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Given the enormous advances in the prevention of perinatal transmission of human immunodeficiency virus (HIV), it is clear that early identification and treatment of all pregnant women with HIV is the best way to prevent neonatal infection and also improve women's health. Furthermore, new evidence suggests that early initiation of antiretroviral therapy in the course of infection is beneficial for individuals infected with HIV and reduces the rate of sexual transmission to partners who are not infected. Screening should be performed after women have been notified that HIV screening is recommended for all pregnant patients and that they will receive an HIV test as part of the routine panel of prenatal tests unless they decline (opt-out screening). Human immunodeficiency virus testing using the opt-out approach, which is currently permitted in every jurisdiction in the United States, should be a routine component of care for women during prepregnancy and as early in pregnancy as possible. Repeat HIV testing in the third trimester, preferably before 36 weeks of gestation, is recommended for pregnant women with initial negative HIV antibody tests who are known to be at high risk of acquiring HIV infection; who are receiving care in facilities that have an HIV incidence in pregnant women of at least 1 per 1,000 per year; who are incarcerated; who reside in jurisdictions with elevated HIV incidence; or who have signs and symptoms consistent with acute HIV infection (eg, fever, lymphadenopathy, skin rash, myalgias, arthralgias, headache, oral ulcers, leukopenia, thrombocytopenia, or transaminase elevation). Rapid screening during labor and delivery or during the immediate postpartum period using the opt-out approach should be done for women who were not tested earlier in pregnancy or whose HIV status is otherwise unknown. Results should be available 24 hours a day and within 1 hour. If a rapid HIV test result in labor is reactive, antiretroviral prophylaxis should be immediately initiated while waiting for supplemental test results. If the diagnosis of HIV infection is established, the woman should be linked into ongoing care with a specialist in HIV care for comanagement.