Retrospective AC2 test result data from Laboratory Corporation of America Holdings (Labcorp) was examined to assess trends in the CT positivity rate over time. The weekly positivity rate for 18,476,856 specimens tested by AC2 during December 24, 2017-December 25, 2019 was determined and plotted by annual quarters using R (13).

Labcorp identified a subset of specimens tested using the AC2 at its Burlington, North Carolina (NC) location over a 5-week period during late December 2019 to early February 2020 for further molecular characterization at CDC. Approximately 470,915 specimens were tested by AC2 using the Panther^® (Hologic, Inc.) instrument during this timeframe. Only NG-negative specimens were selected. Based on previous studies examining the prevalence of the Fl-nvCT variant in other countries, CT-negative or equivocal specimens with RLU ≥15–99 were considered to most likely include potential variants (5, 7, 9). All specimens (N=50) within this group were selected for further characterization (see Supplemental Figure 1 for testing workflow). A further cohort of 300 CT-negative specimens with RLU <15 were additionally selected, as were 51 specimens (CT-positive N=47, with N=2 each CT-negative or CT-equivocal) with RLU ≥100. Specimen types included urine, rectal, endocervical, vaginal, and oropharyngeal swabs. De-identified patient information, including geographic (state) location of collection, anatomical site, patient gender and age, and date of collection was also provided. Specimens included in this study represented individuals in nearly 30 states; approximately half (~51%) were from Virginia, Illinois, Georgia, South Carolina, and North Carolina. This study was determined to not be human subject research through review by the National Center for HIV/AIDS, Viral Hepatitis, STD, and Tuberculosis Prevention.

Genomic DNA (gDNA) was isolated from all specimens (N=401) using 200 μL aliquots processed with the QIAsymphony DSP DNA Mini Kit (Qiagen, Inc., Germantown, MD) on the QIAsymphony (Qiagen, Inc.) automated platform. All specimens were tested using a lab-developed duplex real-time PCR (CT PCR) targeting the CT cryptic plasmid and human ribonuclease P (RNP) as an internal control for a valid clinical specimen on a Rotor-Gene real-time PCR instrument (Qiagen, Inc.) as described previously (14). This in-house assay was previously validated on rectal swab specimens with a limit of detection of ≤100 copies per reaction and analytic sensitivity and specificity of the CT target of 93% and 100%, respectively (14).

All specimens from the ≥15–99 RLU category (N=50) and a subset (N=5) from each of the other RLU categories were tested using a nested end-point PCR assay targeting a portion of the CT 23S rRNA gene with primers previously published (6). Sequencing of the 23S variable region 2 (V2) in purified amplicons proceeded using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Products were analyzed on an Applied Biosystems 3500xL Genetic Analyzer. Reads were assembled and analyzed in Geneious Prime software (Biomatters Ltd., Auckland, New Zealand) for sequence determination.

Additionally, a total of 64 specimens consisting of the entire ≥15–99 RLU category, five from the <15 RLU category, and nine from the ≥100 RLU category (including the five sequenced as described above along with the four AC2 CT negative or equivocal specimens), were re-tested on the Hologic Panther^® platform using the updated AC2 assay, which we obtained as an RUO formulation as the updated formulation was not yet FDA-cleared. Results between the original in vitro diagnostic (IVD) and the updated RUO AC2 assays were compared to assess the performance of this new assay on these specimens.

Among specimens tested during December 24, 2017–December 25, 2019, the average weekly CT positivity rate was 5.5% with a range of 4.6–6.6% (Fig 1). We did not observe any downward trends in positivity as may be expected if large numbers of false-negative tests were occurring due to widespread circulation of diagnostic escape mutants. Notably, weeks with high CT positivity rates (>6.5%) were observed and may likely be associated with repeat testing of specific specimens (e.g., retesting of specimens with an initial equivocal result).

A total of 401 remnant specimens were collected for further evaluation. These specimens were grouped into three different categories based on their initial RLU signal in the AC2 assay: RLU <15, RLU ≥15–99, and RLU ≥100. For each category, the majority of specimens were collected from females (Table 1). The largest proportion of specimens in the <15 RLU category were vaginal swabs while both the ≥15–99 and ≥100 RLU categories had large proportions (~40%) of specimens from unknown anatomical sites. The median ages of individuals from whom specimens were collected was ≤30.

DNA was isolated from all 401 remnant specimens and tested with a lab-developed real-time PCR to determine the suitability of this genetic material for downstream applications, including Sanger sequencing. As expected, all CT-negative specimens with low RLU values (i.e., <15 RLU category) were CT PCR negative (a single specimen was determined to be invalid due to a failure to detect human DNA) (Fig 2). Despite previous AC2 results from specimens in the ≥100 RLU category demonstrating CT positivity in 47/51 specimens (92%), we were only able to detect CT DNA by real-time PCR in 17 of those 47 (36.2%). In the ≥15–99 RLU category, which included the range of RLU values with the greatest probability of containing diagnostic-escape variants, we detected seven CT-positive specimens by real-time PCR, indicating those specimens might contain genetic variance within the 23S rRNA target region.

We attempted Sanger sequencing of 23S rRNA AC2 assay target region for all 50 specimens in the ≥15–99 RLU category group. As controls, we attempted sequencing in a subset of specimens from the <15 RLU (N=5) and ≥100 RLU (N=5) categories. Not surprisingly, there was no amplification and no sequencing results for those tested from the <15 RLU category. All five specimens from the ≥100 RLU category revealed wild-type 23S rRNA sequences. Of the 50 specimens from the ≥15–99 RLU category, six specimens (which were also CT PCR positive) were successfully sequenced. Analysis of the genetic sequence in those six specimens revealed four with wild-type 23S rRNA sequences and two with variations (A1518G and G1526A) in the AC2 assay target region.

We further evaluated the updated RUO AC2 assay using the 60 remnant specimens initially tested with the commercially available IVD AC2 assay and on which sequencing was attempted in this study. In addition, four CT-negative/equivocal specimens from the ≥100 RLU category were examined due to their RLU values from the IVD AC2 assay (between 118 and 226) which often is associated with an algorithmic determination of positive CT status. Specimens in the <15 RLU category again tested negative for CT using the RUO reagents (Fig 3). One of the CT-equivocal specimens from the ≥100 RLU category was classified as CT-positive when re-tested with the updated AC2 reagents (initial RLU of 243 whereas the updated RLU using the updated reagents was 779) and all of the initially CT-positive specimens producing the same results in the RUO assay. The other CT-equivocal specimen from this category became CT-negative as determined by the assay’s algorithm (original RLU of 130 and new RLU of 123) while the two CT-negative specimens from this category remained negative (original RLU value of 118 for both and new RLUs of 10 or 11). In the ≥15–99 RLU category, all 50 specimens were re-tested and, of these, nine originally CT-negative and 13 originally CT-equivocal were CT-positive upon re-test using the RUO assay. These 22 encompassed various specimen types including endocervical, vaginal, oropharyngeal, and rectal swabs as well as urine and included the six real-time PCR CT-positive specimens that were successfully sequenced. The seventh CT PCR positive specimen (specimen type not provided) from this category which we were unable to sequence remained CT-negative using the updated AC2. In total, 23 specimens CT-negative/equivocal by the IVD AC2 assay were CT-positive using these RUO reagents.

We further explored the specimens in the ≥15–99 RLU category that were re-tested using the RUO AC2 assay by stratifying across RLU values produced in the initial IVD AC2 assay. Among the specimens originally testing negative for CT, most (81.3%) of those with initial RLU values between 15–32 were also negative in the RUO assay (Table 2). The proportion of initially CT-negative specimens testing positive in the RUO assay did not increase with higher RLU values from the IVD assay. Notably, none of the specimens in the ≥15–99 RLU category originally testing equivocal for CT were found to be equivocal in the RUO assay; most (≥70%) of these specimens tested positive in the RUO assay.