b WHO-endorsed phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are assumed to be the gold standard for identifying rifampin (RMP) resistance. However, previous results indicated that low-level, yet probably clinically relevant, RMP resistance linked to specific rpoB mutations is easily missed by some growth-based methods. We aimed to compare the level of resistance detected on Löwenstein-Jensen (LJ) medium with resistance detected by the Bactec MGIT 960 automated DST (MGIT-DST) system for various rpoB mutants. Full agreement between LJ and MGIT-DST was observed for mutations located at codons 513 (Lys or Pro) and 531 (Leu, Trp), which were always resistant by both methods. For mutations 511Pro, 516Tyr, 533Pro, 572Phe, and several 526 mutations, LJ and MGIT results were highly discordant, with MGIT-DST failing to give a result or declaring the strains susceptible. Our data show that phenotypic RMP resistance testing of M. tuberculosis is not a binary phenomenon for some rpoB mutations and that the widely used automated MGIT 960 system is prone to miss some RMP resistance-conferring mutations, while careful DST on LJ missed hardly any. Given the association of these mutations with poor clinical outcome, our findings suggest that the gold standard for rifampin resistance should be reconsidered, in order to address the present confusion caused by discrepancies between phenotypic and genotypic results. The impacts of these mutations will depend on the frequency of their occurrence, which may vary from one setting to another.

New rpoB gene primers for detecting Rif r in Mycobacterium tuberculosis complex bacteria achieved 100% specificity and 88% (fresh sputa) and 92% (ethanol-preserved sputa) diagnostic sensitivity and detected up to 4 CFU/sample. Of the 99 Rif r isolates examined, 97% had mutations within cluster I, 2% at codon 176, and 1% at codon 497.Molecular detection of rifampin-resistant (Rif r ) Mycobacterium tuberculosis usually relies on amplification of the hotspot zone for resistance-conferring mutations (cluster I, covering codons 432 to 458 according to the M. tuberculosis nomenclature) (7) of the rpoB gene (4,8,10,12,13,17). Previous studies have shown that 94 to 98% of Rif r M. tuberculosis strains show a mutation in cluster I (10,11,14,15). Resistance-associated mutations have also been described for cluster II (codons 496 and 497) and for codons 176, 486, 558, and 598 (1, 6, 7).We describe and evaluate new primers, covering the entire region with all currently known significant mutations in a single assay.Oligonucleotides were designed on the basis of an alignment of the rpoB gene sequence from the H37Rv M. tuberculosis reference strain (NC 000962; NCBI bank), some relevant nontuberculous mycobacteria (NTM), and nonmycobacterial species using ClustalX (version 1.83.1) software. Amplify software (version 1.2; University of Wisconsin-Madison) was used to estimate the stabilities and binding capacities of the selected oligonucleotides and to simulate PCRs. Figure 1 shows the relative locations of the selected primers.A single PCR with primers rpoBgeneSA (5Ј-GGTTCGCCGC GCTGGCGCGAAT-3Ј) and rpoBgeneRB (5Ј-GACCTCCTCG ATGACGCCGCTTTCT-3Ј) was used for bacterial suspensions, whereas a nested PCR with primers rpoBgeneSAnew (5Ј-GCAA AACAGCCGCTAGTCCTAGTCCGA-3Ј) and rpoBgeneRA (5Ј-GCGCCATCTCGCCGTCGTCAGTACAG-3Ј) for the first run, and rpoBgeneSA and rpoBgeneRB as inner primers, was used to amplify clinical specimens.The first run of the nested PCR was performed with a final volume of 50 l containing 10 mM Tris-HCl (pH 8.6), 50 mM KCl, 1.65 mM MgCl 2 , 200 M of each deoxynucleoside triphosphate, 12.5 pmol of each primer, 1.5 U Taq polymerase (Promega, Madison, WI), and 5 l of DNA extract from clinical specimens. PCR was performed using a PTC 100 MJResearch thermocycler (Whaltham, MA) as follows: a hot start (90°C) followed by 5 min at 94°C; 45 cycles of 45 s at 94°C, 1 min 30 s at 66°C, and 45 s at 72°C; and a final extension of 10 min at 72°C. The second run was performed with a final volume of 25 l enzyme mixture with 0.5 U Taq polymerase and 0.25 l of the first PCR amplicon as follows: a hot start (90°C) followed by 5 min at 94°C, 29 cycles of 45 s at 94°C, 1 min 45 s at 72°C (annealing and extension), and a final extension of 10 min at 72°C. For bacterial suspensions, a single PCR was run under similar conditions but using a 50-l enzyme mixture and 45 cycles. Amplicons were analyzed with a 2% (wt/vol) agarose gel.DNA was extracted from sputum by an adapted Boom extraction method (16) and from bacterial suspensions in 1ϫ TE bu...

The TDR-TB Strain Bank is a high quality bioresource for basic science, supporting the development of new diagnostics and drug-resistant detection tools and providing reference materials for laboratory quality management programmes.

HighlightsFirst insight into resistance levels and genetic diversity of TB in Guinea.Rapid expansion of drug-resistance prone LAM10 Cameroon family.Population structure reveals less ‘ancestral’ TB than in surrounding countries.Knowledge of genetic diversity is relevant for tuberculosis control programs.

Abstract. We report our experience in managing 13 consecutive clinically suspected cases of Buruli ulcer on the face treated at the hospital of the Institut Médical Evangélique at Kimpese, Democratic Republic of Congo diagnosed during [2003][2004][2005][2006][2007]. During specific antibiotherapy, facial edema diminished, thus minimizing the subsequent extent of surgery and severe disfigurations. The following complications were observed: 1) lagophthalmos from scarring in four patients and associated ectropion in three of them; 2) blindness in one eye in one patient; 3) disfiguring exposure of teeth and gums resulting from excision of the left labial commissure that affected speech, drinking, and eating in one patient; and 4) dissemination of Mycobacterium ulcerans infection in three patients. Our study highlights the importance of this clinical presentation of Buruli ulcer, and the need for health workers in disease-endemic areas to be aware of the special challenges management of Buruli ulcer on the face presents.