Acute respiratory infections (ARIs) are one of the major public health problems in developing countries like Nepal. Besides the influenza, several other pathogens are responsible for acute respiratory infection in children. Etiology of infections is poorly characterized at the course of clinical management, and hence empirical antimicrobial agents are used. The objective of this study was to characterize the influenza and other respiratory pathogens by real-time PCR assay. A total of 175 throat swab specimens of influenza-positive cases collected at National Influenza Center, Nepal, during the 2015/16 winter season were selected for detecting other respiratory copathogens. Total nucleic acid was extracted using Pure Link viral RNA/DNA mini kit (Invitrogen), and multiplex RT-PCR assays were performed. Influenza A and B viruses were found in 120 (68.6%) and 55 (31.4%) specimens, respectively, among which coinfections were found in 106 (60.6%) specimens. Among the influenza A-positive cases, 25 (20.8%) were A/H1N1 pdm09 and 95 (79.2%) were A/H3 subtypes. Viruses coinfected frequently with influenza virus in children were rhinovirus (26; 14.8%), respiratory syncytial virus A/B (19; 10.8%), adenovirus (14; 8.0%), coronavirus (CoV)-HKU1 (14; 8.0%), CoV-OC43 (5; 2.9%), CoV-229E (2; 1.1%), metapneumovirus A/B (5; 2.9%), bocavirus (6; 3.4%), enterovirus (5; 2.9%), parainfluenza virus-1 (3; 1.7%), and parainfluenza virus-3 (2; 1.1%). Coinfection of
ARIs are one of the major causes of mortality and morbidity in children especially in developing countries [
The most common etiology of ARI worldwide includes influenza virus (InfV), respiratory syncytial virus (RSV), rhinovirus (RV), metapneumovirus (MPV), bocavirus (BV), adenovirus (AV), enterovirus (EV),
In tropical and subtropical countries in the South East Asia such as Thailand, Singapore, Malaysia, and China, the etiologic agents associated with ILI have been well characterized. However, epidemiology and etiology for ILI is poorly understood in Nepal. A laboratory-based ILI surveillance system is well established in Nepal and has greatly contributed to outbreak investigation, surveillance, and timely response since the emergence of pandemic influenza in 2009. The influenza viruses circulating in Nepal have many similarities with our neighboring countries and the regions. We reported year-round transmission of influenza with a peak activity during the rainy and winter seasons is similar to Thailand, Northern Vietnam, and Lao PDR [
This was a laboratory-based descriptive cross-sectional study conducted from October 2015 to February 2016. A total of 394 throat swab specimens were collected from children aged two months to 12 years with symptoms of influenza-like illness (ILI) during the winter season visiting at National Influenza Center (NIC), National Public Health Laboratory, Kathmandu, Nepal. Of the total, 175 throat swab specimens positive for influenza virus were further tested for detection of other respiratory copathogens. Influenza-like illness was defined as an individual with an acute respiratory infection with history of fever (≥38°C), cough, and/or sore throat with onset within the last 10 days [
The collected data included patient’s demographic characteristics (age and sex); geographic location, type, and subtypes of influenza virus confirmed by real-time polymerase chain reaction (rRT-PCR) assay.
Total nucleic acid (RNA/DNA) was extracted from the throat swab using the PureLink™ Viral RNA/DNA mini kit (Invitrogen, Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions. An internal control (IC) was added to each extraction tube in order to access the quality of extraction at the end of amplification. Finally, nucleic acid was eluted in 50
Statistical analysis was performed using SPSS version 11.5. Descriptive statistics, frequency, and percent were generated. Age-wise distribution of coinfection cases, influenza single-infection, and coinfection with other pathogens were described.
This study was approved by Nepal Health Research Council (reg. no. 180/2015).
One hundred seventy-five throat swab specimens positive for any influenza virus were tested to identify coinfection with other possible etiology of ARI. Influenza A virus was detected in 120 (68.6%) specimen, of which 25 (20.8%) were influenza A/H1N1 pdm09 and 95 (79.2%) were influenza A/H3 subtype. Similarly, influenza B virus was identified in 55 (31.4%) specimens. Among those, 175 children positive for any influenza A or B virus; RSV A-B, rhinovirus, adenovirus, metapneumovirus A-B, parainfluenza virus 1 and 3, bocavirus,
Details of respiratory pathogen coinfection in influenza-positive cases.
Pathogen | Influenza A (%) |
Influenza B (%) |
Total positive (%) |
---|---|---|---|
Rhinovirus | 22 (12.6) | 4 (2.3) | 26 (14.8) |
RSV A/B | 15 (8.6) | 4 (2.3) | 19 (10.8) |
Adenovirus | 13 (7.4) | 1 (0.6) | 14 (8.0) |
Metapneumovirus A/B | 4 (2.3) | 1 (0.6) | 5 (2.9) |
Bocavirus | 2 (1.1) | 4 (2.3) | 6 (3.4) |
|
4 (2.3) | 1 (0.6) | 5 (2.9) |
Enterovirus | 5 (2.8) | 0 (0.0) | 5 (2.9) |
Parechovirus | 0 (0.0) | 0 (0.0) | 0 (0) |
Parainfluenza virus-1 | 2 (1.1) | 1 (0.6) | 3 (1.7) |
Parainfluenza virus-2 | 0 (0.0) | 0 (0.0) | 0 (0) |
Parainfluenza virus-3 | 1 (0.6) | 1 (0.6) | 2 (1.1) |
Parainfluenza virus-4 | 0 (0.0) | 0 (0.0) | 0 (0) |
Coronavirus (OC43) | 3 (1.7) | 2 (1.1) | 5 (2.9) |
Coronavirus (NL63) | 0 (0.0) | 0 (0.0) | 0 (0) |
Coronavirus (229E) | 2 (1.1) | 0 (0.0) | 2 (1.1) |
Coronavirus (HKU1) | 10 (5.7) | 4 (2.3) | 14 (8.0) |
In this study, influenza-positive children cases (
Distribution pattern of respiratory pathogen coinfection among children in influenza A-positive cases (
Pathogens | Age group (year) | Total (%) | ||
---|---|---|---|---|
<5 (%) | 6–10 (%) | 10–12 (%) | ||
RV | 10 (5.7) | 6 (3.4) | 6 (3.4) | 22 (12.6) |
RSV A/B | 8 (4.6) | 4 (2.3) | 3 (1.7) | 15 (8.6) |
AV | 9 (5.1) | 3 (1.7) | 1 (0.6) | 13 (7.4) |
EV | 3 (1.7) | 1 (0.6) | 1 (0.6) | 5 (2.8) |
MPV A/B | 3 (1.7) | 1 (0.6) | 0 (0.0) | 4 (2.3) |
|
2 (1.1) | 1 (0.6) | 1 (0.6) | 4 (2.3) |
BV | 1 (0.6) | 1 (0.6) | 0 (0.0) | 2 (1.1) |
PIV-1 | 1 (0.6) | 1 (0.6) | 0 (0.0) | 2 (1.1) |
PIV-3 | 1 (0.6) | 0 (0.0) | 0 (0.0) | 1 (0.6) |
CoV-OC43 | 2 (1.1) | 1 (0.6) | 0 (0.0) | 3 (1.7) |
CoV-229E | 1 (0.6) | 1 (0.6) | 0 (0.0) | 2 (1.1) |
CoV-HKU1 | 7 (4.0) | 2 (1.1) | 1 (0.6) | 10 (5.7) |
RV, rhinovirus; RSV A/B, respiratory syncytial virus A-B; AV, adenovirus; EV, enterovirus; MPV A/B, metapneumovirus A-B;
Distribution pattern of respiratory pathogen coinfection among children in influenza B-positive cases (
Pathogen | Age group (year) | Total (%) | ||
---|---|---|---|---|
<5 (%) | 6–10 (%) | 10–12 (%) | ||
RV | 3 (1.7) | 1 (0.6) | 0 (0.0) | 4 (2.3) |
BV | 2 (1.1) | 1 (0.6) | 1 (0.6) | 4 (2.3) |
RSV A/B | 2 (1.1) | 1 (0.6) | 1 (0.6) | 4 (2.3) |
AV | 1 (0.6) | 0 (0.0) | 0 (0.0) | 1 (0.6) |
MPV A/B | 1 (0.6) | 0 (0.0) | 0 (0.0) | 1 (0.6) |
|
1 (0.6) | 0 (0.0) | 0 (0.0) | 1 (0.6) |
PIV-1 | 1 (0.6) | 0 (0.0) | 0 (0.0) | 1 (0.6) |
PIV-3 | 1 (0.6) | 0 (0.0) | 0 (0.0) | 1 (0.6) |
CoV-OC43 | 2 (1.1) | 0 (0.0) | 0 (0.0) | 2 (1.1) |
CoV-HKU1 | 2 (1.1) | 1 (0.6) | 1 (0.6) | 4 (2.3) |
RV, rhinovirus; BV, bocavirus; RSV A/B, respiratory syncytial virus A-B; AV, adenovirus; MPV A/B, metapneumovirus A-B;
The proportion of coinfections with rhinovirus 10 (5.7%), respiratory syncytial virus A/B 8 (4.6%), adenovirus 9 (5.1%), and CoV-HKU1 7 (4.0%) viruses was more common in less than five-year-old children with influenza A compared to influenza B-positive cases. Similarly, the rate of coinfections with rhinovirus 6 (3.4%), respiratory syncytial virus A/B 4 (2.3%), adenovirus 3 (1.7%), and CoV-HKU1 2 virus (1.1%) was comparatively found lower among 6–10 year-old children with influenza A and influenza B, respectively (Tables
Similarly, 38 (21.7%) influenza-positive specimens had been coinfected with two respiratory pathogens; 21 (12.0%) specimens contained three respiratory viruses and 9 (5.2%) specimens contained coinfections of four respiratory pathogens (Table
Details of pathogens in single and multiple respiratory infections with influenza-positive cases.
Viruses | Positive number (%) |
---|---|
Influenza A/H1N1 pdm09 | 18 (10.3) |
Influenza A/H3 | 51 (29.1) |
Influenza B | 38 (21.7) |
|
|
pdm09 + HPIV1 | 2 (1.1) |
pdm09 + RSV A/B | 3 (1.7) |
pdm09 + MPV A/B | 1 (0.6) |
A/H3 + MPV A/B | 1 (0.6) |
A/H3 + |
1 (0.6) |
A/H3 + RSV A/B | 7 (4) |
A/H3 + AV | 4 (2.3) |
A/H3 + EV | 1 (0.6) |
A/H3 + RV | 3 (1.7) |
A/H3 + HKU1 | 4 (2.3) |
B + BoV | 3 (1.7) |
B + HKU1 | 1 (0.6) |
B + RV | 1 (0.6) |
B + |
1 (0.6) |
B + MPV A/B | 2 (1.1) |
B + RSV A/B | 3 (1.7) |
|
|
pdm09 + RV + EV | 1 (0.6) |
A/H3 + RV + AV | 6 (3.4) |
A/H3 + RV + COR43 | 3 (1.7) |
A/H3 + RV+ BV | 1 (0.6) |
A/H3 + RV + RSV A/B | 2 (1.1) |
A/H3 + HKU1 + PIV-3 | 1 (0.6) |
A/H3 + RV + EV | 1 (0.6) |
B + BoV + AV | 1 (0.6) |
B + HKU1 + PIV1 | 1 (0.6) |
B + HKU1 + RSV A/B | 1 (0.6) |
B + RV + COR43 | 2 (1.1) |
B + HKU1 + PIV-3 | 1 (0.6) |
|
|
pdm09 + HKU1 + |
1 (0.6) |
A/H3 + HKU1 + |
1 (0.6) |
A/H3 + HKU1 + COR229 + PIV1 | 1 (0.6) |
A/H3 + HKU1 + |
2 (1.1) |
A/H3 + RV+ MPV A/B + EV | 1 (0.6) |
A/H3 + RV + COR229E + RSV | 1 (0.6) |
H3 + RV + BV + AV | 1 (0.6) |
H3 + RV + MPV + EV | 1 (0.6) |
|
|
Monoinfection of influenza A/H1N1 pdm09, influenza A/H3, and influenza B was 10.3, 29.1 and 21.7%, respectively. Coinfection of influenza A and B with other single pathogen was found in 21.7% cases, double pathogen was found in 12% cases, and three or more pathogens were found in 17.2% cases (Table
Respiratory virus is a major cause of acute respiratory infection, of which influenza is one of the major public health burdens in developed and developing countries like Nepal. In this study, a total of 175 influenza-positive specimens were investigated for detection of potential respiratory pathogens from October 2015 to February 2016. We identified the coinfection of 9 noninfluenza respiratory pathogens with influenza virus. Of them, the rate of coinfection with rhinovirus, RSV A/B, adenovirus, and CoV-HKU1 viruses was higher. A little information is available concerning the prevalence and seasonality of these viruses, mainly in developing countries like Nepal, where the possibilities of carrying out this type of study on a regular basis are unusual.
During the winter season of 2015/16, an increased number of influenza virus cases (44.4%) were detected and all influenza-positive cases were further screened for other respiratory pathogens by rRT-PCR using FTD respiratory pathogens 21 kit. The spectrum of the pathogens and their positivity rate could vary between country to country and over the time [
Rhinovirus, RSV A-B, adenovirus, and CoV-HKU1 viruses were most frequently detected as coinfecting pathogens in influenza-positive specimens. Similar to our findings, a study conducted by Koul et al. has shown that influenza and rhinovirus were most commonly detected in respiratory samples [
Our study detected bocavirus, coronavirus, and enterovirus coinfections in influenza-positive cases. Most deaths from pneumonia in children less than 5 years of age occur in developing countries, where information about the clinical impact and severity of viral causes of respiratory infections is limited [
Influenza coinfected with
In this study, respiratory pathogens could be categorized into four groups: single-infection of influenza A (39.4%), influenza B (21.7%), influenza, and multiple coinfections with two pathogens (21.7), three pathogens (12.0%), and four pathogens (5.2%). Studies conducted in Vietnam [
To the best of our knowledge, this could be the first study undertaken with multiplex RT-PCR kit on 21 respiratory pathogen for detection of influenza A, A/H1N1, influenza B, RV, RSV A-B, PIV 1–4, coronavirus OC43, NL63, 229E, and HKU1, MPV A-B, BV,
An increased level of multiple pathogens coinfection with a wide range of respiratory viruses was detected in influenza-positive cases. To the best of our knowledge, findings are new and it was not expected in advance. This study has demonstrated that the wide range of respiratory pathogens is responsible for coinfection in influenza-positive cases in Nepal. However, nothing can be said about the proportion or incidence of other viral infections than influenza as this study was done in a selected group of influenza-positive children. The implication of these findings should be carefully considered in clinical diagnosis and management. In addition, the impact of mono versus multiple coinfections of respiratory pathogens in relation to the severity of disease urges for a complete study in future.
There were several limitations of this study such as very short study period, limited number of samples, and diagnostic reagents. Because of financial constraints, we could not continue our study throughout the year. Hence, the finding of this study does not reflect the whole year scenario. Also, our study could not explore clinical pictures in detail, for example, the severity of illness, period of hospital stay, and its outcome among the single and multiple coinfections, which demands a comprehensive study in future.
Our findings gave baseline information of respiratory viruses and the distribution pattern within the different age groups of children which would help better therapeutic approaches and effective prevention strategies. Furthermore, meticulous attention should be paid to viral infections in younger children.
Influenza is one of the leading causes of ARIs in children during the winter season in Nepal. In addition to influenza, nine different respiratory viruses were identified. These findings are expected to give better understanding of respiratory viruses, as well as strategies of appropriate case management and minimizing the use of antimicrobial agents in Nepal.
The data used to support the findings of this study are included within the article.
The authors declare that there are no conflicts of interest.
The authors are extremely grateful to all the staff working at National Influenza Center, National Public Health Laboratory, Teku, Kathmandu, for their technical assistance during the study period.