Friday, December 23, 2016 Written by Ellen Jo Baron, Ph.D., D(ABMM), Prof. Emerita, Stanford University Director of Medical Affairs, Cepheid

Flu Season 2011-2012

Laboratories performing tests for respiratory viruses can attest to the changing epidemiology of this formerly reliable season. For many years, like clockwork, new influenza strains, having mixed and matched up genetic material from pigs, birds, and humans, originated in Southeast Asia. In the past, the first influenza cases of the year appeared in Australia in June, peaked in late July, and petered out around December.1

In Europe, influenza season typically started in late August, peaked in October, and ended before Christmas.A Flu cases usually appeared on the East Coast of the U.S. in October and spread westward, reaching California in late October or early November, peaking in February, and finally disappearing by end of March.1,B But something totally unexpected happened in 2009. Both the United States, possibly in one of the U.S.'s large factory pig farms, and Asia (particularly South Korea and Thailand, where people and pigs live in close proximity) are thought to be areas where the genetic reassortment leading to the most recent pandemic influenza recombinant virus, novel H1N1 2009 strain, originated.2 And the source of the new strain was not the only surprise; this strain swept through Mexico, moved up to the Southwest U.S. and from there spread to the rest of the world starting in April, after northern hemisphere laboratories had traditionally finished their flu season and were putting their reagents away for another year (Figures 1 and 2).

What has happened with regard to influenza epidemiology since then? Although the new strain, H1N1 novel 2009, dominated the population in its first year, it has since dropped its prevalence back to become just another contributor to the circulating strains of influenza in the world. In fact, it may be only a minor agent of influenza disease this year; approximately 5% of all subtyped influenza A strains as of the first week of 2012.C Is it still important to identify this strain in a particular patient? Most authorities would say "yes," partly because it has a predilection to quickly move from its initial colonization site in the upper respiratory epithelium into deeper lung sites, causing more severe disease, especially in some vulnerable patient populations.3 The influenza A H1N1 novel strain infected younger people more vehemently than the influenza's usual victim, the elderly, and in greater numbers than previously circulating influenza A strains (Figure 3).4 And some novel populations, such as diabetic patients and pregnant women, also seemed to be at increased risk.5 Equally important was the differential susceptibility of various influenza virus strains to the commonly used antiviral treatments (Table 1). This year, in contrast, all 53 strains (including only 2 influenza B) tested so far have been susceptible to both of the neuraminidase inhibitors, oseltamavir and zanamivir.

Two unexpected benefits of the 2009 influenza season were the knowledge gained about the relatively poor performance of commonly used rapid antigen enzyme-immunoassays and the availability of new, accurate molecular tests for respiratory viruses. With the increased need to correctly identify and triage patients with influenza, both for individual patient care needs and for infection control purposes, the reliability of laboratory tests gained importance. Unfortunately, the currently available tests were not up to the job.6 The largest concentration of influenza patients in the U.S., centered on Long Island, provided an excellent resource for comparing test performance. By chance, a large group of high school boys had been visiting Mexico City when the influenza hit. They returned to the New York City area and promptly spread the disease throughout the community. Dr. Christine Ginocchio was in the perfect place to study the laboratory diagnosis of influenza. She received >6,000 samples within a few short months and used them for comparison testing.7 She found that performance varied widely among test types but that rapid antigen tests performed most poorly (Table 2). A retrospective meta-analysis of influenza A point-of-care tests (all rapid antigen format) compared with various high complexity diagnostic assays, primarily molecular assays, for detection of influenza A (novel H1N1 2009) performed by scientists from Johns Hopkins University showed pooled sensitivity to be 68% and specificity to be 81%. The ranges of results among published studies were great (sensitivities varied from 10 to 88% and specificities from 51 to 100%).8

Multiplex molecular platforms are considered the most inclusive and sensitive for diagnosis of respiratory tract infections (Tables 2 and 3). The classic xTAG Respiratory Viral Panel by Luminex Molecular Diagnostics can detect 10 types of viruses after initial extraction of the sample, an off-board nucleic acid amplification, and processing in the Luminex platform. The test is highly complex and requires considerable expertise and 2.5-3 hours of hands-on time to perform; results are available in 5-6 hours, which effectively limits testing to once/day, with an actual average turnaround time of approximately 22-47 hours.9, 10 A newer version, RVP Fast, showed slightly decreased sensitivity for some viruses but 1.5 hours less of hands-on time with a time to results of 8 hours from specimen receipt.11 This assay has been expanded to detect 18 viruses, including several human coronavirus strains, 8 of which have received FDA clearance. The ProFlu test by Prodesse/Gen-Probe also showed good results for detection of RSV and influenza viruses.12 A newer multiplex test, which also detects 18 viruses, the FilmArray from Idaho Technologies, has a dramatically decreased hands-on time (3-5 minutes) and yields results from a single sample in about one hour.10 Each instrument can run a single test packet in an hour, so the current system is geared to medium-to-small volume laboratories.

Whether highly multiplexed tests are appropriate in all situations has not been fully evaluated. In fact, a recent study evaluated the clinical impact of reporting the presence of respiratory viral pathogens in a normal pediatric population within one day of admission versus standard results.13 Among 583 patients less than 12 years old presenting with acute respiratory tract illness, half had results generated using nucleic acid amplification tests (NAATs) for 17 viruses reported to clinicians 12-36 hours after sample collection and the other half had reports of results determined by the usual direct fluorescent antibody (DFA) method. Not unexpectedly, DFA detected fewer pathogens overall. However, there was no statistically significant difference between the groups with regard to hospitalizations, antibiotic administration, or length of stay. Surprisingly, antibiotics were started significantly more often in the group who received rapid reports. One reason given for lack of shorter courses of antibiotics in patients with positive viral NAATs was the fear of bacterial superinfection. As these authors noted, a patient population with significant co-morbidities, including asthma, organ transplantation, and other immunocompromising conditions, would be more likely to benefit from relatively rapid detection of respiratory viruses, including the presence of more than one virus in the sample.

Thus, given the cost, time, and potential reimbursement challenges, it is unclear what the role of the multiplex platforms should be. Two cost analyses, one Canadian and one American, evaluated a multiplex respiratory virus test compared with the cost of a DFA plus culture, i.e., their standard protocols. The Canadian study showed cost-effectiveness when prevalence of disease was >11%.14 The American study did not make such a connection, but >30% of samples tested were positive.9

A recent (September, 2011) update on diagnosis and treatement of upper respiratory tract infections (URI) on MedscapeD suggests "Targeted therapy is not available for most viruses that cause URI. Therefore, viral testing is rarely indicated for uncomplicated viral URIs in the outpatient setting. However, confirmation of a viral condition such as influenza may reduce inappropriate use of antibiotics."

Dr. David Persing, Chief Medical and Technology Officer at Cepheid, suggests that "the real value of these tests will be in the next 5-10 years when new antivirals (for rhinoviruses and others) gain FDA approval, and there is more than one antiviral treatment decision to be made on the basis of the results. In general, the field is moving toward Precision Medicine — giving the right medicines to the right patients at the right time — and Next-Gen molecular methods will play a critical role in the decision support process."

The Cepheid Xpert® Flu assay detects influenza A, the influenza A subtype H1N1 2009 strain, and influenza B with approximately 2-3 minutes of hands-on time; results are available within 75 minutes. Both nasopharyngeal swabs and nasal aspirates/washes can be tested. The current Xpert Flu test is substantially different from the original Emergency Use Authorization (EUA) assay for influenza A H1N1 2009, which was available in 2009 and early 2010 only. Earlier publications that evaluated the performance of the EUA version are no longer relevant to the existing assay. The random access nature of the GeneXpert® instrument allows multiple assays to be initiated within the same time frame. Results of comparison studies of the Xpert Flu assay have been presented at national meetings, including the 2011 Clinical Virology Symposium in Daytona, FL. One poster from the University of Virginia Health System (Seaner et al) compared the Xpert Flu and Prodesse's ProFlu. They found the Xpert assay to be dramatically easier to perform, and sensitivities for detection of seasonal influenza A H1/H3, influenza A H1N1 2009, and influenza B to be 97.3%, 100%, and 94.4%, respectively, with specificities all 100% when compared to the Prodesse product. Although the laboratory had previously been running a rapid antigen test, they soon discontinued it in favor of the Xpert Flu assay. Dr. Melinda Poulter, senior author of the study, noted that both physician and patient satisfaction were enhanced due to shorter wait times. In fact, Mendy said "…almost heaven. I've even had docs call to thank me… never had that happen before." Although actual documentation is now in progress for a publication, it appeared that costs were decreased due to better bed management, and that more targeted drug therapy seems to have reduced both costs and adverse events.

The University of North Carolina also evaluated the Xpert Flu assay.15 The authors reported the sensitivity of the Xpert Flu assay (compared with their in-house laboratory developed PCR test) was best for influenza A (H1 and H3) and influenza B, with 100% detected from retrospective samples and with 100% of the influenza A strains and >95% of the influenza B strains detected from prospective samples. For H1N1 2009 novel strains, the Xpert showed ≥87% sensitivity overall but better performance on prospective samples and nasopharyngeal (np) samples. Lower sensitivity was observed with non-np swabs, as expected, although only 8 such samples were tested. Subsequent analysis showed that the samples that were not detected by Xpert Flu had very low copy numbers of viruses. The authors noted that their laboratory-developed test, even when performed 3-4 times daily during influenza season, had a turnaround time of 8 to 24 hours, which they deemed "unacceptable," leading to their conclusion that the GeneXpert Flu, with hands-on time of 2 minutes and results available 75 minutes later was an "attractive approach."15

REFERENCES

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9. Dundas, N.E., et al., A lean laboratory: operational simplicity and cost effectiveness of the Luminex xTAG respiratory viral panel. J Mol Diagn. 13(2): p. 175-9.
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11. Pabbaraju, K., et al., Comparison of the Luminex xTAG respiratory viral panel with xTAG respiratory viral panel fast for diagnosis of respiratory virus infections. J Clin Microbiol, 2011. 49(5): p. 1738-44.
12. Selvaraju, S.B. and R. Selvarangan, Evaluation of three influenza A and B real-time reverse transcription-PCR assays and a new 2009 H1N1 assay for detection of influenza viruses. J Clin Microbiol, 2010. 48(11): p. 3870-5.
13. Wishaupt, J.O., et al., Clinical impact of RT-PCR for pediatric acute respiratory infections: a controlled clinical trial. Pediatrics. 128(5): p. e1113-20.
14. Mahony, J.B., et al., Cost analysis of multiplex PCR testing for diagnosing respiratory virus infections. J Clin Microbiol, 2009. 47(9): p. 2812-7.
15. Popowitch, E.B., E. Rogers, and M.B. Miller, Retrospective and prospective verification of the Cepheid Xpert influenza virus assay. J Clin Microbiol. 49(9): p. 3368-9.

A. European Centre for Disease Prevention and Control Epidemiological Data site
B. http://www.cdc.gov/flu/weekly/fluactivitysurv.htm
C. http://www.cdc.gov/flu/weekly/index.htm#whomap
D. http://emedicine.medscape.com/article/302460-workup