laboratory diagnosis of parasitic infections lecturer. mohamed el-sakhawy 1

LABORATORY LABORATORY DIAGNOSIS DIAGNOSIS

OF OF PARASITIC PARASITIC

INFECTIONSINFECTIONS

Lectu

rer.

Moh

am

ed

El-

Sakh

aw

y

1

Case diagnosisCase diagnosis

History (Age, occupation, residency, History (Age, occupation, residency, previous infection)previous infection)

ComplaintComplaint Clinical examinationClinical examination InvesigationsInvesigations - - Laboratory investigationsLaboratory investigations - Radiology- Radiology - Surgical intervention - Surgical intervention

(Exploratory)(Exploratory)

Provisional Provisional diagnosisdiagnosis

Confirm the Confirm the diagnosisdiagnosis

2

DIAGNOSISDIAGNOSIS

DIRECTDIRECT INDIRECTINDIRECT MOLECULARMOLECULAR

UrineStool

SputumBiopsyBlood

Aspirates

UrineStool

SputumBiopsyBlood

Aspirates

PCRDNA probes

PCRDNA probes

IHATLATIFATELISACFT

DEIDT

IHATLATIFATELISACFT

DEIDT

3

URINE EXAMINATION

MACROSCOPIC MICROSCOPIC

colour

white smokySedimentation

concentration

Membrane filtration

Chyluria Blood

Filaria S. haematobium

Acetic acid

RBC haemolysis

Clear ova

Ether

Dissolve fat

M.f4

URINE EXAMINATIONURINE EXAMINATION

SEDIMENTATIONSEDIMENTATION CONCENTRATIONCONCENTRATION

15-20 min Centrifuge (2 min)

Clean conical Clean conical glass glass

receptaclereceptacle

5

URINE EXAMINATIONURINE EXAMINATIONMembrane filtration Membrane filtration

techniquetechnique

air

10 ml urine

Nucleopore filter

Eggs of Schistosoma

+ +SalineSaline

6

URINE EXAMINATION

HELMINTHES PROTOZOA ARTHROPODES

• S. haem.egg• E. vermic. egg• S. mansoni egg• Micrfilaria (Ov, Wb)• H sand

Tricomonas. Vaginalis troph

• Pthirus pubis• L. higher deptera

7

URINE EXAMINATIONURINE EXAMINATION

Egg viability

Live eggs Dead eggs

•Well defined miracidium•Flickering F cells

•Hatching moving miracidium •Dark colour•Granulated

8

STOOL EXAMINATION

MACROSCOPIC MICROSCOPIC OTHERS

•Consistency•Colour•Composition

•Culture•Cellophane tape•Baeremann tech.•Ova quantitaion (Stoll & Kato)

TemproryPermanent

Diect saline smear Iodine smear Concentration techniques

Sedimentation Floatation

Saline Formol ether Sat saline Zinc sulphate Sheather’s sugar

9

MACROSCOPIC EXAMINATIONMACROSCOPIC EXAMINATION

COLOURCOLOUR CONSISTENCYCONSISTENCY COMPOSITIONCOMPOSITION Adult PARASITESAdult PARASITES

Pale=Steatorrhea( G.l)

Pale=Steatorrhea( G.l)

-Liquid (Troph)-Formed (Cyst)-Semi formed (Cyst)

-Liquid (Troph)-Formed (Cyst)-Semi formed (Cyst)

??Blood ?? Mucus(dysentry)

??Blood ?? Mucus(dysentry)

*Ascaris worm*E. vermicularis

*T. saginata

Ascaris worm*E. vermicularis*

*T. saginata

STOOL EXAMINATIONSTOOL EXAMINATION

10

STOOL EXAMINATION



•Culture•Cellophane tape•Baeremann tech.•Ova quantitaion •(Stoll & Kato)

TemproryPermanent




11

STOOL EXAMINATIONSTOOL EXAMINATIONTemporaryTemporary

Saline smearSaline smear Iodine smearIodine smear

salinesaline Iodine Iodine 1%1%

Huge number of:Huge number of:

•EggsEggs

• Protozoal troph. Protozoal troph. Motility Motility

(Amoeb, flagellates)(Amoeb, flagellates)

Huge number of:Huge number of:

•Cyst morphological Cyst morphological detailsdetails

12

Staining the saline preparation with methylene blue 13

Lugol iodine–acetic acid solution causes the Lugol iodine–acetic acid solution causes the trophozoite forms to become nonmotile. trophozoite forms to become nonmotile.

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

If a drop of eosin solution is added, the whole field If a drop of eosin solution is added, the whole field becomes stained except for the protozoa (particularly becomes stained except for the protozoa (particularly amoebae), which remain colourless and are thus easily amoebae), which remain colourless and are thus easily recognized.recognized.

14

STOOL EXAMINATIONSTOOL EXAMINATIONScanty infectionScanty infection

Concentration techniquesConcentration techniques

SedimentatioSedimentationn

FloatationFloatation

• Heavy eggs (Ascaris egg)Heavy eggs (Ascaris egg)

• Operculated eggs Operculated eggs (Trematodes)(Trematodes)

• Larvae (Strong sterc.)Larvae (Strong sterc.)

• CystsCysts

• Non Operculated eggs

Trematodes ( S. m.)

Cestode Nematode(Hookworms,TrichosHookworms,Trichostong)tong)

• Cysts 15

STOOL EXAMINATION




TemproryPermanent




16

17

STOOL EXAMINATIONSTOOL EXAMINATIONSaline sedimentationSaline sedimentation

1010 g stoolg stool

SalineSaline

Mesh wire gauzeMesh wire gauze

Conical flaskConical flask

SedimentSediment

EmulsifyEmulsify

STOOL EXAMINATIONSTOOL EXAMINATION Formol Ether Sed. Conc Formol Ether Sed. Conc . .

10%10% FormalinFormalin

11 g stoolg stool

SedimentSediment

formalinformalin

debrisdebris

EtherEther

Thorough mixingThorough mixing

EtherEther

• Ether adsorbs fecal debris & floats.Ether adsorbs fecal debris & floats.

• Formalin fixes & preserves the specimen.Formalin fixes & preserves the specimen.

Conical flask centrif. tubeConical flask centrif. tube

18

STOOL EXAMINATION




TemproryPermanent




19

STOOL EXAMINATIONSTOOL EXAMINATION

Floatation concentrationFloatation concentration

Sat salineSat saline Zn sulphateZn sulphate Sheather’s sugarSheather’s sugar

• Cestode eggs (non op)Cestode eggs (non op)•Nematode eggs?????Nematode eggs?????•Hookworms???????Hookworms???????•TrichostongTrichostong؟؟؟؟؟؟؟؟؟؟؟؟؟؟؟؟؟؟؟؟؟؟

•Egg of S.m.Egg of S.m.•Eggs of small tapewormsEggs of small tapeworms•CystsCysts

• Crypto, Iso. oocystsCrypto, Iso. oocysts

Tin Tin containercontainer

2020 minmin Centrif. 2 Centrif. 2 minmin

SeiveSeive

Clean light eggs & Clean light eggs & cystscysts

20

STOOL EXAMINATION




TemproryPermanent




21

STOOL EXAMINATIONSTOOL EXAMINATIONPermanent Stained smearsPermanent Stained smears

Iron haematoxylin stainIron haematoxylin stain Trichrome stainTrichrome stain Modified Ziehl Neelsen stain Modified Ziehl Neelsen stain

(Crptosporidum.)(Crptosporidum.)

22

STOOL EXAMINATION



• Cellophane tape• Culture•Baeremann tech.•Ova quantitaion •(Stoll & Kato)

TemproryPermanent




23

STOOL EXAMINATION STOOL EXAMINATION Kato techniqueKato technique

Mesh screenMesh screen

TemplateTemplate

HoleHole

Remove the Remove the templatetemplate

Cellophane soaked by Cellophane soaked by glycerin (clears faecesglycerin (clears faeces((

Egg count/ g stoolEgg count/ g stool

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

24

25

STOOL EXAMINATION STOOL EXAMINATION StollStoll’’s techniques technique

NaOHNaOH

44 g Stoolg Stool

Erlynmeyer flaskErlynmeyer flask

5656 CCCC

6060 CCCC

Shake wellShake well 0.150.15 CCCC

Egg count/ slideEgg count/ slide

Eggs/1g= Eggs/slideX100Eggs/1g= Eggs/slideX100

Egg/day=Eggs/1g X stool wt/g in 24 Egg/day=Eggs/1g X stool wt/g in 24 hrshrs

2424 hr stoolhr stool

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

STOOL EXAMINATION STOOL EXAMINATION BaermannBaermann’’s techniques technique

WarmWarm waterwater

Stool/soilStool/soil

seiveseive

Glass funnelGlass funnel

clampclamp

3030 minmin

25-5025-50 CCCC

centrifugecentrifuge

Detec. Of Nematode L. /stool, soilDetec. Of Nematode L. /stool, soil26

STOOL EXAMINATIONSTOOL EXAMINATION Cultures for Nematode Cultures for Nematode

larvaelarvaeFilter paper Filter paper cultureculture

Scanty infectionScanty infection

Larvae of:Larvae of:

• St. stercoralis St. stercoralis (A,L)(A,L)

• HookwormsHookworms

• TrichostrongTrichostrong

WaterWaterSealed petri Sealed petri

dishdish

Filter paperSlide

27

DIAGNOSISDIAGNOSIS


Urine

StoolSputumBiopsy

AspiratesBlood

Urine

StoolSputumBiopsy

AspiratesBlood

PCR

DNA probesPCR

DNA probes

IHAT

LATIFAT

ELISACFT

DEIDT

IHAT

LATIFAT

ELISACFT

DEIDT

28

SPUTUM EXAMINATIONSPUTUM EXAMINATION

MACROSCOPICMACROSCOPIC MICROSCOPICMICROSCOPIC

AppearanceAppearance ConcentrationConcentration

Bloody (Parag)Bloody (Parag)

Rusty brown (Parag)Rusty brown (Parag)

NaOHNaOH

SputumSputum

CentfifugeCentfifuge

29

Parasites/sputumParasites/sputum

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

• P. westermani eggsP. westermani eggs• St. stercoralisSt. stercoralis• AscarisAscaris• Hookworm (filariform L)Hookworm (filariform L)

• Hydatid cyst (sand)Hydatid cyst (sand)• Amoebic abcess (troph)Amoebic abcess (troph)

30

BIOPSY SPECIMENBIOPSY SPECIMEN

SKIN SNIPSKIN SNIP MUSCLE BIOPSYMUSCLE BIOPSY RECTAL BIOPSYRECTAL BIOPSY

O. Volvulus mfO. Volvulus mf T. Spiralis larvaeT. Spiralis larvae Schistosoma eggSchistosoma egg

• Raise skin by needleRaise skin by needle• Slice by scissorsSlice by scissors• Put snip in normal salinePut snip in normal saline• ExamineExamine

Muscle digestion with HCl + pepsinMuscle digestion with HCl + pepsin

31

ASPIRATES EXAMINATIONASPIRATES EXAMINATION

CSFCSF Duodenal aspiratesDuodenal aspirates BM aspiratesBM aspirates Cutanoeus uclerCutanoeus ucler

• Afr. Tryp. (trypom)Afr. Tryp. (trypom)• FLA (troph)FLA (troph)• M.f. of loa loaM.f. of loa loa• L. of T spiralisL. of T spiralis

• G. lamb trophG. lamb troph• Crypto oocystCrypto oocyst• St sterc. Rh L.St sterc. Rh L.• Fasciola eggsFasciola eggs

• L. donovani a,ast.L. donovani a,ast.•T. cruzi amast.T. cruzi amast.•P. falciparum.P. falciparum.

•LeishmaniasisLeishmaniasis

D. intubationD. intubation

D capsule (Enterotest)D capsule (Enterotest)

Direct stain (amast)Direct stain (amast)

NNN (promast)NNN (promast)

•Lumbar puncutreLumbar puncutre• CentrifugeCentrifuge•Examine sed.Examine sed.

floorfloor

EdgeEdge32

DIAGNOSISDIAGNOSIS


Urine

StoolSputumBiopsy

AspiratesBlood

Urine

StoolSputumBiopsy

AspiratesBlood

PCR

DNA probesPCR

DNA probes

IHAT

LATIFAT

ELISACFT

DEIDT

IHAT

LATIFAT

ELISACFT

DEIDT

33

BLOOD EXAMINATION

Blood films Buffy coat films QBC technique Knott’s conc. tech.

Thin Thick

34

BLOOD EXAMINATIONBLOOD EXAMINATIONBLOOD FILMSBLOOD FILMS

ThinThin ThickThickBld drop

spread

Air dry

methyl alcohol

Geimsa

Air dry

Geimsa

Circular motion

Malaria, Babesia, Filaria, Tryp. 35

BLOOD EXAMINATIONBLOOD EXAMINATIONBuffy coat filmBuffy coat film


RBCRBC

WBC (BC)WBC (BC)

plasmaplasma

Citrated bldCitrated bld

3030 minmin

Air dryAir dry FixFix

spreadspread GeimsaGeimsa

Tryp., L. donovani

36

BLOOD EXAMINATIONBLOOD EXAMINATIONQBC techniqueQBC technique


RBCRBC

RBC +parasiteRBC +parasite

Microhaematocrit tubeMicrohaematocrit tube

Acridine orangeAcridine orange

Malaria, Filaria, Trypanosomes37

BLOOD EXAMINATIONBLOOD EXAMINATIONKNOTTKNOTT’’S CONC. TECHNIQUES CONC. TECHNIQUE

1010 mlml

11 mlml

Air dryAir dry fixfix GeimsaGeimsa

Citrated Citrated bldbld

Formalin 2Formalin 2% % sedimentsediment

22 minmin


Filaria38

INDIRECT IMMUNOLOGICAL INDIRECT IMMUNOLOGICAL METHODSMETHODS

Scanty infection.Scanty infection. Tissue parasite no portal of exit Tissue parasite no portal of exit

(Hydatid dis.)(Hydatid dis.) Migratory stage (Fasciola)Migratory stage (Fasciola) Chronic infection fibrosis Chronic infection fibrosis

(Bilharziasis)(Bilharziasis)

39

INDIRECT IMMUNOLOGICAL METHODSINDIRECT IMMUNOLOGICAL METHODS

Antigen detection Antibody detection

• More specific More specific • More accurate.More accurate.• Active infectionActive infection• EarlyEarly• QuantitativeQuantitative

Ab remain in serum forAb remain in serum for months even after curemonths even after cure

40


IHATIHAT LATLAT

++

SensitizedSensitized SheepSheep’’s RBCs RBC

((OO––veve))

AgAg

PatientPatient’’s serums serum ??( ??(ABAB))

AgglutinationAgglutination

++

AgglutinationAgglutination

AgAg

Latex particleLatex particlePatientPatient’’s serums serum

??( ??(ABAB))

41


INDIRECT FLUORESCENT ANTIBODY INDIRECT FLUORESCENT ANTIBODY TESTTEST

parasiteparasite

PatientPatient’’s serums serum

??( ??(ABAB))

Anti human ABAnti human AB

fluoresceinfluorescein

42


ELISAELISA

OPDOPD

OPDOPD

Flat bottom plastic micrititre plateFlat bottom plastic micrititre plate

AgAg


Anti human ABAnti human AB

Peroxidase EPeroxidase E

ABAB

43


CFTCFT

AgAg


complement

Anti sheep ABAnti sheep AB

Sheep’s RBC

-ve Ab

+

ve Ab

haemolysis

No Sheep

RBChaemolysis

Tube / Tube / microplatemicroplate

ABAB

44


Double Electro Immuno Double Electro Immuno DiffusionDiffusion

+ve

-ve

Ag Ab

Buffered gel

Electric current

Line of ppt

45


Immunodiagnostic Strip Test (Dip Stick Test) Immunodiagnostic Strip Test (Dip Stick Test) Ag Ag

Nitrocellulose strip

Monoclonal Ab

Coloured dye

Pt bld (?Ag)

+ve

-ve

Malaria, Filaria, African tryp.

46

DIAGNOSISDIAGNOSIS


Urine

StoolSputumBiopsyBlood

Aspirates

Urine

StoolSputumBiopsyBlood

Aspirates

PCR

DNA probesPCR

DNA probes

IHAT

LATIFAT

ELISACFT

DEIDT

IHAT

LATIFAT

ELISACFT

DEIDT

47

MOLECULAR BIOLOGICAL MOLECULAR BIOLOGICAL TECHNIQUESTECHNIQUESDNA ProbesDNA Probes

DNA Probe

Commercially prepared DNA sequence

Radio active material

Nitrocellulose paper

Sample (Serum/ stool)

??parasite

+ve parasiteHybridizatio

n

Radioactivity

48

MOLECULAR BIOLOGICAL MOLECULAR BIOLOGICAL TECHNIQUESTECHNIQUES

Polymerase Chain Reaction (PCR) Polymerase Chain Reaction (PCR)

Single stranded DNA Single stranded DNA

ReplicationReplication

DetectionDetection T cruzi, T gondiiT cruzi, T gondii49

10 X Objective

50

Relative sizes of helminth eggs as seen in microscope field using the 10 objective (with 10 eye-pieces). Eggs are asseen 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. Hookwormegg, 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.

53

Non-parasitic structures found Non-parasitic structures found in faeces: Carein faeces: Care

must be taken not to report as parasites those structures must be taken not to report as parasites those structures that can be normally found in faeces such as:that can be normally found in faeces such as:

muscle fibres, vegetable fibres, starch cells (stain muscle fibres, vegetable fibres, starch cells (stain blue-black blue-black with iodine), pollen grains, fatty acid crystals, soaps, spores, with iodine), pollen grains, fatty acid crystals, soaps, spores, yeasts, and hairs .yeasts, and hairs .

Large numbers of fat globules may be seen in faeces when Large numbers of fat globules may be seen in faeces when there is malabsorption. there is malabsorption.

Charcot Leyden crystals (breakdown products of eosinophils) Charcot Leyden crystals (breakdown products of eosinophils) can sometimes be seen in faeces (also in sputum) in parasitic can sometimes be seen in faeces (also in sputum) in parasitic infections. They appear as slender crystals with pointed infections. They appear as slender crystals with pointed ends, about 30–40m in lengthends, about 30–40m in length

54

Structures found in faeces that Structures found in faeces that required differentiation from required differentiation from

parasitesparasites..

Structures found in faeces that required differentiation from parasites. 55

Image illustrating Yeast Cells in slide preparationNote similarity to parasitic oocysts.

Image illustrating Vegetable cell in slide preparation.

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

slide preparation

56

Image illustrating Vegetable cell in slide preparation.

Image illustrating a Vegetable Spiral in slide preparation. Such spirals may appear similar to proglottids.

Image illustrating Vegetable Spiral in slide preparation.

57

Image illustrating pollen in slide preparation using a color filter

Image illustrating pollen in slide preparation that could be mistaken

for a Taenia egg. The shell is thinner, of non-uniform thickness, and no

hooks are visible.

Image illustrating pollen resembling a Hymenolepis nana egg. Hooks and

polar filaments are not visible.

Image illustrating geranium pollen cells in slide preparation.

58

Image illustrating peach hair in slide preparation. Note the similarity to

Strongyloides stercoralis.

Image illustrating vegetable hairs in slide preparation.

59

laboratory diagnosis of parasitic infections lecturer. mohamed el-sakhawy 1

Documents

saginata stool examination

saline preparation

consistency liquid troph

formed cyst composition

motility protozoal troph

case diagnosis history

eggseggs protozoal troph

ova quantitaion stoll