Fully automated detection and differentiation of pandemic and endemic coronaviruses (NL63, 229E, HKU1, OC43 and SARS-CoV-2) on the Hologic Panther Fusion

Abstract

The Hologic Panther Fusion (PF) platform provides fully automated CE marked diagnostics for respiratory viruses, including recently SARS-coronavirus 2 by a transcription mediated amplification (TMA) assay, but not for the endemic human coronaviruses (hCoV). Therefore, a laboratory developed multiplexed RT-PCR protocol (LDT) that detects and differentiates the four hCoV NL63, 229E, HKU1 and OC43 was adapted on the PF. The novel CE marked Aptima SARS-CoV-2 TMA and the LDT for hCoV were validated with 321 diagnostic specimens from the upper and lower respiratory tract in comparison to two SARS-CoV-2 RT-PCRs (PF E-gene LDT and genesig RT-PCR, 157 specimens) or the R-GENE hCoV / hParaFlu RT-PCR (164 specimens), respectively. For the endemic hCoV, results were 96.3% concordant with two specimens discordantly positive in the PF and four specimens discordantly positive in the R-GENE assay. All discordantly positive samples had Ct values between 33 and 39. The PF hCoV LDT identified 23 hCoV positive specimens as NL63, 15 as 229E, 15 as HKU1 and 25 as OC43. The Aptima SARS-CoV-2 TMA gave 99.4 % concordant results compared to the consensus results with a single specimen discordantly positive. Moreover, 36 samples from proficiency testing panels were detected and typed correctly by both novel methods. In conclusion, the SARS-CoV-2 TMA and the LDT for hCoV enhanced the diagnostic spectrum of the PF for all coronaviruses circulating globally for a multitude of diagnostic materials from the upper and lower respiratory tract.

Evaluation of the analytical performance and specificity of a SARS-CoV-2 transcription-mediated amplification assay

“…With a LoD of 150 copies/ml the 95% detection probability of the Aptima assay can be considered as sufficiently sensitive. This is consistent with data from others, who found a 100% LoD of 62.5 copies/ml (synthetic RNA ( Smith et al, 2020 )) and a 95% LoD of 288 copies/ml (AccuPlex SARS-CoV-2 Verification Panel ( Cordes et al, 2020 )).…”

“…The validation data of clinical and proficiency panel samples demonstrate a very good test concordance of the Aptima SARS-CoV-2 TMA assay (Hologic) and the RealStar SARS-CoV-2 RT-PCR (Altona), with a PPA of 95.7% and NPA of 100%. Similar agreement values have been described for the Aptima SARS-CoV-2 TMA test with the Panther Fusion SARS-CoV-2, the BioFire Defense COVID-19 or other RT-PCR assays ( Cordes et al, 2020 ; Gorzalski et al, 2020 ; Kuo et al, 2021 ; Smith et al, 2020 ; Trémeaux et al, 2020 ). The three samples with discordant results (positive in the RealStar but negative in the Aptima assay) all had high Ct values corresponding to low viral loads: for two samples virus concentrations clearly below the LoD of the Aptima assay can be assumed, while the third sample which originated from a health-care worker without hospital admission had a viral load of approximately 3800 copies/ml (Real-Star E-Gene RT-PCR).…”

Investigating the optimum sample type and target genes for SARS-CoV-2 detection

“…The ORF 1ab gene and N gene are unique to SARS-CoV-2, but the E fragment is also present in other coronaviruses, such as 229E, OC43, NL63, HKU1, SARS, and MERS. 6-8 To our knowledge, research on whether the inclusion of the E gene is necessary in a large-scale population screening and whether it has an effect on the specificity of the test have been lacking in studies with larger sample sizes. In the present study, no significant effect of excluding the E gene fragment on the interpretation of nucleic acid detection was observed, and only 5 out of 1003 cases showed slight differences in the detection results.…”

Toward a next-generation diagnostic tool: A review on emerging isothermal nucleic acid amplification techniques for the detection of SARS-CoV-2 and other infectious viruses

“…The Hologic Panther system uses a TMA-based SARS-CoV-2 assay [ 297 ], and it is a large-scale clinical testing system capable of running over 1000 tests in a day. This fully automated platform has been used to sensitively detect different human coronaviruses (hCoVs) [ 298 ], and one study even found that its analytical performance was higher than that of RT-PCR [ 299 ].…”