Viral testing:
For viral testing, nasopharyngeal aspirates (NPA) were taken by a trained health personnel within 12 hours of admission by passing 6-8 Fr feeding tube into the nasopharynx and applying gentle suction with a syringe. The secretions were rinsed into viral transport medium (VTM) and transported under cold chain to Regional viral research and diagnostic laboratory (VRDL), Department of Virology for testing of RSV, rhinovirus, influenza A, PIV 2, PIV 3, and hMPV. The samples were subjected to nucleic acid extraction using QIAamp Viral RNA Mini Kit (Qiagen, Heidelberg) and extracted RNAs were reverse transcribed utilizing high capacity cDNA reverse transcription kits (Applied Biosystems). Matrix gene of Influenza A and nucleocapsid gene of RSV, PIV 2 and PIV 3 were targeted to screen the respective RNA according to the protocol by Bharaj et al5. Amplification of Influenza A and RSV were done on monoplex single tube format whereas for the amplification of PIV 2 and PIV 3, multiplexing PCR was used. Viral genome of hMPV was detected in clinical samples by using primers as described by Bouscambert-Duchamp et al16. For the detection of human rhinovirus, highly conserved 5’ un-translated region of the genome was amplified using a previously described nested PCR strategy according to Wisdom et al17. The amplified DNA fragments were identified on a 2% agarose gel with ethidium bromide and visualized under UV transilluminator. For the confirmation, PCR amplified products were purified and sequenced bi-directionally using BigDye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Foster, CA) with an ABI 3500xL genetic analyzer (PE Applied Biosystems Inc., Foster City, CA) and further checked by BLAST tool with already available reference database of NCBI website.
Our study was carried out with the aim to describe the clinical and virological profile, treatment details, intensive care needs, and outcome of infants with AVB.