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Chapter 2
Chapter 2
Chapter 2
2. GENERAL LABORATORY
DIAGNOSIS OF PARASITIC
DISEASES
Acknowledgement
Morphological diagnosis
Immunological diagnosis
Molecular diagnosis
Other techniques
Specimens for parasitological diagnosis
Learning objective
Upon completion of this unit, the student will be able to:
List the common techniques used for the detection of
parasites in fecal, blood and other specimens.
List the types of microscopic methods used in parasitological
investigations.
Discuss the importance of microscope calibration
Describe the difference between immunodiagnostic techniques
Discuss the different parasite concentration methods
Discuss the significance of molecular diagnosis in the
diagnosis parasitic diseases.
2. General laboratory diagnosis of parasitic diseases
4. Molecular diagnosis
5. Culture
6. Animal inoculation
7. Xenodiagnosis
2.1 Morphological diagnosis
Laboratory procedures detect organisms within clinical
specimens using morphological criteria,
1. Fixatives
2. Preparatory reagents
3. Stains
Parasitology staining …..
Method
Result
Chromidial Bars Fluoresce - bright green
Stains
2.Auramine Phenol (Lempert)
Method
Results
Oocysts appear as bright yellow discs against a dark
background.
3. Field Stain A & Field Stain B
Enables rapid staining of fixed thin films.
This particular method is very useful for staining films of
unformed feces, fecal exudates, duodenal aspirates etc.
Method
6. Examine the film using the oil immersion objective and immersion
oil
Results
Parasite nuclei and structures containing chromatin - red
Cytoplasm - bluish-grey
Leukocyte nuclei - purple
Yeasts and bacteria - dark blue
4. Giemsa Stain - Rapid
Giemsa stain can also be used to stain films of unformed
feces, fecal exudate, duodenal aspirates etc.
Method
1. Make a thin film of faeces/exudate. Allow to dry.
2. Fix in methanol for 1 min.
3. Tip off the methanol and flood the slide with Giemsa stain
diluted 1:10 with buffered distilled water pH 7.2 The diluted
stain must be freshly prepared each time.
4. Stain for 20-25 min.
Method…
5. Run tap water on to the slide to float off the stain and
prevent deposition of precipitate on to the film. Allow to drain
dry.
6. Examine the film using the oil immersion objective and
immersion oil.
Results
Parasite nuclei and structures containing chromatin - red
Cytoplasm - bluish-grey
Leucocyte nuclei - purple
Yeasts and bacteria - dark blue.
5. Lugol’s Iodine (Aqueous)
Temporary Stain for Protozoa.
Method
1. Make wet preparations following concentration by the
formol-ether method with Parasep®.
2. Add an equal volume of Lugol's Iodine to 25% glacial acetic
acid.
3. Place a drop of the wet preparation on a slide and add a
drop of the Iodine/acetic acid mixture prepared in 2 above.
4. Cover slip and examine.
Method…
Result
Iodine Stains:
Glycogen - brown
Nuclear Chromatin of Amoebic Cysts - brown/black.
6. Iron Haematoxylen Solution A/B
Method
1. Make smears from unconcentrated stool specimens.
2. Fix in methanol for 5 mins.
3. Stain in modified Trichrome stain for 90 mins.
4. Rinse in acid alcohol for 10 sec then in 95% alcohol.
5. Place in 95% alcohol for 5 mins.
Interpretation
Microsporidial spores are ovoid, refractile and the spore wall
stains bright pinkish-red.
The background debris & bacteria are stained faint green.
7.2. Trichrome For Protozoa
Method
For use:
Add 5 ml glacial acetic acid to 95 ml of the stock solution.
Prepare faecal smears without allowing the smears to dry
and place them immediately in Schaudinn’s fixative for 1
hour.
For use…
The reagents used in the preparation of this fixative are
hazardous and should be handled with care.
The working reagents should be prepared fresh for use.
This fixative is particularly good for making permanently
stained faecal smears of protozoan trophozoites.
6. Polyvinyl alcohol (PVA)
95% ethyl alcohol in Glycerol with Schaudinn’s fixative and
Polyvinyl alcohol powder.
To use:
Emulsify 1 part of faeces in 3 parts of PVA solution.
This method will preserve ova, larvae and trophozoites well,
but cysts may show some distortion.
However some ova and cysts do not concentrate well when
preserved in PVA.
To use…
Faecal smears made from the faeces/PVA mixture and
allowed to dry can be used for the permanent staining of
trophozoites.
Before staining, the slides must be placed in 70% ethyl
alcohol containing 10 drops of Lugol’s iodine to remove the
mercuric chloride.
7. Sodium acetate-acetic formalin acid
Solution (SAF)
Principle:
The stool sample is mixed with a saturated solution of
sodium chloride (increasing the specific gravity). The eggs
are lighter in weight and float to the surface where they can
be collected
Method:
Principle:
A strip of filter-paper is partially submerged in a test-tube
containing water.
Any larvae of Strongyloides stercoralis present in the
specimen migrate against the current of water that rises by
capillary action and accumulate at the bottom of the tube.
Harada Mori…
Method
2. Plug the tube with cotton wool or, preferably, seal with
cellophane tape and keep for 7–8 days at room
temperature.
3. Look for the larvae at the bottom of the tube. Stain with
iodine solution for 1 minute and then examine under the
microscope, using the x 10 objective.
Harada Mori…
The larvae usually seen in fresh stool specimens are the
rhabditiform (first-stage) larvae of S. stercoralis.
However, if the stool was passed more than 12 hours
earlier, the larvae may have hatched into filariform
(infective-stage) larvae.
These must be differentiated from larvae of Ancylostoma
duodenale and Necator americanus (hookworm), which
may also hatch in stools 12–24 hours after passage.
Harada Mori…
The appearance of filariform larvae of S. stercoralis may
indicate a systemic hyperinfection.
The genital primordium will be more visible in preparations
stained with iodine. The iodine kills the larvae and makes
the features easier to see. You will need to use the x 40
objective to see these structures.
If you see a larva with a short mouth opening and a
prominent (clearly visible) genital primordium, it is S.
stercoralis.
Harada Mori…
If you see a larva with a long mouth opening and do not see
a genital primordium, it is A. duodenale or N. americanus.
Anal swabs for pin worm
Anal swabs are used to detect the presence of pinworms
(Enterobius vermicularis).
Pinworms are more common in children than adults. Often,
however, if one child in a family has pinworms other members of
the family will be infected.
Therefore, if a child is found positive, it is desirable to examine
swabs from all members of the family group, especially the
children
Anal swabs…
Pinworm eggs are usually found in the folds of skin around
the anus. They rarely appear in the stool.
Collection of specimen:
Materials for method A
Centrifuge
Cotton swabs
Microscope slides
Pipettes, Pasteus, with rubber bulbs
Saline solution to moisten cotton swabs
Anal swabs…
Saline solution to moisten cotton swabs
Test tubes, 100 x 13 mm
Technique – Method A
Spread buttocks apart, and rub cotton swab over the area
around the anus, but do not insert into the arus. Place the
cotton swab in the tube.
Materials for Method B
Transparent adhesive tape
Tongue depressor /plastic spoon with handle 10 cm long
Microscope slide
Anal swabs…
Technique – Method B
1. Fold a strip of transparent adhesive tape over the end of a
spoon handle or tongue depressor
2. Separate the patient’s buttocks with the other hand. Press
the end of the spoon covered with the tape against the skin
around the anus in several places
3. Place the tape with the sticky side down on a microscope
slide. Before examining the slide, lift the tape up & place a
drop of immersion oil under the middle of the tape and
replace the tape.
Anal swabs…
This will improve the transparency of the tape
Wash your hands after sample collection; otherwise, eggs
that may have contaminated your hands could get into your
mouth and lead to infection.
In order to increase the chances of picking up eggs, the
swab should be taken between 22hr and midnight, or early
in the morning before the patient urinates defecates, or
batches. It may be necessary to collect several swabs
before a positive diagnosis can be made.
Anal swabs…
Tape – swab slides may be examined directly. For cotton
swabs, proceed as follows:
Technique
Care must be taken during collection of stool
specimens. Always wear gloves to avoid
contamination of the fingers
11. Do not lift the slide straight up. The cellophane may
separate. Gently slide the microscope slide
sideways holding the cellophane.
Chemical test for occult blood in stools
This test is used for screening for parasitic infection, for
example intestinal schistosomiasis, or for detection of
bleeding in the intestine caused by polyps, tumours or
inflammation.
It was originally developed using benzidine. However, the
use of benzidine is no longer recommended because it has
been shown to be carcinogenic.
Chemical test…
Principle
Oxygen is produced when the haemoglobin in blood comes
into contact with hydrogen peroxide. The liberated oxygen
reacts with aminopyrine (aminophenazone) to yield a blue
colour.
Chemical …
Results
If the reaction is positive a red colour appears between the
two layers of liquid.
Report the results as follows:
Pale red = positive reaction (+ )
Red = strong positive reaction (++ )
Dark red = very strong positive reaction (+++)
No change in colour = negative reaction (- ).
Specimen processing…
C. Specimens preserved in PVA are mostly used for permanent
staining with trichrome. Prior to staining, they are processed
as follows:
1. Insure that the specimen is well mixed.
2. Prepare a smear using 2 to 3 drops of the specimen
depending on density.
3. Heat fix on slide warmer set at 60oC for 5 minutes or air dry
completely at room temperature.
4. Slides may be trichrome stained or kept for several months in
a protective slide tray or box for future staining.
Methods of stool examination
I. Macroscopic examination
II. Microscopic examination
I. Calibration of ocular micrometer
II. Wet mount preparation
III. Stained slide preparation
IV. UV Fluorescence Microscopy Procedure
Methods of stool examination
Recovery and identification 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)
I. Macroscopic examination
A. Color
B. Consistency
C. Abnormal elements
D. Odor
E. Shape and size
A. Color of stool
Normal:
Adult: brown
New born infants: Black (meconium: babys’ first stool)
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.
Yellow
Runny
Small seed like objects in the stool
Often called baby poop mustard
Color of stool …
Malabsorption of fats,
Diet high in milk and milk products and low in meat
B. Consistency of stool
Staining problem
Problems associated with Wright staining
Too acidic Wright stain - may be due to
Insufficient staining time
Prolonged buffering or washing
Too acidic stain buffer or water which may be due to
exposure of stain or buffer to acid fumes or it may be old
stain the methyl alcohol has slowly oxidized to formic acid.
Method
1. The blood smear is fixed by paling in coplin jar of anhydrous
acetone free methyl alcohol for 1-2 min, the purpose of
fixation is to preserve and prevent disruption by sub sequent
procedures
2. The concentrated giemsa stain is diluted by adding 1-
volume of stain to 9ml volumes of buffered diluted water.
3. The fixed blood smear is place either on a level staining
rack or in a second coplin jar.
4. The 1.10 diluted giemsa stain is added and allowed to
stand for 10 minutes The stain is washed off with a stream
of distilled water.
Quantifying parasites:
In some cases (especially malaria) quantification of
parasites yields clinically useful information.
If this information is needed by the physician, malaria
parasites can be quantified against blood elements such
as RBCs or WBCs.
To quantify malaria parasites against RBCs, count the
parasitized RBCs among 500 - 2,000 RBCs on the thin
smear and express the results as % parasitemia.
% Parasitemia = (parasitized RBCs/total RBCs) × 100
If the parasitemia is high (e.g., >10%) examine 500 RBCs;
If it is low (e.g., <1%) examine 2,000 RBCs (or more);
count asexual blood stage parasites and gametocytes
separately. Only the former are clinically important and
gametocytes of P. falciparum can persist after elimination
of asexual stages by drug treatment.