Mycobacterium Tuberculosis

Question 1

Mycobacterium General Charcateristics

Answer 1

• Gram + wall structure
• mycolic acids
• Lipoarabinomannan (LAM), a lipid polysaccharide complex extends from the plasma membrane to the surface.
• acid fast

Question 2

Mycobacterium - LAM

Answer 2

•LAM is structurally and functionally analogous to the lipopolysaccharide of gramnegative bacteria. These elements give the mycobacteria a cell wall with unusually high lipid content, which accounts for many of their biologic characteristics. It can be thought of as a waxy coat that makes them hardy, impenetrable, and hydrophobic

Question 3

Mycobacterium Growth

Answer 3

• The most important pathogen, M tuberculosis (MTB), is an obligate aerobe.
• Other nutritional requirements vary among species and range from the ability of some nonpathogens to multiply on the washers of water faucets to the strict intracellular parasitism of M leprae.
• Mycobacteria grow more slowly than most human pathogenic bacteria in part due to their hydrophobic cell surface, which causes them to clump and limits permeability of nutrients into the cell.

Question 4

Mycobacterium Disease

Answer 4

• Diseases caused by mycobacteria usually develop slowly, follow a chronic course, and elicit a granulomatous response.
• Infectivity of pathogenic species is high, but virulence (disease production) for healthy humans is moderate. Disease following infection with MTB is the exception rather than the rule.

Question 5

Mycobacteria do not / do produce classic exotoxins or endotoxins.

Answer 5

Mycobacteria do not / do produce classic exotoxins or endotoxins.

Question 6

Two Host Responses that Cause Disease Process

Answer 6

1. a delayed-type hypersensitivity (DTH) reaction to mycobacterial proteins, results in the destruction of nonactivated macrophages containing multiplying organisms. It is detected by intradermal injections of purified proteins from the mycobacteria.
2. cellmediated immunity (CMI) activates macrophages enabling them to destroy mycobacteria contained within their cytoplasm.

The balance between these two responses determines the pathology and clinical response to a mycobacterial infection.

Question 7

M. tuberculosis Organism

Answer 7

• MTB is a slim, strongly acid–alcohol–fast rod. It frequently shows irregular beading in its staining, appearing as connected series of acid-fast granules.
• It grows at 37oC, but not at room temperature, and it requires enriched or complex media for primary growth.
• The classic medium (Löwenstein–Jensen) contains homogenized egg in nutrient base with dyes to inhibit the growth of nonmycobacterial contaminants.
• Growth is slow

Question 8

M. tuberculosis Cell Wall

Answer 8

• Because of its hydrophobic lipid surface, MTB is unusually resistant to drying, to most common disinfectants, and to acids and alkalis. Tubercle bacilli are sensitive to heat, including pasteurization, and individual organisms in droplet nuclei are susceptible to inactivation by ultraviolet light. As with other mycobacteria, the MTB cell wall structure is dominated by mycolic acids and LAM. Its antigenic makeup includes many protein and polysaccharide antigens, of which tuberculin is the most studied.
• Tuberculin consists of heat-stable proteins released into liquid culture media.
• A purified protein derivative (PPD) of tuberculin is used for skin testing for hypersensitivity.

Question 9

M. tuberculosis Epidemiology

Answer 9

Question 10

M. tuberculosis Pathogenesis

Answer 10

Question 11

M. tuberculosis Primary TB

Answer 11

• MTB is a facultative intracellular pathogen whose success depends on avoiding the killing mechanisms of professional phagocytes.
• Primary tuberculosis is the initial infection in which inhaled droplet nuclei containing tubercle bacilli are deposited in the peripheral respiratory alveoli, most frequently those of the well-ventilated middle and lower lobes. At the earliest stages an attachment protein (ESAT-6) may facilitate binding to laminin in the basement membrane of alveolar epithelial cells. In the alveoli the bacteria are recognized by alveolar macrophage complement receptors (CR1, CR3, CR4) and phagocytosed.
• This inaugurates a two-stage battle within the macrophage, which may be resolved in weeks or last for decades.

Question 12

M. tuberculosis Primary TB Stage 1

Answer 12

•The first stage is with the phagosome/lysosome digestive mechanisms of the macrophage. In this process, MTB has the upper hand through its ability to interfere with the acidification of the phagosome, which renders the lysosomal enzymes (which require acidic pH) less effective. This allows the bacteria to multiply freely in the phagosome of the nonactivated macrophage.

Question 13

M. tuberculosis Primary TB Stage 2

Answer 13

• The second stage is the triggering of TH1 immune responses, beginning with digestion and surface presentation of mycobacterial components and ending with cytokine activation of the macrophages.
• The short- and long-term outcomes of the infection depend on the ability of the macrophage activation process to overcome the intracellular edge that MTB has as a result of its ability to block acidification of the phagosome.

Question 14

Early Stages of M. tuberculosis Infection

Answer 14

•In the early stages of infection, MTB-laden macrophages are transported through lymphatic channels to the hilar lymph nodes draining the infected site. From there, a low-level bacteremia disseminates the bacteria to a number of tissues, including the liver, spleen, kidney, bone, brain, meninges, and apices or other parts of the lung. Although the primary site of infection and enlarged hilar lymph nodes can often be detected radiologically (Ghon complex), the distant sites usually have no findings. In fact, the primary evidence for their existence is reactivation at nonpulmonary sites later in life. Tuberculous meningitis is the most serious of these infections.

Question 15

Later Stages of M. tuberculosis Infection

Answer 15

• In the primary lesion as MTB cells multiply, macrophages and dendritic cells release cytokines (tumor necrosis factor, interleukin 12, interferon gamma [IFN-γ]), which attract T cells and other inflammatory cells to the site.
• The recruited CD4 T cells initiate the TH1-type immune response over the following 3 to 9 weeks in which IFN-γ is the primary activator of macrophages.
• This process is highly specific involving only the T cells and the above mentioned cytokine mediators. As the bacteria multiply, they release mycobacterial proteins which elicit a cellular hypersensitivity (DTH) response with its inflammatory fluids, phagocytes, and release of digestive enzymes.
• This adds a diffuse destructive component to the process, which is much less specific than the macrophage activation process. The tissue destruction caused by this chronic inflammatory process is the sole known source of injury in tuberculosis.

Question 16

M. tuberculosis and DTH

Answer 16

• The magnitude of the DTH depends on the size of the MTB population.
• If the TH1 immune process is effective, MTB, the source of antigenic stimulation is controlled and with it the chronic DTH inflammation. As this is the only source of injury, the disease resolves and the patient recovers.
• The mycobacterial protein-specific DTH sensitization remains, and its elicitation is the basis of the tuberculin skin test.

Question 17

M. tuberculosis and Granulomas

Answer 17

• The mixture of the TH1 immune and DTH responses is manifest in a microscopic structure called a granuloma, which is composed of lymphocytes, macrophages, epithelioid cells (activated macrophages), fibroblasts, and multinucleated giant cells all in an organized pattern.
• As the granuloma grows, the destructive nature of the hypersensitivity component leads to necrosis usually in the center of the lesion. This is termed caseous necrosis because of the cheesy, semisolid character of material at the center of large gross lesions, but the term fits the smooth glassy appearance of microscopic granulomas as well.

Question 18

M. tuberculosis and Latency

Answer 18

• In tuberculosis, some of the organisms when faced with oxygen and nutrient deprivation instead of dying enter a prolonged dormant state called latency.
• Specific factors facilitating survival are not known but the waxy nature of the MTB cell wall must be of aid as it is in the environment.
• These organisms lie in the lung and elsewhere waiting for reactivation months, years, or decades later. For the vast majority (>90%) of persons who undergo a primary infection this never happens

Question 19

M. tuberculosis and Reactivation

Answer 19

• The new foci are usually located in body areas of relatively high oxygen tension that would favor growth of the aerobe MTB.
• The apex of the lung is the most common, with spreading, coalescing granulomas, and large areas of caseous necrosis.
• Necrosis often involves the wall of a small bronchus from which the necrotic material is discharged, resulting in a pulmonary cavity and bronchial spread.
• Frequently, small blood vessels are also eroded.
• The destructive nature of these lesions cannot be directly attributed to any products or structural components of MTB. The damage is due to the failure of the host to control growth of MTB and thus the rising load of mycobacterial proteins which stimulate the autodestructive DTH inflammatory response.

Question 20

M. tuberculosis and Tuberculosis

Answer 20

• Humans generally have a high innate immunity to development of disease. Only 10% of immunocompetent persons infected with MTB develop any form of active disease in their lifetime.
• Adaptive immunity to tuberculosis is primarily related to the development of reactions mediated through CD4 T lymphocytes via TH1 pathways. Intracellular killing of MTB by macrophages activated by IFN-γ and other cytokines is the essential step.
• Cytotoxic CD8 T cells are also generated during infection and may play some role.
• Although antibodies are formed in the course of disease, there is no evidence they play any role in immunity.

Question 21

M. tuberculosis Symptoms

Answer 21

•Primary

• asymptomatic or fever and malaise
• Ghon complex, inflitrates in mid zone of lung

•Reactivation

• cough (first dry then with sputum, maybe some blood)
• fever, malaise, fatigue, weight loss, sweating
• inflitrates in apices of lungs —> cavities
• can first appear in other organs, such as the kidneys, bones, lymph nodes, brain, meninges, bone marrow, and bowel.
• Disease at nonpulmonary sites ranges from a localized tumor-like granulomas (tuberculomas) to a fatal chronic meningitis.

Question 22

M. tuberculosis Diagnosis

Answer 22

• Intradermal MTB proteins (PPD)

– Induration >15 mm - 48-72 hrs.

• Demonstrates DTH

– Resolved infection (including treated)

– Latent infection

– Active disease

– BCG immunization (<15mm)

– Infection with other mycobacterial species (<15mm)

Question 23

M. tuberculosis Direct Smears

Answer 23

Question 24

M. tuberculosis Culture

Answer 24

Question 25

M. tuberculosis Diagnosis Quantiferon Gold

Answer 25

Question 26

M. tuberculosis Prevention

Answer 26

Public health measures – USA * Routine PPDs or chest radiographs * Chemoprophylaxis of skin test converters – To prevent reactivation – Single drug (isoniazid) for 6-9 months BCG vaccine – worldwide * Live attenuated strain of M. bovis * Performance variable, particularly in adults * Converts PPD * New recombinant versions – virulence genes added

Question 27

M. leprae

Answer 27

* Mycobacterium leprae, the cause of leprosy (Hansen disease), is an acid-fast bacillus that has not been grown in artificial medium or tissue culture. It will grow slowly (doubling time 14 days) in some animals (mice, armadillos). * Although lack of in vitro growth severely limits study of the organism, the structure and cell wall components appear to be similar to those of other mycobacteria.

Question 28

M. leprae Epidemiology

Answer 28

* The exact mode of transmission is unknown but appears to be by generation of small droplets from the nasal secretions from cases of lepromatous leprosy. * Traumatic inoculation through minor skin lesions or tattoos is also possible. * central reservoir is humans, but infection can come from environmental sources (biting insects) * The infectivity of M leprae is low. Most new cases have had prolonged close contact with an infected person.

Question 29

M. leprae Pathogenesis

Answer 29

* M leprae is an obligate intracellular bacterium that must multiply in host cells to persist. In humans, the target is Schwann cells, the glial cells of the peripheral nervous system. * One mycoside (PGL-1) and a laminin-binding protein facilitate both invasion of Schwann cells and binding to basal lamina of the peripheral nerve axon units. This leads to cell injury and demyelination of peripheral nerves which precedes but is enhanced by the DTH immune response to M. leprae. * This invasion and demyelination of peripheral sensory nerves causes local anesthesia and other changes in the skin depending on the location and degree of immune response. * Individual variability in the extent of immune response is responsible for two major forms of leprosy with a spectrum of illness in between.

Question 30

M. leprae - Tuberculoid

Answer 30

•In the tuberculoid form, few M leprae are seen in lesions with well-formed granulomas, abundant CD4 T cells extensive epithelioid cells, giant cells, and lymphocytic infiltration.

Question 31

M. leprae - Lepromatous

Answer 31

•In lepromatous leprosy there is a lack of CD4+ T cells, numerous CD8+ T cells, foamy macrophages, and dense infiltration with leprosy bacilli.

Question 32

M. leprae - Immunity

Answer 32

* Immunity to M leprae is T-cell–mediated. Tuberculoid cases have minimal disease and evidence of TH1 immune responses including production of typical cytokines (IL-2, IFNγ). * Lepromatous cases have progressive disease and lack TH1 mediators.

Question 33

M. leprae - Tuberculoid Leprosy

Answer 33

* Tuberculoid leprosy involves the development of macules or large, flattened plaques on the face, trunk, and limbs, with raised, erythematous edges and dry, pale, hairless centers. * When the bacterium has invaded peripheral nerves, the lesions are anesthetic. * The disease is indolent, with simultaneous evidence of slow progression and healing. * Because of the small number of organisms present, this form of the disease is usually noncontagious.

Question 34

M. leprae - Lepromatous Leprosy

Answer 34

* In lepromatous leprosy, skin lesions are infiltrative, extensive, symmetric, and diffuse, particularly on the face, with thickening of the looser skin of the lips, forehead, and ears. * Damage may be severe, with loss of nasal bones and septum, sometimes of digits, and testicular atrophy. Peripheral neuropathies may produce deformities or nonhealing painless ulcers. * The organism can spread systemically, with involvement of the reticuloendothelial system.

Question 35

M. leprae Diagnosis

Answer 35

* Leprosy is primarily a clinical diagnosis confirmed by demonstration of AFB in stained scrapings or biopsies of infected tissue, particularly nasal mucosa or ear lobes. * Because M. leprae is not as resistant to decolorization as MTB a variant of the standard acid-fast procedure (Fite stain) must be employed to avoid false-negative results.

Question 36

M. leprae Treatment

Answer 36

* The drugs used for treatment of leprosy are dapsone, rifampin, and clofazimine. * Their deployment is based on disease severity measurements which correspond to the lepromatous and tuberculoid forms. * Those with negative AFB smears and 5 lesions and positive smears (multibacillary) are treated with all 3 drugs

Question 37

Mycobacterium avium–intracellulare Complex

Answer 37

* Mycobacterium avium–intracellulare complex (MAC) includes two closely related mycobacteria, M. avium and M. intracellulare, that grow only slightly faster than M tuberculosis. Among them are organisms that cause tuberculosis in birds (and sometimes swine), but rarely lead to disease in humans. * The most common infection in humans is cavitary pulmonary disease, often superimposed on chronic bronchitis and emphysema. * Cervical lymphadenitis, chronic osteomyelitis, and renal and skin infections also occur. * The organisms in this group are substantially more resistant to anti-tuberculous drugs than most other species, and treatment with the three or four agents found to be most active often requires supplementation with surgery.

Question 38

Mycobacterium avium–intracellulare Complex and AIDS

Answer 38

•Disseminated MAC infections, once rare, are now a common systemic bacterial infection in patients with AIDS. They usually develop when the patient’s general clinical condition and CD4 T cell concentrations are declining. Histologically, granuloma formation is muted, and there are aggregates of foamy macrophages containing numerous intracellular AFB. The diagnosis is most readily made by blood culture, using a variety of specialized cultural techniques. Response to chemotherapeutic agents is marginal, and the prognosis is grave. MAC infections have declined with advances in chemotherapy of AIDS.