An International External Quality Assessment Scheme to Assess the Diagnostic Performance of Polymerase Chain Reaction Detection of Acanthamoeba Keratitis.

PURPOSE

The purpose of this study was to assess the variation in methods and to determine whether an External Quality Assessment Scheme (EQAS) for polymerase chain reaction (PCR) detection of Acanthamoeba keratitis is valuable for the diagnostic process.

METHODS

A multicenter EQAS was introduced, covering 16 diagnostic laboratories. Using Acanthamoeba castellanii ATCC strain 30010, 3 sets of samples were prepared, containing different amounts of DNA, cysts, or trophozoites. Samples were masked and sent to the participants with instructions for use and a questionnaire concerning the applied methodologies. Special attention in this questionnaire was given to the used pretreatment methods to assess existing variations in these procedures.

RESULTS

A large variation in the methodologies and substantial differences in the diagnostic performance were found between participants. In contrast to the DNA samples where all participants had a perfect score, several false negative results were reported for the samples containing cysts or trophozoites. Only 9 participants had an optimal score, whereas one participant reported all samples as negative, one participant reported failures due to inhibition, and the other 5 reported in total 7 false negative results. A clear correlation was noticed between the PCR detection rate and the number of cysts or trophozoites in the sample.

CONCLUSIONS

The results indicate that a pretreatment procedure can be a risky step in PCR-based detections of Acanthamoeba , but it improves the sensitivity and reliability, especially of samples containing cysts. Therefore, participation in an EQAS is informative for routine diagnostic laboratories and can assist in improving the laboratory procedures used for the diagnosis of Acanthamoeba keratitis.