Parafilm as an efficient transport matrix for corneal scrapings.

Abstract

Introduction: Acanthamoeba spp. are free-living parasites increasingly implicated in causing Acanthamoeba keratitis (AK). AK is diagnosed by demonstration of parasites in corneal samples by direct microscopy, culture, and nucleic acid amplification. Most commonly, corneal scrapings are sent to the laboratory smeared between two glass slides. These scrapings are suitable for direct microscopy but less suitable for culture and polymerase chain reaction (PCR) which, in turn, are more sensitive for the diagnosis of AK. Aim: The aim of the study was to explore better alternatives for transporting corneal scrapings from the point-of-care eye center to the concerned laboratories. Materials and Methods: The study used small Parafilm (Bemis Company Inc., USA) squares (PSs) of 1 cm each prepared by cutting Parafilm using a surgical blade under sterile conditions. Each of the four different dilutions of Acanthamoeba suspension (15, 30, 60, and 120 cells) was used in this study. Each dilution was added onto the surface of 36 PSs and kept at room temperature for 24-h, 48-h, and 72-h incubation. The PSs for one particular time point and dilution were used for calcofluor white staining, its inoculation onto the surface of nonnutrient agar having a lawn of Escherichia coli, and Acanthamoeba-specific PCR amplification. In addition, two PSs inoculated with 30 cells and incubated for 24 h and 72 h were used for scanning electron microscopy (SEM). Results and Conclusion: All three diagnostic techniques, i.e. microscopy, culture, and PCR, detected the presence of Acanthamoeba at all the tested concentrations and time points. However, the growth pattern on culture changed directly in proportion to increased incubation periods and increased concentration of inoculum. In addition, the adherence of Acanthamoeba to the Parafilm was confirmed by SEM; these results suggest the use of these PSs as a suitable matrix for the transport of corneal scrapings.