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Indian Journal of Medical Microbiology, (2014) 32(1): 13-18
Original Article
Viral aetiology of acute lower respiratory tract illness in hospitalised paediatric patients of a tertiary hospital: One year prospective study AK Singh, *A Jain, B Jain, KP Singh, T Dangi, M Mohan, M Dwivedi, R Kumar, R A S Kushwaha, JV Singh, AC Mishra, MS Chhaddha
Abstract 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. Key words: Acute lower respiratory tract infection, respiratory syncytial virus, real-time polymerase chain reaction, respiratory viruses
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.[1] 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,[1-3] accounting for 30-70% of ALRI with respiratory syncytial *Corresponding author (email: ) Departments of Microbiology (AKS, AJ, BJ, KPS, TD, MM, MD), Pediatrics (RK), Pulmonary Medicine (RAK), Community Medicine (JVS), NIV, Influenza division, King George’s Medical University, Lucknow, Uttar Pradesh, National Institute of Virology (Director of NIV (ACM), Influenza Division, NIV (MSC)), Pune, Maharashtra, India Received: 16-11-2012 Accepted: 16-09-2013 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.124288
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.
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ALRI definition 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.[4,5] Sample 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
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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 Tables 1 and 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
Table 1: Details of primers and probes used for respiratory viruses identification by monoplex real time RT-PCR for Influenza and measles Viruses Sequences of primers and probes Target Final con. of primers genes and probes (nM) Internal control F AGATTTGGACCTGCGAGCG RNaseP 800 R GAGCGGCTGTCTCCACAAGT RNaseP 800 Probe FAM/TTCTGACCTGAAGGCTCTGCGCG/3BHQ_1/ 200 Inf A F GACCRATCCTGTCACCTCTGAC M 800 R AGGGCATTYTGGACAAAKCGTCTA M 800 Probe FAM/TGCAGTCCTCGCTCACTGGGCACG/3BHQ_1/ 200 Inf B F TCCTCAAYTCACTCT TCGAGCG M 800 R CGGTGCTCTTGACCA AATTGG M 800 Probe FAM/CCAATTCGAGCAGCTGAAACTGCGGTG/3BHQ_1/ 200 Inf A Hum. H1 F AACTACTACTGGACTCTRCTKGAA HA 800 R CCATTGGTGCATTTGAGKTGATG HA 800 Probe FAM/TGAYCCAAAGTCTACTCAGTGCGAAC/3BHQ_1/ 200 Inf A F AAGCATTCCYAATGCAAACC HA 800 R ATTGCRCCRAATATGCCTCTAGT HA 800 Hum. H3 Probe FAM/CAGGATCACATATGGGSCCTGTCC CAG/3BHQ_1/ 200 Inf A Swine H1 F GTG CTATAAACACCAGCCTYCCA HA 800 R CGGGATATTCCTTAATCCTGTRGC HA 800 Probe FAM/CAGAATATACATCCRGTCACAATTGGARAA/3BHQ_1/ 200 Measles FP CCCTGAGGGATTCAACATGATTCT N 900 RP ATCCACCTTCTTAGCTCCGAATC N 900 Probe FA M/TCTTGCTCGCAAAGGCGGTTACGG/BHQ_1 250 Degenerate primer: R: A/G, Y: C/T, K: G/T, S: C/G. M: Matrix, N: Nucleoprotein, HA-NA: Haemagglutinin-neuraminidase, Hum: Human. FAM: 6-carboxyfluorescein, BHQ: Black hole quencher, F: Forward primer, R: Reverse primer, P: Probe www.ijmm.org
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Singh, et al.: Viral etiology of ALRI in children
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].
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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 Tables 1 and 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 identification by multiplex real time RT-PCR Viruses Sequences of primers and probes Target Final con. of primers genes and probes (nM) Panel 1 RSV F1 TGGAAACATACGTGAACAARCTTCA N 500 R1 GCACCCATATTGTWAGTGATGCA N 500 Probe1 JOEN/CGAAGGCTCCACATACACAGCWGCTGT/3BHQ_1/ 150 hMPV F2 TCATATAAGCATGCTATATTAAAAGAGTCTCA N 700 R2 CCTATYTCTGCAGCATATTTGTAATCAG N 500 Probe2 FAM/ACAACAACTGCAGTGACACCCTCATCATT/3BHQ_1/ 100 Panel 2 PIV-1 F1 ACCTACAAGGCAACAACATC HA-NA 500 R1 CTTCCTGCTGGTGTGTTAAT HA-NA 500 Probe1 FAM/GCTGCCCAAACGATGGCTGAAAAAGGGAGGCAGC/3B 340 HQ_1/ PIV-2 F2 CCATTTACCTAAGTGATGGAA HA-NA 600 R2 CCATTTACCTAAGTGATGGAA HA-NA 600 Probe2 JOEN/GCTGCCAATCGCAAAAGCTGTTCAGTCACGGCAGC/3B 340 HQ_1/ PIV-3 F3 GGAGCATTGTGTCATCTGTC HA-NA 600 R3 TAGTGTGTAATGCAGCTCGT HA-NA 600 Probe3 /5TexRd-XN/CGCGCTACCCAGTCATAACTTACTCAACAGCAACA 340 GCGCG/3BHQ_2/ PIV-4 F4 CCTGGAGTCCCATCAAAAGT HA-NA 600 R4 GCATCTATACGAACACCTGCT HA-NA 600 Probe4 /5Cy5/GCTGCCGTCTCAAAATTTGTTGATCAAGACAATACAATTG 340 GCAGC/3BHQ_2/ Panel 3 ADV F1 GGACGCCTCGGAGTACCTGAG H 250 R1 GTGGGGTTTCTGAACTTGTT H 250 Probe1 FAM/CTGGTGCAGTTCGCCCGTGCCA/3BHQ_1/ 250 HBoV F2 TGCAGACAACGCYTAGTTGTTT N 200 R2 CTGTCCCGCCCAAGATACA N 200 Probe2 JOEN/CCAGGATTGGGTGGAACCTGCAAA/3BHQ_1/ 200 Panel 4 RSV A F1 GCTCTTAGCAAAGTCAAGTTGAATGA N 500 R1 TGCTCCGTTGGATGGTGTATT N 500 Probe1 JOEN/ACACTCAACAAAGATCAACTTCTGTCATCCAGC/3BHQ_1/ 200 RSV B F2 GATGGCTCTTAGCAAAGTCAAGTTAA N 500 R2 TGTCAATATTATCTCCTGTACTACGTTGAA N 500 Probe2 FAM/TGATACATTAAATAAGGATCAGCTGCTGTCATCCA/3BHQ_1/ 200 Degenerate primer: R: A/G, W: A/T, Y: C/T, K. N: Nucleoprotein, HA-NA: Haemagglutinin-neuraminidase, H: Hexon, Hum: Human. FAM: 6-carboxyfluorescein, JOEN: 4-5 dichlorocarboxy fluorescein, TXR: Texas red, Cy-5: Indodicarbocyanine, BHQ: Black hole quencher, F1/F2/F3/F4=Forward primer: R1/R2/R3/R4/= Reverse primer, P1/P2/P3/P4=Probe www.ijmm.org
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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). 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.
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Table 3: Viral pathogens detected in children hospitalised with ALRI (N=188) Viruses Total positive cases (%) RSV 40 (21.3) RSV-A 38 (20.2) RSV-B 02 (1.1) Influenza 17 (9.07) Influenza A 14 (7.4) Influenza B 03 (1.6) Measles 16 (8.5) ADV 10 (5.3) hMPV 02 (1.1) HBoV 01 (0.5) Total 86 (45.7) RSV: Respiratory syncytial virus, ALRI: Acute lower respiratory tract infection, ADV: Adeno virus, hMPV: Human metapneumo virus, HBoV: Human boca virus, Percentages were calculated based on the fraction of study population within each age group
Table 4: Age group distribution of children presenting with viral ALRI Virus detected n (%) 5-14 years (N=70) (N=85) (N=33) RSV (A and B) 24 (34) 15 (18) 01 (03) Influenza (A and B) 04 (06) 08 (9.4) 05 (15) ADV 03 (4.2) 04 (4.7) 03 (09) hMPV (A and B) 01 (1.4) 01 (1.1) HBoV 01 (1.1) Measles 05 (7.1) 08 (9.4) 03 (09) Positive cases 37 (52.8) 37 (43.5) 12 (36.3) RSV: Respiratory syncytial virus, ALRI: Acute lower respiratory tract infection, ADV: Adeno virus, hMPV: Human metapneumo virus, HBoV: Human boca virus
Table 5: Clinical features seen in children presenting with ALRI Variables Virus positive Virus negative P value (n=86) (n=102) Median age 2Y (1 month 3Y (1 month (IQR) to 14 years) to 14 years) Male M:F 61:25 (2.4:1) 71:42 (1.7:1) Post illness day 2.3 days 4.2 days Chills and rigor 04 (4.0) 08 (8.0) Ear discharge 02 (2.3) Cough 78 (90.6) 75 (73.5) 0.003 Sore throat 10 (12.0) 16 (15.9) Expectoration 25 (29.3) 27 (27.4) Nasal discharge 59 (69.3) 50 (48.7) 0.007 Vomiting 15 (17.3) 17 (16.8) Diarrhoea 10 (11.6) 03 (2.9) 0.023 IQR: Interquartile Range, ALRI: Acute lower respiratory tract infection
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Singh, et al.: Viral etiology of ALRI in children
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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.[2]
Figure 1: Relation of meteorological factors with respiratory virus infections
Discussion Respiratory viruses are major cause of ALRI.[2,3,13,14] 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.[1,3,14] 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.[14] 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[1] 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.[15] Influenza virus, due to tremendous antigenic heterogeneity evades the herd immunity[16] 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.[17]
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[18] and change in climate, that is low ambient temperature and relatively low humidity, which promote viral growth.[19] 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.[15] 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.[20] 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.[21] We found high prevalence of ADV. One study has shown 0.8% positivity of ADV, which is much lower than our report (5.3%).[22] 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
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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)].
References 1.
Tregoning JS, Schwarze J. Respiratory viral infections in infants: Causes, clinical symptoms, virology, and immunology. Clin Microbiol Rev 2010;23:74-98. 2. Beck ET, Henrickson KJ. Molecular diagnosis of respiratory viruses. Future Microbiol 2010;5:901-16. 3. Broor S, Parveen S, Bharaj P, Prasad VS, Srinivasulu KN, Sumanth KM, et al. A prospective three-year cohort study of the epidemiology and virology of acute respiratory infections of children in rural India. PLoS One 2007;2:e491. 4. WHO guidance for the surveillance of human infection with new influenza A (H1N1) virus posted on WHO website on 29 April 2009. Based on Global surveillance during an influenza pandemic. Available from: http://www.who.int/csr/resources/ publications/swineflu/surveillance/en/index.html [Last updated on 2011 Dec 30]. 5. WHO, Human Infection with Pandemic (H1N1) 2009 Virus: Updated Interim WHO Guidance on Global Surveillance. Available from: http://www.who.int/csr/disease/swineflu/WHO_ case_definition_swine_flu_2009_04_29.pdf [Last accessed on 2012 Aug 01]. 6. Hu A, Colella M, Tam JS, Rappaport R, Cheng SM. Simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus A and B by real-time PCR. J Clin Microbiol 2003;41:149-54. 7. Hubschen JM, Kremer JR, Landtsheer S, Muller CP. A multiplex TaqMan PCR assay for the detection of measles and rubella virus. J Virol Methods 2008;149:246-50. 8. Jokela P, Piiparinen H, Luiro K, Lappalainen M. Detection of human metapneumovirus and respiratory syncytial virus by duplex real-time RT-PCR assay in comparison with direct fluorescent assay. Clin Microbiol Infect 2010;16:1568-73. 9. Jothikumar N, Cromeans TL, Hill VR, Lu X, Sobsey MD, Erdman DD. Quantitative Real-Time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41. Appl Environ Microbiol 2005;71:3131-6. 10. Lu X, Chittaganpitch M, Olsen SJ, Mackay IM, Sloots TP, Fry AM, et al. Real-time PCR assays for detection of bocavirus
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in human specimens. J Clin Microbiol 2006;44:3231-5. 11. Potdar VA, Chadha MS, Jadhav SM, Mullick J, Cherian SS, Mishra AC. genetic characterization of the influenza a Pandemic (H1N1) 2009 virus isolates from India. Plos One 2010;5:e9693. 12. Templeton KE, Scheltinga SA, Beersma MF, Kroes AC, Claas EC. Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4. J Clin Microbiol 2004;1564-9. 13. Bharaj P, Sullender WM, Kabra SK, Mani K, Cherian J, Tyagi V, et al. Respiratory viral infections detected by multiplex PCR among pediatric patients with lower respiratory tract infections seen at an urban hospital in Delhi from 2005 to 2007. Virol J 2009;5:901-16. 14. He J, Gong Y, Zhong WJ, Xu L, Liu Y, Qian FX, et al. Study on the viral etiology of acute respiratory tract infections in the Shanghai area during 2009-2010. J Microbes Infect 2011;6:90-6. 15. Khor CS, Sam IC, Hooi PS, Quek KF, Chan YF. Epidemiology and seasonality of respiratory viral infections in hospitalized children in Kuala, Lumpur, Malaysia: A retrospective study of 27 years. BMC Pediatr 2012;12:32. 16. De Jong JC, Rimmelzwaan GF, Fouchier RA, Osterhaus AD. Influenza virus: A master of metamorphosis. J Infect 2000;40:218-28. 17. Joanne ML. Adenoviruses. Pediatr Rev 2005;26:244-8 18. Cannell JJ, Vieth R, Umhau JC, Holick MF, Grant WB, Madronich S, et al. Epidemic influenza and vitamin D. Epidemiol Infect 2006;134:1129-40. 19. Shaman J, Kohn M. Absolute humidity modulates influenza survival, transmission, and seasonality. Proc Natl Acad Sci USA 2009;106:3243-8. 20. Zaman RU, Alamgir AS, Rahman M, Azziz Baumgartner E, Gurley ES, Sharker MA, et al. Influenza in outpatient ILI case-patients in national hospital-based surveillance, Bangladesh, 2007-2008. PLoS One 2009;4:e8452. 21. Yun BY, Kim MR, Park JY, Choi EH, Lee HJ, Yun CK. Viral etiology and epidemiology of acute lower respiratory tract infections in Korean children. Pediatr Infect Dis J 1995;14:1054-9. 22. Vijay Simha, Anand Neelkanth Pandit. Respiratory viruses in acute respiratory tract infections in Western India. The Indian Journal of Pediatrics 2008;75:341-5. How to cite this article: Singh AK, Jain A, Jain B, Singh KP, Dangi T, Mohan M, Dwivedi M, Kumar R, Kushwaha R, Singh JV, Mishra AC, Chhaddha MS. 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. Source of Support: Nil, Conflict of Interest: None declared.
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Indian Journal of Medical Microbiology, (2014) 32(1): 13-18
Original Article
Viral aetiology of acute lower respiratory tract illness in hospitalised paediatric patients of a tertiary hospital: One year prospective study AK Singh, *A Jain, B Jain, KP Singh, T Dangi, M Mohan, M Dwivedi, R Kumar, R A S Kushwaha, JV Singh, AC Mishra, MS Chhaddha
Abstract 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. Key words: Acute lower respiratory tract infection, respiratory syncytial virus, real-time polymerase chain reaction, respiratory viruses
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.[1] 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,[1-3] accounting for 30-70% of ALRI with respiratory syncytial *Corresponding author (email: ) Departments of Microbiology (AKS, AJ, BJ, KPS, TD, MM, MD), Pediatrics (RK), Pulmonary Medicine (RAK), Community Medicine (JVS), NIV, Influenza division, King George’s Medical University, Lucknow, Uttar Pradesh, National Institute of Virology (Director of NIV (ACM), Influenza Division, NIV (MSC)), Pune, Maharashtra, India Received: 16-11-2012 Accepted: 16-09-2013 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.124288
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.
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ALRI definition 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.[4,5] Sample 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
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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 Tables 1 and 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
Table 1: Details of primers and probes used for respiratory viruses identification by monoplex real time RT-PCR for Influenza and measles Viruses Sequences of primers and probes Target Final con. of primers genes and probes (nM) Internal control F AGATTTGGACCTGCGAGCG RNaseP 800 R GAGCGGCTGTCTCCACAAGT RNaseP 800 Probe FAM/TTCTGACCTGAAGGCTCTGCGCG/3BHQ_1/ 200 Inf A F GACCRATCCTGTCACCTCTGAC M 800 R AGGGCATTYTGGACAAAKCGTCTA M 800 Probe FAM/TGCAGTCCTCGCTCACTGGGCACG/3BHQ_1/ 200 Inf B F TCCTCAAYTCACTCT TCGAGCG M 800 R CGGTGCTCTTGACCA AATTGG M 800 Probe FAM/CCAATTCGAGCAGCTGAAACTGCGGTG/3BHQ_1/ 200 Inf A Hum. H1 F AACTACTACTGGACTCTRCTKGAA HA 800 R CCATTGGTGCATTTGAGKTGATG HA 800 Probe FAM/TGAYCCAAAGTCTACTCAGTGCGAAC/3BHQ_1/ 200 Inf A F AAGCATTCCYAATGCAAACC HA 800 R ATTGCRCCRAATATGCCTCTAGT HA 800 Hum. H3 Probe FAM/CAGGATCACATATGGGSCCTGTCC CAG/3BHQ_1/ 200 Inf A Swine H1 F GTG CTATAAACACCAGCCTYCCA HA 800 R CGGGATATTCCTTAATCCTGTRGC HA 800 Probe FAM/CAGAATATACATCCRGTCACAATTGGARAA/3BHQ_1/ 200 Measles FP CCCTGAGGGATTCAACATGATTCT N 900 RP ATCCACCTTCTTAGCTCCGAATC N 900 Probe FA M/TCTTGCTCGCAAAGGCGGTTACGG/BHQ_1 250 Degenerate primer: R: A/G, Y: C/T, K: G/T, S: C/G. M: Matrix, N: Nucleoprotein, HA-NA: Haemagglutinin-neuraminidase, Hum: Human. FAM: 6-carboxyfluorescein, BHQ: Black hole quencher, F: Forward primer, R: Reverse primer, P: Probe www.ijmm.org
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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].
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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 Tables 1 and 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 identification by multiplex real time RT-PCR Viruses Sequences of primers and probes Target Final con. of primers genes and probes (nM) Panel 1 RSV F1 TGGAAACATACGTGAACAARCTTCA N 500 R1 GCACCCATATTGTWAGTGATGCA N 500 Probe1 JOEN/CGAAGGCTCCACATACACAGCWGCTGT/3BHQ_1/ 150 hMPV F2 TCATATAAGCATGCTATATTAAAAGAGTCTCA N 700 R2 CCTATYTCTGCAGCATATTTGTAATCAG N 500 Probe2 FAM/ACAACAACTGCAGTGACACCCTCATCATT/3BHQ_1/ 100 Panel 2 PIV-1 F1 ACCTACAAGGCAACAACATC HA-NA 500 R1 CTTCCTGCTGGTGTGTTAAT HA-NA 500 Probe1 FAM/GCTGCCCAAACGATGGCTGAAAAAGGGAGGCAGC/3B 340 HQ_1/ PIV-2 F2 CCATTTACCTAAGTGATGGAA HA-NA 600 R2 CCATTTACCTAAGTGATGGAA HA-NA 600 Probe2 JOEN/GCTGCCAATCGCAAAAGCTGTTCAGTCACGGCAGC/3B 340 HQ_1/ PIV-3 F3 GGAGCATTGTGTCATCTGTC HA-NA 600 R3 TAGTGTGTAATGCAGCTCGT HA-NA 600 Probe3 /5TexRd-XN/CGCGCTACCCAGTCATAACTTACTCAACAGCAACA 340 GCGCG/3BHQ_2/ PIV-4 F4 CCTGGAGTCCCATCAAAAGT HA-NA 600 R4 GCATCTATACGAACACCTGCT HA-NA 600 Probe4 /5Cy5/GCTGCCGTCTCAAAATTTGTTGATCAAGACAATACAATTG 340 GCAGC/3BHQ_2/ Panel 3 ADV F1 GGACGCCTCGGAGTACCTGAG H 250 R1 GTGGGGTTTCTGAACTTGTT H 250 Probe1 FAM/CTGGTGCAGTTCGCCCGTGCCA/3BHQ_1/ 250 HBoV F2 TGCAGACAACGCYTAGTTGTTT N 200 R2 CTGTCCCGCCCAAGATACA N 200 Probe2 JOEN/CCAGGATTGGGTGGAACCTGCAAA/3BHQ_1/ 200 Panel 4 RSV A F1 GCTCTTAGCAAAGTCAAGTTGAATGA N 500 R1 TGCTCCGTTGGATGGTGTATT N 500 Probe1 JOEN/ACACTCAACAAAGATCAACTTCTGTCATCCAGC/3BHQ_1/ 200 RSV B F2 GATGGCTCTTAGCAAAGTCAAGTTAA N 500 R2 TGTCAATATTATCTCCTGTACTACGTTGAA N 500 Probe2 FAM/TGATACATTAAATAAGGATCAGCTGCTGTCATCCA/3BHQ_1/ 200 Degenerate primer: R: A/G, W: A/T, Y: C/T, K. N: Nucleoprotein, HA-NA: Haemagglutinin-neuraminidase, H: Hexon, Hum: Human. FAM: 6-carboxyfluorescein, JOEN: 4-5 dichlorocarboxy fluorescein, TXR: Texas red, Cy-5: Indodicarbocyanine, BHQ: Black hole quencher, F1/F2/F3/F4=Forward primer: R1/R2/R3/R4/= Reverse primer, P1/P2/P3/P4=Probe www.ijmm.org
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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). 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.
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Table 3: Viral pathogens detected in children hospitalised with ALRI (N=188) Viruses Total positive cases (%) RSV 40 (21.3) RSV-A 38 (20.2) RSV-B 02 (1.1) Influenza 17 (9.07) Influenza A 14 (7.4) Influenza B 03 (1.6) Measles 16 (8.5) ADV 10 (5.3) hMPV 02 (1.1) HBoV 01 (0.5) Total 86 (45.7) RSV: Respiratory syncytial virus, ALRI: Acute lower respiratory tract infection, ADV: Adeno virus, hMPV: Human metapneumo virus, HBoV: Human boca virus, Percentages were calculated based on the fraction of study population within each age group
Table 4: Age group distribution of children presenting with viral ALRI Virus detected n (%) 5-14 years (N=70) (N=85) (N=33) RSV (A and B) 24 (34) 15 (18) 01 (03) Influenza (A and B) 04 (06) 08 (9.4) 05 (15) ADV 03 (4.2) 04 (4.7) 03 (09) hMPV (A and B) 01 (1.4) 01 (1.1) HBoV 01 (1.1) Measles 05 (7.1) 08 (9.4) 03 (09) Positive cases 37 (52.8) 37 (43.5) 12 (36.3) RSV: Respiratory syncytial virus, ALRI: Acute lower respiratory tract infection, ADV: Adeno virus, hMPV: Human metapneumo virus, HBoV: Human boca virus
Table 5: Clinical features seen in children presenting with ALRI Variables Virus positive Virus negative P value (n=86) (n=102) Median age 2Y (1 month 3Y (1 month (IQR) to 14 years) to 14 years) Male M:F 61:25 (2.4:1) 71:42 (1.7:1) Post illness day 2.3 days 4.2 days Chills and rigor 04 (4.0) 08 (8.0) Ear discharge 02 (2.3) Cough 78 (90.6) 75 (73.5) 0.003 Sore throat 10 (12.0) 16 (15.9) Expectoration 25 (29.3) 27 (27.4) Nasal discharge 59 (69.3) 50 (48.7) 0.007 Vomiting 15 (17.3) 17 (16.8) Diarrhoea 10 (11.6) 03 (2.9) 0.023 IQR: Interquartile Range, ALRI: Acute lower respiratory tract infection
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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.[2]
Figure 1: Relation of meteorological factors with respiratory virus infections
Discussion Respiratory viruses are major cause of ALRI.[2,3,13,14] 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.[1,3,14] 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.[14] 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[1] 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.[15] Influenza virus, due to tremendous antigenic heterogeneity evades the herd immunity[16] 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.[17]
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[18] and change in climate, that is low ambient temperature and relatively low humidity, which promote viral growth.[19] 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.[15] 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.[20] 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.[21] We found high prevalence of ADV. One study has shown 0.8% positivity of ADV, which is much lower than our report (5.3%).[22] 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
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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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Tregoning JS, Schwarze J. Respiratory viral infections in infants: Causes, clinical symptoms, virology, and immunology. Clin Microbiol Rev 2010;23:74-98. 2. Beck ET, Henrickson KJ. Molecular diagnosis of respiratory viruses. Future Microbiol 2010;5:901-16. 3. Broor S, Parveen S, Bharaj P, Prasad VS, Srinivasulu KN, Sumanth KM, et al. A prospective three-year cohort study of the epidemiology and virology of acute respiratory infections of children in rural India. PLoS One 2007;2:e491. 4. WHO guidance for the surveillance of human infection with new influenza A (H1N1) virus posted on WHO website on 29 April 2009. Based on Global surveillance during an influenza pandemic. Available from: http://www.who.int/csr/resources/ publications/swineflu/surveillance/en/index.html [Last updated on 2011 Dec 30]. 5. WHO, Human Infection with Pandemic (H1N1) 2009 Virus: Updated Interim WHO Guidance on Global Surveillance. Available from: http://www.who.int/csr/disease/swineflu/WHO_ case_definition_swine_flu_2009_04_29.pdf [Last accessed on 2012 Aug 01]. 6. Hu A, Colella M, Tam JS, Rappaport R, Cheng SM. Simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus A and B by real-time PCR. J Clin Microbiol 2003;41:149-54. 7. Hubschen JM, Kremer JR, Landtsheer S, Muller CP. A multiplex TaqMan PCR assay for the detection of measles and rubella virus. J Virol Methods 2008;149:246-50. 8. Jokela P, Piiparinen H, Luiro K, Lappalainen M. Detection of human metapneumovirus and respiratory syncytial virus by duplex real-time RT-PCR assay in comparison with direct fluorescent assay. Clin Microbiol Infect 2010;16:1568-73. 9. Jothikumar N, Cromeans TL, Hill VR, Lu X, Sobsey MD, Erdman DD. Quantitative Real-Time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41. Appl Environ Microbiol 2005;71:3131-6. 10. Lu X, Chittaganpitch M, Olsen SJ, Mackay IM, Sloots TP, Fry AM, et al. Real-time PCR assays for detection of bocavirus
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in human specimens. J Clin Microbiol 2006;44:3231-5. 11. Potdar VA, Chadha MS, Jadhav SM, Mullick J, Cherian SS, Mishra AC. genetic characterization of the influenza a Pandemic (H1N1) 2009 virus isolates from India. Plos One 2010;5:e9693. 12. Templeton KE, Scheltinga SA, Beersma MF, Kroes AC, Claas EC. Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4. J Clin Microbiol 2004;1564-9. 13. Bharaj P, Sullender WM, Kabra SK, Mani K, Cherian J, Tyagi V, et al. Respiratory viral infections detected by multiplex PCR among pediatric patients with lower respiratory tract infections seen at an urban hospital in Delhi from 2005 to 2007. Virol J 2009;5:901-16. 14. He J, Gong Y, Zhong WJ, Xu L, Liu Y, Qian FX, et al. Study on the viral etiology of acute respiratory tract infections in the Shanghai area during 2009-2010. J Microbes Infect 2011;6:90-6. 15. Khor CS, Sam IC, Hooi PS, Quek KF, Chan YF. Epidemiology and seasonality of respiratory viral infections in hospitalized children in Kuala, Lumpur, Malaysia: A retrospective study of 27 years. BMC Pediatr 2012;12:32. 16. De Jong JC, Rimmelzwaan GF, Fouchier RA, Osterhaus AD. Influenza virus: A master of metamorphosis. J Infect 2000;40:218-28. 17. Joanne ML. Adenoviruses. Pediatr Rev 2005;26:244-8 18. Cannell JJ, Vieth R, Umhau JC, Holick MF, Grant WB, Madronich S, et al. Epidemic influenza and vitamin D. Epidemiol Infect 2006;134:1129-40. 19. Shaman J, Kohn M. Absolute humidity modulates influenza survival, transmission, and seasonality. Proc Natl Acad Sci USA 2009;106:3243-8. 20. Zaman RU, Alamgir AS, Rahman M, Azziz Baumgartner E, Gurley ES, Sharker MA, et al. Influenza in outpatient ILI case-patients in national hospital-based surveillance, Bangladesh, 2007-2008. PLoS One 2009;4:e8452. 21. Yun BY, Kim MR, Park JY, Choi EH, Lee HJ, Yun CK. Viral etiology and epidemiology of acute lower respiratory tract infections in Korean children. Pediatr Infect Dis J 1995;14:1054-9. 22. Vijay Simha, Anand Neelkanth Pandit. Respiratory viruses in acute respiratory tract infections in Western India. The Indian Journal of Pediatrics 2008;75:341-5. How to cite this article: Singh AK, Jain A, Jain B, Singh KP, Dangi T, Mohan M, Dwivedi M, Kumar R, Kushwaha R, Singh JV, Mishra AC, Chhaddha MS. 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. Source of Support: Nil, Conflict of Interest: None declared.
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