Treatment of Giardiasis and Drug Resistance
Joachim Müller, Andrew Hemphill and Norbert Müller
Abstract
Giardia lamblia (also known as G. duodenalis or
G. intestinalis), a flagellated protozoan, is the most
common causative agent of persistent diarrhea worldwide. The life cycle includes motile, flagellated trophozoites parasitizing the upper intestine and
thick-walled cysts forming in the lower intestine,
which are subsequently shed with the feces. Chemotherapy is directed against the trophozoite stage. The
current antigiardial therapy of choice is metronidazole. In the case of resistance, alternatives are other
nitroimidazoles, quinacrine, albendazole, and nitazoxanide. Here, we present a review of current antigiardial drugs and of their modes of action.
USA for the treatment of persistent diarrhea due to
cryptosporidiosis and giardiasis in people older than
12 years (reviewed by Hemphill et al., 2006). Moreover, in vitro effects of natural compounds such as
isoflavones, polyenes or saturated fatty acids against
trophozoites have been described, but clinical data are
lacking. An overview of antigiardial compounds with
references is given in Table 1.
In this chapter, we will focus on the mode of action
of these drug families (so far as known) and put some
new light on resistance mechanisms.
Mode of Action and Drug Targets
General Remarks
Introduction
For the treatment of giardiasis, chemotherapy is the
method of choice. After the introduction of quinacrine
as the first antigiardial drug in the 1940s, metronidazole (commercially known as Flagyl®) and other nitroimidazoles such as tinidazole have been used as a
therapy of choice for more than five decades. In the
case of resistance formation, substitution therapies
include benzimidazole albendazole, the acridine derivative quinacrine – historically the first antigiardial
drug –, or the aminoglycoside paromomycin alone or
in combination with metronidazole (see for review
Upcroft and Upcroft, 2001 and for a recent clinical
study Mørch et al., 2008). In the mid-1990s, a new
antiparasitic agent, the thiazolide nitazoxanide (commercially known as Alinia®), has been introduced in
the market. Nitazoxanide has been approved in the
Norbert Müller () Institute of Parasitology, University of
Bern, Länggass-Strasse 122, 3012 Bern, Switzerland
The ideal situation for the mode of action of antiparasite drugs consists of binding to a target protein (enzyme or receptor) resulting in the inhibition of
essential cellular functions. Such targets are identified by analyzing structural and metabolic alterations
of the parasite due to the drug followed by the isolation of drug binding proteins or the analysis of resistant vs. sensitive parasites. The isolated targets are
then validated by inhibition studies in vitro, structural
analysis of protein-ligand complexes, and – if possible – knock-out and overexpression studies of the
corresponding gene in the target organism (see e.g.
Wang, 1997).
In an ideal situation, the drug-target interaction depends on distinct structural features of the target. In
this case, point mutations causing the presence or
absence of a single amino acid or nucleotide (in the
case of rRNA as a target) discriminate between resistance or susceptibility. In reality, the situation is more
complex. In resistant organisms, the “ideal” target
may still be present, but the drug cannot access it due
J. Müller et al.
Table 1 Overview of antigiardial drugs. With regard to the efficacy, the order of magnitude of the IC50 from in vitro studies and
– if available – the dose for patients are given
Class
Acridines
Aminoglycosides
Benzimidazoles
Compound
Quinacrine
Paromomycin
Albendazole
Bis-benzimidazoles
Efficacy
Reference
–7
In vitro (10 M)
Bell et al., 1991; Bénéré et al., 2007
In vivo (100 mg tid + MET 750 mg
tid for 2–3 weeks)
Nash et al., 2001; Mørch et al., 2008
–4
–5
In vitro (10 –10 M)
Edlind, 1989
In vivo: 500 mg tid for 1 week
Mørch et al., 2008
–8
In vitro (10 M)
Katiyar et al., 1994, Upcroft and
Upcroft, 2001; Cruz et al., 2003
In vivo (400 mg bid + MET 250 mg
bid or tid for 1 week)
Mørch et al., 2008
–8
–6
In vitro (10 –10 M)
–5
Fatty acids
Dodecanoic acid
In vitro (10 M)
Fluoroquinolones
Ciprofloxacine
In vitro (10 M)
Rayan et al., 2005
–4
Sousa and Poiares-da-Silva, 2001
–6
Isoflavones
Genistein
Formononetin
In vitro (10–8 M)
In vitro (10 M)
Nitrofuranes
Furazolidone
In vitro (10 –10 M)
–5
Bell et al., 1993
Khan et al., 2000; Sterck et al., 2008
–6
–6
Cedillo-Rivera and Munoz, 1992
Nitroimidazoles
Metronidazole
Tinidazole
In vitro (10 M); in vivo
Diaminidines
Pentamidine and derivatives
In vitro (10 –10 M)
Bell et al., 1991
Anthony et al., 2005
–8
–6
Polyenes
Allicin and derivatives
In vitro
Thiazolides
Nitazoxanide
In vitro (10 M)
In vivo
to uptake limitation or detoxification mechanisms. A
search for drug targets (e.g. by protein binding studies)
would thus yield detoxification enzymes, transporters
or even irrelevant drug binding proteins besides the
“true” target. Conversely, susceptibility to a drug may
be triggered by the ability to convert a (ineffective)
prodrug to the (effective) drug. In this case, search for
targets have to be performed with the effective molecule, otherways, the transforming enzyme would be
identified rather than the target itself.
In the case of antiparasite drugs (as well as other
antibiotics), the situation is rendered more complicated by the fact that the drug acts in the vicinity or
even inside the host cell (in the case of intracellular
pathogens). Therefore, the mode of action of the drug
may be modulated if not depend on the susceptibility
of the host cell and not the parasite itself. In the following paragraphs, we will discuss some examples
within the framework of antigiardial drugs.
–6
Gardner and Hill, 2001
Fox and Saravolatz, 2005; Hemphill
et al., 2006
Methods for the Determination of Drug
Efficacy
In order to determine the efficacy of antigiardial drugs
(e.g. in terms of IC50 values), a couple of methods are
currently in use. The most classical way is counting
trophozoites grown axenically in the presence of
drugs (see e.g. Müller et al., 2006). The incorporation
of 3H-thymidine is less subjective (see e. g. Adagu
et al., 2002). Both methods are time-consuming or
need high-level equipment. An elegant alternative is
the use of the vital staining resazurin (Alamar Blue;
Bénéré et al., 2007). Viable cells reduce resazurin
forming a pink compound absorbing at 550 nm and
fluorescing at >590 nm. Although it is possible to use
spectrophotometry for quantifying the product, the
best sensitivity is obtained with fluorimetry. Fluorimeters are, however, expensive and therefore not present
in many labs. Moreover, all these methods cannot be
Treatment of Giardiasis and Drug Resistance
used when trophozoites are not grown axenically, but
in a coculture system with intestinal cells (e.g. Caco
2). In this system, trophozoites may be quantified using real-time PCR with a Giardia-specific target
(Müller et al., 2006).
A suitable alternative would be the use of a reporter strain as shown for instance for Toxoplasma gondii
expressing β-galactosidase (McFadden et al., 1997;
see for a recent application Müller et al., 2009a). In
the case of Giardia, a reporter strain expressing
glucuronidase A from E. coli has been created (Müller
et al., 2009b). This strain is well suited for the determination of IC50 values and for high-throughput drug
screening using simple colorimetric assays. Moreover, it can be used in a coculture system with host
cells thus allowing the determination of the “therapeutical window”, i.e. the gap between drug efficacy
and host cell toxicity in one screen.
Nucleic Acids as Targets
Early studies have shown that the acridine derivative
quinacrine, the first antigiardial drug, interacts with
DNA inhibiting replication and RNA and protein biosynthesis in prokaryonts (Ciak and Hahn, 1967). Like
other acridine derivatives, quinacrine is an intercalating agent binding to DNA with a preference for
(A+T)-rich regions and thereby blocking DNA replication and RNA biosynthesis (Wilson et al., 1994). In
studies concerning pentamidine derivatives (Bell
et al., 1991) and bis-benzimidazoles (Bell et al.,
1993), very nice correlations between the binding affinities of these compounds to calf thymus DNA and
their efficacies against Giardia lamblia have been
pointed out. Since DNA binding is by far not species
specific and therefore prone to side effects on host
cells, the relevance of a DNA binding agent as an antimicrobial drug resides in other – more species specific – properties like a differential uptake or
metabolism or the presence of other targets besides
DNA. For instance, pentamidines and a variety of
other DNA binding compounds such as etoposide induce the cleavage of Trypanosoma DNA minicircles
in a pattern that resembles the action of topoisomerase
II inhibitors (Shapiro and Englund, 1990). DNA modifying enzymes may thus constitute more selective
targets for antimicrobial agents.
Fluoroquinolones such as ciprofloxacine inhibit
DNA-gyrases and topoisomerases from prokaryonts
(Kidwai et al., 1998) and are highly effective against
G. lamblia as shown in a descriptive study (Sousa and
Poiares-da-Silva, 2001). It is, however, unclear, which
type of enzyme is inhibited there since more pronounced studies are lacking. A phylogeny analysis of
type II topoisomerases shows that the gene of
G. lamblia (GlTop2) is eukaryont-like and not closely
related to prokaryont topoisomerases (He et al.,
2005).
Aminoglycosides bind to the small subunit of ribosomes, more exactly to distinct features of the 16SrRNA secondary structure and thereby inhibit protein
biosynthesis (see e.g. Brodersen et al., 2000 for a detailed structural analysis). Therefore, specific mutations in this region confer resistance to various
aminoglycosides in various prokaryonts. G. lamblia
has a 16S-rRNA structure similar to prokaryonts with
a primary sequence suggesting that only paromomycin
and hygromycin are effective, other well known aminoglycosides such as kanamycin are not. This pattern
correlates well with observed susceptibility or resistance to a panel of aminoglycosides (Edlind, 1989).
Cytoskeleton Proteins as Targets
Early studies with benzimidazoles such as albendazole showed effects on the cytoskeleton, especially in
the region of the ventral disk (Fig. 1; see e.g. Chavez
et al., 1992; Hemphill et al., 1996). Molecular genetics has revealed that sensitivity to albendazole (or
other benzimidazoles) in evolutionary distant organisms such as fungi, nematodes, platyhelminthes, and
various protozoa including G. lamblia is correlated to
the presence of specific alleles of β-tubulin-genes
(Kirk-Mason et al., 1988; Driscoll et al., 1989; Katiyar
et al., 1994; Kwa et al., 1995; Henriquez et al., 2008),
the substitution of Phe to Tyr in position 200 being
sufficient for switching from benomyl sensitivity to
resistance as shown in nematodes (Kwa et al., 1995).
Molecular modeling of the putative albendazole binding site in albendazole resistant Acanthamoeba
β-tubulin indicates that 4 of 13 residues are different
from tubulins of sensitive organisms. Resistance is
conferred when, besides Phe200, Phe167 is replaced
(Henriquez et al., 2008).
J. Müller et al.
A: Albendazole
B: Control
Fig. 1 Examples of ultrastructural changes induced by albendazole and visualized by transmission electron microscopy, A,
albendazole; B, control; N, nucleus
Drugs Interfering with Intermediary
Metabolism
The nitroimidazole metronidazole is clearly the best
studied compound affecting intermediary metabolism. When trophozoites are treated with metronidazole the cell looses motility within a few hours
followed by appearance of large zones of lesions on
the dorsal shield and swelling of the cell body (Fig. 2;
see e.g. Müller et al., 2006). According to a widely
referred theory, metronidazole (as a prodrug)
is reduced to a nitro radical by electrons coming
from the enzyme pyruvate:flavodoxin/ferredoxin oxidoreductase (PFOR), a protein lacking in higher eukaryotic cells (Horner et al., 1999). The radical causes
irreversible damages. Results obtained with the microaerophilic parasite Trichomonas vaginalis suggest,
however, another mode of action. By comparing 2-Dprotein electrophoresis patterns from treated and untreated cells, the authors suggest that metronidazole
binds to the sulfhydryl group in the active center of
various enzymes including thioredoxin reductase
thereby impairing essential cellular functions (Leitsch
et al., 2009). Also in this model, an activated form of
metronidazole would act on multiple targets, the
number being limited to enzymes with sulfhydryl
groups in their active center.
A mode of action similar to nitroimidazoles has
been postulated for nitrothiazolides. Upon oral uptake, nitazoxanide, the best studied nitrothiazolide, is
rapidly deacetylated to tizoxanide and further metabolized to tizoxanide-glucuronide (Fox and
Saravolatz, 2005). Tizoxanide has been reported to
display antimicrobial activity similar to nitazoxanide,
while tizoxanide-glucuronide is largely inactive
against a number of pathogens including Giardia.
Thiazolides without the thiazole-associated nitro
group have been shown to exhibit decreased or no
efficacy against Giardia (Adagu, et al., 2001; Müller
et al., 2006). Therefore, an involvement of the nitro
group in the mechanism of action, with the participation of PFOR similar to metronidazole, is likely
(Wright et al., 2003). The nitro group is also required
for in vitro activity against anaerobic bacteria
(Pankuch and Apelbaum, 2006). Moreover, nitazoxanide inhibits activities of recombinant PFOR from
various bacteria and parasites including G. lamblia
(Sisson et al., 2002). A reduction of nitazoxanide,
however, has never been observed in vivo. Instead,
there is some evidence that unreduced nitazoxanide
blocks an intermediate step of the reaction thus inhibiting PFOR in a non-competitive manner (Hoffman
et al., 2007). When G. lamblia trophozoites are treated with nitrothiazolides, ultrastructural analyses
Treatment of Giardiasis and Drug Resistance
A: Control
B: NTZ
C: MTZ
D: MTZ
Fig. 2 Examples of ultrastructural changes induced by nitazoxanide and metronidazole and visualized by scanning electron microscopy, A, control; B, nitazoxanide (NTZ); C, D, metronidazole (MTZ)
reveal severe damages of the ventral disk instead of
damages of the dorsal surface membrane as observed
with metronidazole (Fig. 2; see e.g. Müller et al.,
2006). In G. lamblia, a nitroreductase as a nitazoxanide-binding protein was identified and characterized. Nitroreductase is inhibited by nitazoxanide,
and, as for PFOR, there is no evidence for a reduction
by nitroreductase (Müller et al., 2007b). Overexpression of this nitroreductase is, however, correlated
with increased sensitivity to metronidazole and nitazoxanide (Nillius et al., 2011). Besides nitroreductase, protein disulfide isomerases from G. lamblia
(Müller et al., 2007a) and Neospora caninum (Müller
et al., 2008b) are potential targets for nitazoxanide.
Recent studies have shown that not only nitazoxanide, but also derivatives lacking the nitro group (reviewed in Hemphill et al., 2007) exhibit in vitro
activity against the intracellular apicomplexan parasites N. caninum, Cryptosporidium parvum and
Besnoitia besnoiti (Hemphill et al., 2006; Cortes
et al., 2007) and as well as proliferating host cells
(Müller et al., 2008c). Therefore, it cannot be excluded that the efficacy of nitazoxanide against giardiasis
and cryptosporidiosis in patients is partly due to effects on intestinal cells. In vivo studies on these diseases with non-nitro-thiazolides are lacking. In
proliferating host cells (Caco2), inhibition of glutathione-S-Transferase P1 (GSTP1) by nitazoxanide
J. Müller et al.
and some non-nitro-thiazolides is well correlated
with their efficacy to induce apoptosis. Overexpression and downregulation of GSTP1 is correlated with
an increase, or decrease, of sensitivity, respectively
(Müller et al., 2008c).
Compounds of Plant Origin with
Miscellaneous Targets
In order to improve the situation in chemotherapy of
metronidazole resistant giardiasis, new potential
pharmaceuticals from natural sources have been evaluated regarding their antigiardial activity in vitro. In
general, crude extracts from traditional medicinal
herbs are tested with respect to antigiardial activities
and then fractionated in order to identify the active
principles (see for review Anthony et al., 2005).
Essential oils are a well explored source for antimicrobial compounds. A good example for a study on
antigiardial properties in oregano extracts is given by
Ponce-Macotela et al. (2006). Often, essential oils
have a broad spectrum of efficacy and therefore a potential toxicity to host cells. A good example is a
study where active compounds have been identified
from Allium extracts (Harris et al., 2000). In this
study, the compound with the lowest IC50 is allyl alcohol, a compound with an unspecific toxicity and a potential cancerogene. Allicin, another molecule
effective against Giardia and other parasites such as
Entamoeba, bind to SH-groups from cysteine proteases and other SH-proteins including thioredoxin
reductase (see Ankri et al., 1997; Ankri and Mirelman, 1999), a mode of action recently postulated for
metronidazole (Leitsch et al., 2009).
Another well studied group are isoflavones. Isoflavones are mainly found in Leguminosae where they
have anti-oxidant, anti-microbial and signalling functions (e. g. reviewed in Dakora and Phillips, 1996;
Dixon and Steele, 1999). Within this group of compounds, Khan et al. (2000) identified formononetin, a
major isoflavone of Ononis sp, as the most potent antigiardial agent. In vitro, formononetin has an IC50
value in the range of 10–7 M, thus one order of magnitude lower than the non-methylated daidzein and
genistein, two isoflavones of soybean (Sterk et al.
2008). By daidzein-affinity chromatography, a nucleoside hydrolase was identified as daidzein-binding
protein. Nucleoside hydrolase activity is inhibited by
the three isoflavones mentioned above in a range of
concentrations where daidzein and genistein inhibit
trophozoite growth in vivo. In Crithidia (Parkin et al.,
1991), Leishmania (Shi et al., 1999) or Trypanosoma
(Parkin, 1996), the function of nucleoside hydrolase
is central in salvaging purine nucleosides from the
host and essential since the parasites lack purine de
novo synthesis. Inhibition would thus lead ultimately
to a block of DNA synthesis and thus cell division.
This effect would be rather slow. Formononetin acts
faster by stopping motility and inducing detachment
within less than 2 h (Lauwaet et al., 2010, and own,
unpublished data) and is inhibitory at ten times lower
concentrations suggesting other mechanisms to be involved in addition such as depolarization of the membrane due to uncoupling effects or direct inhibition of
membrane ATPases. However, experimental evidence
for any of these possibilities is lacking.
Resistance Formation
Besides the search for drug binding proteins, another
way to elucidate the action of drugs is the comparison
of resistant vs. non-resistant trophozoites. Resistant
trophozoites are created by selecting growing cells in
the presence of increasing drug concentrations followed by cloning and characterization of the resistant
clones vs. wildtype clones. Moreover, in some cases,
drug (mainly metronidazole) resistant strains have
been isolated from patients and compared to non-resistant strains. Table 2 gives an overview of selected
studies.
Resistance to metronidazole and other nitroimidazoles has been induced in vitro; and various genotypically distinct isolates with reduced drug susceptibility
have also been found in human patients (Gardner and
Hill, 2001; Upcroft and Upcroft, 1993). Formation of
resistance to metronidazole is associated with an altered uptake of the drug (Boreham et al., 1988; see
also Upcroft et al., 1996 for quinacrine), downregulation of the PFOR activity (Upcroft and Upcroft 2001).
This is consistent with the model claiming an involvement of PFOR in metronidazole activation (Townson
et al., 1996; Sisson et al., 2002). Recent findings in
T. vaginalis suggest, however, that metronidazole and
other nitroimidazoles covalently bind and thereby
Treatment of Giardiasis and Drug Resistance
Table 2 Overview of studies on resistance formation in G. lamblia
Drug
Source of resistance
Method
Important findings
Reference
Albendazole
Induction of resistance
in vitro in WB
Comparison to wildtype
with respect to growth
Resistance at 4.5 µM for
several weeks, normal
cyst formation
Lindquist, 1996
Induction of resistance
in vitro in WB 1B
Molecular genetics,
ultrastructural analysis
Induced resistance not
related to β-tubulin
modifications
Upcroft et al., 1996
Patient isolates
Comparison of isolates
with respect to growth
Isolates exhibit
variations in their
sensitivities prior to drug
exposition
Argüello-García et al.,
2004
Induction of resistance
in vitro in WB 1
Representational
difference analysis of
gene expression
Induced resistance not
related to β-tubulin
modifications; antigenic
variation
Argüello-García et al.,
2009
Quinacrine
Induction of resistance
in vitro in 7 different
lines
Comparison to sensitive
lines with respect to
growth, investigation
of quinacrine uptake by
fluorescence
Quinacrine excluded
from resistant lines,
multidrug resistance in
some lines
Upcroft et al., 1996
Metronidazole
Patient isolates with
different susceptibilities
Analysis of uptake
growth
Resistant isolates have a
lower uptake
Boreham et al., 1988
Patient isolates,
induction of resistance
formation in vitro
PFOR assay
PFOR activity is lower
in resistant isolates
Upcroft and Upcroft
1993, 2001 and
references therein
Patient isolates
Comparison of isolates
with respect to growth
Isolates exhibit
variations in their
sensitivities prior to drug
exposition
Argüello-García et al.,
2004
Induction of resistance
in vitro in WB 1
Representational
difference analysis of
gene expression
PFOR expression higher
in resistant clones;
antigenic variation
Argüello-García et al.,
2009
Induction in vitro
T. vaginalis
Comparison to wildtype
by proteomics
Metronidazole binds to
proteins of thioredoxin
pathway
Leitsch et al., 2009
Nitazoxanide
Induction of resistance
formation in vitro in
WB C6
Comparison to
wildtype with respect to
expression of selected
genes and by microarray
Cross-resistance to
metronidazole. Complex
changes of gene
expression including
antigenic variation
and overexpression of
chaperones in resistant
clone
Müller et al., 2007a,
2008a
Isoflavones
Induction of resistance
formation in vitro in
WB C6
Comparison to
wildtype with respect to
expression of selected
genes and by microarray
Most pronounced
changes of gene
expression concern
antigenic variation
Sterk et al., 2007
Unpublished data
J. Müller et al.
inactivate proteins related to the thioredoxin reductase
pathway. Resistant cells overcome this blocking by
reregulating other enzymes involved in oxidoreductive processes, such as PFORs. In this model, downregulation of PFOR would be a consequence rather
than a prerequisite of resistance formation (Leitsch
et al., 2009). In another study, even a positive correlation between metronidazole-resistance formation and
PFOR gene transcription activity has been observed
(Argüello-Garcia et al., 2009). Furthermore, possible
giardial metronidazole-resistance mechanisms involving reduced uptake of the drug (Boreham et al.,
1988) or complex changes in the gene expression pattern including alterations of those genes (encoding
variant surface proteins, VSPs) that mediate antigenic
variation of the parasite (Müller et al., 2007; 2008;
see also below) have been described.
In order to elucidate the biochemical nature of resistance formation against nitazoxanide metronidazole, and formononetin, G. lamblia WB C6 clones
resistant to these drugs were generated (Müller et al.,
2007a, 2008a; Sterck et al., 2007) and compared to
the wildtype WB C6 with respect to their growth behavior and to the expression pattern of genes that are
potentially involved in resistance formation. In all
cases, resistance formation occurred after a small
number of selection cycles. The pattern of up- and
downregulated mRNAs was completely different in a
nitazoxanide/metronidazole double resistant line, a
line resistant for metronidazole only and a formononetin resistant line. The only annotated ORFs downregulated in all screens encoded the major
surface-labeled trophozoite antigen 417 encoding the
major surface antigen C6 (TSA417; Müller et al.
2007a, 2008a; Sterk et al., 2007 and unpublished
data). In the nitazoxanide/metronidazole double resistant line, genes potentially involved in protein
phosphorylation and network formations, especially
ankyrins, and chaperonins (especially the cytosolic
heat shock protein 70) are upregulated. The roles of
these chaperonins in Giardia are not well understood.
In human cells, molecular chaperones such as HSP72
and HSP27 prevent cells from apoptosis induced by
heat shock (Gabai and Sherman, 2002). If similar
mechanisms occur in Giardia, a general response to
stress via induction of chaperonins could be responsible for thermotolerance as well as for drug resistance. Interestingly, a similar change in gene
expression patterns has been found upon expression
of neomycin phosphotransferase as a resistance marker and a subsequent selection for puromycin resistant
trophozoites (Su et al., 2007).
As mentioned above, nitazoxanide inhibits protein
disulfide isomerases (PDIs) from G. lamblia (Müller
et al., 2007a) and N. caninum (Müller et al., 2008b).
Inhibition of PDIs in vivo could lead to the formation
of badly folded proteins and thus to an impairment of
central metabolic and regulatory processes. Cells that
overexpress chaperonins and proteins involved in the
stabilisation of subcellular structures such as ankyrins
could overcome the deleterious effects due to PDI
inhibition.
Such indirect, stabilizing effects due to chaperonins or related proteins could also explain why artificially generated albendazole resistance (Upcroft
et al., 1996; Argüello-Garcia et al., 2009) is not correlated to specific mutations of the β-tubulin gene as
found in other albendazole resistant organisms. In
this context, it is noteworthy that antigenic variation
was also found to be a striking phenomenon associated with in vitro resistance formation against albendazole (Argüello-Garcia et al., 2009).
Giardia populations seem to have very flexible
mechanisms in order to respond to changes in the environment such as drug pressure, presence of antibodies or even changes in medium composition and to
changes of the internal milieu by introduction of
transgenes (Su et al., 2007) in a way that complex
gene expression patterns seem to be fine-tuned in order to find an optimal compromise between speed of
growth and survival. How can these changes be
explained? Individual trophozoites in cultures of
G. lamblia (clone WB C6) exhibit substantial variations in their degree of susceptibility/resistance to different drugs even in absence of any previous drug
pressure (Argüello-Garcia et al., 2004). This suggests
that resistance formation within a G. lamblia in vitro
culture seems to have its origin in a drug-independent
process of continuous growth competition between
relatively drug-susceptible and drug-resistant trophozoite subpopulations. The differences in these subpopulations may be explained by genetic effects, i.e.
random point mutations all over the genome and selection of resistant phenotypes upon drug application
or by epigenetic effects (transcriptional or posttranscriptional) resulting in subpopulations with different
Treatment of Giardiasis and Drug Resistance
gene expression patterns. In the latter case, exposure
of the trophozoites to a drug in sublethal concentrations might even enhance conversion from a sensitive
to a resistant phenotype.
The only common finding in all resistant lines is
the change in variable surface protein gene expression (where investigated) resulting in antigenic variation (Müller and Gottstein, 1998; Svärd et al., 1998;
Prucca and Lujan, 2009). Antigenic variation in
G. lamblia allows the parasite to escape the host immune response (Müller and Gottstein, 1998; Müller,
and von Allmen, 2005; Roxström-Lindquist et al.,
2006). Epigenetic mechanisms at the transcriptional
level involving histone acetylation (Kulakova et al.,
2006) and at the post-transcriptional level involving
RNA interference (Prucca et al., 2008) are considered
as controlling antigenic variation (Prucca and Lujan,
2009). Thus, common epigenetic mechanisms may
control both antigenic variation and drug resistance in
Giardia.
The role of epigenetics in antigenic variation and
drug resistance in Giardia and other parasites is – of
course – far from being understood, but may be of a
broader interest since evolution of antibiotic resistance initiated by increasing sublethal doses of various antibiotics has been attributed to epigenetic
inheritance also in bacteria (Adam et al., 2008). The
overview in Table 2 shows that most studies have
been performed with the strain WB C6 showing a
high degree of antigenic variation (Nash et al., 1990b).
Drug resistance studies from other strains with little
variation such as GS-M-83-H7 or WB A6 (Nash et al.,
1990a) are lacking but would be most useful in putting the light on the relationship between antigenic
variation and drug resistance formation.
Conclusions
After more than 7 decades of antigiardial chemotherapy, metronidazole is still the treatment of choice.
Novel drugs such as nitazoxanide or plant-derived
compounds do not offer obvious advantages with respect to efficacy or resistance formation. A couple of
studies concerning resistance formation suggest a
more complex mechanism than the mere mutation of
a target protein. Where investigated, resistance formation is correlated with antigenic variation suggesting
a common, underlying mechanism far from being understood. Future approaches aimed at transgenic upor down-regulation of putative drug target proteins in
G. lamblia are expected to provide conclusive insights
into both the mode of action of anti-giardial drugs and
the mechanisms that mediate resistance against these
drugs.
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