Rabies is a zoonotic viral disease that induces severe, progressive, and ultimately fatal encephalomyelitis in all mammals, including humans (Fisher et al., 2018). Primarily transmitted via infected animals' saliva, particularly dogs and bats, this preventable disease remains a global public health concern, causing around 59,000 human deaths annually, mainly in low-income countries (Hampson et al., 2015). Once symptoms manifest, rabies is nearly universally fatal, highlighting the urgency of rapid, cost-effective, and accurate diagnosis to enable timely post-exposure prophylaxis (PEP) (Wunner; Conzelmann, 2020).

Rabies is caused by an enveloped, negative-sense, non-segmented, single-stranded RNA virus belonging to the Lyssavirus genus of the Rhabdoviridae family and Mononegavirales order (ICTV, 2023). The Lyssavirus genus comprises seventeen species, and rabies-like diseases from Lyssavirus species (excluding Lyssavirus rabies - RABV) are rare and geographically restricted (Johnson et al., 2010, Weir et al., 2014). Currently, only the RABV species has been reported in Brazil (Cunha et al., 2023).

Antigenic and genetic research on RABV isolates from Latin America has elucidated the disease’s epidemiological profile and identified potential hosts and reservoirs for its diverse epidemiological cycles (Favoretto et al., 2002, Oliveira et al., 2010). RABV’s adaptation to several hosts over time has led to genetic diversity due to the non-corrective insertion of nucleotide bases (Kissi et al., 1999). Consequently, distinct RABV variants have emerged in Brazil. Domestic dogs (Canis lupus familiaris) host antigenic variants AgV1 and AgV2, while vampire bats (Desmodus rotundus) and fruit-eating bats (Artibeus spp.) host AgV3. AgV4 affects free-tailed bats (Tadarida spp.), AgV6 affects hoary bats (Lasiurus spp.), and other species such as white-tufted marmoset (Callithrix jacchus), crab-eating foxes (Cerdocyon thous), and various bat species (Molossus spp., Histiotus spp., Nyctinomops spp., Eptesicus spp., Myotis spp.) host distinct genetic lineages (Favoretto et al., 2002, Oliveira et al., 2010).

The gold standard for rabies diagnosis, recommended by the World Health Organization (WHO), is the direct fluorescent antibody test (dFAT), which exhibits high sensitivity and specificity (Dean et al., 1996, WOAH, 2023). However, false-negatives may occur due to degraded samples, low viral titers, poor quality laboratory supplies, or subjective slide interpretation (Dean et al., 1996, Dupuis et al., 2015, Centoamore et al., 2020). Additionally, the expense of dFAT is related to high-quality fluorescence microscopy and anti-rabies conjugates (Silva et al., 2013, Centoamore et al., 2020).

An erroneous result, such as a false negative, could have severe consequences, including human death due to inadequate medical intervention during rabies PEP (Centoamore, 2017). Rabies diagnosis directly influences preventive measures for humans, patient treatment, and the control strategies in cases of positive rabies focus at municipal centers for zoonosis control (Meslin and Kaplan, 1996, Rupprecht et al., 2018).

Over time, several RT-qPCR assays with hydrolysis probes (such as TaqMan probes) targeting the nucleoprotein gene 3’ terminus have evolved to detect and differentiate Lyssavirus species (Black et al., 2002, Wakeley et al., 2005, Orlowska et al., 2008, Hoffmann et al., 2010). The LN34 Pan-Lyssavirus RT-qPCR assay, developed by Wadhwa et al. (2017), employs primers and a locked nucleic acid (LNA) probe for universal Lyssavirus detection, targeting a highly conserved genome region (Wadhwa et al., 2017).

Gigante et al. (2018) conducted a multi-laboratory study comparing the gold standard dFAT with the LN34 Pan-Lyssavirus RT-qPCR assay, testing 2978 samples across four continents (Africa, America, Asia, and Europe) and various animal species. The LN34 assay confirmed the majority of dFAT-positive samples, providing definitive results even for samples in an advanced autolytic state that were inconclusive with dFAT. Furthermore, the LN34 assay demonstrated no false-negative results and yielded fewer false-positive or inconclusive results (Gigante et al., 2018).

Several public health laboratories already employed RT-qPCR to detect other pathogens, including the influenza virus, Zika virus, HIV, dengue virus, and SARS-CoV-2 virus. These laboratories possess the necessary resources to implement this assay for rabies diagnosis. The routine use of RT-qPCR as a confirmatory test could enhance sensitivity and rapidly detect rabies, overcoming limitations of other techniques (Dettinger et al., 2021).

As the gold standard for rabies diagnosis requires a fast, specific, sensitive, and accurate confirmatory test, this study aimed to assess the performance of the LN34 Pan-Lyssavirus RT-qPCR assay using samples representing various Brazilian variants of the RABV. The goal is to determine if the assay is consistent and reliable enough to serve as a confirmatory technique for rabies surveillance diagnosis.