Fungal Keratitis in Northern Thailand: Spectrum of Agents, Risk Factors and Putative Virulence Factors

Abstract

Fungal keratitis (FK) is a serious ocular infection that can result in various degrees of vision loss, including blindness. The aim of the study was to identify and retrospectively review all FK cases diagnosed between August 2012 and December 2020 at a tertiary care hospital in northern Thailand with a specific focus on epidemiologic features, including season, patient sex and age, the spectrum of pathogens, and presence of certain putative virulence factors. Of 1237 patients with corneal ulcers, 294 (23.8%) were confirmed by direct microscopic examination and/or fungal culture. For the positive cases, direct examinations of Calcofluor white (CW) stains and KOH mounts were found in 97.3% (286/294) and 76.5% (225/294), respectively (p < 0.05). Of the cases diagnosed by microscopy and culture, fungi were isolated in 152 (51.7%), with Fusarium spp. being the most frequently identified (n = 69, 45.5%) followed by dematiaceous fungi (n = 45, 29.6%) and Aspergillus spp. (n = 18, 11.8%). The incidence of FK was higher in the rainy season of July to October. The mean age was 54.4 ± 14.4 (SD) years, with a range of 9–88 years. Males (75.8%) were affected significantly more than females (24.2%) (p < 0.05). Of 294 patients, 132 (44.9%) were middle-aged adults (41–60 years) and 107 (36.4%) were older than 60 years. Trauma to the eye by soil or vegetative matter were the most common preceding factors (188/294; 64.0%). We assessed two virulence factors. First, 142 of the 152 culture-positive FK cases were due to molds, indicating that hyphal morphogenesis is extremely important in disease. We also demonstrated that fungal melanization occurs in the molds during the course of FK by applying a melanin-specific monoclonal antibody (MAb) that labeled fungal elements in corneal samples of patients, and melanin particles derived from the hyphae were also recovered after treatment of the samples with proteolytic enzymes, denaturant and hot concentrated acid. In summary, we demonstrate that northern Thailand has a high rate of FK that is influenced by season and males engaged in outside activities are at highest risk for disease. Moulds are significantly more commonly responsible for FK, in part due to their capacity to form hyphae and melanins. Future studies will examine models of fungal corneal interactions and assess additional factors of virulence, such as secreted enzymes, to more deeply decipher the pathogenesis of FK.

Efficacy and Rapidity of Potassium Hydroxide Mount and Modified Chicago Sky Blue 6B Stain with Potassium Hydroxide in Fungal Keratitis Detection

“…In contrast, the leading risk factor in Asia is trauma [ 1 , 21 , 24 ]. Our results resemble recent reports from Northern Thailand [ 23 , 25 ] that trauma is the leading risk factor for fungal keratitis. In addition, none of our patients were immunocompromised.…”

The Case for Fungal Keratitis to Be Accepted as a Neglected Tropical Disease

“…The sensitivity of microscopy has been reported to be 61–94% using potassium hydroxide, 85% using lactophenol blue and 36–50% using a traditional Gram stain [ 27 ]. If a fluorescence microscope is available, Calcofluor white (which highlights fungal cell walls) may be more sensitive than conventional microscopy, depending on the experience of the microscopist [ 28 ]. Calcofluor white is said to be a mainstay of diagnosis, and when combined with potassium hydroxide stains, sensitivity has been shown to rise to 98.3% [ 29 ].…”

Interaction of Talaromyces marneffei with free living soil amoeba as a model of fungal pathogenesis

“…The method for preparing T. marneffei infected A. castellanii , MOI, fixation and permeabilization were carried out as detailed above. After permeabilization, the cell suspensions were incubated with 10 mg/ml of MAb 8D6 (anti–fungal melanin monoclonal antibody) for 2 h at 37°C ( Youngchim et al., 2004 ; Chongkae et al., 2021 ). Then, the samples were washed five times with 2% BSA-PBS and then incubated for 2 h with Alexa Fluor 488 conjugated goat anti-mouse IgM antibody (Molecular Probes, Eugene, OR, USA) in a 1:500 dilution.…”