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Tuberculosis

basics
Dr.T.V.Rao MD

Dr.T.V.Rao MD

HISTORY of

Tuberculosis
Tuberculosis Is
an Ancient
Disease
Spinal
Tuberculosis in
Egyptian
Mummies
History
dates to 1550
1080 BC
Dr.T.V.Rao MD

Robert
Koch
Discoverer
of
Mycobacter
ium
Tuberculosi
s
Dr.T.V.Rao MD

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

Classification of
4. Atypical
Mycobacteria Mycobacteria
1.

2.

3.

Tubercle bacilli

a)
b)
c)
d)
e)

Human MTB
Bovine M. bovis
Murine M. microti
Avian M. avium
Cold blooded M.
marinum

Lepra bacilli

a)
b)

Human M. leprae
Rat M. leprae
murium

Mycobacteria
causing skin ulcers

a)
b)

M. ulcerans
M. belnei

Dr.T.V.Rao MD

(Runyon Groups)

a)
b)
c)
d)

Photochromogens
Scotochromogens
Nonphotochromogens
Rapid growers

5. Johnes bacillus
M. paratuberculosis

6. Saprophytic
mycobacteria
a)
b)
c)
d)
e)

M. butyricum
M. phlei
M. stercoralis
M. smegmatis
Others

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

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

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.)
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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

M.bovis
Primarily infection among
the cattle
M.bovis infects Tonsils,
Cervical nodes, can
produce Scrofula.
Enter through Intestines
infects the Ileocecal
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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.phlei
M smegmatis present in
Smegma
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Atypical
Mycobacterium
1 Photochromogens
2 Scotochromogens
3 Non
Photochromogens
4 Rapid growers
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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.
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Poverty and Crowded living


spreads Tuberculosis

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Tuberculosis infects Famous


people too

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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).
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General characters of
the genus
Slender rods
Resist staining
but once stained,
resist
decolonization by
dilute mineral
acids; hence
called ACID FAST
BACILLI (AFB)
Dr.T.V.Rao MD

Aerobic, Non-motile,
Non-sporing, Noncapsulated.
Growth generally
slow
Genus includes
Obligate parasites
Opportunist
pathogens
Saprophytes
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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
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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
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Important
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
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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.
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Acid Fast Bacilli seen in a specimen


of Sputum

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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.
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The
character
Why they are
Acid
Fast

Dr.T.V.Rao MD

of Acid
fastness is due
to presence of
Unsapnofiable
wax ( My
colic acid and
semi
permeable
membrane
around the
24

MTB : Cultural
characters MTB grows more

Grow slowly.
luxuriantly (eugonic)
Generation
than M. bovis
time 14-15 hrs
(dysgonic).
Colonies
Addition of 0.5%
appear after 2
Glycerol supports
weeks or at 6-8 growth of human
strains. No effect or
weeks
inhibitory effect on
MTB - Obligate
bovine strains.
aerobe
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Culturing Acid Fast


Bacilli
Slow to grow ,
Generation time is
14 15 hours
> 2 weeks minimal
required period
Grows at 370c do
not grow below
250c
Ph between 6.4 to
7.0
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Eight Week Growth of Mycobacterium tuberculosis


on Lowenstein-Jensen Agar

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Nature of Media
Used

Helps the growth needs


Solid Medium is
commonly used
Lowenstein Jensens
medium
Petrangini
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Lowenstein Jensens
Medium
Contain
coagulated
egg
Mineral salt
solution
Asparagine's
Malachite
green
Agar
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Other Medium

Middle brook
Sula's medium
But not
routinely used
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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.
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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
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Lowenstein Jensen Medium

Selective. Always in screw capped bottle. Bluish Green.


Contains Egg protein Solidifying agent
Mineral salts Mg Sulphate, Mg citrate
Asparagine
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Malachite Green Selective agent

33

On Liquid Medium
Appear as long
serpentine cords in
liquid medium
Virulent strains
grow in a more
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Resistance of
Mycobacterium
Mycobacterium are killed at 600c 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
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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,
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Biochemical Tests on
Mycobacterium spp

Niacin test
10%
cyanogen's
bromide and
4% Aniline in
96% ethanol
are added to
suspension of
C canary
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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
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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
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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
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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
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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
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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
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Genome of Mycobacterium
tuberculosis

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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.
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Tuberculosis spread by
Respiratory route

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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
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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.
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Spread of Tuberculosis

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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
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Predisposing
Factors

Genetic basis,
Age
Stress,
Nutrition,
Co existing infections Eg
HIV
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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.
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Mechanisms of
Infection

Within 10 days of entry of


Bacilli clones of Antigen
specific T Lymphocytes are
produced
Can actively produce
Cytokines,
Interferon which activate
Macrophages form cluster or
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Tubercle with Caseous


Necrosis
Tubercle bacilli

Lymphocyte

Giant cells

Fully activated

Partially activated

macrophage

macrophage
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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
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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.

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Tubercle
discharging
Bronchial tree

TNF-

TNF-
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Immunity in
Tuberculosis.

CD4 T Lymphocytes with Th 1 or Th 2


secrete - 1 Cytokines,2 Interleukin 1,and 2 ,
3 Interferon's ,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.
.
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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.
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Lesions in
Tuberculosis

Exudative and
Productive

Exudative Acute
inflammatory reaction with
edema fluid contains
PolymorphsLymphocytes later
Mononuclear cells.
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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 Ghons focus.
The Hilar Lymph nodes are also involved
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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/spores
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
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Primary complex
Ghons 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.
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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
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Kochs
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 Kochs Phenomenon.
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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.
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Majority of the Tuberculosis


are Pulmonary

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Multiorgan Involvement
in Tuberculosis.

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Complication of
Tuberculosis.
1. Meningitis.
2. Pleurisy,
3. Involvement of Kidney,
4. Spine ( Potts spine )
5. Bone Joints,
6. Miliary tuberculosis
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Symptoms and Sings of


Tuberculosis

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Clinical Illness with


Tuberculosis
Pulmonary Disease
Major
manifestation with
involvement of
Lungs
Hemoptysis, Chest
pain Fever sweets
Anorexia
Cavity formation in
Lungs
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Tuberculosis Pneumothorax

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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
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Tuberculosis Lymphadenitis

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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.
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Diagnosis of
Tuberculosis

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Types of specimens:
-Sputum.
- BAL.
-Pleural effusions
- Urine
- Stool
-CSF
-Aspiration ( gastric cold abscess)
- Blood in case of haematogenous TB
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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.
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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
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3- Cultures on L J media
Lowenstein Jensen medium is an egg based media
with addition of salts, 5 % glycerol, Malachite green &
penicillin.
Advantages: - Specificity about 99 %
- More sensitive (need lower no. of bacilli 10100 / 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 )
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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
*

* False negative reactions may be due to:


- Sever tuberculosis infection (Miliary T.B.)
Hodgkins disease
- Corticosteroid therapy
- Malnutrition
- AIDS
* Children below 5 years of age with no exposure history:
- Positive test must be regarded suspicious
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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.)
- Hodgkins disease

- Corticosteroid therapy
Malnutrition
- AIDS
* Children below 5 years of age with no
exposure history:

- Positive test must be regarded


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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 CO 2
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.

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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)
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Tuberculosis and HIV


infection

HIV association has


become a threat to the
developed countries too
HIV association will lead
to rapid spread of
tuberculosis
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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
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MDR tuberculosis
Multidrug resistant tuberculosis has
become a global threat.
In 1993 WHO declared Tuberculosis a
Global emergency
Animals shed the bacilli in Milk,
humans get infected after drinking
the unsterilized Milk
Pasteurization has reduced the
incidence of Bovine tuberculosis.
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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.
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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)
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Immuno-prophylaxis
Intradermal injection of live
attenuated vaccine Bacille CalmetteGuerin (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%
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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
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Programme created by
Dr.T.V.Rao MD for
Medical and
Paramedical students in
the Developing World
Email
doctortvrao@gmail.com
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