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Viruses causing severe acute respiratory infections (SARI) in children ≤5 years of age at a tertiary care hospital in Rajasthan, India

Severe acute respiratory infections (SARIs) are defined as an acute respiratory illness of recent onset (within seven days) manifested by fever (≥38°C), cough and shortness of breath or difficulty in breathing requiring hospitalization1. The ARIs are the leading cause of deaths among children across the globe2 and Viruses causing severe acute respiratory infections (SARI) in children ≤5 years of age at a tertiary care hospital in Rajasthan, India

SARIs are usually diagnosed clinically and treated by antibiotics as per bacterial culture and susceptibility tests/empirically and supportive care in the absence of facilities for diagnosing viruses. Diagnosis of specific respiratory viral infection can help in proper management of patients by initiation of antiviral drug oseltamivir for influenza, discontinuation of unnecessary antibiotics, enhance supportive therapy, reduce the cost related to unnecessary investigations and reduce the hospital stay 9,10 . SARI, particularly in children being a serious condition demands rapid identification of causative agent.
Similar clinical signs and symptoms are caused by various respiratory viral pathogens; hence, rapid simultaneous identification of different viruses is of major epidemiological and clinical interest 4 . Many methods are available for diagnosis of respiratory viruses such as uniplex polymerase chain reaction (PCR), multiplex PCR, immunofluorescence assay, serology, etc. Immunofluorescence detects viral antigen rapidly, but lacks sensitivity and may require confirmation by viral culture 11 . Serology is also not useful clinically as antibodies appear after one week and require testing of paired serum samples for confirmation of infection. Multiplex real-time PCR assays allow simultaneous amplification of several viruses in a single mixture as compared to monospecific uniplex PCR assay in which separate amplification of each target has to be done which turns out to be more expensive, resource intensive and time consuming 12 . The present study was undertaken to rapidly identify the viruses causing SARI in children admitted to a hospital using the multiplex real-time reverse transcription-PCR (RT-PCR) method.

Material & Methods
The present study was conducted in the J. K. Lone Hospital, a paediatric hospital attached to SMS Medical College, Jaipur, India, over a period of 13 months (March, 2012& March, 2013. Consecutive children aged less than or equal to five years, presenting with fever, cough, shortness of breath, sore throat and nasal catarrh to the hospital were included in the study. Those (n=61) with chronic respiratory ailments, nonconsenting caregivers, with history of hospitalization in preceding 14 days, on antibiotics, having positive bacterial culture report or not admitted in hospital, and children aged greater than five years were excluded.
Considering the prevalence of ARIs in under five children in Rajasthan to be 7 per cent as reported by National Family Health Survey-3 (NFHS-3) 13 , the sample size was calculated as 104 using the formula n=4 pq/l 2 [where n=total number of samples; 4 is the factor to achieve the confidence level of 95%; p=known prevalence; q=100-p and l=allowable permissible (absolute) error, set at 5%].
Sample collection and transportation: A total of 155 throat swab samples were collected from equal number of consecutive patients with SARI using a sterile nylon flocked swabs and placed in viral transport medium, labelled and transported on ice at the earliest to the laboratory. Informed consent was obtained from the parents/guardians of the patients. The study protocol was approved by the institutional ethics committee.
Nucleic acid extraction: Viral nucleic acid was extracted from 200 µl sample using NucliSENS EasyMAG automated nucleic acid extractor (Biomeuriex, France) as per manufacturer's instructions. At the final step, the nucleic acid was eluted in a volume of 55 µl and processed immediately for multiplex PCR. Internal control was added to each sample during nucleic acid extraction to validate the process of extraction and PCR technique used in the study. Statistical analysis: To determine the significance of the data, Chi-square test was employed.

Results
Details of samples collected as per age, months and sex are given in Table I; highest number of samples was collected between September and March (Table I).
Of the 155 children, 113 (72.9%) were positive for at least one of the respiratory viruses, among whom co-infections were detected in 22 (19.5%) children. Single viral infections were observed in 91 (80.5%) children (Table II) (Table II). HMPV was found to be predominant in single infections whereas HRV was found to be predominant in co-infections. No human parechovirus (HPeV) was identified in the study. Four samples (2.6%) were found to be positive for bacterial agent Mycoplasma pneumoniae.

Severe acute respiratory infection (SARI) co-infections:
Among the 22 children with co-infections, no co-infections with more than three viruses were observed (Table III). A total of six (27.3%) cases infected with coronaviruses were detected along with other respiratory viruses. Four (18.2%) children were infected with HRV and HAdV. Of the 27 children with influenza A (H1N1) pdm09 infection, co-infections were observed in six samples, with HBoV as the predominant virus.
Clinical features among different viruses: Most of the patients presented with fever and cough followed by nasal catarrh, sore throat and shortness of breath (Table IV). The HMPV was predominant in cases presenting with fever and cough followed by influenza A (H1N1)pdm09 as compared to other viruses in the study. A total of 56 (41.2%) viruses were found in the samples collected within three days of illness, 68 (50%) in three to six days of illness and 12 (8.8%) in samples collected after more than six days of illness (Table V).
The HMPV (P<0.001) and HAdV were found to be predominantly positive in the age group of 49-60 months (P<0.05) whereas rhinovirus was predominant in the age group 0-12 months (P=0.01) stating that the infections caused were highly significant among the children when age-wise intergroup comparisons were made for each virus (Fig. 1).
Seasonal trends of respiratory viruses: HMPV was the predominant isolate in the study with peak incidence in February 2013. HAdV was found to be circulating

Gender-wise occurrence of respiratory viral infections:
The occurrence of respiratory viruses in males was found to be higher (75/136; 55.1%) compared to that in females (61/136; 44.8%). Human metapneumovirus was found to be predominantly occurring in males as compared to females. In contrast, influenza A (H1N1) pdm09, HRV, HAdV, RSV, HCoV-HKU1, HCoV-229E, HCoV-OC43 were found to be more in females than in males (Table VI).

Discussion
The predominant isolate in the present study was found to be HMPV with 25.7 per cent of children showing positivity. Studies by Maggi et al 14

from
Italy reported that HMPV infections ranged between 7 and 43 per cent over a period of three years, whereas  other studies revealed that HMPV infections were secondary to other ARI, and had low positivity of 8.03 per cent infections 3 and 9.4 per cent in hospitalised children 15 . Influenza A (H1N1)pdm09 was the second most predominant virus detected in the present  Abbreviations as given in Table II. Coronavirus infections in the present study were found to be 8.8 per cent. Papenburg et al 15 in hospitalized patients with acute respiratory tract infections revealed a total of 6 per cent coronaviral infections. The parainfluenza virus infection was seen in 17 (12.5%) children. The results from another study revealed similar pattern with a total of 11.8 per cent children infected with parainfluenza viruses 16 . However, an Indian study reported no positivity of HPIV1-4 in hospitalized children with ALRI 20 . A total of 22 (19.5%) co-infections were identified in the present study which was identical with an earlier study 20 . The frequency of respiratory viral co-infections varied widely in different studies globally (10 -41%) [22][23][24][25] .
Fever was the major clinical feature in the present study followed by cough and nasal catarrh as also reported in other studies 5,26 . However, respiratory infections caused by these viruses usually present clinical features that are nearly indistinguishable 5,27 .
There were seasonal variations in the trends of infections of viruses, with two peaks in a year. The present study revealed the circulation of HRV throughout the year which was similar to an earlier study 6 . However, the seasonal trends of the respiratory viruses seen in the present study varied from other studies 28,29 . The variations in the seasonal distribution of different respiratory viruses may be attributed to the different geographical locations, viral aetiological agents and the methodologies used in the study.
Most of the respiratory viruses were found in the age group 49-60 months followed by 25-36 months old children. Similar findings have been made previously with predominant infections in the children of age less than five years 21 . The distribution of respiratory viruses in males was found to be higher as compared to females. Khor et al 29 also showed higher proportion of infection in male children over females.
The present study had a few limitations, such as detailed clinico-virological correlation was not done with the severity of disease, duration of hospitalization, use of invasive or non-invasive ventilator, outcome of treatment, mortality, etc. in relation to virus identified. SARI can also be caused by various bacterial agents that are fastidious and not isolated in routine bacterial culture such as Chlamydia pneumoniae, Legionella spp., Bordetella spp. etc. 30 ; moreover, Streptococcus pneumoniae and Haemophilus influenzae may fail to grow on culture very often. Therefore, testing all these bacteria also by PCR may help in increasing yield and rapid diagnosis. Other rare viruses such as influenza C, cytomegalovirus may also be tested in samples negative for the first panel. Though throat swab has been reported to give good yield for all viruses, positivity can be further improved by also taking nasopharyngeal swab, nasal swab, tracheal aspirate, bronchoalveolar lavage 21 .
The present results provided preliminary understanding of the viral agents associated with SARI. High positivity of 72.9 per cent was found, detecting 17 viruses with HMPV being the predominant virus, Abbreviations as given in Table II. followed by HRV and HAdV and overall co-infectivity of 19.5 per cent. There is a need for conducting further studies to detect broader range of viruses and other fastidious organisms, to undertake detailed clinical work up for better clinico-virological correlation, and to carry out additional follow up bacterial culture to understand role of secondary bacterial infection. Females, Males, Females, Males, Females, Males, Females, Males, Females, Abbreviations as given in Table II.