Single-reaction, multiplex, real-time rt-PCR for the detection, quantitation, and serotyping of dengue viruses.

open15siThis research was supported by the National Institutes of Health grant 1 RC4 TW008781-01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Background: Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction. Methodology/Principal Findings: An rRT-PCR assay targeting the 5 9 untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log 10 cDNA equivalents/ m L for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/ m L for serotypes 1–4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested. Conclusions/Significance: This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.openWaggoner JJ;Abeynayake J;Sahoo MK;Gresh L;Tellez Y;Gonzalez K;Ballesteros G;Pierro AM;Gaibani P;Guo FP;Sambri V;Balmaseda A;Karunaratne K;Harris E;Pinsky BAWaggoner JJ;Abeynayake J;Sahoo MK;Gresh L;Tellez Y;Gonzalez K;Ballesteros G;Pierro AM;Gaibani P;Guo FP;Sambri V;Balmaseda A;Karunaratne K;Harris E;Pinsky B

Evaluation of an automated system for the quantitation of human Herpesvirus-6 DNA from clinical specimens

Background: Quantitation of human herpesvirus-6 (HHV-6) DNA in clinical specimens is important for the diagnosis and management of HHV-6-associated infection and reactivation in immunocompromised patients, particularly transplant recipients. Methods: The analytical performance of the Altona RealStar ASR HHV-6 qPCR on the semi-automated AltoStar AM16 system was assessed using HHV-6 reference material in plasma and cerebral spinal fluid (CSF). Qualitative and quantitative agreement was determined using 123 clinical EDTA plasma specimens tested using a laboratory-developed HHV-6 qPCR. Results: The 95% Lower Limit of Detection was 20 IU/mL [95% confidence interval (CI): 10 to 29] in plasma and 78 IU/mL (95% CI: 55 to 146) in CSF. The assay was linear from 7.0 to 2.0 log10 IU/mL in both matrices. Overall agreement of the RealStar ASR HHV-6 qPCR on the AltoStar AM16 with a laboratory-developed test was 95.9% (95% CI: 90.8 to 98.7). Passing-Bablok analysis of specimens quantifiable by both methods and at levels >1000 copies/mL revealed a regression line of Y = 1.00*X–0.20, with neither systematic (95% CI Y-intercept: −0.66 to 0.26) nor proportional (95% CI slope: 0.89 to 1.10) bias compared to the reference. Conclusions: The RealStar ASR HHV-6 qPCR on the AltoStar AM16 provides accurate quantitation for clinical monitoring of HHV-6 in immunocompromised hosts

Sensing magnetic flux of Langmuir-Blodgett films of a molecular magnetic system using superconducting films and nano-SQUID devices

We report a study on the response of superconducitng micro-tracks and quantum interference devices (SQUIDs) to a proximal SMM film. As a test case, Langmuir-Blodgett $Mn_{12}$-ac SMM films have been grown on 2 $\mu$m wide Nb tracks and Nb nano-SQUIDs to observe the proximity effect of magnetic moment and magnetization tunneling, respectively. The superconducting critical temperature of thin Nb tracks (thinner than the coherence length of Nb) were found to decrease by the magnetic moment of $Mn_{12}$-ac SMM. Following the thermally activated flux flow (TAFF) model, we found an increase in the vortex unbinding energy of the SMM coated Nb tracks, near critical temperature. More importantly, the random alignment of moments of the $Mn_{12}$-ac molecules at low fields seemed to have the enhancing effect on vortex unbinding energy rather than the saturated state of $Mn_{12}$-ac molecules at high fields. In the fully superconducting state, on the other hand, the vortex pinning effects were found to be more effective in the saturated state of the $Mn_{12}$-ac molecules, as seen from magnetoresistance and field dependent critical current measurements. In a separate experiment, a Langmuir-Blodgett film of SMM was grown on a nano-SQUID to look for local changes in magnetization arising from magnetizatin tunnelling phenomenon in SMMs. Upon magnetizing the SMM (deposited on SQUIDs) at 2 K along the plane of the film and allowing it to relax, we found occasional jumps in the underlying SQUID voltage, unlike bare nano-SQUIDs, which did not show any such jumps over several hours. Therefore, we believe that the jumps in the SQUID voltage are the signatures of random tunneling of magnetization in the SMM layer.Comment: Bibekananda Das and Tapas Senapati contributed equally to this wor

Deep Sequencing-Identified Kanamycin-Resistant Paenibacillus Sp. Strain Ks1 Isolated From Epiphyte Tillandsia Usneoides (Spanish Moss) In Central Florida, Usa

Paenibacillus sp. strain KS1 was isolated from an epiphyte, Tillandsia usneoides (Spanish moss), in central Florida, USA. Here, we report a draft genome sequence of this strain, which consists of a total of 398 contigs spanning 6,508,195 bp, with a G+C content of 46.5% and comprising 5,401 predicted coding sequences

Deep Sequencing-Identified Kanamycin-Resistant Paenibacillus sp. Strain KS1 Isolated from Epiphyte Tillandsia usneoides (Spanish Moss) in Central Florida, USA

Paenibacillus sp. strain KS1 was isolated from an epiphyte, Tillandsia usneoides (Spanish moss), in central Florida, USA. Here, we report a draft genome sequence of this strain, which consists of a total of 398 contigs spanning 6,508,195 bp, with a G+C content of 46.5% and comprising 5,401 predicted coding sequences

Transcription Factor NF-κB: An Update on Intervention Strategies

The nuclear factor (NF)-κB family of transcription factors are ubiquitous and pleiotropic molecules that regulate the expression of more than 150 genes involved in a broad range of processes including inflammation, immunity, cell proliferation, differentiation, and survival. The chronic activation or dysregulation of NF-κB signaling is the central cause of pathogenesis in many disease conditions and, therefore, NF-κB is a major focus of therapeutic intervention. Because of this, understanding the relationship between NF-κB and the induction of various downstream signaling molecules is imperative. In this review, we provide an updated synopsis of the role of NF-κB in DNA repair and in various ailments including cardiovascular diseases, HIV infection, asthma, herpes simplex virus infection, chronic obstructive pulmonary disease, and cancer. Furthermore, we also discuss the specific targets for selective inhibitors and future therapeutic strategies.Fil: Rajendran, Arvind. Louisiana State University; Estados UnidosFil: Inda, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; ArgentinaFil: Bagam, Prathyusha. Southern University and A&M College; Estados UnidosFil: Sahoo, Malaya K.. Stanford University School of Medicine; Estados UnidosFil: Osorio, Diana. Louisiana State University; Estados UnidosFil: Batra, Sanjay. Louisiana State University; Estados Unidos. Southern University and A&M College; Estados Unido

Electrocatalytic Activity of Pd_{20–<i>x</i>}Ag_<i>x</i> Nanoparticles Embedded in Carbon Nanotubes for Methanol Oxidation in Alkaline Media

Low Pd content and highly active bimetallic PdAg electrocatalytic nanoparticles supported on functionalized multiwalled carbon nanotubes (CNT) have been developed for the electrochemical methanol oxidation reaction (MOR). The polyol synthesis procedure is used to substitute Ag into Pd particles in a Pd/CNT matrix. The Pd_{20–<i>x</i>}Ag_<i>x</i>/CNT samples (<i>x</i> wt % = 0, 5, 10, and 15) are characterized by XRD, XPS, Raman spectroscopy, and microscopy methods. The electrocatalytic activities and stabilities of Pd_{20–<i>x</i>}Ag_<i>x</i>/CNT catalysts were examined by cyclic voltammetry (CV), chronoamperometry (CA), and chronopotentiometry (CP) measurements for electrochemical oxidation of methanol in alkaline media. The Pd_{20–<i>x</i>}Ag_<i>x</i>/CNT with <i>x</i> = 10 wt % is found to be a semialloyed sample and shows the best catalytic activity (731 mA mg^–1) compared to the Pd/CNT catalyst (408 mA mg^–1) and other samples. This semialloyed sample also shows long stability and high resistance toward CO poisoning. The semialloyed Pd₁₀Ag₁₀ particles with optimized metal ratio on CNT matrix would be a promising catalyst material for direct methanol fuel cell applications

Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing

Submitted by sandra infurna ([email protected]) on 2016-05-10T17:33:26Z No. of bitstreams: 1 ilana_balanssiano_etal_IOC_2015.PDF: 353524 bytes, checksum: 6a7c79be5b2c2b6591eef9056de35171 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2016-05-10T17:49:31Z (GMT) No. of bitstreams: 1 ilana_balanssiano_etal_IOC_2015.PDF: 353524 bytes, checksum: 6a7c79be5b2c2b6591eef9056de35171 (MD5)Made available in DSpace on 2016-05-10T17:49:31Z (GMT). No. of bitstreams: 1 ilana_balanssiano_etal_IOC_2015.PDF: 353524 bytes, checksum: 6a7c79be5b2c2b6591eef9056de35171 (MD5) Previous issue date: 2015Stanford University School of Medicine.Department of Medicine. Division of Infectious Diseases and Geographic Medicine. Stanford, California, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas, Centro de Referência Nacional para Leptospirose, Coleção de Leptospira, WHO/PAHO Centro Colaborador para Leptospirose. Rio de Janeiro, RJ, Brasil.Stanford University School of Medicine. Department of Pathology. Stanford, California, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Zoonoses Bacterianas, Centro de Referência Nacional para Leptospirose, Coleção de Leptospira, WHO/PAHO Centro Colaborador para Leptospirose. Rio de Janeiro, RJ, Brasil.Stanford University School of Medicine. Department of Pathology. Stanford, California, USA.Stanford University School of Medicine.Department of Medicine. Division of Infectious Diseases and Geographic Medicine. Stanford, California, USA / Stanford University School of Medicine. Department of Pathology. Stanford, California, USA.Background Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens. Methods/Principal Findings 478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%). Conclusions/Significance This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize case detection

Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

Made available in DSpace on 2015-06-17T12:05:20Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) ilana_balassiano2etal_IOC_2014.pdf: 564052 bytes, checksum: 80570b9162fb99d11eb720869f4a7548 (MD5) Previous issue date: 2014Stanford University School of Medicine. Division of Infectious Diseases and Geographic Medicine. Department of Medicine. Stanford, CA, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. WHO/PAHO Centro Colaborador para Leptospirose. Coleção de Leptospira. Centro de Referência Nacional para Leptospirose. Laboratório de Zoonoses Bacterianas. Rio de Janeiro, RJ, Brasil.Stanford University School of Medicine. Department of Pathology. Stanford, CA, USA.Stanford University School of Medicine. Department of Pathology. Stanford, CA, USA.Stanford University School of Medicine. Department of Pathology. Stanford, CA, USA.Stanford University School of Medicine. Department of Pathology. Stanford, CA, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. WHO/PAHO Centro Colaborador para Leptospirose. Coleção de Leptospira. Centro de Referência Nacional para Leptospirose. Laboratório de Zoonoses Bacterianas. Rio de Janeiro, RJ, Brasil.Stanford University School of Medicine. Division of Infectious Diseases and Geographic Medicine. Department of Medicine. Department of Pathology. Stanford, CA, USA.Background: Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species. Methodology/Principal Findings: For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p,0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay. Conclusions/Significance: The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests