Rabies Diagnostic Testing
The direct fluorescent antibody (DFA) test on brain tissue is the gold standard for rabies diagnosis and requires euthanasia of the animal and submission of the head or brain (excluding brain stem) to a qualified laboratory designated by local or state health departments. 1
Postmortem Diagnosis (Animals)
The DFA test must be performed according to the established national standardized protocol by a qualified laboratory. Key procedural requirements include:
Specimen preparation: Animals should be euthanized in a manner that maintains brain integrity. For all animals except very small ones (e.g., bats), only the head or brain (including brain stem) should be submitted 1
Storage and shipping: Specimens must be kept under refrigeration (not frozen) to facilitate prompt testing. Freezing delays testing as specimens must be thawed. Chemical fixation should be avoided as it causes substantial delays and may preclude reliable testing 1
Emergency testing: Should be available to expedite exposure management decisions. CDC's rabies laboratory can provide confirmatory results within 24 hours for inconclusive results, unusual species, or mass exposures 1
Alternative Field Testing
Direct Rapid Immunohistochemical Tests (DRIT) can be used by trained field personnel for surveillance specimens not involving human or domestic animal exposures. However, all positive DRIT results must be confirmed by DFA testing at a qualified laboratory 1
Antemortem Diagnosis (Humans)
No reliable antemortem rabies tests are commercially available for routine use in animals 1. For humans with suspected rabies, antemortem diagnosis requires multiple sample types and tests:
Recommended Antemortem Testing Approach
Combine detection of viral RNA with antibody testing in cerebrospinal fluid (CSF) for optimal diagnostic yield:
Viral RNA detection: Real-time TaqMan PCR on CSF (78.5% sensitivity postmortem, 45.4% antemortem), saliva (85.7% sensitivity), and nuchal skin biopsy (60% sensitivity) 2
Antibody detection: Rabies viral neutralizing antibodies (RVNA) in CSF using rapid fluorescent focus inhibition test (RFFIT) in unvaccinated individuals 2, 3
Combined approach: Real-time PCR alone achieved antemortem diagnosis in 84.6% of cases; when combined with RVNA detection in CSF, diagnosis was achieved in 100% of cases 2
Multiple Sample Collection Strategy
For suspected human rabies cases, collect:
- CSF (for both viral RNA and antibody testing)
- Saliva (preferably 3 samples at 6-hour intervals for higher yield) 4
- Nuchal skin biopsy (hair follicles from nape of neck)
- Serum (for antibody testing)
This multi-sample approach is essential because viral shedding is intermittent and varies over time 1, 3
Critical Limitations and Pitfalls
Unlicensed rapid test kits: No USDA-licensed rapid test kits are commercially available. Unlicensed tests should never be used because sensitivity/specificity are unknown, they haven't been validated against standard methods, and results would still require DFA confirmation 1
Serology alone is insufficient: Rabies antibody titers indicate response to vaccine or infection but do not directly correlate with protection. They should never substitute for current vaccination status in managing exposures 1
Timing matters: For animals that bite humans, healthy dogs, cats, or ferrets should be confined and observed for 10 days rather than immediately tested, as they would not have been shedding virus at the time of bite if they remain healthy 5
Human antemortem diagnosis remains challenging with only 40.6% of cases confirmed antemortem even with combined laboratory assays 3, emphasizing the importance of clinical suspicion and prompt initiation of post-exposure prophylaxis when indicated.