MAJOR ARTICLE

Recovery of Drug-Resistant Influenza Virus

from Immunocompromised Patients: A Case Series
Michael G. Ison,1,a Larisa V. Gubareva,1 Robert L. Atmar,3 John Treanor,4 and Frederick G. Hayden1,2
1
Division of Infectious Diseases and International Health, Department of Medicine, and 2Department of Pathology, University of Virginia Health
Sciences Center, Charlottesville; 3Departments of Medicine and Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas;
4
Division of Infectious Diseases, University of Rochester, Rochester, New York

(See the editorial commentary by McCullers and the article by Ison et al., on pages 7513 and 76572, respectively.)

Influenza virus with resistance to antiviral drugs emerges with increased frequency in immunocompromised
patients and can limit the benefit of M2 and neuraminidase (NA) inhibitors. We document 3 cases of influenza
in severely immunocompromised patients from whom virus variants with molecular markers of resistance to
anti-influenza drugs were recovered. Virus variants recovered from 2 patients had mutations in the M2, NA
(with a previously recognized Glu119Val NA substitution), and hemagglutinin genes. We describe a novel
Asp198Asn NA mutation in an influenza B virus and its decreased susceptibility to both oseltamivir and
zanamivir.

Influenza causes significant morbidity and mortality in
patients with compromised immune systems. Influenza
virus infection in transplant recipients is associated with
a higher rate of pulmonary complications (particularly
viral pneumonia), extrapulmonary manifestations, an
increased risk of graft dysfunction and rejection, and
high attributable mortality [1]. In addition, transplant
recipients have prolonged shedding of influenza virus,
often despite antiviral therapy [2], which promotes the
emergence of antiviral resistance [3]. This is a well-
recognized problem with the use of the M2 inhibitors
amantadine and rimantadine [1] but is an uncommon
complication with the use of the neuraminidase (NA)
inhibitors zanamivir and oseltamivir in adults [4]. In
vitro resistance to NA inhibitors can be mediated by
changes in hemagglutinin (HA), which confer cross-
resistance to the class in cell culture; changes in NA,
which confers resistance that is NA subtype specific and
drug specific; or changes in both [4]. Mutations in HA
typically reduce HA binding efficiency, thus reducing
the viruss dependency on its NA activity [4, 5]. In the
present article, we describe the clinical and virologic
characteristics of 3 immunocompromised patients who
had progressive influenza virus infection despite NA in-
hibitor therapy with or without concurrent M2 inhibi-
tor therapy.

Received 17 May 2005; accepted 4 October 2005; electronically published 13 METHODS

February 2006.
Presented in part: 5th International Symposium on Respiratory Viral Infections,
Cells, virus isolates, and compounds. MDCK cells
Casa de Campo, Dominican Republic, 58 December 2002 (abstract LB15); 1st
European Influenza Conference, St.-Julians, Malta, 2023 October 2002 (abstract grown in Eagles MEM (EMEM) supplemented with
W4-1); Options for the Control of Influenza V Conference, Okinawa Japan, 711 10% fetal bovine serum, antibiotics, and l-glutamine
October 2003 (abstract W05-6).
Potential conflicts of interest: L.V.G. has received grant support from GlaxoWellcome were used for virus propagation and for virus plaque
R&D; F.G.H. has received grant support from and is an ad hoc consultant for Roche; purification (described below). Viruses were propagated
all other authors have no conflicts of interest to report.
Financial support: Public Health Service (grant AI45782 to L.V.G.). by standard procedures, and aliquots were stored at
a
Present affiliation: Transplant Infectious Diseases Service, Northwestern 70C [6].
University Feinberg School of Medicine, Chicago, Illinois.
Reprints or correspondence: Dr. Frederick G. Hayden, University of Virginia Health
The clinical isolates (described below) were isolated
System, P.O. Box 800473, Private Clinics Bldg., Rm. 6577b, Charlottesville, VA initially in MDCK cells and passaged 12 times before
22908 (fgh@virginia.edu).
analysis. The previously characterized NA inhibitorre-
The Journal of Infectious Diseases 2006; 193:7604
 2006 by the Infectious Diseases Society of America. All rights reserved.
sistant mutants Glu119Gly, Glu119Asp, and Arg292Lys
0022-1899/2006/19306-0004$15.00 of the influenza A/turkey/Minnesota/833/80 (H4N2) vi-

760 JID 2006:193 (15 March) Ison et al. (I)

 Table 1. Patient 1: susceptibility testing of isolates recovered from the patient with influenza B/
Rochester/02/2001.

IC50 in the
NA-inhibition
Mutations in assay, nmol/L
Antiviral
Isolate date treatment HA NA O Z
15 Feb 2001 O Referencea Referenceb 37 1.4
23 Feb 2001 O/R ND None 32 1.1
28 Feb 2001 O ND None 42 1.1
1 Mar 2001 O ND None 33 0.9
3 Mar 2001 O ND None 39 1.1
8 Mar 2001 O ND Asp/Asn198c 86 2.2
8 Mar 2001 (clone) Ser285Alad Asp198Asn
e
304 15
12 Mar 2001 O ND None 30 1.1
31 Mar 2001 O ND None 35 1.0
1 Apr 2001 O None None 33 1.2
Resistant control B/Memphis/20/96 Thr198Ile Arg152Lys 1500 220

NOTE. HA, hemagglutinin; NA, neuraminidase; ND, not done; O, oseltamivir; R, rimantadine; Z, zanamivir. None
indicates that there were no mutations in comparison with the first isolate.
a
GenBank accession no. AY947469.
b
GenBank accession no. AY947471.
c
There was a mixture of viruses, some with Asp and others with Asn at aa 198.
d
GenBank accession no. AY947470.
e
GenBank accession no. AY947472.

rus [7, 8] and the mutant Arg152Lys of the influenza B/Memphis/
20/96 [9] were from the virus repository of the Respiratory Dis-
ease Study Unit at the University of Virginia. Stock solutions of
zanamivir (GlaxoSmithKline) and oseltamivir carboxylate (Hoff-
man-La Roche) were prepared in distilled water and frozen in
aliquots at 20C until they were thawed immediately before
use.
NA-inhibition assay. Evaluation of virus susceptibility to
NA inhibitor was assessed in the NA enzymatic activity inhi-
bition assay, as described elsewhere [4]. The IC50 of NA inhib-
itors was calculated by plotting the percentage inhibition of
NA activity against the inhibitor concentration.
Sequence analysis of the HA, NA, and M genes. Viral RNA
was extracted from either original samples or cell culture su-
pernatants, and reverse-transcription polymerase chain reac-
tions and sequence analysis of the HA, NA, and M genes were
performed as described elsewhere [4]. The sequences of prim-
ers used for PCR amplification are available on request. The
sequence alignments were performed by use of the Influenza
Sequence Database [6]. The nucleotide sequences were depos-
ited into the GenBank database (accession numbers AY947469
AY947478).
RESULTS
Patient 1. This 2-year-old white female patient developed
myelomonocytic leukemia at 15 months of age and was treated
by splenectomy and, later, by unrelated cord-blood marrow
transplantation (on 25 January 2001). While receiving antithy-
mocyte globulin and cyclosporine, the patient was started on
a regimen of oseltamivir prophylaxis (10 mg orally twice daily)
on posttransplantation day 15, after her mother received a clin-
ical diagnosis of influenza. Four days after starting the oseltamivir
regimen (day 21), the patient developed nasal congestion, non-
productive cough, and rhinorrhea; a nasopharyngeal swab cul-
ture was found to be positive for influenza B virus (designated
B/Rochester/02/2001). The patient received 30 mg of oseltamivir
twice daily for 2 weeks (until day 46), after which her dose was
decreased to 20 mg twice daily for an additional 4 weeks. The
patient had continued rhinorrhea and cough throughout but had
transient resolution of fever during the second to third weeks of
illness. She received aerosolized ribavirin from day 27 until day
33 and then intravenous ribavirin during the last 10 days of her
illness (days 6979). Despite therapy, she experienced progressive
respiratory distress and gastrointestinal bleeding, and she even-
tually died.
Nine isolates of influenza B virus from the patient, recovered
over a period of 1.5 months, were tested for susceptibility to NA
inhibitors (table 1). Virus recovered on day 42 (8 March 2001)
had an 3-fold increase in the average IC50 (table 1) for osel-
tamivir, suggesting the presence of a subpopulation of resistant
species. Twelve randomly selected individual viral plaques dem-
onstrated IC50 values ranging from 40 nmol/L to 200 nmol/L.
The plaque-purified variant with the highest IC50 had an average
IC50 of 304 nmol/L for oseltamivir (an 10-fold increase) and
of 15 nmol/L for zanamivir (an 10-fold increase). This clone

Drug-Resistant Influenza in Immunocompromised Patients JID 2006:193 (15 March) 761

had both Asp198Asn (N2 subtype numbering) NA and Ser285Ala
HA mutations.
Patient 2. This 63-year-old female patient with chronic lym-
phocytic leukemia was treated in 2001 with fludarabine, cyclo-
phosphamide, and rituximab, followed by alemtuzumab. On 24
January 2002, she developed a respiratory illness with low-grade
fever, cough, and nasal congestion. She experienced increasing
cough and wheezing over the next week, and influenza A virus
(designated A/Texas/131/2002 [H3N2]) was recovered in cul-
ture. Starting on illness day 8, she was treated with a 5-day
course of oral oseltamivir and then with 2 serial 5-day courses
of oral oseltamivir plus rimantadine. Initial chest radiographs
showed no abnormality, but 1 on illness day 22 showed min-
imal patchy opacity in the lower lobe of the left lung. She
continued to have persistent cough and nasal congestion after
discontinuation of therapy. Beginning on 5 June 2002, the com-
bination of inhaled zanamivir plus oral rimantadine was given
for 15 days because of persistent detection of influenza A virus.
Her respiratory symptoms persisted into the fall of 2002 but
eventually resolved, and her respiratory virus culture was doc-
umented to be negative in April 2003.
Among the 5 isolates available for testing, the first was sus-
ceptible to both NA inhibitors (table 2). NA gene analysis dem-
onstrated a mixed population of wild-type virus and Glu119Val
mutants during the second therapy course. The Glu119Val mu-
tant was dominant in the next isolate and had 1100-fold in-
creased IC50 against oseltamivir but no significant change in IC50
against zanamivir. This variant also had 3 amino acid substitu-
tions in the HA, 2 of which (Arg142Gly and Tyr195Phe) reside
near the receptor-binding site [10]. These mutant NA and HA
genes were not detectable in later isolates. A Ser31Asn substi-
tution in M2 persisted in all tested variants after 27 March 2002.
Patient 3. This 60-year-old white female patient underwent
cadaveric renal transplantation for diabetic nephropathy on 6
November 2003 and was maintained on a regimen of tacrolimus,
mycophenolate mofetil, and prednisone. At posttransplantation
week 6, she developed fever, malaise, myalgias, arthralgias, min-
imally productive cough, and a pruritic rash. Evaluation revealed
that the patient had both graft-versus-host disease with pancy-
topenia and influenza A virus (designated A/Charlottesville/03/
2004 [H3N2]) infection determined by culture on 27 December
2003. Oral oseltamivir treatment (75 mg twice daily) was started,
and the dose was subsequently increased to 150 mg twice daily
for a total course of 18 days. However, she had progressive re-
spiratory compromise and developed pneumonia by day 9 of
therapy. Rimantadine was added to her treatment, but she had
continued detection of influenza virus with respiratory failure
and died after a hemorrhagic stroke.
The 6 influenza isolates collected during her illness dem-
onstrated a 1100-fold increase in the IC50 against oseltamivir
between the first (0.3 nmol/L) and the last (82.8 nmol/L) isolate,
as well as a 3-fold increase in IC50 against zanamivir (table 3).
Two mutationsGlu119Val in NA and Ser31Asn in M2were
detected in the last isolate. The HA of the oseltamivir-resistant
mutant contained a substitution at aa 226 (ValrIle).
DISCUSSION
Antiviral therapy with either M2 inhibitors or NA inhibitors
appears to reduce the duration of viral shedding, the risk of
progression to pneumonia, and, possibly, the mortality asso-
ciated with influenza in immunocompromised patients [1, 2,
11]. Unfortunately, some individuals have persistent viral rep-
lication despite antiviral therapy, with the resultant risks of
antiviral resistance emergence and persistent illness [4, 12]. In
this case series, we documented the recovery of dual NA in-

Table 2. Patient 2: susceptibility testing of isolates recovered from the patient with influenza A/Texas/131/2002 (H3N2).

IC50 in the
NA-inhibition
Mutations in assay, nnol/L
Antiviral
Isolate date treatment M2 HA NA O Z
a b c
1 Feb 2002 O Reference Reference Reference 0.8 2.1
11 Feb 2002 O/R None ND Glu/Val119d
27 Mar 2002 Ser31Asn Arg142Gly, Tyr195Phe, Ile239Arg Glu119Val 90 2
17 Jun 2002 Z/R Ser31Asn ND None 1.5 2.5
10 Jul 2002 Ser31Asn None None 1.0 2.2
Sensitive control, A/turkey/MN/833/80 None None 0.4 2.0
Resistant control, A/turkey/MN/833/80 None Glu119Asp 2.1 700

NOTE. On 13 Feb 2002, 17 Apr 2002, and 4 Jun 2002, virus presence was detected, but those samples were not available for drug susceptibility testing.
HA, hemagglutinin; NA, neuraminidase; O, oseltamivir; R, rimantadine; Z, zanamivir. None indicates that there were no mutations in comparison with the first
isolate.
a
GenBank accession no. AY947478.
b
GenBank accession no. AY947476.
c
GenBank accession no. AY9474767.
d
There was a mixture of viruses, some with Glu and others with Val at aa 119.

762 JID 2006:193 (15 March) Ison et al. (I)

Table 3. Patient 3: susceptibility testing of isolates recovered from the patient with influenza A/Charlottesville/03/2004 (H3N2).

IC50 in the
NA-inhibition
Mutations in assay, nmol/L
Antiviral
Isolate date treatment M2 HA NA O Z
a b c
27 Dec 2003 O Reference Reference Reference 0.3 1.0
1 Jan 2004 O None Val/Ile226d None 0.3 1.1
4 Jan 2004 O None Val/Ile226d None 1.0 1.4
5 Jan 2004 O/R None Val/Ile226d e
Glu/Val119, Glu/Lys76
f
39.9 2.8
f
9 Jan 2004 O/R None Val226Ile Glu119Val, Glu/Lys76 39.4 2.8
11 Jan 2004 O/R Ser31Asn Val226Ile Glu119Val 82.8 2.7
Sensitive control A/turkey/MN/833/80 None None None 0.5 2.0
Resistant control 1, A/turkey/MN/833/80 None None Glu119Asp 0.9 593.5
Resistant control 2, A/turkey/MN/833/80 None None Arg292Lys 11000 13.9

NOTE. HA, hemagglutinin; NA, neuraminidase; O, oseltamivir; R, rimantadine; Z, zanamivir. None indicates that there were no mutations in comparison
with the first isolate.
a
GenBank accession no. AY947475.
b
GenBank accession no. AY947474.
c
GenBank accession no. AY947473.
d
There was a mixture of viruses, some with Val and others with Ile at aa 226.
e
There was a mixture of viruses, some with Glu and others with Val at aa 119.
f
There was a mixture of viruses, some with Glu and others with Lys at aa 76.

hibitor and M2 inhibitorresistant mutants in 2 patients; a
novel influenza B virus mutation conferring reduced NA in-
hibitor susceptibility; and the first instance, to our knowledge,
of oseltamivir prophylaxis failure related to the detection of a
resistant variant.
Viruses in all 3 patients had transient changes in the NA
genes. Two patients shed virus with the previously recognized
Glu119Val mutation, which confers oseltamivir resistance in N2-
containing viruses but does not affect susceptibility to zanamivir
[4, 12, 13]. One patient was infected with influenza B virus that
had an Asp198Asn mutation, which conferred reduced suscep-
tibility to both oseltamivir and zanamivir [4]. Because oseltamivir
is less active against influenza B virus NA, modest reductions in
susceptibility may confer clinical resistance and may account for
the prophylaxis failure in this patient. In 2 patients infected with
influenza A virus, mutations indicative of dual resistance to
oseltamivir and M2 inhibitors emerged. Both shed viruses with
the Ser31Asn mutation, the most common mutation recognized
in influenza A/H3N2 [14]. However, NA inhibitor resistance
disappeared after cessation of oseltamivir treatment, whereas
M2 inhibitor resistance was persistent. As was seen in patient 2,
shedding of M2 inhibitorresistant virus for months has been
documented previously [12]. These findings are consistent with
previous reports that NA inhibitorresistant variants are less fit
than are wild-type viruses [13], whereas M2 inhibitorresistant
variants retain fitness.
We also documented that all 3 NA inhibitorresistant viruses
had changes in HA. The aa 226 substitution has the potential
to reduce the efficiency of virus binding to cells, because it
belongs to the HA receptorbinding site [10]. The other sub-
stitutions, at aa 142 and 195, are close to the receptor-binding
site and have the potential to alter the virus requirement for
NA activity. The clinical relevance of substitutions in HA is
uncertain, and they have been uncommonly documented pre-
viously [9]. In cell culture, HA changes reduce virus suscep-
tibility to NA inhibitors by lowering requirements for NA
activity.
Interestingly, there were mixed populations of mutant and
wild-type virus isolated from all 3 patients in the present study.
This might indicate that the virus variant that emerged did not
have a strong growth advantage while exposed to drug. Alter-
natively, it is also possible that propagation of the virus in cell
culture altered the original ratio between resistant and wild-
type variants.
Our findings provide potential insights into the management
of immunocompromised patients with influenza [4]. All 3 pa-
tients were severely immunocompromised, which suggests that
the degree of immunosuppression contributed to the lack of vi-
ral clearance. None of the patients had documented clearance
of influenza virus before discontinuation of antiviral therapy.
If possible, immunosuppressed patients should be managed
with a tailored approach directed by sequential monitoring for
viral replication and resistance testing when replication persists.
Combination NA inhibitor and M2 inhibitor therapy shows en-
hanced antiviral activity in vitro and in vivo [15], but it remains
to be determined whether it reduces resistance emergence. New
surveillance data [16] indicate that 92% of this seasons H3N2
isolates and 25% of this seasons H1N1 isolates are resistant to
M2 inhibitors because of a Ser31Asn mutation in the M2 protein.
Consequently, therapy with M2 inhibitors alone or in combi-
nation would not be appropriate. Other combinations to con-
sider in immunocompromised hosts include dual NA inhibi-

Drug-Resistant Influenza in Immunocompromised Patients JID 2006:193 (15 March) 763

tors or, possibly, an NA inhibitor and ribavirin, although fur-
ther studies in animal models are needed. Theoretically, us-
ing higher oseltamivir doses (150 mg twice daily) may be as-
sociated with greater antiviral effects and, possibly, may re-
duce the emergence of resistance by providing higher blood
concentrations [1]. In addition, vaccination of transplant re-
cipients and their close contacts and consideration of seasonal
antiviral chemoprophylaxis in highly immunocompromised
persons might prevent a potentially fatal influenza virus in-
fection; patients 1 and 3 were not vaccinated, which suggests
that further work is needed to increase vaccination of these
at-risk populations.
Acknowledgments
Special thanks go to Douglas W. Schallon and Kimberly M. Underwood
for their technical assistance with this project.
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