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Bright RA, Shay DK, Shu B, Cox NJ, Klimov AI. Adamantane Resistance Among Influenza A Viruses Isolated Early During
the 2005-2006 Influenza Season in the United States. JAMA. 2006;295(8):891–894. doi:10.1001/jama.295.8.joc60020
Context The adamantanes, amantadine and rimantadine, have been used as first-choice
antiviral drugs against community outbreaks of influenza A viruses for many
years. Rates of viruses resistant to these drugs have been increasing globally.
Rapid surveillance for the emergence and spread of resistant viruses has become
critical for appropriate treatment of patients.
Objective To investigate the frequency of adamantane-resistant influenza A viruses
circulating in the United States during the initial months of the 2005-2006
Design and Setting Influenza isolates collected from 26 states from October 1 through December
31, 2005, and submitted to the US Centers for Disease Control and Prevention
were tested for drug resistance as part of ongoing surveillance. Isolates
were submitted from World Health Organization collaborating laboratories and
National Respiratory and Enteric Virus Surveillance System laboratories.
Main Outcome Measures Using pyrosequencing and confirmatory assays, we identified viruses
containing mutations within the M2 gene that are known to confer resistance
to both amantadine and rimantadine.
Results A total of 209 influenza A(H3N2) viruses isolated from patients in 26
states were screened, of which 193 (92.3%) contained a change at amino acid
31 (serine to asparagine [S31N]) in the M2 gene known to be correlated with
adamantane resistance. Two of 8 influenza A(H1N1) viruses contained the same
mutation. Drug-resistant viruses were distributed across the United States.
Conclusions The high proportion of influenza A viruses currently circulating in
the United States demonstrating adamantane resistance highlights the clinical
importance of rapid surveillance for antiviral resistance. Our results indicate
that these drugs should not be used for the treatment or prophylaxis of influenza
in the United States until susceptibility to adamantanes has been reestablished
among circulating influenza A isolates.
Conclusions Published online February 2, 2006 (doi:10.1001/jama.295.8.joc60020).
Influenza A viruses are a major cause of morbidity and mortality in
the United States. They infect, on average, 10% to 15% of the population annually.1,2 It has been estimated that influenza
A viruses are associated with approximately 31 000 US deaths annually,
with 90% of these deaths occurring among elderly persons.3 Although
annual vaccination is the primary strategy for preventing influenza infections,
influenza antiviral drug therapy has been shown to be an effective means of
preventing and treating influenza.
Two antiviral drugs, adamantane derivatives amantadine and rimantadine,
licensed for antiviral indications in the United States in 1966 and 1993,
respectively, are used for prophylaxis and treatment of influenza, especially
for controlling outbreaks in settings such as nursing homes and long-term
care facilities.4,5 Amantadine
is also licensed for use in the treatment of Parkinson disease.
For influenza, these drugs bind to and block the function of the influenza
A virus M2 ion channel protein, preventing virus replication within the infected
cell.6 The effectiveness of prophylaxis with
these drugs is between 80% and 90%, and when treatment is begun within 48
hours of symptom onset, their use can reduce the duration of illness by 1.5
Resistance to adamantanes can emerge during treatment. A single point
mutation in the sequence coding for the amino acid at position 26, 27, 30,
31, or 34 of the M2 protein confers resistance to the adamantanes.10,11 Replication and transmission of adamantane-resistant
viruses are not hindered by these mutations.10,12-14 A
recent report describing global surveillance for adamantane-resistant influenza
viruses showed a significant increase in resistance from below 2% in 1995-2002
to 12.3% in 2004.15 In the United States, the
frequency of resistance increased significantly from 1.9% in 2004 to 14.5%
during the first 6 months of the 2004-2005 influenza season (October 2004
through March 2005).15 The rate of resistance
in the United States for the entire 2004-2005 season was 11% (R.A.B., CDC,
unpublished data, 2006). The rapid emergence of adamantane-resistant viruses
highlights the need to use results from antiviral susceptibility surveillance
to guide therapeutic decisions for patients with laboratory-confirmed influenza
A pyrosequencing method described previously permits rapid analysis
for mutations within the M2 gene associated with resistance to adamantanes.15 In this study, we used this approach to screen influenza
A viruses isolated early during the 2005-2006 influenza season in the United
States for resistance to adamantanes.
All influenza isolates collected in the United States and submitted
to the Influenza Branch, Centers for Disease Control and Prevention (CDC),
from October 1 through December 31, 2005, were included in this study. This
group of isolates came from 26 states, distributed throughout the country
(Table). Isolates were selected and
submitted by state public health laboratories participating in the World Health
Organization collaborating laboratories or the National Respiratory and Enteric
Virus Surveillance System. Each laboratory is requested to submit isolates
representative of those circulating early in the influenza season. Laboratories
that participate in the World Health Organization influenza surveillance program
were provided clear guidelines for submitting early season influenza isolates
that are representative of those circulating in the state. No effort was made
at the state level to bias the sample selection to a specific population.
Every isolate submitted to the CDC during this period was screened for drug
resistance and therefore there was no intentional or systematic bias in the
sample selection process. No antiviral susceptibility screening was done prior
to submission of isolates to the CDC. Antiviral testing was performed using
isolates submitted to the CDC, prior to further propagation or handling. All
viral isolates were submitted as part of ongoing influenza surveillance. The
National Center for Infectious Diseases has determined that these surveillance
activities are public health practice and not research; thus, they are exempt
from institutional review board regulations.
Viruses were propagated in primary rhesus monkey kidney or Madin-Darby
canine kidney (MDCK) cells. Virus hemagglutinin subtypes were determined by
state public health or other local laboratories either by hemagglutination
inhibition assay or by direct immunofluorescence assay and were confirmed
at the CDC.
Viral RNA was extracted from 100 μL of virus culture, using a total
nucleic acid extraction kit with a MagNaPure instrument (Roche Diagnostics,
Mannheim, Germany). Reverse transcription polymerase chain reaction (PCR)
for complementary DNA synthesis and PCR for DNA amplification were previously
described to amplify a 264–base pair product encompassing nucleotides
764 to 1027 of the influenza A matrix gene.15 Pyrosequencing
was performed according to the manufacturer's (Biotage, Uppsala, Sweden) protocol
as previously described to sequence a 44–base pair region that included
nucleotides 784 to 827 of the matrix gene, which encodes amino acids 25 to
38 of the M2 protein.15 Confirmatory sequencing
by conventional Sanger methods was performed on the same region of the M2
gene as previously described.15 Sequences were
aligned using DNAStar analysis programs (DNAStar, Madison, Wis).
A biological assay, previously described,15 was
used to confirm antiviral resistance results on a blinded group of viruses.
Briefly, monolayers of MDCK cells were pretreated with 0, 0.2, 2.0, or 20 μg/mL
of rimantadine for 30 minutes. Viruses diluted at 1:2 or 1:20 in culture medium,
containing 2 μg/mL of TPCK (L-1-tosylamide-2-phenylethyl
chloromethyl ketone)-trypsin, were added to cell monolayers. Following a 1-hour
adsorption period, virus medium was replaced by culture medium containing
the respective concentration of rimantadine. After a 24-hour incubation period,
virus replication was determined by measuring hemagglutination titers of the
supernatant. Viruses sensitive to rimantadine were identified by a 4-fold
or greater reduction in hemagglutination titer compared with the control cells
Influenza A isolates were collected from October 1 through December
31, 2005, from patients living in 26 states. A total of 193 (92.3%) of 209
influenza A(H3N2) and 2 (25%) of 8 influenza A(H1N1) viruses analyzed contained
point mutations resulting in a serine-to-asparagine change at amino acid 31
(S31N) of the M2 protein that conferred adamantane resistance (Table). No other amino acid change associated with influenza resistance
to adamantanes was detected in these viruses. Six H3N2 isolates contained
a valine-to-isoleucine change at amino acid 27 (V27I) in conjunction with
the S31N mutation; viruses with this combination have been shown to retain
their drug-resistance phenotype in a biological assay (R.A.B., CDC, unpublished
Conventional sequencing was performed on a blinded subset of 40 viruses
to confirm results obtained using the pyrosequencing method. In addition,
a biological assay was used on a blinded subset of 10 isolates (4 sensitive
and 6 resistant) to evaluate the drug-resistance phenotype, and these results
correlated 100% with genotypic results obtained with pyrosequencing.
Adamantane-resistant H3N2 isolates were collected from 26 states, showing
a wide distribution across the United States (Table). Fourteen isolates were obtained from 2 outbreaks within
long-term care facilities. The median age of patients from whom H3N2 isolates
were screened was 23 years, ranging from younger than 1 to 93 years (Table). Patient age was not reported for 10 isolates
A small number of isolates collected from other countries in North America
and submitted to the CDC also were screened for resistance. Ten of 10 H3N2
isolates collected from patients in Mexico and 3 of 3 isolates tested from
Canada also contained the S31N mutation conferring drug resistance.
Influenza antiviral drugs play an important role in a comprehensive
approach to controlling influenza illness and transmission. The frequency
of resistance to amantadine and rimantadine among circulating influenza A
viruses has increased dramatically over the past few years.15 As
part of increased surveillance for the emergence of drug-resistant viruses
at the CDC, all isolates submitted through December 31, 2005, were screened
for resistance to adamantanes by using a pyrosequencing method. Of the 209
A(H3N2) viruses screened, we found an alarmingly high adamantane resistance
rate of 92%. These viruses were isolated from patients residing in 26 states,
representing all regions of the United States. This rate was much higher than
the rate found among viruses collected within the United States during previous
influenza seasons.15 We found identical resistance
among all H3N2 viruses submitted for screening from other North American countries.
Canada recently reported that among H3N2 isolates tested for the 2005-2006
influenza season, 43 of 47 (91%) contained this same mutation, showing that
adamantane-resistant influenza viruses are circulating in other regions of
North America.16 In addition, preliminary and
limited data indicate that the rate of resistance for the 2004-2005 influenza
season continued to increase over the previous report for some counties in
Asia (China, 96%; Hong Kong, 72%; South Korea, 36%; Singapore, 42%) (R.A.B.,
CDC, unpublished data, 2006). Data have not been analyzed for the 2005-2006
influenza season in these countries.
It is known that influenza viruses resistant to amantadine and rimantadine
can emerge quickly in nursing homes and long-term care facilities when these
drugs are being used to control influenza outbreaks.13,17-22 Among
isolates described in this report, the median patient age was 23 years (range,
<1-93 years) for isolates for which the age of a patient was indicated
and most were not residents of long-term care facilities. In fact, only a
small proportion of the isolates (n = 14) were from such facilities.
Therefore, use of these drugs for outbreak control cannot explain the high
rate of resistance. It is most likely that patients were infected by H3N2
viruses circulating in the community already containing the S31N drug-resistant
mutation in the M2 protein. Indeed, this same mutation was the most common
change observed in drug-resistant viruses in previously published studies
that examined prior years' influenza seasons.15,23 In
these studies, in which additional patient information was obtained, adamantane
treatment was rarely documented among persons outside institutional settings.
A limitation of the current study conducted during the initial months of the
2005-2006 influenza season is that additional clinical and epidemiological
data are not yet available.
Amantadine and rimantadine have been considered first-line drugs for
the treatment of community-acquired influenza virus infections. The high frequency
of adamantane-resistant viruses reported here shows that surveillance and
rapid identification of resistant viruses have become critical in planning
for the appropriate treatment and control of influenza infections in the community
and in closed settings. Based on our results of an earlier analysis of 109
of the isolates in this report, the CDC has recommended that neither amantadine
nor rimantadine be used for treatment or prophylaxis of influenza for the
remainder of the 2005-2006 influenza season.24 A
second class of antiviral drugs for influenza is the neuraminidase inhibitors,
including oseltamivir and zanamivir. These drugs, licensed in the United States
in 1999, work by binding to the active site of the influenza A and B virus
neuraminidase proteins and preventing viral release from infected cells. Recent
reports of resistance to this class of drugs have shown very few resistant
influenza A or B viruses.25-27
We have shown that pyrosequencing represents a rapid and accurate high-throughput
screening assay for mutations known to confer antiviral resistance among influenza
viruses. Thus, rapid assessments of drug resistance within a state or large
community are possible if this technology can be introduced into regional
reference laboratories. This information can be relayed quickly to health
care personnel making critical decisions about the prevention and control
of influenza. Our results highlight the importance of continued surveillance
for the emergence and transmission of influenza viruses resistant to antiviral
drugs. They serve as a warning to the medical community of the speed at which
resistant influenza viruses can become predominant circulating strains and
spread throughout a continent.
Corresponding Author: Rick A. Bright, PhD,
CDC Influenza Branch, 1600 Clifton Rd NE, Mailstop G16, Atlanta, GA 30333.
Published Online: February 2, 2006 (doi:10.1001/jama.295.8.joc60020).
Author Contributions: Dr Bright had full access
to all of the data in the study and takes responsibility for the integrity
of the data and the accuracy of the data analysis.
Study concept and design: Bright, Shay, Cox.
Acquisition of data: Bright, Shu.
Analysis and interpretation of data: Bright,
Shay, Shu, Cox, Klimov.
Drafting of the manuscript: Bright, Shay.
Critical revision of the manuscript for important
intellectual content: Bright, Shay, Shu, Cox, Klimov.
Statistical analysis: Shay.
Administrative, technical, or material support:
Shay, Cox, Klimov.
Study supervision: Bright, Cox, Klimov.
Financial Disclosures: None reported.
Disclaimer: The findings and conclusions in
this report are those of the authors and do not necessarily represent the
views of the Centers for Disease Control and Prevention.
Acknowledgment: We thank the National Influenza
Centers, state public health laboratories, and participating laboratories
in the WHO global surveillance program for their submission of isolates. We
also acknowledge Jenna Achenbach, MS, Amanda Balish, BS, Angela Foust, MA,
Rebecca Garten, PhD, Henrietta Hall, BS, Jan Mabry, BSFR, Gilda Perez-Oronoz,
MSPH, and Teresa Wallis, MS, from the CDC Influenza Branch for their contributions
to this project.
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