Our validation study revealed that the kit successfully identified pathogens in the CSF samples collected from 44 of the 78 (56.4%) patients who were clinically diagnosed with acute meningitis. Only 6 of the 44 pathogens identified using the Seeplex Meningitis ACE Detection kit were also identified by other laboratory methods, with the pathogen being HEV in 4 cases and HSV-1 and LM in the remaining 2 cases. Six CSF samples were simultaneously positive for 2 pathogens, suggesting co-infection or contamination rather than false-positivity, because subsequent sequencing of PCR products yielded 98-100% matches with sequences in the BLAST database. Of the 12 pathogens assayed, HEV had the highest prevalence and was detected in 23 samples by the Seeplex Meningitis ACE Detection kit. Of the bacterial strains, SP was the most frequently detected; SP was detected in 5 samples, although it was detected along with viruses, including HEV, HSV-1, and VZV, in 4 samples.