Role of viral and bacterial pathogens in causing pneumonia among Western Australian children: a case–control study protocol

Bhuiyan, Mejbah Uddin; Snelling, Thomas L; West, Rachel; Lang, Jurissa; Rahman, Tasmina; Borland, Meredith L; Thornton, Ruth; Kirkham, Lea-Ann; Sikazwe, Chisha; Martin, Andrew C; Richmond, Peter C; Smith, David W; Jaffe, Adam; Blyth, Christopher C

After collection, the NPS specimen is kept on ice and processed within 4h at the research laboratory of the PMH Children's Clinical Research Facility (CCRF). The NPS specimen is vortexed for 30 seconds before divided into two aliquots with 500 µl each for bacterial and viral assessment, and the vials stored at -80°C. Specimens are batched and periodically sent to reference laboratories for PCR testing. The NPS specimens are transported to the reference laboratories on ice on the same day of testing to avoid an additional freeze-thaw cycle. . A nucleic acid standard is developed using purified plasmid DNA or in vitro transcribed RNA, and the concentration of the DNA and/or RNA standard is quantified using fluorescent dyes. Following this, a standard curve is generated from serial 10-fold dilutions of the DNA and/or RNA standards mentioned above. The viral load in the NPS specimen is quantified by interpolating from the appropriate standard curve. The viral load is reported in viral copies/mL.