Enhanced DNA and RNA pathogen detection via metagenomic sequencing in patients with pneumonia

Abstract

Background Metagenomic next-generation sequencing (mNGS) is an important supplement to conventional tests for pathogen detections of pneumonia. However, mNGS pipelines were limited by irregularities, high proportion of host nucleic acids, and lack of RNA virus detection. Thus, a regulated pipeline based on mNGS for DNA and RNA pathogen detection of pneumonia is essential. Methods We performed a retrospective study of 151 patients with pneumonia. Three conventional tests, culture, loop-mediated isothermal amplification (LAMP) and viral quantitative real-time polymerase chain reaction (qPCR) were conducted according to clinical needs, and all samples were detected using our optimized pipeline based on the mNGS (DNA and RNA) method. The performances of mNGS and three other tests were compared. Human DNA depletion was achieved respectively by MolYsis kit and pre-treatment using saponin and Turbo DNase. Three RNA library preparation methods were used to compare the detection performance of RNA viruses. Results An optimized mNGS workflow was built, which had only 1-working-day turnaround time. The proportion of host DNA in the pre-treated samples decreased from 99 to 90% and microbiome reads achieved an approximately 20-fold enrichment compared with those without host removal. Meanwhile, saponin and Turbo DNase pre-treatment exhibited an advantage for DNA virus detection compared with MolYsis. Besides, our in-house RNA library preparation procedure showed a more robust RNA virus detection ability. Combining three conventional methods, 76 (76/151, 50.3%) cases had no clear causative pathogen, but 24 probable pathogens were successfully detected in 31 (31/76 = 40.8%) unclear cases using mNGS. The agreement of the mNGS with the culture, LAMP, and viral qPCR was 60%, 82%, and 80%, respectively. Compared with all conventional tests, mNGS had a sensitivity of 70.4%, a specificity of 72.7%, and an overall agreement of 71.5%. Conclusions A complete and effective mNGS workflow was built to provide timely DNA and RNA pathogen detection for pneumonia, which could effectively remove the host sequence, had a higher microbial detection rate and a broader spectrum of pathogens (especially for viruses and some pathogens that are difficult to culture). Despite the advantages, there are many challenges in the clinical application of mNGS, and the mNGS report should be interpreted with caution.

Unified metagenomic method for rapid detection of bacteria, fungi and viruses in clinical samples

“…Median human DNA depletion in 29 lower respiratory tract samples was ten cycle thresholds (IQR 4-12). Overall, across upper and lower respiratory tract samples the human DNA depletion was eight (IQR [4][5][6][7][8][9][10][11][12] representing an approximate 256-fold reduction (Table S.2 and S.3).…”

Combined host depletion and metagenomics sequencing significantly improves the diagnosis of pulmonary tuberculosis.

“…Jin et al 19 reported that the sensitivity of mNGS without removing host DNA was 59.01% and 43.75% for con rmed TB cases and clinical TB cases, which was consistent with our mNGS pipeline without human DNA depletion. But, metagenomic sequencing combing host DNA depletion can dramatically shift the diagnostic performance 24 . Combing our data, mNGS-DH and nanopore-DH could improve the detection e ciency of MTB.…”

“…And in de nite PTB, the sensitivity of mNGS-DH was 86.00%, which was superior to culture, Xpert, mNGS and nanopore-DH. These reasons may be caused by decreasing host DNA and shifting microbial reads 24 . And some studies 23,25 have demonstrated that metagenomic sequencing combining host DNA depletion could shift the detection rate of pathogens.…”

Combination of Whole Genome Sequencing and Metagenomics for Microbiological Diagnostics

“…Standardization of the methodological and analytical workflows has just started [ 109 , 110 ]. Proof-of-concept studies have shown the potential clinical impact in pathogen identification within culture-negative samples of, e.g., meningitis and encephalitis [ 111 ], sepsis [ 112 , 113 , 114 ], pneumonia [ 115 , 116 ], and prosthetic joint infections [ 117 , 118 ]. The detection limit of shotgun metagenomics is affected by slow-growing microorganisms or if the potential pathogen is present in low abundance.…”