First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark (ECDC, edited)

[Giuseppe]

First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark (ECDC, edited)

ECDC THREAT ASSESSMENT

First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark

[Original Document: LINK. EDITED.]


01 July 2009


Source and date of request

Internal request following an EWRS message from Denmark health authorities.


Public health issue

Isolation of a mutant A(H1N1)v virus containing a genetic marker of resistance to oseltamivir.


Consulted experts

Internal experts.


Event background information

Event reported by Danish EWRS Focal point on 29 June 2009:
In the context of tracing contacts of a cluster of four cases infected with influenza A(H1N1)v, including two imported cases in Denmark, a female contact, who initially tested negative on PCR, was given prophylaxis with oseltamivir (75mg per day). Five days later, despite reportedly having complied with treatment, she developed flu-like symptoms and was tested positive for A(H1N1)v. Sequencing of the virus showed a single mutation H275Y (H274Y in N2 numbering system) in the neuraminidase gene. The presence of the resistance marker and the phenotypic (in vitro) resistance was confirmed by a WHO collaborating centre, UK. The virus is not a re-assortant and remains susceptible to zanamivir (another neuraminidase inhibitor). No other virus isolated as part of this cluster investigation, including the presumed source patient, showed the mutation.


ECDC threat assessment for the EU

To date, community isolates of the A(H1N1)v virus related to the ongoing pandemic have been found sensitive to oseltamivir and zanamivir and resistant to adamantanes (M2 blockers). This event is the first observation of resistance to oseltamivir in a pandemic A(H1N1)v virus.

Available epidemiological and virological data do not allow determining whether the resistant variant in Denmark arose in an already treated prior case and then transmitted to the patient with documented resistance or in the context of post-exposure prophylaxis.

Importantly, based on clinical criteria, there is no evidence that the resistant virus has subsequently transmitted to other persons.

The mutation consists of the substitution of histidine to tyrosine at amino acid position 275 (H275Y) in the neuraminidase segment gene (position 274 in N2 numbering system). This mutation has been described in the past, associated with so-called secondary resistance to oseltamivir acquired during treatment of both H1N1 and H5N1 virus infections. In Japan, where the drug was used more commonly than in Europe1, the mutation was detected in up to 16% of oseltamivir-treated children, perhaps associated with underdosing(2). However, a recent study in the United Kingdom also confirmed emergence of resistant seasonal H1N1 viruses with this mutation in children treated with standard, approved doses(3).

Consequently, it is likely that such oseltamivir resistance mutation will be observed again in individuals taking oseltamivir (treatment or chemoprophylaxis). Furthermore, the theoretical possibility exists that oseltamivir-resistant neuraminidase might be acquired through reassortment in the future.

The current event does highlight the increased likelihood of such event arising from widespread use of oseltamivir and the need for continued surveillance.

The affected patient recovered without complication. However, in more seriously ill patients, like in those with influenza viral pneumonia, the emergence of resistance might be associated with antiviral treatment failure. Of note, emergence of oseltamivir resistance in the context of treating H5N1 illness has been temporally associated with failure to clear virus and fatal outcome in two patients(4). Such findings emphasise the importance of serial virologic sampling in severely ill patients whenever possible and monitoring for resistance when prolonged shedding of virus is detected.

Depending on the specific H1N1 virus studied, some past studies have shown that seasonal H1N1 virus containing this mutation may be less infectious, although still transmissible in ferrets(5) and reduced in replication and pathogenicity(6), as demonstrated in animal studies. However, oseltamivir-resistant A/Brisbane/59/2007-like viruses with this mutation have spread globally since the 2007?2008 season in the apparent absence of selective drug pressure and displaced other seasonal A(H1N1) viruses in most countries (98% resistance in EU in 2008?2009)(7). These resistant seasonal H1N1 viruses are no less replication competent or virulent in humans than closely-related oseltamivir susceptible seasonal H1N1 viruses.

Thus, the effects of this H275Y mutation on neuraminidase function, viral replication, virulence, and transmissibility in animal models or humans depend on the specific N1 neuraminidase under study. These data need to be ascertained for the pandemic (H1N1) 2009 virus with this oseltamivir-resistant mutation.


Conclusions and recommendations

The emergence of resistance while on antiviral treatment is a well-recognised phenomenon in influenza viruses. Emergence of resistant viruses is always a concern for individual patients as well as for the potential public health threat that they pose if they retain sufficient transmissibility. Surveillance for emergence of resistant viruses in treated persons, particularly those with severe disease or immunocompromised status, and in community isolates is of importance for monitoring this potential clinical and public health problem in the context of the current H1N1 pandemic.

Contact: support@ecdc.europa.eu


References

1. Whitley RJ, Hayden FG, Reisinger K, Young N, Dutkowski R, Ipe D, et al. Oral oseltamivir treatment of influenza in children. Ped Infect Dis J. 2001;20(2):127?33.
2. Ward P, Small I, Smith J, Suter P, Dutkowski R. Oseltamivir (Tamiflu?) and its potential for use in the event of an influenza pandemic. J Antimicrob Chemother. 2005;55(suppl_1):i5?21.
3. Stephenson I et al. Neuraminidase Inhibitor resistance after oseltamivir treatment of acute influenza A and B in children. Clin Infect Dis 2009 (in press)
4. de Jong MD et al. Oseltamivir resistance during treatment of influenza A(H5N1) infection. New Engl J Med 2005; 353: 2667-72
5. Herlocher ML, Truscon R, Elias S, et al. Influenza viruses resistant to the antiviral drug oseltamivir: transmission studies in ferrets. J Infect Dis. 2004;190(9):1627?30.
6. Ives JA, Carr JA, Mendel DB, et al. The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo. Antiviral Res. 2002;55(2):307?17.
7. Meijer A, Lackenby A, Hungnes O, Lina B, van der Werf S, Schweiger B, et al. Oseltamivir-resistant influenza A(H1N1) virus, Europe, 2007?08 season. Emerg Infect Dis. 2009;15(4):552?60.

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[St Michael]

Re: First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark (ECDC, edited)

Related thread:

Japan Reports Tamiflu-Resistant H1N1 Influenza Case


One must wonder if this news is related to the recent fire sale of Tamiflu to third world nations:

Roche To Broaden Tamiflu Access

[wotan]

Re: First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark (ECDC, edited)

Available epidemiological and virological data do not allow determining whether the resistant variant in Denmark arose in an already treated prior case and then transmitted to the patient with documented resistance or in the context of post-exposure prophylaxis.

I believe that is what niman has been saying. Finally others are having to agree.

[pjie2]

Re: First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark (ECDC, edited)

I believe that is what niman has been saying. Finally others are having to agree.

Not quite. Niman appears to be saying that the lack of epidemiological data constitute proof that the resistant strain is circulating in the community and was not due to prophylaxis, and (moreover) was due to recombination and not de novo mutation. I freely confess that I didn't follow his logic for either assertion.

[HenryN]

Re: First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark (ECDC, edited)

Not quite. Niman appears to be saying that the lack of epidemiological data constitute proof that the resistant strain is circulating in the community and was not due to prophylaxis, and (moreover) was due to recombination and not de novo mutation. I freely confess that I didn't follow his logic for either assertion.

H274Y has a VERY well descibed HISTORY of hopping from one H1N1 background to another (in hosts NOT taking Tamiflu). It is in the Denmark isolate, and almost certainly in the Japan isolate, reported ONE day apart (with more to come) and although both patients were taking Tamiflu, it is NOT obvious that the H274Y was from the treatment, and the treatment may have just REVEALED the presence of H274Y, because the patients did not respond to treatment.

Tamiflu causes resistance at multiple positions in multiple sero-types. However, ALL recent (last 3 years) Tamiflu resistance in humans has been H1N1 (only) involving H274Y (only).

The epidemiological and genetic stories are VERY clear.

Earlier predictions were not that Tamiflu resisatnce in general would develop, or that resisatance would be due to reassortment, but rather that H274Y would be ACQUIRED via recombination with seasonal H1N1.

[St Michael]

Re: First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark (ECDC, edited)

H274Y has a VERY well descibed HISTORY of hopping from one H1N1 background to another (in hosts NOT taking Tamiflu). It is in the Denmark isolate, and almost certainly in the Japan isolate, reported ONE day apart (with more to come) and although both patients were taking tamiflu, it is NOT obvious that the H274Y was from the treatment, and teh treatment may have just REVEALED the presence of H274Y, because the patients did not respond to treatment.

Tamiflu causes resistance at multiple positions in multiple sero-types. However, ALL recent (last 3 years) Tamiflu resistance in humans has been H1N1 (only) involving H274Y (only).

The epidemiological and genetic stories are VERY clear.

Aside from the obvious - resistance to Tamiflu and evolutionary fitness - are there any known changes in virulence associated with H274Y acquisition? Wasn't there evidence that Tamiflu resistance actually diminishes virulence in some other influenza strains?

[St Michael]

Re: First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark (ECDC, edited)

The Evolution of Influenza Resistance and Treatment

David M. Weinstock, MD; Gianna Zuccotti, MD

JAMA. 2009;301(10)doi:10.1001/jama.2009.324).

In February 2006, the US Centers for Disease Control and Prevention (CDC) reported that 92.3% of the circulating influenza A(H3N2) at that time was resistant to the adamantanes (amantadine and ramantidine), 1 of 2 pharmacological classes available for the treatment of influenza.1 The resistant viruses harbored an S31N amino acid substitution in the influenza M2 protein that confers resistance but does not affect virulence. Although resistance to adamantanes increased to 14.5% in the prior year,2 the dramatic increase in 2005-2006 came as a shock to both the medical and scientific communities and the public.3

At the time, it was confidently held that influenza was unlikely to develop similar resistance to neuraminidase inhibitors (oseltamivir and zanamivir), the second class of influenza-directed agents.4-5 The neuraminidase inhibitors were designed with an understanding of the structural interaction between neuraminidase and its natural substrate sialic acid. Because these antivirals closely resemble sialic acid, any mutation within neuraminidase that reduced affinity for the pharmacological inhibitors was believed to invariably compromise viral fitness.

The available evidence supported this notion. In vitro selection of neuraminidase inhibitor?resistant mutants is generally difficult, which argues that resistance-associated mutations compromise viability. In a 2002 study,5 the H274Y (histidine-to-tyrosine at codon 274 in N2 nomenclature) mutation in neuraminidase significantly reduced the replication of H1N1 strains and their virulence in mice and ferrets, regardless of whether the mutation was derived by site-directed mutagenesis, through serial passage in culture under drug pressure or during treatment in humans. In clinical trials of oseltamivir, resistance was relatively rare (0.32% in adults and 4.1% in children)4 and the few oseltamivir-resistant isolates recovered during treatment seemed clinically unimportant. As recently as the 2006-2007 influenza season, transmission of oseltamivir-resistant influenza was exceedingly uncommon.6-7

Nevertheless, studies by Dharan et al8 and Gooskens et al9 in this issue of JAMA demonstrate that change was in the air. On December 19, 2008, the CDC reported that nearly all cases of influenza A(H1N1), the predominant circulating strain for the season thus far, are resistant to oseltamivir.10 Dharan et al8 expand that analysis to 268 H1N1 isolates, of which 264 (98.5%) are oseltamivir resistant. Of all influenza isolates typed by the CDC this season, approximately 55% are oseltamivir-resistant H1N1.11 These isolates carry the neuraminidase H274Y mutation that confers resistance to oseltamivir but does not affect susceptibility to zanamivir. The oseltamivir-resistant H1N1, which is now circulating on all major continents,12 is similar to the A/Brisbane/59/2007 strain that circulated during the 2007-2008 season13 and is included in the 2008-2009 influenza vaccines.

The studies by Dharan et al8 and Gooskens et al9 dispel the notion that oseltamivir resistance compromises virulence. Four of the 142 patients (2.8%) with oseltamivir-resistant H1N1 isolates submitted to the CDC during the 2007-2008 influenza season died of influenza. In a comparison between 99 oseltamivir-resistant cases and 182 matched oseltamivir-susceptible cases, there were no discernible differences in the predisposing factors, clinical symptoms, or complications related to influenza infection.

Gooskens et al9 describe a typical nosocomial outbreak of influenza at their center during the 2007-2008 season with an alarming new wrinkle?the outbreak strain was oseltamivir-resistant H1N1. At least 3 patients who were immunocompromised acquired influenza from the index case, each developed pneumonia and 2 died. Five health care workers also developed an influenza-like illness but were not tested for H1N1.

The widespread belief that oseltamivir would retain activity against epidemic influenza strains has crumbled, but this should come as no surprise. Global and regional surveillance networks have tracked the incidence of neuraminidase-inhibitor resistance among circulating influenza for several years. The available epidemiology indicated that oseltamivir resistance was increasing on a trajectory that precisely paralleled the surge of adamantane resistance 3 years earlier (Figure). During the 2007-2008 season, 10.9% of H1N1 viruses tested in the United States were oseltamivir resistant.13 Even higher rates were reported elsewhere, including Canada (26%), Europe (25%), and Hong Kong (12%).14 The highest rate of oseltamivir resistance (67.3%) was in Norway, a country where oseltamivir can only be acquired with a prescription and is rarely used.14 In a study of the cases in Norway, oseltamivir resistance did not affect the phenotype of influenza infection,15 similar to the study by Dharan et al.8 During the summer of 2008, H1N1 strains related to A/Brisbane/59/2007 predominated in the Southern hemisphere and 100% of the H1N1 isolates in South Africa were resistant to oseltamivir.14


Figure 1
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Figure. Frequency of Resistance to Adamantane Among Influenza A(H1N1) Isolates and Oseltamivir Among Influenza A(H3N2) Isolates Submitted to the Centers for Disease Control and Prevention in the United States

Frequency of resistance among isolates submitted to the US Centers for Disease Control and Prevention.1-2,7-8,11, 13 NA indicates not available. The number of H1N1 isolates tested for oseltamivir resistance in 2005-2006 and the number of adamantane resistant H3N2 isolates in 2006-2007 were not available. The oseltamivir resistance before the 2007-2008 influenza season was less than 1%.


If the epidemiology was not sufficiently compelling, newer studies of H1N1 virulence have confirmed that H274Y does not necessarily compromise substrate binding, enzyme activity, viral replication, or virulence in animal models.16-18 Binding of neuraminidase to oseltamivir involves a conformational change in the side chain of the glutamic acid residue at codon 276 (E276). Importantly, the same conformational change is not required for zanamivir binding. Substitution of a tyrosine, which is bulkier than histidine, at codon 274 pushes the carboxyl group of E276 further into the binding site.18 Group 1 neuraminidases (N1, N4, N5, N8) have another tyrosine below the active site at codon 252. The combination of the 2 tyrosines (Y252 and Y274) disrupts the pocket that normally accommodates oseltamivir. In contrast, group 2 neuraminidases (N2, N3, N6, N7, N9) have a smaller threonine at codon 252 and can still accommodate oseltamivir in the binding pocket in the presence of Y274.18

The structural study offers several insights. First, resistance to oseltamivir among strains with group 1 neuraminidases was predictable. Second, this resistance was less likely to affect strains with group 2 neuraminidases, consistent with the complete susceptibility of H3N2 strains to oseltamivir this season.11 Third, zanamivir will maintain activity against some oseltamivir-resistant strains. Fourth, new inhibitors can be intelligently crafted to overcome specific mutations within neuraminidase.19 Fifth, oseltamivir resistance is likely to develop during the treatment of other N1-containing strains, including avian influenza A(H5N1).20 Thus, stockpiles to mitigate an influenza pandemic should not be limited to oseltamivir.

For an influenza strain to achieve global predominance, it must outcompete other strains. A study by Rameix-Welti et al16 supports the intriguing notion that the H274Y mutation provides a competitive advantage to the circulating H1N1 strain that is completely independent of its effect on oseltamivir. The neuraminidase from this strain is distinguished by a unique combination of at least 8 amino acid substitutions, exclusive of H274Y. Both the enzymatic activity and substrate affinity of this neuraminidase (with or without H274Y) are significantly higher compared with neuraminidase proteins from strains that circulated during previous years, although the affinity is reduced somewhat by the H274Y mutation.16 The latter point is especially noteworthy. The "fitness" of an influenza strain is affected by the functional balance between its hemagglutinin and neuraminidase proteins. The additional mutations within the circulating H1N1 strain greatly enhance neuraminidase function, which may throw off the balance. The H274Y mutation, by attenuating neuraminidase substrate affinity, could bring the relative activities of hemagglutinin and neuraminidase back into balance, thereby enhancing the strain's overall fitness.16

In an accompanying editorial to the 2006 report of adamantane resistance, we stated that "an axiom of good infectious disease practice [is that] inappropriate use of anti-infectives invariably results in resistance."3 But if drug-resistant strains can outcompete susceptible ones in the absence of anti-infective pressure, it follows that for this organism and this agent, the most basic "truth" about anti-infective resistance may be wrong.

Between 2002 and 2007, the antigenic and genetic evolution of influenza A(H3N2) at distant locations around the world was remarkably homogenous.21 This homogeneity supports a model in which epidemic viruses circulate globally rather than persisting within a region and evolving locally. In fact, there is a continuous network of temporally overlapping H3N2 epidemics in East and Southeast Asia. The epidemics within temperate regions are seeded from this network, generally first in Oceania, North America and Europe, and then later in South America.21 Reseeding back into East and Southeast Asia is uncommon, such that once H3N2 strains leave East and Southeast Asia they rarely contribute to viral evolution. If the global dissemination of H1N1 follows similar patterns to those for H3N2, the widespread use of oseltamivir is not the cause of widespread resistance. Instead, oseltamivir use outside of East and Southeast Asia, appropriate or not, is unlikely to affect the epidemics within East and Southeast Asia, and thus the next wave of global dissemination.

This new understanding of influenza dynamics helps to explain the discordance between regional frequencies of oseltamivir resistance and oseltamivir use during the 2007-2008 influenza season. For example, oseltamivir resistance among H1N1 strains was 67.3% in Norway, where oseltamivir is rarely used, but was only 3% in Japan, the country with the highest per capita oseltamivir use.14

While rates of oseltamivir-resistant H1N1 remain high, patients who are candidates for influenza treatment or chemoprophylaxis should receive either zanamivir or the combination of oseltamivir and rimantidine (or amantidine, if rimantidine is not available).10 Essentially all influenza A(H3N2) strains circulating this year are resistant to adamantanes but susceptible to both neuraminidase inhibitors. Thus, single-agent oseltamivir is only appropriate for the rare patient with infection that is known to be caused by influenza A(H3N2) or influenza B.

Considering the need for dual-therapy or inhaled zanamivir, which can be difficult to administer and tolerate, the risk/benefit calculus for empirically treating a healthy adult with an influenza-like illness needs to be assessed on a case-by-case basis. Rapid assays to diagnose influenza are insensitive, so the decision should primarily rest on the patient's risk for influenza-related complications and the local influenza epidemiology.

Vaccination remains the cornerstone of global efforts to control the dissemination of influenza. A study by Wang et al22 provides some additional insight into the absolute and relative benefits of the trivalent inactivated vaccine (TIV) and live attenuated influenza vaccine (LAIV). The authors reviewed the health care encounters for influenza and pneumonia among more than a million active-duty members of the US military between 2004 and 2007. In this young healthy population with annual vaccination rates between 50% and 80%, TIV was associated with reduced influenza and pneumonia encounters by 30% to 55% compared with placebo. Those outcomes were similar to previous studies and were better than LAIV, which only outperformed placebo for vaccinees who were relatively vaccine-naive.

The authors speculate that because the attenuated vaccine strains in LAIV must replicate within the recipient to effectively stimulate influenza-specific immunity, the presence of neutralizing antibodies from either previous vaccination or natural infection may prevent this replication and thereby compromise the efficacy of LAIV. Both a randomized study23-24 and a meta-analysis25 have compared TIV with LAIV in adults and both reported an advantage for TIV. Not surprisingly, LAIV is equally or possibly more efficacious in children, who presumably lack preexisting influenza-specific immunity.26-27

How these new findings by Wang et al22 will affect recommendations for influenza vaccination remain to be seen. During typical influenza seasons, it may be prudent to use TIV in patients who were vaccinated at least once in the previous 2 years. On the other hand, live attenuated vaccines against pandemic strains may be more effective than inactivated vaccines, as the population will presumably lack preexisting immunity.

The understanding of influenza biology and epidemiology has advanced markedly; however, the global dissemination of oseltamivir-resistant influenza came as a great surprise. Undoubtedly, new surprises await in the perpetual struggle with influenza as one thing is certain?the organism will continue to evolve. Anticipating the rapid and endless changes in influenza biology and dynamics will require faster diagnostics to molecularly characterize specimens, extensive surveillance among humans and animals, and more rapid and malleable systems for translating basic and epidemiological discoveries into clinically applicable interventions. For now, the best tools to mitigate influenza infection are tried-and-true?vaccination, social distancing, hand washing, and common sense.

[HenryN]

Re: First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark (ECDC, edited)

Aside from the obvious - resistance to Tamiflu and evolutionary fitness - are there any known changes in virulence associated with H274Y acquisition? Wasn't there evidence that Tamiflu resistance actually diminishes virulence in some other influenza strains?

Yes. In fact the argument was made that influenza with H274Y would not transmit and could only emerge in patients taking tamiflu (which killed off the wide type) because it wasn't evolutionarily fit.

[HenryN]

Re: First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark (ECDC, edited)

Not quite. Niman appears to be saying that the lack of epidemiological data constitute proof that the resistant strain is circulating in the community and was not due to prophylaxis, and (moreover) was due to recombination and not de novo mutation. I freely confess that I didn't follow his logic for either assertion.

I think the resistance (almost certainly H274Y) in Hong Kong in patient (from San Franciso) who was NOT taking Tamiflu eliminates a de novo "mutation" and supports more hithhiking via recombination (as predicted).

[pjie2]

Re: First isolation of a secondary oseltamivir-resistant A(H1N1)v strain in Denmark (ECDC, edited)

If the resistant Hong Kong and Denmark strains prove to be most closely related to each other, it suggests that a resistant strain arose once and has spread quite widely before detection. This would be bad news.

If the two strains are not closely related, but instead branch off from different parts of the cluster of swine flu sequences, then it suggests that they have independently acquired resistance.

In neither case would there be enough evidence to say whether the resistance was acquired through recombination or through de novo mutation. To demonstrate recombination, then the sequence flanking the resistance marker should be more closely related to seasonal H1N1 than to swine H1N1. In that case (and if the conclusion were statistically sound - super_flu will know more :-) ), then it would demonstrate recombination between seasonal H1N1 and swine H1N1.