DIAGNOSTIC PARASITOLOGY
College of Medical Laboratory Science
Our Lady of Fatima University
PARA 311
Laboratory Diagnosis
-accurate diagnosis of parasitic infections can help
decrease the prevalence and incidence of a parasitic
infection.
1. Confirm clinical impression
2. Rule out diagnosis
3. Aid a clinician in the choice of proper medication
4. Help in monitoring the effect of treatment
regimen
Diagnostic parasitology is done by:
A. Demonstration of parasites (e.g., eggs, larvae,
adults, cysts, oocysts, trophozoites)
B. Detection of host immune response to the
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parasites (e.g., Abs and Ags)
OUTLINE OF TECHINIQUES (stool)
1. Direct Fecal Smear
2. Kato-thick Smear
3. Concentration
a. Sedimentation Techniques
b. Floatation Techniques
4. Stool Culture
5. Egg counting procedures
6. Perianal Swab
7. Staining stool specimen
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MACROSCOPIC EXAMINATION OF THE STOOL
A. Consistency
* reflects the level of hydration
* gives an indication as to which organisms are present
B. Color
C. Gross examination
*tapeworm proglottids
* adult nematodes (Ascaris or
Enterobius)
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MICROSCOPIC EXAMINATION OF THE STOOL
A. Ocular micrometer
* specially designed ocular piece equipped with measuring
scale
* Must be calibrated to ensure accurate measurement
* Expressed in microns (µ or µm) defined as 0.001 [10-3]
millimeter, or 10-6 meter
* Calibration is aided with the use of a stage micrometer
containing a calibrated scale divided into 0.01-mm units.
* The ocular micrometer is a disk equipped with a line
evenly
divided into 50 or 100 units
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MICROSCOPIC EXAMINATION OF THE STOOL
No. of microns = (no. of stage micrometer units X 1000) /
(no. of ocular micrometer units)
= (0.4 x 1000) / 60
= 6. 67 um or 7 um
Suggested ranges of the micron value per
ocular unit by magnification: (Zeibig, 2013)
10x: 7.5-10 um
40x: 2.5-5 um
100x: 1 um 6
1. DIRECT FECAL SMEAR (DFS)
• Routine method of stool examination
• Employs use of approx. 2 mg of stool and 0. 85% NSS
• Primarily useful in detection of motile protozoan trophozoites
• Trophozoites are pale and transparent
• Nair ’s buffered methylene blue (BMB) – demonstrate nuclear
morphology of trophozoites
a. Entamoeba cytoplasm: light blue
b. Entamoeba nucleus : dark blue
• Lugol’s iodine – temporary stains nuclei of protozoan cysts
a. cytoplasm : golden yellow
b. nucleus: pale and refractile
c. glycogen: deep brown
* Light infections may not be detected
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2. K ATO THICK SMEAR
• Employs use of 50-60 mg of stool (size of two mongo
beans)
• Uses cellophane paper soaked in a mixture of glycerine
and
malachite green solution
• Simple and economical
• Very good in detecting eggs with thick shells (e.g., Ascaris
and Trichuris) but not thin shells
* Usefulness is limited in diarrheic and watery stools
* Not able to detect protozoan cyst and trophozoite
3. CONCENTRATION TECHNIQUES
• Useful in cases of light infections
A. Sedimentation Procedures
1. Acid Ether Concentration Technique (AECT)
a. 40% HClà dissolves albuminous material
b. ether à dissolves neutral fats
- recommended for recovery Trichuris, Capillaria and trematode
eggs specially Schistosoma.
* Drawbacks: loss of parasite to the plug of debris and
possible destruction of protozoan cyst
2. Formalin Ether Concentration Technique (FECT)
a. 10% Formalin as preservative
b. ether à dissolves neutral fats (highly explosive and flammable)
*Ethyl acetate as alternative to ether (not as efficient as ether in
the extraction of fat or mucoidal material from the stool)
- more efficient in recovering cestode eggs and Giardia cysts
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3. CONCENTRATION TECHNIQUES
• Useful in cases of light infections
B. Floatation Procedures
1. Zinc Sulfate (ZnSO 4) Floatation
- 33% ZnSO4 with specific gravity of 1.18-1.20
- If parasites are exposed to high specific gravity,
distortion and shrinkage of protozoan cysts and thin-
walled nematode eggs may occur.
2. Brine Floatation
- uses Table salt solution
- no need for centrifugation since helminth eggs rise
from the surface of the solution.
-Schistosoma become badly shrunken
- NOT useful for operculated eggs like Clonorchis,
Opistorchis and heterophyids because these do not
float in brine solution. 11
3. CONCENTRATION TECHNIQUES
• Useful in cases of light infections
3. Sheather ’s Sugar Floatation
- employs use of boiled sugar solution preserved with
phenol
- best for recovery of coccidian oocysts
(e.g., Cryptosporidium, Cyclospora and Isospora)
- better visualization can be appreciated through the use
of a phase-contrast microscope
4. STOOL CULTURE METHODS
• Useful for hookworm identification
• Stools positive for hookworm ova and/or Strongyloides
rhabditiform larva can be cultured until the filariform larvae
develop
A. Coproculture
- positive stools mixed with moistened soil and granulated
charcoal
- Larvae are harvested using Baermann procedure
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BAERMANN TECHNIQUE
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4. STOOL CULTURE METHODS
B. Harada-Mori or the Test tube Culture Method
- employs use of test tubes and filter paper strips
- also used for cultivation of intestinal protozoan
- Filariform larva will move downwards against the
upward capillary movement of water and can be
recovered from the water at the bottom of the tube
- Strongyloides larvae move upwards and accumulate at
the upper end of the filter paper strip
7cc of boiled water or distilled
water
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5. EGG COUNTING PROCEDURES
• Used to correlate the severity of clinical disease with the
intensity of infection or worm burden
A. Kato-Katz Method or Cellophane Covered Thick Smear
B. Stoll Egg Count
WHO classification of intensity of infections with STH and
Schistosomiasis
Organism Light Intensity Moderate Intensity Heavy Intensity
A. lumbricoides 1-4,999 epg 5,000-49,999 epg ≥ 50,000 epg
T. trichiura 1-999 epg 1,000-9,999 epg ≥ 10,000 epg
Hookworm 1-1,999 epg 2,000-3,999 epg ≥ 4,000 epg
S. japonicum and 1-99 epg 100-399 epg ≥ 400 epg
S. mansoni
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5. EGG COUNTING PROCEDURES
A. KATO-KATZ METHOD or CELLOPHANE COVERED THICK
SMEAR
• Uses a measured amount of stool which has been sieved
through a wire mesh and pressed under cellophane paper
soaked in malachite green solution
• Uniform amount of stool is examined using a template with a
uniform sized hole in the middle
• Consistency is the main determinant of the sensitivity of this
technique
• For ID of Schistosoma ova, 1% eosin can be layered over the
cellophane paper
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5. EGG COUNTING PROCEDURES
B. STOLL EGG COUNT
• 0.1 N NaOH and a stool displacement flask calibrated at 56
mL and 60 mL
• Sodium hydroxide serve as stool diluent, it saponifies fat and
free eggs from fecal debris
• Uses Stoll pipettes calibrated at 0.075 mL and 0.15 mL to
measure amount of diluted stool
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6. PERIANAL SWAB (Cellulose Tape or Scotch Tape Method)
• Used to recover eggs of E. vermicularis and Taenia spp.
• In some laboratories, a drop of toluene or xylene solution
helps in the visualization of eggs
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7. STAINING OF STOOL SAMPLES
• Performed specifically for the examination of the nuclear
characteristics of amoeba.
• Also useful for ID of other intestinal protozoans such as
Balantidium coli and Giardia spp.
a. Iron- hematoxylin
b. Trichome
c. Chlorazol Black E
• Kinyoun staining for coccidians
