Tuberculosis Teaching Basics

1. Dr.T.V.Rao MD 1

2. HISTORY of
Tuberculosis
Tuberculosis Is an
Ancient Disease
Spinal Tuberculosis
in Egyptian
Mummies
History dates to
1550 – 1080 BC
Identified by
PCR
Dr.T.V.Rao MD 2

3. Robert Koch
Discoverer of
Mycobacterium
Tuberculosis
Dr.T.V.Rao MD 3

4. What are Mycobacteria?
• Obligate aerobes growing most
successfully in tissues with a high
oxygen content, such as the lungs.
• Facultative intracellular pathogens
usually infecting mononuclear
phagocytes (e.g. macrophages).
Dr.T.V.Rao MD 4

5. Classification of Mycobacteria
1. Tubercle bacilli
a) Human – MTB
b) Bovine – M. bovis
c) Murine – M. microti
d) Avian – M. avium
e) Cold blooded – M.
marinum
2. Lepra bacilli
a) Human – M. leprae
b) Rat – M. leprae murium
3. Mycobacteria causing
skin ulcers
a) M. ulcerans
b) M. belnei
4. Atypical Mycobacteria
(Runyon Groups)
a) Photochromogens
b) Scotochromogens
c) Nonphotochromogens
d) Rapid growers
4. Johne’s bacillus
M. paratuberculosis
6. Saprophytic mycobacteria
a) M. butyricum
b) M. phlei
c) M. stercoralis
d) M. smegmatis
e) Others
Dr.T.V.Rao MD 5

6. Mycobacterium differ from other
routinely isolated Bacteria
• Slow-growing with a generation time of 12 to
18 hours (c.f. 20-30 minutes for Escherichia
coli).
• Hydrophobic with a high lipid content in the
cell wall. Because the cells are hydrophobic
and tend to clump together, they are
impermeable to the usual stains, e.g. Gram's
stain
Dr.T.V.Rao MD 6

7. Acid fast bacilli
• Known as “Acid-fast bacilli"
because of their lipid-rich cell
walls, which are relatively
impermeable to various basic
dyes unless the dyes are
combined with phenol.
Dr.T.V.Rao MD 7

8. How they are Acid fast
• Once stained, the cells resist
decolourization with acidified
organic solvents and are therefore
called "acid-fast". (Other bacteria
which also contain mycolic acids,
such as Nocardia, can also exhibit
this feature.)
Dr.T.V.Rao MD 8

9. Mycobacterium tuberculosis complex
• Includes Human and Bovine
mycobacterium
• M .Africanism Tropical Africa
• M.microti do not cause human
infections but in small
mammals
Dr.T.V.Rao MD 9

10. M.bovis
• Primarily infection among the
cattle
• M.bovis infects Tonsils, Cervical
nodes, can produce Scrofula.
• Enter through Intestines – infects
the Ileocecal region.
Dr.T.V.Rao MD 10

11. What are atypical MycobacteriumWhat are atypical Mycobacterium
• Infects birds, cold blooded animals worm
blooded animals
• Present in environment
• Opportunistic pathogens
• Others – Saprophytic bacteria
M butryicum present in butter
M.phlei
M smegmatis – present in Smegma
Dr.T.V.Rao MD 11

12. Atypical Mycobacterium
• 1 Photochromogens
• 2 Scotochromogens
• 3 Non Photochromogens
• 4 Rapid growers
Dr.T.V.Rao MD 12

13. MOST IMPORTANT AMONG
INFECTIOUS DISEASES
• Tuberculosis (TB) is the leading
cause of death in the world from a
bacterial infectious disease. The
disease affects 1.8 billion
people/year which is equal to one-
third of the entire world population.
Dr.T.V.Rao MD 13

14. Poverty and Crowded living spreads
Tuberculosis
Dr.T.V.Rao MD 14

15. Tuberculosis infects Famous people too
Dr.T.V.Rao MD 15

16. What are Mycobacteria?
• Obligate aerobes growing most
successfully in tissues with a high
oxygen content, such as the lungs.
• Facultative intracellular pathogens
usually infecting mononuclear
phagocytes (e.g. macrophages).
Dr.T.V.Rao MD 16

17. General characters of the genus
• Slender rods
• Resist staining but
once stained, resist
decolonization by
dilute mineral acids;
hence called ACID
FAST BACILLI (AFB)
• Aerobic, Non-motile,
Non-sporing, Non-
capsulated.
• Growth generally slow
• Genus includes
– Obligate parasites
– Opportunist pathogens
– Saprophytes
Dr.T.V.Rao MD 17

18. Morphology of Mycobacterium
tuberculosis
• Straight, slightly
curved Rod shaped 3
x 0.3microns
• May be single, in
pairs or in small
clumps
• On conditions in
growth appears as
filamentous, club
shaped, or in
Branched forms. Dr.T.V.Rao MD 18

19. ACID FAST BACILLI
• Known as “Acid-fast bacilli"
because of their lipid-rich cell
walls, which are relatively
impermeable to various basic
dyes unless the dyes are
combined with phenol.
Dr.T.V.Rao MD 19

20. Important MycobacteriumImportant Mycobacterium
• Mycobacterium tuberculosis, along with
M. bovis, M. africanum, and M. microti
all cause the disease known as
tuberculosis (TB) and are members of
the tuberculosis species complex. Each
member of the TB complex is
pathogenic, but M. tuberculosis is
pathogenic for humans while M. bovis is
usually pathogenic for animalsDr.T.V.Rao MD 20

21. Avian Tuberculosis
• Transmitted by ingestion and inhalation of aerosolized
infectious organisms from feces.
• Oral ingestion of food and water contaminated with
feces is the most common method of infection.
• Once ingested, the organism spreads throughout the
bird's body and is shed in large numbers in the feces.
• If the bacterium is inhaled, pulmonary lesions and skin
invasions may occur
• transmission of avian TB is from bird to human not
from human to human.
Dr.T.V.Rao MD 21

22. Acid Fast Bacilli seen in a specimen of
Sputum
Dr.T.V.Rao MD 22

23. Acid fast bacilli
Dr.T.V.Rao MD 23

24. Acid fast Bacilli seen as in Florescent
Microscope
• After staining with Ziehl
Neelsen method or
Fluorescent method
( Auramine or Rhodamine
they resist decolonization
by 20% Sulphuric acid and
absolute alcohol for 10 mt,
• So called as Acid and
Alchool fast.
Dr.T.V.Rao MD 24

25. Why they are Acid Fast
• The character of
Acid fastness is
due to presence of
Unsapnofiable
wax ( Mcolic acid
and semi
permeable
membrane around
the cell)Dr.T.V.Rao MD 25

26. MTB : Cultural characters
• Grow slowly.
Generation time
14-15 hrs
• Colonies appear
after 2 weeks or at
6-8 weeks
• MTB - Obligate
aerobe
• MTB grows more
luxuriantly (eugonic)
than M. bovis
(dysgonic).
• Addition of 0.5%
Glycerol supports
growth of human
strains. No effect or
inhibitory effect on
bovine strains.
Dr.T.V.Rao MD 26

27. Culturing Acid Fast Bacilli
• Slow to grow ,
• Generation time is 14 –
15 hours
• > 2 weeks minimal
required period
• Grows at 370
c do not
grow below 250
c
• Ph between 6.4 to 7.0
Dr.T.V.Rao MD 27

28. Eight Week Growth of Mycobacterium tuberculosis
on Lowenstein-Jensen Agar
Dr.T.V.Rao MD 28

29. Nature of Media Used
• Helps the growth needs
• Solid Medium is commonly
used
• Lowenstein Jensen’s medium
• Petrangini
• Middle brook mediumDr.T.V.Rao MD 29

30. Lowenstein Jensen’s Medium
• Contain
coagulated egg
• Mineral salt
solution
• Asparagine's
• Malachite green
• Agar
Dr.T.V.Rao MD 30

31. Other Medium
•Middle brook
•Sula's medium
•But not routinely
used
Dr.T.V.Rao MD 31

32. Nature of Growth CharactersNature of Growth Characters
• M tuberculosis is obligate aerobe
• M.bovis Microaerophilic
• M.tuberculosis growth luxierently
• M.tuberculosis eugonic
• M bovis is dysgonic
• When grown on 0.5% glycerin M tuberculosis growth
improves
• Sodium pyruvate improves the growth of both
organism.
Dr.T.V.Rao MD 32

33. On L J Medium
• M.tuberculosis appear
dry, rough raised
irregular colonies
• Appear wrinkled
• They appear creamy
white
• Become yellowish
• M.bovis appear as flat
smooth, moist, white
and break up easily
Dr.T.V.Rao MD 33

34. Lowenstein Jensen Medium –
Selective. Always in screw capped bottle. Bluish Green.
Contains – Egg protein – Solidifying agent
Mineral salts – Mg Sulphate, Mg citrate
Asparagine
Malachite Green – Selective agent
Dr.T.V.Rao MD 34

35. On Liquid Medium
•Appear as long
serpentine cords in
liquid medium
•Virulent strains grow in
a more dispersedDr.T.V.Rao MD 35

36. Resistance of Mycobacterium
• Mycobacterium are killed at 600
c in 15 – 20 mt
• In sputum they survive for 10 – 30 mt
• Relatively resistant to several chemicals
including Phenol 5 %
• Sensitive to Glutaraldehyde and
Formaldehyde
• Ethanol is suitable application to superficial
surfaces and skin gloves
Dr.T.V.Rao MD 36

37. Resistance to several agents
• Bacilli survive in Droplets for 8 – 10 days
• Survive in
5% phenol,
15% Sulphuric acid
3% Nitric acid,5% oxalic acid,
4% Sodium hydroxide
Dr.T.V.Rao MD 37

38. Biochemical Tests on Mycobacterium
spp
• Niacin test – 10%
cyanogen's
bromide and 4%
Aniline in 96%
ethanol are added
to suspension of –
C canary yellow
color indicates
positive test. Dr.T.V.Rao MD 38

39. Other Tests
• Aryl sulphatase test – Positive in Atypical
Mycobacterium
• Bacilli grown in 0.001 tripotassium phenolpthalein
disulphide / 2 N. Sodium hydroxide added drop by
drop a pink color develops
• Catalase peroxidase test –
Differentiates Atypical from Typical
Most Atypical are strongly Catalase positive
Tubercle bacilli are weakly positive
Tubercle bacilli are peroxidase positive – not atypical
INH resistant strains are negative for testDr.T.V.Rao MD 39

40. Catalase Test
• 30 Vol of H2O2 and 0.2 % alcohol in
distilled water is added to 5 ml of test
culture
• Effervescence indicates Catalase positive
• Other test
Amidase test
Nitrate reduction test
Dr.T.V.Rao MD 40

41. Antigenic Characters
• Group specificity due to Polysaccharides
• Type specificity to protein antigens
• Delayed hypersensitivity to proteins
• Related to each other species
• Some relation between lepra and tubercle bacilli
• Serology – Tests not useful
Antigenic homogeneity between < bovis and
M.microti
Dr.T.V.Rao MD 41

42. Bacteriophages
• There are 4 Bacteriophages A B C D
• A worldwide
• B. Europe and -American
• C rare
• I type nature between A and B and common in
India
• Phage 33 D M tuberculosis and not in BCG
strains
Dr.T.V.Rao MD 42

43. Molecular Typing
• DNA finger printing
differentiates different
strains of Mycobacterium
species
• Treating the organism with
Restriction endonuclease
yields Nucleic acid
fragments of varying length
and strain specific
• Use in epidemiological
studies
Dr.T.V.Rao MD 43

44. Finger printing Methods
• Finger printing is done with
Chromosomal insertion
sequence IS 6110 present in
most strains of Tubercle
bacilli
• Now entire genome of M
tuberculosis is sequenced
• Several Molecular methods
are available for studies
Dr.T.V.Rao MD 44

45. Genome of Mycobacterium
tuberculosis
Dr.T.V.Rao MD 45

46. How tuberculosis
spreads• Tuberculosis (TB) is a contagious disease. Like
the common cold, it spreads through the air.
Only people who are sick with TB in their
lungs are infectious. When infectious people
cough, sneeze, talk or spit, they propel TB
germs, known as bacilli, into the air. A person
needs only to inhale a small number of these
to be infected.
Dr.T.V.Rao MD 46

47. Tuberculosis spread by Respiratory
route
Dr.T.V.Rao MD 47

48. Tuberculosis highly Communicable
Disease.
• Someone in the world is newly infected with
TB bacilli every second.
• Overall, one-third of the world's population is
currently infected with the TB bacillus.
• 5-10% of people who are infected with TB
bacilli (but who are not infected with HIV)
become sick or infectious at some time during
their life. People with HIV and TB infection are
much more likely to develop TB.
Dr.T.V.Rao MD 48

49. In India 1 death / Minute
• Half a million
people die
from disease
every year in
India one
death every
minute
Dr.T.V.Rao MD 49

50. Pathology and Pathogenesis of
Tuberculosis
• Source of Infection – Open case of Pulmonary
Tuberculosis.
• Every open case has potential to infect 20 – 25
healthy persons before cured or dies
• Coughing , Sneezing, or Talking.
• Each act can spill 3000 infective nuclei in the
air,
• Infective particles are engulfed by Alveolar
Macrophages.
Dr.T.V.Rao MD 50

51. Spread of Tuberculosis
Dr.T.V.Rao MD 51

52. 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
Dr.T.V.Rao MD 52

53. Dr.T.V.Rao MD 53

54. Predisposing Factors
• Genetic basis,
• Age
• Stress,
• Nutrition,
• Co existing infections Eg HIV
Dr.T.V.Rao MD 54

55. 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.
• CD4Cell plays role in Immune Mechanisms.
Dr.T.V.Rao MD 55

56. Mechanisms of Infection
• Within 10 days of entry of Bacilli
clones of Antigen specific T
Lymphocytes are produced
• Can actively produce Cytokines,
Interferon γ which activate
Macrophages form cluster or
Granuloma Dr.T.V.Rao MD 56

57. Tubercle with Caseous Necrosis
Giant cells
Tubercle bacilli
Partially activated
macrophage
Lymphocyte
Fully activated
macrophage
Dr.T.V.Rao MD 57

58. Basis of Tubercle formation.
• 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
Dr.T.V.Rao MD 58

59. Diagram of a
Granuloma
NOTE: ultimately a fibrin layer
develops around granuloma
(fibrosis), further “walling off” the
lesion.
Typical progression in pulmonary
TB involves caseation,
calcification and cavity
formation.
Dr.T.V.Rao MD 59

60. Tubercle discharging
Bronchial tree
TNF- αTNF- α
Dr.T.V.Rao MD 60

61. Immunity in Tuberculosis.
• CD4 T Lymphocytes with Th 1 or Th 2 secrete - 1
Cytokines,2 Interleukin 1,and 2 , 3 Interferon's γ ,
4.Tumor necrosis factor.
• The mechanisms with Th 1 secrete
Cytokines Activate Macrophages
Results in protective Immunity,
and contain Infection.
Th 2 manifests with Delayed Hypersensitivity
DTH causes Tissue destruction. and disease will
progress.
. Dr.T.V.Rao MD 61

62. Immunity in Tuberculosis
Activated Macrophages - Epitheliod cells
Forms cluster a granuloma
Activated macrophages turn into Giant cells.
Granuloma contains necrotic tissue Dead
macrophages cheese like caseation.
Apoptosis of bacteria laden cells
Contribute to protective immunity.
Dr.T.V.Rao MD 62

63. Lesions in Tuberculosis
• Exudative – and Productive
• Exudative – Acute inflammatory
reaction with edema fluid – contains
Polymorphs-
Lymphocytes – later Mononuclear cells.
Bacilli are virulent - Host responds with
DTH Injurious.
Productive Type protective ImmunityDr.T.V.Rao MD 63

64. Primary Tuberculosis
• Initial response
• In Endemic countries Young children
• Events of Primary complex
1 Bacilli are engulfed by Alveolar Macrophages
2 Multiply and give raise to Sub pleural focus of
Tuberculosis,Pneumonia,involve lower lobes and
lower part of upper lobes.
Called as Ghon’s focus.
The Hilar Lymph nodes are also involved
Dr.T.V.Rao MD 64

65. Primary complex
• This is known as the primary complex or
primary infection. The patient will heal and a
scar will appear in the infected loci. There will
also be a few viable bacilli/ may remain in
these areas (particularly in the lung). The
bacteria at this time goes into a dormant
state, as long as the person's immune system
remains active and functions normally this
person isn't bothered by the dormant
bacillus.
Dr.T.V.Rao MD 65

66. Primary complex
• Ghon’s focus with Enlarged lymph nodes appear
after 3- 8 weeks after infection.
• Heals in 2 – 6 months calcified,
• Some bacteria remain alive and produce latent
infections.
• Infection activated in Immunosuppressed conditions
Eg. HIV infections and AIDS
• Can produce Meningitis, Miliary tuberculosis, other
disseminated Tuberculosis.
Dr.T.V.Rao MD 66

67. Reactivation of Tuberculosis
• When a person's
immune system is
depressed., a
secondary
reactivation occurs.
85-90% of the cases
seen which are of
secondary
reactivation type
occurs in the lungs. Dr.T.V.Rao MD 67

68. Koch’s Phenomenon
• Tuberculosis infected Guinea pig if injected
with Living Tubercle bacilli
• The site around the injection becomes
necrotic.
• Koch found the same reaction when injected
with old Tuberculin ( heated and
concentration of the tubercle bacilli )
• It has produced the same reaction
• This is called as Koch’s Phenomenon.
Dr.T.V.Rao MD 68

69. Post Primary Tuberculosis
• Mainly occurs due to Reactivation of Latent
infection.
• May also due to Exogenous reinfection
• Differs from Primary Infection.
• Leads to –
Cavitation's of Lungs, Enlargement of
Lymph nodes,
Expectoration of Bacteria laden sputum
Dissemination into Lungs and other extra
pulmonary areas.
Dr.T.V.Rao MD 69

70. Majority of the Tuberculosis
are Pulmonary
Dr.T.V.Rao MD 70

71. Multiorgan Involvement
in Tuberculosis.
Dr.T.V.Rao MD 71

72. Complication of Tuberculosis.
1. Meningitis.
2. Pleurisy,
3. Involvement of Kidney,
4. Spine ( Potts spine )
5. Bone Joints,
6. Miliary tuberculosis
Dr.T.V.Rao MD 72

73. Symptoms and Sings of
Tuberculosis
Dr.T.V.Rao MD 73

74. Clinical Illness with Tuberculosis
• Pulmonary Disease –
Major manifestation
with involvement of
Lungs
Hemoptysis, Chest pain
Fever sweets
Anorexia
Cavity formation in
Lungs
Dr.T.V.Rao MD 74

75. Tuberculosis - Pneumothorax
Dr.T.V.Rao MD 75

76. 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
Dr.T.V.Rao MD 76

77. Tuberculosis - Lymphadenitis
Dr.T.V.Rao MD 77

78. Multidrug-resistant tuberculosis
(MDR-TB)
• Drug resistance arises due to improper
use of antibiotics in chemotherapy of
drug-susceptible TB patients. This
improper use is a result of a number of
actions including, administration of
improper treatment regimens and failure
to ensure that patients complete the
whole course of treatment.
Dr.T.V.Rao MD 78

79. Definition of MDR Tuberculosis
• MDR-TB is defined as resistance to
isoniazid and rifampicin, with or without
resistance to other first-line drugs (FLD).
XDR-TB is defined as resistance to at
least isoniazid and rifampicin, and to any
fluoroquinolone, and to any of the three
second-line injectables (amikacin,
capreomycin, and kanamycin).
Dr.T.V.Rao MD 79

80. MDR tuberculosis dangerous to
Society too
• Essentially, drug
resistance arises in
areas with weak TB
control programmes. A
patient who develops
active disease with a
drug-resistant TB strain
can transmit this form
of TB to other
individuals
Dr.T.V.Rao MD 80

81. X- MDR
• The term ‘totally drug resistant’ has not
been clearly defined for tuberculosis.
XDR-TB severely reduces the options for
treatment although they have not been
studied in large cohorts. Treatment
options for XDR-TB patients who have
resistance to additional second-line anti-
TB drugs are even more limited.
Dr.T.V.Rao MD 81

82. Epidemiology
• An ancient disease, called as white plague
• 1/3 of the world population is infected
• 2 billion infected
• Each year 9 lakhs to 1 million are infected
• Poor nations phase the burnt of the disease.
• In developing world > 4o% of the population is
effected
• 15 million suffer the disease
• 3 million are highly infective.
Dr.T.V.Rao MD 82

83. Diagnosis of Tuberculosis
Dr.T.V.Rao MD 83

84. Types of specimens:
-Sputum.
- BAL.
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric – cold abscess)
- Blood in case of haematogenous TB
Dr.T.V.Rao MD 84

85. 85
Sputum Collection
• Sputum specimens are essential to confirm
TB
– Specimens should be from lung secretions, not
saliva
• Collect 3 specimens on 3 different days
• Spontaneous morning sputum more
desirable than induced specimens
• Collect sputum before treatment is
initiated

86. 86
Culture
• Used to confirm diagnosis of TB
• Culture all specimens, even if smear is
negative
• Initial drug isolate should be used to
determine drug susceptibility

87. Laboratory Diagnosis
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.
Dr.T.V.Rao MD 87

88. 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
Dr.T.V.Rao MD 88

89. Quantitation of AFB in Sputum
Smears
• No of Bacilli No of Fields Report as
• 0 300 Negative
• 1-2 300 Doubtful
• 1- 9 100 1+
• 1- 9 10 2+
• 1-9 1 3+
• 10 or >10 1 4+
Dr.T.V.Rao MD 89

90. Lowenstein–Jensen medium
• When grown on LJ medium,
M. tuberculosis appears as
brown, granular colonies
(sometimes called "buff,
rough and tough"). The
media must be incubated
for a significant length of
time, usually four weeks,
due to the slow doubling
time of M. tuberculosis
Dr.T.V.Rao MD 90

91. 3- Cultures on L J media
Lowenstein –Jensen medium is an egg based media
with addition of salts, 5 % glycerol, Malachite green.
Advantages: - Specificity about 99 %
- More sensitive (need lower no. of bacilli 10-100 /
ml)
- Can differentiate between TB complex &
NTM using biochemical reactions
- Sensitivity tests for antituberculous drugs
( St, INH, Rif., E)
Disadvantages: Slowly growing ( up to 8 weeks )
Dr.T.V.Rao MD 91

92. Detection and identification of mycobacteria
directly
from clinical samples
• Genotypic Methods :
• 􀂄 PCR
• 􀂄 LAMP
• 􀂄 TMA / NAA
• 􀂄 Ligase chain reaction
• 􀂄 Phenotypic Methods :
• 􀂄 FAST Plaque TB
Dr.T.V.Rao MD 92

93. • Essentially PCR is a
way to make millions
of identical copies of
a specific DNA
sequence , which may
be a gene, or a part of
a gene, or simply a
stretch of nucleotides
with a known DNA
sequence, the
function of which
may be unknown
Polymerase Chain Reaction (PCR)
Dr.T.V.Rao MD 93

94. • Interferon-γ assays
measure cell-
mediated immunity
by quantifying IFN-γ
released from
sensitized T cells in
whole blood/PBMCs
incubated with TB
antigens.
Quantiferon-GOLD
Dr.T.V.Rao MD 94

95. Tuberculin Test
Interpretation:
* A positive test indicates previous exposure and
carriage of T.B.
* A negative tuberculin test excludes infection in
suspected persons
* Tuberculin positive persons may develop
reactivation type of T.B.
* Tuberculin negative persons are at risk of gaining
new infection
* False positive reactions are mainly due to:
- Infection with nontuberculous mycobacteria
*
Dr.T.V.Rao MD 95

96. * False negative reactions may be due to:
-
• False negative reactions may be due to:
• - Sever tuberculosis infection (Miliary T.B.)
- Hodgkin’s disease
• - Corticosteroid therapy - Malnutrition
- AIDS
• * Children below 5 years of age with no
exposure history:
• - Positive test must be regarded suspicious
Dr.T.V.Rao MD 96

97. Tuberculin Test
( Mantoux Test )
• Test to be interpreted
in relation to clinical
evaluation.
• Even the induration of 5
mm to be considered
positive when tested on
HIV patients.
• Lacks specificity.

98. Tuberculin Testing - Limitations
• False positive reactions are mainly due to:
• - Infection with nontuberculous mycobacteria
• * False negative reactions may be due to:
• - Sever tuberculosis infection (Miliary T.B.) -
Hodgkin’s disease
• - Corticosteroid therapy - Malnutrition -
AIDS
• * Children below 5 years of age with no exposure
history:
• - Positive test must be regarded suspicious
Dr.T.V.Rao MD 98

99. 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.
Dr.T.V.Rao MD 99

100. III Polymerase Chain Reaction
(PCR) & Gene probe
Nucleic acid probes & nucleic acid amplification
tests in which polymerase enzymes are used to
amplify ( make many copies of specific DNA or
RNA sequences extracted from mycobacterial
cells.
Advantages:
- Rapid procedure - High sensitivity (1-10
( 3 – 4 hours) bacilli / ml sputum)
Dr.T.V.Rao MD 100

101. Tuberculosis and HIV infection
• HIV association has become a
threat to the developed
countries too
• HIV association will lead to
rapid spread of tuberculosis
Dr.T.V.Rao MD 101

102. HIV Considerations
• HIV is the strongest risk factor for
progression to active disease
• HIV kills CD4+
T Helper cells which
normally inhibit M. tuberculosis
• HIV interferes with PPD skin test
• Protease inhibitors interfere with
rifampin
Dr.T.V.Rao MD 102

103. MDR tuberculosis
• Multidrug resistant tuberculosis has become a
global threat.
• In 1993 WHO declared Tuberculosis a Global
emergency
• Animals shed the bacilli in Milk, human’s get
infected after drinking the unsterilized Milk
• Pasteurization has reduced the incidence of
Bovine tuberculosis.
Dr.T.V.Rao MD 103

104. Second Line Drug Treatment (SLD’s)
Less effective, more costly and more toxic, 50% cure rate
• Four months intensive phase (5 drugs)
– Kanamycin
– Ethionamide
– Pyrazinamide
– Ofloxacin
– Cycloserine or Ethambutol
• 7X times p/w in hospital
» Followed by

105. Why Tuberculosis continues to be
Important
• Someone in the world is newly infected with TB
bacilli every second.
• Overall, one-third of the world's population is
currently infected with the TB bacillus.
• 5-10% of people who are infected with TB bacilli (but
who are not infected with HIV) become sick or
infectious at some time during their life. People with
HIV and TB infection are much more likely to develop
TB.
Dr.T.V.Rao MD 105

106. March 24th
World TB Day
Dr.T.V.Rao MD 106

107. 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
* Noncompliance (failure to complete the course):
Directly observed therapy (DOT)
Health care workers observe the medication
Dr.T.V.Rao MD 107

108. Directly Observed Therapy – Short
Course
• DOTS (directly observed treatment, short-
course), is the name given to the World
Health Organization-recommended
tuberculosis control strategy that combines
components:
• Case detection by sputum smear microscopy
• Standardized treatment regimen directly
observed by a healthcare worker or
community health worker for at least the first
two months Dr.T.V.Rao MD 108

109. Immuno-prophylaxis
• Intradermal injection of live attenuated
vaccine Bacille Calmette-Guerin (BCG).
• The strain causes self limited lesion and
induces hypersensitivity & immunity.
• Coverts tuberculin negative person to positive
reactor.
• Immunity lasts for 10-15 years. Immunity 60-
80%
• Some studies proved BCG is doubtful value in
prevention of Tuberculosis
Dr.T.V.Rao MD 109

110. Bacillus Calmette-Guérin (BCG)
• Bacillus Calmette-Guérin (BCG) is a
vaccine against tuberculosis that is
prepared from a strain of the attenuated
(weakened) live bovine tuberculosis
bacillus, Mycobacterium bovis, that has
lost its virulence in humans by being
specially subcultured (230 passages) in
an artificial medium for 13 years,
Dr.T.V.Rao MD 110

111. BCG
• Given at birth without tuberculin testing
• Protects against TB, the disease runs
milder course in protected, prevents
skeletal, meningeal & miliary forms.
• Also found useful in leprosy, leukaemias
and other malignancies by non-specific
stimulation of RE system.
Dr.T.V.Rao MD 111

112. • Programme Created by Dr.T.V.Rao MD
for Medical and Paramedical Students in
the Developing World
• Email
• doctortvrao@gmail.com
Dr.T.V.Rao MD 112