Pathogenesis

The pathogenesis of tuberculosis caused by organisms of the M. tuberculosis complex is discussed in Chapter 69. Inhalation of a single viable organism has been shown to lead to infection, although close contact is usually necessary. Of those who become infected with M. tuberculosis, 15% to 20% develop disease. The disease usually occurs some years after the initial infection, when the patient’s immune system breaks down for some reason other than the presence of tuberculosis bacilli in the lung. In a small percentage of infected hosts, the disease becomes systemic, affecting a variety of organs.

After ingestion of milk from infected cows, Mycobacterium bovis may penetrate the gastrointestinal mucosa or invade the lymphatic tissue of the oropharynx. An attenuated strain of M. bovis, bacillus Calmette-Guérin (BCG), has been used extensively in many parts of the world to immunize susceptible individuals against tuberculosis. Because mycobacteria are the classic examples of intracellular pathogens and the body’s response to BCG hinges on cell-mediated immunoreactivity, immunized individuals are expected to react more aggressively against all antigens that elicit cell-mediated immunity. In rare cases, an unfortunate individual’s immune system is so compromised that it cannot handle the BCG, and systemic BCG infection may develop.

Photochromogens

The photochromogens (Table 43-3) are slow-growing NTM that produce colonies that require light to form pigment.

Scotochromogens

The scotochromogens (Table 43-4) are slow-growing NTM that produce pigmented colonies whether grown in the dark or the light. The epidemiology of the potentially pathogenic scotochromogens has not been definitively described. In contrast to potentially pathogenic nonphotochromogens, these agents are rarely recovered in the clinical laboratory.

Nonphotochromogens

The nonphotochromogens (Table 43-5) are slow-growing NTM that produce unpigmented colonies whether grown in the dark or the light. Of the organisms in this group, M. terrae complex (M. terrae, M. triviale, and M. nonchromogenicum) and M. gastri are considered nonpathogenic for humans. The other nonphotochromogens are considered potentially pathogenic, and many are frequently recovered in the clinical laboratory. The nonphotochromogens belonging to Mycobacterium avium complex are frequently isolated in the clinical laboratory and are able to cause infection in the human host.

General Characteristics

The large group of organisms that constitute the RGM is divided into six major groups of potentially pathogenic species, based on pigmentation and molecular studies (see Box 43-1). Unlike the majority of other mycobacteria, most rapid-growers can grow on routine bacteriologic media and on media specific for cultivation of mycobacteria. On Gram staining, these organisms appear as weakly gram-positive rods resembling diphtheroids.

Epidemiology and Pathogenesis

The rapidly growing mycobacteria considered potentially pathogenic can cause disease in either healthy or immunocompromised patients. Like many other NTM, these organisms are ubiquitous in the environment and are present worldwide. They have been found in soil, marshes, rivers, and municipal water supplies (tap water) and in marine and terrestrial life forms. Infections caused by rapidly growing mycobacteria can be acquired in the community from environmental sources. They also can be nosocomial infections, resulting from medical interventions (including bone marrow transplantation), wound infections, and catheter sepsis. These organisms may be commensals on the skin. They gain entry into the host by inoculation into the skin and subcutaneous tissues as a result of trauma, injections, or surgery, or through animal contact.

The RGM also can cause disseminated cutaneous infections. The description of chronic pulmonary infections caused by rapidly growing mycobacteria suggests a possible respiratory route for acquisition of organisms present in the environment. Of the potentially pathogenic, rapidly growing NTM, M. fortuitum, M. chelonae, and M. abscessus are commonly encountered; these three species account for approximately 90% of clinical disease. Little is known about the pathogenesis of these organisms.

Spectrum of Disease

The spectrum of disease caused by the most commonly encountered rapid-growers is summarized in Table 43-6. The most common infection associated with RGM is posttraumatic wound infection. An increase in wound infections has been associated with planktonic M. abscessus, which can be identified as a rough colonial phenotype on artificial media; these organisms are capable of infecting macrophages. The smooth colonial phenotype typically is identified in biofilms and lacks infectivity.

General Characteristics

M. leprae has not yet been cultivated in vitro, although it can be cultivated in the armadillo and in the footpads of mice. Molecular biologic techniques have provided most of the information about this organism’s genomic structure and its various genes and their products. Although polymerase chain reaction (PCR) assays have been used to detect and identify M. leprae in infected tissues, the technique thus far has not proved as effective diagnostically as anticipated in indeterminate or paucibacillary (few organisms present) disease. Therefore, diagnosis of leprosy is based on distinct clinical manifestations, such as hypopigmented skin lesions and peripheral nerve involvement, in conjunction with a skin smear that tests positive for acid-fast bacilli.

Epidemiology and Pathogenesis

Understanding of the epidemiology and pathogenesis of leprosy is hampered by the inability to grow the organism in culture. In tropical countries, where the disease is most prevalent, it may be acquired from infected humans; however, infectivity is very low. Prolonged close contact and the host’s immunologic status play roles in infectivity.

Spectrum of Disease

Based on the host’s response, the spectrum of disease caused by M. leprae ranges from subclinical infection to intermediate stages of disease to full-blown and serious clinical manifestations involving the skin, upper respiratory system, testes, and peripheral nerves. The two major forms of the disease are a localized form, called tuberculoid leprosy, and a more disseminated form, called lepromatous leprosy. Patients with lepromatous leprosy are anergic to M. leprae because of a defect in their cell-mediated immunity. Because the organisms’ growth is unimpeded, these individuals develop extensive skin lesions containing numerous acid-fast bacilli; the organisms can spill over into the blood and disseminate. In contrast, individuals with tuberculoid leprosy do not have an immune defect, so the disease is localized to the skin and nerves; few organisms are observed in skin lesions. Most of the serious sequelae associated with leprosy are the result of this organism’s tropism for peripheral nerves.

Pulmonary Specimens

Pulmonary secretions may be obtained by any of the following methods: spontaneously produced or induced sputum, gastric lavage, transtracheal aspiration, bronchoscopy, and laryngeal swabbing. Most specimens submitted for examination are sputum, aerosol-induced sputum, bronchoscopic aspirations, or gastric lavage samples. Spontaneously produced sputum is the specimen of choice. To raise sputum, patients must be instructed to take a deep breath, hold it momentarily, and then cough deeply and vigorously. Patients must also be instructed to cover the mouth carefully while coughing and to discard tissues in an appropriate receptacle. Saliva and nasal secretions should not be collected, nor should the patient use oral antiseptics during the collection period. Sputum specimens must be free of food particles, residues, and other extraneous matter.

The aerosol (saline) induction procedure can best be done on ambulatory patients who are able to follow instructions. Aerosol-induced sputum specimens have been collected from children as young as 5 years of age. This procedure should be performed in an enclosed area with appropriate airflow. Operators should wear particulate respirators and take appropriate safety measures to prevent exposure. The patient is told that the procedure is being performed to induce coughing to raise sputum that the patient cannot raise spontaneously and that the salt solution is irritating. The patient is instructed to inhale slowly and deeply through the mouth and to cough at will, vigorously and deeply, coughing and expectorating into a collection tube. The procedure is discontinued if the patient fails to raise sputum after 10 minutes or feels any discomfort. Ten milliliters of sputum should be collected; if the patient continues to raise sputum, a second specimen should be collected and submitted. Specimens should be delivered promptly to the laboratory and refrigerated if processing is delayed.

Sputum collection guidelines recommend collection of an early morning specimen for 3 consecutive days. In many cases the third specimen demonstrates minimal recovery of organisms, and this collection may not be recommended in some laboratories. Pooled specimens are unacceptable because of an increased risk of contamination.

Gastric Lavage Specimens

Gastric lavage is used to collect sputum from patients who may have swallowed sputum during the night. The procedure is limited to senile, nonambulatory patients; children younger than 3 years of age (specimen of choice); and patients who fail to produce sputum by aerosol induction. The most desirable gastric lavage is collected at the patient’s bedside before the patient arises and before exertion empties the stomach. Gastric lavage cannot be performed as an office or clinic procedure.

The collector should wear a cap, gown, and particulate respirator mask and should stand beside (not in front of) the patient, who should sit up on the edge of the bed or in a chair, if possible. The Levine collection tube is inserted through a nostril, and the patient is instructed to swallow the tube. When the tube has been fully inserted, a syringe is attached to the end of the tube and filtered distilled water is injected into the tube. The syringe is then used to withdraw 5 to 10 mL of gastric secretions, which is expelled slowly down the sides of the 50-mL conical collecting tube. Samples should be adjusted to a neutral pH. The laboratory may choose to provide sterile receptacles containing 100 mg of sodium carbonate to reduce the acidity; this improves the recovery of organisms. The top of the collection tube is screwed on tightly, and the tube is held upright during prompt delivery to the laboratory. Three specimens should be collected over a period of consecutive days. Specimens should be processed within 4 hours.

Bronchial lavages, washings, and brushings are collected and submitted by medical personnel. These are the specimens of choice for detecting nontuberculous mycobacteria and other opportunistic pathogens in patients with immune dysfunction.

Urine Specimens

The incidence of urogenital infections shows little evidence of decreasing. About 2% to 3% of patients with pulmonary tuberculosis show urinary tract involvement, but 30% to 40% of patients with genitourinary disease have tuberculosis at some other site. The clinical manifestations of urinary tuberculosis, which are variable, include frequency of urination (most common), dysuria, hematuria, and flank pain. Definitive diagnosis requires recovery of acid-fast bacilli from the urine.

Early morning voided urine specimens (40 mL minimum) in sterile containers should be submitted daily for at least 3 days. The collection procedure is the same as for collecting a clean-catch midstream urine specimen (see Chapter 73). The 24-hour urine specimen is undesirable because of excessive dilution, higher contamination, and difficulty in concentrating. Catheterization should be used only if a midstream voided specimen cannot be collected.

Fecal Specimens

Acid-fast staining or culture of stool (or both) from patients with AIDS has been used to identify patients who may be at risk for developing disseminated M. avium complex disease. The clinical utility of this practice remains controversial; however, if screening stains and/or cultures are positive, dissemination often follows. Feces should be submitted in a clean, dry, wax-free container without preservative or diluent. Contamination with urine should be avoided.

Tissue and Body Fluid Specimens

Tuberculous meningitis is uncommon but occurs in both immunocompetent and immunosuppressed patients. A sufficient quantity of specimen is crucial for isolation of acid-fast bacilli from CSF. Very few organisms may be present in the spinal fluid, which makes their detection difficult. At least 10 mL of CSF is recommended for recovery of mycobacteria. Similarly, as much as possible of other body fluids (10 to 15 mL minimum), such as pleural, peritoneal, and pericardial fluids, should be collected in a sterile container or syringe with a Luer-tip cap. Tissues may be immersed in saline or wrapped in gauze. Swabs are discouraged, because the recovery of organisms is decreased.

Blood Specimens

Immunocompromised patients, particularly those infected with HIV, can have disseminated mycobacterial infection; most of these infections are caused by M. avium complex. A blood culture positive for MAC is always associated with clinical evidence of disease. Recovery of mycobacteria is improved with blood collection in either a broth or the Isolator lysis-centrifugation system (see Chapter 68). Some studies have indicated that the lysis-centrifugation system is advantageous, because quantitative data can be obtained with each blood culture; in patients with AIDS, quantitation of such organisms can be used to monitor therapy and determine the prognosis. However, the necessity of quantitative blood cultures remains unclear.

Blood for culture of mycobacteria should be collected as for routine blood cultures. Blood collected in regular phlebotomy procedures in anticoagulants such as sodium polyanethol sulfonate (SPS), heparin, and citrate may be used to inoculate cultures for the recovery of Mycobacterium species. Conventional blood culture collection systems are unacceptable for the isolation of Mycobacterium spp. However, specialized automated systems are available for growth of Mycobacterium spp., including the Bactec MGIT 960 system (Becton-Dickinson, Franklin Lakes, N.J.), and the BacT/ALERT 3D (Biomerieux, Durham, N.C.).

Wounds, Skin Lesions, and Aspirates

An aspirate is the best type of specimen for culturing of a skin lesion or wound. The skin should be cleansed with alcohol before aspiration of the material into a syringe. If the volume is insufficient for aspiration, pus and exudates may be obtained on a swab and then placed in a transport medium, such as Amie’s or Stuart’s medium (dry swabs are unacceptable). However, a negative culture of a specimen obtained on a swab is not considered reliable, and this should be noted in the culture report.