Original Article

In Vitro Activity of Pentamidine Isethionate against Trophozoite and Cyst of Acanthamoeba


Background: Acanthamoebae are a causative agent of Acanthamoeba keratitis (AK) in immunocompetent individuals. Since access to propamidine isethionate (Brolene®) as a first-line treatment has been limited in recent years, in the current study, we examined the effects of pentamidine isethionate against trophozoite and cyst forms of Acanthamoeba.

Methods: This experimental study was conducted in the Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, during 2019-2020. Pentamidine isethionate at concentrations of 50, 100, 200, 400, 600, 800, and 1000 µM were tested against trophozoites and cyst stages of T4 genotype, at 24- and 48-hour incubation period, and the viability was determined by trypan blue staining. In addition, the cytotoxic effect of the drug was examined in Vero cells using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.

Results: The 50% inhibitory concentration (IC50) of pentamidine isethionate on trophozoite after 24 and 48h were 97.4 µM and 60.99 µM. These results on cyst after 24 and 48h were 470 µM and 175.5 µM, respectively. In MTT assay, the drug showed an inhibitory effect on Vero cell growth with IC50 values of 115.4 µM and 87.42 µM after 24h and 48h, respectively.

Conclusion: Pentamidine isethionate exhibited an inhibitory effect on trophozoite and cyst. Given that the trophozoicidal activity of the drug is in the safe dose, it could be suggested as an alternative in patients with AK; however, further investigation is needed in an animal model to confirm the data.

1. Siddiqui R, Khan NA. Biology and pathogenesis of Acanthamoeba. Parasites & vectors. 2012;5(1):1-3.
2. Coronado-Velázquez D, Silva-Olivares A, Castro-Muñozledo F, et al. Acanthamoeba mauritaniensis genotype T4D: An environmental isolate displays pathogenic behavior. Parasitol Int. 2020;74:102002.
3. Ledee DR, Iovieno A, Miller D, et al. Molecular identification of T4 and T5 genotypes in isolates from Acanthamoeba keratitis patients. Journal of Clinical Microbiology. 2009;47(5):1458-62.
4. Maghsood AH, Sissons J, Rezaian M, et al. Acanthamoeba genotype T4 from the UK and Iran and isolation of the T2 genotype from clinical isolates. J Med Microbiol. 2005;54(Pt 8):755-759.
5. Carrijo-Carvalho LC, Sant'ana VP, Foronda AS, et al. Therapeutic agents and biocides for ocular infections by free-living amoebae of Acanthamoeba genus. Surv Ophthalmol. 2017;62(2):203-218.
6. Moore MB, McCulley JP, Luckenbach M, et al. Acanthamoeba keratitis associated with soft contact lenses. Am J Ophthalmol. 1985;100(3):396-403.
7. Auran JD, Starr MB, Jakobiec FA. Acanthamoeba keratitis. A review of the literature. Cornea. 1987;6(1):2-26.
8. Casemore DP. Sensitivity of Hartmannella (Acanthamoeba) to 5-fluorocytosine, hydroxystilbamidine, and other substances. J Clin Pathol. 1970;23(8):649-652.
9. Nagington J. Isolation of amoebae from eye infections in England. Trans Ophthalmol Soc U K. 1975;95(2):207-9.
10. Warhurst DC, Stamm WP, Phillips EA. Acanthamoeba from a new case of corneal ulcers. Trans Roy Soc Trop Med and Hyg. 1976;170:279.
11. Duma RJ, Finley R. In vitro susceptibility of pathogenic Naegleria and Acanthamoeba speicies to a variety of therapeutic agents. Antimicrob Agents Chemother. 1976;10(2):370-376.
12. Ferrante A, Rowan-Kelly B, Thong YH. In vitro sensitivity of virulent Acanthamoeba culbertsoni to a variety of drugs and antibiotics. Int J Parasitol. 1984;14(1):53-6.
13. Moore MB. Acanthamoeba keratitis. Arch Ophthalmol. 1988;106(9):1181-3.
14. Moore MB, McCulley JP. Acanthamoeba keratitis associated with contact lenses: six consecutive cases of successful management. Br J Ophthalmol. 1989;73(4):271-275.
15. Moore MB, McCulley JP, Kaufman HE, et al. Radial keratoneuritis as a presenting sign in Acanthamoeba keratitis. Middle East Afr J Ophthalmol. 2011;18(3):252-5.
16. Moore MB, McCulley JP, Newton C, et al. Acanthamoeba keratitis: a growing problem in soft and hard contact lens wearers. Ophthalmology. 1987;94:1654-61.
17. Hajialilo E, Rezaeian M, Niyyati M, et al. Molecular characterization of bacterial, viral and fungal endosymbionts of Acanthamoeba isolates in keratitis patients of Iran. Exp Parasitol. 2019. 200:48-54.
18. Khan NA. Pathogenicity, morphology, and differentiation of Acanthamoeba. Curr Microbiol. 2001;43(6):391-5.
19. Walochnik J, Obwaller A, Gruber F, et al. Anti-Acanthamoeba efficacy and toxicity of miltefosine in an organotypic skin equivalent. J Antimicrob Chemother. 2009;64(3):539-45.
20. Alizadeh H, Silvany RE, Meyer DR, et al. In vitro amoebicidal activity of propamidine and pentamidine isethionate against Acanthamoeba species and toxicity to corneal tissues. Cornea. 1997;16(1):94-100.
21. Huang, J, Ozaki, H, Umeda N, et al. Clinical outcomes and prognostic factors associated with Acanthamoeba keratitis treated with pentamidine isethionate. Invest Ophthalmol Vis Sci. 2013;54(15):879.
22. Siddiqui R, Syed A, Tomas S, et al. Effect of free versus liposomal-complexed pentamidine isethionate on biological characteristics of Acanthamoeba castellanii in vitro. J Med Microbiol. 2009;58(3):327-330.
IssueVol 16 No 4 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijpa.v16i4.7868
Acanthamoeba Pentamidine Isethionate Trophozoite Cyst In vitro

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Behnia M, Latifi A, Rezaian M, Kharazi S, Mohebali M, Yasami S, Saghafi S, Chahardoli R, Anasori N, Torkian H, Soleimani M, Niyyati M, Kazemirad E. In Vitro Activity of Pentamidine Isethionate against Trophozoite and Cyst of Acanthamoeba. Iran J Parasitol. 2021;16(4):560-566.