Antiviral Therapy 2010 15:1059–1063 (doi: 10.3851/IMP1657)

Case report Prolonged shedding of - and oseltamivir- resistant A(H3N2) virus with dual mutations in an immunocompromised infant

Guillermo Ruiz-Carrascoso1*, Inmaculada Casas1, Francisco Pozo1, Marta González-Vincent2, Pilar Pérez-Breña1

1Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain 2Hospital Infantil Universitario Niño Jesús, Madrid, Spain

*Corresponding author e-mail: [email protected]

In this study, we report a case of multidrug-resistant contained the substitutions E119V in and influenza A(H3N2) virus isolated from an immunosup- V27A in M2, which produce resistance to oseltamivir and pressed patient with prolonged viral shedding. We also , respectively. This is the first report of this describe the genetic characterization of the haemagglu- dual mutation pattern in multidrug-resistant influenza tinin, neuraminidase and M2 influenza genes. The virus A(H3N2) virus.

Introduction

Influenza, which causes significant morbidity and daily because of neurological toxicity). After receiving ­mortality in immunocompromised patients, is often fludarabine and melphalan as conditioning therapy, he associated with prolonged viral shedding [1,2] underwent allogenic non-identical peripheral blood regardless of vaccination and antiviral therapy. The transplantation from his mother in May 2005 (day 0). ­multidrug-resistant influenza viruses, including pan- Total lymphocyte cell (TLC) count prior to transplan- demic A(H1N1) and seasonal A(H1N1) and A(H3N2), tation was 1,340 cells/mm3 (25%). On day 10, fever as reported to date in treated immunocompromised was observed and oxygen therapy was required because patients [1,3–6], have presented the E119V, R292K or of respiratory insufficiency. On day 12, Grade II acute H275Y mutations in the neuraminidase (NA) gene and graft-versus-host disease (GVHD) that affected the skin the S31N mutation in the M2 gene, which confer osel- and was observed, and he was treated with corticos- tamivir and resistance, respectively. In this teroids and tacrolimus. On day 19, respiratory difficulty study, we report the prolonged shedding of influenza persisted and a bronchoalveolar lavage (BAL) sample A(H3N2) virus with an unusual combination of muta- (sample A) was sent to our laboratory (National Influ- tions conferring multidrug resistance to oseltamivir and enza Center, Madrid, Spain) for further testing. Using amantadine in an immunocompromised infant. previously described PCR assays for respiratory viruses A 6-month-old male with a previous history of influ- [7,8], the tested BAL sample was positive for influenza A. enza A infection detected by immunochromatographic On day 26 post-transplant and after 33 days of antiviral antigen detection in March 2005 that developed into therapy, treatment was discontinued and the patient was interstitial chronic , also presented a T-cell- discharged from hospital with the influenza A infection negative, natural-killer-cell-negative and B-cell-positive apparently resolved. On day 38, fever, rash and upper severe combined immunodeficiency disease, which was respiratory symptoms reappeared (TLC 1,577 cells/ diagnosed 1 month after initial detection. The chest mm3; 9%). A nasal antigen detection swab showed that X-ray showed interstitial bilateral infiltration and the the influenza A infection remained unresolved. Because antigen detection nasal swab tested positive for influ- of the persistence of the respiratory symptoms and the enza A. The patient (weight 7 kg) received treatment prolonged shedding of influenza A, the patient was again with oseltamivir (12 mg twice daily) and amantadine treated with oseltamivir (12 mg once daily) and aman- (15 mg once daily, which was reduced to 7.5 mg once tadine (7.5 mg once daily) for 15 days. On day 86, a

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reactivation of acute cutaneous and intestinal Grade elsewhere [9]. Additional M2 segment amplification III GVHD accompanied by rhinorrhea and crepitation was performed using the outer primers for eight influ- in the right hemithorax with an absence of fever was enza A viral RNA segments [9] and the inner ­primers observed. The culture and PCR results of a nasal swab designed for the generic amplification of the M2 seg- were positive for influenza A (sample B). The dosage of ment: sense 5′-CCTAYCAGAARCGRATGGG-3′ and corticosteroids was increased and the GVHD resolved. antisense 5′-TTCCTITCGRTAYTCYTCCC-3′. The The respiratory symptoms disappeared with supplemen- sequences of the NA, M2 transmembrane domain tal oxygen. In September 2005 (day 132), another epi- and HA1 domain of HA were directly amplified and sode of fever with upper and lower respiratory symptoms sequenced from the clinical samples. Comparisons occurred (TLC 665 cells/mm3; 10%) and a BAL sample of the sequences were made with reference to A/ tested positive for in culture and PCR Panama/2007/1999 numbering and the assays (sample C). Antiviral treatment with oseltamivir mutations with reference to the vaccine strains for the (12 mg twice daily) and amantadine (7.5 mg twice daily) 2004–2005 (A/California/7/2004) and 2005–2006 (A/ was reinitiated and continued until November 2005 (day Wisconsin/67/2005) influenza seasons in the Northern 180), when a throat swab sample (sample D) still tested Hemisphere. positive for influenza A on both culture and PCR assays The sequencing of the NA gene revealed an E119V despite the patient being asymptomatic. After progressive substitution (GenBank accession numbers EU652319, immune reconstitution (TLC 1.876 cells/mm3; 39%), the EU652320, EU652321 and EU652322), which was culture and PCR assays results of the nasal swab and associated with resistance to oseltamivir in the four BAL samples (samples E and F, respectively) were finally samples obtained from the reported case (Figure 2A). negative for influenza A in December 2005 (day 222). The segment encoding the M2 transmembrane The courses of antiviral treatment and dates of sample domain showed the V27A mutation (GenBank acces- collection are summarized in Figure 1. sion numbers EU652323, EU652324, EU652325 and All four samples (A, B, C and D) sent to our laboratory EU652326), which was related with the acquisition of (National Influenza Center) and tested between June resistance to adamantanes, in the same samples con- and November 2005 were identified as subtype H3N2 taining the E119V substitution (Figure 2B). The C50F by haemagglutinin (HA) and NA PCR as described substitution was found in M2 in the four samples. This

Figure 1. Course of treatment and sample collection in a 6-month-old infant with severe combined immunodeficiency disease with persistent A(H3N2) influenza infection

Sample A Sample B Sample C Sample D Samples E and F (T: +19) (T: +86) (T: +139) (T: +180) (T: +222) NA: E119V NA: E119V NA: E119V NA: E119V Influenza-A- ICT: M2: V27A ICT: M2: V27A M2: V27A M2: V27A negative ICT: Influenza- Influenza- Influenza- A-positive A-positive A-positive (T: -7) (T: +38)

Mar Apr May June July Aug Sept Oct Nov Dec

Admission (T: -9)

Peripheral blood transplantation (T: 0)

Oseltamivir + Oseltamivir + Oseltamivir + amantadine amantadine amantadine (T: -7 to +26) (T: +38 to +53) (T: +139 to +180)

The dotted lines indicate antiviral treatment course. ICT, immunochromatographic antigen detection; M2, M2 gene; NA, neuraminidase gene; T, days before (-) or after (+) transplantation.

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Figure 2. N2 and M2 amino acid alignment with the A(H3N2) influenza prototype and vaccine strains

A A 71 | #A/Panama/2007/1999 TTIEKEICPK LAEYRNWSKP QCKITGFAPF SKDNSIRLSA GGDIWVTREP YVSCDPDKCY QFALGQGTTL NNRHSNDTVH DRTPYRTLLM #A/California/7/2004 ...... D...... V...... #A/Wisconsin/67/2005 ...... N...... V...... #Sample A (EU652319) ...... D...... V...... V...... #Sample B (EU652320) ...... D...... V...... V...... #Sample C (EU652321) ...... D...... V...... V...... #Sample D (EU652322) ...... D...... V...... V......

#A/Panama/2007/1999 NELGVPFHLG TKQVCIAWSS SSCHDGKAWL HVCVTGHDEN ATASFIYDGR LVDSIGSWSK KILRTQESEC VCINGTCTVV MTDGSASGRA #A/California/7/2004 ...... D.K...... N...... V.... E...... K. #A/Wisconsin/67/2005 ...... D.K...... N...... V.... E...... K. #Sample A (EU652319) ...... D.K...... N...... V.... E...... K. #Sample B (EU652320) ...... D.K...... N...... V.... E...... A...... K. #Sample C (EU652321) ...... D.K...... N...... V.... E...... A...... K. #Sample D (EU652322) ...... D.K...... N...... V.... E...... A...... K.

308 | #A/Panama/2007/1999 DTKILFIEEG KIVHTSKLSG SAQHVEECSC YPRYPGVRCV CRDNWKGSNR PIVDINVK #A/California/7/2004 ...... T...... I. #A/Wisconsin/67/2005 ...... T...... L...... I. #Sample A (EU652319) ...... T...... I. #Sample B (EU652320) ...... T...... I. #Sample C (EU652321) ...... T...... I. #Sample D (EU652322) ...... T...... I.

B 23 69 | | #A/Panama/2007/1999 SDPLVVAASI IGILHLILWI LDRLFFKCIY RLFKHGLKRG PSTEGVP #A/California/7/2004 ...... V...... #A/Wisconsin/67/2005 ...... N...... V...... #Sample A (EU652323) ....A...... FV...... S...... #Sample B (EU652324) ....A...... FV...... S...... #Sample C (EU652326) ....A...... FV...... S...... #Sample D (EU652325) ....A...... FV...... S......

(A) Alignment of N2 amino acid sequences with the A/Panama/2007/1999 prototype strain, the vaccine strains used in the 2004–2005 (A/California/7/2004) and 2005– 2006 (A/Wisconsin/67/2005) influenza seasons, and the samples A, B, C and D. The GenBank numbers are presented in parentheses. Dots indicate identity. Bold type amino acids highlight the mutations conferring resistance to neuraminidase inhibitors identified in the viruses obtained from the patient.(B) Alignment of M2 amino acid sequences with the A/Panama/2007/1999 prototype strain, the vaccine strains used in the 2004–2005 (A/California/7/2004) and 2005–2006 (A/Wisconsin/67/2005) influenza seasons, and the samples A, B, C and D. The GenBank numbers are presented in parentheses. Dots indicate identity. Bold type amino acids highlight the mutations conferring resistance to adamantanes.

cysteine in position 50 of wild-type influenza viruses the dual mutation pattern E119V in N2 and V27A in is post-translationally modified by palmitoylation M2. Most of the reported cases of prolonged influenza through a thioether linkage [10]. The mutated residues A virus shedding in immunosuppressed patients describe associated with resistance in NA and M2 did not show resistance to neuraminidase inhibitors (NAI), adaman- evidence of mixed viral populations. tanes or both [1,5,12]. The E119V mutation detected in The amino acid changes found in the HA1 domain the immunocompromised infant with prolonged shed- (Figure 3) that affected epitope changes in antigenic ding described here confers oseltamivir resistance but sites were S189K/R (site B), N216D and N246S (site D; does not affect susceptibility to [13]. Most GenBank accession numbers EU982291, EU982292, of the multidrug-resistant influenza A(H3N2) viruses EU982293 and EU982294). The N246S mutation also that have been genetically characterized contain either leads to the loss of an N-linked potential glycosyla- the E119V or R292K mutation in the NA gene and the tion site [11]. No mutations were found in the receptor S31N mutation in the M2 segment [1,5]. After amanta- binding sites. dine treatment, the V27A substitution was the most fre- quent resistance mutation during 1999–2001 in H1N1 Discussion subtype strains [14]; current data for the 2004–2005 influenza seasons indicate that 1.6% of the H3N2 sub- In this report, we describe the first documented case of type viruses contained this mutation [15]. The major- multidrug resistance in an influenza A(H3N2) virus with ity of A(H3N2) viruses from 2005–2006, including the

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Figure 3. H3 amino acid alignment with the A(H3N2) influenza prototype and vaccine strains

36 | #A/Panama/2007/1999 VTNATELVQS SSTGRICDSP HQILDGENCT LIDALLGDPH CDGFQNKEWD LFVERSKAYS NCYPYDVPDY ASLRSLVASS GTLEFNNESF #A/California/7/2004 ...... G...... Q ...... K...... #A/Wisconsin/67/2005 ...... G...... Q ...... K...... D... ##Sample A (EU982291) ...... G...... Q ...... K...... ##Sample B (EU982292) ...... G...... Q ...... K...... ##Sample C (EU982293) ...... G...... Q ...... K...... ##Sample D (EU982294) ...... G...G...... Q ...... K......

#A/Panama/2007/1999 NWTGVAQNGT SSACKRRSNK SFFSRLNWLH QLKYKYPALN VTMPNNEKFD KLYIWGVHHP STDSDQISIY AQASGRVTVS TKRSQQTVIP #A/California/7/2004 .....T...... S...... N ...... T H..F...... G.NN....L. T.....I...... #A/Wisconsin/67/2005 .....T...... N ...... T H..F...... G..N...FLH ...... I...... ##Sample A (EU982291) .....T...... N ...... T H..F...... G..K....L...... I...... ##Sample B (EU982292) .....T...... N ...... T H..F...... G..K....L...... I...... ##Sample C (EU982293) .....T...... N ...... T H..F...... G..K....L...... I...... ##Sample D (EU982294) .....T...... N ...... T H.RF...... G..R....L. T.....I......

#A/Panama/2007/1999 NIGSSPWVRG VSSRISIYWT IVKPGDILLI NSTGNLIAPR GYFKIRSGKS SIMRSDAPIG KCNSECITPN GSIPNDKPFQ NVNRITYGAC #A/California/7/2004 ....R.R..D IP...... #A/Wisconsin/67/2005 ....R.RI.N IP...... ##Sample A (EU982291) D...R.R..D IP...... S...... ##Sample B (EU982292) D...R.R..D IP...... S...... ##Sample C (EU982293) D...R.R..D IP...... S...... ##Sample D (EU982294) D...R.R..D IP...... S...... S......

329 | #A/Panama/2007/1999 PRYVKQNTLK LATGMRNVPE KQTR #A/California/7/2004 ...... #A/Wisconsin/67/2005 ...... ##Sample A (EU982291) ...... ##Sample B (EU982292) ...... ##Sample C (EU982293) ...... ##Sample D (EU982294) ......

Alignment of H3 amino acid sequences corresponding to the HA1 domain with the A/Panama/2007/1999 prototype strain, the vaccine strains used in the 2004–2005 (A/California/7/2004) and 2005–2006 (A/Wisconsin/67/2005) influenza seasons, and the samples A, B, C and D. The The GenBank numbers are presented in parentheses. Dots indicate identity. Bold type amino acids highlight the mutations in antigenic sites detected in the viruses obtained from the patient.

vaccine strain A/Wisconsin/67/2005 (Figure 2B) and the is near the HA receptor binding site D190 and might strains from the more recent influenza seasons, contain have contributed to the persistence of the E119V var- the S31N mutation in M2, which confers resistance to iant, partially compensating for potentially reduced adamantanes. The absence of this mutation in the virus NA activity produced by decreased HA affinity [4]. of this reported case indicates that the virus is geneti- The loss of potential N246 glycosylation in the anti- cally linked to the influenza viruses circulating during genic site B of the HA might be an evolutionary adap- the 2004–2005 influenza season. tation by the virus in a patient with a deficient anti- The C50 position on the M2 segment is highly con- body response. served among human H3N2 subtype viruses, but not in Despite the combination therapy applied in this human H1N1 subtype viruses [16]. Interestingly, the loss patient, viral clearance was documented in two sam- of this palmitoylation site (C50F) was observed in the ples after 42 days of therapy interruption and appeared H3N2 subtype viruses in this reported case. Neverthe- to have been associated with the immunological recon- less, the post-translational modifications did not affect stitution. Oseltamivir and amantadine are not formally M2 ion channel activity [10] or viral replication [16]. recommended in children <1 year old; however, severe Continuous E119V virus shedding was confirmed immunosuppression is a life-threatening comorbid con- for >5 months, both during and after the discontinu- dition, and antiviral treatment of influenza infection ation of oseltamivir therapy. Viral shedding persisted would be advisable in this circumstance. At present, after 89 days of cumulative oseltamivir therapy (Fig- the US Food and Drug Administration authorizes ure 1, sample D). As previously described [1], the treatment with oseltamivir for the A(H1N1) presence of the NA mutation, even in the absence of infection in children <1 year old (dosing recommenda- antiviral pressure (Figure 1, samples B and C), might tion 3 mg/kg twice daily). Currently, the use of aman- indicate that the E119V mutation is stable in untreated tadine is not recommended for children <1 year old immunocompromised patients. The S189K/R change (5 mg/kg/day twice daily in children ≥1 year old). It

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3. Centers for Disease Control and Prevention. Oseltamivir- is possible that the prolonged subtherapeutic doses of resistant novel influenza A (H1N1) virus infection in two both antivirals might have contributed to the devel- immunosuppressed patients – Seattle, Washington, 2009. opment of resistance and the lack of viral clearance. MMWR Morb Mortal Wkly Rep 2009; 58:893–896. 4. Gooskens J, Jonges M, Claas EC, Meijer A, Kroes AC. The family of the infant did not receive antiviral treat- Prolonged influenza virus infection during lymphocytopenia ment and, although it is possible that the patient was and frequent detection of drug-resistant viruses. J Infect Dis infected with an already amantadine- or oseltamivir-re- 2009; 199:1435–1441. sistant virus, no isolates before initiation of drug treat- 5. Ison MG, Gubareva LV, Atmar RL, Treanor J, Hayden FG. Recovery of drug-resistant influenza virus from ment were available for analysis. In addition, patients immunocompromised patients: a case series. J Infect Dis infected with A(H3N2) viruses containing the E119V 2006; 193:760–764. mutation should be treated with inhaled zanamivir or 6. Weinstock DM, Gubareva LV, Zuccotti G. Prolonged shedding of multidrug-resistant influenza A virus in intravenous because this mutation retains an immunocompromised patient. N Engl J Med 2003; susceptibility to both. 348:867–868. In conclusion, because pandemic A(H1N1) and 7. Coiras MT, Aguilar JC, Garcia ML, Casas I, Perez-Brena P. Simultaneous detection of fourteen respiratory viruses in contemporary A(H3N2) viruses are resistant to ada- clinical specimens by two multiplex reverse transcription mantanes and the wide use of NAI can lead to the nested-PCR assays. J Med Virol 2004; 72:484–495. emergence of resistant viruses, antiviral resistance 8. Lopez-Huertas MR, Casas I, Acosta-Herrera B, Garcia ML, Coiras MT, Perez-Brena P. Two RT-PCR based assays testing (particularly NAI) is advisable in immunosup- to detect human metapneumovirus in nasopharyngeal pressed and severely ill patients, particularly when aspirates. J Virol Methods 2005; 129:1–7. positive results on culture and PCR assays suggest a 9. Ruiz-Carrascoso G, Casas I, Pozo F, Pérez-González C, Reina J, Pérez-Breña P. Development and implementation persistent infection after or during the course of anti- of influenza A virus subtyping and detection of genotypic viral treatment. resistance to neuraminidase inhibitors. J Med Virol 2010; 82:843–853. 10. Holsinger LJ, Shaughnessy MA, Micko A, Pinto LH, Acknowledgements Lamb RA. Analysis of the posttranslational modifications of the influenza virus M2 protein. J Virol 1995; 69:1219–1225. This work was supported by the Dirección General 11. Matrosovich M, Gao P, Kawaoka Y. Molecular mechanisms de Salud Pública of the Spanish Ministerio de Sani- of serum resistance of human influenza H3N2 virus and their involvement in virus adaptation in a new host. J Virol dad (grant number DGVI-1429/05-8-B). The authors 1998; 72:6373–6380. express gratitude to Manuela López-Valero, Mónica 12. Boivin G, Goyette N, Bernatchez H. Prolonged Sánchez, Noelia Reyes and Ana María Calderón for of amantadine-resistant influenza a virus quasi species after cessation of antiviral therapy in an immunocompromised their technical assistance. patient. Clin Infect Dis 2002; 34:E23–E25. 13. Gubareva LV. Molecular mechanisms of influenza virus Disclosure statement resistance to neuraminidase inhibitors. Virus Res 2004; 103:199–203. 14. Saito R, Sakai T, Sato I, et al. Frequency of amantadine- The authors declare no competing interests. resistant influenza A viruses during two seasons featuring cocirculation of H1N1 and H3N2. J Clin Microbiol 2003; References 41:2164–2165. 15. Deyde VM, Xu X, Bright RA, et al. Surveillance of 1. Baz M, Abed Y, McDonald J, Boivin G. Characterization of resistance to adamantanes among influenza A(H3N2) and multidrug-resistant influenza A/H3N2 viruses shed during A(H1N1) viruses isolated worldwide. J Infect Dis 2007; 1 year by an immunocompromised child. Clin Infect Dis 196:249–257. 2006; 43:1555–1561. 16. Grantham ML, Wu WH, Lalime EN, Lorenzo ME, 2. Rocha E, Cox NJ, Black RA, Harmon MW, Harrison CJ, Klein SL, Pekosz A. Palmitoylation of the influenza A virus Kendal AP. Antigenic and genetic variation in influenza A M2 protein is not required for virus replication in vitro but (H1N1) virus isolates recovered from a persistently infected contributes to virus virulence. J Virol 2009; 83:8655–8661. immunodeficient child. J Virol 1991; 65:2340–2350.

Accepted for publication 21 April 2010

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