Cysticidal Activity of Antifungals against Different Genotypes of

Acanthamoeba
Alfonso Iovieno,a,b Darlene Miller,a Dolena R. Ledee,a Eduardo C. Alfonso,a
Bascom Palmer Eye Institute, University of Miami—Miller School of Medicine, Miami, Florida, USAa; Department of Ophthalmology, Arcispedale Santa Maria
Nuova—IRCCS, Reggio Emilia, Italyb

Antifungal drugs have been proposed as a novel treatment for Acanthamoeba keratitis. The cysticidal activity of several antifun-
gal compounds was tested against different genotypes of culture collection and clinical isolates of Acanthamoeba. Only voricona-
zole and posaconazole were found to be cysticidal, with no differences in activity observed between clinical and culture collec-
tion isolates.

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A canthamoeba keratitis (AK) is a rare and sight-threatening
corneal infection that affects primarily contact lens wearers
(1). Acanthamoeba is a ubiquitous free-living protozoan that ex-
weeks to obtain complete encystment. PYG flasks were then
placed on ice for 10 s and gently shaken to dislodge the cysts from
the flask walls, and 0.100-␮l aliquots of PYG containing 105 cysts
ists in two forms, a vegetative trophozoite and a dormant cyst per ml were then prepared by manual counting with a hemocy-
form (2). The incidences of AK can differ greatly in different coun- tometer.
tries, ranging from 0.15 per million in the United States to 1.4 per The Sensititre Yeastone plate (TREK Diagnostic Systems,
million in the United Kingdom (1, 2). Cleveland, OH) is a microtiter broth dilution plate for antifungal
The increase in the number of cases of AK that have been re- sensitivity testing (17, 18). The plate contains seven dried antifun-
ported worldwide in recent years poses a problem with diagnosis, gal drugs at 2-fold dilutions (Table 2). Serial dilutions of voricona-
sterilization techniques, contact lens solutions, and the efficacy of zole and posaconazole (1, 5, 10, 20, 40, 80, 160, and 320 ␮g/ml)
antiamoebic treatments (3). Many antiamoebic drugs have poor were prepared separately.
cysticidal activity, and none of the ones currently in use (bigua- Cyst aliquots in PYG were incubated with antifungal drugs at
nides, diamidines) is indeed commercially available (4). 35°C for 48 h. Subsequently, the aliquots were plated on nonnu-
Previous studies have investigated the antiamoebic properties trient agar (NNA) plates layered with Escherichia coli, incubated at
of antifungal drugs, with various results (5–9). Among these com- 35°C, and observed with an inverted microscope daily for 1
pounds, voriconazole has been proven effective in some patients week to detect growth. Growth was defined as the presence of
with AK (10–14). excysted trophozoites found growing on the culture plates. At the
In this study, we tested the cysticidal activity of several antifun- 3-day time point, a fresh inoculum of Escherichia coli (Pro-Lab
gal agents against different Acanthamoeba genotypes. Diagnostics, Round Rock, TX) (about 900 ⫻ 106 CFU/ml; McFar-
A total of 10 Acanthamoeba clinical isolates from patients with land turbidity standard) was applied on the plates to ensure a
keratitis and 8 culture collection isolates (American Tissue Cul-
ture Collection, Manassas, VA) were used for this study. For the
clinical isolates, DNA of axenic cultures was extracted using the Received 24 February 2014 Returned for modification 21 March 2014
UNSET or Chelex method and then PCR amplification of the ge- Accepted 1 July 2014
nus-specific ASA:S1 amplicon was used for genotyping (15, 16). Published ahead of print 7 July 2014
The clinical isolates were all T4 genotypes, whereas the culture Address correspondence to Alfonso Iovieno, alfonsoiovieno@hotmail.com.
collection isolates were classified as genotype T1, T2, T4, T7, T8, Copyright © 2014, American Society for Microbiology. All Rights Reserved.
T9, T10, or T12 (Table 1). The isolates were grown in peptone- doi:10.1128/AAC.02635-14
yeast extract-glucose (PYG) flasks under axenic conditions for 2
TABLE 1 Acanthamoeba culture collection and clinical isolates used in the study and their genotypes
Clinical
ATCC isolate ATCC no. Genotype isolatea Source Genotype
A. castellanii 50494 T1 BP:P7:LCL Contact lens T4
A. pustulosa 50252 T2 BP:P9:RCL Contact lens T4
A. castellanii Neff 30010 T4 BP:P10:RCB Corneal button T4
A. astronyxis 30137 T7 BP:P13:CB Corneal button T4
A. tubyiashi 30867 T8 BP:P3:RCS[2] Corneal scrape T4
A. comandoni 30135 T9 BP:P10:RCS Corneal scrape T4
A. culbertsoni 30171 T10 BP:P11:RCS Corneal scrape T4
A. healyi 1283 T12 BP:P16:RCS Corneal scrape T4
BP:P18:LCS Corneal scrape T4
BP:P22:LCS Corneal scrape T4
a
RCL and LCL, right and left contact lens; (R)CB, (right) corneal button; RCS and LCS, right and left corneal scrape.

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 Acanthamoeba Antifungal Sensitivity

TABLE 2 Antifungals drugs contained in the YeastOne microtiter plate TABLE 3 Voriconazole and posaconazole minimal cysticidal
Antifungal drug Concn range (␮g/ml)
concentration for Acanthamoeba culture collection and clinical isolatesa

Amphotericin B 0.12–8 MCC MCC

Clinical
5-Flucytosine 0.06–64 ATCC isolate V P isolate V P
Anidulafungin 0.015–8
A. castellanii 40 40 BP:P7:LCL 40 40
Caspofungin 0.008–8
A. pustulosa 10 40 BP:P9:RCL 5 40
Micafungin 0.008–8
A. castellanii Neff 40 80 BP:P10:RCB 20 40
Fluconazole 0.12–256
A. astronyxis 40 40 BP:P13:CB 20 80
Itraconazole 0.015–16
A. tubiashi 40 80 BP:P3:RCS[2] 20 40
A. comandoni 80 40 BP:P10:RCS 40 80
robust and sufficient nutrient supply. The minimal cysticidal con- A. culbertsoni 40 20 BP:P11:RCS 40 10
centration (MCC) for each drug was defined as the lowest concen- A. healyi 80 80 BP:P16:RCS 80 20
tration of the drug that resulted in no excystment and trophozoite BP:P18:LCS 10 20
replication within 7 days of incubation. Experiments were per- BP:P22:LCS 40 20
formed in triplicate. Data are expressed as means ⫾ standard de-

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viations. A t test for unpaired data was used to compare MCC
values among groups (SPSS software, SPSS Inc., Chicago, IL).
None of the antifungals in the Sensititre Yeastone plate (am-
photericin B, 5-flucytosine, anidulafungin, caspofungin, mica-
fungin, fluconazole, and itraconazole) exhibited cysticidal activity
at any of the tested concentrations.
The MCCs for voriconazole were 33.13 ⫾ 22.83 ␮g/ml for
clinical isolates and 46.25 ⫾ 23.26 ␮g/ml for culture collection
strains (the mode value for both groups was 40 ␮g/ml). The MCCs
for posaconazole were 43.75 ⫾ 25.04 ␮g/ml for clinical isolates
and 52.5 ⫾ 23.75 ␮g/ml for culture collection strains (the mode
value for both groups was 40 ␮g/ml) (Fig. 1 and Table 3). No
significant differences between clinical and culture collection iso-
lates in the average cysticidal concentrations for voriconazole and
posaconazole were found (P ⬎ 0.05) (Fig. 1).
Our study showed that newer triazoles such as voriconazole
and posaconazole do possess cysticidal activity and could repre-
sent a potential therapeutic agent for treatment of AK. These
drugs inhibit the synthesis of ergosterol, which is a major compo-
nent of the Acanthamoeba cell membrane. Our results are in line
with what previously reported by other authors in previous stud-
ies (6, 8, 9). In the studies by Cabello-Vilchez and Martín-Na-
varro, the voriconazole 90% inhibitory concentration (IC) for
cysts was shown to be around 5 to 15 ␮g/ml for the tested isolates.
The higher inhibitory concentration found in our study might be
a consequence of the different methodology and the higher num-
ber of isolates tested. The cysticidal activity of posaconazole
against Acanthamoeba had never previously been tested.
FIG 1 Minimal cysticidal concentration (MCC) for voriconazole (clinical
isolates, 33.13 ⫾ 22.83 ␮g/ml; ATCC isolates, 46.25 ⫾ 23.26 ␮g/ml; P ⬎ 0.05)
and posaconazole (clinical isolates, 43.75 ⫾ 25.04 ␮g/ml; ATCC isolates,
52.5 ⫾ 23.75 ␮g/ml; P ⬎ 0.05). Data are expressed as means ⫾ standard
deviations.
Mean 46.25 52.50 33.13 43.75
SD 23.26 23.75 22.83 25.04
Mode 40 40 40 40
a
Concentrations are expressed as ␮g/ml. MCC, minimal cysticidal concentration; V,
voriconazole; P, posaconazole.
None of the other antifungal drugs tested in the Sensititre Yeas-
tone microdilution plate showed any cysticidal activity. A cysti-
cidal effect could have been evident only at concentrations much
higher than the ones resulting in activity against filamentary fungi
or yeasts. Among all the antifungal drugs, only caspofungin was
previously identified as a potential antiamoebic agent and tested
against cysts in vitro. Echinocandins impair the synthesis of ␤-glu-
can, which is expressed at high levels during encystment. In a
previous study, caspofungin was cysticidal only at a concentration
of 500 ␮g/ml (7).
In conclusion, our in vitro findings support the potential use of
newer triazoles as antiamoebic agents. Further in vivo studies
would be needed to confirm the potential role of these compounds
in the treatment of AK.
ACKNOWLEDGMENT
None of us has any financial interests relevant to this study.
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