Molecular Diagnostics of Infectious Diseases (eBook)

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Harald H. Kessler, Medical University of Graz, Graz, Austria.

Preface 5
Contents 7
Authors Index 13
Contributors Index 15
1. Choice of adequate sample material 17
1.1 Viruses 17
1.2 Bacteria 35
1.3 Fungi 41
1.4 Protozoa 43
2. Stability of the specimen during preanalytics 45
2.1 Sample integrity during collection 45
2.1.1 Blood 45
2.1.2 Urine 46
2.1.3 Stool 46
2.2 Degradation of DNA 46
2.3 Degradation of RNA 47
2.4 Inhibitors of PCR 48
2.5 How can contamination during specimen collection and in the laboratory be avoided? 49
2.6 How can the sample identity be ensured? 50
2.7 Transport of diagnostic material 50
2.7.1 Category A Infectious Substances 50
2.7.2 Category B Infectious Substances 51
2.7.3 Exempt patient specimens 52
2.8 Stability of nucleic acids of selected pathogens during preanalytics 52
2.8.1 Human immunodeficiency virus type 1 (HIV-1) RNA 52
2.8.2 Hepatitis B virus (HBV) DNA 53
2.8.3 Hepatitis C virus (HCV) RNA 54
2.8.4 Chlamydia trachomatis and Neisseria gonorrhoeae DNAs 54
2.8.5 Viral pathogens producing respiratory tract infections 55
2.8.6 Pathogens in stool specimens 55
2.9 Take home messages 56
2.10 Further reading 56
3. Quality assurance and quality control 57
3.1 Accreditation issues 57
3.2 Standardization of diagnostic tests or test systems 58
3.3 Validation and verification work 60
3.4 Components of validation work 60
3.4.1 Internal and external quality controls 60
3.4.2 Proficiency testing 63
3.4.3 Validation of employee competency 64
3.4.4 Instrument maintenance and calibration 64
3.4.5 Correlation with clinical findings 65
3.5 Components of verification work 65
3.5.1 Components of verification work for IVD/CE labeled and/or FDA-approved or -cleared tests or test systems 66
3.5.2 Components of verification work for laboratory-developed tests or test systems 69
3.6 Take home messages 70
3.7 Further reading 71
4. Extraction of nucleic acids 73
4.1 Manual nucleic acid extraction protocols 73
4.2 Automated nucleic acid extraction platforms 74
4.2.1 Technology principle 74
4.2.2 Desirable features of automated platforms 75
4.3 Preparation of qPCR mixes and addition of eluates (qPCR assay setup) 76
4.4 Currently frequently used commercially available platforms 76
4.5 Take home messages 78
4.6 Further reading 78
5. Amplification and detection methods 79
5.1 Nucleic acid-based tests 80
5.2 Target amplification methods 81
5.2.1 Real-time polymerase chain reaction (qPCR) 82
5.2.2 Isothermal amplification techniques 91
5.2.3 Next generation sequencing (NGS) 94
5.3 Signal amplification methods 95
5.3.1 Branched DNA (bDNA) 96
5.3.2 Hybrid capture assay 96
5.4 What are the key challenges for the future? 97
5.5 Take-home messages 98
5.6 Further reading 99
6. Interpreting and reporting molecular diagnostic tests 101
6.1 Detection of viral infections 101
6.2 Detection of bacterial infections 102
6.3 Quantitative endpoint PCR 103
6.4 Real-time PCR (qPCR) 104
6.5 Reporting results 105
6.5.1 Genetic names 107
6.5.2 Recommendations for reporting results of molecular tests 107
6.5.3 Recommendations for the contents of the molecular test report 108
6.6 Interpretation 109
6.7 Important issues when clinically interpreting molecular diagnostic results 111
6.8 Take home messages 112
6.9 Further reading 112
7. Human immunodeficiency virus 113
7.1 Major symptoms 115
7.1.1 Untreated individuals 115
7.1.2 Treated individuals 116
7.2 Preanalytics 116
7.2.1 Specimen collection 116
7.2.2 Clinical circumstances for using NAT to diagnose HIV infection 117
7.2.3 Clinical circumstances for using NAT to monitor HIV infection 118
7.3 Analytics 119
7.3.1 Main technologies for NAT 119
7.3.2 HIV RNA assays 120
7.3.3 HIV DNA assays 121
7.3.4 HIV drug resistance assays 122
7.3.5 HIV tropism assays 124
7.3.6 Assays for minority HIV variants 125
7.4 Postanalytics 126
7.4.1 Molecular diagnosis of HIV infection 126
7.4.2 Monitoring HIV infection 126
7.5 Take-home messages 127
7.6 Further reading 128
8. Hepatitis viruses 129
8.1 Major symptoms 129
8.2 Preanalytics 129
8.3 Analytics 131
8.3.1 Adenoviruses 132
8.3.2 HAV 133
8.3.3 HBV 133
8.3.4 HCV 134
8.3.5 HDV 135
8.3.6 HEV 136
8.3.7 Herpes viruses 136
8.3.8 Yellow fever virus and hemorrhagic fever viruses 137
8.4 Postanalytics – interpretation of results 137
8.4.1 HAV/HEV 137
8.4.2 HBV 137
8.4.3 HDV 138
8.4.4 HCV 138
8.5 Take-home messages 139
8.6 Further reading 139
9. Pathogens relevant in transplantation medicine 141
9.1 Clinical manifestations 144
9.2 Preanalytics 144
9.2.1 Adenoviruses 145
9.2.2 CMV 145
9.2.3 EBV 146
9.2.4 HHV-6 146
9.2.5 HHV-8 146
9.2.6 VZV 147
9.2.7 BKPyV 147
9.2.8 JCPyV 147
9.3 Analytics 147
9.3.1 Sample preparation 147
9.3.2 Nucleic acids amplification and detection 148
9.4 Postanalytics – interpretation of results 156
9.4.1 Adenoviruses 156
9.4.2 CMV 156
9.4.3 EBV 157
9.4.4 HHV-6 157
9.4.5 HHV-8 157
9.4.6 VZV 157
9.4.7 BKPyV 158
9.4.8 JCPyV 158
9.5 Take-home messages 158
9.6 Further reading 159
10. Pathogens in lower respiratory tract infections 161
10.1 Clinical importance of different etiologic agents 161
10.2 Specimen collection 164
10.2.1 S. pneumoniae 164
10.2.2 M. pneumoniae, L. pneumophila, and C. pneumoniae 166
10.2.3 B. pertussis 166
10.2.4 Respiratory viruses 167
10.3 Diagnostic procedures 167
10.3.1 Sample preparation and nucleic acid extraction 167
10.3.2 Amplification and detection methods for individual agents 168
10.3.3 Multiplex NAATs 174
10.4 External quality control 186
10.5 The clinical usefulness and implementation of NAATs 187
10.6 Concluding remarks 188
10.7 Further reading 189
11. Molecular diagnosis of gastrointestinal pathogens 191
11.1 Clinical manifestations 194
11.2 Preanalytics 194
11.3 Analytics 196
11.4 Postanalytics 202
11.4.1 Clinical sensitivity and diagnostic specificity 202
11.4.2 Interpretation of results 207
11.5 Further reading 208
12. Pathogens relevant in the central nervous system 209
12.1 Clinical manifestations 213
12.1.1 Viral meningitis 213
12.1.2 Acute community-acquired bacterial meningitis 213
12.1.3 Mycobacterium tuberculosis 216
12.1.4 Encephalitis 217
12.2 Preanalytics 217
12.2.1 Goals of etiological investigations 217
12.2.2 Specimens and handling 217
12.2.3 Time of lumbar puncture during the course of illness and quantity of CSF required 218
12.2.4 Transport and storage of specimens 219
12.3 Analytics 220
12.3.1 Sample preparation 220
12.3.2 Nucleic acid amplification and detection 220
12.4 Postanalytics 224
12.4.1 Workflow and testing schedules for molecular tests 224
12.4.2 Limitations of molecular tests 226
12.4.3 Viral CNS infections 227
12.4.4 Bacterial CNS infections 227
12.4.5 Which pathogens should we look for? 228
12.5 Conclusion 229
12.6 Take-home messages 229
12.7 Acknowledgment 230
12.8 Further reading 230
13. Pathogens relevant in sexually transmitted infections 233
13.1 Symptoms and clinical manifestations 233
13.2 Preanalytics 237
13.3 Analytics 237
13.4 Postanalytics 240
13.5 Further reading 241
Index 243