Chapter 23: Mycobacteria

The mycobacteria are rod-shaped, aerobic bacteria that do not form spores. Although they do not stain readily, after being 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 nontuberculous mycobacteria (NTM) frequently infect patients with AIDS, are opportunistic pathogens in other immunocompromised persons, and occasionally cause disease in patients with normal immune systems. There are more than 200 Mycobacterium species, including many that are saprophytes. The mycobacteria that infect humans are listed in Table 23-1.

Morphology and Identification

A. Typical Organisms

In tissue, tubercle bacilli are thin, straight rods measuring about 0.4 × 3 μm (Figure 23-1). 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. When stained by basic dyes, they cannot be decolorized by alcohol, regardless of treatment with iodine. True tubercle bacilli are characterized by “acid fastness”—that is, 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 used for identification of acid-fast bacteria. The method is detailed in Chapter 47. In smears of sputum or sections of tissue, mycobacteria can be demonstrated by yellow-orange fluorescence after staining with fluorochrome stains (eg, auramine, rhodamine). The ease with which acid-fast bacteria can be visualized with fluorochrome stains makes them the preferred stains for clinical specimens (Figure 23-1B). The availability of ultrabright light-emitting diode microscopes, some of which do not require electricity, has advanced fluorescence microscopy in resource-limited countries.

B. 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. Agar-based (solid) media are useful for observing colony morphology, for detection of mixed cultures, for antimicrobial susceptibility testing, and can also provide some indication of the quantity of organisms in a particular specimen.

1. Semisynthetic agar media—These media (eg, Middle-brook 7H10 and 7H11) contain defined salts, vitamins, cofactors, oleic acid, albumin, catalase, and glycerol; the 7H11 medium also contains casein hydrolysate. The albumin neutralizes the toxic and inhibitory effects of fatty acids in the specimen or medium. Large inocula yield growth on these media in several weeks. Because large inocula may be necessary, these media may be less sensitive than other media for primary isolation of mycobacteria.

2. Inspissated egg media—These media (eg, Löwenstein-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 3–6 weeks.

These media with added antibiotics (Gruft and Mycobactosel) are used as selective media.

3. Broth media—Broth media (eg, Middlebrook 7H9 and 7H12) support the proliferation of small inocula. Ordinarily, mycobacteria grow in clumps or masses because of the hydrophobic character of the cell surface. If tweens (water-soluble esters of fatty acids) are added, they wet the surface and thus permit dispersed growth in liquid media. Growth is often more rapid than on complex media. There are several commercial sources of these media that are used in many clinical and reference laboratories. These include the MGIT system (Becton Dickinson, Sparks, MD), VersaTREK^® Culture System (ThermoFisher Scientific, Houston, TX), and MB Redox (Heipha Diagnostica Biotest, Eppelheim, Germany).

C. 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. The doubling time of tubercle bacilli is about 18 hours. Saprophytic forms tend to grow more rapidly, to proliferate well at 22–33°C, to produce more pigment, and to be less acid fast than pathogenic forms.

D. 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) or antibacterial agents (eg, penicillin) that are bacteriostatic to other bacteria can be incorporated into media without inhibiting the growth of tubercle bacilli. Acids and alkalies permit the survival of some exposed tubercle bacilli and are used to help eliminate contaminating organisms and for “concentration” of clinical specimens. Tubercle bacilli are resistant to drying and survive for long periods in dried sputum.

E. Variation

Variation can occur in colony appearance, pigmentation, virulence, optimal growth temperature, and many other cellular or growth characteristics.

F. 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, but fowl and cattle are resistant. M tuberculosis and Mycobacterium bovis are equally pathogenic for humans. The route of infection (respiratory vs intestinal) determines the pattern of lesions. In developed countries, M bovis has become very rare. Some “atypical” mycobacteria, now designated as NTM (eg, Mycobacterium kansasii), produce human disease indistinguishable from tuberculosis; others (eg, Mycobacterium fortuitum) cause only surface lesions or act as opportunists.

Constituents of Tubercle Bacilli

The constituents listed as follows are found mainly in cell walls. 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.

A. Lipids

Mycobacteria are rich in lipids. These include mycolic acids (long-chain fatty acids C78–C90), waxes, and phosphatides. In the cell, the lipids are largely bound to proteins and polysaccharides. Muramyl dipeptide (from peptidoglycan) complexed with mycolic acids can cause granuloma formation; phospholipids induce caseous necrosis. Lipids are to some extent responsible for acid fastness. Their removal with hot acid destroys acid fastness, which depends on both the integrity of the cell wall and the presence of certain lipids. Acid fastness is also lost after sonication of mycobacterial cells. Analysis of lipids by gas chromatography reveals patterns that aid in classification of different species.

Virulent strains of tubercle bacilli form microscopic “serpentine cords” in which acid-fast bacilli are arranged in parallel chains. Cord formation is correlated with virulence. A “cord factor” (trehalose-6,6′-dimycolate) has been extracted from virulent bacilli with petroleum ether. It inhibits migration of leukocytes, causes chronic granulomas, and can serve as an immunologic “adjuvant.”

B. 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. They can also elicit the formation of a variety of antibodies.

C. 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 emitted in droplets smaller than 25 μm in diameter when infected persons cough, sneeze, or speak. The droplets evaporate, leaving organisms that are small enough, when inhaled, to be deposited in alveoli. Inside the alveoli, the host’s immune system responds by release of cytokines and lymphokines that stimulate monocytes and macrophages. Mycobacteria begin to multiply within macrophages. Some of the macrophages develop an enhanced ability to kill the organism, but others may be killed by the bacilli. Pathogenic lesions associated with infection appear in the lung 1–2 months after exposure. Two types of lesions as described later under Pathology may develop. Resistance and hypersensitivity of the host greatly influence development of disease and the type of lesions that are seen.

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 type of host and immune response.

A. Two Principal Lesions

1. Exudative type—This consists of an acute inflammatory reaction with edema fluid; polymorphonuclear leukocytes; and, later, monocytes around the tubercle bacilli. This type is seen particularly in lung tissue, where it resembles bacterial pneumonia. It may heal by resolution so that the entire exudate becomes absorbed; it may lead to massive necrosis of tissue or may develop into the second (productive) type of lesion. During the exudative phase, the tuberculin test result becomes positive.

2. Productive (proliferative) 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. A caseous tubercle may break into a bronchus, empty its contents there, and form a cavity. It may subsequently heal by fibrosis or calcification.

B. 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.

In the first infection, tubercle bacilli always spread from the initial site via the lymphatics to the regional lymph nodes. The bacilli may spread farther and reach the bloodstream, which in turn distributes bacilli to all organs (miliary distribution). The bloodstream can be invaded also by erosion of a vein by a caseating tubercle or lymph node. If a caseating lesion discharges its contents into a bronchus, they are aspirated and distributed to other parts of the lungs or are swallowed and passed into the stomach and intestines.

C. Intracellular Site of Growth

When mycobacteria establish themselves in tissue, they reside principally intracellularly in monocytes, reticuloendothelial cells, and giant cells. The intracellular location is one of the features that makes chemotherapy difficult and favors microbial persistence. Within the cells of immune animals, multiplication of tubercle bacilli is greatly inhibited.

Primary Infection and 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 (Ghon lesion). (3) The tuberculin test result becomes positive.

As described in the early 20th century, primary infection type occurred usually in childhood, and involved any part of the lung but most often the mid-lung fields or the base. Enlarged hilar and mediastinal lymph nodes are frequently observed.

The reactivation type is usually caused by tubercle bacilli that have survived in the primary lesion. Reactivation tuberculosis is characterized by chronic tissue lesions, the formation of tubercles, caseation, and fibrosis. Regional lymph nodes are only slightly involved, and they do not caseate. The reactivation type almost always begins at the apex of the lung, where the oxygen tension (PO₂) is highest.

These differences between primary infection and reinfection or reactivation are attributed to (1) resistance and (2) hypersensitivity induced by the first infection. It is not clear to what extent each of these components participates in the modified response in reactivation tuberculosis.

Immunity and Hypersensitivity

During the first infection with tubercle bacilli, a certain resistance is acquired, and there is an increased capacity to localize tubercle bacilli, retard their multiplication, limit their spread, and reduce lymphatic dissemination. This can be attributed to the development of cellular immunity, with evident ability of mononuclear phagocytes to limit the multiplication of ingested organisms and even to destroy them.

In the course of primary infection, the host also acquires hypersensitivity to the tubercle bacilli. This is made evident by the development of a positive tuberculin reaction (see later discussion). Tuberculin sensitivity can be induced by whole tubercle bacilli or by tuberculoprotein in combination with the chloroform-soluble wax D of the tubercle bacillus but not by tuberculoprotein alone. Hypersensitivity and resistance appear to be distinct aspects of related cell-mediated reactions.

Tuberculin Test

A. Material

Old tuberculin is a concentrated filtrate of broth in which tubercle bacilli have grown for 6 weeks. In addition to the reactive tuberculoproteins, this material contains a variety of other constituents of tubercle bacilli and of growth medium. A purified protein derivative (PPD) is obtained by chemical fractionation of old tuberculin. PPD is standardized in terms of its biologic reactivity as tuberculin units (TU). By international agreement, the TU is defined as the activity contained in a specified weight of Siebert’s PPD Lot No. 49608 in a specified buffer. This is PPD-S, the standard for tuberculin against which the potency of all products must be established by biologic assay (ie, by reaction size in humans). First-strength tuberculin has 1 TU, intermediate-strength has 5 TU, and second-strength has 250 TU. Bioequivalency of PPD products is not based on the weight of the material but on comparative activity.

B. Dose of Tuberculin

A large amount of tuberculin injected into a hypersensitive host may give rise to severe local reactions and a flare-up of inflammation and necrosis at the main sites of infection (focal reactions). For this reason, tuberculin tests in surveys use 5 TU in 0.1 mL solution; in persons suspected of extreme hypersensitivity, skin testing is begun with 1 TU. The volume is usually 0.1 mL injected intracutaneously, usually on the volar aspect of the forearm. The PPD preparation must be stabilized with polysorbate 80 to prevent adsorption to glass.

C. Reactions to Tuberculin

After the tuberculin skin test is placed, the area is examined for the presence of induration no later than 72 hours after placement. It is imperative that a person trained in the accurate reading of these tests examine the area in question. Erythema alone should not be interpreted as a reactive test result. The Centers for Disease Control and Prevention (CDC) has established three different cut points defining a positive test result, considering both the sensitivity and specificity of the test and the prevalence of tuberculosis in various populations. For patients at the highest risk of developing active disease (eg, HIV-infected persons, people who have had exposure to persons with active tuberculosis) 5 mm or larger of induration is considered positive; larger than 10 mm is considered positive for persons with increased probability of recent infection. This category might include individuals such as recent immigrants from high-prevalence countries, injection drug users, and health care workers with exposure to tuberculosis. For persons at low risk for tuberculosis, 15 mm or larger of induration is considered a positive test result. In an individual who has not had contact with mycobacteria, there is generally no reaction to PPD-S. Positive test results tend to persist for several days. Weak reactions may disappear more rapidly.

The tuberculin test result becomes positive within 4–6 weeks after infection (or injection of avirulent bacilli). It may be negative in the presence of tuberculous infection when “anergy” develops because of overwhelming tuberculosis, measles, Hodgkin disease, sarcoidosis, AIDS, or immunosuppression. A positive tuberculin test result may occasionally revert to negative upon isoniazid (INH) treatment of a recent converter. After bacillus Calmette-Guérin (BCG) vaccination, people convert to a positive test result, but this may last for only 3–7 years. Only the elimination of viable tubercle bacilli results in reversion of the tuberculin test result to negative. However, persons who were PPD positive years ago and are healthy may fail to give a positive skin test result. When such persons are retested 2 weeks later, their PPD skin test result—“boosted” by the recent antigen injection—will give a positive size of induration again.

A positive tuberculin test result indicates that an individual has been infected in the past. It does not imply that active disease or immunity to disease is present. Tuberculin-positive persons are at risk of developing disease from reactivation of the primary infection, but tuberculin-negative persons who have never been infected are not subject to that risk, although they may become infected from an external source.

D. Interferon-Gamma Release Assays for Detection of Tuberculosis

Sometimes the results of the tuberculin skin test are equivocal, particularly in persons who have been vaccinated with BCG or who live in areas where NTM are highly prevalent in the environment. In an effort to improve diagnostic accuracy, whole-blood interferon-γ release assays (IGRAs) have been commercially developed. These assays are based on the host’s immune responses to specific M tuberculosis antigens ESAT-6 (early secretory antigenic target-6), CFP-10 (culture filtrate protein-10), and TB7.7, which are absent from most NTM and BCG. The tests detect interferon-γ that is released by sensitized CD4 T cells in response to these antigens. Currently, two commercial assays are available in the United States. The Quantiferon-Gold In-Tube test (QFT-GIT) (Cellestis Limited, Carnegie, Victoria, Australia) is an enzyme-linked immunosorbent assay (ELISA) that detects interferon-γ in whole blood. The T-SPOT-TB (Oxford Immunotec, Oxford, UK) is an ELISA ImmunoSpot assay that uses purified peripheral blood mononuclear cells. Results for both tests are reported as positive, negative, or indeterminate. These assays are still undergoing extensive evaluation. They are susceptible to biological variation in the immune response. However, multiple studies have shown that these assays are comparable to the tuberculin skin test in evaluating latent infection, particularly in persons who have received BCG. However, they should not be used in severely immunocompromised hosts or in very young children (< 5 years of age). The CDC has drafted updated guidelines summarizing recommendations on the use of the IGRAs (see Mazurek et al, 2010).

Patients that who have newly converted from having a negative to a positive result by skin test or IGRA as well as others who have had a positive test result and meet certain criteria for increased risk of active disease if infected are usually given prophylaxis with INH daily for 9 months. Recently, the CDC has published new recommendations for treatment of latent tuberculosis that significantly shorten the length of therapy to 12 weeks. The new regimen consists of once-weekly treatment with INH and rifapentine by directly observed therapy. The new regimen was shown to be equivalent to the old treatment in three randomized controlled trials.

Clinical Findings

Because the tubercle bacillus can involve every organ system, its clinical manifestations are protean. Fatigue, weakness, weight loss, fever, and night sweats 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 result does not prove the presence of active disease caused by tubercle bacilli. Isolation of tubercle bacilli provides such proof.

A. Specimens

Specimens consist of fresh sputum, gastric washings, urine, pleural fluid, cerebrospinal fluid, joint fluid, biopsy material, blood, or other suspected material.

B. Decontamination and Concentration of Specimens

Specimens from sputum and other nonsterile sites should be liquefied with N-acetyl-L-cysteine decontaminated with NaOH (kills many other bacteria and fungi), neutralized with buffer, and concentrated by centrifugation. Specimens processed in this way can be used for acid-fast stains and for culture. Specimens from sterile sites, such as cerebrospinal fluid, do not need the decontamination procedure but can be directly centrifuged, examined, and cultured.

C. Smears

Sputum, exudate, or other material is examined for acid-fast bacilli by 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 traditional acid-fast stains, such as Ziehl-Neelsen, and is the preferred method for clinical material. If acid-fast organisms are found in an appropriate specimen, this is presumptive evidence of mycobacterial infection.

D. 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 (see earlier discussion). The selective broth culture often is the most sensitive method and provides results most rapidly. A selective agar media (eg, Löwenstein-Jensen or Middlebrook 7H10/7H11 biplate with antibiotics) should be inoculated in parallel with broth media cultures. Incubation is at 35–37°C in 5–10% CO₂ for up to 8 weeks. If culture results are negative in the setting of a positive acid-fast stain or if slowly growing NTM (see later) are suspected, then a set of inoculated media should be incubated at a lower temperature (eg, 24–33°C) and both sets incubated for 12 weeks.

Blood for culture of mycobacteria (usually MAC) should be anticoagulated and processed by one of two methods: (1) commercially available lysis centrifugation system or (2) inoculation into commercially available broth media specifically designed for blood cultures. It is medically important to characterize and separate M tuberculosis complex from all the other species of mycobacteria. Isolated mycobacteria should be identified as to species. Conventional methods for identification of mycobacteria include observation of rate of growth, colony morphology, pigmentation, and biochemical profiles. The conventional methods often require 6–8 weeks for identification and are rapidly becoming of historical interest because they are inadequate to identify the expanding numbers of clinically relevant species. Most laboratories have abandoned reliance on these biochemical tests. Growth rate separates the rapid growers (growth in ≤7 days) from other mycobacteria (Table 23-2). Photochromogens produce pigment in light but not in darkness, scotochromogens develop pigment when growing in the dark, and nonchromogens (nonphotochromogens) are nonpigmented or have light tan or buff-colored colonies. Molecular probe methods are available for four species (see later) and are much faster than the conventional methods. The probes can be used on mycobacterial growth from solid media or from broth cultures. DNA probes specific for ribosomal RNA (rRNA) sequences of the test organism are used in a hybridization procedure. There are approximately 10,000 copies of the rRNA per mycobacterial cell, providing a natural amplification system, enhancing detection. Double-stranded hybrids are separated from unhybridized single-stranded probes. The DNA probes are linked with chemicals that are activated in the hybrids and detected by chemiluminescence. Probes for the M tuberculosis complex (M tuberculosis, M bovis, Mycobacterium africanum, Mycobacterium caprae, Mycobacterium microti, Mycobacterium canetti, and Mycobacterium pinnipedii), MAC (M avium, M intracellulare, and closely related mycobacteria), M kansasii, and Mycobacterium gordonae are in use. The use of these probes has shortened the time to identification of clinically important mycobacteria from several weeks to as little as 1 day.

In the United States, these four groups (M tuberculosis complex, M avium complex, M kansasii, and M gordonae) make up 95% or more of clinical isolates of mycobacteria.

For species that cannot be identified by DNA probes, many laboratories with molecular capabilities have implemented 16S rRNA gene sequencing to rapidly identify probe-negative species or send such organisms to a reference laboratory with sequencing capability.

High-performance liquid chromatography (HPLC) has been applied to identification of mycobacteria. The method is based on development of profiles of mycolic acids, which vary from one species to another. HPLC to speciate mycobacteria is available in reference laboratories.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is not yet FDA cleared for identification of Mycobacterium species recovered in culture although progress is being made. It is anticipated that this method will be available in the near future. Susceptibility testing of mycobacteria is an important adjunct in selecting drugs for effective therapy. A standardized broth culture technique can be used to test for susceptibility to first-line drugs. The complex and more arduous conventional agar-based technique usually is performed in reference laboratories; first- and second-line drugs can be tested by this method. A modification of liquid broth cultures involves inoculating mycobacteria on a multi-well plate with and without addition of antibiotics (Microscopic Observation Drug Susceptibility, MODS assay) and examining for cording that is characteristic of M tuberculosis complex. This method is largely used outside the United States.

E. Nucleic Acid Amplification Tests (NAATs)

NAATs are available for the rapid and direct detection of M tuberculosis in clinical specimens. An advance over the in-lab–developed PCR tests and the existing FDA-cleared commercial assays is the GeneXpert MTB/RIF test (Cepheid, Sunnyvale, CA), a real-time multiplex PCR method that both identifies the Mtb complex and also detects genes that encode rifampin resistance. One of the earlier publications on this method (see reference Boehme) reported a sensitivity for smear positive respiratory specimens of 98.2% and for smear negative samples, 72.5%. Overall specificity was 99.2%. In terms of the detection of rifampin resistance, the assay does detect the common mutations, but discrepancies between phenotypic test results and genotypic results still challenge complete reliance on this component of the test. This assay is not yet widely available in the United States but is available in other countries.

The characterization of specific strains of M tuberculosis can be important for clinical and epidemiologic purposes. It facilitates tracking transmission, analysis of outbreaks of tuberculosis, and demonstration of reactivation versus reinfection in individual patients. DNA fingerprinting is done using a standardized protocol based on restriction fragment length polymorphism. Many copies of the insertion sequence 6110 (IS6110) are present in the chromosome of most strains of M tuberculosis, and these are located at variable positions. DNA fragments are generated by restriction endonuclease digestion and separated by electrophoresis. A probe against IS6110 is used to determine the genotypes. Other useful methods for strain characterization include spoligotyping, a PCR-based technique that targets the direct repeat locus of M tuberculosis and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) analysis. The latter method is slowing replacing IS6110 typing. Genotyping is done at the CDC, at some state health department laboratories, and in research laboratories.

Treatment

The primary treatment for mycobacterial infection is specific chemotherapy. The drugs for treatment of mycobacterial infection are discussed in Chapter 28. Two cases of tuberculosis are presented in Chapter 48.

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 greater than 95%.

The two major drugs used to treat tuberculosis are INH and RMP. The other first-line drugs are pyrazinamide (PZA) and ethambutol (EMB). 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 kanamycin, capreomycin, ethionamide, cycloserine, ofloxacin, and ciprofloxacin.

A four-drug regimen of INH, RMP, PZA, and EMB is recommended for persons in the United States who have a slight to moderate risk for being infected with drug-resistant tubercle bacilli. The risk factors include recent emigration from Latin America or Asia, persons with HIV infections or who are at risk for HIV infection and live in an area with a low prevalence of multidrug-resistant tubercle bacilli, and persons who were previously treated with a regimen that did not include RMP. These four drugs are continued for 2 months. If the isolate is susceptible to INH and RMP, PZA and EMB can be discontinued, and the remaining treatment with INH and RMP is continued to complete a 6-month course. In patients with cavitary disease or in whom the sputum culture results are still positive after 2 months of treatment, an additional 3 months of therapy (total course duration of 9 months) should be given to prevent relapse. In noncompliant patients, directly observed therapy is important.

Drug resistance in M tuberculosis is a worldwide problem. Mechanisms explaining the resistance phenomenon for many, but not all, of the resistant strains have been defined. INH resistance has been associated with deletions or mutations in the catalase-peroxidase gene (katG); these isolates become catalase negative or have decreased catalase activity. INH resistance has also been associated with alterations in the inhA gene, which encodes an enzyme that functions in mycolic acid synthesis. Streptomycin resistance has been associated with mutations in genes encoding the ribosomal S12 protein and 16S rRNA, rpsL and rrs, respectively. RMP resistance has been associated with alterations in the B subunit of RNA polymerase, the rpoB gene. Mutations in the DNA gyrase gene gyrA have been associated with resistance to fluoroquinolones. The possibility that drug resistance is present in a patient’s M tuberculosis isolate must be taken into account when selecting therapy.

Multidrug-resistant M tuberculosis (resistant to both INH and RMP) is a major problem in tuberculosis treatment and control. Such strains are prevalent in certain geographic areas and certain populations (eg, hospitals, prisons). There have been many outbreaks of tuberculosis with multidrug-resistant strains. They are particularly important in persons with HIV infections in resource-poor countries. Persons infected with multidrug-resistant organisms or who are at high risk for such infections, including exposure to another person with such an infection, should be treated according to susceptibility test results for the infecting strain. If susceptibility results are not available, the drugs should be selected according to the known pattern of susceptibility in the community and modified when the susceptibility test results are available. Therapy should include a minimum of three and preferably more than three drugs to which the organisms have demonstrated susceptibility.

Extensively drug-resistant (XDR) strains are now globally recognized. These are defined by the World Health Organization (WHO) as isolates of M tuberculosis with resistance to INH and RMP; any fluoroquinolone; and at least one of three injectable second-line drugs such as amikacin, capreomycin, or kanamycin. The true prevalence of XDR tuberculosis is underestimated in resource-limited countries because of the lack of available diagnostic and susceptibility tests. Factors that have contributed to the global epidemic include ineffective tuberculosis treatment; lack of proper diagnostic testing; and most importantly, poor infection control practices. Persons infected with XDR tuberculosis have a poorer clinical outcome and are 64% more likely to die during treatment than persons infected with susceptible strains. In 2006, the WHO Global Task Force on XDR-TB issued multifaceted and comprehensive recommendations to address the XDR-TB epidemic (available at http://www.who.int/tb/features_archive/global_taskforce_report/en/).

Epidemiology

The most frequent source of infection is humans who excrete, 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 tuberculin-negative people, the risk of acquiring tubercle bacilli depends on exposure to sources of infectious bacilli, principally sputum-positive patients. This risk is proportionate to the rate of active infection in the population, crowding, socioeconomic disadvantage, and inadequacy of medical care.

The development of clinical disease after infection may have a genetic component (proven in animals and suggested in humans by a higher incidence of disease in those with HLA-Bw15 histocompatibility antigen). It is influenced by age (high risk in infancy and in elderly adults); by undernutrition; and by immunologic status, coexisting diseases (eg, silicosis, diabetes), and other individual host resistance factors.

Infection occurs at an earlier age in urban than in rural populations. Disease occurs only in a small proportion of infected individuals. In the United States at present, active disease has several epidemiologic patterns in which individuals are at increased risk, including minorities, predominantly African Americans and Hispanics; immigrants from countries of high endemicity; HIV-infected patients; homeless persons; and very young and very old individuals. The incidence of tuberculosis is especially high in minority persons with HIV infections. Primary infection can occur in any person exposed to an infectious source. Patients who have had tuberculosis can be infected exogenously a second time. Endogenous reactivation tuberculosis occurs most commonly among persons with AIDS immunosuppression and elderly malnourished or alcoholic destitute men.

Prevention and Control

1. Prompt and effective treatment of patients with active tuberculosis and careful follow-up of their contacts with tuberculin tests, radiographs, and appropriate treatment are the mainstays of public health tuberculosis control.

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. 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, corticosteroids) or infection. HIV infection is a major risk factor for tuberculosis.

4. Various living avirulent tubercle bacilli, particularly BCG (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 vaccines are inadequate from many technical and biologic standpoints. Nevertheless, BCG is given to children in many countries. Statistical evidence indicates that an increased resistance for a limited period follows BCG vaccination.

5. Eradication of tuberculosis in cattle and pasteurization of milk have greatly reduced M bovis infections.

Concept Checks

• Mycobacteria are rod-shaped, aerobic organisms that are “acid-fast” positive because of the complex nature of their cell walls that include mycolic acids.

• Mycobacteria grow more slowly than other bacteria. Both nonselective and selective media in solid and liquid forms are used to recover organisms from clinical material.

• Although there are more than 200 species of Mycobacteria, the slow-growing M tuberculosis complex is of most importance to humans and public health.

• The hallmark of infections with M tuberculosis is granulomas. Granulomas have a concentric structure that consists of a central necrotic center (caseous necrosis) surrounded by a zone of multinucleated giant cells, monocytes, and histiocytes and an outer ring of fibrosis.

• Humans acquire tuberculosis from inhalation of infected droplet nuclei.

• The tuberculin skin test or the IGRAs can be used to screen persons for latent tuberculosis infection.

• Diagnosis of tuberculosis requires acid-fast smear and culture; nucleic acid amplification tests when performed on smear-positive specimens can be very helpful.

• The mainstay of therapy is an initial four-drug regimen of INH, RMP, PZA, and EMB followed by 4 months of INH and RMP. Acceptable alternative regimens are also available. Multidrug-resistant and XDR tuberculosis have become a global health care crisis.

In addition to tubercle bacilli (eg, M tuberculosis, M bovis), other mycobacteria of varying degrees of pathogenicity have been grown from human sources in past decades. These “atypical” mycobacteria were initially grouped according to speed of growth at various temperatures and production of pigments (see earlier discussion). Several are now identified using DNA probes or by DNA sequencing. Most of them occur in the environment, are not readily transmitted from person to person, and are opportunistic pathogens (see Table 23-1).

Species or complexes that are significant causes of disease are outlined.

Mycobacterium avium Complex

The M avium complex is often called the MAC or MAI (M avium intracellulare) complex. These organisms grow optimally at 41°C and produce smooth, soft, nonpigmented colonies. They are ubiquitous in the environment and have been cultured from water, soil, food, and animals, including birds.

MAC organisms infrequently cause disease in immunocompetent humans. However, in the United States, disseminated MAC infection is one of the most common opportunistic infections of bacterial origin in patients with AIDS. The risk of developing disseminated MAC infection in HIV-infected persons is greatly increased when the CD4-positive lymphocyte count declines to below 100/μL. (See Case 17 in Chapter 48.) Gender, race, ethnic group, and individual risk factors for HIV infection do not influence the development of disseminated MAC infection, but prior Pneumocystis jirovecii infection, severe anemia, and interruption of antiretroviral therapy may increase the risk.

During the first 15 years of the AIDS epidemic, approximately 25% and perhaps as high as 50% of HIV-infected patients developed MAC bacteremia and disseminated infection during the course of AIDS. Subsequently, the use of highly active antiretroviral therapy (HAART) and the use of azithromycin or clarithromycin prophylaxis have greatly decreased the incidence of disseminated MAC infection in AIDS patients.

Other patients at risk include those with cystic fibrosis and pulmonary alveolar proteinosis. Pulmonary MAC disease has also been described in middle-aged to elderly women in the absence of chronic lung disease and has been referred to as “Lady Windermere” syndrome. This form of the disease is indolent and over time is characterized by nodules in the middle lobes and lingula that progress to cavitation. Cervical lymphadenitis is the most common presentation in young children (< 5 years of age). The major manifestation is unilateral, firm adenopathy; fever is generally absent.

Environmental exposure can led to MAC colonization of either the respiratory or gastrointestinal tract. Transient bacteremia occurs followed by invasion of tissues. Persistent bacteremia and extensive infiltration of tissues result in organ dysfunction. Any organ can be involved. In the lung, nodules, diffuse infiltrates, cavities, and endobronchial lesions are common. Other manifestations include pericarditis, soft tissue abscesses, skin lesions, lymph node involvement, bone infection, and central nervous system lesions. The patients often present with nonspecific symptoms of fever, night sweats, abdominal pain, diarrhea, and weight loss. The diagnosis is made by culturing MAC organisms from blood or tissue.

MAC organisms routinely are resistant to first-line antituberculosis drugs. Treatment with either clarithromycin or azithromycin plus EMB is a preferred initial therapy. Other drugs that may be useful are rifabutin (Ansamycin), clofazimine, and fluoroquinolones. Amikacin and streptomycin have activity but are less desirable because of toxicity. Multiple drugs often are used in combination. Therapy should be continued for life. Therapy results in decreasing counts of MAC organisms in blood and amelioration of clinical symptoms.

Mycobacterium kansasii

M kansasii is a photochromogen that requires complex media for growth at 37°C. It can produce pulmonary and systemic disease indistinguishable from tuberculosis, especially in patients with impaired immune responses. Sensitive to RMP, it is often treated with the combination of RMP, EMB, and INH with a good clinical response. The source of infection is uncertain, and communicability is low or absent.

Mycobacterium scrofulaceum

This is a scotochromogen occasionally found in water and as a saprophyte in adults with chronic lung disease. It causes chronic cervical lymphadenitis in children and, rarely, other granulomatous disease. Surgical excision of involved cervical lymph nodes may be curative, and resistance to antituberculosis drugs is common. (Mycobacterium szulgai and Mycobacterium xenopi are similar.)

Mycobacterium marinum and Mycobacterium ulcerans

These organisms occur in water, grow best at low temperature (31°C), may infect fish, and can produce superficial skin lesions (ulcers, “swimming pool granulomas”) in humans. Surgical excision, tetracyclines, RMP, and EMB are sometimes effective.

Mycobacterium fortuitum Complex

These are saprophytes found in soil and water that grow rapidly (3–6 days) in culture and form no pigment. They can produce superficial and systemic disease in humans on rare occasions. The organisms are often resistant to antimycobacterial drugs but may respond to amikacin, doxycycline, cefoxitin, erythromycin, or RMP.

Mycobacterium chelonae-abscessus

These rapid growers should be differentiated because the types and severity of disease are different and because therapy for Mycobacterium chelonae is easier as it is more susceptible to antimicrobial agents. Both species are capable of causing skin, soft tissue, and bone infections after trauma or surgery, which can disseminate in immunocompromised patients. Mycobacterium abscessus is also frequently recovered from patients with respiratory disease in the United States, especially in southeastern regions. The individuals most commonly infected are elderly, white, female nonsmokers. Patients with cystic fibrosis are also at risk and may succumb to a fulminant, rapidly progressive form of the disease. M chelonae is typically susceptible to tobramycin, clarithromycin, linezolid, and imipenem. Clarithromycin, amikacin, and cefoxitin are usually used for treatment of M abscessus, although drug resistance is a major problem with this organism.

Other Mycobacterium Species

The high risk for mycobacterial infection in patients with AIDS has resulted in increased awareness of mycobacterial infections in general. Species previously considered to be curiosities and extremely uncommon have been more widely recognized (see Table 23-1). Mycobacterium malmoense has been reported mostly from Northern Europe. It causes a pulmonary tuberculosis-like disease in adults and lymphadenitis in children. Mycobacterium haemophilum and Mycobacterium genavense cause disease in patients with AIDS. The importance of these two species is not fully understood.

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. Globally the majority of cases occur in Brazil and the Indian subcontinent.

Typical acid-fast bacilli—singly, in parallel bundles, or in globular masses—are regularly found in scrapings from skin or mucous membranes (particularly the nasal septum) in patients with 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 the footpads of mice, local granulomatous lesions develop with limited multiplication of bacilli. Inoculated armadillos develop extensive lepromatous leprosy, and armadillos naturally infected with leprosy have been found in Texas and Mexico. M leprae from armadillo or human tissue contains a unique o-diphenoloxidase, perhaps an enzyme characteristic of leprosy bacilli.

Clinical Findings

The onset of leprosy is insidious. The lesions involve the cooler tissue of the body, including the skin, superficial nerves, nose, pharynx, larynx, eyes, and testicles. The skin lesions may occur as pale, anesthetic macular lesions 1–10 cm in diameter; diffuse or discrete erythematous, infiltrated nodules 1–5 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 the digits. The disfigurement caused by 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 (see the Ridley-Jopling classification system). In the lepromatous type, the course is progressive and malignant, 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 result. 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 a small number of macular skin lesions containing few bacilli, severe asymmetric nerve involvement of sudden onset, and a positive lepromin skin test result. 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 patients with leprosy.

Treatment

Sulfones such as dapsone (see Chapter 28) are first-line therapy for both tuberculoid and lepromatous leprosy. RMP and/or clofazimine generally are included in the initial treatment regimens. Other drugs active against M leprae include minocycline, clarithromycin, and some fluoroquinolones. Regimens recommended by the WHO are practical. Several years of therapy may be necessary to adequately treat leprosy.

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 2–10 years. Without prophylaxis, about 10% of exposed children may acquire the disease. Treatment tends to reduce and abolish the infectivity of patients. The naturally infected armadillos found in Texas and Mexico probably play no role in transmission of leprosy to humans.

Prevention and Control

In the United States, the current recommendations for prevention of leprosy include a thorough examination of household contacts and close relatives. This should include a complete skin examination and an examination of the peripheral nervous system. The U.S. Public Health Service National Hansen’s Disease Program does not recommend routine dapsone prophylaxis. A therapeutic trial may be indicated for patients whose signs and symptoms are suggestive of leprosy but who do not have a definitive diagnosis.

BCG does provide some protection against leprosy especially among household contacts of cases.

Concept Checks

• The NTM are a diverse group of organisms commonly found in the environment, and the group includes both saprophytes and human pathogens.

• The NTM can be further classified into the rapid growers (grow in < 7 days) and slow growers. Each group can be subdivided on the basis of pigment production.

• Members of MAC are among the most frequently isolated NTM. They are responsible for significant disease in patients with AIDS and others with chronic lung disease.

• M kansasii causes pulmonary infections that mimic tuberculosis. It responds to therapy with INH, RIF, and EMB.

• The rapid growers are diverse. M fortuitum complex, M chelonae, and M abscessus are the most prevalent. M abscessus causes the most severe disease and is often multidrug resistant.

• M leprae causes the disease leprosy. The organism is not cultivatable, so diagnosis is difficult. Treatment with dapsone, RMP, and clofazimine is often prolonged for many years.