In the clinical microbiology laboratory advances in nucleic acid detection, quantification, and sequence analysis have led to considerable improvements in the diagnosis, management, and monitoring of infectious diseases. Molecular diagnostic methods are routinely used to make clinical decisions based on when and how to treat a patient as well as monitor the effectiveness of a therapeutic regime and identify any potential drug resistant strains that may impact on the long term patient treatment program. Therefore, confidence in the reliability of the result provided by the laboratory service to the clinician is essential for patient treatment. Hence, suitable quality assurance and quality control measures are important to ensure that the laboratory methods and service meet the necessary regulatory requirements both at the national and international level. In essence, the modern clinical microbiology laboratory ensures the appropriateness of its services through a quality management system that monitors all aspects of the laboratory service pre- and post-analytical-from patient sample receipt to reporting of results, from checking and upholding staff competency within the laboratory to identifying areas for quality improvements within the service offered. For most European based clinical microbiology laboratories this means following the common International Standard Organization (ISO9001) framework and ISO15189 which sets out the quality management requirements for the medical laboratory (BS EN ISO 15189 (2003) Medical laboratories-particular requirements for quality and competence. British Standards Institute, Bristol, UK). In the United States clinical laboratories performing human diagnostic tests are regulated by the Centers for Medicare and Medicaid Services (CMS) following the requirements within the Clinical Laboratory Improvement Amendments document 1988 (CLIA-88). This chapter focuses on the key quality assurance and quality control requirements within the modern microbiology laboratory providing molecular diagnostics.
"However, the development of increasingly sensitive, specific and high throughput techniques such as realtime quantitative PCR (qPCR), microarrays, digital PCR (dPCR), next generation sequencing (NGS) and mass spectrometry has led to their use in a wide variety of applications in a broad range of biological and clinical subjects. This continuous expansion of molecular technologies has swiftly resulted in a demand for more rigorous quantification of nucleic acids, proteins and small molecules in, for example, molecular diagnostics . However, compared with a qualitative yes/no result, reliable, reproducible and biologically relevant quantification poses significant problems in terms of sample preparation and quality control, assay design, optimisation and validation, collection of data, their analysis and, importantly, prudent and transparent reporting . "
[Show abstract][Hide abstract] ABSTRACT: In the twenty-first century, the clinical microbiology laboratory plays a central part in optimizing the management of infectious diseases and surveying local and global epidemiology. This pivotal role is made possible by the adoption of rational sampling, point-of-care tests, extended automation and new technologies, including mass spectrometry for colony identification, real-time genomics for isolate characterization, and versatile and permissive culture systems. When balanced with cost, these developments can improve the workflow and output of clinical microbiology laboratories and, by identifying and characterizing microbial pathogens, provide significant input to scientific discovery.
[Show abstract][Hide abstract] ABSTRACT: Escherichia fergusonii is an emerging pathogen that has been isolated from a wide range of infections in animals and humans. Primers targeting specific genes including yliE (conserved hypothetical protein of the cellulose synthase and regulator of cellulose synthase island), EFER_1569 (hypothetical protein, putative transcriptional activator for multiple antibiotic resistance) and EFER_3126 (putative triphosphoribosyl-dephospho-CoA) were designed for the detection of E. fergusonii by conventional and real-time PCR methods. Primers were screened by in silico PCR against 489 bacterial genomic sequences and by both PCR methods on 55 reference and field strains. Both methods were specific and sensitive for E. fergusonii showing amplification only for this bacterium. Conventional PCR required a minimum bacterial concentration of approximately 10(2) cfu/ml while real-time PCR required a minimum of 0.3 pg of DNA for consistent detection. Standard curves showed an efficiency of 98.5% with an R(2) value of 0.99 for the real-time PCR assay. Cecal and cloacal contents from 580 chickens were sampled from broiler farms located in the Fraser Valley (British Columbia, Canada). Presumptive E. fergusonii isolates were recovered by enrichment and plating on differential and selective media. Of 301 total presumptive isolates, 140 (46.5%) were identified as E. fergusonii by biochemical profiling with the API 20E system and 268 (89.0%) using PCR methods. E. fergusonii detection directly from cecal and cloacal samples without pre-enrichment was achieved with both PCR methods. Hence the PCR methods developed in this work significantly improve the detection of E. fergusonii.
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