Tuberculosis Diagnostic Methods Research Articles

Acid-Fast Positive and Acid-Fast Negative Mycobacterium tuberculosis: The Koch Paradox.

Acid-fast (AF) staining, also known as Ziehl-Neelsen stain microscopic detection, developed over a century ago, is even today the most widely used diagnostic method for tuberculosis. Herein we present a short historical review of the evolution of AF staining methods and discuss Koch's paradox, in which non-AF tubercle bacilli can be detected in tuberculosis patients or in experimentally infected animals. The conversion of Mycobacterium tuberculosis from an actively growing, AF-positive form to a nonreplicating, AF-negative form during the course of infection is now well documented. The mechanisms of loss of acid-fastness are not fully understood but involve important metabolic processes, such as the accumulation of triacylglycerol-containing intracellular inclusions and changes in the composition and spatial architecture of the cell wall. Although the precise component(s) responsible for the AF staining method remains largely unknown, analysis of a series of genetically defined M. tuberculosis mutants, which are attenuated in mice, pointed to the primary role of mycolic acids and other cell wall-associated (glyco)lipids as molecular markers responsible for the AF property of mycobacteria. Further studies are now required to better describe the cell wall reorganization that occurs during dormancy and to develop new staining procedures that are not affected by such cell wall alterations and that are capable of detecting AF-negative cells.

Drug-Resistant Tuberculosis and Modern Approaches towards its Diagnosis

The review of the literature has shown that tuberculosis as a socially conditioned disease remains a serious problem in Kazakhstan and worldwide in general, currently taking the first position among the infectious diseases causing mortality. One of the main reasons of poor effectiveness of treatment and heavy mortality caused by tuberculosis is prevalence of multiple resistance of tuberculosis mycobacteria to antituberculotics. Modern immunological and molecular genetic tuberculosis diagnostic methods, in comparison with the traditional methods, are more sensitive and specific and allow within a short time detecting the presence of tuberculosis mycobacteria in bio-substrates and their resistance to drugs, which reduces the time of abacillation among the patients, increases effectiveness of the treatment, prevents prevalence of drug-resistant strains of tuberculosis mycobacteria, should allow decreasing the tuberculosis infection rate in general and incidence of Multiple/Extensive Drug-Resistant tuberculosis in particular.

New directions using reporter enzyme fluorescence (REF) as a tuberculosis diagnostic platform

Although tuberculosis (TB) is one of the most common causes of morbidity and mortality in humans worldwide and diagnostic methods have been in place for more than 100 years, diagnosis remains a challenge. The main problems with diagnosis relate to the time needed to obtain a definitive result, difficulty in obtaining sputum, the primary clinical material used, and the ability of the causative agent, Mycobacterium tuberculosis, to cause disease in nearly any tissue within the body. In order to decrease incidence of TB, discovery of a novel interventions will be required, since current technologies have only been able to control numbers of infections, not reduce them. Diagnostic innovation is particularly needed because there are no effective pediatric or extrapulmonary TB diagnostic methods and multiple-drug resistance is only identified in less than 25% of those patients that are thought to have it. The most common diagnostic method worldwide remains acid-fast stain on sputum, with a threshold of ∼10,000 bacteria/ml that is only reached ∼5-6 months after development of symptoms. In order to obtain definitive diagnostic results earlier during the disease process, we have developed a diagnostic method designated reporter enzyme fluorescence (REF) that utilizes BlaC produced by M.tuberculosis and custom substrates to produce a specific fluorescent signal with as few as 10 bacteria/ml in clinical samples. We believe that the unique biology of the REF technique will allow it to contribute new diagnostic information that is complementary to all existing diagnostic tests as well as those currently known to be in development.

Detecting Antibody-Labeled BCG MNPs Using a Magnetoresistive Biosensor and Magnetic Labeling Technique

Tuberculosis is still a major global health concern, causing the estimated death of 1.5 million people per year and being associated with high morbidity. The development of point-of-care diagnostic tools for tuberculosis is mandatory, especially because the fast and accurate detection of the slow-growing Mycobacterium tuberculosis by the conventional diagnostic tests is difficult.The objective of this work was to develop the first steps to achieve a portable method for the diagnosis of tuberculosis, by a sandwich-immunoassay combined with magnetoresistive biochip technology.With the purpose of conjugating 250 nm streptavidin-coated magnetic nanoparticles with anti- M.tuberculosis biotinylated antibodies, Mycobacteriumbovis Bacillus Calmette-Guérin was used as a surrogate for M. tuberculosis bacteria. After magnetic capture, target bacteria were brought in contact with the surface of the magnetoresistive biochip previously functionalized with a secondary anti-M. tuberculosis antibody. Magnetically labeled cells were detected by an array of spin-valve sensors, which change their electrical resistance in the presence of the fringe field of the magnetic particles. Optimization studies on the efficiency of the magnetic capture and further recognition of the bacteria by the secondary antibody on the biochip surface were conducted. The results on the magnetoresistive biochip showed a clear difference in the signal between specific and control (non-specific) sensors, suggesting the usefulness of this technique as a potential biorecognition tool for the development of a point-of-care diagnostic method for tuberculosis.

Tuberculosis diagnostic methods in buffaloes

São crescentes as perdas econômicas com baixa na produtividade dos rebanhos bubalinos e a condenação de carcaças em matadouros devido à tuberculose, uma vez que os animais não podem ser tratados e devem ser descartados em abates sanitários. A doença tem impacto econômico e social para a cadeia produtiva do agronegócio brasileiro, além de ser uma zoonose de ampla distribuição. Assim como os bovinos, o búfalo é sensível ao Mycobacterium bovis, principal agente da tuberculose zoonótica. A ocorrência da tuberculose nessa espécie tem sido relatada em diversos países, inclusive no Brasil. Para controle e erradicação dessa enfermidade entre os bovinos e bubalinos no Brasil, o Ministério da Agricultura Pecuária e Abastecimento (MAPA) instituiu o Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose (PNCEBT), cujo principal objetivo é, por meio de métodos confiáveis, obter significativo número de rebanhos livres dessas doenças, em todo o território nacional. Esta revisão reuniu os principais dados referentes ao histórico da ocorrência de M. bovis nos rebanhos do Brasil e aos métodos diagnósticos da doença em búfalos. Na literatura faltam dados relativos à tuberculose bubalina e com o incremento da criação desses animais e de sua importância econômica, notou-se evidente necessidade de estudos e pesquisas relativos à ocorrência e identificação da tuberculose nessa espécie.

Detecting Antibody-Labeled BCG MNPs Using a Magnetoresistive Biosensor and Magnetic Labeling Technique

Tuberculosis is still a major global health concern, causing the estimated death of 1.5 million people per year and being associated with high morbidity. The development of point-of-care diagnostic tools for tuberculosis is mandatory, especially because the fast and accurate detection of the slow-growing Mycobacterium tuberculosis by the conventional diagnostic tests is difficult.The objective of this work was to develop the first steps to achieve a portable method for the diagnosis of tuberculosis, by a sandwich-immunoassay combined with magnetoresistive biochip technology.With the purpose of conjugating 250 nm streptavidin-coated magnetic nanoparticles with anti- M.tuberculosis biotinylated antibodies, Mycobacteriumbovis Bacillus Calmette-Guérin was used as a surrogate for M. tuberculosis bacteria. After magnetic capture, target bacteria were brought in contact with the surface of the magnetoresistive biochip previously functionalized with a secondary anti-M.tuberculosis antibody. Magnetically labeled cells were detected by an array of spin-valve sensors, which change their electrical resistance in the presence of the fringe field of the magnetic particles. Optimization studies on the efficiency of the magnetic capture and further recognition of the bacteria by the secondary antibody on the biochip surface were conducted. The results on the magnetoresistive biochip showed a clear difference in the signal between specific and control (non-specific) sensors, suggesting the usefulness of this technique as a potential biorecognition tool for the development of a point-of-care diagnostic method for tuberculosis.

Tuberculosis in HIV-infected infants, children, and adolescents in Latin America

ObjectiveTo evaluate the occurrence, clinical presentations and diagnostic methods for tuberculosis in a cohort of HIV-infected infants, children and adolescents from Latin America. MethodsA retrospective analysis of children with tuberculosis and HIV was performed within a prospective observational cohort study conducted at multiple clinical sites in Latin America. ResultsOf 1114 HIV-infected infants, children, and adolescents followed from 2002 to 2011, 69 that could be classified as having confirmed or presumed tuberculosis were included in this case series; 52.2% (95% CI: 39.8–64.4%) had laboratory-confirmed tuberculosis, 15.9% (95% CI: 8.2–26.7%) had clinically confirmed disease and 31.9% (95% CI: 21.2–44.2%) had presumed tuberculosis. Sixty-six were perinatally HIV-infected. Thirty-two (61.5%) children had a history of contact with an adult tuberculosis case; however information on exposure to active tuberculosis was missing for 17 participants. At the time of tuberculosis diagnosis, 39 were receiving antiretroviral therapy. Sixteen of these cases may have represented immune reconstitution inflammatory syndrome. ConclusionsOur study emphasizes the need for adequate contact tracing of adult tuberculosis cases and screening for HIV or tuberculosis in Latin American children diagnosed with either condition. Preventive strategies in tuberculosis-exposed, HIV-infected children should be optimized.

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.

Rapid Diagnosis of Mediastinal Tuberculosis With Polymerase Chain Reaction Evaluation of Aspirated Material Taken by Endobronchial Ultrasound–Guided Transbronchial Needle Aspiration

BackgroundEndobronchial ultrasound–guided transbronchial needle aspiration (EBUS-TBNA) is a diagnostic method for tuberculosis (TB). This study was conducted to determine the efficiency of polymerase chain reaction (PCR) testing for detecting TB...

Improving sensitivity for serodiagnosis of tuberculosis using TB16.3-echA1 fusion protein

This study aimed at developing and assessing the fusion proteins with enhanced sensitivity to detect antibodies in plasma as a diagnostic method for tuberculosis. DNA fragments encoding TB16.3 and echA1 gene regions corresponding to proteins TB16.3 and echA1 from Mycobacterium tuberculosis were amplified through PCR. Through a series of restrictions and ligations two novel fusion constructs TB16.3-echA1 and TB16.3-tnPstS1 were produced and expressed in Escherichia coli. These were screened for detection of antibodies in human plasma. The individual antigens TB16.3, echA1 and tnPstS1 and the fusion protein TB16.3-tnPstS1 and TB16.3-echA1 showed sensitivities of 29%, 25.5%, 42.8%, 40.0% and 47.2%, respectively. Lower sensitivity in case of TB16.3-tnPstS1 seems to be due to the structural arrangement between the two proteins, which is likely to mask several of their epitopes. The higher sensitivity of TB16.3-echA1 appears to be due to lesser interaction between the two proteins thus allowing free availability of epitopes for binding antibodies. 64% of TB patients were found positive for either one of the two fusion proteins TB16.3-echA1 and TB16.3-tnPstS1. This study indicates that the novel fusion protein TB16.3-echA1 has a potential in serodiagnosis of TB with improved sensitivity and reliability.

Frequency of Regulatory T-Cells in the Peripheral Blood of Patients with Pulmonary Tuberculosis from Shanxi Province, China

BackgroundTuberculosis (TB) is a disease caused by the chronic and continuous infection of the pathogen Mycobacterium tuberculosis (M. tuberculosis). M. tuberculosis is an intracellular bacterial pathogen and is eliminated mainly through CD4+ effector Th cells. M. tuberculosis induces regulatory T lymphocytes (Tregs) that mediate immune suppression by cell-to-cell contact or by secreting cytokines such as transforming growth factor-β (TGF-β). To understand the role of regulatory T-cells in the pathogenesis of TB, we have measured the in vivo frequency of regulatory T-cells and associated in vivo cytokine production in pulmonary tuberculosis patients.Methodology/Principal FindingsIn this study, we analyzed blood samples from 3 different populations (Group 1: patients with active TB, Group 2: patients recovered from TB and Group 3: healthy controls). We measured natural regulatory T-cell expression in peripheral blood using flow cytometry, and levels of blood serum IFN-γ and TGF-β1 using ELISA. The in vivo function of inductive regulatory T cells was mainly indicated by the expression of IFN-γ, TGF-β1, etc. Frequencyof natural regulatory T cells and inductive regulatory T cells in the peripheral blood samples from Group 1 patients were all significantly higher (P<0.05) than those from Groups 2 and 3.Conclusion/SignificanceOur results indicate that frequency of natural regulatory T cells and inductive regulatory T cells are significantly higher in the peripheral blood of patients with active pulmonary tuberculosis. These findings have potential application in improving TB diagnostic methods.

Early Specific Host Response Associated with Starting Effective Tuberculosis Treatment in an Infection Controlled Placebo Controlled Mouse Study

Recently we proposed exploring the potential of treatment stimulated testing as diagnostic method for tuberculosis (TB). An infection controlled placebo controlled mouse study was performed to investigate whether serum cytokine levels changed measurably during the early phase of TB chemotherapy. Serum was collected prior to and during the first 3 weeks of isoniazid (INH) and rifampicin (RIF) chemotherapy, and levels of 23 selected cytokines/chemokines were measured using a liquid bead array. The serum levels of IFNγ, IP-10, MIG, MCP-1, IL-17 and IL-6 were elevated in the TB infected mice compared to non-infected mice at least at 1 time point measured. In infected mice, IFNγ, IP-10, MIG and MCP-1 levels decreased within 7 days of treatment with RIF+INH compared to placebo. Treatment of non-infected mice in the absence of tuberculosis infection had no effect on these cytokines. IL-17 and IL-6 had decreased to baseline in all infected mice prior to the initiation of treatment. This study demonstrates that systemic levels of some cytokines, more specifically IFNγ, IP-10, MIG and MCP-1, rapidly and specifically change upon starting TB chemotherapy only in the presence of infection in a mouse model. Thus, IFNγ, IP-10, MIG and MCP-1 are promising ‘Treat-to-Test’ targets for the diagnosis of TB and deserve further investigation in a study on human TB suspects.

Detection limit for differentiating between various Mycobacterium species and Pseudomonas aeruginosa using gas chromatography-mass spectrometry (GC-MS) metabolomics: A comparison of two extraction methods

Previously, we investigated the capacity of a metabolomics research approach to characterise and differentiate between various infectious Mycobacterium species andPseudomonas aeruginosa, and compared two extraction procedures, prior to gas chromatography mass spectrometry (GC-MS) and statistical data analyses. In the current investigation, we report that the minimum sample material required (detection limit) for this speciation is 250 cells, using the total metabolome extraction method, and 2500 cells, using the fatty acid metabolome extraction procedure. Considering these detection limits, both methods compared significantly well with the currently used tuberculosis diagnostic methods and have the potential to be implemented clinically. Key words: Detection limit, tuberculosis, metabolomics

Nucleic acid testing for tuberculosis at the point-of-care in high-burden countries

Early diagnosis of tuberculosis (TB) facilitates appropriate treatment initiation and can limit the spread of this highly contagious disease. However, commonly used TB diagnostic methods are slow, often insensitive, cumbersome and inaccessible to most patients in TB endemic countries that lack necessary resources. This review discusses nucleic acid amplification technologies, which are being developed for rapid near patient TB diagnosis, that are in the market or undergoing clinical evaluation. They are based on PCR or isothermal methods and are implemented as manual assays or partially/fully integrated instrument systems, with associated tradeoffs between clinical performance, cost, robustness, quality assurance and usability in remote settings by minimally trained personnel. Unmet needs prevail for the identification of drug-resistant TB and for TB diagnosis in HIV-positive and pediatric patients.

New Diagnostic Methods for Tuberculosis

Correspondence to Jae Ho Lee, M.D. Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine and Lung Institute of Medical Research Center, Seoul National University College of Medicine, Department of Internal Medicine, Seoul National University Bundang Hospital, 82 Gumi-ro 173beon-gil, Bundang-gu, Seongnam 463-707, Korea Tel: +82-31-787-2120, Fax: +82-31-787-2204, E-mail: jhlee7@snubh.org 결핵의 새로운 진단법

Plasma Antibody Profiles as Diagnostic Biomarkers for Tuberculosis

Two billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), worldwide. Ten million to 20 million of the infected individuals develop disease per year. TB is a treatable disease, provided that it is diagnosed in a timely manner. The current TB diagnostic methods are subjective, inefficient, or not cost-effective. Antibody-based blood tests can be used efficiently and cost-effectively for TB diagnosis. A major challenge is that different TB patients generate antibodies against different antigens. Therefore, a multiplex immunoassay approach is needed. We have developed a multiplex panel of 28 M. tuberculosis antigen-coated microbeads. Plasma samples were obtained from over 300 pulmonary TB patients and healthy controls in a country where TB is endemic, Pakistan. Multiplex data were analyzed using computational tools by multivariate statistics, classification algorithms, and cluster analysis. The results of antibody profile-based detection, using 16 selected antigens, closely correlated with those of the sputum-based diagnostic methods (smear microscopy and culture) practiced in countries where TB is endemic. Multiplex microbead immunoassay had a sensitivity and specificity of approximately 90% and 80%, respectively. These antibody profiles could potentially be useful for the diagnosis of nonpulmonary TB, which accounts for approximately 20% of cases of disease. Since an automated, high-throughput version of this multiplex microbead immunoassay could analyze thousands of samples per day, it may be useful for the diagnosis of TB in millions of patients worldwide.

Construction and production of Ag85B:hFcγ1 recombinant protein for designing vaccine and new diagnostic methods for tuberculosis

Construction and production of Ag85B:hFcγ1 recombinant protein for designing vaccine and new diagnostic methods for tuberculosis

A Proteome-Scale Identification of Novel Antigenic Proteins inMycobacterium tuberculosistoward Diagnostic and Vaccine Development

Tuberculosis (TB) remains to be a major infectious disease throughout the world. However, the current vaccine for TB has variable protective efficacy, and there is no commercially available serodiagnostic test for this disease with acceptable sensitivity and specificity for routine laboratory use. One of the potential strategies in developing a new diagnostic method and in improving the TB vaccine involves the identification of novel antigenic candidates. This paper aims to identify systematically the novel antigenic proteins with the greatest potential as protective or diagnostic antigens by using the differential response of Mycobacterium tuberculosis proteins to serum from TB patients and healthy individuals. Approximately 87% of the open reading frames of M. tuberculosis were successfully cloned into IPTG-inducible expression vectors. The clone sets were expressed in Escherichia coli, purified under denatured conditions, and tested for antigenicity using a mixture of sera from 15 TB patients. Out of the 3480 proteins screened, 249 proteins had significant reactions with the serum samples. Among the 249 proteins, 20 proteins were identified as most reactive. Compared with the commercial test kits, 3 novel antigens from the top 20 proteins, namely, Rv1987, Rv3807c, and Rv3887c, provided better sensitivity and accuracy. These newly identified antigenic proteins may be used as candidates for serodiagnostic application and vaccine development. Overall, this study's findings may serve as an essential reference for developing new TB diagnostic methods and more effective tuberculosis vaccines.

Tuberculosis Epidemiology, Diagnosis and Infection Control Recommendations for Dental Settings: An Update on the Centers for Disease Control and Prevention Guidelines

Although rates of tuberculosis (TB) in the United States have decreased in recent years, disparities in TB incidence still exist between U.S.-born and foreign-born people (people living in the United States but born outside it) and between white people and nonwhite people. In addition, the number of TB outbreaks among health care personnel and patients has decreased since the implementation of the 1994 Centers for Disease Control and Prevention (CDC) guidelines to prevent transmission of Mycobacterium tuberculosis. In this article, the authors provide updates on the epidemiology of TB, advances in TB diagnostic methods and TB infection control guidelines for dental settings. In 2008, 83 percent of all reported TB cases in the United States occurred in nonwhite people and 17 percent occurred in white people. Foreign-born people had a TB rate about 10 times higher than that of U.S.-born people. New blood assays for M. tuberculosis have been developed to diagnose TB infection and disease. Changes from the 1994 CDC guidelines incorporated into CDC's "Guidelines for Preventing the Transmission of Mycobacterium tuberculosis in Health-Care Settings, 2005" include revised risk classifications, new TB diagnostic methods, decreased frequencies of tuberculin skin testing in various settings and changes in terminology. Although the principles of TB infection control have remained the same, the changing epidemiology of TB and the advent of new diagnostic methods for TB led to the development of the 2005 update to the 1994 guidelines. Dental health care personnel should be aware of the modifications that are pertinent to dental settings and incorporate them into their overall infection control programs.

New diagnostic methods for tuberculosis

During the last decade, laboratory tests for the detection of Mycobacterium tuberculosis (Mtb) have improved dramatically. Improvements in the ability to detect latent infection with Mtb, disease associated with Mtb, and strains resistant to commonly used antibiotics are reviewed. Advances in the detection of Mtb include light-emitting diode fluorescence microscopy, nucleic acid amplification of Mtb and drug-resistant strains, and more rapid liquid culture with adjunct drug susceptibility testing. In the detection of latent tuberculosis infection, interferon [gamma] release assays offer improved accuracy over the tuberculin skin test. The past 10 years have seen the most rapid growth in new diagnostics for Mtb in over a century. Although these tests offer improvements in the ability to detect Mtb, drug-resistant isolates, and those with latent tuberculosis infection, these improvements are counter-balanced by the need to deploy these tests in areas where Mtb burden is highest.