Lab diagnosis of Trematodes and

Nematodes
 GENERAL CHARATRISTICS
• Helminths are elongated fl at or round worm
like parasites

• They are eukaryotic multicellular and

bilaterally symmetrical
• They belongs to two phyla

• Phylum Platyhelminths (flat worms)

• Phylum: Nemathelminths
 Phylum Platyhelminths (flat worms)

• It includes three classes:

• Class: Cestoidea (tapeworms)

• Class: Trematodea (flukes
• Class: Monogenea (ectoparasite of fishes,
don’t infect man)
 MORPHOLOGY
• Helminths exist in three morphological forms
—
1. Adult form (or theworm)
2. Larval form
3. Eggs
 Larvae form
 In cestodes: Cysticercus, hydatid cyst,
cysticercoid, coracidium, procercoid and
plerocercoid forms.

 In trematodes: Cercaria, metacercaria, redia,

miracidium and sporocyst.

 In nematodes: Rhabditiform larva, filariform

larva and microfilaria.
 Eggs
Oviparous (laying egg) -
EAT

• Enterobius
• Ascaris
• Ancylostoma
• Trichuris
 Viviparous
Producing larvae

• Trichinella
• Wuchereria
• Bulgia
• Dracunculus
 Ovoviviparous
• Laying eggs containing fully formed larvae

• Strongyloides
 Specimen Collection
Specimen
• Stool specimens should be collected in a wide-
mouthed, clean, leak–proof, screw capped
containers.

Specimens other than stool

• Scotch tape method- for Enterobius vermiculais
(pin worm or thread worm)
• Perianal swabs (cellophane tape or NIH
swab):
• Useful for detecting Eggs of Enterobius
vermicularis deposited on the surface of
perianal skin.
• It is also used for eggs of Schistosoma mansoni
and Taenia species
Entero test/ Duodenal capsule test
• The entero-test is useful for parasites such as
protozoa, Strongyloides stercoralis
 Timing

• Specimen should be collected before starting

antiparasitic drugs and closer to the onset of
symptoms
Frequency

• At least three stool specimens collected on

alternate days are adequate to make the
diagnosis of intestinal parasitic diseases
 When to examine
• Liquid stool specimens should be examined
within 15–30 minutes after collection
• Semisolid stools within 1 hour after collection
• Formed stools up to 24 hours after collection.
 Preservatives
• Formalin Two concentrations are commonly
used: 5%, which is recommended for
preservation of protozoan cysts, and 10%, which
is recommended for helminth eggs and larvae.

• Sodium acetate formalin (SAF)

• Merthiolate-iodine formalin (MIF)
• Schaudinn’s fluid
• Polyvinyl alcohol (PVA)
 Macroscopic Examination

• Consistency – formed , Semisolid stools, liquid

• Color – yellow, green, Dark red stool
• Ordor - Frothy pale offensive stool found in
giardiasis
• Presence of blood
• Presence of mucus
• Presence of Adult worms like round worm, thread
worm or segments of tapeworm.
 Microscopic Examination
Direct wet mount (saline and iodine mount)
Drops of saline and Lugol’s iodine are placed on
two corners of a slide

Small amount of feces is mixed by a stick to

form a uniform smooth suspension.

Cover slip is placed on the mount and examined

under low power objective (10X); followed by
high power objective (40X).
 Structures can be visualized by
microscopic examination of stool specimen

Findings
• yeast cells, bacteria, epithelial cells, fat
globules, Pus cells , red blood cells, Charcot
Leyden crystals (diamond shaped), ova , cyst,
trophozoites
Elliptical with sharp Elliptical with sharp Oval to almost spherical;
terminal spine lateral spine rudimentary lateral knob
 Eggs are oval, bile
stained,
unembryonated
and operculated

Fasciola hepatica
 Barrel shaped
(with mucus plugs
at the ends)

Trichuris trichiura
 planoconvex, flattened
along one side, Double
layered egg shell containing a
larva inside,

Egg of Enterobius vermicularis

 Saline mount

• Advantages
• Useful in the detection of trophozoites and
cysts of protozoa and eggs and larvae of
helminths
• Motility of trophozoites and larvae can be
demonstrated in acute infection
• Bile staining property can be appreciated—
bile stained eggs appear golden brown and
non bile-stained eggs appear colorless.
Bile stained Non - Bile stained
 How to remember
Bile stained egg
FAT(3)
• F- Fasciola hepatica
• A- Ascaris lumbricoid
• T- Trichuris trichura,Taenia solium & Taenia
saginata
 Non bile stained egg
NEHA

• N- Necator americanus
• E- Enterobius vermicularis
• H- Hymenolepis nana
• A - Ancylostoma duodenale
 Iodine mount

Advantages
• Nuclear details of cysts, helminthic eggs and
larvae are better visualized
Disadvantages
• Iodine immobilizes and kills parasites, hence
motility of the protozoan, trophozoites and
helminthic larvae cannot be appreciated.

• Bile staining property cannot be appreciated.

 Types of iodine stains

• Lugol’s iodine

• D’Antoni’s iodine

• Dobeil’s iodine
 Concentration Techniques
• If the parasite output is low in feces (egg,
cysts, trophozoites and larvae) and direct
examination may not be able to detect
the parasites, then the stool specimens
need to be concentrated.
 Sedimentation method
• The feces is suspended in a solution with low
specific gravity, so that the eggs and cysts get
sedimented at the bottom
 Sedimentation techniques

• Formalin-ether concentration technique

• Formalin-ethyl acetate concentration

technique

• Formalin-acetone sedimentation technique

 Advantages

• The sensitivity of detecting the ova or cysts

increases by 8–10 folds
• The size and shape of the parasitic structures are
maintained
• Inexpensive, easy to perform
• Fecal odor is removed
• As formalin kills the fecal parasites , no risk
of acquiring laboratory acquired infection.
 Disadvantages

• Trophozoite forms are killed and hence not

detected in this method.
 Floatation method
• The feces is suspended in a solution of high
specific gravity, so that parasitic eggs and cysts
float up and get concentrated at the surface.
 Floatation method
• Saturated salt flotation technique

• Zinc sulphate flotation concentration-

• Sugar floatation technique- cryptosporidia

 Disadvantages

• Flotation technique is not useful for heavier

eggs that do not float in the salt solution
Eggs that float in saturated salt solution

FEATHer
• F – fertilized egg of ascaris
• E- Enterobius vermicularis
• A- Ancylostoma duodenale
• T- Trichuris trichura
• H- Hymenolepis nana
 Eggs that do not float in saturated salt
solution
STUF
S- Strongyloides stercoralis
T - Taenia
U- Unfertilized egg of Ascaris
F – Fasciola hepatica
 Egg Counting (Egg quantifi cation)
Methods

• Direct smear counting method of beaver

• Kato’s cellophane tape

• Stoll’s method or dilution egg counting method

• McMaster’s egg counting

 Stool culture

Hook worm and Strongyloides stercoralis

• Harada Mori filter paper tube method

• Petridish (slant culture) technique
• Baermann funnel technique
• Charcoal culture method
• Agar Plate technique
Identify the eggs