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Mycobacteriology William H. Benjamin, Jr.

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

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Mycobacteriology William H. Benjamin, Jr.

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

  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

  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

  4. Mycobacterium arabinogalactan

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

  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

  7. 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

  8. Tuberculosis cases and deaths US

  9. U. S. Tuberculosis Cases

  10. Alabama Tuberculosis Cases

  11. Examples of high prevalence countries

  12. Transmission of Tuberculosis • Transmission is by infectious droplet • droplet diameter 1 - 5 mm • 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

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

  14. Mantoux skin test mm of induration

  15. 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 mm Early, immunosuppressed • HIV positive or HIV risk factors HIV status unknown • Chest film consistent with old nonreactive tuberculosis • Recent close contact with infectious tuberculosis case

  16. QUANTIFERON-GoldInterferon-gamma release assay • 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-g release

  17. T-SPOT TB Elispot (Enzyme-linked immunospot)

  18. 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)

  19. Tuberculosis Risk Factors • AIDS - CD4 < 250 • Iatrogenic immunosuppression • TNFa inhibitors –Humira, Enbrel, Remicade, Cimzia • corticosteroids • Age • young • old • Alcoholism/malnourishment • Diabetes • Genetics

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

  21. Types of Disease caused by M. tuberculosis

  22. Tuberculosis prevention • Environmental - decrease exposure • Avoid crowded conditions • Air changes • UV irradiation • Chemoprophylaxis – after positive skin test • RULE 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

  23. 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 (62oC 30 min or 71.7oC 15 sec) • HEPA filters

  24. 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

  25. UV Exposed Culture Plate 0 min1 min

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

  27. M. tuberculosis direct smear

  28. AFB Cultures at UAB

  29. Diagnostic M. tuberculosis Cultures

  30. Number of Patients Each Species of Mycobacteria Were Isolated from at UAB 2000 to 2010

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

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

  33. M. tuberculosis concentrated smear

  34. 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 (14CO2 release from 14C palmitic acid) • MB/BacT (CO2 production) • ESP (O2 utilization - pressure change) • MGIT (O2 utilization - quenching of fluorescence)

  35. Colony Morphology of M. gordonae and M. tuberculosis

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

  37. Growth in Liquid Medium: M. tuberculosis and M. fortuitum

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

  39. Hybridization Protection Assay (GenProbe) Denatured (heat) Acridinium-labeled probe Alkaline hydrolysis Inactivated probe Substrate light

  40. 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

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

  42. M. marinumlymphocutaneous

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

  44. 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

  45. 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

  46. 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 Asia 50/50 • Mexico 10/90

  47. 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

  48. 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

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