Molecular Diagnostics: Current Research and Applications | Book
"I would highly recommend this book" (Doodys)
"useful new insights" (Aus. J. Med. Sci.)
Caister Academic Press
Jim Huggett and Justin O'Grady
LGC, Teddington, UK and Norwich Medical School, University of East Anglia, Norwich, UK; respectively
xii + 248
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The application of molecular technology in clinical diagnosis is a rapidly developing area and is predicted to greatly improve the speed, efficiency and accuracy of diagnostic medicine.
The editors of this book have commissioned an excellent series of chapters representing two key molecular diagnostic areas: cancer and infectious diseases. The cancer section deals with the challenges in identifying genetic, epigenetic and transcriptomic biomarkers. The infectious disease section describes the current clinical applications of molecular diagnostics for the detection of viral, bacterial and fungal pathogens as well as an example of the use of molecular diagnostics outside the clinic environment. A cautionary tale describing what can go wrong when molecular methods are applied incorrectly is also provided and makes fascinating reading. A substantial component of the book is dedicated to the process of translating a preclinical test to the bedside and describes the progress in the near patient point-of-care molecular diagnostics market. This is a fundamental consideration for successful translation of diagnostics tests from bench to bedside and is crucial for molecular diagnostics to have an impact on patient care. The final chapter offers a prediction of future trends in the molecular diagnostics of infectious diseases.
This volume is essential reading for anyone involved in the development or application of molecular diagnostics and is recommended for all clinical diagnostics laboratories.
"This book provides a broad overview of today's clinical applications of molecular diagnostics ... an abundance of wise advice and cautionary notes regarding establishing and clinically validating molecular diagnostic assays ... I would highly recommend this book ... one of the best reviews and guidelines for development of point of care testing devices using molecular diagnostics." from Doodys (2014)
"an interesting book for those working in molecular diagnostics ... useful up to date information ... useful new insights" from Aus. J. Med. Sci. (2015) 36: 26-27.
"... focuses on molecular tests that have been implemented, the advantages of these new technologies over old methods but also highlights potential pitfalls ... This book would suit clinicians and microbiologists making decisions on use of diagnostic tests, and researchers working on development of novel tests." from Clinical Infectious Diseases (2015) 60: 1877.
Table of contents
1. Molecular Diagnostics: An Introduction
Jim F. Huggett, Siobhan Dorai-Raj, Agnieszka M. Falinska and Justin O'Grady
A brief introduction to the field of clinical molecular diagnostics. Molecular diagnostics is defined here as diagnostic/prognostic approaches that employ the measurement of nucleic acids (DNA and/or RNA) in clinical situations.
2. Transcriptome-based Biomarkers: A Road Map Exemplified for Peripheral Blood-based Biomarker Discovery, Development and Clinical Use
Joachim L. Schultze
By using high-throughput genomic technologies an enormous number of novel biomarker candidates have been suggested for all the major diseases including cancer, metabolic disorders, autoimmune diseases and infections. Biomarkers are a prerequisite for developing truly personalized medicine. Yet the enormous efforts of biomarker discovery are not paralleled by similarly efficient biomarker development. In fact, only a very small number of biomarkers have made it into clinical practice in recent years. There are many reasons for the lack of translation of discovery into development and clinical practice. Using blood transcriptomics as an example, recent and current development, but also pitfalls and potential solutions for biomarker development are outlined here.
3. Development of Methylation Biomarkers for Clinical Applications and Methylation-sensitive High-resolution Melting (MS-HRM) Technology
Tomasz K. Wojdacz
Aberrations of locus specific methylation are a well-established hallmark of cancer. These epigenetic changes, in principle, cause or contribute to the pathological process, but at the same time can be utilized as biomarkers in clinical disease management. Within clinical disease management there are four fields of biomarker application: prevention, diagnosis, prognosis/prediction and treatment management. Methylation biomarkers fulfil criteria of applicability for each of these fields. However, current use of methylation biomarkers in clinical practice is very limited. The inadequate translation of research findings into clinical use in the field of methylation biomarkers can be mainly attributed to poor standardisation of the technologies for methylation biomarker discovery and screening as well as a paucity of large-scale clinical validation studies of potential biomarkers. Methylation sensitive high resolution melting (MS-HRM) is an established method enabling in-tube cost and labour efficient locus specific methylation screening. The method has been successfully utilised in clinical methylation biomarker validation studies, hence the potential application of this method in diagnostic settings is very promising. This chapter addresses the use of MS-HRM for methylation biomarker development and clinical validation, as well as potential diagnostic applicability of the method.
4. Genetic and Epigenetic Biomarkers of Colorectal Cancer
Stephen A. Bustin and Jamie Murphy
Colorectal cancer (CRC) is one of the most prevalent cancers in the western world. Around 75% of primary CRC diagnoses are in patients with no apparent risk factor other than age, with the remaining 25% of patients having a family history of CRC that suggests a genetic contribution. Although some genetic alterations have been identified as the cause of inherited cancer risk in some families, they account for only around 6% of CRC cases. Hence additional, as yet undiscovered, genes and background genetic factors may drive the development of CRC. CRC arises and progresses through the acquisition of genetic and epigenetic alterations that drive the transformation from normal colon epithelium to adenocarcinoma. The clinical behaviour of cancer cells depends on complex and dynamic interactions between the effects of these alterations and the individual genetic and environmental host contexts. This results in significant variability in the response of individual patients to chemotherapy treatments that are not accurately predicted by conventional prognostic stratification and adjuvant therapy selection procedures. Consequently, there has been keen interest in the identification and functional characterisation of molecular biomarkers, with the expectation that they may facilitate accurate disease diagnosis, have prognostic potential for the individual, or predict patient-specific responses to chemotherapy. Undoubtedly, they will play a role in the future diagnosis and management of CRC; however, DNA-based biomarkers are currently not widely used by physicians and most potential markers are still in the discovery phase waiting to undergo clinical validation.
5. Molecular Diagnosis in Medical Microbiology: The Horizon Draws Near
G.L. Vanstone, R. Gorton, B.M. Charalambous and Tim McHugh
Diagnostic microbiology is at the threshold of a new era; the advent of molecular tools, increased automation and use of informatics to allow interrogation of complex data sets has presented the opportunity to rethink our approach to diagnosis of bacterial and fungal infections. In this review we consider the advances that are in practice today in clinical laboratories. Molecular tools have huge potential in improving patient management and public health. We must be alert to the the danger that the technology blinds us to the underlying biology, and it is essential that we consider the new technologies with the same critical rigour that we would apply to conventional assays and in the context of the clinical setting. The horizon is close and full of opportunity.
6. Molecular Diagnostics: Current Research and Applications
Sophie Collot-Teixeira, Philip Minor and Robert Anderson
Molecular techniques, based on the detection of viral genomes, are revolutionising the field of diagnostic virus detection. They are currently replacing more traditional detection methods, such as cell culture, because they offer the multiple advantages of speed, sensitivity and specificity, which together, are not possible with other established techniques. Molecular methods afford the opportunity to detect viral genome(s), quantify viral load and detect different viral genotypes or mutations. Instruments and chemistries are becoming reasonably affordable and enclosed automated nucleic acid extraction/detection techniques are becoming more and more available increasing the throughput, the reproducibility and confidence in the results.
7. XMRV: A Cautionary Tale
Xenotropic murine leukaemia virus-related virus (XMRV) was discovered in 2006 in tumour tissues from patients with a familial form of prostate cancer. The discovery was made using a powerful molecular detection technique involving random PCR amplification and a high density microarray composed of oligonucleotides from the most conserved sequences of all known viral families. XMRV was claimed to be the first example of a gammaretrovirus to infect humans and its discovery was followed by several studies that confirmed its presence in prostate cancer patients. The publication in 2009 of a highly controversial paper in Science associating the virus with chronic fatigue syndrome (CFS) brought XMRV to the attention of the worldwide public. Its detection in 3.7% of healthy controls sparked fears that millions might be infected with a novel retrovirus of unknown pathogenic potential. However, almost all recent efforts to confirm the association between XMRV, CFS and prostate cancer have failed and XMRV is now thought much more likely to represent a contamination related laboratory artefact rather than a genuine human infection. This chapter reviews the spectacular rise and fall of XMRV and attempts to draw useful lessons from this painful episode in the history of molecular diagnostics.
8. Ancient DNA and the Fingerprints of Disease: Retrieving Human Pathogen Genomic Sequences from Archaeological Remains Using Real-time Quantitative Polymerase Chain Reaction
G. Michael Taylor
It is almost 20 years since the first reports of the amplification of pathogen DNA sequences from human archaeological remains. Diseases, such as tuberculosis and leprosy have been productive areas of endeavour due to their association with characteristic skeletal lesions and the robust nature of the mycobacterial cell wall. A number of other pathogens apart from mycobacteria have been successfully amplified from human remains, including the microorganisms responsible for Malaria, Leishmaniasis, Brucellosis and plague (Black Death). Information from dated contexts can further our understanding of past epidemics, both challenging and informing evolutionary models of microorganism phylogeny and global dissemination. This chapter considers the practical aspects of extracting and amplifying degraded fragments of pathogen DNA that may persist in archival tissues. It includes a discussion of the measures needed to prevent contamination and to authenticate findings, in particular, considering the many advantages of using real-time quantitative PCR methods.
9. Point-of-Care Nucleic Acid Testing: User Requirements, Regulatory Affairs, and Quality Assurance
Angelika Niemz, Tanya M. Ferguson and David S. Boyle
Nucleic acid testing (NAT) for in vitro diagnostic (IVD) use has moved beyond high-complexity reference laboratories to moderate complexity centralised laboratories, and to certain professional point of care (POC) settings in developed and developing countries. To develop, launch, and sustainably implement a POC NAT system requires compliance with regulatory affairs and quality assurance requirements, and long term demonstration of the system's clinical utility. This report discusses the pros and cons of POC versus centralised laboratory testing, general clinical utility considerations, use environment, and user requirements in developed versus developing countries. The report further provides an overview of the applicable regulatory requirements in different regions around the globe, and describes quality assurance considerations during test execution by the end user, which can be especially challenging for POC testing.
10. Point-of-Care Nucleic Acid Testing: Clinical Applications and Current Technologies
Angelika Niemz, Tanya M. Ferguson and David S. Boyle
Nucleic acid testing (NAT) has greatly enhanced the diagnosis of infectious diseases, but typically requires a central laboratory with highly skilled operators and complex equipment. This paradigm is not ideal for indications that require a rapid turn-around or for settings where a complex laboratory infrastructure is not available. In the past 10 years, novel assays and hardware solutions have been developed that have advanced NAT into peripheral laboratories. We anticipate that NAT in the near future will move to near patient settings in hospitals and physician's offices in developed countries, and to low resource primary care settings in developing countries. In this review we discuss selected clinical applications that can benefit from disseminated NAT, and highlight emerging diagnostic technologies for sample preparation, nucleic acid amplification and detection. We further describe systems that integrate several or all of these components to enable highly sensitive, rapid, and user friendly NAT for infectious disease diagnosis outside of the traditional laboratory.
11. From Bench to Bedside: Development of Polymerase Chain Reaction Integrated Systems in the Regulated Markets
Martin Lee, Diane Lee and Phillip Evans
The evolution of the polymerase chain reaction (PCR) as a revolutionary molecular biology tool has been extremely rapid since its inception in 1983. This may be attributed, in part, to the simplicity of the process for the general molecular biologist and also because of the open licensing strategy provided for underpinning products by key stakeholders. The development of commercial products for the applied markets such as food, veterinary, and defence has been greater than for human diagnostics. This sector has been restrictive for most system developers because of the high value of upfront and running royalty licences. In addition, the human diagnostic area is highly regulated in the US (FDA) and the EU (IVDD). The core patents covering the PCR process are approaching their termination dates in key territories, largely negating the requirement for such licences. The opportunity for those developers of Nucleic Acid Tests (NAT) on highly automated laboratory devices and/or simple to use integrated point-of-use PCR devices (iNAT) to "tap into" the content developed within this broad research sector is enormous. This text is written to provide both technical and commercial advice to those involved in this technology transfer opportunity. The text is presented as a number of short key-notes in the following areas: 1) PCR formulation; 2) assay stabilisation; 3) the sample extraction reagent interface; 4) nucleic acid extraction 5) signalling and automated analysis; 6) suppliers and GMP; 7) system validation; and 8) the regulatory process. These notes are intended to provide guidance to the key issues rather than a prescriptive manual. The author will direct the reader to the appropriate authority. It will be of most use for individuals and organisations either intending to provide, or to receive, product content. It is based upon the author's experiences in developing integrated systems for the applied and diagnostics sectors over the last 15 years.
12. Future of Molecular Diagnostics: The Example of Infectious Diseases
Eoin Clancy, Kate Reddington, Thomas Barry, Jim Huggett and Justin O'Grady
Advances in molecular diagnostics technologies offer clinicians improved infectious diseases tests for use in a hospital setting with regards to specificity, sensitivity and turn-around time to results. These advances have the potential to contribute in a positive way to patient care owing to improved decision making on optimal therapeutic approaches. Such advances may be used independent of traditional culture based methods, or in some instances complementing culture. In this chapter we review a number of emerging technologies which can be used for both targeted and non-targeted molecular diagnostics in the clinical setting which may play a central role in the future including fully integrated "sample in - result out" nucleic acid based tests, digital PCR, mass spectrometry and next generation sequencing.
How to buy this book
(EAN: 9781908230416 9781908230645 Subjects: [microbiology] [bacteriology] [virology] [medical microbiology] [molecular microbiology] [pcr] )