The PPT is mainly all about Mycobacterium Tuberculosis. Agents causing the disease Tuberculosis, pathogenesis, laboratory diagnosis, treatment and prophylaxis. It was made for both BSc and MSc students.
Mary MwingaMedical Microbiologist en JSS University (Jagadguru Sri Shivarathreeshwara University),Mysore
• Tuberculosis- Is an Ancient Disease
-Spinal Tuberculosis in Egyptian Mummies
- History dates to 1550 – 1080 BC.
• Robert Koch
- Discoverer of Mycobacterium Tuberculosis (Koch’s disease)
• M. tuberculosis
• major human disease
– Affects healthy people
– association with AIDS
– multiple drug-resistance
– Chronic disease
– Prolonged treatment
3. What are Mycobacteria?
• Obligate aerobes growing most successfully in tissues with a high
oxygen content, such as the lungs.
• Facultative intracellular pathogens : infect mononuclear
phagocytes (e.g. macrophages).
4. 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
d) Rapid growers
5. Johne’s bacillus
6. Saprophytic mycobacteria
a) M. butyricum
b) M. phlei
c) M. stercoralis
d) M. smegmatis
6. General characters of the genus
• Slender rods
• Resist staining
but once stained, resist decolorization 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
7. Mycobacterium tuberculosis complex
• Includes Human and Bovine mycobacterium
• M .Africanism Tropical Africa
• M. microti do not cause human infections but in small mammals
• Primarily infection among the cattle
• M.bovis infects Tonsils, Cervical nodes, can produce Scrofula.
• Enter through Intestines – infects the Ileocecal region.
8. What 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 smegmatis – present in Smegma
10. Mycobacterium tuberculosis
• One of the most serious infectious diseases in the developing
• One third of world’s population infected with M. tuberculosis
• Thirty million people have active disease
• Nine million new cases occur
• Three million people die of the disease, each year.
11. Mycobacterium tuberculosis (MTB)
• Morphology –Straight or slightly curved rods
• 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.
12. Mycobacterium tuberculosis (MTB)
• Ziehl – Neelsen stain – Once stained by Carbol fuchsin, resist decolorization
by 20% Sulphuric acide and absolute alcohol.
Acid & Alcohol Fast (AFB)
• Fluorescent dyes like Auramine O or Rhodamine also stain and the
decolorization is resisted.
• Reason for Acid & Alcohol fastness –
• Presence of unsaponifiable wax Mycolic acid
• Semi permeable membrane around the cell
• Property of cell wall and related to integrity of the cell wall
• Staining may be uniform or granular
13. MTB : Cultural characters
• Slow-growing: generation time of 12 to 18 hours (c.f. 20-30 minutes
for Escherichia coli).
• Colonies appear after 2 weeks or at 6-8 weeks
• 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.
• MTB - Obligate aerobes.
• MTB grows more luxuriantly than M. bovis.
• Addition of 0.5% Glycerol supports growth of human strains.
• No effect or inhibitory effect on bovine strains.
• Not specifically resistant to heat: destroyed by 60° C x 20 min.
• In sputum: can survive 20-30 hrs
• Relatively resistant to disinfectants. Survives exposure to
• 5 % Phenol
• 15 % Sulphuric acid
• 3 % Nitric acid
• 5 % Oxalic acid
• 4 % NaOH
16. Biochemical reactions
• Niacin test – Human MTB produces niacin when grown in egg
• Aryl Sulphatase test – Enzyme Aryl sulphatase formed by only
• Neutral red test – Virulent strains of tubercle bacilli bind neutral
red in alkaline solution while avirulent strains can not.
• Catalase–paroxidase test – Most atypical mycobacteria are
strongly catalase positive while MTB is weakly positive.
- MTB is strongly peroxidase positive while atypical mycobacteria
• Nitrate reduction test – Positive in MTB and negative in M.
• Tuberculosis (TB) is a contagious disease. Like the common cold
spreads through the air.
Only people who are sick with TB in their lungs are infectious.
M.O.T: cough, sneeze, talk or spit propels TB germs (bacilli) into the air.
INFECTION: inhaling a small number of bacilli.
Clinical picture :
* Low grade fever
* Weight loss
* Night sweats
* Cough & hemoptysis
18. 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
• People with HIV and TB infection are much more likely to develop
In India 1 death / Minute
• Half a million people die from disease every year in India (one death
19. Pathology and Pathogenesis of Tuberculosis
• Source of Infection – Open case of Pulmonary Tuberculosis.
• Has potential to infect 20 – 25 healthy persons before cured or dies
• Through Coughing , Sneezing, or Talking.
• Each act can spill 3000 infective nuclei in the air,
• Infective particles are engulfed by Alveolar Macrophages.
20. 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
26. Mechanisms of Infection
• Within 10 days of entry of Bacilli - clones of Antigen specific T
Lymphocytes are produced
• Can actively produce Cytokines, Interferon γ activate Macrophages
to form cluster or Granuloma.
Tubercle with Caseous Necrosis 26
27. Basis of Tubercle formation (Granuloma).
• Tubercle is an Avascular granuloma Containing central zone of giant
cells with or without caseation and peripheral zone of Lymphocytes
• Produce lesions may be
Exudative or Productive
28. Diagram of a
NOTE: ultimately a fibrin layer
develops around granuloma
(fibrosis), further “walling off” the
Typical progression in pulmonary
TB involves caseation,
calcification and cavity formation.
30. Immunity in Tuberculosis.
• CD4 T Lymphocytes with Th 1 or Th 2 secrete - Cytokines,
Interleukin 1,and 2 , Interferon's γ and Tumor necrosis factor.
• 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.
31. Immunity in Tuberculosis
• Activated Macrophages - Epithelioid cells Forms cluster a
• Activated macrophages turn into Giant cells.
• Granuloma contains: necrotic tissue, dead macrophages, cheese like
• Apoptosis of bacteria laden cells contribute to protective immunity.
32. Lesions in Tuberculosis
• Exudative and Productive.
1. Exudative – Acute inflammatory reaction with edema fluid –
contains Polymorphs- Lymphocytes – later Mononuclear cells.
-Bacilli are virulent - Host responds with DTH Injurious.
2. Productive Type protective Immunity
* Lesions, healing or progression of infection depend upon
1- Dose of infecting mycobacteria
2- Resistance and hypersensitivity of host
* Virulence :
- Glycolipids on the outer surface of bacteria
- Enhance granuloma formation
- Inhibit migration of polymorphonuclear
- Help survival of tubercle bacilli inside
A- Primary infection:
* An exudative lesion :
- spread to regional lymph nodes
- A scar of healing may later calcify (Ghon’ focus)
- Lymph nodes caseate and then calcify
- Bacilli in the lesion slowly die
- Tuberculin test becomes positive
- The person is immune & hypersensitive
36. There are multiple light areas (opacities) of varying size that run
together (coalesce). Arrows indicate the location of cavities within
these light areas. The appearance is typical for chronic pulmonary
B- Reactivation type :
* Activation of tubercle bacilli due to immunity
* Formation of tubercles that caseate
- open into a bronchus (open tuberculosis)
* Tubercle bacilli erode a blood vessels
- Infect any organ (Miliary T.B.)
38. Koch’s Phenomenon
• Tuberculosis infected Guinea pig if injected with Living Tubercle
-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.
39. Post Primary Tuberculosis
• Due to Reactivation of Latent infection.
• 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.
42. Clinical Illness with Tuberculosis
• Pulmonary Disease – Major
manifestation with involvement of
-Hemoptysis, Chest pain Fever sweets
-Cavity formation in Lungs
43. 2. 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
44. Multidrug-resistant tuberculosis (MDR-TB)
• DEF: MDR-TB is resistance to isoniazid and rifampicin, with or
without resistance to other first-line drugs (FLD).
• XDR-TB is resistance to at least isoniazid and rifampicin, and to any
fluoroquinolone, and to any of the three second-line injectables
(amikacin, capreomycin, and kanamycin).
• Drug resistance is due to improper use of antibiotics in
chemotherapy of drug-susceptible TB patients.
• As 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.
45. Laboratory diagnosis M. tuberculosis
• Acid fast bacteria demonstration in sputum smear.
• Culture on L J media
• Biochemical identification
• Antibiotic sensitivity test
• Skin testing
• delayed hypersensitivity
• protein purified derivative (PPD)
* According to site of infection :
- Sputum - Urine - Body fluids
- Gastric lavage - Blood - Tissue biopsy
* Specimens need appropriate processing
Liquefaction with N-acetyl-L- cysteine
Sputum Decontamination with NaOH
47. Laboratory Diagnosis
• Pulmonary TB –
• Specimen – ZN
• Sputum – Early morning, if scanty 24 hrs, three consecutive day samples.
• Laryngeal swabs or gastric lavage in children.
• Microscopy –See at least 100 field / 10 minutes.
• Grading –
• 1+ -> 3-9 bacilli in entire smear
• 2+ -> 10 or more in entire smear
• 3+ -> 10 or more bacilli seen in most oil immersion fields
Advantage: - cheap – rapid
- Easy to perform
- High predictive value > 90%
- Specificity of 98%
- sputum ( need to contain 5000-10000 AFB/ ml.)
- Young children, elderly & HIV infected persons may not produce cavities & sputum
49. Detecting AFB by fluorochrome stain using fluorescence microscopy:
• The smear is stained by auramine-O dye.
• TB bacilli are stained yellow against dark background & easily visualized
using florescent microscope.
- More sensitive
- Hazards of dye toxicity
- more expensive
- must be confirmed by Z-N stain
51. 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+
Nature 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.
Lowenstein Jensen Medium –
- Selective medium.
- Always in screw capped bottle.
- Color: Bluish-Green.
-Contains – Egg protein – Solidifying agent
-Mineral salts – Mg sulphate, Mg citrate
-Malachite Green – Selective agent
•GROWTH: grows very slowly: several weeks
– non-pigmented colonies
– niacin production: differentiates from other
Sterilized by - Inspissation
54. On L J Medium
• MTB appear as dry, rough raised irregular colonies.
-Appear wrinkled, creamy white
• M. bovis appear as flat smooth, moist, white and break
• Advantages: - Specificity about 99 %
- More sensitive (need lower no. of bacilli 10-100 / ml)
- Can differentiate between TB complex & NTM using
- Sensitivity tests for antituberculous drugs ( St, INH,
• Disadvantages: Slowly growing ( up to 8 weeks )
55. Laboratory Diagnosis
• Extra -Pulmonary TB –
• Specimen –
• CSF – in suspected meningitis
• Pleural fluid & other exudates
• 2-3 days urine in renal TB
• Biopsy material.
56. Concentration methods
• Purpose – Homogenization & Concentration in sputum & other
• Methods –
• Useful for microscopy, culture & animal inoculation
• Petroff’s method
• Most widely used
• Equal volumes of Sputum + 4% NaOH incubated at 37C X 20 min. Centrifuge at 3000
rpm X 30 min. Sediment neutralized by N/10 HCl.
• Can be used for smear, culture, animal inoculation
• Simpler method
• To avoid centrifugation & Neutralization
• Equal volumes of sputum + solution of Cetrimonium bromide 20 g + NaOH 40 g in one
litre. Allow to stand for five minutes. Inoculate Acid Buffered LJ Medium with swab.
57. Antigenic Characters
• Group specificity due to Polysaccharides.
• Type specificity due 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
58. 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
• Used in epidemiological studies
59. Finger printing Methods
• 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
60. Tuberculin Test (Mantoux test)
• Delayed hypersensitivity skin test to assay:
-cell mediated immunity to tubercle bacillus
Material: A purified protein derivative (PPD)
Dose : 0.1 ml of (PPD) is injected
- Induration equal or greater than 10 mm
- Develop 48-72 hours after injection
Negative test: induration less than 10 mm.
• Positive test interpretation:
indicates exposure to organism
does not indicate active disease
61. Tuberculin Test
* 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
62. 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
63. 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
- Growing mycobacteria utilize the acid, releasing radioactive CO2
which is measured as growth index (GI) in the BACTEC
-The daily increase in GI output is directly proportional to the rate &
amount of growth in the medium.
64. 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.
- Rapid procedure - High sensitivity (1-10
( 3 – 4 hours) bacilli / ml sputum)
65. Tuberculosis and HIV infection
• HIV association has become a threat to the developed countries too
• HIV association will lead to rapid spread of tuberculosis
• 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
66. MDR tuberculosis
• MDR TB is 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.
Drugs used :
1- First line drugs :
- Isoniazid - Rifampicin - Pyrazinamide
- Ethambutol - Streptomycin
2- Second line drugs (more toxic and less effective):
- Kanamycin - capreomycin - Cycloserine
- ethionamide - ciprofloxacin - Ofloxacin
* Noncompliance (failure to complete the course):
- Directly observed therapy (DOT)
-Health care workers observe the medication
• Chemoprophylaxis – INH for one year
- Domicilliary treatment preferred
Use multiple drug therapy to prevent emergence of resistant mutants
* Long duration treatment (6-18 months)
* Four drugs are usually started in initial therapy due to:
- Intracellular location of bacilli
- Slow growth rate of bacilli
- Caseous material blocks penetration of drugs
- Some bacilli persist in a metabolically inactive state
* Sputum becomes non-infective 2-3 weeks after starting therapy
• Intradermal injection of live attenuated vaccine Bacille Calmette-
• The strain causes self limited lesion and induces hypersensitivity &
• Coverts tuberculin negative person to positive reactor.
• Immunity lasts for 10-15 years. Immunity 60-80%
• 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, leukemias and other malignancies by
non-specific stimulation of RE system.