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Therapeutic review

Therapeutic agents and biocides for

ocular infections by free-living amoebae of
Acanthamoeba genus

Linda Christian Carrijo-Carvalho, PhD, Viviane Peracini Sant’ana, PhD,

Annette Silva Foronda, MD, PhD, Denise de Freitas, MD, PhD,
Fabio Ramos de Souza Carvalho, PhD*
Department of Ophthalmology and Visual Sciences, Paulista School of Medicine, Federal University of São Paulo, São
Paulo, Brazil

article info abstract

Article history: Acanthamoeba keratitis is a sight-threatening infectious disease. Resistance of the cystic
Received 1 March 2016 form of the protozoan to biocides and the potential toxicity of chemical compounds to
Received in revised form 26 October corneal cells are the main concerns related to long-term treatment with the clinically
2016 available ophthalmic drugs. Currently, a limited number of recognized antimicrobial
Accepted 28 October 2016 agents are available to treat ocular amoebic infections. Topical application of biguanide
Available online 9 November 2016 and diamidine antiseptic solutions is the first-line therapy. We consider the current
Steven Teich, Editor challenges when treating Acanthamoeba keratitis and review the chemical properties,
toxicities, and mechanisms of action of the available biocides. Antimicrobial therapy using
Keywords: anti-inflammatory drugs is controversial, and aspects related to this topic are discussed.
Acanthamoeba keratitis Finally, we offer our perspective on potential improvement of the effectiveness and safety
biguanides of therapeutic profiles, with the focus on the quality of life and the advancement of indi-
PHMB vidualized medicine.
chlorhexidine ª 2016 Elsevier Inc. All rights reserved.
diamidines
propamidine
hexamidine
eye drops
cornea

1. Introduction human cornea, Acanthamoeba keratitis (AK). The increasing

incidence of AK, which occurs mainly in immunocompetent
Free-living amoebas of Acanthamoeba genus are the etiologic young adults, is a public health problem with serious conse-
agent of a sight-threatening infectious disease that affects the quences for vision considering the lack of a standard therapy

* Corresponding author: Fabio Ramos de Souza Carvalho, PhD, Department of Ophthalmology and Visual Sciences, Paulista School of
Medicine, Federal University of São Paulo, Rua Botucatu 821, Vila Clementino, Sao Paulo 04023-062, Brazil.
E-mail address: [email protected] (F.R. de Souza Carvalho).
0039-6257/$ e see front matter ª 2016 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.survophthal.2016.10.009
204 s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 2 0 3 e2 1 8

and the complexity of the disease prognosis.21,49,80 Most cases Currently, the first-line therapy for AK is topical instillation
of AK have been associated with poor hygiene and/or of organic compounds from the chemical classes of bigua-
improper handling of contact lenses, among other risk fac- nides and diamidines.26 Nevertheless, clinical concerns
tors.49,61 Corneal infections caused by Acanthamoeba species include the varying therapeutic responses among patients
(spp) also have been reported in relation to corneal trauma, with AK, drug toxicity on corneal tissue, optimization of
mainly with organic material such as insects, plants, and cellular targets for drug activation focusing on the protozoan,
ocular surface exposure to contaminated water without as- and increased drug resistance of the cystic forms,26,80 all
sociation with contact lens wear, especially in some devel- relevant issues to be investigated within translational
oping countries.21,26,72 research and individualized medicine. Translational research
The protozoan presents 2 morphologic cellular forms: aims to apply the knowledge and technologies from benchtop
trophozoites and cysts (Fig. 1).125 Trophozoites, active cells at research to improve laboratory diagnostic procedures and
the metabolic, physiologic, and morphologic levels, are early therapeutic interventions, while the selection of an
responsible for the infective stages and virulence of the pro- appropriate therapy with minimal side effects for each patient
tozoan pathogen, whereas cysts are characterized by and maximum efficacy against the pathogen-associated
dormancy and resistance to antimicrobial agents. Both forms infection is an essential aspect to be provided by individual-
are immunogenic, but may have mechanisms to evade im- ized medicine. The development of recrudescent infective
mune attack.94 The pathophysiology of AK is triggered by episodes after treatment failure also may be associated with
initial adhesion of trophozoites to the corneal epithelial cells acquired resistance of the protozoan to antimicrobial agents,
mediated by mannose-binding protein, a cellular surface re- increased risk of visual loss, and need for corneal trans-
ceptor composed of 130-kDa subunits belonging to the lectin plantation.37,62,74,138 Alternatively, other procedures based on
family.106 Subsequent steps in the infectious cascade of physicochemical inactivation of ocular pathogens (corneal
amoebic keratitis involve deeper stromal invasion, degrada- cross-linking, photodynamic therapy, and excimer laser
tion of extracellular matrix components, and induction of phototherapeutic keratectomy) have been proposed as adju-
host cellular death.106,121 Extracellular proteins secreted by vant treatments for infectious keratitis, including
virulent Acanthamoeba strains, designated as secretomes or AK56,63,120,129; however, controversies exist concerning the
exoproteomes, are comprised mainly of proteases and phos- application and standardization of these techniques as safe
pholipases.89,103 The enzymatic activities of these proteins are and optimized therapeutic procedures in the routine treat-
related to the corneal tissue digestive process and the ment of AK.13,57,114 Keratoplasty may be necessary for specific
different patterns of infectious severity observed in patients.27 therapeutic purposes to avoid corneal perforation and to

Fig. 1 e Morphologic characteristics of cysts and trophozoites of Acanthamoeba species isolated from cases of severe
infection. A and B: The pleomorphism of cysts varies from A: a round to B: a stellate shape with a mature and prominent
cellulose-containing double-walled structure. C and D: Trophozoites have a broad hyaline zone (arrowhead) with a large
number of specialized spine-like projections (arrow) called acanthopodia. Magnification, 3400.
 s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 2 0 3 e2 1 8 205

eliminate the parasite load in the corneal tissue, especially in infiltrate (Fig. 2B) and perineural infiltrates, also known as
cases resistant to therapy6,23,91,112,116; however, a case series radial keratoneuritis (Fig. 2C ), are characteristic signs of
showed that the use of keratoplasty in AK patients results in AK.26,112,130 Although radial keratoneuritis is a strongly sug-
better clinical outcomes when performed for visual rehabili- gestive sign of AK in contact lensewearing patients,91 it has
tation.116 Thus, whenever possible, keratoplasty should be also been reported in bacterial keratitis.118 In addition, scler-
avoided during the active phase of infection to ensure a more itis, anterior uveitis, hypopyon, glaucoma, mydriasis, and
favorable prognosis for transplant success.116 Control of the cataract occur in severe cases or late stages of the disease, as
inflammatory process in the corneal tissue may decrease the well as corneal abscess, melting, and perforation.26,91 Delayed
risk of graft rejection, and the eradication of protozoan is diagnosis of AK usually occurs in patients initially mis-
important to avoid the recurrence of infection. diagnosed as having other infectious keratitis, especially
Corneal infection by the free-living protozoan of Acantha- corneal infections caused by Herpes simplex virus.23,26,91,130 The
moeba genus was first described in the early 1970s. An asso- differentiation between AK and herpetic keratitis is supported
ciation of contact lens wear as the main risk factor in by biomicroscopic findings of herpetic dendrites, which are
development of the disease was not recognized until the characterized by their branching pattern and the presence of
mid-1980s.54,98,101 Since the early 1990s, Osato and col- terminal bulbs.147 Abuse of topical anesthetics may cause
leagues105 discussed the need for an optimal therapeutic corneal toxicity that translates clinically into signs of epithe-
regimen for AK and the challenge of cystic eradication in lial defects, superficial punctate keratitis, inflammation, and
their review of in vitro evaluation of biocides against Acan- ring infiltrate, which resemble the signs of AK.107 Accurate
thamoeba. More than 4 decades later, treating AK remains a diagnosis and early treatment initiation are crucial for
major challenge. The absence of a specific therapeutic improving the disease outcome and avoiding blindness.23 Dart
regimen to eradicate the cysts and trophozoites of the pro- and colleagues26 elegantly reviewed the clinical aspects and
tozoan and the undesirable side effects of broad-spectrum management of AK.
antimicrobials associated with the need for long-term ther-
apy are issues worthy of a specific and translational debate.
2.2. Diagnosis of AK
For example, the application of biotechnology and molecular
biology tools in genotypic characterization of the protozoan
The routine laboratory diagnosis of AK consists of culture of
may allow targeted treatment. We review the chemical
corneal scrapings on nonnutrient or Foronda agar plate
properties and modes of activity and toxicity of biocides and
seeded with avirulent and heat-inactivated bacteria, Escher-
other topical drugs used to treat AK and discuss the main
ichia coli preferably, and molecular analysis through DNA
aspects of AK therapy and the properties of alternative anti-
amplification by polymerase chain reaction.14,21,60 Acantha-
Acanthamoeba compounds.
moeba spp among the 19 genotypes previously described
(T1eT19) are characterized according to the nucleotide
sequence of the 18S rDNA gene.84 The T4 genotype is mainly
2. Clinical management of AK and drug associated with ocular infections.84,115,123 To provide early
therapy diagnosis, examination of the corneal smear using calcofluor
white fluorescence staining is a rapid and specific detection
2.1. Clinical findings of AK method (Fig. 3). This technique allows the direct microscopic
visualization of double-walled cellular structures with typical
Corneal infections caused by Acanthamoeba spp can cause morphological characteristics of Acanthamoeba cysts.142 Spe-
blindness. The most frequently described symptoms are cific staining of Acanthamoeba cysts and trophozoites is also
foreign body sensation, redness, tearing, photophobia, accomplished by antibody-based techniques, such as indirect
decreased visual acuity, and in some cases, severe pain not immunofluorescence and immunoperoxidase staining.35,128
predicted by clinical findings.23,26,91,130 Clinical signs include In vivo confocal microscopy is a clinical diagnostic method
punctate epithelial erosions, pseudodendritic lesions (Fig. 2A), that may be helpful in the management of AK as it allows
subepithelial or stromal infiltrates and perineural in- observing a wide range of structural changes in the corneal
filtrates.23,112,130 The presence of a ring-shaped corneal tissue from the onset of protozoan infection and during

Fig. 2 e Clinical signs of Acanthamoeba keratitis. A: Corneal pseudodendrite. B: Typical ring infiltrate. C: Perineural infiltrates
(radial keratoneuritis).
206 s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 2 0 3 e2 1 8

Fig. 3 e Calcofluor white staining of corneal smear showing Acanthamoeba cysts. AeC: Direct examination of corneal
scraping visualized by A: fluorescence microscopy, B: light microscopy, or C: combined light and fluorescence microscopy.

therapy.140 The development of new complementary tech- propamidine isethionate (PP) was prescribed, but half of the
niques to the current diagnostic methods is important to cases had a poor therapeutic response due to cystic
assist in the identification and differentiation of the infecting resistance.127,144
pathogen and thus support the establishment of individual-
ized therapy. The proton nuclear magnetic resonance spec- 2.3. First-line therapies
troscopy, for example, is a promising technique that may
prove to be useful for AK diagnosis.42 No commercially available drugs currently are licensed to
When a diagnosis of AK is confirmed based on clinical treat AK. The main biocides used to treat AK are the biguanide
presentation, the patient’s history often associated with derivatives, that is, polyhexamethylene biguanide hydro-
contact lens use, and a positive culture of Acanthamoeba spp chloride (PHMB) and chlorhexidine gluconate (CLX) or acetate
from corneal scrapings, drug therapy should begin immedi- salt, and diamidine derivativesdthat is, PP and hexamidine
ately. A reduced parasitic load and inhibition of protozoan isethionate (HX). Early therapeutic intervention involves
growth and invasion through the host tissue are the imme- topical instillation of a sterile solution containing the anti-
diate goals of biocide therapy. A medical cure for AK depends microbial compound on the corneal surface. The dosage is
on eradication of the protozoan, trophozoites, and cysts from based on clinical reports and laboratory analyses.26 The bio-
the cornea. Before introduction of the biguanides to treat AK cides formulated as an eye drop solution of biguanide (0.02%
in the 1990s,43,73 1% neomycin combined with 0.1% PHMB or 0.02% CLX) and diamidine (0.1% PP or 0.1% HX) are

Fig. 4 e Treatment of Acanthamoeba infections. Acanthamoeba keratitis, with red eye and classical stromal inflammation in a
form of a ring infiltrate A: during the acute phase of the disease and B: after successful treatment. Acanthamoeba scleritis,
with a severe inflammation C: during acute phase of the disease and D: after treatment, revealing choroid pigments giving a
blue-violet color to the sclera due to its consumption and thinning.
 s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 2 0 3 e2 1 8 207

instilled hourly day and night for the first 2 to 3 days (Fig. 4). anterior segment of the eye may also play a role or lead to the
After the first 72 hours, the eye drops are usually prescribed development of iris atrophy, mydriasis, cataract, and glau-
hourly when awake for additional weeks, and then 4 times a coma during AK therapy.30,44 Alternatively, topical or oral
day. Owing to the chemical toxicity of the biocides in corneal voriconazole (VRC), a triazole antifungal, is used as an adju-
tissue, patients are followed closely after treatment initiation vant compound or second-line therapy in patients unre-
until a gradual reduction of the parasitic load occurs.29,130 sponsive to conventional therapy.8,10,135 The VRC regimen,
Drugs are tapered depending on clinical response and ocular especially the oral and systemic forms, is expensive for
surface toxicity. Treatment may take months and requires routine treatment of AK and requires constant medical su-
long-term follow-up. Biguanides are the preferred biocides to pervision for side effects.3,134 In view of the systemic side ef-
be used as monotherapy. A randomized controlled trial fects, topical use of VRC is desirable, but additional studies are
comparing PHMB and CLX monotherapies in 56 eyes with AK77 needed to support the use of this compound in the treatment
suggested that both biguanides are equally effective as mon- of AK as monotherapy or in combination with biguanides and
otherapy. Although CLX therapy showed slightly higher per- diamidines. Although a published study showed evidence of
centage of cases with successful treatment and improvement clinical improvement in AK patients with the use of topical
of visual acuity in comparison to PHMB, the differences were VRC, only a few cases were evaluated with limited therapeutic
not statistically significant.77 The diamidines are not recom- success.10
mended as monotherapy for AK because of the possible
development of resistance of the Acanthamoeba cysts to the
drug from the beginning of or during treatment.74,126 Combi- 3. Mechanisms of Acanthamoeba resistance
nation therapy of biguanides and diamidines108,127 is to biocides
commonly used to avoid resistant and persistent infections.
Furthermore, antimicrobial therapy regimens based on 3.1. Cystic resistance
concomitant administration of the 2 biguanide compounds
and an increase in the drug concentration to 0.04% or 0.06% The invasion and encystment of the protozoan into the deep
are alternative treatment modalities for resistant and unre- corneal layers may underlie the mechanism of Acanthamoeba
sponsive cases.10,36,70,88 A synergism between PHMB and CLX resistance to topical biocides in vivo103,106 and explain the lack
results in loss of cystic viability with minimal toxicity to the of correlation between the observed clinical responses and
human endothelial cells.83 in vitro sensitivity testing.31,108 The recrudescence of AK is a
Although biguanides and diamidines are commonly used clinical concern. Persistent and recurrent infections112,113,133
to treat AK, side effects caused by drug toxicity and sensitivity may result from encystment and cystic resistance to bio-
reactions on corneal tissue (Fig. 5) could represent an addi- cides and low levels of drug penetration into the deep corneal
tional complexity character on clinical signs.26,39,45 Ulti- stroma.2,26 Epithelial infections (first-stage disease) may be
mately, the impact of the toxicity caused by exposure to the mild-to-moderate in severity and are correlated with a better
drugs over an extended period of daily use has resulted in the treatment response and prognosis, whereas deep stromal in-
absence of corneal tissue regeneration and a worse post- fections (second-stage disease) may be associated with a
therapeutic outcome. For example, the toxicity of topical worse prognosis and delayed response to topical bio-
medications used for AK therapy may contribute to compli- cides.85,103 Cystic resistance to the available chemical drugs
cations reported in the ocular anterior segment, such as raises a question about the effective dose of antimicrobial
cataract, iris atrophy, and corneal ulceration.30,44,100 In addi- drugs, which limits the therapeutic window for AK therapy
tion to drug toxicity, chronic and severe inflammation of the and requires development of new biocides. The physical bar-
rier of the cystic wall provided by acid-insoluble proteins and
alkali-insoluble polysaccharides, the main components of the
ectocysts and the endocysts, respectively, mostly accounts for
the resistance to biocides,138 leading to a lower level of drug
absorption by cysts than trophozoites.137 Turner and col-
leagues138 studied biocide susceptibility during the differen-
tiation process of Acanthamoeba castellanii and observed that
the time of emerging resistance during encystment varies for
different biocides. Resistance to minimal amebicidal concen-
trations of PHMB and propamidine occurred 24 hours after
initiation of the encystment process. Resistance to CLX
developed during 36 hours. The smaller molecular size of CLX
compared with PHMB may contribute to the prolonged uptake
of the biguanide derivative in immature cysts.

3.2. Drug-resistant strains and acquired resistance

Fig. 5 e Ocular surface toxicity during treatment of Increased resistance to biocides may be correlated with some
Acanthamoeba keratitis translated clinically into signs of strains and species of Acanthamoeba.76,92,131,133 Despite the fact
superficial punctate keratitis. that T4 genotypes currently are associated with Acanthamoeba
208 s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 2 0 3 e2 1 8

pathogenic strains, non-T4 genotypes also have been identi- may require higher concentrations than CLX to achieve cys-
fied in severe cases of AK refractory to current medical ther- ticidal activity (Table 1). Additional studies are necessary to
apy.8,51 Acquired resistance to biocides during AK therapy also assess the efficacy of alexidine against different Acanthamoeba
may occur. Ledee and colleagues74 reported development of isolates. This biocide consists of a hexamethylene chain
resistance to propamidine, possibly related to emergence of a linking 2 biguanides attached to the ethylhexyl end groups.
temperature-sensitive phenotype unable to grow at 37
C that The chemical structure of alexidine is similar to that of CLX,
characterizes a more virulent strain.37 Moreover, a correlation with aliphatic chains in place of the chlorophenyl end groups.
between the clinical response and in vitro susceptibility test in The compound is formulated as a salt (alexidine hydrochlo-
a persistent culture-positive case with a good initial thera- ride, C26H56N10.2HCl).
peutic response to biguanide suggested development of
resistance during therapy.133 4.3. Polyhexamethylene biguanide

PHMB, also known as polyhexanide, is a linear polymer con-

4. Biguanides sisting of cationic biguanide groups connected by hexam-
ethylene chains (Fig. 6C ) with the chemical formula (C8H17N5)
4.1. Ophthalmic use n.xHCl, n  40. This compound is synthesized as a hetero-
disperse mixture of polymers with molecular weights ranging
Bioactive biguanides are organic compounds that are de- from 500 to 6000 g/mol.41 PHMB is commercially available in
rivatives of guanylguanidine (C2H7N5) (Fig. 6A).32 CLX and concentrated aqueous solutions (20%) such as pool care san-
PHMB are cationic biguanides with wide-spectrum antimi- itizer or as a disinfectant ingredient and preservative for
crobial activities that generally are used as cationic disinfec- cosmetic products under the trade names Baquacil, Vantocil,
tants, antiseptics, and preservatives.96 Both biguanides are and Cosmocil CQ (Arch Chemicals, Inc., Norwalk, CT, USA).
used as preservatives in eye drops17,39,58 or components of The average length of polymer chains is considered to be n ¼
contact lens disinfecting solutions.22,67 Once the trophocidal 12, with a reported average molecular weight of about 2400 g/
and cysticidal efficacy of the biguanides was recognized mol.1 Some feel that the PHMB activity is favored by long chain
against the Acanthamoeba spp, PHMB and CLX were used lengths (n > 10),41,59 but optimal biocidal activity of PHMB may
widely as first-line therapy for AK.43,73,81,139 Biguanide eye be achieved by the synergistic action of a mixture of small-
drops are unavailable commercially and usually are prepared and long-chain polymers. Small molecules are likely to be
at a compounding pharmacy from stock solutions (20%). absorbed easily, which destabilizes the microbial surface and
facilitates uptake of larger molecules of PHMB exhibiting
4.2. Bisbiguanides greater biocidal activity.55 The advantages of a polymeric
biocide are its nonvolatile property, chemical stability, and
4.2.1. Chlorhexidine gluconate long-term antimicrobial activity.59 The cysticidal activity of
CLX is a bisbiguanide (1,10 -Hexamethylenebis(5-(p-chlor- PHMB is time and concentration dependent.75,131 Several
ophenyl)biguanide)) (Fig. 6B). The chemical structure consists studies have reported the minimal amebicidal concentrations
of a hydrophobic hexamethylene chain linking 2 cationic of PHMB against the Acanthamoeba spp in vitro (Table 1). Aside
chlorophenylbiguanide groups.41 The molecular formula of from the polymer chain lengths, drug exposure times, strains
the CLX base moiety is C22H30Cl2N10 (molecular weight, 505 g/ used, and assay methods may account for differences in
mol), which forms a salt with 2 molecules of acetic acid or minimal amebicidal concentrations. Most reports have
gluconic acid. The activity and microbial uptake of CLX considered PHMB equal to or more effective than CLX against
depend on the pH and biocide concentration.96 Chlorhexidine trophozoites and cysts of the Acanthamoeba spp.64,65,69,75,131
forms micelles in aqueous solutions above the critical micelle Conversely, some studies have reported that CLX was more
concentration.122,148 The solubility of chlorhexidine salts is effective.102,138 These conflicting results may be affected by
gluconate > acetate > chloride. Solutions of chlorhexidine the strains used and in vitro assay conditions. Despite the
gluconate form aggregates, possibly by association of the differences, there were no wide variations in the effective
hexamethylene chain and a phenyl group of 2 adjacent mol- concentrations of PHMB and CLX, suggesting that both are
ecules, which increases drug solubility.148 The overall lower equally effective against Acanthamoeba in vitro.
molecular weight of CLX compared to PHMB may facilitate
absorption into the corneal stroma, but no data support this. 4.4. Ocular toxicity
Wide variations in the effective concentrations of CLX against
different isolated strains of Acanthamoeba spp have been re- The higher cysticidal activity of CLX and PHMB against Acan-
ported102 in different studies to assess the minimal amebici- thamoeba spp among other biocides126 allows use of low con-
dal concentrations against trophozoites and cysts (Table 1). centrations of the compounds to avoid corneal toxicity.
Cysticidal activity is time and concentration dependent.75,131 Generally, PHMB is safe and well tolerated for a wide range of
clinical applications47; however, data on the ocular toxicity of
4.2.2. Alexidine PHMB are scarce compared with CLX. Analysis of the ocular
Alexidine (1,10 -Hexamethylenebis(5-(2-ethylhexyl)biguani- tolerance of CLX, mainly its use as a preservative in contact lens
dedmolecular weight, 509 g/mol)), another antimicrobial disinfecting solutions, showed that it increases corneal
bisbiguanide used in contact lens disinfecting solutions, also permeability by affecting the corneal integrity and may cause
is effective against Acanthamoeba pathogenic strains,4,68 but contact allergic reactions, ocular redness, and photophobia. At a
 s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 2 0 3 e2 1 8 209

Fig. 6 e Chemical structures of compounds and selected biocides that display toxic activities against Acanthamoeba species.
A: Biguanide. B: Chlorhexidine. C: Polyhexamethylene biguanide. D: Propamidine. E: Hexamidine. F: Benzalkonium chloride.
G: Miltefosine. The counterions are omitted.
210 s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 2 0 3 e2 1 8

Table 1 e Minimal amoebicidal concentrations (MACs) of biocides against cysts and trophozoites of Acanthamoeba genus
Type Biocide MACsa (mg/L) on MACsa (mg/L) Species/number of References
trophozoites on cysts strains tested (no.)
28
First-line therapy (biguanides) Chlorhexidine <1e2 <1e16 CI (13)
57
12 25e50 Ac (1)
118
8 27 Ac (1)
67
nd w16e256 CI (19)
89
nd <2e100 CI (19)
66
nd 3e43 Ac (1), Alu (1)
112
nd <200 CI (56)
28
Polyhexamethylene <1e4 <1e4 CI (13, 23)
57
biguanide 12 25e50 Ac (1)
2 >500b Ac (1) 118

67
nd w16e256 CI (19)
89
nd 25e100 CI (19)
66
nd 2e13 Ac (1), Alu (1)
112
nd <200 CI (56)
28
First-line therapy Propamidine <1 2e500 CI (13, 23)
(diamidines) 18 >500b Ac (1) 57

67
nd w1e16 CI (19)
112
nd <1000 CI (56)
2
Hexamidine 5e7 200e205 Ap (3)
67
nd w4e256 CI (19)
Antifungal drugs Amphotericin B nd >256b CI (19) 67

Voriconazole >40b nd Ac (1), Ap (2) 106

nd >10,000b CI (56) 112

45
nd 5e80 CI (10)
112
Natamycin nd <5000 CI (56)
13
Caspofungin 250 500 Ac (1), Ap (1)
4
Other drugs and antimicrobial Alexidine <10 100 Ac (1), Ap (1), Ar (1)
28
compounds Pentamidine <1 1e500 CI (13)
15 >500b Ac (1) 118

67
nd w16e256 CI (19)
28
Neomycin 4e31 250e>500 CI (13, 23)
67
nd w64e256 CI (19)
57
Benzalkonium chloride 25e50 50e100 Ac (1)
118
12 40 Ac (1)
112
nd <500 CI (56)
Povidone-iodine nd >256b CI (19) 67

112
nd <1000 CI (56)
Miltefosine <16 >65b Ac (1), Ap (1), Ale (1) 121

106
16e33 nd Ap (2)
81
13 nd Ac (1), Ap (1)
86
13e204 nd Ac (1), Alu (1)

Ac, Acanthamoeba castellanii; Alu, Acanthamoeba lugdunesis; Ale, Acanthamoeba lenticulata; Ap, Acanthamoeba polyphaga; Ar, Acanthamoeba rhysodes;
CI, clinical isolates; nd, not determined.
a
The measured values vary among the assays used and the Acanthamoeba strains. For clarity, the values have been rounded to whole numbers.
b
Unable to kill Acanthamoeba spp up to the concentration tested.

high concentration (4%), CLX induced corneal injury, edema, the cytotoxicity of the biguanides seems dependent on the time
desquamation, and delayed healing. At a low (0.005%) concen- of cellular contact with the biocides. Short-term exposure of the
tration, CLX caused mild irritation and corneal opacities in an keratocyte culture to the minimal amebicidal concentrations of
experimental model of contact lens wear.39 Contact lens use PHMB and CLX resulted in viability rates above 90%.75
may worsen the adverse effects of the biguanides because CLX
and PHMB bind to soft contact lenses.39,111 Contact lenses
possibly transfer the compounds from the contact lens dis-
infecting solution to the ocular surface and increase retention of 5. Diamidines
the biocides in contact with the cornea. In vitro analyses have
shown that CLX has a toxic effect on conjunctival and corneal 5.1. Chemical properties and ophthalmic use
epithelial cells4,146; the effects of PHMB were not evaluated. A Aromatic diamidines are comprised of 2 benzene rings linked
comparative study of toxicity of the biguanides on corneal by an alkyl chain of variable lengths.96 Propamidine (4,40 -(1,3-
keratocytes suggested that CLX is less toxic than PHMB Propanediyl Bis(oxy))bis-benzene carboximidamide) (Fig. 6D)
regarding loss of cellular viability and morphology; however, and hexamidine (4,40 -(1,6-Hexane Diyl Bis(oxy))bis-benzene
 s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 2 0 3 e2 1 8 211

carboximidamide) (Fig. 6E ) contain a spacer chain of 3 (C3) and 6 plasma membranes, and consequent cytoplasmic leakage and
(C6) carbons, respectively. The chemical formulas of the PP and cellular death.59 Gilbert and Moore41 reviewed the detailed
HX base moieties are C17H20N4O2 and C20H26N4O2, yielding mechanisms of antimicrobial action of the biguanides and
molecular weights of 312 g/mol and 354 g/mol, respectively. other cationic biocides. The modes of action of PHMB and CLX
Diamidines are formulated as a salt of PP or HX and 2 molecules are distinct in some aspects. Each of the 2 cationic groups of
of isethionic acid. Diamidine eye drops are commercially chlorhexidine binds to a negatively charged head group of
available and indicated to treat minor bacterial eye infections in acidic phospholipids on the plasma membrane, establishing a
adults and children, in the form of PP 0.1% (Brolene, Patheon UK rigid link provided by the hexamethylene chain between
Ltd., Swindon, UK) or HX 0.1% (Desomedine, Chauvin Labora- neighboring phospholipids. Consequently, the biocide affects
tory, Montpellier, France). The diamidines have been used off- membrane fluidity with progressive loss of function of
label for AK therapy because the first successful treatment membrane-associated activities. At high CLX concentrations,
was reported using combination therapy of dibromopropami- the plasma membrane has a liquid crystalline state, which
dine isethionate ointment, neomycin, and propamidine eye disrupts the membrane bilayer.41 Leakage of cellular compo-
drops.144 Subsequently, Brasseur and colleagues16 reported nents has a biphasic dose response. At a first stage, leakage
that hexamidine also has in vitro anti-Acanthamoeba activity and increases with increasing CLX concentration, which affects
effectively treated patients with AK. The minimal amebicidal cellular membrane integrity and concurrently promotes
concentrations of PP and HX against cysts and trophozoites of internalization of chlorhexidine. Once CLX enters the cyto-
Acanthamoeba spp have varied among different studies plasm, it causes coagulation of proteins and nucleic acids,
(Table 1). Some authors have reported that pentamidine ise- leading to reduced leakage.96 The various cationic biguanide
thionate, a diamidine with an alkyl chain of 5 carbon atoms, has groups in the PHMB molecule allow binding to multiple
comparable or superior amebicidal activity to that of prop- phospholipids on the plasma membrane, leading to seques-
amidine.5,109 Another study found that PP had more potent tration of acidic phospholipids into clusters and consequent
activity against Acanthamoeba spp than pentamidine isethio- phase separation of membrane lipids.41,55 The coexistence of
nate and HX.76 Pentamidine is used clinically to treat trypano- domains in the fluid and gel phases in the plasma membrane
somiasis and leishmaniasis.11 This drug has also been used in leads to destabilization and disruption of membrane integ-
the treatment and prophylaxis of Pneumocystis pneumonia in rity.79 Hydrolysis of the phosphate ester bonds is another
immunocompromised patients.97 Currently, there is no clinical possible mechanism triggered by PHMB through interaction
evidence regarding the use of pentamidine isethionate as a with acidic phospholipids.55 PHMB also binds to microbial
topical treatment for AK. genomic DNA, altering gene expression at low doses, and
causing DNA precipitation at higher doses.7
5.2. Ocular toxicity The results of studies that described the anti-Acanthamoeba
activity of the biguanides, particularly the ultrastructural
Evaluation of propamidine and pentamidine toxicity in vitro changes in cysts and trophozoites, are consistent with the
showed that the drugs have minor toxic effects on corneal mechanisms of action described previously. In summary, the
epithelial and endothelial cells with short-term exposure. effects of both biguanides were disruption of membrane
Cellular contact with the diamidines for prolonged times at integrity with shortening of acanthopodia, leakage of cellular
concentrations effective against Acanthamoeba spp produced components, and shrinkage. CLX caused discrete rupture of
cytotoxic effects.5 There are no comparative data on the effect plasma membranes and membrane blebbing. PHMB produced
of hexamidine on corneal cells. Abnormal corneal signs more severe structural damage such as membrane disinte-
resulting from propamidine toxicity can mimic the signs of gration, dense precipitates on the cellular surface, and leakage
AK. Thus, abuse of propamidine eye drops may hinder diag- of intranuclear content. Cystic wall swelling also was
nosis of AK and therapeutic follow-up.53,66 In this context, observed.65,75 A proposed mechanism by which the biguanides
there is a lack of data on the safety assessment of diamidines overcome the barrier of double-walled cysts is through inter-
used to treat AK. Evaluation of ocular toxicity over the long action of the cationic groups with mucopolysaccharides on the
term should provide data on the safety of the diamidines to opercula, which mediates internalization of the biocide
treat corneal infections caused by Acanthamoeba spp. through the ostioles in the cystic wall and allows the biguanides
to bind to plasma membranes.126 Analyses of the cell contents
of Acanthamoeba cysts after exposure to PHMB by Raman
6. Mechanisms of action of the biguanides microspectroscopy suggested an internalization process of the
and diamidines PHMB compound in the cyst, which could elicit a cellular death
mechanism preceded by variations in intracellular biochemical
6.1. Biocidal activity of biguanides components, such as appearance of intracellular lipid vesicles,
degradation or denaturation of a-helix proteins, and nuclear
Knowledge of the molecular and cellular events involved in fragmentation followed by DNA degradation.119
the biocidal activity of the biguanide compounds has emerged
primarily from bacterial studies. Electrostatic interactions 6.2. Biocidal activity of diamidines
between the positive charges of the biguanides and negatively
charged biomolecules have an important role in the antimi- Most studies on the biocidal activity of the diamidines were
crobial activity of these compounds, which involves adsorp- performed in microorganisms other than Acanthamoeba spp.
tion to a negatively charged cellular surface, disruption of Similar to the biguanides, the diamidines are expected to have
212 s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 2 0 3 e2 1 8

the same modes of action among different microorganisms, drop formulations.136 The synthetic compound, also named
targeting key biomolecules of bacteria, fungi, and protozoa. alkyl dimethyl benzyl ammonium chloride (Fig. 6F ), is a
Although the mechanisms of action are not fully understood, quaternary ammonium salt with the linear formula
some processes involved in the antimicrobial activity of the C6H5CH2N(CH3)2RCl (R¼CnH2nþ1) containing an alkyl chain (R)
diamidines have been elucidated. The diamidines act as that varies in length. This biocide corresponds to a heteroge-
cationic surface-active agents and cause membrane disrup- neous mixture of homologues (n ¼ 8, 10, 12, 14, 16, 18). BAK is a
tion, leakage of amino acids, and inhibited oxygen uptake.96 cationic surface-active agent used as a surfactant and a broad-
Propamidine displays antifungal activity by affecting mito- spectrum antimicrobial agent. Disruption of the plasma
chondrial membrane integrity.145 Propamidine and pentami- membrane and increased cellular permeability are the main
dine interact with DNA molecules, binding reversibly to mechanisms involved in the antimicrobial action of the
adenine-thymine base pairs in the minor groove of double- biocide. The amphipathic property of BAK mediates its inter-
stranded DNA.52,143 Both biocides inhibit the enzyme S-ade- action with biologic membranes (for a review of quaternary
nosyl-L-methionine decarboxylase involved in the polyamine ammonium biocides, see Gilbert and Moore41). Ocular irrita-
metabolism of Acanthamoeba and promote encystment.48,126 tion, inflammatory reaction, and toxicity of the conjunctival
Interactions of the diamidines with other possible molecular cells and corneal epithelium are common adverse ef-
targets were summarized elsewhere.46 Perrine and col- fects.34,38,146 BAK decreases the transepithelial electric resis-
leagues109 proposed that the biocidal activity of the dia- tance in the cornea because of loss of intercellular tight
midines on Acanthamoeba involves electrostatic interaction junctions and acute toxicity.71 In vivo studies have shown that
with lipids on the plasma membrane, increased membrane the quaternary ammonium compound is more cytotoxic to
permeability and leakage, drug diffusion through the plasma the cornea than chlorhexidine.18 The propamidine formula-
membrane mediated by lipophilic interactions, and denatur- tion contains BAK as a preservative, which can increase
ation of intracellular proteins. According to this mechanism, corneal and conjunctival toxicity but may increase corneal
the biocide action of the diamidines is associated directly with drug absorption and the amebicidal effect of the eye drop
the cationic groups, while elongation of the alkyl chain to solution. Polyquaternium-1, commonly named Polyquad, is
increase lipophilicity may favor diamidine internalization into another quaternary ammonium compound used in
Acanthamoeba. Thus, this study suggested that hexamidine ophthalmic solutions as artificial tear preservative and con-
has higher amebicidal activity than propamidine. tact lens disinfectant. Recently, a case series demonstrated
that exposure to polyquaternium-1 caused a dendritiform
keratopathy, which may be misdiagnosed as AK.90
7. Other biocides
7.3. Voriconazole
7.1. Other biocides with amebicidal activity
VRC is a wide-spectrum triazole (C16H14F3N5Odmolecular
In addition to biguanides and diamidines, other compounds weight, 349 g/mol) effective against yeasts and molds134 that
have been evaluated for their in vitro antiprotozoal activity displays its antifungal activity by inhibiting ergosterol synthe-
against Acanthamoeba spp, including the wide-spectrum anti- sis, thus affecting the cell membrane. Ergosterol is an important
septics povidone-iodine and benzalkonium chloride (BAK), as constituent of the cell membrane of Acanthamoeba cysts and
well as antifungal drugs, for example, natamycin and vor- trophozoites and VRC may have the same mechanism of action
iconazole. The in vivo efficacy of these compounds for treating on the metabolism of the protozoan pathogen. A study showed
AK, along with ocular toxicity and bioavailability, must be that VRC induces programmed cell death in A castellanii.87
examined carefully. In a comparative study of the antiseptics,
antifungal drugs, propamidine, and biguanides on various 7.4. Miltefosine
clinical isolates, natamycin, povidone-iodine, and BAK were the
most effective cysticidal compounds.131 Previous studies have Miltefosine (hexadecylphosphocholine, C21H46NO4P) (Fig. 6G),
shown the antiamoebic properties of natamycin, povidone- another broad-spectrum biocide that displays cytotoxic ac-
iodine, and BAK.40,105,136 The efficacy of voriconazole against tivity against Acanthamoeba spp,25 is a phospholipid analogue
Acanthamoeba is controversial. For example, clinical and with a molecular weight of 408 g/mol obtained by chemical
experimental data suggested that VRC is effective against clin- modification of a quaternary ammonium compound by addi-
ical strains of Acanthamoeba,9,10,19,50,86,135 whereas other studies tion of an ethyl phosphate group.82 Miltefosine is a commer-
showed that VRC has a reversible amoebostatic effect and cially available anticancer drug for topical treatment of skin
showed low cysticidal activity.69,124,131 The minimal amebicidal metastases (Miltex, Baxter, UK) and an antiparasitic drug to
concentrations of these biocides and other drugs against Acan- treat leishmaniasis (Impavido, Aeterna Zentaris, Quebec,
thamoeba spp are summarized in Table 1. The main properties of Canada). The alkylphosphocholine inserts into the outer
some of these compounds are discussed in the following. leaflet of the plasma membrane. The mode of action involves
inhibiting several enzymes on the plasma membrane and
7.2. Benzalkonium chloride intracellularly hampering phospholipid metabolism and
affecting mitochondrial membrane potential.12,150 The inter-
Benzalkonium chloride, one of the most important pre- nalization process in the parasite cell observed in Leishmania
servatives used in eye drop medications,39,104 showed anti- spp is mediated by an aminophospholipid translocase.12
Acanthamoeba activity at the low concentrations present in eye Exposure of Acanthamoeba trophozoites to miltefosine
 s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 2 0 3 e2 1 8 213

resulted in cellular detachment, vacuolization, leakage of reassessment of the effective role of topical instillation of
cytoplasmic contents, and membrane disruption culminating corticosteroids in the inflammatory process during a corneal
in complete cellular lysis.141 Although complete eradication of infection by the protozoan. In addition to the devastating
Acanthamoeba cysts did not occur, the number of viable cysts protozoan effects on the cornea, the side effects by contin-
decreased by at least 99.9%.141 The susceptibility of Acantha- uous use of corticosteroids have to be considered. The use of
moeba spp to miltefosine is presented in Table 1. Some strains steroids to minimize the inflammatory reaction and pain is
had lower susceptibility to this biocide.99 Combination ther- controversial because it may increase Acanthamoeba patho-
apy for AK of miltefosine and PHMB was proposed based on genicity95 and be associated with worse disease outcomes,117
experimental results from in vivo assays.110 The phospholipid possibly due to increased susceptibility to protozoan inva-
analog was toxic to corneal epithelial cells.93 Further in- sion caused by host immunosuppression. Clinical findings
vestigations are needed to prove the efficacy of this compound revealed that, despite the potential benefits of topical corti-
for treating AK. Considering that systemic administration of costeroids during the course of AK therapy, the use of these
miltefosine causes toxicity and teratogenicity,28,132 studies of anti-inflammatory drugs before diagnosis of AK and initiation
the ocular toxicity of this drug should be undertaken to eval- of treatment results in worse outcomes.20,117
uate its use as a topical treatment for AK. Alternatively, nonsteroidal anti-inflammatory drugs emerge
as a promising therapy to control the inflammatory process of
7.5. Caspofungin recurrent and severe infection, which is characterized by in-
vasion of trophozoites into the deeper corneal layers and in-
Caspofungin is an antifungal drug from the class of echino- duction of a heavy inflammatory response by the host activated
candins that consists of a cyclic lipohexapeptide of 1093 Da by the immunogenic and antigenic characteristics of the Acan-
(C52H88N10O15). The drug caspofungin acetate (1213 g/mol) is thamoeba cysts.94,127 For example, flurbiprofen (phenylalkanoic
administered intravenously (Cancidas; Merck and Co., acid) is a nonsteroidal drug that provides anti-inflammatory
Whitehouse Station, NJ, USA). Caspofungin inhibits b-1,3- effects by specifically inhibiting the enzymatic activity of
glucan synthases by a competitive mechanism and impairs cyclooxygenase type 2 from the metabolic breakdown of
the polysaccharide synthesis, hindering cellular growth and arachidonic acid in the endocannabinoid system pathway of
triggering lysis of the microbial cell.33 Bouyer and colleagues15 the host. Furthermore, oral flurbiprofen in combination with
reported the efficacy of caspofungin against trophozoites and topical corticosteroids has been proposed as a therapeutic
cysts of Acanthamoeba spp. alternative to manage AK by controlling acute inflammatory
reactions, disproportionate pain, and severe scleritis.108

8. Effects of steroidal and nonsteroidal anti-

inflammatory agents on AK 9. Conclusions and future perspectives

The use of steroidal anti-inflammatory drugs to treat AK re- Considering the current knowledge about the safety and effi-
mains controversial and a critical issue in clinical practice. cacy of the available therapeutic compounds for treating AK,
Several implications based on basic and clinical investigations the biguanides are the first option, although the benefits of
into the effects of topical steroids on the metabolism of the choosing between CLX and PHMB remain unclear. The molec-
protozoan and in the inflammatory cascade of the patient ular weight affects drug permeability through the cornea,149
have been described previously.95,117 Available synthetic ste- facilitating absorption of smaller compounds into the deep
roids used clinically in ophthalmology to control serious and corneal layers, which may explain better clinical outcomes with
persistent inflammatory reactions of the disease include flu- chlorhexidine. It is difficult, however, to determine the efficacy
orometholone, hydrocortisone, prednisolone, and dexameth- of CLX for treating deep corneal stromal infections. In a clinical
asone eye drop solutions. Interestingly, the application of prospective study comparing both biguanides as monotherapy
prednisolone solutions as a chemical formulation of acetate or for AK, Lim and colleagues77 showed slightly better outcomes
phosphate to treat AK has been described.44 Although pred- with CLX than PHMB. The results were not statistically signifi-
nisolone acetate seems to be the favored anti-inflammatory cant, although the study power was too low to detect clinically
agent for treating keratitis in general, further research is meaningful differences.6 The authors concluded that both
needed to investigate each chemical formulation of prednis- drugs were efficacious as monotherapy. Long-term use of CLX
olone acetate and prednisolone phosphate to determine drug along with propamidine isethionate and benzalkonium chlo-
penetration through the epithelial and stromal layers, tissue ride as preservative may be associated with toxicity in patients
bioavailability, and cytotoxicity in the management of ocular with AK, such as progressive ulcerative keratitis and inflam-
inflammation during treatment development. Recently, Liv- mation of the anterior segment of the eye.30,44,100
ingstone and colleagues78 showed that an interaction between It is unclear whether the combined therapy of biguanide
chlorhexidine and dexamethasone eye drops formed an and diamidine is more effective than monotherapy. A syner-
amorphous glass-like precipitate, seen on confocal micro- gistic effect may potentiate the activity of these drugs against
scopy as crystal-like opacities in the corneal stroma of a pa- Acanthamoeba; however, induction of encystment by the dia-
tient during AK therapy. Corticosteroids are able to suppress midines through inhibition of polyamine metabolism may
macrophage phagocytic activity in the infection site and have have a negative effect on protozoan eradication. Differentia-
an inhibitory effect on neutrophil activation. The impairment tion of the protozoan into cysts can increase resistance to
of such innate host defense mechanisms has led to a critical delivered drugs and cause persistent infections especially in
214 s u r v e y o f o p h t h a l m o l o g y 6 2 ( 2 0 1 7 ) 2 0 3 e2 1 8

invasive forms of the disease characterized by deep stromal of Higher Education Personnel (CAPES grant number
infections. Efforts to improve therapy selection and to develop 23038.001063/2012-01). The sponsor or funding organization
novel chemotherapies for AK through translational research had no role in the design or conduct of this research.
will contribute to successful management of complex forms
of the disease. The effects of topical anti-inflammatory drugs
in AK management and drug interactions are other important references
issues worthy of future investigations.
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Linda Christian Carrijo-Carvalho is a postdoctoral fellow of 14. Boggild AK, Martin DS, Lee TY, et al. Laboratory diagnosis of
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for different types of clinical specimens. J Clin Microbiol.
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