A high-throughput, image-based assay to assess drug sensitivity of Acanthamoeba castellanii cysts
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Abstract
Pathogenic free-living amoebae (FLA) such as Acanthamoeba castellanii are present in soil and water worldwide. A. castellanii causes systemic infections with very high mortality rates, yet drugs specifically targeting this pathogen are not available. Methods to reliably generate and assay cysts, which drive infection recurrence and drug resistance, are unavailable in a high-throughput format suitable for drug screening and testing. In this study, we developed a robust and reproducible protocol for encysting A. castellanii as well as a high-throughput, quantitative cyst viability assay using fluorescent live/dead staining coupled with microscopy and automated image analysis. These methods were used to screen the cysticidal activity of 17 clinically relevant drugs and disinfectants and identified five agents, including caspofungin, as active against cysts.
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Discussion
Thank you for developing what appears to be a really useful set of tools! I appreciate how you've made the techniques accessible and reproducible. The 96-well format and automated analysis should make it relatively straightforward for other researchers to adopt these methods.
I'm particularly grateful for your commitment to open science - making both your code and data freely available on GitHub is exactly what the research community needs more of. This transparency will help other labs build on your work and get the most value from your efforts.
Thank you for the thorough work and for sharing it so openly with the community!
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45 media formulations (Table S2) had high absorbance (≥2.0) by SRB assay for all replicate wells at 48-72h of incubation (Fig S4), with four media leading to mean absorbance values ≥2.5 at 72 hours of incubation (Fig 1A).
It would be helpful to add a short line here like "..indicating more successful encystment"
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ll FEM
I know you define this later in the results section, but it would be helpful to define it as encystment media here as well. As a reader with no direct experience with A. castellanii, the experimental design was confusing without the context.
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