Tuberculosis

Mycobacterium tuberculosis-importance of TB day,classification of Mycobacterium species,Details on Mycobacterium tuberculosis-morphology,culture,resistance,biochemical reactions,antigenic characters,mode of transmission,pathogenesis,complications,lab diagnosis,treatment,DOTS Strategy and prophylaxis

1. TUBERCULOSIS Mrs.VINDHYA .V.V ASST.POFESSOR Microbiology

2. IMPORTANCE OF TB DAY Tuberculosis is a bacterial disease caused by the bacteria MYCOBACTERIUM TUBERCULOSIS TB day is celebrated to raise the common public health awareness about the epidemic disease of tuberculosis its causes,prevention ,and cure ,as well as the efforts done inorder to totally eradicate this disease. As around world 1.7 million of the people are dying of this disease every year. Timely TB identification and treatment are essential for succesful recovery

3. MYCOBACTERIA Mycobacteria are acid fast bacilli They do not stain readly,but once stained they resist decolorisation due to the presence of mycolic acid in the cell wall.so they are called as acid fast bacilli They appear in filamentous form like fungi,hence called as Mycobacteria They are Obligate aerobes growing most successfully in tissues with a high oxygen content, mainly in the lungs

4. History Robert Koch isolated Mycobacterium Tuberculosis in 1882

5. Classification of Mycobacteria 1 .Obligate pathogen Mycobacterium tuberculosis complex M.tuberculosis M.bovis M.africanum M.leprae 2 .Oppertunistic pathogens Atypical mycobacteria - M.kansai - M.avium

6. MYCOBACTERIUM TUBERCULOSIS

7. MORPHOLOGY Straight, slightly curved Acid fast Rod shaped 3 x 0.3microns size Gram reaction is positive May be single, in pairs or in small clumps Non motile Non sporing,Non capsulated

8. MTB : Cultural characters MTB - Obligate aerobe Grow slowly. Generation time 14-15 hrs Colonies appear after 2 weeks or at 6-8 weeks Grows at 37 c Ph between 6.4 to 7.0 Addition of 0.5%glycerol improves its growth.

9. Nature of Media Used Helps the growth needs Solid Medium is commonly used. SOLID MEDIA Lowenstein Jensen’s medium,Petrangini,Dorset [contain egg] Tarshis media [blood] Loeffler [serum] Pawlosky [potato] Most commonly used is Lowenstein Jensen’s Medium Contain coagulated egg Mineral salt solution Asparagine's Malachite green[selective agent]

10. Liquid media Dubos middlebrook’s Proskauer Beck’s Sula’s

11. On L J Medium M.tuberculosis appear dry, rough raised irregular colonies Appear wrinkled They appear creamy white Become yellowish

12. ON LIQUID MEDIA Growth begins at the bottom,creeps up sides and forms prominantsurface pellicle formation

13. Resistance of Mycobacterium Mycobacterium are killed at 60 c in 15 – 20 mt In sputum they survive for 20 – 30hours Relatively resistant to several chemicals including Phenol 5 % Sensitive to Glutaraldehyde and Formaldehyde Bacilli survive in Droplets for 8 – 10 days

14. Biochemical Tests on Mycobacterium spp Niacin test – positive for Mycobacterium Aryl sulphatase test – Negative Neutral red test-Positive Catalase and peroxidase test – Differentiates Atypical from Typical Most Atypical are strongly Catalase positive Tubercle bacilli are weakly positive Tubercle bacilli are peroxidase positive – not atypical Nitrate reduction test-Positive

15. Antigenic Characters Group specificity due to Polysaccharides Type specificity to protein antigens Delayed hypersensitivity to proteins

16. How tuberculosis spreads Tuberculosis (TB) is a contagious disease. Source of Infection – Open case of Pulmonary Tuberculosis. Coughing , Sneezing, or Talking. Each act can spill 3000 infective nuclei in the air, Infective particles are engulfed by Alveolar Macrophages.

17. Tuberculosis spread by Respiratory route

18. Poverty and Crowded living spreads Tuberculosis

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. Dr.T.V.Rao MD 20

21. Predisposing Factors Genetic basis, Age Stress, Nutrition, Co existing infections Eg . HIV Ingestion of infected milk

22. Pathway of pathogenesis Inhalation of bacteria Reach lung Ingested by alveolar macrophages Multiplication in macrophages Ghon focus in lower lobe Hilar lymph node

23. Primary complex formation Healing and calcification Haematogenous Cause latent infection miliary , meningeal disseminated tb Reactivation or exogenous infection Secondary tb Usually pulmonary tb

24. 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

25. Multiorgan Involvement in Tuberculosis.

26. Mechanisms of Infection Mycobacterium do not produce toxins. Allergy and Immunity plays the major role. Only 1/10 of the infected will get disease. Cell Mediated Immunity plays a crucial role. Humoral Immunity – not Important. CD 4 Cell plays role in Immune Mechanisms .

27. Lesion –known as Tubercle Tubercle is a Avascular granuloma Contain central zone of giant cells with or without caseation and peripheral zone of Lymphocytes and Fibroblasts. Produce lesions may be Exudative or Productive

28. Tubercle with Caseous Necrosis Dr.T.V.Rao MD 28 Giant cells Tubercle bacilli Partially activated macrophage Lymphocyte Fully activated macrophage

29. Lesions in Tuberculosis Exudative – and Productive Exudative – Acute inflammatory reaction with edema fluid – contains Polymorphs- Lymphocytes – later Mononuclear cells. Bacilli are virulent . Productive Type protective Immunity

30. Dr.T.V.Rao MD 30 Majority of the Tuberculosis are Pulmonary

31. Symptoms and Signs of Tuberculosis Dr.T.V.Rao MD 31

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

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

34. Dr.T.V.Rao MD 34 Tuberculosis - Lymphadenitis

35. Diagnosis of Tuberculosis

36. Types of specimens : -Sputum. - BAL. -Pleural effusions - Urine - Stool -CSF -Aspiration ( gastric – cold abscess) - Blood in case of haematogenous TB

37. METHODS MICROSCOPY CULTURE SEROLOGY MOLECULAR METHODS BACTERIOPHAGE BASED ANALYSIS MYCOLIC ACID CHARACTERISATION ANIMAL INOCULATION

38. Concentration of specimens When AFB not concentrated by direct microscopy then specimens are concentrated by different techniques.2methods are there 1.petroff’s method 2.modified petroff’s 1.petroff’s method- - equal volumes of sputum sample and 4%sodium hydroxide are mixed incubated at 370c for 20-30 minutes and sediment used for microscopy and culture .

39. MICROSCOPY 1- Sputum smears stained by Z-N stain Three morning successive mucopurulent sputum samples are needed to diagnose pulmonary TB . 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.

40. GRADING BY RNTCP Number of bacilli observed Grade ZERO -- 1-2bacilli/300fields -- 1-9bacilli/100field -- 1-9bacilli/10field --- 1-9bacilli/field -- >9bacilli/field -- Negative Positive 1+ 2+ 3+ 4+

41. 2 - Detecting AFB by fluorochrome stain using fluorescence microscopy : The smear may be stained by aura mine-O dye. In this method the 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

42. Acid fast Bacilli seen as in Florescent Microscope

43. 3- Culture on L J media

44. Tuberculin Test[ mantoux test] It is typeIV hypersensitivity reaction Purified protein derivative is inoculated intradermally on forearm Site observed after 72hrs for appearance of area of erythema and induration About 10mm indicate positive test Below 5mm indicate negative Between 5&9 mm indicates doubtful test

45. 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.

46. III Polymerase Chain Reaction (PCR) & Gene probe

47. Other rapid identification methods are MGIT— Mycobacteria Growth Indicator Tube BACTEC 9000MB BACTEC MGIT 960 ESP CULTURE SYSTEM 2 SEPTICHECK AFB SYSTEM[Manual]

48. Sero diagnosis ANTIGEN DETECTION Latex particle agglutination Dipstick ELISA Sandwich ELISA Capture ELISA

49. ANTIBODY DETECTION ELISA INSTA TEST TB

50. ANIMAL INOCULATION The conc.sample is inoculated intra muscularily into the thigh of 2 healthy Guinea pigs about 12 wks old. The animals are weighed b4 inoculation and intervels thereafter Progressive loss of weight is an indication of the infection Positive animal will show a caseous lesion at the site of inoculation.

51. Treatment Drugs used : 1- First line drugs : - Isoniazid - Rifampicin - Pyrazinamide - Ethambutol - Streptomycin 2- Second line drugs (more toxic and less effective): - Kanamycin - capreomycin - Cycloserin - ethionamide - ciprofloxacin - Ofloxacin Mycobacteria develops drug resistance.Reason for this is –improper prescription,delayed initiation of effective therapy.and HIV infections

52. So as a control measure DOTS strategy becomes important DOTS-Directly Observed Treatment Short course ‘This ensures that,the patient takes medicines regularly untill they are cured’-during the intensive phase a health worker watches the patient taking drug in his or her presence.

53. 5 ELEMENTS OF DOTS ARE Government commitment Case detection through quality assured bacteriology Standardised treatment with patient support and supervision An effective drug supply and management system Monitoring and evaluation system

54. Immuno -prophylaxis BCG Intradermal injection of live attenuated vaccine Bacille Calmette -Guerin (BCG). The strain causes self limited lesion and induces hypersensitivity & immunity. Immunity lasts for 10-15 years. Immunity 60-80% Some studies proved BCG is doubtful value in prevention of Tuberculosis