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Background: Internet-based Chlamydia Screening Implementation (chlamydia screening programme) was introduced in the Netherlands in 2008-2010 to detect and treat asymptomatic infections and to limit ongoing transmission through annual testing and treatment of Chlamydia trachomatis in young people (16-29 years). This population-based screening may be less effective when addressing individuals who are already covered by regular care, instead of addressing a hidden key population without chlamydia testing experience in regular care. This study had two aims: (1) to assess the rate and determinants of newly reached (i.e. not previously tested in 2006-2010) participants in the chlamydia screening programme, and (2) to assess the chlamydia positivity in these newly reached participants. Methods: This observational matching study included all chlamydia tests performed in subjects aged 16-29 years in eastern South Limburg in the Netherlands (population 16-29 years: 41,000) between 2006-2010. Testing was conducted during the systematic chlamydia screening programme (2008-2010), at a sexually transmitted infections clinic (STI clinic), by general practitioners (GPs), and by medical specialists as reported by the medical laboratory serving the region. Data were matched between testing services on individual level. The study population included all participants who were tested at least once for chlamydia by the chlamydia screening programme. Participants were included at their first chlamydia screening participation. Results: In the chlamydia screening programme, 80.7% (4298/ 5323) of participants were newly reached, others were previously tested by the STI clinic (5.7%, n=304), GPs (6.2%, n=328), medical specialists (3.5%, n=187) or a combination of providers (3.9%, n=206). Chlamydia prevalence was similar in newly reached participants (4.8%, 204/4298) and participants previously tested (4.5%, 46/1025, P=0.82). Independent determinants for being a newly reached participant were male gender (men OR 2.9; 95% CI 2.5-3.4) and young age <21 years (versus 25-29 years OR 1.8; 95% CI 1.5-2.2). Conclusions: The majority of the chlamydia screening programme participants have not been tested by regular care, and show similar chlamydia prevalence as those previously tested. Thereby population-based chlamydia screening adds to the existing regular care by testing young individuals hidden to current regular care.