Liver and Biliary Tree Parasites: Echinococcus granulosus and Schistosoma japonicum

LIVER
AND
BILIARY TREE
PARASITES

CESTOIDEA
Order: Cyclophyllidea

Echinococcus granulosus
Echinococcus multilocularis
Echinococcus vogeli

Echinococcus granulosus infection has a world-
wide distribution with a higher prevalence in
South-America (Argentina, Uruguay), Europe
(mediterranean bassin), Northern Africa, Middle
East, South-Central and East Asia.

Echinococcus granulosus: hydatidosis is caused by
the larval stage of E.granulosus.
After ingestion of eggs the onchospheres penetrate
the intestinal mucosa and reach host organs
(mainly liver and lung) where they encyst within a
week reaching 1 cm in diameter in about 5 months.

Echinococcus granulosus: the cysts (2 to 30 cm)
are constituted by an external acellular cuticule
and an inner cellular "germinal" layer (10-25 µ)
that produces the brood capsules containing 6-12
protoscolices or single protoscolices. (Germinal
layer with a protoscolex).

Echinococcus granulosus: the larvae (scolices)
develop from the germinal layer.
The protoscolices are at first evaginated and
measure 120-220 by 70-120 µ.

Echinococcus granulosus: the mature rotoscolices
have 4 suckers and a rostellum with hooklets and
can be observed in the hydatid fluid.

Echinococcus granulosus: detail of the rostellum.

Echinococcus granulosus: the protoscolices then
become invaginated and measure 90-140 by 70-120
µm.They can transform into daughter cysts.
These cysts can proliferate both internally and
externally giving exogenous cysts.Spontaneous or
surgical rupture of the cyst can originate a
secondary hydatidosis.

Echinococcus granulosus: the liver is the most
common site of development of cysts (50-75%).
Lesions can be detected by CT scan or
echography;a septate structure is a characteristic
of active cysts.
Treatment is based on surgical and/or medical
therapy (albendazole)

Echinococcus granulosus: definitive diagnosis is
obtained by means of serologic tests (EIA, IHA,
CIEP/Western Blot);the last two are confirmatory
tests and are useful for the follow-up of treated
patients.
-Detail of liver lesion, CT-scan with septa.
-Western blot analysis: both Ag5 (55 and 65 Kd)
and AgB (8, 16, 24 Kd) bands are present.

Echinococcus granulosus: pulmonary infection is
observed in about 20-30% of patients.
Roentgenografic examination shows round mass
lesions and CT scan demonstrates the fluid
content of the lesion.
Serology has a lower sensitivity in extrahepatic
hydatidosis.

Echinococcus granulosus: any other organ can be
affected:nervous system, heart, bones, spleen
eyes, muscles are the most common sites.
Multiple involvement is frequent.Symptoms and
signs depend on the size,the site and the pressure
of the cyst on host structures.
-CT scan of a spleen cyst.
-MRI scans of a muscular cyst.

Echinococcus granulosus: medullary hydatidosis
is a severe form of the infection.In this case the
mechanical pressure of host tissues caused
paraplegia.The surgical treatment allowed
resolution of symptoms.The infection relapsed
and responded partially to medical treatment.

Echinococcus granulosus: MRI imaging can
demonstrate the relationship between the cyst
and the medulla on the longitudinal axis.
The serology is often negative in infections in sites
other than liver or lung.(Medullary hydatidosis)

ECHINOCOCCUS MULTILOCULARIS

( 1 ) E.multilocularis is a small tapeworm (1,2-4,5 mm in
lenght)that parasites red and arctic foxes (dogs and cats are
the definitive hosts).Definitive hosts are always carnivores.
( 2 ) In the definitive hosts the adult tapeworm, consisting of 2
to 6 proglottids, lives attached to the luminal surface of the
small intestine.The terminal proglottid contains mature eggs
(ovoid, 30-40 µm in diameter).
( 3 ) The embryonated eggs, the infectious stage, are long-lived
and highly resistant to high and low temperature (more than
50° C and down to -40° C).The mature eggs are shed with faeces
and are spread in the environment.It is assumed that the
intermediate host acquires the infections through the ingestion
of contaminated fruits and vegetables.
( 4 ) When the intermediate hosts (predominantly rodents
or other small mammals, or, accidentaly, humans) ingest
eggs,the onchosphere hatches from the egg in the duodenum.
( 5 ) The activated oncosphere penetrates the small intestine,
enters blood vessels and reaches primarly the liver via the portal
vein;In the liver the oncosphere proliferates into the
metacestode surrounded by an inner germinative membrane and
an outer laminated layer.
( 6 ) The lifecycle is completed when an intermediate host,
carrier of viable protoscolices within the cysts, is devoured by a
definite host.

Geographical distribution

Human AE is prevalent in North America (Alaska
and northern Canada), in Europe (France,
Switzerland, Austria and Germany),in Asia (from
the White Sea to the Behring strait in the north and
from Turkey, through Afghanistan, Iran, India,
China, Mongolia to north Japan in the south).
The annual incidence of human disease varies from
28 cases/100.000 inhabitants in Western Alaska
(St. Lawrence Island included),to 0.18-4,4/100.000
in Central Europe.

The disease

The liver is the organ primarily affected;metastases
are mainly observed in cases of advanced disease
and may affect almost any organ.
The disease either spreads via direct contact or via
blood vessels.Secondary AE mostly affects the brain,
the lungs, soft tissue, the spine and other bony
structures.The disease is primarily characterized by
an expansive and infiltrative growth in the liver.
Clinical features may be absent for many years
and mostly become apparent in advanced disease.
They may include hepatomegaly, jaundice,
abdominal pain, weight loss,fever and
manifestations of secondarily affected organs.

Diagnosis
For diagnosing AE the clinician mainly relies on
morphological criteria together with serology and
epidemiological aspects.

On ultrasound a typical lesion demarcates as
a heterogeneous hypoechoic lesion with irregularly
shaped margin and often contains focal areas of
calcification ( 50% of cases). The appearance on
ultrasound is highly variable between cases as can
be appreciated on the images above.It is the ideal
method for screening purposes and short-term
follow-up.

Computed
tomography (CT) and
magnetic-resonance-
imaging (MRI)are
used for further
characterization of
the lesion.They are
indispensable for the
evaluation of
extrahepatic
affection in AE and
they are used for a
preoperative
evaluation.CT best
depicts the typical
calcifications and it is
used for follow-up
examinations at
longer intervalls. For
serology an ELISA
was established
based on the purified
E.multilocularis
carbohydrate antigen
Em2 (derived from
the laminated layer).
It is the reference
test for diagnosis and
it may allow
discrimination of AE
from E.granulosus
infection.However, in
a significant
percentage of cases
the two species can
not be differentiated
simply by serological
means.

Treatment

The only curative treatment for AE to date
is total surgical resection combined with
chemotherapy. Drugs used for the treatment
of AE are benzimidazoles (mebendazole
50mg/KG and albendazole 10-15mg/KG).
Chemotherapy is mainly parasitostatic and
may therefore not be considered curative.
In inoperable or incompletely resected cases
chemotherapy has to be administered for
extended periods of time and often results in
life-long treatment.

ECHINOCOCCUS VOGELI

Echinococcus vogeli: E.vogeli is the agent of the
polycystic hydatidosis.The larval stage proliferates
externally from the germinal layer and forms septa
within the cyst generating microcysts.Endemic in
Central and South America.(Cysts, macro)

Echinococcus vogeli: protoscolices similar to that
of E.granulosus are present in the cyst's fluid.

TREMATODA
Order: Strigeata

-Schistosoma japonicum
-Schistosoma mansoni

Schistosoma spp.: cercarae are the infective forms.
They measure about 500 micron. After encountering
the skin,the cercariae penetrate and lose the tail
transforming into schistosomulae.

S.japonicun: geographic distribution.
S.japonicum occurs in Southeast Asia and western
Pacific countries(including China, the Philipines
and Indonesia).S.mekongi has been reported from
Cambodia and Laos.

S.japonicum: adult schistosomes live in pairs
in the portal system and in mesenteric venules;
adults of S.japonicum are bigger than adults of
S.mansoni.Males are 12-20 mm in lenght and 0,5
wide,and have a ventral infolding from the ventral
sucker to the posterior end forming the
gynecophoric canal.Adult male with female in the
copulatory groove.

Females are slender ( 0,3 mm in diameter)
and longer (up to 26 mm in length),and are held
in the gynecophoric canal during copulation.Each
female may lay up to 2.000-3.000 eggs per day.

S.japonicum egg: eggs measure 70-90 my 55-60
µm in diameter,are oval to round in shape with
subterminal spine.(Formol-ether concentration).

S.japonicum egg: eggs are usually round and have
a small spine or no spine.Other small knobby-
spined or not spined schistosomes that affect
humans are S.mekongi and S.malaysiensis.

S.japonicum: intermediate host of S.japonicum are
snails of the genus Onchomelania, hupensis spp.

Schistosoma spp.: cercarae are the infective forms.
They measure about 500 micron. After encountering
the skin,the cercariae penetrate and lose the tail
transforming into schistosomulae.

Cercaria of Schistosoma mansoni from snail.

S.mansoni: intermediate host of S. mansoni
are snails of the genus Biomphalaria.

S.mansoni: geographical distribution.S.mansoni is
endemic in 43 countries in Africa and occurs in
the americas in Brazil,Suriname, Venezuela and
in the Caribbean.

S.mansoni: adult schistosomes live in pairs in the
portal system and in the mesenteric venules;males
are shorter (7-12 mm in lenght and 2 mm wide)and
have a ventral infolding from the ventral sucker
to the posterior end forming the gynecophoric
canal.
Adult male with female in the copulatory groove.

Adult male and female of S.mansoni.

S.mansoni : Females are slender (1 mm in
diameter)and longer (9-17 mm in length),and are
held in the ginecophoric canal during copulation.
Each female lays about 300 eggs per day.
Adult male with female in the copulatory groove.
Adult of S.mansoni in mesenteric veins of hamster.

S.mansoni egg: S.mansoni eggs measure 110-175
by 45-70 µm;the colour is yellow, with a thin
transparent shell and a strong lateral spine.Fresh
examination of intestinal biopsy with one egg in
the mucosa.

S.mansoni egg: viable eggs contain the motile
larva, the miracidium.After breaking the shell the
ciliated miracidium moves in the water and
reaches the mollusca.
Fresh examination.

S.mansoni egg: egg with typical spine in stools
(formol-ether concentration). Demonstration of
eggs in faeces and urine is the standard method
of diagnosis of schistosomiasis.Sensitivity of one
stool examination does not exceed 60%.

S.mansoni egg: lateral spine at higher magnification.
Other diagnostic methods include intestinal or liver
biopsy.Serology is useful in travellers from endemic
areas before shedding of eggs or in extraintestinal
forms (spinal) but not in natives.

S.mansoni: hepatosplenic schistosomiasis occurs
in S.mansoni and S.japonicum infections; it
results by eggs embolization in hepatic venules
with formation of granulomas and portal fibrosis.
Epatosplenomegaly, bleeding oesophageal varices
and hepatic insufficiency are the more severe
manifestations. Praziquantel is the drug of choice.

Liver biopsy: egg surrounded by granuloma and
fibrosis of portal space.

Polyposis due to S.mansoni infection.

Egyptian with Brazilian with portal
splenomegaly due to hypertension and
infection with ascites due to
S.mansoni. S.mansoni.

S.mansoni: different schistosome stages are used
as antigen source(cercariae, schistosomula,
adults, eggs) for standard immunodiagnostic
tests:enzyme linked immunosorbent assay
(ELISA), indirect immunofluorescence test
(IFAT), radioimmunoassay (RIA), indirect
haemoagglutination (IHA), circumovale precipitin
assay.Serological tests may be useful for
travellers returning from endemic areas and in
patients with light or ectopic infection, with no
detectable eggs in the faeces,urine or intestinal
biopsies (i.e. hepatic, CNS infections).On the
contrary, in patients living in endemic areas, the
positive test may reflect previous exposure to the
agent rather than an active infection;a slow
decrease in titer after effective treatment is
usually observed.Recently, new tests for the
detection of schistosome antigens have been
prepared using monoclonal antibodies.The larval
stage of S.mansoni used as antigen in the
indirect fluorescence test.

TREMATODA
Order: Echinostomata

-FASCIOLA HEPATICA

FASCIOLA HEPATICA

F.hepatica infection is found in rural areas of
temperate and tropical regions, related to cattle
herding.High prevalence is described in Europe
and Latin America.

F.hepatica, adult worm, macroscopic examination:
adults measure 2-5 cm by 8-13 mm, are flat, oval
in shape with a cephalic cone containing the oral
sucker.The adults live in biliary ducts for up to 10
years.

Fasciola hepatica, living adult in bile duct of sheep.

F.hepatica, adult worm, macroscopic examination:
higher magnification: particular of the cephalic
cone with the oral sucker.

F.hepatica, adult worm, liver biopsy: after
excistation in the small intestine, metacercariae
penetrate the intestinal wall and the Glisson
capsule, cross the liver parenchima to the bile
ducts.Eggs can be found in faeces 3-4 months
after penetration.

F.hepatica, adult worm: the diagnosis is confirmed
by the presence of eggs in faeces.Repeated
examinations and concentration techniques are
recommended.Serology is useful when the clinical
picture is compatible and eggs are not found.

F.hepatica, egg: eggs measure 140 by 80 µm and
are operculated. The colour is yellow to brown.
(Formol-ether concentration).

F.hepatica, egg: the opercular end is more visible at
higher magnification;sometimes it can present a
shell irregularity.

F.hepatica, egg: the open
operculum at higher
magnification.

F.hepatica, egg: the operculum can be open.Eggs
are unembrionated and contain a granular
material.

Fasciola hepatica: although direct diagnosis by
observation of eggs in faecal smears it the
reference method, indirect diagnostic tests such as
IF may allow diagnosis when direct observation is
negative.
Immunodiagnosis by indirect mmunofluorescence.

Antigen: frozen sections of Fasciola hepatica.

TREMATODA
Order:Opisthorchiata

-Clonorchis sinensis / Opisthorchis
viverrini
-Opisthorchis felineus

CLONORCHIS SINENSIS /
OPISTHORCHIS VIVERRINI

Clonorchis sinensis/Opisthorchis viverrini:
geographic distribution.

Clonorchis sinensis, liver biopsy:
Clonorchis sinensis adults are 10-25 mm by 3-5
mm,O.viverrini is 5,4-10 by 0,8-1,9 mm.The adults
live in the distal bile ducts and may survive for
30-40 years, causing irritation to biliary cells and
inflammation.

Clonorchis sinensis adult.

Clonorchis sinensis, liver biopsy: most infections
are asymptomatic.Clinical manifestations can be
observed in adults due to obstruction and
dilatation of biliary ducts, cholangitis and in
some cases cholangiocarcinoma.

Cholangiocarcinoma caused by chronic infection
with C.sinensis.

Clonorchis sinensis/ O.viverrini egg: eggs of the
two species are similar.They measure 30-35 by
12-20 µm, are operculated at one end and have a
small knob on the other end. The colour is yellow.

OPISTHORCHIS FELINEUS

Opisthorchis felineus: an estimated 17 million of
people on our planet are infected with fishborne
Opisthorchiidae trematode infections:
Opisthorchis felineus, O.viverrini, Clonorchis
sinensis [1].Despite being preventable fishborne
trematode infection Opisthorchis felineus is
widespread in the Russia.
Opisthorchis felineus: adult fluke

Opisthorchis felineus was first found in 1884
in cat liver in the Northern Italy by Rivolta and
in 1891 in man in Siberia by the Russian
scientist K.N.Vinogradov who named it
“Siberian liver fluke”
Opisthorchis felineus: adult fluke, detail

Opisthorchis felineus (Rivolta, 1884) is the most
prevalent food-borne liver-fluke infection of man
in the Russia, Ukraine and Kazahstan.
Estimated number of persons infected with
O.felineus in Russia is about 1,500,000 [2].

Opisthorchis felineus: opisthorchiasis is most
prevalent in Western Siberian region in the Ob and
Irtish river valleys where the prevalence amongst
local natives (Hanti, Mansi, Nensi - Mongoloid
race)in some settlements of this region reaches
100% and up to 80 amongst nonaborigene
indigenous population [3,4].In the European
Russia the endemic area is located between
Volga and Kama rivers and in some other regions
where prevalence of this infection varies from
sporadic cases to 10% [2].

Opisthorchis felineus: first intermediate hosts are
freshwater snails - Bithyniidae; second
intermediate hosts are freshwater fish -Cyprinidae.

In Russia the most important second intermediate
hosts are Leuciscus idus L., Leuciscus leuciscus L.
and Rutilus rutilus L..Main second intermediate
hosts from Ob and Irtish river valleys:
Leuciscus idus L. (in the middle); Leuciscus
leuciscus L.(at the bottom)
and Rutilus rutilus L. (at the top).

Opisthorchis felineus: final hosts are dogs, cats and
other fish-eating mammals.People in Siberia and some
European regions acquire infection by consumption of
raw, slightly salted and frozen fish (a locally so-called
“stroganina”)because of it natural availability and
because freezing is the most easy and cheap method of
preserving fish in the North [3].Metacercaria in muscle
tissue of Leuciscus idus;compression between two
slides.
(o.s.- oral suker; v.s.- ventral suker; e.v. - excretory
visicle).

Opisthorchis felineus: pathological manifestations
of initial phase of O.felineus infection are multiple
and vary in both quality and intensity from non-
apparent form and acute cases with clinical
manifestations.The major pathology in O.felineus
infection is chronic inflammation of the bile ducts.
Opisthorchis felineus. Metacercarias in culture
(artificial digestion procedure).

Opisthorchis felineus: opisthorchiasis varies in severity from
asymptomatic infection to severe illness with appreciable
morbidity and mortality.In heavily infected patients recurrent
pyogenic cholangitis, liver abscesses,cholecystitis,
pancreatitis, biliary stones may occur.The absence of
pathognomic clinical manifestations and confounding of
diagnosis with other prevalent diseases lead to under-
reporting [3,5].Opisthorchiasis is linked to holangiocarcinoma,
but the pathogenesis is still unclear and liver cancer is one of
most common malignancies that occurs in endemic areas [1].
The outcome in patients with opisthorchiasis is dependent on
early treatment and hence the early detection of infection is
important.Opisthorchiasis parasitological techniques:
- stool and duodenal fluid surveys ,
examination of suspected fish
- artificial digestion procedure ,tissue compression between
two slides .Praziquantel is the drug of choice for treatment of
opisthorchiasis and clonorchiasis.Opisthorchis felineus eggs
(at x 400 magnification) in duodenal fluid on the transparent
polycarbonate Nucleopore membrane (arrow indicates a filter
pore (8 mm) )
Duodenal fluid was obtained by duodenal aspiration.

Liver and Biliary Tree Parasites: Echinococcus granulosus and Schistosoma japonicum

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