A Metagenomics Method for the Quantitative Detection of Pathogens Causing Ventilator-Associated Pneumonia

Abstract

Background The management of ventilator-associated and hospital-acquired pneumonia requires rapid and accurate quantitative detection of the infecting pathogen(s). To do it, we propose a metagenomics next-generation sequencing (mNGS) assay that includes an internal sample processing control (SPC) for the quantitative detection of 20 relevant bacterial species of interest (SOI) from bronchoalveolar lavage (BAL) samples. Results To avoid very major errors in identification of respiratory pathogens due to “false negative” cases, each sample was spiked with Bacillus subtilis, at a precisely defined concentration, using rehydrated BioBall®. This SPC ensured detection and quantification of the pathogen(s) at defined minimum concentrations. In the presented mNGS workflow, absolute quantification of Staphylococcus aureus was as accurate as quantitative PCR. We defined a metagenomics threshold at 5.3E+3 genome equivalent unit per milliliter of the sample for each SOI, to distinguish colonization from higher amounts of pathogens that may be associated with infection. Complete mNGS process and metrics were assessed on 40 clinical samples, showing 100 % sensitivity compared to microbial culture. However, 19 out of the 29 (66 %) SOI detections above the metagenomics threshold were not associated with bacterial growth above classical culture-based clinical thresholds. Taxonomic classification of 7 (37 %) of these “false positive” detections were confirmed by finding specific 16S/MetaPhlAn2 markers, the 12 other (63 %) “false positive” detections did not yield enough reads to check their taxonomic classification. Conclusions Our SPC design and analytical workflow allowed efficient detection and absolute quantification of pathogens from BAL samples, even when the bacterial DNA quantity was largely below manufacturer’s recommendations for NGS. The frequent "false positive" detection suggested the presence of non culturable cells within the tested BAL samples. Finally, mNGS detected mixed infections including bacterial species that were not reported by routine cultures.

Effect of bacterial DNA enrichment on detection and quantification of bacteria in an infected tissue model by metagenomic next-generation sequencing

“…The organism(s) used as calibrators for bacterial quantification should not be members of the host microbiome. Environmental and plant-associated species showed suitability for calibrating the ratios of absolute abundances of endogenous bacteria in metataxonomic (16S rRNA gene based) analyses of unenriched stool samples [40] and metagenomic analysis of liquid (BAL) samples [41]. From a practical point of view, frozen aliquots of a calibrator suspension are convenient as they can be used over longer times than freshly prepared ones.…”