UW MEDICINE │ PATIENTS ARE FIRST
BATTLING BUGS:
INROADS IN
INFECTIOUS DISEASES
UW MINI-MEDICAL SCHOOL
Brad T. Cookson M.D., Ph.D.
February 11, 2014
FEVER: THE HOST RESPONDS
“Humanity has but three great enemies:
fever, famine and war; of these by far the
greatest, by far the most terrible, is
fever.”
Sir William Osler, 1896
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“HOUSTON, WE’VE HAD A PROBLEM.”
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“HOUSTON, WE’VE HAD A PROBLEM.”
Four Leading Causes of
Morbidity and Mortality:
• Heart Disease
• Stroke
• Cancer
• Infection
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DIAGNOSIS: KEY TO EFFECTIVE TREATMENT
Diagnosis. The determination of the
nature of a disease. [G. a deciding] SYN:
diacrisis.
Diacrisis SYN: diagnosis [G. dia-,
through, + krisis, a judgment]
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DIAGNOSIS: KEY TO EFFECTIVE TREATMENT
“Listen to your patient, he is telling you
the diagnosis.”
Sir William Osler (1849 – 1919)
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DIAGNOSIS: A SCIENTIFIC APPROACH
Hypothesis testing:
•
•
•
•
History
Physical Exam
(imaging)
Laboratory Testing
(Laboratory Medicine)
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DEPARTMENT OF LABORATORY MEDICINE
• Chemistry
• Hematology
• Transfusion Medicine
• Immunology
• Microbiology
• Virology
• Medical Informatics
• Molecular Diagnosis Program
(Molecular Microbiology Laboratory)
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DEPARTMENT OF LABORATORY MEDICINE
• Chemistry
Google: UW Medical
Laboratory Science
• Hematology
• Transfusion Medicine
• Immunology
• Microbiology
• Virology
• Medical Informatics
• Molecular Diagnosis Program
(Molecular Microbiology Laboratory)
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IDENTIFY ETIOLOGICAL AGENTS
Determine the nature of disease:
• Predict course and potential outcome(s)
of infection
• Tailor therapy
• Exclude non-infectious cause(s) of
symptoms
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IDENTIFY ETIOLOGICAL AGENTS
Determine the nature of disease:
• Predict course and potential outcome(s)
of infection
• Tailor therapy
• Exclude non-infectious cause(s) of
symptoms
What you can’t see, can kill you!
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TRADITIONAL APPROACH
• If it grows, it can usually be identified
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TRADITIONAL APPROACH
• If it grows, it can usually be identified
• Acquire patient specimen
• blood, urine, CSF
• Microscopic examination
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TRADITIONAL APPROACH
• If it grows, it can usually be identified
• Acquire patient specimen
• blood, urine, CSF
• Microscopic examination
• Isolate
• Amplify
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ISOLATE & AMPLIFY
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ISOLATE & 109 AMPLIFICATION
(Google: Scale of universe;
Powers of Ten)
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IDENTIFY ETIOLOGICAL AGENTS
Phenotype
(physical expression of genotype)
• Identification
• Antibiotic resistance
Gene products
(proteins, enzymes, complex structures)
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IDENTIFY ETIOLOGICAL AGENTS
Phenotype
(physical expression of genotype)
• Identification
• Antibiotic resistance
Gene products
(proteins, enzymes, complex structures)
Genotype
(blueprint for phenotype)
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TRADITIONAL APPROACH
• If it grows, it can usually be identified
What if…
• it grows very slowly?
• it does not grow in the lab?
• it has disguised usual characteristics?
• it has never been seen before?
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TRADITIONAL APPROACH
• If it grows, it can usually be identified
What if…
• it grows very slowly?
• it does not grow in the lab?
• it has disguised usual characteristics?
• it has never been seen before?
• If it grows, it can usually be identified
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IDENTIFY ETIOLOGICAL AGENTS
Phenotype
(physical expression of genotype)
• Identification
• Antibiotic resistance
Gene products
(proteins, enzymes, complex structures)
Genotype
(blueprint for phenotype)
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GENOMIC APPROACH: ISOLATE & AMPLIFY
Target: Species-identifying DNA sequences
flanked by conserved primer binding sites
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GENOMIC APPROACH: ISOLATE & AMPLIFY
Target: Species-identifying DNA sequences
flanked by conserved primer binding sites
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CASE 1
•
•
•
•
•
79-yr-old male
Soft-tissue excision
Inflammatory tissue
No microbial elements
Cultures were negative
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CASE 1
“The differential diagnosis includes:
• infectious process (possible _______
infection),
• inflammatory process such as rheumatoid
nodule or a
• neoplastic process (epithelioid carcinoma
can present with foci of necrosis, however
the histopathological features do not favor
same).”
• PCR and DNA sequencing were performed
on a PET specimen.
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CASE 2
23-yr-old man with refractory seizures
MRI: Vasogenic edema
Gadolinium enhancing
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CASE 2
• Exhaustive serological testing was
negative
• Cultures were negative
• Surgically excised lesion
• Mixed inflammatory cell infiltrate without
presence of microbial elements
• PCR and DNA sequencing were
performed on a fresh surgical specimen.
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CASE 3
• Young person with AML
• Lymph node biopsy
• Cultures negative
PCR and DNA sequencing
were performed.
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GENOMIC APPROACH: ISOLATE & AMPLIFY
Target: Species-identifying DNA sequences
flanked by conserved primer binding sites
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GENOMIC APPROACH: ISOLATE & AMPLIFY
Target: Species-identifying DNA sequences
flanked by conserved primer binding sites
What if…
• infection occurs at a site with normal
microbiota?
• more than one pathogen is present?
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NORMAL MICROBIOTA
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POLYMICROBIAL INFECTION
Clin. Microbiol. Rev. 2012, 25(1):193.
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NEXT GENERATION SEQUENCING
“Sequencing the genome at 109 base
pairs per second…”
Dr. Toby Russell, assisted by Dr. Beverly
Crusher, 2368. Genitronic replication of
Worf’s new spinal column, Episode #115,
Star Trek, The Next Generation
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NGS: ISOLATE & AMPLIFY
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•
•
•
Capture single DNA molecules
Cluster formation: amplify ~1,000 copies
Parallel DNA sequencing of clusters
Read millions of clusters per flow cell!
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BACTERIAL VAGINOSIS
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CYSTIC FIBROSIS
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CASE
• 35-yr-old male with meningitis
• Found to have brain abscess
• CSF cultures were negative
• Traditional PCR + DNA sequencing
revealed polymicrobial infection
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CASE
• 35-yr-old male with meningitis
• Found to have brain abscess
• CSF cultures were negative
• Traditional PCR + DNA sequencing
revealed polymicrobial infection
• Next Generation Sequencing was
performed
• Antibiotic regimen was optimized
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MOLECULAR MICROBIOLOGY LABORATORY
Mission
Support delivery of the best possible
patient care by providing excellence in the
laboratory science of diagnosing
infectious diseases.
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Google: UW Molecular Microbiology
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