antibiotic therapy. Corneal cultures were obtained and sent to our facility's clinical microbiology laboratory for analysis. Same-day in vivo confocal microscopy revealed fungal elements. Nucleic acid sequencing performed on the isolated determined it to be a member of the entomopathogenic genus Metarhizium. Over the course of 3 months, the patient's corneal infiltrate ultimately resolved following topical natamycin 5 % therapy. This is the first reported case to have originated in the Caribbean and to utilize in vivo confocal microscopy to aid diagnosis. Our case also supports previous reports of success with natamycin therapy in treatment of Metarhizium sp. keratitis.

A unique implementation of Hantzsch reaction for determination of natamycin in Yoghurt: Hyphenated with Box-Behnken-design for optimization.

This study aims to develop a novel and selective method for the detection of natamycin (E235) in yoghurt. The suggested method adopts an application of Hantzsch reaction to turn on the fluorescence behavior of natamycin (blue fluorescence), allowing its sensitive and selective determination in yoghurt samples without any overlapping at 485 nm. The originality of the research lies in the fact that this application takes place for the first time, also the detection (LOD) and quantification (LOQ) limits were very low (0.02 and 0.06µg mL-1, respectively) with a linear concentration range of 0.1-1.0 µgmL-1. Moreover, the developed method was employed for the detection of E235 in yoghurt sample with a good recoveries (98.80 ± 1.20-99.20 ± 1.15 (%), over a concentration range of 0.5-1.0 µgmL-1, (LOD = 0.04 and LOQ = 0.12 µgmL-1). Furthermore, the specificity and convenient application of our intended method is an attempt to determine E235 in milk anddairy products with easily followable steps.

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.

Nanomicelles empower natamycin in treating fungal keratitis: An in vitro, ex vivo and in vivo study.

Fungal infections of cornea are important causes of blindness especially in developing nations with tropical climate. However, the challenges associated with current treatments are responsible for poor outcome. Natamycin is the only FDA-approved antifungal drug to treat fungal keratitis, but unfortunately due to its poor water solubility, it is available as suspension. The marketed suspension (5% Natamycin) has rapid precorneal clearance, poor corneal permeability, a higher frequency of administration, and corneal irritation due to undissolved suspended drug particles. In our study, we developed clear and stable natamycin-loaded nanomicelles (1% Natcel) to overcome the above challenges. We demonstrated that 1% Natcel could permeate the cornea better than 5% suspension. The developed 1% Natcel was able to provide sustained release for up to 24 h. Further, it was found to be biocompatible and also improved the mean residence time (MRT) than 5% suspension in tears. Therefore, the developed 1% Natcel could be a potential alternative treatment for fungal keratitis.

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.

Mesoporous zinc oxide-based drug delivery system offers an antifungal and immunoregulatory strategy for treating keratitis.

Fungal keratitis is a refractory eye disease that is prone to causing blindness. Fungal virulence and inflammatory responses are two major factors that accelerate the course of fungal keratitis. However, the current antifungal drugs used for treatment usually possess transient residence time on the ocular surface and low bioavailability deficiencies, which limit their therapeutic efficacy. In this work, natamycin (NATA)-loaded mesoporous zinc oxide (Meso-ZnO) was synthesized for treating Aspergillus fumigatus keratitis with excellent drug-loading and sustained drug release capacities. In addition to being a carrier for drug delivery, Meso-ZnO could restrict fungal growth in a concentration-dependent manner, and the transcriptome analysis of fungal hyphae indicated that it inhibited the mycotoxin biosynthesis, oxidoreductase activity and fungal cell wall formation. Meso-ZnO also promoted cell migration and exhibited anti-inflammatory role during fungal infection by promoting the activation of autophagy. In mouse models of fungal keratitis, Meso-ZnO/NATA greatly reduced corneal fungal survival, alleviated tissue inflammatory damage, and reduced neutrophils accumulation and cytokines expression. This study suggests that Meso-ZnO/NATA can be a novel and effective treatment strategy for fungal keratitis.

Fabrication of antimicrobial PCL/EC nanofibrous films containing natamycin and trans-cinnamic acid by microfluidic blow spinning for fruit preservation.

Fruit is susceptible to various postharvest pathogens; thus, the development of multifunctional preservation materials that can achieve the broad-spectrum inhibition of different pathogens is a current research hotspot. Here, microfluidic blow spinning was used to create a biodegradable polycaprolactone/ethyl cellulose (PCL/EC) nanofibrous film that incorporated two naturally-sourced compounds, natamycin and trans-cinnamic acid, resulting in multi-microbial inhibition. The PCL/EC-based film had a smooth and even morphology, indicating the favorable integration of PCL and EC. After the incorporation of ingredients, the film exhibited good inhibitory activity against Escherichia coli, Staphylococcus aureus, and Botrytis cinerea, and it had finer fiber diameters, higher permeability, and antioxidant properties. We further demonstrated that strawberries that were padded with the film had good resistance to Botrytis cinerea. Also, the film did not interference with the qualities of the strawberries during storage. The study demonstrates a promising application for multi-antimicrobial and bio-friendly packaging materials in postharvest fruit preservation.

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.

Development, characterization and functional properties of sodium alginate-based films incorporated with Schisandra chinensis extract-natamycin complex.

Because of the impact of petroleum-based polymers on environmental deterioration and the need for safe, efficient, and functional packaging films, a sodium alginate (SA)-based film incorporating a Schisandra chinensis extract (SCE)-natamycin (NA) complex was developed for the desired physical and functional properties. The incorporation of SCE-NA into SA-based films decreased the water vapor transmission rate (WVTR), moisture content (MC), and hydrophilicity of the films and improved their opacity, elongation at break (EAB), and thermal stability. Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and X-ray Diffraction (XRD) analyses showed that SA, SCE, and NA had positive interactions and compatibility. In addition, the antimicrobial activity analysis indicated that the SA-SCE-NA film-forming solutions had satisfactory antimicrobial activity against Staphylococcus aureus, Escherichia coli, Saccharomyces cerevisiae, and Aspergillus niger. SA-based composite films have been used to coat cucumbers and blueberries to extend their shelf life. Compared to the neat SA film, the shelf life of cucumbers treated with the SA-SCE-NA film increased by 6 days compared to that in the untreated group at 28 °C, and the shelf life of blueberries increased by 5 days at 4 °C, revealing its potential utilization in food packaging.

Clinical and in vivo confocal microscopy characteristics of Candida keratitis following keratoplasty.

BACKGROUND: We present six patients who developed Candida keratitis postoperatively. The clinical features, diagnostic testing including in vivo confocal microscopy, and outcomes are presented. METHODS: Six patients who developed Candida keratitis following penetrating and endothelial keratoplasty, were referred to Tianjin Medical University Eye Hospital between 2018 to 2021.The diagnosis was established following cultures of either corneal scraping or biopsy. In vivo confocal microscopy examination was also performed to confirm the diagnosis and characterize the morphology, distribution and the depth of Candida spp. All patients were treated with topical voriconazole (VCZ) 1% and natamycin (NTM) 5%. Patients with mid/deep stromal keratitis or interface infection were treated additionally with intrastromal or interface VCZ irrigation (0.05 mg/0.1mL). RESULTS: The cultures of corneal scrapings (4 cases) or biopsies (2 cases) were all positive for Candida spp. In vivo confocal microscopy examination was positive for fungal elements in five of the six patients. The infection resolved in five of the six patients. The patients' final uncorrected visual acuity (UCVA) ranged from hand movements (HM) to 20/80. CONCLUSION: In vivo confocal microscopy is a useful non-invasive clinical technique for confirming the diagnosis of Candida keratitis. Intrastromal and interface irrigated VCZ injections are effective treatment options.

Rapid microwave synthesis of N and S dual-doped carbon quantum dots for natamycin determination based on fluorescence switch-off assay.

Green, one-pot, quick, and easily synthesized nitrogen and sulfur co-doped carbon quantum dots (N,S-CDs) were obtained from cheap and readily available chemicals (sucrose, urea, and thiourea) using a microwave-assisted approach in about 4 min and utilized as a turn-off fluorescent sensor for estimation of natamycin (NAT). First, the effect of N and S doping on the microwave-synthesized CDs' quantum yield was carefully studied. CDs derived from sucrose alone failed to produce a high quantum yield; then, to increase the quantum yield, doping with heteroatoms was carried out using either urea or thiourea. A slight increase in quantum yield was observed upon using thiourea with sucrose, while an obvious enhancement of quantum yield was obtained when urea was used instead of thiourea. Surprisingly, using a combination of urea and thiourea together results in N,S-CDs with the highest quantum yield (53.5%), uniform and small particle size distribution, and extended stability. The fluorescent signal of N,S-CDs was quenched upon addition of NAT due to inner filter effect and static quenching in a manner that allowed for quantitative determination of NAT over a range of 0.5-10.0µg ml-1(LOD = 0.10µg ml-1). The N,S-CDs were applicable for determination of NAT in aqueous humor, eye drops, different environmental water samples, and bread with excellent performance. The selectivity study indicated excellent selectivity of the prepared N,S-CDs toward NAT with little interference from possibly interfering substances. In-silico toxicological evaluation of NAT was conducted to estimate its long-term toxicity and drug-drug interactions. Finally, the preparation of N,S-CDs, and analytical procedure compliance with the green chemistry principles were confirmed by two greenness assessment tools.