Conceived and designed the experiments: AC GP MTR. Performed the experiments: AC MTR. Analyzed the data: MFA AC RCJ GP MTR. Contributed reagents/materials/analysis tools: AA MFA AC HK. Wrote the paper: GP MTR.
The authors have declared that no competing interests exist.
The neglected tropical disease Buruli ulcer (BU) caused by
We have conducted a detailed histopathological and immunohistochemical analysis of tissue specimens from two patients developing multiple new skin lesions 12 to 409 days after completion of antibiotic treatment. Lesions exhibited characteristic histopathological hallmarks of Buruli ulcer and AFB with degenerated appearance were found in several of them. However, other than in active disease, lesions contained massive leukocyte infiltrates including large B-cell clusters, as typically found in cured lesions.
Our histopathological findings demonstrate that the skin lesions emerging several months after completion of antibiotic treatment were associated with
Buruli ulcer (BU) is a chronic necrotizing skin disease presenting with extensive tissue destruction and local immunosuppression. Standard treatment recommended by the WHO includes 8 weeks of rifampicin/streptomycin and, if necessary, wound debridement and skin grafting. In some patients satellite lesions develop close to the primary lesion or occasionally also at distant sites during effective antibiotic treatment of the primary lesion. We performed a detailed analysis of tissue specimens from lesions that emerged in two BU patients from Benin 12 to 409 days after completion of chemotherapy. Histopathology revealed features of tissue destruction typically seen in BU and degenerated acid-fast bacilli. In addition, lesions contained organized immune infiltrates typically found in successfully treated BU lesions. Secondary lesions emerging many months after completion of chemotherapy may have been caused by immune response-mediated paradoxical reactions. However, the late onset may also indicate that they were associated with new infection foci spontaneously resolved by adaptive immune responses primed by antibiotic treatment of the primary lesions.
Buruli ulcer (BU) is a chronic necrotizing infection of subcutaneous tissue caused by
Clinical diagnosis of BU can be confirmed by insertion sequence 2404 (
Reported rates of recurrence after surgical treatment alone range between 6% and 47% because even wide surgical excision of lesions may not remove all bacilli
In active BU disease, a protective cloud of mycolactone around the mycobacterial clusters is thought to both destroy infiltrating leukocytes and hinder them from passing pro-inflammatory signals to other cells. It is most likely, but still remains to be formally proven, that mycolactone production is reduced or abolished early after the onset of R/S chemotherapy due to impairment of mycolactone synthesis, bacterial growth arrest and/or bacterial cell death, reflected by ‘beaded’ appearance of AFBs (MT Ruf; unpublished results). Declining toxin levels allow leukocytes to reach the extracellular mycobacteria, leading to their phagocytosis and destruction
Ethical approval for analyzing patient specimens was obtained from the ethical review board of the Ministry of Health of Benin. Written informed consent from the guardians of the patients was obtained before surgical specimens were used for reconfirmation of BU as well as a detailed immunohistological analysis.
Both patients, two six year old boys, included in this study were laboratory-confirmed BU cases with one primary lesion. Both received a combination of rifampicin (10 mg/kg body weight) and streptomycin (15 mg/kg body weight) administered daily over 8 weeks at the Centre de Diagnostic et de Traitement de l'Ulcère de Buruli (CDTUB) in Pobè, Benin according to the WHO recommendations. Both patients developed several new lesions at different parts of the body, 12 – 409 days after completion of antibiotic treatment. These lesions were removed by limited excision and no additional antibiotic treatment was administered. Excised tissue from a number of these new lesions became available for histopathological analysis (
Patient | Primary lesion | Time span (days) between completion of antibiotic treatment and occurrence of secondary lesion | Time span (days) between occurrence of secondary lesion and surgical excision | Nature of satellite lesion | Location of satellite lesion | Distance of satellite from primary lesion |
1 | ulcer at the right upper arm/elbow | 75 | 4 | ulcer 1 | right axilla | 10 cm |
275 | 1 |
|
back | 25 cm | ||
409 | 2 |
|
right shoulder | 20 cm | ||
409 | 2 |
|
thorax | 30 cm | ||
2 | ulcer at the right upper leg/knee | 12 | 6 | nodule 1 | right upper leg | 5 cm |
46 | 33 |
|
right lower leg | 15 cm | ||
54 | 25 |
|
right upper leg | 5 cm | ||
176 | 1 | nodule 4 | right foot | 30 cm |
Lesions from which tissue samples became available for histopathological analysis are written in bold letters.
Tissue specimens analyzed are listed in
In the present report we describe clinical and histopathological observations in two BU patients that have developed series of new skin lesions (
Patient 1, a six year old boy, presented at the Centre de Diagnostic et de Traitement de l'Ulcère de Buruli (CDTUB) in Pobè, Benin with a 15×15 cm ulcerated plaque lesion at the right forearm and elbow with undermined edges characteristic for BU (
A: Initial ulcerated lesion at the right arm, reaching from the elbow to the forearm. B: Nodule1 appearing on the back, 275 days after end of antibiotic treatment. Both, nodule 2 on the thorax (C) and an ulcerated plaque on the right shoulder (D) had appeared 409 days after completion of antibiotic treatment.
75 days after completion of chemotherapy a first new ulceration 0,5×0,5 cm (ulcer 1) in the axilla of the right arm emerged. After performing some débridement, this lesion had healed 35 days later and the patient was discharged from hospital. 275 days after completion of chemotherapy the patient was readmitted with a non ulcerated fluctuant nodule 1,5×1,5 cm (nodule 1) on the back (
Patient 2, also a six year old boy, presented at the CDTUB with a 20×15 cm ulcerated lesion on the interior side of his right upper leg and knee. Undermined edges as well as ‘cotton wool’ appearance of necrotic tissue at the center of the lesion were characteristic for BU
One week after discharge (46 days after completion of antibiotic treatment) the patient was readmitted with a second nodule (1,5×1,5 cm) located at the lower right leg about 15 cm distal of the border of the primary lesion. Again eight days later (54 days after completion of antibiotic treatment) a third nodule (nodule 3) (3×2 cm) had emerged at the upper right leg located 5 cm proximal of the initial wound. These two nodules were excised 93 days after completion of the antibiotic therapy. While AFB staining, as well as
After surgical excision and healing of the satellite lesions the patient was discharged, but re-admitted 176 days after completion of antibiotic treatment with a fourth nodule (nodule 4) 2×2 cm on the right foot. A minimal surgical intervention was performed and the patient was discharged 10 days later and no further relapses were observed after 10 months of follow-up (February 2011).
Histopathological and immunohistochemical analyses were performed with nodule 1, nodule 2 and ulcer 2 from patient 1, and nodule 2 and 3 of patient 2 (
Features characteristic for BU pathology, such as fat cell ghosts, necrotic soft tissue, hemorrhages, and epidermal hyperplasia were present in all specimens analyzed. As shown in
Histological sections (nodule 2 of patient 2) were stained either with Ziehl-Neelsen (counterstain methylenblue; A, F, G) or with antibodies against cell surface or cytoplasmic markers (counterstain haematoxylin; B–E). A: Overview over excised tissue specimen revealing typical BU pathology features like fat cell ghosts, necrosis, epidermal hyperplasia and AFB (region 2) as well as a strong mixed infiltration typically observed in successfully treated BU lesions (region 1). B: CD14 staining of macrophages/monocytes; C: CD3 staining of T-cells; D: Elastase staining of neutrophils. In the necrotic region 2 large numbers of elastase-positive neutrophilic debris (E) and small clumps of AFB (F) with a beaded appearance (G) were observed.
Clusters of CD20 positive B-cells, another hallmark of ectopic lymphoid tissue developing in BU lesions after successful treatment
A: Band of CD20 positive B-cells in sections of ulcer 2 of patient 1. B–E: serial sections of nodule 3 of patient 2 with a small dense cluster of CD20 positive B-cells (B) surrounded by CD14 positive macrophages/monocytes (C) and few interspersed CD3 positive T-cells (D) from which the majority was CD8 negative (E). Higher magnification (F–I) revealed a very dense package of the B-cells.
In some parts of the lesions small uninfiltrated necrotic areas surrounded by belts of leucocytes still remained. Immunohistochemical analyses gave indications for sequential infiltration of these areas by different types of leucocytes (
Serial sections of nodule 2 of patient 1 with a necrotic area surrounded by a belt of CD14 positive monocytes/macrophages (A) and a more external second belt of CD3 positive T-cells (B). The necrotic core contained N-elastase positive neutrophilic debris (C), but no intact neutrophils (D insert). Clusters of CD20 positive B-cells were found away from the necrotic core (D).
In this report we describe the development of series of new skin lesions in two BU patients 12 – 409 days after completion of antibiotic treatment. The newly emerging nodules and ulcerations were located either at some distance from the initial lesion at the same extremity or at other body locations. Detection of
Detailed immunohistochemical analyses showed that residual necrotic areas were surrounded by an outer belt of T-lymphocytes and an inner belt of macrophages/monocytes with appendices reaching into the necrotic tissue. These belts of intact leucocytes seem to reflect ongoing efforts of the immune system to resolve the necrotic areas. In contrast, remains of neutrophils found inside the necrotic areas seem to be leftovers of early acute neutrophilic infiltration waves. These are also observed in early phases of
Recently O'Brien et al have described the occurrence of paradoxical reaction in two Australian BU patients during R/S treatment of BU
In the case of the two patients described here, new lesions developed at prolonged periods of time after completion of antibiotic treatment. These lesions may represent secondary
We thank Vincent Romanet, Caroline Stork, Melanie Sticker-Jantscheff, Elvira Stillhart and Dr. Masato Murakami from Novartis Basel for excellent technical support and providing access to lab equipment for histopathology. We also thank Dr. Laurent Marsollier and Prof. Jane Cottin from the Centre Hospitalier Universitaire, Angers, France for mycobacteriologic (PCR and culture) results of patient specimen.