As no vaccine was available when the influenza A(H1N1) 2009 pandemic started, NAIs, particularly oseltamivir, were the first-line strategy to combat the virus. Oseltamivir has thus been used as primary pharmacologic intervention for managing ill patients or their contacts. We showed in a severely immunocompromised patient, the selection of the H275Y mutation in the NA of the H1N1pdm09 virus 6 days after the initiation of oseltamivir treatment that was preceded by two days of oseltamivir chemoprophylaxis. While the appropriate oseltamivir dose for chemoprophylaxis in immunocompromised hosts is uncertain, the use of prophylaxis despite evidence of upper respiratory tract infection was probably a trigger for oseltamivir resistance selection. The H275Y mutation induced high-level resistance to oseltamivir which was consistent with previous reports 
. This suggests that oseltamivir monotherapy is likely not effective enough to control influenza shedding in severely immunocompromised patients and may account for resistance selection. Despite no selective drug pressure, the 275Y mutation remained detectable for 54 days.
Although H1N1pdm09 H275Y mutants remain susceptible to zanamivir, the use of this drug had no impact on viral shedding in this patient. Until recently, there have been only few reports of significant zanamivir resistance associated with mutations in the NA gene amongst human influenza viruses, in part due to a more limited use of this molecule: R152K for B virus in an immunocompromised patient 
and Q136K/L and R371K for A or B viruses in tissue culture and in vivo
. For the H1N1pdm09 virus, two reports have shown the emergence of an I223R mutation in the NA subsequently in two immunocompromised patients upon failure of treatment with NAIs 
. In one case, the patient received oseltamivir and then intravenous zanamivir therapy following the emergence of the H275Y mutation that conferred resistance to oseltamivir 
. The I223R mutation was detected upon relapse of virus excretion after discontinuation of zanamivir therapy at a time when the H275Y mutation could no longer be detected and persisted during the second course of zanamivir therapy. In the second case, the I223R mutation was detected along with the H275Y mutation following oseltamivir therapy and prior to the initiation of intravenous zanamivir therapy 
. It is not known whether both mutations were simultaneously present on the same virus and the fate of the mutations after cessation of oseltamivir therapy and during zanamivir treatment was not documented. In the two cases, the occurrence of I223R was associated with an increase of the oseltamivir and zanamivir 50% inhibitory concentrations, relative to wild-type and H275Y mutant viruses, respectively.
In the case reported here, during oseltamivir treatment and before the initiation of zanamivir therapy, the I223R mutation in the NA, was detected in a minor population and became predominant when zanamivir was given. Substitutions of residue I223 for V, T or M were observed among seasonal influenza A and B or A(H5N1) viruses under oseltamivir pressure especially in immunocompromised patients 
or after passages in vitro 
. Alone these three substitutions caused a minor decrease in oseltamivir susceptibility with no impact on zanamivir susceptibility but in combination either with H275Y or E119V they conferred major resistance to oseltamivir. In H5N1 viruses, under oseltamivir pressure, an I223M substitution in the NA was selected in vitro
in combination with the H275Y mutation 
. This dual mutation had a greater impact on the resistance to oseltamivir than H275Y alone but did not confer resistance to zanamivir and showed reduced susceptibility to peramivir. An I223V substitution was detected in the NA of H1N1pdm09 isolates in two summer campers who had received oseltamivir chemoprophylaxis and presented also with the H275Y mutation 
. Engineered viruses harbouring the mutation I223V in their NA showed a decreased sensitivity to oseltamivir and peramivir and enhanced resistance to both NAIs in the presence of the mutation H275Y 
. An I223K mutation was also reported to reduce susceptibility to NAIs 
. These limited data suggest that amino acid substitutions occurring at position 223 may be selected for by oseltamivir along with the H275Y mutation. Here, the association of the I223R mutation with the reduction in zanamivir susceptibility (5 to 7-fold) was demonstrated in vitro
on viral isolates and viruses produced by reverse genetics. In addition the I223R mutation conferred reduced susceptibility to oseltamivir (9 to 18-fold). These results suggest that the I223R substitution was selected for at low levels under oseltamivir treatment and eventually became predominant after zanamivir treatment. However, the modest level of resistance to zanamivir cannot explain per se
the treatment failure considering that the local concentrations of zanamivir are expected to be more than 337- and 135-fold higher than the IC50s for influenza virus NAs respectively in nasal mucosa and pulmonary epithelium after a 10 mg dose given twice daily 
. The relapse of leukaemia with profound immunodeficiency and absence of antibody response is also likely to account for lack of viral control.
In contrast to the H275Y mutation, the I223R mutation persisted at least 19 days after the last zanamivir inhalation but its disappearance thereafter cannot be excluded as viral follow-up was ceased. Potentially reduced viral fitness of 223R viruses was suggested by the reduction in plaque diameter and in virus titers achieved upon growth on MDCK-SIAT1 cells in agreement with a recent report 
but in contrast to another study which showed that 223R viruses grew to higher titers on ST6GalI-MDCK cells 
. In the patient studied here, persistence of 223R viruses was associated with a rebound of influenza virus shedding concomitantly with worsening fever, cough and pansinusitis. Upon prolonged virus shedding, emergence of additional mutations which persisted in the HA and in the NA was observed indicating that the viruses evolved with increasing viral population complexity. These additional mutations did not confer a higher level of resistance to zanamivir. Whether they actually improved viral fitness, and thus enabled the persistence of the I223R mutation in H1N1pdm09 viruses deserves further analysis. Viruses harboring both I223R and H275Y mutations produced by reverse genetics showed increased resistance to both zanamivir and oseltamivir compared to single mutants as also shown by Pizzorno et al. 
. Furthermore, the simultaneous presence of both mutations resulted in further reduced plaque diameter and reduced virus growth on MDCK–SIAT1 cells, as compared to single mutants suggesting that the fitness of the double mutants was significantly impaired. This was in contrast to the observations made by Pizzorno et al. which showed similar growth for wild-type and double mutant viruses on ST6GalI-MDCK cells 
. However, the likely reduced fitness for the viruses harboring both I223R and H275Y mutations is supported by the fact that double mutants were not detected in the clinical sample positive for both mutations.
This study clearly demonstrates that the mutation I223R alone confers reduced susceptibility to both oseltamivir and zanamivir and was primarily selected by oseltamivir. Despite zanamivir cessation, I223R mutants persisted along with additional mutations in the NA and HA. This highlights the potential for evolution of H1N1pdm09 viruses resistant to NAIs and the risk for possible emergence of sufficiently fit resistant strains able to widely spread. As prophylactic use (1×75 mg per day) has been associated with the selection of the H275Y mutation and potentially could be involved in the selection of the I223R mutation that confers resistance to both oseltamivir and zanamivir, the therapeutic dose of oseltamivir (2×75 mg per day) for prophylaxis should be carefully considered especially in immunocompromised hosts. Restricting the use of neuraminidase inhibitors only to treatment of patients that have ILI symptoms could also be an option to reduce the potential of generating resistance.
Because immunocompromised patients are at risk for sustained influenza virus shedding and for resistance selection, close monitoring and reporting of resistance to NAIs are essential. To limit the spread of any new resistant strain from this population, the promotion of prophylactic measures including influenza vaccination of relatives and healthcare providers should be encouraged as well as reinforcement of infection control practices. This study along with other reports of an I223R mutation reminds the need to develop new drugs with different viral targets.