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Dengue virus (DENV) is a mosquito-borne human pathogen endemic in 129 countries. In Italy, presence of the competent vector increases the risk of autochthonous transmission from imported cases, and a surveillance national plan for Arboviruses (PNA 2020-2025) is in place. The results of laboratory diagnosis of DENV in Italy from November 2015 to November 2021 are presented. Samples from 502 suspected DENV cases were tested by both molecular/antigen NS1 and serological assays, including Plaque Reduction Neutralization Test (PRNT). Samples were tested in parallel for DENV, Chikungunya (CHIKV)and Zika virus (ZIKV). Cases were classified based on laboratory results according to the Italian National Plan for Arboviruses (PNA) criteria. After evaluation of laboratory results, there were 140/502 confirmed and 7/502 probable cases (DENV cases). A positive molecular test was found in 27.1% of cases and 66.7% had a positive antigen NS1 test. The lag time from symptoms to collection was 4 and 6 days, respectively, with a higher range of day detection limit for antigen NS1. DENV cases gave 87.8% IgM and 82.9% PRNT positive/border line results: samples positivity of 72.20% and 33.9% in IgM and PRNT, respectively, was observed within the first 7 days of disease. Overall, 10.9% IgM positive results were not confirmed as positive in PRNT or molecular test among excluded cases (274/502). 26/502 suspected cases were both DENV and ZIKV positive by IgM and/or PRNT, making it difficult to provide a final differential diagnosis. None had a possible co-infection of DENV and CHIKV. DENV diagnosis by molecular/antigen tests is the gold standard but the sample collection time is a limitation. Serological tests are thus necessary. However, IgM results need PRNT confirmation. Co-circulation of DENV and ZIKV increases diagnostic difficulty. Continuous evaluation of diagnostic strategies is essential against future DENV autochthonous outbreaks. [ABSTRACT FROM AUTHOR] more...