Epidemiology and Molecular Characterization of Seasonal Influenza Viruses in Iraq

Abstract

Abstract
The importance of influenza viruses in respiratory infections in the Middle East, including Iraq, has been
historically overlooked. Nowadays, with the pandemic of corona virus disease 2019, the importance of
prevention from other respiratory diseases, such as seasonal influenza, can be a critical step in the health
management system. Therefore, this study aimed to evaluate the prevalence and seasonal occurrence of
influenza viruses in the Iraqi population presented with influenza‐ like illness (ILI) or severe acute respiratory
infection (SARI)within2015-2017. Moreover, this study was conducted to identify the periods with increased
influenza transmission for vaccination recommendations in Iraq. In the present study, we presented the cases of
infection by influenza A or B viruses. To test influenza virus types A (H1N1 and H3N2) and B, 1,359 throat and
nasal swabs were collected from patients with ILI or SARI. Ribonucleic acid was extracted and amplified using
a set of primers and probes. The frequency rates of infection were obtained at 1,616 (45%) and 1974 (55%) in
females and males, respectively. The mean age of the participants was estimated at 31.71±22.68 with a
minimum and maximum ages of 1 month and 96 years, respectively. It was revealed that influenza virus type A
was the most predominant with an incidence of 16.2%, followed by type B with 0.33% incidence. It was also
found that December was the most prevalent month of being infected by influenza viruses types A and B
(30.02% and 0.48%, respectively). Vaccination in September would likely protect the highest number of
patients. It was clear that the influenza A virus was predominant over type B. In Iraq, influenza A and B viruses
were found in a large percentage of ILI and SARI cases. Additionally, males were reported to be more likely to
become infected than females.
Keywords: Real-Time RT-PCR, Influenza type A, Influenza type B

Full text

Archives of Razi Institute, Vol. 76, No. 4 (2021) 871-877 Copyright © 2021 by
Razi Vaccine & Serum Research Institute
DOI: 10.22092/ari.2021.355950.1748
1. Introduction
Influenza A and B are the most common influenza
viruses that cause epidemic human disease and are
further divided into subtypes (for A viruses) and
lineages (for B viruses) based on antigenic differences.
Subtypes of influenza A viruses have already been
identified. Since point mutations and recombination
events may occur during viral replication, resulting in
frequent antigenic change (i.e., antigenic drift), new
influenza viruses may emerge (1).
Influenza viruses are enveloped viruses with a
segmented ribonucleic acid (RNA) genome and are
members of the Orthomyxoviridae family. Influenza is
a respiratory illness that is caused by a virus. The most
common signs and symptoms related to influenza,
which can include all or some of them, are fever,
Original Article
Epidemiology and Molecular Characterization of Seasonal
Influenza Viruses in Iraq
Aufi, I. M1 *, Khudhair, A. M2, Ghaeb AL-Saadi, L3, Almoneem Ahmed, M. A1, Mahdi
Shukur, F. M1
1. Department of Virology, Central Public Health Laboratory, Ministry of Health, Public Health Directorate, Baghdad, Iraq
2. Department of Microbiology, College of Medicine, Al-Iraqi University, Baghdad, Adhamiyah, Iraq
3. Department of Biology, College of Science, Al-Mustansiriyah University, Baghdad, Iraq
Received 11 September 2021; Accepted 27 September 2021
Corresponding Author: anfamone@gmail.com
Abstract
The importance of influenza viruses in respiratory infections in the Middle East, including Iraq, has been
historically overlooked. Nowadays, with the pandemic of corona virus disease 2019, the importance of
prevention from other respiratory diseases, such as seasonal influenza, can be a critical step in the health
management system. Therefore, this study aimed to evaluate the prevalence and seasonal occurrence of
influenza viruses in the Iraqi population presented with influenza‐like illness (ILI) or severe acute respiratory
infection (SARI)within2015-2017. Moreover, this study was conducted to identify the periods with increased
influenza transmission for vaccination recommendations in Iraq. In the present study, we presented the cases of
infection by influenza A or B viruses. To test influenza virus types A (H1N1 and H3N2) and B, 1,359 throat and
nasal swabs were collected from patients with ILI or SARI. Ribonucleic acid was extracted and amplified using
a set of primers and probes. The frequency rates of infection were obtained at 1,616 (45%) and 1974 (55%) in
females and males, respectively. The mean age of the participants was estimated at 31.71±22.68 with a
minimum and maximum ages of 1 month and 96 years, respectively. It was revealed that influenza virus type A
was the most predominant with an incidence of 16.2%, followed by type B with 0.33% incidence. It was also
found that December was the most prevalent month of being infected by influenza viruses types A and B
(30.02% and 0.48%, respectively). Vaccination in September would likely protect the highest number of
patients. It was clear that the influenza A virus was predominant over type B. In Iraq, influenza A and B viruses
were found in a large percentage of ILI and SARI cases. Additionally, males were reported to be more likely to
become infected than females.
Keywords: Real-Time RT-PCR, Influenza type A, Influenza type B

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headache, myalgia, prostration, coryza, sore throat, and
cough (1). The influenza virus is divided into three
types, namely A, B, and C, which are considered
different genera. In humans, the sickness is known as
"flu", and type A is the most frequent, resulting in
influenza disease, followed by type B, which is spread
through airdrops from infected people or intimate
contact with infected animals (2). Influenza A is the
most frequent strain and kills more people than
influenza B (3). The surface antigens hemagglutinin
and neuraminidase determine the subtypes of influenza
A (4).
An increase in mortality is observed during typical
influenza seasons when influenza viruses are
transmitted. Despite the fact that not all additional
occurrences occurring during these times can be traced
directly to influenza, the assessments of mortality
incidence during influenza seasons are valuable for
tracking influenza-related outcomes from season to
season. The estimates that only include outcomes
contribute to pneumonia and influenza and are likely to
overlook a number of serious diseases that are at least
partially attributed to influenza since neither the deaths
caused by the aggravation of dependent cardiac nor
pulmonary diseases linked to influenza infection are not
included in this grade (5). There isn't a specific
diagnostic test available; although anecdotal reports
work out on false-negative test results, rapid antigen-
based tests for influenza appear to be suitable for
pandemic H1N1 influenza and others (6).
In developing countries, acute viral respiratory tract
infection is the leading cause of hospitalization for
newborns and young children and the primary reason
for death (7, 8). Because of quicker reversal times and
greater sensitivity, nucleic acid testing by reverse
transcription polymerase chain reaction (RT-PCR) has
replaced classical virus culture in the clinical diagnosis
of influenza (9). Since influenza activity and influenza-
like illness (ILI) are widespread across the country, it is
critical to recognize both the differences between these
conditions and the most appropriate treatment. In
addition, it is necessary to diagnose the pathogen that is
the most common cause of influenza.
2. Material and Methods
Influenza surveillance was carried out at the Central
Public Health Laboratory from 2015 to 2017. A total of
3,561 specimens were obtained from 3,561 patients
who had developed respiratory symptoms. Patients'
symptoms ranged from mild upper respiratory tract
infections, such as fever above 38°C, common cold,
cough, coryza, sore throat, and shortness of breath, to
lower respiratory tract infections, such as laryngitis,
bronchiolitis, and pneumonia. All information
concerning these patients, including their medical
history, was considered in the report. Throat swab or
nasopharyngeal swab specimens were collected and
directly immersed into a sterile tube, and Viral
Transport Media (VTM, Copan, USA) were used for
maintaining viral viability during transportation and
until its arrival to the laboratory. Specimens were
transported to the Virology Department, National
Influenza Center at the Central Public Health
Laboratory, in a cool box and stored at -80°Ctill the
analysis time.
2.1. Ribonucleic Acid Extraction
For all 3,561 respiratory specimens, viral RNA was
extracted using the QIAamp Viral RNA Mini Kit
(Qiagen, GmbH, Hilden, Germany) according to the
manufacturer's instructions, and the specimens were
then kept at -80°C until use.
All the clinical specimens were tested for influenza A
and B using real-time RT-PCR CDC Influenza Virus
Real-Time RT-PCR A/B Typing Panel kit (Atlanta,
USA). The master mix was prepared using a Super Script
III platinum one-step RT-PCR kit (Invitrogen, USA). A
25-µl master mix contained 12.5 µl reaction buffer (5x),
0.5 µl SuperScript TM III RT/Platinum TM Taq mix, 0.5
µl of each primer and probe (40 µM concentrationfor each
primer and 10 µM concentration for probe), 5.5 µl PCR
water, 0.5 µl Rox dye (1/10 dilution), and 5 µl of
specimen RNA template. Subsequently, amplification and

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detection were performed with Fast 7500 Real-Time PCR
system (Applied Biosystems) as follows: RT step
activation at 50°C for 5 min, initial denaturation at 95°C
for 2 min, followed by 45 cycles: 95°C for 3sec and 55°C
for 30 sec.
Specimens positive for influenza A were subjected to
subtyping with CDC Influenza Virus Real-Time RT-
PCR Subtyping Influenza A(H3/H1pdm09) Panel kit
(Atlanta, USA). The master mix and RT-PCR thermal
profile are described above.
2.2. Statistical Analysis
The statistical analysis system was analyzed in IBM
SPSS Statistics version 25. All values and proportions
and their frequencies were checked by applying the
Pearson Chi-square (X2) and cross tab test to
investigate the significant comparison between viral
infection percentages in different investigating markers
of the study population.
3. Results
3.1. Demographic and Clinical Characteristics of
Enrolled Patients
Active surveillance was conducted on patients
presenting with ILI referring to the Central Public
Health Laboratory within January 2015-December
2017, which rendered for the collection of 3,561
specimens. Out of the total number of patients, 1,616
(45%) and 1,974 (55%) cases were female and male,
respectively. The mean age of the enrolled patients was
obtained at 31.71±22.68 with a minimum and
maximum ages of 1and 96 years, respectively.
3.2. Etiological Characteristics
Out of the total surveyed population (n=3,950), 582
(14.73%) cases were positive for influenza A virus, 12
(0.3%) subjects were positive for influenza B virus, and
2,996 (75.84%) patients were negative.
Further detection for subtyping of influenza A was
conducted; the results of which showed the
predominance of H1N1(14.60%), H3N2 (5.26%), and
(0%) for each of H5N1 and H7N9 (Figure 1).
The comparison of age groups with infection revealed
that younger people were more susceptible in getting
infection. 1-10 years old individuals were significantly
infected with influenza A compared with older cohorts
(Figure 2).
Figure 3 shows that when the month is used as a
parameter for case distribution, December is the month
with the largest prevalence of infection by all types A,
with 30.02%, while type B has the highest frequency
in October, with 0.48%.
The results of bivariate analysis by gender and year
indicated that a higher proportion of men (n=1,974,
55%) were infected than women (n=1616, 45%). High
significance is apparent in these results (Table 1 and 2).
Figure 1. Percentage of influenza A subtypes

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Figure 2. Rate of infection according to age groups
Figure 3. Rate of infection according to months
Table 1. Comparison between males and females in influenza-like illness infection in three years of study
Year
2015
2016
2017
Total
Gender
Female
938
442
236
1,616
47.3%
41.6%
43.5%
45.0%
Male
1,047
620
307
1,974
52.7%
58.4%
56.5%
55.0%
Pearson Chi-
square Tests
Chi-square9.497
Sig.0.009
Chi-square=9.497 Sig.=0.009

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4. Discussion
The first step in controlling transmissible diseases is
to guarantee precise and reliable diagnosis. The fact
that several distinct organisms can cause respiratory
illnesses with identical clinical signs makes a
physician's diagnosis of influenza problematic.
Molecular approaches applied directly to clinical
materials play an essential role in the diagnosis and
surveillance of influenza viruses. According to the
Centers for Disease Control and Prevention's annual
vital statistics report, between 12 and 32 million
occurrences occur each year. Due to reduced
turnaround times and higher sensitivity, RT-PCR
nucleic acid testing has largely supplanted classical
virus culture in the clinical diagnosis of influenza (7).
Some results (e.g., influenza illness validated by viral
culture or PCR) are more specific than others (e.g., ILI
defined by a clinical case definition, without definite
diagnostic testing). Clinical mortality rates have been
found to be high in several studies (10).
Specimens were collected in all seasons ofa3-year
investigation to provide an impression of infection in
all seasons and a broader concept of the distribution of
infection and its types throughout the year. As
influenza is considered a highly contagious sickness.
According to the infection rate statistics over the three-
year study period, negative cases accounted for 82.65%
of total cases, which could be a good result due to the
increased influenza vaccination use in Iraq, as well as
greater awareness of prevention and transmission.
The exact dates of the onset, peak, and end of
influenza activity vary from season to season and
cannot be predicted with certainty. Annual influenza
epidemics, however, are more common in the autumn
and winter in various nations. Influenza frequently
begins to spread (11), which is supported by our
findings, demonstrating that, based on the seasonal
distribution of infection, the winter months had the
highest rates, peaking in December, November,
Table 2. Comparison between significances
Age groups
Chi-square
95.319
df
18
Sig.
0.000*
FLUA
Chi-square
107.339
df
2
Sig.
0.000*
FLUB
Chi-square
109.672
df
2
Sig.
0.000*
Outcome
Chi-square
1.989
df
2
Sig.
0.370
FLUA: Influenza A virus; FLUB: Influenza B virus

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January, and February in descending order. This
outcome is consistent with the findings of another
investigation (12).
Influenza is contagious in people of all ages and is
difficult to be assessed precisely since numerous, if not
the majority, of those afflicted, do not require medical
attention, and therefore, are not diagnosed (13). Among
the population of this study (n=3,950), 582 (16.2%)
cases were positive for influenza A virus by RT-PCR,
12 (0.3%) subjects were positive for influenza B virus,
and 29 (0.8%) were positive. The results of comparing
males and females regarding the development of
influenza infection showed that males were more
affected than females (55% vs.45%, respectively). The
mechanisms that determine the differences between
genders are complex and can include hormonal,
immunological, behavioral, and genetic factors. It has
been revealed that females generate higher adaptive and
innate immune responses, compared to males. The
uneven susceptibility of females and males to infectious
diseases has been attributed to mating competition and
diet as behavioral and environmental factors (14).
Based on the findings of the present study, after
vaccination, females were better protected against
lethal challenges with new influenza virus strains than
males. The death and alive outcomes of infection were
recorded as the highest result with 7.7% in 2015 and
2017, compared to 1.1% in 2016. The reason for this
difference might be explained by influenza being
regarded as a non-lethal infection, except for specific
types (15). In the current study, the most common form
of influenza virus was reported to be type A,
accounting for 16.2%, followed by type B with 0.33%
frequency. The remaining percent (82.65%) was related
to negative cases, which was left out of the typing.
According to the results of a study (16), considering the
most common forms of influenza virus, type A was the
most prevalent (16.2%), followed by the Middle East
Respiratory Syndrome virus (0.81%) and type B
(0.33%). The remaining 25% was for negative
situations, with 82.65% being eliminated from typing.
Authors' Contribution
Study concept and design: I. M. A.
Acquisition of data: A. M. K.
Analysis and interpretation of data: L. G. A.
Drafting of the manuscript: M. A. A. A.
Critical revision of the manuscript for important
intellectual content: F. M. M. S.
Statistical analysis: I. M. A.
Administrative, technical, and material support: I. M.
A.
Ethics
All the procedures were approved by the Ethics
Committee at the Public Health Directorate, Baghdad,
Iraq. Under the project number 2021-54789-78411.
Conflict of Interest
The authors declare that they have no conflict of
interest.
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