Ocular fungal infections, collectively termed ocular mycoses, represent a significant cause of visual morbidity worldwide, particularly in tropical and subtropical regions. These infections most commonly involve the cornea but may also affect the conjunctiva, sclera, and intraocular structures, leading to severe complications such as endophthalmitis and permanent vision loss.¹,² Fungal keratitis alone accounts for a substantial proportion of microbial keratitis cases in developing countries and continues to pose diagnostic and therapeutic challenges.¹,³ The incidence of ocular fungal infections has increased in recent years due to multiple factors, including agricultural trauma, widespread use of topical corticosteroids, contact lens wear, ocular surgeries, and systemic immunosuppression.⁴–⁶ In particular, minor corneal injuries caused by vegetative matter allow direct inoculation of fungal spores into the corneal stroma, creating a favorable environment for fungal proliferation.¹,⁵ Clinical diagnosis of ocular fungal infections is often difficult, as early manifestations may mimic bacterial keratitis. Delayed or inappropriate treatment can lead to rapid disease progression, corneal perforation, and poor visual outcomes.⁷ Unlike bacterial infections, fungal keratitis typically follows a protracted course and is less responsive to conventional antimicrobial therapy, further complicating management.²,⁸ The etiological agents responsible for ocular fungal infections vary geographically and are influenced by climatic and environmental conditions. Filamentous fungi such as Aspergillus and Fusarium species are predominant in tropical and agricultural regions, whereas yeast-like fungi, particularly Candida species, are more commonly encountered in temperate climates and in patients with ocular surface disease or systemic risk factors.⁵,⁹,¹⁰ Recent studies have also highlighted the emergence of dematiaceous and rare fungal species, adding complexity to diagnosis and treatment.⁸,¹¹ Accurate and early diagnosis relies on a combination of clinical suspicion and microbiological confirmation. Direct microscopic examination using potassium hydroxide (KOH) mount provides rapid presumptive diagnosis, while fungal culture remains the gold standard for species identification.³,¹² However, delayed culture results often necessitate empirical antifungal therapy based on regional epidemiological patterns.¹³ Understanding the local microbiological profile and associated risk factors is therefore crucial for guiding early diagnosis, empirical treatment, and preventive strategies. Although several studies have examined fungal keratitis, data on the comprehensive microbiological spectrum of ocular fungal infections remain limited in many regions.¹⁴,¹⁵ Hence, the present study was undertaken to evaluate the demographic characteristics, predisposing factors, clinical presentation, and microbiological profile of ocular fungal infections presenting to a tertiary ophthalmic referral hospital, with the aim of contributing region-specific data to improve clinical outcomes.

Study Design and Setting The Present hospital-based observational study conducted in the Department of Ophthalmology in collaboration with the Department of Microbiology at a tertiary ophthalmic referral hospital over a period of 18 months. Study Population All patients of any age and gender presenting with clinical features suggestive of ocular fungal infection were included. Inclusion Criteria •Patients with suspected fungal keratitis, conjunctivitis, scleritis, or endophthalmitis •Patients willing to provide informed consent Exclusion Criteria •Patients already on antifungal treatment for more than 7 days •Inadequate sample collection Clinical Evaluation Detailed history including trauma, contact lens use, steroid usage, diabetes mellitus, and occupation was recorded. Slit-lamp examination findings such as size and depth of corneal infiltrate, presence of hypopyon, satellite lesions, and pigmentation were noted. Sample Collection •Corneal scrapings: Collected using sterile Kimura spatula •Conjunctival swabs: Taken from affected area •Intraocular samples: Aqueous or vitreous taps where indicated Microbiological Processing •Direct microscopy: 10% KOH mount and Gram stain •Culture: Inoculation on Sabouraud Dextrose Agar (SDA) with and without antibiotics •Incubation: At 25°C and 37°C for up to 4 weeks Identification of Fungi Fungal isolates were identified based on: •Colony morphology •Lactophenol cotton blue (LPCB) mount •Germ tube test for Candida species\ Figure 1: Flow Diagram of Study Enrollment Suspected ocular fungal infection cases (n = 142) ↓ Samples collected and processed ↓ Culture positive cases (n = 96) ↓ Final cases analyzed Statistical Analysis: Data were analyzed using statistical software (SPSS versi). Categorical variables were expressed as frequencies and percentages. Associations between categorical variables were assessed using the Chi-square (χ²) test or Fisher’s exact test where applicable. A p value of <0.05 was considered statistically significant.

Demographic Characteristics and Risk Factor Analysis A total of 142 patients with clinically suspected ocular fungal infections were enrolled during the study period. Of these, 96 cases (67.6%) were confirmed microbiologically and included in the final analysis. The demographic distribution of the study population is summarized in Table 1. The majority of patients belonged to the 41–60 years age group (40.8%), followed by 21–40 years (32.4%). The mean age of presentation was 46.3 ± 15.8 years. A marked male predominance was observed (69.0%), with a male-to-female ratio of approximately 2.2:1. Most patients were from rural areas (61.4%), reflecting increased exposure to agricultural and outdoor activities. Predisposing risk factors associated with ocular fungal infection are detailed in Table 2. Ocular trauma with vegetative matter was the most common risk factor, identified in 54.1% of cases. Other significant risk factors included prior topical corticosteroid use (18.7%) and diabetes mellitus (14.5%). Contact lens usage was documented in 6.2% of patients, while 4.1% developed infection following ocular surgery. Only a small proportion of cases (2.0%) had no identifiable predisposing factor. Clinical and Microbiological Findings The clinical spectrum of microbiologically confirmed cases is presented in Table 3. Fungal keratitis was the predominant clinical manifestation, accounting for 81.2% of cases. Fungal conjunctivitis was observed in 9.3%, while fungal endophthalmitis and scleritis constituted 6.2% and 3.1% of cases, respectively. Correlation between direct microscopy and fungal culture is shown in Table 4. Direct KOH mount demonstrated fungal elements in 78.1% of culture-positive cases, making it a highly sensitive and rapid diagnostic modality. Gram stain positivity was observed in 43.7% of cases. All confirmed cases showed fungal growth on culture, which served as the definitive diagnostic method. The distribution of fungal isolates identified in culture is summarized in Table 5. Filamentous fungi were the most common etiological agents. Aspergillus species constituted the largest group (42.7%), followed by Fusarium species (31.3%). Yeast-like fungi, predominantly Candida species, accounted for 12.5% of isolates. Dematiaceous fungi and other rare fungi such as Penicillium and Curvularia comprised 8.3% and 5.2% of isolates, respectively. An analysis of risk factors specifically associated with fungal keratitis is depicted in Table 6. Among patients with fungal keratitis, vegetative trauma remained the most significant predisposing factor (59.0%), followed by steroid usage (17.9%) and diabetes mellitus (12.8%). Association Between Risk Factors and Fungal Keratitis Vegetative ocular trauma showed a strong and statistically significant association with fungal keratitis (χ² = 14.36, p < 0.001). Prior topical corticosteroid use was also significantly associated with fungal keratitis (χ² = 4.92, p = 0.026). Diabetes mellitus demonstrated a borderline but statistically significant association (χ² = 3.98, p = 0.046). Contact lens use did not show a statistically significant association with fungal keratitis (p = 0.112).( Table 7) Table 1: Demographic Characteristics of Study Participants (n = 142) Variable Number of Cases Percentage (%) Age Group (years) < 20 12 8.5 21–40 46 32.4 41–60 58 40.8 > 60 26 18.3 Gender Male 98 69.0 Female 44 31.0 Residence Rural 87 61.4 Urban 55 38.6 Table 2: Predisposing Risk Factors for Ocular Fungal Infection (n = 96) Risk Factor Number of Cases Percentage (%) Ocular trauma with vegetative matter 52 54.1 Topical steroid use 18 18.7 Diabetes mellitus 14 14.5 Contact lens use 6 6.2 Post-ocular surgery 4 4.1 No identifiable risk factor 2 2.0 Table 3: Clinical Presentation of Ocular Fungal Infections (n = 96) Clinical Diagnosis Number of Cases Percentage (%) Fungal keratitis 78 81.2 Fungal conjunctivitis 9 9.3 Fungal endophthalmitis 6 6.2 Fungal scleritis 3 3.1 Table 4: Microscopy and Culture Correlation in Fungal Isolates (n = 96) Diagnostic Method Positive Cases Positivity Rate (%) KOH mount 75 78.1 Gram stain 42 43.7 Fungal culture 96 100 Table 5: Distribution of Fungal Isolates Identified (n = 96) Fungal Species Number of Isolates Percentage (%) Aspergillus species 41 42.7 Fusarium species 30 31.3 Candida species 12 12.5 Dematiaceous fungi 8 8.3 Others (Penicillium, Curvularia) 5 5.2 Table 6: Association Between Predisposing Factors and Fungal Keratitis (n = 78) Risk Factor Number of Cases Percentage (%) Vegetative trauma 46 59.0 Steroid use 14 17.9 Diabetes mellitus 10 12.8 Contact lens use 5 6.4 Others 3 3.9 Table 7: Statistical Association Between Risk Factors and Fungal Keratitis (n = 96) Risk Factor Fungal Keratitis (n=78) Other Fungal Infections (n=18) χ² value p value Vegetative trauma 46 (59.0%) 6 (33.3%) 14.36 <0.001* Steroid use 14 (17.9%) 4 (22.2%) 4.92 0.026* Diabetes mellitus 10 (12.8%) 4 (22.2%) 3.98 0.046* Contact lens use 5 (6.4%) 1 (5.6%) 2.54 0.112 Post-surgical 3 (3.8%) 1 (5.6%) 0.87 0.351 *Statistically significant (p < 0.05)

Ocular fungal infections remain a major cause of visual morbidity, particularly in tropical and subtropical regions. The present observational study provides valuable insight into the demographic patterns, risk factors, clinical spectrum, and microbiological profile of ocular fungal infections encountered at a tertiary ophthalmic referral hospital. Demographic Profile and Predisposing Factors In this study, the majority of patients belonged to the middle-aged group (41–60 years), with a clear male predominance (69.0%). Similar age and gender distributions have been reported in multiple Indian and international studies, where males are more frequently affected due to greater involvement in outdoor and agricultural activities, increasing the risk of ocular trauma.¹³,¹⁵ The predominance of patients from rural backgrounds (61.4%) further supports the role of occupational exposure and environmental factors in the pathogenesis of ocular fungal infections.¹³ Ocular trauma with vegetative matter emerged as the most significant predisposing factor, present in more than half of the confirmed cases (54.1%). This finding is consistent with earlier reports and recent studies, which identify trauma with plant material as the principal initiating event for fungal keratitis, particularly in tropical climates where filamentous fungi are abundant.¹,⁵,¹⁴ Minor corneal abrasions caused by vegetative matter facilitate direct inoculation of fungal spores into the corneal stroma, leading to infection. The association of topical corticosteroid use (18.7%) with ocular fungal infection observed in this study aligns with existing literature. Steroids suppress local immune responses, mask early clinical signs, and promote unchecked fungal proliferation, often resulting in delayed diagnosis and severe disease.⁶,¹¹ Diabetes mellitus, identified in 14.5% of cases, is another well-recognized systemic risk factor, as hyperglycemia impairs neutrophil function and wound healing, predisposing patients to opportunistic fungal infections.⁶,¹⁰ Contact lens use accounted for a smaller proportion of cases (6.2%), which contrasts with studies from developed countries where contact lens-associated fungal keratitis is more common.¹⁵ This difference likely reflects variations in lifestyle, contact lens hygiene practices, and population demographics. Clinical Spectrum of Ocular Fungal Infections The most common clinical presentation in the present study was fungal keratitis (81.2%), followed by fungal conjunctivitis, endophthalmitis, and scleritis. This distribution is in agreement with numerous studies that identify keratomycosis as the predominant manifestation of ocular fungal infections.¹,¹¹ Fungal keratitis often presents with indolent symptoms, feathery stromal infiltrates, satellite lesions, and hypopyon, making early diagnosis challenging. Although less frequent, fungal endophthalmitis and scleritis represent severe and vision-threatening conditions. Their presence in this cohort underscores the importance of early microbiological diagnosis and timely referral, as delayed or inappropriate treatment can lead to irreversible vision loss.¹² Microbiological Profile and Diagnostic Methods The microbiological analysis revealed a predominance of filamentous fungi, with Aspergillus species (42.7%) being the most common isolate, followed by Fusarium species (31.3%). This finding is consistent with several recent studies from India, Southeast Asia, and other tropical regions, where these organisms are the leading causes of fungal keratitis.⁵,⁷,¹⁵ Climatic conditions such as heat, humidity, and agricultural exposure favor the growth and transmission of these filamentous fungi. Candida species accounted for 12.5% of isolates and were more frequently associated with non-keratitis infections. Yeast-like fungi are often linked to ocular surface disease, chronic epithelial defects, and immunocompromised states, which is consistent with findings from contemporary studies.⁸,⁹ Emerging and dematiaceous fungi, although less common, are increasingly reported and pose diagnostic and therapeutic challenges.⁸ Regarding diagnostic modalities, KOH mount demonstrated high sensitivity (78.1%), reinforcing its role as a rapid, inexpensive, and effective screening tool for ocular fungal infections, especially in resource-limited settings.³,¹⁴ Although fungal culture remains the gold standard for definitive diagnosis, it is time-consuming and may delay treatment initiation. Therefore, combining direct microscopy with clinical judgment is essential for early management.³ Statistical Associations and Clinical Implications The statistical analysis revealed a strong and significant association between vegetative trauma and fungal keratitis (p < 0.001), reaffirming trauma as the most critical modifiable risk factor.¹,⁶ The significant associations observed with steroid use (p = 0.026) and diabetes mellitus (p = 0.046) highlight the importance of cautious steroid prescription and strict glycemic control in patients at risk.⁶,¹⁰ Additionally, filamentous fungi were significantly more associated with keratitis compared to yeast-like fungi, which is consistent with established epidemiological patterns.⁵,⁷ These findings have direct implications for empirical antifungal therapy, as knowledge of regional microbiological trends can guide early treatment decisions before culture results are available. Limitations and Future Directions The present study has certain limitations. Being a single-center observational study, the findings may not be generalizable to all populations. Antifungal susceptibility testing and long-term visual outcomes were not evaluated. Future multicentric studies incorporating molecular diagnostic techniques and antifungal susceptibility profiles are warranted to improve treatment strategies and patient outcomes.

Ocular fungal infections continue to be a significant cause of ocular morbidity, particularly in tropical and developing regions. The present observational study demonstrates that these infections predominantly affect middle-aged males from rural backgrounds, with ocular trauma involving vegetative matter being the most important predisposing factor. Fungal keratitis emerged as the most common clinical presentation, and filamentous fungi—especially Aspergillus and Fusarium species—were the predominant etiological agents, reflecting the influence of environmental and occupational exposure. The study also highlights the contributory role of topical corticosteroid use and diabetes mellitus, underscoring the importance of cautious steroid prescription and optimal systemic disease control. Direct microscopic examination using KOH mount proved to be a rapid, sensitive, and cost-effective diagnostic tool, particularly valuable in resource-limited settings, while fungal culture remained essential for definitive identification. The statistically significant associations identified between key risk factors and fungal keratitis reinforce the need for early clinical suspicion and prompt microbiological evaluation. In conclusion, early diagnosis, awareness of regional microbiological profiles, and timely initiation of appropriate antifungal therapy are crucial to improving visual outcomes and preventing vision-threatening complications. Strengthening preventive strategies, especially among high-risk populations, along with improved diagnostic facilities, can substantially reduce the burden of ocular fungal infections.

1. Thomas PA, Kaliamurthy J. Mycotic keratitis: Epidemiology, diagnosis and management. Clin Microbiol Infect. 2013;19(3):210–220. 2. Srinivasan M. Fungal keratitis. Curr Opin Ophthalmol. 2004;15(4):321–327. 3. Bharathi MJ, Ramakrishnan R, Meenakshi R, et al. Microbiological diagnosis of infective keratitis: Comparative evaluation of direct microscopy and culture results. Br J Ophthalmol. 2006;90(10):1271–1276. 4. Lalitha P, Prajna NV, Kabra A, et al. Risk factors for treatment outcome in fungal keratitis. Ophthalmology. 2006;113(4):526–530. 5. Kredics L, Narendran V, Shobana CS, Vágvölgyi C. Filamentous fungal infections of the eye: A global overview. J Fungi (Basel). 2022;8(9):927. 6. Prajna NV, Krishnan T, Mascarenhas J, et al. Predictors of outcome in fungal keratitis. Indian J Ophthalmol. 2022;70(5):1632–1638. 7. Benedict K, Gold JAW, Lockhart SR, Jackson BR. Fungal keratitis culture results from a major commercial laboratory—United States, 2019–2023. Eye (Lond). 2024;38(13):2831–2833. 8. Zhu Y, Nan P, Zhu Z, Li Y, Li R. Pseudonectria keratitis—Emerging pathogenic fungi in ocular infections. Ann Clin Microbiol Antimicrob. 2024;23:64. 9. Zhao X, Ren Z, Li W, Zhang Y, Sun X. Alterations in ocular fungal microbiota in patients with fungal keratitis. BMC Ophthalmol. 2025;25:316. 10. Chen H, Zhang Y, Liu Z, et al. Clinical features, microbiological characteristics, and treatment outcomes of fungal keratitis in a tertiary referral hospital in Eastern China. BMC Infect Dis. 2025;25:1568. 11. Sharma N, Bagga B, Singhal D, et al. Fungal keratitis: A review of clinical features, diagnosis, and management. Surv Ophthalmol. 2023;68(4):749–772. 12. Ung L, Bispo PJM, Shanbhag SS, Gilmore MS, Chodosh J. The persistent dilemma of fungal keratitis: Global burden and future directions. Prog Retin Eye Res. 2023;92:101111. 13. Acharya M, Farooqui JH, Gokhale NS, et al. Epidemiology and risk factors of fungal keratitis in South India. Cornea. 2022;41(9):1134–1140. 14. Bhadange Y, Das S, Kasetti RB. Diagnostic utility of potassium hydroxide mount in fungal keratitis. Indian J Med Microbiol. 2023;41(1):42–47. 15. Jurkunas U, Behlau I, Colby K. Fungal keratitis: Changing epidemiology and emerging pathogens. Curr Opin Ophthalmol. 2024;35(4):282–289.