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,
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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 m. Adagu et al12
have shown the mean 50 per cent inhibitory concentration
(IC50) value to metronidazole as 18.47 m for the most
susceptible isolates of E. histolytica with a >30 m
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 g/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

117

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.
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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: indurc@sify.com