2. 1 Mycobacteriology William H. Benjamin, Jr. William H. Benjamin, PhD Department of Pathology UAB

3. 2 Mycobacteria sp. Acid Fast Bacilli (AFB) Mycolic acids (C78 - 91) Waxes Obligate aerobes Slow growing –days to weeks to form colonies –18 hour doubling time for M. tuberculosis

4. 3 Identification of Acid Fast Bacilli Mycobacterium sp. - are identified by the acid fast stain Mycobacteria predate animal life 140 named Mycobacterial species –More than 40 have infected humans AIDS other immunocompromised

5. 4 Mycobacterium arabinogalactan

6. 5 Obligate Pathogenic Mycobacteria Mycobacterium tuberculosis –First bacteria shown to cause disease –1882 Koch’s postulates M. leprae –causes Hansen’s disease or leprosy

7. 6 Prehistory of Tuberculosis 17,000 BPE (before present era) bison in Wyoming USA –IS6110 and Spoligotype confirmed M. tuberculosis 10,000 BPE in Germany skeletal evidence 3500 - 3000 BPE Egypt Potts disease –PCR positive for Mtb sequence 1300 BPE 8 year old Inca boy –Pott’s disease, AFB smear positive, IS6110 PCR positive

8. 7

9. 8 Mycobacterium tuberculosis Humans are the only natural host 1/3 of the world population is infected 9.2 million cases of tuberculosis/year –disease 1.7 million deaths caused by tuberculosis/year

10. Tuberculosis cases and deaths US

11. 10 U. S. Tuberculosis Cases

12. Alabama Tuberculosis Cases 11

13. 12 NamibiaZambiaUS Population (million)1.811.5300 Tuberculosis cases16,15678,00013,676 Tuberculosis rate/100,0008226804.2 Tuberculosis death rate61?0.22 Examples of high prevalence countries

14. Transmission of Tuberculosis Transmission is by infectious droplet –droplet diameter 1 - 5  m –droplet contains 1 - 3 bacilli –droplets settle 9 mm/min in still air –infectious dose is 5 - 200 infectious droplets –average patient exhales 1.25 infectious droplets/hour –some cases produce 150 - 200 infectious droplets/hour

15. 14 Exposure close contact No infection >50% Infection 50% Primary active TB 5% Latent TB 95% Never reactivate 90% Reactivation TB 5% lifetime HIV+ 5-10% per year

16. 15 Mantoux skin test mm of induration

17. 16 Significant Induration on Mantoux 15 mm Always indicates infection 10 mm population at risk –low income, minority races, IV drug users –foreign born - from high prevalence countries –Institution populations - prisons, nursing homes, mental institutions –silicosis, diabetes mellitus, malignancies, immunosuppressive agents 5 mmEarly, immunosuppressed –HIV positive or HIV risk factors HIV status unknown –Chest film consistent with old nonreactive tuberculosis –Recent close contact with infectious tuberculosis case

18. 17 Blood test to detect M. tuberculosis infection Antigens used are not in BCG (vaccine strain) –ESAT6, CFP-10, TB7.7 Used to detect latent infection No second visit Blood mixed with Mtb antigen Incubate overnight Detect Mtb specific INF-  release QUANTIFERON-Gold Interferon-gamma release assay

19. T-SPOT TB Elispot (Enzyme-linked immunospot) 18

20. M. tuberculosis infection without disease Inhalation of infectious droplet Hilar and peribronchiolar lymph nodes –4 - 6 weeks Lymphohematogenous dissemination –6 - 8 weeks Tubercle formation Granulomatous inflammation: caseous necrosis Dystrophic calcification (Ghon complex)

21. Tuberculosis Risk Factors AIDS - CD 4 < 250 Iatrogenic immunosuppression –TNF  inhibitors –Humira, Enbrel, Remicade, Cimzia –corticosteroids Age –young –old Alcoholism/malnourishment Diabetes Genetics

22. Types of Tuberculous disease Childhood tuberculosis “Adult” or reactivation disease Acute tuberculous pneumonia (AIDS) Miliary tuberculosis Cold abscess Addison’s disease (Adrenal insufficiency)

23. 22 Types of Disease caused by M. tuberculosis

24. Tuberculosis prevention Environmental - decrease exposure –Avoid crowded conditions –Air changes –UV irradiation Chemoprophylaxis – after positive skin test RULE OUT DISEASERULE OUT DISEASE –INH - after Tuberculin skin test conversion 9 months of daily oral INH BCG vaccine (Bacille-Calmette-Guerin) –Causes positive skin test –Used in much of the world, except US

25. 24 Environmental Resistance of M. tuberculosis Survives drying Susceptible to UV irradiation (2 hours in sunlight) Resistant to many disinfectants –susceptible to chlorine and phenols Pasteurization kills (62 o C 30 min or 71.7 o C 15 sec) HEPA filters

26. 25 Germicidal Ultraviolet Light UV light at 254 nm is effective in killing infectious agents –In air ducts –In upper room irradiation Also used in biological safety cabinets –Demonstration of effectiveness of killing M. tuberculosis on Middlebrook 7H11 plate

27. 26 UV Exposed Culture Plate 0 min1 min

28. Diagnosis of Tuberculosis AFB smear Tuberculin skin test (Mantoux test) Chest radiograph AFB culture

29. 28 M. tuberculosis direct smear

30. AFB Cultures at UAB

31. 30 Diagnostic M. tuberculosis Cultures

32. Number of Patients Each Species of Mycobacteria Were Isolated from at UAB 2000 to 2010 M. avium cx520 M. fortuitum353 M. tuberculosis197 M. chelonae/abscessus91 M. kansasii89 M. mucogenicum18 M. lentiflavum15 M. marinum8 M. terrae7 Grp II scotochromogen7 M. xenopi6 M. scrofulaceum3 M. szulgai3 M. brumae3 M. triplex3 M. gastri3 M. smegmatis2 M. simiae2 M. cooki2 M. asiaticum2 M. interjectum2 M. phlei2 Grp III nonphotochromogen 2 M. austroafricanum1 M. peregrinum1 M. nonchromogenicum1 M. gordonae627

33. 32 Microbiological Diagnosis of Tuberculosis Digestion - mucolytic agents Decontamination Concentration Acid fast Stain Cultivation of Mycobacteria –solid media –liquid culture Anti-Mycobacterial susceptibilities

34. 33

35. 34 Decontamination and Concentration of AFB Cultures Mucolytic agent (N-acetyl-L-cysteine) 2% NaOH –1% oxalic acid Centrifuge 3000 x g for 30 min.

36. 35 M. tuberculosis concentrated smear

37. 36 Mycobacteria Culture Media Solid media - 21 - 26 days to detection –Loewenstein-Jensen (egg based) –Middlebrook 7H11 (agar based) Liquid media - 8 to 14 days to detection –BACTEC 460 ( 14 CO 2 release from 14 C palmitic acid) –MB/BacT (CO 2 production) –ESP (O 2 utilization - pressure change) –MGIT (O 2 utilization - quenching of fluorescence)

38. 37 Colony Morphology of M. gordonae and M. tuberculosis

39. 38 VersaTREK® Mycobacteria MGIT BD Liquid Culture Systems MB/BacT

40. 39 Growth in Liquid Medium: M. tuberculosis and M. fortuitum

41. 40 Identification of Mycobacteria Biochemical tests –2 to 3 weeks GenProbe - DNA - RNA hybridization –2 hours HPLC - high performance liquid chromatography –1 hour

42. 41 Hybridization Protection Assay (GenProbe) Denatured (heat) Acridinium-labeled probe Alkaline hydrolysis Substratelight Inactivated probe

43. Mycobacterium avium complex (MAC) found in soil and water - tap water transmission through either respiratory or GI tract pulmonary disease like tuberculosis disseminated disease in AIDS patients –50% of autopsies resistant to many anti-mycobacterial drugs slow growing non-pigmented colonies

44. Other Important MOTT M. kansasii - Photochromogen –Tuberculosis like disease M. marinum - Photochromogen –Found in water - fish tanks and surface water –30 to 33 o C optimum temperature M. scrofulaceum - –granulomatous cervical lymphadenitis in children M. fortuitum - M. chelonei complex –Rapid growers - colonies in less than 7 days –Skin infections, pulmonary disease

45. 44 M. marinum lymphocutaneous

46. 45 M. marinum Fresh or salt water or no water exposure Photochromogen 1-2 patients/year at UAB 9 finger patients 30 o C optimum temperature colonies form in 10-14 days

47. Mycobacterium leprae Hansen’s disease humans and armadillos are the only natural hosts 12 million cases worldwide 6,000 registered cases in US, 112 - 350 cases/year transmitted by inhalation or skin contact with contaminated respiratory secretions of lepromatous patients incubation period is 3 months to 3 years

48. Leprosy diagnosis –does not grow on artificial media –will grow in nude mice or armadillo –AFB stain of nasal secretions –lepromin test - skin test treatment –dapsone and rifampicin - at least 1 year prevention –isolation of acute lepromatous cases –vaccines under development

49. 48 M. leprae epidemiology World wide –14 million cases treated 20 years –Eliminated from 113/122 countries (<1/10,000 population) Ratio of tuberculoid/lepromatous –Africa and India 90/10 –Southeast Asia50/50 –Mexico10/90 Leprosy cases 19855,200,000 1995805,000 1999753,000 2004286,000

50. 49 US Leprosy Western Gulf - Texas, Louisiana 1975 found wild armadillos infected 1968 experimental infection of armadillo 1961 tissue diagnosis in stored armadillo 16% of armadillos infected in one survey 20 – 40 cases/ year of US contracted Hansen’s disease

51. Clinical Types of Leprosy Tuberculoid leprosy –intact cell mediated response to M. leprae –organisms rare in tissue –organisms grow in nerves in cooler parts of the body –cutaneous loss of sensation - nerve damage due to cell mediated immunity

52. Lepromatous Leprosy depressed CMI response specific for M. leprae bacteremia with localization in nerves and skin high numbers of organisms in macrophages less loss of nerve function leonine facies other organs involved - testes, spleen and liver

53. 52 Lepromatous leprosy

54. 53 Tuberculoid leprosy Non-progressive disease intact cell mediated response organisms rare in tissue macular lesions predominate organisms invade nerves and form granulomas cutaneous loss of sensation - nerve damage due to CMI

55. 54 Tuberculous leprosy