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7/3/18 1 THE EMERGENCE OF PATHOGEN GENOMICS IN DIAGNOSTIC LABORATORIES ERIN H. GRAF, PHD, D(ABMM) DIRECTOR, INFECTIOUS DISEASE DIAGNOSTICS LABORATORY 2 OVERVIEW Metagenomics in Infectious Disease Diagnostics Metagenomics in Outbreak Investigation Workforce Development Strategies 3 EVOLUTION OF CULTURE-INDEPENDENT DIAGNOSTICS Relatively unbiased Inexpensive Slow Insensitive Extremely sensitive Fast Biased Limit to # targets Unbiased All pathogens in one test Expensive* Culture Molecular (e.g. PCR) Metagenomics 4 METAGENOMICS Taxonomic (genetic) composition of a community Dependent upon sampling Not a novel field Environmental Micro/Ecology Wooley et al, PLoS Comput Biol 2010 5 METAGENOMICS Clinical Microbiology Descriptive Pathogen Discovery Diagnostics Library Preparation Extract and Shear 6 BIOINFORMATIC ANALYSIS: TAXONOMIC ASSIGNMENT S. aureus Staphylococcus Firmicutes Human Each of these equals 1 read Sum of reads= Metagenomic Data Metagenomics

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Page 1: ASCLS talk · OVERVIEW •Metagenomics in Infectious Disease Diagnostics •Metagenomics in Outbreak Investigation •Workforce Development Strategies 3 EVOLUTION OF CULTURE-INDEPENDENT

7/3/18

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THE EMERGENCE OF PATHOGEN GENOMICS IN DIAGNOSTIC LABORATORIES

ERIN H. GRAF, PHD, D(ABMM)DIRECTOR, INFECTIOUS DISEASE DIAGNOSTICS LABORATORY

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OVERVIEW

• Metagenomics in Infectious Disease Diagnostics• Metagenomics in Outbreak Investigation• Workforce Development Strategies

3

EVOLUTION OF CULTURE-INDEPENDENT DIAGNOSTICS

Relatively unbiased Inexpensive

SlowInsensitive

Extremely sensitiveFast

BiasedLimit to # targets

UnbiasedAll pathogens in one test

Expensive*

Culture

Molecular (e.g. PCR)

Metagenomics

4

METAGENOMICS

• Taxonomic (genetic) composition of a community• Dependent upon sampling

• Not a novel field

EnvironmentalMicro/Ecology

Wooley et al, PLoS Comput Biol 2010

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METAGENOMICS

Clinical Microbiology

DescriptivePathogenDiscovery Diagnostics

Library PreparationExtract and Shear

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BIOINFORMATIC ANALYSIS: TAXONOMIC ASSIGNMENT

S. aureus

Staphylococcus

Firmicutes

Human

Each of these equals 1 read

Sum of reads= Metagenomic Data

Metagenomics

Page 2: ASCLS talk · OVERVIEW •Metagenomics in Infectious Disease Diagnostics •Metagenomics in Outbreak Investigation •Workforce Development Strategies 3 EVOLUTION OF CULTURE-INDEPENDENT

7/3/18

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METAGENOMICS FOR ID DIAGNOSTICS

Goal: Improve pathogen detection in culture-negative tissues

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METAGENOMICS FOR ID DIAGNOSTICS

Goal: Improve pathogen detection in formalin-fixed tissuesIssues with reference method:• Inhibition of PCR

• Metagenomics can detect lower levels of pathogen DNA • Smaller/many amplicons will improve sensitivity

• Cross-amplification of human DNA• Bioinformatic subtraction of human DNA will improve pathogen

detection• Contamination from many sources

• Metagenomics can decode mixed sequences

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TARGETED METAGENOMICS

Bioinformatics• Commercially available pipeline

Sanger (reference method)

Multi Locus-16s-NGS:Pool APool B

Start with culture-positive fixed tissues

NEGATIVE CONTROL (FIXED TISSUE, NO INFECTION)

• Controls have LOTS of bacterial sequence• Reagents• Fixation process• Normal flora

• High diversity

CULTURE POSITIVE FIXED TISSUE

• Lung with Gordonia sp.

• Sequence now dominated by pathogen

Input OneCodex picture

Page 3: ASCLS talk · OVERVIEW •Metagenomics in Infectious Disease Diagnostics •Metagenomics in Outbreak Investigation •Workforce Development Strategies 3 EVOLUTION OF CULTURE-INDEPENDENT

7/3/18

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TARGETED METAGENOMICS OUTPERFORMS REFERENCE METHOD

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Source Culture result Reference method result

Metagenomics result % pathogen reads per total

Heart valve S. aureus S. aureus S. aureus 63.8%

Heart valve Viridans Strep Acinetobacter sp. Strep mutans 29%

Heart valve Corynebacterium sp. neg Corynebacterium sp. 54.5%

Heart valve S. aureus S. aureus S. aureus 83.3%

Heart valve Viridans Strep Viridans Strep Streptococcus mitis/oralis 70.3%

Stomach tissue Campylobacter jejuni neg Campylobacter jejuni 46.2%

Lung tissue Gordonia sp. neg Gordonia sp. 76.6%

Synovium E. coli neg E. coli 83.3%

Synovium Aggregatibacteraphrophilus

neg A. aphrophilus 2% by manual analysis

Larynx tissue Strep mitis/oralis P. acnes Strep mitis/oralis 31.3%

Leg tissue Clostridium sp. Clostridium sp. Clostridium sp. 87.8%

Skin tissue S. aureus neg S. aureus 83.4%

Leg tissue S. aureus neg S. aureus 38.3%

Metagenomics = 100% sensitiveReference method = 31% sensitive

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CULTURE NEGATIVE FIXED TISSUE: CASE EXAMPLE• 15 year old with a chronic submandibular draining lesion• Abscess formation reported by Pathologist• Multiple negative cultures• Treated with Penicillin and Clindamycin• ID ready to treat for MAI

• 6 months of Clarithromycin and Rifampin

>56% of reads

Prevotella

26% S to Pen47% S to Clinda 16

TARGETED METAGENOMICS FOR DIAGNOSTICS• Great way to get our technologists’ “feet wet” with NGS

• Small data files • Only looking for bacteria

• Option for specimens not submitted for culture• Improved yield in culture-negative cases• Informatically subtract “noise” to clean up reporting

• Evaluating bioinformatic approaches • Expand to fungal and mycobacterial targets

• Amplicons can be pooled and sequenced with bacterial

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METAGENOMICS IN OUTBREAK INVESTIGATION

Goal: Support Infection Prevention and Control team through molecular investigation

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CASE EXAMPLE• Increased PCR positivity for Adenovirus noted in the Neonatal

Intensive Care Unit• Babies had respiratory symptoms and/or conjunctivitis prompting

panel testing

0

5

10

15

20

25

NICU PICU ED Oncology 8 South 4W CSH

Adenovirus (+) by unit for the prior week

Hospital AcquiredCommunity Acquired

Page 4: ASCLS talk · OVERVIEW •Metagenomics in Infectious Disease Diagnostics •Metagenomics in Outbreak Investigation •Workforce Development Strategies 3 EVOLUTION OF CULTURE-INDEPENDENT

7/3/18

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CASE EXAMPLE• Infection control discovered that all babies had received a recent

ophthalmologic exam• Cleaning protocol was in place prior to this outbreak for any equipment

touching the babies• All equipment was swabbed and tested for Adenovirus by PCR• Headsets and hand-held lenses test positive-

this equipment does not come in to contactwith the babies

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METAGENOMICS FOR OUTBREAK INVESTIGATION

• Shotgun sequence clinical and ophthalmologic equipment samples• Generate whole Adenovirus genome• 35,000 base pairs

• Compare whole genome sequences between cases and controls from same time period

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Reference genomeCoverage depth

Consensus sequence

METAGENOMICS FOR OUTBREAK INVESTIGATION

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METAGENOMICS FOR OUTBREAK INVESTIGATION Ophthalmologic exam lens

NICU 9 NICU 7 NICU 5 NICU 3 NICU 16 NICU 14 NICU 12 NICU 10 HCW 2 HCW 1

AdV 3 Ref Seq NICU 8 NICU 6 NICU 4 NICU 2 NICU 15 NICU 13 NICU 11 NICU 1

Control 2 Control 1

Control 4 Control 3

AdV 6 Ref Seq AdV 2 Ref Seq

0.5

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METAGENOMICS FOR OUTBREAK INVESTIGATION

Outcome of investigation:• No secondary cases of transmission!!!• Changes to exam procedure• Disinfect all equipment with bleach before first patient exam, and in

between each patient• Lenses must be stored in a cleanable container

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PROMISE AND CHALLENGES OF METAGENOMICS• Sequencing has many applications in clinical microbiology• Diagnostics• Infection Control• Pathogen Discovery

• NGS approaches are in their infancy• Massive amounts of data• Contamination vs. flora vs. pathogen• Antimicrobial susceptibility

• Lack of standardization and limited validation guidance• FDA draft guidance• ASM clinical validation guidance

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7/3/18

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OUR APPROACH TO WORKFORCE DEVELOPMENT

Wet bench• Hands-on library preparation training for staff from vendor• Shadowing Genomics laboratory procedures

Dry bench (bioinformatics)• Sent technologists to 3-day workshop on bioinformatics software• Sent technologists to 3-day workshop at the Institute for Genome

Sciences• Sending technologist to ASM NGS workshop

Easy gap to fill

Hard gap to fill

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BIOINFORMATICS- CLOSING THE GAP

• Staff buy-in is easy• Advanced skills• High profile clinical cases

• Commercially available pipelines• Train wet bench staff to be bioinformaticians?• Need working knowledge for interpretation- contamination vs. flora vs.

pathogen• Will masters and PhD level bioinformaticians become embedded

in lab?

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RESOURCESFree:• Educational webinars (archived)

• APHL/CDC • Illumina and other vendors

• Software demos with tutorials• Manual alignment: Bionumerics, CLC workbench, Geneious• Data-to-answer pipelines: OneCodex, Taxonomer, CosmosID

• Colleagues in Human Genomics Laboratories

Require funding*:• Workshops

• IGS, ASM NGS, Bionumerics28

ACKNOWLEDGEMENTS

• Julia Sammons, MD and CHOP IP&C• Julia Was• Caitlin Dougherty• Jeffrey Fink• Kayla Molitoris• Zahra Kradi

• APHL