Mycobacterium Tuberculosis.pptx

MYCOBACTERIA
 They are slender rods
 Sometimes branching resembling fungal mycelium.
 In liquid cultures, they form a mould like pellicle.
 Aerobic, Noncapsulated, Nonsporing, Nonmotile
 The genus ' mycobacterium ' includes obligate parasites, opportunistic
pathogens, saprophytes.
Introduction
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History
 First to be identified is Lepra bacillus
discovered by Hansen in 1868.
 ROBERT KOCH in 1882 isolated the
mammalian tubercle bacillus and proved its
causative role in Tuberculosis by satisfying
KOCH'S POSTULATES.
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Koch’s Postulates
A micro organism can be accepted as the causative agent of an Infectious
Disease only if the following conditions are satisfied.
1. The bacterium should be constantly associated with the lesions of the disease.
2. It should be possible to isolate the bacterium in pure culture from the lesions.
3. Inoculation of such pure culture into suitable laboratory animals should
reproduce the lesions of the disease.
4. It should be possible to re-isolate the bacterium in pure culture from the
lesions produced in the experimental animals.
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Classification of Mycobacteria
1. MTC – Human, Bovine, Africanum, Microti
2. M. leprae
3. Atypical mycobacteria
4. Saprophytic mycobacteria
Human infections are primarily caused by:
1. MTC
2. MOTT
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 Straight or slightly curved 3 x 0.3 μm in size
 Occurring singly, in pairs or in small clumps.
 Size depends on conditions of growth.
 Long filamentous, club shaped, branching forms may sometimes be seen.
 M. bovis is straighter, shorter and stouter
M. tuberculosis
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Staining Characteristics
 Ziehl Neelsen staining - Acid fast bacilli
– Acid fastness – Resisting decolorization by weak mineral acids like H2SO4 / HCL
– Both by ZN method and Fluorescent staining, Acid fastness is due to the presence
of lipid rich waxy cell wall [Mycolic acid] or due to the semi permeable membrane
around the cell.
– Beaded or barred forms are frequently seen in M. tuberculosis
– Cell wall is thick, composed of three layers enclosing a trilaminar plasma
membrane.
– Spheroplasts and L forms are formed when grown in the presence of lysozymes
7

Lipid-Rich Cell Wall of Mycobacterium
Mycolic acids
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Cultural Characteristics
 Bacilli grow slowly
 Generation time- 14 – 18 hours & optimum temperature- 37oC
 Growth does not occur below 25oC and above 40oC .
 Obligate aerobes, Optimum PH 6.4 – 7
 Addition of 0.5 % glycerol improves the growth.
 Sodium pyruvate also helps in growth
 Highly susceptible to toxic substances: fatty acids
 Colonies appear in 2-8 weeks.
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 On solid media, forms
– Dry
– Rough
– Raised
– Irregular colonies with wrinkled
surface.
 On further incubation forms
– Creamy white
– Yellowish
– Buff coloured colonies
 Not easily emulisifiable
Colony Morphology
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 Common solid media LOWENSTEIN -
JENSEN medium
 Consists of
– Coagulated hen’s egg
– Mineral salt solution
– Asparagine
– Malachite green
Solid Culture Media
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 On liquid media without dispersing agents forms a prominent surface
pellicle which may extend along the sides above the medium –
Hydrophobic nature of cell wall
 Virulent strains tend to form long serpentine cords. [Cord Factor]
 Sula’s, Middlebrook’s 7H9, Kirchner’s and Dubos’
Liquid Culture Media
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 Killed at 60oC in 15 - 20 minutes.
 Sputum - Remain alive for 20 - 30 hrs.
 Droplet nuclei - Retain viability for 8 - 10 days
 Cultures - Viable at room temperature for 6 - 8 months and may be
stored for up to 2 yrs at -20oC.
 Sensitive to Formaldehyde and Glutaraldehyde.
 Destroyed by Tincture of iodine in 5 min and by 80% Ethanol in 2 - 10
min.
Resistance
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1. Bacteriophage types:
A, I, B, C
2. Bacteriocin types:
2 types
3. Molecular typing:
Restriction endonuclease – RFLP
Entire genome of tubercle bacillus has been
sequenced
Types
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1. Highly infectious for guinea pigs and hamsters
2. Natural infection in humans, dogs qnd primates
3. Non pathogenic for rabbits, cats, goats, bovines and fowl
4. Mice are moderately susceptible
5. M. bovis – Cattle, dogs, cats, badgers, swine, parrots, birds and
humans but nonpathogenic for fowl
6. M. africanum – Africa
7. M. microti - Voles
Hosts
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Antigenic Properties
 Cell wall contains four major antigens
– Lipids
» Long-chain fatty acids called mycolic acids: responsible for granuloma formation
» Pattern on gas chromatography used to classify different species
» Cord factor is also responsible for virulence
– Lipoarabinomannan
» Cell-wall associated glycolipid
» Inhibit macrophage activation, leading to its persistence
– Proteins
» Delayed type hypersensitivity reaction: Tuberculin reaction
– Polysaccharide
» Group specificity 21

Pathogenesis
 Source – Open case of TB
 Sputum – 10000 Bacilli/ml
 Infect 25 cases before death or cure
 Aerosols – 3000 infectious nuclei/cough
 Mode of infection- direct inhalation of aerosolized bacilli contained in droplet
nuclei of expectorated sputum
 Rarely – Infected milk, inoculation
 Inhaled bacilli reach lungs and phagocytosed by alveolar macrophages
(Trojan Horse phenomenon)
 Escaped killing by inhibiting phagosomal vacuole and modulate macrophage
apoptosis
22

TB mechanism for cell entry
– The tubercle bacillus can bind directly to mannose receptors on macrophages
via the cell wall-associated mannosylated glycolipid (LAM)
TB can grow intracellularly
– Once TB is phagocytosed, it can inhibit phagosome-lysosome fusion
Slow generation time
– Immune system cannot recognize TB, or cannot be triggered to eliminate TB
Mechanisms of Virulence
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High lipid concentration in cell wall
– Accounts for impermeability and resistance to antimicrobial agents
Antigen 85 complex
– It is composed of proteins secreted by TB that can bind to fibronectin
– These proteins can aid in walling off the bacteria from the immune system
Cord factor
– Associated with virulent strains of TB
– Toxic to mammalian cells
Mechanisms of Virulence
24

Pulmonary Tuberculosis
 TB infection begins when the mycobacteria reach the pulmonary
alveoli where they are ingested by and replicate in alveolar
macrophages.
 Immunity – Cell mediated by CD4+T helper cells [Th1 and Th2 cells]
and their cytokines like Interferon γ, IL 1, 2 and TNF α.
– Th1 – Cytokines activate macrophages, containment, protective.
– Th2 – Cytokines lead to DTH, tissue destruction, progression of
disease.
 The lesion which is produced is called a Granuloma or a Tubercle
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Pulmonary Tuberculosis
 Essential pathology is Ghon focus in lower lung
 Characteristic lesion, tubercle, seen post-primary TB
 It is an avascular granuloma composed of central zone containing
giant cells, with or without caseation, and a peripheral zone of
lymphocytes and fibroblasts
 Depending on time of infection and type of response TB can be
– Primary
– Post-primary (Secondary or Adult)
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Granuloma
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 Primary tuberculosis:
– Initial infection
– In endemic areas, in young children more susceptible.
– Alveolar macrophage engulf the bacilli, multiply intracellularly
and give rise to a subpleural focus of tuberculous pneumonia
commonly located in lower lobe or lower part of upper lobe
(Ghon focus)
– Hilar lymph nodes are involve
– Primary complex: Ghon focus + Lymph nodes
– Formed in 3-8 weeks of infection, heals in 2-6 months by
calcification.
– In children with impaired immunity, lesion may enlarge and
cause miliary or meningeal tuberculosis
Tuberculosis
29

 Post primary or Adult or Secondary TB:
– Reactivation of latent infection
– Exogenous reinfection
– Affects mainly the upper lobes, lesions undergoing necrosis and tissue
destruction, leading to cavitation, LN are not involved usually.
– Open cases of TB – Spread of TB
– In the immunodeficient – No cavity formation, but widespread infection
in many organs and systems
– Post primary lesion heals with scarring (Simon’s focus)
Tuberculosis
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Ghon focus & TB Lung
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19-09-2023 09:22:28 32
Cavitation in TB

Lesions
Primarily of two types
 Exudative type
– Acute inflammatory reaction with accumulation of edema fluid,
polymorphonuclear leucocytes, and later, of lymphocytes and
mononuclear cells
– Typically seen when plenty of virulent bacilli and host response is
DTH
 Productive type
– Lesion is predominantly cellular
– Associated with protective immunity
33

Extrapulmonary Tuberculosis
The most common sites affected:
 Lymph nodes (TB lymphadenitis)
 Bones (Spinal TB)
 Serous membranes (TB abdomen and pelvic organs)
 Most serious forms of spread are disseminated TB and
tuberculous meningitis
 Pleural TB and TB pericarditis
 Generally associated with HIV, other immunocompromised
conditions and diabetes
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Epidemiology
 Tuberculosis is an ancient disease .
 Evidence of spinal tuberculosis is present in some Egyptian
mummies.
 Tubercle bacillus DNA has been detected by molecular analysis in a
mummy dated circa 1550 - 1080 B.C.
 Tuberculosis has been for many centuries the most important of
human infections, in its global prevalence, Devastating morbidity and
massive mortality.
 It has been called the '' White Plague '' and '‘The captain of all
the men of death ''
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Epidemiology
 Poverty
 Lesser in affluent nations, More in developing
 TB- HIV– Worsened the scenario
 Multi-drug resistance
 WHO 1993 – As a global emergency
 Human infection with M. bovis is worldwide
– Through aerosolised route between animals
– To human by MILK
– Can affect any system
– Prevented by pasteurization
– No Human-Human transmission
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Laboratory Diagnosis
 Microscopy
 Culture
 Demonstration of hypersensitivity to tuberculo-protein [Mantoux
test]
 Molecular diagnostic methods
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Specimen for diagnosis of TB
Respiratory
 Sputum: Best collected in morning before any meal. Two samples
recommended by NTEP
If sputum unavailable:
 Broncho-alveolar lavage
 Laryngeal aspirates
 Gastric washing (Mainly used for children)
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Specimens
Non respiratory
 Fluid examination (Cerebrospinal, Ascitic, Pleural,
Pericardial, Synovial fluid, Urine)
 Tissue biopsy (From affected site, Bone marrow liver may
be diagnostic in disseminated disease)
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Decontamination & Concentration of specimens
In order to inhibit organisms other than mycobacteria (in class
IIb BSC)
 Petroff’s method
– Sputum incubated with equal volume of 4% NaOH soln at 37C
with frequent shaking until it becomes clear (20 min).
– Centrifuge at 3000 rpm for 20 min and supernatant poured off
– Neutralise the sediment with N/10 HCl and use for culture
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Decontamination & Concentration of specimens
 NALC combined with 2% NaOH
– Better than Petroff’s
– N-acetylcysteine is used for liquefaction of sputum
– NaOH kills the contaminating bacteria
– Neutralise with buffer and concentrated by centrifugation
– Compatible with automated systems
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Microscopy
 Single most reliable method in diagnosis
 Smear - Thick purulent part of sputum.
 Smears are dried, heat fixed and stained by ZIEHL-NEELSEN
technique.
 Under oil immersion objective, acid fast bacilli are seen as bright red
rods, background is blue, yellow or green depending on the counter
stain used.
 Minimum bacilli:10000/ml required
 Positive report - 2 or more typical bacilli have been seen.
 When several smears are to be examined - Fluoroscent microscopy
using fluorescent dye such as auramine-rhodamine.
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1. Prepare the smear, Fix
2. Carbol fuchsin
3. Acid-Alcohol
4. Loeffler’s methylene blue
5. Wash and dry
6. Microscopy – Single most reliable
method for diagnosis and treatment
7. Ehrlich, Kinyoun’s, Spores etc.
8. Fluorescent staining
Ziehl Neelsen Staining
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ZN Smear Grading – WHO/NTEP
Criterion
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Culture
 Gold standard, detecting as few as 10 - 100 bacilli per ml.
 Culture media: Lowenstein Jensen Medium
 Incubation: aerobically at 37C
 Duration: 8-12 weeks
Newer methods of cultivation
 BacT/ALERT: Uses colorimetry for growth detection, measured on
basis of CO2 production as a result of bacterial metabolism
 BACTEC-MGIT: Automated mycobacteria growth indicator tube
– Uses 7H9 Middlebrook medium with fluorometric technology for detection of
O2 consumption
– Incorporation of pyrazinamide in the medium detects the resistance
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Tests for Identification
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Species
Glycerol
Enhanced
Pyruvate
Enhanced
Niacin
Production
Pyrazinamide
Sensitivity
O2
Preference
Pathogenicity
M. tuberculosis YES YES YES SENSITIVE AEROBIC PATHOGENIC
M. bovis NO YES NO RESISTANT MICRO PATHOGENIC
M. africanum NO YES Variable SENSITIVE MICRO PATHOGENIC
M. microti Variable YES YES SENSITIVE AEROBIC
NON
PATHOGENIC
BCG YES YES NO RESISTANT AEROBIC OPPORTUNIST

Drug Sensitivity Tests
 Absolute concentration method
Number of media containing serial concentrations of drugs are
inoculated and the minimum inhibitory concentrations calculated.
 Resistance ratio method
2 sets of media containing graded concentrations of drugs are inoculated,
One set with the test strain and the other with a standard strain of known
sensitivity.
 Proportion method
Indicates the average sensitivity of the strain, taking into account the fact
that any population will contain cells with varying degrees of sensitivity
to a drug.
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Nucleic Acid Technology
 Nucleic acid amplification by PCR and LCR
 Rapid and detect drug resistance simultaneously
 Gene Xpert MTB/RIF assay:
– Full automated
– Simultaneously detect MTB and RIF resistance
 Trunat
– Indigenous detection method developed in India
– Similar to CBNAAT
 Transcription mediated amplification (TMA)
– Targets ribosomal RNA, improvement of PCR based DNA amplification
 Line probe assay (LPA)
– Detects MTC and both resistance to INH and RIF
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Immunodiagnosis
 Not useful in diagnosis, especially in endemic areas and are not
recommended
 Tuberculin test (Mantoux test)
– Demonstration of hypersensitivity to PPD to detect exposure to bacilli
– Positive test denotes prior exposure or immunization with BCG
– Becomes positive after 4-6 weeks of exposure or immunization and
wanes off gradually
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Mantoux Test
0.1ml Purified Protein Derivative (PPD)
of Tubercle bacillus antigen --- 5 T.U
Intradermally in
flexor aspect of
Lt fore arm
RAISED WHEAL Read at 48 -72 hours
Positive
Induration is 10 mm OR more
Negative 5mm or less
Equivocal 6 to 9mm
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 A positive tuberculin test indicates hypersensitivity to tuberculoprotein
- Infection with tubercle bacillus or BCG immunisation, recent or past,
with or without a clinical disease.
 The test becomes positive 4-6 weeks after infection or immunisation.
 Tuberculin allergy wanes in 4-5 years.
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False negative tests (anergy):
 Miliary TB
 New born & elderly
 HIV
 Recent infection
 Malnutrition
 Immunosuppressive therapy
 Lymphoreticular malignancy
 Sarcoidosis
 Measles
False positive tests:
 In infections with some related mycobacteria (Atypical mycobacteria)
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1. Microscopy, Culture, Animal inoculation
2. CSF – PCR, DNA Probes
3. Blood, Bone Marrow & Liver biopsy specimen –
Miliary, HIV Co-infection
4. Renal tuberculosis – 3 to 6 morning samples
5. Fluids – Centrifugation
Extra Pulmonary TB
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BCG Vaccine
 Bacillus Calmette Guerin 1906 - 13 Years
 Dosage: Usual strength is 0.1mg/0.1ml volume.
 Dosage: Infants <4 Weeks 0.05ml.
 Local abscess formation and enlarged regional lymph nodes.
 BCG should not be administered after the age of 2 years.
 BCG should not be given to infants and children with active HIV
disease, though it may be given with benefit to asymptomatic HIV
positives.
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Complications Of BCG
 LOCAL: Abscess, indolent ulcer, keloid,
tubrculides, confluent lesions, lupoid lesions,
lupus vulgaris.
 REGIONAL: Enlargement and suppuration of
draining lymph nodes.
 GENERAL: Fever, mediastinal adenitis,
erythema nodosum, tendency to keloid
formation after wounding at other sites and
very rarely nonfatal meningitis.
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Directly Observed Treatment, Short Course (DOTS)
 DOTS – Provides most effective medicine
 Confirms that it is taken
INTENSIVE PHASE:
 Health worker watches as the patient
swallows drug in his presence
CONTINUATION PHASE:
 The patient is issued medicine for 1 week
of which first dose is taken in the presence
of health worker.
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Drug resistance
 Due to mutations, with an approximate rate of 1 in 108 cell
divisions
 Factors:
– Lapses in prescribing practices
– Drug delivery
– Patient compliance
 WHO classification of drug resistance
– Resistance in newly treated
– Resistance in previously treated
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Drug-resistant TB
 Multidrug-resistant tuberculosis (MDR-TB)
– Resistance to rifampicin (R) and isoniazid (H)
– With or without resistance to one or more other drugs
– May be primary or acquired
– Dangerous in those with HIV co-infection
 Extensively drug resistant tuberculosis (XDR-TB)
– Resistant to first line (MDR) plus any flouroquinolone and
– At least one of three injectable second-line drugs (amikacin,
kanamycin and capreomycin)
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Prevalence of TB
 Global incidence rate = 128 cases/100,000
 1.4 million TB-related deaths
 8.8 million new cases of TB
 80% of the TB burden is in 22 countries
 TB/HIV co-infection and TB drug resistance are key barriers to
progress
 TB/HIV: ~2.5 million people with HIV;
– About 5% of TB patients estimated to be HIV positive
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Tuberculosis Control Programs
1. NTP: Since 1962
Permanent countrywide program
Integrated with health delivery systems both at urban, rural levels
To reduce the problem of tuberculosis, so that its no more a public
health hazard
2. RNTCP: India+WHO+World Bank [1992]
↑ 85% Cure rate - DOTS
↑ 70% Case detection
Involvement of NGOs
3. STOP TB 2006-2015 – DOTS
4. NTEP from Jan 2020: promote quality of life and health by
preventing, controlling and finally eliminating TB 78

THANK YOU!
19-09-2023 09:22:28 85

Mycobacterium Tuberculosis.pptx

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