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DNA Typing–Based Validation and a Simple Detection Assay

Savitri Sharma, MD; Gunisha Pasricha, MS; Debashish Das, MPhil; Ramesh K. Aggarwal, PhD

Arch Ophthalmol. 2004;122:1430-1434.

Objectives  To establish that the protozoan Acanthamoeba is one of the causative organisms associated with non–contact lens–related keratitis in the Indian population and to develop a simple and sensitive diagnostic assay for clinical testing.

Design  DNA sequencing of nuclear 18S and 26S ribosomal DNA motifs was performed and compared with the reference Acanthamoeba strains, to establish the genetic identity of the putative amoeba isolates obtained from the corneal scrapings of non–contact lens–wearing patients with keratitis. Ribosomal DNA typing of clinical corneal scrapings from the patients with keratitis was performed by means of a simple agarose gel–based multiplex polymerase chain reaction assay, to detect the cases of Acanthamoeba keratitis.

Results  The ribosomal DNA analysis of 15 putative amoeba isolates obtained from the corneal scrapings of 14 patients with keratitis and 1 from the patients' environment established the isolates to be pathogenic formsof Acanthamoeba belonging to type T4 ribosomal DNA genotype. Multiplex polymerase chain reaction assay was specific and sensitive enough to detect as low as 5 pg of Acanthamoeba DNA. Its utility as a reliable diagnostic assay was demonstrated directly with the use of 34 additional corneal scrapings.

Conclusions  Acanthamoeba is one of the causative organisms of keratitis in Indian patients with no history of contact lens usage. Moreover, the Acanthamoeba infection can be easily detected in the clinical samples by means of the simple multiplex polymerase chain reaction assay based on ribosomal DNA typing.

Clinical Relevance  This study suggests the need and means to determine the incidence and prevalance of Acanthamoeba keratitis in India and elsewhere. Moreover, the polymerase chain reaction assay would help in early and definitive diagnosis, leading to better prognosis of Acanthamoeba keratitis condition.

From the Jhaveri Microbiology Centre, Hyderabad Eye Research Foundation, L. V. Prasad Eye Institute, Hyderabad, India (Dr Sharma, Ms Pasricha, and Mr Das); and Centre for Cellular and Molecular Biology, Hyderabad (Dr Aggarwal). The authors have no relevant financial interest in this article.

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