Module Two

MEDICAL PROTOZOLOGY
Dr Kenneth Lado
Department of Microbiology
 LEARNING OBJECTIVES
By the end of this module, students will be able to:
 Discuss the classification of medically important protozoa.
 Discuss the pathogenesis and clinical aspects of infections.
 Describe the general epidemiological aspects and
transmission patterns of diseases caused by protozoa.
 Identify the methods and procedures of laboratory
diagnosis of pathogenic protozoa in clinical specimens.
 Discuss treatment options for protozoan infections.
 Implement the preventive and control measures of
protozoan infection.
 General characteristics
• Protozoa (singular, protozoan), from the Greek ‘protos’ and
‘zoon’ meaning “first animal”, are members of eukaryotic
protists.
• They may be distinguished from other eukaryotic protists
by their ability to move at some stage of their life cycle and
by their lack of cell wall.
• Protozoa are single-celled eukaryotic microorganisms
belonging to kingdom Protista.
• The body wall is covered by cell membrane. Its cytoplasm is
made up of ectoplasm and endoplasm. The nucleus is
usually single but may be double or multiple.
• Reproduction can be asexual (e.g. binary fission,
schizogony, endodyogeny) or sexual (e.g. gametogony).
 Protozoa can be divided into the
following groups:
1. Amoebae (Has pseudopodia as a 3. Apicomplexa (Has a structure called
apical complex which serves as the
mean of locomotion) organ ofattachment to host cells. They
Amoebae of medical importance: have an alternating sexual and asexual
life cycle)
• Amoeba in the large intestine: Apicomplexa of medical importance:
Entamoeba histolytica • Blood: Plasmodium, Babesia
• Free-living amoebae in CNS and • Tissue: Toxoplasma gondii, Sarcocystis
eye: Naegleria, Acanthamoeba • Gastrointestinal: Cryptosporidium,
Cystoisospora, Cyclospora
2. Flagellates (Has flagella as organ 4. Ciliate (Has cilia for locomotion)
of locomotion) Ciliate of medical importance:
Flagellates of medical importance: • Gastrointestinal: Balantidium coli
5. Microsporidia
• Hemoflagellates: Trypanosoma, Microsporidia of medical importance:
Leishmania • Gastrointestinal: Enterocytozoon
• Gastrointestinal: Giardia lamblia bieneusi

• Urogenital: Trichomonas vaginalis

Protozoa of medical importance to
human
Occurrence of protozoa
• Protozoa are found in all moist habitats. They
are common in sea, in soil and in fresh water.
These organisms occur generally as a single
cell. Colonies of protozoa might also occur in
which individual cells are joined by
cytoplasmic threads and form aggregates of
independent cells.
Distinct features of some protozoa:
• Resistant cyst (non-motile) stage to
survive adverse environmental conditions, such as
desiccation, low nutrient supply, and
even anaerobiosis.
For example, the soil amoeba, Naegleria is a
resistant cyst in dry weather, a naked amoeba in
moist soil, and becomes flagellated when flooded
with water.
Morphology of protozoa
• Most prominent microscopic, ranging in size from
2 to more than 100μm.
They have within a mass of protoplasm, consisting
of a true membrane:bound nucleus and cytoplasm.
• The nucleus contains clumped or dispersed
chromatin and central nucleolus or karyosome,
which are useful structures to distinguish
protozoan species from one another based on
the shape, size and distribution of these
structures.
 Importance of protozoa
• Vital link in the food chain and ecological balance
of many communities in wetland & aquatic
environments.
• Biological sewage treatment, which involves both
anaerobic digestion and/or aeration.
• Use as laboratory organisms in research areas, by
which their asexual reproduction enables clones
to be established with the same genetic make-up.
These are useful in the study of cell cycles and
nucleic acid biosynthesis during cell division
 Reproduction and regeneration of
protozoa
• Generally, protozoa multiply by asexual
reproduction.
• But some parasitic forms may have an asexual
phase in one host and a sexual phase in
another host.
 Transmission
• In most parasitic protozoa, the developmental stages are often
transmitted from one host to another within a cyst.
• The reproduction process is also related to the formation
of the cyst.
• Asexual reproduction of some ciliates and flagellates is associated
with cyst formation, and sexual reproduction of Sporozoa invariably
results in a cyst.
• Pathogenic protozoa can spread from one infected person to
another by:
• Faecal – oral transmission of contaminated foods and water.
• Insect bit inoculums or rubbing infected insect faeces on the site of
bite.
• Sexual intercourse.
 Pathogenesis
Factors that are important for pathogenecity
include:
• Attachment to the host tissue followed by
replication to establish colonization.
• Toxic products released by parasitic protozoa.
• Shifting of antigenic expression to evade the
immune response and inactivate host
defence.
 Antiprotozoal agents
• Generally the antiprotozoal agents target
relatively rapidly proliferating, young, growing
cells of the parasite.
• Most commonly, these agents target nucleic
acid synthesis, protein synthesis, or specific
metabolic pathways (e.g. folate metabolism)
unique to the protozoan parasites.
 END

FOR
• SUKURAN • PATIENCE
• ASANTE SANA • PARTICIPATING • TOLERANCE
• CONTRIBUTING

THANK YOU LISTENING

 Module Three
AMOEBIASIS
INTRODUCTION
Definition:
• Amoebiasis, or amoebic dysentery, is a gastrointestinal illness
caused by a microscopic parasite called Entamoeba histolytica,
which is spread through human feces.
Amoebas primitive unicellular microorganisms with a relatively simple
life cycle which can be divided into two stages:
• Trophozoite – actively motile feeding stage.
• Cyst – quiescent, resistant, infective stage.
Their reproduction is through binary fission, e.g. splitting of the
trophozoite or through the development of numerous trophozoites
with in the mature multinucleated cyst.
• Motility is accomplished by extension of pseudopodia (“false foot”)
 Entamoeba histolytica
History
• Entamoeba histolytica was discovered in 1875 by Losch in
the dysenteric feces of a patient in St Petersburg, Russia.
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.’
Epidemiology:
Amebiasis is an infectious disease caused by Entamoeba
histolytica . Each year, amoebiasis affects an estimated 50
million people worldwide and causes 100,000 deaths.
• [Link] has a worldwide distribution. Although it is found in
cold areas, the incidence is highest in tropical and subtropical
regions that have poor sanitation and contaminated water.
• About 90% of infections are asymptomatic, and the remaining
produces a spectrum of clinical syndrome.
• Patients infected with [Link] pass noninfectious trophozotes
and infectious cysts in their stools.
• Therefore, the main source of water and food contamination is the
symptomatic carrier who passes cysts.
• Symptomatic amoebiasis is usually sporadic.
• The epidemic form is a result of direct person-to-person faecal-oral
spread under conditions of poor personal hygiene.
• Morphological features
Entamoeba histolytica occurs in 3 forms.
1. Trophozoite
2. Precyst
3. Cyst
• Trophozoite is the vegetative form of the parasite and
the only form present in tissues.
• It is irregular in shape and varies in size from 12 to 60
μm; average being 20 μm . It has a cytoplasm which
consists of ectoplasm and endoplasm.
• Ectoplasm is clear and transparent. Endoplasm is finely
granular and contains nucleus, food vacuoles and
phagocytosed erythrocytes.
• In the case of dysentery, RBCs may be visible in the
cytoplasm, and this feature is diagnostic for
[Link] are finger-like projections
formed by movements of ectoplasm in one direction.
• Its nucleus is spherical and contains central karyosome.
The nuclear membrane is lined by a rim of evenly
distributed chromatin. It reproduces by binary fission.
It is killed by drying, heat and chemical sterilization.
• The trophozoites undergo encystment in the intestinal
lumen. Before encystment, the trophozoite extrudes its
food vacuoles and rounds up to form a precystic
stage, measuring 10–20 μm in size. It contains a large
glycogen vacuole and chromatoid bars.
It secretes a cyst wall to become cyst.
The cyst is spherical in shape. Immature cyst contains a single
nucleus, a glycogen vacuole and chromatoid bars which are
cigar shaped with rounded ends.
The chromatoid bars are visible in saline. With iron
haematoxylin stain, nuclear chromatin and chromatoid bodies
appear deep blue or black. When stained with iodine, the
glycogen mass appears golden brown while the nuclear
chromatin and karyosome bright yellow.
Mature cyst contains 4 nuclei. It measures 10–20 μm in size.
The glycogen mass and chromatoid bars disappear in mature
cyst.
The cyst wall is highly resistant to gastric juice and
unfavourable environmental conditions.
 Life Cycle
(1) The cysts (usually found in formed stools) and
trophozoites (in loose stools) are passed out in faeces of
infected human. (2) Cysts are ingested via contaminated
food or water. (3) In the intestine, the cysts undergo
excystation and form trophozoites (4).
(5) As the trophozoite passes down the intestine, it undergoes
encystation and is excreted in the faeces.
• Entamoeba histolytica completes its life cycle in human
host. In the majority of cases, E. histolytica remains as a
commensal in the large intestine.
• They are carriers or asymptomatic cyst passers and are
responsible for maintenance and transmission of infection
in the community.
Pathogenesis and Clinical Features
• Entamoeba histolytica causes intestinal and
extraintestinal amoebiasis.
The lumen-dwelling amoebae do not cause any
illness. They cause disease only when trophozoites
invade the intestinal tissues. The trophozoite
penetrates the epithelial cells in the colon.
Virulent factors:
Its movements and histolysin, a tissue lytic
enzyme,which damages the mucosal epithelium.
Amoebic lectin mediates adherence.
• Mucosal penetration produces discrete ulcers
with pinhead centre and raised edges.
• Sometimes, the invasion remains superficial and
heals spontaneously.
• The ulcers are multiple and are confined to the
colon, being most numerous in the caecum and
recto-sigmoidal region.
The intervening mucous membrane between the
ulcers remains healthy
• The amoebic ulcer is flask shaped in cross-
section.
• Multiple ulcers may coalesce to form large
necrotic lesions with ragged and undermined
edges and are covered with brownish slough.
• The ulcers generally do not extend deeper
than submucosal layer. Amoebae are seen at
the periphery of the lesions and extending
into the surrounding healthy tissues.
 Summary
• 80% of patients infected with Entamoeba
histolytica will be asymptomatic.
• When E. histolytica becomes pathogenic, it
invades the crypts of colonic glands and
burrows down into the submucosa.
• It is stopped by the muscularis propria, so
then burrows laterally, and with the resulting
inflammation and necrosis a flask-shaped
ulcer is formed.
Multiple mucosal ulcers at caecum &
Ascending colon
• Clinical manifestations:
The incubation period is highly variable, from 4 days to a
year or longer. On an average it is from 1 to 4 months.
The clinical course is characterised by prolonged
latency, relapses and intermissions.
Symptoms:
• Diarrhoea, vague abdominal, flatulence, and cramping
and dysentery.
• Dysentery is an infection in intestine causing bloody
diarrhea.
Sites affected in amoebiasis
Amoebiasis can be classified as intestinal and extraintestinal
amoebiasis.
INTESTINAL AMOEBIASIS
• The ulcers may involve the muscular and serous coats of
the colon, causing perforation and peritonitis.
• Blood vessel erosion may cause haemorrhage.
• Deep ulcers form scars and may lead to strictures and
partial obstruction.
• A granulomatous pseudotumoral growth may develop on
the intestinal wall from a chronic ulcer.
• This is called amoebic granuloma or amoeboma and may
be mistaken for a malignant tumour.
 Differential diagnosis of amoebic and
Bacillary dysentry
• Bacillary dysentry is a bacterial infection of the
intestines that causes severe diarrhoea, fever,
and abdominal pain.
EXTRAINTESTINAL AMOEBIASIS
• Liver involvement is the most common
extraintestinal complication of intestinal
amoebiasis.
• About 5–10% of patients with intestinal
amoebiasis will develop amoebic liver abscess
(ALA).
• ALA arises from haematogenous spread of
amoebic trophozoites from colonic mucosa or by
direct extension.
• ALA patients do not present with bowel
symptoms.
• Liver damage may not be directly caused by the
amoebae, but by lysosomal enzymes and
cytokines from the inflammatory cells
surrounding the trophozoites.
• The centre of the abscess contains thick brown
pus (anchovy sauce), which is liquefied necrotic
liver tissue free of amoeba.
• The trophozoite is in the wall of the abscess.
• Liver abscess may be multiple or more often
solitary, usually located in the upper right lobe
of the liver.
• Jaundice develops only when lesions are
multiple or when they press on the biliary
tract. Large, untreated abscess may rupture
into the lungs and pericardium.
• The incidence of liver abscess is more
common in adult males.
Other complications of intestinal
amoebiasis
• Immunity
• Infection with invasive strains will activate
both humoral and cellular immune responses.
Systemic antibodies can be demonstrated
within a week of invasive infection.
• Infection confers some degree of protection
against the recurrence of invasive colitis and
liver abscess in endemic areas.
 Diagnosis
1. Diagnosis of intestinal amoebiasis
(a) Microscopic examination
• Demonstration of cysts or trophozoites in stool sample. Since
excretion of cysts in the stool is often intermittent, at least 3
consecutive specimens should be examined.
• Trophozoite and cyst of E. histolytica have similar morphology to E.
dispar and E. moshkovskii which are non-pathogens. Molecular
technique can differentiate these 3 species.
• Examination of a fresh dysenteric faecal specimen or rectal scraping
fortrophozoite stage. (Motile amoebae containing red cells are
diagnostic of amoebicdysentery). Examination of formed or
semiformed faeces for cyst stage
• Fixed stool smear can be stained with trichrome to demonstrate
cysts and trophozoites.
(b) Sigmoidoscopy for mucosal scrapings
Direct wet mount and iron haematoxylin staining to demonstrate
trophozoites.
(c) Stool culture
Stool culture is a sensitive method in diagnosing chronic and asymptomatic
intestinal amoebiasis. However, it is not a routine method of diagnosis.
(d) Serodiagnosis
Serological test is positive only in invasive amoebiasis.
(e) Molecular diagnosis
Polymerase chain reaction (PCR) to detect E. histolytica in stool and to
differentiate between the other species that are non-pathogens (E. dispar and
E. moshkovskii).
2. Diagnosis of extraintestinal amoebiasis
(a) Microscopic examination
Demonstration of trophozoites in pus aspirated from the
wall of liver abscess. The pus obtained from the centre of
the abscess may not contain amoebae as they are
confined to the wall of the abscess. Cysts are not found in
extraintestinal lesions.
Stool examination rarely can detect E. histolytica cyst.
(b) Molecular diagnosis
PCR of pus aspirated from ALA
(c) Serodiagnosis
 Treatment
[Link] amoebicides:
Diloxanide furoate(500mg tablet every 8 hrs for 10days;
children:20mg/kg), iodoquinol, paromomycin and tetracycline
act in the intestinal lumen but not in tissues.
2. Tissue amoebicides:
Emetine and chloroquine are effective in systemic infection,
but less effective in the intestine.
3. Both luminal and tissue amoebicides:
Metronidazole (750–800 mg 3 times daily for 5–10 days);
Tinidazole(2g/day for 3-5 days) and Ornidazole(1,500mg as a
single dose in the evening for 1-2 days) act on both sites.
• Carriers should also be treated because of the risk of
transmitting the infection to others. Paromomycin or
iodoquinol should be used in these cases.
• Metronidazole and Tinidazole are both luminal and
tissue amoebicides, neither of them reach adequate
levels in the gut lumen.
• Therefore, patients with ALA should also receive
treatment with a luminal agent to ensure eradication
of infection.
• Paromomycin (25–35 mg/kg/day, divided into 3 doses
for 7 days) is the drug of choice.
Summary for chemotherapy
 Prevention and Control
1. Boil drinking water
2. Wash fruits and vegetables in clean water
before eating
3. Detection and treatment of carriers and
prohibit them from food handling
4. Health education
 Pathogenic Free-Living Amoebae
(FLA)
• Among the numerous types of FLA found in
water and soil, a few are potentially
pathogenic and can cause human infections.
1. Naegleria fowleri causes primary amoebic
meningoencephalitis (PAM)
2. Acanthamoeba spp. cause granulomatous
amoebic encephalitis (GAE) and amoebic
keratitis (AK).
 Naegleria fowleri
Distribution
• Naegleria fowleri is a thermophilic amoeba
that thrives in warm water ([Link])
and soil. It has a worldwide distribution.
Habitat
• In human, N. fowleri is found in the central
nervous system (CNS).
Morphology
It occurs in 3 forms:
1. Trophozoite (2 forms)
(a) Amoeboid
(b) Flagellate
2. Cyst
The trophozoites can withstand moderate heat (45
°C), but die at chlorine levels of 2 ppm and salinity
of 0.7%. The amoeboid form is about 10–20 μm
with rounded pseudopodia (lobopodia), a spherical
nucleus and a big endosome.
It is the invasive and the infective form of the
parasite.
The flagellate form is biflagellated, pear-shaped and
occurs when trophozoites are transferred to
distilled water.
The flagellate can revert to the amoeboid form.
Trophozoites encyst due to unfavourable conditions
like food deprivation, desiccation and cold
temperature.
The cyst is 7–10 μm in diameter and has a smooth
double wall. They are the resting or the dormant
form and can resist unfavourable conditions, such
as drying and chlorine up to 50 ppm.
Cysts of N. fowleri have never been found in
cerebrospinal fluid (CSF).
 Life Cycle
(1) Cyst. (2) Trophozoite. (3) Flagellated form
showing flagella. (4) The trophozoite replicates
by promitosis. (5) The trophozoite penetrates
the nasal mucosa. (6) The trophozoite migrates
to the brain via the olfactory nerves.
Infection occurs when humans go swimming or
diving in warm freshwater (lakes and rivers),
hotsprings, heated pools, or nasal irrigation
using contaminated tap water.
Pathogenesis and Clinical Features
• Primary amoebic meningoencephalitis (PAM) is usually
reported in previously healthy young adults or
children.
• During contact with contaminated water, the amoebae
invade the nasal mucosa and pass through the
olfactory nerves in the cribriform plate into the
meninges and brain to initiate an acute meningitis and
encephalitis.
The incubation period varies from 2 days to 2 weeks. The
disease progresses rapidly, causing fever, headache,
vomiting, stiff neck, ataxia, seizure and coma and is
almost always fatal.
 Diagnosis
1 Cerebrospinal fluid (CSF) examination
The CSF is cloudy to purulent, with prominent neutrophils, elevated protein
and low glucose, resembling pyogenic meningitis.
Wet film examination of CSF may show motile trophozoites. Fixed smear can
be stained with Giemsa or a modified trichrome stain for identification. Cysts
are not found in CSF or brain.
Histological examination of the brain following autopsy may show the
presence of trophozoites.
2 Culture
Naegleria fowleri in CSF can be grown on non-nutrient agar plates coated
with Escherichia coli.
Both trophozoites and cysts can be detected in culture.
3 Molecular diagnosis
PCR on CSF specimen.
 Treatment
The drug of choice is intravenous amphotericin
B (1.5 mg/kg/day in 2 divided doses for 3 days
followed by 1 mg/kg/day once daily for 11 days).
It can also be given intrathecally. Treatment
combining miconazole and sulfadiazine has
shown limitedsuccess.
Majority of cases are fatal despite treatment.
 Prevention and Control
• Chlorination of swimming pools
 Acanthamoeba Species
Acanthamoeba culbertsoni is the species most
often responsible for human infection.
Other species like A. polyphaga, A. castellanii,
and A. astronyxis have also been reported.
Distribution
It is an opportunistic pathogen found worldwide
in the environment, water and soil.
Habitat
In human, it is found in the CNS and eye.
Morphology
It occurs in 2 forms:
1. Trophozoite
2. Cyst
Trophozoite measures 20–50 μm in size and is
characterized by spine-like
pseudopodia(acanthopodia).
It does not have a flagellate stage. The cyst has a
polygonaldouble walled and is highly resistant. It
measures 10–20 μm. Cyst is found in brain tissue.
 Life Cycle
(1) Cyst. (2) Trophozoite showing spinous acanthopodia.
(3) The trophozoite replicates by mitosis.
(4) The cyst and trophozoite enter humans (5) through
the eye, (6) through nasal passages and (7) through
ulcerated or broken skin.
Both trophozoites and cysts are infective. Humans acquire
infection by inhalation of cyst or trophozoite, or via
broken skin or eyes.
Upon reaching the lungs after inhalation, the
trophozoites enter the blood circulation and invade the
CNS, producing granulomatous amoebic encephalitis
(GAE).
Pathogenesis and Clinical Features
[Link] amoebic encephalitis (GAE)
GAE usually occurs in patients who are immunodeficient.
The parasite spreads haematogenously to the CNS.
Invasion of the connective tissue and induction of
proinflammatory responses lead to neuronal damage that
can be fatal within days.
Clinical features are that of intracranial space-occupying
lesions with seizures, paresis and mental deterioration.
Autopsy of the brain reveals severe oedema and
haemorrhagic necrosis. In immunocompromised states
like AIDS, disseminated disease occurs with a widespread
infection affecting skin, lungs, sinuses and other organs.
2. Acanthamoeba keratitis
An infection of the eye that occurs in healthy persons and
develops from the entry of the amoebic cyst through
abrasions on the cornea.
Most cases have been associated with the use of contact
lenses. The clinical features resemble that of severe
herpetic keratitis.
The eye is severely painful in amoebic infection.
Unilateral photophobia, excessive tearing, redness and
foreign body sensation are the early signs and symptoms.
Keratitis can result in permanent visual impairment
or blindness.
 Diagnosis
1. Diagnosis of GAE
Demonstration of trophozoites and cysts in brain
biopsy, culture (non-nutrient agar plate coated with
E. coli), or immunofluorescence microscopy using
monoclonal antibodies.
CSF examination can reveal motile trophozoite
forms.
2. Diagnosis of amoebic keratitis
Demonstration of the cyst in corneal scrapings by
wet mount, histology or culture.
 Treatment
No effective treatment is available for GAE.
In acanthamoeba keratitis, therapy involves
topical application of biguanide or
chlorhexidine. When vision is threatened,
keratoplasty can be done. Multidrug
combinations which include pentamidine,
sulfadiazine, rifampicin and fluconazole are
being used with limited success.
 Prevention and Control
• Tap water should not be used to rinse contact
lenses
Different types of Amoebae
 END

FOR
• SUKURAN • PATIENCE
• ASANTE SANA • PARTICIPATING • TOLERANCE
• CONTRIBUTING

THANK YOU LISTENING