A Framework of Critical Considerations in Clinical Exome Reanalyses by Clinical and Laboratory Standards Institute.

Exome reanalysis is useful for providing molecular diagnoses for previously uninformative samples. However, challenges exist in implementing a practical solution for clinicians and laboratories. This study complements the current literature by providing practical considerations for patient-level and cohort-level reanalyses. The Clinical and Laboratory Standards Institute assembled the Document Development Committee and an interpretation working group that developed the framework for reevaluation of exome-based data. We describe two distinct but complementary approaches toward exome reanalyses: clinician-initiated patient-level reanalysis, and laboratory-initiated cohort-level reanalysis. We highlight the advantages and constraints for both approaches, and provide a high-level conceptual guide for ordering clinicians and laboratories through the critical decision pathways. Because clinical exome sequencing continues to be the standard of care in genetics, exome reanalysis would be critical in increasing the overall diagnostic yield. A systematic guide will facilitate the efficient adoption of reevaluation of exome data for laboratories, health care professionals, genetic counselors, and clinicians.

Collaborative Development and Implementation of a Video-Based Curriculum Expansion to Improve Refugee Women's Birth Experiences in the United States.

Many refugee and immigrant women in the United States experience cultural and language barriers when seeking pregnancy-related medical care. Such barriers may delay needed care and adversely impact birth outcomes. Embrace Refugee Birth Support (Embrace) in Clarkston, Georgia, supports pregnant refugee women by offering birth education classes in the women's primary languages. Our academic-practice partnership designed and implemented a series of birth education videos for Embrace participants. Based on input from former participants, the partnership team created video scenarios that could be embedded into Embrace's existing didactic curriculum. The videos addressed common challenges and learning needs identified by previous participants. All videos were filmed in the participant's primary languages (Swahili and Kinyarwanda) and featured actual Embrace graduates who spoke the languages. Then, Embrace trainers used the video scenarios to augment teaching on birth preparedness and foster participant discussion during class sessions. After implementation, a focus group with participants in the video-expanded class reported the videos were well received, understood, and practically related to their pregnancy needs. Overall, participants reported that video scenarios were an important part of their learning and skill development, as well as a positive experience. Embrace has plans to continue creating native language educational videos for additional languages and birth-related topics. The academic partner's attempts to measure video impact with standardized quantitative instruments at baseline were terminated. The substantive revisions in data collection strategies highlight the need for cross-cultural flexibility and the potential for unforeseen barriers when using quantitative research tools among non-English-speaking participants.

Practical Guidance to Implementing Quality Management Systems in Public Health Laboratories Performing Next-Generation Sequencing: Personnel, Equipment, and Process Management (Phase 1).

Quality standards as part of an effective quality management system (QMS) are the cornerstone for generating high-quality test results. Next-generation sequencing (NGS) has the potential to improve both clinical diagnostics and public health surveillance efforts in multiple areas, including infectious diseases. However, the laboratories adopting NGS methods face significant challenges due to the complex and modular process design. This document summarizes the first phase of quality system guidance developed by the Centers for Disease Control and Prevention (CDC) NGS Quality Workgroup. The quality system essentials of personnel, equipment, and process management (quality control and validation) were prioritized based on a risk assessment using information gathered from participating CDC laboratories. Here, we present a prioritized QMS framework, including procedures and documentation tools, to assist laboratory implementation and maintenance of quality practices for NGS workflows.

Cross-platform evaluation of commercial real-time reverse transcription PCR master mix kits using a quantitative 5'nuclease assay for Ebola virus.

Selection of optimal reaction master mix reagents is essential to obtain the best performance with diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Every year the number of commercially available master mix kits increases, so it is prudent to periodically evaluate kits on the market. In this study we evaluated five commercial real-time RT-PCR master mix kits, the RealMasterMix RT-PCR ROX kit, the AgPath-ID One-Step RT-PCR kit, the SuperScript III Platinum One-step Quantitative RT-PCR system, the QuantiTect Probe RT-PCR kit, and the LightCycler RNA HybProbe amplification kit, using a 5'nuclease assay for Ebola virus. The kits were evaluated using the manufacturer's recommended conditions, as well as conditions which have been used with the Ebola virus assay during outbreaks. When evaluated for use in Ebola virus RNA detection, the AgPath-ID kit resulted in the greatest sensitivity in comparison to the other four kits. The efficacy of the AgPath-ID kit was instrument-independent in the five real-time PCR platforms tested. This study demonstrated that Ebola virus RNA detection was not equivalent among the master mix reagents studied and, thus, that this variable can affect real-time RT-PCR assay sensitivity. Furthermore, this study rates the master mix reagents for their suitability, providing diagnostic laboratories the option to select from these kits to suit their specific laboratory needs for real-time RT-PCR.

Evaluation of automated and manual commercial DNA extraction methods for recovery of Brucella DNA from suspensions and spiked swabs.

This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean microbial DNA isolation kit. These six extraction methods were performed upon three pathogenic Brucella species: B. abortus, B. melitensis, and B. suis. Viability testing of the DNA extracts indicated that all six extraction methods were efficient at inactivating virulent Brucella spp. Real-time PCR analysis using Brucella genus- and species-specific TaqMan assays revealed that use of the MasterPure kit resulted in superior levels of detection from bacterial suspensions, while the MasterPure kit and MagNA Pure Compact performed equally well for extraction of spiked swab samples. This study demonstrated that DNA extraction methodologies differ in their ability to recover Brucella DNA from PBS bacterial suspensions and from swab specimens and, thus, that the extraction method used for a given type of sample matrix can influence the sensitivity of real-time PCR assays for Brucella.