Asunto(s)

RESUMEN

SETTING: Studies of US populations have produced conflicting findings about the impact of diabetes mellitus (DM) on tuberculosis (TB) treatment outcomes. OBJECTIVE: To investigate the association between DM and all-cause mortality among patients on anti-tuberculosis treatment in California, USA. DESIGN: Using TB surveillance data, we conducted a retrospective analysis of California patients with culture-confirmed TB who started anti-tuberculosis treatment during 2010-2014. We used Cox proportional hazards models to estimate the association of DM with all-cause mortality and conducted a sensitivity analysis to estimate the attenuating effect of unmeasured confounding by body mass index. RESULTS: Among 8461 patients with TB, 2124 (25.1%) had DM and 713 (8.4%) died during anti-tuberculosis treatment. A higher proportion of TB-DM patients died (13.1% vs. 6.8% TB-no DM). After adjusting for confounders, DM was associated with mortality (adjusted hazards ratio [aHR] 1.35, 95%CI 1.15-1.57). There was effect modification by human immunodeficiency virus (HIV) status, with HIV-positive patients having an aHR of 5.33 (95%CI 1.76-16.12). CONCLUSION: TB patients with DM had a greater hazard of death during anti-tuberculosis treatment than those without DM. Further investigation into the impact of HIV on the relation of DM to death is necessary.

Asunto(s)

RESUMEN

SETTING: California, United States. OBJECTIVE: To determine the frequency of tuberculosis (TB) patients at risk for relapse who received at least 9 months of anti-tuberculosis treatment (extended treatment) and to identify factors associated with not receiving extended treatment. DESIGN: We analyzed characteristics of culture-confirmed pulmonary TB patients reported to the California TB Registry during 2004-2009. Patients with cavities on initial chest radiograph and delayed culture conversion (⩾70 days) were at 'high risk of relapse', and anti-tuberculosis treatment of ⩾270 days was 'extended treatment'. We used a generalized linear model to identify independent risk factors for absence of extended treatment in the high risk of relapse group. RESULTS: Among 5680 TB patients, 483 (8.5%) were at high risk of relapse: 372 (77%) received extended treatment but 111 (23%) did not. Factors associated with absence of extended treatment included negative sputum smears (adjusted prevalence ratio [aPR] 2.62, 95%CI 1.69-4.05), residence in three specific counties (aPR 1.71, 95%CI 1.19-2.46) and Black race (aPR 1.56, 95%CI 1.03-2.38). CONCLUSIONS: Nearly a quarter of TB patients at high risk of relapse did not receive extended treatment. Increased efforts are needed to ensure that all patients who may benefit from extended anti-tuberculosis treatment receive it.

Asunto(s)

RESUMEN

OBJECTIVE: To estimate the burden of tuberculosis (TB) contact investigations in California, assess outcomes and effectiveness, and identify performance gaps. METHODS: Aggregate program management reports were used to examine contact investigations conducted for pulmonary TB cases reported between 1 July 1999 and 30 June 2000 in California. Findings were compared to national objectives, and performance gaps were identified. Costs were estimated, and effectiveness of TB case detection and prevention was assessed. RESULTS: A total of 2032 acid-fast bacilli sputum smear-positive and sputum smear-negative/culture-positive cases was reported; 17774 contacts were elicited, and 15582 (88%) contacts were evaluated. TB disease and latent tuberculosis infection (LTBI) were diagnosed in 111 (<1%) and 4609 (30%) contacts, respectively; 1958 (43%) contacts with LTBI completed treatment. Costs of contact investigations were estimated at dollars 4.8 million; 81% of expected TB cases were detected, but only 35% of cases expected to occur within 2 years following the investigation were prevented. CONCLUSIONS: California's performance did not meet national objectives for contact evaluation or treatment completion; improved effectiveness of contact investigations in California is needed. Although analysis of existing contact investigation surveillance data provided a macro-level view of performance gaps, expanded surveillance data are required to inform interventions.

Asunto(s)

RESUMEN

This study assessed the extent to which laboratory methods recommended by the Centers for Disease Control and Prevention were used in tuberculosis testing of patients in California in 1998. While recommended methods were used for most patients, there was room for improvement by hospital and independent non-health maintenance organization laboratories.

Asunto(s)

RESUMEN

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a tropical ulcerative skin disease. One of the most intriguing aspects of this disease is the presence of extensive tissue damage in the absence of an acute inflammatory response. We recently purified and characterized a macrolide toxin, mycolactone, from M. ulcerans. Injection of this molecule into guinea pig skin reproduced cell death and lack of acute inflammatory response similar to that seen following the injection of viable bacteria. We also showed that mycolactone causes a cytopathic effect on mouse fibroblast L929 cells that is characterized by cytoskeletal rearrangements and growth arrest within 48 h. However, these results could not account for the extensive cell death which occurs in Buruli ulcer. The results presented here demonstrate that L929 and J774 mouse macrophage cells die via apoptosis after 3 to 5 days of exposure to mycolactone. Treatment of cells with a pan-caspase inhibitor can inhibit mycolactone-induced apoptosis. We demonstrate that injection of mycolactone into guinea pig skin results in cell death via apoptosis and that the extent of apoptosis increases as the lesion progresses. These results may help to explain why tissue damage in Buruli ulcer is not accompanied by an acute inflammatory response.

Asunto(s)

RESUMEN

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom.

Asunto(s)

RESUMEN

Salmonella typhi and Salmonella gallinarum phenotypes correlated with mouse host restriction have been identified by using in vitro and in vivo systems. S. typhi is capable of entering the murine intestinal epithelium via M cells, as is Salmonella typhimurium, which causes systemic infection in the mouse. But, unlike S. typhimurium, S. typhi does not destroy the epithelium and is cleared from the Peyer's patches soon after M-cell entry. S. gallinarum appears to be incapable of entering the murine Peyer's patch epithelium. Our in vitro evidence suggests that S. gallinarum is taken up in murine phagocytic cells by a mechanism different from that of S. typhimurium. S. typhimurium is taken up at a higher frequency and is maintained at higher viable counts throughout a 24-h time course in a murine macrophage-like cell line than are S. gallinarum and S. typhi.

Asunto(s)

RESUMEN

Enteric microbial pathogens interact with the gut epithelium to establish infection. Recently, it has become clear that many microorganisms that colonize or traverse the intestinal mucosa do so via the specialized M cells. Recent work has shown that Shigella flexneri and Salmonella typhimurium specifically target M cells to initiate infection of the host.

Asunto(s)

RESUMEN

Tuberculosis continues to be responsible for the deaths of millions of people, yet the virulence factors of the causative pathogens remain unknown. Genetic complementation experiments with strains of the Mycobacterium tuberculosis complex have identified a gene from a virulent strain that restores virulence to an attenuated strain. The gene, designated rpoV, has a high degree of homology with principal transcription or sigma factors from other bacteria, particularly Mycobacterium smegmatis and Streptomyces griseus. The homologous rpoV gene of the attenuated strain has a point mutation causing an arginine-->histidine change in a domain known to interact with promoters. To our knowledge, association of loss of bacterial virulence with a mutation in the principal sigma factor has not been previously reported. The results indicate either that tuberculosis organisms have an alternative principal sigma factor that promotes virulence genes or, more probably, that this particular mutant principal sigma factor is unable to promote expression of one or more genes required for virulence. Study of genes and proteins differentially regulated by the mutant transcription factor should facilitate identification of further virulence factors.

Asunto(s)

RESUMEN

Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra.

Asunto(s)

RESUMEN

Diaminopimelic acid (DAP) is a major component of the peptidoglycan layer of the mycobacterial cell wall. The mycobacterial cell wall has been implicated as a potential virulence factor and is highly immunogenic. The pathway for biosynthesis of DAP may serve as a target in the design of antimycobacterial agents and construction of in vivo selection systems. Despite its significance, this biosynthetic pathway is poorly understood in mycobacteria. In order to develop a better understanding of mycobacterial DAP biosynthesis, the aspartate semialdehyde dehydrogenase (asd) genes of Mycobacterium smegmatis, bacille Calmette-Guerin (BCG), Mycobacterium avium, Mycobacterium leprae, and Mycobacterium tuberculosis were isolated. The M. smegmatis asd gene was isolated by complementation in Escherichia coli. This gene was then used to isolate the asd genes from other mycobacteria. The asd-complementing fragments from BCG and M. smegmatis were sequenced. An open reading frame upstream of the mycobacterial asd gene was identified as the mycobacterial aspartokinase gene (ask). Primer extension analysis revealed that the only transcriptional start in this region is found 5' of the ask gene. This observation indicates that the mycobacterial ask and asd genes are in an operon.

Asunto(s)

RESUMEN

The purpose of this work was to develop a system to identify virulence determinants of M. tuberculosis by genetic complementation. The ability to grow in mouse spleen and/or lung was defined as a potential phenotype for virulence. Enrichment for growing recombinant clones from a pool of H37Ra transformants containing the integrating pYUB178::H37Rv cosmid library was accomplished by in vivo selection. A molecular strategy was devised to isolate and clone the 25-kb H37Rv genomic fragment ivg that conferred in vivo growth advantage to H37Ra. This study is a first step toward understanding the genetics of virulence in M. tuberculosis. A detailed description of these experiments has been submitted for publication in Infection and Immunity.

Asunto(s)

RESUMEN

We have developed a novel method for screening a Mycobacterium bovis (BCG) cosmid library in Mycobacterium smegmatis for the detection of immunostimulatory T-cell antigens (Ag). Distinctive protein banding patterns were demonstrated in culture filtrates of three of 30 recombinant M. smegmatis clones: pBCCS13 (41 and 73 kDa); pBCCS221 (30, 50 and 68 kDa); pBCCS223 (100 kDa). Western immunoblots indicated that monoclonal antibodies (mAb) directed to the previously characterized 19-, 30-, 38-, 65- and 71-kDa mycobacterial Ag were not reactive with the distinctive recombinant proteins. Furthermore, T-cell Western blots demonstrated that fractions containing the distinctive proteins were immunostimulatory. A given tuberculin-positive donor expressed unique patterns of blastogenic reactivity to protein fractions isolated from each of the three recombinant clones. Restriction enzyme digests of the three recombinant BCG inserts revealed distinctive DNA-banding patterns. The immunostimulatory Ag, therefore, are most likely encoded within different regions of the BCG genome, as contained within three distinct inserts. T-cell Western blots further indicated a heterogeneity in the repertoire of BCG-responsive T cells since tuberculin-positive donors varied in the pattern of reactivity to protein fractions isolated from the same recombinant filtrate. Most likely, immunity to M. tuberculosis results from activation of a heterogeneous array of T cells targeted to multiple immunostimulatory Ag. The method we describe should greatly enhance our ability to define the full spectrum of T-cell Ag encoded by mycobacteria, particularly those which are secreted proteins.

Asunto(s)

RESUMEN

Bacteria within the Mycobacterium avium complex are prominent in the environment and are a source of serious disseminated infections in patients with AIDS. Serovars of the M. avium complex are distinguished from all other mycobacteria and from one another by the presence of highly antigenic glycolipids, the glycopeptidolipids, on their surfaces. A genomic library of DNA from serovar 2 of the M. avium complex was constructed in the Escherichia coli-Mycobacterium shuttle cosmid, pYUB18, and used to clone and express in Mycobacterium smegmatis the genes responsible for the biosynthesis of the oligosaccharide segment of the M. avium serovar 2-specific glycopeptidolipid. The responsible gene cluster was mapped to a 22- to 27-kb functional region of the M. avium genome. The recombinant glycolipid was also isolated by high-pressure liquid chromatography and chemically characterized, by gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry, to demonstrate that the lipopeptide core originated in M. smegmatis, whereas the oligosaccharide segment arose from the cloned M. avium genes. This first-time demonstration of the cloning and expression, in a nonpathogenic mycobacterium, of the genes encoding complex cell wall glycoconjugates from a pathogenic mycobacterium presents a new approach for studying the role of such products in disease processes.

Asunto(s)

RESUMEN

Mycobacteriophage L5, a temperate phage of mycobacteria, integrates site-specifically into the Mycobacterium smegmatis chromosome. We have identified the int gene and attP site of L5, characterized the chromosomal attachment site (attB), and constructed plasmid vectors that efficiently transform M. smegmatis through stable site-specific integration of the plasmid into the bacterial genome. These integration-proficient plasmids also efficiently transform slow-growing mycobacteria such as the pathogen Mycobacterium tuberculosis and the vaccine strain bacille Calmette-Guérin (BCG). The ability to easily generate stable recombinants in these slow-growing mycobacteria without the requirement for continual selection is of particular importance for the construction of recombinant BCG vaccines and for the isolation and characterization of mycobacterial pathogenic determinants in animal model systems. Integration vectors of this type should be of general use in a number of additional bacterial systems where temperate phages have been identified.

Asunto(s)

Asunto(s)