Giardia and giardiasis

1
Giardia and giardiasis Trichomonas and
Trichomoniasis Medical Parasitology
Course February 19, 2008 University of
Georgia Silvia N.J.Moreno
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PROTOZOA
Protozoa are one-celled animal-like organisms
Most protozoa are too small to be seen with the
naked eye (between 1050 µm to 0.5 mm. They can
be observed with a microscope. Protozoa are
ubiquitous throughout aqueous environments and
the soil, and play an important role in their
ecology. Some protozoa can form cysts for
protection against harsh conditions, allowing to
survive extreme temperatures or harmful chemicals
or lack of food, water, or oxygen. For parasitic
species the cyst will be important for
transmission.
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PROTOZOA
Because of the size of protozoan they were
undetected until Antony van Leeuwenhoek
(1632-1723) invented the first magnifying glass.
He had no fortune and received no higher
education or university degrees.
A Dutch tradesman van Leeuwenhoek is known as the
Father of Microbiology. He made this first
known observation of (what is most likely)
Giardia intestinalis when examining his own feces
under his microscope. Van Leeuwenhoek did not,
however, make the connection between this
observed creature and any pathology.
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HOW PROTOZOA MOVE
FLAGELLA a whip like extension found singly or
in pairs with undulating motion which allows
cells to swim through liquid media CILIA hair
like projections shorter than flagella. Capable
of rhythmical motion and act in almost synchrony
creating whip-like patterns. PSEUSOPODIA A
temporary projection of the cytoplasm. This is
observed in amoebas.
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THE FLAGELLUM
Flagella are microtubule-based structures that
propel cells through surrounding fluid.
The internal structure is composed of a central
axoneme and an outer sheath that is the
continuation of the plasma membrane. The axoneme
consists of nine peripheral (doublets) and one
central pair of microtubules (singlets).
The force for propulsion is provided by dynein
(motor proteins) anchored in rows along one side
of each doublet, which can walk along the
microtubule of the adjacent doublet. In order to
produce coordinated bending of the flagellum,
these dynein motors organized into multi-headed
complexes called the inner and outer dynein arms
must produce their power strokes in synchrony.
http//www.northland.cc.mn.us/biology/Biology1111/
animations/flagellum.swf
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Reproduction
Reproduction in protozoa may be sexual or
asexual. Most common asexual reproduction is by
Binary Fission. Other types are multiple fission
(merogony, schizogony, sporogony), budding.
Paramecium dividing. Observe that the plane of
fission is traverse
A protozoa cell dividing by binary fission with
the plane of fission longitudinal
Plasmodiun schizont. The nucleus divide
repeatedly before cytokinesis and a large number
of daughters are produced.
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Endodyogeny An example of internal budding in
which only two daughters cells are formed. This
happens during the asexual reproduction of
Toxoplasma gondii
The daughter cells begin forming within their
cell membranes, distributed throughout the
cytoplasm of the mother cell rather than at the
periphery. Toxoplasma parasites divide by
endodyogeny. This parasite also uses sexual
reproduction during its life cycle.
http//morrissettelab.bio.uci.edu/FrontpageLabpage
FINAL.html
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METABOLISM
Animal-like protists are all heterotrophic,
requiring their energy in the form of complex
carbon molecules and their nitrogen in the form
of a mixture of preformed aminoacids. Endocytosis
allows protozoa the ingestion of particles or
fluid droplets. Protozoa carry out the many
reactions of glycolysis, krebs cycle,
pentose-phosphate shunt, electron transport,
lipid oxidations and syntheses, and other
metabolic events. ATP is the most common form of
usable energy, although a few parasites also use
pyrophosphate.
The process by which a cell takes in
extracellular fluid by the invagination of the
cell membrane. The pocket then pinches off to
form a vesicle, which subsequently ruptures,
releasing its contents into the cytosol.
Pinocytosis ("cell drinking") is a form of
endocytosis.
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METABOLISM
Important biological factors Many parasites
occupy environments in which neither oxygen or
glucose are limited. These organisms derive their
energy from glycolysis mainly. The partially
oxidized products can be excreted as waste. They
still need to deal with the oxidation of the
reduced NAD. In some parasites the electrons
are transferred to the pyruvate forming ethanol
or lactic acid.
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METABOLISM
PYRUVATE
Pyruvate CoASH
Ferredoxin (ox)
Xred
Pyruvate ferredoxin oxidoreductase
Acetyl- CoA
Ferredoxin (red)
Xox
Some protozoan parasites have no mitochondria
(ex. amebas, trichomonas and giardia). The final
acceptor can be pyruvate. These protozoa take up
oxygen but their availability makes little or no
difference in their energy metabolism. Pyruvate
can be further oxidized by a pyruvate ferredoxin
oxidoreductase. This is an enzyme that is not
present in the cells of the hosts.
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Vilem Lambl
Two hundred years after van Leeuwenhoek had seen
Giardia parasites, in 1859, a Czech physician
named Vilem Lambl observed G. intestinalis in the
stools of children with diarrhea, but believed
the protazoa to be commensal and not responsible
for the pathology. The species was then named
Giardia lamblia in his name. The question as to
whether giardia was a pathogen or commensal was
debated for many decades during the first part of
the twentieth century.
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GIARDIASIS
Infection with the protozoan parasite Giardia
lamblia

• Giardia duodenalis most common name used. G.
  intestinales and G. lamblia are also used.
• Giardia spp. can parasitize the intestinal tract
  of a wide range of vertebrates, including humans.
• Disease is prevalent in children attending day
  care centers.
• In addition, domestic dogs and certain wild
  animals serve as hosts.
• Transmission of Giardia is predominantly by
  ingestion of food or water contaminated with
  cysts.

Trophozoite
Cyst
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• Only one host. Simple life cycle
• Cysts are ingested and in the small intestine,
  excystation releases trophozoites (each cyst
  produces two trophozoites).
• Trophozoites multiply by longitudinal binary
  fission, remaining in the lumen of the proximal
  small bowel where they can be free or attached to
  the mucosa by a ventral sucking disk. 
• Encystation occurs as the parasites transit
  toward the colon. 
• The cyst is the stage found most commonly in
  nondiarrheal feces

LIFE CYCLE
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Three Giardia Cysts
A Giardia trophozoite
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The trophozoite of Giardia duodenalis

• Active metabolizing, motile form, lives in the
  upper two-thirds of the small intestine (duodenum
  and jejunum)
• 12 to 15 µm long, shaped like a pear
• Two nuclei
• Equal DNA content
• Both transcriptionally active
• Both divide at the same time
• Four pairs of flagella 8 flagella
• Ventral Adhesive Disc
• Reproduction by binary fission-asexual
  reproduction

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Scanning electron micrographs of trophozoites of
G. lamblia.
Observe the typical pear-shaped body. a Dorsal
view of G. lamblia. Observe the lateral flange
(arrowhead) and the caudal or posterior (P) and
the anterior (A) flagella. b A ventral view of
the parasite. The arrowhead indicates the
presence of the lateral flange, which surrounds
the whole cell. The ventral flagella (V) are seen
in the region of the ventral groove and the
adhesive or striated disk (SD) is clearly
observable. Bars a 2.7 ?m and b 2.0 ?m.
Microscopy and Microanalysis (2004), 10 513-527
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The Giardia cyst
After cyst wall formation the parasite undergoes
one round of nuclear division without cytokinesis
resulting in four nuclei. These four nuclei (Nu)
are located at the anterior end. The flagella and
adhesive disk are lost, but the axonemes (Ax) and
median bodies (MB) persist. Cysts are oval shaped
and measure 11-14 µm in length and 6-10 µm wide.
They have a well-defined wall (CW) which is often
set apart from the cytoplasm of the parasite. The
cysts are passed in the feces and can survive for
up to three months under appropriate temperature
and moisture conditions.
SEM illustrating the filamentous nature of the
cyst wall
Clinical Microbiology Reviews, 13, p. 35-54
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Encystation and Excystation
Encystation Trophozoite gradually round up and
detach, lose motility and become refractile.
Encystation-specific secretory vesicles (ESV) are
formed In vivo occurs in the lumen of the small
intestine. Trophozoites starts synthesizing cyst
wall components and transport them to the outer
surface. In vitro can be induced by High pH
and high bile salts concentration
Excystation Cysts pass through the stomach where
they are exposed to gastric acid. In the
duodenum, the gastric chyme is neutralized by
bicarbonate. Excystation probably occurs in the
upper small intestine. Motile trophozoites emerge
in the small intestine. In vitro can be induced
by low pH exposure of the cyst and transfer to
pH 8 and protease treatment
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GIARDIA METABOLISM
G. Lamblia is an aerotolerant anaerobe Parasite
have no mitochondria Tricarboxylic acid and
cytochrome system is absent Parasite uses glucose
and stores glycogen
PFO
GLUCOSE PYRUVATE
ACETYL-COA
NADH oxidase
NADH NAD
O2
H2O
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PATHOGENESIS

• Many cases of infection show no evidence of
  disease
• Mechanism still unknown
• Giardia colonizes the upper small intestine
• Villous blunting
• Lymphocytic infiltration
• Malabsorption
• No tissue invasion-high number of trophozoites
  present in the duodenal crypts

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PATHOGENESIS Possible Mechanisms

• Trophozoites released in the upper part of the
  SI move to the duodenum and jejunum and attach.
  Suction force may damage microvilli.
• Large number of parasite may interfere
  mechanically with digestion.
• Symptoms may result from inflammation of the
  mucosal cells of the small intestine.
• AIDS patients do not appear to be more
  susceptible to giardiasis

22
Adhesion of the trophozoite to the epithelial
cells of the small intestine.
Note the impression left on the epithelia where a
trophozoite has detached (upper left) and the
interaction between the ventral disk of the
parasite and the brush boarder cells of the
intestines (lower right).
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Antigenic Variation
The ability of a parasite to change the proteins
it presents to the hosts immune system.

• The surface of the Giardia trophozoite is covered
  by the Variant Specific Protein (VSP)- only one
  is expressed per trophozoite.
• Expression of different VSPs results in the
  phenomenon of Antigenic Variation
• Over 150 different VSP genes are encoded in the
  Giardia genome.
• Mechanisms not known

The entire surface of the trophozoite is covered
by the variant-specific surface protein (VSP).
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Is re-infection by Giardia due to antigenic
switching after differentiation?
VSP changes Re-infection
New VSP expressed Colonization
VSP-1 expressed Encystation
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GIARDIASIS
CLINICAL FEATURES Incubation period 1-2
weeks Majority of infected individual are
asymptomatic SYMPTOMS Diarrhea with loose,
fool-smelling stools Flatulence Abdominal cramps
and bloating Nausea anorexia Malaise Weight
loss Prolonged symptoms Malabsorption
Marked spasm and irritability of the duodenum and
jejunum with resultant poor filling of the
duodenal C-loop and proximal and mid-jejunum.
Marked thickening, edema, and spiking of mucosal
folds throughout the proximal small bowel . The
loops appear widely separated and the lumen
appears narrow in many segments due to the
combination of irritability and edema. The barium
column is segmented and flocculated from spasm
and increased fluid within the upper small bowel.
(Courtesy of the late Dr. Richard Marshak, New
York City).
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GIARDIASIS

• DIAGNOSIS
• Identification of cysts or trophozoites in fecal
  specimens
• May need to be repeated
• Detection of Giardia antigens in feces.
• Serodiagnosis is not useful

The Cypress Diagnostics Giardia kit is an enzyme
linked immunosorbent assay (ELISA) intended for
the detection of Giardia antigen in fecal
specimen.
Imaging DiagnosisThe combination of mucosal
edema, irritability, rapid transit of barium
through the proximal small bowel, abnormal
clumping of barium with evidence of increased
secretions, narrowing of the bowel lumen, and
localization of the pathological features to the
duodenum and jejunum is typical of giardiasis.
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GIARDIASIS
TREATMENT Metronidazole not FDA approved but
used (250 mg x 3 for 5-7 d) Quinacrine-
discontinued in 1992 Tinidazole- not approved in
the US. Single dose of 2 g. Furazolidone-
approved by the FDA for giardiasis Paranomycin-
used for pregnant women Nitazoxanide recently
approved for giardiasis and cryptosporidosis in
children
NitazoxanideAlinia (nitazoxanide)
Tablets (nitazoxanide) for Oral Suspension
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A GIARDIA VACCINE Giardia Vax Finally there is
a vaccine!!!         Now there is a vaccine to
prevent signs of disease associated with Giardia
infection in dogs. The first vaccine to prevent
disease and reduce cyst shedding caused by
Giardia infection in dogs. Giardia Vax is safe
and effective for healthy dogs eight weeks of age
or older.           To completely clear your
Beagle kennel of the protozoan giardia once and
for all, give all Beagles (adults and pups) a 5
day treatment of Metronidazole (Flagyl), followed
up by a shot of Giardia Vax. Then give your
Beagles an annual booster of Giardia Vax and
giardiasis will be gone for good from your
hounds. I highly recommend that all Beaglers
order this vaccine and do the shots yourself.
Giardiasis has been the most common protozoan
infection in North America for the past 20 years.
It is a very, highly contagious intestinal
infection and is carried by both humans and all
warm-bloodied animals. Be on the safe side and
have your Beagle(s) vaccinated against this
troublesome organism.
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GIARDIA TRANSMISSION

• The resistant cysts is responsible for
  transmission.
• The cysts can survive several months in cold
  water. 
• Reservoir-human and possibly other mammals
• 10-25 cysts sufficient. Possible sources of
  cysts
• Feces for fertilizer
• Defective piping
• Flyes
• Soiled hands

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EPIDEMIOLOGY OF GIARDIASIS

• Worldwide distribution
• Common in areas with poor sanitary conditions
• Seasonal with peak during late summer in UK, USA
  and Mexico
• Prevalence 2-5 in industrialized countries
• 20-30 in developing countries
• Travelers, hikers, campers are at risk.
  Swallowing water while swimming, drinking tap
  water, eating lettuce.
• Risk groups in the US travelers, children in
  day cares and homosexual men

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GIARDIASIS PREVENTION

• Avoid drinking water from sources associated with
  outbreaks
• Water filtration
• Chlorination is not effective
• In day cares wash hands and proper disposing of
  diapers
• Hikers and backpackers should be warned to boil
  or filter water
• Swimming pools are specially vulnerable

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Trichomonas vaginalis

• Cause of trichomoniasis, a sexually-transmitted
  (rare instance of neonatal infections acquired at
  birth)
• Common cause of vaginitis
• 200 million cases/yr worldwide
• 3-5 million cases/yr US
• Single form - trophozoite (no cyst form)
• Found in urogenital tract (vagina urethra in
  females urethra, seminal vesicles prostate in
  males)
• Extracellular - adheres to epithelia

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LIFE CYCLE
Trichomonas vaginalis resides in the female lower
genital tract and the male urethra and prostate ,
where it replicates by binary fission .  The
parasite does not appear to have a cyst form, and
does not survive well in the external
environment.  Trichomonas vaginalis is
transmitted among humans, its only known host,
primarily by sexual intercourse.
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A modern drawing of Trichomonas vaginalis,
showing essentials of morphology
Five flagella, four anterior and fifth
incorporated within the UM Nucleus is
anterior Axostylecommences at the nucleus and
bisects the parasite Granules hydrogenosomes,
important for metabolism AF, anterior flagella
AX, axostyle CO, costa HY, hydrogenosome N,
nucleus PB, parabasal body PG, parabasal body
and Golgi apparatus RF, recurrent flagellum.
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MORPHOLOGY
A T. vaginalis parasite as seen in broth
culture. The axostyle, undulating membrane, and
flagella are clearly visible. B T. vaginalis on
the surface of a vaginal epithelial cell prior to
ameboid transformation. C Ameboid morphology of
T. vaginalis as seen in cell culture. Note that
the side opposite the undulating membrane adheres
to the vaginal epithelial cell. Bars, 5 µm.
Clin. Microbiol. Rev. 11300
36

• Trichomonas vaginalis
• Relies on host for synthesis of essential
  molecules
• (nucleotides, fatty acids, amino acids)
• Ancient eukaryote
• Facultative anaerobe
• Lacks mitochondria
• Contains hydrogenosomes (involved in carbohydrate
  metabolism and drug therapy)

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HYDROGENOSOME

• Structure
• 0.5-2 ?m double membrane organelle
• no genetic material
• Present in anaerobic/aerotolerant organisms
• Trichomonas
• rumen-dwelling ciliates
• free-living ciliates
• rumen fungi

Surrounded by two unit membranes (arrowheads).
Membrane invaginations and vesicles fusing with
hydrogenosome membranes are depicted (Arrows).
Detection of carbohydrates in T. foetus using
periodic acid-thiosemicarbazide- silver
proteinate technique. Reaction product is on
membranes of hydrogenosomes (H). Bulbous
structure in the hydrogenosome (arrow) as well
endoplasmic reticulum membranes (arrowheads),
nuclear envelope (N), Golgi complex (G), and
Glycogen granules (GL) were labeled.
38
BIOCHEMISTRY
Pyruvate from the cytosol is oxidizes do acetyl
coA by the Pyruvate Ferredoxin Oxidoreductase
(PFO (1)) in the Hydrogenosome. The enzyme
Hydrogenase (4) uses the electrons from
ferredoxin and transfer them to H to form
hydrogen gas. Acetyl coA can be further
metabolized by the acetatesuccinate CoA
transferase (2) to form acetate and succiniyl-CoA
(2) which could be hydrolyzed into CoA and
succinate and the energy released used to make
ATP by the succinate thiokinase (3).
The PFO is the target for Metronidazole
In the presence of metronidazole, electrons
generated by PFO are transported by ferredoxin
2Fe2S to the drug (bold arrow) and not to
their natural acceptor hydrogenase (HY).
Metronidazole is reduced with one electron
forming a nitro anion free radical. The cytotoxic
radicals (RNO2-) are formed as intermediate
products of the drug reduction.
Int. J. Parasitol. 1999, 29 199
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PATHOGENESIS

• Molecular mechanisms of pathogenesis
• Adhesion play an important role probably
  involves complex interactions. Parasite cell
  surface play main role. T, pH and time are
  important
• Excretion of soluble factors extracellular
  proteinases
• Hemolysis
• Contact independent mechanisms a soluble toxin?
• A possible double-stranded RNA virus?

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CLINICAL MANIFESTATIONS

• T. vaginalis infects the squamous epithelium in
  the genital tract
• More persistent in females
• Acute
• Diffuse vulvitis
• Yellow or green discharge
• Hemorragic spots in the vaginal mucosa
• Chronic
• Mild symptoms with pruritus
• Vaginal discharge mixed with mucus
• 25-50 women are asymptomatic
• Complications in women 1) increased HIV
  transmission and infectivity 2) greater risk of
  tubal infertility and atypical pelvic
  inflammatory disease (PID) 3) increased risk of
  cervical cancer 4) association with preterm
  birth and premature rupture of membrane and low
  birth weight infants
• In men 1) increased HIV transmission and
  infectivity 2) common cause of nongonoccocal
  urethritis and 3) a factor in male infertility as
  a result of decreases in both sperm motility and
  viability.

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Increased shedding of HIV
The Increased shedding of HIV could be the result
of the local inflammation produced by the STD.
Increased susceptibility to HIV as a result of
breaks in the mucosal tissues because of the STD.
A higher prevalence of STDs among HIV-infected
individuals. An increased susceptibility to STDs
as a result of immunosuppression associated with
HIV
HIV seropositive men with symptomatic urethritis
and Trichomonas had a six-fold higher
concentration of HIV RNA in semen than
HIV-positive men with urethritis in the absence
of trichomoniasis.
A 2001 study reported that treatment of
trichomoniasis with a 10-day course of
metronidazole resulted in a 4.2-fold reduction in
mean HIV-1 virus in vaginal secretions.
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EPIDEMIOLOGY OF TRICHOMONIASIS

• Cosmopolitan distribution
• More than 170 M cases worldwide with 2-3 million
  symptomatic infections annually in the USA
• Up to one-third of cases of T. vaginalis in
  females is asymptomatic. However, complications
  are more frequently described (pelvic
  inflammatory disease in cases with HIV
  co-infection, cervical intraepithelial neoplasia
  and reproductive health problems)
• High-risk groups are African- American, prison
  inmates, drug users and sex workers
• Prevalence increases with age

Global incidence of 8 STDs
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DIAGNOSIS

• Clinical presentation discharge, strawberry
  cervix
• Visualization of the motile parasites in vaginal
  secretions (45-60 sensitivity)
• Culture of parasites in special media.
  (InPouch). This method will identify up to 95 of
  infections.
• Antigen detection test from Genzyme Corp.
  (78.5-98.6 sensitivity)
• Oligonucleotide probe test (80-90 sensitivity)
• PCR-based test under development

InPouch, Biomed Diagnostics. A two-chambered
plastic bag culture system to improve
OSOM Trichomonas Rapid Test A rapid test for the
detection of Trichomonas Vaginalis Easy - to -
Run - Dipstick simple, one reagent. Accurate -
95 agreement against Composite Reference
Standard (culture and wet mount). Unaffected by
other common infections.
44
TREATMENT

• METRONIDAZOLE 2g orally in a single dose (97
  cure rate)
• Brand name Flagyl. Sexual partner should also be
  treated
• TINIDAZOLE recently licensed for use in the
  USA. Useful for resistant infections

Sexual education helps prevent the spread of this
disease
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COMPARING GIARDIA TRICHOMONAS AND E. HISTOLYTICA
46
Giardia Entamoeba Trichomonas Common
Characteristics

• Early-diverging Eukaryotes
• Facultative Anaerobes
• Carbohydrate metabolism is anaerobic
• Lack mitochondria
• Extracellular parasites
• Do not invade host cells
• Adhere to plasma membrane of host cells
• Contact-dependent cytotoxicity
• Mechanical damage to host tissues
• Replicate and divide within the host by binary
  fission
• All are killed by treatment with Metronidazole,
  a drug that selectively kills anaerobic cells