|Year : 2018 | Volume
| Issue : 2 | Page : 163-171
Profile of respiratory pathogens causing acute respiratory infections in hospitalised children at Rajasthan a 4 year's study
M Anjaneya Swamy1, Bharti Malhotra2, PV Janardhan Reddy3, Jitendra Tiwari2
1 Department of Microbiology and Immunology, Sawai Man Singh Medical College, Jaipur; Department of Microbiology, Ananta Institute of Medical Sciences and Research Center, Rajsamand, Rajasthan, India
2 Department of Microbiology and Immunology, Sawai Man Singh Medical College, Jaipur, Rajasthan, India
3 Department of Microbiology and Immunology, Sawai Man Singh Medical College, Jaipur, Rajasthan; Department of Microbiology, Genomix Carl Private Limited, Pulivendula, Andhra Pradesh, India
|Date of Web Publication||7-Aug-2018|
Dr. Bharti Malhotra
Department of Microbiology and Immunology, Sawai Man Singh Medical College, Jaipur - 302 004, Rajasthan
Source of Support: None, Conflict of Interest: None
Introduction: Various pathogens cause respiratory tract infections in children of <5 years of age causing severe morbidity and mortality. The profile of causative agents varies from place to place. Aims: The objectives of our study were to detect the profile and trends of respiratory pathogens causing acute respiratory tract infection in children using a custom multiplex real-time polymerase chain reaction (RT-PCR) and to develop a diagnostic algorithm. Materials and Methods: A total of 997 children with clinical manifestations of respiratory infections were included in the study. Their nasopharyngeal aspirate and throat swab samples were subjected to nucleic acid extraction followed by multiplex RT-PCR for eighteen viruses and six bacteria. Statistical Analysis Used: Chi-square test was employed to study the P value of different viruses and bacteria. Results: A total of 765 (76.73%) samples were found to be positive for one of the respiratory pathogens. Viruses were detected in 598 (59.98%) and bacteria in 167 (41.85%) samples, respectively. The prevalence of single and co-infections among viruses and bacteria were 77.76% and 22.24%, 81.44% and 18.56% each, respectively. Respiratory syncytial virus (RSV) A/B and Streptococcus pneumoniae were the most predominant pathogens detected in the study and were associated with lower respiratory tract infections. Conclusion: RSV and S. pneumoniae were the most common pathogens detected, higher prevalence was observed in children <1 year of age. Viruses were predominant during winter months. The study helped to prepare diagnostic algorithm which will help in reducing diagnostic costs. However, further studies are required to assess whether viruses are bystander or real pathogens and include larger panel of bacteria and viruses for diagnosis.
Keywords: Acute respiratory infection, hospitalised children, multiplex real-time polymerase chain reaction, viruses
|How to cite this article:|
Swamy M A, Malhotra B, Janardhan Reddy P V, Tiwari J. Profile of respiratory pathogens causing acute respiratory infections in hospitalised children at Rajasthan a 4 year's study. Indian J Med Microbiol 2018;36:163-71
|How to cite this URL:|
Swamy M A, Malhotra B, Janardhan Reddy P V, Tiwari J. Profile of respiratory pathogens causing acute respiratory infections in hospitalised children at Rajasthan a 4 year's study. Indian J Med Microbiol [serial online] 2018 [cited 2019 May 21];36:163-71. Available from: http://www.ijmm.org/text.asp?2018/36/2/163/238700
| ~ Introduction|| |
Respiratory infections pose major public health problem worldwide. About 13 million children of <5 years of age, die every year, 95% of them are from developing countries and one-third of the total deaths are due to acute respiratory infections (ARIs). ARI accounts for 30%–50% of the total paediatric outpatient visits and 20%–30% of the paediatric admissions. ARIs are mainly self-limited, but can lead to complications requiring hospitalisation due to severe acute respiratory infections (SARI) which can be fatal at times, cause high economic burden on health care systems and to family of patients.
The causative agents include mainly viruses, up to 60%, (e.g., Respiratory syncytial virus (RSV), Influenza A, Rhinovirus, Adenovirus); bacteria (e.g., Streptococcus pneumoniae, Mycoplasma pneumoniae and Staphylococcus aureus) and fungi (e.g., Pneumocystis jirovecii)., The severity of the disease can be affected by type of infecting agent and predisposing factors such as age, immune status of the host, single or mixed infections and virulence mechanisms of the viral agents. It is important to know the profile and trends of various agents causing RTI in children so as to initiate appropriate therapy. Moreover, due to lack of identification of viruses antibiotics are given which cause undue side effects and also increase antimicrobial drug resistance. There are only few studies enumerating the profile of pathogens causing ARI in hospitalised patients. Most of the respiratory pathogens produce similar symptoms as a result it is difficult to identify them clinically. Various commercial and in-house multiplex real-time polymerase chain reaction (RT-PCR) assays are being used for simultaneous amplification of multiple pathogens in a single-reaction mixture, which makes them rapid and cost-effective., Hence, the present study was undertaken to identify the profile and trends of various respiratory pathogens in hospitalised children using a validated customised multiplex RT-PCR.
| ~ Materials and Methods|| |
This study was conducted at the Indian Council of Medical Research (ICMR)/DHR Grade-I Virology reference Laboratory-Advanced Research Laboratory, Department of Microbiology and Immunology, S. M. S. Medical College, Jaipur during 2012–2016. The study was approved by Institutional Ethics Committee.
All the patients enroled in the study were freshly diagnosed and hospitalised cases. Any previous history of respiratory infections was excluded from the study to prevent the detection of residual nucleic acid. Written consent was obtained from the parent/guardian. Clinical signs and symptoms of children enroled in the study were noted in pro forma for fever, cough, nasal catarrh, shortness of breath, sore throat, wheezing, bronchiolitis and pneumonia.
Sample collection and transportation
Nasopharyngeal aspirate and throat swabs from 997 children (age range 1–60 months) with acute respiratory illness were collected in 3 ml of viral transport media. Samples were labelled and transported on ice packs at the earliest to the laboratory. On reaching the laboratory, samples were registered and given a unique identification number before aliquoting for processing and storage.
Nucleic acid extraction
Nucleic acid from samples was extracted using NucliSENSE EasyMAG (Biomeurex) automated extractor according to the manufacturer's instructions, 110 μl of nucleic acid was eluted from a volume of 400 μl sample.
Previously standardised customised multiplex PCR was used for identification of various viruses, bacterial PCR standardisation was done using the same protocol but tested in duplex in samples negative for any virus [Table 1].
|Table 1: Customised primers and probes for the detection of respiratory viruses and bacteria using real time reverse transcription - polymerase chain reaction (multiplex and duplex)|
Click here to view
Chi-square test was employed to study the P value of different viruses and bacteria.
| ~ Results|| |
Viruses detected in children
Out of 997 children enroled, 598 (59.98%) children were positive for at least one of the respiratory viruses of which co-infections were detected in 133 (22.24%) children. Single infection was observed in 465 (77.76%) children [Table 2]. The most common virus detected in the study (including single and co-infection) was RSV A/B in 279 (37.35%), followed by human rhinoviruses (HRV) 106 (14.19%) and HAdV 83 (11.11%). The profile of viruses causing ARIs is mentioned in [Table 2]. Details of co-infections of different viruses are mentioned in [Table 3].
|Table 2: Age-wise distribution of the respiratory viruses and the incidence of single and co-infections|
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Clinical features and association with respiratory viruses
Cough and fever were the most common clinical manifestation followed by shortness of breath, pneumonia and bronchiolitis [Table 4]. The RSV A/B was predominant in cases presenting with pneumonia and bronchiolitis.
|Table 4: Clinical features in association with different respiratory viruses|
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Viruses among different age groups
Positivity for respiratory viruses was found to be more in the age group of 1–12 months; 495 (66.26%) cases, followed by 13–24 months' age patients with 101 (13.52%) cases and 25–36 months' age patients, 65 (8.70%). Most of the viruses were found predominantly in the age group of 1–12 months as compared to other age groups [Table 2].
Seasonal trends of respiratory viruses
During the study, respiratory viruses were predominant during the winter months [Figure 1], RSV A/B had its high peaks in December and January while human metapneumovirus (HMPV) A/B and Pandemic H1N1 had only single peak in February 2013 and for HAdV in December 2014 and 2015 only. HRV was found to be circulating throughout the year irrespective of the seasons, having high peaks in March, May and December.
|Figure 1: Month-wise activity of different respiratory viruses from March-2012 to March 2016|
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On Chi-square test, P value was found to be significant for some viruses such as RSV (P < 0.0001), HMPV A/B (P = 0.001), pandemic H1N1 (P = 0.0022), Flu A (P = 0.0165) and EV (
P = 0.0167) when age-wise intergroup comparisons were made for each virus.
Bacteria causing ARI
Among samples negative for respiratory viruses, 167 (41.85%) were positive for bacteria. Among these, S. pneumoniae was the most common, positive in 127 (62.18%) cases followed by Moraxella More Details catarrhalis in 60 (29.85%) cases, [Table 5]. Predominance of bacterial agents was also found in the age group of 1–12 months. P < 0.05 was observed for S. pneumoniae and M. catarrhalis.
|Table 5: Age-wise distribution of the respiratory bacteria and the incidence of single and co-infections|
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Based on the viruses/bacteria detected in the study and the severity of the disease that they cause, a diagnostic algorithm was developed [Figure 2].
| ~ Discussion|| |
Respiratory infections claim many lives globally every year. Profile and trends of pathogens causing respiratory infection varies from place to place and as per se ason and geographic conditions, data from India are very limited hence the present study was done.
In the present study, the overall positivity for viral and bacterial pathogens was found to be 76.73%, whereas a study from Dhaka  reported a positivity of 82.5%. This could be due to many reasons such as low-sample size etc. Predominant virus detected in the present study was RSV A/B (37.35%), as also reported from New Zealand (39%), while wide variation was reported from China (13.06%) and Malaysia (70.6%). The variations in detection rates by various studies may be due to sampling methods, genetic variability among the populations, number of samples enroled in the study, samples collected during different seasons of the year and the method used for detection. RSV is known to be an important cause of hospitalisation in children younger than 1 year of age, this may be due to lower immunity in infants.,
Higher numbers of HRV infections were seen in children of 1–12 months in our study than other age groups as also reported from Latin America. Tregoning and Schwarze 2010 reported that RSV and HRV are the most common causes of acute respiratory viral infections as also seen in present study. The positivity of other respiratory viruses, i.e., HAdV, HMPV, HPIV-1 to HPIV-4, HCoV-OC43, HCoV-229E, HCoV-NL 63, HCoV-HKU1, EV, HBoV, Pandemic H1N1, Flu A, Flu B and HPeV were between 11.11%–0.13% [Table 2]. An Indian study by Singh et al., 2014 reported no positivity of HPIV 1–4 and 1.10% for HBoV and 7.74% for Influenza (Flu A and Flu B) in hospitalised children with ALRI.
The prevalence of respiratory pathogens were found to be higher in the age group of <1 year with more predilection to RSV A/B and S. pneumoniae and higher co-infection as also reported by Bhuyan et al., 2017. Most of the viruses in our study were associated with both upper and lower respiratory tract infections. However, RSV A/B and S. pneumoniae were mostly associated with lower respiratory tract infections (i.e. bronchiolitis and pneumonia) in our study and at Dhaka too. On comparing seasonal variation, in our study RSV A/B showed its highest peak in winters, during January similarly a study from Madagascar reported RSV A/B peak in February. HMPV A/B also had highest peak in February as reported from Karachi also. Thereafter, no peaks of HMPV A/B were observed and HMPV A/B was replaced by RSV A/B as the predominant virus which continued in the entire study as the major virus. Continuous positivity with a peak of pandemic H1N1 was observed from December 2012 to April 2013. This may be due to the high prevalence of pandemic H1N1 in the study region. HRV was found to be circulating almost entire year with peaks in March, May and December.
Positivity of the respiratory viruses was not continuous throughout the years. Some studies reported circulation of the respiratory viruses throughout the year, whereas others show distinct seasonality, which could be due to seasonal variations of host immune response, climatic changes, which may promote the viral growth. Low temperatures may favour the circulation of the viruses. The information on seasonal positivity of respiratory pathogens can help public and clinicians to take necessary preventive measures to control respiratory infections.
Timely identification of virus etiology in such patients can prevent misuse of antibiotics and emergence of multidrug-resistant bacteria. Moreover, the detection of fastidious bacteria by RT-PCR could help patient by initiating appropriate therapy timely. S. pneumoniae was the predominant bacterial agent as also reported from Dhaka, who report it to be an important cause of mortality in children <5 years of age. The overall positivity for the bacterial agents was found to be 41.85%. Although the detection rate of Mycoplasma pneumoniae was only 1.99% out of which 1.49% were associated with pneumonia. Even though M. catarrhalis was second-most predominant bacteria detected in the study its role in respiratory infections needs to be evaluated further as it is a known commensal. In children, <5 years high prevalence of both bacterial and viral pathogens was observed. Immature immune system may be the reason behind this. Although bacteria cause secondary infections after viral infections, they should not be excluded from screening.
A diagnostic algorithm was prepared based on the severity of the disease caused and frequency of detection of pathogens. This will help in rapid economic diagnosis. However, there were some limitations in our study, only viral negative samples were screened for bacteria, screening of all samples would have given better picture; moreover, only limited panel of bacteria were tested and antibiotic sensitivity testing was also not done. Further studies are required including these parameters to answer all questions.
| ~ Conclusion|| |
The RSV A/B, HRV, HAdV and HMPV A/B respiratory viruses were more prevalent than others and that too in winter months. Children aged <1 year were more vulnerable to respiratory viruses and bacteria, diagnostic algorithm developed can help provide rapid economic diagnosis. Rapid early identification of viruses and fastidious bacteria by PCR can help start proper therapy timely and prevent misuse of antibiotics and development of antimicrobial drug resistance. However, inclusion of all bacteria responsible for respiratory infections and the drug resistance markers can further help in patient care, additional studies are required to know whether the agent identified is a bystander or a true pathogen. This will also help in planning strategies for vaccination.
The authors acknowledge the financial support from ICMR to BM for setting up ICMR Grade-I/DHR Viral Research and diagnostic Laboratory and Senior Research Fellowship to MAS.
Financial support and sponsorship
This study was supported by the Indian Council of Medical Research, New Delhi, India.
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Prajapati B, Talsania N, Sonaliya KN. A study on prevalence of acute respiratory tract infections (ARI) in under five children in urban and rural communities of Ahmedabad district, Gujarat. Natl J Community Med 2011;2:255-9.
Selvaraj K, Chinnakali P, Majumdar A, Krishnan IS. Acute respiratory infections among under-5 children in India: A situational analysis. J Nat Sci Biol Med 2014;5:15-20.
Lee JH, Chun JK, Kim DS, Park Y, Choi JR, Kim HS, et al.
Identification of adenovirus, influenza virus, parainfluenza virus, and respiratory syncytial virus by two kinds of multiplex polymerase chain reaction (PCR) and a shell vial culture in pediatric patients with viral pneumonia. Yonsei Med J 2010;51:761-7.
Bicer S, Giray T, Çöl D, Erdağ GÇ, Vitrinel A, Gürol Y, et al.
Virological and clinical characterizations of respiratory infections in hospitalized children. Ital J Pediatr 2013;39:22.
Bhuyan GS, Hossain MA, Sarker SK, Rahat A, Islam MT, Haque TN, et al.
Bacterial and viral pathogen spectra of acute respiratory infections in under-5 children in hospital settings in Dhaka city. PLoS One 2017;12:e0174488.
Sanghavi SK, Bullotta A, Husain S, Rinaldo CR. Clinical evaluation of multiplex real-time PCR panels for rapid detection of respiratory viral infections. J Med Virol 2012;84:162-9.
Malhotra B, Swamy MA, Reddy PV, Kumar N, Tiwari JK. Evaluation of custom multiplex real – Time RT – PCR in comparison to fast – Track diagnostics respiratory 21 pathogens kit for detection of multiple respiratory viruses. Virol J 2016;13:91.
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;42:1564-9.
Hammitt LL, Kazungu S, Welch S, Bett A, Onyango CO, Gunson RN, et al.
Added value of an oropharyngeal swab in detection of viruses in children hospitalized with lower respiratory tract infection. J Clin Microbiol 2011;49:2318-20.
Esposito S, Molteni CG, Daleno C, Valzano A, Tagliabue C, Galeone C, et al.
Collection by trained pediatricians or parents of mid-turbinate nasal flocked swabs for the detection of influenza viruses in childhood. Virol J 2010;7:85.
Cui LJ, Zhang C, Zhang T, Lu RJ, Xie ZD, Zhang LL, et al.
Human coronaviruses HCoV-NL63 and HCoV-HKU1 in hospitalized children with acute respiratory infections in Beijing, China. Adv Virol 2011;2011:129134.
Kwofie TB, Anane YA, Nkrumah B, Annan A, Nguah SB, Owusu M, et al.
Respiratory viruses in children hospitalized for acute lower respiratory tract infection in Ghana. Virol J 2012;9:78.
Nix WA, Maher K, Johansson ES, Niklasson B, Lindberg AM, Pallansch MA, et al.
Detection of all known parechoviruses by real-time PCR. J Clin Microbiol 2008;46:2519-24.
Wolffs PF, Bruggeman CA, van Well GT, van Loo IH. Replacing traditional diagnostics of fecal viral pathogens by a comprehensive panel of real-time PCRs. J Clin Microbiol 2011;49:1926-31.
Greiner O, Day PJ, Bosshard PP, Imeri F, Altwegg M, Nadal D, et al.
Quantitative detection of Streptococcus pneumoniae
in nasopharyngeal secretions by real-time PCR. J Clin Microbiol 2001;39:3129-34.
Kodani M, Yang G, Conklin LM, Travis TC, Whitney CG, Anderson LJ, et al.
Application of TaqMan low-density arrays for simultaneous detection of multiple respiratory pathogens. J Clin Microbiol 2011;49:2175-82.
Greiner O, Day PJ, Altwegg M, Nadal D. Quantitative detection of Moraxella catarrhalis
in nasopharyngeal secretions by real-time PCR. J Clin Microbiol 2003;41:1386-90.
Chien YW, Vidal JE, Grijalva CG, Bozio C, Edwards KM, Williams JV, et al.
Density interactions among Streptococcus pneumoniae
, Haemophilus influenzae
and Staphylococcus aureus
in the nasopharynx of young Peruvian children. Pediatr Infect Dis J 2013;32:72-7.
Mitchell SL, Budhiraja S, Thurman KA, Lanier Thacker W, Winchell JM. Evaluation of two real-time PCR chemistries for the detection of Chlamydophila pneumoniae
in clinical specimens. Mol Cell Probes 2009;23:309-11.
Yang G, Benson R, Pelish T, Brown E, Winchell JM, Fields B, et al.
Dual detection of Legionella pneumophila
and legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region. Clin Microbiol Infect 2010;16:255-61.
Trenholme AA, Best EJ, Vogel AM, Stewart JM, Miller CJ, Lennon DR, et al.
Respiratory virus detection during hospitalisation for lower respiratory tract infection in children under 2 years in South Auckland, New Zealand. J Paediatr Child Health 2017;53:551-5.
Li H, Wei Q, Tan A, Wang L. Epidemiological analysis of respiratory viral etiology for influenza-like illness during 2010 in Zhuhai, China. Virol J 2013;10:143.
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.
Singh AK, Jain A, Jain B, Singh KP, Dangi T, Mohan M, et al.
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.
] [Full text]
Reiche J, Schweiger B. Genetic variability of group A human respiratory syncytial virus strains circulating in Germany from 1998 to 2007. J Clin Microbiol 2009;47:1800-10.
Figueras-Aloy J, Carbonell-Estrany X, Quero J; IRIS Study Group. Case-control study of the risk factors linked to respiratory syncytial virus infection requiring hospitalization in premature infants born at a gestational age of 33-35 weeks in Spain. Pediatr Infect Dis J 2004;23:815-20.
Garcia J, Espejo V, Nelson M, Sovero M, Villaran MV, Gomez J, et al.
Human rhinoviruses and enteroviruses in influenza-like illness in Latin America. Virol J 2013;10:305.
Tregoning JS, Schwarze J. Respiratory viral infections in infants: Causes, clinical symptoms, virology, and immunology. Clin Microbiol Rev 2010;23:74-98.
Hoffmann J, Rabezanahary H, Randriamarotia M, Ratsimbasoa A, Najjar J, Vernet G, et al.
Viral and atypical bacterial etiology of acute respiratory infections in children under 5 years old living in a rural tropical area of Madagascar. PLoS One 2012;7:e43666.
Ali A, Khowaja AR, Bashir MZ, Aziz F, Mustafa S, Zaidi A, et al.
Role of human metapneumovirus, influenza A virus and respiratory syncytial virus in causing WHO-defined severe pneumonia in children in a developing country. PLoS One 2013;8:e74756.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]