Diagnostic tests in virology

Diagnostic Methods in Virology

n          Direct detection

n          Examination of clinical specimen for virus particles, virus antigen, or viral nucleic acids

n          Indirect examination

n          Isolation and growth virus

n          Serology

n          Detection of rising titers of antibody or of IgG

n          Diagnosis of common viral infections

Direct examination

p   Called rapid diagnostic methods

n   Result within same or next day

p   Useful when patient care depends on lab results

n   Diagnosis of RSV infection in neonates

n   CMV infections in immunocompromised patients

1.     Antigen Detection

n     immunofluorescence, ELISA etc.

2. Electron Microscopy         

n     morphology of virus particles

n     immune electron microscopy

3. Light Microscopy

n     histological appearance of infected cells

n     inclusion bodies within infected cells

4. Viral Genome Detection

n     hybridization with specific nucleic acid probes

n     polymerase chain reaction (PCR)

Antigen detection:

p        immunofluorescence testing for respiratory viruses

n         RSV, flu A, flu B, and adenoviruses

p        detection of rotavirus antigen in feces

p        pp65 CMV antigenaemia test

p        detection of HSV and VZV in skin scrapings

p        detection of HBsAg in serum.

Advantages and Disadvantages

Advantages

p    Assays are rapid to perform

p    Result available quickly, usually within a few hours.

Potential Problems

p    Sensitivity and specificity may be poor.

p    Requires good specimens for test to work properly.

p    Procedures involved are often tedious and time-consuming and thus expensive in terms of laboratory time.

Immunofluorescense

CMV pp65 antigenaemia test

Electron Microscopy (EM)

p   Virus particles are detected and identified on the basis of morphology.

p   Mainly used for diagnosis of viral gastroenteritis

n   detect viruses in feces

p  rotavirus, adenovirus, astrovirus, calicivirus and Norwalk-like viruses.

p   Occasionally used for detection of viruses in vesicles and other skin lesions

n   herpesviruses and papillomaviruses.

Electron Microscopy

p10⁶ virus particles per ml required for visualization

p 50,000 - 60,000 magnification used.

pViruses may be detected in the following specimens.

Feces                                      Rotavirus, Adenovirus

                                                Norwalk like viruses

                                                Astrovirus, Calicivirus                                       

Vesicle Fluid                        HSV

                                                VZV

Skin scrapings                    papillomavirus, orf

                                                molluscum contagiosum

Electron micrographs

Immune Electron Microscopy

p       The sensitivity and specificity of EM enhanced by immune electron microscopy

p       There are two variants:-

p       Classical Immune electron microscopy (IEM)

n       Sample treated with specific anti-sera.

n       Viral particles agglutinated and congregated together by antibody.

p       Solid phase immune electron microscopy (SPIEM)

n       grid is coated with specific anti-sera.

n       Virus particles in sample will be absorbed onto grid by the antibody.

Problems with Electron Microscopy

p    Expensive equipment

p    Expensive maintenance

p    Require experienced observer

p    Sensitivity often low (large vral count required)

Light Microscopy

p   Replicating virus produce histological changes in infected cells.

n   changes characteristic or non-specific.

p   Viral inclusion bodies

n   collections of replicating virus particles

n   Examples of inclusion bodies include:

p  the negri bodies found in rabies

p  cytomegalic inclusion bodies (CMV infections).

p   Histology serve as useful adjunct in diagnosing viral infections

Viral Genome Detection

Molecular techniques for direct detection of viral genome

p       Classical molecular techniques:

n       Dot-blot and Southern-blot

p      Use specific DNA or RNA probes for hybridization.

p      Specificity depends on conditions used for hybridization.

p      May allow for quantification of DNA/RNA in specimen.

p       Newer molecular techniques:

n       Polymerase chain reaction (PCR) 

p      amplification of target nucleic acid, or the signal itself.

p      extremely sensitive technique: sensitivity down to 1 DNA molecule

n       PCR has many problems

p      Contamination - give false positive result.

p      Positive result may not indicate disease

§      Particularly with latent viruses 

Indirect Examination

Involves isolation and growth of virus:

1. Cell Culture               cytopathic effect  (CPE) 

                                      hemabsorption

                                      immunofluorescence

2. Eggs                          pocks on CAM

                                      hemagglutination

                                      inclusion bodies

3. Animals                     disease or death

Virus Isolation

p       Cell Cultures are most widely used for virus isolation

p       3 types of cell cultures:

p     Primary cells - Monkey Kidney

§       best cell culture systems; support the widest range of viruses.

§       very expensive; often difficult to obtain a reliable supply.

p     Semi-continuous cells - Human embryonic kidney and skin fibroblasts

p     Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCK

§      most easy to handle; range of viruses supported is often limited

Identification of growing virus

Growing virus is detected by:

n   Cytopathic Effect (CPE)

p  ballooning of cells or syncytia formation

p  may be specific or non-specific

§    HSV and CMV produces a specific CPE, whereas enteroviruses do not.

n   Hemadsorption

p  cells acquire ability to stick to mammalian red blood cells.

p  mainly used for detection of influenza and parainfluenza viruses.

n   Confirm identity of virus using:

p  neutralization, hemadsorption- inhibition, immunofluorescence, or molecular tests.

Cytopathic Effect (1)

Cytopathic Effect (2)

Hemadsorption

Problems with cell culture

n         Long period (up to 4 weeks) required for result.

n         Often very poor sensitivity

p       sensitivity depends on the condition of the specimen.

n         Susceptible to:

p       bacterial contamination.

p       toxic substances which may be present in the specimen.

n         Many viruses will not grow in cell culture

p       Hepatitis B, diarrheal viruses, parvovirus, papillomavirus.

Rapid Culture Techniques

p         viral antigens are detected 2 to 4 days after inoculation.

p         CMV DEAFF test is the best example

p      cell sheet is grown on individual cover slips in a plastic bottle.

p      Following inoculation, bottle is spun at a low speed for one hour (to speed up the adsorption of the virus) and then incubated for 2 to 4 days.

p      cover slip is taken out and examined for the presence of CMV early antigens by immunofluorescence.  

DEAFF test for CMV

Viruses Isolated by Cell Culture

Serology

p  mainstay of viral diagnosis.

p  primary humoral immune response to antigen.

n   Following exposure, first antibody to appear is IgM, followed by a much higher titer of IgG.

n   Reinfection

p level of specific IgM either remain the same or rises slightly.

p IgG shoots up rapidly and far more earlier than in a primary infection.

Typical Serological Profile After Acute Infection

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

Serology

Detection of rising titers of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection.

Serology

p   Types of serological tests available.

n   EIA and RIA - look specifically for IgM or IgG,

n   CFT and HAI - can only detect total antibody, which comprises mainly IgG.

p   Some of these tests are much more sensitive than others:

n   EIAs and radioimmunoassays - most sensitive tests available,

n   CFT and HAI tests - not so sensitive.

n   Newer techniques - EIAs offer better sensitivity, specificity and reproducibility

p   Sensitivity and specificity of assays depend greatly on the antigen used.

Serology

Criteria for diagnosing Primary Infection

p    A significant rise in titer of IgG or total antibody between acute and convalescent sera

p    Presence of IgM – rapid means of diagnosis

p    Seroconversion – antibody negative state to a positive state

p    A single high titer of IgG (or total antibody) - very unreliable because hard to define cut off

Serology

Criteria for diagnosing Reinfection

n    significant increase in titer of IgG or total antibody between acute and convalescent sera

n    Absence or slight increase in IgM

p   Often difficult to differentiate re-infection/re-activation from a primary infection.

n   Important in cases such as rubella infection in pregnant women in first trimester

p  Primary infection – associated with high risk of fetal damage whereas re-infection is not

Complement Fixation Test

ELISA for HIV antibody

Microplate ELISA for HIV antibody:

pcolored wells indicate reactivity (darker the color, the higher the reactivity)

Western Blot

HIV-1 Western Blot

p    Lane1: Positive Control

p    Lane 2: Negative Control

p    Sample A: Negative

p    Sample B: Indeterminate

p    Sample C: Positive

Limitations of serological diagnosis

p    How useful a serological result is depends on the individual virus.

n    rubella and hepatitis A

p   onset of clinical symptoms coincide with the development of antibodies.

p   detection of IgM or rising titers of IgG in the serum of the patient would indicate active disease.

n    many viruses often produce clinical disease before the appearance of antibodies

p   respiratory and diarrheal viruses

p   any serological diagnosis would be retrospective and therefore will not be that useful.

n    Some viruses produce clinical disease months or years after seroconversion

p   HIV and rabies

p   mere presence of antibody is sufficient to make a definitive diagnosis.

Problems associated with serology:

p     long length of time required for diagnosis for paired acute and convalescent sera

p     mild local infections may not produce a detectable humoral immune response

n    HSV genitalis

p     immunocompromised patients often give a reduced or absent humoral immune response.

p     Extensive antigenic cross-reactivity between related viruses may lead to false positive results

n    HSV and VZV

n    Japanese B encephalitis and Dengue

p     Patients with infectious mononucleosis and those with connective tissue diseases may react non-specifically giving a false positive result

p     Patients given blood or blood products may give a false positive result due to the transfer of antibody.

Antibody in the CSF

p   In a healthy person, there should be little or no antibodies in the CSF.

n   With viral meningitis or encephalitis, antibodies may be produced against the virus by lymphocytes in the CSF.

n   The

CSF antibodies

p         Used mainly for the diagnosis of herpes simplex and VZV encephalitis

p         CSF normally contain little or no antibodies

p         presence of antibodies suggest meningitis or meningoencephalitis (produced by lymphocytes)

CSF antibody titer  _>  _1_  is indicative of meningitis

    ^{Serum antibody titer    100}

p         This depends on an intact blood-brain barrier.

Rapid Diagnosis Based on the Detection of Viral Antigens

Nasopharyngeal Aspirate          RSV

                                                Influenza A and B

                                                Parainfluenza

                                                Adenovirus

Feces                                       Rotaviruses

                                                Adenoviruses

                                                Astrovirus

Skin                                         HSV

                                                VZV

Blood                                       CMV (pp65 antigenaemia test)