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Identification of sexually transmitted infections (STI) is limited by the infrequent assessment of rectal STI. This study assesses usability of self-collected rectal swabs (SRS) in diagnosing rectal Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG).In 2006 to 2007, clients of the Amsterdam and South Limburg STI outpatient clinics reporting receptive anal intercourse were asked to fill out a questionnaire and provide SRS. A standard provider-collected rectal swab (PRS) was also taken, and both were tested for CT and NG by a nucleic acid amplification tests. SRS performance was compared with PRS as to agreement, sensitivity, and specificity.Prevalence of rectal CT was 11% among the 1458 MSM and 9% among the 936 women. Rectal NG prevalence was 7% and 2%. In 98% of both MSM and women, SRS and PRS yielded concordant CT test results, for NG agreement was 98% for MSM and 99.4% for women. SRS performance for CT and NG diagnosis was good in both groups and was comparable for both study regions. Slightly more (57% of MSM, 62% of women) preferred SRS to PRS or had no preference; 97% would visit the STI clinic again if SRS was standard practice.Because anal sex is a common practice for MSM and women, and anal STI are frequently present, rectal screening should be an essential part of an STI consultation. SRS is a feasible, valid, and acceptable alternative for MSM and women attending STI clinics, and hence should be considered for other settings as well.