Rabies Virus Diagnostic Testing—Evidence towards updating tissue sampling DFA testing

Introduction/Background

Rabies is a fatal zoonotic disease of public health concern. The direct fluorescent antibody (DFA) test is internationally recognized as the gold standard for postmortem rabies diagnosis, with some variations in sampling recommendations depending on the reference standard used. The United States National Rabies Diagnostic protocol recommends sampling both the brainstem and cerebellum for testing. Our study aimed to assess whether brainstem tissue alone is sufficient for reliable DFA diagnosis by comparing brainstem and cerebellar results from routine case submissions.

Methods

We reviewed 437 DFA-positive rabies cases submitted between January 2013 and January 2026 from multiple species [skunks (323), cats (41), bovines (41), horses (10), dogs (9), foxes (7), raccoons (2), sheep (1), goat (1), donkey (1), and llama (1)]. Brainstem and cerebellar impression smears were fixed in cold acetone, stained with fluorescein isothiocyanate, and examined for apple-green fluorescence indicating rabies antigen. Antigen distribution was graded on a scale of 1 to 4 based on abundance. Tissue scores were statistically compared using paired student t-tests and Pearson correlation.

Results

Brainstem and cerebellum scores demonstrated a high correlation (r = 0.93; N = 437; t = 52.40; DF = 435; P = 4.98 × 10⁻¹⁹⁰). The paired (two-tailed) Student’s t-test showed a small but significant difference (P = 0.0005; t = 3.48; DF = 436; mean difference = 0.03; 95% CI: 0.01–0.05) between brainstem (3.87 ± 0.39) and cerebellum (3.84 ± 0.51) scores.

Conclusions

Brainstem and cerebellum scores show a strong positive correlation indicative of consistent antigen detection. Brainstem showed higher mean scores compared to cerebellum. These findings provide supporting evidence towards revising minimum tissue sampling standards to allow for independent brainstem testing when optimal tissue collection is not feasible. This will improve diagnostic efficiency and biosafety without compromising diagnostic accuracy or public health protection.