DIAGNOSTIC METHODS IN

PARASITOLOGY
EX A. Collection of stool Specimen.
A B. Macroscopic examination: Stool should be examined macroscopically for its
MI consistency, color, odor and presence of blood and mucous. Sometime
NA helminthes worm like roundworm, hookworm and tapeworm proglottids can
TI be observed.
O C. Microscopic examination: Microscopic examination of stool contribute for
N the findings of Charcot-Leyden Crystals and cellular exaudates such as pus
OF cells, RBCs and macrophages. It is also used for the detection of protozoa
ST (cysts & trophozoites), helminthes (ova and larva).
O The methods includes: Wet mounts, thick smears, Permanent stained
OL preparation and various concentration methods.
1. Wet mounts:
a. Wet saline mount: It is made by emulsifying a small quantity of stool with
a drop of(0.85%) saline. It is used for detecting live trophozoites (motility). Ova
and larvae of different parasites can be observed.
b. Iodine staining: Lugols iodine (5g iodine, 10g potassium iodide and 100ml
distilled water) helps to confirm the identity of cysts as it stains the glycogen
vacuoles and nuclei. Motility cannot be observed by iodine wet mount.
c. Eosin staining: 1% Eosin solution can be used for staining the trophozoites
and cysts, helminthes ova and larvae.
2. Thick smear:
Not useful for routine examination. Used for the survey for the intestinal
helminths egg. Kato thick smear technique is used however not useful for the
diagnosis of protozoa or helminths larvae.

3. Permanent Stained Smear:

2 methods commonly used are Iron-hematoxylin stain and Wheatley’s
trichrome stain. They are used for the confirmation of intestinal protozoan, both
trophozoites and cysts. Helminths eggs and larvae take up too much stain and
usually cannot be identified.
Other methods like Modified Ziehl-Neelsen (acid-fast) stain (Hot method) is
used for the detection of Oocysts of Cryptosporidium and Isospora. 5% Sulphuric
acid is used as a decolorizer.
4. Concentration methods:
When the parasites are scanty in stools, routine microscopic examination may
fail to detect them. In such case concentration methods are used. It can be done for
freshly passed and preserved stools. 2 methods are commonly used. They are
Floatation methods and Sedimentation methods. In floatation methods stools
sample is suspended in a solution of high specific gravity so that the parasitic egg
and cysts float up whereas in sedimentation method stool is suspended in a solution
with low specific gravity so that the egg and cysts sedimented at the bottom.
a. Floatation methods:
i. Saturated salt solution technique:
Procedure:
- 1g of the stool is emulsified with 2ml saturated salt solution of NaCl
(specific gravity of 1.2) in a flat bottom tube.
- The tube is then filled completely to the brim with the salt solution.
- A slide is then placed in the container so that it contact with the surface of
the solution without any air bubbles. After 20-30 minutes the slide is removed and
observed under the microscope.
- This allows the detection of eggs of common nematodes such as
roundworm, hookworms and whipworm but is not applicable for the eggs of
tapeworm, unfertilized egg of Ascaris, egg of trematodes and protozoan cysts.
ii. Zinc Sulphate Centrifugal floatation:
Procedure:
- Make a fine suspension of about 1g of stool in 10ml of water and strain through gauze to
remove coarse particles.
- Collect the liquid in a small test tube and centrifuge for 1 min at 2500 rpm/min. Pour off
the supernatant, add water, resuspend and centrifuge in the same manner repeating the process till
the supernatant is clear.
- Pour off the clear supernatant, add a small quantity of zinc sulphate solution (specific
gravity 1.18 – 1.2), and resuspend the sediment well.
- Add zinc sulphate solution to a little below the brim and centrifuge at 2,500 rpm/min for
1 min.
- Take sample carefully from the surface using a wire loop, transfer to slide and examined
under the microscope.
This technique is useful for protozoan cysts and egg of nematodes and small tapeworms
but it does not detect unfertilized roundworm eggs, nematodes larvae and eggs of most
trematodes and large tapeworms.
b. Sedimentation methods:
i. Formal-ether sedimentation technique:
- It is the most widely used sedimentation method.
Procedure:
- Emulsify 1-2g stool in 10ml of water and strained through the gauge piece. Take the filtrate in
15ml of tube and spin at 2500rpm/min for 2-3mins.
- The supernatant is discarded and the sediment is resuspended in 10ml of physiological saline
and centrifuged again.
- Discard the supernatant. Add 7ml of 10% formalin-saline, mix well and let it stand for 10 mins.
- Add 3ml of ether or Ethyl acetate and shake well. Spin at 2500 rpm/min for 2-3mins. Four
layers will form-ether layer, a plug of derbis/fat, the formalin-saline layer and the sediment at
the bottom.
- Carefully discard the above 3 layers and suspend the sediment in a few drop of fluid and
examine wet and iodine preparation.
This method is useful for all helminthes egg and protozoan cysts.
EG
G
CO Semi quantitative method assessment of the worm burden can be made
UNby estimating the number of eggs passed in stools. This is done by egg count.
TI However, these are at the best approximations and only a rough indications of
NGworm. Egg count help to classify the helminths infections as heavy, moderate
and light. Following are the different methods used in egg count.
ME
TH
O
DS
1. The standard wet mount gives rough indication of the number of egg. 1-2mg of the
stool is used for the smear preparation and if all the eggs are counted then the
number of egg per gm of stool can be assessed.
2. The modified Kato thick smear technique using 50mg of stool cleared by
glycerine-soaked cellophane coverslip can be used for egg counting.
3. MacMaster’s egg counting chamber can also be used. In this method eggs in 20 mg
of stool is concentrated by salt floatation on the square grid on the roof of the
chamber which can be counted.
4. Stoll’s dilution technique: 4 gms of stool is mixed thoroughly with 56ml of N/10
NaOH using beads in a rubber stoppered glass tube. Exactly 0.15ml of the
emulsion is removed by a measuring pipette and placed in the slide and all the eggs
are counted. The number of eggs per gm of the stool is obtained by multiplying the
count of two such preparations by 100. The figure requires to be corrected for the
consistency of feces by multiplying 1.5 for hard formed stool, by 2 for mushy
formed stool, by 3 for loose stools, by 4 for liquid stool and by 5 for watery stool.
5. Scotch Cellulose Tape Method: In this method a piece of transparent
adhesive tape is pressed firmly against perianal skin and the adhesive
surface of the tape is spread on a glass slide. The slide is then placed
under the microscope and observed for the parasitic eggs. A drop of
toluene or xylol may be placed between the tape and the slide to clear the
preparation.
 F Fecal culture is not that much of importance for routine
E diagnosis but is important of the species differentiation. Following
C are some of the culture methods applied.
A 1. Haroda and Mori Filter Paper Strip Culture: This method uses a
L strip of filter paper on which the feces is smeared in the middle third.
C The paper strip are kept in the centrifuged tube with water at the
U bottom in which the strip dips. The tubes are incubated at room
temperature for 7-10 days at dark. During this period the larvae
LT
develops and fall in the water at the bottom from which they can be
U collected. This method can be applied for the detection of
R Hookworm, Strongyloides.
E
2. Agar plate culture for Strongyloides: Approx. 2g of stool is inoculated into agar
plates and the plates are sealed with tape. Then these plates are incubated at
room temperature for 2 days. In positive cases larvae will crawl over the agar
making visible tracks over it. For confirmation the larvae the plates are
examined microscopically.
3. Boeck and Drbohlav’s method (Blood serum and Egg albumin) B.D media and
NIH (National Institute of Health) media are used to culture E. histolytica.
EX
AMI
NAT Blood examination is the routine diagnostic method in malaria,
filariasis, African trypanosomiasis and bebesiosis. It is sometime positive in
IONChaga’s disease and rarely in Kala-azar and toxoplasmosis. Blood
OF examination is done in the following ways.
BLO
OD
1. Examination of Malarial Parasites.
The standard diagnostic method in malaria is the examination of stained blood stain-both thin
and thick smears.
a. Collection of the blood.
b. Thin smear:
- A small drop (10-15µl) of blood is spread in a clean grease-free slide with a spreader
to give a uniform smear, ideally a single cell smear.
- The slide is then air dried and then stained with Giemsa or Leishman stain.
- For Giemsa staining the smear is fixed with methanol for 3-5min. After drying
Giemsa stain is applied for 30-45mins. The slide is then washed with tap water, dried and
examined under the oil immersion objective.
- For Leishman stain prior fixation is not necessary as stain is an alcoholic solution
which fixes as it stains. Stain is applied for 1-2 mins and diluted with twice its volume of
buffered water and kept for 10-15mins. Then smear is then washed, dried and then examined
under the oil immersion.
c. Thick smear:
- Since the large volume of blood is used, it allow the parasite to be detected even with
low parasitemia. It is about 30 times more sensitive than the thin smear and can detect about 20
parasites/µl of blood.
- A big drop of blood (20-30µl) of blood is kept in a clean grease-free side and spread with
the corner of another clean slide to form a uniform thick smear of about 1cm 2 .
-
The smear is then air dried and then dehemoglobinized before staining.
-
Giemsa staining or Leishman staining can be performed. Wright’s staining can also be
performed.
-
Smear is then observed under the oil immersion field for the presence of trophozoites,
schizonts and gametocytes of Plasmodium species.
2. Examination for Microfilaria:
a. Collection of blood.
b. Wet mount:
- 2 to 3 drops of blood are kept in a clean side and mixed with 2 drops of
water to lyse the RBCs.
- The preparation is then covered with coverslip and sealed.
- It is then observed under the microscope at low power for the motile
microfilariae.
c. Stained smear:
- Thick smear is prepared as of Malaria and stained.
- Stained smear has an advantage that the morphology of microfilariae can be
studied and species identification can be done.
3. Examination of Sputum:
- Collection of the specimen.
- Sputum examination is commonly done for the demonstration of ova of
Paragonimus westermani and sometime for the detection of trophozoites of E.
histolytica. Rarely the larval stage of hookworm, A. lumbricoides and S. stercoralis
can detected. Direct normal saline mount is done from the sputum sample.
4. Urine or Body fluid examination:
- Urine examination is performed for the detection of eggs of Schistosoma and
Trichomonas vaginalis. Microfilaria may be detected from chylous urine in lymphatic filariasis.
- Large volume of the urine is collected and allowed to settle down.
- Centifuge is done from the settled part of the urine and microscopy (wet mount) is
performed from the deposit.
5. Tissue biopsy:
- Tissue biopsy and Fine Needle Aspirations are taken for the diagnosis of
Trypanosomiasis or Leishmaniasis.
- Wet mount preparation of lymphnode aspiration and chancre fluid are used
for the rapid method for the demonstration of trypanosomes.
- Biopsy from liver, spleen, bone marrow and lymphnodes are taken in
visceral leishmaniasis for demonstration of trypanosomes.
- Adult filarial worms can be sometimes be found in section of biopsied
lymphnode.
6. Muscle biopsy:
- Spiral larva of Trichinella spiralis, larva of Taenia (Cysticercus) and
amastigote form of Trypanosoma cruzi can be demonstrated in skeletal muscle of
biopsy.

7. Urogenital specimen:
- Vaginal and uretheral discharge can be used for the detection of
Trichomonas vaginalis.
- Specimen should be collected in a 0.85% NaCl and sent to lab as soon as
possible for the detection of motile trophozoites.
8. Immunological diagnosis:
- Several serological diagnosis have been developed for the detection of
antigen and antibodies to the parasites.
- In general Indirect hemagglutination (IHA), Enzyme Linked
Immunosorbent Assay (ELISA), Counter Immune Electrophoresis (IF), Direct
agglutination test (DAT), Complement fixation test (CFT) are used for the detection
of different parasitic infection.
9. Skin test:
- Casoni’s test: It is used for the diagnosis of hydatid disease.
- Leishmanin test: This test is used to measured the delayed hypersensitivity
to Leishmaniasis.
- Failey’s test: Used for the detection of Schistosomiasis.
10. Molecular methods:
- Nucleic acid probes and amplification technique like polymerase chain
reaction (PCR) and multiplex PCR, and DNA hybridization technique are used for
the detection of parasite in specimens.
 It is an immediate hypersensitivity skin test which was introduced
by Casoni in 1911. Antigen from the Casoni test is sterile hydatid fluid
drawn from unilocular hydatid cysts from sheep, pig, cattle or man. The
fluid is filtered, tested for sterility and stored in sealed ampoules under
refrigeration.
For the test 0.2 ml of the antigen is injected intradermally in one
arm. For control an equal amount of sterile normal saline is injected
intradermally on the other arm. The controls fades almost immediately
while the tested sites in positive case develops a large wheal measuring
5cm or more in diameter with multiple pseudopodia within 30 mins. The
test has low sensitivity (55-70%) and gives false positive reactions in
patients suffering from other cestode infection.

CASONI TEST:
 This test performed and a non specific immunological
test for the detection of kala-azar. A drop of full strength
(40%) formalin is added to 1ml of serum. A positive test is
indicated by the rapid and complete coagulation of the
serum. The test indicates greatly increased serum gamma
globulin and thus is non-specific. This test is widely used
and is not positive till the disease is of a three month
duration.

ALDEHYDE TEST: