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Molecular Diagnostics of Infectious Diseases 2nd revised
edition Edition Harald H. Kessler (Editor) Digital Instant
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Author(s): Harald H. Kessler (editor)
ISBN(s): 9783110278927, 3110278928
Edition: 2nd revised edition
File Details: PDF, 2.77 MB
Year: 2012
Language: english
Molecular Diagnostics of Infectious Diseases
Edited by Harald H. Kessler
Molecular Diagnostics
of Infectious Diseases
2nd Revised Edition
Edited by Harald H. Kessler
DE GRUYTER
Editor
Professor Dr. med. Harald H. Kessler
Molecular Diagnostics Laboratory and Research Unit Molecular Diagnostics
IHMEM, Medical University of Graz
Universitätsplatz 4
8010 Graz, Austria
E-Mail: [email protected]
The book contains 19 figures and 49 tables.
ISBN 978-3-11-027883-5
e-ISBN 978-3-11-027892-7
Library of Congress Cataloging-in-Publication data

A CIP catalog record for this book has been applied for at the Library of Congress.
Bibliographic information published by the Deutsche Nationalbibliothek
The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie;

detailed bibliographic data are available on the Internet at http://dnb.d-nb.de.
© 2012 Walter de Gruyter GmbH & Co. KG, Berlin/Boston.

The publisher, together with the authors and editors, has taken great pains to ensure that all infor-
mation presented in this work (programs, applications, amounts, dosages, etc.) reflects the stan-
dard of knowledge at the time of publication. Despite careful manuscript preparation and proof
correction, errors can nevertheless occur. Authors, editors and publisher disclaim all responsibility
and for any errors or omissions or liability for the results obtained from use of the information, or
parts thereof, contained in this work.
The citation of registered names, trade names, trademarks, etc. in this work does not imply, even in
the absence of a specific statement, that such names are exempt from laws and regulations protect-
ing trademarks etc. and therefore free for general use.
Printing and binding: Hubert & Co. GmbH & Co. KG, Göttingen
Cover image: © Chepko Danil/iStockphoto
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Printed in Germany
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Preface to the Second Edition
Many interesting and exciting developments have occurred since the publication of the
first edition that has been out of stock within little more than a year. Due to those new
developments, portions of the book have been revised and additional material of inter-
est is presented to the readers in this new edition.
Molecular diagnostics based on the direct detection of specific genetic material in
a specimen through nucleic acid amplification techniques (NAT) has largely replaced
antigen testing by immunoassays and become the leading technology. The new molecu-
lar test systems are more specific and sensitive. They are able to detect the presence of
a pathogen faster than immunoassays and thus mainly used for screening or diagnosing
patients for numerous diseases. Furthermore, NAT provide the possibility for detection
of genetic variations allowing for further characterization of pathogens including geno-
and subtypes and drug-resistant variants.
Historically, the use of molecular methods was constrained mainly by their labor-
intensive nature. They were error-prone and their operation required skilled laboratory
personnel. Today, improved molecular diagnostics instrumentation automates many of
the assay steps allowing for high-volume testing. New molecular assays with the majo-
rity of them being IVD/CE-labeled and/or FDA-approved/-cleared are regularly intro-
duced to the market by several manufacturers. Consequently, molecular diagnostics
is indicated in many areas of health-care including infectious diseases, hematology/
oncology, genetic disorders, and forensic medicine with molecular diagnostics of infec-
tious diseases making up the major part of the molecular IVD market.
This book is intended to give a timely overview about molecular diagnostics of infec-
tious diseases. In the general part (chapters 1–6), preanalytical, analytical and postana-
lytical issues are discussed. Furthermore, special attention is drawn on quality assur-
ance/quality control issues. In the special part (chapters 7–13), NAT for detection of
pathogens producing infectious diseases are discussed in detail. Each chapter focuses
on infectious diseases targeting a specific body tract or system. All together, this book
can be used as a starting point when one needs to evaluate which molecular method,
test system, or instrument may be considered or chosen for diagnostics of infectious
diseases.
I wish to acknowledge outstanding assistance from all authors and contributors in
the preparation of this book, without whom this new edition would not have been pos-
sible. Furthermore, I wish to thank all helpful people from the publisher involved in the
production of this book.
This book tries to cover the whole field and provides a maximum timely content.
However, the reader should consider that this field is still changing rapidly. Suggestions
for further improvements to be considered in a future edition are highly appreciated.
Please contact the editor at [email protected].
Enjoy reading this new edition!
Harald H. Kessler
Table of Contents
Preface ................................................................................................................ v
1 Choice of adequate sample material............................................................ 1

1.1 Viruses ............................................................................................... 1
1.2 Bacteria ............................................................................................. 17
1.3 Fungi ................................................................................................. 22
1.4 Protozoa ............................................................................................ 24
2 Stability of the specimen during preanalytics .............................................. 25

2.1 Degradation of DNA .......................................................................... 25
2.2 Degradation of RNA .......................................................................... 26
2.3 Inhibitors of PCR ................................................................................ 27
2.4 How can contamination during specimen collection
and in the laboratory be avoided? ...................................................... 28
2.5 How can the sample identity be ensured? .......................................... 28
2.6 Transport of diagnostic material ......................................................... 28
2.6.1 Category A Infectious Substances ......................................... 29
2.6.2 Category B Infectious Substances ......................................... 29
2.6.3 Exempt patient specimens .................................................... 30
2.7 Stability of nucleic acids of selected pathogens during preanalytics ... 30
2.7.1 Human immunodeficiency virus type 1 (HIV-1) RNA ........... 30
2.7.2 Hepatitis B virus (HBV) DNA ............................................... 31
2.7.3 Hepatitis C virus (HCV) RNA................................................ 31
2.7.4 Chlamydia trachomatis and Neisseria gonorrhoeae DNAs.... 32
2.7.5 Viral pathogens producing respiratory tract infections .......... 32
2.7.6 Pathogens in stool specimens ............................................... 33
2.8 Take-home messages.......................................................................... 33
2.9 Further reading .................................................................................. 33
3 Quality assurance and quality control ......................................................... 35

3.1 Accreditation issues ........................................................................... 35
3.2 Validation and verification work ........................................................ 36
3.3 Components of validation work ......................................................... 36
3.3.1 Internal and external quality controls ................................... 36
3.3.2 Proficiency testing ................................................................ 39
3.3.3 Validation of employee competency .................................... 40
3.3.4 Instrument maintenance and calibration .............................. 40
3.3.5 Correlation with clinical findings ......................................... 40
viii Table of Contents
3.4 Components of verification work ....................................................... 41

3.4.1 Components of verification work for IVD/CE labeled
and/or FDA-approved or -cleared tests or test systems .......... 42
3.4.2 Components of verification work for laboratory-developed
tests or test systems .............................................................. 44
3.6 Further reading .................................................................................. 46
4 Extraction of nucleic acids........................................................................... 47

4.1 Manual nucleic acid extraction protocols .......................................... 47
4.2 Automated nucleic acid extraction platforms ..................................... 47
4.2.1 Technology principle ........................................................... 48
4.2.2 Desirable features of automated platforms............................ 48
4.3 Preparation of PCR mixes and addition of eluates .............................. 50
4.4 Currently frequently used commercially available platforms
for nucleic acid extraction ................................................................. 50
4.6 Further reading .................................................................................. 52
5 Amplification and detection methods .......................................................... 53

5.1 Nucleic acid-based tests .................................................................... 54
5.2 Target amplification methods ............................................................. 51
5.2.1 Real-time polymerase chain reaction (qPCR) ........................ 56
5.2.2 Isothermal amplification techniques ..................................... 62
5.2.3 Next generation sequencing (NGS) ...................................... 65
5.3 Signal amplification methods ............................................................. 65
5.3.1 Branched DNA (bDNA)........................................................ 66
5.3.2 Hybrid capture assay............................................................ 66
5.4 What are the key challenges for the future? ........................................ 67
5.6 Further reading .................................................................................. 68
6 Interpreting and reporting molecular diagnostic tests ................................. 69

6.1 Qualitative and quantitative detection of viral infections .................... 69
6.2 Detection of bacterial infections ........................................................ 69
6.3 Quantitative endpoint PCR ................................................................ 70
6.4 Real time PCR .................................................................................... 72
6.5 Reporting results ................................................................................ 72
6.5.1 Genetic names ..................................................................... 74
6.5.2 Recommendations for reporting results of molecular tests .... 74
6.5.3 Recommendations for the contents of the molecular
test report ............................................................................. 75
6.6 Interpretation ..................................................................................... 76
6.7 Important issues when clinically interpreting molecular
diagnostic results ............................................................................... 77
6.9 Further reading .................................................................................. 78
Table of Contents ix
7 Human immunodeficiency virus .................................................................. 79

7.1 Major symptoms ................................................................................ 81
7.1.1 Untreated individuals ........................................................... 81
7.1.2 Treated individuals ............................................................... 82
7.2 Preanalytics ....................................................................................... 82
7.2.1 Specimen collection ............................................................ 82
7.2.2 Clinical circumstances for using NAT to diagnose
HIV infection ....................................................................... 83
7.2.3 Clinical circumstances for using NAT to monitor
HIV infection ....................................................................... 84
7.3 Analytics ............................................................................................ 85
7.3.1 Main technologies for NAT .................................................. 85
7.3.2 HIV RNA assays ................................................................... 85
7.3.3 HIV DNA assays .................................................................. 87
7.3.4 HIV resistance assays ........................................................... 87
7.3.5 HIV tropism assays ............................................................... 89
7.3.6 Assays for minority HIV quasispecies ................................... 90
7.4 Postanalytics ...................................................................................... 90
7.4.1 Molecular diagnosis of HIV infection ................................... 90
7.4.2 Monitoring HIV infection ..................................................... 91
7.6 Further reading .................................................................................. 92
8 Hepatitis viruses .......................................................................................... 93

8.1 Major symptoms ................................................................................ 93
8.2 Preanalytics ....................................................................................... 93
8.3 Analytics ............................................................................................ 95
8.3.1 Adenoviruses ....................................................................... 96
8.3.2 HAV ..................................................................................... 97
8.3.3 HBV ..................................................................................... 97
8.3.4 HCV .................................................................................... 98
8.3.5 HDV .................................................................................... 100
8.3.6 HEV ..................................................................................... 100
8.3.7 Herpesviruses ...................................................................... 100
8.3.8 Yellow fever virus and hemorrhagic fever viruses ................. 100
8.4 Postanalytics – interpretation of results ............................................... 100
8.4.1 HAV/HEV ............................................................................. 101
8.4.2 HBV ..................................................................................... 101
8.4.3 HDV .................................................................................... 101
8.4.4 HCV .................................................................................... 102
8.6 Further reading .................................................................................. 102
9 Pathogens relevant in transplantation medicine........................................... 105

9.1 Clinical manifestations ....................................................................... 107
9.2 Preanalytics ....................................................................................... 107
9.2.1 Adenoviruses ....................................................................... 108
x Table of Contents
9.2.2 BKV ..................................................................................... 108

9.2.3 CMV .................................................................................... 108
9.2.4 EBV ...................................................................................... 109
9.2.5 HHV-6 ................................................................................. 109
9.2.6 HHV-8 ................................................................................. 109
9.2.7 VZV ..................................................................................... 109
9.3 Analytics ............................................................................................ 110
9.3.1 Sample preparation .............................................................. 110
9.3.2 Nucleic acids amplification and detection ........................... 110
9.4 Postanalytics – interpretation of results ............................................... 116
9.4.1 Adenoviruses ....................................................................... 116
9.4.2 BKVyV ................................................................................. 116
9.4.3 CMV .................................................................................... 118
9.4.4 EBV ...................................................................................... 118
9.4.5 HHV-6 ................................................................................. 119
9.4.6 HHV-8 ................................................................................. 119
9.4.7 VZV ..................................................................................... 119
9.6 Further reading .................................................................................. 120
10 Pathogens in lower respiratory tract infections ............................................ 121

10.1 Clinical importance of different etiologic agents ................................ 121
10.2 Specimen collection .......................................................................... 124
10.2.1 S. pneumoniae ..................................................................... 125
10.2.2 M. pneumoniae, C. pneumoniae, and L. pneumophila ........ 125
10.2.3 B. pertussis ........................................................................... 126
10.2.4 Respiratory viruses ............................................................... 126
10.3 Diagnostic procedures ....................................................................... 126
10.3.1 Sample preparation and nucleic acid extraction ................... 127
10.3.2 Amplification and detection methods for individual agents .. 127
10.3.3 Multiplex NAATs.................................................................. 133
10.4 External quality control ...................................................................... 141
10.5 The clinical usefulness and implementation of NAATs ....................... 142
10.6 Concluding remarks ........................................................................... 143
10.7 Further reading .................................................................................. 143
11 Molecular diagnosis of gastrointestinal pathogens....................................... 145

11.2 Preanalytics ....................................................................................... 148
11.3 Analytics ............................................................................................ 150
11.4 Postanalytics ...................................................................................... 160
11.4.1 Clinical sensitivity and diagnostic specificity ........................ 160
11.4.2 Interpretation of results ........................................................ 162
11.5 Further reading .................................................................................. 163
Table of Contents xi
12 Pathogens relevant in the central nervous system ........................................ 165

12.1.1 Viral meningitis .................................................................... 168
12.1.2 Acute community-acquired bacterial meningitis .................. 171
12.1.3 Mycobacterium tuberculosis ................................................ 171
12.1.4 Encephalitis ......................................................................... 171
12.2 Preanalytics ....................................................................................... 172
12.2.1 Goals of etiological investigations ........................................ 172
12.2.2 Specimens and handling ...................................................... 172
12.2.3 Time of lumbar puncture during the course of illness
and quantity of CSF required ................................................ 173
12.2.4 Transport and storage of specimens ...................................... 174
12.3 Analytics ............................................................................................ 175
12.3.1 Sample preparation .............................................................. 175
12.3.2 Nucleic acid amplification and detection ............................. 175
12.4 Postanalytics ...................................................................................... 178
12.4.1 Workflow and testing schedules for molecular tests.............. 178
12.4.2 Limitations of molecular tests ............................................... 180
12.4.3 Viral CNS infections ............................................................. 181
12.4.4 Bacterial CNS infections ...................................................... 181
12.4.5 Which pathogens should we be looking for? ........................ 182
12.5 Conclusion ........................................................................................ 182
12.7 Acknowledgment ............................................................................... 184
12.8 Further reading .................................................................................. 184
13 Pathogens relevant in sexually transmitted infections .................................. 185

13.1 Symptoms and clinical manifestations................................................ 185
13.2 Preanalytics ....................................................................................... 188
13.3 Analytics ............................................................................................ 188
13.4 Postanalytics ...................................................................................... 191
13.5 Further reading .................................................................................. 192
14 Index ........................................................................................................... 193

Authors Index
Corinne F.L. Amar Chapter 11 Dieter Hoffmann Chapter 8

FoodBorne Pathogens Reference Unit Institute of Virology
61 Colindale Avenue Technische Universitaet Muenchen
NW9 5EQ LONDON Trogerstrasse 30
UK 81675 MUENCHEN
Phone: +44(20)83277341 Germany
[email protected] Phone: +49(89)41406825
[email protected]
Stephen A. Bustin Chapter 5 Margareta Ieven Chapter 10

3rd Floor Alexandra Wing Vaccine & Infectious Disease Institute
The Royal London Hospital University of Antwerp
LONDON E1 1BB Wilrijkstraat 10
UK 2650 EDEGEM
Phone: +44(20)78828748 Belgium
[email protected]
Marco Ciotti Chapter 9 Jacques Izopet Chapter 7

U.O.C. di Virologia Molecolare Laboratoire de Virologie
Fondazione Policlinico Universitatio Institut Federatif de Biologie
Tor Vergata 330 Avenue de Grande Bretagne, TSA 40031
Viale Oxford 31059 TOULOUSE Cedex 9
81-00133 ROMA France
Italy Phone: +33(5)67690424
Phone: +39(6)20902087 [email protected]
[email protected]
Harald H. Kessler Chapter 4
Center for Applied Biomedicine
Georg Endler Chapter 2 Medical University of Graz
Central Laboratory Universitaetsplatz 4
Municipal Hospital Wilhelminen 8010 GRAZ
Montlearstrasse 37 Austria
1090 WIEN Phone: +43(316)3804363
Austria [email protected]
Phone: +43(1)491503308
[email protected]
Helene Peigue-Lafeuille Chapter 12
Laboratoire de Virologie
Suzanne M. Garland Chapter 13 Centre de Biologie
The Royal Women´s Hospital CHRU Clermont-Ferrand
Locked Bag 300 58, rue Montalembert
PARKVILLE 3052 63003 Clermont-Ferrand
Australia France
Phone: +61(3)83453671 Phone:+33(4)73754850
[email protected] [email protected]
xiv Authors Index
Holger F. Rabenau Chapter 1 Ranjini Valiathan Chapter 6

Institute for Medical Virology Laboratory for Clinical and Biological Studies
JWG-University Frankfurt/Main University of Miami – Miller School of Medicine
Paul-Ehrlich-Strasse 40 1550 NW 10th Avenue, Fox Cancer Building,
60596 FRANKFURT/MAIN Suite 118
Germany MIAMI, FL 33136
Tel.: +49(69)63015312 USA
[email protected]
Reinhard B. Raggam Chapter 3
Clinical Institute of Medical and Chemical
Laboratory Diagnostics
Medical University of Graz
Auenbruggerplatz 15
8036 GRAZ
Austria
Phone: +43(316)38580243
[email protected]
Contributors Index
Deshratn Asthana Jamie Murphy

Laboratory for Clinical and Biological Studies 3rd Floor Alexandra Wing
University of Miami – Miller School of Medicine The Royal London Hospital
1550 NW 10th Avenue, Fox Cancer Building, LONDON E1 1BB
Suite 118 UK
MIAMI, FL 33136 Phone: +44(20)78828748
USA [email protected]
Phone: +1(305)2432010
[email protected]
Korinna S. Nadas
Institute of Virology
Cecile Henquell Technische Universitaet Muenchen
Laboratoire de Virologie Trogerstrasse 30
Centre de Biologie 81675 MUENCHEN
CHRU Clermont-Ferrand Germany
58, rue Montalembert Phone: +49(89)41406825
63003 CLERMONT-FERRAND [email protected]
France
Phone: +33(4)73754850
Ulrike Protzer
[email protected]
Institute of Virology
Technische Universitaet Muenchen
Margit Hübner Trogerstrasse 30
Center for Applied Biomedicine 81675 MUENCHEN
Medical University of Graz Germany
Universitaetsplatz 4 Phone: +49(89)41406821
8010 GRAZ [email protected]
Austria
Phone: +43(316)3804380
Georg Slavka
[email protected]
Central Laboratory
Municipal Hospital Wilhelminen
Katherine Loens Montlearstrasse 37
Vaccine and Infectious Disease Institute 1090 WIEN
University of Antwerp Austria
Wilrijkstraat 10 Phone: +43(1)491503308
2650 EDEGEM [email protected]
Belgium
Phone: +32(3)8202751
Sepehr N. Tabrizi
[email protected]
The Royal Women´s Hospital
Locked Bag 300
PARKVILLE 3052
Australia
Phone: +61(3)83453671
[email protected]
1 Choice of adequate sample material
Holger F. Rabenau; Reinhard B. Raggam; Margit Hübner
Nucleic acid amplification testing (NAT) has gained major impact on the detection of
pathogens with molecular assays being widely used in the routine diagnostic labora-
tory. Reliable molecular diagnostics strongly depends on preanalytical issues including
the choice of adequate sample material, optimal sampling time regarding the course of
disease, and both time and conditions of the sample transport to the laboratory.
This chapter focuses on the choice of adequate sample materials for molecular
diagnostics of viruses, bacteria, fungi, and protozoa. Pathogens with epidemiological
and clinical significance for which molecular diagnostics plays an important role are
discussed in alphabetical order.
1.1 Viruses
Adenoviruses (Family: Adenoviridae; approx. 50 human serotypes, subgenera A–F)

Epidemiology: worldwide distribution.
Transmission: droplets and smear infection; entrance gates are eyes and the oropharynx.
Incubation period: 5–12 days.
Clinical presentation: adenovirus infections are often asymptomatic or cause respiratory
tract infections, gastroenteritis, and epidemic keratoconjunctivitis.
Complications: meningoencephalitis in children, disseminated, sepsis-like adenoviral
infection with multiple organ manifestations in immunosuppressed patients.
Indication and choice of the adequate sample material for NAT:
Clinical presentation Sample material

Epidemic keratoconjunctivitis Conjunctival swab
Pneumonia Bronchoalveolar lavage (BAL), EDTA whole blood
Hemorrhagic cystitis Urine
Encephalitis Cerebrospinal fluid (CSF)
Gastroenteritis, diarrhea Stool
Pre-emptive monitoring/suspected EDTA whole blood, nasopharyngeal swab or
adenovirus infection under aspirate, throat washing, urine
immunosuppression
Astrovirus (Family: Astroviridae)

Epidemiology: occasional outbreaks, e.g. in nursing homes or nosocomial outbreaks in
hospitals.
2 1 Choice of adequate sample material
Transmission: smear infections or through contaminated food and water.

Clinical presentation: gastroenteritis with fever, vomiting and abdominal pain.

Gastroenteritis, diarrhea Stool
Bocavirus (BoV) (Family: Parvoviridae; 4 species: BoV1–BoV4)

Epidemiology: worldwide distribution, in 2–19% of patients with upper or lower respi-
ratory tract disease predominantly during winter and spring, very common during early
childhood, co-infections with other respiratory viruses frequently observed, BoV2 through
BoV4 mainly in stool (enteric species), associated with gastroenteritis, co-infections
with other gastrointestinal viruses in up to 100% of stool specimens.
Transmission: Transmission routes unknown; however, most likely transmitted by inha-
lation or contact with infectious sputum, feces, or urine.
Incubation period: Unknown.
Clinical presentations: BoV1: Respiratory tract infection with cough and wheeze, rhin-
orrhea, tachypnea, and fever. BoV2 through BoV4: Gastroenteritis.
Complications: Rash or exanthema, thrombopenia, pneumonia, sepsis (rarely).
Respiratory tract infection, pneumonia Nasopharyngeal swab or aspirate, throat washing, BAL
Gastroenteritis Stool
Note: BoV may persist in the respiratory or gastrointestinal tract as a bystander without symptoms
resulting in frequent detection of BoV.
Coronaviruses (Family: Coronaviridae; 3 genera: group I includes CoV-229E and CoV-

NL63, group II includes CoV-OC43, CoV-HKU1, and SARS-CoV, and group III includes
only avian pathogens.)
Epidemiology: worldwide distribution, according to genogroup high prevalence already
in childhood.
Transmission: droplets and smear infection.
Incubation period: 2–5 days (SARS 2–20 days).
Clinical presentation: Respiratory tract infections. SARS disease with fever, cough, short-
ness of breath, pneumonia, and bronchiolitis; CoV-NL63 occurs especially in children
with disorders of the upper respiratory tract, pneumonia, and bronchiolitis.

Upper respiratory tract infection Nasopharyngeal swab or aspirate, throat washing,
induced sputum
Pneumonia, bronchiolitis BAL
Suspected SARS infection Nasopharyngeal swab or aspirate, throat washing,
(induced) sputum, BAL, EDTA whole blood
1.1 Viruses 3
Cytomegalovirus (CMV) (Family: Herpesviridae)

Epidemiology: worldwide distribution, seroprevalence 50–100%.
Transmission: oro-oral contact (kissing) through saliva, and sexually through genital
secretions, rarely droplets or smear infection; pre- and perinatal; possibly iatrogenic.
Clinical presentation: primary infection usually asymptomatic, mononucleosis-like
symptoms may occur, rarely hepatitis. Re-activation is usually asymptomatic but in the
immunocompromised it is potentially life threatening.
Complications: pneumonia, encephalitis, retinitis, enterocolitis and/or hepatitis; acute/
chronic graft rejection; bacterial and fungal super-infections; acceleration of underlying
viral infections.

Pneumonia BAL, EDTA whole blood
Encephalitis CSF, EDTA whole blood
Disseminated CMV infection BAL, CSF, EDTA whole blood
Colitis Stool
Hepatitis EDTA whole blood
Retinitis Aqueous humor
Pre-emptive monitoring/suspected CMV EDTA whole blood, bone marrow, throat
infection under immunosuppression washing, urine, BAL, CSF, stool
Prenatal infection Amniotic fluid, fetal EDTA whole blood
Perinatal infection EDTA whole blood, urine
Dengue viruses (Family: Flaviviridae; 4 serotypes)

Epidemiology: distribution of the virus in almost all tropical and subtropical regions.
Transmission: bite through Aedes mosquitoes.
Clinical presentation: two peaked febrile illness, followed by arthralgia and rash.
Complications: re-infection can lead to dengue hemorrhagic fever (DHF) or dengue
shock syndrome (DSS).

Unclear serological constellation/serological EDTA whole blood, serum
confirmation
Suspicion or exclusion of dengue infection EDTA whole blood, serum
DHF, DSS EDTA whole blood, serum
Enteroviruses (Family: Picornaviridae; approx. 70 human types, including Polio-,

Coxsackie- and ECHO viruses)
Epidemiology: worldwide distribution, infections occur mainly in summer.
Transmission: fecal-oral.
Incubation period: 2–14 (rarely up to 35) days.

Clinical presentation: frequently asymptomatic course or non-specific feverish infec-
tion (‘summer flu’). Depending on the virus type, different diseases and symptoms are
observed, e.g. respiratory tract infections, herpangina, acute hemorrhagic conjunctivitis,
aseptic meningitis, meningoencephalitis, paralysis, eruptions, Hand-Foot-Mouth disease,
myocarditis, pericarditis, hepatitis, and epidemic pleurodynia (Bornholm’s disease).
Complications: severe systemic disease of newborns with meningitis, meningoenceph-
alitis and myocarditis.

Aseptic meningitis, meningoencephalitis CSF, stool
Myocarditis, pericarditis, pleurodynia, hepatitis Organ biopsy, stool
Conjunctivitis Conjunctival swab
Respiratory tract infections, herpangina, Nasopharyngeal swab or aspirate, skin swab,
Hand-Foot-Mouth disease throat washing
Systemic disease Stool
Note: shedding of enteroviruses in the stool can persist for months.
Epstein-Barr virus (EBV) (Family: Herpesviridae)

Epidemiology: worldwide distribution, seroprevalence in adults usually >90%, virus
persistence with frequent (subclinical) reactivation and virus excretion.
Transmission: oro-oral contact (kissing) through saliva, and sexually through genital
secretions, rarely droplets or smear infection.
Clinical presentation: before puberty mainly asymptomatic, afterwards infectious mono-
nucleosis.
Complications: meningitis, encephalitis, Guillain-Barré syndrome, hepatitis, splenic
rupture, hemolytic and aplastic anemia, thrombocytopenia. EBV infection is also associ-
ated with Burkitt’s lymphoma, nasopharyngeal carcinoma, lymphoproliferative disease,
lymphoma in immunocompromised patients (e.g. post-transplant lymphoproliferative
syndrome [PTLD]), X-linked lymphoproliferative syndrome, oral hairy cell leukoplakia,
and Hodgkin’s lymphoma.

Meningitis, encephalitis CSF
Mononucleosis, aplastic anemia, Guillain-Barré syndrome EDTA whole blood
Pre-emptive monitoring/suspected EBV infection under EDTA whole blood
immunosuppression
PLTD risk patients EDTA whole blood
Oral hairy cell leukoplakia Biopsy
1.1 Viruses 5
Hantaviruses (Family: Bunyaviridae; approx. 20 hantavirus species)

Transmission: through infectious, aerosolic feces of chronically infected rodents (viral
reservoir).
Clinical presentation: hemorrhagic fever with renal syndrome (HFRS) mainly caused by
Hantaan, Dobrava and Seoul species; nephropathia epidemica caused by Puumala spe-
cies; Hantavirus pulmonary syndrome (HPS) caused by Sin Nombre virus.

HFRS, nephropathia epidemica EDTA whole blood, Urine, organ biopsy
HPS EDTA whole blood, organ biopsy
Note: the diagnosis is usually based on serological antibody testing.
Hepatitis A virus (HAV) (Family: Picornaviridae)

Epidemiology: worldwide distribution, in industrialized countries the prevalence is rela-
tively low.
Transmission: fecal-oral.
Incubation period: 3–5 weeks.
Clinical presentation: infection in childhood often asymptomatic. The risk of a symp-
tomatic course of the disease increases with age. Unspecific symptoms such as nausea,
loss of appetite, malaise, and aversion to fatty foods are followed by viral hepatitis with
jaundice.

Suspected HAV infection Stool
Note: the diagnosis is usually based on serological antibody testing.
Hepatitis B virus (HBV) (Family: Hepadnaviridae)

Epidemiology: worldwide distribution, approximately 350 million chronic carriers.
Transmission: parenteral, vertical, sexual.
Incubation period: 1–7 months, depending on the mode of transmission and the infec-
tive dose.
Clinical presentation: often inapparent HBV infection or unspecific symptoms such as
malaise, anorexia, arthralgia, and vasculitis.
Complications: tendency for development of chronic HBV infection (approximately
10%, for perinatal infection exceeding 90%) with chronic active hepatitis, liver fibrosis,
liver cirrhosis, and hepatocellular carcinoma.

Suspected acute or chronic HBV infection EDTA plasma, serum
Therapy decision-making and monitoring EDTA plasma, serum
Hepatitis C virus (HCV) (Family: Flaviviridae; at least 6 genotypes with numerous

subtypes)
Epidemiology: worldwide distribution, approximately 170 million chronic carriers.
Transmission: mainly parenteral, through contaminated blood, e.g. ‘needle sharing’among
intravenous drug abusers. Rarely through needle-stick injuries in healthcare settings,
sexual contact, or mother-to-child transmission. Infection through blood and blood
products occurred frequently before introduction of antibody testing in 1991.
Clinical presentation: mainly asymptomatic or mild and unspecific symptoms with leth-
argy, anorexia, less than 10% are icteric.
Complications: high tendency for development of chronic HCV infection (exceeding
70%), with chronic active hepatitis, liver fibrosis, liver cirrhosis and hepatocellular
carcinoma.

Suspected acute or chronic HCV infection EDTA plasma, serum
Therapy decision making and monitoring EDTA plasma, serum
Note: HCV genotyping is mandatory before starting antiviral therapy.
Hepatitis D virus (HDV) (Genus: Delta virus, no family; 3 genotypes)

Epidemiology: worldwide distribution, with high prevalence in South America and low
prevalence in central and northern Europe.
Transmission: mostly parenteral; sexual transmission is possible. Two possible ways of
infection, either as a co-infection (HBV and HDV are transmitted simultaneously), or as
a super-infection (HDV infection of an already HBV-infected individual).
Incubation period: 4 weeks to 8 months.
Clinical presentation: acute HBV-HDV co-infections often cause a severe acute hepa-
titis with significant mortality. The super-infection of a chronic HBV carrier with HDV
usually leads to the formation of a chronic co-infection and is often more severe than
HBV mono-infection.

Suspected acute or chronic HDV infection EDTA plasma, serum
Note: monitoring of HDV therapy may be done through monitoring of HBV load.
1.1 Viruses 7
Hepatitis E virus (HEV) (Family: Hepeviridae, Genus: Hepevirus; 4 genotypes)

Epidemiology: most countries in the developing world (especially Southeast Asia), with
only a few indigenous cases from industrialized countries. Genotypes 1 and 2 are re-
stricted to humans (associated with large outbreaks in developing countries), while
genotypes 3 and 4 infect humans, pigs and other animal species.
Transmission: fecal-oral, through contaminated water, or food (e.g. consumption of
insufficient cooked shell fish, wild boar or deer liver meat).
Clinical presentation: similar to HAV, self-limiting disease, no chronic course. However,
sometimes prolonged fecal excretion. Occasionally severe liver disease (fatality rate
1–4%). Especially in pregnant women, lethality is up to 25%.

Suspected HEV infection Stool
Note: due to the frequently false-positive serological test results, PCR testing for HEV is strongly
recommended in cases of suspected HEV infection.
Herpes simplex virus type 1 and type 2 (HSV-1, HSV-2) (Family: Herpesviridae)
Epidemiology: worldwide distribution, seroprevalence in adults 75–95% (HSV-1) and
15–25% (HSV-2), virus persistence with frequent reactivation and excretion (in up
to 50%).
Transmission: mainly through oro-oral contact (HSV-1) and through intimate contact
(HSV-2), rarely droplets or smear infection.
Clinical presentation: primary HSV-1 based infection is usually asymptomatic, some-
times referred to as gingivostomatitis. Primary genital HSV-2 based infection is often
associated with blistering and ulceration, pain, fever and dysuria. Reactivation of HSV-1
and HSV-2 typically leads to painful vesicular eruptions (asymptomatic reactivation
with virus excretion is possible).
Complications: conjunctivitis, herpes simplex dermatitis, eczema herpeticum, general-
ized HSV infection with hepatitis or pneumonia, meningitis, herpes encephalitis, Bell’s
palsy, herpes of the neonate.

Encephalitis, meningoencephalitis CSF, EDTA whole blood
Retinitis Aqueous humor
Bell’s palsy Saliva
Pneumonia BAL
(Continued)
(Continued)

Herpes of the neonate CSF, EDTA whole blood, serum, conjunctival swab,
nasopharyngeal swab or aspirate, skin swab
Generalized HSV infection EDTA whole blood
Active HSV-Infection Swabs (herpetic lesions)
Note: detection of HSV DNA in saliva or mucosal swabs is possible even in clinically healthy
individuals through asymptomatic viral shedding. If there is clinical evidence of herpes encephalitis,
a negative HSV DNA result should not be the sole criterion for antiviral treatment discontinuation.
CSF sampling should always be done before starting antiviral treatment. If vesicular eruptions
are present, vesicular fluid should always be taken using swabs. Regarding suspected diagnosis
of herpes of the neonate, testing for HSV DNA is also meaningful in the absence of herpetic
lesions.
Human herpes virus 6 (HHV-6) (Family: Herpesviridae; 2 types, A and B)

Epidemiology: worldwide distribution, seroprevalence in childhood approximately 95%.
Transmission: mainly through saliva, through sexual contact or perinatal.
Clinical presentation: exanthema subitum (roseola infantum – 3-day fever) with fever
and volatile rash.
Complications: in rare cases, encephalitis, meningitis, (fulminant) hepatitis. In immuno-
suppressed patients, virus reactivation possible with interstitial pneumonia and organ
rejection.

Suspected active HHV-6 infection EDTA whole blood, serum
Interstitial pneumonia BAL, EDTA whole blood, serum
Encephalitis, meningitis CSF, EDTA whole blood, serum
Hepatitis (fulminant) Liver biopsy, EDTA whole blood, serum
Pre-emptive monitoring/suspected HHV-6 EDTA whole blood, serum
infection under immunosuppression
Note: detection of HHV-6 DNA in peripheral blood lymphocytes, lymphatic tissue and biopsies is
of limited clinical significance because of virus persistence.
Human herpes virus 7 (HHV-7) (Family: Herpesviridae)

Transmission: mainly through saliva, probably through droplets.
Clinical presentation: exanthema subitum (roseola infantum – 3-day fever) with fever
and volatile rash.
1.1 Viruses 9
Complications: febrile seizure, diarrhea, vomiting. In immunosuppressed patients, virus

reactivation is possible with interstitial pneumonia and organ rejection.

Suspected active HHV-7 infection EDTA whole blood, serum
Note: detection of HHV-7 DNA in peripheral blood lymphocytes, lymphatic tissue and biopsies is
of limited clinical significance because of virus persistence.
Human herpes virus 8 (HHV-8) (Family: Herpesviridae; Kaposi’s sarcoma-associated

herpes virus)
Epidemiology: worldwide distribution, endemic Kaposi’s sarcoma (KS) in Africa, iatro-
genic (in organ transplant recipients) and HIV-associated KS. Seroprevalence in North-
ern Europe and the U.S. approximately 3%, with higher prevalence in risk groups (male
homosexuals and bisexuals).
Transmission: mainly through sexual contacts or iatrogenic.
Incubation period: a few weeks to a few months.
Clinical presentation: Kaposi’s sarcoma, Castleman’s disease.

Suspected Kaposi’s sarcoma EDTA whole blood, serum, biopsy
Castleman’s disease EDTA whole blood, serum
Human immunodeficiency virus type 1 (HIV-1) and 2 (HIV-2) (Family: Retroviridae;

HIV-1 subgroup M, with subtypes A–K, subgroups O, N and circulating recombinant
forms; HIV-2 subtypes A–E)
Epidemiology: worldwide distribution of HIV-1, specific risk groups include male ho-
mosexuals, intravenous drug abusers (‘needle sharing’), and professional sex workers.
HIV-2 occurs mainly in Western Africa and India.
Transmission: parenteral through blood, sexual contacts, intravenous drug abuse or
perinatal (including breastfeeding).
Incubation period: 2–8 weeks until primary symptoms; 2–10 years (and longer) until
AIDS is established.
Clinical presentation: primary symptoms include mononucleosis-like illness, non-
specific feverish infection, sometimes a maculopapular rash, and acute neurological
symptoms. AIDS is characterized by a cellular immune defect resulting in opportunistic
infections and tumors.

Unclear serology/borderline results of immunoblot EDTA plasma
confirmation
Suspected acute infection (serological window) EDTA plasma
Unclear neurological symptoms, encephalopathy CSF, EDTA plasma
Clarification of mother-to-child transmission EDTA plasma taken from the newborn
Sick newborn of HIV-positive mother EDTA plasma
Monitoring of HAART EDTA plasma
Suspected development of drug resistance, EDTA plasma
failure of HAART
Note: if unclear serology (repeatedly reactive ELISA with negative or borderline immunoblot
confirmation) exists, HIV RNA should always be tested. The newborn of an HIV-positive mother
should be tested on HIV RNA immediately after birth; if HIV RNA is undetectable retesting should
be done after 6–8 weeks and after 12–16 weeks. If failure of HAART is suspected, sequencing to
check for antiretroviral drug resistance should be performed.
Influenza viruses (Family: Orthomyxoviridae; 3 genera, A, B and C)

Epidemiology: worldwide distribution, annual epidemics in some countries and spo-
radic pandemics.
Clinical presentation: systemic and respiratory disease. A sudden onset of fever, with
headache, dry cough, myalgia and sore throat are observed.
Complications: pneumonia (often bacterial super-infection), myocarditis, myositis with
myoglobinuria, Reye’s syndrome.

Suspected influenza infection Nasopharyngeal swab or aspirate,
throat washing
Tracheobronchitis Nasopharyngeal swab or aspirate,
throat washing, BAL
Pneumonia BAL
Measles virus (Family: Paramyxoviridae)

Epidemiology: worldwide distribution, significant importance in developing countries
(particularly Africa) with a relatively high mortality rate.
Transmission: droplets infection.
Incubation period: 10–14 days (infectivity from approx. 5 days before the onset of rash
until their healing).
1.1 Viruses 11
Clinical presentation: typical symptoms with conjunctivitis, Koplik’s spots and maculo-
papular (morbilliform) rash.
Complications: otitis media, diarrhea, Hecht’s giant cell pneumonia, bacterial super-
infections, central nervous system involvement including subacute measles encephalitis
(SME), acute post-measles encephalitis (APME) and subacute sclerosing panencepha-
litis (SSPE).

Suspected acute measles infection, unclear EDTA whole blood, nasopharyngeal swab or
serological constellation aspirate, throat washing, urine
Hecht’s giant cell pneumonia BAL
Encephalitis CSF, brain biopsy
Note: for the diagnosis of APME and SSPE, the detection of antibodies in CSF is relevant while the
use of PCR is not.
Metapneumovirus (MPV) (Family: Paramyxoviridae; 2 subtypes, A and B)

Epidemiology: worldwide distribution, seroprevalence in children >95%.
Clinical presentation: disorders of the upper respiratory tract, bronchiolitis, pneumonia.

Upper respiratory tract infections Nasopharyngeal swab or aspirate, throat
washing, induced sputum
Pneumonia, bronchiolitis BAL
Mumps virus (Family: Paramyxoviridae)

Epidemiology: worldwide distribution, increased incidence in winter and spring.
Clinical presentation: one- or two-sided parotitis; unilateral orchitis.
Complications: sterility (men), meningitis, rarely pancreatitis or diabetes mellitus.

Suspected mumps meningitis CSF
Parotitis Saliva, urine
Norovirus (Family: Caliciviridae)

Epidemiology: increased incidence in winter (‘winter vomiting’) with some tenacious
nosocomial outbreaks.
Transmission: fecal-oral, aerosols, smear infection, contaminated food.

Incubation period: 10–50 h.
Clinical presentation: gastroenteritis with nausea, vomiting, diarrhea, fever, headache
and myalgia.
Sample material
Gastroenteritis (suspected nosocomial outbreak) Stool, vomit
Papillomaviruses (HPV) (Family: Papillomaviridae; approx. 150 genotypes)

Transmission: through direct or intimate skin/mucosal contact.
Clinical presentation: often inapparent, infections of the skin and mucous membranes,
depending on the virus type, ‘low risk’ types (6, 11, 42, 43, 44, 54, 61, 70, 72, 81) which
cause mainly benign diseases and ‘high-risk’ types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56,
58, 59, 68) which have the potential for causing malignant diseases. Diseases include:
Verruca plantaris, epidermodysplasia verruciformis (EV) with skin cancer and condylo-
mata acuminata plana, conjunctival papilloma, cervical intraepithelial neoplasia (CIN),
Butchers warts, M. Bowen, cervix-, penis-, anus-, throat- and oral cavity-carcinoma.
Condylomata acuminata, epidermodysplasia Cell containing swabs, biopsy

verruciformis, conjunctival papilloma, Butchers warts,
CIN, HPV-associated carcinomas
Note: pathogen detection and typing is relevant for risk assessment of cervical neoplasia.
Parainfluenzavirus type 1–4 (Family: Paramyxoviridae; 4 serotypes)

Epidemiology: worldwide distribution, type 4 mainly found in America. In temperate
latitudes, annual outbreaks occur in the winter months, mainly in children less than 3
years of age (seroprevalence in childhood approx. 90%).
Clinical presentation: acute respiratory tract disorders, mainly in young children. In
adults, infection is usually mild or unapparent.

Suspected acute parainfluenza infection Nasopharyngeal swab or aspirate, throat washing,
induced sputum
Pneumonia BAL
1.1 Viruses 13
Parvovirus B19 (Family: Parvoviridae)

Epidemiology: worldwide distribution, seroprevalence in adults approximately 70%.
Transmission: droplets, possibly through blood and blood products, transplacental
infection.
Clinical presentation: Erythema infectiosum (Fifth disease).
Complications: in immunocompetent patients lymphadenopathy and arthralgia; in im-
munocompromised patients chronic infection with chronic anemia, and thrombocy-
topenia. Further, meningitis, encephalopathy, myocarditis, vasculitis and hepatitis can
be associated with Parvovirus B19 infection. Vertical Parvovirus B19 infection can lead
to hydrops fetalis (10%), with pre-existing anemia and risk of aplastic crisis.

Anemia EDTA whole blood, bone marrow
Aplastic crisis EDTA whole blood
Meningitis, encephalopathy EDTA whole blood, CSF
Myocarditis, hepatitis, vasculitis EDTA whole blood, biopsy
Prenatal infection EDTA whole blood
Suspected hydrops fetalis Amniocyte biopsy, amniotic fluid
Note: Parvovirus B19 can persist in the bone marrow without clinical symptoms.
Polyomaviruses (BKPyV, JCByV) (Family: Polyomaviridae)

Epidemiology: worldwide distribution, seroprevalence in adults approximately 70%.
Transmission: probably through the respiratory tract.
Incubation period: unknown.
Clinical presentation: only in immunocompromised patients, BKPyV causes infection of
the urinary tract with (hemorrhagic) cystitis and ureter stenosis or systemic disease (BKV
associated nephropathy in 1–10% of renal-transplant recipients and sporadic in other
solid-organ transplant patients). JCPyV causes progressive multifocal encephalopathy
(PML), especially in AIDS patients.

Systemic disease EDTA whole blood, urine
PML CSF, brain biopsy
Preemptive monitoring after kidney transplantation, EDTA whole blood, urine
suspected BKV associated nephropathy in renal-
transplant recipients, hemorrhagic cystitis in allogenic
bone marrow transplant recipients
Note: increased risk of systemic infection with BKV after kidney transplantation. If urine is tested, it
has to be considered that intermittent excretion in clinically healthy individuals is also possible.
Rabies virus (Family: Rhabdoviridae)

Epidemiology: worldwide distribution, annually approximately 60 000 cases, mainly in
developing countries.
Transmission: through a bite from, or narrow (mucosal) contact with an infected animal.
Incubation period: usually 3–12 weeks, rarely a few days up to 6 years.
Clinical presentation: in approximately 70% rabies-related encephalitis with headache,
followed by tonic spasms of the pharyngeal-, laryngeal- and respiratory muscles, in-
creased salivation, extreme hydrophobia, death (100%) as a result of heart paralysis.
In approximately 30% paralytic rabies (‘silent rage’) similar to that of Guillain-Barré
syndrome can be observed.

Suspected rabies Skin biopsy
Suspected rabies encephalitis CSF, brain tissue
Respiratory syncytial virus (RSV) (Family: Paramyxoviridae; 2 types, A and B)

Epidemiology: worldwide distribution, epidemic infections in late autumn and winter.
Clinical presentation: fever with rhinitis, laryngitis, bronchiolitis and pneumonia. For
infants <4 months, RSV infection is partially life threatening. Older children and adults
mainly have milder symptoms such as rhinitis and tracheobronchitis.
Complications: otitis media, apnea.

Rhinitis, laryngitis, tracheobronchitis, bronchiolitis, Nasopharyngeal swab or aspirate, BAL
pneumonia
Otitis media Nasopharyngeal swab or aspirate
Rhinovirus (Family: Picornaviridae; more than 100 serotypes; 3 species: RV-A, RV-B,
RV-C)
Epidemiology: RV causes respiratory illnesses, including the so called ‘common cold’.
Distribution is worldwide and affects all age groups. Prevalence is throughout the year
with peaks in early fall and spring.
Transmission: Droplets and smear infection.
Incubation period: 12 hours to 3 days.
Clinical presentations: Rhinitis, rhinosinusitis, pharyngitis, acute otitis media, bronchi-
olitis, pneumonia.
Complications: Asthma exacerbation, acute exacerbation of chronic obstructive pul-
monary disease and cystic fibrosis, fatal pneumonia in immunocompromised patients
after solid organ/bone marrow transplantation and in hematopoetic stem cell transplant
recipients.
1.1 Viruses 15

Indication/Suspected diagnosis Sample material
Respiratory tract infection, pneumonia Nasopharyngeal swab or aspirate, throat
washing, induced sputum, BAL
Otitis media Middle ear fluid specimen
Notes: Rhinovirus shedding normally stops within 11 to 21 days. Persistence may represent serial
infections.
Rotavirus (Family: Reoviridae, serogroups A–F)

Epidemiology: worldwide distribution, in developing countries every year approximately
800 000 rotavirus-related deaths in children.
Transmission: oral-fecal.
Clinical presentation: severe gastroenteritis with diarrhea and vomiting, fever and dehy-
dration, especially during infancy.
Complications: encephalitis, exsiccosis.

Severe gastroenteritis Stool
Suspected rotavirus encephalitis CSF
Note: the detection of viral antigen in stool is currently the diagnostic tool of choice. For rapid
confirmation of nosocomial outbreaks, the use of automated repetitive-sequence-based PCR is
recommended.
Rubella virus (Family: Togaviridae)

Transmission: droplets, transplacental infection.
Clinical presentation: flu-like symptoms with nuchal lymphadenopathy and small dot-
ted exanthema; in adults, partially volatile arthritis.
Complications: vertical rubella infection in the first trimester of pregnancy can lead to
embryopathy with spontaneous stillbirth (20%); further to ‘classic’ Gregg’s triad with
deafness, cataract and heart defects and sometimes even mental retardation.

Suspected vertical rubella infection EDTA whole blood, chorionic villi biopsy,
amniotic fluid, fetal blood
Suspected congenital rubella infection Urine, nasopharyngeal swab or aspirate, lens
material, EDTA whole blood, CSF
Cataract of the newborn Aqueous humor, lens material
(Continued)
(Continued)
Suspected acute rubella infection, unclear EDTA whole blood, saliva, nasopharyngeal swab
serological constellation or aspirate, throat washing
Note: if vertical transmission of rubella infection is suspected, a chorionic villi biopsy can be used
during weeks 11–18 of pregnancy for NAT testing (besides maternal EDTA whole blood testing).
Amniotic fluid should be tested during weeks 18–22 of pregnancy; afterwards IgM antibody testing
of fetal blood is recommended.
Tick-borne encephalitis virus (TBEV) (Family: Flaviviridae; 3 subtypes)

Epidemiology: TBEVs are endemic to forest areas in the majority of European countries.
TBE is the most important arthropod-transmitted viral disease in Europe.
Transmission: tick-bite.
Clinical presentation: frequently biphasic illness, starting with flu-like symptoms,
fever prior to neurological symptoms. Of those infected, 10–30% develop a severe
neurological disease, such as meningitis, meningoencephalitis, or meningoencepha-
lomyelitis.
Complications: long-term sequelae (e.g. flaccid paralysis); the case-fatality rate is up
to 5%.
Aseptic meningitis, meningoencephalitis, meningomyelitis CSF (EDTA whole blood)
Note: the detection of specific antibodies in serum and in CSF is currently the diagnostic method
of choice.
Varicella zoster virus (VZV) (Family: Herpesviridae)

Epidemiology: worldwide distribution, seroprevalence in adults more than 95%.
Transmission: droplets or mucous membrane contact, transplacental infection.
Clinical presentation: primary infection is also referred to as chickenpox. During child-
hood, it usually shows a milder course; in adults, especially immunocompromised
patients, secondary bacterial infections, sepsis, hemorrhagic chickenpox, pneumonia or
encephalitis can occur. The VZV reactivation leads to herpes zoster (shingles).
Complications: post-zoster neuralgia, secondary bacterial infections, encephalitis,
generalized zoster infection with septicemia, zoster ophthalmicus, Ramsey-Hunt syn-
drome. In cases of primary infection between week 13 and week 20 of pregnancy
the varicella congenital syndrome can be observed (in approx. 2%) with skin lesions
in dermatomal distribution, neurologic defects, eye diseases and skeletal anomalies.
A perinatal infection results in chickenpox of the neonate with significant lethality
up to 35%.
1.2 Bacteria 17

Encephalitis CSF, EDTA whole blood
Hemorrhagic chickenpox Skin swab, skin biopsy, EDTA whole blood
Ramsey-Hunt syndrome Saliva, skin swab
Chickenpox of the neonate Skin swab, EDTA whole blood
Clarification of a florid varicella-zoster virus infection Skin swab
Generalized varicella zoster infection Skin swab, EDTA whole blood
Note: sampling should always be done before starting antiviral treatment. If vesicular eruptions
are present, vesicular fluid should always be taken using swabs for sampling. If saliva is tested,
it has to be considered that viral shedding into the oral cavity can also be observed in healthy
individuals.
West Nile virus (WNV) (Family: Flaviviridae)

Epidemiology: distribution in Africa, parts of Europe, India, Israel, USA.
Transmission: bite through Culex spp. mosquitoes. Isolated vertical transmission possible.
Clinical presentation: mostly asymptomatic course (80%), fever with flu-like symptoms,
headache and eruptions.
Complications: encephalitis, menigoencephalitis, flaccid paralysis (especially in per-
sons over 70 years).

Encephalitis, meningoencephalitis, flaccid paralysis CSF, EDTA whole blood
Suspected West Nile virus infection EDTA whole blood
1.2 Bacteria
Bordetella spp. (Family: Proteobacteria; Aerobes; 3 species, Bordetella pertussis, Borde-

tella parapertussis, Bordetella bronchiseptica)
Epidemiology: worldwide distribution with seasonal increases in wintertime.
Transmission: droplets.
Clinical presentation: whooping cough, starting with an initial catarrhal phase with
symptoms similar to those of the common cold, proceeding with a dry and persistent
cough.
Complications: subconjunctival bleedings, otitis media, pneumonia, apnea.
Whooping cough Nasopharyngeal swab or aspirate
Note: the combination of real-time PCR and single-serum serology (IgA) are currently the most
efficient diagnostic tools. The molecular assay should be able to distinguish between B. pertussis
and B. parapertussis.
Borrelia burgdorferi (Family: Spirochaetaceae)

Epidemiology: most common species in Europe and USA, mostly in the warm months
of the year. In endemic areas, 2–50% of the ticks are infected.
Transmission: tick-bite.
Incubation period: depending on the stadium of the disease, stadium I: 1–16 weeks,
stadium II: months, stadium III: years.
Clinical presentation: stadium I: erythema chronicum migrans (ECM); Stadium II: facial
palsy, meningoencephalitis, myocarditis, lymphadenosis cutis benigna; Stadium III:
arthritis, acrodermatitis chronica atrophicans, polyneuropathia; neuroborreliosis.

Arthritis Joint puncture fluid
Neuroborreliosis CSF
Note: high analytic sensitivity required because of low bacterial concentration.
Chlamydia trachomatis (Family: Chlamydiaceae)

Epidemiology: worldwide distribution, genital chlamydia infection is the most frequent-
ly diagnosed bacterial sexually transmitted infection worldwide, infection does not pre-
vent against re-infection.
Transmission: Chlamydia trachomatis is transmitted during unprotected vaginal, anal
or oral sex and can be passed from an infected mother to the newborn during vaginal
delivery.
Clinical presentation: urethritis, cystitis, cervicitis, lymphogranuloma venerum,
inclusion-body conjunctivitis.
Complications: pelvic inflammatory disease, prostatitis, epididymitis, sterility, arthritis
(Mb. Reiter), perinatal infection with trachoma and blindness of the newborn.

Cervicitis Cervical swab (cell containing)
Urinary tract infections, prostatitis, epididymitis, Urethral swab (males), urine
arthritis dysenterica (Reiter’s syndrome)
1.2 Bacteria 19
Chlamydophila pneumoniae (Family: Chlamydiaceae)

Epidemiology: worldwide distribution, Chlamydophila pneumoniae affects all age
groups and is most common among the older teenage-age and the 60–79-year-old
groups. Re-infection is common after a short period of immunity. The incidence is
1/1000 per year. Chlamydophila pneumoniae causes 10% of community-acquired
pneumonias.
Clinical presentation: respiratory tract infections including pharyngitis, laryngitis,
pneumonia.
Respiratory tract infections, pneumonia Induced sputum, BAL
Enterococcus, vancomycin-resistant (VRE) (Family: Enterococcaceae)

Epidemiology: Enterococcus faecalis (90–95%) and Enterococcus faecium (5–10%) are
common commensal microorganisms in the intestines of humans: the most important
feature of this genus is the high level of endemic antibiotic resistance. In the last two
decades, particularly virulent strains of Enterococcus that are resistant to vancomycin
(VRE) have emerged in nosocomial infections of hospitalized patients.
Clinical presentation: important clinical infections caused by enterococci include
wound infections, urinary tract infections, bacteremia, bacterial endocarditis, diverticu-
litis with diarrhea and meningitis.
Complications: sepsis with an overall rate of lethality up to 58%.

Wound infections Swab
Meningitis CSF
Endocarditis, septicemia EDTA whole blood
Note: the phenotypes VanA and VanB are the most common acquired VREs. For infection control
purposes, the identification of species level is mandatory to distinguish from non-transferable
intrinsic VanC resistance.
Legionella spp. (Family: Legionellaceae, Aerobes; approx. 50 species and 70 serogroups)

Epidemiology: worldwide distribution, common sources of Legionella include cooling
towers, air conditioning systems, domestic hot water systems, fountains and similar dis-
seminators that draw upon a public water supply; natural sources include freshwater ponds
and creeks. Legionella pneumophila is the most epidemiologically relevant species.
Transmission: aerosols.
Clinical presentation: legionellosis with multifocal necrotizing pneumonia; pontiac
fever (without pneumonia).
Complications: fatality rate exceeding 20%.


Legionellosis, pneumonia BAL
Mycobacterium spp. (Family: Mycobacteriaceae, Aerobes; approx. 100 species)

Epidemiology: worldwide distribution, approximately 15 million infections/year, classi-
fied into several major groups for the purpose of diagnosis and treatment: M. tuberculosis
complex (MTBC) including M. tuberculosis, M. bovis, M. africanum, M. microti,
M. canetti, M. pinnepedi, M. caprae, and the vaccination strain Bacillus Calmette-
Guerin (BCG); nontuberculous mycobacteria (NTM) or atypical mycobacteria, which
can cause pulmonary disease similar to tuberculosis, lymphadenitis, skin disease, or
disseminated disease.
Transmission: aerosols, aerogen (pulmonary tuberculosis); gastrointestinal ingestion and
skin lesions (extrapulmonary tuberculosis).
Incubation period: 4–12 weeks, sometimes years.
Clinical presentation: unapparent infection, B-symptomatic, cough.
Complications: pleuritis, pneumonia, miliary TB, meningitis, arthritis, osteomyelitis,
urogenital TB, sepsis landouzy (rare).

Pulmonary tuberculosis Induced sputum, BAL, pleural effusion
Extrapulmonary tuberculosis CSF, urine, bone marrow, organ tissues, lymphatic
tissue, joint puncture fluid
Note: molecular assays should include detection of atypical mycobacteria. NAT may provide rapid
identification of different clinically relevant mycobacteria.
Mycoplasma pneumoniae (Family: Mycoplasmataceae, Aerobes)

Clinical presentation: respiratory tract infections including pharyngitis, tracheobronchi-
tis, interstitial pneumonia, bronchiolitis.
Complications: subsegmental and segmental atelectasis of the lung; otitis media.
Respiratory tract infections, pneumonia Induced sputum, BAL
Note: NAT enables epidemiological monitoring of macrolide resistance to M. pneumoniae.

1.2 Bacteria 21
Neisseria spp. (Family: Neisseriaceae, Aerobes; 2 human pathogenic species, Neisseria

meningitidis and Neisseria gonorrhoeae)
Transmission: droplets (N. meningitidis); sexually transmitted or perinatal (N. gonor-
rhoeae).
Incubation period: 2–10 days (N. meningitidis); 2–7 days, sometimes weeks (N. gonor-
rhoeae).
Clinical presentation: for N. meningitides: meningitis, sepsis; for N. gonorrhoeae: gon-
orrhea with purulent (or pus-like) discharge from the genitals which may be foul smell-
ing, inflammation, redness and swelling of the outer genitals, acute urethritis.
Complications: for N. gonorrhoeae: pelvic inflammatory disease, bartholinitis, sterility
(women); prostatitis, proctitis, epididymitis, sterility (men); disseminated gonococcal infec-
tion with sepsis, endocarditis, meningitis; perinatal infection with conjunctivitis purulenta.

Meningitis CSF
Gonorrhea, urethitis, prostatitis Urethral swab, urine (men)
Cervicitis Cervical swab
Pelvic inflammatory disease, bartholinitis, proctitis Vaginal/cervical/anal swab
Sepsis EDTA whole blood, CSF
Note: always note time of specimen collection.
Methicillin-resistant Staphylococcus aureus (MRSA) (Family: Staphylococcaceae,

Aerobes)
Epidemiology: worldwide distribution. MRSA is sub-categorized as community-
acquired MRSA (CA-MRSA) or healthcare-associated MRSA (HA-MRSA).
Transmission: smear infection.
Incubation period: highly variable.
Clinical presentation: wound infections, pneumonia.
Complications: necrotizing pneumonia (especially CA-MRSA), endocarditis and sepsis.

Wound infection Swab
Pneumonia BAL
Bacteremia, endocarditis EDTA whole blood
Note: detection is mainly based on the mecA gene. Blood culture is still mandatory for the diagnosis
of bacteremia.
Group B Streptococcus (GBS) (Family: Streptococcaceae, Aerobes)

Epidemiology: worldwide distribution, GBS is part of the normal flora of the gut and gen-
ital tract and is found in 20–40% of women, carriers of the organism are asymptomatic.
Transmission: perinatal infection.
Incubation period: a few hours.
Clinical presentation: neonatal infections including pneumonia, meningitis, septicemia.
Complications: sepsis, hearing loss as long-term sequelae of GBS-meningitis.

Suspected GBS infection Vaginal or cervical swab, anal swab
Meningitis CSF
Bacteremia, sepsis EDTA whole blood
Note: NAT for rapid intrapartum screening.
Streptococcus pneumoniae (Family: Streptococcaceae, Aerobes)

Epidemiology: worldwide distribution, Streptococcus pneumoniae can be part of the
normal upper respiratory tract flora (5–10% of healthy adults and 20–40% of healthy
children) with the potential to become pathogenic.
Transmission: droplets, smear infection, endogenous infection.
Incubation period: highly variable.
Clinical presentation: respiratory tract infections, sinusitis, pneumonia, otitis media,
meningitis, bacteremia.
Complications: sepsis, brain abscess.

Respiratory tract infections, pneumonia Nasopharyngeal swab, induced sputum, BAL,
pleural effusion
Meningitis CSF
Bacteremia, sepsis EDTA whole blood
Note: blood culture is still mandatory for the diagnosis of bacteremia.
1.3 Fungi
Aspergillus spp. (Group: Molds; more than 100 species)

Epidemiology: worldwide distribution. Approximately 10 species are medically relevant
including Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus and Aspergillus
flavus.
Transmission: spore inhalation.
1.2 Bacteria 23
Clinical presentation: pulmonary aspergillosis includes allergenic bronchopulmonal

aspergillosis (ABPA), aspergilloma in the lung and pneumonia (immunocompromised
patients). Extrapulmonary aspergillosis includes rhino sinusitis, allergic fungal sinusitis
(AFS), aspergilloma in the brain and endocarditis.
Complications: invasive aspergillosis, sepsis.

Pulmonary aspergillosis Lung tissue, BAL, EDTA whole blood
Aspergilloma, AFS, endocarditis Fungus ball, sinunasal mucus, organ tissue, CSF
Acute invasive aspergillosis EDTA whole blood, CSF
Note: detection of aspergillus DNA in EDTA whole blood samples is not necessarily associated
with invasive aspergillosis. Monitoring including a molecular assay (in addition to galactomannan
testing) could be useful for patients at risk.
Candida spp. (Group: Yeasts; more than 150 species)

Epidemiology: worldwide distribution. Approximately 7 species are medically relevant
including Candida albicans, the most significant pathogenic species, Candida tropi-
calis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida dubliniensis
and Candida lusitaniae.
Transmission: candida is commonly present in humans, and its growth is normally lim-
ited by the human immune system and by other microorganisms, such as bacteria oc-
cupying the same locations (niches) in the human body. A weakened or undeveloped
immune system or metabolic illnesses such as diabetes are significant predisposing
factors of candidiasis.
Clinical presentation: in immunocompetent persons, candidiasis usually presents as a
localized infection of the skin or mucosal membranes, including the oral cavity (thrush),
the pharynx or esophagus, the gastrointestinal tract, the urinary bladder, or the genitalia,
causing vaginal irritation, vaginitis or balanitis.
Complications: in immunocompromised patients Candida spp. has the potential to be-
come systemic, causing candidemia and sepsis.

Mucocutaneous candidiasis Oral swab, vaginal swab
Pneumonia BAL, lung tissue, bronchial/tracheal secretion
Invasive candidiasis EDTA whole blood
Note: the diagnosis of pulmonary candidiasis is based on histological demonstration of the yeast in
lung tissue with associated inflammation. Early detection of candida DNA in whole blood samples
enables earlier commencement of antifungal therapy. However, the use of beta-D-glucan testing in
serum may be superior for the diagnosis and therapy monitoring of candidemia.
1.4 Protozoa
Toxoplasma gondii (Family: Sarcocystidae)

Epidemiology: worldwide distribution, estimated seroprevalence between 30% and
65%, with large variations between countries. Primary host is the felid (cat) family.
Transmission: ingestion of raw or partly cooked meat containing Toxoplasma cysts,
hand-to-mouth contact after handling undercooked meat or contaminated cat feces,
contaminated drinking water, transplacental infection or receiving an infected organ
transplant or blood transfusion.
Clinical presentation: mostly asymptomatic with mild fever and swollen lymph nodes.
Complications: in immunocompromised patients and following congenital infection,
severe toxoplasmosis with encephalitis and necrotizing retinochoroiditis.

Suspected infection CSF, amniotic fluid, EDTA whole blood,
aqueous humor
2 Stability of the specimen during preanalytics
Georg Endler; Georg Slavka
Although sample processing is usually well standardized and covered by established

standards in the molecular diagnostic laboratory, preanalytic procedures outside the
laboratory usually follow unwritten traditions with considerable interindividual vari-
ability. Studies have shown that preanalytical errors make up to 85% of all laboratory
errors, 95% of them occurring outside the laboratory. In particular, molecular assays are
sensitive to suboptimal preanalytic conditions. False-negative results may occur due to
degradation of nucleic acids during transport or polymerase chain reaction (PCR) inhib-
itors, resulting in delayed or misdiagnosis of infections. However, contamination may
cause false-positive results, which could have severe consequences. The main issues of
concern during sample transport and storage include nucleic acid integrity, contamina-
tion, sample identity, and the risk of environmental hazards due to infectious material.
2.1 Degradation of DNA
In biological matrices, DNA may be degraded rapidly due to the presence of desoxy-
ribonucleases (DNases). In vivo, DNases play a major role to ensure individual genomic
integrity. Thus, it is not surprising that DNases also represent a major defense mechanism
against biological pathogens in the induction of apoptosis. Apoptosis is characterized
by cell shrinkage, nuclear condensation, and internucleosomal DNA cleavage. Besides
the central role of caspases and other proteases, cell death triggers DNA degradation so
that DNases have an active role in apoptotic cell death. The best-characterized apop-
totic DNase is CAD, a neutral Mg-dependent endonuclease. Its activity is regulated
by its inhibitor, ICAD which is cleaved by caspases. Other neutral DNases such as
endonuclease G and GADD have been shown to cleave nuclear DNA in apoptotic
conditions. In cells, the cytosolic pH is maintained at 7.2, mostly due to the activity of
the Na+/H+ exchanger. In many apoptotic conditions, a decrease in the intracellular pH
has been shown. This decrease may activate different acid DNases, mostly when the pH
decreases below 6.5. Three acidic DNases II are known at present: DNase IIa, DNase
IIβ and L-DNase II. Apart from dedicated DNases, several proteins also have DNase
activity including, for example lactoferrin. Thus, DNases are present in all body fluids
fulfilling a variety of physiological functions. In patients with cystic fibrosis, inhalatory
DNases have been successfully applied to reduce the viscoelasticity of sputum and to
enhance the clearance of secretions.
Although essential in vivo, DNases are the main cause of in vitro DNA degradation
in the biologic matrix, which can lead to a false-negative result. Because all DNases are
either Mg2+ or Ca2+ dependent and usually activated at a pH of 6.5 or lower, inactiva-
tion of DNases can be achieved easily through adequate strategies (򐂰Tab. 2.1). In daily
26 2 Stability of the specimen during preanalytics
Tab. 2.1: Strategies for inactivation of DNases.
Depletion of Mg2+ and Ca2+ ions through chelating agents (e.g. EDTA)
Adjustment of pH to 7.5
Storage at –20°C or lower
Use of dried specimens
laboratory practice, it is advisable to store native specimens intended for DNA diagnos-
tics at –20°C or lower for long-term storage. In contrast, dried specimens can be stored
at ambient temperature.
Purified DNA can be stored safely in Tris–EDTA buffer at room temperature for 6
months or at 2–8°C for at least 1 year in the absence of DNases. Storage may be ex-
tended to up to 7 years at –20°C and to a longer period at –70°C or lower. Samples of
questionable purity should always be stored at or below –20°C to ensure DNA integrity.
The freezers used to store purified DNA should not be the ‘frost-free’ type because
this type of freezer cycles temperatures continually which may lead to deterioration of
nucleic acids by shearing.
2.2 Degradation of RNA
Ribonuclease (RNase) is a type of nuclease that catalyzes the degradation of RNA into
smaller components. All organisms studied contain several RNases of different classes
showing that RNA degradation is a very ancient and important process. Besides clean-
ing of cellular RNA that is no longer required, RNases play a key role in the maturation
of RNA molecules including messenger RNAs and non-coding RNAs. In addition, active
RNA degradation systems are a first defense against RNA viruses and provide the un-
derlying machinery for more advanced cellular immune strategies such as RNAi. Some
cells also secrete large quantities of non-specific RNases. RNases are thus extremely
common, resulting in a very short lifespan for any RNA that is not in a protected envi-
ronment. It is worth noting that all intracellular RNAs are protected from RNase activity
by a number of strategies including 5‘ end capping, 3‘ end polyadenylation, and folding
within an RNA protein complex (ribonucleoprotein particle or RNP).
RNA analysis and quantification require specimens with completely intact RNA to
produce optimal results. Although nonenzymatic hydrolysis of phosphodiester bonds is
favored by high temperature or pH and the presence of divalent cations (Mg2+, Mn2+),
an RNA sample is most likely to be degraded rapidly by a contaminating RNase. Com-
plete removal or inactivation of RNases during RNA extraction procedures has proven
to be very difficult. In fact, RNases may even be introduced into the sample accidentally
during handling. There are several possible sources for introduction of RNases in the
laboratory. RNases are ubiquitous in the environment and are found in pollen, dust, and
fingerprint grease.
If no specific RNase inhibitors are used, long-term storage of diagnostic RNA
samples should be done preferably at –80°C or lower to inhibit RNase activity, as
RNase may limit the stability of RNA even in frozen samples at –20°C. RNase inhibitors
such as concentrated formamide or 4M-guanidine isothiocyanate have been applied
2.3 Inhibitors of PCR 27
successfully for long-term storage of RNA up to 18 months; however, they may interact
with downstream applications such as RNA isolation or cDNA synthesis. Recently,
ready-to-use RNA stabilization solutions have been brought on the market allowing
storage of diagnostic RNA samples at 37°C for 1 day, 21°C for 1 week, 4°C for 1
month, and –20°C for long-term storage.
2.3 Inhibitors of PCR
For as long as the technique of PCR has been used, inhibition has been an obstacle
to success. All who use PCR are likely to be impacted by inhibitors at some time with
the wide range of non-blood specimens used for detection of pathogens and the often
suboptimal sampling conditions making PCR based assays for pathogen detection in
non-blood specimens especially vulnerable. Inhibitors generally exert their effects
through direct interaction with DNA or interference with DNA polymerases. Direct
binding of agents to single-stranded or double-stranded DNA may prevent amplification
and facilitate co-purification of the inhibitor and the DNA. Inhibitors may also interact
directly with the DNA polymerase to block enzyme activity. Furthermore, the cofactor
requirements of DNA polymerases may be the target of inhibition. Magnesium is a criti-
cal cofactor and agents that reduce Mg2+ availability or interfere with binding of Mg2+ to
the DNA polymerase can inhibit PCR. The presence of inhibitors in specimens has been
described in many publications. Common specimen types known to contain inhibi-
tors include blood, sputum, urine, feces, and tissues (򐂰Tab. 2.2). Additional sources of
inhibitors may be materials and reagents that are exposed to samples during process-
ing or DNA purification. These include excess potassium chloride, sodium chloride
and other salts, ionic detergents such as sodium deoxycholate, sarkosyl and sodium
dodecylsulfate, ethanol and isopropanol, and phenol.
The best way to avoid PCR inhibition is to prevent the inhibitor from being processed
with the sample. For inhibitors that are inherent to the sample, as is the case for blood
and certain body fluids, this is not always possible. In particular, vessels containing
heparin should be avoided. Heparin represents one of the most potent PCR inhibitors
being usually not removed completely through standard nucleic acid purification pro-
tocols. When a nucleic acid purification protocol is introduced, the method’s ability to
obtain efficiently inhibitor-free DNA from a wide range of sample types should be evalu-
ated. Furthermore, techniques proven to eliminate inhibitors from the purified template
DNA should be favored. There are several options to avoid effects from inhibitors not
Tab. 2.2: Specimen types and inhibitors.
Type of specimen Possible inhibitors

Blood Heme, hemoglobin, immunoglobulin G, lactoferrin, heparin
Urine Urea
Respiratory Acidic polysaccharides
Feces Bile salts, complex polysaccharides
Tissue Collagen, myoglobin
28 2 Stability of the specimen during preanalytics
eliminated during extraction. Firstly, the choice of the DNA polymerase may have a
significant impact on avoidance of inhibition. For instance, AmpliTaq® DNA polymerase
which is the standard for use with several commercial PCR kits is known to be among
those most sensitive to inhibition. This underscores the importance of sample handling
and extraction. Secondly, increasing the amount of DNA polymerase or using additives
such as bovine serum albumin which provides some resistance to inhibitors in blood
are proven methods. Finally, adding less DNA template to the amplification can often
improve performance significantly, if the assay sensitivity is sufficient to obtain valid
results from templates that contain inhibitors.
2.4 How can contamination during specimen collection and in the

laboratory be avoided?
During specimen collection, standard precautions as used for standard microbiology

tests are usually sufficient to minimize the risk of contamination. However, it is essential
that all vessels used are suitable for molecular tests and are free of nucleic acids and
nucleases.
Due to the extreme sensitivity of PCR, sample contamination in the laboratory re-
sulting in false-positive results is another important issue. Contamination of the PCR
reaction mixture may happen through previously amplified sequences or nucleic acids
from the PCR operator or other positive samples. It is therefore indispensable that the
sample remains tightly capped and not used for any other test prior to molecular testing.
Especially, pipettors in clinical chemistry analyzers have been reported to be a possible
source of cross contamination. Therefore, it would be preferable to perform molecular
testing on blood samples out of unopened primary blood tubes.
2.5 How can the sample identity be ensured?
Most errors in sample identification occur at the time of specimen collection. Thus, it
is highly recommended that the laboratory provides well-defined standard operating
procedures to minimize errors due to wrong patient identification.
The patient and the patient’s specimen must be clearly identified at the time of col-
lection. Specimens for molecular testing must be identified with a firmly attached label
bearing at least an identification number, the patient’s full name and date of birth (the
patient’s name alone is not sufficient for uniquely identifying a patient), and the speci-
men source (i.e. the type of tissue from which the specimen was taken). As suggested by
the NCCLS, each sample should be identified by two independent personal identifiers
of which at least one should be readable without technical equipment (e.g. barcode
readers).
2.6 Transport of diagnostic material
Because diagnostic material should be considered as potentially infectious, trans-

port outside the laboratory and the hospital is subject to national and international
2.6 Transport of diagnostic material 29
regulations. For road-bound transport in Europe, the European Agreement concerning

the International Carriage of Dangerous Goods by Road (ADR) applies. In parallel,
regulations issued by the International Air Transport Association (IATA) apply for the
air-bound transport of diagnostic material.
2.6.1 Category A Infectious Substances

The list of Category A Infectious Substances includes infectious substances capable of
causing permanent disability or life-threatening or fatal disease in otherwise healthy
humans. Samples are assigned UN 2814 – Infectious Substances based on the known
medical history and symptoms of the human source, endemic local conditions, and
professional judgment concerning individual circumstances. If there is any doubt as to
whether or not a pathogen may fall within this category, it should be transported accord-
ing to the Category A Infectious Substance transport regulations.
For transportation of any Category A Infectious Substance, packaging that meets
UN performance requirements for Class 6.2 substances as shown by design type test-
ing must be used. This is known as UN type-approved packaging and is certified and
labeled accordingly. The packaging must include an inner packaging, an itemized list
of contents, and an outer packaging. The inner packaging must comprise one or more
leak-proof primary receptacle(s) and leak-proof secondary packaging. For liquid speci-
mens, an absorbent material in sufficient quantity to absorb the entire contents must
be placed between the primary receptacle and the secondary packaging; if multiple
fragile primary receptacles are placed in a single secondary packaging, they must be
either individually wrapped or separated to prevent contact between them. The prima-
ry receptacle(s) or the secondary packaging must be capable of withstanding without
leakage an internal pressure producing a pressure differential of 95 kPa and tempera-
tures in the range –40°C to +55°C. Furthermore, the inner package must be capable of
successfully passing a drop test from a height of 1.2 m. There must be no leakage from
the primary receptacle(s) and these must remain protected by the absorbent material if
required.
Between the secondary packaging and the outer packaging, an itemized list of con-
tents must be enclosed. Finally, a rigid outer packaging of adequate strength for its
capacity, mass, and intended use must be added with the smallest external dimension
being not less than 100 mm. The outer packaging may contain refrigerants such as dry
ice. In that case, the primary receptacle(s) and the secondary packaging must maintain
their integrity at the temperature of the refrigerant used.
2.6.2 Category B Infectious Substances

The list of Category B Infectious Substances includes infectious substances that do
not meet the criteria for inclusion in Category A Infectious Substances. Samples are
assigned UN 3373 – Biological Substances, Category B. In clinical practice, the major-
ity of diagnostic material will be assigned category B.
For transportation of any Category B Infectious Substance, packing instructions are
essentially identical to those for any Category A Infectious Substance, except that
there is no need to use formally UN type-approved packaging. The outer packag-
ing must be clearly labeled on the external surface of the outer packaging using a
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*** START OF THE PROJECT GUTENBERG EBOOK BLUE AND

PURPLE ***
CONTENTS
BLUE AND PURPLE
SONGS TO A WIFE
BLUE AND PURPLE
FAR HORIZONS
HEBE’S EYES
SWEET FACE, I SEE THEE SHINE
TWO FLOWERS
THE MUSIC OF MY HEART
THE TRYST
NATURE’S LOVELINESS
YOU
THE LAST LIGHT
WHEN YOU WERE BORN
FORTUNE, YOU HAVE NAUGHT I NEED
LET US MAKE A GARDEN
SANCTUARY
STARS
REJUVENATION
A SONG
HEBE
SPRING
THE FAY
A SONG
THE GARDENER
REVELATION
THE KEEPER OF THE KISSES
MUSIC IN HADES
THE DREAM
THE BOON
JACK O’LANTERN
OH, TRANQUIL NIGHT
DESPAIR
TO A PHOTOGRAPH
SONG
HELL
ALONE
ROAMING
STORM
THE VOID
ABSENCE
WANDERING
DESTINY
EAST WIND
LULLABY
RESURRECTION
LAUGHTER
ALCHEMY
SURRENDER
WHAT IS DAY WITHOUT THE SUN?
THE MORN
THE GARDEN MADE FOR ME
TO A REPEATER
THE MUSIC OF A DREAM
A FLOWER
WHAT WOULD YOU DO?
HER SOUL’S SWEET HEART
I LOVE YOU SO!
LOVE’S LAST QUEST
CONSECRATION
BLUE AND PURPLE

FRANCIS NEILSON
NEW YORK: B. W. HUEBSCH

MCMXX
COPYRIGHT, 1920, BY
B. W. HUEBSCH
SONGS TO A WIFE
My love is beautiful and sweet; she is like a pale pink
rose full of the glory of dew and sun. Sharon’s garden
knows not a bloom so fair as she. Persia holds not a
fragrance so heavenly in its perfumed bowers. Oh, my
wondrous love, pour thy scented charm into the chalice of
my longing heart; fill with thy fresh splendour the air I
breathe; and give me youth to spend on thee, my well-
beloved. I am the gardener, born to tend one flower. My
flower is the radiance of a dawn in June. Like a veil of
glowing pearls my love spreads her light; she is my
morning, my joy of perfect hours. I will sing to her the
song fresh roses raise from their delicious petals when
night departs and they rejoice, sun-kissed, when all the
east is rich in gold. Lovely is my bloom. Her soul is the
first blossom given by Him who made the loveliness of
Spring.
BLUE AND PURPLE
IN BLUE AND PURPLE CLAD
A pearl set in the hollow of a stone,

Wrought deftly by an artist of great skill;
A sapphire ’twas that bore the pearl so still
Within its bosom; taking from its tone
Those fires of deep delight to Asia known.

Blent in an amethyst, the priceless twain
Enthronèd were, o’er glowing worlds to reign,
In gladness richer than the morn has shown.
She, like a regal lily of the field,

On which the sunset colours softly lay,
Forgot that life was sometime dark and sad;
She smiled, and bade all sorrow’s wounds be healed;
Then she was lovelier than heav’n’s best day—
Ethereal, in blue and purple clad.
FAR HORIZONS
We stand upon the barren shore,
And look far out to sea,
The crooning waves their burden pour
On you and me.
Our longing eyes, full of our mind,

On far horizons lie—
There, where our joy we hope to find
Before we die.
How fair the tempting journey seems—

Smooth lake of mystery—
How frail the craft, our forethought deems,
For such a sea!
For you and me, my lovely one,

And all our mighty hopes;
One step, dear love, and we have done,
And—cut the ropes?
Lashed to the past we stand, and fear

To leave our ties and pain;
Though (speaks the soul, if we would hear)
Our loss is gain.
Fear blurs the vision of our dream,

Fear fills our hearts with dread,
Soon we shall find upon life’s stream
Our souls are dead.
We stand upon the shore and mourn;

We grieve, despairingly,
To leave the fetters we have borne—
So patiently.
Or, do we grieve that we are weak,

Lack courage to be free,
And spurn the liberty we seek
For slavery?
Doubts lie—like pebbles on this strand—

In our sad souls, my mate.
Before us lies the promised land,
Behind us—fate.
Then, let us here together bide,

With faces toward the sea,
And hope that some fair morning’s tide
Take you and me.
HEBE’S EYES
The light of Hebe’s eyes

Gives colour to the skies,
It makes the azure dome
A radiant place,
Where love might find a home,
Sweet as her face.
Ethereal are the hues

Where birds a-wing would lose
Themselves in heavenly bliss;
As I would do—
If I might soar to kiss
Her eyes so blue!
SWEET FACE, I SEE THEE SHINE
Sweet face, I see thee shine

Out of the bosom of the east at morn;
Thy tenderness, divine,
Lies mirrored in the pearly dew at dawn.
The flower that smiles at me,

Holds in its cup the picture of your face;
In rivulets I see
The flowing charm of your abiding grace.
The sapling tells me how

Your body’s symmetry grows strong and straight;
The winds which whisper now,
Tell me your love and trust will not abate.
The steadfast stars above

Reflect the fervour of your constant mind,
Your deep unwav’ring love—
The rarest jewel eager man can find!
In nature’s soul thou art—

I see thee, hear thee, feel thee, ever near;
Dear love, thou art the heart
Of those eternal joys our souls revere.
TWO FLOWERS
I saw a bloom,
So beautiful,
My sad heart lost its gloom,
And cares that dull
The senses, soon passed far away—
The bloom brought joy into the day.
I saw her face

When she bent down
And kissed the bloom. Then grace
Was Hebe’s crown
Of loveliness, and there! upon
Her brow the light of heaven shone!
THE MUSIC OF MY HEART
The soft night, like a silent child

Before some wondrous thing,
Withholds its breath, as if beguiled
By songs the fairies sing.
It seems to stand and listen, still

As statue in a grove—
Perhaps it hears a fairy trill
A strain Titania wove.
Ah, no, the night hears not her song,

For it would then be glad;
And I have listened here so long,
I know the night is sad.
Now if it be a song that keep

The hour when night should part,
Then night must hear from my soul’s deep,
The music of my heart.
THE TRYST
My love is coming through green fields to me—
Why does she tarry so?
She knows I wait on cliffs above the sea,
And dare not to her go;
For I am prisoned to the spot where love
Has chained my feet, and must not call or move.
My love is gath’ring harebells, where the mead

Is starred with flowers to kiss
Her ling’ring feet; there sedges intercede,
And whisper runes of bliss—
Beseeching her to stay and heed me not—
For she can make a heaven of any spot!
My love is list’ning to the skylark’s song,

Delight is in her ears.
She cannot know her lover yearns so long,
And drinks his salty tears
To quench his thirst for all her winsome grace—
Her absence makes a desert of the place.
My love is drinking in the air which blows

The perfumes of the sea,
The journeying breeze wafts past me—well she knows—
Though me she cannot see!
Her lovely eyes, the yearning west would woo,

Look not on me while blooms in green fields sue.
She knows ’tis deathless love that holds me fast,

Chained to this rock so grim;
That I shall wait for her, until the last
Sun sets o’er ocean’s rim.
That flowers shall die and green fields fade and sear,
Ere I forsake the tryst to greet her here.
NATURE’S LOVELINESS
Yes, everywhere I go
I see the constant flow
Of nature’s loveliness—
But, oh, if I could see
These scenes, my love, with thee,
How bright would be their dress!
I can no more rejoice

Without your gracious voice
Exulting in my ear,
And nature, too, requires
Your soulful, ardent fires,
To beautify the year.
The tender blooms turn pale

When I, alone, through vale
And gully, searching pass;
They seem to say to me,
“Where is your mate? for we
Bloom only for your lass.”
My worship in the glen

Goes up for naught, dear, when
I stand alone in prayer;
The sea, the dunes, the trees,
Chide me, and every breeze
Sings lamentation there.
No, nothing in this world

Where gales and snows have whirled
A joyous tempest down—
Which spread a carpet fine
For thee to tread, can shine
As your belovèd crown.
They do not envy you

They do not envy you,
They love the sweet, the true—
They know you are sincere
As morning’s spark of light
In dew orbs shining bright,
When heaven is blue and clear.
They want your merry laugh,

Like rain for them to quaff;
They want to kiss your feet;
They want to see your eyes—
Full glory of blue skies—
Your smile they yearn to greet.
Come to the woods, my own,

With every blessing known
To man, which you can bring;
Here is your royal goal,
Come, with your joyous soul,
And make all nature sing!
YOU
What is this mystery?
This subtle wonder—you?
Which fills my soul with ecstasy,
My eyes with dew?
What are you, influence, so mild?
As subtle as the air which sways
The stalwart pine. What child
Of nature are you?
Soul obeys your slightest motion.
Mind is set in deep commotion—
By your presence—
By your absence—
Being thrills beneath your glance!
A smile will all my thought enhance.
Touch my lips, and every bliss
Seeks heaven’s glory in a kiss!
You! sweet influence, what art
God used in fashioning you apart
From His renownèd mould,
In the marvellous days of old?
Why, all the elements combined
In making you
The dearest mystery refined,
The ages through!
Yet, what are you? with power
So great to bind my will,
Fast in strong chains each hour;
And every action fill
With echoes of one name,
Resounding in love’s hall of fame?
You! Unlike your kind—
An essence of God’s mind.
An attribute of His deep joy,
When in his toil of love
He fashioned you without alloy,
y y,
The masterpiece to prove,
With every splendid gift—replete.
You—complete!
My earth, sky, sea, and air;
My fruit, flower, jewel rare;
My every need of day and night—
Sun, moon, stars, space; my soul’s delight!
Your name whose syllables are wings
Which waft me high,
Above the fragrant air which brings
Faint eastern aromatics to the sky.
Ever a mystery of art to be,
A subtle influence subjecting me.
Like, fair Hamadryad, created anew—
Ineffable, mystical, wonderful—you!
THE LAST LIGHT
The foothills of Nebraska shine

In a disc of sunset gold;
The cornstalks glisten like pale wine—
But the wind is bitter cold.
Around my love a radiance lies,

’Tis the glow of her soul’s sun;
’Twill light a vision in my eyes—
When the long day’s work is done.
WHEN YOU WERE BORN

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Molecular Diagnostics of Infectious Diseases

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Molecular Diagnostics of Infectious Diseases 3rd revised

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Nucleic Acids as Molecular Diagnostics 1st Edition Andreas

Edited by Harald H. Kessler

The book contains 19 ﬁgures and 49 tables.

Library of Congress Cataloging-in-Publication data

Bibliographic information published by the Deutsche Nationalbibliothek

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliograﬁe;

© 2012 Walter de Gruyter GmbH & Co. KG, Berlin/Boston.

1 Choice of adequate sample material............................................................ 1

2 Stability of the specimen during preanalytics .............................................. 25

3 Quality assurance and quality control ......................................................... 35

3.4 Components of veriﬁcation work ....................................................... 41

4 Extraction of nucleic acids........................................................................... 47

5 Ampliﬁcation and detection methods .......................................................... 53

6 Interpreting and reporting molecular diagnostic tests ................................. 69

7 Human immunodeﬁciency virus .................................................................. 79

8 Hepatitis viruses .......................................................................................... 93

9 Pathogens relevant in transplantation medicine........................................... 105

9.2.2 BKV ..................................................................................... 108

10 Pathogens in lower respiratory tract infections ............................................ 121

11 Molecular diagnosis of gastrointestinal pathogens....................................... 145

12 Pathogens relevant in the central nervous system ........................................ 165

13 Pathogens relevant in sexually transmitted infections .................................. 185

14 Index ........................................................................................................... 193

Corinne F.L. Amar Chapter 11 Dieter Hoffmann Chapter 8

Stephen A. Bustin Chapter 5 Margareta Ieven Chapter 10

Marco Ciotti Chapter 9 Jacques Izopet Chapter 7

Holger F. Rabenau Chapter 1 Ranjini Valiathan Chapter 6

Deshratn Asthana Jamie Murphy

Adenoviruses (Family: Adenoviridae; approx. 50 human serotypes, subgenera A–F)

Clinical presentation Sample material

Astrovirus (Family: Astroviridae)

Transmission: smear infections or through contaminated food and water.

Clinical presentation Sample material

Bocavirus (BoV) (Family: Parvoviridae; 4 species: BoV1–BoV4)

Clinical presentation Sample material

Coronaviruses (Family: Coronaviridae; 3 genera: group I includes CoV-229E and CoV-

Clinical presentation Sample material

Cytomegalovirus (CMV) (Family: Herpesviridae)

Clinical presentation Sample material

Dengue viruses (Family: Flaviviridae; 4 serotypes)

Clinical presentation Sample material

Enteroviruses (Family: Picornaviridae; approx. 70 human types, including Polio-,

Incubation period: 2–14 (rarely up to 35) days.

Clinical presentation Sample material

Note: shedding of enteroviruses in the stool can persist for months.

Epstein-Barr virus (EBV) (Family: Herpesviridae)

Clinical presentation Sample material

Hantaviruses (Family: Bunyaviridae; approx. 20 hantavirus species)

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material