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TECHNICAL METHODS FOR DIAGNOSIS OF PARASITIC

INFECTIONS/DISEASES
▪ The investigation of parasitic infection depends on the biologic behavior of the parasites, their
habitat, life cycle, mode of transmission and sources.
▪ Diagnosis of most parasitic infections and diseases are dependent upon laboratory examination
of the different specimens taken from the infected individuals.
▪ Materials to be examined include stool, urine, sputum, blood, spinal fluid, and tissue biopsy of
the host.
▪ Successful laboratory identification of parasites requires the knowledge and practice of
laboratory procedures such as handling and collection of satisfactory specimens.
▪ It also requires experience in the different characteristics of the various species of parasites and
their stages of development which may include the egg stage, larval stage/s, adult/s, cyst,
trophozoite as well as familiarity with pseudoparasitic forms and artifacts that maybe mistaken for
parasites.
▪ The detection and identification of any parasitic forms will depend on proper collection,
transportation, preservation and proper preparation of materials for their microscopic studies
both in the living state and in stained preparation.
▪ It is important to know the approximate size of the various parasites. Whenever possible, materials
should be examined in the fresh state and in as natural a medium as possible.
▪ The implementation of a reliable laboratory processing techniques/methods is a very important step
in successful laboratory diagnosis.

COLLECTION, TRANSPORT AND PROCESSING OF SPECIMENS STOOL:

✓ Stool is the most frequently examined specimen since most parasites inhabit the intestine.
✓ Both Protozoa and Helminths maybe detected from a properly collected and prepared stool
specimen.
✓ Different parasitic forms maybe recovered from such sample such as eggs, larva, adult, cyst and
trophozoite.
✓ OVA and PARASITE EXAMINATION: The MOST COMMON PROCEDURE performed in the
area of parasitology is the examination of a stool specimen for ova and parasites (abbreviated as
O&P), where OVA refers to the egg stage of select parasites and parasites encompasses the other
morphologic forms that may be present.
✓ Morphologic forms of protozoa and helminths may be detected from a properly collected and
prepared stool specimen.
✓ Trophozoites and cysts, Helminth stages such as eggs, larvae, proglottids, and adult worms.
✓ The typical stool collection protocol consists of THREE SPECIMENS, one specimen collected
every other day or a total of three collected in 10 days.
✓ One exception is in the diagnosis of amebiasis, in which up to SIX SPECIMENS IN 14 DAYS
is acceptable.

GENERAL CONSIDERATIONS FOR PROPER COLLECTION AND PRESERVATION OF

STOOL:
1) Stool should be collected in a clean, dry, sterile wide-mouthed container with a tightly fitted lid to
maintain adequate moisture.
2) A sufficient quantity of stool should be collected such that repeated examination can be done if
necessary. The smallest acceptable amount of stool required for parasitic study is 2 to 5 grams
or pea or thumb size.
3) Urine should not be allowed to contaminate the stool specimen because it has been known to
destroy some parasites like trophozoites of protozoa.
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4) Stool should NOT be retrieved from the toilet bowl water because free living protozoa and
nematodes may be confused with human parasites making laboratory diagnosis inaccurate.
In addition, water may destroy some parasites such as Schistosoma eggs and amoebic
trophozoites.
5) Presence of toilet paper in the stool sample may mask parasites or make examination of the sample
difficult.
6) Certain medications and substances may interfere with the detection of parasites.
7) Specimen must be obtained prior to administration of any type of drug therapy such as antimicrobial
agents/antibiotics, anti-helminthic, anti-diarrheal drugs, antacids, laxatives, soap and hypertonic
salt enemas may suppress the parasites giving a negative result. Two to three weeks should be
allowed to elapse after treatment before re-examining the stool for the presence of parasites.
8) Stool samples from patients whose therapy includes barium, bismuth, mineral oil, kaolin may
interfere with stool examination. These interfering substances may produce unknown objects or
may mask possible parasites during examination. Five to seven days should be allowed to elapse
after treatment before re-examining the stool for the presence of parasites.
9) Saline cathartics like Buffered Sodium Phosphate maybe used only to obtain purged specimens but
before purgation is resorted to, normally passed specimens should be obtained as some parasites
are passed irregularly. Purged stool specimen is used only if negative results are obtained after
several stool specimens had been examined.
10) When handling all specimens, gloves, mask and laboratory gown must be worn at all times.
11) A freshly collected stool sample (which is immediately submitted to the laboratory within one
hour after collection) is the ideal specimen for parasitic examination.
12) Specimens should be examined as soon as possible and it should not be kept warm. When it is not
possible, the sample must be preserved to maintain its integrity.
13) The SPECIMEN CONTAINER should be labeled with the patient’s name and identification
number, the physician’s name, and the date and time of sample collection.
14) Some form of REQUISITION, paper or computer-based, should accompany the specimen
indicating the test(s) requested.
15) OTHER INFORMATION, such as suspected diagnosis, travel history, and clinical findings, is
helpful, but may not be provided.
16) Specimen should be placed into a ZIP LOCK PLASTIC BAG for transport to the laboratory.
17) TROPHOZOITES are usually found in LIQUID STOOL, it is recommended that liquid
specimens be EXAMINED WITHIN 30 MINUTES OF PASSAGE.
18) It is advisable to examine three samples before excluding parasites. The typical stool collection
protocol for screening a patient suspected of a parasitic infection/disease consists of three total
samples one every other or third day.

TWO GENERAL METHODS OF PRESERVATION:

1. Physical Preservation – placed the sample inside the refrigerator (DO NOT FREEZE)
2. Chemical Preservation – use of preservatives/fixatives

FIXATIVES FOR PRESERVATION OF STOOL SAMPLE ARE THE FOLLOWING:

A. FORMALIN
B. PVA
C. SAF

1. FORMALIN
▪ Most commonly used stool preservatives;
▪ Required Volume: FOR 1 GRAM OF STOOL SAMPLE, ADD 3 ML OF FORMALIN;
▪ All-purpose fixative and two concentrations are usually used:
A. 5% - USED FOR PROTOZOAN CYST
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B. 10% - USED FOR HELMINTH EGGS AND LARVAE

ADVANTAGES:
1. Maybe routinely used for direct examinations and concentration procedures.
2. Easy to prepare.
3. Preserve specimens for up to several years
4. Long shelf life
DISADVANTAGES:
1. Can not be used for permanent smears/stains
2. Can not be used for preservation of trophozoites
3. Morphologic details of cyst and eggs may fade with time

2. POLYVINYL ALCOHOL (PVA)

▪ Best preservative and is especially effective when combined with Schaudinn’s solution;
▪ Often referred to as “Gold standard”
▪ Composition:
o PVA – polyvinyl alcohol, distilled water, glycerol
o Schaudinn’s solution – Mercuric chloride, glacial acetic acid, ethanol
ADVANTAGES:
1. Easy to mix with the specimen
2. Serves as a good adhesive, which helps to glue the specimen on the slide. Can be used for permanent
smear/stain
3. Long shelf life when stored at room temperature
4. Cysts, trophozoites, helminth eggs can be preserved better
DISADVANTAGES:
1. Not easy to prepare
2. Presence of large amount of mercury which is toxic/poisonous

3. SODIUM-ACETATE-FORMALIN (SAF)
▪ Composition: 40% Formaldehyde, glacial acetic acid, Sodium Acetate, distilled water
ADVANTAGES:
1. Can preserve protozoan cyst and trophozoite
2. Eliminates the use of mercury
3. Inexpensive
4. Easy to prepare
5. Long shelf life
6. Contains buffering capabilities to decrease the distortion of protozoa
DISADVANTAGES:
1. Adhesive property is not good so addition of albumin on the slides is necessary for adhesion
2. Contain a large amount of water diluting the specimen and makes it easy to miss the parasites.

PROCESSING OF STOOL SAMPLE

A. MACROSCOPIC/GROSS EXAMINATION:
✓ Stool specimens submitted for parasitic study should be first examined grossly to determine the
color and consistency of the sample. In addition, the specimen should be screened for the presence
of gross abnormalities.
✓ Freshly passed stool is the sample of choice for parasitic study because it is the best specimen for
accurate detection of the delicate protozoan trophozoite and flagellates.
✓ The color of stool is important because it may indicate the condition of the patient, that is whether
the patient has recently had undergone a special procedure such as Barium enema or if the patient
is on antibiotic therapy. However, color maybe diet or foods taken by the patient may affect the
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color of the stool.

✓ Possible color includes dark brown, black, clay, yellow, red, green and white.
✓ The consistency or degree of moisture in a stool specimen may serve as indicator of what possible
stage/s of parasite is present in the sample.
✓ Possible consistency includes hard/well-formed (cyst, helminth eggs and larvae), soft/semi-formed
(cyst, trophozoite, helminth eggs and larvae) and watery/liquid/diarrheic (trophozoite).
✓ Stool containing mucus or blood should be specifically selected for microscopic study.
✓ In some instances, wherein a number of specimens are received, examine first the fecal specimens
that are watery or liquid for they may contain motile amoeba.
✓ Gross abnormalities possibly found in stool include adult worms, proglottids, pus and mucus.
✓ First the surface of the stool should be examined for parasites such as proglottids or adult.
The sample should be broken up with applicator stick and examined once more for parasites.
✓ Samples containing portions or the entire adult worms maybe carefully washed with NSS through
a wire screen. This process allows retrieval and examination of the parasites for identification
purposes.
✓ The presence of abnormalities other the actual parasites may have parasitic indications.
✓ Blood/mucus in loose or liquid stool may suggest the presence of amebic ulcerations in the large
intestine.
✓ Bright red blood on the surface of a formed stool is usually associated with irritation and bleeding.

MICROSCOPIC EXAMINATION:
✓ The microscopic examination of stool as well as of other specimen types involves several steps:
calibrating the ocular microscope, processing of stool samples and examining the slides
microscopically.
✓ Calibration and use of Micrometer
o Parasites are measured in units known as microns by the use of an ocular micrometer.
o It is an eyepiece that is equipped with a numbered scale 1 to 50 or 1 to 100 units.
o Calibration of the ocular micrometer is necessary to define how many microns are
equivalent to each of these so-called units.
✓ Techniques In Processing Stool Sample – Microscopic Examination:
o DIRECT FECAL SMEAR (DFS)
This is the simplest and rapid of all fecal examination techniques.
This technique is easy to perform and the unstained preparation is particularly
valuable for study of living parasites such as motile protozoa trophozoite, helminth
eggs and nematode larvae.
The iodine film is employed primarily to study the diagnostic features of protozoan
cysts.
The disadvantage of this method is that light infection may not be detected because
small quantity of fecal sample is used.
o KATO THICK OR KATO-KATZ (CELLOPHANE COVERED THICK SMEAR)
This technique is simple and economical.
This is suitable for large scale fecal examination for all common types of helminth
eggs.
This may be used to count the number of helminth eggs (Kato-katz).
The disadvantage of this technique is that it is unsuitable for diarrheic stool and
examination of feces containing excessive amount of fiber or gas.
This technique cannot be used to diagnose protozoan cyst and trophozoite.
o CONCENTRATION TECHNIQUES
This technique is done to separate the parasite from the bulk of materials or
undigested food particles present in the sample such as bacteria, undigested food
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elements, fibers and others.

This technique is done to ensure proper diagnosis in cases of light infection.
However, this technique cannot be used diarrheic stool containing for protozoan
trophozoites.
Methods employed in concentration technique:
• SEDIMENTATION
• FLOTATION
• CENTRIFUGAL FLOATATION

SEDIMENTATION
Principle: Parasite has a higher specific gravity than the solution so the parasite will sink to the bottom.
a. Simple sedimentation
b. Centrifugation
b.1 Acid Ether Concentration Technique (AECT)
b.2 406 MGL/Formalin Ether Concentration Technique (FECT)
b.3 Merthiolate Iodine Formaldehyde Concentration Technique (MIFCT)

FLOTATION
Principle: Parasite has a lower specific gravity than the solution used therefore it will float on the surface
of the solution.
a. Brine Floatation Technique

CENTRIFUGAL FLOATATION
This method combines the principle of gravitational and flotation. This technique provides high
concentration of parasitic objects practically free or detritus.
a. Zinc Sulfate Centrifugal Flotation Techniques (ZSCFT)

ARTIFACTS AND CONFUSERS

✓ These are structures that closely resemble parasites but in reality are not parasites. Such artifacts
and confusers maybe the result of disease processes, medications and/or dietary habits (foods).
✓ Most common artifacts and confusers present in stool sample:

POLLEN GRAINS
o Resemble the eggs of Taenia species only are smaller measuring 12 to 20 micron in size.
o They may either appear round or symmetrically lobed.
o Unlike Taenia, there are no internal structures.

VEGETABLE CELLS
o May easily be confused with helminth eggs.
o These are typically large and roundish-oval to irregularly round in shape may measure up
to 150 micron in size.
o Thick shell walls are usually present.
o The interior portion of vegetable cells is unorganized and often appears to consists of
primarily large vacuoles.

VEGETABLE SPIRALS
o Often resemble helminth larvae in shape and size. Unlike helminth larvae, vegetable spirals
do not have a head or tail region.
o Vegetable spirals are readily distinguished from parasitic forms by their “ladder-like”
appearance. The “ladder” consists of a series of rungs that are spaced closely together.
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PLANT HAIR
o May resemble helminth larvae in size and shape and appear to have a nondescript internal
structure.
o It does not have diagnostic structures, such as buccal cavity, esophagus, intestine or genital
primordium and no head or tail region.

PLANT MATERIAL
o Resemble helminth eggs particularly an unfertilized Ascaris lumbricoides egg in size and
shape.
o This artifact is typically round to oval in shape and may or may not have a definite cell
wall.
o Plant material is often rough in appearance and may have hairs (psuedocilia) extending
from its periphery. The interior of the cell looks like a cluster of odd-shaped vacuoles.

WHITE BLOOD CELLS

o Often mistaken for amebic cyst, especially those of Entamoeba histolytica.
o These cells are usually present in patients suffering from ulcerative colitis, bacterial
dysentery or intestinal Ameobiasis.
o These cells have a two to four lobed nucleus similar in appearance to E. histolytica nucleus.
o Nuclear inclusions such as karyosome and peripheral chromatin are absent in ABCs.

YEAST
o Round or oval in shape and maybe confused with protozoan cyst.
o Yeast cells typically show no definite internal structures.

STARCH CELLS/GRANULES
o Round or oval and elongated in shape with irregular outline.
o The shell is thick, very irregular with cracks.
o They may appear as similar to Protozoan cyst.
o They lack internal structures and with a nondescript mass located inside the cells and may
resemble a nucleus.
o These granules are residue of starchy food such as potatoes, beans, yams and cassava.

UNDIGESTED MEAT FIBERS

o Oval or rectangular with rounded corners.
o These are transparent with no granules inside.
o May resemble helminth eggs.

SOAPS
o Round, oval or irregular in shape with lines radiating from the center and visible near the
rim.
o Resemble helminth eggs.

BUBBLES/OIL DROPLETS
o Perfectly round with no internal structures.
o Resemble helminth eggs.

REFERENCE:

LABORATORY GUIDE IN PARASITOLOGY BY EDISON D. RAMOS, RMT, MPH, MSMT