Next-generation Sequencing in the Clinical Microbiology ...the Clinical Microbiology Lab: Experiences at UCLA Holly Huse, Ph.D., M(ASCP)CM Clinical Microbiology Post-doctoral fellow

Next-generation Sequencing in the Clinical Microbiology Lab:

Experiences at UCLAHolly Huse, Ph.D., M(ASCP)CM

Clinical Microbiology Post-doctoral fellow

Department of Pathology and Laboratory Medicine

University of California, Los Angeles

Disclosures

• I have no financial relationships with any commercial interests related to the content of this talk.

Objectives

• Describe next-generation sequencing technologies

• Summarize applications of next-generation sequencing in the clinical microbiology laboratory

• Discuss the implementation of next-generation sequencing in the clinical microbiology lab at UCLA

• Analyze cases in which next-generation sequencing has been used to diagnose infectious diseases

Objectives





Why should I care about next-generation sequencing (NGS)?

NGS Applications in the Clinical Lab

• FDA approved next-generation diagnostic tests:

• Oncomine Dx Target Test• Non-small cell lung cancer• Analyze alterations in a panel of genes that predict treatment

response• Example: Deletion in EGFR exon 19 > respond better Gilotrif®

• FoundationOne CDx™• Ovarian cancer• BRCA1/2 alterations > respond better to Rubraca®

• MiSeqDx Cystic Fibrosis 139-Variant Assay• Identify mutations associated with Cystic Fibrosis

NGS Outside the Lab

History of DNA Sequencing

1953Structure of

DNA discovered

1977Federick Sanger develops

dideoxy sequencing

1995Genome of Haemophilus influenzae

is sequenced

1996Next-generation

sequencing developed

2001Human

genome is sequenced

1996-presentExplosion of high-

throughput techniques for DNA sequencing

The Beginning of DNA Sequencing

• “Sanger sequencing”

Heather, James M., and Benjamin Chain Genomics 107.1 (2016): 1-8.

“... [A] knowledge of sequences could contribute much to our understanding of

living matter.”Frederick Sanger

Two-time Nobel Prize Laureate in

Chemistry

DNA: A Review

• DNA is the basis for heredity

• Consists of two chains twisted around each other > double helix

• Composed of 4 nucleotides: A, T, C, G

• Sequences > genes

• All the genes > genome

Genetic mutations

Sanger Sequencing

• Chain termination dideoxy method

• Make 4 reaction mixtures with fluorescently labeled replication-stopping nucleotides

• Random incorporation of chain-terminating nucleotides during DNA synthesis

Sanger Sequencing

• Thousands of DNA fragments are sorted by size

• Sequential nucleotides are determined by the last incorporated nucleotide

• The sequence is then depicted as a chromatogram

Applications in clinical microbiology

• Sequence single strands of DNA with a high degree of certainty

• Microbial Identification

• Sequence a gene common to all organisms

• 16S ribosomal RNA gene for bacteria

• Housekeeping genes (rpoB, hsp) for Mycobacterium spp.

• ITS, D1/D2 regions for fungi

• Microbial typing

• Sequencing multiple conserved genes to determine relatedness

Typical UCLA Workflow

MALDI-TOF MS

Mycobacterium sp.

Rare or fastidious bacteria

No identificationCulturedisolate

rpoBgene

Why Do We Need New Sequencing Technology?• More demand for sequencing: need a higher throughput

solution

• Increasing need for detection of rare mutations

• Sanger is too expensive: $2,500-$5,000 to sequence one E. coli genome (5,000,000 base pairs)

• “Next-generation sequencing” is the answer!

• Blanket term for sequencing technologies that produce large amount of sequence data in one run

Let’s go over some sequencing terminology!

NGS Terminology Explained With Shotgun Sequencing Workflow

• Shotgun sequencing: need to shear DNA into smaller pieces

Commins, Jennifer, Christina Toft, and Mario A. Fares. Biological procedures online 11.1 (2009): 52.

BANG!

BANG!

DNA to be sequenced

Sheared pieces > librarySequence the library

Reads: ATCG’s of certain lengths

Computer puts together overlapping reads

Contig

NGS Terminology Explained With Shotgun Sequencing Workflow

• Assembly: computer analysis where reads with overlaps are put together into contigs

• Coverage: Number of reads match a particular DNA region

Commins, Jennifer, Christina Toft, and Mario A. Fares. Biological procedures online 11.1 (2009): 52.

Coverage of region in the box = 4

More coverage = Assembly easier and better quality of

sequences

Next-Generation Sequencing: How It Works

Shendure, Jay, and Hanlee Ji. Nature biotechnology 26.10 (2008): 1135-1145.

• This is just one of many different methods

• Large pieces of DNA are fragmented

• Small DNA pieces called adapters are attached to each DNA fragment

• Adapters allow DNA fragments to attach to detection surfaces

• Flow cell

Next-Generation Sequencing: How It Works

• Fragments are distributed onto the detection surface

• PCR performed on the whole surface to generate copies of each immobilized DNA fragment

• 1 fragment is amplified into many identical copies to form “spots”

• Spots are big enough for the instrument to “see”

Next-generation sequencing

• Each “spot” undergoes cyclic sequencing to figure out the sequence

• A,T,C,G are color coded

• Each cycle, one nucleotide is added to each complementary strand at each spot

• Photo is taken and the signal is measured

• Machine recognizes which nucleotide was added based on the color

• Millions of spots are sequenced simultaneously

• At end of run: sequences of millions of fragments!

Advantages of Next-Generation Sequencing Over Sanger Sequencing

• Cost per base sequenced is much lower

• You can sequence 20+ E. coli genomes/run• < $100/genome compared to $5000

• Generates WAY more data in a much shorter time• Illumina MiSeq: up to 50 million reads per run in 65

hours!

• Allows for more complex analyses• De novo assembly (assemble with no reference sequence)

• Variant detection

• Quantitation (copy number comparison)

50 Million Reads?!! The Instrument Must Be Gigantic!

Benchtop sequencers

Illumina MiSeq Illumina MiniSeq

Ion ProtonOxford Nanopore

Objectives





Applications of NGS in Clinical Microbiology

• Culture-dependent

• Whole genome sequencing• Outbreak

investigations

• Identify genes that confer antimicrobial resistance

Applications of NGS in Clinical Microbiology

• Culture-independent

• Sequence DNA directly from a clinical specimen

• Detects all organisms: bacteria, viruses, fungi, and parasites• Culture-negative infections• Non-cultivable organisms• Fastidious organisms

• UCSF: CSF

• Karius: plasma

CSF Plasma

Pneumonia 15-25%

Meningitis & Encephalitis 60-80%

Sepsis > 20%

Endocarditis & Myocarditis10-20%

Diseases with unknown etiologies

Culture-independent

Bacteria, viruses, yeast, molds, protozoa

Unbiased

Antimicrobial susceptibilities

Blood cultureflags positive

Gram stainPhone call

ID

1-3 days…1-2 days…

Traditional Blood Stream Infection Diagnostics

< 1 hour!MALDI-TOF MS

[antibiotic]

Sub-culture

1-6 weeks…

AbR

Strain Typing

Multi Locus Sequence Typing

Serotyping

Pulse Field Gel Electrophoresis Outbreak!

What’s Wrong?•Slow: 3-5 days total

•Wrong empiric therapy = 5x higher mortality1

•25-40% of patients receive ineffective empiric therapy 2

•Up to 60% of septic patients have negative blood cultures

•What about fastidious, uncultivable, or unusual organisms?

1. 2006 Crit Care Med 34:1589

2. Chest 2000:118;146-55

Traditional Bloodstream Infection Diagnostics

Purify DNA Prepare sequencing library

Blood culturebottle

ATGCATCTTAGC

GCGCATCGTACG

GGCAAACGTTTTCAGG

GCAATTCTTCGGGCATT

Raw sequencing readsGCGCATCGTACG

Sequence

GCGCATCGTACG

GCAATTCTTCGGGCATT

DNA sequence analysis

IDAbR

1-12 hours!1-2 days…

NGS Blood Stream Infection Diagnostics

Outbreak!

Direct from specimen

Case Study

• Duke University Medical Center

• 60-year-old man presented to the ER with fever, hypotension, and altered mental status

• Developed chills, vomiting, disorientation, and a dusky discoloration of his face and extremities

• His past medical history was significant for splenectomy in 1974

• No medications, recent travel, or sick contacts

• He suffered several bites and scratches from the family German shepherd

• Quickly progressed to acute respiratory distress and septic shock with kidney failure

Abril, et al. OFID 2016. 3(3).

Timeline of events

Day 1 2 3 4 5 6 23 31 55

LEVO VANC

CFPM

DOXY, ACYV, AMP

PIPTZ

CIPR

Bloodcultures

GNRs inBuffy coat

NGSKarius

NGSresults 1 blood culture +GNRs

Dischargefrom ICU Hospice

16S rRNAPCR results


Results from NGS


Capnocytophaga canimorsus

No β-lactamasedetected

Summary

• C. canimorsus: fastidious Gram-negative bacteria foundin cat and dog saliva

• Causes fulminant sepsis in individuals with dog bites• Greatest risk: asplenia, functional asplenia, cirrhosis

• Traditional methods• MALDI-TOF: unable to provide ID

• 16S rRNA Sanger sequencing: ID confirmed on 3rd attempt

• NGS performed directly from patient specimenprovided results within 24 hours• Culture-independent, unbiased, antibiotic resistance


Objectives





“Superbug” Outbreak at UCLA Medical Center

Carbapenem resistant Enterobacteriaceae (CRE)

Examples of commonly used β-lactam antibiotics

PenicillinsAmoxicillinAmpicillin

β-lactam/β-lactamase inhibitor combinationsAmoxicillin-clavulanate AKA Augmentin

CephalosporinsCefazolin (1st)Cefuroxime (2nd) Ceftazidime (3rd)Ceftriaxone (3rd)Cefepime (4th)

CarbapenemsDoripenemErtapenemImipenemMeropenem

Broadest spectrum of activity

CRE are resistant to all β-lactam agents

Mode of action

Outer Membrane

Periplasm

Inner membrane

Porin Channel

β-lactam

Penicillin binding proteins(PBPs)

Mechanisms of carbapenem resistance

Porin channel obstructed

Usually with ESBL or AmpC Carbapenemase producer

Carbapenemase

β-lactam β-lactam

β-lactamase

KPC, NDM-1, IMP, VIM, SME, OXA-48

Superbugs: Carbapenem resistant Enterobacteriaceae (CRE)

•Resistant to any carbapenem or positive for a carbapenemase

•Urgent public health threat

•50% mortality for serious infections

•Often multi-drug resistant

•Mobile genetic elements carry other antibiotic resistance genes

•Asymptomatic carriers

UCLA CRE Baseline 2009-2015

0

20

40

60

80

100

2009 2010 2011 2012 2013 2014 2015

# P

atie

nts

CRE < 1% of all Enterobacteriaceae

What happened at UCLA?• September 2014-January 2015

• 48-year-old woman with end-stage liver disease due to cirrhosis of unknown cause

• In September, she received a liver transplant at UCLA, which had been complicated by a bile leak

• She had a stent placed by endoscopic retrograde cholangiopancreatography (ERCP)

• Over several weeks, she developed sepsis and an intra-abdominal infection

• CRE isolated from abdominal fluid and blood cultures

• Expired 2 months after her liver transplant

CRE-infected patient

• MALDI-TOF ID: Klebsiella pneumoniae

• Laboratory developed PCR test for carbapenemases:• Negative: KPC, NDM-1, IMP, VIM, SME, OXA-48

• At that time, UCLA was just starting NGS on the Illumina Miseq platform

• This is an interesting isolate:

• Unknown mechanism of carbapenem resistance

• Let’s put this on our next run!

AmikacinGentamicinTobramycin

>32>10>10

RRR

Aztreonam, Cefepime, Cetazidime, Ceftriaxone

>32>32>32>32

RRRR

ErtapenemImipenemMeropenem

>82

>16

RIR

CiprofloxacinLevofloxacin

>2>8

RR

Piper-tazo >128 R

Trim-sulfa >4/80 R

MinocyclineTigecycline

>32 4

RI

Colistin ≤0.5 S

Susceptibilities

Pathogen ID

Strain typing

Antibiotic resistance determination

Data Analysis

Klebsiella pneumoniae

Strain XH209 from China

blaOXA-232!

What is this gene and how did it get here?

Whole genome sequencing

OXA-232

• Novel carbapenemase identified in 2011 in 3 patients who had moved from India to France

• 1st U.S. case: 2013 in Pittsburgh from a patient from India

• Our patient had no significant travel history

• Remember: not identified by our laboratory-developed PCR for carbapenemases

Searching for more OXA-232• Reviewed our PCR

negative CRE

• Designed a PCR to identify OXA-232

• All PCR-negative CRE since first isolate had OXA-232!

• Notified infection prevention

• All subsequent CRE screened using new OXA-232 PCR 0

1

2

3

4

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan

Nu

mb

er o

f p

atie

nts

wit

h C

RE

KPC SME NDM-1 Neg/Unknown

2014 2015

Hemarajata et al. AACC 2015. 59(9): 5574-5580.

Response and Outbreak investigation

• OXA-232 isolates identified from 7 patients

• Linked to exposure to 2 duodenoscopes used in ERCP

• Diagnose and treat problems of the biliary or pancreaticductal systems

Response and Outbreak investigation

• Procedure immediately stopped

• Notified Los Angeles County Public Health

• All scopes sent for routine ethylene oxide sterilization

• Patients who underwent ERCP during the outbreak period were screened for OXA-232

• Total:

• 9 patients with active infection

• 179 patients exposed, 150 screened

• 7 healthy patients with colonization

• 2 deaths attributable to CRE infection

Epidemiological Data Shows that Outbreak Followed Endoscopy Procedures

• Also identified 7 patients that were exposed to scopes A and B that were colonized with OXA-232 CRE (not shown)

Sa

Cx+

SaCx+ Sb

Cx+

Cx+

Cx+

Cx+

Cx+

Cx+

Cx+

Sb

Sb

Sa

Sa

Sa

Sa

Sa

Pt0

Pt1

Pt2

Pt3

Pt4

Pt5

Pt6

Pt7

Pt8

9/2014 10/2014 11/2014 12/2014 1/2015

Hospital stay

Cx+ Culture positive

Sa Scope a

Sb Scope b

Key

• Patient 0: Patient with previous hospital stay in India

Single Nucleotide Variant (SNV) Analysis

• When bacteria replicate, mutations in the genome accumulate over time

• Compare changes to a reference genome > SNVs

• Result: strains with shared SNVs are related

Ref

1

2

3

4

5

Relatedness Based on SNV Analysis

• 32 isolates

• 17 patients

Yang et al. CID 2017. 64: 894-901.

Reference genome

• Only patient exposed to scope B!• Was not exposed through Patient 1

200-100 0 100 300 Days

Patient 1 isolates

24

5

8

7

c6

c7

10

9

66

6

6

c4

c1

c5

c26

6c3

3

0 Index patient

Transmitted from 1 to other patients through scope A

Transmission from 0 1

Summary

• WGS provided the first evidence of the presence of OXA-232 which was missed by our laboratory developed PCR

• WGS allowed for prompt development of a specific screening test for OXA-232 and infection prevention intervention

• SNV and molecular clock analyses provided insight into possible transmission events

Objectives





Case Study

• 68 year old male with a history of lymphoma• Presents to UCLA with altered mental status, fatigue,

and generalized weakness• Vital signs were within normal ranges• Upon admission, his EEG showed abnormal discharge

pattern• Could be infectious or non-infectious• Multiple tests were performed:• Bacterial, fungal, viral serology . . .• For 2 weeks, testing continued with all negative results• CSF was sent for metagenomics testing

NGS results

St. Louis Encephalitis Virus (SLEV)

• Spherical, enveloped RNAvirus

• Flaviviridae• Dengue Virus• West Nile Virus• Yellow Fever• Zika Virus

• Spread by Culexmosquitoes

• Diagnosed by serology

Clinical Manifestations

• Less than 1% of infections are clinically apparent

• Incubation period ranges from 5 to 15 days

• Abrupt onset of fever, headache, dizziness, nausea, and malaise

• Symptoms intensify over a period of several days to a week

• Some patients spontaneously recover after this period

• Other patients develop signs of central nervous system infections, including stiff neck, confusion, dizziness, and tremors

• About 40% of children and young adults develop only fever and headache

• Almost 90% of elderly persons develop encephalitis

• Case-fatality ratio is 5 to 15%

SLEV neuroinvasive disease cases, 2007–2016

Average 7 cases annually

Why is this Result Significant?

• For the patient:• Diagnosis was established• No specific treatment available• Patient expired• Family given a diagnosis

• For public health:• Outbreak studies and surveillance• Mosquito surveillance detected

SLEV a few months previous

• Last known human case of SLEV in Orange County area was in 1984

Case Study

• 14-year-old boy with severe combined immunodeficiency (SCID)

• In July 2013, he presented with headache and fevers, diffuse weakness, myalgias, nausea, and vomiting

• Multiple admissions prior to this with pretty much same symptoms

• 1 year earlier: missionary trip to Puerto Rico• Swam in a river and the ocean• 17-year-old fellow traveler had been hospitalized for 4

days with fever and hematuria• 4 months earlier: vacation in Florida, swam in a pool at

a resort where there were a number of feral cats

Wilson, Michael R., et al. New England Journal of Medicine 370.25 (2014): 2408-2417.

Summary of Hospital Course

• Evidence of meningitis in CSF

• MRI

• Brain biopsy

• All cultures negative

• Treated as non-infectious process with medications to boost his immune system

• No improvement

Dr: What is happening to this poor kid???!!!


Meanwhile…..

• Condition continued to decline with new-onset psychiatric symptoms

• Ventricular drain placed due to worsening hydrocephalus

• Started on Cefuroxime to prevent drain infection

• Placed in a medically induced coma to control new-onset seizures

• Desperate times call for desperate measures!

• Decided to try unbiased metagenomic sequencing directly from CSF


Data Analysis


Leptospirosis

• Bacterial disease that affects humans and animals

• Caused by bacteria of the genus Leptospira, spirochetes

• Spread through the urine of infected animals, which can get into water or soil• Cattle, Pigs, Horses, Dogs, Rodents, Wild animals

• Infected animals may have no signs or symptoms

• Humans can become infected through:• Contact with urine from infected animals• Contact with water, soil, or food contaminated with the

urine of infected animals

Clinical manifestations

• Symptoms:• High fever, Headache, Chills, Muscle aches, Vomiting,

Jaundice, Red eyes, Abdominal pain, Diarrhea, Rash

• Incubation period: 2 days to 4 weeks

• Symptoms may occur in 2 phases: • First phase: fever, chills, headache, muscle aches,

vomiting, or diarrhea

• The patient may recover for a time but become ill again

• If a second phase occurs: kidney failure, liver failure, or meningitis

Diagnosis

• Challenging!

• Dark field microscopy• Visualize organism• Not routinely performed

• Culture• Specialized media• Up to 3 months!

• PCR• Rapid• Urine or serum• Acute illness

• Serology• Antibodies detectable 6-10 days of disease• Reach peak levels within 3-4 weeks

Back to the patient

• Treatment was changed to high-dose penicillin

• Probably acquired while swimming in freshwater in Puerto Rico

• Gradually recovered over the next 7 days

• Discharged in September!


New York Times

Advantages and Disadvantages

• Advantages• Decrease turn

around time

• Unbiased

• Identify uncultivable, fastidious, or novel organisms

• Disadvantages• Culturing isolates

• Normal flora vs. pathogens

• Microbes can evolve novel resistance mechanism

• Cost

• Validation

Key points


• Technology that produces large amounts of DNA sequence data in one run


• Outbreak investigations

• Detection of antimicrobial resistance determinants

• Direct from specimen ID

Key points

• Discuss the implementation of next-generation sequencing in the clinical microbiology lab at UCLA• Identified outbreak of CRE associated with endoscopes• Working with reference labs for direct from specimen ID:

CSF and plasma

• Analyze cases in which next-generation sequencing has been used to diagnose infectious diseases• SLEV from CSF• Leptospirosis, CSF• C. canimorsus, plasma

Acknowledgements

• Dr. Romney Humphries

• Dr. Omai Garner

• Dr. Peera Hemarajata

• Dr. Shaun Yang

• UCLA Clinical Microbiology

Documents

Next-generation Sequencing in the Clinical Microbiology ...the Clinical Microbiology Lab: Experiences at UCLA Holly Huse, Ph.D., M(ASCP)CM Clinical Microbiology Post-doctoral fellow