@article {13, title = {A comparison of DNA extraction procedures for the detection of Mycobacterium ulcerans, the causative agent of Buruli ulcer, in clinical and environmental specimens.}, journal = {J Microbiol Methods}, volume = {76}, year = {2009}, month = {2009 Feb}, pages = {152-8}, abstract = {

Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease in humans after tuberculosis and leprosy. Although the disease is associated with aquatic ecosystems, cultivation of the bacillus from the environment is difficult to achieve. Therefore, at the moment, research is based on the detection by PCR of the insertion sequence IS2404 present in M. ulcerans and some closely related mycobacteria. In the present study, we compared four DNA extraction methods for detection of M. ulcerans DNA, namely the one tube cell lysis and DNA extraction procedure (OT), the FastPrep procedure (FP), the modified Boom procedure (MB), and the Maxwell 16 Procedure (M16). The methods were performed on serial dilutions of M. ulcerans, followed by PCR analysis with different PCR targets in M. ulcerans to determine the detection limit (DL) of each method. The purity of the extracted DNA and the time and effort needed were compared as well. All methods were performed on environmental specimens and the two best methods (MB and M16) were tested on clinical specimens for detection of M. ulcerans DNA. When comparing the DLs of the DNA extraction methods, the MB and M16 had a significantly lower DL than the OT and FP. For the different PCR targets, IS2404 showed a significantly lower DL than mlsA, MIRU1, MIRU5 and VNTR6. The FP and M16 were considerably faster than the MB and OT, while the purity of the DNA extracted with the MB was significantly higher than the DNA extracted with the other methods. The MB performed best on the environmental and clinical specimens. This comparative study shows that the modified Boom procedure, although lengthy, provides a better method of DNA extraction than the other methods tested for detection and identification of M. ulcerans in both clinical and environmental specimens.

}, keywords = {Animals, Buruli Ulcer, DNA Transposable Elements, DNA, Bacterial, DNA, Plant, Environmental Monitoring, Humans, Mycobacterium ulcerans, Polymerase Chain Reaction, Reagent Kits, Diagnostic, Sensitivity and Specificity, Soil Microbiology, Water Microbiology}, issn = {0167-7012}, doi = {10.1016/j.mimet.2008.10.002}, author = {Durnez, Lies and Stragier, Pieter and Roebben, Karen and Ablordey, Anthony and Leirs, Herwig and Portaels, Fran{\c c}oise} } @article {8, title = {Identification of a new variable number tandem repeat locus in Mycobacterium ulcerans for potential strain discrimination among African isolates.}, journal = {Clin Microbiol Infect}, volume = {13}, year = {2007}, month = {2007 Jul}, pages = {734-6}, abstract = {

Intra-species discrimination in the highly clonal pathogen Mycobacterium ulcerans was studied in a diverse collection of isolates by PCR amplification of a short sequence repeat locus containing heterogeneous arrays of tri-nucleotide repeats with an ACC consensus pattern. Post-amplification analysis indicated excellent typeability and identified five M. ulcerans alleles, including a unique Angolan type differentiated for the first time among a genetically conserved cluster of African isolates. These results are consistent with previously investigated independent markers, and provide an additional locus for variable number tandem repeat-based typing of M. ulcerans.

}, keywords = {Africa, Bacterial Typing Techniques, DNA, Bacterial, Humans, Minisatellite Repeats, Mycobacterium Infections, Nontuberculous, Mycobacterium ulcerans, Polymerase Chain Reaction, Sequence Alignment, Sequence Analysis, DNA, Species Specificity}, issn = {1198-743X}, doi = {10.1111/j.1469-0691.2007.01716.x}, author = {Ablordey, A and Fonteyne, P-A and Stragier, P and Vandamme, P and Portaels, F} } @article {10, title = {First cultivation and characterization of Mycobacterium ulcerans from the environment.}, journal = {PLoS Negl Trop Dis}, volume = {2}, year = {2008}, month = {2008}, pages = {e178}, abstract = {

BACKGROUND: Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin.

METHODOLOGY/PRINCIPAL FINDINGS: One culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on L{\"o}wenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5{\textquoteright} end of 16S rRNA gene of 00-1441 was 100\% identical to M. ulcerans and M. marinum, and the sequence of the 3{\textquoteright} end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone.

CONCLUSION: Strain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.

}, keywords = {Animals, Bacterial Toxins, Cells, Cultured, Environmental Microbiology, Female, Foot, Genotype, Hemiptera, Macrolides, Macrophages, Mass Spectrometry, Mice, Mice, Inbred BALB C, Mycobacterium ulcerans, Phylogeny, Polymerase Chain Reaction, RNA, Ribosomal, 16S}, issn = {1935-2735}, doi = {10.1371/journal.pntd.0000178}, author = {Portaels, Fran{\c c}oise and Meyers, Wayne M and Ablordey, Anthony and Castro, Ant{\'o}nio G and Chemlal, Karim and de Rijk, Pim and Elsen, Pierre and Fissette, Krista and Fraga, Alexandra G and Lee, Richard and Mahrous, Engy and Small, Pamela L C and Stragier, Pieter and Torrado, Eg{\'\i}dio and Van Aerde, Anita and Silva, Manuel T and Pedrosa, Jorge} } @article {11, title = {[Follow-up of the first case of Mycobacterium ulcerans infection documented by PCR, genotyping and culture in the Republic of Congo-Brazzaville].}, journal = {Med Trop (Mars)}, volume = {68}, year = {2008}, month = {2008 Apr}, pages = {137-43}, abstract = {

This article presents follow-up data from the first patient in whom Mycobacterium ulcerans infection (MUI) was documented by PCR, genotyping and culture in the Republic of Congo-Brazzaville. Findings show the importance of regular clinical and microbiological evaluation for the disseminated form of the disease. The patient was probably infected in Pointe Noire where MUI has been described but never documented. Culture of specimens collected before antibiotic treatment showed that the bacterium was sensitive to the antibiotics being administered (streptomycin and rifampin) and was identical to isolates from Atlantic-coast regions of West Africa where MUI is endemic. The patient was treated with streptomycin and rifampin for 12 weeks in association with surgery. During treatment clinical examination was performed every day and microbiological analysis every two weeks. The duration of follow-up from the end of specific antibiotic treatment was 26 months. Medical treatment failed to prevent bone involvement and fistulae that were treated by surgery. However medical treatment may have limited dissemination of the disease. Serial microbiological evaluation was useful to detect bone involvement in this patient, but persistent positive gene amplification is not a proof of active disease. This study confirms that MUI is still endemic in the region of Pointe Noire. This finding underlines the need to optimize epidemiologic surveillance, laboratory diagnostic capabilities, and therapeutic management in the Republic of Congo-Brazzaville.

}, keywords = {Adult, Anti-Bacterial Agents, Biopsy, Buruli Ulcer, Congo, DNA, Bacterial, Follow-Up Studies, Genotype, Humans, Male, Mycobacterium ulcerans, Polymerase Chain Reaction, Skin}, issn = {0025-682X}, author = {Kibadi, K and Stragier, P and Muyembe-Tamfum, J J and Pedrosa, J and Portaels, F} }