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.