Acanthamoeba sp are free-living protozoa found worldwide, in soil, freshwater, tap water and hot springs. It can cause a painful vision-threatening disease in the human cornea, acanthamoeba keratitis (AK). The major risk factors for AK are the use of contact lenses, predominantly the extended-wear of soft lenses and the use of homemade saline or wearing contact lenses while swimming. The “gold” standard for laboratory diagnosis of AK is the isolation of the organism from a non-nutrient agar plate seeded with E coli. Encystations may occur after incubation of the culture plate for 5-10 days when growth is detected. A PCR assay was used to detect Acanthamoeba cysts in corneal specimens with a faster turnaround time than culture.
Our in-house Acanthamoeba PCR assay was validated previously against culture. DNA was extracted by Roche MagNA Pure machine and detected by Light Cycler II without the use of Internal Controls. The new method includes Bioline Internal Controls, extracted by Siemens Kingfisher machine and detected by Roche LC480 platform. Validation was conducted against samples previously tested by culture and PCR.
Sensitivity of the new method compared with the previous method was 100%, specificity was 98.15% PPV was 75% and NPV was 100%. Measurement of precision and limit of detection were determined in this validation.
The addition of internal controls to the sample extraction ensures that PCR inhibitors are recognized. The Real Time PCR test can be used reliably for the detection of Acanthamoeba sp. in eye and corneal specimens.