TRADITIONAL DIAGNOSTIC TOOLS USED IN VIRAL DISEASES.

VIRAL CULTURES

- Cell cultures are derived from dispersed cells taken from original tissue and disaggregated by enzymatic, mechanical or chemical means.

- Cell cultures gained interest in virus isolation in the 1950s’, largely due to the discovery that poliovirus would proliferate in cell cultures that were not of neutral origin.

VIRAL CULTURES

-Cell cultures:

i) Are convenient and less expensive than egg and animal

ii) Are convenient for microscopic examination for cell proliferation.

iii) Provide a desirable environment for the detection and identification of many human viral pathogens.

VIRAL CULTURES

-Virus isolation in cell cultures has been the gold standard as a method for virus detection, but in recent years powerful tools have been discovered for viral detection, from development of monoclonal antibodies to the introduction of molecular diagnostics.

-The above modalities are sensitive and highly specific in viral identification.

VIRAL CULTURES

- Cell cultures can be prepared in standard 16- by 125-mm glass or plastic round-bottom screw-cap tube.

- Well known cell types found in virology laboratories include: primary rhesus monkey kidney (RhMK) cells, primary rabbit kidney cells, human lung fibroblasts (MRC-5), human foreskin fibroblasts, human epidermoid carcinoma cells (HEp-2), human lung carcinoma cells (A549), and others.

VIRAL CULTURES

-Once a clinical sample is obtained, its inoculated into several cell lines, so as to provide a suitable host for whichever virus that may be present.

-For successful virus isolation, you need to employ the appropriate selection, collection, transport, and processing of clinical samples.

VIRAL CULTURES

-You need to collect the sample with the highest titer, and preserve that sample while maintaining the viral titer and infectivity.

- In general, if you are sending specimens for virus culture, send them in virus transport medium. Do not send dry swabs for virus investigations – they will almost always be discarded.

VIRAL CULTURES

- The sample should be kept cool(2-8 degrees or on wet ice). This preserves the viral infectivity and increases the virus recovery, especially for labile viruses.

- Once the sample arrives in the lab, the liquid medium is centrifuged. Viruses tend to remain dispersed in the liquid while bacteria, fungi, cells, blood, mucus and fibers move to the bottom of the spun bottle.

VIRAL CULTURES

- The processed inoculum may be added to the culture by either simply adding 0.2 ml or 0.3 ml of the sample to each tube or by adsorption inoculation.

- In adsorbtion inoculation, the cell culture medium is decanted from the cell culture monolayer.

- The inoculum is applied directly to the monolayer. It’s incubated for 30-90 minutes in a horizontal position at 35-37degrees.

VIRAL CULTURES

- Excess inoculum is discarded and fresh cell culture medium is added.

- This process enhances adsorbtion of the viral particles and recovery of some viruses.

- The viral tube cultures are incubated for day to weeks, depending on the specimen and virus

VIRAL CULTURES

- Microscopic examination is done daily for the first week of incubation to maximize the detection of viral growth and on alternate days for the remainder of the incubation period.

CYTOPATHIC EFFECTS(CPE)

- These are morphological changes on the cell caused by viral infections.

- Some CPE can be observed in unfixed unstained cells under low power, some require fixation and staining to see the manifestation of the viral infection.

- There are several types of CPE. Note however that a virus may not conform to the norm for its family or may produce different CPE in different host cell types.

CPE Cont’d

- The rate of CPE can also be used to identify viruses;

i) Rapid viruses- the CPE appears within 1-2 days in cultures inoculated at low multiplicity of infection(<0.1).

ii) Slow viruses- the CPE appears within 4-5days in cultures inoculated at low multiplicity of infection(MOI).

CPE

- At a high MOI, all CPE can occur rapidly. Therefore, decisions about the rate of CPE should be based on the lowest MOI that produces CPE.

TYPES OF CPE

a) Total destruction

- All cells in the monolayer are destroyed. They shrink, become dense and detach(death) from the glass within 72hours.

b) Subtotal destruction

- There is detachment(death) of some of the cells but not all.

TYPES OF CPE

c) Focal degeneration

- In this case the viruses show localised areas of infection(foci).

- This focal nature is due to the direct cell to cell transfer rather than diffusion through the extracellular medium.

- Eventually the entire monolayer may be involved.

TYPES OF CPE

d) Swelling and clumping

- The infected cells enlarge and clump together in ‘grape-like’ clusters.

e) Foamy degeneration(vacuolization)

- Here the virus causes production of large and/or numerous vacuoles.

- Vacuolization is difficult to visualize without staining.

TYPES OF CPE

f) Cell fusion (syncytium or polykaryon formation)

- The plasma membrane of four or more cells fuse producing a multinucleated cell with four or more nuclei.

- Small syncytia are seen only after staining.

- The cytoplasm needs to be examined carefully so as to distinguish between cell fusion and cell clumping, where each cell has its distinct plasma membrane.

TYPES OF CPE

g) Inclusion bodies

- Indicates areas of the cell where nucleic acid or viral protein are being synthesized or where virions are being assembled.

- The areas have altered staining and cannot be seen on live cell cultures.

- -The inclusions may be single or multiple, large or small, round or irregular in shape, intracellular or intracytoplasmic, eosinophilic or basophilic, depending on the causative virus.

SEROLOGY ANTIBODY ANTIGEN TESTS

- Serological techniques that detect antibody are based on the principle of adding specific viral antigen(s) to patient serum.

- If virus-specific antibody is present in the serum then it will bind to the antigen to form an antigen/antibody complex.

- The complex is then detected using an indicator system.

SEROLOGY ANTIBODY ANTIGEN TESTS

- These tests can be reversed to detect the presence instead of viral antigen, such as hepatitis B surface antigen, in the patient’s serum.

- They include:

• Complement fixation test (CFT)

• Enzyme-linked immunosorbent assays (EIA or ELISA)

• Immunofluorescence (IF)

• Latex and gel particle agglutination

• Western blot (WB) and line assays (LIA)

Enzyme-linked immunosorbent assays (EIA or ELISA)

- These are the most widely used serological assays in routine diagnostic laboratories.

- The process:

i) Antigen is attached to the base of a plastic microtitre well (solid phase).

ii) Patient’s serum is added, if specific antibody is present in the serum it will attach to the antigen on the solid phase. Excess serum is washed off.

ELISA

iii) Anti-human antibody coupled to an enzyme is added to bind to this antibody/antigen complex. Excess enzyme is washed off.

iv) An enzyme for the substrate is added. Colorchange shows a positive reaction due to action of the enzyme attached to the complex to the substrate.

- Color change can be seen by the naked eye or measured in a spectrophotometer.

- Intensity of the color shows how much antibody is in the serum.

ELISA

- The assay can be constructed to detect either IgM or IgG, depending upon whether the anti-human antibody is directed to the IgM or IgGclass.

- ELISA can also be reversed to detect viral antigens, by coating the solid phase by antibody (mono- or polyclonal) specific for the antigen to be tested.

ELISA

- Its advantages include: they are rapid(done within 2-3hours), can be easily automated and are objective in that they can be read using a spectrophotometer.

ELISA

IMMUNOFLUORESENCE (IF)TESTS

- Use the same principle as ELISA.

- Can also be used to detect viral antigen or antibody in the patients’ serum.

- The enzyme/substrate detector used however to detect a positive reaction is fluorescein-labelled anti-human antibody which appears as apple-green fluorescence under a light microscope.

Immunofluoresence tests

- To look for viral antigen, cells from the patient’s secretions are fixed to a spot on the glass slide and fluorescein-labelled monoclonal antibody against the virus is added.

- Note that, a mixture of these monoclonal antibodies can be added at the same time to detect a panel of viruses.

Immunofluoresence tests

- Their advantage is that they are rapid serological tests, however interpretation is subjective, therefore depend on operator expertise.

LATEX AGGLUTINATION(LA) AND GELATIN PARTICLE AGGLUTINATION

TEST(GPAT)- The antigen or antibody is adsorbed on latex or

gelatin, once the patients’ serum is added, a positive reaction is noted when there’s agglutination of the particles.

Latex agglutination

Gelatin particle agglutination test

COMPLEMENT FIXATION TEST(CFT)

- Is based on the principle that an antibody/antigen complex once formed will bind complement.

- Complement activity is measured by the capacity of serum to lyse antibody optimally sensitized red blood cells.

-In presence of a titrated complement level, sensitized SRBC are lysed. When complement-activating antibodies, IgM or IgG, are present complement is fixed and unavailable to lyse the sensitized SRBC.

- It has been used in the past, but is complex and insensitive, so its being replaced with ELISA.

HEMAGGLUTINATION AND HEMAGLUTINATION INHIBITION

TESTS- They detect antibodies to viruses (rubella,

influenza) that possess a haemagglutininantigen.

- Red cells (RBC) are chemically coupled with an antigen. They are reacted with the patients’ serum, if the specific antibodies to the antigen are present, clumping of the cells occurs.

- They are relatively insensitive and give non-specific results.

HEMAGGLUTINATION AND HEMAGLUTINATION INHIBITION

TESTS- In hemagglutination inhibition tests, there’s

inhibition of agglutination of antigen coated

erythrocytes by a homologous antigen.

- Antigen is detected and its quantity determined by the degree of inhibition of agglutination following incubation with low concentrations of the antibody.

NEUTRALIZATION TESTS

- If there are virus-specific neutralizing antibodies in the serum, then the virus will be neutralized and will not be able to grow in culture.

- The patients’ serum is incubated with antibodies of known viral specificity. This mixture is inoculated into cell culture.

NEUTRALIZATION TESTS

- Presence of CPE shows that the antibodies didn’t bind, inactivate or neutralize the virus, but a lack of CPE shows that the antibodies bound, inactivated or neutralized the virus.

- Its sensitive, but labour intensive and technically demanding.

WESTERN BLOT

- Specific viral proteins are transferred on blotting paper either from a gel (western blot) or produced by recombination or peptide synthesis (line immunoassays).

- The patient serum is added and the viral antigen band on the blotting paper develops colour if specific antibody to that particular antigen is present in the serum.

- Its advantage is that its very specific and is able to distinguish antibody directed against specific virus proteins.