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Objectives Estonia has one the highest number of new HIV diagnoses in the European Union, mainly among injecting drug users and heterosexuals. Little is known of HIV incidence, which is crucial for limiting the epidemic. Using a recent HIV infection testing algorithm (RITA) assay, we aimed to estimate HIV incidence in 2013. Methods All individuals aged ≥18 years newly-diagnosed with HIV in Estonia January- December 2013, except blood donors and those undergoing antenatal screening, were included. Demographic and clinical data were obtained from the Estonian Health Board and the Estonian HIV-positive patient database. Serum samples were tested for recent infection using the LAg-avidity EIA assay. HIV incidence was estimated based on previously published methods. Results Of 69,115 tested subjects, 286 (0.41%) were newly-diagnosed with HIV with median age of 33 years ( IQR: 28-42) and 65% male. Self-reported routes of HIV transmission were mostly heterosexual contact ( n = 157, 53%) and injecting drug use ( n = 62, 21%); 64 (22%) were with unknown risk group. Eighty two (36%) were assigned recent, resulting in estimated HIV incidence of 0.06%, corresponding to 642 new infections in 2013 among the non-screened population. Incidence was highest (1.48%) among people who inject drugs. Conclusions These high HIV incidence estimates in Estonia call for urgent action of renewed targeted public health promotion and HIV testing campaigns. [ABSTRACT FROM AUTHOR]