This paper describes an action framework for countries with low tuberculosis (TB) incidence (<100 TB cases per million population) that are striving for TB elimination. The framework sets out priority interventions required for these countries to progress first towards “pre-elimination” (<10 cases per million) and eventually the elimination of TB as a public health problem (less than one case per million). TB epidemiology in most low-incidence countries is characterised by a low rate of transmission in the general population, occasional outbreaks, a majority of TB cases generated from progression of latent TB infection (LTBI) rather than local transmission, concentration to certain vulnerable and hard-to-reach risk groups, and challenges posed by cross-border migration. Common health system challenges are that political commitment, funding, clinical expertise and general awareness of TB diminishes as TB incidence falls. The framework presents a tailored response to these challenges, grouped into eight priority action areas: 1) ensure political commitment, funding and stewardship for planning and essential services; 2) address the most vulnerable and hard-to-reach groups; 3) address special needs of migrants and cross-border issues; 4) undertake screening for active TB and LTBI in TB contacts and selected high-risk groups, and provide appropriate treatment; 5) optimise the prevention and care of drug-resistant TB; 6) ensure continued surveillance, programme monitoring and evaluation and case-based data management; 7) invest in research and new tools; and 8) support global TB prevention, care and control. The overall approach needs to be multisectorial, focusing on equitable access to high-quality diagnosis and care, and on addressing the social determinants of TB. Because of increasing globalisation and population mobility, the response needs to have both national and global dimensions.

Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-α, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-γ-inducing cytokines, IL-12 and IFN-α, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-γ production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-α, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-α and IL-18 play a significant role in enhancing IFN-γ synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.

The objectives of this study were to investigate the origin of highly discordant rifampin (rifampicin) (RMP) drug susceptibility test results obtained for Mycobacterium tuberculosis strains during proficiency testing. Nine Supra-National Tuberculosis Reference Laboratories tested the RMP susceptibilities of 19 selected M. tuberculosis strains, using standard culture-based methods. The strains were classified as definitely resistant (R) ( n = 6) or susceptible (S) ( n = 2) or probably resistant (PR) ( n = 8) or susceptible (PS) ( n = 3) based on rpoB mutations and treatment outcome. All methods yielded a susceptible result for the two S and three PS strains lacking an rpoB mutation and a resistant result for one R strain with a Ser531Leu mutation and one PR strain with a double mutation. Although the remaining 12 R and PR strains had rpoB mutations (four Asp516Tyr, three Leu511Pro, two Leu533Pro, one each His526Leu/Ser, and one Ile572Phe), they were all susceptible by the radiometric Bactec 460TB or Bactec 960 MGIT methods. In contrast, only one was susceptible by the proportion method on Löwenstein-Jensen medium and two on Middlebrook 7H10 agar. Low-level but probably clinically relevant RMP resistance linked to specific rpoB mutations is easily missed by standard growth-based methods, particularly the automated broth-based systems. Further studies are required to confirm these findings, to determine the frequency of these low-level-resistant isolates, and to identify technical improvements that may identify such strains.

Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains represent a major threat for tuberculosis (TB) control. Treatment of MDR-TB patients is long and less effective, resulting in a significant number of treatment failures. The development of further resistances leads to extensively drug-resistant (XDR) variants. However, data on the individual reasons for treatment failure, e.g. an induced mutational burst, and on the evolution of bacteria in the patient are only sparsely available. To address this question, we investigated the intra-patient evolution of serial MTBC isolates obtained from three MDR-TB patients undergoing longitudinal treatment, finally leading to XDR-TB. Sequential isolates displayed identical IS6110 fingerprint patterns, suggesting the absence of exogenous re-infection. We utilized whole genome sequencing (WGS) to screen for variations in three isolates from Patient A and four isolates from Patient B and C, respectively. Acquired polymorphisms were subsequently validated in up to 15 serial isolates by Sanger sequencing. We determined eight (Patient A) and nine (Patient B) polymorphisms, which occurred in a stepwise manner during the course of the therapy and were linked to resistance or a potential compensatory mechanism. For both patients, our analysis revealed the long-term co-existence of clonal subpopulations that displayed different drug resistance allele combinations. Out of these, the most resistant clone was fixed in the population. In contrast, baseline and follow-up isolates of Patient C were distinguished each by eleven unique polymorphisms, indicating an exogenous re-infection with an XDR strain not detected by IS6110 RFLP typing. Our study demonstrates that intra-patient microevolution of MDR-MTBC strains under longitudinal treatment is more complex than previously anticipated. However, a mutator phenotype was not detected. The presence of different subpopulations might confound phenotypic and molecular drug resistance tests. Furthermore, high resolution WGS analysis is necessary to accurately detect exogenous re-infection as classical genotyping lacks discriminatory power in high incidence settings.

The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis , but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine- N -oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O ₂ · ⁻ and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O ₂ · ⁻ , with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O ₂ · ⁻ -mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.

The virulence of a Mycobacterium tuberculosis H37Rv sigE mutant was studied in immunodeficient and immunocompetent mice. The mutant was strongly attenuated in both animal models and induced formation of granulomas with different characteristics than those induced by the wild-type strain.