Highlights
Rapid and accurate lab diagnosis of rabies in humans and animals is crucial for timely administration of rabies related medical care, which is also called postexposure prophylaxis (PEP). A diagnostic lab can determine if an animal has rabies within a few hours. These results can help decide if costly PEP is needed and potentially save patients from unnecessary physical, emotional, and financial stress.
Diagnosis in animals
Animals showing signs of rabies should be immediately euthanized by a professional and specimens submitted to a qualified rabies laboratory for testing. A diagnosis of rabies can be made after detection of rabies virus from any part of the affected brain, but to rule out rabies, the test must include a full cross-section of tissue from both the brain stem and cerebellum. Rabies testing requires that the animal be euthanized. There are no approved methods for ante-mortem rabies testing of animals.
In the U.S., the results of a rabies test are typically available within 24 to 72 hours after an animal is euthanized. While suspected rabies exposure requires urgent medical attention, most people with suspected rabies exposure can delay post-exposure prophylaxis until the results of this test are returned. Rabies testing of animals is reportable to state health departments and CDC.
Diagnosis in humans
Several tests are necessary to diagnose rabies antemortem (before death) in humans; no single test is sufficient. Tests are performed on samples of saliva, serum, spinal fluid, and skin biopsies of hair follicles at the nape of the neck. Postmortem (after death) testing requires the collection of brainstem and cerebellum tissues.
Due to the risk of rabies exposure to healthcare workers and close community contacts, deceased patients suspected of rabies should always undergo an autopsy and post-mortem rabies testing. Rabies testing in humans is reportable to state health departments and CDC.
Diagnostic testing methods
Detection of rabies virus antigen or RNA by any of the below methods can confirm a rabies infection, but gold standard tests should be used to rule out infection. Ruling out infection also requires that appropriate tissues are tested, which include a full cross-section of the brainstem and cerebellum for post-mortem human and animal testing. Ante-mortem human rabies testing that is negative is a strong indication that the patient does not have rabies, however, post-mortem confirmatory testing should be pursued if an alternative diagnosis is not identified.
Direct fluorescent antibody (DFA) test
The DFA test is based on the observation that animals infected by rabies virus have rabies virus proteins (antigens) present in their tissues. Because rabies is present in nervous tissue (and not blood like many other viruses), the ideal tissue to test for rabies antigen is brain tissue. Other innervated tissues may have antigens; however, tests with these tissues are less accurate at detecting rabies when compared to brain tissues.
The most important part of a DFA test is fluorescently labeled anti-rabies antibody. When labeled antibody is incubated with rabies-suspect brain tissue, it will bind to rabies antigen. Unbound antibodies can be washed away and areas where antigen is present can be visualized as fluorescent-apple-green areas using a fluorescence microscope. If rabies virus is absent there will be no staining.
Because of its high sensitivity and specificity, the DFA test is one of several "gold standard" diagnostic methods for detecting rabies and has been rigorously evaluated by international, national, and state health laboratories.
Positive DFA
Negative DFA
Direct rapid immunohistochemistry test (DRIT)
The DRIT for rabies functions similarly to the DFA test in detecting the presence of rabies virus antigen in animal tissues. Like DFA, DRIT relies on the observation that rabies virus proteins are present in infected tissues, particularly in the nervous system. The preferred tissue for DRIT testing is brain tissue due to its high concentration of rabies antigen. However, other innervated tissues may also contain the antigen, albeit with slightly lower accuracy compared to brain tissue.
The key component of the DRIT test is the rapid immunohistochemical staining with anti-rabies antibodies. These antibodies are labeled with a fluorescent or chromogenic marker and, when incubated with suspect tissue, bind specifically to rabies antigen. Excess antibodies are then washed away, and areas containing rabies antigens appear fluorescent or in color under a microscope.
Conversely, the absence of staining indicates the absence of rabies virus. Similar to DFA, DRIT boasts high sensitivity and specificity, making it a reliable diagnostic tool for rabies. It has been thoroughly evaluated by international, national, and state health laboratories, cementing its status as a "gold standard" method for rabies diagnosis.
Realtime Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
The LN34 PCR test for rabies is a newer diagnostic test that uses real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) methodology to detect the presence of rabies virus genetic material.
The best tissue for rabies testing by any test is the brain, and rabies rule-out by the LN34 test requires a full cross-section of the brainstem and representative samples from the cerebellum. Skin biopsy from the nape of the neck and saliva samples can be tested by LN34 to detect rabies in suspect antemortem human rabies cases. This innovative test targets a highly conserved region of the virus genome including the leader region and nucleoprotein gene, which ensures robust detection across all viruses that can cause rabies.
The LN34 test works by a single-tube reaction where viral genetic material is amplified into many copies and detected by a fluorescent probe. The LN34 PCR test offers numerous advantages, including its exceptional sensitivity, specificity, and rapid turnaround time. Moreover, its versatility allows for testing on a variety of sample types, including decomposed or formalin-fixed tissues, which may not be viable for other diagnostic techniques.
As with the DFA test, the best tissue for rabies testing by the LN34 test is brain tissue, and rabies rule-out requires a full cross-section of the brainstem and representative samples from the cerebellum. The World Health Organization (WHO) and World Organization for Animal Health (WOAH) recognize the LN34 test as a "gold standard" test, and it is increasingly recognized and adopted globally for rabies diagnosis and surveillance.
Immunohistochemistry (IHC)
IHC methods are sensitive and specific for the detection of rabies virus antigen in formalin-fixed tissues. Tissues fixed in formalin must first be processed by routine histologic methods, embedded in paraffin, and sectioned to formalin-fixed paraffin-embedded slides.
Rabies virus antigen is detected using specific anti-rabies monoclonal or polyclonal antibodies. IHC testing is more sensitive and specific than histologic staining methods, such as hematoxylin and eosin (H&E) and Sellers stains.
Rabies-infected neuronal cells of the brain with intracytoplasmic inclusions. The red stain indicates the presence of rabies virus antigen using the Streptavidin-biotin complex staining method.
Brain tissue testing negative for rabies by the IHC method, meaning no rabies virus antigen was detected.
Histologic examination
General histopathology
Histologic 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 rabies virus-infected animals and humans are stained with a histologic stain, such as hematoxylin and eosin, evidence of encephalomyelitis may be recognized by a trained microscopist. This method is nonspecific and not considered diagnostic for rabies.
Before current diagnostic methods were available, rabies diagnosis was made using this method in addition to the clinical case history. Most of the significant histopathologic features, or changes in tissue caused by disease, of rabies infection were described in the last quarter of the 19th century. After Louis Pasteur's successful experiments with rabies vaccination, scientists were motivated to identify the pathologic lesions of rabies virus.
Histopathologic evidence of rabies encephalomyelitis (inflammation) in brain tissue and meninges includes the following:
- Mononuclear infiltration
- Perivascular cuffing of lymphocytes or polymorphonuclear cells
- Lymphocytic foci
- Babes nodules consisting of glial cells
- Negri bodies
Perivascular cuffing or inflammation around a blood vessel. Perivascular inflammatory cell infiltrates in hematoxylin & eosin stained brain tissue. (100x Magnification)
Babes Nodules
Perivascular cuffing or inflammation around a blood vessel. Perivascular inflammatory cell infiltrates in hematoxylin & eosin stained brain tissue. (200x Magnification)
Blood vessel without inflamatory cells (200x magnification). A = Red blood cells. B = Squamous epithelial cells
Rabies serology
Who should receive routine rabies virus serological testing?
Typically, rabies virus-neutralizing antibody tests, such as the rapid fluorescent focus inhibition test, are used to monitor antibody levels in people that may have an occupational risk of rabies virus exposure (e.g., veterinarians, rabies virus laboratory workers, etc.).
In some cases, such serological testing is used to check the immune response of a person undergoing rabies postexposure prophylaxis when major deviations in the vaccination schedule occur, or there are concerns about a patient's immune status. CDC's National Rabies Reference Laboratory offers serological testing for people and animals on a case-by-case basis. Approval before submission of serum is required.
For most people and animals completing an approved rabies vaccination regimen, routine serological testing is not necessary to document seroconversion, unless:
- the person is immunosuppressed.
- significant deviations of the prophylaxis schedule have occurred [link to deviation tool].
- the patient-initiated vaccination internationally with a product of questionable quality; or
- the person's antibody status is being monitored routinely due to occupational exposure to rabies virus.
Regulation of diagnostic test kits
Recently, point-of-care diagnostic tests have become increasingly available to the public. Promising studies conducted in Africa, Asia, and the United States have found that certain point-of-care tests can demonstrate high sensitivity and specificity. However, no point-of-care test for rabies has been rigorously validated and none are approved for use by USDA, CDC's National Rabies Reference Laboratory, WHO, or WOAH.
In general, if a given diagnostic product contains all materials needed to run a particular test, including instructions on the interpretation of results, and if the intent of the manufacturer is to provide a self-contained, point-of-care product, such an application, may be considered a diagnostic kit, and is under regulation by the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), Center for Veterinary Biologics.
More information is available at the Center for Veterinary Biologics website.