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Alcohol use disorders (AUD) among tuberculosis patients: a study from Chennai, South India.
PUBLISHED: 04-08-2011
Alcohol Use Disorders (AUDs) among tuberculosis (TB) patients are associated with nonadherence and poor treatment outcomes. Studies from Tuberculosis Research Centre (TRC), Chennai have reported that alcoholism has been one of the major reasons for default and mortality in under the DOTS programme in South India. Hence, it is planned to conduct a study to estimate prevalence of alcohol use and AUDs among TB patients attending the corporation health centres in Chennai, India.
Authors: Thomas Bodmer, Angelika Ströhle.
Published: 04-09-2012
Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2 Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in > 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2 Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10
18 Related JoVE Articles!
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Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray
Authors: Yvonne Linger, Alexander Kukhtin, Julia Golova, Alexander Perov, Peter Qu, Christopher Knickerbocker, Christopher G. Cooney, Darrell P. Chandler.
Institutions: Akonni Biosystems, Inc..
Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.
Immunology, Issue 86, MDR-TB, gel element microarray, closed amplicon, drug resistance, rifampin, isoniazid, streptomycin, ethambutol
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The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis
Authors: Mark F Brady, Jorge Coronel, Robert H Gilman, David AJ Moore.
Institutions: The Warren Alpert Medical School of Brown University, Universidad Peruana Cayetano Heredia, Johns Hopkins Bloomberg School of Public Health, Imperial College London .
Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections. Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains. The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB. The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB. In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.
Microbiology, Issue 18, tuberculosis, TB, multidrug resistant tuberculosis, MDRTB, culture, diagnostic
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A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)
Authors: Sandra Newton, Adrian Martineau, Beate Kampmann.
Institutions: Imperial College London , Barts & The London School of Medicine and Dentistry.
Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays. Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date. Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model. Note: Definitions/Abbreviations BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. CFU = Colony Forming Unit (a measure of mycobacterial viability).
Immunology, Issue 55, M.tuberculosis, BCG, whole blood assay, lux reporter genes, immune responses, tuberculosis, host pathogen interactions
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Enzyme-linked Immunospot Assay (ELISPOT): Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens
Authors: Isfahan R. Chambers, Tiffany R. Cone, Kyra Oswald-Richter, Wonder P. Drake.
Institutions: Vanderbilt University School of Medicine, Vanderbilt University School of Medicine.
Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.
Immunology, Issue 45, ELISPOT, Th-1 Immune Response, interferon gamma, T cell, adaptive immunity
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Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format
Authors: Leslie Hall, Kurt P. Jude, Shirley L. Clark, Nancy L. Wengenack.
Institutions: Mayo Clinic .
The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioMérieux, Hazelwood, MO). The agar proportion method, while considered the “gold” standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is ≥1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.
Immunology, Issue 52, Mycobacterium tuberculosis, MIC, antimicrobial susceptibility testing, first and second line drugs, microtiter plate, broth dilution
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Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model
Authors: Mary Zimmerman, Xiaolin Hu, Kebin Liu.
Institutions: Medical College of Georgia.
Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.
Immunology, Issue 45, Metastasis, CTL adoptive transfer, Lung, Tumor Immunology
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Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat
Authors: Daniel E. Bradford, Katherine P. Magruder, Rachel A. Korhumel, John J. Curtin.
Institutions: University of Wisconsin-Madison.
Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., “startle probes”). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.
Behavior, Issue 91, Startle; electromyography; shock; addiction; uncertainty; fear; anxiety; humans; psychophysiology; translational
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Utility of Dissociated Intrinsic Hand Muscle Atrophy in the Diagnosis of Amyotrophic Lateral Sclerosis
Authors: Parvathi Menon, Steve Vucic.
Institutions: Westmead Hospital, University of Sydney, Australia.
The split hand phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A novel split hand index (SI) was developed to quantify the split hand phenomenon, and its diagnostic utility was assessed in ALS patients. The split hand index was derived by dividing the product of the compound muscle action potential (CMAP) amplitude recorded over the abductor pollicis brevis and first dorsal interosseous muscles by the CMAP amplitude recorded over the abductor digiti minimi muscle. In order to assess the diagnostic utility of the split hand index, ALS patients were prospectively assessed and their results were compared to neuromuscular disorder patients. The split hand index was significantly reduced in ALS when compared to neuromuscular disorder patients (P<0.0001). Limb-onset ALS patients exhibited the greatest reduction in the split hand index, and a value of 5.2 or less reliably differentiated ALS from other neuromuscular disorders. Consequently, the split hand index appears to be a novel diagnostic biomarker for ALS, perhaps facilitating an earlier diagnosis.
Medicine, Issue 85, Amyotrophic Lateral Sclerosis (ALS), dissociated muscle atrophy, hypothenar muscles, motor neuron disease, split-hand index, thenar muscles
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Growth of Mycobacterium tuberculosis Biofilms
Authors: Kathleen Kulka, Graham Hatfull, Anil K. Ojha.
Institutions: University of Pittsburgh, University of Pittsburgh.
Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7. In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species. Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.
Immunology, Issue 60, Mycobacterium tuberculosis, tuberculosis, drug tolerance, biofilms
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Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development
Authors: Russell A. Morton, Marvin R. Diaz, Lauren A. Topper, C. Fernando Valenzuela.
Institutions: University of New Mexico Health Sciences Center.
Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.
Medicine, Issue 89, fetal, ethanol, exposure, paradigm, vapor, development, alcoholism, teratogenic, animal, mouse, model
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The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Authors: Sara Tremblay, Vincent Beaulé, Sébastien Proulx, Louis-Philippe Lafleur, Julien Doyon, Małgorzata Marjańska, Hugo Théoret.
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
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Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation
Authors: Amanda K. Rizk, Rima Wardini, Emilie Chan-Thim, Barbara Trutschnigg, Amélie Forget, Véronique Pepin.
Institutions: Concordia University, Concordia University, Hôpital du Sacré-Coeur de Montréal.
Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to exercise training recommendations. The study of exercise adherence is thus a key step towards the optimization of PR programs. To date, mostly indirect measures, such as rates of participation, completion, and attendance, have been used to determine adherence to PR. The purpose of the present protocol is to describe how continuous data tracking technology can be used to measure adherence to a prescribed aerobic training intensity on a second-by-second basis. In our investigations, adherence has been defined as the percent time spent within a specified target heart rate range. As such, using a combination of hardware and software, heart rate is measured, tracked, and recorded during cycling second-by-second for each participant, for each exercise session. Using statistical software, the data is subsequently extracted and analyzed. The same protocol can be applied to determine adherence to other measures of exercise intensity, such as time spent at a specified wattage, level, or speed on the cycle ergometer. Furthermore, the hardware and software is also available to measure adherence to other modes of training, such as the treadmill, elliptical, stepper, and arm ergometer. The present protocol, therefore, has a vast applicability to directly measure adherence to aerobic exercise.
Medicine, Issue 81, Data tracking, exercise, rehabilitation, adherence, patient compliance, health behavior, user-computer interface.
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A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening
Authors: Christophe. J Queval, Ok-Ryul Song, Vincent Delorme, Raffaella Iantomasi, Romain Veyron-Churlet, Nathalie Deboosère, Valérie Landry, Alain Baulard, Priscille Brodin.
Institutions: Université de Lille.
Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read out inferring on the pathogenesis of Mtb within host cells.
Infection, Issue 83, Mycobacterium tuberculosis, High-content/High-throughput screening, chemogenomics, Drug Discovery, siRNA library, automated confocal microscopy, image-based analysis
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An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei
Authors: Ann-Kristin Mueller, Jochen Behrends, Jannike Blank, Ulrich E. Schaible, Bianca E. Schneider.
Institutions: University Hospital Heidelberg, Research Center Borstel.
Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.
Infectious Diseases, Issue 84, coinfection, mouse, Tuberculosis, Malaria, Plasmodium berghei, Mycobacterium tuberculosis, natural transmission
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An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Authors: Justen Manasa, Siva Danaviah, Sureshnee Pillay, Prevashinee Padayachee, Hloniphile Mthiyane, Charity Mkhize, Richard John Lessells, Christopher Seebregts, Tobias F. Rinke de Wit, Johannes Viljoen, David Katzenstein, Tulio De Oliveira.
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
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Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages
Authors: Maruti Nandan Rai, Sapan Borah, Gaurav Bairwa, Sriram Balusu, Neelima Gorityala, Rupinder Kaur.
Institutions: Centre for DNA Fingerprinting and Diagnostics, Andhra Pradesh, India, Fiers-Schell-Van Montagu Building, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium.
A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.
Immunology, Issue 82, Candida glabrata, THP-1 macrophages, colony forming unit (CFU) assay, fluorescence microscopy, signature-tagged mutagenesis
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Electroporation of Mycobacteria
Authors: Renan Goude, Tanya Parish.
Institutions: Barts and the London School of Medicine and Dentistry, Barts and the London School of Medicine and Dentistry.
High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.
Microbiology, Issue 15, Springer Protocols, Mycobacteria, Electroporation, Bacterial Transformation, Transformation Efficiency, Bacteria, Tuberculosis, M. Smegmatis, Springer Protocols
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Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model
Authors: Denise Lawrie, George Janossy, Maarten Roos, Deborah K. Glencross.
Institutions: National Health Laboratory Services (NHLS-SA), University of Witwatersrand, Lightcurve Films.
We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing > 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.
Immunology, Issue 44, Human Immunodeficiency virus (HIV); CD4 lymphocyte count, white cell count, CD45, panleucogating, lymphocyte activation, CD38, HIV viral load, antiretroviral therapy (ART), internal quality control
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