Rabies is an acute zoonotic viral disease that is almost always fatal once clinical symptoms appear [1]. Caused by a virus of the Lyssavirus genus and belonging to the Rhabdoviridae family, it exhibits a marked tropism for nerve cells [2][3]. Rabies is mainly transmitted through the bites or scratches of infected animals, particularly dogs, which account for about 99% of human cases and represent the main reservoir of the virus [4][5]. Although rabies can be controlled in some European countries through the vaccination of foxes, it remains endemic in many regions of the world, particularly in Africa and Asia, where it continues to pose a major public health problem [5][6].

The rabies virus consists of a single-stranded, negative-sense RNA genome [5]. This 12 kb genome includes five main genes: N (nucleoprotein), P (phosphoprotein), M (matrix protein), G (glycoprotein), and L (large protein, involved in replication) [3]. The G protein plays a crucial role in infection by allowing the virus to bind to cellular receptors and is often targeted during vaccine development [7]. The genetic variability of the rabies virus is relatively low; however, it exists in the form of several Lyssavirus subtypes, which mainly correspond to different animal species that serve as reservoirs for the virus [8]. This genetic variability is an important factor for epidemiological surveillance, as it can influence both transmission dynamics and the effectiveness of vaccination strategies [8].

Each year, rabies kills approximately 60,000 people worldwide, with a particularly high number of victims in Africa, where the disease accounts for about 44% of global deaths [9]. Burkina Faso, like other West African countries, faces an endemic situation in which thousands of cases of attacks by infected animals are reported each year, mainly in the capital city, Ouagadougou [10][11]. The vaccination of domestic animals, particularly dogs, is the main method of prevention, as it breaks the cycle of virus transmission to humans [12][13].

Despite the progress made in vaccination, many challenges remain, particularly regarding access to post-exposure treatments and the implementation of reliable diagnostic methods such as Direct Immunofluorescence (DFA) and RT-PCR [13]. In recent years, research efforts have intensified; however, many areas—particularly rural and remote ones—still lack the infrastructure and resources needed to effectively combat this disease. The present study focuses on the molecular epidemiology of rabies, particularly the use of RT-PCR as a diagnostic method, in order to better understand the spread of the virus and to propose strategies adapted to local contexts, especially in Burkina Faso.

The present study, conducted on brain tissue samples from biting animals collected in Burkina Faso between 2018 and 2019, evaluated the diagnosis of rabies using Direct Immunofluorescence (DFA) and conventional RT-PCR. The objective was to describe the epidemiological characteristics and to compare the performance of these two diagnostic approaches. Data analysis revealed a considerable proportion of missing information regarding the geographical origin, age, and vaccination status of the animals. This lack of information, which has also been reported in other African countries [15], reflects the predominance of stray dogs, which are often culled after biting incidents without any known background information [16]. This incompleteness limits the ability to assess epidemiological patterns accurately and may introduce information bias in subgroup analyses. As a result, some associations may be underestimated or overestimated, and the generalizability of these findings to the broader animal population in Burkina Faso is potentially reduced. Most of the samples originated from Kadiogo Province, which is likely explained by its proximity to the national reference laboratory, facilitating the transport of animal heads for analysis—a pattern also observed in Namibia [17]. This geographical concentration of samples may represent a source of selection bias, potentially limiting the generalizability of the findings to the entire country. The canine species accounted for 94.8% of cases, confirming its major role as a reservoir and vector of rabies in African countries [6]. Only 4% of the animals were vaccinated, a proportion comparable to that reported in Niger [18] e and Mali [19], but lower than the 13% observed in Burkina Faso by Savadogo et al. [20].

The overall infection rate obtained by DFA was 88.6%, similar to that reported in the Central African Republic [21] but lower than the 96% observed by Dao et al. [22]. The infection rate was higher among unvaccinated animals and those with unknown vaccination status compared to vaccinated animals, confirming the importance of vaccination in prevention. However, the relatively high percentage of vaccinated but positive animals suggests possible vaccine failures. Several factors may contribute to this situation, including improper vaccine administration, an incomplete or irregular vaccination schedule, poor vaccine quality, inadequate storage conditions (cold-chain breaches), or exposure to the virus shortly before or shortly after vaccination, before protective immunity could be established. A relationship between aggressiveness and infection was also observed: all animals that had bitten two or more people tested positive, compared to 86.4% of those that had bitten only one person. This finding supports the classical description of aggressiveness as a common sign of rabies in animals. Finally, a lower infection rate was observed in young animals under three months of age, possibly due to maternal antibody protection or lower exposure risk.

The overall infection rate detected by RT-PCR was 36.1%, considerably lower than that obtained by DFA. Several factors may explain this result: degradation of viral RNA due to prolonged sample storage, exacerbated by power outages disrupting the cold chain [23], variable RNA quality, sometimes with low concentrations and insufficient purity; or a lower viral load in some vaccinated animals, leading to non-detection by PCR. The primers used targeted conserved regions of the N gene and can detect various Lyssavirus species [14], however, tissue preservation conditions appear to have had a major impact on the sensitivity of PCR under our study conditions.

The concordance between the two methods was low, with a Cohen’s Kappa coefficient of 0.13. Out of 97 samples, only 46 showed concordant results (35 positive and 11 negative). More than half of the samples that tested positive by DFA were negative by PCR, although no DFA-negative sample tested positive by PCR. These findings contrast with those of De Benedictis et al. [14], who reported almost perfect agreement between the two methods. In our study, the discrepancy is likely attributable to sample preservation conditions and the variable quality of extracted RNA. Nevertheless, both methods showed similar trends, with higher infection rates observed in unvaccinated animals and those older than three months.

This study is noteworthy for having used both a classical method (DFA) and a molecular method (RT-PCR) to assess rabies in many samples collected over two consecutive years, thereby providing reliable documentation of virus circulation in an African context, where data remain scarce. It highlights the strong involvement of dogs as the main reservoir, the importance of vaccination, and aggressiveness as a predominant clinical sign. However, certain limitations should be noted: the lack of available information on the animals (age, origin, vaccination status), the prolonged storage of samples in a context of cold chain disruptions, and the sometimes-insufficient quality of extracted RNA likely affected the sensitivity of RT-PCR, thereby explaining the low concordance observed between the two diagnostic methods.

This study confirmed the predominant role of dogs in the transmission of rabies in Burkina Faso and revealed a high infection rate among biting animals, particularly those that were unvaccinated. Direct Immunofluorescence (DFA) demonstrated greater sensitivity than conventional RT-PCR under our working conditions, the latter likely being limited by the quality and preservation of the samples. Despite the low concordance between the two methods, the observed trends were similar, highlighting the relevance of their combined use in resource-limited settings. These findings underscore the need to strengthen vaccination programs for domestic animals, especially dogs, to reduce the risk of human transmission. The study also emphasizes the importance of improving epidemiological data collection and sample preservation conditions to enhance the reliability of molecular diagnosis. Introducing RNA-stabilizing reagents (e.g., RNAlater® or equivalent) at the point of sample collection for tissues destined for PCR analysis would help maintain RNA integrity during transport and storage. Further investigations using real-time RT-PCR, which offers higher sensitivity, along with genetic sequencing of circulating strains, would refine the molecular epidemiology of rabies in Burkina Faso. Finally, better integration of veterinary and medical surveillance within a “One Health” approach represents a key perspective for progress toward the elimination of human rabies by 2030, in line with WHO objectives.