Experimental Parasitology xxx (2014) xxx–xxx

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Experimental Parasitology

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Combined drug therapy in the management of granulomatous amoebicencephalitis due to Acanthamoeba spp., and Balamuthia mandrillaris

http://dx.doi.org/10.1016/j.exppara.2014.03.0250014-4894/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Address: Department of Biological and BiomedicalSciences, Aga Khan University, Stadium Road, Karachi, Pakistan. Fax: +92 (0)213493 4294.

E-mail address: Naveed5438@gmail.com (N.A. Khan).1 Both authors contributed equally to this manuscript.

Please cite this article in press as: Kulsoom, H., et al. Combined drug therapy in the management of granulomatous amoebic encephalitis due to Amoeba spp., and Balamuthia mandrillaris. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.03.025

Huma Kulsoom 1, Abdul Mannan Baig 1, Ruqaiyyah Siddiqui, Naveed Ahmed Khan ⇑Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan

h i g h l i g h t s

� Granulomatous amoebic encephalitisalmost always results in death.� Combinations of clinical drugs were

tested against brain-infectingamoebae.� At 100 lM, prochlorperazine with

either loperamide or apomorphinewere amoebicidal.� Procyclidine plus loperamide also

showed potent amoebicidal effects.� These findings may provide novel

strategies for therapy against GAE.

g r a p h i c a l a b s t r a c t

.

a r t i c l e i n f o

Article history:Received 27 November 2013Received in revised form 19 March 2014Accepted 26 March 2014Available online xxxx

Keywords:Acanthamoeba castellaniiBalamuthia mandrillarisClinical drugsIn vitroAmoebicidalEncephalitis

a b s t r a c t

Granulomatous amoebic encephalitis (GAE) is caused by two protist pathogens, Acanthamoeba spp., andBalamuthia mandrillaris. Although rare, it almost always results in death. In the present study, amoebaewere treated with various combinations of clinically-approved drugs, targeting vital cellular receptorsand biochemical pathways. The results revealed that among the seven different combinations tested,three proved highly effective against both Acanthamoeba castellanii as well as B. mandrillaris at aconcentration of 100 lM. These combinations included (i) prochlorperazine plus loperamide; (ii)prochlorperazine plus apomorphine; and (iii) procyclidine plus loperamide. In viability assays, none ofthe drug-treated amoebae emerged as viable trophozoites, suggesting irreversible amoebicidal effects.Four combinations of drugs tested showed varied potency against A. castellanii and B. mandrillaris at100 lM. The combination of haloperidol and loperamide was highly effective against A. castellanii at100 lM, but potent effects against B. mandrillaris were observed only at 250 lM. Digoxin and amlodipinewere effective against A. castellanii and B. mandrillaris at 100 lM and 250 lM, respectively. In contrast,the combination of apomorphine and haloperidol was effective against B. mandrillaris and A. castellaniiat 100 lM and 250 lM, respectively. At 100 lM, the combination of procyclidine and amiodarone waseffective against neither A. castellanii nor B. mandrillaris. In this case, amoebicidal properties wereobserved at 750 lM for A. castellanii, and 950 lM for B. mandrillaris. As these drugs are used clinicallyagainst non-communicable diseases, the findings reported here have the potential to be tested in aclinical setting against amoebic encephalitis caused by A. castellanii and B. mandrillaris.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

Granulomatous amoebic encephalitis (GAE) is a rare but serioushuman disease leading almost always to death (Martinez andVisvesvara, 1997; Visvesvara et al., 2007). It is caused by two protist

cantha-

2 H. Kulsoom et al. / Experimental Parasitology xxx (2014) xxx–xxx

pathogens, Acanthamoeba spp., and Balamuthia mandrillaris. Earlydiagnosis, followed by aggressive treatment using a combinationof drugs is a pre-requisite in successful treatment, but even then,the prognosis remains poor (>90% case fatality rate). Existing drugshave limitations due to a high degree of toxicity associated with del-eterious side effects and limited ability to cross the blood–brain bar-rier (Schuster and Visvesvara, 2004).

The clinical symptoms of GAE include headache, fever, behav-ioral changes, lethargy, stiff neck, aphasia, ataxia, nausea, cranialnerve palsies, confused state, seizures, coma, which finally leadto death. Patients exhibit haemorrhagic necrotizing lesions or brainabscess (detected by neuroimaging scans) with severe meningealirritation and encephalitis (Martinez and Visvesvara, 1997). Postmortem examination often shows severe oedema and haemor-rhagic necrosis. The granulomatous response may be absent orminimal in patients with a severely impaired immune system(Martinez and Visvesvara, 1997; Visvesvara et al., 2007). AlthoughGAE can occur in healthy individuals, immunocompromised ordebilitated patients due to HIV infection, diabetes, immunosup-pressive therapy, malignancies, malnutrition, and alcoholism areparticularly at risk (Visvesvara et al., 2007). At present, there isno recommended treatment for GAE and the majority of casesare identified at the post-mortem stage. Current therapeutic agentsinclude a combination of drugs including ketoconazole, fluconazole,albendazole, itraconazole, sulfadiazine, pentamidine isethionate, flu-cytosine, trimethoprim/sulfa-methoxazole, clarithromycin, rifampinand azithromycin (Schuster and Visvesvara, 2004; Visvesvara et al.,2007), while miltefosine, phenothiazines-thioridazine, trifluoperazinehave shown promise in the experimental studies (Diaz, 2011; Kottinget al., 1992; Walochnik et al., 2002). It is distressing to note thatdespite advances in antimicrobial chemotherapy and supportive care,the fatality rate associated with GAE due to Acanthamoeba spp., and B.mandrillaris has remained >90%. Recently, we screened clinicallyapproved drugs, targeting vital cellular receptors and biochemicalpathways against Acanthamoeba spp., and B. mandrillaris. Whentested against B. mandrillaris, the findings revealed that amlodipine,apomorphine, haloperidol, loperamide, prochlorperazine, and procy-clidine showed potent amoebicidal effects, while amiodarone, anddigoxin exhibited minimal cytotoxic effects. When tested againstAcanthamoeba castellanii, the findings revealed that amlodipine, pro-chlorperazine, loperamide, amiodarone, procyclidine, digoxin, apo-morphine exhibited amoebicidal effects, while haloperidol exhibited

Table 1Clinically available drugs tested in combination in the present study and their known mo

Drug Mode of action

Amiodarone � Class III antiarrhythmic agent, amiodarone shows beta blockelike actions� Binding to the nuclear thyroid receptor

Amlodipine � Calcium antagonist that inhibits the transmembrane influx of ccle and cardiac muscle� Acts as a functional inhibitor of acid sphingomyelinase sphing

tion and apoptosis, or cell deathApomorphine � Non-selective (D) dopaminergic receptor agonist

� An antagonist at 5-HT and a-adrenergic receptorsDigoxin � Binds to sodium/potassium-transporting ATPase alpha-1 chain

cells and other tissues� Inhibitor of cholesterol side-chain cleavage enzyme, mitochon

Haloperidol � Dopamine antagonist� Muscarinic receptor antagonist� Histamine antagonist

Loperamide � l-Opioid receptors agonist� Calmodulin binder

Prochlorperazine � Dopamine (D2) receptor antagonist� Muscarinic receptor antagonist� Histamine antagonist

Procyclidine � Blocks the neurotransmitter acetylcholine in the central and thrinic receptors

Please cite this article in press as: Kulsoom, H., et al. Combined drug therapy inmoeba spp., and Balamuthia mandrillaris. Exp. Parasitol. (2014), http://dx.doi.or

minimal cytotoxic effects. The present study was designed to deter-mine synergistic effects of drugs. Drugs targeting distinct pathwayswere given in combination at micromolar concentrations and theiramoebicidal properties were elucidated.

2. Materials and methods

All chemicals were purchased from Sigma (Poole, Dorset, UK),unless otherwise stated. Among the various drugs tested, amlodip-ine, apomorphine, and loperamide were purchased from Sigma;procyclidine was purchased from Auden McKenzie Pharma; halo-peridol was purchased from Searle Pharma Ltd.; amiodarone andprochlorperazine were purchased from Sanofi-Aventis, and digoxinwas purchased from GlaxoSmithKline. All drugs tested, their modeof action and proposed combination are indicated in Table 1.

2.1. Human brain microvascular endothelial cell culture

Primary human brain microvascular endothelial cells weregrown in RPMI-1640 containing 10% foetal bovine serum, 10%NuSerum, 2 mM glutamine, 1 mM pyruvate, penicillin (100 U/ml),streptomycin (100 U/ml), non-essential amino acids and vitaminsas previously described (Stins et al., 1997; Alsam et al., 2003).

2.2. Acanthamoeba cultures

A. castellanii belonging to the T4 genotype (American Type Cul-ture Collection, ATCC 50492), was grown in 10 mL of PYG medium[0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract, and 1.5%(w/v) glucose] in T-75 tissue culture flasks at 37 �C without shak-ing as described previously (Khan and Siddiqui, 2009). The mediawere refreshed 15–20 h prior experiments. A. castellanii adheringto flasks represented the trophozoite form and were collected byplacing the flasks on ice for 30 min with gentle agitation and usedin all experiments.

2.3. Cultures of B. mandrillaris

B. mandrillaris, originally sourced from the brain of a mandrillbaboon were obtained from American Type Culture Collection,ATCC 50209. For routine culturing, 106 amoebae (suspended in

de of action.

Combinations of drugs tested in thepresent study

r-like and potassium channel blocker- 1. Haloperidol + loperamide2. Prochlorperazine + loperamide3. Procyclidine + loperamide4. Prochlorperazine + apomorphine5. Digoxin + amlodipine6. Apomorphine + haloperidol7. Procyclidine + amiodarone

alcium ions into vascular smooth mus-

omyelin is involved in signal transduc-

to inhibit its function at heart muscle

drial

e peripheral nervous system at Musca-

the management of granulomatous amoebic encephalitis due to Acantha-g/10.1016/j.exppara.2014.03.025

H. Kulsoom et al. / Experimental Parasitology xxx (2014) xxx–xxx 3

10 mL of RPMI-1640) were inoculated on HBMEC monolayers inT-75 tissue culture flasks. The flasks were incubated in a 5%CO2 incubator at 37 �C. The amoebae consumed HBMEC within48 h and produced approximately 5–8 � 106 amoebae (>95%trophozoites), which were subsequently used for experiments(Siddiqui et al., 2007).

2.4. Amoebicidal assays

For amoebicidal effects, A. castellanii were incubated with dif-ferent combination of drugs (Table 1) at various concentrations(100 lM to 1 mM) in phosphate buffered saline (PBS) in 24-wellplates (5 � 105 amoebae/mL/well). Plates were incubated at 37 �Cfor 24 h. After this incubation, amoebae were centrifuged for10 min at 1000�g and supernatant were aspirated, followed bythe addition of 0.5 mL of PBS. This process was repeated 3X toremove extracellular drug. Finally, amoebae were resuspended inPYG and inoculated in 24-well plates (0.5 mL PYG/well). Plateswere incubated at 30 �C for 48 h and emergence of trophozoiteswas considered as viable amoebae, and the absence of viableamoebae was considered as an amoebicidal effect. In some exper-iments, plates were incubated for up to a week to observe theemergence of viable trophozoites.

To determine amoebicidal activity of drugs against B. mandril-laris trophozoites, amoebae (3 � 105 amoebae/0.5 mL/well) wereincubated with different combination of drugs (Table 1) at variousconcentrations (100 lM to 1 mM) in RPMI-1640. Plates were incu-bated in a 5% CO2 incubator at 37 �C for 24 h. After this incubation,amoebae were centrifuged for 10 min at 1000�g and supernatantwere aspirated, followed by the addition of 0.5 mL of PBS. This pro-cess was repeated 3X to remove any extracellular drug. Finally, B.mandrillaris were resuspended in RPMI-1640 and inoculated in24-well plates containing HBMEC monolayers as food source.Plates were incubated in a 5% CO2 incubator at 37 �C for up to48 h and emergence of trophozoites was considered as viableamoebae, and absence of amoebae as well as intact HBMEC mono-layers was considered as non-viable amoebae. In some experi-ments, plates were incubated for up to a week to observe theemergence of viable trophozoites.

3. Results

3.1. At micromolar concentration, the combined drug therapy showedup to 100% kill of B. mandrillaris

Our previous studies have shown that at 500 lM concentration,amlodipine, apomorphine, haloperidol, loperamide, prochlorpera-zine, and procyclidine show potent amoebicidal effects, while ami-odarone, and digoxin exhibited minimal cytotoxic effects against B.mandrillaris. In the present study, amoebae were treated with var-ious combinations of drugs, targeting distinct biochemical path-ways. The results revealed that 100 lM prochlorperazine plus100 lM loperamide show potent amoebicidal effects (Table 2 and

Table 2The effects of clinically available drugs tested in different combinations against B. mandrillaramoebae were washed to remove extracellular drugs and inoculated in growth medium. Ggrowth was considered as potent irreversible amoebicidal effects.

Drugs combination (100 lM) Growth

Haloperidol + loperamide +Prochlorperazine + loperamide �Procyclidine + loperamide �Prochlorperazine + apomorphine �Digoxin + amlodipine +Apomorphine + haloperidol �Procyclidine + amiodarone +

Please cite this article in press as: Kulsoom, H., et al. Combined drug therapy inmoeba spp., and Balamuthia mandrillaris. Exp. Parasitol. (2014), http://dx.doi.or

Fig. 1). To further confirm their effect on viability, drug-treatedamoebae were inoculated on HBMEC. No viable amoebae wereobserved post-treatment, and HBMEC monolayers remained intact,indicating the irreversible effects of drugs (Fig. 1). Even longerincubations of up to 4 days did not allow the re-emergence ofamoebae trophozoites. Nonetheless, viable B. mandrillaris tropho-zoites emerged in control wells (Fig. 1). Similarly, 100 lM procycli-dine plus 100 lM loperamide showed 100% kill rate and no viableamoebae were observed post-treatment (Table 2 and Fig. 1). At100 lM concentration, the combinations of prochlorperazine plusapomorphine, and apomorphine plus haloperidol were equallyeffective (Table 2 and Fig. 1). In contrast, combinations of haloper-idol plus loperamide, digoxin plus amlodipine, and procyclidineplus amiodarone at 100 lM were ineffective, as viable amoebaewere observed. When higher concentrations of these drug combi-nations were tested, the results revealed that the combination ofhaloperidol plus loperamide was effective at 250 lM; Digoxin plusamlodipine was effective at 250 lM, while the combination of pro-cyclidine plus amiodarone was effective at 950 lM (Table 2 andFig. 1).

3.2. High sensitivity of A. castellanii to micromolar concentrations ofcombined drug therapy

At 500 lM concentration, amlodipine, prochlorperazine andloperamide showed more than 95% kill rate, while amiodarone,procyclidine, digoxin, and apomorphine exhibited up to 50% amoe-bicidal effects (Baig et al., 2013). In contrast, haloperidol did notaffect amoebae viability. In the present study, the results revealedthat 100 lM haloperidol plus 100 lM loperamide exhibited potentamoebicidal effects (Table 3). To further confirm viability, drugs-treated amoebae were inoculated in the growth medium, PYG.No viable amoebae were observed, post-treatment, indicating irre-versible effects of drugs. Similarly, 100 lM prochlorperazine plus100 lM loperamide showed 100% kill rate and no viable amoebaewere observed post-treatment (Table 3). At 100 lM concentration,the combination of procyclidine plus loperamide; prochlorperazineplus apomorphine, and digoxin plus amlodipine were equallyeffective. In contrast, combinations of apomorphine plus haloperi-dol, and procyclidine plus amiodarone at 100 lM were ineffectiveas viable amoebae were observed. When higher concentrations ofdrugs were tested, the results revealed that the combination ofapomorphine plus haloperidol is effective at 250 lM, while thecombination of procyclidine plus amiodarone is effective at750 lM (Table 3).

4. Discussion

The current treatment regimen for GAE involves a mixture ofdrugs to provide additive/synergistic effects and even so themortality remains more than 90% (Visvesvara et al., 2007; Matinet al., 2008; Diaz, 2011). Recently, we tested several drugsthat are already in approved clinical use for non-communicable

is. Amoebae were treated with different combinations of drugs for 24 h. Subsequently,rowth was observed after 48 h. (+) indicates growth, and (�) indicates no growth. No

Drugs combination Growth

Haloperidol (250 lM) + loperamide (250 lM) �

Digoxin (250 lM) + amlodipine (250 lM) �

Procyclidine (950 lM) + amiodarone (950 lM) �

the management of granulomatous amoebic encephalitis due to Acantha-g/10.1016/j.exppara.2014.03.025

Fig. 1. B. mandrillaris (3 � 105 amoebae) were incubated with seven different combinations of drugs including, (i) haloperidol plus loperamide; (ii) prochlorperazine plusloperamide; (iii) procyclidine plus loperamide; (iv) prochlorperazine plus apomorphine; (v) digoxin plus amlodipine; (vi) apomorphine plus haloperidol; and (vii)procyclidine plus amiodarone for 24 h as described in ‘‘Section 2’’. In negative controls, HBMEC alone remained intact (A). When B. mandrillaris were added, viabletrophozoites were observed, while no HBMEC monolayer was observed (B). At 100 lM concentration, the combination of prochlorperazine plus loperamide proved highlyeffective in killing amoebae, as no viable trophozoites were observed and the HBMEC remained intact (C). Similarly, at 100 lM concentration, the combinations ofprocyclidine plus loperamide (D); prochlorperazine plus apomorphine (E); and apomorphine plus haloperidol (F) showed potent amoebicidal effects. In contrast,combinations of haloperidol plus loperamide at 100 lM were ineffective, as viable amoebae were observed and HBMEC monolayer was disrupted (G). When higherconcentrations of these drug combinations were tested, the results revealed that the combination of haloperidol plus loperamide was effective at 250 lM (H). Similarly, thecombination of digoxin plus amlodipine at 100 lM was ineffective, as viable amoebae were observed and HBMEC monolayer was disrupted (I). When higher concentrationsof these drug combinations were tested, the results revealed that the combination of digoxin plus amlodipine was effective at 250 lM (J). The combination of procyclidineplus amiodarone at 100 lM was ineffective (K), but proved amoebicidal at 950 lM (L).

4 H. Kulsoom et al. / Experimental Parasitology xxx (2014) xxx–xxx

diseases by targeting vital cellular receptors/biochemical pathways(Baig et al., 2013). Although receptors/biochemical pathways beingtargeted by these drugs have not been characterized in amoebae,the results revealed that clinically available drugs exhibited potentanti-amoebic effects in vitro at 0.5 mM (Baig et al., 2013). In thepresent study, several combinations of drugs with known modeof actions were tested at micromolar concentrations as describedin Table 1. Briefly, the combination of haloperidol and loperamidewas used to target the calcium-dependent intracellular proteinssynergistically. The combination of prochlorperazine and lopera-mide was used to target Dopamine and Muscarinic receptors. Thecombination of procyclidine and loperamide was used to affect

Please cite this article in press as: Kulsoom, H., et al. Combined drug therapy inmoeba spp., and Balamuthia mandrillaris. Exp. Parasitol. (2014), http://dx.doi.or

Bcl2 concentration inside cells (pro-apoptotic stimuli). The combi-nation of prochlorperazine and apomorphine was used to targetDopamine, Muscarinic, a-adrenergic receptors and 5HT receptors.The combination of digoxin and amlodipine was used to raiseintracellular calcium to toxic levels. The combination of apomor-phine and haloperidol was used to block the Dopamine receptorby latter drug and observe the effects of apomorphine agonistaction on the aforementioned receptor. The combination of procy-clidine and amiodarone was used to affect sodium and potassiumions imbalance inside the cell. For B. mandrillaris, several drugscombinations were effective at 100 lM concentrations, albeit somewere used at higher concentrations (Table 2). When tested alone,

the management of granulomatous amoebic encephalitis due to Acantha-g/10.1016/j.exppara.2014.03.025

Fig. 1 (continued)

Table 3The effects of clinically available drugs tested in different combinations against A. castellanii. Amoebae were treated with different combinations of drugs for 24 h. Subsequently,amoebae were washed to remove extracellular drugs and inoculated in growth medium. Growth was observed after 48 h. (+) indicates growth, and (�) indicates no growth. Nogrowth was considered as potent irreversible amoebicidal effects.

Drugs combination (100 lM) Growth Drugs combination Growth

Haloperidol + loperamide + Haloperidol (250 lM) + loperamide (250 lM) �Prochlorperazine + loperamide �Procyclidine + loperamide �Prochlorperazine + apomorphine �Digoxin + amlodipine + Digoxin (250 lM) + amlodipine (250 lM) �Apomorphine + haloperidol �Procyclidine + amiodarone + Procyclidine (950 lM) + amiodarone (950 lM) �

H. Kulsoom et al. / Experimental Parasitology xxx (2014) xxx–xxx 5

digoxin and amiodarone were ineffective against B. mandrillaris.Digoxin binds to sodium/potassium-transporting ATPase alpha-1chain and inhibits its function, while amiodarone also shows potas-sium channel blocker-like actions (Eichhorn and Gheorghiade, 2002;Millan et al., 2002; Sweetman, 2011). In the present study, digoxinwas combined with amlodipine (calcium antagonist) and this com-bination resulted in potent amoebicidal effects (Brunton et al., 2011;Sweetman, 2011). When combined with procyclidine (M-receptor

Please cite this article in press as: Kulsoom, H., et al. Combined drug therapy inmoeba spp., and Balamuthia mandrillaris. Exp. Parasitol. (2014), http://dx.doi.or

antagonist) (Millan et al., 2002; Jevtovic-Todorovic et al., 2003;Brunton et al., 2011; Sweetman, 2011), amiodarone showedamoebicidal effects.

For A. castellanii, amiodarone, procyclidine, digoxin, and apo-morphine exhibited limited amoebicidal effects, while haloperidolhad minimal toxic effects (Baig et al., 2013). In the present study,haloperidol was combined with apomorphine (Non-selective[D] dopaminergic receptor agonist and antagonist at 5-HT and

the management of granulomatous amoebic encephalitis due to Acantha-g/10.1016/j.exppara.2014.03.025

6 H. Kulsoom et al. / Experimental Parasitology xxx (2014) xxx–xxx

a-adrenergic receptors) and this combination resulted in potentamoebicidal effects at micromolar concentrations. When combinedwith procyclidine (M-receptor antagonist), amiodarone showedamoebicidal effects.

Among the seven different combinations tested, three provedhighly effective against both A. castellanii as well as B. mandrillarisat 100 lM. This suggests that biochemical pathways/cellularreceptors are conserved for both free-living amoebae and are vitalfor their viability. These combinations included (i) prochlorpera-zine plus loperamide; (ii) prochlorperazine plus apomorphine;and (iii) procyclidine plus loperamide. These findings are of greatvalue as they offer potential drugs of choice in GAE, especially inthe absence of the correct identification of the causative agent.

Four combinations of drugs tested showed varied potencyagainst A. castellanii and B. mandrillaris at 100 lM. For example,the combination of haloperidol and loperamide was highly effec-tive against A. castellanii at 100 lM of each drug, but potent effectsagainst B. mandrillaris were observed only at 250 lM. Similarly, thecombination of digoxin and amlodipine was highly effectiveagainst A. castellanii at 100 lM of each drug, but potent effectsagainst B. mandrillaris were observed only at 250 lM. In contrast,the combination of apomorphine and haloperidol was highly effec-tive against B. mandrillaris at 100 lM of each drug, but potenteffects against A. castellanii were observed only at 250 lM. At100 lM, the combination of procyclidine and amiodarone waseffective against neither A. castellanii nor B. mandrillaris. In thiscase, amoebicidal properties were observed at 750 lM for A. castel-lanii, and 950 lM for B. mandrillaris. As both A. castellanii and B.mandrillaris are targeted, albeit at varied concentrations, thesefindings suggest conserve targets/pathways in both free-livingamoebae. However, both pathogens exhibit variability in drug sus-ceptibility, which may be due to the drug degrading and modifyingenzymes, reduced membrane permeability for the drugs, expres-sion of drug efflux pumps, over production and alteration of drugtargets, and bypassing inhibitory pathway (Butler and Buss,2006; Powers, 2004; Coates et al., 2011; D’Costa et al., 2011;Donadio et al., 2010; Lee et al., 2011). One or more of the afore-mentioned strategies may explain reduced drug susceptibilitymechanisms. Thus drugs should be used with caution forprolonged clinical use. Although the aforementioned concentra-tions are well within the physiological limits (reviewed in Baiget al., 2013), there is a long way to go before proving their clinicalusefulness in GAE that will require determination of pharmacoki-netics profiles, safety margins etc. However, we feel that thepresent study will open avenues for further research that may pro-vide strategies for therapy against this difficult to treat infection.

In summary, it is believed that the findings reported here willbe of potential value in the rational development of therapeuticinterventions and open up new fields of research. These findingsmust be explored and followed up in clinical practice asdrugs (tested in combinations) in the present study are FDAapproved for use against non-communicable diseases with knownmechanisms of action on target cells.

Please cite this article in press as: Kulsoom, H., et al. Combined drug therapy inmoeba spp., and Balamuthia mandrillaris. Exp. Parasitol. (2014), http://dx.doi.or

Acknowledgment

This work was funded by the Aga Khan University, Karachi,Pakistan.

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