Elsevier

The Lancet

Volume 364, Issue 9436, 28 August–3 September 2004, Pages 759-765
The Lancet

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Resistant influenza A viruses in children treated with oseltamivir: descriptive study

https://doi.org/10.1016/S0140-6736(04)16934-1Get rights and content

Summary

Background

Oseltamivir is an effective inhibitor of influenza virus neuraminidase. Although viruses resistant to oseltamivir emerge less frequently than those resistant to amantadine or rimantadine, information on oseltamivir-resistant viruses arising during clinical use of the drug in children is limited. Our aim was to investigate oseltamivir resistance in a group of children treated for influenza.

Methods

We analysed influenza A viruses (H3N2) collected from 50 children before and during treatment with oseltamivir. We sequenced the genes for neuraminidase and haemagglutinin and studied the mutant neuraminidases for their sensitivity to oseltamivir carboxylate.

Findings

We found neuraminidase mutations in viruses from nine patients (18%), six of whom had mutations at position 292 (Arg292Lys) and two at position 119 (Glu119Val), which are known to confer resistance to neuraminidase inhibitors. We also identified another mutation (Asn294Ser) in one patient. Sensitivity testing to oseltamivir carboxylate revealed that the neuraminidases of viruses that have an Arg292Lys, Glu119Val, or Asn294Ser mutation were about 104–105-fold, 500-fold, or 300-fold more resistant than their pretreatment neuraminidases, respectively. Oseltamivir-resistant viruses were first detected at day 4 of treatment and on each successive day of the study. More than 103 infectious units per mL of virus were detected in some of the patients who did not shed drug-resistant viruses, even after 5 days of treatment.

Interpretation

Oseltamivir-resistant mutants in children being treated for influenza with oseltamivir arise more frequently than previously reported. Furthermore, children can be a source of viral transmission, even after 5 days of treatment with oseltamivir.

Introduction

Influenza viruses continue to cause an unacceptable number of deaths and substantial economic losses worldwide. The last pandemic of influenza was in 1968, but a new pandemic virus will certainly arise. In addition to vaccines, two classes of antiviral agents have been used to treat influenza—the M2 ion channel inhibitors and the neuraminidase inhibitors. The M2 inhibitors amantadine and rimantadine inhibit influenza A viruses but drug-resistant mutants are present in about 30% of treated patients; resistant variants are genetically stable and can be transmitted from person to person.1 Furthermore, we have shown2 that the frequency of resistant viruses in children admitted and treated with amantadine could be as high as 80%.

Over the past few years, inhibitors of the viral enzyme neuraminidase have been used to treat influenza in clinical settings.3 Whereas the viral haemagglutinin binds to cell-surface sialyloligosaccharide receptors and initiates virus infection, the enzymatic activity of neuraminidase sialidase removes sialic acids from the oligosaccharides, promoting the release of viruses from infected cells.3 The importance of the neuraminidase in viral replication has made it an attractive target for the development of effective antiviral drugs. Knowledge of the conserved active sites for type A and type B neuraminidase molecules4, 5, 6 led to the production of two specific and potent neuraminidase inhibitors, namely inhaled zanamivir and oral oseltamivir, which are therapeutically useful in various clinical situations.7, 8 As with any antiviral compound, the therapeutic importance of the neuraminidase inhibitors can be compromised by the emergence of drug-resistant mutants. In cell culture with a neuraminidase inhibitor, mutations are introduced into the surface proteins of influenza viruses.9, 10, 11 Although such inhibitors act mainly on neuraminidase, viruses with haemagglutinin substitutions are usually selected first.12 These mutations reduce the sialic acid-binding activity of the haemagglutinin and the virus becomes less dependent on the sialidase activity of the neuraminidase, whereas subsequent neuraminidase mutations render the virus fully drug resistant. Most of the neuraminidase mutations detected so far result in low or unstable neuraminidase activity.10, 13, 14, 15

The neuraminidase substitutions in drug-resistant viruses include aminoacid residues 119, 152, 274, and 292 of the enzyme's active centre (N2 numbering system).3, 15 The arginine to lysine mutation (Arg292Lys) is the most typical substitution noted in N2-containing viruses isolated after treatment with oseltamivir in vivo and in vitro.14, 15, 16 Induction of neuraminidase inhibitor resistance needs multiple passages in cell culture, by contrast with amantadine resistance, which typically needs only one passage.14, 17, 18 Thus, the low frequency of oseltamivir resistance in controlled clinical trials, about 4% for children aged 1–12 years and 0·4–1% for adults,1, 19, 20 is not surprising. Our aim was to investigate such resistance in a group of children treated for influenza in Japan.

Section snippets

Patients

We obtained samples for influenza virus analysis from patients admitted to hospital and from outpatients during February to March, 2002 (Nippon Kokan Hospital), and January to February, 2003 (Keiyu Hospital, Kawasaki Municipal Hospital, Isehara Kyodo Hospital), all of whom were treated with oseltamivir. Treatment with oseltamivir (4 mg/kg daily in divided doses twice a day, for 2 [patient number 26], 3 [patients 27, 49], 4 [patients 16, 20], or 5 days [all remaining patients]) was not started

Results

We obtained samples for influenza virus analysis from 14 patients admitted and from one outpatient during February to March, 2002, and from 15 patients admitted and 20 outpatients during January to February, 2003. Patients ranged in age from 2 months to 15·8 years (median 3·7), and 80% were younger than age 5 years (table 1). Only seven (14%) of 50 individuals had received influenza vaccine. Although several children had pre-existing conditions, none was immunocompromised or receiving

Discussion

The difficulty of generating viruses resistant to neuraminidase inhibitors in cell culture,14 and the low frequency of such viruses in patients treated in clinical trials,11, 15, 16 have led to the impression that the development of resistance is a minor difficulty compared with the emergence of amantadine or rimantadine resistant viruses. However, in our study, about a fifth of children developed resistance by day 4 or later during treatment with oseltamivir. Furthermore, just over a quarter

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