Clinical Characteristics and Treatment Outcomes of Pediatric Fungal Keratitis

Introduction

Fungal keratitis is relatively rare in children.¹ There was only one case (5.9%) with fungal infection in a five-year study of childhood infectious keratitis in Canada.² In a 23-year study of childhood infectious keratitis in the United States, 12 cases of fungal infection occurred.³ Recently, neutrophil-to- lymphocyte ratio (NLR) has been used as a marker for systemic inflammatory status.⁴ The NLR is the total count of neutrophils divided by those of lymphocytes. It has been recognized as a valuable marker of systemic inflammation, which is significantly increased in many eye disorders, such as keratoconus, glaucoma, and pterygium.^5,6 NLR in childhood differs from that in adulthood. It shows higher values directly after birth, then gradually decreases, and starts to slowly increase around the age of 3, gradually approaching adult levels.⁷ In this study, we retrospectively review the medical records of pediatric patients with fungal keratitis who were treated in our hospital between 1996 and 2021. We also investigated the potential association between NLR and outcomes of fungal keratitis.

Fungal keratitis can lead to severe visual impairment. It accounts for about 20% to 60% of all microbial keratitis cases,⁸ but seems to be less common in children.^2,3 Our understanding of the unique characteristics of fungal keratitis in children is limited due to its low incidence. The lack of knowledge can result in delayed, incorrect, or inadequate treatment, with long-lasting and devastating consequences. Childhood blindness is a life-long impairment that imposes significant emotional and financial burdens on families and society. Therefore, it is crucial to prioritize the study of PFK to ensure timely and appropriate interventions.

Methods

Study Design and Population

The medical records of children (<18 years old) who were diagnosed with fungal keratitis from 1996 to 2021 were reviewed. The patient’s demographic characteristics, risk factors, disease characteristics, treatment, and outcomes were recorded. An informed consent was obtained from the parents and/or legal guardians. This study was approved by the Ethics Committee of Qingdao Eye Hospital of Shandong First Medical University ([2020]19) and adhered to the tenets of the Declaration of Helsinki.

Diagnostic Criteria for Fungal Keratitis

Corneal scrapings were subjected to 10% KOH mount. Corneal scrapings were also plated on Sabouraud’s Dextrose Agar for culture and strain identification. In addition, patient’s corneas were examined using confocal microscopy. The diagnosis of fungal keratitis was confirmed by the presence of fungal elements in the KOH preparations or confocal microscopic images, or positive culture results.^1,9

Ocular Examinations

A detailed clinical history was obtained followed by measurement of visual acuity and intraocular pressure. Slit-lamp examination was performed to note the characteristics of the corneal infiltrate. Clinical photographs were obtained on a slit lamp mounted camera (BX900, Haag-Streit) under cobalt blue light after staining with fluorescein dye. Confocal microscopy was performed by an experienced technician (HRT3, Heidelberg).

Treatment Strategy

Once the diagnosis of fungal keratitis was confirmed, antifungal agents, including topical voriconazole 1%, natamycin 5% or amphotericin B 0.25% were administered hourly. Subsequent adjustments were made based on the drug susceptibility results of each patient. If the drug therapy was not effective after one week, either limited superficial keratectomy (for anterior infiltrates) or keratoplasty (for deep infiltrates) was performed. All patients followed this treatment strategy.

Anti-Fungal Drug Sensitivity Tests and Determination of MIC

Anti-fungal drug sensitivity tests were performed in accordance with National Committee for Clinical Laboratory Standards (NCCLS) standard M38-A guidelines as described in previous studies.¹⁰ The control strain was Candida parapsilosis (ATCC22019). In brief, seven kinds of anti-fungal drugs were packaged on the 96 well plates in the antifungal drug sensitivity test kits (Jinzhang Medical and New Technological Institute, Tianjin, China), which include amphotericin B, ketoconazole, miconazole, itraconazole, fluconazole, 5-fluorocytosine, and terbinafine. The strains to be examined were inoculated onto PDA media, cultured for two to seven days, and the inoculums were diluted to (0.5–5)×10⁴ CFU/mL. Values of minimum inhibitory concentrations (MIC) were read after 24 to 48 hours of incubation at 27 °C.

Statistical Analysis

All data were processed using SPSS 22.0 software. A Chi-squared test was used to compare the incidence at different times and the positive rate of different tests. A comparison of visual acuity between the groups was performed with a Mann–Whitney test. Spearman correlation analysis was used to assess the correlation between the two sets of data. Factors associated with the surgical treatment were assessed using logistic regression analysis. The difference was statistically significant at p < 0.05.

Results

Epidemiological Characteristics

A total of 47 children (48 eyes) received treatment for fungal keratitis, representing 16.3% (48/294) of all children with infectious keratitis. Among them, 24.1% (35/145) were reported between 1996 and 2008, and 8.7% (13/149) between 2009 and 2021 (p = 0.001, X²-test). During the same period, 3223 adult patients (3225 eyes) were diagnosed with fungal keratitis, accounting for 59.3% (3225/5435) of all adult patients with infectious keratitis. The incidence of fungal keratitis was significantly lower in children compared to adults (p < 0.001, X²-test).

Out of the 47 enrolled patients, 31 were male (66.0%) and 16 were female (34.0%). The mean age at onset was 11.8±4.1 years (range: 1–17 years). Among them, children aged ≤5 years constituted 6.4% (3/47), those aged 6–12 years accounted for 38.3% (18/47), and those aged 13–17 years accounted for 55.3% (26/47). A total of 93.6% (44/47) of patients were from rural areas. No children with a history of diabetes were identified.

70.2% (33/47) of patients were diagnosed between April and September (spring and summer). Additionally, 51.1% (24/47) of the patients visited the hospital within one week of symptoms onset, and only one patient (2.2%) with contact lens-induced fungal keratitis had symptoms lasting for more than one month.

The leading cause of fungal keratitis in children was trauma due to dust or foreign body in the eye (29.2%, 14/48), or trauma with vegetative matter (25.0%, 12/48). There was one case where a 17-year-old boy developed fungal infection after undergoing LASIK surgery for myopia (Table 1). Unexplained fungal keratitis accounted for 25.0% (12/48) of the cases.

Table 1 Risk Factors for Fungal Keratitis in Children at the Shandong Eye Institute, 1996–2021

A total of 91.7% eyes (44/48) received initial treatment at a local hospital. Of these, 22/44 (50.0%) received a correct diagnosis and received topical antifungals treatment. 27.3% (12/44) of the eyes were treated as bacterial keratitis and 2.3% (1/44) were treated for viral keratitis. Additionally, 20.5% (9/44) of eyes remained undiagnosed or had unclear history about prior topical medication history. 11.4% (5/44) eyes had a definitive history of topical corticosteroid use.

Clinical Features

During the initial visit to the hospital, 31.3% (15/48) of the eyes had a hypopyon, 6.3% (3/48) had corneal perforation, and 6.3% (3/48) had endophthalmitis. The average diameter of the corneal ulcer at the initial visit to our hospital was 4.7±2.7 mm (range: 1.5–12.0 mm). 25.0% (12/48) of the eyes had small ulcers (diameter < 3 mm), while 41.7% (20/48) had large ulcers (diameter > 5 mm). The average NLR value was 2.3±1.1 (range: 0.7 to 7.0). NLR was found to be positively correlated with the age of onset (r = 0.437, p =0.002) and the diameter of the corneal ulcer (r = 0.298, p = 0.04).

Microbiology Results

Fungal smear results were positive in 81.3% (39/48) of cases, whereas fungal culture was positive in 79.2% (38/48) of cases. Confocal microscopy showed a positive result in 85.7% (18/21) of the examined cases. There was no statistical difference among these detection methods (p = 0.815, X²-test). In 62.5% (30/48) infected eyes, the fungal organism was identified. Among these identified fungi, Fusarium infection was found in 41.7% (20/48) of cases while Aspergillus was responsible for 20.8% (10/48) of cases. In 37.5% (18/48) of cases, the causative agent remained uncertain. Four patients had bacterial co-infections with Staphylococcus epidermidis, Bacillus subtilis, Enterococcus, and Hafnia (Table 2). There was no significant difference in the clinical features of corneal infections between Fusarium and Aspergillus (Table 3).

Table 2 Fungal Spectrum of Pediatric Fungal Keratitis at the Shandong Eye Institute, 1996–2021

Table 3 Comparison of Clinical Features Between Fusarium and Aspergillus Keratitis at the Shandong Eye Institute, 1996–2021

Drug Susceptibility

The mean MIC for Fusarium was 1.231 for voriconazole and 1.932 for amphotericin B. In contrast, the mean MIC for itraconazole against Aspergillus was the lowest (0.250), followed by voriconazole (0.380) (Table 4).

Table 4 Pediatric Fungal Keratitis and Commonly Used Antifungal Drugs at the Shandong Eye Institute, 1996–2021 (Minimum Inhibitory Concentrations, g/Ml)

Based on the results of the drug susceptibility tests, the sensitivity of several antifungal drugs against Fusarium and Aspergillus was evaluated. Voriconazole exhibited the highest sensitivity against Fusarium, with a rate of up to 65%, followed by Natamycin and amphotericin B with a sensitivity of 50%. Fluconazole, ketoconazole, and terbinafine showed higher rates of resistance against Fusarium, with resistances of 85%, 75%, and 75%, respectively.

For Aspergillus, voriconazole demonstrated the highest sensitivity, with a rate of up to 80.0%. Ketoconazole and itraconazole exhibited sensitivities of 70.0%, respectively. In contrast, natamycin exhibited complete resistance (90%) to Aspergillus, while amphotericin B and fluconazole showed resistance of 70%. The summarized drug susceptibility results can be found in Table 5.

Table 5 Results of Susceptibility in Children With Fungal Keratitis at the Shandong Eye Institute, 1996–2021

Treatment and Outcomes

Overall, only 16.7% (8/48) were successfully treated with medical management alone. Seven eyes (14.6%) underwent superficial keratectomy due to worsening of the disease (increase in corneal ulcer area or deepening of infiltration) during medical treatment and were subsequently cured. 66.7% (32/48) of the eyes required keratoplasty, with 21 eyes receiving PKP and 11 eyes receiving LKP. Enucleation was required in one eye (2.1%). Logistic regression analysis revealed a significant association between NLR and the need for surgical treatment (odds ratio = 117.926, p = 0.019) (Table 6). Additionally, the diameter of the corneal ulcer was found to be significantly associated with the need for keratoplasty (odds ratio = 3.556, p = 0.014) (Table 7).

Table 6 Results of the Regression Analysis of the Various Risk Factors for Surgical Treatment for Pediatric Fungal Keratitis

Table 7 Results of the Regression Analysis of the Various Risk Factors for Keratoplasty for Pediatric Fungal Keratitis

In the Fusarium group, 45% (9/20) of the cases had LKP, 40% (8/20) had PKP, 10% (2/20) had excision of lesion, and 5% (1/20) were treated with topical medications alone. In the Aspergillus group, 20% (2/10) had LKP, 70% (7/10) had PKP, and 10% (1/10) had superficial keratectomy.

The recurrence rate of keratitis after keratoplasty was 3.1% (1/32) during the follow-up period of 4.0 ± 3.9 (0.6–13.9) years. In one patient with Aspergillus infection, the infiltration recurred three days after LKP (9.1%, 1/11), and a PKP was required. During a follow-up period of 3.8 ± 4.1 (1.2–13.9) years, there were three instances of graft rejection (14.3%) after PKP, between 1 and 23 months after the surgery. However, all cases responded well to topical treatment with corticosteroids.

The median best corrected visual acuity (BCVA) after PKP was 20/50 (20/200 – 20/25), with 42% of the eyes achieving a BCVA of ≥20/40. After LKP, the median BCVA was 20/50 (20/100 – 20/40), with 36.4% of the eyes achieving a BCVA ≥ 20/40. The median BCVA after medical treatment was 20/50 (20/400 – 20/20), 26.7% of the eyes achieved ≥20/40. There was no significant difference between BCVA between PKP and LKP eyes (p = 0.913). Additionally, no statistically significant difference was found between BCVA after medical treatment and corneal transplantation (p = 0.951). However, the BCVA after treatment was lower in children ≤8 years [median 20/60 (20/400 – 20/40)] compared to children >8 years [median; 20/50 (20/200 – 20/20); p = 0.032].

Discussion

This study represents the largest investigation to date focusing on pediatric fungal keratitis. Fungal keratitis is the predominant cause of infectious corneal ulcers in Northern China, mainly related to injuries with vegetative matter.¹ In this study, most of the participants were from rural areas, where the risk of injury is higher compared to urban regions. It is noteworthy that over the last 12 years, the incidence of fungal keratitis in children has dropped to 8.7%, which is comparable to that in the United States.³

Medical management of fungal keratitis involves the use of antifungal drugs including polyenes (amphotericin B, natamycin, and nystatin), azoles (ketoconazole, miconazole, econazole, fluconazole, itraconazole, voriconazole, and posaconazole), allylamine (terbinafine) and echinocandins (caspofungin). Suboptimal corneal penetration of these drugs is a major limitation. The newer triazoles such as voriconazole have exhibited excellent corneal penetration even through an intact corneal epithelium. Voriconazole is potent against a wide spectrum of fungi, such as C. albicans, C. parapsilosis, C. tropicalis, A. fumigatus, A. flavus, and F. solani. In our study, voriconazole was the most effective antifungal drug with the most favorable MIC values (Table 5). Early administration of the correct medication is particularly crucial for fungal keratitis. Incorrect topical steroid use can rapidly worsen the condition.

Corneal transplantation in children is associated with a higher incidence of complications compared to adult surgery.¹¹ However, in this study, where the average age of the patients was 12 years old, the incidence of postoperative complications was not significantly different from that observed in adults.^12–14 The growth patterns of Fusarium and Aspergillus are different in the cornea, with about 90% of Fusarium hyphae aligning parallel to the corneal stromal lamellae, while Aspergillus exhibits more vertical growth.¹⁵ To mitigate rejection and the long-term complications such as corneal endothelial failure, LKP is recommended for corneal ulcers caused by Fusarium injection.⁹ However, the oblique growth pattern of Aspergillus contributes to its greater erosive potential and the subsequent need for PKP.^10,13 Monocular vision abnormalities in childhood can lead to amblyopia, which explains the lower BCVA observed in children aged ≤8 years compared to older children.

The etiology, onset time and seasonal characteristics of pediatric fungal corneal ulcers are similar to those in adults. Fungal corneal ulcers occurring in children do not exhibit greater severity than those in adults, despite their immature immune defenses. In addition, a low NLR in children may serve as a protective factor against fungal corneal ulcers. In certain cases, an excessive activation of immune cells leads to the release of large quantities of cytokines, resulting in an uncontrollable cascade or a cytokine storm. This severe immune response can cause significant damage to human tissues and organs.^16,17 In fungal corneal ulcers, neutrophils can play a dual role by both combating fungal pathogens and causing corneal damage through the release of reactive oxygen species (ROS) and proteases.^18–20 In this study, the association of a higher NLR in the peripheral blood with a larger corneal ulcer diameter, supports the dual role theory. Currently, there are a limited number of studies on lymphocytes and infectious eye diseases, highlighting the need for further investigations in this area.

In summary, our study identified that the incidence of fungal keratitis is lower in children compared to adults. A low NLR in childhood might serve as a protective factor against severe ulcers. Corneal transplantation remains a viable option to provide the long-term improvement in BCVA.