Severe Flu Season Provides an Opportunity for Molecular POC

It’s late January, 2018. It’s the centenary of the opening salvos of the “Spanish” Flu Pandemic, the unsung (and misnomered) villain of the later days of The Great War.  With cases on the rise right now, the 2017-’18 flu season has yet to reach its peak[1], surpassing that of the 2012-’13 season, and young people are being hit particularly hard. Let’s talk flu diagnostics.

(Especially, as Kalorama has just completed a study on molecular point-of-care systems, which is one of several technologies used for flu testing).

The typical way to diagnose an infection by the influenza virus in emergency facilities is with lateral flow immunochromatographic assays; they’re simple, they’re quick, and most importantly, they’re inexpensive. The problem with these tests (also called rapid influenza diagnostic tests, or RIDTs), however, is that they aren’t good. Their sensitivity has been deemed “sub-optimal” by the Centers for Disease Control: in a letter to the New England Journal of Medicine[2], one of their studies evaluated one such test at 51%, and cited in the letter was a study[3] that found another RIDT’s sensitivity rate to be a staggering 27%. While the latter figure seems to be an extreme example, a meta-analysis[4] conducted in 2012 found that RIDTs had, on average, sensitivities of 62.3%.

The gold standard for diagnostics is real-time PCR, but this has a few things going against it. Actually, it’s the diametric opposite of RIDTs: PCR is complex, requiring a lot of preparation and technical knowledge to operate; PCR is time-consuming, because of the preparation and the complexity of its mechanism; PCR is also expensive because of its complexity, but also in terms of patient isolation and management, because of the time it takes to get results.

So for a few years now, big biotech firms have been working on better solutions, employing nucleic acid amplification techniques that make PCR so accurate and engineering them into relatively tiny and simple packages, and as it turns out, they really are much better than RIDTs, and much faster and simpler than PCR. The devices are cartridge-based, requiring minimal input from the user; they turn results out in well under an hour, with most in the 15-30 minute range.

Alere’s Influenza A&B assay for its isothermal i platform, was among the first of its kind to be given a CLIA waiver, allowing the test to be performed at the point of care, where RIDTs are the usual go-to diagnostic solution. The i uses a technique called Nicking Enzyme Amplification Reaction (NEAR), which depends on, as the name implies, nicking enzyme to replicate the nucleic acid at a constant temperature range of 55-59°C. thus eliminating the need for the thermal cycler necessary in PCR. The device is therefore less complex, and the process consumes less time and energy as there is no need to denature DNA into single strands. The Influenza A&B assay produces results from nasal swab in approximately 15 minutes, and Alere reports sensitivities of 97.9% and 92.5% for influenza A (IFV-A) and influenza B (IFV-B), respectively, compared to cell culture.[5]

Cepheid’s Xpert Xpress Flu cartridge, for the GeneXpert platform, was granted a CLIA waiver just weeks ago, on 9 January. Cepheid also produces a joint flu/RSV Xpert assay, on which the studies reviewed for this article focus. The assay, Xpert Xpress Flu/RSV, has been CLIA-waived since 2015, and requires samples to be collected via nasopharyngeal swab (the newer IFV-only is indicated for sample collection with a less invasive nasal swab). Cepheid reports 98.2% and 100% positive agreement for IFV-A and IFV-B, respectively, between the Xpert Xpress Flu/RSV and conventional PCR; positive results are reported in twenty minutes.[6] The GeneXpert system is based on more conventional real-time PCR methodology than the aforementioned isothermal Alere i, but uses solid state heating and forced-air cooling, operating at a temperature range of 50-95°C.[7] There are currently four variants of the system, each named for the number of cartridges that can be simultaneously analyzed – I, II, IV, and XVI – and the Omni, a fully portable system that can operate solely on battery power, is anticipated for a second quarter 2018 launch.

Last, but not least, is Roche’s cobas Liat, which was developed from IQuum’s Lab-in-a-Tube analyzer. Like the GeneXpert, the cobas Liat operates on the more conventional real-time PCR methodology, and while the reagents of the system’s assays are in a compartmentalized tube, it operates on the same principle as the cartridge-based systems, in that the assays are fully self-contained and require minimal user interaction. The cobas Influenza A/B assay was granted a CLIA waiver in September 2015; results from nasopharyngeal swabs are reported in twenty minutes or less. Roche reports 100% sensitivity for both IFV-A and -B, and 96.8% and 94.1% specificity, respectively.[8]

Clinical researchers have conducted evaluations of each of these instruments, and in the literature reviewed for this entry, there is consensus that the devices compare favorably with conventional PCR methodology and are much more reliable than RIDTs.

  • Using PCR as the sole reference of comparison, Cepheid’s Xpert Flu/RSV test was found to have an overall sensitivity of 97.8% (A: 97.0%, B: 100%) and 100% specificity across the board.[9] One 2016 study compared the Xpert Flu/RSV to bioMérieux’s BioFire FilmArray, with an overall positive agreement of 97.3% (100% and 92.3% for IFV-A and IFV-B, respectively),[10] with the investigators concluding that the Xpert Xpress would be useful for diagnosing immunocompetent patients during peak flu season, while the FilmArray, being a more extensive multiplex panel diagnostic, should be designated for sicker patients and those coming in during decreased flu prevalence.

Concern over decreasing test sensitivity due to viral genetic drift in some commercially available flu diagnostics was the basis of a 2016 study, which looked at the Xpert Flu/RSV in addition to a multiplex PCR test that had been reformulated in the wake of the 2014-’15 flu season to be more sensitive. Cepheid’s test was found to have 100% sensitivity and 94.3% specificity for IFV when compared to PCR, though no distinction was made between A and B types.[11]

Finally, a 2017 review compared the Xpert Flu/RSV test to the performance of the ARIES Flu A/B & RSV test by Luminex; the BioFire FilmArray was the reference test used in the comparison.[12] Both assays were found to have 100% sensitivity to both A and B virus types; Cepheid’s test fell slightly short of the ARIES in test specificity, with 95.4%for IFV-A. However, the report’s authors did state that the Xpert has an edge over the ARIES for its substantially quicker turnaround time (20min vs two hours), though both devices require the same hands-on time and are equally easy to use.

  • A direct comparison in 2016 of Roche’s cobas Liat and Alere’s i platforms (using the BioFire FilmArray for reference) found the cobas test to be 100% sensitive and specific across the board in detecting both influenza types.[13] Alere had a relatively poor showing, with 71.3% sensitivity for IFV-A; however, this is likely attributed to low viral load due to samples being collected late in patients’ illnesses. The researchers found the cobas Liat to be a robust platform with performance that favors comparably with that of the FilmArray, though it should be noted that bioMérieux’s multiplex-capable assay detected some comorbidities with respiratory syncytial virus (RSV) and other respiratory pathogens that neither of the other platforms would have picked up. This is significant, as RSV could potentially impact treatment and management of influenza infection.

Another 2016 study also found the cobas Flu A/B test to have high sensitivity and specificity specs. The test detected only two false negatives, both for IFV-A, which were caught by the Cepheid Xpert Flu assay. The errors were likely due to low viral load resulting from the freeze-thaw cycle in sample preparation. The investigators noted two shortcomings of the platform and test: one, only a single test can be conducted at a time, and two, the test is not capable of IFV-A subtyping, unable to distinguish between the various hemagglutinin and neuraminidase proteins found on the viruses’ surfaces.[14]

A larger-scale (n > 1600), multicenter evaluation conducted in 2017[15] also regarded the cobas Flu A/B highly, with >99% sensitivity for both virus types, with the researchers’ summary basically being that the use of POC devices can be a potential boon for patient care as improvement in turnaround times provide a great potential to reduce time to treatment and to reduce length of stay, which will in turn reduce costs to care facilities.

  • A 2014 multicenter evaluation (n > 500) of the Alere i Influenza A&B test analyzed direct nasal swab samples taken from seven sites during the 2012-’13 flu season, compared against R-mix shell vial viral culture.[16] After discrepancy analysis, the i Influenza A&B was reported to have 99.3% and 97.6% sensitivities and 98.1% and 100% specificities for influenza types A and B, respectively.

In 2015, performance of the i was tested using Cepheid’s flu assay for reference,[17] but the results were not favorable to Alere’s test: the i had 80% sensitivity compared to the Xpert for IFV-A, and it fared worse with IFV-B, resulting with 33.3% compared to Xpert and 46.4% compared to conventional PCR. The author of the article said that the i is reasonably sensitive, but that low-viral load specimens contributed to poor detection, especially in the IFV-B test, due possibly to an additional freeze-thaw cycle to which the samples were exposed, resulting in degraded viral DNA, and that it should also be noted that the RIDTs with which these assays are ultimately meant to compete would have returned much worse performance as they are typically reported to show sensitivities in the 10-80% themselves.

An evaluation conducted in 2016 returned better results (93.8%/100% sensitivity for types A and B, respectively)[18] and was more positive in its review, adding that previous studies that have reported poor showings by the assay and others of its type were likely to have done so due to a lack of pediatric samples, which are well known to have higher viral loads than adult samples, and also because many of these studies were undertaken using previously frozen samples, which will have some degree of nucleic acid decay. Sensitivities should be considerably better than what is reported in these studies as care facilities will be using fresh patient samples.

In 2017 a cost analysis was conducted that compared the cost of patient isolation and treatment across multiple treatment centers in the United Kingdom using the Alere i platform and conventional PCR.[19] While sensitivity for the i averaged 77% for both viral types across the four centers, the speed of the results made the real difference; PCR results average an additional 1.1 days over the POC device without proper diagnosis (viz. false positive or negative), meaning 1.1 extra days of isolation and treatment for patients misdiagnosed as being infected, or 1.1 extra days without management for those misdiagnosed as being clear of infection. All told, the Alere i was determined to save £43,000 per 1,000 patients compared to PCR; if assuming 100% accuracy in patient isolation, that figure potentially increases to £261,600. The relatively low sensitivity reflected in the results appeared to be due to human error, involving the use of throat swabs and, to some extent, the storage and handling of reagents.

It can be concluded that molecular POC solutions have great potential over the more commonly used RIDTs, as they are far superior in sensitivity, on par with conventional PCR but with all the speed. There do seem to be caveats, however. These devices are touted to be so simple to use that they require no laboratory expertise. But there appears to be an ongoing theme in these studies that the devices still do require a degree of care to be taken when operating them, as handling reagents and samples a certain way can indeed have a negative impact on an assay’s efficacy, which was especially apparent with the Alere i flu test. Of course, some of this can be mitigated by using fresh patient specimens, which will most often be the case, but it still appears that there yet needs to be a bit of education in protocol, as fool-proof as the tests are supposed to be.

The other issue is cost effectiveness. Frederick Nolte, director of clinical laboratories at the Medical University of South Carolina, and the author of the 2016 report on the direct comparison between Alere’s and Roche’s offerings, said in a recent interview[20] that POC tests are “essentially one hundred percent sensitive” compared to the 80% sensitivity of lateral flow test, but they “[come] at a cost that is four times as much.” There was a cost-benefit study with the Alere i published just last year, but it was in a comparison against conventional PCR, not against the RIDTs that the i is supposed to replace. With the time it takes to prepare and operate a PCR run, it’s almost a given that these tests are going to be more cost effective, this is the wrong contest to hold. Because while RIDTs are not quite the gold standard that PCR is, they are so commonly used because they are cheap, fast, and good enough. These molecular tests are undoubtedly far better than their immunochromatographic counterparts, but the question is, are they better enough? Comparisons against lateral flow tests is where the cost analyses need to be focused. Until then, clinical facilities may not be convinced, even if the science guys are.

[1] United States Centers for Disease Control and Prevention (CDC). 2017-2018 Influenza Season Week 2 ending January 13, 2018.Updated 19 January 2018.

[2] Faix DJ, Sherman SS, Waterman SH. (2009) Rapid-Test Sensitivity for Novel Swine-Origin Influenza A (H1N1) Virus in Humans. New England Journal of Medicine; 361, 728-9. DOI: 10.1056/NEJMc0904264

[3] Uyeki TM, Ramakrishna P, Vukotich C, Stebbins S, Rinaldo CR, Ferng Y, Morse SS, Larson EL, Aiello AE, Davis B, and Monto AS. (2009) Low Sensitivity of Rapid Diagnostic Test for Influenza. Clinical Infectious Diseases; 48(9), e89-e92. DOI: 10.1086/597828

[4] Chartand C, Leeflang MM, Minion J, Brewer T, and Pai M. (2012) Accuracy of Rapid Influenza Diagnostic Tests: A Meta-analysis. Annals of Internal Medicine; 156(7), 500-11. DOI: 10.7326/0003-4819-154-7-201204030-00403

[5]Alere i Influenza A & B product page

[6]Xpert Xpress Flu/RSV datasheet

[7]GeneXpert product brochure

[8]Roche cobas Liat product page

[9] Salez N, Nougairede A, Ninove L, Zandotti C, de Lamballerie X, and Charrel RN. (2015) Prospective and retrospective evaluation of the Cepheid Xpert® Flu/RSV XC assay for rapid detection of influenza A, influenza B, and respiratory syncytial virus. Diagnostic Microbiology and Infectious Disease; 81(4), 256-8. DOI: 10.1016/j.diagmicrobio.2015.01.008

[10] Wahrenbrock MG, Matushek S, Boonlayangoor S, Tesic V, Beavis KG, and Charnot-Katsikas A. (2016) Comparison of Cepheid Xpert Flu/RSV XC and BioFire FilmArray for Detection of Influenza A, Influenza B, and Respiratory Syncytial Virus. Journal of Clinical Microbiology; 54(7), 1902-3. DOI: 10.1128/JCM.00084-16

[11] Huzly D, Korn K, Bierbaum S, Eberle B, Falcone V, Knöll A, Steininger P, and Panning M. (2016) Influenza A virus drift variants reduced the detection sensitivity of a commercial multiplex nucleic acid amplification assay in the season 2014/15. Archives of Virology; 161(9), 2417-23. DOI: 10.1007/s00705-016-2930-8

[12] McMullen P, Boonlayangoor S, Charnot-Katsikas A, Beavis KG, and Tesic V. (2017) The performance of Luminex ARIES® Flu A/B & RSV and Cepheid Xpert® Flu/RSV XC for the detection of influenza A, influenza B, and respiratory syncytial virus in prospective patient samples. Journal of Clinical Virology; 95, 84-5. DOI: 10.1016/j.jcv2017.08.018

[13] Nolte FS, Gauld L, and Barrett SB. (2016) Direct Comparison of Alere i and cobas Liat Influenza A and B Tests for Rapid Detection of Influenza Virus Infection. Journal of Clinical Microbiology; 54(11), 2763-6. DOI: 10.1128/JCM.01586-16.Editor

[14] Melchers WJG, Kuijpers J, Jackson Sickler J, and Rahamat-Langendoen J. (2016) Lab-in-a-tube: Real-time molecular point-of-care diagnostics for influenza A and B using the cobas® Liat® system. Journal of Medical Virology; 89, 1382-6. DOIL 10.1002/jmv.24796

[15] Gibson J, Schecter-Perkins EM, Mitchell P, Mace S, Tian Y, Williams K, Luo R, and Yen-Lieberman B. (2017) Multi-center evaluation of the cobas® Liat® Influenza A/B & RSV assay for rapid point-of-care diagnosis. Journal of Clinical Virology; 95, 5-9. DOI: 10.1016/j.jcv.2017.08.004

[16] Bell J, Bonner A, Cohen DC, Birkhahn R, Yogev R, Triner W, Cohen J, Palavecino E, and Selvarangan R. (2014) Multicenter clinical evaluation of the Alere™ i Influenza A&B isothermal nucleic acid amplification test. Journal of Clinical Virology; 61, 81-6. DOI: 10.1016/j.jcv.2015.06.001

[17] Jokela P, Vuorinen T, Waris M, and Manninen R. (2015) Performance of the Alere i influenza A&B assay and mariPOC test for the rapid detection of influenza A and B viruses. Journal of Clinical Virology; 70, 72-6. DOI: 10.1016/j.jcv.2015.07.294

[18] Nguyen Van JC, Caméléna F, Dahoun M, Pilmis B, Mizrahi A, Lourtet J, Behillil S, Enouf V, and Le Monnier A. (2016) Prospective evaluation of the Alere i Influenza A&B nucleic acid amplification versus XPert Flu/RSV. Diagnostic Microbiology and Infectious Disease; 85, 19-22. DOI: 10.1016/j.diagmicrobio.2015.11.012

[19] Davis S, Allen AJ, O’Leary R, Power M, Price DA, Simpson AJ, Tunbridge A, Vale L, Whiteside M, Evans C, and Raza M. (2017) Diagnostic accuracy and cost analysis of the Alere i Influenza A&B near-patient test using throat swabs. Journal of Hospital Infection; 97(3), 301-9. DOI: 10.1016/j.jhin.2017.05.017

[20] Johnson M. “Cepheid’s CLIA-Waived Multi-Module System Ups Competition in POC MDx; Barriers to Uptake Linger.” GenomeWeb. Published online 11 January 2018.