Battling Bugs: Inroads in infectious Diseases

1. Battling Bugs: Inroads in infectious Diseases UW Mini-Medical School Brad T. Cookson M.D., Ph.D. February 11, 2014

2. 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

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: key to effective treatment Diagnosis. The determination of the nature of a disease. [G. a deciding] SYN: diacrisis. DiacrisisSYN: diagnosis[G. dia-, through, + krisis, a judgment]

6. Diagnosis: key to effective treatment “Listen to your patient, he is telling you the diagnosis.” Sir William Osler (1849 – 1919)

7. Diagnosis: a scientific approach Hypothesis testing: • History • Physical Exam • (imaging) • Laboratory Testing (Laboratory Medicine)

8. Department of Laboratory Medicine • Chemistry • Hematology • Transfusion Medicine • Immunology • Microbiology • Virology • Medical Informatics • Molecular Diagnosis Program (Molecular Microbiology Laboratory)

9. Department of Laboratory Medicine Google: UW Medical Laboratory Science • Chemistry • Hematology • Transfusion Medicine • Immunology • Microbiology • Virology • Medical Informatics • Molecular Diagnosis Program (Molecular Microbiology Laboratory)

10. 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

11. 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!

12. Traditional approach • If it grows, it can usually be identified

13. Traditional approach • If it grows, it can usually be identified • Acquire patient specimen • blood, urine, CSF • Microscopic examination

14. Traditional approach • If it grows, it can usually be identified • Acquire patient specimen • blood, urine, CSF • Microscopic examination • Isolate • Amplify

15. isolate & amplify

16. isolate & 109 amplification (Google: Scale of universe; Powers of Ten)

17. Identify etiological agents Phenotype • Identification • Antibiotic resistance (physical expression of genotype) Gene products (proteins, enzymes, complex structures)

18. Identify etiological agents Phenotype • Identification • Antibiotic resistance (physical expression of genotype) Gene products (proteins, enzymes, complex structures) Genotype (blueprint for phenotype)

19. 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 neverbeen seenbefore?

20. 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 neverbeen seenbefore? • Ifit grows, it can usuallybe identified

21. Identify etiological agents Phenotype • Identification • Antibiotic resistance (physical expression of genotype) Gene products (proteins, enzymes, complex structures) Genotype (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. Case 1 • 79-yr-old male • Soft-tissue excision • Inflammatory tissue • No microbial elements • Cultures were negative

25. Case 1 “The differential diagnosis includes: • infectious process (possible _______ infection), • inflammatory process such as rheumatoid nodule or a • neoplastic process (epithelioidcarcinoma can present with foci of necrosis, however the histopathological features do not favor same).” • PCR and DNA sequencing were performed on a PET specimen.

26. Case 2 23-yr-old man with refractory seizures MRI: Vasogenic edema Gadolinium enhancing

27. 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.

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

29. Genomic approach: isolate & amplify Target: Species-identifying DNA sequences flanked by conserved primer binding sites

30. Genomic approach: isolate & amplify What if… • infection occurs at a site with normal microbiota? • more than one pathogen is present? 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. Next Generation Sequencing Dr. Toby Russell, assisted by Dr. Beverly Crusher, 2368. Genitronic replication of Worf’s new spinal column, Episode #115, Star Trek, The Next Generation “Sequencing the genome at 109 base pairs per second…”

34. NGS: isolate & amplify • Capture single DNA molecules • Cluster formation: amplify ~1,000 copies • Parallel DNA sequencing of clusters • Read millions of clusters per flow cell!

35. Bacterial vaginosis

36. Cystic fibrosis

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

38. 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

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

40. Google: UW Molecular Microbiology