International Journal for Parasitology 37 (2007) 11–20

www.elsevier.com/locate/ijpara

Review Article

Irritable bowel syndrome: A review on the role of intestinal

protozoa and the importance of their detection and diagnosis
a,*
D. Stark , S. van Hal a, D. Marriott a, J. Ellis b, J. Harkness a

a
St. Vincent’s Hospital, Department of Microbiology, Victoria St., Darlinghurst, NSW2010, Sydney, Australia
b
University of Technology Sydney, Department of Medical and Molecular Biosciences, Sydney, Australia

Received 31 July 2006; received in revised form 6 September 2006; accepted 19 September 2006

Abstract

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder in which abdominal pain is associated with a defect or a
change in bowel habits. Gut inflammation is one of the proposed mechanisms of pathogenesis. Recent studies have described a possible
role for protozoan parasites, such as Blastocystis hominis and Dientamoeba fragilis, in the etiology of IBS. Dientamoeba fragilis is known
to cause IBS-like symptoms and has a propensity to cause chronic infections but its diagnosis relies on microscopy of stained smears,
which many laboratories do not perform, thereby leading to the misdiagnosis of dientamoebiasis as IBS. The role of B. hominis as an
etiological agent of IBS is inconclusive, due to contradictory reports and the controversial nature of B. hominis as a human pathogen.
Although Entamoeba histolytica infections occur predominately in developing regions of the world, clinical diagnosis of amebiasis is
often difficult because symptoms of patients with IBS may closely mimic those patients with non-dysenteric amoebic colitis. Clinical man-
ifestations of Giardia intestinalis infection also vary from asymptomatic carriage to acute and chronic diarrhoea with abdominal pain.
These IBS-like symptoms can be continuous, intermittent, sporadic or recurrent, sometimes lasting years without correct diagnosis. It is
essential that all patients with IBS undergo routine parasitological investigations in order to rule out the presence of protozoan parasites
as the causative agents of the clinical signs.
Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Irritable bowel syndrome; IBS; Protozoa; Dientamoeba fragilis; Blastocystis hominis

1. Introduction
Irritable bowel syndrome (IBS) is defined as a functional
group of bowel disorders in which abdominal pain is asso-
ciated with defecation or alterations in bowel habit in the
absence of an organic cause (Brandt et al., 2002). It is
one of the most commonly diagnosed gastrointestinal ill-
nesses with prevalence rates of 10–15% in North America
and Europe (Brandt et al., 2002; Mertz, 2003 ) leading to
an estimated cost to the United States of 1.7 billion dollars
in 2000 (Mertz, 2003). Similar rates have been documented
in developing countries; although it is difficult to know how
aggressively alternative diagnoses were excluded in these
populations (Olubuyide et al., 1995; Curioso et al., 2002;
*
Corresponding author. Tel.: +61 2 8382 9196; fax: +61 2 8382 2989.
E-mail address: dstark@stvincents.com.au (D. Stark).
Lule and Amayo, 2002; Gwee, 2005; Kang, 2005; OKeke
et al., 2005). Irritable bowel syndrome affects all ages and
all sexes with a 2:1 female predominance.
The pathophysiology of IBS remains elusive and no
mechanism is unique to, or characterises, IBS. There are
probably several interconnected factors which occur to
varying degrees in patients that account for the clinical
symptoms of IBS. These include altered gut reactivity
(colonic and/or small bowel motility) in response to lumi-
nal or psychological stimuli, visceral afferent hypersensitiv-
ity, and a hypersensitive gut with enhanced visceral
perception and pain (Thompson et al., 2000; Mertz,
2003). It is unclear whether this hypersensitivity is medi-
ated via the local or central nervous system or a dysregula-
tion of the brain–gut axis. In addition, hereditary,
environmental and dietary factors probably play a role
(Levy et al., 2001; Brandt et al., 2002).

0020-7519/$30.00 Ó 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijpara.2006.09.009
12 D. Stark et al. / International Journal for Parasitology 37 (2007) 11–20
Persistent low-grade inflammation may play a role in
IBS. It is estimated that 7–31% of patients with infec-
tious gastroenteritis go on to develop IBS (post-infec-
tious IBS) (McKendrick and Read, 1994; Neal et al.,
1997; Rodriguez and Ruigomez, 1999). This translates
into a 14-fold increase in the overall risk of developing
IBS following acute diarrhoea compared with the general
population. Risk factors include longer duration of
symptoms, young age and being female. The immune
activation is characterised by increased intra-epithelial
and lamina propria lymphocytes. These changes have
been documented to persist for more than a year follow-
ing the resolution of the infectious agent (McKendrick
and Read, 1994; Neal et al., 1997; Rodriguez and Ruigo-
mez, 1999). Inflammation may also occur in deeper lay-
ers of the gut. In a small IBS cohort, full-thickness
biopsies revealed normal mucosa but intra- and peri-gan-
glionic lymphocytic infiltration at the region of the
myenteric plexus (Tornblom et al., 2002). Furthermore,
a correlation was revealed with the numbers of mast
cells in close proximity to enteric nerves and the intensity
of symptoms (Barbara et al., 2004). Most patients recov-
er. However, in a subset of patients, persistent immune
activation results in ongoing symptoms. Possible mecha-
nisms include a genetic predisposition, continuous anti-
genic exposure (bacterial, parasitic or dietary) or
molecular mimicry.
As there are no biological markers for IBS, diagnosis is
based on a cluster of clinical symptoms (Rome II criteria;
Saito et al., 2000) which include abdominal pain or discom-
fort with associated changes in bowel frequency and/or
stool form. Symptoms may be relieved by defecation. A
central feature that supports IBS is the presence of contin-
uous or recurrent symptoms for a minimum of 3 months.
Passage of mucous and bloating or sensation of abdominal
distension may also occur.
There are no firm recommendations about the extent
and type of testing required to exclude other organic
pathology. Investigation of stool for ova, cyst and parasites
is generally recommended when diarrhoea is the major
manifestation of IBS. The intestinal protozoa associated
with IBS-like symptoms are shown in Table 1. The role
that protozoan parasites may play in this complex disease
has not been fully investigated.
2. Blastocystis hominis and IBS
Blastocystis hominis is an enteric unicellular protozoan
parasite that inhabits the human intestinal tract and is
the most frequently reported protozoan in human faecal
samples (Stenzel and Boreham, 1996). The taxonomy of
B. hominis remains controversial. Although criteria by
which to define the species have not been agreed upon,
extensive genetic diversity was documented using numer-
ous molecular techniques. At least 10 B. hominis subspecies
(genotypes) have being identified. (Clark, 1997; Abe, 2004;
Yoshikawa et al., 2004; Yan et al., 2006).
High prevalence rates of infection with B. hominis are
seen in both developed and developing regions of the world
(Suresh et al., 2005; Yaicharoen et al., 2005). Acquisition of
B. hominis is thought to occur as a result of frequent ani-
mal–human, human–human and human–animal transmis-
sion. Animal carriage was recognised in non-human
primates including: pigs and cattle, and birds, rodents, rep-
tiles, amphibians and insects with similar molecular diver-
sity (Tan, 2004; Tanizaki et al., 2005).
The role of B. hominis in human intestinal disease is con-
troversial. Several clinical and epidemiological studies
implicate B. hominis as a pathogen while others dismiss it
as a commensal (Chen et al., 2003; Leder et al., 2005; Ros-
signol et al., 2005; Tan and Suresh, 2006). However, the lit-
erature remains anecdotal and most of the studies were
uncontrolled. As no recognised animal model exists for
B. hominis, Koch’s postulate is unable to be fulfilled in
order to confirm the nature of B. hominis as a pathogen.
The role of B. hominis in IBS is likewise limited by the
uncertainty surrounding its pathogenicity. However, symp-
toms that were attributed to infection with B. hominis are
non-specific, IBS-like and include diarrhoea, abdominal
pain, cramps or discomfort and nausea (Babb and Wagen-
er, 1989; Dawes et al., 1990; Doyle et al., 1990; Zaki et al.,
1991; Rossignol et al., 2005; Graczyk et al., 2005). Further-
more, chronic excretion with persistent symptoms has been
reported (Zaki et al., 1991).
Low grade inflammation is one proposed mechanism of
IBS through persistent antigenic exposure as in persistent
carriage/infection. Immune activation was demonstrated
by Hussain et al. (1997) who showed that IgG antibody lev-
els to B. hominis in patients with IBS were significantly ele-
vated compared with asymptomatic controls. Endoscopic
biopsies have demonstrated oedema and inflammation in
the colon and small bowel from IBS patients (Gallagher
and Venglarick, 1985; Guglielmetti et al., 1989; Al-Tawil
et al., 1994). Furthermore, exacerbation of ulcerative colitis
may occur with the acquisition of B. hominis (Jeddy and
Farrington, 1991). Zuckerman et al. (1994) demonstrated
that inflammation leads to impaired intestinal permeability
with reduced excreted radioactive markers.
Yakoob et al. (2004) demonstrated faecal carriage of B.
hominis to occur more frequently in patients with IBS
(46%) than the control group (7%). In contrast, Tungtrong-
chitr et al. (2004) found no association between faecal
carriage of B. hominis and IBS. Clearly, more extensive
case-controlled studies are required to clearly define the
potential pathogenicity of B. hominis in intestinal disease
and IBS.
3. Dientamoeba fragilis and IBS
Dientamoeba fragilis is a trichomonad parasite and
humans are probably the definitive host. However, there
are no recent studies on the organism’s host distribution
and there have been few reports on D. fragilis in species other
than humans. Non-human primates including macaques
Table 1
Intestinal protozoa associated with chronic diarrhoea and Irritable bowel syndrome-like symptoms
Species
Balantidium coli
Blastocystis hominis
Cryptosporidium parvum
Cyclospora cayetanensis
Dientamoeba fragilis
Entamoeba histolytica
Giardia intestinalis
Isospora belli
Global distribution
Developing regions particularly
warm and temperate climates,
rare in developed regions
Worldwide
Worldwide
Mainly developing regions
Worldwide
Mainly developing regions, rare
in developed Western Counties
Worldwide
Mainly tropical developing
regions, rare in developed
countries
Diagnosis
Wet preparations, concentrates
and/or permanent stains
Wet preparation, permanent
stain, culture techniques
Modified acid fast stain, multiple
specimens
Modified acid fast stain, multiple
specimens
Permanent stain – fixation of
faecal material essential to
prevent degeneration of
trophozoites, multiple specimens
Permanent stain, multiple
specimens
Permanent stain, multiple
specimens
Wet prep on fresh faecal material
or from concentrated material;
oocysts easily overlooked,
multiple specimens
Clinical symptoms
Asymptomatic, diarrhoea or
dysentry
Controversial cause of diarrhoea
Diarrhoea, abdominal cramps,
nausea, 5–10 bowel movements,
often self-limiting
Similar to C. parvum
Diarrhoea, abdominal pain, and
nausea
Asymptomatic, diarrhoea, colitis,
dysentery
Asymptomatic, diarrhoea
Diarrhoea, which may last for
long periods, weight loss,
abdominal colic, with 6–10 bowel
movements per day
Duration and other information
Diarrhoea may persist for weeks
D. Stark et al. / International Journal for Parasitology 37 (2007) 11–20
to months
Asymptomatic carriage, acute
diarrhoea chronic infections
(weeks–months)
Dependant on immune status,
chronic infections seen in
immunosuppressed
Self-limiting (3–4 days) or
intermittent (2–3 weeks)
Immunosuppressed patients >12
weeks
Days to years, chronic infection
common
Weeks, years, chronic infections
reported
Acute infection is often followed
by a subacute or chronic phase
Chronic infections can develop
(mainly in the
immunosuppressed); oocysts can
be shed for several months to
years
13
14 D. Stark et al. / International Journal for Parasitology 37 (2007) 11–20
(Hegner and Chu, 1930; Knowles and Das Gupta, 1936) and
baboons (Myers and Kuntz, 1968) were reported as having
D. fragilis trophozoites in their stools and it was also report-
ed in a sheep (Noble and Noble, 1952). Given this, coupled
with the high incidence of D. fragilis in humans, we must
assume that humans are the primary host for this organism.
Furthermore, as D. fragilis diagnostic procedures are not
commonly performed in most laboratories the true preva-
lence of this trichomonad is probably significantly under-re-
ported (Girginkardesler et al., 2003; Crotti et al., 2005).
Dientamoeba fragilis is pathogenic and causes acute
gastrointestinal disease in children and adults (Windsor
et al., 1998; Crotti et al., 2005; Stark et al., 2005b). In
addition, chronic infections have been well documented.
The most frequent clinical symptoms associated with D.
fragilis infection are diarrhoea (50%), abdominal pain
(83%) and looseness of stools (Windsor and Johnson,
1999; Stark et al., 2005b). The duration of symptoms
in chronic infection has varied from 1 month to 2 years
(Windsor et al., 1998). In a prospective Australian study
conducted over a 30 month period, 32% of patients with
D. fragilis presented with chronic diarrhoea (>2 weeks to
years in duration). Chemotherapy directed against D.
fragilis results in clinical improvement and subsequent
faecal clearance has been documented in symptomatic
patients. Tetracycline, doxycycline, iodoquinol, metroni-
dazole and secnidazole have all been used with varying
success (Priess et al., 1991; Borody et al., 2002;
Girginkardesler et al., 2003).
There is little understanding of the pathogenesis and
pathology resulting from D. fragilis infection. Inflamma-
tion and fibrosis were described in the vermiform appendix
(Burrows et al., 1954; Burrows and Swerdlow, 1956).
Eosinophilic colitis (Cuffari et al., 1998) and ulcerative coli-
tis (Shein and Gelb, 1983; Ring et al., 1984) have also been
reported. However, there are no pathological features that
are unique to D. fragilis. Furthermore, animal experiments
in rats (Kean and Malloch, 1966) and recent reports, have
failed to demonstrate invasion and ulceration (Cerva et al.,
1991). Elucidation of the exact pathology of D. fragilis
infection is hampered by the lack of a suitable animal mod-
el (Dobell, 1940; Knoll and Howell, 1945; Kean and
Malloch, 1966).
As D. fragilis causes a chronic illness with symptoms
similar to IBS, it is not surprising that some patients with
D. fragilis are misdiagnosed as having IBS (Borody et al.,
2002). Furthermore, in this study treatment with iodoqui-
nol and doxycycline resulted in successful elimination of
D. fragilis and resolution of IBS symptoms (Borody
et al., 2002). Potentially important studies such as this
one need to be reported in more detail in the peer-reviewed
scientific literature.
4. Entamoeba histolytica and IBS
Entamoeba histolytica is a non-flagellated amoeboid
protozoan parasite. The genus Entamoeba comprises six
species (E. histolytica, Entamoeba dispar, Entamoeba mosh-
kovskii, Entamoeba poleki, Entamoeba coli and Entamoeba
hartmanni), that colonise the intestinal lumen. Humans are
the primary reservoir (Stauffer et al., 2006). All these spe-
cies are considered commensal organisms and cause no
intestinal disease with the exception of E. histolytica.
Faecal carriage of E. dispar is far more common than E.
histolytica. Entamoeba dispar is morphologically identical
to E. histolytica and genetic differences have confirmed
the separation of these two as independent species (Dia-
mond and Clark, 1993). Entamoeba histolytica is an inva-
sive pathogen and the causative agent of amebiasis with
approximately 50 million cases annually, usually acquired
in the developing world. However most humans infected
with E. histolytica are asymptomatic (Benetton et al.,
2005).
When clinical symptoms develop they are usually limit-
ed to the gastrointestinal tract. The incubation period of
invasive disease can vary greatly, ranging from days to
years. The symptoms of abdominal pain, tenderness and
diarrhoea with >10 bowel movements/day correspond with
colitis and ulcerative disease on histopathological examina-
tion. Bowel complications occur in 1–4% of patients. Inva-
sive or extraintestinal disease is uncommon and may be
present in 0.1–1% of symptomatic patients, with the liver
being the most common site involved (>50%) (Stauffer
and Ravdin, 2003).
The factors that control the pathogenesis of E. histolyti-
ca are not completely understood. However, key features
are the ability of the organism to lyse host cells and cause
tissue destruction. Furthermore, E. histolytica has also
been shown to induce both cellular and humoral immune
responses in extra-intestinal disease (Ackers and Mirelman,
2006).
Amoebiasis may form part of the differential diagnosis
of patients with IBS, especially in those with an acute pre-
sentation or acute exacerbations of IBS symptoms. A travel
history is helpful, as cases are generally limited to those
patients who have visited an endemic area. Early studies
implicated amoebic dysentery in the development of IBS
among British soldiers returning from Egypt at the end
of World War II (Stewart, 1950; Chaudhary and Truelove,
1962). Despite these initial reports, several studies from
India have suggested that exposure to E. histolytica did
not predispose patients to IBS symptoms (Nanda et al.,
1984; Anand et al., 1997; Sinha et al., 1997, 1999) and all
patients spontaneously eradicated the organism.
5. Giardia intestinalis and IBS
Giardia intestinalis is a common and ubiquitous flagel-
lated protozoan parasite, with a worldwide distribution.
Giardia species are parasites of mammals and other
animals, including reptiles and birds (Ali and Hill, 2003;
Hamnes et al., 2006; Castro-Hermida et al., 2006). Humans
become infected by ingestion of cysts, which develop into
trophozoites after excystation. Infections occur in both
 D. Stark et al. / International Journal for Parasitology 37 (2007) 11–20
developed and developing regions of the world (Savioli
et al., 2006).
After acquisition, approximately 50% of people clear
Giardia without any untoward effects; 5–15% of people
shed cysts asymptomatically while the remainder develop
an acute and/or chronic infection (Ali and Hill, 2003; Sav-
ioli et al., 2006). The acute infection lasts for days to weeks
and is accompanied by nausea and the sudden onset of
explosive, watery, foul smelling diarrhoea. The acute phase
is often followed by a sub-acute or chronic phase. Chronic
symptoms may last for years and be continuous, intermit-
tent, sporadic or recurrent with episodes of diarrhoea or
loose stools. Between bouts of diarrhoea the patient may
have normal stools. However, abdominal discomfort may
be continuous and independent of the alteration in bowel
habits (Vandenberg et al., 2006).
Giardia infection, especially chronic infection, can
resemble IBS and must be excluded by diagnosis. Further-
more, evidence for post-infectious IBS secondary to low
grade inflammation was demonstrated by D’Anchino
et al. (2002). In this study, the persistent low grade inflam-
mation was associated with faecal persistence of Giardia. In
a recent study of 137 patients with symptoms of IBS who
underwent duodenal biopsies and stool examinations for
parasites, Giardia infection was diagnosed in 6.5% of
patients with IBS (Grazioli et al., 2006), indicating Giardia
may be a common cause of IBS-like symptoms.
6. Other enteric protozoa causing chronic infections
Balantidium coli is the only pathogenic ciliate to infect
humans. The distribution is limited to warm tropical cli-
mates and infections are closely associated with pigs.
Human infections are rare in temperate climates and in
Western industrialised counties. Clinical presentations
include a spectrum of disease from asymptomatic carriage
to severe dysentery. Symptoms include nausea, vomiting
and diarrhoea or dysentery (Garcia, 2001). The diarrhoea
may persist for weeks to months with dysenteric symptoms
resembling those of amebiasis. As these infections predom-
inate in developing countries the role of B. coli in IBS has
not been investigated.
Other intestinal protozoa that are associated with
chronic infections are the coccidian parasites: Cryptospror-
idium parvum, Cyclospora cayetanensis and Isospora belli.
Cryptosporidium parvum has a worldwide distribution,
while C. cayetanensis and I. belli are restricted in their dis-
tribution to developing regions (Garcia, 2001). Isospora
belli is further limited to mainly tropical regions. The dura-
tion and type of symptoms associated with infections by
coccidian parasites are dependent on the immune status
of the patient. Immunocompetent patients generally have
a self-limited diarrhoea while chronic infection occurs in
immunosuppressed patients (Lewthwaite et al., 2005).
Cyclospora cayetanensis is an important emerging gastroin-
testinal pathogen with a number of outbreaks reported in
the USA and Canada (Fleming et al., 1998; Herwaldt
15
and Beach, 1999). Diarrhoea is generally rapid in onset
with frequent relapses which may last from 4 to 7 weeks
and may be prolonged if the patient is immunosuppressed
(Hodge et al., 1995; Fleming et al., 1998). Even though coc-
cidian protozoa may cause chronic infection with IBS-like
symptoms it is unlikely they play any role in true IBS cases.
7. Importance of diagnostic tests
As discussed above, intestinal protozoa must be includ-
ed in the differential diagnosis of IBS because they cause
symptoms resembling IBS (Benetton et al., 2005; Stark
et al., 2005b; Savioli et al., 2006) or they may be innocent
bystanders, as occurs with asymptomatic carriage or infec-
tions by non-pathogenic protozoa. They may cause signif-
icant flares of IBS with acquisition. Furthermore, they may
lead to IBS, secondary to ongoing low-grade inflammation.
Regardless of the mechanism or association, the impor-
tance of accurately performed diagnostic tests for intestinal
protozoa is crucial for diagnostic, therapeutic and investi-
gational decisions.
Optimal laboratory detection of intestinal protozoa
depend on several factors including the number and type
of specimen collected, the processing methods employed
and the experience and training of laboratory staff involved
in the identification of these organisms. A number of
patients may intermittently shed protozoa in their stool.
Similarly, some protozoa (G. intestinalis and D. fragilis)
have been shown to have highly variable and intermittent
shedding (van Gool et al., 2003). Therefore, a single stool
examination has a low sensitivity for detecting parasites.
Hiatt et al. (1995) compared the sensitivity of examining
one stool specimen with that of three specimens. They
found that many infections were not detected in the first
specimen submitted but were detected with subsequent
stool examinations. In patients with E. histolytica, up to
four to nine separate faecal examinations were required
to make the diagnosis. With additional stool examinations,
the diagnostic yield increases 22.7% for E. histolytica,
11.3% for G. intestinalis and 31.1% for D. fragilis (Hiatt
et al., 1995). These features suggest that a single stool spec-
imen examination will miss many pathogenic protozoa and
that for several protozoa a series of five to six separate
stool samples is required (Garcia, 2001).
The identification and diagnosis of protozoa with wet
mounts are a challenge, even for the most experienced
microscopist. The characteristics used to make a definitive
diagnosis of pathogenic protozoa are shown in Table 2 and
the photomicrographs of the organisms are shown in
Fig. 1. Organisms such as D. fragilis may be missed using
this method as prompt fixation and permanent staining is
required as trophozoites degenerate rapidly within hours
of being passed (Yang and Scholten, 1977). Faecal concen-
tration methods are routinely used in laboratories to detect
low numbers of protozoa. However, B. hominis can be dis-
rupted and destroyed by concentration techniques (Miller
and Minshew, 1988).
 16
Table 2
Characteristics to aid identification of protozoa
Organism Size Shape Nuclear characteristics Other features
Blastocystis hominis 6–40 lm Generally round Multiple small nuclei surrounding central body Different forms exist. Central body form is the
(looks like a vacuole) most common form seen, amoebic form (may be
difficult to identify as it may be confused with
yeast cells), and thin and thick walled cysts also

D. Stark et al. / International Journal for Parasitology 37 (2007) 11–20

occur
Cryptosporidium Oocysts 4–6 lm Generally round No visible nucleus, mature oocysts contain Oocyst is the diagnostic stage in stool
sporozoites
Cyclospora Oocysts 8–10 lm Generally round No nucleus visible, they resemble cryptosporidium Acid fast variable and may appear having no
but are larger and more acid fast variable colour, light pink or dark red; may appear
wrinkled in permanent stains; auto fluoresce with
epifluorescence
Dientamoeba fragilis 5–15 lm Amoeboid May have 1 or 2 nuclei; majority of forms Even though the organism is classified as a
binucleate (60%); nuclear structure not visible in flagellate it does not posses flagella; cytoplasm
unstained preparations; no peripheral chromatin; usually finely granular +/ ingested material/
fragmented karyosome (4–8 granules) vacuolated; size and shape varies greatly
Entamoeba histolytica Trophozoite: 12–20 lm Amoeboid 1 nucleus, difficult to see in unstained Cytoplasm finely granular, clear differentiation of
preparations; peripheral chromatin present in fine ectoplasm and endoplasm
granules with uniform size and distribution;
karyosome small compact and centrally located
Cyst: 10–20 lm Spherical Mature cyst, 4 nuclei; immature 1 or 2; nuclear Chromatoidal bodies may be present, bodies
structure difficult to see on wet preparations; usually elongated with blunt, round, smooth edges
peripheral chromatin present in fine granules with (round or oval); glycogen may be present;
uniform size and distribution; karyosome small clumped chromatin may be present in early cysts
compact and centrally located
Giardia intestinalis Trophozoite:10–20 lm long 5–15 lm wide Pear shaped Trophozoites: 2 nuclei, not visible in unstained Falling leaf motility, 4 lateral, 2 ventral and 2
preparations caudal flagella
Cyst: 8–19 lm long, 7–10 lm wide Oval, ellipsoidal Cysts: 4 nuclei, not distinct in unstained Longitudinal fibers in cysts, deep staining median
or round preparations bodies usually lie across longitudinal fibres;
shrinkage may occur in cysts as the cytoplasm
pulls away from the cyst wall; dehydrating
reagents in permanent stains may cause shrinkage
to the outside of cell wall causing a ‘‘halo’’ effect
 D. Stark et al. / International Journal for Parasitology 37 (2007) 11–20
Fig. 1. Microphotographs of protozoa. (A) Blastocystis hominis, (B) Dientamoeba fragilis, (C) Giardia intestinalis, (D) Entamoeba histolytica, (E)
Cryptosporidium parvum, (F) Cyclospora. Scale bar is 10 lm.
Multiple different culture techniques exist for luminal
parasites (Clark and Diamond, 2002) and these are diffi-
cult, time consuming, costly and often unrewarding. As
such, these techniques are usually restricted to specialist
parasitology laboratories and are not offered by routine
diagnostic laboratories. However, several studies have
shown that culture techniques are more sensitive than per-
manent stains in the recovery of B. hominis and D. fragilis
(Sawangjaroen et al., 1993) with a modified Robinson
medium dramatically increasing the detection rate for D.
fragilis (Windsor et al., 2003). One limitation of culture sys-
tems is that specimens need to be inoculated promptly as
reduced temperatures and refrigeration adversely affect
protozoa, especially trophozoites of D. fragilis, which
degenerate rapidly (Hakansson, 1936; Wenrich, 1944;
Sawangjaroen et al., 1993).
Serodiagnosis of invasive amebiasis has been demon-
strated to be both sensitive and specific. Several antigen
detection kits (enzyme immunoassays) are available for
the detection of E. histolytica, Giardia and C. parvum in
faecal specimens. These tests have good reported sensitivi-
ties (>90%) and specificities (>90%) (Garcia, 2001).
However, they should be used in conjunction with light
microscopy of permanent stains as patients may have
multiple pathogens.
Conventional and real-time PCR assays exist for several
parasites. These tests have high sensitivity compared with
antigen-based detection tests and morphological stool
examinations by microscopy (Bialek et al., 2002; Tanriver-
17
di et al., 2002). Compared with conventional PCR, real-
time PCR has several advantages: high sensitivity, no
requirement for post-amplification analysis which minimis-
es the risks for laboratory contamination and presentation
of results in numerical form, which are easier to interpret
than visual stained gel analysis. However, real-time or con-
ventional PCR are costly and therefore are only slowly
being introduced into clinical laboratories. Molecular
methods have recently been developed for the detection
of D. fragilis in human faeces (Stark et al., 2005a, 2006).
Methods for the simultaneous detection of multiple
pathogens in a single assay include real-time multiplex
PCR and more recently the use of oligonucleotide micro-
array technology (Wang et al., 2004). With real-time mul-
tiplex PCR there is a limit to the number of sites that can
be amplified in a single PCR reaction without loss of
sensitivity secondary to interference. This limitation is cir-
cumvented with microarrays as multiple specific oligonu-
cleotide probes are immobilised on a microchip with
subsequent hybridisation of amplified target DNA. Detec-
tion therefore combines powerful DNA amplification and
specific oligonucleotide probes directed at multiple target
sequences. Microarrays are highly sensitive, specific and a
high-throughput platform for multiple pathogen detection
and differentiation (Wang et al., 2004). Such methods
would ideally be suited for the detection of protozoan par-
asites as low numbers of organisms and/or multiple organ-
isms are often present in a single environmental or clinical
sample. This technology offers the greatest potential for
18 D. Stark et al. / International Journal for Parasitology 37 (2007) 11–20
future multi-pathogen testing but is expensive and proba-
bly beyond the capacity of many laboratories. Future
advances in this technology may, however, change the out-
look for this technology in the diagnostic laboratory.
Clinicians need to be made aware of the tests for faecal
protozoa routinely performed by their laboratory and to
ensure the correct methods are used in diagnosis. Parasites
may not be detected if permanent staining is not performed
or if only combination antigen-based kits are used. This in
turn may result in the misdiagnosis of IBS. We recommend
that to maximise recovery of protozoan parasites a mini-
mum of three faecal specimens should be collected over a
time frame of no more than 10 days and that a permanent
stained (trichrome or iron-haematoxylin stain) examina-
tion should always be undertaken by interested and well
trained laboratory staff (Garcia, 2001).
8. Conclusion
Irritable bowel syndrome is a complicated, multifaceted
heterogeneous disorder, as opposed to a single entity dis-
ease. As the exact pathogenesis of IBS is under investiga-
tion, we can only postulate as to what, if any, roles
protozoan parasites play in this condition. However, it is
essential that all patients with IBS undergo routine parasi-
tological investigations to rule out protozoan parasites as
the causative agents of the clinical signs. Blastocystis hom-
inis was reported as a possible etiological agent in IBS. Giv-
en the controversial nature of B. hominis as a bona fide
pathogen the weight of evidence suggests it seems unlikely
that B. hominis plays a major role in IBS. The presence of
B. hominis in stool samples from patients with IBS does not
necessarily imply that the symptoms are due to this organ-
ism and other infective and non-infectious causes should be
investigated. Dientamoeba fragilis was also reported in
patients diagnosed with IBS. Despite this brief report, there
is no other convincing evidence of note to suggest that D.
fragilis is associated with IBS, although the association is
probably worth investigating further. Dientamoebiasis in
its own right can cause IBS-like symptoms and since it
may persist as a long-term infection, a correct diagnosis
and treatment should be given. Both Giardia and E. histoly-
tica can also produce symptomology consistent with IBS. It
is essential that correct testing procedures are utilised by
laboratory staff and correct test ordering is undertaken
by clinicians to ensure that parasitic protozoa are defini-
tively excluded in patients with IBS. Further study is
required not only on the pathophysiological effects these
protozoa have on the intestinal mucosa of humans, but
also large scale prospective trials assessing the role intesti-
nal protozoa play in IBS need to be rationally performed.
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