Several tests are necessary to confirm or rule out rabies in a human intra-vitam. Tests are performed on samples of serum, spinal fluid, skin biopsies from the nape of the neck, and saliva. Routinely, serum and spinal fluid are tested for antibodies to rabies virus. Saliva maybe tested by virus isolation or nested reverse transcription polymerase chain reaction (RT-PCR) methods.
Although it is a nonspecific and less-sensitive method of diagnosing rabies, histological examination of biopsy or autopsy tissues is occasionally useful in diagnosing unsuspected cases of rabies that have not been tested by routine methods. When brain tissue from a rabies virus-infected animal is stained with a histological stain, such as hematoxylin and eosin, a trained microscopist may recognize evidence of encephalomyelitis.
Histopathologic evidence of rabies encephalomyelitis in brain tissue and meninges includes the following:
1. Mononuclear infiltration
2. Perivascular cuffing of lymphocytes or polymorphonuclear cells
3. Lymphocyte foci
4. Babes nodules consisting of glial cells
5. Negri bodies
Rabies inclusions were detected in 1903. Dr. Adelchi Negri reported that he had identified what he believed was the etiologic agent of rabies, the Negri body. This was a round or oval inclusion body sometimes observed within the cytoplasm of nerve cells of animals infected with rabies. Negri bodies may vary in size from 0.25 to 27 µm. These inclusions are found most frequently in the pyramidal cells of Ammon’s horn, in the Purkinje cells of the cerebellum, and in the cells of the medulla and various ganglia. With Seller’s stain, Negri bodies appear magenta in color and have small (0.2 µm to 0.5 µm), dark-blue interior basophilic granules. The histological technique may detect Negri bodies in only 50% of known positive samples in contrast to the DFA which detects these inclusion bodies in essentially 99% of samples.
Mouse inoculation test
Mouse inoculation test, in spite of its simplicity, depends greatly on the accuracy of its performance for dependable results. White mice of both sexes are equally susceptible to rabies virus. Mice of all ages are susceptible to intracerebral inoculation of rabies virus, but suckling mouse up to 3 days are more sensitive. Single mouse dose for intracerebral inoculation is 0,03 ml 10% suspension of suspected brain. Observation period should be 28 days after inoculation. If mouse showing symptoms of rabies, should be killed and prepared for DFA test.
Other tests for diagnosis of rabies
Other tests for diagnosis and research, such as electron microscopy (EM), immunohistochemistry (IHC), RT-PCR, and isolation in cell culture are useful tools for studying the virus structure, molecular typing, and virulence of rabies viruses.
When samples contain a small amount of rabies virus, it may be difficult to detect the virus by routine methods. Virus isolation in cell cultures, such as mouse neuroblastoma cells (MNA) or baby hamster kidney (BHK) cells, increases the sensitivity of detection by increasing the virus concentration as virus replicates in the cell cultures. These are excellent methods for confirming the DFA test results.
Another method of amplification of rabies virus uses biochemical methods to produce many copies of the rabies virus nucleic acid. Rabies RNA can be copied into a DNA molecule using reverse transcriptase (RT). The DNA copy of rabies can be amplified using the technique of polymerase chain reaction (PCR). With this method, rabies virus RNA can be enzymatically amplified as DNA copies. Forty cycles of amplification, can produce up to 1012 times the original amount of nucleic acid in the virus sample. This technique is used to confirm DFA results and to detect rabies virus in saliva and skin biopsy samples that contain minute amounts of rabies virus.