How Modern Tests Are Revolutionizing Respiratory Infection Diagnosis
When a patient arrives at a clinic with a fever, cough, and fatigue, doctors face a familiar diagnostic puzzle. These symptoms could signal SARS-CoV-2, influenza, respiratory syncytial virus (RSV), or even a common cold. Until recently, determining the exact culprit required multiple tests, valuable time, and significant resources—luxuries that clinicians often don't have when making urgent treatment decisions. The COVID-19 pandemic not only highlighted this challenge but accelerated the development of an ingenious solution: multiplex molecular testing that can detect multiple pathogens simultaneously from a single sample 6 .
Fever, cough, and fatigue can indicate multiple respiratory viruses, making clinical diagnosis challenging without testing.
Traditional single-target tests require separate runs, delaying results when timely treatment decisions are critical.
At its core, multiplex PCR operates on the same fundamental principles as conventional polymerase chain reaction—it amplifies specific genetic sequences to detect the presence of pathogens—but with a crucial enhancement. Where standard PCR identifies just one target per test, multiplex PCR can detect several different targets simultaneously in the same reaction tube. This simultaneous detection is made possible by designing multiple primer and probe sets that target unique genetic sequences of each virus, with each probe labeled with a different fluorescent dye that can be distinguished by the detection instrument 5 .
Nasopharyngeal swab collected from patient
RNA/DNA extracted from the sample
Multiple primer sets simultaneously amplify target sequences
Different colored probes detect each pathogen
Software interprets fluorescence patterns to identify pathogens
Limited sample quantities can be used more efficiently
Combined reactions lower per-test expenses
Single setup reduces hands-on time and error risk
Clinicians receive complete diagnostic information more quickly
| Parameter | Single-Target PCR | Multiplex PCR | Practical Impact |
|---|---|---|---|
| Targets per test | 1 | 3+ (e.g., SARS-CoV-2, Flu A/B, RSV) | More comprehensive diagnostic information |
| Sample required | High (multiple aliquots) | Low (single aliquot) | Better for pediatric or limited samples |
| Time to result | Several hours per target | Simultaneous detection | Faster treatment decisions |
| Cost per target | Higher (separate reactions) | Lower (combined reaction) | More efficient resource use |
| Workflow complexity | Multiple setups | Single setup | Reduced technician time and error risk |
To understand how different multiplex assays perform in real-world conditions, researchers conducted a retrospective comparative study published in 2025 that evaluated three commercial systems: the STANDARD™ M10 Flu/RSV/SARS-CoV-2 assay, the Savanna® Respiratory Viral Panel-4 assay, and the Xpert® Xpress SARS-CoV-2/Flu/RSV test, which served as the reference method 1 3 . All three are RT-PCR tests suitable for point-of-care testing, representing the cutting edge of rapid molecular diagnostics.
Flu/RSV/SARS-CoV-2 Assay
Respiratory Viral Panel-4
SARS-CoV-2/Flu/RSV (Reference)
| Virus Target | Assay | Sensitivity (%) | Specificity (%) | Retest Rate |
|---|---|---|---|---|
| Influenza A | STANDARD M10 | 100.0 | >99 | 0% |
| Savanna | 92.6 | >99 | 5.0% | |
| Influenza B | STANDARD M10 | 95.7 | >99 | 0% |
| Savanna | 95.7 | >99 | 5.0% | |
| RSV | STANDARD M10 | 97.1 | >99 | 0% |
| Savanna | 100.0 | 94.2 | 5.0% | |
| SARS-CoV-2 | STANDARD M10 | 97.0 | >99 | 0% |
| Savanna | 90.9 | 94.3 | 5.0% |
| Virus Target | Xpert Xpress | STANDARD M10 | Savanna | LabTurbo |
|---|---|---|---|---|
| Influenza A | Most sensitive 1 | Intermediate | Intermediate | 3,333 copies/mL 6 |
| Influenza B | Most sensitive 1 | Intermediate | Intermediate | 6,667 copies/mL 6 |
| RSV | Most sensitive 1 | Intermediate | Intermediate | 8,333 copies/mL 6 |
| SARS-CoV-2 | Most sensitive 1 | Intermediate | Intermediate | 8,333 copies/mL 6 |
Creating reliable multiplex PCR tests requires specialized reagents and equipment designed to handle the complexity of simultaneously detecting multiple targets. The research evaluated in our featured study, along with similar investigations, relies on a suite of specialized tools that form the modern molecular laboratory's diagnostic toolkit.
The comprehensive evaluation of these three multiplex assays reveals both the impressive capabilities and current limitations of simultaneous detection systems for major respiratory viruses. The STANDARD M10 assay emerges as a consistently reliable option with high sensitivity and specificity across all targets, while the Savanna assay shows promise but requires further refinement to address its false positive issues and higher retest rate 1 3 . The superior analytical sensitivity of the Xpert reference method highlights the ongoing importance of detection limits in test selection 1 3 .
The multiplex PCR kit market is expected to grow at a compound annual growth rate of 8-10% between 2025 and 2033 7 .
Development of miniaturized and portable PCR systems for point-of-care testing 7 .
Creation of more customizable and automated kits to streamline workflow 7 .
These findings have significant implications for clinical practice. Rapid, accurate multiplex testing enables more targeted patient management—appropriate antiviral therapy for influenza, isolation precautions for COVID-19, and supportive care for RSV 6 . This specificity is particularly valuable in emergency departments, urgent care settings, and during seasonal outbreaks when respiratory infections surge. The efficiency of testing for multiple pathogens simultaneously also offers operational benefits to clinical laboratories struggling with increasing test volumes and limited resources.
Looking ahead, several trends are likely to shape the next generation of multiplex respiratory virus assays. The successful evaluation of the LabTurbo multiplex real-time RT-PCR kit in a separate study—demonstrating 100% agreement with reference methods—suggests that the performance bar for these tests continues to rise 6 . The development of in-house multiplex assays for other viral targets, such as one achieving 100% sensitivity for HSV, VZV, and EBV in transplant patients, shows how this technology is expanding into new clinical applications 8 .
As these technologies evolve, the ideal multiplex assay of the future will likely combine the analytical sensitivity of the Xpert system, the consistent clinical performance of the STANDARD M10, and the versatility to detect an even broader panel of respiratory pathogens. Such advances will continue to transform how clinicians diagnose and manage respiratory infections, ultimately improving patient outcomes and strengthening our defenses against seasonal outbreaks and future pandemics alike.