Influenza is considered one of the leading causes of mortality globally with an estimated 650,000 deaths occurring annually according to the new WHO estimates. It severely affects the high-risk group like the young children, the elderly, pregnant women and those with certain underlying medical conditions such as heart disease, asthma and chronic lung disease. Currently, there are 13 rapid flu tests used in the U.S with an acceptable clinical performance of which 6 are antigen-based rapid flu tests (also known as rapid influenza diagnostic tests (RIDTs) and 7 are nucleic-acid based rapid molecular tests available in primary care.

Diagnosing influenza

As the majority of the influenza cases are diagnosed clinically, the challenge of it continues as it could also be indistinguishable from other infections with respiratory syncytial virus, adenovirus and others. Therefore, a laboratory test is required to confirm a definitive diagnosis to help with the management of the case and permit monitoring of disease spread which is important for public health. The current diagnostic tests available for influenza include serology, viral culture, reverse transcription polymerase chain reaction (RT-PCR), viral culture, rapid molecular assays and immunofluorescence assays. Also, the proper collection and storage of the specimens are important for the detection of the influenza infections. The sensitivity and the specificity could vary based on the type of test, the type of specimen used and illness onset to the time the specimen was collected. The preferred samples for influenza testing are nasal swab and nasal wash based on the kind of test used and the specimen should be collected within 3-4 days. Except for the RIDT, other forms of diagnosis require sophisticated laboratory capacity equipped with skilled laboratory staff. Rapid influenza diagnostic tests (RIDTs) can be used in physician’s offices or clinics without the need for laboratory services and can be interpreted within 5-15 minutes.

Potential roles of RIDT for disease management

Individual patient management: Rapid influenza diagnostic tests (RIDTs) can assist with the diagnosis and the management of patients presenting influenza-like illness (ILI). However, the limitations of the commercial RIDTs is that it cannot specifically confirm pandemic influenza A (H1N1) 2009 virus and a positive results does not differentiate between pandemic (H1N1) virus or exclude the infection with seasonal influenza A viruses. Therefore, the general use of RIDTs among hospitalized patients should not be encouraged when alternative diagnostics such as RT-PCR or Immunofluorescence assay are available.

Semi-closed community outbreak management: RIDTs can assist in detecting influenza A among semi-closed communities and institutions such as schools that report high numbers of ILI which can help with the interventions for institutional control outbreaks.

Reclassification of antigen detection test systems by FDA

As of January 2017, antigen-based RIDT systems have been reclassified into class II by FDA to improve the overall rating of testing. This was done mainly because of the poor sensitivity of some of the antigen-based RIDTs which has resulted in the number of misdiagnosed cases. By this reclassification, FDA has established controls for antigen-based RIDTs so that the accuracy and reliability could be achieved. Accordingly, the manufacturers had time until January 12, 2018 to ensure their tests comply with the new regulation. This is therefore expected to minimize the rate of misdiagnosed infections by promoting the development of improved devices that can identify the influenza virus.

The Clinical laboratory improvement amendments (CLIA) waived Rapid Flu tests;

Available CLIA-Waived, Antigen-based rapid flu tests:

  • Alere BinaxNOW Influenza A and B Card 2 (Reader)
  • BD Veritor System for Rapid Detection of Flu A+B (Reader)
  • Princeton Biomeditech BioSign Flu A+B
  • Quidel QuickVue Influenza A+B
  • Quidel Sofia Influenza A+B FIA on the Sofia FIA Analyzer (Reader)
  • Quidel Sofia Influenza A+B FIA on the Sofia 2 FIA Analyzer (Reader)

Available CLIA-Waived, Nucleic Acid-Based Rapid Flu Tests:

  • Alere I Flu A/b
  • BioMerieux BioFire FilmArray RP EZ
  • Cepheid Xpert Flu + RSV Xpress Assay performed on the GeneXpert Xpress System (GXI)
  • Cepheid Xpert Xpress Flu Assay performed on the GeneXpert Xpress System (GXII and GXIV)
  • Mesa Biotech.Inc.Accula Flu A/Flu B Test performed on the Accula Dock
  • Roche Cobas Liat Influenza A/B Assay
  • Roche Cobas Liat Influenza A/B +RSV Assay

Microfluidic Chip to detect influenza

The microfluidic thermoplastic chip has the ability to amplify influenza A virus from nasopharyngeal specimens and aspirates and upon loading the sample, the device sequentially performs on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. The chip is designed as disposable with one-time use, thus reducing the possibility of specimen cross-contamination.

Nano Microarray-based approaches

Microarray-based approaches have been useful for the detection and subtyping the influenza viruses. The semiconductor-based influenza A microarray that was developed can detect all known subtypes of influenza A viruses under five hours. However, the multiple steps involved in various assays makes them expensive, susceptible to contamination, labor intensive and can also make them prone to false negative results. Over the years, there has been an increased interest in the use of nanoparticles coupled with silver staining for diagnostic applications. A nanoparticle-based genomic microarray assay (nanomicroarray) which can detect the H5N1 viral nucleic acid and also identifies the subtype of influenza A in the absence of RT-PCR amplification procedures. The nanomicroarray assay could also be used to detect influenza A and B viruses and also differentiate influenza A virus strains from seasonal influenza. It therefore provides a tool for accurate diagnosis and rapid identification of the influenza viruses during seasonal epidemics or pandemics.

Next-Generation Sequencing (NGS)

In a study titled ‘Evaluation of unbiased Next-Generation Sequencing of RNA (RNA-seq) as a Diagnostic Methods in Influenza Virus-Positive Respiratory Samples’, it illustrates that unbiased RNA sequencing by NGS from diagnostic samples could be better than the current diagnostic technologies as it has the potential to identify known as well as the unknown pathogen. With the decline in the costs of NGS methods, it enables the use of this technique in routine diagnostic settings. This pilot study shows that unbiased RNA sequencing as a valuable tool for complementing the current diagnostics particularly in clinical settings.

Digital immunoassays test (DIA)

A study conducted titled ‘Comparison of two generation influenza rapid diagnostic tests with instrument-based digital readout systems for influenza virus detection’ was published in pubmed. The study method used two types of digital influenza RDTs using a digital readout system and one conventional RDT which were compared using 314 specimens. The results showed that the digital-based readout systems can be used for more sensitive diagnosis than the conventional RDTs in clinical settings.

References

https://www.fda.gov/downloads/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/UCM596063.pdf

http://apps.who.int/iris/bitstream/handle/10665/44304/9789241599283_eng.pdf?sequence=1

https://jcm.asm.org/content/53/7/2238

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3671660/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848591/

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033176

https://www.ncbi.nlm.nih.gov/pubmed/27327199