CLINICAL PARASITOLOGY LABORATORY

INTRODUCTION OF PARASITOLOGY
 Ectoplasm: outer (non-granulated), typically watery
OUTILNE  Homogenous and serves as an organ for motility and
At the end of the session, the student must be able to engulfment of food by producing pseudopodia 
learn:  Helps in respiration, discharging waste material and
I.Protozoa providing protective covering
A.Composition 3.Structures for locomotion 
B. Classification of Protozoan Parasite  Psuedopodia: fingerlike  temporary prolongation/
C. General Rule for Amebae fingerlike projections
II. Entamoeba histolytica  Flagella: Tail-like 
A. Life Cycle of E. histolytica  Cilla: Hair-like  surrounding or covering the body
B. Morphologic comparison between E. of the protozoan
histolytica and E. coli  Undulating membrane an organ as well to support
C. Pathogenesis the movement or the mobility of the parasite
D. Pathology Ang mga structure na ito ay used for the locomotion or
movement of the parasites
E. Pathogenic Determinants/Virulence Factor
F. Laboratory Diagnosis 4.Plasma membrane  controls secretions and excretions
G. Treatment 5. Cytosome 
H. Prevention and Control  cell mouth
III. Non-Pathogenic species 6.Chromatoidal bodies 
A. Entamoeba coli  storage for glycogen protein
B. Entamoeba dispar B. CLASSIFICATION OF PROTOZOANS
C. Entamoeba hartmanni PARASITES
D. Entamoeba polecki
E. Entamoeba gingivalis
F. Entamoeba moshkovskii
G. Endolimax nana
H. Iodamoeba butschlii
IV. Free Living Pathogenic Amoeba
A. Acanthamoeba spp. (Acathamoeba
castellani)
B. Naegleria fowleri
V. Phylum ciliophora cilates
A. Balantidium coli

PROTOZOA
 Proto (first), Zoa (animals)  single celled or
unicellular organism, considered as eukaryotic cell
because they possess true nucleus.
 Unicellular organism that performs all the functions:
reproduction, digestion, respiration, excretion, etc.
A. COMPOSITION

1.Nucleus 
usually single but may be double or multiple;
contains one or more nucleoli or a central
karyosome; DNA containing body.
Karyosome situated peripherally or centrally of the
nucleus and is found in the intestinal amoeba. Subphylum Sarcodina – intestinal / extra-intestinal
Karyosome is important din for us to identify our amoeba as well as the pre living pathogenic amoeba.
intestinal amoeba, kasi yung position ng karyosome Came from word SARCOS means FLESH/BODY.
can be used to identify your amoeba, if they are Includes parasites which have no permanent locomotory
situated centrally that is for Entamoeba histolytica, organ but they move with the aid of temporary
and for peripherally or nasa side is for entamoeba prolongations of the body called as the pseudopodia.
coli
2.Cytoplasm  Subphylum mastigophora - from t he word MASTIX
 Endoplasm: inner (often granulated), dense part.  means whip or flagellum, may tail like projections on their
 Granulated because it shows number of structures body, includes protozoa which possess whip like flagella
such as golgi bodies. endoplasmic reticulum, food ( atrial flagellates, reproductive organ flagellates, blood
vacuoles, and contractile vacuoles  and tissue flagellates)
 Contractile vacuoles regulate osmotic pressure Phylum ciliophora - has 1 parasite. Motile by means of
cillia or the hair like projections covering the entire body
between the parasite and its environment 
surface. The only human parasite in this group is
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BALANTIDIUM COLI, and rarely causes decentered. (gums) 
 Amebiasis— presence of amoeba in any part of the
Phylum Apicomplexa – formerly known as sporozoa,the body (exclusively applied to E. histolytica) 
members of this group at some stage of their life cycle  Asexually multiplies through binary fission.
possess Apical complex, called Apicomplexa of the
structure called the Apical complex. The Apical complex is ENTAMOEBA HISTOLYTICA
used by the parasite for the attachment to the host cell,
para dumikit sya sa host cell kailangan nya ng Apical MORPHOLOGIC FORMS
complex
1.Tropozoite: divides through "binary fission", capable
Phylum microspora – frequently cause disease in immuno of encystation (overpopulation, pH change, food supply,
deficient subject. availability of oxygen) 
 Trophozoite undergo encystation in intestinal lumen
There are 2 stages commonly encountered in protozoan or rectum
and they are the cyst and the trophozoites 2.Precyst: contains large glycogen vacuole and two
chromatid bars and then secretes a highly retractile cyst
2 processes of formation of cyst and the trophozoites wall around it and becomes cyst.
3. Cyst: with protective thick cell wall (hyaline), capable
 Encystation of excystation 
Stage forming a cyst or becoming enclosed to a capsule,  Cyst found on contaminated food and water could
this event takes place in the rectum of the host as feces withstand the acidic pH of our stomach because of
are dehydrated or soon after the feces have been excreted. its thick cell wall made up of hyaline.
Process of becoming a cyst (from trophozoites developing 4.Metacyst: liberated quadrinucleate amoeba during
into a cyst). excystation 
 Excystation
 No morphologic difference among other Entamoeba
Escape from cyst or envelope, produces a trophozoite
spp. such as E. moshkovskii and E. dispar. However,
from the cyst stage, and it takes place in the large
they can be differentiated through isoenzyme
intestine of the host after the cyst has been ingested.
analysis, PCR, and monoclonal antibody typing.
Counterpart o kabaliktaran ng encystation (from cyst
going or developing to trophozoites). EX means dati
syang cyst. For example, you have ingested a INEFECTIVE STAGE
contaminated water that contains the cyst inside the
intestine the cysts will transform to become the  mature quadrinucleate cyst passed in feces
trophozoites
MODE OF TRANSMISSION

 Ingestion of contaminated food and/or water with E.

histolytica cyst. 
 Primary route is fecal-oral 
 Venereal transmission 
 Direct colonic inoculation through contaminated
enema equipment.

TROPHOZOITE CYST
Vegetative and motile Non-motile, feeding
C. COMPOSITION stage (feeding stage) stage
Found in fresh watery, Found in soft to formed
 All Entamoeba are commensal except for soft or semi-formed stool stool
Entamoeba histolytica.  can benefit to the host Fragile Resistant to acidic pH
without giving harm to the host, if all entamoeba is
commensal they are not pathogenic
 With pseudopodium (false feet): finger-like
structures for movement formed by sudden jerky
movements of the ectoplasm in one direction. 
 Undergoes ENCYSTATION except for E. gingivalis
and Dientamoeba fragilis. 
 E. gingivalis and Dientamoeba fragilis do not have a
cyst form and stays in trophozoite form. 
 Inhabits the large intestine except for E. gingivalis
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Movement Unidirectional,Progressive Sluggish, non
progressive
and non
directional

Shape of Finger-like Blunted

pseudopodia

Manner of One at a time/explosive Several at a

release of time
pseudopodia
Nucleus Uninucleated (central Uninucleated
karyosome) (eccentric
karyosome)

Inclusion RBC Bacteria,

yeast, debris

Cytoplasm Clean looking Dirty looking

Size Bigger Smaller

B.CYST
Point of E. histolytica E. coli
Differentiation
No. of nuclei Quadrinucleated More than 4
Chromatoidal Sausage, rod, Broomstick,
bar cigar shaped splinter-like
Manner of Thin Thick
release of
pseudopodia

ENTAMOEBA HISTOLYTICA (A. TROPHOZIOTE

B.PRECYSTIC STAGE C. UNICLEATE CYST D. BINUCLEATE
CYST E. MATURE QUADRINUCLE CYST)

MORPHOLOGIC COMPARISON BETWEEN E.

HISTPLYTICA AND E. COLI TROPHOZIOTE

A.TROPHOZIOTE
POINT OF E. HYSTOLYTICA E.COLI
DIFFERENT
ATION
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AMOEBIC BACILLAR
DYSENTER Y
Y DYSENTER
ENTAMOEBA ENTAMOEBA Y
HYSTOLYTICA COLI
Onset Gradual Acute
Signs/Symptoms No Fever ans
CYST significant usually
fever or vomiting
vomiting
Odor of feces Offensive, Odorless
Fishy odor
Blood and mucus (+) Often watery
and bloody
pH Acidic Alkaline
PATHOLOGY Pus Few Numerous
cell/PMN/Nuetroph
ils
Cellular exudates Scant Massive
Pykonotic Numerous Few
residue Present Absent
Charcot Leyden Present Absent
cystals
Pathogenuc Few Numerous
Amoeba
Macrophages Absent Present
Common associated disease: Intestinal amebiasis,
amebic colitis, amebic dysentery, extraintestinal
amebiasis 
 Amebic colitis- gradual onset of abdominal pain and
diarrhea with or without blood and mucus on the
stool 
ENTAMOEBA ENTAMOEBA
HYSTOLYTICA COLI  Ameboma – mass-like lesions with abdominal pain
and history of dysentery. It may be mistaken for
carcinoma or malignant tumor.
PATHOGENEIS
Symptoms 
 Gradual onset of abdominal pain   
 Diarrhea (with or without blood)
 In children: bloody diarrhea, fever and abdominal
pain
 Abscess formation > Amoebic liver abscess 
Pathology (Intestinal amebiasis) 
 Amoebic dysentery vs. Bacillary Dysentery 
 Presence of Charcot-Leyden crystals, product from
metabolism of eosinophils, found microscopically in Can cause ulceration "flask-shaped ulcer" in the intestines
the stool in cases of amoebic dysentery (cecum, ascending colon and sigmoid)
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1.Galactose-inhibitable adherence lectin (Gal Lectin):

receptor mediated adherence of amoeba to target cells PATHOGENIC DETERMINANTS/VIRULENCE
2. Amoeba ionophore: cell lysis and tissue invasion  FACTOR
 Microscopic detection of cyst and trophozoite is the
LABORATORY DIAGNOSIS
standard method of parasitological diagnosis. 
Ionophore can attract calcium (anion), the calcium helps
 Minimum of 3 stools specimen on different days
the Gal lectin so the parasite can adhere on the target cells
should be examined (one stool sample for each day)
3. Cystein proteinase: most important, tissue invading
 For the detection of trophozoite, fresh stool
factor
specimen should be examined 30 mins after
EXTRAINTESTINAL AMOEBIASIS defecation. 
 Detection of E. histolytica trophozoite with ingested
 Through the portal vein (liver), trophozoite reach
RBC under saline solution is diagnostic of
other parts of the body (liver, brain, lungs and
amebiasis.
kidneys). 
1. Direct Fecal Smear 
 Metastatic amoebiasis- involvement of distant
 saline solution: trophozoite motility 
organs by hematogenous spread or through
 saline + methylene blue: Entamoeba spp. stain blue
lymphatic resulting to abscesses in the kidney, brain,
(differentiate Entamoeba spp. from WBC) 
spleen, and adrenals 
 saline + iodine: nucleus of E. histolytica can be
 Amoebic hepatitis – repeated invasion in the liver
observed (differentiate E. histolytica from
can cause inflammation 
nonpathogenic amoeba)
 Amoebic liver abscess – most common
2.Concentration Techniques 
extraintestinal form of amoebiasis; fever, upper right  In case of light infection, cyst and trophozoite may
quadrant pain; thick chocolate brown pus (liquefied not be detected in direct fecal smear. 
necrotic liver tissue)   Formalin Ether/ Ethyl Acetate Concentration
 Amoebic appendicitis and peritonitis  Technique (FECT) Merthiolate Iodine Formalin
 Pulmonary amoebiasis  Concentration (MIFC) – Sedimentation technique
 Cerebral amoebiasis  3.Culture 
 Splenic abscess   More sensitive than stool microscopy but not
 Cutaneous amoebiasis  routinely available (Ex: Robinson's and Inoki
 Genitourinary amoebiasis –destructive ulcerative medium, Boeck and Drbohlav media, NIH
lesions may resemble carcinoma  polygenic media, Craig's medium and Nelson's
 Asymptomatic carriers: cysts becomes unnoticed, medium
ameba reproduce but infected individual shows no 4.Serologic Testing 
clinical symptoms.   ELISA (Enzyme-linked Immunosorbent Assay),
 Diagnostic Stage: identification of the cyst or CIE (Counter Immunoelectrophoresis), AGD (Agar
trophozoite  Gel Diffusion), IHAT (Indirect Hemeagglutination
 Sample for ID: stool (examined within 30 minutes Test) and IF-AT (Indirect Fluorescent Antibody
from collection) Test) 
 IHAT and IFAT considered as gold standard in
detecting E. histolytica infection
5.Molecular Testing : PCR 
 In case of extraintestinal amoeba, CT-scan and MRI
may be used to detect amebic liver abscess.
To cure invasive disease at both intestinal and
TREATMENT extraintestinal site and to eliminate passage of cyst from
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intestinal lumen 
 Metronidazole: drug of choice for invasive
amebiasis (Tinidazole and secnidazole are also
effective) 
 Diloxanide furoate: drug of choice for
asymptomatic cyst passers 
 Percutaneous drainage of the liver abscess:
Patients who do not respond to metronidazole and
need prompt relief of severe pain

4. ENTAMOEBA POLECKI

 Parasite of the pigs and monkeys (rarely infect

humans) 
 Proper hygiene   Humans are accidental/incidental host 
 Provision for sanitary disposal of human feces  PREVENTION AND CONTROL
 Improve access to clean and safe drinking water  
 Good food preparation practices  Entamoeba chattoni: found in apes and monkeys,
 Avoid using "night soil"  identical to E. polecki, identification via isoenzyme
 Food handler should be examined for cyst carriage analysis
 Health education and promotion

NON-PATHOGENIC SPECIES

Harmless inhabitant
1.ENTAMOEBA COLI of the colon 
 Cysts: Size (10 — 35 microns) 
 Larger than E. histolytica   
 Consists of 8 nuclei with very diffuse karyosomes
 May become hypernucleated with 16-32 nuclei May
also contain needle-like chromatoidal bodies with
irregular fragmented/sharp/splintered ends 
 Trophozoites: Size (15-50 microns) 
 Smaller than E. histolytica 
 Has one nucleus containing large, diffuse 5.ENTAMOEBA GINGIVALIS
karyosomes  
 Not capable of encystation. Trophozoite form only
 Peripheral chromatin is usually dense and irregular
 Can be found in the mouth (gum and teeth surfaces)
 Cytoplasm is usually rough and contain few to many
ingested debris  Abundant in cases of oral diseases 
 No cyst stage, does not inhabit the intestines 
 Transmission through kissing, droplet spray, sharing
utensils 
 May ingest RBC (rarely), associated on lesions
inside the mouth

2. ENTRAMOEBA DISPAR 6. ENTAMOEBA MOSHHOVSKII

 Morphologically similar to E. histolytica, but with
different DNA and RNA.  Morphologically indistinguishable from those of the
disease causing species E. histolytica and the non-
3 ENTAMOEBA HARTMANNI pathogenic E. dispar, but differs from them
 Similar to E. histolytica except much smaller and no biochemically and genetically. Although sporadic
RBC inclusions  cases of human infection with E. moshkovskii have
 "small-race E. histolytica” been reported, the organism is considered primarily
a free-living amoeba. 
 physiologically unique: osmotolerant, able to grow
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at room temperature and able to survive at 0-41 o c
7. ENDOLIMAX NANA
 "Smallest amoeba" 
 "Cross eyed cyst" — 4 eccentric nuclei 
 Blot-like karyosome

SPECIMEN
 Discharges, exudates and tissue secretions
PATHOGENESIS
Granulomatous Amoebic Encephalitis (GAE)
 destructive encephalopathy and associated
meningeal irritation 
 Disease of immunocompromised (AIDS) 
 Laboratory diagnosis: made by demonstration of
trophozoites and cysts in brain biopsy (post-mortem
8.IODAMOEBA BUTSCLII in most cases), culture, and immunofluorescence
microscopy-using monoclonal antibodies. 
 "iodine-cyst" because of its affinity to iodine   CSF shows lymphocytic pleocytosis (abnormal
 Large glycogen vacuole/ body which stains deeply increase in the number of lymphocyte in the CSF),
with iodine  slightly elevated protein levels, and normal or
 Uninucleated — resembling a "basket of flowers" slightly decreased glucose levels. 
shape  CT scan of brain provides inconclusive findings.

Granulomatous Amoebic Encephalitis (GAE)

 perforation of the cornea and results to subsequent

loss of vision 
 Laboratory diagnosis: made by demonstration of
the cyst in corneal scrapings by wet mount,
histology, culture (growth can be obtained from
FREE LIVING PATHOGENIC AMOEBA
corneal scrapings inoculated on nutrient agar,
1. ACANTHAMOEBA SPP. (ACANTHAMOEBA overlaid with live or dead Escherichia coli and
 Ubiquitous, free-living ameba  incubated at 300 C), demonstration of cyst and
CASTELLANI
 With an active trophozoite stage with characteristic trophozoites in stool and PCR.
prominent "thorn-like" appendages (acanthopodia)
and resilient cyst stage 
 Aquatic organism, can survive in contact lens
cleaning solutions 
 Most common ameba of freshwater and soil 
 No flagellate state
MODE OF TRANSMISSION
 Aspiration or nasal inhalation: use of contaminated
swimming pools, deep well, etc.  2. NAEGLERIA FOWLERI
 Direct invasion of the eye: contaminated saline
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 Free-living protozoan with two vegetative forms: an coli, (+) both trophozoites and cysts. 
ameba (trophozoite form) and a flagellate Molecular Diagnosis: Polymerase chain reaction (PCR)
(swimming form) 
 "brain-eating amoeba"  PREVENTION
 Thermophilic organism that thrive best in hot  Frequent cleaning 
springs and other warm aquatic environment   Chlorination 
 True pathogen   Salination
 Incubation period varies from 2 days to 2 weeks. 
 Disease almost ends fatally within a week

PHYLUM CILIOPHORA CILATES

1. BALANTIDIUM
 Causative agentCOLIof "balantidiasis or balantidial
dysentery", similar to amoebic dysentery 
 Largest protozoan parasite 
 Only parasitic ciliate 
 Primarily associated with pigs

PATHOGENESIS
Fatal Primary Amoebic Encephalitis (PAM)
 patients initially complain of fever, headache, sore
throat, nausea and vomiting 
 Hemorrhagic necrosis in post mortem examination
of infected brain

MORPHOLOGY
 Has trophozoite and cyst stage 
Parts: 
 Cytosome: entry of food 
“Kernig;s sign”  Cytophyge: excretes waste 
 Two dissimilar nucleus: Large kidney-shaped
 diagnostic sign for meningitis where the patient is
macronucleus and micronucleu 
unable to fully straighten his or her leg when the hip
 One or two contractile vacuoles
is flexed at 90 degrees because of hamstring
stiffness

MODE OF TRANSMISSION PATHOGENIC DETERMINANT

 Oral and intranasal routes while swimming in  Hyaluronidase: causes the ulceration, secreted by
contaminated pools, rivers and lakes trophozoite 
 Laboratory diagnosis:  Ulceration is described as flask-shaped ulcer but
CSF examination  with rounded base and wider neck. 
 cloudy to purulent     Unlike like Entamoeba histolytica, Balantidium coli
 neutrophilic leukocytosis does not invade the liver or other extraintestinal site.
 elevated protein and low glucose r LABORATORY DIAGNOSIS
 esembling pyogenic meningitis   Stool examination: microscopic demonstration of
Wet film examination of CSF: (+) trophozoites  cyst and trophozoite in direct 
Autopsy: (+) trophozoites in immunofluorescent staining  Biopsy: specimens and scrapings from intestinal
Culture: can be grown in several kinds of liquid axenic ulcers can be examined for presence of trophozoites
media or non-nutrient agar plates coated with Escherichia and cysts. 
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 Culture: can also be cultured in vitro in Locke's egg
albumin medium or NIH polyxenic medium like
Entamoeba histolytica, but it is rarely necessary.

MODE OF TRANSMISSION
 ingestion of food/water contaminated with B. coli
cyst
INFECTIVE STAGE
 Cyst 
TREATMENT
 Tetracycline is the drug of choice. Alternatively
Doxycycline can be given. 
 Metronidazole and nitroimidazote have also been
reported to be useful in some cases.
PREVENTION
 Avoidance of contamination of food and water with
human or animal feces. 
 Prevention of human-pig contact. 
 Treatment of infected pigs. 
 Treatment of individuals shedding B. coli cysts.