Tuberculosis Teaching Basics

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Tuberculosis Teaching Basics

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MS Pゴシック:

Tuberculosis Teaching Basics Dr.T.V.Rao MD 1 Dr.T.V.Rao MD

Calibri:

HISTORY of Tuberculosis Tuberculosis Is an Ancient Disease Spinal Tuberculosis in Egyptian Mummies History dates to 1550 – 1080 BC Identified by PCR Dr.T.V.Rao MD 2

Wingdings:

Robert Koch Discoverer of Mycobacterium Tuberculosis Dr.T.V.Rao MD 3

Tahoma:

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 4

Blue Highway Linocut:

Classification of Mycobacteria Tubercle bacilli Human – MTB Bovine – M. bovis Murine – M. microti Avian – M. avium Cold blooded – M. marinum Lepra bacilli Human – M. leprae Rat – M. leprae murium Mycobacteria causing skin ulcers M. ulcerans M. belnei Atypical Mycobacteria (Runyon Groups) Photochromogens Scotochromogens Nonphotochromogens Rapid growers Johne ’ s bacillus M. paratuberculosis Saprophytic mycobacteria M. butyricum M. phlei M. stercoralis M. smegmatis Others Dr.T.V.Rao MD 5

Times New Roman:

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 6

Symbol:

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 7

Office Theme:

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.) Dr.T.V.Rao MD 8

Tuberculosis Teaching Basics :

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 9

HISTORY of Tuberculosis:

M.bovis Primarily infection among the cattle M.bovis infects Tonsils, Cervical nodes, can produce Scrofula. Enter through Intestines – infects the Ileocecal region. Dr.T.V.Rao MD 10

Robert Koch Discoverer of Mycobacterium Tuberculosis:

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 Dr.T.V.Rao MD 11

What are Mycobacteria? :

Atypical Mycobacterium 1 Photochromogens 2 Scotochromogens 3 Non Photochromogens 4 Rapid growers Dr.T.V.Rao MD 12

Classification of Mycobacteria:

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. Dr.T.V.Rao MD 13

Mycobacterium differ from other routinely isolated Bacteria:

Poverty and Crowded living spreads Tuberculosis Dr.T.V.Rao MD 14

Acid fast bacilli:

Tuberculosis infects Famous people too Dr.T.V.Rao MD 15

How they are Acid fast:

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 16

Mycobacterium tuberculosis complex:

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, Non-capsulated. Growth generally slow Genus includes Obligate parasites Opportunist pathogens Saprophytes Dr.T.V.Rao MD 17

M.bovis:

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. Dr.T.V.Rao MD 18

What are atypical Mycobacterium:

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 19

Atypical Mycobacterium:

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 Dr.T.V.Rao MD 20

MOST IMPORTANT AMONG INFECTIOUS DISEASES:

Avian Tuberculosis T ransmitted 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. Dr.T.V.Rao MD 21

Poverty and Crowded living spreads Tuberculosis:

Acid Fast Bacilli seen in a specimen of Sputum Dr.T.V.Rao MD 22

Tuberculosis infects Famous people too:

Acid fast bacilli Dr.T.V.Rao MD 23

What are Mycobacteria? :

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. Dr.T.V.Rao MD 24

General characters of the genus:

Why they are Acid Fast The character of Acid fastness is due to presence of Unsapnofiable wax ( Mcolic acid and semi permeable membrane around the cell) Dr.T.V.Rao MD 25

Morphology of Mycobacterium tuberculosis:

MTB : Cultural characters Grow slowly. Generation time 14-15 hrs Colonies appear after 2 weeks or at 6-8 weeks MTB - Obligate aerobe MTB grows more luxuriantly (eugonic) than M. bovis (dysgonic). Addition of 0.5% Glycerol supports growth of human strains. No effect or inhibitory effect on bovine strains. Dr.T.V.Rao MD 26

ACID FAST BACILLI:

Culturing Acid Fast Bacilli Slow to grow , Generation time is 14 – 15 hours > 2 weeks minimal required period Grows at 37 0 c do not grow below 25 0 c Ph between 6.4 to 7.0 Dr.T.V.Rao MD 27

Important Mycobacterium:

Eight Week Growth of Mycobacterium tuberculosis on Lowenstein-Jensen A gar Dr.T.V.Rao MD 28

Avian Tuberculosis:

Nature of Media Used Helps the growth needs Solid Medium is commonly used Lowenstein Jensen ’ s medium Petrangini Middle brook medium Dr.T.V.Rao MD 29

Acid Fast Bacilli seen in a specimen of Sputum:

Lowenstein Jensen ’ s Medium Contain coagulated egg Mineral salt solution Asparagine's Malachite green Agar Dr.T.V.Rao MD 30

Acid fast bacilli:

Other Medium Middle brook Sula's medium But not routinely used Dr.T.V.Rao MD 31

Acid fast Bacilli seen as in Florescent Microscope:

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. Dr.T.V.Rao MD 32

Why they are Acid Fast:

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 Dr.T.V.Rao MD 33

MTB : Cultural characters:

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 Dr.T.V.Rao MD 34

Culturing Acid Fast Bacilli:

On Liquid Medium Appear as long serpentine cords in liquid medium Virulent strains grow in a more dispersed manner. Dr.T.V.Rao MD 35

PowerPoint Presentation:

Resistance of Mycobacterium Mycobacterium are killed at 60 0 c 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 Dr.T.V.Rao MD 36

Nature of Media Used:

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 Dr.T.V.Rao MD 37

Lowenstein Jensen’s Medium:

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. Dr.T.V.Rao MD 38

Other Medium:

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 Dr.T.V.Rao MD 39

Nature of Growth Characters:

Catalase Test 30 Vol of H 2 O 2 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 Dr.T.V.Rao MD 40

On L J Medium:

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 Dr.T.V.Rao MD 41

PowerPoint Presentation:

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 Dr.T.V.Rao MD 42

On Liquid Medium:

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 Dr.T.V.Rao MD 43

Resistance of Mycobacterium:

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 Dr.T.V.Rao MD 44

Resistance to several agents:

Genome of Mycobacterium tuberculosis Dr.T.V.Rao MD 45

Biochemical Tests on Mycobacterium spp:

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. Dr.T.V.Rao MD 46

Other Tests:

Tuberculosis spread by Respiratory route Dr.T.V.Rao MD 47

Catalase Test:

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. Dr.T.V.Rao MD 48

Antigenic Characters:

In India 1 death / Minute Half a million people die from disease every year in India one death every minute Dr.T.V.Rao MD 49

Bacteriophages:

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. Dr.T.V.Rao MD 50

Molecular Typing:

Spread of Tuberculosis Dr.T.V.Rao MD 51

Finger printing Methods:

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 Dr.T.V.Rao MD 52

Genome of Mycobacterium tuberculosis:

Dr.T.V.Rao MD 53

How tuberculosis spreads:

Predisposing Factors Genetic basis, Age Stress, Nutrition, Co existing infections Eg HIV Dr.T.V.Rao MD 54

Tuberculosis spread by Respiratory route:

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. CD 4 Cell plays role in Immune Mechanisms . Dr.T.V.Rao MD 55

Tuberculosis highly Communicable Disease.:

Mechanisms of Infection Within 10 days of entry of Bacilli clones of Antigen specific T Lymphocytes are produced Can actively produce Cytokines, Interferon γ which activate Macrophages form cluster or Granuloma Dr.T.V.Rao MD 56

In India 1 death / Minute :

Tubercle with Caseous Necrosis Giant cells Tubercle bacilli Partially activated macrophage Lymphocyte Fully activated macrophage Dr.T.V.Rao MD 57

Pathology and Pathogenesis of Tuberculosis:

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 Dr.T.V.Rao MD 58

Spread of Tuberculosis:

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 . Dr.T.V.Rao MD 59

Generation of Droplet Nuclei:

Tubercle discharging Bronchial tree TNF- a TNF- a Dr.T.V.Rao MD 60

PowerPoint Presentation:

Immunity in Tuberculosis. CD 4 T Lymphocytes with Th 1 or Th 2 secrete - 1 Cytokines,2 Interleukin 1,and 2 , 3 Interferon's γ ,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. . Dr.T.V.Rao MD 61

Predisposing Factors:

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. Dr.T.V.Rao MD 62

Mechanisms of Infection :

Lesions in Tuberculosis Exudative – and Productive Exudative – Acute inflammatory reaction with edema fluid – contains Polymorphs- Lymphocytes – later Mononuclear cells. Bacilli are virulent - Host responds with DTH Injurious. Productive Type protective Immunity Dr.T.V.Rao MD 63

Mechanisms of Infection:

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 Ghon ’ s focus. The Hilar Lymph nodes are also involved Dr.T.V.Rao MD 64

Tubercle with Caseous Necrosis:

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/ 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. Dr.T.V.Rao MD 65

Basis of Tubercle formation.:

Primary complex Ghon ’ s 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. Dr.T.V.Rao MD 66

PowerPoint Presentation:

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. Dr.T.V.Rao MD 67

Tubercle discharging:

Koch ’ s 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 Koch ’ s Phenomenon. Dr.T.V.Rao MD 68

Immunity in Tuberculosis.:

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. Dr.T.V.Rao MD 69

Immunity in Tuberculosis :

Majority of the Tuberculosis are Pulmonary Dr.T.V.Rao MD 70

Lesions in Tuberculosis:

Multiorgan Involvement in Tuberculosis. Dr.T.V.Rao MD 71

Primary Tuberculosis:

Complication of Tuberculosis . Meningitis. Pleurisy, Involvement of Kidney, Spine ( Potts spine ) Bone Joints, Miliary tuberculosis Dr.T.V.Rao MD 72

Primary complex:

Symptoms and Sings of Tuberculosis Dr.T.V.Rao MD 73

Primary complex:

Clinical Illness with Tuberculosis Pulmonary Disease – Major manifestation with involvement of Lungs Hemoptysis, Chest pain Fever sweets Anorexia Cavity formation in Lungs Dr.T.V.Rao MD 74

Reactivation of Tuberculosis:

Tuberculosis - Pneumothorax Dr.T.V.Rao MD 75

Koch’s Phenomenon:

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 Dr.T.V.Rao MD 76

Post Primary Tuberculosis:

Tuberculosis - Lymphadenitis Dr.T.V.Rao MD 77

Majority of the Tuberculosis are Pulmonary:

Multidrug-resistant tuberculosis (MDR-TB) Drug resistance arises due to improper use of antibiotics in chemotherapy of drug-susceptible TB patients. This improper use is a result of a number of actions including, administration of improper treatment regimens and failure to ensure that patients complete the whole course of treatment. Dr.T.V.Rao MD 78

Multiorgan Involvement in Tuberculosis.:

Definition of MDR Tuberculosis MDR-TB is defined as resistance to isoniazid and rifampicin, with or without resistance to other first-line drugs (FLD). XDR-TB is defined as resistance to at least isoniazid and rifampicin, and to any fluoroquinolone, and to any of the three second-line injectables (amikacin, capreomycin, and kanamycin). Dr.T.V.Rao MD 79

Complication of Tuberculosis.:

MDR tuberculosis dangerous to Society too Essentially, drug resistance arises in areas with weak TB control programmes. A patient who develops active disease with a drug-resistant TB strain can transmit this form of TB to other individuals Dr.T.V.Rao MD 80

Symptoms and Sings of Tuberculosis:

X- MDR The term ‘ totally drug resistant ’ has not been clearly defined for tuberculosis. XDR-TB severely reduces the options for treatment although they have not been studied in large cohorts. Treatment options for XDR-TB patients who have resistance to additional second-line anti-TB drugs are even more limited. Dr.T.V.Rao MD 81

Clinical Illness with Tuberculosis:

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. Dr.T.V.Rao MD 82

Tuberculosis - Pneumothorax:

Diagnosis of Tuberculosis Dr.T.V.Rao MD 83

Extra pulmonary Tuberculosis:

Types of specimens: -Sputum. - BAL. -Pleural effusions - Urine - Stool -CSF -Aspiration ( gastric – cold abscess) - Blood in case of haematogenous TB Dr.T.V.Rao MD 84

Tuberculosis - Lymphadenitis:

85 Sputum Collection Sputum specimens are essential to confirm TB Specimens should be from lung secretions, not saliva Collect 3 specimens on 3 different days Spontaneous morning sputum more desirable than induced specimens Collect sputum before treatment is initiated

Multidrug-resistant tuberculosis (MDR-TB):

86 Culture Used to confirm diagnosis of TB Culture all specimens, even if smear is negative Initial drug isolate should be used to determine drug susceptibility

Definition of MDR Tuberculosis:

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. Dr.T.V.Rao MD 87

MDR tuberculosis dangerous to Society too:

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 Dr.T.V.Rao MD 88

X- MDR:

Quantitation of AFB in Sputum Smears No of Bacilli No of Fields Report as 0 300 Negative 1-2 300 Doubtful 1- 9 100 1+ 1- 9 10 2+ 1-9 1 3+ 10 or >10 1 4+ Dr.T.V.Rao MD 89

Epidemiology:

Lowenstein–Jensen medium When grown on LJ medium, M. tuberculosis appears as brown, granular colonies (sometimes called "buff, rough and tough"). The media must be incubated for a significant length of time, usually four weeks, due to the slow doubling time of M. tuberculosis Dr.T.V.Rao MD 90

Diagnosis of Tuberculosis :

3- Cultures on L J media Lowenstein –Jensen medium is an egg based media with addition of salts, 5 % glycerol, Malachite green. 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 ) Dr.T.V.Rao MD 91

PowerPoint Presentation:

Detection and identification of mycobacteria directly from clinical samples Genotypic Methods : 􀂄 PCR 􀂄 LAMP 􀂄 TMA / NAA 􀂄 Ligase chain reaction 􀂄 Phenotypic Methods : 􀂄 FAST Plaque TB Dr.T.V.Rao MD 92

Sputum Collection:

Essentially PCR is a way to make millions of identical copies of a specific DNA sequence , which may be a gene, or a part of a gene, or simply a stretch of nucleotides with a known DNA sequence, the function of which may be unknown Polymerase Chain Reaction (PCR) Dr.T.V.Rao MD 93

Culture:

Interferon-γ assays measure cell-mediated immunity by quantifying IFN-γ released from sensitized T cells in whole blood/PBMCs incubated with TB antigens. Quantiferon-GOLD Dr.T.V.Rao MD 94

Laboratory Diagnosis:

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 * Dr.T.V.Rao MD 95

PowerPoint Presentation:

* False negative reactions may be due to: - False negative reactions may be due to: - Sever tuberculosis infection (Miliary T.B.) - Hodgkin ’ s disease - Corticosteroid therapy - Malnutrition - AIDS * Children below 5 years of age with no exposure history: - Positive test must be regarded suspicious Dr.T.V.Rao MD 96

Quantitation of AFB in Sputum Smears:

Tuberculin Test ( Mantoux Test ) Test to be interpreted in relation to clinical evaluation. Even the induration of 5 mm to be considered positive when tested on HIV patients. Lacks specificity.

Lowenstein–Jensen medium:

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.) - Hodgkin ’ s disease - Corticosteroid therapy - Malnutrition - AIDS * Children below 5 years of age with no exposure history: - Positive test must be regarded suspicious Dr.T.V.Rao MD 98

3- Cultures on L J media Lowenstein –Jensen medium is an egg based media with addition of salts, 5 % glycerol, Malachite green. :

Recent Methods for Diagnosis I – BACTEC 460 ( rapid radiometric culture system ) specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C 14 – 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. Dr.T.V.Rao MD 99

Detection and identification of mycobacteria directly from clinical samples:

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) Dr.T.V.Rao MD 100

Polymerase Chain Reaction (PCR):

Tuberculosis and HIV infection HIV association has become a threat to the developed countries too HIV association will lead to rapid spread of tuberculosis Dr.T.V.Rao MD 101

Quantiferon-GOLD:

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 Dr.T.V.Rao MD 102

Tuberculin Test:

MDR tuberculosis Multidrug resistant tuberculosis has become a global threat. In 1993 WHO declared Tuberculosis a Global emergency Animals shed the bacilli in Milk, human ’ s get infected after drinking the unsterilized Milk Pasteurization has reduced the incidence of Bovine tuberculosis. Dr.T.V.Rao MD 103

* False negative reactions may be due to: - :

Second Line Drug Treatment (SLD ’ s) Less effective, more costly and more toxic, 50% cure rate Four months intensive phase (5 drugs) Kanamycin Ethionamide Pyrazinamide Ofloxacin Cycloserine or Ethambutol 7X times p/w in hospital Followed by

Tuberculin Test ( Mantoux Test ):

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. Dr.T.V.Rao MD 105

Tuberculin Testing - Limitations:

March 24 th World TB Day Dr.T.V.Rao MD 106

Recent Methods for Diagnosis:

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 Dr.T.V.Rao MD 107

III Polymerase Chain Reaction (PCR) & Gene probe:

Directly Observed Therapy – Short Course DOTS (directly observed treatment, short-course), is the name given to the World Health Organization-recommended tuberculosis control strategy that combines components: Case detection by sputum smear microscopy Standardized treatment regimen directly observed by a healthcare worker or community health worker for at least the first two months A regular drug supply Dr.T.V.Rao MD 108

Tuberculosis and HIV infection:

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 60-80% Some studies proved BCG is doubtful value in prevention of Tuberculosis Dr.T.V.Rao MD 109

HIV Considerations:

Bacillus Calmette-Guérin (BCG) Bacillus Calmette-Guérin (BCG) is a vaccine against tuberculosis that is prepared from a strain of the attenuated (weakened) live bovine tuberculosis bacillus, Mycobacterium bovis, that has lost its virulence in humans by being specially subcultured (230 passages) in an artificial medium for 13 years, Dr.T.V.Rao MD 110

MDR tuberculosis:

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. Dr.T.V.Rao MD 111

Second Line Drug Treatment (SLD’s):

Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students in the Developing World Email doctortvrao@gmail.com Dr.T.V.Rao MD 112

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