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In vitro susceptibility of Acanthamoeba to antifungal drugs

In vitro susceptibility of Acanthamoeba to antifungal drugs. A. Iovieno, MD; D. Miller, DHSc ; E.C. Alfonso, MD. Bascom Palmer Eye Institute University of Miami - Miller School of Medicine . Authors have no financial interest. Introduction.

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In vitro susceptibility of Acanthamoeba to antifungal drugs

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  1. In vitro susceptibility of Acanthamoeba to antifungal drugs A. Iovieno, MD; D. Miller, DHSc; E.C. Alfonso, MD Bascom Palmer Eye Institute University of Miami - Miller School of Medicine Authors have no financial interest

  2. Introduction • Acanthamoebakeratitis (AK) is a painful, sight-threatening and recalcitrant corneal infection caused by pathogenic Acanthamoeba • Acanthamoeba is a free-living ubiquitous protozoa, and exists in a vegetative (trophozoite) and a resistant cystic form CYST TROPHOZOITE • Acanthamoeba has recently been classified in 15 genotypes (T1-T15) based on 18S rRNA sequencing. Pathogenic Acanthamoebae are usually T4 genotype, with few exceptions identified as T3, T5, T6, T11, and T15 (Chong, 2007; Di Cave ,2008; Khan, 2000; Ledee , 1996; Spanakos , 2006; Stothard , 1998; Visvesvara, 2007)

  3. Standard anti-amoebic medications (diamidines, biguanides) have low penetration in ocular tissues and are highly toxic • Several antifungal drugs have been tested in vitro and in vivo against Acanthamoeba with contradictory results • The presence of ergosterol in the amoeba membrane would theorically justify the use of triazole drugs (ergosterol synthesis inhibitors) against Acanthamoeba • In particular, voriconazole, a new triazole compound, has been suggested as an additional therapy for AK (Bouyer ,2007; Ishibashi, 1990; Lee, 2007; Masselam, 2008; Rain, 1996; Schuster, 2006)

  4. Purpose • To test the cysticidal properties of several antifungal drugs against clinical and environmental isolates of Acanthamoeba • To analyze the influence of amoeba genotype on the susceptibility to new triazole antifungal medications (voriconazole, posaconazole)

  5. Methods • 15 Acanthamoeba isolates (100 µl of a 104/ml solution of cysts) obtained from AK patients were exposed for 48 hours to increasing concentrations of 8 different commercially available antifungals on a YeastOne® assay 96 well-plate

  6. The Minimal Cysticidal Concentration (MCC) was defined as the lowest concentration of drug that resulted in no excistment and trophozoites replication in 10 days • MCC was assessed by using YeastOne® colorimetric assay • Viability of amoeba isolates was further tested by plating the isolates on non-nutrient agar-agar plates layered with Escherichia Coli

  7. The cysticidal activity of high concentration voriconazole and posaconazole ( 1-320 µg/ml) was tested on 2 clinical T4 genotype isolates (T4-1 and T4-2) and 2 non-pathogenic environmental isolates (A. pustulosa, T2 genotype; A. tubiashii, T8 genotype) A. tubiashii – T8 A. pustulosa – T2 Clinical isolate – T4

  8. Results • None of the tested antifungal drugs in Yeastone ® plate at any concentration was able to efficiently inhibit Acanthamoebaexcistment and growth • Higher concentrations of posaconazole showed no effect on Acanthamoeba clinical isolates; voriconazolewas cysticidal in T4-2 isolate only at high concentrations (MCC: 180 µg/ml) • Cysts of environmental isolates were more susceptible in vitro to triazoles ( A. pustulosa: voriconazole MCC 40 µg/ml, posaconazole MCC 20 µg/ml; A. tubiashii: voriconazole MCC 80 µg/ml, posaconazole MCC 20 µg/ml)

  9. T4-1 Posaconazole, 320 µg/ml Voriconazole, 320 µg/ml T4-2 Posaconazole, 320 µg/ml Voriconazole, 180 µg/ml Viable trophozoite (green arrow) were recovered from high concentration voriconazole and posaconazole in clinical isolates. Notice motility of trophozoites from the trackings in agar-agar (red arrow)

  10. A. pustolosa Voriconazole, 40 µg/ml Posaconazole, 20 µg/ml A. tubiashii Voriconazole, 80 µg/ml Posaconazole, 20 µg/ml Environmental isolates were more sensitive to antifungal drugs. Notice co-existence of cysts (red arrow) and trophozoite (green arrow)

  11. Conclusions • Clinical isolates of Acanthamoeba were highly resistant to commercially available antifungal drugs, including new triazoles • Voriconazole and posaconazole exhibited cysticidal activity against environmental non-pathogenic isolates at high concentrations • Acanthamoeba genotype influences not only pathogenicity but may determine resistance to medical treatment • Further studies are needed to analyze the molecular and genetic bases of antifungals resistance.

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