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

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“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

FEVER: THE HOST RESPONDS

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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. The determination of the nature of a disease. [G. a deciding] SYN: diacrisis. Diacrisis SYN: diagnosis [G. dia-, through, + krisis, a judgment]

DIAGNOSIS: KEY TO EFFECTIVE TREATMENT

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“Listen to your patient, he is telling you the diagnosis.”

Sir William Osler (1849 – 1919)

DIAGNOSIS: KEY TO EFFECTIVE TREATMENT

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Hypothesis testing:

• History• Physical Exam• (imaging)• Laboratory Testing

(Laboratory Medicine)

DIAGNOSIS: A SCIENTIFIC APPROACH

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• Chemistry• Hematology• Transfusion Medicine• Immunology• Microbiology• Virology• Medical Informatics• Molecular Diagnosis Program

(Molecular Microbiology Laboratory)

DEPARTMENT OF LABORATORY MEDICINE

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• Chemistry• Hematology• Transfusion Medicine• Immunology• Microbiology• Virology• Medical Informatics• Molecular Diagnosis Program

(Molecular Microbiology Laboratory)

DEPARTMENT OF LABORATORY MEDICINE

Google: UW Medical Laboratory Science

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Determine the nature of disease:• Predict course and potential outcome(s) of infection• Tailor therapy• Exclude non-infectious cause(s) of symptoms

IDENTIFY ETIOLOGICAL AGENTS

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Determine the nature of disease:• Predict course and potential outcome(s) of infection• Tailor therapy• Exclude non-infectious cause(s) of symptoms

IDENTIFY ETIOLOGICAL AGENTS

What you can’t see, can kill you!

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• If it grows, it can usually be identified

TRADITIONAL APPROACH

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• If it grows, it can usually be identified• Acquire patient specimen

• blood, urine, CSF• Microscopic examination

TRADITIONAL APPROACH

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• If it grows, it can usually be identified• Acquire patient specimen

• blood, urine, CSF• Microscopic examination• Isolate• Amplify

TRADITIONAL APPROACH

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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

Gene products

(physical expression of genotype)• Identification• Antibiotic resistance

(proteins, enzymes, complex structures)

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IDENTIFY ETIOLOGICAL AGENTS

Phenotype

Gene products

Genotype

(physical expression of genotype)• Identification• Antibiotic resistance

(proteins, enzymes, complex structures)

(blueprint for phenotype)

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• 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?

TRADITIONAL APPROACH

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• 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

TRADITIONAL APPROACH

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IDENTIFY ETIOLOGICAL AGENTS

Phenotype

Gene products

Genotype

(physical expression of genotype)• Identification• Antibiotic resistance

(proteins, enzymes, complex structures)

(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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• 79-yr-old male• Soft-tissue excision• Inflammatory tissue• No microbial elements• Cultures were negative

CASE 1

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“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.

CASE 1

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23-yr-old man with refractory seizures

CASE 2

MRI: Vasogenic edema Gadolinium enhancing

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• 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.

CASE 2

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CASE 3

PCR and DNA sequencing were performed.

• Young person with AML• Lymph node biopsy• Cultures negative

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GENOMIC APPROACH: ISOLATE & AMPLIFY

Target: Species-identifying DNA sequences flanked by conserved primer binding sites

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What if…• infection occurs at a site with

normal microbiota? • more than one pathogen is

present?

GENOMIC APPROACH: ISOLATE & AMPLIFY

Target: Species-identifying DNA sequences flanked by conserved primer binding sites

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NORMAL MICROBIOTA

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POLYMICROBIAL INFECTION

Clin. Microbiol. Rev. 2012, 25(1):193.

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Dr. Toby Russell, assisted by Dr. Beverly Crusher, 2368. Genitronic replication of Worf’s new spinal column, Episode #115, Star Trek, The Next Generation

NEXT GENERATION SEQUENCING

“Sequencing the genome at 109 base pairs per second…”

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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!

NGS: ISOLATE & AMPLIFY

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BACTERIAL VAGINOSIS

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CYSTIC FIBROSIS

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• 35-yr-old male with meningitis• Found to have brain abscess• CSF cultures were negative• Traditional PCR + DNA sequencing revealed polymicrobial infection

CASE

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• 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

CASE

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Mission

Support delivery of the best possible patient care by providing excellence in the laboratory science of diagnosing infectious diseases.

MOLECULAR MICROBIOLOGY LABORATORY

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Google: UW Molecular Microbiology