Mycobacterium tuberculosis
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  • 1. Mycobacterium tuberculosis Dr. Pendru Raghunath Reddy
  • 2. Mycobacteia are slender rods that sometimes show branching, filamentous forms resembling fungal mycelium Classification The genus Mycobacterium contains three groups 1.Obligate parasites 2.Opportinistic pathogens 3.Saprophytes
  • 3. Obligate parasites Mycobacterium tuberculosis complex Contains M. tuberculosis, M. bovis, M. africanum, M. microti, M. canetti, M. caprae and M. pinnipedii Mycobacterium leprae
  • 4. Opportunistic pathogens Non-tuberculous mycobacteria (NTM) This group contains mixed group of isolates from diverse sources: birds, cold-blooded and warm-blooded animals, from skin ulcers, and from soil, water and other environmental sources They are opportunistic pathogens and can cause many types of disease
  • 5. Mycobacterium tuberculosis • long, slender, straight or curved, about (3 x 0.3 µm in size) • Aerobe • Acid fast bacilli • Intracellular • Mycolic acid, waxes & lipids in cell wall • Slow growing (Doubling time: 15 – 20 hours)
  • 6. The Nobel Prize in Physiology or Medicine 1905 In 1882 while working in Berlin he discovered the tuberculosis bacteria and the means of culturing it
  • 7. Pathogenesis Source of infection Open case of pulmonary tuberculosis Mode of infection Direct inhalation of aerosolised bacilli contained in the droplet nuclei of expectorated sputum Infection also occurs infrequently by ingestion for example, through infected milk, and rarely by inoculation
  • 8. Transmission of M. tuberculosis  Millions of tubercle bacilli in lungs (mainly in cavities)  Coughing projects droplet nuclei into the air that contain tubercle bacilli • One cough can release 3,000 droplet nuclei • One sneeze can release tens of thousands of droplet nuclei
  • 9. M. tuberculosis does not spread by: • Sharing dishes and utensils • Using towels and linens • Handling food • Sharing cell phones • Touching computer keyboard
  • 10. The initial infection with M. tuberculosis is referred to as a primary infection Subsequent disease in a previously sensitized person, either from an exogenous source or by reactivation of a primary infection is known as postprimary tuberculosis Both forms exhibit quite different pathological features
  • 11. Primary tuberculosis It is the initial infection by tubercle bacilli in a host The site of the initial infection is usually the lung These bacilli engulfed by alveolar macrophages, multiply and give rise to a subpleural focus of tuberculous pneumonia Which is commonly located in the lower lobe or lower part of the upper lobe to form the initial lesion or Ghon focus Some bacilli are carried to the hilar lymphnodes through macrophages, where additional foci of infection develops
  • 12. The Ghon focus, together with the enlarged hilar lymphnodes, form the primary complex M. tuberculosis multiply within the alveolar macrophages Th-1 cells produce cytokines to activate these macrophages Activated macrophages effectively destroy most of the tubercle bacilli However, some bacilli escape the macrophage- mediated destruction and induce the hypersensitivity reaction A hard tubercle or granuloma is formed due to the hypersensitivity reaction
  • 13. When fully developed, tubercle/granuloma consists of 3 zones 1. A central area of large, multinucleated giant cells containing tubercle bacilli 2. A mid zone of pale epitheloid cells, often arranged radially 3. A peripheral zone of fibroblasts, lymphocytes and monocytes  Later, peripheral fibrous tissue develops, and the central area undergoes caseation necrosis  A caseous tubercle may break into a bronchus, empty its contents there, and form a cavity  It may subsequently heal by fibrosis or calcification
  • 14. Tubercle or granuloma formation in tuberculosis
  • 15. Postprimary (secondary) tuberculosis It is due to reactivation of latent infection or exogenous reinfection and differs from the primary type in many respects It is characterised by chronic tissue lesions, the formation of tubercles, caseation and fibrosis Regional lymphnodes are only slightly involved, and they do not caseate Postprimary tuberculosis always begins at the apex of the lung, where the oxygen tension is highest The necrotic materials break out into the airways, leading to expectoration of bacteria-laden sputum, which is the main source of infection to contacts
  • 16. Differences beween primary and postprimary tuberculosis Characteristics Site Local lesion Cavity formation Lymphatic involvement Infectivity* Local spread *Pulmonary cases Primary Postprimary Any part of lung Apical region Small Large Rare Frequent Yes Minimal Uncommon Uncommon Usual Frequent
  • 17. Immunology Tubercle bacilli do not contain or secrete a toxin The exact basis of their virulence is not understood, but seems to be related to their ability to survive and multiply in macrophages Humoral immunity appears to be irrelevant The only specific immune mechanism effective is the CMI
  • 18. The key cell is the activated CD4+ helper T cell which can develop along two different paths: The Th1 and Th2 cells Th1 dependent cytokines activate macrophages, resulting in protective immunity and containment of the infection Th2 cytokines induce delayed type hypersensitivity (DTH), tissue destruction and progressive disease
  • 19. Koch’s phenomenon Koch’s phenomenon is a combination of hypersensitivity and immunity It is the response of a tuberculous animal to reinfection When a healthy guinea pig is inoculated subcutaneously with virulent tubercle bacilli, the puncture site heals quickly After 10-14 days, a nodule appears at the site of injection which ulcerates and the ulcer persists till the animal dies of progressive tuberculosis
  • 20. If on the other hand, virulent tubercle bacilli are injected in a guinea pig, which had received a prior injection of tubercle bacilli 4-6 weeks earlier, an indurated lesion appears at the site of injection in a day or two which undergoes necrosis to form a shallow ulcer This ulcer heals rapidly without involvement of the regional lymphnodes or tissues. This is called Koch’s phenomenon Koch’s phenomenon has got three components 1. A local reaction of induration and necrosis 2. A focal response in which there occurs acute congestion and even hemorrhage around the tuberculous foci in tissues 3. A systemic response of fever that may sometimes be fatal
  • 21. Laboratory diagnosis Specimen collection Type of lesion Specimen Pulmonary tuberculosis Sputum Laryngeal swabs or bronchial washings Gastric lavage Renal tuberculosis Urine Tuberculous meningitis CSF  Early morning sputum samples should be collected for 3 consecutive days in a sterile container  In case of renal tuberculosis, 3-6 morning urine samples should be collected
  • 22. Concentration of specimens Concentration of a specimen is done to achieve; 1. Homogenisation of the specimen 2. Decontamination i.e. to kill commensal bacteria 3. Concentrate the bacilli in the specimen without inactivation  The concentrate is used for smear preparation, cultutre and animal inoculation  Petroff’s method is used to concentrate sputum specimens
  • 23. Diagnostic Methods
  • 24. Direct Methods
  • 25. Direct Microscopy  Ziehl-Neelsen staining (hot staining method)  Kinyoun’s method (cold staining method)  Acid fast bacilli resist decolourisation with acid and alcohol once they have been stained with carbolfuchsin  AFB appear as pink, long, slender bacilli with beaded appearance
  • 26. Fluorescent staining by Auramine O or auramine rhodamine  Mycobacterium spp. will fluoresce yellow against dark background under fluorescent microscope
  • 27. Diagnosis of pulmonary tuberculosis under RNTCP DOTS: Directly observed treatment short-course
  • 28. Culture  Concentrated specimen is inoculated on Lowenstein – Jensen’s medium and incubated at 370C for 2 – 8 weeks M. tuberculosis  Colonies appear as buff coloured, dry, irregular colonies with wrinkled surface and not easily emulsifiable (Buff, rough and tough colonies)  Colonies are creamy white to yellow colour with smooth surface and easily emulsifiable M. bovis
  • 29. Differentiating features of M. tuberculosis and M. bovis
  • 30. Biochemical reactions Niacin test  M. tuberculosis lacks the enzyme that converts Niacin to Niacin ribonucleotide due to this large amount of Niacin accumulates in the culture medium  Niacin is detected by addition of 10% cyanogen bromide and 4% aniline in 96% ethanol  Positive reaction – canary yellow  M. tuberculosis – Positive  M. bovis - Negative
  • 31. Nitrate reduction test  M. tuberculosis produce an enzyme nitro reductase which reduces nitrate to nitrite  This detected by colorimetric reaction by addition of sulphanilamide and n-naphthyl- ethylene diamine dihydrochloride  Positive reaction – pink or red colour  M. tuberculosis – Positive  M. bovis - Negative
  • 32. M. tuberculosis is resistant to TCH (Thiophene - 2 - carboxylic acid hydrazide); hence, growth occurs Growth in presence of TCH M. bovis is susceptible; therefore, does not grow M. tuberculosis M. bovis
  • 33. Rapid culture methods 1. BACTEC 2. Mycobacterial growth indicator tube (MGIT) 3. Bac T/ Alert 3D system BACTEC system  Average time to detect Mycobacterium growth is 8 days  Radio metric method  Detects the presence of Mycobacteria based on their metabolism rather than visible growth
  • 34.  0.5 ml of processed sample is added to 4 ml of Middlebrook 7H12 broth containing C14 radio labelled palmitic acid  Mycobacteria metabolises C14 radio labelled palmitic acid and release radio actively labeled 14CO2  BACTEC 460 instrument measures 14CO and reports in terms of growth 2 index (GI)  A growth index of 10 or more is considered positive  More sensitive than traditional method  Problem of disposal of radio active waste
  • 35. Animal inoculation  0.5 ml of concentrated specimen is inoculated intramuscularly into the thigh of two healthy guineapigs  The animals are weighed prior to inoculation and thereafter at weekly intervals  Tuberculin test is done after 3 – 4 weeks  Progressive loss of weight and positive tuberculin skin reaction indicates infection  One animal is killed after 4 weeks and autopsied, if it shows no evidence of tuberculosis the other animal is autopsied after 8 weeks
  • 36. Autopsy shows 1. Caseous lesion at the site of inoculation 2. Enlarged caseous inguinal lymph nodes 3. Tubercles may be seen in spleen, lungs, liver, or peritoneum 4. Kidneys are not affected
  • 37. Allergic tests  Tuberculosis infection leads to the development of delayed hypersensitivity to M. tuberculosis antigen, which can be detected by Mantoux test Mantoux test (tuberculin test)  0.5 ml of PPD containing 5 TU is injected intradermally on flexor aspect of fore arm
  • 38.  Site is examined after 48 – 72 hrs  Induration of 10 mm or more is considered positive  Positive tuberculin test indicates hypersensitivity to tuberculoprotein denoting infection with tuercule bacilli or BCG immunisation, recent or past with or without clinical disease
  • 39. Uses 1. To diagnose active infection in infants and young children 2. To measure the prevalence of infection in community 3. Indication of successful BCG vaccination
  • 40. Detection of antibodies  Various methods such as enzyme linked immunosorbent assay (ELISA), radio immunoassay (RIA), latex agglutination assay have been employed for detection of antibodies in patient serum  However, diagnostic utility of these methods is doubtful  WHO has recommended that these tests should not be used for diagnosis of active tuberculosis
  • 41. Quantiferon-Gold  Is an in vitro assay that measures the cell mediated immune -response in the infected individuals through the levels of interferon gamma (IFN-γ) released by the sensitised T- lymphocytes after stimulation by M. tuberculosis antigens
  • 42. Molecular methods 1. Polymerase chain reaction (PCR) 2. LAMP 3. Ligase chain reaction PCR  Rapid method to detect M. tuberculosis directly in clinical samples based on DNA amplification  IS6110 sequence is generally targeted for detection M. tuberculosis complex
  • 43. Prophylaxis General measures Adequate nutrition, good housing and health education are as important as specific antibacterial measures Immunoprophylaxis The BCG (Bacille Calmette-Guerin) vaccine (0.1 ml), administered soon after birth by intradermal Injection failing which it may be given at any time during the first year of life This is a strain of M. bovis attenuated by 239 serial subcultures in a glycerine-bile-potato medium over a period of 13 years
  • 44. Bacille Calmette-Guérin = BCG! Albert Calmette Camille Guérin
  • 45. Chemoprophylaxis This is the administration of antituberculous drugs (usually only isoniazid) 1. To persons with latent tuberculosis (asymptomatic tuberculin positive) 2. To persons with a high risk of developing active tuberculosis 3. To the infant whose mother with active tuberculosis 4. To the children living with a case of active tuberculosis in the house  Isoniazid 5 mg/kg daily for 6 – 12 months is the usual course