Diagnostic
Parasitology
MedBio4: Medical Parasitology
1st Semester, A.Y. 24-25
Laboratory Diagnosis
A parasitology laboratory should be
able to:
confirm a clinical impression that
the condition has a parasitic nature;
rule out differential diagnoses;
aid a clinician in the choice of
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proper medication; and
help in monitoring the effect of a
treatment regimen.
Laboratory Diagnosis
Example:
Dysentery is a disease that is
characterized by diarrhea and contains
both blood and mucus (macroscopic
examination).
Diagnosis through clinical signs and Stool
stated symptoms, but confirmed by
microscopic examination.
Laboratory Diagnosis
Example:
Dysentery is a disease that is
characterized by diarrhea and Bacillary dysentery Amoebic dysentery
contains both blood and mucus
(macroscopic examination). Feces: Feces:
no forms of trophozoites
bacteria are present
Diagnosis through clinical signs
identified as
and stated symptoms, but
trophozoites
confirmed by microscopic
examination.
Laboratory Diagnosis
factors that generate reliable results:
proper collection, handling, and processing of specimens prior to
examination
the skill of the laboratory analyst (examiner)
the quality of equipment used in the examination.
Laboratory Diagnosis
There are two ways of diagnosing
parasitic infections:
Taenia saginata
1. Demonstration of parasite or
parasite components -
definitive diagnosis
egg scolex mature proglottid
2. Detection of host immune
response (humoral) to the
parasites - presumptive
evidence of infection
Laboratory Diagnosis
factors that generate reliable results:
proper collection, handling, and processing of specimens prior to
examination
the skill of the laboratory analyst (examiner)
the quality of equipment used in the examination.
Laboratory Protocols
in Specimen Collection
MedBio4: Medical Parasitology
1st Semester, A.Y. 24-25
Specimen Collection
Protocols for parasitologic study depend on type of sample and examination
When this part is compromised, the results can be affected dramatically
Proper specimen selection and processing are crucial to parasite recovery
Collection and Processing:
Environmental Samples
a. Water Samples
Cryptosporidium sp. and
Giardia sp.
Procedures:
EPA Method 1623
Modified Method 1623
Elution – the process of extracting one material from another by washing
with a solvent
b. Soil Samples
Protozoan and helminths
hookworms (filariform larvae)
Procedure:
Collect using auger/spade -
20 cm deep
Put in sterile plastic bags
Sent to lab for immediate
processing
Amphizoic - organism that can exist either
as a parasite or as free-living organism
Collection and Processing:
Human Biological Samples
a. Urine
Trichomonas vaginalis (trophozoites)
and Schistosoma haematobium (eggs)
Terminal urine specimen (last 10-
20mL) - 24-hrs
Excretion of eggs - highest around
midday
In case of delay: 0.5 mL of 10% formalin
to prevent eggs from hatching Trichomonas vaginalis
a. Urine
Microfilariae can sometimes
be found in the urine of patients with a
heavy filarial infection.
Schistosoma haematobium
b. Stool
Most common (intestinal in origin)
3-5 grams in sterile containers
Prevent contamination w/ urine
Immediate processing
In case of delay: Preservatives
b. Stool - Macroscopic Examination
Consistency:
Watery/Loose - diarrheic stool (trophozoites)
Formed/Hard or Semi-formed/Soft - cysts,
helminth eggs, and larvae
b. Stool - Macroscopic Examination
Presence of:
Tapeworm proglottids, and adult worms
Blood, mucus, and pus
Fresh (bright red) - acute lower GI
bleeding or irritation
Bloody w/ mucus - amebic ulceration in
the colon or large intestine
b. Stool - Microscopic Examination
1. Ocular Micrometer Calibration
2. Processing of Stool Samples
3. Examining the mounted slides
microscopically
Egg of Trichuris sp. measured with an ocular micrometer.
b. Stool - Microscopic Examination
1. Ocular Micrometer Calibration
size difference (microns)
2. Processing of Stool Samples
DFS
Kato-thick
Concentration Techniques
3. Examining the mounted slides
microscopically
Egg of Trichuris sp. measured with an ocular micrometer.
c. Perianal swabs
Enterobius vermicularis
scotch tape
tongue depressor
glass slide
PPE (gloves and mask) - Highly infective
Collect early in the day, before taking a bath
Collection of E. vermicularis eggs using perianal
swab (scotch tape method)
c. Perianal swabs
Enterobius vermicularis
scotch tape
tongue depressor
glass slide
PPE (gloves and mask) - Highly infective
Collect early in the day, before taking a bath
Collection of E. vermicularis eggs using perianal
swab (scotch tape method)
d. Sputum
Paragonimus westermani
Lung migration (larval stages of ASH):
Ascaris lumbricoides
Strongyloides stercolaris
Hookworms
E. histolytica (trophozoite)
Echinococcus granulosus (Scolex)
Collect early morning and put deep sputum Procedure for deep sputum: rinse mouth with
water and expel the sputum by breathing and
in sterile containers coughing at 2-minutes interval
Why deep sputum? High sens/concentrated
d. Sputum
Direct wet mount using saline or iodine
N-acetyl-L-cysteine (mucolytic)
Centrifugation
Paragonimus westermani egg
e. Aspirates
Body fluids smeared and stained on glass
slides
Duodenal - Giardia lamblia, Strongyloides
stercoralis
Nasogastric intubation
Entero Test or Nasogastric intubation
Liver and Lung - E. histolytica
Entero test or String test
f. Blood
Leishmania donovani and
Trypanosoma spp.
Plasmodium and Babesia spp.
Trypanosoma cruzi
Thick and thin blood smear using
Giemsa stain
Serum for serologic tests to detect
antibodies in a patient
g. CSF
Naegleria fowleri
Acanthamoeba spp. (also obtained in
corneal scrapings)
Cultured on non-nutrient agar seeded Naegleria fowleri
with Escherichia coli.
Other pathogen recovered form central
nervous system
Trypanosoma spp. (trypomastigote)
Toxoplasma gondii
Taeniasolium solium cysticercus larvae Acanthamoeba spp.
g. CSF
Lumbar Puncture Naegleria fowleri
Centrifuged
Examined in wet mount for
trophozoites or permanent stained
smears
Acanthamoeba spp.
Laboratory Diagnossis
of Parasitic Diseases
MedBio4: Medical Parasitology
1st Semester, A.Y. 24-25
Outline
1. Parasitic (morphological) diagnosis
Macroscopic
Microscopic
2. Immunological diagnosis
Antibody detection
Antigen detection
3. Molecular diagnosis
4. Culture
5. Animal inoculation (Xenodiagnosis)
Morphological Diagnosis
Macroscopic and Microscopic
Morphological Diagnosis
A. Macroscopic examination
Ex. stool specimen examined
with the naked eye for:
occult blood
parasite component
color
consistency
B. Microscopic examination
Ex. blood, stool, urine (stained or
unstained preparation)
Morphological Diagnosis
B. Microscopic examination
Elements that may be found in stool specimens
White blood cells
Polymorphonuclears (PMNs)
Eosinophils
Macrophages
Red blood cells
Charcot-Leyden crystals
Epithelial cells
Eggs of arthropods
Fungal spores
Elements of plant origin
Animal hairs
Morphological Diagnosis
Special stains and corresponding parasites
Temporary stain Permanent stain in histopathologic slides
Giemsa/field
Eosin
stain
Thompson’s stain Trichrome stain
Iron-Hematxylin
Lugol’s iodine solution
stain
Modified
Sargeaunt’s stain
Ziehl-Neelson stain
Phenol-Auromine
Burrow’s stain
method
Acridine orange
Permanent Stain
PVA-preserved specimen
Iron-hematoxylin Stain
stains intestinal protozoa Images captured from a trichrome stained smear were submitted
helminth egg and larvae may be obscured to DPDx for diagnostic assistance. The smears were made from a
good stain for fresh, PVA, or SAF-preserved polyvinyl-alcohol (PVA) preserved fecal specimen, but no other
fecal smears patient or specimen information was given. What is your
diagnosis? Based on what criteria? What valuable information
Wheatley's Trichrome Stain would have been useful if provided?
one of the most commonly used stain for
intestinal protozoa
stain for fresh and PVA-preserved but not
w/ SAF-preserved smears
Acid-Fast Stain
Cryptosporidium,
Isospora,
Cyclospora
Immunodiagnosis
A. Antibody detection
serum
Ab is produced in response to a
particular parasitic infection
Ab may persist for a long period of
time in the serum after an infection
has ended
unable to distinguish between past
or present infection
Immunodiagnosis
A. Antibody detection
Microscopy - gold standard
However, parasites can be low in numbers during pre-patent and chronic periods
of infection, hence, microscopic examination may yield false negative results.
Specimen preparation for microscopic examination can also be laborious and
tedious when a lot of samples need to be examined during epidemiologic
investigations.
Immunodiagnosis
Immunodiagnostic techniques are
required when:
Parasites live in the tissue of internal
organ and cannot be 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 Trichinella spiralis
stage.
Immunodiagnosis
Immunodiagnosis is of particular value for:
South American trypanosomiasis Trichinosis
(Chronic stage) Toxoplasmosis
African trypanosomiasis (when Toxocarisis
parasitaemia is low) Hydatid disease
Leishmaniasis Schistosomiasis
Filariasis Malaria
Amoebic liver abscess
Culture Methods
Harada-Mori or the Test Tube Culture
Method
Hookworm and S. stercoralis
presence of rhabditiform larvae
distinguish bet. Strongyloides sp. and
hookworm
development of into filariform stage
filariform larvae will generally move
downwards
Culture Methods
Culture Methods
Hemoflagellates: Novy-MacNeal-Nicolle (NNN)
NNN slant + 1 drop of blood or ground tissue
Animal Inoculation
Xenodiagnosis
Lab-cultured bug/reduviid bug/kissing
bug takes a blood meal on the patient
The bugs are dissected after 20-25 days
to examine for T. cruzi epimastigote
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.
Examination of
Stool Sample
Laboratory Diagnosis
Ova and Parasite (O&P) Examination
stool
eggs, larvae, adults, trophozoites, cysts
urine
blood
sputum
cerebrospinal fluid
tissue aspirates
tissue biopsies
orifice swabs
What is Feces?
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
Examination of Stool
A. Macroscopic examination
Ex. stool specimen examined with
the naked eye for:
occult blood
parasite component
color
consistency
B. Microscopic examination
Ex. blood, stool, urine, mucus
(stained or unstained preparation)
Examination of Stool
B. Microscopic examination
Elements that may be found in stool specimens
White blood cells
Polymorphonuclears (PMNs)
Eosinophils
Macrophages
Red blood cells
Charcot-Leyden crystals
Epithelial cells
Eggs of arthropods
Fungal spores
Elements of plant origin
Animal hairs
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).
Collection of Stool or Fecal Sample
clean, wide-mouthed
containers with tight
fitting lid
retain moisture, prevent
spillage, and
contamination
3 specimens within 10
days (Amebiasis - 6 in
14 days)
Collection of Stool or Fecal Sample
After treatment, stools are
rechecked:
protozoan: 3 - 4 weeks
helminth: 1 - 2 weeks
Taenia sp.: 5 - 6 weeks
How many stool samples should be collected when following the
typical O&P collection protocol?
A. 1
B. 2
C. 3
D. 4
Important factors in stool analysis
A. Intake of drugs/medicinal
All of these drugs have been
substances
found to leave crystalline
1. antacids residues.
2. anti-diarrheals
3. barium - samples should be collected a
4. bismuth week after
5. laxatives
A barium enema is a type of x-ray that allows your doctor to see your colon
and rectum. It is also called a colon x-ray or lower GI exam. Barium enemas
can help diagnose changes to your large intestine, such as your colon and
rectum.
Important factors in stool analysis
B. Intake of antibiotics decreases the number of
protozoans for several weeks
C. Amount of stool dictated
routine stool examination -
by techniques
thumb/walnut size (formed) or 5-6
tbsp (watery)
D. Contamination w/ toilet
destroy trophozoites and may
water, urine, or soil
contain free-living organisms
Important factors in stool analysis
E. Age of stool sample Liquid specimen should be
examined within 30mins and
formed within 1 hour
F. Delay in examination may ensure that parasites are present
require preservation in identifiable stage
E. Temporary storage of fecal prolonged refrigeration =
samples in refrigerator (3-5℃) desiccation
trophozoites are killed
eggs, cysts survive
Important factors in stool analysis
Gross/Macroscopic - Color
Normal:
Adult: brown
New born infants: Black (meconium)
Breast feed infants: scrambled egg
Infant feed on animal milk: “curd like”
CONTENT WARNING:
POOP PICS AHEAD!!!
Gross/Macroscopic - Color
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.
Gross/Macroscopic - Color
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
Gross/Macroscopic - Color
Breastfed Stool
Yellow
Runny
Small seed like objects in the stool
Often called baby poop mustard
Gross/Macroscopic - 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.
Gross/Macroscopic - Color
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)
Gross/Macroscopic - 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
Gross/Macroscopic - Consistency
1 - resists puncture
2 - can be punctured
3 - can be cut with applicator
4 - can be reshaped
5 - shaped into container
6 - can flow
7 - can pour
Gross/Macroscopic - 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)
Cleary watery, loose mixed with mucus and blood
Gross/Macroscopic - Consistency
Classification of the form, (appearance in a toilet) of feces 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.
Gross/Macroscopic - 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
Gross/Macroscopic
Amount
Normal:
varies with diet
about 100 to 400 g per day
Size
Normal:
healthy piece of feces is about one foot long.
Gross/Macroscopic
Frequency
Normal:
three times a day to once every three days.
average person poops about once a day.
Odor
Normal:
aromatic, affected by ingested food and person’s own bacterial flora
Abnormal
Pungent (infection, blood, sloughed tissue)
Gross/Macroscopic - 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
Gross/Macroscopic - 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
Gross/Macroscopic - Constituents
Normal:
water (about 75%).
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)
Abnormal:
pus: bacterial infection
mucus: inflammatory condition
blood: gastrointestinal bleeding
large quantities of fat: malabsorption
foreign objects: accidental ingestion
Techniques
A. Direct Fecal Smear (DFS)
B. Kato Thick Smear
C. Concentration Techniques
Techniques
A. Direct Fecal Smear (DFS)
Techniques
B. Kato Thick Smear
STH
Mass epidemiologic
studies
Techniques
C. Concentration Techniques
Sedimentation
Flotation
Preservation
Formalin
5% - protozoan cyst
10% - helminth eggs and larvae
FECT (formalin-ether/ethyl acetate
concentration technique)
Preservation
Schaudinn’s solution
fresh stool for staining the stool
smear
mercuric choride (toxic)
Preservation
Polyvinyl alcohol (PVA)
protozoan cysts and trophozoites for
permanent staining
mercuric chloride
FECT
Preservation
Merthiolate-iodine-formalin (MIF)
intestinal protozoans, helminth eggs, and larvae
merthiolate and iodine (stain)
formalin (preservative)
wet mount using preserved stools
Sodium acetate-acetic acid formalin (SAF)
liquid fixative w/ long shelf-life
does not contain mercuric chloride
image not as sharp as PVA or Schaudinn’s
Preservation
