Preface p. v
Authors Index p. xiii
Contributors Index p. xv
Choice of adequate sample material p. 1
Viruses p. 1
Bacteria p. 19
Fungi p. 25
Protozoa p. 27
Stability of the specimen during preanalyses p. 29
Sample integrity during collection p. 29
Blood p. 29 Urine p. 30 Stool p. 30 Degradation of DNA p. 30 Degradation of RNA p. 31 Inhibitors of PCR p. 32
How can contamination during specimen collection and in the laboratory be avoided? p. 33
How can the sample identity be ensured? p. 34
Transport of diagnostic material p. 34
Category A Infectious Substances p. 34
Category B Infectious Substances p. 35
Exempt patient specimens p. 36
Stability of nucleic acids of selected pathogens during preanalyses p. 36
Human immunodeficiency virus type 1 (HIV.1) RNA p. 36
Hepatitis B virus (HBV) DNA p. 37
Hepatitis C virus (HCV) RNA p. 38
Chlamydia trachomatis and Neisseria gonorrhoeae DNAs p. 38
Viral pathogens producing respiratory tract infections p. 39
Pathogens in stool specimens p. 39
Take home messages p. 40
Further reading p. 40
Quality assurance and quality control p. 41
Accreditation issues p. 41
Standardization of diagnostic tests or test systems p. 42
Validation and verification work p. 44
Components of validation work p. 44
Internal and external quality controls p. 44
Proficiency testing p. 47
Validation of employee competency p. 48
Instrument maintenance and calibration p. 48
Components of verification work p. 49 Components of verification work for IVD/CE labeled and/or FDA-approved or-cleared
tests or test systems
p. 50 Components of verification work for laboratory-developed tests or test systems p. 53
Take home messages p. 54
Further reading p. 55
Extraction of nucleic acids p. 57
Manual nucleic acid extraction protocols p. 57
Automated nucleic acid extraction platforms p. 58
Technology principle p. 58
Desirable features of automated platforms p. 59
Preparation of qPCR mixes and addition of etuates (qPCR assay setup) p. 60 Currently frequently used commercially available platforms p. 60
Take home messages p. 62
Further reading p. 62
Amplification and detection methods p. 63
Nucleic acid-based tests p. 64
Target amplification methods p. 65
Real-time polymerase chain reaction (qPCR) p. 66
Isothermal amplification techniques p. 75
Next generation sequencing (NGS) p. 78
Signal amplification methods p. 79
Branched DNA (bDNA) p. 80
Hybrid capture assay p. 80
What are the key challenges for the future? p. 81
Take-home messages p. 82
Further reading p. 83
Interpreting and reporting molecular diagnostic tests p. 85
Detection of viral infections p. 85
Detection of bacterial infections p. 86
Quantitative endpoint PCR p. 87
Real-time PCR (qPCR) p. 88
Reporting results p. 89
Genetic names p. 91
Recommendations for reporting results of molecular tests p. 91 Recommendations for the contents of the molecular test report p. 92
Interpretation p. 93
Important issues when clinically interpreting molecular diagnostic results p. 95
Take home messages p. 96
Further reading p. 96
Human immunodeficiency virus p. 97
Untreated individuals p. 99
Treated individuals p. 100
Preanalyses p. 100
Specimen collection p. 100
Clinical circumstances for using NAT to diagnose HIV infection p. 101 Clinical circumstances for using NAT to monitor HIV infection p. 102
Analytics p. 103
Main technologies for NAT p. 103
HIV RNA assays p. 104
HIV DNA assays p. 105
HIV drug resistance assays p. 106
HIV tropism assays p. 108
Assays for minority HIV variants p. 109
Postanalytics p. 110
Molecular diagnosis of HIV infection p. 110
Monitoring HIV infection p. 110
Take-home messages p. 111 Further reading p. 112 Hepatitis viruses p. 113 Major symptoms p. 113 Preanalyses p. 113 Analytics p. 115 Adenoviruses p. 116 HAV p. 117 HEV p. 117 HCV p. 118 HDV p. 119 HEV p. 120 Herpes viruses p. 120
Yellow fever virus and hemorrhagic fever viruses p. 121
Postanalytics - interpretation of results p. 121
HAV/HEV p. 121 HBV p. 121 HDV p. 122 HCV p. 122 Take-home messages p. 123 Further reading p. 123
Pathogens relevant in transplantation medicine p. 125
Clinical manifestations p. 128
Preanalyses p. 128
CMV p. 129 EBV p. 130 HHV-6 p. 130 HHV-8 p. 130 VZV p. 131 BKPyV p. 131 JCPyV p. 131 Analytics p. 131 Sample preparation p. 131
Nucleic acids amplification and detection p. 132
Postanalytics - interpretation of results p. 140
Adenoviruses p. 140 CMV p. 140 EBV p. 141 HHV-6 p. 141 HHV-8 p. 141 VZV p. 141 BKPyV p. 142 JCPyV p. 142 Take-home messages p. 142 Further reading p. 143
Pathogens in lower respiratory tract infections p. 145
Clinical importance of different etiologic agents p. 145
Specimen collection p. 148
S. pneumoniae p. 148
M. pneumoniae, L. pneumophila, and C. pneumoniae p. 150
B. pertussis p. 150
Respiratory viruses p. 151
Diagnostic procedures p. 151
Sample preparation and nucleic acid extraction p. 151
Amplification and detection methods for individual agents p. 152
Multiplex NAATs p. 158
External quality control p. 170
The clinical usefulness and implementation of NAATs p. 171
Concluding remarks p. 172
Further reading p. 173
Molecular diagnosis of gastrointestinal pathogens p. 175
Clinical manifestations p. 178
Preanalytics p. 178
Analytics p. 180
Clinical sensitivity and diagnostic specificity p. 186
Interpretation of results p. 191
Further reading p. 192
Pathogens relevant in the central nervous system p. 193
Clinical manifestations p. 197
Viral meningitis p. 197
Acute community-acquired bacterial meningitis p. 197
Mycobacterium tuberculosis p. 200
Encephalitis p. 201
Preanalyses p. 201
Goals of etiological investigations p. 201
Specimens and handling p. 201
Time of lumbar puncture during the course of illness and quantity of CSF required p. 202
Transport and storage of specimens p. 203
Analytics p. 204
Sample preparation p. 204
Nucleic acid amplification and detection p. 204
Postanalytics p. 208
Workflow and testing schedules for molecular tests p. 208
Limitations of molecular tests p. 210
Viral CNS infections p. 211
Bacterial CNS infections p. 211
Which pathogens should we look for? p. 212
Conclusion p. 213
Take-home messages p. 213
Acknowledgment p. 214
Further reading p. 214
Pathogens relevant in sexually transmitted infections p. 217
Symptoms and clinical manifestations p. 217
Preanalytics p. 221
Analytics p. 221
Postanalytics p. 224
Further reading p. 225
Index p. 227
