- Mycobacterium tuberculosis causes tuberculosis and infects around 1.7 million people annually, causing over 9 million new cases and 1.7 million deaths per year. An estimated 500,000 people are infected with multidrug resistant strains. - Risk of infection and disease is highest among socioeconomically disadvantaged people with poor housing and nutrition. Tuberculosis is transmitted via respiratory aerosols from people with active, untreated tuberculosis. - Laboratory diagnosis involves microscopy, culture, and molecular techniques using sputum, gastric washings, urine, tissues or other clinical samples. Staining methods like Ziehl-Neelsen identify acid-fast bacilli. Culturing is needed for species identification and drug

1. Mycobacterium tuberculosis
Dr. Rubaiya Binte Kabir
Mphil Part-1
DMP-15th

2. Epidemiology
• Mycobacterium tuberculosis causes tuberculosis.
• Each year it is estimated that 1.7 million people die of
tuberculosis and that 9 million new cases occur.
• An estimated 500,000 people are infected with a
multidrug resistant strain of M. tuberculosis.
• Risk of infection and disease is highest among
socioeconomically disadvantaged people with poor
housing and poor nutrition.

3. Global burden of TB , 2015

5. Traditional Runyon Classification
Classification Organism
1. Tuberculosis complex - M. tuberculosis
- M. africanum
- M. bovis
- M. leprea
2. Photocromogens - M. kansassi
- M. marinum
3. Scotochromogens - M. scrofulecium
- M. gordonae
4. Nonchromogens - M. avium complex
5. Rapid growers - M. fortuitum-chelonae complex
- M. abscessus
- M. smegmatis
- M. mucogenicum

6. Morphology
• In tissue, they are thin, straight rods measuring about
0.4X3 µm; on artificial media, filamentous forms are
seen with variable morphology from one species to
another.
• They can occur singly, in pairs or as small clumps.

7. • They are obligate aerobic, non-motile, non-capsulated
and non-spore forming.
• Cell wall : rigid, rich in lipids, hydrophobic, resistant
to many disinfectants, and stains, detergents,
antibiotic, host immune response.
• They are acid and alcohol fast.
• Grow slowly (doubling time 18hours.)

8. Resistance
• Relatively resistance to acid and alkali (5% phenol,
15% H₂SO₄, 3% nitric acid, 4% NaOH).
• Resistance to dehydration; so can survive in dried
expectorated sputum ( for 20-30 hours and in droplet
nuclei for 8-10 days)which is important for
transmission by aerosol.
• Destroyed by 80% ethanol in 2 minutes, by iodine in
5 minutes.
• Sensitive to formaldehyde, glutaraldehyde.
• Killed at 60˚C for 10-15minutes.
• Culture may be killed by direct sunlight for 2 hours.

9. Constituents of Tubercle Bacilli

10. • Lipids :
1. Mycolic acid :
a long chain fatty acids C78-C90.
Muramyl dipeptide from peptidoglycan complexed with
mycolic acid
Form granuloma.

11. 2. Waxes : Delayed hypersensitivity.
3. Phosphatides : causes caseous necrosis.
4. Cord factor ( trehalose-6,6-dimycolate): an important
virulent factor. Virulent strains grow in characteristic
‘serpentine’ cord like pattern in which the acid fast
bacilli are arranged in parallel chains.
Inhibit migration of leukocyte
Chronic granuloma and acts as immunogenic adjuvant.

12. 5. Phthiocerol Dimycocerosate (DIM) :
- major virulent factor.
- participate both in receptor dependent
phagocytosis and prevention of phagosomal
acidification.
- controls invasion of macrophage by the bacilli by
targeting lipid organization in host membrane.
Modify its biophysical properties (DIM induced change
in lipid ordering)

13. 1. Favors the efficiency of receptor mediated
phagocytosis of M.TB.
2. Contribute to the control of ph driving bacilli in a
protective niche.

14. • Proteins :
1. Lipoarabinomannan (LAM) : functionally related to
O-antigenic LPS present in other bacteria.
i) Inhibit the activation of macrophage by IFN-γ
ii) Induce macrophage to release TNF- β which causes
fever, wt. loss and tissue damage & IL-10 which
suppress mycobacteria induce T-cell proliferation.
2. Phosphotidylinositol mannoside .
3. Exported repetitive protein : prevents the
phagosome from fusing with the lysosome allowing
the organism to escape the degradative enzymes of
lysosome.
** proteins are anchored in plasma membrane.

15. **Purified protein derivatives (PPDs) : extracted and
purified preparations of cell wall protein derivatives
which are transport protein and porins interspersed
throughout the cell layers, combining with chloroform
soluble wax D of tubercle bacilli, forms PPDs.
Biologically important antigens.
Stimulate patient’s immune response
Interact with toll like receptor on surface of macrophage

16. Production of IL-12 by macrophage.
Differentiation of naïve helper T cell to form Th1 cell
Delayed hypersensitivity.
They are used in Tuberculin test.

17. • Polysaccharide : induce immediate hypersensitivity
and can serve as antigen in reaction with sera of
infected person.

18. Transmission
• Transmitted from person to person by respiratory
aerosol produced by coughing of smear positive
people.
• Source of the organism is the cavity in the lung that
has erode into the bronchus.
• Portal of entry is respiratory tract and initial site of
infection is the lungs.
• In tissues, it resides chiefly within the
reticuloendothelial cells (macrophage).
• M. bovis which is found in unpasteurized cow’s milk,
can also cause GI tuberculosis in human.

20. Pathogenesis
• Virulence factor :
1. No exotoxin, no endotoxin.
2. Capability to escape killing by macrophage and
delayed type of hypersensitivity are achieved by –
a. Cord factor.
b. Sulfatides.
c. LAM.
d. Heat shock protein.

21. The natural history and spectrum of tuberculosis

22. • Steps of pathogenesis :
Infection with M.TB
No immune host (usually child)
Primary tuberculosis
organism enters the resp. airway and infectious particles
penetrate to the alveoli.
Phagocytized by alveolar macrophage
1. M.TB prevents phagolysosome (by exported
repetitive protein).
2. Evade macrophage killing.

23. i) Acute exudative lesion develops and rapidly spread
to lymphatics and regional lymph nodes
ii) Lymph node undergoes massive caseation with
calcification (ghon lesion)
i)+ii)-Ghon focus (usually lower lobe)

24. Sequence of events in Primary pulmonary TB and development of cell mediated
immunity with positive tuberculin test.

25. • Fate of ghon focus :
A. Heals by fibrosis : tuberculin test positive due to
delayed hypersensitivity.
B. Patients with ed production of IFN-γ, TNF-α or
defect in the receptor for these cytokines(as in HIV,
malnutrition)
Progressive lung disease
Death.

26. C. Spread of organism in the host :
Spread from initial site via the lymphatics to the
regional lymphnodes
Spread further reach bloodstream ( erosion of a vein by
caseating tubercle or lympnode can also occur)
Severe bacteremia
Distribute bacilli to all organs
Miliary tuberculosis
death

27. D. Lymphatic or hematogenous dissemination:
Chronic granuloma (productive/proliferative lesion)
i) If small : macrophage is stimulated bacteria is
killed.
ii) If large :
Encapsulated with fibrin, central area undergoes
caseous necrosis, forming tubercle.
Protect bacteria from macrophage killing.
Dormant tubercle bacilli (latent tuberculosis)

28. Reactivation when
- Old age,
- Immunosuppressive disease/therapy.
caseous tubercle break into bronchus, empty its content
Form cavitary lesion (usually upper lobe)

29. • Fate of cavitary lesion :
A. Fibrosis and calcification.
B. Extra pulmonary TB :
- CNS : parenchymal tuberculoma,
meningitis.
- vertebral body : pott’s disease.
- lymphadenitis.
- renal TB
- GI TB
- adrenal TB.

30. Immunity and hypersensitivity
• During 1st infection with tubercle bacilli, a certain
resistance is acquired by cellular immunity:
1. Increased capacity to localize tubercle bacilli.
2. Retard their multiplication.
3. Limit their spread.
4. Reduce lymphatic dissemination.
5. Acquire hypersensitivity to the tubercle bacilli.
This can be attributed to the development of
cellular immunity, with evident ability of mononuclear
phagocytes to limit the multiplication of ingested
organisms and even to destroy them.

31. • A gene called Nramp determines natural resistance to
tuberculosis. The NRAMP protein is located in the
membrane of phagosome in macrophage and play a
role in killing the organism within the phagosome.
• Patient deficient in cellular immunity ( e. g -AIDS)
are at risk of life threatening tuberculosis.
• Mutation in the IFN-γ receptor gene leads to lack of
activation of macrophage thus predispose to severe
tuberculosis.
• Rheumatoid arthritis patient, when treated with TNF
antagonist, has increased risk of tuberculosis
reactivation.

32. Tuberculin test
• Material :
- PPD is used as antigen.
- 1st strength tuberculin has 1 TU, intermediate
strength has 5 TU, 2nd strength has 250 TU.
• Dose of tuberculin :
- in surveys 5 TU is used in 0.1 ml solution.
- 0.1ml containing 0.04µml
tuberculin PPD is injected
intracutaneously on the volar
aspect of the forearm.

33. - The PPD preparation must be stabilized with
polysorbate 80 to prevent adsorption to glass.
• Reaction to Tuberculin :
- the area of injection
is to be examined within 72
hours for the presence of
induration.
-The skin test is evaluated by measuring
the diameter of induration and not the
erythema.

35. • Interpretation :
- a positive skin test indicates
1. previous infection by the organism but not
necessarily active disease.
2. After BCG vaccination (usually 5-10mm and tends
to decrease with time).
3. 15mm or more are assumed to be infected (even if
they have received the BCG vaccine)

36. • False negative : it may be negative in presence of
tuberculosis infection due to immunosuppression in
conditions like –
1. Overwhelming TB
2. Miliary TB
3. Measles
4. Sarcoidosis
5. Scarlet fever
6. Brucellosis
7. Hodgkin’s disease
8. Leprosy
9. malnutrition
10. Administration of
immunosuppressive drug.
11. Immunosuppressive
disease.
12. Before 6 months of age.
13. Before 4 weeks of
infection

37. • False positive : infection by atypical mycobacteria.
• Reversion to negative : upon Isoniazid treatment of a
recent converter; only the elimination of viable
tubercle bacilli results in reversion.
• Tuberculin positive persons are at risk of developing
disease from reactivation; but tuberculin negative
persons are not, although may become infected from
external source.

38. Interferon gamma release assays
• Based on :
1. ESAT – 6 (early secretory antigenic target - 6)
2. CFP – 10 (culture filtrate protein - 10)
3. TB7.7
The test detect IFN-γ that is released by
sensitized CD4 T-cell in response to these antigens
which are absent in BCG and NTM.
• Two commercial assays are available :
1. Quantiferon Gold In-Tube test ( QFT-GIT) :
ELISA that detect IFN-γ in whole blood.

40. 2. T-SPOT-TB : ELISA Immunospot that detect
IFN-γ in purified peripheral blood mononuclear cells.
• Results are reported as positive, negative or
indeterminate.
• They evaluate latent infection, particularly in persons
who have received BCG vaccine.
• Should not be used in :
1. severely immunocompromised hosts.
2. young children (< 5 years of age.)

41. Interpretation of IGRAs results

43. Phase 1 – Latent TB Infection Phase 2 – Active TB Disease
TB germs are “asleep” in host. This phase
can last for a very long time – even many
years.
TB germs are active and spreading.
They are damaging tissue in host.
Patients do not look or feel sick; chest x-ray
is usually normal.
usually feel sick.
Can not spread TB to other people. If the TB germs are in lungs, it can
spread TB to other people by coughing,
sneezing, talking, or singing.
Usually treated by taking one medicine for 9
months.
Treated by taking 3 or 4 TB medicines
for at least 6 months.
Latent TB infection vs. Active TB disease

44. Clinical findings
• Common features are : fever, fatigue, weight loss.
Symptoms of Tuberculosis

46. • Pulmonary : cough and haemoptysis.
• Scrofula : cervical lymphadenitis.
• Erythema nodosum.
• Miliary TB.
• Tuberculous meningitis : headache (intermittent or
persistent for 2-3weeks); progress to coma over a
period of days to weeks.
• Tuberculous osteomyelitis : back pain, stiffness,
lower extremity paralysis.

47. • Gastrointestinal TB : abdominal pain (mimic PUD- if
in duodenum or stomach); diarrhoea, hematochezia –
if in colon; malabsorbtion; intestinal obstruction and
haemorrhage may occur.
• Oropharyngeal TB : non-healing painless ulcer of
mouth; difficulty in swallowing accompanied by local
adenopathy.
• Renal TB : sterile pyuria; dysuria, haematuria, flank
pain.
• Tubercular acute pericarditis : chest pain; can lead to
cardiac temponade or constriction.

48. • Tuberculous arthritis : usually involve only one joint.
Hip and knee( most common) followed by ankle,
elbow, wrist, shoulder.
• Genitourinary TB : women- mimic PID; men- painful
scrotal mass, prostatitis, orchitis, epididymitis; flank
pain, dysuria, frequent urination.
• In old age (poor immune response) : active TB may
manifest as nonresolving pneumonitis.

49. Laboratory diagnosis
• Chest X- Ray :
- typical cavitary or calcified lesions in lungs are
detected radio-graphically.
Advanced tuberculosis; infection in
both lungs is indicated with white
arrows, and formation of cavity is
marked by black arrows.
Diffuse, uniformly distributed,
small miliary shadows indicating
miliary TB.

50. • Specimens :
1. Fresh sputum
2. Gastric washing
3. Urine
4. Pleural fluid
5. CSF
6. Joint fluid
7. Biopsy material
8. Blood
9. Others.

51. • Container :
- should be strong and leak proof.
- sterile plastic universal container which is
suitable for sputum, pus, bronchial washing, tissues,
feces etc.

52. • Sample collection :
1. Sputum :
- sputum and not the saliva is to be collected.
- 3 or more consecutive samples should be
examined, collected first thing in the morning.

53. Procedure of sputum collection

54. 2. Urine :
- 3 consecutive early morning urine (EMU) should
be collected ( whole sample should be sent).
3. Pus :
- should be collected in universal container or into
syringe.
- a swab should be used only if the sample is very
small; only culture can be done.
4. Aspirates :
- pleural or ascitic fluid; in containers containing
anticoagulant (e.g.-Tri sodium citrate).
-as much as possible should be sent.

55. 5. CSF :
- sterile container without anticoagulant.
6. Tissue :
- sterile container or larger plastic or glass
container.
7. In HIV infected patients, AFB may be detected from
buffy coat smears prepared from EDTA anticoagulated
blood.
*** formalin or other fixatives must never be added.

56. • Isolation of mycobacteria from clinical samples :
1. Homogenization : liquefaction of nonsterile samples
with N-acetyl-L-lysine .
2. Decontamination : by 4% NaOH followed by
neutralization with 14% potassium dihydrogen
orthophosphate buffer (color change red to orange
yellow) or 3% oxalic acid followed by neutralization
with 2% NaOH (color change yellow to
orange).[indicator-phenol red]
3. Centrifugation : 1500-2000g for 20 minutes.
4. Neutralization.
5. Inoculation of culture media.

57. • Microscopy :
1. Zeil-Neelsen staining :
Requires :
I. Heated carbol fuchsin for the ZN stain.
II. 3% acid alcohol or 20% H₂SO₄ for decolorizing ZN
stain.
III. Methylene blue for ZN counter stain.
Reporting :
I. Red bacilli in blue background : AFB positive.
II. If no AFB after examining 100 fields : No AFB
seen.

58. Acid fast AFB is stained red due to presence of mycolic
acids which form a complex with carbol fuchsin and can not
be removed by acid in the decolorizing reagent.
The organism may appear beaded.
*** sputum smear need 5000-10000 AFB/ml to be detected
which may be found in the samples from patients’ with
cavity lesion which may be absent in young children,
elderly, HIV patients.

59. 2. Auramine – phenol technique :
Requires :
I. Auramine – phenol for staining.
II. 1% acid alcohol for decolorizing.
III. Potassium permanganate for darkening the
background.
Reporting :
I. Fluorescent AFB : AFB positive.
II. No fluorescent rod : no AFB seen.
*** flurochrome stained organisms are detected by their
fluorescent glow which makes them larger.

60. AFB are white yellow rods glowing against a dark
background.
Auramine fluoresces ,which binds to mycolic acids of
AFB, when illuminated by blue violet or ultra violet
light thus demonstrates presence of AFB.

61. ** Reporting Acid- Fast smears (according to WHO) :
ZN stain Fluorochrome stain Reporting
1. 0/300 fields 0-70/ fields AFB not seen
2. 1-2/300 fields 1-2/70 fields Doubtful (+/-);
examine another
sample
3. 1-9/100 fields 2-18/50 fields 1+
4. 1-9/10 fields 4-36/10 fields 2+
5. 1-9/fields 4-36/field 3+
6. >9/fields >36/field 4+

62. • Culture of mycobacteria :
- media for primary culture should include a non
selective and selective medium.
- selective medium contains antibiotics (PANTA)
to prevent the overgrowth of contaminants and fungi.
- incubation is at 35-37˚C in 5-10% CO₂ for up to
8 weeks.
- optimum pH is 6.4-7.
- The addition of 0.5% glycerol improves the
growth of M. tuberculosis but have no effect on or even
impair the growth of M. bovis whereas sodium pyruvate
helps the growth of both types.

63. - if culture results are negative or slowly growing
NTM are suspected, then a set of inoculated media
should be incubate at 24-33˚C and both sets incubated
12 weeks.

64. 1. Lӧwenstein-Jensen media :
- inspissated egg media.
- contains : defined salts ,
glycerol/sodium pyruvate ,
complexed organic
substances ( fresh eggs,
potato flour etc.)
malachite green (inhibit
commensals)
- is made selective by adding antibiotics.

65. - incubate at 35-37˚C in 5-10% CO₂ for up to 8
weeks.
- pH : 6.95; solid slope is made by inspissating
the container at 85˚C for 1 hour.
- buff, rough, tough colony of M. tuberculosis is
seen.

67. 2. Middlebrook 7H9 Broth media :
- more often used for sensitivity testing than
isolation.
- if tweens ( water soluble esters of fatty acid) are
added : wet the surface and permit dispersed growth.
- contains glycerol, oleic acid, dextrose, albumin :
allow growth of all mycobacteria except M. bovis.
- commercial sources :
i) the MGIT system ( BACTEC medium)
ii) versa TREK culture system
iii) MB Redox.

68. - in BACTEC medium : radioactive metabolites
are present, and growth can be detected by production
of radioactive ¹⁴C in about 2 weeks.
Antibiotic cocktail (PANTA) is also added.

69. - In liquid media, the growth begins at the bottom,
creeps up and forms prominent pellicle at the surface.
Top : demonstration of M.TB
growth in middlebrook 7H9
from CSF sample from TBM
and non-TBM patients.
Bottom : demonstration of
growth in nutrient broth from
positive samples.

70. 3. Semisynthetic agar media :
- middlebrook 7H10 and 7H11( Dubos media).
- contains defined salts, vitamins, oleic acid,
albumin, catalase, glycerol, dextrose (in enrichment
media).
- middlebrook 7H11 also contain casein
hydrosylate ( growth stimulant for M.TB).
- NaCl : osmotic equilibrium.
- biotin : revival of damaged organism and
involve in carboxylation and decarboxylation.
- oleic acid : utilized in metabolism of the
organism.

71. - albumin : neutralize the toxic and inhibitory
effects of fatty acids in media.
- catalase : destroy any peroxidase in media if
present.
- glycerol : source of carbon and energy for
M.TB.
- dextrose : source of energy.
- PANTA is added to inhibit contaminants.
- used as enrichment media.

72. M. tuberculosis M. fortuitum
M. scrofulaceum M. kansasii

73. • Sensitivity testing :
1. Resistance ratio method :
- performed either on solid media-egg or
middlebrook agar (conventional agar based technique)
or liquid media-middlebrook or dubos broth
(Standardized broth culture)
- standardized broth culture can be used for
susceptibility to 1st line drugs. Test results are available
3-4 weeks after inoculation giving approx. 105 cfu/ml.
cultures are examined for growth at 2, 3, 4 weeks.
- conventional agar based technique can be used
for both 1st and 2nd line drugs.

74. - the MIC for test strain is compared with the modal
MIC of control strains to a given antibiotic and the
resistance ratio is expressed as :
MIC for test / modal MIC of control.
A ratio of 4 or more for a test strain indicates
resistance to a given organism.
2. 1% proportion method :
- quantitative test.
- degree of growth in antibiotic - free and
antibiotic - containing media are compared.

75. - the growth of more than 1% of the inoculum on
antibiotic containing media when compared to
antibiotic- free one indicates resistance to that conc. of
antibiotic.
- determines : antibiotic MICs, breakpoint
sensitivity.
- can not be done in liquid media except
BACTEC.
3. Absolute concentration method :
- determines test strains MICs.
- usually performed on middlebrook’s 7H10
medium.

76. 4. Disc diffusion sensitivity :
agar plates are flooded-sealed with test organism
Antibiotic discs are applied
Plate then placed in a sealed bag
Incubated at appropriate temp. for up to 3 days.
Zone size recorded and reported.
- used in rapid growers.
- if no growth within 3 days, result may be
invalid.

77. 5. MODS (Microscopic Observation Drug
Susceptibility) assay :
- modification of liquid broth culture in multi-
well plate with or without antibiotic.
- examine for cording of M. tuberculosis
complex.
6. Nitrate reductase assay (NRA)
- depends on ability to reduce nitrate into nitrite.
- bacteria in inoculated in lӧwenstein-jesen media
KNO₃ incorporated with or without 1st line drugs.

78. - after incubation for 7, 10, 14 days, reagent is
added and nitrate reduction, indicating growth could be
detected by color change.
- if the color change in drug containing media is
more than drug free one, an isolate is considered
resistant.
Left : sensitive to all 4 drugs. Right : resistant to SM and RIF
(GC-growth control; SM-streptomycin 4µg/ml; INH-isoniazid 0.2µg/ml; RIF-
rifampicin 40µg/ml ; EMB-ethambutol 2.0µg/ml )

79. • Biochemical tests :
1. Niacin test.
2. Catalase- peroxidase test.
1. Niacin test :
- Niacin is detected by
addition of 10% cyanogen bromide
and 4% aniline in 96% ethanol to a
suspension of the culture.
- Positive reaction – Canary
Yellow color.
- M. tuberculosis – Positive.
- M. bovis – Negative.
3. Pyrazinamide test.
4. Nitrate reduction test.

80. 2. catalase- peroxide test :
- Equal volumes of H2O2 and
0.2% catechol in distilled water are
added to 5ml of test culture and allowed
to stand.
- Effervescence – Catalase positive.
- Browning – Peroxidase positive.
- Atypical mycobacteria – Catalase
positive and Peroxidase negative.
- M. tuberculosis – Catalase
and Peroxidase positive.

81. 3. Pyrazinamide test :
- Enzyme pyrazinamidase hydrolyses pyrazinamide
to ammonia and pyrazionic acid which is detected by
adding ferric ammonium sulphate.
- Positive reaction – Pink color band.
- M. tuberculosis – Positive.
- M. bovis – Negative.

82. 4. Nitrate reduction test :
- Sulphanilamide and n-naphthyl-ethylene is added
to bacterial suspension in nitrate medium.
- Positive reaction – Red color.
- M. tuberculosis – Positive.
- M. bovis – Negative.

83. Others :
Neutral Red Test :
-Virulent strains of tubercle bacilli
can bind to neutral red in alkaline buffer
solution, avirulent strains cannot bind.
Positive : M. tuberculosis, M. bovis, M.
avium, M. ulcerans
Tween 80 Hydrolysis test :
- Tween 80 is added to the medium.
- Positive reaction – Colour change
from yellow to red at pH 7.
Positive : M. kansasii, M. gordonae.
Negative : M. scrofulaceum

84. Aryl Sulphatase Test :
- The bacilli is grown in a
medium containing 0.001M
tripotassium phenolphthalein
disulphate.
- 0.2N NaOH is added,
drop by drop.
- Positive reaction – Pink
color.
- Only positive for atypical
mycobacteria.

85. • Identification of mycobacteria :
A. Conventional method : includes-
1. Observation of rate of growth.
2. Colony morphology.
3. Test for pigmentation.
4. Biochemical profile.
B. DNA probe test .
C. Others : a) HPLC (high performance liquid
chromatography)
b) MALDI-TOF MF (Matrix Assisted
Laser Desorbsion Ionization Time Of Flight Mass
Spectrometry)

86. A. Conventional method :
- identification of species within the M. tuberculosis
complex is given below :
Species Glycerol
enhanced
Pyruvate
enhanced
Niacin
production
PZA
sensitivity
Oxygen
preference
Pathogenic
ity
1. M.
tuberculosis
Yes Yes Yes S Aerobic P
2. M. bovis No Yes No R Micro P
3 M.
africanum
No Yes Variable S Micro P
4. M.
microti
Variable Yes Yes S Aerobic N
5. BCG Yes Yes No R Aerobic O

87. B. DNA probe assay :
- specific for rRNA. Sequence of the test organism.
- used in a hybridization procedure.
- DNA probes are linked with chemicals that are
activated in the hybrids and detected by
chemiluminescence.
- identification can be done in 1 day.
- probes for M. tuberculosis complex, MAC, M.
kansasii, M. gordonae are in use.
- 16S rRNA gene sequencing is used in probe
negative cases to identify rapidly in lab with molecular
capabilities.

88. D. HPLC :
- based on development of profiles of mycolic acids
which vary from one species to another.
• Nucleic acid amplification tests (NAATs) :
- rapid and direct detection of M.TB in clinical
specimens.
- GeneXpert MTB/RIF test is a real time multiplex
PCR method that both identify M.TB complex and also
detect gene that encode RIF resistance.

89. GeneXpert MTB/RIF test

90. • Molecular Typing :
1. IS6110 RFLP Typing : Restriction endonuclease
treatment yields nucleic acid fragments of varying
lengths, the pattern of which are strain specific.
2. Spoligotyping (spacer oligotyping) : PCR based
technique. This is based on polymorphism in the
direct repeat (DR) locus MIRU-VNTR analysis.

91. Treatment
First line drugs :
1. Rifampicin.
2. Isoniazid.
3. Pyrazinamide.
4. Ethambutol.
Second line drugs :
1. Cycloserine.
2. Ethionamide.
3. Paraaminosalicylic
acid.
4. Streptomycin.
5. Capreomycin.
6. Levofloxacin.
7. Moxifloxacin.
8. Kanamycin.
9. Gatifloxacin.

92. • Mechanism of action of 1st line drugs :
1. Isoniazid- inhibits mycolic acid synthesis.
2. Rifampicin- blocks RNA synthesis by blocking
DNA dependent RNA polymerase.
3. Pyrazinamide- bactericidal-slowly metabolizing
organism within acidic environment of
phagocyte or caseous granuloma.
4. Ethambutol - Bacteriostatic
- Inhibition of Arabinosyl Transferase
***Streptomycin - Inhibition of Protein synthesis by
disruption of ribosomal function.

94. DOTS
• Directly observed treatment, short-course
• DOT means that a trained health care worker or
other designated individual provides the prescribed
TB drugs and watches the patient swallow every
dose.

95. Multi-Drug Resistance TB
• TB caused by strains of Mycobacterium tuberculosis
that are resistant to at least isoniazid and rifampicin,
the most effective anti- TB drug.
• Globally, 3.6% are estimated to have MDR-TB.
• Almost 50% of MDR-TB cases worldwide are
estimated to occur in China and India.

96. Treating Drug Resistant TB

97. Extensively drug resistance TB
• Extensively drug-resistant TB (XDR-TB) is a form of
TB caused by bacteria that are resistant to isoniazid
and rifampicin (i.e. MDR-TB) as well as any
fluoroquinolone and any of the second-line anti-TB
injectable drugs (amikacin, kanamycin or
capreomycin).

98. Treating Extensively Drug Resistant TB

99. Standardized regimens
• Bangladesh NTP has adopted Standardized regimens
for new and retreatment cases.
• Treatment phases :
1. Initial phase :
- new cases : 2 months.
- retreatment cases : 3 months.
** infectious patients quickly become non-infectious
within 2 weeks.

100. 2. Continuation phase : according to categories.
TB diagnostic
category
Patient category Treatment regimen
Intensive case
(daily)
Continuation
phase (daily)
I • New smear positive.
• New smear negative PTB.
• Extrapulmonary TB.
• Concomitant/ associated HIV/
AIDS.
2 (HRZE) 4 (HR)
II • Sputum smear positive PTB
with h/o treatment of more than
1 month.
• Relapse.
• Treatment failure after cat-1.
• Treatment after default.
• Others.
2 (HRZE)S/
1 (HRZE)
5 (HR)E

101. • FDCs : Fixed Dose Combinations.
- 4FDC : rifampicin - 150mg
isoniazid - 75mg
pyrazinamide - 400mg
ethambutol - 275mg.
- 2FDC : rifampicin - 150mg
isoniazid - 75mg.

102. • Dosage of FDC tablets for adults :
1. Category-1 :
2.Category-2 :
Pre-treatment weight (kg) Intensive phase Continuation phase
Daily (1st 2 months) Daily (next 4 months)
Num. of 4FDC tab Num. of 2 FDC tab
30-37
38-54
55-70
>70
2
3
4
5
2
3
4
5
Pre-treatment
weight
Intensive phase Continuation phase
Daily (1st 3months) Daily (1st 2months) Daily (next 5 months)
Num. of 4FDC tab Inj. streptomycin Num. of
2FDC tab
Ethambutol
400mg (num. of
tab)
30-37
38-54
55-70
>70
2
3
4
5
500mg
750mg
1gm
1gm
2
3
4
5
2
3
3
4

103. Side effects and management
• Minor :
Side effects Drugs Management
1. anorexia,
nausea, abdominal
pain
Pyrazinamide,
rifampicin
Drug with after
meal.
2. Joint pain Pyrazinamide NSAID.
3. Burning
sensation in the
feet
Isoniazid Pyridoxine 100mg
daily.
4. Red urine Rifampicin Reassurance
5. Itching with
minor skin rash
All drugs Antihistamine

104. Side effects Drugs Management
1. Itching with skin rash All drugs • Stop anti-TB drugs.
• Identify offending drug.
2. Deafness Streptomycin Stop and never use again.
3. Dizziness (vertigo and
nystagmus)
Streptomycin Stop and never use again.
3. Jaundice, hepatitis Most (mainly isoniazid,
pyrazinamide, rifampicin)
Stop all until jaundice
resolves.
4. Vomiting and confusion
(acute liver failure)
Most • Stop all until jaundice
resolves.
• Urgent LFT and PT.
5. Visual impairment Ethambutol Stop and never use again.
6. Shock syndrome,
purpura, ARF, acute
hemolytic anemia
Rifampicin Stop and never use again.
• Major :

105. Prevention and control

106. 1. Prompt and effective treatment of patients.
2. Immunoprophylaxis by BCG vaccine :
- Bacille Calmette- Guerin vaccine.
- when injected intradermally it can confer
tuberculin hypersensitivity and an enhanced ability to
activate macrophage that kill the organism.
- this gives rise to delayed hypersensitivity and
cell mediated immunity.
- Dose : 0.1ml reconstituted BCG vaccine
intradermally just after birth.

107. 3. Chemoprophylaxis :
- usually only with isoniazid.
- recommended for : 1. latent TB.
2. high risk of developing TB.
3. uninfected (HIV +ve/-ve)
exposed to high risk M.
tuberculosis infection.
4. Eradication of tuberculosis in cattle and
pasteurization of milk have greatly reduced M. bovis
infection.

108. Thank you….