s3 lab virological tests

8/8/2019 S3 Lab Virological Tests

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S3 Laboratory: Virological Tests (Overview)by Micro-Para Department

DIAGNOSTIC METHODS IN VIROLOGY

y Direct Examinationo Antigen Detection

Immunofluorescence ELISA

o Electron Microscopy Morphology Of Virus Particles Immune Electron Microscopy

o Light Microscopy Histological Appearance Inclusion Bodies

o Viral Genome Detection Hybridization With Specific Nucleic Acid

Probes

Polymerase Chain Reaction (PCR)y Indirect Examination (Virus Isolation)

o Cell Culture Cell Culture Eggs Animals

o Eggs Pocks on Cam Haemagglutination Inclusion Bodies

o Animals Disease Or Death

y Serologyo Detection of rising titres of antibody between acute

and convalescent stages of infection, or the detection

of IgM in primary infection.

Classical Techniques Newer Techniques

1. Complement fixation tests(CFT)

1. Radioimmunoassay (RIA)2. Haemagglutination inhibition

tests2. Enzyme linked

immunosorbent assay (EIA)3. Immunofluorescence

techniques (IF)3. Particle agglutination

4. Neutralization tests 4. Western Blot (WB)5. Counter-

immunoelectrophoresis5. RIBA, Line immunoassay

y Cell Cultures are most widely used for virus isolationy 3 types of cell cultures:

1. Primary cells - Monkey Kidney2. Semi-continuous cells - Human embryonic kidney and

skin fibroblasts

3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCKy Primary cell culture are widely acknowledged as the best cell

culture systems available since they support the widest range of

viruses. However, they are very expensive and it is often

difficult to obtain a reliable supply. Continuous cells are the

most easy to handle but the range of viruses supported is often

limited.

y Growing virus may produce1. CYTOPATHIC EFFECT (CPE) - such as the ballooning

of cells or syncytia formation, may be specific or non-

specific.

Cytopathic effect of enterovirus 71 and HSV in cell culture.Note the BALLOONING OF CELLS (B)

(Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)

CELL CULTURES

VIRUS ISOLATION

g

y

g

f

py

y

g

g

p

B

RSV

Measles Virus

S

S


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2. HAEMADSORPTION- cells acquire the ability to stickto mammalian red blood cells.

y Confirmation of the identity of the virus may be carried outusing neutralization, haemadsorption-inhibition or

immunofluorescence tests.

PROBLEMS WITH CELL CULTURE:

y Long period (up to 4 weeks) required for result.y Often very poor sensitivity, sensitivity depends on a large extent

on the condition of the specimen.

y Susceptible to bacterial contamination.y Susceptible to toxic substances which may be present in the

specimen.

y Many viruses will not grow in cell culture e.g. Hepatitis B,Diarrheal viruses, parvovirus, papillomavirus.RAPID CULTURE TECHNIQUES

y Available whereby viral antigens are detected 2 to 4 days afterinoculation.

y CMV DEAFF test : the best example, wherebyo The cell sheet is grown on individual cover slips in a

plastic bottle.

o Following inoculation, the bottle then is spun at a lowspeed for one hour (to speed up the adsorption of the

virus) and then incubated for 2 to 4 days.

o The cover slip is then taken out and examined for thepresence of CMV early antigens byimmunofluorescence.

VIRUSES ISOLATED BY CELL CULTURE

Viruses readily isolated Viruses less frequently isolated Herpes Simplex Varicella-Zoster Cytomegalovirus Measles Adenoviruses Rubella Polioviruses Rhinoviruses Coxsackie B viruses Coxsackie A viruses Echoviruses Influenza Parainfluenza Mumps Respiratory Syncytial Virus

106 virus particles per ml required for visualization, 50,000 - 60,000

magnification normally used. Viruses may be detected in the following

specimens.

y Faeceso Rotaviruso Adenoviruso Norwalk like viruseso Astroviruso Calicivirus

y Vesicle Fluido HSVo VZV

y Skin scrapingso Papillomaviruso Orfo Molluscum contagiosum

IMMUNE ELECTRON MICROSCOPY

y The sensitivity and specificity of EM may be enhanced byimmune electron microscopy. There are two variants:

o Classical Immune electron microscopy (IEM) - the sampleis treated with specific anti-sera before being put up for

EM. Viral particles present will be agglutinated and thus

congregate together by the antibody.

o Solid phase immune electron microscopy (SPIEM) - thegrid is coated with specific anti-sera. Virus particles

(courtesy of Linda Stannard, University of Cape Town, S.A.)

DEAFF test for CMV(Virology Laboratory, Yale-

New Haven Hospital)

Syncytial Formation(S) caused by mumps virus and Haemadsorption(H)of erythrocytes onto the surface of the cell sheet.


SYNCYTIUM FORMATION(S) in cell culture caused by RSV (top), andmeasles virus (bottom).


S

H H

Adenovirus

Rotavirus


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present in the sample will be absorbed onto the grid by

the antibody.

PROBLEMS WITH EM

y Expensive equipmenty Expensive maintenancey Require experienced observery Sensitivity often lowy Criteria for diagnosing Primary Infection

o 4 fold or more increase in titre of IgG or total antibodybetween acute and convalescent sera

o Presence of IgMo Seroconversiono A single high titre of IgG (or total antibody) - very

unreliable

y Criteria for diagnosing Reinfectiono fold or more increase in titre of IgG or total antibody

between acute and convalescent sera

o Absence or slight increase in IgMTypical Serological Profile After Acute Infection

COMPLEMENT FIXATION TEST

ELISA FOR HIV ANTIBODY

WESTERN BLOT

USEFULNESSOF SEROLOGICAL RESULTS

HIV-1 Western BlotLane1: Positive ControlLane 2: Negative ControlSample A: NegativeSample B: IndeterminateSample C: Positive

Microplate ELISA for HIV antibody:Colored wells(C) indicate REACTIVITY

Complement Fixation Test in Microtiter Plate.Rows 1 and 2 exhibit complement fixation obtained with

acute and convalescent phase serum specimens,respectively. (2-fold serum dilutions were used) Theobserved 4-fold increase is significant and indicates

Note that during reinfection, IgM may be absent orpresent at a low level transiently

SEROLOGY

c


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y How useful a serological result is depends on the individualvirus.

y For example, for viruses such as rubella and hepatitis A, theonset of clinical symptoms coincide with the development of

antibodies. The detection of IgM or rising titres of IgG in the

serum of the patient would indicate active disease.

y However, many viruses often produce clinical disease beforethe appearance of antibodies such as respiratory and diarrhoeal

viruses. So in this case, any serological diagnosis would be

retrospective and therefore will not be that useful.y There are also viruses which produce clinical disease months or

years after seroconversion e.g. HIV and rabies. In the case of

these viruses, the mere presence of antibody is sufficient to

make a definitive diagnosis.

PROBLEMS WITH SEROLOGY

y Long period of time required for diagnosis for paired acute andconvalescent sera.

y Mild local infections such as HSV genitalis may not produce adetectable humoral immune response.

y Extensive antigenic cross-reactivity between related viruses e.g.HSV and VZV, Japanese B encephalitis and Dengue, may lead to

false positive results.y immunocompromised patients often give a reduced or absent

humoral immune response.

y Patients with infectious mononucleosis and those withconnective tissue diseases such as SLE may react non-

specifically giving a false positive result.

y Patients given blood or blood products may give a false positiveresult due to the transfer of antibody.

y Used mainly for the diagnosis of herpes simplex and VZVencephalitis

y CSF normally contain little or no antibodiesy presence of antibodies suggest meningitis or

meningoencephalitis

CSF antibody titre >_1_is indicative of Meningitis

Serum antibody titre100

y Diagnosis depends on the presence of an intact blood-brainbarrier

y Nasopharyngeal Aspirateo RSVo Influenza A and Bo Parainfluenzao Adenovirus

y Faeceso Rotaviruseso Adenoviruseso Astrovirus

y Skino HSV

o VZVy Blood

o CMV (pp65 antigenaemia test)

CMV pp65 Antigenaemia Test

(Virology Laboratory, Yale-New Haven

Positive immunofluorescence test for

rabies virus antigen. (Source: CDC)

IMMUNOFLUORESCENSE

RAPID DIAGNOSIS BASED ON THE DETECTION OF VIRAL ANTIGENS

CSF ANTIBODIES


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ADVANTAGES

y Result available quickly, usually within a few hours.DISADVANTAGES

y Often very much reduced sensitivity compared to cell culture,can be as low as 20%. Specificity often poor as well.

y Requires good specimens.y The procedures involved are often tedious and time-consuming

and thus expensive in terms of laboratory time

Clinical Category BloodThroatswab

Faeces CSF Other

1. Meningitis + + + +2. Encephalitis + + + + Brain biopsy3. Paralytic

disease+ + + +

4. Respiratoryillness

+ +Nasopharyngeal

aspirate

5. Hepatitis +6. Gastroenteritis +7. Congenital

diseases+ Urine, saliva

8. Skin lesions + + Lesion sample e.g.vesicle fluid, skinscrapping

9. Eye lesions Eye swab10. Myocarditis + Pericardial fluid11. Myositis + +12. Glandular fever +13. Post Mortem + Autopsy

After use, swabs should be broken into a small bottle containing 2 ml

of virus transport medium. Swabs should be sent to the laboratory as

soon as possible without freezing. Faeces, CSF, biopsy or autopsy

specimens should be put into a dry sterile container.

y Methods based on the detection of viral genome are alsocommonly known as molecular methods. It is often said that

molecular methods is the future direction of viral diagnosis.

y However in practice, although the use of these methods isindeed increasing, the role played by molecular methods in a

routine diagnostic virus laboratory is still small compared to

conventional methods.

y It is certain though that the role of molecular methods willincrease rapidly in the near future.

CLASSICAL MOLECULAR TECHNIQUES

y Dot-blot, Southern blot, in-situ hydridization are examples ofclassical techniques. They depend on the use of specificDNA/RNA probes for hybridization.

y The specificity of the reaction depends on the conditions usedfor hybridization. However, the sensitivity of these techniques is

not better than conventional viral diagnostic methods.

y However, since they are usually more tedious and expensivethan conventional techniques, they never found widespread

acceptance.

POLYMERASE CHAIN REACTION

y PCR allows the in vitro amplification of specific target DNAsequences by a factor of 106 and is thus an extremely sensitive

technique.

y It is based on an enzymatic reaction involving the use ofsynthetic oligonucleotides flanking the target nucleic sequence

of interest.

y These oligonucleotides act as primers for the thermostableTaq polymerase. Repeated cycles (usually 25 to 40) of

denaturation of the template DNA (at 94oC), annealing of

primers to their complementary sequences (50o

C), and primerextension (72oC) result in the exponential production of the

specific target fragment.

y Further sensitivity and specificity may be obtained by thenested PCR.

y Detection and identification of the PCR product is usuallycarried out by agarose gel electrophoresis, hybridization with a

specific oligonucleotide probe, restriction enzyme analysis, or

DNA sequencing.

y Advantages of PCR:o Extremely high sensitivity, may detect down to one

viral genome per sample volume

o Easy to set upo Fast turnaround time

y Disadvantages of PCRo Extremely liable to contaminationo High degree of operator skill requiredo Not easy to set up a quantitative assay.o A positive result may be difficult to interpret,

especially with latent viruses such as CMV, where any

seropositive person will have virus present in their

blood irrespective whether they have disease or not.

y These problems are being addressed by the arrival ofcommercial closed systems such as the Roche Cobas Amplicor

which requires minimum handling. The use of synthetic internal

competitive targets in these commercial assays has facilitated

the accurate quantification of results. However, these assays

are very expensive.

Schematic of PCR

Each cycle doubles the copy number of the target

MOLECULAR METHODS

SPECIMENSFOR ROUTINE TESTS


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OTHER NEWER MOLECULAR TECHNIQUES

y Branched DNA is essentially a sensitive hydridization techniquewhich involves linear amplification. Whereas exponential

amplification occurs in PCR.

y Therefore, the sensitivity of bDNA lies between classicalamplification techniques and PCR. Other Newer molecular

techniques depend on some form of amplification.

y Commercial proprietary techniques such as LCR, NASBA, TMAdepend on exponential amplification of the signal or the target.

y Therefore, these techniques are as susceptible to contaminationas PCR and share the same advantages and disadvantages.

y PCR and related techniques are bound to play an increasinglyimportant role in the diagnosis of viral infections.

y DNA chip is another promising technology where it would bepossible to detect a large number of viruses, their pathogenic

potential, and their drug sensitivity at the same time.

COMPARISON BETWEEN PCR AND OTHER NUCLEIC ACID

AMPLIFICATION TECHNIQUES

MethodTarget

AmplificationSignal

Amplification Thermocycling SensitivityCommercialExamples

PCRExponential

No Yes HighRocheAmplicor

LCR NoExponential

Yes HighAbbotLCX

NASBAExponentia

lNo No High

Organon

Teknika

TMAExponential

No No High Genprobe

Q-Replicase

NoExponential

No High None

BranchedDNA No Linear No Medium

ChironQuantiple

x

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