Corneal Ulcer Due to the Fungus Lasiodiplodia theobromae in an Ecuadorian Farmer

Case Report 

Corneal Ulcer Due to the Fungus Lasiodiplodia theobromae in an Ecuadorian Farmer

Corresponding author Dr. Jeannete Zurita, Unidad de Investigaciones en Biomedicina. Zurita & Zurita Laboratorios, Av. De la Prensa N49-221 y Manuel Valdivieso Quito-Ecuador, Tel: 593-2-3-945-120; Email:jzurita@zuritalaboratorios.com

Abstract

We describe a case of a corneal ulcer as a result of traumatic implantation caused by the fungus Lasiodiplodia theobromae. It was isolated from a corneal lesion in a 35-year-old farmer in Puerto Quito (tropical forest of Ecuador). After a month of incubation, no sporulation was observed; the isolate was identified by DNA sequencing. The initial treatment with topic and intrastromal voriconazole failed. The patient was treated by conjunctival flap covering surgery and needs a corneal transplant.

Keywords: Corneal Ulcer; Ecuador; Fungal Keratitis; Lasiodiplodia theobromae
Introduction
Microbial keratitis remains the fifth cause of blindness worldwide [1], with 6% to 60% of cases involving fungal species [2]. These infections are more prevalent in tropical and subtropical regions of low and middle-income countries and are related to agricultural activities [1,2]. The corneal abrasion after an ocular trauma often occurs prior to the acquisition of infection, but other risk factors of fungal keratitis stems from the use of contact lenses and ocular surface diseases [1-3]. Environmental filamentous fungi, including Fusarium spp. and Aspergillus spp., are the most frequent etiological agents. However, other species of fungi could cause human keratitis [2].
Lasiodiplodia theobromae is a pleomorphic ascomycete that is mostly distributed in tropical and subtropical areas [4]. This fungus is a plant pathogen but has unfrequently been involved in human mycosis. Fungal keratitis has rarely been described as a cause of mycotic keratitis with approximately 6% of cases [1,2]. Nevertheless, the prevalence of keratitis caused by L. theobromae remains largely unknown and mostly based on information from case reports [1, 2, 5]. In this case, we described a fungal keratitis caused by L. theobromae in Ecuador and compared the results with previous reports.
Case
In October 2015, a 35-year-old man attended the Ophthalmology Department of a hospital in Quito complaining of pain in his red-coloured left eye. The patient was a farmer in Puerto Quito, a zone located in the tropical forest of Ecuador. He had a traumatic exposure of white powder in his left eye while he was harvesting cacao (day 0). After this accident he had pain, conjunctival hyperaemia, and pruritus with a progressive decrease in visual acuity. Antibiotic eye drops were prescribed for a week by the local health care centre (day 6). The patient with no clinical improvement and a marked decrease in visual acuity (hand motion) attended the emergency service in a hospital in Quito, where a 7 x 5 mm corneal ulcer was diagnosed using fluorescein (Figure 1a). Fusarium spp. was considered as the presumptive etiologic agent. The treatment started with 5% voriconazole and 1% vancomycin drops every hour in addition to 200 mg of voriconazole and 1000 mg of ceftriaxone administered intravenously every 12 hours (day 14).
Clinical specimens were collected and handled according to standard protocols and biosafety. The samples were inoculated in blood and chocolate agar and incubated at 35°C. The mold grew as cotton-like colonies that became dark-grey within 7 days (day 21) (Figure 1b). The fungal isolate was dematiaceous and grew up rapidly. Subsequently, it was sub-cultured in Sabouraud dextrose agar, Mycosel agar, potato dextrose agar and cornmeal agar and incubated at 30°C. A duplicate Sabouraud dextrose agar plate was incubated at room temperature (Figure 1c). There was no growth on Mycosel agar and cornmeal agar. Sub-cultures were initially white and fluffy with grey and black areas turning black and woolly over time with a black reverse (day 27). Lactophenol cotton blue staining microscopic examination showed brown septate hyphae with a width of 6 μm (Figure 1d). The fungus was not sporulated. The strain was called H467Z&Z.
L. theobromae species identification was achieved by sequencing (day 28).
Figure 1. Phenotypic findings: a) corneal ulcer stained with fluorescein; b) growth of L. theobromae strain H467Z&Z in blood agar; c) growth in Saboraud dextrose agar; d) microscopy was performed using blue lactophenol preparation and septate hyphae were noted (40x). No sporulation was observed after a month; e) flap covering surgery.
Briefly, total DNA was isolated using a High Pure PCR Template Preparation kit (Roche Diagnostic, Schweiz, Switzerland). The internal transcribed spacer (ITS) was amplified using the universal primers ITS1 and ITS4. The PCR was carried out at an annealing temperature of 52 °C. The 586 bp product was visualized in 1% agarose gel electrophoresis. The sequencing was performed at Macrogen Inc. (Seoul, South Korea). The ITS sequence was analysed using Genious v9.1 software and BLAST. An UPGMA analysis was conducted by comparing the H467Z&Z and L. theobromae ITS sequences in GenBank which were described as causes of eye infections (Fig. 2). The H467Z&Z ITS sequence was deposited in GenBank [accession No. KU291531]. The analysis showed 100% similarity and identity with strains that caused keratitis in Peru, Thailand, Vietnam, and India and with strains isolated from plants in Ecuador.
In spite of medication, the infection continued to progress into an 8 x 9 mm corneal ulcer in 80% of the eye on day 25. An intrastromal injection of voriconazole was administered on day 29, with no clinical improvement. Conjunctival flap covering surgery was made on day 34. The patient was injected with 200 mg of voriconazole intravenously every 12 hours for 12 days, and a corneal transplant was programmed in about six months.
Figure 2. UPGMA tree of the ITS sequence of the H467Z&Z isolate and sequences of L. theobromae reported as causing keratitis and isolated from plants in Ecuador (*).The sequences were downloaded from the GenBank database. Sequences of 385 bp were used to build the tree in Genious v9.1. We used the Tamura Nei genetic distance model with 500 replicates and no out-group was included. The H467Z&Z strain is shown in red.
Discussion
Ecuador is an agricultural tropical country located in the Andean Region of South America with a high risk of acquisition of fungal infections. However, underreporting of cases drives a lack of the knowledge about etiological agents and prevalence of these infections. There are a small number of cases of  keratitis caused by L. theobromae worldwide, with approximately 25 cases being reported in the scientific literature [2]. Although the species description was made in Ecuador by Patouillard et al. in 1892, as a plant pathogen [6], this report presents the first description of keratitis caused by L. theobromae in our country.

Case reports show L. theobromae as a cause of complicated and difficult-to-treat keratitis [2,5], with approximately 80% of the cases requiring surgical intervention [2]. Voriconazole is the first option against keratitis with filamentous fungi [5]. In this report, the initial therapy was topic voriconazole, with no clinical improvement. This initial failure is in accordance to a previous study [7] where the infection with a sensitive strain was not reduced by applying topic voriconazole, most probably because the therapy reached a concentration below the MIC of the strain. Additionally, strains of L. theobromae showed resistance in vitro to azoles and natamicyn [8]. The resistance was not tested in the H467Z&Z strain because it is not a routine procedure. In our case, after the failure of topic voriconazole the treatment included intrastromal injection of voriconazole without the expected result. This observation contrasted with Lekhanont et al., where the intrastromal injection of voriconazole was applied successfully [9]. The failure of intrastromal voriconazole injection observed in our case could be the result of the advanced stage of the infection (day 29). On the other hand, natamicyn is the antimycotic of choice for keratitis in several countries [10], but in Ecuador, as in other countries this drug is not available or difficult to find. Still, natamicyn could have poor penetration in the eye tissue [11].Additionally, a rapid and opportune confirmatory diagnosis by the laboratory is needed, especially in non-sporulating moulds, such as L. theobromae. PCR-based DNA sequencing techniques are rapid, reliable, and valuable tools [12]. Unfortunately, delayed diagnosis is common because of the lack of clinical suspicion and poor diagnostic laboratories in rural areas. These factors can affect the initial identification of infection. Moreover, compared to bacterial and viral keratitis, fungal infections progress slowly, thus resulting in delayed medical consultation. In this context, prevention of infection among rural health and agricultural workers plays an important role in reducing the incidence of keratitis in high risk areas. Such interventions have already been shown to minimize the problem in other tropical areas by using low-cost methods for detecting corneal erosions, use of antibiotics and antifungal ointments, and community training. These methods have resulted in the reduction of microbial keratitis incidence [1].

More reports describing the therapeutics aspects of keratitis infection caused by L. theobromae are needed to develop better strategies to manage these infections.

Acknowledgements
We would like to thank Dr Hector Martínez, Subdirector de Docencia e Investigación of Hospital Eugenio Espejo, for authorizing the publication of this case report. The molecular study was supported by Zurita & Zurita Laboratorios, Biomedical Research Unit.
Conflict of Interests
The authors declared no conflict of interests.

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