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

“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 2

“HOUSTON, WE’VE HAD A PROBLEM.” 3

Four Leading Causes of Morbidity and Mortality: Heart Disease Stroke Cancer Infection 4

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 5

“Listen to your patient, he is telling you the diagnosis.” Sir William Osler (1849 – 1919) DIAGNOSIS: KEY TO EFFECTIVE TREATMENT 6

Hypothesis testing: History Physical Exam (imaging) Laboratory Testing (Laboratory Medicine) DIAGNOSIS: A SCIENTIFIC APPROACH 7

Chemistry Hematology Transfusion Medicine Immunology Microbiology Virology Medical Informatics Molecular Diagnosis Program (Molecular Microbiology Laboratory) DEPARTMENT OF LABORATORY MEDICINE 8

Chemistry Hematology Transfusion Medicine Immunology Microbiology Virology Medical Informatics Molecular Diagnosis Program (Molecular Microbiology Laboratory) DEPARTMENT OF LABORATORY MEDICINE 9 Google: Google: UW Medical Laboratory Science

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 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 What you can’t see, can kill you!

growsidentified If it grows, it can usually be identified TRADITIONAL APPROACH 12

growsidentified If it grows, it can usually be identified Acquire patient specimen Acquire patient specimen blood, urine, CSF blood, urine, CSF Microscopic examination Microscopic examination TRADITIONAL APPROACH 13

growsidentified If it grows, it can usually be identified Acquire patient specimen Acquire patient specimen blood, urine, CSF blood, urine, CSF Microscopic examination Microscopic examination Isolate Isolate Amplify Amplify TRADITIONAL APPROACH 14

ISOLATE & AMPLIFY 15

ISOLATE & 10 9 AMPLIFICATION 16 Google (Google: Scale of universe; Powers of Ten)

IDENTIFY ETIOLOGICAL AGENTS 17 Phenotype Gene products (physical expression of genotype) Identification Antibiotic resistance (proteins, enzymes, complex structures)

IDENTIFY ETIOLOGICAL AGENTS 18 Phenotype Gene products Genotype (physical expression of genotype) Identification Antibiotic resistance (proteins, enzymes, complex structures) (blueprint for phenotype)

growsidentified 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 19

growsidentified 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 usually If it grows, it can usually be identified TRADITIONAL APPROACH 20

IDENTIFY ETIOLOGICAL AGENTS 21 Phenotype Gene products Genotype (physical expression of genotype) Identification Antibiotic resistance (proteins, enzymes, complex structures) (blueprint for phenotype)

GENOMIC APPROACH: ISOLATE & AMPLIFY 22 Target: Species-identifying DNA sequences flanked by conserved primer binding sites

GENOMIC APPROACH: ISOLATE & AMPLIFY 23 Target: Species-identifying DNA sequences flanked by conserved primer binding sites

79-yr-old male Soft-tissue excision Inflammatory tissue No microbial elements Cultures were negative CASE 1 24

“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 25

23-yr-old man with refractory seizures CASE 2 26 MRI: Vasogenic edemaGadolinium enhancing

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 27

CASE 3 28 PCR and DNA sequencing were performed. Young person with AML Lymph node biopsy Cultures negative

GENOMIC APPROACH: ISOLATE & AMPLIFY 29 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? GENOMIC APPROACH: ISOLATE & AMPLIFY 30 Target: Species-identifying DNA sequences flanked by conserved primer binding sites

NORMAL MICROBIOTA 31

POLYMICROBIAL INFECTION 32 Clin. Microbiol. Rev. 2012, 25(1):193.

Dr. Toby Russell, assisted by Dr. Beverly Crusher, Genitronic replication of Worf’s new spinal column, Episode #115, Star Trek, The Next Generation NEXT GENERATION SEQUENCING 33 “Sequencing the genome at 10 9 base pairs per second…”

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 34

BACTERIAL VAGINOSIS 35

CYSTIC FIBROSIS 36

35-yr-old male with meningitis Found to have brain abscess CSF cultures were negative Traditional PCR + DNA sequencing revealed polymicrobial infection CASE 37

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 38

Mission Support delivery of the best possible patient care by providing excellence in the laboratory science of diagnosing infectious diseases. MOLECULAR MICROBIOLOGY LABORATORY 39

40 Google: Google: UW Molecular Microbiology