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Viability test exclusively is not adequate to evaluate the T4 Acanthamoeba keratitis’ treatment

Journal of Microbiology & Experimentation
Geisa Bernardes,1 Daniella de Sousa Mendes Moreira Alves,1 Daianny Costa da Silva,1 Luciano Moreira Alves,2 Ana Maria de Castro,1 Marina Clare Vinaud1

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Acanthamoeba keratitis is a serious corneal infection and can lead to blindness. Cysts and recurrence of infection represent a challenge in the treatment of keratitis due to its high resistance to adverse conditions and to most medications. This intriguing property can lead to late diagnosis and can explain absence of cyst removal during and after therapy regimen. So the evaluation of Acanthamoeba cysts viability after exposure to drugs used in Acanthamoeba keratitis treatment is so important.
Material and methods: Cysts (105 /mL) of two clinical and an environmental T4 isolates were separately incubated with polyhexamethylene biguanide (0.02%), chlorhexidine digluconate (0.02%) or propamidine isethionate (0.1%) at 37 °C for a period of 1h or 24h. Cysts were quantified, stained with trypan blue and inoculated in agar plates covered with inactivated Escherichia coli.
Results: None of the treatment regimens induced complete cysts elimination. The environmental isolate had no reduction on cysts quantification compared to clinical isolates. The trypan blue exclusion method demonstrated non-viable cysts in the treatment with chlorhexidine digluconate and propamidine isethionate. In the treatment with polyhemamethylene biguanide, viable and non-viable cysts were observed after 24h of incubation. The cultures were positive for all treatment protocols except for clinical isolate ATCC 30461 with chlorhexidine digluconate regimen after 24 h.
Conclusions: The presence of cysts observed in cultures reiterates cysts viability even after treatment demonstrating the demonstrating the need to follow up this form of development during treatment. The trypan blue staining is not adequate to evaluate the Acanthamoeba cysts viability due to its recovery in culture. With these results, we conclude that the viability test exclusively is not adequate to evaluate the Acanthamoeba keratitis treatment and that its always necessary to perform culture technique.


Acanthamoeba, viability, culture, polyhexamethylene biguanide, chlorhexidine digluconate, propamidine isethionate, trypan blue