Research article Open Access
Like 0


Antiviral treatment of immunocompromised patients with prolonged influenza virus infection can lead to multidrug resistance. This study reveals the selection of antiviral resistance mutations in influenza A(H1N1)pdm09 virus in an immunocompromised patient during a 6-month period. The patient was treated with two courses of oseltamivir (5 days and 2 months, respectively), with the first course starting at symptom onset, and subsequently zanamivir (2 months and 10 days, respectively). Respiratory samples were investigated by Sanger and next generation sequencing (NGS) and, for NGS data, low-frequency-variant-detection analysis was performed. Neuraminidase-inhibition tests were conducted for samples isolated in Madin-Darby canine kidney cells. In a sample collected 15 days after the end of the first treatment with oseltamivir (Day 20 post-symptom onset), oseltamivir resistance was detected (mutation H275Y with 60.3% frequency by NGS). Day 149 when the patient had almost completed the second zanamivir treatment, mixes of the following resistance mutations were detected; H275Y(65.1%), I223R(9.2%), and E119G(89.6%), accompanied by additional mutations, showing a more complex viral population in the long-term treated patient. Two samples obtained on Day 151 from bronchoalveolar lavage (BAL) and nasopharyngeal swab, respectively, showed different mutation profiles, with a higher frequency of antiviral resistance mutations in BAL. The results emphasise the importance of timely antiviral resistance testing both for treatment of individual patients as well as for preventive measures to control the development and transmission of antiviral resistant viruses.


Article metrics loading...

Loading full text...

Full text loading...



  1. LeGoff J, Rousset D, Abou-Jaoudé G, Scemla A, Ribaud P, Mercier-Delarue S, et al. I223R mutation in influenza A(H1N1)pdm09 neuraminidase confers reduced susceptibility to oseltamivir and zanamivir and enhanced resistance with H275Y. PLoS One. 2012;7(8):e37095.  https://doi.org/10.1371/journal.pone.0037095  PMID: 22936969 
  2. Alonso M, Rodríguez-Sánchez B, Giannella M, Catalán P, Gayoso J, López Bernaldo de Quirós JC, et al. Resistance and virulence mutations in patients with persistent infection by pandemic 2009 A/H1N1 influenza. J Clin Virol. 2011;50(2):114-8.  https://doi.org/10.1016/j.jcv.2010.10.007  PMID: 21074490 
  3. Bruminhent J, Deziel PJ, Wotton JT, Binnicker MJ, Razonable RR. Prolonged shedding of pandemic influenza A (H1N1) 2009 virus in a pancreas-after-kidney transplant recipient. J Clin Virol. 2014;61(2):302-4.  https://doi.org/10.1016/j.jcv.2014.07.004  PMID: 25081940 
  4. Gooskens J, Jonges M, Claas ECJ, Meijer A, Kroes ACM. Prolonged influenza virus infection during lymphocytopenia and frequent detection of drug-resistant viruses. J Infect Dis. 2009;199(10):1435-41.  https://doi.org/10.1086/598684  PMID: 19392620 
  5. World Health Organization (WHO). Influenza Vaccines. Geneva: WHO. [Accessed 20 May 2016]. Available at: http://www.who.int/influenza/vaccines/en/
  6. World Health Organization (WHO). Recommended composition of influenza virus vaccines for use in the 2014-15 northern hemisphere influenza season. Geneva: WHO. [Accessed 20 May 2016]. Available at: http://www.who.int/influenza/vaccines/virus/recommendations/201402_recommendation.pdf?ua=1
  7. O’Shea D, Widmer LA, Stelling J, Egli A. Changing face of vaccination in immunocompromised hosts. Curr Infect Dis Rep. 2014;16(9):420.  https://doi.org/10.1007/s11908-014-0420-2  PMID: 24992978 
  8. Zbinden D, Manuel O. Influenza vaccination in immunocompromised patients: efficacy and safety. Immunotherapy. 2014;6(2):131-9.  https://doi.org/10.2217/imt.13.171  PMID: 24491087 
  9. Ison MG. Influenza prevention and treatment in transplant recipients and immunocompromised hosts. Influenza Other Respi Viruses. 2013;7(Suppl 3):60-6.  https://doi.org/10.1111/irv.12170  PMID: 24215383 
  10. Eliakim-Raz N, Vinograd I, Zalmanovici Trestioreanu A, Leibovici L, Paul M. Influenza vaccines in immunosuppressed adults with cancer. Cochrane Database Syst Rev. 2013;10(10):CD008983. PMID: 24166741 
  11. Beck CR, McKenzie BC, Hashim AB, Harris RC, Zanuzdana A, Agboado G, et al. Influenza vaccination for immunocompromised patients: summary of a systematic review and meta-analysis. Influenza Other Respi Viruses. 2013;7(Suppl 2):72-5.  https://doi.org/10.1111/irv.12084  PMID: 24034488 
  12. van der Velden AMT, Mulder AHL, Hartkamp A, Diepersloot RJA, van Velzen-Blad H, Biesma DH. Influenza virus vaccination and booster in B-cell chronic lymphocytic leukaemia patients. Eur J Intern Med. 2001;12(5):420-4.  https://doi.org/10.1016/S0953-6205(01)00149-2  PMID: 11557327 
  13. Bright RA, Medina MJ, Xu X, Perez-Oronoz G, Wallis TR, Davis XM, et al. Incidence of adamantane resistance among influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: a cause for concern. Lancet. 2005;366(9492):1175-81.  https://doi.org/10.1016/S0140-6736(05)67338-2  PMID: 16198766 
  14. Dong G, Peng C, Luo J, Wang C, Han L, Wu B, et al. Adamantane-resistant influenza a viruses in the world (1902-2013): frequency and distribution of M2 gene mutations. PLoS One. 2015;10(3):e0119115.  https://doi.org/10.1371/journal.pone.0119115  PMID: 25768797 
  15. Hurt AC. The epidemiology and spread of drug resistant human influenza viruses. Curr Opin Virol. 2014;8(0):22-9.  https://doi.org/10.1016/j.coviro.2014.04.009  PMID: 24866471 
  16. Nguyen HT, Fry AM, Gubareva LV. Neuraminidase inhibitor resistance in influenza viruses and laboratory testing methods. Antivir Ther. 2012;17(1 Pt B):159-73.  https://doi.org/10.3851/IMP2067  PMID: 22311680 
  17. Pozo F, Lina B, Andrade HR, Enouf V, Kossyvakis A, Broberg E, et al. , Community Network of Reference Laboratories for Human Influenza in Europe. Guidance for clinical and public health laboratories testing for influenza virus antiviral drug susceptibility in Europe. J Clin Virol. 2013;57(1):5-12.  https://doi.org/10.1016/j.jcv.2013.01.009  PMID: 23375738 
  18. Takashita E, Fujisaki S, Shirakura M, Nakamura K, Kishida N, Kuwahara T, et al. , Influenza Virus Surveillance Group of Japan. Influenza A(H1N1)pdm09 virus exhibiting enhanced cross-resistance to oseltamivir and peramivir due to a dual H275Y/G147R substitution, Japan, March 2016. Euro Surveill. 2016;21(24):30258.  https://doi.org/10.2807/1560-7917.ES.2016.21.24.30258  PMID: 27336226 
  19. World Health Organization (WHO) Global Influenza Surveillance Network. Manual for the laboratory diagnosis and virological surveillance of influenza. Geneva: WHO press; 2011.
  20. World Health Organization. Meetings of the WHO working group on surveillance of influenza antiviral susceptibility – Geneva, November 2011 and June 2012. Wkly Epidemiol Rec. 2012;87(39):369-74. PMID: 23061103 
  21. Ives JAL, Carr JA, Mendel DB, Tai CY, Lambkin R, Kelly L, 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.  https://doi.org/10.1016/S0166-3542(02)00053-0  PMID: 12103431 
  22. Pizzorno A, Abed Y, Bouhy X, Beaulieu E, Mallett C, Russell R, et al. Impact of mutations at residue I223 of the neuraminidase protein on the resistance profile, replication level, and virulence of the 2009 pandemic influenza virus. Antimicrob Agents Chemother. 2012;56(3):1208-14.  https://doi.org/10.1128/AAC.05994-11  PMID: 22203589 
  23. Tramontana AR, George B, Hurt AC, Doyle JS, Langan K, Reid AB, et al. Oseltamivir resistance in adult oncology and hematology patients infected with pandemic (H1N1) 2009 virus, Australia. Emerg Infect Dis. 2010;16(7):1068-75.  https://doi.org/10.3201/eid1607.091691  PMID: 20587176 
  24. European Centre for Disease Prevention and Control (ECDC)/WHO Regional Office for Europe. Flu News Europe, Joint ECDC–WHO weekly influenza update, week 20/2015. ECDC–WHO; 2015.
  25. Correia V, Santos LA, Gíria M, Almeida-Santos MM, Rebelo-de-Andrade H. Influenza A(H1N1)pdm09 resistance and cross-decreased susceptibility to oseltamivir and zanamivir antiviral drugs. J Med Virol. 2015;87(1):45-56.  https://doi.org/10.1002/jmv.23986  PMID: 25042157 
  26. Rogers MB, Song T, Sebra R, Greenbaum BD, Hamelin M-E, Fitch A, et al. Intrahost dynamics of antiviral resistance in influenza A virus reflect complex patterns of segment linkage, reassortment, and natural selection. MBio. 2015;6(2):e02464-14.  https://doi.org/10.1128/mBio.02464-14  PMID: 25852163 
  27. To KK, Hung IF, Li IW, Lee KL, Koo CK, Yan WW, et al. Delayed clearance of viral load and marked cytokine activation in severe cases of pandemic H1N1 2009 influenza virus infection. Clin Infect Dis. 2010;50(6):850-9.  https://doi.org/10.1086/650581  PMID: 20136415 
  28. Zhirnov OP, Vorobjeva IV, Saphonova OA, Poyarkov SV, Ovcharenko AV, Anhlan D, et al. Structural and evolutionary characteristics of HA, NA, NS and M genes of clinical influenza A/H3N2 viruses passaged in human and canine cells. J Clin Virol. 2009;45(4):322-33.  https://doi.org/10.1016/j.jcv.2009.05.030  PMID: 19546028 

Data & Media loading...

Submit comment
Comment moderation successfully completed
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error