A REVIEW LECTURE FOR MEDICAL TECHNOLOGISTS

NAPOLEON Y. MORON, RMT

PRE-TEST

Identify the following

parasites.
1
2
3
4

5
6
7
8
9
ANSWERS
 Trichomonas vaginalis
trophozoites
Blastocystis hominis
cyst

1
Balantidium coli 2
trophozoite
3
 Entamoeba
histolytica/dispar
cyst

Acanthamoeba 5
cyst
Entamoeba coli 6
cyst
 Cryptosporidium
oocyst
Giardia lamblia
trophozoite

7
Isospora belli 8
oocyst
9
KINGDOM PROTISTA
PHYLUM SARCOMASTIGOPHORA
PHYLUM CILIOPHORA
PHYLUM APICOMPLEXA
PHYLUM MICROSPORA
 PHYLUM SARCOMASTIGOPHORA
SUBPHYLUM SARCODINA SUBPHYLUM MASTIGOPHORA
Giardia
Entamoeba
Chilomastix
Iodamoeba
Trichomonas
Acanthamoeba
Dientamoeba
Endolimax
Trypanosoma
Naegleria
Leishmania
PHYLUM CILIOPHORA

Balantidium coli
PHYLUM APICOMPLEXA

Plasmodium
Babesia
Toxoplasma
Isospora
Cyclospora
Cryptosporidium
PHYLUM Microspora

Microsporidium
Enterocytozoon
Encephalitozoon
 AMOEBAE (Subphylum Sarcodina)
INTESTINAL AMOEBA
Entamoeba histolytica Entamoeba polecki
Entamoeba dispar Endolimax nana
Entamoeba hartmanni Entamoeba coli
Iodamoeba butchlii (*Entamoeba gingivalis)
EXTRAINTESTINAL AMOEBA
Acanthamoeba spp. Naegleria fowleri
Entamoeba histolytica
 only pathogenic amoeba; associated with intestinal
and extraintestinal infections (colitis and liver abcess)
 Schaudinn (1903) gave the name E. histolytica
because of its ability to lyse human tissues.
 Emile Brumpt postulated that there were two
morphologically indistinguishable species of E.
histolytica (1) E. dysenteriae – causing disease and (2)
E. dispar – harmless commensal
Entamoeba histolytica
INFECTIVE STAGE:
CYST

DIAGNOSTIC STAGE:
CYST AND
TROPHOZOITE
Entamoeba histolytica
CYST STAGE
SIZE 12-15 um
SHAPE Round
NUCLIE 4 in mature cyst, immature with 1 or 2 occasionally seen
Thin, regular, circular membrane
Small, compact, central karyosome (Black Dot) – “BULL’S EYE”
CYTOPLASM granular; “dirty” appearance
CHROMATOID Oblong, rounded at ends (sausage shaped or cigar shaped); not
BODIES found in all cysts
VACUOLE Sometimes a large glycogen vacuole in young cysts with one or
two nuclei.
Entamoeba histolytica
TROPHOZOITE STAGE
SIZE 15-20 um (usually the size of 3-4 erythrocytes)
SHAPE AND Elongated and changing when moving
MOTILITY Round when not moving
Progressive/ Unidirectional movement
Pseudopodia (finger-like) may be seen
NUCLIE 1, not visible in unstained preparation, clearly seen to have a
regular membrane and a small dense karyosome (Black Dot) –
“BULL’S EYE”
CYTOPLASM Finely granular, with ingested RBC, noninvasive organism may
contain bacteria
Entamoeba histolytica
Majority of cases are asymptomatic (cyst carriers)
Virulence Factors:
1. Gal/Gal NAc LECTIN- adherence
2. AMEBAPORES- Pore Formers
3. CYSTEINE PROTEINASE- used in invading tissues,
cytopathic for host tissues
Entamoeba histolytica
PATHOLOGY
Amoebiasis
a. Asymptomatic – cyst passer
b. Symptomatic – trophozoite in stool
Amoebic Colitis
Ameboma
Intestinal ulceration – “BOTTLE NECK ULCER”
Amoebic Liver Abscess (ALA) – most common extraintestinal form
Amoebic Hepatitis
Cutaneous Amoebiasis
 AMOEBIC DYSENTERY BACILLARY DYSENTERY
ONSET Gradual Acute
SIGNS/ SYMPTOMS No significant fever or vomiting Fever and usually vomiting
ODOR (feces) Offensive/ Fishy odor Odorless
BLOOD AND MUCUS POSITIVE Often watery and bloody
pH Acidic Alkaline
PUS Few Numerous
CELLS/PMN/NEUTROPHIL
CELLULAR EXUDATES Scant Massive
PYKNOTIC RESIDUES Numerous Few
CHARCOT LEYDEN CRYSTAL Present Absent
PATHOGENIC AMOEBAE Present Absent
BACTERIA Few Numerous
MACROPHAGES Absent Present
Entamoeba histolytica
DIAGNOSIS
Stool Exam
-3 stool specimens collected in different days
-Consistency – bloody, mucoid diarrhea
-Ammonia in urine-contaminated stool can kill
trophozoites
Entamoeba histolytica
DIAGNOSIS
DFS – best for recovery of trophozoites (within 30
minutes)
-Lugol’s Iodine – Nucleus of Cysts/ Karyosome
-Methylene Blue – Nucleus of Trophozoite
-Iodine can kill Trophozoites
-Trophozoite with ingested RBCs is diagnostic of
amoebiasis
- Polymorphonuclear leukocytes may be mistaken as
E. histolytica/dispar
Entamoeba histolytica
DIAGNOSIS
Conc. Methods – FECT, MIFC : more sensitive for detection
of cyst
Stool Culture – Robinson’s & Inoki Medium
Permanent stain
a. Iron Hematoxylin
b. Trichrome staining
c. Chlorazon
Serology : IHAT, IFAT, CIE, AGD, ELISA
Entamoeba histolytica
TREATMENT
Metronidazole – drug of choice
COMMENSAL AMOEBAE
All species have the following stages
1. trophozoite
2. precyst
3. cyst
4. metacystic trophozoite
“EXCEPT Entamoeba gingivalis w/c has NO CYST STAGE”
 In stool exam:
- Cysts – formed stool
- concentration techniques (FECT, ZnSO4)
- Trophozoite – watery or semi-formed stool
- DFS
 Ingestion of mature cyst

Cyst passes through acidic stomach

Excystation in lower small intestine

(alkaline environment)

Metacystic trophozoites colonize large intestines

(reproduce by binary fission)

Encystation in the lower colon

(dehydrated environment)

Mature cyst/ trophozoite is passed through the

environment
Entamoeba dispar
Morphologically similar to Entamoeba histolytica
DNA, Ribosomal RNA, isoenzyme patterns are
different

 Reporting in stool :
“Positive for Entamoeba histolytica/ dispar cyst”
Entamoeba hartmanni
Similar to E. histolytica
Smaller, does not ingest RBC, more sluggish in
movement
Mature cyst measures 5- 10 um, quadrinucleated
with coarse cytoplasm
Immature cyst have short chromatoidal bars with
tapered ends, or thin and bar like
Entamoeba coli
CYST
POINT OF Entamoeba Entamoeba coli
DIFFERENTIATION
histolytica
Size 12-15 um 12-25 um
Shape Round Round/ Slightly Oval
Nuclei 1-4 nuclei, 4-8 nuclei,
central karyosome large diffuse eccentric karyosome
Chromatoidal Bars Sausage, cigar, coffin lid Broomstick, needlestick, splinter-
shape like
Entamoeba histolytica cyst

Entamoeba coli cyst

Entamoeba coli
TROPHOZOITE
POINT OF Entamoeba histolytica Entamoeba coli
DIFFERENTIATION
Size 15-20 um 20-25 um
Shape Elongated and changing Oval or elongated
Motility Progressive, unidirectional Nonprogressive, nondirectional, sluggish
Nuclei Mononucleate Mononucleate,
central karyosome Eccentric karyosome
Pseudopodia Finger-like, released one at a Broader and blunt in shape
time
Cytoplasmic With ingested RBCs Dirty looking with bacteria, NO RBCs
inclusion
Entamoeba polecki
Parasite of pigs and monkeys
Rarely infects humans
Cyst is consistently mononucleated
Nuclear Membrane and karyosome are very
prominent, in stained fecal smear
Entamoeba gingivalis
Found in the mouth – gum, teeth surface, gum
pockets, tonsillar crypts
Transmitted by kissing, droplets and sharing utensils
Trophozoite – 10-20 um
 NO CYST STAGE
 Moves quickly (w/ numerous blunt pseudopodia)
 Numerous food vacuoles containing cellular debris
and bacteria, and sometimes WBCs
Endolimax nana/nanus
Smallest amoeba
Trophozoite (8-10 um)
-sluggish movement, with many small rounded
pseudopodia
 Cyst (6-8 um)
- Quadrinucleate
- Large irregular karyosome – “BLOT-LIKE”
Iodamoeba butschlii
CYST (10-12 um)
- mononucleated, with very large karyosome
pressed against a cluster of granules
- with Large GLYCOGEN BODY which stains
deeply with iodine
TROPHOZOITE (12-15 um)
- sluggish, nonprogressive
- has large vesicular nucleus with a large
endosome surrounded by achromatic granules.
 AMOEBAE (Subphylum Sarcodina)
INTESTINAL AMOEBA
Entamoeba histolytica Entamoeba polecki
Entamoeba dispar Endolimax nana
Entamoeba hartmanni Entamoeba coli
Iodamoeba butchlii *Entamoeba gingivalis
EXTRAINTESTINAL AMOEBA
Acanthamoeba spp. Naegleria fowleri
EXTRAINTESTINAL AMOEBA
Acanthamoeba spp. Naegleria fowleri
Acanthamoeba
Ubiquitous small free living ameba
Characterized by active trophozoite stage and
dormant cyst stage
 Reproduce by binary fission
 A. castellani, A. culbertsoni, A. hutchetti, A.
polyphaga, A. rhysoides
Acanthamoeba
Trophozoite
 has a single large nucleus w/
centrally located densely staining
nucleolus, large endosome, finely
granulated cytoplasm and a large
contractile vacuole.
Acanthapodia – small, spiny
filament for locomotion
Sluggish, polydirectional motility
Acanthamoeba
CYST
Double-walled outer
wrinkled wall and inner
polygonally shaped wall.
Pores or ostioles are seen at
the point of contact between
the two walls
Acanthamoeba
Acanthamoeba

GRANULOMATOUS AMEBIC
ENCEPHALITIS (GAE)
Associated with the use of soft contact
lenses
Acanthamoeba
Diagnosis of GAE is made only after death in
most cases
High incidence in AIDS patients
Epithelial biopsy for Acanthamoeba keratitis
 Culture: PYGC (Proteose-peptone, Yeast extract,
Glucose and Cystine) plus antibiotic
PCR
Naegleria
Free-living ameboflagellate
-cyst
-amoebic trophozoite –inside the body
-flagellate (swimming form) – outside the body
 Naegleria fowleri – pathogenic
 Naegleria gruberi – nonpathogenic
 Naegleria philippinensis – locally occurring
species
Naegleria
 Trophozoite has lobose
monopseudopodium and a very
prominent nucleus with a
centrally located nucleolus
 Pear-shaped biflagellated form
has a directional motility
Naegleria
TRANSMISSION:
-Oral/ Intranasal
routes while
swimming in
contaminated pools,
lakes and rivers
Naegleria
 Can cause Primary Amebic Meningoencephalitis
(PAM), gastritis, diarrhea
 Can survive in up to 46 degrees Celcius and up
to 0.5 ug/mL of hyperchlorinated water
Other Intestinal Parasite
Blastocystis hominis
Initially describe by Prowasek and
Alexeieff, then named by Brumpt in 1912
Formerly classified as yeast
(Schizosaccharomyces, then Blastomyces)
Inhabitant of the lower intestinal tract
Not a commensal but a potential
pathogen
 Multiplies by binary fission
Blastocystis hominis
4 morphological forms: (polymorphic)
- Vacoulated (spherical, 5-10 µm
diameter, with large central vacuole
pushing the cytoplasm and nuclei to
the cell periphery; predominant)
- Amoeba-like (exhibit active extension
or retraction of pseudopodia; ingests
bacteria; occasionally observed)
Blastocystis hominis
4 morphological forms:
- Garnular form (mainly observed in
old cultures; granular contents
develop into daughter cells of the
ameba form when the cell
ruptures)
- Multiple fission (arise from the
vacuolated forms)
Blastocystis hominis
Cause Blastocystosis, but considering it
as a cause of gastrointestinal pathology
is controversial
Diagnosis
- DFS, Concentration techniques
- Stained smears using Hematoxylin and
Trichrome
- Stool culture (Boeck and Drbohlav’s or
Nelson and Jones media
Blastocystis hominis

Harbored also by pig-tailed

macaques, chickens, dogs,
ostriches, lizards and cockroaches
 PHYLUM SARCOMASTIGOPHORA
SUBPHYLUM SARCODINA SUBPHYLUM MASTIGOPHORA
Giardia
Entamoeba
Chilomastix
Iodamoeba
Trichomonas
Acanthamoeba
Dientamoeba
Endolimax
Trypanosoma
Naegleria
Leishmania
Parts of a Flagellate
 Flagella – for locomotion
 Parabasal Body – energy body
 Cytosome (mouth)
 Undulating membrane – extension of the plasma
membrane
 Axostyle – support
 Costa – rib-like support
Giardia lamblia
 also known as Giardia intestinalis, Giardia
duodenalis, Lamblia duodenalis, or Lamblia
intestinalis
 discovered by Anton van Leeuwenhoek (1681)
in his own stool
 first described by Lambl in 1859 who called it
Cercomonas intestinalis
 Renamed by Stiles (1915) as Giardia lamblia
Giardia lamblia
 Intestinal parasitic flagellate
 lives in the duodenum, jejunum, upper ileum
 acquired through ingestion of water or food
contaminated with the mature cyst
Giardia lamblia
CYST STAGE
8-12 µm long by 7-10 µm wide
Ovoid and double walled with 2-4 nuclei
Has flagella retracted into axonemes, and deeply
stained curve fibrils surrounded by a tough hyaline
cyst wall.
Giardia lamblia
TROPHOZOITE STAGE
9-12 µm long by 5-15 µm wide
Pyriform/teardrop/ pear shaped
Pair of ovoid nuclei on each side of the axostyle
(medial line); 2 ventral sucking disk; 4 pairs of flagella
“Old Man Face” appearance on permanently stained
smears
Erratic tumbling/falling leaf/ Kite-like motility
Variant-specific proteins (VSPs) covers the entire
surface of the parasite
Giardia lamblia
Giardia lamblia
 Giardiasis/ Lambliasis
- Ingestion of contaminated food (as few as 10 cysts)
- Attaches to the duodenal epithelium by sucking
disks causing enterocyte apoptosis
- Incubation period: 1-4 weeks
- Stomach cramps, diarrhea, excessive flatus with an
odor of hydrogen sulfide – “rotten eggs”
- Steatorrhea in chronic infection
- Traveller’s diarrhea
- Gay-Bowel syndrome
Giardia lamblia
DIAGNOSIS
DFS – trophozoites and/or cysts
Duodeno-Jejunal aspiration or biopsy
Entero-test/ Beale’s String test
- Patient swallows a gelatin capsule containing a nylon
string with one end attached to the patient’s cheek; then
removed after 6-8 hours and the adherent fluid is
examined for presence of trophozoites
Immunochromatography in stool, ELIZA, IFT, Direct
fluorescent antibody assay
Chilomastix mesnili
Nonpathogenic
Cyst stage
- 10 µm with 1 nucleus
- Lemon shaped/ Nipple shaped
Trophozoite stage
- 15 µm, uninucleated, 4 flagella (3 anterior + 1
flagellar cytosome)
- spiral groove – “cork-screw motility”
- assymetrical, Shepherds crook appearance
Chilomastix mesnili
Trichomonads
 Trichomonas hominis - diarrhea
 Trichomonas tenax - gingivitis
 Trichomonas vaginalis - Urogenital
Trichomonas vaginalis
 No cyst stage
 Trophozoite
- 10-20 µm, long and broadly oval to pear shaped
with anterior nucleus
- Long axostyle and short undulating membrane
- Jerky, tumbling, twitching motility
 only parasite with siderophil granule
Trichomonas vaginalis
Trichomonas vaginalis
 Trichominiasis
- vaginitis, prostatitis, urethritis, associated with other STDs
like gonorrhea
 In females
- Frothy yellow green vaginal discharge that smells bad
- Itchy, irritated genitals with lower abdominal pain
- Pain during sex/ urination
- Vaginitis – Strawberry cervix
In males
- asymptomatic, self-limiting and less persistent
Trichomonas vaginalis
DIAGNOSIS
 Microscopy of urine/ vaginal swabs
 Culture – in pouchculture
- Daimond’s Modified Culture medium
- Feinbergwittington culture medium
- Trypticase liver serum culture medium
 Whift Test – 20% KOH – Fishy odor
Dientamoeba fragilis
 originally described as amoeba but actually a
flagellate
 No cyst stage
7-12 µm with 1 or 2 rosette-shaped nuclei
 resembles Trichomonas
 lives in the mucosal crypts of the cecum and the
upper colon
Transmitted via fecal-oral route of via the
transmission of Enterobius vermicularis egg
Dientamoeba fragilis
Dientamoeba fragilis
Dientamoeba fragilis
 can be asymptomatic, or symptomatic with
colicky abdominal pain, loss of appetite,
intermittent mucoid diarrhea
 Diagnosis is through demonstration of a
binucleate trophozoite in stool (fixed and stained)
Not detected by stool concentration methods
Fixatives: PVA, Schaudinn’s fixative
Blood and Tissue Flagellates
Trypanosoma cruzi
Etiologic agent of “Chagas’ Disease” or
“American trypanosomiasis”.
Discovered by Carlos Chagas; the only parasite
discovered and studied before it was known to
cause disease.
Trypanosoma cruzi
An intracellular parasite
Exhibits 4 stages:
- Amastigote
(muscles/tissuesof humans)
- Promastigote (midgut of
triatomine bugs)
- Epimastigote (midgut of
triatomine bugs)
- Trypomastigote (human
bloodstream)
Trypanosoma cruzi
Trypomastigote
- long, slender :16-20 µm
- Short, stumpy : 15 µm
- posterior end is usually pointed
- Characteristically C-shaped or U or S-shaped
Amastigote
- Develop in muscles and other tissues
- Round or ovoid with 1.5-4 µm diameter
- Found in small groups of cyst-like tissues
Trypanosoma cruzi
 Intermediate hosts:
- REDUVIID/ TRIATOMINE BUGS :
- Triatoma (Kissing Bug)
- Rhodnius, (Cone Nosed Bug)
- Panstrongylus (Assasin Bug)
Trypanosoma cruzi
Trypanosoma cruzi
Chagoma – local inflammation producing a small
painful, reddish nodule
Romaňa’s sign – edema of the eyelid and
conjunctiva; unilateral swelling of
face
Chagas’ disease is serious and often fatal in
young children while less severe and chronic in
older children and adults involving cardiomypathy
Trypanosoma cruzi
Diagnosis:
- Demonstration of trypanosomes in blood,
cerebrospinal fluid, fixed tissues or lymph.
- T. cruzi can be seen by direct examination or thick
blood smears only in the first 2 months of acute
disease.
- Blood culture, Xenodiagnosis, Serologic tests and PCR
Treatment: Nifurtimox and Benznidazole – partially
effective
Trypanosoma brucie complex
Composed of T. brucei gambiense and T.
brucei rhodesiense
Causative agent of “African sleeping
sickness”
- Gambian/ West African sleeping sickness (chronic
form)
- Rhodesian/ East African sleeping sickness (acute
type)
Trypanosoma brucie complex
Exhibits only epimastigote and trypomastigote
 Vector: “Glosinna spp. (Tsetse Fly)”
- Trypomastigotes in the salivary gland of the tsetse fly is
inoculated to humans during blood meal)
In humans, T. brucei live in the blood, reticular
tissues of lymph and spleen, and cerebrospinal fluid
Trypanosomes evades immune response through
antigenic variation
Trypanosoma brucie complex
Chancre – earliest sign of African trypanosomiasis
(local, hard, painful lesion at site of inoculation)
 Gambian trypanosomiasis
- Winterbottom’s sign : enlargement of posterior cervical
lymph nodes
- Kerandel’s sign : hyperesthesia/ delayed sensation to
pain
Rhodesian trypanosomiasis
- More rapid and fatal
Somnolence : excessive sleeping
Trypanosoma brucie complex
Diagnosis
- (+) trypomastigote in Giemsa-stained blood, lymph
node aspirate and cerebrospinal fluid.
- Buffy coat concentration method
- Serologic tests
Leshmanias
Leishmania tropica
Leishmania braziliensis
Leishmania donovani
 Vector: “sandlfy” – Phlebotomus spp.
-- Lutsomyia spp.
Exhibit amastigote and promastigote(infective
stage) forms
Leshmanias
Leshmanias

promastigote
Leshmanias
Leishmania tropica
- Invades the lymphoid tissues of the skin
- Causes Cutaneous Leishmaniasis characterized by skin
ulcers
- Parasites are found in macrophages and histiocytes
Leishmania braziliensis
- Invades the skin and mucous membrane
- Causes American/ Mucocutaneous Leishmaniasis
- Espundia – metastatic spread of to the oronasal and
pharyngeal mucosa causing “Tapir nose”
Leshmanias
Leishmania donovani
- Invades the visceral organs
- Causes Visceral Leishmaniasis or Kala-azar
- Marked hyperplasia of reticular cells and marked
increase in the vascularity of the tissues
Leshmanias
Diagnosis
- Tissue biopsy (skin, bone marrow, spleen and lymph
nodes)
- Serologic tests
- Culture (Novey, McNeal, Nicolle media)
- Montenegro Skin test – Leshmanin skin test
Leshmanias
Hemoflagellate Amastigote Promastigote Epimastigote Trypomastigote
T. Cruzi + + + +
T. brucei rhodesiense - - + +
T. Brucei gambiense - - + +
Leishmania + + - -
PHYLUM CILIOPHORA

Balantidium coli
Balatidium coli
Causative agent of Balantidias or balantidial
dysentery
Largest protozoan parasite in humans
Only ciliate known to cause human disease
Reservoir host: pigs
Exhibits both trophozoite and cyst stages
Balatidium coli
Trophozoite
- 30-300 µm by 30-100 µm
- Has cytosome (acquiring food) and an cytopyge (excrete
waste)
- 2 dissimilar nuclei (macro and micronucleus)
- Mucocyst – beneath cell membrane, probably for adhesion
- Rotary motion
Cyst (infective stage)
- 40-60 µm diameter, spherical and ovoid, covered with thick
spherical wall
Balatidium coli
Balatidium coli
B. coli is a tissue
invader
Creates characteristic
ulcer with a round base
and wide neck, due to
hyaluronidase produced
by trophozoites
Diarrhea with bloody
mucoid stool
Balatidium coli
Diagnosis
- DFS, Concentration techniques
- Biopsy
PHYLUM APICOMPLEXA

Plasmodium
Babesia
Toxoplasma
Isospora
Cyclospora
Cryptosporidium
PHYLUM Microspora

Microsporidium
Enterocytozoon
Encephalitozoon