Transcript Epidemiology of Influenza A Virus Circulating In Kansas

SWACM
2009
EPIDEMIOLOGY OF INFLUENZA A VIRUS CIRCULATING IN KANSAS CITY: SUBTYPES AND RESISTANCE
TO ANTIVIRAL DRUGS (2007 - 2009)
Suresh B. Selvaraju and Rangaraj Selvarangan
Children’s Mercy Hospitals and Clinics, Kansas City, MO 64108
Contact: [email protected]
ABSTRACT
MATERIALS AND METHODS
Background: Influenza viruses are the major causative agents of human acute
respiratory disease world-wide. This year CDC reported an increase in antiviral
drug resistance to oseltamivir among H1N1 strains. Further, H3N2 strains retained
resistance to adamantanes. The alarming increase in drug resistance among
evolving subtypes of influenza A viruses warrant regional investigation on
epidemiology of these resistant strains.
Aim: The aim of this study was to investigate the oseltamivir and adamantane
resistance among H1 and H3 strains of influenza viruses in Kansas City during
three subsequent years, 2007 to 2009.
Methods: A total of 450 Respiratory specimens positive for Influenza A antigen (150
each year,) by rapid antigen test were used for this study. RNA was extracted, cDNA
was prepared and tested on ABI 7500 real-time PCR system. Influenza A positive
specimens were sub-typed as A/H1 or A/H3 types by Taqman-based real-time PCR.
Influenza A/H1 and A/H3 strains were tested for their susceptibility to Oseltamivir
(H274Y mutation) and adamantanes (S31N mutation) using genotyping assays.
Results: The influenza A H1/H3 typing results showed that H1 type was
predominant during the 2007 (n=74; 49.3%) and 2009 (n=119; 79.3%) seasons when
H3 type dominated the 2008 (n=82; 54.7%) season. The Oseltamivir resistance
(H274Y) in the A/H1 type was 100% (113/113) for 2009 strains, which is similar to the
99.8% of nationwide surveillance report by the CDC. Interestingly, only 0.3%
(n=1/32) of neuraminidase inhibitor resistance was observed during 2008 season
when CDC reported about 10.9%. During 2007 season 100% oseltamivir
susceptibility (n=69/69) was observed among A/H1 strains. Adamantane resistance
was 100% (n=2/2) among 2007 A/H3 strains and 94% (77/82) during 2008 season.
One A/H3 strain was typed during 2009 season and it was susceptible to
adamantanes.
Conclusions: Our regional antiviral surveillance study helped in understanding the
epidemiology of drug resistance among influenza viruses in Kansas City area. The
simple and rapid real-time PCR-based methods validated for typing influenza A
H1/H3 strains and for detection of both oseltamivir (H274Y mutation) and
adamantane (S31N) resistance will be useful for routine monitoring of antiviral
resistance in clinical laboratories. The demographic and clinical data of patients
infected with antiviral drug resistant and susceptible influenza A viruses are being
reviewed.
Specimens. A total of 450 (150 each year) specimens that were characterized by rapid
antigen tests as positive for influenza A strains were selected for this study.
Controls. The oseltamivir resistant and susceptible reference strains, A/New Jersey/15/07
(H1N1) and A/California/27/07(H1N1), respectively, were obtained from Dr. Larisa V.
Gubareva, CDC, Atlanta. These reference strains were included as positive and negative
controls in real-time PCR experiments.
Influenza A-H1/H3 typing. Nucleic acid was extracted from 200 µl of specimen by using
either NucliSENS easyMAG (bioMerieux Inc., Durham, NC) automated extraction or
QuickGene-Mini80 (AutoGen, Holliston, MA) semi-automated extraction systems. cDNA was
prepared using TaqMan reverse transcription reagents as per the manufacturer’s
recommendation (Applied Biosystems, Foster City, CA). Influenza A positive specimens
were sub-typed for A/H1 and A/H3 types by Taqman probe-based real-time PCR using 7500
real-time PCR system as described earlier (Suwannakarn, et al. 2008) with a little
modification in A/H1-specific forward primer (Table 1).
H274Y and S31N mutation detection. All A/H1 strains were tested for oseltamivir resistance
(H274Y mutation) and A/H3 strains were tested for adamantane resistance (S31N mutation).
Genotyping-based real-time PCR reactions were performed using two oligonucleotide
probes with single-base variation for resistant and susceptible strains, respectively, as
described by Carr, et al. 2008 and Cheng, et al. 2009. The primers and probes sequences
used for this study are given in Table 1.
INTRODUCTION
Influenza viruses are classified into types A, B and C, based on antigenic
differences in their nucleoprotein (NP) and matrix (M) protein. Influenza viruses A
and B are the major causative agents of human acute respiratory disease worldwide. Infants, elderly persons, and individuals with compromised cardiac,
pulmonary, or immune function are at the greatest risk of serious complications
from these viruses. Most influenza pandemics are associated with type A which
represents the most widespread human pathogen. Influenza A viruses can be
classified further into subtypes H1N1 and H3N2 based on the antigenic variations.
Adamantane derivatives (amantadine and rimantadine) and oseltamivir were
recommended drugs for antiviral treatment however, adamantanes were not
preferred now due to the high proportion of resistance among all circulating strains
(Bright et al. 2005). Later, oseltamivir is used widely as an antiviral drug for
influenza that potentially inhibit neuraminidase (NA) protein of influenza virus (Kim
et al. 1997). Until recently, oseltamivir resistance occurred in <1% of circulating
viruses globally. An increased number of influenza A viruses (H1N1) with resistance
to oseltamivir was first reported by Norway in 2008. The viruses carried specific
Histidine-to-Tyrosine mutation at position 274 in the NA protein that confers
resistance to oseltamivir (Lackenby et al. 2008). Most recently, CDC has observed
an increase (98% in 2009 as compared to 10.9% in 2008) in oseltamivir antiviral
drug resistance among H1N1 strains of influenza virus in the USA. Further, these
oseltamivir resistant strains reverted to being adamantine susceptible, while the
H3N2 strains retain adamantine resistance and oseltamivir susceptibility. The
alarming increase and diversity in drug resistance among evolving strains of
influenza A viruses warrants careful investigation on epidemiology of these
resistant strains.
3. Number of A/H1-A/H3 strains typed for three subsequent years
Drug resistance
A
Sequences
5’-AYTACTGGACYYTGCTDRAA -3’
FluA-H1-R
5’-AAGCCTCTACTCAGTGCGAA-3’
5’-FAM-TTGAGGCAAATGGAAATCTAATAGC ’-BHQ-1 -3
This study and
Suwannakarn,
et al. 2008
OR-Genotyping
FluAN1-H275Y-F
5’-CCGCCTCGTACAAAATCTTCAAGA-3’
FluAN1-H275Y-R
5’-CAGTGTCTGGGTAACAGGAACATT-3’
FluAN1-H275Y-OR
5’-FAM-CCTCATAATAAAAATTG-MGBNFQ-3’
FluAN1-H275Y-OS
5’-VIC-CTCATAATGAAAATTG-MGBNFQ-3’
Carr, et al. 2008
S31N-Genotyping
H3DR-F
5’- TGCAGATGCAACGATTCAAGTG -3’
H3DR-R
5’- AAAAGACGATCAAGAATCCACAATATCA -3’
H3-31N-P
5’-FAM-CCGCGAATATCATTGGGA-MGBNFQ-3’
H3-31S-P
5’-VIC-CCGCGAGTATCATTGGGA- MGBNFQ-3’
Cheng, et al.
2009
2. Number of A/H1-A/H3 strains typed for three subsequent years
2007
A/H1
2008
2009
74/150 (49.3%)
32/150 (21.3%)
119/150 (79.3%)
2175 (26%)
334 (21.3%)
2007
0/74 (0%)
4/588 (0.7%)
2/2 (100%)
Not available
2008
1/32 (3.1%)
111/1020 (10.9)
77/82 (94%)
524/525 (99.8%)
2009
113/113 (100%)
1141/1146 (99.6%)
0/2 (0%)
245/245 (100%)
A/H1
Year
2006’07
2007’08
A/H3
NY
WI
NY
WI
0/37
(0%)
0/44
(0%)
0/107
(0%)
1/42
(2.4%)
38/42
(90.5%)
101/101
(100%)
29/39
(74.4%)
44/44
(100%)
Drug resistance
Oseltamivir
Adamantanes (H3N2)
(H1N1)
NY (2001
NY
WI
WI
– ‘07)
0/18
Not
30/35
Not
(0%)
available (85.7%)
available
5/45
8/46
Not
Not
(11.1%) (17.4%) available available
 During 2006-07 season A/H1 type was predominant (50%) in our region which
agreed with CDC annual report (62%). Interestingly, A/H3 strain was
predominant type in NY (90%) and WI (74%) regions. Both regions did not
observe any A/H1 type during that season.
 During 2007-08 season 54.7% of typed strains were A/H3 strain in our region
and it is lower than CDC report (74%). However, NY and WI region observed
100% of A/H3 strains in their population when A/H1 was only 2.4%. In our
region 21% of types strains were A/H1 and it is similar to CDC report of 26%.
In 2008-09, A/H1 population was predominant before novel H1N1s were
reported in the USA.
 All A/H1 strains were susceptible or showed very less level of resistance
(0.7%; CDC) to oseltamivir during 2007 season. All A/H3 strains were resistant
to adamantanes in KC (100%) and 85.7% of strains were resistant in NY region.
During 2008 season only 0.3% (1/32) of oseltamivir-resistant A/H1 was
observed in KC region while CDC and NY region reported about 10-11% and
WI region showed 17%. During 2009 season, all A/H1 strains have become
resistant to oseltamivir in KC region and it is similar to CDC report.
 The untypeable strains vary from 20% (n=30) in 2009 to 49% (n=74) in 2007.
Similarly, in NY region 54.5% of the influenza strains were untypeable.
 The observed differences in both typing and antiviral drug resistance among
circulating influenza strains encourage regional based studies for better
understanding of epidemiology and drug resistance.
REFERENCES
A/H3
3912 (62.3%)
CDC
A/H1-H3 typing
A/H1-H3 typing
CDC
Kansas City
 The demographic and clinical data of patients infected with antiviral drug
resistant and susceptible influenza A strains are being reviewed.
RESULTS
Kansas City
CDC
References
FluA-H1-F
Year
Kansas City
CONCLUSIONS
A/H1-A/H3 typing
Flu-H1-P
Adamantanes (A/H3 strains)
4. New York (NY) and Wisconsin (WI) regional surveillance study
1. Primers and probes sequences used in this study*
PCR reaction
Oseltamivir (A/H1 strains)
Year
Kansas City
2/150 (1.3%)
82/150 (54.7%)
2/150 (1.3%)
CDC
2368 (37.7%)
6115 (74%)
300 (19.1)
1.
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