MYCOBACTERIA: INTRODUCTION

Mycobacteria are aerobic, acid-fast bacilli (rods). They are neither gram-positive nor gram-negative; i.e., they are stained poorly by the dyes used in Gram stain. They are virtually the only bacteria that are acid-fast. (One exception is Nocardia asteroides, the major cause of nocardiosis, which is also acid-fast.) The term "acid-fast" refers to an organism s ability to retain the carbolfuchsin stain despite subsequent treatment with an ethanol-hydrochloric acid mixture. The high lipid content (approximately 60%) of their cell wall makes mycobacteria acid-fast.
The major pathogens are Mycobacterium tuberculosis, the cause of tuberculosis, and Mycobacterium leprae, the cause of leprosy.
MYCOBACTERIUM TUBERCULOSIS
Disease
This organism causes tuberculosis.
Important Properties
M. tuberculosis grows slowly (i.e., it has a doubling time of 18 hours, in contrast to most bacteria, which can double in number in 1 hour or less). Because growth is so slow, cultures of clinical specimens must be held for 6–8 weeks before being recorded as negative. M. tuberculosis can be cultured on bacteriologic media, whereas M. leprae cannot. Media used for its growth (e.g., L?wenstein-Jensen medium) contain complex nutrients (e.g., egg yolk) and dyes (e.g., malachite green). The dyes inhibit the unwanted normal flora present in sputum samples. M. tuberculosis is an obligate aerobe; this explains its predilection for causing disease in highly oxygenated tissues such as the upper lobe of the lung and the kidney. Its cell wall contains several complex lipids: (1) long-chain (C78–C90) fatty acids called mycolic acids, which contribute to the organism s acid-fastness; (2) wax D, one of the active components in Freund s adjuvant, which is used to enhance the immune response to many antigens in experimental animals; and (3) phosphatides, which play a role in caseation necrosis.
Cord factor (trehalose dimycolate) is correlated with virulence of the organism. Virulent strains grow in a characteristic "serpentine" cordlike pattern, whereas a virulent strains do not. The organism also contains several proteins, which, when combined with waxes, elicit delayed hypersensitivity. These proteins are the antigens in the PPD (purified protein derivative) skin test (also known as the tuberculin skin test). A lipid located in the bacterial cell wall called phthiocercol dimycoserosate is required for pathogenesis in the lung. M. tuberculosis is relatively resistant to acids and alkalis. NaOH is used to concentrate clinical specimens; it destroys unwanted bacteria, human cells, and mucus but not the organism. M. tuberculosis is resistant to dehydration and so survives in dried expectorated sputum; this property may be important in its transmission by aerosol.
Transmission & Epidemiology
M. tuberculosis is transmitted from person to person by respiratory aerosol, and its initial site of infection is the lung. In the body, it resides chiefly within reticuloendothelial cells, e.g., macrophages. Humans are the natural reservoir of M. tuberculosis; there is no animal reservoir. Most transmission occurs by aerosols generated by the coughing of "smear-positive" people, i.e., those whose sputum contains detectable bacilli in the acid-fast stain. However, about 20% of people are infected by aerosols produced by the coughing of "smear-negative" people.
Pathogenesis
M. tuberculosis produces no exotoxins and does not contain endotoxin in its cell wall. In fact, no mycobacteria produce toxins. The organism preferentially infects macrophages and other reticuloendothelial cells. M. tuberculosis survives and multiplies within a cellular vacuole called a phagosome. The organism produces a protein called "exported repetitive protein" that prevents the phagosome from fusing with the lysosome, thereby allowing the organism to escape the degradative enzymes in the lysosome. Lesions are dependent on the presence of the organism and the host response. There are two types of lesions:
1. Exudative lesions, which consist of an acute inflammatory response and occur chiefly in the lungs at the initial site of infection.
2. Granulomatous lesions, which consist of a central area of giant cells containing tubercle bacilli surrounded by a zone of epithelioid cells. These giant cells, called Langhans giant cells, are an important pathologic finding in tuberculous lesions. A tubercle is a granuloma surrounded by fibrous tissue that has undergone central caseation necrosis. Tubercles heal by fibrosis and calcification. The primary lesion of tuberculosis usually occurs in the lungs. The parenchymal exudative lesion and the draining lymph nodes together are called a Ghon complex. Primary lesions usually occur in the lower lobes, whereas reactivation lesions usually occur in the apices. Reactivation lesions also occur in other well-oxygenated sites such as the kidneys, brain, and bone. Reactivation is seen primarily in immunocompromised or debilitated patients.
Spread of the organism within the body occurs by two mechanisms:
1. A tubercle can erode into a bronchus, empty its caseous contents, and thereby spread the organism to other parts of the lungs, to the gastrointestinal tract if swallowed, and to other persons if expectorated.
2. It can disseminate via the bloodstream to many internal organs. Dissemination can occur at an early stage if cell-mediated immunity fails to contain the initial infection or at a late stage if a person becomes immunocompromised.
Immunity & Hypersensitivity
After recovery from the primary infection, resistance to the organism is mediated by cellular immunity, i.e., by CD4-positive T cells and macrophages. Circulating antibodies also form, but they play no role in resistance and are not used for diagnostic purposes. Patients deficient in cellular immunity, such as AIDS patients, are at much higher risk for disseminated, life-threatening tuberculosis. Mutations in the -interferon receptor gene are another cause of defective cellular immunity that predisposes to severe tuberculosis. This emphasizes the importance of activation of macrophages by -interferon in the host defense against M. tuberculosis.
Prior infection can be detected by a positive tuberculin skin test result, which is due to a delayed hypersensitivity reaction. PPD is used as the antigen in the tuberculin skin test. The intermediate-strength preparation of PPD, which contains 5 tuberculin units, is usually used. The skin test is evaluated by measuring the diameter of the induration surrounding the skin test site. Note that induration (thickening), not simply erythema (reddening), must be observed. The diameter required to judge the test as positive varies depending upon the status of the individual being tested. Induration of 15 mm or more is positive in a person who has no known risk factors. Induration of 10 mm or more is positive in a person with high-risk factors, such as a homeless person, intravenous drug users, or nursing home residents. Induration of 5 mm or more is positive in a person who has deficient cell-mediated immunity, e.g., AIDS patients, or has been in close contact with a person with active tuberculosis.
The tuberculin test becomes positive 4–6 weeks after infection. Immunization with BCG vaccine can cause a positive test, but the reactions are usually only 5–10 mm and tend to decrease with time. People with PPD reactions of 15 mm or more are assumed to be infected with M. tuberculosis even if they have received the BCG vaccine. A positive skin test reverts to negative in about 5–10% of people.
Tuberculin reactivity is mediated by the cellular arm of the immune system; it can be transferred by CD4-positive T cells but not by serum. Infection with measles virus can suppress cell-mediated immunity, resulting in a loss of tuberculin skin test reactivity . A gene called Nramp determines natural resistance to tuberculosis. People who have mutations in the Nramp gene have a much higher rate of clinical tuberculosis than those with a normal allele. The NRAMP protein is located in the membrane of the phagosome in macrophages and plays an important role in killing the organism within the phagosome.
Clinical Findings
Many organs can be involved. 1-Fever, fatigue, night sweats, and weight loss are common.
2-Pulmonary tuberculosis causes cough and hemoptysis.
3-Scrofula is mycobacterial cervical adenitis that presents as swollen nontender lymph nodes, usually unilaterally.
4- Both M. tuberculosis and Mycobacterium scrofulaceum cause scrofula. Erythema nodosum, characterized by tender nodules along the extensor surfaces of the tibia and ulna, is a manifestation of primary infection seen in patients who are controlling the infection with a potent cell-mediated response. Miliary tuberculosis is characterized by multiple disseminated lesions that resemble millet seeds. Tuberculous meningitis and tuberculous osteomyelitis, especially vertebral osteomyelitis (Pott s disease), are important disseminated forms.
Gastrointestinal tuberculosis is characterized by abdominal pain and diarrhea accompanied by more generalized symptoms of fever and weight loss. Intestinal obstruction or hemorrhage may occur. The ileocecal region is the site most often involved. Tuberculosis of the GI tract can be caused by either M. tuberculosis when it is swallowed after being coughed up from a lung lesion or by M. bovis when it is ingested in unpasteurized milk products. Oropharyngeal tuberculosis typically presents as a painless ulcer accompanied by local adenopathy.
In renal tuberculosis, dysuria, hematuria, and flank pain occur. "Sterile pyuria" is a characteristic finding. The urine contains white blood cells, but cultures for the common urinary tract bacterial pathogens show no growth. However, mycobacterial cultures are often positive.
Laboratory Diagnosis
Acid-fast staining of sputum or other specimens is the usual initial test. For rapid screening purposes, auramine stain, which can be visualized by fluorescence microscopy, can be used.
After digestion of the specimen by treatment with NaOH and concentration by centrifugation, the material is cultured on special media, such as L?wenstein-Jensen agar, for up to 8 weeks. It will not grow on a blood agar plate. In liquid BACTEC medium, radioactive metabolites are present and growth can be detected by the production of radioactive carbon dioxide in about 2 weeks. A liquid medium is preferred for isolation because the organism grows more rapidly and reliably than it does on agar. If growth in the culture occurs, the organism can be identified by biochemical tests. For example, M. tuberculosis produces niacin, whereas almost no other mycobacteria do. It also produces catalase. More rapid identification tests using DNA probes are also available.
Because drug resistance, especially to INH , is a problem, susceptibility tests should be performed. However, the organism grows very slowly and susceptibility tests usually take several weeks, which is too long to guide the initial choice of drugs. The luciferase assay, which can detect drug-resistant organisms in a few days, is a distinct improvement. Luciferase is an enzyme isolated from fireflies that produces flashes of light in the presence of adenosine triphosphate (ATP). If the organism isolated from the patient is resistant, it will not be damaged by the drug; i.e., it will make a normal amount of ATP, and the luciferase will produce the normal amount of light. If the organism is sensitive to the drug, less ATP will be made and less light produced.
There are two approaches to the diagnosis of latent infections. One is the PPD skin test. Because there are problems both in the interpretation of the PPD test and with the person returning for the skin test to be read, a quantifiable laboratory-based test is valuable. This laboratory test is the interferon-gamma release assay called QuantiFERON-TB. In this assay, blood cells from the patient are exposed to antigens from M. tuberculosis and the amount of interferon-gamma released from the cells is measured.

Treatment & Resistance
Multidrug therapy is used to prevent the emergence of drug-resistant mutants during the long (6- to 9-month) duration of treatment. (Organisms that become resistant to one drug will be inhibited by the other.) Isoniazid isonicotinylhydrazide (INH), a bactericidal drug, is the mainstay of treatment. Treatment for most patients with pulmonary tuberculosis is with three drugs: INH, rifampin, and pyrazinamide. INH and rifampin are given for 6 months, but pyrazinamide treatment is stopped after 2 months. In patients who are immunocompromised ,who have disseminated disease, or who are likely to have INH-resistant organisms, a fourth drug, ethambutol, is added and all four drugs are given for 9–12 months. Although therapy is usually given for months, the patient s sputum becomes noninfectious within 2–3 weeks.

Prevention
prevention of the spread of the organism depends largely on the prompt identification and adequate treatment of patients who are coughing up the organism. The use of masks and other respiratory isolation procedures to prevent spread to medical personnel is also important. Contact tracing of individuals exposed to patients with active pulmonary disease who are coughing should be done.
An important component of prevention is the use of the PPD skin test to detect recent converters and to institute treatment for latent infections
BCG vaccine can be used to induce partial resistance to tuberculosis. The vaccine contains a strain of live, attenuated M. bovis called bacillus Calmette-Guérin. The vaccine is effective in preventing the appearance of tuberculosis as a clinical disease, especially in children, although it does not prevent infection by M. tuberculosis. It is used primarily in areas of the world where the incidence of the disease is high.. BCG vaccine should not be given to immunocompromised people because the live BCG organisms can cause disseminated disease. BCG vaccine is also used to treat bladder cancer. The vaccine is instilled into the bladder and serves to nonspecifically stimulate cell-mediated immunity, which can inhibit the growth of the carcinoma cells. Pasteurization of milk and destruction of infected cattle are important in preventing intestinal tuberculosis.










Mycobacterium leprae
This organism causes leprosy.
Important Properties
M. leprae has not been grown in the laboratory, either on artificial media or in cell culture. It can be grown in the mouse footpad or in the armadillo. The optimal temperature for growth (30°C) is lower than body temperature; it therefore grows preferentially in the skin and superficial nerves. It grows very slowly, with a doubling time of 14 days. This makes it the slowest-growing human bacterial pathogen. One consequence of this is that antibiotic therapy must be continued for a long time, usually several years.
Transmission
Infection is acquired by prolonged contact with patients with lepromatous leprosy, who discharge M. leprae in large numbers in nasal secretions and from skin lesions
Pathogenesis
The organism replicates intracellularly.
There are two distinct forms of leprosy
tuberculoid
lepromatous—.
Clinical Findings
The incubation period averages several years, and the onset of the disease is gradual. In tuberculoid leprosy, hypopigmented macular or plaque-like skin lesions, thickened superficial nerves, and significant anesthesia of the skin lesions occur. In lepromatous leprosy, multiple nodular skin lesions occur, resulting in the typical leonine (lionlike) facies.
Laboratory Diagnosis
In lepromatous leprosy, the bacilli are easily demonstrated by performing an acid-fast stain of skin lesions or nasal scrapings. Lipid-laden macrophages called "foam cells" containing many acid-fast bacilli are seen in the skin.
In the tuberculoid form, very few organisms are seen and the appearance of typical granulomas is sufficient for diagnosis. Cultures are negative because the organism does not grow on artificial media. No serologic tests are useful.
Treatment
dapsone, but because sufficient resistance to the drug has emerged, combination therapy is now recommended, e.g., dapsone, rifampin, and clofazimine for lepromatous leprosy and dapsone and rifampin for the tuberculoid form.
Treatment is given for at least 2 years or until the lesions are free of organisms..
Prevention
Isolation of all lepromatous patients, coupled with chemoprophylaxis with dapsone for exposed children, is required. There is no vaccine