Specimen Collection and Procedures in Clinical Parasitology

FECAL SPECIMEN EXAMINATION

Most common method of diagnosis of intestinal parasites (Protozoan & Metazoan)

Recommended time for examination

* Liquid and Soft Stools

Within 30 mins

Recommended time for examination

* Fully Formed Stools

Within 3-4 hours

May contain protozoan trophozoites

Liquid and Soft Stools

May contain protozoan cysts

Fully Formed Stools

How many specimens should be collected everyday for Fecal Specimen Examination

3

SUBSTANCES THAT INTERFERE IN STOOL EXAMINATIONS

* Urine Contamination
* Castor/ Mineral Oil
* Barium, Bismuth, Kaolin, Milk of Magnesia, Antacids
* Antibiotics/Antimalarials
* Enema

EXAMINATION OF FECAL SPECIMENS

1. Wet Mount Technique
2. Concentration Techniques
3. Permanently Stained Smears
4. Culture
5. Immunoassay (Antigen Detection)

a. Guardia spp.

b. Cryptosporidium spp.

c. Entamoeba histolyca

Recommended ratio of **Fixative to Stool**

3:1

Formalin

* All-purpose fixative
* Advantages: Easy to prepare, long shelf life
* Disadvantages: Not suitable for permanent stain

Formalin concentration:

* 5% =

Protozoans

Formalin concentration:

* 10% =

Helminths

Schaudinn’s Solution

* For permanent stain
* Toxic because it contains Mercuric Chloride
* Causes disposable problems

Polyvinyl Alcohol

* Acts as an adhesive for stool specimen when preparing a slide for staining
* Combined with schaudinn’s fixative (replaced by *copper sulfate and zinc sulfate*)

Merthiolate-Iodine-Formalin

* Useful fixation for parasite
* Cannot be used in permanent stain

Components of MIF:

* **Merthiolate**

* Staining component
* AKA Thimerosal

Components of MIF:

* **Iodine**

Staining component

Components of MIF:

* **Formalin**

Preservative

Sodium-Acetate-Formalin

* Used for permanent stains, direct wet mounts, and concentration procedures
* Does not contain mercury
* The quality is not good

Two-Vial Techniques

* One portion of specimens is fixed in three parts of 5%-10% buffered **formalin**
* One portion in the three parts is the **polyvinyl alcohol (PVA)** fixative

Macroscopic Findings:

* Macroscopic Parasite

Possible Eitology:

* Pinworms
* Tapeworm proglottids
* Helminths

Macroscopic Findings:

* Blood-tinged mucus (formed specimens)

Possible Eitology:

* Trophic amoebae

Macroscopic Findings:

* Bloody mucus

Possible Eitology:

* Amoebic ulcerations in the large intestine

Macroscopic Findings:

* Bright red blood (formed specimens)

Possible Eitology:

* Bleeding Hemorrhoids

Macroscopic Findings:

* Occult Blood

Possible Eitology:

* Other gastrointestinal disorder

Direct Wet Mount/Direct Fecal Smear (DFS)

* Most common
* Most easily formed parasitologic test
* Most useful when fresh specimen, especially

liquid stools or duodenal aspirates, are examined for **motile trophozoites** or **helminth larvae**

Stains used for DFS

1. 0.85% Normal Saline Solution: Basic viewing of cysts and trophozoites


2. Lugol’s Iodine: Can only visualize cysts
3. Nair’s Methylene Blue: Used to visualize trophozoites

Alternative stains for DFS

* Modified D’Antoni’s
* Iodine solution
* Gram’s Iodine

Can be seen using NSS

* Eggs
* Protozoan trophozoites
* Larvae

Iodine is toxic to

Trophozoites

Concentration Techniques

More sensitive in detecting cyst, larvae, and eggs

Sedimentation (Concentration Techniques)

The heavier parasites settle at the bottom because of gravity or centrifugation

Floatation (Concentration Techniques)

Lighter parasite cyst and eggs rise to the surfaced solution of high specific gravity

Formalin Ether Concentration Technique (FECT)

Recommended for protozoa and helminth larvae and ova

Acid Ether Concentration Techniques (AECT)

Recommended for Trichuris, Capillaria, and Trematode eggs

Reagents used in FECT

10% Formalin, Ether

Reagents used in AECT

40% HCl, Ether

Reagents used in Mini Parasep SF Kit

10% Formalin, **Triton X**

Disadvantage of Floataion Technique

Eggs and Cysts distortion

Zinc Sulfate (𝑍𝑛𝑆𝑂4) Flotation

* **Reagent:** 33% 𝑍𝑛𝑆𝑂4 33%
* **Specific Gravity:** 1.18-1.20

Brine Solution

* **Reagent:** Saturated Table Salt (NaCl)
* Not Suitable for operculated eggs

Sheather’s Sugar Flotation

* **Reagent:** Boiled sugar with phenol
* For **coccidian oocyte**

Wheatley’s Trichrome Stain

* Widespread acceptance because of its simplicity, reliability and cost-effectiveness
* Appropriate specimens: **fixed in Schaudinn’s fixative or PVA fixative**; SAF-or MIF-preserved specimens may be stained with trichrome, but the results are less satisfactory
* May appear in different shades of blue, green, or purple

Iron Hematoxylin Stain

* More difficult to perform than the trichrome stain
* May stain clearer and sharper than trichrome
* Modified iron hematoxylin stain that may prove useful by **incorporates carbol fuchsin,** allowing concurrent staining of acid-fast organisms

Acid-fast organisms

* *Cryptosporidium*
* *Cyclospora*
* *Cystoisospora*

Modified Acid-Fast Stain

* Detects the oocysts of acid-fast organisms
* Sensitive but lack specificity

Acid-fast staining techniques

1. Modified Kinyoun method
2. Modified Acid-Fast Dimethyl

Sulfoxide
3. Auramine-O

Stain for *Microsporidia*

* Microsporidia (especially *Enterocytozoon bieneusi and Encephalitozoon intestinalis*)
* Modified trichrome stain uses an increased (10-fold) concentration of chromotrope 2R with increased staining time
* Stains that are fungi related and has a spore forming ability

Cellulose Tape Technique for Pinworms

* *Enterobius vermicularis*, migrates from the cecum to the perianal skin (deposits typical embryonated egg)
* Enterobius vermicularis also known as society worm or pinworms
* Detects Eggs or adults (occasionally)

Specimens from Cellulose Tape Technique should be collected:

* Late at night OR
* First thing in the morning before

bathing or defecation

Nocturnal Pruritus Ani

* A symptom for Enterobiasis, it is the itching of the perianal skin at night
* It is the time the female *E. vermicularis* travels lay its eggs

*E. vermicularis eggs* are known for their

D-shaped appearance

Kato Thick Smear

* Qualitative method helps in finding out if the parasite are present or absent in the stool sample
* 50-60 mg of stool or approximately 2 Mongo beans
* Simple technique, for mass stool examinations
* Good for detecting eggs with thick eggshells (not recommended for protozoans and used for helminth eggs)
* Cellophane soaked in a mixture of glycerine and malachite green solution

Malachite Green

Decreases the brightness of the microscope

Glycerine

Acts as the clearing solution

Kato-Katz Smear

* Uses measured amount of stool
* Quantitative method that allows to determine the number of eggs in a gram of stool
* Uses a template with a standardized hole

1\.0 mm (Thickness)

9\.0 mm (Hole Diameter)

50 mg (Amount of Stool)

EPG = count x 20

1\.5 mm (Thickness)

6\.0 mm (Hole Diameter)

41\.7 mg (Amount of Stool)

EPG = count x 24

0\.5 mm (Thickness)

6\.5 mm (Hole Diameter)

20 mg (Amount of Stool)

EPG = count x 50

Stoll Egg Count

* Reagent: 0.1N NaOH (sodium hydroxide)

Egg Hatching Method

* Analysis of schistosomiasis
* Detects the presence of eggs in light infections and determines their viability
* Urine/ Stool + water + Erlenmeyer Flask
* Hatched *miracidia* (larvae) are positively phototropic and move towards/near the light

Nematode Culture and Recovery Techniques (Coproculture)

Stool is incubated in a humid environment to encourage egg hatching

Harada-Mori Filter Paper Strip Culture

* Larvae migrate from the feces into a

water phase, where they may be

readily detected
* If the parasite moves **downwards** to

the water, it is a **hookworm**
* If the parasite moves **upwards** to the

paper, it is a Strongyloides

Charcoal Culture

* Larvae first migrates into a dampened gauze pad
* Then placed in water, allowing the larvae to settle out

Baermann Funnel Technique

* Sensitive and reliable method for recovery of Strongyloides and other nematode larvae from stool
* Fecal sample is placed on several layers of gauze on top of a wire screen that is suspended in a funnel
* Bottom of the funnel is clamped off, and water is added to the level of the gauze
* Larvae actively migrate through the gauze and settle to the bottom of the funnel

Artifacts

Objects that might resemble Enteric Parasites

(ARTIFACTS) White Blood Cells/ Macrophages/ Squamous and Columnar Epithelial Cells

Amoeba

(ARTIFACTS) Yeast and Starch Granules

Protozoan Cyst

(ARTIFACTS) Pollen and Fungal Conidia

Helminth eggs

(ARTIFACTS) Plant Fibers

Nematode Larvae

Pieces of vegetables or vegetable skins

Adult worms or proglottids

Charcot-Leyden Crystals

* Hexagonal bipyramidal crystal
* Localized in granules and cytoplasm of eosinophil and basophil

The parasites we can detect or isolate in blood examinations are

* Plasmodium Species
* Babesia microti
* Trypanosoma
* Filariae-tissue nematodes

Thick and Thin Blood Smear

* Commonly performed method when identifying blood parasite
* Thin Smear: Species identification; spread over the slide in a thin layer, yielding instact, non overlapping cellular elements
* Thick Smear: Preferred for **diagnosis**; concentrated in a small area that is many cell layers deep

Blood for examination may be obtained by

* Fingerstick: blood should flow freely
* Earlobe puncture
* Venipuncture: the first drop of blood (anti-coagulant free) from the needle is used to prepare the films at the bedside

If collection tubes are needed, which is the preferred tube to be used in venipuncture for the preparation of slides?

EDTA

* Anticoagulant may distort the parasites

Best Stain for Blood Smear

Giemsa Stain

* Host cell and parasite chromatin stains vividly but the hemoglobin in erythrocytes is only pale red
* The only stain that allows visualization of the erythrocyte stippling that occurs with infection by certain malarial parasites

Alternative stain for Blood Smear

Wright’s Stain

* Stains parasites less well than Giemsa
* Stains erythrocytes, producing a busier background

Fresh Giemsa stain must be made ____ by diluting stock solution into phosphate - buffered water

Each day of use

Buffered water must be maintained at

6\.8- 7.2 pH

Buffy Coat Smears

Diagnosis of Leishmania, Trypanosomes, Microfilariae

Membrane FIltration

Lysed blood + 5 um Membrane filter

Knott’s Concentration

* Blood + 2% Formalin =

Centrifuge

Knott’s Concentration

* Sediment

Check for Microfilariae

Urine

* *Trichomonas vaginalis*
* *Schistosoma haematobium*

Cerebrospinal Fluid (CSF)

* *Trypanosoma spp.*
* *Naegleria fowleri*
* *Angiostrongylus*
* *Taenia solium* (cysticercus larvae)

Duodenal material

* Entero test (Enteric capsule test)
* *Giardia lamblia*
* *Strongyloides stercoralis*

Sigmoidoscopy material

*E. histolytica*

Vaginal and urethral discharges, prostatic secretions

*Trichomonas vaginalis*

Sputum

* *Paragonimus westermani*
* *Pneumocystis jiroveci*

Biopsy:

* Skin

* *Onchocerca sp.*
* *Mansonella*

*spp.*

Biopsy:

* Muscle

*Trichinella spp.*

Biopsy:

* Rectal

Schistosomes eggs

Biopsy:

* Lymph Node:

Adult filarial worms

Biopsy:

* Tissue Culture

* *Leishmania spp.*
* *Trypanosoma spp.*

Skin:

* Bachman Skin Test

*T. Spiralis*

Skin:

* Casoni Skin Test

*E. granulosus*

Skin:

* Dr. Montenegro Intradermal Test

*Leishmania spp.*

IMMUNODIAGNOSTIC TECHNIQUES

* Only detects for the antigen related to the parasite
* Enzyme immunoassay (EIA)
* Indirect Immunofluorescence Assay (IFA)
* Direct Fluorescence Antibody Assay (DFA)
* Western Blot
* Radioimmunoassay
* Immunodiffusion