LABORATORY

Lecturer. Mohamed El-Sakhawy

DIAGNOSIS
OF
PARASITIC
INFECTIONS

1
 Case diagnosis
History (Age, occupation, residency, previous
infection)
Complaint Provisional diagnosis
Clinical examination
Invesigations Confirm the diagnosis

- Laboratory investigations
- Radiology
- Surgical intervention (Exploratory)

2
 DIAGNOSIS
DIRECT INDIRECT MOLECULAR
Urine IHAT
Stool LAT
Sputum IFAT PCR
Biopsy ELISA DNA probes
Blood CFT
Aspirates DEIDT

3
 URINE EXAMINATION

MACROSCOPIC MICROSCOPIC

colour

Sedimentation
white smoky
concentration
Chyluria Blood
Filaria S. haematobium Membrane filtration

Ether Acetic acid

Dissolve fat RBC haemolysis

M.f Clear ova 4

 URINE EXAMINATION
SEDIMENTATION CONCENTRATION

Clean conical
glass receptacle

15-20 min Centrifuge (2 min)

5
 URINE EXAMINATION
Membrane filtration technique

air

10 ml urine

Nucleopore filter

+ Saline

Eggs of Schistosoma
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 URINE EXAMINATION

HELMINTHES PROTOZOA ARTHROPODES

S. haem.egg
E. vermic. egg
Tricomonas. Vaginalis Pthirus pubis
S. mansoni egg
troph L. higher deptera
Micrfilaria (Ov, Wb)
H sand

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 URINE EXAMINATION

Egg viability

Live eggs Dead eggs

Well defined miracidium

Flickering F cells Dark colour
Granulated
Hatching moving miracidium

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 STOOL EXAMINATION

MACROSCOPIC MICROSCOPIC OTHERS

Culture
Consistency Cellophane tape
Colour Permanent Temprory Baeremann tech.
Composition Ova quantitaion (Stoll & Kato)

Diect saline smear Iodine smear Concentration techniques

Floatation
Sedimentation

Saline Formol ether Sat saline Zinc sulphate Sheathers sugar

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 STOOL EXAMINATION

MACROSCOPIC EXAMINATION

COLOUR CONSISTENCY COMPOSITION Adult PARASITES

-Liquid (Troph) *Ascaris worm

Pale=Steatorrhea ?? Blood ?? Mucus
-Formed (Cyst) *E. vermicularis
(G.l) (dysentry)
-Semi formed (Cyst) *T. saginata

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 STOOL EXAMINATION

MACROSCOPIC MICROSCOPIC OTHERS

Culture
Consistency Cellophane tape
Baeremann tech.
Colour Permanent Temprory Ova quantitaion
Composition (Stoll & Kato)

Diect saline smear Iodine smear Concentration techniques

Floatation
Sedimentation

Saline Formol ether Sat saline Zinc sulphate Sheathers sugar

11
 STOOL EXAMINATION
Temporary
Saline smear Iodine smear
saline Iodine 1%

Huge number of:

Eggs Huge number of:

Protozoal troph. Motility Cyst morphological details

(Amoeb, flagellates) 12
Staining the saline preparation with
methylene blue 13
 Lugol iodineacetic acid solution causes the
trophozoite forms to become nonmotile.

Using a fine Pasteur pipette, allow a drop of

methylene blue solution to run under the coverslip
over the saline preparation (Fig. 7). This will stain the
nuclei of any cells present and distinguish the lobed
nuclei of polymorphs from the large single nuclei of
mucosal cells.

If a drop of eosin solution is added, the whole field

becomes stained except for the protozoa (particularly
amoebae), which remain colourless and are thus
easily recognized.
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 STOOL EXAMINATION
Scanty infection
Concentration techniques

Sedimentation Floatation

Heavy eggs (Ascaris egg) Non Operculated eggs

Operculated eggs (Trematodes) Trematodes ( S. m.)

Larvae (Strong sterc.) Cestode

Nematode(Hookworms,Trichostong)
Cysts
Cysts

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 STOOL EXAMINATION

MACROSCOPIC MICROSCOPIC OTHERS

Culture
Consistency Cellophane tape
Baeremann tech.
Colour Permanent Temprory Ova quantitaion
Composition (Stoll & Kato)

Diect saline smear Iodine smear Concentration techniques

Floatation
Sedimentation

Saline Formol ether Sat saline Zinc sulphate Sheathers sugar

16
 STOOL EXAMINATION
Saline sedimentation

Mesh wire gauze

Saline Emulsify
Conical flask

10 g stool
Sediment

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 STOOL EXAMINATION
Formol Ether Sed. Conc.

Ether

Ether debris

10% Formalin formalin

1 g stool
Sediment
Thorough mixing Conical flask centrif. tube

Ether adsorbs fecal debris & floats.

Formalin fixes & preserves the specimen. 18
 STOOL EXAMINATION

MACROSCOPIC MICROSCOPIC OTHERS

Culture
Consistency Cellophane tape
Baeremann tech.
Colour Permanent Temprory Ova quantitaion
Composition (Stoll & Kato)

Diect saline smear Iodine smear Concentration techniques

Floatation
Sedimentation

Saline Formol ether Sat saline Zinc sulphate Sheathers sugar

19
 STOOL EXAMINATION
Clean light eggs &
cysts
Floatation concentration

Sat saline Zn sulphate Sheathers sugar

Cestode eggs (non op)

Egg of S.m.
Nematode eggs?????
Eggs of small tapeworms Crypto, Iso. oocysts
Hookworms???????
Cysts
Trichostong

Tin container
Seive

20 min Centrif. 2 min

20
 STOOL EXAMINATION

MACROSCOPIC MICROSCOPIC OTHERS

Culture
Consistency Cellophane tape
Baeremann tech.
Colour Permanent Temprory Ova quantitaion
Composition (Stoll & Kato)

Diect saline smear Iodine smear Concentration techniques

Floatation
Sedimentation

Saline Formol ether Sat saline Zinc sulphate Sheathers sugar

21
 STOOL EXAMINATION
Permanent Stained smears

Iron haematoxylin stain

Trichrome stain
Modified Ziehl Neelsen stain (Crptosporidum.)

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 STOOL EXAMINATION

MACROSCOPIC MICROSCOPIC OTHERS

Cellophane tape
Consistency Culture
Baeremann tech.
Colour Permanent Temprory Ova quantitaion
Composition (Stoll & Kato)

Diect saline smear Iodine smear Concentration techniques

Floatation
Sedimentation

Saline Formol ether Sat saline Zinc sulphate Sheathers sugar

23
 STOOL EXAMINATION
Kato technique
Mesh screen

Hole
Template
Remove the template

Cellophane soaked by glycerin

(clears faeces(

Egg count/ g stool

Egg quant. Of: Ascaris, T. trich., Hookworms, S. mansoni

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 STOOL EXAMINATION
Stolls technique
Egg quant. Of: Ascaris, T. trich., Hookworms, S. mansoni
24 hr stool

60 CC
4 g Stool
56 CC
Shake well 0.15 CC

NaOH

Egg count/ slide

Eggs/1g= Eggs/slideX100

Erlynmeyer flask Egg/day=Eggs/1g X stool wt/g in 24 hrs

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 STOOL EXAMINATION
Baermanns technique
Stool/soil

seive
25-50 CC
Warm water

Glass funnel 30 min centrifuge

clamp

Detec. Of Nematode L. /stool, soil

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 STOOL EXAMINATION
Cultures for Nematode larvae
Filter paper culture

Filter paper
Slide
Sealed petri dish
Water

Scanty infection
Larvae of:
St. stercoralis (A,L)
Hookworms
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Trichostrong
 DIAGNOSIS
DIRECT INDIRECT MOLECULAR
Urine IHAT
Stool LAT
Sputum IFAT PCR
Biopsy ELISA DNA probes
Aspirates CFT
Blood DEIDT

28
 SPUTUM EXAMINATION

MACROSCOPIC MICROSCOPIC NaOH

Sputum
Appearance Concentration

Bloody (Parag) Centfifuge

Rusty brown (Parag)

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 Parasites/sputum

P living in lung P migrating in lung P resulting from rupture of

St. stercoralis
Hydatid cyst (sand)
P. westermani eggs Ascaris
Amoebic abcess (troph)
Hookworm (filariform L)

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 BIOPSY SPECIMEN

SKIN SNIP MUSCLE BIOPSY RECTAL BIOPSY

O. Volvulus mf T. Spiralis larvae Schistosoma egg

Raise skin by needle

Slice by scissors
Muscle digestion with HCl + pepsin
Put snip in normal saline
Examine

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 ASPIRATES EXAMINATION

CSF Duodenal aspirates BM aspirates Cutanoeus ucler

Afr. Tryp. (trypom)

G. lamb troph
FLA (troph) L. donovani a,ast.
Crypto oocyst
M.f. of loa loa T. cruzi amast. Leishmaniasis
St sterc. Rh L.
L. of T spiralis P. falciparum.
Fasciola eggs

Lumbar puncutre
Centrifuge D. intubation Direct stain (amast
Examine sed.

D capsule (Enterotest) NNN (promast)

floor

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Edge
 DIAGNOSIS
DIRECT INDIRECT MOLECULAR
Urine IHAT
Stool LAT
Sputum IFAT PCR
Biopsy ELISA DNA probes
Aspirates CFT
Blood DEIDT

33
 BLOOD EXAMINATION

Blood films Buffy coat films QBC technique Knotts conc. tech.

Thin Thick

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 BLOOD EXAMINATION
BLOOD FILMS
Thin Thick
Bld drop

spread Circular motion

Air dry Air dry

methyl alcohol
Geimsa

Geimsa
Malaria, Babesia, Filaria, Tryp.
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 BLOOD EXAMINATION
Buffy coat film

plasma

WBC (BC)
centrifuge Air dry Fix

30 min
RBC
spread Geimsa

Citrated bld
Tryp., L. donovani

36
 BLOOD EXAMINATION
QBC technique

RBC +parasite

Acridine orange
centrifuge

RBC

Microhaematocrit tube

Malaria, Filaria, Trypanosomes

37
 BLOOD EXAMINATION
KNOTTS CONC. TECHNIQUE
Citrated bld

1 ml

centrifuge
10 ml Air dry fix Geimsa
2 min

Formalin 2 % sediment

Filaria
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 INDIRECT IMMUNOLOGICAL
METHODS
Scanty infection.
Tissue parasite no portal of exit (Hydatid dis.)
Migratory stage (Fasciola)
Chronic infection fibrosis (Bilharziasis)

39
 INDIRECT IMMUNOLOGICAL METHODS

Antigen detection Antibody detection

More specific
More accurate.
Active infection
Ab remain in serum for
Early
months even after cure
Quantitative

40
 INDIRECT IMMUNOLOGICAL METHODS

IHAT LAT
Ag
Ag
+
+
Patients serum
Latex particle
(?? AB)
Patients serum
Sensitized
(?? AB)
Sheeps RBC
(Ove)

Agglutination Agglutination
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 INDIRECT IMMUNOLOGICAL METHODS
INDIRECT FLUORESCENT ANTIBODY TEST

fluorescein

Anti human AB

Patients serum
(?? AB)

parasite

42
 INDIRECT IMMUNOLOGICAL METHODS
ELISA

OPD
Peroxidase E
OPD

Anti human AB

Patients serum
(?? AB) AB
Ag

Flat bottom plastic micrititre plate

43
 INDIRECT IMMUNOLOGICAL METHODS
CFT

Sheeps RBC

Anti sheep AB

AB
complement

Patients serum
(?? AB)

Ag

Tube / microplate

44
 INDIRECT IMMUNOLOGICAL METHODS
Double Electro Immuno Diffusion
Line of ppt

Electric current

Ag Ab

Buffered gel

45
 INDIRECT IMMUNOLOGICAL METHODS
Immunodiagnostic Strip Test (Dip Stick Test) Ag

Pt bld (?Ag)

Coloured dye

Monoclonal Ab
Nitrocellulose strip
Malaria, Filaria, African46tryp.
 DIAGNOSIS
DIRECT INDIRECT MOLECULAR
Urine IHAT
Stool LAT
Sputum IFAT PCR
Biopsy ELISA DNA probes
Blood CFT
Aspirates DEIDT

47
MOLECULAR BIOLOGICAL TECHNIQUES
DNA Probes

Radio active material

Commercially prepared DNA sequence

DNA Probe
Hybridization
+ve parasite

Nitrocellulose paper

Sample (Serum/ stool) Radioactivity

?? parasite

48
 MOLECULAR BIOLOGICAL TECHNIQUES
Polymerase Chain Reaction (PCR)

Single stranded DNA

Replication

Detection T cruzi, T gondii 49

10 X Objective

50
Relative sizes of helminth eggs as seen in microscope
field using the 10 objective (with 10 eye-pieces).
Eggs are as
seen in a saline preparation.
1. E.vermicularis, 2. A.lumbricoides, 3. S.stercoralis
larva (motile), 4. Hookworm, 5. T.trichiura, 6.
D.latum, 7. O.sinensis,
8. Fasciola sp, 9. S.mansoni, 10. Paragonimus sp, 11.
S.japonicum, 12. S.intercalatum, 13.Taenia sp,
14.V.nana, 15. H.dimunuta.

51
40 X Objective

52
Relative sizes of trophozoites and cysts of intestinal
protozoa, common nematode eggs and larva of
Strongyloides as seen in microscope field using the 40
objective (with 10 eyepieces).
1. I.belli oocyst, 2. A lumbricoides egg, 3. Leucocytes, 4.
E.histolytica/E.dispar cyst, 5. E.histolytica trophozoite
(motile),
6. Red cells, 7. S.stercoralis larva (motile), 8. E.coli cyst
(mature), 9. G.lamblia cyst, 10. C.mesnili cyst, 11.
Hookworm
egg, 12. G.lamblia trophozoite (motile).
Iodine preparation: 13. E.coli cyst, 14. I.buetschlii cyst, 15.
E.histolytica/E.dispar cyst, 16. V.nana cyst, 17. T.trichiura
egg,
18. Blastocystis hominis, 19. G.lamblia cyst.
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 Non-parasitic structures found in faeces:
Care
must be taken not to report as parasites those
structures that can be normally found in faeces such as:
muscle fibres, vegetable fibres, starch cells (stain blue-
black with iodine), pollen grains, fatty acid crystals,
soaps, spores, yeasts, and hairs .
Large numbers of fat globules may be seen in faeces
when there is malabsorption.
Charcot Leyden crystals (breakdown products of
eosinophils) can sometimes be seen in faeces (also in
sputum) in parasitic infections. They appear as slender
crystals with pointed ends, about 3040m in length
54
 Structures found in faeces that required
differentiation from parasites.

Structures found in faeces that required differentiation from

parasites. 55
 Image illustrating Red Blood Cells in slide
Image illustrating Fat Globules in slide
preparation.
preparation

Image illustrating Yeast Cells in slide preparation Image illustrating Vegetable cell in slide
56
Note similarity to parasitic oocysts. preparation.
Image illustrating Vegetable Spiral in slide
preparation.

Image illustrating a Vegetable Spiral in slide

Image illustrating Vegetable cell in slide preparation. Such spirals may appear similar to
57
preparation. proglottids.
Image illustrating pollen in slide preparation that Image illustrating pollen resembling a Hymenolepis
could be mistaken for a Taenia egg. The shell is nana egg. Hooks and polar filaments are not visible.
thinner, of non-uniform thickness, and no hooks
are visible.

Image illustrating geranium pollen cells in slide

Image illustrating pollen in slide preparation preparation.
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using a color filter
Image illustrating peach hair in slide preparation. Image illustrating vegetable hairs in slide
Note the similarity to Strongyloides stercoralis. preparation.

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