key: cord-0915624-oi5dod5t
authors: Newsom, Kimberly; Zhang, Yuan; Chamala, Srikar; Martinez, Katherine; Clare‐Salzler, Michael; Starostik, Petr
title: The Hologic Aptima SARS‐CoV‐2 assay enables high ratio pooling saving reagents and improving turnaround time
date: 2021-07-02
journal: J Clin Lab Anal
DOI: 10.1002/jcla.23888
sha: a0e991d3d801a2c4cfb9b8082e164268e7353bbc
doc_id: 915624
cord_uid: oi5dod5t

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.

trying to stay opened, expanding testing capacity through accurate, easy to use, and high-throughput molecular diagnostic methods becomes urgent.

So far, more than 160 molecular diagnostic assays for the detection of SARS-CoV-2 have been approved by the Food and Drug Administration (FDA) under the Emergency Use Authorization (EUA). In May 2020, the Hologic ® Aptima SARS-CoV-2 assay based on transcription-mediated amplification (TMA) received FDA approval (EUA200734) (Aptima™, Hologic ® Panther System). TMA is an isothermal, auto-catalytic target amplification method. The assay shows high sensitivity for SARS-CoV-2 detection and high correlation with the CDC RT-PCR assay. 2, 3 Most importantly, the Hologic ® Panther platform is a complete sample-to-result automated instrument for testing up to 1200 samples/day/instrument. It thus enables high-throughput testing with minimum manual labor involvement.

With the ever-increasing demand for SARS-CoV-2 testing capacities, pooling of samples for testing becomes a viable alternative in an environment strapped for resources, reagents, and consumables.

Sample pooling implies that several samples are mixed together at a given ratio and tested as one single pool. Only positive pools have to be deconvoluted and individual neat samples tested. Otherwise, negative pools are assumed to contain negative samples. The practice of pooling biological samples together for testing is not a new technique, it can be traced back to at least 1940s 4 when it was used for syphilis outbreak testing. It is suggested that testing of pooled samples is better to use for surveillance samples from populations with low prevalence of an infectious agent. 5 Different sample pooling methods were validated to be used in SARS-CoV-2 testing by many laboratories. [6] [7] [8] [9] [10] However, all the previously validated sample pooling methods used the RT-PCR assay.

Recently, testing pooled samples with the Hologic Aptima™ TMA assay has been approved by the FDA (https://www.fda.gov/medic aldevic es/coron aviru s-disea se-2019-covid -19-emerg ency-use-autho rizat ions-medic al-devic es/vitro -diagn ostic s-euas#indiv idual -molec ular). In this study, we evaluated the performance of the Aptima™ TMA assay from Hologic ® pooling 10 samples together. We explored pooling in a screening setting, testing asymptomatic individuals returning to work or school when the prevalence of positive cases in the population tested was 0.3%-0.7%.

Total of over 50,000 nasopharyngeal (NP) swabs in viral transportation medium (VTM) were processed for clinical SARS-CoV-2 testing at University of Florida (UF) Pathology Laboratories from both symptomatic and asymptomatic subjects between May and September 2020. Residual specimens of these samples which were stored in −80°C were used to validate the described pooling experiments using the Aptima assay.

This FDA-approved TMA-based assay detects two different conserved regions within the ORF1ab section of the SARS-CoV-2 viral genome using the Hologic ® Panther platform. The testing was performed following manufacturer's (Hologic Inc.) instructions. Cut-off value for positive results was set at 580 kRLU for neat samples and 324 for pooled samples.

The 

FDA-approved Xpert Xpress SARS-CoV-2 assay was run on the GeneXpert instrument (Cepheid). The test incorporates sample preparation, nucleic acid extraction, amplification, and detection of viral nucleic acid targets N2 and E.

Hamilton Microlab next-generation sequencing (NGS) STAR liquid handling system (Hamilton Co.) was used to create sample pools using a proprietary program. Briefly, unscrewed collection tubes containing samples were placed into racks that were pulled onto the robot deck, barcodes on the tubes were read and 50 µl of VTM from each of the 10 sequential samples was pipetted into a barcoded Hologic lysis tube. The pooled samples were then loaded onto the Hologic Panther platform for the Aptima assay. Informatics part of the pooling process was described previously. 11 

To assess the effect of pooling on the Aptima assay performance, 34 SARS-CoV-2-positive samples from the analyzed population were combined with negative previously tested samples to generate 10 to 1 (10/1) pools. To assure that the whole positive range from strong positive to weak positive was covered, neat positive samples were also analyzed with the CDC RT-PCR assay that generates quantification cycle (Cq) values corresponding to the number of viral copies in the sample in linear fashion ( Table 1) 

To better validate the performance of positive 10/1 pools with a weak signal, additional 23 positive samples from previous Aptima testing were employed. Before the pools were prepared, the neat samples underwent testing with the Cepheid RT-PCR assay to better determine viral copy numbers. Immediately afterwards, these same samples were pooled, run with the Aptima assay in triplicate, and the results plotted ( Figure 4C ). We decided to run triplicates, as previous We ran the neat samples with the Cepheid RT-PCR assay right before pooling in order to better assess the viral load in the samples and to make sure the viral RNA did not degrade during storage, as the original Aptima results for neat samples were several weeks old. 

To calculate the PPA of the pooled samples, we combined data on pools tested, shown in Figures 3 and 4 . Altogether, we had 57 pools known to contain a positive neat sample (Table 2) is optimal for 0.1%-2% positivity rate. 7 Pooling of samples has been proven to work with RT-PCR, [6] [7] [8] 26 while there is much less experience about pooling for a TMA assay.

It is amenable to pooling exactly like a PCR-based assay with the caveat that the assay results measured in kRLU do not show a strict linear correlation with viral copy numbers when a sample is diluted.

We applied pooling to screening a population of asymptomatic UF employees and students returning to campus. With a positivity rate of less than 0.5% we were able to pool 10/1 achieving very considerable savings in reagents, consumables, labor, and shortening the TAT.

As an academic lab well-equipped to run NGS and Taqman-based assays, we had already available the instrumentation and human capital needed to accomplish the task. The process of pooling thousands of samples is not trivial and requires both pipetting robot capabilities and informatics support as described elsewhere. 11 The Aptima SARS-CoV-2 assay is highly sensitive, nevertheless, pooling at such high ratio leads inevitably to loss of sensitivity. To improve the sensi- Results for 500 positive samples tested by the CDC RT-PCR SARS-CoV-2-modified assay between July and September 2020 were plotted. The frequency of the corresponding N2 Cq values is shown 10/1 pooled sample testing by the Aptima TMA assay is an effective method for screening saving valuable resources.

The authors would like to thank the medical staff from the Division of Molecular Pathology, Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine.

The authors declare that they have no competing interests.

All the data generated during and/or analyzed during the current study are available from the corresponding author (PS) upon reasonable request.

Petr Starostik https://orcid.org/0000-0002-8992-1230

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How to cite this article

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

TA B L E 2 57 SARS-CoV-2-positive samples and their 10/1 pools tested with Aptima SARS-CoV-2 Assays