Molecular Diagnostics of Infectious Diseases

Molecular Diagnostics of Infectious Diseases

by Harald H. Kessler (Editor)

Hardcover(3rd revised edition)

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Product Details

ISBN-13: 9783110327885
Publisher: De Gruyter
Publication date: 05/13/2014
Edition description: 3rd revised edition
Pages: 253
Product dimensions: 6.90(w) x 9.60(h) x 0.70(d)
Age Range: 18 Years

About the Author

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

Table of Contents

Preface v

Authors Index xiii

Contributors Index xv

1 Choice of adequate sample material Holger F. Rabenau Reinhard B. Raggam Margit Hübner Eva Leitner 1

1.1 Viruses 1

1.2 Bacteria 19

1.3 Fungi 25

1.4 Protozoa 27

2 Stability of the specimen during preanalyses Georg Endler Georg Slavka Markus Exner 29

2.1 Sample integrity during collection 29

2.1.1 Blood 29

2.1.2 Urine 30

2.1.3 Stool 30

2.2 Degradation of DNA 30

2.3 Degradation of RNA 31

2.4 Inhibitors of PCR 32

2.5 How can contamination during specimen collection and in the laboratory be avoided? 33

2.6 How can the sample identity be ensured? 34

2.7 Transport of diagnostic material 34

2.7.1 Category A Infectious Substances 34

2.7.2 Category B Infectious Substances 35

2.7.3 Exempt patient specimens 36

2.8 Stability of nucleic acids of selected pathogens during preanalyses 36

2.8.1 Human immunodeficiency virus type 1 (HIV.1) RNA 36

2.8.2 Hepatitis B virus (HBV) DNA 37

2.8.3 Hepatitis C virus (HCV) RNA 38

2.8.4 Chlamydia trachomatis and Neisseria gonorrhoeae DNAs 38

2.8.5 Viral pathogens producing respiratory tract infections 39

2.8.6 Pathogens in stool specimens 39

2.9 Take home messages 40

2.10 Further reading 40

3 Quality assurance and quality control Reinhard B. Raggam John Saldanha Harald H. Kessler 41

3.1 Accreditation issues 41

3.2 Standardization of diagnostic tests or test systems 42

3.3 Validation and verification work 44

3.4 Components of validation work 44

3.4.1 Internal and external quality controls 44

3.4.2 Proficiency testing 47

3.4.3 Validation of employee competency 48

3.4.4 Instrument maintenance and calibration 48

3.4.5 Correlation with clinical findings 49

3.5 Components of verification work 49

3.5.1 Components of verification work for IVD/CE labeled and/or FDA-approved or-cleared tests or test systems 50

3.5.2 Components of verification work for laboratory-developed tests or test systems 53

3.6 Take home messages 54

3.7 Further reading 55

4 Extraction of nucleic acids Harald H. Kessler 57

4.1 Manual nucleic acid extraction protocols 57

4.2 Automated nucleic acid extraction platforms 58

4.2.1 Technology principle 58

4.2.2 Desirable features of automated platforms 59

4.3 Preparation of qPCR mixes and addition of etuates (qPCR assay setup) 60

4.4 Currently frequently used commercially available platforms 60

4.5 Take home messages 62

4.6 Further reading 62

5 Amplification and detection methods Stephen A. Bustin Harald H. Kessler 63

5.1 Nucleic acid-based tests 64

5.2 Target amplification methods 65

5.2.1 Real-time polymerase chain reaction (qPCR) 66

5.2.2 Isothermal amplification techniques 75

5.2.3 Next generation sequencing (NGS) 78

5.3 Signal amplification methods 79

5.3.1 Branched DNA (bDNA) 80

5.3.2 Hybrid capture assay 80

5.4 What are the key challenges for the future? 81

5.5 Take-home messages 82

5.6 Further reading 83

6 Interpreting and reporting molecular diagnostic tests Ranjini Valiathan Deshratn Asthana 85

6.1 Detection of viral infections 85

6.2 Detection of bacterial infections 86

6.3 Quantitative endpoint PCR 87

6.4 Real-time PCR (qPCR) 88

6.5 Reporting results 89

6.5.1 Genetic names 91

6.5.2 Recommendations for reporting results of molecular tests 91

6.5.3 Recommendations for the contents of the molecular test report 92

6.6 Interpretation 93

6.7 Important issues when clinically interpreting molecular diagnostic results 95

6.8 Take home messages 96

6.9 Further reading 96

7 Human immunodeficiency virus Jacques Izopet 97

7.1 Major symptoms 99

7.1.1 Untreated individuals 99

7.1.2 Treated individuals 100

7.2 Preanalyses 100

7.2.1 Specimen collection 100

7.2.2 Clinical circumstances for using NAT to diagnose HIV infection 101

7.2.3 Clinical circumstances for using NAT to monitor HIV infection 102

7.3 Analytics 103

7.3.1 Main technologies for NAT 103

7.3.2 HIV RNA assays 104

7.3.3 HIV DNA assays 105

7.3.4 HIV drug resistance assays 106

7.3.5 HIV tropism assays 108

7.3.6 Assays for minority HIV variants 109

7.4 Postanalytics 110

7.4.1 Molecular diagnosis of HIV infection 110

7.4.2 Monitoring HIV infection 110

7.5 Take-home messages 111

7.6 Further reading 112

8 Hepatitis viruses Dieter Hoffmann Thomas Michler Ulrike Protzer 113

8.1 Major symptoms 113

8.2 Preanalyses 113

8.3 Analytics 115

8.3.1 Adenoviruses 116

8.3.2 HAV 117

8.3.3 HEV 117

8.3.4 HCV 118

8.3.5 HDV 119

8.3.6 HEV 120

8.3.7 Herpes viruses 120

8.3.8 Yellow fever virus and hemorrhagic fever viruses 121

8.4 Postanalytics - interpretation of results 121

8.4.1 HAV/HEV 121

8.4.2 HBV 121

8.4.3 HDV 122

8.4.4 HCV 122

8.5 Take-home messages 123

8.6 Further reading 123

9 Pathogens relevant in transplantation medicine Marco Ciotti Harald H. Kessler 125

9.1 Clinical manifestations 128

9.2 Preanalyses 128

9.2.1 Adenoviruses 129

9.2.2 CMV 129

9.2.3 EBV 130

9.2.4 HHV-6 130

9.2.5 HHV-8 130

9.2.6 VZV 131

9.2.7 BKPyV 131

9.2.8 JCPyV 131

9.3 Analytics 131

9.3.1 Sample preparation 131

9.3.2 Nucleic acids amplification and detection 132

9.4 Postanalytics - interpretation of results 140

9.4.1 Adenoviruses 140

9.4.2 CMV 140

9.4.3 EBV 141

9.4.4 HHV-6 141

9.4.5 HHV-8 141

9.4.6 VZV 141

9.4.7 BKPyV 142

9.4.8 JCPyV 142

9.5 Take-home messages 142

9.6 Further reading 143

10 Pathogens in lower respiratory tract infections Margareta Ieven Katherine Loens 145

10.1 Clinical importance of different etiologic agents 145

10.2 Specimen collection 148

10.2.1 S. pneumoniae 148

10.2.2 M. pneumoniae, L. pneumophila, and C. pneumoniae 150

10.2.3 B. pertussis 150

10.2.4 Respiratory viruses 151

10.3 Diagnostic procedures 151

10.3.1 Sample preparation and nucleic acid extraction 151

10.3.2 Amplification and detection methods for individual agents 152

10.3.3 Multiplex NAATs 158

10.4 External quality control 170

10.5 The clinical usefulness and implementation of NAATs 171

10.6 Concluding remarks 172

10.7 Further reading 173

11 Molecular diagnosis of gastrointestinal pathogens Corinne F.L. Amar 175

11.1 Clinical manifestations 178

11.2 Preanalytics 178

11.3 Analytics 180

11.4 Postanalytics 186

11.4.1 Clinical sensitivity and diagnostic specificity 186

11.4.2 Interpretation of results 191

11.5 Further reading 192

12 Pathogens relevant in the central nervous system Helene Peigue-Lafeuille Cécile Henquell 193

12.1 Clinical manifestations 197

12.1.1 Viral meningitis 197

12.1.2 Acute community-acquired bacterial meningitis 197

12.1.3 Mycobacterium tuberculosis 200

12.1.4 Encephalitis 201

12.2 Preanalyses 201

12.2.1 Goals of etiological investigations 201

12.2.2 Specimens and handling 201

12.2.3 Time of lumbar puncture during the course of illness and quantity of CSF required 202

12.2.4 Transport and storage of specimens 203

12.3 Analytics 204

12.3.1 Sample preparation 204

12.3.2 Nucleic acid amplification and detection 204

12.4 Postanalytics 208

12.4.1 Workflow and testing schedules for molecular tests 208

12.4.2 Limitations of molecular tests 210

12.4.3 Viral CNS infections 211

12.4.4 Bacterial CNS infections 211

12.4.5 Which pathogens should we look for? 212

12.5 Conclusion 213

12.6 Take-home messages 213

12.7 Acknowledgment 214

12.8 Further reading 214

13 Pathogens relevant in sexually transmitted infections Suzanne M. Garland Sepehr N. Tabrizi 217

13.1 Symptoms and clinical manifestations 217

13.2 Preanalytics 221

13.3 Analytics 221

13.4 Postanalytics 224

13.5 Further reading 225

Index 227

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