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Histopathology of the lepromatous skin biopsy
Clinics in Dermatology (2015) 33, 38–45
Histopathology of the lepromatous skin biopsy Cesare Massone, MD a,⁎, Workalemahu Alemu Belachew, MD b , Antonio Schettini, MD c a
Department of Dermatology, Medical University of Graz, Graz, Austria Mekelle University, College of Health Science, Mekelle, Ethiopia c Fundation Alfredo Da Matta, Manaus, Amazonas, Brazil b
Abstract The histopathology of lepromatous skin varies according to the cell-mediated immunity of the host against Mycobacterium leprae. In tuberculoid and borderline tuberculoid leprosy, epithelioid noncaseating granulomas predominate, and acid-fast bacilli (AFB) are absent or only rarely present. In borderline lepromatous and lepromatous leprosy, the infiltrate is composed of macrophages with a vacuolar cytoplasm, lymphocytes, and plasma cells. AFB are numerous. Edema inside and outside the epithelioid granulomas, together with the appearance of large giant cells, are the main features of type 1 reactions. A conspicuous neutrophilic infiltrate in the subcutis with or without vasculitis is found in erythema nodosum leprosum. The main histopathologic features of leprosy and its particular forms are discussed in this review. © 2015 Elsevier Inc. All rights reserved.
The main feature of the vast majority of leprosy biopsy specimens is a granulomatous infiltrate that has different features according to the form of leprosy, the time and site of the biopsy, the presence of a leprosy reaction, and therapy. The clinical spectrum of leprosy correlates in most of cases (but not in all) directly with histopathologic results, reflecting the different grade of cell-mediated immune response (CMI) of the host against Mycobacterium leprae.1–4 Although leprosy is a routine diagnosis and well-known issue in endemic areas, recognition may become difficult in nonendemic areas, where leprosy may be misdiagnosed as many different inflammatory dermatoses. In fact, due to increased immigration from endemic areas, leprosy cases might be more commonly observed in nonendemic areas, such as Europe.5 To formulate a correct diagnosis and classification of leprosy, three cardinal points are needed. First, histopathologic diagnosis of leprosy is impossible without exact
⁎ Corresponding author. Tel.: +43 316 385 13235; fax: +43 316 385 14957. E-mail address: [email protected] (C. Massone). http://dx.doi.org/10.1016/j.clindermatol.2014.10.003 0738-081X/© 2015 Elsevier Inc. All rights reserved.
clinical data, such as anamnesis (origin or travels to endemic countries; contact with leprosy patients; household leprosy cases); number, distribution, and type of the lesions; presence or absence of anesthesia; therapies; and other pertinent signs and symptoms (e.g., of type 1 or type 2 reaction). The final diagnosis and classification of a leprosy patient in a referral center should always be formulated on a detailed clinical-pathologic correlation. Second, the skin biopsy has to be correctly performed: It must be deep (at least 4 mm punch biopsy including part of the subcutaneous fat) and without artifacts (squeeze).6 Lastly, a representative part of the lesion must be biopsied. This means that in indeterminate leprosy (IL) the center of a small hypopigmented lesion (or better, the center of the anesthetic area) or the border of an annular macule has to be biopsied. In tuberculoid (TT), borderline tuberculoid (BT), and borderline borderline leprosy (BB) the infiltrated margins are diagnostic; in borderline lepromatous (BL) and lepromatous leprosy (LL), the center of a macule, plaque, or nodule. If a type 1 reaction (T1R) is suspected, the most edematous and infiltrated area of the lesion has to be biopsied, whereas in cases of erythema nodosum leprosum (ENL) the biopsy must be deep to include the subcutaneous fat.6,7
Histopathology of the lepromatous skin biopsy Hematoxylin and eosin (H&E) and Fite-Faraco (FF) stains must be performed on each biopsy specimen by an experienced laboratory technician. Serial sections are sometimes needed. Immunohistochemistry of the lymphocytic infiltrate is never necessary for the diagnosis. Polymerase chain reaction (PCR) for M. leprae is useful only in a limited number of cases. FF is the method of choice for staining AFB; control sections should be used; and “whenever there is doubt, there should be no hesitation in staining further sections.”1–4 There are several recipes for modified stain for AFB.1–4,6 AFB are assessed on histopathologic sections exactly in the same way as in the smear, according to the following logarithmic indices (bacterial index of granuloma [BIG]): 0: Absence of bacilli 1 +: 1-10 bacilli in 100 high-power fields (HPFs) 2 +: 1-10 bacilli in 10 HPFs 3 +: 1-10 bacilli for HPFs 4 +: 10-100 for HPFs 5 +: 100-1000 for HPFs 6 +: Many clumps, “globi” (N 1000 bacilli) Unfortunately, the Ridley-Jopling clinical classification does not always correlate with the histologic features, and in some cases, there is a discrepancy between clinical and histopathologic classification. Different authors found an overall agreement between clinical type and histologic type, 58% and 70%, respectively, with the major disparity observed in borderline cases.1–4
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Tuberculoid leprosy The granulomatous infiltrate is more often multifocal, with a typical periadnexal (with involvement of the arrector pili muscle and sweat glands) and perineural distribution, both in the superficial and deep portions of the dermis. Skin adnexa may be also infiltrated and destroyed by the granulomas (Figure 4). Extension of the infiltrate to the subcutis is possible. The granulomas are noncaseating and formed by mature epitheliod cells, “encapsulated” by many lymphocytes (Figures 1 and 2). Numerous large Langhans cells are pathognomonic. Multinucleated giant cells may be present, whereas plasma cells are absent. Fibrin in the center of the granuloma is not observed. The epidermis is often atrophic and “eroded” by the granulomas present in the superficial dermis. There is no clear subepidermal zone. Nerves are enlarged and swollen. The perinerium is intact and may be surrounded by lymphocytes. In other cases, nerves are infiltrated by the granulomas, and they may be destroyed beyond recognition. Rarely, a patch of fibrinoid necrosis may be observed. A careful search may reveal few remnants of nerve tissue in the granuloma. Some authors report that S100 stain is superior to H&E in identifying nerve fragmentation in TT, but S100 staining is usually not needed. AFB are usually absent (AFB: 0 to 1 +) and PCR is negative in most cases.1–4 The main differential diagnoses are sarcoidosis, tuberculosis, leishmaniasis, and secondary syphilis. The presence of plasma cells should suggest consideration of leishmaniasis. The presence of prominent epidermal hyperplasia favors deep mycoses, tuberculosis, and leishmaniasis.9,10
Histopathologic patterns of leprosy The inflammatory cells present in leprosy lesions are epithelioid cells, macrophages, lymphocytes, plasma cells, and in specific cases also neutrophils and mast cells.8 According to CMI response to M. leprae, different types of granulomatous reaction can be observed. Epithelioid cells are usually seen in TT and BT, whereas foamy macrophages characterize the infiltrate of BL and LL. The infiltrate can often “touch” the epidermis in TT, but only rarely in BT, and typically spares the epidermis in BB, BL, and LL. Rarely the infiltrate may be found in the superficial dermis only; more usually it involves all the dermis and sometimes also the subcutis. Some cases may be characterized by a prominent lymphocytic infiltrate, resulting in a diagnostic pitfall. In each biopsy the nerves have to be carefully checked. The presence of AFB inside the nerve is diagnostic of leprosy. Although the polar forms TT and LL are quite easily diagnosed and classified, borderline cases may sometimes present overlapping features. For this reason, combinations of symbols, such as TT-BT, BT-BB, BB-BL and BL-LL, are used for those cases that show intermediate features among two groups.1–4
Fig. 1 Tuberculoid (TT). Multifocal granulomatous infiltrate in the superficial and deep dermis, periadnexal and perineural. The granulomas are focally in contact with the epidermis.
40
Fig. 2 Tuberculoid (TT) granulomas are characterized by noncaseating mature epithelioid cells and are surrounded by many lymphocytes.
Borderline tuberculoid leprosy BT presents a similar infiltrate to TT. The main differences reside in the lesser maturation of epithelioid cells and in particular the presence of the lymphocytes within and not only around the granulomas; moreover, granulomas are less organized and tubercle formation is less prominent (Figure 3). Undifferentiated, medium-sized giant cells are typical of BT. The epidermis may or may not be “eroded” by granulomas. Nerves typically are moderately swollen and have perineural and intraneural granulomas. The perineurium is infiltrated by lymphocytes. AFB are 0 to 2 +. PCR is positive in almost half of cases.1–4 The main differential diagnoses are sarcoidosis, tuberculosis, leishmaniasis, and secondary syphilis.9,10
Borderline borderline leprosy BB is also characterized by granulomas with immature epithelioid cells but without the formation of well-defined
Fig. 3 In borderline tuberculoid (BT) granulomas the epithelioid cells are less mature than in BT and lymphocytes are not only around but also infiltrate the granulomas.
C. Massone et al.
Fig. 4 Borderline lepromatous (BL). Macrophage granulomas that infiltrate the superficial and deep dermis, also with periadnexal and perineural distribution, but that are always separated by the epidermis by a typical narrow zone (Unna band).
epithelioid granulomas. Giant cells are not present, and lymphocytes are diffusely distributed. Macrophages represent a fairly significant proportion of cells. There is often edema. The epidermis is atrophic. The subepidermal zone is not involved. Nerves are not swollen but are infiltrated by epithelioid cells and lymphocytes and partly destroyed. A lamination of the perineurium (reactive proliferation of perineural cells) may be observed. AFB are 3 to 4 +. PCR is positive in only almost all cases.1–4
Borderline lepromatous leprosy The infiltrate in BL is characterized by the presence of macrophages (so-called macrophage granuloma) admixed with lymphocytes. In some cases, lymphocytes predominate over macrophages (Figures 4-7). A solitary clump of
Fig. 5 Borderline lepromatous (BL). The macrophages have small vacuoles in the cytoplasm, and there are several lymphocytes invading the granuloma. Plasma cells are present.
Histopathology of the lepromatous skin biopsy
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Fig. 6 Borderline lepromatous (BL). In some cases lymphocytes predominate over macrophages. In these cases the diagnosis might be a challenge.
epithelioid cells among the macrophages may be found. The infiltrate can be diffuse, patchy nodular, perivascular, or periadnexal but always separated from the epidermis by a typically narrow zone (Unna band) of collagen. Macrophages have a more or less prominent foamy cytoplasm, especially in regressing lesions, but no large vacuoles are usually found. Plasma cells are present. Nerves have an onion-skin perineurium with lymphocytes forming a cuff around a nerve bundle. AFB (4 to 5 +) are present inside macrophages, Schwann cells, endothelial cells, and arrector pili muscles.1–4
Lepromatous leprosy
Fig. 8 Lepromatous leprosy (LL). Patchy nodular infiltrate of macrophages in the all dermis without granuloma formation. A clear grenz zone in the superficial dermis between the infiltrate and the epidermis is typically present.
LL is characterized by collections and sheets of macrophages diffusely distributed in the dermis (but also a “patchy nodular” arrangement can be found). Lymphocytes and plasma cells are diffusely scattered or in a clump. There are no epithelioid cells. The epidermis is atrophic, and a clear grenz zone (Unna band) in the superficial dermis between the infiltrate and the epidermis is typically present (Figure 8). Skin adnexa are surrounded by macrophages and are
atrophic. The infiltrate can extend to the deep dermis and subcutaneous fat. Macrophages present as a gray cytoplasm with foamy changes (lepra cells, Virchow cells; Figure 9) in variable amount from mild foamy appearance in early lesions to large vacuoles in older or regressing lesions (occurring also in multinucleated giant cells; Figure 10).
Fig. 7 Borderline lepromatous (BL) (same case as Figure 6). A predominately lymphocytic infiltrate is observed also around the eccrine glands and nerves.
Fig. 9 Lepromatous leprosy (LL). Macrophages present a gray cytoplasm with mild foamy changes (lepra cells, Virchow cells in early lesions). Plasma cells are present.
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C. Massone et al.
Fig. 10 Lepromatous leprosy (LL). In older or regressing lesions, large vacuoles appear.
Fig. 12 Acid-fast bacilli (AFB) are granular or fragmented in patients while receiving or shortly after multidrug therapy (MDT).
Nerves have a perineural collection of macrophages and may have an onion-skin perineurium but without significant infiltration, or they may be fairly normal. Nerves may be somewhat hyaline or fibrosed but not swollen. Numerous AFB (5-6 +) are present within macrophages, sweat and sebaceous glands, hair follicles, arrector pili muscles, Schwann cells, perineural cells, and vascular endothelium. AFB may be arranged in parallel array, or forming clusters, or densely packed in large masses known as “globi” (Figure 11). AFB have a solid appearance in untreated patients, whereas they are granular or fragmented in patients during or shortly after multidrug therapy (MDT; Figure 12).PCR is obviously positive in both LL and BL and is not necessary for the diagnosis.1–4 The histopathologic differential diagnoses of both LL and BL mainly include other diseases that produce foamy macrophages, such as xanthomas and xanthogranulomas, post–kala-azar dermal leishmaniasis, paraffinoma, and rarely infections caused by other nontuberculous (atypical) mycobacteria.9,10
Indeterminate leprosy Indeterminate leprosy (IL) is characterized by a superficial or more often superficial and deep perivascular and periadnexal lymphohistiocitic infiltrate. In some cases, the infiltrate is more prominent around the adnexa (sweat and sebaceous glands, hair follicles) than around the vessels. Mast cells are increased, whereas granulomas are absent. The epidermis may present with focal vacuolar degeneration of basal keratinocytes and exocytosis of lymphocytes. A perineural infiltrate can be seen in one third of cases, and lymphocytes “cuffing” the nerve fibrils are a good hint for the diagnosis. Nerves, however, are not enlarged. AFB are difficult to find, and FF serial sections are needed to find single or small groups of AFB in a nerve. The subepidermal zone just beneath the basal layer and the arrector pili muscles are sites where AFB might be also found. PCR is positive only in a small proportion of cases, being in these cases of some help for the diagnosis.1–4,11 Diagnosis of IL without detailed clinical information is not possible, because the histopathologic differential diagnosis includes the large group of dermatides with a perivascular and periadnexal lymphohistiocytic infiltrate.9,10
Leprosy reactions Type 1 reaction
Fig. 11 Lepromatous leprosy (LL). Numerous solid acid-fast bacilli (AFB) are present solitarily or in globules.
T1R occur mainly in BT and BB and sometimes also in BL patients. On the background of a BT, BB, or BL, histopathologic result, the histologic features that characterize T1R are the presence of edema in the superficial dermis and edema in the granuloma with disorganization of the granuloma, the appearance of foreign body giant cells (sometimes with vacuoles due to intracellular edema), large Langhans giant cells with epidermal erosion with spongiosis (Figures 13 and 14), and fibroplasia in the dermis. In severe
Histopathology of the lepromatous skin biopsy
Fig. 13 Borderline tuberculoid (BT) with type 1 reaction (T1R). Edema in the papillary dermis but also inside the granuloma with disorganization of the granuloma and presence of large Langhans giant cells.
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Fig. 15 Erythema nodosum leprosum (ENL). A neutrophilic infiltrate is present together with foamy macrophages.
The term erythema nodosum leprosum is a misnomer that communicates misinformation to clinicians and histopathologists alike. The definition of ENL is inappropriate, mainly as regards its clinical similarity with true EN: The term type 2 leprosy reaction would be more appropriate. ENL can occur in both LL and (less commonly) BL. Two different variants have been described histopathologically. One has been reported by Ridley as “pink node type” or classic ENL
(or a mild form of ENL; Figure 15). Typically, the infiltrate is centered on small granulomas in the subcutis with clusters of neutrophils around old foamy macrophages. Eosinophils, plasma cells, and mast cells are present. Even if in histopathologic textbooks ENL has been classified as a mostly lobular panniculitis with vasculitis, it should be stressed that vasculitis is not a conspicuous feature of classic ENL. In fact, classic features of small or medium vessel vasculitis, necrotizing changes, and thrombus formation have been reported in classic ENL lesions in almost 25% of cases. ENL is a dynamic process and of course the time of the biopsy influences the histopathologic features. In fact, vasculitic changes are more often seen in really early lesions. Opposed to this concept, in necrotizing ENL (or severe ENL; Figure 16) the vasculitic changes with neutrophilic infiltrate, hemorrhages, and thrombi can be severe and may produce degeneration of the collagen with necrosis of both dermis and epidermis (clinically ulcerated lesions). In these cases ENL may resolve with dermal fibrosis. Solid AFB will be found in lesions coming from untreated patients, whereas granular or fragmented AFB are usually seen in patients undergoing therapy. In cases occurring long after MDT, FF can be negative.1–4,12
Fig. 14 Borderline tuberculoid (BT) with type 1 reaction (T1R). Edema in the granuloma with disorganization of the granuloma and presence of many lymphocytes and of large Langhans giant cells.
Fig. 16 Necrotizing erythema nodosum leprosum (ENL) or severe ENL. In this case a leukocytoclastic vasculitis with neutrophils and hemorrhages coexists with Virchow cells.
reactions, breakdown of the granuloma and even necrosis or ulceration can occur, and few collections of neutrophils may be found. As T1R subside, there is a reduction in the edema and formation of well-organized tubercles. The five key features are dermal edema, intragranuloma edema, giant cell size, giant cell numbers, and HLA-DR expression.12,13 If T1Rs occur in BL, epithelioid granulomas with giant cells and edema are observed together with Virchow cells and macrophage granuloma.1–4,12,13
Erythema nodosum leprosum
44 The main histologic differential diagnoses include LucioAlvarado phenomenon (see below), true EN, erythema induratum Bazin, Sweet syndrome, pyoderma gangrenosum, and deep mycotic infections.9,10
Particular forms of leprosy Histoid leprosy The term histoid leprosy (HL) was coined by Wade in 1963 to describe firm, reddish, or skin-colored, dome-shaped or oval papules and nodules, regular in contour with translucent shiny and stretched overlying skin, that on histologic examination have a circumscribed macrophage granuloma with the predominance of spindle-shaped cells or polygonal cells and unusually large numbers of AFB. Some cases may resemble a fibrohystiocytic tumor. A small collection of foamy macrophages may be present. HL is a clinical and pathologic variant of multibacillary leprosy usually occurring in patients receiving long-term diaminodiphenylsulfone therapy, with initial improvement followed by relapse.1–4,14
Lucio-Latapi leprosy Lucio-Latapi leprosy is a distinct form of lepromatous leprosy reported mainly in Mexico but also in areas of Central America and South America. It is characterized by a diffuse nonnodular infiltration of the skin. Histopathologically, it is similar to LL, with diffuse infiltration of Virchow cells.15
Lucio-Alvarado phenomenon Patients with Lucio-Latapi leprosy may develop fever, arthralgias, myalgias, and very painful red or purpuric macules, of irregular shapes, angulated or “stellar,” on the lower legs, thighs, hip, trunk, and upper limbs. Histopathologically, there is epidermal necrosis, ulceration, features of diffuse lepromatous leprosy in the dermis with numerous AFB, and a panvasculitis of both superficial and deep vessels. In particular, a lobular panniculitis is found, with the mediumsized arteries infiltrated by macrophages with the consequent narrowing of their lumens, occlusion, and ischemic changes.16
Regressive lesions During or after MDT can be confirmed to have had successful treatment by histopathologic conformation; however, clearance of the infiltrate and of AFB may take several years, depending on granuloma fraction and the BIG. Perineural granulomas in TT may persist 18 months after the cessation of effective therapy.1–4
C. Massone et al. The epithelioid granulomas gradually disappear in TT and BT. They may assume the picture of nonspecific perivascular chronic inflammation, mainly constituted by lymphocytes. BL and LL regressive lesions have a “fatty degeneration” with the appearance of more and larger vacuoles (even giant vacuoles, especially in LL) and with a diffuse lymphocytic infiltrate. AFB are granular or fragmented, and their number may also shrink. Skin adnexa are atrophic and disappear without fibrosis. Nerve bundles are replaced by fibrosis. Foamy cells disappear slowly, leaving a perivascular lymphocytic infiltrate with few foamy cells. After successful MDT, lesions can histologically clear in 2 to 5 years or more, but in some patients, a few foamy cells may persist for their lifetime for unexplained reasons.1–4
Relapse Relapse may take place at the site of a preexisting lesion, in a fully resolved site, or in a previously not involved site. In relapsing TT and BT, epithelioid granulomatous Langerhan giant cells reappear with the same distribution as in active lesions. Differentiating relapse from T1R is extremely difficult. In BL and LL relapse, in addition, small and large foci of newly arrived lymphocytes and spindle-shaped macrophages with pink granular cytoplasm are identified along with a few small clumps of foamy macrophages. The main feature is the reappearance of solid-staining AFB in biopsy specimens. The coexistence of granular or fragmented AFB is also possible. It is not possible to distinguish histologically between a relapse and a reinfection.1–4,17
Diagnostic clues Histologic features that should always induce consideration of leprosy and exclusion through biopsy in a patient coming from endemic areas are as follows: • Epithelioid granulomas with multifocal distribution • “Erosion” of the epidermis by epithelioid granulomas and without epidermal hyperplasia • Perineural and periadnexal granulomatous infiltrate • Swollen and enlarged nerves • Necrosis of a nerve • Perineural lymphocytic infiltrate with elongated shape at the dermal-epidermal junction • A lyphmo histioxyxtic infiltrate that is more prominent around the adnexa than around the vessels • Macrophage granuloma with foamy cells with a grenz zone • Foamy cells with large vacuoles • Onion-skin infiltrate around nerves • Neutrophils in a macrophage granuloma with foamy cells
Histopathology of the lepromatous skin biopsy • A plasma cell with 6 Ls: lues, lupus vulgaris, Lyme/ borelliosis, leprosy, leishmaniasis, lymphoma9 • The pale infiltrate: lues, lupus vulgaris, leprosy, leishmaniasis9
References 1. Ridley DS. Pathogenesis of Leprosy and Related Diseases. London: Wright. 1988250. 2. Ridley DS. Histological classification and the immunological spectrum of leprosy. Bull World Health Organ. 1974;51:451. 3. Job CK. Pathology of leprosy. In: Hastings RC, ed. Leprosy. 2nd ed. Edinburgh: Churchill Livingstone; 1994. p. 193-224. 4. Massone C. Histopathology of the skin. In: Nunzi E, Massone C, eds. Leprosy, A Practical Guide. Berlin: Springer; 2012. p. 115-136. 5. Massone C, Nunzi E, Cerroni L. Histopathologic diagnosis of leprosy in a nonendemic area. Am J Dermatopathol. 2010;32:417-419. 6. Singh A, Weng X, Nath I. Skin biopsy in leprosy. In: Khopkar U, ed. Skin Biopsy—Perspectives. Rijeka, Croatia: InTech; 2011. p. 73-86. 7. Ridley DS. Skin Biopsy in Leprosy. Basel, Switzerland: Ciba-Geigy. 1977:63.
45 8. Mahaisavariya P, Jiamton S, Manonukul J, Khemngern S. Mast cells in leprosy and leprosy reaction. Int J Dermatol. 2000;39:274-277. 9. Weedon D. Skin Pathology. 3rd ed. London: Elsevier. 2010. 10. Ackerman AB. Histologic Diagnosis of Inflammatory Skin Diseases. 2nd ed. Baltimore: Williams & Wilkins. 1997:522. 11. Liu TC, Yen LZ, Ye GY, Dung GJ. Histology of indeterminate leprosy. Int J Lepr Other Mycobact Dis. 1982;50:172-176. 12. Ridley DS. Reactions in leprosy. Lepr Rev. 1969;40:77-81. 13. Lockwood DN, Lucas SB, Desikan KV, Ebenezer G, Suneetha S, Nicholls P. The histological diagnosis of leprosy type 1 reactions: Identification of key variables and an analysis of the process of histological diagnosis. J Clin Pathol. 2008;61:595-600. 14. Pradeep NS, Nanda Kumar G. A clinical and histopathological study of histoid leprosy. Int J Dermatol. 2013;52:580-586. 15. Magaña M. Lucio–Latapi leprosy. In: Nunzi E, Massone C, eds. Leprosy, A Practical Guide. Berlin: Springer; 2012. p. 111-114. 16. Magaña M. Lucio’s phenomenon. In: Nunzi E, Massone C, eds. Leprosy, A Practical Guide. Berlin: Springer; 2012. p. 241-244. 17. Shetty VP, Wakade AV, Ghate SD, Pai VV. Clinical, bacteriological and histopathological study of 62 referral relapse cases between Jan 2004 and Dec 2009 at the Foundation for Medical Research, Mumbai. Lepr Rev. 2011;82:235-243.
Histopathology of the lepromatous skin biopsy Cesare Massone, MD a,⁎, Workalemahu Alemu Belachew, MD b , Antonio Schettini, MD c a
Department of Dermatology, Medical University of Graz, Graz, Austria Mekelle University, College of Health Science, Mekelle, Ethiopia c Fundation Alfredo Da Matta, Manaus, Amazonas, Brazil b
Abstract The histopathology of lepromatous skin varies according to the cell-mediated immunity of the host against Mycobacterium leprae. In tuberculoid and borderline tuberculoid leprosy, epithelioid noncaseating granulomas predominate, and acid-fast bacilli (AFB) are absent or only rarely present. In borderline lepromatous and lepromatous leprosy, the infiltrate is composed of macrophages with a vacuolar cytoplasm, lymphocytes, and plasma cells. AFB are numerous. Edema inside and outside the epithelioid granulomas, together with the appearance of large giant cells, are the main features of type 1 reactions. A conspicuous neutrophilic infiltrate in the subcutis with or without vasculitis is found in erythema nodosum leprosum. The main histopathologic features of leprosy and its particular forms are discussed in this review. © 2015 Elsevier Inc. All rights reserved.
The main feature of the vast majority of leprosy biopsy specimens is a granulomatous infiltrate that has different features according to the form of leprosy, the time and site of the biopsy, the presence of a leprosy reaction, and therapy. The clinical spectrum of leprosy correlates in most of cases (but not in all) directly with histopathologic results, reflecting the different grade of cell-mediated immune response (CMI) of the host against Mycobacterium leprae.1–4 Although leprosy is a routine diagnosis and well-known issue in endemic areas, recognition may become difficult in nonendemic areas, where leprosy may be misdiagnosed as many different inflammatory dermatoses. In fact, due to increased immigration from endemic areas, leprosy cases might be more commonly observed in nonendemic areas, such as Europe.5 To formulate a correct diagnosis and classification of leprosy, three cardinal points are needed. First, histopathologic diagnosis of leprosy is impossible without exact
⁎ Corresponding author. Tel.: +43 316 385 13235; fax: +43 316 385 14957. E-mail address: [email protected] (C. Massone). http://dx.doi.org/10.1016/j.clindermatol.2014.10.003 0738-081X/© 2015 Elsevier Inc. All rights reserved.
clinical data, such as anamnesis (origin or travels to endemic countries; contact with leprosy patients; household leprosy cases); number, distribution, and type of the lesions; presence or absence of anesthesia; therapies; and other pertinent signs and symptoms (e.g., of type 1 or type 2 reaction). The final diagnosis and classification of a leprosy patient in a referral center should always be formulated on a detailed clinical-pathologic correlation. Second, the skin biopsy has to be correctly performed: It must be deep (at least 4 mm punch biopsy including part of the subcutaneous fat) and without artifacts (squeeze).6 Lastly, a representative part of the lesion must be biopsied. This means that in indeterminate leprosy (IL) the center of a small hypopigmented lesion (or better, the center of the anesthetic area) or the border of an annular macule has to be biopsied. In tuberculoid (TT), borderline tuberculoid (BT), and borderline borderline leprosy (BB) the infiltrated margins are diagnostic; in borderline lepromatous (BL) and lepromatous leprosy (LL), the center of a macule, plaque, or nodule. If a type 1 reaction (T1R) is suspected, the most edematous and infiltrated area of the lesion has to be biopsied, whereas in cases of erythema nodosum leprosum (ENL) the biopsy must be deep to include the subcutaneous fat.6,7
Histopathology of the lepromatous skin biopsy Hematoxylin and eosin (H&E) and Fite-Faraco (FF) stains must be performed on each biopsy specimen by an experienced laboratory technician. Serial sections are sometimes needed. Immunohistochemistry of the lymphocytic infiltrate is never necessary for the diagnosis. Polymerase chain reaction (PCR) for M. leprae is useful only in a limited number of cases. FF is the method of choice for staining AFB; control sections should be used; and “whenever there is doubt, there should be no hesitation in staining further sections.”1–4 There are several recipes for modified stain for AFB.1–4,6 AFB are assessed on histopathologic sections exactly in the same way as in the smear, according to the following logarithmic indices (bacterial index of granuloma [BIG]): 0: Absence of bacilli 1 +: 1-10 bacilli in 100 high-power fields (HPFs) 2 +: 1-10 bacilli in 10 HPFs 3 +: 1-10 bacilli for HPFs 4 +: 10-100 for HPFs 5 +: 100-1000 for HPFs 6 +: Many clumps, “globi” (N 1000 bacilli) Unfortunately, the Ridley-Jopling clinical classification does not always correlate with the histologic features, and in some cases, there is a discrepancy between clinical and histopathologic classification. Different authors found an overall agreement between clinical type and histologic type, 58% and 70%, respectively, with the major disparity observed in borderline cases.1–4
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Tuberculoid leprosy The granulomatous infiltrate is more often multifocal, with a typical periadnexal (with involvement of the arrector pili muscle and sweat glands) and perineural distribution, both in the superficial and deep portions of the dermis. Skin adnexa may be also infiltrated and destroyed by the granulomas (Figure 4). Extension of the infiltrate to the subcutis is possible. The granulomas are noncaseating and formed by mature epitheliod cells, “encapsulated” by many lymphocytes (Figures 1 and 2). Numerous large Langhans cells are pathognomonic. Multinucleated giant cells may be present, whereas plasma cells are absent. Fibrin in the center of the granuloma is not observed. The epidermis is often atrophic and “eroded” by the granulomas present in the superficial dermis. There is no clear subepidermal zone. Nerves are enlarged and swollen. The perinerium is intact and may be surrounded by lymphocytes. In other cases, nerves are infiltrated by the granulomas, and they may be destroyed beyond recognition. Rarely, a patch of fibrinoid necrosis may be observed. A careful search may reveal few remnants of nerve tissue in the granuloma. Some authors report that S100 stain is superior to H&E in identifying nerve fragmentation in TT, but S100 staining is usually not needed. AFB are usually absent (AFB: 0 to 1 +) and PCR is negative in most cases.1–4 The main differential diagnoses are sarcoidosis, tuberculosis, leishmaniasis, and secondary syphilis. The presence of plasma cells should suggest consideration of leishmaniasis. The presence of prominent epidermal hyperplasia favors deep mycoses, tuberculosis, and leishmaniasis.9,10
Histopathologic patterns of leprosy The inflammatory cells present in leprosy lesions are epithelioid cells, macrophages, lymphocytes, plasma cells, and in specific cases also neutrophils and mast cells.8 According to CMI response to M. leprae, different types of granulomatous reaction can be observed. Epithelioid cells are usually seen in TT and BT, whereas foamy macrophages characterize the infiltrate of BL and LL. The infiltrate can often “touch” the epidermis in TT, but only rarely in BT, and typically spares the epidermis in BB, BL, and LL. Rarely the infiltrate may be found in the superficial dermis only; more usually it involves all the dermis and sometimes also the subcutis. Some cases may be characterized by a prominent lymphocytic infiltrate, resulting in a diagnostic pitfall. In each biopsy the nerves have to be carefully checked. The presence of AFB inside the nerve is diagnostic of leprosy. Although the polar forms TT and LL are quite easily diagnosed and classified, borderline cases may sometimes present overlapping features. For this reason, combinations of symbols, such as TT-BT, BT-BB, BB-BL and BL-LL, are used for those cases that show intermediate features among two groups.1–4
Fig. 1 Tuberculoid (TT). Multifocal granulomatous infiltrate in the superficial and deep dermis, periadnexal and perineural. The granulomas are focally in contact with the epidermis.
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Fig. 2 Tuberculoid (TT) granulomas are characterized by noncaseating mature epithelioid cells and are surrounded by many lymphocytes.
Borderline tuberculoid leprosy BT presents a similar infiltrate to TT. The main differences reside in the lesser maturation of epithelioid cells and in particular the presence of the lymphocytes within and not only around the granulomas; moreover, granulomas are less organized and tubercle formation is less prominent (Figure 3). Undifferentiated, medium-sized giant cells are typical of BT. The epidermis may or may not be “eroded” by granulomas. Nerves typically are moderately swollen and have perineural and intraneural granulomas. The perineurium is infiltrated by lymphocytes. AFB are 0 to 2 +. PCR is positive in almost half of cases.1–4 The main differential diagnoses are sarcoidosis, tuberculosis, leishmaniasis, and secondary syphilis.9,10
Borderline borderline leprosy BB is also characterized by granulomas with immature epithelioid cells but without the formation of well-defined
Fig. 3 In borderline tuberculoid (BT) granulomas the epithelioid cells are less mature than in BT and lymphocytes are not only around but also infiltrate the granulomas.
C. Massone et al.
Fig. 4 Borderline lepromatous (BL). Macrophage granulomas that infiltrate the superficial and deep dermis, also with periadnexal and perineural distribution, but that are always separated by the epidermis by a typical narrow zone (Unna band).
epithelioid granulomas. Giant cells are not present, and lymphocytes are diffusely distributed. Macrophages represent a fairly significant proportion of cells. There is often edema. The epidermis is atrophic. The subepidermal zone is not involved. Nerves are not swollen but are infiltrated by epithelioid cells and lymphocytes and partly destroyed. A lamination of the perineurium (reactive proliferation of perineural cells) may be observed. AFB are 3 to 4 +. PCR is positive in only almost all cases.1–4
Borderline lepromatous leprosy The infiltrate in BL is characterized by the presence of macrophages (so-called macrophage granuloma) admixed with lymphocytes. In some cases, lymphocytes predominate over macrophages (Figures 4-7). A solitary clump of
Fig. 5 Borderline lepromatous (BL). The macrophages have small vacuoles in the cytoplasm, and there are several lymphocytes invading the granuloma. Plasma cells are present.
Histopathology of the lepromatous skin biopsy
41
Fig. 6 Borderline lepromatous (BL). In some cases lymphocytes predominate over macrophages. In these cases the diagnosis might be a challenge.
epithelioid cells among the macrophages may be found. The infiltrate can be diffuse, patchy nodular, perivascular, or periadnexal but always separated from the epidermis by a typically narrow zone (Unna band) of collagen. Macrophages have a more or less prominent foamy cytoplasm, especially in regressing lesions, but no large vacuoles are usually found. Plasma cells are present. Nerves have an onion-skin perineurium with lymphocytes forming a cuff around a nerve bundle. AFB (4 to 5 +) are present inside macrophages, Schwann cells, endothelial cells, and arrector pili muscles.1–4
Lepromatous leprosy
Fig. 8 Lepromatous leprosy (LL). Patchy nodular infiltrate of macrophages in the all dermis without granuloma formation. A clear grenz zone in the superficial dermis between the infiltrate and the epidermis is typically present.
LL is characterized by collections and sheets of macrophages diffusely distributed in the dermis (but also a “patchy nodular” arrangement can be found). Lymphocytes and plasma cells are diffusely scattered or in a clump. There are no epithelioid cells. The epidermis is atrophic, and a clear grenz zone (Unna band) in the superficial dermis between the infiltrate and the epidermis is typically present (Figure 8). Skin adnexa are surrounded by macrophages and are
atrophic. The infiltrate can extend to the deep dermis and subcutaneous fat. Macrophages present as a gray cytoplasm with foamy changes (lepra cells, Virchow cells; Figure 9) in variable amount from mild foamy appearance in early lesions to large vacuoles in older or regressing lesions (occurring also in multinucleated giant cells; Figure 10).
Fig. 7 Borderline lepromatous (BL) (same case as Figure 6). A predominately lymphocytic infiltrate is observed also around the eccrine glands and nerves.
Fig. 9 Lepromatous leprosy (LL). Macrophages present a gray cytoplasm with mild foamy changes (lepra cells, Virchow cells in early lesions). Plasma cells are present.
42
C. Massone et al.
Fig. 10 Lepromatous leprosy (LL). In older or regressing lesions, large vacuoles appear.
Fig. 12 Acid-fast bacilli (AFB) are granular or fragmented in patients while receiving or shortly after multidrug therapy (MDT).
Nerves have a perineural collection of macrophages and may have an onion-skin perineurium but without significant infiltration, or they may be fairly normal. Nerves may be somewhat hyaline or fibrosed but not swollen. Numerous AFB (5-6 +) are present within macrophages, sweat and sebaceous glands, hair follicles, arrector pili muscles, Schwann cells, perineural cells, and vascular endothelium. AFB may be arranged in parallel array, or forming clusters, or densely packed in large masses known as “globi” (Figure 11). AFB have a solid appearance in untreated patients, whereas they are granular or fragmented in patients during or shortly after multidrug therapy (MDT; Figure 12).PCR is obviously positive in both LL and BL and is not necessary for the diagnosis.1–4 The histopathologic differential diagnoses of both LL and BL mainly include other diseases that produce foamy macrophages, such as xanthomas and xanthogranulomas, post–kala-azar dermal leishmaniasis, paraffinoma, and rarely infections caused by other nontuberculous (atypical) mycobacteria.9,10
Indeterminate leprosy Indeterminate leprosy (IL) is characterized by a superficial or more often superficial and deep perivascular and periadnexal lymphohistiocitic infiltrate. In some cases, the infiltrate is more prominent around the adnexa (sweat and sebaceous glands, hair follicles) than around the vessels. Mast cells are increased, whereas granulomas are absent. The epidermis may present with focal vacuolar degeneration of basal keratinocytes and exocytosis of lymphocytes. A perineural infiltrate can be seen in one third of cases, and lymphocytes “cuffing” the nerve fibrils are a good hint for the diagnosis. Nerves, however, are not enlarged. AFB are difficult to find, and FF serial sections are needed to find single or small groups of AFB in a nerve. The subepidermal zone just beneath the basal layer and the arrector pili muscles are sites where AFB might be also found. PCR is positive only in a small proportion of cases, being in these cases of some help for the diagnosis.1–4,11 Diagnosis of IL without detailed clinical information is not possible, because the histopathologic differential diagnosis includes the large group of dermatides with a perivascular and periadnexal lymphohistiocytic infiltrate.9,10
Leprosy reactions Type 1 reaction
Fig. 11 Lepromatous leprosy (LL). Numerous solid acid-fast bacilli (AFB) are present solitarily or in globules.
T1R occur mainly in BT and BB and sometimes also in BL patients. On the background of a BT, BB, or BL, histopathologic result, the histologic features that characterize T1R are the presence of edema in the superficial dermis and edema in the granuloma with disorganization of the granuloma, the appearance of foreign body giant cells (sometimes with vacuoles due to intracellular edema), large Langhans giant cells with epidermal erosion with spongiosis (Figures 13 and 14), and fibroplasia in the dermis. In severe
Histopathology of the lepromatous skin biopsy
Fig. 13 Borderline tuberculoid (BT) with type 1 reaction (T1R). Edema in the papillary dermis but also inside the granuloma with disorganization of the granuloma and presence of large Langhans giant cells.
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Fig. 15 Erythema nodosum leprosum (ENL). A neutrophilic infiltrate is present together with foamy macrophages.
The term erythema nodosum leprosum is a misnomer that communicates misinformation to clinicians and histopathologists alike. The definition of ENL is inappropriate, mainly as regards its clinical similarity with true EN: The term type 2 leprosy reaction would be more appropriate. ENL can occur in both LL and (less commonly) BL. Two different variants have been described histopathologically. One has been reported by Ridley as “pink node type” or classic ENL
(or a mild form of ENL; Figure 15). Typically, the infiltrate is centered on small granulomas in the subcutis with clusters of neutrophils around old foamy macrophages. Eosinophils, plasma cells, and mast cells are present. Even if in histopathologic textbooks ENL has been classified as a mostly lobular panniculitis with vasculitis, it should be stressed that vasculitis is not a conspicuous feature of classic ENL. In fact, classic features of small or medium vessel vasculitis, necrotizing changes, and thrombus formation have been reported in classic ENL lesions in almost 25% of cases. ENL is a dynamic process and of course the time of the biopsy influences the histopathologic features. In fact, vasculitic changes are more often seen in really early lesions. Opposed to this concept, in necrotizing ENL (or severe ENL; Figure 16) the vasculitic changes with neutrophilic infiltrate, hemorrhages, and thrombi can be severe and may produce degeneration of the collagen with necrosis of both dermis and epidermis (clinically ulcerated lesions). In these cases ENL may resolve with dermal fibrosis. Solid AFB will be found in lesions coming from untreated patients, whereas granular or fragmented AFB are usually seen in patients undergoing therapy. In cases occurring long after MDT, FF can be negative.1–4,12
Fig. 14 Borderline tuberculoid (BT) with type 1 reaction (T1R). Edema in the granuloma with disorganization of the granuloma and presence of many lymphocytes and of large Langhans giant cells.
Fig. 16 Necrotizing erythema nodosum leprosum (ENL) or severe ENL. In this case a leukocytoclastic vasculitis with neutrophils and hemorrhages coexists with Virchow cells.
reactions, breakdown of the granuloma and even necrosis or ulceration can occur, and few collections of neutrophils may be found. As T1R subside, there is a reduction in the edema and formation of well-organized tubercles. The five key features are dermal edema, intragranuloma edema, giant cell size, giant cell numbers, and HLA-DR expression.12,13 If T1Rs occur in BL, epithelioid granulomas with giant cells and edema are observed together with Virchow cells and macrophage granuloma.1–4,12,13
Erythema nodosum leprosum
44 The main histologic differential diagnoses include LucioAlvarado phenomenon (see below), true EN, erythema induratum Bazin, Sweet syndrome, pyoderma gangrenosum, and deep mycotic infections.9,10
Particular forms of leprosy Histoid leprosy The term histoid leprosy (HL) was coined by Wade in 1963 to describe firm, reddish, or skin-colored, dome-shaped or oval papules and nodules, regular in contour with translucent shiny and stretched overlying skin, that on histologic examination have a circumscribed macrophage granuloma with the predominance of spindle-shaped cells or polygonal cells and unusually large numbers of AFB. Some cases may resemble a fibrohystiocytic tumor. A small collection of foamy macrophages may be present. HL is a clinical and pathologic variant of multibacillary leprosy usually occurring in patients receiving long-term diaminodiphenylsulfone therapy, with initial improvement followed by relapse.1–4,14
Lucio-Latapi leprosy Lucio-Latapi leprosy is a distinct form of lepromatous leprosy reported mainly in Mexico but also in areas of Central America and South America. It is characterized by a diffuse nonnodular infiltration of the skin. Histopathologically, it is similar to LL, with diffuse infiltration of Virchow cells.15
Lucio-Alvarado phenomenon Patients with Lucio-Latapi leprosy may develop fever, arthralgias, myalgias, and very painful red or purpuric macules, of irregular shapes, angulated or “stellar,” on the lower legs, thighs, hip, trunk, and upper limbs. Histopathologically, there is epidermal necrosis, ulceration, features of diffuse lepromatous leprosy in the dermis with numerous AFB, and a panvasculitis of both superficial and deep vessels. In particular, a lobular panniculitis is found, with the mediumsized arteries infiltrated by macrophages with the consequent narrowing of their lumens, occlusion, and ischemic changes.16
Regressive lesions During or after MDT can be confirmed to have had successful treatment by histopathologic conformation; however, clearance of the infiltrate and of AFB may take several years, depending on granuloma fraction and the BIG. Perineural granulomas in TT may persist 18 months after the cessation of effective therapy.1–4
C. Massone et al. The epithelioid granulomas gradually disappear in TT and BT. They may assume the picture of nonspecific perivascular chronic inflammation, mainly constituted by lymphocytes. BL and LL regressive lesions have a “fatty degeneration” with the appearance of more and larger vacuoles (even giant vacuoles, especially in LL) and with a diffuse lymphocytic infiltrate. AFB are granular or fragmented, and their number may also shrink. Skin adnexa are atrophic and disappear without fibrosis. Nerve bundles are replaced by fibrosis. Foamy cells disappear slowly, leaving a perivascular lymphocytic infiltrate with few foamy cells. After successful MDT, lesions can histologically clear in 2 to 5 years or more, but in some patients, a few foamy cells may persist for their lifetime for unexplained reasons.1–4
Relapse Relapse may take place at the site of a preexisting lesion, in a fully resolved site, or in a previously not involved site. In relapsing TT and BT, epithelioid granulomatous Langerhan giant cells reappear with the same distribution as in active lesions. Differentiating relapse from T1R is extremely difficult. In BL and LL relapse, in addition, small and large foci of newly arrived lymphocytes and spindle-shaped macrophages with pink granular cytoplasm are identified along with a few small clumps of foamy macrophages. The main feature is the reappearance of solid-staining AFB in biopsy specimens. The coexistence of granular or fragmented AFB is also possible. It is not possible to distinguish histologically between a relapse and a reinfection.1–4,17
Diagnostic clues Histologic features that should always induce consideration of leprosy and exclusion through biopsy in a patient coming from endemic areas are as follows: • Epithelioid granulomas with multifocal distribution • “Erosion” of the epidermis by epithelioid granulomas and without epidermal hyperplasia • Perineural and periadnexal granulomatous infiltrate • Swollen and enlarged nerves • Necrosis of a nerve • Perineural lymphocytic infiltrate with elongated shape at the dermal-epidermal junction • A lyphmo histioxyxtic infiltrate that is more prominent around the adnexa than around the vessels • Macrophage granuloma with foamy cells with a grenz zone • Foamy cells with large vacuoles • Onion-skin infiltrate around nerves • Neutrophils in a macrophage granuloma with foamy cells
Histopathology of the lepromatous skin biopsy • A plasma cell with 6 Ls: lues, lupus vulgaris, Lyme/ borelliosis, leprosy, leishmaniasis, lymphoma9 • The pale infiltrate: lues, lupus vulgaris, leprosy, leishmaniasis9
References 1. Ridley DS. Pathogenesis of Leprosy and Related Diseases. London: Wright. 1988250. 2. Ridley DS. Histological classification and the immunological spectrum of leprosy. Bull World Health Organ. 1974;51:451. 3. Job CK. Pathology of leprosy. In: Hastings RC, ed. Leprosy. 2nd ed. Edinburgh: Churchill Livingstone; 1994. p. 193-224. 4. Massone C. Histopathology of the skin. In: Nunzi E, Massone C, eds. Leprosy, A Practical Guide. Berlin: Springer; 2012. p. 115-136. 5. Massone C, Nunzi E, Cerroni L. Histopathologic diagnosis of leprosy in a nonendemic area. Am J Dermatopathol. 2010;32:417-419. 6. Singh A, Weng X, Nath I. Skin biopsy in leprosy. In: Khopkar U, ed. Skin Biopsy—Perspectives. Rijeka, Croatia: InTech; 2011. p. 73-86. 7. Ridley DS. Skin Biopsy in Leprosy. Basel, Switzerland: Ciba-Geigy. 1977:63.
45 8. Mahaisavariya P, Jiamton S, Manonukul J, Khemngern S. Mast cells in leprosy and leprosy reaction. Int J Dermatol. 2000;39:274-277. 9. Weedon D. Skin Pathology. 3rd ed. London: Elsevier. 2010. 10. Ackerman AB. Histologic Diagnosis of Inflammatory Skin Diseases. 2nd ed. Baltimore: Williams & Wilkins. 1997:522. 11. Liu TC, Yen LZ, Ye GY, Dung GJ. Histology of indeterminate leprosy. Int J Lepr Other Mycobact Dis. 1982;50:172-176. 12. Ridley DS. Reactions in leprosy. Lepr Rev. 1969;40:77-81. 13. Lockwood DN, Lucas SB, Desikan KV, Ebenezer G, Suneetha S, Nicholls P. The histological diagnosis of leprosy type 1 reactions: Identification of key variables and an analysis of the process of histological diagnosis. J Clin Pathol. 2008;61:595-600. 14. Pradeep NS, Nanda Kumar G. A clinical and histopathological study of histoid leprosy. Int J Dermatol. 2013;52:580-586. 15. Magaña M. Lucio–Latapi leprosy. In: Nunzi E, Massone C, eds. Leprosy, A Practical Guide. Berlin: Springer; 2012. p. 111-114. 16. Magaña M. Lucio’s phenomenon. In: Nunzi E, Massone C, eds. Leprosy, A Practical Guide. Berlin: Springer; 2012. p. 241-244. 17. Shetty VP, Wakade AV, Ghate SD, Pai VV. Clinical, bacteriological and histopathological study of 62 referral relapse cases between Jan 2004 and Dec 2009 at the Foundation for Medical Research, Mumbai. Lepr Rev. 2011;82:235-243.