Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.
24 Related JoVE Articles!
A Simple Chelex Protocol for DNA Extraction from Anopheles spp.
Institutions: Malaria Institute at Macha, Johns Hopkins Bloomberg School of Public Health.
Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5
. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8
. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl.
from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2
vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl.
mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 μl of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 μl was retained while the other 10 μl was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10
. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11
, a PCR for identification of Anopheles gambiae
and a nested PCR for typing of Plasmodium falciparum
. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum
detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.
Infection, Issue 71, Immunology, Infectious Diseases, Genetics, Molecular Biology, Microbiology, Parasitology, Entomology, Malaria, Plasmodium falciparum, vector, Anopheles, Diptera, mosquitoes, Chelex, DNA, extraction, PCR, dissection, insect, vector, pathogen
Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test
Institutions: University of Bern, MCL Laboratories Inc..
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
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
Immunology, Issue 62, tuberculosis, drug resistance, rifampicin, rapid diagnosis, Xpert MTB/RIF test
An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei
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
Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites
Institutions: National Institutes of Health, Xiamen University.
Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3
. The malaria parasite Plasmodium yoelii
is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9
. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum
. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii
that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10
Genetic crosses in P. yoelii
and other rodent malaria parasites are conducted by infecting mice Mus musculus
with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11
. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.
Infectious Disease, Issue 47, Genetic cross, genetic mapping, malaria, rodent
Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice
Institutions: The Walter and Eliza Hall Institute of Medical Research.
We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei
ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum
-mediated severe malaria in humans 1,2
. Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3
and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6
, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.
Immunology, Issue 71, Infection, Infectious Diseases, Pathology, Hematology, Molecular Biology, Cellular Biology, Mouse, Brain, Intravascular inflammation, leukocytes, Plasmodium berghei, parasite, malaria, animal model, flow cytometry
An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells
Institutions: CHUL (CHUQ), Quebec City, Quebec, Canada.
, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1
. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3
. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro
studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum
schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5
. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7
, but that it decreased HIV-1 infection of macrophages8
. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum
-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env
gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.
Immunology, Issue 66, Infection, Medicine, Malaria, HIV-1, Monocyte-Derived Macrophages, PBMC, Red blood cells, Dendritic Cells, Co-infections, Parasites, Plasmodium falciparum, AIDS
2D and 3D Chromosome Painting in Malaria Mosquitoes
Institutions: Virginia Tech.
Fluorescent in situ
hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae
. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae
. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.
Immunology, Issue 83, Microdissection, whole genome amplification, malaria mosquito, polytene chromosome, mitotic chromosomes, fluorescence in situ hybridization, chromosome painting
Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow
Institutions: Barcelona Centre for International Health Research, University of Barcelona- Scientific and Technological Centers, Institució Catalana de Recerca i Estudis Avançats (ICREA).
The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2
. Thus, in vivo
imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3
, the complete development of the parasite in the skin 4
, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5
. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6
. Thus, intravital imaging has radically changed our view on key events in Plasmodium
development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii
in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii
17X as opposed to infections with the P.yoelii
17XL lethal parasite line 7
. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo
. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen.
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.
Immunology, Issue 59, intravital microscopy, GFP, malaria, spleen, mobility, adhesion, Plasmodium yoelii, Balb/c mice
Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation
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.
The Multiple Sclerosis Performance Test (MSPT): An iPad-Based Disability Assessment Tool
Institutions: Cleveland Clinic Foundation, Cleveland Clinic Foundation, Cleveland Clinic Foundation, Cleveland Clinic Foundation.
Precise measurement of neurological and neuropsychological impairment and disability in multiple sclerosis is challenging. We report a new test, the Multiple Sclerosis Performance Test (MSPT), which represents a new approach to quantifying MS related disability. The MSPT takes advantage of advances in computer technology, information technology, biomechanics, and clinical measurement science. The resulting MSPT represents a computer-based platform for precise, valid measurement of MS severity. Based on, but extending the Multiple Sclerosis Functional Composite (MSFC), the MSPT provides precise, quantitative data on walking speed, balance, manual dexterity, visual function, and cognitive processing speed. The MSPT was tested by 51 MS patients and 49 healthy controls (HC). MSPT scores were highly reproducible, correlated strongly with technician-administered test scores, discriminated MS from HC and severe from mild MS, and correlated with patient reported outcomes. Measures of reliability, sensitivity, and clinical meaning for MSPT scores were favorable compared with technician-based testing. The MSPT is a potentially transformative approach for collecting MS disability outcome data for patient care and research. Because the testing is computer-based, test performance can be analyzed in traditional or novel ways and data can be directly entered into research or clinical databases. The MSPT could be widely disseminated to clinicians in practice settings who are not connected to clinical trial performance sites or who are practicing in rural settings, drastically improving access to clinical trials for clinicians and patients. The MSPT could be adapted to out of clinic settings, like the patient’s home, thereby providing more meaningful real world data. The MSPT represents a new paradigm for neuroperformance testing. This method could have the same transformative effect on clinical care and research in MS as standardized computer-adapted testing has had in the education field, with clear potential to accelerate progress in clinical care and research.
Medicine, Issue 88, Multiple Sclerosis, Multiple Sclerosis Functional Composite, computer-based testing, 25-foot walk test, 9-hole peg test, Symbol Digit Modalities Test, Low Contrast Visual Acuity, Clinical Outcome Measure
Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology
Institutions: University of Exeter, Queen Mary University of London, St. Bartholomew's Hospital and The London NHS Trust.
Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1
. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2
. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus
in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3
, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus
is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus
galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 →3)-β-D-glucan, but not in the mucorales that lack this component in their cell walls1,4
. Issues surrounding the accuracy of these tests1,4-6
has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5
recently described the generation of an Aspergillus
-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5
. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus
spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 → 3)-β-D-glucan assays7
Here, we present a simple LFD procedure to detect Aspergillus
antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.
Immunology, Issue 61, Invasive pulmonary aspergillosis, acute myeloid leukemia, bone marrow transplant, diagnosis, monoclonal antibody, lateral-flow technology
Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites
Institutions: Royal Military College of Canada, Queen's University.
The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g.
carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.
Environmental Sciences, Issue 93, biochar, characterization, carbon sequestration, remediation, International Biochar Initiative (IBI), soil amendment
High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays
Institutions: Walter and Eliza Hall Institute of Medical Research, University of Melbourne.
merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum
biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies.
Immunology, Issue 89, Parasitic Diseases, malaria, Plasmodium falciparum, hemozoin, antibody, Fc Receptor, opsonization, merozoite, phagocytosis, THP-1
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
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
Tilt Testing with Combined Lower Body Negative Pressure: a "Gold Standard" for Measuring Orthostatic Tolerance
Institutions: Simon Fraser University .
Orthostatic tolerance (OT) refers to the ability to maintain cardiovascular stability when upright, against the hydrostatic effects of gravity, and hence to maintain cerebral perfusion and prevent syncope (fainting). Various techniques are available to assess OT and the effects of gravitational stress upon the circulation, typically by reproducing a presyncopal event (near-fainting episode) in a controlled laboratory environment. The time and/or degree of stress required to provoke this response provides the measure of OT. Any technique used to determine OT should: enable distinction between patients with orthostatic intolerance (of various causes) and asymptomatic control subjects; be highly reproducible, enabling evaluation of therapeutic interventions; avoid invasive procedures, which are known to impair OT1
In the late 1980s head-upright tilt testing was first utilized for diagnosing syncope2
. Since then it has been used to assess OT in patients with syncope of unknown cause, as well as in healthy subjects to study postural cardiovascular reflexes2-6
. Tilting protocols comprise three categories: passive tilt; passive tilt accompanied by pharmacological provocation; and passive tilt with combined lower body negative pressure (LBNP). However, the effects of tilt testing (and other orthostatic stress testing modalities) are often poorly reproducible, with low sensitivity and specificity to diagnose orthostatic intolerance7
Typically, a passive tilt includes 20-60 min of orthostatic stress continued until the onset of presyncope in patients2-6
. However, the main drawback of this procedure is its inability to invoke presyncope in all individuals undergoing the test, and corresponding low sensitivity8,9
. Thus, different methods were explored to increase the orthostatic stress and improve sensitivity.
Pharmacological provocation has been used to increase the orthostatic challenge, for example using isoprenaline4,7,10,11
or sublingual nitrate12,13
. However, the main drawback of these approaches are increases in sensitivity at the cost of unacceptable decreases in specificity10,14
, with a high positive response rate immediately after administration15
. Furthermore, invasive procedures associated with some pharmacological provocations greatly increase the false positive rate1
Another approach is to combine passive tilt testing with LBNP, providing a stronger orthostatic stress without invasive procedures or drug side-effects, using the technique pioneered by Professor Roger Hainsworth in the 1990s16-18
. This approach provokes presyncope in almost all subjects (allowing for symptom recognition in patients with syncope), while discriminating between patients with syncope and healthy controls, with a specificity of 92%, sensitivity of 85%, and repeatability of 1.1±0.6 min16,17
. This allows not only diagnosis and pathophysiological assessment19-22
, but also the evaluation of treatments for orthostatic intolerance due to its high repeatability23-30
. For these reasons, we argue this should be the "gold standard" for orthostatic stress testing, and accordingly this will be the method described in this paper.
Medicine, Issue 73, Anatomy, Physiology, Biomedical Engineering, Neurobiology, Kinesiology, Cardiology, tilt test, lower body negative pressure, orthostatic stress, syncope, orthostatic tolerance, fainting, gravitational stress, head upright, stroke, clinical techniques
A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting
Institutions: Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Liverpool School of Tropical Medicine, New York University School of Medicine.
causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum
-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum
has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1
. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2
and restrict their environment to a more favorable low oxygen pressure 3
. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro
, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5
. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6
. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8
has also been associated with severe disease 9
One of the most recently described P. falciparum
cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro
. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10
. Whether clumping occurs in vivo
remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11
. In addition, the ability of clinical isolate cultures to clump in vitro
was directly linked to the severity of disease in Malawian 12
and Mozambican patients 13
, (although not in Malian 14
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15
. Here, we present a method for in vitro
platelet-mediated clumping of P. falciparum
with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.
Infection, Issue 75, Infectious Diseases, Immunology, Medicine, Microbiology, Molecular Biology, Cellular Biology, Parasitology, Clumping, platelets, Plasmodium falciparum, CD36, malaria, malarial infections, parasites, red blood cells, plasma, limited resources, clinical techniques, assay
Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication
Institutions: University of Sydney, University of Hull, Hull and East Yorkshire Hospitals, Addenbrookes Hospital.
Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual's use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual's postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.
Medicine, Issue 82, Posture, Computerized dynamic posturography, Ankle brachial pressure index, Peripheral arterial disease, Intermittent claudication, Balance, Posture, EquiTest, Sensory Organization Test, Motor Control Test
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
Light/dark Transition Test for Mice
Institutions: Graduate School of Medicine, Kyoto University.
Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.
Neuroscience, Issue 1, knockout mice, transgenic mice, behavioral test, phenotyping
Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window
Institutions: University of California, San Diego, La Jolla Bioengineering Institute.
This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo
fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei
ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.
Neuroscience, Issue 45, Brain hemodynamics, blood flow, microcirculation, vascular morphology, leukocyte adherence, cerebral malaria
Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection
Institutions: Cornell University, Cornell University.
Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.
Plant Biology, Issue 15, Annual Review, Aphids, Plant Virus, Potato Leaf Roll Virus, Microinjection Technique
Population Replacement Strategies for Controlling Vector Populations and the Use of Wolbachia pipientis for Genetic Drive
Institutions: Johns Hopkins University.
In this video, Jason Rasgon discusses population replacement strategies to control vector-borne diseases such as malaria and dengue. "Population replacement" is the replacement of wild vector populations (that are competent to transmit pathogens) with those that are not competent to transmit pathogens. There are several theoretical strategies to accomplish this. One is to exploit the maternally-inherited symbiotic bacteria Wolbachia pipientis. Wolbachia is a widespread reproductive parasite that spreads in a selfish manner at the extent of its host's fitness. Jason Rasgon discusses, in detail, the basic biology of this bacterial symbiont and various ways to use it for control of vector-borne diseases.
Cellular Biology, Issue 5, mosquito, malaria, genetics, infectious disease, Wolbachia
Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes
Institutions: University of California, Irvine (UCI).
In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.
Cellular Biology, Issue 5, mosquito, malaria, dengue fever, genetics, infectious disease, Translational Research
Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes
Institutions: Johns Hopkins University.
In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.
Cellular Biology, Issue 5, Translational Research, mosquito, malaria, virus, dengue, genetics, injection, RNAi, transgenesis, transgenic