A metagenomics method for the quantitative detection of bacterial pathogens causing hospital-associated and ventilator-associated pneumonia

The management of ventilator-associated and hospital-acquired pneumonia requires rapid and accurate quantitative detection of the infecting pathogen(s). To achieve this, we propose a metagenomics next-generation sequencing (mNGS) assay that includes the use of an internal sample processing control (SPC) for the quantitative detection of 20 relevant bacterial species of interest (SOI) from bronchoalveolar lavage (BAL) samples. To avoid very major errors in the 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 the 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 5.3 x 10(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 >99.9% 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 the "false-positive" detections was confirmed by finding specific 16S/MetaPhlAn2 markers, the 12 other "false-positive" detections did not yield enough reads to check their taxonomic classification. 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 the manufacturer's recommendations for NGS. The frequent "false-positive" detections suggested the presence of nonculturable cells within the tested BAL samples. Furthermore, mNGS detected mixed infections, including bacterial species not reported by routine cultures.