Molecular Diagnostics of Infectious Diseases (eBook)

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

Preface 5
1 Choice of adequate sample material 17
1.1 Viruses 17
1.2 Bacteria 33
1.3 Fungi 38
1.4 Protozoa 40
2 Stability of the specimen during preanalytics 41
2.1 Degradation of DNA 41
2.2 Degradation of RNA 42
2.3 Inhibitors of PCR 43
2.4 How can contamination during specimen collection and in the laboratory be avoided? 44
2.5 How can the sample identity be ensured? 44
2.6 Transport of diagnostic material 44
2.6.1 Category A Infectious Substances 45
2.6.2 Category B Infectious Substances 45
2.6.3 Exempt patient specimens 46
2.7 Stability of nucleic acids of selected pathogens during preanalytics 46
2.7.1 Human immunodeficiency virus type 1 (HIV-1) RNA 46
2.7.2 Hepatitis B virus (HBV) DNA 47
2.7.3 Hepatitis C virus (HCV) RNA 47
2.7.4 Chlamydia trachomatis and Neisseria gonorrhoeae DNAs 48
2.7.5 Viral pathogens producing respiratory tract infections 48
2.7.6 Pathogens in stool specimens 49
2.8 Take-home messages 49
2.9 Further reading 49
3 Quality assurance and quality control 51
3.1 Accreditation issues 51
3.2 Validation and verification work 52
3.3 Components of validation work 52
3.3.1 Internal and external quality controls 52
3.3.2 Proficiency testing 55
3.3.3 Validation of employee competency 56
3.3.4 Instrument maintenance and calibration 56
3.3.5 Correlation with clinical findings 56
3.4 Components of verification work 57
3.4.1 Components of verification work for IVD/CE labeled and/or FDA-approved or -cleared tests or test systems 58
3.4.2 Components of verification work for laboratory-developed tests or test systems 60
3.5 Take-home messages 61
3.6 Further reading 62
4 Extraction of nucleic acids 63
4.1 Manual nucleic acid extraction protocols 63
4.2 Automated nucleic acid extraction platforms 63
4.2.1 Technology principle 64
4.2.2 Desirable features of automated platforms 64
4.3 Preparation of PCR mixes and addition of eluates 66
4.4 Currently frequently used commercially available platforms for nucleic acid extraction 66
4.5 Take-home messages 67
4.6 Further reading 68
5 Amplification and detection methods 69
5.1 Nucleic acid-based tests 70
5.2 Target amplification methods 71
5.2.1 Real-time polymerase chain reaction (qPCR) 72
5.2.2 Isothermal amplification techniques 78
5.2.3 Next generation sequencing (NGS) 81
5.3 Signal amplification methods 81
5.3.1 Branched DNA (bDNA) 82
5.3.2 Hybrid capture assay 82
5.4 What are the key challenges for the future? 83
5.5 Take-home messages 84
5.6 Further reading 84
6 Interpreting and reporting molecular diagnostic tests 85
6.1 Qualitative and quantitative detection of viral infections 85
6.2 Detection of bacterial infections 85
6.3 Quantitative endpoint PCR 86
6.4 Real time PCR 88
6.5 Reporting results 88
6.5.1 Genetic names 90
6.5.2 Recommendations for reporting results of molecular tests 90
6.5.3 Recommendations for the contents of the molecular test report 91
6.6 Interpretation 92
6.7 Important issues when clinically interpreting molecular diagnostic results 93
6.8 Take-home messages 93
6.9 Further reading 94
7 Human immunodeficiency virus 95
7.1 Major symptoms 97
7.1.1 Untreated individuals 97
7.1.2 Treated individuals 98
7.2 Preanalytics 98
7.2.1 Specimen collection 98
7.2.2 Clinical circumstances for using NAT to diagnose HIV infection 99
7.2.3 Clinical circumstances for using NAT to monitor HIV infection 100
7.3 Analytics 101
7.3.1 Main technologies for NAT 101
7.3.2 HIV RNA assays 101
7.3.3 HIV DNA assays 103
7.3.4 HIV resistance assays 103
7.3.5 HIV tropism assays 105
7.3.6 Assays for minority HIV quasispecies 106
7.4 Postanalytics 106
7.4.1 Molecular diagnosis of HIV infection 106
7.4.2 Monitoring HIV infection 107
7.5 Take-home messages 108
7.6 Further reading 108
8 Hepatitis viruses 109
8.1 Major symptoms 109
8.2 Preanalytics 109
8.3 Analytics 111
8.3.1 Adenoviruses 112
8.3.2 HAV 113
8.3.3 HBV 113
8.3.4 HCV 114
8.3.5 HDV 116
8.3.6 HEV 116
8.3.7 Herpesviruses 116
8.3.8 Yellow fever virus and hemorrhagic fever viruses 116
8.4 Postanalytics - interpretation of results 116
8.4.1 HAV/HEV 117
8.4.2 HBV 117
8.4.3 HDV 117
8.4.4 HCV 118
8.5 Take-home messages 118
8.6 Further reading 118
9 Pathogens relevant in transplantation medicine 121
9.1 Clinical manifestations 123
9.2 Preanalytics 123
9.2.1 Adenoviruses 124
9.2.2 BKV 124
9.2.3 CMV 124
9.2.4 EBV 125
9.2.5 HHV-6 125
9.2.6 HHV-8 125
9.2.7 VZV 125
9.3 Analytics 126
9.3.1 Sample preparation 126
9.3.2 Nucleic acids amplification and detection 126
9.4 Postanalytics - interpretation of results 132
9.4.1 Adenoviruses 132
9.4.2 BKVyV 132
9.4.3 CMV 134
9.4.4 EBV 134
9.4.5 HHV-6 135
9.4.6 HHV-8 135
9.4.7 VZV 135
9.5 Take-home messages 135
9.6 Further reading 136
10 Pathogens in lower respiratory tract infections 137
10.1 Clinical importance of different etiologic agents 137
10.2 Specimen collection 140
10.2.1 S. pneumoniae 141
10.2.2 M. pneumoniae, C. pneumoniae, and L. pneumophila 141
10.2.3 B. pertussis 142
10.2.4 Respiratory viruses 142
10.3 Diagnostic procedures 142
10.3.1 Sample preparation and nucleic acid extraction 143
10.3.2 Amplification and detection methods for individual agents 143
10.3.3 Multiplex NAATs 149
10.4 External quality control 157
10.5 The clinical usefulness and implementation of NAATs 158
10.6 Concluding remarks 159
10.7 Further reading 159
11 Molecular diagnosis of gastrointestinal pathogens 161
11.1 Clinical manifestations 164
11.2 Preanalytics 164
11.3 Analytics 166
11.4 Postanalytics 176
11.4.1 Clinical sensitivity and diagnostic specificity 176
11.4.2 Interpretation of results 178
11.5 Further reading 179
12 Pathogens relevant in the central nervous system 181
12.1 Clinical manifestations 184
12.1.1 Viral meningitis 184
12.1.2 Acute community-acquired bacterial meningitis 187
12.1.3 Mycobacterium tuberculosis 187
12.1.4 Encephalitis 187
12.2 Preanalytics 188
12.2.1 Goals of etiological investigations 188
12.2.2 Specimens and handling 188
12.2.3 Time of lumbar puncture during the course of illness and quantity of CSF required 189
12.2.4 Transport and storage of specimens 190
12.3 Analytics 191
12.3.1 Sample preparation 191
12.3.2 Nucleic acid amplification and detection 191
12.4 Postanalytics 194
12.4.1 Workflow and testing schedules for molecular tests 194
12.4.2 Limitations of molecular tests 196
12.4.3 Viral CNS infections 197
12.4.4 Bacterial CNS infections 197
12.4.5 Which pathogens should we be looking for? 198
12.5 Conclusion 198
12.6 Take-home messages 199
12.7 Acknowledgment 200
12.8 Further reading 200
13 Pathogens relevant in sexually transmitted infections 201
13.1 Symptoms and clinical manifestations 201
13.2 Preanalytics 204
13.3 Analytics 204
13.4 Postanalytics 207
13.5 Further reading 208
14 Index 209