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This study aimed to examine the agreement between sexually transmitted infection (STI) screening using self-collected specimens and physician-collected specimens, and to investigate the acceptability of self-collection for screening in an 18-month study of female sex workers in a high-risk, low-resource setting.A total of 350 female sex workers in Nairobi, Kenya, participated in a prospective study from 2009 to 2011. Women self-collected a cervicovaginal specimen. Next, a physician conducted a pelvic examination to obtain a cervical specimen. Physician- and self-collected specimens were tested for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium (MG) using Aptima nucleic acid amplification assays (Hologic). Specimens were collected at 3-month intervals over 18-month follow-up. κ Statistics measured agreement of positivity between self-collection and physician collection.Baseline STI prevalence was 2.9% for N. gonorrhoeae, 5.2% for C. trachomatis, 9.2% for T. vaginalis, and 20.1% for MG in self-collected samples, and 2.3%, 3.7%, 7.2%, and 12.9%, respectively, in physician-collected samples. κ Agreement was consistently strong (range, 0.66–1.00) for all STIs over the 18-month study period, except for MG, which had moderate agreement (range, 0.50–0.75). Most participants found self-collection easy (94%) and comfortable (89%) at baseline, with responses becoming modestly more favorable over time.Self-collected specimen screening results showed strong agreement to clinical-collected specimens, except for MG, which was consistently detected more commonly in self-collected than in physician-collected specimens. Acceptability of the self-collection procedure was high at baseline and increased modestly over time. In high-risk, low-resource settings, STI screening with self-collected specimens provides a reliable and acceptable alternative to screening with physician-collected specimens.