|Year : 2014 | Volume
| Issue : 1 | Page : 13-18
Viral aetiology of acute lower respiratory tract illness in hospitalised paediatric patients of a tertiary hospital: One year prospective study
AK Singh1, A Jain1, B Jain1, KP Singh1, T Dangi1, M Mohan1, M Dwivedi1, R Kumar2, R A S Kushwaha3, JV Singh4, AC Mishra5, MS Chhaddha6
1 Department of Microbiology, NIV, Infl uenza division, King George's Medical University, Lucknow, Uttar Pradesh, India
2 Department of Pediatrics, NIV, Infl uenza division, King George's Medical University, Lucknow, Uttar Pradesh, India
3 Department Pulmonary Medicine, NIV, Infl uenza division, King George's Medical University, Lucknow, Uttar Pradesh, India
4 Department of Community Medicine, NIV, Infl uenza division, King George's Medical University, Lucknow, Uttar Pradesh, India
5 Department of Community Medicine, National Institute of Virology (Director of NIV, Pune, Maharashtra, India
6 Influenza Division, NIV, Pune, Maharashtra, India
|Date of Submission||16-Nov-2012|
|Date of Acceptance||16-Sep-2013|
|Date of Web Publication||4-Jan-2014|
Department of Microbiology, NIV, Infl uenza division, King George's Medical University, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Context: Acute lower respiratory tract infections (ALRI), ranked as the second leading cause of death are the primary cause of hospitalisation in children. Viruses are the most important causative agents of ALRI. Aim: To study the viral aetiology of ALRI in children at a tertiary care hospital. Setting and Design: One year prospective observational study in a tertiary care hospital of King George's Medical University, Lucknow. Material and Methods: Nasopharyngeal aspirate (NPA) was collected from children admitted with signs and symptoms of ALRI who were aged 0-14 years. Samples were transported to the laboratory at 4°C in viral transport media and processed for detection of respiratory syncytial virus (RSV) A and B, influenza virus A and B, adenovirus (ADV), human Boca virus (HBoV), human metapneumo virus (hMPV) and parainfluenzavirus 1, 2, 3 and 4 using mono/multiplex real-time polymerase chain reaction (RT-PCR). STATA was used for statistical analysis. Results: In one year, 188 NPAs were screened for respiratory viruses, of which 45.7% tested positive. RSV was most commonly detected with 21.3% positivity followed by measles virus (8.5%), influenza A virus (7.4%), ADV (5.3%), influenza B virus (1.6%), hMPV (1.1%) and HBoV (0.5%). Month wise maximum positivity was seen in December and January. Positivity rate of RSV was highest in children aged < 1 year, which decreased with increase in age, while positive rate of influenza virus increased with increasing age. Conclusion: The occurrence of viral predominance in ALRI is highlighted.
Keywords: Acute lower respiratory tract infection, respiratory syncytial virus, real-time polymerase chain reaction, respiratory viruses
|How to cite this article:|
Singh A K, Jain A, Jain B, Singh K P, Dangi T, Mohan M, Dwivedi M, Kumar R, Kushwaha R, Singh J V, Mishra A C, Chhaddha M S. Viral aetiology of acute lower respiratory tract illness in hospitalised paediatric patients of a tertiary hospital: One year prospective study. Indian J Med Microbiol 2014;32:13-8
|How to cite this URL:|
Singh A K, Jain A, Jain B, Singh K P, Dangi T, Mohan M, Dwivedi M, Kumar R, Kushwaha R, Singh J V, Mishra A C, Chhaddha M S. Viral aetiology of acute lower respiratory tract illness in hospitalised paediatric patients of a tertiary hospital: One year prospective study. Indian J Med Microbiol [serial online] 2014 [cited 2019 Jun 20];32:13-8. Available from: http://www.ijmm.org/text.asp?2014/32/1/13/124288
| ~ Introduction|| |
All over the world, acute lower respiratory tract infections (ALRIs) are a leading cause of morbidity, mortality and hospital admission in the paediatric age group.  World Health Organisation (WHO) estimates that ALRI causes nearly four million deaths per year, at a rate of more than 60 deaths/100,000 population. Viruses are known to be a very important cause of respiratory tract infections, ,, accounting for 30-70% of ALRI with respiratory syncytial virus (RSV), influenza virus (Inf.), measles virus, adenovirus (ADV), human Boca virus (hBoV), human metapneumo virus (hMPV) and parainfluenza virus (PIV) on the top of list. Viral ALRI usually presents with clinical features that do not vary considerably from virus to virus. Therefore, the responsibility of early and correct aetiological diagnosis comes on laboratory in order to avoid unnecessary expensive and disruptive empiric treatment and to provide public health measures for controlling spread of the diseases. Seasonality of different respiratory viruses also varies dramatically.
The present study was planned to study the viral aetiology of ALRI in paediatric hospitalised patients over a period of one year to gain better understanding of the seasonality (including association with meteorological factors), epidemiology and clinical profiles associated with viral ALRI.
| ~ Material and Methods|| |
Children aged upto 14 years presenting with ALRI and hospitalised in paediatric wards of King George Medical University, Lucknow, were recruited prospectively from June 2011-May 2012, using standardised case definition of WHO. [4,5] Clinical and epidemiological data were collected using structured questionnaire. Written informed consent was obtained from parents or legal guardians of children enrolled in the study.
For children aged > 1 week and < 2 months: Admission to the paediatric ward with any of the following: Respiratory rate > 60 per min, severe chest in drawing, nasal flaring, grunting, fever ≥ 38°C, hypothermia < 35.5°C or pulse oxygenation < 90%.
For children aged 2 months to < 5 years: Cough or difficulty in breathing and any one of the following: Respiratory rate > 50/min for infants aged 2 months to < 1 year, > 40/min for children aged 1 to < 5 years, chest in drawing or stridor in a calm child, unable to drink or breast feed, vomiting, convulsions, lethargic or unconscious or pulse oxygen saturation < 90%.
For children aged ≥ 5 years: Fever ≥ 38°C, cough or sore throat, and shortness of breath or difficulty in breathing.
Hospitalisation was a mandatory part of the ALRI case definition in all the age groups. ,
Nasopharyngeal aspirates (NPA) were collected by inserting a suction catheter into the posterior nasopharyngeal space via the nostril with the patients in the recumbent position. A low suction force was applied to collect approximately 0.5 ml fluid, which is then transferred into 3 ml of viral transport medium (VTM with penicillin [100 U/ml], streptomycin [100 mg/ml] and amphotericin B [20 mg/ml]). NPA was immediately transported to the Virology Laboratory, maintaining cold chain and without delay centrifuged at 1000 rpm for 3 min. Supernatant was immediately stored at -80°C till further use.
Nucleic acid extraction
Total nucleic acid was extracted by using High Pure Viral Nucleic Acid Kit (Roche Kit) according to the manufacturer's instructions. Briefly, 200 μl of sample was used and finally the nucleic acid was eluted in 60 μl of elution buffer. RNA extracts were used immediately for real-time polymerase chain reaction (RT-PCR) or stored at -80°C for long-term use. A negative extraction control was always extracted parallel using molecular grade sterile water as sample.
Real time PCR
Details of probes and primers are mentioned in [Table 1] and [Table 2]. RT-PCR for RSV, influenza A and B viruses, measles virus, ADV, hMPV, hBoV and PIV1, 2, 3 and 4 was done using already published primer probe sequences.[6-12] A series of monoplex and multiplex RT-PCRs were performed for detection of respiratory viruses. Monoplex reactions were run for influenza A, influenza B and measles virus. If sample was positive for influenza A then subtyping into human H1N1, human H3N2 and swine H1N1 was done using monoplex RT-PCR reaction. The panels of multiplex PCR reactions were RSV and hMPV (panel 1); PIV1, 2, 3 and 4 (panel 2) and ADV with hBoV (panel 3). If sample was positive for RSV then typing of RSV into RSV A and RSV B was done using multiplex reaction (panel 4). RNase P gene was used as internal control to check sample quality [Table 1].
|Table 1: Details of primers and probes used for respiratory viruses identifi cation by monoplex real time|
RT-PCR for Infl uenza and measles
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All RT-PCR detection was carried out in ABI 7500 Real-Time PCR Instrument (Applied Biosystems). Each PCR reaction mixture consisted of 25 μl total volume with 12.5 μl of 2 × ABI RT-PCR buffer with 1μl of 25 × RT-enzyme (AgPath-IDTM One Step RT-PCR Kit) for viral targets. The final concentrations of primers and probes in reaction mix are detailed in [Table 1] and [Table 2]. The amount of viral template that had been added was 5 μl and the total volume of 25 μl was made-up by adding nuclease free water.
|Table 2: Details of primers and probes used for respiratory virus identifi cation by multiplex real time RT-PCR|
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Thermal cycling conditions of amplification by monoplex RT-PCR were as follows: Reverse transcription at 50°C × 30 min, initial denaturation at 95°C × 15 min; 45 amplification cycles of 95°C × 30s and annealing of 55°C × 45 s for RNase P, influenza A and influenza B and 60°C × 30 s for measles virus.
Thermal cycling conditions for amplification by multiplex RT-PCR were as follows: Reverse transcription at 50°C - 30 min and initial denaturation at 95°C - 15 min for panel no. 1, 2 and 4. There was no RT step for panel-3. Amplification cycles (45 in number) were as follows: 94°C - 20 s and 60°C - 1 min for panel-1; 95°C - 30 s and 55°C - 1 min for panel-2; 94°C - 1 min and 60°C - 1 min for panel-3; 95°C - 15 s and 60°C - 1 min for panel-4.
| ~ Results|| |
A total of 188 children fulfilling inclusion criteria of ALRI were enrolled from June 2011 to May 2012. Viral pathogens were detected in samples from 86/188 (45.7%) cases [Table 3]. Of all respiratory viruses, RSV turned out to be the most commonly detected virus with 21.3% positivity (40/188) followed by measles (16/188, 8.5%), influenza A (14/188, 7.4%), ADV (10/188, 5.3%), influenza B (3/188, 1.6%), hMPV (2/188, 1.1%) and hBoV (1/188, 0.5%). PIV 1, 2, 3 and 4 were not detected in any sample. All influenza A viruses were human H3N2. RSV type A was detected in 95% (38/40) instances and RSV type B was detected in 5% (2/40) of instances. Only two samples showed co-infection with more than one virus, that is RSV B with hMPV (one case) and Influenza A virus with ADV (one case).
|Table 3: Viral pathogens detected in children hospitalised with ALRI (N=188)|
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Analysis of individual virus positivity according to patient age group is detailed in [Table 4]. Positivity rate of RSV was highest in children aged < 1 year, which decreased with increase in age, while positive rate of influenza virus increased with increasing age.
Clinical characteristics of the patients with respiratory virus infection are summarised in [Table 5]. Common symptoms remain chills and rigors, ear discharge, expectoration, nasal discharge and vomiting. Few patients also presented with diarrhoea. The clinical presentations of ALRI patients testing positive for respiratory viruses were compared with those of cases testing negative for respiratory viruses. Patients testing positive for viruses showed significantly high percentage of cough, nasal discharge and diarrhoea (P < 0.05, software used STATA 11.0).
The seasonal distribution of respiratory viruses was also studied and is shown in [Figure 1]. RSV infection showed the peak activity during winters and inverse correlation with temperature and rainfall. Maximum positivity of influenza virus was seen in rainy months, which showed a direct correlation with rainfall and humidity. Measles and ADVs were present throughout the year.
|Figure 1: Relation of meteorological factors with respiratory virus infections|
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| ~ Discussion|| |
Respiratory viruses are major cause of ALRI .,,, In the present study, RSV was the most frequently detected virus from hospitalised children presenting as ALRI followed by measles virus, influenza virus and ADV. Our findings are consistent with other reports especially from India and other part of country indicating that RSV and influenza viruses are prominent causes of severe respiratory tract infections .,, Somehow, we did not detect any PIV in this time frame. To rule out any technical fault, every run had a valid positive control.
Our study found a slight male preponderance, which is commonly reported finding in ALRI.  In concordance to our study, most of the other studies have also reported that infants aged < 1 year are most commonly positive for viruses, especially RSV  and influenza virus infection is common in older children. Protection by maternal antibodies against RSV infection in infants remains questionable. As the child grows, his immunity against this virus builds up due to repeated exposures and provides protection . Influenza virus, due to tremendous antigenic heterogeneity evades the herd immunity  and does not give lasting immunity for any particular strain. Therefore, as the child grows, he/she become more vulnerable for influenza virus infection. ADV is uncommon in the first six postnatal months, where maternal antibody confers protection . Neutralising antibodies develop in early childhood after asymptomatic and symptomatic infection, which occur with equal frequency. It is very difficult to characterise individual respiratory viruses on the basis of clinical presentation of the patients. However, cough, nasal discharge and diarrhoea were observed more significantly in virus-positive cases rather than virus-negative cases, which is consistent with previous report. 
India is geographically located at 28°36.8'N and 77°12.5'E in the northern hemisphere of the globe. India being a tropical country has diversity in climates from North to South and East to West. This particular study is representative of north India where average temperature is 25.3°C while warmest average max/high temperature is 41°C in May and coolest average min/low temperature is 7°C in January. Mean relative humidity for an average year is recorded as 49.2% and on a monthly basis it ranges from 25% in April and May to 73% in August and September. North of India receives on average 715 mm (28.1 inches) of precipitation annually with the driest weather in November when on balance 1 mm (0.0 in) of rain falls across 1 day and with the wettest weather as July when on balance 211 mm (8.3 in) of rain falls across 14 days. In temperate countries most respiratory viral infections occur in winters probably due to seasonal variation in host immune response  and change in climate, that is low ambient temperature and relatively low humidity, which promote viral growth.  Seasonal trends in tropical countries are more diverse. Some studies showed that respiratory virus infections occur round the year, while some show clearer seasonality. We found remarkable differences in seasonal pattern of ALRI along with specific virus activity, which might indicate varied transmission mode in different sub-regions of the continent. As shown in present study, RSV has shown inverse relation with temperature in a study from tropical Kualalumpur.  We observed increase in influenza activity in rainy season (maximum positivity seen in August, 2011-2012), which do not coincide with the influenza seasonality described in different studies. 
We are monitoring influenza activity in the patients presenting not only as ALRI but also as influenza like illness (ILI), and found remarkable increase of influenza activity in rainy season. Influenza A virus, H3N2 type was found to be circulating in ILI as well as in ALRI during 2011-2012 (data not shown). For ADV, most studies show sporadic occurrence without any seasonal trend.  We found high prevalence of ADV. One study has shown 0.8% positivity of ADV, which is much lower than our report (5.3%).  Seasonality is less clear for hMPV and hBoV, which may be due to the smaller number of positive cases.
Taken together, our study contributes critical baseline data on viral aetiology of ALRI in north India, and elucidates the importance of RSV, influenza and ADV as dominant viral aetiology of paediatric ALRI. High prevalence of ADV is shown in this study. The epidemiology of respiratory viruses needs to be studied further to increase the effectiveness of a planned vaccination and prophylaxis programmes.
| ~ Acknowledgment|| |
Dr. Shashi Krishna, Ms. Preeti Sharma, staff of influenza project and VDL staff for support in lab work and collection of samples. Indian Council of Medical Research, New Delhi for financial support [5/8/7/14/2009-ECD-1(Vol. II)].
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]