Mycobacterium tuberculosis

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 Obligate parasites Opportinistic pathogens 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. In 1882 while working in Berlin he discovered the tuberculosis bacteria and the means of culturing it The Nobel Prize in Physiology or Medicine 1905

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 One cough can release 3,000 droplet nuclei One sneeze can release tens of thousands of droplet nuclei Millions of tubercle bacilli in lungs (mainly in cavities) Coughing projects droplet nuclei into the air that contain tubercle bacilli

9. M. tuberculosis does not s pread b y: 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. Characteristics Primary Postprimary Site Any part of lung Apical region Local lesion Small Large Cavity formation Rare Frequent Lymphatic involvement Yes Minimal Infectivity* Uncommon Usual Local spread Uncommon Frequent *Pulmonary cases Differences beween primary and postprimary tuberculosis

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 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 Type of lesion Specimen Pulmonary tuberculosis Sputum Laryngeal swabs or bronchial washings Gastric lavage Renal tuberculosis Urine Tuberculous meningitis CSF

22. Concentration of specimens Concentration of a specimen is done to achieve; Homogenisation of the specimen Decontamination i.e. to kill commensal bacteria 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 37 C for 2 – 8 weeks 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 M. tuberculosis

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 M. bovis is susceptible; therefore, does not grow M. bovis M. tuberculosis Growth in presence of TCH

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 C 14 radio labelled palmitic acid Mycobacteria metabolises C 14 radio labelled palmitic acid and release radio actively labeled 14 CO 2 BACTEC 460 instrument measures 14 CO 2 and reports in terms of growth 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 Caseous lesion at the site of inoculation Enlarged caseous inguinal lymph nodes Tubercles may be seen in spleen, lungs, liver, or peritoneum 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 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 Polymerase chain reaction (PCR) LAMP 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 Gu érin

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