Evaluation of LN34 Pan-Lyssavirus RT-qPCR assay for rabies diagnosis in Brazil

Rabies, a fatal zoonotic viral disease affecting mammals, including humans, remains a significant global health concern, particularly in low-income countries. The disease, primarily transmitted through infected animal saliva, prompts urgent diagnosis for timely post-exposure prophylaxis (PEP). The gold standard diagnostic test, direct fluorescent antibody test (dFAT), while sensitive, suffers from limitations such as subjective interpretation and high costs. As a confirmatory technique, the LN34 Pan-Lyssavirus RT-qPCR assay has emerged as a promising tool for universal Lyssavirus detection. This study evaluated its performance using 130 rabies virus isolates representing eleven Brazilian variants and 303 clinical samples from surveillance operations. The LN34 assay demonstrated 100% sensitivity and 98% specificity compared to dFAT. Additionally, it detected all samples, including those missed by dFAT, indicating superior sensitivity. The assay's specificity was confirmed through Sanger nucleotide sequencing, with only a minimal false-positive rate. Comparative analysis revealed higher accuracy and concordance with dFAT than traditional rabies tissue culture infection tests (RTCIT). False-negative RTCIT results were attributed to low viral load or suboptimal sampling. These findings underscore the LN34 assay's utility as a confirmatory technique, enhancing rabies surveillance and control in Brazil. Its widespread adoption could significantly improve diagnostic sensitivity, crucial for effective PEP and public health interventions.