Microbes and Infectious Diseases Rapid diagnosis of fungal keratitis in patients with corneal ulcer using calcofluor white stain

complications, diagnosing and treating fungal keratitis is crucial. This study aims to measure the efficacy of a calcofluor white (CFW) stain for the quick diagnosis of fungal keratitis and to contrast the positive rates, sensitivity, and specificity with a 10% potassium hydroxide (KOH)-based smear and culture technique. Methods: From individuals with clinically suspected corneal ulcers, 30 corneal scrapings had been collected. Data on demographics had been analyzed. Results: Of the 30 patients, 40% were women and 60% were men. There was a 1.5:1 man-to-woman ratio. The age of patients ranged from 29 to 71 years (mean 46.67 ± 10.90). The age presentation of those between the ages of 41 and 50 years was the most frequent (36.7%). The majority of cases were farmers (43.3%). Trauma was the most common predisposing factor (46.6%). Twenty-four (80%) cases were culture positive. Eleven (36.7%) were fungal, 13 (43.3%) were bacterial and 6 (20%) showed no growth. Fusarium was the most common fungal isolate (36.4%), followed by Aspergillus (27.3%). While Staphylococcus aureus was the most common bacterial isolate (46.2%), followed by Pseudomonas (38.4%). The sensitivity of KOH wet mount and CFW stain was 72.7% and 90.9%, respectively. The specificity of both KOH wet mount and CFW stain was 100%. Conclusion: The early diagnosis of fungal keratitis can be made rapidly by direct microscopic examination of fungal elements using CFW stain. When diagnosing fungal keratitis, CFW has higher sensitivity to KOH.


Introduction
Fungal keratitis is one of the primary causes of serious ocular morbidity as well as blindness [1]. It is prevalent worldwide, but it is more common in the tropics and subtropics [2]. The incidence of fungal keratitis in Egypt is 20-55% (mean 40%) [3]. Increases in the lowest temperature and the high levels of atmospheric humidity in the area are causing this incidence to increase in proportion to these climatic changes [4]. Early detection and antifungal treatment are essential to prevent further complications, which include the formation of hypopyon, endophthalmitis, or vision loss [5]. Fusarium spp. and Aspergillus spp. are the most commonly recognized causative agents of fungal keratitis [6]. Several factors; such as the widespread use of antibiotics and steroids, contact lens usage, ocular trauma with agricultural materials, systemic immunosuppression, diabetes mellitus, and postoperative infection, are linked to an increase in the frequency of fungal keratitis [7].
The diagnosis of fungal keratitis remains problematic to make. Numerous clinical features are not exclusive to fungal ulcers, so antifungal treatment has to be withheld till a diagnosis has been established through laboratory tests. The most critical step in the early therapy of suspected fungal keratitis is obtaining corneal material for guided smears and media inoculation. Smears are employed to gain quick information on the disease. Even though culture aids in accurate identification and diagnosis, direct microscopic detection of fungal elements in scrapings of the cornea allows for a quick presumptive diagnosis [4].
This study aims to measure the efficacy of a calcofluor white (CFW) stain for the fast diagnosis of fungal keratitis and to contrast the positive rates, sensitivity, and specificity with a 10% potassium hydroxide (KOH)-based smear and culture technique.

Study settings and population
A total of 30 patients with clinically suspected corneal ulcers attending an ophthalmology clinic in Ain Shams University Hospitals over the period from May 2021 to March 2022 had been involved in the research. Everyone who participated provided their informed consent. Ages, sex, occupations, history of trauma, the mode of trauma, history of prior treatment, usage of steroid eye drops or contact lenses, any related systemic illnesses like diabetes mellitus, and ocular abnormalities or disorders had all been recorded in the patient's demographic profile.

Study design
A cross-sectional descriptive study.

Ethical approval
The Faculty of Medicine at Ain Shams University's Ethics and Research Committee gave the study acceptance with approval No. MS 246/2021. Informed consent was obtained from each patient before enrollment. Identification codes had been utilized for data administration to preserve information confidentiality and privacy; these findings had been employed only for scientific purposes.

Inclusion criteria
The study included patients attending the ophthalmology clinic who had signs and symptoms suggestive of corneal ulcer. It also included patients who had a resistant corneal ulcer and were unresponsive to treatment.

Exclusion criteria
History of autoimmune disease and impending perforated corneal ulcer.

Specimen collection
Corneal scrapings were collected with a No.15 Bard Parker blade by an experienced Ophthalmologist under complete aseptic conditions following 4% lignocaine instillation. Scraping was done from the leading edge and the base of each ulcer and was collected in a sterile container and sent to the central Microbiology lab in Ain Shams University Hospitals. The samples were subjected to 10% KOH wet mount examination, CFW examination, and cultivation on blood agar and Sabouraud dextrose agar in a C-shaped manner.

KOH wet mount examination
The scraping material was put onto a spotless glass slide. One or two drops of sterile 10% KOH (Alpha Chemika Laboratories, Mumbai, India) were applied and covered using a clean coverslip to avoid the formation of air bubbles. The slide was left for 5-10 minutes and was examined under the microscope for the presence of hyphal elements or budding yeasts (Figure 1).

Calcofluor white stain examination
The specimen was added to a clean glass slide. Then a drop of CFW stain was added. This stain is composed of 0.5 g/l Evans blue dye and 1 g/l Calcofluor white stain M2R from Sigma-Aldrich in St. Louis, Mo., USA. A cover slip was placed on the specimen and then was left for 1 minute. The slide was covered with a paper towel and was gently pressed to remove any excess fluid and was examined under UV light at x100 to x400 magnifications. A filamentous structure was thought to be a fungal filament if it showed branching and septation, seen in the center of the smear, was identified at low power (× 100) and was confirmed at higher power (× 400 or × 1000).
Fungal elements appear as bright apple-green fluorescence with typical morphology (Figure 2).

Culture
The scraping material was inoculated directly onto the blood agar plates (Oxoid, UK) and Sabouraud dextrose agar (SDA) plates (Oxoid, UK) in a Cshaped manner. The SDA plates were incubated for up to 2 weeks at 28°C and 37°C [8] and were examined daily during the first week and twice a week during the second week [9]. Failure of growth after two weeks was diagnosed as negative for fungal growth and the plates were discarded [10]. The blood agar plates were incubated at 37°C for 48 hours and were checked daily for fungal and bacterial growth before being discarded after 48 hours [10].
Following growth on plates, isolated fungi were identified based on the rate of growth, macroscopic morphological characteristics (colony color on the obverse and reverse side, colony textures, and submerged hyphae), and microscopic characteristics (such as conidiogenous cells and conidiophores, the shape and organization of conidia, macromicroconidia and blastoconidia and the absence or presence of chlamydospores) (Figure 3).
Positive microbial culture was diagnosed if semiconfluent growth was seen at the inoculation site on a solid medium, it was compatible with clinical findings, and smear findings were compatible with that of the culture.

Statistical analysis
A Statistical Package for Social Science had been employed to perform the statistical analysis (SPSS version 23.0 for Windows; SPSS Inc., Chicago, IL, USA). In terms of sensitivity and specificity, the findings of direct microscopy analysis of smear specimens had been compared to those of fungal culture employing the Chi-square (χ2) test. p-values lower than 0.05 had been regarded as significant (S). The staining methods' sensitivity (true positive rate), specificity (true negative rate), PPV, NPV, and accuracy had been computed employing standard statistical formulas.

As seen in figures
The culture technique had been regarded as the gold standard to diagnose fungal keratitis. We discovered that the positive rates of direct microscopic inspection of 30 corneal scraping samples for fungal components by 10%KOH and CFW stain were 26.7% and 33.3%, respectively. These results were compared to the two different quick detection techniques utilized for diagnosing fungal keratitis. When the direct microscopic inspection was compared to culture on SDA for sensitivity and specificity, CFW had greater sensitivity (90.9%) and specificity (100%), followed by 10% KOH sensitivity (72.7%) and specificity (100%). In addition, when direct microscopic inspection and culture on SDA were compared for accuracy, CFW exhibited better accuracy (96.7%) followed by 10% KOH (90.0%) as reliable tools in the diagnosis of fungal keratitis as illustrated in table (7)

Discussion
Microbial keratitis is a frequent and possibly vision-threatening ocular infection that is caused by fungi, viruses, bacteria, or parasites. The eyes are normally protected by many natural defense mechanisms from invasion by these microorganisms. However, predisposing variables, including contact lens usage, corneal trauma, and ocular abnormalities might change the eye's resistance and allow fungi to invade the cornea [11]. A Fungal corneal ulcer is an important ophthalmologic problem, especially in developing countries. It is responsible for the significant prevalence of morbidity and blindness worldwide. Fungi, however, are one of the most elusive as well as difficult organisms to treat and diagnose among the organisms that cause keratitis [12].
Men's predominance had been seen in this research (men: 60% and women: 40%). This was in accordance with results obtained by Khadka et al. The most affected age group in our study was 41-50 years (36.7%). This was in concordance with the study done by Satpathy et al. [19] (17.9%) and Nathiya [20] (32%). This may be explained as people of the age group of 40-50 years are the primary force in manual work, particularly in agricultural work, and are more engaged in outdoor activities [4]. However, Khor et al. [15] and Bou et al. [21] reported discrepant results as they found that the peak age group presentation among infectious keratitis cases was from 21 to 30 years.
The most frequent predisposing factor related to infectious keratitis in the current research was corneal trauma, with 14 (46.6%) cases, followed by steroid use, with 6 (20%), and DM, with 3 (10%). This came in agreement with a study conducted by Harbiyeli et al. [27], Alekhya et al. [28], and Mahmoud et al. [29]. On the other hand, other publications showed different results as a study conducted by Gorski et al. [30] who found that among 155 patients with microbial keratitis, contact lens wear was (39%) followed by corneal trauma (8%). Another study conducted in South Texas by Puig et al. [31] found that contact lens wearing (32.4%) is the most common risk factor related to infectious keratitis. These results could be explained by the fact that usage of contact lenses is more common in females and ocular trauma is more in males most probably because of their occupation [24].
Our study revealed that the most common mode of trauma was a vegetative matter, which was seen in (28.6%) of cases. This came in line with the findings of Chidambaram et al. [25] that reported that the source of trauma was vegetative matter (46%), among keratitis cases.
Total culture positivity was 80%. This was similar to a study by Manikandan et al.
[6] (82.2%) and different from other studies done by Acharya et al. [32], Termote et al. [33], and Tuft et al. [34] who reported a lower rate of isolation, 62.9%, 37.5% and 51.6% of culture-positive cases, respectively. Positive corneal ulcer cultures range between 43% and 77%. The differences between studies might be due to variations in the techniques employed to determine positivity; sample collection techniques; prior empirical topical antibiotics; and the inability to employ selective media for isolation of the organisms [29].  [24] found that Candida spp. (62.3%) was the commonest isolate among fungal keratitis patients.
Isolation is a conclusive technique for diagnosing fungal keratitis. Much of the research deemed SDA culture to be the gold standard. Direct microscopy using KOH wet mount and CFW stain, when compared with clinical presentation, may, according to reports, be able to detect more instances than culture alone. Although, PCR is able to detect fungal DNA in a high proportion of culture negative cases, it is difficult to be used as a routine diagnostic test in our hospitals due to the economic reasons and time-consuming. So, as a quick, simple, reliable, easy visibility at low power, with high sensitivity and specificity and inexpensive if fluorescent microscope is available in the laboratory, we heartily recommend the use of Calcofluor white stain.

Conclusion
One rapid and efficient way to make an early diagnosis of fungal keratitis is by performing a direct microscopic examination of fungal elements in corneal scrapings using a Calcofluor white stain. When diagnosing fungal keratitis, 10% potassium hydroxide mount is less sensitive than calcofluor white stain with fluorescence microscopy.

Conflict of interest:
There aren't any conflicting interests.