Cytopathogenicity of acanthamoeba, vahlkampfia and hartmannella: quantative & qualitative in vitro studies on keratocytes.

OBJECTIVES

To compare the cytopathogenicity of Vahlkampfia and Hartmannella clinical isolates with a type culture of Acanthamoeba castellanii.

METHODS

The cytopathic effect produced during 24 h co-incubation with cultured keratocytes was assessed at set time intervals. Formal quantative studies involved image analysis of the area of cells remaining after 6 h. The mechanism of cytopathogenicity was elucidated using time-lapse video, light and scanning electron microscopy. The ability to produce cell damage in the absence of physical contact was studied using the transwell apparatus. The role of apotosis was also investigated.

RESULTS

All three isolates produced near destruction of the keratocyte monolayer within 24 h, although initial cell destruction was more rapid with Acanthamoeba. For all three genera, the mechanism of cell damage involved physical attack and trogocytosis: cytopathic products were also implicated as cell damage was produced in the absence of physical contact, but apoptosis was not demonstrated.

CONCLUSIONS

While the results do not prove that Vahlkampfia and Hartmannella are pathogens, they provide important evidence supporting the thesis that they cause keratitis by demonstrating that their ability to produce a cytopathic effect on keratocytes in vitro is similar in magnitude and mechanism to that of the known pathogen Acanthamoeba castellanii. The mechanisms by which small free-living amoebae produce cell damage is poorly understood. The ability of genera of amoebae other than Acanthamoeba to produce corneal infection remains controversial. In this study, the cytopathogenicity of Vahlkampfia and Hartmannella isolated from a case human keratitis are compared both quantitatively and qualitatively with that to the known pathogen Acanthamoeba castellanii. The results suggest that representatives of each of the 3 genera produce a similar degree of cytopathic effect on keratocytes after 24h of co-incubation and that a combination of physical and chemical factors are responsible.