Jpn.J.Leprosy 69, 93-100(2000)

Clinico-histopathological Findings of Buruli Ulcer

Milanga Mwanatambwe'), Yukiko Fukunishi2), Mikihisa Yajima3), Keiji Suzuki3), Kingsley Asiedu4), Samuel Etuafel5)., Nobutaka Yamada') and Goro Asano') 1)Department of Pathology, Nippon Medical School,Japan 2)National Sanatorium, Matsuoka Hoyo-en, Aomori, Japan 3)National (Sanatorium, Tama-Zenshoen,) Higashi murayama, Japan 4)Global Tuberculosis Program, WHO, Geneva, Switzerland 5)St. Martin Hospital, Agroyesum, Ashanti Country, Ghana

[Received:20 Apr 2000/ Accepted:6 June 2000]

Keywords: BuruliUlcer, Histopathology, Mycobacterium ulcerans, Acid-fast bacilli (AFB)

Here,we reportthe clinico-pathologicalfindings of Buruliulcer. The patientswere 2 females,9 and 23 yearsof age and one male,47 yearsof age fromthe Ashanti Country of Ghana.Clinically, cutaneous lesions wereclassified as nodular,ulcero-nodular and ulcerative.Histopathologically, lesions involved cutaneous and subcutaneoustissue, which showed lympho-epithelioid cell proliferationand panniculitiswith characteristic fat necroticchanges. Vascular inflammation, with the nervetissue involvement, are prominent features on the chronologicalspectrum of the 3 cases.In all butthe earlycase, Mycobacterium ulcerans could be visualized fromthe mid dermalarea to the subcutisby Fite-Faracoand Haradastain. The ulceratedlesions were also immunoreactiveto phenolicglycolipid-I(PGL-I). These findings suggest Mycobacterium ulcerans infection withlesions of differentages. Further,we also showthe needto identifydistinct caracteristics for differential diagnosiswith lesions caused by othermycobacteria.

Introduction world, especially in western Africa (Fig. 1). The dis ease is caused by Mycobacterium ulcerans, which is Buruli ulcer is a skin ulcerating disease, affect closely related to the mycobacteria of tuberculosis ing children and adults of both sex, without distinc and leprosy. Buruli ulcer is the third most frequent tion of age, but particularly prevalent in children be mycobacterial disease after tuberculosis and leprosy. tween 2 and 14 years of age. The disease, described In endemic areas, only experienced medical practi a century ago in Ouganda Buruli county', is re tioners would be able to make accurate differential emerging at an alarming rate in some parts of the diagnosis between lesions of Buruli ulcer, and those caused by other microorganisms like, cutaneous lep 'Corresponding author : rosy, cutaneous tuberculosis, spirochetiosis, Department of Pathology Nippon Medical School, Bunkyo-ku, Sendagi 1-1-5 diphteriosis, actinomycosis, fusobacillosis, filariosis Tokyo 113 0086, Japan and leishmaniosisz). The culture of Mycobacterium

93 ulcerans or the confirmation of its genomic sequence by Buruli ulcer's patients. The 3 patients did not re by PCR will not be a practical diagnostic method in ceive any other medication prior to the surgical pro most endemic countries in the near future. Histopa cedure. From the surgically removed materials, the thology, if supported by well defined criteria, will specimen for histopathology were taken from the remain the gold standard diagnostic method. most active area of the lesions. Specimens from each patient were then divided in 2 separate portions. One Materials and Methods set of specimen was fixed in 10% neutral formalin and the other in 2.5% glutaraldehyde solution in 0.1M The skin specimen in this study, were obtained in cacodylate buffer at pH 7.4. The formalin fixed speci the course of skin reparative surgery, from 3 patients mens were routinely processed and stained with He admitted at the Agroyesum missionary hospital in matoxylin-eosin(H&E), Fite-Faraco and Harada re the Ashanti Country in Ghana. The clinical data of agents. For the phenolyc glycolipid-1(PGL-1) the 3 patients are summarized in Table 1. The surgi immunostaining, paraffin blocks of the formalin cal excisions were performed under local anesthesia fixed specimen were sectioned into 3 um sections according to a surgical procedure in use atAgroyesum and processed for PGL-1 immunostaining using the St. Martin Hospital. All the cases were admitted to ABC method. this hospital with the presumptive diagnosis of Buruli ulcer. These presumptive diagnoses were based on Results the endemicity of the area, the clinical history of the diseases, the physical examination of lesions and the The specimens from all 3 cases showed characteris experience of the 3 medical practitioners in charge tic histopathologic changes as a result of Mycobac of this 80 beds' hospital of which 1/3 are occupied terium ulcerans infection. In case 1, macroscopically

Worlddistribution ofBuruli Ulcer (-), withthe highest prevalence inAfrica inthe last decade().

Fig.i. World distribution of Buruli Ulcer, showing the highest prevalence in Africa

94 Jpn.J.Leprosy 69, 93-100(2000)

a single nodule with firm consistency was seen on served in perivascular spaces from the papillary der the posterior aspect of the left elbow (Table 1). Mi mis down the reticular dermis. In the subcutis, in croscopically, inflammatory cell infiltration was ob flammatory cells were scantly scattered in the adi

Table I. Summary of patients' clinical data

Table II. Summary of histopathological findings of the 3 cases

95 pose tissue (Fig.2a). Ultrastructurally, blood vessels able inflammatory cell infiltration was evident in fatty showed hypertrophic endothelial cells with vacuoliza tissue (Fig 3b). Some blood vessels showed the reca tion and perivascular increase of collagen fibrils (Fig. nalization of thrombi with collagenous degeneration 2b). In case 2, macroscopically an ulcerated nodule of their walls. In the perivascular fibrotic region, on the upper, anterior aspect of the left arm was noted. slight inflammatory cell infiltration was evident. Histologically, the reticular dermis showed coagula Scattered acid-fast bacilli (AFB) were also present tive necrosis of the connective tissue along with pan in these blood vessel walls (Fig.3c). Macroscopically, niculitis (Fig 3a). Perivascular edema with remark case 3 showed an ulcer on the lateral aspect of the

Fig.2a Inflammatory cell infiltrationis observed in fatty tissue of Fig.3a.Coagulative necrosis of the connective tissue and the subcutis in case 1.(H&Estain x 200) panniculitis are shown in this picture of the reticular Fig.2b.Bloodvessels showing hypertrophic endothelial cells with dermis of case 2. (H&E stain x 200) vacuolization and increase perivascular collagen fibrils Fig.3b. Remarkable perivascular edema and lesions of vas in this ultrastructural picture of case1.(x 3600) culitis are demonstrated in the adipose tissue of this evolutive case 2.( H&E stain x 200) Fig.3c.A blood vessel shows slight inflammatory cell infiltra tion in perivascular fibrotic region in case 2.(Fite Faraco stain x 200)

96 Jpn.J.Leprosy 69, 93-100(2000)

right upper arm. Microscopically, coagulative necro ters of AFB in the necrotic reticular dermis and the sis of all layers of the dermis in the ulcer bed was subcutis (Fig.4b). Ultrastructurally, various degen evident. Ghost adipocytes were scattered in the sub erative changes in the bundles of peripheral nerves cutis. Fibrotic granulomatous lesions, including were observed, associated with thickening of multinucleated giant cells, epithelioid cells, histio Schwann cell basement membrane and vacuolization cytes and lymphocytes were observed in the margins of axons. Mast cell infiltration was also visible of ulcer bed (Fig.4a). AFB stains (Fite-Faraco and (Fig.4c). Also, the lumen of some blood vessels Harada) demonstrated small globi and confluent clus showed blood stasis, endothelial degeneration and

Fig.4a.The margins of the ulcerative lesion of case 3 show a Fig.4c. Ultrastructure of peripheral nerve in case 3: De granulomatous lesion comprising multinuclear giant generative change of peripheral nerve, consistent cells, hystiocytes, epithelioid cells and lymphocytes with thickening of Schwann cell basement mem (H&E stain x 200). brane and axons' vacuolization, associated with Fig.4b.ln the reticular dermis of case 3, the main confluent mast cell infiltration(x 4300). cluster of AFB are seen in foamy structures (Harada Fig.4d.Ultrastructure of blood vessel in case 3: The lumen stain x 200). of a blood vessel of case 3, shows blood stasis and endothelial degeneration and Mycobacterium ulcerans can be visualized as round-shape dense bodies (x 5800; arrow).

97 contained, Mycobacterium ulcerans, as round-shape of mycobacterial infection, are rarely visualized in dense bodies (Fig.4d, arrow). The histopathological lupus vulgaris like in early cases of Buruli ulcer'. In findings of the 3 cases are summarized in Table 11. advance cases of Buruli ulcer, most of the well ac Immunohistochemically, PGL-1 was positive in the tive looking AFB were organized in confluent globi subcutis in case 2 and case 3. However, it was nega like formations or what has been designated foamy tive in case 1. structures. Small tufts or individual AFB could be seen up to the reticular dermis by the Harada stain. Discussion They were not found by either of our AFB stains (Fite Faraco and Harada stains) in case I of our report. On a chronologic spectrum, lesions described in these The absence of AFB in early cases and, the re-epi 3 cases suggest a continuous degenerative process, thelialization stage of any form of Buruli ulcer, like advancing toward an extended necrosis of tissue, from in case 3, with fibrosis and granuloma, may render superficial to deep dermal layers down to the subcu the differential diagnosis even more difficult. tis. As suggested by the vascular lesions in case 1, Early lesions of Buruli ulcer may be histologi nodules, along with plaques and papules are prob cally confused with several findings of cases of lep ably among the early forms of Buruli ulcer. But, de rosy. Aggregates of mononuclear cells surrounding finitive conclusions will still await larger series of blood vessels along with intercellular edema, are fre studies. One of the big concern of pathologists here quently described in leprosy lesions. These findings should reside in the facts that Buruli ulcer like many resemble alterations seen in case 2 and 3 of our re other skin diseases, has been shown to be diseases of port. But, in contrast to leprosy, in early cases of poor rural areas in endemic countries". Cutaneous Buruli ulcers, vacuolated histiocytes and AFB are tuberculosis (scrofuloderma, tuberculous gumma and frequently absent, as in case 1 of this report6>.In typi lupus vulgaris) and leprosy presenting as nodules, cal ulcerated cases of Buruli ulcer, early or late, as in plaques or ulcers are highly prevalent in rural areas". case 2 and 3, the fatty necrosis surrounding septal To start with the clinical level, it would take years of and fascial structures, would certainly favor the di experience for doctors to be capable of accurate se agnosis of infection by Mycobacterium ulcerans. lective differential diagnosis. As a routine tool for Alterations described in the cases of erythema diagnosis, histopathological findings are important. nodosum leprosum are beyond the scope of our dis Histopathologically, in lupus vulgaris, scrofuloderma cussion. In a larger series of study, issues like vascu and tuberculous gumma, various necrotic alterations lar thrombotic alterations should deserve more at of the basal layer of the epidermis are likely to make tention. In case 2, blood vessels of various sizes, the difference with lesions of Buruli ulcer. In early showed edematous and sclerotic changes with par stages of Buruli ulcer, as in case I and to some ex tial to almost complete thrombotic obliteration. Simi tent in case 2, epidermal alterations are strikingly lar changes have been described in the cases of both absent. Hyperkeratosis, acanthosis and papillomato tuberculosis and leprosy'-". In contrast, we could sis have been well described in some cases of cuta observe the presence of AFB in the wall of altered neous tuberculosis). But these were not seen in the blood vessels in case 2 and 3. The coagulative necro 3 cases of our report and, have been an occasional sis of connective tissues in the dermis, with special finding in Buruli ulcer 5).Foci of necrosis, frequently attention to collagen fibers, has been described in described in the papillary dermis of various forms of both tuberculosis, leprosy and Buruli ulcers'. Periph cutaneous tuberculosis are not common findings in eral nerve alterations, accepted under the generic the cases of Buruli ulcer. Also, the presence of AFB, denomination of "nerve involvement " by some that may be considered a pathognomonic indication mycobacteriologists, have been frequent findings in

98 Jpnj.Leprosy 69, 93-100(2000)

the cases of our report. These alterations may prob HA: A new mycobacterial infection in man. J ably have a link, with the lack of pain generally de Pathol Bacteriol. 60: 93-122 (1948). scribed in the cases of Buruli ulcer and, should be 2) Van der Werf TS, Van der Graaf WTA, Tappero addressed after further observations. Also, if a larger JW, Asiedu K: Mycobacterium ulcerans infec series of studies confirm the presence of PGL-1 in tion. Lancet.1354: 1013-1018 (1999). tissues infected by Mycobacterium ulcerans, new 3) Meyers WM, Connor DH, Me Cullough B, considerations will be opened in the pathogenic ap Bourland J, Moris R, Proos L: Distribution of proaches of lesions caused by Mycobacterium mycobacterium ulcerans infections in Zaire, in ulcerans. If proved so, beside mycolactone, the toxin cluding new foci. Ann Soc beige Med Trop. 54: incriminated in the pathogenesis of tissue alterations 147-157 (1974) in Buruli ulcer", some glycolipids of the mycobac 4) Santa Cruz DJ, Strayer DS: The histologic spec terial wall should deserve more scrutiny. To sum trum of the cutaneous mycobacterioses. Hum marize the findings of our report, we have drawn the Pathol.13 : 485-495 (1982) impression that pathognomonic or major findings of 5) Connor DH, Lunn HE Buruli Ulceration. A clini Buruli ulcer, are deeply located in the distal dermis copathologic study of 38 Ugandans with myco and the subcutis, while lesions of tuberculosis and bacterium infection. Archiv Pathol. 81: 183-199 leprosy are more proximally situated. This, as far as (1966) advanced cases of Buruli ulcer are concerned. In early 6) Portaels F, Aguiar J, Fissette K, Fonteyene P, De cases, lines of separation may probably not be easy Beenhouwer H, De Rijk P, Guedenon A, Lemans to draw, unless, septal panniculitis prove to be the R, Steunou C, Zinsou C, Dumonceau JM, Meyers hallmark in a study with a larger series of early cases. WM: Direct detection and identification of my This is not the easy part of the problem, when one cobacterium ulcerans in clinical specimens by recall the difficulties met by most authors in com PCR and oligonucleotide-specific capture plate pelling anamnestic data of Buruli ulcer patients10'. hybridization. J Clin Microbiol. 35: 1097-1100 With all these considerations, it is our view that a (1997) standardized criteria would be of valuable help to 7) Degitz K, Steidl M, Thomas P, Plewig G, the histopathological differential diagnosis. We pro Volkenandt M: Aetiology of tuberculids. Lan pose a standardized evaluation system with a scor cet. 341: 239-240 (1993) ing scale that will separate diagnostic features and 8) Yajima M, Murata J, Yamada N, Asano G: Ultra suggestive features. structural observations of small blood vessels in leprosy patients. Jap J Leprosy. 60: 121-127 Aknowledgment (1991) 9) Pahlevan AA, Wright DJ, Andrews C, George The authors wish to thank Mr. Takenori Fujii for KM, Small PL, Foxwell BM: The inhibitory technical assistance. The histopathological review action of mycobacterium ulcerans soluble fac with Dr. Munehiro Yokoyama, was of valuable con tors on monocytes /T cell cytokine production tribution. and NF-kappaB function. J Immunol. 163:3928 3935(1999) References 10) Hayman J: Out of Africa: Observations on the histopathology of mycobacterium ulcerans infec 1) Mac Callum P, Tolhurst JC, Buckle G, Sissons tion. J Clin Pathol. 46: 5-9 (1993)

99 ブル リー潰瘍 の臨床組織病理学 的所見

ム ワ ナ タ ン ブ ェ ミ ラ ン ガi>福 西 征 子2)、 矢 島 幹 久3)、 鈴 木 慶 治3) ア シ デ ュ キ ン グ ス レ イ4)、 エ ツ ァ フ ェ ル サ ム エ ル5)、 山 田 宣 孝1)、 浅 野 伍 朗1) 1)日 本 医科 大 学病 理 学教 室 2)国 立療 養 所 松 丘 保 養 園 3)国 立 療 養 所 多 磨 全 生 園 4)WHO本 部 、 ジ ュ ネ ー ブ 、 ス イ ス 5)聖 マ ー チ ン病 院 、 ガ ー ナ 国

〔受 付:2000年4月20日 、 受 理:2㎜ 年6月6日 〕

キ ー ワ ー ド:ブ ル リ ー 潰 瘍 、 組 織 病 理 学 、 ミ コ バ ク テ リ ア 、 好 酸 性 球 菌

こ こ に3例 の ブ ル リ ー 潰 瘍 の 臨 床 病 理 学 的 知 見 に つ き報 告 し ま す 。 症 例 は ガ ー ナ 国 出 身 の9才 と23才 の 女 性 と47才 の 男 性 で 、 臨 床 的 に は 皮 膚 に 結 節 状 の 病 変 と 潰 瘍 病 変 に 加 え て 潰 瘍 性 病 変 を 伴 う 結 節 性 病

変 が み ら れ た 。 病 理 組 織 学 的 に は 真 皮 内 に リ ンパ 球 や 類 上 皮 細 胞 の 増 殖 と 脂 肪 壊 死 の み ら れ る 脂 肪 織 炎 を 認 め た 。 ま た3症 例 に 共 通 し た 病 変 と して 神 経 病 変 と 共 に 血 管 炎 が 観 察 さ れ た 。 特 に 進 行 例 で は ミ コ バ ク テ リ ア が 真 皮 層 と皮 下 組 織 に 局 在 し原 田 氏 法 で 確 認 さ れ 、PGL-1に 陽 性 反 応 を 示 し た 。 今 後 、 潰 瘍 形 成 性 の ミ コ バ ク テ リ ア の 感 染 が 他 の ミ コ バ ク テ リ ア に よ り発 生 す る 病 巣 と ど の よ う に 異 な る か に つ い て の 解 析 が 必

要 で あ る 。

'Correspondingauthor 〒113東 京 都 文 京 区千 駄 木1-1-5 日本 医 科 大 学 病 理 Te1．03-3822-2131Fax．03-5685-3067

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