To date, there are no tests available to diagnose human rabies infection ante-mortem, or before the onset of clinical disease. However, rabies should be included in the differential diagnosis of all patients who present with unexplained, acute, progressive viral encephalitis, even in areas where the disease is not endemic.

WHO defines a clinical case of rabies as a subject presenting with an acute neurological syndrome (i.e. encephalitis) dominated by forms of hyperactivity (i.e. furious rabies) or paralytic syndromes (i.e. dumb rabies), progressing towards coma and death, usually by cardiac or respiratory failure, typically within 7-10 days after the first sign, if no intensive care is instituted.

As diagnosis based on clinical ground alone is difficult and often unreliable; it is recommended to confirm a clinical case of rabies through the use of laboratory-based techniques. For post mortem diagnosis, the gold-standard diagnostic technique is to detect rabies virus antigen in infected tissues, preferably brain smears or touch impressions collected from a biopsy, by fluorescent antibody test (FAT). FAT is recommended by WHO and in 95-99% of cases, gives reliable results on fresh specimens within a few hours. Other methods for detection of lyssavirus antigens such as direct rapid immunohistochemistry tests are proven to have sensitivity and specificity comparable to the FAT. WHO recommends further development of direct rapid immunohistochemistry tests as an alternative to the FAT for improved decentralized laboratory-based surveillance in endemic areas.

Ante-mortem diagnosis, or diagnosis of rabies during life (by intra-vitam techniques) is difficult and dependent on widespread dissemination of virus through the nervous system. It is strongly discouraged for rabies diagnosis in animals as sensitivity varies widely according to the stage of the disease, immunological status, intermittent viral excretion and training of the technical staff.

Fluorescent antibody (FA) test

A quick and easy procedure for the diagnosis of rabies is the use of a suitable dye for the detection of Negri bodies. Histopathological techniques have, however, been replaced in most laboratories by the fluorescent antibody (FA) test, which was first developed in 1958 by Goldwasser & Kissling.

The FA test is now the most widely used method for diagnosing rabies infection in animals and humans. It is based on microscopic examination, under ultraviolet light, of impressions, smears or frozen sections of brain or nervous tissue after treatment with antirabies serum or globulin conjugated with fluorescein isothiocyanate. The test is accurate and results can often be obtained within 30 minutes of receipt of the specimen, although for routine purposes a period of 2-4 hours is desirable for the fixation in cold acetone.

Apart from an appropriate microscope, the two main requirements for success in using this technique are well trained personnel and conjugated antiserum or globulin of good quality. After one year’s experience, most laboratories find over 99% agreement between the FA test and the mouse inoculation (MI) test. In the first year, however, some laboratories may miss up to 10% or even 20% of the positives with the FA test and for this reason, both tests (MI and FA) should be run in parallel during this period.

Stringent control of the labelled antirabies antibodies should be carried out to determine the specificity of the fluorescence and to minimize the number of false-positives. Appropriate tissue sampling is also important. Examination of impressions or smears of tissue samples from Ammon’s horn and brain stem are recommended. Labelled rabies antibodies can be prepared against the whole rabies virus. More highly potent antisera can be prepared using purified and concentrated rabies virus or virion components such as ribonucleoprotein.

Conjugated monoclonal rabies antibodies are being increasingly used in routine diagnosis. The specificity of these latter conjugates is greater than those prepared against the whole virion or virion components. A conjugate composed of two labelled monoclonal antibodies is now widely used and is available commercially. Panels of monoclonal antibodies are also used in studying the epidemiology of rabies.

The FA technique is a highly sensitive method for detecting rabies antigen in fresh specimens. However, it may also be performed on fixed specimens. The specimen should be treated with one or more proteolytic enzymes such as trypsin or pepsin before staining to unmask the antigenic sites. The sensitivity of the test using fixed specimens has been reported to be 90-100% of that obtained using fresh specimens. However, it is recommended that fresh tissue be examined where possible.

When specimens are received in 50% glycerol-saline, it is imperative that the tissue be washed several times in saline before staining.

Fixed rabies viruses can grow in a wide variety of cells. Successful in vitro cultivation of rabies virus was first reported in 1936. This property has been used extensively in research on rabies. However, it is only recently that techniques for the isolation of street rabies from suspect material in cell cultures have been developed. Tests for the isolation of street rabies in cell culture were first carried out in the mid 1970s using baby hamster kidney cells, line 21 (BHK-21), and chick embryo-related (CER) and neuroblastoma cells.

These studies demonstrated that rabies infection could be detected by immunofluorescence from as early as 4-5 hours up to 5 days following inoculation. Furthermore, it was found that BHK-21 cells were comparable in sensitivity to mice, whereas neuroblastoma cells were more sensitive than mice to infection by street rabies virus. The difference in sensitivity between neuroblastoma cells and BHK-21 cells and other cell lines was reported to be associated with the neural origin of the former. However, Webster & Casey suggested that the difference may also be related to viral strain differences, as well as to cell type.

In routine rabies diagnosis, positive specimens contain amounts of antigen that can easily be detected by the FA test, the MI test or virus isolation in neuroblastoma cells. In the latter case, the result is obtained within 18-24 hours, although rabies antigen may be detected in these cells by FA as early as 4-5 hours after inoculation.

Furthermore, virus isolation in cell culture has been shown to be as efficient as the FA test and the MI test for demonstrating small amounts of rabies virus. However, specimens containing a small amount of rabies virus and which are negative by FA and subsequently positive by virus isolation in cell culture require an incubation period of 4 days after inoculation of the cells.

In view of the usually short delay in obtaining the result, isolation of rabies virus in cell culture should replace intracerebral mouse inoculation whenever possible. It should, however, be borne in mind that only laboratories where cell-culture techniques are currently used can successfully maintain neuroblastoma cells for diagnosis.

In the rabies field the enzyme-linked immunosorbent assay (ELISA) was initially developed for the titration of rabies virus-neutralizing antibodies. The technique was applied to the quantification of rabies antigen by Atanasiu et al using fluorescein-labelled IgG to the purified nucleocapsid. Subsequently, Perrin et al developed an ELISA called rapid rabies enzyme immunodiagnosis (RREID), which was based upon the detection of rabies virus nucleocapsid antigen in brain tissue. In this test, microplates are coated with purified IgG and an IgG-peroxidase conjugate is used to react with immunocaptured antigen.

This technique was compared with the FA test in a collaborative study involving six laboratories in Europe and North America. The study showed a good correlation between the FA test and RREID, although the latter test was less sensitive.

A further study was organized to evaluate the RREID under conditions prevailing in rabies laboratories in developing countries. The study found over 96% agreement between the FA test and RREID. Similar results were obtained in a study on more than 3000 specimens. It should be noted, however, that in view of its lower sensitivity, RREID should not replace FA in laboratories where FA is already performed.

RREID is a simple and relatively cheap technique, which can be especially useful for epidemiological surveys. It may be used to examine partially decomposed tissue specimens for evidence of rabies infection, but it cannot be used with specimens that have been fixed in formalin. Since the antigen can be visualized with the naked eye, the test can be carried out in laboratories that do not have the necessary equipment for FA tests.

Monoclonal antibodies are produced by hybridomas of fused mouse myeloma cells and splenocytes from mice immunized with either the rabies virus or rabies-related viruses. They were first produced by Wiktor et al in 1978. These hybridomas secreted monoclonal antibodies directed against the glycoprotein (G protein) or nucleocapsid of rabies virus.

Detailed information on the production and use of monoclonal antibodies displayed specific reactivity patterns which were used to characterize and classify rabies and rabies-related viruses into groups corresponding to antigenic determinants. Since then, other hybridomas have been produced and different panels of monoclonal antibodies have been established to allow differentiation of rabies virus isolates from terrestrial and bat host species in the USA, western Europe, and, to a lesser extent, Africa, Asia, eastern Europe and Latin America. Between 1982 and 1990, WHO- coordinated collaborative studies on the use of monoclonal antibodies in rabies diagnosis and research.

These studies led to the establishment of two panels of monoclonal antibodies, allowing identification of the various lyssavirus serotypes and the differentiation of major virus strains used for vaccine production from field virus isolates. An additional panel of monoclonal antibodies was also selected to differentiate rabies viruses isolated from terrestrial animal species from those isolated from European bat species.

Although monoclonal antibodies are mainly used for epidemiological investigations, they were found to be very useful for rabies diagnosis in certain circumstances, such as imported cases of human rabies and rabies associated with uncertain exposure, and also routinely in countries where large-scale programmes for oral vaccination of foxes are under way to establish that no infections are caused by the vaccine strain.

In addition to the brain and spinal cord, rabies virus antigen can be detected by FA in the peripheral nerves, salivary glands, saliva, and also in the cornea and skin during the final stages of the disease. Intra vitam diagnosis of rabies by FA in corneal impressions was first described by Schneider in animals and by Cifuentes et al in humans. However, a study of the reliability of corneal impressions for rabies diagnosis showed that, especially when sampling is done under field conditions, a negative result could not rule out the diagnosis of rabies.

Examination of skin biopsy material was shown to be a valuable technique for intra vitam diagnosis of rabies in animals and humans. Anderson et al showed that rabies antigen may be detected in skin biopsies from humans at the onset of clinical signs. In contrast, Blenden et al found that only some patients (25-50%) showed positive results during the early phase of clinical illness and that the proportion of positive results increased as the disease progressed.

Specimens for intra vitam diagnosis should be of a good quality. They should be refrigerated immediately after collection and until the test is carried out. This is important, since partially autolysed specimens will reduce the percentage of positive results and contamination of the material may lead to false-positive results. Examination of skin biopsies may also be used for post-mortem diagnosis in countries where opening of the skull of the dead person is not accepted by relatives on cultural or religious grounds.

Reference test

Serum neutralization assays are used to determine the potency of rabies serum and immunoglobulins for post-exposure treatment, and to evaluate the immunogenicity of human and, to a lesser degree, animal rabies vaccines. The standard procedures recommended at the seventh meeting of the WHO Expert Committee on Rabies were the mouse neutralization test (MNT) and the plaque reduction assay.

Since then, plaque reduction methods have been superseded by fluorescent focus inhibition tests, which are more convenient. Although the MNT is still widely used as a reference test, the rapid fluorescent focus inhibition test (RFFIT) has become the test of choice in most modern laboratories. The RFFIT has been shown to be at least as sensitive as the MNT in measuring virus-neutralizing antibodies.