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Identification of Acanthamoeba cysts and trophozoites in cases of keratitis is traditionally done with microbiological techniques such as smear examination with 10% potassium hydroxide (KOH) and culture. Double walled cyst with hexagonal inner wall is characteristic of Acanthamoeba. We report a unique case of a 9 year old boy who presented with dense anterior corneal stromal infltration, which on smear examination showed atypical double walled spherical cysts, leading to a diagnostic dilemma. An 18S rRNA gene-based PCR done on the growth on culture, subsequently identifed a rarely reported species of Acanthamoeba. The patient was advised combination therapy with polyhexamethylene biguanide (PHMB 0.02%) and chlorhexidine (0.02%) eye drops. Three weeks post treatment, the keratitis resolved with scarring and vascularisation and visual acuity improved to 20/60. At 8 weeks follow up Best corrected visual acuity further improved to 20/30 with contact lens.