UNIT – TWO

LABORATORY DIAGNOSIS OF
PARASITIC DISEASES

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 OUTLINE

– Macroscopic examination

– Microscopic diagnosis

– Immunological diagnosis

– Molecular diagnosis

– Other techniques

– Specimens for parasitological diagnosis

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 Learning objective
Upon completion of this unit of instruction and lecture, the
student will be able to:
•  List the common techniques used for the detection of
parasites in fecal and blood specimens.
• Discuss the importance of microscopic adjustment in
parasitology.
• List the types of microscopic methods used in parasitological
investigations.
• Describe the difference between immunodiagnostic techniques
and detection of antigens in parasitology specimens.
• Discuss the significance of molecular methods in the diagnosis
of parasitic diseases.

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 2.1. General laboratory techniques

1. Parasitic (morphological) diagnosis

– Macroscopic
– Microscopic
2. Immunological diagnosis
– Antibody detection
– Antigen detection
3. Molecular diagnosis
4. Culture
5. Animal inoculation
6. Xenodiagnosis

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 1-Parasitic (morphological) diagnosis
• Laboratory procedures detect organisms within clinical
specimens using morphological criteria,
A. Macroscopic-using our naked eye
• Eg: Stool specimen can be examined with the naked eye
for presence of some adult worms
– E.g. Ascaris, Taenia species, E.vermicularis and gravid
Taenia species

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B. Microscopic
• the majority of intestinal, blood, urinary and skin parasites are
usually detected microscopically
• Use different body specimen such as blood, stool, urine, CSF etc
– either directly or following concentration techniques in
stained or unstained preparation

What is staining? What is purpose of staining? (reading)

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Systematic microscopic techniques - adjusting
the microscope

• Select appropriate objectives

• Adjust optimal illumination

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 Types of stain
Temporary stain
• Eosin
• Thomson’s stain
• Lugol’s iodine solution
• Sargeant’s stain
• Burrow’s stain
• Acridine orange

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 Permanent stain
• Giemsa/field stain

• Trichrome stain

• Iron-hematxiline stain

• Modified ziehl-neelson stain

• Phenol-auromine method

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 2-Immunodiagnosis:-

• Is based on the detection of :

A. Antibody detection in person's serum
• Ab is produced in response to a particular parasitic infection.
• The Ab. may persist for a long period of time in the serum after an
infection has ended
• antibody tests are unable to distinguish between past or present
infection

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B. Antigen detection
– Ag. is excreted by parasites and can be found in the serum,
urine, CSF, feces or other specimens.
– Antigen tests provide evidence of present infection

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 When do Immunodiagnostic techniques are
required?

• Parasites live in the tissue of internal organ and can not easily
obtained for examination.
• Parasites can be found in specimens only in certain stages of
infection,
– e.g., in the acute stage not in the chronic stage.
• Parasites are present intermittently or in too few numbers to be
easily detected in the specimens.
• The techniques used to detect parasites are complex or time
consuming.

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 Immunodiagnosis is of particular value for:

• South American trypanosomiasis, Chronic stage

• African trypanosomiasis, when parasitaemia is low
• Leishmaniasis
• Filariasis
• Amoebic liver abscess
• Trichinosis
• Toxoplasmosis
• Toxocarisis
• Hydatid disease
• Schistosomiasis
• Malaria

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 3-Culture

• Growth medium is provided for a specific parasite.

• A specimen is tested for the presence of an infectious agent able to grow
within that medium
• Culture allows identification of infectious organisms by examining their
– microscopic features,
– by detecting the presence of substances produced by pathogens, and
– by directly identifying an organism by its genotype
• Relatively few of protozoa and helminths parasites, can be cultured.
– eg. E.histolytica, T.vaginalis, T.cruzi and Leishmania species

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 4. Xenodiagnosis (XD)

• XD is a diagnostic procedure which uses the vector which acts as a

biological culture medium for the detection of T. cruzi in the blood of
infected man and other mammals.
• Routine or natural XD consists in the use of one cylindrical pot containing
7-10 nymphs of unfed triatomine bugs which suck blood from the skin of
the individual to be examined
• After an incubation period, the abdominal contents of the utilized nymphs
are examined by means of different techniques (compression of the
abdomen, dissection, grinding and homogenization of the intestine, and
liquefaction of the whole insect).

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 4. Xenodiagnosis (XD)

• Eg. In the XD non-infected laboratory reared nymphs of triatomines

are used.
• The triatomines unfed in the previous 3-4 weeks, is applied, to the
skin surface (upper limb) of the individual to be examined for 20-30
min, .
• After this time, the nymphs become engorged and kept in
entomological laboratory conditions.
• After about 30 days, excreta (feces and/or urine) of the insects are
microscopically examined for moving T. cruzi trypanomastigotes.

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 5. Molecular Diagnosis
• Microscopic examination is still considered the “gold standard”
for the diagnosis of parasitic diseases. 
• the stool specimen can be analyzed using molecular techniques
such as polymerase chain reaction (PCR). 
• PCR amplified fragments can be analyzed by:
– using restriction fragment length polymorphisms (RFLP) or
– DNA sequencing if further characterization is needed.

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Types of specimen for parasitological
tests

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 Types of Specimen Used For
Parasitological Examination
• • Liver aspirates
Stool
• Spleen aspirates
• Blood
• Muscle biopsy
• Urine
• Rectal scraping
• Sputum • Duodenal aspirates
• Skin • Bronchial biopsy
• CSF • Perianal swab
• Bone marrow
• Lymph gland aspirates

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 Types of Specimen…
 Stool :-.e. g., intestinal nematodes, cestodes, trematodes
and protozoa.
 Blood :- e.g., Haemoparasites
 Urine :- e.g., S. hematobium, T. vaginalis,
 Sputum :- e.g., P. westermani.
 Skin :- e.g., L. aethopica, O. volvulus, D. medinensis and
E. vermiculari
 Cerebro-Spinal fluid:- e.g., Trypanosoma rhodisense and
Naegleria fowleri.
 Bone marrow:- e.g., L. donovani and T.gondii
 Lymphgland aspirates:- e.g Trypanosoma rhodisense,
L..donovani and T. gondii
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 Types of Specimen…
 Liver aspirate :e.g. E.histolytica, L.donovani and T.gondii

 Spleen aspirate:- e.g., L.donovani and T.gondii

 Muscle biopsy:- e.g., T. spiralis

 Rectal scraping:- e.g., Schistosoma species

 Duodenal aspirate:- e.g., G. lamblia, F. hepatica and S. stercoralis

 Bronchial biopsy :- e.g., P.carnii

 Perianal swab:- e.g., E.vermicularis

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Fecal (stool) specimens

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 WHAT IS FECES ( STOOL)

• FECES (STOOL)
– waste product or substance formed in the digestive tract and
excreted out through the rectum (rear end).
• WHY IS IT CALLED FECES?
– Feces comes from the Latin word "faex,“
– It means "dregs." Dregs means the most undesirable part.
• FECES ARE ALSO KNOWN AS STOOL .
– Stool comes from the Anglo Saxon word "stol," which means
"seat”

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 Examination of stool
• Purpose of examining stool:
– To identify intestinal parasitic infection associated with
» Severe anemia especially in pregnant & child
» Series ill-health
» Persistent diarrhea
» Weight loss, mal-absorption
» Impairment of development etc
– To identify chronic infection with serious complication if
untreated
– To identify parasitic causes of blood and mucus
– To assist in surveillance &control of parasitic infection

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 HOW ARE FECES EXAMINED TO DIAGNOSE MEDICAL
PROBLEMS?

• Macroscopic examination of stool

– color
– consistency
– Abnormal elements
– smell,
– quantity
• Microscopy examination of stool
– for blood, mucus, fat, or parasites
• Immunological
– Ag-detection

• Molecular technique

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 Biosafety
• potential risks with stool specimens includes:
– ingestion of eggs or cysts,
– skin penetration by infective larvae, and
– infection by non-parasitic agents found in stool and biologic
fluids. 
• These risks can be minimized:
– by adopting universal precautions, and
– Following standard microbiological laboratory practices
(using of Biosafety  Level 2)  

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• Wear protective safety glasses, gloves and laboratory coat when
processing specimens.
• Use biological safety cabinets as needed.
• Do not eat, drink, smoke, apply cosmetics or manipulate contact
lenses in work area.
• Decontaminate work surface at least once a day and after any spill of
potentially infectious material.
• If you have cuts or abrasions on the skin of your hands, cover them
with adhesive dressing.
• If you use any sharp instruments, dispose of them in a “sharps”
container for decontamination.
• Remove gloves and wash your hands after completing any task
involving the handling of fecal material.

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 Collection of fecal specimens

• Recovery and Identification depends on:

– proper collection and fixation
– Proper container
• clean, dry, water-proof, wide-mouth container
– Sufficient amount
• About 20-40grams of well-formed stool, or
• 5-6 table spoonfuls of watery stool (10ml)

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 Precautions

• NO contamination with H2O - could bring free-living

protozoans
• NO contamination with urine - destroys trophozoites

• Collect before barium enema - obscures organisms (7+ days)

• Collect before antibiotics - may decrease number of organisms

• Series of 3 specimens; 2 normal, 1 purged (if necessary)

– within 10 days on alternate days

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 Collection …..continued

• Recheck:
– protozoan: 3 - 4 weeks
– helminth: 1 - 2 weeks
– Taenia sp: 5 - 6 weeks

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 Stool: Color

• Normal:
– Adult: brown
– New born infants:- Black (meconium)
– Breast feed infants:- scrambled egg
– Infant feed on animal milk:- “ curd like”

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 Meconium: Baby’s first stool

• First stool a baby will pass

thick
• Green, tar-like substance
• Lines the intestines of the
fetus
• First bowel movement within
a few hours after birth.

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 Transitional Stool - Stage One

• Newborn slowly begins to

pass the meconium after
birth
• Meconium will begin to
change in consistency
• Slightly lighter in color than
meconium.

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 Transitional Stool - Stage Two

• Stool is lighter in color and

slightly less thick than
meconium.

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 Transitional Stool - Stage Three

• Much lighter and thinner

than meconium.
• Occurs just before regular
stooling begins

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 Breastfed Stool

• Yellow
• Runny
• Small seed like objects in the
stool
• Often called baby poop mustard

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 Stool: Color
– Changes in the color, consistency, and frequency of bowel
movements is known as a "change in bowel habits.
– In some cases, an unusual stool color is harmless and can
be attributed to a particular food or medication
– Changes in stool color that persist can be a serious matter
and should always be investigated

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 When should you worry about the color of your
stool?
• Abnormal:

– Clay or white:
• Absence of bile pigment (bile obstruction), or
• diagnostic study using barium
– Black or tarry:
• Drug (e.g., iron),
• bleeding from upper gastrointestinal tract (e.g.,
stomach, small intestine),
• diet high in red meat and
• dark green vegetables (e.g., spinach)

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 Stool: Color
• Abnormal:
– Red:
• Bleeding from lower gastrointestinal tract (e.g., rectum),
• hemorrhoids
• some foods
–red gelatin,
–tomato juice or soup
–large amounts of beets
– Pale:
• Malabsorption of fats,
• diets high in milk and milk products and low in meat

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 B- Consistency of stool
• Varies due to diet but provides information on the stage
of protozoa that is present
Hard- resists puncture
formed- can be punctured
Soft-can be cut with applicator
Mushy- can be reshaped
loose- shaped in to container
Diarrhea- can flows
Watery- can pour

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 Stool: Consistency
• Normal: Formed, soft, semisolid or mushy
• Abnormal:
– Hard, dry, constipated stool
• Dehydration, decreased intestinal motility resulting
from lack of fiber in diet, lack of exercise,
emotional upset, laxative abuse
– Diarrhea
• Increased intestinal motility (e.g., irritation of the
colon by bacteria)
• watery, loose mixed with mucus and blood

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 Bristol stool form scale

• Classification of the form,( appearance in a toilet) of faeces into

seven groups.
• The form of the stool depends on the time it spends in the colon.
• Chart breakdown
– Types 1 and 2 indicate constipation;
– Types 3 and 4 are usually the most comfortable to pass,
– Types 5-6 tend to be associated with urgency, while
– Type 7 is diarrhea.

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 Stool: Shape
• Normal: Cylindrical (contour of rectum) about 2.5 cm (1
inch) in diameter in adults

• Abnormal: Narrow, pencil-shaped, or string like stool

– Obstructive conditional of the rectum

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 Stool: Amount and size
• Normal:
– Amount
• Varies with diet
• About 100 to 400g per day
– Size
• A healthy piece of feces is about one foot long.
• Shorter sized feces suggest that the colon is not able
to process the food correctly, and
• the feces produced does not have the correct
amount of moisture in it.

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 Stool: Frequency

• HOW OFTEN DOES THE AVERAGE PERSON

POOP?
• Normal range
– three times a day to once every three days.
– average person poops about once a day.
• Abnormal
– four times a day or more and
• the stool has a liquid consistency – diarrhea
– less than two or three days a week and
• the stool is hard, dry, and difficult to pass-constipation

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 Stool: Odor
• WHAT MAKES FECES SMELL SO BAD?
– The distinctive odor of feces is due to bacterial action.
– Specifically, the bacteria produce various compounds
and gases that lead to the infamous smell of feces.
– Gut flora produce compounds such as indole, skatole,
and thiols (sulfur containing compounds), as well as
the inorganic gas hydrogen sulfide
– The bad smell of feces will usually be reduced by
eating more natural foods that do not contain any
artificial flavors or chemicals

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 Stool: Odor

• Normal: Aromatic, affected by ingested food and

person’s own bacterial flora

• Abnormal: Pungent
– Infection, blood

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 Stool: Constituents
• Normal:
– water (about 75%).
– Rest constitute:
• dead bacteria that helped us digest our food, living
bacteria,
• undigested food residue (known as fiber),
• cellular linings, sloughed epithelial cells
• substances released from the intestines (such as mucus)
and the liver.
• fat, protein, dried constituents of digestive juices (e.g.,
bile pigments),
• inorganic matter (e.g., calcium, phosphates)
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 Stool: Constituents

• Abnormal:
– Pus: bacterial infection
– Mucus: inflammatory condition
– Parasites
– Blood: gastrointestinal bleeding
– Large quantities of fat: malabsorption
– Foreign objects: accidental ingestion

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 Microscopic examination

• Direct smear
• Smear after concentration
• Permanent stained smear

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 Direct mount
Methods in Parasitology
Fresh Stool Examination (Wet mount)
• Contents
1.1 Materials
1.2 Preparing wet mount
1.3 Examining wet mount
Materials:
• Microscope slides
• Cover slips
• Sodium chloride solution bottle with pipette
• Wooden stick
• Fresh stool
• Gloves

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1-Use cleaned microscope Slides

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2-Place a drop of saline

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3-Take a small amount of stool with a
wooden stick

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4-Mix stool with saline

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Note: Mistake:Too much stool

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5-Place coverslip; Avoid air bubbles!

6-Examination of helminth ova/larva/cyst/trophozoites

: Use 10x objective
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• Ova, cysts, trophozoites and adult worms can be
identified as per their characteristic features

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 Preservation

• Required if not delivered to lab immediately

• Preserve protozoan morphology
• Prevents development of worm eggs/larvae
• Commercial kits - vials for PVA (Polyvinyl Alcohol) &
formalin
• 3 parts fixative to 1 part stool
• Record time collected and placed in preservative

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 Preservation ……continued

• Polyvinyl mercuric chloride (PVA)

– mercury chloride: fixative
– polyvinyl alcohol: resin aids adherence
• Copper-based: not as good as mercury
• Zinc-based - morphology better than copper

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 Preservation

• 5 - 10% formalin - wet mount/concentrate; cysts, eggs,

larvae preserved long time.
• Sodium-acetate-acetic acid formalin (SAF) -
concentration & permanent stains; albumin helps
adherence; good for iron hematoxylin stain
• Merthiolate-iodine-formalin (MIF)
– preserves protozoan/worms in wet mount &
concentration (NOT permanent stain)

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 Examination of fecal specimen

• Consistency (aids ID of protozoan)

– Liquid
• 1o motile protozoan trophozoites
• examine within 1/2 hour of passage
• preserve for permanent smear
– Semi-formed
• also some cysts (semi-formed)

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 Fecal exam ….continued

• Formed stool
– protozoan cysts
– examine on day of passage
– refrigerate up to 24 hours
– in formalin for concentration procedures
– in PVA for permanent smears
• NOT in incubator - destroys parasites, increases bacteria

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 Fecal exam continued

• Color
– Brown: normal
– Dark: possible bleeding of upper GI tract
– Fresh blood: possible bleeding lower GI tract
• Select areas of stool with blood

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 Microscopic exam

• General guide in microscopic examination

– correct illumination
– ocular micrometer - not interchangeable after
calibration
– scan edges of cover slip
– observe entire slide (tedious)
– use references: pictures, size charts, stained positive
slides

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 Direct wet mount

• 1o motile protozoans in liquid/soft stools

• PVA preservation not acceptable - cloudy
• Wet preparation - small amount of stool in
– saline: detect worm eggs/larvae & refractile protozoan
cysts
– iodine: shows nuclear detail
• Low objective (high if suspicious object), low light

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 Concentration

• Parasites in small amount of fluid

• Removes most of debris
• Based on difference in specific gravity between parasites
and concentrating solution
• Protozoan trophozoites do not survive
• Detects protozoan cysts, worm larvae/eggs

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 Concentration Procedures

• Fecal concentration techniques are used to concentrate

and quantify fecal parasites when a direct wet mount of a
fecal material fails to reveal the presence of parasitic
organisms
• The methods concentrate the parasites using gravity or
centrifugation
• Fecal concentration techniques include:
– The sedimentation method
– The floatation method

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 Concentration Procedures

• Sedimentation method
– Uses the principle of gravity or centrifugation to allow for the
recovery of all protozoa, eggs and larvae present
– The sediment preparation contains more fecal debri
– Example:  the formalin-ether method

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 Concentration Procedures

• Floatation method
– Separates protozoan cysts and certain helminth eggs through
the use of a liquid with a high specific gravity
– Provides a clearer preparation without much debri compared
to the sedimentation method
– Examples:
 zinc sulfate floatation technique
 saturated sodium chloride method

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 Concentration Procedures

• Field survey techniques

– Kato-Katz
 developed for the semi-concentration and semiquantitative
estimation of shistosome eggs in feces
– Formol detergent
 is more sensitive than the Kato-Katz technique because
more feces is used
 uses formalin which kills fecal pathogens

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 Sedimentation

• Easiest procedure
• Centrifugation or gravity - recovers all organisms and
stages
• Uses formalin-ethyl acetate

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 Flotation

• Yields less fecal debris

• Organisms suspended in zinc sulfate (S.G. = 1.180)
• Recovers most parasites in surface film
• Opens/collapses operculated eggs
• Distorts protozoan cysts
• Heavy eggs may be missed - sink to bottom
• Examine soon for maximum recovery

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 Permanent stains

• Made from all stools to identify protozoans

• Trichrome - stain of choice
– fresh stool in PVA
– blue-green cytoplasm of trophozoites and cysts
– purple/red nuclear chromatin, karyosome,
chromatoidal bars, RBCs
– eggs/larva: red; debris/yeast: green

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 Iron hematoxylin stain

• More time-consuming
• Harder to read
– Organisms: gray-black
– Background: light blue-gray

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 Microscopic examination

• 10 - 15 minutes/slide in selected areas

• Scan thick and thin areas at 10x or 40x
• Scan thin areas with 100x objective

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 Other specimens examined for
intestinal parasites
• Duodenal aspirates - e.g., Strongyloides sp., Giardia
lamblia
• Sigmoidoscopy specimens - e.g., ameba,
Cryptosporidium
• Urine - Schistosoma hematobium & Enterobius
vermicularis ova
• Cellophane tape-Enterobius vermicularis ova

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 Other specimens continued

• Vaginal/urethral discharge - Trichomonas vaginalis

trophozoites
• Sputum
– Strongyloides larvae
– Paragonimus westermani eggs
– Entamoeba histolytica (from pulmonary abscess)

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