Laboratory Diagnosis The diagnosis of animal and human rabies can be made by 4 methods: (1) histopathology (2) virus

cultivation (3) Serology (4) virus antigen detection. Although each of the first 3 methods have distinct advantages, none provide a rapid definitive diagnosis. Tissues infected with rabies virus are currently identified most rapidly and accurately by means of immunofluorescence or immunoperoxidase staining using antirabies 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. 1. Histopathology - A definitive pathologic diagnosis of rabies can be based on the finding of Negri bodies in the brain or the spinal cord. They are sharply demarcated, more or less spherical, and 210 m 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. Negri bodies are pathognomonic of rabies. However, Negri bodies are only present in 71% of cases.

Figure: Negri body in body of neuron

2. Virus cultivation - Available tissue 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-day) 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. The most definitive means of diagnosis is by virus cultivation from infected tissue. Tissue culture lines, such as WI-38, BHK-21, or CER. Since rabiesvirus induce

minimal CPE, IF is routinely used to detect the presence of rabiesvirus Ag in the tissue culture. The more commonly used method for virus isolation is by the inoculation of saliva, salivary gland tissue and brain tissue intracerebrally into infant mice. The mice should develop paralysis and death within 28 days. Upon death, the brains are examined for the presence of the virus by immunofluorescence. 3. Serology - Serum antibodies to rabies can be detected by immunofluorescence or 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. Circulating antibodies appear slowly in the course of infection but they are usually present by the time of onset of clinical symptoms. The most commonly used serological tests were the mouse infection neutralization test (MNT) or the rapid fluorescent focus inhibition test (RFFIT). These tests have now been largely superseded by EIAs. Serology had been reported to be the most useful method for the diagnosis of rabies.

Figure: Positive IF test for rabies antigen (Source: CDC)

4. Rapid virus antigen detection - in recent years, virus antigen detection by IF had become widely used. The potentially infected tissue is incubated with fluorescein-labeled antibody. The cells are examined by fluorescent microscopy for the presence of fluorescent intracytoplasmic inclusions. The specimens which are usually used are corneal impressions (obtained by gently abrading the cornea with a microscopic slide) or neck skin biopsy (the cells examined are the sensory nerves). In an American series, IF of corneal impressions or neck skin impressions was

diagnostic only in 50% of cases early in the course of the clinical illness. 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 humanely 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.