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. 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
a. M.tuberculosis
b. M.bovis
c. M.africanum
d. M.leprae
2.Oppertunistic
pathogens
a. Atypical mycobacteria
-M.kansai
-M.avium

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 370c
 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
1. Lowenstein Jensen’s
medium,Petrangini,Do
rset[contain egg]
2. Tarshis media [blood]
3. Loeffler[serum]
4. 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
1. Dubos
2. middlebrook’s
3. Proskauer
4. Beck’s
5. 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 600c 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 tuberculosisspreads
 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.
CD4 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.
1. Meningitis.
2. Pleurisy,
3. Involvement of Kidney,
4. Spine ( Potts spine )
5. Bone Joints,
6. 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
1. MICROSCOPY
2. CULTURE
3. SEROLOGY
4. MOLECULAR METHODS
5. BACTERIOPHAGE BASED ANALYSIS
6. MYCOLIC ACID CHARACTERISATION
7. 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 C14 – labeled palmitic acid & PANTA
antibiotic mixture.
Growing mycobacteria utilize the acid, releasing radioactive
CO2 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.

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

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

52. Sero diagnosis
ANTIGEN DETECTION
a. Latex particle agglutination
b. Dipstick ELISA
c. Sandwich ELISA
d. Capture ELISA

53. ANTIBODY DETECTION
a. ELISA
b. INSTA TEST TB

54. 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.

55. 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

56. 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.

57. 5 ELEMENTS OF DOTS ARE
1. Government commitment
2. Case detection through quality assured bacteriology
3. Standardised treatment with patient support and
supervision
4. An effective drug supply and management system
5. Monitoring and evaluation system

58. 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