Mycobacterium tuberculosis clinical isolates of the Beijing and East-African Indian lineage induce fundamentally different host responses in mice compared to H37Rv

Substantial differences exist in virulence among Mycobacterium tuberculosis strains in preclinical TB models. In this study we show how virulence affects host responses in mice during the first four weeks of infection with a mycobacterial strain belonging to the Beijing, East-African-Indian or Euro-American lineage. BALB/c mice were infected with clinical isolates of the Beijing-1585 strain or the East-African Indian (EAI)-1627 strain and host responses were compared to mice infected with the non-clinical H37Rv strain of the Euro-American lineage. We found that H37Rv induced a 'classical' T-cell influx with high IFN-γ levels, while Beijing-1585 and EAI-1627 induced an influx of B-cells into the lungs together with elevated pulmonary IL-4 protein levels. Myeloid cells in the lungs appeared functionally impaired upon infection with Beijing-1585 and EAI-1627 with reduced iNOS and IL-12 expression levels compared to H37Rv infection. This impairment might be related to significantly reduced expression in the bone marrow of IFN-γ, TNF-α and IFN-β in mice infected with Beijing-1585 and EAI-1627, which could be detected from the third day post infection onwards. Our findings suggest that increased virulence of two clinical isolates compared to H37Rv is associated with a fundamentally different systemic immune response, which already can be detected early during infection

Dynamics of interferon-gamma release assay and cytokine profiles in blood and respiratory tract specimens from mice with tuberculosis and the effect of therapy

There are limitations on diagnostic methods to differentiate between active and latent tuberculosis (TB), and the prediction of latent progression to TB disease is yet complex. Traditionally, tuberculosis-specific host immune response was visualized using the tuberculin skin test. Nowadays, IFN-γ release assays (IGRA) provide a more specific and sensitive tool, by which exposure to Mtb could be determined. However, the merit of IGRA aids in diagnosing active TB is yet unclear. We adapted IGRA for use in mice, and quantifying bead-based flow cytometry techniques were used to assess cytokine profiles during the course of untreated infection and to investigate the value of IGRA and cytokines as biomarkers for therapy response. High variability of IGRA results during progression of active TB infection related to various phases of infection was obtained. However, a significant decrease in IGRA results and in levels of IFN-γ, IL-17, IP-10 or MIG was observed and appeared to be associated with successful therapy. This outcome does not support the value of IGRA to accurately diagnose active TB or to monitor infection progression. However, IGRA proved to be a useful biomarker to monitor therapy success. In addition, different cytokines might serve as biomarkers

A novel approach : the propensity to propagate (PTP) method for controlling for host factors in studying the transmission of Mycobacterium tuberculosis

RATIONALE: Understanding the genetic variations among Mycobacterium tuberculosis (MTB) strains with differential ability to transmit would be a major step forward in preventing transmission. OBJECTIVES: To describe a method to extend conventional proxy measures of transmissibility by adjusting for patient-related factors, thus strengthening the causal association found with bacterial factors. METHODS: Clinical, demographic and molecular fingerprinting data were obtained during routine surveillance of verified MTB cases reported in the Netherlands between 1993 and 2011, and the phylogenetic lineages of the isolates were inferred. Odds ratios for host risk factors for clustering were used to obtain a measure of each patient's and cluster's propensity to propagate (CPP). Mean and median cluster sizes across different categories of CPP were compared amongst four different phylogenetic lineages. RESULTS: Both mean and median cluster size grew with increasing CPP category. On average, CPP values from Euro-American lineage strains were higher than Beijing and EAI strains. There were no significant differences between the mean and median cluster sizes among the four phylogenetic lineages within each CPP category. CONCLUSIONS: Our finding that the distribution of CPP scores was unequal across four different phylogenetic lineages supports the notion that host-related factors should be controlled for to attain comparability in measuring the different phylogenetic lineages' ability to propagate. Although Euro-American strains were more likely to be in clusters in an unadjusted analysis, no significant differences among the four lineages persisted after we controlled for host factors.Portuguese Foundation for Science and Technology (FCT) (SFRH/BD/33902/2009 to HN-G). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Characteristics of pncA mutations in multidrug-resistant tuberculosis in Taiwan

<p>Abstract</p> <p>Background</p> <p>Pyrazinamide (PZA) is an important first-line drug in multidrug-resistant tuberculosis (MDRTB) treatment. However, the unreliable results obtained from traditional susceptibility testing limits its usefulness in clinical settings. The detection of <it>pncA </it>gene mutations is a potential surrogate of PZA susceptibility testing, especially in MDRTB isolates. The impact of genotypes of <it>M. tuberculosis </it>in <it>pncA </it>gene mutations also remains to be clarified.</p> <p>Methods</p> <p>MDRTB isolates were collected from six hospitals in Taiwan from January 2007 to December 2009. <it>pncA </it>gene sequencing, pyrazinamidase activity testing, and spoligotyping were performed on all of the isolates. PZA susceptibility was determined by the BACTEC MGIT 960 PZA method. The sensitivity and specificity of <it>pncA </it>gene analysis were estimated based on the results of PZA susceptibility testing.</p> <p>Results</p> <p>A total of 66 MDRTB isolates, including 37 Beijing and 29 non-Beijing strains, were included for analysis. Among these isolates, 36 (54.5%) were PZA-resistant and 30 (45.5%) were PZA-susceptible. The PZA-resistant isolates were more likely to have concomitant resistance to ethambutol and streptomycin. Thirty-seven mutation types out of 30 isolates were identified in the <it>pncA </it>gene, and most of them were point mutations. The sensitivities of <it>pncA </it>gene sequencing for PZA susceptibility in overall isolates, Beijing and non-Beijing strains were 80.6%, 76.2%, and 86.7% respectively, and the specificities were 96.7%, 93.8%, and 100% respectively.</p> <p>Conclusions</p> <p>More than half of the MDRTB isolates in this study are PZA-resistant. Analysis of <it>pncA </it>gene mutations helped to identify PZA-susceptible MDRTB isolates, especially in non-Beijing strains.</p

Contribution of Efflux to the Emergence of Isoniazid and Multidrug Resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment

Complement and the Alternative Pathway Play an Important Role in LPS/D-GalN-Induced Fulminant Hepatic Failure

Fulminant hepatic failure (FHF) is a clinically severe type of liver injury with an extremely high mortality rate. Although the pathological mechanisms of FHF are not well understood, evidence suggests that the complement system is involved in the pathogenesis of a variety of liver disorders. In the present study, to investigate the role of complement in FHF, we examined groups of mice following intraperitoneal injection of LPS/D-GalN: wild-type C57BL/6 mice, wild-type mice treated with a C3aR antagonist, C5aR monoclonal antibody (C5aRmAb) or CR2-Factor H (CR2-fH, an inhibitor of the alternative pathway), and C3 deficient mice (C3−/− mice). The animals were euthanized and samples analyzed at specific times after LPS/D-GalN injection. The results show that intraperitoneal administration of LPS/D-GalN activated the complement pathway, as evidenced by the hepatic deposition of C3 and C5b-9 and elevated serum levels of the complement activation product C3a, the level of which was associated with the severity of the liver damage. C3a receptor (C3aR) and C5a receptor (C5aR) expression was also upregulated. Compared with wild-type mice, C3−/− mice survived significantly longer and displayed reduced liver inflammation and attenuated pathological damage following LPS/D-GalN injection. Similar levels of protection were seen in mice treated with C3aR antagonist,C5aRmAb or CR2-fH. These data indicate an important role for the C3a and C5a generated by the alternative pathway in LPS/D-GalN-induced FHF. The data further suggest that complement inhibition may be an effective strategy for the adjunctive treatment of fulminant hepatic failure

Optimization of the rifampin dosage to improve the therapeutic efficacy in tuberculosis treatment using a murine model

Item does not contain fulltextRationale: The dosage of 10 mg/kg/d rifampin, as currently used in the treatment of tuberculosis (TB), is not an optimal dose. Shortening of treatment duration might be achievable using an increased rifampin dose. Objectives: Determination of optimal rifampin dosage in mice, resulting in maximum therapeutic effect and without adverse effects. Assessment of associated pharmacokinetic parameters and pharmacokinetic/pharmacodynamic indices. Methods: A murine TB infection using a Beijing genotype Mycobacterium tuberculosis strain was established by intratracheal bacterial instillation followed by proper inhalation, while keeping mice in a vertical position. We assessed dose-dependent activity of rifampin in single-drug treatment during 3 weeks. The maximum tolerated dosage, pharmacokinetic parameters, and pharmacokinetic/pharmacodynamic index were determined. Therapeutic efficacy of a range of rifampin (R) dosages added to a regimen of isoniazid (H) and pyrazinamide (Z) was assessed. Measurements and Main Results: Maximum tolerated dosage of rifampin in the murine TB was 160 mg/kg/d. Pharmacokinetic measurement in HR(10)Z and HR(160)Z therapy regimens showed for rifampin a Cmax of 16.2 and 157.3 mg/L, an AUC0-24h of 132 and 1,782 h.mg/L, and AUC0-24h/minimum inhibitory concentration ratios of 528 and 7129, respectively. A clear dose-effect correlation was observed for rifampin after 3-week single-drug treatment. Administration of HR(80)Z allowed 9-week treatment duration to be effective without relapse of infection. Conclusions: Our findings indicate that the currently used rifampin dosage in the therapy of TB is too low. In our murine TB model a rifampin dosage of 80 mg/kg/d enabled a significant reduction in therapy duration without adverse effects