Fungal keratitis caused by Pseudallescheria boydii: clinical and mycological characteristics

Background Pseudallescheria keratitis is rare but important type of fungal keratitis because of the inherently resistance of the organism to many existing antifungal agents. Methods Slit-lamp and confocal microscopy were used for clinical examinations. Fungal isolates were identified based on morphological characteristics and DNA sequence of the internal transcribed spacer region (ITS). In vitro antifungal susceptibility testing for fungal isolates was performed according to the Clinical and Laboratory Standards Institute (CLSI, M38-A2). Result All patients had a history of ocular trauma. In clinical examination hypopion were seen in three patients. The main antifungal medications were topical voriconazole. After treatment the visual acuity of all patients improved in 2–3 weeks. Conclusion All four patients of Pseudallescheria keratitis had similar clinical features. Accurate and rapid identification of species should be helpful in treating p. boydii keratitis.


Introduction
Fungal keratitis is not life threatening but certainly cause ocular morbidity and sight threatening all over the world [1]. It has been caused by fungal species that capable to colonize and invade the ocular surface and corneal stroma [2]. The etiologic and epidemiologic pattern of fungal keratitis varies depending on patient status, geographic location, and climate [3]. Fungal keratitis can cause by at least 35 genera of moulds and yeasts [3,4]. Pseudallescheria boydii (previously Petriellidium boydii, Allescheria boydii, Monosporium apiospermum) belongs to the group of septate filamentous fungi, which are the most common pathogenic fungi causing opportunistic human infections associated with trauma [5,6]. This saprophytic fungus frequently isolated from soil, polluted water, sewage, decaying organic substances, manure, and potted plants throughout the world [7]. P. boydii implicated in a wide range of infections (mycetoma, pneumonia, osteomyelitis, arthritis, sinusitis, endocarditis, meningitis, and brain abscess) in both immunecompromised and immune-competent patients [7]. Several reports have documented eye infections such as keratitis, scleritis, sclerokeratitis, chorioretinitis, and endophthalmitis due to P. boydii following injury, herpetic keratitis and operation [8]. The first keratitis caused by Scedosporium species reported in 1955 [9]. The prognosis of fungal keratitis caused by P. boydii is poor [7]. Treatment of these pathologies is difficult because of the inherently resistance of the organism to many existing antifungal agents [8,9]. We present the clinical characteristics, risk factors, treatment, and prognosis of four patients with P. boydii keratitis, and also present the antifungal sensitivities of the isolated strain.

Subjects
Slit-lamp and in vivo confocal microscopy (IVCM) (Heidelberg Engineering, Heidelberg, Germany) were used for clinical examinations. The clinical characteristics of four patients with Pseudallescheria keratitis were reviewed.

Sample collection and processing
Corneal scraping material was collected after anaesthetising the cornea with instillation of drops of 0.5% proparacaine and cleaning the cornea with sterile normal saline to remove all necrotic exudates. After 5 min with the help of blade no. 15 scraping was done under slit lamp illumination by the ophthalmologist. Corneal scrapings were sent to Medical Mycology laboratory, Tehran University of Medical Sciences, for mycology studies. Gram staining, 10% Potassium Hydroxide (KOH) mount, culture on sabouraud dextrose agar with chloramphenicol and slide culture was carried out to identify the isolate up to the genus level.

Molecular identification
One colony of each fungal species scraped and DNA was extracted using a DNA isolation kit (Gene All DNA extraction kit; Gene All, Germany) according to the manufacturer's instructions. To confirm the Pseudallescheria species identification, the Internal Transcribed Spacer (ITS) region was sequenced [10]. ITS 1 (5′-CGC TGC GTT CTT CAT CG-3′) and ITS 2 (5′-TCG ATG AAG AAC GCA GCG-3′) primers were used for amplification. Yielded sequence was subjected to Basic Local Alignment Search Tool (BLAST) program (http://www. blast.ncbi.nlm.nih.gov/Blast).

Clinical characteristics
Data regarding age, sex, occupation, detailed clinical history, clinical presentation and symptoms are summarized in Table 1.

Patient 1
The patient was a 46-year-old woman who felt dust particles blow into her right eye during farm work. On clinical examination, her best-corrected visual acuity (BCVA) was counting fingers (CF) from 50 cm. On slitlamp examination she had a grayish, superficial stromal infiltration with epithelial defect and indistinct margins. Hypopyon was detected in the anterior chamber (Fig. 1A). Examination of the cornea with the IVCM showed a mass of interlocking and branching white lines in the area of the infiltrate, this feature suggesting filamentous fungus keratitis ( Fig. 2A). Based on clinical picture and the result of corneal scraping treatment were begun with oral itraconazole (200 mg/day), topical 1% voriconazole hourly, topical 0.1% amphotricin B 2 time/ day and topical 0.5% levofloxacin 4 times/day. After medications the hypopyon resolved in a few days, the focal infiltrate resolved within 2 weeks and the BCVA in her right eye improved to 20/160 within 2 months.

Patient 2
A 69-year-old man attended our hospital with a sudden loss of visual acuity in left eye after trauma with plant material. On clinical examination, her BCVA was CF from 50 cm. Slit-lamp examination showed corneal ulcer and associated hypopyon (Fig. 1B). Examination of the cornea with the IVCM showed branching white lines in the area of the infiltrate, this feature suggesting filamentous fungus keratitis (Fig. 2B). Based on clinical picture and the result of corneal scraping the patient was treated with application topical 1% voriconazole hourly and

Patient 3
The patient was a 30-year-old man whose left eye was injured by a wood particle during woodworking. He had history of pain, redness, sensation of foreign body, excessive tearing and diminution of vision in the left eye. On initial examination, her BCVA was CF from 50 cm. On slit-lamp examination he had grayish white lesion involving the corneal endodermis, pyoderma in the endothelium, and peripheral corneal edema. Hypopyon was detected in the anterior chamber (Fig. 1C). Examination of the cornea with the IVCM showed branching white lines in the area of the infiltrate, this feature suggesting filamentous fungus keratitis (Fig. 2C). Based on clinical picture and the result of corneal scraping treatment were begun with topical 1% voriconazole hourly. After medications the hypopyon resolved in a few days, the focal infiltrate resolved within 2 weeks and the BCVA in her left eye improved to 20/19 within 2 months.

Patient 4
A 37-year-old man whose right eye was injured by a branch of a tree during farm work visited ophthalmology outpatient unit at our tertiary referral center. He had progressive pain, sensation of foreign body, excessive tearing and redness in the right eye. On initial examination, the BCVA of the right eye was found to be 20/60. Slit lamp examination of the affected eye revealed superficial corneal stromal infiltrate with gray-white in color and feathery edged margins. Satellite lesions and hypopyon were not seen. Based on clinical picture and the result of corneal scraping treatment were begun with topical 1% voriconazole hourly. After medications the focal infiltrate resolved within 10 days and the BCVA in her light eye improved to 20/16 within 1 month.

Mycology characteristics
Direct microscopic examination of scrapes using KOH and gram staining reveals septate hyphae. Gray-white fungal colonies grew on sabouraud dextrose agar after 3 days' incubation at 30°C. Identification was subsequently made from microscopic examination of the colonies that showed ovoid conidia with hyphae which are features characteristic of P. boydii. Using NCBI BLAST, the fungi isolates were identified as P. boydii with 100% identity in the GenBank database. The Table 2 shows the MICs of various antifungal drugs against P. boydii isolates. The P. boydii isolates were sensitive to voriconazole, itraconazole, posaconazole, caspofungin and Chlor hexidin, while resistant to amphotericin B, 5-flucytosine, fluconazole and natamycin.

Discussion
P. boydii and its asexual form (S. apiospermum) belong to the group of septate filamentous fungi are uncommon cause of mycotic keratitis in humans. A review of the literature showed that P. boydii is the most common species of Scedosporium boydii complex (S. aurantiacum, P. minutispora, S. dehoogii, S. dehogii, and P. boydii) that reported as Pseudallescheria keratitis [12,13]. To the best of our knowledge, fungal keratitis caused by P. boydii has only been reported in the literature as single cases [14][15][16][17]. For the first time, we describe the clinical and mycological features of four fungal keratitis caused by P. boydii over a one-year period in a training hospital. Rathi et al. reported that the principal risk factor for Pseudallescheria keratitis was ocular trauma [8]. The Pseudallescheria species are commonly found in soil, water, and sewage [9]. Thus, the principal risk factor for Pseudallescheria keratitis is ocular trauma and contamination by concomitant soil contamination [7,9]. All of our patients (3 patient injured by plant material and 1 by dust particles) also gave a similar history of trauma.
Treatment of fungal keratitis remains a difficult problem for each ophthalmologist because there is no ideal antimycotic drug [12]. A delayed diagnosis and the use of conventional antifungals often lead to poor prognosis in Pseudallescheria keratitis [6]. Treatment is often based on clinical response [7]. Voriconazole was reported to be effective against S. apiospermum [7,18]. However, it seems to be less effective against another Scedosporium species [19]. Viconazole treatment failure has sometimes resulted in the surgical procedure such as penetrating keratoplasty (PKP) [20]. Surgical procedure can result in complications such as pain, postoperative inflammation and graft rejection [21]. Hernandez et al. and Nulens et al. have reported voriconazole to be effective against fungal keratitis caused by S. apiospermum [22,23]. Ramakrishnan and colleagues reported in their series, natamycin and fluconazole combination therapy succeeded in seven of 10 cases of ocular infections caused by S. apiospermum [13]. Patients in our study were prescribed voriconzole alone without needing for surgical interventions and PKP. All patients showed complete healing of their ulcers with this monotherapy. In our antifungal susceptibility tests, all four strains displayed in vitro resistance to amphotericin B and fluconazole and susceptible to voriconazole and itraconazole. In vitro sensitivity tests for caspofungin displayed three isolates resistance and one isolate susceptible to this antifungal agent. There is few investigation of the antifungal susceptibility against P. boydi isolated from keratitis. Sicilia and colleagues have reported the isolated strain displayed in vitro resistance to all the antifungal agents (amphotericin B, fluconazole, itraconazole, ketoconazole and 5 -fluorocytosine) tested [7]. These results are some similar to our results. The chlorhexidine MICs of all isolates of P. boydi tested were 0.008 μg/ml. Four patients with Fusarium keratitis were successfully treated with chlorhexidine 0.02% eye drops by Oliveira dos Santos et al. [24]. The efficacy of chlorhexidine for treating fungal keratitis has been confirmed by studies [24,25]. There is a need for further research on the use of chlorhexidine in Pseudallescheria keratitis.
In conclusion, these patients bold the importance of determining causative organism of fungal keratitis and their antibiotic susceptibility. Culture findings are limited in identifying organisms. Sequencing of polymerase chain reaction-amplified DNA is good for accurate and rapid identification of species that can be helpful for optimize treatment.