Lecture 1 : Medical Parasitology

Medical Parasitology is the study of parasites and as such that does not include
bacterial, fungal or viral parasites. Human parasites are separated into intestinal
and blood borne parasites. For a parasite to be defined as intestinal it must have
an intestinal life cycle stage, though it may have life- cycle stages in the heart,
blood vessels, and lungs in the humans, other animals or the environment.

The association between two organisms may be one of the following:

Mutualism: mutual benefit is derived from the association.
Symbiosis: mutual benefit, but the two organisms cannot live
independently.
Commensalism: one partner benefits (commensal) while the other (host) is
unaffected. It may be called a non-pathogenic parasite.
When an animal lives on another organism from which it receives food and
shelter without any compensation to it, and then this association is called
parasitism. The animal, which enjoys advantages, is the parasite. All animals
have parasites; hence there are more parasites than free-living animals. The
habitat occupied by a parasite is very different from the environment of its free-
living ancestors, hence it has either to adapt itself to this new habitat or perish.
Parasitism: one organism (parasite) lives at the expense of the other (host). The
latter usually suffers from the association with pathogenic parasite).
Parasitism is the form of mutual relations between organisms of various kinds,
from which one (parasite) uses another (host) as environment for living, and
from which it obtains food causing him damage (disease).

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Classification of Parasites
Each parasite belongs to a phylum, class, order, family, genus and species; the
scientific designation of a parasite is binomial, a generic name (genus) and a
specific name (species).
The parasites of humans in the phylum protozoa are now classified under three
subphyla: Sarcomastigophora (containing the amoebae and flagellates);
Apicomplexa (containing the sporozoan); and Ciliophora (containing the
ciliates). The important human parasites are found within these great groups.
1. Subphylum (Sarcodina) is typically amoeboid and in represented in
humans by class of Entamoeba, Endolimax, lodamoeba, Naegleria, and
Acanthamoeba.
2. Subphylum Zoomastigophora, the flagellates, have one or more whip-
like flagella and, in some cases, an undulating membrane (e.g.,
trypanosomes). These include intestinal and genitourinary flagellates
(Giardia, Trichomonas, Dientamoeba, Chilomastix) and blood tissue
flagellates (Trypanosoma, Leishmania).
3. Subphylum Sporozoa undergoes a complex life cycle with alternating
sexual and asexual reproductive phases, usually involving two different
hosts (e.g., arthropod and vertebrate, as in the blood forms). The subclass
Coccidia contains the human parasites Isospora, Toxoplasma, and others.
One of these, Cryptosporidium, has been implicated as a cause of
intractable diarrhea among the immunosuppressed. Among the

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Haemosporina (blood sporozoan) are the malaria parasite (Plasmodium
species) and the subclass Piroplasmia, which includes Babesia species.
Pneumocystis has recently been shown to be a member of the Fungi rather than
the Protozoa. It is another opportunistic parasite of immunosuppressed
individuals.
4. Subphylum Ciliata is a complex protozoan bearing cilia distributed in
rows or patches, with two kinds of nuclei in each individual. Balantidium
coli, a giant intestinal ciliate of humans and pigs, is the only human
parasite representative of this group.
The Parasitic Worms, or helminths, of a human being, belong to two
Subphyla:
1. Subphylum Platyhelminths (flatworms) lack a true body cavity (celom)
and are characteristically flat in dorsoventral section. Medically
important species belong to the classes Cestoda (tapeworms) and
Trematoda (flukes). The tapeworms of humans are band-like and
segmented; the flukes are typically leaf-shaped, and the schistosomes are
narrow and elongate. The important tissue and intestinal cestodes of
humans belong to the genera Diphyllobothrium, Spirometra, Taenia,
Echinococcus, Hymenolepis, and Dipylidium. Medically important
trematode genera include Schistosoma, Paragonimus, Clonorchis,
Opistorchis, Heterophyes, Metagonimus, Fusciolopsis, and Fasciola.
2. Subphylum Nemathelminths (worm-like, separate-sexed, insegmented
roundworms) include many parasitic species that infect humans.

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 Phylum Protozoa
General Features
The single protozoal cell performs all functions. Most of the protozoa are
completely nonpathogenic but few may cause major diseases such as malaria,
leishmaniasis, and sleeping sickness. Protozoa like Cryptosporidium parvum
and Toxoplasma gondii are being recognized as opportunistic pathogens in
patients affected with human immunodeficiency virus (HIV) and in those
undergoing immunosuppressive therapy. Protozoa exhibit a wide range of a size
(1- 150 µm), shape, and structure; yet all possess essential common features.
Single-celled microorganisms belonging to the animal kingdom are classified as
Protozoa (Greek Protos—first; zoon—animal). Within its single cell, the
protozoon contains all structures required for performing its various functions.
Some free-living protozoa resemble plants containing green plastids that enable
them to perform photosynthesis. It is believed that these represent the earliest
forms of animal life. Numerous varieties of protozoa have evolved to suit all
manner of environmental conditions.
Free-living protozoa are found in all habitats—in the deep ocean or in shallow
freshwaters, in hot springs or in ice, under the soil, or in the snow on mountain
tops. Parasitic protozoa have however adapted to different host species, with
more restricted physicochemical requirements.
Protozoa exhibit a wide range of size, shape, and structure, yet all possess
certain essential common features. The typical protozoan cell is bounded by a
trilaminar unit membrane, supported by a sheet of contractile

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fibrils that enable the cell to change its shape and to move. The cytoplasm can
often be differentiated into an outer rim of relatively homogeneous ectoplasm
and a more granular inner endoplasm.
The ectoplasm serves as the organ of locomotion and for engulfment of food
materials by putting forth pseudopodial processes. It also functions in
respiration, discharging waste materials, and also as a protective covering for
the cell. Within the endoplasm is the nucleus within a tough nuclear membrane.
The nucleus is usually single but maybe double or multiple, some species
having as many as a hundred nuclei in one cell. The nucleus contains one or
more nucleoli or a central endosome or karyosome. The chromatin may be
distributed along the inner surface of the nuclear membrane (peripheral
chromatin) or as condensed masses around the karyosome. The endoplasm
shows a number of structures-the endoplasmic reticulum, mitochondria, and
Golgi bodies. Contractile vacuoles may be present which serve to regulate the
osmotic pressure. Several food vacuoles also may be seen.
The active feeding and growing stage of the protozoa are called the trophozoite
(G.trophos-nourishment). The cell may obtain nourishment from the
environment by diffusion or by active transport across the plasma membrane.
Larger food particles are taken in by phagocytosis through pseudopodia. Some
species ingest food through
Protozoa: General Features of special mouth-like structures or cytostomes.
Minute droplets of food may also enter by pinocytosis. Several species possess
a resting or resistant cystic stage which enables prolonged survival under
unfavorable conditions. The cystic stage may also involve reproduction by the
nucleus dividing once or more to give rise to daughter

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trophozoites on excystation. The cyst is usually the infective stage for the
vertebrate host.
Reproduction is usually asexual. The most common method is binary fission by
the mitotic division of the nucleus, followed by the division of the cytoplasm. In
amoebae, division occurs along any plane, but in flagellates, the division is
along the longitudinal axis and in ciliates in the transverse plane. Some
protozoa, as for instance the malaria parasites exhibit schizogony in which the
nucleus undergoes several successive divisions within the schizont to produce a
large number of merozoites. Sexual stages are seen in ciliates and Sporozoa. In
ciliates, the sexual process is conjugation in which two organisms join together
and reciprocally exchange nuclear material. In Sporozoa, male and female
gametocytes are produced, which after fertilization form the zygote giving rise
to numerous sporozoites by sporogony.
SubPhylum Sarcomastigophora
Simple protozoa that have no fixed shape. They are classified under the
Phylum-. The cytoplasm is bounded by a unit membrane and can be
differentiated into an outer ectoplasm and an inner endoplasm. Pseudopodia are
formed by the ectoplasm thrusting out, being followed by the endoplasm
flowing in, to produce blunt projections. Pseudopodial processes appear and
disappear, producing quick changes in the shape of the cell. These are
employed for locomotion and engulfment of food by phagocytosis. Amoebae
may be free-living or parasitic. A few of the free-living amoebae can, on
occasion act as human pathogens, producing meningoencephalitis and other
infections. Some of them can act as carriers of pathogenic bacteria. The
parasitic amoebae inhabit the alimentary canal.

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Parasitic Amoebae
Parasitic amoebae belong to the following genera: Genus Species:
1. Entamoeba:
a) E.histolytica.
b) Entamoeba dispar.
c) E.coli.
d) E.polecki.
e) E.hartmanni.
f) Entamoeba gingivalis.
2. Endolimax.
a) E.nana.
3. Iodamoeba I.butschlii.
4. Dientamoeba.
a) D.fragilis.
lecture2 Entamoeba histolytica
Is an important human pathogen, causing amoebic dysentery as well as hepatic
amoebiasis and other extraintestinal lesions. E.hartmanni is nonpathogenic,
though it resembles E. histolytica very closely except for its smaller size and
was therefore known as the ‘small race’ of E. histolytica. E.polecki a natural
parasite of pigs and monkeys may sometimes infect humans causing diarrhoea.
E. coli is a common commensal in the colon and its importance is that it may be
mistaken for E.histolytica. E.gingivalis is present in the mouth, being found in
large numbers when the oral hygiene is poor. It has no cystic stage and so the
trophozoites depend for transmission on direct oral contact as in kissing, air-
borne spread through salivary
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droplets and fomites such as shared drinking and eating utensils.
It is generally nonpathogenic, though it has been claimed that it contributes to
periodontal disease. All the genera of intestinal amoebae other than Entamoeba
are nonpathogenic commensals, except D.fragilis, which may occasionally
cause chronic, but mild intestinal symptoms. Intestinal amoebae can be
differentiated based on their morphological features.
History
History Entamoeba histolytica was discovered in 1875 by Losch in the
dysenteric feces of a patient in St Petersburg, Russia. He also observed it in
colonic ulcers at autopsy and produced dysentery in a dog by inoculation
through the rectum. In 1890, William OsIer reported the case of a young man
with dysentery who later died of liver abscess. Councilman and Lafleur in 1891
established the pathogenesis of intestinal and hepatic amoebiasis and
introduced the terms ‘amoebic dysentery’ and ‘amoebic liver abscess.
Geographical Distribution E. histolytica is world-wide in prevalence. It is much
more common in the tropics than elsewhere, but it has been found wherever
sanitation is poor, in all climatic zones, from Alaska (61° N) to the Straits of
Magellan (52°S). It has been reported that about 10 per cent of the world’s
population and 50 per cent of the inhabitants of some developing countries may
be infected with the parasite. The infection is not uncommon even in affluent
countries, about 1 per cent of Americans being reported to be infected. While
the large majority of the infected are asymptomatic, invasive amoebiasis causes
disabling illness in an estimated 50 million persons and death in 50,000
annually, mostly in the tropical belt of Asia. Africa and Latin America. It is the
third leading parasitic cause of mortality, after malaria and schistosomiasis. E.
histolytica is found in the human colon.

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Natural infection also occurs in monkeys, dogs and probably in pigs also but
these animals do not appear to be relevant as sources of human infection.
Infection is mostly asymptomatic. It commonly occurs in the lumen of the colon
as a commensal, but sometimes invades the intestinal tissues to become a
pathogen.

Morphology
E. histolytica occurs in three forms:
a. Trophozoite.
b. Precystic.
c. Cystic stages.

(Figs. 1 A to E) Entamoeba histolytica: (A) Trophozoite; (B) Precystic stage; (C) Uninucleate.
(D) Binucleate cyst; and (E) Mature quadrinucleate cyst.

Trophozoite
Trophozoite is the vegetative or growing stage of the parasite. It is the only
form present in tissues. It is irregular in shape and varies in size from 12- 60
µm; average being 20 µrn. It is large and actively motile in freshly- passed
dysenteric stool, while smaller in convalescents and carriers.The parasite, as it
occurs free in the lumen as a commensal is generally smaller in size, about 15-
20 µm and has been called the minuta form.
Cytoplasm: Outer ectoplasm is clear, transparent and refractile. Inner
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endoplasm is finely granular, having a ground glass appearance. The endoplasm
contains nucleus, food vacuoles, erythrocytes, occasionally leukocytes and
tissue debris.
Pseudopodia are finger-like projections formed by sudden jerky movements of
ectoplasm in one direction, followed by the streaming in of the whole
endoplasm.
Typical ameboid motility is a crawling or gliding movement and not a free
swimming one. The direction of movement may be changed suddenly, with
another pseudopodium being formed at a different site, when the whole
cytoplasm flows in the direction of the new pseudopodium. The cell has to be
attached to some surface or particle for it to move. In culture tubes, the
trophozoites may be seen crawling up the side of the glass tube.
Pseudopod formation and motility are inhibited at low temperatures.
Nucleus is spherical 4- 6 µm in size and contains central karyosome,
surrounded by clear halo and anchored to the nuclear membrane by fine
radiating fibrils called the Linin network, giving a cartwheel appearance. The
nucleus is not clearly seen in the living trophozoites, but can be clearly
demonstrated in preparations stained with iron hematoxylin. Nuclear membrane
is lined by a rim of chromatin distributed evenly as small granules. The
trophozoites from acute dysenteric stools often contain phagocytosed
erythrocytes. This feature is diagnostic as phagocytosed red cells are not found
in any other commensaJ intestinal amebae. The trophozoites divide by binary
fission in every 8 hours.Trophozoiles survive up to 5 hours at 37°C and are
killed by drying, heat and chemical sterilization. Therefore, the infection is not
transmitted by trophozoites. Even if live trophozoites from freshly-passed
stools are ingested, they are

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rapidly destroyed in stomach and cannot initiate infection.

Precystic Stage
Trophozoites undergo encystment in the intestinal lumen. Encystment does not
occur in the tissues nor in feces outside the body. Before encystment, the
trophozoite extrudes its food vacuoles and becomes round or oval, about 10- 20
µmin size. This is the precystic stage of the parasite. It contains a large
glycogen vacuole and two chromatid bars. It then secretes a highly retractile
cyst wall around it and becomes cyst.
Cystic Stage
The cyst is spherical in shape about 10-20 µmin in size. The early cyst contains
a single nucleus and two other structures a mass of glycogen, and 1- 4
chromatoid bodies or chromatoid bars, which are cigar-shaped. The chromatoid
bodies are so-called because they stain with hematoxylin, like chromatin. As the
cyst matures, the glycogen mass and chromidial bars disappear and the nucleus
undergoes two successive mitotic divisions to form two and then four nuclei.
1he mature cyst is, thus quadrinucleate. The cyst wall is a highly refractile
membrane, which makes it highly resistant to gastric juice and unfavorable
environmental conditions.The nuclei and chromidial bodies can be made out in
unstained films, but they appear more prominently in stained preparations. With
iron hematoxylin stain, nuclear chromatin and chromaroid bodies appear deep
blue or black, while the glycogen mass appears unstained. When stained with
iodine, the glycogen mass appears golden brown, the nuclear chromatin and
karyosome bright yellow, and the chromatoid bodies appear as clear space,
being unstained.
Life Cycle

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Entamoeba histolytica passes its life cycle only in one host (man).
Cysts and trophozoites are typically found in diarrheal stool. Infection by
Entamoeba histolytica occurs by ingestion of mature cysts in fecally
contaminated food, and water. Excitation occurs in the small intestine and
trophozoites are released, which migrate to the large intestine. The trophozoites
multiply by binary fission and produce cysts, and both stages are passed in the
feces. Because of the protection conferred by their walls, the cysts can survive
days to weeks in the external environment and are responsible for transmission.
Trophozoites passed in the stool are rapidly destroyed once outside the body,
and if ingested would not survive exposure to the gastric environment. The
cysts passing in stool of infected individuals. In some patients, the
trophozoites invade the intestinal mucosa or, through the bloodstream,
extraintestinal sites such as the liver, brain, and lungs, with resultant pathologic
manifestations.

Figure ( 2): Life cycle of E. histolytica.

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Infective Form
Mature quadrinucleate cyst passed in feces of convalescents and carriers. The
cysts can remain viable under moist conditions for about I0 days.

Mode of Transmission
Man acquires infection by swallowing food and water contaminated with cysts.
Excystation: Is hapened when the cyst reaches cecum or lower part of the
ileum, due to the alkaline medium, the cyst wall is damaged by trypsin.
Metacyst mean the cytoplasm gets detached from the cyst wall and ameboid
movements appear causing a tear in the cyst wall, through which quadrinucleate
ameba is liberated.
Metacystic Trophozoites mean the nuclei in the metacyst immediately undergo
division to form eight nuclei, each of which gets surrounded by its own
cytoplasm to become eight small amebulae or metacystic trophozoites.
Pathogenesis and Clinical Features
E. histolytica causes intestinal and extraintestinal amebiasis. Incubation period
is highly variable. On an average, it ranges from 4 days to 4 months. Amebiasis
can present in different forms and degree of everity, depending on the organ
affected and the extent of damage caused.

Intestinal Amebiasis
The lumen-dwelling amebae do not cause any illness. They cause disease only
when they invade the intestinal tissues. This happens only in about 10% of
cases of infection, the remaining 90% being

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Asymptomatic.
Not all strains of E. hislolylica are pathogenic or invasive. Differentiation
between pathogenic and nonpathogenic strains can be made by susceptibility to
complementmediated lysis and phagocytic activity or by the use of genetic
markers or monoclonal antibodies and zymodeme analysis.
Amebic ulcer
Is the typical lesion seen in intestinal amebiasis.The ulcers are multiple and are
confined to the colon, being most numerous in the cecum and next in the
sigmoidorectal region. The intervening mucous membrane between the ulcers
remains healthy. Ulcers appear initially on the mucosa as raised nodules with
pouting edges measuring pinhead to l inch. They later break down discharging
brownish necrotic material containing large numbers of trophozoites.

Figure (3): Flask-shaped amebic ulcer

The typical amebic ulcer is flask-shaped in cross section, with mouth and neck
being narrow and base large and rounded. Multiple ulcers may coalesce to form
large necrotic lesions with ragged and undermined edges and are covered
with brownish slough.

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Ameboma
Occasionally, a granulomatous pseudotumoral growth may develop on the
intestinal wall by rapid invasion from a chronic ulcer. This amebic granuloma
or ameboma may be mistaken for are malignant tumor. Amebomas are most
frequent at cecum and rectosigmoid junction.
Laboratory diagnosis of Entamoeba histolytica
Stool examination: Bloody and\or mucoid stool sample to see trophozoite and
cyst stages by normal saline and Lugol's iodine direct smear respectively.
The sample (stool) should be collected into a wide mouth container and
examined without delay, and should be inspected via: Macroscopy and
Microscopy.
Macroscopy: The stool is characterized by:
Foul-smelling, copious, semiliquid, brownish -black in color, intermingled with
blood and mucus, and it does not adhere to the container.
Microscopy
Saline preparation:
 The cellular exudate is scanty and consists of only the nuclear masses
(pyknotic bodies) of a few pus cells, epithelial cells and macrophages.
 The RBCs are in clumps and yellow or brown -red in color.
 Charcot-Leyden crystals are often present. These are diamond-shaped,
clear and refractile crystals.
 Actively motile trophozoites throwing pseudopodia can be demonstrated
in freshly-passed stool. Presence of ingested RBCs clinches the identity
of E. hislolytica. Nucleus is not visible but a faint outline may be
detected.

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  Cyst has a smooth and thin cell wall and contains round refractile
chromatoid bars. Glycogen mass is not visible.
Iodine preparation: For the demonstration of cysts or dead trophozoites,
stained preparations may be required for the study of the nuclear character.
Iodine-stained preparation is commonly employed for this purpose. The
trophozoite of E. histolytica stains yellow to light brown. The nucleus is clearly
visible witl1 a central karyosome. The cytoplasm of the cystic stage shows a
smooth and hyaline appearance. Nuclear chromatin and karyosome appear
bright yellow. Glycogen masses stain golden brown and chromatoid bars are
not stained.
The trichrome stain is useful to demonstrate intracellular features of both
trophozoites and cysts. Since the excretion of cysts in the stool is often
imminent, at least three consecutive samples should be examined.
Mucosal scrapings: Scraping obtained by sigmoidoscopy is often contributory.
The examination method includes a direct wet mount and iron hematoxylin and
immunofluorescent staining with anti-E.hislolytica antibodies.
Stool culture: Stool culture is a more sensitive method in diagnosing chronic
and asymptomatic intestinal amebiasis. The culture of stools gives higher
positivity for E. histolytica as compared to direct examination.
Media used for stool culture include to diagnosis the E. histolytica: Boeck and
Drbohlav's biphasic medium. NIH polygenic medium, Craig's medium, Nelson's
medium, Robinson's medium, and Balamuth's medium.
Serodiagnosis: Serological tests become positive only in invasive amebiasis.

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Antibody detection: Amebic antibodies appear in serum only in the late stages
of intestinal amebiasis. Tests for antibodies in scrum help in the diagnosis of
mainly extraintestinal infections. Serological tests include indirect
hemagglutination assay (IHA), indirect fluorescent antibody (IFA), enzyme-
linked immunosorbent assay (ELISA), counter-current immunoelectrophoresis
(CIEP), and latex agglutination tests.
Serum with an antibody titer of 1:256 or more by IHA and 1:200 by IFA are
considered to be significant.
Amebic antigen detection: Amebic antigen in serum are detected only in
patients with active infections and disappears after clinical cure. Antigen like
Lipophosphoglycan (LPG) amebic lectin, serine-rich E. histolytica protein
(SREHP) are detected using monoclonal antibodies by ELISA. Molecular
diagnosis: Recently, deoxyribonucleic acid (DNA) probes and
radioimmunoassay have been used to detect E. histolytica in the stool. It is a rapid
and specific method.
Treatment
Both luminal and tissue amebicides: Metronidazole and related compounds
like tinidazole and om imidazole act on both sites and are the drug of choice for
treating amebic colitis and amebic liver abscess.

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 Lecture3 Entamoeba moshkovskii
Entamoeba moshkovskii is also morphologically indistinguishable from E.
histolytica and E. dispar (may be the third subspecies of E. histolytica).
This species was first described from Moscow sewage by Tshalaia in 1941 and
was thereafter reported to occur in many different countries including India. It
can be distinguished from E. histolytica by isoenzyme analysis, molecular
methods, and detection of lectin antigen.Though it is a non-pathogen harboring
in the intestine recent studies from Bangladesh and India have reported E.
moshkovskii as a sole potential pathogen in patients presenting with
gastrointestinal symptoms and/or dysentery, highlighting the need for further
study to investigate the pathogenic potential of this organism.

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Entamoeba moshkovskii is found worldwide and is generally considered to be a
free-living ameba. Based on microscopic morphology, this organism is
indistinguishable from E. histolytica and E. dispar, except in cases of invasive
disease when E. histolytica contains ingested RBCs. Although first isolated
from sewage, E. moshkovskii can also be found in clean riverine sediments to
brackish coastal pools. Apparently, there are some differences that separate this
organism from E. histolytic and E. dispar. However, these differences pertain
to physiology rather than morphology; E. moshkovskii is osmotolerant, can be
cultured at room temperature, and is resistant to emetine.

Life Cycle and Morphology

The life cycle is essentially identical to that of E. dispar, and morphological
differences are minimal to none. In wet preparations, trophozoites usually range
in size from 15 to 20 μm and cysts normally range in size from 12 to 15 μm. It
is important to remember that on the permanent stained smear there is a certain
amount of artificial shrinkage due to dehydration; therefore, all of the
organisms, including pathogenic E. histolytica, may be somewhat smaller (from
1 to 1.5 μm) than the sizes quoted for the wet-preparation measurements.
Morphology of Trophozoites
Trophozoites do not ingest RBCs, and the motility is similar to that of both E.
histolytica and E. dispar. Nuclear and cytoplasmic characteristics are very
similar to those seen in E. histolytica; however, trophozoites of E. moshkovskii
do not contain ingested RBCs.

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Morphology of Cysts: Nuclear characteristics and chromatoidal bars are
similar to those in E. histolytica and E. dispar.

Entamoeba Coli
Entamoeba coli was first described by Lewis (1870) and Cunningham (1871)
in Kolkata and its presence in healthy persons was reported by Grassi (1878). It
is worldwide in distribution and a nonpathogenic commensal intestinal ameba.
It is a larger than E. histolytica about 20-50 µm with sluggish motility and
contains ingested bacteria but no red cells. The nucleus is clearly visible in
unstained films and has a large eccentric karyosome and thick nuclear
membrane lined with coarse granules of chromatin. Cysts are large, 10- 30 µm
in size, with a prominent glycogen mass in the early stage. The chromatoid
bodies are splinter-Like and irregular. The mature cyst has eight nuclei.

Life cycle
The life cycle is the same as in E. histolytica except that it remains a luminal
commensal without tissue invasion and is nonpathogenic.

Figuer (4)A to C: Schematic diagram of the morphological forms of Entamoeba coli.

(A) Vegetative form; (B) Binucleate cyst; and (C) Eight-nucleate cyst

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Entamoeba hartmanni
Entamoeba hartmanni occurs wherever E. histolytica is found. It is now
considered to be a separate species of nonparhogenic commensal intestinal
ameba. It is much smaller than E. histolytica, the trophozoirc measuring 4- 12
µm and cyst 5-10 µmin size. Trophozoites do nor ingest red cells and their
motility is less vigorous. The cyst resembles that of Endolimax nana.

Figure (5): Trophozoite of Entamoeba hartmanni.

Entamoeba Gingivalis
Entamoeb gingivalis was the first ameba of humans, discovered by Gros in
1849. It is global in distribution. Only the trophozoite is found; the cystic stage
being apparently absent. The trophozoite is about 10-20 µm, actively motile
with multiple pseudopodia. The cytoplasm contains food vacuoles with ingested
bacteria, leukocytes, and epithelial cells. Nucleus is round with central
karyosome lined by coarse chromatin granules. The ameba lives in gingival
tissues and is abundant in unhygienic mouths. It is a commensal and is not
considered to cause any disease. It is transmitted by

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direct oral contact. E. gingivalis have been found in bronchial washings and
vaginal and cervical smears, where it can be mistaken for E.histolytica.

lecture4 Endolimax Nana

This common commensal ameba is widely distributed. It lives in the human
intestine.The trophozoite is small (nana: small), less than 10 µm in size with
sluggish motility.The nucleus has a conspicuous karyosome connected to the
nuclear membrane by one or none coarse strands. The cyst is small, oval, and
quadrinucleate with glycogen mass and chromidial bars, which are
inconspicuous or absent. It is nonpathogenic.

Figure (6): Endolimax nana. (A) Vegetative form: and (B) Quadrinucleate cyst.

Iodamoeba Butschlii
This is widely distributed, though less common than E. coli and E. nana.
Trophozoite is small, 6- 12 µm, with a conspicuous nucleus. The prominent
karyosome is half the size of the nucleus, having a bull's eye appearance. The
cyst is oval, uninucleate and has a prominent iodine staining glycogen mass
(iodophilic body). Hence, the name lodamoeba. It is nonpathogenic. The
comparative morphology of amebae infecting humans is illustrated in.

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Figure (7): Iodamoeba butschlii. (A) Vegetative form: and (B) Cyst.

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 lecture 5 ;Subphylum Mastigophora
Parasitic protozoa which possess whip-like flagella as their organs of
locomotion are classified under the Phylum-Sarcomastigophora, Subphylum-
Mastigophora, class Zoomastigophorea (from mastix-whip, photos-bearing).
Depending on their habitat, they can be considered under two headings:
1. Lumen-dwelling flagellates (Intestinal, Oral and Genital Flagellates):
Flagellates found in the alimentary and urogenital tracts.
2. Haemoflagellates: Flagellates found in blood and tissues.

Flagellates classified as:

Subphylum: Mastigophora
Class: Zoomastigophora (mastix: whip)
Depending on their habitat, they can be considered under:
Lumen-dwellingflagellates: Flagellates found in the alimentary tract and
urogenital tract.
Hemoflagellates: Flagellates found in blood and tissues.
Most luminal flagellates are nonpathogenic commensals.

25
Two of them cause clinical diseases: (1) Giardia lamblia, which can cause
diarrhea, and (2) Trichomonas vaginalis, which can produce vaginitis and
urethritis.

Intestinal Flagellates
Most luminal flagellates are nonpathogenic commensals. Two of them cause
clinical disease, Giardia lamblia which can cause diarrhea, and Trichomonas
vaginalis which can produce vaginitis and urethritis. Intestinal flagellates found
in humans are listed below, with the sites affected by them shown in
parenthesis.
1. Giardia lamblia (duodenum, jejunum).
2. Trichomonas vaginalis (vagina, urethra).
3. Trichomonas. tenax (mouth).
4. Trichomonas. hominis (caecum).
5. Chilomastix mesnili (caecum).
6. Dientamoeba fragilis (See Chapter-2).

26
 Lecture5; Giardia Lamblia
History and Distribution
This flagellate was observed by Leeuwenhoek (1681) in his own stools and was
thus one of the earliest of protozoan parasites to have been recorded. It is
named Giardia after Professor Giard of Paris and lamblia after Professor Lambl
of Prague who gave a detailed description of the parasite. Worldwide in
distribution, it is the most common intestinal protozoan pathogen. Infection
may be asymptomatic or cause diarrhoea.
Giardiasis (gee-ar-die-a-sis with a soft "G") is an infection of the small
intestine that is caused by the parasite, Giardia duodenalis, also known as
Giardia lamblia and Giardia intestinalis.
It is the most common cause of parasitic gastrointestinal disease; it is estimated
that 20,000 cases of giardiasis occur each year in the U.S., and there is a 20%
to 40% prevalence in the world's population.
Giardia lamblia exists in two forms, an active form called a trophozoite,
and an inactive form called a cyst. The active trophozoite attaches to the lining
of the small intestine with a "sucker" and is responsible for causing the signs
and symptoms of giardiasis.
The trophozoite cannot live long outside of the body, therefore it cannot spread
the infection to others. The inactive cyst, on the other hand, can exist for
prolonged periods outside the bod.
When it is ingested, stomach acid activates the cyst, and the cyst develops into
the disease-causing trophozoite. It takes ingestion of only ten cysts to cause
infection. Trophozoites are important not only because they cause the
symptoms of giardiasis, but also because they produce the cysts that exit the
body in the feces and spread the infection to others.
27
Cysts of Giardia are present in the feces of infected persons. Thus, the
infection is spread from person to person by contamination of food with feces,
or by direct fecal-oral contamination. Cysts also survive in water, for example
in fresh water lakes and streams. As a result, giardiasis is the mostcommon
cause of water-borne, parasitic illness in the U.S.
Domestic mammals (for example, dogs, cats, calves) and wild mammals (for
example, beavers) can become infected with Giardia; however, it is not clear
how often domestic or wild mammals transmit giardiasis to humans.

Morphology
It occurs in two forms: trophozoite and cyst.

Figure (8): Giardia lamblia (schematic diagram): A- trophozoite front view; B- trophozoite
lateral view; C-cyst.

Trophozoite
The trophozoite has a falling leaf-like motility, usually measures 10–20 µm in
length and 5–15 µm in width.

28
Shape: In front view, it is pear shaped (or tear drop or tennis racket shaped)
with rounded anterior end and pointed posterior end. Laterally, it appears as a
curved portion of a spoon (sickle shaped).
It is convex dorsally while the ventral surface has a concavity bearing a bilobed
adhesive disc. Hence, it appears as sickle-shaped in the lateral view.
Trophozoite is bilaterally symmetrical; on each side from the midline it bears.
One pair of nuclei, Pair of median bodies. Four pairs of basal bodies or
blepharoplast (from which the axoneme arises).
Four pairs of flagella—two lateral, one ventral and one caudal pair of flagella,
pair of parabasal bodies (connected to basal bodies through which the axoneme
passes) and pair of axoneme or axostyle (the intracellular portion of the
flagella).

Cyst
Giardia cyst is oval shaped, measures 11–14 µm in length and 7–10 µm
in width, it contains four nuclei and remnants of axonemes, basal bodies and
parabasal bodies and it is the infective form as well as the diagnostic form of
the parasite.

Life cycle
Giardia lamblia possesses a simple life cycle which is composed of 2 stages: (1)
the trophozoite and (2) the cyst.

29
 Figure (9): Life cycle of Giardia lamblia

Host: Giardia lamblia life cycle in one host.

Infective form: Mature cyst.
Mode of transmission: Man acquires infection by ingestion of food and water
contaminated with mature cysts or rarely by sexual route (mainly in
homosexuals).

Development of Life cycle in Man:

Excystation: Two trophozoites are released from each cyst in the duodenum
within 30 minutes of entry.
 Multiplication: Trophozoites multiply by longitudinal binary fission in
the duodenum.

30
  Adhesion: Trophozoites adhere to the duodenal mucosa by the bilobed
adhesive ventral disc.
This is achieved by the microtubules of median bodies, contractile proteins and
lectins present on the surface of adhesive disc that binds to the intestinal
receptors.
 In active stage of the disease, sometimes the trophozoites are excreted in
diarrhea stool.
 Encystation: Gradually when the trophozoites pass down to the large
intestine, encystation begins.

Pathogenicity and Clinical Features

Giardia lamblia is typically seen within the crypts of duodenal and jejunal
mucosa. It does not invade the tissue but remains tightly adhered to the
intestinal epithelium by means of the Sucking-disk.
They may cause abnormalities of villous architecture by cell apoptosis and
increased lymphatic infiltration of lamina propria. Loss of brush border
epithelium of the intestine leads to a deficiency of enzymes including
disaccharides.
.
Often they are asymptomatic, but in some cases, Giardia may lead to mucus
diarrhea, fat malabsorption (steatorrhea), dull epigastric pain, belching, and
flatulence. The stool contains excess mucus and fat but no blood.
Children may develop chronic diarrhea, malabsorption of fat, vitamin A,
vitamin B12, folic acid, protein, sugars like xylose disaccharides, weight loss
and sprue-like syndrome.

31
Signs and symptoms usually appear one to three weeks after exposure and may
include: Watery, sometimes foul-smelling diarrhea that may alternate with soft,
greasy stools, fatigue or malaise, abdominal cramps and bloating, gas or
flatulence, nausea and weight loss.

Laboratory diagnosis of Giardia lamblia

- Stool examination-detects cysts and trophozoites.
- Entero-test.
Antigen detection in stool (copro-antigen) by ELISA and rapid
immunochromatography test (ICT).
Antibody detection in serum by ELISA and IFA, culture, molecular method—
PCR, and radiological findings—barium meal, X-ray.

Stool examination
Multiple stool samples (at least 3) should be tested before a negative result is
reported.
Wet Mount In bright-field microscopy, cysts appear ovoid to ellipsoid in shape
and usually measure 11 to 14 µm (range: 8 to 19 µm). Immature and mature
cysts have 2 and 4 nuclei, respectively. Intracytoplasmic fibrils are visible in
cysts.
Giardia cysts can be demonstrated by iodine and saline wet mount preparations
but they cannot differentiate active disease from carriers.

32
Fig .( 10) Wet mount for Giardia lamblia cyst stage with saline and iodine

Demonstration of the trophozoites with falling leaf like motility by saline mount
indicates active stage of the disease. Giardia adheres firmly to the duodenal
mucosa by adhesive disc leading to intermittent shedding. Hence, repeated stool
examination (at least three consecutive samples) should be done.
Sensitivity varies from 60% to 80% with one stool and more than 90% after
three stools examination.
Concentration techniques like zinc sulfate floatation or formalin ether
sedimentation methods are employed to increase the chance of detection.
Duodenal sampling: If stool examination is negative, then direct duodenal
samples like aspirates (obtained by entero-test) or biopsy (done by endoscopy)
should be processed.

33
Fig .( 11) Trophozoite of Giardia lamblia (A) saline mount front view; (B) Giemsa stain
Entero-test
It uses a gelatin capsule attached to a thread.

Antigen Detection in Stool (Copro-antigen)

The enzyme-linked immunosorbent assay (ELISA) and direct fluorescent
antibody tests (DFA) are available using labeled monoclonal antibodies against
cyst wall protein antigens.

Molecular methods
Detection of Giardia nucleic acid by polymerase chain reaction (PCR) or by
gene probes is highly sensitive and specific

Treatment
Several drugs can be used to treat Giardia infection. Effective treatments
include metronidazole, tinidazole, and nitazoxanide. Other medications include
paromomycin, quinacrine, and furazolidone.

Lecture6; Trichomonas vaginalis

Geographical Distribution: It is encountered in all climates and all social

groups.
Habitat: In female it is found mainly in vagina and in male it is in
urethra.

34
Morphology: It is found only in trophozoite form which bears following
characters, pear shaped measuring 10 to 30 µ × 5 to 10 µ, it has short
undulating membrane which comes up to the middle of the body,
possesses 4 anterior flagellae, a prominent axostyle which bifurcates the body
into two and projects posteriorly, there is a costa, parabasal body, rounded
nucleus (anteriorly), chromatin granules are present all over, more densely near
costa and axostyle, flagellae give characteristic webbing or rotatory motility,
and it multiplies by binary fission in longitudinal axis.

Fig.( 12 )Trichomonas vaginalis

Mode of Transmission
It is primarily a venereal disease in which transmission can also be from
person-to-person contact. However, newborns may get infected during birth.
Fomites (such as clothes, utensils, and furniture) also form another way of
transmission of infection.
Incubation Time: It varies from 4 to 30 days.

Pathogenesis and Pathology

Within a few days following the introduction of viable Trichomonas vaginalis
into the vagina, the proliferating colonies of this flagellate cause
35
degeneration and desquamation of the vaginal epithelium. It is followed by
leukocytic inflammation of the tissue layer. Very large numbers of
trichomonads and leukocytes are now present in the vaginal secretion, which is
liquid, greenish, or yellow, and covers the mucosa down to the urethral orifice,
vestibular glands, and clitoris. As the acute condition changes to the chronic
stage, the secretion loses its purulent appearance due to a decrease in the
number of trichomonads and leukocytes, Trichomonas vaginalis in male
genitalia may be symptomless or may be responsible for an irritating, persistent,
or recurring urethritis.

Clinical Picture
The vaginal secretion is extremely irritating, almost unbearable and is
constantly flowing. The symptoms may continue from a few days to many
months. After each menstruation there is a tendency for acute stage to recur.
The chronic condition transforms into latent one and secretions become normal
with no manifestation although trichomonas are still present. Difference in the
intensity of symptoms may be due to differences in virulence of strains of this
organism. In male patients it may be symptomless or may cause urethritis and
prostatitis.

Laboratory Diagnosis
In female patient, Trichomonas vaginalis maybe demonstrated in sedimented
urine, vaginal secretion, or from vaginal scraping.
In male patient Trichomonas vaginalis maybe found in the centrifuged urine and
prostatic secretions following massage of the prostatic gland.

36
However, care should be taken to prevent contamination of the specimen with
feces, since Trichomonas hominis maybe has seen and thus misdiagnosed as
Trichomonas vaginalis. The smear is stained using Giemsa, PAS, Papanicolaou,
Leishman, Diff Quick and acridine orange.
Treatment
Trichomoniasis is usually treated quickly and easily with antibiotics. Most
people are prescribed an antibiotic called metronidazole which is very effective
if taken correctly. You'll usually have to take metronidazole twice a day, for 5
to 7 days. Sometimes this antibiotic can be prescribed in a single, larger dose.

Trichomonas tenax
Harmless commensal of the oral cavity, periodontal area, carious cavities of the
tooth, tonsillary crypts, etc. Measures 5 to 10 µ, i.e. smaller in size as compared
to Trichomonas vaginalis. Transmission is through fomites, salivary droplets,
and kissing.

37
 lecture 7;Hemoflagellate

Including genus: Leishmania and Trypanosoma (blood tissue species): There

are four morphological forms of clinical significance associated with the
hemoflagellates: Amastigote, promastigote, epimastigote and trypomastigote.

General characteristics
1- They live in the blood and tissues of man and other vertebrate hosts and
in the gut of the insect vector.
2- Members of this family have a single nucleus, a kinetoplast and a single
flagellum.
3- Nucleus is round or oval and is situated in the central part of the body.
4- Kineloplast consists of a deeply staining parabasal body and adjacent
dot-Like blepharoplast.
5- The parabasal body and blepharoplast are connected by one or more thin
fibr.
6- Flagellum is a thin, hair-like structure, which originate from the
blepharoplast.
7- The portion of the flagellum ,which is inside the body of the parasite and
extends from the blepharoplasl to surface of the body is known as
axoneme.
8- A free flagellum at the anterior end traverses on the surface of the
parasite as a narrow undulating membrane.
9- Hemoflagellates exist in two or more of four morphological stages.

38
The transmission of hemoflagellates
Is accomplished by the bite of an arthropod vector. Flagellate protozoa found in
blood or tissues of human and there are two genera of medical importance
(Leishmania and Trypanosoma). The major difference between these two
genera is that primary diagnostic form found in Leishmania is the amastigote,
whereas that of Trypanosoma is the trypomastigote
1. Amastigotes: It is Roundish to oval in shape, Consist of a nucleus and
kinetoplast. The large single nucleus is typically located off-center .The
dotlike blepharoplast is attached to a small axoneme, this axoneme
extends to the edge of the organism.The single parabasal body is located
adjacent to the blepharoplast.
2. Promastigotes: It is Long and slender in appearance. The large single
nucleus is located in or near the center .The kinetoplast is located in the
anterior end of the organism .A single free flagellum extends anteriorly
from the axoneme.
3. Epimastigotes: It is long and slightly wider than promastigote form.The
large single nucleus is located in posterior end .The kinetoplast located
anterior to the nucleus .Undulating membrane extending half of the body
length .A single free flagellum extends anteriorly from the axoneme.
4. Trypomastigotes: It is C or U shape in stained blood films .Long and
slender in appearance .One nucleus located anterior to the kinetoplast
.The kinetoplast is located in the posterior end of the organism
.Undulating membrane extending entire body length .A single free flagellum
extends anteriorly from the axoneme when present

39
 Table(1): Morphological form of hemofagellate

Genus Leishmania:
Leishmaniasis There are many different species of Leishmania and the disease
that they cause. directly linked to the species of Leishmania with which a
person Several species of Leishmania are pathogenic for man: L. donovani
causes visceral leishmaniasis (Kala-azar, black disease, dumdum fever); L.
tropica cause cutaneous leishmaniasis (oriental sore, Delhi ulcer, Aleppo, Delhi
or Baghdad boil); and L. braziliensis (also, L. mexicana and
L. peruviana) are etiologic agents of mucocutaneous leishmaniasis .

Life Cycle
Leishmania are transmitted by arthropod. In this case it is a small biting fly
known as a sand fly. Leishmaniae spend part of their life cycle in the gut of the
sandfly, but their life cycle is completed in a vertebrate host. Within the sandfly
gut, the protozoa are carried as extracellular promastigotes, these
parasites multiply in the gut and migrate toward the pharynx. Sandflies
40
transferred promastigotes to the vertrebrate host when the sandfly takes a meal
blood by expelling leishmaniae into the bite wound of the mammalian host.
From where they pass into the blood and tissues of the human host.

Figure (12): Life Cycle of leishmamia spp.

Pathogensis and clinical finding

Leishmaniasis is a parasitic disease caused by several species of genus
Different species of leishmania cause different disease.
A. L. donovani causes visceral leishmaniasis also called Kala-azar and Dum
Dum fever. Spleenomegaly & hepatomegaly the infection is generalized
and the parasite is distributed in the internal organs. The parasite may
also cause a variety of skin lesions (dermal leishmaniasis) without any
visceral manifestations.

41
Laboratory Diagnosis 1. Giemsa-stained slides of blood , bone marrow
, lymph node aspirates and biopsies of the infected areas for the diagnosis of
amastigote forms.
2. Culture of blood, bone marrow and other tissues these samples show
the promastigote forms.
3. Serological tests.
B. L. tropica: causes tropic sore or Baghdad boil, oriental sore and
cutenaeous
Leishmianiasis. The infection is limited to a local lesion of the skin and
subcutaneous tissues.

Figure (13): L. tropica and L.major amastigote a-intracellular b-intercellular.

Laboratory Diagnosis

42
 1. The specimen of choice for identify the amastigotes of Leishmania
braziliensis is a biopsy of the infected ulcer.
2. Microscopic examination of the Giemsa-stained preparations should
reveal the typical amastigotes. Promastigotes may be present when the
sample is collected immediately after introduction into the patient.
3. Culturing the infected material, which often demonstrated the
promastigote stage.
4. Serological tests.

Treatment
Pharmacologic therapies include the following:
 Pentavalent antimony (sodium stibogluconate or meglumine
antimonate): Used in cutaneous leishmaniasis

 Liposomal amphotericin B (AmBisome): Effective against pentavalent

antimony ̶ resistant mucocutaneous disease and visceral leishmaniasis

 lecture 8 SubPhylum Apicomplexa

 Coccidia
 Coccidia are small protozoans (one-celled organisms), which are
members of the class Sporozoa, an exclusively parasitic group that
typically requires alternation of sexual and asexual reproduction in the
life cycle.
The coccidia are characterized by a thick walled oocyst stage that is typically
excreted with the feces. Some coccidia (Cryptosporidium, Cyclospora,
Isospora) carry out their entire life cycle within the intestinal epithelial cells of
the host and are transmitted by the fecal-oral route. Other

43
coccidia (Sarcocystis, Toxoplasma) have a more complicated life cycle
involving tissue cysts and multiple hosts (ie, heteroxenous).
1- Locomotive organelles absent, the flagella present only in male game.
2- Life cycles are complex, with well-developed a sexual(which
produce merozoites , some of these merozoites differentiate into
macro and microgametes) and sexual stages (which produce oocysts.
3- Sporozoa produce special spore like cells called sporozoites.
4- It is intracellular parasite with complex cycle alternating between
humans and mosquitoes as in malaria, while in T.gondii which causes an
acute infection in human is acquired from cats and other animals.
5- Have similar independent gametocytes ,the male or microgametocyte
and female or macrogametocyte. The female produce a single
macrogamete and the male produce multiple gametes , followling an
oocyst is formed after fertilization.

44
 Lecture 9; Plasmodium spp

Figure (14): Life cycle of plasmodium spp.

Plasmodium falciparum
Plasmodium falciparum is the most important malaria parasite, found in the
tropics and sub-tropics, being responsible for approximately 50% of all malaria
cases. The incubation period of P. falciaprum malaria is the shortest, between 8
and 11 days and has a periodicity of 36 – 48 hours. It can be differentiated from
the other species by the morphology of different stages found in the
peripheral blood. In infections with Plasmodium
falciparum usually only young trophozoites and gametocytes are seen in
45
peripheral blood smears, the schizonts are usually found in capillaries sinuses
of internal organs and in the bone marrow. The disease runs an acute course and
often has lethal outcome. It is a significant cause of abortions and stillborns and
even death of non-immune pregnant women.

Life cycle
The aspects of the life cycle, which are specific to P. falciparum, are as follows:
a) It attacks all ages of erythrocytes so that a high density of parasites can
be reached quickly. In extreme cases up to 48% of the red blood cells can
be parasitised.
b) Multiple infections resulting in several ring forms in a corpuscle are not
uncommon.
c) The latter stages in the asexual cycle do not occur in the peripheral blood
as in other forms of malaria, so that only rings and crescents are found in
blood films. After 24 hours the ring forms and older trophozoites show a
tendency to clump together and adhere to the visceral capillary walls.
They become caught up in the vessels of the heart, intestine, brain or
bone marrow in which the later sexual stages are completed.

Morphology of Trophozoites
Red blood cells in Plasmodium falciparum infections are not enlarged and they
may have a spiky outline, common in cells, which have dried slowly. The
typical arrangement of cytoplasm in young trophozoites is the well-known ring
formation, which thickens and invariably contains several vacuoles as the
trophozoite develops. Chromatin is characteristically found

46
as a single bead, but double beads and small curved rod forms frequently
occur.

Maurer’s dots are slow to appear and are first seen as minute purplish dots, 6 or
less in number. The points become spots, still few in number and are now
characteristic enough to be recognised. Maurer describes them as fine ringlets,
loops or streaks. They are seldom absent from the red blood cells containing
large rings but the staining of the spots is very sensitive to pH and is seldom
seen if the pH falls below 6.8.
Trophozoites of P. falciparum can be found on the edge of the red blood cells.
These are known as acole

47
Gametocytes
Gametocytes are the sexual stage of the malaria parasite. Plasmodium
falciparum gametocytes appear in the peripheral circulation after 8 – 11 days of
patent parasitaemia and they have assumed their typical crescentic shapes. They
soon reach their peak density, and then decline in numbers, disappearing in
about 3 months as a rule.
The female form, or macrogametocyte, is usually slenderer and somewhat
longer than the male, and the cytoplasm takes up a deeper blue colour with
Giemsa stain. The nucleus is small and compact, staining dark red, while the
pigment granules are closely aggregated around it. The male form, or
microgametocyte, is broader than the female and is more inclined to be
sausage shaped. The cytoplasm is either pale blue or tinted with pink and the
nucleus, which stains dark pink, is large and less compact than in the female,
while the pigment granules are scattered in the cytoplasm around it. In humans,
gametocytes neither multiply, nor cause symptoms but they are the forms,
which are infective to the mosquito. When a female Anopheline mosquito takes
a blood meal, the male and female gametocytes continue their sexual
development.

Schizonts
Schizonts are rarely seen in the peripheral blood and their presence may
indicate a potentially serious parasitaemia. Schizonts have 8 – 36 merozoites
and a large mass of golden brown pigment (haemozoin) seen in the pre-
schizont and schizont stage.

48
Clinical Signs of Disease
Symptoms include headache, photophobia, muscle aches and pains, anorexia,
nausea and vomiting. Complications include severe anaemia cerebral malaria,
renal disease, black water fever, dysentery, pulmonary oedema and tropical
splenomegaly syndrome.

Plasmodium vivax
Introduction
Plasmodium vivax is found almost in all places, where malaria is endemic and
is the most predominant of malaria parasites. Causing 43% of all cases of
malaria in the world, it also has the widest geographical distribution. Although
the disease itself is not usually life threatening, it can cause severe acute illness.
Plasmodium vivax does not infect West Africans due to the fact that West
Africans do not possess the Duffy Antigen on the red blood cells, which the
parasite requires to enter the red blood cell. It has an incubation period of
between 10 and 17 days, which is sometimes prolonged to months or years due
to the formation of hypnozoites. It has a periodicity of 48 hours. Plasmodium
vivax infections are usually characterised by the presence of more than one
developmental stage in the peripheral blood film. The parasites parasitise young
enlarged erythrocytes and Schüffner’s dots develop on the erythrocyte
membrane.

Life cycle
The aspects of the life cycle, which are specific to P. vivax, are as follows:

49
 a) The degree of infectivity is low, only the young immature corpuscles are
infected; about 2% of erythrocytes are parasitised.
b) The periodicity of the asexual cycle is closely synchronised.
c) Hypnozoites develop in the liver, so that relapses may occur.

Morphology of Trophozoites
Most trophozoites of P. vivax are already several hours old when they appear in
peripheral blood and by that time the Schüffner’s dots are already visible. The
trophozoites are actively amoeboid and contain single or sometimes double
chromatin dots that are either circular or ovoid. As the trophozoites mature, the
Schüffner’s dots increase in number and size and the parasite changes from
large irregular rings to rounded or ovoid forms in mature trophozoites.

Gametocytes
Mature female gametocytes are large rounded parasites, which fill or nearly fill
the host cell. The cytoplasm is blue and fairly homogenous. The nuclear
chromatin is a single, well-defined purplish mass, varied in form and usually
peripheral in distribution. Mature male gametocytes can be distinguished from
females by the large, loose and ill-defined mass of chromatin and by their paler
colour and smaller mass.

Schizonts
The parasitised red cells are much enlarged containing Schüffner’s dots. The
parasites are large, filling the enlarged red cell. There are between 12-24
merozoites in the schizonts (usually16). The pigment is a golden
brown central loose mass.

50
Clinical Signs of Disease
Symptoms include headache, photophobia, muscle aches and pains, anorexia,
nausea and vomiting. Complications due to P. vivax are relatively rare and arise
due do a previous debility or pre-existing disease.

lecture 10 ;Plasmodium ovale

Introduction
Plasmodium ovale is widely distributed in tropical Africa especially the west
coast, despite that it is a species that is rarely encountered. It has also been
reported in South America and Asia. It has an incubation period of 10
– 17 days, which is sometimes prolonged to months or years due to the
formation of hypnozoites. It has a periodicity of 48 hours; the fever it produces
is milder than the benign tertian P. falciparum.

Life cycle
The features of the life cycle, which are specific to P. ovale, are as follows:
a) It morphologically resembles P. malariae in most of its stages;
b) The changes produced in the erythrocytes in general are similar to those
produced by P. vivax, but Schüffner’s dots appear considerably earlier
(in the ring stage) and are coarser and more numerous;
c) In the oocyst the pigment granules are (usually) characteristically
arranged in two rows crossing each other at right angles;
d) Hypnozoites develop in the liver so that relapses may occur.

51
Morphology
Parasites of P. ovale are usually found in enlarged and stippled red blood cells
(James’s dots), similar to those found in P. vivax infections. Host cells show an
oval shape, particularly those containing younger stages of the parasites and the
host cell may also show “spiking” or fimbriation.

Trophozoites
Young trophozoites are found as compact rings in cells containing Schüffner’s
dots. The trophozoite rings remain compact as they develop and show little of
the amoeboid features common in P. vivax. Small, scattered pigment granules
can be seen in developing trophozoites, which disperse as the trophozoite
matures. Late trophozoites are round and consolidated with an increase in
cytoplasm, they are very similar to P. vivax at this stage.

Gametocytes
The mature gametocytes are round, filling two thirds of the red cell. The red
blood cell is slightly enlarged and stippled and contains pigment, which has a
distinct arrangement of concentric rods, mostly at the periphery.

Schizonts
The parasite is smaller than red blood cells and contains 6-12 merozoites,
usually 8 in a single ring. The pigment is a brown / greenish central clump. The
red cell is slightly enlarged, stippled, frequently oval and fimbriated.

Clinical Signs of Disease

Symptoms, like those of P. vivax, include headache, photophobia,

52
muscle aches and pains, anorexia, nausea and vomiting. Complications due to
P. ovale are relatively rare and arise due do a previous debility or pre- existing
disease.

Plasmodium malariae
Introduction
Plasmodium malariae occurs mainly in the subtropical and mild areas where P.
falciparum and P. vivax occur. However, it is less frequently seen, responsible
for approximately 7% of all malaria in the world. It has an incubation period of
18 – 40 days and a periodicity of 72 hours.

Life cycle
The features of the life cycle, which are specific to P. malariae, are as follows:
a) Infected erythrocytes are not larger than uninfected ones and sometimes
even smaller;
b) Mature erythrocytes are attacked and rarely reticulocytes, so that the
density of parasites is very low; about 0.2% of erythrocytes are
parasitised;
c) It is often difficult to distinguish between a large trophozoite and an
immature gametocyte.

Morphology
Parasites of P. malariae are typically compact heavily pigmented parasites,
which are usually smaller and more deeply stained than normal. They tend to
parasitise small, old red blood cells, they do not contain any inclusion dots and
the parasitaemia is usually low.

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Trophozoites
Trophozoites are found as fairy large fleshy rings with a single chromatin dot.
These can be much distorted and can often take the form of bands across the
cell. All trophozoites have a single chromatin dot and contain pigment.

Gametocytes
Gametocytes contain large amounts of black pigment, with chromatin present
as a compact mass in females and diffuse in males. They occupy less than two
thirds of the red blood cell.

Schizonts
Schizonts are usually few in numbers with 6 – 12 large merozoites in a single
ring. Pigment is usually present as a central black mass. The parasites present
are generally only found at one stage of schizogony development.

Clinical Signs of Disease

Symptoms include headache, photophobia, muscle aches and pains, anorexia,
nausea and vomiting. Plasmodium malariae, like P. vivax and P. ovale are
relatively benign. However, chronic infections in children may lead to
nephrotic syndrome due to immune complexes depositing on the glomerular
wall.

Diagnosis of malaria parasites

The definitive diagnosis of malaria infection is still based on finding malaria
parasites in blood films. In thin films the red blood cells are fixed so

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the morphology of the parasitised cells can be seen. Species identification can
be made, based upon the size and shape of the various stages of the parasite and
the presence of stippling (i.e. bright red dots) and fimbriation (i.e. ragged ends).
However, malaria parasites may be missed on a thin blood film in case of low
parasitaemia. Therefore, examination of a thick blood film is recommended.
With a thick blood film, the red cells are approximately 6 – 20 layers thick
which results in a larger volume of blood being examined.

Thick Blood Films

In examining stained thick blood films, the red blood cells are lysed, so
diagnosis is based on the appearance of the parasite. In thick films, organisms
tend to be more compact and denser than in thin films.

Thin Blood Films

When examining thin blood films for malaria you must look at the infected red
blood cells and the parasites inside the cells

Serological diagnosis
Various test kits are available to detect antigens derived from malaria parasites.
Such immunologic (“immunochromatographic”) tests most often use a dipstick
or cassette format, and provide results in 2-15 minutes. These “Rapid
Diagnostic Tests” (RDTs) offer a useful alternative to microscopy in situations
where reliable microscopic diagnosis is not available. Malaria RDTs are
currently used in some clinical settings and programs. The World Health
Organization is conducting comparative performance evaluations of

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many of the RDTs which are commercially available worldwide based on a
panel of parasites derived from a global network of collection sites. Results of
this testing is available at:

Molecular Diagnosis
Parasite nucleic acids are detected using polymerase chain reaction (PCR).
Although this technique may be slightly more sensitive than smear microscopy,
it is of limited utility for the diagnosis of acutely ill patients in the standard
healthcare setting. PCR results are often not available quickly enough to be of
value in establishing the diagnosis of malaria infection. PCR is most useful for
confirming the species of malarial parasite after the diagnosis has been
established by either smear microscopy or RDT.

Figure (15 ): Differences between stages of Plasmodium spp

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Laboratory Diagnosis
1. Microscopic examination of giemsa-stained slides of aspiration of fluid
underneath the ulcer bed for typical amastigotes.
2. Culture of the ulcer tissue may also reveal the promastigote forms.
3. Serological tests such as Indirect Fluorescent Antibody (IFA) also
vailable.
C. L. brasiliensis causes Espundia Mucocutaneous leishmaniasis. The
infection is limited to a local lesion of the skin but may metastasise to
other areas of skin and oro-nasal mucosa.The primary lesion often
disappears spontaneously, followed by mucocutaneous lesions that destroy
the mucosal surface of the nose, pharynx, and larynx. If the condition is
untreated, potentially fatal secondary bacterial infections and
disfigurement may occur.

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