Buruli ulcer - Diagnosis of Mycobacterium ulcerans disease
Histopathological methods for diagnosis
Objectives
This chapter will assist you to understand the various histopathological methods for the diagnosis of Mycobacterium ulcerans disease.
What you should know
A detailed history and description of the lesion that has been excised is very important for a meaningful evaluation and for archival purposes. Name, age, sex, laboratory or hospital number and site of lesion are absolutely essential.
Selection of site for biopsy specimen
Excisional specimens are advised. Specimens taken by punch are often unsatisfactory.
Non-ulcerative lesions
Specimens should be obtained from the presumed centre of the lesion and include all levels of the skin and subcutaneous tissue down to fascia.
Ulcerative lesions
Specimens should be taken from the edge of the ulcer and include the entire thickness of the skin and subcutis down to fascia.
Fixation of tissue
Optimally, the tissue should be fixed in neutral or buffered 10% formalin (pH 7.4). Ideally, the tissue should be fixed in a volume of formalin 10 times the volume of tissue for at least 24 hours before shipping. After fixation, the tissue can be shipped in smaller volumes of fixative. Care should be taken to identify the tissue with permanent markings on the container label. Bone must be decalcified before sectioning.
Preparation of histopathological sections
Routine processing of fixed tissue is sufficient. Sections should be cut at 4-5 microns and stained by: 1) haematoxylin and eosin; 2) Ziehl-Neelsen for AFB; 3) Grocott methenamine-silver for fungi; and 4) tissue Gram’s stain for other bacteria (see Annex 6). Other stains are employed as indicated.
Gross changes
Surface changes of non-ulcerated lesions often show loss of topographic markings and discolouration. Cut sections show changes in colouration, necrosis and mineralization. Lymph nodes show soft greyish-tan cut surfaces. After decalcification, cut sections of bone show yellowish necrosis of the marrow and often, thinning of the cortex.
Histopathological changes
Skin changes
Necrotic (active) stage: non-ulcerated lesions - The epidermis is intact, but is often hyperplastic. The upper dermis is usually intact but may show various stages of degeneration with infiltration of small numbers of inflammatory cells. There is contiguous coagulation necrosis of the lower dermis, subcutaneous tissue and underlying fascia (Fig. 17–19). There is oedema with remarkably few inflammatory cells, unless the lesion is infected secondarily by pyogenic bacteria. Adipose cells swell, but may lose their nuclei and retain their cell wall (fat cell ghosts—Fig. 20).
Vasculitis is common in the subcutaneous tissue, often with occlusion of vessels by thrombi (Fig. 21 and Fig. 22).
Varying degrees of mineralization are seen, especially in African patients. The ZN stain classically reveals large numbers of extracellular acid-fast bacilli (AFB); often in clusters and confined to the necrotic areas (Fig. 23).
Most bacilli are in the deeper areas of the specimen but may invade the interstitium of the adipose tissue and lobular septa of the subcutaneous tissue (Fig. 24).
Continuing necrosis of the dermis usually leads to degeneration of the epidermis and ultimate ulceration. Necrosis, however, may spread laterally with proliferation of AFB in the subcutaneous tissue and fascia (Fig. 25). Ulceration of the epidermis in such cases is often a very late event. The spread of disease in this manner leads to the plaque and oedematous forms of the disease.
Necrotic (active) stage: ulcerative lesions - Ulcers are undermined with reepithelialization of the edges of the lesion and undersurface of the overlying flap of the dermis (Fig. 26). Adjacent epidermis is usually hyperplastic. The base of the pristine ulcer contains a necrotic slough of cellular debris and fibrin, sometimes with a central eschar. There is contiguous coagulation necrosis of the subcutaneous tissue and fascia similar to that described for non-ulcerated lesions (Fig. 25 and Fig. 27). AFB are located in the base of the central slough and necrotic subcutaneous tissue. The disease rarely extends into underlying muscle. Vasculitis and mineralization are seen often (Fig. 21).
Organizing (early granulomatous stage) - Early healing is characterized by a poorly organized granulomatous response in the dermis and subcutaneous tissue (Fig. 28). The granulomatous infiltration comprises swollen macrophages (epithelioid cells), Langhans’ giant cells and lymphocytes. These eventually form organized tuberculoid granulomas. Foamy macrophages, lymphocytes and plasma cells are sometimes seen at the margin of necrotic fat. AFB are scarce or absent.
Healing stage - As healing advances, granulation tissue forms followed by fibrosis and a depressed scar (Fig. 29 and Fig. 30). AFB are seldom seen.
Lymph nodes
Although clinical lymphadenopathy is rarely appreciated, significant lymphadenitis is often seen histopathologically, both in lymph nodes adjacent to lesions and in regional nodes. Those adjacent to lesions may show marked invasion of the capsule by AFB (Fig. 31). The parenchyma is often markedly necrotic with destruction of cortical lymphoid tissue (Fig. 32). In such cases the entire node may be invaded by AFB. Regional lymph nodes, however, may show only sinus histiocytosis. Granulomatous changes are usually not seen, and AFB are rarely seen in regional nodes.
Bone changes
Bone may be affected by direct extension from an overlying lesion of Buruli ulcer, or at a site distant from recognized lesions, presumably by haematogenous spread of M. ulcerans (Fig. 33). Histopathologically, the marrow is extensively necrotic and the bone trabeculae are eroded (Fig. 34). AFB are present in varying numbers, most often in the necrotic marrow (Fig. 35 and Fig. 36). Although some lesions in bone seem to be purely an effect of the M. ulcerans in the bone, approximately 50% of the osteomyelitic lesions are coinfected by pyogenic organisms such as streptococci, staphylococci and Corynebacterium sp. In such instances, there is suppuration and the organisms may be visible in Gram’s stained sections. Well formed granulomas may develop producing a chronic osteomyelitis that is probably caused by M. ulcerans.
Patients with extensive disease (comment): Patients with aggressive oedematous lesions involving large body areas often have widespread oedema and impaired renal function, or other evidence suggesting visceral organ involvement. Such patients sometimes die early in the course of the disease. While some authorities suspect that these events are attributable to a systemic effect of the toxin, this question can only be resolved by increased efforts to study the pathophysiology of such patients and by the study of autopsy specimens.
Notes on interpretation of laboratory tests
As with all laboratory tests, the quality of the results produced depends on the quality and prompt delivery of the samples. It is not advisable to conclude that a patient does not have Buruli ulcer even if all tests are negative. Such situations may arise if biopsies or swabs are taken from areas where no organisms are present, or transport times are prolonged.
For example, in one series, 500 patients were considered to have proven Buruli ulcer using a strict definition of having at least two of the following tests positive: culture, PCR, histology or ZN. When each diagnostic method was considered alone, the following sensitivities were obtained: ZN 40–80%, culture 20–60%, histology > 90%, PCR > 90%. ZN and culture in particular are dependent on the type of clinical lesion. For example ZN sensitivity for nodules was 40%, for ulcers 60% and plaques 80%. For culture, bone specimens were positive in only 20% of cases, 50% for ulcers and 60% for plaques. Sensitivity of culture may be further improved by initial passage in mice (up to 75%). Clearly it is not advisable to exclude the diagnosis or to conclude that the patient has Buruli ulcer based on any one laboratory test.
Although uncommon, false positive ZN or PCR results or even false positive culture results have occurred. If the result from the laboratory does not fit with the clinical presentation, or is questioned by the clinician, laboratory tests should be repeated on freshly collected specimens.
Ideally, results obtained using several modalities and multiple samples should be considered together. However, in practice, in endemic areas, experienced clinicians commonly make accurate presumptive diagnosis on clinical grounds alone, or by using a combination of clinical appearances and a ZN stained smear.