INNOVATION,%20ARRAYS%20AND%20INFECTIONS:%20E%20Pluribus%20Unum

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INNOVATION, ARRAYSAND INFECTIONSE Pluribus Unum

• James Versalovic, M.D., Ph.D., FCAP
• Director, Division of Molecular Pathology
• Director, Microbiology Laboratories
• Texas Childrens Hospital
• Associate Professor of Pathology
• Baylor College of Medicine

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Texas Medical Center, Houston
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Transplantation
Infectious Diseases
Solid Tumors, Sarcomas
Coagulopathies Hemoglobinopathies
Leukemias, Lymphomas
Patient Sample Blood, Tissue
Neurological disorders, Neuropathology
Drug Metabolism
Molecular Pathology
Translational research
Training
Diagnosis
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Slow Roads to FreewaysE Pluribus Unum

• From Many, One
• Many DNA Probes One Signal
• Many Questions One Answer
• One Diagnosis

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THE PROBLEM Common Challenges in Diagnosing
Human Infections

• Variety of Etiologies to Consider
• Bacterial, Viral, Parasitic
• New, Re-Emerging, or Under-Appreciated Agents
• Colonization versus Infection
• Human Microbiota and Microbiome
• Bacteria and Viruses
• Example of C. difficile Colonization in Infants
• Limitations of Current Stool-Based Strategies
• Bacteriologic Culture
• Antigen Detection

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The Human Microbiome Project The Indigenous
Microbiota and Metagenomics
Eckburg P et al. Science (2005)
3081635-1638 gt60 novel bacteria gt80
nonculturable bacteria
Mixed Microbial Communities
Human Colon 800-1000 species Firmicutes Bacteroide
tes
Firmicutes include Lactobacillus spp.
S. Macfarlane et al. Appl Environ Microbiol
(2005) 717483-7492.
Turnbaugh PJ et al. Nature (2007) 449804-810.
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• Childrens Hospital and Regional Medical Center,
  Emergency Medicine Department, Seattle, USA
• Children all ages
• Including early adulthood (gt20 yo)
• Children presenting with diarrhea
• Stool specimen or rectal swab
• 4799 patients discharged with diagnosis of
  diarrhea or gastroenteritis
• 1626 samples/patients enrolled in study (33.9)
• 47 of stool samples that underwent complete
  testing yielded a specific etiologic agent
• 372 samples with sufficient quantities of
  material for complete testing (bacterial, viral,
  and parasitic)

EJ Klein et al. (2006) Clin. Infect Dis
43807-813.
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Diagnosis of Acute Gastroenteritis in Children,
by Etiologic Agent
STEC and Salmonella were most common bacterial
pathogens Did not look for EPEC or ETEC
Viral detection for adenovirus, astrovirus,
rotavirus by EIA did not look for norovirus
EJ Klein et al. (2006) Clin Infect Dis 43807-813
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Challenge Diagnosis of Respiratory Tract
Infections

• Continually expanding repertoire of respiratory
  viruses
• Complexity of bacterial colonization in
  ventilator-associated infections

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BACTERIAL/FUNGAL IDENTIFICATION
DNA SEQUENCING Today
PYROSEQUENCING Today
ARRAYS AND MICROARRAYS Have arrived
REAL-TIME PCR Today
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SOLUTIONS Arrays and Microarrays

• Arrays refer to any parallel detection system
  that facilitates multi-analyte detection with
  sets of antibodies or probes
• Liquid bead arrays
• Microarrays refer to solid phase-based high
  density arrays
• Highly parallel technologies
• Opportunities for multi-analyte (multi-gene,
  multi-pathogen, and multi-mutation) detection in
  clinical specimens

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Liquid Bead Arrays
Antibodies
Proteins
Peptides
Oligonucleotides
Luminex-100
Panomics Inc.
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Respiratory viral panel (Luminex) was
FDA-approved on Jan. 3, 2008
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Liquid Bead Arrays Different Molecular
Strategies
TSPE
Different Methods for Linking DNA Targets with
Probes/Beads Signal Generation by Fluorescence
(Flow Cytometry)
RMA
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Array-Based Respiratory Virus Detection

• Liquid bead arrays by Respiratory Multi-code-PLx
  Assay (RMA) and microsphere flow cytometery
  (Luminex)
• RMA detected respiratory viruses in 71.8 versus
  23.3 of clinical specimens by DFA and viral
  culture
• Nasal wash samples from 5 year-old children

Lee W-M et al. (2007) J Clin Microbiol
452626-2634
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Array-Based Respiratory Virus Detection

• Liquid bead arrays by target-specific primer
  extension (TSPE) and microsphere flow cytometry
  (RVP Luminex)
• The RVP test yielded 98.5 sensitivity versus
  68.8 of clinical specimens by DFA and viral
  culture
• Nasopharyngeal swabs from regional virology lab
  (mostly adults)

Mahony J et al. (2007) J Clin Microbiol
452965-2970
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Sepsis and Bloodstream Pathogens Molecular
Strategies

• POSITIVE BLOOD CULTURES

Real-Time PCR
Gram Stain
Pyrosequencing
Liquid Bead Arrays
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Anticipated Workflow of Sepsis Panel
Positive Blood Culture
Gram Stain and Subculture
Bacterial DNA Extraction (manual and automated)
DNA Amplification
(16S rRNA V1, V3, and V6 regions)
Hybridization with Luminex DNA beads
(29 beads 20mer probes)
Detection on Luminex100
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Bacterial Pathogen Identification by Array-Based
Detection of 16S rRNA Gene Sequences
V1
V3
V6
C3
C4
C5
C6
C7
C8
C9
V2
V4
V5
V7
V8
V9
C1
C2
Primers
Primers
Primers
Sequencing
Sequencing
Sequencing
primer
primer
primer

• Ribosomal RNA genes are universal and highly
  conserved.
• These genes contain variable regions with
  specific sequence targets
• that enable bacterial pathogen identification.

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Organism Detection by Liquid Bead Arrays
Yellow positive signals specific for pathogenic
genus or species
16S rRNA V3-based bead arrays detected

• Enterococcus sp.
• Staphylococcus aureus
• Staphylococcus epidermidis
• Streptococcus mitis/pneumoniae

• Streptococcus mutans
• Pseudomonas aeruginosa
• Escherichia coli
• Enterobacter cloacae/ Klebsiella pneumoniae

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DNA Microarrays for Pathogen Detection in a
Microbial World
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Virus Discovery and Detection

• Microarray-based hybridization for viral
  detection in human specimens
• Computer algorithms can automate viral
  identification
• Comparing hybridization patterns with theoretical
  energy profiles

Urisman A. et al (2005) Genome Biology 6R78
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The ViroChip

• Microarray-based viral detection
• 70mer spotted arrays
• ViroChip microarray recognizes greater than 140
  viruses
• Human rhinovirus detected post-infection (panel
  A)
• Nasal lavage
• Human rhinovirus detection in natural colds
  (panel B)
• Nasal lavage
• Human parainfluenza virus 1 detected (panel C)
• Nasal lavage

Wang D et al. (2002) Proc Natl Acad Sci
9915687-15692.
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• PhyloChip high-density oligonucleotide
  microarray for bacterial detection
• More than 8,000 taxa / chip
• At least 11 probes per taxon
• 16S rRNA gene sequencing detected reduction from
  16.2 to 5.6 (mean number of) bacterial species
  with antibiotic therapy

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• Bacterial PhyloChip studies in human
  endotracheal aspirates
• intubated ICU patients
• Sampling at beginning of parenteral antibiotic
  versus 4-10 days of therapy
• Loss of bacterial diversity was correlated with
  ventilator-associated pneumonia during antibiotic
  therapy

Flanagan JL et al. (2007) J Clin Microbiol
451954-1962.
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Diagnostic Pathology in the Era of Molecular and
Personalized Medicine

• Multiparameter Diagnostics
• Predict disease susceptibility
• Stratify disease by pathogen and host immune
  response
• Stage disease
• Monitor treatment response
• Predict response to therapy

Weston AD and Hood L. Systems biology,
proteomics, and the future of health care toward
predictive, preventative, and personalized
medicine. J Proteome Res 2004 3179196.
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Molecular Diagnostics The Next 10-15 Years

• Blood diagnostic window for disease analysis
• Multiparameter testing 1000(s) markers
  measured and analyzed simultaneously
• Highly parallel diagnostic testing
• Gene expression and proteomic profiling of body
  fluids, tissues, and single cells
• Clinical genome sequencing (1000 lt one hour)
• Nanotechnology-based diagnostics

Hood L, et al. Systems biology and new
technologies enable predictive and preventative
medicine. Science 2004 306640643. Weston AD and
Hood L. Systems biology, proteomics, and the
future of health care toward predictive,
preventative, and personalized medicine. J
Proteome Res 2004 3179196.
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Conclusions and Future Trends

• Targeted molecular methods will continue to be
  useful for common infections
• Real-time PCR
• Viral load testing
• Shift to multi-analyte and global molecular
  strategies highly parallel diagnostic
  strategies
• Solid Phase Microarrays
• Liquid Bead Arrays
• Gene expression, proteomic or metabolomic
  profiling of host immune responses
• Predict disease susceptibility
• Follow patterns of acute or chronic infections
• Examine patterns of treatment response or recovery

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Acknowledgments
Ruth Ann Luna
Renee Webb
Milton J. Finegold, TCH Pathology Sherry Dunbar,
Luminex Corp. Grant-A-Starr Foundation
Ebony Courtney
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TRANSFORMING PATHOLOGYEmerging technology
driving practice innovation