Download Acanthamoeba - Posters On Demand 2009

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
 Acanthamoeba keratitis (AK) is a
painful, sight-threatening and
recalcitrant corneal infection
caused by pathogenic
Acanthamoeba
 Acanthamoeba is a free-living
CYST
TROPHOZOITE
ubiquitous protozoa, and exists in
a vegetative (trophozoite) and a
resistant cystic form
 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)
 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)
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)
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
Drug
Concentration range (µg/ml)
anidulafungin
0.015-8
micafungin
0.008-8
caspofungin
0.008-8
5-flucytosine
0.6-64
posaconazole
0.008-8
voriconazole
0.008-8
itraconazole
0.015-16
fluconazole
0.12-256
amphotericin B
0.12-8
 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
 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
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)
T4-1
Posaconazole, 320 µg/ml
Voriconazole, 180 µg/ml
Posaconazole, 320 µg/ml
T4-2
Voriconazole, 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 agaragar (red arrow)
A. pustolosa
Posaconazole, 20 µg/ml
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)
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.