LABORATORY DIAGNOSIS

OF PARASITIC DISEASES
 OUTLINE
Macroscopic examination

Microscopic diagnosis

Immunological diagnosis

Molecular diagnosis

Other techniques

Specimens for parasitological diagnosis

 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 microscope calibration 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 diagnosis in the
diagnosis parasitic diseases.
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
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

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
Systematic microscope technique
–1
Types of stain
Temporary stain
 Eosin
 Thomson’s stain
 Lugol’s iodine solution
 Sargeant’s stain
 Burrow’s stain
 Acridine orrange
Permanent stain
Giemsa/field stain
Trichrome stain
Iron-hematxiline stain
Modified ziehl-neelson stain
Phenol-auromine method
2-Immunodiagnosis:-
Is based on the detection of :
A. Antibody detection 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
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
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.
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
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
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 cylindric 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).
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.
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.
WHAT IS FECES ( STOOL)
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
Examination of stool
Purpose of examining stool:
To identify intestinal parasitic infection associated
 Severe anemia especially in pregnant & child
 Series ill-health

 Persistent diarrhea

 Weight loss, malabsorbtion

 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
HOW ARE FECES EXAMINED TO DIAGNOSE MEDICAL
PROBLEMS?

Macroscopic examination of stool

 color
 consistency.
 Abnormal elements
 smell,
 quantity, and

Microscopy examination of stool

 for blood, mucus, fat, or parasites
Immunological
 Ag-detection

Molecular technique
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 as well as
standard microbiological laboratory practices
(Biosafety Level 2).
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.
Collection of fecal specimens
recovery and ID depends on:
proper collection and fixation
Proper container
 clean, dry, water-proof, wide-mouth container
Sufficient amount
 About 20-40 grams of well-formed stool or 5-6 table spoonfuls of
watery stool (10ml)
Prequation
NO contamination with H O - could bring free-living
2
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
Collection continued
Recheck:
protozoan: 3 - 4 weeks
helminth: 1 - 2 weeks
Taenia sp.: 5 - 6 weeks
Stool: Color
Normal:
Adult: brown
New born infants:- Black(meconium)
Breast feed infants:- scrambled egg
Infant feed on animal milk:- “ curd like”
 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.
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.
Transitional Stool - Stage Two
Stool is lighter in
color and slightly less
thick than
meconium.
Transitional Stool - Stage
Three
Much lighter and
thinner than
meconium.
Occurs just before
regular stooling
begins
Breastfed Stool
Yellow
Runny
Small seed like
objects in the stool
Often called baby
poop mustard
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 by a
physician.
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)
Stool: Color
Abnormal:
Red:
 Bleeding from lower gastrointestinal tract (e.g., rectum),
 hemorrhoids

 some foods
 red gelatin,
 tomato juice or soup

 large amounts beets

Pale:
 Malabsorption of fats,
 diet high in milk and milk products and low in meat
B- Consistency of stool
Varies due to diet but provides information on the
stage of protozoa that is present
 1-Hard- resists puncture
 2-formed- can be punctured

 3- Soft-can be cut with applicator

 4- Mushy- can be reshaped

 5- loose- shaped in to container

 6- Diarrhea- can flows

 7-Watery- can pour

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) Normal stool is formed, soft, or mushy in consistency
Cleary watery, loose mixed with mucus and blood
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.
Stool: Shape
Normal: Cylindrical (contour of rectum) about 2.5
cm (1 inch) in diameter in adults

Abnormal: Narrow, pencil-shaped, or stringlike stool

Obstructive conditional of the rectum
Stool: Amount and size
Normal:
Amount
 Varies with diet
 About 100 to 400 g 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.
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
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
Stool: Odor
Normal: Aromatic, affected by ingested food and
person’s own bacterial flora

Abnormal: Pungent
Infection, blood
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)
Stool: Constituents
Abnormal:
Pus: bacterial infection
Mucus: inflammatory condition
Parasites
Blood: gastrointestinal bleeding
Large quantities of fat: malabsorption
Foreign objects: accidental ingestion
Microscopic examination
Direct smear
Smear after concentration
Permanent stained smear
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
1-Use cleaned microscope Slides
2-Place a drop of saline
3-Take a small amount of stool with
a wooden stick
4-Mix stool with saline
Note: Mistake:Too much stool!
 5-Place coverslip Avoid air bubbles!

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

: Use 10x objective
Ova, cysts, trophozoites and adult worms can be
identified as per their characteristic features
Preservation
Required if not delivered to lab immediately
Preserve protozoan morphology
Prevents development of worm eggs/larvae
Commercial kits - vials for PVA & formalin
3 parts fixative to 1 part stool
Record time collected and placed in preservative
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
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)
Examination of fecal specimen
Consistency (aids ID of protozoan)
Liquid
1 o
motile protozoan trophozoites
examine within 1/2 hour of passage

preserve for permanent smear

Semi-formed
also some cysts (semi-formed)
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
Fecal exam continued
Color
Brown: normal
Dark: possible bleeding upper GI tract
Fresh blood: possible bleeding lower GI
tract
Select areas of stool with blood
Microscopic exam
General
correct illumination
ocular micrometer - not interchangeable
after calibration
scan edges of coverslip
observe entire slide (tedious)
use references: pictures, size charts,
stained positive slides
Direct wet mount
1o motile protozoans in liquid/soft stools
PVA preservation not acceptable - cloudy
Wet prep - 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
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
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
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
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
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
Sedimentation
Easiest procedure
Centrifugation or gravity - recovers all organisms and
stages
Uses formalin-ethyl acetate
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
Permanent stains
Made from all stools to ID 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
Iron hematoxylin stain
More time-consuming
Harder to read
Organisms: gray-black
Background: light blue-gray
Microscopic examination
10 - 15 minutes/slide in selected areas
Scan thick and thin areas at 10x or 40x
Scan thin areas with 100x objective
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
Other specimens continued
Vaginal/urethral discharge - Trichomonas vaginalis
trophozoites
Sputum
Strongyloides
larvae
Paragonimus westermani eggs
Entamoeba histolytica (from pulmonary
abscess)