laboratory diagnosis of virus infection

8/16/2019 Laboratory Diagnosis of Virus Infection

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LABORATORY DIAGNOSIS OF VIRUSLABORATORY DIAGNOSIS OF VIRUS

INFECTIONINFECTION

Laboratory technologyLaboratory technology is changing andis changing andautomated techniques are now replacingautomated techniques are now replacingtraditional, but often labour intensive, methods.traditional, but often labour intensive, methods.

Rapid methods:Rapid methods: based on detection of virusbased on detection of virusantigen or of virus IgM are now widely used toantigen or of virus IgM are now widely used togive a faster turn-round of report to clinicians.give a faster turn-round of report to clinicians.

irus diseases are diagnosed in the laboratoryirus diseases are diagnosed in the laboratorybyby ::

!.!. "erology"erology, i.e. demonstration of virus antibody, i.e. demonstration of virus antibody#or antigen$#or antigen$

%.%. Isolation of virusIsolation of virus

More rarely, virus particles can be demonstratedMore rarely, virus particles can be demonstrated

in a specimen by electron microscopy.in a specimen by electron microscopy.


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LABORATORY DIAGNOSIS OF VIRUS INFECTIONLABORATORY DIAGNOSIS OF VIRUS INFECTION continuecontinue

"&R'L'()"&R'L'()

*ow the principal diagnostic technique in virology.*ow the principal diagnostic technique in virology.+I(*'"I"+I(*'"I"

ntibody is detected in patients serum by reaction with nown virusntibody is detected in patients serum by reaction with nown viruspreparation: now increasingly used for antigen detection usingpreparation: now increasingly used for antigen detection usingnown antibody.nown antibody.

ssaysssays

/he reaction is detected by: /he reaction is detected by:!.!. L0&LLI*(L0&LLI*( one of the detecting reagents with:one of the detecting reagents with:

i.i. EnzymeEnzyme #en1yme-lined immunoassay or &LI"$#en1yme-lined immunoassay or &LI"$ when tested with chromogenic substrate awhen tested with chromogenic substrate a

colour develops.colour develops.

ii.ii. Radioactive isotope ( Radioactive isotope ( radioimmunoassay or RI$radioimmunoassay or RI$

the labelled reagent is demonstrated in a radioactivitythe labelled reagent is demonstrated in a radioactivitycounter or by e2posure on 3-ray 4lm.counter or by e2posure on 3-ray 4lm.

iii.iii. 5luorescein5luorescein : 6uorescence is detected under 7 light: 6uorescence is detected under 7 light

microscopically.microscopically.


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&LI"&LI"

*ow the most widely used serological test.*ow the most widely used serological test.

7sed mainly for testing for antibody=IgM or Ig(.7sed mainly for testing for antibody=IgM or Ig(.

ith some viruses, used for testing for antigen directly inith some viruses, used for testing for antigen directly inpatiens sample.patiens sample.

/he principles of an &LI" are illustrated diagrammatically in /he principles of an &LI" are illustrated diagrammatically in

under it.under it.

&LI"s can be varied by :&LI"s can be varied by :

i. 7se of monoclonal virus antibody=to increase speci4city.i. 7se of monoclonal virus antibody=to increase speci4city.

ii. 7se of class-speci4c antibody #e.g. anti Ig(, anti IgM or antiii. 7se of class-speci4c antibody #e.g. anti Ig(, anti IgM or anti

Ig$ to detect one class of antibody in patiens serum.Ig$ to detect one class of antibody in patiens serum.iii. 8ompetitive assay in which antibody is detected byiii. 8ompetitive assay in which antibody is detected bycompeting with nown #labelled$ antibody for combining sitescompeting with nown #labelled$ antibody for combining siteson the nown virus preparation.on the nown virus preparation.


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Diagram for ELISA test for irus Ig! anti"o#$Diagram for ELISA test for irus Ig! anti"o#$

irusirus >> 9atients serum 9atients serum

dd en1yme-labelleddd en1yme-labelled

anti-human IgM antiserumanti-human IgM antiserum

IncubateIncubatestop reactionstop reaction

add substrateadd substrate

Measure reaction by colour intensityMeasure reaction by colour intensity

in optical density readerin optical density reader

8alculate as positive or negative8alculate as positive or negative

reaction by comparison with controlsreaction by comparison with controls


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LABORATORY DIAGNOSIS OF VIRUS INFECTION continueLABORATORY DIAGNOSIS OF VIRUS INFECTION continue

&n1yme substrates used in &LI"s&n1yme substrates used in &LI"s

include:include: i. ?orseradish pero2idase and ortho-i. ?orseradish pero2idase and ortho-

phenyldiamine.phenyldiamine.

ii. laline phosphatase andii. laline phosphatase andp-p-

nitrophenyl phosphate.nitrophenyl phosphate.

*ote:*ote: con@ugation of antibody withcon@ugation of antibody with

biotin and substrate with avidin orbiotin and substrate with avidin orstreptavidin greatly increasesstreptavidin greatly increasesbinding and so the sensitivity of thebinding and so the sensitivity of theassay.assay.


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Auantitation:Auantitation: 'ne disadvantage is that antibody'ne disadvantage is that antibody

cannot be titrated by &LI", so thecannot be titrated by &LI", so the

traditional diagnostic criterion of four-traditional diagnostic criterion of four-fold rise in antibody titre #see below$fold rise in antibody titre #see below$

cannot be used: this is not necessarycannot be used: this is not necessary

in the case of IgM detection, whith byin the case of IgM detection, whith byitself proves recent infectionitself proves recent infection


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RADIOI!!UNOASSAY RADIOI!!UNOASSAY

0ased on similar principles to &LI", but0ased on similar principles to &LI", butseldom used because of diBculty in handlingseldom used because of diBculty in handlingradioisotopes in diagnostic laboratories.radioisotopes in diagnostic laboratories.

IMM7*'5L7'R&"8&*8&IMM7*'5L7'R&"8&*8& Antibody detection Antibody detection

+ilutions of patiens serum are added to spots+ilutions of patiens serum are added to spotsof virus infected cells on microscope slide.of virus infected cells on microscope slide.

9ositive reaction is detected by addition of9ositive reaction is detected by addition of6uorescein-labelled anti-human Ig( #or anti-6uorescein-labelled anti-human Ig( #or anti-IgM$ with 6uorescence seen under 7 light.IgM$ with 6uorescence seen under 7 light.


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Com%&ement Fi'ation TestCom%&ement Fi'ation Test

"till used in must virus laboratories,"till used in must virus laboratories,but being phased out and replacedbut being phased out and replacedby more sensitive techniques lieby more sensitive techniques lie

&LI" that can be automated.&LI" that can be automated.

*ote:*ote: positive reaction is indicated bypositive reaction is indicated by

lac of lysis of the indicator systemlac of lysis of the indicator systemof sheep red cells sensiti1ed withof sheep red cells sensiti1ed withanti red cell antibodyanti red cell antibody


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Com%&ement Fi'ation TestCom%&ement Fi'ation Test continuecontinue

'ther rarer tests for virus antibody include:'ther rarer tests for virus antibody include:

i.i. ?aemagglutination-inhibition test:?aemagglutination-inhibition test: Many virusesMany viruses

haemagglutinate erythrocytes, but virus antibodyhaemagglutinate erythrocytes, but virus antibodyblocs this. ntibody can be detected in a patientsblocs this. ntibody can be detected in a patientsserum by inhibition of virus haemagglutination.serum by inhibition of virus haemagglutination.

ii.ii. Radial immune haemolysis:Radial immune haemolysis: useful qualitative test for useful qualitative test forantibody detection=but not titration=withantibody detection=but not titration=withhaemagglutinating viruses. idely used as a screenhaemagglutinating viruses. idely used as a screen

test for immunity to rubella. irus and erythrocytes aretest for immunity to rubella. irus and erythrocytes aremi2ed in an agar gel in plate with added complement.mi2ed in an agar gel in plate with added complement.9atients sera are added to wells cut in the agar: if9atients sera are added to wells cut in the agar: ifantibody is present, 1ones of haemolysis appear roundantibody is present, 1ones of haemolysis appear roundthe wells on incubation.the wells on incubation.

iii.iii. *eutrali1ation:*eutrali1ation: ntibody prevents virus infection ofntibody prevents virus infection ofcells. ntibody can be detected by neutrali1ation ofcells. ntibody can be detected by neutrali1ation ofvirus cytopathic eCect #89&$ tissue culture. /ime-virus cytopathic eCect #89&$ tissue culture. /ime-consuming and labour-intensive, so not widely used.consuming and labour-intensive, so not widely used.

iv.iv. estern blot:estern blot: 9atients serum is tested for reaction with9atients serum is tested for reaction withindividual virus proteins separated by gelindividual virus proteins separated by gelelectrophoresis on a strip of membrane.electrophoresis on a strip of membrane.


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Virus Iso&ationVirus Iso&ation continuecontinue

!.!. /issue culture /issue culture

/issue culture is really cell culture in vitro and consists /issue culture is really cell culture in vitro and consistsof a single layer #monolayer$ of actively metaboli1ingof a single layer #monolayer$ of actively metaboli1ingcells adherentto glass or plastic surface in a test tube,cells adherentto glass or plastic surface in a test tube,petri plate or on one side bottle.petri plate or on one side bottle.

%.%. 8hic embryo8hic embryo

Rarely used now for virus diagnosis. 7seful forRarely used now for virus diagnosis. 7seful forpreparation of bul virus, e.g. for antigen or vaccinepreparation of bul virus, e.g. for antigen or vaccineproduction.production.

E.E. Laboratory animalsLaboratory animals"ome viruses can only be isolated by inoculation of"ome viruses can only be isolated by inoculation oflaboratory animals, usually mice. fter inoculation thelaboratory animals, usually mice. fter inoculation theanimals are observed for signs of disease or death. /heanimals are observed for signs of disease or death. /heviruses are identi4ed by testing for neutrali1ation ofviruses are identi4ed by testing for neutrali1ation oftheir pathogenicity for animals by standard antiviraltheir pathogenicity for animals by standard antiviral

serasera


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Direct Demonstration Of VirusDirect Demonstration Of Virus

rapid method of virus diagnosis. irus or virus antigen is rapid method of virus diagnosis. irus or virus antigen isdetected in lesions, 6uids, tissues, or e2cretions fromdetected in lesions, 6uids, tissues, or e2cretions fromthe patient and a result can be obtained within an hourthe patient and a result can be obtained within an houror two of receipt of the specimen.or two of receipt of the specimen.

/he main techniques are: /he main techniques are:

1.1. Serological:Serological: preferably with monoclonal antiviralpreferably with monoclonal antiviralantibody. /he most populer method isantibody. /he most populer method isimmuno6uorescence, but &LI" now also used for this,immuno6uorescence, but &LI" now also used for this,especially useful for rapid diagnosis of respiratory virusespecially useful for rapid diagnosis of respiratory virusinfection.infection.

1.1. Electron microscopy:Electron microscopy: virus particles are detected andvirus particles are detected andprovisionally identi4ed #but not serologically typed$ onprovisionally identi4ed #but not serologically typed$ onthe basis of morphology. Mainly used for detection ofthe basis of morphology. Mainly used for detection ofthe faecal viruses that cause gastroenteritis.the faecal viruses that cause gastroenteritis.


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Direct Demonstration Of VirusDirect Demonstration Of Virus continuecontinue

3.3. Probes:Probes: Radiolabelled virus +* sequences canRadiolabelled virus +* sequences can

be used to detect virus genome or mR* inbe used to detect virus genome or mR* intissues and 6uids by molecular hybridi1ation.tissues and 6uids by molecular hybridi1ation.

4.4. Polymerase chain reactionPolymerase chain reaction #98R$#98R$ techniquetechniqueprovides a powerful tool for ampli4cation ofprovides a powerful tool for ampli4cation of

virus nucliec acid in tissues, cells, body 6uids:virus nucliec acid in tissues, cells, body 6uids:an e2tremely sensitive technique with greatan e2tremely sensitive technique with greatpromise, although not yet much used inpromise, although not yet much used indiagnosis.diagnosis.

ncl!tion bodies:ncl!tion bodies: are virus-induced masses seen inare virus-induced masses seen inthe nucleus or cytoplasm of infected cells. iththe nucleus or cytoplasm of infected cells. itha few e2eptions, e.g. rabies too non-speci4c toa few e2eptions, e.g. rabies too non-speci4c tobe useful in diagnosis.be useful in diagnosis.

laboratory diagnosis of virus infection

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