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Mycobacteria slides for lecture

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Mycobacteria slides for lecture

  1. 1. MYCOBACTERIA OF MEDICAL IMPORTANCE Prof M.I.N. Matee, PhD Department of Microbiology and Immunology, MUCHS
  2. 2. Mycobacteria: overview <ul><li>Introduction and Definitions </li></ul><ul><li>Epidemiology of Mycobacterioses </li></ul><ul><li>TB: Pathogenesis and clinical presentation </li></ul><ul><li>TB: Diagnosis </li></ul><ul><li>TB: Treatment </li></ul><ul><li>TB: Prevention </li></ul><ul><li>Conclusions </li></ul>
  3. 3. MYCOBACTERIUM <ul><li>THIS GENUS IS COMPOSED OF: </li></ul><ul><li>Strictly aerobic, acid-fast rods, does not </li></ul><ul><li>stain well (gram stain indeterminant), </li></ul><ul><li>DNA has high g+c content, unique cell wall, </li></ul><ul><li>Mycolic acid carbon chain length > c60 </li></ul><ul><li>Relatively slow growth (two groups) </li></ul><ul><li>RAPID GROWERS (Visible colonies in <5 days) </li></ul><ul><li>SLOW GROWERS (Visible colonies in > 5 days) </li></ul><ul><li>TYPE SPECIES: Mycobacterium tuberculosis </li></ul>
  4. 4. TB: Epidemiology <ul><li>transmitted in respiratory aerosols </li></ul><ul><li>Occasionally from animals (e.g. milk) </li></ul><ul><li>With pasteurization, most TB now due to human - human transmission </li></ul><ul><li>1/3 world’s population carry M. tuberculosis : not infectious </li></ul><ul><li>8 million cases / year: contagious </li></ul><ul><li>3 million deaths / year </li></ul>
  5. 5. Burden of TB in Tanzania <ul><li>Tanzania is among the 22 countries with highest TB burden despite :- </li></ul><ul><ul><li>implementing DOTS strategy since early 1980’s </li></ul></ul><ul><ul><li>Having national coverage of DOTS </li></ul></ul><ul><ul><li>Having a well functioning TB control programme </li></ul></ul><ul><ul><li>High Government and International commitment to TB control in the country </li></ul></ul><ul><li>Notification rate total TB cases for 2002 was about 188/100,000 </li></ul><ul><li>Underlying course is the HIV/AIDS epidemic in the country </li></ul><ul><li>TB is 3 rd leading cause of adult mortality </li></ul>
  6. 6. Estimated Global TB incidence rates, 2000 The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. Global Tuberculosis Control. WHO Report 2002 . WHO/CDS/TB/2002.295 25 - 49 50 - 99 100 - 300 0 - 9 10 - 24 <ul><ul><ul><li>300 </li></ul></ul></ul>No estimate Africa > 300 /100,000
  7. 7. Prevalence of HIV among TB patients <ul><li>WHO supported surveys to establish prevalence of HIV among SM+ TB cases in 2 phases: 1991-1993; 1994-1998 </li></ul><ul><li>Total of 6,084 and 10,612 TB cases surveyed in phase 1 and 2 respectively </li></ul><ul><li>TB/HIV co-infection in phase 1 was 32% (range 14%-59%) and 44% in phase 2 (range 21%-77%) </li></ul><ul><li>HIV accounted for 60% of increase in Smear positive TB notification rate from 54/100,000 in phase 1 to 74/100,000 in phase 2. </li></ul>NTLP - MOH
  8. 9. Tanzania: TB Cases - trends, 1979 to 2001
  9. 10. Impact of HIV on the Epidemiology of Tuberculosis
  10. 11. Impact HIV in treatment outcomes High Mortality in HIV Co-infected patients van den Broek & Mfinanga, 1998 NTLP reports 2003
  12. 13. THE TUBERCULOSIS COMPLEX (Organisms that resemble M. tuberculosis; Causing a similar type of disease in humans) <ul><li>M. tuberculosis </li></ul><ul><li>M. bovis </li></ul><ul><li>M. africanum </li></ul>
  13. 14. THE RUNYON GROUPING: An older idea that Remains useful for differentiating mott
  14. 15. RUNYON GROUPS I,II,III: Slow growing mycobacterium Visible colonies on solid media After more than 5 days incubation GROUP I: PHOTOCHROMOGENS Produce pigment When grown in the light but not the dark EXAMPLES: M. kansasii ; M. marinum ; M. simiae GROUP II: SCOTOCHROMOGENS Pigment when grown in both light and dark EXAMPLES: M. scrofulaceum ; M. szulgai ; M. xenopi GROUP III: NONCHROMOGENS No pigment when grown in both light and dark EXAMPLES: M. avium-intracellulare ; M. genevense M. ulcerans and M. leprae do not fit in this scheme
  15. 16. RUNYON GROUP IV: Rapidly growing mycobacterium Visible colonies on solid medium In less than 5 days incubation EXAMPLES: M. fortuitum ; M. chelonae ; M. abscessus
  16. 17. Robert Koch 1843-1910
  17. 18. MYCOBACTERIUM TUBERCULOSIS Prototypic facultative intracellular pathogen Primary human pathogen Transmitted from human to human Closely related to M. bovis It is an intracellular pathogen (typically inside alveolar macrophages). This organism does not secrete an identifiable exotoxin. Host immunity to M tuberculosis appears to be cell mediated (CMI).
  18. 19. CELL ASSOCIATED TOXINS MAY BE IMPORTANT VIRULENCE FACTORS FOR MANY MYCOBACTERIA The glycolipids, such as trehalose dimycolates, of Mycobacterium tuberculosis and related organisms appear to be related to their virulence .
  19. 20. Bacterial cell wall structure Gram + Gram - Mycobacterium Lipid Peptido- Lipid + Porins Mycolate Acyl LAM arabino- Bilayer glycan LPS Lipids Lipo-arabino galactan mannin
  20. 22. Biochemical properties <ul><ul><ul><li>Niacin (+) </li></ul></ul></ul><ul><ul><ul><li>Nitrate reduction(+) </li></ul></ul></ul><ul><ul><ul><li>(–) for catalase at 68 0 C </li></ul></ul></ul><ul><ul><ul><li>Tween hydrolysis, </li></ul></ul></ul><ul><ul><ul><li>Arylsulfatase production, </li></ul></ul></ul><ul><ul><ul><li>Tellurite reduction, </li></ul></ul></ul><ul><ul><ul><li>Salt tolerance, </li></ul></ul></ul><ul><ul><ul><li>pyrazinamidase (pyr) production </li></ul></ul></ul>
  21. 23. Reaction to physical and chemical agents <ul><li>Resistant to chemical agents – malachite green, penicillins </li></ul><ul><li>In liquid media – grow in clumps – hydrophobic </li></ul><ul><li>Resistant to acids and alkalines – helps to decontaminate and concentrate samples </li></ul>
  22. 24. Constituents of tubercle bacilli <ul><li>Lipids – mycolic acids – long chain fatty acids C78-C90) –responsible for acid fastness </li></ul><ul><li>Lipids linked to proteins and polysaccharides </li></ul><ul><li>Muramly dipeptide </li></ul><ul><li>Surpentine cords – cord factor – trehalose-6,6-dimycolate </li></ul>
  23. 25. Virulence factors ( M. tb .) <ul><ul><li>Cord factor – is a glycolipid, trehalose 6,6’ dimycolate, </li></ul></ul><ul><ul><li>t is responsible for the serpentine growth (filaments or cords) </li></ul></ul><ul><ul><li>It is toxic to leukocytes, antichemotactic, interferes with mitochondrial function in mice and plays a role in the development of granulomatous lesions </li></ul></ul><ul><ul><li>Iron capturing ability – required for survival inside phagocytes </li></ul></ul><ul><ul><li>Sulfolipids prevent phagosome-lysosome fusion so that the organisms are not exposed to lysosomal enzymes (is important in intracellular survival) </li></ul></ul>
  24. 26. Tuberculosis – primary infection route
  25. 27. Tuberculosis in humans <ul><li>A key issue is to understand why individuals infected with M. tuberculosis experience different clinical outcomes </li></ul>INTRACELLULAR pathogen (facultative extracellular) Exposed Infected (2 billion, 8 million new cases per year) Primary TB Latent TB Reactivation 30% 80-90% 5-10% 5-10% Clearance 70% Death (2 million)
  26. 28. Pathogenesis <ul><li>Inhalation of droplets (1-5 µm) </li></ul><ul><li>Reach alveoli </li></ul><ul><li>Disease due to proliferation and host interaction </li></ul><ul><li>Resistance and hypersensitivity influence disease </li></ul>
  27. 29. T helper Cell Differentiation <ul><li>Type 1 response </li></ul><ul><li>immunity to mycobacteria </li></ul><ul><li>inflammation </li></ul><ul><li>rheumatoid arthritis, diabetes </li></ul><ul><li>Type 2 response </li></ul><ul><li>IgE antibody responses </li></ul><ul><li>Immunity to some parasites </li></ul><ul><li>allergic diseases </li></ul>IFN  TNF  IL4 IL13 IL10 Th0 Th2 Th1
  28. 34. Pathology <ul><li>Two principal lesions: </li></ul><ul><ul><li>Exudative type – resembles bacterial pneumonia </li></ul></ul><ul><ul><ul><li>Acute inflammatory reaction </li></ul></ul></ul><ul><ul><ul><li>Edema </li></ul></ul></ul><ul><ul><ul><li>Later, monocytes </li></ul></ul></ul><ul><ul><ul><li>Heals by fibrosis </li></ul></ul></ul><ul><ul><ul><li>May develop to productive type </li></ul></ul></ul><ul><ul><ul><li>Tuberculin test is positive </li></ul></ul></ul>
  29. 35. <ul><li>Productive type </li></ul><ul><ul><li>Chronic granuloma, consisting of three zones: </li></ul></ul><ul><ul><ul><li>A central large area – multinucleate giant cells with bacilli </li></ul></ul></ul><ul><ul><ul><li>A mid zone area – epitheliod cells </li></ul></ul></ul><ul><ul><ul><li>Peripheral zone – fibroblasts, lymphocytes and monocytes </li></ul></ul></ul><ul><ul><ul><li>Later the central area – caseation necrosis, and peripheral area becomes fibrous -> turbecle </li></ul></ul></ul><ul><ul><ul><ul><li>May break into a bronchus </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Or heal by fibrosis and calcification </li></ul></ul></ul></ul>
  30. 36. Large caseating tubercle Miliary tubercles HUMAN LUNG HUMAN LUNG TUBERCULOSIS
  31. 37. Spread of organisms in the host <ul><li>Tubercle spreads by direct extension to lymphatics -> lymph nodes -> blood stream -> various organs </li></ul><ul><li>Blood stream involvement also due to erosion of a vein </li></ul><ul><li>If contents of tubercle are swallowed could be passed to stomach and intestine </li></ul>
  32. 38. Tuberculosis – hematogenous extension Extrapulmonary Lymph nodes Liver Bones
  33. 39. Primary and reaction TB <ul><li>When host has first contact with TB bacilli the following features will occur: </li></ul><ul><ul><li>1. an exudative lesion develops and rapidly spreads to lymphatics and regional lymph noded – Ghon complex </li></ul></ul><ul><ul><li>2. the lymph node undergo massive caesation, which ussually calcifies </li></ul></ul><ul><ul><li>3. tuberculin test becomes positive </li></ul></ul><ul><li>Primary TB may occur on any part of the lung but more frequently on the base </li></ul>
  34. 40. Reactivation TB <ul><li>Caused by bacilli that have survived in the primary lesion </li></ul><ul><li>Chronic tissue necrosis </li></ul><ul><li>Formation of tubercles, caesation and fibrosis </li></ul><ul><li>Almost always begins at the apex of the lung </li></ul>
  35. 41. M. tuberculosis
  36. 42. Symptoms of TB Disease <ul><li>Prolonged cough (may produce sputum)* </li></ul><ul><li>Chest pain* </li></ul><ul><li>Hemoptysis* </li></ul><ul><li>Fever </li></ul><ul><li>Chills </li></ul><ul><li>Night sweats </li></ul><ul><li>Fatigue </li></ul><ul><li>Loss of appetite </li></ul><ul><li>Weight loss/failure to gain weight </li></ul><ul><li>*commonly seen in cases of pulmonary TB </li></ul>
  37. 43. Clinical Manifestations of TB <ul><li>Organ specific </li></ul><ul><ul><li>pneumonia: cough, sputum +/- blood </li></ul></ul><ul><ul><li>scrofula: swollen lymph nodes </li></ul></ul><ul><ul><li>meningitis: headache, obtundation </li></ul></ul><ul><ul><li>miliary TB: no obvious source </li></ul></ul><ul><ul><li>genitourinary: sterile pyuria </li></ul></ul>
  38. 44. <ul><li>Evidence of infection </li></ul><ul><ul><li>Chest x-ray - hilar lymphadenopathy calcification of primary focus/LN </li></ul></ul><ul><ul><li>Delayed hypersensitivity response to purified protein derivative (PPD) MANTOUX /HEAF TEST </li></ul></ul><ul><li>Evidence of active disease </li></ul><ul><ul><li>Sputum for AFB positive </li></ul></ul><ul><li>Evidence of active disease </li></ul><ul><ul><li>Indirect evidence of infection (Mantoux) </li></ul></ul><ul><ul><li>Direct evidence of infection PCR / culture </li></ul></ul><ul><ul><li>Histo-pathological evidence </li></ul></ul>DIAGNOSIS
  39. 45. Chest Radiograph Abnormalities often seen in apical or posterior segments of upper lobe or superior segments of lower lobe May have unusual appearance in HIV-positive persons Cannot confirm diagnosis of TB Arrow points to cavity in patient's right upper lobe.
  40. 46. Diagnosis of tuberculosis <ul><li>Tuberculin test </li></ul><ul><ul><li>intradermal injection of PPD (5 TU) </li></ul></ul><ul><ul><li>Induration, edema, erythema </li></ul></ul><ul><ul><li>Read after 48-72 h </li></ul></ul><ul><ul><li>Positive: induration≥ 10 mm </li></ul></ul><ul><ul><li>It is DTH </li></ul></ul>
  41. 47. Administering Tuberculin Skin Test <ul><li>Inject intradermally 0.1 ml of 5 </li></ul><ul><li>TU PPD tuberculin </li></ul><ul><li>Produce wheal 6 mm to 10 mm </li></ul><ul><li>in diameter </li></ul><ul><li>Do not recap, bend, or break </li></ul><ul><li>needles, or remove needles from syringes </li></ul>
  42. 48. Reading the Tuberculin Skin Test <ul><li>Read reaction 48-72 hours </li></ul><ul><li>after injection </li></ul><ul><li>Measure only induration </li></ul><ul><li>Record reaction in millimeters </li></ul>
  43. 49. Factors that affect the PPD Reaction Type of Reaction Possible Cause False-positive Nontuberculous mycobacteria BCG vaccination Anergy False-negative Recent TB infection Very young age (< 6 months old) Live-virus vaccination Overwhelming TB disease
  44. 56. <ul><ul><ul><ul><li>Kinyon – uses a higher content of phenol (organic solvent) in the carbol fuchsin primary stain to allow penetration of the stain without the need to apply heat. Also uses acid alcohol to destain and methylene blue as the counterstain. </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Auramine-rhodamine fluorochrome (a fluorescent stain) requires a fluorescent microscope, but allows one to scan the slide on high dry so that slides may be read much faster </li></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Stain with auramine-rhodamine for 10 minutes (phenol in the solution allows for penetration) </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Destain with acid alcohol </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Counterstain with acridine orange </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>A positive result is a bright yellow fluorescence. </li></ul></ul></ul></ul></ul>
  45. 57. Mycobacteria: auramine stain
  46. 58. Nucleic Acid Hybridization <ul><li>Ability of DNA strands from one organism to hybridize with the DNA strands of another organism </li></ul><ul><li>Southern blotting </li></ul>
  47. 59. DNA fingerprinting Steps: 1. Digest DNA into smaller fragments 2. Separate fragments by size 3. Visualize bands 4. Each band corresponds to 1 fragment size 5. Unique pattern of bands identifies donor of DNA
  48. 60. Mycobacteria colonies on LJ medium
  49. 61. MGIT System <ul><li>Middlebrook media </li></ul><ul><li>Fluorescent compound on bottom that is quenched by oxygen in the tube. </li></ul><ul><li>As mycobacteria consume oxygen, fluorescence can be detected. </li></ul><ul><li>Most of MAC detected in nine days while MTB by 14 days. </li></ul><ul><li>Follow up with acid fast stain. </li></ul>
  50. 64. Tuberculosis and the Immune Reconstitution Inflammatory Syndrome (IRIS)
  51. 65. Definition <ul><li>‘ a paradoxical inflammatory reaction against a foreign antigen (alive or dead) in patients who have started antiretroviral therapy and who have undergone a reconstitution of their immune responses against this antigen’ </li></ul>
  52. 66. Names <ul><li>Immune reconstitution inflammatory syndrome (IRIS) </li></ul><ul><li>Immune restoration disease (IRD) </li></ul><ul><li>Paradoxical reactions </li></ul>
  53. 67. Pathogenesis <ul><li>Increased lymphoproliferative response to mycobacterium antigens in vitro </li></ul><ul><li>Restoration of cutaneous response to Tuberculin </li></ul><ul><li>Associated with TNFA, IL6 </li></ul>
  54. 68. Risk factors for TB/IRIS <ul><li>Starting ARV’s within 6 weeks of TB treatment </li></ul><ul><li>Disseminated, extra-pulmonary disease </li></ul><ul><li>Low base line CD4 count </li></ul><ul><li>Rise in CD4 % </li></ul><ul><li>Fall in viral load </li></ul><ul><li>High bacillary burden? </li></ul>
  55. 69. Types of TB IRIS <ul><li>Patient unknown to have TB at the start of HAART </li></ul><ul><li>Patient on TB treatment before or at the start of HAART </li></ul>
  56. 70. Timing of IRIS <ul><ul><li>Mean of 15 days after starting HAART </li></ul></ul><ul><ul><li>Up to months (years) </li></ul></ul><ul><ul><li>Syndrome lasts for 10-40+ days </li></ul></ul>
  57. 71. Prevention and control of tuberculosis 1. Prompt and effective treatment of patients with active TB 2. Careful follow-up of contacts – tuberculin test, X-rays etc 3. Prophylaxis of asymptomatics, tuberculin positives 4. Correct underlying cause of immune suppression 5. Immunization with live-attenuated tubercle bacilli e.g. BCG 6. Eradication of TB in cattle and pasteurization of milk
  58. 72. OTHER IMPORTANT MYCOBACTERIA <ul><li>1. Mycobacterium avium-intracelluare complex </li></ul><ul><li>causes TB like disease in birds </li></ul><ul><li>opportunistic pathogen in humans </li></ul><ul><li>Very prominent cause of disease in AIDS patients </li></ul><ul><li>has been decreased following HAART </li></ul><ul><li>Not easily transmitted </li></ul><ul><li>Difficult to treat ( drug of choice is rifabutin) </li></ul>
  59. 73. Mycobacterium ulcerans <ul><li>Causes buruli ulcer </li></ul><ul><li>Infection limited to fatty tissue beneath dermis </li></ul><ul><li>Does not grow above 33oc </li></ul>
  60. 74. Mycobacterium marinum <ul><li>Extrapulmonary ulcerative lesions </li></ul><ul><li>Growth of organism restricted to 34 0 c </li></ul><ul><li>Disease called “swimming pool granuloma” </li></ul><ul><li>Does not respond well to therapy </li></ul>
  61. 75. <ul><li>Mycobacterium kansasii </li></ul><ul><li>Pulmonary and disseminated disease similar to tuberculosis (organisms do not produce niacin) </li></ul><ul><li>does not respond well to antimicrobials </li></ul><ul><li>(no response to anti-tuberculosis therapy) </li></ul><ul><li>opportunistic pathogen </li></ul><ul><li>Runyon group I (photochromogen) </li></ul>
  62. 76. Mycobacterium scrofulaceum <ul><li>Causes scrofula (cervical lymphadentis) </li></ul><ul><li>drug resistant </li></ul><ul><li>Runyon group II (scotochromogen) </li></ul>
  63. 77. Mycobacterium fortuitum complex <ul><li>causes chronic abscesses(often wound associated) </li></ul><ul><li>can be confused with M. tuberculosis </li></ul><ul><li>often drug resistant </li></ul><ul><li>rapidly growing (Runyon group IV) </li></ul>
  64. 78. HANSEN’S DISEASE (Leprosy) caused by M. leprae Hansen’s disease is a chronic, slowly progressive Granulomatous Disease involve ectodermally derived tissues e.g. skin and peripheral nerves. Usually limited to the cooler parts of the body e.g. skin, nose and upper respiratory tract. It rarely affects internal organs such as the brain, liver, spleen, kidneys, and bones. It has a specific predilection for peripheral nerves.
  65. 79. Mycobacterium leprae is an acid-fast rod that grows in macrophages, nerve cells, the foot pads of thymectimized mice, and the nine banded armadillo. It has never been grown in artificial medium, an obligate intracellular pathogen. has a generation time of 12 to 14 days, the incubation period for onset of disease is prolonged, therapy must also be prolonged (perhaps the lifetime of the individual).
  66. 80. There are 3 forms of the disease: <ul><li>A. Lepromatous: </li></ul><ul><ul><ul><li>Most progressive many acid fast bacteria in bundles called globi. </li></ul></ul></ul><ul><ul><ul><li>No well formed granulomas (T-cell deficiency) </li></ul></ul></ul><ul><li>High serum antibody levels </li></ul><ul><li>Low Th1 response to M. leprae </li></ul><ul><li>Th2 response-->humoral immunity (not protective) </li></ul><ul><li>B. Tuberculoid : (Less contagious) Few bacteria present with multiple granulomas. </li></ul><ul><li>Organisms present at low levels </li></ul><ul><li>Low infectivity </li></ul><ul><li>Strong Th1 response to M. leprae antigens </li></ul><ul><li>C. borderline: Has mixed characteristics of both lepromatous and tuberculoid forms </li></ul>
  67. 81. <ul><li>HANSEN’S DISEASE is treatable </li></ul><ul><li>with outpatient therapy. </li></ul><ul><li>DRUGS OF CHOICE: </li></ul><ul><li>SULFONES (i.e. Dapsone) </li></ul><ul><li>PHENAZIDES (Lampren) </li></ul><ul><li>RIFAMPIN </li></ul><ul><li>CLOFAZAMINE </li></ul>
  68. 82. ~ 500,000 cases Worldwide Treatment – Dapsone (1940s-1970s) Rifampicin (1970s-) Multidrug Therapy- Dapsone, Rifampicin, Clofazamine

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