Mycobacterium Tuberculosis

The PPT is mainly all about Mycobacterium Tuberculosis. Agents causing the disease Tuberculosis, pathogenesis, laboratory diagnosis, treatment and prophylaxis. It was made for both BSc and MSc students.

1. MYCOBACTERIA TUBERCULOSIS (MTB) Mary Mwinga

2. Tuberculosis Tuberculosis- Is an Ancient Disease -Spinal Tuberculosis in Egyptian Mummies - History dates to 1550 – 1080 BC. Robert Koch - Discoverer of Mycobacterium Tuberculosis (Koch’s disease) M. tuberculosis major human disease Affects healthy people problems association with AIDS multiple drug-resistance Chronic disease Prolonged treatment

3. What are Mycobacteria? Obligate aerobes growing most successfully in tissues with a high oxygen content, such as the lungs. Facultative intracellular pathogens : infect mononuclear phagocytes (e.g. macrophages). 3

4. Classification of Mycobacteria Tubercle bacilli Human – MTB Bovine – M. bovis Murine – M. microti Avian – M. avium Cold blooded – M. marinum Lepra bacilli Human – M. leprae Rat – M. leprae murium Mycobacteria causing skin ulcers M. ulcerans M. belnei Atypical Mycobacteria (Runyon Groups) Photochromogens Scotochromogens Nonphotochromogens Rapid growers Johne’s bacillus M. paratuberculosis Saprophytic mycobacteria M. butyricum M. phlei M. stercoralis M. smegmatis Others

5. General characters of the genus Slender rods Resist staining  but once stained, resist decolorization by dilute mineral acids  hence called ACID FAST BACILLI (AFB) Aerobic, Non-motile, Non-sporing, Non-capsulated. Growth generally slow Genus includes Obligate parasites Opportunist pathogens Saprophytes

6. Mycobacterium tuberculosis complex Includes Human and Bovine mycobacterium M .Africanism Tropical Africa M. microti do not cause human infections but in small mammals M. bovis Primarily infection among the cattle M.bovis infects Tonsils, Cervical nodes, can produce Scrofula. Enter through Intestines – infects the Ileocecal region. 7

7. What are atypical Mycobacterium Infects birds, cold blooded animals worm blooded animals Present in environment Opportunistic pathogens Others – Saprophytic bacteria -M butryicum present in butter -M. phlei -M smegmatis – present in Smegma 8

8. Atypical Mycobacterium 1 Photochromogens 2 Scotochromogens 3 Non Photochromogens 4 Rapid growers 9

9. Mycobacterium tuberculosis Epidemiology One of the most serious infectious diseases in the developing world One third of world’s population infected with M. tuberculosis Thirty million people have active disease Nine million new cases occur Three million people die of the disease, each year.

10. Mycobacterium tuberculosis (MTB) Morphology –Straight or slightly curved rods 3 x 0.3microns May be single, in pairs or in small clumps On conditions in growth appears as filamentous, club shaped, or in Branched forms.

11. Mycobacterium tuberculosis (MTB) Ziehl – Neelsen stain – Once stained by Carbol fuchsin , resist decolorization by 20% Sulphuric acide and absolute alcohol.  Acid & Alcohol Fast (AFB) Fluorescent dyes like Auramine O or Rhodamine also stain and the decolorization is resisted. Reason for Acid & Alcohol fastness – Presence of unsaponifiable wax Mycolic acid Semi permeable membrane around the cell Property of cell wall and related to integrity of the cell wall Staining may be uniform or granular

12. MTB : Cultural characters Slow-growing: generation time of 12 to 18 hours (c.f. 20-30 minutes for Escherichia coli ). Colonies appear after 2 weeks or at 6-8 weeks Hydrophobic with a high lipid content in the cell wall. Because the cells are hydrophobic and tend to clump together, they are impermeable to the usual stains, e.g. Gram's stain. MTB - Obligate aerobes. MTB grows more luxuriantly than M. bovis. Addition of 0.5% Glycerol supports growth of human strains. No effect or inhibitory effect on bovine strains.

13. MTB : Culture media Solid media – Egg containing Lowenstein-Jensen Medium Petragnini medium Dorset Blood containing – Tarshi’s Serum containing – Loeffler’s serum slope Potato containing – Pawlowsky’s Liquid media – Dubos’ Middlebrooks Proskauer & Beck’s Sula’s Sauton’s

14. Resistance Not specifically resistant to heat: destroyed by 60° C x 20 min. In sputum: can survive 20-30 hrs Relatively resistant to disinfectants. Survives exposure to 5 % Phenol 15 % Sulphuric acid 3 % Nitric acid 5 % Oxalic acid 4 % NaOH

15. Biochemical reactions Niacin test – Human MTB produces niacin when grown in egg medium. Aryl Sulphatase test – Enzyme Aryl sulphatase formed by only atypical mycobacteria. Neutral red test – Virulent strains of tubercle bacilli bind neutral red in alkaline solution while avirulent strains can not. Catalase– paroxidase test – Most atypical mycobacteria are strongly catalase positive while MTB is weakly positive. - MTB is strongly peroxidase positive while atypical mycobacteria are negative. Nitrate reduction test – Positive in MTB and negative in M. bovis

16. Tuberculosis Tuberculosis (TB) is a contagious disease. Like the common cold spreads through the air. Only people who are sick with TB in their lungs are infectious. M.O.T: cough, sneeze, talk or spit propels TB germs (bacilli) into the air. INFECTION: inhaling a small number of bacilli. Clinical picture : * Low grade fever * Weight loss * Night sweats * Fatigue * Cough & hemoptysis

17. Tuberculosis highly Communicable Disease. Someone in the world is newly infected with TB bacilli every second. Overall, one-third of the world's population is currently infected with the TB bacillus. 5-10% of people who are infected with TB bacilli (but who are not infected with HIV) become sick or infectious at some time during their life. People with HIV and TB infection are much more likely to develop TB. In India 1 death / Minute Half a million people die from disease every year in India (one death every minute) 18

18. Pathology and Pathogenesis of Tuberculosis Source of Infection – Open case of Pulmonary Tuberculosis. Has potential to infect 20 – 25 healthy persons before cured or dies Through Coughing , Sneezing, or Talking. Each act can spill 3000 infective nuclei in the air, Infective particles are engulfed by Alveolar Macrophages. 19

19. Generation of Droplet Nuclei One cough produces 500 droplets The average TB patient generates 75,000 droplets per day before therapy This falls to 25 infectious droplets per day within two weeks of effective therapy 20

20. Predisposing Factors Genetic basis, Age Stress, Nutrition, Co existing infections Eg HIV 21

21. Modes of infection 1- Droplet infection - Person to person by inhalation aerosols - Mycobacterium tuberculosis (Pulmonary tuberculosis) 2- Ingestion of milk - Infected cattle - Mycobacterium bovis (Intestinal tuberculosis) 3- Contamination of abrasion - Laboratory workers (Skin infection)

22. Pathogenesis of tuberculosis Pulmonary tuberculosis. Extra-pulmonary tuberculosis.

23. Pathogenesis of tuberculosis 1. Pulmonary tuberculosis infects lungs distributed within macrophages facultative intracellular pathogen inhibits phagosome-lysosome fusion resists lysosomal enzymes other MTB factors: Mycobactin - siderophore Cord factor - damages mitochondria

24. Pathogenesis * Inhalation of tubercle bacilli * They multiply in the alveolar macrophages * An early tubercle (granuloma) is formed

25. Mechanisms of Infection Within 10 days of entry of Bacilli - clones of Antigen specific T Lymphocytes are produced Can actively produce Cytokines, Interferon γ  activate Macrophages to form cluster or Granuloma. Tubercle with Caseous Necrosis 26 Giant cells Tubercle bacilli Partially activated macrophage Lymphocyte Fully activated macrophage

26. Basis of Tubercle formation (Granuloma). Tubercle is an Avascular granuloma Containing central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts. Produce lesions may be Exudative or Productive 27

27. Diagram of a Granuloma NOTE: ultimately a fibrin layer develops around granuloma (fibrosis) , further “walling off” the lesion. Typical progression in pulmonary TB involves caseation , calcification and cavity formation . 28

28. Tubercle discharging Bronchial tree TNF- a TNF- a 29

29. Immunity in Tuberculosis. CD 4 T Lymphocytes with Th 1 or Th 2 secrete - Cytokines, Interleukin 1,and 2 , Interferon's γ and Tumor necrosis factor. Th 1: secrete Cytokines  Activate Macrophages  Results in protective Immunity, and contain Infection. Th 2 manifests with Delayed Hypersensitivity  DTH causes Tissue destruction.  and disease will progress. . 30

30. Immunity in Tuberculosis Activated Macrophages - Epithelioid cells  Forms cluster a granuloma Activated macrophages turn into Giant cells . Granuloma contains: necrotic tissue, dead macrophages, cheese like caseation. Apoptosis of bacteria laden cells contribute to protective immunity. 31

31. Lesions in Tuberculosis Exudative and Productive. 1. Exudative – Acute inflammatory reaction with edema fluid – contains Polymorphs- Lymphocytes – later Mononuclear cells. -Bacilli are virulent - Host responds with DTH Injurious. 2. Productive Type protective Immunity 32

32. Pathogenesis * Lesions, healing or progression of infection depend upon 1- Dose of infecting mycobacteria 2- Resistance and hypersensitivity of host * Virulence : - Glycolipids on the outer surface of bacteria - Enhance granuloma formation - Inhibit migration of polymorphonuclear leucocytes - Help survival of tubercle bacilli inside macrophages

33. Pathogenesis A- Primary infection: * An exudative lesion : - spread to regional lymph nodes - A scar of healing may later calcify ( Ghon’ focus ) - Lymph nodes caseate and then calcify - Bacilli in the lesion slowly die - Tuberculin test becomes positive - The person is immune & hypersensitive

34. Classical tubercular lesion – Granuloma with typical Langhan’s giant cells, epithelioid cells, lymphocytes and fibrosis.

35. There are multiple light areas (opacities) of varying size that run together (coalesce). Arrows indicate the location of cavities within these light areas. The appearance is typical for chronic pulmonary tuberculosis.

36. Pathogenesis B- Reactivation type : * Activation of tubercle bacilli due to immunity * Formation of tubercles that caseate - fibrosis - open into a bronchus ( open tuberculosis ) * Tubercle bacilli erode a blood vessels - Infect any organ (Miliary T.B.)

37. Koch’s Phenomenon Tuberculosis infected Guinea pig if injected with Living Tubercle bacilli: -The site around the injection becomes necrotic. Koch found the same reaction when injected with old Tuberculin ( heated and concentration of the tubercle bacilli ) It has produced the same reaction This is called as Koch’s Phenomenon. 38

38. Post Primary Tuberculosis Due to Reactivation of Latent infection. Also due to Exogenous reinfection Differs from Primary Infection. Leads to – Cavitation's of Lungs, Enlargement of Lymph nodes, Expectoration of Bacteria laden sputum Dissemination into Lungs and other extra pulmonary areas. 39

39. Complication of Tuberculosis . Meningitis. Pleurisy, Involvement of Kidney, Spine ( Potts’ spine ) Bone Joints, Miliary tuberculosis 40

40. Symptoms and Sings of Tuberculosis 41

41. Clinical Illness with Tuberculosis Pulmonary Disease – Major manifestation with involvement of Lungs -Hemoptysis, Chest pain Fever sweets -Anorexia -Cavity formation in Lungs 42

42. 2. Extra pulmonary Tuberculosis Bacteria on circulation leads to bacteremia leads to involvement of -GUT, Genito urinary system, Meningitis -Gastro Intestinal system, skin, Lymph nodes Bone marrow. -Spinal infection Potts spine, Arthritis 43

43. Multidrug-resistant tuberculosis (MDR-TB) DEF: MDR-TB is resistance to isoniazid and rifampicin, with or without resistance to other first-line drugs (FLD). XDR-TB is resistance to at least isoniazid and rifampicin, and to any fluoroquinolone, and to any of the three second-line injectables (amikacin, capreomycin, and kanamycin). Drug resistance is due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients. As a result of a number of actions including, administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment. 44

44. Laboratory diagnosis M. tuberculosis Acid fast bacteria demonstration in sputum smear. Culture on L J media Biochemical identification Antibiotic sensitivity test Skin testing delayed hypersensitivity tuberculin protein purified derivative (PPD) X-ray PCR

45. SPECIMENS * According to site of infection : - Sputum - Urine - Body fluids - Gastric lavage - Blood - Tissue biopsy * S pecimens need appropriate processing Liquefaction with N-acetyl-L- cysteine Sputum Decontamination with NaOH Centrifugation

46. Laboratory Diagnosis Pulmonary TB – Specimen – ZN Sputum – Early morning, if scanty 24 hrs , three consecutive day samples. Laryngeal swabs or gastric lavage in children. Microscopy –See at least 100 field / 10 minutes. Grading – 1+ -> 3-9 bacilli in entire smear 2+ -> 10 or more in entire smear 3+ -> 10 or more bacilli seen in most oil immersion fields Advantage : - cheap – rapid - Easy to perform - High predictive value > 90% - Specificity of 98% Disadvantages: - sputum ( need to contain 5000-10000 AFB/ ml.) - Young children, elderly & HIV infected persons may not produce cavities & sputum containing AFB.

47. Acid fast bacilli 48

48. Detecting AFB by fluorochrome stain using fluorescence microscopy: The smear is stained by auramine-O dye . TB bacilli are stained yellow against dark background & easily visualized using florescent microscope. Advantages: - More sensitive - Rapid Disadvantages: - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

49. Acid fast Bacilli seen as in Florescent Microscope 50

50. Quantitation of AFB in Sputum Smears No of Bacilli No of Fields Report as 0 300 Negative 1-2 300 Doubtful 1- 9 100 1+ 1- 9 10 2+ 1-9 1 3+ 10 or >10 1 4+ 51

51. CULTURE: Nature of Growth Characters M tuberculosis is obligate aerobe. M.bovis Microaerophilic M.tuberculosis growth luxierently M.tuberculosis eugonic M bovis is dysgonic When grown on 0.5% glycerin M tuberculosis growth improves Sodium pyruvate improves the growth of both organism. 52

52. Culture Lowenstein Jensen Medium – Selective medium. Always in screw capped bottle. Color: Bluish-Green. -Contains – Egg protein – Solidifying agent -Mineral salts – Mg sulphate, Mg citrate -Asparagine -Malachite Green – Selective agent GROWTH : grows very slowly: several weeks non-pigmented colonies niacin production: differentiates from other mycobacteria Sterilized by - Inspissation

53. On L J Medium MTB appear as dry, rough raised irregular colonies. -Appear wrinkled, creamy white -Become yellowish. M. bovis appear as flat smooth, moist, white and break up easily. Advantages : - Specificity about 99 % More sensitive (need lower no. of bacilli 10-100 / ml) - Can differentiate between TB complex & NTM using biochemical reactions - Sensitivity tests for antituberculous drugs ( St, INH, Rif., E) Disadvantages : Slowly growing ( up to 8 weeks ) 54

54. Laboratory Diagnosis Extra -Pulmonary TB – Specimen – CSF – in suspected meningitis Pleural fluid & other exudates 2-3 days urine in renal TB Biopsy material.

55. Concentration methods Purpose – Homogenization & Concentration in sputum & other specimens. Methods – Useful for microscopy, culture & animal inoculation Petroff’s method Most widely used Equal volumes of Sputum + 4% NaOH incubated at 37C X 20 min. Centrifuge at 3000 rpm X 30 min. Sediment neutralized by N/10 HCl . Can be used for smear, culture, animal inoculation Simpler method To avoid centrifugation & Neutralization Equal volumes of sputum + solution of Cetrimonium bromide 20 g + NaOH 40 g in one litre . Allow to stand for five minutes. Inoculate Acid Buffered LJ Medium with swab.

56. Antigenic Characters Group specificity due to Polysaccharides. Type specificity due to protein antigens. Delayed hypersensitivity to proteins Related to each other species Some relation between lepra and tubercle bacilli Serology – Tests not useful Antigenic homogeneity between < bovis and M.microti 57

57. Molecular Typing DNA finger printing differentiates different strains of Mycobacterium species Treating the organism with Restriction endonuclease yields Nucleic acid fragments of varying length and strain specific Used in epidemiological studies 58

58. Finger printing Methods Done with Chromosomal insertion sequence IS 6110 present in most strains of Tubercle bacilli. Now entire genome of M. tuberculosis is sequenced Several Molecular methods are available for studies 59

59. Tuberculin Test (Mantoux test) Delayed hypersensitivity skin test to assay: -cell mediated immunity to tubercle bacillus Material: A purified protein derivative (PPD) Dose : 0.1 ml of (PPD) is injected intradermal Reading: Positive test- - Induration equal or greater than 10 mm - Develop 48-72 hours after injection Negative test : induration less than 10 mm. Positive test interpretation:  indicates exposure to organism  does not indicate active disease

60. Tuberculin Test Interpretation: * A positive test indicates previous exposure and carriage of T.B. * A negative tuberculin test excludes infection in suspected persons * Tuberculin positive persons may develop reactivation type of T.B. * Tuberculin negative persons are at risk of gaining new infection

61. Tuberculin Testing - Limitations False positive reactions are mainly due to: Infection with nontuberculous mycobacteria * False negative reactions may be due to - Sever tuberculosis infection (Miliary T.B.) - Hodgkin ’ s disease Corticosteroid therapy - Malnutrition - AIDS Children below 5 years of age with no exposure history: - Positive test must be regarded suspicious 62

62. Recent Methods for Diagnosis I – BACTEC 460 ( rapid radiometric culture system ) -specimens are cultured in a liquid medium (Middle brook7H9 broth base) containing C 14 – labeled palmitic acid & PANTA antibiotic mixture. - Growing mycobacteria utilize the acid, releasing radioactive CO 2 which is measured as growth index (GI) in the BACTEC instrument. -The daily increase in GI output is directly proportional to the rate & amount of growth in the medium. 63

63. III Polymerase Chain Reaction (PCR) & Gene probe - Nucleic acid probes & nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from mycobacterial cells. Advantages: - Rapid procedure - High sensitivity (1-10 ( 3 – 4 hours) bacilli / ml sputum) 64

64. Tuberculosis and HIV infection HIV association has become a threat to the developed countries too HIV association will lead to rapid spread of tuberculosis Considerations: HIV is the strongest risk factor for progression to active disease HIV kills CD4 + T Helper cells which normally inhibit M. tuberculosis HIV interferes with PPD skin test Protease inhibitors interfere with rifampin 65

65. MDR tuberculosis MDR TB is a global threat. In 1993 WHO declared Tuberculosis a Global emergency Animals shed the bacilli in Milk, human’s get infected after drinking the unsterilized Milk Pasteurization has reduced the incidence of Bovine tuberculosis. 66

66. TREATMENT -- Drugs - Rifampicin - Isoniazid   isonicotinylhydrazide (INH) -- Bactericidal -Pyrazinamide -Streptomycin -Ethambutol -Ethionamide -Thioacetazone/ amithiozone -- Bacteriostatic -Paraminosalicylic acid -Cycloserine

67. Treatment Drugs used : 1- First line drugs : - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective): - Kanamycin - capreomycin - Cycloserine - ethionamide - ciprofloxacin - Ofloxacin * Noncompliance (failure to complete the course): - Directly observed therapy (DOT) -Health care workers observe the medication

68. Treatment Chemoprophylaxis – INH for one year - Domicilliary treatment preferred Use multiple drug therapy to prevent emergence of resistant mutants * Long duration treatment (6-18 months) * Four drugs are usually started in initial therapy due to: - Intracellular location of bacilli - Slow growth rate of bacilli - Caseous material blocks penetration of drugs - Some bacilli persist in a metabolically inactive state * Sputum becomes non-infective 2-3 weeks after starting therapy

69. Immuno-prophylaxis Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (BCG). The strain causes self limited lesion and induces hypersensitivity & immunity. Coverts tuberculin negative person to positive reactor. Immunity lasts for 10-15 years. Immunity 60-80%

70. BCG Given at birth without tuberculin testing Protects against TB,  the disease runs milder course in protected, prevents skeletal, meningeal & miliary forms. Also found useful in leprosy, leukemias and other malignancies by non-specific stimulation of RE system.