Retrospective Evaluation of Potassium Hydroxide Mounts and Fungal Cultures in Suspected Mycotic Keratitis at a Tertiary Eye Care Center in South India

Introduction

Mycotic keratitis is a leading cause of corneal blindness, particularly in developing countries like India, where factors such as environmental exposure, agricultural activities, and inadequate healthcare access contribute to its prevalence.^1,2 Ocular trauma, contact lens use are important predisposing factors in the development of infective keratitis.³

The significant impact of mycotic keratitis on the agrarian population, primarily due to increased exposure to environmental pathogens and risk factors associated with agricultural practices, underscores the need for urgent attention from public health specialists.⁴ Corneal blindness ranks second only to cataract-related blindness, with a prevalence ranging from 0.25% to 1.75%, presenting a significant health and economic burden in densely populated countries such as India.⁵ This issue not only places a considerable burden on physicians who are tasked with diagnosing and treating these infections, but also highlights the necessity for comprehensive public health strategies aimed at prevention, education, and resource allocation in vulnerable communities.^6,7 In addition to that, the emerging trends of antifungal resistance of species like Fusarium is of significant concern as noted in few studies.⁸

The clinical diagnosis of mycotic keratitis is based on the features of an indolent ulcer which progresses slowly over weeks, usually following a history of trauma from vegetative matter like a stick, thorn, paddy leaf, soil, groundnut husk, onion peel, etc. The slit lamp features of a dry looking ulcer, a thick exudation in the anterior chamber (Figure 1), minimal congestion, satellite lesions, aid in the clinical diagnosis which when supported by laboratory techniques, is essential to minimize further ocular morbidity.

Common laboratory methods for identifying microbial agents responsible for corneal infections include direct microscopy by Potassium Hydroxide (KOH) wet mount examination and culture. While these methods are valuable, special stains such as Gomori’s methenamine silver, periodic acid-Schiff, and calcofluor white are often time-consuming, expensive, and require specialized equipment, such as a compound microscope, limiting their accessibility in many clinical settings. Gram stains, and Giemsa stains are widely used for microbial detection; however, they can be subject to misinterpretation, presence of artifacts, and a lack of sensitivity for detecting Candida and other yeasts. KOH examination, in contrast, is a simple and rapid diagnostic tool for mycotic keratitis that can be performed in an office setting, making it more practical for immediate clinical use.⁹ There are several studies on the species identification of mycotic keratitis, sensitivity and specificity patterns of KOH wet mounts, but few studies exist on correlation of these both and their impact on public health.

The etiological agents of mycotic keratitis vary among patient populations, geographic regions, and climatic conditions, and they can also change over time. This variation underscores the importance of understanding region-specific etiological agents for effective management. Previous studies have indicated that environmental fungi, such as Fusarium and Aspergillus, are commonly isolated in tropical climates, highlighting the need for local data to device treatment strategies.^10,11

Therefore, the purpose of this study was to retrospectively analyze the accuracy of KOH wet mounts and fungal cultures, as well as the types of fungal species isolated in patients presenting at a referral center in South India. This information is critical for improving diagnostic approaches and tailoring treatment protocols in the region.

Materials and Methods

This retrospective analysis involved the medical records of 150 eyes from 150 patients with suspected mycotic keratitis, whose corneal scrapings were analyzed by a trained microbiologist between November 2016 and November 2017. The corneal scrapings of all patients with clinical suspicion of mycotic keratitis diagnosed by a single ophthalmologist on slit-lamp examination in the above period were included. Corneal scrapings were obtained from the leading edge and base of the ulcers using a sterile 24-gauge needle and were inoculated on culture plates. The scrapings from patients with history of previous ocular surgery, those with bacterial /mixed infections and those on antifungal treatment for more than three days were excluded.

A portion of the sample was subjected to a 10% KOH wet mount (Figure 2a) for direct microscopic examination. Corneal scraping samples were also inoculated directly into two Sabouraud Dextrose Agar (SDA) tubes. The procedures were conducted under a laminar flow hood. The SDA tubes were incubated at 27°C, with daily examinations during the first week and biweekly examinations for the subsequent three weeks. Tubes were discarded at three weeks if no growth was observed.

Fungal growth that was positive was examined both macroscopically (Figure 2b) and microscopically to identify the species using Lactophenol cotton blue. Considering the missing/lost samples as Missing completely at random data (MCAR) which has no relationship with observed/unobserved variables, the results were statistically analyzed using SPSS software version 18.0. The Pearson Chi-Square test was applied when appropriate, with a P-value of <0.05 considered statistically significant.

Results

Data from 150 suspected cases of mycotic keratitis were analyzed. The patients' ages ranged from 2 to 89 years, with the majority (n=65) being over 60 years (Table 1).

Male patients (n=90) outnumbered female patients (n=60).

KOH test

Of the 150 samples, KOH testing was performed on 87 (74.6%) patients, of which 24 (33.3%) tested positive, while 63 (41.3%) tested negative. KOH testing was not conducted in 63 (41.3%) cases due to factors such as lost samples, or sample drying before reaching the lab. Of the 87 patients tested, 53 (60.9%) were male, and 34 (39.1%) were female. KOH positivity was observed in 16 males (30.2%) and 8 females (23.5%), but the difference in positivity rates between genders was not statistically significant (P=0.498) (Table 2).

Fungal culture

The fungal cultures were positive in 84 (56%) patients and negative in 66 (44%) patients. Among those with positive fungal cultures, 42 (46.7%) were male, and 24 (40.0%) were female, a difference that was not statistically significant. Of the 63 patients where KOH was not performed, fungal cultures were positive in 27 (42.9%) (Table 3).

A higher positivity rate was noted in 43.6% (17/39) of cases where both KOH and fungal cultures were positive, compared to 14.6% (7/48) where KOH was positive, but the fungal culture was negative. This association between KOH and fungal culture positivity was statistically significant (P=0.003, odds ratio = 4.53, 95% confidence interval: 1.630-12.57). The sensitivity of the KOH test was 70.8%, and its specificity was 65.07%.

Fungal species distribution

Among the 84 positive fungal cultures, the species identified included Aspergillus fumigatus (13 cases, 19.7%), Fusarium solani (25 cases, 37.9%), Aspergillus flavus (8 cases, 12.1%), Aspergillus niger (7 cases, 10.6%), Acremonium (3 cases, 4.5%), Seidosporium (2 cases, 3.0%), and Geotrichum, Non-albicans Candida, Cephalosporium, Cladosporium, and Curvularia (1 case each, 1.5%). Contaminants were found in nine cases (13.6%), and one patient had both Aspergillus flavus and Aspergillus fumigatus (Table 4). The most commonly isolated species was Fusarium, which is concurrent with the study findings of Srinivasan et al., in southern India.

Aspergillus fumigatus was most commonly isolated in the 18-35 year age group (16.7%), while Fusarium was most frequent in patients over 60 years (52.3%). The overall positivity rate ranged from 33.3% to 52.3% across age groups, though the difference in positivity rates was not statistically significant (P=0.301). Male patients had a higher positivity rate compared to female patients in the middle-aged group, though the difference was not statistically significant.

Discussion

Fungal corneal ulcers, or mycotic keratitis, can affect individuals across a wide age range, as seen in our study, where patient ages spanned from 2 to 89 years. However, we found that the highest incidence occurred in patients over 60 years, likely due to age-related decline in ocular surface immunity and increased exposure to risk factors such as ocular surface disease and prolonged medication use. Similar age-related patterns have been observed in other studies, highlighting the vulnerability of older adults to fungal infections.

We also observed a higher prevalence of mycotic keratitis in male patients (n=90) when compared to female patients (n=60), consistent with the findings of Bandyopadhyay S et al.¹² This gender disparity may be explained by greater outdoor exposure among men, particularly in agricultural settings, where fungal pathogens like Fusarium and Aspergillus are common in organic matter. Trauma to the cornea from such materials is a significant risk factor for fungal infections. However, some studies, such as the one by Kotigadde S et al., reported a higher incidence in women, suggesting that local environmental factors and cultural roles may influence these trends.¹³ Although men were more affected in our study, KOH positivity rates between men and women were not significantly different.

KOH wet-mount preparation, a commonly used diagnostic tool, demonstrated a sensitivity of 70.8% and a specificity of 65.07% in our study. These figures are lower than those reported in other studies, such as the Hyderabad study and the study conducted by Sharma et al., where sensitivity and specificity reached 81.2% and 83.8%, respectively.⁹ The variability in diagnostic accuracy may be due to differences in sample collection techniques, fungal species involved, or lab conditions. While KOH provides a rapid diagnosis, its moderate sensitivity means that a negative result does not exclude the presence of fungal infection.¹⁴ Thus, clinicians should always correlate KOH findings with clinical suspicion.

One of the notable findings in our study was the discrepancy between KOH and fungal culture results. While 14.6% of KOH-positive samples were culture-negative, 43.6% of samples that were positive in both KOH and fungal culture had a higher positivity rate, suggesting a strong correlation between the two methods in cases of significant fungal load. These findings align with the concept that culture-negative results, despite positive KOH findings, could result from insufficient sample size, prior antifungal treatment, or degradation of the sample during transport to the lab. Chander et al., reported a higher culture negativity rate (46.86%), indicating that sample quality and prior treatment significantly impact culture results.¹⁵

Our study revealed that Fusarium species were the predominant pathogens identified in cases of mycotic keratitis, accounting for 37.9% of all fungal infections. Notably, this pathogen was particularly prevalent among patients over 60 years of age, where the prevalence soared to 52.3%. This finding is consistent with previous research conducted by Bharathi et al., in Tirunelveli, which also identified Fusarium as the most common causative agent of fungal keratitis in tropical regions.¹⁶ The correlation between age and the incidence of Fusa-rium infections may be attributed to several factors, including age-related decline in ocular immunity, increased likelihood of trauma, and greater exposure to environmental pathogens, particularly in older adults who may have more outdoor activities.

Tropical climates, characterized by high humidity and frequent rainfall, create ideal conditions for the growth and proliferation of fungal organisms, particularly Fusarium. These environmental factors facilitate fungal spore dispersal and colonization, leading to an increased incidence of infections. Understanding the epidemiology of fungal keratitis in tropical regions is critical, as it informs clinicians about potential pathogens and risk factors associated with the disease.

In contrast, different geographical areas have reported varying pathogen profiles. For instance, a study by Katara et al., found that Aspergillus species were more prevalent in western Gujarat, suggesting regional differences in environmental conditions and agricultural practices that influence the types of fungi present.¹⁷ Similarly, Kotigadde et al., identified Aspergillus fumigatus as the leading pathogen in coastal Karnataka, emphasizing that local factors, such as the types of crops grown and the agricultural practices employed, play a significant role in determining the spectrum of fungal pathogens in a given area. These differences underscore the importance of localized epidemiological studies to effectively guide treatment protocols.

The association observed between KOH mount results and fungal culture positivity in our study is clinically significant. When both tests yield positive results, the likelihood of a fungal infection is considerably heightened, which can aid clinicians in making prompt and informed treatment decisions. This finding reinforces the value of KOH microscopy as a rapid screening tool, particularly in resource-limited settings where access to advanced culture facilities may be limited. The ability to quickly identify potential fungal infections allows for timely intervention, which is crucial in preventing complications associated with mycotic keratitis.

However, it is essential to note that in cases where clinical suspicion for fungal keratitis remains high even in the face of negative KOH and culture results clinicians should not hesitate to initiate empirical antifungal treatment. Given the potentially sight-threatening nature of fungal infections, prompt treatment is critical, as delays can lead to irreversible ocular damage and loss of vision. Empirical therapy based on clinical judgment, combined with continued monitoring and follow-up, can significantly improve patient outcomes and reduce the risk of complications associated with mycotic keratitis.

Clinical implications

Our findings underscore the importance of integrating clinical judgment with laboratory results in the diagnosis of fungal keratitis.¹⁸ While KOH is a useful and quick diagnostic tool, its moderate sensitivity means that clinicians cannot rely on it exclusively. Fungal cultures, though more specific, require time for results and can be affected by factors like sample size and prior antifungal treatment. Thus, in cases where both KOH and fungal culture are negative, clinicians should still consider empirical antifungal therapy based on clinical suspicion.

Challenges and future directions

The diagnosis of fungal keratitis is often challenging due to the limitations of current diagnostic methods. KOH microscopy, although rapid, lacks sufficient sensitivity, while fungal cultures are slow and may yield false negatives. There is a growing need for advanced diagnostic methods such as polymerase chain reaction (PCR) and next-generation sequencing (NGS), which could offer faster and more accurate detection of fungal pathogens.¹⁹ However, these techniques are not yet widely available, especially in resource-limited settings. The development of point-of-care diagnostic tools that are both sensitive and accessible could significantly improve outcomes for patients with mycotic keratitis.

In terms of treatment, prolonged antifungal therapy is often required, and drug delivery to the cornea remains a challenge. Future research should focus on improving drug penetration, exploring alternative delivery methods and optimizing treatment protocols to reduce the duration of infection and prevent complications.^20,21

Public health implications

Fungal keratitis is a significant public health concern, especially in regions with high agricultural activity, where eye trauma from organic matter is more common. The limitations in diagnostic tools such as KOH and culture mean that delayed or inaccurate diagnoses are more likely, contributing to higher rates of vision impairment and blindness. Limited access to advanced diagnostic tools, such as PCR or next-generation sequencing, in low-resource settings exacerbates the issue, leading to poorer patient outcomes. Public health initiatives should focus on improving awareness of fungal keratitis among at-risk populations, enhancing the availability of diagnostic resources, and training healthcare workers to recognize early symptoms. Additionally, preventive measures, such as the use of protective eyewear in high-risk occupations, can help reduce the incidence of fungal keratitis.

Conclusion

Fungal keratitis poses challenges in diagnosis and treatment. KOH microscopy has moderate sensitivity (70.8%) and specificity (65.07%), and a negative result does not rule out infection. Patients with high clinical suspicion often respond to antifungal treatment, even if KOH and culture are negative, emphasizing clinical judgment and early intervention.

Fusarium species are the main cause of fungal corneal ulcers, particularly in tropical regions and older adults linked to agricultural exposure. Early diagnosis and treatment are crucial, as Fusarium infections are more aggressive and harder to treat than Aspergillus.

KOH microscopy is a quick tool in resource-limited settings but has limited sensitivity. Fungal cultures, though more specific, are slower and can give false negatives due to small samples or prior antifungal use. Optimizing sample collection can improve accuracy. For high-risk patients, empirical antifungal treatment should begin without waiting for culture results. Public health efforts like promoting eye protection and early eye care are essential to reduce fungal keratitis cases. Future research should focus on developing faster, reliable diagnostic methods, such as PCR, and more effective treatments, especially for resistant Fusarium species.

The primary limitation of our study is its retrospective nature and reliance on existing medical records. Prospective studies comparing and correlating KOH microscopy and fungal cultures are recommended to better understand their accuracy and to facilitate the integration of KOH microscopy into primary care settings for the early diagnosis of mycotic keratitis.

Conflicts of interest

None