Sensitivity, specificity, inclusivity and exclusivity of the updated Aptima Combo 2 assay, which provides detection coverage of the new diagnostic-escape Chlamydia trachomatis variants

Abstract

Background Four new variants of Chlamydia trachomatis (nvCTs), detected in several countries, cause false-negative or equivocal results using the Aptima Combo 2 assay (AC2; Hologic). We evaluated the clinical sensitivity and specificity, as well as the analytical inclusivity and exclusivity of the updated AC2 for the detection of CT and Neisseria gonorrhoeae (NG) on the automated Panther system (Hologic). Methods We examined 1004 clinical AC2 samples and 225 analytical samples spiked with phenotypically and/or genetically diverse NG and CT strains, and other potentially cross-reacting microbial species. The clinical AC2 samples included CT wild type (WT)-positive (n = 488), all four described AC2 diagnostic-escape nvCTs (n = 170), NG-positive (n = 214), and CT/NG-negative (n = 202) specimens. Results All nvCT-positive samples (100%) and 486 (99.6%) of the CT WT-positive samples were positive in the updated AC2. All NG-positive, CT/NG-negative, Trichomonas vaginalis (TV)-positive, bacterial vaginosis-positive, and Candida-positive AC2 specimens gave correct results. The clinical sensitivity and specificity of the updated AC2 for CT detection was 99.7 and 100%, respectively, and for NG detection was 100% for both. Examining spiked samples, the analytical inclusivity and exclusivity were 100%, i.e., in clinically relevant concentrations of spiked microbe. Conclusions The updated AC2, including two CT targets and one NG target, showed a high sensitivity, specificity, inclusivity and exclusivity for the detection of CT WT, nvCTs, and NG. The updated AC2 on the fully automated Panther system offers a simple, rapid, high-throughput, sensitive, and specific diagnosis of CT and NG, which can easily be combined with detection of Mycoplasma genitalium and TV.

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

“…Hologic has recently developed an updated version of the AC2 assay, which includes an additional CT 23S rRNA target that should provide detection coverage for the nvCTs identified to date and this revised AC2 assay has replaced the previously used assay in Europe. [19] Ideally all diagnostic platforms should employ more than one diagnostic target to prevent mutants from emerging. Further work is required to assess the true impact NG positivity has on specimens which co-harbour CT and are tested on the AC2, as well as considering the impact of sensitivity on other multi-plex platforms.…”

A molecular screening assay to identify Chlamydia trachomatis and distinguish new variants of C. trachomatis from wild‐type

“…The mutational events in specific target binding sequences of C. trachomatis can raise the possibility of false‐negative results, which led to increasing the spread of nvCT, especially in high‐frequency transmitting populations (Unemo and Clarke, 2011). Though an updated AC2 assay containing dual‐target detection that detects all known nvCT strains have been FDA‐cleared in the United States and clinically validated (Unemo et al ., 2020) and CE‐Marked in Europe, knowledge regarding the spread of the nvCT is relatively limited (Unemo et al ., 2019a) because the majority of laboratories are still not able to identify these nvCTs. Enhanced and continued surveillance based on screening C. trachomatis samples (negative results using the AC2 platform and positive results using other detection platforms based on targets without 23S rRNA) by this assay will be very useful in addressing these concerns.…”

The Impact of Selected Bacterial Sexually Transmitted Diseases on Pregnancy and Female Fertility

“…Another variant connected with false-negative results of the Aptima Combo 2 assay was single-nucleotide polymorphism (SNP) in the bacterial gene encoding 23S rRNA (Finnish new variant C. trachomatis - nvCT). However, the problem has now been resolved and a new version of the test detects Finnish nvCT [ 62 , 63 , 66 ]. N. gonorrhoeae false-negative results caused by genetic variations of the target site are less common and concerns only some tests produced by laboratories (home-made tests) [ 64 ].…”