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

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

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

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 Eventually escape into the cytoplasm Requires a T-cell mediated immune response for infection control/eradication Granuloma formation

Removal of M. tuberculosis Depends on the Cell-Mediated Arm of Host Defense

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

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

Lung Tuberculosis

Extrapulmonary Tuberculosis

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

Transmission of Tuberculosis Inhalation of aerosols containing mycobacteria Bacteria can float in the air for several hours Ingestions (M. bovis) According to CDC, TB is not spread by Handshaking Sharing food or a drink\touching bed lines or toilet seats Sharing tooth brushes Kissing

Principle of the Tuberculin Test

QuantiFERON®-TB Gold Test Measure IFNg 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

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

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)

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

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)

Worldwide Threats by M. tuberculosis Large fraction of the world population is infected 1/3 of world population is infected ~ (1.7 billion people) Each year ~ 9 million new cases world wide 5 – 10 % will develop active tuberculosis (TB) Worldwide almost 2 million deaths from TB TB is the leading killer of people who are HIV infected Even though TB has is declining in the US, the decline of the average annual percentage rate is slowing down From 1993 – 2000 ~ 6.6% decline /year From 2003 – 2008 only ~ 3.4% decline/year In the US decline of cases In 2008 12,904 TB cases reported

Worldwide Threats by M. tuberculosis Multidrug resistant TB Case rates have also declined (407 cases in 1993 and 86 cases in 2008) Result from sequential mutations Extremely drug-resistant TB First reported in 2005 3 cases reported in the US in 2006 Increase in Europe observed 7.3% of all MDR strains

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.

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

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

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.

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, 11th ed, 2006. Immunobiology, by Janeway,, Travers, Walport, and Shlomchik, Garland Science, 6th 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#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&tbnw=94&prev=/images%3Fq%3Dmycobacterium%2Btuberculosis%2Bcell%2Bwall%26gbv%3D2%26hl%3Den (accessed 4 15 08)