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
• 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
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
• 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
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
Abril, et al. OFID 2016. 3(3).
Results from NGS
Abril, et al. OFID 2016. 3(3).
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
Abril, et al. OFID 2016. 3(3).
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
“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
• 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
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???!!!
Wilson, Michael R., et al. New England Journal of Medicine 370.25 (2014): 2408-2417.
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
Wilson, Michael R., et al. New England Journal of Medicine 370.25 (2014): 2408-2417.
Data Analysis
Wilson, Michael R., et al. New England Journal of Medicine 370.25 (2014): 2408-2417.
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!
Wilson, Michael R., et al. New England Journal of Medicine 370.25 (2014): 2408-2417.
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
• Describe next-generation sequencing technologies
• Technology that produces large amounts of DNA sequence data in one run
• Summarize applications of next-generation sequencing in the clinical microbiology laboratory
• 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