The Journal of molecular diagnostics : JMD
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The coronavirus disease 2019 (COVID-19) pandemic now has >2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. ⋯ Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay to enable higher throughput.
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The increasing incidence of bloodstream infections including sepsis is a major challenge in intensive care units worldwide. However, current diagnostics for pathogen identification mainly depend on culture- and molecular-based approaches, which are not satisfactory regarding specificity, sensitivity, and time to diagnosis. Herein, we established a complete diagnostic workflow for real-time high-throughput sequencing of cell-free DNA from plasma based on nanopore sequencing for the detection of the causative agents, which was applied to the analyses of eight samples from four septic patients and three healthy controls, and subsequently validated against standard next-generation sequencing results. ⋯ Moreover, an extrapolation of real-time sequencing performance on a cohort of 239 septic patient samples revealed that more than 90% of pathogen hits would have also been detected using the optimized MinION workflow. Reliable identification of pathogens based on circulating cell-free DNA sequencing using optimized workflows and real-time nanopore-based sequencing can be accomplished within 5 to 6 hours following blood draw. Therefore, this approach might provide therapy-relevant results in a clinically critical timeframe.
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Comparative Study
Comparison of Real-Time Quantitative PCR and Digital Droplet PCR for BCR-ABL1 Monitoring in Patients with Chronic Myeloid Leukemia.
Real-time quantitative PCR (qPCR) is routinely used to detect minimal residual disease in chronic myeloid leukemia patients. The absolute quantification with droplet digital PCR (ddPCR) could reduce the inherent variability of qPCR. We established a duplex ddPCR assay using the Europe against Cancer (EAC) primer/probe system for breakpoint cluster region protein-tyrosine-protein kinase ABL1 (BCR-ABL1) and ABL1 and compared the results with qPCR. cDNA samples (n = 230) from patients with chronic myeloid leukemia were analyzed using both procedures. ⋯ In conclusion, using the EAC protocol for BCR-ABL1 quantification with ddPCR is feasible and shows low intra-assay and interassay variation but requires a cutoff that reduces sensitivity. The commercial ddPCR assay is highly sensitive and specific for BCR-ABL1. The use of either ddPCR assay resulted in a shift to lower molecular response classes compared with qPCR aligned to international scale.
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This editorial highlights the article by Schreier et al that emphasizes on reforming the process for regulating laboratory developed testing.
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Developments in diagnostics reform legislation in the United States are occurring at a rapid pace. The framework for future regulatory oversight of clinical laboratory testing is currently under intensive debate among stakeholders that represent patients, practitioners, laboratories, diagnostic manufacturers, and regulators. The importance of clinical laboratory test standardization is a key component of any plan for regulatory reform. ⋯ Several important clinical laboratory test standardization projects are currently under way. The US Food and Drug Administration has acknowledged the need for such standardization. In its most recent draft guidance, the agency requested input from the scientific community as to actions that can be taken to facilitate standardization.