How Do They Do Rabies Test

Rabies testing is a critical procedure for diagnosing this deadly viral disease, which affects the central nervous system. The methodologies used are complex and require specialized laboratory settings to ensure accurate results. This article explores the detailed processes of how rabies tests are conducted, providing insights into the scientific underpinnings and practical applications of these vital diagnostic tools.

The Importance of Rabies Testing

Rabies is a zoonotic disease, meaning it can be transmitted from animals to humans. The rabies virus, primarily spread through the saliva of infected animals, poses a significant threat due to its almost invariably fatal outcome if left untreated. Early and accurate diagnosis is paramount in preventing the virus from progressing to the point where it causes irreversible neurological damage. Rabies testing serves several crucial purposes:

• Confirmation of Infection: Testing confirms the presence of the rabies virus in animals suspected of carrying the disease, enabling prompt intervention and preventing further spread.
• Public Health Surveillance: Surveillance programs rely on testing to monitor the prevalence of rabies in animal populations, guiding public health strategies and vaccination campaigns.
• Post-Exposure Prophylaxis (PEP) Decisions: Testing results influence decisions regarding PEP for individuals potentially exposed to the virus, helping to prevent the onset of the disease in humans.

Sample Collection for Rabies Testing

The accuracy of rabies testing hinges on the quality of the sample collected. The preferred sample type varies depending on whether the test is performed on an animal suspected of having rabies or on a human.

Animal Samples

For animals, the gold standard for rabies testing is the brain tissue. The primary reason for this is that the rabies virus has a predilection for the central nervous system. Here are the steps and considerations involved in collecting brain tissue:

1. Euthanasia: Animals suspected of having rabies must be humanely euthanized. This step is necessary because testing requires direct access to brain tissue, which cannot be obtained from a living animal.
2. Brain Removal:
   • The head is carefully separated from the body to facilitate brain removal.
   • Using appropriate personal protective equipment (PPE), such as gloves and face shields, a trained professional makes an incision to access the skull.
   • The skull is opened using specialized tools to expose the brain.
   • The entire brain is carefully removed, ensuring minimal damage to the tissue.
3. Sample Preservation:
   • The brain tissue is typically divided into multiple samples to allow for various testing methods or for reference purposes.
   • One of the most common methods of preservation is freezing. Samples are placed in sterile containers and frozen at -20°C or lower.
   • Another method is to preserve the tissue in a fixative solution, such as 10% buffered formalin. This method is used when histological examination is required.
4. Shipping:
   • Samples must be packaged and shipped in accordance with international regulations for transporting infectious substances.
   • Triple packaging is generally required, including a primary leak-proof container, a secondary leak-proof container, and a rigid outer packaging.
   • Appropriate labels, such as "Biological Substance, Category B," must be affixed to the package, along with the necessary documentation.

Human Samples

In humans, rabies testing is performed on samples collected from living individuals or during post-mortem examinations. The types of samples and methods of collection differ to accommodate these circumstances.

1. Ante-Mortem Samples:
   • Saliva: Saliva samples can be collected using a sterile swab. The swab is inserted into the oral cavity to collect saliva, which is then placed in a sterile container.
   • Skin Biopsy: A small skin biopsy is taken from the nape of the neck. This area is chosen because rabies virus antigens can often be detected in the cutaneous nerves at the base of hair follicles.
   • Cerebrospinal Fluid (CSF): CSF is collected via lumbar puncture. This procedure is invasive and is performed only when necessary. CSF can be used to detect rabies antibodies or viral RNA.
   • Serum: Blood samples are collected to obtain serum, which is tested for the presence of rabies antibodies.
2. Post-Mortem Samples:
   • Brain Tissue: As with animal samples, brain tissue is the preferred sample for post-mortem rabies diagnosis. Samples are collected and preserved following similar procedures as those used for animals.

Rabies Testing Methods

Several laboratory methods are used to detect the rabies virus. These methods vary in their sensitivity, specificity, and the type of information they provide. Here are some of the most commonly used rabies testing methods:

1. Direct Fluorescent Antibody (DFA) Test

The DFA test is the gold standard for rabies diagnosis. It is rapid, sensitive, and highly specific. The test is based on the principle of detecting rabies virus antigens in brain tissue using fluorescent-labeled antibodies.

1. Sample Preparation:
   • A small section of brain tissue is placed on a microscope slide.
   • The tissue is fixed, typically with acetone, to preserve the cellular structure and prevent degradation.
2. Antibody Staining:
   • The fixed tissue is incubated with fluorescent-labeled antibodies that are specific to the rabies virus.
   • These antibodies bind to rabies virus antigens, if present in the tissue.
3. Microscopy:
   • The slide is examined under a fluorescent microscope.
   • If rabies virus antigens are present, they will appear as bright green or yellow-green fluorescent particles against a dark background.
4. Interpretation:
   • The presence of fluorescent particles indicates a positive result, confirming the presence of the rabies virus.
   • The absence of fluorescence indicates a negative result.

2. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

RT-PCR is a molecular technique used to detect the presence of the rabies virus RNA in various types of samples. It is highly sensitive and specific, making it a valuable tool for rabies diagnosis.

1. RNA Extraction:
   • RNA is extracted from the sample (e.g., brain tissue, saliva, CSF) using commercially available kits.
   • The extracted RNA is purified to remove any contaminants that could interfere with the PCR reaction.
2. Reverse Transcription:
   • The extracted RNA is converted into complementary DNA (cDNA) using reverse transcriptase enzyme.
   • This step is necessary because PCR requires DNA as the template.
3. PCR Amplification:
   • The cDNA is amplified using PCR with primers specific to the rabies virus genome.
   • The PCR reaction involves repeated cycles of heating and cooling to amplify the target DNA sequence.
4. Detection:
   • The amplified DNA is detected using various methods, such as gel electrophoresis or real-time PCR.
   • In gel electrophoresis, the PCR product is separated on an agarose gel and visualized using a fluorescent dye.
   • Real-time PCR allows for the quantification of the amplified DNA in real-time, providing a more sensitive and accurate result.
5. Interpretation:
   • The presence of the amplified DNA indicates a positive result, confirming the presence of the rabies virus RNA.
   • The absence of the amplified DNA indicates a negative result.

3. Virus Isolation

Virus isolation involves culturing the rabies virus from a sample. This method is more time-consuming and labor-intensive than DFA and RT-PCR but can be useful for characterizing the virus and for research purposes.

1. Sample Preparation:
   • The sample (e.g., brain tissue, saliva) is homogenized and diluted in a cell culture medium.
   • The diluted sample is filtered to remove any bacteria or fungi.
2. Cell Culture:
   • The filtered sample is inoculated onto a susceptible cell line, such as mouse neuroblastoma cells or BHK-21 cells.
   • The cells are incubated at 37°C in a humidified atmosphere containing 5% CO2.
3. Observation:
   • The cells are monitored daily for signs of viral infection, such as cytopathic effects (CPE).
   • CPE include changes in cell morphology, such as rounding, detachment from the culture vessel, and cell lysis.
4. Confirmation:
   • If CPE are observed, the presence of the rabies virus is confirmed using other methods, such as DFA or RT-PCR.
5. Interpretation:
   • The isolation of the rabies virus indicates a positive result, confirming the presence of the virus in the sample.
   • The absence of virus isolation indicates a negative result.

4. Mouse Inoculation Test

The mouse inoculation test (MIT) is one of the oldest methods for rabies diagnosis. It involves inoculating laboratory mice with the sample and observing them for signs of rabies.

1. Sample Preparation:
   • The sample (e.g., brain tissue, saliva) is homogenized and diluted in a sterile solution.
2. Inoculation:
   • Laboratory mice are inoculated intracerebrally with the diluted sample.
   • The mice are typically 3-4 weeks old and are highly susceptible to rabies virus infection.
3. Observation:
   • The mice are observed daily for up to 28 days for signs of rabies, such as paralysis, tremors, and behavioral changes.
4. Confirmation:
   • If the mice develop signs of rabies, their brains are examined using DFA to confirm the presence of the rabies virus.
5. Interpretation:
   • The development of rabies in the inoculated mice indicates a positive result, confirming the presence of the virus in the sample.
   • The absence of rabies in the inoculated mice indicates a negative result.

5. Histopathology

Histopathology involves examining brain tissue under a microscope to look for characteristic signs of rabies virus infection.

1. Sample Preparation:
   • A small section of brain tissue is fixed in formalin and embedded in paraffin.
   • The paraffin-embedded tissue is sectioned into thin slices using a microtome.
   • The tissue sections are mounted on microscope slides and stained with histological stains, such as hematoxylin and eosin (H&E).
2. Microscopy:
   • The stained tissue sections are examined under a light microscope.
   • The pathologist looks for characteristic signs of rabies, such as:
     • Negri bodies: Eosinophilic (pink-staining) inclusion bodies in the cytoplasm of nerve cells, particularly in the hippocampus and cerebellum.
     • Inflammation: Perivascular cuffing and neuronal degeneration.
3. Interpretation:
   • The presence of Negri bodies is highly suggestive of rabies, although they are not always present in all cases.
   • Inflammation and neuronal degeneration can also support a diagnosis of rabies.

6. Serology

Serological tests are used to detect antibodies against the rabies virus in serum or CSF. These tests can be useful for diagnosing rabies in humans and for assessing the immune response to rabies vaccination.

1. Rapid Fluorescent Focus Inhibition Test (RFFIT):
   • The RFFIT is considered the gold standard for measuring rabies virus-neutralizing antibodies.
   • Serum samples are incubated with a known amount of rabies virus.
   • The mixture is then added to susceptible cells in culture.
   • If antibodies are present in the serum, they will neutralize the virus and prevent it from infecting the cells.
   • The cells are examined under a fluorescent microscope to determine the number of infected cells.
   • The titer of neutralizing antibodies is determined based on the dilution of serum that inhibits infection by 50% (50% fluorescent focus reduction neutralization test, or FRNT50).
2. Enzyme-Linked Immunosorbent Assay (ELISA):
   • ELISA is a more rapid and high-throughput method for detecting rabies antibodies.
   • Serum samples are added to microplates coated with rabies virus antigens.
   • If antibodies are present, they will bind to the antigens.
   • The bound antibodies are detected using an enzyme-labeled secondary antibody.
   • The enzyme substrate is added, and the resulting color change is measured using a spectrophotometer.
   • The amount of color change is proportional to the amount of antibody in the sample.

Interpreting Rabies Test Results

The interpretation of rabies test results must be done carefully and in conjunction with the clinical history and epidemiological data. A positive result indicates the presence of the rabies virus or antibodies, while a negative result indicates the absence of the virus or antibodies. However, false positives and false negatives can occur, so confirmatory testing may be necessary.

Factors Affecting Test Results

Several factors can affect the accuracy of rabies test results:

• Sample Quality: Poorly collected or preserved samples can lead to false negatives.
• Timing of Sample Collection: The stage of infection can affect the amount of virus present in the sample. Samples collected early in the infection may have low viral loads, leading to false negatives.
• Laboratory Technique: Improperly performed tests or contaminated reagents can lead to false positives or false negatives.
• Vaccination Status: Vaccination can affect serological test results. Vaccinated individuals may have detectable rabies antibodies, which can complicate the interpretation of test results.

Advances in Rabies Testing

Advances in molecular diagnostics and immunology have led to the development of new and improved rabies testing methods. Some of these advances include:

• Next-Generation Sequencing (NGS): NGS allows for the rapid and comprehensive sequencing of the rabies virus genome. This information can be used to track the spread of the virus, identify new variants, and develop more effective vaccines.
• Monoclonal Antibodies: Monoclonal antibodies are highly specific antibodies that can be used to detect and differentiate between different strains of the rabies virus.
• Point-of-Care (POC) Tests: POC tests are rapid and easy-to-use tests that can be performed at the point of care, such as in the field or in a clinic. These tests can provide results in minutes, allowing for rapid decision-making.

Conclusion

Rabies testing is a critical component of rabies prevention and control. Accurate and timely diagnosis is essential for preventing the spread of the virus and for guiding post-exposure prophylaxis decisions. The testing methods described in this article provide a comprehensive overview of the techniques used to diagnose rabies in animals and humans. As technology advances, new and improved testing methods will continue to improve our ability to detect and control this deadly disease.