The esat-6 and cfp-10 genes are essential for virulence in Mycobacterium tuberculosis. Among nontuberculous mycobacteria, we found these genes only in M. kansasii, M. szulgai, M. marinum, and M. riyadhense, with unique sequences. This adds a phylogenetic and taxonomical characteristic and may represent a virulence factor for nontuberculous mycobacteria.The 6-kDa early secretory antigenic target (ESAT-6) and 10-kDa culture filtrate protein (CFP-10) of Mycobacterium tuberculosis are potent T-cell antigens (2, 12). The genes encoding ESAT-6 and CFP-10 are situated within "region of difference 1" (RD1) of Mycobacterium tuberculosis but are also present in some nontuberculous mycobacteria (NTM) (2, 12). Deletion of RD1 in M. tuberculosis significantly decreases its virulence in animal models (8), suggesting that genes residing in RD1 are involved in pathogenesis. In M. tuberculosis, the RD1 proteins effect translocation to the cytosol, a mechanism for survival within macrophages (15). Therefore, RD1 in NTM may also play a crucial role in virulence.To improve our understanding of the pathogenesis of NTM disease and the possible role of RD1 in virulence, we screened a wide diversity of NTM for the presence of RD1.From our laboratory database, we retrieved isolates of all five Mycobacterium kansasii subtypes based on 16S-23S internal transcribed spacer sequencing (n ϭ 15),M. riyadhense (n ϭ 1), and M. tuberculosis H37Rv.The selection of these strains was based on their phylogenetic relationship with the M. tuberculosis complex in the multigene taxonomical model published by Devulder et al. (3).To establish the presence of an RD1-like element and sequences of the esat-6 and cfp-10 genes, we used the Esa-12 (CATGACAGAGCAGCAGTG) and Esa-303 (5Ј-GCCCT ATGCGAACATCCC-3Ј) primers for esat-6 and the opBR78 (5Ј-GTAGCCCGGGATGGCAGAGATGAAGACCGATGC C-3Ј) and opBR103 (5Ј-TCAGAAGCCCATTTGCGAGGAC AGC-3Ј) primers for cfp-10 (1). The M. smegmatis esat-6 and cfp-10 gene sequences were extracted from the whole-genome sequence in the GenBank database (accession number CP000480).Using these primers, we were able to demonstrate an RD1 (1). None of the PCRnegative species hybridized with either probe (data not shown). The presence of RD1, characterized by an esat-6-like gene and an cfp-10-like gene, is a phylogenetic characteristic among the NTM. It is mainly found only among slowly growing NTM species that are phylogenetically related to the M. tuberculosis complex based on the multigene taxonomical model by Devulder et al. (3) and in the more distantly related rapid grower M. smegmatis. Possibly, the presence of RD1 reflects phylogenetic relationships to the M. tuberculosis complex.Previous authors have recorded the presence of RD1 in M. flavescens (1). We were unable to demonstrate it in four reference strains (ATCC 23008, ATCC 23033, ATCC 23035, and ATCC 23039) and a clinical isolate. Thus, with its presence of an RD1 region, M. smegmatis still stands out among the rapid growing NTM.Gey van Pittius et al. have demonstrated the presence ...

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.

The mycobacterium growth indicator tube (MGIT960) automated liquid medium testing method is becoming the international gold standard for second-line drug susceptibility testing of multidrug-and extensively drug-resistant Mycobacterium tuberculosis complex isolates. We performed a comparative study of the current gold standard in the Netherlands, the Middlebrook 7H10 agar dilution method, the MGIT960 system, and the GenoType MTBDRsl genotypic method for rapid screening of aminoglycoside and fluoroquinolone resistance. We selected 28 clinical multidrug-and extensively drug-resistant M. tuberculosis complex strains and M. tuberculosis H37Rv. We included amikacin, capreomycin, moxifloxacin, prothionamide, clofazimine, linezolid, and rifabutin in the phenotypic test panels. For prothionamide and moxifloxacin, the various proposed breakpoint concentrations were tested by using the MGIT960 method. The MGIT960 method yielded results 10 days faster than the agar dilution method. For amikacin, capreomycin, linezolid, and rifabutin, results obtained by all methods were fully concordant. Applying a breakpoint of 0.5 g/ml for moxifloxacin led to results concordant with those of both the agar dilution method and the genotypic method. For prothionamide, concordance was noted only at the lowest and highest MICs. The phenotypic methods yielded largely identical results, except for those for prothionamide. Our study supports the following breakpoints for the MGIT960 method: 1 g/ml for amikacin, linezolid, and clofazimine, 0.5 g/ml for moxifloxacin and rifabutin, and 2.5 g/ml for capreomycin. No breakpoint was previously proposed for clofazimine. For prothionamide, a division into susceptible, intermediate, and resistant seems warranted, although the boundaries require additional study. The genotypic assay proved a reliable and rapid method for predicting aminoglycoside and fluoroquinolone resistance.The emergence of multidrug-resistant tuberculosis (MDR-TB) in the 1990s, and more recently, extensively drug resistant tuberculosis (XDR-TB), has revealed the need for new drugs and alternative, second-line treatment regimens. Many of these second-line drugs are either old drugs that had not been frequently used because of side effects or unproven efficacy or newer drugs intended primarily for treatment of other infections (3). Their use necessitated an evaluation of drug susceptibility testing (DST) and a determination of the critical concentrations of these alternative drugs.A variety of techniques are now available for second-line DST, of which the mycobacterium growth indicator tube automated liquid culture system (MGIT960) is probably the most used and best validated at this moment (13). The latest addition to second-line DST are genotypic methods, which detect mutations in the gyrA gene of Mycobacterium tuberculosis that are associated with fluoroquinolone resistance and mutations in the rrs operon that are associated with resistance to capreomycin and the aminoglycosides (2-5).Despite the arrival of these novel tools, many unce...

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bIn the Netherlands, clinical isolation of nontuberculous mycobacteria (NTM) has increased over the past decade. Proper identification of isolates is important, as NTM species differ strongly in clinical relevance. Most of the currently applied identification methods cannot distinguish between all different Mycobacterium species and complexes within species. rpoB gene sequencing exhibits a promising level of discrimination among rapidly and slowly growing mycobacteria, including the Mycobacterium avium complex. In this study, we prospectively compared rpoB gene sequencing with our routine algorithm of reverse line blot identification combined with partial 16S rRNA gene sequencing of 455 NTM isolates. rpoB gene sequencing identified 403 isolates to species level as 45 different known species and identified 44 isolates to complex level, and eight isolates remained unidentifiable to species level. In contrast, our reference reverse line blot assay with adjunctive 16S rRNA gene sequencing identified 390 isolates to species level (30 distinct species) and identified 56 isolates to complex level, and nine isolates remained unidentified. The higher discriminatory power of rpoB gene sequencing results largely from the distinction of separate species within complexes and subspecies. Also, Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium interjectum were separated into multiple groupings with relatively low sequence similarity (98 to 94%), suggesting that these are complexes of closely related species. We conclude that rpoB gene sequencing is a more discriminative identification technique than the combination of reverse line blot and 16S rRNA gene sequencing and could introduce a major improvement in clinical care of NTM disease and the research on the epidemiology and clinical relevance of NTM.

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The MTBDRplus and MTBDRsl assays may aid in decision making in tuberculosis treatment in low-level drug resistance settings and should preferably be used to exclude resistance.

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