The Hologic Aptima SARS‐CoV‐2 assay enables high ratio pooling saving reagents and improving turnaround time

Abstract

Background The Hologic Aptima™ TMA SARS‐CoV‐2 assay was employed to test pooled nasopharyngeal (NP) samples to evaluate the performance of pooled sample testing and characterize variables influencing results. Methods Results on 1033 previously tested NP samples were retrieved to characterize the relative light units (RLU) of SARS‐CoV‐2‐positive samples in the tested population. The pooling strategy of combining 10 SARS‐CoV‐2 samples into one pool (10/1) was used in this study. The results were compared with neat sample testing using the same Aptima™ TMA SARS‐CoV‐2 assay and also the CDC RT‐PCR and the Cepheid SARS‐CoV‐2 assays. Results The Aptima assay compares favorably with both CDC RT‐PCR and the Cepheid SARS‐CoV‐2 assays. Once samples are pooled 10 to 1 as in our experiments, the resulting signal strength of the assay suffers. A divide opens between pools assembled from strong‐positive versus only weak‐positive samples. Pools of the former can be reliably detected with positive percent agreement (PPA) of 95.2%, while pools of the latter are frequently misclassified as negative with PPA of 40%. When the weak‐positive samples with kRLU value lower than 1012 constitute 3.4% of the total sample profile, the assay PPA approaches 93.4% suggesting that 10/1 pooled sample testing by the Aptima assay is an effective screening tool for SARS‐CoV‐2. Conclusion Performing pooled testing, one should monitor the weak positives with kRLU lower than 1012 or quantification cycle (Cq) value higher than 35 on an ongoing basis and adjust pooling approaches to avoid reporting false negatives.

A precise review on NAATs‐based diagnostic assays for COVID‐19: A motion in fast POC molecular tests

“…Transcription‐mediated amplification (TMA) is an isothermal, exponential auto‐catalytic target amplification method that is almost identical to NASBA. 12 , 73 This method has been utilized to amplify cellular mRNA and viral RNA using RT and T7 RNA polymerase in a short time. 74 , 75 Two primers that are designed to perform the current method are a promoter primer with a 5′ flanking sequence for recognition of RNA polymerase and a nonpromoter primer.…”

“…The Aptima SARS‐CoV‐2 assay, known as Panther system, is a NAAT‐based detection method that is developed by Hologic Panther in May 2020. 12 It got FDA approval for testing COVID‐19 patients for EUA only on 14 May 2020. 76 The Aptima assay targets two virus unique sequences located on the ORF1ab gene of the SARS‐CoV‐2 and is conducted on the automated Panther platform.…”

“… 76 , 81 Therefore, the assay with a performance rate of up to 1200 samples per day is considered as a high‐throughput, fully automated molecular test. 12 Drawbacks for this assay include the need to train personnel and only use a nasopharyngeal swab for collecting samples. Negative results do not mean a lack of SARS‐CoV‐2 infection and should not be considered as final decisions.…”

“… 9 Up to now, over 160 molecular diagnostic methods for the detection of 2019‐nCoV have been approved by US Food and Drug Administration (FDA) and Emergency Use Authorizations (EUA). 12 Conforming to the World Health Organization (WHO), the instant precedence for emerging of COVID‐19 clinical diagnostics methods should be based on detection at the rapid point‐of‐care (POC). 13 POC tests are affordable, hand‐held molecular diagnostic assays operated at or near the sample collection site, and results are presented within minutes not hours.…”

Performance of the Procleix SARS-CoV-2 transcriptase-mediated amplification (TMA) assay for the diagnosis of COVID-19 in nasopharyngeal sample pools. Small pilot study.

“…Las tres muestras original-mente positivas por TMA, pero subsiguientemente negativas en dilución 1/16 habían sido negativas para el gen N por RTPCR, lo que sugiere que podrían corresponderse con muy bajas cargas víricas. Previamente se ha observado que señales de Ct en RT-PCR superiores a 35 pueden condicionar la aparición de resultados falsos negativos en mezclas de muestras estudiadas por TMA [ 10 ].…”