Background/aims The aim of this study was to correlate the various forms of Acanthamoeba on ex vivo confocal microscopy (EVCM) with in vivo confocal microscopy (IVCM) and findings from cultured positive cases of Acanthamoeba keratitis.

Methods Acanthamoeba live, dead and empty cysts, and live trophozoites were prepared in vitro and inoculated into porcine cornea using a sterile 26-gauge needle and examined ex vivo using the Heidelberg Retina Tomograph II/Rostock Corneal Module. IVCM images from 12 cultured positive Acanthamoeba cases, obtained using the same instrument, were compared with EVCM findings. Phase contrast images were also obtained to compare with both EVCM and IVCM findings. The change in cyst morphology with depth was evaluated by imaging the same cysts over a defined cornea depth measurement.

Results EVCM morphologies for live cysts included four main types—hyper-reflective central dot with hyper-reflective outer ring, hyper-reflective central dot with hyporeflective outer region, stellate shaped hyper-reflective centre with hyporeflective outer region and hyper-reflective round/polygonal shaped cyst; one main type for dead cysts—hyper-reflective central dot with hyporeflective outer region; two main types for empty cysts— hyper-reflective central dot with hyper-reflective outer ring/hyporeflective outer region; and one main type for trophozoites—large coarse speckled area of heterogeneous hyper-reflective material. Matching IVCM images show good correlation with EVCM. Cyst morphology altered when imaged at different depths.

Conclusion EVCM demonstrated the various forms of Acanthamoeba cyst and trophozoites can be used as a reference to identify similar structures on IVCM.