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Influenza A and B viruses cause significant morbidity and mortality worldwide each year. The neuraminidase inhibitors (NAIs) are the most commonly used class of influenza antiviral drugs for the treatment of infected patients. In vitro studies have shown that influenza B viruses are significantly less susceptible to oseltamivir and other neuraminidase inhibitors compared with influenza A viruses. Following analysis of published clinical studies, we show that oseltamivir does appear to have lower effectiveness in patients infected with influenza B virus compared with influenza A infected patients, but due to insufficient studies on zanamivir, laninamivir or peramivir, it was not possible to conclude the relative effectiveness of these drugs against influenza A virus compared with B virus.

Relatively little is known about the burden of influenza in tropical countries. The seroprevalence of pandemic influenza A (H1N1) 2009, seasonal H1N1 and H3N2 was determined in Kuala Lumpur, Malaysia. Pre- and post-pandemic residual laboratory sera were tested by hemagglutination-inhibition. The seroprevalence of A(H1N1)pdm09 increased from 3.7% pre-pandemic to 21.9% post-pandemic, giving an overall cumulative incidence of 18.1% (95% CI, 13.8-22.5%), mainly due to increases in those <5, 5-17, and 18-29 years old. In contrast with findings from USA, Europe, and Australia, pre-existing seroprevalence to A(H1N1)pdm09 was low at 5.6% in the elderly age group of >55 years. A(H1N1)pdm09 affected almost a third of those <30 years in Kuala Lumpur. Pre-pandemic seroprevalence was 14.7% for seasonal H1N1 and 21.0% for H3N2, and these rates did not change significantly after the pandemic. Seasonal and pandemic influenza cause a considerable burden in tropical Malaysia, particularly in children and young adults., (Copyright © 2013 Wiley Periodicals, Inc.)

Influenza A H1N1 2009 virus caused the first pandemic in an era when neuraminidase inhibitor antiviral drugs were available in many countries. The experiences of detecting and responding to resistance during the pandemic provided important lessons for public health, laboratory testing, and clinical management. We propose recommendations for antiviral susceptibility testing, reporting results, and management of patients infected with 2009 pandemic influenza A H1N1. Sustained global monitoring for antiviral resistance among circulating influenza viruses is crucial to inform public health and clinical recommendations for antiviral use, especially since community spread of oseltamivir-resistant A H1N1 2009 virus remains a concern. Further studies are needed to better understand influenza management in specific patient groups, such as severely immunocompromised hosts, including optimisation of antiviral treatment, rapid sample testing, and timely reporting of susceptibility results., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Neuraminidase inhibitors (NAIs) represent a newer class of anti-influenza drugs. Widespread natural or acquired resistance to NAIs is a major public health concern as it limits pharmaceutical options available for managing seasonal and pandemic influenza virus infections. Molecular-based methods, such as pyrosequencing, sequencing, and PCR are rapid techniques for detecting known genetic markers of resistance, but they are unable to identify novel mutations that may confer resistance, or subtle differences in the susceptibility of viruses to the NAIs. This chapter describes the chemiluminescent neuraminidase (NA) inhibition (NI) assay, a functional method used for assessing influenza virus susceptibility to NAIs. The assay generates IC(50) values (drug concentration needed to reduce the NA enzymatic activity by 50%) which are determined by curve-fitting analysis. Test viruses showing elevated IC(50) values relative to those of NAI-sensitive reference viruses of the same antigenic type and subtype are further analyzed by pyrosequencing or conventional sequencing to identify known markers of NAI resistance or new changes in the NA. The criteria for NAI resistance are currently not well defined and tend to vary by laboratory and NI assay, therefore harmonization of NI assay conditions and interpretation of results across surveillance laboratories is necessary to improve the NAI susceptibility testing and analysis.

Neuraminidase inhibitors (NAIs) are presently the only effective antiviral drugs for treatment and chemoprophylaxis of influenza A and B infections, due to the high prevalence of resistance to the adamantane class of drugs among influenza A(H3N2) and A(H1N1) viruses, including the 2009 pandemic H1N1 strain. The limited pharmaceutical options currently available for control of influenza infections underscore the critical need for surveillance on NAI susceptibility of influenza viruses circulating globally. This chapter describes the fluorescent neuraminidase (NA) inhibition (NI) assay, a functional method used for assessing influenza virus susceptibility to NAIs. The IC(50) (drug concentration needed to reduce the NA enzymatic activity by 50%) values generated in this assay are used to evaluate the NAI-susceptibility of test viruses relative to those of sensitive reference viruses of the same antigenic type and subtype. Test viruses with significantly elevated IC(50)s are further analyzed by pyrosequencing or conventional sequencing to identify known markers of NAI resistance or novel changes in the NA. The harmonization of NI assay conditions and interpretation of results across surveillance laboratories is necessary to improve NAI susceptibility testing and analysis.

Using an in vivo ferret model, we investigated the development of resistance to oseltamivir and zanamivir for two different influenza A(H5N1) viruses (A/Vietnam/1203/2004, haemagglutinin phylogenetic clade 1, and A/Chicken/Laos/26/2006, haemagglutinin phylogenetic clade 2.3) by treating the animals with doses equivalent either to the recommended human treatment dose or a range of sub-optimal drug doses. No resistance was observed in oseltamivir-treated ferrets, but analysis of nasal washes from zanamivir-treated ferrets infected with influenza A/Vietnam/1203/2004 revealed one viral isolate (from a ferret receiving the highest dose of zanamivir, 1.0mg/kg twice daily) with a zanamivir IC(50) that was 350-fold higher than the other isolates tested. The same virus also demonstrated a 26-fold increase in oseltamivir IC(50). The isolate with reduced susceptibility was taken from a ferret 8 days post-infection that was being treated with the recommended human zanamivir dose. Sequence analysis of the resistant virus revealed a glutamine (Q) to leucine (L) mutation at residue 136 of the neuraminidase. This is the first report of this mutation being associated with neuraminidase inhibitor susceptibility and one of the few reported mutations that confer zanamivir resistance, and as such should be closely monitored in influenza A(H5N1) and other N1 viruses in the future. Further animal studies and human clinical trials are necessary to optimize neuraminidase inhibitor dosing strategies for the treatment of influenza A(H5N1) infections., (2010 Elsevier B.V. All rights reserved.)

Along with influenza vaccines, the world is currently almost completely dependent on two licensed drugs for the treatment or prevention of seasonal (influenza A and B viruses) and pandemic influenza (influenza A viruses). These drugs - oseltamivir (Tamiflu) and zanamivir (Relenza) - are classified as neuraminidase inhibitors (NAIs) because they act by inhibiting one of the key surface proteins of the influenza virus, the neuraminidase, which in turn reduces the ability of the virus to infect other respiratory cells. Our dependence on these drugs has arisen because of high levels of resistance with seasonal influenza viruses to the older class of anti-influenza drugs, the adamantanes (amantadine and rimantadine), combined with the lack of activity of these drugs against influenza B viruses. Recently, however, significant levels of oseltamivir-resistant influenza A(H1) seasonal influenza viruses have also been encountered, which has been associated with a single amino acid change in the viral neuraminidase (H274Y). Oseltamivir is the most widely used and stockpiled NAI and, while these A(H1) viruses are still sensitive to zanamivir, it highlights the ease with which the influenza virus can mutate and reassort to circumvent available drugs. Fortunately, the current pandemic A(H1N1) 2009 virus, which is circulating globally, remains largely sensitive to both NAIs, although a small number of oseltamivir-resistant viruses have been isolated from patients to date, again with the H274Y mutation. Clearly there is a need to use the NAI drugs prudently to ensure they remain an effective defence against future seasonal and pandemic influenza viruses, along with careful monitoring of levels of resistance in the circulating viruses combined with the further development of new anti-influenza drugs.

The neuraminidase inhibitors are a class of antiviral drugs used for both the prophylaxis and treatment of influenza infections. Clinical trials of these inhibitors detected a low level of resistant viruses from treated individuals, although a higher frequency was detected in children (5%-6%) compared to adults (1%-4%). In addition, there have been some previous reports of NA inhibitor resistant viruses being isolated from untreated individuals. Here we report on the NA inhibitor sensitivity of over 1,000 influenza isolates collected through the World Health Organization (WHO) global influenza surveillance program. Of the total number of viruses analysed, only 2 (0.2%) strains (an A(H1N1) strain and an influenza B strain) were considered to have a significant reduction in sensitivity to at least one of the neuraminidase inhibitor drugs. Interestingly, both of these strains were isolated from untreated patients in the youngest age cohort (less than 2 years). Although the influenza B strain is unlikely to be clinically resistant, the A(H1N1) virus contained the same His274Tyr neuraminidase mutation that has been observed in resistant mutants following oseltamivir treatment. Given these results it may be important to enhance neuraminidase inhibitor susceptibility testing of viruses from patients in the less than two years cohort.

During annual influenza epidemics, influenza B viruses frequently co-circulate with influenza A viruses and in some years, such as 2005, large outbreaks have occurred while in other years, the virus virtually disappears. Since 1987 there have been two lineages of influenza B viruses co-circulating in various countries and causing disease in humans. The proportions of these two lineages vary from year to year and country to country. For example, in 2005, the B/Victoria/2/87 lineage was predominant in New Zealand while in Australia the B/Yamagata/16/88 lineage was more common. Antigenic and genetic analysis has revealed gradual movement in the both lineages. Careful monitoring of the two virus lineages is important, as they are antigenically distinct. This is an important consideration for influenza vaccine formulation decisions, as only one influenza B component is traditionally included in the annual trivalent influenza vaccine.

The rapid and accurate detection of influenza A and B in a hospital setting is useful to confirm infection, exclude other diseases and assist in the management of patient illness including the possible use of specific antiviral therapy. We evaluated the use of the Directigen Flu A+B in a paediatric hospital laboratory in comparison with the established diagnostic tests direct immunofluorescence, viral culture and reverse transcriptase-polymerase chain reaction. A total of 193 respiratory specimens were examined and the Directigen test detected positive samples with an 80.8 per cent sensitivity and a specificity of 100 per cent. This study confirms other paediatric studies which have found the Directigen Flu A+B to be less sensitive than traditional laboratory tests but nevertheless to have a potential role in patient management especially when a positive result is obtained.

Two hundred and forty-five human influenza A and B viruses isolated in Australia between 1996 and 2003 were tested for their sensitivity to the NA inhibitor drugs, zanamivir and oseltamivir using a fluorescence-based neuraminidase inhibition assay. Based on mean IC50 values, influenza A viruses (with neuraminidase subtypes N1 and N2) were more sensitive to both the NA inhibitors than were influenza B strains. Influenza A viruses with a N1 subtype and influenza B strains both demonstrated a greater sensitivity to zanamivir than to oseltamivir carboxylate, whereas influenza A strains with a N2 subtype were more susceptible to oseltamivir carboxylate. A comparison of IC50 values for viruses isolated before and after the release of the NA inhibitors in Australia, found there was no significant difference in the sensitivity of strains to either neuraminidase inhibitor and none of the isolates tested showed clinically significant resistance.