Lecture 18 and 19 TUBERCULOSIS (Mycobacterium tuberculosis- also called the tubercle bacillus)
Reading Assignments: (1) Text Chapter 23; Ch. 59, pp. 553-563. (2) Gilligan, P.H., M.L. Smiley, and D.S. Shapiro. 1997. Cases in Medical Microbiology and Infectious Diseases (2nd ed.), (Case #8), pp. 41-44 and (Case #55), pp. 251-253. American Society for Microbiology, Washington, D.C.
1. INTRODUCTION History and Perspective
A. Discovered as the etiological agent by Robert Koch in 1882.
B. In the early 1800's, epidemic in the U.S., Europe and England (annual death rate of 1% of the population.)
C. In the developed countries, as living conditions improved, the incidence of tuberculosis began to decline. (Cure discovered 1950's; development of inexpensive screening tests (skin tests, chest X-rays); public health programs aggresively pursued; required to accept treatment) Case numbers dropped to all time.
D. In the developing countries, tuberculosis remained a major cause of death.
E. Early 1970's, anti-tuberculosis programs dismantled and funds redirected. For the next decade, tuberculosis cases continued to decline; then a sudden increase in cases in the mid-1980's. The number of new cases has continued to increase every year since. Factors that account for this reemergence include:
1. Deterioration of the health care infrastructure
2. Association of tuberculosis with the AIDS epidemic
3. Immigrants coming to the U.S. from areas where tuberculosis is endemic.
4. Transmission in congregate settings (nursing homes, correctional facilities, and homeless shelters).
5. The development of multiply resistant strains.
6. Suspension of research by pharmaceutical companies on new anti- TB drugs because they had been assured that tuberculosis was a disease of the past.
2. PROPERTIES OF THE GENUS Mycobacterium (including M. tuberculosis)
A. The genus includes a variety of closely related organisms ranging from nonpathogenic soil saprophytes to species always pathogenic in humans and animals (M. tuberculosis, M. bovis and M. leprae). Genus Name: Mycobacterium (fungus-like bacterium) - moldlike pellicles form when members of the genus are grown in liquid medium.
B. Mycobacterium spp. Have a Unique Cell Wall
1. Mycobacterium spp.(including M. tuberculosis)- obligately aerobic rod-shaped bactera with a Gram-positive cell wall. However, unlike the cell walls of other Gram-positive bacteria, the cell wall of Mycobacterium spp. consists of peptidoglycan plus a number of unusual glycolipids including arabinogalactan, lipoarabinomannan, and mycolic acid. (Mycolic acids are unusual in bacteria and are found only in the cell walls of Mycobacterium spp. and Corynebacterium spp.) Lipids make up more than 10% of the total weight of the cell of mycobacteria.
2. The complex, lipid-rich cell wall:
a. protects the bacteria in vivo from the effects of phagolysosomal contents and allows mycobacteria to survive in normal, unactivated macrophages.
b. makes them resistant to chemical agents (such as disinfectants) and to drying (which contributes to their transmission). (The lipids do not protect the cells from heat; mycobacteria are killed by pasteurization. They are also inactivated by UV light.)
c. is stimulatory to the mammalian immune system. (Immunologists enhance the antibody response to protein antigens with Freund's complete adjuvant which contains mycobacterial cell walls.)
C. Mycobacteria are "Acid Fast"
1. They cannot be stained by the Gram stain because of their high lipid content.
2. Acid fast staining is used to stain mycobacteria. Bacteria are treated with a red dye (fuchsin) and steamed. (This drives the stain into the cell and because of the lipid, they become difficult to destain.) Once stained they resist decolorization with 3% hydrochloric acid and 95% alcohol ("acid fast").
D. Mycobacteria Grow Very Slowly Mycobacteria grow slowly (doubling time = 24 h) because of the high concentration of lipids in their cell walls. Their hydrophobic surfaces cause them to clump together and inhibits the transport of nutrients into the cell. On agar- the colonies appear after 3-6 weeks as irregular, waxy, and raised..
4. THE DISEASE TUBERCULOSIS (Pathogenesis, Clinical Findings, and Host Response)
A. Representative Case Studies # 8 and in the Text p. 231-232)
2. Predisposing factors
B. Encounter and Entry into the Host (Transmission) Inhalation of droplet nuclei in aerosols (see Prescott p. 707) or by breathing dust particles. The likelihood of infection is related to the (1) number or organisms being coughed up, (2) the frequency of coughing, (3) the closeness of contact and (4) the adequacy of ventilation. Tuberculosis also can be spread via the gastrointestinal tract or from skin lesions.
C. Spread, Multiplication, and Damage
SUMMARY OF PATHOGENESIS
Tubercle bacilli produce no toxins. They are invasive intracellular pathogens whose virulence is linked to their ability to live and grow inside of normal macrophages. Only activated macrophages can kill M. tuberculosis. The specific cell-mediated immune response may either (1) lead to protective immunity and resolution of the disease or (2) to tissue-destroying hypersensitivity reactions and progression of the disease. Clinical symptoms of disease are linked to the uncontrolled, progressive, and chronic inflammatory response (Type IV Delayed Type Hypersensitivity) which the host against the organisms. (Note: If cell-mediated immunity (delayed-type hypersensitivity) is such a double edged sword, does it really benefit the host?
1. To date, not a single virulence gene has been defined, and the molecular basis for invasion of, and multiplication within macrophages remains obscure.
2. Virulence is associated with:
a. the ability of mycobacteria to survive in vivo under the variety of physical and chemical conditions (such as low pH, high lactic acid, high CO2, etc.) present in the developing lesions (specific details unknown).
b. the ability of M. tuberculosis to survive inside of unactivated macrophages (Review strategies for intracellular survival inside of phagocytes - Lecture 7). Mycobacteria:
1. inhibit lysosomal fusion with the phagocytic vacuole (associated with a group of lipids, the sulfolipids),
2. resist killing and digestion inside of phagolysosome (due to the waxy cell surface????)(Mycobacteria also suppress production of superoxide anion)
c. Toxicity of the mycolic acids from the cell wall; toxic when injected into animals and known to stimulate the inflammatory response.
THE DISEASE TUBERCULOSIS: STEP BY STEP
1. Primary Tuberculosis
a. Individual not previously infected inhales the tubercle bacilli in droplet nuclei which travel down the bronchi and reach the lungs.
b. Mycobacteria are engulfed by the alveolar macrophages, but not killed. They reproduce inside the macrophages. Neutrophils and other macrophages are called into the area as part of an inflammatory response, and the mycobacteria begin to multiply within these macrophages as well.
c. The mycobacteria are carried (inside the macrophages) to the bronchopulmonary (hilar) lymph nodes that drain the infected site. (Host response: An inflammatory response dominated by TH cells is initiated at the original site of infection, in the lymph vessels, and in the hilar lymph nodes but this takes time to develop.)
d. From the hilar lymph nodes, the mycobacteria are carried in the bloodstream to multiple sites in the body including the liver, spleen, kidney, bone, brain, meninges, and other parts of the lung. What's happening to the patient???? Infection or Disease????? During this time, signs and symptoms in the patient at this stage may be absent or manifest as a mild influenza-like disease, however the primary site of infection may be seen on an X-ray as an opaque area. The tuberculin skin test becomes positive.
HOW DOES THE IMMUNE SYSTEM FIGHT BACK???
As cell-mediated immunity develops (2-6 weeks after infection), it begins to curb the proliferation of the organisms and limit their spread. (Review Lecture 9 for the mechanism.) "Activated" TH lymphocytes (TH1 subset; also called TDTH cells) mediate the response and the cytokines they produce, including gamma interferon, "activate" the macrophages to produce additional cytokines (such as IL-1 and TNF). These cytokines mediate further inflammation and help the "activated macrophages" kill the mycobacteria within them. Nitrous oxide and other reactive nitrogen intermediates contribute to the destruction of the mycobacteria inside the "activated macrophages".
Note: Two symptoms in the patient with advanced disease (either disseminated primary infections or secondary infections) are the direct result of macrophage activation. Fever is associated with the release of IL-1 from the macrophages, and severe weight loss is the result of TNF released by the macrophages.
WHAT'S HAPPENING IN THE TISSUES???? -
THE FORMATION OF GRANULOMAS CALLED TUBERCLES
In the tissues where the inflammatory response is occurring, macrophages in the vicinity of the bacteria fuse to form giant cells (called epithelioid cells) and a layer of macrophages and T cells forms around them walling off the organisms. The walled off lesion is called a tubercle. (The cell- mediated (T cell plus phagocyte) response that produces tubercles is called a granulomatous response because the tubercles appear macroscopically as granulomas.) In time, the center of the lesions become necrotic forming a cheese-like consistency called caseous necrosis.
OUTCOMES OF PRIMARY TUBERCULOSIS
i. Complete Resolution- the cell mediated immune response clears the infection. This is rare.
(a) Dormant (Latent) Tuberculosis- the mycobacteria multiply initially, but are held in check by cell-mediated immunity. Tubercles become calcified and mycobacteria cease to grow. This is the usual outcome.
1. May be stable for life
2. May be reactivated (see secondary tuberculosis below)
(b) Disseminated Miliary Tuberculosis- the mycobacteria continue to multiply and are not held in check by cell-mediated immunity. Tubercles liquify and mycobacteria disseminate throughout the body. Numerous small tubercles can be found throughout the body (millet seed-like lesions), and fatal infections may result. This outcome is rare in an imunocompetent person, but usual in imunocompromised individuals.
2. Seconday Tuberculosis: SECONDARY TUBERCULOSIS IS THE RESULT OF:
a. Reactivation of previous disease- caused by temporary immunosuppresion (corticosteroids cancer chemotherapy, AIDS) or other predisposing factors (malnutrition, alcoholism, diabetes, old age, loss of spouse- seen in men over 50).
b. Reinfection with externally acquired tubercle bacilli - leads to the same manifestations as reactivation disease. Reactivation usually occurs in body areas of high oxygen tension and low lymphatic drainage, most often in the apex of the lung. The lesions show spreading, coalescing tubercles (containing numerous mycobacteria), and large areas of caseous necreosis. Caseous lesions liquify, discharge their contents into bronchi, resulting in a pulmonary cavity and bronchial spread. Small blood vessels are frequently eroded and the sputum in these patients is often blood stained. The disease is characterized by chronic fever and weight loss, night sweats, productive coughing, and hemoptysis. In advanced disease, blood vessels may rupture leading to death due to hemorrhage. Less commonly, reactivation tuberculosis occurs in other organs such as the kidneys, bones, lymph nodes, brain meninges, and bone marrow. These are important complications in AIDS patients.
5. DIAGNOSIS OF TUBERCULOSIS
A. Medical History
B. Physical Examination
C. Tuberculin Skin Testing (PPD test or Mantoux test) - a skin test used to screen individuals for M. tuberculosis infection or disease. PPD is a M. tuberculosis antigen (purified protein derivative) that is injected in the forearm with a tuberculin syringe. The forearm is examined at the site of injection 48 to 72 h later. A positive reaction is induration (thickening) and erythema (redness) at the site of injection. No induration is a negative reaction. A positive reaction indicates that the individual has been infected at some time with M. tuberculosis (and has made a CELLULAR IMMUNE RESPONSE to M. tuberculosis antigens - PLEASE NOTE: ANTIBODY IS NOT INVOLVED!). A positive test does not tell you about the activity of the infection, which may have occurred years earlier. A negative test may not rule out disease. For example, patients with AIDS may lose their tuberculin sensitivity and become anergic (unreactive to the antigen). The tuberculin skin test is a valuable tool, but it is not perfect. It is most useful in populations where a high proportion of the population is not tuberculin positive. Infection with mycobacteria other than M. tuberculosis and vaccination with bacille Calmette-Guerin (BCG) may lead to false-positive reactions.
D. Radiographic Examination (Opaque lesions on X-ray)
E. Diagnostic Microbiology
1. Specimen Collection
Persons suspected of pulmonary tuberculosis should have at least 3 sputum specimens examined by smear and culture. Bronchoscopy can be used for patients who cannot cough up a good sputum specimen. Health workers must take precautions when collecting sputum specimens because of the infectious aerosols produced. A variety of specimens may be submitted from other body sites.
2. Laboratory Protocol
a. Direct examination of Sputum smears
(1) Detection of acid-fast bacilli in sputum smears (Ziehl-Neelson stain) - presumptive dx (may be mycobacteria other than M. tuberculosis)
(2) Fluorescent staining of sputum smears with Auramine O is faster and more sensitive.
(3) Direct detection and identification of M. tuberculsosis in sputum specimens by PCR is still experimental.
b. Culture of specimen on laboratory media (takes weeks but must be done to confirm M. tuberculosis).
(1) Solid media (Lowenstein Jensen) and liquid media are inoculated.
(2) After colonies are seen, identification is by conventional biochemical tests, or by nucleic acid probes specific for M. tuberculosis, M avium, and M. intracellulare . High performance liquid chromatography (HPLC) is also used to speciate isolates based on the spectrum of mycolic acids in the cell wall.
c. Restriction fragment length polymorphism (RFLP), a method of DNA fingerprinting, can be used to identify specific strains of M. tuberculosis and thus track transmission of tuberculosis during outbreaks.
d. Antibiotic sensitivity testing is very important because of multiple resistance seen with M. tuberculosis.
A. Treatment of Tuberculosis Infection
Preventive therapy for treatment of tuberculosis infection substantially reduces the risk that infection will progress to disease. People with positive skin tests or who are in high risk groups are given daily isoniazid for 6 to 12 months.
B. Treatment of Tuberculosis Disease
The initial regimen for treating tuberculosis disease includes multiple drugs: isoniazid, rifampin, pyrazinamide, and either ethambutol or streptomycin. Two to four drugs are used because resistance to any one drug occurs with high frequency. When two or more drugs are used simultaneously, each helps prevent the emergence of tubercle bacilli resistant to the others. Multiple drugs also work synergistically (see Text p. 239). This means that drugs administered in combination are more effective than a single drug alone. Contacts of tuberculosis cases may also be treated prophylactically.
Tuberculosis must be treated for a long time (6-24 months) compared with many other infectious diseases. Noncompliance is a major problem with tuberculosis treatment (and control). Multiple pills must be taken and side effects (like nausea) may occur. On recommended regimen within 12 months. Inadequate treatment can lead to relapse, continued transmission, and the development of drug resistance. One way to ensure that patients adhere to therapy is to use directly observed therapy (DOT). DOT means that a health care worker or other designated person watches the patient swallow each dose of medication.
7. CONTROL OF TUBERCULOSIS
A. Community Tuberculosis Control
All new tuberculosis cases and suspected cases should be reported by physicians to the health department. Reporting tuberculosis is required by law in every state. Early reporting is essential for evaluating persons who have been in contact with tuberculosis patients. Although tuberculosis care and treatment are often provided by other medical care providers, the health department has the ultimate responsibility for ensuring the tuberculosis patients do not transmit the disease to others. The health department carries out surveillance, conducts contact and outbreak investigations, monitors patients for adverse reactions and adherence to therapy (DOT). If necessary, quarantine measures may be imposed.
B. Worldwide Control of Tuberculosis
(1) The history of tuberculosis indicates that it can be controlled by sanitary measures and improved standards of living.
(2) Vaccine: There is no available vaccine made from M. tuberculosis, but BCG (Bacille Calmette-Guerin), a live attenuated strain of M. bovis is used as a vaccine in Europe and other parts of the world (not in the United States). There is little evidence that it confers protection against M. tuberculosis. A drawback of BCG vaccination is that the vaccine recipient converts to tuberculin positive on the skin test. This negates the value of the PPD skin test as an indicator of infection.
8. AIDS AND MYCOBACTERIAL INFECTIONS
(Read Text p. 239 and Case # 55- Be able to summarize M. tuberculosis disease in AIDS patients and discuss disease caused by the atypical mycobacteria Mycobacterium avium complex (MAC) and Mycobacterium kansasii.)
Material in this handout was taken from:
1. Salyers, A. and D. Whitt. 1994. Bacterial Pathogenesis - a Molecular Approach. ASM Press, Washington, D.C.
2. Ryan, K.J. 1994. Sherris Medical Microbiology, 3rd ed. Appleton and Lange, Norwalk, CT.
3. Core Curriculum on Tuberculosis. 1994. Centers for Disease Control and Prevention, Atlanta, GA.