Proficiency tests were organised on an annual basis from 2009 to 2017. No proficiency test was organised in 2018 as it was decided to hold the trial every 2 years from 2017. Upon registration, each participating laboratory had to choose the tests it wanted to apply out of FAT, RTCIT [2,21,25,26], real-time RT-PCR [2,21,2729] or conventional RT-PCR [2,21,30] and the mice inoculation test (MIT) [2,21,31]. MIT was assessed in 2009 only and real-time RT-PCR has been assessed since 2011 with the exception of 2017. The participating laboratories had to evaluate the tests they were using routinely according to their routine protocols, so no protocols were consequently sent with the samples. As multiple diagnostic tests were assessed on the same samples (FAT and RTCIT, or FAT, RTCIT and conventional RT-PCR, or FAT and real-time RT-PCR, etc.) a global diagnostic conclusion was also requested per sample from 2017 onwards to mimic as closely as possible the usual conditions under which rabies is diagnosed, i.e. for each sample, all the laboratories were invited to conclude on the animal’s infection status (infected if positive/not infected if negative) on the basis of their results.

The laboratories were advised to store lyophilised samples at 5±3°C upon their reception, and to perform the tests as soon as possible thereafter. Each laboratory had to check the samples’ condition on arrival and to fill out an acknowledgement of receipt. If panels arrived damaged or in a suspect condition, they could be replaced upon the participating laboratory’s request. Because freeze-dried samples were sent, they had to be reconstituted by adding 1 mL of sterile distilled water under a biosafety cabinet. After adding water, a 30-minute waiting period was necessary to rehydrate the brain homogenates. Each participant had to state on the result form the results for each sample of each technique evaluated (positive or negative, detected or not detected). Furthermore, in order to allow a fair evaluation of participants, each laboratory had to state which strains the laboratory technique was able to detect. In this way, any wrong results for strains that the laboratory’s techniques were not supposed to amplify were not taken into consideration.

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