Influenza is a disease of the respiratory tract caused by the influenza virus. The main types of influenza viruses in the human population are influenza A and influenza B. Two subtypes, or lineages, of both virus types circulate in the human population: the influenza A virus subtypes H1N1 and H3N2, and the influenza B virus Yamagata and Victoria lineages. Infections with these viruses generally cause a self-limiting respiratory disease in healthy individuals, characterized by headache, sore throat, muscle ache, fatigue, and fever.
While the course of illness usually takes one week, complete recovery to normal daily activities may take weeks. Influenza virus infection in risk groups, e.g., individuals over 65 years of age, children under two years of age, pregnant women, obese patients, and patients with concomitant morbidity, may lead to more serious complications, including acute respiratory distress syndrome, sometimes associated with septicemia and multiple organ failure.
Another well-known and feared complication is superinfection with Pneumococcus and Staphylococcus aureus, leading to increased mortality. In avian species, such as waterfowl, multiple influenza A virus subtypes have been identified, which are characterized by their specific hemagglutinin and neuraminidase proteins on the virus membrane. Cross species transmission of avian influenza A viruses such as H5N1 and the recent H7N9 strains to humans is characterized by mortality rates as high as 60%.1,2
Currently, vaccination is the most successful protection against disease from seasonal influenza viruses. However, a number of challenges remain. Due to viral genetic changes (antigenic drift), vaccines largely protect against homologous strains only. The available vaccines need to be updated yearly, based on information of the antigenicity of the influenza virus strains circulating in humans. Vaccine efficacy in specific populations, such as immune compromised individuals, is low. And not all individuals from groups at increased risk for severe course of disease are vaccinated each year. Even more worrisome is the fact that timely vaccination against avian influenza virus strains that are newly introduced into the human population is currently not possible.3
Antiviral agents against influenza A and B viruses are therefore still urgently needed. Worldwide, approved drugs for the treatment of influenza are the adamantanes amantadine and rimantadine and the neuraminidase inhibitors (NAIs) oseltamivir, zanamivir, and peramivir. Adamantanes are rarely used in clinical practice due to the natural resistance that has developed in circulating influenza A viruses, and the lack of an antiviral effect against influenza B viruses. Although the usefulness of NAIs has been a subject of debate, clinical trials have clearly shown that treatment with NAIs may result in earlier resolution of symptoms and clearance of infectious virus, if treatment is initiated early during the course of disease (within 48 hours after the onset of first symptoms).4 However, in the more “heterogeneous” patient groups, the benefits of neuraminidase inhibitors are less apparent. Antiviral efficacy observational trials and reports on special populations, e.g., immunocompromised patients, are often confounded by late initiation of antiviral treatment.5
RNA viruses such as HIV-1, HCV, and the influenza virus are characterized by their high mutation rate. As a consequence, treatment with single antiviral agents often results in the rapid selection of less susceptible variants. Treatment with multiple antiviral agents simultaneously, or with drugs with a high genetic barrier to resistance development, will lead to better outcomes and lower resistance rates. In combination with normal functional immune responses, treatment of influenza with NAIs will rapidly clear the virus, and development of drug resistance in healthy individuals is therefore very low.4 Increased resistance rates are found in treated individuals with prolonged virus shedding, such as populations with an immature or compromised immune system.6,7 But unpredictable events have also been contributing to the development of drug-resistant viruses. For instance, from 2006 to 2009 there was an unexplained increase of oseltamivir-resistant influenza A H1N1 strains, from approximately 1% to >99%.8,9 Thus, resistance monitoring in both treated and untreated patients remains important, particularly in patient groups with increased risk for development of resistance, including immunocompromised individuals and children of less than five years of age.
Diagnostics, surveillance, and efficacy testing
Nowadays, virus culture has largely been replaced by RT-PCR as the preferred method for the detection of influenza virus by routine diagnostic laboratories (see below). However, culture assays still provide valuable quantitative information on the infectious nature of viruses detected by RT-PCR. Virus culture, and especially quantitative virus culture, requires a significant level of expertise, as well as awareness of evolutionary changes in characteristics of viruses that may affect their detection. For example, the standard method to detect influenza virus grown in culture is based on agglutination of virus particles and virus receptor-bearing red blood cells (hemagglutination). Yet, the majority of currently circulating H3N2 strains fail to agglutinate red blood cells of various species efficiently, if at all10 (Van Baalen et al, in prep). To circumvent this problem, a read-out method based on detection of viral nucleoprotein by ELISA has recently been developed and validated, which detects influenza A viruses independent of their hemagglutination capacity (Van Baalen et al, in prep). Virus culture is also essential for the assessment of phenotypic characteristics, including susceptibility to antiviral compounds, using enzymatic assays for NAIs or plaque assays for other antivirals.
Historically, serological assays for detecting antibodies that inhibit influenza virus hemagglutination or that neutralize infection in vitro were used to diagnose influenza virus infection in patients. Proper diagnosis requires a follow-up sample several weeks after the first visit to show a serum antibody titer rise, which makes this assay unsuitable for patient management. Serological analyses are, however, essential for epidemiological studies and monitoring antigenic drift. A titer rise of hemagglutination inhibition (HI) antibodies, and in many cases also virus neutralization (VN) antibodies, are primary end points in clinical trials that assess the efficacy of the yearly updated whole inactivated vaccines (WIV), live attenuated vaccines (LAIV), and novel vaccination strategies.
RT-PCR has in the past ten years largely taken over the role as the gold standard for clinical influenza diagnostics. To cover the large number of commercial and noncommercial systems is beyond the scope of this article (it is reviewed elsewhere11). The methods can largely be divided into commercial and in-house systems, and qualitative and quantitative assays. Commercial assays are in general easy to use, with some expertise required, but not to the same extent as quantitative culture or in-house developed RT-PCR methods. They are often considered expensive, although additional costs of in-house developed assays, such as more expensive skilled personnel, development cost, and quality control testing of new batches of chemicals are often left out of the earlier equation. Due to regulations, updates of assays to new potential pandemic strains may take some time to reach routine diagnostic laboratories, however.
The most widely used in-house assay system for the detection of influenza virus is real-time RT-PCR.12 Despite its drawbacks (little multiplex ability, expensive read-out equipment), real-time RT-PCR has significant advantages (easily quantifiable, relatively fast result out), making it attractive for generating results for patient management. Quantitative results may aid in monitoring the effect of treatment. Assays may be rapidly adapted to fit influenza strains newly introduced in the human population like H7N9 and H5N1, which does however, require significant knowledge on mismatch tolerance of primers and probe in combination with the mastermix used.13,14 At little extra cost within the same run as the screening assay, real-time RT-PCRs may be run on the same sample, allowing typing of the influenza strain or discrimination between influenza strains susceptible or resistant to antiviral treatment. The latter has the advantage of being able to detect minority resistant species within a patient down to 5%, with high sensitivity at low virus concentrations. Where culture and subsequent antiviral resistance phenotyping or highly sensitive sequencing techniques (see below) still require at least 5,000 viral particles per milliliter (approximately Ct 32 in real-time RT-PCR), resistance mutation specific real-time RT-PCR can detect up to 10- to 100-fold lower numbers of viral particles.14
Given the need for timely management of antiviral treatment, having a resistance profile determined at the same time as the result of the influenza screening RT-PCR may significantly benefit patient care, especially in patients with increased risk of developing severe disease. Other widely used rapid genotyping methods for detection of single mutations which correlate with reduced antiviral efficacy are high resolution melt analyses and pyrosequencing techniques.15,16 These assays are highly sensitive and quantitative. The disadvantage of these assays is their sensitivity to contamination, and as a result the need for stringent separation of PCR rooms used for these techniques from other PCR rooms. Furthermore, these assays require an additional RT-PCR, and thus are more expensive and time-consuming. In addition, Sanger sequencing and next generation sequencing (NGS) techniques are widely used to determine genotypic resistance.
Currently available protocols for both Sanger sequencing and NGS are almost exclusively based on a prior virus expansion step in culture before sequencing can be performed because of the high viral loads required for these protocols. In general Ct values of 22 to 25 (translating to approximately 5 million virus particles per mL) are required for direct sequencing by either the Sanger or NGS methods, on original materials without culture step. In our experience, samples with Ct values up to 32 taken within one day after onset of symptoms can efficiently be cultured with approximately 98% of the samples being culture positive. This percentage, however, dramatically drops at later time points (70% at day 3, 45% at day 6, and 10% at day 10). Hence sensitivity of sequencing either directly from original material or with a prior culture step is very low during later time points in the disease process.
A Sanger sequencing technique has therefore recently been developed that is based on nested RT-PCR and that allows sequencing of samples up to Ct 32 at later time points during infection, using original material with no prior culture amplification. It also allows NGS for the gene segments HA, NA, PA, PB1, and PB2 of all currently circulating seasonal influenza strains (2009H1N1, H3N2, B Victoria, B Yamagata). When properly executed, the Sanger sequencing allows detection of minority species down to 10% to 20%, or even down to 5% with prior knowledge at which positions resistance associated mutations may occur (Figure 1). Throat swab samples from oseltamivir treated patients that contained (according to 275Y and 275H specific realtime RT-PCRs) minority mixtures of approximately 5% to 10% 275Y (Samples 1-3). In the case of Sample 4, an equal mixture was sequenced with our sensitive nested RT-PCR sequencing protocol. NGS using 454 (percentages given below the electropherograms) confirmed the percentages of 275Y in the samples. With prior knowledge on the positions where resistance may occur, the minority resistant species could also be detected within the electropherograms of the Sanger sequencing result (red T peak at position 823 [boxed red]in the electropherograms).
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Other methods for the detection of antiviral resistant viruses include phenotypic assays.17 Phenotypic resistance tests have the advantage of detecting all potential resistant strains, while genotyping assays will detect only previously described resistant strains, as they require prior knowledge of which mutations in the viral genome induce resistance. On the other hand, sensitivity of virus culture required for phenotyping resistance is low at later time points during infection when resistance may occur. Phenotypic resistance may further be underestimated when resistant strains are present as minority species, or when they are compromised in their in vitro replication kinetics, e.g., as clearly shown for H3N2 292K oseltamivir resistant strains.18,19
Aims of diagnostic testing
The majority of patients with respiratory illness suspected for influenza virus infection at the offices of general practitioners and general hospitals are uncomplicated patients who are very likely to recover with minimal intervention by physicians. Interventions on the basis of results of a testing algorithm are therefore relatively minor with respect to the outcome of the infection. Influenza virus-infected patients with pneumonia are often given antibiotic treatment because Pneumococcal and/or Staphylococcus aureus infection can usually not be ruled out.20 Rapid tests for influenza are well suited for this purpose.
It should be kept in mind, however, that a physician’s clinical diagnosis based on seasonality and influenza-specific symptoms without in vitro diagnostics has a positive predictive value around 60%, and the addition of a rapid test result will increase this percentage only to around 80%. In academic and general hospitals with sufficient capacity for quarantining patients, influenza virus- and RSV-infected patients may be separately isolated from other patients to prevent nosocomial transmission. It may be argued that rapid tests are also sufficient for this purpose because RT-PCR diagnosed patients missed by rapid tests often carry low viral loads and may not easily transmit virus. Most laboratories will, however, offer fast RT-PCR- based panel testing to prevent misinterpretation of rapid test results.
Patients with an increased risk for developing severe disease, such as children with underlying disease and immunocompromised patients, are in general referred to hospitals with high expertise both in the clinical setting and in the laboratory. The diagnostic test armamentarium of routine diagnostic laboratories serving these hospitals requires assays that enable monitoring of the influenza viral load during antiviral treatment. Viral load assays may further be used to estimate which pathogen is the causative agent when multiple pathogens are detected in a patient. Both quantitative culture and real-time RT-PCR give quantitative results. Interpretation and technical performance of such tests require a high level of expertise. The advantage of quantitative culture over real-time RT-PCR is its ability to give information on the amount of infectious virus particles, while real-time RT-PCR detects both infectious and non-infectious virus particles as well as viral breakdown products. Especially in intubated patients with serious lung damage, very high viral loads may be detected by real-time RT-PCR in lower respiratory tract samples that cannot be cultured, in which resistance often does not develop despite long-term persistence, and therefore may be non-infectious viral debris.21
Quantitative culture is, however, relatively expensive and time-consuming and requires high-level expertise for even the most academic diagnostic laboratories. These assays are therefore for diagnostic purposes, in general only performed in the context of clinical trials. A clear example in which quantitative culture may have significant benefit is clinical trials with antivirals, such as favipiravirs, which increase the production of non-infectious particles.22 In clinical trials with antivirals that directly inhibit virus production, quantitative culture may give information on the duration of infectious virus shedding and the ability to reduce transmissibility. Given the fact that data generated on a limited number of patients in a clinical trial has a significant impact on the health of large numbers of patients when a drug is licensed, relatively expensive assays requiring a high level of expertise that provide important information on antiviral drug potency and resistance development, including quantitative real time RT-PCR, quantitative culture and even NGS, are needed and likely to be cost-effective. In summary, the various levels of influenza diagnostics applied in different settings are illustrated in Figure 2.
Martin Schutten, PhD, is head of the Unit Clinical Virology of the Viroscience Lab at the Erasmus MC Rotterdam, the Netherlands, and senior consultant to Viroclinics Biosciences B.V. Paul Zoeteweij, PhD, is Director, Clinical Services, at Viroclinics Biosciences B.V. Carel van Baalen, PhD, is Director, Assay Development, at Viroclinics Biosciences B.V. Pieter Fraaij, MD, PhD, is pediatric infectious disease specialist at the Department Pediatrics and Viroscience Lab at the Erasmus MC.
- To KK, Chan JF, Chen H, Li L, Yuen KY. The emergence of influenza A H7N9 in human beings 16 years after influenza A H5N1: a tale of two cities. Lancet Infect Dis. 2013;13(9):809-821
- Fraaij PL, Heikkinen T. Seasonal influenza: the burden of disease in children. Vaccine. 2011;29(43):7524-7528.
- Fraaij PL, Bodewes R, Osterhaus AD, Rimmelzwaan GF. The ins and outs of universal childhood influenza vaccination. Future Microbiol. 2011;6(10):1171-1184.
- Whitley RJ, Boucher CA, Lina B, et al. Global assessment of resistance to neuraminidase inhibitors, 2008-2011: The Influenza Resistance Information Study (IRIS). Clin Infect Dis. 2013;56(9):1197-1205.
- Muthuri SG, Myles PR, Venkatesan S, Leonardi-Bee J, Nguyen-Van-Tam JS. Impact of neuraminidase inhibitor treatment on outcomes of public health importance during the 2009-2010 influenza A(H1N1) pandemic: a systematic review and meta-analysis in hospitalized patients. J Infect Dis. 2013;207(4):553-563.
- Van der Vries E, Stittelaar KJ, van Amerongen G, et al. Prolonged influenza virus shedding and emergence of antiviral resistance in immunocompromised patients and ferrets. PLoS Pathog. 2013;9(5):e1003343.
- Van der Vries E, Schutten M, Fraaij P, Boucher C, Osterhaus A. Influenza virus resistance to antiviral therapy. Adv Pharmacol. 2013;67:217-246.
- Hauge SH, Dudman S, Borgen K, Lackenby A, Hungnes O. Oseltamivir-resistant influenza viruses A (H1N1), Norway, 2007-08. Emerg Infect Dis. 2009;15(2):155-162.
- Meijer A, Lackenby A, Hungnes O, et al. Oseltamivir-resistant influenza virus A (H1N1), Europe, 2007-08 season. Emerg Infect Dis. 2009;15(4):552-560.
- Lin YP, Xiong X, Wharton SA, et al. Evolution of the receptor binding properties of the influenza A(H3N2) hemagglutinin. Proc Natl Acad Sci U S A. 2012;109(52):21474-21479.
- Kumar S, Henrickson KJ. Update on influenza diagnostics: lessons from the novel H1N1 influenza A pandemic. Clin Microbiol Rev. 2012;25(2):344-361.
- Van der Vries E, Schutten M. Satisfying the need for rapid diagnosis of new variant influenza A H1N1. Expert Rev Mol Diagn. 2010;10(3):251-253.
- Stadhouders R, Pas SD, Anber J, Voermans J, Mes TH, Schutten M. The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5’ nuclease assay. J Mol Diagn. 2010;12(1):109-117.
- Van der Vries E, Anber J, van der Linden A, et al. Molecular assays for quantitative and qualitative detection of influenza virus and oseltamivir resistance mutations. J Mol Diagn. 2013;15(3):347-354.
- Arvia R, Corcioli F, Azzi A. High resolution melting analysis as a tool to detect molecular markers of antiviral resistance in influenza A viruses. J Virol Methods. 2013;189(2):265-270.
- Deyde VM, Sheu TG, Trujillo AA, et al. Detection of molecular markers of drug resistance in 2009 pandemic influenza A (H1N1) viruses by pyrosequencing. Antimicrob Agents Chemother. 2010;54(3):1102-1110.
- Okomo-Adhiambo M, Sheu TG, Gubareva LV. Assays for monitoring susceptibility of influenza viruses to neuraminidase inhibitors. Influenza Other Respi Viruses. 2013;7 (suppl 1):44-49.
- Yen HL, McKimm-Breschkin JL, Choy KT, et al. Resistance to neuraminidase inhibitors conferred by an R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population. MBio. 2013;4(4):e00396-13.
- Hurt AC, Nor’e SS, McCaw JM, et al. Assessing the viral fitness of oseltamivir-resistant influenza viruses in ferrets, using a competitive-mixtures model. J Virol. 2010;84(18):9427-9438.
- van de Pol AC, Wolfs TF, Tacke CE, et al. Impact of PCR for respiratory viruses on antibiotic use: theory and practice. Pediatr Pulmonol. 2010.
- Fraaij PL, van der Vries E, Beersma MF, et al. Evaluation of the antiviral response to zanamivir administered intravenously for treatment of critically ill patients with pandemic influenza A (H1N1) infection. J Infect Dis. 2011;204(5):777-782.
- Baranovich T, Wong SS, Armstrong J, et al. T-705 (favipiravir) induces lethal mutagenesis in influenza A H1N1 viruses in vitro. J Virol. 2013;87(7):3741-3751.