LABORATORY DIAGNOSIS OF PARASITIC INFECTION

SPECIMEN COLLECTION AND PROCESSING

1. Diagnosis of parasite infections often depend on observing parasite forms that include protozoa ova, larva or adult forms

2. Specimen includes stool, tissue, urine, sputum and blood

A. Stool sample should be free from antimicrobial agents that can inhibit parasitic growth. Barium from enemas can obscure parasites during microscopic examination
- at least 3 grams of fecal sample on 3 consecutive days
- because urea and acidic pH inhibit some parasite and distort their morphology, stool should be free of urine
- liquid stools best for trophozoites detection and formed stool for cyst and ova detection

B. Stool preservatives
- stool should not be preserve for longer hours
- 5 to 10% formalin for concentration procedures
- PVA for stained smear preparation
- Sodium Acetate Formalin = concentration procedures and stained smear preparation

C. Fecal Concentration Methods
a. Formalin-Ethyl Acetate Sedimentation
b. Zinc Sulfate Flotation
c. Sheather Sugar Flotation

D. Blood Concentration Methods

Collection Methods

a. Cellophane / Scotch tape method (pinworm)
b. Entero Test – string test
c. Sigmoidoscopy – to collect colon material

Cellophane / Scotch tape method (pinworms)

Collection Method (pinworm)

Entero Test

– string test

Sigmoidoscopy

– to collect colon material

Sample Types and associated parasites

a. Feces: Giardia, Cryptosporadium, Entamoeba, Ascaris, Enterobius, etc
b. Blood: Plasmodium, Leishmania, Trypanosoma, Microfilariae
c. Skin: Onchocerca
d. Vaginal or urethral: Trichomonas
e. Eye scraping: Acanthamoeba
f. Tissue: Naegleria, Acanthamoeba, & Leishmania
g. Urine: Schistosoma & Trichomonas
h. Sputum: Ascaris & Strongyloides

MICROSCOPIC EXAMINATION

A. Direct wet preparation or direct wet mount
B. Concentration Methods
1. Sedimentation (Formalin-Ethyl Acetate Sedimentation Procedure)
2. Zinc Sulfate Floatation Technique
C. Permanent Stains

Direct wet preparation or direct wet mount

Purpose: To detect the presence of motile protozoan trophozoites; other stages detected include cysts, oocysts, ova and larvae of worms.

Principle: A small portion of unfixed stool is mixed w/ saline or iodine then studied under the microscope.

Concentration Methods

- can both used on both fresh and preserved specimens. It can be used to detect cysts, oocysts and larvae of nematodes

Purposes:
1. To aggregate parasites present into a small volume of the sample that enables the detection of small numbers of parasites that might not be detected in direct wet preparation. 2. To remove debris and other contaminants that might interfere with the microscopic examination.

Sedimentation (Formalin-Ethyl Acetate Sedimentation Procedure)

Principle: Based on the specific gravity- parsites are heavier than the solution than the fecal debris.

Zinc Sulfate Floatation Technique

Principle: Based on the differences in specific gravity and the sample debris (heavier than the parasites). The zinc sulfate has a specific gravity of 1.18 – 1.20 and is used as the concentrating solution.

Permanent Stains

- serves as a final step in the microscopic examination for the detection of parasites. It is designed to confirm the presence of cysts and/ or trophozoites of protozoans.

OTHER SPECIMENS & LABORATORY PROCEDURES

1. Duodenal Material
2. Sigmoidoscopy Material
3. Cellophane tape or Scotch Tape Preparation
4. Blood
5. Cerebrospinal Fluid
6. Tissue and Biopsy Specimens
7. Genitourinary Secretions

Duodenal Material

- this may be collected using nasogastric tube (NGT) or through the enteric capsule test (Entero test).

- the collected fluid must be examined immediately to prevent rapid deterioration of trophozoites, if there is any. Less than 2ml volume is recommended for this procedure. The sample undergoes centrifugation prior to microscopic examination of the sediments.

- in the Entero- test, patient is advised to swallow gelatin capsule that cointains a coil of yarn that is weighted, which will be released to the duodenum as the capsule dissolves in the stomach. The free end of the yarn is attached to the neck or cheek of the patient and pulled out after 4 hours of incubation. The bile stained material attached to the string is then examined under the microscope by wet preparation followed by permanent stain application.

Sigmoidoscopy Material

- this is done by examination of the colon and collection of the material, which can be used in biopsy examination. It is helpful in the diagnosis of Entamoeba histolytica infection.

Cellophane tape or Scotch Tape Preparation

- this is done to detect the presence of pinworm, Enterobius vermicularis. The female parasite migrates to the anus and lays it eggs.
- done in the morning before defacation or washes.
- used to detect tapeworm eggs of Taenia spp.

Blood

- for blood-borne parasites (leishmania, trypanosoma, plasmodium and filarial worms)
- thin (spp identification) and thick (number) blood smear, blood from earlobe or fingertip.
- stain used: wright’s or giemsa

Cerebrospinal Fluid

- used to detect amebic infections.
- immediate examination for motility detection
- wet preparations to detect morphological characteistics (Naegleria, Acanthamoeba, Trypanosoma, Toxoplasma gondii, Taenia solium (cystercosis) and Echinococcus.

Tissue and Biopsy Specimens

- utilized to detect the presence of Leishmania, Toxoplasma gondii, Trypanosoma, Taenia solium, Trichinella spiralis.
- specimen of choice: liver abscess (if suspected amoebic liver abscess

Genitourinary Secretions

- to detect blood fluke in urine (Schistosoma haematobium), Trichomonas vaginalis
- cotton swab collection, centrifuged urine sediments
- saline wet preparations for trophozoites demonstration.