Report of the laboratory confirmation of cases from the CDTUB Pobé, Benin

In 2009, 420 diagnostic samples (corresponding to 301 patients suspected of M. ulcerans infection) from the Centre de Dépistage et de Traitement de l’Ulcère de Buruli of Benin were subjected to IS2404 PCR at the Centre Hospitalier Universitaire of Angers. 180 samples (corresponding to 141 patients) were confirmed positive to M. ulcerans infection by PCR

A multiplex kindred with severe Buruli Ulcer dis playing Mendelian inheritance

Buruli Ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacteriosis worldwide after tuberculosis and leprosy, and has been flagged in 1998 by the World Health Organization as an emerging neglected infectious disease.  The physiopathology of Mycobacterium ulcerans infection primarily involves the lipidic toxin mycolactone, a unique feature among mycobacteria. The resulting extensive skin ulcers and/or osteomyelitis cause pathologic scarring responsible for severe life-lasting functional disabilities in the affected population, mainly composed of children of less than 15 year of age.  Buruli ulcer mainly strikes in Western Sub-Saharan Africa but cases have been reported in more than 30 countries worldwide. A common characteristic of the endemic countries consists in the extreme clustering of BU cases in families living in the vicinity of slow-flowing or stagnant waters in rural areas.  However, only a fraction of these heavily exposed individuals develop Buruli ulcer, which leads us to hypothesize a genetic etiology accounting for this variability.    To tackle this issue, we adopted an extreme-phenotype strategy, which consisted in recruiting the most severe of  the  >1,500 BU cases diagnosed and treated during the last 7 years at the Centre de Détection et de Traitement de l\u27Ulcère de Buruli in Pobè, Benin. We report here the analysis of a single highly-informative consanguineous family in which two siblings were affected with exceptionally severe PCR-confirmed BU. The index case suffered from a multifocal edematous form of BU, which disseminated under treatment and involved the four limbs, eventually requiring amputation to heal. Her sister suffered from an edematous form which affected the right arm from shoulder to fingers.  Blood was obtained from the 2 parents, 2 affected and 3 unaffected children. DNA was processed for the genotyping of >900,000 Single Nucleotide Polymorphisms by the Affymetrix Genome-Wide 6.0 array.  After quality control procedures, 120,156 independent SNPs were used for linkage analysis by homozygosity mapping. Three regions, on chromosome 5 and 8, cosegregated with the affected status following a Mendelian recessive inheritance mode, i.e. were shared homozygous by descent by the 2 affected individuals but not the 3 unaffected siblings (yielding the maximum possible LOD score given the pedigree. Sequencing of genes in these regions is currently ongoing and show promising results.  This first description of a genetic etiology for extremely severe BU will have far reaching biological and medical implications. 

Fine needle aspiration for the diagnosis of M. ulcerans infection and for mycolactone detection

Over recent years, management of Buruli ulcer patients has considerably changed with advances in antibiotherapy. Antibiotherapy is particularly effective on nonulcerative forms. However, the bacteriological diagnosis in the early forms is difficult because simple and non-invasive methods are not available. In this study, the diagnostic effectiveness of the Fine Needle aspiration was evaluated on early lesions. Our results showed that PCR from FNA samples, unlike Ziehl-Neelsen staining, is very sensitive on nonulcerative forms like other standard sampling methods (biopsy and punch biopsy). Furthermore, mycolactone was detected in aspirated liquid from lesions in mouse experimentally infected by M. ulcerans and in FNA from Buruli ulcer patients. This is a crucial observation to encourage the development of diagnosis test based on mycolactone detection. Moreover, mycolactone was never detected in aspirated liquid from a patient treated by antibiotherapy. To conclude, fine needle aspiration is a simple, fast, painless, accurate and inexpensive method of sampling and could be used for diagnosis of M. ulcerans infection

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

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 lesio