CLINICAL PARASITOLOGY

LECTURER: MISS DEBBIE KATES SANTISTEBAN, RMT I Topic 2

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● Stool samples from patients whose therapy includes
barium, bismuth, or mineral oil should be collected prior
SPECIMEN COLLECTION AND PROCESSING
to therapy or not until 5 to 7 days after the completion of
therapy.
● Successful laboratory identification of parasites requires ● Taken during therapy, mask possible parasites during
the knowledge and practice of laboratory testing in the examination.
preanalytic, analytic, and postanalytic steps. ● Collection of specimens from patients who have taken
antibiotics or malarial medications should be delayed
for 2 weeks following therapy.
STOOL FOR OVA AND PARASITE EXAMINATION
● Stool specimens should be collected in a clean,
watertight container with a tight-fitting lid.
● Most common procedure performed in the area of ● Peanut size stool
parasitology is the examination of a stool specimen ● Avoid contaminants - urine, water, tissue.
for ova and parasites (O&P). ● The specimen container should be labeled properly
● Ova-egg Stage select parasites and parasites consists of the ff:
encompasses the other morphologic forms that may be ○ patient’s name
present. ○ identification number
● Two general components associated with this routine ○ physician’s name
parasitology: ○ date and time of sample collection
○ Microscopic Examination
○ Macroscopic Examination
TIME FRAME FROM SAMPLE COLLECTION AND STOOL
CONSISTENCY
MICROSCOPIC EXAMINATION COMPONENTS

● Collection ● Time frame from sample collection to receipt and

● Transport examination in the laboratory.
● Fixatives for preservation ● Fresh specimen is required to demonstrate the motility
of protozoan trophozoites.
● The trophozoite stage is sensitive to environmental
COLLECTION AND TRANSPORT changes and, on release from the body, disintegrates
rapidly.
● Morphologic forms of protozoa and helminths may be ● Trophozoites are usually found in liquid stool (examined
detected from a properly collected and prepared stool within 30 minutes)
specimen. ● Semiformed stool - mixture of protozoan cysts and
● Helminth stages - eggs, larvae, proglottids, and adult trophozoites (evaluated within 1 hour)
worms, because parasites are often shed (i.e., enter ● Formed stool specimens are most likely doesn’t contain
and subsequently passed in the stool) trophozoites (kept for 24 hours)
● Intermittently, they may not appear in a stool specimen
on a daily basis; therefore, multiple specimens are FIXATIVES FOR PRESERVATIVES
recommended for adequate detection.
● The routine stool collection - consists of 3 specimens, 1
specimen collected every other day, a total of three ● Freshly collected sample - ideal specimen for parasitic
collected in 10 days. examination.
● Exception - diagnosis of amebiasis - six specimens in ● Fixatives - substances that preserve the morphology of
14 days is acceptable. protozoa and prevent further development of certain
helminth eggs and larvae.
● Important factors to be considered during collection:
○ Manner of collection FORMALIN
○ Time frame collection
● Medications and substances may interfere with the
● Use of formalin has a potential health hazard
detection of parasites.

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CLINICAL PARASITOLOGY
LECTURER: MISS DEBBIE KATES SANTISTEBAN, RMT I Topic 2
______________________________________________________________________
● It has been used for many years as an all-purpose ● Can be used for performing concentration techniques
fixative for the recovery of protozoa and helminths. and permanent stained smears.
● Two concentrations of formalin are commonly used
○ 5% concentration - protozoan cysts ADVANTAGES
○ 10% concentration - helminth eggs and larvae
● Formalin can be routinely used for directly examinations ● Only requires a single vial and it is mercury-free.
and concentration procedures, but not for permanent ● Long shelf
smears. ● Used for preparing smears for staining with the modified
acid-fast stain to detect coccidian oocysts
ADVANTAGES
DISADVANTAGES
● Easy to prepare
● Preserves specimens for up to several years ● Adhesive properties of SAF are not good
● Long shelf life ● Protozoa morphology from SAF-preserved specimens
is not as clear in permanent stains
DISADVANTAGES

MODIFIED POLYVINYL ALCOHOL

● Does not preserve parasite morphology adequately for
permanent smears
● Trophozoites usually cannot be recovered and ● Other alternatives to mercury-based PVA are the use of
morphologic details of cysts and eggs may fade with substitute compounds containing copper sulfate or zinc
time sulfate.

ADVANTAGES
POLYVINYL ALCOHOL

● Used for concentration methods

● Comprised of a plastic powder that acts as an adhesive ● Permanent stained smears
for the stool specimen when preparing slides for
staining DISADVANTAGES
● Most often combined with Schaudinn solution, which
usually contains zinc, copper sulfate, or mercuric
chloride as a base. ● Do not provide the same quality of preservation for
adequate protozoan morphology on a permanent
ADVANTAGES stained slide as the mercury-based fixatives

● Trophozoites and cysts of the protozoa, as well as most ALTERNATIVE SINGLE-VIAL SYSTEMS
helminth eggs, are detected using this fixative.
● Used for preparation of a permanent stained smear ● Several manufacturers have developed nontoxic
● Long shelf life (room temperature) fixatives.

DISADVANTAGES
PROCESSING

● Concentration techniques is not as effective as formalin

● Once a stool specimen has been received in the
laboratory, the analytic phase begins, also known as
SODIUM ACETATE FORMALIN processing.
● Examined in two perspectives: macroscopic and
● Viable alternative to the use of PVA and Schaudinn microscopic
fixative is sodium acetate formalin (SAF).

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CLINICAL PARASITOLOGY
LECTURER: MISS DEBBIE KATES SANTISTEBAN, RMT I Topic 2
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saline or iodine and subsequent examination of the
MACROSCOPIC EXAMINATION resultant mixture under the microscope
● to detect the presence of motile protozoan trophozoites
● Examined macroscopically - determine the color and
consistency of the sample. DIRECT SALINE WET PREPARATION
● Should be screened and examined for the presence of
gross abnormalities ● made by placing a drop of 0.85% saline on a glass slide
and using a wooden stick or another mixing tool.
MICROSCOPIC EXAMINATION ● The resulting slide should be thin enough for
newspaper print to be read.
● To detect the presence of parasites in a stool specimen ● 22-mm square cover slip is placed on the slide
● The microscopic examination of stool for ova and
parasites involves three distinct procedures: DIRECT IODINE WET PREPARATION
○ Direct wet preparations
○ Concentrated stool ● Made to enhance the detail of protozoan cysts.
○ Permanently stained smear ● Is made as using a drop of iodine (Lugol’s
● or D’Antoni’s formula)
● Since iodine kills any trophozoites present, it is
MICROSCOPE CONSIDERATIONS
recommended to use saline and iodine wet mount side
by side
● Microscope - is the important piece of equipment in the
parasitology laboratory.
● Good features and good optics are critical to the CONCENTRATION METHODS
successful detection of parasites.
● Size is an important diagnostic feature in parasitology, it ● Concentration techniques provide the ability to detect
is necessary for the microscope to have an ocular small numbers of parasites that might not be detected
micrometer. using direct wet preparations.
● Diagnostic stages of parasites detected microscopically ● The purpose of concentration is to aggregate parasites
are measured in units called microns. present into a small volume of the sample and to
remove as much debris as possible that might hinder
the laboratory technician’s ability to see any parasites
present clearly.
● It can be formed on fresh or preserved stool specimens.
● Allows the laboratory technician to detect protozoan
cysts, oocysts, helminth eggs, and larvae.
● Protozoan trophozoites do not usually survive the
procedure.
● Two types of concentration methods:
○ Sedimentation
FIGURE 2-1: OCULAR MICROMETER ○ Flotation’
● These techniques use differences in specific gravity and
Ocular micrometer is a disk that is inserted into the eyepiece of centrifugation to separate the parasites from the fecal
the microscope. debris and increase their recovery.

DIRECT WET PREPARATION SEDIMENTATION TECHNIQUES

● Also known as a direct wet mount ● Parasites are concentrated in the sediment of the tube
● Defined as a slide made by mixing a small portion of following centrifugation and the sediment is examined
unfixed stool (stool with no added preservatives) with microscopically.

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CLINICAL PARASITOLOGY
LECTURER: MISS DEBBIE KATES SANTISTEBAN, RMT I Topic 2
______________________________________________________________________
● Formalin–Ethyl Acetate Sedimentation Procedure -
is the most widely used sedimentation technique.
APPEARANCE OF MICROSPORIDIA ON MODIFIED
TRICHROME STAIN
FLOTATION

● The parasites are less dense than the solutions used

and, during centrifugation, they float to the surface.
● Material from the surface film is examined
microscopically.
● Zinc Flotation Technique - is the most widely used
technique.

PERMANENT STAINS
CELLOPHANE TAPE PREPARATION
● The final procedure in the O&P examination is the
preparation and examination of a permanent stained ● It is the specimen of choice for the detection of
smear. Enterobius vermicularis (pinworm) eggs.
● These slides are considered permanent because after ● Adult female pinworms may also be seen.
staining, they are typically cover-slipped and sealed, ● Recovery of Taenia species eggs
thus allowing them to remain intact long term.
● Critical portion since it is designed to confirm the
presence of protozoa cysts and/or trophozoites. BLOOD
● Two common stains:
● Systemic or blood-borne parasitic infections are
TRICHROME (WHEATLEY MODIFICATION) diagnosed by demonstrating the diagnostic stage(s) of
the responsible parasite(s).
● The most widely used permanent stain ● A thick and thin blood smear is prepared.
● It uses reagents with a relatively long shelf life and the ● Giemsa stain is used.
procedure is easy. ● E.g. Leishmania donovani and Trypanosoma spp.,
● Shows distinct color differences among the cytoplasmic Plasmodium and Babesia spp., and microfilariae
and nuclear structures

IRON HEMATOXYLIN

● May be used instead of the trichrome technique

● Considered to be time-consuming yet reveals excellent
morphology of the intestinal protozoa.

APPEARANCE OF SELECT PROTOZOAN STRUCTURES

AND BACKGROUND MATERIAL ON IRON HEMATOXYLIN
STAIN

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