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Background: Despite limited prospective data, it is commonly believed that human immunodeficiency virus (HIV) and hepatitis infections are widespread in the penetrating trauma population, placing healthcare workers at risk for occupational exposure. Our primary study objective was to measure the prevalence of HIV (anti-HIV), hepatitis B (HB surface antigen [HBsAg]), and hepatitis C virus (anti-HCV) in our penetrating trauma population., Methods: We prospectively analyzed penetrating trauma patients admitted to Temple University Hospital between August 2008 and February 2010. Patients (n = 341) were tested with an oral swab for anti-HIV and serum evaluated for HBsAg and anti-HCV. Positives were confirmed with western blot, neutralization immunoassay, and reverse transcription polymerase chain reaction, respectively. Demographics, risk factors, and clinical characteristics were analyzed., Results: Of 341 patients, 4 patients (1.2%) tested positive for anti-HIV and 2 had a positive HBsAg (0.6%). Hepatitis C was the most prevalent measured infection as anti-HCV was detected in 26 (7.6%) patients. Overall, 32 (9.4%) patients were tested positive for anti-HIV, HBsAg, or anti-HCV. Twenty-eight (75%) of these patients who tested positive were undiagnosed before study enrollment. When potential risk factors were analyzed, age (odds ratio, 1.07, p = 0.031) and intravenous drug use (odds ratio 14.4, p < 0.001) independently increased the likelihood of anti-HIV, HBsAg, or anti-HCV-positive markers., Conclusions: Greater than 9% of our penetrating trauma study population tested positive for anti-HIV, HBsAg, or anti-HCV although patients were infrequently aware of their seropositive status. As penetrating trauma victims frequently require expedient, invasive procedures, universal precautions are essential. The prevalence of undiagnosed HIV and hepatitis in penetrating trauma victims provides an important opportunity for education, screening, and earlier treatment of this high-risk population.