Mycobacterium tuberculosis - PowerPoint Presentation

Slide1

Mycobacterium tuberculosis

Dr.

Pendru

Raghunath

ReddySlide2

Mycobacteia

are slender rods that sometimes show branching,

filamentous forms resembling fungal mycelium

Classification

The genus Mycobacterium contains three groups

Obligate parasites

Opportinistic

pathogens

SaprophytesSlide3

Obligate parasites

Mycobacterium tuberculosis complex

Contains

M. tuberculosis

,

M.

bovis

,

M.

africanum

,

M.

microti

,

M.

canetti

,

M.

caprae

and

M.

pinnipedii

Mycobacterium

lepraeSlide4

Opportunistic pathogens

Non-

tuberculous

mycobacteria

(NTM)

This group contains mixed group of isolates from diverse

sources: birds, cold-blooded and warm-blooded animals,

from skin ulcers, and from soil, water and other

environmental sources

They are opportunistic pathogens and can cause many types

of diseaseSlide5

Mycobacterium tuberculosis

long, slender, straight or curved,

about (3 x 0.3 µm in size)

Aerobe

Acid fast

bacilli

Intracellular

Mycolic acid, waxes & lipids in cell wallSlow growing (Doubling time: 15 – 20 hours)Slide6

In 1882 while working in Berlin

he discovered

the tuberculosis bacteria

and the means of culturing it

The Nobel Prize in Physiology or Medicine

1905Slide7

Pathogenesis

Source of infection

Open case of pulmonary tuberculosis

Mode of infection

Direct inhalation of

aerosolised

bacilli contained in the

droplet nuclei of expectorated sputumInfection also occurs infrequently by ingestion for example,through infected milk, and rarely by inoculationSlide8

Transmission of

M. tuberculosis

One cough can release 3,000 droplet nuclei

One sneeze can release tens of thousands of droplet nuclei

Millions of tubercle bacilli in lungs (mainly in cavities)

Coughing projects droplet nuclei into the air that contain tubercle bacilliSlide9

M. tuberculosis

does not s

pread

b

y:

Sharing dishes and utensils

Using towels and linens

Handling foodSharing cell phonesTouching computer keyboardSlide10

The initial infection with

M. tuberculosis

is referred to as a

primary infection

Subsequent disease in a previously sensitized person, either

from an exogenous source or by reactivation of a primary

infection is known as

postprimary tuberculosisBoth forms exhibit quite different pathological featuresSlide11

Primary tuberculosis

It is the initial infection by tubercle bacilli in a host

The site of the initial infection is usually the lung

These bacilli engulfed by alveolar macrophages, multiply

and give rise to a

subpleural

focus of

tuberculous pneumoniaWhich is commonly located in the lower lobe or lower part of the upper lobe to form the initial lesion or Ghon focus

Some bacilli are carried to the hilar lymphnodes through macrophages, where additional foci of infection developsSlide12

The

Ghon

focus, together with the enlarged

hilar

lymphnodes

, form the primary complexM. tuberculosis multiply within the alveolar macrophagesTh-1 cells produce cytokines to activate these macrophagesActivated macrophages effectively destroy most of the tubercle bacilliHowever, some bacilli escape the macrophage- mediated destruction and induce the hypersensitivity reactionA hard tubercle or

granuloma is formed due to the hypersensitivity reactionSlide13

When fully developed, tubercle/

granuloma

consists of 3 zones

1. A central area of large, multinucleated giant cells containing

tubercle bacilli

2. A mid zone of pale

epitheloid

cells, often arranged radially3. A peripheral zone of fibroblasts, lymphocytes and monocytesLater, peripheral fibrous tissue develops, and the central area undergoes caseation necrosis

A caseous tubercle may break into a bronchus, empty its contents there, and form a cavityIt may subsequently heal by fibrosis or calcificationSlide14

Tubercle or

granuloma

formation in tuberculosisSlide15
Slide16

Postprimary (secondary) tuberculosis

It is due to reactivation of latent infection or exogenous

reinfection

and differs from the primary type in many respects

It is

characterised

by chronic tissue lesions, the formation of tubercles, caseation and fibrosisRegional lymphnodes are only slightly involved, and they do not caseatePostprimary tuberculosis always begins at the apex of the lung,

where the oxygen tension is highestThe necrotic materials break out into the airways, leading to expectoration of bacteria-laden sputum, which is the main source of infection to contactsSlide17

Characteristics

Primary

Postprimary

Site

Any

part of lung

Apical region

Local lesion SmallLarge

Cavity formationRareFrequent

Lymphatic involvement

Yes

Minimal

Infectivity*

Uncommon

Usual

Local spread

Uncommon

Frequent

*Pulmonary cases

Differences

beween

primary and

postprimary

tuberculosisSlide18

Immunology

Tubercle bacilli do not contain or secrete a toxin

The exact basis of their virulence is not understood, but

seems to be related to their ability to survive and multiply

in macrophages

Humoral immunity appears to be irrelevant

The only specific immune mechanism effective is the CMISlide19

The key cell is the activated CD4+ helper T cell which can

develop along two different paths: The Th1 and Th2 cells

Th1 dependent cytokines activate macrophages, resulting

in protective immunity and containment of the infection

Th2 cytokines induce delayed type hypersensitivity (DTH),

tissue destruction and progressive diseaseSlide20

Koch’s phenomenon

Koch’s phenomenon is a combination of hypersensitivity

and immunity

It is the response of a

tuberculous

animal to

reinfectionWhen a healthy guinea pig is inoculated subcutaneously with virulent tubercle bacilli, the puncture site heals quicklyAfter 10-14 days, a nodule appears at the site of injection which ulcerates and the ulcer persists till the animal dies of progressive tuberculosisSlide21

If on the other hand, virulent tubercle bacilli are injected in a

guinea pig, which had received a prior injection of tubercle bacilli

4-6 weeks earlier, an

indurated

lesion appears at the site of injection

in a day or two which undergoes necrosis to form a shallow ulcer

This ulcer heals rapidly without involvement of the regional

lymphnodes or tissues. This is called Koch’s phenomenon Koch’s phenomenon has got three components1. A local reaction of induration and necrosis

2. A focal response in which there occurs acute congestion and even hemorrhage around the tuberculous foci in tissues3. A systemic response of fever that may sometimes be fatalSlide22

Laboratory diagnosis

Specimen

collection

Early

morning

sputum samples should be collected for 3 consecutive days in a sterile container In case of renal tuberculosis, 3-6 morning urine samples

should be collected

Type of lesion

Specimen

Pulmonary tuberculosis

Sputum

Laryngeal

swabs or

bronchial washings

Gastric

lavage

Renal tuberculosis

Urine

Tuberculous

meningitis

CSFSlide23

Concentration of specimens

Concentration of a specimen is done to achieve;

Homogenisation

of the specimen

Decontamination i.e. to kill

commensal

bacteria

Concentrate the bacilli in the specimen without inactivationThe concentrate is used for smear preparation, cultutre and

animal inoculationPetroff’s method is used to concentrate sputum specimens Slide24

Diagnostic MethodsSlide25

Direct MethodsSlide26

Direct Microscopy

Ziehl-Neelsen

staining

(hot staining method)

Kinyoun’s

method (cold staining method) Acid fast bacilli resist decolourisation with acid and alcohol once they have been stained with carbolfuchsin AFB appear as pink, long, slender bacilli with beaded appearanceSlide27

Fluorescent staining by

Auramine

O or

auramine

rhodamine

Mycobacterium spp. will

fluoresce yellow against dark background under fluorescent microscopeSlide28

Diagnosis of pulmonary tuberculosis

under RNTCP

DOTS: Directly observed treatment short-courseSlide29

Culture

Concentrated specimen is inoculated

on Lowenstein – Jensen’s medium and

incubated at 37

0

C for 2 – 8 weeks

Colonies appear as buff coloured, dry, irregular colonies with wrinkled surface and not easily emulsifiable (Buff, rough and tough colonies)

Colonies are creamy white to yellow colour with smooth surface and easily emulsifiable

M.

bovis

M. tuberculosisSlide30

Differentiating features of

M. tuberculosis

and

M.

bovis

Slide31

Biochemical

reactions

Niacin test

M. tuberculosis

lacks the enzyme that converts Niacin to

Niacin ribonucleotide due to this large amount of Niacin accumulates in the culture medium Niacin is detected by addition of 10% cyanogen bromide and

4% aniline in 96% ethanol Positive reaction – canary yellow M. tuberculosis – Positive

M.

bovis

- NegativeSlide32

Nitrate reduction test

M. tuberculosis

produce an enzyme nitro

reductase

which

reduces nitrate to nitrite

This detected by colorimetric reaction

by addition of sulphanilamide and n-naphthyl- ethylene diamine dihydrochloride Positive reaction – pink or red colour

M. tuberculosis – Positive M. bovis - NegativeSlide33

M. tuberculosis

is resistant to TCH (Thiophene - 2

- carboxylic acid hydrazide); hence, growth occurs

M. bovis

is susceptible; therefore,

does not grow

M. bovis

M. tuberculosis

Growth in presence of TCHSlide34

Rapid culture methods

1. BACTEC

2.

Mycobacterial

growth indicator tube (MGIT)

3.

Bac T/ Alert 3D system BACTEC system Average time to detect Mycobacterium growth is 8 days Radio metric method Detects the presence of Mycobacteria based on their metabolism rather than

visible growthSlide35

0.5 ml of processed sample is added to 4 ml of Middlebrook

7H12 broth containing C

14

radio labelled palmitic acid

Mycobacteria metabolises C

14

radio labelled palmitic acid and

release radio actively labeled 14CO2 BACTEC 460 instrument measures 14CO2 and reports in terms of growth index (GI) A growth index of 10 or more is considered positive More sensitive than traditional method

Problem of disposal of radio active wasteSlide36

Animal inoculation

0.5 ml of concentrated specimen is

inoculated intramuscularly into the

thigh of two healthy

guineapigs

The animals are weighed prior to

inoculation and thereafter at weekly intervals Tuberculin test is done after 3 – 4 weeks Progressive loss of weight and positive tuberculin skin reaction indicates infection One animal is killed after 4 weeks and autopsied, if it shows no evidence of tuberculosis the other animal is autopsied after 8 weeksSlide37

Autopsy shows

Caseous

lesion at the site of inoculation

Enlarged

caseous

inguinal lymph nodes

Tubercles may be seen in spleen, lungs, liver, or peritoneum

Kidneys are not affectedSlide38

Allergic tests

Tuberculosis

infection leads to the development of delayed

hypersensitivity to

M. tuberculosis

antigen, which can be detected by Mantoux test Mantoux test (tuberculin test) 0.5 ml of PPD containing 5 TU is injected intradermally on flexor aspect of fore armSlide39

Site is examined after 48 – 72 hrs

Induration of 10 mm or more is

considered positive

Positive tuberculin test indicates

hypersensitivity to tuberculoprotein

denoting infection with tuercule bacilli

or BCG immunisation, recent or past

with or without clinical diseaseSlide40

Uses

To diagnose active infection in infants and young children

2. To measure the prevalence of infection in community

3.

Indication of successful BCG vaccinationSlide41

Detection of antibodies

Various methods such as enzyme linked

immunosorbent

assay

(ELISA), radio immunoassay (RIA), latex agglutination assay

have been employed for detection of antibodies in

patient serum However, diagnostic utility of these methods is doubtful WHO has recommended that these tests should not be used for diagnosis of active tuberculosis Slide42

Quantiferon

-Gold

Is an in vitro assay that measures the cell mediated immune

-response in the infected individuals through the levels of

interferon gamma (IFN-

γ

) released by the sensitised T- lymphocytes after stimulation by M. tuberculosis antigensSlide43

Molecular methods

Polymerase chain reaction (PCR)

LAMP

Ligase

chain reaction

PCR

Rapid method to detect

M. tuberculosis directly in clinical samples based on DNA amplificationIS6110 sequence is generally targeted for detection

M. tuberculosis complexSlide44

Prophylaxis

General measures

Adequate nutrition, good housing and health education are as

important as specific antibacterial measures

Immunoprophylaxis

The BCG (

Bacille Calmette-Guerin) vaccine (0.1 ml), administered soon after birth by intradermal Injection failing which it may be given at any time during the first year of life

This is a strain of M. bovis attenuated by 239 serial subcultures in a glycerine-bile-potato medium over a period of 13 yearsSlide45

Bacille

Calmette-Guérin

= BCG!

Albert

Calmette

Camille

Gu

érinSlide46

Chemoprophylaxis

This is the administration of

antituberculous

drugs

(usually only

isoniazid

)

1. To persons with latent tuberculosis (asymptomatic tuberculin positive)2. To persons with a high risk of developing active tuberculosis3. To the infant whose mother with active tuberculosis4. To the children living with a case of active tuberculosis in the house

Isoniazid 5 mg/kg daily for 6 – 12 months is the usual courseSlide47

References:

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