Diagnostic Methods in
Parasitology
[Link]. Baraa abdulsalam Alattabi
INTRODUCTION
Laboratory procedures play an important role in the
diagnosis of parasitic infections, both for confirmation of
clinical suspicion and for identifying unsuspected infections.
The principles of laboratory diagnosis are the same as in
bacterial and viral infections, but the relative importance of
the different methods varies greatly.
• While isolation of the infecting agent and detection of
specific antibodies are the major methods in bacteriology
and virology, they are of much less importance in
parasitology than morphological identification of the
parasite by microscopy
• Compared to bacteria and viruses, parasites are very large and
possess distinctive shape and structure, which enables their specific
diagnosis on morphological grounds.
• Due to their complex antigenic structure and extensive
cross-reactions, serological diagnosis is of limited value in parasitic
infections.
• Although many pathogenic parasites can be grown in laboratory
cultures, this method is not suitable for routine diagnosis because of
its relative insensitivity and the delay involved.
:Diagnosis of parasitic diseases depends on
A- history and Clinical features : like fever, pain
B- Laboratory diagnosis include:
1-Microscopy 2-Culture
3- Serological test 4-Skin test
5-Molecular method 6- Animal inoculation
7-Xenodiagnosis 8-Imaging
9- Hematology
Morphological diagnosis of parasites
:consists of two steps
(1) detection of the parasite or its parts in clinical samples
(2) its identification.
l. Detection depends on collection of the appropriate
samples and their examination by suitable techniques.
2. Identification requires adequate skill and expertise
in recognizing the parasite in its various stages and its
differentiation from morphologically similar artifacts.
A description of the common diagnostic techniques in
parasitology is given here.
EXAMINATION OF STOOL
Collection of Fresh Stool Specimen
• All stool specimens should be collected in a suitable, clean, wide mouthed container like a
plastic container with a light-fitting lid, waxed cardboard box.
• All fresh specimens should be handled carefully because each specimen represents a
potential source of infectious material.
• The specimen should not be contaminated with water, urine, or disinfectants. Liquid
stools should be examined or preserved within 30 minutes of passage. Soft stools should be
examined or preserved within 1 hour of passage and formed stool should be examined or
preserved with in 24 hours of passage.
• Normally passed stools are preferable.
• Examination of fresh specimens is necessary for observing motility of protozoan parasites.
• Stool should be examined for its consistency, color, odor and presence of blood or
mucus.
• in some instances, parasites may be seen on gross inspection, as in the case of
roundworm, pinwonn , or tapeworm proglottids.
Microscopic Examination
• the microscope should be equipped with a
micrometer eyepiece, as it is often essential to
measure the size of parasites. For example, the
differentiation between cysts of the pathogenic
Entamoeba histolytica and the nonpathogenic E.
hartmanni is based entirely on their sizes.
Wet Mounts
• Unstained [Link]: The unstained wet film is the
standard preparation and is made by emulsifying a small
quantity of stool in a drop of (0.85%) saline placed on a
slide and applying a coverslip (22 mm x 22 mm) on top,
avoiding air bubbles. Any suspicious object may then be
examined with the high-power objective.
• Wet saline mounts: Wet saline mounts are particularly
useful for detecting live motile trophozoites of E.
histolytica, Balantidium coli and Giardia lamblia. Eggs of
helminths are also readily seen. Rhabditiform larvae of
Strongyloides stercoralis are detected in freshly passed
stool.
Iodine staining: Iodine staining or wet mounts is another
standard method of examination. Either Lugol's iodine
diluted (5 g iodine, 10 g potassium iodide and 100 mL of
distilled water). Iodine helps to confirm the identify of
cysts, as it prominently stains the glycogen vacuoles and
nuclei. Protozoan cyst stained with iodine show yellow
gold cytoplasm, brown glycogen material and pale
refractile nuclei.
PROCEDURE
1. Prepare a direct smear of the specimen by mixing a small portion (2 mg)
of feces with a drop of sterile physiological (0.85%) saline on a clean
glass slide.
2. Place a coverslip over the sample and examine the wet mount
preparation for the presence of motile protozoa. The organisms are very pale
and transparent and are more easily observed under low light intensity.
3. Once the wet mount has been thoroughly examined, a drop of Lugol’s
Iodine (working solution) can be placed at the edge of the coverslip, or a new
mount can be prepared using iodine alone. The prepared slide can be sealed
if desired.
4. Examine the slide for the presence of brown parasitic structures.
Interpretation of Results
The majority of parasitic organisms and cysts adopt a brown coloration when
stained with Lugol’s Iodine. When stained correctly, protozoan cysts contain
yellow-gold cytoplasm, brown glycogen material, and paler refractile nuclei.
Trophozoite of E. histoyltica cyst of E. histoyltica
Trophozoite of G.
lamblia
: Modified Ziehl-Neelsen ( acid-fast) stain
1) Faecal smears are made either directly from the stool sample or from the
concentration deposit.
2) Allow to air dry.
3) Fix in methanol for 3 minutes.
4) Stain with strong carbol fuchsin for 15-20 minutes.
5) Rinse thoroughly in tap water.
6) Decolorize in acid alcohol (1% HCl in methanol) for 15-20 seconds.
7) Rinse thoroughly in tap water.
8) Counterstain with 0.4% malachite green (or methylene blue) for 30-60
seconds.
9) Rinse thoroughly and air dry.
10) Examine using x40 and x100 objectives.
Results
Oocysts are seen as acid fast (bright pink) oval to round bodies (4 to 6 mm in),
diameter), surrounded by a colourless halo.
Modified AFB
stain showing oocyst of [Link]
IMMUNOLOGICAL DIAGNOSIS
Serology
• diagnosis is attempted by serological demonstration of parasitic antigens in blood,
tissues, or secretions of suspected patients. Virtually, all types of serological reactions
have been used. However, serodiagnosis in parasitic infections has only limited value
due to various factors:
• Parasites are complex antigenically and exhibit wide range of cross-reactions, so that
serological tests are not sufficiently specific.
• Another difficulty is indistinguishing between past and current infections. This has
been solved partly by looking for immunoglobulin M (IgM) antibody, as in amebiasis
and toxoplasmosis.
In general, indirect hemagglutination (IHA),
enzyme linked immunosorbent assay
(ELISA) indirect immunofluorescence (IF),
direct agglutination test (DAT) and
complement fixation test (CFT) are
moderately sensitive; and simple
precipitation in gel and coated particle
agglutination tests are least sensitive.
Serology:
has not been very useful in the diagnosis
of individual cases, but has been valuable
as a screening method in epidemiological
surveys. However, in some infections
where parasites are seldom demonstrable
in patients, for example in toxoplasmosis
and hydatidosis, serology is of great help.
Laboratory Diagnosis of Blood
and tissue parasite
MOLECULAR METHODS
In parasitology, routine laboratory diagnosis involves conventional
methods, such as optical microscopy, used for the morphological
identification of parasites. Currently, molecular biology techniques
are increasingly used to diagnose parasite structures in order to
enhance the identification and characterization of parasites.
• nucleic acid-based diagnostic tests are mainly available in
specialized or reference centers. Nucleic acid probes and
amplification techniques such as polymerase chain reaction (PCR)
and multiplex PCR, western blot and deoxyribonucleic acid (DNA)
hybridization techniques are increasingly used to detect parasites
in specimens of blood, stool, or tissue from patients.
• These test are useful for detecting subspecies or strain level
identification which is important for epidemiological
studies and are also used to detect parasitic drug resistance.
MOLECULAR METHODS
• Deoxyribonucleic acid probe is a highly sensitive
method
for the diagnosis of malaria. It can detect even less than
10 parasite/ μL of blood.
• B1 gene of T. gondii can be detected by PCR of the
amniotic fluid in case of congenital toxoplasmosis. PCR
have been developed for detection of filarial DNA from
patients blood.
If parasite cannot be identified by microscopy,
amplification of SS ( 18S )ribonucleic acid (RNA) by
PCR is recommended.
Procedure of molecular detection
1. Clinical Sampling
The first step is to collect, prepare, concentrate and sort the samples.
2. Nucleic Acid Extraction
During the second step, the nucleic acid is isolated from the sample to be
studied. No matter what downstream analysis is performed (RT-PCR or
sequencing), without removing contaminants or inhibitors from the sample, a
good quality analysis is not possible.
3. Reaction Setup
The third step is the configuration of the reaction. Polymerase chain reaction
(PCR) is a fundamental technique in molecular biology due to its exceptional
ability to amplify and precisely quantify extremely low levels of nucleic acids.
Many steps can be automated with ready-to-use kits and automated liquid
handlers.
Procedure of molecular detection
4. Analyze
Finally, during the 4th step, the analysis of the prepared nucleic acid
can be carried out. Nucleic acid detection techniques are currently
based on fluorescence, colorimetry or chemiluminescence.
As an example, you can use: capillary electrophoresis systems, gel
imagers, real-time PCR instruments or other nucleic acid analyzers.