Comparison of a PCR-based method with culture and direct examination for diagnosis of Acanthamoeba keratitis

ABSTRACT

The aim was to compare three different methods [direct examination, culture and PCR methods] for the diagnosis of Acanthamoeba keratitis [AK] in corneal scrapes. Twenty eight corneal scrapes and contact lenses were collected from keratitis patients and referred to the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences. Corneal scrapes were divided in three parts for direct examination, culture on non-nutrient agar and PCR analysis. PCR analysis was also performed using a 18S rRNA gene primer pair [DF3 region]. DF3 [Diagnostic fragment 3] is a region of the nuclear small subunit ribosomal RNA gene which is specific for detecting Acanthamoeba strains. Acanthamoeba was the causative agent of keratitis in 50% of the patients. Direct smear of all prepared corneal scrapes in AK patients was negative and culture was positive in only 14.3% of the isolates. PCR analysis was positive in 71.4% of AK patients. These three methods were negative in corneal scrapes of non-AK patients. The sensitivity and specificity of PCR technique for the detection of Acanthamoeba sp. were calculated as 71.4% and 100%, respectively. According to high sensitivity and specificity of PCR-based method, this study confirmed that PCR using 18S rRNA gene primers [DF3 region] is more useful for detecting AK cases compare to culture and direct microscopy methods: