Background

Acute respiratory infections (ARIs) are one of the illnesses of highest morbidity and mortality in children worldwide [1-3]. The pathogens causing ARIs vary geographically and by season, but globally viruses play a major role. Respiratory syncytial virus (RSV) is by far the most common pathogen associated with severe respiratory diseases as bronchiolitis, exacerbation of asthma, or pneumonia in early life, and is a leading cause of hospitalization in children under two [4]. Influenza viruses have the greatest potential to cause severe respiratory diseases in the very young, the elderly and those with underlying chronic conditions [5]. Enteroviruses including human rhinoviruses (HRV) and human enteroviruses (EV), previously identified in childhood upper respiratory tract infections, are commonly associated with milder ARIs and have been suspected as major etiological agents of lower respiratory tract infections leading to bronchiolitis and pneumonia in infants [6]. It has also been reported that human metapneumovirus (hMPV) causes approximately 5-10% of all ARIs in children and adults [7] and adenoviruses (ADV) account for 5-15% of respiratory infections in children [8]. Respiratory illnesses can be attributable to other viruses such as parainfluenza viruses (PIV) and human coronaviruses hCoV-229E, OC43 [7]. With rapid progress in molecular diagnostics, newly discovered viruses including human bocavirus (hBoV), human coronaviruses (hCoV-NL63, hCoV-HKU1), human parechoviruses (hPeV), and polyomaviruses WU (WUPyV) and KI (KIPyV) have also been detected in children with respiratory infections, with varying levels of proof of causation [9].

Hospital-based studies in children published over the last decade worldwide have identified viruses in up to 95% of ARI episodes, with a single virus found in 40-60% and multiple viruses in 1-40% of infected patients [7,8,10]. Co-infection is reportedly related to the time of year when circulations of multiple viruses occur [11]. Some studies have shown that the prevalence of co-infections is not related to the absolute prevalence of individual viruses [12]. Factors such as young age, male gender, and history of immunosuppression are associated with an increased chance of viral co-infections [11,13,14]. There could be likely interactions between climatic, environmental, and behavioral factors, and complex interplay between circulating viruses and population-level immunity regarding viral co-infections. Understanding these factors may help us prevent transmission of these infections.

Recent etiologic studies on pediatric respiratory infections mostly report the prevalence in hospitalized children and the seasonality of viruses without elaborating viral co-infection. Therefore, the significance of the detection of multiple viral pathogens in ARIs is unclear. Here, we investigated fourteen common respiratory viruses among pediatric outpatients in southern China during 2010–2011 and their associations with meteorological factors.

Methods

Study location

This study was conducted at the Pediatric Outpatient Walk-in Clinics, the First Affiliated Hospital of Shantou University Medical College. The Pediatric Department provides both primary and tertiary care (common practice in China) for approximately 35,000 children per year in the Chaoshan region of southern China. The Chaoshan region is in the subtropical zone with an average annual temperature of 21.3°C and excellent to lightly polluted air quality levels (air quality index, AQI: 17–142, in 2012–2013).

Study design

Based on modified WHO standard case definition of ARIs [7], eligible participants were defined as a child 0–16 years of age presenting within 3 days of onset of illness with at least two of the following: fever, sore throat, cough, rhinorrhea, nasal congestion, and hoarseness of voice. Patients with any condition preventing swab collection were excluded. We recruited eligible patients in the morning, during which approximately 70% of patient visits are made, on a daily basis except public holidays from December 2010 to November 2011. Participants’ demographic details and clinical features are shown in Table 1. Paired nose and throat-flocked swabs (Copan, Brescia, Italy, Cat. no. 503CS01 and 502CS01) were collected from each participant, combined in one tube, and stored within 3 h of collection at −80°C until further processing.

Table 1 Demographic and clinical characteristics, and virus positivity of pediatric outpatients with acute respiratory infections (n = 1,074)
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Laboratory procedure

Multiplex real-time PCR was performed using Roche, Lightcycler 480 II (Roche Diagnostics, Penzberg, Germany) to identify the following 14 respiratory viruses: influenza A (FluA), influenza B (FluB), respiratory syncytial viruses A and B (RSV), human coronaviruses 229E, OC43, HKU1 and NL63 (hCoV), human metapneumoviruses A and B (hMPV), human parainfluenza virus types 1, 2 , 3, and 4 (PIV1, PIV2, PIV3, and PIV4), human rhinoviruses A, B, and C (HRV), human enteroviruses (EV), human adenoviruses (ADV), human bocavirus (hBoV), and human parechoviruses (hPeV).

Nucleic acid extraction was performed using the QIAamp Viral RNA Mini Kit (QIAGEN GmbH, Hilden, Germany, Cat. no. 52906). Reverse transcription and Real-time PCR assays were performed as described previously [15], except for the primers and/or probes for HRV, hPeV, and internal control equine arteritis virus (EAV, see the sequences of 14 viruses in Additional file 1). Due to known cross-reactivity between enteroviruses [16-18], HRV was detected using two sets of primers and probes: HRV-v1 (version 1) for screening and HRV-v2 (version 2) for confirmation. Real-time PCR results were interpreted as described previously [15]. The PCR was considered positive or negative when the Cp value was less than 40 cycles or exceeded 40 cycles, respectively, and the positive control showed the expected Cp value, negative control was negative, and internal control showed the expected Cp value. A negative internal control signal was accepted in case of a positive target sequence with correct positive and negative control signals.

Meteorological data

Meteorological data, including the average daily temperature (°C), the average daily humidity (%), and the average daily wind velocity (km/h), were collected from the official website of Shantou Meteorology, TuTiempo.net (http://www.tutiempo.net/en/Climate/Shantou/2011/593160.htm).

Statistical analysis

We used Chi-square test to compare differences in the distribution of categorical variables, ANOVA and Kruskall Wallis tests to compare medians, and the Pearson correlation analysis to evaluate the associations between the meteorological factors and viruses and among viruses. The variables with significant associations were further analyzed in multivariate logistic regression models, in which symptoms and positivity of viruses were treated as dependent and independent variables to assess virus-symptom associations; and individual viruses were treated as dependent variables with meteorological factors or other viruses as independent variables to investigate meteorological factor-virus and virus-virus associations. A two-tailed p-Value of <0.05 was considered significant. All these analyses were performed with SPSS Statistics version 17.0.

Ethics

The study was approved by the Ethics Committee of the First Affiliated Hospital of Shantou University Medical College and the Oxford University Tropical Research Ethical Committee (OxTREC). Written informed consent was obtained from parents or legal guardians of children enrolled in the study.

Results

Of 1,074 children (62.3% male) recruited, 43.6% (468/1,074) were >2-5 years old (Table 1). At least one virus was identified in 82.3% (884/1,074) of the patients, with single virus in 43.7% (469/1,074) and multiple viruses in 38.6% (415/1,074). hPeV was not detected. Compared with virus-negative patients, virus-positive patients were less likely to have fever (OR: 0.7, 95% CI: 0.5-1.0, p = 0.05). Patients with multiple viruses were more likely to have rhinorrhea than those with single virus (OR: 1.4, 95% CI: 1.1-1.9, p < 0.05, Table 1). hCoV (OR: 1.6, 95% CI: 1.0-2.4) and PIV4 (OR: 1.6, 95% CI: 1.0-2.4) were more prevalent in the >5 year age group than in the ≤5 year group (all p ≤ 0.05), while hBoV (OR: 0.3, 95% CI: 0.1-0.7) and RSV (OR: 0.4, 95% CI: 0.2-1.0) were less frequently found in the >5 year group (all p < 0.05). Chi-square test and multivariate logistic regression analysis showed that cough was positively associated with HRV and RSV, and negatively with EV; rhinorrhea was positively associated with HRV, PIV3, and hBoV, and negatively with EV; fever was positively associated with EV, and negatively with HRV and PIV3; and nasal congestion was positively associated with RSV, and negatively with EV and hCoV (all p < 0.05, Table 2).

Table 2 Multivariate logistic regression analysis of associations between viruses and clinical presentations
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Viruses detected alone or co-detected with other viruses are shown in Table 3. The most frequently detected virus was EV (42.3%, 374/884), followed by HRV (39.9%, 353/884), and hCoV (17.5%, 155/884). EV and HRV were most commonly co-detected with other viruses (Table 3) and also the most commonly co-detected pair of viruses (23.1%, 96/415, see the distribution pattern of viruses in Additional file 2). Screening with HRV-v1 identified 298 cases co-positive for HRV and EV, and subsequent confirmation with HRV-v2 primers/3 probes [15] resulted in only 96 positive cases (32.2%, 96/298).

Table 3 Detection of viruses from nasal/throat swabs of pediatric outpatients with acute respiratory infections, ARIs (n = 884)
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Seasonality and meteorological factors

The temporal circulation and co-circulation patterns of viruses are shown in Figures 1 and 2. There were overlap** seasonal trends of many viruses throughout the year, with dual peaks for EV in July and September and single peaks for the other viruses. Both EV and HRV circulated throughout the year. hCoV and PIV4 circulated predominantly between April and May but sporadically throughout the year. PIV3, RSV, FluA, and ADV peaked in January, while hBoV peaked in March. FluB circulated mostly from February to July with a peak in April. Co-detection of 5–7 viruses occurred all in May (see Additional file 3). The optimal average daily temperature, humidity, and wind velocity for these viruses are shown in Table 4.

Figure 1
figure 1

Temporal circulation pattern of respiratory viruses (n = 1,074). EV, enterovirus; HRV, human rhinovirus; hCoV, human coronavirus; PIV1-4, parainfluenza 1–4; hBoV, human bocavirus; RSV, respiratory syncytial virus; FluA, influenza A; FluB, influenza B; ADV, adenovirus. % = individual-virus-positive cases/total cases tested.

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Figure 2
figure 2

Temporal co-circulation pattern of respiratory viruses (n = 1,074). EV, enterovirus; HRV, human rhinovirus; hCoV, human coronavirus; PIV1-4, parainfluenza 1–4; hBoV, human bocavirus; RSV, respiratory syncytial virus; FluA, influenza A; FluB, influenza B; ADV, adenovirus. % = individual-virus-positive cases/total cases tested.

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Table 4 The optimal average daily temperature, humidity, and wind velocity for some circulating viruses
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Virus-meteorological and virus-virus associations

Table 5 shows the multivariate logistic regression models for independent associations between the viruses and meteorological factors and between the viruses. EV was positively associated with the average temperature and humidity and the presence of hCoV and PIV4, but negatively with HRV, PIV3, and hBoV. HRV was negatively associated with the presence of EV and PIV3. hCoV was positively associated with the average temperature and humidity and the presence of EV and PIV4. PIV3 was positively associated with the average humidity and the presence of RSV and FluA, but negatively with the average temperature and wind velocity, and the presence of EV, HRV, and hBoV. PIV4 was positively associated with the average temperature and the presence of hCoV and RSV, however, negatively with the wind velocity. hBoV was positively associated with RSV and FluA, but negatively with the average temperature and humidity and the presence of EV and PIV3. RSV was positively associated with the presence of PIV3-4, hBoV, and FluA, but negatively with the average temperature and wind velocity. FluA was positively associated with the presence of PIV3-4, hBoV, and RSV, but negatively with the average temperature.

Table 5 Multivariate logistic regression analysis of virus-meteorological and virus-virus associations
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Discussion

This is the first prospective study reporting the associations between meteorological parameters and co-circulation patterns of 14 common respiratory viruses. The viral detection rate among pediatric outpatients with ARIs in this study (82.3%, 884/1,074) was higher than those reported from Nan**g, China (16 viruses, 50.6%, 248/490) [19] and other countries, including Honduras (16 viruses, 75.4%, 260/345) [20] and Greece (17 viruses, 70.0%, 428/611) [6] in the same study period. Enteroviruses (EV, 34.8% and HRV, 32.9%) were most frequently detected in our outpatient children. Influenza viruses and RSV, the leading pathogens in pediatric outpatients in similar studies [6,21-23], were detected in 10.4% and 7.0% of our cases, with hCoVs (229E, OC43, HKU1, and NL63) in 14.4%, and relatively recently discovered viral pathogens hBoV and hMPV in 7.3% and 0.3% of cases, respectively (Table 3).

The viral co-detection rate (38.6%, 415/1,074) was also high among our study population. Reported rates of co-detection vary widely, from 6.1% among pediatric patients with influenza-like illness [19] to 62% among infants with acute bronchiolitis [24]. Detection of dual viruses is common, and co-detection of five [25] or even six viruses [26] is not anecdotal. All the cases with 5–7 viruses in this study were in May, the end of the cold season in the Chaoshan region. This may be in part due to past viral infections, as some viruses can still be detectable by PCR several weeks after infection [15,16]. Most studies have shown that RSV is the predominant respiratory pathogen co-detected in hospitalized children, followed by HRV, PIV, hMPV, hBoV, and FluA [25,27]. In this study, EV, HRV, hCoV, and PIV 3–4 were involved in the majority of co-detections, with EV-HRV as the most frequently co-detected pair (23% of co-detections). EV and HRV were included in the panels in many studies globally [6,7,10,11,24,28-34], and the EV-HRV pair was the most commonly detected pair among outpatient children with ARIs in Finland (19.6% of co-detections) [28] and infants with acute bronchiolitis in Brazil [10]. The co-detection rate of EV-HRV in this study is similar to that in Finland [28].

Varying detection rates of multiple viruses in different studies may reflect the differences in the study period and location, study population, environmental factors, the number of respiratory pathogens tested, and/or the diagnostic methods/techniques used. Likely reasons behind high detection rates of single and multiple viruses in this study could be due to improved recovery of viruses by using flocked swabs [35] and/or combined nasal and throat swabs [16].

There are advantages and disadvantages of multiplex PCR technique in diagnosing respiratory viral infections. While its high sensitivity and specificity facilitate simultaneous detection of a large spectrum of viruses, including those difficult to be identified by traditional methods [32], its capacity to detect low amounts of viral nucleic acids in some cases during viral incubation period, asymptomatic infection, or post-infectious shedding makes it difficult to interpret the results [30,32]. The development and validation of standardized quantitative PCR with clinically relevant cutoff values [30] or combining qPCR with serology could be helpful for etiologic understanding of simultaneous presence of multiple viruses.

Certain host-specific risk factors may predispose a child to respiratory co-infection. Younger age [11,

Abbreviations

ARIs:

Acute respiratory infections

FluA:

Influenza A

FluB:

Influenza B

RSV:

Respiratory syncytial virus

hCoV:

Human coronavirus

hMPV:

Human metapneumovirus

PIV:

Parainfluenza virus

HRV:

Human rhinovirus

EV:

Enteroviruses

ADV:

Adenoviruses

hBoV:

Human bocavirus

hPeV:

Human parechoviruses

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Acknowledgements

We would like to thank the pediatricians from the Pediatric Department, the First Affiliated Hospital of Shantou University Medical College for their generous support, the children and their guardians for participation in this study, Richard Molenkamp at the University of Amsterdam, Academic Medical Center for technique and knowledge transfer to set up the multiplex real-time PCR, Jieling Chen at the Shantou-Oxford Clinical Research Unit for technical assistance, and staff in the International Institute of Infection and Immunity, Shantou University Medical College for their assistance with real-time PCR.

This study was supported by the Li Ka Shing Foundation, Shantou University Medical College, and the University of Oxford (grant No. B9RSRT0-14). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Pediatric Department, the First Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, P.R. China

    Binglin Cui & Beiyan Wu

  2. The Research Center of Translational Medicine, the Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, P.R. China

    Dangui Zhang

  3. Shantou-Oxford Clinical Research Unit, Shantou University Medical College, Shantou, Guangdong, P.R. China

    Hui Pan & William Ba-Thein

  4. Oncology Research Laboratory, Cancer Hospital of Shantou University Medical College, Shantou, Guangdong, P.R. China

    Fan Zhang

  5. Oxford University Clinical Research Unit, Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Ho Chi Minh City, Viet Nam

    Jeremy Farrar & H Rogier van Doorn

  6. Consultant Office, Shantou University Medical College, Shantou, Guangdong, P.R. China

    Frieda Law

  7. Department of Microbiology and Immunology, Shantou University Medical College, Shantou, Guangdong, 515041, P.R. China

    William Ba-Thein

Authors
  1. Binglin Cui
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  2. Dangui Zhang
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  3. Hui Pan
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  4. Fan Zhang
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  8. Beiyan Wu
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  9. William Ba-Thein
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Corresponding author

Correspondence to William Ba-Thein.

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Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

BLC and HP designed and performed the experiments, analyzed the data, and wrote the paper. DGZ designed and performed the experiments, and analyzed the data. FZ analyzed the data. JF conceived and designed the experiments, and analyzed the data. FL analyzed the data, and facilitated the study. HRvD analyzed the data. BYW facilitated the study. WB-T designed the experiments, analyzed the data, and wrote the paper. All authors read and approved the final manuscript.

Additional files

Additional file 1:

Primers and probes of 14 viruses.

Additional file 2:

The distribution pattern of viruses in pediatric outpatients with acute respiratory infections, ARIs (n=415).

Additional file 3:

The number of viral co-positive cases by month.

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Cui, B., Zhang, D., Pan, H. et al. Viral aetiology of acute respiratory infections among children and associated meteorological factors in southern China. BMC Infect Dis 15, 124 (2015). https://doi.org/10.1186/s12879-015-0863-6

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  • DOI: https://doi.org/10.1186/s12879-015-0863-6

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