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1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
✉
Corresponding author.
This is a publication of the U.S. Government. This publication is in the public domain and is therefore without copyright. All text from this work may be reprinted freely. Use of these materials should be properly cited.
Most oseltamivir-resistant pandemic (H1N1) 2009 viruses have been isolated from
immunocompromised patients. To describe the clinical features, treatment,
outcomes, and virologic data associated with infection from pandemic (H1N1) 2009
virus with H275Y mutation in immunocompromised patients, we retrospectively
identified 49 hematology–oncology patients infected with pandemic (H1N1)
2009 virus. Samples from 33 of those patients were tested for H275Y genotype by
allele-specific real-time PCR. Of the 8 patients in whom H275Y mutations was
identified, 1 had severe pneumonia; 3 had mild pneumonia with prolonged virus
shedding; and 4 had upper respiratory tract infection, of whom 3 had prolonged
virus shedding. All patients had received oseltamivir before the H275Y mutation
was detected; 1 had received antiviral prophylaxis. Three patients excreted
resistant virus for >60 days. Emergence of oseltamivir resistance is frequent
in immunocompromised patients infected with pandemic (H1N1) 2009 virus and can
be associated with a wide range of clinical disease and viral kinetics.
Keywords: Influenza, oseltamivir, immunocompromised host, diagnosis, treatment outcome, antiviral drug resistance, oncology, viruses, research
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All other clinicians completing this activity will be issued a certificate of
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Release date: March 23, 2011; Expiration date: March 23, 2012
Learning Objectives
Upon completion of this activity, participants will be able to:
Distinguish clinical characteristics associated with treatment-resistant
pandemic (H1N1) 2009
Analyze outcomes of patients infected with treatment-resistant pandemic
(H1N1) 2009
Identify the rate of H275Y mutation development among patients with pandemic
(H1N1) 2009 infection in the current study
Evaluate the virology of treatment-resistant pandemic (H1N1) 2009
MDSCAPE CME EDITOR
Karen L. Foster, Technical Writer/Editor, Emerging Infectious
Diseases. Disclosure: Karen L. Foster has disclosed no relevant financial
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MEDSCAPE CME AUTHOR
Charles P. Vega, MD, Associate Professor; Residency Director, Department
of Family Medicine, University of California, Irvine. Disclosure: Charles P.
Vega, MD, has disclosed no relevant financial relationships.
AUTHORS
Disclosures:Christian Renaud, MD, MSc; Alexandre A. Boudreault; Jane Kuypers, PhD;
Kathryn H. Lofy, MD;andLawrence Corey, MD,have disclosed no relevant financial relationships.Michael J. Boeckh, MD,has disclosed the following relevant financial relationships: received
grants for clinical research from Adamas, GlaxoSmithKline, and Roche; served as
an advisor or consultant for Baxter HealthCare Pharmaceuticals and
Roche/Genentech, Inc.Janet A. Englund, MD,has disclosed the following relevant financial relationships: received
grants for clinical research from Adamas, ADMA, Chimerix, MedImmune Inc., and
Novartis Pharmaceuticals Corporation; served as an advisor to US Attorney
General, District of Alaska.
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association.
1. You are seeing a 50-year-old man being treated for acute myeloid leukemia.
He complains of 3 days of fever, chills, fatigue, and wet cough. You suspect
influenza, which has been more prevalent in your area over the last month, but
you are concerned regarding possible antiviral resistance in this high-risk
patient. Which of the following statements regarding the clinical
characteristics of patients in the current study who have resistant pandemic
(H1N1) 2009 is most accurate?A. Only adults, not children, had pandemic
(H1N1) 2009 with the H275Y mutationB. Most cases of resistant pandemic (H1N1) 2009
were generated from contact with other infected patientsC. Most cases of resistant
pandemic (H1N1) 2009 were diagnosed more than 5 days after the onset of symptomsD.
Lymphopenia was present in nearly half of patients with the H275Y mutation2. A
rapid polymerase chain reaction (PCR) test is positive for the patient in
question number 1. Which of the following statements regarding outcomes of
patients with the H275Y mutation in the current study is most accurate?A.
No patients diedB. The H275Y mutation was associated with higher levels of
virulenceC. Viral shedding was prolonged, even after clinical recoveryD. No patient
receiving only oseltamivir experienced clinical recovery3. What was the rate
of mutation development among all infected patients in the current
study?A. 4%B. 16%C. 28%D. 50%4. Which of the following statements
regarding the virology of cases in the current study is most accurate?A.
Pandemic (H1N1) 2009 was detected in all tissues in the patient with the most severe
illnessB. There was generally no discrepancy between genotyping from nasal wash and
bronchoalveolar lavage samplesC. The combination of oseltamivir plus rimantadine
prevented the development of the H275Y mutationD. The initial viral load had no
effect on the emergence of resistance
Activity Evaluation
1. The activity supported the learning objectives.
Strongly Disagree
Strongly Agree
1
2
3
4
5
2. The material
was organized clearly for learning to occur.
Strongly Disagree
Strongly Agree
1
2
3
4
5
3. The content
learned from this activity will impact my practice.
Strongly Disagree
Strongly Agree
1
2
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4. The activity
was presented objectively and free of commercial bias.
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
1Author affiliations: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA (C. Renaud, A.A. Boudreault, L. Corey, M.J. Boeckh, J.A. Englund);
2Seattle Children’s Hospital, Seattle (C. Renaud, J.A. Englund);
3University of Washington, Seattle (J. Kuypers, L. Corey, M.J. Boeckh, J.A. Englund);
4Washington State Department of Health, Shoreline, Washington, USA (K.H. Lofy)
✉
Address for correspondence: Janet A. Englund,
Children’s Hospital and Regional Medical Center, 4800 Sand Point Way
NE #8G-1, Seattle, WA 98105 USA; email: janet.englund@seattlechildrens.org
✉
Corresponding author.
This is a publication of the U.S. Government. This publication is in the public domain and is therefore without copyright. All text from this work may be reprinted freely. Use of these materials should be properly cited.
Most oseltamivir-resistant pandemic (H1N1) 2009 viruses have been isolated
from immunocompromised patients. To describe the clinical features,
treatment, outcomes, and virologic data associated with infection from
pandemic (H1N1) 2009 virus with H275Y mutation in immunocompromised
patients, we retrospectively identified 49 hematology–oncology
patients infected with pandemic (H1N1) 2009 virus. Samples from 33 of those
patients were tested for H275Y genotype by allele-specific real-time PCR. Of
the 8 patients in whom H275Y mutations was identified, 1 had severe
pneumonia; 3 had mild pneumonia with prolonged virus shedding; and 4 had
upper respiratory tract infection, of whom 3 had prolonged virus shedding.
All patients had received oseltamivir before the H275Y mutation was
detected; 1 had received antiviral prophylaxis. Three patients excreted
resistant virus for >60 days. Emergence of oseltamivir resistance is
frequent in immunocompromised patients infected with pandemic (H1N1) 2009
virus and can be associated with a wide range of clinical disease and viral
kinetics.
The development of antiviral drug resistance in influenza viruses affects patient
care. Concerns for worldwide spread of resistant virus are growing (1). Approximately 300
patients with oseltamivir-resistant pandemic (H1N1) 2009 virus have been reported to
the World Health Organization, with the complexity of treatment and consequences of
infection well described (2–5). Millions of oseltamivir doses have been
stockpiled worldwide, representing one of the major interventions to contain and
mitigate the impact of influenza and potentially offer treatment to large numbers of
patients (6,7). The efficacy and cost of pharmacologic
interventions to contain oseltamivir-resistant virus are of major concern.
Most resistant pandemic (H1N1) 2009 viruses have been detected in immunocompromised
patients who received neuraminidase inhibitors, and all but 1 had the H275Y
neuraminidase mutation (2). This mutation had already been observed before the
pandemic (H1N1) 2009 outbreak, for example, it was detected worldwide in healthy
patients who had not received antiviral drugs and were infected with seasonal (H1N1)
virus during the 2008–09 influenza season (8). Efforts are under way to characterize and
detect the H275Y mutation, but data on clinical impact and viral fitness (i.e.,
replicative capacity in vitro and in vivo that can further be correlated with
transmissibility and virulence) associated with this mutation are still needed. We
describe in detail the clinical features, treatment, outcomes, and virologic data
associated with infection caused by pandemic (H1N1) 2009 virus with H275Y mutation
in immunocompromised patients.
Materials and Methods
Hematology–oncology patients who were infected with pandemic (H1N1) 2009
virus and received care at adult and pediatric Seattle Cancer Care Alliance
(Seattle, WA, USA) units or clinics during May 1, 2009–April 30, 2010,
were identified by using infection control data and laboratory databases. All
samples with pandemic (H1N1) 2009 virus detected by in-house real-time reverse
transcription–PCR targeting the matrix, and hemagglutinin genes were
retrospectively tested by our allele-specific real-time PCR (ASPCR) for H275Y
genotype (9,10). ASPCR uses 2 allele-specific
forward primers (wild-type and mutant) and a common reverse primer and probe.
Wild-type and mutant genotypes were defined by the difference in PCR cycle
threshold values (∆cycle thresholdmutant –wild type)
between the mutant primer and the wild-type primer amplification curves for the
same sample. The ASPCR directed toward the H275Y mutation only was designed and
validated in our laboratory, with good correlation demonstrated by
pyrosequencing (9).
ASPCR also can provide an accurate quantitative result of mutant percentage in a
mixed population, as described (11). Samples were not tested for adamantanes
resistance because pandemic (H1N1) 2009 virus was considered to be uniformly
resistant to adamantanes.
Specimens collected from inpatients or outpatients were either nasal wash (NW)
samples or bronchoalveolar lavage (BAL) samples. NW samples were tested by using
multiplex real-time reverse transcription–PCR for respiratory syncytial
virus, parainfluenza virus 1–4, human metapneumovirus, adenovirus,
bocavirus, coronavirus (OC43, 229E, HKU1, and NL63), and rhinovirus at the same
time as influenza A subtyping and influenza B. BAL samples were tested for the
same respiratory viruses and for bacterial, mycobacterial, viral, and fungal
cultures, fungal PCR, galactomanan, cytomegalovirus by shell vial, respiratory
syncytial virus by shell vial, respiratory virus direct immunofluorescence
assay, and Pneumocystis spp. direct immunofluorescence assay.
NW samples were collected by instilling 5 mL of normal saline in each nare and
having the patient blow his or her nose directly into a sterile cup. For younger
children, suction was used to collect nasal secretions. BAL samples were
obtained according to a standardized protocol. Samples were refrigerated within
4 h after collection and transported to the molecular virology laboratory. All
available samples were kept frozen at –80°C for up to 8 months
after initial clinical testing. Total nucleic acid was extracted as described
(12).
Institutional review board approval was obtained from our institutional
committee. A chart review of all patients with mutant H275Y virus was
retrospectively performed by using standardized case record forms.
Results
ASPCR Results
We identified 49 adult and pediatric hematology–oncology or
hemopoietic cell transplant (HCT) patients who were infected by pandemic
(H1N1) 2009 virus during May 1, 2009–April 30, 2010. Of these
patients, 16 had no specimen available for genotyping of position H275 by
ASPCR because the initial diagnostic test was performed in another
laboratory (12 patients), no residual sample was available (3 patients), or
the viral load was too low to genotype (1 patient). For 33 patients, at
least their first sample, obtained before treatment, was available for
genotyping. One of the 33 first samples collected had the H275Y mutation.
For 17 patients, repeat samples were obtained for clinical reasons, but only
12 patients had sufficient viral load for genotyping. The H275Y mutation
developed in 7 (58%) patients. The H275Y mutation was identified in 8
patients; 3 of these patients (patients 1, 2, and 6) have been described
(5,13).
Clinical Characteristics
Five of the 8 patients with H275Y mutation had undergone allogenic HCT or
were receiving conditioning for future allogenic HCT (Table 1). Two patients had malignancies (acute
lymphoblastic leukemia and osteosarcoma), and 1 had aplastic anemia. Four
were children and 4 were adults. Three patients had severe lymphopenia, with
lymphocyte counts persistently <200 × 103 cells/L.
Clinical characteristics are presented in Table 2; viral kinetics and treatment are presented in the Figure for each patient. No patients
had contact with another infected patient in the hospital or the clinic, and
no evidence was found of nosocomial transmission of resistant strains.
Table 1. Demographic characteristics and underlying conditions in 8
patients with H275Y mutation of pandemic (H1N1) 2009 virus, Seattle
Cancer Care Alliance, Seattle, Washington, USA, May 1,
2009–April 30, 2010*.
Patient no.
Age, y/sex
BMI
Underlying
disease
State of
disease
HCT
Recent
immunosuppressive therapy
Concurrent
illness
Lymphocytes,
× 103 cells/L
1†
51/M
20.5
AML
Remission
2 y
post–allo-HCT
MMF, tacrolimus
GVHD, renal failure
110
2†
47/F
22.9
AML
Relapse
3 y
post–allo-HCT
Chemotherapy 1 mo before
influenza dx and 1 d after-dx
None
540
3
50/M
23.3
CML
Remission
9 mo
post–allo-HCT
TBI,
cyclophosphamide, tacrolimus
GVHD
1,470
4
7/F
18.9
ALL
Relapse
NA
Chemotherapy 2 d after influenza
diagnosis; prednisone 1.5 mg/kg/d
*BMI, body mass index; HCT, hemopoietic cell transplant; AML, acute
myeloblastic leukemia; allo, allogenic; MMF, mycophenolate mofetil;
GVHD, graft-versus-host disease; NA, not applicable; CML, chronic
myeloblastic leukemia; TBI, total body irradiation; ALL, acute
lymphoblastic leukemia; CSA, cyclosporine; CMV, cytomegalovirus;
ATG, antithymocyte globuline. †Patients previously
reported in references (5,13).
Table 2. Clinical characteristics and outcomes of 8 patients with H275Y
mutation of pandemic (H1N1) 2009 virus, Seattle Cancer Care
Alliance, Seattle, Washington, USA, May 1, 2009–April 30,
2010*.
Patient no.
Symptoms of
URTI
Signs of
LRTI
Radiology
results
Antiviral drug
therapy before resistance
Co-pathogens
Outcome
1
24 h before diagnosis:
congestion, headache
Hypoxemia; positive BAL result
on d 5
Bilateral ground glass
opacity
Oseltamivir 150 mg 2×/d
followed by peramivir
None
Death related to
influenza
2
48 h before diagnosis:
congestion, wet cough, sore throat, fever (24 h)
Hypoxemia; positive BAL result
on d 25
Bilateral ground glass
opacity
Oseltamivir 150 mg 2×/d
+ rimantadine 100 mg 2×/d
Pneumocystis
spp. (DFA + in BAL)
Alive
3
5 d before diagnosis: fever (24
h), wet cough
Hypoxemia; positive BAL
result on d 2
Multiple nodules with halo
sign
Oseltamivir 150 mg
2×/d
Aspergillus (PCR and GM
result positive in BAL sample); Staphylococcus
aureus; PIV3
Alive
4
<24 h before diagnosis: fever
(5 d), cough
Hypoxemia
Bilateral infiltrates
Oseltamivir 2 mg/kg
2×/d
None
Alive
5
<24 h before diagnosis:
cough, rhinorrhea, congestion
No
Bronchial thickening
Oseltamivir 150 mg
2×/d
None
Alive
6
<24 h before diagnosis:
rhinorrhea, fever (24 h), cough
No
None
Oseltamivir 150 mg
2×/d
None
Alive
7
24 h before diagnosis: sore
throat, fever (24 h), cough
No
CXR stable
Oseltamivir 75 mg 2×/d
+ rimantadine 100 mg 2×/d
Rhinovirus (PCR result
positive in NW sample)
Alive
8
24 h before diagnosis: sore
throat, fever (24 h), chills
No
CXR normal
Prophylaxis: oseltamivir 45 mg
2×/d for 10 d, ended 3 d before influenza
diagnosis
Viral kinetics in nasal washes and treatment data of 7 patients
(patient nos. 1–7 shown top to bottom) with H275Y mutation of
pandemic (H1N1) 2009 virus, Seattle Cancer Care Alliance, Seattle,
Washington, USA, May 1, 2009–April 30, 2010. Black lines,
pandemic (H1N1) 2009 viral load; red line, % H275Y mutant. OTV,
oseltamivir; RBV, ribavirin; RTM, rimantadine; ZNV, zanamivir; PMV,
peramivir; BAL, bronchoalveolar lavage.
Severe Pneumonia
Severe pneumonia followed by acute respiratory distress syndrome
developed in 1 of the 8 patients (patient 1). This patient was initially
treated with oseltamivir for 4 days; then treatment was changed to
intravenous peramivir because of the patient’s inability to
tolerate oral therapy. Just before initiation of peramivir, a BAL sample
showed the absence of H275Y mutation in the viral population that were
present in the lung but no concomitant NW sample was available for
testing. After 7 days of intravenous peramivir, an NW sample showed
H275Y mutation in 100% of the viral population. This patient
subsequently received triple-combination antiviral drug therapy (i.e.,
oseltamivir, rimantadine, and oral ribavirin) while awaiting intravenous
zanamivir that was administered during days 18–25. The patient
died of severe pneumonia and multiorgan failure after several days of
mechanical ventilation. Autopsy showed necrotizing pancreatitis with
bilateral pulmonary consolidation, pulmonary hemorrhage, diffuse
alveolar damage, and patchy fibrosis. Pandemic (H1N1) 2009 virus was
proven by PCR in the NW sampled at autopsy but not in the lung tissue or
pancreas. Influenza viruses detected in the NW samples were completely
wild-type at autopsy.
Mild Lower Respiratory Tract Infection with Prolonged
Shedding
In 3 patients (patients 2–4), lower respiratory tract infection
quickly developed, but the patients recovered without complications. Two
of these patients initially were treated with oseltamivir alone, and 1
was treated with oseltamivir and rimantadine. Viral H275Y mutation was
detected at days 23, 8, and 17 for patients 2, 3, and 4, respectively.
All 3 patients had prolonged mild upper respiratory tract symptoms,
consisting mainly of rhinorrhea and dry cough, accompanying a variable
duration of viral shedding. Influenza virus was detected in the NW
sample for 93, 8, and 47 days in patients 2, 3, and 4, respectively.
Patient 2 had shedding of fully resistant viral population documented
for at least 26 days after antiviral drug therapy was stopped. Patient 3
recovered after 17 days of oseltamivir therapy, even though a small
percentage of H275Y mutations (10%) were present in his NW sample at day
8. In patient 4, the percentage of H275Y mutants declined slightly after
treatment was stopped. Two of these 3 patients had substantial
concurrent pathogens (Pneumocystis jiroveci and
Aspergillus fumigatus) in BAL samples at the same
time as pandemic (H1N1) 2009 virus infection, making the diagnosis of
pandemic (H1N1) 2009–related pneumonia less certain.
Upper Respiratory Tract Infection with Prolonged Shedding
Three patients (patients 5–7) had symptoms of only upper
respiratory tract disease. None had hypoxemia or infiltrate on chest
radiograph, but all 3 had a cough and 2 had fever for 24 hours. They all
had profound lymphopenia (0–366 × 103 cells/L)
and 1 had graft-versus-host disease. Two patients were initially treated
with oseltamivir alone and 1 with oseltamivir and rimantadine. One
hundred percent mutant virus developed in patients 5–7; these
patients shed influenza virus for 65, 75, and 12 days, respectively.
Patient 5 maintained a 100% mutant viral population while receiving
peramivir and continued to maintain this percentage while on prolonged
oseltamivir therapy. Patient 6 remained infected with a fully resistant
viral population many days after oseltamivir was stopped. Patient 7
improved rapidly, but because of underlying lung disease, PCR was
repeated on day 12 and showed continual viral shedding with a low viral
load. No other viral testing was performed, and this patient recovered
completely with a second 5-day course of oseltamivir.
Prophylaxis
One young patient (patient 8) had 85% H275Y mutant viral population
detected in the first NW sample obtained on day 2 of illness. This
patient had been in contact with 2 family members who had documented
influenza infection but did not receive antiviral drug therapy. The
patient then received oseltamivir prophylaxis (45 mg 2×/d) for 10
days; 3 days after prophylaxis was completed symptoms developed,
including fever, chills and sore throat. The patient was treated with 10
days of oseltamivir and had a mild course of disease with rapid clinical
resolution.
Initial Therapy
In all patients, except patient 8, virus was fully wild-type when the initial
specimen was collected. All patients had received antiviral drug therapy
before the H275Y mutation was detected. One patient had received oseltamivir
prophylaxis, 4 had received only oseltamivir treatment, 2 had received
combination oseltamivir/rimantadine therapy, and 1 had received oseltamivir
followed by intravenous peramivir.
Virologic Data
In 3 of the 8 patients (patients 4–6), viral loads increased after
H275Y viruses were detected. In 2 patients (patients 2 and 4), viral loads
declined after start of either intravenous or inhaled zanamivir. In 1
patient, viral load declined during 17 days of oseltamivir therapy, even
though 10% of the viral population was documented to be H275Y mutants. Five
of the 8 patients were found at some time to have mixed, wild-type and
mutant, populations. We did not identify any discrepancy between BAL sample
and NW sample genotyping results, but both types of samples were collected
in close proximity (within 2–4 days) in only 3 patients. Wild-type
virus was detected in 2 patients in paired NW and BAL samples; mutant virus
was detected in paired NW and BAL samples in 1 patient. The 3 patients with
the highest initial viral load (>6 log10 copies/reaction) had
a substantial percentage of H275Y mutants at days 8, 9, and 11 (range
35%–100%). Patients with lower initial viral loads were not tested a
second time until 12–23 days later. These long intervals do not
enable us to compare the timing of resistance emergence according to initial
viral load.
Discussion
During the pandemic (H1N1) 2009 outbreak, neuraminidase-resistant viruses emerged
rapidly in immunocompromised patients. Our retrospective analysis suggests a
high rate of oseltamivir resistance conferred by the H275Y mutation in treated
immunocompromised patients. The rate of mutation development ranged from 8 (16%)
of 49 patients, with all infected patients as denominator, to 7 (58%) of 12 on
the basis of samples obtained after antiviral drug therapy began. Two other
studies have reported similar rates. One study from Scotland reported a 50% rate
(5/10) of H275Y mutation in immunocompromised patients with samples available
after oseltamivir therapy began (14). The other study, in Australia, reported a
13.3% rate (4/30) of H275Y mutation in treated immunocompromised patients (not
all of them tested) or 57% rate (4/7) in only treated patients for whom samples
were available after treatment began (15).
The 8 patients in our study who had H275Y mutant virus demonstrated a wide range
of clinical disease, from benign upper respiratory tract symptoms to severe and
fatal respiratory insufficiency. Whether the H275Y mutation is associated with
higher rates of death or severe disease is unclear. The small number of
immunocompromised patients infected with resistant pandemic (H1N1) 2009 virus
did not enable us to compare them with patients infected with sensitive pandemic
(H1N1) 2009 virus. However, our case series highlights the rapid emergence of
resistant viruses in the context of mild to severe influenza disease. In 7 of
the 8 cases in our study, resistance was not associated with long-term
consequences and was not even suspected in 4 of the 7 cases. This observation
could suggest that resistant viruses are not more virulent than wild-type
viruses.
Influenza resistance kinetics as provided in this study by the percentage of
H275Y mutants enable a better determination of the timing of emergence of
resistant virus and the possible subsequent clearance of this resistant viral
population. Our results suggest that elevated viral loads seen early in disease
might be associated with more rapid emergence of resistance, but more complete
and regular testing after the initial sample is necessary to confirm that
hypothesis.
In this case series, some patients conserved the resistant viral population after
antiviral pressure removal, whereas another patient (patient 1) cleared his
resistant viral population to recover a fully wild-type virus. Similarly, the
H275Y mutation described in another case report disappeared after antiviral drug
therapy was completed, while others showed conserved resistant viral population
(14,16). Those 2 different evolutions of
mutant viral population suggest the possibility of quasispecies with different
fitness. The H275Y neuraminidase mutation had affected in vitro viral fitness
when incorporated in seasonal (H1N1) virus, but data looking at 2008–09
seasonal (H1N1) virus showed that the circulating H275Y mutant strain had
recovered its fitness, possibly explaining its sustainable transmission (17). Recently,
permissive secondary mutations in the neuraminidase gene leading to fitness
recovery have been proposed in seasonal (H1N1) viruses (18). Substitutions V234M and R222Q
buffer deficiencies in neuraminidase folding or stability caused by H275Y and
simultaneously allow the virus to keep its neuraminidase/hemagglutinin balance,
which is implicated in viral fitness (19). Further molecular and in vitro analysis of
viral strains from patients who cleared and did not clear the mutant viral
population might provide more input about permissive secondary mutations in
pandemic (H1N1) 2009 virus.
We were not able to provide information about whether an H275Y mutant viral
population can develop in the upper respiratory tract and not in the lower
respiratory tract or the converse scenario. The limited data we described on
paired NW and BAL samples suggest that the upper and lower respiratory tracts
are likely to be infected with the same viral H275Y genotype. Others have shown
that quasispecies harboring the D222G hemagglutinin mutation that confers
improved pneumocyte receptors binding could be found specifically or in larger
proportions in endotracheal aspirates than in paired nasopharyngeal aspirates
(20). Analysis of
D222G mutant virus has not yet been reported in immunocompromised patients, a
condition in which mutations develop more readily.
We have demonstrated that the H275Y mutation can develop in immunocompromised
patients under different antiviral drug treatment regimens. Oseltamivir is the
antiviral agent most associated with the emergence of H275Y. Peramivir selects
for the H275Y mutation in seasonal (H1N1) by successive passages in vitro, but
data have not clearly confirmed this observation in vivo (21). One patient in our study had
received oseltamivir and peramivir before resistance detection. We believe that
if peramivir was not the drug that selected the initial mutation, it did not
prevent establishment of dominant H275Y virus population (13). Because peramivir is rarely used
as frontline therapy, data to support this hypothesis are likely to be difficult
to obtain. Combination therapy with oseltamivir and rimantadine did not prevent
development of H275Y mutation in 2 of the patients in our study. The issue of
antiviral drug resistance in immunocompromised patients makes determination of
optimal initial therapy necessary. Inhaled zanamivir is often contraindicated
because the patients may have respiratory failure or underlying lung disease,
and novel agents (e.g., DAS181) are not yet available. Triple-combination
therapy (i.e., oseltamivir, rimantadine, and oral ribavarin) potentially could
reduce emergence of resistance, but more data are needed to support this
hypothesis (22,23).
This case series has some limitations. It does not provide a totally accurate
incidence of resistance in immunocompromised patients. Some cases of pandemic
(H1N1) 2009 might have been missed, particularly if symptoms were mild or if the
patient was not identified by infection control surveillance. Also, repeat
testing was performed in only 17 patients, in whom 5 viral loads were too low to
genotype. Systematic testing of infected patients at 5 or 6 days after initial
pandemic (H1N1) 2009 diagnosis might have caught even more minor mutant viral
populations. Our ASPCR was aimed only at detection of the H275Y mutation.
Presence of other rare mutations as the I223R could have influenced our results
(24,25). However, our data highlight the
need for surveillance and clinical testing for resistant mutations in
immunocompromised patients by using sensitive and rapid molecular diagnostic
tests.
Acknowledgments
We thank the respiratory virus clinical technologists at the University of
Washington Molecular Virology Laboratory, the Seattle Cancer Care Alliance
infection control practitioners, and the Seattle Children’s Hospital
infection control practitioners for their help and support.
M.J.B. received grant support from the National Institutes of Health CA18029,
CA15704, HL93294, research support from Roche, Glaxo-Smith-Kline, and Adamas,
and consulting fees from Roche/Genentech; he served on a data safety monitoring
board for and influenza vaccine study funded by US government funds from the
Office for Preparedness and Response, Biomedical Advanced Research and
Development Authority, under contract to DynPort Vaccine Company. J.A.E.
received research support from Novartis, MedImmune, Adamas, and ADMA.
Biography
Dr Renaud is a medical microbiologist-pediatric infectious diseases physician
working at Fred Hutchinson Cancer Research Center, Seattle, USA. His main
interests include clinical virology and respiratory virus infections.
Footnotes
Suggested citation for this article: Renaud C, Boudreault
AA, Kuypers J, Lofy KH, Corey L, Boeckh MJ, et al. H275Y mutant pandemic
(H1N1) 2009 virus in immunocompromised patients. Emerg Infect Dis [serial on
the Internet]. 2011 Apr [date cited]. http://dx.doi.org/10.3201/eid1704.101429
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