Drug Resistance Updates 7 (2004) 41–51

Review
Opportunistic amoebae: challenges in prophylaxis and treatment
Frederick L. Schuster a,∗ , Govinda S. Visvesvara b
a Viral and Rickettsial Disease Laboratory, California Department of Health Services, Richmond, CA 94804, USA
b Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA

Received 29 December 2003; received in revised form 10 January 2004; accepted 10 January 2004

Abstract
This review focuses on free-living amoebae, widely distributed in soil and water, causing opportunistic and non-opportunistic infections
in humans: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. Diseases include primary amoebic
meningoencephalitis (N. fowleri), granulomatous amoebic encephalitis, cutaneous and nasopharyngeal infections (Acanthamoeba spp.,
Balamuthia mandrillaris, S. diploidea), and amoebic keratitis (Acanthamoeba spp). Acanthamoeba, Balamuthia, and Naegleria have been
repeatedly isolated; S. diploidea has been reported only once, from a brain infection. Antimicrobial therapy for these infections is gener-
ally empirical and patient recovery often problematic. N. fowleri is highly sensitive to the antifungal agent amphotericin B, but delay in
diagnosis and the fulminant nature of the disease result in few survivors. Encephalitis and other infections caused by Acanthamoeba and
Balamuthia have been treated, more or less successfully, with antimicrobial combinations including sterol-targeting azoles (clotrimazole,
miconazole, ketoconazole, fluconazole, itraconazole), pentamidine isethionate, 5-fluorocytosine, and sulfadiazine. The use of drug com-
binations addresses resistance patterns that may exist or develop during treatment, ensuring that at least one of the drugs may be effective
against the amoebae. Favorable drug interactions (additive or synergistic) are another potential benefit. In vitro drug testing of clinical
isolates points up strain and species differences in sensitivity, so that no single drug can be assumed effective against all amoebae. Another
complication is risk of activation of dormant cysts that form in situ in Acanthamoeba and Balamuthia infections, and which can lead to
patient relapse following apparently effective treatment. This is particularly true in Acanthamoeba keratitis, a non-opportunistic infection
of the cornea, which responds well to treatment with chlorhexidine gluconate and polyhexamethylene biguanide, in combination with
propamidine isothionate (Brolene), hexamidine (Désomodine), or neomycin. Acanthamoeba spp. may also be carriers of endosymbiotic
bacteria (Legionella and Legionella-like pathogens) and have been implicated in outbreaks of pneumonias in debilitated hosts. As with
other infectious diseases, recovery is dependent not only on antimicrobial therapy, but also on patient’s immune status, infective dose and
virulence of the ameba strain, and on how early the disease is diagnosed and drug therapy initiated.
© 2003 Elsevier Ltd. All rights reserved.
Keywords: Amoebic encephalitis; Amoebic keratitis; Acanthamoeba spp.; Balamuthia mandrillaris; Naegleria fowleri; Sappinia diploidea

1. Introduction granulomatous amoebic encephalitis, as well as dissemi-

Free-living amoebae belonging to the genera Acan-
thamoeba, Balamuthia, and Naegleria are responsible for
opportunistic and non-opportunistic infections in humans
and other animals (Martinez and Visvesvara, 1997). These
organisms can be isolated from soil and water, and are
widely spread in the outdoor and home (flowerpots, aquaria,
water taps and sink drains, humidifiers) environments. In
the soil habitat, they feed on bacteria, except for Balamuthia
which probably feeds on other amoebae. Persons devel-
oping infections include both immunocompromised and
immunocompetent individuals. Acanthamoeba spp. cause
∗ Corresponding author. Tel.: +1-510-307-8651.
E-mail addresses: fschuste@dhs.ca.gov (F.L. Schuster),
gsv1@cdc.gov (G.S. Visvesvara).
nated, cutaneous and nasopharyngeal infections, primarily
in immunocompromised individuals; they also cause amebic
keratitis in immunocompetent persons. Balamuthia man-
drillaris, a close relative of Acanthamoeba, causes similar
infections in both immunocompromised and immunocom-
petent hosts, the latter being mostly children. Naegleria
fowleri is the causal agent of primary amoebic menin-
goencephalitis, a non-opportunistic infection in otherwise
healthy children and young adults. There is no universal
cure for these amoebic infections. With few exceptions, an-
timicrobial therapy is largely empirical, and optimal therapy
has yet to be determined. The mouse serves as a useful ani-
mal model for study of amoebic encephalitides; the animal
is inoculated intranasally or intracranially with a suspension
of amoebae and the murine disease parallels the human
infection (Janitschke et al., 1996; Jarolim et al., 2000;

1368-7646/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.drup.2004.01.002
42 F.L. Schuster, G.S. Visvesvara / Drug Resistance Updates 7 (2004) 41–51
Martinez et al., 1973). Most drug testing, however, is carried
out in vitro.
2. Acanthamoeba spp.
The Acanthamoeba life-cycle consists of trophic and cys-
tic stages, with the latter being more resistant to antimi-
crobials. Approximately 17 species of Acanthamoeba are
recognized based on morphology and genomic sequencing
(Stothard et al., 1998), several of which cause opportunis-
tic infections (Marciano-Cabral and Cabral, 2003). Many of
the infections have been in HIV/AIDS and organ-transplant
patients, or persons in a debilitated state of health.
A broad array of drugs has been used to treat acan-
thamoebic infections and some successes have been reported
(Schuster and Visvesvara, 2003). Most cases, however, es-
pecially in hosts weakened by opportunistic infections or
made vulnerable by immunosuppressive medications, are fa-
tal. For amoebic encephalitis, the problem of finding opti-
mal antimicrobial therapy is compounded by difficulty in
diagnosing the infection. Lacking specific pathognomonic
symptoms, recognition of the infection is often at autopsy.
Even then, infections may be missed in routine histopatho-
logical reviews due to lack of familiarity with the amoebae.
In contrast to systemic infections caused by Acan-
thamoeba which have an insidious and subclinical course,
amoebic keratitis, a non-opportunistic infection, will cause
the patient to seek help because of pain, tearing, dimin-
ished vision, and photophobia. Diagnosis is by corneal
scraping and staining to visualize amoebae, or by culti-
vation of the scrapings for isolation and identification of
the amoeba (Schuster, 2002). Unlike protocols for bacte-
rial susceptibility testing, there are no reference strains of
Acanthamoeba or other amoebae to be used as standards
for testing amoebic susceptibility. During in vitro testing, it
is also important to distinguish between amoebastatic and
amoebicidal drugs. With the former, amoebae that appear
to be non-dividing or dead upon microscopic examination
recover following transfer to drug-free medium. Drugs must
also be cysticidal to prevent recurrence of infection from
activation of dormant cysts that have survived antimicrobial
treatment. Because of variable sensitivities, each isolate re-
quires individual testing for susceptibility to drugs and drug
concentrations, leading to sometimes contradictory results
from different laboratories.
2.1. Antimicrobial treatment for systemic Acanthamoeba
infections
Drugs that have been used in treating nasopharyngeal,
disseminated, and central nervous system infections in-
clude ketoconazole, fluconazole, itraconazole, pentamidine
isethionate, trimethoprim-sulfamethoxazole (cotrimoxa-
zole), sulfadiazine, and 5-fluorocytosine (flucytosine). Cuta-
neous infections have been treated with topical applications
of chlorhexidine gluconate and ketoconazole cream. Lo-
calized infections (e.g. cutaneous lesions) can develop into
disseminated infections through hematogenous spread. In
spite of several recoveries following drug therapy, the out-
look for most patients remains grim, especially those weak-
ened by multiple opportunistic infections. Some successful
treatment regimens have included pentamidine isethion-
ate, flucytosine, itraconazole, topical chlorhexidine and
ketoconazole cream (for skin lesions) in a lung transplant
patient with disseminated acanthamebiasis without central
nervous system (CNS) involvement (Oliva et al., 1999);
trimethoprim-sulfamethoxazole, rifampin, and ketoconazole
in two pediatric patients with CNS infections (Singhal et al.,
2001); cotrimoxazole, flucytosine, sulfadiazine in a pediatric
patient with CNS infection (Karande et al., 1991); intra-
venous pentamidine, topical chlorhexidine, and ketocona-
zole cream in a renal transplant patient with disseminated
infection (Slater et al., 1994); sulfadiazine, and fluconazole
in an HIV/AIDS patient with granulomatous encephalitis
(Martinez et al., 2000); flucytosine therapy following inef-
fective ketoconazole treatment in an HIV/AIDS patient with
cutaneous infection (Helton et al., 1993); multidrug therapy
for treatment of amoebic rhino-sinusitis in an HIV/AIDS pa-
tient (Rivera and Padhya, 2002). A combination of penicillin
and chloramphenicol was apparently successful in treating
a patient with meningitis in which Acanthamoeba culbert-
soni was recovered from the CSF (Lalitha et al., 1985); no
post-isolation in vitro testing of the amoeba was performed
to confirm sensitivity. A chronic CNS infection with Hart-
mannella (Acanthamoeba) rhysodes improved but was not
cured following sulfamethazine treatment (Cleland et al.,
1982). Acanthamoeba castellanii, isolated from skin nodules
of a fatal cutaneous infection in an HIV/AIDS patient, was
resistant in vitro to metronidazole, amphotericin B, rifampin,
pentamidine, 5-fluorcystosine, and itraconazole (Hunt et al.,
1995). Amphotericin B was found ineffective in treating a
fatal case of disseminated acanthamoebiasis (Gullet et al.,
1979). Recurrence of disease in cases that were success-
fully treated with antimicrobials has not been reported, but
the possibility exists that cysts in brain and other tissues
might reactivate to give rise to trophic amoebae. Table 1
provides a summary of selected successful antimicrobial
therapies that have been used in systemic Acanthamoeba
infections.
A number of other antimicrobials have been tested in
vitro for activity against Acanthamoeba isolates, but have
not yet been tried in the clinic. Azithromycin was inhibitory
for up to 98% of brain, skin, and corneal isolates, while the
related macrolides clarithromycin and erythromycin were
ineffective (Schuster and Visvesvara, 1998). Several phe-
nothiazines (e.g. chlorpromazine, trifluoperazine) showed
activity against Acanthamoeba clinical isolates (Schuster
and Visvesvara, 1998). Miltefosine (hexadecylphospho-
choline), an anti-cancer drug used for treating visceral
leishmaniasis (Sundar et al., 2002) is also active against En-
tamoeba histolytica (Seifert et al., 2001) and Acanthamoeba
 F.L. Schuster, G.S. Visvesvara / Drug Resistance Updates 7 (2004) 41–51 43

Table 1
in halting an infection is determined by a number of factors,
Examples of successful antimicrobial treatments of Acanthamoeba infec-
tions including the immune status of the host, the progression of
the disease and the time at which drug intervention is initi-
Antimicrobials Dosage Reference
ated, the infectious dose of amoebae, and the virulence and
Amebic encephalitis drug sensitivity profile of the particular strain causing the
Sulfamethazine 1 g qid Cleland et al. (1982)
infection.
Cotrimoxazole 75 mg/kg q12h IV Karande et al. (1991) Variations in both virulence and antibiotic sensitivity are
5-Fluorocytosine 150 mg/kg q6h encountered among clinical isolates of Acanthamoeba, even
Sulfadiazine 150 mg/kg q6h
between strains of a single species. With regard to differ-
Penicillin G 2 × 106 U q3h IV Lalitha et al. (1985) ences in virulence, some species and/or strains produce a
Chloramphenicol 500 mg q6h po
higher mortality in the mouse model or cause a more pro-
Sulfadiazine 500 mg qid Martinez et al. (2000)a nounced cytopathic effect in tissue cultures (De Jonckheere,
Pyrimethamine 50 mg qd 1980). A brain isolate (A. healyi) was more virulent than
Fluconazole 200 mg bid
isolates grown in tissue culture (A. culbertsoni) or those
Ketoconazole 5 mg/kg qd Singhal et al. (2001) from the environment when tested in an immunodeficient
Rifampin 10 mg/kg qd
mouse model (Kong et al., 1998). Likewise, mouse brain
TMP-SMX 20 mg/kg qd
and keratitis isolates were more cytopathogenic in tissue
Cutaneous infection cultures than isolates from tap water or contact lens cases
Ketoconazole 200 mg q8h IV Helton et al. (1993)a
5-Fluorocytosine 40 mg/kg q8h
(Walochnik et al., 2000). With regard to differences in
antimicrobial susceptibility, a comparative study of four
Pentamidine 4 mg/kg q24h IV Slater et al. (1994)
species of Acanthamoeba, found variations in sensitiv-
Topical chlorhexidine Cleansing bid
Ketoconazole cream 2% following ity to clotrimazole, with Acanthamoeba polyphaga being
cleansing most resistant and A. rhysodes most sensitive to the drug
Nasopharyngeal infection
(Stevens and Willaert, 1980). In a comparative study of
Pentamidine 4 mg/kg qd IV Rivera and Padhya azoles, clotrimazole showed the best inhibition against a
Levofloxacin 500 mg qd IV (2002)a different strain of A. polyphaga: clotrimazole (up to 99% in-
Amphotericin B 550 mg qd IV hibition) > bifonazole > ketoconazole > itraconazole > flu-
5-Fluorocytosine 2 gm q6h conazole (no inhibition) (Schuster, 1993). Even so, at the
Rifampin 600 mg bid po
Itraconaole 200 mg bid po
concentrations tested, the azoles were amoebastatic but not
amoebacidal.
Disseminated infection
The cell membrane is the target of azole compounds and
Pentamidine Dosages not given Oliva et al. (1999)
5-Fluorocytosine of amphotericin B (AMB). The azoles interfere with syn-
Itraconazole thesis of sterols that are incorporated into the membrane,
Topical chlorhexidine leading to defective membrane architecture, increased per-
Ketoconazole cream meability, and leakage of ions from the cell (Sande and
Abbreviations: qxh, every x hours; qd, every day; bid, twice a day; qid, Mandell, 1985). Ergosterol and 7-dehyrostigmasterol are
four times each day; po, oral administration; IT, intrathecal administration; major sterol membrane components of the non-pathogenic
IV, intravenous administration. A. castellanii (Smith and Korn, 1968) and the pathogenic A.
a HIV/AIDS patient.
culbertsoni strain A-1 (Mehdi et al., 1988). Cycloartenol is
the ergosterol precursor for A. polyphaga, whose conversion
isolates (Walochnik et al., 2002). Although not tested clini- is presumably blocked by azole activity (Raederstorff and
cally against Acanthamoeba, miltefosine may have potential Rohmer, 1985). The imidazoles (clotrimazole, bifonazole,
for treatment of systemic infections as well as amoebic ketoconazole) are more effective against Acanthamoeba than
keratitis (Seal, 2003). Ability of antimicrobials to enter the triazoles (itraconazole, fluconazole) (Schuster, 1993).
the brain and CSF are also important considerations. Mil- The polyene AMB acts by preferentially binding to ergos-
tefosine and the phenothiazines penetrate the blood-brain terol and producing pores in the cell membrane (Sande and
barrier as does azithromycin, although accumulation of the Mandell, 1985). It is not the drug of choice for acanthamoe-
latter in cerebrospinal fluid is reportedly low (0.015 ␮g/ml) bic infections, since many isolates are neither inhibited
(Jaruratanasirikul et al., 1996). nor killed by it (Duma and Finley, 1976). The pathogenic
Because of a multidrug treatment approach, there is lit- N. fowleri, however, is highly sensitive to AMB (see
tle or no evidence of clinical resistance to any of the drugs below).
used in treating Acanthamoeba infections. Drug combina-
tions might give rise to synergistic or additive effects that are 2.2. Antimicrobial treatment for Acanthamoeba keratitis
not apparent from in vitro testing of individual drugs. Drug
testing in the mouse model, simulating a clinical situation, Amoebic keratitis can result from corneal trauma or, more
is infrequent. The value of a drug or combination of drugs likely, the transfer of Acanthamoeba spp. either in their
44 F.L. Schuster, G.S. Visvesvara / Drug Resistance Updates 7 (2004) 41–51

Table 2
vegetative state or as dormant cysts to the corneal surface
Successful antimicrobial treatment regimens for Acanthamoeba keratitis,
through the use of improperly or poorly maintained contact listed chronologically
lenses. The contact lens storage case, for lack of proper
Antimicrobials Dosage Reference
cleaning, can support growth of bacterial biofilms, which
can turn the case into a culture vessel for the bacteria-eating Ketoconazole 200 mg bid po Hirst et al. (1984)
amoebae. Amoebae are transferred to the eye when the con- Miconazole drops 10 mg/ml qh
Gentamicin drops qid
tact lens is placed upon the corneal surface. Once established
there, they penetrate the stroma, from which they are difficult Propamidine isethionate 0.1% qh Wright et al. (1985)
to eradicate. Because there is a greater likelihood for diagno- Dipropamidine ointment 0.15% q4h
Neomycin q4h
sis of amoebic keratitis than for systemic infections, antimi-
crobial treatment can be initiated early in the course of infec- Brolenea eyedrops qds Yeoh et al. (1987)
Brolene ointment qds
tion and, with compliant and intensive drug use (often hourly
applications), prospects for recovery are excellent. Drug use, Clotrimazole drops 1% qh Driebe et al. (1988)
however, can induce encystment (Kilvington et al., 1990) Propamidine qh
Ketoconazole 200 mg bid po
and once drug levels have declined, can lead to excystation Topical N-PB-Gb qh
and flare-up of the infection. In order to preclude recurrence,
Itraconazole 50 mg qd po Ishibashi et al. (1990)
treatment must be continued for 3–4 months (Kosrirukvongs
Topical miconazole
et al., 1999; Ficker et al., 1990). Some of the drugs initially
used in treating these infections were marginally amoe- Neosporinc drops qid Sharma et al. (1990)
Miconazole drops 10 mg/ml qh
bacidal or cysticidal and allowed eventual recovery from
inhibition. The cyst is more likely to survive treatment ow- PHMBd 0.02% qh to q3h Larkin et al. (1992)
ing to its thick wall and impermeability (Aksozek et al., Chlorhexidine 0.02% in saline qh Seal et al. (1995)
2002). Distinctly higher drug concentrations are required Propamidine 0.1% qh
to destroy cysts than are needed to destroy trophic amoe- PHMB 0.02% qh to qid Murdoch et al. (1998)
bae (Kilvington et al., 1990; Turner et al., 2000), and the ±Propamidine 0.1% qh
process of encystation is accompanied by development of ±Chlorhexidine 0.1% qid
±Désomedinee
drug resistance (Lloyd et al., 2001; Larkin et al., 1992; Lim
et al., 2000; Turner et al., 2000; Wright et al., 1985). Re- Chlorhexidine 0.006% qh Kosrirukvongs
currence of infection from excysting amoebae may lead to et al. (1999)
repeated corneal transplants (keratoplasty) in attempts to PHMB 0.02% qh Pérez-Santonja
eliminate or reduce numbers of amoebae and restore vision. ±Chlorhexidine 0.02% qh et al. (2003)
±Brolene 0.1% qh
In more chronic situations or where stubborn drug resis-
±Désomedine 0.1% qh
tance has been encountered, enucleation (surgical removal
of the eye from its socket) is a last resort to halting the The dosages and scheduling indicated in the table are generally for initial
treatment, and drug applications taper off over time and with improvement.
infection. In some of the referenced papers, several case histories are presented with
Substantially more literature exists on the drug sensi- minor differences in treatments.
tivity of Acanthamoeba strains and species involved in a Brolene: propamidine isethionate and dipropamidine.
b N-PB-G: neomycin–polymyxin B–gramicidin.
amoebic keratitis, than in systemic infections. Some of the
c Neosporin: neomycin–polymyxin B–bacitracin.
earlier chemotherapies employed were topical neomycin– d PHMB: polyhexamethylene biguanide.
polymyxin B–bacitracin (Neosporin) with or without mi- e Désomodine (Desmodine): hexamidine.
conazole or ketoconazole (Sharma et al., 1990); oral itra-
conazole and topical miconazole (Ishibashi et al., 1990);
systemic ketoconazole and topical miconazole (Hirst 1993). Its use, however, is discouraged because of resis-
et al., 1984); topical clotrimazole plus other antimicro- tance of cysts, and possible neurotoxicity (Murdoch et al.,
bials (Driebe et al., 1988). Brolene eyedrops (an over-the 1998; Seal, 2003). Selected examples of successful an-
counter pharmaceutical containing propamidine isethionate timicrobial regimens for treatment of amoebic keratitis are
and dibromopropamidine) and ointment, available in the presented in Table 2. Anti-inflammatory agents (topical
United Kingdom, have been used successfully in treating steroids) are generally added to the anti-amoebic treatment
keratitis cases (Yeoh et al., 1987), but are not well-tolerated regimens (Larkin et al., 1992; Pérez-Santonja et al., 2003),
in the eye when used over long periods of time (Murdoch but caution has been suggested in their use because of sup-
et al., 1998; Wright et al., 1985; Yeoh et al., 1987). Re- pression of macrophage activity (Berger et al., 1990; Seal,
sistance to propamidine was found in six out of eight 2003).
clinical isolates of Acanthamoeba (minimal inhibitory A major improvement in treating amoebic keratitis came
concentration ≥ 500 ␮g/ml) (Pérez-Santonja et al., 2003). about with the use of two cationic antiseptics, chlorhexi-
Neomycin was effective against trophic amoebae in vitro, dine gluconate and polyhexamethylene biguanide (PHMB;
but less so against cysts (Hay et al., 1994; Varga et al., Bacquacil) (Elder and Dart, 1995; Larkin et al., 1992).
 F.L. Schuster, G.S. Visvesvara / Drug Resistance Updates 7 (2004) 41–51
These have become the drugs of choice for treating ker-
atitis. Chlorhexidine may have an advantage over PHMB
(Seal et al., 1995; but see Elder and Dart, 1995 for a dif-
ferent view). Used at a concentration of 0.02% or lower,
the two drugs have been shown to be clinically effective
against both vegetative amoebae and dormant cysts, and
are well-tolerated in the eye (Kosrirukvongs et al., 1999).
PHMB is a more potent cysticidal agent than propami-
dine (Larkin et al., 1992; Varga et al., 1993). PHMB was
used with or without other antimicrobials (chlorhexidine,
Brolene, and hexamidine, a diamidine compound marketed
in France as Désomedin) (Pérez-Santonja et al., 2003).
Chlorhexidine and PHMB have been used alone, or in
combination with propamidine isethionate and/or neomycin
(Hay et al., 1994; Lindquist, 1998), or with 0.1% hexami-
dine (Murdoch et al., 1998); they were also used in com-
bination (Murdoch et al., 1998), although the two together
might prove to be toxic (Seal, 2003).
Development of drug resistance during prolonged treat-
ment of infections is understandably a concern. Unlike the
situation for systemic infections where it is difficult to as-
sess drug resistance, it is more readily apparent in keratitis
when treatment does little to alleviate symptoms. Repeated
amoeba isolation from a persistent keratitis case demon-
strated resistance development to Brolene (propamidine) and
to an experimental arsenic compound (R6/56) in the course
of the infection, as shown by an increase in the minimal in-
hibitory drug concentration for the two compounds (Ficker
et al., 1990). Two keratitis cases were reported to be re-
fractory to PHMB, even though the isolated amoebae were
demonstrably sensitive to the compound in vitro (Murdoch
et al., 1998). The rationale for using combination drug ther-
apy is to minimize risk of selection for resistance during
treatment of an infection.
Other drugs for use in amoebic keratitis have been
tested in vitro. Magainins, a group of natural and syn-
thetic membrane-active peptides, tested favorably when
combined with silver nitrate (Schuster and Jacob, 1992).
Povidone-iodine (Betadine) was reported to be a better
amoebicidal agent than chlorhexidine when tested against
corneal isolates of Acanthamoeba in vitro (Gatti et al., 1998).
Activity of seven different diamidine compounds, including
propamidine and hexamidine, against Acanthamoeba was
shown to be proportional to the length of the alkyl chain
of the diamidine molecule, relating to its lipophilicity and
its ability to penetrate the amoeba cell membrane (Perrine
et al., 1995). Hexamidine was found to be more effec-
tive than propamidine, though but this has been recently
disputed (Seal, 2003). Myristamidopropyl dimethylamine
(MAPD) has been found to be effective in vitro against
both trophic and cystic stages of Acanthamoeba (Kilvington
et al., 2002), and did not select for resistance in amoeba
populations that survived exposure to low concentrations of
MAPD (Schuster et al., 2003). But for many drugs tested
against amoeba isolates, in vitro activity was no guarantee
of in vivo efficacy in amoebic keratitis (Elder et al., 1994;
45
Ficker et al., 1990; Lim et al., 2000; Pérez-Santonja et al.,
2003; Seal, 2003).
Animal models have been developed to study amoebic
keratitis, but they have not been used to evaluate antimicro-
bial efficacy (Badenoch et al., 1990; He et al., 1992; van
Klink et al., 1993). A readily available model for in vivo
testing has yet to be established. Corneal and other isolates
of amoebae produce cytopathogenicity in tissue cultures,
which can be used as an index of strain virulence (Badenoch
et al., 1995; Niszl et al., 1998). The ability of antimicrobials
to block cytopathic destruction of tissue culture monolayers
has, in turn, been used as a technique for drug evaluation
(Schuster and Visvesvara, 1998).
2.3. Acanthamoeba as carrier of pathogenic bacteria
Acanthamoeba isolates from environmental water and soil
samples (Newsome et al., 1998) as well as clinical samples
(Fritsche et al., 1993; Rowbotham, 1983) have been found
to harbor endosymbiotic bacteria. The most prominent of
these are Legionella spp. or Legionella-like bacteria, which
have had a role in community-acquired and nosocomial
pneumonias. Amoebae found in tap water, ventilating and
cooling systems and humidifiers are lysed by these bacteria,
and the bacteria are subsequently released in aerosols. Oc-
currences in hospitals and office buildings of legionellosis,
Pontiac and humidifier fevers, and atypical pneumonias have
been associated with Acanthamoeba spp. and pose a partic-
ular problem for elderly or immunocompromised patients.
Endosymbionts may also influence outcomes of cases of
amoebic keratitis (Murdoch et al., 1998). In addition to Le-
gionella spp., bacteria with disease potential found naturally
in Acanthamoeba and other amoebae are Parachlamydia
acanthamoeba (Maurin et al., 2002), Neochlamydia hart-
mannellae (Horn et al., 2000), and Rickettsia-like organisms
(Fritsche et al., 1999). Furthermore, Acanthamoeba can
be readily infected in vitro with bacteria including Afipia
felis, Burkholderia cepacia, Escherichia coli O157, Liste-
ria monocytogenes, Mycobacterium avium, Mycobacterium
bovis, Simkania negevensis, and Vibrio cholerae.
Little has been done to determine antimicrobial sensi-
tivities of naturally occurring endosymbionts, despite their
potential as etiologic agents of pneumonia. This is due in
part to the difficulty in culturing some of the endosymbiotic
bacteria. In a survey of patients with community-acquired
pneumonia, antibodies were detected to Legionella-like
bacterial and Parachlamydia strains associated with Acan-
thamoeba. Erythromycin plus a second antimicrobial (ce-
furoxime or ceftazidime) were effective in patient treatment
(Marrie et al., 2001). An in vitro study of antimicrobial
susceptibility of two strains of P. acanthamoeba derived
from amoebae compared the amoeba isolates with other
Chlamydia spp. The acanthamoebal isolates were resistant
to ␤-lactam and fluoroquinolone antibiotics, but were sen-
sitive to aminoglycosides and cotrimoxazole (Maurin et al.,
2002).
46 F.L. Schuster, G.S. Visvesvara / Drug Resistance Updates 7 (2004) 41–51

Table 3
3. Naegleria fowleri
Successful therapy in treatment of primary amoebic meningoencephalitis
caused by Naegleria fowleri
Over 30 species of Naegleria have been identified on the
Antimicrobials Dosage Reference
basis of sequencing data (De Jonckheere, 2002). Only one
species, N. fowleri, has been isolated from human infections, Amphotericin B 0.75 mg/kg qd IV Apley et al. (1970)
although several others are pathogenic in the mouse model. Amphotericin B 0.1 mg 5 times Brown (1991)
The amoeba has a life-cycle that includes amebic and cys- per day IT
tic stages and, for most species, a transient flagellate stage Rifampin 600 mg q12h
that develops from the vegetative ameba. The organism is Amphotericin B 1 mg/kg qd Jain et al. (2002)
the causal agent of primary amoebic meningoencephaltis Rifampin 450 mg qd po
(PAM) in immunocompetent children and young adults. The Ornidazole 500 mg q8h
infection is acquired by swimming or bathing in warm fresh Amphotericin B 1.5 mg/kg q12h IV Seidel et al. (1982)
waters, such as lakes, ponds, hot springs, and thermally pol- 1.5 mg per day IT
Miconazole 350 mg/m2 q8h IV
luted rivers or streams. Trophic amoebae, cysts (or even
10 mg per day IT
flagellates; Singh and Das, 1972) in the water enter into the Rifampin 10 mg/kg q8h po
nasal passages, penetrate the nasal epithelium, and migrate Sulfisoxazole 1 g q6h IV
along the olfactory nerves to the brain. PAM is a fulminant Amphotericin B 60 mg qd Wang et al. (1993)
disease, almost invariably fatal in a matter of days. Because Rifampin 450 mg qd
the disease has a short incubation period, and runs its course Chloramphenicol 1 g qid
within 7–10 days, early diagnosis and initiation of antimi- Dosages noted in table are initial drug levels used in treatment and were
crobial therapy are critical to patient survival. Diagnosis of reduced with time.
PAM is by microscopic identification and/or cultivation of
amoebae from freshly drawn CSF (Schuster, 2002).
treating PAM follow. In a rare instance of PAM in an
3.1. Antimicrobial therapy for PAM immunocompromised patient with systemic lupus erythe-
matosus, high concentrations of IV and IT AMB started on
The drug of choice in treating PAM is the antifungal hospital day 9 (IV at 0.5, and IT at 1.0 mg/kg) cleared the
polyene antibiotic AMB. Naegleria amoebae are highly CSF of amoebae, but the patient died on hospital day 40
sensitive to the drug, with a minimal amoebicidal concen- with amoebae present in the CSF (Shrestha et al., 2003).
tration of 0.026–0.078 ␮g/ml (Duma et al., 1971). But drug Similar clearing of amoebae from the CSF of an adult male
treatment must be started early in order to be effective and followed IT and IV administration of AMB not until hospi-
there are only a handful of recoveries published in the litera- tal day 3, and the patient expired on day 9 with numerous
ture out of an estimated 200 cases (Anderson and Jamieson, amoebae having reappeared in the CSF (Lawande et al.,
1972; Apley et al., 1970; Brown, 1991; Jain et al., 2002; 1980). Intravenous AMB (0.6 mg/kg of body weight) and
Seidel et al., 1982; Wang et al., 1993). In a well-documented ceftriaxone (100 mg/kg) were used unsuccessfully to treat a
recovery, the patient was given intravenous (IV) and in- pediatric case of PAM (Shenoy et al., 2002). In none of these
trathecal (IT) AMB, IV and IT miconazole, and oral ri- three cases was follow-up in vitro testing of the amoeba
fampin (Seidel et al., 1982). In vitro testing of the isolate done.
from the patient indicated a synergistic or additive effect of In addition to AMB, other antifungal drugs have been
AMB and miconazole, but found no efficacy for rifampin tested against Naegleria isolates in vitro and in mice in-
(Seidel et al., 1982). Amphotericin B, rifampin, and ornida- fected with the amoebae. Miconazole and ketoconazole were
zole were used successfully to treat a suspected PAM case shown in vitro to be active against N. fowleri, but not in the
(Jain et al., 2002). Unlike Acanthamoeba (and Balamuthia, mouse model (Elmsly et al., 1980). As noted above, micona-
see following section), which encyst in tissues, Naegleria zole, which is not available in the United States, was used
remains in the trophic form in brain tissue and, once amoe- in conjunction with AMB in successful treatment of PAM
bae are destroyed by effective antimicrobial treatment, the (Seidel et al., 1982), but it is not clear if it was the combi-
infection should not recur. Antimicrobial regimens for sev- nation of the two drugs or AMB alone that was effective.
eral cases of successful treatment of PAM are summarized in AMB, clotrimazole, and miconazole were found effective in
Table 3. vitro against N. fowleri (Duma and Finley, 1976). A com-
In cases where intensive AMB therapy was employed, parison of AMB with ketoconazole, fluconazole, and itra-
death of the patient is assumed to be due to the pathophys- conazole in vitro, found clinical isolates of N. fowleri to be
iology of disease or to delay in starting therapy, and not to most sensitive to ketoconazole and AMB, but less so to the
resistance to AMB. Naegleria isolates from deceased PAM triazoles itraconazole and fluconazole (Tiewcharoen et al.,
patients have all shown sensitivity to amphotericin B when 2002). A drawback in the use of the AMB is the risk of
tested in vitro (Duma and Finley, 1976; Tiewcharoen et al., impaired kidney function following drug use. In comparing
2002). Several examples of unsuccessful use of AMB in AMB with liposomal AMB, a less toxic form of the drug,
 F.L. Schuster, G.S. Visvesvara / Drug Resistance Updates 7 (2004) 41–51
the MIC for AMB was 0.1 ␮g/ml, while that of liposomal
AMB was 1 ␮g/ml (Goswick and Brenner, 2003b). Liposo-
mal AMB, however, was found less effective in the mouse
model and in vitro testing than the more toxic form of AMB.
AMB methyl ester, another AMB derivative less toxic than
the parent compound, was also found to be less effective in
the mouse model (Ferrante, 1982).
AMB targets the plasma membrane, as noted in the sec-
tion on Acanthamoeba. The drug acts by binding to ergos-
terol in the membrane, producing pores and causing loss of
small molecules (Sande and Mandell, 1985). Few studies
have been done on the sterol components of the Naegleria
membrane, and none on the membrane of N. fowleri. Cy-
cloartenol is the ergosterol precursor in N. lovaniensis; in
N. gruberi, the precursors are cycloartenol, lanosterol, and
parkeol (Raederstorff and Rohmer, 1987). In an ultrastruc-
tural study of the effect of AMB on N. fowleri and N. gruberi,
the drug produced distortions of nuclear shape, increase in
cytoplasmic membranes (rough and smooth varieties), mito-
chondrial abnormalities, appearance of autophagic vacuoles,
and plasma membrane blebbing (Schuster and Rechthand,
1975). These changes in the cell became more pronounced
with duration of exposure and increased concentration of
AMB.
The macrolide azithromycin has both in vitro and in
vivo (mouse model) efficacy against pathogenic Naegleria
(Goswick and Brenner, 2003a; Schuster et al., 2001). Clar-
ithromycin was less effective than azithromycin in vitro,
and erythromycin was minimally effective (Schuster et al.,
2001). As noted earlier, azithromycin levels in the cere-
brospinal fluid have been found to be low (Jaruratanasirikul
et al., 1996).
Like clinical Acanthamoeba isolates, variations in viru-
lence exist among N. fowleri isolates. Cytopathogenicity for
tissue culture cells is often regarded as an indicator of viru-
lence (John and John, 1989, 1994). Differences in virulence
are also reflected in mortality in the mouse model (Wong
et al., 1977). The amoeba isolated from the successfully
treated PAM case mentioned above (Seidel et al., 1982), was
found to be among the least cytopathic for monkey kidney
cell cultures among several N. fowleri isolates, with 7 days
required for destruction of the tissue culture monolayer as
compared to 2–6 days for others (John and John, 1989).
Furthermore, it was also the most sensitive to phenothiazine
compounds of several strains tested in vitro (Schuster and
Mandel, 1984). A possible conclusion is that a combination
of low virulence and greater drug sensitivity of the isolate
were factors in survival of the patient in that particular
case.
4. Balamuthia mandrillaris
Balamuthia, like Acanthamoeba, has trophic and cystic
stages in its life-cycle (Visvesvara et al., 1993). The amoeba
was first isolated from the brain of a pregnant mandrill ba-
47
boon that had died in a zoological park in ∼1985, and was
soon recognized in human and other animals as the cause
of cutaneous, nasopharyngeal, and central nervous system
infections (Martinez and Visvesvara, 1997). All isolates are
morphologically and genomically alike, and are members of
a single species. The earliest reported cases were in immuno-
suppressed, immunocompromised, or debilitated hosts, but
later cases have occurred in immunocompetent children. We
are aware of more than 100 cases on record at the Centers
for Disease Control and in the literature since the organism’s
recognition, all but a few being fatal. Like acanthamoebi-
asis, balamuthiasis is an insidious disease taking up to 2
years to develop in the host. Diagnosis is difficult and is
based upon finding amoebae in biopsied brain and other tis-
sues, or by immunofluorescence staining for Balamuthia an-
tibodies in patient serum (Schuster et al., 2001; Visvesvara
et al., 1993). The amoeba has also been isolated from biop-
sied brain tissue into tissue culture using monkey kidney or
other cell-types as a feeder layer (Schuster, 2002). Because
of lack of familiarity with the amoeba, it is likely that cases
are undiagnosed and the disease underreported. Most diag-
noses are made postmortem.
4.1. Antimicrobial therapy for Balamuthia infections
There are few successful treatments for Balamuthia
amoebic encephalitis (BAE). If the infection can be rec-
ognized early in its course, there is an opportunity for
antimicrobial intervention. Two such cases reported in the
literature had successful outcomes, both having used sim-
ilar combinations of antimicrobial agents (Deetz et al.,
2003). The drugs used included flucytosine, pentamidine
isethionate, fluconazole, sulfadiazine, a macrolide antibi-
otic (azithromycin or clarithromycin), and phenothiazines
(thioridazide or trifluoperazine). Amoebae, however, were
not isolated from either patient for in vitro drug sensitiv-
ity testing. In vitro results on drug activity against other
clinical isolates of Balamuthia found pentamidine isethion-
ate > azithromycin > amphotericin B to be most effective in
that order, while fluconazole, flucytosine, and sulfadiazine
had limited efficacy (Schuster and Visvesvara, unpublished
observations). It may be that in vivo the drugs have syner-
gistic effects that are not apparent in vitro. A concern in
these two cases was for recurrence of the infection as a
result of reactivation of dormant cysts in brain lesions. Both
patients, however, have shown no evidence of reactivation
of disease over periods of about 5 and 7 years, although they
remain on fluconazole plus sulfadiazine or clarithromycin
(Deetz et al., 2003). The drug regimens for the two cases, a
64-year-old male and a 5-year-old female, are summarized
in Table 4.
Pentamidine isethionate has been used in treatment of
CNS infections by Acanthamoeba (see above) as well as
Balamuthia. While demonstrably effective in vitro against
Balamuthia amoebae, it was reported to have relatively poor
penetration into the CNS when tested in HIV/AIDS patients
48 F.L. Schuster, G.S. Visvesvara / Drug Resistance Updates 7 (2004) 41–51
Table 4
Successful treatment of two patients with confirmed Balamuthia amoebic
encephalitis
Antimicrobials Dosage
Patient: 64-year-old male
Flucytosine 2 g q6h
Fluconazole 400 mg qd
Pentamidine isethionatea 4 mg/kg qd IV
Sulfadiazine 1.5 g q6h
Azithromycin, clarithromycinb 500 mg qd
Trifluoperazine 10 mg q12h
Patient: 5-year-old female
Flucytosine 110 mg/kg qd
Fluconazole 14 mg/kg qd
Pentamidine isethionatea 1 mg/kg qd
Azithromycin, clarithromycinb 14 mg/kg qd
Thioridizine 1 mg/kg qd
For detailed account of drug therapy and side effects, see Deetz et al.
(2003).
a Discontinued because of developing hyperglycemia.
b Switched from azithromycin to clarithromycin because of concern
about poor penetration into the CNS, and side effects (elevated creatinine
level, possible nephritis).
(Donnelly et al., 1988). Its use had to be discontinued in
both cases cited because of side effects. In its favor, pen-
tamidine was shown to concentrate within hemoflagellate
and malaria parasites through selective membrane transport
mechanisms and, despite extensive prophylactic use of the
drug in parts of Africa, development of resistance was rare
(Bray et al., 2003). Diamidines with improved ability to
cross the blood-brain barrier and lower toxicity would be
highly desirable in treating amoebic and other CNS infec-
tions (Bray et al., 2003).
There is no information on the membrane composition
of Balamuthia amoebae. Because of its close relationship
to Acanthamoeba (Booton et al., 2003), it may also have
similar membrane sterols (ergosterol and its precursor cy-
cloartenol) (Raederstorff and Rohmer, 1985). Clinical iso-
lates show variable in vitro susceptibility to amphotericin B,
but are relatively insensitive to the triazole compound, flu-
conazole (Schuster, unpublished observations). More testing
with other azoles is needed to obtain a sensitivity profile of
these membrane-active compounds.
By far the majority of cases (∼98%) on record, whether
BAE, cutaneous or nasopharyngeal infections, have been
fatal. Even with antemortem diagnosis of BAE, initiation of
effective antimicrobial therapy may come too late to help
the patient (Bakardjiev et al., 2003). The use of flucytosine,
fluconazole, pentamidine, and clarithromycin at regular or
high dosages was ineffective in four Argentinean pediatric
BAE cases (Galarza et al., 2002). Miltefosine (hexade-
cylphosphocholine) was tested against clinical isolates of
Balamuthia and has minimal inhibitory concentrations of
30–40 ␮M (Schuster, unpublished data). Its ability to cross
the blood-brain barrier would make it a potential drug for
treating BAE.
5. Sappinia diploidea
A unique case of CNS infection with S. diploidea in an im-
munocompetent individual was treated with azithromycin,
pentamidine, itraconazole, and flucytosine following surgi-
cal excision of the necrotic lesion. The infection may have
developed from an earlier sinus infection. The patient re-
covered, apparently without neurologic impairment (Gelman
et al., 2003).
6. Conclusions
Progress has been made in treatment of encephalitis and
related infections caused by the free-living amoebae Acan-
thamoeba and Balamuthia. While new drugs and combina-
tions of drugs have been tried in clinical situations, these
amoebic diseases continue to have a high mortality. Part of
the reason is due to difficulty in diagnosing the diseases,
and the resultant delay in initiating effective therapy. More
often than not, diagnosis is made at autopsy. It is likely that
amoebic encephalitides are underreported, given the lack of
familiarity of physicians and pathologists with the diseases
and their causal agents. For the most part, combinations of
drugs have been used in treatment and, while mortality re-
mains high, successful recoveries have been published in the
literature. Any indications of drug resistance are hidden by
the multidrug approach used in treating infections. Naegleria
fowleri, the causal agent of meningoencephalitis, is sensi-
tive to amphotericin B and other antifungal compounds, but
the fulminant nature of the disease, coupled with diagnostic
delay, is often a critical factor in determining outcome.
Acanthamoeba keratitis is a discrete infection that is more
amenable to antimicrobial treatment than systemic infec-
tions. More readily recognized because of its immediate ef-
fect on the comfort and well-being of patients, the infection
responds favorably to treatment with cationic disinfectants
(chlorhexidine, polyhexamethylene biguanide), which have
now become the drugs most frequently used in treatment.
Still, new compounds with amoebacidal and cysticidal ac-
tivity are needed for use in the few situations where drug
resistance is encountered. Myristamidopropyl dimethy-
lamine and hexadecylphosphosphocholine (miltefosine) are
two compounds that show promise for use in recalcitrant
keratitis infections, but they have yet to be tested clinically.
The recognition of an unusual soil amoeba, S. diploidea,
as the cause of a brain lesion indicates that other free-living
amoebae are capable of causing infections in humans, and
these can pose challenges to the development of effective
antimicrobial therapy.
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