Mycobacteria

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  • Mycobacteria
    • long, slender rods
    • nonmotile
    • do not form spores
    • Mycolic acid in cell wall (MCQ)
      • cell walls are unusual in that they are approximately 60 percent lipid contain an unique class of very long–chain (75 to 90 carbons), b-hydroxylated fatty acids (mycolic acids).
      • creates a waxy cell surface (MCQ)
      • makes mycobacteria strongly hydrophobic
      • accounts for their acid-fast staining characteristic. (MCQ)
      • make mycobacteria impervious to many chemical disinfectants
      • convey resistance to the corrosive action of strong acids or alkalis. (MCQ)
    • Cord factor (MCQ)
      • a mycoside that contains two molecules of mycolic acid esterified to the disaccharide trehalose.
      • It is found in virulent mycobacteria(MCQ)
      • its presence is responsible for a phenomenon in which the individual bacteria grow parallel to each other, forming large, serpentine cords.
      • responsible for the cachexia observed in tuberculosis patients(MCQ)
      • responsible for the fever and pulmonary necrosis that is characteristic of tuberculosis.
    • Sulfatides(MCQ)
      • A group of glycolipids similar to cord factor
      • are multiacylated trehalose 2-sulfates.
      • inhibit phagosome-lysosome fusion
      • enhance survival of phagocytosed mycobacteria. (MCQ)
    • Wax D (MCQ)
      • complicated mycoside
      • 15 to 20 molecules of mycolic acid are esterified to a large polysaccharide composed of arabinose, galactose, mannose, glucosamine, and galactosamine—all of which seem to be linked to the peptidoglycan of the cell wall
      • Wax D acts as an adjuvant to increase the antibody response to an antigen,
      • it is the active component in Freund’s complete adjuvant, which employs intact tubercle bacilli emulsified with water, oil, and antigen. (MCQ)
    • Lipoarabinomannan (LAM). (MCQ)
      • Lipopolysaccharide in Mycobacteria
      • inducer of tumor necrosis factor-alpha synthesis by monocytes and macrophages. (MCQ)
    • Mycobacteria are also resistant to drying but not to heat or ultraviolet irradiation.
    • Mycobacteria are strictly aerobic. (MCQ)
    • Most species grow slowly with generation times of 8 to 24 hours. (MCQ)
    • organisms can remain viable as droplet nuclei suspended in room air for at least 30 minutes
    • The principal mode of contagion is person-to-person transmission by inhalation of the aerosol.
    • virulence of M. tuberculosis rests with its ability to survive and grow within host cells
    • when engulfed by macrophages, bacterial sulfolipids inhibit the fusion of phagocytic vesicles with lysosomes(MCQ)
    • The ability of M. tuberculosis to grow even in immunologically activated macrophages and to remain viable within the host for decades is a unique characteristic of the pathogen.
    • M. tuberculosis stimulates both a humoral and a cell- mediated immune response.
    • cellular immunity (CD4+ T cells) and the accompanying delayed hypersensitivity directed against a number of bacterial protein antigens, contribute to both the pathology of and immunity to the disease.
    • Tuberculin reaction:
      • a manifestation of delayed hypersensitivity to protein antigens of M. tubercu- losis. (MCQ)
      • Although such tests can be used to document contact with the tubercle bacillus,
      • It does not confirm that the patient currently has active disease. (MCQ)
    • Mantoux test(MCQ)
      • purified protein derivative (PPD) is prepared from culture filtrates of the organism and biologically standardized.
      • Activity is expressed in tuberculin units.
      • In the routine procedure (Mantoux test), a measured amount of PPD is injected intradermally (MCQ)
      • It is read 48 to 72 hours later for the presence and size of an area of induration (hardening) at the site of injection(MCQ)
      • Dose
        •  5 TU is usual dose – if test negative; increase dose to 250 TU(MCQ)
      •  Positive test(MCQ)
        • The test is read after 48 – 72 hours. (MCQ)
        • Induration of ≥ 10 mm with erythema. (MCQ)
        • Induration of 5-9 mm – low level sensitization with tubercle bacilli or cross reacting mycobacteria.
        • In AIDS patients: 5 mm induration – positive test.
      •  A positive reaction usually develops 4 to 6 weeks after initial contact with the organism. (MCQ)
      • It remains positive for life, although it may wane after some years or in the presence of immunosuppression by medications or dis- ease.
      • Interpretation of tuberculin test
        • Positive Test(MCQ)
          • Person infected by M. tuberculosis at sometime in life
          • Person infected with strongly cross reacting other mycobacteria
          • Previous vaccination with BCG
          • Child < 5 years if not vaccinated: active disease.
        • Negative Test(MCQ)
          • No induration or < 5 mm
          • In healthy individual – not infected with MTB
          • Pre-hypersensitivity stage of primary infection
        • False negative test (MCQ)
          • Early TB (test becomes positive after 4-6 weeks of infection)
          • Immunosuppression (AIDS).
          • Steroid therapy

 Laboratory identification:

    • Diagnosis of active pulmonary tuberculosis includes
      • demonstration of clinical symptoms
      • abnormal chest radiographs
      • confirmation by isolation of M. tuberculosis from relevant clinical material.
    • Identification in clinical specimens:
      • A microscopic search for acid-fast bacilli using techniques such as the Ziehl-Neelsen stain is the most rapid test for mycobacteria(MCQ)
      • M. tuberculosis cannot be reliably distinguished on morphologic grounds from other pathogens in the genus
      • A definitive identifcation of M. tuberculosis can only be obtained by
        • culturing the organism
        • by using one of the newer molecular methods
    • Culture
      • 2 to 8 weeks is  required to culture the tubercle bacillus because of its slow growth (MCQ)
      • it is essential for determining its antibiotic sensitivity
      • required to confirming the specific identity of the bacillus by growth and biochemical characteristics.
      • The organisms grow on selective media,
        • coagulated serum, glycerin agar
        • glycerin potato, glycerin broth
        • egg media (Petroffs, Petragnani’s, Dorset’s, Loewenstein’s, Lubenau’s, Vinogradov’s, etc.)
      • They may be cultured on Soton’s synthetic medium which contains asparagine, glycerin, iron citrate, potassium phosphate, and other substances. (MCQ)
      • The best and quickest (on the sixth-eighth day) growth is obtained on Petroffs egg medium which consists of egg yolk, meat extract, agar, glycerin, and gentian violet. (MCQ)
      • Certain levels of vitamins (biotin, nicotinic acid, riboflavin. etc.) are necessary for the growth of M. tuberculosis. (MCQ)
      • Pryce’s microculture method is the most effective. (MCQ)
        • Virulent mycobacteria produce convoluted strands in the microcultures,
        • non-virulent strains form amorphous clusters.
      • The virulent and non-virulent M. tubercu-losis strains are differentiated by their growth on butyrate albumin agar (Middlebrook-Dubos test). (MCQ)
        • virulent strains grow in the form of pleats
        • non-virulent strains form irregular clusters.
      • Stain the smears with neutral red
        •  has an affinity for virulent mycobacteria and stains them purple-pink
        • Non-virulent strains are stained yellow)
      • Colony morphology: Bread-crumbs appearance.
        •  Lowenstein Jensen Medium – growth in 4-6 weeks(MCQ)
        •  Middlebrook Agar – growth 2-3 weeks(MCQ)
    • Biochemical Identification
      • Niacin test: (MCQ)
        •  Only M. tuberculosis and M. simiae are positive for this test
        • To differentiate M. tuberculosis from M. simiae. M.simiae produces pigment while M. tuberculosis does not.
      • Nitrate reduction test  +ve
      • Urease test –   + ve
      • Tellurite reduction test   -ve
      • Sodium chloride tolerance test     -ve
      • Pigment production test   -ve
      • Catalase test  – +ve
      • Arylsulfate test   -ve
      • Growth on MacConkey agar –ve

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    • Nucleic acid amplification
      • Amplified M. tuberculosis direct test
        • uses enzymes that rapidly make copies of M. tuberculosis 16S ribosomal RNA, which can be detected using genetic probes. (MCQ)
        • sensitivity of the test ranges from 75 to 100 percent
        • specificity of 95 to 100 percent
        • it is used for patients whose clinical smears are positive for acid-fast bacilli and whose cultures are in progress.
      • Molecular techniques have the potential to shorten the time required to detect and identify M. tuberculosis in clinical specimens
      • Polymerase chain reaction (PCR),
        • amplifies a small portion of a predetermined target region of the M. tuberculosis DNA.
        • Using human sputum, commercial PCR kits can confirm the diagnosis of tuberculosis within 8 hours, with a sensitivity and specificity that rivals culture tech- niques.
        • PCR analysis facilitates DNA fingerprinting of specific strains, allowing studies of the progress of epidemics.
    • Vaccines:
      • produced from Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis. (MCQ)
      • injected intradermally(MCQ)
      • confer tuberculin hypersensitivity and an enhanced ability to activate macrophages that kill the pathogen.
      • 80 percent protective against serious forms of tuberculosis, such as meningitis in children(MCQ)
      • used in mass immunization campaigns
      • results in conversion from PPD negative to PPD positive


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