WS13.02 Comparison of qPCR and 16S rRNA marker-gene Next-Generation Sequencing (NGS) for the detection and quantification of respiratory pathogens in the sputum of people with cystic fibrosis (PwCF)

Objectives: Non-culture methods are increasingly used for the detection and quantification of pathogens [1Forbes et al.Front Microbiol. 2017; 8: 1069Crossref PubMed Scopus (182) Google Scholar]. qPCR allows absolute quantification of target genes, while 16S rRNA marker-gene MiSeq sequencing (NGS) provides taxonomic resolution to a genera level [2Janda JM Abbott SL J Clin Microbiol. 2007; 45: 2761-2764Crossref PubMed Scopus (1223) Google Scholar], with the relative abundance of each of these bacterial genera determined within a sample. The aim of this study was to compare qPCR and relative abundance of 16S rRNA amplicons for the detection and quantification of bacteria within sputum samples from PwCF. Methods: Sputum samples (n = 179) were collected as part of the Real-world Orkambi cohort Cork study (ROCK). Microbial community composition was determined by NGS and qPCR was carried out for selected bacteria. Correlation between methods was determined by calculation of Cohen’s Kappa coefficient. TableDetection of target bacterial pathogens by qPCR and NGS and agreement between detection methodsGeneTarget bacterial pathogenNo. of samples positive by qPCRNo. of samples positive by NGSCohen's kappa coefficient16S rRNAAll bacteria1791791oprLPseudomonas aeruginosa1281560.51ecfX1340.49hpDHaemophilus influenzae17850.18smbB210.21lytAStreptococcus pneumoniae271760.04femAStaphylococcus aureus101970.84 Open table in a new tab Results: There was almost perfect agreement for detection of S. aureus between methods, with moderate agreement for P. aeruginosa, slight agreement for H. influenzae and no agreement for S. pneumoniae. There was a positive correlation between oprL (r = 0.93, p < 0.001) and ecfX (r = 0.94, p < 0.001) copy number and the relative abundance of Pseudomonas spp. determined by NGS and between femA (r = 0.93, p < 0.001) and Staphylococcus spp. relative abundance. There was a weaker positive correlation between hpD (r = 0.4, p < 0.001) and smpB (r = 0.42, p < 0.001) copy number and the relative abundance of Haemophilus spp.. Conclusion: Both qPCR and NGS can be used to detect pathogens in respiratory samples. Discordance between methods may be due to detection of species from the same genera by NGS which are not detected by species specific qPCR assays. Supported by EU/EFPIA IMI iABC grant n° 115721 We would also like to acknowledge funding from the European Commission for CFMATTERS, Grant agreement 603038