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Molecular Diagnostics of Infectious Diseases 3rd revised
edition Edition Harald H. Kessler (Editor) Digital Instant
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Author(s): Harald H. Kessler (editor)
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Harald H. Kessler (Ed.)
Molecular Diagnostics of Infectious Diseases
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Molecular Diagnostics
of Infectious Diseases

Edited by
Harald H. Kessler

3rd fully revised edition

DE GRUYTER
Editor
Prof. Dr. med. Harald H. Kessler
Medizinische Universität Graz
Zentrum für Angewandte Biomedizin
Universitätsplatz 4
8010 Graz, Österreich
E-Mail: [email protected]

ISBN 978-3-11-032788-5
e-ISBN 978-3-11-032812-7
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Preface to the Third Edition

A period of only two years elapsed between the appearance of the first and second edi-
tions of this book. This third edition has been produced after just another two years
since almost all copies of the second edition of this book were sold within slightly
more than a year.
Interesting and exciting developments have occurred since the publication of the
second edition that made it appropriate to revise portions of the book and to present
additional material of interest to the readers. All of these new findings and achieve-
ments are reflected in this third edition while the themes that initially inspired the
creation of this book were continued. The book maintains its tradition of clearly arran-
ged chapters with an emphasis on clinical practicality along with a detailed review of
the technical background of methods described. In the general part (chapters 1–6),
preanalytical, analytical, and postanalytical issues are discussed. Furthermore, special
attention is drawn on quality assurance/quality control and standardization issues. In
the special part (chapters 7–13), nucleic acid testing for detection of pathogens pro-
ducing infectious diseases is discussed in detail. Each chapter focuses on infectious
diseases targeting a specific body tract or system. Throughout the text, practice points
are found that highlight clinical scenarios requiring targeted molecular diagnostics.
In this third edition, the whole text has been carefully reviewed; all chapters have
been updated and several new sections added including those about standardization
of molecular assays and the polyomavirus JC as another pathogen relevant in immuno-
suppression. Furthermore, new tables and figures have been added. All authors and
contributors have been commissioned to ensure that the material is fresh, up to date,
and relevant.
The overall philosophy and approach of the book is unchanged. As with the pre-
vious edition, the standard style throughout the text was maintained. I hope that the
uniformity achieved will make the text appreciably easier to read and assimilate.
Altogether, 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 the third edition of this book would not
have been possible. I would especially like to extend my sincere gratitude to the very
helpful staff at De Gruyter for smoothing the path towards publication. I trust that the
readers will enjoy this new edition and benefit from the additional material. While
this book tries to cover the whole field and to provide a maximum timely content,
the reader should always 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].

Harald H. Kessler
Contents

Preface v

Authors Index xiii

Contributors Index xv

1 Choice of adequate sample material 1

Holger F. Rabenau, Reinhard B. Raggam, Margit Hübner and Eva Leitner
1.1 Viruses 1
1.2 Bacteria 19
1.3 Fungi 25
1.4 Protozoa 27

2 Stability of the specimen during preanalytics 29

Georg Endler, Georg Slavka and Markus Exner
2.1 Sample integrity during collection 29
2.1.1 Blood 29
2.1.2 Urine 30
2.1.3 Stool 30
2.2 Degradation of DNA 30
2.3 Degradation of RNA 31
2.4 Inhibitors of PCR 32
2.5 How can contamination during specimen
collection and in the laboratory be avoided? 33
2.6 How can the sample identity be ensured? 34
2.7 Transport of diagnostic material 34
2.7.1 Category A Infectious Substances 34
2.7.2 Category B Infectious Substances 35
2.7.3 Exempt patient specimens 36
2.8 Stability of nucleic acids of selected pathogens
during preanalytics 36
2.8.1 Human immunodeficiency virus type 1 (HIV-1) RNA 36
2.8.2 Hepatitis B virus (HBV) DNA 37
2.8.3 Hepatitis C virus (HCV) RNA 38
2.8.4 Chlamydia trachomatis and Neisseria gonorrhoeae DNAs 38
2.8.5 Viral pathogens producing respiratory tract infections 39
2.8.6 Pathogens in stool specimens 39
2.9 Take home messages 40
2.10 Further reading 40
viii Contents

3 Quality assurance and quality control 41

Reinhard B. Raggam, John Saldanha and Harald H. Kessler
3.1 Accreditation issues 41
3.2 Standardization of diagnostic tests or test systems 42
3.3 Validation and verification work 44
3.4 Components of validation work 44
3.4.1 Internal and external quality controls 44
3.4.2 Proficiency testing 47
3.4.3 Validation of employee competency 48
3.4.4 Instrument maintenance and calibration 48
3.4.5 Correlation with clinical findings 49
3.5 Components of verification work 49
3.5.1 Components of verification work for IVD/CE labeled
and/or FDA-approved or -cleared tests or test systems 50
3.5.2 Components of verification work for
laboratory-developed tests or test systems 53
3.6 Take home messages 54
3.7 Further reading 55

4 Extraction of nucleic acids 57

Harald H. Kessler
4.1 Manual nucleic acid extraction protocols 57
4.2 Automated nucleic acid extraction platforms 58
4.2.1 Technology principle 58
4.2.2 Desirable features of automated platforms 59
4.3 Preparation of qPCR mixes and addition of
eluates (qPCR assay setup) 60
4.4 Currently frequently used commercially available platforms 60
4.5 Take home messages 62
4.6 Further reading 62

5 Amplification and detection methods 63

Stephen A. Bustin and Harald H. Kessler
5.1 Nucleic acid-based tests 64
5.2 Target amplification methods 65
5.2.1 Real-time polymerase chain reaction (qPCR) 66
5.2.2 Isothermal amplification techniques 75
5.2.3 Next generation sequencing (NGS) 78
5.3 Signal amplification methods 79
5.3.1 Branched DNA (bDNA) 80
5.3.2 Hybrid capture assay 80
 Contents ix

5.4 What are the key challenges for the future? 81

5.5 Take-home messages 82
5.6 Further reading 83

6 Interpreting and reporting molecular diagnostic tests 85

Ranjini Valiathan and Deshratn Asthana
6.1 Detection of viral infections 85
6.2 Detection of bacterial infections 86
6.3 Quantitative endpoint PCR 87
6.4 Real-time PCR (qPCR) 88
6.5 Reporting results 89
6.5.1 Genetic names 91
6.5.2 Recommendations for reporting results
of molecular tests 91
6.5.3 Recommendations for the contents of the molecular
test report 92
6.6 Interpretation 93
6.7 Important issues when clinically interpreting molecular
diagnostic results 95
6.8 Take home messages 96
6.9 Further reading 96

7 Human immunodeficiency virus 97

Jacques Izopet
7.1 Major symptoms 99
7.1.1 Untreated individuals 99
7.1.2 Treated individuals 100
7.2 Preanalytics 100
7.2.1 Specimen collection 100
7.2.2 Clinical circumstances for using NAT to diagnose
HIV infection 101
7.2.3 Clinical circumstances for using NAT to monitor
HIV infection 102
7.3 Analytics 103
7.3.1 Main technologies for NAT 103
7.3.2 HIV RNA assays 104
7.3.3 HIV DNA assays 105
7.3.4 HIV drug resistance assays 106
7.3.5 HIV tropism assays 108
7.3.6 Assays for minority HIV variants 109
7.4 Postanalytics 110
x Contents

7.4.1 Molecular diagnosis of HIV infection 110

7.4.2 Monitoring HIV infection 110
7.5 Take-home messages 111
7.6 Further reading 112

8 Hepatitis viruses 113

Dieter Hoffmann, Thomas Michler and Ulrike Protzer
8.1 Major symptoms 113
8.2 Preanalytics 113
8.3 Analytics 115
8.3.1 Adenoviruses 116
8.3.2 HAV 117
8.3.3 HBV 117
8.3.4 HCV 118
8.3.5 HDV 119
8.3.6 HEV 120
8.3.7 Herpes viruses 120
8.3.8 Yellow fever virus and hemorrhagic fever viruses 121
8.4 Postanalytics – interpretation of results 121
8.4.1 HAV/HEV 121
8.4.2 HBV 121
8.4.3 HDV 122
8.4.4 HCV 122
8.5 Take-home messages 123
8.6 Further reading 123

9 Pathogens relevant in transplantation medicine 125

Marco Ciotti and Harald H. Kessler
9.1 Clinical manifestations 128
9.2 Preanalytics 128
9.2.1 Adenoviruses 129
9.2.2 CMV 129
9.2.3 EBV 130
9.2.4 HHV-6 130
9.2.5 HHV-8 130
9.2.6 VZV 131
9.2.7 BKPyV 131
9.2.8 JCPyV 131
9.3 Analytics 131
9.3.1 Sample preparation 131
9.3.2 Nucleic acids amplification and detection 132
9.4 Postanalytics – interpretation of results 140
 Contents xi

9.4.1 Adenoviruses 140

9.4.2 CMV 140
9.4.3 EBV 141
9.4.4 HHV-6 141
9.4.5 HHV-8 141
9.4.6 VZV 141
9.4.7 BKPyV 142
9.4.8 JCPyV 142
9.5 Take-home messages 142
9.6 Further reading 143

10 Pathogens in lower respiratory tract infections 145

Margareta Ieven and Katherine Loens
10.1 Clinical importance of different etiologic agents 145
10.2 Specimen collection 148
10.2.1 S. pneumoniae 148
10.2.2 M. pneumoniae, L. pneumophila, and C. pneumoniae 150
10.2.3 B. pertussis 150
10.2.4 Respiratory viruses 151
10.3 Diagnostic procedures 151
10.3.1 Sample preparation and nucleic acid extraction 151
10.3.2 Amplification and detection methods for individual agents 152
10.3.3 Multiplex NAATs 158
10.4 External quality control 170
10.5 The clinical usefulness and implementation of NAATs 171
10.6 Concluding remarks 172
10.7 Further reading 173

11 Molecular diagnosis of gastrointestinal pathogens 175

Corinne F.L. Amar
11.1 Clinical manifestations 178
11.2 Preanalytics 178
11.3 Analytics 180
11.4 Postanalytics 186
11.4.1 Clinical sensitivity and diagnostic specificity 186
11.4.2 Interpretation of results 191
11.5 Further reading 192

12 Pathogens relevant in the central nervous system 193

Helene Peigue-Lafeuille and Cécile Henquell
12.1 Clinical manifestations 197
12.1.1 Viral meningitis 197
xii Contents

12.1.2 Acute community-acquired bacterial meningitis 197

12.1.3 Mycobacterium tuberculosis 200
12.1.4 Encephalitis 201
12.2 Preanalytics 201
12.2.1 Goals of etiological investigations 201
12.2.2 Specimens and handling 201
12.2.3 Time of lumbar puncture during the course of illness
and quantity of CSF required 202
12.2.4 Transport and storage of specimens 203
12.3 Analytics 204
12.3.1 Sample preparation 204
12.3.2 Nucleic acid amplification and detection 204
12.4 Postanalytics 208
12.4.1 Workflow and testing schedules for molecular tests 208
12.4.2 Limitations of molecular tests 210
12.4.3 Viral CNS infections 211
12.4.4 Bacterial CNS infections 211
12.4.5 Which pathogens should we look for? 212
12.5 Conclusion 213
12.6 Take-home messages 213
12.7 Acknowledgment 214
12.8 Further reading 214

13 Pathogens relevant in sexually transmitted infections 217

Suzanne M. Garland and Sepehr N. Tabrizi
13.1 Symptoms and clinical manifestations 217
13.2 Preanalytics 221
13.3 Analytics 221
13.4 Postanalytics 224
13.5 Further reading 225

Index 227
Authors Index

Corinne F. L. Amar Chapter 11 Suzanne M. Garland Chapter 13

FoodBorne Pathogens Reference Unit The Royal Women’s Hospital
PHE Colindale Locked Bag 300
61 Colindale Avenue PARKVILLE 3052
NW9 5EQ LONDON Australia
UK Phone: +61(3)83453671
Phone: +44(20)82004400 [email protected]
[email protected]
Dieter Hoffmann Chapter 8
Stephen A. Bustin Chapter 5 Institute of Virology
Faculty of Health, Social Care & Education Technische Universitaet Muenchen
Anglia Ruskin University Trogerstrasse 30
Bishop Hall Lane 81675 MUENCHEN
CHELMSFORD CM1 1SQ Phone: +49(89)41406825
UK [email protected]
Phone: +44(845)1964845
[email protected] Margareta Ieven Chapter 10
Vaccine & Infectious Disease Institute
Marco Ciotti Chapter 9 University of Antwerp
U.O.C. di Virologia Molecolare Wilrijkstraat 10
Fondazione Policlinico Universitatio Tor 2650 EDEGEM
Vergata Belgium
Viale Oxford Phone: +32(3)8213644
81-00133 ROMA [email protected]
Italy
Phone: +39(6)20902087 Jacques Izopet Chapter 7
[email protected] Laboratoire de Virologie
Institut Federatif de Biologie
Georg Endler Chapter 2 330 Avenue de Grande Bretagne, TSA 40031
Gruppenpraxis Labors.at 31059 TOULOUSE Cedex 9
Praterstrasse 22 France
1020 WIEN Phone: +33(5)67690424
Austria [email protected]
Phone: +43(1)260530
[email protected]
xiv Authors Index

Harald H. Kessler Chapter 4 Reinhard B. Raggam Chapter 3

Center for Applied Biomedicine Clinical Institute of Medical and
Medical University of Graz Chemical Laboratory Diagnostics
Universitaetsplatz 4 Medical University of Graz
8010 GRAZ Auenbruggerplatz 15
Austria 8036 GRAZ
Phone: +43(316)3804363 Austria
[email protected] Phone: +43(316)38580243
[email protected]
Helene Peigue-Lafeuille Chapter 12
Laboratoire de Virologie Ranjini Valiathan Chapter 6
Centre de Biologie Laboratory for Clinical and
CHRU Clermont-Ferrand Biological Studies
58, rue Montalembert University of Miami – Miller School
63003 CLERMONT-FERRAND of Medicine
France 1550 NW 10th Avenue, Fox Cancer Building,
Phone:+33(4)73754850 Suite 118
[email protected] MIAMI, FL 33136
USA
Holger F. Rabenau Chapter 1 Phone: +1(305)2432010
Institute for Medical Virology [email protected]
JWG-University Frankfurt/Main
Paul-Ehrlich-Strasse 40
60596 FRANKFURT/MAIN
Germany
Tel.: +49(69)63015312
[email protected]
Contributors Index

Deshratn Asthana Eva Leitner

Laboratory for Clinical and Biological Center for Applied Biomedicine
Studies Medical University of Graz
University of Miami – Miller School Universitaetsplatz 4
of Medicine 8010 GRAZ
1550 NW 10th Avenue, Fox Cancer Building, Austria
Suite 118 Phone: +43(316)3804383
MIAMI, FL 33136 [email protected]
USA
Phone: +1(305)2432010 Katherine Loens
[email protected] Vaccine and Infectious Disease Institute
University of Antwerp
Markus Exner Wilrijkstraat 10
Gruppenpraxis Labors.at 2650 EDEGEM
Praterstrasse 22 Belgium
1020 WIEN Phone: +32(3)8202751
Austria [email protected]
Phone: +43(1)260530
[email protected] Thomas Michler
Institute of Virology
Cecile Henquell Technische Universitaet Muenchen
Laboratoire de Virologie Trogerstrasse 30
Centre de Biologie 81675 MUENCHEN
CHRU Clermont-Ferrand Phone: +49(89)41406825
58, rue Montalembert [email protected]
63003 CLERMONT-FERRAND
France Jamie Murphy
Phone: +33(4)73754850 3rd Floor Alexandra Wing
[email protected] The Royal London Hospital
LONDON E1 1BB
Margit Hübner UK
Center for Applied Biomedicine Phone: +44(20)78828748
Medical University of Graz [email protected]
Universitaetsplatz 4
8010 GRAZ
Austria
Phone: +43(316)3804380
[email protected]
xvi Contributors Index

Ulrike Protzer Georg Slavka

Institute of Virology Central Laboratory
Technische Universitaet Muenchen Municipal Hospital Wilhelminen
Trogerstrasse 30 Montleartstrasse 37
81675 MUENCHEN 1160 WIEN
Phone: +49(89)41406821 Austria
[email protected] Phone: +43(1)491503308
[email protected]
John Saldanha
John Saldanha Consultancy Sepehr N. Tabrizi
Oakland, CA The Royal Women´s Hospital
USA Locked Bag 300
Phone: +1(510)6194713 PARKVILLE 3052
[email protected] Australia
Phone: +61(3)83453671
[email protected]
1 Choice of adequate sample material
Holger F. Rabenau, Reinhard B. Raggam,
Margit Hübner and Eva Leitner

Nucleic acid amplification testing (NAT) has gained major impact on the detection of
pathogens. Today, NAT is widely used in the routine diagnostic laboratory. It is emplo-
yed in special situations including the very early stage of infection before production
of antibodies and in patients lacking antibody production due to immunosuppres-
sion. Furthermore, NAT is the method of choice to detect/exclude vertical transmis-
sion and to monitor therapy.
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 respira-
tory tract infections, gastroenteritis, and epidemic keratoconjunctivitis.
Complications: meningoencephalitis in children, disseminated, sepsis-like adenovi-
ral 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

Upper respiratory tract infection Nasopharyngeal swab or aspirate, throat
washing, induced sputum
Pneumonia Bronchoalveolar lavage (BAL), EDTA whole blood
Hemorrhagic cystitis Urine
Encephalitis Cerebrospinal fluid (CSF)

(Continued)
2 1 Choice of adequate sample material

(Continued)

Clinical presentation Sample material

Gastroenteritis Stool
Pre-emptive monitoring/suspected adenovirus EDTA whole blood, nasopharyngeal swab or
infection under immunosuppression aspirate, throat washing, urine

Astrovirus (Family: Astroviridae)

Epidemiology: occasional outbreaks, e.g. in nursing homes or nosocomial outbreaks
in hospitals.
Transmission: smear infections or through contaminated food and water.
Incubation period: 1–3 days.
Clinical presentation: gastroenteritis with fever, vomiting and abdominal pain.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Gastroenteritis Stool

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

Epidemiology: worldwide distribution, in 2–19% of patients with upper or lower res-
piratory 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
inhalation or contact with infectious sputum, feces, or urine.
Incubation period: Unknown.
Clinical presentations: BoV1: Respiratory tract infection with cough and wheeze, rhi-
norrhea, tachypnea, and fever. BoV2 through BoV4: Gastroenteritis.
Complications: Rash or exanthema, thrombopenia, pneumonia, sepsis (rarely).

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Upper respiratory tract infection Nasopharyngeal swab or aspirate, throat washing

Pneumonia 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; 4 genera: alpha including the human

CoVs229E and NL229E, beta including the human CoVsOC43, HKU1, MERS-CoV, and
SARS-CoV, gamma including only avian pathogens, and the provisional delta genus)
 1.1 Viruses 3

Epidemiology: worldwide distribution depending on genus, high prevalence already

in childhood.
Transmission: droplets and smear infection.
Incubation period: 2–5 days (SARS 2–20 days, MERS 5-12 days).
Clinical presentation: Respiratory tract infections. MERS/SARS disease with fever,
cough, shortness of breath, pneumonia, and bronchiolitis; CoV-NL229E occurs espe-
cially in children with disorders of the upper respiratory tract, pneumonia, and
bronchiolitis.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Upper respiratory tract infection Nasopharyngeal swab or aspirate, throat

washing, induced sputum
Pneumonia, bronchiolitis BAL
Suspected MERS/SARS infection Nasopharyngeal swab or aspirate, throat
washing, induced sputum, BAL

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.
Incubation period: 20–60 days.
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.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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 infection under EDTA whole blood, bone marrow,
immunosuppression throat 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.
4 1 Choice of adequate sample material

Incubation period: 3–7 days.

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).

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Unclear serological constellation/serological confirmation EDTA whole blood, serum

Suspicion or exclusion of dengue infection EDTA whole blood, serum
DHF, DSS EDTA whole blood, serum

Enteroviruses (Family: Picornaviridae; more than 100 human pathogenic 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 con-
junctivitis, 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, meningoence-
phalitis and myocarditis.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Aseptic meningitis, meningoencephalitis CSF, stool

Myocarditis, pericarditis, pleurodynia, hepatitis Organ biopsy, stool
Conjunctivitis Conjunctival swab
Respiratory tract infection, 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.
 1.1 Viruses 5

Incubation period: 30–50 days.

Clinical presentation: before puberty mainly asymptomatic, afterwards infectious
mononucleosis.
Complications: meningitis, encephalitis, Guillain-Barré syndrome, hepatitis, splenic
rupture, hemolytic and aplastic anemia, thrombocytopenia. EBV infection is also
associated with Burkitt’s lymphoma, nasopharyngeal carcinoma, lymphoprolifera-
tive disease, lymphoma in immunocompromised patients (e.g. post-transplant lym-
phoproliferative disease [PTLD]), X-linked lymphoproliferative syndrome, oral hairy
leukoplakia, and Hodgkin’s lymphoma.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Meningitis, encephalitis CSF

Mononucleosis, aplastic anemia, Guillain-Barré syndrome EDTA whole blood
Pre-emptive monitoring/suspected EBV infection under EDTA whole blood
immunosuppression
PTLD EDTA whole blood
Pneumonia BAL, EDTA whole blood
Oral hairy leukoplakia Biopsy

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

Epidemiology: worldwide distribution.
Transmission: through infectious, aerosolic feces and urine of chronically infected
rodents (viral reservoir).
Incubation period: 5–35 days.
Clinical presentation: hemorrhagic fever with renal syndrome (HFRS) mainly caused
by Hantaan, Dobrava and Seoul species; nephropathia epidemica caused by Puumala
species; Hantavirus pulmonary syndrome (HPS) caused by Sin Nombre virus.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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
relatively low.
Transmission: fecal-oral.
Incubation period: 3–5 weeks.
6 1 Choice of adequate sample material

Clinical presentation: infection in childhood often asymptomatic. The risk of a

symptomatic 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.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Suspected HAV infection EDTA plasma, serum, 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 fibro-
sis, liver cirrhosis, and hepatocellular carcinoma.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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 vertical. Infection through blood and blood products
occurred frequently before introduction of antibody testing in 1991.
Incubation period: 2–26 weeks.
Clinical presentation: mainly asymptomatic or mild and unspecific symptoms with
lethargy, 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.
 1.1 Viruses 7

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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 hepati-
tis 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.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Suspected acute or chronic HDV infection EDTA plasma, serum

Note: monitoring of HDV therapy may be done through monitoring of HBV load.

Hepatitis E virus (HEV) (Family: Hepeviridae, Genus: Hepevirus; 4 genotypes)

Epidemiology: most countries in the developing world (especially Southeast Asia),
indigenous cases in industrialized countries. Genotypes 1 and 2 are restricted to
humans, 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).
Incubation period: 3–8 weeks.
Clinical presentation: similar to HAV, usually self-limiting disease but chronic hepa-
titis E may occur, especially in immunocompromised patients. Sometimes prolonged
fecal excretion. Occasionally severe liver disease (fatality rate 1–4%). Especially in
pregnancy, lethality up to 25%.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Suspected HEV infection EDTA plasma, serum, stool

Note: Frequently false-positive serological test results; PCR testing for HEV is thus strongly
recommended in cases of suspected HEV infection.
8 1 Choice of adequate sample material

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.
Transmission: mainly through oro-oral contact and through intimate contact, rarely
droplets or smear infection.
Incubation period: 2–12 days.
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 reacti-
vation with virus excretion is possible).
Complications: conjunctivitis, herpes simplex dermatitis, eczema herpeticum, gene-
ralized HSV infection with hepatitis or pneumonia, meningitis, herpes encephalitis,
Bell’s palsy, herpes of the neonate.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Encephalitis, meningoencephalitis CSF, EDTA whole blood

Retinitis Aqueous humor
Conjunctivitis Conjunctival swab
Bell’s palsy Saliva
Pneumonia BAL, EDTA whole blood
Herpes of the neonate CSF, EDTA whole blood, serum, conjunctival
swab, nasopharyngeal swab or aspirate,
skin swab
Generalized HSV infection EDTA whole blood
Herpetic lesions Swab

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.
Incubation period: 5–15 days.
Clinical presentation: exanthema subitum (roseola infantum – 3-day fever) with fever
and volatile rash.
 1.1 Viruses 9

Complications: in rare cases, encephalitis, meningitis, (fulminant) hepatitis. In

immunosuppressed patients, virus reactivation possible with interstitial pneumonia
and organ rejection.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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)

Epidemiology: worldwide distribution.
Transmission: mainly through saliva, probably through droplets.
Incubation period: 5–15 days.
Clinical presentation: exanthema subitum (roseola infantum – 3-day fever) with fever
and volatile rash.
Complications: febrile seizure, diarrhea, vomiting. In immunosuppressed patients,
virus reactivation is possible with interstitial pneumonia and organ rejection.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Suspected active HHV-7 infection EDTA whole blood, serum

Pre-emptive monitoring/suspected HHV-7 infection under EDTA whole blood, serum
immunosuppression

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,
iatrogenic (in organ transplant recipients) and HIV-associated KS. Seroprevalence
in Northern Europe and the U.S. approximately 3%, with higher prevalence in risk
groups (male homosexuals and bisexuals).
Transmission: mainly through sexual contact or iatrogenic.
10 1 Choice of adequate sample material

Incubation period: a few weeks to a few months.

Clinical presentation: Kaposi’s sarcoma, Castleman’s disease.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Suspected Kaposi’s sarcoma EDTA whole blood, serum, biopsy

Castleman’s disease EDTA whole blood, serum
Pre-emptive monitoring/suspected HHV-8 EDTA whole blood, serum
infection under immunosuppression

Human immunodeficiency virus type 1 (HIV-1) and 2 (HIV-2) (Family: Retroviri-

dae; HIV-1 subgroup M, with subtypes A–K, subgroups O, N and circulating recom-
binant forms; HIV-2 subtypes A–E)
Epidemiology: worldwide distribution of HIV-1, specific risk groups include male
homosexuals, intravenous drug abusers (“needle sharing”), and professional sex
workers. HIV-2 can be found mainly in Western Africa and India.
Transmission: sexual contact, intravenous drug abuse, exposure to contaminated
blood, and vertical (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 maculopapular rash and acute neurological
symptoms. AIDS is characterized by a cellular immune defect resulting in opportuni-
stic infections and tumors.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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
Newborn of HIV-positive mother EDTA plasma
Monitoring of HAART EDTA plasma
Suspected development of drug resistance, failure EDTA plasma
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.
 1.1 Viruses 11

Human T-lymphotropic virus type 1 (HTLV-1) and 2 (HTLV-2) (Family: Retroviridae)

Epidemiology: worldwide distribution of HTLV-1, specific risk groups include intra-
venous drug abusers (“needle sharing”), male homosexuals, and professional sex
workers. HTLV-2 is mainly found in Native Americans and South American Indians
but also in Asian countries, commonly in Japan and Korea.
Transmission: sexual contact, intravenous drug abuse, exposure to contaminated
blood, and vertical (including breastfeeding).
Incubation period: decades, 15–20 years for the development of adult T-cell leukemia/
lymphoma (ATLL).
Clinical presentation: ATLL (with end-stage organomegaly), HTLV-associated myelo-
pathy (HAM)/tropical spastic paraparesis (TSP) including motor and sensory changes
in the extremities, spastic gait in combination with weakness of the lower limbs,
clonus, and bladder dysfunction, opportunistic infections and tumors through altera-
tions in the host’s immune functions.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

ATLL, HAM, TSP, therapy monitoring EDTA whole blood

Note: HTLV is predominantly cell-associated. Quantitation of HTLV proviral DNA in lymphocytes has
high prognostic relevance.

Influenza viruses (Family: Orthomyxoviridae; 3 genera, A, B and C)

Epidemiology: worldwide distribution, annual epidemics in some countries and spo-
radic pandemics.
Transmission: droplets and smear infection.
Incubation period: 1–3 days.
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.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Tracheobronchitis Nasopharyngeal swab or aspirate, throat

washing, BAL
Pneumonia BAL

Measles virus (Family: Paramyxoviridae)

Epidemiology: worldwide distribution, significant importance in developing coun-
tries (particularly Africa) with a relatively high mortality rate.
12 1 Choice of adequate sample material

Transmission: droplet infection.

Incubation period: 10–14 days (infectivity from approx. 5 days before the onset of
rash).
Clinical presentation: typical symptoms with conjunctivitis, Koplik’s spots and macu-
lopapular (morbilliform) rash.
Complications: otitis media, diarrhea, Hecht’s giant cell pneumonia, bacterial super-
infections, central nervous system involvement including subacute measles ence-
phalitis (SME), acute post-measles encephalitis (APME) and subacute sclerosing
panencephalitis (SSPE).

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Suspected acute measles infection, unclear EDTA whole blood, nasopharyngeal swab or
serological constellation aspirate, oral fluid, 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%.
Transmission: droplet infection.
Incubation period: 3–7 days.
Clinical presentation: disorders of the upper respiratory tract, bronchiolitis,
pneumonia.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Upper respiratory tract infection Nasopharyngeal swab or aspirate, throat

washing, induced sputum
Pneumonia, bronchiolitis BAL

Mumps virus (Family: Paramyxoviridae)

Epidemiology: worldwide distribution, increased incidence in winter and spring.
Transmission: droplet infection.
Incubation period: 18–21 days.
Clinical presentation: one- or two-sided parotitis; unilateral orchitis.
Complications: sterility, meningitis, rarely pancreatitis or diabetes mellitus.
 1.1 Viruses 13

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Suspected mumps meningitis CSF

Parotitis Oral fluid, urine

Norovirus (Family: Caliciviridae; 5 genogroups: GI, GII, and GIV relevant for humans,
GIII infecting bovine species, GV infecting mice)
Epidemiology: increased incidence in winter with some tenacious nosocomial out-
breaks.
Transmission: fecal-oral, aerosols, smear infection, contaminated food.
Incubation period: 10–50 h.
Clinical presentation: gastroenteritis with nausea, vomiting, diarrhea, fever,
headache and myalgia.

Indication and choice of the adequate sample material for NAT:

Sample material

Gastroenteritis Stool, vomit

Papillomaviruses (HPV) (Family: Papillomaviridae; approx. 150 genotypes)

Epidemiology: worldwide distribution.
Transmission: through direct or intimate skin/mucosal contact.
Incubation period: 21–28 days.
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.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Condylomata acuminata, epidermodysplasia verruciformis, Cell containing swab, biopsy

conjunctival papilloma, Butchers warts, CIN, HPV-associated
carcinomas

Note: pathogen detection and typing is relevant for risk assessment of cervical neoplasia.
14 1 Choice of adequate sample material

Parainfluenza virus 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%).
Transmission: droplets infection.
Incubation period: 3–6 days.
Clinical presentation: acute respiratory tract disorders, mainly in young children. In
adults, infection is usually mild or unapparent.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Upper respiratory tract infection Nasopharyngeal swab or aspirate, throat

washing, induced sputum
Pneumonia BAL

Parvovirus B19 (Family: Parvoviridae)

Epidemiology: worldwide distribution, seroprevalence in adults approximately 70%.
Transmission: droplets, possibly through blood and blood products, transplacental
infection.
Incubation period: 7–10 days.
Clinical presentation: Erythema infectiosum (Fifth disease).
Complications: in immunocompetent patients, lymphadenopathy and arthralgia; in
immunocompromised patients, chronic infection with chronic anemia, and thrombo-
cytopenia. 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.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Anemia EDTA whole blood, bone marrow

Aplastic crisis EDTA whole blood
Meningitis, encephalopathy EDTA whole blood, CSF
Myocarditis, hepatitis, vasculitis EDTA whole blood, biopsy
Suspected hydrops fetalis Amniotic fluid

Note: Parvovirus B19 can persist in the bone marrow without clinical symptoms.

Polyomaviruses (BKPyV, JCPyV) (Family: Polyomaviridae)

Epidemiology: worldwide distribution, seroprevalence in adults up to 90%.
Transmission: probably through respiratory fluids, urine, or contaminated water.
 1.1 Viruses 15

Incubation period: unknown.

Clinical presentation: usually asymptomatic. In immunocompromised patients,
BKPyV causes infection of the urinary tract with (hemorrhagic) cystitis. In severe
cases, clinical manifestations may include ureter stenosis and even renal dysfunc-
tion. JCPyV can pass the blood-brain barrier and may lead to the fatal progressive
multifocal leukoencephalopathy (PML) in immunosuppressed individuals, especially
in AIDS patients. However, it can also cause infection of the urinary tract.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

PML CSF, brain biopsy

Preemptive monitoring after kidney transplantation, suspected EDTA whole blood, urine
BKPyV associated nephropathy in renal-transplant recipients,
hemorrhagic cystitis in allogenic bone marrow transplant
recipients

Note: increased risk of systemic infection with BKPyV 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 head-
ache, followed by tonic spasms of the pharyngeal-, laryngeal- and respiratory
muscles, increased salivation, extreme hydrophobia, death (100%) as a result of heart
paralysis. In approximately 30% paralytic rabies (“silent rage”) similar to that of Guil-
lain-Barré syndrome can be observed.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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.
Transmission: droplets and smear infection.
Incubation period: 3–7 days.
16 1 Choice of adequate sample material

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.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Rhinitis, laryngitis, tracheobronchitis, bronchiolitis, Nasopharyngeal swab or aspirate,

pneumonia BAL
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, bron-
chiolitis, pneumonia.
Complications: Asthma exacerbation, acute exacerbation of chronic obstructive pul-
monary disease and cystic fibrosis, fatal pneumonia in immunocompromised pati-
ents after solid organ/bone marrow transplantation and in hematopoetic stem cell
transplant recipients.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Respiratory tract infection, pneumonia Nasopharyngeal swab or aspirate, throat

washing, induced sputum, BAL
Otitis media Nasopharyngeal swab or aspirate

Notes: Rhinovirus shedding normally stops within 11 to 21 days. Persistence may represent serial
infections.

Rotavirus (Family: Reoviridae, serogroups A–G)

Epidemiology: worldwide distribution, in developing countries every year more than
450 000 rotavirus-related deaths in children.
Transmission: fecal-oral.
Incubation period: 1–3 days.
 1.1 Viruses 17

Clinical presentation: severe gastroenteritis with diarrhea and vomiting, fever and
dehydration, especially during infancy.
Complications: encephalitis, exsiccosis.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Gastroenteritis Stool
Suspected rotavirus encephalitis CSF

Rubella virus (Family: Togaviridae)

Epidemiology: worldwide distribution.
Transmission: droplets, transplacental infection.
Incubation period: 10–21 days.
Clinical presentation: flu-like symptoms with nuchal lymphadenopathy and small
dotted exanthema; in adults, partially volatile arthritis.
Complications: vertical rubella infection in the first trimester of pregnancy can lead to
embryopathy with spontaneous stillbirth (20%) or to the congenital rubella syndrome
with sensorineural deafness, eye abnormalities (especially retinopathy, cataract, and
microphthalmia), and congenital heart disease (especially patent ductus arteriosus)
and sometimes even mental retardation.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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
Suspected acute rubella infection, unclear EDTA whole blood, oral fluid, nasopharyngeal
serological constellation swab 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 coun-
tries. TBE is the most important arthropod-transmitted viral disease in Europe.
Transmission: tick-bite.
Incubation period: 7–21 days.
18 1 Choice of adequate sample material

Clinical presentation: frequently biphasic illness, fever prior to neurological

symptoms. Of those infected, 10–30% develop a severe neurological disease, such as
meningitis, meningoencephalitis, or meningoencephalomyelitis.
Complications: long-term sequelae (e.g. flaccid paralysis); the case-fatality rate is up
to 5%.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Neurological symptoms CSF

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, mucous membrane contact, transplacental infection.
Incubation period: 10–23 days.
Clinical presentation: primary infection is also referred to as chickenpox. During child-
hood, it usually shows a milder course; in adults, especially in immunocompromised
patients, secondary bacterial infections, sepsis, hemorrhagic chickenpox, pneumo-
nia, or encephalitis can occur. 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%.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Encephalitis CSF, EDTA whole blood

Pneumonia BAL, EDTA whole blood
Hemorrhagic chickenpox Skin swab, skin biopsy, EDTA whole blood
Ramsey-Hunt syndrome Oral fluid, skin swab
Chickenpox of the neonate Skin swab, EDTA whole blood
Clarification of a florid varicella-zoster virus Skin swab
infection
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 oral fluid is tested, it has
to be considered that viral shedding into the oral cavity can also be observed in healthy individuals.
 1.2 Bacteria 19

West Nile virus (WNV) (Family: Flaviviridae)

Epidemiology: distribution in Africa, parts of Europe, India, Israel, USA.
Transmission: bite through Culex spp. mosquitoes. Vertical transmission possible.
Incubation period: 3–14 days.
Clinical presentation: mostly asymptomatic course (80%), fever with flu-like symp-
toms, headache and eruptions.
Complications: encephalitis, menigoencephalitis, flaccid paralysis (especially in
persons aged over 70 years).

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Encephalitis, meningoencephalitis, flaccid CSF, EDTA whole blood

paralysis
Suspected West Nile virus infection EDTA whole blood

1.2 Bacteria

Bordetella spp. (Family: Proteobacteria; aerob; 3 species, Bordetella pertussis, Borde-

tella parapertussis, Bordetella bronchiseptica)
Epidemiology: worldwide distribution with seasonal increases in wintertime.
Transmission: droplets.
Incubation period: 7–20 days.
Clinical presentation: whooping cough, starting with an initial catarrhal phase with sym-
ptoms similar to those of the common cold, proceeding with a dry and persistent cough.
Complications: subconjunctival bleedings, otitis media, pneumonia, apnea.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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.
20 1 Choice of adequate sample material

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.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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 fre-
quently diagnosed bacterial sexually transmitted infection worldwide, infection does
not prevent 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.
Incubation period: 10–24 days.
Clinical presentation: urethritis, cystitis, cervicitis, lymphogranuloma venerum,
inclusion-body conjunctivitis.
Complications: pelvic inflammatory disease, prostatitis, epididymitis, sterility, arth-
ritis (Mb. Reiter), perinatal infection with trachoma and blindness of the newborn.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Cervicitis Cervical swab (cell containing)

Urinary tract infections, prostatitis, epididymitis, arthritis Urethral swab (males), urine
dysenterica (Reiter’s syndrome)

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.
Transmission: droplets.
Incubation period: 7–28 days.
Clinical presentation: respiratory tract infections including pharyngitis, laryngitis,
pneumonia.
 1.2 Bacteria 21

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Respiratory tract infections, pneumonia Induced sputum, BAL

Clostridium difficile (Family: Clostridiaceae, anaerob)

Epidemiology: distribution in industrialized countries, responsible for both hospital-
acquired and community-acquired diarrhea leading to a major public health problem.
Transmission: fecal-oral route.
Risk factors: healthcare environment, antimicrobial treatment, age ≥ 65, immunosup-
pression, chronic underlying disease, proton pump inhibitors.
Clinical presentation: diarrhea.
Complications: life threatening bowel complications.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Diarrhea Unformed stool

Note: Only Clostridium difficile producing toxins (A/B) are leading to infection; therefore, the NAT
should target toxin genes.

Enterococcus, vancomycin-resistant (VRE) (Family: Enterococcaceae, aerob)

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, diver-
ticulitis with diarrhea and meningitis.
Complications: sepsis with an overall rate of lethality up to 58%.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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.
22 1 Choice of adequate sample material

Helicobacter pylori (Family: Helicobacteriaceae, microaerophilic)

Epidemiology: worldwide distribution. Helicobacter pylori infects over 50% of the
world’s population but only a small subset of infected people develop Helicobacter
pylori-associated disease.
Transmission: oral-oral, gastro-oral or fecal-oral route.
Clinical presentation: abdominal pain (stomach ache), nausea.
Complications: chronic gastritis, peptic ulcer disease, mucosa-associated lymphoid
tissue (MALT) lymphoma, gastric carcinoma.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Gastritis Biopsy

Legionella pneumophila (Family: Legionellaceae, aerob)

Epidemiology: worldwide distribution, common sources of Legionella include cooling
towers, air conditioning systems, domestic hot water systems, fountains and similar
disseminators that draw upon a public water supply; natural sources include fresh-
water ponds and creeks.
Transmission: aerosols.
Incubation period: 2–10 days.
Clinical presentation: legionellosis with multifocal necrotizing pneumonia; pontiac
fever (without pneumonia).
Complications: fatality rate exceeding 20%.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Legionellosis, pneumonia BAL

Mycobacterium tuberculosis complex (Family: Mycobacteriaceae, aerob)

Epidemiology: worldwide distribution, approximately 15 million infections/year, clas-
sified into M. tuberculosis, M. bovis, M. africanum, M. microti, M. canetti, M. pinnepedi,
M. caprae, M. mungi, and the vaccination strain Bacillus Calmette-Guerin (BCG).
Transmission: mainly droplets.
Incubation period: 4–12 weeks, sometimes years.
Clinical presentation: unapparent infection, fever, night sweats, and weight loss, cough.
Complications: pneumonia, pleuritis, miliary tuberculosis, meningitis, arthritis,
osteomyelitis, urogenital tuberculosis, sepsis landouzy (rare).
 1.2 Bacteria 23

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Pulmonary tuberculosis Induced sputum, BAL, pleural effusion

Extrapulmonary tuberculosis CSF, urine, bone marrow, organ tissues,
lymphatic tissue, joint puncture fluid

Mycoplasma pneumoniae (Family: Mycoplasmataceae, aerob)

Epidemiology: worldwide distribution.
Transmission: droplets.
Incubation period: 7–28 days.
Clinical presentation: respiratory tract infections including pharyngitis, tracheobron-
chitis, interstitial pneumonia, bronchiolitis.
Complications: subsegmental and segmental atelectasis of the lung; otitis media.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Respiratory tract infections, pneumonia Induced sputum, BAL

Note: NAT enables epidemiological monitoring of macrolide resistance to M. pneumoniae.

Neisseria species (Family: Neisseriaceae, aerob; 2 human pathogenic species, Neis-

seria meningitidis and Neisseria gonorrhoeae)
Epidemiology: worldwide distribution.
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. meningitidis: meningitis, sepsis; for N. gonorrhoeae:
gonorrhea with purulent (or pus-like) discharge from the genitals which may be foul
smelling, 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
infection with sepsis, endocarditis, meningitis; perinatal infection with conjunctivitis
purulenta.
24 1 Choice of adequate sample material

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Meningitis CSF
Gonorrhea, urethitis, prostatitis Urethral swab, urine (men)
Cervicitis Cervical swab
Pelvic inflammatory disease, bartholinitis, Vaginal/cervical/anal swab
proctitis
Conjunctivitis Conjunctival swab
Sepsis EDTA whole blood, CSF

Methicillin-resistant Staphylococcus aureus (MRSA) (Family: Staphylococcaceae,

aerob)
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.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Wound infection Swab

Pneumonia BAL
Bacteremia, endocarditis EDTA whole blood
Screening Nasal swab

Note: detection is mainly based on the mecA gene. Blood culture is still mandatory for the diagnosis
of bacteremia.

Group B Streptococcus (Family: Streptococcaceae, aerob)

Epidemiology: worldwide distribution, Group B Streptococcus is part of the normal
flora of the gut and genital 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.
 1.3 Fungi 25

Complications: sepsis, hearing loss as long-term sequelae of Group B Streptococcus-

meningitis.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Suspected Group B Streptococcus infection Vaginal or cervical swab, anal swab

Meningitis CSF
Bacteremia, sepsis EDTA whole blood

Note: NAT for rapid intrapartum screening.

Streptococcus pneumoniae (Family: Streptococcaceae, aerob)

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, endogenous infection.
Incubation period: highly variable.
Clinical presentation: respiratory tract infections, sinusitis, pneumonia, otitis media,
meningitis.
Complications: sepsis, brain abscess.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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 sepsis.

1.3 Fungi

Aspergillus species (Group: Molds; more than 100 species)

Epidemiology: worldwide distribution. Approximately 10 species are medically rele-
vant including Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, and Asper-
gillus flavus.
Transmission: spore inhalation.
26 1 Choice of adequate sample material

Clinical presentation: pulmonary aspergillosis includes allergenic bronchopulmonal

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

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

ABPA BAL, lung tissue

Aspergilloma, AFS, encephalitis, endocarditis Fungus ball, sinunasal mucus, CSF, organ tissue
Acute invasive aspergillosis, sepsis EDTA whole blood, CSF

Note: detection of aspergillus DNA in EDTA whole blood samples is not necessarily associated
with invasive aspergillosis. In addition to serum aspergillus antigen (galactomannan) testing for
monitoring, molecular testing can enhance diagnostic sensitivity in patients at risk.

Candida species (Group: Yeasts; more than 150 species)

Epidemiology: worldwide distribution. Approximately 7 species are medically rele-
vant including Candida albicans, the most significant pathogenic species, Candida
tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida dublinien-
sis and Candida lusitaniae.
Transmission: endogenous infection.
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
become systemic, causing candidemia and sepsis.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

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 27

Pneumocystis jirovecii (former carinii) (Family: Pneumocystidaceae)

Epidemiology: worldwide distribution. Pneumocystis jirovecii can be found in lungs of
healthy people and as a source of opportunistic infection.
Transmission: airborne route, endogenous infection.
Clinical presentation: pneumonia
Complications: life-threatening pneumonia in immunocompromised patients (e.g.
AIDS patients).

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Pneumonia BAL, lung tissue

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.
Incubation period: 1–2 weeks.
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.

Indication and choice of the adequate sample material for NAT:

Clinical presentation Sample material

Suspected infection CSF, amniotic fluid, EDTA whole blood, aqueous

humor
2 Stability of the specimen during preanalytics
Georg Endler, Georg Slavka and Markus Exner

Although sample processing is usually well standardized and covered by estab-

lished standards in the molecular diagnostic laboratory, preanalytical procedures
outside the laboratory usually follow unwritten traditions with considerable inter-
individual variability. Studies have shown that preanalytical errors make up to 85%
of all laboratory errors, 95% of them occurring outside the laboratory. In particu-
lar, molecular assays are sensitive to suboptimal preanalytical conditions. False-
negative results may occur due to degradation of nucleic acids during transport or
polymerase chain reaction (PCR) inhibitors, 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, contamination, sample identity, and the risk
of environmental hazards due to infectious material.

2.1 Sample integrity during collection

Prior to sample collection standardized procedures might help to reduce frequent pre-
analytical errors in clinical practice. Standard procedures to reduce frequent errors
do not differ significantly from standard sample collection procedures and have been
discussed extensively in various consensus documents. These include correct patient
identification, infection control, and exclusive use of single use disposals (including
needles and collection devices). Since sample collection for these rather specialized
tests is usually not done routinely by the staff, it is advisable to provide a short one
page summary to facilitate sample collection including the following steps:
1. Selection of appropriate sample collection devices
2. Correct identification of the patient (in up to 2% of all samples, wrong patient
identification can be suspected) and appropriate labeling of the collection tubes
3. Sample collection
4. Safe disposal of needles and other potentially infectious items
5. Storage and shipment procedures

The following general sampling considerations for selected materials may be helpful.

2.1.1 Blood

Blood sampling for viral nucleic acid testing does not differ significantly from stan-
dard phlebotomies. Due to DNase inhibition in EDTA, viral nucleic acids are more
Exploring the Variety of Random
Documents with Different Content
The men said, “We did not break any law. We did not go near the
mines and you know it. We were on the public road.”
“Well,” said the deputy, “we are going to arrest you anyway.”
They defied him to arrest them, insisting they had not violated the
law. They gave him twenty-five minutes to leave town. They sent for
his brother, who was the company doctor, and told him to take him
out.
That night I went to hold a meeting with them. They told me what
had happened.
I said, “Boys, it would have been better if you had surrendered,
especially as you had the truth on your side and you had not been
near the mines.”
After the meeting I went to a nearby camp—Montgomery—where
there was a little hotel and the railway station. Before leaving, the
boys, who came to the edge of the town with me said, “You will be
coming back soon, Mother?”
I had no idea how soon it would be.
The next morning I went to the station to get an early train. The
agent said to me, “Did you hear what trouble they had up in
Stanford Mountain last night?”
“I think you are mistaken,” I answered, “for I just came down from
there myself last night.”
“Well,” he said, “they have had some trouble there, all the same.”
“Anyone hurt?”
“Yes; I was taking the railway messages and couldn’t get all the
details. Some shooting.”
I said, “Take back my ticket. I must go up to those boys.”
I took the short trail up the hillside to Stanford Mountain. It seemed
to me as I came toward the camp as if those wretched shacks were
huddling closer in terror. Everything was deathly still. As I came
nearer the miners’ homes, I could hear sobbing. Then I saw
between the stilts that propped up a miner’s shack the clay red with
blood. I pushed open the door. On a mattress, wet with blood, lay a
miner. His brains had been blown out while he slept. His shack was
riddled with bullets.
In five other shacks men lay dead. In one of them a baby boy and
his mother sobbed over the father’s corpse. When the little fellow
saw me, he said, “Mother Jones, bring back my papa to me. I want
to kiss him.”
The coroner came. He found that these six men had been murdered
in their beds while they peacefully slept; shot by gunmen in the
employ of the coal company.
The coroner went. The men were buried on the mountain side. And
nothing was ever done to punish the men who had taken their lives.
 CHAPTER X
The March of the Mill Children

In the spring of 1903 I went to Kensington, Pennsylvania, where

seventy-five thousand textile workers were on strike. Of this number
at least ten thousand were little children. The workers were striking
for more pay and shorter hours. Every day little children came into
Union Headquarters, some with their hands off, some with the
thumb missing, some with their fingers off at the knuckle. They were
stooped little things, round shouldered and skinny. Many of them
were not over ten years of age, although the state law prohibited
their working before they were twelve years of age.
The law was poorly enforced and the mothers of these children often
swore falsely as to their children’s age. In a single block in
Kensington, fourteen women, mothers of twenty-two children all
under twelve, explained it was a question of starvation or perjury.
That the fathers had been killed or maimed at the mines.
I asked the newspaper men why they didn’t publish the facts about
child labor in Pennsylvania. They said they couldn’t because the mill
owners had stock in the papers.
“Well, I’ve got stock in these little children,” said I, “and I’ll arrange a
little publicity.”
We assembled a number of boys and girls one morning in
Independence Park and from there we arranged to parade with
banners to the court house where we would hold a meeting.
A great crowd gathered in the public square in front of the city hall. I
put the little boys with their fingers off and hands crushed and
maimed on a platform. I held up their mutilated hands and showed
them to the crowd and made the statement that Philadelphia’s
mansions were built on the broken bones, the quivering hearts and
drooping heads of these children. That their little lives went out to
make wealth for others. That neither state or city officials paid any
attention to these wrongs. That they did not care that these children
were to be the future citizens of the nation.
The officials of the city hall were standing in the open windows. I
held the little ones of the mills high up above the heads of the crowd
and pointed to their puny arms and legs and hollow chests. They
were light to lift.
I called upon the millionaire manufacturers to cease their moral
murders, and I cried to the officials in the open windows opposite,
“Some day the workers will take possession of your city hall, and
when we do, no child will be sacrificed on the altar of profit.”
The officials quickly closed the windows, just as they had closed
their eyes and hearts.
The reporters quoted my statement that Philadelphia mansions were
built on the broken bones and quivering hearts of children. The
Philadelphia papers and the New York papers got into a squabble
with each other over the question. The universities discussed it.
Preachers began talking. That was what I wanted. Public attention
on the subject of child labor.
The matter quieted down for a while and I concluded the people
needed stirring up again. The Liberty Bell that a century ago rang
out for freedom against tyranny was touring the country and crowds
were coming to see it everywhere. That gave me an idea. These
little children were striking for some of the freedom that childhood
ought to have, and I decided that the children and I would go on a
tour.
I asked some of the parents if they would let me have their little
boys and girls for a week or ten days, promising to bring them back
safe and sound. They consented. A man named Sweeny was
marshal for our “army.” A few men and women went with me to help
with the children. They were on strike and I thought they might as
well have a little recreation.
The children carried knapsacks on their backs in which was a knife
and fork, a tin cup and plate. We took along a wash boiler in which
to cook the food on the road. One little fellow had a drum and
another had a fife. That was our band. We carried banners that said,
“We want more schools and less hospitals.” “We want time to play.”
“Prosperity is here. Where is ours?”
We started from Philadelphia where we held a great mass meeting. I
decided to go with the children to see President Roosevelt to ask
him to have Congress pass a law prohibiting the exploitation of
childhood. I thought that President Roosevelt might see these mill
children and compare them with his own little ones who were
spending the summer on the seashore at Oyster Bay. I thought, too,
out of politeness, we might call on Morgan in Wall Street who owned
the mines where many of these children’s fathers worked.
The children were very happy, having plenty to eat, taking baths in
the brooks and rivers every day. I thought when the strike is over
and they go back to the mills, they will never have another holiday
like this. All along the line of march the farmers drove out to meet us
with wagon loads of fruit and vegetables. Their wives brought the
children clothes and money. The interurban trainmen would stop
their trains and give us free rides.
Marshal Sweeny and I would go ahead to the towns and arrange
sleeping quarters for the children, and secure meeting halls. As we
marched on, it grew terribly hot. There was no rain and the roads
were heavy with dust. From time to time we had to send some of
the children back to their homes. They were too weak to stand the
march.
We were on the outskirts of New Trenton, New Jersey, cooking our
lunch in the wash boiler, when the conductor on the interurban car
stopped and told us the police were coming down to notify us that
we could not enter the town. There were mills in the town and the
mill owners didn’t like our coming.
I said, “All right, the police will be just in time for lunch.”
Sure enough, the police came and we invited them to dine with us.
They looked at the little gathering of children with their tin plates
and cups around the wash boiler. They just smiled and spoke kindly
to the children, and said nothing at all about not going into the city.
We went in, held our meeting, and it was the wives of the police
who took the little children and cared for them that night, sending
them back in the morning with a nice lunch rolled up in paper
napkins.
Everywhere we had meetings, showing up with living children, the
horrors of child labor.
At one town the mayor said we could not hold a meeting because he
did not have sufficient police protection. “These little children have
never known any sort of protection, your honor,” I said, “and they
are used to going without it.” He let us have our meeting.
One night in Princeton, New Jersey, we slept in the big cool barn on
Grover Cleveland’s great estate. The heat became intense. There
was much suffering in our ranks, for our little ones were not robust.
The proprietor of the leading hotel sent for me. “Mother,” he said,
“order what you want and all you want for your army, and there’s
nothing to pay.”
I called on the mayor of Princeton and asked for permission to speak
opposite the campus of the University. I said I wanted to speak on
higher education. The mayor gave me permission. A great crowd
gathered, professors and students and the people; and I told them
that the rich robbed these little children of any education of the
lowest order that they might send their sons and daughters to places
of higher education. That they used the hands and feet of little
children that they might buy automobiles for their wives and police
dogs for their daughters to talk French to. I said the mill owners take
babies almost from the cradle. And I showed those professors
children in our army who could scarcely read or write because they
were working ten hours a day in the silk mills of Pennsylvania.
“Here’s a text book on economics,” I said, pointing to a little chap,
James Ashworth, who was ten years old and who was stooped over
like an old man from carrying bundles of yarn that weighed seventy-
five pounds. “He gets three dollars a week and his sister who is
fourteen gets six dollars. They work in a carpet factory ten hours a
day while the children of the rich are getting their higher education.”
That night we camped on the banks of Stony Brook where years and
years before the ragged Revolutionary Army camped, Washington’s
brave soldiers that made their fight for freedom.
From Jersey City we marched to Hoboken. I sent a committee over
to the New York Chief of Police, Ebstein, asking for permission to
march up Fourth Avenue to Madison Square where I wanted to hold
a meeting. The chief refused and forbade our entrance to the city.
I went over myself to New York and saw Mayor Seth Low. The
mayor was most courteous but he said he would have to support the
police commissioner. I asked him what the reason was for refusing
us entrance to the city and he said that we were not citizens of New
York.
“Oh, I think we will clear that up, Mr. Mayor,” I said. “Permit me to
call your attention to an incident which took place in this nation just
a year ago. A piece of rotten royalty came over here from Germany,
called Prince Henry. The Congress of the United States voted
$45,000 to fill that fellow’s stomach for three weeks and to entertain
him. His brother was getting $4,000,000 dividends out of the blood
of the workers in this country. Was he a citizen of this land?”
“And it was reported, Mr. Mayor, that you and all the officials of New
York and the University Club entertained that chap.” And I repeated,
“Was he a citizen of New York?”
“No, Mother,” said the mayor, “he was not.”
“And a Chinaman called Lee Woo was also entertained by the
officials of New York. Was he a citizen of New York?”
“No, Mother, he was not.”
“Did they ever create any wealth for our nation?”
“No, Mother, they did not,” said he.
“Well, Mr. Mayor, these are the little citizens of the nation and they
also produce its wealth. Aren’t we entitled to enter your city?”
“Just wait,” says he, and he called the commissioner of police over to
his office.
Well, finally they decided to let the army come in. We marched up
Fourth Avenue to Madison Square and police officers, captains,
sergeants, roundsmen and reserves from three precincts
accompanied us. But the police would not let us hold a meeting in
Madison Square. They insisted that the meeting be held in Twentieth
Street.
I pointed out to the captain that the single taxers were allowed to
hold meetings in the square. “Yes,” he said, “but they won’t have
twenty people and you might have twenty thousand.”
We marched to Twentieth Street. I told an immense crowd of the
horrors of child labor in the mills around the anthracite region and I
showed them some of the children. I showed them Eddie Dunphy, a
little fellow of twelve, whose job it was to sit all day on a high stool,
handing in the right thread to another worker. Eleven hours a day he
sat on the high stool with dangerous machinery all about him. All
day long, winter and summer, spring and fall, for three dollars a
week.
And then I showed them Gussie Rangnew, a little girl from whom all
the childhood had gone. Her face was like an old woman’s. Gussie
packed stockings in a factory, eleven hours a day for a few cents a
day.
We raised a lot of money for the strikers and hundreds of friends
offered their homes to the little ones while we were in the city.
The next day we went to Coney Island at the invitation of Mr. Bostick
who owned the wild animal show. The children had a wonderful day
such as they never had in all their lives. After the exhibition of the
trained animals, Mr. Bostick let me speak to the audience. There was
a back drop to the tiny stage of the Roman Colosseum with the
audience painted in and two Roman emperors down in front with
their thumbs down. Right in front of the emperors were the empty
iron cages of the animals. I put my little children in the cages and
they clung to the iron bars while I talked.
I told the crowd that the scene was typical of the aristocracy of
employers with their thumbs down to the little ones of the mills and
factories, and people sitting dumbly by.
“We want President Roosevelt to hear the wail of the children who
never have a chance to go to school but work eleven and twelve
hours a day in the textile mills of Pennsylvania; who weave the
carpets that he and you walk upon; and the lace curtains in your
windows, and the clothes of the people. Fifty years ago there was a
cry against slavery and men gave up their lives to stop the selling of
black children on the block. Today the white child is sold for two
dollars a week to the manufacturers. Fifty years ago the black babies
were sold C. O. D. Today the white baby is sold on the installment
plan.
“In Georgia where children work day and night in the cotton mills
they have just passed a bill to protect song birds. What about the
little children from whom all song is gone?
“I shall ask the president in the name of the aching hearts of these
little ones that he emancipate them from slavery. I will tell the
president that the prosperity he boasts of is the prosperity of the
rich wrung from the poor and the helpless.
“The trouble is that no one in Washington cares. I saw our
legislators in one hour pass three bills for the relief of the railways
but when labor cries for aid for the children they will not listen.
“I asked a man in prison once how he happened to be there and he
said he had stolen a pair of shoes. I told him if he had stolen a
railroad he would be a United States Senator.
“We are told that every American boy has the chance of being
president. I tell you that these little boys in the iron cages would sell
their chance any day for good square meals and a chance to play.
These little toilers whom I have taken from the mills—deformed,
dwarfed in body and soul, with nothing but toil before them—have
never heard that they have a chance, the chance of every American
male citizen, to become the president.
“You see those monkeys in those cages over there.” I pointed to a
side cage. “The professors are trying to teach them to talk. The
monkeys are too wise for they fear that the manufacturers would
buy them for slaves in their factories.”
I saw a stylishly dressed young man down in the front of the
audience. Several times he grinned. I stopped speaking and pointing
to him I said, “Stop your smiling, young man! Leave this place! Go
home and beg the mother who bore you in pain, as the mothers of
these little children bore them, go home and beg her to give you
brains and a heart.”
He rose and slunk out, followed by the eyes of the children in the
cage. The people sat stone still and out in the rear a lion roared.
The next day we left Coney Island for Manhattan Beach to visit
Senator Platt, who had made an appointment to see me at nine
o’clock in the morning. The children got stuck in the sand banks and
I had a time cleaning the sand off the littlest ones. So we started to
walk on the railroad track. I was told it was private property and we
had to get off. Finally a saloon keeper showed us a short cut into the
sacred grounds of the hotel and suddenly the army appeared in the
lobby. The little fellows played “Hail, hail, the gang’s all here” on
their fifes and drums, and Senator Platt when he saw the little army
ran away through the back door to New York.
I asked the manager if he would give the children breakfast and
charge it up to the Senator as we had an invitation to breakfast that
morning with him. He gave us a private room and he gave those
children such a breakfast as they had never had in all their lives. I
had breakfast too, and a reporter from one of the Hearst papers and
I charged it all up to Senator Platt.
We marched down to Oyster Bay but the president refused to see us
and he would not answer my letters. But our march had done its
work. We had drawn the attention of the nation to the crime of child
labor. And while the strike of the textile workers in Kensington was
lost and the children driven back to work, not long afterward the
Pennsylvania legislature passed a child labor law that sent thousands
of children home from the mills, and kept thousands of others from
entering the factory until they were fourteen years of age.
 CHAPTER XI
Those Mules Won’t Scab Today

Lattimer was an eye-sore to the miners. It seemed as if no one

could break into it. Twenty-six organizers and union men had been
killed in that coal camp in previous strikes. Some of them had been
shot in the back. The blood of union men watered the highways. No
one dared go in.
I said nothing about it but made up my mind that I was going there
some night. After the raid of the women in Coaldale in the Panther
Creek, the general manager of Lattimer said that if I came in there I
would go out a corpse. I made no reply but I set my plans and I did
not consult an undertaker.
From three different camps in the Panther Creek I had a leader bring
a group of strikers to a junction of the road that leads into Lattimer.
There I met them with my army of women again.
As I was leaving the hotel the clerk said, “Mother, the reporters told
me to ring their bell if I saw you go out.”
“Well, don’t see me go out. Watch the front door carefully and I will
go out the back door.”
We marched through the night, reaching Lattimer just before dawn.
The strikers hid themselves in the mines. The women took up their
position on the door steps of the miners’ shacks. When a miner
stepped out of his house to go to work, the women started mopping
the step, shouting, “No work today!”
Everybody came running out into the dirt streets. “God, it is the old
mother and her army,” they were all saying.
The Lattimer miners and the mule drivers were afraid to quit work.
They had been made cowards. They took the mules, lighted the
lamps in their caps and started down the mines, not knowing that I
had three thousand miners down below ground waiting for them and
the mules.
“Those mules won’t scab today,” I said to the general manager who
was cursing everybody. “They know it is going to be a holiday.”
“Take those mules down!” shouted the general manager.
Mules and drivers and miners disappeared down into the earth. I
kept the women singing patriotic songs so as to drown the noise of
the men down in the mines.
Directly the mules came up to the surface without a driver, and we
women cheered for the mules who were the first to become good
union citizens. They were followed by the miners who began running
home. Those that didn’t go up were sent up. Those that insisted on
working and thus defeating their brothers were grabbed by the
women and carried to their wives.
An old Irish woman had two sons who were scabs. The women
threw one of them over the fence to his mother. He lay there still.
His mother thought he was dead and she ran into the house for a
bottle of holy water and shook it over Mike.
“Oh for God’s sake, come back to life,” she hollered. “Come back and
join the union.”
He opened his eyes and saw our women standing around him.
“Shure, I’ll go to hell before I’ll scab again,” says he.
The general manager called the sheriff who asked me to take the
women away. I said, “Sheriff, no one is going to get hurt, no
property is going to be destroyed but there are to be no more
killings of innocent men here.”
I told him if he wanted peace he should put up a notice that the
mines were closed until the strike was settled.
The day was filled with excitement. The deputies kept inside the
office; the general manager also. Our men stayed up at the mines to
attend to the scabs and the women did the rest. As a matter of fact
the majority of the men, those with any spirit left in them after years
of cowardice, wanted to strike but had not dared. But when a hand
was held out to them, they took hold and marched along with their
brothers.
The bosses telephoned to John Mitchell that he should take me and
my army of women out of Lattimer. That was the first knowledge
that Mitchell had of my being there.
When the manager saw there was no hope and that the battle was
won by the miners, he came out and put up a notice that the mines
were closed until the strike was settled.
I left Lattimer with my army of women and went up to Hazelton.
President Mitchell and his organizers were there. Mr. Mitchell said,
“Weren’t you afraid to go in there?”
“No,” I said, “I am not afraid to face any thing if facing it may bring
relief to the class that I belong to.”
The victory of Lattimer gave new life to the whole anthracite district.
It gave courage to the organization. Those brave women I shall
never forget who caused those stone walls to fall by marching
around with tin pans and cat calls.
Soon afterward, a convention was called and the strike was settled.
The organizers got up a document asking every miner to subscribe
so much to purchase a $10,000 house for John Mitchell. The
document happened to come into my hands at the convention which
was called to call off the victorious strike. I arose and I said:
“If John Mitchell can’t buy a house to suit him for his wife and for his
family out of his salary, then I would suggest that he get a job that
will give him a salary to buy a $10,000 house. Most of you do not
own a shingle on the roof that covers you. Every decent man buys a
house for his own wife first before he buys a house for another
man’s wife.”
I was holding the petition as I spoke and I tore it up and threw the
bits on the floor. “’Tis you men and your women who won the
strike,” I said, “with your sacrifice and your patience and your
forbearance through all these past weary months. ’Tis the sacrifice
of your brothers in other trades who sent the strike benefits week in
and week out that enabled you to make the fight to the end.”
From then on Mitchell was not friendly to me. He took my attitude as
one of personal enmity. And he saw that he could not control me. He
had tasted power and this finally destroyed him. I believe that no
man who holds a leader’s position should ever accept favors from
either side. He is then committed to show favors. A leader must
stand alone.
 CHAPTER XII
How the Women Mopped Up Coaldale

In Lonaconia, Maryland, there was a strike. I was there. In Hazelton,

Pennsylvania, a convention was called to discuss the anthracite
strike. I was there when they issued the strike call. One hundred and
fifty thousand men responded. The men of Scranton and Shamokin
and Coaldale and Panther Creek and Valley Battle. And I was there.
In Shamokin I met Miles Daugherty, an organizer. When he quit work
and drew his pay, he gave one-half of his pay envelope to his wife
and the other half he kept to rent halls and pay for lights for the
union. Organizers did not draw much salary in those days and they
did heroic, unselfish work.
Not far from Shamokin, in a little mountain town, the priest was
holding a meeting when I went in. He was speaking in the church. I
spoke in an open field. The priest told the men to go back and obey
their masters and their reward would be in Heaven. He denounced
the strikers as children of darkness. The miners left the church in a
body and marched over to my meeting.
“Boys,” I said, “this strike is called in order that you and your wives
and your little ones may get a bit of Heaven before you die.”
We organized the entire camp.
The fight went on. In Coaldale, in the Hazelton district, the miners
were not permitted to assemble in any hall. It was necessary to win
the strike in that district that the Coaldale miners be organized.
I went to a nearby mining town that was thoroughly organized and
asked the women if they would help me get the Coaldale men out.
This was in McAdoo. I told them to leave their men at home to take
care of the family. I asked them to put on their kitchen clothes and
bring mops and brooms with them and a couple of tin pans. We
marched over the mountains fifteen miles, beating on the tin pans as
if they were cymbals. At three o’clock in the morning we met the
Crack Thirteen of the militia, patrolling the roads to Coaldale. The
colonel of the regiment said “Halt! Move back!”
I said, “Colonel, the working men of America will not halt nor will
they ever go back. The working man is going forward!”
“I’ll charge bayonets,” said he.
“On whom?”
“On your people.”
“We are not enemies,” said I. “We are just a band of working women
whose brothers and husbands are in a battle for bread. We want our
brothers in Coaldale to join us in our fight. We are here on the
mountain road for our children’s sake, for the nation’s sake. We are
not going to hurt anyone and surely you would not hurt us.”
They kept us there till daybreak and when they saw the army of
women in kitchen aprons, with dishpans and mops, they laughed
and let us pass. An army of strong mining women makes a
wonderfully spectacular picture.
Well, when the miners in the Coaldale camp started to go to work
they were met by the McAdoo women who were beating on their
pans and shouting, “Join the union! Join the union!”
They joined, every last man of them, and we got so enthusiastic that
we organized the street car men who promised to haul no scabs for
the coal companies. As there were no other groups to organize we
marched over the mountains home, beating on our pans and singing
patriotic songs.
Meanwhile President Mitchell and all his organizers were sleeping in
the Valley Hotel over in Hazelton. They knew nothing of our march
onto Coaldale until the newspaper men telephoned to him that
“Mother Jones was raising hell up in the mountains with a bunch of
wild women!”
He, of course, got nervous. He might have gotten more nervous if
he had known how we made the mine bosses go home and how we
told their wives to clean them up and make decent American citizens
out of them. How we went around to the kitchen of the hotel where
the militia were quartered and ate the breakfast that was on the
table for the soldiers.
When I got back to Hazelton, Mitchell looked at me with surprise. I
was worn out. Coaldale had been a strenuous night and morning
and its thirty mile tramp. I assured Mitchell that no one had been
hurt and no property injured. The military had acted like human
beings. They took the matter as a joke. They enjoyed the morning’s
fun. I told him how scared the sheriff had been. He had been talking
to me without knowing who I was.
“Oh Lord,” he said, “that Mother Jones is sure a dangerous woman.”
“Why don’t you arrest her?” I asked him.
“Oh Lord, I couldn’t. I’d have that mob of women with their mops
and brooms after me and the jail ain’t big enough to hold them all.
They’d mop the life out of a fellow!”
Mr. Mitchell said, “My God, Mother, did you get home safe? What did
you do?”
“I got five thousand men out and organized them. We had time left
over so we organized the street car men and they will not haul any
scabs into camp.”
“Did you get hurt, Mother?”
“No, we did the hurting.”
“Didn’t the superintendents’ bosses get after you?”
“No, we got after them. Their wives and our women were yelling
around like cats. It was a great fight.”
 CHAPTER XIII
The Cripple Creek Strike

(1903)
The state of Colorado belonged not to a republic but to the Colorado
Fuel and Iron Company, the Victor Company and their dependencies.
The governor was their agent. The militia under Bell did their
bidding. Whenever the masters of the state told the governor to
bark, he yelped for them like a mad hound. Whenever they told the
military to bite, they bit.
The people of Colorado had voted overwhelmingly for an eight-hour
day. The legislature passed an eight-hour law but the courts had
declared it unconstitutional. Then when the measure was submitted
directly to the people, they voted for it with 40,000 votes majority.
But the next legislature, which was controlled by the mining
interests, failed to pass the bill.
The miners saw that they could not get their demands through
peaceful legislation. That they must fight. That they must strike. All
the metal miners struck first. The strike extended into New Mexico
and Utah. It became an ugly war. The metal miners were anxious to
have the coal miners join them in their struggle.
The executive board of the United Mine Workers was in session in
Indianapolis and to this board the governor of Colorado had sent a
delegation to convince them that there ought not to be a strike in
the coal fields. Among the delegates, was a labor commissioner.
I was going on my way to West Virginia from Mount Olive, Illinois,
where the miners were commemorating their dead. I stopped off at
headquarters in Indianapolis. The executive board asked me to go to
Colorado, look into conditions there, see what the sentiments of the
miners were, and make a report to the office.
I went immediately to Colorado, first to the office of The Western
Federation of Miners where I heard the story of the industrial
conflict. I then got myself an old calico dress, a sunbonnet, some
pins and needles, elastic and tape and such sundries, and went
down to the southern coal fields of the Colorado Fuel and Iron
Company.
As a peddler, I went through the various coal camps, eating in the
homes of the miners, staying all night with their families. I found the
conditions under which they lived deplorable. They were in practical
slavery to the company, who owned their houses, owned all the
land, so that if a miner did own a house he must vacate whenever it
pleased the land owners. They were paid in scrip instead of money
so that they could not go away if dissatisfied. They must buy at
company stores and at company prices. The coal they mined was
weighed by an agent of the company and the miners could not have
a check weighman to see that full credit was given them. The
schools, the churches, the roads belonged to the Company. I felt,
after listening to their stories, after witnessing their long patience
that the time was ripe for revolt against such brutal conditions.
I went to Trinidad and to the office of the Western Federation of
Miners. I talked with the secretary, Gillmore, a loyal, hard-working
man, and with the President, Howell, a good, honest soul. We sat up
and talked the matter over far into the night. I showed them the
conditions I had found down in the mining camps were heart-
rending, and I felt it was our business to remedy those conditions
and bring some future, some sunlight at least into the lives of the
children. They deputized me to go at once to headquarters in
Indianapolis.
I took the train the next morning. When I arrived at the office in
Indianapolis, I found the president, John Mitchell, the vice-president,
T. L. Lewis, the secretary, W. B. Wilson of Arnot, Pennsylvania, and a
board member, called “old man Ream,” from Iowa. These officers
told me to return at once to Colorado and they would call a strike of
the coal miners.
The strike was called November 9th, 1903. The demand was for an
eight hour day, a check weighman representing the miners, payment
in money instead of scrip. The whole state of Colorado was in revolt.
No coal was dug. November is a cold month in Colorado and the
citizens began to feel the pressure of the strike.
Late one evening in the latter part of November I came into the
hotel. I had been working all day and into the night among the
miners and their families, helping to distribute food and clothes,
encouraging, holding meetings. As I was about to retire, the hotel
clerk called me down to answer a long distance telephone call from
Louisville. The voice said, “Oh for God’s sake, Mother, come to us,
come to us!”
I asked what the trouble was and the reply was more a cry than an
answer, “Oh don’t wait to ask. Don’t miss the train.”
I got Mr. Howell, the president, on the telephone and asked him
what was the trouble in Louisville.
“They are having a convention there,” he said.
“A convention, is it, and what for?”
“To call off the strike in the northern coal fields because the
operators have yielded to the demands.” He did not look at me as he
spoke. I could see he was heart sick.
“But they cannot go back until the operators settle with the southern
miners,” I said. “They will not desert their brothers until the strike is
won! Are you going to let them do it?”
“Oh Mother,” he almost cried, “I can’t help it. It is the National
Headquarters who have ordered them back!”
“That’s treachery,” I said, “quick, get ready and come with me.”
We telephoned down to the station to have the conductor hold the
train for Louisville a few minutes. This he did. We got into Louisville
the next morning. I had not slept. The board member, Ream, and
Grant Hamilton, representing the Federation of Labor, came to the
hotel where I was stopping and asked where Mr. Howell, the
president was.
“He has just stepped out,” I said. “He will be back.”
“Well, meantime, I want to notify you,” Ream said, “that you must
not block the settlement of the northern miners because the
National President, John Mitchell, wants it, and he pays you.”
“Are you through?” said I.
He nodded.
“Then I am going to tell you that if God Almighty wants this strike
called off for his benefit and not for the miners, I am going to raise
my voice against it. And as to President John paying me ... he never
paid me a penny in his life. It is the hard earned nickels and dimes
of the miners that pay me, and it is their interests that I am going to
serve.”
I went to the convention and heard the matter of the northern
miners returning to the mines discussed. I watched two shrewd
diplomats deal with unsophisticated men; Struby, the president of
the northern coal fields, and Blood, one of the keenest, trickiest
lawyers in the West. And behind them, John Mitchell, toasted and
wined and dined, flattered and cajoled by the Denver Citizens’
Alliance, and the Civic Federation was pulling the strings.
In the afternoon the miners called on me to address the convention.
“Brothers,” I said, “You English speaking miners of the northern
fields promised your southern brothers, seventy per cent of whom
do not speak English, that you would support them to the end. Now
you are asked to betray them, to make a separate settlement. You
have a common enemy and it is your duty to fight to a finish. The
enemy seeks to conquer by dividing your ranks, by making
distinctions between North and South, between American and
foreign. You are all miners, fighting a common cause, a common
master. The iron heel feels the same to all flesh. Hunger and
suffering and the cause of your children bind more closely than a
common tongue. I am accused of helping the Western Federation of
Miners, as if that were a crime, by one of the National board
members. I plead guilty. I know no East or West, North nor South
when it comes to my class fighting the battle for justice. If it is my
fortune to live to see the industrial chain broken from every
workingman’s child in America, and if then there is one black child in
Africa in bondage, there shall I go.”
The delegates rose en masse to cheer. The vote was taken. The
majority decided to stand by the southern miners, refusing to obey
the national President.
The Denver Post reported my speech and a copy was sent to Mr.
Mitchell in Indianapolis. He took the paper in to his secretary and
said, pointing to the report, “See what Mother Jones has done to
me!”
Three times Mitchell tried to make the northern miners return to the
mines but each time he was unsuccessful. “Mitchell has got to get
Mother Jones out of the field,” an organizer said. “He can never lick
the Federation as long as she is in there.”
I was informed that Mitchell went to the governor and asked him to
put me out of the state.
Finally the ultimatum was given to the northern miners. All support
for the strike was withdrawn. The northern miners accepted the
operators’ terms and returned to work. Their act created practical
peonage in the south and the strike was eventually lost, although
the struggle in the south went on for a year.
Much of the fighting took place around Cripple Creek. The miners
were evicted from their company-owned houses. They went out on
the bleak mountain sides, lived in tents through a terrible winter
with the temperature below zero, with eighteen inches of snow on
the ground. They tied their feet in gunny sacks and lived lean and
lank and hungry as timber wolves. They received sixty-three cents a
week strike benefit while John Mitchell went traveling through
Europe, staying at fashionable hotels, studying the labor movement.
When he returned the miners had been lashed back into the mines
by hunger but John Mitchell was given a banquet in the Park Avenue
Hotel and presented with a watch with diamonds.
From the day I opposed John Mitchell’s authority, the guns were
turned on me. Slander and persecution followed me like black
shadows. But the fight went on.
One night when I came in from the field where I had been holding
meetings, I was just dropping to sleep when a knock—a loud knock
—came on my door. I always slept in my clothes for I never knew
what might happen. I went to the door, opened it, and faced a
military chap.
“The Colonel wants you up at headquarters.”
I went with him immediately. Three or four others were brought in:
War John and Joe Pajammy, organizers. We were all taken down to
the Santa Fe station. While standing there, waiting for the train that
was to deport us, some of the miners ran down to bid me good-bye.
“Mother, good-bye,” they said, stretching out their hands to take
mine.
The colonel struck their hands and yelled at them. “Get away from
there. You can’t shake hands with that woman!”
The militia took us to La Junta. They handed me a letter from the
governor, notifying me that under no circumstances could I return to
the State of Colorado. I sat all night in the station. In the morning
the Denver train came along. I had no food, no money. I asked the
conductor to take me to Denver. He said he would.
“Well,” I said, “I don’t want you to lose your job.”
I showed him the letter from the governor. He read it.
“Mother,” he said, “do you want to go to Denver?”
“I do,” said I.
“Then to Hell with the job;” said he, “it’s to Denver you go.”
In Denver I got a room and rested a while. I sat down and wrote a
letter to the governor, the obedient little boy of the coal companies.
 Mother Jones Heading Protest Procession of Strikers at
Denver
“Mr. Governor, you notified your dogs of war to put me out of the
state. They complied with your instructions. I hold in my hand a
letter that was handed to me by one of them, which says ‘under no
circumstances return to this state.’ I wish to notify you, governor,
that you don’t own the state. When it was admitted to the
sisterhood of states, my fathers gave me a share of stock in it; and
that is all they gave to you. The civil courts are open. If I break a
law of state or nation it is the duty of the civil courts to deal with
me. That is why my forefathers established those courts to keep
dictators and tyrants such as you from interfering with civilians. I am
right here in the capital, after being out nine or ten hours, four or
five blocks from your office. I want to ask you, governor, what in Hell
are you going to do about it?”
I called a messenger and sent it up to the governor’s office. He read
it and a reporter who was present in the office at the time told me
his face grew red.
“What shall I do?” he said to the reporter. He was used to acting
under orders.
“Leave her alone,” counselled the reporter. “There is no more
patriotic citizen in America.”
From Denver I went down the Western Slope, holding meetings,
cheering and encouraging those toiling and disinherited miners who
were fighting against such monstrous odds.
I went to Helper, Utah, and got a room with a very nice Italian
family. I was to hold a meeting Sunday afternoon. From every
quarter the men came, trudging miles over the mountains. The shop
men were notified not to come but they came anyhow. Just as the
meeting was about to open, the mayor of the little town came to me
and said that I could not hold a meeting; that I was on company
ground. I asked him how far his jurisdiction extended. He said as far
as the Company’s jurisdiction. He was a Company mayor.
So I turned to the audience and asked them to follow me. The
audience to a man followed me to a little tent colony at Half Way
that the miners had established when they had been evicted from
their homes.
When the meeting closed I returned to Helper. The next day,
although there was no smallpox in town, a frame shack was built to
isolate smallpox sufferers in. I was notified that I had been exposed
to smallpox and must be incarcerated in the shack. But somehow
that night the shack burned down.
I went to stay in Half Way because the Italian family were afraid to
keep me longer. Another Italian family gave me a bare room in their
shack. There was only a big stone to fasten the door. No sooner was
I located than the militia notified me that I was in quarantine
because I had been exposed to smallpox. But I used to go out and
talk to the miners and they used to come to me.
One Saturday night I got tipped off by the postoffice master that the
militia were going to raid the little tent colony in the early morning. I
called the miners to me and asked them if they had guns. Sure, they
had guns. They were western men, men of the mountains. I told
them to go bury them between the boulders; deputies were coming
to take them away from them. I did not tell them that there was to
be a raid for I did not want any bloodshed. Better to submit to
arrest.
Between 4:30 and 5 o’clock in the morning I heard the tramp of feet
on the road. I looked out of my smallpox window and saw about
forty-five deputies. They descended upon the sleeping tent colony,
dragged the miners out of their beds. They did not allow them to put
on their clothing. The miners begged to be allowed to put on their
clothes, for at that early hour the mountain range is the coldest.
Shaking with cold, followed by the shrieks and wails of their wives
and children, beaten along the road by guns, they were driven like
cattle to Helper. In the evening they were packed in a box car and
run down to Price, the county seat and put in jail.
Not one law had these miners broken. The pitiful screams of the
women and children would have penetrated Heaven. Their tears
melted the heart of the Mother of Sorrows. Their crime was that
they had struck against the power of gold.
The women huddled beneath the window of the house where I was
incarcerated for smallpox.
“Oh Mother, what shall we do?” they wailed. “What’s to become of
our little children!”
“See my little Johnny,” said one woman, holding up a tiny, red baby
—new born.
“That’s a nice baby,” I said.
“He sick. Pretty soon he die. Company take house. Company take
my man. Pretty soon company take my baby.”
Two days after this raid was made, the stone that held my door was
suddenly pushed in. A fellow jumped into the room, stuck a gun
under my jaw and told me to tell him where he could get $3,000 of
the miners’ money or he would blow out my brains.
“Don’t waste your powder,” I said. “You write the miners up in
Indianapolis. Write Mitchell. He’s got money now.”
“I don’t want any of your damn talk,” he replied, then asked:
“Hasn’t the president got money?”
“You got him in jail.”
“Haven’t you got any money?”
“Sure!” I put my hand in my pocket, took out fifty cents and turned
the pocket inside out.
“Is that all you got?”
“Sure, and I’m not going to give it to you, for I want it to get a jag
on to boil the Helen Gould smallpox out of my system so I will not
inoculate the whole nation when I get out of here.”
“How are you going to get out of here if you haven’t money when
they turn you loose?”
“The railway men will take me anywhere.”
There were two other deputies outside. They kept hollering for him
to come out. “She ain’t got any money,” they kept insisting. Finally
he was convinced that I had nothing.
This man, I afterward found out, had been a bank robber, but had
been sworn in as deputy to crush the miners’ union. He was later
killed while robbing the post office in Prince. Yet he was the sort of
man who was hired by the moneyed interests to crush the hopes
and aspirations of the fathers and mothers and even the children of
the workers.