Tuberculosis

Outline of Lecture
• Epidemiological Importance of Tuberculosis
• Taxonomy and morphology
• Growth characteristics
• Clinical manifestations
 Tuberculosis – 2018 Status

https://www.who.int/tb/publications/global_report/en/
New TB Cases in 2018 - Global
TB Deaths in 2018 - Global
 Putting things in perspective
New
TB
Cases
in the
World
In
2018:
~ 10 million

10 million is roughly the population of the whole of Sweden

TB
Deaths
in the
World
In
2018:
~ 1.5 million

1.5 million is the approximate population of Bahrain

Tuberculosis Status 2018 - India

j d
Putting things in perspective again
New
TB
Cases
in
India
In
2018:
~ 2.7 million

2.7 million is roughly the total population of Kanpur

TB
Deaths
in
India
In
2018:
~ 4.5 lakhs

4.5 lakhs is the approximate population of Udaipur

 Outline of Lecture
• Epidemiological Importance of Tuberculosis
• Taxonomy and Morphology
• Growth characteristics
• Clinical manifestations
 Genus
Mycobacterium
 Genus Mycobacterium
The name
‘Mycobacterium ‘ was
derived from the Greek
prefix ‘myco’ meaning
fungus

The name came from

•Their growth on the
surface of liquid media,
and
•Branching shape of
cells in some species Mycobacterium Fungus
 Genus Mycobacterium
Includes important human pathogens, i.e.
M. tuberculosis
M. bovis always pathogenic
M. leprae
and
M. kansasii
M. avium potentially pathogenic
 Common Characteristics of
Genus Mycobacterium
•Rod-shaped, rarely
filamentous
•Aerobic or
microaerophilic.
•Non-encapsulated

•Non-sporing

•Non-motile
 Taxonomy
Domain: Bacteria

No need to memorize
Phylum: Actinobacteria
Order: Actinomycetales
Family: Mycobacteriaceae
Genus: Mycobacterium
Species: Mycobacterium tuberculosis
 Taxonomy
Mycobacterium tuberculosis belongs to a group
of species known as the Mycobacterium
tuberculosis complex.
The complex includes
• M. tuberculosis
• M. bovis
• M. africanum
• M. canettii
and several other species
 Mycobacterium tuberculosis

Mycobacterium tuberculosis was discovered by Robert Koch.

 Staining
properties
 Gram stain
This picture is of a
sputum smear stained
by Gram’s technique.

Cells of M. tuberculosis
do not take up either
crystal violet or
safranine, and appear as
unstained slits against a
pink background.

These unstained slits

are jokingly called
‘Gram ghosts’

Although mycobacteria have a Gram-positive cell wall structure,

technically they cannot be stained by Gram’s staining technique
because the lipids attached to the cell wall form a hydrophobic barrier.
 Ziehl-Neelsen stain

Mycobacteria can be stained by the acid-fast method in which heat,

phenol, and alcohol are used to drive a dye across the lipid-rich cell
wall. Later, a dilute mineral acid is used to remove the dye from other
structures except mycobacterial cells. That is why, mycobacteria are
called ‘acid-fast’.
 Ziehl-Neelsen staining technique

Step 1 Step 2 Step 3 Step 4

In this schematic diagram, the bacilli are acid-fast and the cocci are non-acid-fast.
Step 1. Cold carbon fuchsin is applied. Only the non acid-fast cocci get stained pink.
Step 2 The slide and stain are heated. The acid-fast bacilli get stained pink too.
Step 3 Dilute acid is applied. The cocci get decolorized. Acid-fast bacilli remain pink.
Step 4 Methylene blue is applied. Non-acid-fast cocci stain blue. The AFB stay pink.
Smear with acid-fast and non-acid-fast bacteria

In this photograph, ‘A’ are non-acid-fast cocci while ‘B’ are acid-fast
bacilli.
Note: Please note that there is no rule that bacilli have to be acid-
fast, or that cocci have to be non-acid-fast.
 Auramine stain - Fluorescent

Instead of Carbon-fuchsin, acid-fast bacilli can also be stained with a fluorescent dye
called Auramine O using the same principle of heat-aided entry of stain into
mycobacterial cell, followed by selective decolorization with a dilute mineral acid.

The brilliant contrast between bright bacilli and a dark background allows slides to be
scanned at lower magnifications. As a result, it takes much less time to examine slides.
 Acid-fast
Stain needs to be driven inside a mycobacterial cell with heat,
phenol and alcohol

Once stained by Ziehl-Neelsen technique, it is difficult to

decolorize the cell with dilute mineral acids

Therefore they are called acid-fast

Difficult to stain; difficult to decolorize.

Cells of M. tuberculosis resist decolorization with alcohol too.

That is why it is called acid and alcohol fast.
Mycolic acid – The Basis of acid-fastness
Acid-fastness or acid-and-alcohol-fastness is due to a thick waxy
cell wall with high molecular weight mycolic acids.

Mycolic acids are high molecular weight, branched-chain fatty

acids. In the mycobacterial cell wall, there are covalently linked to
arabinogalactan which is covalently linked to peptidoglycan
Mycobacterial Cell Wall
 Mycobacterial Cell Wall

Compared to the cell walls of most other bacteria, mycobacterial cell

walls have lots of lipids and also lots of sugars, especially mannose,
galactose, arabinose, and trehalose.
Think of it as Son Halva, full of ghee and sugar.
 Outline of Lecture
• Epidemiological Importance of Tuberculosis
• Taxonomy and morphology
• Growth characteristics
• Clinical manifestations
Growth in vitro
Growth requirements: M. tuberculosis
Mycobacterium tuberculosis has simple growth
requirements

It is an obligate aerobe, but extra CO2 helps

growth

Optimal growth temperature: 35 – 37°C

Growth is slow; doubling time is about 18 hours

Culture media – 3 basic types
Egg-based, e.g., Lowenstein-Jensen
(Eggs, asparagine, glycerol, various salts, malachite
green)

Agar-based, semi-synthetic, e.g., Middlebrook

7H10
(Casein peptone, glycerol, vitamins, various salts,
glutamic acid, plus oleic acid, albumin, glucose and
catalase)

Liquid, e.g., Kirschner’s, Middlebrook 7H12

Egg based: Löwenstein-Jensen (L-J) Medium

M. tuberculosis colonies visible on the surface of medium

Colonies on M. tuberculosis on L-J medium,
magnified
Agar based: Middlebrook 7H10 Medium
Colonies of M. tuberculosis on Middlebrook 7H10,
magnified
Liquid: Kirschner’s Medium
Liquid: Middlebrook 7H12 broth in MGIT tubes

Mycobacterial Growth Indicator Tubes (MGIT) have a polymer

septum that contains a compound which fluoresces when
exposed to ultraviolet rays only if the tube contains carbon
dioxide released during the growth of mycobacteria.
 Outline of Lecture
• Epidemiological Importance of Tuberculosis
• Taxonomy and morphology
• Growth characteristics
• Evolution of Infection and Clinical
manifestations
Natural History of M. tuberculosis Infection
Infection does not always lead to disease.
Some people can eliminate infection at the earliest
stages with the help of innate immunity.
Others eliminate infection a little later after acquired
immunity is activated.
In others, infection is not eliminated. However,
bacterial replication is arrested inside granulomas
which are maintained by T-cell help (Latent TB)
Only in some people, do the bacteria continue to
replicate and cause disease (Active TB)
Natural History of M. tuberculosis Infection
Latent Infection

Active Disease

Latent Infection
 Natural History of M. tuberculosis Infection

Active screening of exposed individuals has given rise to the concept of ‘Subclinical TB’ which
lies midway between Latent TB and Active TB (Disease). Subclinical TB is characterized by
radiological and microbiological evidence of infection in the absence of symptoms of TB.
‘Incipient TB’ is currently only a concept, a state in which Latent TB is progressing towards
Subclinical TB. However, currently there is no laboratory test to identify it.
Organs affected by Tuberculosis Disease

Disease involves the lungs in about 90% of patients

Organs affected in Extrapulmonary Tuberculosis

Lymph nodes, the pleura, bones and joints, genitourinary tract, and
the meninges are the commonest organs involved
Lecture 2 will deal with the following
• Laboratory diagnosis
• Treatment
• Epidemiology and Prevention
Thank you