P064 Evaluation of Chlamydia trachomatis positive specimens escaping detection in the Aptima Combo 2 assay in the United States

Background The Aptima Combo 2® (AC2, Hologic) assay detects Chlamydia trachomatis (CT) and Neisseria gonorrhoeae in clinical specimens. The assay target for CT is a region within the 23S ribosomal RNA (rRNA). Recent international reports highlighted a failure to detect CT variants with nucleotide substitutions in the AC2 target region. We explored the presence of CT 23S variants in the United States and whether a redesigned AC2 assay would successfully detect them. Methods A total of 50 specimens (tested using the AC2 assay between December 2019 – February 2020) with CT-negative or equivocal results (and having relative light unit (RLU) signal ≥15–99) were selected for further analysis. Additional specimens (RLU 100 [n=51]) were also examined. A custom real-time PCR was used to detect CT DNA in these samples and a portion of the CT 23S rRNA gene was amplified and sequenced. Samples were then tested using a redesigned AC2 assay provided by the manufacturer as research use only (RUO) reagent. Results All samples with AC2 results Conclusions Our findings show the redesigned AC2 assay is able to detect 23S rRNA point mutation variants that were not detected by its predecessor. Neither of these variant strains were associated with recent reports of diagnostic escape mutants in Europe.