Official American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention Clinical Practice Guidelines: Diagnosis of Tuberculosis in Adults and Children.

BACKGROUND: Individuals infected with Mycobacterium tuberculosis (Mtb) may develop symptoms and signs of disease (tuberculosis disease) or may have no clinical evidence of disease (latent tuberculosis infection [LTBI]). Tuberculosis disease is a leading cause of infectious disease morbidity and mortality worldwide, yet many questions related to its diagnosis remain. METHODS: A task force supported by the American Thoracic Society, Centers for Disease Control and Prevention, and Infectious Diseases Society of America searched, selected, and synthesized relevant evidence. The evidence was then used as the basis for recommendations about the diagnosis of tuberculosis disease and LTBI in adults and children. The recommendations were formulated, written, and graded using the Grading, Recommendations, Assessment, Development and Evaluation (GRADE) approach. RESULTS: Twenty-three evidence-based recommendations about diagnostic testing for latent tuberculosis infection, pulmonary tuberculosis, and extrapulmonary tuberculosis are provided. Six of the recommendations are strong, whereas the remaining 17 are conditional. CONCLUSIONS: These guidelines are not intended to impose a standard of care. They provide the basis for rational decisions in the diagnosis of tuberculosis in the context of the existing evidence. No guidelines can take into account all of the often compelling unique individual clinical circumstances.

Official American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention Clinical Practice Guidelines: Diagnosis of Tuberculosis in Adults and Children.

BACKGROUND: Individuals infected with Mycobacterium tuberculosis (Mtb) may develop symptoms and signs of disease (tuberculosis disease) or may have no clinical evidence of disease (latent tuberculosis infection [LTBI]). Tuberculosis disease is a leading cause of infectious disease morbidity and mortality worldwide, yet many questions related to its diagnosis remain. METHODS: A task force supported by the American Thoracic Society, Centers for Disease Control and Prevention, and Infectious Diseases Society of America searched, selected, and synthesized relevant evidence. The evidence was then used as the basis for recommendations about the diagnosis of tuberculosis disease and LTBI in adults and children. The recommendations were formulated, written, and graded using the Grading, Recommendations, Assessment, Development and Evaluation (GRADE) approach. RESULTS: Twenty-three evidence-based recommendations about diagnostic testing for latent tuberculosis infection, pulmonary tuberculosis, and extrapulmonary tuberculosis are provided. Six of the recommendations are strong, whereas the remaining 17 are conditional. CONCLUSIONS: These guidelines are not intended to impose a standard of care. They provide the basis for rational decisions in the diagnosis of tuberculosis in the context of the existing evidence. No guidelines can take into account all of the often compelling unique individual clinical circumstances.

Monocarbonyl analogs of curcumin inhibit growth of antibiotic sensitive and resistant strains of Mycobacterium tuberculosis.

Tuberculosis (TB) is a major public health concern worldwide with over 2 billion people currently infected. The rise of strains of Mycobacterium tuberculosis (Mtb) that are resistant to some or all first and second line antibiotics, including multidrug-resistant (MDR), extensively drug resistant (XDR) and totally drug resistant (TDR) strains, is of particular concern and new anti-TB drugs are urgently needed. Curcumin, a natural product used in traditional medicine in India, exhibits anti-microbial activity that includes Mtb, however it is relatively unstable and suffers from poor bioavailability. To improve activity and bioavailability, mono-carbonyl analogs of curcumin were synthesized and screened for their capacity to inhibit the growth of Mtb and the related Mycobacterium marinum (Mm). Using disk diffusion and liquid culture assays, we found several analogs that inhibit in vitro growth of Mm and Mtb, including rifampicin-resistant strains. Structure activity analysis of the analogs indicated that Michael acceptor properties are critical for inhibitory activity. However, no synergistic effects were evident between the monocarbonyl analogs and rifampicin on inhibiting growth. Together, these data provide a structural basis for the development of analogs of curcumin with pronounced anti-mycobacterial activity and provide a roadmap to develop additional structural analogs that exhibit more favorable interactions with other anti-TB drugs.

Evaluation of the Cepheid Xpert MTB/RIF assay.

The lack of capacity to provide laboratory confirmation of a diagnosis of tuberculosis disease (TB) is contributing to enormous gaps in the ability to find, treat and follow TB patients. WHO estimates that globally only about 57% of the notified new cases of pulmonary TB in 2012 and about 19% of rifampicin-resistant TB cases were laboratory confirmed. The Cepheid Xpert(®) MTB/RIF assay has been credited with revolutionizing laboratory testing to aid in the diagnosis of TB and rifampicin-resistant TB. This semi-automated test can detect both the causative agent of TB and mutations that confer rifampicin resistance from clinical specimens within 2 h after starting the test. In this article, we review the performance of the test, its pathway to regulatory approval and endorsement, guidelines for its use and lessons learned from the implementation of the test in low-burden, high-resource countries and in high-burden, low-resource countries.

Aminoglycoside cross-resistance in Mycobacterium tuberculosis due to mutations in the 5' untranslated region of whiB7.

Since the discovery of streptomycin's bactericidal activity against Mycobacterium tuberculosis, aminoglycosides have been utilized to treat tuberculosis (TB). Today, the aminoglycosides kanamycin and amikacin are used to treat multidrug-resistant (MDR) TB, and resistance to any of the second-line injectable antibiotics, including kanamycin, amikacin, or capreomycin, is a defining characteristic of extensively drug-resistant (XDR) TB. Resistance to kanamycin and streptomycin is thought to be due to the acquisition of unlinked chromosomal mutations. However, we identified eight independent mutations in the 5' untranslated region of the transcriptional activator whiB7 that confer low-level resistance to both aminoglycosides. The mutations lead to 23- to 145-fold increases in whiB7 transcripts and subsequent increased expression of both eis (Rv2416c) and tap (Rv1258c). Increased expression of eis confers kanamycin resistance in these mutants, while increased expression of tap, which encodes an efflux pump, is a previously uncharacterized mechanism of low-level streptomycin resistance. Additionally, high-level resistance to streptomycin arose at a much higher frequency in whiB7 mutants than in a wild-type (WT) strain. Although whiB7 is typically associated with intrinsic antibiotic resistance in M. tuberculosis, these data suggest that mutations in an uncharacterized regulatory region of whiB7 contribute to cross-resistance against clinically used second-line antibiotics. As drug resistance continues to develop and spread, understanding the mechanisms and molecular basis of antibiotic resistance is critical for the development of rapid molecular tests to diagnose drug-resistant TB strains and ultimately for designing regimens to treat drug-resistant cases of TB.

GeneXpert for TB diagnosis: planned and purposeful implementation.

Xpert MTB/RIF is a major advance for TB diagnostics, especially for multidrug-resistant (MDR) TB and HIV-associated TB. But implementation concerns including cost, technical support requirements, and challenging demands of providing second-line TB drugs for diagnosed MDR-TB cases call for gradual, careful introduction based on country circumstances.

Imatinib-sensitive tyrosine kinases regulate mycobacterial pathogenesis and represent therapeutic targets against tuberculosis.

The lengthy course of treatment with currently used antimycobacterial drugs and the resulting emergence of drug-resistant strains have intensified the need for alternative therapies against Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis. We show that Mtb and Mycobacterium marinum use ABL and related tyrosine kinases for entry and intracellular survival in macrophages. In mice, the ABL family tyrosine kinase inhibitor, imatinib (Gleevec), when administered prophylactically or therapeutically, reduced both the number of granulomatous lesions and bacterial load in infected organs and was also effective against a rifampicin-resistant strain. Further, when coadministered with current first-line drugs, rifampicin or rifabutin, imatinib acted synergistically. These data implicate host tyrosine kinases in entry and intracellular survival of mycobacteria and suggest that imatinib may have therapeutic efficacy against Mtb. Because imatinib targets host, it is less likely to engender resistance compared to conventional antibiotics and may decrease the development of resistance against coadministered drugs.

Cellular immune responses to nine Mycobacterium tuberculosis vaccine candidates following intranasal vaccination.

BACKGROUND: The identification of Mycobacterium tuberculosis vaccines that elicit a protective immune response in the lungs is important for the development of an effective vaccine against tuberculosis. METHODS AND PRINCIPAL FINDINGS: In this study, a comparison of intranasal (i.n.) and subcutaneous (s.c.) vaccination with the BCG vaccine demonstrated that a single moderate dose delivered intranasally induced a stronger and sustained M. tuberculosis-specific T-cell response in lung parenchyma and cervical lymph nodes of BALB/c mice than vaccine delivered subcutaneously. Both BCG and a multicomponent subunit vaccine composed of nine M. tuberculosis recombinant proteins induced strong antigen-specific T-cell responses in various local and peripheral immune compartments. Among the nine recombinant proteins evaluated, the alanine proline rich antigen (Apa, Rv1860) was highly antigenic following i.n. BCG and immunogenic after vaccination with a combination of the nine recombinant antigens. The Apa-induced responses included induction of both type 1 and type 2 cytokines in the lungs as evaluated by ELISPOT and a multiplexed microsphere-based cytokine immunoassay. Of importance, i.n. subunit vaccination with Apa imparted significant protection in the lungs and spleen of mice against M. tuberculosis challenge. Despite observed differences in the frequencies and location of specific cytokine secreting T cells both BCG vaccination routes afforded comparable levels of protection in our study. CONCLUSION AND SIGNIFICANCE: Overall, our findings support consideration and further evaluation of an intranasally targeted Apa-based vaccine to prevent tuberculosis.

Mycobacterium tuberculosis components stimulate production of the antimicrobial peptide hepcidin.

We investigated the in vitro production of the antimicrobial peptide hepcidin by cells of the innate immune system that harbor Mycobacterium tuberculosis. Stimulation of mouse lung macrophages with M. tuberculosis or IFN-γ + M. tuberculosis induced hepcidin mRNA. In human alveolar A549 epithelial cells, lipoglycans of M. tuberculosis, in particular mannose-capped lipoarabinomannan and phosphatidyl-myo-inositol mannosides, were strong inducers of hepcidin mRNA. In mouse dendritic cells, hepcidin mRNA was increased by subcellular fractions and culture filtrate proteins of M. tuberculosis and by TLR2 and TLR4 agonists, but not by TLR9 agonists, IL-1α, IL-6 or TNF-α. Flow cytometry evaluation of human peripheral blood mononuclear cells demonstrated that CD11c(+) myeloid dendritic cells stimulated with killed M. tuberculosis or live M. bovis BCG produced hepcidin. The production of the antimicrobial peptide hepcidin by cells that interact with M. tuberculosis suggests a host defense mechanism against mycobacteria.

Core gene set as the basis of multilocus sequence analysis of the subclass Actinobacteridae.

Comparative genomic sequencing is shedding new light on bacterial identification, taxonomy and phylogeny. An in silico assessment of a core gene set necessary for cellular functioning was made to determine a consensus set of genes that would be useful for the identification, taxonomy and phylogeny of the species belonging to the subclass Actinobacteridae which contained two orders Actinomycetales and Bifidobacteriales. The subclass Actinobacteridae comprised about 85% of the actinobacteria families. The following recommended criteria were used to establish a comprehensive gene set; the gene should (i) be long enough to contain phylogenetically useful information, (ii) not be subject to horizontal gene transfer, (iii) be a single copy (iv) have at least two regions sufficiently conserved that allow the design of amplification and sequencing primers and (v) predict whole-genome relationships. We applied these constraints to 50 different Actinobacteridae genomes and made 1,224 pairwise comparisons of the genome conserved regions and gene fragments obtained by using Sequence VARiability Analysis Program (SVARAP), which allow designing the primers. Following a comparative statistical modeling phase, 3 gene fragments were selected, ychF, rpoB, and secY with R2>0.85. Selected sets of broad range primers were tested from the 3 gene fragments and were demonstrated to be useful for amplification and sequencing of 25 species belonging to 9 genera of Actinobacteridae. The intraspecies similarities were 96.3-100% for ychF, 97.8-100% for rpoB and 96.9-100% for secY among 73 strains belonging to 15 species of the subclass Actinobacteridae compare to 99.4-100% for 16S rRNA. The phylogenetic topology obtained from the combined datasets ychF+rpoB+secY was globally similar to that inferred from the 16S rRNA but with higher confidence. It was concluded that multi-locus sequence analysis using core gene set might represent the first consensus and valid approach for investigating the bacterial identification, phylogeny and taxonomy.

Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis.

The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the -10 and -35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20-180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 microg/mL < minimum inhibitory concentration (MIC)

Mutations at embB codon 306 are an important molecular indicator of ethambutol resistance in Mycobacterium tuberculosis.

Ethambutol resistance in clinical Mycobacterium tuberculosis isolates is associated primarily with missense mutations in the embB gene. However, recent reports have described the presence of embB mutations, especially those at embB codon 306, in isolates susceptible to ethambutol. To clarify the role of embB mutations in ethambutol resistance, we sequenced the ethambutol resistance-determining region in spontaneous ethambutol-resistant mutants. In our study, 66% of spontaneous mutants contained a single point mutation in embB, with 55% of these occurring at embB 306. The MIC of ethambutol for spontaneous mutants was increased two- to eightfold relative to the pansusceptible M. tuberculosis strains from which the mutants were generated. To further characterize the role of embB 306 mutations, we directly introduced mutant alleles, embB(M306V) or embB(M306I), into pansusceptible M. tuberculosis strains and conversely reverted mutant alleles in spontaneous ethambutol-resistant mutants back to those of the wild type via allelic exchange using specialized linkage transduction. We determined that the MIC of ethambutol was reduced fourfold for three of the four spontaneous ethambutol-resistant embB 306 mutants when the mutant allele was replaced with the wild-type embB allele. The MIC for one of the spontaneous mutants genetically reverted to wild-type embB was reduced by only twofold. When the wild-type embB allele was converted to the mutant allele embB(M306V), the ethambutol MIC was increased fourfold, and when the allele was changed to M306I, the ethambutol MIC increased twofold. Our data indicate that embB 306 mutations are sufficient to confer ethambutol resistance, and detection of these mutations should be considered in the development of rapid molecular tests.

Complete rpoB gene sequencing as a suitable supplement to DNA-DNA hybridization for bacterial species and genus delineation.

DNA-DNA hybridization (DDH), the gold standard for bacterial species delineation, is a laborious method and the alternative, average nucleotide identity (ANI), a genomic sequence-derived parameter, is not applicable to non-sequenced species. A universal cut-off value to delineate bacterial species does not exist, yet a DDH value <70 % and ANI <95+/-0.5 % have proved useful in selected examples. We herein compare published values for DDH and ANI with sequence similarity of rpoB gene sequences retrieved from GenBank for strains of 230 bacterial species representative of 45 genera. Intraspecific rpoB sequence similarity was 98.2-100 %. We observed that an rpoB gene sequence similarity

Genome-wide exploration of the drug action of capreomycin on Mycobacterium tuberculosis using Affymetrix oligonucleotide GeneChips.

OBJECTIVE: Multi-drug resistance and latent infection are two major issues in current tuberculosis (TB) control and management. Capreomycin is an important drug used for TB with multi-drug resistance. A recent study also indicates that this drug possesses unique bactericidal activity against non-replicating TB bacilli among known anti-TB drugs. Thus, there is an urgent need for investigating the full-spectrum action of capreomycin. METHODS: Here we conduct the first microarray-based study on capreomycin using the high-resolution Affymetrix oligonucleotide GeneChip system. RESULTS: The results indicate that capreomycin primarily acts on the information pathways but it also significantly affects cell wall, cell processes, intermediate metabolism and respiration in Mycobacterium tuberculosis. CONCLUSIONS: This study not only transcriptionally validates the specific molecular target, 16S rRNA, but also discovers potential new targets of capreomycin, including genes operating at the DNA level, such as Rv0054 (ssb) and Rv3715c (recR), as well as genes involved in cell division like Rv3260c (whiB2). In addition, the nuo gene cluster and the ATP synthase gene cluster are repressed.

Understanding the action of INH on a highly INH-resistant Mycobacterium tuberculosis strain using Genechips.

The availability of the complete sequence of Mycobacterium tuberculosis genome coupled with microarray technology has enabled a high-throughput approach to the pharmacogenomics of this organism. Isoniazid (INH) is a first-line drug for the treatment of tuberculosis and the microarray approach has generated new insight into the action of INH on a drug-susceptible strain. It has also shown that INH does not induce any significant change in gene expression when applied to a catalase-negative INH-resistant strain, which is expected because catalase activity is required to convert the prodrug INH to its active form. But it has yet to be determined how a partially resistant strain responds to INH. In this study, we explore the mechanism of INH against a highly INH-resistant strain, compare drug-induced gene-expression profiles between resistant and susceptible strains, and determine whether or not and how the resistant strain responds to INH at low and high concentrations. The global gene-expression profiles of the resistant strain in response to INH treatments were obtained using the Affymetrix oligonucleotide GeneChips. The results showed that the resistant strain did not exhibit the characteristic gene-expression signature of type II fatty acid synthase (FAS-II) inhibition when exposed to low-level INH, but it responded with that specific pattern under high-level INH, although the response profile was somewhat shrunken relative to that for a susceptible strain. We found that INH acted on the FAS-II pathway in both resistant and susceptible strains, and little evidence suggested that INH might kill resistant bacteria via other mechanisms. This suggests that there may be potential benefit of treating INH-resistant bacteria with INH at a level that is effective and safe.

The acid-induced operon Rv3083-Rv3089 is required for growth of Mycobacterium tuberculosis in macrophages.

The Rv3083-Rv3089 operon of Mycobacterium tuberculosis has been shown to be induced 17-33-fold when tubercle bacilli were exposed in vitro to acidic conditions which may mimic those that the bacilli encounter early during the infection and it is induced during growth in macrophages. To understand the role of this operon in intracellular survival, we constructed a knockout of the operon in the M. tuberculosis H37Rv strain. No differences were observed in the growth of mutant and wild-type mycobacteria on axenic media. Though the uptake of mutant and wild-type bacteria by eukaryotic cells was similar, the mutant failed to grow subsequently. By 192h post-infection, the fold differences between the wild-type and mutant bacteria were significant thus leading to the conclusion that the mutant is defective for intracellular growth in these cell lines. Complementation of the knockout restored intracellular growth to wild-type levels. During the first 24-48h post-infection, mutant bacteria also stimulated production of significantly less IL-1beta, IL-6, IL-8, RANTES, and MCP-1 by THP-1 cells than wild-type bacteria. Overall, the data indicate that the operon plays an important role in the ability of M. tuberculosis to grow inside host cells.

Tuberculosis subunit vaccine development: impact of physicochemical properties of mycobacterial test antigens.

Tuberculosis caused by Mycobacterium tuberculosis continues to be one of the major public health problems in the world. The eventual control of this disease will require the development of a safe and effective vaccine. One of the approaches receiving a great deal of attention recently is subunit vaccination. An efficacious antituberculous subunit vaccine requires the identification and isolation of key components of the pathogen that are capable of inducing a protective immune response. Clues to identify promising subunit vaccine candidates may be found in their physicochemical and immunobiological properties. In this article, we review the evidence that the physicochemical properties of mycobacterial components can greatly impact the induction of either protective or deleterious immune response and consequently influence the potential utility as an antituberculous subunit vaccine.