key: cord-0875816-xszjp5gn
authors: Fernández-Huerta, Miguel; Salmerón, Paula; Hernández-Hermida, Yolanda; Andrés, Cristina; Niubó, Jordi; Calatayud, Laura; Angeles Domínguez, M; Pumarola, Tomàs; Ardanuy, Carmen; Antón, Andrés; Càmara, Jordi
title: Multicenter clinical evaluation of a novel transcription-mediated amplification assay for SARS-CoV-2 molecular testing
date: 2022-02-14
journal: Enferm Infecc Microbiol Clin
DOI: 10.1016/j.eimc.2022.01.014
sha: c6297b3e52dc0d16aee562a6b82529ce797bb460
doc_id: 875816
cord_uid: xszjp5gn

Introduction: The onset and spread of COVID-19 pandemic has forced clinical laboratories to rapidly expand testing capacity for SARS-CoV-2. This study evaluates the clinical performance of the TMA Procleix SARS-CoV-2 assay in comparison to the RT-PCR assay AllplexTM SARS-CoV-2 for the qualitative detection of SARS-CoV-2 RNA. Methods: Between November 2020 and February 2021, 610 upper-respiratory specimens received for routine SARS-CoV-2 molecular testing were prospectively collected and selected at the Hospital Universitari Vall d’Hebron and the Hospital Universitari Bellvitge in Barcelona, Spain. All samples were processed in parallel with the TMA and the RT-PCR assays, and results were compared. Discrepancies were retested by an additional RT-PCR method and the clinical history of these patients was reviewed. Results: Overall, the level of concordance between both assays was 92.0% (κ, 0.772). Most discordant results (36/38, 94.7%) corresponded to samples testing positive with the TMA assay and negative with the RT-PCR method. Of these discrepant cases, most (28/36, 77.8%) were finally classified as confirmed or probable SARS-CoV-2 cases according to the discrepant analysis. Conclusion: In conclusion, the TMA Procleix SARS-CoV-2 assay performed well for the qualitative detection of SARS-CoV-2 RNA in a multisite clinical setting. This novel TMA assay demonstrated a greater sensitivity in comparison to RT-PCR methods for the molecular detection of SARS-CoV-2. This higher sensitivity but also the qualitative feature of this detection of SARS-CoV-2 should be considered when making testing algorithm decisions.

The onset and rapid spread of COVID-19 pandemic has forced clinical laboratories to rapidly expand testing capacity for SARS-CoV-2. 1 between both methods is conducted to assess the relevance of these cases in the management of SARS-CoV-2 infected individuals.

Between November 2020 and February 2021, 154 and 456 upper-respiratory specimens received for routine SARS-CoV-2 molecular testing were prospectively collected and randomly selected for the current evaluation at the Hospital Universitari Vall d'Hebron (HUVH) and the Hospital Universitari Bellvitge (HUB) in Barcelona, Spain;

respectively. The selection included nasopharyngeal and nasal swabs, placed in viral transport medium (VTM) from various manufacturers, collected from symptomatic and asymptomatic individuals, with no bias towards age or gender.

The detailed methodology for the current evaluation was conducted as follows ( Figure   S1 ). VTM aliquots from selected fresh specimens were manually transferred into sterile tubes containing lysis buffer in a 1:1 proportion. If available, surplus specimen material was stored at -80 for subsequent analysis. SARS-CoV-2 molecular testing was first performed with the TMA Procleix SARS-CoV-2 assay. Using the same inactivated aliquot, nucleic acid extraction was performed with automated systems recommended by the manufacturer and SARS-CoV-2 detection was sequentially executed using the RT-PCR assay Allplex TM SARS-CoV-2.

In case of discrepant results, stored samples were thawed, inactivated and processed with an additional RT-PCR technique: the Alinity m SARS-CoV-2 assay (Abbott Molecular, US) or the RT-PCR cobas® SARS-CoV-2 test (Roche Molecular Systems, US). Also, discrepant cases were further analyzed through an exhaustive review of the medical record.

were interpreted according to the manufacturer´s criteria.

The TMA Procleix SARS-CoV-2 assay is a novel multiplex NAAT based on TMA methodology for the qualitative molecular detection of SARS-CoV-2 virus. 4 The assay, EUA granted by the FDA, incorporates an exogenous internal control, and specifically targets the viral N gene. The test is ready to be run on the fully automated all-in-one

Procleix Panther system (Grifols International, S.A.) using 750 µL material from upperrespiratory swabs. In this regard, the assay provides a ratio value that corresponds to an analytical parameter based on chemiluminescense with a qualitative interpretation.

Values ≥1.00 are considered reactive/positive, according to the manufacturer´s criteria.

Statistical analyses were performed using GraphPad Prism version 8.0.1 (GraphPad Software Inc., USA). The Cohen´s kappa statistic (κ) was used to evaluate the agreement between both assays, and 95% confidence intervals (CI) were calculated by exact methods.

Minor disagreement was defined as a positive test with one assay and an inconclusive test with the other. In the current comparison, only the RT-PCR assay Allplex TM SARS-CoV-2 could yield inconclusive results, defined by the manufacturer as those cases testing positive for the E target, but negative for both the N and the RdRP/S targets.

Major disagreement was considered that reverting from positive to negative depending on the study assay. If available, discrepant specimens were retested with an additional RT-PCR method and managed as follows. A confirmed case of SARS-CoV-2 was considered that discrepancy testing positive with the additional RT-PCR assay. A probable case was considered that testing negative with the additional RT-PCR J o u r n a l P r e -p r o o f 9 technique, but having epidemiological or clinical data suggestive and compatible with recent history of SARS-CoV-2 infection. Unresolved cases were reported as inconclusive.

Institutional Review Board approval (PR(AG)259/2020) was obtained from the HUVH Clinical Research Ethics Committee.

The qualitative comparison of the 610 study samples is displayed in Table 1 . Overall, the level of concordance between both assays was 92.0% (95% CI, 89.5%-94.0%), with a κ value of 0.772 (0.715-0.830). Of the 38 major discrepancies, 36 (94.7%) corresponded to samples testing positive with the TMA Procleix SARS-CoV-2 assay and negative with the RT-PCR assay Allplex TM SARS-CoV-2.

A comprehensive analysis of discrepant results is detailed in Tables 2A and 2B. While   Table 2A focuses on the analytical approach of this evaluation, Table 2B provides an indepth analysis of discrepant cases by incorporating some key epidemiological features from the medical record. The main conclusions of this assessment are summarized in Table 3 . Overall, from the 36 discrepancies mentioned above, 28 (77.8%) were finally classified as confirmed (11/36) or probable (17/36) SARS-CoV-2 cases. On the other hand, both major discrepancies testing positive with the RT-PCR assay Allplex TM SARS-CoV-2 but negative with the TMA Procleix SARS-CoV-2 assay remained inconclusive. Finally, eight of the 11 minor disagreements (72.7%), were at last reported as confirmed (6/11) or probable (2/11) SARS-CoV-2 cases.

The current wide availability of commercial assays to address the critical need for largescale SARS-CoV-2 testing also requires comprehensive analytical, clinical and real-life experiences to better understand the strengths and limitations of each diagnostic Overall, the TMA Procleix SARS-CoV-2 assay performed well for the detection of SARS-CoV-2 compared to the RT-PCR assay AllplexTM SARS-CoV-2, with a substantial agreement between assays (κ, 0.772). Most major discrepancies (95%) corresponded to samples testing positive with the TMA Procleix SARS-CoV-2 assay, but reverting to negative results with the RT-PCR test. When pondering the discrepant analyses, many of these cases (31%) were analytically confirmed to be positive for SARS-CoV-2, while many others (47%) were classified as probable positive according to the epidemiological and clinical history. These results suggest that the TMA Procleix SARS-CoV-2 assay may be more sensitive for the detection of SARS-CoV-2 RNA compared to RT-PCR methods, similar to what has been reported for this and other TMA tests such as the Aptima SARS-CoV-2 assay (Hologic, Inc., US). [4] [5] [6] [7] On the other hand, most minor discrepancies, these were specimens testing positive with the TMA test but inconclusive with the RT-PCR assay Allplex TM SARS-CoV-2, were also reported as confirmed or probable SARS-CoV-2 cases in accordance with the hypothesis of an inherent higher sensitivity of the TMA assay. In this regard, findings from an in vitro supplemental experiment, using 10-fold dilutions in five SARS-CoV-2 positive samples, analytically reinforce this conclusion ( Figure S2 ).

Our study evidences that the TMA Procleix SARS-CoV-2 assay is highly sensitive. The methodology used strongly suggests that this feature, compared to RT-PCR methods, is not a matter of pre-analytical variability rather the intrinsic chemistry behind TMA technology. 8, 9 Despite the presumptively high specificity of this technique, the increased sensitivity and the lack of quantitative/semi-quantitative parameters as a rough measure The study does require some considerations. First, information is missing in some discrepant cases due to the retrospective nature of the epidemiological and clinical data collection, and some specimens were unavailable for discrepant analytical testing. Also, at some point of the study, the TMA Procleix SARS-CoV-2 assay was used to actively select positive results, thus biasing the true positivity rate during that period. Inconclusive cases are considered: i) those testing positive for the E target, but negative for both the N and the RdRP/S targets with the RT-PCR Allplex TM SARS-CoV-2 assay, according to the manufacturer´s criteria, ii) those testing positive for the E target, but negative for the ORF-1ab target with the RT-PCR cobas® SARS-CoV-2 assay, according to the manufacturer´s criteria, and iii) those unresolved after the discrepant analytical testing.

c The two SARS-CoV-2-specific probes are labeled with the same fluorophore, so the test provides a unique Ct value for both RdRP and N targets. d Specimens unavailable for discrepant analytical testing.

A minor disagreement is considered that discrepancy testing positive with one assay but inconclusive with the other study technique. Contrary, a major disagreement is considered that reverting from positive to negative depending on the study assay.

Abbreviations: TMA, transcription-mediated amplification; RT-PCR, real-time reverse-transcriptase PCR; Ct, cycle-threshold; P, positive; N, negative; I, inconclusive; NA, not amplified; ND, not determined. 

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