—  SYMPOSIUM #19  —

Mycobacterial Diseases: Past, Present and Future
Moderator: Gary W. Procop

Section 3 - Clinicopathologic classification of Mycobacterium ulcerans disease (Buruli ulcer)

W. M. Meyers, M. Maleombho-Usher, F. M. Abalos, J. Aguiar, R. C. Johnson, M. Debacker, D. S. Walsh, and F. Portaels
Armed Forces Institute of Pathology, Washington DC, USA;
Centre Sanitaire et Nutritionnel Gbemoten, Benin;
Programme National de Lutte contre l'Ulcère de Buruli, Cotonou, Bénin;
Walter Reed Army Institute of Research, Silver Spring, Maryland, USA,
and Institute of Tropical Medicine, Belgium


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Definition and Etiology

M. ulcerans is a slow-growing environmental mycobacterium that infects the skin, subcutaneous tissues, and bone, often giving rise to indolent ulcers. After tuberculosis and leprosy, Buruli ulcer (BU) is the third most common mycobacteriosis. M. ulcerans grows optimally at 32° C and elaborates a necrotizing immunosuppressive cytotoxin called mycolactone. The etiologic agent was isolated in 1948 in Australia by MacCallum et al.

Transmission
Sources of the etiologic agent are closely associated with tropical wetlands. Most likely, human skin becomes contaminated with M. ulcerans by contact with wetland environment (mud, water, plants, fish, etc.) or putatively by insects (waterbugs). Trauma to skin at sites of contamination may introduce the etiologic agent into skin and/or subcutaneous tissue. Size of inoculum, depth of inoculation and virulence of M. ulcerans strain may influence disease form. For example, superficial inoculation may provoke strong cell-mediated immunity (CMI), leading to localized disease; deep inoculation of large numbers of M. ulcerans organisms may lead to early necrosis, limiting local CMI. Innate host variables may determine type of disease; for example, HLA type and immunologic conditioning by other environmental mycobacteria.

Spectrum of M. ulcerans Disease
Based on clinicopathologic studies of several thousand M. ulcerans disease patients accessioned at the AFIP, we propose the following schema for the spectrum of active forms of the disease and their possible natural history:



Clinical features of the forms of M. ulcerans disease include:

Nodular: movable, painless but often pruritic, subcutaneous nodule approximately 2-4 cm in diameter.

Minor ulcerative: small, sharply limited ulcer with slight undermining, self-healing.

Major ulcerative: small to large ulcer, wide undermining, well demarcated but initially with edematous perimeter, with late healing but occasional dissemination.

Contiguous disseminated: small or large edematous indurated plaque that may ulcerate late.

Metastatic: spread from initial lesion to distant site, usually in skin and/or bone.

Note: The early pustular form described in Australia has a natural history similar to major ulcerative forms; however, we have no examples of this type and do not include it in this classification.

Clinicohistopathologic correlations of forms of M. ulcerans disease include:

Nodular: coagulation necrosis of lower dermis and subcutaneous tissue, with acid-fast bacilli (AFB) in center.

Minor ulcerative: ulcer small, AFB in central necrotic slough, with scarring of surrounding dermis.

Major ulcerative: AFB in necrotic base and adjacent areas, with wide undermining.

Contiguous disseminated: widespread contiguous coagulation necrosis, with AFB in panniculus and fascia.

Metastatic: typical changes of M. ulcerans disease at distant skin sites, or M. ulcerans- specific osteomyelitis.

Healing (early organization): loosely arranged lymphocytes, epithelioid and giant cells. AFB scarce or absent.

Healing (late organization): well organized delayed-type hypersensitivity granuloma, and scarring.

Pathogenesis
After inoculation into the skin, M. ulcerans proliferates and elaborates a toxin(s) causing necrosis of dermis, panniculus, and fascia. One well-defined toxin of M. ulcerans is a plasmid related, polyketide-derived macrolide (mycolactone) that is a virulence factor for Buruli ulcer. M. ulcerans promotes apoptosis in lesions. Early lesions are closed, but as necrosis spreads, the overlying skin ulcerates, leaving undermined margins and a necrotic slough in the ulcer base. Histopathologic sections reveal contiguous coagulation necrosis of the deep dermis and panniculus, with destruction of local nerves, appendages, and blood vessels. Clumps of extracellular AFB are plentiful and frequently limited to the base of the ulcer and adjacent necrotic subcutaneous tissue. In early infections and in cellular infiltrates at the periphery of developed lesions, M. ulcerans may be found intracellularly. In contiguous disseminated cutaneous lesions, the M. ulcerans organisms tend to spread along the fascial planes.

The etiologic agent may spread, presumably by lymphatic and hematogenous pathways, to distant foci, usually to skin and bone. Specific, severe osteomyelitis develops in 10% TO 15% of BU patients. Hematogenous spread to bones seems to start at the epiphyseal/metaphyseal level. Histologically there may be local and regional necrotizing lymphadenitis with invasion by M. ulcerans. In active lesions, inflammatory cells are conspicuously few, perhaps as a result of the immunosuppression by the toxin. With healing, there are granulomas, and the ulcer is eventually replaced by a depressed scar.

While information on the topic remains limited, HIV infection may not predispose to M. ulcerans infection, but renders infection with M. ulcerans more aggressive.

Histopathologic Stages of BU
We propose the following histopathologic classification of stages of M. ulcerans infection: I, Necrosis; II, Organizing; III, Healing; IV, Combined stages.

In a histopathologic study of biopsy specimens of skin from more than 1000 patients, using the above staging plan, we obtained the following results in laboratory confirmed lesions (AFB in the specimen, and PCR or culture positive for M. ulcerans): Necrosis 70%, organizing 2%, healing 8%, combined (necrosis, organizing and healing) 20%. Ten percent had osteomyelitis.

Complex grading systems have been proposed for lesions suspected of being M. ulcerans disease, but we prefer a simple 3-tiered system:
  1. Not M. ulcerans disease

  2. Consistent with M. ulcerans disease

  3. M. ulcerans disease

Differential Diagnosis
We have noted the following primary diagnoses in specimens submitted to the AFIP with a clinical impression of, or suspicious for, M. ulcerans disease:

Infections : staphylococcal abscess, tropical phagedenic ulcer, tuberculosis, atypical mycobacteriosis (other than M. ulcerans), Majocchi's granuloma, epididymitis, phycomycosis, phaeohyphomycosis, African histoplasmosis, filariasis, leishmaniasis; necrotizing fasciitis;

Neoplastic conditions:
Malignant: carcinoma of breast, squamous cell carcinoma, basal cell carcinoma, Kaposi's sarcoma, lymphoma, osteosarcoma; Benign: dermatofibroma, lipofibroma, lipoma, leiomyoma, angiolipoma, hidradenoma, pilomatricoma;

Miscellaneous: stasis ulcer, calcinosis cutis, epidermal cyst, gouty tophus, foreign body granuloma.


Specimen Preparation for Histopathologic Diagnosis
Biopsy specimens that include the necrotic base, the undermined edge of lesions, subcutaneous tissue and fascia are optimal for diagnosis. Specimens from nonulcerated disease are best obtained from the presumed center of the lesion. Punch specimens are often nonproductive. Specimens should be approximately 1 cm thick and as long as is convenient. Specimens are best fixed in buffered 10% formalin (4% formaldehyde). If molecular studies are planned, an additional specimen should be fixed in 70% ethanol.

References
  1. Abalos FMV, Aguiar J, Guédénon A, Portaels F, Meyers WM (2000) Mycobacterium ulcerans infection (Buruli ulcer): A case report of the disseminated nonulcerative form. Ann Diagn Pathol 4:386-390

  2. Ablordey A, Swings J, Hubans C, Chemlal K, Locht C, Portaels F, Supply P (2005) Multilocus variable-number tandem repeat typing of Mycobacterium ulcerans. J Clin Microbiol 43:1546-1551

  3. Adusumilli S, Mve-Obiang A, Sparer T, Meyers W, Hayman J, Small PL (2005) Mycobacterium ulcerans toxic macrolide, mycolactone, modulates the host immune response and cellular location of M. ulcerans in vitro and in vivo. Cell Microbiol 7:1295-1304

  4. Aguiar J, Domingo MC, Guédénon A, Meyers W, Steunou C, Portaels F (1997) L'ulcère de Buruli, une maladie mycobactérienne importante et en récrudescence au Bénin. Bull. Séanc Acad Roy Sci Outre-Mer 43:325-358

  5. Amofah G, Bonsu F, Tetteh C, Okrah J, Asamoa K, Asiedu K, Addy J (2002) Buruli ulcer in Ghana : results of a national case search. Emerg Infect Dis 8:167-170

  6. Bretzel G, Siegmund V, Racz P, van Vloten F, Ngos F, Thompson W, Biason P, Adjei O, Fleischer B, Nitschke J (2005) Post-surgical assessment of excised tissue from patients with Buruli ulcer disease: progression of infection in macroscopically healthy tissue. Trop Med Int Health 10:1199-1206

  7. Chemlal K, de Ridder K, Fonteyne PA, Meyers WM, Swings J, Portaels F (2001) The use of IS2404 restriction fragment length polymorphisms suggests the diversity of Mycobacterium ulcerans from different geographical areas. Am J Trop Med Hyg 64:270-273

  8. Connor DH, Lunn F (1966) Buruli ulceration. A clinicopathologic study of 38 Ugandans with Mycobacterium ulcerans infection. Arch Pathol 81:183-189

  9. Connor DH, Meyers WM, Krieg RE (1976) Infection by Mycobacterium ulcerans. In, Binford CH, Connor DH (eds), Pathology of Tropical and Extraordinary Diseases. Washington DC: Armed Forces Institute of Pathology. Vol I, pp 226-235

  10. Debacker M, Aguiar J, Steunou C, Zinsou C, Meyers WM, Guédénon A, Scott JT, Dramaix M, Portaels F (2004) Trends in Mycobacterium ulcerans disease (Buruli ulcer) patients as seen in a rural hospital of southern Benin, 1997-2001. Emerg Infect Dis 10:1391-1398

  11. Debacker M, Aguiar J, Steunou C, Zinsou C, Meyers WM, Portaels F (2005) Buruli ulcer recurrence, Benin . Emerg Infect Dis 11:584-589

  12. Debacker M, Aguiar J, Steunou C, Zinsou C, Meyers WM, Scott JT, Dramaix M, Portaels F (2004) Mycobacterium ulcerans disease: role of age and gender in incidence and morbidity. Trop Med Int Health 9:1297-1304

  13. Eddyani M, Adjei O, Teugels G, De Weirdt D, Boakye D, Meyers WM, Portaels F (2004) Potential role for fish in transmission of Mycobacterium ulcerans disease (Buruli ulcer): An environmental study. Appl Environ Microbiol 70:5679-5681

  14. Etuaful S, Carbonnelle B, Grosset J et al (2005) Efficacy of the combination rifampin- streptomycin in preventing growth of Mycobacterium ulcerans in early lesions of Buruli ulcer in humans. Antimicrob Agents Chemother 49:3182-3186

  15. Evans MRW, Mawdsley J, Bull R, Lockwood DNJ, Thangaraj H, Shanahan D, Rajakula-singam K (2003) Buruli ulcer in a visitor to London. Br J Dermatol 149:907-909

  16. Fenner F (1974) The "Bairnsdale bacillus": Mycobacterium ulcerans. Papua New Guinea Med J 17:127-128

  17. George KM, Chatterjee D, Gunawardana G, Welty D, Hayman J, Lee R, Small PLC (1999) Mycolactone: A polyketide toxin from Mycobacterium ulcerans required for virulence. Science 283:854-857

  18. Gooding TM, Johnson PDR, Campbell DE, Hayman JA, Hartland EL, Kemp AS , Robins-Browne RM (2001) Immune response to infection with Mycobacterium ulcerans. Infect Immun 69:1704-1707

  19. Guarner J, Bartlett J, Spotts Whitney EA, Raghunathan PL, Stienstra Y, Asamoa K, Etuaful S, Klutse E, Quarshie E, van der Werf TS, van der Graaf WTA, King CH, Ashford DA (2003) Histopathologic features of Mycobacterium ulcerans infection. Emerg Infect Dis 9:651-656

  20. Hayman J (1993) Out of Africa : observations on the histopathology of Mycobacterium ulcerans infection. J Clin Pathol 46:5-9

  21. Horsburgh CR Jr, Meyers WM (1997) Buruli ulcer. In, Pathology of Emerging Infections. Horsburgh CR Jr, Nelson AM (eds). Washington DC: American Society for Microbiology. Chapter 7, pp 119-134

  22. Huygen K (2003) Prospects for vaccine development against Buruli disease. Expert Rev Vaccines 2:561-569

  23. Johnson PD, Stinear T, Small PL et al (2005) Buruli ulcer (M. ulcerans infection): new insights, new hopes for disease control. PLoS Med 2:e108

  24. Johnson RC, Ifebe D, Hans-Moevi A, Kestens L, Houessou R, Guédénon A, Meyers WM, Portaels F (2002) Disseminated Mycobacterium ulcerans disease in an HIV-positive patient: a case study. AIDS 16:1704-1705

  25. Johnson RC, Sopoh GE, Boko M, Zinsou C, Gbovi J, Makoutode M, Portaels F (2005) Distribution of Mycobacterium ulcerans (Buruli ulcer) in the district of Lalo in Benin . Trop Med Int Health 10:863-871 (in French)

  26. Kiszewski AE, Becerril E, Aguilar LD, Kader ITA, Meyers WM, Portaels F, Hernàndez Pando R (2006) The local immune response in ulcerative lesions of Buruli disease. Clin. Expl. Immunol. 143:445-451

  27. MacCallum P, Tolhurst JC, Buckle G, Sissons HA (1948) A new mycobacterial infection in man. J Pathol Bacteriol 60:93-122

  28. Marsollier L, Aubry J, Coutanceau E et al (2005) Colonisation of the salivary glands of Naucoris cimcioides by Mycobacterium ulcerans requires host plasmocytes and a macrolide toxin, mycolactone. Cell Microbiol 7:935-943

  29. Marsollier L, Honore N, Legras P et al (2003) Isolation of three Mycobacterium ulcerans strains resistant to rifampin after experimental chemotherapy of mice. Antimicrob Agents Chemother 47:1228-1232

  30. Marsollier L, Robert R, Aubry J, Saint Andre JP, Kouakou H, Legras P, Manceau AL, Mahaza C, Carbonnelle B (2002) Aquatic insects as a vector for Mycobacterium ulcerans. Appl Environ Microbiol 68:4623-4628

  31. Meyers WM (1995) Mycobacterial infections of the skin. In, Doerr W, Seifert G (eds). Tropical Pathology, 2nd ed. Berlin: Springer-Verlag. Vol 8, pp 291-377

  32. Meyers WM, Portaels F (2006) Mycobacterium ulcerans infection (Buruli ulcer). In, Tropical Infectious Diseases: Principles, Pathogens, and Practice. 2nd ed. (two volumes). Guerrant, Walker & Weller, eds. New York: Churchill Livingstone (Elsevier). Vol. 1, Chapter 37, pp. 428-435

  33. Meyers WM, Shelly WM, Connor DH (1974) Heat treatment of Mycobacterial ulcerans infections without surgical excision. Am J Trop Med Hyg 23:924-929

  34. Meyers WM, Shelly WM, Connor DH, Meyers EK (1974) Human Mycobacterium ulcerans infections developing at sites of trauma to skin. Am J Trop Med Hyg 23:919-923

  35. Meyers WM, Tignokpa N, Priuli GB, Portaels F (1996) Mycobacterium ulcerans infection (Buruli ulcer): first reported patients in Togo. Br J Dermatol 134:1116-1121

  36. Mve-Obiang A, Lee RE, Portaels F, Small PLC (2003) Heterogeneity of mycolactone toxins produced by Mycobacterium ulcerans: Implications on virulence. Infect Immun 71:774-783

  37. Mve-Obiang A, Lee RE, Umstot ES et al (2005) A newly discovered mycobacterial pathogen isolated from lethal infections in laboratory colonies of Xenopus species produces a novel form of the M. ulcerans macrolide toxin, mycolactone. Infect Immun 73:3307-3312

  38. Novales-Santa Coloma J, Navarrete-Franco G, Iribe P, López-Cepeda LD (2005) Ulcerative cutaneous mycobacteriosis due to Mycobacterium ulcerans: Report of two Mexican cases. Int J Lepr Other Mycobact Dis 73:5-12

  39. Pahlevan AA, Wright DJM, Andrews C, George KM, Small PLC, Foxwell BM (1999) The inhibitory action of Mycobacterium ulcerans soluble factor on monocyte/T cell cytokine production and NF-kB function. J Immunol 163:3928-3935

  40. Phillips R, Adjei O, Lucas S, Benjamin N, Wansbrough-Jones M (2004) Pilot randomised double-blind trial of treatment of Mycobacterium ulcerans disease (Buruli ulcer) with topical nitrogen oxides. Antimicrob Agents Chemother 48:2866-2870

  41. Pimsler M, Sponsler TA, Meyers WM (1988) Immunosuppressive properties of the soluble toxin from Mycobacterium ulcerans. J Infect Dis 157:577-580

  42. Portaels F, Aguiar J, Debacker M, Guédénon A, Steunou C, Zinsou C, Meyers WM (2004) Mycobacterium bovis BCG vaccination as prophylaxis against Mycobacterium ulcerans osteomyelitis in Buruli ulcer disease. Infect. Immun. 72: 62-65

  43. Portaels F, Chemlal K, Elsen P, Johnson PDR, Hayman JA, Kirkwood R, Meyers WM (2001) Mycobacterium ulcerans in wild animals. Rev Sci Tech Off Int Epiz 20: 252-264

  44. Portaels F, Elsen P, Guimaraes-Peres A, Fonteyne PA, Meyers WM (1999) Insects in the transmission of Mycobacterium ulcerans infection. Lancet 353:986

  45. Pszolla N, Sarkar MR, Strecker W, Kern P, Kinzl L, Meyers WM, Portaels F (2003). Buruli ulcer: a systemic disease. Clin. Infect. Dis. 37:e78-82

  46. Raghunathan PL, Whitney EA, Asamoa K et al (2005) Risk factors for Buruli ulcer disease (Mycobacterium ulcerans infection): results from a case-control study in Ghana . Clin Infect Dis 40:1445-1453

  47. Read JK, Heggie CM, Meyers WM, Connor DH (1974) Cytotoxic activity of Mycobacterium ulcerans. Infect Immun 9:1114-1122

  48. Rondini S, Horsfield C, Mensah-Quainoo E, Junghanss T, Lucas S, Pluschke G Contiguous spread of Mycobacterium ulcerans in Buruli ulcer lesions analysed by histopathology and real-time PCR quantification of mycobacterial DNA. J Pathol (2005 in press, 10 pages)

  49. Rondini S, Mensah-Quainoo E, Troll H, Bodmer T, Pluschke G (2003) Development and application of real-time PCR assay for quantification of Mycobacterium ulcerans DNA. J Clin Microbiol 41:4231-4237

  50. Siegmund V, Adjei O, Racz P et al (2005) Dry-reagent-based PCR as a novel tool for laboratory confirmation of clinically diagnosed Mycobacterium ulcerans associated disease in areas in the tropics where M. ulcerans is endemic. J Clin Microbiol 43:271-276

  51. Sizaire V, Nackers F, Comte E, Portaels F (2006) Mycobacterium ulcerans infection: control, diagnosis, and treatment. Lancet Infect Dis 6:288-296

  52. Smith PG, Revill WD, Lukwago E, Rykushin YP (1976) The protective effect of BCG against Mycobacterium ulcerans disease: a controlled trial in an endemic area of Uganda. Trans R Soc Trop Med Hyg 70:449-457

  53. Stienstra Y, van der Graaf WTA, Asomoa K, van der Werf TS (2002) Beliefs and attitudes toward Buruli ulcer in Ghana. Am J Trop Med Hyg 67:207-213

  54. Stinear T, Davies JK, Jenkin GA, Hayman JA, Oppedisano F, Johnson PDR (2000) Identification of Mycobacterium ulcerans in the environment from regions in Southeast Australia in which it is endemic with sequence-capture PCR. Appl Environ Microbiol 66:3206-3213

  55. Stinear T, Mve-Obiang A, Small PLC, Frigui W, Pryor MJ, Brosch R, Jenkin GA, Johnson PD, Davies JK, Lee RE, Adusumilli S, Garnier T, Haydock SF, Leadlay PF, Cole ST (2004) Giant plasmid-encoded polyketide syntheses produce the macrolide toxin of Mycobacterium ulcerans. Proc Natl Acad Sci USA 101:1345-1349

  56. Stinear T, Ross SC, Davies JK, Marino L, Robins-Browne RM, Oppedisano F, Sievers A, Johnson PD (1999) Identification and characterization of IS2404 and IS2606: Two distinct repeated sequences for detection of Mycobacterium ulcerans by PCR. J Clin Microbiol 37:1018-1023

  57. Stragier P, Ablordey A, Bayonne LM, Lugor YL, Sindani IS, Suykerbuyk P, Wabinga H, Meyers WM, Portaels F (2006) Heterogeneity among Mycobacterium ulcerans isolates from Africa. (Dispatches.) Emerg Infect Dis 12:844-847

  58. Stragier P, Ablordey A, Meyers WM, Portaels F (2005) Genotyping Mycobacterium ulcerans and Mycobacterium marinum by using mycobacterial interspersed repetitive units. J Bacteriol 187:1639-1647

  59. Tanghe A, Content J, Van Vooren JP, Portaels F, Huygen K (2001) Protective efficacy of a DNA vaccine encoding antigen 85A from Mycobacterium bovis BCG against Buruli ulcer. Infect Immun 69:5403-5411

  60. Thangaraj HS, Phillips RO, Evans MR, Wansbrough-Jones MH (2003) Emerging aspects of Buruli ulcer. Expert Rev Anti Infect Ther 1:217-222

  61. Toll A, Gallardo F, Ferran M, Gilaberte M, Iglesias M, Gimeno JL, Rondini S, Pujol RM (2005) Aggressive multifocal Buruli ulcer with associated osteomyelitis in an HIV- positive patient. Clin Exp Dermatol 30:649-651

  62. Trott KA, Stacy BA, Lifland BD, Diggs HE, Harland RM, Khokha MK, Grammer TC, Parker JM (2004) Characterization of a naturally occurring infection of a Myco- bacterium ulcerans -like strain in a colony of African tropical clawed frogs (Xenopus tropicalis). Comp Med 54:309-317

  63. Wagner D, Young LS (2004) Nontuberculous mycobacterial infections: a clinical review. Infection 32:257-270

  64. Wansbrough-Jones M, Phillips R (2006) Buruli ulcer: emerging from obscurity. Lancet 367:1849-1858

  65. WHO. Fact sheet number 199. (accessed 2006) Mycobacterium ulcerans infection. http://www.who.int/mediacentre/factsheets/fs199/en/print.html