laboratory diagnosis of parasitic infections

LABORATORY LABORATORY DIAGNOSIS DIAGNOSIS

OF OF PARASITIC PARASITIC

INFECTIONSINFECTIONS

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

DIAGNOSISDIRECT INDIRECT MOLECULARUrineStool

SputumBiopsyBlood

Aspirates

PCRDNA probes

IHATLATIFATELISACFT

DEIDT

URINE EXAMINATION

MACROSCOPIC MICROSCOPIC

colour

white smokySedimentation

concentration

Membrane filtration

Chyluria BloodFilaria S. haematobium

Acetic acid

RBC haemolysis

Clear ova

Ether

Dissolve fat

M.f

URINE EXAMINATIONURINE EXAMINATIONSEDIMENTATIONSEDIMENTATION CONCENTRATIONCONCENTRATION

15-20 min Centrifuge (2 min)

Clean conical Clean conical glass glass

receptaclereceptacle

URINE EXAMINATIONURINE EXAMINATIONMembrane filtration Membrane filtration

techniquetechnique

air

10 ml urine

Nucleopore filter

Eggs of Schistosoma

+ +SalineSaline

URINE EXAMINATION

HELMINTHES PROTOZOA ARTHROPODES

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

• T. Vag troph • Pthirus pubis• L. higher deptera

URINE EXAMINATIONURINE EXAMINATION

Egg viabilityLive eggs Dead eggs

•Well defined miracidium•Flickering F cells•Hatching moving miracidium

•Dark colour•Granulated

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

MACROSCOPIC EXAMINATION

COLOUR CONSISTENCY COMPOSITION Adult PARASITES

Pale=Steatorrhea( G.l)

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

??Blood ?? Mucus(dysentry)

*Ascaris worm*E. vermicularis

*T. saginata

STOOL EXAMINATIONSTOOL EXAMINATION

STOOL EXAMINATION



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

TemproryPermanent




STOOL EXAMINATIONSTOOL EXAMINATIONTemporaryTemporary

Saline smearSaline smear Iodine smearIodine smearsalinesaline 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

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

STOOL EXAMINATION




TemproryPermanent




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 stoolSedimentSediment

formalinformalin

debrisdebrisEtherEther

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

STOOL EXAMINATION




TemproryPermanent




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

STOOL EXAMINATION




TemproryPermanent




STOOL EXAMINATIONSTOOL EXAMINATIONPermanent Stained smearsPermanent Stained smears

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

(Crypto.)(Crypto.)

STOOL EXAMINATION



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

TemproryPermanent




STOOL EXAMINATION STOOL EXAMINATION Kato techniqueKato technique

Mesh screenMesh screen

TemplateTemplateHoleHole

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

STOOL EXAMINATION STOOL EXAMINATION Stoll’s techniqueStoll’s 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 Baermann’s techniqueBaermann’s 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, soil

STOOL EXAMINATIONSTOOL EXAMINATION Cultures for Nematode Cultures for Nematode

larvaelarvaeFilter paper Filter paper cultureculture

Scanty infectionScanty infectionLarvae of:Larvae of:• St. stercoralis St. stercoralis (A,L)(A,L)• HookwormsHookworms• TrichostrongTrichostrong

WaterWaterSealed petri Sealed petri

dishdish

Filter paperSlide

DIAGNOSISDIRECT INDIRECT MOLECULAR

UrineStool

SputumBiopsy

AspiratesBlood

PCRDNA probes

IHATLATIFATELISACFT

DEIDT

SPUTUM EXAMINATIONSPUTUM EXAMINATION

MACROSCOPICMACROSCOPIC MICROSCOPICMICROSCOPIC

AppearanceAppearance ConcentrationConcentration

Bloody (Parag)Bloody (Parag)

Rusty brown (Parag)Rusty brown (Parag)

NaOHNaOH

SputumSputum

CentfifugeCentfifuge

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)

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

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

EdgeEdge


UrineStool

SputumBiopsy

AspiratesBlood

PCRDNA probes

IHATLATIFATELISACFT

DEIDT

BLOOD EXAMINATION

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

Thin Thick

BLOOD EXAMINATIONBLOOD EXAMINATIONBLOOD FILMSBLOOD FILMS

ThinThin ThickThickBld drop

spread

Air dry

methyl alcohol

Geimsa

Air dry

Geimsa

Circular motion

Malaria, Babesia, Filaria, Tryp.

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

BLOOD EXAMINATIONBLOOD EXAMINATIONQBC techniqueQBC technique


RBCRBC

RBC +parasiteRBC +parasite

Microhaematocrit tubeMicrohaematocrit tube

Acridine orangeAcridine orange

Malaria, Filaria, Trypanosomes

BLOOD EXAMINATIONBLOOD EXAMINATIONKNOTT’S CONC. TECHNIQUEKNOTT’S CONC. TECHNIQUE

1010 mlml

11 mlml

Air dryAir dry fixfix GeimsaGeimsa

Citrated Citrated bldbld

Formalin 2Formalin 2% % sedimentsediment

22 minmin


Filaria

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)

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


IHATIHAT LATLAT

++

SensitizedSensitized Sheep’s RBCSheep’s RBC

((O–veO–ve))

AgAg

Patient’s serumPatient’s serum ??( ??(ABAB))

AgglutinationAgglutination

++

AgglutinationAgglutination

AgAg

Latex particleLatex particle Patient’s serumPatient’s serum ??( ??(ABAB))


INDIRECT FLUORESCENT ANTIBODY INDIRECT FLUORESCENT ANTIBODY TESTTEST

parasiteparasite


Anti human ABAnti human AB

fluoresceinfluorescein

INDIRECT IMMUNOLOGICAL INDIRECT IMMUNOLOGICAL METHODSMETHODSELISAELISA

OPDOPD

OPDOPD

Flat bottom plastic micrititre plateFlat bottom plastic micrititre plate

AgAg


Anti human ABAnti human AB

Peroxidase EPeroxidase E

ABAB


CFTCFT

AgAg


complement

Anti sheep ABAnti sheep AB

Sheep’s RBC

-ve Ab

+ve Ab

haemolysis

No Sheep

RBChaemolysis

Tube / Tube / microplatemicroplate

ABAB


Double Electro Immuno Double Electro Immuno DiffusionDiffusion

+ve

-ve

Ag Ab

Buffered gel

Electric current

Line of ppt


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

Nitrocellulose stripMonoclonal

Ab

Coloured dye

Pt bld (?Ag)

+ve

-ve

Malaria, Filaria, African tryp.


UrineStool

SputumBiopsyBlood

Aspirates

PCRDNA probes

IHATLATIFATELISACFT

DEIDT

MOLECULAR BIOLOGICAL MOLECULAR BIOLOGICAL TECHNIQUESTECHNIQUESDNA ProbesDNA Probes

DNA Probe

Commercially prepared DNA sequence

Radio active material

Nitrocellulose paper

Sample (Serum/ stool)

??parasite

+ve parasite Hybridization

Radioactivity

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 gondii

laboratory diagnosis of parasitic infections

Documents

faecesegg count g stool

hrs24 hr stool egg

soilstool examination

eggs protozoal troph

eggs1g x stool wtg

flagellateshuge number

ccegg count slideeggs1g

conical flask centrif