Detection of Oseltamivir Drug Resistance in Seasonal and Pandemic Influenza A 2009
H1n1 Virus From Patients at JaipurResearch Protocol
For Registration for Ph.D. Programme
Submitted by
KHUSHBU TRIVEDI
Under the Guidance of
Dr. Seema Bhadauria
Department of Microbiology
JECRC University
Dr. Bharti Malhotra
Department of Microbiology
SMS Medical College
2014
JECRC UNIVERSITY, JAIPUR
1
CONTENTS
1. INTRODUCTION
2. PART REVIEW OF LITERATURE
3. STATEMENT OF THE RESEARCH PROBLEM
4. OBJECTIVES OF THE STUDY
5. SCOPE OF THE STUDY
6. SIGNIFICANCE OF THE STUDY
7. LIMITATIONS
8. METHODOLOGY
9. MONTH WISE PLAN OF WORK
10.MONTH WISE PLAN OF WORK(GRAPHICAL REPRESENTATION)
11.REFERENCES
2
INTRODUCTION
Respiratory illnesses due to influenza virus infection are a major cause of morbidity
and mortality worldwide and a leading cause of deaths in developing countries.
Globally influenza is responsible for 250,000 to 500,000 deaths annually.1 Influenza
viruses are single-stranded, negative-sense enveloped RNA viruses of the family
Orthomyxoviridae, are of three types: A, B and C; types B and C are generally only
found in humans. Type A influenza viruses are responsible for most seasonal
influenza epidemic morbidity and mortality, and all influenza pandemic strains. Type
A is further divided into subtypes by antigenic characterization of the two surface
glycoproteins hemagglutinin (HA) and neuraminidase (NA) and can be classified into
sixteen HA and nine NA subtype.2 Influenza A viruses evolve rapidly due to both
antigenic drift and reassortment (antigenic shift), and in humans, new antigenic
variants emerge constantly to give rise to yearly epidemics.3 When an antigenically
novel strain emerges and circulates widely in people it can cause a pandemic. Three
influenza pandemics have been reported in past history A/H1N1 in 1918, A/H2N2
in1957 and A/H3N2 in1968 and the recent 2009 pandemic A/H1N1leading to high
mortality and morbidity.4
The recent pandemic Influenza A HINI 2009 virus emerged in humans in early April
2009 in Mexico and California. The H1N1 strain then quickly spread worldwide
through human-to-human transmission, though the swine flu pandemic was declared
over by WHO on August 10, 2010. The initial death count found that there were
18,500 H1N1 casualties between April 2009 and August 2010, but as these figure
were based on laboratory-confirmed tests of victims, it is likely that these figures are
vastly underestimated than the true number of deaths. The recent CDC report puts
the estimate at 151,700 to 575,400 deaths during the first year of the flu virus
spread, with a median of about 284,500(CDC).5
As per the current status based on Flu Net reporting (as of 27 August 2013, 09:20
UTC), during weeks 32 to 33 (4 August 2013 to 17 August 2013), National Influenza
Centres (NICs) and other national influenza laboratories from 64 countries, areas or
territories reported data. The WHO GISRS laboratories tested more than 12286
specimens. 1224 were positive for influenza viruses, of which 1009 (82.4%) were
type3d as influenza A and 215 (17.6%) as influenza B. Of the sub-typed influenza A
3
viruses, 402 (43.8%) were influenza A (H1N1) pdm09 and 516 (56.2%) were
influenza A (H3N2). Of the characterized B viruses, 10 (66.7%) belong to the B-
Yamagata lineage and 5 (33.3%) to the B-Victoria lineage. India is also still having
Influenza A 2009 H1N1 (swine flu) patients in 2013 with significant morbidity and
mortality, especially in North India with Delhi, Rajasthan, Gujarat and Haryana
among the worst-hit states. While Delhi has had 1,267 cases, Gujarat has reported
over 260 cases, followed by Haryana with over 300 cases. 6
As per the most recent data from Rajasthan total of 4029 cases were tested
during January 1 to September 6 2013 among which 865 were positive for
pandemic influenza 265 died (the highest in the country) (IDSP, Jaipur)
Oseltamivir and zanamivir are the two primary antiviral agents recommended for the
prevention and treatment of influenza.7 Oseltamivir and zanamivirare neuraminidase
inhibitors targeting the viral surface protein neuraminidase and are effective against
Influenza A and B when given within 48 hours of patient becoming symptomatic.
Therapeutic options are presently limited to 2 neuraminidase (NA) inhibitors,
oseltamivir and zanamivir, because this virus has a swine-origin matrix 2 (M2) gene,
which contains a mutation associated with resistance to M2 ion channel blockers
amantadine and rimantadine. Oseltamivir is a prodrug that is hydrolyzed by the liver
to its active metabolite, oseltamivir carboxylate, with an elimination half-life of about
6-10 h. Oseltamivir is a neuraminidase inhibitor, serving as a competitive inhibitor of
sialic acid, found on the surface proteins of normal host cells. By blocking the activity
of the neuraminidase, oseltamivir prevents new viral particles from being released by
infected cells.8 Although oseltamivir has been widely used in persons infected with
pandemic (H1N1) 2009 virus, resistance was not observed until recently. Three
unrelated cases of resistance to oseltamivir were observed in Denmark, Japan, and
Hong Kong.9
Resistance to oseltamivir can occur due to a point mutation in any of several regions
of the neuraminidase protein of the virus.10 A single nucleotide mutation in the NA
gene from C to T results in an amino acid change from histidine (H) to tyrosine (Y) at
N1 position 275 i.e.H275 to Y275 by N1 nomenclature, or His274 Tyr by universal
N2 nomenclature this change is known to be associated with oseltamivir resistance
in H1N1 Influenza virus.
4
During the 2007–2008 influenza seasons, a significant rise in the percentage of
oseltamivir-resistant isolates with the H275Y mutation was observed among
seasonal Influenza A H1N1 strains in Europe, Asia and the USA.11 This virus has
now become the dominant virus population globally, overtaking oseltamivir
susceptible seasonal influenza A virus (H1N1). The molecular basis for the 274Y
variant in seasonal influenza A virus (H1N1) virus and the mechanism by which this
resistant variant became the dominant population remain unknown.12 A histidine to
tyrosine substitution in the NA active site (H275Y) has previously been documented
as a genetic marker of resistance to oseltamivir among patients with either seasonal
Influenza A H1N1 or Influenza A H5N1 virus infections.13
In response to this development, pyrosequencing and real-time PCR have become
the most applicable high-throughput methods to detect oseltamivir resistance
mutations.14 Growing virus in embryonated chicken eggs and or tissue culture can
generate a large quantity of virus for further characterization, but it is time-consuming
and labor-intensive.
Traditionally Sanger sequencing had been used to evaluate whether
neuraminidase mutations are present. This method has a sensitivity of ca. 20%, and
the initial screening for rare resistant species in patient samples has therefore been
performed in enriched cultured specimens. Recently, more sensitive methods have
been developed to identify the His275Tyr mutation in pandemic H1N1 Influenza A
virus. Probe-based real-time PCR detects and distinguishes His275 and Tyr275 in
patient samples. Pyrosequencing detects all variants in the PCR-amplified region,
including His275 and Tyr275, High resolution melting curve analysis is used to
screen for any mutations in the concerned region. With these methods, the
sensitivities of rare resistant variant detection are as low as ca. 5% and 3 to 5%
respectively.
As Sanger sequencing is cumbersome, time consuming, expensive, requires
technical skill, equipment is also not available in most labs moreover it has sensitivity
of ca.20% only but can identify mutations in whole NA gene while allele specific real
time PCR can rapidly identify the most common mutation conferring drug resistance
directly in patient samples and is readily available in most of the labs.15 As
5
monitoring mutations conferring the antiviral resistance in influenza is critical to
public health epidemiology and pandemic preparedness activities.16
The present study is planned to detect the presence or absence of the H275Y
mutation which confer resistance to oseltamivir, in seasonal and pandemic (H1N1)
influenza viruses by allele specific real time PCR and confirm by sequencing.
Part Review of Literature
Influenza viruses are single-stranded, negative-sense enveloped RNA viruses
of the family Orthomyxoviridae, are of divided into three types: A, B and C; types B
and C are generally only found in humans. Type A influenza viruses are responsible
for most seasonal influenza epidemic morbidity and mortality, and all influenza
pandemic strains. Type A is further divided into subtypes by antigenic
characterization of the two surface glycoproteins hemagglutinin (HA) and
neuraminidase (NA). Influenza A virus contains eight gene segments and encodes
10–11 proteins, including HA, NA, matrix proteins (M1 and M2), nucleoprotein (NP),
nonstructural proteins (NS1 and NS2) and a polymerase complex (PA, PB1, PB2
and PB1F2). Based on the antigenic characterization of the HA and NA, the strain
can be classified into sixteen HA and nine NA subtypes. Aquatic birds are the natural
reservoirs of Influenza A viruses and all sixteen HA and all nine NA subtypes in most
of the possible combinations have been isolated from these avian species. Only a
small subset of Influenza A virus subtypes has become established in mammalian
species, including humans.2 Influenza A viruses evolve rapidly due to both antigenic
drift and reassortment (antigenic shift), and in humans, new antigenic variants
emerge constantly to give rise to yearly epidemics.3 When an antigenically novel
strain emerges and circulates widely in people it can cause a pandemic. Novel
Influenza A viruses are unpredictable and occasionally cause pandemics, including
the pandemics of 1918 (an H1N1 subtype virus), 1957 (H2N2) and 1968 (H3N2).
The 1918 pandemic virus caused approximately 675,000 deaths in the USA and
killed approximately 40 million people worldwide.4
Recently in 2009 pandemic swine-origin Influenza A H1N1 has been demonstrated
to have emerged by reassortment between two established swine influenza virus
lineages.17 Initially it was difficult to predict the impact of this pandemic or how the
6
virus will evolve; this novel virus had already spread globally and rapidly.18 Till August
2010, worldwide more than 214 countries had reported laboratory confirmed cases
of pandemic influenza H1N1 2009, including over 18449 deaths. India confirmed its
first case on 16 May 2009, when a man travelling from New York via Dubai and Delhi
tested positive for the H1N1 Influenza virus in Hyderabad. The second case was
reported by the National Institute of Virology (NIV) Pune in a mother and son duo
from Chennai on 1 June 2009.19
In India, till August 8, 2010, a total of 15, 4259 persons were tested for H1N1
Influenza And 23.4% were found to be positive including 1833 deaths. Transmission
was intense in western States of Maharashtra and Gujarat.20 Though the pandemic is
over but even now as per WHO report from 1st Jan 2013 to 6st Feb 2013 there have
been 132 deaths and 708 cases of swine flu across the India (2013) the worst hit
state has been Rajasthan with 236 cases and 65 deaths, followed by Haryana.21
The Transmission to humans H1N1 virus can be spread in three main ways by direct
transmission when an infected person sneezes mucus into the eyes, nose or mouth
of another person; through people inhaling the aerosols produced by infected people
coughing, sneezing and spitting; and through hand-to-mouth transmission from
either contaminated surfaces or direct personal contact, such as a hand-shake. The
most common symptoms of the disease are chills, fever, sore throat, muscle pain
severe headache, coughing, weakness, fatigue, and general discomfort. Sore throat,
fever and coughs are the most frequent symptoms. People who work with poultry
and swine, especially people with intense exposures, are at increased risk of
zoonotic infection with influenza virus endemic in these animals, and constitute a
population of human hosts in which zoonosis and reassortment can co‐occur.22
Drug resistance
Oseltamivir, zanamivir, peramivir, and A-315675, amantadine and rimantadine are
the various antiviral drugs available for Influenza. Oseltamivir and zanamivir are the
two primary antiviral agents recommended for the prevention and treatment of
influenza A.9
7
o Tiffany G. Sheu, et al in 2008 detected a rise in the incidence of oseltamivir
resistance in Influenza A (H1N1) viruses carrying the H274Y mutation in the
United States and in other countries in 2007 to 2008. The frequency of
resistance among Influenza A (H1N1) viruses in March 2008 was found to be
8.6% (50/579 isolates) in the United States. Total of 3,261 influenza A virus
isolates collected worldwide were screened for susceptibly to oseltamivir and
zanamivir during the three seasons (2004 to 2008). The number of viruses
tested per season during this period rose from 371 to 648 to 2,242,
respectively. For the ongoing 2007 to 2008 season, 1,309 influenza A virus
isolates were tested. The mean IC50 for A (H1N1) viruses was found to be
low to both oseltamivir (0.91 nM) and zanamivir (1.06 nM).23
o Chen et al in 2009 studied the resistance to oseltamivir in Influenza A
pandemic (H1N1) 2009 virus isolated from an untreated person in Hong
Kong, China. Investigations showed a resistant virus with the neuraminidase
(NA) 274Y genotype in quasi-species from a nasopharyngeal aspirate.
Therefore it was concluded that the monitoring for the naturally occurring NA
274Y mutation in this virus is necessary.9
o Zaraket et al in 2010 demonstrated the emergence and widespread
occurrence of drug-resistant seasonal human influenza A viruses, especially
oseltamivir-resistant A/H1N1 virus. In their study, seasonal influenza A/H1N1
viruses, including oseltamivir-resistant and susceptible viruses, obtained from
several areas in Japan during the 2007 to 2009 were analyzed. Phylogenetic
analysis revealed that the oseltamivir-resistant strain evolved from a
reassortant oseltamivir-susceptible strain (clade 2B) which circulated in the
2007–2008 season by acquiring the H275Y resistance-conferring mutation in
the NA gene. The oseltamivir-resistant lineage represented 100% of the
H1N1 isolates from the 2008–2009 seasons. Therefore, a reassortment
event involving two distinct oseltamivir-susceptible lineages, followed by the
H275Y substitution in the NA gene and other mutations elsewhere in the
genome, contributed to the emergence of the oseltamivir- resistant lineage.24
8
o Nguyen T jack et al. in 2010 studied the in vitro activity of a triple combination
antiviral drug (TCAD) regimen composed of drugs with different mechanisms
of action against drug- resistant seasonal and 2009 H1N1 influenza viruses.
Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested
against amantadine- and oseltamivir-resistant Influenza A viruses using an in
vitro infection model in MDCK cells. Their data demonstrated that the TCAD
regimen composed of amantadine, ribavirin, and oseltamivir is highly
synergistic against resistant viruses, including 2009 H1N1. The TCAD
regimen overcomes baseline drug resistance to both classes of approved
Influenza Antiviral, and thus may represent a highly active antiviral therapy
for seasonal and pandemic influenza.25
MATERIALS AND METHODS
Specimen:Nasopharyngeal swab, aspirate, nasal wash, Nasal/throat swab.
Specimen Collection :
Label the specimen collection vial containing VTM with identification no. and date.
Under good illumination, ask the patient to open his/her mouth. Take a sterile swab,
partially dip in a vial containing normal saline and swab both the tonsils and the
posterior pharynx vigorously, till the patient gags. Repeat with a second swab. Place
both the swab in same tube containing viral transport medium. Place the tube in the
icebox with wet ice.
Transportion& Storage :
Throat swab or nasal swab specimen will be transported to the laboratory as soon as
possible in Viral Transport Medium (VTM) maintaining cold chain. Hold at 2-8°C if
delay is anticipated (< 1 week). If delay > 1 week is expected, store at-70°C.
Sample processing:
9
RNA extraction: it will be carried out using automated Nuclisense easyMAG
extraction method as per manufacturers’ protocol and will be stored at -70°C.
Allelic discrimination PCR: This allelic discrimination real time reverse
transcriptase (RT)-PCR will be carried out for detection of the H275Y mutation
(N1 numbering) in the NA gene of seasonal and pandemic (H1N1) influenza
viruses using specific primers and probes.26
NA gene Amplification: NA gene will be amplified using specific primers to
detect H275Y mutation.26
Gel electrophoresis: The amplified product of NA gene will be gel
electrophoresed.
NA gene Sequencing: Direct sequencing of PCR products will be performed
by using a Big- Dye Terminator v3.1 cycle sequencing reaction kit on an ABI
3500 DX Genetic Analyzer (Applied Bio).27
Cell culture: determination of drug resistance to oseltamivir by phenotypic
method by cell culture in MDCK cell line.28
Tools/Instrument for collecting data:
ABI 7500 Dx, Real time PCR for detecting drug resistence.
ABI Thermal cycler for Amplification
ABI 3500 DX Genetic Analyzer for NA gene sequencing.
INCLUSION CRITERIA
A written consent will be taken from the patient at the time of sample collection for
the enrolment of the patient in the study. Influenza A and pandemic Influenza A
sample positive by PCR but with low Ct value and clinical details.
STATEMENT OF THE RESEARCH PROBLEM
Detection of oseltamivir drug resistance in seasonal and pandemic influenza A 2009
H1N1 virus from patients at Jaipur.
DRUG RESISTENCE: Drug resistence is the reduction in effectiveness of a drug
such as an antimicrobial in curing a disease or condition. When the drug is not
10
indented to kill or inhibit a pathogen, then the term is equivalent to doses failure or
drug tolerance.
OBJECTIVES OF THE STUDY
The study will be undertaken with the following aims and objectives:
1. To detect oseltamivir drug resistance in seasonal and pandemic influenza viruses
by real time PCR.
2. To compare detection of drug resistance by real time PCR and NA gene
sequencing method.
3. Drug resistance will be detected in representative number of samples by
phenotypic method.
SCOPE OF THE STUDY The study will identify the presence of drug resistance to oseltamivir in
seasonal and pandemic Influenza A virus in Jaipur region.
Compare sensitivity and specificity of real time PCR and sequencing
technologies for detecting drug resistance.
SIGNIFICANCE OF THE STUDYThe data obtained from the study will assist in formulation of new therapeutic
strategies based on prevalence of resistance to the drug. Characterization of virus
both by in vitro and in vivo method will help in monitoring drug resistance and
detection of mutation in treated as well as untreated patients.
LIMITATIONSThis proposed cross-sectional study is a laboratory based approach to detect the
presence of drug resistance in seasonal and pandemic Influenza A virus in patient
samples reporting at our lab. The screening method would be real time PCR which
identifies only a single allele specific mutation with good sensitivity of 5% while
direct PCR-based DNA sequencing can detect all mutations in the concerned gene
but has low sensitivity and a particular mutant is detected only if it is present >20%
of the total quasi species pool.
11
MONTH WISE PLAN OF WORK
Activity Time taken (in months)
Review of literature Throughout the study
Preparation of research proposal Initial 5 to 6 months
Collection of samples from patients 9 months
Experimentation 12 months
Analysis and interpretation of data and
Verification of the results
6months
Preparation and submission of report 3months
Total time = 36 months
MONTH WISE PLAN OF WORK (Graphical representation)
Activity
/ Month
Review of
literature
Preparation of
research
proposal
Collection of
samplesExperiment
Analysis and
interpretation of
data
Preparation
and submission
of report
12
0 to 6
6 to 15
15 to 27
27 to 33
33 to 36
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