Thousands of pathogens are known to infect humans, but only a fraction are readily identifiable using current diagnostic methods. Microbial cell-free DNA sequencing offers the potential to non-invasively identify a wide range of infections throughout the body, but the challenges of clinical-grade metagenomic testing must be addressed. Here we describe the analytical and clinical validation of a next-generation sequencing test that identifies and quantifies microbial cell-free DNA in plasma from 1,250 clinically relevant bacteria, DNA viruses, fungi and eukaryotic parasites. Test accuracy, precision, bias and robustness to a number of metagenomics-specific challenges were determined using a panel of 13 microorganisms that model key determinants of performance in 358 contrived plasma samples, as well as 2,625 infections simulated in silico and 580 clinical study samples. The test showed 93.7% agreement with blood culture in a cohort of 350 patients with a sepsis alert and identified an independently adjudicated cause of the sepsis alert more often than all of the microbiological testing combined (169 aetiological determinations versus 132). Among the 166 samples adjudicated to have no sepsis aetiology identified by any of the tested methods, sequencing identified microbial cell-free DNA in 62, likely derived from commensal organisms and incidental findings unrelated to the sepsis alert. Analysis of the first 2,000 patient samples tested in the CLIA laboratory showed that more than 85% of results were delivered the day after sample receipt, with 53.7% of reports identifying one or more microorganisms.
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The core software used as part of the Karius test is described in the Clinical-grade microbial cfDNA sequencing for infectious disease section in Methods, under the sub-sections Sequence data processing and alignment, Microorganism abundance estimation and Pathogen detection. The open source software includes the following external tools: bcl2fastq v184.108.40.206, Trimmomatic v0.32, Bowtie v2.2.4 and BLAST v2.2.30. A description of all open source code is included in Methods and further details are available on request. The proprietary portions of the code are not available.
The data that support the findings of this study are available from the corresponding author on request. Sequencing data that support the finding of this study (with human reads removed) have been deposited in NCBI SRA and can be accessed with the BioProject identifier PRJNA507824.
Christensen, K. L. et al. Infectious disease hospitalizations in the United States. Clin. Infect. Dis. 49, 1025–1035 (2009).
Barlam, T. F. et al. Implementing an antibiotic stewardship program: guidelines by the Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America. Clin. Infect. Dis. 62, e51–e77 (2016).
Liesenfeld, O., Lehman, L., Hunfeld, K. P. & Kost, G. Molecular diagnosis of sepsis: new aspects and recent developments. Eur. J. Microbiol. Immunol. 4, 1–25 (2014).
Kumar, A. et al. Initiation of inappropriate antimicrobial therapy results in a fivefold reduction of survival in human septic shock. Chest 136, 1237–1248 (2009).
Fenollar, F. & Raoult, D. Molecular diagnosis of bloodstream infections caused by non-cultivable bacteria. Int. J. Antimicrob. Agents 30, S7–S15 (2007).
Mancini, N. et al. The era of molecular and other non-culture-based methods in diagnosis of sepsis. Clin. Microbiol. Rev. 23, 235–251 (2010).
Fishman, J. A. Infection in solid-organ transplant recipients. N. Engl. J. Med. 357, 2601–2614 (2007).
Tomblyn, M. et al. Guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective. Biol. Blood Marrow Transplant. 15, 1143–1238 (2009).
Paul, M. et al. Systematic review and meta-analysis of the efficacy of appropriate empiric antibiotic therapy for sepsis. Antimicrob. Agents Chemother. 54, 4851–4863 (2010).
Kumar, A. An alternate pathophysiologic paradigm of sepsis and septic shock: implications for optimizing antimicrobial therapy. Virulence 5, 80–97 (2014).
Ramanan, P., Bryson, A. L., Binnicker, M. J., Pritt, B. S. & Patel, R. Syndromic panel-based testing in clinical microbiology. Clin. Microbiol. Rev. 31, 1–28 (2018).
Schreckenberger, P. C. & McAdam, A. J. Point–counterpoint: large multiplex PCR panels should be first-line tests for detection of respiratory and intestinal pathogens. J. Clin. Microbiol. 53, 3110–3115 (2015).
Kothari, A., Morgan, M. & Haake, D. A. Emerging technologies for rapid identification of bloodstream pathogens. Clin. Infect. Dis. 59, 272–278 (2014).
Simner, P. J., Miller, S. & Carroll, K. C. Understanding the promises and hurdles of metagenomic next-generation sequencing as a diagnostic tool for infectious diseases. Clin. Infect. Dis. 66, 778–788 (2018).
Greninger, A. L. et al. Clinical metagenomic identification of Balamuthia mandrillaris encephalitis and assembly of the draft genome: the continuing case for reference genome sequencing. Genome Med. 7, 113 (2015).
Wilson, M. R. et al. Actionable diagnosis of neuroleptospirosis by next-generation sequencing. N. Engl. J. Med. 370, 2408–2417 (2014).
Schlaberg, R. et al. Validation of metagenomic next-generation sequencing tests for universal pathogen detection. Arch. Pathol. Lab. Med. 141, 776–786 (2017).
Naccache, S. N. et al. Diagnosis of neuroinvasive astrovirus infection in an immunocompromised adult with encephalitis by unbiased next-generation sequencing. Clin. Infect. Dis. 60, 919–923 (2015).
Stokowski, R. et al. Clinical performance of non-invasive prenatal testing (NIPT) using targeted cell-free DNA analysis in maternal plasma with microarrays or next generation sequencing (NGS) is consistent across multiple controlled clinical studies. Prenat. Diagn. 35, 1243–1246 (2015).
Song, K., Musci, T. J. & Caughey, A. B. Clinical utility and cost of non-invasive prenatal testing with cfDNA analysis in high-risk women based on a US population. J. Matern. Fetal Neonat. Med. 26, 1180–1185 (2013).
Fan, H. C., Blumenfeld, Y. J., Chitkara, U., Hudgins, L. & Quake, S. R. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc. Natl Acad. Sci. USA 105, 16266–16271 (2008).
Schutz, E. et al. Graft-derived cell-free DNA, a noninvasive early rejection and graft damage marker in liver transplantation: a prospective, observational, multicenter cohort study. PLoS Med. 14, e1002286 (2017).
Bloom, R. D. et al. Cell-free DNA and active rejection in kidney allografts. J. Am. Soc. Nephrol. 28, 2221–2232 (2017).
De Vlaminck, I. et al. Noninvasive monitoring of infection and rejection after lung transplantation. Proc. Natl Acad. Sci. USA 112, 13336–13341 (2015).
De Vlaminck, I. et al. Circulating cell-free DNA enables noninvasive diagnosis of heart transplant rejection. Sci. Transl. Med. 6, 241ra277 (2014).
Snyder, T. M., Khush, K. K., Valantine, H. A. & Quake, S. R. Universal noninvasive detection of solid organ transplant rejection. Proc. Natl Acad. Sci. USA 108, 6229–6234 (2011).
Aravanis, A. M., Lee, M. & Klausner, R. D. Next-generation sequencing of circulating tumor DNA for early cancer detection. Cell 168, 571–574 (2017).
Lanman, R. B. et al. Analytical and clinical validation of a digital sequencing panel for quantitative, highly accurate evaluation of cell-free circulating tumor DNA. PLoS ONE 10, e0140712 (2015).
Bettegowda, C. et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci. Transl. Med. 6, 224ra224 (2014).
Dawson, S. J. et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N. Engl. J. Med. 368, 1199–1209 (2013).
Abril, M. K. et al. Diagnosis of Capnocytophaga canimorsus sepsis by whole-genome next-generation sequencing. Open Forum Infect. Dis. 3, ofw144 (2016).
Hong, D. K. et al. Liquid biopsy for infectious diseases: sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease. Diagn. Microbiol. Infect. Dis. 92, 210–213 (2018).
Lefterova, M. I., Suarez, C. J., Banaei, N. & Pinsky, B. A. Next-generation sequencing for infectious disease diagnosis and management: a report of the association for molecular pathology. J. Mol. Diagn. 17, 623–634 (2015).
Dunne, W. M. Jr, Westblade, L. F. & Ford, B. Next-generation and whole-genome sequencing in the diagnostic clinical microbiology laboratory. Eur. J. Clin. Microbiol. Infect. Dis. 31, 1719–1726 (2012).
Kim, D. et al. Optimizing methods and dodging pitfalls in microbiome research. Microbiome 5, 52 (2017).
Salter, S. J. et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12, 87 (2014).
Weiss, S. et al. Tracking down the sources of experimental contamination in microbiome studies. Genome Biol. 15, 564 (2014).
Naccache, S. N. et al. The perils of pathogen discovery: origin of a novel parvovirus-like hybrid genome traced to nucleic acid extraction spin columns. J. Virol. 87, 11966–11977 (2013).
Infectious Disease Next Generation Sequencing Based Diagnostic Devices: Microbial Identification and Detection of Antimicrobial Resistance and Virulence Markers. Draft Guidance for Industry and Food and Drug Administration Staff (Food and Drug Administration, 2016).
Chang, C. P. et al. Elevated cell-free serum DNA detected in patients with myocardial infarction. Clin. Chim. Acta 327, 95–101 (2003).
Lo, Y. M., Rainer, T. H., Chan, L. Y., Hjelm, N. M. & Cocks, R. A. Plasma DNA as a prognostic marker in trauma patients. Clin. Chem. 46, 319–323 (2000).
Vincent, J. L., Martinez, E. O. & Silva, E. Evolving concepts in sepsis definitions. Crit. Care Nurs. Clin. North. Am. 23, 29–39 (2011).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinform. 10, 421 (2009).
Xia, L. C., Cram, J. A., Chen, T., Fuhrman, J. A. & Sun, F. Accurate genome relative abundance estimation based on shotgun metagenomic reads. PLoS ONE 6, e27992 (2011).
Bennett J. E., Dolin, R. & Blaser, M. J. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases 8th edn (Saunders, Philadelphia, 2015).
Burnham, P. et al. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma. Sci. Rep. 6, 27859 (2016).
Kalia, V. C. et al. Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium. BMC Genomics 12, 18 (2011).
Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline—Second Edition. NCCLS document EP5-A2 (NCCLS, 2004).
Dellinger, R. P. et al. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock, 2012. Intensive Care Med. 39, 165–228 (2013).
Bagdasarian, N., Rao, K. & Malani, P. N. Diagnosis and treatment of Clostridium difficile in adults: a systematic review. JAMA 313, 398–408 (2015).
Focosi, D., Antonelli, G., Pistello, M. & Maggi, F. Torquetenovirus: the human virome from bench to bedside. Clin. Microbiol. Infect. 22, 589–593 (2016).
Karius, Inc. Pathogen List https://www.kariusdx.com/pathogen-list/3.1.1 (2018).
The authors would like to thank S. Sinha for assistance with the preparation of the manuscript, as well as H. Quach, R. Davila, S. Madan, V. Baichwal, C. Ho, H. Seng, R. Aquino, A. Parham, R. Mann, I. Brown, P. Callagy, A. Visweswaran, C. Keller, C. Bucsit and A. Araya for their contributions to these validation studies.
This study was funded by Karius, Inc. and describes the validation of a product developed by Karius, Inc. All authors (excepting S.T., J.V.Q. and S.Y.) are current or former employees and/or share -holders of Karius, Inc. This does not alter our adherence to Nature Microbiology policies on sharing data and materials.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figures 1–9, Supplementary Tables 1–4 and Supplementary Table 8.
Subjects with definite and probable infections identified by NGS.
Aetiology of infection for patients with positive NGS results adjudicated as possible.
Subjects with ‘unlikely’ infections identified by NGS.
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Blauwkamp, T.A., Thair, S., Rosen, M.J. et al. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol 4, 663–674 (2019). https://doi.org/10.1038/s41564-018-0349-6
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