No widespread dissemination of Chlamydia trachomatis diagnostic-escape variants and the impact of Neisseria gonorrhoeae positivity on the Aptima Combo 2 assay

Abstract

ObjectivesA Finnish Chlamydia trachomatis (CT) new variant was detected in 2019 that escaped detection in the Hologic Aptima Combo 2 (AC2) assay due to a C1515T mutation in the CT 23S rRNA target region. Reflex testing of CT-negative/CT-equivocal specimens as well as those positive for Neisseria gonorrhoeae (NG) with the Hologic Aptima CT (ACT) assay was recommended to identify any CT variants.MethodsFrom June to October 2019, specimens with discrepant AC2/ACT CT results were submitted to Public Health England and screened for detectable CT DNA using an inhouse real-time (RT)-PCR. When enough DNA was present, partial CT 23S rRNA gene sequencing was performed. Analysis of available relative light units and interpretative data was performed.ResultsA total of 317 discordant AC2/ACT specimens were collected from 315 patients. Three hundred were tested on the RT-PCR; 53.3% (n=160) were negative and 46.7% (n=140) were positive. Due to low DNA load in most specimens, sequencing was successful for only 36 specimens. The CT 23S rRNA wild-type sequence was present in 32 specimens, and two variants with C1514T or G1523A mutation were detected in four specimens from three patients. Of the discordant specimens with NG interpretation, 36.6% of NG-negative/CT-negative AC2 specimens had detectable CT DNA on the inhouse RT-PCR vs 53.3% of NG-positive/CT-negative specimens.ConclusionsNo widespread dissemination of AC2 diagnostic-escape CT variants has occurred in England. We however identified the impact of NG positivity on the discordant AC2/ACT specimens; a proportion appeared due to NG positivity and the associated NG signal, rather than any diagnostic-escape variants or low DNA load. Several patients with gonorrhoea may therefore receive false-negative AC2 CT results. Single diagnostic targets and multiplex diagnostic assays have their limitations such as providing selection pressure for escape mutants and potentially reduced sensitivity, respectively. These limitations must be considered when establishing diagnostic pathways.

Chlamydia trachomatis Variants Escaping Detection in the Aptima Combo 2 Assay in the United States

“…Although we did not detect any Fl-nvCT variants among US specimens, these variants add to other known AC2 diagnosis escape mutants described in other recent studies 8,9 . Similar to our findings, researchers in England did not detect the Fl-nvCT variant among specimens collected for testing during a 5-month period in 2019 10,11 . However, 2 other variants (C1514T, G1523A) were detected among false CT-negative/equivocal specimens.…”

“…8,9 Similar to our findings, researchers in England did not detect the Fl-nvCT variant among specimens collected for testing during a 5-month period in 2019. 10,11 However, 2 other variants (C1514T, G1523A) were detected among false CT-negative/equivocal specimens.…”

“…Subsequent studies have also revealed the presence of other diagnostic-escape mutants (C1514T, C1522T, and G1523A) 8–10 . Notably, a study in England conducted in 2019 failed to detect the Fl-nvCT variant, but the C1514T and G1523A variants were detected 10,11 …”

“…[8][9][10] Notably, a study in England conducted in 2019 failed to detect the Fl-nvCT variant, but the C1514T and G1523A variants were detected. 10,11 Considering the potential for a prevalence of the FI-nvCT variants circulating in the United States, we collected potentially false-negative or equivocal specimens initially tested with the AC2 assay submitted to a large commercial testing laboratory. Additional molecular characterization of these specimens was conducted at the CDC.…”