Mycobacteria:
IntroductionThe mycobacteria are rod-shaped, aerobic bacteria that do not form spores. Although they do not stain readily, once stained they resist decolorization by acid or alcohol and are therefore called "acid-fast" bacilli. Mycobacterium tuberculosis causes tuberculosis and is a very important pathogen of humans. Mycobacterium leprae causes leprosy. Mycobacterium avium-intracellulare (M avium complex, or MAC) and other atypical mycobacteria frequently infect patients with AIDS, are opportunistic pathogens in other immune-compromised personsMycobacterium tuberculosis
Morphology & Identification
Typical Organisms
In tissue, tubercle bacilli are thin straight rods measuring about 0.4 x 3m. On artificial media, coccoid and filamentous forms are seen with variable morphology from one species to another. Mycobacteria cannot be classified as either gram-positive or gram-negative. Once stained by basic dyes they cannot be decolorized by alcohol, regardless of treatment with iodine. True tubercle bacilli are characterized by "acid-fastness"ie, 95% ethyl alcohol containing 3% hydrochloric acid (acid-alcohol) quickly decolorizes all bacteria except the mycobacteria. Acid-fastness depends on the integrity of the waxy envelope. The Ziehl-Neelsen technique of staining is employed for identification of acid-fast bacteria. In smears of sputum or sections of tissue, mycobacteria can be demonstrated by yellow-orange fluorescence after staining with fluoro-chrome stains (eg, auramine, rhodamine).
Culture
The media for primary culture of mycobacteria should include a nonselective medium and a selective medium. Selective media contain antibiotics to prevent the overgrowth of contaminating bacteria and fungi. There are three general formulations that can be used for both the nonselective and selective media.
Semisynthetic Agar Media
These media (eg, Middlebrook 7H10 and 7H11) contain defined salts, HYPERLINK "javascript:showDrugClass('vitamins');" vitamins, cofactors, oleic acid, HYPERLINK "javascript:showDrugInfo(10);" albumin, catalase, glycerol, HYPERLINK "javascript:showDrugInfo(2458);" glucose, and malachite green; the 7H11 medium contains casein hydrolysate also, these media may be less sensitive than other media for primary isolation of mycobacteria.
Inspissated Egg Media
These media (eg, Lwenstein-Jensen) contain defined salts, glycerol, and complex organic substances (eg, fresh eggs or egg yolks, potato flour, and other ingredients in various combinations). Malachite green is included to inhibit other bacteria. Small inocula in specimens from patients will grow on these media in 36 weeks.These media with added antibiotics are used as selective media.
Broth Media
Broth media (eg, Middlebrook 7H9 and 7H12) support the proliferation of small inocula. Growth is often more rapid than on complex media.
Growth Characteristics
Mycobacteria are obligate aerobes and derive energy from the oxidation of many simple carbon compounds. Increased CO2 tension enhances growth. Biochemical activities are not characteristic, and the growth rate is much slower than that of most bacteria
Reaction to Physical and Chemical Agents
Mycobacteria tend to be more resistant to chemical agents than other bacteria because of the hydrophobic nature of the cell surface and their clumped growth. Dyes (eg, malachite green) can be incorporated into media without inhibiting the growth of tubercle bacilli. Acids and alkalies permit the survival of some exposed tubercle bacilli . Tubercle bacilli are resistant to drying and survive for long periods in dried sputum.
Pathogenicity of Mycobacteria
There are marked differences in the ability of different mycobacteria to cause lesions in various host species. Humans and guinea pigs are highly susceptible to M tuberculosis infection, whereas fowl and cattle are resistant. M tuberculosis and Mycobacterium bovis are equally pathogenic for humans.
Constituents of Tubercle Bacilli
Mycobacterial cell walls can induce delayed hypersensitivity and some resistance to infection and can replace whole mycobacterial cells in Freund's adjuvant. Mycobacterial cell contents only elicit delayed hypersensitivity reactions in previously sensitized animals.
Lipids
Mycobacteria are rich in lipids. These include mycolic acids (long-chain fatty acids C78C90), waxes, and phosphatides. In the cell, the lipids are largely bound to proteins and polysaccharides. Lipids are to some extent responsible for acid-fastness.
Proteins
Each type of mycobacterium contains several proteins that elicit the tuberculin reaction. Proteins bound to a wax fraction can, upon injection, induce tuberculin sensitivity such as purified protein derivative (PPD). They can also elicit the formation of a variety of antibodies.
Polysaccharides
Mycobacteria contain a variety of polysaccharides. Their role in the pathogenesis of disease is uncertain. They can induce the immediate type of hypersensitivity and can serve as antigens in reactions with sera of infected persons.
Pathogenesis
Mycobacteria are inhaled in droplets and reach alveoli. The disease results from establishment and proliferation of virulent organisms and interactions with the host. Injected avirulent bacilli (eg, BCG) survive only for months or years in the normal host. Resistance and hypersensitivity of the host greatly influence the development of the disease.
Pathology
The production and development of lesions and their healing or progression are determined chiefly by (1) the number of mycobacteria in the inoculum and their subsequent multiplication, and (2) the resistance and hypersensitivity of the host.
Two Principal Lesions
Exudative Type
This consists of an acute inflammatory reaction, with edema fluid, poly-morphonuclear leukocytes, and, later, monocytes around the tubercle bacilli. This type is seen particularly in lung tissue, where it resembles bacterial pneumonia
Productive Type
When fully developed, this lesion, a chronic granuloma, consists of three zones: (1) a central area of large, multinucleated giant cells containing tubercle bacilli; (2) a mid zone of pale epithelioid cells, often arranged radially; and (3) a peripheral zone of fibroblasts, lymphocytes, and monocytes. Later, peripheral fibrous tissue develops, and the central area undergoes caseation necrosis. Such a lesion is called a tubercle.
Spread of Organisms in the Host
Tubercle bacilli spread in the host by direct extension, through the lymphatic channels and bloodstream, and via the bronchi and gastrointestinal tract.
Once mycobacteria establish themselves in tissue, they reside principally intra-cellularly in monocytes, reticulo-endothelial cells, and giant cells. The intracellular location is one of the features that makes chemotherapy difficult and favors microbial persistence.
Primary Infection & Reactivation Types of Tuberculosis
When a host has first contact with tubercle bacilli, the following features are usually observed: (1) An acute exudative lesion develops and rapidly spreads to the lymphatics and regional lymph nodes. The exudative lesion in tissue often heals rapidly. (2) The lymph node undergoes massive caseation, which usually calcifies. (3) The tuberculin test becomes positive.
This primary infection type occurred in the past, usually in childhood but now frequently in adults who have remained free from infection and therefore tuberculin-negative in early life. In primary infections, the involvement may be in any part of the lung but is most often at the base.
Clinical Findings
Since the tubercle bacillus can involve every organ system, its clinical manifestations are protean. Fatigue, weakness, weight loss, and fever may be signs of tuberculous disease. Pulmonary involvement giving rise to chronic cough and spitting of blood usually is associated with far-advanced lesions. Meningitis or urinary tract involvement can occur in the absence of other signs of tuberculosis. Bloodstream dissemination leads to miliary tuberculosis with lesions in many organs and a high mortality rate.
Diagnostic Laboratory Tests
A positive tuberculin test does not prove the presence of active disease due to tubercle bacilli. Isolation of tubercle bacilli provides such proof.
Specimens
Specimens consist of fresh sputum, gastric washings, urine, pleural fluid, cerebrospinal fluid, joint fluid, biopsy material, blood, or other suspected material.
Smears
Sputum, exudates, or other material is examined for acid-fast bacilli by Ziehl-Neelsen staining. Stains of gastric washings and urine generally are not recommended, because saprophytic mycobacteria may be present and yield a positive stain. Fluorescence microscopy with auramine-rhodamine stain is more sensitive than acid-fast stain.
Culture, Identification, and Susceptibility Testing
Processed specimens from nonsterile sites and centrifuged specimens from sterile sites can be cultured directly onto selective and nonselective media . The selective broth culture often is the most sensitive method and provides results most rapidly. A selective agar media (eg, Lwenstein-Jensen or Middlebrook 7H10/7H11 biplate with antibiotics) should be inoculated in parallel with broth media cultures. Incubation is at 3537 C in 510% CO2 for up to 8 weeks. If cultures are negative in the setting of a positive acid-fast stain or slowly growing atypical mycobacteria are suspected, then a set of inoculated media should be incubated at a lower temperature (eg, 2433 C) and both sets incubated for 12 weeks.
Molecular probes provide a rapid, sensitive, and specific method to identify mycobacteria. The probes can be used on mycobacterial growth from solid media or from broth cultures.
Susceptibility testing of mycobacteria is an important adjunct in selecting drugs for effective therapy. A standardized radiometric broth culture technique can be used to test for susceptibility to first-line drugs.
DNA Detection, Serology, and Antigen Detection
The polymerase chain reaction holds great promise for the rapid and direct detection of M tuberculosis in clinical specimens. The overall sensitivity is 5590% with a specificity of about 99%. The test has the highest sensitivity when applied to specimens that have smears positive for acid-fast bacilli; the PCR test is approved for this use on sputum specimens that are acid-fast stain-positive.
Enzyme immunoassays have been used to detect mycobacterial antigens, but the sensitivity and specificity are less than with other methods.
Treatment
Between one in 106 and one in 108 tubercle bacilli are spontaneous mutants resistant to first-line antituberculosis drugs. When the drugs are used singly, the resistant tubercle bacilli emerge rapidly and multiply. Therefore, treatment regimens use drugs in combination to yield cure rates of > 95%.
The two major drugs used to treat tuberculosis are HYPERLINK "javascript:showDrugInfo(326);" isoniazid and HYPERLINK "javascript:showDrugInfo(545);" rifampin. The other first-line drugs are HYPERLINK "javascript:showDrugInfo(529);" pyrazinamide, HYPERLINK "javascript:showDrugInfo(232);" ethambutol, and HYPERLINK "javascript:showDrugInfo(575);" streptomycin. Second-line drugs are more toxic or less effective (or both), and they should be used in therapy only under extenuating circumstances (eg, treatment failure, multiple drug resistance). Second-line drugs include HYPERLINK "javascript:showDrugInfo(1397);" kanamycin, HYPERLINK "javascript:showDrugInfo(1228);" capreomycin, HYPERLINK "javascript:showDrugInfo(1331);" ethionamide, HYPERLINK "javascript:showDrugInfo(157);" cycloserine, HYPERLINK "javascript:showDrugInfo(450);" ofloxacin, and HYPERLINK "javascript:showDrugInfo(127);" ciprofloxacin.
Epidemiology
The most frequent source of infection is the human who excretes, particularly from the respiratory tract, large numbers of tubercle bacilli. Close contact (eg, in the family) and massive exposure (eg, in medical personnel) make transmission by droplet nuclei most likely.
Susceptibility to tuberculosis is a function of the risk of acquiring the infection and the risk of clinical disease after infection has occurred. For the tuberculin-negative person, the risk of acquiring tubercle bacilli depends on exposure to sources of infectious bacilliprincipally sputum-positive patients. This risk is proportionate to the rate of active infection in the population, crowding, socioeconomic disadvantage, and inadequacy of medical care.
Prevention & Control
(1) Prompt and effective treatment of patients with active tuberculosis and careful follow-up of their contacts with tuberculin tests, x-rays, and appropriate treatment are the mainstays of public health tuberculosis control. Resurgence of tuberculosis implies that these control measures have not been done adequately.
(2) Drug treatment of asymptomatic tuberculin-positive persons in the age groups most prone to develop complications (eg, children) and in tuberculin-positive persons who must receive immunosuppressive drugs greatly reduces reactivation of infection.
(3) Individual host resistance: Nonspecific factors may reduce host resistance, thus favoring the conversion of asymptomatic infection into disease. Such factors include starvation, gastrectomy, and suppression of cellular immunity by drugs (eg, HYPERLINK "javascript:showDrugClass('corticosteroids');" corticosteroids) or infection. HIV infection is a major risk factor for tuberculosis.
(4) Immunization: Various living avirulent tubercle bacilli, particularly BCG (bacillus Calmette-Gurin, an attenuated bovine organism), have been used to induce a certain amount of resistance in those heavily exposed to infection. Vaccination with these organisms is a substitute for primary infection with virulent tubercle bacilli without the danger inherent in the latter. The available HYPERLINK "javascript:showDrugClass('vaccines');" vaccines are inadequate from many technical and biologic standpoints. Nevertheless, BCG is given to children in many countries. In the United States, the use of BCG is suggested only for tuberculin-negative persons who are heavily exposed (members of tuberculous families, medical personnel). Statistical evidence indicates that an increased resistance for a limited period follows BCG vaccination.
(5) The eradication of tuberculosis in cattle and the pasteurization of milk have greatly reduced M bovis infections.Mycobacterium leprae
Although this organism was described by Hansen in 1873 (9 years before Koch's discovery of the tubercle bacillus), it has not been cultivated on nonliving bacteriologic media. It causes leprosy. There are more than 10 million cases of leprosy, mainly in Asia.
Typical acid-fast bacillisingly, in parallel bundles, or in globular massesare regularly found in scrapings from skin or mucous membranes (particularly the nasal septum) in lepromatous leprosy. The bacilli are often found within the endothelial cells of blood vessels or in mononuclear cells. When bacilli from human leprosy (ground tissue nasal scrapings) are inoculated into footpads of mice, local granulomatous lesions develop with limited multiplication of bacilli..
Clinical Findings
The onset of leprosy is insidious. The lesions involve the cooler tissue of the body: skin, superficial nerves, nose, pharynx, larynx, eyes, and testicles. The skin lesions may occur as pale, anesthetic macular lesions 110 cm in diameter; diffuse or discrete erythematous, infiltrated nodules 15 cm in diameter; or a diffuse skin infiltration. Neurologic disturbances are manifested by nerve infiltration and thickening, with resultant anesthesia, neuritis, paresthesia, trophic ulcers, and bone resorption and shortening of digits. The disfigurement due to the skin infiltration and nerve involvement in untreated cases may be extreme.
The disease is divided into two major types, lepromatous and tuberculoid, with several intermediate stages. In the lepromatous type, the course is progressive and malign, with nodular skin lesions; slow symmetric nerve involvement; abundant acid-fast bacilli in the skin lesions; continuous bacteremia; and a negative lepromin (extract of lepromatous tissue) skin test. In lepromatous leprosy, cell-mediated immunity is markedly deficient and the skin is infiltrated with suppressor T cells. In the tuberculoid type, the course is benign and nonprogressive, with macular skin lesions, severe asymmetric nerve involvement of sudden onset with few bacilli present in the lesions, and a positive lepromin skin test. In tuberculoid leprosy, cell-mediated immunity is intact and the skin is infiltrated with helper T cells.
Systemic manifestations of anemia and lymphadenopathy may also occur. Eye involvement is common. Amyloidosis may develop.
Diagnosis
Scrapings with a scalpel blade from skin or nasal mucosa or from a biopsy of earlobe skin are smeared on a slide and stained by the Ziehl-Neelsen technique. Biopsy of skin or of a thickened nerve gives a typical histologic picture. No serologic tests are of value. Nontreponemal serologic tests for syphilis frequently yield false-positive results in leprosy.
Treatment
Sulfones such as HYPERLINK "javascript:showDrugInfo(168);" dapsone are first-line therapy for both tuberculoid and lepromatous leprosy. HYPERLINK "javascript:showDrugInfo(545);" Rifampin or HYPERLINK "javascript:showDrugInfo(134);" clofazimine generally is included in the initial treatment regimens. Other drugs active against M leprae include HYPERLINK "javascript:showDrugInfo(407);" minocycline, HYPERLINK "javascript:showDrugInfo(131);" clarithromycin, and some fluoroquinolones.
Epidemiology
Transmission of leprosy is most likely to occur when small children are exposed for prolonged periods to heavy shedders of bacilli. Nasal secretions are the most likely infectious material for family contacts. The incubation period is probably 210 years. Without prophylaxis, about 10% of exposed children may acquire the disease.
Prevention & Control
Identification and treatment of patients with leprosy are the keys to control. Children of presumably contagious parents are given chemoprophylactic drugs until treatment of the parents has made them noninfectious. If any member of a domestic group has lepromatous leprosy, such prophylaxis is required for children in the group. Experimental BCG vaccination and an M leprae vaccine are also being explored for family contacts and possibly for community contacts in endemic areas.