Experimental Parasitology 197 (2019) 29–35

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Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr

Evaluation of the sensitivity to chlorhexidine, voriconazole and itraconazole T

of T4 genotype Acanthamoeba isolated from Mexico
Dolores Hernández-Martíneza, María Reyes-Batlleb, Ismael Castelan-Ramíreza,
Perla Hernández-Olmosa, Virginia Vanzzini-Zagoc, Elizabeth Ramírez-Floresa, Inés Sifaouib,
José E. Piñerob, Jacob Lorenzo-Moralesb, Maritza Omaña-Molinaa,∗
a
Faculty of Superior Studies Iztacala, UNAM. Tlalnepantla, State of Mexico, Mexico
b
University Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, Tenerife, Canary Island, Spain
c
Hospital Para Evitar la Ceguera en México, “Luis Sánchez Bulnes”, Mexico

A R T I C LE I N FO A B S T R A C T

Keywords: Free-living amoebae of the genus Acanthamoeba are the etiological agents of cutaneous lesions, granulomatous
Acanthamoeba, Mexico amoebic encephalitis (GAE) and amoebic keratitis (AK), which are chronic infections with poor prognosis if not
Chemotherapy assays diagnosed promptly. Currently, there is no optimal therapeutic scheme to eradicate the pathologies these pro-
Voriconazole tozoa cause. In this study we report the morphological and molecular identification of three species of the genus
Chlorhexidine
Acanthamoeba, belonging to T4 group; A. polyphaga isolated from the corneal ulcer of a patient sample of AK
Itraconazole
case; A. castellanii isolated from the contact lens of an AK patient and A. palestinensis obtained from a soil sample.
The in vitro activity of chlorhexidine, itraconazole and voriconazole drugs against trophic stage was also eval-
uated through a colorimetric assay based on the oxidation-reduction of alamar blue. The strains in the study
were sensitive to the evaluated drugs; although when determining the 50% inhibitory concentration (IC50)
statistically significant differences were observed. A. castellanii showed to be highly sensitive to voriconazole
(0.66 ± 0.13 μM) but the least sensitive to chlorhexidine and itraconazole (8.61 ± 1.63 and 20.14 ± 4.93 μM,
respectively), A. palestinensis showed the highest sensitivity to itraconazole (0.502 ± 0.11 μM) and A. polyphaga
expressed moderate sensitivity to chlorhexidine and itraconazole and lower sensitivity to voriconazole
(10.10 ± 2.21 μM). These results showed that species of the genus Acanthamoeba express different sensitivity to
the tested drugs, which could explain the problems surrounding the establishment of a treatment of choice in the
infections caused by these amoebae. We consider that although chlorhexidine and itraconazole show good ac-
tivity on these amoebae and have been used in cases of AK in Mexico with acceptable results, voriconazole
should be considered as the first therapeutic option of future Acanthamoeba infections that will be diagnosed in
our country.

1. Introduction identified using morphological features (Page, 1988), molecular clas-

Free living amoebae of the genus Acanthamoeba are ubiquitously
distributed in the environment and have been isolated worldwide from
natural habitats such as soil, water-bodies, as well as man-made en-
vironments (Marciano-Cabral and Cabral, 2003). These amoebae play
an important ecological and clinical role, since they control bacterial
populations (Bryant et al., 1982), and are the etiological agents of
human and animal pathologies. Acanthamoeba species have two stages
in its life cycle, an active trophozoite stage that exhibits vegetative
growth and a highly resistant cyst stage with minimal metabolic ac-
tivity (Visvesvara et al., 2007). Though, Acanthamoeba strains can be
sification of this genus is now widely accepted and 20 genotypes are
known so far; T1–T20 based on the analysis sequence differences of the
diagnostic fragment 3 region of the 18S rRNA gene (Stothard et al.,
1998; Visvesvara et al., 2007; Nuprasert et al., 2010; Corsaro et al.,
2015). Until now, of the 20 genotypes, T4 is the most abundant and
includes pathogenic strains (Lorenzo-Morales et al., 2015). Ad-
ditionally, others genotypes such as T3, T11, and T13 have also been
related to eye infection (Scheid et al., 2008; Lorenzo-Morales et al.,
2011; Grün et al., 2014; Derda et al., 2015; Heredero-Bermejo et al.,
2015; Omaña-Molina et al., 2016).
It is well known that several Acanthamoeba species are the

∗
Corresponding author. UNAM FES Iztacala, Avenida de los Barrios No. 1. Los Reyes Iztacala, Tlalnepantla, Mexico State, 54090, Mexico.
E-mail address: maritzaomanam@yahoo.com.mx (M. Omaña-Molina).

https://doi.org/10.1016/j.exppara.2019.01.006
Received 11 October 2018; Received in revised form 12 December 2018; Accepted 11 January 2019
Available online 12 January 2019
0014-4894/ © 2019 Published by Elsevier Inc.
D. Hernández-Martínez et al.
etiological agents of cutaneous lesions, sinus infections and granulo-
matous amoebic encephalitis (GAE), a disease with close to 90% mor-
tality. These amoebae also provoke a vision-threatening amoebic ker-
atitis (Marciano-Cabral and Cabral, 2003; Khan, 2006; Visvesvara et al.,
2007). An early diagnosis followed by an effective treatment is essential
to assure a successful prognosis. However, in many cases infections
caused by Acanthamoeba are difficult to treat, usually because the
clinical course is not specific, it can easily be confused with bacterial or
viral infections, not all laboratories implement reliable diagnostic tests
(Visvesvara, 2013), and these problems are compounded by the limited
efficacy of anti-amoebic drugs to cross the blood-brain barrier
(Heredero-Bermejo et al., 2016). In addition to, the virulence of each
species, the high resistance of their cysts forms (Perez-Santonja et al.,
2003; Siddiqui and Khan, 2012; Lorenzo-Morales et al., 2013) as well as
differences in the sensitivity of strains to the diverse drugs used
(Martín-Navarro et al., 2008, 2013; Cabello-Vílchez et al., 2014).
Several therapeutic agents have been used alone or in several
combinations schemes, but none of these treatments have proven to be
always effective or some of them are not recommended because of their
adverse side effects (Schuster and Visvesvara, 2004). Nevertheless good
results have been obtained in patients with cutaneous infections treated
with chlorhexidine gluconate, topical ketoconazole and itraconazole,
amphotericin B, voriconazole, pentamidine and 5-fluorocytosine
(Martínez, 1985; Slater et al., 1994; Oliva et al., 1999; Walia et al.,
2007). In GAE cases some of the drugs prescribed include, pentamidine
B, pentamidine isethionate, sulfadiazine, flucytosine, fluconazole, tri-
methoprim-sulfamethoxazole, rifampicin and miltefosine (Martínez,
1985; Martínez and Visvesvara, 1997; Aichelburg et al., 2008; Webster
et al., 2012). The combined prescription of these drugs, mainly in some
immunocompetent patients, has allowed the remission of the infection
(Nehete et al., 2018). Consequently, in patients with AK, some of these
drugs from the group of pentamidines, azoles, antibiotics and bigua-
nides have also been successfully used; polyhexamethylene biguanide
(PHMB) is used as a disinfectant in some cleaning contact lenses solu-
tions (Schuster and Visvesvara, 2004). However, although the use of
some of these drugs has been reported with good results, in some cases,
treatments are prolonged, consequently, the need for searching drugs
and therapeutic schemes of choice in infections caused by Acantha-
moeba is necessary.
In Mexico studies have been conducted to determine the presence
and distribution of potentially pathogenic free-living amoebae from
different habitats, reporting the presence of Acanthamoeba spp. from
diverse aquatic environments (Rivera et al., 1983, 1989; 1993; Bonilla-
Lemus et al., 2010, 2014; Ramírez et al., 2014), the atmosphere (Rivera
et al., 1994) and even from the nasal cavity and nasopharynx of dental
patients (Rivera et al., 1986). In the medical area there are only two
cases reported of GAE infection caused by Acanthamoeba; one of them
correspond to a child with a mixed cerebral infection caused by Acan-
thamoeba sp. and Aspergillus sp. who died in spite of being treated with
amphotericin B, clarithromizine, ketoconazole, pentamidine, flucyto-
sine, fluconazole and transfer factor. In the second case an adult patient
who was treated with rifampicin, trimethoprim-sulfamethoxazole, flu-
conazole and metronidazole survived the infection of these amoebae
(Becerril, 2014). In 2016, Omaña-Molina et al. reported two cases of
AK; in which A. griffini and A. royreba were the ethiological agents. The
drugs prescribed were chlorhexidine in the first case and topical ne-
tilmicin and oral itraconazole in the second; in both cases the treat-
ments were prolonged for several months with good prognosis.
Currently, chlorhexidine, itraconazole and voriconazole are drugs
that are used and recommended in other countries for Acanthamoeba
pathologies (Arnalich-Montiel et al., 2012; Cabello-Vílchez et al., 2014;
Martín-Navarro et al., 2013; Rocha-Cabrera et al., 2015). In Mexico
these therapeutic schemes are not always considered as the first line of
treatment. In this work we report the morphological and molecular
identification of 3 strains of Acanthamoeba isolated from different
sources in Mexico and evaluate their sensitivity in vitro to
30
Experimental Parasitology 197 (2019) 29–35
chlorhexidine, itraconazole and voriconazole.
2. Material and methods
2.1. Acanthamoeba in study
This work was carried out with three strains of the genus
Acanthamoeba previously isolated from different sources. Two of these
were obtained from “El Hospital para Prevenir la Ceguera en México,
Luis Sánchez Bulnes”. Strain A was obtained from a corneal ulcer of an
AK patient contact lens user, strain B was isolated from contact lens of
an AK patient and strain C was isolated from a soil sample. The
amoebae were grown in axenic conditions in PYG medium (0.75% (w/
v) proteose-peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glu-
cose) supplemented with 40 μg/ml of gentamicin and incubated at 30 °C
(optimal temperature) in culture bottles of 25 cm2. The subsequent
assays were performed with trophozoites harvested at the end of the
logarithmic phase of growth.
2.2. Morphological identification
Amoebae were identified taking in account morphological char-
acterizes of trophozoite and cyst, using the morphological taxonomic
criteria of Page (1988).
2.3. Molecular identification
Acanthamoeba DNA extraction was obtained by placing 1–2 mL of
axenic cultures directly into the Maxwell 16 Tissue DNA Purification Kit
sample cartridge (Promega). Genomic DNA was purified using the
Maxwell 16 Instrument as described in the Maxwell 16 DNA
Purification Kits. Technical Manual #TM284 (Promega). DNA yield and
purity were determined using the Nano Drop 1000 spectrophotometer
(Fisher Scientific). DNA amplification reactions were performed using
genus-specific markers for Acanthamoeba. A volume of 30 μl containing
approximately 40 ng template DNA, buffer (1 × ) without MgCl2,
2.5 mM MgCl2, 200 μM dNTP, 2.5 pmol of each primer pair, and 1.25
units of Taq DNA polymerase (Applied Biosystems, NJ), pH 8.3, was
used for amplification in an Artik Cycler Thermocycler (Thermo
Scientific). The cycling conditions were as follows: initial denaturation
of 95 °C for 5 min; 40 repetitions of denaturation at 95 °C for 45 s, an-
nealing phase at 50 °C for 45 s, and elongation at 72 °C for 45 s; and
final elongation at 72 °C for 7 min. Amplification products were frac-
tionated using 2% agarose electrophoresis stained with a solution of
20,000 × of REALSAFE Nucleic Acid Staining Solution (Durviz, Madrid,
Spain) and visualized under UV light. A type strain, Acanthamoeba
castellanii Neff ATCC 30010 from the American Type Culture Collection
(ATCC), was used as a positive control and distilled water added to the
reaction mixture (instead of DNA) as the negative control.
Genotyping of the amoebic strain was performed by amplification of
the Diagnostic Fragment 3 (DF3 region) of the rRNA gene as previously
described (Booton et al., 2002; Niyyati et al., 2009). PCR products were
purified using the Qiaquick PCR purification kit (Qiagen, Hilden, Ger-
many) and sequenced using a MegaBACE 1000 automatic sequencer
(Healthcare Biosciences, Barcelona, Spain) in the University of La La-
guna Sequencing Services (Servicio de Secuenciación SEGAI, University
of La Laguna, Spain). The obtained sequences were aligned using Mega
5.0 software program. Genotype identification was based on sequence
analysis of the DF3 region as previously described by comparison with
the available Acanthamoeba DNA sequences in the GenBank database.
2.4. Drug susceptibility testing
The activity of chlorhexidine, voriconazole and itraconazole, used
against infections caused by Acanthamoeba spp., was evaluated at dif-
ferent concentrations against the strains of studied Acanthamoeba.
D. Hernández-Martínez et al.
Chlorhexidine (chlorhexidine digluconate, Alfa Aesar, Germany) is a
standard antiseptic belonging to the biguanide family that is commonly
used in contact lens maintenance solutions. Itraconazole and vor-
iconazole (Sigma) are inhibitors of ergosterol synthesis that has been
highly effective against clinical strains of Acanthamoeba (Arnalich-
Montiel et al., 2012; Cabello-Vílchez et al., 2014; Martín-Navarro et al.,
2013; Rocha-Cabrera et al., 2015). The activity of the drugs was based
on the oxido-reduction of Alamar Blue assay (McBride et al., 2005;
Martín-Navarro et al., 2008), it was used for the determination of drug
efficacy against the trophozoite stage of the Acanthamoeba strains.
Briefly, 2 × 104 trophozoites of the strains under study were placed in
96-well plates in PYG medium, allowed to incubate for 3 h and the
drugs were freshly prepared and added at a concentration range of 100
to 0.15 μM to the wells containing cells; these concentrations were
chosen as a routine standard in our laboratory as per described in
Martín-Navarro et al., (2008). The test plates were incubated for 96 h
under normal culture conditions. At 6 h prior to the end of incubation,
10 μl of Alamar Blue reagent was added to each of the test wells of the
96-well plate. Subsequently, the plates were analyzed on an EnSpire
Multimode Plate Reader (Perkin Elmer) using a test wavelength of
570 nm and a reference wavelength of 620 nm. The percentage of in-
hibition and median inhibitory concentration (IC50) were calculated by
linear regression analysis using a 95% confidence limit. All experiments
were performed three times each in triplicate and the mean values were
derived. Two-way analysis of variance and Tukey test were used for
analysis of the data. Values of P < 0.05 were considered significant.
The inhibition curves of the statistical analysis were developed using
Sigma Plot 12.0 software program (Systat Software).
3. Results
3.1. Morphological identification
Amoebae isolated from different source were grown in axenic con-
ditions in PYG medium and were morphologically identified as A.
polyphaga (A), A. castellanii (B) and A. palestinensis (C) (Fig. 1).
3.2. Molecular identification
PCR amplification and sequence analysis of the DF3 of the
Acanthamoeba small subunit rRNA gene revealed that the three
Acanthamoeba strains belong to genotype T4. These strains showed >
95% homology of the DF3 region when compared with the other
Acanthamoeba genotype T4 sequences available in the GenBank data-
base.
3.3. Drug susceptibility testing
A. castellanii, A. polyphaga and A. palestinensis were sensitive to
chlorhexidine, itraconazole and voriconazole (Fig. 2A). Of the nine
determinations made, eight had an IC50 less than 10.1 μM. The most
active compounds were voriconazole for A. castellanii and A. palesti-
nensis, while itraconazole for A. palestinensis and A. polyphaga, with an
IC50 between 0.502 and 3.311 μM. Regarding chlorhexidine our results
showed promising activity with IC50 between 4.76 and 8.61 μM for
three species evaluated. Microscopic analysis of the in vitro interaction
of the amoebae with the drugs showed that the IC50 induced the en-
cystment of a small amount of trophozoites (unquantified data). How-
ever, as the concentration of the drugs increased, the total destruction
of the trophozoites was observed.
The IC50 obtained from pharmaceutical products evaluated showed
significant differences depending on the drug and/or the amoebae
(Fig. 2B). For example, A. castellanii showed a significantly higher
sensitivity to voriconazole in comparison to other evaluated drugs.
Contrary, A. polyphaga was significantly less sensitive to voriconazole.
The analysis of the sensitivity of A. palestinensis to voriconazole and
31
Experimental Parasitology 197 (2019) 29–35
itraconazole indicated that there was no significant difference between
them (p > 0.05, Tukey test). Besides, A. palestinensis was significantly
more sensitive to chlorhexidine as compared to the other species. A.
palestinensis and A. polyphaga are considerably more sensitive to itra-
conazole than A. castellanii, which, in opposition, showed little sensi-
tivity to this drug, with an IC50 of 20.114 μM, the highest of all de-
terminations.
4. Discussion
In this study we describe the morphological and molecular identi-
fication of three species of the genus Acanthamoeba, as well as the
evaluation of the effect of drugs on these. The strains used in this study
belong to T4 genotype and were morphologically identified as A.
polyphaga, A. castellanii and A. palestinensis which were sensitive to the
3 drugs evaluated; with differences in the mean inhibitory concentra-
tions (IC50) and sensitivity to the drugs.
A. polyphaga and A. castellanii, isolated from corneal ulcer and
contact lenses of patients with AK, are species commonly found en-
vironmentally as well as etiological agent of human pathologies. Both
species have been related with GAE cases (Visvesvara et al., 2007), and
have been reported as the main etiological agents of AK (Dart et al.,
2009). Historically, A. polyphaga was the specie reported in the first
case of AK in the United States (Jones et al., 1975). Contrarily, few
cases of GAE and AK are related with A. palestinensis (Willaert and
Stevens, 1976; Maghsood et al., 2005). Nevertheless, it is important the
study these strains, considering their pathogenic potential of amoebae
of this genus; despite its lower abundance in the environment or low
incidence in clinical cases.
In addition to the morphological diagnosis, sequencing of the 18S
rRNA gene is currently used to differentiate Acanthamoeba strains and
to establish their phylogenetic relationships. The strains in this study
were placed in the T4 genotype. Strains of Acanthamoeba isolated from
clinical cases and the environment are frequently situated in this gen-
otype (Booton et al., 2002, 2005; Zhang et al., 2004; Cabello-Vílchez
et al., 2013; Rocha-Cabrera et al., 2015; Wagner et al., 2016). This
suggests that this group has a greater abundance in the environment
and a greater virulence. Nevertheless, Arnalich-Montiel et al. (2014)
reported that AK cases of non-T4 genotype, T2, T3, T7 and T11, did not
show a good response to medical therapy, and needed surgical inter-
vention. The effect of these genotypes resulted in extra corneal inva-
sion, as well as a remarkably poor visual outcome compared with those
of T4 genotype. Also, in a study comparing sensitivity to multi-purpose
solutions for contact lens of T3, T4, T5 and T11 isolates, non-T4 types
were more resistant (Shoff et al., 2007). More studies are needed to
determine if these relationships exist.
In Mexico, only the genotype of 2 isolates from AK cases has been
reported, one was identified as A. griffini (T3) and other as A. royreba
(T4); both infections responded promptly to antiamoebic treatment,
although remission took more than 3 months. Nevertheless, the patient
infected with the T3 genotype needed a keratoplasty in order to im-
prove the visual acuity (Omaña-Molina et al., 2016). Both strains
showed to be low virulent but expressed invasive capacity to reach the
CNS in the GAE murine model (González-Robles et al., 2013, 2014).
In agreement with the results reported by Omaña-Molina et al.
(2016) and those presented in this work, in Mexico the T4 genotype
seems to be the most frequently associated with clinical cases and
perhaps more abundant in the environment, however it is important to
continue with the genotyping of Acanthamoeba isolates to know their
prevalence and distribution in our country.
Although the first report of a clinical case caused by Acanthamoeba
was made half a century ago (Callicott, 1968), these amoebae are still
not recognized in some medical circles, often not considered within the
first diagnosis, and in the majority of the countries where infections
occurred, there is no treatment of choice. In addition, variable results of
sensitivity and drug resistance have been reported. In this work,
D. Hernández-Martínez et al. Experimental Parasitology 197 (2019) 29–35

Fig. 1. Phase contrast microscopy. Images of trophozoites and cysts of the amoebae in study. A, B) A. polyphaga isolated from a corneal ulcer; C, D) A. castellanii from
the contact lens of a patient with AK and E, F). A. palestinensis obtained from a soil sample. Trophozoites (A, C, E) show main morphological characteristics of
Acanthamoeba: Nucleus (N), nucleolus (n), contractile vacuole(cv), acantopodia(a). Cysts (B, D, F) showing endocyst (en), exocyst (ex) Bar 10 μM.

trophozoites of A. castellanii, A. polyphaga and A. palestinensis were
sensitive to the drugs evaluated; although the IC50 was variable be-
tween species and drugs, with results similar to those reported in other
studies (Martín-Navarro et al., 2008; Cabello-Vílchez et al., 2014;
Rocha-Cabrera et al., 2015; Taravaud et al., 2017). However, the 50
inhibitory concentration of all drugs induced the encystment of a small
number of amebae. Although the cysticidal activity of chlorhexidine has
been documented (Lee et al., 2007; Abjani et al., 2016), more studies
are required to know the sensitivity of these forms of amoebic re-
sistance to different drugs.
Among the drugs evaluated, the most active compounds against
trophozoies in study were voriconazole and itraconazole, demon-
strating that in vitro antiamoebic interaction of both azoles was similar,
except for itraconazole and A. castellanii, with an IC50 significantly
higher than the others. Aqeel et al. (2016) suggest the emergence of
Acanthamoeba strains resistant to azoles, as seen in Candida albicans.
Ergosterol and 7-dehyrostigmasterol are the mains sterols of the
membrane in trophozoites of Acanthamoeba (Smith and Korn, 1968;
Mehdi et al., 1988), voriconazole and itraconazole inhibit ergosterol
synthesis by binding to sterol 14α-demethylase enzyme (CYP51) of A.
castellanii and A. polyphaga (Lamb et al., 2015). It has also been
determined that voriconazole and itraconazole have a significantly
higher affinity for the CYP51 enzyme of C. albicans (Warrilow et al.,
2013) and A. castellanii than for its human homolog and found that
voriconazole is 8–16 times more potent than itraconazole (Lamb et al.,
2015). This correlates with our results.
It has been documented that voriconazole induces programmed cell
death in A. castellanii (Martín-Navarro et al., 2015), suggesting that this
drug is able to cause the death of these amoebae by non-necrotic me-
chanisms, which is convenient during the treatment of infections
caused by Acanthamoeba by reducing the inflammatory process. These
results show that voriconazole should be one of the main options for the
treatment of infections caused by Acanthamoeba due to its high se-
lectivity for CYP51 of the pathogen, its greater potency and its ability to
induce programmed cell death.
Regarding chlorhexidine, our results also showed interesting and
promising activity with IC50 less than 10 μM for the species in study.
Chlorhexidine is a biguanide compound used as an antiseptic; it has a
positive charge and the plasmatic membrane of the parasite is nega-
tively charged, which produces structural and permeability changes
that can cause cell damage and death (Siddiqui et al., 2016). It has been
documented that PHMB and chlorhexidine also have cysticidal effect

32
D. Hernández-Martínez et al.
Fig. 2. A) IC50 (μM) (mean values ± 95% confidence interval) of chlorhexidine, voriconazole and itraconazole tested against three T4 genotype isolated of
Acanthamoeba. B) For a given species or treatment, data containing similar alphabets are not significant (p > 0.05, Tukey test).
(Lee et al., 2007; Abjani et al., 2016), both are capable of binding to the
mucopolysaccharide plug of the ostiole resulting in penetration of the
cyst to amoeba causing damage, cell lysis and death (Lim et al., 2008).
The first reports of the use of chlorhexidine in AK cases were in the
90's (Booth and Morrell, 1994; Hay et al., 1994). Since then, some
studies have been conducted to evaluate their activity on these
amoebae in vitro and in vivo, also finding variable results; as mono-
therapy in vitro with good results (Martín-Navarro et al., 2008; Ortillés
et al., 2017; Cabello-Vílchez et al., 2014; Rocha-Cabrera et al., 2015),
but in AK patients variable results are reported (Lim et al., 2008). In
vivo combined therapy with propamidine, have shown good results
(Hay et al., 1994; Booth and Morrell, 1994; Seal et al., 1996). In
Mexico, a case of AK (Omaña-Molina et al., 2016) was treated for
months with moxifloxacin, netilmicin, natamycin and chlorhexidine
with poor visual outcomes, since keratoplasty implemented.
The infections caused by Acanthamoeba are usually of chronic
course thus the drugs are prescribed for a long time, therefore it is very
important to evaluate their side effects and their toxicity. Itraconazole
and voriconazole are triazoles that have relatively low toxicity and
fewer side effects in patients, compared with amphotericin B (Lamb
et al., 2015), another drug used systemically in these infections. Seal
et al. (1996) reported the successful treatment of 12 patients with
chlorhexidine combined with pentamidine, without clinical evidence of
toxicity. More recently it has been reported that chlorhexidine is less
toxic to keratocytes than PHMB (Lee et al., 2007) and propamidine
isethionate (Fernández-Ferreiro et al., 2017); two compounds that are
used regularly in some countries to treat AK.
We consider that itraconazole, voriconazole and chlorhexidine are
good therapeutic options in order to treat infections caused by
Acanthamoeba, in our country. Both azoles are available in Mexico in
tablets or injectable solutions and are able to be used in systemic or
local infections. In addition, voriconazole usually is prepared in
33
Experimental Parasitology 197 (2019) 29–35
hospitals or pharmacies for ophthalmic purposes.
Another important factor that must be considered to establish a
therapeutic scheme is the cost of the drugs. In Mexico, itraconazole is a
drug used for the treatment of amoebic keratitis which is inexpensive
and easily accessible, however, it is prescribed orally for prolonged
periods, on the other hand it has been proven that chlorhexidine is
effective against the trophozoites of Acanthamoeba, although it is dif-
ficult to find in our country. The use of voriconazole is a promising
therapeutic strategy nevertheless expensive, which limits its prescrip-
tion especially if it is used for prolonged periods.
In this work we report the isolation of T4 genotype strains
Acanthamoeba in Mexico that show in vitro sensitivity to chlorhexidine,
voriconazole and itraconazole. We consider that although chlorhex-
idine and itraconazole show good activity on these amoebae and have
been used in cases of AK in Mexico with acceptable results, vor-
iconazole should also be considered as the first therapeutic option of
future Acanthamoeba infections in our country, implemented either as
monotherapy or in combined therapy.
Finally, we consider that it is very important to communicate clin-
ical Acanthamoeba cases and the pathogenic potential of amoebae iso-
lated from diverse sources, as well as to suggest the possibility of using
more effective drugs that benefit patients affected by the disease.
Acknowledgments
We appreciate the technical support, as well as the suggestions to
the manuscript provided by the biologist Lizbeth Salazar Villatoro.
This work was supported by the grants PI18/01380, Fondo Europeo
de Desarrollo Regional (FEDER) and RICET [project no. RD16/0027/
0001 of the programme of Redes Temáticas de Investigación
Cooperativa, FIS], Spanish Ministry of Science, Innovation and
Universities, Madrid, Spain.JEPB was funded by Proyectos puente al
D. Hernández-Martínez et al.
Plan Estatal de I + D + I. Plan Propio 2018 Universidad de La Laguna.
Also we appreciate the support of “Programa de becas estudiantiles
SEP-UNAM-FUNAM-2017“. Beca de capacitación en Métodos de
Investigación. Primera Fase.
Finally, we acknowledge to SSS Sarma and S. Nandini for their
comments on the manuscript and the support during PASD, DGAPA,
UNAM course.
References
Abjani, F., Khan, N.A., Yousuf, F.A., Siddiqui, R., 2016. Targeting cyst wall is an effective
strategy in improving the efficacy of marketed contact lens disinfecting solutions
against Acanthamoeba castellanii cysts. Contact Lens Anterior Eye 39 (3), 239–243.
http://doi:10.1016/j.clae.2015.11.004.
Aichelburg, A.C., Walochnik, J., Assadian, O., Prosch, H., Steuer, A., Perneczky, G.,
Visvesvara, G.S., Aspöck, H., Vetter, N., 2008. Successful treatment of disseminated
Acanthamoeba sp. infection with miltefosine. Emerg. Infect. Dis. 14 (11), 1743–1746.
http://doi:10.3201/eid1411.070854.
Aqeel, Y., Siddiqui, R., Anwar, A., Shah, M.R., Khan, N.A., 2016. Gold nanoparticle
conjugation enhances the antiacanthamoebic effects of chlorhexidine. Antimicrob.
Agents Chemother. 60, 1283–1288. https://doi.org/10.1128/AAC.01123-15.
Arnalich-Montiel, F., Martín-Navarro, C.M., Alió, J.L., López-Vélez, R., Martínez-
Carretero, E., Valladares, B., Piñero, J.E., Lorenzo-Morales, J., 2012. Successful
monitoring and treatment of intraocular dissemination of Acanthamoeba. Arch.
Ophthalmol. 130, 1474–1475.
Arnalich-Montiel, F., Lumbreras-Fernández, B., Martín-Navarro, C.M., Valladares, B.,
Lopez-Velez, R., Morcillo-Laiz, R., Lorenzo-Morales, J., 2014. Influence of
Acanthamoeba genotype on clinical course and outcomes for patients with
Acanthamoeba keratitis in Spain. J. Clin. Microbiol. 52 (4), 1213–1216. https://doi.
org/10.1128/JCM.00031-14.
Becerril, F.M.A., 2014. Parasitología Médica, fourth ed. Mcgraw-Hill Interamericana
Editores, México, pp. 437.
Bonilla-Lemus, P., Ramírez-Bautista, G.A., Zamora-Muñoz, C., Ibarra-Montes, M.R.,
Ramírez, E., Hernández-Martínez, M.D., 2010. Acanthamoeba spp. in domestic tap
water in houses of contact lens wearers in the metropolitan area of Mexico City. Exp.
Parasitol. 126 (1), 54–58. http://doi:10.1016/j.exppara.2009.11.019.
Bonilla-Lemus, P., Caballero, V.A.S., Carmona, J.J., Lugo, V.A., 2014. Occurrence of free-
living amoebae in streams of the Mexico Basin. Exp. Parasitol. 145 (Suppl. S), 28–33.
http://doi:10.1016/j.exppara.2014.07.001.
Booth, A., Morrell, A.J., 1994. Treatment of Acanthamoeba keratitis. Eye 8 (6), 719–720.
http://doi:10.1038/eye.1994.191.
Booton, G.C., Kelly, D.J., Chu, Y.W., Seal, D.V., Houang, E., Lam, D.S.C., Byers, T.J.,
Fuerst, P.A., 2002. 18S ribosomal DNA typing and tracking of Acanthamoeba species
isolates from corneal scrape specimens, contact lenses, lens cases, and home water
supplies of Acanthamoeba keratitis patients in Hong Kong. J. Clin. Microbiol. 40,
1621–1625.
Booton, G.C., Visvesvara, G.S., Byers, T.J., Kelly, D.J., Fuerst, P.A., 2005. Identification
and distribution of Acanthamoeba species genotypes associated with nonkeratitis in-
fections. J. Clin. Microbiol. 43, 1689–1693.
Bryant, R.J., Woods, L.E., Coleman, D.C., Fairbanks, B.C., McClellan, J.F., Coleii, C.V.,
1982. Interactions of bacterial and amoebal populations in soil microcosms with
fluctuating moisture content. Appl. Environ. Microbiol. 43, 747–752.
Cabello-Vílchez, A.M., Martín-Navarro, C.M., López-Arencibia, A., Reyes-Batlle, M.,
González, A.C., Guerra, H., Gotuzzo, E., Valladares, B., Piñero, J.E., Lorenzo-Morales,
J., 2013. Genotyping of potentially pathogenic Acanthamoeba strains isolated from
nasal swabs of healthy individuals in Peru. Acta Trop. http://doi:10.1016/j.
actatropica.2013.10.006.
Cabello-Vílchez, A.M., Martín-Navarro, C.M., López-Arencibia, A., Reyes-Batlle, M.,
Sifaoui, I., Valladares, B., Piñero, J.E., Lorenzo-Morales, J., 2014. Voriconazole as a
first-line treatment against potentially pathogenic Acanthamoeba strains from Peru.
Parasitol. Res. 113 (2), 755–759. http://doi.org/10.1007/s00436-013-3705-8.
Callicott, J.H., 1968. Amebic meningoencephalitis due to free-living amebas of the
Hartmannella (Acanthamoeba- Naegleria) group. Am. J. Clin. Pathol. 49, 84.
Corsaro, D., Walochnik, J., Köhsler, M., Rott, M.B., 2015. Acanthamoeba misidentification
and multiple labels: redefining genotypes T16, T19, and T20 and proposal for
Acanthamoeba micheli sp. nov. (genotype T19). Parasitol. Res. 114 (7), 2481–2490.
https://doi.org/10.1007/s00436-015-4445-8.
Dart, J.,K., Saw, V.P., Kilvington, S., 2009. Acanthamoeba keratitis:diagnosis and treat-
ment update2009. Am. J. Ophthalmol. 148 (4), 487–499. http://doi:10.1016/j.ajo.
2009.06.009.
Derda, M., Solarczyk, P., Cholewiński, M., Hadaś, E., 2015. Genotypic characterization of
amoeba isolated from Acanthamoeba keratitis in Poland. Parasitol. Res. 114 (3),
1233–1237. http://doi:10.1007/s00436-015-4319-0.
Fernández-Ferreiro, A., Santiago-Varela, M., Gil-Martínez, M., González-Barcia, M.,
Luaces-Rodríguez, A., Díaz-Tome, V., Pardo, M., Méndez, J.B., Piñeiro-Ces, A.,
Rodríguez-Ares, M.T., Lamas, M.J., Otero-Espinar, F.J., 2017. In vitro evaluation of
the ophthalmic toxicity profile of chlorhexidine and propamidine isethionate eye
drops. J. Ocul. Pharmacol. Therapeut. 33 (3), 202–209. http://doi:10.1089/jop.
2016.0053.
González-Robles, A., Salazar-Villatoro, L., Omaña-Molina, M., Lorenzo-Morales, J.,
Martínez-Palomo, A., 2013. Acanthamoeba royreba: morphological features and in
vitro cytopathic effect. Exp. Parasitol. 133 (4), 369–375. http://doi:10.1016/j.
exppara.2013.01.011.
34
Experimental Parasitology 197 (2019) 29–35
González-Robles, A., Salazar-Villatoro, L., Omaña-Molina, M., Reyes-Batlle, M., Martín-
Navarro, C.M., Lorenzo-Morales, J., 2014. Morphological features and in vitro cyto-
pathic effect of Acanthamoeba griffini trophozoites isolated from a clinical case. J.
Parasitol. Res. 2014, 1–10. http://doi:10.1155/2014/256310.
Grün, A.L., Stemplewitz, B., Scheid, P., 2014. First report of an Acanthamoeba genotype
T13 isolate as etiological agent of a keratitis in humans. Parasitol. Res. 113 (6),
2395–2400. http://doi:10.1007/s00436-014-3918-5.
Hay, J., Kirknessi, C.M., Seal, D.V., Wright, P., 1994. Drug resistance and Acanthamoeba
keratitis: the quest for alternative antiprotozoal chemotherapy. Eye 8, 555–563.
Heredero-Bermejo, I., Criado-Fornelio, A., De Fuentes, I., Soliveri, J., Copa-Patiño, J.L.,
Pérez-Serrano, J., 2015. Characterization of a human pathogenic Acanthamoeba
griffini isolated from a contact lens wearing keratitis patient in Spain. Parasitology
142 (2), 363–373. http://doi:10.1017/S0031182014001140.
Heredero-Bermejo, I., Sánchez-Nieves, J., Soliveri, J., Gómez, R., De la Mata, F.J., Copa-
Patiño, J.L., Pérez-Serrano, J., 2016. In vitro anti-Acanthamoeba synergistic effect of
chlorhexidine and cationic carbosilane dendrimers against both trophozoite and cyst
forms. Int. J. Pharm. 509 (1–2), 1–7. http://doi:10.1016/j.ijpharm.2016.04.075.
Jones, D.B., Visvesvara, G.S., Robinson, N.M., 1975. Acanthamoeba polyphaga keratitis
and Acanthamoeba uveitis associated with fatal meningoencephalitis. Trans.
Ophthalmol. Soc. U. K. 95 (2), 221–232.
Khan, N.A., 2006. Acanthamoeba: biology and increasing importance in human health.
FEMS Microbiol. Rev. 30 (4), 564–595.
Lamb, D.C., Warrilow, A.G.S., Rolley, N.J., Parker, J.E., Nes, W.D., Smith, S.N., Kelly,
D.E., Kelly, S.L., 2015. Azole antifungal agents to treat the human pathogens
Acanthamoeba castellanii and Acanthamoeba polyphaga through inhibition of sterol
14α-Demethylase (CYP51). Antimicrob. Agents Chemother. 59 (8), 4707–4713.
http://doi:10.1128/AAC.00476-15.
Lee, J.E., Oum, B.S., Choi, H.Y., 2007. Cysticidal effect on Acanthamoeba and toxicity on
human keratocytes by polyhexamethylene biguanide and chlorhexidine. Cornea 26,
736–741. http://doi.org/10.1097/ICO.0b013e31805b7e8e.
Lim, N., Goh, D., Bunce, C., 2008. Comparison of polyhexamethylene biguanide and
chlorhexidine as monotherapy agents in the treatment of Acanthamoeba keratitis. Am.
J. Ophthalmol. 145, 130–135.
Lorenzo-Morales, J., Morcillo-Laiz, R., Martín-Navarro, C.M., Lopez-Velez, R., López-
Arencibia, A., Arnalich-Montiel, F., Maciver, S.K., Valladares, B., Martínez-Carretero,
E., 2011. Acanthamoeba keratitis due to genotype T11 in a rigid gas permeable
contact lens wearer in Spain. Contact Lens Anterior Eye 34, 83–86. http://doi:10.
1016/j.clae.2010.10.007.
Lorenzo-Morales, J., Martín-Navarro, C.M., López-Arencibia, A., Arnalich-Montiel, F.,
Piñero, J.E., Valladares, B., 2013. Acanthamoeba keratitis: an emerging disease
gathering importance worldwide? Trends Parasitol. 29, 181–187.
Lorenzo-Morales, J., Khan, N.A., Walochnik, J., 2015. An update on Acanthamoeba ker-
atitis: diagnosis, pathogenesis and treatment. Parasite 22, 10. http://doi:10.1051/
parasite/2015010.
Maghsood, A.H., Sissons, J., Rezaian, M., Nolder, D., Warhurst, D., Khan, N.A., 2005.
Acanthamoeba genotype T4 from the UK and Iran and isolation of the T2 genotype
from clinical isolates. J. Med. Microbiol. 54, 755–759.
Marciano-Cabral, F., Cabral, G., 2003. Acanthamoeba spp. as agents of disease in humans.
Clin. Microbiol. Rev. 16, 273–307.
Martín-Navarro, C.M., Lorenzo-Morales, J., Cabrera-Serra, M.G., Rancel, F., Coronado-
Alvarez, N.M., Piñero, J.E., Valladares, B., 2008. The potential pathogenicity of
chlorhexidine-sensitive Acanthamoeba strains isolated from contact lens cases from
asymptomatic individuals in Tenerife, Canary Islands, Spain. J. Med. Microbiol. 57,
1399–1404.
Martín-Navarro, C.M., López-Arencibia, A., Arnalich-Montiel, F., Valladares, B., Piñero,
J.E., Lorenzo-Morales, J., 2013. Evaluation of the in vitro activity of commercially
available moxifloxacin and voriconazole eye-drops against clinical strains of
Acanthamoeba. Graefes Arch. Clin. Exp. Ophthalmol. 251, 2111–2117. http://doi:10.
1007/s00417-013-2371-y.
Martín-Navarro, C.M., López-Arencibia, A., Sifaoui, I., Reyes-Batlle, M., Valladares, B.,
Martínez-Carretero, E., Piñero, J.E., Maciver, S.K., Lorenzo-Morales, J., 2015. Statins
and voriconazole induce programmed cell death in Acanthamoeba castellanii.
Antimicrob. Agents Chemother. 59 (5), 2817–2824. http://doi:10.1128/AAC.
00066-15.
Martínez, A.J., 1985. Free-living Amebas: Natural History, Prevention, Diagnosis,
Pathology, and Treatment of Disease. CRC Press, Inc, Boca Raton, Florida.
Martínez, A.J., Visvesvara, G.S., 1997. Free-living, amphizoic and opportunistic amebas.
Brain Pathol. 7, 583–598.
McBride, J., Ingram, P.R., Henríquez, F.L., 2005. Development of colorimetric microtiter
plate assay for assessment of antimicrobials against Acanthamoeba. J. Clin. Microbiol.
43, 629–634.
Mehdi, H., Garg, H.S., Garg, N.K., Bhakuni, D.S., 1988. Sterols of Acanthamoeba culbert-
soni strain A-1. Steroids 51, 551–558. http://doi.org/10.1016/0039128X(88)
90051-7.
Nehete, L.S., Kumar, A., Chavali, P., A., R.P, Devi, B.I., 2018. Fulminant acanthamoebic
meningoencephalitis in immunocompetent patients: an uncommon entity. Br. J.
Neurosurg. 23, 1–4. http://doi./10.1080/02688697.2018.1485873.
Niyyati, M., Lorenzo-Morales, J., Rezaie, S., Rahimi, F., Mohebali, M., Maghsood, A.H.,
Motevialli-Haghi, A., Martín-Navarro, C.M., Farnia, S., Valladares, B., Rezaeian, M.,
2009. Genotyping of Acanthamoeba isolates from clinical and environmental speci-
mens in Iran. Exp. Parasitol. 121 (3), 242–245.
Nuprasert, W., Putaporntip, C., Pariyakanok, L., Jongwutiwes, S., 2010. Identification of a
novel T17 genotype of Acanthamoeba from environmental isolates and T10 genotype
causing keratitis in Thailand. J. Clin. Microbiol. 48, 4636–4640. http://doi.org/10.
1128/JCM.01090-10.
Oliva, S., Jantz, M., Tiernan, R., Cook, D.L., Judson, M.A., 1999. Successful treatment of
D. Hernández-Martínez et al.
widely disseminated acanthamoebiasis. South. Med. J. 92 (1), 55–57.
Omaña-Molina, M., Vanzzini-Zago, V., Hernandez-Martinez, D., Gonzalez-Robles, A.,
Salazar-Villatoro, L., Ramirez-Flores, E., Oregon-Medina, E., Lorenzo-Morales, J.,
Martinez-Palomo, A., 2016. Acanthamoeba genotypes T3 and T4 as causative agents
of amoebic keratitis in Mexico. Parasitol. Res. 115 (2), 873–878. http://doi:10.1007/
s00436-015-4821-4.
Ortillés, Á., Belloc, J., Rubio, E., Fernández, M.T., Benito, M., Cristóbal, J.Á., Calvo, B.,
Goñi, P., 2017. In-vitro development of an effective treatment for Acanthamoeba
keratitis. Int. J. Antimicrob. Agents 50 (3), 325–333. http://doi:10.1016/j.
ijantimicag.2017.03.033.
Page, F.C., 1988. A New Key to Freshwater and Soil Gymnamoebae with Instructions for
Culture. Culture Collections of Algae and Protozoa. Freshwater Biological
Association. Ambleside, Cumbria, England, pp. 92–96.
Perez-Santonja, J.J., Kilvington, S., Hughes, R., Tufail, A., Metheson, M., Dart, J.K.G.,
2003. Persistently culture positive Acanthamoeba keratitis; in vivo resistance and in
vitro sensitivity. Ophthalmol. Times 110, 1593–1600.
Ramírez, E., Robles, E., Martinez, B., Ayala, R., Sainz, G., Martinez, M.E., Gonzalez, M.E.,
2014. Distribution of free-living amoebae in a treatment system of textile industrial
waste water. Exp. Parasitol. 145 (Suppl. l), S34–S38. http://doi:0.1016/j.exppara.
2014.07.006.
Rivera, F., Ramírez, P., Vilaclara, G., Robles, E., Medina, F., 1983. A survey of pathogenic
and free living- amoebae inhabiting swimming pool water in Mexico City. Environ.
Res. 32 (1), 205–211.
Rivera, F., Rosas, I., Castillo, M., Chávez, M., Gómez, R., Chío, R.E., Islas, J., 1986.
Pathogenic and free-living protozoa cultured from nasopharyngeal and oral regions
of dental patients: II. Environ. Res. 39 (2), 364–371.
Rivera, F., Lares, F., Gallegos, E., Ramírez, E., Calderon, A., Martinez, J.J., Rodriguez, S.,
Alcocer, J., 1989. Pathogenic amoeba in natural termal waters of three resorts of
Hidalgo, Mexico. Environ. Res. 50 (2), 289–295.
Rivera, F., Ramírez, E., Bonilla, P., Calderon, A., Gallegos, E., Rodriguez, S., Ortiz, R.,
Zaldívar, B., Ramírez, P., Durán, A., 1993. Pathogenic and free-living amoebae iso-
lated from swimming pools and physiotherapy tubs in Mexico. Environ. Res. 62 (1),
43–52.
Rivera, F., Bonilla, P., Ramírez, E., Calderón, A., Gallegos, E., Rodríguez, S., Ortíz, R.,
Hernández, D., Rivera, V., 1994. Seasonal distribution of air-borne pathogenic and
free-living amoebae in Mexico city and its suburbs. Water. Air and Soil Poll 74,
65–87.
Rocha-Cabrera, P., Reyes-Batlle, M., Martín-Navarro, C.M., Dorta-Gorrín, A., López-
Arencibia, A., Sifaoui, I., Martínez-Carretero, E., Piñero, J.E., Martín-Barrera, F.,
Valladares, B., Lorenzo-Morales, J., 2015. Detection of Acanthamoeba on the ocular
surface in a Spanish population using the Schirmer strip test: pathogenic potential,
molecular classification and evaluation of the sensitivity to chlorhexidine and vor-
iconazole of the isolated Acanthamoeba strains. J. Med. Microbiol. 64 (8), 849–853.
http://doi:10.1099/jmm.0.000103.
Scheid, P., Zöller, L., Pressmar, S., Richard, G., Michel, R., 2008. An extraordinary en-
docytobiont in Acanthamoeba sp. isolated from a patient with keratitis. Parasitol. Res.
102 (5), 945–950. http://doi:10.1007/s00436-007-0858-3.
Schuster, F.L., Visvesvara, G.S., 2004. Opportunistic amoebae: challenges in prophylaxis
and treatment. Drug Resist. Updates 7, 41–51.
Seal, D., Hay, J., Kirkness, C., Morrell, A., Booth, A., Tullo, A., Ridgway, A., Armstrong,
35
Experimental Parasitology 197 (2019) 29–35
M., 1996. Successful medical therapy of Acanthamoeba keratitis with topical chlor-
hexidine and propamidine. Eye 10 (Pt 4), 413–421.
Shoff, M., Rogerson, A., Schatz, S., Seal, D., 2007. Variable responses of Acanthamoeba
strains to three multipurpose lens cleaning solutions. Optom. Vis. Sci. 84, 202–207.
Siddiqui, R., Khan, N.A., 2012. Biology and pathogenesis of Acanthamoeba. Parasites
Vectors 5, 6. http:doi.org/10.1186/1756-3305-5-6.
Siddiqui, R., Aqeel, Y., Khan, N.A., 2016. The development of drugs against Acanthamoeba
infections. Antimicrob. Agents Chemother. 60 (11), 6441–6450. http://doi:10.1128/
AAC.00686-16.
Slater, C.A., Sickel, J.Z., Visvesvara, G.S., Pabico, R.C., Gaspari, A.A., 1994. Brief report:
successful treatment of disseminated Acanthamoeba infection in an im-
munocompromised patient. N. Engl. J. Med. 331 (2), 85–87.
Smith, F.R., Korn, E.D., 1968. 7-Dehydrostigmasterol and ergosterol: the major sterols of
an amoeba. J. Lipid Res. 9 (4), 405–408.
Stothard, D.R., Schroede-Diedrich, J.M., Awwad, M.H., Gast, R.J., Ledee, D.R., Rodríguez-
Zaragoza, S., Dean, D.L., Fuerst, P.A., Byers, T.J., 1998. The evolutionary history of
the genus Acanthamoeba and the identification of eight new 18S rRNA gene sequence
types. J. Eukaryot. Microbiol. 45, 45–54. http://doi.org/10.1111/j.1550-7408.1998.
tb05068.x.
Taravaud, A., Loiseau, P.M., Pomel, S., 2017. In vitro evaluation of antimicrobial agents
on Acanthamoeba sp. and evidence of a natural resilience to amphotericin B. Int. J.
Parasitol. Drugs. Drug. Resist. 7 (3), 328–336. http://doi:10.1016/j.ijpddr.2017.09.
002.
Visvesvara, G.S., Moura, H., Schuster, F.L., 2007. Pathogenic and opportunistic free-living
amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia
diploidea. FEMS Immunol. Med. Microbiol. 50, 1–6.
Visvesvara, G.S., 2013. Infections with free-living amebae. Handbook of clinical neu-
rology 14. In: Garcia, H.H., Tanowitz, H.B., Del Brutto, O.H. (Eds.),
Neuroparasitology and Tropical Neurology. Elsevier B. V, pp. 153–168.
Walia, R., Montoya, J.G., Visvesvera, G.S., Booton, G.C., Doyle, R.L., 2007. A case of
successful treatment of cutaneous Acanthamoeba infection in a lung transplant re-
cipient. Transpl. Infect. Dis. 9 (1), 51–54.
Wagner, C., Reyes-Batlle, M., Vethencourt, Y.M.A., Galindo, P.M.V., Guzmán, R.C., Nessi,
P.A.J., Dorta, P.A., López-Arencibia, A., Sifaoui, I., Pérez, G.M.V., Pérez, S.E.,
Martínez-Carretero, E., Valladares, B., Piñero, J.E., Lorenzo-Morales, J., 2016.
Genotyping of clinical isolates of Acanthamoeba genus in Venezuela. Acta Parasitol.
61 (4), 796–801. http://doi.org/10.1515/ap-2016-0110.
Warrilow, A.G., Parker, J.E., Kelly, D.E., Kelly, S.L., 2013. Azole affinity of sterol 14α-
demethylase (CYP51) enzymes from Candida albicans and Homo sapiens. Antimicrob.
Agents Chemother. 57 (3), 1352–1360. http://doi:10.1128/AAC.02067-12.
Webster, D., Umar, I., Kolyvas, G., Bilbao, J., Guiot, M., Duplisea, K., Qvarnstrom, Y.,
Visvesvara, G.S., 2012. Case Report: treatment of granulomatous amoebic en-
cephalitis with voriconazole and miltefosine in an immunocompetent soldier. Am. J.
Trop. Med. Hyg. 87 (4), 715–718. https://doi.org/10.4269/ajtmh.2012.12-0100.
Willaert, E., Stevens, A.R., 1976. Indirect immunofluorescent identification of
Acanthamoeba causing meningoencephalitis. Pathol. Biol. 24, 545–547.
Zhang, Y., Sun, X., Wang, Z., Li, R., Luo, S., Jin, X., Deng, S., Chen, W., 2004.
Identification of 18S ribosomal DNA genotype of Acanthamoeba from patients with
keratitis in North China. Invest. Ophthalmol. Vis. Sci. 45, 1904–1907.