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The purpose of this study was to develop a simple procedure for cell lysis and DNA extraction for direct detection of Mycobacterium ulcerans in aquatic insects, gills and intestinal contents of fish, molluscs and human tissue samples using a nested PCR method specific for the insertion sequence IS2404. The simultaneous action of sodium N-lauroyl sarcosine, guanidinium isothiocyanate, chloroform and Tris-saturated phenol on mycobacteria, followed by a DNA purification method using mini-columns fitted with silica-cellulose membranes was successfully employed to extract DNA from cultured bacteria, environmental and human tissue samples. All specimens were collected from Buruli ulcer endemic regions. M. ulcerans DNA was detected in 11 of 57 aquatic insects, one of six molluscs and three of 15 fish, supporting the hypothesis that the fauna of major Buruli ulcer endemic foci in swampy terrain of tropical and subtropical regions can be a source of M. ulcerans infection.

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We compared various diagnostic tests for their abilities to detect Mycobacterium ulcerans infection in specimens from patients with clinically active disease. Specimens from 10 patients from the area of Zangnanado (Department of Zou, Benin) with advanced, ulcerated active M. ulcerans infections were studied by direct smear, histopathology, culture, PCR, and oligonucleotide-specific capture plate hybridization (OSCPH). A total of 27 specimens, including 12 swabs of exudate collected before debridement and 15 fragments of tissue obtained during debridement, were submitted to bacteriologic and histopathologic analysis. The histopathologic evaluation of tissues from all six patients so tested revealed changes typical of those caused by M. ulcerans infection. Five specimens were contaminated, and M. ulcerans was cultivated on Löwenstein-Jensen medium from 12 of the remaining 22 (54.5%) specimens. Detection of mycobacteria was performed by PCR, and M. ulcerans was detected by OSCPH with a new probe (5-CACGGGATTCATGTCCTGT-3) reacting with M. ulcerans and Mycobacterium marinum. In 10 of 22 (45.5%) specimens, M. ulcerans was identified by PCR-OSCPH. There was no statistically significant difference between the detection of M. ulcerans by culture and by PCR-OSCPH (P > 0.05). This is the first demonstration of an amplification system (PCR-OSCPH) with a sensitivity similar to that of culture for the direct and rapid recognition of M. ulcerans in clinical specimens. This system is capable of identifying M. ulcerans, even in paucibacillary lesions. Our findings suggest that PCR-OSCPH should be used in the quest for the elusive environmental reservoir(s) of M. ulcerans.

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