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.

Invasive punch or incisional skin biopsy specimens are currently employed for the bacteriological confirmation of the clinical diagnosis of Buruli ulcer (BU), a cutaneous infectious disease caused by Mycobacterium ulcerans. The efficacy of fine-needle aspirates (FNA) using fine-gauge needles (23G by 25 mm) for the laboratory confirmation of BU was compared with that of skin tissue fragments obtained in parallel by excision or punch biopsy. In three BU treatment centers in Benin, both types of diagnostic material were obtained from 33 clinically suspected cases of BU and subjected to the same laboratory analyses: i.e., direct smear examination, IS2404 PCR, and in vitro culture. Twenty-three patients, demonstrating 17 ulcerative and 6 nonulcerative lesions, were positive by at least two tests and were therefore confirmed to have active BU. A total of 68 aspirates and 68 parallel tissue specimens were available from these confirmed patients. When comparing the sensitivities of the three confirmation tests between FNA and tissue specimens, the latter yielded more positive results, but only for PCR was this significant. When only nonulcerative BU lesions were considered, however, the sensitivities of the confirmation tests using FNA and tissue specimens were not significantly different. Our results show that the minimally invasive FNA technique offers enough sensitivity to be used for the diagnosis of BU in nonulcerative lesions.

ulcerans, as detected by culture, when specimens remained in semisolid transport medium for long periods of time (up to 26 weeks). We can conclude that the method with semisolid transport medium is very robust for clinical specimens from patients with Buruli ulcer that, due to circumstances, cannot be analyzed in a timely manner. This transport medium is thus very useful for the confirmation of a diagnosis of Buruli ulcer with specimens collected in the field.

Mycobacterium africanum is an important cause of tuberculosis (TB) in West Africa. So far, two lineages called M. africanum West African 1 (MAF1) and M. africanum West African 2 (MAF2) have been defined. Although several molecular studies on MAF2 have been conducted to date, little is known about MAF1. As MAF1 is mainly present in countries around the Gulf of Guinea we aimed to estimate its prevalence in Cotonou, the biggest city in Benin. Between 2005–06 we collected strains in Cotonou/Benin and genotyped them using spoligo- and 12-loci-MIRU-VNTR-typing. Analyzing 194 isolates, we found that 31% and 6% were MAF1 and MAF2, respectively. Therefore Benin is one of the countries with the highest prevalence (37%) of M. africanum in general and MAF1 in particular. Moreover, we combined our data from Benin with publicly available genotyping information from Nigeria and Sierra Leone, and determined the phylogeographic population structure and genotypic clustering of MAF1. Within the MAF1 lineage, we identified an unexpected great genetic variability with the presence of at least 10 sub-lineages. Interestingly, 8 out of 10 of the discovered sub-lineages not only clustered genetically but also geographically. Besides showing a remarkable local restriction to certain regions in Benin and Nigeria, the sub-lineages differed dramatically in their capacity to transmit within the human host population. While identifying Benin as one of the countries with the highest overall prevalence of M. africanum, this study also contains the first detailed description of the transmission dynamics and phylogenetic composition of the MAF1 lineage.

T uberculosis (TB) is among the top 10 causes of death in the global ranking (1). Although Peru accounts for only 3% of the population of the Americas, it has 9.5% of the region's TB cases. According to the World Health Organization (WHO), 21,916 new cases of pulmonary TB were reported in Peru between January and November 2013 (2). Urban areas are more affected, with 59% of all Peruvian TB cases, 82% of multidrug-resistant (MDR) TB cases, and 93% of extensively drug-resistant (XDR) TB cases occurring in Lima. Within the capital area, TB cases are heterogeneously distributed. The most affected districts are located in the northeast and together represent 86% of the reported cases in the capital (3, 4). San Juan de Lurigancho is the most populated district in this area, with 1,004,339 inhabitants (5), and reports a pulmonary TB incidence rate of 193 cases per 100,000 inhabitants, a smear-positive TB incidence rate of 126 cases per 100,000 inhabitants (6), and an overall MDR prevalence of 7% (7), exceeding the national averages for the three indicators of 103 cases per 100,000 inhabitants, 62 cases per 100,000 inhabitants, and 5.3%, respectively (8). The HIV prevalence among TB patients in this setting is similar to the national prevalence, which in 2008 was 2.6% (9).Peru has been considered a good example of the beneficial effects of implementing directly observed therapy short course (DOTS) in a country's health system (10). Nevertheless, national surveys from 1996 and 2006 have shown increases in MDR rates from 2.4% to 5.3% among new cases and from 15.7% to 23.6% among previously treated cases (11). This paradox of improved TB management and worsening resistance prevalence has been explained by increases in the notified cases, i.e., in case detection, although treatment outcomes remain poor (12). Basically, the observed increases in MDR-TB rates may result from two factors, i.e., transmission of MDR-TB and acquired resistance due to ineffective TB treatment selecting for spontaneous mutations in specific genes associated with drug resistance (13,14).Molecular strain typing (genotyping) has significantly contributed worldwide to the understanding of TB epidemiology and transmission dynamics (15,16), by confirming outbreaks (17) and identifying the clonal spread of successful strains, including MDR strains (18,19). Furthermore, molecular typing has shown that the Mycobacterium tuberculosis complex (MTBc) has a diverse population structure, being composed of seven lineages of human