PROF. DR.

Abdalwahab Bdewi Hussain

ENTAMOEBA COLI
•It is a worldwide parasite. It lives freely in the
lumen of large intestine of man and is
nonpathogenic.
•Like E. histolytica, it exists in three stages —
trophozoite, precyst and cyst.
ENTAMOEBA POIECKI
• E. polecki is a common parasite of the caecum and
colon of pigs and has also been found as human
parasite.
• The trophozoite resembles that of E. coli and measures
12—18 µm in diameter.
• The cyst normally has only a single nucleus even when
mature.
• The nuclear endosome is quite large; glycogen granules
and chromidial bodies may or may not be present.
ENTAMOEBA GINGIVALIS
• E. gingivalis is the first parasitic amoeba to be
recognized.
• It was described by Gros in 1849 in the soft tartar
between the teeth.
• It is unusual among the Entamoeba in two
respects — first, it inhabits the mouth rather
than the large bowel; secondly, no cyst of
gingivalis has ever been found.
ENTEROMONAS HOMINIS
• Enteromonas hominis is a rare species living as a harmless
commensal in the lumen of the caecum and other parts of the
large intestine of humans and primates.
• It was first discovered by de Fonseca (1915) in diarrhoeic stool
in Brazil. It has been reported from both warm and temperate
climates.
• It exists in two forms — trophozoite and cyst.
• Trophozoite is pear-shaped, rounded or ovoidal measuring
4—10 µm in length and 3—6 µm in width.
• It possesses four flagella. By means of its three anterior
flagella it has jerky forward movement.
The fourth flagellum is adherent to the body of the trophozoite and then extends posteriorly as free flagellum.

The cytoplasm is finely vacuolated and contains numerous bacteria. The nucleus is situated at the anterior end.
There is no cytostome.

Cyst is oval, 6—8 µm in length and 4—6 µm in

breadth and contains one to four nuclei. E. hominis is
a harmless commensal. It does not produce any
symptoms. It is transmitted by ingestion of food and
water contaminated with cysts.

Diagnosis is made by demonstration of trophozoites

and cysts in fresh faecal films and haematoxylin-
stained preparations.
 RETORTAMONAS INTESTINALIS

• Retortamonas intestinalis, also known as Embadomonas intestinalis is a

rare harmless commensal in the caecum of man.
• Wenyon and O'Connor first observed this parasite in the stool in Egypt. It
has been reported from both warm and temperate areas of the world. It
exists in two forms — trophozoite and cyst.
• Trophozoite is oval, small, measuring 6—9 µm in length and 34 urn in
breadth.
• The cytoplasm is finely granular and vacuolated.
• The spherical nucleus with central karyosome is situated anteriorly and by
its side lies the cytostome.
• It has two flagella which originate from two minute blepharoplasts
present near the nucleus
One of the flagella is longer and directed anteriorly and the other is slightly shorter,
passes posteriorly through the cytostome before becoming free. The trophozoites
multiply by longitudinal binary fission.

Cyst is pyriform, measures 4—7 µm in length and 3—4 µm in breadth. Like trophozoite,
it also has single nucleus. The infection is acquired by ingestion of food or water
contaminated with cysts of R. intestinalis in the stools ofan infected individual. It is
nonpathogenic, although it is commonly discovered only in diarrhoeic stools.

Diagnosis is made by demonstration of trophozoites and cysts in fresh faecal films and
haematoxylin- stained preparations.
DIENTAMOEBA FRAGILIS
• Dientamoeba fragilis was initially thought to be an amoeba.
• It is now classified as a flagellate even though flagella are not observed with
light microscope.
• In practice, this organism may appear as an amoebic trophozoite, rather than
as flagellate in microscopic mounts.
• The generic name is derived from the binucleate nature of the trophozoite and
species name from the fragmented appearance of its nuclear chromatin.
• Wenyon (1909) first discovered this parasite. Jepps and Dobell (1918)
described it.
• It is a cosmopolitan parasite. It lives in the lumen of human colon along with
the true amoebae.
• Only the trophozoite stage is known. The cyst stage has not been confirmed to
date. It is small, varying from 6—12 µm in diameter. It is
Trophozoite measures 10—25 11m in diameter
and is actively motile by multiple pseudopodia.
The cytoplasln is differentiated into clear ectoplasm and granular endoplasm in which digested
leucocytes and epithelial cells are found within food vacuoles.

At time bacteria and rarely red blood cells are also seen.

Nucleus is spherical, 2-4 µm in diameter.

It has a central karyosome and nuclear Inembrane is lined with closely packed chromatin granules.
Since morphologically E. gingivalis is very similar to the trophozoite of E. histolytica,

it is important to make the correct identification from a sputum specimen, that is, E. gingivalis,
which is considered to be a nonpathogen rather than E. histolytica from a pulmonary abscess.
E. gingivalis occurs as a commensal in the gingival tissue around the teeth,
particularly if there is suppuration, as in pyorrhoea alveolaris, but it also occurs
in apparently hygienic mouth and on dental plates if they are not kept clean.

Occasionally, this amoeba has been identified from the crypts and in histologic
sections of diseased tonsils and in vaginal and cervical smears from women
using intrauterine devices.

It is transmitted for person-to-person by close contact like kissing or from

contaminated drinking utensils.

Diagnosis can be established by demonstration of trophozoites of E. gingivalis

in the material removed from gingival margin of the gums, from between teeth
or from denture.
ENDOLIMAX NANA

• It is cosmopolitan, small (nana, small) amoeba found in the

lumen of the large intestine of humans, primates and pigs.
• It has all the stages described for E. histolytica and E. coli,
i.e., trophozoite, precyst and cyst.
• Trophozoites are small, measuring 6- 15 µm (average 10
µm ) in diameter.
• The cytoplasm has an inner finely granular endoplasm and
a clear outer ectoplasm. It exhibits sluggish motility by
means of short, blunt, hyaline pseudopodia. Cytoplasmic
inclusions contain bacteria, small vegetable cells, and
crystals but never red blood cells.
Nucleus is minute spherical with a large irregular karyosome lying eccentrically from
which several achromatic strands extend to the nuclear membrane. There is normally no
peripheral chromatin on the nuclear membrane.

Cyst is oval measuring 8—10 µm in diameter.

The number of nuclei varies from 1—4 but mature cyst is quadrinucleate. Chromidial bars
and glycogen vacuole are absent.

E. nana is transmitted from man-to-man by ingestion of viable cysts in polluted water or

food. It is normally regarded as nonpathogenic although there are isolated reports of
gastrointestinal symptoms in AIDS patients for which no other cause could be found.
IODAMOEBA BÜTSCHLII
I. biitschlii is worldwide in its distribution. It lives as harmless commensal in the
lumen of large intestine Of man, monkeys and pigs.

Trophozoites vary in size from 6-20 µm in diameter and are fairly active in freshly
evacuated unformed stools and show sluggish movement in older stools.

The clear ectoplasm is not usually well differentiated from denser endoplasm that
contains coarse and fine granules and has bacteria and east cells in food vacuoles.

Occasionally, a discrete glycogen vacuole which stains golden brown with iodine.
Nucleus is relatively large measuring 2.0—3.5 µm In diameter.

The karyosome is a large circular mass, central in position and surrounded by refractile
globules.
Peripheral chromatin lining is absent.

Cysts of I. bütschlii are ovoid or irregularly pyriform in shape, uninucleate and measure 8—15
µm in longest diameter.
The cytoplasm contains a large glycogen vacuole. Chromidial bars are absent.

The natural habitat of I. bütschlii is the lumen of large intestine.

Here the trophozoite feeds on enteric bacteria.

It is transmitted from man-to-man when the viable cysts are ingested in polluted water or food.
It is nonpathogenic luminal parasite of large intestine.
OPPORTUNISTIC AMOEBAE
• Free-living amoebae of the genera Naegleria, Acanthamoeba and
Balamuthia are facultative parasites of man.
• They are ubiquitous in nature, found commonly in soil and water
(swimming pools, tap water, and heating and air-conditioning units) where
they feed on bacteria, but as opportunists, they may produce serious
infection of the central nervous system and the eye. Therefore, they are
known as opportunistic amoebae.
• These amoebae may be natural hosts for Legionella as well as for other
bacteria such as Listeria monocytogenes, Vibrio cholerae, Mycobacterium
leprae, Burkholderia cepacia, and Pseudomonas aeruginosa. Because of
their well known resistance to chlorine, the amoebic cysts are considered
to be vectors for these intracellular bacteria
 This can have tremendous significance for any hospital where
the water source is contaminated with free-living amoebae.

These amoeba have also been found to be acceptable hosts for

Chlamydia pneumoniae, echoviruses and polioviruses.

Free living amoebae belong to phylum Sarcomastigote sub

phylum Sarcodina super class rizopoda class lobosea and order
amoeba and schizopyridia.

The nulclei of oppurtonistic amoebae posses a latge nucleolus or

karyosome surrounded by a halo and nuclear membrane without
chromatin granules these features help to differentiate these
amoeba from E. histolytica.
Morphology:
• N. fowleri ahs two stages motile trophozoite and non motile cysts
• Trophozoite occur in two forms amoebid and flagellate. Amoebid form is elongate
• It is actively motile by means of eruptive blunt pseudopodia called lopopodia
• Measures 15 -30 µm average 22 µm in length.
• It has distinctive phagocytic structures known as amoebostoma used for engulfment. In
tissue trophozoite ingest erythrocytes leucocytes and cause distinction of tissue.
• The nucleus is small 3 µm in diameter and has a large central karyosome surrounded by
halo and no peripheral nuclear chromatin reproduction in Nagelaria is by simple binary
fission of the trophozoite.
• Cysts of N. fowleri are uninucleate, spherical, 7-15 µm in diameter and are surrounded by
a relatively thin cyst wall, a feature that makes Nagelaria cysts susceptible to desiccation,
Under electron microscopy they travel 1—2 mucoid plugged pores or ostioles,
• Amoebae encyst sorn conditions are appropriate and later, excyst In a favourable
environments Cysts and flagellate forms of N fowleri have never been found in or CSE.
Lite cycle and pathogenicity
• The amoeboid form of N fowleri is the Invasive stage of the parasite
Man acquires infection by nasal on during swimming In freshwater
lars pones or swimming pools containing infective forms Infection
may also be acquired by inhalation of dust containing forms.
• It is likely that flagellate forms or cysts of N. fowleri could enter the
nose However since the amoeboid forms is the invasive stage of the
parasite
• therefore, it appears that flagellate forms and the amoeboid forms
and the amoeboid forms escape from the cysts in the nose.
• The amoeboid forms invade the nasal mucosa, cribriform plate and
travel along the olfactory nerves, to brain.
They invade olfactory bulbs and then spread to the more posterior regions of the brain
leading to a rapidly fatal infection known as primary amoebic meningoencephalitis (PAM).

It occurs in healthy children and young adults with a recent history of swimming in
freshwater.

Patient develops severe frontal headache, fever (390—400C), anorexia, nausea, vomiting
and signs of meningeal irritation, frequently evidenced by a positive Kernig's sign.

Involvement of the olfactory lobes may lead to disturbances in smell or taste.

Patient may also develop visual disturbances, confusion, irritability, seizures and coma.

The disease usually results in death within 72 hours of the onset of symptoms.
The period between contact with the organism and onset of clinical symptoms vary from 2—3 days
to as long as 7—15 days.

PAM may resemble acute purulent bacterial meningitis, and these conditions may be difficult to
differentiate particularly in the early stage.

Clinical patient histories indicate exposure to the organism via fresh water lakes or swimming pools
shortly before onset; patients had been previously healthy with no specific underlying problems.

Pathogenic Naegleria organisms have also been isolated from nasal passages Of individuals with
no history of water exposure, thus suggesting the possibility of airborne exposure.
Laboratory diagnosis
• The diagnosis of PAM can be made by microscopic identification of living or
stained amoebae in CSF.
• Motile amoebae with characteristic morphology can be readily demonstrated
in simple wet-mount preparation of fresh CSF specimen. Refrigeration of CSF is
not recommended because this may kill the amoebae.
• When centrifuging the CSFs low speed (150g for 5 minutes) should be used so
that the trophozoites are not damaged.
• CSF smear may be stained with Wright or Giemsa stains. With these stains
amoebae have considerable amount of sky-blue cytoplasm and relatively
small, delicate pink nuclei.
• These can be differentiated from mononuclear leucocytes which have a small
amount of sky-blue cytoplasm and large purplish nuclei.
In centrifuged deposit of CSF, the amoebae tend to be rounded and flattened without
pseudopodia.

Amoebae can also be demonstrated by fluorescent antibody staining of the CSF and in the
histologic sections of the brain biopsied tissue by immunofluorescence and
immunoperoxidase methods.
The bacterial stain like Gram staining is of little value because heat fixing destroys the
amoebae and causes them to stain poorly and appear as degenerating cells.

As in case of fulminating bacterial meningitis, the CSF is purulent or sanguinopurulent with

leucocyte counts (predominantly neutrophils) varying from a few hundreds to cells/gl.

CSF protein content is generally increased and glucose level is low. No bacterium is
detected by Gram staining or culture of CSF.
N. fopeleri may be cultivated by placing some of the CSF on non-nutrient agar (l .5%) spread
with a lawn of washed Escherichia coli or Entembacter aerogenes and incubated at 37 C.

The amoebae will grow on the moist agar surface and will use the bacteria as food, producing
plaques as they clear the bacteria.

As colonies grow and expand, cysts that survive moderate desiccation are formed; thus, stmins
can be maintained by transfer of either trophic or cystic forms.

Treatment

•At present there is no satisfactory treatment for PAM.

•Antibacterial antibiotics and antiamoebic drugs are ineffective. Amphotericin B,
a drug of considerable toxicity, is the antinaeglerial agent for which there is
evidence of clinical effectiveness. In experimental infections, tetracycline acts
synergistically with amphotericin B to protect mice.
ACANTHAMOEBA SPECIES
• Although only one species of Naegleria (N.fovvleri) is known to cause
disease in man, several Acanthamoeba species including A.
culbertsoni, A. castellanii, A. polyphaga, A. astronyxis, A. healyi.

Morphology
• Acanthatnoeba exists as active trophoyoites and resistant cysts.
• There is no flagellate form. of Acanthamoeba are larger than those of
Naegleria and meaqlre 24 56 in length.
• They have irregular appearance With spike- like pseudopodia known
as acanthopodia; hence the name Acanthcvnocba (acanth, spine or
thorn).
It has a single nucleus with a large dense central prominent nucleolus
sutTounded by a halo.

Cysts of Acanthamoeba are double-walled and, therefore. quitc resistant in

the environment.

These are spherical. about 20 ltm or more in diameter. The cyst wall is
made up of an outcr wrinkled ectocyst and an inner endocyst.

Acanthamoeba differes from Naegleria in not having a flagellate stage and in

forming cysts in tissues, Trophozoite of Acanthamoeba move slowly while
those of Naegleria arc actively motile.
Life cycle and pathogenicity
• Man acquires in infection by inhalation of aerosol or dust containing
trophozoite and cysts.
• The trophozoites reach the lower respiratory tract and from there
they invade central nervous system through the blood stream.
• The infection may also be acquired by direct invasion through broken
or ulcerated skin or eye.
• Although Acanthamoeba is able to invade the nasal mucosa and
cause fatal CNS disease in experimental animals, this is not thought
to be the usual route of Invasion in human Infection.
• In contrast to Nagleria cysts of Acanthamoeba are also formed in
tissues.
Acanthamoeba causes granulomatous amoebic encephalitis (GAE).

It occurs in persons who are debilitated or immunosuppressed

including patients with AIDS and those undergoing immunosuppressive
therapy.

The disease is usually of gradual onset and takes a prolonged chronic

course lasting weeks, months or years, and is characterized by focal
granulomatous lesions. This is in sharp contrast to Naegleria infection.
 In healthy persons, Acanthamoeba may cause keratitis.

Infection occurs by direct contact through minor corneal trauma by exposure to

contaminated water or contact lenses.

Amoebae attach to the contact lenses stored in contaminated solutions and are
transferred to the eye when lenses are placed over the cornea. Amoebae become
established as part of the corneal flora and may invade the corneal stroma
through a break in the epithelium or through the intact epithelium.

They produce an infection that progresses to Acanthamoeba keratitis, which

usually develops over a period of weeks to months.
It causes severe ocular pain, affected vision and a stromal infiltrate that is
frequently ring-shaped and composed predominantly of neutrophils.

If not properly managed it may lead to loss of vision.

Laboratory diagnosis
•Diagnosis of GAE is made by demonstration of trophozoites of Acanthamoeba
in CSF or trophozoites and cysts in brain tissue.
•Diagnosis of Acanthamoeba keratitis may be made by identifying trophozoites
and cysts in corneal scrapings.
•Wet mount preparation of the corneal scraping shows motile trophozoites.
•Trophozoites and cysts can be demonstrated in histopathological preparations
of corneal tissue stained with haematoxylin and eosin, Giemsa, Heidenhain's
haematoxylin, Gomori's chromium haematoxylin, periodic acid-Schiff, Bauer
chromic acid-Schiff, silver methenamine and indirect fluorescent antibody
technique (IFAT).
Rapid diagnosis of Acanthamoeba keratitis may be made by identifying
amoebae or cysts In corneal scraping using procedures for Giemsa
staining, calcofluor white staining and IFAT As in case of N. fowleri,
Acanthamoeba may be cultured on non-nutrient agar spread with
washed E. coli or E. aerogenes and incubated at 30 ⁰ C instead Of 37⁰C.

Acanthamoeba does not have a flagellate stage but its trophozoites are
identified by small spiky acanthopodia and cysts are readily identified
by their double walled wrinkled appearance.
Species identification may be made by IFAT.
Treatment

• There is no satisfactory treatment for GAE.

• Total excision of the mass and treatment with ketoconazole,
penicillin and chloramphenicol has been claimed to be useful.
• Acanthamoeba keratitis may be managed by use of
combination of dibromopropamidine and propamidine
isethionate ointment or drops and ketoconazole; topical
miconazole and neosporin with epithelial debridement;
topical clotrimazole; oral Itraconazole with topical miconazole
and surgical debridement; and topical polyhexamethylene
biguanide have also been claimed to be successful for the
treatment of Acanthamoeba keratitis.
BALAMUTHIA MANDRILLARIS
B. mandrillaris is a newly described amoeba. Like Acanthamoeba spp. and unlike N. fowleri, it
does not have a flagellate stage.

Trophozoites of B. mandrillaris are irregular or branching in shape.

Their length ranges from 12—60 µm .

They are sluggishly motile. In tissue culture, broad pseudopodia are usually seen; however, as
the monolayer cells are destroyed, the trophozoites develop fingerlike pseudopodia.

Cysts are spherical 6—30 µm in diameter with two walls.

The inner wall is thin and spherical and the outer wall is thick and wrinkled much like the cysts
of Acanthamoeba. Both trophozoites and cysts of B. mandrillaris are found in tissue.
 Flagellates
The flagellates
A central
belong to the
supporting rod,
Flagellates are phylum
known as
protozoa that These arise Sarcomastigop
These are axostyle, and
bear one to from The nuclear hora,
known as an undulating
several long, bleplvaroplasts characters are subphylum
flagella membrane
delicate, and are distinctive in Mastigophora,
(singular, supported at
thread-like organelles of every species. class
flagellum). the base by a
extensions of locomotion. Kinetoplastide
basal fibre are
the cytoplasm. a and order
observed in
Trypanosomati
some species.
dae .
According to their habitat, the flagellates
are classified into two broad groups:
• (i) intestinal, oral and genital flagellates; and
• (ii) blood and tissue flagellates.
INTESTINAL, ORAL AND GENITAL

FLAGELLATES

GIARDIA LAMBLIA
• Giardia was discovered by Leeuwenhoek in 1681 in his own stool but was not
described until 1859 by Lambl. The organism was named after Professor A. Giard of
Paris and Professor F. Lambl of Prague.

Geographical distribution
• Giardia lamblia is a cosmopolitan parasite. The highest prevalence of G lamblia occurs
in tropics and subtropics where sanitation is poor.
• Giardia infects 200 million people worldwide and may produce symptoms in 500,000
individuals every year. Infections seem to be more common in children than adults.
Habitate:
• It inhabits duodenum and the upper part of jejunum of man.

Morphology

It exists in two forms:

• • Trophozoite
• Cyst
Trophozoite
• It is pear-shaped with rounded antemor and pointed posterior end (Figs. 4.1 and 4.2).
It measures 10-20 µm in length and 5—15 µm in width. The dorsal surface is convex
while on the ventral surface it has a shallow posteriorly notched concavity (sucking
disc) that embraces anterior half of the organism. It acts as an organelle of
attachment.
It is bilaterally symmetrical and has one pair of nuclei, one on each side of the midline,
one pair of axostyles, one pair of parabasal bodies present on the axostyles, four pairs of
flagella and probably four pairs of blepharoplasts from which the flagella arise.

Two pairs of blepharoplasts (one lateral and one more median) are situated on each side
of the midline, between and slightly anterior to the two nuclei.

Two axonemes (also known as axostyles) arise from the median pair of blepharoplasts.
These axonemes pass out from the posterior end of the body and give rise to posterior
(caudal) pair of flagella.

From the two lateral blepharoplasts there originate two axonemes that proceed forward
in a curved course towards the midline, cross each other, then describe a wide arc and
give rise to lateral pair of crossed flagella
• Third pair of blepharoplasts lies near the centre of the sucking disc. They give rise
to short axonemes and central pair of flagella.

• Fourth pair of blepharoplasts has not been located, but the axonemes that arise
from them have been traced up to the notch of the sucking disc.

This pair of blepharoplasts gives rise to lateral pair of uncrossed flagella.

The trophozoites are motile by rapid movement of the flagella. The nuclei are
rounded and possess a central karyosome.

The nuclear membrane is delicate and is not lined by chromatin material.

By rapid movement Of the flagella, the trophozoites move from place to place, and
by applying their sucking discs to epithelial surfaces they become firmly attached.
Cyst
• Mature cyst is oval in shape and measures 11—14 µm x 7—10
µm in size.
• It has two pairs of nuclei which may remain clustered at one end
or lie in pairs at Flagellates opposite ends.
• The remains of the flagella and margins of the sucking disc may
be seen inside the cytoplasm of the cyst.

Culture
• G. lamblia can be grown axenically in Diamond's medium, the
medium also used for axenic cultivation of E. histolytica.
Life cycle
• It passes its life cycle in a single host, the man-
• No intermediate host is required.
• Mature cyst is the infective form of the parasite.
• Man acquires infection by ingestion of cysts in
contaminated water and food. Infection may occur
through ingestion of as few as 10 cysts.
• Infection may also be acquired by anal-oral
• sexual practices among male homosexuals. Within 30
minutes of ingestion excystation occurs in the duodenum.
The cyst hatches out two trophozoites, which then multiply to form enormous
numbers and colonize in the duodenum and upper part of jejunum.
To avoid acidity of duodenum, it may localize in biliary tract.

By means of the concavity on its ventral surface the trophozoite attaches to the
mucosal surface of the duodenum and jejunum.
In frankly diarrhoeic stools. it is usual to find only the trophozoites.

Encystation occurs commonly in transit down the colon where the intestinal
contents lose moisture and patient starts passing formed stools.
The trophozoites retract the flagella into the axonemes, the cytoplasm becomes
condensed and a thin tough hyaline cyst wall is secreted.
As the cyst matures, the internal structures are doubled, so that when
excystation occurs, the cytoplasm divides, thus producing two trophozoites.
Antigenic variation
• G lamblia is known to undergo surface antigenic variation.
• The antigens involved belong to a group of variant-
specific surface proteins that are unique cysteine-rich zinc
finger proteins.
• This may provide a mechanism enabling the organism to
escape the host's immune response.
Pathogenicity
• The presence of G lamblia in the glandular crypts of duodenal-jejunal mucosa may
not cause any pathology.
• These flagellates do not invade the tissues, but feed on mucous secretions.
• With the help of sucking disc the parasite attaches itself to the surface of the
epithelial cells in the duodenum and jejunum and in an appreciable number of
cases it may cause duodenal and jejunal itTitation leading to duodenitis and
jejunitis.
• Patient may complain ofdull epigastric pain, flatulence and chronic diarrhoea of
steatorrhoea type.
• The stool is voluminous, foul smelling and contains large amount of mucus and fat
but no blood.
• This is due to malabsorption since the parasites are coated on the mucosa, thus
absorption suffers.
• Patient loses weight. When the parasite localizes in the biliary tract, it may lead to
chronic cholecystitis and jaundice.
Giardiasis is one of the more common causes of traveller's diarrhoea.

Visitors to areas endemic for Giardia are more likely to present with symptoms than individuals
who live in the area.

This difference is probably due to the development of immunity from prior, and possibly continued,
exposure to the organism.

Various conditions that have been associated with giardiasis in the compromised patient include
hypogammaglobulinaemia, protein or caloric malnutrition, histocompatibility antigen HLA YB- 12,
gastric achlorhydria, blood group A, and reduced secretory IgA levels in the gut.

Although patients with HIV infection have also been found to have giardiasis, the infection does not
appear to be more severe among this group, regard-
Immune response

• Studies with humans and experimental animals have confirmed the presence of
both humoral and cellular immune responses to Giardia.
• The majority of infected patients produce detectable levels of Giardia-specific
antibodies; however, the biological role that these antibodies play in the host
immune response to the infection is unclear.
• IgM anti-Giardia antibodies are short-lived and IgG antibodies may remain at high
titres for many months after the patient has been treated and cured.
• Giardia-specific IgA may also be important in both defence against and clearance of
the parasite. degree of protection of breast-fed infants against
• Giardia infection has also been shown to depend the level of IgA-specific antibodies
in breas't milk. In the intestine, IgA antibodies may influence the local immune
response by inhibiting parasite adherence Experimental animal studies provide
evidence that T lymphocytes and Peyer's patch helper T lymphocyte play a role in
the host immune response.
• In athyrnic mice, which are deficient in both T lymphocytes and Peyer's patch
helper T lymphocytes, inoculation with G muris results in a chronic infection with
large number oforganisms. In contrast, immunocompetent mice clear the parasite
and develop resistance to reinfection.
 • Giardiasis can be diagnosed by identification of cysts of G lamblia
in the formed stools and the trophozoites of the parasite in
diarrhoeal stools by normal saline and iodine preparation, and
Laboratory diagnosis

iron haematoxylin stain as in case of E. histolytica.

• Because the parasites are attached firmly to the mucosa by
means of sucking disc, a series of even five or six consecutive
stool specimens may not show any parasite.
• These parasites also tend to be passed in the stool on a cyclic
basis. Trophozoites of G. lamblia may be detected in the bile
aspirated from duodenum by intubation and by duodenal capsule
technique (Enterotest).
• Motility of the trophozoites on wet preparations may be difficult
to see because the organisms are caught up in mucus. It may be
represented by nothing more than a slight flutter of flagella.
For the detection of G lamblia in faecal specimens. a fluorescent method using monoclonal
antibodies is extremely sensitive and specific.

ELISA test has been developed for the detection of Giardia antigen in faeces.

After multiple stool examinations, examinal ion of bile aspirated from duodenum and Enterotest ire
negative, biopsy from multiple duodenal and jejunal sites may confirm the diagnosis of giardiasis.

To the preparations can be air dried, fixed in methanol, stained with Giemsa stain. Trophozoites may
be s attached to the microvillous border within the cryps.

They appear purple and epithelial cells appear pink Routine histological procedures should also
performed, but trophozoites are very difficult to and may be present in very few of the sections.

Anti-Giardia antibodies, in the patient serum, may detected by ELISA and indirect fluorescent
antibody tests.

However. these may indicate present or past infection. Axenically cultured G lamblia trophozoites
are used as antigens in these tests.
Treatment
• Treatment of giardiasis is carried out with metronidazole. tinidazole and furazolidone.
Metronidazole very effective but has potential carcinogenicity in rats and produces mutagenic
changes in bacteria. It is recommended for pregnant women. Tinidazole has proven more
effective than metronidazole as a single dose. Furazolidone is often used for treating children.
Prevention
• • improved water supply,
• • proper disposal of human faeces,
• • improved personal hygiene,
• • routine hand-washing,
• • proper storage of food and water,
• • control of insects which may come in contact With infected stools and then contaminate
food or water, and
• • treatment of symptomatic and asymptomati individuals. Prospects are poor for the
development of a potential vaccine.
TRICHOMONAS
Genus Trichomonas contains three species which occur in humans:

• T. tenax
• T. hominis
• T. vaginalis.

These flagellates exist only in trophozoite stage.

Cystic stage is absent.

They have four anterior flagella and one lateral flagellum which is attached to the surface of the parasite to form
undulating membrane.

The undulating membrane is supported at the base by a rod-like structure known as costm The axostyle runs down
the middle of the body and ends in the pointed tail-like extremity.

A round nucleus is located in the anterior portion.

 TRICHOMONAS TENAX

• It is a pyriform flagellate. It measures 5—12 pm in length and 5—10 µm in

width.
• It is a harmless commensal of the human mouth, living in the tartar around the
teeth, in cavities of carious teeth, in necrotic mucosal cells in the gingival
margins of gums and in pus pockets in tonsillar follicles.
• It is transmitted by kissing, salivary droplets and fomites.
• Although T. tenax is considered to be harmless commensal in the mouth. there
are reports of respiratory infections and thoracic abscesses.
• The majority of these cases have been reported from Western Europe. Diagnosis
can be made by demonstration of T tenax in the tartar by microscopy, and no
therapy is indicated. Better oral hygiene will rapidly eliminate the infection.
TRICHOMONAS HOMINIS
• It is pyriform, measuring 5—14 µm in length and 7—10 µm in width.
• It inhabits the caecum of man in humans and several other primate species and feeds on
enteric bacteria.
• It does not invade the intestinal mucosa.
• Though it has occasionally been found in the diarrhoeic stools, its pathogenicity is yet to
be established.
• In freshly passed specimens, particularly in unformed stools, the motility may be visible.
• In wet preparation, look for the flagellar movement from the undulating membrane and
the presence of the axostyle.
• The undulating membrane extends the entire length of the body, in contrast to that seen
in T vaginalis, which extends along only half the body.
• Since there is n known cyst stage, transmission probably occurs in the trophic form.
• If ingested in a protected substance such as milk, these organisms can apparently survive
passage through the stomach and small intestine in patients with achlorhydria.
 • Trichomonas vaginalis was first observed by Donne in 1836.
• It has worldwide distribution with higher prevalence among persons
with multiple sexual partners or other venereal diseases.
• Morphologically, it resembles T. tenar but it is larger than this. It
TRICHOMONAS measures 7—23 µm in length and 5—15 µm in width.
VAGINALIS • In a wet mount the trophozoite has a characteristic jerky motility.
• The normal habitat of the parasite is the vagina and urethra of
women, and the urethra, seminal vesicles and prostate of man. It
may also be found in the Bartholin's glands and in urinary bladder in
females.
Pathogenicity

• The parasite lives on the mucosa feeding on bacteria and leucocytes.

• T vaginalis is an obligate parasite.
• It cannot live without close association with the vaginal urethral or prostatic
tissues.
• Human trichomoniasis is a widely prevalent sexually transmitted disease of
worldwide importance.
• Approximately 10% of vulvovaginitis is due to infection with T vaginalis.
Asymptomatic infections have been observed in 50% of infected female patients.

The organism is responsible for a mild vagnitis with discharge.

Vaginal discharge contains a large number of parasites and leucocytes and is liquid, greenish or
yellow.

It covers the mucosa down to the urethral orifice, vestibular glands and clitoris.

Male patients usually have mild or asymptomatic infections. They may develop itching and
discomfort inside penile urethra, especially during urination.

The parasite is transmitted by sexual intercourse. It has been postulated that, in male patients, high
concentration of zinc in prostatic fluid in urogenital tract may have lytic effect on the parasite.
The exact mechanism of pathogenesis is still not
elucidated and appears to be multifactorial
depending upon the inherent virulence of the
parasite and host factors.

The main mechanisms postulated seem to be

mediated by cell to cell adhesion, haemolysis,
excretion of soluble proteinases, pore-forming
proteins and cell detaching factor.

Presence of double-stranded RNA virus in few

strains has been reported and the parasite is
able to undergo phenotypic variation in its
presence.
Laboratory diagnosis

Microscopy
• The diagnosis can be made by demonstration of trophozoites of T.
vaginalis in wet mounts of the sedimented urine, vaginal secretions or
vaginal scrapings by bright field, dark-field, or phase-contrast
microscopy.
• In males it may be found in urine or prostatic secretions.
• Fixed smears may be stained with Papanicolaou, Giemsa, Leishman and
periodic acid-Schiff stain and seen under light microscope.
• The parasites may also be detected by fluorescent microscopy by
staining with fluorescein-labelled monoclonal antibody.
 Culture
T vaginalis can be isolated from urethral and vaginal exudates on several commercially
available media.

Trussell and Johnson's medium is a simple medium that gives good growth.

It consists of proteose peptone, sodium chloride, sodium thioglycollate and normal human
serum.

Simplified trypticase serum medium is also suitable for the isolation of T vaginalis.

It is also used to maintain bacteria-free cultures of the flagellate. It grows best at 350—37 OC
under anaerobic conditions and less well aerobically.
The optimal pH for growth is 5.5—6.0. Culture is very sensitive (95%) procedure for diagnosis
of trichomoniasis. It is recommended when direct smear is negative.
Antibody and antigen detection
• Several types of ELISA have been developed for T vaginalis, either to measure antibodies or to
detect antigen of T vaginalis in clinical samples.

Molecular methods
• • Nucleic acid hybridization methods for detection of T. vaginalis have sensitivity and
specificity as good as culture methods.
• • Polymerase chain reaction (PCR) for the diagnosis of trichomoniasis has also been
developed.

Prevention
• Since infection is contracted through sexual intercourse, therefore, the preventive measures
include:
• • detection and treatment of cases, both males and females;
• • avoidance ofsexual contact with infected persons; and
• • use of condoms. There is no vaccine currently available for use against T vaginalis.
Treatment
• Metronidazole is highly effective therapeutic agent.
• It is given orally 250 mg three times daily for seven days or 2 gram
orally as a single dose.
• It is contraindicated in pregnancy.
• In this situation topical therapy with clotrimazole 100 mg daily for
seven days is recommended. Simultaneous treatment of sexual
partners is essential to prevent recurrence of infection.
 Chilomastix mesnili is a common flagellate living as a harmless commensal in the
CHILOMASTIX caecum and colon of man.
MESNILI It belongs to the order Retortamonadida. Davaine (1854) observed this parasite for the
first time in the stool.

It has a cosmopolitan distribution but is more prevalent in warm than in cool climates.
It has well-defined trophozoite and cystic stages.

Trophozoite is pear-shaped measuring 6—20 µm in length and 3—10 um in breadth.

The posterior end of the trophozoite is drawn out into a long cone. The spherical
nucleus, measuring 3—4 µm in diameter is situated anteriorly.

It has a small distinct, central karyosome and a few achromatic fibrils extending to the
nuclear membrane, and chromatin plaques lining the membrane.

A large conspicuous cytostome (mouth) is seen on one side of the nucleus. It has three
free anterior flagella, a delicate flagellum lying within the cytostome and two that
support the edges of the cytostome.
The cytoplasm is finely granular and contains numerous food vacuoles.
Cyst is lemon-shaped with a small projection at the anterior end.

It measures 7—10 µm in length and 4— 6 µm in breadth and is surrounded by a thick tough cyst wall.

The cytoplasm is densely granular and separated from the cyst wall at the narrower end of the cyst. The
single nucleus lies near 'the centre.
Remnants of the cytostome are also visible. C. mesnili is a normal inhabitant of the caecum and colon of
man.
The trophozoites live on enteric bacteria and multiply by binary fission.

In freshly passed liquid stools, only trophozoites are seen, in semi-formed stools, both trophozoites and
cysts may be observed, and in well-formed stools, only cysts are present.
Transmission of the parasite, from one person to another, takes place by ingestion of food or water
contaminated with cysts of C. mesnili in the stools of an infected individual.
C. mesnili is a harmless commensal and does not produce any symptom. The diagnosis can be made by
detection of trophozoites and cysts of C mesnili in the faecal smear.
ENTEROMONAS HOMINIS
• Enteromonas hominis is a rare species living as a harmless
commensal in the lumen of the caecum and other parts of the
large intestine of humans and primates.
• It has been reported from both warm and temperate climates.
• It exists in two forms — trophozoite and cyst.
• Trophozoite is pear-shaped, rounded or ovoidal measuring 4—10
µm in length and 3—6 µm in width.
• It possesses four flagella. By means of its three anterior flagella it
has jerky forward movement.
• The fourth flagellum is adherent to the body of the trophozoite
and then extends posteriorly as free flagellum.
The cytoplasm is finely vacuolated and contains numerous bacteria.

The nucleus is situated at the anterior end.

There is no cytostome. Cyst is oval, 6—8 µm in length and 4—6 µm in breadth and
contains one to four nuclei. E. hominis is a harmless commensal. It does not produce any
symptoms. It is transmitted by ingestion of food and water contaminated with cysts.

Diagnosis is made by demonstration of trophozoites and cysts in fresh faecal films and
haematoxylin- stained preparations.
RETORTAMONAS INTESTINALIS
• Retortamonas intestinalis, also known as Embadomonas intestinalis is
a rare harmless commensal in the caecum of man.
• It has been reported from both warm and temperate areas of the
world. It exists in two forms — trophozoite and cyst.
• Trophozoite is oval, small, measuring 6—9 µm in length and 34 urn in
breadth.
• The cytoplasm is finely granular and vacuolated.
• The spherical nucleus with central karyosome is situated anteriorly
and by its side lies the cytostome.
• It has two flagella which originate from two minute blepharoplasts
present near the nucleus.
One of the flagella is longer and directed anteriorly and the other is slightly shorter,
passes posteriorly through the cytostome before becoming free. The trophozoites
multiply by longitudinal binary fission.

Cyst is pyriform, measures 4—7 µm in length and 3—4 µm in breadth. Like

trophozoite, it also has single nucleus. The infection is acquired by ingestion of food or
water contaminated with cysts of R. intestinalis in the stools ofan infected individual. It
is nonpathogenic, although it is commonly discovered only in diarrhoeic stools.

Diagnosis is made by demonstration of trophozoites and cysts in fresh faecal films and
haematoxylin- stained preparations.
DIENTAMOEBA FRAGILIS
• Dientamoeba fragilis was initially thought to be an amoeba. It is now classified as
a flagellate even though flagella are not observed with light microscope.
• In practice, this organism may appear as an amoebic trophozoite, rather than as
flagellate in microscopic mounts. The generic name is derived from the binucleate
nature of the trophozoite and species name from the fragmented appearance of
its nuclear chromatin.
• It is a cosmopolitan parasite. It lives in the lumen of human colon along with the
true amoebae.
• Only the trophozoite stage is known.
• The cyst stage has not been confirmed to date.
• It is small, varying from 6—12 µm in diameter.
• It actively motile by means of hyaline pseudopodia that may be lobose or angular
Food vacuoles containing bacteria may be present.

It can be easily differentiated from intestinal amoebae as it contains two and sometimes up to four nuclei and a
flagellate structure (parabasal body) near the nuclei.

The karyosome is large and consists of four or more discrete granules.

The nuclear membrane is not lined by chromatin. The life cycle of D. fragilis is
not completely under- stood.

Because D. fragilis does not have an identified cyst state, direct foodborne or
waterborne transfer from host to host is less likely.

The nine times higher incidence of D. fragilis in patients with pinworm

infection suggests that the Enterobius vermicularis eggs may be infected with
the flagellate and serve as the chief vector for transfer to humans.
 This possibility may also explain why almost 50% of reported cases of
dientamoebiasis occur in patients under 20 years of age.

It is mildly pathogenic in about 25% of infected individuals, who may

develop intermittent diarrhoea, abdominal pain, flatulence, nausea,
vomiting, anorexia, malaise, weight loss, and unexplained eosinophilia.

Infection can be diagnosed by detection of trophozoites in permanently

stained smears (e.g., trichrome).

The trophozoites are pale staining and their nuclei may resemble those of
Endo/imax nana or Entamoeba hartmani.

The drugs of choice in the treatment of symptomatic cases include

tetracycline and metronidazole.
BLOOD AND TISSUE FLAGELLATES
• Blood and tissue flagellates belong to the family Trypanosomatidae, class
Kinetoplastidea and order Trypanosomatida.
• They possess a single nucleus, a single kinetoplast and a single flagellum.
• The kinetoplast consists of parabasal body and an adjacent dot like
blepharoplast. The blepharoplast and parabasal body are connected by
one or more delicate fibrils.
• The flagellum arises from the blepharoplast. The portion of the flagellum
extending from the blepharoplast to the surface of the body of the parasite
is known as axoneme.
• Family Trypanosomatidae consists of six genera, of which Leishmania and
Trypanosoma are pathogenic to man. Species of this family may exist in
two or more forms.
LEISHMANIA
• The genus Leishmania is widely distributed in nature. It has a number of species
that are nearly identical morphologically.
• Differentiation is, therefore, on a number ofbiochemical and epidemiological
criteria, use of monoclonal probes to detect specific antigens, promastigote
growth patterns in vitro in the presence of antisera, vectors and reservoir hosts.
• The parasites of the Old World leishmaniasis (L. donovani, L. ifantum, L tropica, L
major and L. aethiopica) are transmitted to humans by the bite of female sandflies
of the genus Phlebotomus;
• while those of the New World leishmaniasis (L. peruviana, L. chagasi, L. mexicana
complex and L. braziliensis complex) are carried by sandflies of the genera
Lutzomyia and Psychodopvgus.
• The term 'New World' refers to the Americas and the 'Old World' is used for the
rest of the world. Leishmanias pass their life cycle in two hosts — invertebrate
hosts and vertebrate hosts.
Former are the sandflies and the latter are mammals in which the parasites
reside within the phagolysosomal system of mononuclear phagocytic cells,
typically macrophages.

However, in the invertebrate hosts, the parasites are extracellular,

development occurs exclusively in the gut and transmission is via the
mouthparts during blood feeding.

These morphologically similar parasites living in a single series of cells, cause

diversity of diseases.

Leishmaniasis is a collection of diseases, each with its own clinical

manifestations and epidemiology. It is mainly a zoonosis, although in certain
areas of the world there is primarily human-vector-human transmission.
Animal inoculation
• The hamster is the laboratory animal of choice for the isolation of any form of
Leishmania spp.
• Young (2-4 months old) hamsters of either sex are inoculated intraperitoneally
with aspirates or biopsy material obtained under sterile conditions from
cutaneous ulcers, lymph nodes, spleen, liver, bone marrow, buffy coat cells or
spinal fluid.
• It results in a generalized infection.
• Spleen impression smears should be examined for the presence of organisms.
• The infection develops slowly in hamsters. Several months may be required to
produce a detectable infection. For this reason, Culture procedures are
usually selected as more rapid means of parasite recovery.
• Animals should be kept for 9—12 months before a negative report is given.
 OLD WORLD LEISHMANIASIS

LEISHMANIA DONOVANI
•Sir William Leishman in 1900 discovered this parasite in spleen smear of a soldier
who had died of Dum Dum fever' or kala-azar contracted at Dum Dum Kolkata.
•Leishman reported this finding in 1903, in which year Donovan also reported the
same parasite in spleen smear of a patient from Chennai.
•The name Leishmania donovani was therefore given to this parasite.

Habitat
•It is an obligate intracellular parasite of reticuloendothelial cells, predominantly of
liver, spleen, bone marrow and lymph nodes of man and other vertebrate hosts (dog
and hamster) where it occurs in amastigote form.
Morphology

The parasite exists in two morphological forms:

• • Amastigote
• • Promastigote.
Amastigote
• In the amastigote form the parasite resides in the cells of
reticuloendothelial system (macrophages, monocytes,
polymorphonuclear leucocytes, or endothelial cells) of
vertebrate hosts. It is non-motile,
round or oval body measuring 2—4 µm in length along the longitudinal axis

Cell membrane is delicate and can be demonstrated in fresh specimens only.

Nucleus is round or oval, less than I µm in diameter. It is situated in the middle of the cell or along the side of
the cell wall.

Kinetoplast consists of parabasal body and blepharoplast which are connected by one or more delicate fibrils.
It lies tangentially or at right angle to the nucleus.

The axoneme arises from the blepharoplast and extends to the margin of the body. It represents the
intracellular portion of the flagellum. Alongside the axoneme lies a clear unstained space known as vacuole.

In preparations stained with Giemsa or Wright stain, the cytoplasm

appears pale blue, the nucleus red, parabasal body deep red and kinetoplast bright red.
Promastigote
• Promastigotes are found in the digestive tract of insect vector (sandfly)
and in the culture media.
• These are elongated, motile, extracellular stage of the parasite.
• Fully developed promastigotes measure 15—25 µm in length and 1.5—
3.5 µm in breadth. Nucleus is situated centrally.
• Kinetoplast lies transversely near the anterior end. In front of the
kinetoplast lies a pale staining Vacuole. From the blepharoplast arises
the axoneme which projects from the anterior end of the parasite as
Flagellates
• free flagellum. Flagellum may be of the same length as the body of the
parasite or longer. It does not curve round the body of the parasite,
therefore, there is no undulating membrane (Fig. 4.10).
Cultivation

NNN medium
• L. donovani can be cultured on NNN medium which was first introduced by Novy and McNeal
(1904) and later modified by Nicolle (1908).
• It consists of two parts of salt agar and one part of defibrinated rabbit blood.
• The tubed salt agar medium is melted and then cooled to 48⁰C.
• To each tube of medium one-third of its volume of sterile defibrinated rabbit blood is added
and mixed thoroughly by rotating the tubes.
• The tubes are slanted and allowed to cool preferably on ice, as more water of condensation is
obtained. Blood, aspirates, or small biopsy samples from spleen, liver, or bone marrow
obtained aseptically are inoculated into water of condensation of the medium and incubated
at 22⁰—25 ⁰ C.
• The amastigote form changes into pro- mastigote form which then multiplies rapidly by
longitudinal fission to produce a large number of flagellates, particularly in the water of
condensation at the bottom of the tube.
Hockmeyer's medium
• This liquid medium consists of Schneider's commercially prepared insect
cell culture medium, supplemented by the addition of 30% heat
inactivated foetal calf serum and with 100 IU penicillin and 100 gg
streptomycin per ml.
• The medium is inoculated with the specimen, incubated at 22 ⁰—25 ⁰ C,
and examined microscopically daily tor the presence of promastieotes.
• The latter can usually be detected after 2—3 days of incubation but the
cultures should be held for four weeks.
 Life cycle

• L. donovani passes its life cycle in two hosts — man and also dog in some areas are
the vertebrate hosts, and female sandfly of the genus Phlebotomus is the
invertebrate host, Indian kala-azar is considered to be a non-zoonotic infection with
man as the sole reservoir.
• Important sandfly hosts include P. argentipes, R orientalis and P martini. Of these R
argentipes is the Indian vector.
• Amastigotes of the parasite are present in the blood stream of the patient, both
free as well as phagocytosed by polymorphonuclear leucocytes and monocytes.
• These are taken up by the sandfly in a blood meal and reach midgut of the insect.
Here the parasite transforms into promastigotes and multiplies producing
enormous numbers.
• The parasites then proceed forwards to the pharynx and buccal cavity which they
block between the 6th and 9th day of its infective blood meal.
Because of this, the sandfly has difficulty in getting a blood meal, nevertheless, it pricks the skin of the
victim and regurgitates the promastigotes in the wound caused by its proboscis.

These are engulfed by nearby fixed macrophages and change into amastigotes within the cytoplasm of
these host cells.

Here the amastigotes multiply slowly and may remain more or less quiescent for weeks or months.

Thereafter, parasitized macrophages are set free into the blood stream and are carried from the skin to
spleen, liver, bone marrow, and other centres of reticuloendothelial activity.

The amastigotes are now taken up by fixed macrophages such as Kupffer's cells in the liver, and multiply
by simple binary fission till the cells become packed with the parasites (50—200 or more in the
cytoplasm of the infected cell).
The infected cell ruptures and the parasites are liberated into the circulation.

These are taken up by other reticuloendothelial cells followed by multi-

plication of the parasites and rupture of the cells.

In this way the entire reticuloendothelial system becomes progressively

infected. In the blood stream, some of the free amastigotes are phagocytosed
by polymorpho- nuclear leucocytes and monocytes.

A blood-sucking insect draws these free amastigotes, as well as those within

the cells during its blood meal and the cycle is repeated.
Pathogenicity
• L. donovani causes visceral leishmaniasis or kala (kala meaning black and azar meaning disease),
• Dum fever or tropical splenomegaly. In India, kala azar.
• Incubation period generally varies from months, but it may be as short as 10 days or as longas 2
years.
• The parasite spreads from the site of inoculation to multiply in reticuloendothelial cells, in the
spleen, liver, lymph nodes, and bone marrow
• This leads to progressive enlargement of these organs.
• The host cellular and humoral defence mechanisms are stimulated. The former results in marked
proliferation of macrophages.
• These cellular elements make up a large part of the bone marrow, compromising both the
erythropoietic and granulocytic activity. The effect of this in the peripheral blood is leucopenia with
granulocytopenia and relative monocytosis, anaemia (usually normocytic) and thrombocytopenia.
• The spleen and liver become markedly enlargd and hypersplenism contributes to the production of
anaemia. It has been suggested that the erythrocytes adsorb immune complexes and become
subject to enhanced phagocytosis by the macrophages of the liver and spleen. Lymphadenopathy is
also produced.
Production of globulin is greatly increased.

This leads to reversal of the albumin: globulin ratio. The disease is reticuloendotheliosis
due to invasion of reticuloendothelial cells by L. donovani. Reticulo- endothelial cells of
various organs are proliferated and are packed with amastigote forms of the parasite.

The disease manifests clinically with fever, malaise, head- ache, progressive enlargement
of spleen, liver, and lymph nodes, anaemia, leucopenia and emaciation.

Skin changes are often seen on the face, hands, feet, and abdomen, particularly in India,
where patients acquire an earth-gray colour. If left untreated 75-95% patients die within 2
years.
Death in kala-azar is due to secondary infections. Visceral leishmaniasis and human
immuno- deficiency virus (HIV) together are synergistic infections because visceral
leishmaniasis accelerates the development of acquired immunodeficiency syndrome
(AIDS) and the presence of HIV infection enhances the spread of visceral disease. #

HIV may either activate subclinical leishmaniasis or make the patientFlagellates

susceptible to a new infection. Pisceral leishmaniasis has emogedas one ofthe most
important opportunistic infections in HIV-infected patients.

The presentation of visceral leishmaniasis in the presence of HIV infection is ven

atypical and serological tests may be negative.
Immunity
• In contrast to cutaneous leishmaniasis, cell-mediated immunity is impaired in
active kala-azar patients who consequently lack a delayed type
hypersensitivity response, but this can be demonstrated after cure.
Laboratory diagnosis
• Various tests which can be carried out for the laboratory diagnosis of kala-azar

NON-SPECIFIC LABORATORY TESTS

• • Blood count (pancytopenia mainly neutropenia and decreased erythrocyte
count)
• • Haemoglobin estimation (anaemia)
• • Estimation of serum proteins (raised serum proteins with reversal of
albumin: globulin ratio due to greatly increased IgG levels)
PARASITOLOGICAL DIAGNOSIS

• Peripheral blood by thick film method (amastigote form)

• Needle biopsy/aspiration
• — Lymph node
• — Bone marrow
• — Liver
• — Spleen
By touch preparation or smear stained with Giemsa stain
(amastigote form)

• Culture of blood and needle biopsy/aspiration material

(promastigote form)

• Molecular methods

• Animal inoculation
IMMUNOLOGICAL TESTS

Non-specific tests:
• • Aldehyde test Indicate greatly increased serum proteins.
• Antimony test
• - • Complement fixation test with W.K.K. antigen

Specific tests:
• • Direct agglutination test (DAT)
• • Indirect haemagglutination test (IHA)
• • Indirect fluorescent antibody test (IFAT)
• Enzyme-linked immunosorbent assay (ELISA)
LEISHMANIN OR MONTENEGRO SKIN TEST

Non-specific laboratory tests

These include:
• 1. Blood count: Total and differential leucocyte count reveals
pancytopenia, mainly neutropenia and decreased erythrocyte count.
The average total count of leucocytes is 3,000/gl of blood. During the
course of the disease, the count may fall to 1,000/gl of blood or even
below. Erythrocytes are also decreased in number.
• 2. Haemoglobin estimation: It reveals anaemia.
• 3. Estimation of serum proteins: It reveals raised serum proteins with
reversal of the albumin: globulin ratio due to greatly raised IgG levels.
Parasitological diagnosis

Diagnosis of leishmaniasis can be confirmed by:

• 1. Peripheral blood film: Amastigote form of the parasite may be demonstrated
inside circulating monocytes and less often in neutrophils, in the stained
peripheral blood film by thick film method. Owing to the small number of
Leishmania parasites present in the peripheral blood, an examination ofa thin
film is often negative.
• 2. Needle biopsy/aspiration: Deeper tissues, e.g., lymph node, bone marrow, liver
and spleen may be sampled by needle biopsy/aspiration. Amastigote forms of
the parasite can be demonstrated in touch prepa- rations or smears stamed with
Giemsa stain. Spleen aspirate is the most reliable material for demonstrating
parasites in kala-azar. However, bleeding might continue from the puncture
wound in the soft and enlarged spleen, resulting in death. Therefore, spleen
puncture should not be performed in a patient with haemorrhagic diathesis and
leukaemia.
 3. Culture: Whatever material is collected (blood and biopsy/aspiration material from various
organs), it should be inoculated on NNN medium or Hockmeyer's medium and incubated at
220—250 C and examined microscopically twice a week for first 2 weeks and once a week
thereafter for up to 4 weeks before they are reported as negative. Promastigote stages can be
detected microscopically in wet mounts taken from centrifuged culture fluid. The material can
also be stained with Giemsa stain to facilitate observation at a higher magnification.

4. Molecular methods: A number of molecular methods have been developed for species
identification of the promastigotes, including the use of DNA probes, isoenzyme analysis, PCR, and
monoclonal antibodies.

5. Animal inoculation: For special purpose the pathogenic material may be inoculated into a
hamster which is the most susceptible experimental animal.
Immunological tests

These include non-specific and specific tests:

1. Non-specific tests

• Aldehyde test
• Aldehyde test: A drop of full strength (40%) formalin is added to 1 ml of serum. A positive test is indicated by the rapid
and complete coagulation of the serum. This serum test merely indicates greatly increased serum gamma globulin and
thus is non- specific. This test is widely used and is not positive till the disease is ofat least three months duration.

• Antimony test
• Antimony test: This test also depends upon a rise of serum gamma globulin. When a 4% urea stibamine solution in
distilled water is mixed with serum from a patient with kala-azar, it leads to the formation of a profuse flocculent
precipitate. It is less reliable than aldehyde test.

• Complement fixation test with W.K.K. antigen

• Complementßation test with W.K.K. antigen: Complement fixation test may be carried out for detection ofserum
antibodies in visceral leishmaniasis. The antigen originally used was prepared from human tubercle bacillus by
Witebsky, Kleingenstein and Kuhn hence known as W.K.K. antigen. Since the antigen is not prepared from L. donovani,
therefore, this test is non-specific.
2. Specific tests
• There are currently four tests in use, the direct agglutination test (DAT), indirect fluorescent antibody test (IFAT),
counterimmunoelectrofluoresis (CIE), and enzyme-linked immunosorbent assay (ELISA). DAT is very specific,
and does not require expensive equipment or reagents. ELISA using species-specific mono- clonal antibodies,
and DNA probes have been successful in the direct detection of Leishmania antigen.

Leishmanin or Montenegro test

• It is a delayed hypersensitivity reaction to intradermal crude Leishmania antigen. It was first introduced in the
South America by Montenegro, In this test, 0.2 ml of killed suspension of promastigotes of L. donovani,
containing 6—10 million of the promastigotes per ml of 0.5% phenol saline, is injected intradermally. The test is
read after 48—72 hours. A positive test shows an area of erythema and induration 5 mm or more in diameter.
The test becomes positive 6—8 weeks after cure from kala-azar. Cell-mediated immunity is impaired in active
kala-azar patients who consequently lack a delayed hypersensitivity response. Therefore, leishmanin test is
negative in active cases of kala-azar This test is of great value in epidemiological studies but is of little clinical
use.
• Diagnosis of visceral leishmania.sis in thepresence of HIV infection is particularly difficult as the presentation
may be very atypical and serological tests may be negative.

Differential diagnosis
• Kala-azar must be differentiated from malaria, trypanosomiasis, schistosomiasis, liver abscess, tropical
splenomegaly, histoplasmosis, brucellosis, tuberculosis, relapsing fever, myeloid leukaemia- lymphoma, cirrhosis
of liver, thalassaemia and 'various gammopathies.
POST KAI-A-AZAR DERMAL LEISHMANIASIS
• Post kala-azar dermal leishmaniasis (PKDL) first described in patients with visceral leishmaniasis
caused by L. donovani in India.
• In India, skin lesions may appear 2—10 Fars after successful therapy for visceral leishmaniasis In
East Africa, lesions appear within a few months.
• It is caused by the reversal of L donovani from viscerotropic to dermatotropic.
• It is not associated with L infantum infection. PKDL is occasionally seen in patients with no history of
visceral disease but only in places where L. donovani is transmitted, this is probably a sequel to
subclinical infection.
• Macules and papules usually appear first around the mouth and spread to the nick and then to
extensor surfaces of the arms, the trunk, and sometimes the legs In the beginning they look like
small hypopigmented patches; these then enlarge and may progress to nodules.
• The lesions are son, granulomatous of vatying sizes and unless traumatized, do ulcerate When they
are abundant, the clinical appearance may resemble that of lepromatous leprosy, Diagnosis of P K
DL can be established by demonstration of amastigote form of L by a micrcopicopic examination of
Leishman-stained smear prepared from the biopsy material obtained from nodular lesions.
• Direct smear examination from the hypopigmented macules does not generally reveal any parasite,
LEISHMANIA INFANTUM
• Main vertebrate host is the domestic dog. which develops an
acute or chronic disease. Canine visceral leishmania presents
abundant parasites in the skin.
• Invertebrate hosts or vector of L. infantum are P. ariasi and P.
pernicious.
• In man It causes infantile leishmaniasis classically restricted
to children. especially those below the age of 2 years.
However. It may also Involve adults, particularly those
Infected with Ill V. PKDL is not seen with L infantum infection.
LEISHMANIA TROPICA

Habitat
• It occurs inside reticuloendothelial cells (clasmatocytes) of the skin,

Morphology
• The amastigote form occurs in man, whereas promastigote form is found in sandfly
and in cultures. Morphologically, both these forms of L tropica are indistinguishable
from those of L donovani-

Cultivation
• L tropica,like L donovani, can be cultured on NNN medium and Hockmeyer's medium.
Susceptible animal As in case of L. donovani, hamster is susceptible to L tropica
infection.
• The susceptible animal is hamster.
Life cycle
• Vertebrate host is man and invertebrate host is sandfly (P,
sergenli).
• life cycle of L. tropica, in vertebrate and invertebrate
hosts. Is similar to that of L donovani except that man
amastigote form of the former resides in the
reticuloendothelial cells of skin and and not in viscera
• One factor restricting the parasite causing cutaneous
leishmaniasis is temperature in skin because some species
are sensitive
Pathogenicity:
• Leishmania tropica causes urban anthroponotic cutaneous leishmaniasis or oriental sore or delhi
boil.
• Infection transmitted to man either by direct inoculation of promastigote of L. tropica through bites
sand fly or by crushing of the infected sand fly into the punctured wound caused by the bite.
• At the site of inoculation promastigote are phagocytosed by reticuloendothelial cells of the skin and
are transformed into mastigote.
• Acutanoeus lesion or leishmanioma develops at site of infective sandfly bite.
• It is characterised by chronic infective granuloma with fibrosis in the early stage.
• The lesion due to the proliferation of endothelial cells of skin that contain large number of
amastigotes. Later. round cell infilteration (lymphocyte) and plasma cell) associated With a marked
in the number of parasite and development of delayed hypersensitivity skin reaction (leishmania
reaction) occur,
• Incubation period varies from a few weeks to 6 months and in some cases it may years- Clinically,
the lesion begins as a raised papule about 2.5 cm in diameter. In majority of cases. It ulcerates.
• The ulcer has clean-cut margin with a raised indurated edge, surrounded by red areola At this stage.
• parasite is found along the red rnargin and not on floor of the ulcer. The ulcer heals spontaneously
in about 6 months, leaving a depressed scar and solid immunity.
There is a marked development of reactions but a weak antibody although specific antibodies
can be detected.

Although specific antibodies can be detected

The cell mediated reactions are responsible for a marked delayed type hypersensitivity to
Leishmanian in active and cured cases.

The sores are distributed on the exposed parts of the body, particularly on the face
extremities.

the average number of sores is around two.

Oriental sore is not associated with systemic manefistation although there may be
enlargement of the draining lymph nodes.
Laboratory diagnosis
• Diagnosis of L. tropica infection is made by the microscopic examination of material
obtained by puncture of the indurated edge of the sore and stained with Giemsa or
Wright stain.
• Amastigote form of the parasite will be seen in large numbers inside the macrophages.
• Smears made by scraping the floor of the ulcer are often negative because amastigote-
infected macrophages are destroyed in the presence of secondary bacterial infection.
• If smears are negative, biopsy from the margin of the ulcer at times provides specific
proof of infection.
• Isolation of promastigote of L. tropica may be made from the aspirates of the ulcer by
culture in NNN medium and Hockmeyer's medium.
• The specimen for culture is obtained by injecting a little volume of sterile physiological
saline in the indurated margin of the ulcer and then aspirating it.
• A few drops of the aspirate are then inoculated into each medium. Leishmanin skin test
Intradermal injection of leishmanin (killed promasti- gotes of L. tropica in 0.5% phenol
saline) shows a marked delayed 'type hypersensitivity response.
LEISHMANIA MAJOR
• Important mammalian hosts of L. major are great gerbil (Rhombomys opimus) and fat sand rat
(Psammomys obesus), and important sandfly vectors are P. papatasi, P. dubosqi and P salehi.
• L. major infection in humans occurs in epidemics in groups of people entering sparsely
inhabited zoonotic foci. Major outbreaks have been reported in military groups and in
workers on development projects.
• In sub-saharan Africa oubreaks are much less frequent and human cases occur sporadically.
Trans- mission between humans without a mammalian reservoir host has not been well
established.
• L. major causes rural, zoonotic, cutaneous leishmaniasis or Oriental sore. It causes "wet" lesion
which becomes necrotic and exudative, forming a loose crust above a granulomatous base
that eventually produces the characteristic scar, The lesions may number more than 100.
• Lymphatic spread may occur in L. major infections, with subcutaneous nodules in a linear
distribution. As in case of L. tropica, spontaneous cure of L. major infection usually results in a
solid immunity and marked delayed type hypersensitivity response to intradermal inoculation
of leishmanin. The latter may also be detected before healing.
• Method of diagnosis of L. major infection is similar to that caused by L. tropica.
LEISHMANIA AETHIOPICA
• Important mammalian hosts of this parasite are rock hyraxes (Heterohyrax brucei and
Procavia spp.), and important sandfly hosts are P. longipes and P. pedifen In man it
may cause cutaneous leishmaniasis and diffuse cutaneous leishmaniasis (DCL).
• L. aethiopica lesions are more swollen and less necrotic than those of L. tropica. They
are frequently barely exudative, with gradual scaling or exfoliation of the dermis at
the centre.
• These lesions may last for years before healing.
• DCL is a rare form of disease caused by L. aethiopica.
• The parasites are restricted to the skin, but become widely distributed over much of
the surface, in large, swollen plaques and nodules.
• This condition resembles lepromatous leprosy. However, the abundant parasites in
the nodules provide easy distinction. In this condition, neither humoral nor cell-
mediated immune responses are activated. It is diffcult to treat and this condition
may last for the rest of the life of the patient.
Treatment of the Old World leishmaniasis
• Various drugs which may be used for the treatment of the Old World leishmaniasis.
• Single lesions caused by L. major and L. tropica heal naturally in a few months and
leave the patient immune for life.
• Therefore, chemical treatment in such cases may not be justified. However, if the
lesions are multiple or disfiguring, the chemotherapy is required.
• With L. donovani infection, patients have a high risk pharyngeal and laryngeal
mucosae.
• These lesions may appear within a few months of the original skin lesion, or years
later when the patient appears to have been cured of his initial infection.
• The pathology of skin lesion is similar to that of L. tropica. L. mexicana complex may
also cause 'anergic diffuse cutaneous leishmaniasis' (ADCL). Histological examination
of the skin and mucous lesion shows infiltration of lymphocytes, plasma cells and
large mononuclear cells and necrosis of tissues.
• The amastigotes of the parasites are 'found in large numbers inside the
clasmatocytes and monocytes at the periphery of the lesion.
Laboratory diagnosis
• The diagnosis is established by demonstration of amastigote forms of the parasites in the
material obtained by puncture of the nodule or edge of the ulcer.
• It is stained with Giemsa or Wright stain, cultured on NNN medium and inoculated into
hamester.
• Antileishmanial antibodies may be detected in the patient serum by indirect fluorescent
antibody (IFA) test using fixed amastigotes as antigens.
• It gives positive results in 89—95% of cases. IFA titre falls after successful chemotherapy.
Enzyme-linked immuno- sorbent assay is also positive in 85% of cases. Leishmanin or
Montenegro skin test is positive in cutaneous and mucocutaneous leishmaniasis.

LEISHMANIA PERUVIANA
• The dog, Canis familaris, is the only known mammalian host other than humans, but this
animal 's role in the epidemiology of the human disease remains obscure.
• This parasite causes simple cutaneous leishmaniasis without invasion of oro-nasal mucosa. It
is particularly common in school children, commonly resulting in extensive facial scars. Ulcers
are usually self-healing and impart a solid immunity to reinfection with the same parasite.
LEISHMANIA CHAGASI
• Mammalian hosts include human and domestic dog,
Canis familaris. Among wild animals, the foxes (Lycalopex
vetulus and Cerdocyon thous )seem to be important
natural reservoirs. Lutzomyia longipalpis is visceral
leishmaniasis (AVL). Unless treated it has a fatal outcome.
• Treatment and prophylaxis of the New World
leishmaniasis Treatment and prophylaxis of the New
World leishmaniasis is much the same as that for the Old
World leishmaniasis.
TRYPANOSOMES
TRYPANOSOMES
• Trypanosomes are lvaemoflagellates that live in the blood and tissues or their
human hosts. Trypanosoma brucei has three subspecies — Trypanosoma brucei
brucei, T h. gambiense and T b. rhodesiense.
• The first one is an animal pathogen and the other two cause African
trypanosomiasis or sleeping sickness in man, T cruzi causes South American
trypanosomiasis or Chagas' disease. T rangeli infects humans without causing
disease and must, therefore, be carefully distinguished from the pathogenic
species, T brucei was first demonstrated by Bruce in horses and cattle suffering
from 'nagana' during the year 1895.
• Forde, 1902 was first to demonstrate motile trypanosomes in the blood of a man
suffering from fever.
• This parasite was subsequently named as T gambiense by Dutton in 1902. Kleine,
1909 demonstrated the development of the parasite in the vector, tsetse fly.
TRYPANOSOMA BRUCEI GAMBIENSE

Habitat

It inhabits:
• • connective tissue spaces of the various organs;
• • the reticular tissue of the lymph nodes and spleen;
• • the intercellular spaces in the brain;
• • lymph channels throughout the body;
• • blood; and cerebrospinal fluid.
Morphology
• In the blood of the vertebrate host, T b. gambiense exists in trypomastigote form. It occurs in three
forms a long slender form having a flagellum, a short stumpy one without a flagellum, and an
intermediate one
• Because of these morphologic differences in the of the vertebrate host, this trypanosome is said to
polymorphic. The first form to appear is a long slender one and later hen the Infection is established,
the short stumpy form together with the intermediate one appear.
• In fresh blood the trypanosomes may be seen as motile, colourless, spindle-shaped bodies with a
blunted posterior end and a finely pointed anterior end.
• The long slender and short stumpy forms measure 20 x 3 µm and 10 x 5 11m respectively.
• The nucleus is large, oval and central in position. Kinetoplast (parabasal body and blepharoplast) is
situated on the posterior end. From the blepharoplast arises the flagellum. It curves round the
body in the form of an undulating membrane and then continues beyond the anterior end as a free
flagellum.
• The undulating membrane is thrown into 3 or 4 folds. The intracellular portion of the flagellum is
known as axoneme. In Giemsa or Wright stained preparations, the cytoplasm and the undulating
membrane appear pale blue, the nucleus reddish purple or red and the kinetoplast and the
flagellum dark red.
Antigenic variation
• One of the most interesting aspects of the trypanosomes is their ability to vary the
antigenic nature of their surface coat. Within their genome, trypanosomes contain over I
,000 different genes coding for different variant surface glycoproteins (VSG).
• It appears that during the growth of trypanosomes new variant antigen types (VAT) are
constantly being produced by mutations, additions, deletions and recombination.
• New surface coat variants appear at frequencies of I in 106 cells. Each wave of
parasitaemia caused by a new variant emerges at 5—10 days interval and is
accompanied by fever and followed by monocytosis and production of antibody against
the VSG This vanant specific antibody is involved in the rernoval or clearance of majority
of the trypanosomes from the blood and other body fluids.
• This process ends each wave of parasitaemia. However, there preexists within that
population a rnmor population of trypanosomes with a different VSG. This minor
population then gives rise to the next wave of parasitaemia. Antibody, therefore, acts as a
selecting agent removing the major VSG population and allowing the other variant
antigen types that exist in the population to develop.
Cultivation
• T b. gambiense can be grown in Weinman's medium. This medium
consists of 100 ml of citrated human plasma and human
haemoglobin (made by mixing I part of blood and 3 parts of distilled
water), 900 ml of distilled water and 8 gram of sodium chloride. The
medium is adjusted to pH 7.4—7.5 and dispensed in rubber-
stoppered test tubes. It is inoculated and incubated at 260—280 C.
• The culture becomes positive in 7—10 days. Long slender forms of
trypomastigote, similar to midgut forms of tsetse fly, are
encountered in culture. In contrast to T b. rhodesiense, laboratory
animals such as mice, rats and guinea pigs are less susceptible to T b.
gambiense infection.