Mycobacteria

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Mycobacteria

  1. 1. LECTURE : MYCOBACTERIUM Diseases: Tuberculosis & Leprosy. 1. Mycobacterium tuberculosis Complex. 2. Mycobacterium tuberculosis: M. Africanum. M. Asian. M. bovis. M. Avium. Prof. Abbas Hayat
  2. 2. Historical Background • 8000-4000 B.C. M. bovis causing TB in animals. • 5000-1000 B.C, domestication of cattle, human infection by M. bovis likely through milk ingestion. 1000 BC, widespread pulmonary TB emerged. • M.tuberculosis, a specialized form of M.bovis developed among milk-drinking Indo-Europeans who then spread the disease during their migration into Western Europe and Eurasia. • After 1000 B.C. M.TB causing pulmonary TB had spread throughout the known world. • 668-626 BC. The classic TB signs--cough, expectoration, hemoptysis, wasting of the body, were well recognized. The earliest written evidence of pulmonary TB was from the library of the Assyrian king Assurbanipal (668-626 BC):
  3. 3. • 1600 TB responsible for 20% deaths in London. • 1800 over 30 % deaths in Paris. • 1865, French military doctor Jean-Antoine Villemin transmitted organism from one animal to other. • 1882 Robert Koch isolated tubercle bacillus. • 1890 Koch found tuberculin now used to identify existence of TB. • 1905, Koch won Nobel Prize for tuberculin. • 1943. Streptomycin, purified from Streptomycesgriseus. • 1949 Following streptomycin, p-amino salicylic • 1952 isoniazid 1954 pyrazinamide, 1955 cycloserine, 1962 ethambutol 1963 a rifampicin; were introduced as anti-TB agents. . • At time of discovery of bacillus, 1/5 people developed TB during their lifetime.
  4. 4. KOCH`PHENOMENON 1. When a guinea pig is injected subcutaneously with virulent tubercle bacilli, the puncture wound heals quickly, but a nodule forms at the site of injection in two weeks. This nodule ulcerates and the ulcer does not heal. The regional lymph nodes develop tubercles and caseates massively. 2. When the same animal is injected with tubercle bacilli in another part of the body, 6-8 weeks after there is rapid necrosis of skin and tissue, but the ulcer heals rapidly and regional lymph nodes do not become infected.
  5. 5. INTRODUCTION to Tuberculosis. • Someone infected with tuberculosis every second. • One third of the world's population infected with tuberculosis complex bacteria. • Left untreated, one person with tuberculosis will infect 10-15 people per year. . • Great concern: New strains; .WHO reports death with "multi-drug resistant TB" M.D.R.T.in the U.S. was approximately70 percent. • Diagnosis to death: four to sixteen weeks. • 90 million new tuberculosis cases and 30 million deaths worldwide. • It strikes people of all races, ages, and income levels. Higher risk. HIV infection, Close contacts with infectious TB; • Poor; Homeless; Prisons; Alcoholics; Elderly & Health care workers.
  6. 6. AT PRESENT TB is a global emergency according to the W.H.O. 3 million cases in Pakistan.
  7. 7. • MORPHOLOGY IDENTIFICATION & CULTURAL CHARACTERISTICS. • Zeihl Nelson Staining • Acid Fast Bacilli: Retain Carbol Fuschin stain & are not decolorized by Ethanol – HCL mixture, • Counter stain is Methylene blue, (RED RODS AGAINST BLUE BACKGROUND) • High lipid content in cell wall 66 % make them acid fast and heating required for penetration. • Neither Gram positive nor negative.
  8. 8. Drop suspension onto slide Air dry slide 10 minutes at 60 °C, heat-fix slide 10 minutes at 90 °C Flood slide with Carbol Fuchsin Hold flame beneath the slide until steam appears but do not allow it to boil Allow hot slide to sit for 3 to 5 minutes, rinse with tap water Flood slide with 30% hydrochloric acid in isopropol alcohol Allow to sit 1 minute, rinse with tap water Flood slide with Methylene Blue Allow to sit 1 minute, rinse with tap water Blot dry View under oil immersion lens
  9. 9. B. Auramine Florescence Stain. Fluorescent microscope and Rhodamin and auramine stains.
  10. 10. Important Properties: • Grow slowly. Doubling time 18 hours; Cultures require 6-8 weeks. • CULTURE: Lowenstein –Jenson medium (L.J) (egg yolk and Malachite green dye). • Obligate Aerobe: Predilection for upper lobe lungs, and Kidney. Contains complex lipids 1. Mycolic acids : Acid fastness 2. Wax D: Freunds adjuvant 3. Phospholipids: Caseation necrosis. 4. Glycolipid : Cord factor.
  11. 11. • Proteins + Waxes: elicit delayed hypersensitivity response (PPD) • Resistant to Acids and Alkalis and Drying.
  12. 12. PATHOGENESIS
  13. 13. "Resistance vs. Susceptibility`` • Tissue destruction results from Cell-mediated hypersensitivity. • Mycobacteria  inflammatory lesion  granulomatous lesion characterized by a mononuclear cell infiltrate surrounding a core of degenerating epithelioid and multinucleated giant (Langhans) cells.  This lesion (called a tubercle)  fibroblasts,  center progresses to caseous necrosis. Liquefaction of caseous material → erosion of the tubercle → cavitations and the release of massive numbers of bacilli into the sputum.
  14. 14. • In resistant host, the tubercle → calcified. • Early in infection, Mycobacteria → the lymphatics to the hilar or mediastinal lymph nodes. →thoracic duct → blood stream, or directly into the circulation by erosion of the developing tubercle into a pulmonary vessel. • Extra pulmonary hematogenous dissemination. → (e.g., spleen, liver, and kidneys) and, eventually, reinoculation of the lungs. • GHON COMPLEX (Primary lesion + Draining Lymph nodes) • GHON LESION (Reinfection or Reactivation without involvement of draining lymph nodes)
  15. 15. PRIMARY & REACTIVATION DISEASE • Tissue destruction results from presence of Organism & Host response (cell-mediated hypersensitivity) Two types of lesions: 1. Exudative Lesions: Acute inflammatory response mainly polymorphonuclear. Primary lesion: lower lobes in lungs: Parenchymal exudative lesion and the draining lymph nodes are called GHON COMPLEX. 2. Granulomatous Lesions: Central area of Giant cells containing tubercle bacilli, surrounded by epitheloid cells. Reactivation lesions: In apices, also Kidney Brain & Bones.
  16. 16. INGESTION OF UNPASTEURIZED MILK (M.Bovis) gastrointestinal tract.
  17. 17. Host Defenses • Susceptibility is influenced by genetic and ethnic factors. Acquired resistance is mediated by T lymphocytes, which lyses infected macrophages directly or activate them via soluble mediators (e.g., gamma interferon) to destroy intracellular bacilli; antibodies play no protective role. Clinical Manifestations. • Clinical signs and symptoms develop in only a small proportion (5-10 percent) of infected healthy people. • Pulmonary disease; prominent symptoms are chronic, productive cough, low-grade fever, night sweats, easy fatigability, and weight loss. • Extra pulmonary manifestations.
  18. 18. • Lymphadenitis; kidney, bone, or joint involvement; meningitis; or disseminated (miliary) disease. ``The patient coughs frequently; his sputum is thick and sometimes contains blood. His breathing is like a flute. His skin is cold, but his feet are hot. He sweats greatly and his heart is much disturbed. When the disease is extremely grave, he suffers from diarrhea.`` • Tuberculosis primarily affects the lower respiratory system and is characterized by a chronic productive cough, low-grade fever, • Night sweats, and weight loss.
  19. 19. DIAGNOSIS.
  20. 20. • The Mantoux test: intradermal injection of a measured volume (0.1 ml) containing a specified quantity (5 tuberculin units) of PPD. The transverse diameter of induration is measured 48 to 72 hours later. • Interpretation varies, as shown in MANTOUX TEST. • 5 T.U. 25 T.U. 250 T.U intradermal injection. • 48 to 72 hours later 5-10 mm of induration
  21. 21. Interpretation: Positive Test means a person has been exposed, Negative means Anergy, non exposure defective Cell Mediated Immunity or Miliary Tuberculosis.
  22. 22. CLINICAL SPECIMENS: • Sputum, Bronchial, Gastric washings, Pleural fluid, urine, cerebrospinal fluid • Biopsy material: endometrial; lymph nodes, other tissues etc. • Stained and cultured for acid-fast bacilli. Culture and identification of Mycobacteria in such specimens are mandatory for diagnosis.
  23. 23. CULTURE:
  24. 24. • Lowenstein-Jensen medium. Egg yolk media with malachite green in screw capped. 2. Dubos Medium 3. Synthetic Media. Require 6- 8 weeks for growth `` `RUFF BUFF & TOUGH COLONIES``
  25. 25. 2. Rapid Broth. Bactec. System
  26. 26. 3. Commercial chemiluminescent DNA probes, gas-liquid chromatography, high- performance liquid chromatography, and thin-layer chromatography allow identification of a few species of mycobacteria within hours after sufficient growth is present on solid or in a liquid medium
  27. 27. 4. Polymerase chain reaction (PCR)
  28. 28. Treatment and Control
  29. 29. • Aggressive prophylactic chemotherapy in tuberculin converters. • In individuals with clinical disease, short term (6-9 month) ambulatory therapy with so-called first-line anti-mycobacterial drugs, such as isoniazid, rifampin, pyrazinamide, and ethambutol, results in disappearance of viable tubercle bacilli from the sputum, rendering the patient noninfectious. • Directly observed therapy (DOT) has been instituted in high prevalence areas, especially among non compliant patients, as the only reliable means of ensuring that patients complete their treatment successfully. • the patient is cured. • the spread of disease is stopped. • MDR-TB is prevented.
  30. 30. • DOTS has been tested in New York, Tanzania, Indonesia, Peru, and China with good results. According to a report published in the March 10 issue of the Archives of Internal Medicine, the Program resulted in a 52% decrease in patients with MDR- TB in New York between 1991 and 1994.
  31. 31. Development of MDRT • 1960s, 1-2% of isolates were resistant to 2+ drugs. • 1970s, 3-5% of isolates were resistant to 2+ drugs. • 1986, no more national drug-resistance surveys. • 1991, 33% of isolates resistant to 1+ drugs, 13% resistant to the 4 front-line drugs. • When resistance to two or more of the first line drugs is detected, additional drugs (ethionamide, streptomycin, ciprofloxacin) may be added to the regimen.
  32. 32. COMBINATION THERAPY • Following streptomycin, p-amino salicylic acid (1949), isoniazid(1952), pyrazinamide (1954), cycloserine (1955), ethambutol (1962) and rifampin(rifampicin; 1963) were introduced as anti-TB agents. Amino glycosides such ascapreomycin, viomycin, kanamycin and amikacin, and the newer quinolones (e.g.ofloxacin and ciprofloxacin) are only used in drug resistance situations.
  33. 33. • Two properties of anti-TB drugs are important: antibacterial activity, highest in • Isoniazid Rifampin Streptomycin • and their capacity to inhibit the development of resistance, the most effective drugs being • Isoniazid Rifampin Ethambutol • The multiple drug regimen described earlier is very effective-->90% cure rate- if taken for 6-8 months.
  34. 34. PREVENTION& CONTROL • ``TEST & SLAUGHTER`` Policy • 1950 – PPD testing of cattle herds and slaughtering of animals resulted in elimination of this disease from United States. • Improved host resistance, • Better housing, • Prophylaxis with Isoniazid. • In patients exposed to infectious patients or recent converters and less than 45 years old.
  35. 35. BCG Vaccination. • A viable, attenuated strain of M bovis, called bacilli Calmette-Guérin (BCG), after the French microbiologists. Used in 120 countries. • First developed in 1920`s but strain got contaminated with pathogenic mycobacterium, vaccination stopped, till WW II. • Requires about 200 passages on potato medium culture.
  36. 36. Nontuberculous Mycobacteria, earlier known as Anonymous then Atypical Mycobacteria, now MYCOBACTERIA OTHER THAN TUBERCULOSIS(MOTT)
  37. 37. Epidemiology • A crucial difference between M tuberculosis and Nontuberculous Mycobacteria is lack of transmission of the latter from patient to patient. • No evidence that infections are contagious. • Organisms exist saprophytically in the soil or water, occasionally in association with some infected-animal reservoir (e.g., poultry infected with M avium). Inhalation or ingestion of viable Mycobacteria or introduction of bacilli through skin abrasions initiates the infection. • PATHOGENESIS Pathogenesis is similar to Mycobacteria. There may be granuloma formation
  38. 38. SIGNS & SYMPTOMS • Patients exhibit lower respiratory disease similar to tuberculosis (M kansasii, M avium-intracellulare), cervical lymphadenitis. • (M scrofulaceum), skin and soft tissue infections (M ulcerans, M marinum), or disseminated disease in persons infected with HIV.
  39. 39. CLINICAL PRESENTATION OF MYCOBACTERIA OTHER THAN TUBERCULOSIS (MOTT) Group 1 (Photocromogens) • M. kansasii: resembles tuberculosis • M. marinium: granulomatous ulcerative lesion ``Swimming pool granuloma`` treatment by tetracycline effective.
  40. 40. Group II (Scotochromogens) • M. scorfulaceum: cause scrofula; graunulomatous cervical adenitis in children. Surgical excision can cure.
  41. 41. Group III (Nonchromogens) • M. avium- intracellularae pulmonary disease indistinguishable from tuberculosis esp. in immunocompromised and HIV; highly resistant to anti tuberculous drugs 06 drug combination may be required.
  42. 42. Group IV ( Rapid Growing Mycobacteria) • M. fortuitum-chelonei complex: rarely cause human disease except in 1. Immunocompromised 2.prosthetic heart valves and hip joints... Frequently resistant may require multiple drug therapy and surgical excision. • M.Smegmetis : Non pathogenic but confuses in A.F.B. smears.
  43. 43. Treatment and Control • Many Nontuberculous Mycobacteria are resistant to commonly used drugs • Antibiotic regimens may require several (five or six) drugs including rifampin, which is quite effective against M kansasii, or clarithromycin, which has marked activity against the M avium- intracellulare complex. • Surgical resection is occasionally recommended with or without chemotherapy. • In treating disseminated infections in AIDS patients, a regimen of five or six drugs, including clarithromycin, ethambutol and perhaps rifampicin, should be considered.
  44. 44. Patience of Deaf Children
  45. 45. Mycobacterium Leprosy
  46. 46. ARMEDILLO
  47. 47. TWO TYPES OF DISEASE 1. LEPROMATOUS LEPROSY: course progressive and malign with nodular skin lesions, symmetric nerve involvement abundant acid fast bacilli in scrapings continuous bacteremia and a negative lepromin skin test
  48. 48. • 2. TUBERCULOID LEPROSY: course is benign and nonprogressive, with macular skin lesions , severe asymmetric nerve involvement of sudden onset and few bacilli present in lesions, and a Positive lepromin skin test. • Delayed hypersensitive is markedly defective in lepromatous leprosy.
  49. 49. CLINICAL FINDINGS • Insidious onset. • Involve cooler tissues of body i.e. skin superficial nerves , nose, pharynx, eyes, and testicles. • Skin lesions as pale anesthetic macular lesions 1-10 cm in dia, diffuse or discreet erythematous, infiltrated nodules 1-5 cm. or a diffuse skin infiltration.
  50. 50. • Neurological involvement manifested by nerve infiltration and thickening, resultant anesthesia, neuritis, parasthesia , trophic ulcers, and bone reabsorption with shortenening of digits. • Disfiguration may be extreme ``Lioness facies``
  51. 51. DIAGNOSIS • Scraping from skin, nasal mucosa, or from biopsy of the ear lobe skin are smeared on slide and stained by ZIEHL NELSON technique. • Biopsy of skin or thickened nerve gives characteristic histological picture.
  52. 52. TREATMENT • Sulphones i.e dapsone and Rifampin suppress growth if given for many months. • Sulphone resistance emerging . • Amithiazone substitute drug.
  53. 53. PREVENTION & CONTROL • In endemic areas removal of young children from infected families is employed with some success. • Chemotherapy of infected cases good prophylaxis for the community. • Chemoprophylaxis of contacts with Sulphones. • Experimental B.C.G. has been used with possible benefits.
  54. 54. Thank you for your patience

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