Abstract
Purpose :
Acanthamoeba species are free-living microorganisms found in a variety of environments which can cause a severe and sight-threatening infection of the cornea, Acanthamoeba keratitis. Currently, conventional culture and molecular methods are used for the detection of Acanthamoeba. Culture-based methods are time-consuming, complex and may not detect cysts that do not excyst or trophozoïtes that are damaged during sample preparation. Molecular methods on the other hand, cannot distinguish between viable and non-viable Acanthamoeba. To overcome these challenges, we developed a rapid and sensitive viability PCR (v-PCR) assay capable of differentiating between viable and non-viable Acanthamoeba within one day. In viability PCR, samples are pre-treated with a photoreactive dye such as propidium monoazide (PMAxx) that selectively binds to DNA from non-viable Acanthamoeba and prevents DNA amplification. This approach provides valuable insights for monitoring treatment and disease control.
Methods :
A clinical Acanthamoeba strain isolated from an ocular sample was cultured on nonnutrient agar plates supplemented with E. coli and harvested using a cell scraper in Page’s amoeba saline solution. A culture of 105 cells/mL was used for the experiments. For assessment of viability, samples were analyzed both with routine PCR and with v-PCR. The results from both PCRs were compared to assess the percentage viability. For validation, cultured Acanthamoeba were heat-killed and viable and non-viable Acanthamoeba were mixed in pre-defined ratios. The disinfection efficacy of Menicon Progent was also evaluated using the v-PCR assay. All experiments were performed in duplicate.
Results :
The results showed that PMAxx could effectively prevent DNA amplification of non-viable Acanthamoeba, where a 4.8 log reduction of the PCR signal could be achieved.
To illustrate the use of v-PCR, the effectiveness of Menicon Progent solution was demonstrated against Acanthamoeba. After pretreating samples with Menicon Progent, both viable and non-viable Acanthamoeba were not detected.
Conclusions :
In the current study, a rapid viability RT-PCR assay has been developed that can distinguish between viable and non-viable Acanthamoeba species based on the difference between routine PCR and v-PCR.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.