Received: 26 June 2020 | Accepted: 24 July 2020

DOI: 10.1002/jmv.26364

RESEARCH ARTICLE

High prevalence of SARS‐CoV‐2 and influenza A virus (H1N1)

coinfection in dead patients in Northeastern Iran

Seyed A. Hashemi MD1 | Saghar Safamanesh MSc2 |

Hamed Ghasemzadeh‐moghaddam2 | Majid Ghafouri MD1 | Amir Azimian PhD2

1
Department of Infectious Diseases, School of
Medicine, North Khorasan University of
Medical Sciences, Bojnurd, Iran
2 In the last months of 2019, an outbreak of fatal respiratory disease started in
Department of Pathobiology and Laboratory
Sciences, School of Medicine, North Khorasan
University of Medical Sciences, Bojnurd, Iran
Correspondence
Amir Azimian, PhD, Department of
Pathobiology and Laboratory Sciences, School
of Medicine, North Khorasan University of
Medical Sciences, Bojnurd 74877‐94149, Iran.
Email: amir_azimian2003@yahoo.com
Funding information
North Khorasan University of Medical
Sciences, Grant/Award Number: 980002
Abstract
Wuhan, China, and quickly spread to other parts of the world. It was named COVID‐
19, and to date, thousands of cases of infection and death are reported worldwide.
This disease is associated with a wide range of symptoms, which makes accurate
diagnosis of it difficult. During previous severe acute respiratory syndrome (SARS)
pandemic in 2003, researchers found that the patients with fever, cough, or sore
throat had a 5% influenza virus‐positive rate. This finding made us think that the
wide range of symptoms and also relatively high prevalence of death in our patients
may be due to the coinfection with other viruses. Thus, we evaluated the coinfection
of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) with other re-
spiratory viruses in dead patients in North Khorasan. We evaluated the presence of
influenza A/B virus, human metapneumovirus, bocavirus, adenovirus, respiratory
syncytial virus (RSV), and parainfluenza viruses in 105 SARS‐CoV‐2 positive dead
patients, using polymerase chain reaction (PCR) and reverse transcription PCR tests.
We found coinfection with influenza virus in 22.3%, RSV, and bocavirus in 9.7%,
parainfluenza viruses in 3.9%, human metapneumovirus in 2.9%, and finally adeno-
virus in 1.9% of SARS‐CoV‐2 positive dead cases. Our findings highlight a high
prevalence of coinfection with influenza A virus and the monopoly of coinfection
with Human metapneumovirus in children.

KEYWORDS
adenovirus, bocavirus, coinfection, human metapneumovirus, influenza virus, parainfluenza
virus, respiratory syncytial virus, SARS‐Cov‐2

1 | INTRODUCTION
In December 2019 an outbreak of cases of pneumonia with un-
known etiology emerged in Wuhan, China, and most affected pa-
tients complained of symptoms related to viral respiratory illness
including fever, cough, headache, and breathlessness while some
showed respiratory failure, shock, and acute respiratory distress
syndrome.1,2 Evaluation of human airway epithelial cells, laboratory
tests, and genome sequencing led to confirming a novel β‐
coronavirus, which belonged to the Coronaviridae family; and on
7 January 2020, the World Health Organization named this disease
as COVID‐19.3
This virus called SARS‐CoV‐2, and is highly transmissible and led
to thousands of deaths in the world.
The accurate and rapid diagnosis of the etiological agent in pa-
tients is a key to survival, as based on numerous evidence, there are
no significant symptoms for making COVID‐19 distinguishable from
influenza infection. Consequently, it may mislead us when it is vital to
decide and choose the best treatment for patients. Notably, we found
the mortality rate surpassed the global average in our region.

1008 | © 2020 Wiley Periodicals LLC wileyonlinelibrary.com/journal/jmv J Med Virol. 2021;93:1008–1012.

HASHEMI ET AL.
So, simultaneous considering other viruses and early diagnosis to
apply the best treatment is vital. This way, we can save people with
COVID‐19‐like symptoms even though the other respiratory viruses
such as influenza maybe the main culprit.
2 | METHODS
We evaluated samples of 105 dead COVID‐19 positive patients. The
chief complaint in most of them was an acute respiratory syndrome.
Most of them had a wide range of signs, including fever, cough,
gastrointestinal discomfort, and joints pain. Clinical presentation,
laboratory findings, and radiological features were collected from
electronic medical records. We assessed their samples to detect
some important respiratory viruses, including the influenza virus,
parainfluenza virus (A–D), bocavirus, adenovirus, human metapneu-
movirus, and respiratory syncytial virus (RSV). The nasopharyngeal
and throat swabs of suspected patients were evaluated for COVID‐
19 using the LightMix Modular SARS‐CoV‐2 probe and primers (TIB
molbiol, Berlin, Germany) and addbio one‐step RT master mix (ADD
BIO Inc, Daejeon, Republic of Korea). The other respiratory viruses
4‐6
were evaluated using previously described methods. We also got
other related information such as laboratory tests and chest com-
puted tomography scan results and physical examination data from
patient's files with the license of the ethics committee of North
Khorasan University of Medical Sciences (No.: 980002).
3 | RESULTS
We evaluated 3446 samples from 02 March 2020 to 20 April 2020 in
North Khorasan Province. Of these, 1899 cases had negative, and
1444 cases had positive SARS‐COV‐2 reverse transcription poly-
merase chain reaction test, and finally, 105 cases had died in hospi-
tals, mortality rate 7.27%. Figure 1 shows the rate of coinfection with
other respiratory viruses in dead patients. We found a high pre-
valence of coinfection of SARS‐CoV‐2 with influenza A virus in dead
F I G U R E 1 The rate of coinfection with other respiratory viruses
in SARS‐CoV‐2 positive dead patients. SARS‐CoV‐2, severe acute
respiratory syndrome coronavirus 2
| 1009
patients (Figure 1). Most of the coinfected patients were men more
than 60‐year‐old (Table 1). They were coinfected with the H1N1 type
of influenza A virus. Among 105 cases of death, 23 cases (22.3%)
were coinfected with the influenza A virus. Of these, 78.3% were
H1N1, and 21.7% were non‐H1N1. Figure 2 shows the distribution of
underlying diseases in different age ranges of SARS‐CoV‐2 positive
dead patients. Underlying health conditions were frequently ob-
served in more than 40 years old, and heart disease was the most
common. None of 14 to 40 years old dead patients had an underlying
disease, and convulsion was the only underlying disease observed in
this age group.
4 | D IS C U S S I O N
The COVID‐19 outbreak started in December 2019 in Wuhan. To
date, this disease has spread in many countries and has resulted in
millions of cases of infection and thousands of deaths all over the
world.7 Recently published reports of concurrent infections of other
respiratory viruses such as influenza virus8‐12 and human me-
tapneumovirus13,14 with SARS‐CoV‐2 infection have suggested that
coinfection could affect the morbidity and mortality ratio. In addition
to these, due to the wide range of symptoms in patients and rela-
tively higher mortality in comparison with the global rate,15 we
decided to evaluate the coinfection of SARS‐CoV‐2 with other viral
respiratory tract pathogens in our patients. Influenza spreads across
Iran for over 6 months. It usually peaks in October and subsides in
April, and our samples were collected from March 02, 2020, to April
20, 2020, when the influenza virus was in circulation. Limited studies
were performed regarding the prevalence of different influenza virus
types in Iran, and we do not have exact information about the in-
fluenza epidemiology in Iran in 2019 to 2020, but in some pub-
lished papers last year, authors have reported that the influenza
A subtypes H1N1 and H3N2 are the most prevalent influenza types
in Iran.16‐20
In our samples, SARS‐CoV‐2 was coinfected with influenza A
virus at a higher rate (22.3%) in comparison with other respiratory
tract viruses (Table 1), this ratio was 19.3% in living patients. The
occurrence of SARS‐CoV‐2 and influenza A coinfection is relatively
similar in living and dead patients, and it may be due to the high
circulation of seasonal influenza and low usage of influenza vacci-
nation in that area. Some researchers found that the influenza vac-
cination leads to a decrease of death due to COVID‐19, and have
assumed that some cases of death in COVID‐19 patients may be
related to SARS‐CoV‐2 and influenza coinfection.21 The first re-
port by Wu et al on SARS‐CoV‐2 and influenza virus coinfection was
published on June 2020.8,22 The report was about coinfection in a
69‐year‐old man. According to this, most of our coinfected cases
were men aged more than 60 years (Table 1). The above mentioned
patient did not develop any underlying disease, but our cases had
some illnesses such as heart disease, asthma, diabetes, and chronic
neurological disease (Table 2). In total, the most prevalent underlying
diseases in our SARS‐Cov‐2/influenza positive dead patients were
1010 | HASHEMI ET AL.

T A B L E 1 The prevalence of viruses in various age range and genders

Age range (number/%) Gender (number/%)

Virus Type Subtype 0‐14 14‐40 40‐60 >60 Male Female Total

Human metapneumovirus 3 (100%) 0 0 0 2 (66%) 1 (33%) 3 (6.1%)

Bocavirus 1 (11.1%) 2 (22.2) 4 (44.4%) 2 (22.2%) 5 (55.5%) 4 (44.5%) 9 (18.4%)

Adenovirus 0 0 1 (50%) 1 (50%) 0 2 (100%) 2 (4.1%)

Parainfluenza virus 0 0 0 4 (100%) 3 (75%) 1 (25%) 4 (8.2%)

RSV 0 1 (12.5%) 1 (12.5%) 6 (75%) 4 (50%) 4 (50%) 8 (16.4%)

Influenza virus A H1N1 1 (5.5%) 0 3 (16.7%) 14 (77.8%) 11 (61.1%) 7 (38.9%) 18 (36.8%)

Non‐H1N1 0 0 2 (40%) 3 (60%) 3 (60%) 2 (40%) 5 (10.2%)
B 0 0 0 0 0 0 0

heart disease, asthma, diabetes, and chronic neurological diseases,

F I G U R E 2 The distribution of underlying diseases in different age
ranges of SARS‐CoV‐2 positive dead patients. 1, hypertension; 2,
diabetes; 3, chronic neurological disease/convulsion; 4, chronic
pulmonary disease/asthma; 5, severe obesity; 6, cancer; 7, hepatitis;
8, kidney disease; 9, heart disease. SARS‐CoV‐2, severe acute
respiratory syndrome coronavirus 2
respectively. In concordance with our findings, Yue et al found that
coinfection with SARS‐CoV‐2 and influenza virus was common at
their center. They worked on 307 SARS‐CoV‐2 infected patients and
reported that the influenza B virus coinfection leads to a higher risk
of developing an adverse prognosis.23
Contrary to our findings, Kim et al reported a shallow rate of
SARS‐CoV‐2 coinfection with the influenza A/B virus. They report
only one case of influenza A positive test in 116 SARS‐CoV‐2 positive
patients, and it should be noted that like our findings, they did not
find the influenza B virus in their positive samples.24 On the other
hand, in their report, other respiratory viruses including rhinovirus/
enterovirus, RSV, and non‐SARS coronaviruses were detected at high
rates. Besides, in another study, Wang et al evaluated 8274 patients
with 2745 SARS‐CoV‐2 positive cases. 5.8% of SARS‐CoV‐2 positive
and 18.4% of SARS‐CoV‐2 negative cases were positive for other
viruses.25 Furthermore, influenza virus, RSV, bocavirus, parainfluenza
virus, Human metapneumovirus, and adenovirus, were detected in

T A B L E 2 The distribution of coinfected viruses in SARS‐CoV‐2 positive patients with various underlying diseases

Human Parainfluenza Influenza virus

H1N1 metapneumovirus Bocavirus Adenovirus virus RSV Non‐H1N1 Total

Hypertension 0 0 0 0 0 0 0 0

Diabetes 0 0 1 (50%) 0 0 1 (5.5%) 1 (20%) 2 (8.7%)

Chronic neurological disease/ 1(33.3%) 0 0 0 0 1 (5.5%) 1 (20%) 2 (8.7%)

convulsion

Chronic pulmonary disease/ 0 1 (11.1%) 0 0 0 2 (11%) 0 2 (11%)

asthma

Severe obesity 0 0 0 0 0 0 0 0

Cancer 0 0 0 0 0 0 0 0

Hepatitis 0 0 0 0 0 0 0 0

Kidney disease 0 0 0 0 0 0 0 0

Heart disease 0 2 (22.2%) 2 (100%) 3 (75%) 2 (50%) 5 (27.8%) 1 (20%) 6 (26.1%)

Total 3 9 2 4 8 18 5 23
HASHEMI ET AL.
our positive samples (Figure 1). We consider, our study has two
significant findings. The first and the most important is the high rate
of coinfection with influenza A virus (H1N1). We cannot comment
which virus had first infected the patients, serological tests may help
judge this. The second important finding is the limitation of coin-
fection with human metapneumovirus in children (Table 1). It seems
that we confront additional challenges in patients with coinfections,
especially in cold seasons because other respiratory viruses such as
influenza are more common. It becomes significant when the patients
have the signs of COVID‐19 infection, but their tests are negative for
SARS‐CoV‐2 but positive for another virus.
In summary, our study suggests that COVID‐19 might be un-
derdiagnosed because of false‐negative tests or coinfection with
other respiratory viruses. More extensive viral testing might be
needed for accurate etiologic identification, particularly if it affects
clinical management decisions.
A C K N O W L E D GE M E N TS
This study is part of the national COVID‐19 screening program of
Iran. The Primer and Probes were supported by the Ministry of
Health of Iran and other laboratory kits provided by North Khorasan
University of Medical Sciences (Grant No.: 990002).
CO NFLICT OF I NTERE STS
The authors declare that there are no conflict of interests.
A UT HO R C ONT RI BU TIO NS
SAH conceptualized and designed the study, coordinated and su-
pervised data collection, carried out the initial analyses, and reviewed
and revised the manuscript. SS collected the laboratory data, carried
out the PCR and RT‐PCR tests, and edited and revised the manu-
script. MG contributed to data collection, carried out the initial
analyses, and reviewed and revised the manuscript. AA con-
ceptualized and designed the study, carried out the PCR and RT‐PCR
tests, drafted the initial and final version of the manuscript. All au-
thors approved the final manuscript as submitted and agreed to be
accountable for all aspects of the work.
D A T A A V A I L A B I LI TY S TA T E ME N T
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
OR CID
Amir Azimian http://orcid.org/0000-0002-2532-8198
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