Acanthamoeba on Sabouraud's agar from a patient with keratitis.

A 25-year-old transgender patient came with complaints of watery discharge, red eye and photophobia in the left eye since 2 days. The patient had a history of wearing colored contact lenses since 4 years and cleaning the lens with tap water. Culture of lenses on Mac Conkey and blood agar yielded Klebsiella pneumoniae and Pseudomonas aeruginosa. Sabouroud's agar showed yeast cells and double-walled cysts of Acanthamoeba species. On further incubation of Sabouroud's agar, the cysts transformed to trophozoites. Parallel results were obtained on tap water agar. The previous therapy of moxifloxacin was changed to local Neosporin application.

INTRODUCTION

Acanthamoeba species are ubiquitous, freeliving protozoan parasites that can be isolated from diverse habitats like soil, stagnant water, fresh water ponds etc.[12] They can infect immunocompromised as well as healthy persons. Keratitis is the most common human infection caused by Acanthamoeba. Acanthamoeba keratitis accounts for profound morbidity and significant loss in visual acuity in about 16% of infected corneal ulcer patients,[3] and thereby the quick and correct etiological diagnosis is essential. The diagnosis is mostly done by wet mount, culture on tap water agar, histopathology, and PCR.[4]

We cite a case of keratitis in which the etiological diagnosis was missed in the primary wet mount and was negative for parasites but was culture positive for Acanthamoeba on Sabouraud's agar.

CASE REPORT

A 25-year-old transgender using colored contact lens for professional reasons presented with watery discharge, red eye, photophobia, and blepharospasm in the left eye. The patient had been using contact lens for the past 4 years, and regularly changed them every 6 months. He had used tap water for moistening his left lens as the fluid in the lens box had dried up and he had run out of lens fluid.

On examination, he had a corneal ulcer with hypopyon and ring infiltration in the left eye, conjuctival congestion, and a hazy left fundus [Figure 1]. Corneal scrapping sent to the microbiology lab grew Klebsiella pneumoniae and Pseudomonas aeruginosa on MacConkey agar and blood agar and Candida on Sabouraud's agar. The direct wet mount from scrapings and lens fluid did not reveal any parasites.

Figure 1

Corneal ulcer with hypopion and ring infiltration

The Gram stain from Sabouraud's agar on the third day showed rounded structures which were confused with pus cells. As the patient was a contact lens user, there was suspicion of parasitic etiology. On wet mount examination from the Sabouraud's agar, cysts of Acanthamoeba were seen [Figure 2]. After two more days of incubation, the cysts transformed to trophozoites [Figure 3]; this was also confirmed by culture on tap water agar enriched with Escherichia coli and Candida.

Figure 2

Wet mount showing double-walled cysts of Acanthamoebasp. (×400)

Figure 3

Wet mount showing trophozoite of Acanthamoeba sp. with spiny acanthopodia (×400)

The patient was initially treated with moxifloxacin, tobramycin drops, and artificial tears. On receiving the microbiology report, the patient was treated with local neosporin and improvement was seen after 1 week.

DISCUSSION

The diagnosis of Acanthamoeba keratitis is done by wet mount, culture on tap water agar, histopathology, and PCR.[4] Zeybek et al. in 2010[5] used proteose peptone–yeast, proteose peptone–glucose, proteose peptone–yeast extract–glucose, Jones's medium, Chang's serum–casein–glucose yeast extract medium, tryptone soya broth (Oxoid), RPMI (Sigma), and soil extract with salts to cultivate free-living amoeba from water samples but were successful only on proteose peptone–yeast and proteose peptone–yeast extract–glucose. Growth on Sabouraud's dextrose agar has been reported rarely[6] but is not routinely used.

Our cultivation of Acanthamoeba on Sabouraud's agar showed parallel results with tap water agar seeded with E. coli. It could be used as a cultivation medium for Acanthamoeba in places where tap water agar or non-nutrient E. coli agar overlaid with Page's saline are not available. Therefore, a repeat wet mount from the Sabouraud's agar after 2 days and 4 days is recommended as the diagnosis is often missed in the primary wet and KOH mount from corneal scrapping, especially when facilities for Acanthamoeba culture and PCR are not available.

Also stains like Giemsa and KOH mount are used for the detection of the morphological form of Acanthamoeba sp. A simple Gram stain could also give a clue to the diagnosis as it is more commonly done.

Hence this study throws light on Acanthamoeba as an important pathogen causing keratitis in patients using contact lens. Simple diagnostic methods should be used for the laboratory identification of the parasite.