Can singleplex assays be more efficient than multiplexed (multiple target) assays?

Singleplex Assays Can Be More Efficient Than Multiplexed Assays in Specific Clinical Scenarios

Yes, singleplex assays can be more efficient than multiplexed assays for detecting certain respiratory pathogens, particularly when targeting specific viruses that require higher analytical sensitivity or when dealing with emerging pathogens. 1

Advantages of Singleplex Assays

Superior Analytical Sensitivity

• Some targets on commercial multiplex panels are detected more efficiently by singleplex assays 1
• Specific examples where singleplex assays demonstrate superior sensitivity:
  • Respiratory syncytial virus type A (RSVA)
  • Influenza A (FLUA)
  • Adenovirus (ADV), particularly group E 1
  • Emerging viral pathogens that may be missed by commercial multiplex panels 1

Better Detection of Specific Pathogens

• Singleplex assays can be optimized for a single target without competition for reagents or primer/probe interactions 1
• For adenovirus detection, in-house singleplex NAATs often show higher sensitivity than multiplex panels 1
• Singleplex assays can be rapidly developed and deployed for emerging pathogens that may not be included in existing multiplex panels 1

Improved Differentiation of Similar Viruses

• Singleplex assays can better differentiate closely related viruses that multiplex panels may struggle to distinguish 1
• Example: Distinguishing between enterovirus (EV) and rhinovirus (RV), which some multiplex panels cannot differentiate 1

When to Choose Singleplex Over Multiplex

Target-Specific Considerations

• When testing for specific viruses known to have decreased sensitivity in multiplex formats 1
• When detection of emerging viral strains is needed (e.g., enterovirus D68) 1
• When differentiation between closely related viruses is clinically important 1

Patient Population Considerations

• For immunocompromised patients where detection of specific non-respiratory adenovirus species (A, D, F) is important 1
• When monitoring viral clearance or response to antiviral therapy in individual patients over time 1

Limitations of Multiplex Panels

• Inability to differentiate closely related viruses 1
• Uneven detection sensitivity across different targets 1
• Limited ability to detect emerging viral pathogens due to proprietary nature of primers and probes 1
• Detection of only respiratory species of adenovirus (B, C, E), missing non-respiratory species (A, D, F) 1
• Higher cost compared to targeted singleplex testing 1

Algorithmic Approach to Choosing Between Singleplex and Multiplex Testing

1. Determine clinical question:

   • If testing for a specific suspected pathogen → Consider singleplex
   • If broad pathogen detection needed → Consider multiplex

2. Assess patient population:

   • Immunocompromised patients at risk for specific pathogens → May benefit from singleplex assays
   • Transplant recipients, ICU patients, pediatric patients with underlying conditions → Often benefit from multiplex panels 2

3. Consider pathogen characteristics:

   • For adenovirus, RSV-A, or influenza A detection → Singleplex may offer better sensitivity 1, 3
   • For detection of multiple potential pathogens → Multiplex provides broader coverage

4. Evaluate resource constraints:

   • Limited resources/targeted testing needed → Singleplex may be more cost-effective
   • Need for comprehensive testing → Multiplex reduces overall testing time 1

Practical Considerations

• Singleplex assays can be more efficient when the pre-test probability for a specific pathogen is high 2
• Multiplex panels may be more efficient when testing for multiple potential pathogens, despite higher cost per test 1
• For certain viruses like influenza A/H3, influenza B, parainfluenza virus 2, and rhinovirus, some multiplex platforms may actually demonstrate better analytical sensitivity than singleplex assays 3

In clinical practice, the choice between singleplex and multiplex testing should be guided by the specific clinical scenario, target pathogens, and available resources to optimize both diagnostic accuracy and efficiency.