Un agradecimiento especial para el Servicio Nacional de Salud Laboratorio (NHLS) de la República de Sudáfrica para la organización nacional de los servicios de flujo de diagnóstico del VIH relacionadas (http: \ \ www.nhls.ac.za y http://www.plgcd4 . net). Los estudios se han realizado destacados en el Departamento de Hematología y Medicina Molecular, Universidad de Witwatersrand (Johannesburgo, Sudáfrica, con el apoyo del Jefe de Departamento, el profesor Wendy Stevens). Los autores no han recibido subvenciones para la publicación de las sociedades comerciales o las agencias de financiamiento. Las opiniones expresadas son las de los autores no necesariamente reflejan las opiniones de las instituciones de la Universidad de Witwatersrand o el NHLS de Sudáfrica.

<ol>
	<li>Mugyenyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+versus+clinically+driven+laboratory+monitoring+of+HIV+antiretroviral+therapy+in+Africa+(DART):+a+randomized+non-inferiority+trial.">Routine versus clinically driven laboratory monitoring of HIV antiretroviral therapy in Africa (DART): a randomized non-inferiority trial.</a> Lancet. 375, 123-131 (2009).</li><li>Donnell, D., Kiarie, J., Thomas, K., Baeten, J., Lingappa, J., Cohen, C., Celum, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ART+and+risk+of+heterosexual+HIV-1+transmission+in+HIV-1+serodiscordant+African+couples:+A+Multinational+Prospective+Study.">ART and risk of heterosexual HIV-1 transmission in HIV-1 serodiscordant African couples: A Multinational Prospective Study.</a> , (2010).</li><li>Montaner, J., Wood, E., Kerr, T., Yip, B., Lima, V., Shannon, K., Harrigan, R., Hogg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Expanded+HAART+Coverage+with+a+Decrease+in+New+HIV+Diagnoses,+Particularly+among+Injection+Drug+Users+in+British+Columbia,+Canada.">Association of Expanded HAART Coverage with a Decrease in New HIV Diagnoses, Particularly among Injection Drug Users in British Columbia, Canada.</a> , (2010).</li><li>Petti, C. A., Polage, C. R., Quinn, T. C., Ronald, A. R., Sande, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+medicine+in+Africa:+a+barrier+to+effective+health+care.">Laboratory medicine in Africa: a barrier to effective health care.</a> Clin Infect Dis. 42, 377-382 (2006).</li><li>Brown, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+health:+Great+expectations.">Global health: Great expectations.</a> BMJ. 334, 874-876 (2007).</li><li>Crisp, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+the+World+Upside+Down.">Turning the World Upside Down.</a> Royal Society of Medicine Press. , (2009).</li><li>Hecht, R., Bollinger, L., Stover, J., McGreevey, W., Muhib, F., Madavo, C. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunophenotyping+of+acute+leukemia+by+flow+cytometric+analysis.+Use+of+CD45+and+right-angle+light+scatter+to+gate+on+leukemic+blasts+in+three-color+analysis.">Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis.</a> Am J Clin Pathol. 100, 534-540 (1993).</li><li>Mokone, G. N., Lawrie, D., Coetzee, L., Glencross, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+of+CD45+patterns+in+the+PanLeucoGating+CD4+test+can+be+used+for+diagnosis+of+white+cell+abnormalities.">Interpretation of CD45 patterns in the PanLeucoGating CD4 test can be used for diagnosis of white cell abnormalities.</a> Poster presentation. , (2007).</li><li>Lawrie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wide-spread+clinical+uses+of+PanLeucoGating+technology+in+the+HIV+diagnosis+oriented+immunohaematology+laboratory.">The wide-spread clinical uses of PanLeucoGating technology in the HIV diagnosis oriented immunohaematology laboratory.</a> PhD Thesis, in preparation. , (2010).</li><li>Badri, M., Lawn, S. D., Wood, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+CD4+cell+counts+for+early+prediction+of+virological+failure+during+antiretroviral+therapy+in+a+resource-limited+setting.">Utility of CD4 cell counts for early prediction of virological failure during antiretroviral therapy in a resource-limited setting.</a> BMC Infect Dis. 8, 89 (2008).</li><li>Peter, T., Badrichani, A., Wu, E., Freeman, R., Ncube, B., Ariki, F., Daily, J., Shimada, Y., Murtagh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+implementing+CD4+testing+in+resource-limited+settings.">Challenges in implementing CD4 testing in resource-limited settings.</a> Cytometry B Clin Cytom. 74, S123-S130 (2008).</li><li>Denny, T. N., Gelman, R., Bergeron, M., Landay, A., Lam, L., Louzao, R., Mandy, F. F., Schmitz, J., Spira, T., Wilkening, C., Glencross, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+North+American+multilaboratory+study+of+CD4+counts+using+flow+cytometric+panLeukogating+(PLG):+a+NIAID-DAIDS+Immunology+Quality+Assessment+Program+Study.">A North American multilaboratory study of CD4 counts using flow cytometric panLeukogating (PLG): a NIAID-DAIDS Immunology Quality Assessment Program Study.</a> Cytometry B Clin Cytom. 74, S52-S64 (2008).</li><li>Janossy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changing+pattern+of+&#34;smart&#34;+flow+cytometry+(S-FC)+to+assist+the+cost-effective+diagnosis+of+HIV,+tuberculosis,+and+leukemias+in+resource-restricted+conditions.">The changing pattern of &#34;smart&#34; flow cytometry (S-FC) to assist the cost-effective diagnosis of HIV, tuberculosis, and leukemias in resource-restricted conditions.</a> Biotechnol J. 3, 32-42 (2008).</li></ol>

Los derechos de patentes internacionales para el método de CD4 se describe aquí se llevan a cabo por el NHLS de Sudáfrica y en la actualidad bajo la licencia de BCI, Miami, Fl, EE.UU..

En los últimos años los programas impulsados ​​por la caridad y la subvención concedida, han ayudado a la creación de unas excelentes prácticas clínicas en áreas de recursos limitados del planeta con alta carga de VIH. Los programas de arte han tenido éxito en la reducción de la mortalidad y ahora se están iniciando en el recuento de CD4 superior 2,3. Ya hay indicios de que estas nuevas medidas será disminuir la propagación de la infección por VIH 4,5, a pesar de la falta de una vacuna específica contra el VIH. En el video se documenta que al seguimiento de los pacientes en tratamiento antirretroviral, estas técnicas pueden ser mejor enfocado y ser más rentable que la observada en la práctica de rutina en los laboratorios occidentales (Lista 1). Tal enfoque dirigida por África y puede ser esencial para apoyar la modernización de los servicios de laboratorio en un momento en que la infección por VIH, tratados con ART, se está convirtiendo en una enfermedad crónica. Como un seguimiento de la terapia antirretroviral requiere CD4 preciso contar y de carga viral relacionados con la tecnología, una tarea más exigente que para llevar a cabo un simple recuento de CD4-para colocar a los pacientes en grupos arbitrarios con el fin de ayudar a una decisión sobre el inicio ART 16. Con la ayuda de estos diagnósticos, es decir, mediante la prueba de CD8/CD38, es posible reducir los costos de supervisión VL-relacionados en&gt; 60% de los pacientes en tratamiento antirretroviral 16,19. También es importante destacar que es la prueba de CD8/CD38 16,17, y no el recuento de CD4 de 37 años, que es probable que recoger los primeros signos de rebotes VL y / o la falta de cumplimiento del paciente en la toma de la medicación. Un elemento esencial de una estrategia coordinada rentable servicio central es la disponibilidad de una red a gran escala nacional o regional con el apoyo de expertos y asesores académicos que están familiarizados con los aspectos técnicos y médicos de la operación. Esto se demuestra por las actividades de la red de Sudáfrica NHLS y los programas de formación CD4-PLG.net 33, 36, que proporcionan el estímulo, la educación y la evaluación de la calidad de los centros más pequeños y estos también pueden ser &quot;el punto de atención&quot; de servicios. Es importante destacar que las operaciones de los laboratorios centrales y puntos de atención son complementarias. Mientras que el primero tiene la doble función de proporcionar una solución rentable operación a gran escala de servicios, así como la formación básica / avanzada para el periférico laboratorios de 33,38, en que éste contribuye a contener la propagación de la terapia antirretroviral mediante el reclutamiento de los pacientes VIH + que son elegibles para ART a partir de una base más amplia, incluyendo las clínicas rurales. Aquí cierta cautela es necesaria porque el punto de atención de diagnóstico puede ser mucho más costoso y no garantiza automáticamente la precisión 38. Por tanto, es razonable mantener el sistema de dos niveles y para facilitar la coordinación entre los dos niveles sin permitir que ninguno de los brazos a disminuir. En conclusión, en el ámbito del control diagnóstico del manejo clínico de las enfermedades infecciosas es necesario un ahorro de costes se puede lograr por los servicios de apt que utilizan la tecnología moderna más barato y mejor coordinados con una calidad mucho mejor 13,39. Bien organizados nacionales (y regionales) las disposiciones de diagnóstico de servicio debe recibir un mayor apoyo por 6-8 evitar un escenario en que prácticamente todo el apoyo de subvención se pierde en los métodos caros y / o no probadas tan pronto como éstos son importados de los países ricos sin un cuidado pre- escrutinio constructivo preventivo. Del mismo modo, un diseño más flexible de los ensayos clínicos terapéuticos con la plena participación de los médicos locales y el personal sanitario debe conducir a la reducción de la frecuencia innecesariamente y / o demasiado ambiciosos pruebas de laboratorio 3. Esta política le daría más oportunidad de un mejor aprovechamiento de los resultados disponibles de diagnóstico preciso en una evaluación coordinada clínica / laboratorio de los pacientes en tratamiento antirretroviral. Estos diagnósticos inteligentes también puede ser iniciado por los países de escasos recursos-8 en las áreas de la tuberculosis y las leucemias con una plataforma existente flujo de citometría de 40 familiares.

comprehensive &amp; cost effective laboratory monitoring of hiv/aids: an african role model

Presentamos el video de ayudar a la terapia antirretroviral (ART) por un servicio de laboratorio apt - lo que representa un modelo sudafricano para las pruebas de diagnóstico de gran escala económica. En los países de bajos ingresos económicos ART ha transformado las perspectivas para la supervivencia de los pacientes seropositivos al VIH, pero existen dudas sobre si es necesario para la vigilancia de laboratorio de arte y en lo que los costos - en situaciones en las que la calidad general de los servicios de patología todavía puede ser muy bajo. La respuesta adecuada es el establecimiento de servicios económicamente viables, con una mejor coordinación y más estricta evaluación de la calidad interna que se ve en los países occidentales. Este video, fotografiado en el lugar de los Servicios de Laboratorio Nacional de Salud (NHLS-SA) en la Universidad de Witwatersrand, Johannesburgo, Sudáfrica, proporciona un esquema coordinado ampliación de la original de dos colores CD4-CD45 estrategia PanLeucoGating (PLG). Así, los seis módulos de la presentación del vídeo revelan la sencillez de un ensayo de flujo de 4 colores citometría de combinar pruebas hematológicas, inmunológicas y virológicas relacionadas en un solo tubo. Estos módulos de vídeo son los siguientes: (i) la puesta en marcha de instrumentos, (ii) la preparación de la muestra, (iii) pruebas de recuentos absolutos y monitoreo de la calidad de cada muestra de cordón-cuenta-tasa, (iv) la prueba de heamatological CD45 de células blancas cuenta las diferencias y, (v) el recuento de células CD4, y (vi) la activación de células T CD8 + CD38 medido por la pantalla, un parámetro relacionado con la carga viral. El potencial de ahorro de costes es notable. Este acuerdo es un claro ejemplo de la factibilidad de realizar&gt; 800-1000 pruebas por día, con un control de calidad más estrictas que las aplicadas en los laboratorios occidentales, y también con la transferencia de tecnología a otros laboratorios dentro de una red NHLS-SA. Expertos asesores, directores de laboratorios y responsables de las políticas que llevan a la obligación de tomar decisiones sobre la introducción de la tecnología médica moderna no suele estar en condiciones para ver los últimos detalles técnicos que lleva a cabo en los grandes laboratorios regionales con enormes cargas de trabajo. Por lo tanto, este video muestra los detalles de estos nuevos desarrollos.

Watch this Scientific Journal Video about Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model at JoVE.com

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

La terapia antirretroviral para tratar el VIH / SIDA se controla en el sur de África a gran escala. La citometría de flujo se combina para hematología (CD45), la inmunología (CD4) y ​​carga viral ensayo relacionado CD38. Grabado en NHLS-SA laboratorios, Johannesburgo, estos métodos modernos son costo-eficientes, con mayor control local de calidad interno, que sirven como modelos a imitar por los limitados recursos de diagnóstico.

Supervisión efectiva de laboratorio integral y costo del VIH / SIDA: un modelo a seguir de África

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

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Research

JoVE Journal

Immunology and Infection

16.6K vistas.  National Health Laboratory Services (NHLS-SA). La terapia antirretroviral para tratar el VIH / SIDA se controla en el sur de África a gran escala. La citometría de flujo se combina para hematología (CD45), la inmunología (CD4) y ​​carga viral ensayo relacionado CD38. Grabado en NHLS-SA laboratorios, Johannesburgo, estos métodos modernos son costo-eficientes, con mayor control local de calidad interno, que sirven como modelos a imitar por los limitados recursos de diagnóstico.

Video: Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Un modelo ortotópico de cáncer de ovario seroso en ratones inmunocompetentes para In vivo Imágenes y seguimiento de tumores de las respuestas inmunitarias del tumor

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Investigación

Inmunología e Infección

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

Una analítica Caja de Herramientas para la Evaluación Integral de bioquímica, estructural y transcriptoma de biopelículas oral mediada por Streptococcus mutans

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Inferencia genotípica del VIH-1 tropismo utilizando la población basada en la secuencia de V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Método rentable para el Rastreo de uso microbiana específica virus humanos y animales

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

La inducción de la enfermedad de injerto contra huésped y<em&gt; En Vivo</em&gt; T Monitoreo Celular Utilizando un modelo murino MHC de concordancia

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

El uso de proteínas fluorescentes de Seguimiento glicosoma Dinámica en el tripanosoma africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

Medidas de seguridad y procedimientos de operación en una (A) BSL-4 de laboratorio: 1. Nivel de Bioseguridad 4 Traje Laboratorio de Entrada y Salida Procedimientos suite

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

Medidas de seguridad y procedimientos de operación en una (A) BSL-4 de laboratorio: 3. Aerobiología

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Imágenes de bioluminiscencia para detectar la infección en fase tardía de la tripanosomiasis africana

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Cefoperazona tratados modelo de ratón clínicamente relevante<em&gt; Clostridium difficile</em&gt; R20291 Strain

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...