1. MICR 454L Emerging and Re-Emerging Infectious Diseases Lecture 7: M. tuberculosis Dr. Nancy McQueen & Dr. Edith Porter

2. Overview M. tuberculosis Morphology Growth and metabolic characteristics Virulence factors Diseases Diagnosis Culture PCR Immune response Therapy Threats

3. Mycobacterium tuberculosis

4. M. tuberculosis Acid fast rods Lipid-rich cell wall Mycolic acids Lowenstein Jensen agar Eggs Potatoes Malachite green Slow growth Up to 6 weeks http://www.ann-clinmicrob.com/content/figures/1476-0711-4-18-5.jpg

5. M. tuberculosis: Virulence Factors Lipid-rich cell wall Mycolic acids Resistant to host defense Intracellular survival in macrophages Iron capturing ability Sulfolipids prevent phagsome-lysosome fusion Requires a T-cell mediated immune response for infection control/eradication Granuloma formation

8. The Course of TB Infection and Disease Airborne Infection No symptoms Not sick Cannot spread disease Chest X Ray and sputum are normal AIDS increases susceptibility Reactivation (secondary) TB Untreated: Severe illness, Death Symptoms Can sp[read infection Positive skin test Possible abnormal chest X ray Positive sputum smear or culture Dissemination Latent TB TB Disease 10 %90 %

9. M. tuberculosis: Diseases General Symptoms Feelings of sickness or weakness Weight loss Fever Night sweats Lung tuberculosis Coughing Chest pain Hemoptysis Extrapulmonary Depends on localization

10. Lung Tuberculosis

11. Extrapulmonary Tuberculosis

12. M. tuberculosis: Diagnosis History Physical exam Mantoux Skin test (tuberculin test with purified protein derivative) QuantiFERON®-TB Gold Test Chest radiograph Sputum smear Culture

13. Principle of the Tuberculin Test

14. QuantiFERON®-TB Gold Test Measure IFN  production by patient peripheral blood leukocytes in response to M. tuberculosis antigens (protein antigens ESAT-6 and CFP-10) Rapid T cell response only in primed individuals

15. Interpretation of Tuberculin and QuantiFERON Positive = previous contact with M. tuberculosis Positive DOES NOT mean TB disease

16. M. tuberculosis: Therapy Isoniazid (INH) Rifampin (RIF) Ethambutol Pyrazinamide DOTS Direct observational therapy short course At least 2 in combination (INH + RIF) Prolonged time (at least 6 months)

17. Anti-Tuberculosis Drug Targets Mycolic acid INH inhibits mycolic acid synthesis Ethambutol inhibits mycolic acid incorporation into the cell wall Fatty acid synthetase I (FASI) Pyrazinamide inhibits fatty acid synthesis RNA synthesis Inhibited by rifampin

18. Resistance of M. tuberculosis Mutations in codon 306 of the embB gene (ethambutol) are discussed as marker and predictor of resistance development to multiple antibiotics Not all mutated strains are resistant but resistant strains are mutated. Alterations in RNA polymerase (Rifampicin)

19. Worldwide Threats by M. tuberculosis 1/3 of world population is infected Each year ~ 9 million new cases 5 – 10 % will develop active tuberculosis (TB) Worldwide ~ 1.5 million deaths from TB However: In 2006, a total of 13,767 tuberculosis (TB) cases (4.6 per 100,000 population) in the US 3.2% decline from 2005 Co-infection with HIV Multidrug resistant TB Extremely drug-resistant TB 3 cases reported in the US in 2006

20. Extremely Drug-Resistant M. tuberculosis XDR TB Resistant to almost all drugs used to treat TB, including the two best first-line drugs: isoniazid and rifampin Resistant to the best second-line medications: fluoroquinolones (DNA gyrase mutations) And at least one of three injectable drugs (i.e., amikacin, kanamycin, or capreomycin; mutations in 16sRNA and ribosomal protein genes). Possibly involvement of drug efflux pumps. 49 cases of XDR TB have been reported between 1993 and 2006 in the US

21. Reported Cases of XDR in the US MMWR Weekly, March 23rd, 2007

22. New Drugs are Needed Immune modulators IL-2, IFN-gamma, GM-CSF, IL-12 New chemicals targeting essential genes of M. tuberculosis

23. Take Home Message One third of the world population is infected with M. tuberculosis but only 10% develop active disease. The lipid rich cell wall and slow growth contribute to resistance to host defense and difficulties in antibiotic treatment. The emergence of extremely drug resistant tuberculosis strains poses a great threat to the public.

24. Additional Resources The Microbial Challenge, by Krasner, ASM Press, Washington DC, 2002. Brock Biology of Microorganisms, by Madigan and Martinko, Pearson Prentice Hall, Upper Saddle River, NJ, 11 th ed, 2006. Immunobiology, by Janeway,, Travers, Walport, and Shlomchik, Garland Science, 6 th ed, 2005. Malak Kotb Genetics of Susceptibility to Infectious Diseases Volume 70, Number 10, 2004 / ASM News Y 457-463 htttp://www.cdc.gov/ulcer/keytocure.htm#whatistp://www.cdc.gov/ulcer/keytocure.htm#whatis http://dermatlas.med.jhmi.edu/derm/resultNoCache.cfm Zager and McNerney (2008) Multidrug-resistant tuberculosis. BMC Infectious Disease. 8: 10. Safi H, Sayers B, Hazbón MH, Alland D. (2008) Antimicrob Agents Chemother. Mar 31 [Epub ahead of print] Transfer of embB306 mutations into clinical Mycobacterium tuberculosis alters susceptibility to ethambutol, isoniazid and rifampin. Zimhony O et al. (2000) Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis. Nat Med. Sep;6(9):1043-7. http://images.google.com/imgres?imgurl=http://www.biozentrum.uni- wuerzburg.de/fileadmin/REPORT/BIOTE/pic/biote016_img_0.jpg&imgrefurl=http://www.biozentrum.uni- wuerzburg.de/fileadmin/REPORT/BIOTE/biote016.htm&h=734&w=488&sz=82&hl=en&start=4&tbnid=_R4zGz7SCgf70M:&tbnh=141&tbn w=94&prev=/images%3Fq%3Dmycobacterium%2Btuberculosis%2Bcell%2Bwall%26gbv%3D2%26hl%3Den (accessed 4 15 08)