Rabies diagnosis is critical for both animal and human health, requiring precise and timely testing to manage potential exposures and prevent the spread of this deadly virus. As there are no approved ante-mortem rabies tests for animals, suspected cases presenting clinical signs necessitate immediate euthanasia by trained professionals, followed by laboratory submission of specimens for definitive diagnosis. Accurate diagnosis relies on detecting the rabies virus within brain tissues, specifically requiring examination of both the brain stem and cerebellum to effectively rule out rabies.
Rabies Diagnosis in Animals: Post-Mortem Testing is Key
In veterinary medicine, rabies testing is exclusively performed post-mortem. When an animal exhibits symptoms suggestive of rabies, the standard protocol involves humane euthanasia by animal control or veterinary experts. Following euthanasia, specific tissue samples must be collected and submitted to a certified rabies laboratory. To ensure diagnostic accuracy and to definitively rule out rabies, testing protocols mandate the inclusion of a complete cross-section of tissue from both the brain stem and cerebellum. It is crucial to understand that ante-mortem rabies testing in animals is not currently possible with approved methods.
Turnaround time for rabies test results in the U.S. typically ranges from 24 to 72 hours post-euthanasia. While suspected rabies exposure is a medical emergency demanding prompt attention, individuals with potential exposure can generally defer post-exposure prophylaxis until test results confirm or exclude rabies in the animal involved. Importantly, all animal rabies testing is a reportable condition, mandated to be communicated to state health departments and the Centers for Disease Control and Prevention (CDC).
Rabies Diagnosis in Humans: Ante-Mortem and Post-Mortem Approaches
Diagnosing rabies in humans is a complex process, often requiring multiple tests, especially when performed ante-mortem (before death). No single test can definitively confirm rabies in a living person. Diagnostic samples for ante-mortem testing in humans include saliva, serum, spinal fluid, and skin biopsies, specifically from hair follicles at the nape of the neck. Post-mortem diagnosis, similar to animal testing, involves the collection of brainstem and cerebellum tissues.
Given the significant risk of rabies exposure to healthcare personnel and close contacts within the community, autopsy and post-mortem rabies testing are mandatory for deceased patients suspected of having rabies. Like animal cases, human rabies diagnoses must be reported to state health departments and the CDC to facilitate public health surveillance and response.
Diagnostic Testing Methods for Rabies Detection
Confirmation of rabies infection hinges on the detection of rabies virus antigen or RNA through various laboratory methods. However, to confidently rule out rabies, employing gold standard tests and ensuring the examination of appropriate tissues are paramount. For both post-mortem human and animal testing, this necessitates a complete cross-section of the brainstem and cerebellum. While a negative ante-mortem rabies test in humans strongly suggests the absence of rabies, confirmatory post-mortem testing remains essential if an alternative diagnosis is not established.
Direct Fluorescent Antibody (DFA) Test: A Gold Standard for Rabies Diagnosis
The Direct Fluorescent Antibody (DFA) test is a cornerstone in rabies diagnosis. It operates on the principle that rabies-infected tissues contain rabies virus proteins, known as antigens. As rabies primarily affects the nervous system, brain tissue is the optimal sample for DFA testing. While other innervated tissues may contain antigens, brain tissue analysis provides superior accuracy.
The core reagent in the DFA test is a fluorescently labeled anti-rabies antibody. When this labeled antibody is applied to rabies-suspect brain tissue, it binds to any rabies antigen present. After washing away unbound antibodies, areas with rabies antigen become visible as fluorescent apple-green regions under a fluorescence microscope. The absence of fluorescence indicates the absence of rabies virus.
The DFA test’s high sensitivity and specificity have established it as a gold standard diagnostic method for rabies, validated extensively by international, national, and state health laboratories.
Positive Direct Fluorescent Antibody (DFA) Test for Rabies
Negative Direct Fluorescent Antibody (DFA) Test for Rabies
Direct Rapid Immunohistochemistry Test (DRIT): Rapid and Reliable Rabies Detection
The Direct Rapid Immunohistochemistry Test (DRIT) mirrors the DFA test in its approach to detecting rabies virus antigen in animal tissues. DRIT, like DFA, leverages the presence of rabies virus proteins within infected tissues, particularly in the nervous system. Brain tissue remains the preferred sample for DRIT testing due to its high antigen concentration. Though other innervated tissues can be used, brain tissue offers greater diagnostic accuracy.
DRIT employs rapid immunohistochemical staining using anti-rabies antibodies labeled with a fluorescent or chromogenic marker. These antibodies selectively bind to rabies antigen in suspect tissues. Following incubation and removal of excess antibodies, areas containing rabies antigens are visualized under a microscope, appearing fluorescent or colored depending on the marker used. Conversely, no staining signifies the absence of rabies virus. DRIT, with its high sensitivity and specificity, is a dependable rabies diagnostic tool, recognized as a gold standard method by international, national, and state health laboratories.
Real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR): Advanced Molecular Rabies Diagnosis
The LN34 PCR test represents a cutting-edge diagnostic approach for rabies, utilizing real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) to detect rabies virus genetic material.
Brain tissue is the preferred sample for rabies testing regardless of the method, and the LN34 test for rabies rule-out necessitates a complete cross-section of the brainstem along with representative samples from the cerebellum. In living humans suspected of rabies, skin biopsies from the nape of the neck and saliva samples can be analyzed using LN34. This innovative assay targets a highly conserved region of the rabies virus genome, encompassing the leader region and nucleoprotein gene, ensuring broad detection across all rabies-causing viruses.
The LN34 test operates as a single-tube reaction, amplifying viral genetic material into numerous copies, which are then detected by a fluorescent probe. The LN34 PCR test offers significant advantages, including exceptional sensitivity, specificity, and rapid results. Its adaptability extends to testing diverse sample types, including decomposed or formalin-fixed tissues, which may be unsuitable for other diagnostic techniques.
Similar to the DFA test, brain tissue is optimal for LN34 testing, and ruling out rabies requires examination of a full cross-section of the brainstem and cerebellum samples. The World Health Organization (WHO) and the World Organisation for Animal Health (WOAH) recognize the LN34 test as a gold standard, with increasing global adoption for rabies diagnosis and surveillance.
Immunohistochemistry (IHC): Rabies Antigen Detection in Fixed Tissues
Immunohistochemistry (IHC) methods offer sensitive and specific detection of rabies virus antigen in formalin-fixed tissues. Formalin-fixed tissues require processing through routine histologic methods, embedding in paraffin, and sectioning into formalin-fixed paraffin-embedded slides before IHC analysis.
Rabies virus antigen detection in IHC relies on specific anti-rabies monoclonal or polyclonal antibodies. IHC testing surpasses traditional histologic staining methods like hematoxylin and eosin (H&E) and Sellers stains in both sensitivity and specificity for rabies diagnosis.
Positive Immunohistochemistry (IHC) Test for Rabies: Rabies-infected neuronal cells of the brain showing intracytoplasmic inclusions (red stain indicates rabies virus antigen).
Negative Immunohistochemistry (IHC) Test for Rabies: Brain tissue showing no rabies virus antigen detected.
Histologic Examination: Traditional Rabies Diagnostic Insights
General Histopathology for Rabies
Histologic examination of biopsy or autopsy tissues can occasionally aid in diagnosing unsuspected rabies cases that have not undergone routine rabies-specific testing. When brain tissue from rabies-infected animals and humans is stained with histologic stains such as hematoxylin and eosin, a trained microscopist may identify signs of encephalomyelitis. However, this method is non-specific and not considered a definitive rabies diagnostic test.
Before the advent of current advanced diagnostic techniques, rabies diagnosis relied on this method in conjunction with clinical history. Significant histopathologic features of rabies infection were largely described in the late 19th century, spurred by Louis Pasteur’s rabies vaccination success and the subsequent drive to understand the disease’s pathological lesions.
Histopathologic evidence of rabies encephalomyelitis (brain inflammation) in brain tissue and meninges includes:
- Mononuclear cell infiltration
- Perivascular cuffing of lymphocytes or polymorphonuclear cells
- Lymphocytic foci
- Babes nodules (glial cell clusters)
- Negri bodies (pathognomonic inclusions, though not always present)
Perivascular Cuffing in Rabies Encephalomyelitis (100x Magnification): Inflammatory cell infiltrates around a blood vessel in hematoxylin & eosin stained brain tissue.
Babes Nodules in Rabies Encephalomyelitis
Perivascular Cuffing in Rabies Encephalomyelitis (200x Magnification): Inflammatory cell infiltrates around a blood vessel in hematoxylin & eosin stained brain tissue.
Normal Blood Vessel in Brain Tissue (200x magnification): Absence of inflammatory cells. A = Red blood cells. B = Squamous epithelial cells.
Rabies Serology: Monitoring Antibody Levels
When is Rabies Virus Serological Testing Necessary?
Rabies virus-neutralizing antibody tests, such as the rapid fluorescent focus inhibition test, are primarily used to monitor antibody levels in individuals at occupational risk of rabies exposure (e.g., veterinarians, rabies laboratory personnel).
Serological testing may also be indicated to assess immune response following rabies post-exposure prophylaxis, particularly when significant deviations from the standard vaccination schedule occur or if there are concerns about a patient’s immune status. The CDC’s National Rabies Reference Laboratory offers serological testing for humans and animals on a case-by-case basis, requiring prior approval before serum submission.
Routine serological testing is generally not needed for most individuals and animals who complete a standard rabies vaccination regimen to confirm seroconversion, except in specific scenarios:
- Immunocompromised individuals
- Significant deviations from the recommended prophylaxis schedule
- Vaccination initiated internationally with products of uncertain quality
- Routine monitoring of antibody status due to occupational rabies virus exposure
Regulation of Rabies Diagnostic Test Kits
Point-of-care rabies diagnostic tests are becoming increasingly accessible. While promising studies in Africa, Asia, and the United States suggest high sensitivity and specificity for certain point-of-care tests, no rabies point-of-care test has undergone rigorous validation or received approval from the USDA, CDC’s National Rabies Reference Laboratory, WHO, or WOAH.
Diagnostic products containing all necessary materials for testing, including result interpretation instructions, intended for self-contained, point-of-care use, are classified as diagnostic kits. In the United States, these kits are regulated by the United States Department of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), Center for Veterinary Biologics. Further details are available on the Center for Veterinary Biologics website.
