This chapter reviews the epidemiology, pathogenesis, and animal models of mycobacterial eye diseases and summarizes the clinical features, laboratory diagnosis, and treatment of these diverse diseases.

The cell wall of mycobacteria, like that of Nocardia and Corynebacterium, contains type A peptidoglycan, in which there is direct cross-linking between m-diaminopimelic acid and D-alanine. The peptidoglycan is covalently linked to a polysaccharide composed of arabinose and galactose. The presence of arabinogalactan results in immunologic cross-reactivity among the three genera.

Mycobacteria are characterized by large amounts of lipid and true waxes (up to 40% to 60% of the total dry weight of mycobacteria, compared to 20% in gram-negative bacteria and 1% to 4% in gram-positive bacteria) in their cell walls. The high lipid content accounts for their unusual growth and staining characteristics. After staining with carbol fuchsin, mycobacteria are resistant to decolorization with acid alcohol and are therefore termed “acid-fast.” One class of lipids, the mycosides, is composed of mycolic acids bound to carbohydrates as glycolipids and is unique to acid-fast organisms.¹ Mycolic acid is a large α-branched, β-hydroxy fatty acid that varies in size from one species of mycobacterium to another. Wax D (not a true wax) is a large mycoside in which 15 to 20 molecules of mycolic acid are esterified to a large polysaccharide composed of arabinose, galactose, mannose, glucosamine, and galactosamine. Wax D, the active ingredient in Freund's complete adjuvant, acts to increase the antibody response to an antigen. As a result, antigens that by themselves are poorly immunogenic, such as tuberculoprotein, induce host delayed-type hypersensitivity.

Unlike some bacteria, the virulence of pathogenic mycobacteria is not related to the secretion of toxins or the presence of a capsule. Cord factor is a mycoside in which two molecules of mycolic acid are esterified to the disaccharide 6,6'-dimycolytrehalose. It is found in virulent mycobacteria and is responsible for the phenomenon in which individual bacteria grow parallel to each other to form large serpentine cords. Cord factor inhibits the migration of polymorphonuclear leukocytes in vitro. Nonvirulent mycobacteria do not grow in cords, and when cord factor is extracted from cells they lose their virulence. Cord factor is not the only component of mycobacteria that has been correlated with virulence, however, as other lipids are also toxic to macrophages.

M. leprae contains a species-specific cell wall component known as phenolic glycolic lipid 1 (PGL-1), which enhances intracellular survival and limits antimicrobial penetration.² Both PGL-1 and the glycoprotein lipoarabinomannan may induce immunologic unresponsiveness of lymphocytes and macrophages in lepromatous leprosy.³

Mycobacteria are slow-growing organisms (particularly M. leprae), with doubling times measured on the order of days to weeks. This slow growth accounts for the long incubation periods for tuberculosis and leprosy as well as the indolent course of most nontuberculous mycobacterial keratitides. Most mycobacteria grow best at 37°C. M. tuberculosis and the nontuberculous mycobacteria can be grown on artificial media, but no in vitro method of culturing M. leprae exists. Runyon and others classified the nontuberculous mycobacteria into four groups based on pigment production, rate of growth, and colony morphology:

• Runyon group I consists of the photochromogens, which are slow-growing species that produce carotenoid pigments when exposed to light.
  
• Runyon group II consists of slow-growing scotochromogens, which produce a yellow-orange pigment in either light or dark.
  
• Runyon group III consists of nonphotochromogens that may contain white, tan, or pale-yellow pigment.
  
• Runyon group IV consists of “rapid-growing” mycobacteria and contains the species most responsible for nontuberculous mycobacterial ocular infection.
  

Differentiation of species by morphologic, physiologic, and biochemical characteristics is listed in Table 1. Mycobacterium avium and Mycobacterium intracellulare are not distinguishable by these tests and are therefore referred to together as M. avium complex.

TABLE 56-1. Distinguishing Laboratory Characteristics of Medically Important Mycobacteria

Tubercle bacilli can survive but cannot multiply within the anoxic and acidic caseating necrosis. Cell-mediated immunity, driven primarily by interferon-γ and possibly other cytokines secreted by activated lymphocytes, helps to destroy any bacilli that escape areas of caseation. Both CD4+ (helper) T lymphocytes and CD8+ (suppressor) T lymphocytes are involved.⁵

Nontuberculous mycobacteria are found in soil, water, and dust. They may infect tissue if local resistance is compromised. In the eye, this may occur as a result of trauma, surgery (e.g., penetrating keratoplasty or radial keratotomy), or contact lens wear. Pulmonary infections have been reported, but person-to-person transmission does not occur. Inoculation of the corneal stroma stimulates both acute and chronic granulomatous inflammation, which limits spread of the infection.⁶ The use of topical corticosteroids suppresses the granulomatous response and worsens the keratitis.

There is a wide range to both the clinical and histopathologic manifestations of leprosy.⁷ At one end of the spectrum is tuberculoid leprosy, characterized by strong cell-mediated immunity and little humoral immune response. Affected patients have cutaneous and peripheral nerve trunk involvement; histopathologically, there is a granulomatous reaction with only a few organisms noted. At the other end of the spectrum is lepromatous leprosy, characterized by disseminated infection with minimal cell-mediated immunity. Currently, six classes of leprosy are recognized: indeterminate (I), tuberculoid (T), borderline tuberculoid (BT), borderline (BB), borderline lepromatous (BL), and lepromatous (LL). The World Health Organization also uses skin smear results to classify patients. Paucibacillary patients have negative skin smears for acid-fast bacilli and encompass the I, T, and BT classes. Multibacillary patients have positive skin smears and are classified as BB, BL, or LL. Finally, acute reactional (inflammatory) states may occur in leprosy and may cause extensive tissue destruction. The type I lepra (reversal) reaction represents a delayed hypersensitivity reaction against bacillary antigens. It may occur after initiation of treatment causes an increase in cell-mediated immunity (“upgrading reaction”) or in untreated patients in whom borderline leprosy shifts toward the lepromatous pole (“downgrading reaction”). The type II lepra reaction (erythema nodosum leprosum) is an immune complex reaction that follows a release of bacillary antigens from macrophages, triggering antigen-antibody reaction, complement fixation, and neutrophil infiltration. Although the skin is primarily affected in type II reactions, iridocyclitis, arthritis, proteinuria, orchitis, and lymphadenopathy can also occur.

The mode of transmission of leprosy is unclear. Overcrowding, poverty, and poor hygiene are risk factors, so that the prevalence approaches 1 case per 1000 population in developing countries (15 million cases worldwide), compared to only about 7000 affected patients in the United States. Leprosy is only mildly infectious and causes a slow granulomatous reaction; most manifestations of the disease are a result of the host's immune response. Although person-to-person spread by nasal droplet infection remains the most likely source, soil contamination, insect vectors, and contact with infected armadillos all may contribute to infection in certain areas of the world. There are wide differences in the percentages of tuberculoid and lepromatous leprosy in different parts of the world, as well as in the ocular sequelae. Corneal disease is more common in Africa and India, whereas iridocyclitis is more common in Southeast Asia.⁸ Further, there are genetic and hormonal factors that affect disease expression. Lepromatous leprosy, for example, is more common in men than in women and is associated with HLA-MTI. Tuberculoid leprosy has been associated with HLA-DR3. As with tuberculous infection, most infected patients do not manifest clinical disease.

Susceptibility of the guinea pig to infection with the human tubercle bacilli is comparable to that of human infants or immunodeficient adults. A model using low-dose aerosol inoculation to approximate more closely the conditions for human infection has been developed.⁹ About 10 virulent tubercle bacilli are delivered via the respiratory route to the lungs. Twenty-eight days after infection, lesions are visible by x-ray and organisms can be recovered from excised lesions. The guinea pigs develop high tuberculin sensitivity and show considerable caseous necrosis on histopathology, which parallels the condition in immunocompetent humans. All animals infected with virulent organisms eventually die from their infection. This animal model has been used extensively to evaluate the response to chemotherapy⁹ and to investigate the protective effect of potential tuberculosis vaccine candidates.¹⁰

The mouse has a resistance to tuberculosis similar to that of immunocompetent humans. Unlike humans, mice develop only low degrees of tuberculin sensitivity and show little caseous necrosis. The mouse model is useful in the study of immunity and immunopathology associated with tuberculosis. Some mouse models use immunocompetent animals¹¹; others use immunodeficient animals such as the nude mouse,¹² the severe combined immunodeficiency mouse,¹¹ or animals rendered immunodeficient by thymectomy followed by CD4+ T-cell depletion by means of the administration of purified anti-CD4 monoclonal antibody.¹³ Both aerosol and intravenous inoculations have been used. These models have been used to define the role of T cells, macrophages, and cytokines necessary for the development of acquired immunity.

Because adult reactivation tuberculosis accounts for the majority of clinical tuberculosis cases in the world, an animal model that leads to the reactivation of dormant bacteria followed by the development of tuberculosis is desirable. The best documented animal model of dormancy is the Cornell model,¹⁴ in which mice are infected intravenously and then treated with isoniazid and pyrazinamide. After 12 weeks, the spleen and lungs are sterile. If the animals are left untreated, the disease will reactivate in 60%, who will develop tuberculosis 3 to 4 months later. This model may be used to evaluate potential therapeutic vaccines that target persons who are already infected with M. tuberculosis but do not have clinical disease.

With the recognition of the impact of M. avium complex infections in patients with AIDS, an animal model for this disease has been desirable. The beige mouse is naturally immune deficient and develops uncontrolled infection when intravenously inoculated with M. avium complex organisms,¹⁵ but it is not clear whether the efficacy of drug therapy based on this model accurately predicts the therapeutic response in humans.

Animal models of M. fortuitum⁶ and M. chelonae¹⁶ keratitis have been described that closely mimic human disease in their clinical and histopathologic features. These models are useful because of the limited correlation between in vitro drug sensitivities and clinical efficacy and have demonstrated that different species of atypical mycobacteria have different susceptibilites to various antimicrobial agents.

The nine-banded armadillo, which has a low basal temperature that permits replication of M. leprae, is the most widely used animal model of leprosy.¹⁷ A full discussion of this model is beyond the scope of this chapter.

Nontuberculous mycobacterial ocular disease usually takes the form of keratitis after trauma, surgery, or contact lens wear.^21,22 Clinically, infection is usually apparent 2 to 8 weeks after exposure, but it may take as long as 2 years, by which time empiric therapy with corticosteroids or antibiotics may have modified the presentation. The keratitis is usually indolent, similar to that caused by fungi, anaerobic bacteria, or herpes simplex virus. Satellite lesions are common, but suppuration is usually only mild and corneal perforation is rare. A “cracked glass” appearance from separation of corneal lamellae by spreading organisms may occur early in the disease. Dendriform epithelial ulceration, ring infiltrates, and crystalline keratopathy have been reported. Scleritis, canaliculitis, dacryocystitis, orbital granuloma, and endophthalmitis have rarely been reported. M. avium complex species are frequently noted in the choroid of HIVinfected patients at autopsy but rarely cause clinical disease.

Ocular involvement is common in leprosy.^8,23,24 The ocular adnexae, external eye, and anterior segment are preferentially affected because their temperatures are lower than other parts of the eye. The cornea may be invaded by intraneural spread along limbal ciliary nerves or by hematogenous spread along pannus vessels. Acute hypopyon uveitis may occur during erythema nodosum leprosum. In chronic uveitis, progressive iris atrophy and scarring may occur in the absence of pain or redness. Iris pearls (small white or yellow clusters of bacilli within mononuclear cells without surrounding inflammatory reaction) are pathognomonic for leprosy. Secondary complications of acute and chronic uveitis include glaucoma and cataract. In addition, leprosy may cause loss of eyebrows and eyelashes (madarosis), corneal hypoesthesia with secondary infection and scarring, facial nerve palsy with lagophthalmos, decreased basal and reflex tearing, and trichiasis. Beading of corneal nerves, scleritis, and epiphora caused by nasolacrimal duct obstruction from nasal collapse may occur in lepromatous disease. Five percent to 10% of patients with ocular leprosy are blind.⁸

Tubercles are the characteristic lesions of primary tuberculosis. They begin as an acute inflammation marked by accumulation of polymorphonuclear leukocytes. After the development of hypersensitivity, the leukocytes are replaced by lymphocytes and macrophages as a granulomatous reaction develops. Central caseation is surrounded by a ring of epithelioid cells, which may fuse to form multinucleated giant cells (Fig. 1). Peripherally are found lymphocytes, macrophages, and proliferating fibroblasts.

Skin biopsies from leprosy patients reveal numerous acid-fast bacilli, which may form clumps called globi. Hematoxylin and eosin staining of lepromatous skin biopsies show numerous foam cells, highly vacuolated macrophages laden with acid-fast bacilli. Skin biopsies from tuberculoid patients show few or no acid-fast bacilli, but granulomatous invasion of dermal nerves, with or without caseation, is pathognomonic. Biopsies of ocular tissues (Fig. 2) show numerous organisms inside macrophages, endothelial cells, nerves, pigment epithelium, and smooth muscle cells. There is infiltration of the conjunctiva, cornea, iris, and ciliary body with macrophages, lymphocytes, and plasma cells. Miliary lepromata are seen in iris pearls and around corneal nerves.⁸

As noted above, no in vitro culture medium has been developed for M. leprae. Cultures of other mycobacteria may be obtained using either egg-based media (e.g., Löwenstein-Jensen) or agar-based media (e.g., Middlebrook-Cohn 7H10 or 7H11). Malachite green dye is typically added to the agar to inhibit growth of nonmycobacterial contaminants. Agar-based media support growth more quickly and offer better visualization of colony morphology. The high lipid content of mycobacteria gives the cultures a waxy, dry, and wrinkled surface (Fig. 3). Virulent and avirulent strains cannot be distinguished by colony morphology. Cultures usually take 2 to 4 weeks to grow, although Runyon group IV mycobacteria may grow as early as 3 to 7 days. Cultures are at least 10-fold more sensitive than stains.

More recently, radiometric detection of growth in 1 to 2 weeks has been achieved with Middlebrook 7H12 broth, available as BACTEC 12B vials (Becton Dickinson Diagnostic Instrument Systems, Cockeysville, MD). Nonetheless, a culture should be reported as negative only if the Löwenstein-Jensen slants show no growth after 8 weeks of incubation.

DNA probes are used in many laboratories to speciate the mycobacteria. Commercially available probes have been used to identify M. tuberculosis, M. avium complex, Mycobacterium kansasii, and Mycobacterium gordonae, but the sensitivity, reproducibility, and predictive value of these tests remain uncertain. Molecular epidemiology, using DNA fingerprint analysis of M. tuberculosis isolates by restriction fragment length polymorphisms, has been used to characterize tuberculosis transmission patterns in large populations, to document false diagnoses of tuberculosis caused by laboratory cross-contamination, and to distinguish reinfection from relapse.²⁵

Skin testing with purified protein derivative is used to determine tuberculous infection. Tuberculin hypersensitivity is mediated by T lymphocytes and determined in part by the patient's immunologic status. Although a full discussion of skin testing is beyond the scope of this chapter, it must be understood that results based on the amount of induration should not be interpreted simply as positive or negative. The American Thoracic Society and the Centers for Disease Control and Prevention have issued guidelines that take into account various risk factors, including concurrent infection with HIV. Cross-reactivity with nontuberculous mycobacteria can occur. The usefulness of nontuberculous mycobacterial skin tests, especially in patients with ocular disease, is limited.

Diagnosis of ocular leprosy is made by microscopic evaluation of tissues.^7,8 M. leprae is unique in that it loses its acid-fastness by pyridine extraction and has dopa oxidase activity. It grows best at temperatures less than 37°C, in part accounting for its predilection for the cooler areas of the body and its growth in the cold-blooded nine-banded armadillo. Viable M. leprae organisms stain brightly, whereas dead bacilli stain irregularly. The morphologic index, which measures the percentage of solidly staining bacilli in dermal skin smears and tissue sections, provides a measure of viability of the organism. The bacteriologic index, a logarithmic measurement of the numbers of acid-fast bacilli in the dermis, is useful in determining response to therapy. Although M. leprae does not grow in tissue culture, short-term cultures are available that allow determination of antimicrobial sensitivity.²⁶ The lepromin intradermal skin test, which uses heat-killed M. leprae, is not diagnostically useful but is useful for classification. It is positive in tuberculoid patients and some treated lepromatous patients but negative in untreated lepromatous patients and falsely positive in some patients from endemic regions. A positive Fernandez reaction (induration detectable at 24 to 48 hours) indicates delayed-type hypersensitivity to M. leprae. A positive Mitsuda reaction (induration detectable after 3 to 4 weeks) indicates cell-mediated immunity.

Recommendations for the treatment of nontuberculous mycobacterial keratitis are limited by lack of experience and the relatively poor correlation between in vitro sensitivity profiles and clinical response. In general, topical amikacin 1% to 2% solution, one drop every half hour, should be started³¹ and may be supplemented by subconjunctival amikacin (20 mg in 0.5 ml). Topical fluoroquinolones such as ciprofloxacin have shown promise in animal models of M. fortuitum keratitis but are less effective against M. chelonae.¹⁶ Macrolide antibiotics such as clarithromycin have also shown promise because of their intraocular penetration. There are insufficient data to make specific recommendations for keratitis caused by nontuberculous mycobacteria other than M. fortuitum or M. chelonae, or for noncorneal infections. Debridement and local excision are usually indicated; therapeutic penetrating keratoplasty may be necessary in unresponsive keratitis. There is a theoretical concern that the use of ointments on the eye may enhance the virulence of nontuberculous mycobacteria because of reduced phagocytosis.

As with tuberculosis, treatment of ocular leprosy requires systemic therapy, based on the disease type and the degree of resistance.⁷ Dapsone and rifampin are the initial drugs of choice; clofazimine is used in dapsone-resistant disease. Type I lepra reactions should be treated with oral corticosteroids. Thalidomide is the drug of choice for erythema nodosum leprosum but must not be used in women of childbearing age because of teratogenicity. Local ocular complications of leprosy such as keratopathy, trichiasis, iritis, and glaucoma must also be addressed. Topical and oral corticosteroids are helpful in acute iritis but less so in chronic iritis.³²

1. Lederer E: The mycobacterial cell wall. Pure Appl Chem 25:135, 1971

2. Hunter SW, Fujiwara T, Brennan PJ: Structure and antigenicity of the major specific glycolipid antigens of Mycobacterium leprae. J Biol Chem 257:15072, 1982

3. Kaplan G, Gandhi RR, Weinstein DE et al: Mycobacterium leprae antigen-induced suppression of T cell proliferation in vitro. J Immunol 138:3028, 1987

4. Vassalli P: The pathophysiology of tumor necrosis factor. Ann Rev Immunol 10:411, 1992

5. Kaufmann SHE: Immunity to intracellular bacteria. In Paul WE (ed): Fundamental Immunology. 3d ed. New York, Raven Press, 1993

6. Paschal JF, Holland GN, Sison RF et al: Mycobacterium fortuitum keratitis: Clinicopathologic correlates and corticosteroid effects in an animal model. Cornea 11:493, 1992

7. Gelber RH: Leprosy (Hansen's disease). In Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. New York, Churchill-Livingstone, 1995

8. Hussein N, Ostler HB: Hansen disease. In Pepose JS, Holland GN, Wilhelmus KR (eds): Ocular Infection and Immunity. St. Louis, Mosby-Year Book, 1996

9. Smith DW: A guinea pig model of experimental airborne tuberculosis for evaluation of the response to chemotherapy: The effect on bacilli in the initial phase of treatment. Tubercle 72:223, 1991

10. Balasubramanian V, Wiegeshaus EH, Smith DW: Mycobacterial infection in guinea pigs. Immunobiology 191:395, 1994

11. North RJ, Izzo AA: Mycobacterial virulence. Virulent strains of Mycobacterium tuberculosis have faster in vivo doubling times and are better equipped to resist growth-inhibiting functions of macrophages in the presence and absence of specific immunity. J Exp Med 177:1723, 1993

12. Ueda K, Yamazaki S, Yamamoto S, Someya S: Spleen cell transfer induces T cell-dependent granulomas in tuberculous nude mice. J Reticuloendothel Soc 31:469, 1982

13. Dunn PL, North RJ: Virulence ranking of some Mycobacterium tuberculosis and Mycobacterium bovis strains according to their ability to multiply in the lungs, induce lung pathology, and cause mortality in mice. Infect Immun 63:3428, 1995

14. McCune RM, Feldmann FM, McDermott W: Microbial persistence. II. Characteristics of the sterile state of tubercle bacilli. J Exp Med 123:469, 1996

15. Gangadharam PRJ, Perumal VK, Farhi DC, LaBreccque J: The beige mouse model for Mycobacterium avium complex (MAC) disease: Optimal conditions for the host and parasite. Tubercle 70:257, 1989

16. Lin R, Holland GN, Helm CJ et al: Comparative efficacy of topical ciprofloxacin for treating Mycobacterium fortuitum and Mycobacterium chelonae keratitis in an animal model. Am J Ophthalmol 117:657, 1994

17. Kircheimer WF, Storrs EE: Attempts to establish the armadillo (Dasyptus novemcinctus Linn.) as a model for study of leprosy. Int J Lepr 693, 1971

18. Dunn JP, Helm CJ, Davidson PT: Tuberculosis. In Pepose JS, Holland GN, Wilhelmus KR (eds). Ocular Infection and Immunity. St. Louis, Mosby-Year Book, 1996

19. Helm CJ, Holland GN: Ocular tuberculosis. Surv Ophthalmol 38:229, 1993

20. Dutt AK, Moers D, Stead WW: Short-course chemotherapy for extrapulmonary tuberculosis: Nine years' experience. Ann Intern Med 104:7, 1986

21. O'Brien TP, Matoba AY: Nontuberculous mycobacterial diseases. In Pepose JS, Holland GN, Wilhelmus KR (eds): Ocular Infection and Immunity. St. Louis, Mosby-Year Book, 1996

22. Bullington RH Jr, Lanier JD, Font RL: Nontuberculous mycobacterial keratitis: Report of two cases and review of the literature. Arch Ophthalmol 110:519, 1992

23. Sugar J, Hill C: Leprosy. In Mannis MJ, Macsai MS, Huntley AC (eds): Eye and Skin Disease. Philadelphia, Lippincott-Raven, 1996

24. Dana M-R, Hochman MA, Viana MAG et al: Ocular manifestations of leprosy in a noninstitutionalized community in the United States. Arch Ophthalmol 112:626, 1994

25. Ross BC, Raios K, Jackson K, Dwyer B: Molecular cloning of a highly repeated DNA element from Mycobacterium tuberculosis and its use as an epidemiologic tool. J Clin Microbiol 30:942, 1992

26. Franzblau SG: Drug susceptibility testing of Mycobacterium leprae in the BACTEC 460 system. Antimicrob Agents Chemother 33:2115, 1989

27. Centers for Disease Control: Initial therapy for tuberculosis in the era of multidrug resistance: Recommendations of the advisory council for the elimination of tuberculosis. MMWR 42:1, 1993

28. Alwood K, Keruly J, Moore-Rice K et al: Effectiveness of supervised, intermittent therapy for tuberculosis in HIV-infected patients. AIDS 8:1103, 1994

29. Woods AC, Wood R, Senterfit LB: Studies in experimental ocular tuberculosis. Arch Ophthalmol 59:559, 1958

30. Nanda M, Pflugfelder SC, Holland S: Mycobacterium tuberculosis scleritis. Am J Ophthalmol 108:736, 1989

31. Swenson JM, Wallace RJ Jr, Silcox VA, Thornsberry C: Antimicrobial sensitivity of five subgroups of Mycobacterium fortuitum and Mycobacterium chelonae. Antimicrob Agents Chemother 28:807, 1985

32. Esperitu CG, Gelber R, Ostler HB: Chronic anterior uveitis in leprosy: An insidious cause of blindness. Br J Ophthalmol 75:273, 1991