Contiguous spread of Mycobacterium ulcerans in Buruli ulcer lesions analysed by histopathology and real-time PCR quantification of mycobacterial DNA.

The distribution of M. ulcerans in Buruli ulcer lesions was analysed by IS2404 real-time PCR quantification of M. ulcerans DNA and by semi-quantitative microscopic assessment of the number of acid-fast bacilli (AFB). Mycobacterial burden was compared with histopathological changes. Focal distribution of tissue destruction extending into areas with high and low mycobacterial burden was a feature in all lesions analysed. Even where most of the mycobacteria were washed out of ulcerative lesions, peaks of mycobacterial DNA and AFB in the necrotic base of the ulcers still marked the position of the primary infection focus. Significant amounts of mycobacterial DNA and microcolonies were also present in samples from more peripheral regions and occasionally in excised margins of macroscopically and histologically healthy-appearing excised tissue margins. Additional peaks of mycobacterial DNA clearly marked sites where satellite lesions were developing. Even when granulomas provided evidence for the development of cell-mediated immunity, development of satellite lesions by contiguous spreading was not completely prevented. Areas free of mycobacterial DNA were found between primary and secondary infection foci and around scarring tissue of healing lesions. These results demonstrate that IS2404 real-time PCR analysis is a better tool than the less sensitive and only semi-quantitative microscopic enumeration of AFB for studying the dynamics of M. ulcerans infection in situ.