Comparison of nucleic acid extraction platforms for detection of select biothreat agents for use in clinical resource limited settings

High-quality nucleic acids are critical for optimal PCR-based diagnostics and pathogen detection. Rapid sample processing time is important for the earliest administration of therapeutic and containment measures, especially in the case of biothreat agents. In this context, we compared the Fujifilm QuickGene-Mini80 to Qiagen\u27s QIAamp Mini Purification kits for extraction of DNA and RNA for potential use in austere settings. Qiagen (QIAamp) column-based extraction is the currently recommended purification platform by United States Army Medical Research Institute for Infectious Diseases for both DNA and RNA extraction. However, this sample processing system requires dedicated laboratory equipment including a centrifuge. In this study, we investigated the QuickGene-Mini80, which does not require centrifugation, as a suitable platform for nucleic acid extraction for use in resource-limited locations. Quality of the sample extraction was evaluated using pathogen-specific, real-time PCR assays for nucleic acids extracted from viable and γ-irradiated Bacillus anthracis, Yersinia pestis, vaccinia virus, Venezuelan equine encephalitis virus, or B. anthracis spores in buffer or human whole blood. QuickGene-Mini80 and QIAamp performed similarly for DNA extraction regardless of organism viability. It was noteworthy that γ-irradiation did not have a significant impact on real-time PCR for organism detection. Comparison with QIAamp showed a less than adequate performance of the Fujifilm instrument for RNA extraction. However, QuickGene-Mini80 remains a viable alternative to QIAamp for DNA extraction for use in remote settings due to extraction quality, time efficiency, reduced instrument requirements, and ease of use

The CTSA Consortium's Catalog of Assets for Translational and Clinical Health Research (CATCHR)

The 61 CTSA Consortium sites are home to valuable programs and infrastructure supporting translational science and all are charged with ensuring that such investments translate quickly to improved clinical care. Catalog of Assets for Translational and Clinical Health Research (CATCHR) is the Consortium's effort to collect and make available information on programs and resources to maximize efficiency and facilitate collaborations. By capturing information on a broad range of assets supporting the entire clinical and translational research spectrum, CATCHR aims to provide the necessary infrastructure and processes to establish and maintain an open‐access, searchable database of consortium resources to support multisite clinical and translational research studies. Data are collected using rigorous, defined methods, with the resulting information made visible through an integrated, searchable Web‐based tool. Additional easy‐to‐use Web tools assist resource owners in validating and updating resource information over time. In this paper, we discuss the design and scope of the project, data collection methods, current results, and future plans for development and sustainability. With increasing pressure on research programs to avoid redundancy, CATCHR aims to make available information on programs and core facilities to maximize efficient use of resources.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106893/1/cts12144.pd

The CTSA Consortium's Catalog of Assets for Translational and Clinical Health Research (CATCHR): The Ctsa Consortium's Catchr

The 61 CTSA Consortium sites are home to valuable programs and infrastructure supporting translational science and all are charged with ensuring that such investments translate quickly to improved clinical care. CATCHR (Catalog of Assets for Translational and Clinical Health Research) is the Consortium’s effort to collect and make available information on programs and resources to maximize efficiency and facilitate collaborations. By capturing information on a broad range of assets supporting the entire clinical and translational research spectrum, CATCHR aims to provide the necessary infrastructure and processes to establish and maintain an open-access, searchable database of consortium resources to support multi-site clinical and translational research studies. Data is collected using rigorous, defined methods, with the resulting information made visible through an integrated, searchable web-based tool. Additional easy to use web tools assist resource owners in validating and updating resource information over time. In this article, we discuss the design and scope of the project, data collection methods, current results, and future plans for development and sustainability. With increasing pressure on research programs to avoid redundancy, CATCHR aims to make available information on programs and core facilities to maximize efficient use of resources

Characterization of the EsaI/EsaR quorum sensing system: A model for quorum sensing repression

Gram-negative bacteria utilize the conserved quorum sensing regulatory strategy for the population dependent control of gene expression. In the Gram-negative bacterium Pantoea stewartii subsp. stewartii, the EsaI/EsaR quorum sensing regulatory pair governs the cell density dependent expression of an exopolysaccharide virulence factor, which is essential for Stewart\u27s wilt disease in maize. The EsaI protein is a conserved acyl-homoserine lactone signal synthase; EsaR is the cognate transcription factor. This dissertation research focuses on the characterization of the EsaI and EsaR proteins, and the mechanism for EsaI/EsaR quorum sensing regulation of exopolysaccharide biosynthesis: Prior genetic data indicate that EsaR functions as a transcriptional repressor that is responsive to inducing aryl-homoserine lactone concentrations for derepression. This dissertation provides additional genetic and extensive biochemical evidence to confirm the repressor function of EsaR in the control of its own regulation and the repression of the rcsA gene. This research further defines two 20-base pair imperfect palindromes, which serve as specific EsaR DNA binding sites. The position of these two elements within the regulated promoters is consistent with an EsaR repressor role, in that, bound EsaR sterically inhibits RNA-polymerase/promoter interactions under acyl-homoserine lactone limiting conditions. Inducing signal concentrations neutralize EsaR repressor activity and permit RNA polymerase to initiate transcription. Accordingly, this research provides unequivocal proof for a quorum sensing regulatory model by transcriptional repression. ^ This dissertation further establishes that the mechanism for quorum sensing regulation of exopolysaccharide synthesis in Pantoea stewartii follows a hierarchical pathway. EsaR represses the rcsA gene under signal-limiting conditions, while allowing expression at inducing concentrations. The rcsA gene encodes the essential RcsA co-activator, which, in complex with the RcsB protein, activates the exopolysaccharide biosynthesis pathway. More significantly, this study provides new information indicating that quorum sensing regulation may serve as an important switch to direct the expression of different surface polymers depending on the cell-density or the developmental state of the bacterial population. This dissertation research therefore achieves the goal to characterize essential elements for the EsaI/EsaR quorum sensing system, and provides a foundation for research aimed at defining the biological role of quorum sensing regulation in Pantoea stewartii.

Detection of 16S rRNA and KPC Genes from Complex Matrix Utilizing a Molecular Inversion Probe Assay for Next-Generation Sequencing

Abstract Targeted sequencing promises to bring next-generation sequencing (NGS) into routine clinical use for infectious disease diagnostics. In this context, upfront processing techniques, including pathogen signature enrichment, must amplify multiple targets of interest for NGS to be relevant when applied to patient samples with limited volumes. Here, we demonstrate an optimized molecular inversion probe (MIP) assay targeting multiple variable regions within the 16S ribosomal gene for the identification of biothreat and ESKAPE pathogens in a process that significantly reduces complexity, labor, and processing time. Probes targeting the Klebsiella pneumoniae carbapenemase (KPC) antibiotic resistance (AR) gene were also included to demonstrate the ability to concurrently identify etiologic agent and ascertain valuable secondary genetic information. Our assay captured gene sequences in 100% of mock clinical samples prepared from flagged positive blood culture bottles. Using a simplified processing and adjudication method for mapped sequencing reads, genus and species level concordance was 100% and 80%, respectively. In addition, sensitivity and specificity for KPC gene detection was 100%. Our MIP assay produced sequenceable amplicons for the identification of etiologic agents and the detection of AR genes directly from blood culture bottles in a simplified single tube assay

A highly multiplexed broad pathogen detection assay for infectious disease diagnostics.

Rapid pathogen identification during an acute febrile illness is a critical first step for providing appropriate clinical care and patient isolation. Primary screening using sensitive and specific assays, such as real-time PCR and ELISAs, can rapidly test for known circulating infectious diseases. If the initial testing is negative, potentially due to a lack of developed diagnostic assays or an incomplete understanding of the pathogens circulating within a geographic region, additional testing would be required including highly multiplexed assays and metagenomic next generation sequencing. To bridge the gap between rapid point of care diagnostics and sequencing, we developed a highly multiplexed assay designed to detect 164 different viruses, bacteria, and parasites using the NanoString nCounter platform. Included in this assay were high consequence pathogens such as Ebola virus, highly endemic organisms including several Plasmodium species, and a large number of less prevalent pathogens to ensure a broad coverage of potential human pathogens. Evaluation of this panel resulted in positive detection of 113 (encompassing 98 different human pathogen types) of the 126 organisms available to us including the medically important Ebola virus, Lassa virus, dengue virus serotypes 1-4, Chikungunya virus, yellow fever virus, and Plasmodium falciparum. Overall, this assay could improve infectious disease diagnostics and biosurveillance efforts as a quick, highly multiplexed, and easy to use pathogen screening tool