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

2

“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

3

“HOUSTON, WE’VE HAD A PROBLEM.”

4

“HOUSTON, WE’VE HAD A PROBLEM.”

Four Leading Causes of Morbidity and Mortality:

• Heart Disease

• Stroke

• Cancer

• Infection

5

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

6

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

Sir William Osler (1849 – 1919)

DIAGNOSIS: KEY TO EFFECTIVE TREATMENT

7

Hypothesis testing:

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

(Laboratory Medicine)

DIAGNOSIS: A SCIENTIFIC APPROACH

8

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

(Molecular Microbiology Laboratory)

DEPARTMENT OF LABORATORY MEDICINE

9

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

(Molecular Microbiology Laboratory)

DEPARTMENT OF LABORATORY MEDICINE

Google: UW Medical Laboratory Science

10

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

11

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!

12

• If it grows, it can usually be identified

TRADITIONAL APPROACH

13

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

• blood, urine, CSF• Microscopic examination

TRADITIONAL APPROACH

14

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

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

TRADITIONAL APPROACH

15

ISOLATE & AMPLIFY

16

ISOLATE & 109 AMPLIFICATION

(Google: Scale of universe;Powers of Ten)

17

IDENTIFY ETIOLOGICAL AGENTS

Phenotype

Gene products

(physical expression of genotype)

• Identification• Antibiotic resistance

(proteins, enzymes, complex structures)

18

IDENTIFY ETIOLOGICAL AGENTS

Phenotype

Gene products

Genotype

(physical expression of genotype)

• Identification• Antibiotic resistance

(proteins, enzymes, complex structures)

(blueprint for phenotype)

19

• 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

20

• 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

21

IDENTIFY ETIOLOGICAL AGENTS

Phenotype

Gene products

Genotype

(physical expression of genotype)

• Identification• Antibiotic resistance

(proteins, enzymes, complex structures)

(blueprint for phenotype)

22

GENOMIC APPROACH: ISOLATE & AMPLIFY

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

23

GENOMIC APPROACH: ISOLATE & AMPLIFY

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

24

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

CASE 1

25

“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

26

23-yr-old man with refractory seizures

CASE 2

MRI: Vasogenic edema Gadolinium enhancing

27

• 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

28

CASE 3

PCR and DNA sequencing were performed.

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

29

GENOMIC APPROACH: ISOLATE & AMPLIFY

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

30

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

31

NORMAL MICROBIOTA

32

POLYMICROBIAL INFECTION

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

33

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…”

34

• 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

35

BACTERIAL VAGINOSIS

36

CYSTIC FIBROSIS

37

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

CASE

38

• 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

39

Mission

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

MOLECULAR MICROBIOLOGY LABORATORY

40

Google: UW Molecular Microbiology