specimen collection and handling Flashcards
most common procedure performed in parasitology
Stool for Ova and Parasites (O&P) examination.
What is the most common clinical sample in parasitology?
Stool specimens.
- as the parasites shed intermittently in stool
- parasites invade the body, complete their life cycle, and are subsequently passed in the stool.
What does “Ova” refer to in O&P testing?
egg stage of a parasite.
What are the key factors to remember when studying the life cycle of a parasite?
Mode of transmission
Infective stage
Diagnostic stage
What does “Parasites” refer to in O&P testing?
Other morphologic forms of a parasite.
What are the two general components of the O&P examination?
Macroscopic examination
Microscopic examination
What are the key pre-analytical phases in O&P testing?
Collection
Transport
Preservation
- these are important as it affects specimen integrity; improper handling can lead to organism disintegration and misidentification.
goal of O&P examination?
correctly identify the causative agent of parasitic infection.
Why are parasites not always detected in a single stool specimen?
Parasites are shed intermittently, so they may not be present in every sample.
Hence, collect multiple stool specimens to increase the likelihood of detecting parasites in stool specimens.
- collect 3 stool specimen
typical stool collection protocol for O&P examination?
three stool specimens collected every other day over 10 days.
collected every other day as the parasites are shed intermittently and may not be present in every sample.
What is the exception to the typical stool collection protocol?
Amebiasis—suspected cases require six stool specimens over 14 days.
How can medications affect stool sample testing for parasites?
can mask the detection of parasites = false-negative results.
When should stool specimens be collected if a patient is on medication?
Before starting medications or after completing the medication course to avoid interferences.
after 5 to 7 days:
mineral oil
antacids
kaolin
barium
bismuth
after 2-3 weeks
antibiotic or antimalarial medications
after 3 weeks:
gallbladder dyes
How should stool specimens be transported to the laboratory?
placed in a Ziplock plastic bag for secure transport.
- requisition form should be separated from the specimen container to prevent contamination.
- PPE must be worn and universal precaution must be exhibited at all times.
Why must trophozoites be examined quickly after stool passage?
sensitive to environmental changes rapidly disintegrate outside the body
How soon must liquid stool specimens be examined?
Within 30 minutes of passage to prevent trophozoite disintegration.
Which parasitic forms can survive longer outside the host?
Protozoan cysts, helminth eggs, and larvae can remain viable for extended periods.
Why is the time frame of specimen collection important in parasite testing?
maintain specimen integrity and demonstrate parasite motility for accurate identification.
Why is stool consistency important in O&P examination?
determine the morphologic forms of parasites present in the sample.
What parasite form is usually found in
1. liquid stool?
2. semi-formed
3. formed stool
1.trophozoites—must be examined within 30 minutes to prevent disintegration.
- Both trophozoites and cysts —within 1 hour.
- cysts—can be held for up to 24 hours before examination
Substances that preserve the morphology of protozoa and prevent further development of certain helminth eggs and larvae.
fixative
- must be fixed in the preservative for at least 30 minutes before processing begins
What is the correct fixative-to-stool ratio?
3 parts fixative to 1 part stool
What is an example of a commonly used fixative in O&P testing?
Total-Fix fixative—a single vial system for fecal specimen preservation.
What tests can be performed on preserved fecal specimens?
Formalin sedimentation concentration
Permanent stained smears (trichrome, iron-hematoxylin)
Fecal immunoassays
DFA for Cryptosporidium spp. and Giardia lamblia
Who is responsible for collecting stool specimens and transferring them into vials?
Patients
- they must be properly instructed on the correct procedure.
- appropriate volume of stool to fill on the line on a fixative vial indication
- lab preference based on the testing method
What does the fill line on a fixative vial indicate?
The appropriate volume of stool specimen to be added.
All purpose fixative for the recovery of protozoa and helminths.
formalin
commonly used for:
✔ Direct examinations
✔ Concentration procedures
❌ Not for permanent smears
the two commonly used formalin concentrations and their purposes:
5% Formalin → Preserves protozoan cysts
10% Formalin → Preserves helminth eggs and larvae
advantages and disadvantages of formalin
✔ Easy to prepare
✔ Preserves specimens for several years
✔ Has a long shelf life
does not preserve parasite morphology well, causing trophozoites to degrade and morphologic details to fade over time.
- trophozoites usually cannot be recovered from formalin-preserved specimens as formalin is toxic to trophozoites
- chemical hazard and poses health risk
Comprised of a plastic powder that acts as an adhesive for the stool specimen when preparing slides for staining.
Polyvinyl alcohol
- most combined with:
Schaudinn Solution which contains:
Zinc sulfate
Copper sulfate or mercuric chloride
advantages and disadvantages of PVA
- can easily detect
✔ Trophozoites
✔ Protozoan cysts
✔ Most helminth eggs - allows the preparation of permanent stained smears
- long shelf life when stored at room temperature.
- recovery of certain parasites is less effective compared to formalin.
(uses 2 vial system
Formalin vial → for concentration techniques
PVA vial → for permanent stained smears) - contains mercuric chloride,
Viable alternative for PVA and Schuadinn fixative.
Sodium acetate formalin
✔ Concentration techniques
✔ Permanent stained smears
- Can be used for preparing smears for staining with the modified acid-fast stain to detect coccidian oocysts
advantage and disadvantages of SAF
✔ Requires only a single vial
✔ Mercury-free (safer alternative)
✔ Easy to prepare and has a long shelf life
Poor adhesive properties, making it less effective for slide preparation.
Protozoa morphology in SAF-preserved specimens is less clear in permanent stains compared to mercury-based fixatives.
Alternative to mercury-based PVA are the use of substitute compounds containing copper sulfate or zinc sulfate.
Modified PVA (Polyvinyl alcohol)
✔ Concentration techniques
✔ Permanent stained smears
advantage and disadvantages of modified PVA
Advantages:
- Can be used for concentration methods and permanent stained smears.
- Mercury free.
- Zinc Sulfate fixatives provide better results than copper sulfate reagents.
Disadvantages:
- Substitute products do not provide the same quality of preservation for adequate protozoan morphology on a permanent stained slide as the mercury-based fixatives.
- Modified PVA fixatives are more likely to be negatively affected if proper protocol is not followed. (e.g stool-to-fixative ratio, adequate mixing)
Alternative nontoxic fixatives.
Alternative single-vial systems
- Free of formalin and mercury.
advantages and disadvantage of alternative single-vial systems
Advantages
- Can be used for concentration techniques and permanent stained smears.
- Some can be used for performing fecal immunoassays.
Disadvantages:
- Does not provide the same quality of preservation as mercury based fixatives and organism identification will be more difficult from permanent stained slides.
stool preservatives and applicable laboratory procedures
index card
Begins when a stool specimen has been received in the laboratory.
analytical phase
Also referred to as “Processing”
*macroscopic examination (unaided eye) before the microscopic examination (make use of microscope).
Macroscopic Examination determines
(index card)
- Consistency and color of the sample.
- Presence of gross abnormalities.
*Performed with a fresh, unpreserved stool specimen.
how to perform macroscopic examination
1st: Samples are broken up using an applicator stick and examined for macroscopic parasites (adult helminths).
2nd: Samples containing adult worms are carefully washed through a wire screen.
possible descriptive terms for microscopic examination of stool
transes
Some lab only make use of these terms:
Formed
Semi-formed
Watery
Microscopic examination detects
- Presence of parasites in a stool specimen using a microscope.
- Involves 3 distinct procedures:
Direct wet preparations
Concentrated technique
Permanently stained smear
*Performed with a fresh stool specimen.
Unit of measurement for microscopic detection of diagnostic stages of parasites.
microns
Measuring scale that is necessary for the ocular piece of the microscope.
Ocular micrometer
- Must be calibrated to ensure accurate measurement.
Unit of measurement: microns
calibration and the use of an ocular micrometer
transes
formula to find the no. of microns
no. of stage micrometer units x 1000 / no. of ocular micrometer units
Made by mixing a small portion of unfixed stool with saline and subsequent examination under the microscope.
direct wet preparation/ direct wet mount
- To detect the presence of motile protozoan trophozoites.
- can observe:
Protozoan cysts, oocysts, helminth eggs and larvae
Made to enhance the detail of protozoan cysts.
direct iodine wet preparation
- A drop of iodine (Lugol’s or D’Antoni’s formula) is used in place of saline.
- Light adjustment of the microscope is critical for the detection of protozoa.
- The light should be reduced using the iris diaphragm to provide contrast between the cellular elements in the specimen.
- Lowering the condenser is often recommended to lower the light and allow for otherwise transparent structures to be seen.
Provide the ability to detect small numbers of parasites that might not be detected using direct wet preparations.
concentration methods
- Purpose: To aggregate parasites and to remove as much debris as possible. = increase sensitivity to detect the parasites.
Can be performed on fresh or preserved stool specimens.
Allows detection of protozoan cysts, oocysts, helminth eggs, and larvae.
Protozoan trophozoites do not usually survive
2 types of concentration methods
Sedimentation concentration technique
- Parasites are concentrated in the sediment of the tube following centrifugation and the sediment is examined microscopically.
Flotation concentration technique
- Parasites are less dense than the solutions used.
- Parasites float to the surface.
Most widely used sedimentation technique.
formalin-ethyl acetate
Based on specific gravity.
Ethyl acetate is added to a saline-washed formalin-fixed sample and the tube is then centrifuged.
Parasites settle in the sediment of the tube.
Fecal debris rises up to the upper layers of the test tube.
Tube is then decanted and the sediment is examined in a wet prep, unstained (i.e., with saline) and with iodine.
advantages and disadvantages of formalin-ethyl acetate
Advantage:
good recovery of most parasites and is easy to perform.
Disadvantage:
preparation contains more fecal debris than a flotation technique and is more challenging to the microscopist.
What is the Zinc Sulfate Flotation Technique based on?
based on differences in specific gravity between parasites and fecal debris.
- Fecal debris is heavy and sinks to the bottom of the test tube.
- Parasites are lighter and float toward the top of the tube.
What is the specific gravity (SG) range of zinc sulfate in this technique?
1.18 to 1.20
advantage and disadvantages of the Zinc Sulfate Flotation Technique?
+ It removes more fecal debris, yielding a cleaner preparation for easier microscopic examination.
- Some helminth eggs are too dense to float, leading to missed parasite detection.
A microscope slide that contains a fixed sample that has been allowed to dry and subsequently stained.
Permanent Stained Smear
- to confirm the presence of protozoa cysts and/or trophozoites.
- Allows observation of detailed features of protozoa by staining intracellular organelles.
Common Stains Used for Routine O&P Testing:
Specialized Stains:
(index card)
Common Stains Used for Routine O&P Testing:
Trichrome (Wheatley modification)
Iron hematoxylin
Specialized Stains:
Modified Acid-Fast
Modified Trichrome Stain
What are the three types of rapid methods for parasite detection?
Enzyme Immunoassay (EIA)
Direct Fluorescent Antibody (DFA)
Membrane Flow Cartridge
purpose: detect antigens in patient specimens.
What do rapid test kits contain for parasite detection?
Monoclonal antibodies, which are cloned using a single cell line.
Can rapid tests detect multiple parasites at once?
Some products detect a single parasite antigen, while others test for more than one.
What is the main advantage and major limitation of rapid methods?
+ highly sensitive and specific for parasite detection.
- usually detect only one or two pathogens at a time.
How is duodenal material collected for parasite examination?
via nasogastric intubation or the Enterotest (enteric capsule test).
can observe:
✔ Giardia intestinalis (trophozoites)
✔ Cryptosporidium spp.
✔ Isospora belli
✔ Strongyloides stercoralis
✔ Eggs of Fasciola hepatica and Clonorchis sinensis
What parasite is detected using sigmoidoscopy material?
Entamoeba histolytica (often found in ulcer material).
- collected and examined by:
✔ Obtained by aspiration or scraping
✔ Examined using direct wet preparations and permanent stains
What is the specimen of choice for detecting Enterobius vermicularis (pinworm)?
Cellophane tape preparation is used to detect pinworm eggs.
other specimens and lab techniques
Blood
Thick and thin smears
Permanent stains
Knott’s technique
Buffy coat slides
Culture
CSF
Tissue and biopsy specimens
Urine and genital secretions
Eye specimens
Mouth scrapings nasal discharge
Skin nips
Culture methods
Animal inoculation and xenodiagnosis
thick vs thin smear
Thick Smear
- For screening purposes.
- Typically have a much higher concentration of parasites.
- Disadvantage: RBCs have been lysed
Thin Smear
- Recommended for species identification
what are the 2 permanent stain
Wright’s Stain:
- Fixative + Stain
- Yields satisfactory results
Giemsa Stain:
- Fixative and Stain are separate.
- Preferred Stain (Allows detection of parasite details necessary for species identification)
this technique is designed to concentrate blood specimens suspected of containing low numbers of microfilariae.
Knott Technique
- consists of combining 1ml of blood and 10 ml of 2% formalin in a centrifuge tube then the mixture should be thoroughly mixed and spawned for 1 min at 500 centrifugal force or relative centrifugal force.
What is the Buffy Coat and how is it extracted?
white blood cell layer extracted from blood specimens using a capillary pipette after centrifugation.
- uses Giemsa stain
- detects:
✔ Leishmania spp.
✔ Trypanosoma spp.
What type of blood is used for Buffy Coat preparation?
Oxalated or citrated blood
What is the process for Buffy Coat extraction?
Place blood in a Wintrobe tube
Centrifuge at 100 x g for 30 minutes
Blood separates into three layers:
RBCs (bottom)
Buffy coat (middle, white cell layer)
Plasma (top)
What types of samples can be cultured for parasitic infections?
Blood, bone marrow, and tissue samples
What is the Novy-MacNeal-Nicolle (NNN) Medium used for?
used to culture Leishmania spp. and Trypanosoma cruzi.
- Slant is observed under 400x magnification.
- Negative cultures should be held for 1 month.
Reveals excellent morphology of the intestinal protozoa.
Iron Hematoxylin
*Nuclear detail: Stained clearer and sharper than with trichrome.
cytoplasm - blue to purple
nuclear material - dark blue to dark purple
debris and background material - light blue, sometime w pink tint
Used for detection of oocysts of Cryptosporidium, Isospora, and Cyclospora.
Modified Acid Fast
spores of microsporidia - pink to red w clear interior
polar tube - red horizontal or diagonal bar
bacteria, yeast, debris - pink to red
background - green
Most widely used permanent stain
Trichrome (Wheatley modification)
*Uses reagents with a relatively long shelf life and the procedure is easy to perform.
cytoplasm of entamoeba histolytic trophozoites and cysts - light pink or blue-green
cytoplasm of entamoeba coli cysts - purple tint
nuclear karyosomes - bright red to red purple
degenerated parasites - light green
background - green
CSF Wet Preparation
parasites and organisms that can be detected in the CSF:
Naegleria fowleri
Acanthamoeba spp.
Trypomastigote stages of Trypanosoma spp.
Toxoplasma gondii
Microsporidia
Taenia solium cysticercus larvae
Special stains for CSF
Giemsa
Trichrome
Modified trichrome stains
Other Sterile Fluids
Fluid present in cysts
Aspirates
Peritoneal fluid
Pleural fluid
Bronchial washings
[fluids found in different parts of the body that are usually free from bacteria.]
Nutrient Agar Seeded with Escherichia coli
Used to culture Naegleria fowleri and Acanthamoeba spp.
Nutrient agar provides food for growing microorganisms like amoebas.
Procedure for Culturing with Nutrient Agar
CSF sediment is added to the agar medium.
The sample is sealed and incubated at 35°C.
After incubation, the culture plate is examined for evidence of amoeba growth.
Parasites Detected with Tissue Biopsy
Leishmania spp.
Toxoplasma gondii
Free-living amoeba
Trypanosoma spp.
Trichinella spiralis
Microsporidia
Entamoeba histolytica
[Used when parasites are difficult to detect in stool or fluid.]
Sputum Specimen Considerations
Collected early in the morning for better quality.
Collected into a wide-mouthed container.
Avoid mixing saliva with the specimen.
Can be examined directly or concentrated with chemicals like N-acetylcysteine.
[Sputum is a mucus sample from the lungs]
Parasitic Infections Found in Sputum
Paragonimus westermani
Strongyloides stercoralis
Microsporidia
Entamoeba histolytica
Entamoeba gingivalis
Ascaris lumbricoides
Hookworm
[Helps detect parasitic infections in the lungs and digestive system.]
Urine and Genital Secretions Specimen Collection
Collected in a clean container with a watertight lid.
Centrifuged on arrival at the lab.
Microscopic examination of sediment for parasites.
Parasites in Urine and Genital Secretions
Schistosoma haematobium eggs
Trichomonas vaginalis trophozoites
Microfilariae (in heavy infections)
*Examined for Trichomonas vaginalis trophozoites.
Saline wet preparations are used for detecting motile trophozoites.
Eye Specimens Collection
Corneal scrapings, contact lenses, and contact lens solution for Acanthamoeba keratitis.
Keep small tissue samples moist with sterile saline.
Store specimens in an airtight container.
Culture on Agar Plate for Eye Specimens
Seed with Gram-negative bacteria.
Examine daily for 1 week.
Trophozoites: Appear in 4 days.
Cysts: Appear in 4 to 5 days.
Eye Specimen Scraping Examination
Transferred to glass slides.
Stained with calcofluor white stain.
Examined under fluorescent microscopy.
Histologic Methods for Eye Specimens
detect Acanthamoeba, T. gondii, Microsporidia, and Loa loa.
Mouth Scrapings and Nasal Discharge
Sample of choice for the detection of:
- Entamoeba gingivalis
- Trichomonas tenax
Nasal discharge specimens:
- For the recovery of Naegleria fowleri
(Material obtained via mouth scrapings and nasal discharge should be placed in a clean airtight collection container, such as on a swab or in a cup.)
Animal Inoculation Method
Uses mice, guinea pigs, and hamsters.
Suitable specimens: Blood, lymph node aspirates, CSF, and bone marrow from patients suspected of Leishmania, Trypanosoma, or Toxoplasma infections.
Parasites That Can Be Isolated with Culture
Entamoeba histolytica
Trichomonas vaginalis
Leishmania spp.
Trypanosoma cruzi
Toxoplasma gondii
Technique used for the diagnosis of Chagas’ disease
Xenodiagnosis
- An uninfected reduviid bug is allowed to take a blood meal from the patient and the bug’s feces is then examined to observe for the presence of T. cruzi.
The phase after completing the analytic testing where the results are interpreted and reported.
Post-Analytical Phase
- Reporting a Positive Specimen
- Reporting the Presence of Certain Cells in the Specimen
1.
Scientific name of the parasite.
Stage of the parasite (e.g., cyst, trophozoite, larvae, eggs).
2.
Method: Reported qualitatively (presence or absence).
- Should include a comment indicating that this procedure does not detect Cryptosporidium spp., Cyclospora cayetanensis, and Microsporidia.
In situations in which quantitation is important are as follows:
Blastocystis hominis
Helminth eggs - Trichuris trichiura
Clonorchis sinensis
Schistosoma spp.
Plasmodium
Babesia spp.
*Charcot-Leyden crystals
Also reported when found and can be quantitated.
