Laboratory diagnosis of viral infection

LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

 

Specimen collection and transport:

 

            Viral shedding is usually greatest during the early stages of infection, so the best specimens are those collected as early as possible. Specimens should be collected as aseptically as possible. Aspirated secretions are often preferable, but swabs are easier to use for collection. Tissue samples must be kept moist. Viral transport medium can be used. It is optimal to process viral specimens for culture immediately. However, if specimens cannot be processed immediately after collection, they should be stored at 40 C. Specimens should not be frozen unless a significant delay (greater than 4 days) in processing occurs. In that case, specimens should be frozen and held in a –700 C freezer. Do not freeze specimens at –200 C. This temperature facilitates the formation of ice crystals, which disrupts the host cells and result in significant loss of viral viability.

 

Appropriate Specimens:

 

A commonsense approach in selecting specimens for isolation is to collect the specimens from the infected site. However, if systemic, congenital, or generalized disease is involved, then specimens from multiple sites, including blood and CSF, as well as from the portals of entry or exit are appropriate.

 

Methods in Diagnostic virology:

            Three major methods are used to diagnose viral infections:

  • Direct detection of the virus in the clinical specimen.
  • Serologic antibody assays to detect viral antibodies.
  • Isolation of the virus in culture.

 

  1. Direct detection:

Direct detection methods are generally not as sensitive as culture methods.

Immunostaining:

Immunofluorescence can be a flexible tool used to detect various viral agents directly in clinical specimens. Some tests use direct fluorescence antibody, in which fluorescence labeled antibodies are incubated on fixed smears of host cells. A more specific test is the indirect immunofluorescent antibody test, which uses an unlabeled antibody to the virus that is then detected by a second immunolabeled antibody.

Enzyme immunoassay:

Various tests are available, but EIA test are often less sensitive than culture or fluorescent immunoassays (FA), so negative results are confirmed with culture, FA or gene amplification.

 

Optical immunoassay:

OIAs use silicon wafers coated with antibody as a means of viral detection. Virus combines with antibodies on the wafer and produces a reflection change in the silicon wafer.

Nucleic acid probes:

Nucleic acid hybridization tests can detect viruses from various clinical specimens.

Gene amplification assays:

Various gene amplification techniques are available for the amplification and detection of viruses. Different technologies include PCR, branched DNA (bDNA) and Nucleic acid sequence based amplification (NASBA).

Electron microscopy:

It is now becoming rare asset in clinical laboratories, yet it can detect noncultivable viruses from clinical specimens.

 

 

 

Light microscopy:

Many viruses form characteristic inclusion bodies in infected cells. These inclusions can be detected in cell scrapings from infected sites, e.g., Negri bodies in rabies.

 

  1. Serologic assays:

Viral serology provide limited information and certain problems are inherent in the methods.

  1. Serologic assays measure host response rather than detect virus.
  2. The antibody producing capabilities of human host varies widely.
  3. The antibody level does not necessarily correlate with the acuteness or activity of the infection.

Paired sera demonstrating seroconversion or a four-fold rise in titer are

required to establish a diagnosis of recent infection.

 

  1. Viral isolation:

In clinical virology, isolating virus is still the gold standard technique.

Traditionally three methods are used for isolation of viruses in diagnostic virology: cell culture, animal inoculation, and embryonated eggs. Cell culture techniques are most commonly used. Animal inoculation is extremely costly and used only as a special resource and in reference or research laboratories. Embryonated eggs are rarely used.

 

Cell cultures for viral isolation:

Cell cultures can be divided into three categories: primary, diploid, and continuous. Primary cell culture is obtained from tissue removed from an animal. The tissue is finely minced and then treated with an enzyme to further disperse individual cells. The cells are then seeded onto a surface to form a monolayer. With primary cell lines, only very limited cell division occurs.  The commonly used primary cell culture is primary monkey kidney (PMK). Diploid cell cultures can divide, but passage is limited to 50 generations. Human neonatal lung (HNL) is a standard diploid cell culture used in clinical virology. Continuous cell cultures have variable numbers of chromosomes and are capable of indefinite passage. Hep2 (human laryngeal carcinoma), A549 (human lung cancer) and Vero (monkey kidney) are example of continuous cell lines used in diagnostic virology.

 

Cytopathic effects on cell cultures for presumptive identification of viral agents:

Cell cultures can be used for presumptive identification because of a characteristic CPE that viruses produce on certain cells.

 

Virus

Cell culture CPE
Herpes simplex virus Hep2 Large, rounded cells
Cytomegalovirus Human diploid

Fibroblast

Large, rounded cells
Varicella zoster virus Human diploid

Fibroblast

Foci or rounded cells;

possible syncytia

Enterovirus Hep2 Refractile, round cells in clusters
Adenovirus Hep2 Large, rounded cells in clusters
RSV Hep2 Syncytia

 

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