Article Text

Utility of Fungiflora Y stain in rapid diagnosis of Acanthamoeba keratitis
  1. TOMOYUKI INOUE
  1. Department of Ophthalmology, Osaka University Medical School, Osaka, Japan
  2. Clinical Laboratory, Osaka University Hospital
  3. Osaka, Japan
  4. Department of Ophthalmology, Osaka University Medical School, Osaka, Japan
  1. SEISHI ASARI,
  2. KAZUKO TAHARA
  1. Department of Ophthalmology, Osaka University Medical School, Osaka, Japan
  2. Clinical Laboratory, Osaka University Hospital
  3. Osaka, Japan
  4. Department of Ophthalmology, Osaka University Medical School, Osaka, Japan
  1. AKIRA KIRITOSHI,
  2. YOSHITSUGU INOUE,
  3. YOSHIKAZU SHIMOMURA
  1. Department of Ophthalmology, Osaka University Medical School, Osaka, Japan
  2. Clinical Laboratory, Osaka University Hospital
  3. Osaka, Japan
  4. Department of Ophthalmology, Osaka University Medical School, Osaka, Japan
  1. Tomoyuki Inoue, MD, Department of Ophthalmology, Osaka University Medical School, 2-2 Yamadaoka, Suita Osaka, 565 Japan.

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Editor,—Acanthamoebakeratitis is a serious and often misdiagnosed corneal infection that can lead to severe loss of vision. Signs and symptoms ofAcanthamoeba keratitis may also occur with bacterial, viral, or other fungal corneal disease, complicating the diagnosis of early stage Acanthamoebakeratitis. Since advancedAcanthamoeba keratitis is difficult to treat, early and accurate diagnosis is imperative for treatment. Therefore, the easy and rapid method to detect the pathogens is needed.

Recently, Fungiflora Y staining solution has been developed to detect fungi.1 This staining solution, like calcofluor white,2 has a specific affinity for chitin and cellulose which are components of fungal cell wall.1 The endocysts of Acanthamoeba also contain cellulose3 and has been ascertained to be stained with Fungiflora Y.4 However, the clinical utility of Fungiflora Y in the diagnosis of Acanthamoeba keratitis has not yet been reported.

We report a case of severe Acanthamoebakeratitis diagnosed with the help of Fungiflora Y staining. We also discuss the possible clinical use of Fungiflora Y staining to diagnose other cases of Acanthamoeba keratitis.

CASE REPORTS

A 55 year old woman, who had worn soft contact lenses for 1 month, presented in February 1994 with a 2 week history of foreign body sensation and photophobia in the left eye. These symptoms gradually became worse and she was diagnosed with herpes simplex keratitis in another clinic because of a pseudodendriform corneal epithelial lesion. Despite topical applications of antibiotics, corticosteroids, and aciclovir ointment four times a day, no improvement was observed and the patient experienced severe eye pain and progressive decrease in vision in the left eye.

When the patient first visited the department of ophthalmology at Osaka University Medical School in early April 1994, her visual acuity was 0.1 in the left eye and slit lamp examination revealed stromal disciform keratitis with a central ulceration (Fig 1). Her right eye was normal.

Figure 1

Slit lamp examination revealed stromal disciform keratitis with a central ulceration.

We obtained a deep corneal scraping for cytopathological examination and culturing. Light microscopic examination failed to disclose any Gram and Giemsa stained organisms. Furthermore, with haematoxylin and eosin staining, no pathogen could be found. However, Fungiflora Y staining to detect fungus unexpectedly demonstrated fluorescent Acanthamoebacysts, which in turn led to the diagnosis ofAcanthamoeba keratitis. The indication of the steps and time using the Fungiflora Y staining kit (Biomate Co, Ltd, Tokyo, Japan) was as follows. The scrape was transferred onto slide glass, and a drop of the counterstaining reagent (A solutuon) was added. After 2 minutes, the slide was rinsed with running water and stained with the staining reagent (B solution) for 5 minutes. The slide was rinsed under running water and observed under a fluorescent microscope at a wavelength 405 nm.Acanthamoeba cysts appeared as clear yellowish-green fluorescence and were easily identified (Fig2).Subsequently, cultures of the corneal scrapings were found to be positive for Acanthamoeba cysts and trophozoites. When mitochondrial DNA was digested by restriction enzyme it was identified as AcanthamoebaMa.5

Figure 2

Acanthamoeba cysts stained with Fungiflora Y (×400). The staining procedure was simple and rapid and required no complex skills. Acanthamoeba cysts were readily identified under low magnification fluorescence microscopy.  

The appropriate treatment agents and doses were determined by in vitro sensitivity testing, which showed thatAcanthamoeba trophozoites were sensitive to miconazole and pimaricin and cysts were sensitive to pimaricin only.

The patient received the following treatment for 1 month: 0.1% miconazole eye drops six times a day, pimaricin eye drops four times a day, and ofloxacin eye drops four times a day, in addition to a daily 400 mg intravenous dose of miconazole for 4 weeks. Eye pain disappeared and the lesions improved slowly. Central stromal infiltration gradually became less dense, but the occurrence of stromal scarring led to the need for penetrating keratoplasty in December 1995. Visual acuity returned to 0.5 in the left eye at 2 years postoperatively, and she has been free of recurrent disease.

COMMENT

In this case, no pathogens were detected on examination of corneal tissue samples, prepared with conventional stains even after repeated evaluation. To identify fungi, we examined smears of corneal scrapings stained with Fungiflora Y and unexpectedly found theAcanthamoeba cysts stained fluorescently. This confirmation of our diagnosis ofAcanthamoeba keratitis allowed us to initiate the appropriate treatment with confidence.

To our knowledge, this is the first clinical case report ofAcanthamoeba keratitis diagnosed with Fungiflora Y stain.

The procedure for staining with Fungiflora Y is simple and rapid, requiring no experience and complex skill,4 and in our case Acanthamoeba cysts were easily identified under low magnification fluorescence microscope. Furthermore, when we observed this slide after 3 months,Acanthamoeba cysts were well stained fluorescently and easily identified. Consequently, fluorescent intensity of Fungiflora Y stain is stable and fluorescent attenuation is much smaller than the FITC labelled antibody or other fluorescence staining methods. The results in this case indicate that the staining with Fungiflora Y may be a useful additional technique for identifyingAcanthamoeba cysts in keratitis specimens. Further evaluation is required to establish how this technique compares with the other available techniques.

References

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