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Transgender inclusive and gender affirmative healthcare includes asking patients about gender identity and sex assigned at birth through what is known as the ‘two-step’ methodology. In May 2016, the STD clinic in Seattle, WA switched from using a one-step to a two-step gender identity question. We aimed to determine if the updated questions increased ascertainment of transgender and gender nonconforming (TGNC) patients, and used the improved gender identity data to describe the HIV/STD risk profile of TGNC patients.We conducted a pre-post analysis comparing the proportion of patients that identified as TGNC during the year prior to and following implementation of the two-step questions. Gender identity and medical history questions were ascertained using a computer-assisted self-interview. The two-step question included two new gender response options: non-binary/genderqueer and write-in.Institution of the two-step question resulted in a 4.8-fold increase in patients who were identified as TGNC: 36 of 6,635 (0.5%) to 172 of 7,025 (2.4%) of patients (p<0.001). After implementation, 89 patients identified as non-binary/genderqueer (51.7% of TGNC patients). The proportion of patients identified as transgender men and women increased from 0.2% to 0.5% (p=0.002) and 0.4% to 0.6% (p=0.096), respectively. Non-binary patients’ HIV/STI risk profile was distinct from that of transgender and cisgender men who have sex with men (MSM), suggesting that distinguishing sub-populations within the TGNC population is important for risk stratification.Using a two-step gender identity question and including non-binary/genderqueer options increased our clinic’s ascertainment of TGNC patients and more accurately captured gender identity among STD clinic patients.