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Tuberculosis

M.Sc. Biotechnology Part-II (Sem III)Paper II - Unit II

Mumbai UniversityBy: Mayur D. Chauhan

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Introduction

• Tuberculosis, MTB, or TB in the past also called phthisis, phthisis pulmonalis, or consumption, is a widespread, and in many cases fatal, infectious disease caused by various strains of mycobacteria, usually Mycobacterium tuberculosis.

• Tuberculosis typically attacks the lungs, but can also affect other parts of the body.

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Types of Tuberculosis Infection

• Infection mainly affects the lungsPulmonary

• Pleura, CNS, Genitourinary, Lymphatic system, Bones and Joints etc

Extra-pulmonary

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Extra-pulmonary Sites

• Pleura (Tuberculous pleurisy) • Central nervous system (Tuberculous meningitis)• Lymphatic system (Scrofula of the neck) • Genitourinary system (Urogenital tuberculosis)• Bones and joints (Pott disease of the spine). When it

spreads to the bones, it is also known as "osseous tuberculosis“, a form of osteomyelitis.

• A potentially more serious, widespread form of TB is called "disseminated" TB, commonly known as miliary tuberculosis.

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Symptoms of TB

• General signs and symptoms include fever, chills, night sweats, loss of appetite, weight loss, and fatigue.

• Significant nail clubbing may also occur.

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Causative Agent and it’s Properties• Mycobacterium tuberculosis is the causative agent for TB

and it belongs to the group of Mycobacteriaceae.• M. tuberculosis organisms are straight or slightly curved rods• occurring singly, in pairs, or in small clumps.• They measure about 3 0.3 m in size; sometimes long

filamentous forms are also seen. M. bovis is usually shorter and stouter than M. tuberculosis.

• They are Gram positive but are difficult to stain. They stain poorly with Gram staining.

• They are nonmotile, nonsporing, and noncapsulated

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Mycobacterium tuberculosis Complex

• M. bovis• M. cannettii

• M. tuberculosi

s

• M. microti

Bacillus- Calmette

GuerinM. caprae

M. africannum

M. pinnipedii

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Acid Fastness of Mtb

• M. tuberculosis is acid fast and alcohol fast; it resists decolorization by 20% sulfuric acid and absolute alcohol.

• This acid-fast staining is the most important property of the bacteria and is used widely for identification of the bacteria.

• Ziehl–Neelsen (ZN)-staining procedure is used to demonstrate acid fastness of the bacteria.

• With this stain, M. tuberculosis stains bright red, while the tissue cells and other organisms are stained blue.

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C] Ziehl- Neelsen Staining and Grading

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• These bacilli can also be stained by Auramine• O stain and examined under a fluorescent

microscope. They• appear as bright fluorescent rods against a

dark background

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Culturing Mtb

• M. tuberculosis is an obligate aerobe and characteristically grows very slowly in media.

• Optimum temperature is 37°C, and the bacteria do not grow below 25°C or above 40°C.

• Optimum pH is 6.4–7.0. M. bovis is microaerophilic on primary isolation, which subsequently becomes aerobic on subculture

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• M. tuberculosis is a slow-growing bacillus with an average generation time of 14–15 hours

• Prolonged incubation is therefore necessary for demonstrating growth of the bacteria.

• The colonies usually appear in almost 2 weeks, but sometimes require incubation up to 8 weeks to appear.

• M. tuberculosis can grow on a wide range of enriched solid and liquid media.

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Culture on solid media

• The examples of solid media are (i) egg-containing media (Lowenstein–Jensen [LJ] medium,Petragnani and Dorset egg medium)(ii) blood-containing media (Tarshis medium), serum-combining media (Loeffler’s serum slope), (iii) potato-based media (Pawlowsky medium).

• On these media, M. tuberculosis produces dry, rough, raised, and irregular colonies with a wrinkled surface.

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Lowenstein-Jensen Medium• LJ medium without starch is most widely used and is also

recommended by the International Union against Tuberculosis (IUAT).

• The LJ medium consists of coagulated whole egg, asparagines, malachite green, mineral salt, and glycerol or sodium pyruvate.

• Malachite green inhibits growth of bacteria other than mycobacterium.

• Addition of 0.75% glycerol enhances growth of M. tuberculosis, but is inhibitory to growth of M. bovis.

• On the LJ medium after 6–8 weeks of incubation, M. tuberculosis produces yellowish or buff-colored colonies. They are tenacious not easily emulsified

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Culture in liquid media

• The commonly used liquid media include Soloac’s solutions, Dubos medium, and Middlebrook’s and Beck’s medium.

• In liquid media, M. tuberculosis produces growth that appears first at the bottom and then grips up t the sides and produces a surface pellicle that may extend along the sides above the medium.

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• The liquid media are not used for routine culture of the bacilli.

• They are usually used for (i) preparation of mycobacterial antigens for vaccines and (ii) to assess antibiotic sensitivity of M. tuberculosis

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Biochemical reactions• Niacin test is an important test to identify niacin-positive human

strains of M. tuberculosis and differentiate them from niacin-negative M. bovis strains. M. tuberculosis human strains are niacin test positive. M. simiae and a few strains of M. chelonae are also niacin positive. They produce niacin as a metabolic by-product when grown on an egg-containing solid medium.

• M. tuberculosis is usually catalase positive. They lose their catalase activity when they become resistant to isoniazid (INH). Catalase-negative strains of M. tuberculosis are not virulent for guinea pigs.

• M. tuberculosis is amidase positive. It produces amidase enzymes, such as nicotinamidase and pyrazinamidase, which split various amide substrates.

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• M. tuberculosis is positive for nitrate reduction test. It produces the enzyme nitrate reductase, thereby reducing nitrate to nitrite. M. bovis and M. avium lack the enzyme nitroreductase and therefore are negative for nitrate reduction test.

• M. tuberculosis as well as M. bovis are neutral red test positive. They have the property to bind neutral red in alkaline buffer solution.

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• M. tuberculosis is arylsulfate test negative. If arylsulfatase is produced, it splits the phenolphthalein substrate, releasing free phenolphthalein, which turns pink to red when alkali is added to the medium.

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Other properties• Mycobacteria are killed by heating at 60°C for 15–20

minutes. • Killing of the bacteria is dependent on the nature of the

clinical specimen in which the bacteria are present.• Mycobacteria in the sputum may survive for 20–30 hours,

but in dried sputum protected from the sunlight they may survive longer, up to 6 months.

• In droplet nuclei, they may remain alive for 8–10 days.• Bacteria are killed when exposed to direct sunlight for 2

hours, but they remain viable at room temperature for 6–8 months.

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• Tubercle bacilli are sensitive to formaldehyde and glutaraldehyde.

• They are destroyed by tincture of iodine in 5 minutes and by 80% ethanol in 2–10 minutes.

• They are generally more resistant to chemical disinfectants than other nonsporeforming bacilli.

• They can survive exposure to 5% phenol, 15% sulfuric acid, 3% nitric acid, 5% oxalic acid, and 4% sodium hydroxide.

• M. tuberculosis is susceptible to pyrazinamide, while M. bovis and other mycobacteria are resistant. M. tuberculosis is resistant to thiophene-2-carboxylic acid hydrazide (TCH), while M. bovis is susceptible.

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Cell Wall of Mtb

• The cell wall of M. tuberculosis consists of four layers—(i) peptidoglycan layer,

(ii) arabinogalactan layer, (iii) mycolic acid layer, (iv) mycosides

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• The peptidoglycan layer is the innermost layer covalently linked with arabinogalactan (polysaccharide) and its terminal ends are linked to mycolic acid.

• This layer is overlaid with polypeptides and a layer of mycolic acid consisting of free lipids, glycolipids, and peptidoglycolipids. These lipids constitute nearly 60% of the dry weight of the cell wall.

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• The peptide chains present in the outer layer are important antigens, which stimulate cell-mediated immunity (CMI) in infected humans.

• PPDs are extracted and partially purified preparation of these proteins, which are used as antigens in tuberculosis skin test.

• The mycolic acid fraction of the lipids of the cell wall is responsible for many of the characteristic properties of the bacilli. These include (a) acid fastness, (b) slow growth by delaying permeation of nutrients, (c) resistance to commonly used antibiotics, (d) resistance to detergents, (e) unusual resistance to killing by acids and alkalies, and (f) clumping or cord formation.

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Antigenic Structure

• Mycobacteria possess two types of antigens: (a) cell wall insoluble polysaccharide antigens and (b) cytoplasmic soluble protein antigens.

• Cell Wall Antigens include arabinogalactans, lipoarabinomannan, and also lipids, glycolipids and peptidoglycolipids.

• Cytoplasmic antigens include antigen 5, antigen 6, antigen 14, antigen 19, antigen 33, antigen 38, an antigen 60

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Transmission of Tuberculosis

• Person to Person• Present in Air-borne

particles termed as Droplet Nuclei.

• 1-5 µm in size• Generated when

infected patients cough or sneeze

• Bacteria may remain viable in the environment uptill 8 months.

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Pathogenesis of Tuberculosis

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Virulence factors• The factors determining the virulence of M. tuberculosis

are poorly understood. M. tuberculosis does not produce any toxin.

• Although cord factor and sulfolipids are toxic substances produced by mycobacterium, their existence as virulence factors is doubtful.

• Cord factor: Cord factor was so called because of the false belief that it is responsible for producing serpentine cords typically found on the surface of liquid or on solid media by M. tuberculosis. The cord factor was originally thought to be a virulent factor, which no longer holds true.

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• Sulfolipids: Sulfolipids are of doubtful virulence factor. Their exact role in pathogenesis of the disease is not known.

• They are found to be associated with virulence of tubercle bacilli by preventing fusion of phagosome and lysosome inside the macrophages, thereby allowing the bacteria to multiply within the macrophages.

• The main pathology in the infected tissue caused by mycobacterial infection is primarily due to responses of the host to M. tuberculosis infection rather than any virulence factor produced by it.

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Host Immunity

• M. tuberculosis infection in an infected host induces CMI.

• The CMI is manifested either as delayed tuberculin hypersensitivity or as resistance to infection.

• The course of infection is determined by the interaction of hypersensitivity or immunity

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Tuberculin hypersensitivity reaction

• This was first described by Robert Koch in experimentally infected animals,such as guinea pigs.

• Demonstration of this tuberculin reactivity in guinea pigs is known as Koch’s phenomenon.

• Koch phenomenon is demonstrated by subcutaneous injection of pure culture of virulent tubercle bacilli in a normal guinea pig.

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• Initially, no immediate visible reaction is observed at the site of inoculation in the guinea pigs.

• But after 10–14 days, a hard nodule appears at the site of inoculation, which soon breaks down to from an ulcer that persists till the animal dies of progressive tuberculosis.

• The regional lymph nodes draining the region become enlarged and caseous.

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• In contrast, when a guinea pig already inoculated 4–6 weeks earlier by tubercle bacilli is injected with tubercle bacilli, an individual lesion develops at the site of second inoculation within 24–48 hours.

• The lesion undergoes necrosis in another day to produce a shallow ulcer that heals rapidly, involving the regional lymph nodes and other tissues.

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• The DTH can be induced by live attenuated and killed bacilli, bacillary products, and tubercular protein.

• Usually live o killed bacilli or tubercular protein (tuberculin) are employed for demonstration of hypersensitivity reaction

• This hypersensitivity reaction can be transferred passively by cells, but not by serum.

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Tuberculin test

• Tuberculin test is performed to demonstrate delayed type IV (DTH) or cell-mediated (CMI) hypersensitivity reaction to tubercle bacilli.

• Originally, the tuberculin test was performed by using a protein known as old tuberculin (OT) prepared by Robert Koch.

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• The OT is a protein component of tubercle bacilli prepared from a 6–8 week culture filtrate of M. tuberculosis cultured in 5% glycerol, which is concentrated 10-fold by evaporation on a steam bath.

• The OT is a crude protein, which consists of protein as a constituent but varies from batch to batch in its purity and potency.

• This has now been replaced by the use of PPD of tubercle bacilli.

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Humoral Immunity

• Humoral immunity is characterized by the development of antibodies in serum, but they do not play any role in conferring immunity against the bacteria.

• Antibodies against polysaccharide, proteins, and phosphate antigens of tubercle bacilli have been demonstrated in serum of patients with tuberculosis.

• These serum antibodies, however, are not protective.

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Habitat

• M. tuberculosis inhabits primarily the respiratory tract of the infected human host

• The droplet nuclei consisting of M. tuberculosis have been found in the terminal air spaces of the lung.

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Reservoir, source, and transmissionof infection

• Human beings are the only source and reservoir for M. tuberculosis infection.

• The infectiousness of the source is of primary importance, which determines the possibility of transmission of the disease.

• This depends on bacillary load of lesions and also on the morphology of the lesion. Lesions with cavities have 100–10,000 bacilli; therefore, cases with cavitary lesions are potentially highly infectious.

• Cases treated with antitubercular therapy are less infectious than those who are not treated with any antitubercular drugs.

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• The decrease in infectiousness is primarily due to reduction in the bacillary load in the lungs.

• Humans acquire M. tuberculosis infection most frequently by inhalation of infectious aerosolized droplets.

• These infective droplets are usually coughed or sneezed into environment by a patient suffering from pulmonary tuberculosis.

• The acts of coughing, sneezing, and speaking release a large number of droplets containing as many as 3000 infectious airborne droplet nuclei per cough.

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• The droplet nucleus is small, measures 5 μm or less, and may contain approximately 1–10 tubercle bacilli.

• Theoretically, although a single tubercle bacillus may cause disease, in practice 5–200 inhaled bacilli are essential for infection.

• These droplets by virtue of their small size remain suspended in the air for a very long period of time.

• The infection is acquired rarely by inoculation.• M. bovis infection is transmitted to humans by ingestion of raw

milk of the cows infected with M. bovis. • The infection among animals is spread by aerosolized bacilli in

moist cough sprays. • The infected animals usually excrete the bacilli in their milk.

Person-to-person transmission of M. bovis usually does not occur.

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References

• Textbook of Microbiology and Immunology by Subhash Parija

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