Comprehensive Molecular Testing for Respiratory Pathogens in Community-Acquired Pneumonia

Gadsby, Naomi J.; Russell, Clark D.; McHugh, Martin P.; Mark, Harriet; Conway Morris, Andrew; Laurenson, Ian F.; Hill, Adam T.; Templeton, Kate E.

LRT specimens were cultured according to national standard protocols to detect common respiratory bacteria [35] . Isolates were identified using standard biochemical methods and/or matrix-assisted laser desorption/ionization time-of-flight (Bruker, Coventry, United Kingdom). Specimens with any named bacterial species identified by culture were considered to be culture positive. Following routine culture, LRT specimens were centrifuged, and mucopurulent material was stored at −80°C for up to 15 months. As part of the study protocol, specimens and associated clinical data were collected and anonymized before molecular testing in accordance with local ethical approval (South East Scotland Scottish Academic Health Sciences Collaboration Human Annotated BioResource reference no. 10/S1402/33). Thawed specimens were homogenized by vortexing with sterile glass beads, and a 200-μL aliquot was treated with lysozyme at 37°C followed by proteinase K at 56°C for 1 hour each. Total nucleic acid was then extracted using the automated nucliSENS easyMAG (BioMérieux, Basingstoke, United Kingdom) instrument with an off-board lysis protocol, including the addition of phocine herpes virus as an internal extraction control. Extracts were stored at 4°C for up to 5 days or longer at −80°C. Fast multiplex real-time PCR was performed using a combination of multiplex assays developed or adapted in-house for the following [10] [11] [12] [13] [14] [15] 33] . As previously described [33] , bacterial loads for S. pneumoniae, H. influenzae, M. catarrhalis, S. aureus, E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii were expressed as colony forming units (CFUs) per milliliter of purulent material and calculated from standard curves generated by target gene plasmid dilution series on each PCR run. Quality control reactions (quantitative real-time PCR for GAPDH human gene and qualitative real-time PCR for gB phocine herpes virus internal control [IC]) were carried out on all specimens [33] . Quantitative GAPDH PCR was used as an indication of the cell content of each specimen because the GAPDH gene is present in human DNA. Negative (no template) controls were included on every extraction and PCR run to check purity of reagents. Runs were accepted if the negative controls were negative and the standard curves were linear; runs that failed quality control were repeated. Qualitative results were accepted if the internal control was positive or if at least 1 other target was positive. Quantitative results were accepted if the internal control quantification cycle (Cq) value fell within the range ±1 log Cq difference to negative extraction controls. Outside of this range, quantitative results were not accepted due to the potential for partial PCR inhibition leading to inaccuracy of measurement.