diagnosis_of viral infection.ppt

8/18/2019 Diagnosis_of Viral infection.ppt

1/38

DIAGNOSIS OF VIRAL

INFECTION

An Overview


2/38

Diagnostic Methods in

Virology1. Direct Examination

2. Indirect Examination (Virus

Isolation)

3. Serology


3/38

Direct Examination1. Antigen Detection immunofluorescence, ELISA etc.

2. Electron Microscopy morphology of virus particlesimmune electron microscopy

3. Light Microscopy histological appearance

inclusion bodies

4. Viral Genome Detection hybridization with specific

nucleic acid probes

polymerase chain reaction

!"#$


4/38

Indirect Examination1. Cell Culture cytopathic effect "!E$

haemabsorption

immunofluorescence

2. Eggs poc%s on "A&

haemagglutination

inclusion bodies

3. Animals disease or death


5/38

Serology'etection of rising titers of antibody between acute and convalescent

stages of infection, or the detection of Ig& in primary infection.


6/38

Virus Isolation"ell "ultures are most widely used for virus isolation, there are (

types of cell cultures)

*. !rimary cells + &on%ey idney-. Semi+continuous cells + uman embryonic %idney and s%in

fibroblasts

(. "ontinuous cells + eLa, /ero, ep-, LL"+&-, &'"

!rimary cell culture are widely ac%nowledged as the best cell culturesystems available since they support the widest range of viruses.

owever, they are very e0pensive and it is often difficult to obtain a

reliable supply. "ontinuous cells are the most easy to handle but the range

of viruses supported is often limited.


7/38

Cell Cultures1rowing virus may produce

*. "ytopathic Effect "!E$ + such as the ballooning of cells or

syncytia formation, may be specific or non+specific.

-. aemadsorption + cells ac2uire the ability to stic% to

mammalian red blood cells.

"onfirmation of the identity of the virus may be carried out usingneutralization, haemadsorption+inhibition or immunofluorescence

tests.


8/38

Cytoathic E!ect (1)

"ytopathic effect of enterovirus 3* and S/ in cell culture) note the ballooning of cells./irology Laboratory, 4ale+5ew aven ospital, Linda Stannard, 6niversity of "ape 7own$


9/38

Cytoathic E!ect (2)

Syncytium formation in cell

culture caused by #esp.

Syncytial /irus top$, and

measles virus bottom$.courtesy of Linda Stannard, 6niversity of "ape

7own, S.A.$


10/38

"aemadsortion

Syncytial formation caused by mumps virus and

haemadsorption of erythrocytes onto the surface of the cell

sheet.courtesy of Linda Stannard, 6niversity of "ape 7own, S.A.$


11/38

#ro$lems %ith cell culture Long period up to 8 wee%s$ re2uired for result.

Often very poor sensitivity, sensitivity depends on a

large e0tent on the condition of the specimen.

Susceptible to bacterial contamination.

Susceptible to to0ic substances which may be present in

the specimen. &any viruses will not grow in cell culture e.g. epatitis

9, 'iarrhoeal viruses, parvovirus, papillomavirus.


12/38

&aid Culture 'echniues#apid culture techni2ues are available whereby viral antigens

are detected - to 8 days after inoculation. 7he "&/ 'EA::

'etection of Early Antigen :luorescent :oci$ test is the best

e0ample, whereby

7he cell sheet is grown on individual cover slips in a plastic

bottle.

:ollowing inoculation, the bottle then is spun at a low speedfor one hour to speed up the adsorption of the virus$ and then

incubated for - to 8 days.

7he cover slip is then ta%en out and e0amined for the

presence of "&/ early antigens by immunofluorescence.

http://sunzi1.lib.hku.hk/hkjo/view/21/2100433.pdfhttp://sunzi1.lib.hku.hk/hkjo/view/21/2100433.pdf


13/38

DE** test +or CMV

/irology Laboratory, 4ale+5ew aven ospital$


14/38

Viruses Isolated $y Cell

CultureViruses readily isolated y cell culture Less !re"uently isolated #iruses

erpes Simple0 /aricella+;oster

"ytomegalovirus &easles

Adenoviruses #ubella

!olioviruses #hinoviruses

"o0sac%ie 9 viruses "o0sac%ie A viruses

Echoviruses

Influenza

!arainfluenza

&umps

#espiratory Syncytial /irus


15/38

Electron Microscoy*


16/38

Electronmicrograhs

Adenovirus #otavirus

courtesy of Linda Stannard, 6niversity of "ape 7own, S.A.$

Astroviruses


17/38

Immune Electron

Microscoy 'he sensiti,ity and seci-city o+ EM may $e enhanced$y immune electron microscoy. 'here are t%o,ariants/

"lassical Immune electron microscopy IE&$ + the sample is

treated with specific anti+sera before being put up for E&. /iral

particles present will be agglutinated and thus congregate

together by the antibody.Solid phase immune electron microscopy S!IE&$ + the grid is

coated with specific anti+sera. /irus particles present in the

sample will be absorbed onto the grid by the antibody.


18/38

#ro$lems %ith Electron

Microscoy Exensi,e euiment

Exensi,e maintenance

&euire exerienced o$ser,er

Sensiti,ity o+ten lo%


19/38

Serology"riteria for diagnosing !rimary Infection

8 fold or more increase in titre of Ig1 or total antibody between

acute and convalescent sera !resence of Ig&

Seroconversion

A single high titre of Ig1 or total antibody$ + very unreliable

"riteria for diagnosing #einfection

fold or more increase in titre of Ig1 or total antibody between acute

and convalescent sera

Absence or slight increase in Ig&


20/38

'yical Serological #ro-le +ter cuteIn+ection

5ote that during reinfection, Ig& may be absent or present at a low level transiently


21/38

Comlement *ixation 'est

"omplement :i0ation 7est in &icrotiter !late. #ows * and - e0hibit complement

fi0ation obtained with acute and convalescent phase serum specimens,

respectively. -+fold serum dilutions were used$


22/38

E0IS +or "IV anti$ody

&icroplate ELISA for I/ antibody) colored wells indicate reactivity


23/38

estern lot

I/+* ?estern 9lot

Lane*) !ositive "ontrol Lane -) 5egative "ontrol

Sample A) 5egative

Sample 9) Indeterminate

Sample ") !ositive


24/38

se+ulness o+ Serological

&esults ow useful a serological result is depends on the individual virus.

:or e0ample, for viruses such as rubella and hepatitis A, the onset of

clinical symptoms coincide with the development of antibodies. 7he

detection of Ig& or rising titres of Ig1 in the serum of the patient would

indicate active disease.

owever, many viruses often produce clinical disease before the

appearance of antibodies such as respiratory and diarrhoeal viruses. So in

this case, any serological diagnosis would be retrospective and thereforewill not be that useful.

7here are also viruses which produce clinical disease months or years

after seroconversion e.g. I/ and rabies. In the case of these viruses, the

mere presence of antibody is sufficient to ma%e a definitive diagnosis.


25/38

#ro$lems %ith Serology Long period of time re2uired for diagnosis for paired acute and convalescent

sera.

&ild local infections such as S/ genitalis may not produce a detectale

humoral immune response. E0tensive antigenic cross+reactivity between related viruses e.g. S/ and

/;/, @apanese 9 encephalitis and 'engue, may lead to !alse positi#e results.

immunocompromised patients often give a reduced or absent humoral

immune response.

!atients with infectious mononucleosis and those with connective tissue

diseases such as SLE may react non+specifically giving a false positive result.

!atients given blood or blood products may give a false positive result due to

the transfer of antibody.


26/38

CS* anti$odies 6sed mainly for the diagnosis of herpes

simple0 and /;/ encephalitis

"S: normally contain little or no antibodies

presence of antibodies suggest meningitis or

meningoencephalitis


27/38

&aid Diagnosis ased on

the Detection o+ Viralntigens 5asopharyngeal Aspirate #S/

Influenza A and 9

!arainfluenza

Adenovirus

:aeces #otaviruses

Adenoviruses

Astrovirus

S%in S/

/;/

9lood "&/ pp=> antigenaemia test$


28/38

Immuno4uorescense

!ositive immunofluorescence test for

rabies virus antigen. Source) "'"$

/irology Laboratory, 4ale+5ew

aven ospital$


29/38


30/38

d,antages andDisad,antages

Advantages

#esult available 2uic%ly, usually within a few hours.

!otential !roblems

Often very much reduced sensitivity compared to cell culture,

can be as low as -


31/38

Secimens +or &outine

'estsClinical Category 'lood (hroat

s)a

$aeces C%$ *ther

*. &eningitis B B B B

-. Encephalitis B B B B 9rain biopsy

(. !aralytic disease B B B B

8.

#espiratory illness B B 5asopharyngeal aspirate>. epatitis B

=. 1astroenteritis B

3. "ongenital diseases B 6rine, saliva

C. S%in lesions B B Lesion sample e.g. vesicle

fluid, s%in scrapping

D. Eye lesions Eye swab

*


32/38

Molecular Methods &ethods based on the detection of viral genome are also

commonly %nown as molecular methods. It is often said that

molecular methods is the future direction of viral diagnosis.

owever in practice, although the use of these methods is

indeed increasing, the role played by molecular methods in a

routine diagnostic virus laboratory is still small compared to

conventional methods.

It is certain though that the role of molecular methods will

increase rapidly in the near future.


33/38

Classical Molecular

'echniues 'ot+blot, Southern blot are e0amples of classical techni2ues.

7hey depend on the use of specific '5A#5A probes for

hybridization. 7he specificity of the reaction depends on the conditions used

for hybridization. owever, the sensitivity of these techni2ues

is not better than conventional viral diagnostic methods.

owever, since they are usually more tedious and e0pensive

than conventional techni2ues, they never found widespread

acceptance.


34/38

#olymerase Chain &eaction

(1) !"# allows the in vitro amplification of specific target '5A se2uences by a

factor of * to 8


35/38

#olymerase Chain &eaction

(2) Advantages of !"#)

E0tremely high sensitivity, may detect down to one viral genome per sample volume

Easy to set up

:ast turnaround time

'isadvantages of !"#

E0tremely liable to contamination

igh degree of operator s%ill re2uired

5ot easy to set up a 2uantitative assay.

A positive result may be difficult to interpret, especially with latent viruses such as"&/, where any seropositive person will have virus present in their blood

irrespective whether they have disease or not.

/ery e0pensive.


36/38

Schematic of !"#

Each cycle doubles the copy number of the target


37/38

7ther 8e%er Molecular

'echniues 9ranched '5A is essentially a sensitive hybridization techni2ue which involves

linear amplification. ?hereas e0ponential amplification occurs in !"#.

7herefore, the sensitivity of b'5A lies between classical amplificationtechni2ues and !"#. Other 5ewer molecular techni2ues depend on some form of

amplification. "ommercial proprietary techni2ues such as L"# ligase chain reaction$, 5AS9A

5ucleic Acid Se2uence 9ased Amplification$ , 7&A 7issue &icro Arrays$depend on e0ponential amplification of the signal or the target.

7herefore, these techni2ues are as susceptible to contamination as !"# and sharethe same advantages and disadvantages.

!"# and related techni2ues are bound to play an increasingly important role inthe diagnosis of viral infections.

'5A chip is another promising technology where it would be possible to detect alarge number of viruses, their pathogenic potential, and their drug sensitivity at

the same time.


38/38

Method

(arget

Ampli!ication

%ignal

Ampli!ication (hermocycling %ensiti#ity

Commercial

E+amples

,C- E0ponential 5o 4es .igh #oche Amplicor

LC- 5o E0ponential 4es .igh Abbot L"F

.A%'A E0ponential 5o 5o .igh Organon 7e%ni%a

(MA E0ponential 5o 5o .igh 1enprobe

/01-eplicase 5o E0ponential 5o .igh 5one

'ranched D.A 5o Linear 5o &edium "hiron Guantiple0

"omparison between !"# and other nucleic

acid Amplification 7echni2ues

diagnosis_of viral infection.ppt

Documents