Drug resistance in amoebiasis

Devendra Bansal, Nancy Malla & R.C. Mahajan

Department of Parasitology, Post Graduate Institute of Medical Education & Research, Chandigarh, India

Received April 8, 2005

Amoebiasis caused by Entamoeba histolytica, is a major public health problem in developingcountries. Morphologically similar E. dispar is non pathogenic. Because of the redefinition of E.histolytica and E. dispar, and the limited number of antiamoebic drugs available, a new approachto treat such individuals is necessary. The cost of treating asymptomatic individuals is highlyexorbitant and not justifiable. The indiscriminate use of antiamoebic drugs can result in increasedminimum inhibitory concentration (MIC) values against Entamoeba species, and treatment failuremay emerge as an important public health problem. Development of new antiamoebic drugs is stillin infancy and vaccine development appears to be distant dream. In future, the development ofdrug resistance may seriously affect the control of disease. This review discusses the factors involvedin drug resistance mechanisms developed by the parasite.

Key words Amoebiasis - emetine - in vitro sensitivity - metronidazole - multidrug resistance mechanism

Review Article

Entamoeba histolytica, associated with high morbidityand mortality continues to be a major public healthproblem throughout the world. Asymptomatic individualsaccount for almost 90 per cent of the infections1. Poverty,ignorance, overcrowding, poor sanitation and malnutritionfavour transmission and increased disease burden2.Prevalence varies from country to country and within acountry3-6. In India, it was estimated to be 2-55 per centin early nineties7. Identification of two morphologicallysimilar Entamoeba sp., pathogenic E. histolytica and nonpathogenic E. dispar responsible for asymptomaticinfection, have raised the question of treating all suchcases8. The recommendation is that E. histolytica shouldbe specifically identified, treated with antiamoebic drugs.In individuals with only E. dispar, the treatment isunnecessary. Treating asymptomatic individuals

indiscriminately may lead to drug resistance. Though itdoes not appear to be a serious problem now, reports offailed treatment with metronidazole9 and differences indrug susceptibilities10-13 do suggest that this couldprobably herald the development of drug resistanceclinically. Therefore, understanding the mechanismsinvolved in multi drug resistance of different antiamoebicdrugs is essential.

Antiamoebic drugs

Antiamoebic drugs are classified into three groups:luminal, tissue, and mixed amoebicides. Metronidazoleis the major drug of choice and other nitroimidazolederived compounds like tinidazole, secnidazole andornidazole are equally effective. Diloxanide furoate,

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diiodohydroxyquin, paromomycin, emetine andchloroquine have also been used as alternate drugs.Diloxanide furoate is the mainstay for treatingasymptomatic cyst carriers14. Chloroquine could be usedalong with metronidazole/emetine in cases of hepaticamoebiasis. However, emetine is rarely used on accountof its toxicity. Metronidazole, tinidazole and other 5-nitroimidazole agents which kill the trophozoites byalterations in the protoplasmic organelles of the amoebaare ineffective in treating cyst passers. Whereas,chloroquine acts on the vegetative forms of the parasiteand kills it by inhibiting DNA synthesis, emetine killsthe trophozoites mainly by inhibiting protein synthesis15.

In vitro drug sensitivity

To understand the magnitude of drug resistance, drugsensitivity of clinical isolates of E. histolytica is important.Recent studies have shown differences in drug sensitivityin E. histolytica isolates, indicating that there might be asmall percentage of amoebae which are either resistantor may eventually become resistant due to indiscriminateuse of antiamoebic agents10-13. Upcroft and Upcroft11 havereported minimum inhibitory concentration (MIC) ofmetronidazole ranging from 12.5-25 µm. Adagu et al12

have shown the mean 50 per cent inhibitory concentration(IC

50) value to metronidazole as 18.47 µm for the most

susceptible isolates of E. histolytica with a >30 µmvalue as the cut-off value for resistance. Burchard andMirelman10, on examining the in vitro sensitivity tometronidazole and emetine of non-pathogeniczymodemes showed that all were equally sensitive to theboth drugs (1-10 µg/ml). Recently, we have reportedsignificantly higher IC

50 value of all the four drugs

(metronidazole, chloroquine, tinidazole and emetine) forthe clinical isolates compared to the reference strain ofE. histolytica (HM1: IMSS). Interestingly, E. disparisolates showed higher IC

50 value than the E. histolytica

or the reference strain13.

Drug resistance mechanisms in E. histolytica

Resistance in clinical isolates is less defined inbiochemical terms because parasite populations are oftenheterogenous. Parasite may evade drug action by hidingin sanctuaries. So far the mechanisms of drug resistancehypothesized in protozoan parasite are decrease of druguptake because of loss of a transporter required for

uptake, the efflux of drugs from the parasite either bythe P-glycoproteins (Pgp) or by ATPases, the alterationof drug target, and loss of drug activation16.

Metronidazole and related nitroimidazole, tinidazole(which is not available in some countries), are the onlyeffective drugs for the treatment of trichomoniasis andgiardiasis. In the latter cases, clinical resistance to thesedrugs has been documented17-19. Laboratory inducedmetronidazole resistance in E. histolytica has beenreported and metronidazole resistant E. histolyticastrains have been maintained indefinitely in medium20.

The models available to study the mechanism of drugresistance in E. histolytica include in vitro inducedmetronidazole resistant cell lines of E. histolytica20

and emetine resistant mutant clones derived fromvirulent HM1: IMSS strain, which was selected aftermutagenesis with alkylating agent, ethylmethanesulphonate21. Expression of iron containingsuperoxide dismutase (fe-SOD) and peroxiredoxin hasbeen shown to be increased three to five fold inmetronidazole resistant parasites and this has beenconfirmed at the gene expression level with decrease inthe expression of ferredoxin and flavin reductase22. Thecritical involvement of fe-SOD and peroxiredoxin wasconfirmed by episomal transfection of these antioxidantenzymes into metronidazole susceptible isolates, whichwas associated with reduced drug susceptibility22.

Though the mechanisms of multi drug resistance(MDR) in Entamoeba emetine mutants have not beenstudied in detail, MDR in mammalian tumour cells iswell characterized. Tumour cells become resistant tosimple chemotherapeutic drugs in vitro23. The drugresistance is usually due to increased efflux of the drugfrom the tumour cells, which is energy dependent andis inhibited by calcium ion channel blockers24. The MDRgene encoding for the Pgp (170 kDa protein), has beenisolated from drug resistant cancer cells25. This consistsof two homologous halves containing six membranespanning alpha-helices and a cytoplasmic ATP bindingsite for efflux of drugs26,27, with the ATP binding siteshowing an extensive amino acid sequence homologueswith bacterial transport proteins25,28. P-glycoprotein alsohas a binding protein for chemotherapeutic agents suchas vinblastin suggesting that it is directly involved inthe drug transport mechanism in MDR cells29. The

116 INDIAN J MED RES, FEBRUARY 2006

overexpression of surface Pgp also produces the MDRphenotype in protozoan parasites30 like Plasmodium31,Trichomonas32, Giardia17, Leishmania33, andEntamoeba34.

E. histolytica has several features common with MDRphenotype described in mammalian tumour cells35. Theseare cross-resistance to unrelated drugs, increased effluxand decreased accumulation of radiolabelled drugs, reversalof resistance by calcium channel blockers like verapamil,and overexpression of 4.5 kb long mRNAs homologous tothe mammalian P-glycoprotein. So far, six Pgp like genes(EhPgp1-EhPgp6) have been cloned and sequenced, butcopy number has not been described in laboratorymutants36,37. Of these, four were clearly expressed in drugresistant line (EhPgp1, EhPgp2, EhPgp5, EhPgp6), whilethe remaining two were pseudogenes (EhPgp3, EhPgp4)38.

Studies on genetic relatedness suggest that EntamoebaP-glycoproteins are more related to the human and mouseP-glycoprotein than to the Plasmodium and LeishmaniaP-glycoprotein. The amino acid sequence of amoebicMDR like PCR products were 46 to 97 per cent identicalwith each other, 46 to 50 per cent identical to humanMDR sequence and 30-35 per cent to P. falciparum MDRlike sequence36. Differential gene expression in drugresistant mutants has been detected by using specificprobes for each EhPgp gene. The differential expressionof EhPgp gene was initiated by characterizing theupstream flanking regions of EhPgp1 and EhPgp5 genes.The upstream sequence showed 53 and 66 per centpositional identity to Dicotyostelium discoideumpromoters39. Growing emetine resistant clones have anincreased expression of EhPgp1 and EhPgp5 mRNAwhen compared to the wild type cloned amoebae37.Recently we have found that MDR gene wasoverexpressed in emetine resistant clone of E. histolytica,whereas no expression of mRNA was seen in the clinicalisolates of E. histolytica and E. dispar and also in thesensitive strain of E. histolytica (HM1: IMSS)(unpublished data), indicating thereby that more than onemechanism might be operating in the development ofresistance in clinical isolates of E. histolytica. Most ofthe studies carried out so far have only tried to analyzethe mechanism of resistance in emetine resistant clones.However, further studies to evaluate MDR phenotype inclinical isolates of E. histolytica, are desired. Further, dataon resistance to other antiamoebic agents including

metronidazole are also lacking and therefore theantiamoebic susceptibility of pathogenic clinical isolatesneeds to be investigated to help in developing strategiesto increase the efficacy and the life span of the limitednumber of currently available antiamoebic drugs.

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Reprint requests: Dr R.C. Mahajan, Emeritus Professor, Department of ParasitologyPost Graduate Institute of Medical Education & Research, Chandigarh 160012, Indiae-mail: indurc@sify.com