Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal
Protozoan Diseases
Saul Tzipori and Justyna J Jaskiewicz, Tufts Cummings School of Veterinary Medicine, North Grafton, MA, USA
Ó 2017 Elsevier Inc. All rights reserved.
This article is an updated version of the previous edition article by Ynes R. Ortega, Mark L. Eberhard, volume 5, pp. 354–366, Ó 2008, Elsevier Inc.
Cryptosporidiosis
Gastrointestinal cryptosporidiosis affects people of all ages. It is
characterized by profuse watery diarrhea, which is most severe
in children, the elderly, and individuals with compromised
immunity.
Biology
Although Cryptosporidium has been identified primarily in the
gastrointestinal tract as a cause of diarrheal illness in humans,
AIDS patients have also been reported with infections of the
gallbladder and the respiratory tract. In the past, infection
was considered to be caused by a unique species of Cryptospo-
ridium parvum. This was later differentiated into genotype I
(human genotype) and II (bovine genotype). Genotype I was
later renamed Cryptosporidium hominis and its transmission is
considered to be almost exclusively anthroponotic. Cryptospo-
ridium parvum, which now refers to genotype II, is still consid-
ered the zoonotic group and consists of various genotypes.
Of the 15 species of Cryptosporidium described as infecting
reptiles, fish, birds, and mammals, nine can also infect humans:
C. hominis (humans), C. parvum and Cryptosporidium andersoni
(cattle), Cryptosporidium canis (dogs), Cryptosporidium felis
(cats), Cryptosporidium meleagridis (turkeys), Cryptosporidium
muris (rodents), Cryptosporidium suis (of pigs), and Cryptospo-
ridium cervine genotype (deer and sheep) (Fayer, 2010; Xiao
et al., 2001). Most of these are morphologically similar
(Table 1); therefore molecular assays are needed to differen-
tiate between them. Although most widely used are the geno-
typic tools detecting small subunit of 18S rRNA region, other
genetic targets such as oocyst wall protein and heat-shock
protein could also differentiate between Cryptosporidium
species. Additionally, full genome sequences are available for
C. parvum (Abrahamsen et al., 2004) and C. hominis (Xu
et al., 2004).
Life Cycle
Oocysts are excreted in the feces of an infected individual or,
in the case of immunocompromised people suffering from
Table 1 Differential characteristics of coccidian intestinal parasites
Characteristics Cryptosporidium Cyclospora
Oocyst size 4–6 mm 8–10 mm
Number of sporocysts 0 2
Sporozoites/sporocysts 4/oocyst 2 4
Infectious stage Oocyst Oocyst
Sporulation time N/A 7–15 days
Autofluorescence No Yes
Treatment of choice Nitazoxanide Trimethoprim/sulfamethoxazole
International Encyclopedia of Public Health, 2nd edition, Volume 6 http://dx.doi.org/10.1016/B978-0-12-803678-5.00358-1
respiratory cryptosporidiosis, with the sputum. These oocysts
are already infectious and can infect susceptible individuals.
When ingested or inhaled, oocysts undergo the process of
excystation, in which four sporozoites are released from
each oocyst. These sporozoites are invasive stages, which
target epithelial cells of the gastrointestinal (preferentially
ileum) or respiratory epithelial cells. Upon excystation,
while gliding in search for the target host cell, sporozoites
release proteins from their apical complex, which enable
host cell invasion. The series of proteins enabling locomo-
tion through the mucus, adhesion, and invasion have been
described as important virulence factors (Bouzid et al.,
2013). Once sporozoite attachment has occurred, the cell
engulfs the parasite, which now remains in an intracellular
but extracytoplasmic parasitophorous vacuole. A feeding
organelle located between the parasite and the cytoplasmic
membrane allows for selective transport of substances. This
is crucial for the parasite, which seems to have poor biosyn-
thetic capacity as inferred from compaction of its genome.
For example, Cryptosporidium has three salvage enzymes for
pyrimidines, which make it entirely dependent on the
host. Two of these, uridine kinase-uracil phosphoribosyl-
transferase and thymidine kinase, are unique to C. parvum
within the phylum Apicomplexa and may account in part
for the organism’s resistance to a broad range of drugs.
The unique localization within the cell protects the parasite
from the host’s cellular defense mechanisms and enables
resistance to drug therapy.
Once in the host cell, Cryptosporidium undergoes asexual and
then sexual multiplication. Firstly, sporozoites differentiate
into trophozoites, and then mitotic division initiates formation
of the type I meront consisting of six to eight merozoites.
Sporozoite-resembling merozoites escape meronts and rein-
vade the host cell to form type II meronts or recreate type I mer-
onts and intensify infection via repeated cycles of asexual
reproduction. Conversely, merozoites released from the type
II meront initiate sexual reproduction by reinvading the host
cells and differentiating into micro- and macrogamonts.
Sixteen microgametes bud out from the microgamont, fertilize
the adjacent macrogamont, and result in formation of a diploid
zygote. The zygote undergoes sporogony, matures, and
Isospora Sarcocystis
20
33 mm by 10–19 mm 9 by 15 mm sporocysts
2 2
4
Oocyst Sporocyst and oocyst
Up to 48 h None
Yes Yes
Trimethoprim/sulfamethoxazole Not indicated
79
80 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases
differentiates into either thin-walled oocysts (20%), which can
continue reinfection in the intestine, or thick-walled oocysts
(80%), which are shed during defecation (Figure 1). All the
intracellular developing stages remain in the parasitophorous
vacuole in the apical part of the host cell. Oocysts shed with
feces are resistant to environmental conditions and can survive
for months in water or contaminated foods with high water
activity.
While in immunocompetent patients, the ileum is the
main site of infection, in HIV-infected people distribution
of the parasite is more widespread within the intestinal tract
(Kelly et al., 1998). Among immunocompromised patients,
Cryptosporidium can also localize to the hepatobiliary tract
(French et al., 1995), pancreas, lungs (Travis et al., 1990),
stomach (Rossi et al., 1998), and middle ear (Dunand
et al., 1997). The pathology in immunosuppressed patients
is variable as it can be complicated by coinfections;
however, malabsorption and villi atrophy have been
correlated in some AIDS patients with parasite burden
(Goodgame et al., 1995). Inflammatory damage to the
small intestine was demonstrated in pediatric AIDS patients
(Guarino et al., 1997) and malnourished children
Figure 1 Life cycle of Cryptosporidium parvum. Reproduced from www.cdc.gov/dpdx/cryptosporidiosis/index.html.
(Kirkpatrick et al., 2002), which suggests diarrhea of
secretory origin as pathology.
Epidemiology
Children and the elderly are most affected. Children younger
than 2 years of age are at highest risk of development of severe
diarrhea. Additionally, underlying malnutrition increases
frequency of chronic infection, regardless of immunocompe-
tence status (Mondal et al., 2012; Tumwine et al., 2003). Simi-
larly, AIDS patients with low CD4þ T cell count have persistent
diarrhea and are not able to spontaneously clear infection.
Transmission of gastrointestinal cryptosporidiosis occurs
via the fecal–oral route. The infectious dose sufficient to infect
a healthy person is as little as 10–83 oocysts of C. hominis
(Chappell et al., 2006), and 132 of C. parvum (Dupont et al.,
1995). Contaminated water or food usually serves as a source
of infection; however, transmission may also occur via
person-to-person contact (within households, among children
in day-care centers, between sexual partners) or by contact with
animals excreting Cryptosporidium oocysts. Transmission of
pulmonary cryptosporidiosis via inhalation can also occur
 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases
Table 2 Description of some large food- and waterborne outbreaks of Cryptosporidium
Year Location Population infected
1987 Carrollton, GA 12 960
1992 Jackson County, OR 15 000
1993 Milwaukee, WI 403 000
1995 Minnesota 50
1996 Connecticut, New York 66; 1 death
2003 Maine 287
2010 (Winderstrom et al., 2014) Östersund, Sweden 27 000
2013 (Gertler et al., 2013) Halle, Germany 167
(Sponseller et al., 2014). Petting zoos have been implicated in
cryptosporidiosis outbreaks involving children who came in
contact with animals. Similarly, small outbreaks involving
veterinary students have been recorded (Preiser, 2003).
Human infections are characterized by an incubation period
of 1–12 days.
In the United States, investigations of food-borne outbreaks
have incriminated consumption of fresh vegetables and
prepared foods as common modes of transmission (Table 2).
Vegetables and produce eaten raw may be contaminated by irri-
gation water at the farm level, by water used to keep produce
fresh and hydrated at the market level, or during food prepara-
tion at the consumer level. In other instances, manipulation of
foods by food handlers with cryptosporidiosis or improper
hygienic practices has initiated food-borne outbreaks. Crypto-
sporidium has also been isolated from various species of animals
other than their natural host. Flies, rotifers, and free-living
nematodes may serve as transport vectors of viable oocysts.
Mussels, clams, and oysters can concentrate Cryptosporidium
oocysts in their gills and hemolymph and have been isolated
from shellfish worldwide; however, to date, outbreaks of cryp-
tosporidiosis associated with consumption of shellfish have
not been reported.
In parts of the developing world, where access to clean water
and adequate sanitation are lacking, cryptosporidiosis is
endemic, most severely affecting populations of young children
and HIV-infected people. In fact, Cryptosporidium is the second
most frequent contributor to the incidence of severe diarrhea
among infants in Africa and Asia (Kotloff et al., 2012). In the
rural setting, livestock significantly contributes to
environmental contamination with oocysts of zoonotic
strains (Daniels et al., 2015).
Clinical Manifestations
In immunocompetent individuals, self-limiting diarrhea lasts,
on average, 15 days; however, oocyst shedding in the feces
can continue for more than 30 days in an intermittent fashion
(Shepherd et al., 1988). Unlike otherwise healthy patients,
immunocompromised patients suffer from voluminous,
chronic, and life-threatening diarrhea (Abubakar et al., 2007).
Impaired absorption and enhanced secretion contribute to
stunting among pediatric patients. A Prolonged intestinal
damage can also result in long-term cognitive damage, reduced
immune response, and decreased vaccine efficacy (Guerrant
et al., 1999).
81
Outbreak Comments
Waterborne/drinking Spring/river
Waterborne/drinking Spring/river
Waterborne/drinking Lake
Food-borne Chicken salad
Food-borne Fresh apple cider
Food-borne Fresh apple cider
Waterborne/drinking Municipal water treatment failure
Waterborne/recreational Summer/river
Diagnosis
The most commonly used diagnostic method is the detection
of oocyst or their antigens in stool or sputum samples. Oocysts
measure 4–6 mm in diameter and can be detected using micro-
scopic methods: least costly modified acid-fast stain and more
expensive direct and indirect immunofluorescence (Figure 2).
Sensitivity of both microscopic methods can be affected by
intermittent shedding and sample consistency. Other
antibody-based assays targeting Cryptosporidium antigen in feces
are commercially available for use with clinical and environ-
mental samples. Molecular assays such as genetic fingerprinting
and genotyping are also used for diagnosis; however, these are
mainly applied in epidemiological studies and outbreak
investigations.
Treatment
Many drugs have been evaluated against Cryptosporidium, but
only a handful is effective. In the United States, the only
FDA-approved drug for treatment of cryptosporidiosis is
nitazoxanide (approved for use in children). When
nitazoxanide (500 mg) is given twice daily for 3 days to
children without HIV infection, a 96% clinical recovery is
achieved, and 93% of patients stop shedding Cryptosporidium
oocysts. Paromomycin has also been evaluated with
conflicting results, particularly in AIDS patients. In HIV-
infected persons, highly active antiretroviral treatment is the
most effective means of resolving Cryptosporidium infection as
it aids in the replenishment of CD4þ cells. Relapse of the
infection may occur if treatment is discontinued. Given that
parasite residing in the host cell is not accessible to drugs,
virulence factors enabling adhesion and invasion of
sporozoites and merozoites hold more promise as drug targets.
Multiple in vitro studies and in vivo mice and calf models of
targeted immune therapy against these targets show efficacy in
limitation of infection with C. parvum (Imboden et al., 2010,
2012; Riggs et al., 2002). Also, immune colostrum is known
to provide protection against oral challenge in animal models
(Perryman et al., 1999). Additionally, the possibility of adap-
tive immunity has been studied. Recent studies reported the
successful reduction of parasite load in mice receiving CD4þ
T cells from C. parvum–infected mice (Tessema et al., 2009).
Similarly, evidence of complete protection of rechallenged
piglets after recovery from primary infection has been reported
(Sheoran et al., 2012).
82 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 2 Morphological characteristics of Cryptosporidium parvum, Cyclospora cayetanensis, and Isospora belli. Cryptosporidium: (a) differential inter-
ference contrast microscopy (DIC); (d) modified acid-fast (AF) stain; (g) antibody-based immunofluorescence assay. Cyclospora: (b) DIC; (e) AF stain;
(h) autofluorescence. Isospora: (c) DIC; (f) AF stain; (i) autofluorescence.

Prevention
Cryptosporidium oocysts are highly resistant to chlorine at levels
standardly used in treatment of drinking water or chlorinated
recreational water venues. For that reason, waterborne trans-
mission, either by drinking or by recreational water, has proven
to be the most frequent mode of transmission of this parasite in
the developed world. Both of the largest outbreaks: Milwaukee
in 1993 affecting 400 000 people and Sweden in 2010 affecting
27 000 people, arose from improper water treatment
(Mackenzie et al., 1994; Widerstrom et al., 2014). Outbreaks
resulting from exposure to contaminated recreational water
are usually smaller in number of affected individuals, e.g.,
recent outbreak of C. hominis in Germany following river flood-
ing affected 167 people (Gertler et al., 2015). In the United
States, many Cryptosporidium waterborne outbreaks have been
reported (Table 2). Much emphasis has been placed on pre-
venting parasite transmission by way of tap or drinking water.
The water industry has studied extensively procedures to
adequately remove and inactivate Cryptosporidium oocysts.
Regulations and methods to detect this parasite in water are
available from the U.S. Environmental Protection Agency
(2016).
Cryptosporidiosis can be prevented by maintaining good
hygienic practices, including washing hands after toilet use and
before food handling. Special attention is needed for children
and persons in contact with animals. This is particularly impor-
tant for food handlers. Hikers should avoid drinking river or lake
water unless it is boiled, filtered (<2 mm pore diameter), or,
though more difficult, rendered safe by efficient chemical treat-
ment. Water coagulation/flocculation, sedimentation, filtration,
and disinfection can remove more than 99% of oocysts. UV,
ozone, chlorine, and chlorine dioxide have been examined for
inactivation efficiency in Cryptosporidium with variable results
(Table 3).
DNA vaccines expressing sporozoite surface proteins have
been demonstrated to be effective in eliciting specific responses
against C. parvum in mouse models (Benitez et al., 2009). Thus
far, research on vaccine and immunotherapy holds most
promise in developing effective control strategies of Cryptospo-
ridium infections.
Cyclosporiasis
Cyclospora cayetanensis was first reported in patients with acute
diarrhea in 1979. At that time it was considered to be a cocci-
dian-like, a cyanobacteria-like, blue-green alga or large Crypto-
sporidium organism. In 1992, it was finally fully characterized
and classified within the phylum Apicomplexa as a coccidian.
Unlike other coccidian parasites infectious to humans, Cyclo-
spora requires 7–15 days to fully sporulate and become infec-
tious. Cyclosporiasis is characterized by persistent diarrhea,
anorexia, nausea, abdominal pain, flatulence, fatigue, and in
some instances, fever.
Biology
As of today, C. cayetanensis is only described to infect humans.
Nonhuman primates also harbor other species of Cyclospora:
Cyclospora cercopitheci, Cyclospora colobi, and Cyclospora papionis,
 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases
Table 3 Summary of inactivation procedures of Cryptosporidium and Cyclospora
Parasite Compound/treatment
Cryptosporidium a
Chlorine
Monochloramine
Chlorine dioxide
Ozone
Desiccation
Hydrogen peroxide
UV radiation
Heat
Freezing
Cyclosporab Heat
Freezing
a
Fayer, R, Speer, C.A., Dubey, J.P., 2006. The general biology of Cryptosporidium. In: Fayer, R. (Ed.), Cryptosporidium and Crypto-
sporidiosis. CRC Press, Boca Raton, FL, pp. 1–42.
which are morphologically similar to C. cayetanensis. These
species seem to be also host species–specific. Molecular anal-
ysis of the 18S sRNA gene of C. cayetanensis and the three other
Cyclospora species suggests that they are molecularly different.
The genus Cyclospora is phylogenetically most closely related
to the Eimeria species, although they are morphologically
different. Cyclospora cayetanensis oocysts were also identified
in feces of chickens, ducks, and dogs living in endemic areas
(Chu et al., 2004; Eberhard et al., 1999); however, the role of
animals as reservoirs has not been established. To date, no
animal models have been established to experimentally prop-
agate Cyclospora. Experimental infection of a variety of host
species and human volunteers was not successful (Alfano-
Sobsey et al., 2004; Eberhard et al., 2000).
Life Cycle
Unsporulated oocysts are excreted into the environment with
the feces of infected individuals. After 7–15 days of incuba-
tion at 23–27  C, oocysts differentiate to form two sporocysts
per oocyst, each containing two sporozoites (Figure 2). Cyclo-
spora infection is acquired when contaminated food or water
containing sporulated oocysts is ingested. Upon their excysta-
tion in duodenum and jejunum, released sporozoites infect
epithelial cells of the intestinal tract, particularly the terminal
portion of the jejunum and ileum. Sporozoites multiply asex-
ually producing type I and II meronts. Asexual multiplication
may continue or sexual reproduction may be initiated by
differentiation of type II meronts into sexual stages: micro
and macrogametocytes. As a result of fertilization, oocysts
are formed and excreted into the environment as unsporu-
lated oocysts (Figure 3). Cyclospora oocysts have been also
detected in sputum samples from HIV patients coinfected
with pulmonary tuberculosis (Di Gliullo et al., 2000; Hussein
et al., 2005).
Epidemiology
Biological and epidemiological evidence suggests that C.
cayetanensis is an anthroponotic pathogen transmitted via
the fecal–oral route. Food- and waterborne transmission
seem thus to be the main routes of transmission. The time
83
CT product Result
1
7200 mg min l >99% inactivation
3600 mg min l1 >99% inactivation
1.12 mg min l1 97% inactivation
4.5 mg min l1 99% inactivation
Air dry/4 h 100% inactivation
288 mg l1, 30 min 97.9% inactivation
1.0 mWs cm2 2 log reduction
50–55  C, 5 min Noninfectious
20  C, 3 days Noninfectious
70  C, 15 min No sporulation
15  C, 2 days No sporulation
required for the oocysts to mature and become infectious
in the environment makes person-to-person transmission
unlikely. Consumption of unsanitary water, lack of sanita-
tion, soil contact, and presence of animals in the household
have been associated with increased risk of cyclosporiasis
(Bern et al., 1999; Zerpa et al., 1995). Given unsuccessful
attempts to infect domestic animals, animal reservoirs are
unlikely to play a role; however, a potential role for animals
in transmission cannot be excluded. Detection of Cyclospora
DNA in stool samples from canine and avian species raised
the question whether they serve as a passive transport of
oocysts or play a role in transmission (Chu et al., 2004). In
areas of endemicity, Cyclospora presents a marked seasonality.
The specific conditions favoring oocyst survival in these areas
and seasons are unknown. For example, in Peru, cyclosporia-
sis is highly prevalent in areas near the desert regions of the
Pacific Ocean coast during the warm season (December to
May) when the ambient temperature is high and relative
humidity is low. In Nepal, the highest incidence of Cyclospora
in expatriates occurs during the rainy season from April to
June. In the moderate highlands of Guatemala, the preva-
lence of Cyclospora is between May and August and peaks
in June when the seasonal rains are just beginning and the
mean temperature is descending from its yearly high. Preva-
lence in these regions is high in children between 1.5 and
9 years old, suggesting that a specific immune response is
developed and adults are less susceptible to this infection.
Additionally, young children, immunocompromised people,
and foreign travelers are the most susceptible populations in
developing countries.
By contrast, in the industrialized world, the majority of the
population is susceptible due to lack of immunity. Cyclospor-
iasis is reported usually among travelers or results from food-
borne contamination. In the United States, cyclosporiasis has
been associated with consumption of berries, basil, snow
peas, lettuce, and recently cilantro, imported from countries
where Cyclospora is endemic (Table 4). Irrigation of produce
with contaminated water containing Cyclospora oocysts is
most likely the source of contamination during the food-
borne outbreaks. Waterborne outbreaks have also been
described. In 1990, staff from a Chicago hospital acquired
Cyclospora. An epidemiological investigation revealed that tap
84 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases

Sporulated
oocyst

5 Ingestion of
i
contaminated
food/water

Raspberries

Water

Basil

Sporulated oocysts
4
enter the food chain
Oocyst sporulation
3 in the environment

Environmental
2
contamination
Unsporulated
oocyst

1 Excretion of
d unsporulated
i = Infective stage oocysts in
d = Diagnostic stage the stool

Unsporulated Sexual Asexual

oocyst
7
Excystation
6
Zygote Meront Meront
II I

Figure 3 Life cycle of Cyclospora cayetanensis. Reproduced from www.cdc.gov/dpdx/cyclosporiasis/index.html.

water was the likely source of contamination. In Nepal, British
soldiers acquired the infection by drinking municipal water
with chlorine levels acceptable for survival of Cyclospora
oocysts. Cyclospora can also be concentrated by shellfish and
could be a potential source of contamination when these are
ingested raw.
Clinical Manifestations
Cyclosporiasis manifests with explosive diarrhea with abdom-
inal cramping, nausea, flatulence, anorexia, and weight loss
(Ortega et al., 1997). Young children and the elderly are of
higher risk for the development of severe illness (Behera
et al., 2008). In endemic settings, however, asymptomatic
infections in adults and milder infections of shorter duration
in children are common. The duration and severity of cyclo-
sporiasis are also increased among HIV patients. Biliary disease
is reported as another clinical manifestation among AIDS
patients (de Gorgolas et al., 2001; Sifuentesosornio et al.,
1995).
Diagnosis
Cyclospora oocysts measure c.8–10 mm in diameter, thus can be
visualized using microscopy. Covered with a bilayered wall,
Cyclospora oocysts stain variably using the modified acid-fast
stain. Best staining method for detection of oocysts is the
heated safranin stain protocol, which uniformly stains oocysts
pink (Visvesvara et al., 1997). Oocysts can be easily identified
using phase contrast microscopy or by epifluorescence micros-
copy given their autofluorescence (Figure 2). Additionally,
PCR-based molecular as diagnostic tools show more
sensitivity than conventional microscopy assays (Mundaca
et al., 2008). Because shedding of oocysts is intermittent,
 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases
Table 4 Description of some large food- and waterborne outbreaks of Cyclospora
Year Location
1990 Chicago, IL
1994 Pokhara, Nepal
1996 United States, Canada
1997 United States, Canada
1997 Washington, DC
1998 Ontario, Canada
2004 (Crist, 2004) Philadelphia, PA
2013 (CDC, 2013) Multistate, United States
2015 (CDC, 2015) Multistate, United States
collection of stool samples every 2–3 days increases specificity
and sensitivity of testing (Eberhard et al., 1997).
Treatment
Trimethoprim in combination with sulfamethoxazole (TMP-
SMX) is effective in the treatment of Cyclospora infection.
Treatment of adults with 160–800 mg twice daily for 7 days
and children with 5 mg kg1 twice daily for 7 days ceases
oocyst excretion and resolves symptoms in 1–3 days
posttreatment (Madico et al., 1997). Ciprofloxacin and
nitazoxanide has also been reported as alternative treatment
options in patients allergic to sulfonamides (Verdier et al.,
2000; Zimmer et al., 2007). Because recurrence of infection
in HIV-infected patients is common, prolonged therapy may
be required.
Prevention
Cyclospora oocysts can be effectively inactivated by heating and
freezing (see Table 3). Appropriate water filtration treatment
and proper food preparing hygiene may limit dissemination
of this infection.
Cystoisosporiasis
Cystoisosporiasis caused by an intracellular protozoan, Cystoi-
sospora belli, is a prevalent diarrheal disease in tropical and
subtropical regions (Arora and Arora, 2009). Together with
C. parvum, C. belli accounts for most intestinal infections
among immunocompromised patients (Agholi et al., 2013;
Vignesh et al., 2007). Known before to belong to the Isospora
genus, it was reclassified in 2005 as Cystoisospora (Barta et al.,
2005).
Cystoisospora belli oocysts are of ellipsoidal shape and
measure 25–30 mm by 10–15 mm. Similar to other Cystoiso-
spora, mature oocysts of C. belli are diplosporocystic and tetra-
sporozoic. Human infection occurs when ingested mature
oocysts release sporozoites into the small intestine (Figure 4).
After invasion of the epithelial cell, sporozoites form a perinu-
clear parasitophorous vacuole, in which endogenous stages of
parasite remain (Resende et al., 2014). All development occurs
in the epithelial cells of the distal duodenum and proximal
jejunum. Firstly, the parasite undergoes schizogony resulting
in release of asexual-stage merozoites. As in other coccidians,
infection can propagate via asexual cycles through repeated
85
No. of cases Outbreak Comment
21 Waterborne/drinking Water tank
12 Waterborne/drinking Municipal water
1465 Waterborne/drinking Raspberries/Guatemala
1012 Food-borne Raspberries/Guatemala
341 Food-borne Basil
315 Food-borne Raspberries/Guatemala
50 Food-borne Snow peas
631 Food-borne Cilantro
546 Food-borne Salad mix (cilantro)
release and invasion of merozoites. It is not until sexual stages
emerge, when the life cycle is completed with the release of
immature oocysts. Shed in feces, oocysts fully mature in the
environment during next 48 h (Table 1). During maturation,
a single sporoblast divides into two sporocysts, each containing
four sporozoites.
Homoxenous C. belli is considered to be infectious only to
humans. No animal reservoir has been identified thus far.
Transmission is waterborne and food-borne. Almost all re-
ported cases have occurred in developing countries or among
immigrants from or travelers to developing countries. Cystoiso-
spora belli is more frequently isolated from immunocompro-
mised individuals. It has been reported in institutionalized
care facilities where patients are housed for prolonged periods
and where poor sanitary conditions are present.
Cystoisosporiasis is characterized by diarrhea, steatorrhea
(soft, foamy, and foul-smelling bowel movements), weight
loss, abdominal pain, and in some instances, fever. These
symptoms are most severe in children and immunocompro-
mised patients, particularly HIV/AIDS patients. In immuno-
competent people, if untreated, disease usually resolves after
2–3 weeks. In contrast, infections in immunosuppressed
patients evolve to relapsing, chronic enteritis, leading to malab-
sorption and cachexia. Chronicity and recurrences of infection
are attributed to capacity of C. belli to form unizoic cysts in
extraintestinal sites, where they remain resistant to treatment.
Extraintestinal isosporiasis has thus far been reported in lymph
nodes (Restrepo et al., 1987), the liver and spleen (Michiels
et al., 1994), the gallbladder (Benator et al., 1994), and the
lamina propria of the small intestine (Frenkel et al., 2003).
The role of macrophages is implicated in extraintestinal
dissemination of infection (Resende et al., 2009). Unlike other
protozoan infections, cystoisosporiasis can be associated with
eosinophilia (Certad et al., 2003).
The diagnosis of cystoisosporiasis is usually made by iden-
tification of oocysts in fecal samples. Although oocysts can be
detected using modified acid-fast staining method, their auto-
fluorescence under UV light can greatly increase sensitivity of
detection (see Figure 2). Because of the intermittent nature of
oocysts shedding, analysis of repeated stool samples may be
required for diagnosis.
The treatment of choice is TMP-SMX at 160–800 mg four
times a day for 10 days and then two times a day for
3 weeks. Pyrimethamine and sulfadiazine are also effective,
and other combinations of antimicrobials such as
primaquine phosphate and nitrofurantoin or chloroquine
phosphate have been used.
86 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases

Figure 4 Life cycle of Cystoisospora belli. Reproduced from www.cdc.gov/dpdx/cystoisosporiasis/index.html.

Prevention can be achieved by improvement of personal localized to tissues of prey species, which serve as an interme-
hygiene measures and sanitary conditions to eliminate fecal– diate host.
oral transmission.

Sarcocystis
Parasites in the genus Sarcocystis belong to the Sarcocystidae
family, and in contrast to other coccidia, two hosts are required
to complete its life cycle (Figure 5). Its heteroxenous life cycle is
based on prey–predator host relationship. Sexual reproduction
takes place in the intestinal mucosa of the flesh-eating defini-
tive host, usually a carnivore, while asexual reproduction is
Biology
Numerous Sarcocystis species have been described among
domestic and wildlife animals. They are characterized by
specificity for both intermediate and definitive hosts. Acci-
dental infections of nonspecific species may take place;
however, the parasite is unable to complete its life cycle in
accidental hosts. At least two species affect humans as a defin-
itive host, namely Sarcocystis hominis (also known as Sarcocys-
tis bovihominis) and Sarcocystis suihominis. Humans can also
 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases 87

Figure 5 Life cycle of Sarcocystis hominis. Reproduced from www.cdc.gov/dpdx/sarcocystosis/index.html.

become an accidental intermediate host for several species of
Sarcocystis.
Life Cycle
The transfer stage between definitive and intermediate host is
an environmentally resistant oocyst (Figure 6). Each oocyst
contains two sporocysts, consisting of four infective sporozo-
ites each. The thin wall of oocysts often breaks and free sporo-
cysts can be observed. On the other hand, the transmitting stage
between intermediate and definitive host is the bradyzoite con-
tained within a cyst in the muscle or other tissues of the inter-
mediate host (Figure 7). Oocysts or sporocysts persist in the
environment after being shed in the feces of the definitive
host, e.g., humans or carnivores. When ingested by an interme-
diate host, sporozoites are released in the small intestine and
penetrate through the epithelium to reach endothelial cells of
small arteries, where they undergo their asexual division. As
a consequence, the second generation of merozoites enters
muscle tissue and forms sarcocysts, in which repeated divisions
take place to reach an infectious form containing bradyzoites.
Mature sarcocysts have been found in all striated muscles and
to a lesser extent in the smooth muscle. The intermediate hosts
for S. hominis and S. suihominis are cattle and swine, respec-
tively. Infection of humans with asexual stages is considered
accidental, given that man is unlikely to maintain the life cycle
as an intermediate host. After sporocyst-infested tissues are
eaten up by a susceptible definitive host, infectious bradyzoites
enter the intestinal lamina propria, where sexual reproduction
takes place. Each bradyzoite develops into a gamete. Fusion of
a micro- and macrogamete gives rise to an oocyst, which is
released to the lumen and appears in feces.
88 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases
(a) (b)
Figure 6 Morphological characteristics of Sarcocystis in stool preparations: (a) Oocyst of Sarcocystis hominis with two sporocysts, each with two or
three sporozoites visible, preparation colored with methyl green, differential interference contrast (DIC) microscopy; (b) Sporocyst of S. hominis with
three sporozoites visible, preparation colored with methyl green, DIC microscopy; (c) Oocyst of Sarcocystis sp. showing UV autofluorescence (the
sporocysts are fluorescing). (a) and (b) Courtesy of M. Scaglia, Infectious Diseases Dept., University Hospital IRCCS, San Matteo, Pavia, Italy.
Figure 7 Sarcocystis cysts in the muscle showing numerous merozo-
ites and compartmentalization of the cyst.
Clinical Manifestations
The sporogonic stages in the human intestine are generally re-
ported to be only mildly pathogenic and self-resolving, with
transient fever and diarrhea being reported (Chen et al.,
1999). Other studies have reported more severe acute diarrhea
and vomiting with chills, difficulty breathing, and rapid pulse
lasting up to 48 h (Heydorn, 1977). Experimental infections
in man and other primate hosts have demonstrated that the
prepatent period is on the order of 2 weeks and that sporocyst
shedding may last from a few days up to 6 months in some
cases (Fayer et al., 1979). Most human cases with intramuscular
cysts do not exhibit symptoms of inflammation; however, cases
with severe vasculitis and myositis have been reported (Arness
et al., 1999; McLeod et al., 1980; Van den Enden et al., 1995).
Cyst stages in the muscles of intermediate hosts persist for
months, if not years.
Diagnosis
The diagnosis requires identification of oocysts or free sporo-
cysts in the feces. Concentration methods routinely used for
fecal examination increase sensitivity of diagnosis. The sporo-
cysts are broadly ovoid and measure about 9 by 15 mm for
S. hominis and a bit smaller for S. suihominis, 10 by 13 mm.
The sporozoites are visible through the thin, colorless, trans-
parent sporocyst wall (Figure 6). Detection of human infection
(c)
with cyst stages (bradyzoites enveloped in a protective wall) is
through routine histologic examination of muscle biopsy.
Sarcocystis in meat can be detected by direct observation of
macroscopic sarcocysts.
Treatment and Prevention
Several drugs, including sulfadiazine, tinidazole, and acetyl spi-
ramycin may be effective in treating the intestinal infection, but
because Sarcocystis infections in humans are subclinical or
produce mild transient symptoms, specific therapy is not indi-
cated. Human infection is best prevented by cooking or
freezing meat, which inactivates bradyzoites inside of sarco-
cysts. Heat treatment at 60, 70, and 100  C for 20, 15 and
5 min, respectively, and freezing at 4 and 20  C for 48
and 12 h, respectively, render bradyzoites noninfectious
(Saleque et al., 1990). Bovine and swine infections can be pre-
vented by protecting animal food and water from fecal contam-
ination of human origin.
Giardiasis
Giardiasis is a major cause of waterborne diarrhea around the
world. It is caused by a single species of a flagellated enteric
protozoan, variably named Giardia intestinalis, Giardia lamblia,
or Giardia duodenalis. Giardia inhabits the small intestine of
humans and other animals, including monkeys, rodents,
dogs, cats, horses, goats, cattle, birds, reptiles, and fish. Out
of eight different genotypes described thus far (A–H), only
A and B infect humans (Read et al., 2004). The remaining geno-
types are observed in animals.
Life Cycle
The Giardia life cycle consists of two stages: a trophozoite and
a cyst. The trophozoite, the anaerobic invasive stage of the para-
site, is 10–20 mm long by 5–15 mm wide on average and is flat-
tened laterally, convex dorsally, and flat ventrally. Much of the
ventral surface consists of an adhesive disk, which the organism
uses to attach firmly to the brush border of enterocytes. The
trophozoite has one pair of anteriorly positioned nuclei and
four pairs of flagella, which enable its locomotion and adhe-
sion to the intestinal epithelium (Figures 8 and 9). After intake
 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases 89

Figure 8 Giardia intestinalis in stool preparations: (a) trophozoite in trichrome-stained preparation; (b) cyst in trichrome preparation; (c) Giardia
cysts (and Cryptosporidium oocysts) fluorescing in direct fluorescent antibody (DFA) assay. Giardia cyst is at the top of the image and the three small
Cryptosporidium oocysts are below. (b) Courtesy of T. Orihel, Tulane University.

2
i

Contamination of water, food, or

hands/fomites with infective cysts

Trophozoites are also

passed in stool but
they do not survive in
the environment

1
i = Infective stage i
d
d = Diagnostic stage d Cyst

3 4 5
Cyst Trophozoites

Figure 9 Life cycle of Giardia. Reproduced from www.cdc.gov/dpdx/giardiasis/index.html.

of glucose from the intestinal lumen, trophozoites divide asex-
ually in a process of longitudinal binary fission, which gives rise
to two daughter trophozoites (Figure 9). As new trophozoites
transit down the colon, they prepare for encystation. Encysta-
tion is triggered by environmental changes such as alkaline
pH of bile salts (Lujan et al., 1996). In this process, flagella
are retracted and encystment vesicles are formed in the
cytoplasm. The hyaline membrane components secreted
from the vesicles cover the trophozoite surface and eventually
harden into a cyst wall. Host-to-host transmission is accom-
plished by viable cyst excretion with feces (see Figure 9).
The cysts are oval in shape and measure 8–12 mm in length
by 7–10 mm in width. Mature cysts have four nuclei located
at one end of the cyst. As the cyst matures, internal structures
90 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases

and the adhesion disk are doubled. Upon ingestion by a new however, host specificity of their genotypes disables cross-
host, cysts excyst in the proximal bowel in response to presence species transmission.
of pancreatic enzymes. A single trophozoite is released and Giardiasis can occur year-round in all settings, temperate as
divides into two identical trophozoites, which colonize the well as tropical. There is strong evidence, however, that some sea-
proximal bowel by way of the adhesion disk. sonality occurs in temperate regions, such as the United States,
with increased incidence in the summer months, peaking in
early fall. Increased recreational water activity during summer
Epidemiology months has been postulated to be a reason for this increase in
incidence. Giardiasis can also be a common cause of traveler’s
Due to heavy contamination of surface waters, giardiasis is one of
diarrhea during or shortly after return from a trip abroad.
the most common intestinal parasitic infections around the
world. It infects people of all ages; however, most affected are
young children, young adults, backpackers, campers, hikers, Pathogenesis
and travelers to the endemic areas. In the developing regions of
Pathogenesis of Giardia involves direct damage to the mucosal
the world, giardiasis is one of the most common diarrheal path-
layer of small intestine due to degradation of mucin by the
ogens among children below 2 years of age (Kotloff et al., 2012).
trophozoite (Amat et al., 2015). This damage facilitates trans-
Infection occurs upon ingestion of as few as 10–25 viable
mucosal bacterial translocation, which contributes to the
cysts. Contaminated food and water are the most common
pathology of giardiasis (Chen et al., 2013). Despite the
sources of exposure (Table 5); however, person-to-person
damage, an inflammatory response is not consistently present.
transmission is also possible if personal hygiene is poor.
Seemingly, contrary to genotype A, genotype B isolates are
Outbreaks are often linked to contaminated food, drink, or
known to induce inflammatory infiltration. This correlates
recreational water. Risk factors associated with development
with different severity of symptomology between the two line-
of giardiasis are contact with recreational freshwater, swallow-
ages (Puebla et al., 2014; Pestechian et al., 2014). Additionally,
ing water during swimming, eating lettuce, and drinking tap
downregulation of key chemokines expression in gastrointes-
water (Stuart et al., 2003). High frequencies of infection have
tinal inflammation was observed (Cotton et al., 2014;
been reported among hikers and campers who drank water
Roxstrom-Lindquist et al., 2005). Recent evidence also suggests
from mountain streams. Animals also harbor Giardia species;
that Giardia damages intestinal epithelial cytoskeletal by
cleavage of villin, contributing to disintegration of tight junc-
Table 5 Description of some large food- and waterborne outbreaks tions between epithelial cells (Bhargava et al., 2015). L-arginine
of Giardia and glucose are primary sources of energy for Giardia trophozo-
No. of
ites (Schofield et al., 1990). Depletion from arginine, associ-
Year Location cases Outbreak Comments ated with extensive infection, ceases intestinal epithelial
proliferation and induces intestinal apoptosis (Stadelmann
1990 Colorado 123 Waterborne; drinking Municipal et al., 2012). This multifactorial damage to the epithelial cells
water (surface) water and brush border increases permeability of the intestinal
1990 Connecticut 27 Food-borne; Cafeteria epithelium resulting in fluid secretion and impairment of
vegetables absorption, which results in diarrhea. Both humoral and cell-
1990 Illinois 75 Food-borne; salad bar Hospital
mediated immune responses have been reported to occur in
1992 Idaho 15 Waterborne; drinking Trailer park
water (well)
human giardiasis. Given different infection scenarios between
1992 Nevada 80 Waterborne; drinking Municipal individuals, it is likely that nonimmune factors determine
water (surface) water susceptibility to infection, duration, and severity of the disease.
1993 New Jersey 43 Waterborne; Swim club Identification of Giardia antigens is crucial for development of
recreational water adaptive immunity; however, it remains difficult due to the
1994 Indiana 80 Waterborne; Community trophozoite’s surface antigenic variation during encystation.
recreational water pool
1994 Tennessee 304 Waterborne; drinking Penitentiary
water (surface) Clinical Manifestations
1995 New York 1449 Waterborne; drinking Municipal
Depending on host susceptibility factors and genotype viru-
water (surface) water
1996 Illinois 6 Food-borne; ice cream Fairgrounds
lence, giardiasis can result in asymptomatic disease, acute
1997 Oregon 100 Waterborne; drinking Campground self-limiting diarrhea, or chronic diarrhea with debilitating
water (well) malabsorption (Eligio-Garcia et al., 2005; Haque et al.,
2000 Colorado 27 Waterborne; drinking Resort 2005). Symptomatic infection results in irritation of the
water (surface) duodenum with excess secretion of mucus and dehydration,
2000 New York 82 Food-borne Bowling accompanied by epigastric pain, flatulence, nausea, vomiting,
alley loss of appetite, or chronic diarrhea with steatorrheic stool con-
2001 Florida 6 Waterborne; drinking Household taining large amounts of mucus and fat but no blood. Chronic
water (well) giardiasis tends to occur in immunocompromised individuals,
2004 Tennessee 6 Food-borne; chicken Workplace
who may also experience extraintestinal sequelae such as joint
salad
2004 Washington 19 Waterborne; ice Restaurant
pain, skin and eye reactions, and neurological symptoms
(Halliez and Buret, 2013; Wensaas et al., 2012). Malabsorption
 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases
associated with chronic or recurring diarrhea among children
in developing areas results in stunted growth and decrease in
cognitive function (Al-Mekhlafi et al., 2005).
Diagnosis
Diagnosis of infection is typically by microscopic detection of
cysts in freshly collected stool or trophozoites in acutely diar-
rheic stool. Organisms can occasionally be seen in direct exam-
ination, but a concentration procedure is recommended.
Because of their distinctive shape and localization of the nuclei,
the diagnosis can often be made on wet, unstained samples.
Staining with trichrome may enhance detection and confirma-
tion of infection. Due to intermittent excretion, multiple stool
collections over a period of 3 days are recommended to
increase diagnostic sensitivity. In addition to direct or stained
specimens, commercial direct fluorescent antibody (DFA)
assays are available and often used as the gold standard for
diagnosis (Figure 8). Enzyme-linked immunosorbent assay–
based tests are commercially available and are useful for
screening large numbers of samples. Occasionally, a duodenal
aspirate may be required for diagnosis. It can be obtained using
a string test, in which the patient is asked to swallow a gelatin
capsule containing a string. The string is then retrieved and
examined for attachment of organisms.
Treatment
Two drugs of the 5-nitroimidazole group, namely metronidazole
or tinidazole, are the recommended drugs of choice for treat-
ment of giardiasis, with efficacy ranging from 80% to 90%
(Gardner and Hill, 2001). Alternative agents available for treat-
ment failures are nitazoxanide, albendazole, and paromomycin.
Paromomycin is not absorbed from the gastrointestinal tract and
Figure 10 Life cycle of Dientamoeba fragilis. Reproduced from www.cdc.gov/dpdx/dientamoeba/index.html.
91
is often used during pregnancy. Due to its 90% efficacy but
severe side effects, quinacrine, an antimalaria drug, can be
used as a last resort treatment for drug-resistant giardiasis
(Requena-Mendez et al., 2014). Treatment-refractory giardiasis
is common and requires prolonged treatment with higher doses
of different classes of drugs.
Prevention
Use of chlorine alone at levels routinely used in municipal
treatment facilities does not rapidly inactivate cysts, especially
at lower water temperatures; therefore additional measures
must be in place. Water treatment including flocculation, sedi-
mentation, filtration, and finally, chlorination is required to
free water from Giardia cysts. Water hygiene during camping
or traveling overseas can be achieved by boiling, filtration
through <1 mm pore filter, or adequately long treatment with
chlorine or iodine preparations.
Dientamoeba
Dientamoeba fragilis, until recently erroneously classified as an
ameba, is an intestinal trichomonad parasite, which lost its
flagella. Although it struggles to receive recognition as a signifi-
cant intestinal pathogen, between 6% and 30% of people
suffering from intestinal parasitosis harbor D. fragilis (Rayan
et al., 2007; Vandenberg et al., 2006). In some areas of the
world, the prevalence of dientamoebiasis exceeds that of giardi-
asis (Stark et al., 2010).
In contrast to other intestinal protozoa, which develop
a cystic stage in order to survive in the environment external
to the host, D. fragilis seems to exist only in the form of
a trophozoite (Figure 10). The trophozoite measures from 3
92 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases
to 20 mm in diameter and contains one to four nuclei. Several
reports of pseudocyst, precyst, or cyst stages of D. fragilis have
been deemed inconclusive (Clark et al., 2014; Munasinghe
et al., 2013; Stark et al., 2014). The mode of transmission is
unknown, but there is some evidence that, much like Histomo-
nas of turkeys, which is transmitted via eggs of the nematode
Heterakis gallinae, D. fragilis may be carried inside the egg of
the human pinworm, Enterobius vermicularis. It has been noted
that D. fragilis and pinworm infection occur together more
frequently than would be expected (Ogren et al., 2015;
Girginkardesler et al., 2008; Cerva et al., 1991; Preiss et al.,
1990; Burrows and Swerdlow, 1956). Limited evidence indi-
cates that experimental infection with pinworm eggs also
resulted in infection with D. fragilis (Ockert, 1972). While
DNA of D. fragilis was found with varying frequencies inside
of pinworm eggs, trophozoite cultures could not be established
from these eggs (Ogren et al., 2013; Roser et al., 2013).
Given that the fecal–oral route is the most probable transmis-
sion mode for D. fragilis, poor water sanitation and hygiene
standards increase risk of infection (Millet et al., 1983; Rayan
et al., 2007). In settings with high sanitation standards, children
Figure 11 Life cycle of Entamoeba coli, Entamoeba hartmanni, Entamoeba polecki, Endolimax nana, and Iodamoeba butschlii. Reproduced from www.
cdc.gov/dpdx/intestinalAmebae/index.htm.
and caring adults are at greatest risk of infection due to the close
nature of contact (Ogren et al., 2015; Roser et al., 2013).
Dientamoeba fragilis has not been reported to invade tissues,
but there is some evidence that its presence may, on occasion,
produce irritation of the intestinal mucosa with excess secretion
of mucus and hypermotility of the bowel leading to a mucousy
diarrhea. Infected individuals may suffer from acute or chronic
diarrhea, lasting more than 2 weeks, abdominal pain, nausea,
and rarely fever (Stark et al., 2010). Asymptomatic infections
may be present (Johnson and Clark, 2000).
Standard antiprotozoal therapy including iodoquinol,
paromomycin, tetracycline, or metronidazole appears to
resolve D. fragilis infection (Nagata et al., 2012). Use of metro-
nidazole, however, is associated with treatment failure and the
risk of relapse (Stark et al., 2010).
Nonpathogenic Amebas
A number of protozoa in the ameba group inhabit human
gastrointestinal tract but are not believed to cause significant
 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases 93

preparations may provide diagnostic challenges to distinguish

Figure 12 Trichrome-stained stool preparation illustrating trophozo-
ites of Entamoeba hartmanni (E.h.) and Iodamoeba butschlii (I.b.). Cour-
tesy of T. Orihel, Tulane University.
disease and are often referred to as the nonpathogenic amebas.
These include Entamoeba coli, Entamoeba hartmanni, Entamoeba
polecki, Entamoeba gingivalis, Endolimax nana, and Iodamoeba
butschlii. Identification of these organisms in the stool is signif-
icant for several reasons. Firstly, it indicates exposure to fecal
contamination with inherent risk of exposure to other patho-
genic organisms. Also, the various stages found in microscopic
between one of these organisms and Entamoeba histolytica,
a tissue-invasive ameba.
These organisms share some features of morphology and
life cycle (Figure 11) yet differ in other aspects. All have
a trophozoite stage, which exhibits pseudopod movement,
and, with the exception of E. gingivalis, all form cyst stages in
preparation for evacuation into the environment. Although
trophozoites can be observed in stool preparations (Figure 12),
especially in diarrheic samples, it is the cyst stage that is moder-
ately durable in the environment and can withstand moderate
putrefaction and desiccation. The cyst stage is transmitted
through fecal contamination of food or drink. The trophozoite
stage released upon ingestion initiates infection. No cyst stage
has been identified for E. gingivalis, and transmission is
presumed to occur via droplet spray or other exchange of oral
secretions from the mouth during close contact. Although
most commonly detected in the gingiva and tonsillar crypts,
E. gingivalis has also been recovered from vaginal and cervical
smears in women with intrauterine devices.
The mature cyst of E. coli measures 10–30 mm in diameter
and contains eight nuclei (Figure 13(b) and (c)). Immature
cysts may contain two or four nuclei and can be confused
with the pathogenic E. histolytica. Mature cysts of
E. hartmanni measure 5–10 mm, contain four nuclei
(Figure 13(d)) and can best be distinguished from
E. histolytica by size, but due to similar number of nuclei, the
two are often confused. Entamoeba polecki, a cosmopolitan
parasite of pigs and monkeys, is rarely diagnosed in humans.
It most closely resembles E. coli, although the cysts tend to

(a) (b) (c)

(d) (e) (f)

Figure 13 Various intestinal protozoa in stained stool preparations: (a) Dientamoeba fragilis trophozoite in iron hematoxylin–stained preparation; (b)
Entamoeba coli cyst in wet preparation colored with iodine showing five nuclei; (c) Entamoeba coli cyst in iron hematoxylin–stained preparation with
multiple nuclei in focal plane; (d) Entamoeba hartmanni cyst in trichrome-stained preparation; (e) Endolimax nana in iron hematoxylin–stained prepara-
tion; (f) Iodamoeba butschlii cyst in iron hematoxylin–stained preparation. All images courtesy of T. Orihel, Tulane University.
94 Protozoan Diseases: Cryptosporidiosis, Giardiasis, and Other Intestinal Protozoan Diseases
be smaller measuring 5–11 mm in diameter. Endolimax nana is
a small ameba that typically produces ovoid cysts measuring
5–14 mm in diameter (Figure 13(e)). They can be confused
with cysts of E. histolytica and E. hartmanni because of cyst
size and the presence of four nuclei. Large and distinct nuclear
karyosome, however, distinguishes this ameba from other
species of Entamoeba. Iodamoeba butschlii cyst can be easily
distinguished between other amebas. Its distinctive single
nucleus has a large, prominent karyosome. The cyst measures
6–15 mm in diameter and is of pyriform or ovoid shape. The
most conspicuous feature of the cyst, however, is the large
glycogen vacuole (Figure 13(f)).
All of these amebas are cosmopolitan in distribution, with
infections tending to be more common in developing countries
and in communities with poor hygiene and sanitation. Infec-
tion rates of 30–50% are common and can approach 100%.
Animal reservoir hosts are not believed to play important roles
in human infection, although monkeys and dogs have been
found naturally infected with an ameba species very similar
to E. coli. Many monkeys also harbor an ameba indistinguish-
able from E. nana. Iodamoeba butschlii is a natural parasite of
primates, and the I. suis species of hogs is believed to be the
same species.
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