Outbreak of tuberculosis in a closed setting: views on transmission based on results from molecular and conventional methods.

BACKGROUND: This study describes an outbreak of tuberculosis (TB) in a nursing home for men with mental disorders where residency is lengthy or permanent. This type of setting can provide a model of transmission as contact with the rest of society is extremely limited. AIM: To determine if cases of TB, diagnosed around the same time and in the same place, are linked based on results using molecular and conventional methods. METHODS: The strains of Mycobacterium tuberculosis were analysed by drug resistance testing and mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTRV). Microbiological results were related to clinical history and time of diagnosis. FINDINGS: Nine patients were diagnosed with TB, and strains were recovered from seven of these patients. Unexpectedly, the strains with the same genotype showed different patterns of resistance, and only two strains demonstrated identical patterns. MIRU-VNTR analysis demonstrated that one patient was infected with two different strains. CONCLUSION: Variation between the strains indicates that the outbreak may have arisen from several sources of infection. The variation in resistance indicates that rapid emergence of antimicrobial resistance is possible. As such, several questions are raised concerning source of infection, development of disease, resistance and mixed infections.

Impact of immigration on tuberculosis epidemiology in a low-incidence country.

Mycobacterium tuberculosis strains from 349 patients were isolated in western Sweden during the years 2001-2005. Only 26% of the tuberculosis (TB) patients were born in Sweden. All the others were born in any of 42 different countries; 17% in other European countries, 28% in Africa, 16% in Asia, 11% in the Middle East, and 2% in South America. The mean age of the Swedish-born patients was 67 years, while the mean age among the foreign-born patients was 37 years. The male/female ratio was 1.6 among the Swedes and 0.9 among those born abroad. Extrapulmonary manifestations of TB were most common among patients born in Africa while lung infections without extrapulmonary manifestations were most common in patients born in Europe, including Sweden. Spoligotyping showed that patients with T or Beijing strains had more pulmonary TB than extrapulmonary TB, while patients with EAI and CAS strains had a high proportion of extrapulmonary TB. The ancestral and/or evolutionary older PGG1 strains were more often isolated from the foreign-born patients than from the Swedish-born patients, who had strains generally being of the evolutionary recent genogroups PGG2/PGG3. We conclude that immigration from countries with a high incidence of TB has a strong impact on the TB epidemiology in western Sweden, a finding that should be taken into account by TB control strategists when developing programmes for eradication of TB in low prevalence settings.

Tuberculin skin test reactivity of health care students in a country with a low prevalence of tuberculosis.

SETTING: Health care students in Sweden. OBJECTIVE: To analyse the distribution of tuberculin skin test (TST) reactions and epidemiological factors related to TST reactivity. DESIGN: TST reactivity was analysed in 1190 students. A linear regression model was created for the relative contribution of background factors of TST reactivity. A subgroup of 287 non-vaccinated subjects was comparatively skin-tested with Mycobacterium avium sensitin and tuberculin. RESULTS: Among non-bacille Calmette-Guérin (BCG) vaccinated students, 91% had no TST reaction (0 mm induration) and reactions of ≥ 10 mm were found in 2.9%, whereas 34% of BCG-vaccinated students had no TST reaction and 42% had reactions of ≥ 10 mm. The expected contribution to TST reactivity was 6.0 mm for a history of BCG vaccination, 3.0 mm for a country of birth with medium/high incidence of TB and 1.6 mm per 10 years of age. The sensitin reactions exceeded the TST reactions by ≥ 3 mm in 52% of the comparatively tested subjects with TST reactions of ≥ 1 mm. CONCLUSION: BCG vaccination, cross-reactivity with non-tuberculous mycobacteria, geographic origin and age had a decisive influence on TST reactivity. Most non-vaccinated health care students were non-reactive, which highlights the need to organise preventive measures in settings where TB exposure is expected.

Difference in neutrophil cytokine production induced by pathogenic and non-pathogenic mycobacteria.

This study shows differences between Mycobacterium tuberculosis and Mycobacterium avium (opportunistic) and Mycobacterium smegmatis (non-pathogenic), with respect to their abilities to induce cytokine/chemokine release from human neutrophils. Neutrophils incubated with live cells of M. tuberculosis, M. avium, or M. smegmatis produced and released TNF-alpha, IL-6, and IL-8. No or very small amounts of these cytokines/chemokines were found in resting neutrophils, suggesting that they were newly synthesised. The levels of TNF-alpha, IL-6, and IL-8 produced/released from neutrophils incubated with M. tuberculosis were markedly lower than those of the opportunistic or non-pathogenic bacterial species. The production of TNF-alpha reached a maximum level at a time (4 h) when the production of IL-8 had only just started, and this was true for all three mycobacteria tested. However, the time course for IL-6 production differed between the species, reaching a peak value after 8 h for M. tuberculosis not seen with the other bacteria. It is likely that the relatively high levels of cytokines induced by opportunistic/non-pathogenic mycobacteria are of importance for the induction of an innate immune response through which these organisms are eliminated, while the low levels of cytokines released by neutrophils interfering with M. tuberculosis might help the bacteria to persist.

Priming of human neutrophils by mycobacterial lipoarabinomannans: role of granule mobilisation.

Lipoarabinomannans (LAMs) from mycobacteria were investigated concerning their effect on human neutrophils. Two types of LAM, the mannose-capped ManLAM from the virulent Mycobacterium tuberculosis H37Rv and the mannose-lacking AraLAM from a rapidly growing mycobacterial strain were used. Neither AraLAM nor ManLAM induced any significant direct activation of the NADPH-oxidase. Both LAMs, however, primed the neutrophils so that subsequent stimulation with the peptide chemoattractants fMet-Leu-Phe (fMLF), Trp-Lys-Tyr-Met-Val-DMet (WKYMVm) and the mammalian lactose-binding lectin galectin-3 resulted in a markedly enhanced oxidative response. The LAM-induced priming was accompanied by an increased exposure of complement receptors 1 and 3 as well as the formyl peptide receptor on the neutrophil surface, suggesting that the enhanced oxidative response could be due to upregulation of receptors on the cell surface as a result of granule mobilisation. Since LAM-primed neutrophils released 65% of the cell content of gelatinase but showed no increased release of vitamin B(12)-binding protein, mobilisation of the gelatinase granules rather than the specific granules is concluded to be responsible for the priming effects. This is in agreement with the subcellular localisation of receptors for fMLF, WKYMVm, as well as galectin-3, which are stored in the secretory vesicles and gelatinase granules. The priming effect appeared very similar to that of Escherichia coli lipopolysaccharide, and since no differences in activity could be detected between AraLAM and ManLAM, we hypothesize that the lipid anchor of the LAM is responsible for the priming effects.

The tuberculin skin test in relation to immunological in vitro reactions in BCG-vaccinated healthcare workers.

The aim was to study the tuberculin skin test in relation to immunological in vitro reactions in bacille Calmette-Guerin (BCG)-vaccinated healthcare workers. The present study was performed in Sweden, a country with a low incidence of tuberculosis, a high BCG vaccination efficacy and high tuberculin conversion rates. BCG-vaccinated healthcare workers (n=381) were tuberculin skin tested. From these, 11 subjects with negative tuberculin reactions (<6 mm) were matched for age and sex with 11 subjects with large positive reactions (> or = 15 mm). Lymphocyte transformation and the production of interferon-gamma (IFN-gamma) were analysed after stimulation in vitro of peripheral blood mononuclear cells with tuberculin purified protein derivative, heat-killed tubercle bacilli and a culture filtrate from tubercle bacilli. In the tuberculin-positive group the lymphocyte transformation response was 2-3 times larger, and IFN-gamma production was 7-10 times larger, than in the tuberculin-negative group (p<0.001). The present results suggest that a positive tuberculin skin test in bacille Calmette-Guerin-vaccinated subjects indicates a stronger immune response of the protective T-helper 1-type than does a negative test. In similar settings, the study supports the traditional practice of regarding the tuberculin skin test in bacille Calmette-Guerin-vaccinated subjects as an indicator of a protective immune response against tuberculosis.

[Tuberculosis a threat again. Multiresistance in the Baltic States and in Russia; Nordic countries initiate cooperation to prevent transmission]. / Tuberkulos åter ett hot. Multiresistens i Baltikum och Ryssland; nordiskt samarbete inlett för att hejda spridning.

The incidence of tuberculosis (TB) has more than doubled in the Baltic States during the last decade and is now 10-15 times higher than in Sweden. It is also a serious problem in Russia. Strains resistant to one or several of the anti-tuberculous drugs are common as is multi-drug resistance (MDR), i.e. strains resistant to the two most effective drugs rifampicin and isoniazid. MDR-TB is very difficult to treat; the mortality rate is high. Initiatives have been taken in the Nordic countries in order to help to control and improve the situation by way of supportive measures.

Activation of human neutrophils by mycobacterial phenolic glycolipids.

The interaction between mycobacterial phenolic glycolipids (PGLs) and phagocytes was studied. Human neutrophils were allowed to interact with each of four purified mycobacterial PGLs and the neutrophil production of reactive oxygen metabolites was followed kinetically by luminol-/isoluminol-amplified chemiluminescence. The PGLs from Mycobacterium tuberculosis and Mycobacterium kansasii, respectively, were shown to stimulate the production of oxygen metabolites, while PGLs from Mycobacterium marinum and Mycobacterium bovis BCG, respectively, were unable to induce an oxidative response. Periodate treatment of the M. tuberculosis PGL decreased the production of oxygen radicals, showing the importance of the PGL carbohydrate moiety for the interaction. The activation, however, could not be inhibited by rhamnose or fucose, indicating a complex interaction which probably involves more than one saccharide unit. This is in line with the fact that the activating PGLs from M. tuberculosis and M. kansasii contain tri- and tetrasaccharides, respectively, while the nonactivating PGLs from M. marinum and M. bovis BCG each contain a monosaccharide. The complement receptor 3 (CR3) has earlier been shown to be of importance for the phagocyte binding of mycobacteria, but did not appear to be involved in the activation of neutrophils by PGLs. The subcellular localization of the reactive oxygen metabolites formed was related to the way in which the glycolipids were presented to the cells.

The phagocyte chemiluminescence paradox: luminol can act as an inhibitor of neutrophil NADPH-oxidase activity.

The chemiluminescence system amplified by luminol or isoluminol is a sensitive and widely used method for determination of respiratory burst products generated by the NADPH-oxidase in phagocytes. The present study shows that luminol, but not isoluminol, can inhibit the release of oxygen metabolites generated by human neutrophil NADPH-oxidase. The difference in structure between luminol and isoluminol (rendering luminol more lipophilic than isoluminol, and thereby membrane-permeable), is suggested to determine indirectly whether or not the molecule is inhibitory. Luminol was shown to have an increased inhibitory effect after preincubation of neutrophils on a surface of aggregated IgG, suggesting that the cells can be transferred from a 'luminol-insensitive' to a 'luminol-sensitive' state. Since luminol had no inhibitory effect in a cell-free NADPH-oxidase system, it is likely that it interferes with the signal transduction pathway, leading to assembly and/or activation of the oxidase. As a consequence of the present results, showing that luminol but not isoluminol can inhibit NADPH-oxidase activity, we suggest that isoluminol is used in future studies of superoxide anion release from phagocytes.

Serotaxonomic analysis of glycolipids from Mycobacterium chelonae-M. fortuitum complex and bovine farcy strains.

The antigenicity and cross-reactivity of glycolipids from strains of bovine farcy and the Mycobacterium chelonae-M. fortuitum complex were analyzed using the ELISA technique. Purified alkali-stable glycopeptidolipids (GPLs) with a characteristic dimethylrhamnosyl sugar unit extracted from M. abscessus, M. chelonae, M. peregrinum and M. senegalense, gave very strong reactions with sera against members of the same four species. Particularly strong cross-reactions were evident between M. peregrinum and M. senegalense. These GPLs reacted more weakly with antisera against the other mycobacteria tested, though clear reactions were noticed with M. farcinogenes and M. fortuitum and also with M. bovis BCG, M. phlei, and M. tuberculosis strains. Alkali-labile diacyl trehalose (DAT) and triacyl trehalose (TAT) from M. fortuitum reacted with homologous sera, and with that against M. tuberculosis. Traces of uncharacterized acyl trehaloses isolated from two strains of M. farcinogenes gave comparatively weak reactions. Mycobacteria labeled M. farcinogenes and M. senegalense produced glucosylated trehalose-based glycolipids (GTs) and the studies showed that the major type was antigenic. These glycolipids cross-reacted strongly with M. senegalense NCTC 4524 but not with the type strain of M. senegalense. On the basis of the chemical patterns and the antigenicity of the GPLs it is evident that M. peregrinum and M. senegalense are particularly closely related and these species show a very close affinity to M. abscessus-M. chelonae.

Purification and characterisation of isocitrate dehydrogenase and malate dehydrogenase from Mycobacterium tuberculosis and evaluation of their potential as suitable antigens for the serodiagnosis of tuberculosis.

SETTING: Enzymes from Mycobacterium tuberculosis are potent antigens and might thus be of interest in the serodiagnosis of tuberculosis. OBJECTIVE: The purpose of the study was to purify and characterize the two enzymes isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH) from, M. tuberculosis and to evaluate their potential in the serodiagnosis of tuberculosis. DESIGN: The two enzymes were analysed for specificity by electrophoresis and then purified by means of affinity chromatography using reactive dyes and ion exchange chromatography. The two isolated enzyme fractions were analysed by ELISA, using antisera against related organisms. They were then tested as antigens in ELISA together with sera from tuberculous patients and controls. RESULTS: The electrophoretical analyses showed that the two enzymes each differed markedly from the corresponding enzymes of other mycobacteria. The serological analyses, however, could not distinguish between either IDH or MDH from other mycobacteria, but organisms of other genera, such as Nocardia, gave much weaker responses. When IDH and MDH were tested with sera from tuberculous patients and controls the former gave clearly higher optical density values than the latter. CONCLUSION: The enzymes/antigens IDH and MDH may be of value in developing a serological test for tuberculosis. The latter fraction seemed particularly capable of discriminating patients from controls.

Semantide- and chemotaxonomy-based analyses of some problematic phenotypic clusters of slowly growing mycobacteria, a cooperative study of the International Working Group on Mycobacterial Taxonomy.

During previous cooperative numerical taxonomic studies of slowly growing mycobacteria, the International Working Group on Mycobacterial Taxonomy described a number of strains whose taxonomic status was ambiguous. A new study of DNA, RNA, and proteins from 66 of these organisms was performed to correlate their properties with phenotypic clustering behavior; the results of this study permitted 51 of the strains studied to be assigned to known species. The methods used to characterize the semantides included nucleotide sequencing and assessment of levels of semantide relatedness by affinity binding techniques, including whole DNA-DNA hybridization, probe hybridization, and antibody binding. There was good overall agreement between the phenotypic and chemotaxonomic clusters and the groups of organisms identified by semantide analyses. Our results supported the conclusion that we should continue to rely on polyphasic taxonomy to provide satisfactory systematic resolution of members of the genus Mycobacterium. We identified no single 16S rRNA interstrain nucleotide sequence difference value that unequivocally defined species boundaries. DNA-DNA hybridization remains the gold standard, but common resources are needed to permit DNA-DNA hybridization analyses to be made available to laboratories that are not prepared to use this technology. One of the large novel clusters which we studied corresponds to the recently described species Mycobacterium interjectum, a pathogen that resembles the nonpathogen Mycobacterium gordonae phenotypically. We also identified strains that appear to represent ribovars of Mycobacterium intracellulare which do not react with the commercial diagnostic probes that are currently used for identification of this species. Other branches or clusters consisted of too few strains to permit a decision about their taxonomic status to be made.

Enzymatic and antigenic analyses of strains of Mycobacterium bovis, M. bovis BCG, and M. tuberculosis.

Strains of Mycobacterium bovis, M. bovis BCG, and M. tuberculosis, including a so-called Canetti strain, were analyzed by means of two-dimensional immunoelectrophoresis (2D-IE), 2D-IE combined with enzyme staining, and multilocus enzyme electrophoresis (MEE). The results demonstrated a close antigenic and enzymatic resemblance among all the strains tested, even though the BCG strains could be divided into two groups based on the presence of one precipitinogen. Eight of the precipitinogens were shown to correspond to enzymes in M. bovis BCG and 10 in M. tuberculosis. Thus, catalase, isocitrate dehydrogenase, malate dehydrogenase, peroxidase, and several others were identified. By means of MEE the strains of M. tuberculosis, M. bovis, and M. bovis BCG could be differentiated. The analyses further indicated that the M. tuberculosis strain Canetti was more closely related to M. bovis than to M. tuberculosis.

Stereochemistry of 2,4-dimethyleicos-2-enoate from the pyruvylated glycolipid of Mycobacterium smegmatis.

The absolute stereochemistry of 2,4-dimethyleicos-2-enoic acid, isolated from the pyruvylated glycolipid of Mycobacterium smegmatis, has been determined. The two enantiomers of methyl 2,4-dimethyleicos-2-enoate were synthesised for the first time but could not be separated by gas chromatography on cyclodextrin phases. (E)-2-Methyloctadec-2-enoate, an intermediate in the synthesis, is a characteristic component of acyl trehalose glycolipids from Mycobacterium fortuitum. Ozonolysis of the fatty acid ester mixture, obtained from the pyruvylated glycolipid produced 2-methyloctadecanoate. It was identified as the (S)-enantiomer by comparison with (2R) and (2S)-2-methyloctadecanoic acid, intermediates in the synthesis of (4R)- and (4S)-2,4,-dimethyleicos-2-enoic acid, using enantioselective gas chromatography of the methyl esters with heptakis(2,6-di-O-methyl-3-O-pentyl)-beta-cyclodextrin as a chiral stationary phase. The natural acid was therefore determined to be 2E-(4S)-2,4-dimethyleicos-2-enoic acid.

Thin-layer chromatography and mass spectrometry of pentafluorobenzylidene derivatives of members of the phthiocerol family from Mycobacterium tuberculosis and Mycobacterium marinum.

The difference in relative stereochemistry of the 1.3-diol unit of mycobacterial phthiocerols can be determined by simple thin-layer chromatographic analysis of pentafluorobenzylidene acetal derivatives. The threo phthiocerol acetals from Mycobacterium tuberculosis are composed of equal amounts of two axial-equatorial stereoisomers but the erythro phthiocerols from Mycobacterium marinum form only one acetal with diequatorial substituents. The two acetals formed from a threo phthiocerol can be separated by thin-layer chromatography and mass spectra of the two stereoisomers allowed assignment of their structures.

Fast atom bombardment mass spectrometry of mycobacterial phenolic glycolipids.

Fast atom bombardment mass spectra were successfully recorded for intact glycosylphenolphthiocerol dimycocerosates (phenolic glycolipids, PGLs) from Mycobacterium kansasii, M. leprae, M. tuberculosis, M. marinum, M. bovis and M. haemophilum. Characteristic fragment ions from the loss of the oligosaccharide moiety and one of the long-chain multimethyl-branched mycocerosic acids were observed in most cases. A tandem mass spectrometric experiment was carried out on the PGL from M. tuberculosis, revealing the type of mycocerosic acids esterified to individual homologues. Mass spectra of homologues separated by reversed-phase high-performance liquid chromatography gave information on the substitution pattern in certain cases. The potential of matrix-assisted laser desorption ionization spectroscopy was demonstrated by a successful analysis of the PGL from M. tuberculosis.

Thin-layer chromatographic analysis of glycolipids and mycolic acids from Mycobacterium farcinogenes, Mycobacterium senegalense and related taxa.

Seventy-nine representative strains of Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum and Mycobacterium senegalense were analysed by thin-layer chromatography for diagnostic glycolipid and mycolic acid patterns. On the basis of glycolipid patterns the 16 M. senegalense strains were assigned to four groups (I, II, III and IV); one strain received as M. farcinogenes had glycolipids that corresponded to those given by the M. senegalense group III strain. Representatives of M. fortuitum produced alkalilabile glycolipids whereas the M. peregrinum strains produced alkali-stable glycopeptidolipids related to those of M. senegalense group I. Members of M. senegalense groups I, III and IV and the M. farcinogenes strains gave the characteristic pattern consisting of alpha- and epoxymycolates on thin-layer chromatographic analysis of the products of alkaline hydrolysis with 5% aqueous tetrabutylammonium hydroxide. Extraction of mycolates from the same strains using acid methanolysis revealed alpha-mycolates and characteristic more polar long-chain components originating from epoxymycolates. In contrast, the M. senegalense group II strains contained an additional more polar omega-1 methoxymycolate that was detected from both acid and alkaline hydrolysates; this spot was also seen in some M. fortuitum and M. peregrinum strains. Preliminary investigations, using a combination of thin-layer chromatography and immunostaining, showed that glycopeptidolipids from M. peregrinum and M. senegalense group I strains were antigenic, a cross-reaction between members of these taxa was shown. In contrast, no reaction was detected between glycopeptidolipids extracted from M. peregrinum and M. senegalense group I strains with antisera from either M. farcinogenes or M. fortuitum.

An integrated procedure for the direct detection of characteristic lipids in tuberculosis patients.

An integrated method is described for the sensitive detection of tuberculostearic, mycocerosic and mycolic acids in infected materials from tuberculosis patients. Tuberculostearic acid is analysed by two-dimensional gas chromatography of pentafluorobenzyl esters, the key component being switched from a short non-polar column to a high resolution polar column with final electron capture detection. Mycocerosic acids are identified by simple one-dimensional electron capture gas chromatography of pentafluorobenzyl esters, with the use of negative-ion chemical ionisation gas chromatography in indecisive cases. Conversion of mycolic acids to anthrylmethyl esters produces compounds which are suitable for sensitive detection by fluorescence high-performance liquid chromatography. Application of all three of these methods to sputum samples from tuberculosis patients gave profiles characteristic of Mycobacterium tuberculosis.