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After completing her training in pediatrics and psychiatry at New York City's Mount Sinai Hospital, Levine moved from Manhattan to central Pennsylvania in 1993 where she joined the faculty of the Penn State College of Medicine and the staff at Penn State Hershey Medical Center. During her tenure there, she created Penn State Hershey Medical Center's Division of Adolescent Medicine and the Penn State Hershey Eating Disorders Program. She was in charge of the latter when she was nominated for the position of Pennsylvania physician general in 2015.

Pennsylvania Department of Health
In 2015, Levine was nominated by Pennsylvania Governor-elect Tom Wolf to serve as Pennsylvania's physician general. In one of her most lauded actions as physician general, Levine signed an order allowing law enforcement officers to carry the anti-overdose medication naloxone. In less than a year, this policy saved nearly 1,000 lives in the state.

In July 2017, Governor Wolf appointed Levine as Secretary of Health, and she was unanimously confirmed by the Pennsylvania State Senate.

COVID-19 response
Levine briefing COVID-19 measures with Gov. Tom Wolf at the Penn State Milton S. Hershey Medical Center in June 2020

During 2020 and until January 23, 2021, Levine led the public health response on COVID-19 in Pennsylvania as the state secretary of health. She worked closely on a daily basis with the FEMA director and led a daily press briefing. Along with Gov. Tom Wolfe, Levine faced criticism from a few Republican leaders over her handling of the pandemic, particularly with regard to nursing home patients.

On March 18, 2020, Levine directed Pennsylvania nursing homes to admit new patients, including stable patients recovering from the COVID-19 virus who were released from the hospital. Despite warnings from nursing home trade groups that such policies could unnecessarily cost more lives, there is no evidence that Levine placed COVID-positive patients in nursing home facilities or that her decision resulted in excess death among the elderly. Levine was also criticized for mishandling COVID data reporting and insufficiently addressing the long-standing oversight problems in Pennsylvania nursing homes that were exacerbated by the COVID-19 pandemic. Levine faced further scrutiny in May of 2020, when she moved her own mother out of a nursing home. In defending the move, Levine said, "My mother requested, and my sister and I, as her children, complied to move her to another location during the COVID-19 outbreak" and described her mother as "more than competent to make her own decisions." These issues were momentarily highlighted by Republican lawmakers after President Biden nominated Levine for the post of United States Assistant Secretary for Health. However, Levine's leadership during the COVID-19 pandemic has been widely praised as calm and steadfast by many Pennsylvanians and local media outlets.

Biden administration
Levine with U.S. Secretary of Health and Human Services Xavier Becerra after being sworn in as assistant secretary for health, March 27, 2021

On February 13, 2021, President Joe Biden formally nominated Levine to serve as Assistant Secretary for Health. Her confirmation hearing with the Senate HELP Committee took place on February 25. On March 17, the committee voted 13–9 to advance her nomination for a full Senate vote. On March 24, the Senate voted 52–48, with two Republicans joining all members of the Senate Democratic Caucus to confirm her nomination. Levine is the first openly transgender person to hold an office that requires Senate confirmation. On October 19, 2021, Levine was commissioned as a four-star admiral in the U.S. Public Health Service Commissioned Corps, becoming the first openly transgender four-star officer in any of the United States uniformed services. She is also the first female four-star admiral in the Commissioned Corps.

LGBTQIA+ health disparities

Shortly after her confirmation, Levine told NBC News that LGBTQIA+ youth are topmost in her mind when it comes to addressing health disparities in the United States. She cited bullying, suicide, discriminatory policies, and isolation during the COVID-19 pandemic as pressing issues among LGBTQIA+ youth. Levine has also expressed concerns about vaccine hesitancy among LGBTQIA+ youth who are more likely to experience medical distrust and less likely to seek medical care.

During an April 2022 speech at Texas Christian University, Levine criticized "disturbing - and frankly discriminatory - laws and actions" that many states have implemented that affect the lives of LGBTQIA+ youth. In an interview with NPR, she cited a range of policies, including Florida's "Don't Say Gay" bill and Texas' push to investigate parents who provide gender-affirming care to their transgender children. Arguing that such policies are based on politics rather than public health, Levine encouraged people to contact the Office for Civil Rights when they feel they are being discriminated against and vowed to provide support to those who contact her office.