Pathogen Detection Using Cell-free DNA in Body Fluids. Wei Gu, MD, PhD Clinical Instructor University of California, San Francisco 10/18/18
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1 Pathogen Detection Using Cell-free DNA in Body Fluids Wei Gu, MD, PhD Clinical Instructor University of California, San Francisco 10/18/18
2 Disclosures No financial conflict of interest Testing discussed may be pursued as a laboratory-developed test (LDT) validated in the CLIA-certified UCSF Clinical Microbiology Laboratory and is not FDA-approved. Testing may be marketed and sold by UCSF.
3 Intro Case: 33 year old woman transferred from outside hospital 2 weeks productive cough -> respiratory failure. All blood culture and respiratory cultures were negative at UCSF.
4 Cell-free (cf)dna Workflow Low Microbial Background: eg x less Propionibacterium acnes
5 BAL plasma NGS Miseq: NGS Results BAL cell-free DNA: reads / million (RPM) to Haemophilus influenza Plasma cell-free DNA : 69 reads / million to Haemophilus influenza Subsequently, 1 of 4 blood culture taken before antibiotics at OSH was reportedly positive for H. influenza. Also confirmed by 16S sequencing on BAL.
6 Local Body Fluid vs. Plasma
7 Nanopore NGS Results BAL: RPM 4159 (Hiseq: 11096) Plasma: RPM 37 (Hiseq: 69)
8 mngs Vs. 16S (UPCR) Case A Case B
9 Vs. 16S - Case A 3 year old boy with necrotizing pneumonia 16S - Pleural Fluid: Streptococcus mitis group Improved on vancomycin/meropenem
10 Vs. 16S - Case A Illumina Hiseq Pleural Fluid cfdna WGS -> SURPI:
11 Vs. 16S - Case A Oxford Nanopore
12 Fluorescence Signal Fluorescence Signal Digital PCR Verification Samples 1-10 Samples 1-10 Klebsiella pneumoniae Streptococcus mitis
13 Vs. 16S - Case B ~70 year old man with fever and encephalopathy, placed on empiric antibiotics. Has Deep Brain Stimulator implant for Parkinson s. CSF: WBC 28->760 in 1 month. 16S, ITS sequencing on CSF: negative 2X
14 Vs. 16S - Case B CSF 2 days prior to implant removal: 16S sequencing: negative Enterobacter aerogenes: 316 RPM
15 Vs. 16S Case B Oxford Nanopore
16 Vs. 16S - Case B Brain surgery to remove infected implant Culture: Klebsiella (Enterobacter) aerogenes
17 Fluorescence Signal Digital PCR Verification Klebsiella aerogenes Samples 1-10
18 Bonus Cancer Diagnostics
19 Copy Number Variation Patient Sample (CNV) Detection Reference Sample 99% of data is human! 19
20 CNV of Normal Control Plasma cfdna Tissue FFPE
21 Plasma: Metastatic Neuroendocrine Plasma Cancer Tissue
22 Conclusions Same Sample Same Data One test, any body fluid Pathogens: High Signal, Low Microbial Contamination Cancers: Suggested by Abundant Copy Number Variations 22
23 Acknowledgements Infectious Disease Wayne Deng, Marco Lee Yasemin Sucu, Shaun Arevalo, Allan Gopez, Dianna Ng, Asmeeta Achari, Kevin Reyes, Jeffrey Whitman, Lillian Khan, Eric Chow Kelsey Zorn, Hannah Sample Scot Federman, Doug Stryke CLS s in Micro, Heme, Chem,Immuno Charles Chiu, Joe DeRisi, Steve Miller Oncology Eric Talevich, Iwei Yeh Kelsey Zorn, Hannah Sample Yasemin Sucu, Shaun Arevalo, Allan Gopez CLS s in Micro, Heme, Chem, Immuno Jeff Simko, Michael Wilson, Charles Chiu, Steve Miller, Joe DeRisi, Linlin Wang, John Greenland, Patrick Devine
24 Acknowledgements Funding NIH K08 CA (Gu) NIH R01 HL105701, R21 AI (Chiu) Chan Zuckerberg Initiative / Biohub California Initiative to Advance Precision Medicine Charles and Helen Schwab Foundation George and Judy Marcus Innovation Fund Sandler Foundation UCSF Medical Center (Clinical Laboratories) UCSF Department of Laboratory Medicine
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