Histopathological changes and clinical responses of Buruli ulcer plaque lesions during chemotherapy: a role for surgical removal of necrotic tissue?

Abstract

Background: Buruli ulcer (BU) caused by Mycobacterium ulcerans is a necrotizing skin disease usually starting with a subcutaneous nodule or plaque, which may ulcerate and progress, if untreated, over months and years. During the currently recommended antibiotic treatment with rifampicin/streptomycin plaque lesions tend to ulcerate, often associated with retarded wound healing and prolonged hospital stays.

Methodology/principal findings: Included in this study were twelve laboratory reconfirmed, HIV negative BU patients presenting with plaque lesions at the CDTUB in Allada, Benin. Punch biopsies for histopathological and immunohistochemical analysis were taken before start of treatment and after four to five weeks of treatment. Where excision or wound debridement was clinically indicated, the removed tissue was also analyzed. Based on clinical judgment, nine of the twelve patients enrolled in this study received limited surgical excision seven to 39 days after completion of chemotherapy, followed by skin grafting. Lesions of three patients healed without further intervention. Before treatment, plaque lesions were characterized by a destroyed subcutis with extensive necrosis without major signs of infiltration. After completion of antibiotic treatment partial infiltration of the affected tissue was observed, but large necrotic areas remained unchanged.

Conclusion/significance: Our histopathological analyses show that ulceration of plaque lesions during antibiotic treatment do not represent a failure to respond to antimycobacterial treatment. Based on our results we suggest formal testing in a controlled clinical trial setting whether limited surgical excision of necrotic tissue favours wound healing and can reduce the duration of hospital stays.

Figure 1. Presentation of BU lesions before and during therapy.

Representatives of the different patient groups are depicted. A, B: Patients with lesions that did not ulcerate and which did not require a surgical intervention. C: Patient with a lesion that ulcerated during treatment but did not require a surgical intervention. D: Patient with a lesion that did not ulcerate but was excised; E, F: Patients with lesions that ulcerated during treatment and needed surgical excisions. A: Patient 1; day −1, before start of treatment, punch hole visible. B: Patient 2; day 0, at the start of treatment, punch hole visible. C: Patient 3; C1: day −2 before start of treatment; C2: day 50 after start of treatment; C3: day 300 after end of treatment. D: Patient 5; D1: day −8 before start of treatment; D2: day 10 after end of antibiotic treatment; D3: day 17 after end of antibiotic treatment; D4: day 35 after end of antibiotic treatment. E: Patient 10; E1: day −1 before start of treatment. E2: day 89 after start of treatment. E3: day 39 after end of antibiotic treatment. E4: day 75 after end of antibiotic treatment. F: Patient 12; F1: day 0, at start of treatment; F2: day 26 after start of treatment; F3: day 54 after start of treatment; F4: day 39 after end of antibiotic treatment. F5: day 72 after end of antibiotic treatment.

Figure 2. Characteristic histopathological features of tissue samples taken before start of antibiotic treatment.

Histological sections were stained either with Haematoxylin-Eosin (HE) (A, C–E), Ziehl-Neelsen (counterstain methylenblue) (ZN) (B) or with antibodies against cell surface or cytoplasmic markers (counterstain haematoxylin) (F–H). A: Punch biopsy with large necrotic areas, fat cell ghosts and oedema but relatively intact epidermis and dermis. B: a band of extracellular AFBs is present in a deep layer of the necrotic subcutis. C: epidermis and dermis. D: necrotic region with fat cell ghosts. E: few infiltrating cells around a blood vessel. F: N-elastase staining revealed the presence of neutrophilic debris inside the necrotic regions. G: few intact neutrophils and H: CD68 positive infiltrating macrophages were found.

Figure 3. Characteristic histopathological features of tissue samples taken 26–34 days after start of antibiotic treatment.

Histological sections were stained either with Haematoxylin-Eosin (HE) (A, B, H), Ziehl-Neelsen (counterstain methylenblue) (ZN) (C) or with antibodies against cell surface or cytoplasmic markers (counterstain haematoxylin) (D–G, I). A: Punch biopsy with large necrotic areas, fat cell ghosts and oedema but relatively intact epidermis and dermis. B: Higher magnification of necrotic tissue with large numbers of fat cell ghosts. C: Small numbers of intra and extracellular beaded AFB. D: N-elastase positive intact neutrophils were rare. E: More intact CD68 positive macrophages and F: CD3 positive T-cells were observed in the dermal tissue. Additionally, small CD20 positive B-cell cluster (G), few granulomas (H) and langhans giant cells (I) were found in only few of the samples.

Figure 4. Characteristic histopathological features of tissue surgically excised to support wound healing.

Histological sections were stained either with Haematoxylin-Eosin (HE) (A–C), Ziehl-Neelsen (counterstain methylenblue) (ZN) (L) or with antibodies against cell surface or cytoplasmic markers (counterstain haematoxylin) (D–K). A: Overview over an excised tissue specimen still harbouring large necrotic areas with fat cell ghosts and oedema. B: Overview over an excised tissue specimen presenting with mixed infiltration in the former necrotic region. C: Necrosis and oedema of the dermis of an excised non-ulcerative lesion. D: CD14 (D1) and N-elastase (D2) staining revealing a clear border between infiltration with intact CD14 positive macrophages (D1) and neutrophilic debris inside the necrotic area (D2). Infiltrated tissue areas contained large numbers of CD68 positive macrophages (E) and large numbers of CD3 positive cells (F). These belonged mainly of the CD8 (G) and not of the CD4 (H) subset. Langhans and foreign body giant cells (I) and B-cell cluster (J) were present in the majority of the samples. Accumulations of N-elastase positive cells (K) were occasionally found. AFB were rare, had a beaded appearance and intracellular location (L).