In order to receive their PT panel, laboratories were invited to undertake either the entire pre-exposure prophylaxis (PrEP) or a booster dose according to the staff vaccination history. Data Availability StatementAll relevant data are within the manuscript and its Supporting Information files. Abstract To achieve the goal of eliminating dog-mediated human rabies deaths by 2030, many African countries have agreed to list rabies as a priority zoonotic disease and to carry out both short and long-term control programs. Within this context, reliable local diagnosis is essential for the success of field surveillance systems. However, a harmonized, sustainable and supportive diagnostic offer has yet to be achieved in the continent. We herewith describe the organization and outcome of a proficiency test (PT) for the post-mortem diagnosis of rabies in animals, including thirteen veterinary laboratories and one public health laboratory in Africa. Participants were invited to assess both the performance of the Direct Fluorescent Antibody (DFA) test and of a conventional RT-PCR. From your submitted results, while thirteen laboratories proved to be able to test the samples through DFA test, eleven performed the RT-PCR method; ten applied both techniques. Tlr2 Of note, the number of laboratories able to apply rabies RT-PCR experienced increased from four to ten after the exercise. Importantly, results showed a higher proficiency in applying the molecular test compared to the DFA test (concordance, sensitivity and specificity: 98.2%, 96.97% and 100% for RT-PCR; 87.69%, 89.23% and 86.15% for DFA test), indicating the feasibility of molecular methods to identify animal pathogens in Africa. Another positive end result of this approach was that negative and positive controls were made available for further in-house validation of new techniques; in addition, a detailed questionnaire was provided to collect useful and relevant information around the diagnostic procedures and biosafety steps applied at laboratory level. Author summary Although Africa has the highest per capita Tacalcitol monohydrate death rate from rabies, the incidence of canine rabies around the continent remains a matter that needs to be further investigated; in addition, the lack of accurate information impairs the establishment of long-term actions to progress towards a rabies free status. In this scenario, creating efficient diagnostic facilities is usually of utmost importance for eradicating the disease and implementing effective surveillance programs in endemic areas. With the final objective of eliminating dog-mediated human rabies globally by 2030, sample submission to African veterinary laboratories for rabies diagnostic screening is expected to increase. Furthermore, in compliance with ISO/IEC 17025:2017 standard, participating to proficiency screening (PT) exercises becomes mandatory not only to regularly assess the validity of the in-house protocols but also to harmonize the techniques. Thirteen veterinary laboratories and one public health laboratory in Africa were invited to take part in the exercise in response to an invitation from your Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), Italy, which hosts the FAO rabies Reference Center. This study presents the results obtained by applying the DFA test and the conventional RT-PCR method to the PT panel, which show that even if laboratories preferably apply the DFA test to detect the presence of rabies in animal samples, the overall concordance of results was higher through molecular screening. Importantly, the organization of the exercise proved to be a good opportunity to update the vaccine cover status of laboratory staff. Introduction Rabies computer virus still claims more Tacalcitol monohydrate than 59, 000 human lives every year and affects lower socio-economic groups in resource-poor countries. In Africa, where the highest per-capita death rate is usually accounted for [1], rabies has been Tacalcitol monohydrate listed as a priority zoonotic disease through the national zoonotic disease prioritization process using the Centre for Disease Control (CDC) One Health Zoonotic Disease Prioritization Tool (OHZDPT) in many African countries [2]. The tripartite collaboration of World Health Organization (WHO), World Organisation for Animal Health (OIE) and Food and Agriculture Business of the United Nations (FAO), together with the nonprofit organisation Global Alliance for Rabies Control (GARC) have set up the goal to end dog-mediated human rabies by 2030. The removal of canine rabies in Africa is usually achievable through the firm political will and engagement of local stakeholders, who are required to take on responsibilities and perform actions for the cause [3]. Tools are available to support and guideline countries in developing national programs and strategies for sustainable rabies prevention, rabies control and gradually progress towards rabies removal [4]. In this context, estimating the disease burden relies on accurate surveillance plans in the field. Performant diagnostic facilities require the availability of well-maintained infrastructures and experienced laboratory technicians to perform the recommended protocols. An accurate diagnosis.