Background Influenza A viruses are medically significant pathogens responsible for higher mortality and morbidity throughout the world. Swine influenza is known to be caused by influenza A subtypes H1N1, H1N2, and H3N2, which are highly contagious, and belongs to the family Orthomyxoviridae. Efficient and accurate diagnosis of influenza A in individuals is critical for monitoring of a constantly evolving pandemic. A rapid result is important, because timely treatment can reduce disease severity and duration. Rapid antigen tests were among the first-line diagnostic tools for the detection of pandemic H1N1 (2009) virus infection during the initial outbreak. Current study focuses on the significant approach of the usage of molecular method utilizing real-time PCR for the detection of type A influenza virus (H1N1 subtype) in humans. Methods A total of 2000 mixed nasal/throat swab specimens collected in commercial viral transport from Apollo hospitals, Hyderabad were submitted to Institute of Preventive Medicine for molecular testing by reverse transcriptase polymerase chain reaction (RT-PCR) from 2009 to 2015 from its affiliated primary care clinics. Results Among the 2000 samples collected, 700 samples were positive for Human Inf A, swine Inf A, and Swine Inf H1 (fourth table in the article). One thousand two hundred samples were negative for Human Inf A, swine Inf A, and Swine Inf H1, and 100 samples were positive for Influenza A only. Conclusion The molecular testing of H1N1 patients helped the clinicians in timely diagnosis and treatment of these patients during the pandemic surveillance. The RT-PCR test has higher sensitivity and specificity; hence it is considered to be the best tool to use during the pandemic surveillance, as compared to the any other commercial antigen-based tests, which show a variable performance, with the sensitivities of tests from different manufacturers ranging from 9 to 77%.

1. Molecular diagnosis of H1N1 virus

2. Original Article
Molecular diagnosis of H1N1 virus
Kalal Iravathy Goud a,
*, Kavitha Matam a
, Adi Maha Lakshmi Madasu a
,
Ravi Vempati a
, Sagarika Daripalli a
, Madhuri Pullamula a
,
Sunitha Narreddy b
, Lavanya Nutankalva b
a
Molecular Biology and Cytogenetics Department, Apollo Hospitals, Hyderabad, India
b
Infectious Disease Department, Apollo Hospitals, Hyderabad, India
a p o l l o m e d i c i n e x x x ( 2 0 1 5 ) x x x – x x x
a r t i c l e i n f o
Article history:
Received 6 July 2015
Accepted 18 July 2015
Available online xxx
Keywords:
H1N1 virus
Molecular diagnosis
Swine flu
Influenza A virus
a b s t r a c t
Background: Influenza A viruses are medically significant pathogens responsible for higher
mortality and morbidity throughout the world. Swine influenza is known to be caused by
influenza A subtypes H1N1, H1N2, and H3N2, which are highly contagious, and belongs to
the family Orthomyxoviridae. Efficient and accurate diagnosis of influenza A in individuals
is critical for monitoring of a constantly evolving pandemic. A rapid result is important,
because timely treatment can reduce disease severity and duration. Rapid antigen tests were
among the first-line diagnostic tools for the detection of pandemic H1N1 (2009) virus
infection during the initial outbreak. Current study focuses on the significant approach
of the usage of molecular method utilizing real-time PCR for the detection of type A influenza
virus (H1N1 subtype) in humans.
Methods: A total of 2000 mixed nasal/throat swab specimens collected in commercial viral
transport from Apollo hospitals, Hyderabad were submitted to Institute of Preventive
Medicine for molecular testing by reverse transcriptase polymerase chain reaction (RT-
PCR) from 2009 to 2015 from its affiliated primary care clinics.
Results: Among the 2000 samples collected, 700 samples were positive for Human Inf A,
swine Inf A, and Swine Inf H1 (fourth table in the article). One thousand two hundred
samples were negative for Human Inf A, swine Inf A, and Swine Inf H1, and 100 samples were
positive for Influenza A only.
Conclusion: The molecular testing of H1N1 patients helped the clinicians in timely diagnosis
and treatment of these patients during the pandemic surveillance. The RT-PCR test has
higher sensitivity and specificity; hence it is considered to be the best tool to use during the
pandemic surveillance, as compared to the any other commercial antigen-based tests,
which show a variable performance, with the sensitivities of tests from different manu-
facturers ranging from 9 to 77%.
# 2015 Published by Elsevier B.V. on behalf of Indraprastha Medical Corporation Ltd.
* Corresponding author at: Molecular Biology and Cytogenetics Department, Apollo Hospitals, Jubilee Hills, Hyderabad, India.
Tel.: +91 040 23607777x4012; mobile: +91 9989831655.
E-mail addresses: ira_15@rediffmail.com, driravathy_g@apollohospitals.com (K. Iravathy Goud).
APME-301; No. of Pages 5
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apme.2015.07.005
Available online at www.sciencedirect.com
ScienceDirect
journal homepage: www.elsevier.com/locate/apme
http://dx.doi.org/10.1016/j.apme.2015.07.005
0976-0016/# 2015 Published by Elsevier B.V. on behalf of Indraprastha Medical Corporation Ltd.

3. 1. Introduction
Since the identification of the pandemic influenza (H1N1) 2009
virus and its subsequent antigenic and genetic characteriza-
tion, this new influenza virus strain has rapidly spread
worldwide.1–10
As of December 2009, >600,000 cases and at
least 8768 deaths were reported.11
In June 2009, the outbreak
was officially declared a pandemic by the World Health
Organization (WHO). The pandemic (H1N1) 2009 strain evolved
from the family of swine triple-reassortant viruses, which
contain genes derived from avian, swine, and human
influenza viruses. The pandemic (H1N1) 2009 strain acquired
the hemagglutinin (H) gene from a swine H1N2 virus and the
neuraminidase (N) and matrix protein genes from the Eurasian
swine lineage, and it evolved into a pathogen capable of
sustaining efficient human-to-human transmission.4
Frontline pandemic surveillance relies on rapid diagnosis
of suspected cases and timely treatment of infected individu-
als. The current diagnostic tests for pandemic (H1N1) 2009
virus include qualitative reverse transcriptase polymerase
chain reaction (RT-PCR) and antigen-based assays. The
antigen-based assays provide rapid diagnosis (within
15 min) but with a sensitivity of only 56%, when compared
with the results of RT-PCR (74%), which is more sensitive.12,13
The qualitative RT-PCR analysis has the primers specific for
the hemagglutinin or neuraminidase gene (or both) of the
pandemic virus. Due to its high sensitivity and specificity, RT-
PCR is the preferred diagnostic platform in individual labs or in
centralized lab settings. Nowadays along with the routine
antigen testing, RT-PCR is also accessible in resource-limited
setting and has been used frequently in diagnosis.
This manuscript focuses on the importance of the
molecular tools and its use in point-of-care testing at an
affordable cost in critically needed pandemic surveillance.
Confirmation of novel influenza A (H1N1) infection may be
necessary for surveillance purposes and for special situations,
e.g. severely ill patients, patients with immune-compromising
conditions, and pregnant and breast feeding women, which is
possible by molecular methods only.
2. Materials and methods
2.1. Clinical specimen preparation
A total of 2000 mixed nasal/throat swab specimens were
collected in commercial viral transport Media (Himedia) and
submitted to the Molecular Biology and Cytogenetics Depart-
ment, Apollo hospitals, Hyderabad from 2009 to 2015 from its
affiliated primary care clinics. These included patients, who
attended the emergency medicine department at Apollo
Hospitals, Hyderabad, public and private primary care clinics,
in-patients with influenza-like symptoms, as well as patients
or staff with compatible contact or travel histories. These
samples were submitted to Institute of Preventive Medicine
(IPM), which is the centralized laboratory recognized by the
Telangana and Andhra Pradesh state Governments for
molecular RT-PCR testing.
Patient ages ranged from 19 days to 85 years, and the male-
to-female ratio was 1.2 to 1 (Table 1). A duly filled in form along
with clinical history and the previous vaccination details were
collected and submitted to IPM along with the sample.
Extraction was performed with QIAamp®
Viral RNA Mini
Kit. The primer and probe sequence used are shown in Table 2.
The PCR primers are designed to target these three viruses:
Human Inf A, Swine Inf A, and Swine Inf H1. The PCR
conditions are shown in Table 3. Applied biosystems real time
PCR 7000 equipment was used.
3. Results
Among the 2000 samples collected, 700 samples were positive
for Human Inf A, Swine Inf A, and Swine Inf H1 (Table 4). One
Table 1 – Age groups and gender of the patients.
GenderAge group 0–25 years 25–50 years 50–75 years >75 years
Male 177 589 365 104
Female 123 453 137 52
Mean age (years) 22 35–40 60 79
Table 3 – RT-PCR amplification conditions.
Reaction volume 25 ml
Program the thermo cycler as follows:
Reverse transcription 50 8C for 30 min
Taq inhibitor activation 95 8C for 2 min
PCR amplification (45 cycles) 95 8C for 15 s
55 8C for 30 s (FAM is used for
fluorescence data)
Table 2 – Primer and probe sequence for the Inf A, SW Inf
A and SW H1virus.
Primers and probes Sequence (50
>30
)
Inf A Forward GAC CRA TCC TGT CAC CTC TGA C
Inf A Reverse AGG GCA TTY TGG ACA AAK CGT CTA
Inf A Probe TGC AGT CCT CGC TCA CTG GGC ACG
SW Inf A Forward GCA CGG TCA GCA CTT ATY CTR AG
SW Inf A Reverse GTG RGC TGG GTT TTC ATT TGG TC
SW Inf A Probe CYA CTG CAA GCC CA‘‘T’’' ACA CAC
AAG CAG GCA
SW H1 Forward GTG CTA TAA ACA CCA GCC TYC CA
SW H1 Reverse CGG GAT ATT CCT TAA TCC TGT RGC
SW H1 Probe CA GAA TAT ACA ‘‘T’’CC RGT CAC AAT
TGG ARA A
RnaseP Forward AGA TTT GGA CCT GCG AGC G
RnaseP Reverse GAG CGG CTG TCT CCA CAA GT
RnaseP Probe TTC TGA CCT GAA GGC TCT GCG CG
a p o l l o m e d i c i n e x x x ( 2 0 1 5 ) x x x – x x x2
APME-301; No. of Pages 5
Please cite this article in press as: Iravathy Goud K, et al. Molecular diagnosis of H1N1 virus, Apollo Med. (2015), http://dx.doi.org/10.1016/j.
apme.2015.07.005

4. thousand two hundred samples were negative for Human Inf
A, Swine Inf A, and Swine Inf H1, but 100 samples were positive
for Influenza A only. In 700 positive samples, 5 samples were
false positive and in 1200 negative samples, 10 samples were
false negative. These samples were retested for confirmation,
and the 5 false positive samples after repeating the tests
showed negative, and 10 false negative showed positive. These
were correlated with clinical symptoms of the patients.
4. Discussion
Influenza testing is not needed for all patients with signs and
symptoms of influenza to make antiviral treatment decisions.
Usually flu-associated hospitalizations are very commonly
seen in people of 65 years and children 0–4 years of age, as
these age groups have low immunity and are prone to
infections. In our study, both male and female patients were
equally infected for H1N1, and there was no significant
difference between the age group. Once influenza activity
has been identified in the community or geographic area, a
clinical diagnosis of influenza can be made for outpatients
with signs and symptoms consistent with suspected influen-
za, especially during periods of peak influenza activity in the
community. For most outpatients and emergency room
patients, molecular testing is performed, and results are
available in a timely manner to inform clinical decision-
making. Molecular testing is not needed on all patients with
suspected influenza, but is most appropriate for hospitalized
patients if a positive test would result in a change in clinical
management. Clinicians should be aware of the approved
clinical specimens for the molecular assay.14
If treatment is clinically indicated, antiviral treatment
should not be withheld from patients with suspected influenza
while awaiting testing results during periods of peak influenza
activity in the community, when the likelihood of influenza is
high. Since results from molecular assays will take at least 3–
8 h or sometimes 24 h for the results to be available, antiviral
treatment will be started as soon as possible, because the
greatest benefit is when treatment is initiated as close to
illness onset as possible, especially for patients at high risk of
serious outcomes. In our study, in 2000 patients, similarly 700
patients suspected of H1N1 infection were put on anti-viral
treatment until the RT-PCR reports arrived due to which the
death rate and the spread of infection in our hospital were low.
Once the molecular reports were out, the patients were treated
accordingly. The 100 patients positive for Human influenza A
and negative for swine influenza A and swine H1 virus were
treated accordingly. The families of H1N1 positive patients
were counseled accordingly. These patients were placed in the
isolated wards for a week and treated with great precaution to
minimize the spread of infection. The doctors, nurses, and the
laboratory personals walking closely with these patients were
vaccinated for H1N1.
Hospitalized patients with suspected influenza without
lower respiratory tract disease will have upper respiratory
tract specimens collected for influenza testing. Collection of
lower respiratory tract specimens from hospitalized patients
with suspected influenza and pneumonia can be considered
for influenza testing by RT-PCR, if influenza testing of upper
respiratory tract specimens is negative and if positive testing
would result in a change in clinical management. Hospitalized
patients with suspected influenza and respiratory failure on
mechanical ventilation can have an endotracheal aspirate
specimen collected for influenza testing by RT-PCR, if a
laboratory diagnosis of influenza has not been determined.
Bronchoalveolar lavage fluid, if collected for other diagnostic
purposes, can also be tested by RT-PCR for influenza viruses.15
A number of different laboratory diagnostic tests can be
used for detecting the presence of influenza viruses in
respiratory specimens, including direct antigen detection
tests, virus isolation in cell culture, or detection of influen-
za-specific RNA by real-time RT-PCR. These tests differ in their
sensitivity and specificity in detecting influenza viruses as well
as in their commercial availability, the amount of time needed
from specimen collection until results are available, and the
ability of the tests to distinguish between different influenza
virus types (A versus B), and influenza A subtypes (e.g. novel
H1N1 versus seasonal H1N1 versus seasonal H3N2 viruses).
Serologic tests on paired acute (within 1 week of illness onset)
and convalescent (collected 2–3 weeks later) sera can help to
establish a retrospective diagnosis of influenza virus infection
for epidemiological and research studies. However, such a
serial serological testing is not routinely available through
clinical laboratories.16
Molecular assays such as RT-PCR are particularly useful in
identifying influenza virus infection as a cause of respiratory
outbreaks in institutions (e.g. nursing homes, chronic care
facilities, and hospitals). Positive results from one or more ill
persons with suspected influenza can support decisions to
promptly implement prevention and control measures for
influenza outbreaks. Clinicians should be aware of require-
ments from their public health authorities regarding notifica-
tion of any suspected or confirmed institutional influenza
outbreaks, and on when respiratory specimens should be
collected from ill persons and sent to a public health laboratory
for laboratory confirmation of influenza.14
Many factors can influence influenza testing results.
Influenza viral shedding in the upper respiratory tract
generally declines substantially after 4 days in patients with
uncomplicated influenza. Patients with lower respiratory tract
disease may have prolonged influenza viral replication in the
lower respiratory tract. Molecular tests can detect influenza
viral RNA (positive results) for a longer duration than other
Table 4 – 2000 samples sent for H1N1 testing from 2009 to 2015.
Total samples Human Inf A Swine Inf A Swine Inf H1 Result
700 samples Positive Positive Positive Positive for H1N1
100 samples Positive Negative Negative Negative for H1N1
1200 samples Negative Negative Negative Negative for H1N1
a p o l l o m e d i c i n e x x x ( 2 0 1 5 ) x x x – x x x 3
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5. influenza testing (e.g. antigen testing – immunofluorescence
or rapid influenza diagnostic tests). Although RT-PCR is the
most sensitive influenza test and is highly specific, negative
results can occur in persons with influenza for multiple
reasons; hence, negative RT-PCR results may not always
exclude a diagnosis of influenza. If clinical suspicion of
influenza is high, antiviral treatment continue in patients
with severe illness time from illness onset to collection of
respiratory specimens for testing.16
If testing is delayed or is done at a facility other than where
the patient is hospitalized, specimens are to be placed in
sterile viral transport media, consistent with test specifica-
tions, and refrigerated until transported to the laboratory for
testing as soon as possible. Freezing and thawing are avoided
or minimized to avoid degradation of influenza viruses, if viral
culture will be performed. In our case as well, this test was not
performed in-house. The samples were collected and sent to
IPM on the same day of collection, if the sample was collected
before 5.00 pm in the evening, or refrigerated if collected after
5.00 pm, and then it would be sent to IPM for testing the next
day morning.
Sensitivities and specificities of RT-PCR and other molecu-
lar assays are high compared to other assays, which use
different methods. However, even with RT-PCR, false negative
results can occur due to improper or poor clinical specimen
collection or from poor handling of a specimen after collection
and before testing. A negative result can also occur by testing a
specimen that was collected, when the patient is no longer
shedding detectable influenza virus. Similarly in our study as
well, we had 10 cases, which were false negatives, and we have
retested them, as the clinician was suspecting the H1N1
infection in these patients. The results correlated with the
clinical history, and the patients responded to the therapy.17
False positive results, although rare, can occur (e.g. due to
lab contamination or other factors). A positive result in a
person, who recently received intranasal administration of
live attenuated influenza virus vaccine (LAIV), may indicate
detection of vaccine virus. LAIV contains influenza virus
strains that undergo viral replication in respiratory tissues at
lower temperature (e.g. nasal passages) than internal body
temperature. Since the nasal passages are infected with live
influenza virus vaccine strains during LAIV administration,
sampling the nasal passages within a few days after LAIV
vaccination can yield positive influenza testing results. It may
be possible to detect LAIV vaccine strains up to 7 days after
vaccination, and in rare situations, for longer periods. In our
study, we had 5 false positive cases, and these were retested
again.18
And the result was found to be negative and clinically
correlated. Hence, while collecting samples, clinical history is
to be collected and it is important along with the information
of previous vaccination. Molecular assays for influenza are
increasingly being used in clinical settings.1
RT-PCR is the
molecular assay that can identify the presence of influenza
viral RNA in respiratory specimens.
5. Conclusion
In conclusion, we report that RT-PCR assay provides high
sensitivity and specificity through detection of sequences
specific to the hemagglutinin gene of the pandemic (H1N1)
2009 virus without the need for a secondary confirmation step.
Molecular assays are more sensitive and specific for detecting
influenza and have proven effective in the detection of
pandemic (H1N1). This study focuses on the importance of
modern molecular techniques in providing the appropriate
diagnosis of H1N1 virus.
Conflicts of interest
The authors have none to declare.
Acknowledgments
The authors are grateful to Institute of Preventive Medicine for
processing the samples, and to the management of Apollo
Hospitals, for the financial support.
r e f e r e n c e s
1. Anonymous. Swine influenza A (H1N1) infection in two
children—Southern California. MMWR Morb Mortal Wkly Rep.
2009;58:400–402.
2. Anonymous. Swine influenza: how much of a global threat?
Lancet. 2009;373:1495.
3. Burch J, Corbett M, Stock C, Nicholson K, Elliot AJ, Duffy S.
Prescription of anti-influenza drugs for healthy adults: a
systemic review and meta-analysis. Lancet Infect Dis.
2009;9:537–545.
4. Cohan. Straight from the pig's mouth: swine research with
swine influenzas. Science. 2009;325:140–141.
5. Dineva MA, Candotti D, Fletcher-Brown F, Allain JP, Lee H.
Simultaneous visual detection of multiple viral amplicons
by dipstick assay. J Clin Microbiol. 2005;43:4015–4021.
6. Drexler JF, Helmer A, Kirberg H, et al. Poor clinical sensitivity
of rapid antigen test for influenza A pandemic (H1N1) 2009
virus. Emerg Infect Dis. 2009;15:1662–1664.
7. Ellis J, Iturriza M, Allen R, et al. Evaluation of four real-time
PCR assays for detection of influenza A(H1N1)v viruses. Eur
Surveill. 2009;14:19230.
8. Faix DJ, Sherman SS, Waterman SH. Rapid-test sensitivity
for novel swine-origin influenza A (H1N1) virus in humans.
N Engl J Med. 2009;361:728–729.
9. Fernandez C, Cataletto M, Lee P, Feuerman M, Krilov L. Rapid
influenza A testing for novel H1N1: point-of-care
performance. J Postgrad Med. 2010;122:28–33.
10. Garten RJ, Davis CT, Russell CA, et al. Antigenic and genetic
characteristics of swine-origin A(H1N1) influenza viruses
circulating in humans. Science. 2009;325(5937):197–201.
11. WHO. Clinical features of severe cases of pandemic influenza.
Geneva, Switzerland: WHO; 2009. http://www.who.int/csr/
don/2009_09_11/en/index.html Posting date 11.9.2009.
12. Hermann B, Larsson C, Zweygberg BW. Simultaneous
detection and typing of influenza viruses A and B by a
nested reverse transcription-PCR: comparison to virus
isolation and antigen detection by immunofluorescence
and optical immunoassay (FLU OIA). J Clin Microbiol.
2001;39:134–138.
13. Nicholson KG, Wood JM, Zambon M. Influenza. Lancet.
2003;362:1733–1744.
a p o l l o m e d i c i n e x x x ( 2 0 1 5 ) x x x – x x x4
APME-301; No. of Pages 5
Please cite this article in press as: Iravathy Goud K, et al. Molecular diagnosis of H1N1 virus, Apollo Med. (2015), http://dx.doi.org/10.1016/j.
apme.2015.07.005

6. 14. Ellis JS, Zambon MC. Molecular diagnosis of influenza. Rev
Med Virol. (6):2002;(6):375–389.
15. Mahony JB. Nucleic acid amplification-based diagnosis of
respiratory virus infections. Expert Rev Anti Infect Ther.
2010;11:1273–1292.
16. Wang R, Taubenberger JK. Methods for molecular
surveillance of influenza. Expert Rev Anti Infect Ther. (5):2010;
(5):517–527.
17. Shu B, Wu KH, Emery S, et al. Design and performance of the
CDC real-time reverse transcriptase PCR swine flu panel for
detection of 2009 A (H1N1) pandemic influenza virus. J Clin
Microbiol. 2011;49(7):2614–2619.
18. Block SL, Yogev R, Hayden FG, Ambrose CS, Zeng W, Walker
RE. Shedding and immunogenicity of live attenuated
influenza vaccine virus in subjects 5–49 years of age. Vaccine.
2008;26(38):4940–4946.
a p o l l o m e d i c i n e x x x ( 2 0 1 5 ) x x x – x x x 5
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