Laboratory Diagnosis
of parasitic infections
Professor Sudheer Kher
Learning Objectives
Describe the principles, techniques, standards and recording of
results and interpretation of different methods used in
diagnosis of parasitic diseases
Case diagnosis




History (Age, occupation, residency, previous
infection)
Complaint
Provisional diagnosis
Clinical examination
Confirm the diagnosis
Invesigations
- Laboratory investigations
- Radiology
- Surgical intervention (Exploratory)
DIAGNOSIS
DIRECT
INDIRECT
MOLECULAR
Urine
Stool
Sputum
Biopsy
Blood
Aspirates
IHAT
LAT
IFAT
ELISA
CFT
PCR
DNA probes
Laboratory Diagnosis of Parasitic
Infections
Purpose –
Confirmation of clinical suspicion
Identification of unsuspected infection
Methods same as used in Bacteriology & Virology
but significance of different methods varies.
Isolation least important, morphological
identification very important.
Serology relatively less important
Morphological identification
Examination of faeces –
Gross
Microscopy
Saline mount
Iodine Mount
Thick smears – not commonly used
Permanent stained smears
Iron hematoxylene
Whearley’s trichrome stain
Concentration methods
Floatation techniques
Sedimentation techniques
Morphological identification
Examination of Blood
Thin Smear
Thick smear
Wet mount for microfilaria
Stains used – Leishman / Giemsa
Cultivation of parasites
Culture methods –
Amoeba
Leishmania & Trypanosoma
Malarial parasite
Animal inoculation – Not practical
Xenodiagnosis – Vectors infected experimentally
Immunological diagnosis
Immunological diagnosis
Serology – All tests available
IHA
ELISA
CIEP
IF
CFT
More useful in
Amoebiasis
Leishmaniasis
Malaria
Toxoplasmosis
Trichinosis
Filariasis
Echinococcosis
Skin Tests – Specificity low,
cross reactions common
Casoni’s test
Leishmanin test
BLOOD EXAMINATION
BLOOD FILMS

Thin
Thick

Bld drop
spread
Air dry
Circular motion
Air dry
methyl alcohol
Geimsa
Geimsa
Malaria, Babesia, Filaria, Tryp.
BLOOD EXAMINATION
Buffy coat film
plasma
centrifuge
30 min
WBC (BC)
RBC
Air dry
spread
Fix
Geimsa
Citrated bld
Trypanosoma., L. donovani
STOOL EXAMINATION
MACROSCOPIC
•Consistency
•Colour
•Composition
OTHERS
MICROSCOPIC
Permanent
Direct saline smear
•Culture
•Cellophane tape
Temporary
Iodine smear
Concentration techniques
Floatation
Sedimentation
Saline
Formol ether
Sat saline
Zinc sulphate
STOOL EXAMINATION
MACROSCOPIC EXAMINATION
COLOUR
CONSISTENCY
Pale=Steatorrhea
(Giardia)
-Liquid (Troph)
-Formed (Cyst)
-Semi formed (Cyst)
COMPOSITION
?? Blood ?? Mucus
(dysentry)
Adult PARASITES
*Ascaris worm
*E. vermicularis
*T. saginata
STOOL EXAMINATION
Temporary
Saline smear
Iodine smear
saline
Iodine 1%
Huge number of:
•Eggs
• Protozoal troph. Motility
(Amoeba, flagellates)
Huge number of:
•Cyst morphological details
STOOL EXAMINATION
Scanty infection
Concentration techniques
Sedimentation
• Heavy eggs (Ascaris egg)
Floatation
• Non Operculated eggs
• Operculated eggs (Trematodes)
Trematodes ( S. mansoni)
• Larvae (Strongyloides stercoralis)
Cestode Tape worms
Nematode(Hookworm, Round worms)
• Cysts
• Cysts
URINE EXAMINATION
MACROSCOPIC
MICROSCOPIC
colour
white
Sedimentation
smoky
Chyluria
concentration
Blood
Filaria
S. haematobium
Ether
Acetic acid
Dissolve fat
RBC haemolysis
Microfilaria
Clear ova
Membrane filtration
SPUTUM EXAMINATION
MACROSCOPIC
Appearance
MICROSCOPIC
Concentration
Bloody (Paragonymous)
Rusty brown (Paragonymous)
NaOH
Sputum
Centfifuge
Parasites/sputum
P living in lung
• P. westermani eggs
P migrating in lung
P resulting from rupture of
• St. stercoralis
• Ascaris
• Hookworm (filariform L)
• Hydatid cyst (sand)
• Amoebic abcess (troph)
BIOPSY SPECIMEN
SKIN SNIP
O. volvulus mf
•
•
•
•
Raise skin by needle
Slice by scissors
Put snip in normal saline
Examine
MUSCLE BIOPSY
T. spiralis larvae
RECTAL BIOPSY
Schistosoma egg
Muscle digestion with HCl + pepsin
ASPIRATES EXAMINATION
CSF
Duodenal aspirates
BM aspirates
Cutanoeus ulcer
Edge
INDIRECT IMMUNOLOGICAL
METHODS




Scanty infection.
Tissue parasite no portal of exit (Hydatid dis.)
Migratory stage (Fasciola)
Chronic infection fibrosis (Bilharziasis)
INDIRECT IMMUNOLOGICAL METHODS
Antigen detection
•
•
•
•
•
More specific
More accurate.
Active infection
Early
Quantitative
Antibody detection
Ab remain in serum for
months even after cure
INDIRECT IMMUNOLOGICAL METHODS
IHAT
LAT
Ag
Ag
+
+
Latex particle
Sensitized
Sheep’s RBC
(O–ve)
Patient’s serum
(?? AB)
Agglutination
Patient’s serum
(?? AB)
Agglutination
INDIRECT IMMUNOLOGICAL METHODS
INDIRECT FLUORESCENT ANTIBODY TEST
fluorescein
Anti human AB
Patient’s serum
(?? AB)
parasite
MOLECULAR BIOLOGICAL TECHNIQUES
DNA Probes
Radio active material
Commercially prepared DNA sequence
DNA Probe
+ve parasite
Hybridization
Nitrocellulose paper
Sample (Serum/ stool)
?? parasite
Radioactivity
MOLECULAR BIOLOGICAL TECHNIQUES
Polymerase Chain Reaction (PCR)
Single stranded DNA
Replication
Detection
T cruzi, T gondii