Diagnostic Parasitology Flashcards
Ova refers to
Egg stage of the parasite
Procedure for stool specimen
Ova and Parasite
The typical stool collection protocol consists of ____ specimen.
3 specimen
Microscopic examination consists of three possible
components:
○ Collection
○ Transport
○ Fixatives
How was the 3 specimen collected?
One specimen collected every other day or a total of three collected in 10 days
In cases of Amebiasis, how it was collected?
six (6) specimens in 14 days is acceptable.
Stool samples from patients undergoing therapy that
include:
○ Barium
○ Bismuth or mineral oil
○ Antacids
○ Laxatives
● Should be collected ______________
Prior to therapy or not until 5-7 days after the completion of therapy.
Antibiotics and antimalarial medications
○ Should be _____________
Delayed for two (2) weeks following therapy.
Temporary storage of fecal samples in a refrigerator _________ is acceptable.
(3 to 5°C)
The recommended time for liquid stool is
Within 30 minutes of passage
Semi-formed stool should be examined
within 1 hour of
passage.
Formed stool specimens can be held for ______ following collection.
24 hours
Stool container should be in
Clean, wide-mouthed containers made of waxed
cardboard or plastic, watertight with a tight-fitting lid
how many grams is the acceptable amount of stool required for
parasite study,
2-5 grams
SPECIMEN CONTAINER LABELS
○ Patient’s name and Identification number
○ Age and Sex
○ Physician’s name
○ Date and Time of Collection
○ Other information:
■ Suspected diagnosis
■ Travel History
■ Clinical Findings
Are substances that preserve the morphology of
protozoa and prevent further development of certain
helminth eggs and larvae.
Fixatives
the ratio of fixative to stool specimens.
3:1 ratio
The specimen must be fixed in the preservative for at
____________ before processing begins.
least 30 minutes
○ Most commonly used fixative.
○ All-purpose fixative for the recovery of protozoa and
helminths.
○ May be routinely used for direct examinations and
concentration procedures, but not for permanent
smears.
Formalin
Two concentrations of formalin are commonly used
■ 5% concentration ideally preserves protozoan cysts
■ 10% concentration preserves helminth eggs and larvae
○ Comprised of a plastic powder that acts as an
adhesive for stool specimens when preparing slides
for staining.
○ Most often combined with Schaudinn solution, which
usually contains zinc sulfate, copper sulfate, or
mercuric chloride as a base.
Polyvinyl Alcohol (PVA)
Advantages of PVA
■ Can be used for the preparation of permanent
stained smears.
■ Long shelf life when stored at room temperature.
○ Contains merthiolate (also called thimerosal) and
iodine which act as staining components, while
formalin acts as preservative.
○ Useful for the fixation of intestinal protozoans,
helminth eggs, and larvae.
Merthiolate-Iodine Formalin (MIF)
○ Can be used for performing concentrations
techniques and permanent stained smears.
○ Only requires a single vial and it is mercury-free.
Sodium Acetate Formalin
○ Free of formalin and mercury.
○ Can be used for concentration technique and
permanent stained smears.
Alternative Single-Vial System
Macroscopic Examination
Color
Consistency
Gross abnormalities
Microscopic Examination
○ Three (3) distinctive procedures
■ Direct wet preparations
■ Concentrated techniques
■ Permanently stained smear
ELEMENTS WHICH MAY FOUND IN STOOL SPECIMENS
- White blood cells
- Red Blood cells
- Macrophages
- Charcot Leyden crystals
- Epithelial cells
- Eggs or arthropods, plant nematodes and other spurious parasites
- Fungal spores
- Elements of plant origin which resemble some parasites include:
- Plant and animal hairs may loo like helminth larvae
In actual practice, one can mistake the active macrophages for
Ametic trophozoites
○ Used to measure objects observed microscopically
accurately.
○ Is a disk that is inserted into the eyepiece of the
microscope.
○ Diagnostic stages of parasites detected
microscopically are measured in units known as
microns
○ Calibration is necessary
Ocular Micrometer
○ Also known as direct wet mount
○ Slide may by mixing a small portion (2g) of unfixed
stool with saline or iodine
○ Under the microscope, the presence of motile
protozoan trophozoites is being detected
Direct wet preparation
The reagent choice for direct wet preparation
0.85% saline solution
Trophozoites can be stained to demonstrate the
nuclear morphology using
Nair’s buffered methylene blue solution (BMB)
○ Take a small quantity of stool sample.
○ Transfer onto 2 spots on a glass slide.
○ Add 1 drop of saline solution on one spot and 1 drop
of iodine solution on the other spot.
○ Cover the 2 drops with 2 cover slides and read the
preparation under the microscope.
Direct fecal smear
○ May be made to enhance the detail of the protozoan
cysts
○ Lugol’s D’Antoni’s formula (drop of iodine)
Direct wet Iodine preparation
○ Useful in detecting eggs with thick shells such as in
Ascaris and Trichuris.
○ Not eggs with thin shells (hookworms)
○ If the preparation is too long before the examination,
hookworm eggs become too transparent or distorted
making identification very difficult.
○ Not for protozoan cysts and trophozoites
Kato-Thick Smear
○ Provide the ability to detect small numbers of
parasites that might be detected using wet
preparations.
○ Aggregate the parasites present into a small volume
of the sample.
○ Can be performed on fresh or preserved stool
specimens.
○ Trophozoites DO NOT usually survive the procedure.
Concentration methods
Two types of concentration methods:
Sedimentation and Floatation
○ Most widely used sedimentation technique.
○ Based on specific gravity.
○ Ethyl acetate is added to a saline-washed
formalin-fixed sample and the tube is then
centrifuged.
Formalin-Ethyl Acetate Sedimentation
Advantages and Disadvantages of Formalin-Ethyl Acetate Sedimentation technique
○ Advantages:
■ It provides good recovery of most parasites
■ Easy to perform
○ Disadvantages:
■ The preparation contains more fecal debris than
a flotation technique
○ Recommended for the recovery of Trichuris,
Capillaria, and trematode eggs especially
Schistosoma.
○ Also, the choice of stool material comes from animals
like cats and dogs.
Acid Ether Concentration Technique (AECT)
○ Useful for the recovery of both helminth eggs and
protozoan cysts.
○ Makes use of 10% formalin which is an all-purpose
fixative.
○ Can also be done with formalin-preserved and
PVA-preserved stools.
○ More parasites can be recovered from
formalin-preserved samples.
○ Parasite morphology is also better preserved in
formalin than in PVA.
Formalin Ether Concentration Technique
Zinc Sulfate Floatation Technique main reagent
33% ZnSO4 (zinc sulfate) solution
Zinc sulfate with a specific gravity of ____________-, is
used as the concentrating solution.
1.18 to 1.20
○ Is considered the BEST for the recovery of coccidian
oocysts, mainly Cryptosporidium, Cyclospora, and
Isospora.
○ A boiled sugar solution preserved with phenol is used
in this method.
○ With this procedure, visualization of oocysts can be
better appreciated through the use of a phase
microscope.
Sheather’s Sugar Floatation
○ Use of a saturated table salt solution.
○ Stools are directly mixed with brine solution.
○ Centrifugation is not needed since helminth eggs rise
to the surface of the solution.
○ Low cost and simple but helminth eggs like hookworm
and Schistosoma become badly shrunken.
○ Not useful for operculated eggs like Clonorchis, and
heterophyids because these do not float in brine
solution
Brine Floatation
○ The final procedure in the O&P examination.
○ Defined as a microscope slide that contains a fixed
sample that has been allowed to dry and
subsequently stained.
○ Designed to confirm the presence of protozoa cysts
and/or trophozoites.
○ Allows laboratory technicians to observe detailed
features of protozoa by staining intracellular
organelles
Permanent Stains
Permanent Stains
The slides are reviewed under ___________
oil immersion (100×)
300 fields are reviewed before the slide can be
considered
Negative
Two common stains are used for routine O and P
testing and these include:
■ Trichrome (Wheatly modification)
■ Iron hematoxylin
○ Most widely used permanent stain.
○ It uses reagents with a relatively long shelf life and the
procedure is easy to perform.
○ Distinct color differences among the cytoplasmic and
nuclear structures of selected parasitic forms.
Wheatly Trichrome
○ Time-consuming
○ Excellent morphology of the intestinal protozoa.
○ In some cases, the nuclear detail of these organisms
is considered to be stained clearer and sharper than
when stained with trichrome.
Iron Hematoxylin
○ They do not detect oocysts of the coccidian parasites
or spores of microsporidia.
Specialized Stains
■ Has become an important permanent stain
procedure for the detection of the oocysts of
Cryptosporidium, as well as those of Isospora
and Cyclospora.
Modified Acid-fast stain
○ Has been developed that incorporates a carbol
fuchsin step;
○ This allows for the detection of acid-fast parasites in
addition to the other protozoa normally recovered
using the iron hematoxylin stain.
Modified Acid-fast stain
○ Positive stools are mixed with moistened soil or
granulated charcoal.
○ This simulates environmental conditions in nature.
○ Larvae are harvested using the Baermann
procedure.
Copro culture
○ Use of test tubes and filter paper strips.
Harada-Mori or test tube culture method
will generally move downwards against the upward capillary movement of water, therefore be recovered from the water at the bottom of the tube
Filariform Larvae
may instead move upwards and accumulate at the upper end of the filter paper strip.
Strongyloides Larvae
25C
In dark
7 days
Harada-Mori
Centrifuge the sample Harada-Mori Technique at
1500 rpm for 2 minutes
○ Correlate the severity of clinical disease with the
intensity of infection or worm burden
○ Also done to assess the efficacy of anthelminthics and
the reduction of worm burden following treatment.
Egg counting procedures
Uses a measured amount of stool that has been
sieved through a wire mesh and pressed under
cellophane paper soaked in glycerin-malachite green
solution.
Kato-Katz Method
○ Used for assessing the intensity of infection in
schistosomiasis and common soil-transmitted
helminthiases like ascariasis, trichuriasis, and
hookworm infection.
Kato-Katz Method
The main determinant for the sensitivity of Kato-Katz Method is
Consistency of the stool
TRUE OR FALSE:
In Kato-Katz Method-
○ Can only be done on fresh formed stools and not on
liquid and preserved samples.
True
For the identification of Schistosoma ova, we can used ________
1% eosin solution can be layered over the cellophane paper.
■ Can help in the visualization of the miracidium
STOLL EGG COUNT make used of
0.1N NaOH (Sodium Hydroxide)
Sodium Hydroxide: stool diluent
Used to recover eggs of Enterobius vermicularis
and Taenia spp.
Perianal Swab
○ Often referred to as rapid methods.
○ Can be obtained as kits that contain monoclonal
antibodies.
Stool Screening Methods
○ The specimen may be collected by nasogastric
intubation or by the enteric capsule test
(Enterotest)
Duodenal Material
the simpler method for collecting
duodenal material without requiring intubation
Entero Test
○ Often helpful for detecting E. histolytica.
○ Coccidian parasites and microsporidia may also be recovered.
Sigmoidoscopy Material
○ Is the specimen of choice for the detection of
Enterobius vermicularis (pinworm) eggs.
○ Otherwise known as the Graham Technique.
Cellophane Tape Preparation
The ideal time to collect the sample in Cellophane tape technique
Morning specimen
How many specimens should be collected daily in Cellophane Tape technique>?
5 specimens
Blood Parasites that may recovered in the blood:
○ Leishmania donovani
○ Trypanosoma spp.
○ Plasmodium
○ Babesia spp.
○ Microfilaria
○ Some parasites (e.g., Trypanosoma spp.,
microfilariae) that can be detected by observing
motility under low- and high-power magnification.
○ Species identification is not possible with this method.
Wet/Fresh Preparation
■ Are used in the demonstration of microfilariae
and rapid diagnosis of malarial infection.
Thick smear
■ Are most useful in species identification of
malarial parasites.
Thin smear
3 Stains for Blood Parasites
○ Giemsa Stain
○ Wright’s Stain
○ Delafield Hematoxylin
○ Considered the preferred stain because it allows for
the detection of parasite detail necessary for species
identification
Giemsa Stain
○ Fixation is not needed because it already contains
alcohol
Wright Stain
○ Useful in demonstrating the detailed structures of
microfilariae.
○ In this method, thick films are dehemoglobinized in
2% formalin with 1% acetic acid.
Delafield Hematoxylin
○ Makes use of a capillary tube which is precoated with
acridine orange and potassium oxalate.
Quantitative Buffy Coat
○ Also useful when the density of microfilariae is low.
○ Make use of a syringe attached to a Swinney filter
holder
Membrane Filtration
○ Designed to concentrate blood specimens suspected
of containing low numbers of microfilariae.
Knott’s Concentration
Specimens submitted to culture can be
Blood, Bone Marrow and Tissue
is an example of culture medium designed for the recovery of Leishmania spp. and Trypanosoma cruzi.
Novy-MacNeal-Nicolle (NNN) medium
Parasites found in the CSF (NATToMiTE):
(NATToMiTE):
■ Naegleria fowleri
■ Acantamoeba spp.
■ Trypanosoma spp. Trypomastigotes
■ Toxoplasma gondii
■ Microsporidia
■ Taenia solium cysticercus larvae
■ Echinococcus spp.
○ Specimen should be collected early in the morning.
○ Saliva is not appropriate for examination
○ May be examined directly via wet preps and/or
concentrated using N- acetylcysteine.
○ Wet preps and permanent stains
Sputum
○ Recommended for the recovery of a number of
parasites, including intracellular organisms such as
Leishmania spp. and T. gondii.
○ Surgical removal of the specimen
Tissue and Intestinal Biopsy
Tissue and Intestinal Biopsy
■ Is the preferred method for handling these
samples.
Impression smears
Is the specimen of choice for patients suspected
of liver abscesses caused by E. histolytica
Hepatic Abscess
○ Specimen of choice for the detection of Schistosoma
haematobium eggs
Urine and Genital Specimen
○ Acantamoeba keratitis is best diagnosed by the
collection and examination of corneal scrapings.
Eye specimen
○ Useful in the detection of Onchocerca volvulus
Skin Scrapings
○ Appropriate specimens from patients suspected of
suffering from Leishmania, Trypanosoma and
Toxoplasma
Animal Inoculation and Xenodiagnosis
■ Is a technique used for the diagnosis of Chagas’
disease.
Xenodiagnosis
○ For Schistosome species
■ S. mansoni and S. japonicum
Circumoval Precipitin Test (COPT)
Which of the following is the specimen of choice to
demonstrate intracellular parasites such as Toxoplasma
gondii and Leishmania spp.?
Tissue
