Cysticidal Activity of Antifungals against Different Genotypes of Acanthamoeba

Abstract

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

TEXT

Acanthamoeba 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 exists in two forms, a vegetative trophozoite and a dormant cyst form (2). The incidences of AK can differ greatly in different countries, ranging from 0.15 per million in the United States to 1.4 per million in the United Kingdom (1, 2).

The increase in the number of cases of AK that have been reported worldwide in recent years poses a problem with diagnosis, sterilization techniques, contact lens solutions, and the efficacy of antiamoebic treatments (3). Many antiamoebic drugs have poor cysticidal activity, and none of the ones currently in use (biguanides, diamidines) is indeed commercially available (4).

Previous studies have investigated the antiamoebic properties of antifungal drugs, with various results (5–9). Among these compounds, voriconazole has been proven effective in some patients with AK (10–14).

In this study, we tested the cysticidal activity of several antifungal agents against different Acanthamoeba genotypes.

A total of 10 Acanthamoeba clinical isolates from patients with keratitis and 8 culture collection isolates (American Tissue Culture Collection, Manassas, VA) were used for this study. For the clinical isolates, DNA of axenic cultures was extracted using the UNSET or Chelex method and then PCR amplification of the genus-specific ASA:S1 amplicon was used for genotyping (15, 16).

The clinical isolates were all T4 genotypes, whereas the culture collection isolates were classified as genotype T1, T2, T4, T7, T8, T9, T10, or T12 (Table 1). The isolates were grown in peptone-yeast extract-glucose (PYG) flasks under axenic conditions for 2 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-μl aliquots of PYG containing 10⁵ cysts per ml were then prepared by manual counting with a hemocytometer.

TABLE 1.

Acanthamoeba culture collection and clinical isolates used in the study and their genotypes

ATCC isolate	ATCC no.	Genotype	Clinical 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

^aRCL and LCL, right and left contact lens; (R)CB, (right) corneal button; RCS and LCS, right and left corneal scrape.

The Sensititre Yeastone plate (TREK Diagnostic Systems, Cleveland, OH) is a microtiter broth dilution plate for antifungal sensitivity testing (17, 18). The plate contains seven dried antifungal drugs at 2-fold dilutions (Table 2). Serial dilutions of voriconazole and posaconazole (1, 5, 10, 20, 40, 80, 160, and 320 μg/ml) were prepared separately.

TABLE 2.

Antifungals drugs contained in the YeastOne microtiter plate

Antifungal drug	Concn range (μg/ml)
Amphotericin B	0.12–8
5-Flucytosine	0.06–64
Anidulafungin	0.015–8
Caspofungin	0.008–8
Micafungin	0.008–8
Fluconazole	0.12–256
Itraconazole	0.015–16

Cyst aliquots in PYG were incubated with antifungal drugs at 35°C for 48 h. Subsequently, the aliquots were plated on nonnutrient agar (NNA) plates layered with Escherichia coli, incubated at 35°C, and observed with an inverted microscope daily for 1 week to detect growth. Growth was defined as the presence of excysted trophozoites found growing on the culture plates. At the 3-day time point, a fresh inoculum of Escherichia coli (Pro-Lab Diagnostics, Round Rock, TX) (about 900 × 10⁶ CFU/ml; McFarland turbidity standard) was applied on the plates to ensure a robust and sufficient nutrient supply. The minimal cysticidal concentration (MCC) for each drug was defined as the lowest concentration of the drug that resulted in no excystment and trophozoite replication within 7 days of incubation. Experiments were performed in triplicate. Data are expressed as means ± standard deviations. 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 (amphotericin B, 5-flucytosine, anidulafungin, caspofungin, micafungin, fluconazole, and itraconazole) exhibited cysticidal activity at any of the tested concentrations.

The MCCs for voriconazole were 33.13 ± 22.83 μg/ml for clinical isolates and 46.25 ± 23.26 μg/ml for culture collection strains (the mode value for both groups was 40 μg/ml). The MCCs for posaconazole were 43.75 ± 25.04 μg/ml for clinical isolates and 52.5 ± 23.75 μg/ml for culture collection strains (the mode value for both groups was 40 μg/ml) (Fig. 1 and Table 3). No significant differences between clinical and culture collection isolates in the average cysticidal concentrations for voriconazole and posaconazole were found (P > 0.05) (Fig. 1).

FIG 1.

Minimal cysticidal concentration (MCC) for voriconazole (clinical isolates, 33.13 ± 22.83 μg/ml; ATCC isolates, 46.25 ± 23.26 μg/ml; P > 0.05) and posaconazole (clinical isolates, 43.75 ± 25.04 μg/ml; ATCC isolates, 52.5 ± 23.75 μg/ml; P > 0.05). Data are expressed as means ± standard deviations.

TABLE 3.

Voriconazole and posaconazole minimal cysticidal concentration for Acanthamoeba culture collection and clinical isolates^a

ATCC isolate	MCC		Clinical isolate	MCC
	V	P		V	P
A. castellanii	40	40	BP:P7:LCL	40	40
A. pustulosa	10	40	BP:P9:RCL	5	40
A. castellanii Neff	40	80	BP:P10:RCB	20	40
A. astronyxis	40	40	BP:P13:CB	20	80
A. tubiashi	40	80	BP:P3:RCS[2]	20	40
A. comandoni	80	40	BP:P10:RCS	40	80
A. culbertsoni	40	20	BP:P11:RCS	40	10
A. healyi	80	80	BP:P16:RCS	80	20
			BP:P18:LCS	10	20
			BP:P22:LCS	40	20
Mean	46.25	52.50		33.13	43.75
SD	23.26	23.75		22.83	25.04
Mode	40	40		40	40

^aConcentrations are expressed as μg/ml. MCC, minimal cysticidal concentration; V, voriconazole; P, posaconazole.

Our study showed that newer triazoles such as voriconazole and posaconazole do possess cysticidal activity and could represent a potential therapeutic agent for treatment of AK. These drugs inhibit the synthesis of ergosterol, which is a major component of the Acanthamoeba cell membrane. Our results are in line with what previously reported by other authors in previous studies (6, 8, 9). In the studies by Cabello-Vilchez and Martín-Navarro, the voriconazole 90% inhibitory concentration (IC) for cysts was shown to be around 5 to 15 μg/ml for the tested isolates. The higher inhibitory concentration found in our study might be a consequence of the different methodology and the higher number of isolates tested. The cysticidal activity of posaconazole against Acanthamoeba had never previously been tested.

None of the other antifungal drugs tested in the Sensititre Yeastone microdilution plate showed any cysticidal activity. A cysticidal 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 β-glucan, which is expressed at high levels during encystment. In a previous study, caspofungin was cysticidal only at a concentration of 500 μg/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.