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

2. Introduction Acanthamoeba keratitis (AK) is a painful, sight-threatening and recalcitrant corneal infection caused by pathogenic Acanthamoeba (Chong, 2007; Di Cave,2008; Khan, 2000; Ledee, 1996; Spanakos, 2006; Stothard, 1998; Visvesvara, 2007) 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 Acanthamoeba is a free-living ubiquitous protozoa, and exists in a vegetative (trophozoite) and a resistant cystic form CYST TROPHOZOITE

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 10 4 /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 DrugConcentration range (µg/ml) anidulafungin0.015-8 micafungin0.008-8 caspofungin0.008-8 5-flucytosine0.6-64 posaconazole0.008-8 voriconazole0.008-8 itraconazole0.015-16 fluconazole0.12-256 amphotericin B0.12-8

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. pustulosa – T2 A. tubiashii – T8 Clinical isolate – T4

8. Results None of the tested antifungal drugs in Yeastone ® plate at any concentration was able to efficiently inhibit Acanthamoeba excistment and growth Higher concentrations of posaconazole showed no effect on Acanthamoeba clinical isolates; voriconazole was 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 T4-2 Voriconazole, 320 µg/ml Voriconazole, 180 µg/ml Posaconazole, 320 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 A. tubiashii Voriconazole, 40 µg/ml 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.