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Molecular Microbiology: Diagnostic Principles and Practice, Second Edition
Editors: David H. Persing1, Fred C. Tenover2, Yi-Wei Tang3, Frederick S. Nolte4, Randall T. Hayden5, Alex van Belkum6
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Affiliations:
1: Editor in chief Cepheid Corporation, Sunnyvale, California;
2: Cepheid Corporation, Sunnyvale, California;
3: Vanderbilt University School of Medicine, Nashville, Tennessee;
4: Medical University of South Carolina, Charleston, South Carolina;
5: St. Jude Children’s Research Hospital, Memphis, Tennessee;
6: Erasmus University, Rotterdam, The Netherlands
Content Type: Reference
Format:
Electronic, Hardcover
Publication Year:
2011
Category: Clinical Microbiology; Bacterial Pathogenesis
Print ISBN
:
9781555814977
e-ISBN
:
9781555816834
MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.
The second edition of Molecular Microbiology: Diagnostic Principles and Practice presents the latest molecular diagnostic techniques to support clinical care and basic and clinical research. The authors-all experienced researchers and diagnosticians-have conducted a comprehensive review and evaluation of this rapidly evolving field and provide the new material in an easy-to-read summary format. Moreover, the book offers a broad range of practical advice, such as determining the appropriate type and quantity of a specimen, releasing and concentrating the targets, and eliminating inhibitors.
Molecular Microbiology not only examines techniques to detect individual pathogens, but also explores the growing trend toward a systems approach for diagnosing infectious diseases, with chapters covering gastrointestinal infections, sepsis, meningitis, and encephalitis. In short, this text not only encapsulates the current state of the science, but also points to new avenues for research that will broaden the application and usefulness of molecular diagnostics.
Hardcover, 936 pages, full-color insert, illustrations, index.
Book Summary
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DIAGNOSTIC PRINCIPLES
I. MOLECULAR HYBRIDIZATION FOR MICROBIAL IDENTIFICATION
Chapter 1 : Applications of Fluorescence In Situ Hybridization in Diagnostic Microbiology
- Authors: Stefan Juretschko, Thomas R. Fritsche
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The presence of a sufficient number of target molecules is critical in detecting conferred fluorescence of probe-target hybrids and permits direct visualization of intact organisms by epifluorescence microscopy. This direct approach to visualization of organisms using fluorescence in situ hybridization (FISH) simultaneously provides information on phylogenetic relationships of organisms, their spatial distribution in the sample matrix, their relative abundance, and their relative physiologic activity. An incubator, a water bath, and an epifluorescence microscope are essential, with the microscope being most critical for successful FISH performance. The main goal in designing specific probes is to find a suitable and unique region within the 16S or 23S rRNA that permits discrimination of target from nontarget organisms. Once a probe has been designed and confirmed to be specific for the motif desired by using the appropriate databases, it must be evaluated against a series of reference organisms. In case of a low number of ribosomes, the simultaneous application of two fluorescence-monolabeled probes targeting two different regions of rRNA with the same specificity would theoretically result in a twofold amplification of signal intensity when applied to the test samples. The chapter talks about expanded techniques combined with FISH. FISH, based upon either DNA or PNA oligonucleotide probes, is a rapid diagnostic method capable of challenging traditional culture techniques for the direct and accurate identification not only of nonfastidious pathogens but also of fastidious, slow-growing, and difficult-to-cultivate organisms.
Chapter 2 : DNA Probes for Culture Confirmation and Direct Detection of Bacterial and Fungal Infections: a Review of Current Technologies and Assays
- Authors: Julie Kingery, Karen C. Carroll
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This chapter focuses on nonamplified nucleic acid probes and their current uses in the clinical laboratory. DNA probes are pieces of nucleic acid that are labeled in some way and are designed to seek out and bind to stretches of DNA or RNA that have sequences that are complementary to the probe. In hybridization reactions, a double-stranded DNA molecule is denatured to single strands. Several formats for the hybridization reactions exist: solid phase; in solution (liquid phase); in situ; or by use of a Southern hybridization procedure after gel electrophoresis. In sandwich hybridization assays, one probe is attached to a solid support such as a nitrocellulose filter in single-stranded form and ‘‘captures’’ homologous nucleic acids in liquid samples; a second probe, which recognizes a contiguous area of the nucleic acid, carries the reporter molecule such as a radioisotope or biotin. The target and probe nucleic acids are free to move in solution, maximizing chances that complementary sequences will bind. Southern hybridization involves using purified DNA that is cleaved with restriction endonucleases. There are numerous methods for detecting the binding of probe to target nucleic acid. Commercially available DNA probes used for culture confirmation of bacteria, mycobacteria, and fungi are discussed in the chapter. Of the assays discussed in the chapter, probes for the detection of mycobacteria have had the greatest clinical impact. The chapter describes the utility of nonamplified probes for the diagnosis of sexually transmitted diseases, vaginal infections, and streptococcal pharyngitis.
II. NUCLEIC ACID AMPLIFICATION METHODS
Chapter 3 : In Vitro Nucleic Acid Amplification Techniques
- Authors: Vivekanand Datta, Randall T. Hayden
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Nucleic acid amplification (NAA) techniques have come of age. The specific amplification and detection of an oligonucleotide sequence went from the fictional to the mundane in the span of two decades. By looking at the theoretical basis for each method, NAA techniques can be placed into one of two broad categories: (i) target amplification systems, including PCR, ligase chain reaction (LCR), self-sustaining sequence amplification (3SR), nucleic acid sequence-based amplification (NASBA), transcription-based amplification system (TAS), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and loop-mediated isothermal amplification (LAMP); and (ii) signal amplification systems (including probe amplification methods), such as branched-DNA technologies (bDNA) and cleavage-based signal amplification (cycling probe technologies [CPT] and Invader assays). Proofreading may help maintain fidelity of replication, and its absence can result in a relatively high rate of nucleotide incorporation errors (misincorporation), most relevant when starting with low target numbers. Misincorporation can produce amplicon mismatched to detection probes and can also result in inefficient amplification (especially when longer genetic stretches are targeted), due to primer mismatch in subsequent amplification rounds. Successful ligation relies on contiguous positioning and correct base pairing of the 3' and 5' ends of oligonucleotide probes on a target DNA molecule. The reliability of sequence data has improved, and the taxonomic and cytogenetic characterization of microorganisms has advanced tremendously. These improvements give the tools to allow the rapid and increasingly routine development of molecular methods for the identification, quantification, and characterization of microorganisms.
Chapter 4 : Real-Time PCR and Melting Analysis
- Authors: Carl T. Wittwer, Noriko Kusukawa
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This chapter talks about the fundamentals of real-time PCR and melting analysis. It draws an analogy between bacterial growth and PCR and then considers the kinetic requirements of PCR. It provides an overview of real-time instrumentation and fluorescent indicators. It considers methods for detection, quantification, and melting analysis, including high-resolution melting analysis. Real-time PCR with melting analysis can integrate the detection, quantification, and analysis of microbes in one rapid assay. In real-time PCR and melting analysis, fluorescence acquisition may extend the time required in instruments with high noise and/or low fluorescence sensitivity. All real-time PCR instruments monitor sample fluorescence during thermal cycling and are available from many manufacturers. A wide variety of different instruments, dyes, and probe designs are available for real-time PCR. The chapter discusses some of the methods commonly used for detection, quantification, and melting analysis. It also focuses on melting-curve analysis. Inspection of continuous plots during real-time PCR suggests that hybridization information can be extracted during temperature cycling when dyes or hybridization probes are used. Continued improvements in speed, integration of high-resolution melting analysis, and adoption of simple hybridization probe techniques like unlabeled probe and snapback primers will expand the reach of this powerful technique in the coming years.
Chapter 5 : Quantitative Molecular Methods
- Authors: Donna M. Wolk, Randall T. Hayden
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Quantitative molecular methods provide information about the concentration of microbial nucleic acid target present in a sample. The evolution of various quantitative methods includes PCR and commercial alternatives to PCR. Quantitative methods that rely on target amplification include transcription-mediated amplification (TMA) and nucleic acid sequenced-based amplification (NASBA). Commercially available quantitative signal and probe amplification methods include the branched DNA (bDNA) method and the Invader assay, respectively. Quantitative methods based on PCR include three broad categories: quantitative PCR (Q-PCR) is typically used to determine the microbial density or ‘‘load’’ of DNA in clinical specimens; (ii) quantitative reverse transcriptase PCR (QRT-PCR) is used to determine the density of RNA viruses, an approach commonly called ‘‘viral load’’ testing; and (iii) in what are often referred to as ‘‘gene expression’’ assays, QRT-PCR can be used to determine relative mRNA expression levels for different disease states. This chapter concentrates on the technological features unique to quantitative molecular assays. The advantages and limitations of these methods are reviewed in the chapter. For microbial quantitation, whole-organism comparisons are best but not always feasible; therefore, plasmids or oligonucleotides are often used as substitutes. The chapter talks about general issues for viral load measurement, and other general considerations and tips for quantitative methods. Speed, accuracy, and utilization of results will be paramount to the future of quantitative technology as new methods extend our understanding of pathogenesis and advance our ability to improve diagnosis and disease management.
Chapter 6 : Use of Commercial Amplification Tests in the Clinical Microbiology Laboratory: Test Selection and Quality Assurance
- Authors: Gerri S. Hall, Mark Katanik, Marion Tuohy, Mary Sholtis
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Molecular approaches to the detection of infectious-disease agents in the clinical microbiology laboratory can be performed through the use of commercially available products or by using in-house or “home-brew” procedures. Both types of assays are currently used for the diagnosis of two sexually transmitted organisms, Chlamydia trachomatis and Neisseria gonorrhoeae, for screening of Group B Streptococcus in pregnancy, for nasal screening of methicillin-resistant Staphylococcus aureus (MRSA) and detection of both methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in wounds and positive blood culture vials, for detection of toxigenic Clostridium difficile in stool samples, and for the detection of Mycobacterium tuberculosis. A description of how to choose and implement a commercially available assay for these agents is provided in this chapter. The chapter talks about implementation of the new assay, and use of commercial assays for M. tuberculosis amplification. Three commercial PCR-based amplification assays have been cleared by Food and Drug Administration (FDA) for the detection of the toxin B gene in stool samples. All of the principles for implementation of a molecular test that have been described in this chapter will also need to be performed for C. difficile amplification assays. After implementation of a molecular assay, monitoring the process and results to be assured of the expected performance of the assay should be ongoing.
III. LABORATORY OPERATIONS
Chapter 7 : Nucleic Acid Isolation: Overview of Sample Preparation Methods
- Author: Charles E. Hill
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The type of sample being submitted for analysis determines the method used for nucleic acid isolation. Serum is also an acceptable material and yields similar clinical utility, but viral quantification may be slightly lower than plasma from the same patient due to entrapment of some extracellular virus within the blood clot. Regardless of the source, nucleic acid extraction usually consists of three primary processes: (i) lysis, (ii) denaturation/degradation of other biomolecules, and (iii) separation of the nucleic acids from other constituents in the sample and/or concentration of the DNA or RNA. Although there are several possible ways to classify nucleic acid extraction techniques, the following types of chemistries are the most commonly used: (i) precipitative methods, (ii) liquid-phase extractions, and (iii) solid-phase extractions. Although all three types of extraction methods are used, solid-phase extractions now are the most widely utilized due to easy scalability and the availability of automated instruments for these methods. While precipitative and liquid-phase extraction methods are still widely used, the most commonly performed DNA and RNA extractions involve selective binding of nucleic acids to an immobilized matrix (solid phase). The majority of the alcohol can be removed by essentially drying the matrix with the nucleic acid bound. Automation of the nucleic acid extraction process typically provides a more reproducible yield of DNA and/or RNA.
Chapter 8 : Laboratory Design and Operations
- Authors: P. Shawn Mitchell, Jeffrey J. Germer, Joseph D. C. Yao
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Molecular methods play an increasingly important role in diagnostic microbiologic testing, and this trend will continue with the introduction of new technologies and continued improvement in current assay designs. Individual laboratories considering the implementation of such molecular methods for diagnostic testing face unique challenges in achieving optimal laboratory design and operations that may vary greatly depending on the laboratory setting and the molecular methods employed. Efficient laboratory operation and workflow are closely dependent on optimal design of the laboratory space, and in turn, optimal laboratory design is influenced by the various assay methods and instruments used in the laboratory. Chemical fume hoods and cabinets for storage of hazardous chemicals and flammable liquids should also be incorporated in the overall laboratory design. Efficient clinical laboratory operations providing quality testing services are dependent on several key elements. In addition to the use of dedicated work areas and equipment, regular cleaning and decontamination can also be effective in further reducing the likelihood of amplicon contamination. Specific precautions that should be considered prior to implementation of three molecular diagnostic methods–hybridization-based methods, target amplification-based methods, and target amplification-detection and postamplification analysis methods–have been discussed in the chapter.
IV. MOLECULAR TYPING METHODS
Chapter 9 : Molecular Phylogenetic Analysis
- Authors: Les Dethlefsen, Paul W. Lepp, David A. Relman
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Phylogenetic analysis, in the strictest sense, is the process of testing hypotheses about the descent of species from a common ancestor. This chapter provides an overview of a task essential to all: obtaining a working hypothesis of the evolutionary relationships among a group of organisms, summarized as a phylogenetic tree. Molecular phylogenetic analysis became more powerful and more accessible with the advent of rapid, inexpensive DNA sequencing, eventually leading to a major revision in the understanding of the evolutionary relationships among all living organisms. In order to provide a statistically robust representation of the phylogeny, a phylogenetic marker needs to have a sufficient number of independently evolving positions so that at least several changes differentiate the most closely related taxa of interest. After a phylogenetic marker has been selected, one must obtain sequence data. The core of automated alignment algorithms is optimization of a scoring function. Points are added for identities and similarities at each position, points are subtracted for mismatches, and many points are subtracted for gaps. The chapter emphasizes the use of aligned sequence databases in preference to de novo alignment not only for the savings in time and effort but also for the higher quality. It discusses maximum parsimony and maximum likelihood, the most widely implemented character-based methods, as well as Bayesian phylogenetic inference, a relatively new method rapidly growing in popularity. Although it may be possible to identify an optimal topology, the reader is cautioned that an optimal tree does not guarantee the true phylogeny.
Chapter 10 : Pulsed-Field Gel Electrophoresis: Laboratory and Epidemiologic Considerations for Interpretation of Data
- Authors: Richard V. Goering, Efrain M. Ribot, Peter Gerner-Smidt
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This chapter reviews pulsed-field gel electrophoresis (PFGE) as an epidemiological tool, considering (i) factors that influence the electrophoretic process, (ii) methodological streamlining, (iii) the troubleshooting of common problems, (iv) quality assurance, (v) use of PFGE for continuous surveillance, and (vi) issues of data interpretation. To be suitable for reliable PFGE analysis, intact chromosomal DNA must be isolated in a protected environment free from mechanical, chemical, and enzymatic degradation to yield a clear and reproducible macrorestriction fragment pattern. As PFGE analysis is applied to larger study populations, the need for computer-assisted analysis (CAA) of banding patterns becomes increasingly evident. At the laboratory level the quality assurance/ quality control (QA/QC) system consists of strict adherence to each of the PFGE standard operating procedures (SOPs) as described in the laboratory QA/QC manual. It is important to emphasize that the successful establishment of dynamic databases is dependent on strict adherence to well-defined QA and QC criteria. An important component of the protocol standardization and QA/QC program for PulseNet is the annual update meeting. Molecular typing, along with a variety of other microbiological assays is clearly moving toward sequence-based analysis. However, this approach is still being validated for a variety of applications including strain typing. Thus far, none of the new sequence-based typing methods are as broadly applicable as PFGE. Therefore, while this problem will undoubtedly be solved in the future, at present PFGE will clearly continue to provide meaningful epidemiological data on molecular typing in a variety of important settings for years to come.
Chapter 11 : Strain Typing Using Multiple “Variable Number of Tandem Repeat” Analysis and Genetic Element CRISPR
- Authors: Christine Pourcel, Gilles Vergnaud
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This chapter reviews the current status and future prospects of two very highly promising high-throughput genotyping approaches, i.e., multiple-locus VNTR analysis (MLVA) and clustered regularly interspaced short palindromic repeats (CRISPR) analysis. Tandem repeats (TRs) often are found in families of intergenic elements. Such TRs are frequently used for genotyping because of their high degree of polymorphism. For example, MLVA schemes for M. tuberculosis, Streptococcus pneumoniae, P. aeruginosa, S. aureus, and Leptospira interrogans use TRs within intergenic elements. One potential problem in trying to make strain typing using variable number of tandem repeat (VNTR) readily available in multiple laboratories is that many of the TR alleles are too large for routine capillary electrophoresis. In species of more recent emergence, such as Y. pestis, almost all strains possess the same number of CRISPRs, although the number of spacers and DRs varies, and their position on the chromosome changes due to rearrangements. Comparison of the four available Y. pestis sequenced genomes shows the existence of three CRISPRs, two of which present a different number of spacers. The analysis of a large collection of strains showed that all three loci could vary in size, although one was more polymorphic than the others. In this study, a large group of isolates from outbreaks in Vietnam in the 1960s revealed that the CRISPRs acquired new spacers in a polarized fashion and that these spacers originated either from a bacteriophage or from chromosomal genes.
Chapter 12 : Repetitive Sequence-Based PCR Typing of Bacteria and Fungi
- Authors: Stacie R. Frye, Mimi Healy
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Of the number of molecular typing methods that have been described including multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), DNA sequencing of variable genes, ribotyping, PCR ribotyping, restriction fragment length polymorphism analysis, randomly amplified polymorphic DNA (RAPD) analysis, amplified fragment length polymorphism (AFLP) analysis, and repetitive sequence-based PCR (rep-PCR), this chapter focuses on typing of bacteria and fungi specifically using rep-PCR. The theory of rep-PCR is discussed with a comparison of the workflow for traditional gel-based rep-PCR and an automated commercially available system. It is important that the culture be pure, as rep-PCR primer binding sites are widely distributed among bacterial genera. The repetitive extragenic palindrome (REP) was the first repetitive element described in bacteria. The chromosomal locations of the enterobacterial repetitive intergenic consensus (ERIC) elements differ among species but are conserved throughout the kingdom Bacteria. In addition to bacteria, various fungi have been associated with nosocomial infections. Filamentous fungi, such as Fusarium species and the Zygomycetes, also contribute to the growing list of health care-associated (HA) infections. While the debate continues over the most cost-effective approach for reducing HA infections, it is clear that some form of an active surveillance program is a key component of effective infection control strategies. Future applications for automated rep-PCR include extension of the menu of both bacterial and fungal pathogens and expansion of the online databases.
Chapter 13 : Molecular Typing Methods for Analysis of Extraintestinal Pathogenic Escherichia coli
- Author: James R. Johnson
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Escherichia coli, although perhaps best known as a cause of diarrheal disease, is actually responsible for more morbidity, mortality, and increased health care costs in the developed world as an extraintestinal pathogen. Two main categories of typing methods are relevant for studies involving extraintestinal pathogenic E. coli (ExPEC), including methods that define the strain’s phylogenetic and clonal background (at varying levels of resolution) and those that detect virulence-associated accessory traits. Alternative methods for resolving phylogenetic relationships at the clonal group level that are simpler and cheaper than multilocus sequence typing (MLST) include PCR-based genomic profiling methods, e.g., random amplified polymorphic DNA (RAPD) analysis and repetitive-element (REP) PCR, as performed using the ERIC, BOX, or REP primers. Methods for molecular typing of ExPEC find application in various kinds of studies, including between-population comparisons, assessments of individual isolates for their virulence potential or clonal similarity to other individual isolates, and assessments of colonization and transmission dynamics. The approaches used for statistical analysis of molecular typing data are an important consideration in population level studies involving ExPEC. Molecular typing of ExPEC for phylogenetic and clonal background, as well as accessory traits (e.g., virulence factors) can lead to important new insights into the origins, reservoirs, clinical and commensal behavior, and host group associations of this important group of E. coli. Attention to study design, population selection, specific molecular methods, and appropriate statistical analysis approaches can enhance the quality of typing studies involving ExPEC, which may lead to improvements in human or animal health.
V. DETECTION METHODOLOGY
Chapter 14 : Fluorescence Resonance Energy Transfer
- Authors: J. R. Uhl, Y.-W. Tang, F. R. Cockerill III
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This chapter discusses general fluorescence resonance energy transfer (FRET) probe technology and the use of FRET with single and dual hybridization probes in microbiology. It focuses on hydrolysis and hybridization FRET probes and the many variations used for detection of amplification products in PCR amplification. True multiplex assays simultaneously amplify different nucleic acid targets and result in multiple unique PCR products. These types of assays often have two or more sets of primers and probes and are most commonly used with genomic targets in which the amount of target nucleic acid is normally large and the ratio of the two target nucleic acids is close to unity. Recent applications of FRET probes in bacteriology include assays for Vibrio parahaemolyticus, Clostridium difficile, Brucella, Francisella tularensis, Clostridium perfringens, Listeria monocytogenes, Burkholderia cepacia complex, Campylobacter jejuni and Campylobacter coli, and penA and ponA genotypes in Neisseria gonorrhoeae. The use of homogeneous FRET probe technology for detection of PCR products provides an opportunity for microbiologists to use molecular detection in a closed system. The necessary specificity and sensitivity of many microbiology tests are achievable using PCR with FRET detection. The technology has become widely available, and configurations of instrumentation and FRET design are available for many applications. Hybridization FRET probes provide great sensitivity and specificity to real-time PCR with the benefit of sensitive detection of nucleic acid sequences with unexpected polymorphisms. The hybridization FRET probes also enable multiple formats for robust multiplexing reactions often with just a single set of primers and probes.
Chapter 15 : Detection of Pathogenic Organisms with Multicolor Molecular Beacons
- Authors: Fann Wu, Phyllis Della-Latta, Sanjay Tyagi, Fred Russell Kramer
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The chapter describes a detection method that is based on the sequence-dependent hybridization of fluorogenic reporter molecules called “molecular beacons’’ and its applications. Molecular beacon probes represent a new class of oligonucleotides that can hybridize and report the presence of specific nucleic acids in homogeneous solutions. In addition, improvisations on the basic theme of molecular beacons have also appeared in the literature. Molecular beacons are primarily employed as highly specific amplicon detection probes in homogeneous, real-time, multiplex gene amplification assays. On the other hand, the generation of all five fluorescent colors during PCR amplification indicates that the mycobacteria in the sample are rifampin susceptible. Infection by certain types of human papillomavirus (HPV) can lead to cervical cancer; therefore, accurate identification of these oncogenic HPV genotypes is critical. One assay detects HPV-DNA by SYBR Green® and then distinguishes the seven most prevalent high-risk HPV genotypes by using real-time molecular beacon PCR. They can be used as hybridization detection probes, not only for real-time monitoring of DNA amplification in vitro but also for real-time monitoring of the distribution and transport of mRNAs in living cells. The current available applications of molecular beacons for detection of pathogenic microorganisms are listed in the chapter. Efforts are being made to explore their applications in many areas including genotyping of infectious agents and mutation analysis for the identification of drug-resistant pathogens.
Chapter 16 : Microwell Plate Detection Systems for Amplicon Detection and Characterization
- Authors: Jiang Fan, Michael J. Loeffelholz
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Sequence-specific methods for the detection of the products of nucleic acid amplification procedures (amplicons) have been developed for a variety of solid phases, including nylon membranes, microwell plates, microparticles, and, most recently, microchips (oligonucleotide probe microarrays). This chapter deals with procedures in which the molecules that capture amplicons are immobilized onto the surface of wells of microwell plates. Microwell plate detection systems can be divided into two formats based on the molecule used to capture amplicons: an oligonucleotide probe (sequence-specific capture) and avidin (nonspecific capture). Microwell plate detection systems have been developed in-house and are available commercially. Microwell plate detection procedures are frequently based on capture and detection of biotinylated amplicons. This requires that the primer used in the PCR amplification to generate the amplicon strand complementary to the probe be tagged with biotin. The biotin substituent will allow the amplicon to be either captured or detected by avidin (or streptavidin), depending on the detection format. Polystyrene microwell plates are typically used for amplicon detection because of their high DNA binding capabilities. There is substantial literature demonstrating the excellent analytical performance and clinical utility of microwell plate detection systems for PCR amplicon detection. Microwell plate detection of amplicons is flexible, is compatible with virtually any target, and can easily detect multiple pathogens under common hybridization and wash conditions.
Chapter 17 : Detection Methodology: Pyrosequencing
- Authors: Liying Yan, Robert Slinger
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Pyrosequencing is a non-gel-based, real-time approach to sequence DNA by monitoring DNA polymerase activity using an enzymatic luminometric inorganic pyrophosphate detection assay. Using pyrosequencing for microbiology identification or drug resistance screening involves three main steps: assay design, PCR, and the actual pyrosequencing. To make pathogen identifications, pyrosequencing assays have frequently used the highly conserved targets that are also used in Sanger sequencing, specifically 16S rRNA in bacteria and mycobacteria and the 18S/internal transcribed spacer (ITS) region in fungi, although other targets are used when the conserved regions are inadequate for species identification. 16S rRNA is a bacterial identification target commonly used by many different technologies, including Sanger sequencing. Pyrosequencing is a real-time sequencing technology alternative to Sanger sequencing. The versatility of pyrosequencing allows bacteria to be identified via 16S single-nucleotide polymorphism (SNP) analysis or de novo sequencing of the variable regions. Pyrosequencing enabled the differentiation of Hantaan virus from Seoul virus, the most common hemorrhagic fever with renal syndrome pathogens in Asia, and of Andes virus from Sin Nombre virus, the two viruses that cause hantavirus cardiopulmonary syndrome in the Americas. Pyrosequencing has been compared prospectively to conventional sequencing for the diagnosis and genotyping of the parasite Toxoplasma gondii. Microbial drug resistance is usually developed over time by the introduction of a point mutation or sometimes multiple mutations. The ability to fuse real-time PCR with real-time pyrosequencing may be the catalyst that leads to widespread adoption of pyrosequencing in clinical laboratories.
Chapter 18 : Solid- and Liquid-Phase Array Technologies
- Author: Melissa B. Miller
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This chapter focuses on DNA-based hybridization array technology. The chapter reviews the methodologies of cDNA, oligonucleotide, electronic, and liquid bead arrays. In each of these methodologies, the probe refers to the DNA sequence bound to the solid surface support in the microarray, whereas the target is the ‘‘unknown’’ sequence of interest. Printed microarrays have the advantages of simplicity and relatively low cost compared to synthesized microarrays, which are discussed in the chapter. While two-dimensional microarrays have had an impact on our understanding of the microbial world and the application of this diagnostic potential to infectious diseases, the most widespread and practical application is the use of liquid bead suspension arrays in diagnostic microbiology, which is therefore emphasized when applicable. The potential use of low- or medium-density arrays for the simultaneous detection of large numbers of microbial genetic targets is one of the most promising areas in applying microarray technology to diagnostic microbiology. Broad-range amplification often focuses on the rRNA genes (16S, 18S, 23S, or intergenic transcribed spacers) due to the inherent dichotomy of conserved and polymorphic sequences. Though the range of organisms included was relatively limited (species of Bacillus, Listeria, Staphylococcus, Escherichia, Shigella, Klebsiella, Salmonella, Enterobacter, Ralstonia, Burkholderia, and Pseudomonas), this study provides proof of principle for the use of suspension arrays for the identification of broad-range amplification products. Though broad-range amplification typically focuses on the rRNA genes, other targets have been employed.
VI. NEW TECHNOLOGIES
Chapter 19 : Next-Generation DNA Sequencing and Microbiology
- Authors: Russell Higuchi, Ulf Gyllensten, David H. Persing
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Recent, dramatic increases in the throughput of DNA sequencing instruments provide opportunities for microbiology research that previously were limited to large genome centers. This chapter reviews on the technology behind these “next-generation” platforms and compares the three that are currently the most popular and also reviews applications to date of these new technologies to microbiology research. In the chapter, steps that are similar and shared among the next-generation sequencing platforms are first described generically. The following sections provide a few examples of microbiological applications of deep-sequencing technologies. The study of vaginal flora has been the subject of many culture-based investigations over the years, but recently it has been recognized that many of the organisms actually present are not represented among those that can be recovered by in vitro cultivation. It may be able to simultaneously determine drug resistance, either at the mutational level within specific viral or bacterial genes or by determining the presence of expressed drug resistance genes such as those encoding beta-lactamases or other markers. In all likelihood, deep-sequencing technologies will make their way into clinical laboratories in the next several years, probably starting out with genetic and oncology testing but ultimately moving into molecular microbiology laboratories as well.
Chapter 20 : Raman Spectroscopy for Bacterial Strain Typing
- Authors: Diana Willemse-Erix, Alex van Belkum, Kees Maquelin
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In the past 20 years, phenotypic typing methods have been largely replaced by typing methods based on the comparison of genomic DNA (molecular typing), such as PCR fingerprinting, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). An alternative approach to bacterial typing is based on applying Raman spectroscopy to test subtle differences in the molecular composition of the biomass, reflecting differences in the genomic DNA. The most important advantages of Raman spectroscopy compared to established molecular typing methods are speed, high sample throughput, and ease of use. In a Raman scattering event, an incident photon transfers some of its energy to the molecule, which leads to a lower energy in the scattered photon than in the incident photon. The approach can be as simple as a visual assessment of clearly identifiable spectral features that can only correlate to the biochemical component of interest. A well-known example of a microorganism causing hospital-acquired infections (HAI) is methicillin-resistant Staphylococcus aureus (MRSA). Therefore, the authors used an MRSA reference collection to demonstrate the capabilities of Raman spectroscopy. This reference collection contained 20 well-characterized MRSA isolates that had previously been analyzed by multiple typing techniques. Using Raman spectroscopy as a bacterial typing tool, infection control teams will have a tool for the continuous monitoring of isolates in their hospital, and they will be aware of the need for corrective action earlier, all leading to an accurate, real-time rather than retrospective surveillance approach in combating HAI.
Chapter 21 : Multiplex PCR Product Detection and Discrimination
- Authors: Steven Zink, Nick M. Cirino, Christina Egan
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This chapter examines the current state-of-the-art multiplex PCR and diagnostic platforms that are based on multiplex PCR but contain additional features to enhance sensitivity, multiplexing capability, or ease of use. Advances that aid in the development and optimization of multiplex rtPCR-based assays, such as primer design software and novel rtPCR reagents, are also reviewed. Multiplex PCR is a technique in which the amplification and detection of two or more target DNA or RNA sequences in a single reaction are accomplished through the use of specific primers or a combination of specific primers and probes. Microarrays have been utilized to measure the levels of expression of genes, to identify single-nucleotide polymorphisms, and to genotype organisms. Another technology that is being utilized is the reverse line-blot hybridization assay (mPCR/RLB). This method in combination with multiplex PCR has been reported to provide some benefits over microarray methods. Bead-based suspension array technology is being utilized currently for antigen-, protein-, and nucleotide-based detection assays. New technologies and new platforms for high-throughput DNA sequencing are reaching maturity and should soon be available for routine use in diagnostic laboratories. With concomitant advances in the fields of microfluidics, nucleotide and fluorescent dye chemistries, and information processing, highly multiplexed nucleic acid detection and identification technologies will gradually come to be applied in a vast range of situations in which sensitivity, specificity, and speed are indispensable.
Chapter 22 : Detection of Emerging Antimicrobial Resistance by Use of the Ibis T5000 Universal Biosensor
- Authors: David J. Ecker, Rangarajan Sampath, Christian Massire, Lawrence B. Blyn, Mark W. Eshoo, Thomas A. Hall, Steven A. Hofstadler
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This chapter describes about a new technology for the identification of microbes and molecular identification of drug resistance using a platform known commercially as the Ibis T5000 universal biosensor. The PCR/electrospray ionization-mass spectrometry (ESI-MS) technique was initially developed for the identification of microbes, including previously unknown or unculturable organisms, in original patient specimens or environmental surveillance samples in which multiple microbes may be present. Applications of Ibis T5000 technology can be thought of in an hourglass model. Drug resistance in bacteria and viruses is often mediated by mutations in the genes that encode the proteins that are the targets of the drugs. Many of the most important drug-microbe combinations are of this nature. An important feature of PCR/ESI-MS for detecting emerging drug resistance is that nucleic acid does not need to be isolated from pure colonies of the target microbe. The ability of PCR/ESI-MS to detect a low-abundance nucleic acid amplicon that has a mutation representing an emerging antimicrobial resistance in the presence of a higher-abundance wild-type background is critical to a number of applications. The final eight-primer-pair panel includes two primer pairs that target efp, the gene found to be the most useful in discriminating between different Acinetobacter species by MLST. Generally, drug resistance mutations in HIV arise due to selective pressure in patients with incompletely suppressed virus replication. HIV-1 isolates with drug resistance mutations may also be transmitted to newly infected individuals.
DIAGNOSTIC APPLICATIONS
VII. MOLECULAR DETECTION OF BACTERIAL ORGANISMS
Chapter 23 : Update on the Detection and Characterization of Bacterial Pathogens by Nucleic Acid Amplification
- Authors: K. Loens, H. Goossens, M. Ieven
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Real-time-based platforms currently offer numerous advantages over conventional nucleic acid amplification techniques (NAATs) such as higher speed, less handling of PCR products, decreased risk of false-positive results due to carryover contamination, and the capability to quantify results. This chapter reviews some updates on the detection by NAAT of some relevant individual agents alone, and associated with clinical syndromes, with emphasis on the detection of respiratory agents, particularly the atypical pathogens Mycobacterium pneumoniae, Chlamydophila pneumoniae, Legionella spp., and Bordetella pertussis but also Streptococcus pneumoniae, for which also quantitative real-time amplification is discussed. Given the many alternative amplification protocols proposed for some applications, such studies are clearly needed. Although such studies are often lacking, new-generation molecular techniques are gradually replacing tissue culture and even conventional PCRs as the gold standard for the diagnosis of respiratory infections and for the detection of particular individual bacterial pathogens, as is further reviewed in this chapter. A recent development is the application of PCR for detecting the family of Haemophilus integrating conjugative elements among antibiotic-resistant Haemophilus influenzae type b directly to cerebrospinal fluid (CSF) to diagnose Haemophilus influenzae type b meningitis and predict the organism's susceptibility, irrespective of culture results. The potential of pyrosequencing after broad-range PCR was successfully investigated by Jordan for the identification of bacterial pathogens responsible for sepsis in neonates.
Chapter 24 : Molecular Detection of Chlamydia trachomatis and Neisseria gonorrhoeae
- Authors: Jens K. Møller, Björn Herrmann, Jørgen Skov Jensen, Henrik Westh
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A nucleic acid amplification test (NAAT) may enable the laboratory to detect Chlamydia trachomatis and Neisseria gonorrhoeae with high sensitivity and specificity in traditional urogenital swabs and in different types of samples obtained noninvasively by patients at home or in other settings. This chapter provides guidance in selecting the most appropriate NAATs for C. trachomatis and N. gonorrhoeae among available commercial and in-house assays. The selection of targets for detection of C. trachomatis and N. gonorrhoeae is a major point in determining which assay to use for routine diagnostics. Sequence variation in the target region may lead to false-negative results, whereas the presence of the target gene in other species may lead to false-positive results. Routine diagnostics of C. trachomatis infections is predominantly performed with commercial NAAT high-volume test systems, but there are still applications where in-house-developed methods are useful. The number of samples in a pool depends on the prevalence, and it is calculated from the number of samples, which needs to be tested individually from positive pools. Samples from a negative pool should be reported as negative. The rapid spread of a mutant variant of C. trachomatis in Sweden escaping detection by some NAATs has been a warning that vigilance for drug-resistant mutants should be enforced.
Chapter 25 : PCR Detection of Haemophilus ducreyi, Treponema pallidum, and Mycoplasma genitalium
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This chapter outlines the need for, and application of, molecular tests for Haemophilus ducreyi, Treponema pallidum, and Mycoplasma genitalium, three sexually transmitted diseases (STDs) pathogens for which commercial molecular tests are not currently available. H. ducreyi and T. pallidum are the causative agents of chancroid and syphilis, respectively, which along with herpes simplex virus (HSV) are responsible for the majority of genital ulcer disease (GUD) cases worldwide. The first PCR assays specific for M. genitalium were simultaneously developed in two laboratories and provided a means of detecting M. genitalium in patient specimens. The authors await further application of these quantitative PCR assays to assess other aspects of M. genitalium infection such as the association of M. genitalium burden in cervical, urine, and vaginal specimens and their association with signs and symptoms of infection at these sites. Unlike most PCRs, TMA assays target rRNA, which is present in multiple copies per cell, thus potentially increasing both the analytical and the clinical sensitivity of M. genitalium detection. Similarly, PCR and TMA assays for M. genitalium have allowed studies defining the association of this emerging pathogen with reproductive tract disease in both men and women. Undoubtedly other, previously uncultivated organisms colonizing the reproductive tract will be detected in the future. Their identification and the subsequent development of specific PCR assays and treatment regimens will expand one's knowledge of reproductive tract pathogens.
Chapter 26 : Molecular Detection and Characterization of Mycobacterium tuberculosis
- Author: Betty A. Forbes
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This chapter focuses on nucleic acid amplification based (NAA) assays that detect Mycobacterium tuberculosis directly in clinical specimens; specifics regarding the laboratory diagnosis of infections caused by the nontuberculous mycobacteria are not included. The chapter talks about on NAA assays that detect M. tuberculosis directly in clinical specimens. The salient features of commercially available NAA assays are first briefly described, followed by an overview of in-house-developed assays. Second, similar to commercially available NAA assays, evaluation of in-house NAA assays has used primarily respiratory specimens, but testing has also been performed on nonrespiratory specimens such as pleural fluids, gastric aspirates, formalin-fixed and paraffin-embedded tissues, fresh tissues, cerebrospinal fluids (CSF), and other sterile body fluids. Third, in-house assays have also been developed to detect both M. tuberculosis complex and drug resistance. Finally, other miscellaneous aspects of NAA assays including their use in monitoring response to therapy, cost and cost-effectiveness, and newer approaches and modifications are addressed. Identifications obtained by use of the specific deletion profiles correlated 100% with the original identifications for all M. tuberculosis complex members except M. africanum; further characterization resulted in profiles specific for all members. Subsequent studies using genomic deletions revealed that by testing an isolate for signature deletions, members of the M. tuberculosis complex can be identified.
Chapter 27 : Sequence-Based Identification and Characterization of Mycobacteria
- Authors: Nancy L. Wengenack, Leslie Hall
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Perhaps no other technique has revolutionized the identification and characterization of a single clinically relevant genus as much as the use of sequencing for mycobacterial diagnostics. This chapter provides a comprehensive account of the current use of DNA sequencing for the identification and characterization of mycobacteria in the clinical microbiology laboratory. The problems associated with phenotypic identification of mycobacteria were highlighted in 1996, when Springer and colleagues compared 34 isolates identified by both biochemical testing and 16S rRNA sequencing. There are no FDA-approved platforms at this time designed specifically for the sequence-based identification of microorganisms. However, there are a number of commercially available sequencing platforms, and laboratories are free to establish their own laboratory-developed (home brew) system for mycobacterial sequencing. Molecular determination of drug resistance by sequence analysis has been successfully used for Mycobacterium tuberculosis and, in a limited way, for other mycobacteria. Drug resistance in M. tuberculosis is moderated by mutations in a handful of genes. Direct sequencing remains a challenge due to the paucity of mycobacteria in many specimen types especially in comparison with the amount of other bacteria present in complex matrices like sputum.
Chapter 28 : Molecular Detection of Group B Streptococcus
- Authors: Elizabeth M. Marlowe, Preeti Pancholi
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The advantage direct probe detection offers is a savings of 24 to 48 h in turnaround time (TAT) in detecting group B Streptococcus (GBS). Additionally because the assay is not an amplified molecular assay, there are no template contamination concerns when running the test. Conventional and real-time PCR assays have been developed for the rapid detection of GBS. GBS-PCR tests performed with alternative specimen types including amniotic fluid, neonatal screening swabs, blood, breast milk, urine, and serum have been described in the literature. A rapid and sensitive intrapartum real-time PCR assay offers the advantage of ascertaining the colonization status before delivery. A multiplex PCR-based reverse line-blot hybridization assay and partial sequence of cps gene has been used to compare the distribution of GBS serotypes, serotype III subtypes, and antibiotic resistance-associated genes. Determination of serotype is also epidemiologically important with respect to the proposed development of a preventative conjugate vaccine. Real-time PCR offers a powerful tool for sensitive, specific, and rapid detection of GBS directly from specimens at the time of delivery. It also offers a more sensitive antepartum test with improved TAT over culture.
Chapter 29 : Molecular Detection and Identification of Methicillin-Resistant Staphylococcus aureus
- Authors: Willem van Leeuwen, Alex van Belkum
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This review focuses on the detection and identification of methicillin-susceptible Staphylococcus aureus (MRSA) both from primary microbiological (enrichment) cultures and clinical materials. The current gold standard method to identify S. aureus from cultures is the AccuProbe Staphylococcus aureus Culture Identification Test (Gen-Probe). A major problem in classical MRSA diagnosis is the variable phenotypic expression of the mecA gene-dependent methicillin resistance. With the availability of complete inventories of putative virulence genes, based on whole-genome comparisons, the possibilities for targeted diagnosis will increase in the future. The Velogene Rapid MRSA identification assay is based on a chimeric probe targeting the mecA gene. Pooling of clinical swabs and process automation will reduce the costs in the future. It needs to be emphasized that in principle the issues covered in this review can be extrapolated to species and isolates of each and every other antibiotic-resistant microbial infectious disease agent including, for instance, vancomycin-resistant enterococci. Molecular technology has changed the horizon, and for Chlamydia trachomatis, for instance, molecular detection already is the gold standard technology. That molecular testing will also revolutionize MRSA detection is obvious. It remains to be seen which of the many currently available technologies will in the end be the one that is collectively embraced by the majority of clinical microbiologists.
Chapter 30 : Identification of Bacteria by DNA Target Sequencing in a Clinical Microbiology Laboratory
- Authors: Rosemary C. She, Keith E. Simmon, Cathy A. Petti
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Nucleic acid sequencing of various bacterial genes and other DNA targets has been used for determining the phylogeny of bacteria and for their identification. A brief overview of nucleic acid sequencing is shown in this chapter. DNA targets have conserved regions flanking variable regions that can be used to differentiate closely related bacterial species. The routine use of sequencing can greatly enhance the ability of the clinical microbiology laboratory to identify bacteria on many levels. Of consideration in the routine use of DNA target sequencing is the need for technical expertise and its cost. The chapter addresses preparation of DNA from pure culture. Certain conventional methods such as latex agglutination assays are quicker, simpler, and less expensive than DNA target sequencing for the identification of beta-hemolytic streptococci. Basic conventional methods perform well in identifying common isolates, such as Bacteroides fragilis group, Peptostreptococcus spp., and most Clostridium spp. DNA target sequencing can provide more accurate identifications, especially since databases from conventional methods often are not current and do not reflect the tremendous genetic diversity within anaerobic taxa. For agents of bioterrorism, i.e., Bacillus anthracis, Brucella spp., Clostridium botulinum, Francisella tularensis, and Yersinia pestis, 16S rRNA sequencing has varying utility. Molecular studies have enhanced our knowledge about the taxonomical diversity among bacteria and allowed better definition of the epidemiology of bacterial infections.
Chapter 31 : Broad-Range PCR for Detection and Identification of Bacteria
- Author: Matthias Maiwald
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The two main molecules that are suitable for bacterial broad-range PCR are the 16S rRNA gene, consisting of approximately 1,540 bp (in Escherichia coli, 1,542 bp), and the 23S rRNA gene, consisting of approximately 2,900 bp (in E. coli, 2,904 bp). While the impact of this appears to be somewhat smaller for diagnostic broad-range PCR in cases of suspected monomicrobial infections, it can be dramatic for polymicrobial infections, microbial flora studies, or environmental studies, where certain bacterial taxa may become significantly over- or under-represented after PCR amplification. In addition, the inherent nonselective nature of broad-range PCR makes it susceptible to minute amounts of any bacterial DNA that might be encountered along the various steps of testing. The classical way to detect and identify bacteria by broad-range PCR is via visualization of PCR products by standard gel electrophoresis, followed by sequencing, preferably for both DNA strands of the products. Broad-range PCR offers two potential benefits: it lacks selectivity for particular groups of bacteria, and it can detect as well as identify culture-resistant, fastidious, damaged, and slow-growing microorganisms. Bacterial broad-range PCR has also attracted interest in transfusion medicine, in order to assess blood products for bacterial contamination. Conducting broad-range PCR analysis at a level of high analytical and clinical sensitivity is a complex task and remains one of the most difficult and challenging PCR applications. Sequence-based identification of positive diagnostic broad-range PCR products is generally advisable, even when real-time technology is used.
Chapter 32 : Detection of Antimicrobial Resistance Genes and Mutations Associated with Antimicrobial Resistance in Bacteria
- Authors: Fred C. Tenover, J. Kamile Rasheed
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During the past several years, commercial products for direct detection of antimicrobial-resistant microorganisms, antimicrobial resistance genes, and mutations associated with antimicrobial resistance have been introduced into the clinical laboratory market. In this chapter, a variety of methods of detecting resistant microorganisms are explored and several novel mechanisms of antimicrobial resistance are reviewed. Commercial real-time PCR assays to detect resistant bacteria directly in clinical samples are now used in many laboratories worldwide to detect methicillin-resistant Staphylococcus aureus (MRSA) strains in nasal samples and vancomycin-resistant enterococci (VRE) in stool samples. Detection of mutations associated with resistance can be accomplished by pyrosequencing, line probes, molecular beacons, microarrays, or a variety of other technologies. Phenotypic detection of oxacillin resistance in staphylococci, which is mediated primarily by the mecA determinant, has improved with the substitution of cefoxitin for oxacillin in disk diffusion and MIC testing protocols, particularly with the coagulase-negative strains. While DNA sequencing remains the gold standard for analyzing novel β-lactamase genes, novel microarrays can also be used to identify β-lactamase genes. Additional methods, such as pyrosequencing and microarrays, will soon be available commercially to detect mutations associated with resistance. Thus, rapid detection of antimicrobial-resistant bacteria is likely to become a standard practice in most clinical laboratories, which should have a positive impact on both infection control practices and improvement of anti-infective therapy.
Chapter 33 : Detection of Agents Associated with Bioterrorism
- Authors: Paul Keim, Victor Waddell, David M. Engelthaler
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In this current age of continued threat of large-scale infectious disease outbreaks, whether intentional or natural, the importance of quickly identifying and characterizing the causes of the outbreaks has never been greater. This chapter provides a brief look at the issues and needs related to biothreat detection and the systems and technology developed, or under development, to address them. While anthrax is the most visible and perhaps most likely biothreat disease, others are predicted to have high biothreat potential as well. Real-time PCR has become one of the most widely used tools in diagnostics and biothreat pathogen detection. The focus of confirmatory testing for select agents is real-time PCR technology. BioWatch is an environmental surveillance system set up to continuously monitor air samples for biothreat agents, with the purpose of providing an early-warning system for public policy, safety, and health officials at the local level. There have been a number of positive results reported in the news media, although these are generally classified as background noise from naturally occurring pathogens. There are multiple approaches to next-generation sequencing, but two of the most promising (SOLiD and Illumina GA) are based on very short read lengths that are still suitable for whole-genome genotyping.
VIII. MOLECULAR DETECTION AND CHARACTERIZATION OF VIRUSES
Chapter 34 : Diagnostic Molecular Virology: Current Practice and Future Trends
- Author: Frederick S. Nolte
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Molecular methods are now firmly established as new diagnostic gold standards for most of the clinically important viruses. Molecular methods have changed the face of clinical virology and created new opportunities for laboratories to impact diagnosis and management of patients with viral infections. This chapter discusses the thoughts and impressions on the current practice and future trends in diagnostic molecular virology. Real-time PCR methods have largely replaced conventional, end-point PCR methods in clinical laboratories, with the attendant benefits of speed, broad dynamic range and increased precision for target quantitation, and reduced contamination. Multiplex, real-time amplification methods are limited to three to four different targets by the small number of available fluorescent reporter dyes. These new uses for hepatitis C virus (HCV) viral load measurements provide motivation for patients to complete therapy and permit the individualization of therapy duration. There has also been significant progress in the development of specifically targeted antiviral therapy for hepatitis C, such as NS5 polymerase and NS3/4 protease inhibitors. Phylogenetic genotyping has a more limited role in the clinical management of patients with chronic hepatitis B than it does in patients with chronic hepatitis C .
Chapter 35 : Molecular Detection and Characterization of HIV-1
- Authors: Angela M. Caliendo, Colleen S. Kraft
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This chapter focuses on the available molecular tests for diagnosis, monitoring, and management of HIV-1-infected individuals. It also focuses on molecular methods as they apply to the diagnosis and management of human immunodeficiency virus type 1 (HIV- 1). The guidelines for initiation of therapy based on viral load have changed as one's understanding of disease progression at higher CD4 cell counts has improved. Combination therapy using drugs from multiple classes has been the most effective approach to controlling viral replication. There are several FDA-approved assays for the detection, quantification, and characterization of HIV-1, and this field has expanded recently with the approval of real-time RTPCR viral load tests. A section covers conventional and real-time viral load tests, RNA and proviral DNA tests for the detection of HIV-1, resistance testing, and the tropism assay. The Amplicor monitor test was modified in a study comparing viral load values between the conventional and real-time tests using a different extraction method than recommended by the manufacturer, and it is unclear what impact this may have had on the viral load values. The current recommendations for specimen collection and processing for HIV-1 resistance testing are the same as for viral load testing. Genotypic resistance testing is one of the most complex tests performed in the molecular microbiology laboratory and involves multiple technical steps to generate the sequence as well as interpretation of the sequencing data. Currently, the systems for viral load testing require high capital investment in instrumentation along with reliable electricity and pure water.
Chapter 36 : Molecular Detection and Characterization of Hepatitis C Virus
- Authors: Michael S. Forman, Alexandra Valsamakis
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Hepatitis C virus (HCV) and molecular methods are inextricably linked, in history and in clinical practice. This virus was the first to be identified with molecular methods, and nucleic acid tests (NATs) have become fundamentally important in the diagnosis and therapeutic management of the chronic infections it causes. Rates of spontaneous virus clearance are highest in acutely infected individuals who manifest signs of hepatitis, suggesting that an early immune response may be protective. Risk factors for disease progression include diseases or behaviors that induce additional hepatic injury (such as concomitant hepatitis B virus [HBV] infection and alcohol consumption) or impair antiviral immunity (such as HIV infection). Treatment for HCV infection has undergone significant evolution in regard to available agents, duration, and dosing strategy. Important concepts in regard to testing for acute HCV infection are that (i) serology and RNA assessment should both be performed and (ii) testing should be undertaken at multiple time points before exclusion of the diagnosis. Given the asymptomatic nature of most acute HCV infections, the majority of patients will present symptomatically, in the chronic phase. The approval of STAT-C drugs has the potential to vastly improve treatment efficacy and concomitantly impose a new paradigm for therapeutic monitoring. The effect of telaprevir on the performance of viral load testing and the necessity for additional tests such as resistance assays remains to be determined.
Chapter 37 : Molecular Detection and Characterization of Hepatitis B Virus
- Author: David R. Hillyard
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Hepatitis B virus (HBV) is a small, enveloped DNA virus belonging to the family Hepadnaviridae that causes transient or persistent (chronic) infection of the liver. The appreciation of clinical syndromes of acute and chronic hepatitis laid the foundation for the discovery of each of the human hepatitis viruses. Generation of multiple surface proteins from a single reading frame occurs by translation initiation at separate AUG codons within a transcript rather than by generation and cleavage of precursor proteins (as occurs with hepatitis C virus [HCV]). The focus of molecular testing is instead the characterization, treatment, and management of chronic hepatitis B (CHB) infection. Compared to antibody-based detection methods for other viruses, serologic detection of HBV is relatively sensitive due to overexpression of surface antigen (HBsAg) as noninfectious subviral particles. Nucleic acid testings (NATs) for intrahepatic HBV replication intermediates such as covalently closed circular DNA (cccDNA) have been used to characterize viral replication and evaluate antiviral therapies but are not available for routine clinical use. The earliest examples of commercial HBV NATs were quantitative, reflecting the clinical utility of HBV DNA as a surrogate for measurement of infectious hepatitis B virion burden. Given the extreme genetic variability of the HBV genome, attention will hopefully be focused on development of improved chemistries for accommodation of polymorphisms in viral load assays to reduce the risk of under quantification.
Chapter 38 : Molecular Detection of Human Papillomaviruses
- Authors: Denise I. Quigley, Elizabeth R. Unger
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The importance of human papillomaviruses (HPVs) in biology and medicine was most recently highlighted by the award of the 2008 Nobel Prize in Medicine to Harald zur Hausen in recognition of his discovery of the oncogenic role of HPV in cervical cancer. This chapter provides a brief overview of the biology and natural history of the virus and a description of current clinical applications, laboratory methods, and possible future directions. The major (L1) and minor (L2) capsid proteins make up the viral protein coat. The chapter focuses on the HPV assays that have been used in published studies, been designed to provide clinically relevant information, and are available commercially. The major limitation of targeting the E6/E7 region is the large number of variants, not all of which have been fully described for all types targeted by HPV assays. The Invader HPV16/18 reagents employ probe amplification technology for the Invader HPV screening assay. The research-use-only Linear Array HPV genotyping test is based on PGMY09/11 L1 consensus PCR followed by reverse line probe hybridization for detection of 37 HPV types. It is clear that HPV testing is evolving and many new assays are appearing on the market. Nucleic acid detection assays are often performed in molecular diagnostic or cytology laboratories and necessitate specific training of personnel. The clinical utility of HPV genotyping and implementation of guidelines for appropriate clinical follow-up based on genotype may change the paradigm for HPV testing.
Chapter 39 : Molecular Detection of Respiratory Viruses
- Authors: Richard S. Buller, Max Q. Arens
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This chapter deals with current assays that are commercially available or noncommercial assays that have been well characterized in peer-reviewed literature. Methods for the molecular detection of respiratory viruses have, depending on the capabilities and resources available to the laboratory, a variety of potential clinical applications. Molecular amplification assays for the detection of rhinovirus RNA in clinical specimens often target the 5’ NCR, which contains sequences conserved across the 100 rhinovirus serotypes. In addition to advances in technology, another important step for the future will be the performance of clinical trials to prove the clinical usefulness of respiratory virus panel (RVP) results. Laboratorians and physicians will have to come to terms with co- and multiple infections. Molecular detection of respiratory viruses is currently a rapidly changing field and will likely continue to be for the near future. The good news is that more information about the many and varied causes of respiratory illness are currently acquired and that this information will be an important part of the puzzle in advancing human health.
Chapter 40 : Viral Infections in Transplant Recipients
- Authors: Robin Patel, Frederick S. Nolte
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This chapter discusses the role of nucleic acid tests in the diagnosis and management of herpes simplex virus (HSV), varicella-zoster virus (VZV), human herpesvirus 6 (HHV-6), cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK virus (BKV), parvovirus (erythrovirus) B19, and adenovirus in hematopoietic and solid-organ transplant recipients. HSV most commonly causes reactivation of oral or genital mucocutaneous lesions in transplant recipients. The majority of transplant recipients are VZV seropositive pretransplantation, and at risk for VZV reactivation, most commonly in the form of zoster. Due to the severity of primary VZV infection in transplant recipients, solid-organ transplant candidates should be screened for antibody to VZV prior to transplantation, and consideration should be given to administration of the varicella vaccine prior to transplantation to solid-organ transplant candidates who have no history of prior VZV disease and are VZV seronegative. To complicate the matter, some pediatric liver and heart transplant recipients may exhibit chronic high EBV viral loads. Molecular diagnostics play a paramount role in the diagnosis and management of transplant recipients with infections caused by viruses. These methods have greatly enhanced diagnosis of viral infections due to the increase in speed and sensitivity compared to traditional antigen detection or culture methods. In addition, routine monitoring of transplant recipients for CMV, EBV, and BKV viremia using these methods has become the standard of care at many transplant centers, and this monitoring has facilitated earlier clinical interventions that have dramatically reduced morbidity caused by these viruses.
Chapter 41 : New Virus Discovery in the 21st Century
- Authors: Stacy Finkbeiner, David Wang
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The development of new technologies has propelled virus discovery efforts forward at an unprecedented pace. As this area of research has become increasingly active, a review of virus discovery efforts over the last ~10 years is warranted. This chapter describes the evolution of sequence-independent PCR methods, panviral microarrays, and mass sequencing in the discovery of novel viruses. It also highlights the viruses discovered by these methods, and the importance and novelties of some of these viruses. Sequence-independent PCR strategies and new technologies such as microarrays and mass sequencing, while not without their own limitations, circumvent many of the limitations associated with traditional virus discovery methods and consequently have revolutionized the process of virus discovery. The discovery in 2001 of a novel human pneumovirus, called human metapneumovirus (HMPV), relied upon a sequence-independent PCR approach in conjunction with viral culture to identify a previously unknown virus. The first application of virus discovery based on cDNA-amplified fragment length polymorphism (VIDISCA) to virus discovery resulted in the identification of HCoV-NL63. DNA microarrays first emerged in the mid-1990s as powerful tools to measure gene expression or genomic content changes in various organisms. Dideoxy sequencing or Sanger sequencing, first described in 1977, has been the dominant DNA sequencing technology used during the last ~30 years. With the advent of new technologies, a shift has occurred such that in many instances, the rate-limiting step is no longer discovery but understanding the biological relevance and impact of newly discovered viruses.
IX. DETECTION AND CHARACTERIZATION OF FUNGAL PATHOGENS
Chapter 42 : Molecular Detection and Characterization of Fungal Pathogens
- Author: Stephane Bretagne
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The increasing use of molecular methods for the identification and epidemiological study of fungal pathogens will require accessible and reliable public databases containing comprehensive sequence data. The molecular detection of pathogenic fungi directly in clinical specimens (e.g., blood, tissue, bronchoalveolar lavage [BAL] fluid, and other body fluids) is most often accomplished through PCR-based DNA amplification using Taq polymerase, although alternative methods such as RNA detection or isothermal amplification have also been used. Even with the resolution of such technical issues, there remain several drawbacks in using BAL fluid for the diagnosis of invasive aspergillosis. First, except for in limited studies, BAL is typically performed at a late stage of clinical disease, when the patient already has pneumonia or prolonged fever of unknown origin. Second, some attempts have been made to differentiate carriage, reactivation, and infection for Pneumocystis pneumonia by calculating the ratio of Pneumocystis DNA to human DNA. Among endemic mycoses, genetic probes for culture confirmation of the most commonly encountered dimorphic systemic fungal pathogens, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis/Coccidioides posadasii, have been reported. Genotyping based on multilocus sequence typing (MLST) or microsatellite markers can help one gain insight into the genetic relatedness of fungal isolates. The current trend towards mass sequencing creates the possibility of getting more and more pieces of information on the genome of each microorganism.
Chapter 43 : Sequence-Based Fungal Identification and Classification
- Authors: Jianli Dong, Michael J. Loeffelholz, Michael R. McGinnis
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Identification and classification of fungi from clinical samples are important for antifungal susceptibility testing and epidemiological investigation. Sequence-based molecular techniques are increasingly used in the identification and taxonomy characterization of fungal infections. Phenotypic characteristics used for the recognition and classification of fungi are those that are either easily observed or measured, or a combination of both. An ultimate goal of fungal classification is to draw inferred phylogenetic relationships. In brief, it is sequencing by synthesis, and it is based on the synthesis of cDNA from PCR amplicons. DNA targets that have been used for fungal identification and classification include rDNA, cytochrome b, β-tubulin, calmodulin, enolase, chitin synthase, heat shock protein, and other housekeeping and functional genes. The increased use of gene sequences to recognize different clades within traditional medically important species emphasizes the importance of using sequence data in contrast to species-specific probes to identify particular fungal species. Clinical laboratories are under increasing pressure to provide rapid identification and classification of fungal infections due to the growing number of immunocompromised patients that are susceptible to fungal infection, and the availability of targeted antifungal agents. Sequencing and database comparison of PCR amplicons coupled with phylogenetic methods provide a robust strategy for species recognition, especially for uncommon and emerging pathogenic fungi.
Chapter 44 : Molecular Detection of Antifungal Resistance
- Author: Thomas D. Edlind
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The increasing need for clinically relevant antifungal susceptibility assays is driven by three recent developments in medical mycology: (i) the increasing incidence of fungal infection due to immunosuppression (associated with AIDS, organ and tissue transplantation, and aggressive treatments for cancer and autoimmune disease), (ii) the expanding number of antifungals with both shared and distinct mechanisms of action, and (iii) the recognition of wide variation in susceptibility due to both intrinsic and acquired antifungal resistance. With respect to disease-causing fungi, some of these conditions apply, but clearly in limited and specific ways. First, for most fungal pathogens and antifungals, the development of resistance during treatment is rare. Second, relatively few studies have directly examined the clinical relevance of antifungal susceptibility data. The majority of these mutations involve a single Cyp51A residue, G54, although at least four additional residues have also been implicated. The currently known mutations associated with azole resistance in Candida albicans Erg11 and Aspergillus fumigatus Cyp51A are dispersed over 1.2 kbp of primary sequence. Echinocandins (caspofungin, micafungin, and anidulafungin) are the most recently introduced class of antifungals but are highly promising due to their mechanism-based selective toxicity. A great deal has been learned in recent years regarding molecular mechanisms of antifungal resistance, particularly in the yeasts C. albicans and C. glabrata and the mold A. fumigatus. In addition, simple and cost-effective approaches to measuring RNA expression and sequencing DNA are required.
X. DETECTION AND CHARACTERIZATION OF PARASITIC PROTOZOA
Chapter 45 : Overview of the Development, Utility, and Future of Molecular Diagnostics for Parasitic Diseases
- Authors: Bobbi Pritt, Jon Rosenblatt
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Molecular diagnostics are undoubtedly changing the practice of modern clinical parasitology. Molecular assays have been developed for virtually all parasites causing clinical infections in humans. This chapter focuses on the techniques that have made the greatest impact in the clinical parasitology laboratory to date, namely, monoplex and multiplex real-time PCR. To illustrate the wide-ranging applications of clinical molecular parasitology, the chapter examines the large body of work on malaria. Conventional and real-time PCR assays for the Plasmodium species causing human malaria are now available in several reference laboratories and may be used as stand-alone tests or in conjunction with microscopy for species identification, quantification, and detection of some mixed infections. Serology is widely used but may be of limited utility in circumstances where it is critical to make an accurate diagnosis. Despite the recent progress in molecular diagnostics, there are significant limitations that hinder widespread adoption in the clinical parasitology laboratory. The majority of clinical laboratories do not have the expertise to develop in-house tests and must rely on commercially available assays.
Chapter 46 : Molecular Approaches for Diagnosis of Malaria and Characterization of Genetic Markers of Drug Resistance
- Authors: Laura K. Erdman, Michael Hawkes, Kevin C. Kain
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The diagnosis of imported malaria is complicated by the lack of experience with traditional microscopic detection methods and expertise with clinical malaria among laboratory professionals and clinicians, respectively. The development of powerful molecular techniques has led to extensive genetic and proteomic characterization of the malaria parasite. This chapter reviews the application of these molecular approaches for the improvement of malaria diagnosis and detection of genetic markers of antimalarial resistance. Real-time PCR assays for malaria diagnosis offer advantages in several of these respects. SNP analysis by mass spectrometry has been applied to the characterization and surveillance of various pathogens, including malaria parasites. Molecular methods not only are useful for detecting known resistance markers in parasite isolates but also may identify novel resistance markers. Another approach is based on the observation that drug resistance loci often have little sequence variation in surrounding genomic regions; these regions may not have yet been diversified by genetic drift if the resistance locus was recently acquired, or they may be essential for the resistance phenotype. The power of molecular technologies has led to improvements in the accuracy and rapidity of malaria diagnostic tests and detection of antimalarial resistance markers. Importantly, high-quality artemisinin combination treatments (ACTs) must be made accessible and affordable in regions where malaria is endemic, which will reduce demand for ineffective antimalarials and counterfeit drugs and preserve ACT efficacy.
Chapter 47 : Molecular Approaches for Diagnosis of Chagas’ Disease and Genotyping of Trypanosoma cruzi
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Molecular approaches have been shown to be valuable tools contributing to the diagnosis of congenital Trypanosoma cruzi infection and to the follow-up of infected patients having received trypanocidal drugs, as well as for identifying lineages and sublineages of T. cruzi. In the first part of this chapter molecular analyses are considered as diagnostic tools at the individual and/or epidemiological level; this is followed, in the second part, by detailed protocols for detecting T. cruzi DNA using qualitative PCR and quantitative PCR (qPCR) as well as identifying (sub)lineages by hybridization with specific DNA probes. PCR could help in the diagnosis of T. cruzi infection/Chagas’ disease or in the management of infected patients in three main cases: (i) the diagnosis of congenital infection, (ii) the early detection of a reactivation of Chagas’ disease in immunosuppressed patients, and (iii) the follow-up of infected patients treated with antiparasitic drugs. PCR, particularly qPCR, was also demonstrated to be useful for the early diagnosis of Chagas’ disease reactivation in immunosuppressed patients. The detection of DNA is made with the SYBR Green fluorophore, an intercalating dye, fluorescing only when bound to dsDNA. The fluorescence of each sample is measured after each cycle. The SYBR Green signal measured is proportional to the dsDNA synthesized de novo in the course of the PCR cycles. High specificity of amplification is mandatory and must be assessed at the end of the amplification by electrophoresis or by analysis of the melting curves of the amplicons.
XI. SYSTEMS MICROBIOLOGY
Chapter 48 : Molecular Diagnosis of Gastrointestinal Infections
- Authors: Benjamin A. Pinsky, Niaz Banaei
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Enteric infections cause significant morbidity and mortality and have a significant financial cost worldwide. This chapter discusses the molecular techniques for the clinical identification of the bacteria, viruses, and parasites most commonly responsible for infections of the lower gastrointestinal tract. Numerous studies have reported on the development of PCR assays for the detection of enteric pathogens. Overall, multiplex, real-time PCR assay is favored because of its speed and elimination of postamplification carryover. The chapter summarizes the advancements achieved for the detection of enteric bacterial pathogens by molecular methods and discusses the remaining challenges. It also discusses the special case of hospital-acquired Clostridium difficile infection and the role of molecular methods in its diagnosis. Except for Plesiomonas shigelloides, which causes disease only in some patients, Salmonella, Shigella, and Yersinia enterocolitica are intrinsic enteric pathogens in humans. Importantly, the sequence of the viral capsid protein encoded by open reading frame 2 (ORF2) correlates directly with the serotype, making RT-PCR the most common technique for astrovirus detection and typing. The major pathogenic intestinal coccidia include Cryptosporidium parvum, Cryptosporidium hominis, Cyclospora cayetanensis, and Isospora belli, with Cryptosporidium spp. being the most common. The microsporidia are composed of more than 1,000 species of small, spore-forming, obligate intracellular organisms originally classified as protozoa but now considered fungi.
Chapter 49 : Molecular Approaches to the Diagnosis of Sepsis
- Authors: Paul H. M. Savelkoul, Remco P. H. Peters
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This chapter summarizes the pros and cons of such alternative methods for detection of microorganisms in blood and discusses the perspectives of rapid molecular diagnosis of sepsis. Various methods have been assessed to reduce the time required for identification of microorganisms in blood cultures, including hybridization techniques, PCR-based applications, and spectrometric analysis. Among the hybridization techniques suited to identification of microorganisms in blood cultures are fluorescence in situ hybridization (FISH) and chemiluminescent probe matrices. To further reduce time to diagnosis of bloodstream infection (BSI), molecular methods may be applied directly to blood samples, without prior cultivation of microorganisms. Determination of susceptibility to antimicrobial treatment in sepsis is currently performed on cultured strains by determination of the MICs of a broad spectrum of different antibiotics. Current methods of molecular detection of sepsis concentrate on detection of DNA of the microorganism in the blood by both qualitative and quantitative approaches. In addition, alternative DNA extraction methods and multiplex PCR approaches are a first step towards point-of-care testing. For rapid detection of sepsis this is a promising approach, but a real implementation remains to be seen. Rapid molecular detection of the causative pathogen of sepsis is within reach, but many studies are required before clinical implementation. Several techniques are being tested for this application, but the best technique has not been determined. In general, all these promising tests will add to but not replace conventional blood culture as long as phenotypic susceptibility testing is needed.
Chapter 50 : Molecular Approaches to the Diagnosis of Meningitis and Encephalitis
- Authors: Karen C. Bloch, Yi-Wei Tang
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This chapter reviews various diagnostic tests available for central nervous system (CNS) infections and provides a detailed discussion of specific molecular approaches to the most common organisms causing meningitis and encephalitis in the United States. A vast array of organisms have been associated with encephalitis, including bacteria, fungi, viruses, and protozoa. Just as meningitis and encephalitis localize to distinct compartments of the CNS, the pathogens causing these two syndromes differ, and the respective microbiologies of meningitis and encephalitis are discussed separately. While the chapter focuses on the use of molecular assays for diagnosis of CNS infection, traditional microbiological diagnostic approaches continue to play an important role in pathogen identification and are complementary to nucleic acid amplification techniques. A DNA probe array has been developed for the simultaneous identification of herpesviruses, enteroviruses, and flaviviruses after several PCR amplifications. Nucleic acid amplification techniques have markedly improved the identification of CNS infections caused by viral and fastidious bacterial pathogens. Molecular techniques such as PCR allow rapid diagnosis, with consequent improvement of outcomes and cost savings. Results of molecular diagnostic testing for CNS infections must be interpreted in the context of the individual patient presentation and clinical illness, and close cooperation between the laboratory and the clinician is required for optimal use of these technologies.
XII. THE PATHOGEN/HOST INTERFACE
Chapter 51 : Genomics and DNA Variation: Determinants of Susceptibility and Outcomes in Microbial Diseases
- Authors: Chiea-Chuen Khor, Stephen J. Chapman, Adrian V. S. Hill
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Microbial infection has been one of the leading contributors to morbidity and mortality throughout human history. Even today, diseases such as bacterial pneumonia, cholera, diphtheria, plague, tuberculosis (TB), typhoid fever, and typhus continue to exact their toll, particularly in developing countries. Studies of genetic susceptibility to microbial disease have largely focused on candidate genes. In most cases, the genetic determinants for susceptibility to particular infectious diseases appear to be confined to very specific pathogen-associated molecular patterns (PAMPs). Studies on the host genetics of viral infections have not been limited to the analysis of susceptibility to disease. In the case of HIV infection, the most frequently studied outcomes are mortality following HIV and progression from seroconversion toward full-blown AIDS. The challenge of identifying TB susceptibility loci is substantial, as there is considerable difficulty in correctly classifying TB cases and controls due to the phenomenon of "latent" TB, which ultimately leads to significant loss of statistical power and underestimation of genetic effect sizes. The importance of mannose-binding lectin (MBL) in limiting fungal infections is clear even in small clinical studies; haplotypes resulting in low serum MBL levels confer marked susceptibility to invasive aspergillosis. The immediate future of genetic susceptibility studies of infectious diseases is clear. The elucidation of novel pathways in susceptibility or resistance to malaria and bacteremia could help in the development of more-efficacious vaccines in the distant future.
Chapter 52 : Genetically Polymorphic Cytochrome P450s and Transporters and Personalized Antimicrobial Chemotherapy
- Author: Hong-Guang Xie
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This chapter summarizes a number of functionally important genetic polymorphisms in drug-metabolizing enzymes and drug transporters and their genotyping methodologies, by which individuals susceptible to drug toxicity or loss of efficacy can be identified or predicted easily in the clinical setting. The human cytochrome P450 (CYP) enzyme system contributing to drug metabolism comprises the CYP families CYP1, CYP2, and CYP3. Among these CYPs, proteins encoded by the human CYP3A genes catalyze the metabolism of nearly half of all currently used drugs, and approximately 40% of all marketed drugs are substrates for CYP2C9, CYP2C19, and CYP2D6. Moreover, the CYP2B6, CYP2C9, CYP2C19, and CYP2D6 enzymes exhibit large variations in the levels of their protein expression and enzymatic activity as a result of frequently occurring, functionally important genetic polymorphisms. Typically, in addition to passive diffusion, a drug is transported into cells by uptake (or influx) transporters, such as organic anion transporters (OATs), organic anion transport peptides (OATPs), and organic cation transporters (OCTs), whereas the drug and its metabolite(s) are removed from the cells by efflux transporters, such as P-glycoprotein (P-gp), multidrug resistance proteins (MRPs), and breast cancer resistance protein (BCRP). Anti-infective drugs may be substrates for some uptake transporters, drug-metabolizing enzymes, and efflux transporters.
Chapter 53 : Host Genomic Profiling in Human Immunodeficiency Virus Infection
- Authors: Kevin V. Shianna, Amalio Telenti
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Host data can be presented under the concept of genetics of candidate genes, genome-wide studies, and the more specific areas of comparative and evolutionary genetics, and pharmacogenetics. A parallel development is taking place in the field of pharmacogenetics that aims at the identification of genetic variants that modulate drug response, pharmacokinetics, and toxicity. This chapter emphasizes the technical and technological possibilities in genetics and genomics for the study of host susceptibility to human immunodeficiency virus type 1 infection. Study phenotypes should be easily measurable and reliable (i.e., low intraindividual variability), but variable (i.e., high interindividual variability). In the HIV field these include standardized quantitative traits, such as viral load, CD4 T-cell count, and other biomarkers of disease. Clinical endpoints are best studied if they are not the composite result of multiple biological and environmental factors. All essentially involve a set of probes on an array and require amplification and labeling of cDNA/cRNA followed by hybridization to the probes on the array. Recently, groups have started to perform sequencing experiments on the entire transcriptome. This approach is similar to sequencing an entire human genome, except the input is cDNA rather than genomic DNA. It is too early to measure the full potential of large-scale genome research in HIV-1 infection. However, some general principles can be drawn. The first aspect relates to the fact that the human genome is a canvas that provides reference to multiple types of data. The second aspect concerns the use of new knowledge.
Chapter 54 : Innate Immunity and Host Defense against Microbial Infection
- Authors: Markus Schnare, Salman Qureshi
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A principal function of the innate immune system is to perform broad-based surveillance for abnormal interactions between microbes and their hosts. Activation of innate immunity rapidly triggers a series of direct antimicrobial host defenses while simultaneously generating instructive signals, including chemokine and cytokine gene induction, that alert and mobilize a definitive adaptive immune response. Comparative genomic sequencing has revealed remarkable conservation of Toll-like receptors (TLRs) among diverse vertebrates and underlines a strong selective pressure to recognize pathogen-associated molecular patterns (PAMPs). Adaptive immunity relies on the great variability and capacity for random somatic rearrangement of gene segments to provide narrow, specific recognition of the molecular details of foreign antigens, as exemplified by antigen receptors expressed by B and T cells. Pattern recognition receptors (PRRs) are an expanding group of highly conserved molecules that detect the presence of pathogenic microbes. PRRs are transmitted to subsequent generations through the germ line and consist of various combinations of conserved modular domains that have broad specificities for structures that are unique to microbes termed PAMPs. TLRs are among the best-characterized PRRs and represent an essential first line of host immune defense. Several fungal PAMPs, including phospholipomannan and glucuronoxylomannan, activate MyD88-dependent host defense pathways, while beta-glucan, a major carbohydrate polymer of fungal cell walls, is recognized by Dectin-1, a member of the myeloid cell-expressed, NK cell receptor-like C-type lectin family. Rapid and dramatic advances in the field of innate immunity have considerable implications for the understanding of human immunodeficiency and susceptibility to infection.
XIII. LABORATORY STANDARDIZATION, PROFICIENCY-TESTING PROGRAMS, QUALITY-CONTROL STANDARDS, AND MONITORING
Chapter 55 : Molecular Method Verification
- Authors: Donna M. Wolk, Elizabeth M. Marlowe
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This chapter talks about aspects of molecular method verification as well as the common statistical analyses that are used during this process. Stevenson et al. showed how polymorphisms in the probe target can affect the performance of an herpes simplex virus (HSV) real-time PCR assay. Such polymorphisms can result in decreased sensitivity of an assay, which is critical for a cerebrospinal fluid (CSF) specimen. In this study using the crossing threshold (CT) as a cutoff for assay sensitivity, it was estimated that as many as 15% of the HSV type 1 (HSV-1)-positive and 7% of the HSV-2-positive specimens would be missed when comparing two real-time HSV PCRs based on commercially available analyte specific reagents (ASRs). The author's clinical laboratory research is, in essence, translational research, in which their purpose is to describe, explain, predict outcomes, and control their systems. The chapter focuses on those aspects of biostatistics that are relevant to the verification and validation of new laboratory assays. Clinical Laboratory Improvement Act (CLIA) has clear guidelines on the verification and validation of laboratory-developed tests (LDTs) and other non-FDA-approved/ cleared assays. Choices of statistical methods and methods for drawing conclusions on the accuracy are less defined. Analysis of method verification data may require collaboration with a statistician or use of software specifically designed for CLIA method verification such as EP Evaluator.
Chapter 56 : Molecular Test Validation, Monitoring, and Quality Control
- Author: Matthew J. Bankowski
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Test validation is the ongoing process of ensuring that the expected performance of an assay is consistently met in testing clinical specimens. Test (or assay) validation is an integral part of quality assessment (QA), which includes quality control, quality improvement, and method validation. QA encompasses routine quality control, proficiency testing, technical staff competency, instrument calibration, and clinical correlation. Quality management of molecular testing begins with the test request and continues through specimen collection, transport, processing, analytical testing, result generation, result review, test interpretation, and reporting. This chapter focuses mainly on the analytical phase of testing. The types of molecular testing included in the chapter are qualitative, quantitative, multiplex, and microarray methods. Quantitative molecular testing consists of numeric values with defined units in the test result. Quality assurance includes quality control, quality improvement, and method validation. Validation of the analytical phase not only includes ensuring adequate and acceptable training on the test but also relies upon evaluation by actual observation of the technologist performing the test on a recurrent basis (operator competency assessment). This should include validation of staff adherence to the standard operating procedure (SOP) exactly as stated, biosafety, patient confidentiality, result interpretation, reporting, and quality control documentation. The use of quality controls, proficiency testing, and monitoring of technical staff competency and equipment and instrument performance are all essential parts of this process.
Chapter 57 : External Quality Assessment and Proficiency Testing in Diagnostic Molecular Microbiology
- Author: Michael J. Mitchell
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Proficiency testing (PT) and external quality assurance/assessment (EQA) are considered interchangeable terms; the generally accepted international terminology is ‘‘proficiency testing,’’ which is used in this chapter. The requirement for PT, as discussed in the chapter, applies to the Centers for Medicare and Medicaid Services (CMS)-accredited laboratories and pertains to nonwaived testing. Laboratory-specific aspects of the protocols should include detailed information regarding specimen evaluation, methods and reagents used for sample reconstitution and testing, maximum interval between reconstitution and testing, storage of excess sample, documentation. In order for PT to be relevant to the quality of patient care testing, PT challenge specimens must be tested in the same manner as patient specimens, including the processing, method and extent of testing, and personnel assigned to perform the testing. Among the microbiology surveys, there are pathogen-specific surveys specifically challenging molecular diagnostic strategies, including qualitative detection and identification, quantification, and genotyping. Proficiency testing, along with other Clinical Laboratory Improvement Amendments (CLIA) quality requirements, provides laboratories and accreditation organizations the tools needed to assess and improve the quality of diagnostic testing. The collaborative efforts of external advocacy groups, professional organizations and societies, governmental and nongovernmental agencies, and commercial providers of quality assurance products should result in increasing improvements to the technical quality and clinical utility of molecular diagnostic testing for infectious diseases. These improvements should result in improvements in patient safety and clinical outcomes as well as the efficiency of diagnostic testing for laboratories and health care systems.
Chapter 58 : Laboratory Controls and Standards
- Author: Maurice Exner
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Growing numbers of microbiology laboratories are turning to the wide array of molecular technologies and platforms that are becoming available. With these new systems comes the need for effective ways to gauge their accuracy and reproducibility. This chapter describes different types of controls, along with their design and their potential utility in molecular microbiology applications. Control materials may take the form of run controls, calibrators, or standards. Run controls are used in a number of different embodiments to monitor overall assay performance and to estimate analytical uncertainty. Regardless of the type of control or the final application, an ideal control, calibrator, or standard would be composed of a matrix that is similar to the specimen matrix being tested, and it would contain the agent being targeted in the intact, biological form in which it naturally occurs. While assay-ready control materials can be purchased from a number of vendors, the number of organisms and assays for which they are available is limited. Raw-material components for control construction are available for many analytes, but to use these materials, each laboratory performing a test would have to manufacture its own controls and design reaction specifications to detect the control targets. The concept of a range would imply use for quantitative assays only; however, even qualitative molecular detection methodologies often provide results that lend themselves to some type of quantitative or semiquantitative analysis.
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The Quarterly Review of Biology
04 June 2013
Nucleic acid amplification and related molecular techniques play an increasingly important role in diagnostic microbiology laboratories. Molecular methods are widely used in viral load assays and to detect selected bacteria; many of these tests are now performed with commercial kits.These trends are reflected in the second edition of Molecular Microbiology. The first six sections provide a good overview of molecular techniques, including sample preparation, amplification and detection, strain typing, and new technologies. The remainder of the book focuses on applications in clinical microbiology.
The chapters on HIV, Hepatitis B, and Hepatitis C provide solid reviews on the role of viral load assays in patient management and discuss the current generation of commercial assays as well as methods for detecting mutations that confer resistance to antiviral agents. The chapter on respiratory viruses provides an excellent review of this complex group of organisms and the challenge of developing multiplex assays that simultaneously detect many pathogens, a topic that became particularly important after the emergence of a pandemic strain of H1N1 influenza virus in 2009. Other noteworthy chapters include a discussion of the role of DNA sequencing in bacterial identification and an excellent review on central nervous system infections. The tests discussed in the sections on mycology, parasitology, and host/pathogen interactions are mostly at the investigational stage, although fungal identification by sequencing and amplification tests for malaria are now available in many reference laboratories.
In recent years there has been a marked increase in regulatory oversight of laboratory-developed tests. The terms “verification” and “validation,” which were not listed in the index of the first edition, now have their own chapters and detail the steps required to insure that molecular tests produce accurate results. This volume will be useful for residents, fellows, and laboratory directors who are performing molecular assays or need to help others interpret the results.
The Quarterly Review of Biology
Reviewer: Eric D. Spitzer, Pathology, Stony Brook University, Stony Brook, New York
Review Date: December 2012
Doody's Review Service
02 June 2013
At A Glance
The second edition of Molecular Microbiology: Diagnostic Principles and Practice presents the latest molecular diagnostic techniques to support clinical care and basic and clinical research. The authors all experienced researchers and diagnosticians have conducted a comprehensive review and evaluation of this rapidly evolving field and provide the new material in an easy-to-read summary format. Moreover, the book offers a broad range of practical advice, such as determining the appropriate type and quantity of a specimen, releasing and concentrating the targets, and eliminating inhibitors.
Molecular Microbiology not only examines techniques to detect individual pathogens, but also explores the growing trend toward a systems approach for diagnosing infectious diseases, with chapters covering gastrointestinal infections, sepsis, meningitis, and encephalitis. In short, this text not only encapsulates the current state of the science, but also points to new avenues for research that will broaden the application and usefulness of molecular diagnostics.
New to the Second Edition
This edition offers the most current findings, practices, and technology. There are new chapters on microbial phylogeny, nucleic acid sequence analysis, and pharmacogenomics. The book also features several newly updated sections, including sections dedicated to verification and validation of molecular tests, laboratory operations, molecular typing, and new technologies for nucleic acid detection. In addition, the reader will find the latest information on quality control, quality assurance, and laboratory design to help with running a safe, efficient molecular laboratory.
Key Features
· Presents the latest basic scientific theory underlying molecular diagnostics
· Offers tested and proven applications of molecular diagnostics for the diagnosis of infectious diseases
· Illustrates and summarizes key concepts and techniques with detailed figures and tables
· Advises on the latest quality control and quality assurance measures
· Explores emerging technologies, including the use of molecular typing methods for real-time tracking of infectious outbreaksDescription
With its presentation of the components for using molecular techniques in the diagnostic laboratory, this book is certain to become a well-used standard. There has been a great deal of change in this area since the last edition was published 10 years ago.
Purpose
The book is a source of accurate information on the molecular techniques and practices needed to perform these tests in a diagnostic laboratory. This is an important goal at a time that more clinical laboratories are moving toward molecular testing methods.
Audience
It is designed for clinical laboratory personnel, including doctoral-level directors, pathologists, other physicians, and technical staff. The authors are all accomplished laboratory scientists with many years of experience in the field.
Features
Some of the detailed information this book presents on nucleic acid amplification techniques includes details on instruments and chemistries used to detect amplified product, dyes used for detection, how temperature is used in the process, and different types of probes. There is also an abundance of information on nucleic acid isolation. Several chapters include an appendix with resource information including databases. The largest section includes individual chapters on the detection of specific infectious agents such as monitoring the level of HIV in a patient before and during treatment, detection of methicillin resistant Staphylococcus aureus, and simultaneous detection of multiple respiratory viruses. Each of these chapters includes information on how this test is used in clinical medicine and tips on implementation. Another useful section is the last one, which gives guidance on controls and standards needed in each assay, verification studies, validation and continuous assessment of these diagnostic as ways. These are issues that continue to be of great importance to the development and performance of an accurate, useful diagnostic test.
Assessment
This book should be in any diagnostic laboratory that provides molecular tests for infectious agents. It contains valuable information that is difficult to find in one source.
Doody's Review Service
Reviewer: Rebecca Horvat, PhD, D(ABMM) (University of Kansas Medical Center)
Review Date: Unknown
©Doody’s Review Service
