Volume 11. Babesia, Trypanosomes & Leishmania
Co- Authors: M. Arcari
1, A. Baxendine 1 and C. E. Bennett2
Diasys Ltd 2.
University of Southampton
10. Babesia species
Babesia species are protozoan
parasites of domestic and wild animals. They
belong to the subclass Piroplamsia and are commonly referred to as
‘piroplasms’ due to the pear-like shaped merozoites which live as small
intra-erythrocytic parasites. They commonly infect mammals, particularly
cattle, sheep, goats, hosres, pigs, dogs and cats and occasionally man.
Species infective to humans
are the cattle form Babesia bovis
which can often be fatal and Babesia
microti which is less pathogenic. Until
recently B. microti was confined to
the United States but is becoming known as an important tick-borne disease of
man in temperate climates.
Human infections have been
found in Europe and North America. The infection is known as Babesiosis, it
can also be described as ‘redwater fever’ or ‘tick fever’ and has a
major impact on the livestock industries in many countries.
What is unusual with this
parasite’s life cycle is the development in the vector. They use the
one-host ticks, belonging to the genus Boophilus.
The parasites are passed to the eggs and hence to the larval stages which can
thus become infective after the adult tick dies. This process is known as
The ticks which are known to
carry the parasite of Babesia bovis
are Boophilus microplus, B. decoloratus
and B. annulatus.
babesiosis is a zoonosis, acquired by tick bite when individuals accidentally
interact with the natural life cycle of the parasite.
cycle is best known for B. canis
which infects dogs, but it is generally the same in each of the one-tick hosts
and the definitive host. (Fig. 1)
tick bites, sporozoites are
injected into the blood stream and penetrate the erythrocytes. In contrast to
the malaria life cycle, there is no tissue stage for Babesia
bovis. Babesia multiplies in the red cell by budding in contrast to
schizogony in Plasmodium species.
The red cell ruptures and daughter parasites invade new erythrocytes
for further asexual multiplication. Some
of the sporozoites injected by the
tick vector follow a different path of intra-erythrocytic development, growing
slowly and "folding" to form accordion-like structures which are
destined to undergo further development in the tick vector.
Within the intestine of the tick, the accordion-like stage eventually
fuses with another, to form a zygote.
Further development outside the intestine occurs in a variety of tissues, the
salivary glands and ovaries being especially important for transmission.
Sporozoites in tick salivary
glands are injected into the mammalian host at the next blood meal. Trans-ovarial
transmission of Babesia bovis also
takes place so that newly hatched onto adult stages can then take place.
1. Generalised life cycle of the parasite Babesia,
which causes the disease babesiosis in man. (Adapted and redrawn from Smyth,
small mammal host of Babesia microti,
sporozoites from the tick vector
first enter lymphocytes and undergo merogony,
the daughter parasites of which then enter erythorcytes. (Fig. 2)
Babesia microti do not undergo trans-ovarial
transmission, but once a larva has become infected from a mammalian host they
are able to pass on the infection trans-stadialy to the nymph.
of Babesia species 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). (www.cdfound.to.it)
Babesia bovis -
Patients who are particularly at risk are those who have had a
splenectomy. The patient may feel
vaguely unwell at first but by the time the diagnosis has been made, is
usually very ill, with fever, prostration, jaundice, anaemia and
haemoglobulinuria. Nausea, vomiting and diarrhoea have also been recorded.
Unlike malaria symptoms, the symptoms of babesiosis do not exhibit
Babesia microti - Most human infections are subclinical. Where
clinical illness develops, the incubation period is 1 to 3 weeks, occasionally
up to 6 weeks. The illness usually begins gradually, with anorexia and
fatigue, plus fever (without periodicity), sweating, rigors and generalised
myalgia. Physical examination may reveal only fever, but may also show mild
splenomegaly and occasionally mild hepatomegaly.
diagnosis depends upon finding parasites on blood film examination which can
be detected 2 to 4 weeks after a tick bite.
Hamster inoculation and serology have also been used for diagnosis.
Babesia bovis are
pear shaped, oval or round and may exist in pyriform pairs. There may be 1 to
8 parasites per red cell. Ring
forms can be confused with malaria parasites, especially Plasmodium
falciparum. However, in
contrast to Plasmodium species, Babesia
do not form pigment, do not cause alterations in red cell morphology and
finally do not exhibit the Maurer's clefts of Plasmodium
falciparum, the Schuffner's dots of Plasmodium
vivax, or the James's dots of Plasmodium
The "Maltese cross form" is unique to Babesia but in its absence it may be very difficult to distinguish
young ring forms of Plasmodium
falciparum, from Babesia. The
absence of pigment cannot be relied upon, as young rings of Plasmodium
do not exhibit pigment. Babesia,
are smaller than malaria parasites, and in some of the larger rings there is
white vacuole containing erythrocyte stroma, instead of the pink vacuole seen
in malaria. Babesia
parasites do not form schizonts.
Ring, rod shaped, pyriform, amoeboid, and "Maltese cross"
forms are seen. In heavy infections different stages may be noted in the same
red cell. Intra-erythrocytic stages measure approximately 2 by 1.5mm. In very high parasitaemias,
extracellular merozoites are found singly or as a syncytial structure. Peak
parasitaemia varies between less than 1% to approximately 10%.
The Indirect Fluorescent Antibody Test (IFAT) is available for both B.
bovis and for B. microti and is the most useful serological test for early
This is not routinely used for diagnosis but B. microti grows well in hamsters and can serve as a confirmatory
Toxoplasma gondii, the causative organism of toxoplasmosis, was first observed in 1927 in the gondi, a North African rodent. The first human case of toxoplasmosis was also reported that year. The organism is a coccidian protozoa belonging to the sub-phylum Apicomplexa and has a world wide distribution occurring in all warm-blooded animals.
are the definitive hosts and they become infected by ingesting oocysts or
cysts in tissues of paratenic hosts, such as mice, or transplacentally. Man
becomes infected either by direct ingestion of oocysts from a cat or by eating
raw or undercooked meat. Those
who handle raw meat are particularly at risk. Infection can be transmitted
The development of the entereoepithelial (sexual)
cycle in a cats intestine is brought about by the ingestion of sporulated
oocysts of a mouse with cysts. The pre-patent period up to the shedding of the
oocysts varies with the stage of T.
gondii ingested, for example only 3 – 10 days if the cat has ingested a
mouse containing cysts, but about 19 – 20 days or longer after direct
infection with oocysts or ingestion of a mouse containing only tachyzoites.
Women most at risk of delivering an infected infant are those who acquire the
infection just prior to gestation. (Fig.
3, 4 & 5)
Humans can acquire infection by;
ingestion of the oocyst shed in the cats faeces
of the tachyzoite in infected milk or transplacentally
Ingestion of the tissue cyst
in undercooked or raw meat.
Transplant of an infected
organ in a seronegative recipient
Figure 3. Life
cycle of Toxoplasma gondii, causes
toxoplamosis in man.
Figure 4. Diagrammatic
illustration of a Toxoplasma gondii
trophozoite in a macrophage of a vertebrate. (Adapted and redrawn from Smyth,
evidence has shown that approximately one third of the world's population has Toxoplasma
antibodies. This suggests that
the majority of infections are benign with most people exhibiting few or no
symptoms, but fever and swelling may be seen.
However Toxoplasma gondii can
cause severe illness in congenital infections, acquired infections and in
immunocompromised patients. This may lead to ocular toxoplasmosis and
ultimately to fatal CNS disorders such as encephalitis.
Figure 5. Toxoplasma gondii
tissue cyst containing 8 – 20 parasites (Giemsa stain) (www.cdfound.to.it)
occurs approximately in 1 per 1000 pregnancies.
It can cause severe damage to and even death of the foetus.
Proliferation of tachyzoites leads to intracellular calcification,
corioretinitis, hydrocephaly, psychomotor disturbances and convulsions.
A small, proportion of babies who are asymptomatic at birth develop
retinocoroiditis or mental retardation as children or young adults. When a mother is first exposed to the parasite in later
pregnancy the infant is likely to be less severely damaged or asymptomatic.
with T. gondii are often mild with
flu-like symptoms thus they often go unnoticed. However lympadenopathy is the most easily recognised symptom
and it can be accompanied by fever, headache and myalgia. Toxoplasmosis may also produce infectious mononucleosis like
symptoms. Ocular toxoplasmosis is
also a common manifest however it is not yet proven whether this is due to
congenital or acquired infections. Other
manifestations of Toxoplasma infections
are meningoencephalitis, hepatitis, pneumonitis and myocarditis.
Toxoplasmosis has been shown to occur as an opportunistic pathogen in immunocompromised patients and can cause severe complications. Toxoplasmosis in immunocompromised patients almost always arises from a reactivation of latent infections. Conditions which can predispose to toxoplasmosis are malignancies, organ transplants, leukaemias and patients with acquired immune deficiency syndrome (AIDS). In immunocompromised patients, the central nervous system is primarily involved with diffuse encephalopathy, meningoencephalitis or cerebral mass lesions. Toxoplasma encephalitis has been reported as a life-threatening among patients with AIDS.
detection of toxoplasma specific antibodies is most commonly used in clinical
laboratories. Specific IgG antibodies typically persist for life whereas
specific IgM antibodies begin to decline after several months.
Most laboratories carry out preliminary tests for IgG antibodies and
more definitive tests including IgM and IgA are carried out in reference
laboratories. The Sabin-Feldman Dye Test is the gold standard for detecting
the presence of specific antibodies. It
measures the total amount of specific antibody in a serum which is capable of
participating in antibody-mediated killing of tachyzoites by complement.
This test involves the use of live tachyzoites which are derived from infected mice or rats.
Because of the use of live organisms, this test is not recommended in
the use of routine laboratories and is thus only employed in reference centres.
Culture of parasites in
animals is the best overall method but it can take up to six weeks before the
result is available and is thus a disadvantage.
Tissue culture is more rapid taking three or four days to obtain a
result, but is not as sensitive.
The direct detection of very small amounts of specific nucleic acid has been made possible by the introduction in 1985 of the polymerase chain reaction (PCR). This technique results in the amplification of a specific fragment of DNA from within the parasite genome which is detected by ethidium bromide staining, following gel electrophoresis. PCR is so sensitive it should detect Toxoplasma DNA in one cyst. However this may indicate a latent infection rather than an active infection. However its sensitivity may create problems since it will detect very small amounts of DNA from latent as well as active infections and it does not differentiate between cyst and tachyzoite DNA. Thus samples like blood, CSF, urine and and amniotic fluid should be used as they do not contain the latent stages. PCR shows great promise but as yet is still labour intensive and expensive for routine use in the laboratory.
Trypanosomes are haemoflagellates and three species
of the genus Trypanosoma are
responsible for disease in humans such as sleeping sickness.
Trypanosomes occur in the blood of the majority of
vertebrate animals. The life cycle involves intermediate host, which usually
is an insect. Many species of tyrpanosomes can live in harmony with their
hosts producing no pathogenic effect, but the best known species are those
that are pathogenic to their definitive hosts. The disease in caused by the
pathogenic types is called, trypanosomiasis.
and Trypanosoma brucei gambiense - The
metacyclic trypanosomes are found in the proboscis of the insect vector -
infection is therefore inoculative. The above are the aetiological agents of
African trypanosomiasis, it is a zoonotic species in the fact that it
multiplies in the blood of a range of animals, domestic and wild animals as
well a man.
brucei rhodesiense causes
an acute form of sleeping sickness in East Africa, while T. b. gambiense causes chronic sleeping sickness in West Africa.
These are known as salivarian trypanosomes as they
complete their development in the salivary system (anterior portion of the
vector). Transmission takes place by innoculation of the metacyclic stage.
cruzi - The metacyclic trypanosomes
occupy a posterior position in the gut of the insect vector and are passed out
in the faeces - infection is therefore contaminative.
This is the aetiological agent of South American trypanosomiasis.
These trypanosomes are known as stercocarian as
they complete their development in the posterior region of the vector, so that
the infective forms appear in the insects faeces. Hosts are infected by the
Transmission from one vertebrate to another is
carried out by blood-sucking invertebrates, usually an insect. The vector for
African Trypanosomes is the Tse tse fly, Glossina,
and the species which cause the disease are T. b. gambiense and T. b.
rhodesiense. (Fig. 6 &7)
Metacyclic (infective) trypomastigotes are
inoculated through the skin when a tsetse fly takes a blood meal.
The parasites develop into long slender trypomastigotes which multiply
at the site of inoculation where ulceration occurs. The trypanosomes continue
to develop and then may invade the lymphatic tissues, the heart, various
organs and in later stages, the central nervous system.
Trypomastigotes are taken up by the tsetse fly (male and female) during
a blood meal. The parasites develop in the midgut of the fly where they
multiply. 2-3 weeks later the trypomastigotes move to the salivary glands
transforming from epimastigotes into metacyclic (infective) trypomastigotes.
The tsetse fly remains infective for life i.e. about 3 months.
Figure 6. Generalised life cycle of the Trypanosoma sp. Which cause African trypnaosomiasis.
The mode of transmission mentioned above,
metacyclic transmission, requires to be separated from mechanical
transmission, a process in which trypanosomes survive, for a short time, on
and about mouth parts of an insect and are inoculated into a new host when the
vector bites again, without undergoing any developmental cycle.
Metacyclic transmission requires a lapse of time to
allow the trypanosomes to reach an infective stage by a particular
developmental sequence in the vector, usually a period of several days.
The parasite is an elongated cell with single
nucleus which usually lies near the centre of the cell. Each cell bears a
single flagellum which appears to arise from a small granule - the kinetoplast.
The kinetoplast is a specialised part of the mitochondria and contains DNA.
The length and position of the trypanosome’s flagellum is variable. In
trypanosomes from the blood of a host the flagellum originates near the
posterior end of the cell and passes forward over the cell surface, its sheath
is expanded and forms a wavy flange called an undulating membrane.
Development is characterised by the occurrence of
three types of blood forms (polymorphic), these are:
Slender forms: long and thin, about 29mm
long, free flagellum.
Stumpy forms: thick and short, average
typically no free flagellum, but a short one may be present.
forms: about 23mm
long with a moderately thick body and a
free flagellum of medium length. (Fig. 8 & 9)
8. Diagrammatic illustration of a typical trypanosome.
(Adapted and redrawn from Smyth, J.D, 1994)
Figure 9. Trypanosoma brucei 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.
The early stages of African trypanosomiasis may be
asymptomatic and there is a low grade parasitiaemia. This period may last for
several weeks to several months. The
disease may terminate untreated at this stage or go on to invade the lymph
glands. Invasion of the lymph
glands is usually accompanied by a high irregular fever with shivering,
sweating and an increased pulse rate. The lymph glands near the bite often
become swollen, in T. b. gambiense
the glands at the back of the neck and T.
b. rhodesiense usually the glands under the jaw are affected (Winterbottoms
sign). As the disease progresses
oedema of the eyelids, face and sleeplessness are features along with
increasing lethargy and listlessness.
Trypanosomes may invade the central nervous system
giving symptoms of meningoencephalitis, confusion, apathy, excessive sleeping
and incontinence. At this stage,
the cerebro-spinal fluid (CSF) usually contains mononuclear cells and a few
trypanosomes may be detected. If
untreated, character changes, mental deterioration and coma develops, finally
resulting in death. Such signs
are more commonly seen with gambiense
than in rhodesiense in which
patients often die before these symptoms develop fully.
diagnosis of African trypanosomiasis
Laboratory diagnosis of African trypanosomiasis is
Examination of blood for the
Examination of aspirates from
enlarged lymph glands for the parasites
Examination of the CSF for the
Detection of trypanosomal
antibodies in the serum
Examination of blood
and thin blood films blood film
Thick and thin blood films are
made and stained with Fields stain and examined as for malaria parasites
This method is carried out as
10 ml of citrated blood is centrifuged at 2000 rpm for 5 minutes to pack the
red blood cells.
plasma and white cell layer are removed by a Pasteur pipette and transferred
to a clean centrifuge tube.
is centrifuged for a short time in order to deposit any red blood cells
supernatant fluid is removed by pipette to a clean tube.
is centrifuged at 5000 rpm for 10 minutes.
supernatant fluid is removed with a pipette and discarded.
deposit is examined microscopically for trypanosomes.
anion-exchange centrifugation technique (ref. Transactions Royal Society of
Tropical Medicine and Hygiene. 1979. 73.
Heparinised blood is passed through an anion exchange column. As the blood travels down the column the red cells are adsorbed while the less strongly charged trypanosomes are washed through with saline. The eluate is centrifuged and examined microscopically for motile trypanosomes.
centrifuged in a microhaematoctit tube for 5 minutes.
Parasites can be seen microscopically at the junction of the packed red
cells and plasma.
Examination of lymph gland aspirates
The aspirate can be examined microscopically by
making a wet preparation, or if there is not much material, it can be allowed
to dry on a slide and then stained with either rapid Field’s stain or with
Giemsa and examined microscopically.
Examination of CSF
In the late stages of African trypanosomiasis,
trypanosomes may be found in the CSF together with IgM - containing morula
(Mott) cells, lymphocytes and other mononuclear cells. Once the CSF has been
collected it must be examined as soon
as possible. The parasites are unable to survive for more than 15-20
minutes in CSF once it has been removed. The parasites become inactive, are
rapidly lysed and will not therefore be detected. The CSF should be examined
wet and spun down in a sterile tube and a film made from the deposit. The film
is then stained with rapid Field’s or Giemsa and examined microscopically.
NB. It is impossible to distinguish between T.
b. gambiense from T. b. rhodesiense
on a stained film as the two subspecies which infect man are identical.
cruzi occurs throughout South and
Central America, especially in Brazil, Argentina and Mexico causing the
disease known as Chagas’ disease. It is estimated that over 24 million
people are infected with this species. It is a zoonotic parasite with over 150
species of wild animals known to harbour the parasites, for example opposums,
dogs, rates, pigs and cats.
It is transmitted to man by brightly coloured bugs
belonging to the Reduviidae family, subfamily Triatominae. All stages of these bugs are known to become infected. (Fig.
The bugs live in the crack of the walls and vegetal
roofs of the poorly maintained houses, coming out at night to feed on the
exposed parts of the hosts body.
Figure 10. Insect vector of Trypanosoma cruzi, belongs to colourful insect Triatominae, also
known as the kissing bug.
Metacyclic trypomastigotes are deposited in faeces
on the skin as the triatomine bug
(reduviid bug) feeds. The bug usually bites round the edges of the
mouth and eyes. The trypomastigotes are either rubbed into the skin by
scratching the irritated area or penetrate the
conjunctiva or membranes of the nose and mouth. Trypomastigotes become
amastigotes in localised reticulo endothelial cells and multiply. The amastigotes develop into trypomastigotes which are
released into the blood when the cell ruptures. No multiplication of the
parasite takes place in the blood in its trypomastigote stage. The
trypomastigotes reach tissue cells especially heart muscle, nerves, skeletal
muscle and smooth muscle of the gastrointestinal system by way of the blood
and lymphatic system. The trypomastigotes become amastigotes and multiply
forming pseudocysts. Within the pseudocyst some amastigotes become elongated and develop first into epimastigotes and then trypomastigotes. When the cell ruptures the trypomastigotes are released into the blood and
11. Generalised life cycle of the South American
trypanosomes, Trypanosoma cruzi,
which is known to case Chagas’ disease.
Continue to circulate whilst others invade further
tissue cells. The life cycle
completes when a triatomine bug vector ingests circulating trypomastigotes. In
the vector the trypomastigotes transform and develop into epimastigotes,
multiply by binary fission in the gut of the bug. After about 10 - 15 days,
metacyclic trypomastigotes are formed and can be found in the hindgut of the
bug. (Fig. 11)
Figure 12. Trypanosoma cruzi causes a disease known as Chagas’ disease, and is caused by the haemoflagellate Trypanosoma cruzi. T.cruzi in blood sample (Giemsa stain) (www.cdfound.to.it)
Figure 13. Multiplication of Trypanosoma
cruzi in man 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. (Giemsa stain) (www.cdfound.to.it)
Many people infected with T. cruzi remain asymptomatic and free from Chagas’ disease or
experience only an acute infection without progressing to the chronic stage.
The most severe form of the disease is most commonly seen in children
younger than 5 years of age. Multiplication
of T. cruzi at the site of infection can produce an inflamed swelling (chagoma)
which persists for weeks. Trypomastigotes or amastigotes may be seen in the
aspirate of the chagoma. Regional lymph nodes may become infected which
frequently involve one side of the face. Unilateral oedema of the upper and
lower eyelid may occur along with conjuctivitis. This is known as Romana’s sign.
In the acute stage of infection trypomastigotes can
be found in the blood. Symptoms may pass unnoticed, but there may be fever,
malaise increased pulse rate and enlargement of lymph glands, liver and
possibly spleen. Muscle aches and
pains are characteristic at this stage and parasites may be seen in blood
films. The acute form is most often seen in young children and occasionally
can cause serious damage to the heart and other complications leading to death
caused by central nervous system involvement.
Chronic manifestations include signs of cardiac
muscle damage with a weak and irregular heartbeat, oedema, heart enlargement
leading to heart failure. Dilation of the digestive tract resulting in
megaoesophagus and megacolon may also occur.
About 10% of persons infected with T.
cruzi develop chronic Chagas cardiopathy.
Examination of blood
a) Thick and thin blood films
are made and stained with Fields stain and examined as for malaria parasites.
Wet preparations of blood can also be examined for motile trypanosomes.
Xenodiagnosis is useful in chronic and sub acute
(low parasitaemia) disease. Sterile
bugs are fed on patients by attaching a black bag containing the bugs to the
arm of the patient and allowing them to feed for 30 minutes. Twenty five to thirty days later the bugs are dissected and
the contents of the hindgut and rectum are examined microscopically for the
presence of trypanosomes.
Blood culture is as sensitive as xenodiagnosis but
it requires sterile conditions.
Serology tests include:
(i) IFAT indirect fluorescence antibody test
CFT complement fixation test
indirect haemaglutination test
enzyme linked immunoabsorbent assay
Other lab findings include:
Raised ESR, marked lymphocytosis with atypical
NB. In certain areas of S. America where Trypanosoma
rangeli (non pathogenic species transmitted by Rhodnius bug) is found with
T. cruzi all positive preparations should be checked to confirm T.
is caused by parasites of the genus Leishmania
and is endemic in many parts of Africa, Asia and South America.
It is transmitted by Phlebotomus species, sandfly.
Leishmania species are mainly parasites of man and other animals,
especially dogs and rodents. They cause diseases collectively known as
Leishmaniasis; causing 3 types of disease i.e. visceral leishmaniasis,
cutaneous leishmaniasis and muco-cutaneous leishmaniasis. These are all
debilitating and disfiguring diseases which occur throughout the Old and New
World. The parasites are unusual in that they live entirely within the cells
of the reticulo-endothelial cells, they have become perfectly adapted as the
proteolytic enzymes which attack other foreign bosies in the blood stream do
not destroy them.
visceral leishmaniasis (VL), sometimes known as Kala-azar, is caused by Leishmania
donovani complex; L. donovani
and L. donovani infantum in the Old World and L. donovani chagasi in the New World. The clinical features -azar caused by these species are
similar, but they have different epidemiological features. The parent species L.
donovani occurs in Asia (Northeastern China, India and Iran) and Africa
(primarily Sudan, Kenya and Ethiopia) and can affect people of all ages.
The parasite (L. d. infantum) which causes VL in countries bordering the
Mediterranean, (Southern Europe as well as North Africa) affects young
children as well as infants. It
is now being seen in the immunocompromised. In the New World also, VL is
mainly a disease of young children, with the causative organism L.
d. chagasi being closely related to, but slightly different from, L.
donovani. The main geographical foci of VL in Latin America are in
northern and northeastern Brazil. Small foci are found in northern Argentina,
Columbia and Venezuela. Sporadic
cases are found in central American countries, including Mexico.
leishmaniasis is caused by L. tropica,
L. major and L. aethiopica in
the Old World and L. mexicana complex
in the New World. Leishmania tropica is widely distributed around the Mediterranean
basin, Afghanistan, Kenya, Kenya, Armenia, Azerbaijan, Turkmenistan and
Uzbekistan. Leishmania aethiopica
is seen in the highlands of Ethiopia and L.
major occurs in the Middle East, West Africa, North Africa and Kenya. Leishmania mexicana
complex is found in Central America and the Amazon Basin.
caused by the L. braziliensis
complex and is found in Brazil, Eastern Peru, Bolivia, Paraguay, Ecuador,
Columbia and Venezuela.
forms of infection starts when a female sandfly (Phlebotomus
species) takes a blood meal from an infected host. Small amounts of blood, lymph and macrophages infected with Leishmania
amastigotes are ingested. Once ingested the amastigotes transform to promastigotes
in the sandfly, the non-infective promastigotes
divide and develop into infective metacyclic
promastigotes. These are formed in the midgut of the sandfly and migrate
to the proboscis. When the
sandfly bites the extracellular inoculated promastigotes at the site of the
bite is phagocytosed by macrophages. After
phagocytosis, transformation to dividing amastigotes
occurs within 24 hours. Reproduction
at all stages of the lifecycle is believed to occur by binary fission.
No sexual stage has been identified. (Fig.
14. Generalised life cycle of Leishmania sp., which by using a sandfly as a vector can cause
Leishmaniasis in man,
exist as flagellated extracellular promastigotes
in the Sandfly vector and as a flagellar obligate intracellular amastigotes
within mononuclear phagocytes of their vertebrate hosts. The various species
are not distinguishable morphologically from one another. When stained with Romanowsky stains such as Giemsa, amastigotes
appear as round or oval bodies ranging from 2 - 3mm in diameter with a well defined nucleus and kinetoplast, a rod shaped
specialised mitochondrial structure that contains extranuclear DNA. The
flagellated promastigote form is
spindle shaped, measuring 10 - 20mm in length, not including the length of the flagellum.
As in the amastigote form a nucleus and kinetoplast are clearly
15 & 16)
Figure 15. Amastigote of Leishmaniasp. in a macrophage of a vertebrate host. (Adapted and redrawn form Smyth, J.D, 1994)
16. Leishmania promastigote. This stage of the parasite are
seen in the vector, sandfly.
incubation period of VL may vary between 2 weeks and 18 months. The onset of VL is usually insidious with fever, sweating,
weakness and weight loss. The
most prominent findings are fever, hepatosplenomegaly and anaemia.
The sites mainly affected are the liver, spleen and bone marrow.
Enlargement of the liver is due to hyperplasia of Kupffer cells which
are packed with amastigotes. The
bone marrow is infiltrated with parasitised macrophages.
Some organs, notably the kidneys, may show pathological changes
secondary to deposition of immune complexes. In advanced cases, ascites and
oedema can develop. Deaths are
usually due to secondary bacterial infections such as pneumonia, tuberculosis
Parasites may be found in a splenic aspirate, liver biopsy or bone
marrow biopsy. These techniques,
especially splenic aspirate and liver biopsy, can be hazardous and require
previous expertise in the procedure.
in methanol for 1 minute
with Giemsa 1 in 10 in buffered distilled water pH 6.8 for 30 minutes (or use
the rapid Field’s stain)
the slide in buffered water and drain dry
of leishmania should be seen in positive smears. They are approximately 2-4µm
in size, oval and are frequently seen within the cytoplasm of the macrophage.
The amastigotes possess a nucleus and a rod - shaped kinetoplast within the
cytoplasm. In many samples a very small number of parasites are present.
Extensive searching of the film is necessary.
The aspirates can be cultured in Novy-Nicolle-MacNeal (NNN) or
Schneider's Drosophila medium. In
culture the amastigote stage
converts to the promastigote stage.
However, this is not a rapid technique, as the parasites may take
anything from 10 - 21 days to grow.
VL produces large amounts of specific IgG which can be used for
diagnosis. Currently the most
used sero diagnostic tests are Indirect-immuno Fluorescent Antibody Test (IFAT),
Enzyme Linked Immunosorbent Assay (ELISA) and Direct Agglutination Test (DAT).
Clinical Disease – Cutaneous Leishmaniasis
a bite from an infected sandfly, a small red papule appears at the site of the
bite about 2 – 8 weeks later. The
papule increases in size centrifugally. The
patient then mounts either a hypersensitive response or an anergic response.
In a hypersensitive response, the papule eventually ulcerates, becomes
depressed and then eventually heals through scarring.
The patient is now immune from subsequent bites.
In an anergic response, the nodule grows and spreads over large areas
of skin. This resembles leprosy.
The margin of the lesion contains amastigotes whereas the centre
contains debris and dead skin material. This
the margin of the lesion is aseptically punctured with a hypodermic needle and syringe containing a
small amount of saline. The
aspirate which is drawn up into the needle is examined microscopically and/or
cultured using the method described in visceral leishmaniasis.
Gene amplification techniques are powerful and sensitive methods and are
useful in diagnosis of cutaneous leishmaniasis particularly when organisms
cannot be detected microscopically. It
is also very useful for the speciation of Leishmania
parasites thus the correct treatment can be administered.
Murray, PR, Drew, WL, Koyayashi, GS & Thomson,
JH: Medical Microbiology. Mosby Books
Inc., New York (1990)
Peters, W & Gilles, HM: Tropical Medicine & Parasitology. Wolfe Medical Publications
Jeffrey & Leach: Atlas
of Medical Helminthology and Protozoology. E & S Livingstone Ltd.
Ash, LR & Orihel, TC: Atlas of Human Parasitology. ASCP Press, Chicago.
Garcia, LS & Bruckner, DA: Diagnostic Medical Parasitology. Elsevior Science Publishing Co.
Muller, R & Baker, JR: Medical Parasitology. Gower Medical Publishing.
Smyth, J.D: Introduction
to Animal Parasitology. Cambridge University Press (1994)
Snell, JJS, Farrell, ID & Roberts, C: Quality
Control, Principles and Practice in the Microbiology Laboratory. Public
Health Laboratory Service. ISBN 0 901 144 312.