Antiviral Therapy 2015; 20:97–100 (doi: 10.3851/IMP2811)
Case report Primarily oseltamivir-resistant influenza A (H1N1pdm09) virus evolving into a multidrugresistant virus carrying H275Y and I223R neuraminidase substitutions Sebastian Grund1*, Charikleia Gkioule1, Tahani Termos2, Nico Pfeifer3, Guido Kobbe4, Jens Verheyen5†, Ortwin Adams1† Institute for Virology, Medical Faculty, University of Düsseldorf, Düsseldorf, Germany Institute of Virology, University of Cologne, Cologne, Germany 3 Computational Biology and Applied Algorithmics, Max-Planck-Institute for Informatics, Saarbrücken, Germany 4 Clinic for Hematology, Oncology and Clinical Immunology, University of Düsseldorf, Düsseldorf, Germany 5 Institute of Virology, University Clinics, University Duisburg/Essen, Essen, Germany 1 2
*Corresponding author e-mail: [email protected] Contributed equally to this manuscript
†
Antiviral susceptibility testing and reporting of viruses carrying amino acid substitutions conferring antiviral drug resistance is essential to assess the spread and clinical impact of these viruses. Here, we report on a patient who was infected with a primarily oseltamivir-resistant influenza A (H1N1pdm09) virus following allogeneic stem cell transplantation and rituximab treatment. Under prolonged
virus replication and zanamivir therapy the neuraminidase amino acid substitutions H275Y and I223R were detected conferring high-level resistance to oseltamivir and crossresistance to zanamivir. The emergence of these amino acid changes has been reported rarely worldwide and has been associated with fatal clinical outcomes. The patient survived the influenza infection after 170 days of follow-up.
Introduction The incidence rate of the H275Y (N1 numbering) neuraminidase substitution, conferring resistance to the antiviral neuraminidase inhibitor oseltamivir, has been reported to be 2–3% in influenza A (H1N1pdm09) viruses worldwide based on a recent meta-analysis [1]. The prevalence of influenza isolates harbouring this amino acid change is increased in patients requiring intensive care treatment (39%) [2]. This is due to immunocompromising medical conditions that are present frequently in these patients leading to prolonged virus shedding and to the broad-scale use of oseltamivir [3,4]. The dual amino acid substitution H275Y/I223R in the neuraminidase gene conferring highlevel resistance to oseltamivir and cross-resistance to zanamivir has been described rarely in humans so far and has been associated with fatal clinical outcomes [5–7].
Case presentation A myelodysplastic syndrome was diagnosed in a 55-year-old female patient with an underlying chronic ©2015 International Medical Press 1359-6535 (print) 2040-2058 (online)
AVT-14-CR-3255_Grund.indd 97
obstructive pulmonary disease (COPD) in April 2010. She received an allogeneic stem cell transplant from an unrelated donor following myeloablative conditioning on 15 September 2010 (-132 days prior to the first positive influenza A virus PCR; Figure 1). Following regular haematopoietic reconstitution she developed EBVrelated post-transplant high-grade B-cell lymphoma (EBV+PTLD), which was treated with a single dose of rituximab (1×375 mg/m2) on 14 December 2010 (day -43 prior to the first positive influenza A virus PCR). After treatment she had severe immunodeficiency reflected by peripheral blood lymphopenia. In addition she received 10 mg prednisolone once daily for the treatment of acute graft-versus-host disease grade II. On 25 January 2011 (day 0) she presented with fever and respiratory symptoms and the influenza A (H1N1pdm09) virus was detected in her sputum by PCR (Figure 1). On 2 February (day 8) oseltamivir treatment was initiated with a reduced dosage of 75 mg once daily due to an underlying nephrological 97
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Figure 1. Clinical course and virological follow-up of the patient 0 Oseltamivir Zanamivir
Influenza A/H1N1 PCR (Ct)
10
H275Y /I223R
H275Y
H275Y
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40 2010
2011 Dec
Jan
Feb
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Aug
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8 22/23
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os
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-43
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Days after first positive influenza PCR
-132
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Mar
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Viral load (black and grey dots), antiviral therapy and neuraminidase inhibitor resistance mutations in a patient suffering from a myelodysplastic syndrome who was infected with a primarily oseltamivir-resistant (H275Y) influenza A (H1N1pdm09) virus following allogeneic stem cell transplantation (SCT) and rituximab treatment. Under zanamivir inhalation the virus evolved transiently into a virus carrying the dual amino acid substitution H275Y/I223R (grey dots) that was reported to be highlevel resistant to oseltamivir with cross-resistance to zanamivir. The virus reverted from I223R to I223 (wild-type) while the H275Y mutation persisted until the end of follow-up. Finally the patient recovered from the influenza infection and the underlying myelodysplastic syndrome. The resistance mutations are shown in the grey bar. Ct, PCR threshold cycle of detection; IVIG, intravenous immunoglobulin.
disease (glomerular filtration rate 29 ml/min) and antibiotics were given additionally to avoid secondary bacterial infection. No other bacterial or fungal infections were detected. Symptoms worsened despite treatment and she was admitted to hospital on 16 February (day 22) because of dyspnea requiring supplemental oxygen therapy. As of the 17 February (day 23) inhalative zanamivir (10 mg twice daily) was given additionally. A CT scan on 22 February (day 28) showed diffuse pneumonia but symptoms improved in the further course. Oseltamivir was ceased on 2 March (day 36, after 28 days of therapy) and the patient was discharged from hospital on 10 March (day 44). Inhalation with zanamivir was continued until the end of April 2011 (until day 95, for a total of 72 days). 98
AVT-14-CR-3255_Grund.indd 98
Despite clinical improvement repeated PCR testing for influenza A (H1N1pdm09) virus remained positive until 14 June 2011 (day 140). On that day, the patient received intravenous immunoglobulin (IVIG; 20 g Gammunex™; Grifols, Frankfurt, Germany). Influenza A virus RNA became undetectable on 28 June 2011 (day 154) and remained negative during virological follow-up (until day 170). Respiratory symptoms further decreased but due to pre-existing COPD the patient continued to suffer from chronic airway obstruction and recurrent infectious exacerbations. A CT scan from February 2012 showed chronic emphysema and residues of previous pulmonary infection. She remained in complete remission of the myelodysplastic syndrome and the EBV+PTLD at the end of follow-up (539 days after transplantation). ©2015 International Medical Press
20/02/2015 10:08:01
Multidrug-resistant influenza virus A/H1N1
Virological findings Retrospective Sanger sequencing of the neuraminidase genes in the frozen (-20°C) clinical samples (sputum, nasopharyngeal swabs and pharyngeal lavages) revealed that the patient was infected originally with a primarily oseltamivir-resistant influenza virus strain carrying the H275Y amino acid substitution (Figure 1) [1]. Consistently with this finding, a clinical improvement of the patient was only achieved after zanamivir treatment was initiated as this mutation does not confer crossresistance to this drug [2]. However, in the sputum sample obtained on 29 March 2011 (day 63) the additional amino acid substitution I223R was detected. Influenza viruses carrying this dual amino acid change (H275Y/ I223R) have been found rarely in humans so far [5–7] and they have been shown to demonstrate high-level resistance to oseltamivir and cross-resistance to zanamivir [2,8]. The dual mutant virus population was detected again in a pharyngeal lavage on 12 April 2011 (day 77) while sequencing of the sample from 5 April (day 70) was unsuccessful due to low-level viral load (Figure 1). However, a reversion of H275Y/I223R to H275Y was found in the follow-up respiratory samples with the H275Y mutation persisting until the end of follow-up. No other amino acid changes that have been described to confer resistance to antiviral drugs were detected in the neuraminidase protein [9].
Discussion The selection of the H275Y amino acid substitution, conferring resistance to oseltamivir, has been described most frequently in immunocompromised patients under antiviral treatment [3,4]. However, to the best of our knowledge, the present report is the first of a primarily oseltamivir-resistant influenza A (H1N1pdm09) virus evolving into a virus population bearing the dual amino acid change H275Y/I223R. The original source of infection could not be detected and the patient had not received oseltamivir prophylaxis or therapy prior to the infection. Furthermore, it is the first description of a virus population carrying the dual amino acid substitutions (H275Y/I223R) reverting to the merely oseltamivir-resistant virus H275Y. While the patient survived in the present case, fatal outcomes of infections with this virus have been reported by Nguyen et al. [6] in a 14-year-old girl with systemic lupus erythematosus, by van der Vries et al. [7] in a 5-year-old boy with acute lymphoblastic leukaemia and by LeGoff et al. [5] in a 24-year-old male patient suffering from acute myelogenous leukaemia. One important question to be discussed is if the patient survived the influenza infection because the virus population reverted from H275Y/I223R to Antiviral Therapy 20.1
AVT-14-CR-3255_Grund.indd 99
H275Y. However, firstly it is not possible to distinguish between the effect of antiviral therapy, the treatment of the underlying myelodysplastic syndrome/EBV+PTLD and the decreasing immunocompromising effect of rituximab over time. Secondly it must be called into question if the resistance pattern of the virus population had a major impact on the clinical outcome. As shown in Figure 1 the influenza viral load decreased constantly and the patient improved clinically under zanamivir treatment. Furthermore, the dual amino acid substitution H275Y/I223R was detected only for a short time before the virus population reverted to H275Y. This might be due to a reduced fitness of the virus based on these amino acid changes. Contradictory results have been obtained on the replication capacity of drug-resistant influenza viruses: Pizzorno et al. [8] found an equivalent replication fitness of a virus carrying the H275Y/I223R substitutions compared to wild-type while LeGoff et al. [5] reported on a reduced replication capacity of this virus in MDCK cells. By contrast, it has been shown that influenza viruses carrying merely the H275Y substitution only have a minor reduction in replication fitness compared with wild-type viruses [10]. Therefore, similar to the case presented here, it has been reported by others that the H275Y amino acid change can persist even after the cessation of oseltamivir therapy [11]. The contribution of the IVIG treatment to the clinical outcome is unclear. The impact of IVIG treatment in patients with severe influenza disease has not been studied sufficiently as many of these patients require this therapy anyway due to underlying diseases like autoimmunological disorders or haematological malignancies. However, a beneficial effect on the reduction of mortality was shown for treatment with convalescent plasma preparations that contained neutralizing antibody titres ≥1:160 [12]. From a clinical point of view it would have been desirable to detect the oseltamivir resistance at an early stage of infection and to treat the patient with zanamivir prior to the deterioration of symptoms. However, resistance analysis is not available generally in clinical routine diagnostics so far. As oseltamivir resistance is found more frequently in immunocompromised patients than in the general population, early resistance testing should be considered in these patients, especially under antiviral therapy [2]. One limitation of the case report presented here is that no phenotypic resistance testing was performed as the virus did not grow in MDCK cells after freezing of the clinical samples at -20°C. Another limitation is that genotypic resistance analysis was performed by Sanger sequencing, which is a qualitative assay; therefore, the resistance pattern of the virus population but not the drug resistance of individual virus clones was analysed. 99
20/02/2015 10:08:01
S Grund et al.
Taken together, an infection with a primarily oseltamivir-resistant influenza A (H1N1pdm09) virus was detected in a patient with an underlying myelodysplastic syndrome following allogeneic stem cell transplantation and rituximab treatment for EBV+PTLD. Under zanamivir therapy the virus evolved into a virus population carrying the dual amino acid substitution H275Y/I223R that confers high-level resistance to oseltamivir and crossresistance to zanamivir and has been reported rarely worldwide so far. In the further course the virus reverted to a merely oseltamivir-resistant virus strain (H275Y). The patient survived the influenza infection after 170 days of follow-up. Antiviral resistance testing and reporting of viruses carrying resistance mutations is essential to address the spread and prevalence of these viruses in future.
Acknowledgements We wish to thank the technical staff of the Institutes for Virology of the Universities of Düssedorf and Cologne for their excellent technical assistance.
Disclosure statement The authors declare no competing interests.
References 1. 2.
Thorlund K, Awad T, Boivin G, et al. Systematic review of influenza resistance to the neuraminidase inhibitors. BMC Infect Dis 2011; 11:134. Nguyen HT, Trujillo AA, Sheu TG, et al. Analysis of influenza viruses from patients clinically suspected of infection with an oseltamivir resistant virus during the 2009 pandemic in the United States. Antiviral Res 2012; 93:381–386.
3.
Graitcer SB, Gubareva L, Kamimoto L, et al. Characteristics of patients with oseltamivir-resistant pandemic (H1N1) 2009, United States. Emerg Infect Dis 2011; 17:255–257. 4. Mehta T, McGrath E, Bheemreddy S, et al. Detection of oseltamivir resistance during treatment of 2009 H1N1 influenza virus infection in immunocompromised patients: utility of cycle threshold values of qualitative real-time reverse transcriptase PCR. J Clin Microbiol 2010; 48:4326–4328. 5. LeGoff J, Rousset D, Abou-Jaoude G, 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:e37095. 6. Nguyen HT, Fry AM, Loveless PA, et al. Recovery of a multidrug-resistant strain of pandemic influenza A 2009 (H1N1) virus carrying a dual H275Y/I223R mutation from a child after prolonged treatment with oseltamivir. Clin Infect Dis 2010; 51:983–984. 7. van der Vries E, Stelma FF, Boucher CA. Emergence of a multidrug-resistant pandemic influenza A (H1N1) virus. N Engl J Med 2010; 363:1381–1382. 8. Pizzorno A, Abed Y, Bouhy X, 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; 56:1208–1214. 9. Deyde VM, Sheu TG, Trujillo AA, et al. Detection of molecular markers of drug resistance in 2009 pandemic influenza A (H1N1) viruses by pyrosequencing. Antimicrob Agents Chemother 2010; 54:1102–1110. 10. Brookes DW, Miah S, Lackenby A, et al. Pandemic H1N1 2009 influenza virus with the H275Y oseltamivir resistance neuraminidase mutation shows a small compromise in enzyme activity and viral fitness. J Antimicrob Chemother 2011; 66:466–470. 11. Tramontana AR, George B, Hurt AC, et al. Oseltamivir resistance in adult oncology and hematology patients infected with pandemic (H1N1) 2009 virus, Australia. Emerg Infect Dis 2010; 16:1068–1075. 12. Hung IF, To KK, Lee CK, et al. Convalescent plasma treatment reduced mortality in patients with severe pandemic influenza A (H1N1) 2009 virus infection. Clin Infect Dis 2011; 52:447–456.
Accepted 2 June 2014; published online 18 June 2014
100
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©2015 International Medical Press
20/02/2015 10:08:01
Case report Primarily oseltamivir-resistant influenza A (H1N1pdm09) virus evolving into a multidrugresistant virus carrying H275Y and I223R neuraminidase substitutions Sebastian Grund1*, Charikleia Gkioule1, Tahani Termos2, Nico Pfeifer3, Guido Kobbe4, Jens Verheyen5†, Ortwin Adams1† Institute for Virology, Medical Faculty, University of Düsseldorf, Düsseldorf, Germany Institute of Virology, University of Cologne, Cologne, Germany 3 Computational Biology and Applied Algorithmics, Max-Planck-Institute for Informatics, Saarbrücken, Germany 4 Clinic for Hematology, Oncology and Clinical Immunology, University of Düsseldorf, Düsseldorf, Germany 5 Institute of Virology, University Clinics, University Duisburg/Essen, Essen, Germany 1 2
*Corresponding author e-mail: [email protected] Contributed equally to this manuscript
†
Antiviral susceptibility testing and reporting of viruses carrying amino acid substitutions conferring antiviral drug resistance is essential to assess the spread and clinical impact of these viruses. Here, we report on a patient who was infected with a primarily oseltamivir-resistant influenza A (H1N1pdm09) virus following allogeneic stem cell transplantation and rituximab treatment. Under prolonged
virus replication and zanamivir therapy the neuraminidase amino acid substitutions H275Y and I223R were detected conferring high-level resistance to oseltamivir and crossresistance to zanamivir. The emergence of these amino acid changes has been reported rarely worldwide and has been associated with fatal clinical outcomes. The patient survived the influenza infection after 170 days of follow-up.
Introduction The incidence rate of the H275Y (N1 numbering) neuraminidase substitution, conferring resistance to the antiviral neuraminidase inhibitor oseltamivir, has been reported to be 2–3% in influenza A (H1N1pdm09) viruses worldwide based on a recent meta-analysis [1]. The prevalence of influenza isolates harbouring this amino acid change is increased in patients requiring intensive care treatment (39%) [2]. This is due to immunocompromising medical conditions that are present frequently in these patients leading to prolonged virus shedding and to the broad-scale use of oseltamivir [3,4]. The dual amino acid substitution H275Y/I223R in the neuraminidase gene conferring highlevel resistance to oseltamivir and cross-resistance to zanamivir has been described rarely in humans so far and has been associated with fatal clinical outcomes [5–7].
Case presentation A myelodysplastic syndrome was diagnosed in a 55-year-old female patient with an underlying chronic ©2015 International Medical Press 1359-6535 (print) 2040-2058 (online)
AVT-14-CR-3255_Grund.indd 97
obstructive pulmonary disease (COPD) in April 2010. She received an allogeneic stem cell transplant from an unrelated donor following myeloablative conditioning on 15 September 2010 (-132 days prior to the first positive influenza A virus PCR; Figure 1). Following regular haematopoietic reconstitution she developed EBVrelated post-transplant high-grade B-cell lymphoma (EBV+PTLD), which was treated with a single dose of rituximab (1×375 mg/m2) on 14 December 2010 (day -43 prior to the first positive influenza A virus PCR). After treatment she had severe immunodeficiency reflected by peripheral blood lymphopenia. In addition she received 10 mg prednisolone once daily for the treatment of acute graft-versus-host disease grade II. On 25 January 2011 (day 0) she presented with fever and respiratory symptoms and the influenza A (H1N1pdm09) virus was detected in her sputum by PCR (Figure 1). On 2 February (day 8) oseltamivir treatment was initiated with a reduced dosage of 75 mg once daily due to an underlying nephrological 97
20/02/2015 10:08:00
S Grund et al.
Figure 1. Clinical course and virological follow-up of the patient 0 Oseltamivir Zanamivir
Influenza A/H1N1 PCR (Ct)
10
H275Y /I223R
H275Y
H275Y
20
30
40 2010
2011 Dec
Jan
Feb
Jun
H
Jul
Aug
140 154 170
t
95
tre
at
ce
m
as
en
ed
63 70 77
IV
IG
ir
36 44
O s ta elt l a am d O se Za mi ivir lta na ss m m io Di ivir ivi n sc c r ha ea rg se ho e fr d sp om ita l
8 22/23
pi
m xi tu Ri
0
os
ab
T
-43
SC ic ne ge lo
May
Days after first positive influenza PCR
-132
Al
Apr
iv
Days prior to infection
Mar
m
Nov
na
Oct
Za
Sep
Viral load (black and grey dots), antiviral therapy and neuraminidase inhibitor resistance mutations in a patient suffering from a myelodysplastic syndrome who was infected with a primarily oseltamivir-resistant (H275Y) influenza A (H1N1pdm09) virus following allogeneic stem cell transplantation (SCT) and rituximab treatment. Under zanamivir inhalation the virus evolved transiently into a virus carrying the dual amino acid substitution H275Y/I223R (grey dots) that was reported to be highlevel resistant to oseltamivir with cross-resistance to zanamivir. The virus reverted from I223R to I223 (wild-type) while the H275Y mutation persisted until the end of follow-up. Finally the patient recovered from the influenza infection and the underlying myelodysplastic syndrome. The resistance mutations are shown in the grey bar. Ct, PCR threshold cycle of detection; IVIG, intravenous immunoglobulin.
disease (glomerular filtration rate 29 ml/min) and antibiotics were given additionally to avoid secondary bacterial infection. No other bacterial or fungal infections were detected. Symptoms worsened despite treatment and she was admitted to hospital on 16 February (day 22) because of dyspnea requiring supplemental oxygen therapy. As of the 17 February (day 23) inhalative zanamivir (10 mg twice daily) was given additionally. A CT scan on 22 February (day 28) showed diffuse pneumonia but symptoms improved in the further course. Oseltamivir was ceased on 2 March (day 36, after 28 days of therapy) and the patient was discharged from hospital on 10 March (day 44). Inhalation with zanamivir was continued until the end of April 2011 (until day 95, for a total of 72 days). 98
AVT-14-CR-3255_Grund.indd 98
Despite clinical improvement repeated PCR testing for influenza A (H1N1pdm09) virus remained positive until 14 June 2011 (day 140). On that day, the patient received intravenous immunoglobulin (IVIG; 20 g Gammunex™; Grifols, Frankfurt, Germany). Influenza A virus RNA became undetectable on 28 June 2011 (day 154) and remained negative during virological follow-up (until day 170). Respiratory symptoms further decreased but due to pre-existing COPD the patient continued to suffer from chronic airway obstruction and recurrent infectious exacerbations. A CT scan from February 2012 showed chronic emphysema and residues of previous pulmonary infection. She remained in complete remission of the myelodysplastic syndrome and the EBV+PTLD at the end of follow-up (539 days after transplantation). ©2015 International Medical Press
20/02/2015 10:08:01
Multidrug-resistant influenza virus A/H1N1
Virological findings Retrospective Sanger sequencing of the neuraminidase genes in the frozen (-20°C) clinical samples (sputum, nasopharyngeal swabs and pharyngeal lavages) revealed that the patient was infected originally with a primarily oseltamivir-resistant influenza virus strain carrying the H275Y amino acid substitution (Figure 1) [1]. Consistently with this finding, a clinical improvement of the patient was only achieved after zanamivir treatment was initiated as this mutation does not confer crossresistance to this drug [2]. However, in the sputum sample obtained on 29 March 2011 (day 63) the additional amino acid substitution I223R was detected. Influenza viruses carrying this dual amino acid change (H275Y/ I223R) have been found rarely in humans so far [5–7] and they have been shown to demonstrate high-level resistance to oseltamivir and cross-resistance to zanamivir [2,8]. The dual mutant virus population was detected again in a pharyngeal lavage on 12 April 2011 (day 77) while sequencing of the sample from 5 April (day 70) was unsuccessful due to low-level viral load (Figure 1). However, a reversion of H275Y/I223R to H275Y was found in the follow-up respiratory samples with the H275Y mutation persisting until the end of follow-up. No other amino acid changes that have been described to confer resistance to antiviral drugs were detected in the neuraminidase protein [9].
Discussion The selection of the H275Y amino acid substitution, conferring resistance to oseltamivir, has been described most frequently in immunocompromised patients under antiviral treatment [3,4]. However, to the best of our knowledge, the present report is the first of a primarily oseltamivir-resistant influenza A (H1N1pdm09) virus evolving into a virus population bearing the dual amino acid change H275Y/I223R. The original source of infection could not be detected and the patient had not received oseltamivir prophylaxis or therapy prior to the infection. Furthermore, it is the first description of a virus population carrying the dual amino acid substitutions (H275Y/I223R) reverting to the merely oseltamivir-resistant virus H275Y. While the patient survived in the present case, fatal outcomes of infections with this virus have been reported by Nguyen et al. [6] in a 14-year-old girl with systemic lupus erythematosus, by van der Vries et al. [7] in a 5-year-old boy with acute lymphoblastic leukaemia and by LeGoff et al. [5] in a 24-year-old male patient suffering from acute myelogenous leukaemia. One important question to be discussed is if the patient survived the influenza infection because the virus population reverted from H275Y/I223R to Antiviral Therapy 20.1
AVT-14-CR-3255_Grund.indd 99
H275Y. However, firstly it is not possible to distinguish between the effect of antiviral therapy, the treatment of the underlying myelodysplastic syndrome/EBV+PTLD and the decreasing immunocompromising effect of rituximab over time. Secondly it must be called into question if the resistance pattern of the virus population had a major impact on the clinical outcome. As shown in Figure 1 the influenza viral load decreased constantly and the patient improved clinically under zanamivir treatment. Furthermore, the dual amino acid substitution H275Y/I223R was detected only for a short time before the virus population reverted to H275Y. This might be due to a reduced fitness of the virus based on these amino acid changes. Contradictory results have been obtained on the replication capacity of drug-resistant influenza viruses: Pizzorno et al. [8] found an equivalent replication fitness of a virus carrying the H275Y/I223R substitutions compared to wild-type while LeGoff et al. [5] reported on a reduced replication capacity of this virus in MDCK cells. By contrast, it has been shown that influenza viruses carrying merely the H275Y substitution only have a minor reduction in replication fitness compared with wild-type viruses [10]. Therefore, similar to the case presented here, it has been reported by others that the H275Y amino acid change can persist even after the cessation of oseltamivir therapy [11]. The contribution of the IVIG treatment to the clinical outcome is unclear. The impact of IVIG treatment in patients with severe influenza disease has not been studied sufficiently as many of these patients require this therapy anyway due to underlying diseases like autoimmunological disorders or haematological malignancies. However, a beneficial effect on the reduction of mortality was shown for treatment with convalescent plasma preparations that contained neutralizing antibody titres ≥1:160 [12]. From a clinical point of view it would have been desirable to detect the oseltamivir resistance at an early stage of infection and to treat the patient with zanamivir prior to the deterioration of symptoms. However, resistance analysis is not available generally in clinical routine diagnostics so far. As oseltamivir resistance is found more frequently in immunocompromised patients than in the general population, early resistance testing should be considered in these patients, especially under antiviral therapy [2]. One limitation of the case report presented here is that no phenotypic resistance testing was performed as the virus did not grow in MDCK cells after freezing of the clinical samples at -20°C. Another limitation is that genotypic resistance analysis was performed by Sanger sequencing, which is a qualitative assay; therefore, the resistance pattern of the virus population but not the drug resistance of individual virus clones was analysed. 99
20/02/2015 10:08:01
S Grund et al.
Taken together, an infection with a primarily oseltamivir-resistant influenza A (H1N1pdm09) virus was detected in a patient with an underlying myelodysplastic syndrome following allogeneic stem cell transplantation and rituximab treatment for EBV+PTLD. Under zanamivir therapy the virus evolved into a virus population carrying the dual amino acid substitution H275Y/I223R that confers high-level resistance to oseltamivir and crossresistance to zanamivir and has been reported rarely worldwide so far. In the further course the virus reverted to a merely oseltamivir-resistant virus strain (H275Y). The patient survived the influenza infection after 170 days of follow-up. Antiviral resistance testing and reporting of viruses carrying resistance mutations is essential to address the spread and prevalence of these viruses in future.
Acknowledgements We wish to thank the technical staff of the Institutes for Virology of the Universities of Düssedorf and Cologne for their excellent technical assistance.
Disclosure statement The authors declare no competing interests.
References 1. 2.
Thorlund K, Awad T, Boivin G, et al. Systematic review of influenza resistance to the neuraminidase inhibitors. BMC Infect Dis 2011; 11:134. Nguyen HT, Trujillo AA, Sheu TG, et al. Analysis of influenza viruses from patients clinically suspected of infection with an oseltamivir resistant virus during the 2009 pandemic in the United States. Antiviral Res 2012; 93:381–386.
3.
Graitcer SB, Gubareva L, Kamimoto L, et al. Characteristics of patients with oseltamivir-resistant pandemic (H1N1) 2009, United States. Emerg Infect Dis 2011; 17:255–257. 4. Mehta T, McGrath E, Bheemreddy S, et al. Detection of oseltamivir resistance during treatment of 2009 H1N1 influenza virus infection in immunocompromised patients: utility of cycle threshold values of qualitative real-time reverse transcriptase PCR. J Clin Microbiol 2010; 48:4326–4328. 5. LeGoff J, Rousset D, Abou-Jaoude G, 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:e37095. 6. Nguyen HT, Fry AM, Loveless PA, et al. Recovery of a multidrug-resistant strain of pandemic influenza A 2009 (H1N1) virus carrying a dual H275Y/I223R mutation from a child after prolonged treatment with oseltamivir. Clin Infect Dis 2010; 51:983–984. 7. van der Vries E, Stelma FF, Boucher CA. Emergence of a multidrug-resistant pandemic influenza A (H1N1) virus. N Engl J Med 2010; 363:1381–1382. 8. Pizzorno A, Abed Y, Bouhy X, 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; 56:1208–1214. 9. Deyde VM, Sheu TG, Trujillo AA, et al. Detection of molecular markers of drug resistance in 2009 pandemic influenza A (H1N1) viruses by pyrosequencing. Antimicrob Agents Chemother 2010; 54:1102–1110. 10. Brookes DW, Miah S, Lackenby A, et al. Pandemic H1N1 2009 influenza virus with the H275Y oseltamivir resistance neuraminidase mutation shows a small compromise in enzyme activity and viral fitness. J Antimicrob Chemother 2011; 66:466–470. 11. Tramontana AR, George B, Hurt AC, et al. Oseltamivir resistance in adult oncology and hematology patients infected with pandemic (H1N1) 2009 virus, Australia. Emerg Infect Dis 2010; 16:1068–1075. 12. Hung IF, To KK, Lee CK, et al. Convalescent plasma treatment reduced mortality in patients with severe pandemic influenza A (H1N1) 2009 virus infection. Clin Infect Dis 2011; 52:447–456.
Accepted 2 June 2014; published online 18 June 2014
100
AVT-14-CR-3255_Grund.indd 100
©2015 International Medical Press
20/02/2015 10:08:01
