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. 2024 Oct 22;14(21):2350.
doi: 10.3390/diagnostics14212350.

Performance Assessment of a Multiplex Real-Time PCR Assay for Detection of Viruses Causing Respiratory Tract Infections

Pallavi Upadhyay  1 , Fahida Surur  1 , Vijay Singh  1
Affiliations

Performance Assessment of a Multiplex Real-Time PCR Assay for Detection of Viruses Causing Respiratory Tract Infections

Pallavi Upadhyay et al. Diagnostics (Basel). .

Abstract

Objectives: Following the COVID-19 pandemic, global epidemiological trends demonstrate a return to pre-pandemic levels of respiratory syncytial virus (RSV) and influenza (Flu) A/B viruses. For the appropriate clinical management of viral infections, reliable and timely diagnosis is crucial. The clinical presentation of these respiratory viral infections shows significant overlaps; thus, the syndromic diagnosis of these infections becomes challenging. The goal of this study was to compare the performance of three multiplex real-time PCR-based platforms for the detection of SARS-CoV-2, Flu A, Flu B, and RSV.

Materials and methods: A retrospective study was performed on 200 de-identified nasopharyngeal and oropharyngeal specimens. All samples were tested simultaneously on three PCR-based platforms for the detection of SARS-CoV-2, Flu A, Flu B, and RSV: HealthTrackRx's real-time PCR Open Array® respiratory panel, TrueMark™ SARS-CoV-2, Flu A, Flu B, RSV Select Panel, and BioFire® RP2.1 Panel. The positive and negative predictive value of each test was evaluated at a 95% confidence interval.

Results: Among the 200 tested samples, the TrueMark™ and OpenArray® laboratory-developed tests (LDTs) showed a 100% concordance for the detection of SARS-CoV-2, Flu A, Flu B, and RSV. Overall agreement of 100% was observed for nasopharyngeal samples between the laboratory-developed tests and FDA-approved BioFire® RP2.1 Panel. Diagnostic results for these four respiratory viruses, in clinical samples, between the LDTs and the FDA-approved comparator demonstrated full concordance.

Conclusions: Respiratory viral infections represent one of the major global healthcare burdens. Consequently, the accurate detection and surveillance of these viruses are critical, particularly when these viruses are known to co-circulate. The excellent performance and full concordance of the LDTs, with the BioFire® Respiratory RP2.1 panel, in detecting SARS-CoV-2, Flu A, Flu B, and RSV shows that these tests can be confidently implemented for the clinical testing of respiratory viral infections.

Keywords: RSV; SARS-CoV-2; influenza virus; molecular diagnostics; multiplex RT-PCR; respiratory tract infections; respiratory viruses.

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Conflict of interest statement

The authors declare the following competing interests: P.U., V.S., and F.S. are employees of HealthTrackRx.

Figures

Figure 1
Figure 1
Ct values comparison between OpenArray® and TrueMark™ Select Panel detected for the detection of SARS-CoV-2, Flu A, Flu B, and RSV. Alphabets represent statistically significant differences between the two PCR tests (p < 0.05) in a t-test analysis.
Figure 2
Figure 2
Distribution of Ct values for the positive cohort on OpenArray® and TrueMarkTM-based multiplex PCR laboratory-developed tests for the detection of SARS-CoV-2, Influenza A, Influenza B, and RSV.
Figure 3
Figure 3
Relative prevalence of coinfecting organisms identified using the OpenArray® PCR platform among specimens that tested positive for (A) SARS-CoV-2, (B) Influenza A, (C) Influenza B, or (D) RSV.
See this image and copyright information in PMC

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