Rapid Cultivation of Acanthamoeba spp. Isolated from Environmental Samples Using Nanocomposite and Leech Saliva on Non-Nutrient Agar

Abstract

Objective: Acanthamoeba spp. are microscopic single-celled protozoa commonly found in the environment, particularly in soil, water sources, and dust. These parasites are associated with serious infections such as Acanthamoeba keratitis and granulomatous amebic encephalitis. Recent epidemiological studies have highlighted a significant increase in Acanthamoeba keratitis cases. Current treatment methods are generally effective only in the early stages and show limited success when applied late, emphasizing the urgent need for more effective therapeutic and cultivation approaches. The laboratory cultivation of Acanthamoeba spp. is traditionally performed using axenic or monoxenic cultures. However, these methods have notable drawbacks, including the loss of virulence, reduced encystment capability, errors in bacterial inoculation, and time-consuming procedures. To address these limitations, nanotechnological approaches have been proposed. Nanotechnology offers innovative solutions for developing new drug formulations and diagnosing, preventing, and treating various diseases. Methods: In this study, we developed humic acid-coated magnetic nanocomposites capable of adhering to the cyst wall of Acanthamoeba spp. without causing damage. Experimental results demonstrated that these nanocomposites bind with high affinity to macromolecules on the cyst wall, facilitating the aggregation of parasites in the pellet. Additionally, biologically enriched leech saliva was incorporated into the culture medium to enhance the growth rate. Leech saliva provides a rich source of organic matter and bioactive molecules that promote cell division. The addition of leech saliva resulted in a significant increase in the growth rate of Acanthamoeba spp., with maximum growth density observed at 120 h. Results: These findings indicate that humic acid-coated magnetic nanocomposites and leech saliva -enriched culture media offer a promising alternative to conventional methods for the rapid and efficient cultivation of Acanthamoeba spp. Our study concludes that humic acid-coated magnetic nanocomposites effectively concentrate parasites, increasing their quantitative density, while leech saliva provides a nutrient-rich environment that stimulates trophozoite feeding and division. Conclusion: This study is noteworthy for presenting an innovative and effective method for the rapid laboratory cultivation and potential treatment of Acanthamoeba spp.