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. 2022 Nov 7;17(11):e0276729.
doi: 10.1371/journal.pone.0276729. eCollection 2022.

Validation of SARS-CoV-2 pooled testing for surveillance using the Panther Fusion® system: Impact of pool size, automation, and assay chemistry

Rudolph Park  1   2 , Prabha Chandrasekaran  1   2 , Heather Hernandez  1   2 , Ines Lakhal-Naouar  1   2 , Kristina K Peachman  1 , Holly R Hack  1   2 , Dante Coleman  1   2 , Jason Ouellette  1   2 , Janice M Darden  1   2 , Oussama M'hamdi  1   2 , Victor A Sugiharto  2   3 , Hua-Wei Chen  2   3 , Megan A Schilling  3 , Mark P Simons  3 , Natalie D Collins  4 , Yuliya S Johnson  5 , Linda L Jagodzinski  1 , Sheila A Peel  1
Affiliations

Validation of SARS-CoV-2 pooled testing for surveillance using the Panther Fusion® system: Impact of pool size, automation, and assay chemistry

Rudolph Park et al. PLoS One. .

Abstract

Combining diagnostic specimens into pools has been considered as a strategy to augment throughput, decrease turnaround time, and leverage resources. This study utilized a multi-parametric approach to assess optimum pool size, impact of automation, and effect of nucleic acid amplification chemistries on the detection of SARS-CoV-2 RNA in pooled samples for surveillance testing on the Hologic Panther Fusion® System. Dorfman pooled testing was conducted with previously tested SARS-CoV-2 nasopharyngeal samples using Hologic's Aptima® and Panther Fusion® SARS-CoV-2 Emergency Use Authorization assays. A manual workflow was used to generate pool sizes of 5:1 (five samples: one positive, four negative) and 10:1. An automated workflow was used to generate pool sizes of 3:1, 4:1, 5:1, 8:1 and 10:1. The impact of pool size, pooling method, and assay chemistry on sensitivity, specificity, and lower limit of detection (LLOD) was evaluated. Both the Hologic Aptima® and Panther Fusion® SARS-CoV-2 assays demonstrated >85% positive percent agreement between neat testing and pool sizes ≤5:1, satisfying FDA recommendation. Discordant results between neat and pooled testing were more frequent for positive samples with CT>35. Fusion® CT (cycle threshold) values for pooled samples increased as expected for pool sizes of 5:1 (CT increase of 1.92-2.41) and 10:1 (CT increase of 3.03-3.29). The Fusion® assay demonstrated lower LLOD than the Aptima® assay for pooled testing (956 vs 1503 cp/mL, pool size of 5:1). Lowering the cut-off threshold of the Aptima® assay from 560 kRLU (manufacturer's setting) to 350 kRLU improved the assay sensitivity to that of the Fusion® assay for pooled testing. Both Hologic's SARS-CoV-2 assays met the FDA recommended guidelines for percent positive agreement (>85%) for pool sizes ≤5:1. Automated pooling increased test throughput and enabled automated sample tracking while requiring less labor. The Fusion® SARS-CoV-2 assay, which demonstrated a lower LLOD, may be more appropriate for surveillance testing.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Fusion® and Aptima® vs CDC 2019-nCoV, TaqPath™ and cobas® assay neat test results.
CT results for Fusion® (ORF1ab target) vs (A) CDC 2019-nCoV (N1 and N2 targets), (B) TaqPath™ (ORF1ab) and (C) cobas® (ORF1ab). Linear regression equation shown as y = R2 = Coefficient of regression. kRLU results for Aptima® (ORF1ab) vs (D) CDC 2019-nCoV, (E) TaqPath™ and (F) cobas®. (G) Aptima® vs Fusion® SARS-CoV-2 results with correlation coefficient (r) calculated for high (CT <25), moderate (25<CT<35) and low (CT >35) viral load ranges. Statistical significance (p) calculated by two-sided t-test. (D-G) Aptima® cut-off threshold (560 kRLU) shown in green.
Fig 2
Fig 2. Distribution of Aptima® pooled sample test results.
Samples used in manual (left panel) and automated (right panel) pool generation. Test results are represented as circles. Numbers within bars represent median values. Dashed horizontal line represents cut-off value (560 kRLU). Two-sided t-test, * p<0.03, *** p<0.0001.
Fig 3
Fig 3. Distribution of Fusion® neat and pooled sample test results.
Samples used in manual pool generation (left panel) and automated pool generation (right panel). Test results are represented as circles. Numbers within bars represent median values. Two-sided t-test, ***p<0.0001.
Fig 4
Fig 4. Linearity of the Panther Fusion® SARS-CoV-2 assay for pooled testing.
(A)(C) Bland-Altman plot and (B)(D) Passing-Bablok regression plots are presented for 5:1 or 10:1 pooled testing versus neat with the Panther Fusion® SARS-CoV-2 assay. For the Bland-Altman plots, the area between the dotted lines (green or orange) indicates the 95% limits of agreement. For the Passing-Bablok regression, the confidence interval is shaded (green or orange) and the blue line indicates the line of identity. The slope and intercept of the regression line are reported in the left top of each panel.
Fig 5
Fig 5. Source of RNA impacts LLOD.
LLOD for the Hologic Panther® SARS-CoV-2 assays determined by 5 independent measurements, performed over 5 days in duplicates (n = 10 for each datapoint). Blue: Aptima® results. Orange: Fusion® results. (A) Heat Inactivated Virus target in STM/UTM (B) Genomic RNA target in STM/UTM. The probit (predicted proportion of replicates positive) versus the SARS-CoV-2 RNA concentration identified.
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