Secondary Buruli ulcer skin lesions emerging several months after completion of chemotherapy: paradoxical reaction or evidence for immune protection?

Abstract

Background: The neglected tropical disease Buruli ulcer (BU) caused by Mycobacterium ulcerans is an infection of the subcutaneous tissue leading to chronic ulcerative skin lesions. Histopathological features are progressive tissue necrosis, extracellular clusters of acid fast bacilli (AFB) and poor inflammatory responses at the site of infection. After the recommended eight weeks standard treatment with rifampicin and streptomycin, a reversal of the local immunosuppression caused by the macrolide toxin mycolactone of M. ulcerans is observed.

Methodology/principal findings: 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.

Conclusion/significance: Our histopathological findings demonstrate that the skin lesions emerging several months after completion of antibiotic treatment were associated with M. ulcerans infection. During antibiotic therapy of Buruli ulcer development of new skin lesions may be caused by immune response-mediated paradoxical reactions. These seem to be triggered by mycobacterial antigens and immunostimulators released from clinically unrecognized bacterial foci. However, in particular the lesions that appeared more than one year after completion of antibiotic treatment may have been associated with new infection foci resolved by immune responses primed by the successful treatment of the initial lesion.

Figure 1. Clinical presentation of lesions (Patient 1).

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.

Figure 2. Histopathological presentation of secondary lesions.

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.

Figure 3. Presence of B-cell clusters in the secondary lesions.

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.

Figure 4. Bands of leucocytes surrounding an uninfiltrated necrotic area.

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).