Laboratory Diagnosis

1.         Approaches to Diagnosis of Viral diseases

a.         Virus Isolation:

i.          the clinical virologist provides living systems in which viruses will proliferate.

ii.          cell cultures are the living system used in most clinical laboratories.

iii.         although the viral particles cannot be seen as they replicate in cell cultures, their presence is signaled by changes in the appearance of the virus-infected cells.

iv.         these changes, called cytopathogenic effect (CPE), can be detected when cells are viewed with an ordinary light microscope.

v.         CPE may be detectable in 24 to 48 hours with viruses such as herpes simplex virus and some of the enteroviruses; however, many viruses require 7 to 10 days or more for detection.

b.         Viral antigen detection:

i.          use of immunological techniques, which rely on the specific reactivity of known viral antibodies with their antigens, to demonstrate that viral antigens are present.

ii.          the binding of antibodies and antigens is signaled to the clinical virologist through reactions such as agglutination, fluorescence, or color change, which can be observed visually or measured using a spectrophotometer.

iii.         viral antigens present within infected cells in viral cultures or in samples taken directly from the infected individual can be detected and identified.

iv.         the process usually takes a few hours to complete.

c.         Viral serology:

i.          use of standard serological methods to detect specific antibodies that have been produced by the host in response to viral infection or exposure.

ii.          by demonstrating that antibodies are present or that there is a change to antibody level, the virologist can provide valuable information concerning viral disease status.

iii.         this approach is useful in circumstances in which the virus cannot be conveniently isolated in cell culture nor its antigens detected by routine viral antigen detection methods.

d.         Molecular diagnostics:

i.          through application of molecular probes, which are labeled DNA sequences that are complementary to unique portions of viral DNA, viral nucleic acids within infected cells can be detected and identified.

ii.          this technology paired with polymerase chain reaction, allows detection of minuscule quantities of viral nucleic acids that may be missed by traditional viral assays.

2.         Need for Laboratory Diagnosis

a.         Precise diagnosis needed for prescription of appropriate antiviral chemotherapy.

b.         Management of the patient or prognosis is influenced by the diagnosis – abortion is recommended if rubella is diagnosed in the first trimester of pregnancy.

c.         Infections may demand public health measures to prevent spread to others – blood banks routinely screen for HIV and hepatitis B and C viruses which may be present in blood donated by symptomless carriers.

d.         Surveillance of viral infections may determine their significance, natural history, and prevalence in the community, with a view to establishing priorities and means of control, and monitoring and evaluating immunization programs.

e.         Continuous surveillance of the community may provide evidence of new epidemics, new diseases, new viruses, or new virus-disease associations.

3.         Collection, Packaging and Transport of Specimens

a.         Successful isolation and identification of a viral agent from clinical material depend on proper care in selecting, collecting and transporting the specimen to the laboratory.

b.         Time of Collection:

i.          virus shedding is greatest during the acute stage of illness; therefore, specimens should be collected as soon as possible after the onset of illness.

ii.          the chance of viral recovery is best during the first 3 days after onset and is greatly reduced with many viruses beyond 5 days.

c.         Site of collection:

i.          the selection of the specimen is based on the site of infection or clinical syndrome or the virus suspected.

ii.          specimens such as blood, throat swabs, spinal fluid, stools, vesicle fluid, lesion scrapings, and urine are usually submitted for virus isolation.

iii.         swabs are used for collection of many types of samples, such as those from the nose or throat or from skin or genital lesions.

iv.         cerebrospinal fluid may yield virus in cases of meningitis.

v.         biopsy or autopsy specimens may be taken by needle or knife from any part of the body for virus isolation, or snap-frozen for immunofluorescence.

d.         Collection of Specimens:

e.         Packaging of specimens:

i.          because of the lability of many viruses, specimens intended for virus isolation must always be kept cold and moist.

ii.          immediately after collection the swab should be swirled around in a small screw-capped bottle containing virus transport medium.

iii.         this medium consists of a buffered balanced salt solution, to which has been added protein (e.g. gelatin or albumin) to protect the virus against inactivation, and antibiotics to prevent the multiplication of bacteria and fungi.

iv.         the swab stick is then broken off aseptically into the fluid, the cap is tightly fastened and secured with adherent tape to prevent leakage.

f.          Transport of specimens:

i.          if a transit time of more than an hour is anticipated the container should be sent refrigerated with ‘cold packs’ (4C) or ice in a thermos flask or styrofoam box.

ii.          international or transcontinental transport of important specimens, particularly in hot weather, generally requires that the container be packed in dry ice to maintain the virus in a frozen state.

4.         Sequence of Viral isolation Procedures

5.         Microscopic Identification

a.         Viruses can be detected and identified by direct microscopic examination of clinical specimens such as biopsy material or skin lesions.

b.         Light microscopy:

i.          light microscopy can reveal characteristic inclusion bodies or multinucleated giant cells.

ii.          the Tzanck smear shows herpesvirus-induced multinucleated giant cells in vesicular skin lesions.

iii.         largion virions, e.g. Guarnieri bodies of poxviruses.

iv.         inclusion bodies representing viral aggregates.

v.         detection of viral antigen, e.g. by immunofluorescent mononuclear antibody.

c.         UV microscopy is used for fluorescent-antibody staining of the virus in infected cells.

d.         Electron microscopy detects virus particles, which can be characterized by their size and morphology.

6.         Cytopathogenic Effect

a.         Many viruses infect susceptible cell cultures and produce degenerative cellular changes, which can be observed microscopically – these changes are called cytopathogenic effect (CPE) of the virus.

b.         CPE appears because the virus integrates itself into the cellular nucleic acid and then directs the cell to manufacture viral components rather than the cellular components needed by the cell for its own maintenance.

c.         Without the needed components, the cell degenerates, and the cellular morphology changes – these changes are viral CPE.

d.         The CPE of a virus may involves all parts of the cell and is usually constant for a specific virus and type of cell culture.

e.         Types of CPE:

6.         Detecting Cytopathogenic effect-negative Viruses

a.         Because some viruses may produce CPE slowly or not at all, alternative methods have been devised for detecting the presence of viruses when CPE is absent.

b.         Hemadsorption:

i.          during replication, some viruses may discretely alter cell surfaces.

ii.          viral hemagglutinating proteins, specified by the viral genome, are expressed on the plasma membranes of virus-infected cells.

iii.         these same hemagglutinating proteins are present on the envelope of the infecting viruses and are responsible for the affinity for erythrocytes that occurs in infected cells.

iv.         the hemadsorption procedure is used to test cell culture cells for their affinity for erythrocytes.

c.         Hemadsorption testing:

i.          the cell culture medium is removed from infected cell cultures and replaced with a suspension of erythrocytes (usually guinea pig).

ii.          the culture is refrigerated for 30 minutes at 4C and observed microscopically; then the tubes are incubated at 35C for 30 minutes and observed again.

iii.         if a hemadsorbing virus has altered the surfaces of the cells, the erythrocytes will not adhere to uninfected cells.

iv.         it is used as a screening method for respiratory viruses such as influenza and parainfluenza during seasons when the respiratory viruses are prevalent.

d.         Plaquing:

i.          a viral suspension is added to a monolayer of cultured cells.

ii.          the cell culture is then covered with a nutrient agar mixture containing a vital dye, usually neutral red, which is taken up by living cells.

iii.         as the virus replicates and destroys the cells, the neutral red is released by dead or dying cells, leaving cleared areas called plaques.

e.         Hemagglutination:

i.          some viruses, or an antigen derived from them, are capable of binding to erythrocytes through complementary receptor sites on the erythrocyte surface.

ii.          the binding of virus and erythrocyte is called viral hemagglutination.

iii.         in hemagglutination testing, a viral suspension is diluted and mixed with a suspension of erythrocytes of the appropriate species.

iv.         the mixture is incubated and the erythrocytes settle to the bottom of the tube.

v.         unagglutinated erythrocytes form a pellet in the center of the bottom of the tube; agglutinated erythrocytes make a layer of small clumps, which covers the bottom of the tube.

7.         Enzyme Immunoassay

a.         Enzyme immunoassay (ELA) or enzyme-linked immunosorbent assay (ELISA) are designed in different formats to detect antigen or antibody.

b.         Most are solid-phase ELAs; the ‘capture’ antibody is attached (by simple absorption or by covalent bonding) to a solid substrate, typically the wells of polystyrene or polyvinyl microtiter plates, so facilitating washing steps.

c.         Direct ELA:

i.          virus and soluble viral antigens from the specimen are allowed to adsorbed to the capture antibody.

ii.          after unbound antigen has been washed away, an enzyme-labeled antiviral antibody is added.

iii.         various enzymes can be linked to the antibody; horseradish peroxidase and alkaline phosphatase are commonly used.

iv.         after a final washing step, readout is based on the color change that follows addition of an appropriate organic substrate for the particular enzyme.

v.         the test can be made quantitative by serially diluting the antigen to obtain an end point, or by using spectrophotometry to measure the amount of enzyme-conjugated antibody bound to the captured antigen.

d.         Indirect ELA:

i.          they are widely used because of their greater sensitivity and avoidance of the need to label each antiviral antibody in the laboratory repertoire.

ii.          here, the detector antibody is unlabeled, and a further layer, labeled (species-specific) anti-immunoglobulin, is added as the ‘indicator’ antibody.

e.         Radioimmunoassay:

i.          the only significance is that the label is not an enzyme but a radioactive isotope such as I-125, and the bound antibody is measured in a gamma counter.

ii.          the RIA is a highly sensitive and reliable assay that lends itself well to automation, but the cost of the equipment and the health hazard of working with radioisotopes argue against its use in small laboratories.

8.         Immunofluorescence

a.         Principles:

i.          the antibody is labeled with a fluorochrome.

ii.          the antigen-antibody complex, when excited by light of short wavelength, emits light of a particular longer wavelength.

iii.         the emitted light is visualized as fluorescence in an ordinary microscope when lights of all other wavelengths is filtered out.

b.         Direct Immunofluorescence:

i.          a frozen tissue section, or an acetone-fixed cell smear or monolayer on a coverslip, is exposed to fluorescein-tagged antiviral antibody.

ii.          unbound antibody is then washed away, and the cells are inspected by light microscopy using a powerful ultraviolet / blue light source.

iii.         the apple-green light emitted from the specimen is revealed (against a black background) by incorporating filters in the eyepieces that absorb all the blue and ultraviolet incident light.

c.         Indirect Immunofluorescence:

i.          it differs in that the antiviral antibody is untagged.

ii.          it binds to antigen and is itself recognized by fluorescein-conjugated anti-immunoglobulin.

iii.         the high affinity of avidin for biotin can also be exploited in immunofluorescence by coupling biotin to antibody and flurescein to avidin.

d.         Advantages:

i           .though technically demanding, immunofluorescence has proved to be of great value in the early identification of viral antigens in infected cells taken from patients with diseases with a small number of possible etiological agents.

ii.          there is no difficulty in removing partly detached infected cells from the mucous membrane of the upper respiratory tract, genital tract, eye, or from the skin, simply by swabbing or scraping the infected area with reasonable firmness.

iii.         cells are also present in mucus aspirated from the nasopharynx.

e.         Respiratory infections with paramyxoviruses, orthomyxoviruses, adenoviruses, and herpesviruses are particularly amendable to rapid diagnosis by immunofluorescence.

f.          Immunofluorescence can also be applied to infected tissue, for example, brain biopsies for the diagnosis of herpes simplex encephalitis or measles SSPE.

9.         Virus Neutralization

a.         The neutralization test is based on the principle that a live virus, when acted on by its specific antibody, will be neutralized and thus incapable of infecting susceptible cells.

b.         The neutralization procedure is performed in two stages:

i.          first stage: live virus and antibodies are reacted.

ii.          second stage: aliquots of stage one mixture are inoculated into susceptible cell cultures.

c.         After a suitable incubation period of 5 to 7 days, the host tissue is examined for the presence of cytopathogenic effect.

d.         If antibodies bind to the virus in stage one, the virus is neutralized and prevented from infecting the susceptible cell culture to produce a cytopathogenic effect.

e.         If antibodies do not bind to the virus in stage one, the virus remains active and infects the susceptible cells to produce a cytopathogenic effect.

f.          Neutralization can be used for identification of either unknown virus or viral antibodies:

i.          when an unknown virus is to be identified, a suspension of the unknown virus is mixed with antibodies of known specificity in stage one of testing.

ii.          if unknown antibodies are to be identified, a suspension of known virus is mixed with the patient’s serum that contains the antibodies of unknown specificity.

10.       Complement Fixation

a.         It is used to identify viral antigens or viral antibodies.

b.         The complement fixation assay is based on the principle that complement, a lytic agent, is bound (fixed) by antigen-antibody complexes.

c.         Complement fixation is a two-stage examination:

i.          stage one: antibody, antigen, and complement are mixed and incubated for 18 to 24 hours; if the antibody is specific for the antigen, antigen-antibody complexes form, and the complement is fixed.

ii.          stage two: antibody-coated erythrocytes, which serve as antigen-antibody complexes, are added to the stage one mixture and incubated for 1 to 2 hours.

iii.         if the complement was fixed by antigen-antibody complexes in stage one, it is no longer active or available to act on the antibody-coated erythrocytes, and the erythrocytes remain intact and are not hemolyzed.

d.         Therefore, an absence of hemolysis indicates that the antigen and antibody of stage one were specific for each other.

e.         Clinical Application

i.          when complement fixation is used for identification of unknown antibodies, the antigen in stage one is a suspension of known virus, and the antibody is supplied by adding the patient’s serum.

ii.          it is commonly used for measurement of antibodies against influenza and parainfluenza viruses.

11.       Hemagglutination Inhibition

a.         The hemagglutinating capacity of a virus is blocked when the virus is reacted with specific antibody.

b.         Hemagglutination inhibition assays are two-stage assays.

c.         Stage 1:

i.          the live hemagglutinating virus is mixed with antibody.

ii.          if this antibody is specific for the virus, the antibody attaches to the virus and inhibits it so that it is unable to hemagglutinate.

d.         Stage 2:

i.          erythrocytes are added to the stage one mixture.

ii.          the antibody-coated (inhibited) viruses are unable to hemagglutinate the erythrocytes.

iii.         when the antibodies in stage one are not specific for the virus, there is no binding, and the virus remains active and uninhibited.

iv.         when erythrocytes are added in stage two, the active (uninhibited) virus hemagglutinates the erythrocyte.

e.         This technique is applicable only to viruses with hemagglutinating ability.

f.          The most common application of hemagglutination inhibition is in the identification of viral antibodies, including measles, mumps, and rubella antibodies.

12.       Immunoblotting

a.         Also known as Western blotting, it is the ultimate refinement to test for antibodies against all of the proteins present in a particular virus.

b.         Principles:

i.          combines electrophoretic separation techniques with antibody detection methods.

ii.          blotting refers to the transfer of DNA, RNA, or protein from electrophoretic gels to a membrane.

iii.         blotting is used to prepare the antigen, and an immunoassay method is used to react antibodies with the blotted antigen, to identify, or characterize either the blood antigen or the antibodies.

c.         Steps to Western blotting:

i.          purified virus is solubilized with the anionic detergent sodium dodecyl sulfate (SDS) and the constituent proteins separated into discrete bands according to molecular size by polyacrylamide gel electrophoresis (SDS-PAGE).

ii.          the separated proteins are electrophoretically transferred (‘blotted’) onto a nitrocellulose membrane to immobilize them.

iii.         finally, the test serum is allowed to bind to the viral proteins on the membrane, and their presence is demonstrated using a radio-labeled or enzyme-labeled anti-species antibody.

d.         Evaluation of Results:

i.          reactivity is signaled by the presence of colored bands at appropriate positions and of sufficient intensity on the strip.

ii.          the test bands are compared with bands produced by positive control samples to characterize the bands.

iii.         the immunoblotting technique is used extensively in clinical virology in the confirmatory testing for HIV-1 antibodies.

e.         Immunoblotting permits demonstration of antibodies to some or all of the proteins of any given virus and can be used to:

i.          discriminate between infection with closely related viruses sharing certain antigens.

ii.          monitor the presence of antibodies to different antigens at different stages of infection.

13.       Applications of Serology

a.         A significant rise in antibody titer between serum samples is indicative of recent infection:

i.          a serum sample is obtained as soon as a viral etiology is suspected (acute-phase).

ii.          a second sample is obtained 10-14 days later (convalescent-phase).

iii.         if the antibody titer in the convalescent-phase serum sample is at least 4-fold higher than the titer in the acute-phase serum sample, the patient is considered to be infected.

iv.         an antibody titer on a single sample does not distinguish between a previous infection and a current one.

b.         Because of the necessary interval between the two specimens, a diagnosis is provided only in retrospect.

c.         However, there are two situations when serology is still the diagnostic method of choice despite the delay involved in waiting for a rising titer to make itself apparent:

i.          when it is not practicable to attempt cultivation of viruses that are notoriously difficult to isolate, e.g. some togaviruses and bunyaviruses.

ii.          management of the case is not urgent, e.g. in a woman with a rash during the first 4 months of pregnancy a clear demonstration of a rising antibody titer against rubella virus constitutes a strong indication for abortion.

d.         There are also a number of situations when finding antibody in a single specimen of serum can be diagnostic:

i.          when specific antibody of the IgM class is found.

ii.          when a seriously ill patient recently returned from abroad is found to have antibodies against an exotic virus, such as Lassa or Ebola virus.

iii.         in the case of certain persistent infections, e.g. HIV, where it is known that most or all infections progress, the presence of antibody can be used as a reliable diagnostic marker of ongoing infection.

14.       IgM class-specific Antibody Assays

a.         A rapid diagnosis can be made on the basis of a single acute-phase serum by demonstrating virus-specific antibody of the IgM class.

b.         Because IgM antibodies appear early after infection but drop to low levels within 1-2 months and generally disappear within 3 months, they are diagnostic of recent (or chronic) infection.

c.         Moreover, if found in a newborn baby, they are diagnostic of intrauterine infection, because maternal IgM, unlike IgG, does not cross the placenta.

d.         ELA, RIA, and immunofluorescence are the generally employed immunoassays to render IgM class-specific.

e.         A particular problem with IgM assays is interference by the rheumatoid factor (RF), which is antibody, mainly of the IgM class, directed against the constant region (Fc) of normal IgG.

f.          RF produces false positives in IgM immunoassays, because it binds to antiviral IgG in human serum, forming IgM-IgG complexes, which in turn bind the anti-human IgM employed as detector antibody in the assay format.

g.         To minimize the impact of RF, it is advisable to employ a reverse IgM  assay, in the simplest form of which monoclonal anti-human IgM is used as the capture antibody and labeled virus as the detector/indicator.

15.       Serological Procedures used in Virology

16.       Advantages and Disadvantages of various diagnostic methods