BASICS

OF
DIAGNOSTIC
PARASITOLOGY
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PARASITE DISEASES TRICHOMONIASIS

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LABORATORY
DIAGNOSIS
“To see is to believe…”

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 INTRODUCTION
Most parasitic diseases cannot be
established based on clinical signs and
symptoms alone.
A proper laboratory examination confirms
a suspected parasitic condition.

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 IMPORTANCE of LAB DIAGNOSIS

CONFIRM RULE OUT AID/ HELP

A clinical impression Differential The choice of proper
that the condition diagnoses treatment and
has a parasitic monitor its
nature effectiveness

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DIAGNOSIS OF PARASITIC INFECTION
DEMONSTRATION OF PARASITE
COMPONENTS
Adult eggs, larvae, cysts, oocysts,
trophozoites and antigen

DETECTION OF HOST IMMUNE

RESPONSE
Antibodies

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 https://vle.upm.edu.ph/pluginfile.php/204414/mod_resource/content/1/WHO%20bench%20aids%20for%2

6 0parasitology%20diagnosis.pdf
 SAMPLE COLLECTION
- Most common method of diagnosis of intestinal
parasites is through demonstration of parasite
component in the stool
- Best collected in clean, wide-mouthed containers

https://www.hensomed.com/products/stool-container-60ml/
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 FACTORS TO CONSIDER:
- Drug intake (antacids, anti-diarrheals, laxatives,
barium, bismuth, antibiotics)
- Amount of stool to be collected: routinely a thumb
sized formed stool or 5 - 6 tbsp of watery stool
- Contamination
- Age of stool: submit within 30 mins to 1 hour
- Delay in examination
- Temporary storage: 3-5c is acceptable; never
freeze!

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 STOOL PRESERVATIVES:
FORMALIN
● All purpose fixative
● 5% concentration for protozoan
cysts
● 10% concentration for helminth
eggs and larvae
● May add Sodium phosphate to
enhance morphological
preservation
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SCHAUDINN’S SOLUTION
● Used to preserve fresh stool in
preparation for staining stool
smears
● Contains Mercuric Chloride
● Toxic to humans
● Disposal problems
 STOOL PRESERVATIVES:
POLYVINYL ALCOHOL MERTHIOLATE-IODINE-FORMALIN
● Plastic resin ● Contains Merthiolate
● Serves to adhere stool sample ● Iodine as staining component,
into a slide and should be freshly prepared
● Incorporated with Schaudinn’s ● Formalin as preservative
solution ● For intestinal protozoans,
● Preservation of cysts and helminth eggs and larvae
trophozoites for permanent
smear

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STOOL PRESERVATIVES:
SODIUM ACETATE-ACETIC
ACID-FORMALIN
● No Mercuric Chloride
● Images preserved lose its
sharpness
● Stable and long shelf life

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MICROSCOPIC
EXAMINATION
 Microscopic Examination
● Direct Fecal Smear
● Kato Thick Smear
● Concentration Techniques
○ Sedimentation Procedures
i. Acid Ether Concentration Technique (AECT)
ii. Formalin-Ether/Ethyl Acetate Concentration Technique (FECT)
○ Floatation Procedures
i. Zinc Sulfate Flotation
ii. Brine Flotation
iii. Sheather’s Sugar Flotation
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Direct Fecal Smear

● Routine Stool Examination

● Detects Helminth eggs and larvae
● Trophozoites appear very pale and transparent
● Protozoan cyst can be detected (Lugol’s Iodine
Solution)
● Disadvantage: Light infections may not be
detected
 Kato Thick Smear (Kato-Katz)

Materials and reagents

Simple, cost-effective
1.Wooden applicator sticks.
2.Screen (stainless steel, nylon or plastic: 60-105 µm mesh) (Fig. 1). ●
3.Template (stainless steel, plastic, or cardboard) (Fig. 1). A hole of 9 mm on a
1 mm thick template will deliver about 50 mg of faeces; a hole of 6 mm on a
● Used in mass stool examinations
1.5 mm thick template, 41.7 mg; and a hole of 6.5 mm on a 0.5 mm thick
template, 20 mg. The same size of templates should always be used to ensure
● Detects eggs with thick shells (e.g. Ascaris, Trichuris)
repeatability and comparability of prevalence and intensity data. ● Disadvantages
4.Spatula (plastic) (Fig. 1).
5.Microscope slides (75 x 25 mm). ○ May be unable to detect eggs with thin shells
6.Hydrophilic cellophane (40–50 µm thick, strips 25 x 30 or 25 x 35 mm in
size). (e.g. Hookworm)
○ Limited usefulness in watery stools
7.Flat-bottom jar with lid, forceps and toilet paper or absorbent tissue.
8.Newspaper.

○ Unable to detect protozoan cysts and

9.Glycerol-malachite green (1 mL of 3% aqueous malachite green is added to
100 mL of glycerol and 100 ml of distilled water and mixed well). This solution
is poured onto the cellophane strips in a jar and left for at least 24 hours prior
to use.
trophozoites
 Concentration Techniques
Floatation
Sedimentation Zinc Sulfate
● 33% Zinc Sulfate

Acid Ether Concentration Brine

Technique (AECT) ● Saturated table salt
● Main reagents: 40% HCl, Ether ● No centrifugation
● Recovery of Trichuris, Capillaria, ● Low-cost & simple
Trematode eggs, ● Disadvantage:
● Recommended in examining cat ○ Helminth & Schistosoma
and dog stool material become shrunken.
○ Not useful for
Formalin Ether Concentration operculated eggs (e.g.
Technique (FECT) Clonorchis, Opisthorchis)
● Reagents: 10& formalin, Ether Sheather's Sugar
● Recovery of helminth eggs &
protozoans ● Boiled sugar preserved with
● Better parasite quantity and phenol
morphology preservation than other ● Best for coccidian oocyst (e.g.
preservatives Cryptosporidium, Cyclospora)
● Phase microscope
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STOOL CULTURE
METHODS

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 COPRO CULTURE
● Positive stools are mixed with
moistened stool or granulated
charcoal.
● This simulates environmental
conditions in nature.

PDL - Petri Dish Lid

FCM - Fecal-Charcoal Mixture
MFP - Moist Filter Paper
PD - Petri Dish

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 TEST TUBE CULTURE METHOD
HARADA-MORI
● Positive stool is applied to filter paper and
● placed in a test tube with 7 ml distilled
water
● Filariform larvae will generally move
downwards against the upward capillary
movement of water and can therefore be
recovered from the water at the bottom of
the tube.
● On the other hand, Strongyloides larvae
may instead move upwards and
accumulate at the upper end of the filter
paper strip.

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EGG COUNTING PROCEDURES

● Helps correlate severity of clinical disease

with intensity of infection or worm burden
● Helps assess the efficacy of anti-helminthics
● Procedures:
○ Kato Katz method
○ Stoll Egg count

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 KATO-KATZ METHOD
● Measured amount of stool is sieved thru a
wire mesh and pressed under cellophane
paper soaked in glycerine malachite green
solution
-> all eggs seen in the preparation is
counted
● Useful for assessing intensity of
Schistosoma/Ascaris/Tricuris and
hookworm infections
● Note: well-formed stools yield higher egg
counts

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KATO-KATZ METHOD

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KATO-KATZ METHOD

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 STOLL EGG COUNT
● Reagent: 0.1 N NaOH
○ acts as a stool diluent,
saponifies fat & frees eggs from fecal
debris
● Sensitivity is determined by the
consistency of the stool
○ formed stool can displace
more sodium hydroxide than
liquid stool

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 STAINING OF
STOOL SPECIMEN

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 STAINS
● Staining for amoebae and intestinal ● Kinyoun’s Acid Fast Staining Method
protozoans like Balantidium and ○ For Cryptosporidium, Cyclospora,
Giardia: and Cystoisospora oocysts
○ The oocysts stain pink to red with
1. Iron-Hematoxylin a blue or green background.
2. Trichome
3. Periodic Acid Schiff (PAS)
4. Chlorazol Black E
●

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 PERIANAL SWAB
used to recover eggs of Enterobius vermicularis and
Taenia spp.

Enterobius vermicularis Taenia spp. gravid

● gravid female migrates ● segments can crawl out
out through the anus at of the anus and in the
night time, and deposits process, ova are
eggs on the perianal squeezed out of the
skin. segment and are
deposited on the
perianal skin.

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 CELLULOSE TAPE / SCOTCH TAPE METHOD
used to examine for presence of eggs of Enterobius.

● This is done by sampling the perianal skin using a strip of cellulose

tape attached onto a glass slide.
● The sticky side is applied to the skin.
● The specimen can be collected early in the morning before the
patient has taken a bath or before the patient has washed the
perineum.
● Positive results have also been obtained from swabs collected late
at night when the patient has already slept for several hours.
● Collected specimens are then examined under the microscope for
the presence of eggs or the adult Enterobius

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CELLULOSE TAPE / SCOTCH TAPE METHOD
used to examine for presence of eggs of Enterobius.

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CELLULOSE TAPE / SCOTCH TAPE METHOD
used to examine for presence of eggs of Enterobius.

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EXAMINATION OF
BLOOD

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Finger-prick Blood Samples

1. Wet/Fresh Preparation

-Detects large and motile microfilariae and trypomastigotes

-Species identification is not possible

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Finger-prick Blood Samples

2. Stained Smears

● Thick Films ● Thin Films

> 2-3 three small drops of drop > Thick at one end, thin and feathery at
> Mixed and spread on an area of 2cm in the other end
diameter > Avoid streaks and holes in the film
> Films are thoroughly dried and > Air dried
dehemoglobinized > Fix with methanol
> Staining > Staining

> Used to demonstrate microfilariae > Used in species identification of

malarial parasites
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Finger-prick Blood Samples

Types of Stains

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Finger-prick Blood Samples

3. Capillary Tube Method

-Heparinized capillary tube is used and centrifuged.

-Buffy coat area:

> malaria parasites
> microfilariae
> trypanosomes,
> Babesia

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Finger-prick Blood Samples

3. Capillary Tube Method ● Quantitative Buffy Films

> A capillary tube is precoated with acridine

orange and potassium oxalate.
● Buffy Coat Films
> A cylindrical float is inserted to enlarge the
> The white cell layer can be spread and stained layers.
either with Giemsa or Wright's Stain
> After centrifugation, the tube is read using an
ultraviolet microscope.

> The DNA of the parasites takes up the acridine

orange stain causing fluorescence among the
non-fluorescing red blood cells.

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VENOUS Blood Samples

● Knott’s Concentration ● Membrane Filtration

> Used if low microfilaremia > Also used to recover microfilariae

> Reagent: 10ml of 2% Formalin > 1ml fresh or anticoagulated blood

> Sediment is spread like a thin smear + 10ml distilled water
and stained > Lysed blood through a Swinney
membrane filter

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EXAMINATION OF
SPUTUM

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Best specimen to examine: First morning specimen

Parasites that may be recovered:

1. Migrating larvae of Ascaris Lumbricoides, Strongyloides stercoralis, & hookworms

2. Paragonimus ova
3. Echinococcus granulosus hooklets from
pulmonary hydatid cysts
4. Protozoa such as:
- Entamoeba histolytica trophozoites from pulmonary amebic abscess
- Cryptosporidium parvum oocysts, although very rare
- Non-pathogenic Entamoeba gingivalis and Trichomonas tenax

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Methods:

A. Gross or Macroscopic Examination

1. Consistency:

> Serous, mucoid, purulent, bloody or combination,

should be reported;

2. Color:

> Yellow color may indicate pus

> Greenish tint may indicate Pseudomonas
infection
> Bright red color may indicate a recent
bleeding rust color may indicate breakdown
of hemoglobin. 42
Methods:

B. Microscopic Examination

1. Wet mount using saline or iodine:

protozoan trophozoites

2. Sputum Concentration

> If the sputum is thick or viscous, an equal amount of 3%

NaOH is added, thoroughly mixed, and then centrifuged.

>The supernate is discarded, and the sediment is studied

as a wet mount.

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EXAMINATION OF
URINE

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Best specimen to examine: Urine collected first thing in the morning

Parasites that may be recovered:

1. Trichomonas vaginalis
2. Eggs of Wuchereria bancrofti microfilariae and Schistosoma haematobium.

Method of Urine Sedimentation

1. Thoroughly mix the urine sample with a syringe and fill two 15-mL conical tubes.
2. Centrifuge the tubes at 2000 g for 2 minutes. Alternatively, samples may be left to
sediment for 1 hour.
3. Discard the supernatant by inverting the tubes.
4. Place a few drops of sediment onto a slide, then apply a coverslip.
5. Examine microscopically using the 10x objective.
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Trichomonas vaginalis (left) and Schistosoma haematobium (right)

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 EXAMINATION OF
TISSUE ASPIRATES

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1. Liver

> Most common aspirate submitted for parasite diagnosis in the Philippines
- Margin or the wall of the abscess
- Entamoeba histolytica trophozoites
- Hydatid sand- intact and degenerating scolices of Echinococcus granulosus.

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2. Duodenum

Used to diagnose

1. Giardia lamblia
2. Strongyloides stercoralis

Entero Test/String test

> A capsulated yarn is swallowed by the patient.

> The yarn is expected to reach the duodenum.
> After about 4 hours, the yarn is retrieved and the mucoidal material clinging
to the yarn is examined for the presence of the above mentioned parasites
under the microscope.
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Entero Test/String test

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3. Skin

> Aspirates taken from cutaneous ulcerations-cutaneous leishmaniasis.

> One clinical form of leishmaniasis is cutaneous/Oriental sore.

> Aspirate taken from below the ulcer bed using a sterile needle.
> Smears are prepared and stained with Giemsa when dried.
Positive samples-presence of amastigotes.

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 EXAMINATION OF
CEREBROSPINAL FLUID
(CSF)

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Parasites that may be recovered:

1. Trypomastigotes of Trypanosoma spp

2. Trophozoites of Naegleria
3. Parastrongylus larvae

> Immediate examination of the CSF is required within 20 minutes.

> The CSF must be centrifuged at 7,000 g for 10 minutes
> The supernatant fluid discarded, and the parasites visualized from the
sediment.

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Thank you!

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