mtb classification in orthodontics and dentofacial orthopaedics

mbt class

1. Mycobacterium tuberculosis MTB 1

2. Clinical case 50 year old man presented with history of low grade fever with evening rise of temperature and productive cough for two months. He had blood tinged sputum. History revealed loss of appetite, weight loss of 5 kg. chest X ray revealed nodular infiltrate was seen in the apical area of the upper lobe. Sputum smear was positive for AFB . Provisional diagnosis????? MTB 2

3. Etiological agents of Tuberculosis Mycobacterium tuberculosis complex M.tuberculosis M.bovis M.caprae M.pinnipedii M.microtii M.kanasii M.africanum MTB 3

4. MTB vs NTM MTB NTM(Atypical Mycobacteria) Always pathogenic Found in environment- occasionally cause opportunistic infections in humans Acid as well alcohol fast Only acid fast Responds to anti-tubercular therapy Does not respond to routinely used anti- tubercular drugs MTB 4

5. Need to differentiate between NTM and MTB Clinical picture non specific- Resembles tuberculosis For establishing the species so that specific therapy MTB 5

6. Infections caused by MTB P ulmonary Extra-pulmonary L ymphadenitis Plueral Genito -urinary Renal Skeletal CNS Abdominal Pericarditis Skin lesion Miliary TB Primary Post Primary/ Secondary pulmonary TB pulmonary TB MTB 6

7. Signs and symptoms of pulmonary TB MTB 7

8. Need for laboratory diagnosis To establish the specific etiological agent Initiation of specific anti- tubercular therapy To identify the drug resistant strains To prevent the spread of infection- 1 open case of PTB can spread the infection to 15 individuals Epidemiological purpose MTB 8

9. Approach to Laboratory diagnosis Specimen collection and transport Microscopic examination Culture Identification Molecular I mmunological Drug sensitivity testing MTB 9

10. Presumptive TB diagnosis(TB suspect) Any one of the following- Fever> 2 weeks Cough>2 weeks Haemoptysis significant weight loss Any abnormality in the chest X ray MTB 10

11. Specimen Collection and transport MTB 11

12. Specimen collection and transport 2 sputum specimen One early morning and second spot specimen 2ml – mucopurulent Containers to be labelled on side and not on the lid Examined on the same day In case of delay – specimen can be refrigerated for 48 hrs MTB 12

13. Microscopic examination ZN/Acid-fast stain Primary stain 1 % Carbol fuschin Mordant Heating Decolourizer 25%H 2 SO 4 Secondary stain 0.3% Methylene blue/ malachite green Fluorescent stain Primary stain Auramine O- phenol Decolourizer 1% HCl - Alcohol Secondary stain 0.1% KMnO 4 MTB 13

14. Microscopic examination ZN staining / Acid fast staining Fluorescent staining MTB 14 P ink coloured acid fast Straight or curved bacilli, arranged in singles, pairs or short clumps

15. Microscopic examination Acid fast/ZN stain Advantages- Easy to perform Economical Good specificity in endemic area Disadvantages- Poor sensitivity- 10 4 bacilli/ ml of sputum is required for smear to be positive Fluorescent stain Advantages- Greater sensitivity Less time taking Disadvantage- Expensive Requires expertise MTB 15

16. Microscopic examination Need for grading- Monitoring response to treatment Assessing the disease severity Assessing the infectiousness of the patient MTB 16 No.of bacilli seen No.of fields examined Result Grading No bacilli 100 OIF NEGATIVE 1-10 Bacilli 100 OIF POSITIVE Scanty 10-99 Bacilli 100 OIF POSITIVE 1+ 1-9 Bacilli/ per feild 50 OIF POSITIVE 2+ >10 bacilli/ per feild 20 OIF POSITIVE 3+ Grading of sputum smear as per RNTCP

17. Microscopy Advantage Disadvantage Rapid Cannot identify species Easy to perform Cannot identify viable bacilli Economical Cannot determine the drug susceptibility profile Less sensitive MTB 17

18. Microscopy Overcome the limitation of microscopy culture is performed. Advantage of culture- Sensitivity – 10-100 bacilli/ml Detect only viable bacilli High specificity Hence considered as “ Gold Standard ” MTB 18

19. Digestion and Decontamination Need To liquefy the thick tenacious secretion to release the organisms To inhibit the normal flora To concentrate the organism Methods of Decontamination Petroff’s method(using 4% NaOH ) NACL(N-acetyl-L-Cysteine)+ 2%NaOH MTB 19

20. Culture Solid Agar based methods 7H10 7H11 Potato starch agar Egg based media LJ Liquid Conventional Middlebrook 7H9 Middlebrook 7H11 Dubos’s Automated BACTEC-MB MGIT(mycobacterial growth indicator tube) BACT-T Alert ESP culture MTB 20

21. C ulture LJ media Enriched media Contains Hen’s egg, Mineral salt solution Aspargine Malachite green Sterilized by inspissation MTB 21

22. Growth on LJ Colony morphology of MTB N on pigmented r ough, tough, buff growth Dry rough raised irregular colonies with wrinkled surface MTB 22

23. Growth on LJ Colony morphology of M.bovis White coloured Smooth, flat, moist MTB 23

24. Culture MGIT( Mycobacterial growth indicator tube) RNTCP recommended for culture and drug sensitivity testing Automated method Principle - Uses an oxygen sensitive fluorescent compound dissolved in the broth. Initially , large amount of dissolved oxygen quenches emissions from the compound and no fluorescence can be detected. Later, actively respiring MTB consume oxygen and the absence of oxygen causes fluorescence MTB 24

25. MTB 25

26. Culture BACTEC Automated liquid culture method Principle- Media contains radiolabelled C atoms, MTB as it grows utilizes this radiolabelled C to produce CO2. detection of radiolabelled CO2 helps in detection of growth Not routinely recommended MTB 26

27. MTB 27

28. Automated methods Advantage Disadvantage Faster growth- 2 to 3 weeks Cannot differentiate between species Continuous monitoring Increased chances of contamination Expensive MTB 28

29. Culture method Advantages Disadvantage More sensitive ( can detect 10-100 bacilli) Time consuming( 4-6 weeks for growth and another 4 weeks for drug sensitivity testing) Detect viability Cannot be used for immediate diagnosis and treatment Determine drug resistance MTB 29

30. Biochemical reaction Test to differentiate MTB from M.bovis Niacin test- positive Nitrate reduction - positive Pyranzinamidase test- positive Resistance to thiophene2 carboxylic acid Test to differentiate MTB from NTM Aryl sulphatase – negative Catalase – weakly positive MTB 30

31. Difference between MTB and M.bovis MTB M.bovis Host Humans Bovine Oxygen preference Aerobic Microaerophilic Culture characteristics Eugonic , dry, rough,raised , irregular, wrinkled surface not emulsifiable Dysgonic Flat,smooth , moist,white , breaks up easily Niacin Positive Negative Nitrate reduction Positive Negative Growth in TCH Positive Negative 0.5% glycerol Helps growth No help MTB 31

32. Molecular methods Advantages- Less time taking More sensitive (can detect 1bacilli/ml) Detection of drug resistance Epidemiological typing MTB 32

33. Molecular methods Techniques available Polymerase chain reaction Line probe assay Transcription mediated amplification Strand displacement assay Nucleic acid amplification assay Ligase chain reaction MTB 33

34. Immunological methods Antigen detection Detection of MTB specific Ag ( lipoarabinomannam ) in urine, sputum P erformed using ELISA, Dipstick, latex agglutination test Low sensitivity ( 40-50%), hence not recommended MTB 34

35. Immunological methods Antibody detection N ot recommended Cross reactivity with other environmental mycobacteria Variable antibody response MTB 35

36. Animal innoculation Guinea pig Rabbit MTB 36

37. Drug sensitivity testing Solid culture media- LJ Liquid culture media- MGIT Molecular methods – determine the genetic determinants of drug resistance MTB 37

38. X-pert MTB Rif assay Principle- Closed cartridge based nucleic acid amplification test (CB-NAAT ) Detects presence of MTB and rifampicin resistance simultaneously Molecular beacon assay determining the 81 base pair “core” region of the wild-type rpoB gene associated with rifampicin resistance Done directly on the sample and results are available in < 2 hours MTB 38

39. MTB 39

40. MTB 40

41. Line-probe assay DNA strip test detects tuberculosis resistance to both rifampicin and isoniazid Results are available in 5 hours Highly accurate and cost-effective MTB 41

42. Diagnosis of EPTB Specimen collection- depending on the site infected Microscopic examination of smear is less sensitive Culture and molecular methods preferred Decontamination of specimen is not generally required MTB 42

43. Diagnosis of EPTB Site of infection Diagnostic features Pleural TB Elevated ADA and interferon gamma Renal TB Smear microscopy of e arly morning urine specimen done on consecutive 3-5 days Tuberculous meningitis Cobweb appearance on standing Elevated CSF pressure Raised protein and chloride level Decreased glucose levels MTB 43

44. Tuberculin test Uses- Diagnosis of latent TB infections Antigens used- PPD ( Purified protein derivative) It is purified preparation of active tuberculoprotein prepared by growing MTB in culture media Dosage- 1 TU( 0.00002 mg of PPD) Procedure Mantoux test- 0.1 ml PPD containing 1 TU is injected intradermally into the flexor aspect of the forearm Heaf test- not recommended MTB 44

45. Tuberculin test Reading and interpretation Taken after 24-48 hrs At the site of inoculation, an induration surrounded by erythema ≥10 mm- positive 6-9 mm- equivocal/doubtful <5 mm- negative MTB 45

46. Tuberculin test In adults Indicates past exposure or present exposure Does not confirm active infection In children Indicator of active infection MTB 46

47. Tuberculin test False positive False negative 8-14 weeks after BCG vaccination Early or advanced stages of TB, miliary TB Non tuberculous mycobacterial infection Decreased immunity MTB 47

48. BCG vaccine Discovered by bacillus, Calmette and guerin Developed by 239 serial passages of M.bovis in glycerol potato bile media over a period of 13 years Live attenauted vaccine In I ndia, Danish 1331 used Central BCG laboratory, Guindy Chennai Lyophilized form reconstituted by diluting it in normal saline MTB 48

49. BCG vaccine Administration- 0.1ml intradermal, at the insertion of left deltoid at birth Mantoux becomes positive after 8-14 weeks 80% efficacious In children helps prevents complicated TB Duration of immunity- 15 to 20 years Complications Ulceration and lymphadenitis at the site of inoculation Progressive TB in immunocompromised MTB 49

50. BCG vaccine Contradications to BCG HIV positive children Immunocompromised children Pregnancy Other uses Protection against leprosy Adjunctive therapy in bladder carcinoma MTB 50

51. Treatment of TB Aim of treatment Interrupt transmission by rendering patients non infectious Prevent morbidity and death by curing Patients Prevent emergence of drug resistance Prevent relapse MTB 51

52. Treatment Of TB First line Second Line Isoniazid Ethionamide Rifampicin Quinolones - Ofloxacin , Ciprofloxacin Pyranzinamide Aminoglycoside- kanamycin, Capreomycin,Amikacin Ethambutol Cycloserine Streptomycin Macrolide Para-amino salicylic acid MTB 52

53. Drug resistant TB MDR TB- strain of MTB which is resistant to Rifampicin or Isoniazid or both XDR- strain of MDR TB resistant strain also resistant to fluoroquinolones and atleast one of the injectables aminoglycoside (kanamycin, amikacin, capreomycin ) XXDR(TDR) strain of XDR TB resistant to all the currently available anti-TB drugs MTB 53

54. RNTCP MTB 54

55. List of RNTCP endorsed TB diagnostics Microscopy (for AFB): Sputum smear Ziehl -Nelson staining Sputum smear Fluorescence stains and examined under direct or indirect microscopy with or without LED Culture: Solid (Lowenstein-Jansen) media Liquid media (Middle Brook) using manual, semi-automated or automated e.g., Bactec , MGIT Rapid diagnostic molecular test: Conventional PCR-based LPA for MTB complex Real-time PCR-based Nucleic Acid Amplification Test (NAAT) for MTB complex e.g., Xpert MTB Rif assay

56. Treatment of Tuberculosis as per RNTCP MTB 57