Tuberculosis

basics
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HISTORY of

Tuberculosis
Tuberculosis Is an Ancient Disease Spinal Tuberculosis in Egyptian Mummies History dates to 1550 1080 BC Identified by PCR
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Robert Koch Discoverer of Mycobacterium Tuberculosis

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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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Classification of Mycobacteria
1. Tubercle bacilli
a) b) c) d) e) Human MTB Bovine M. bovis Murine M. microti Avian M. avium Cold blooded M. marinum Human M. leprae Rat M. leprae murium

4. Atypical Mycobacteria (Runyon Groups)

a) b) c) d) Photochromogens Scotochromogens Nonphotochromogens Rapid growers

2. 3.

Lepra bacilli
a) b)

5. Johnes bacillus
M. paratuberculosis

6. Saprophytic mycobacteria
a) b) c) d) e) M. butyricum M. phlei M. stercoralis M. smegmatis Others
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Mycobacteria causing skin ulcers

a) b) M. ulcerans M. belnei
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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
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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 phenol.
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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
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M.bovis
Primarily infection among the cattle M.bovis infects Tonsils, Cervical nodes, can produce Scrofula. Enter through Intestines infects the Ileocecal region.
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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 onethird 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)
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 in Branched forms.

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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 phenol.
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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 for humans while M. bovis is usually pathogenic for animals
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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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Why they are Acid Fast

The character of Acid fastness is due to presence of Unsapnofiable wax ( My colic acid and semi permeable membrane around the cell)

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MTB : Cultural characters

MTB grows more Grow slowly. luxuriantly (eugonic) Generation time than M. bovis 14-15 hrs (dysgonic). Colonies appear Addition of 0.5% after 2 weeks or at Glycerol supports 6-8 weeks growth of human strains. No effect or MTB - Obligate inhibitory effect on aerobe bovine strains.
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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 Middle brook medium
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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 Malachite Green Selective agent Sterilized by - Inspissation
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On Liquid Medium Appear as long serpentine cords in liquid medium Virulent strains grow in a more dispersed manner.
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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, 4% Sodium hydroxide
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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 yellow color indicates positive test.

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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 not atypical INH resistant strains are negative for test
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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 are much more likely to develop TB.
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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 therapy
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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. CD4 Cell plays role in Immune Mechanisms.

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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 Granuloma
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Tubercle with Caseous Necrosis

Tubercle bacilli Lymphocyte

Giant cells

Fully activated macrophage

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Partially activated 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 Productive
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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- a
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TNF- a
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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. Bacilli are virulent - Host responds with DTH Injurious.

Productive Type protective Immunity

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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 person isn't bothered by the dormant bacillus.
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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 occurs in the lungs.

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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. 2. 3. 4. 5. 6. Meningitis. Pleurisy, Involvement of Kidney, Spine ( Potts spine ) Bone Joints, 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 10-100 / 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 suspicious
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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 CO2 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 with rifampin
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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) Health care workers observe the medication
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Immuno-prophylaxis
Intradermal injection of live attenuated vaccine Bacille Calmette-Guerin (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 6080% Some studies proved BCG is doubtful value in prevention of Tuberculosis
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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 stimulation of RE system.
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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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