# From Triplex to Quadruplex: Enhancing CDC’s Respiratory qPCR Assay with RSV Detection on Panther Fusion® Open Access™

## TL;DR

This study improves a respiratory virus detection test by adding RSV to an automated system that already detects influenza and SARS-CoV-2.

## Contribution

The novel contribution is the development of a fully automated quadruplex RT-qPCR assay for detecting four respiratory viruses with high accuracy.

## Key findings

- The LDRA assay achieved amplification efficiencies of 97–105% and detected 32 RSV variants without cross-reactivity.
- The assay demonstrated ≥98% agreement with other diagnostic tests and reliably detected mixed infections.
- The system is suitable for middle-income healthcare settings, offering rapid and accurate detection of four respiratory viruses.

## Abstract

The overlapping circulation of influenza (Flu), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; SC2), and respiratory syncytial virus (RSV) continues to challenge clinical laboratories, particularly in settings with limited automation and fragmented healthcare coverage. This study expanded the CDC Flu-SC2 assay by incorporating a laboratory-developed test (LDT) for RSV A/B detection into a fully automated quadruplex RT-qPCR (LDRA) on the Panther Fusion® Open Access™ system. The design, based on more than 8000 RSV genomic sequences targeting the conserved M gene, achieved optimal amplification efficiencies (97–105%) and full multiplex compatibility. Analytical assessment established limits of detection between 9.6 and 37.8 copies per reaction, absence of cross-reactivity with 30 respiratory pathogens, and inclusivity for 32 viral variants. Commutability and diagnostic performance among the LDRA, CE IVD-marked Allplex™ SARS-CoV-2/FluA/FluB/RSV, and US IVD-marked Panther Fusion® SARS-CoV-2/Flu A/B/RSV Assays were evaluated using 405 nasopharyngeal UTM-preserved swabs. The LDRA demonstrated excellent concordance (overall agreement ≥ 98%, κ > 0.95), strong diagnostic accuracy, and reliable detection of mixed infections. This quadruplex provides a fully automated, rapid, and accurate solution for the simultaneous detection of influenza A, influenza B, SARS-CoV-2, and RSV viruses, enhancing molecular diagnostic capacity and supporting equitable, timely clinical decision-making in middle-income healthcare systems such as that of the Dominican Republic.