73 research outputs found
Culturability of Mycobacterium tuberculosis cells isolated from murine macrophages: a bacterial growth factor promotes recovery
Viewpoint: Scientific dogmas, paradoxes and mysteries of latent Mycobacterium tuberculosis infection
Worldwide, there are nearly 10 million new cases of active TB and 1.8 million associated deaths every year. WHO estimates that one-third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), forming a huge latent Mtb global reservoir. This renders the prospect of ever eliminating Mtb from the human race almost impossible. Several controversial issues regarding host-pathogen interactions and existing prevention and eradication strategies for latent Mtb infections need to be critically re-examined. In this viewpoint, widely held assumptions on Mtb latency and isoniazid monotherapy and chemoprophylaxis are challenged. We highlight the need for future research to resolve these issues and to develop evidence-based strategies for better understanding of equilibrium and escape of Mtb in the human body, eventually leading to global recommendations for elimination of the latent Mtb state through informed policy and practice. Until such strategies and policies are realized, WHO and TB experts will have to settle for global TB control rather than eradication
The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods
Causative agents of melioidosis and glanders are among the most dangerous bacterial pathogens for human. Moreover, Burkholderia pseudomallei and Burkholderia mallei are considered to be potential bioterrorism agents. In connection with this, timely diagnostics of such bacteria is of high importance. In our study, we made an attempt to develop an approach for detecting pathogenic Burkholderia spp. by combining species-specific amplification and strain-specific dot blotting assay with monoclonal antibodies. The following pathogenic Burkholderia strains were used in experiments: B. mallei (C-4, C-5, t-12, B-120, P-1, Мuksuwar-11, Z-12,Zagreb, Ivanovich, 5534), and B. pseudomallei (100, 102, 115, 116, 132, 135, 301, 51274, 60913, 61503). Real-Time PCR (RT-PCR) and dot blotting with monoclonal antibodies against surface Burkholderia epitopes were used to detect such pathogens. RT-PCR was carried out by using primers designed to recognize DNA fragments in B. mallei IS407A-fliP and the gene Orf12 from B. pseudomallei. For this, DNA was isolated from bacterial cells suspended at 1 × 104 microbial cells/ml. accumulation of the end reaction products was visualized by staining with dye SYBR Green I. Specificity of amplification reaction was determined by measuring melting temperature (Tm) for end products followed by running gel electrophoresis. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. Importantly, no signals specific to heterologous microbial DNA (isolated from bacterial cell suspension at concentration of 1 × 107 microbial cells/ml) were detected by using RT-PCR. Thus, RT-PCR provides an opportunity for assessing an inter-species diversity among pathogenic Burkholderia species. A genus-specificity was observed by using monoclonal antibodies 3D3 which bind to both Burkholderia strains, whereas antibodies 2D11 exhibited no selective binding to strain Р1 B. mallei and strain 100 B. pseudomallei, thereby displaying a strain-specific interaction. Thus, it allowed to conclude that combining a species-specific DNA amplification particularly RT-PCR together with immune-based assay such as dot blotting by using a panel of monoclonal antibodies seems to be a promising approach for assessing intra-species diversity among pathogenic Burkholderia
Application of immunomagnetic separation for accelerated detection of <i>F. tularensis</i> cells in soil samples using an immunochromatographic test
Introduction. Epizootological monitoring of the area contamination with the causative agent of tularemia implies the collection and analysis of a variety of field specimens. The analysis of such objects is time- and labour-consuming. In this context, simple and fast diagnostic techniques are needed to analyze specimens under resource-limited conditions.
Aim. To study the possibility of using immunomagnetic separation for accelerated detection of Francisella tularensis cells in soil samples using immunochromatography.
Materials and methods. Immunomagnetic particles (IMPs) were produced by using monoclonal antibodies to lipopolysaccharide (LPS) of the tularemia causative agent. Soil specimens weighing 1 g with preliminary introduced inactivated F. tularensis 15/10 cells were used in the study. The samples were suspended in an extraction buffer (EB) and filtered. Tularemia cells were separated by IMP suspension. The particles were washed, resuspended in EB and heated at 100C for 5 minutes. The supernatant was analyzed with test strips based on F. tularensis IC-test kit.
Results. A combination of the immunomagnetic separation method and the IC test to detect F. tularensis cells identified up to 1 106 cells of the tularemia pathogen in analyzed soil samples, while 1 107 cells were detected in soil washouts in the absence of immunomagnetic separation.
Conclusion. The developed technique combining immunomagnetic separation and IC tests opens up prospects for express diagnostics of soil sample contamination in tularemia foci. The analysis takes about 3 hours, and its sensitivity is 1 106 cells/g of soil. The technique is simple, not requiring sophisticated expensive equipment. It can be easily adapted for testing other specimen types (water, grain, etc.). In addition, separated bacterial cells can be used for F. tularensis detection by other methods
The role of resuscitation promoting factors in pathogenesis and reactivation of Mycobacterium tuberculosis during intra-peritoneal infection in mice
<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis </it>can enter into a dormant state which has resulted in one third of the world's population being infected with latent tuberculosis making the study of latency and reactivation of utmost importance. <it>M. tuberculosis </it>encodes five resuscitation promoting factors (Rpfs) that bear strong similarity to a lysozyme-like enzyme previously implicated in reactivation of dormant bacteria <it>in vitro</it>.</p> <p>We have developed an intraperitoneal infection model in mice, with immune modulation, that models chronic infection with similar properties in mouse lungs as those observed in the murine aerosol infection model. We have assessed the behavior of mutants that lack two or three <it>rpf </it>genes in different combinations in our intraperitoneal model.</p> <p>Methods</p> <p>C57Bl/6 mice were intraperitonealy infected with H37Rv wild type <it>M. tuberculosis </it>or mutant strains that lacked two or three <it>rpf </it>genes in different combinations. After 90 days of infection aminoguanidine (AG) or anti-TNFα antibodies were administrated. Organ bacillary loads were determined at various intervals post infection by plating serial dilutions of organ homogenates and enumerating bacteria.</p> <p>Results</p> <p>We found that the <it>rpf </it>triple and double mutants tested were attenuated in their ability to disseminate to mouse lungs after intraperitoneal administration and were defective in their ability to re-grow after immunosuppression induced by administration of aminoguanidine and anti-TNFα antibodies.</p> <p>Conclusion</p> <p>Rpf proteins may have a significant physiological role for development of chronic TB infection and its reactivation <it>in vivo</it>.</p
Анализ влияния жевательной мускулатуры на череп экспериментального животного при наличии травмирующего/ изменяющего биомеханику мышцы фактора
Most authors recognize that the currently dominant scientific paradigm assumes that genomic rather than functional factors regulate (cause, control) the growth of cranial bones and cranial sutures. In contrast, the authors of this article offer some clarifications to address some unintentional conceptual misconceptions, based primarily on an extensive review of the relevant current literature and their own experience. This article describes the increased modulation of mechanotransduction produced by skeletal muscle activity, to which bone cells respond maximally. The authors describe the actual chain of events that influences the stimulation of bone cell growth. This influence makes it possible to propose a means of controlling these processes and developing new correction methods, including suppression of phenotypic expression. The authors present the results of a study of the effect of mechanical loading of the masticatory muscles with a continuous stretching stimulus to increase the width of extension of the sagittal suture of the experimental animal in vivo. The methodology is described, and objective instrumental control data are presented. The results of statistical processing are also presented. The authors give empirical data in this paper. These experiments prove that chewing load is one of the primary stimuli that generate craniofacial variations, affecting the structure of the cranial suture. The authors conducted the experimental part of the research at the Vivarium of Conventional Animals Collaborative Center of the Federal Research Center of the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences.Признавая, что доминирующая в настоящее время научная парадигма предполагает, что геномные, а не функциональные факторы регулируют (вызывают, контролируют) рост костей черепа и черепных швов, основываясь, прежде всего, на обширном обзоре соответствующей современной литературы и собственном опыте, авторы предлагают ряд уточнений, направленных на устранение некоторых непреднамеренных концептуальных заблуждений. В данной статье описывается процесс повышения модуляции механотрансдукции, продуцируемый мышечной активностью скелетных мышц, на которые максимально реагируют костные клетки. Авторами описанафактическаяцепь событий, оказывающая влияниена стимуляциюроста костных клеток, что позволяет предложить средство контроля данных процессов, разрабатывать новые методики коррекции, включая подавление фенотипической экспрессии. В статье представлены результаты исследования влияния механической нагрузки жевательных мышц с непрерывным стимулом растяжения в направлении увеличения ширины / растяжения сагиттального шва экспериментального животного in vivo. Описана методика и представлены данные объективного инструментального контроля, а также результаты, полученные в результате статистической обработки. Авторы в данной работе представляют экспериментальные доказательства того, что жевательная нагрузка является одним из основных стимулов, который генерирует черепно-лицевые вариации, влияя на структуру черепного шва. Экспериментальная часть исследований проводилась на базе Центра коллективного пользования «Виварий конвенциональных животных» Федерального исследовательского центра Институт цитологии и генетики Сибирского отделения Российской академии наук
The Incidence and Clinical Implication of Sputum with Positive Acid-Fast Bacilli Smear But Negative in Mycobacterial Culture in a Tertiary Referral Hospital in South Korea
Although it is not rare to find sputum that is positive acid-fast bacilli (AFB) smear but subsequent culture fails to isolate mycobacteria in clinical practice, the incidence and clinical implication of those sputa from new patients has not been clearly elucidated. The aim of this study was to determine the incidence and clinical implication of sputum with positive AFB smear but negative in mycobacterial culture. All sputa that were positive AFB smear requested during diagnostic work up for new patients visiting Seoul National University Hospital from 1 January 2005 through 31 December 2006 were included. Sputa producing a positive AFB smear but negative mycobacterial culture were classified into one of four categories: laboratory failure to isolate mycobacteria, false positive AFB smear, pathogen may show a positive AFB smear other than mycobacteria, and indeterminate results. Out of 447 sputa with a positive AFB smear, 29 (6.5%) failed to culture any organism. Among these 29 sputa, 18 were caused by laboratory failure to isolate mycobacteria, six were false positive smears, and five indeterminate. Although most sputum with a positive AFB smear but negative culture could be classified as a laboratory failure, clinicians should consider the possibility of false positive AFB smear
Identification of T-Cell Antigens Specific for Latent Mycobacterium Tuberculosis Infection
BACKGROUND: T-cell responses against dormancy-, resuscitation-, and reactivation-associated antigens of Mycobacterium tuberculosis are candidate biomarkers of latent infection in humans. METHODOLOGY/PRINCIPAL FINDINGS: We established an assay based on two rounds of in vitro restimulation and intracellular cytokine analysis that detects T-cell responses to antigens expressed during latent M. tuberculosis infection. Comparison between active pulmonary tuberculosis (TB) patients and healthy latently M. tuberculosis-infected donors (LTBI) revealed significantly higher T-cell responses against 7 of 35 tested M. tuberculosis latency-associated antigens in LTBI. Notably, T cells specific for Rv3407 were exclusively detected in LTBI but not in TB patients. The T-cell IFNgamma response against Rv3407 in individual donors was the most influential factor in discrimination analysis that classified TB patients and LTBI with 83% accuracy using cross-validation. Rv3407 peptide pool stimulations revealed distinct candidate epitopes in four LTBI. CONCLUSIONS: Our findings further support the hypothesis that the latency-associated antigens can be exploited as biomarkers for LTBI
Capabilities of using ICG/NIR-chromoscopy in performing endovideosurgical liver resections
Endovideoscopic interventions on the liver in combination with intraoperative contrast of blood vessels and bile ducts allow to facilitate performing the manipulation stage of the intervention, diagnose previously hidden pathology and reduce the risk of postoperative complications This clinical observation shows the capabilities of intraoperative ICG/NIR chromoscopy during endovideosurgical left-sided hemihepatectomy in patient with colorectal metastases
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