﻿Detection of Oseltamivir-Resistant Pandemic Influenza
A(H1N1)pdm2009 in Brazil: Can Community Transmission
Be Ruled Out?
Thiago Moreno L. Souza1*
, Paola C. Resende1
, Natalia Fintelman-Rodrigues1
, Tatiana Schaffer
Gregianini2
, Nilo Ikuta6
, Sandra Bianchini Fernandes3
, Ana Luisa Furtado Cury4
, Maria do Carmo Debur
Rosa5
, Marilda M. Siqueira1
1 Laboratório de Vírus Respiratórios e do Sarampo, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil, 2 Laboratório Central de Saúde
Pública do Estado do Rio de Grande do Sul, Fundação Estadual de Produção e Pesquisa em Saúde Seção de Virologia, Porto Alegre, Rio Grando do Sul,
Brazil, 3 Laboratório Central de Saúde Pública do Estado de Santa Catarina, Florianópolis, Santa Catarina, Brazil, 4 Laboratório Central de Saúde Pública do
Estado de Minas Gerais, Instituto Octávio Magalhães Fundação Ezequiel Dias, Belo Horizonte, Minas Gerais, Brazil, 5 Laboratório Central de Saúde Pública do
Estado do Paraná, Curitiba, Paraná, Brazil, 6 Universidade Luterana do Brasil, Porto Alegre, Rio Grande do Sul, Brazil
Abstract
Although surveillance efforts that monitor the emergence of drug-resistant strains of influenza are critical, systematic
analysis is overlooked in most developing countries. We report on the occurrence of strains of pandemic influenza
A(H1N1)pdm09 with resistance and decreased susceptibility to oseltamivir (OST) in Brazil in 2009, 2011 and 2012.
We found 7 mutant viruses, 2 with the mutation S247N and other 5 with the mutation H275Y. Most of these viruses
were from samples concentrated in the southern region of Brazil. Some of these resistant viruses were detected prior
to the initiation of OST treatment, suggesting that community transmission of mutant viruses may exist. Moreover, we
show that one of these OST-resistant (H275Y) strains of A(H1N1)pdm09 was discovered in the tri-border region
between Brazil, Argentina and Paraguay, highlighting that this strain could also be found in other Latin American
countries. Our findings reinforce the importance of enhanced antiviral resistance surveillance in Brazil and in other
Latin American countries to confirm or rule out the community transmission of OST-resistant strains of
A(H1N1)pdm09.
Citation: Souza TML, Resende PC, Fintelman-Rodrigues N, Gregianini TS, Ikuta N, et al. (2013) Detection of Oseltamivir-Resistant Pandemic Influenza
A(H1N1)pdm2009 in Brazil: Can Community Transmission Be Ruled Out? PLoS ONE 8(11): e80081. doi:10.1371/journal.pone.0080081
Editor: Hiroshi Nishiura, The University of Tokyo, Japan
Received April 29, 2013; Accepted October 9, 2013; Published November 11, 2013
Copyright: © 2013 Souza et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was supported by Instituto Oswaldo Cruz/Fiocruz (www.ioc.fiocruz.br) and Brazilian Ministry of Health/Decit and SVS (http://
dtr2001.saude.gov.br/sctie/decit/index.htm). MMS was supported in part by individual research grants from CNPq (www.cnpq.br). MMS and TLMS used, in
part, individual research grants from FAPERJ (www.faperj.br) and CNPq. Thanks are also due to IOC/Fiocruz for providing PCR and NFR fellowships. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: tmoreno@ioc.fiocruz.br
Introduction
Influenza causes respiratory tract infection and is associated
with high rates of morbidity and mortality, which can be more
severe in pandemic periods. Although vaccines against
influenza are available, changes in their antigenic composition
are a necessary consequence of viral escape from immune
response ­ leading to mismatch between the vaccine and
circulating strains [1]. Influenza vaccination is generally
recommended for individuals at higher risk of influenza-
associated complications [2]. Therefore, anti-influenza drugs
are essential for prophylaxis and therapeutic interventions [3].
Neuraminidase inhibitors (NAIs), such as oseltamivir (OST),
are the main anti-influenza drugs in clinical use [3], as
resistance to adamantanes has become common [3]. Before
the 2009 pandemic, OST-resistant strains of seasonal human
influenza A(H1N1) viruses had already been detected [3].
Since the 2009 pandemic, the use of OST has grown,
potentially most importantly imposing selective pressures on
the pandemic influenza A(H1N1)pdm09 virus [3]. Therefore,
enhanced surveillance capacity to detect the emergence of
NAI-resistant strains of A(H1N1)pdm09 should be developed,
especially in developing countries, where the scenario is even
more challenging due to limited resources and/or laboratory/
epidemiological capacities for surveillance. The global
circulation of OST-resistant strains of A(H1N1)pdm09 is
approximately 1 % [4,5], and concerns about community
circulation of these agents have been raised [4,5]. It is
PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e80081
unknown for most of the developing countries if there is
community circulation of these agents. In 2009 and 2010, our
study on immunocompromised, hospitalised or deceased
patients did not detect the circulation of OST-resistant strains
of A(H1N1)pdm09 in Brazil [6,7]. Nevertheless, our more
recent data demonstrated that community transmission of
mutant viruses might occur in Brazil. In this article, we discuss
possibilities of community transmission in the discovery context
of H275Y and S247N viruses.
Material and Methods
Ethics statement
Ethic committee approval and need for informed consent
have been waived for this study because influenza surveillance
is covered by Brazilian public health policies and all data were
analysed in an anonymous fashion. This is in compliance with
Decree 05 of February 21, 2006 the Secretariat of Health
Surveillance of the ministry of health [8]. This states that: "The
results of laboratory tests of disease of immediate notification
listed in Annex III of this Decree shall be notified by the national
reference laboratories". Within Annex III, in sub-item II, human
influenza is listed as a disease for which of results of samples
from research outbreaks should be notified. The mentioned
Decree is update from time to time according to needs imposed
by public health and preceded by the Brazilian Law No. 6,259,
of October 30, 1975 [9]. The second article of this law states
that: "the action of epidemiological surveillance comprehends
the information, investigations and necessary surveys for
planning and evaluation the measures of disease control and
situations of health problems", which is the case when
community transmission of OST-resistant strains of influenza
seems to be occurring.
Patients and data collection
Our laboratory is the National Influenza Center (NIC) in
Brazil, we continuously receive a sub-set of samples from the
influenza surveillance system from individuals with fever
(>37.8°C) and respiratory influenza-like illness [10], who have
been treated according to Brazilian guidelines [11]. The
samples were accompanied by clinical-epidemiological forms
containing at least some basic information, such as patient
initials, gender, age, city/state of onset of illness and the dates
of the beginning of the symptoms and sample collection. Other
clinical characteristics of the patients were collected non-
systematically. It is important to note that we used convenience
samples for our study, which makes our analysis more
convenient for research purposes than as final epidemiological
data.
Sample collection and diagnosis
Nasopharyngeal Dacron swabs or aspirates (NPAs) were
collected, and RNA was extracted using a viral RNA mini kit
(Qiagen, CA), according to the manufacturer's instructions.
RNA was eluted in 10 mM Tris-HCl, pH 8.0, with 1 mM EDTA
(TE buffer) and stored at -70°C. This RNA was used for one-
step Real-time RT-PCR assays for influenza subtyping
according to the World Health Organisation (WHO)
recommendations [10].
Cells and virus isolation
Madin-Darby canine kidney (MDCK) cells were cultured in
Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand
Island, NY) supplemented with 10 % foetal bovine serum (FBS;
Hyclone, Logan, Utah), 100 U/mL penicillin and 100 µg/mL
streptomycin and were incubated at 37 °C in 5 % CO2 [10].
Virus isolation was performed in either 9-day-old embryonated
eggs or in MDCK cells, as previously described [10]. We
confirmed viral isolation using haemagglutination,
neuraminidase activity or real-time RT-PCR assays [10,12,13].
Viruses were passaged no more than three times.
Functional antiviral assay
To determine the IC50 values of our samples to OST
carboxylate, we performed functional antiviral assays using the
NA-StarTM
assay kit (Life Technologies, CA), according to the
manufacturer's instructions. Briefly, the viral isolates obtained
from MDCKs or embryonated eggs were centrifuged at 800 x g
for 10 min to remove cellular debris. Next, the neuraminidase
(NA) activities in these samples were titred as a two-fold
dilution to determine the working dilution of the virus. After that,
the NA activity of the viral isolates was measured in the
presence of different concentrations of OST carboxylate. For
comparison, assays with wild-type and resistant standard
strains of influenza A(H1N1)pdm09 were run in parallel to
validate the IC50 values of our isolates, as recommended [10].
The WT (H275) and resistant (H275Y) strains of
A(H1N1)pdm09 virus A/Perth/265/2009 and A/Perth/261/2009,
respectively, were kindly donated by isirv-AVG (formally the
NISN group).
Molecular antiviral assays
Single nucleotide polymorphisms (SNP) in the NA gene were
analysed by pyrosequencing, as described previously [14]. The
RT-PCR was performed using biotinylated primers and
SuperScript III/ Platinum Taq DNA Polymerase in a one-step
reaction (Invitrogen, CA). The biotinylated RT-PCR products
were then mixed with streptavidin and washed to obtain a
biotinylated single-stranded DNA. This DNA was used as a
template for hybridisation to residue-specific sequencing
primers [14].
Alternatively, the whole NA gene was sequenced by Sanger
sequencing according to a protocol described elsewhere [15].
The amplified RT-PCR products were purified using the
QIAquick PCR Purification kit (QIAGEN, Valencia, CA) and
sequenced using a BigDye Terminator v3.1 Cycle Sequencing
kit (Life technologies, CA). The products were analysed in an
ABI Prism 3130XL genetic analyser (Life technologies). The
dataset generated were assembled in Sequencher 5.0 software
(GeneCodes Corporation, Michigan, USA) with a NA reference
sequence, A/California/4/2009 (GenBank accession number:
FJ966084). Of note, N1 numbering was used for NA
throughout this study.
Oseltamivir-Resistant A(H1N1)pdm09 in Brazil
PLOS ONE | www.plosone.org 2 November 2013 | Volume 8 | Issue 11 | e80081
Results
Sampling and clinical data
From 2011 forward, our laboratory-based influenza antiviral
resistance surveillance became broader than in previous years
[7] because every current A(H1N1)pdm09-positive NPA
received by our laboratory, from patients in 4 out of 5 regions of
Brazil (498 cases in total: 163 from 2011 and 335 from 2012),
was screened for H275Y and S247N mutations in the
neuraminidase (NA) gene. These mutations were chosen for
initial investigation because H275Y can lead to resistance to
OST [16], and S247N was described early in the pandemic as
decreasing viral susceptibility to OST [16,17] and, more
recently, as a potentiator of H275Y-imposed resistance against
OST [18]. In line with the World Health Organization (WHO),
we considered the detection of H275Y and S247N as predictive
of strains with resistance and decreased susceptibility to OST,
respectively [16,17]. We managed to successfully perform
pyrosequencing analysis of 325 and 300 samples to detect the
H275Y and S247N mutations, respectively.
The studied samples belonged to patients with a median age
of 20 years old [ranging from 0 to 79 years old (min. and max.)
or from 3 to 37 years old (quartile deviation)] ­ representing the
majority of individuals not covered by Brazilian policy to receive
influenza vaccination [2]. Approximately 47 % of the individuals
were males. The temporal and geographical distribution of our
samples was representative of influenza activity in Brazil in
2011 and 2012. Samples collected from May to August
(autumn to winter in Brazil) accounted for 70 % of the
specimens. Approximately 80 % and 8 % of the samples were
from the southern and southeastern regions, respectively (the
remaining 12 % were from northern and northeastern regions).
Of the patients with a completed standardised data collection
(231 A(H1N1)pdm09-confirmed patients), 47 % displayed
severe acute respiratory infection (SARI) during the onset of
illness, 21 % had been vaccinated against influenza, 20 %
were hospitalised, 11 % had other predisposing conditions
(HIV/AIDS, cancer, Down syndrome and heavy drug users)
and 6.6 % were deceased. Systematic information about the
use of OST therapy was rarely collected (5.6 %), which may
reflect the absence of this specific field in the standardised data
collection form and negligence concerning a critical issue of
influenza surveillance.
Detection of mutant viruses
As we mentioned above, our sampling was orientated by the
screening of H275Y and S247N mutations by pyrosequencing.
Among these successfully analysed samples, Sanger
sequencing was performed with a representative number of
specimens, 56 (GenBank accession numbers: KC984872-
KC984935). This subset of specimens had WT amino acid
residues at positions V116, I117, E119, Q136, K150, D151,
D199 and I223. Seven mutant viruses were detected, by both
methodologies, 2 S247N and 5 H275Y viruses (Table 1).
Functional assays were successfully performed with 56 virus
isolates. The IC50 values for sensitive and resistant viruses
were 0.8 ± 0.7 nM and 89 ± 23 nM (mean ± SD), respectively
(Figure 1).
The S247N mutation was identified in viruses from 2 patients
(Table 1), including a 37-year-old female from Minas Gerais
(MG) with SARI and diffuse lung infiltrates who was admitted to
the intensive care unit (ICU). She had OST therapy initiated 4
days after the onset of illness, and her NPA sample was
collected before such treatment. She passed away as a
complication of influenza infection. The other patient was a
three-month-old male with Down syndrome and pulmonary
complications, from the state of RS. He required hospitalisation
and died due to influenza infection. No information about OST
use was recorded.
The H275Y A(H1N1)pdm09 virus was found in four other
patients (Table 1), from the Santa Catarina (SC), Parana (PR)
and RS states. The patient from SC, a 36-year-old male,
required ICU admission but had a benign clinical outcome.
Importantly, OST was administered to him 7 days after the
onset of illness, and NPA was collected before that. The patient
from PR, a 12-year-old male, rapidly received OST treatment,
which had been initiated within 24 h after the beginning of the
symptoms. His sample was collected before treatment. He had
a benign clinical outcome. The patients from RS were 28- and
34-year-old males displaying pneumonia. Both patients
survived. These two patients received OST before sample
collection, and 100 % of H275Y quasi-species were observed
in samples from these individuals, both by pyrosequencing and
Sanger sequencing (Table 1). This rapid emergence is very
unusual, suggesting that they might have been primarily
infected by the resistant virus. Of note, all these seven
individuals had not been vaccinated against influenza.
Table 1. Clinical and virological aspects of patients in which OST-resistant strains of influenza were detected.
Patient Mutations Region State Age a Gender Symptoms date Collection date Oseltamivir (Beginning date) Deceased Oseltamivir before sampling
1 H275Y Southern RS 26 M 24-Aug-09 25-Aug-09 NI NI NI
2 S247N Southern RS 3* M NI 2-Jul-11 NI Yes NI
3 H275Y Southern SC 36 M 14-May-12 20-May-12 21-Jul-12 No No
4 H275Y Southern PR 12 M 21-Jun-12 22-Jun-12 22-Jul-12 No No
5 H275Y Southern RS 34 M 28-Jun-12 2-Jul-12 1-Jul-12 No Yes
6 H275Y Southern RS 28 M 22-Jun-12 27-Jun-12 25-Jun-12 No Yes
7 S247N Southeast MG 37 F 1-Jul-12 5-Jul-12 5-Jul-12 Yes NI
a Years old; * months old; NI ­ not informed
doi: 10.1371/journal.pone.0080081.t001
Oseltamivir-Resistant A(H1N1)pdm09 in Brazil
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Considering that the state of RS was more severely affected
during the pandemic [19] and has a temperate climate, we also
studied an additional 100 samples from this state from 2009 to
further evaluate the previous existence of H275Y virus in
Brazil. We indeed found one sample from an
immunocompromised 28-years-old male with the H275Y
mutation. Information on the use of OST or clinical outcome
was not available for this patient. In fact, he passed away a
month after the onset of illness, and reasons other than
influenza cannot be ruled out.
Discussion
In this work, some of the OST-resistant viruses and viruses
with decreased susceptibility to this drug were detected after
the beginning of antiviral treatment, such as for the two patients
from RS and for another from MG (Table 1, patients 5 to 7).
Although these results may suggest the emergence of mutant
virus due to selective pressures imposed by the treatment, the
absence of any sample prior to treatment makes it difficult to
confirm. Moreover, we detected in patients 5 to 7 100 % of
quasi-species in their samples (by both pyrosequencing and
Sanger) at zero to two days after OST initiation. This short time
frame may also jeopardise the conclusion that mutant viruses
were generated by selective pressure. Thus, the suggestion of
community transmission of H275Y virus may be not limited to
patients from PR and SC states, for whom resistant viruses
were detected before antiviral therapy initiation occurred (Table
1, patients 3 and 4) and to the resistant virus found in 2009 in a
patient with scarce medical records. Thus, continuous and
systematic studies on the circulation of NAI-resistant strains of
influenza in Brazil should be conducted to confirm or rule out
this suggestion. In fact, community transmission of H275Y
viruses has been described in other parts of the world [4,5] and
is possible due to a new balance, which favours viral fitness,
between NA and haemagglutinin (HA) activities in this strain
[20].
Figure 1. IC50 values for Brazilian isolates. A total of 56 A(H1N1)pdm09 isolates (at maximum three passages in MDCKs) were
titred, and their IC50 values were determined using NA-StarTM
assay (Brazilian). For comparison, the WT (H275) and resistant
(H275Y) strains of A(H1N1)pdm09 virus A/Perth/265/2009 and A/Perth/261/2009, respectively. The data represent the mean ±
maximum and minimum ranges.
doi: 10.1371/journal.pone.0080081.g001
Oseltamivir-Resistant A(H1N1)pdm09 in Brazil
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Moreover, considering the continental-wide distribution of
Brazil from the equatorial line to sub-tropical areas, the
seasonality and impact of respiratory viruses may be greatly
variable throughout Brazilian territory. The southern states of
Brazil, RS, SC and PR, generally have a temperate climate and
more marked seasonality of influenza than other states. As a
result of that, during the pandemic for example, the mortality
ratio of A(H1N1)pdm09-confirmed deaths for Brazil as a whole
was 1.1 % (1.1 case per 100,000 inhabitants), whereas the
ratio for the southern region was 3.0 % [19]. Thus, high
influenza activities in these states could lead to more rapid viral
evolution, which in the presence of antiviral drugs would lead to
the selection of resistant strains that may be endowed with the
ability for sustained transmission [20]. Considering samples
analysed in this study and in our previous publication [7], over
913 samples of 6303 A(H1N1)pdm09-confirmed cases (> 95 %
confidence interval; CI) were interrogated for the presence of
H275Y mutation from 2009 to 2012 (Table 2). This is the
largest laboratory-based influenza-related study performed in
Brazil in terms of number of cases investigated. We found 4 out
of 5 OST-resistant H275Y in 2012, when nearly all
A(H1N1)pdm09-confirmed cases (> 97 %) were studied for
rresistance emergence (Table 2). In 2012, an incidence of
approximately 1.19 % of OST-resistant strains can be
estimated in Brazil, which is similar to what have been found in
others parts of the world [4,5]. Of note, our study was carried
out with convenience samples received by the National
Influenza Center, rather than being a classical epidemiological
designed investigation.
Another highlight from our study would be the detection of
OST-resistant viruses in Foz do Iguaçu city (Iguaçu Falls) in
the state of PR. This city forms a tri-border with the Argentine
and Paraguayan cities of Puerto Iguazu and Ciudad de Leste,
respectively. Foz do Iguaçu is a cosmopolitan city with over
200,000 inhabitants and more than 1,700,000 inbound and
outbound flights and buses annually at the city's international
airport and bus station [21]. The flow of people between Foz do
Iguaçu and Ciudad de Leste is estimated to be over 16,500
persons daily [21]. Therefore, it seems plausible to believe that
OST-resistant A(H1N1)pdm09 could also be found in
Paraguay. To our knowledge, there are no scientific studies on
influenza resistance to NAIs in Paraguay. In Argentina, few
cases of OST-resistant strains of pandemic virus have been
described in its capital [22]. No information about H275Y virus
strains in the northeastern region of Argentina was found.
Therefore, our data reinforce the importance of enhanced
surveillance on the emergence of NAI-resistant strains of
influenza in developing countries because such emergence
may drive critical decisions in public health, ranging from drug
stockpiling to health interventions.
Author Contributions
Conceived and designed the experiments: TMLS PCR NFR
TSG NI SBF ALFC MCDR MMS. Performed the experiments:
PCR NFR. Analyzed the data: TMLS PCR NFR TSG NI SBF
ALFC MCDR MMS. Contributed reagents/materials/analysis
tools: TMLS PCR NFR TSG NI SBF ALFC MCDR MMS. Wrote
the manuscript: TMLS PCR NFR MMS. Analyzed the data and
performed laboratory-based assays: TMLS PCR NFR MMS.
Performed influenza surveillance data collection: TSG SBF
ALFC NI MCDR.
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