Review Article Indian J Med Res 123, February 2006, pp 115-118
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 developing countries. 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 approach to treat such individuals is necessary. The cost of treating asymptomatic individuals is highly exorbitant and not justifiable. The indiscriminate use of antiamoebic drugs can result in increased minimum inhibitory concentration (MIC) values against Entamoeba species, and treatment failure may emerge as an important public health problem. Development of new antiamoebic drugs is still in infancy and vaccine development appears to be distant dream. In future, the development of drug resistance may seriously affect the control of disease. This review discusses the factors involved in drug resistance mechanisms developed by the parasite.
Key words Amoebiasis - emetine - in vitro sensitivity - metronidazole - multidrug resistance mechanism
indiscriminately may lead to drug resistance. Though it does not appear to be a serious problem now, reports of failed treatment with metronidazole9 and differences in drug susceptibilities 10-13 do suggest that this could probably herald the development of drug resistance clinically. Therefore, understanding the mechanisms involved in multi drug resistance of different antiamoebic drugs is essential.
Entamoeba histolytica, associated with high morbidity and mortality continues to be a major public health problem throughout the world. Asymptomatic individuals account for almost 90 per cent of the infections1. Poverty, ignorance, overcrowding, poor sanitation and malnutrition favour transmission and increased disease burden2. Prevalence varies from country to country and within a country3-6. In India, it was estimated to be 2-55 per cent in early nineties7. Identification of two morphologically similar Entamoeba sp., pathogenic E. histolytica and non pathogenic E. dispar responsible for asymptomatic infection, have raised the question of treating all such cases8. The recommendation is that E. histolytica should be specifically identified, treated with antiamoebic drugs. In individuals with only E. dispar, the treatment is unnecessary. Treating asymptomatic individuals
Antiamoebic drugs Antiamoebic drugs are classified into three groups: luminal, tissue, and mixed amoebicides. Metronidazole is the major drug of choice and other nitroimidazole derived compounds like tinidazole, secnidazole and ornidazole are equally effective. Diloxanide furoate, 115
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diiodohydroxyquin, paromomycin, emetine and chloroquine have also been used as alternate drugs. Diloxanide furoate is the mainstay for treating asymptomatic cyst carriers14. Chloroquine could be used along with metronidazole/emetine in cases of hepatic amoebiasis. However, emetine is rarely used on account of its toxicity. Metronidazole, tinidazole and other 5nitroimidazole agents which kill the trophozoites by alterations in the protoplasmic organelles of the amoeba are ineffective in treating cyst passers. Whereas, chloroquine acts on the vegetative forms of the parasite and kills it by inhibiting DNA synthesis, emetine kills the trophozoites mainly by inhibiting protein synthesis15. In vitro drug sensitivity To understand the magnitude of drug resistance, drug sensitivity of clinical isolates of E. histolytica is important. Recent studies have shown differences in drug sensitivity in E. histolytica isolates, indicating that there might be a small percentage of amoebae which are either resistant or may eventually become resistant due to indiscriminate use of antiamoebic agents10-13. Upcroft and Upcroft11 have reported minimum inhibitory concentration (MIC) of metronidazole ranging from 12.5-25 µm. Adagu et al12 have shown the mean 50 per cent inhibitory concentration (IC50) value to metronidazole as 18.47 µm for the most susceptible isolates of E. histolytica with a >30 µm value as the cut-off value for resistance. Burchard and Mirelman10, on examining the in vitro sensitivity to metronidazole and emetine of non-pathogenic zymodemes showed that all were equally sensitive to the both drugs (1-10 µg/ml). Recently, we have reported significantly higher IC50 value of all the four drugs (metronidazole, chloroquine, tinidazole and emetine) for the clinical isolates compared to the reference strain of E. histolytica (HM1: IMSS). Interestingly, E. dispar isolates showed higher IC50 value than the E. histolytica or the reference strain13. Drug resistance mechanisms in E. histolytica Resistance in clinical isolates is less defined in biochemical terms because parasite populations are often heterogenous. Parasite may evade drug action by hiding in sanctuaries. So far the mechanisms of drug resistance hypothesized in protozoan parasite are decrease of drug uptake because of loss of a transporter required for
uptake, the efflux of drugs from the parasite either by the P-glycoproteins (Pgp) or by ATPases, the alteration of drug target, and loss of drug activation16. Metronidazole and related nitroimidazole, tinidazole (which is not available in some countries), are the only effective drugs for the treatment of trichomoniasis and giardiasis. In the latter cases, clinical resistance to these drugs has been documented17-19. Laboratory induced metronidazole resistance in E. histolytica has been reported and metronidazole resistant E. histolytica strains have been maintained indefinitely in medium20. The models available to study the mechanism of drug resistance in E. histolytica include in vitro induced metronidazole resistant cell lines of E. histolytica20 and emetine resistant mutant clones derived from virulent HM1: IMSS strain, which was selected after mutagenesis with alkylating agent, ethyl methanesulphonate21. Expression of iron containing superoxide dismutase (fe-SOD) and peroxiredoxin has been shown to be increased three to five fold in metronidazole resistant parasites and this has been confirmed at the gene expression level with decrease in the expression of ferredoxin and flavin reductase22. The critical involvement of fe-SOD and peroxiredoxin was confirmed by episomal transfection of these antioxidant enzymes into metronidazole susceptible isolates, which was associated with reduced drug susceptibility22. Though the mechanisms of multi drug resistance (MDR) in Entamoeba emetine mutants have not been studied in detail, MDR in mammalian tumour cells is well characterized. Tumour cells become resistant to simple chemotherapeutic drugs in vitro23. The drug resistance is usually due to increased efflux of the drug from the tumour cells, which is energy dependent and is inhibited by calcium ion channel blockers24. The MDR gene encoding for the Pgp (170 kDa protein), has been isolated from drug resistant cancer cells25. This consists of two homologous halves containing six membrane spanning alpha-helices and a cytoplasmic ATP binding site for efflux of drugs26,27, with the ATP binding site showing an extensive amino acid sequence homologues with bacterial transport proteins25,28. P-glycoprotein also has a binding protein for chemotherapeutic agents such as vinblastin suggesting that it is directly involved in the drug transport mechanism in MDR cells29. The
BANSAL et al: DRUG RESISTANCE IN AMOEBIASIS
overexpression of surface Pgp also produces the MDR phenotype in protozoan parasites30 like Plasmodium31, Trichomonas 32 , Giardia 17 , Leishmania 33 , and Entamoeba34. E. histolytica has several features common with MDR phenotype described in mammalian tumour cells35. These are cross-resistance to unrelated drugs, increased efflux and decreased accumulation of radiolabelled drugs, reversal of resistance by calcium channel blockers like verapamil, and overexpression of 4.5 kb long mRNAs homologous to the mammalian P-glycoprotein. So far, six Pgp like genes (EhPgp1-EhPgp6) have been cloned and sequenced, but copy number has not been described in laboratory mutants36,37. Of these, four were clearly expressed in drug resistant line (EhPgp1, EhPgp2, EhPgp5, EhPgp6), while the remaining two were pseudogenes (EhPgp3, EhPgp4)38. Studies on genetic relatedness suggest that Entamoeba P-glycoproteins are more related to the human and mouse P-glycoprotein than to the Plasmodium and Leishmania P-glycoprotein. The amino acid sequence of amoebic MDR like PCR products were 46 to 97 per cent identical with each other, 46 to 50 per cent identical to human MDR sequence and 30-35 per cent to P. falciparum MDR like sequence36. Differential gene expression in drug resistant mutants has been detected by using specific probes for each EhPgp gene. The differential expression of EhPgp gene was initiated by characterizing the upstream flanking regions of EhPgp1 and EhPgp5 genes. The upstream sequence showed 53 and 66 per cent positional identity to Dicotyostelium discoideum promoters39. Growing emetine resistant clones have an increased expression of EhPgp1 and EhPgp5 mRNA when compared to the wild type cloned amoebae37. Recently we have found that MDR gene was overexpressed in emetine resistant clone of E. histolytica, whereas no expression of mRNA was seen in the clinical isolates of E. histolytica and E. dispar and also in the sensitive strain of E. histolytica (HM1: IMSS) (unpublished data), indicating thereby that more than one mechanism might be operating in the development of resistance in clinical isolates of E. histolytica. Most of the studies carried out so far have only tried to analyze the mechanism of resistance in emetine resistant clones. However, further studies to evaluate MDR phenotype in clinical isolates of E. histolytica, are desired. Further, data on resistance to other antiamoebic agents including
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metronidazole are also lacking and therefore the antiamoebic susceptibility of pathogenic clinical isolates needs to be investigated to help in developing strategies to increase the efficacy and the life span of the limited number of currently available antiamoebic drugs. References 1. Amoebiasis. Wkly Epidemiol Rec 1997; 72 : 97-9. 2. Walsh AL. Prevalence in Entamoeba histolytica infection. In: Ravdin JI, editor. Amoebiasis: human infection by Entamoeba histolytica. New York: John Wiley & Sons, Inc; 1988 p. 93-105. 3. Tanaka J, Kaneda Y. Seroepidemiology of anti-E. histolytica antibody by ELISA in the greater Tokyo area, Tokai. J Expt Clin Med 1991; 16 : 253-8. 4. Haque R, Mollah NA, Ali IK, Alam K, Eubanks A, Lyerly D, et al. Diagnosis of amoebic liver abscess and intestinal infection with the Techlab Entamoeba histolytica II antigen detection and antibody tests. J Clin Microbiol 2000; 38 : 3235-9. 5. Blessmann J, Van Linh P, Nu PA, Thi HD, Muller-Myhsok B, Buss H, et al. Epidemiology of amoebiasis in a region of high incidence of amoebic liver abscess in central Vietnam. Am J Trop Med Hyg 2002; 66 : 578-83. 6. Kang G, Mathew MS, Rajan DP, Daniel JD, Mathan MM, Mathan VI, et al. Prevalence of intestinal parasites in rural southern Indians. Trop Med Int Health 1998; 3 : 70-5. 7. Mahajan RC, Sehgal R, Ganguly NK. Recent developments in amoebiasis research. Indian Rev Life Sci 1991; 11 : 139-67. 8. Pillai DR, Keystone JS, Sheppard DC, MacLean JD, MacPherson DW, Kain KC. Entamoeba histolytica and Entamoeba dispar: epidemiology, and comparison of diagnostic methods in a setting of non endemicity. Clin Infect Dis 1999; 29 : 1315-8. 9. Hanna RM, Dahniya MH, Badr SS, El-Betagy A. Percutaneous catheter drainage in drug-resistant amoebic liver abscess. Trop Med Int Health 2000; 5 : 578-81. 10. Burchard GD, Mirelman D. Entamoeba histolytica: virulence potential and sensitivity to metronidazole and emetine of four isolates possessing nonpathogenic zymodemes. Exp Parasitol 1988; 66 : 231-42. 11. Upcroft P, Upcroft JA. Drug targets and mechanisms of resistance in the anaerobic protozoa. Clin Microbiol Rev 2001; 14 : 150-64. 12. Adagu IS, Nolder D, Warhurst DC, Rossignol JF. In vitro activity of nitazoxanide and related compounds against isolates of Giardia intestinalis, Entamoeba histolytica, Trichomonas vaginalis. J Antimicrob Chemother 2002; 49 : 103-11.
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Reprint requests: Dr R.C. Mahajan, Emeritus Professor, Department of Parasitology Post Graduate Institute of Medical Education & Research, Chandigarh 160012, India e-mail:
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