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

The respiratory viruses and atypical bacterial pathogens such as Mycoplasma pneumoniae and Chlamydophila pneumoniae are detected by multiplex real-time polymerase chain reaction (PCR) at PathWest Laboratories (QEII) using previously described primers and probes [58, 59] . In detail, nucleic acid is extracted from the NPS using the MagNA Pure 96 System (Roche Applied Science, Indianapolis, US). Extracted nucleic acids are tested in Applied Biosystems ViiA7 Real-time PCR Quantitative viral load is assessed on all NPS specimens that are positive for respiratory viruses. The primers, probes and protocols for determining the viral load for a range of viral pathogens in NPS specimens have been developed by PathWest (Sikazwe, C et al unpublished data). 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.