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

Curable sexually transmitted infections (STIs) remain major global public health concerns [1]. Chlamydia trachomatis (CT) infection is the most common bacterial STI with 127 million estimated cases among adults each year globally [1]. In the European Union/European Economic Area (EU/EEA) and in many other international settings, the incidence of CT infections has increased during the past decade [1, 2]. The widespread use of highly sensitive and specific nucleic acid amplification tests (NAATs), particularly in high-income countries, has substantially contributed to the increased detection and incidence. Undetected and untreated CT infections can result in serious complications and sequelae, including infertility. CT infections are frequently asymptomatic and accordingly not detected if appropriate laboratory diagnostics is not performed [3].

Validated and quality-assured CT NAATs are recommended for highly sensitive and specific diagnosis of CT infections. Compared to other CT diagnostic methods, NAATs have highly superior sensitivities with maintained high specificities, use non-invasive specimens, are rapid, and provide opportunities for automation [3]. Initially, CT was considered to be genomically highly conserved. However, in 2006 the Swedish new variant of CT (nvCT) was described [4]. This Swedish nvCT has a deletion in the cryptic plasmid resulting in false negative results using the Roche and Abbott NAATs available at that time [5, 6]. Subsequently, whole genome sequencing of CT provided evidence of relatively substantial recombination and a mutational frequency level similar to the one in many other bacterial species [7, 8]. This showed that CT evolves more than previously predicted and diagnostic-escape CT mutants can emerge.

The sensitivity and specificity of the US FDA-approved Aptima Combo 2 assay (AC2; Hologic Inc., San Diego, CA, USA), based on target capture (TC) and transcription-mediated amplification (TMA) chemistries, for detection of CT (target: 23S rRNA) and Neisseria gonorrhoeae (NG; target: 16S rRNA) have proven excellent in many studies [9‐17]. However, in early 2019, the Finnish nvCT (FI-nvCT) was identified [18, 19]. This variant has a single nucleotide polymorphism (SNP) in the CT 23S rRNA gene, i.e. C1515T (Escherichia coli numbering), resulting in escaped detection by the acridinium ester CT detection probe used in AC2 [18‐24]. The FI-nvCT was initially estimated to represent 6–10% of the CT positive samples in Finland [18, 19]. The FI-nvCT has subsequently been reported in Sweden [20], Norway [21], and Denmark [24]. Three additional AC2 diagnostic-escape nvCTs have been reported in single specimens in Norway [21], the United Kingdom [25], and Japan (the present study), and one of these three nvCTs was found widely spread in Denmark [24]. These three AC2 diagnostic-escape nvCTs, as the FI-nvCT, have a SNP in the AC2 CT probe detection sequence of 23S rRNA, i.e. 23S rRNA C1514T, G1523A [21, 24, 25], or C1522T (the present study). The Aptima C. trachomatis NAAT (ACT; Hologic Inc.), which targets CT 16S rRNA, detects all of these diagnostic-escape nvCTs. Reflex testing of AC2 samples with relative light unit (RLU) values of 15–99 (mostly “high negative” or equivocal results) using ACT was implemented in May–June 2019 in many European countries [22, 26]. However, this ACT reflex testing substantially increases the work load and associated costs in these laboratories, and a more sustainable solution is imperative. Accordingly, an updated version of AC2, which was designed to detect also all these and other diagnostic-escape nvCTs, has now been developed by Hologic Inc. (San Diego, USA). This updated AC2 assay includes one additional CT detection probe targeting a second region of the CT 23S rRNA [27].

The aim of the present study was to evaluate the clinical sensitivity and specificity, as well as the analytical inclusivity and exclusivity of the updated AC2 (provided as research-use-only [RUO] material) for detection of wild-type (WT) CT, nvCTs, and NG on the automated Panther system (Hologic).

The evaluation panel consisted of 1004 clinical, routinely collected samples (from 2015 to 2020) previously diagnosed using AC2 and/or ACT, in accordance with the instructions from the manufacturer (Hologic), and 225 samples spiked with geographically and temporally (from 1971 to 2016) diverse isolates of NG, CT, FI-nvCT, and T. vaginalis (TV), as well as non-NG Neisseria, Moraxella, non-TV Trichomonas, or non-CT Chlamydia.

The results of all testing of AC2 clinical samples and spiked samples with the updated AC2 are summarised in Table 1.

The present study is the first clinical and analytical validation of the updated AC2 NAAT (Hologic), which includes one NG target and two CT targets [27] and most recently was granted US FDA approval (https://​www.​accessdata.​fda.​gov/​scripts/​cdrh/​cfdocs/​cfPMN/​pmn.​cfm?​ID=​K200866). Since the initial report of the FI-nvCT in late-May 2019 in Finland [18, 19] and subsequent reports of FI-nvCT and additional diagnostic-escape nvCTs in Sweden [20], Norway [21], Denmark [24], and the United Kingdom [25], reflex testing with ACT of possible AC2 diagnostic-escape nvCT has been performed in all European diagnostic laboratories [22, 26]. However, Hologic has now developed an updated version of the AC2 assay [27], which was designed to detect also all of the new diagnostic-escape nvCTs [18–25, the present study] and includes one additional CT detection probe targeting a second region of the CT 23S rRNA. In the present study, we show that the updated AC2 assay has a maintained high clinical sensitivity and specificity, as well as an ideal analytical inclusivity and exclusivity, when examining samples spiked in clinically relevant concentrations of microbe, in the detection of WT CT and NG. Furthermore, the updated AC2 assay also effectively detected all of the described AC2 diagnostic-escape nvCTs [18–25, the present study], i.e. clinical AC2 specimens containing the FI-nvCT (n = 114), nvCT-G1523A (n = 46), nvCT-C1522T (n = 6), and nvCT-C1514T (n = 4) were all positive. Thus, the clinical sensitivity and specificity of the updated AC2 for CT detection was 99.7 and 100%, respectively. Despite that samples were preselected as positive with the original version of AC2, only two CT WT-positive samples were missed with the updated AC2 assay (not sufficient samples remaining for confirmation with ACT). It cannot be excluded that a preselection of samples using the updated AC2 assay would provide similar results when retested in the original AC2 assay. Both the clinical sensitivity and specificity of the updated AC2 assay for NG detection was 100%.

The recent emergence of AC2 diagnostic-escape nvCTs [18–25, the present study], spread of the Swedish nvCT escaping detection using the NAATs available from Roche and Abbott at that time [4‐6, 33], and constantly evolving CT [7, 8], illustrate the necessity of dual target diagnostic CT NAATs and surveillance of the stability of NAAT targets in national and international settings. Furthermore, laboratories should be included in appropriate external quality assessments to detect diagnostic-escape nvCTs and it is imperative to adequately survey and analyse incidence, any unexplained shifts in positivity rates, and/or annual collections of samples diagnosed as negative/equivocal using NAATs with different target(s).

Widespread implementation of validated, accurate and quality-assured CT dual target NAATs for diagnosis of CT is crucial internationally. The updated AC2 assay, now including two CT targets and one NG target [27], showed a high clinical sensitivity and specificity, as well as an ideal inclusivity and exclusivity in the detection of WT CT, all described nvCTs [4–6,18–25, the present study], and NG. Nevertheless, international and national surveillance programmes capturing NAAT diagnostic-escape variants for CT as well as other infectious agents are imperative.