Fungal keratitis is a critical ophthalmic condition that critically threatens vision, particularly in regions where warm and humid climates facilitate the growth of pathogenic fungi¹. Characterized by corneal inflammation and ulceration, this condition is often associated with trauma to the eye, especially in individuals involved in agricultural activities, in whom contact with vegetative matter is common. The complexity of fungal keratitis lies in its diverse etiology, with a wide range of fungal species capable of causing infection, including Fusarium, Aspergillus, and Curvularia spp.².

The diagnosis of fungal keratitis presents unique challenges because of the subtle clinical presentation of the condition in the early stages and the need for specialized microbiological investigations to confirm the presence of fungal pathogens. Delays in diagnosis or misdiagnosis can lead to rapid disease progression, resulting in significant corneal damage and potentially irreversible vision loss. Moreover, the treatment of fungal keratitis is often complicated by the limited availability of effective antifungal agents, the need for prolonged therapy, and the potential adverse effects of systemic and topical anti- fungal medications³. Given the high morbidity associated with fungal keratitis, understanding its epidemiological patterns and identifying key risk factors are essential for improving patient outcomes. In regions such as Tamil Nadu, where agri- culture plays a central role in the local economy, the incidence of fungal keratitis is particularly high, underscoring the need for targeted public health interventions⁴.

This study conducted a comprehensive evaluation of the epidemiological characteristics, causative organisms, and clinical outcomes of fungal keratitis among patients in a tertiary care center in Kelambakkam, Tamil Nadu, India. By systematically analyzing demographic data, risk factors such as diabetes and environmental exposure, and treatment responses, this research sought to elucidate the factors con- tributing to the disease's prevalence and improve the diag- nostic and therapeutic approaches to this potentially blinding condition.

Additionally, this study explored the distribution of specific fungal pathogens responsible for keratitis in this region in comparison with global trends. The findings will add to the existing body of knowledge on fungal keratitis and offer practical insights for clinicians in similar tropical and sub- tropical regions with a high disease burden.

This retrospective study was conducted at the Department of Microbiology, Chettinad Hospital and Research Institution, a tertiary care hospital in South India, from December 2019 to December 2023. The study commenced after obtaining approval from the Institutional Human Ethics Committee (reference ID: IHEC II/0102/21). After obtaining informed consent from the patients, 53 patients with suspected fungal keratitis who visited the OPD/IPD of Ophthalmology were included in the study. Samples were collected according to patient availability in the hospital. Patients with ocular in- fections other than fungal keratitis were excluded.

The following clinical variables were recorded: demographic data, medical history, risk factors such as history of ocular trauma with vegetative or non-vegetative material, contact lens use, prior ocular surgery, and clinical presentation. After a clinical assessment by the ophthalmologist, an experienced practitioner conducted a slit-lamp microscopic examination. Corneal scrapings were then collected under strict aseptic conditions using a sterile no. 15 Bard-Parker blade following the administration of topical anesthesia (4% lignocaine). The scrapings were taken from the edges and base of the ulcer, with the eyelids held open to prevent contamination from the lid margins or eyelashes. Scrapings were obtained from both central and peripheral regions of the ulcer using firm but gentle strokes.

Microscopic examination of ocular specimens involved subjecting a heat-fixed smear to Gram staining and pre- paring a wet mount with 10% KOH, which does not damage the cell walls of fungi, which remain intact and visible. This selective action is essential because it dissolves the cellular elements and increases the contrast between the fungi and the surrounding area, making it easier to identify fungal elements.

The collected material was inoculated onto culture medium, blood agar, and chocolate agar to culture bacterial patho- gens and Sabouraud's dextrose agar (SDA) to culture fungal pathogens. Cultures inoculated onto SDA were incubated at 25 and 37℃.

The inoculated media were examined daily for 8 weeks and discarded if no growth was observed. Fungal isolates were identified according to colony morphology on SDA, and lactophenol cotton blue (LPCB) mounts were prepared to identify filamentous fungi based on their microscopic characteristics, such as hyphal structure, septation, and spore morphology. Slide cultures were performed when necessary. To enhance identification accuracy, representative isolates were further confirmed using matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (MALDI-TOF MS) at the Department of Microbiology, JIPMER (Puducherry, India). MALDI-TOF MS was performed using the Bruker Micro- flex LT/SH system (Bruker, Billerica, MA, USA). Fungal isolates were processed using the standard formic acid acetonitrile extraction protocol recommended by the manufacturer for filamentous fungi. Mass spectra were acquired and analyzed using Bruker Biotyper software (version 4.1, Bruker) with the Filamentous Fungi Library (version 3.0, Bruker). The identi- fication confidence score was interpreted as follows: ≥2.0, reliable species-level identification; 1.7-1.99, reliable genus-level identification; and <1.7, unreliable identification.

Only identifications with scores ≥1.7 were considered for reporting. Although common genera such as Fusarium, Aspergillus, and Curvularia were reliably identified because of their presence in the validated library, we acknowledge that MALDI-TOF MS has known limitations in distinguishing certain filamentous fungi species attributable to spectral overlap or database insufficiency. Molecular confirmation (e.g., ITS or β-tubulin gene sequencing) was not performed, which is a recognized limitation, especially for rare or cryptic species such as Basidiobolus and Cylindrocarpon spp.

1. Antifungal susceptibility testing

The antifungal susceptibility of the isolated pathogens was assessed according to Clinical and Laboratory Standards Institute (CLSI) guidelines outlined in document M27 for yeast and M38 for filamentous fungi. To prepare the fungal inocula, first, the fungal isolates were grown on SDA plates at 35℃ for 24-48 h. Then, suspensions of the fungal spores were created in sterile saline or distilled water, and the fungal concentration in each suspension was adjusted to achieve the 0.5 McFarland standard. To prepare antifungal solutions, stock solutions were created for the antifungal agents according to the manufacturer's instructions. Serial 2-fold dilutions of these agents were made in RPMI 1640 medium to achieve the required concentration ranges. In microdilution testing, 0.1 mL of growth medium were dropped into each well of a 96-well microtiter plate. Then, 100 μL of each antifungal dilu- tion were added to the wells, resulting in final concentrations of 0.03125-16 μg/mL, or other concentrations as appropriate for the specific antifungal agent, and then 100 μL of each prepared fungal inoculum were added to the wells. Control wells with a fungal inoculum but no antifungal agents and sterility control wells without a fungal inoculum were also included in the assay. The plates were covered and incubated at 35℃ for 24-48 h for yeast or up to 72 h for filamentous fungi. Fungal growth was visually assessed using a UV spec- trophotometer, and the optical density (OD) was measured at 530 nm. Epidemiological cutoff values (ECVs) were not applied to interpret the minimum inhibitory concentration (MIC) data, primarily because of the absence of established breakpoints for many of the isolated filamentous fungi. As such, the reported MICs are only descriptive and comparative, and they should not be used to infer clinical resistance or susceptibility.

Of the 53 samples collected, 44 were culture-positive. Patients with culture-positive fungal keratitis patients were primarily male (34/44, 77.27%, Table 1). As presented in Fig. 1, patients with fungal keratitis were most commonly aged 40-50 years (28%), followed by 50-60 years (27%).

Figure 1. Age distribution

Modify Delete

Concerning occupations, culture-positive patients were primarily farmers (65.9%). The primary source of infection was contact or trauma caused by vegetable matter (55.5%), with contact lenses being a less frequent cause (4.5%, Table 1). As listed in Table 1, symptoms experienced by patients with fungal keratitis included redness (98%), pain (93%), blurred vision (84%), decreased vision (79.5%), watering (50%), discharge (45.4%), white opacity (22.7%), and a foreign body sensation in the eye (18.1%). Most patients experienced reduced vision, redness, tearing, and pain at the infection site. In addition, 12 patients (27.27%) had co- incident diabetic mellitus, whereas two patients (4.5%) had coincident hypertension (Table 1).

Fungal isolates were identified according to colony morph- ology on SDA, and LPCB mounts (Fig. 2) were prepared to identify filamentous fungi based on their microscopic characteristics. Based on MALDI-TOF MS, Fusarium spp. re-presented the most commonly isolated pathogens (16 isolates, 36.3%), followed by Aspergillus flavus (10 isolates, 22.7%) and Curvularia spp. (7 isolates, 15.9%). Meanwhile, Scedosporium spp. and Alternaria spp. were isolated from two patients each (4.5%), whereas Aspergillus fumigatus, Aspergillus niger, Cladosporium spp., Exserohilum turcicum, Penicillium spp., Basidiobolus spp., and Cylindrocarpon spp. were isolated from one patient each (2.2%, Table 2). The MIC is the lowest concentration of an antifungal agent that completely inhibits visible growth or leads to a significant decrease in OD relative to the control. The evaluation used amphotericin B and voriconazole as standard antifungal agents, with their results tabulated in Table 3. The results were presented as the mean ± standard deviation for quantitative variables and summarized as frequency (percentage) for categorical variables.

Figure 2. Culture staining with lactophenol cotton blue (A. Aspergillus flavus, B. Curvularia, C. Fusarium spp., and D. Cladosporium spp.)

Modify Delete

Fungal keratitis is a significant public health concern, particu- larly in tropical and subtropical regions where environmental factors and socioeconomic conditions contribute to its pre- valence⁵. The observed predominance of fungal keratitis among male patients in this study, particularly those engaged in agricultural activities, reflects broader epidemiological trends observed in similar geographical settings and gender-specific occupational exposures. Men, especially those working in agriculture, construction, and other outdoor occupations, are more frequently exposed to plant material, soil, and organic debris that harbor fungal spores⁶. This increased exposure heightens their risk of sustaining corneal injuries, which represent a common precursor to fungal infections. Moreover, cultural and social factors can influence healthcare-seeking behavior, with men potentially more likely to report or seek treatment for eye injuries. The age distribution, with a peak incidence in people aged 40-50 years, aligns with the results of several epidemiological studies in India, China, and Southeast Asia⁷. In these regions, middle-aged individuals represent the primary workforce in agriculture, leading to higher exposure to risk factors such as trauma from vegetation. The higher susceptibility in this age group could also be linked to cumu- lative exposure to environmental fungi over time, as well as age-related changes in ocular surface immunity and tear film stability, which can predispose individuals to infections⁸. Geographically, fungal keratitis is more prevalent in rural and agricultural regions in which environmental conditions, such as humidity and temperature, favor the growth and dispersion of fungi such as Fusarium and Aspergillus^9,10. The study by Ghosh identified Fusarium spp. as the leading causative agents in this region, consistent with the environ- mental prevalence of these fungi in the soil and organic matter, particularly in tropical climates where fungal spores are more readily dispersed¹¹. Seasonal variations also play a role, with Das et al. reporting that the incidence of fungal keratitis was higher during the rainy season, when agricultural activities peak and injuries related to plant debris are more common¹². Socioeconomic factors are critical determinants in the epidemiology of fungal keratitis. In lower-income rural settings, limited access to healthcare, poor hygiene practices, and a lack of awareness about occupational safety and the serious nature of ocular injuries can delay diagnosis and treatment, leading to higher rates of fungal keratitis¹³. The reliance on traditional remedies and late presentation to healthcare facilities further complicate the management of fungal infections, often resulting in poorer outcomes in rural areas than in urban areas, which have better access to medical care¹⁴ (Chidambaram et al., 2018). The study by Bharathi et al. documented that corneal trauma is a well-established risk factor for fungal keratitis, with minor injuries often acting as the portal of entry for pathogenic fungi. The present study found that a large percentage (56%) of patients had a history of corneal injury, mainly caused by agricultural tools or plant materials, aligning with the findings of other research in comparable rural settings¹⁵. The role of trauma underscores the need for protective eyewear and education about eye safety in high-risk occupations. According to Prajna, in addition to trauma, pre-existing ocular surface diseases and the use of topical corticosteroids are risk factors for fungal keratitis¹⁶. Corticosteroids, commonly used to treat various ocular conditions, can suppress the local immune response, creating a favorable environment for fungal colonization and infection. The current findings should prompt a re-evaluation of the widespread use of topical corticosteroids in fungal keratitis-endemic regions. Fungal keratitis remains a global concern, with its incidence and etiology significantly varying by geographic location, climate, and population demographics. For instance, the incidence of fungal keratitis is lower in tem- perate regions, and the spectrum of causative fungi differs, with Candida spp. being more prevalent in temperate areas, in contrast to the dominance of filamentous fungi such as Fusarium and Aspergillus in tropical climates¹¹. The global distribution and impact of fungal keratitis highlight the need for region-specific public health strategies, including aware- ness campaigns, protective measures for at-risk populations, and the development of accessible and effective antifungal treatments tailored to local epidemiology. Public health initia- tives should focus on preventive measures such as educating the rural workforce about the risks of corneal injuries and the importance of early medical intervention. Additionally, improving access to healthcare and promoting the use of protective eyewear in high-risk occupations could reduce the incidence of fungal keratitis and improve outcomes for affected individuals. Understanding the microbiological profile of fungal pathogens is crucial for effectively diagnosing and treating fungal keratitis. The microbiological profile encom- passes the types of fungi responsible for infections, their prevalence, and their antifungal susceptibility patterns. This profile provides insights into the specific disease-causing pathogens, informs treatment decisions, and facilitates the development of targeted therapeutic strategies¹⁷. In a prior study, Thomas identified Fusarium spp. as the most prevalent pathogens, accounting for 36.36% of the isolated fungi. Fusarium is widely found in soil and plant debris, and it frequently causes fungal keratitis, particularly in tropical and subtropical areas¹⁸. Fusarium spp. are highly pathogenic, and they are often associated with severe ocular infections because of their ability to invade corneal tissue aggressively. The filamentous nature of Fusarium allows it to penetrate deeper into the corneal stroma, leading to more severe disease outcomes¹⁹. Aspergillus flavus and Aspergillus niger were also significant pathogens in this study, representing 22.72% and 2.27% of the isolates, respectively. Aspergillus spp. are found throughout the environment, particularly in decaying plant material and soil. They are known to cause keratitis, often following trauma from organic matter¹⁸. Aspergillus flavus is particularly notorious for producing aflatoxins, which can exacerbate the virulence of infections²⁰. According to Bharathi, Curvularia spp., accounting for 15.9% of the isolates in the present study, comprises another significant group of fungi associated with fungal keratitis. These fungi are found in soil, plant material, and decaying vegetation. Curvularia infections often occur in patients with a history of trauma from plant material, aligning with the findings in this study²¹. Curvularia spp. are known for their ability to cause chronic and recalcitrant infections, often requiring prolonged anti- fungal therapy¹⁵. Other isolated fungi included Alternaria spp., Cladosporium spp., Exserohilum turcicum, Penicillium spp., Scedosporium spp., Basidiobolus spp., and Cylindrocarpon spp., each representing a smaller percentage of the total isolates. Although these fungi are less common, their detec- tion underscores the diversity of fungal pathogens responsible for keratitis. For instance, Scedosporium spp. are often asso- ciated with more resistant infections, and they pose significant treatment challenges because of their inherent antifungal resistance²². Accurate identification of fungal pathogens in keratitis is achieved through a combination of clinical, micro- biological, and molecular techniques. Initial identification is often based on clinical presentation and slit-lamp micro-scopy, which can reveal characteristic features of fungal infections. However, definitive identification requires culture-based methods, in which fungi are isolated and identified according to their morphological and biochemical char- acteristics^18,23. Molecular techniques, such as PCR and DNA sequencing, offer a more precise identification of fungi, and they can detect pathogens that are difficult to culture or identify using traditional methods²⁴. These techniques are especially useful in identifying less common or emerging fungal pathogens, and they can provide insights into the genetic and epidemiological aspects of the infection^21,25. Meanwhile, Ghosh noted that traditional antifungal treat- ments can have varying efficacy depending on the pathogen. For example, Fusarium and Aspergillus spp. are often resistant to certain antifungals, necessitating the use of alternative or combination therapies¹¹. The development of resistance among fungal pathogens poses significant challenges in managing fungal keratitis and underscores the need for continuous surveillance and new antifungal agents. The epidemiological patterns, risk factors (e.g., trauma caused by vegetative material), and predominant fungal species reported in this study are consistent with previous findings from India and other tropical regions. Although the use of MALDI-TOF MS and the identification of rare fungal species (e.g., Basidiobolus spp., Scedosporium spp., Cylindrocarpon spp.) offer some novel insight, the overall findings might not represent a substantial deviation from established regional data. However, this study provides updated evidence from a 4-year post-pandemic period, which could reflect evolving trends in fungal keratitis etiology and antifungal susceptibility. The diverse microbiological profile of fungal pathogens high- lights the necessity for individualized treatment strategies based on the specific fungi isolated from patients. Empirical treatment with broad-spectrum antifungals is often employed initially, but definitive therapy should be tailored according to the susceptibility profiles of the isolated pathogens²⁶.

Some limitations of this study should be acknowledged. This study was conducted retrospectively at a single tertiary care center, and it included a relatively small number of patients (n = 53), which might limit how broadly the findings can be applied to other settings or populations. Although we used MALDI-TOF MS, a rapid and increasingly popular method, for fungal identification, the reliability of this tech- nique depends heavily on the reference database. Because molecular confirmation (such as ITS or β-tubulin sequencing) was not performed, there might be some uncertainty in the species-level identification of certain rare or uncommon fungi. In terms of antifungal susceptibility testing, although we followed CLSI guidelines (M27 for yeast and M38 for fila- mentous fungi), established clinical breakpoints or ECVs could not be applied for most isolates, as these data are not currently available for many filamentous fungi. Thus, the MIC data presented should be viewed as descriptive rather than predictive of clinical outcomes. Finally, although our findings on the most common fungal pathogens and associated risk factors are consistent with previous results from tropical regions, this study contributes updated local data, including rarely reported Cylindrocarpon spp., Scedosporium spp., and Syncephalastrum spp.