Laboratory diagnosis of Rabies Virus Infection

Laboratory diagnosis of human or animal rabies has to be based on the following findings:

  1. Demonstration of virus antigens (Ag) or nucleic acid from brain, spinal cord, salivary glands, saliva, cornea or skin by means of immunofluorescence or polymerase chain reaction (PCR).
  2. Postmortem demonstration of Negri bodies in brain tissue.
  3. Isolation of virus from brain tissue and/or saliva.
  4. Antibodies (Ab) cannot usually be demonstrated before manifestation of disease, and in many cases serological tests are also negative throughout the clinical course.
Rabies antigens or nucleic acid
Tissues infected with rabies virus are currently identified most rapidly and accurately by means of immunofluorescence or immunoperoxidase staining using anti rabies monoclonal antibodies. A biopsy specimen is usually taken from the skin of the neck at the hairline. Impression preparations of brain or cornea tissue may be used.

 A definitive pathologic diagnosis of rabies can be based on the findings of negri bodies in the brain or the spinal cord. They are sharply demarcated, more or less spherical, and 2-10µm in diameter, and they have a distinctive internal structure with basophilic granules in an eosinophilic matrix. Negri bodies contain rabies virus antigens and can be demonstrated by immunofluorescence. Both negri bodies and rabies antigen can usually be found in animals or humans infected with rabies, but they are rarely found in bats.

Fluorescent antibody technique (FAT) on human brain smear positive for rabies
Fluorescent antibody technique (FAT) on human brain smear positive for rabies
Reverse transcription- polymerase chain reaction (RT-PCR) testing can be used to amplify parts of a rabies virus genome from fixed or unfixed brain tissue. Although unusual as a diagnostic test, sequencing of amplified products allows identification of the infecting virus strain.
Virus isolation
Available tissue sample is inoculated intracerebrally into suckling mice. Infection in mice results in encephalitis and death. The central nervous system of the inoculated animal is examined for Negri bodies and rabies antigen. In specialized laboratories, hamster and mouse cell lines can be inoculated for rapid (2 to 4 days) growth of rabies virus;
This is much faster than virus isolation in mice. An isolated virus is identified by fluorescent antibody tests with specific antiserum. Virus isolation takes too long to be useful in making a decision about whether to give vaccine.
Serum antibodies to rabies can be detected by immunofluorescence or Neutralisation( Nt) tests. Such antibodies develop slowly in infected persons or animals during progression of the disease but promptly after vaccination with cell derived vaccines. Antibodies in cerebrospinal fluid are produced in rabies- infected individuals but not in response to vaccination.
Animal observation
All animals considered rabid or suspected rabid should be sacrificed immediately for laboratory examination of neural tissues. Other animals should be held for observation for 10 days. If they show any signs of encephalitis, rabies, or unusual behavior, they should be killed  and the tissues examined in the laboratory. If they appear normal after 10 days, decisions must be made on an individual basis in consultation with public health officials.

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