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Laboratory Diagnosis of Parasitic Diseases
Laboratory Diagnosis of Parasitic Diseases
Laboratory Diagnosis of Parasitic Diseases
PARASITIC DISEASES
OUTLINE
– Macroscopic examination
– Microscopic diagnosis
– Immunological diagnosis
– Molecular diagnosis
– Other techniques
• 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
– 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: 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
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
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
• 1o 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
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
Blood specimen
Parasites that may be detected in blood
specimens include the agents of malaria
(Plasmodium spp.), babesiosis (Babesia spp.),
trypanosomiasis (Trypanosoma spp.),
leishmaniasis (Leishmania donovani), and
filariasis (Wuchereria bancrofti, Brugia malayi,
Loa loa, and Mansonella spp.).
Blood specimen
• The most important techniques to be
performed in the clinical laboratory to assist in
the diagnosis of blood parasites include
preparation, staining, and examination of
thick and thin blood films. Other techniques
used less frequently include the buffy coat
smear and various concentration techniques
reserved for recovery of microfilariae
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