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Background: Coronavirus 2019 disease (COVID-19) is caused by the virus SARS-CoV-2, transmissible both person-to-person and from contaminated surfaces. Early COVID-19 detection among healthcare workers (HCWs) is crucial for protecting patients and the healthcare workforce. Because of limited testing capacity, symptom-based screening may prioritize testing and increase diagnostic accuracy. Methods and findings: We performed a retrospective study of HCWs undergoing both COVID-19 telephonic symptom screening and nasopharyngeal SARS-CoV-2 assays during the period, March 9—April 15, 2020. HCWs with negative assays but progressive symptoms were re-tested for SARS-CoV-2. Among 592 HCWs tested, 83 (14%) had an initial positive SARS-CoV-2 assay. Fifty-nine of 61 HCWs (97%) who were asymptomatic or reported only sore throat/nasal congestion had negative SARS-CoV-2 assays (P = 0.006). HCWs reporting three or more symptoms had an increased multivariate-adjusted odds of having positive assays, 1.95 (95% CI: 1.10–3.64), which increased to 2.61 (95% CI: 1.50–4.45) for six or more symptoms. The multivariate-adjusted odds of a positive assay were also increased for HCWs reporting fever and a measured temperature ≥ 37.5°C (3.49 (95% CI: 1.95–6.21)), and those with myalgias (1.83 (95% CI: 1.04–3.23)). Anosmia/ageusia (i.e. loss of smell/loss of taste) was reported less frequently (16%) than other symptoms by HCWs with positive assays, but was associated with more than a seven-fold multivariate-adjusted odds of a positive test: OR = 7.21 (95% CI: 2.95–17.67). Of 509 HCWs with initial negative SARS-CoV-2 assays, nine had symptom progression and positive re-tests, yielding an estimated negative predictive value of 98.2% (95% CI: 96.8–99.0%) for the exclusion of clinically relevant COVID-19. Conclusions: Symptom and temperature reports are useful screening tools for predicting SARS-CoV-2 assay results in HCWs. Anosmia/ageusia, fever, and myalgia were the strongest independent predictors of positive assays. The absence of symptoms or symptoms limited to nasal congestion/sore throat were associated with negative assays. [ABSTRACT FROM AUTHOR]