Mycobacterium avium subsp. paratuberculosis in an Italian Cohort of Type 1 Diabetes Pediatric Patients.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease in ruminants. Recent studies have linked MAP to type 1 diabetes (T1D) in the Sardinian population. The aim of this study was to investigate the prevalence of MAP infection in a T1D cohort from continental Italy compared with healthy control subjects. 247 T1D subjects and 110 healthy controls were tested for the presence of MAP. MAP DNA was detected using IS900-specific polymerase chain reaction (PCR). The presence of antibodies towards a MAP antigen, heparin binding hemoagglutinin (HBHA), was detected by ELISA. We demonstrated a higher MAP DNA prevalence in plasma samples from T1D patients and a stronger immune response towards MAP HBHA, compared with healthy control subjects. Moreover, in the recent onset patients, we observed an association between anti-MAP antibodies and HLA DQ2 (DQA1 0201/DQB1 0202). These findings taken together support the hypothesis of MAP as an environmental risk factor for the development of T1D in genetically predisposed subjects, probably involving a mechanism of molecular mimicry between MAP antigens and pancreatic islet β-cells.

Disseminated M. avium complex infection in the Swiss HIV Cohort Study: declining incidence, improved prognosis and discontinuation of maintenance therapy.

Introduction of potent antiretroviral combination therapy (ART) has reduced overall morbidity and mortality amongst HIV-infected adults. Some prophylactic regimes against opportunistic infections can be discontinued in patients under successful ART. (1) The influence of the availability of ART on incidence and mortality of disseminated M. avium Complex infection (MAC). (2) The safety of discontinuation of maintenance therapy against MAC in patients on ART. The Swiss HIV-Cohort Study, a prospective multicentre study of HIV-infected adults. Patients with a nadir CD4 count below 50 cells/mm3 were considered at risk for MAC and contributed to total follow-up time for calculating the incidence. Survival analysis was performed by using Kaplan Meier and Cox proportional hazards methods. Safety of discontinuation of maintenance therapy was evaluated by review of the medical notes. 398 patients were diagnosed with MAC from 1990 to 1999. 350 had a previous CD4 count below 50 cells/mm3. A total of 3208 patients had a nadir CD4 count of less than 50 cells/mm3 during the study period and contributed to a total follow-up of 6004 person-years. The incidence over the whole study period was 5.8 events per 100 person-years. In the time period of available ART the incidence of MAC was significantly reduced (1.4 versus 8.8 events per 100 person-years, p < 0.001). Being diagnosed after 1995 was the most powerful predictor of better survival (adjusted hazard ratio for death: 0.27; p < 0.001). None of 24 patients discontinuing maintenance therapy while on ART experienced recurrence of MAC during a total follow-up of 56.6 person-years (upper 95% confidence limit 5.3 per 100 person-years). Introducing ART has markedly reduced the risk of MAC for HIV-infected individuals with a history of very low CD4 counts. Survival after diagnosis of MAC has improved after ART became available. In patients responding to ART, discontinuation of maintenance therapy against M. avium may be safe.

Free-living amoebae: Biological by-passes in water treatment.

Free-living amoebae constitute reservoirs for many bacteria including not only well-known pathogens but also emerging pathogens responsible for respiratory diseases, and contribute to the protection, survival and dissemination of these bacteria in water systems, despite the application of disinfection or thermal treatments. In this article we review the available information on the presence of free-living amoebae and amoebae-resisting bacteria in drinking water systems, on the factors that contribute to their presence in the water and/or the biofilms, on the possible control measures and their effectiveness, and we identify some gaps in current knowledge needing further research.

Mycobacterium tuberculosis aortic graft infection with recurrent hemoptysis: a case report.

INTRODUCTION: Mycobacterium tuberculosis may cause a large variety of clinical presentations due to its ability to disseminate by contiguity or hematogenously. Tuberculosis may remain undiagnosed for years due to the chronic course of the disease, with potentially life-threatening long-term complications. CASE PRESENTATION: In this case report, we describe a tuberculous aortic graft infection in a 72-year-old man documented by polymerase chain reaction and cultures. The patient presented with three episodes of hemoptysis following a remote history of miliary tuberculosis. The infection was treated by graft replacement and prolonged antimycobacterial therapy. CONCLUSION: Tuberculous infection of a vascular graft is an uncommon complication, but should be considered in patients with an intravascular device and a history of previous tuberculosis, especially when hematogenous spread may have occurred a few months after surgery, or when an active mycobacterial infection is present in close proximity to the graft.

Immuno-reactive proteins from Mycobacterium immunogenum useful for serodiagnosis of metalworking fluid hypersensitivity pneumonitis.

Metalworking fluid-associated hypersensitivity pneumonitis (MWF-HP) is a pulmonary disease caused by inhaling microorganisms present in the metalworking fluids used in the industrial sector. Mycobacterium immunogenum is the main etiological agent. Among the clinical, radiological and biological tools used for diagnosis, serological tests are important. The aim of this study was to identify immunogenic proteins in M. immunogenum and to use recombinant antigens for serological diagnosis of MWF-HP. Immunogenic proteins were detected by two-dimensional Western blot and candidate proteins were identified by mass spectrometry. Recombinant antigens were expressed in Escherichia coli and tested by enzyme-linked immunosorbent assay (ELISA) with the sera of 14 subjects with MWF-HP and 12 asymptomatic controls exposed to M. immunogenum. From the 350 spots visualized by two-dimensional gel electrophoresis with M. immunogenum extract, 6 immunogenic proteins were selected to be expressed as recombinant antigens. Acyl-CoA dehydrogenase antigen allowed for the best discrimination of MWF-HP cases against controls with an area under the receiver operating characteristics (ROC) curve of 0.930 (95% CI=0.820-1), a sensitivity of 100% and a specificity of 83% for the optimum threshold. Other recombinant antigens correspond to acyl-CoA dehydrogenase FadE, cytosol aminopeptidase, dihydrolipoyl dehydrogenase, serine hydroxymethyltransferase and superoxide dismutase. This is the first time that recombinant antigens have been used for the serodiagnosis of hypersensitivity pneumonitis. The availability of recombinant antigens makes it possible to develop standardized serological tests which in turn could simplify diagnosis, thus making it less invasive.

Development of a method to investigate the hydrolysis of xenobiotic esters by a Mycobacterium smegmatis homogenate.

One of the main problems in combating tuberculosis is caused by a poor penetration of drugs into the mycobacterial cells. A prodrug approach via activation inside mycobacterial cells is a possible strategy to overcome this hurdle and achieve efficient drug uptake. Esters are attractive candidates for such a strategy and we and others communicated previously the activity of esters of weak organic acids against mycobacteria. However very little is known about ester hydrolysis by mycobacteria and no biological model is available to study the activation of prodrugs by these microorganisms. To begin filling this gap, we have embarked in a project to develop an in vitro method to study prodrug activation by mycobacteria using Mycobacterium smegmatis homogenates. Model ester substrates were ethyl nicotinate and ethyl benzoate whose hydrolysis was monitored and characterized kinetically. Our studies showed that in M. smegmatis most esterase activity is associated with the soluble fraction (cytosol) and is preserved by storage at 5°C or at room temperature for one hour, or by storage at -80°C up to one year. In the range of homogenate concentrations studied (5-80% in buffer), k(obs) varied linearly with homogenate concentration for both substrates. We also found that the homogenates showed Michaelis-Menten kinetics behavior with both prodrugs. Since ethyl benzoate is a good substrate for the mycobacterial esterases, this compound can be used to standardize the esterasic activity of homogenates, allowing results of incubations of prodrugs with homogenates from different batches to be readily compared.

Fas ligand-induced apoptosis of infected human macrophages reduces the viability of intracellular Mycobacterium tuberculosis.

Mycobacterium tuberculosis-specific cytolytic activity is mediated mostly by CD4+CTL in humans. CD4+CTL kill infected target cells by inducing Fas (APO-1/CD95)-mediated apoptosis. We have examined the effect of Fas ligand (FasL)-induced apoptosis of human macrophages infected in vitro with M. tuberculosis on the viability of the intracellular bacilli. Human macrophages expressed Fas and underwent apoptosis after incubation with soluble recombinant FasL. In macrophages infected either with an attenuated (H37Ra) or with a virulent (H37Rv) strain of M. tuberculosis, the apoptotic death of macrophages was associated with a substantial reduction in bacillary viability. TNF-induced apoptosis of infected macrophages was coupled with a similar reduction in mycobacterial viability, while the induction of nonapoptotic complement-induced cell death had no effect on bacterial viable counts. Infected macrophages also showed a reduced susceptibility to FasL-induced apoptosis correlating with a reduced level of Fas expression. These data suggest that apoptosis of infected macrophages induced through receptors of the TNF family could be an immune effector mechanism not only depriving mycobacteria from their growth environment but also reducing viable bacterial counts by an unknown mechanism. On the other hand, interference by M. tuberculosis with the FasL system might represent an escape mechanism of the bacteria attempting to evade the effect of apoptosis.

Combined Use of Mycobacterium tuberculosis-Specific CD4 and CD8 T-Cell Responses Is a Powerful Diagnostic Tool of Active Tuberculosis.

Immune-based assays are promising tools to help to formulate diagnosis of active tuberculosis. A multiparameter flow cytometry assay assessing T-cell responses specific to Mycobacterium tuberculosis and the combination of both CD4 and CD8 T-cell responses accurately discriminated between active tuberculosis and latent infection.

Genome-wide re-sequencing of multidrug-resistant Mycobacterium leprae Airaku-3.

Genotyping and molecular characterization of drug resistance mechanisms in Mycobacterium leprae enables disease transmission and drug resistance trends to be monitored. In the present study, we performed genome-wide analysis of Airaku-3, a multidrug-resistant strain with an unknown mechanism of resistance to rifampicin. We identified 12 unique non-synonymous single-nucleotide polymorphisms (SNPs) including two in the transporter-encoding ctpC and ctpI genes. In addition, two SNPs were found that improve the resolution of SNP-based genotyping, particularly for Venezuelan and South East Asian strains of M. leprae.

Mycobacterium tuberculosis transmission in a country with low tuberculosis incidence: role of immigration and HIV infection.

Immigrants from high-burden countries and HIV-coinfected individuals are risk groups for tuberculosis (TB) in countries with low TB incidence. Therefore, we studied their role in transmission of Mycobacterium tuberculosis in Switzerland. We included all TB patients from the Swiss HIV Cohort and a sample of patients from the national TB registry. We identified molecular clusters by spoligotyping and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis and used weighted logistic regression adjusted for age and sex to identify risk factors for clustering, taking sampling proportions into account. In total, we analyzed 520 TB cases diagnosed between 2000 and 2008; 401 were foreign born, and 113 were HIV coinfected. The Euro-American M. tuberculosis lineage dominated throughout the study period (378 strains; 72.7%), with no evidence for another lineage, such as the Beijing genotype, emerging. We identified 35 molecular clusters with 90 patients, indicating recent transmission; 31 clusters involved foreign-born patients, and 15 involved HIV-infected patients. Birth origin was not associated with clustering (adjusted odds ratio [aOR], 1.58; 95% confidence interval [CI], 0.73 to 3.43; P = 0.25, comparing Swiss-born with foreign-born patients), but clustering was reduced in HIV-infected patients (aOR, 0.49; 95% CI, 0.26 to 0.93; P = 0.030). Cavitary disease, male sex, and younger age were all associated with molecular clustering. In conclusion, most TB patients in Switzerland were foreign born, but transmission of M. tuberculosis was not more common among immigrants and was reduced in HIV-infected patients followed up in the national HIV cohort study. Continued access to health services and clinical follow-up will be essential to control TB in this population.

Mycobacterium tuberculosis-specific CD8+ T cells are functionally and phenotypically different between latent infection and active disease.

Protective immunity to Mycobacterium tuberculosis (Mtb) remains poorly understood and the role of Mtb-specific CD8(+) T cells is controversial. Here we performed a broad phenotypic and functional characterization of Mtb-specific CD8(+) T cells in 326 subjects with latent Mtb infection (LTBI) or active TB disease (TB). Mtb-specific CD8(+) T cells were detected in most (60%) TB patients and few (15%) LTBI subjects but were of similar magnitude. Mtb-specific CD8(+) T cells in LTBI subjects were mostly T EMRA cells (CD45RA(+) CCR7(-)), coexpressing 2B4 and CD160, and in TB patients were mostly TEM cells (CD45RA(-) CCR7(-)), expressing 2B4 but lacking PD-1 and CD160. The cytokine profile was not significantly different in both groups. Furthermore, Mtb-specific CD8(+) T cells expressed low levels of perforin and granulysin but contained granzymes A and B. However, in vitro-expanded Mtb-specific CD8(+) T cells expressed perforin and granulysin. Finally, Mtb-specific CD8(+) T-cell responses were less frequently detected in extrapulmonary TB compared with pulmonary TB patients. Mtb-specific CD8(+) T-cell proliferation was also greater in patients with extrapulmonary compared with pulmonary TB. Thus, the activity of Mtb infection and clinical presentation are associated with distinct profiles of Mtb-specific CD8(+) T-cell responses. These results provide new insights in the interaction between Mtb and the host immune response.

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis.

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.