Blood,bone marrow,spleen parasites (171)

BLOOD,
BONE MARROW,
SPLEEN
PARASITES

BLOOD NEMATODA
Order: Filariata

BRUGIA MALAYI /
WUCHERERIA BANCROFTI

Brugia malayi /Wuchereria bancrofti: B.malayi is
transmitted by mosquitoes of the genus
Mansonia, Anopheles and Aedes.
W.bancrofti is transmitted by mosquitoes of the
genus Culex, Anopheles and Aedes.
Close-up of mosquitoes on human skin.

Lymphatic filariases have a wide geographic
distribution.W.bancrofti and B.malayi infect some
128 milion people,and about 43 milion have
symptoms.Lymphatic filariases have a wide
geographic distribution.B.malayi infection is
endemic in Asia(China, Corea, India, Indonesia,
Malaysia, Philippines, Sri Lanka).B.timori infection
occurs in Indonesia (islands of Alor, Flores, Timor).
W.bancrofti has a larger distribution : Asia
(China, India, Indonesia, Japan, Malaysia,
Philippines, South-East Asia,Sri Lanka, Tropical
Africa, Central and South America, Pacific Islands.

Brugia malayi: microfilariae measure 270 by 8
µm, have a sheath and a tail with terminal
constriction,elongated nuclei and absence of
nuclei in the cephalic space.They have nocturnal
periodicity.(Wet mount preparation).

Brugia malayi: The microfilariae are sheathed and
can be distinguished from W.bancrofti for size
(275-320x7,5-10), location of nuclei and tail
nuclei.(Fresh examination, particular of the
caudal space).

Brugia malayi: detail of the cephalic space.
Microfilariae are usually nocturnally periodic but
sub-periodic strains of B.malayi and W.bancrofti
are observed.(wet mount, detail of the cephalic
space of B.malayi microfilaria).

Brugia malayi: identification of microfilariae in
stained smear is possible by observation of the
stained sheath (W.bancrofti sheath does not
stain).

Microfilaria of Wuchereria bancrofti
(Giemsa stain, x 400)

Brugia malayi: the tail is tapered and present a
constriction.The last two nuclei are divided by the
constriction. The sheath stains pink.
(Caudal space of B.malayi, Giemsa stain).

Brugia malayi: the cephalic space is longer than
broad(in W.bancrofti is as long as broad).
(Detail of the cephalic space of B.malayi microfilaria,
Giemsa stain).

Lymphatic filariasis: adults of B.malayi and
W.bancrofti live in the lymphatic vessels and
lymphnodes where they cause dilatation,
inflammatory infiltrates and, at last, blockage of the
lymphatic circulation.Adenolymphangitis, orchitis,
epididimitis associated with fever are the
commonest manifestation of the acute stage of the
infection;eosinophilia is frequent at this stage.
Lymphoedema particularly of the legs and scrotum,
hydrocoeles and chyluria are the result of the
progression of the disease;genital manifestations
are frequent in W.bancrofti infections while they are
rare during B.malayi infections.
-Lymphatic filariasis: elephantiasis of scrotum.
-Tanzanian with elephantiasis due to W.bancrofti.
-Another Tanzanian patient with elephantiasis due
to W.bancrofti.
-Early hydrocoel in a Tanzanian man with
W.bancrofti infection

Lymphatic filariasis: elephantiasis is the last
consequence of the swelling of limbs and scrotum.
Diethylcarbamazine (DEC), ivermectine and
albendazole used alone or in combination are the
drugs of choice.
-Elephantiasis of the limbs.
-Thai patient with elephantiasis of leg due to
W.bancrofti or Brugia malayi.
-Second Thai patient with elephantiasis due to
lymphatic filariasis.

Loa loa: the infection is endemic in West and
Central Africa,especially in Angola, Cameroun,
Congo, Eq. Guinea, Gabon, Nigeria, RCA, Zaire.

Loa loa: after injection larvae develop into adults
in 6 months and may live for 17 years in the
organism.Microfilariae measure 275 by 5-6 µm
and are present in blood without periodicity.
Count is mandatory before therapy.

Loa loa: microfilariae measure with the sheath
240-300 by 5-6 µm.The sheath doesn't stain with
Giemsa. The nuclei extend from the small cephalic
space to the tip of the tail.(Giemsa stain).

Loa loa: the sheath does not stain and appears
as a virtual space around the larva.
(Giemsa stain).

Loa loa: the nuclei form a continuous row to the
tip of tail.The unstained sheath is well visible.
(Detail of the caudal space, Giemsa stain).

Loa loa: large nuclei extend from the little
cephalic space.
(Detail of the cephalic space, Giemsa stain).

Loa loa: the presence of the unstainded sheath is
clearly visible as a space around the larva.
(Detail of cephalic space with a polymorphonuclear
cell, Giemsa stain).

Loa loa: microfilaria.
Thick film, Mayer's haematoxylin (400 X).

Loa loa: microfilaria, tail area.
Thick film, Mayer's haematoxylin (1.000 X).

Loa loa: microfilariae can be demonstrated in blood
with fluorochromes
(Acridine orange stain).

Loa loa: although direct diagnosis by observation
of microfilariae in blood is the reference method,
indirect diagnostic tests such as IF may allow
diagnosis when direct observation is negative,
especially in subjects who are not resident in
endemic areas.
The frequent cross-reaction with other nematode
infections limits the usefulness of serology in
these patients.
Immunodiagnosis by indirect
immunofluorescence.
Antigen: frozen sections of Dirofilaria immitis.

MANSONELLA OZZARDI
MANSONELLA PERSTANS

M. ozzardi is endemic in Central and South
America.
The adults live in subcutaneous tissues.
Unsheathed microfilariae (200x4-5 µm) released in
blood without periodicity,have a small cephalic
space, a long and slender tail without nuclei to the
end.

M. perstans is endemic in Africa and South
America. The adult lives in body cavities.
Unsheathed microfilariae (200x4-5 µm) released
in blood without periodicity have a small cephalic
space and nuclei to the end of tail.

Mansonella perstans: microfilaria.
Thick film, (400 X).

BLOOD SPOROZOEA
Order: Eucoccidiida

Plasmodium falciparum
Plasmodium malariae
Plasmodium ovale
Plasmodium vivax
Toxoplasma gondii

Plasmodium sp.: geographic distribution.

Plasmodium sp.: the genus Anopheles includes
more than 400 species of mosquitoes.Many
may act as vectors of human diseases such as
malaria,filariasis and some arbovirus.
Eggs present a pair of lateral floats and are laid
singly on the water surface,while larvae lay in
a horizontal position under the water surface.

Plasmodium sp.: the resting position of the adult is
characteristic with the proboscid, head and
abdomen in a straight line at an angle of about 45°
with the surface on which they rest.Only about 60
species can transmit malaria and they greatly
differ in their efficiency as vectors according to
man biting behaviour, survival, fertility, adaptation
to different breeding place.The most efficient
vectors belong to the A.gambiae complex,widely
distributed in tropical Africa, where also important
is A.funestus.In Asia important vectors are
A.culicifaciens, A.dirus, A.sinensis and A.miminus;
in the Pacific area A.farauti and A.maculatus play a
predominant role in malaria transmission. The
main vector in South America in A.albimanus.

P.falciparum: species identification is possible on
the basis of the appearance of parasites of each of
the four malaria species.Shape and size of asexual
parasites and of macro- and microgametocytes,
developmental stages in peripheral blood,
modifications of infected erythrocytes,presence of
dots or clefts on the red blood cells are the main
differential characteristics.

Malaria diagnosis relies on observation of
parasites in Giemsa-stained thin or thick smears
(G-TS).
Alternative techniques for identification of malaria
parasites are based on fluorochromes such as
Acridine Orange (AO), DAPI-PI or BCP.
With these dyes malaria parasites are easily
recognized under UV light, reducing the time spent
reading the slides.Another method, based on
fluorochromes, the quantitative buffy coat (QBC)
(Becton-Dickinson) analysis wich uses AO staining
of centrifuged parasites in a capillary tube
containing a float, has been shown to be rapid and
accurate.

P.falciparum: gametocytes of P.falciparum.
QBC technique (60X).

Recently different immunochromatographic
tests such as the ParaSight F(Becton Dickinson)
and the Malaquick (ICT) wich capture and
detect the histidine rich protein 2 (HRP-2)
antigen,and the OPTIMAL wich detects
Plasmodium lactate dehydrogenase(pLDH)
have been developed and distributed.
The tests are highly sensitive and specific and
are now able to distinguish P.falciparum
infections from non-falciparum infections.
P.falciparum trophozoites, thin smear, Giemsa
stain.

Malaria diagnosis:whereas thin film gives more
informations on parasite morphology and permits
an easier morphologic differentiation,G-TS is
more sensitive allowing a concentration of
plasmodia (10-15 folds) and it is the standard
reference diagnostic test.

P.falciparum trophozoites, thick smear, Giemsa stain.

Malaria diagnosis:
G-TS needs careful stain (2% Giemsa) and experience in
examining slides;reasonable sensitivity is reached by
observing at least 500-1.000 White Blood Cells
(WBC).Quantification of baseline parasitemia is necessary
for monitoring the response to therapy. Parasites must be
counted in parallel with leucocyte and parasitemia
expressed as parasites/µl.
N. of parasites counted x N. of WBC/µl
= N. of
parasites/µl
N. of WBC counted

P. falciparum: trophozoites are small rings with
single or double small chromatin dots, and
regular cytoplasm; multiple infection and
high parasitemia (>5%) are common.
Dots or cleft (Maurer's) can be observed on the
infected RBCs.
stain.

P. falciparum: sometimes trophozoites appear at
the edge of the red blood cell (applique form)
left.Erythrocytes maintain regular shape and
size.
stain.

P. falciparum: late trophozoites and schizonts
usually are not observed in peripheral blood
unless in severe infections.
Cerebral malaria: late trophozoites with a
coarse granule of pigment in peripheral blood.
P.falciparum, thin smear, Giemsa stain.

P.falciparum: micro- and macrogametocytes are
easily recognized by their crescentic, cigar- or
banana-like shape.Microgametocytes have a
diffuse chromatin,while macrogametocytes have
thickened chromatin.
Microgametocyte, Giemsa thin smear.

P.falciparum: in thick films red blood cells are not
visible and leucocytes and parasites appear
smaller than in thin smears.Trophozoites have a
ring or comma shape, with one or two dots of
chromatin.The pigment, when present, is compact.

P.falciparum: trophozoites in Giemsa-stained
thick films have a wide range of shapes.
Maurer's clefts are not visible.

P.falciparum: micro- and macrogametocytes have
an evident malaric pigment,scattered through in
the cytoplasm in the microgametocyte.
Microgametocyte, Giemsa thick smear.

P.falciparum: staining with fluorochromes is rapid
(less than 1 min)and observation of slides can be
performed at low magnification (400X) allowing
rapid screening of smears even with low
parasitemia. P.falciparum (DAPI-PI).

P.falciparum trophozoites. Acridine Orange stain.

P.falciparum: the sensitivity of different isolates
of P.falciparum to drugs can be assessed with the
WHO "in vitro test".
The development to mature schizont in presence
of therapeutic levels of the drug demonstrates
resistance of the isolate.

P.falciparum: severe P.falciparum infections are
clinical forms characterized by potentially fatal
manifestations or complications:cerebral malaria,
defined by a state of unrousable coma in absence
of other causes,is the most common manifestation.

Celebral malaria: parasitized RBCs in brain vessels
(H&E stain).

P.falciparum: rosetting of infected and uninfected
red blood cells and cytoadherence of parasitized
erythrocytes to the vascular endothelium,play a
crucial role in sequestration of parasites and
obstruction of brain vessels.
Induction of host cytokines and soluble mediators
such as oxygen radicals and NO play an important
role in the pathogenesis of the infection.

P.falciparum: the brain appears oedematous,
hyperaemic and with pigment deposition; the
capillaries, expecially of the white matter,
appear dilated and congested and obstructed by
parasitized RBCs.

P.falciparum: renal failure may result from
sequestration of RBCs and alteration of the renal
microcirculation.
Glomerulal and interstitial vessels present RBCs
adhering to the endothelium.

P.falciparum: renal failure may also result from
releasing of compounds secondary to intravascular
haemolysis (not haemoglobin itself) that can cause
acute tubular necrosis especially in presence of
dehydratation and acidosis.

P.falciparum: sequestration and cytoadherence
of parasitized RBCs in heart microcirculation is
frequent but myocardial dysfunctions and
cardiac arrhythmias are uncommon in severe
falciparum malaria.

P.falciparum: jaundice and abnormalities of liver
function tests are frequent findings in severe
falciparum malaria but hepatic failure is rare even
in heavily infected individuals.

P.falciparum: histological abnormalities include
Kuppfer hyperplasia,mononuclear hyperplasia and
sinusoid dilatation;swollen hepatocytes contain
haemosiderin.Kuppfer cells contain a lot of
malaria pigment.

A fatal case of P.falciparum malaria (liver):
malarial pigment within Kupffer cells (H&E X 400)

A fatal case of P.falciparum malaria (liver):
note a parasitized erytrocyte (H&E X1000)

P.falciparum: pulmonary, non specific complications,
such as atypical pneumonia, lobar pneumonia or
bronchopneumonia,frequently occur during malaria
infections.Pulmonary oedema is a specific and severe
complication of P.falciparum infection: 3-10% This
syndrome, wich resembles the Acute Respiratory
Distress Syndrome (ARDS),has a relative late onset
(wich may be abrupt) in the course of the infection and
is often associated with other manifestations of the
severe falciparum malaria.Different pathogenic
mechanisms have been suggested:
-increased capillary membrane permeability[due to
microemboli or to Disseminated Intravascular
Coagulation (DIC)]
-impaired function of the alveolar capillaries;
-severe disfunction of the pulmonary microcirculation;
-allergic phenomena;
-therapeutic fluid overload.
The chest radiograph, in severe cases, shows
widespread bilateral,confluent intraalveolar and
interstitial infiltrates.

Plasmodium sp.: the genus Anopheles includes
more than 400 species of mosquitoes.Many may
act as vectors of human diseases such as malaria,

filariasis and some arbovirus.
Eggs present a pair of lateral floats and are laid
singly on the water surface,while larvae lay in a
horizontal position under the water surface.

Plasmodium sp.: the resting position of the adult is
characteristic with the proboscid, head and abdomen
in a straight line at an angle of about 45° with the
surface on which they rest.Only about 60 species can
transmit malaria and they greatly differ in their
efficiency as vectors according to man biting
behaviour, survival, fertility, adaptation to different
breeding place.The most efficient vectors belong to
the A.gambiae complex,widely distributed in tropical
Africa, where also important is A.funestus.
In Asia important vectors are A.culicifaciens, A.dirus,
A.sinensis and A.miminus;in the Pacific area
A.farauti and A.maculatus play a predominant role
in malaria transmission. The main vector in South
America in A.albimanus.

P.malariae: species identification is possible on the
basis of the appearance of parasites of each of the
four malaria species.Shape and size of asexual

Malaria diagnosis relies mainly on observation of
(G-TS).Alternative techniques for identification of
malaria parasites are based on fluorochromes
such as Acridine Orange (AO), DAPI-PI or BCP.
recognized under UV light,reducing the time spent
reading the slides.Another method, based on
fluorochromes, the quantitative buffy coat (QBC)
(Becton-Dickinson) analysis wich uses AO staining
of centrifuged parasites in a capillary tube
containing a float, has been shown to be rapid and
accurate.

P.falciparum: gametocytes of
P.falciparum. QBC technique (60X).

Recently different immunochromatographic tests
such as the ParaSight F(Becton Dickinson) and the
Malaquick (ICT) wich capture and detect the
histidine rich protein 2 (HRP-2) antigen, and the
OPTIMAL wich detects Plasmodium lactate
dehydrogenase (pLDH) have been developed and
distributed.
The tests are highly sensitive and specific and are
now able to distinguish P.falciparum infections
from non-falciparum infections.
P.malariae trophozoites, thin smear, Giemsa stain.

Malaria diagnosis:
whereas thin film gives more informations on
parasite morphology and permits an easier
morphologic differentiation, G-TS is more sensitive
allowing a concentration of plasmodia (10-15
folds)
and it is the standard reference diagnostic test.

trophozoites,
thick smear

Schizont,
thick smear

Malaria diagnosis:
examining slides;
reasonable sensitivity is reached by observing at least
500-1.000 White Blood Cells (WBC).
Quantification of baseline parasitemia is necessary
for monitoring the response to therapy.
Parasites must be counted in parallel with leucocyte
and parasitemia expressed as parasites/µl.


= N. of
parasites/µl
N. of WBC counted

P. malariae: trophozoites are usually small rings
with a single dot of chromatin or have a compact,
regular cytoplasm that seems to contain the
nucleus.
The pigment in late trophozoites is scattered.
(Thin smear, Giemsa).

P. malariae: trophozoites may assume a band form
typical of the species.Red blood cells are not
enlarged or rather smaller than normal.Multiple
infection is rare. The parasitemia is usually low. No
dots or clefts.(Thin smear, Giemsa).

P.malariae: schizonts are small and with a low
number of merozoites (<12) arranged in regular
forms (rosettes) with a thickened, often central,
pigment.The complete erythrocytic cycle takes
72 hours and ends with the releasing of free
merozoites (c).(Thin smear, Giemsa).

P. malariae: micro- and macrogametocytes are
round, small with chromatin defined; they must
be differentiated from late trophozoites.During
P.malariae infection all stages of development
are present in peripheral blood.
(Microgametocyte, Giemsa stain).

P.ovale: species identification is possible on the
basis of the appearance of parasites of each of the
four malaria species.Shape and size of asexual

Malaria diagnosis relies on observation of
recognized under UV light,reducing the time
spent reading the slides.Another method, based
on fluorochromes, the quantitative buffy coat
(QBC)(Becton-Dickinson) analysis wich uses AO
staining of centrifuged parasites in a capillary
tube containing a float, has been shown to be
rapid and accurate.

such as the ParaSight F (Becton Dickinson) and
the Malaquick (ICT) wich capture and detect
the histidine rich protein 2 (HRP-2) antigen, and
the OPTIMAL wich detects Plasmodium lactate
dehydrogenase (pLDH) have been developed and
distributed.The tests are highly sensitive and
specific and are now able to distinguish
P.falciparum infections from non-falciparum
infections.
P.ovale, thin smear, Giemsa stain.

Malaria diagnosis:
morphologic differentiation, G-TS is more
sensitive allowing a concentration of plasmodia
(10-15 folds)and it is the standard reference
diagnostic test.

P.ovale, thick smear, Giemsa stain.

Malaria diagnosis: G-TS needs careful stain
(2% Giemsa) and experience in examining slides;
reasonable sensitivity is reached by observing at least
500-1.000 White Blood Cells (WBC).
for monitoring the response to therapy.
Parasites must be counted in parallel with leucocyte
and parasitemia expressed as parasites/µl.

= N. of
parasites/µl
N. of WBC counted

Plasmodium ovale: trophozoite

All stages are seen in blood films; prominent
Shuffner's dots are present at all stages.
Trophozoites appear as rings with, usually, a
compact cytoplasm;they do not have ameboid
cytoplasm.The parasites are smaller than P.vivax.

P.ovale: red blood cells are enlarged, ovalized
and distorted with fimbriae at poles.
Schizonts have usually 8-10 merozoites.

P.ovale: micro- and macrogametocytes are
sometimes difficult to differentiate from late
trophozoites;they are round and occupy almost
the entire erythrocyte.Microgametocytes have a
more scattered chromatin.

P.vivax: species identification is possible on the
basis of the appearance of parasites of each of
the four malaria species. Shape and size of
asexual parasites and of macro- and
microgametocytes,
modifications of infected erythrocytes,presence
of dots or clefts on the red blood cells are the
main differential characteristics.

Malaria diagnosis relies mainly on observation of
recognized under UV light, reducing the time
spent reading the slides.Another method, based
on fluorochromes, the quantitative buffy coat
(QBC) (Becton-Dickinson) analysis wich uses AO
staining of centrifuged parasites in a capillary
tube containing a float,has been shown to be
rapid and accurate.

such as the ParaSight F(Becton Dickinson) and
the Malaquick (ICT) wich capture and detect
the histidine rich protein 2 (HRP-2) antigen,
and the OPTIMAL wich detects Plasmodium
lactate dehydrogenase (pLDH)have been
developed and distributed.The tests are highly
sensitive and specific and are now able to
distinguish P.falciparum infections from non-
falciparum infections.
P.vivax trophozoites, GT-s.

Malaria diagnosis:
morphologic differentiation,G-TS is more sensitive
allowing a concentration of plasmodia(10-15 folds)
and it is the standard reference diagnostic test.

P. vivax trophozoites, thick smear, Giemsa stain.

Malaria diagnosis:
examining slides;reasonable sensitivity is reached by
observing at least 500-1.000 White Blood Cells (WBC).
for monitoring the response to therapy.Parasites must be
counted in parallel with leucocyte and parasitemia
expressed as parasites/µl.

= N. of
parasites/µl
N. of WBC counted

P.vivax: young trophozoites are small with single
(rarely double) chromatin,with a loop of thin
cytoplasm.The red blood cell is sligthly enlarged
and a few Shuffner's dots are present.
Parasitemia range form 0.5 to 2%, multiple
infection is rare.

P.vivax: the trophozoites increases in size and the
cytoplasm becomes ameboid with rapid movements
("vivax").The red blood cell enlarges and prominet
Shuffner's dots are present.(Thin smear, Giemsa).

P.vivax: in more advanced stage of development
trophozoites occupy most of the RBC, and have a
large vacuole and fine rods of pigment.The
nucleus increases in size.

P.vivax: late trophozoites have a more dense
cytoplasm, and a large vacuole.

P.vivax: in young schizonts the nucleus divides and
the vacuole disappears;
the cytoplasm is dense.

P.vivax: in about 48hours schizogony is
completed.Mature schizont may contain 12-24
merozoites. In thick smears schizonts look
smaller than in thin smears and the Schuffner's
dots are not always visible.

P.vivax: gametocytes are round or oval without
vacuole; most of the RBC is occupied by the
parasite. Macrogametocytes have a compact
chromatin mass while microgametocytes have a
more diffuse nucleus stained pink.

P.vivax: staining with fluorochromes is rapid (less
than 1 min) and observation of slides can be
performed at low magnification (400X) allowing rapid
screening of smears even with low parasitemia.
P. vivax (DAPI-PI).

T. gondii: T.gondii encephalitis (TE) is the most
common cerebral opportunistic infection in
patients with AIDS.
The typical lesion is an ipodense focal area with
ring contrast-enhancement and edema.
(CT scan of a toxoplasmic encephalitis).

T. gondii: tissue cysts, 100-300 µm, may contain
up to 3.000 bradyzoites.The wall of mature
pseudocysts is believed to represent a
combination of host and parasitic components.

T. gondii: diagnosis of TE is usually presumptive,
based on clinical and radiologic findings and on the
response to treatment; cerebral biopsy sometimes
allows identification of pseudocysts in tissue
sections. (H&E stain).

T. gondii: toxoplasmic pseudocyst within an
inflammatory tissue reaction. (H&E stain).

T. gondii: the pseudocysts of T.gondii can be
observed in tissue sections with monoclonal
antibodies.

T. gondii: direct detection of T.gondii in clinical
specimens is rare;parasites can be isolated from
blood, CSF, amniotic fluid,tissue biopsies on cell lines
(THP-1 or MRC-5).
In clinical specimens the presence of parasites can
also be demonstrated by PCR analysis.

T. gondii: intracellular trophozoites of T.gondii in a
cell culture.
The trophozoites proliferate within the vacuole
developing a pseudocyst.
(Trophozoites in a THP-1 cell, Giemsa stain).

T. gondii: in cell cultures T.gondii proliferates to
form a pseudocyst of 8-20 parasites.

T. gondii: lysis of a THP-1 cell with release of
tachizoites in culture.

T. gondii: microscopical features of tachizoites of
Toxoplasma gondii and peritoneal macrophages
of mouse in peritoneal exudate. (SEM)

T. gondii: microscopical features of tachizoites of
Toxoplasma gondii and peritoneal macrophages of
mouse in peritoneal exudate. (SEM)

T. gondii: the anterior pole of an endozoid in
tangential projection.Several subpellicular
fibrils and their insertion on the anterior polar
ring are visible.

T. gondii: transmision electron microscopic picture.
Longitudinal section of an endozoid.

T. gondii: cross-section through an endozoid
in an advanced stage of endodiogeny.
The daugther cells appear to be surrounded.
In each of these news cells there are two round
bodies that lengthen forming the first rhoptries.

BABESIA CANIS

Babesia spp: babesiosis is a zoonosis that affects
several animals:B.canis (dogs), B.equi (horses),
B.bovis (cattle), B.microti (rodents).Some Babesia
spp. are not host specific and can be transmitted to
humans:B. microti and B.bovis/divergens.The
infection is transmitted by the bite of ticks of the
Family Ixodidae of the genera Dermatocentor,
Ixodes and Rhipicephalus.The main vector of
B.microti is I.dammini, while vector of B.microti is
I.ricinus
B.canis, Giemsa stain.

Babesia spp.: intraerythrocytic organisms in blood
smears

Babesia spp.: after inoculation by the vector, the
trophozoites enter the bloodstream and multiply inside the
erythrocytes by budding, releasing two to fours daughter
parasites and causing hemolytic anemia. Ticks become
infected by ingesting blood of parasitized mammals.
Motile "vermicules" develop and multiply in the tick's gut
and then migrate through the body (salivary glands and
ovaries).In some species transovarial transmission
(B.bovis and B.caballi)or transtadial passage, from larva to
nimph (B.microti) occur.Vermicules of Babesia spp.
(B.caballi ?) obtained from crushed Rhipicephalus
turanicus eggs. Tick collected from horses in a military
farm in Turkey where the prevalence of equine babesiosis
is high.

Babesia spp.: by transovarial transmission "vermicules"
can infect tick eggs;
they multiply in the yolk and in intestinal tissues of the
larva;
pyriform bodies are then observed in the salivary glands of

the haematophage larvae and nimphs.
Vermicules of Babesia spp. (B.caballi ?) obtained from
crushed Rhipicephalus turanicus eggs. Tick collected from
horses
in a military farm in Turkey where the prevalence of
equine babesiosis is high.

B.canis: diagnosis depends on the observation
of the intraerythrocytic organisms in blood
smears.Pear shaped microorganisms (2-5 µm)
and tetrads are the diagnostic shape of the
parasite. (Giemsa stain).

B.canis: intraerythrocytic parasites can be
confused with P.falciparum or P.malariae
trophozoites.
Ring and band forms are sometimes observed.
(Giemsa stain).

B.equi: trophozoites of B.equi can mimic
P.falciparum young ring trophozoites.

ZOOMASTIGOPHOREA
Order: Kinetoplastida

TRYPANOSOMA CRUZI
(Chagas' disease)

T. cruzi: american trypanosomiasis was first
described by Carlos Chagas in Brasil in 1909.
The infection, Chagas' disease, is caused
by the haemoflagellate Trypanosoma cruzi.
tc1: T.cruzi in blood sample, Giemsa.

T. cruzi: the disease is a public health threat in
most Latin American countries,although cases due
to blood derivatives or blood transfusion has been
reported in non-endemic regions.
According to WHO the overall prevalence of human
T.cruzi infection is estimated in 18 million cases
and 100 million people are living at risk.
tc2: T. cruzi: geographical distribution.

T. cruzi: the vectors are reduvidae bugs which are
haematophagus and the most important are
Triatoma infestans(Argentina, Chile, Brazil, Bolivia,
Paraguay, Uruguay, Peru),T. sordida (Argentina,
Bolivia, Brazil, Paraguay),Rhodnius prolixus
(Colombia, Venezuela, Mexico, Central America),
T. dimidiata (Ecuador, Mexico, Central America),
and Panstrogylus megistus (northeast Brazil).

T. cruzi: the transmission by the vector is faecal.
T.cruzi infective metacyclic trypomastigotes are shed
in the faeces of the bug and are inoculated into the
human host by scratching infected faeces into skin
abrasions usually caused by the bug in the process of
feeding (blood-sucking).
T.cruzi metacyclic trypomastigote: scanning electron
microscopy showing T.cruzi trypomastigotes
recovered from an infected Triatoma spp. in Pedro
Carbo, Ecuador.

T. cruzi: infective metacyclic trypomastigotes are
shed in the faeces of the bug and inoculated into
the vertebrate host not only by skin lesions but also
through the mucosa of the mouth and,in humans,
through the conjunctiva of the eyes.

T. cruzi: trypomastigotes can infect most of the
vertebrate cells,polymorphonuclear leucocytes and
macrophages are probably among the first
vertebrate host cells with which T.cruzi interacts in
vivo.
tc7a: In vitro T.cruzi infection of macrophages
showing the presence of amastigotes:
Wright-Giemsa stain, showing replicating T.cruzi
amastigotes within host cell.

T. cruzi: this invasive step is crucial for the life
cycle of the parasite since it has to become
intracellular to multiply.
tc7b: In vitro T.cruzi infection of macrophages
immunofluorescence assay showing T.cruzi
amastigotes after treatment with anti-T.cruzi
polyclonal mouse sera.

T. cruzi: trypomastigotes in the host cell transform
into amastigotes,which multiply intracellularly by
binary division inducing inflammatory and
immunological responses in vivo, and destroy cells
in vitro.
Amastigotes are then released into the blood
stream as trypomastigotes.The latter are
nondividing forms which are able to infect a wide
range of new host cells but muscle and glia seem
most often parasitized,or they have to be ingested
by another reduviid bug in order to continue the
parasite life cycle in the invertebrate host.
tc8: Trypomastigotes reach the myocardial cells
and after penetration they multiply as amastigotes
with formation of a pseudocyst.

T. cruzi: in the Reduvidae bug the bloodstream
derived trypomastigote forms pass along the
digestive tract through irreversible morphological
transformations in sequence;each developmental
stage occurs in a specific portion of the insect's gut.
Thus, in the stomach, most blood trypomastigotes
change into epimastigotes and rounded forms
(sphaeromastigotes).
tc9: T.cruzi epimastigote. Immunofluorescence
studies using antibodies to a T.cruzi protein named
Tc52(immunosuppressive factor which also express a
thiol-transferase activity)and confocal microscopy.
An intense labeling located at the posterior end of
an epimastigote indicate that Tc52 is targeted to the
reservosomes(These organelles are small vesicles
inside multivesicular structures being formed
predominantly at the posterior end of epimastigotes).

T. cruzi: epimastigotes divide actively in the
vector's intestine and reach the rectum where
a final differentiation results in the infective
metacyclic trypomastigotes which are
eliminated in the bug's faeces.
tc10: T.cruzi epimastigote. Epimastigote
reacting with a monoclonal antibody against
T.cruzi.

T. cruzi: some researchers have postulated that
sphaeromastigotes may change either into short
epimastigotes,dividing forms in the intestine, or
into long epimastigotes which are nondividing
forms but are able to reach the rectum where they
transform into the final metacyclic trypomastigote
form.In any case, this hypothesis remains
controversial.
tc10b: T.cruzi epimastigote. Scanning electron
microscopyshowing T.cruzi epimastigote.

T. cruzi: there are three phases of the infection.
The acute phase usually passes unnoticed but
there may be an inflamed swelling or chagoma
at the site of entry of the trypanosomes.
Romanas'sign is when this swelling involves the
eyelids but it occurs only in about 1-2% of the
cases.In the acute phase, mortality is less than 5%
and death may result from acute heart failure
or meningoencephalitis in children less than two
years old.Romana’s sign, clinical manifestation
tipically observed in the acute phase of some
Chagas’ disease patients.

T. cruzi: general symptoms in acute Chagas' disease
may also include fever, hepatosplenomegaly,
adenopathies and myocarditis.Electrocardiographic
changes involve sinus tachycardia, prolongation
of the P-R interval, primary T-wave changes and
low QRS voltage.Chest X-ray can reveal
cardiomegaly of different degrees.
The intermediate phase is clinically asymptomatic
and is detected by the presence of specific
antibodies.No parasites are found in bloostream
smears but xenodiagnosis could be positive in some
cases.
Acute Chagas myocarditis (Haematoxylin and Eosin
X 160)tc12: Posteroanterior chest radiograph
showing enlarged heart due to T.cruzi infection.
tc12a: Acute Chagas' disease myocarditis
(Haematoxylin and Eosin X160)

T. cruzi: the chronic phase of Chagas'disease
develops 10 - 20 years after infection and affects
internal organs such as the heart,oesophagus and
colon as well as the peripheral nervous system.
The lesions of Chagas’ disease are incurable and in
severe cases patients may die as result of heart
failure.

T.cruzi parasitize mainly the cardiac muscle but
any cell type may be parasitized (smooth
muscle cells, hystiocytes): cardiac muscle with
amastigotes, H&E stain.

Chagas' disease megacardia
(slide from the late Prof.Koberle, Brazil)

Apical aneurysm in Chagas' disease
(slide from the late Prof.Koberle, Brazil)

T. cruzi: on the other side, megacolon is associated

with abnormal constipation (weeks).Faecal
impaction and sigmoid volvulus are side-effects of
megacolon.Neurological changes in chronic
Chagas' disease include changes at the level of the
central, peripheral or autonomic nervous system.

Chagasic megacolon with enlargement of the
sigmoid;patient from Morona Santiago province,
southeastern Ecuador

X-ray showing megaoesophagus in Chagas' disease

X-ray showing megacolon in Chagas' disease

T. cruzi: can be observed in the peripheral blood
only in the acute case of the disease.Its presence is
the best definition of the acute phase as all other
signs are variable.
-Wright-Giemsa staining of T.cruzi trypomastigote
in peripheral blood smear from an acute infected
patient.
-T.cruzi in mouse blood (Giemsa stain)

T. cruzi: trypomastigotes have a prominent
subterminal kinetoplast that often distort the
membrane of the cell,an elongated nucleus and
an undulating membrane.
-T.cruzi trypomastigote: blood stream
trypomastigotes are 15-20 µm in length and
appear with a typical C or S-shaped form.

T. cruzi: multiplication only occurs in the
amastigote phase,
which grows in a variety of tissue cells especially
muscle.
-In vitro infected fibroblast showing a large
number of intracellular amastigotes.

T. cruzi: laboratory diagnostic tests based on
serology (IFA, ELISA) and Polymerase Chain
Reaction (PCR) specific for T.cruzi, have been
developed.
-T.cruzi trypomastigotes reacting with monoclonal
Ab.

T. cruzi: serological cross-reactions can occur
with infections such as leprosy, leishmaniasis,
treponematoses, malaria and multiple myeloma.
Trypanosoma rangeli is also an important cause
of false-positive testing, especially in areas where
T.cruzi coexists with T.rangeli.
-In vitro T.cruzi infection of macrophages
confocal microscopy showing T.cruzi amastigotes
after treatment with anti-Tc24 mouse sera.

T. cruzi: two drugs are in common use.
Nifurtimox (Lampit, production was discontinued in
1991)and Benznidazole (Rochagan).
The latter which is now the drug of choice,
is given in an oral dose of 6 mg/kg body weight for
30 or 60 days.Both drugs produce anorexia, weight
loss, headache and dizziness,gastric irritation, and
sometimes peripheral neuritis.Experimental drugs
are under evaluation.Treatment of patients in the
intermediate or chronic phase is controversial.
Congenital Chagas'disease and transfusion-
associated acute disease require Rochagan
therapy.Transfusion infection can be prevented by
donor screening or,by mixing the blood with
gentian violet (0,25 gr./L for 24 hours) to kill
T.cruzi.Vector control programmes involving
insecticide spraying with modern pyretroids and
new tools for delivery in endemic areas is being
carried out in some Latin American countries.
tc20: TEM microphotograph of T.cruzi
epimastigote.

TRYPANOSOMA
BRUCEI RHODESIENSE /
T.B. GAMBIENSE

Sleeping sickness occurs in Africa between the
15° North and the 20° South.
The T.b.rhodesiense form is found in East and
Central-East Africa whereas the T.b.gambiense
infection occurs in Central and West Africa.

The African
trypanosomiasis
is transmitted
by several
species of tse-
tse flies
(Glossina spp.).

Larva and
pupae of
Glossina
morsitans

Adult Glossina
tachinoides in
West Africa

T. b. gambiense and rhodesiense: two forms of
trypomastigote can be seen in peripheral blood:
one is long slender, 30 µm in length,and is
capable of multiplying in the host, the other is
stumpy, not dividing,18 µm in length.

Trypanosoma brucei gambiense and rhodesiense:
trypanosomes appear in the peripheral blood 5 to
21 days after the infecting bite.

Trypanosoma brucei gambiense and rhodesiense:
the terminal stage of the infection ("sleeping
sickness") is the result of a chronic
meningoencephalomyelitis. (H&E stain).

Trypanosoma brucei gambiense and
rhodesiense: the typical pathological lesion of
trypanosomiasis is a perivascular round-cell
infiltration (perivascular cuffing) due to glial
cells, lymphocytes and plasmocytes (Mott cells).
(H&E stain).

Visceral leishmaniasis has a wide geographic
distribution.North-Eastern China, India, Middle-
East, Southern Europe (Mediterranean bassin),
Northern Africa, Central-East Africa and, in foci,
Central and South America(especially Brazil and
Honduras).

The infection is transmitted by various species of
Phlebotomus, the sand fly.

Leishamnia spp. wich affect humans can be
differentiated by geographical distribution, clinical
spectrum, immunological features,isoenzymes and
Kinetoplast DNA (kDNA) characterization.
(Leishmania amastigotes, bone marrow aspirate,
Giemsa stain).

Visceral leishmaniasis (Kala-azar) is caused by
parasites of the genus Leishmania, subgenus
Leishmania, complex donovani (donovani,
infantum, chagasi species).Viscerotropic strains of
L.infantum and L.tropica have been described.
(bone marrow aspirate)

Diagnosis of the infection depends on
identification of amastigotes in tissues (bone
marrow, spleen, liver, limph nodes) or in
blood.Other organs may be affected, expecially in
HIV-1 positive patients
(intestinal and respiratory tract).Amastigotes can
be found inside and outside the reticuloendothelial
cells. They measure 2-5 µm, are oval with a large
nucleus (in red), a kinetoplast (usually
perpendicular, in red to violet) and a pale blue
cytoplasm.(Bone marrow aspirate).

Leishamnia sp.: Cultures (on NNN or Tobie media) of
blood or tissues samples may permit isolation of the
parasite, allowing the subsequent characterisation.
When introduced in culture the amastigotes
transform into promastigotes in 7-21 days.
(Wet mount preparation).

Leishamnia sp.: Leishmania promastigotes measure
15-20 by 1.3-3.5 µm and have a single flagellum,
measuring 15-28 µm.Serologic examination (EIA,
direct Agglutination, IF, WB) is useful in
immunocompetent individuals, not ALWAYS in
HIV-1 positive patients.

Visceral leishmaniasis: liver biopsy can
demonstrate the Leishmania amastigotes
inside the reticuloendothelial cells. The hepatic
structure is preserved.

Visceral leishmaniasis: liver biopsy at higher
magnification with intracellular amastigotes.

Visceral leishmaniasis: spleen biopsy is a very
high sensitive method of diagnosis but it is not
widely used because of the risk of hemorrhage.
Splenic tissue is rich in amastigotes allowing a
rapid and sensitive diagnosis.

Visceral leishmaniasis: spleen biopsy with
intracellular amastigotes.

Blood,bone marrow,spleen parasites (171)

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