The distribution of 20 variable regions resulting from insertiondeletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and ''modern'' strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum3 M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.evolution ͉ diagnostic ͉ identification T he mycobacteria grouped in the Mycobacterium tuberculosis complex are characterized by 99.9% similarity at the nucleotide level and identical 16S rRNA sequences (1, 2) but differ widely in terms of their host tropisms, phenotypes, and pathogenicity. Assuming that they all are derived from a common ancestor, it is intriguing that some are exclusively human (M. tuberculosis, Mycobacterium africanum, Mycobacterium canettii) or rodent pathogens (Mycobacterium microti), whereas others have a wide host spectrum (Mycobacterium bovis). What was the genetic organization of the last common ancestor of the tubercle bacilli, and in which host did it live? Which genetic events may have contributed to the fact that the host spectrum is so different and often specific? Where and when did M. tuberculosis evolve? Answers to these questions are important for a better understanding of the pathogenicity and the global epidemiology of tuberculosis and may help to anticipate future trends in the spread of the disease.Because of the unusually high degree of conservation in their housekeeping genes, it has been suggested that the members of the M. tuberculosis complex underwent an evolutionary bottleneck at the time of speciation, estimated to have occurred roughly 15,000-20,000 years ago (2). Also, it has been speculated tha...

SUMMARY With an estimated 9.4 million new cases globally, tuberculosis (TB) continues to be a major public health concern. Eighty percent of all cases worldwide occur in 22 high-burden, mainly resource-poor settings. This devastating impact of tuberculosis on vulnerable populations is also driven by its deadly synergy with HIV. Therefore, building capacity and enhancing universal access to rapid and accurate laboratory diagnostics are necessary to control TB and HIV-TB coinfections in resource-limited countries. The present review describes several new and established methods as well as the issues and challenges associated with implementing quality tuberculosis laboratory services in such countries. Recently, the WHO has endorsed some of these novel methods, and they have been made available at discounted prices for procurement by the public health sector of high-burden countries. In addition, international and national laboratory partners and donors are currently evaluating other new diagnostics that will allow further and more rapid testing in point-of-care settings. While some techniques are simple, others have complex requirements, and therefore, it is important to carefully determine how to link these new tests and incorporate them within a country's national diagnostic algorithm. Finally, the successful implementation of these methods is dependent on key partnerships in the international laboratory community and ensuring that adequate quality assurance programs are inherent in each country's laboratory network.

We performed a multicenter prevalence study of nontuberculous mycobacteria (NTM) involving 1,582 patients (mean age, 18.9 years; male/female ratio, 1.06) with cystic fibrosis in France. The overall NTM prevalence (percentage of patients with at least one positive culture) was 6.6% (104/1,582 patients), with prevalences ranging from 3.7% (in the east of France) to 9.6% (in the greater Paris area). Mycobacterium abscessus complex (MABSC; 50 patients) and Mycobacterium avium complex (MAC; 23 patients) species were the most common NTM, and the only ones associated with fulfillment of the American Thoracic Society bacteriological criteria for NTM lung disease. The "new" species, Mycobacterium bolletii and Mycobacterium massiliense, accounted for 40% of MABSC isolates. MABSC species were isolated at all ages, with a prevalence peak between 11 and 15 years of age (5.8%), while MAC species reached their highest prevalence value among patients over 25 years of age (2.2%).Nontuberculous mycobacteria (NTM) have emerged as "new" pathogens in cystic fibrosis (CF) patients over the last 2 decades (10). CF centers worldwide have reported isolation of NTM from the respiratory tracts of CF patients, with prevalence values ranging from 5% to 20% (5,6,8,9,13,14,16,19,22,25). Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABSC) species are the most frequently isolated NTM and together account for Ͼ95% of NTM lung diseases affecting CF patients. The MAC, a member of the subgroup comprising slowly growing mycobacteria, ranks first in North America (22), whereas the MABSC, a member of the subgroup comprising rapidly growing mycobacteria, seems to predominate in Western Europe (15,23,25) and is also more prevalent than the MAC in Israel (19).Previous studies have reported isolation of NTM from 6.6 to 9.8% of French CF cohorts (9,23,25). These studies also reported a much higher isolation rate for MABSC than for MAC or other NTM species (23,25). However, these studies were done exclusively in pediatric CF centers in Paris. This may have distorted the results since MABSC species are more prevalent than MAC species in children (23). Moreover, the epidemiology of NTM in Paris does not necessarily reflect the situation in other regions of France. For example, studies involving non-CF patients have reported higher rates of NTM disease in urban areas (20). Moreover, previous French studies were performed before M. abscessus (now M. abscessus sensu lato, or the MABSC) was shown to include at least three distinct species, M. abscessus (sensu stricto) (hereafter referred to as M. abscessus), Mycobacterium massiliense, and Mycobacterium bolletii (1,3). The prevalences of these three species in CF patients in France were therefore unknown.We thus conducted a large, prospective, nationwide study addressing NTM prevalence in CF patients in France. This study shows relatively low prevalence figures for French CF centers. It also provides evidence that MABSC species are currently the most prevalent NTM in the French CF populat...

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536 T , M. massiliense CIP 108297 T , and M. bolletii CIP 108541 T ) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.