Sequence optimized diagnostic assay for Ebola virus detection.

Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013-2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.

Comparison of Illumina and Oxford Nanopore Sequencing Technologies for Pathogen Detection from Clinical Matrices Using Molecular Inversion Probes.

Next-generation sequencing is rapidly finding footholds in numerous microbiological fields, including infectious disease diagnostics. Here, we describe a molecular inversion probe panel for the identification of bacterial, viral, and parasitic pathogens. We describe the ability of Illumina and Oxford Nanopore Technologies (ONT) to sequence small amplicons originating from this panel for the identification of pathogens in complex matrices. The panel correctly classified 31 bacterial pathogens directly from positive blood culture bottles with a genus-level concordance of 96.7% and 90.3% on the Illumina and ONT platforms, respectively. Both sequencing platforms detected 18 viral and parasitic organisms directly from mock clinical samples of plasma and whole blood at concentrations of 104 PFU/mL with few exceptions. In general, Illumina sequencing exhibited greater read counts with lower percent mapped reads; however, this resulted in no effect on limits of detection compared with ONT sequencing. Mock clinical evaluation of the probe panel on the Illumina and ONT platforms resulted in positive predictive values of 0.91 and 0.88 and negative predictive values of 1 and 1 from de-identified human chikungunya virus samples compared with gold standard quantitative RT-PCR. Overall, these data show that molecular inversion probes are an adaptable technology capable of pathogen detection from complex sample matrices on current next-generation sequencing platforms.

Homeplace: Care and resistance among public housing residents facing mixed-income redevelopment.

Low-income communities of color experience significant political, economic, and health inequities and, not unrelatedly, are disproportionately exposed to violent crime than are residents of higher income communities. In an effort to mitigate concentrations of poverty and crime, governmental agencies have partnered with affordable housing developers to redevelop public housing "projects" into mixed-income communities and to do so within a "trauma-informed" framework. The current study analyzes how residents have historically and contemporaneously negotiated, endured, and resisted structural and interpersonal violence in 2 long-standing, predominately African American, public housing communities undergoing a public-private housing redevelopment initiative. Interviews with 44 adult public housing residents (age range = 18-75 years; 82% African American/Black) were conducted during a 2-year period while residents' homes were being demolished and rebuilt into mixed-income communities. Analysis of in-depth interviews used constructivist grounded theory principles to reveal a common theme and basic social process of the ongoing formation of homeplace, with subthemes focusing on the ways homeplace emerges through shared lineage, knowing and caring practices; how homeplace is maintained through networks of protection in unsafe contexts; how homeplace is disrupted as a result of redevelopment activities; and the reclamation of homeplace during redevelopment in the service of hope and healing. These findings offer a nuanced view of resident's lived experiences of place-based trauma and collective resistance and resilience, while also highlighting the place-specific ways in which redevelopment unsettles deeply rooted sociocultural configurations of home and community. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

FDA-ARGOS is a database with public quality-controlled reference genomes for diagnostic use and regulatory science.

FDA proactively invests in tools to support innovation of emerging technologies, such as infectious disease next generation sequencing (ID-NGS). Here, we introduce FDA-ARGOS quality-controlled reference genomes as a public database for diagnostic purposes and demonstrate its utility on the example of two use cases. We provide quality control metrics for the FDA-ARGOS genomic database resource and outline the need for genome quality gap filling in the public domain. In the first use case, we show more accurate microbial identification of Enterococcus avium from metagenomic samples with FDA-ARGOS reference genomes compared to non-curated GenBank genomes. In the second use case, we demonstrate the utility of FDA-ARGOS reference genomes for Ebola virus target sequence comparison as part of a composite validation strategy for ID-NGS diagnostic tests. The use of FDA-ARGOS as an in silico target sequence comparator tool combined with representative clinical testing could reduce the burden for completing ID-NGS clinical trials.

Diagnostic targETEd seQuencing adjudicaTion (DETEQT): Algorithms for Adjudicating Targeted Infectious Disease Next-Generation Sequencing Panels.

Next-generation sequencing (NGS) for infectious disease diagnostics is a relatively new and underdeveloped concept. If this technology is to become a regulatory-grade clinical diagnostic, standardization in the form of locked-down assays and firmly established underlying processes is necessary. Targeted sequencing, specifically by amplification of genomic signatures, has the potential to bridge the gap between PCR- and NGS-based diagnostics; however, existing NGS assay panels lack validated analytical techniques to adjudicate high background and error-prone NGS data. Herein, we present the Diagnostic targETEd seQuencing adjudicaTion (DETEQT) software, consisting of an intuitive bioinformatics pipeline entailing a set of algorithms to translate raw sequencing data into positive, negative, and indeterminate diagnostic determinations. After basic read filtering and mapping, the software compares abundance and quality metrics against heuristic and fixed thresholds. A novel generalized quality function provides an amalgamated quality score for the match between sequence reads of an assay and panel targets, rather than considering each component factor independently. When evaluated against numerous assay samples and parameters (mock clinical, human, and nonhuman primate clinical data sets; diverse amplification strategies; downstream applications; and sequence platforms), DETEQT demonstrated improved rejection of false positives and accuracies >95%. Finally, DETEQT was implemented in the user-friendly Empowering the Development of Genomics Expertise (EDGE) bioinformatics platform, providing a complete, end-to-end solution that can be operated by nonexperts in a clinical laboratory setting.

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

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.

Sequence Optimized Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection of Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus within the family Bunyaviridae. Infection can result in general myalgia, fever, and headache with some patients developing hemorrhagic fever with mortality rates ranging from 5% to 30%. CCHFV has a wide geographic range that includes Africa, Asia, the Middle East, and Europe with nucleotide sequence variation approaching 20% across the three negative-sense RNA genome segments. While phylogenetic clustering generally aligns with geographic origin of individual strains, distribution can be wide due to tick/CCHFV dispersion via migrating birds. This sequence diversity negatively impacts existing molecular diagnostic assays, leading to false negative diagnostic results. Here, we updated a previously developed CCHFV real-time reverse transcription polymerase chain reaction (RT-PCR) assay to include strains not detected using that original assay. Deep sequencing of eight different CCHFV strains, including three that were not detectable using the original assay, identified sequence variants within this assay target region. New primers and probe based on the sequencing results and newly deposited sequences in GenBank greatly improved assay sensitivity and inclusivity with the exception of the genetically diverse strain AP92. For example, we observed a four log improvement in IbAr10200 detection with a new limit of detection of 256 PFU/mL. Subsequent comparison of this assay to another commonly used CCHFV real-time RT-PCR assay targeting a different region of the viral genome showed improved detection, and both assays could be used to mitigate CCHFV diversity for diagnostics. Overall, this work demonstrated the importance of continued viral sequencing efforts for robust diagnostic assay development.

Evaluation of Signature Erosion in Ebola Virus Due to Genomic Drift and Its Impact on the Performance of Diagnostic Assays.

Genome sequence analyses of the 2014 Ebola Virus (EBOV) isolates revealed a potential problem with the diagnostic assays currently in use; i.e., drifting genomic profiles of the virus may affect the sensitivity or even produce false-negative results. We evaluated signature erosion in ebolavirus molecular assays using an in silico approach and found frequent potential false-negative and false-positive results. We further empirically evaluated many EBOV assays, under real time PCR conditions using EBOV Kikwit (1995) and Makona (2014) RNA templates. These results revealed differences in performance between assays but were comparable between the old and new EBOV templates. Using a whole genome approach and a novel algorithm, termed BioVelocity, we identified new signatures that are unique to each of EBOV, Sudan virus (SUDV), and Reston virus (RESTV). Interestingly, many of the current assay signatures do not fall within these regions, indicating a potential drawback in the past assay design strategies. The new signatures identified in this study may be evaluated with real-time reverse transcription PCR (rRT-PCR) assay development and validation. In addition, we discuss regulatory implications and timely availability to impact a rapidly evolving outbreak using existing but perhaps less than optimal assays versus redesign these assays for addressing genomic changes.

Development and evaluation of a panel of filovirus sequence capture probes for pathogen detection by next-generation sequencing.

A detailed understanding of the circulating pathogens in a particular geographic location aids in effectively utilizing targeted, rapid diagnostic assays, thus allowing for appropriate therapeutic and containment procedures. This is especially important in regions prevalent for highly pathogenic viruses co-circulating with other endemic pathogens such as the malaria parasite. The importance of biosurveillance is highlighted by the ongoing Ebola virus disease outbreak in West Africa. For example, a more comprehensive assessment of the regional pathogens could have identified the risk of a filovirus disease outbreak earlier and led to an improved diagnostic and response capacity in the region. In this context, being able to rapidly screen a single sample for multiple pathogens in a single tube reaction could improve both diagnostics as well as pathogen surveillance. Here, probes were designed to capture identifying filovirus sequence for the ebolaviruses Sudan, Ebola, Reston, Taï Forest, and Bundibugyo and the Marburg virus variants Musoke, Ci67, and Angola. These probes were combined into a single probe panel, and the captured filovirus sequence was successfully identified using the MiSeq next-generation sequencing platform. This panel was then used to identify the specific filovirus from nonhuman primates experimentally infected with Ebola virus as well as Bundibugyo virus in human sera samples from the Democratic Republic of the Congo, thus demonstrating the utility for pathogen detection using clinical samples. While not as sensitive and rapid as real-time PCR, this panel, along with incorporating additional sequence capture probe panels, could be used for broad pathogen screening and biosurveillance.

Cross-institute evaluations of inhibitor-resistant PCR reagents for direct testing of aerosol and blood samples containing biological warfare agent DNA.

Rapid pathogen detection is crucial for the timely introduction of therapeutics. Two groups (one in the United Kingdom and one in the United States) independently evaluated inhibitor-resistant PCR reagents for the direct testing of substrates. In the United Kingdom, a multiplexed Bacillus anthracis (target) and Bacillus subtilis (internal-control) PCR was used to evaluate 4 reagents against 5 PCR inhibitors and down-selected the TaqMan Fast Virus 1-Step master mix (Life Technologies Inc.). In the United States, four real-time PCR assays (targeting B. anthracis, Brucella melitensis, Venezuelan equine encephalitis virus [VEEV], and Orthopoxvirus spp.) were used to evaluate 5 reagents (plus the Fast Virus master mix) against buffer, blood, and soil samples and down-selected the KAPA Blood Direct master mix (KAPA Biosystems Inc.) with added Platinum Taq (Life Technologies). The down-selected reagents underwent further testing. In the United Kingdom experiments, both reagents were tested against seven contrived aerosol collector samples containing B. anthracis Ames DNA and B. subtilis spores from a commercial formulation (BioBall). In PCR assays with reaction mixtures containing 40% crude sample, an airfield-collected sample induced inhibition of the B. subtilis PCR with the KAPA reagent and complete failure of both PCRs with the Fast Virus reagent. However, both reagents allowed successful PCR for all other samples-which inhibited PCRs with a non-inhibitor-resistant reagent. In the United States, a cross-assay limit-of-detection (LoD) study in blood was conducted. The KAPA Blood Direct reagent allowed the detection of agent DNA (by four PCRs) at higher concentrations of blood in the reaction mixture (2.5%) than the Fast Virus reagent (0.5%), although LoDs differed between assays and reagent combinations. Across both groups, the KAPA Blood Direct reagent was determined to be the optimal reagent for inhibition relief in PCR.

Evaluation of inhibitor-resistant real-time PCR methods for diagnostics in clinical and environmental samples.

Polymerase chain reaction (PCR) is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at lower signature concentrations. One potential solution is direct PCR assessment without sample processing. Here, we evaluated nine inhibitor-resistant PCR reagents for direct detection of Francisella tularensis in seven different clinical and environmental samples using an established real-time PCR assay to assess ability to overcome PCR inhibition. While several of these reagents were designed for standard PCR, the described inhibitor resistant properties (ex. Omni Klentaq can amplify target DNA samples of up to 20% whole blood or soil) led to our evaluation with real-time PCR. A preliminary limit of detection (LOD) was determined for each chemistry in whole blood and buffer, and LODs (20 replicates) were determined for the top five chemistries in each matrix (buffer, whole blood, sputum, stool, swab, soil, and sand). Not surprisingly, no single chemistry performed the best across all of the different matrices evaluated. For instance, Phusion Blood Direct PCR Kit, Phire Hot Start DNA polymerase, and Phire Hot Start DNA polymerase with STR Boost performed best for direct detection in whole blood while Phire Hot Start DNA polymerase with STR Boost were the only reagents to yield an LOD in the femtogram range for soil. Although not the best performer across all matrices, KAPA Blood PCR kit produced the most consistent results among the various conditions assessed. Overall, while these inhibitor resistant reagents show promise for direct amplification of complex samples by real-time PCR, the amount of template required for detection would not be in a clinically relevant range for most matrices.

A model for outpatient care.

Chaplains have seriously discussed outpatient care for a number of years. This article describes a model of thorough and intentional outpatient care that is practiced in conjunction with an inpatient care to cancer populations. Rationales, values, institutional dynamics, and clinical care are discussed. Although the focus is on outpatient care where slightly more than 50% of personnel resources are invested, the care also occurs in the context of both inpatient care and follow-up care after discharge to home. A general model of spiritual and religious care and more specific models pertinent to hematopoietic stem cell transplant populations and general oncology populations are described. The models attend times of distress and times of "ordinary" and celebratory experiences, attend care of patient, caregiver, and staff.