﻿Published Ahead of Print 10 March 2010.
10.1128/JCM.02540-09.
2010, 48(5):1884. DOI:
J. Clin. Microbiol.
Mendelson
Orzitzer, Roberto Azar, Zehava Grossman and Ella
Meningher, Shira Hirsh, Jana Robinov, Virginia Levy, Sara
Musa Hindiyeh, Daniela Ram, Michal Mandelboim, Tal
Reverse Transcriptase PCR
Resistance Mutation H275Y by Real-Time
(H1N1) 2009 Virus Neuraminidase
Rapid Detection of Influenza A Pandemic
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JOURNAL OF CLINICAL MICROBIOLOGY, May 2010, p. 1884­1887 Vol. 48, No. 5
0095-1137/10/$12.00 doi:10.1128/JCM.02540-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Rapid Detection of Influenza A Pandemic (H1N1) 2009
Virus Neuraminidase Resistance Mutation H275Y by
Real-Time Reverse Transcriptase PCR
Musa Hindiyeh,1
* Daniela Ram,1
Michal Mandelboim,1,2
Tal Meningher,2
Shira Hirsh,2
Jana Robinov,1
Virginia Levy,1
Sara Orzitzer,1
Roberto Azar,1
Zehava Grossman,1,3
and Ella Mendelson1,3
Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Tel-Hashomer,
Israel1
; Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel2
; and Department of Epidemiology and
Preventive Medicine, School of Public Health, Sackler Faculty of Medicine,
Tel-Aviv University, Tel-Aviv, Israel3
Received 31 December 2009/Returned for modification 17 February 2010/Accepted 3 March 2010
The emergence of oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus highlights the need for
rapid oseltamivir resistance screening. We report the development and validation of high-throughput real-time
reverse transcriptase PCR assays for the detection of the H275Y substitution in the neuraminidase 1 gene that
can be accomplished in 3 to 4 h.
The continuous spread of influenza A pandemic (H1N1)
2009 virus [pandemic (H1N1) 2009 virus] is compelling the use
of the neuraminidase inhibitors (NAI) oseltamivir (Tamiflu)
and zanamivir (Relenza) to treat infected patients in order to
minimize further spread of the virus. NAI are the only avail-
able antivirals against this pandemic (H1N1) 2009 virus, be-
cause adamantanes (amantadine and rimantadine) are com-
pletely ineffective (4, 16). The extensive use of these NAI, in
particular oseltamivir, is creating an unprecedented selective
pressure for the emergence and spread of drug-resistant viral
strains. The World Health Organization (WHO) has recom-
mended vigilant monitoring for oseltamivir-resistant viruses,
because the number of documented sporadic resistant cases is
increasing, reaching nearly 100 cases worldwide by 15 Decem-
ber 2009 (18). Recently, the U.S. Centers for Disease Control
and Prevention (CDC) reported the first human-to-human
transmission of oseltamivir-resistant pandemic (H1N1) 2009
virus in two summer campers receiving oseltamivir prophylaxis
(2). In addition, the WHO has announced outbreaks of osel-
tamivir-resistant pandemic (H1N1) 2009 virus in two immuno-
compromised groups, one in North Carolina and the other in
Wales, United Kingdom (18). In both outbreaks, human-to-
human oseltamivir-resistant virus transmission was suspected.
At least two mechanisms contribute to neuraminidase resis-
tance in the seasonal influenza viruses H1N1 and avian influ-
enza H5N1 (12). One mechanism involves reduction of the
binding efficiency of virus hemagglutinin to its receptor. The
other is associated with amino acid substitutions in and around
the NA active site, of which the substitution at position 275
(histidine to tyrosine [H275Y]) is the most common (7, 11, 6).
Sequence analysis of the hemagglutinin gene is not a reliable
indicator of neuraminidase drug resistance phenotype, but his-
tidine-to-tyrosine (H275Y) substitution in the active site of the
NA-1 gene does indicate reduced binding affinity of the neur-
aminidase inhibitor oseltamivir. Recent reports characterizing
the current oseltamivir-resistant pandemic (H1N1) 2009 virus
confirmed the presence of the H275Y mutation (2, 3, 10).
While phenotypic analysis of oseltamivir-resistant influenza
A viruses is widely accepted as the "gold standard" methodol-
ogy for detecting influenza virus drug resistance, genotypic
analysis has been widely utilized to detect a point mutation
(cytosine to thymine) at position 823 of the NA-1 gene that
results in a histidine-to-tyrosine substitution (8, 13). The ge-
notypic assays include sequencing part of the neuraminidase
gene by using the Sanger dideoxy sequencing method or by
pyrosequencing. These assays are labor-intensive, with a long
turnaround time ranging from 24 to 72 h, and require special-
ized equipment and human effort. Moreover, sequencing and
pyrosequencing assays have reduced sensitivities for detecting
low concentrations (15%) of quasispecies present in a pa-
tient's sample (14). Therefore, high-throughput assays with
short turnaround times are needed in order to expedite osel-
tamivir drug resistance detection.
In the present study we validated two real-time reverse
transcriptase PCR (qRT-PCR) assays by utilizing TaqMan
chemistry for the detection of the point mutation (cytosine to
thymine) at position 823 of the NA-1 gene of pandemic
(H1N1) 2009 virus. One set of modified primers and two
probes previously reported by Chutinimitkul et al. were uti-
lized to validate both assays (5). Chutinimitkul et al. initially
validated these primers and probes for the detection of osel-
tamivir resistance in H5N1 isolates (5). The primers utilized in
the present study were modified to increase the assay's sensi-
tivity and specificity to detect pandemic (H1N1) 2009 virus,
while the two minor groove binding (MGB) probes were syn-
thesized as described by Chutinimitkul et al. (5). The modified
primers were forward, 5-GGG GCA GTG GCT GTG TTA-
3, and reverse, 5-AGG GCG TGG ATT GTC TCC-3. The
two MGB probes utilized were sensitive, 5­6-carboxyfluores-
cein (FAM)­TCC TCA TAG TGR TAA TT­3­nonfluorescent
* Corresponding author. Mailing address: Central Virology Labora-
tory, Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel.
Phone: 972-3-530-2066. Fax: 972-3-530-2457. E-mail: hindiyeh@yahoo
.com.

Published ahead of print on 10 March 2010.
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quencher (NFQ), and resistant, 5­FAM­TCC TCA TAG
TAR TAA TT­3­NFQ. The point mutation in the probe was
positioned 7 bases from the 3 end to minimize cross-reactivity
with similar sequences (1). Two qRT-PCR assays were de-
signed, for H275 for the detection of cytosine (sensitive) and
H275Y for the detection of thymine (resistant) at position 823.
This permitted detection of mixed infections of oseltamivir-
resistant and oseltamivir-sensitive viruses. Samples positive for
the H275Y mutation by the TaqMan assay were confirmed by
sequencing part of the neuraminidase gene by utilizing the
same set of primers described above. The sequencing proce-
dures were previously described (9).
Sensitivity of the qRT-PCR assays was optimized by evalu-
ating different concentrations of primers (200, 300, 600, and
900 nM) and probes (100, 200, and 300 nM). The concentra-
tions of primers used in this study that gave the best detection
limits were 600 nM for the primers and 200 nM for the probes.
The AgPath-ID one-step RT-PCR kit (Applied Biosystems-
Ambion, CA) was used to develop both assays. Briefly, two
25-l qRT-PCR mixtures that contained 12.5 l of 2 RT-
PCR buffer, 1 l of 25 RT-PCR enzyme mix, a 600 nM
concentration of each primer, and a 200 nM concentration of
either the sensitive or the resistant probe were prepared to
evaluate 5 l of each patient sample. 5-Carboxy-X-rhodamine,
succinimidyl ester (ROX) present in the 2 RT-PCR buffer
was utilized as an internal reference dye. Amplification and
detection were performed using an Applied Biosystems 7500
platform under the following conditions: 30 min at 48°C for
reverse transcription, 10 min at 95°C to activate AmpliTaq
Gold DNA polymerase, and 50 cycles of 15 s at 95°C and 1 min
at 60°C.
The analytical sensitivity of the H275Y qRT-PCR was de-
termined by 10-fold serial dilution of oseltamivir-resistant pan-
demic (H1N1) 2009 virus RNA isolated from cultured virus of
an Israeli patient [A/Israel/6290/2009(H1N1); accession num-
ber GU371269]. Briefly, viral RNA was extracted from 200 l
of A/Israel/6290/2009(H1N1) virus stock (7.8  105
50% tissue
culture infective doses [TCID50]/ml) with a NucliSENS
easyMAG extractor (bioMe
´rieux, Boxtel, Netherlands). RNA
was eluted in 55 l of elution buffer and stored at 70°C.
Analysis of the 10-fold serial dilutions of viral RNA for the
H275Y mutation in triplicate by qRT-PCR revealed that the
assay's analytical sensitivity was 0.014 TCID50 with 97.2% am-
plification efficiency (Fig. 1A). On the other hand, the analyt-
ical sensitivity of the oseltamivir-sensitive (H275) qRT-PCR
assay after 10-fold serial dilutions of extracted RNA from
3.7  105
TCID50/ml cultured virus from an Israeli patient
[A/Israel/119/2009(H1N1)] was 0.0019 TCID50 (Fig. 1B). The
amplification efficiency of the H275 qRT-PCR assay was 100%.
The H275 assay's analytical sensitivity was confirmed in tripli-
cate with similar results.
The specificities of the two assays were investigated by an-
alyzing one clinical isolate of each of the common human
respiratory viruses that were strongly positive by real-time
PCR analysis (threshold cycle [Ct], 25). These included sea-
sonal influenza A viruses (H1N1 and H3N2), influenza B virus,
human metapneumovirus, respiratory syncytial virus (RSV)
types A and B, and adenovirus type 2. Both assays were spe-
cific, since no positive signals were noted in the analysis of
these viruses (data not shown).
Validation of the two assays, H275 (sensitive) and H275Y
(resistant), was performed on 31 patient samples collected
between 15 June 2009 and 1 December 2009 from 18 patients
with a high risk of developing oseltamivir resistance. This
group included hospitalized patients who were treated with at
least one course of oseltamivir without improvement. Most of
the patients were immunologically suppressed due to various
underlying diseases. All 31 patient samples were positive for
pandemic (H1N1) 2009 virus (Ct, 34) based on qRT-PCR
assay as previously described by Panning et al. (15).
Of the 18 patients, 6 (33%) were positive for the H275Y
mutation by qRT-PCR. Table 1 summarizes the data for all of
these patients. In patient 1, oseltamivir-resistant virus was first
detected in a sample obtained after 9 days of oseltamivir ther-
apy. The first sample of patient 1 tested sensitive for oseltami-
vir on 30 July 2009, and mixed sensitive and resistant viruses
were detected in this patient's second sample when tested on 9
August 2009. Sequence analysis of the neuraminidase genes
amplified from the two samples confirmed the results of the
qRT-PCR assays. Similar results were obtained for patient 4.
Interestingly, only oseltamivir-resistant viruses were detected
in samples from patients 2, 3, and 5. These results were also
confirmed by sequence analysis. Since the initial samples col-
lected from these three patients at the onset of clinical symp-
toms contained resistant viruses, it is highly unlikely that they
were infected with sensitive strains. Rather, it appears that they
were initially infected with resistant viruses as previously re-
ported by the CDC and the WHO (2, 18).
FIG. 1. Linear limits of detection of the H275Y (A) and H275 (B) qRT-PCR assays. Serial (10-fold) dilutions of influenza A pandemic (H1N1)
2009 virus resistant (A) or sensitive (B) to oseltamivir were prepared and tested in qRT-PCR assays. Ct values were obtained for each dilution and
are plotted against the TCID50 value.
VOL. 48, 2010 NOTES 1885
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In patient 6, qRT-PCR results indicated the presence of
mixed sensitive and resistant viruses in the patient's first sam-
ple, while sequence analysis revealed only resistant virus in that
sample (Table 1). qRT-PCR showed that there was a 3-Ct
difference, with 1 log more resistant virus than sensitive virus.
The lower sensitivity of the sequencing assay could in part
explain why the 10% portion of sensitive virus in the pool
containing mostly resistant virus was not detected. Patient 6
was indeed infected with an oseltamivir-sensitive virus, as the
presence of this virus was confirmed both by the qRT-PCR
assay and by sequence analysis of a positive endotracheal as-
pirate sample (Table 1).
The qRT-PCR assays were also used to screen 120 patient
samples that tested positive for pandemic (H1N1) 2009 virus
between 15 June 2009 and 1 December 2009. The purpose was
to determine whether oseltamivir-resistant virus was circulat-
ing in the community and to determine if the H275Y assay
cross-reacted with oseltamivir-sensitive virus in clinical sam-
ples. Patients in this study group had not been treated with
oseltamivir at the time of sample collection. All samples tested
positive in the H275 assay, while none tested positive using the
H275Y qRT-PCR assay. Thus, the H275Y assay was specific to
detecting resistant virus only, and infection with oseltamivir-
resistant pandemic (H1N1) 2009 virus is apparently still a rare
event at this time.
More recently, van der Vries et al. reported the develop-
ment of three highly sensitive qRT-PCR assays for detection of
the pandemic (H1N1) 2009 virus H1 and N1 genes and of the
H275Y oseltamivir resistance mutation (17). However, oselta-
mivir-resistant virus was not detected in the clinical samples
tested; thus, the clinical sensitivity was not evaluated.
One of the drawbacks of any qRT-PCR assay is that it is
sequence specific, and any genetic shift in the primer or probe
locations can alter the assay performance. The current pan-
demic (H1N1) 2009 virus may yet change further in the region
encompassing the H275 location, other than the rare resistance
mutation H275Y, and this assay will not detect such changes.
This limitation can be partially overcome by sequencing or
pyrosequencing, which can detect multiple nucleotide substi-
tutions simultaneously (6). Multiplexing the two described as-
says would improve the flow of work and further reduce the
cost.
The recent report of two oseltamivir-resistant pandemic
(H1N1) 2009 virus outbreaks, in the United Kingdom and the
United States, is a strong reminder that countries should fol-
low the WHO recommendations and develop a vigilant system
for detecting oseltamivir-resistant viruses (18). The qRT-PCR
assay described in this report can be used to screen large
numbers of clinical samples for drug resistance with a very
short turnaround time (3 to 4 h) and at a very low cost. In
addition, this assay detects low quantities of either sensitive or
resistant viruses in a mixed infection, whereas sequencing fails
to detect the minor virus type.
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no.
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(day-mo-yr)
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N*823
sequencing
resultb
Influenza A
pandemic
(H1N1) 2009
H275
(sensitive)
H275Y
(resistant)
Interpretation
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a
Neg, negative result; ND, not determined; WT, wild type.
b
The nucleotide(s) found at position 823 (N*823) in the neuraminidase 1 gene.
1886 NOTES J. CLIN. MICROBIOL.
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