NAOSITE: Nagasaki University's Academic Output SITENAOSITE: Nagasaki University's Academic Output SITE
Viral pathogens associated with acute respiratory infections in
central vietnamese children.
Yoshida, Lay Myint; Suzuki, Motoi; Yamamoto, Takeshi;
Nguyen, Hien Anh; Nguyen, Cat D; Nguyen, Ai T; Oishi,
Kengo; Vu, Thiem D; Le, Tho H; Le, Mai Q; Yanai, Hideki;
Kilgore, Paul E; Dang, Duc Anh; Ariyoshi, Koya
Citation The Pediatric infectious disease journal, 29(1), pp.75-77; 2010
© 2010 Lippincott Williams & Wilkins, Inc. / This is a non-final version of
an article published in final form in The Pediatric infectious disease journal,
29(1), pp.75-77; 2010.
Title: Viral Pathogens Associated with Acute Respiratory Infections in Central
Lay Myint Yoshida, MBBS, PhD,* Motoi Suzuki, MD,* Takeshi Yamamoto, MSc,* Hien
Anh Nguyen, MSc ,# Dinh Lien Cat Nguyen, MD,§ Thi Thuy Ai Nguyen, MD,§ Kengo
Oishi, BSc,* Thiem Vu Dinh, MD,# Le Huu Tho, MD, PhD,‡ Le Mai Quynh, MD, PhD#
Hideki Yanai, MD, Ph.D, * Paul E Kilgore, MPH, MD, *# Duc Anh Dang, PhD, # and
Koya Ariyoshi, MD, PhD*
*Department of Clinical Medicine, Institute of Tropical Medicine, Nagasaki University,
Nagasaki, Japan; #National Institute of Hygiene and Epidemiology, Hanoi, Vietnam;
§Khanh Hoa General Hospital; ‡Khanh Hoa Health Service, Nha Trang, Vietnam;
*#International Vaccine Institute, Seoul, South Korea.
Address for correspondence:
Koya Ariyoshi, MD, PhD, Department of Clinical Medicine, Institute of Tropical
Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki, Japan, 852-8523.
Tel:81-95-819-7842, Fax: 81-95-819-7843, email: firstname.lastname@example.org
Keywords: viral; respiratory pathogens; multiplex PCR; pediatric; Vietnam
Abbreviated title: Viral Pediatric Acute Respiratory Infections in Vietnam
Running title: Pediatric Viral Respiratory Agents, Vietnam
Hospitalized Vietnamese children with acute respiratory infection (ARI) were
investigated for 13 viral pathogens using multiplex-polymerase chain reaction. We
enrolled 958 children of whom 659(69%) had documented viral infection: rhinovirus
(28%), respiratory syncytial virus (23%), influenza virus (15%), adenovirus (5%),
human metapneumo virus (4.5%), parainfluenza virus (5%) and bocavirus (2%). These
Vietnamese children had a range of respiratory viruses which underscores the need for
enhanced ARI surveillance in tropical developing countries.
Acute respiratory tract infections (ARI) are a major cause of morbidity and
mortality in children worldwide (1). Viruses are common cause of lower respiratory
tract disease in infants and young children and represent a major public health problem
in children (2). Because there are limited resources and facilities for virus isolation and
detection in the developing countries, the role of viral respiratory pathogens in the
tropical region has not been well-studied. To investigate the magnitude of viral
respiratory infections among hospitalized infants and children in a tropical country, we
applied a newly developed multiplex-polymerase chain reaction (PCR) technique into
an active ARI surveillance program conducted in south central Vietnam.
The study was conducted from February 1, 2007 to March 31, 2008 at Khanh
Hoa General Hospital (KHGH) which is a tertiary care facility located in Nha Trang city,
Khanh Hoa Province. According to the field site census survey in July 2006, the study
catchment area of 16 communes in Nha Trang city, had 198,729 residents including
13,952 children less than 5 years of age. An ARI case was defined as any child
presenting with cough or/and difficulty in breathing. Before study enrollment, informed
consent was obtained from parents of children who presented with ARI and lived in the
study catchment area. Patient clinical information, chest radiographs (CXR), laboratory
data and nasopharyngeal (NP) samples were collected from all enrolled patients.
Radiograph interpretations were standardized using MiASoft, Ltd. (Faringdon, UK)
radiology training modules. Acute Respiratory Infection patients with normal CXR
were categorized as upper respiratory tract infection (URTI) and patients with abnormal
CXR were categorized as lower respiratory tract infection (LRTI). Bronchiolitis was
defined as ARI patient ≤ 2 years old presenting with wheezing and abnormal CXR
without evidence of pulmonary consolidation.
Nasopharyngeal (NP) samples were collected at the time of admission and viral
nucleic acid was extracted using QIA viral RNA minikit (QIAGEN Inc., Valencia, CA,
USA). Four multiplex-PCR assays (1: influenza A, influenza B, RSV, hMPV; 2: PIV-1,
-2, -3, and -4; 3: rhinovirus, coronavirus 229E, coronavirus OC43; 4: adenovirus and
bocavirus) were performed to detect 13 respiratory viruses in each NP sample. A second
confirmatory-PCR was performed for samples positive on the initial PCR test
(Supplementary table 1., online only). Samples positive for both PCR assays were
defined as positive. Reverse transcription-PCR (RT-PCR) assays were performed using
one-step RT-PCR kit from QIAGEN. For the multiplex PCR and hemi-nested PCR
assays, Taq DNA polymerase (Promega, San Luis Obispo, CA) was used. Twenty-eight
primers used were established in this study and 11 were from the published studies (3-5).
Positive templates were used in each assay for quality control.
During the 14-months study period, a total of 1,014 pediatric patients from the
catchment area were admitted to KHGH, of which 958 (95%) were enrolled in the study.
Males comprised 58% of patients and 94% of the patients were less than 5 years old
(median age: 1.4 years). The results showed that one or more respiratory viruses were
found in 69% of patients: 11% had dual and 1.4% had triple infection. Eighty six
percent of the viral ARI patients were less than 3 years old (detail information of age
breakdown is shown in supplementary table 2, online only).
Major viruses detected were rhinovirus (28%), RSV (23%) and influenza A
(15%). This was followed by adenovirus (5%), hMPV (5%), PIV3 (4%) and bocavirus
(2%). Other viruses (PIV1, PIV2 and influenza B) were detected in a small proportion
(1.5%) of ARI patients. Across age, sex, and case categories, there were no significant
differences between proportion of virus positive and negative patients.
The pattern of virus detection did not differ between URTI and LRTI patients. A
total of 268 radiologically-confirmed pneumonia (RCP) patients and 195 bronchiolitis
patients were identified. PIV3 detection was significantly associated with hospitalized
LRTI (p=0.016) and bronchiolitis (p=<0.001). Similar to previous reports, we found
that RSV infection was significantly associated with bronchiolitis (p=0.002) (6). We
also found that a significantly higher proportion of patients (n=119) with multiple virus
infections had radiological-confirmed pneumonia (p=0.05 by Fisher’s exact test)
(supplementary table 2, online only). None of the cases with multiple virus infection
Influenza A infection predominated during the 2007 cool season, from February
through June 2007 (Figure 1) while RSV patients were detected largely during warmer
months, from July to November with the peak in August 2007. In contrast to influenza
A and RSV, rhinovirus was detected year round with no distinct seasonality. In early
2008, ARI patients were infected with rhinovirus but not influenza A virus.
To calculate the incidence rates, we used only one year data from March 1st
2007 to February 28th 2008 during which 812 children less than 5 years old were
admitted to KHGH (Table 1). The hospitalized ARI incidence for children less than 5
years living in Nha Trang was 58 of 1000 children-year. The highest hospitalized ARI
incidence was observed in children less than 1 year age group (130/ 1000 children-year)
while the highest age group for RCP and LRTI were among 1-2 year age group (39 and
85/ 1000 children-year respectively). The highest incidence of rhinovirus- and
influenza-associated ARI hospitalization were found among 1-2 year age group
(41/1000 children-year and 18/1000 children-year) while highest RSV-associated ARI
incidence (41/1000 children-year) occurred in infant less than 12 months.
In this study, we demonstrated pediatric ARI was associated with a wide variety
of viruses in a tropical developing country setting such as south central region of
Vietnam. Ten different respiratory viruses were associated with approximately
two-thirds of the hospitalized patients with ARI. Recently discovered viruses, including
hMPV (7) and bocavirus (8) were associated with a substantial number of pediatric ARI
hospitalizations. It is plausible that prevalence of viral pathogens associated with ARI
may vary in different countries with different geographic and climatic characteristics.
The spectrum of major respiratory pathogens identified in our study was similar to
previous studies in Germany and South Korea (9, 10). However, there were some
differences in terms of proportion and seasonality of detected viruses. Our study showed
a higher proportion of influenza-associated ARI compared with either Germany or
We detected no viruses in one-third of children hospitalized with ARI. It is
possible that some children may have had infection with other viruses not included in
our assays (e.g., enteroviruses, other types of coronaviruses like NL63 and HKU1). In
addition, some patients may have cleared their viral infection before the collection of
clinical samples or the viruses circulating in our study population may carry
polymorphisms in the primer binding sequences of the our target genes.
Although our study showed a high incidence of respiratory viral infections
among hospitalized ARI patients, we believe that bacterial pathogens account for a
substantial proportion of ARI among hospitalized children. Since this study was only 14
months, we cannot conclude on the seasonality of the viruses that we detected. However
it was interesting that a different pattern of influenza was observed in the last 2 months
compared to the same period of previous year. For the above reasons, we believe that
further studies are warranted to investigate the relation between viral infection and
bacterial ARI, and the interaction among different respiratory viruses, and to elucidate
seasonal patterns and annual variations of viral infections in tropical developing
countries like Vietnam.
In conclusion, a high incidence of respiratory virus-associated ARI was found
among hospitalized children in central Vietnam. In this study, rhinoviruses, RSV and
influenza A viruses were the commonest respiratory viruses detected among children.
The identification of recently identified respiratory pathogens such as human
metapneumovirus and bocavirus in central Vietnam underscore the need to enhance
existing surveillance systems to understand the full burden of respiratory pathogens.
We thank Drs. Takato Odagiri, Masahiro Noda from National Institute of
Infectious Diseases, Tokyo, Dr.Nobuhisa Ishiguro from Hokkaido University and
Dr.Katsumi Mizuta from Yamagata Prefectural laboratory for providing the necessary
control viruses. We thank the parents and participants in this study as well as staff from
Khanh-Hoa Health Service and medical staff from of Khanh Hoa General Hospital for
their support in this study. The primers and multiplex-PCR assays established and used
in this study are pending for patent. This work was supported by the program of
Founding Research Centers for Emerging and Reemerging Infectious Diseases (MEXT)
Japan and Japan Society for Promotion of Science.
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Figure 1. Seasonality of leading viral pathogens among hospitalized children in
February 2007- March 2008
SUPPLIMENTAL DIGITAL CONTENT LEGEND
Supplementary Table 1. Primers and PCR assays for multiplex PCR and hemi-nested
Supplementary Table 2. Characteristics of Hospitalized Pediatric Viral Acute
Respiratory Infection patients, February 2007 through March 2008
Table 1. Hospitalized incidence of ARI and major viral agents among children less than
five years in Nha Trang (incidence per 1000 children, March 1st 2007 to Feb 28th 2008)
0 to 1yr 2250
65.8 (148) 35.1 (79) 30.7 (69)
1 to 2yr 2442 127.4 (311)
2 to 3yr 3319 39.5 (131) 24.1 (80) 10.8 (36) 10.5 (35) 8.7 (29) 6.9 (23)
3 to 4yr 2944 19.7 (58) 9.9 (29) 4.8 (14) 4.4 (13) 4.8 (14) 3.4 (10)
4 to 5yr 2997 6.7 (20) 3.3 (10) 1.3 (4) 1 (3) 0.7 (2) 2 (6)
< 5yrs, 13952 58.2 (812) 34 (474) 16.3 (227) 15.8 (220) 15.3 (214) 8.7 (122)
Highest incidence in each group are shown in bold
ARI: acute respiratory infection, LRI: lower respiratory infection, RCP: radiological
confirmed pneumonia, RSV: respiratory syncytial virus, InfluA: influenza virus type A