Application and Integration of Omics-powered Diagnostics in Clinical and Public Health Microbiology

Various “omics” methods have recently revolutionized molecular diagnostics. Next-generation sequencing (NGS) makes it possible to sequence a human genome in just one day. Whole genome sequencing (WGS) greatly improves the ability to investigate the outbreaks of numerous pathogens. Metagenomics helps to analyze the microbiome, which aids greatly in identifying the pathogenesis of infectious diseases. Proteomic-based methods, namely matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF-MS), have a promising role in identifying myctobacteria and fungi, and predicting antimicrobial resistance. While there are numerous scientific publications on “omics” applications for microbiology, there are relatively few books that review this topic from a clinical diagnostics perspective. This book looks at this field from a holistic viewpoint, instead of limiting by type of “omics” technology, in order to cover the body of knowledge needed for practitioners and academics interested in clinical and public health microbiology. Additionally, it addresses the management, economical, regulatory and operational aspects of integrating these technologies into routine diagnostics. Contents Chapter 1: Introduction to Advanced Diagnostics in Microbiology 1.1 Introduction 1.2 Exploring Novel Diagnostic Techniques 1.3 Introduction to NGS 1.4 Summary and Book Outline References Chapter 2: Overview of Microbial NGS for Clinical and Public Health Microbiology 2.1 Introduction 2.2 Implementation of NGS in Clinical Microbiology Laboratories 2.3 Whole-Genome Sequencing 2.3.1 Outbreak Management and Infection Prevention within the Hospital 2.3.2 Outbreak Management and Infection Prevention within the Region 2.3.3 Transmission of Zoonotic Microorganisms Between Animals and Humans 2.3.4 Antimicrobial Resistance Characterisation Through NGS 2.4 Metagenomics 2.4.1 Amplicon-Based Metagenomics of the 16S–23S rRNA Encoding Region 2.4.2 Shotgun Metagenomics for the Identification and Typing of Microbial Pathogens 2.4.3 Shotgun Metagenomics to Characterise the Gut Microbiome/Resistome 2.5 Clinical Impact of NGS References Chapter 3: WGS for Bacterial Identification and Susceptibility Testing in the Clinical Lab 3.1 Issues to Consider for the Incorporation of WGS in the Routine Workflow of the Clinical Lab 3.2 Bacterial Identification by WGS in the Clinical Laboratory 3.3 Susceptibility Testing by Whole Genome Sequencing 3.4 WGS for the Identification and Susceptibility Testing of Mycobacteria 3.5 Conclusions References Chapter 4: Whole-Genome Sequencing for Bacterial Virulence Assessment 4.1 Introduction 4.1.1 Toxins 4.1.2 Secretion Systems and Their Effectors 4.1.3 Adhesive Factors 4.1.4 Invasive Factors 4.1.5 Resistance to Reactive Oxygen and Nitrogen Species 4.1.6 Immune System Escape 4.1.7 Nutrient Uptake 4.2 Clinical Applications 4.3 Methods and Procedures 4.3.1 Sequencing 4.3.2 Software and Pipelines 4.3.3 Databases 4.3.4 Ontologies 4.3.5 Further Developments 4.4 Interpretation, Validation and Impact 4.5 Future Perspectives References Chapter 5: Epidemiological Typing Using WGS 5.1 Basic Concepts of Bacterial Typing 5.1.1 General Concepts 5.1.2 Purpose/Applications of Typing 5.1.3 Variability 5.2 Established Typing Methods 5.2.1 Pulsed-Field Gel Electrophoresis (PFGE) 5.2.2 Repetitive Extragenetic Palindromic Sequence-Based PCR (REP-PCR) 5.2.3 Variable Number of Tandem Repeat (VNTR) Typing 5.2.4 Amplified Fragment Length Polymorphism Typing (AFLP) 5.2.5 Single Locus Sequence Typing 5.2.6 Multilocus Sequence Typing 5.2.7 Plasmid Typing 5.2.8 Proteomic Alternatives for Typing 5.3 Whole-Genome Sequencing for Typing 5.3.1 Whole-Genome Sequencing (WGS) 5.3.2 SNP Based Versus Allele-Based Approaches 5.3.3 Nomenclature of WGS Data 5.3.4 The Accessory Genome 5.3.5 Backwards Compatibility with Established Methods 5.3.6 Variability and Comparability of WGS Data 5.4 Typing for Infection Control 5.5 Future Perspectives References Chapter 6: Next-Generation Sequencing in Clinical Virology 6.1 Metagenomic Sequencing for Pathogen Detection and Discovery 6.1.1 Pathogen Detection 6.1.2 Virus Discovery 6.2 Virome Analysis 6.2.1 The Human Virome in Different Body Compartments 6.2.2 The Gut Virome 6.2.3 The Blood/Plasma Virome 6.2.4 The Oral and Respiratory Tract Virome 6.2.5 The Genitourinary Virome 6.2.6 The Skin Virome 6.3 Antiviral Susceptibility and Resistance 6.3.1 Herpesviruses 6.3.2 Influenza Viruses 6.3.3 Hepatitis Viruses 6.3.3.1 Hepatitis B Virus (HBV) 6.3.3.2 Hepatitis C Virus (HCV) 6.3.4 Human Immunodeficiency Virus (HIV) 6.4 Future Perspective References Chapter 7: Metagenomic Applications for Infectious Disease Testing in Clinical Laboratories 7.1 Introduction 7.2 Clinical Need for Advanced Testing 7.3 Test Design and Development 7.3.1 Pre-Analytic Factors 7.3.2 Specimen Preparation 7.3.2.1 Nucleic Acid Extraction 7.3.2.2 Pathogen Enrichment 7.3.2.3 Library Preparation 7.3.2.4 Sequencing 7.3.3 Sequence Analysis 7.3.3.1 Sequence Analysis Tools 7.3.3.2 Organism Classification and Result Interpretation 7.3.3.3 Identifying Contamination 7.3.3.4 Result Interpretation 7.3.3.5 Approach to Test Validation 7.3.4 Quality Management 7.3.4.1 Quality Control and Assessment 7.3.4.2 TAT 7.4 Remaining Challenges for NGS in Clinical Diagnostics 7.4.1 Sample Processing 7.4.2 Sequencing and Data Analysis 7.4.3 Test Utilization 7.4.4 Incidental Findings 7.5 Conclusions References Chapter 8: Integrating Metagenomics in the Routine Lab 8.1 Introduction 8.2 Sample Preparation 8.2.1 Human DNA Removal 8.2.1.1 Pre-extraction Depletion 8.2.1.2 Post-extraction Enrichment/Depletion 8.2.2 Direct Sequencing Approaches 8.2.2.1 Use of Whole Genome Amplification for Low-Input Samples, Rare or Single Cells 8.2.2.2 Direct Sequencing of Circulating Cell-Free DNA 8.2.3 Targeted Sequencing or Selective Sequencing 8.2.3.1 On-Target Sequencing (Bait-Capture Approach) 8.2.3.2 Selective Sequencing: The ‘Read Until’ Approach 8.3 Bioinformatic Challenges 8.4 Examples of CMg Applications (Table 8.1) 8.4.1 Bone and Joint Infections (BJI) 8.4.2 Blood Samples 8.4.3 Respiratory Samples 8.4.4 Central Nervous System Infections 8.4.5 Urinary Tract Infections 8.5 Challenges in CMg Implementation 8.6 Future Perspectives References Chapter 9: Advanced Applications of MALDI-TOF MS – Typing and Beyond 9.1 General Introduction 9.1.1 MALDI-TOF MS Functions and Workflow 9.1.2 MALDI-TOF MS Based Identification Process 9.1.3 Resolution of MALDI-TOF MS for Typing 9.2 Current Challenges for Species Identification 9.3 MALDI-TOF Mass Spectrometry-Based Typing 9.4 Different Typing Approaches 9.4.1 Empirical Identification of Marker Peaks 9.4.2 Prediction of Marker Peaks from Bacterial DNA Sequences 9.4.3 Expansion of the Mass Range to Detect Marker Peaks Otherwise Not Accessible 9.4.4 Self-Learning Classification Algorithms 9.5 Sample Preparation Methods Used for Typing 9.6 Examples 9.6.1 Methicillin-Resistant Staphylococcus aureus 9.6.2 Escherichia coli 9.6.3 Clostridioides difficile 9.6.4 Salmonella spp. 9.7 Future Perspectives References Chapter 10: Advanced Applications of MALDI-TOF: Identification and Antibiotic Susceptibility Testing 10.1 Introduction 10.2 Identification of Microorganisms Directly from Clinical Samples (Fig. 10.1) 10.3 Proteomic Approaches to Detect Antibiotic Susceptibility by MALDI-TOF MS 10.3.1 Detection of Antibiotic Susceptibility by Measuring Enzymatic Activity in MALDI-TOF MS 10.3.1.1 Detection of β-Lactamase Activity 10.3.1.2 Detection of Resistant Strains Directly from Clinical Samples 10.3.1.3 Detection of the AAC(6′)-Ib-Cr Enzyme 10.3.2 Detection of Antibiotic Susceptibility by MALDI-TOF MS through Other Techniques 10.4 Automation in Clinical Practice References Chapter 11: Fourier Transform Infrared Spectroscopy (FT-IR) for Food and Water Microbiology 11.1 Introduction 11.2 Fourier-Transform Infrared (FT-IR) Spectroscopy in Microbiology: An Overview 11.3 Principles of FT-IR Spectroscopy Applied to Bacterial Cells 11.4 Experimental Details and Data Analysis 11.5 FT-IR Based Typing at the Species and Infra-Species Level of Bacteria in Food and Water Microbiology 11.5.1 Bacillus sp. 11.5.2 Listeria sp. 11.5.3 Staphylococcus sp. 11.5.4 Lactobacillus sp. 11.5.5 Enterococcus sp. 11.5.6 Other Gram-Positive Bacteria 11.5.7 Escherichia sp. 11.5.8 Salmonella sp. 11.5.9 Yersinia sp. 11.5.10 Campylobacter sp. 11.5.11 Legionella spp. 11.5.12 Vibrio sp. 11.5.13 Mycobacterium sp. 11.6 Future Perspectives References Chapter 12: Omics for Forensic and Post-Mortem Microbiology 12.1 Introduction 12.2 Omics, Databases and Detection of Biological Agents 12.3 Analysis of Pathogen Transmission and Outbreaks 12.4 Omics to Determine Infection as the Cause of Death (COD) 12.5 Post-Mortem Interval Estimation by the Use of Thanatomicrobiome 12.6 Human Body Fluid Identification Using Microbial DNA 12.7 The Human Microbiome to Identify Individuals 12.8 Metagenomics and the Forensic Analysis of Soils 12.9 Omics and the Forensic Analysis of Drowning 12.10 Future Perspectives References