Advanced Techniques in Diagnostic

Microbiology Volume 1 Techniques

Yi-Wei Tang
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Yi-Wei Tang · Charles W. Stratton
Editors

Advanced
Techniques
in Diagnostic
Microbiology
Volume 1: Techniques
Third Edition
Advanced Techniques in Diagnostic Microbiology
Yi-Wei Tang • Charles W. Stratton
Editors

Advanced Techniques in
Diagnostic Microbiology
Volume 1: Techniques

Third Edition
Editors
Yi-Wei Tang Charles W. Stratton
Departments of Laboratory Medicine Department of Pathology, Microbiology
and Internal Medicine and Immunology and Medicine
Memorial Sloan Kettering Cancer Center Vanderbilt University Medical Center
New York, NY, USA Nashville, TN, USA

Department of Pathology and Laboratory

Medicine
Weill Medical College of Cornell
University
New York, NY, USA

ISBN 978-3-319-33899-6    ISBN 978-3-319-33900-9 (eBook)

https://doi.org/10.1007/978-3-319-33900-9

Library of Congress Control Number: 2018952488

© Springer Nature Switzerland AG 2006, 2013, 2018

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
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Preface

Medical microbiology is a branch of medical science that deals with the prevention,
diagnosis, and therapy of infectious diseases. A clinical microbiologist is a profes-
sional within the field of medical microbiology who is knowledgeable about the
characteristics of microbial pathogens, including their modes of transmission as
well as their mechanisms of infection and growth. Clinical microbiologists often
practice in a clinical microbiology laboratory or a public health laboratory where
they may direct these laboratories. Clinical microbiology laboratories are con-
cerned mainly with the diagnostic aspects of the practice of medical microbiology,
whereas public health laboratories are more concerned with the epidemiology of
infectious diseases within given populations. There is, and must be, a strong link
between clinical microbiology laboratories and public health laboratories. Clinical
microbiology laboratories primarily determine whether pathogenic microorgan-
isms are present in clinical specimens collected from individuals with suspected
infections; if such microbial pathogens are found, these microorganisms are identi-
fied and susceptibility profiles, when indicated, are determined. Clinical microbi-
ologists work closely with and serve as consultants with physicians who are caring
for infected individuals. The importance of the field of medical microbiology can
be appreciated by noting that hospitals in the United States annually report over
five million cases of infectious disease-related illnesses.
Diagnostic microbiology within the clinical microbiology laboratory continues
to undergo a quiet revolution that already has resulted in many benefits for micro-
biologists, clinicians, and most importantly patients. This revolution was initially
made possible by the elucidation of the structure of DNA and the genetic code,
which allowed scientific advances centered around hybridization probes, the poly-
merase chain reaction, genomics, transcriptomics, proteomics, and metabolomics.
These technical advances in molecular microbiology over the first decade of the
twenty-first century have profoundly altered every aspect of the clinical microbiol-
ogy laboratory, including their staffing patterns, work flow, and turnaround time.
More recently, fully automated sample processing systems with digital plate read-
ing technology have emerged as a second wave of technical advances, and have
enabled clinical microbiology laboratories to cope with large numbers of samples

v
vi Preface

with improved quality despite limited personnel and financial resources. Moreover,
total laboratory automation in the clinical microbiology laboratory also provides
superior and more rapid detection of microbial growth. The total laboratory auto-
mation system combined with molecular microbiology technical advances such as
MALDI-TOF MS and rapid phenotypic susceptibility methods promises to mark-
edly reduce the turnaround time and ultimately reduce the length of stay for hospi-
talized patients with infections.
The knowledge base required to stay current in the rapidly changing and
advancing technology involved in molecular microbiology, as well as similar
advances in total laboratory automation in the clinical microbiology laboratory,
is enormous. In 2006 and 2013, the first and second editions of Advanced
Techniques in Diagnostic Microbiology were published and were well received.
According to its “Book Performance Report 2017,” since its online publication
on August 06, 2012, there has been a total of 145,240 chapter downloads for the
second edition eBook by the end of 2017 on SpringerLink. This means the second
edition has been one of the top 25% most downloaded eBooks in the relevant
SpringerLink eBook Collection for 5 consecutive years. In order to continue to
provide this kind of relevant and current information for microbiologists, the
third edition of Advanced Techniques in Diagnostic Microbiology has been exten-
sively revised and extended with a total of 55 chapters covering all current state-
of-the art techniques and applications in the field of diagnostic microbiology.
Advanced Techniques in Diagnostic Microbiology thus provides a comprehen-
sive, well-referenced, and up-to-date description of these rapidly evolving
advanced methods for the diagnosis of infectious diseases in the routine clinical
microbiology laboratory.
The third edition is extended to two volumes. The first volume covers the
principles and characteristics of important molecular techniques; these tech-
niques include rapid antigen testing, advanced antibody detection, real-time/
digital nucleic acid amplification techniques, state-of-the-art molecular typing
techniques, and MALDI-TOF MS. New chapters on advanced techniques have
been added; these include chapters on total laboratory automation systems, rapid
phenotypic antimicrobial susceptibility methods, metabolic techniques, and
transcriptomic methods. The second volume describes the application of these
advanced techniques; new and evolving molecular applications such as molecu-
lar detection and characterization of carbapenem-resistant Enterobacteriaceae,
advanced typing techniques applied to molecular epidemiology investigations,
and multiplex approaches for syndromic testing are covered. Like the first two
editions, a diverse team of authors provides authoritative, comprehensive, and
well-referenced information on clinically relevant topics; these include sequence-
based bacterial identification, blood and blood product screening, automated
blood culture systems, molecular diagnosis of sexually transmitted diseases,
advances in the molecular diagnosis of fungal/mycobacterial infections, metage-
nomic sequencing of microbiomes, and application of advanced techniques for
antimicrobial stewardship.
Preface vii

We hope our readers like this technique- and application-based approach and
their feedback is greatly appreciated. We want to again thank the authors who
devoted their time and effort to produce their chapters. We also thank the staff at
Springer. Finally, we continue to appreciate the constant encouragement of our
wives and family members throughout this long effort. They are, indeed, the “wind
in our sails.”

New York, NY, USA Yi-Wei Tang

Nashville, TN, USA Charles W. Stratton
Contents

 utomated Blood Cultures������������������������������������������������������������������������������    1

A
Xiang Y. Han
Laboratory Automation in Clinical Bacteriology�����������������������������������������   15
Antony Croxatto
 iochemical Profile-Based Microbial Identification Systems����������������������   33
B
Safina Hafeez and Jaber Aslanzadeh
 dvanced Phenotypic Antimicrobial Susceptibility Testing Methods��������   69
A
Charles W. Stratton
Rapid Microbial Antigen Tests������������������������������������������������������������������������   99
Sheldon Campbell and Marie L. Landry
Antibody Detection: Principles and Applications ���������������������������������������� 127
Yun F. (Wayne) Wang
 rocalcitonin and Other Host-Response-Based Biomarkers
P
for Evaluation of Infection and Guidance
of Antimicrobial Treatment���������������������������������������������������������������������������� 149
Philipp Schuetz, Ramon Sager, Yannick Wirz, and Beat Mueller
 unctional Assessment of Microbial, Viral, and Parasitic Infections
F
Using Real-Time Cellular Analysis���������������������������������������������������������������� 161
Dazhi Jin, Xiao Xu, Min Zheng, Alex Mira, Brandon J. Lamarche,
and Alex B. Ryder
 ellular Fatty Acid-Based Microbial Identification and Antimicrobial
C
Susceptibility Testing �������������������������������������������������������������������������������������� 199
Nicole Parrish and Stefan Riedel
 ALDI-TOF Mass Spectrometry-Based Microbial Identification
M
and Beyond ������������������������������������������������������������������������������������������������������ 211
Alexander Mellmann and Johannes Müthing

ix
x Contents

 ranscriptomic Techniques in Diagnostic Microbiology������������������������������ 235

T
Zachary E. Holcomb and Ephraim L. Tsalik
 he Use of Microbial Metabolites for the Diagnosis
T
of Infectious Diseases �������������������������������������������������������������������������������������� 261
Mahesh J. Thalavitiya Acharige, Seena S. Koshy, and Sophia Koo
 ucleic Acid Extraction and Enrichment������������������������������������������������������ 273
N
Jeong Hwan Shin
Nonamplified Probe-Based Microbial Detection and Identification ���������� 293
Fann Wu, Tao Hong, and Phyllis Della-Latta
 olecular Typing Techniques: State of the Art�������������������������������������������� 305
M
Richard V. Goering
PCR and Its Variations������������������������������������������������������������������������������������ 327
Eleanor A. Powell and Michael Loeffelholz
Non-PCR Amplification Techniques�������������������������������������������������������������� 347
Rosemary C. She, Ted E. Schutzbank, and Elizabeth M. Marlowe
 eal-Time and Digital PCR for Nucleic Acid Quantification���������������������� 377
R
Alexander J. McAdam
 irect Nucleotide Sequencing for Amplification Product
D
Identification���������������������������������������������������������������������������������������������������� 389
Tao Hong
 olid and Suspension Microarrays for Detection and Identification
S
of Infectious Diseases �������������������������������������������������������������������������������������� 403
Sherry Dunbar, Janet Farhang, Shubhagata Das, Sabrina Ali,
and Heng Qian
 eal-Time Detection of Amplification Products Through
R
Fluorescence Quenching or Energy Transfer������������������������������������������������ 451
Caitlin Otto and Shihai Huang
 CR/Electrospray Ionization-Mass Spectrometry as an Infectious
P
Disease Diagnostic Tool������������������������������������������������������������������������������������ 481
Volkan Özenci and Kristoffer Strålin
 ucleic Acid Amplicons Detected and Identified
N
by T2 Magnetic Resonance������������������������������������������������������������������������������ 491
Jessica L. Snyder, Heather S. Lapp, Zhi-Xiang Luo, Brendan Manning,
and Thomas J. Lowery
Molecular Contamination and Amplification Product Inactivation ���������� 505
Susan Sefers and Jonathan E. Schmitz

Index������������������������������������������������������������������������������������������������������������������ 527
Contributors

Sabrina Ali Luminex Corporation, Toronto, ON, Canada

Jaber Aslanzadeh Division of Clinical Microbiology, Hartford Hospital, Hartford,
CT, USA
Sheldon Campbell Pathology and Laboratory Medicine Service, VA Connecticut,
West Haven, CT, USA
Department of Laboratory Medicine, Yale University School of Medicine, New
Haven, CT, USA
Antony Croxatto Institute of Microbiology, Lausanne University Hospital and
Lausanne University, Lausanne, Switzerland
Shubhagata Das Luminex Corporation, Austin, TX, USA
Phyllis Della-Latta Department of Pathology & Cell Biology, Columbia University
Medical Center, New York-Presbyterian Hospital, New York, NY, USA
Sherry Dunbar Luminex Corporation, Austin, TX, USA
Janet Farhang Luminex Corporation, Austin, TX, USA
Richard V. Goering Department of Medical Microbiology and Immunology,
Creighton University School of Medicine, Omaha, NE, USA
Safina Hafeez Department of Pathology and laboratory Medicine, Divsion of
Clinical Microbiology, Hartford Hospital, Hartford, CT, USA
Xiang Y. Han Department of Laboratory Medicine, Unit 84, The University of
Texas M.D. Anderson Cancer Center, Houston, TX, USA
Zachary E. Holcomb Duke University School of Medicine, Durham, NC, USA
Tao Hong Department of Pathology, Hackensack University Medical Center,
Hackensack, NJ, USA
Shihai Huang Abbott Molecular Inc., Des Plaines, IL, USA

xi
xii Contributors

Dazhi Jin Zhejiang Provincial Center for Disease Control and Prevention,
Hangzhou, Zhejiang, China
Sophia Koo Division of Infectious Diseases, Brigham and Women’s Hospital,
Boston, MA, USA
Harvard Medical School, Boston, MA, USA
Dana-Farber Cancer Institute, Boston, MA, USA
Seena S. Koshy Division of Infectious Diseases, Brigham and Women’s Hospital,
Boston, MA, USA
Harvard Medical School, Boston, MA, USA
Brandon J. Lamarche ACEA Biosciences, Inc, San Diego, CA, USA
Marie L. Landry Department of Laboratory Medicine, Yale University School of
Medicine, New Haven, CT, USA
Heather S. Lapp T2 Biosystems, Lexington, MA, USA
Michael Loeffelholz Department of Pathology, University of Texas Medical
Branch, Galveston, TX, USA
Thomas J. Lowery T2 Biosystems, Lexington, MA, USA
Zhi-Xiang Luo T2 Biosystems, Lexington, MA, USA
Brendan Manning T2 Biosystems, Lexington, MA, USA
Elizabeth M. Marlowe Roche Molecular Systems, Inc., Pleasanton, CA, USA
Alexander J. McAdam Infectious Disease Diagnostics Laboratory, Department of
Laboratory Medicine, Boston Children’s Hospital, Boston, MA, USA
Harvard Medical School, Boston, MA, USA
Alexander Mellmann Institute of Hygiene, University Hospital Münster, Münster,
Germany
Alex Mira FISABIO Foundation; Center for Advanced Research in Public Health,
Valencia, Spain
Beat Mueller Medical University Department, Division of General Internal and
Emergency Medicine, Kantonsspital Aarau, Aarau, Switzerland
University of Basel, Basel, Switzerland
Johannes Müthing Institute of Hygiene, University Hospital Münster, Münster,
Germany
Caitlin Otto SUNY Downstate Medical Center, Brooklyn, NY, USA
Volkan Özenci Department of Clinical Microbiology, Karolinska University
Hospital, Stockholm, Sweden
Contributors xiii

Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska

Institute, Stockholm, Sweden
Nicole Parrish Department of Pathology, Johns Hopkins University, Baltimore,
MD, USA
Eleanor A. Powell Department of Laboratory Medicine, Memorial Sloan Kettering
Cancer Center, New York, NY, USA
Heng Qian Luminex Corporation, Toronto, ON, Canada
Stefan Riedel Harvard Medical School and Beth Israel Deaconess Medical Center,
Boston, MA, USA
Alex B. Ryder University of Tennessee Health Science Center, Memphis, TN,
USA
Ramon Sager Medical University Department, Division of General Internal and
Emergency Medicine, Kantonsspital Aarau, Aarau, Switzerland
University of Basel, Basel, Switzerland
Jonathan E. Schmitz Department of Pathology, Microbiology, and Immunology,
Vanderbilt University Medical Center, Nashville, TN, USA
Philipp Schuetz Medical University Department, Division of General Internal and
Emergency Medicine, Kantonsspital Aarau, Aarau, Switzerland
University of Basel, Basel, Switzerland
Ted E. Schutzbank Ascension – St. John Providence, Grosse Pointe, MI, USA
Susan Sefers Department of Pathology, Microbiology, and Immunology, Vanderbilt
University Medical Center, Nashville, TN, USA
Rosemary C. She Keck School of Medicine of the University of Southern
California, Los Angeles, CA, USA
Jeong Hwan Shin Department of Laboratory Medicine, Busan Paik Hospital, Inje
University College of Medicine, Busan, South Korea
Jessica L. Snyder T2 Biosystems, Lexington, MA, USA
Kristoffer Strålin Department of Infectious Diseases, Karolinska University
Hospital, Stockholm, Sweden
Unit of Infectious Diseases, Department of Medicine Huddinge, Karolinska
Institutet, Stockholm, Sweden
Charles W. Stratton Department of Pathology, Microbiology and Immunology,
Vanderbilt University School of Medicine, Nashville, TN, USA
Mahesh J. Thalavitiya Acharige Division of Infectious Diseases, Brigham and
Women’s Hospital, Boston, MA, USA
Harvard Medical School, Boston, MA, USA
xiv Contributors

Ephraim L. Tsalik Emergency Medicine Service, Durham VAMC, Durham, NC,

USA
Center for Applied Genomics & Precision Medicine, Duke University Medical
Center, Durham, NC, USA
Division of Infectious Diseases, Department of Medicine, Duke University Medical
Center, Durham, NC, USA
Yun F. (Wayne) Wang Emory University and Grady Memorial Hospital, Atlanta,
GA, USA
Yannick Wirz Medical University Department, Division of General Internal and
Emergency Medicine, Kantonsspital Aarau, Aarau, Switzerland
University of Basel, Basel, Switzerland
Fann Wu Department of Pathology & Cell Biology, Columbia University Medical
Center, New York-Presbyterian Hospital, New York, NY, USA
Xiao Xu ACEA Biosciences, Inc, San Diego, CA, USA
Min Zheng State Key Laboratory of Diagnostic and Treatment for Infectious
Diseases, Hangzhou, Zhejiang, China
Automated Blood Cultures

Xiang Y. Han

Introduction

Cultivation or otherwise detecting an infectious agent typically confirms a clinically

suspected infection. Timely identification of a cultured microorganism along with
antimicrobial susceptibility testing is used to ensure effective antimicrobial therapy.
Bloodstream infections, being systemic, are the most severe forms of infection.
They are frequently life-threatening, and blood cultures to detect circulating micro-
organisms have been the diagnostic standards. Much of the scientific and techno-
logic advances for blood culture methods have been made through the 1970s–1990s,
with further refinements and improvements accomplished in the past two decades.
This chapter briefly reviews important aspects of blood culture methodology with
emphasis on automated culturing systems.

Principles

The principles and scientific basis for optimizing the diagnostic yield of blood cul-
tures have been reviewed and summarized (for adult patients) [1, 2]. Most parame-
ters were initially established for manual blood culture systems that used basal
culture media. A recent study addressed some of these parameters for newer auto-
mated culturing systems and media and found them to be still valid for the most part
[3]. Major characteristics of blood cultures are summarized as follows.

X. Y. Han (*)
Department of Laboratory Medicine, Unit 84, The University of Texas M.D. Anderson
Cancer Center, Houston, TX, USA
e-mail: xhan@mdanderson.org

© Springer Nature Switzerland AG 2018 1

Y.-W. Tang, C. W. Stratton (eds.), Advanced Techniques in Diagnostic
Microbiology, https://doi.org/10.1007/978-3-319-33900-9_1
2 X. Y. Han

Host and Microbial Factors Invasion of the bloodstream by microorganisms

reflects the failure of initial host defense, such as the loss of integrity of skin and
mucosa and weakening of the innate and acquired immunity, to prevent such inva-
sion or spread from a localized infection site. Among certain patients having an
intravascular device or using recreational drugs intravenously, direct seeding of the
bloodstream with microorganism is also possible. Once in the bloodstream,
microbes are constantly attacked by host defenses, such as complements, phago-
cytic leukocytes, antibodies, and other factors, and are filtered by the liver and the
spleen. The ability of invading microorganisms to evade host defenses (or antimi-
crobial agents) favors their survival and dissemination in the bloodstream. On the
other hand, if the host defenses are paralyzed, such as seen with leucopenia or
immune suppression by medications or other means, even the least pathogenic
organisms are able to cause fatal infections. Therefore, both the host and microbial
factors determine the occurrence, severity, and duration of septic episodes; these
factors also influence the yield of blood cultures. Moreover, the presence of antimi-
crobial agents in the circulation may reduce the yield of blood cultures.

Timing, Blood Volume, and Frequency of Cultures Timing of the blood draw may
influence the yield of blood cultures. Most of the time, bacteria or fungi are not
constantly distributed or evenly circulated in the bloodstream except in the case of
endocarditis; thus, the host responses, such as rising fever, are likely to herald the
best time to draw a blood culture. Blood culture should also be obtained, if at all
possible, before the initiation of empiric antimicrobial therapy.
For each septic episode, two or three sets of cultures over a 24-h period provide
the maximum recovery for the offending microorganism(s). A set of blood cultures
usually means one aerobic broth bottle and one anaerobic broth bottle with each
inoculated with 10 ml blood for an adult patient and incubated under aerobic and
anaerobic conditions, respectively. This practice requires draws of a total of
40–60 ml blood from two to three venipunctures from different arms. In children
and infants, the volume will be less and should be based upon the weight of the
child/infant (see below). In a culture bottle, the blood sample is diluted by the cul-
ture broth to reach a blood-broth ratio of 1:2.5–1:10, which may dilute inhibitory
blood components to favor microbial growth. It is generally accepted and hence
practiced that 40 ml for two culture sets in adults offers good culture recovery while
maintaining cost-effective microbiology. A few laboratories have recently demon-
strated that a 60 ml blood draw yielded consistently higher culture recoveries than
did lower volume draws [4, 5]. However, drawing higher blood volumes may add to
the cost and increase the likelihood of iatrogenic anemia for the patient, particularly
one who was already anemic.
The need to draw repeat blood cultures hinges on the patient’s response to initial
treatment, the identity and hence expected behavior of the cultured microbe, and
antimicrobial susceptibility test results. It may take a few days for a patient receiv-
ing adequate therapy to show obvious clinical improvement. The patient thus may
still spike a fever for 2–3 days while clearing the killed and dying microorganisms
Automated Blood Cultures 3

from the circulation. The underlying condition of the patient, such as profound
­neutropenia, may also affect the response time to therapy. Persistence of fever dur-
ing therapy is a common reason for repeating blood cultures.
Atmosphere and Length of Incubation Traditionally, both aerobic and anaerobic
blood cultures have been obtained and are thus recommended. However, the declin-
ing proportion of bacteremias due to obligate anaerobes has led to suggestion that
routine anaerobic cultures may not be needed and should be tailored to the needs of
individual institution, patient population, and even the individual patient. Anaerobic
cultures are valuable for patients who have had recent surgery or gynecologic/
obstetric procedures because of the high number of anaerobes in the lower gastroin-
testinal and urogenital tracts.
How long should blood cultures be incubated? Several studies on different cul-
turing systems have shown that an incubation period up to 5 days is sufficient to
recover nearly all significant microorganisms (~99%) [3, 6–9]. The vast majority of
organisms become culture positive in the first 3 days, and most fastidious bacteria
can be recovered during the extra 2 days, including the HACEK organisms
(Aggregatibacter (Haemophilus) aphrophilus, Aggregatibacter (Actinobacillus)
actinomycetemcomitans, Cardiobacterium species, Eikenella corrodens, and
Kingella kingae), Brucella spp., and nutritionally variant streptococci (currently
known as Abiotrophia species and Granulicatella species) [10]. A new species,
Cardiobacterium valvarum, proposed by us, is a cause of endocarditis and can be
cultured within 3 days [11]. The length of culture for Brucella spp. had been contro-
versial until studies done in the past two decades by Bannatyne et al. [12] in which
90 of 97 such bacteremic patients became culture positive within 5 days and by
Baysallar et al. [13] who noted that 30 of 30 were positive within 4 days. Bloodstream
infections due to Francisella tularensis are rare today, with fewer than a dozen such
cases per year in the United States, yet blood cultures usually become positive upon
3–8 days of incubation [10, 14]. Yeasts, such as Candida species that are among the
most common ten microorganisms isolated from blood cultures, can also be isolated
within 5 days of incubation [3, 6, 8].
Considerations of Blood Cultures in Pediatric Patients The blood culture method-
ology has been developed and refined in adult patients who have adequate circulat-
ing blood volumes for evaluation. In pediatric patients, however, the smaller body
blood volume has contributed to far fewer studies. In a systematic review, Bard and
TeKippe summarized current consensus for pediatric blood cultures [15]. Compared
to the blood cultures in adult population, blood cultures in pediatric patients recover
far more contaminants, accounting for 25–69% of all positive blood cultures. In
order to reduce contaminants and to recover true pathogens, adequate blood culture
volumes are important. Yet, for safety consideration, <1% of the circulating blood
volume of a pediatric patient should be cultured. Blood cultures for anaerobic
organisms are less significant than are cultures for aerobes. Therefore, the entire
volume of a blood draw can be cultured in an aerobic broth bottle. In a recent study
of blood culture use in critically ill children, a blood culture decision algorithm was
found to be helpful in reducing unnecessary blood cultures [16].
4 X. Y. Han

Reporting and Interpretation of Positive Culture

Reporting Once a blood culture is signaled positive, it should be confirmed by a

Gram stain and reported immediately to the ordering care provider. This is consid-
ered to be a critical value in most medical centers. A preliminary blood culture
report should include patient identifications and the following specific culture
information:

1. The Gram stain findings, such as a Gram-positive or Gram-negative reaction and

cellular morphology and arrangement.
2. The duration of incubation, usually in hours from inoculation to culture positiv-
ity, which may provide useful information or some correlation with the severity
of infection, microbial load in the bloodstream, and growth rate or fastidiousness
of the microbe.
3. The number and type of positive bottles, such as aerobic bottles and/or anaerobic
bottles, to indicate confidence of true infection versus contaminants and aerobic
organism versus anaerobic organism.
This type of report provides a timely alert that a patient has bloodstream infection
and also may provide clues as to the possible pathogen for altering or narrowing
empiric antimicrobial therapy. For example, aerobic cultivation of Gram-positive
cocci in chains from a patient with pneumonia and a blood culture incubation time
of ~10 h would hint that the cause of the pneumonia will likely be pneumococcus.
Interpretation of Significance Due to the ubiquitous presence of microorganisms
on and within our body as well as in the surrounding environment, a positive blood
culture often requires interpretation, which may not always be straightforward.
Both the isolated microorganism and the host factors need to be considered on a
case-by-case basis. The pathogenic potential of an organism should be considered
and can be roughly divided into four categories:

1. Strict pathogens irrespective of host factors, such as F. tularensis, Mycobacterium

tuberculosis, Brucella species, Yersinia pestis, Bacillus anthracis, and others.
2. Usual pathogens with high pathogenic potential, such as Staphylococcus aureus;
almost all members of the family Enterobacteriaceae, Pseudomonas aerugi-
nosa, and Candida albicans; and most other Gram-negative bacilli.
3. Common occasional pathogens with low pathogenic potential, such as Bacillus
species (excluding B. anthracis) that are common soil dwellers, coagulase-­
negative staphylococci (CoNS), and coryneform bacilli; and Propionibacterium
acne that are common skin flora; and alpha-hemolytic or viridans group strepto-
cocci and enterococci that are common oral or gut flora.
4. Rare occasional pathogens, such as infrequent soil or water organisms, rare
human body site florae, and normal florae from pet or exotic animals. Some
examples are Methylobacterium spp., Rhizobium spp., and Roseomonas spp.
from water or soil, Bartonella spp. from cats, and Pasteurella spp. from dogs.
Automated Blood Cultures 5

Those usual pathogens on this list are common blood isolates and are usually clini-
cally significant when isolated. On the other hand, most occasional pathogens are
common blood culture contaminants, usually become positive late during the 5-day
culturing period, and are most often not clinically significant when isolated. In most
hospitals, isolation of an occasional pathogen from a single broth bottle means con-
taminant, whereas isolation from two or more bottles likely denotes true infection.
Therefore, with more and more patients being immunosuppressed in one manner or
another and/or having intravascular devices that are prone to microbial colonization,
each positive blood culture requires clinical correlation with other findings as well as
sound clinical judgment to make final interpretation, as suggested earlier by others
[17, 18]. For instance, we found that, in patients with cancer who commonly have
mucositis and severe neutropenia or immune suppression, isolation of viridans group
streptococci means true infection most of the time rather than contamination [19].
Isolation of the rare occasional pathogen is more likely related to exposure, car-
riage of intravascular or intra-tissue device, long-term antibiotic use leading to sup-
pression of normal flora with selection of resistant microorganisms, immune defect,
congenital anomaly with an infection-prone niche, or combination thereof. For
example, a bicuspid aortic valve is a congenital anomaly that predisposes the valve
to infection, usually after three decades of life [11]. Methylobacterium radiotoler-
ans, a water organism, is rarely reported to cause infections; yet we have seen such
infections in cancer patients who are neutropenic and also have an intravenous cath-
eter for long periods of time [20].
Rapid Microbial Identification After Culture After a signal indicating a positive
blood culture, it is necessary to subculture the microorganism for purity and quan-
tity on agar plates in order to perform identification and susceptibility testing.
Depending on the growth rate of the organism, these steps may take between 1 and
3 days or even longer, which results in delay in obtaining necessary information for
optimal management of the patient. Thus, efforts have been made in recent years to
more rapidly identify the microorganism in a positive blood culture. Because CoNS
and S. aureus are Gram-positive cocci and common blood isolates yet have mark-
edly different pathogenic potential with therapeutic and prognostic significance, it
is important to rapidly differentiate these staphylococci, particularly methicillin-­
resistant S. aureus, after isolation from blood cultures. Studies have found that dif-
ferentiation of S. aureus and CoNS can be achieved within a few hours after isolation
by a number of methods; these include tube coagulase test, peptide nucleic acid
fluorescence in situ hybridization (PNA-FISH) (AdvanDx, Woburn, Mass.), or API
RAPIDEC staph (API) (bioMerieux, Durham, N.C.) [21–23]. The tube coagulase
test is cheap and easy to perform and can be used in small hospital settings. Recently,
Qian et al. [24] also found that a latex agglutination test based on the detection of
penicillin-binding protein 2a could detect methicillin-resistant S. aureus directly
from positive blood culture bottles as well as from isolated colonies with sensitivity,
specificity, and predictive values all being >90%.
In the past several years, matrix-assisted laser desorption ionization time-of-­
flight (MALDI-TOF) mass spectrometry has been used to identify microorganisms
6 X. Y. Han

directly in positive blood culture bottles. Briefly, a microorganism in a positive broth

is pelleted by centrifugation, washed with a saline solution, lysed with an organic
solvent, and applied to MALDI-TOF. Identity of the organism can be achieved
within 30 min. In several clinical evaluations [25–29], this methodology works well
for most bacteria and fungi in the setting of monomicrobic positive blood cultures.
As such, it is gaining popularity for its speed, simplicity, and accuracy.
Other Trends Some noticeable trends in the past decades are increasing numbers of
as well as increasing life span for immune defective or suppressed patients and,
thus, emergence of more opportunistic or rare pathogens; more frequent use of anti-
biotics and associated resistance, in fact, up to 29% of blood cultures come from
patients with active antimicrobial therapy; increasing use of indwelling devices,
such as intravascular catheters and others; and emergence of more Candida and
other fungal infections in the bloodstream [3, 30].

Automated Culturing Systems

Blood culture methodology has evolved over the past four decades from manual
methods (currently rarely being used) to automated blood culturing systems. The
major advantage of an automated system, such as the earlier BACTEC NR660, is to
automatically detect the growth of microorganisms through the presence and accu-
mulation of CO2 produced by the metabolism of the organisms. The automation
obviates manual inspection or examination that is required periodically to detect
microbial growth. Automated agitation of culture bottles also improves mixing and
aeration to promote the growth of aerobes and facultative anaerobes. These features
make blind subcultures of negative bottles unnecessary, as shown in a few studies
reviewed by Reimer et al. [2] Automation has improved the practice of blood culture
enormously in terms of timely report of positive culture and more laboratory effi-
ciency and consequently better patient care.
Continuously monitoring blood culturing systems (CMBCS) currently are the
most commonly used blood culture methods. Introduced in the early 1990s, CMBCS
have added nearly continuous (every 10–12 min) monitoring of microbial growth to
the automated systems. Currently, three CMBCS are available in the United States,
and they are briefly shown in Table 1. More detailed description can be found else-
where [31].
New versions of these CMBCS, available for the past decade or so, have kept the
key elements from earlier versions while refined the hardware, computer system,
data processing, and report. The trend is to increase user-friendly features for space,
operation, and flexibility. Figure 1 illustrates a culture and detection curve using the
BACTEC FX system.
Numerous studies have been published to evaluate the performance of the
CMBCS and associated media with or without various lytic agents or additives to
remove blood antimicrobics; a number of representative ones are summarized as
Automated Blood Cultures 7

Table 1 Commercial continuously monitoring blood culturing systems (CMBCS) in the United
States
Test
Current interval Microbial detection Initial system since
Manufacturer system, year (min) mechanism early 1990s
BioMerieuxa BacT/Alert 3D, 10 Colorimetric for CO2 BacT/Alert series for
2001 production varying holding
capacity
Becton-­ BACTEC FX, 10 Fluorescent for CO2 BACTEC series for
Dickinson 2009 production varying holding
capacity
Trek diagnostic VersaTREK, 12 Manometric for gas ESP series for varying
systems 2004 (CO2 and other gases) holding capacity
a
A new system from this manufacturer, called Bact/Alert Virtuo, was introduced in Europe in 2014
and is being evaluated in the United States and Canada

Fig. 1 Culture and detection of Klebsiella pneumoniae. The blood culture was set up in BACTEC
FX system with Aerobic Plus broth bottle and the release of CO2 from bacterial growth monitored
every 10 min by fluorescence. The culture turned positive with an incubation of 0 day 12 h and
40 min. A typical sigmoid curve was seen

follows (Table 2). McDonald et al. [32] compared the BacT/Alert standard bottle
with BacT/Alert FAN bottle that contains Ecosorb™, an antimicrobic-absorbing
substance, and they found that FAN bottle recovered significantly more microbes
from all septic episodes, especially S. aureus, CoNS, and members of
Enterobacteriaceae. Along with this, however, recovery of all contaminants, includ-
ing CoNS, was also higher. The performance of the BacT/Alert FAN bottle and
BACTEC Plus aerobic/F bottle (with resins to absorb antimicrobics) was also com-
pared, and the two systems were found to be comparable [33]. In a study comparing
8 X. Y. Han

Table 2 Performance evaluations of automated culturing systems and media with or without lytic
agents or additives
Compared systems and/or Reference no.
media (broth bottle) Main findings (author, year)
BacT/Alert FAN vs. BacT/ BacT/Alert FAN improved recovery of S. [32] (McDonald,
Alert standard aureus, CoNS, and enterics 1996)
BacT/Alert FAN vs. Comparable [33] (Jorgensen,
BACTEC Plus/F 1997)
BacT/Alert FAN vs. TREK BacT/Alert FAN improved recovery of S. [34] (Doern,
ESP 80A aureus, enterics, and non-Pseudomonas 1998)
aeruginosa gram-negative rods
BacT/Alert FAN vs. TREK Overall comparable. BacT/Alert FAN better for [35] (Welby-­
ESP 80A, in pediatric S. aureus and antibiotic-treated samples; ESP Sellenriek, 1997)
patients 80A better for streptococci and enterococci.
BacT/Alert FAN vs. Comparable to detect fungemia. [36] (McDonald,
BACTEC fungal medium 2001)
BACTEC Plus BACTEC Plus Anaerobic/F bottles detected [37] (Wilson,
Anaerobic/F bottles vs. more microorganisms 2001)
Standard Anaerobic/F
bottles
BacT/Alert standard vs. BacT/Alert standard improved recovery of S. [38] (Mirrett,
BACTEC 9240 standard aureus, CoNS, and yeasts 2003)
BacT/Alert FA Medium in Comparable [39, 40] (Petti
plastic vs glass bottles 2005; Riley
2005)
BacT/Alert 3D SA and SN Overall comparable for bacteria and fungi. [41] (Mirrett
vs. VersaTREK Redox VersaTrek better in detecting streptococci and 2007)
media enterococci.
BacT/Alert FA plus and Overall better culture detection and Gram stain [42] (Kirn 2014)
FN plus vs. BacT/Alert FA interpretation with BacT/Alert FA plus and FN
and FN media plus media.
Bact/Alert Virtuo vs Bact/ Similar recovery rates of but significantly [43] (Jacobs
Alert 3D shorter time to detection with Virtuo 2017)

BacT/Alert FAN versus Trek ESP 80A, Doern et al. [34] found that BacT/Alert
FAN recovered more S. aureus, enteric bacilli, and non-Pseudomonas aeruginosa
Gram-negative rods, along with more contaminants too. In a similar study in pedi-
atric patients [35], the two systems were found to be overall comparable with BacT/
Alert FAN recovering more S. aureus and better for antibiotic-containing samples
and ESP 80A recovering more streptococci and enterococci. Since yeasts are an
increasing cause of nosocomial bloodstream infections, McDonald et al. [36] com-
pared BacT/Alert FAN with BACTEC fungal medium for their recovery, and the
two systems were found comparable. The anaerobic culture media have also been
compared; Wilson et al. [37] found that the BACTEC Plus Anaerobic/F bottles
detected more microorganisms and episodes of bacteremia and fungemia than the
BACTEC Standard Anaerobic/F bottles. Mirrett et al. [38] compared BacT/Alert
standard bottle and BACTEC standard bottle and found the former significantly
Automated Blood Cultures 9

improved the recovery of S. aureus, CoNS, and yeasts. Two recent studies found
that, in the BacT/Alert system, the plastic culture bottles were comparable to the
glass bottles [39, 40].
Recent studies have also evaluated the newer versions of CMBCS and their
media. Mirrett et al. [41] compared the BacT/Alert 3D standard media and
VersaTREK Redox media and found that they were overall comparable. The same
team [42] recently compared the BacT/Alert FA plus broth and the FN plus broth
with the BacT/Alert FA and FN media; they found slightly better performance of
the newer FA plus broth and FN plus broth that contain adsorbent polymeric beads.
A recent BacT/Alert system, BacT/Alert Virtuo, that was introduced in 2014 in
Europe, was evaluated in the United States and Canada in 2017 [43]. This clinical
trial compared BacT/Alert Virtuo with BacT/Alert 3D and found nearly identical
recovery rates for the systems yet significantly shorter time to detection, by a mean
of 1.8 h, with Virtuo.
Together, these data show that CMBCS, each with similar cost, strengths, and
weaknesses, perform well overall in delivering timely and accurate diagnosis of
bloodstream infections. Incorporation of lytic or antimicrobic-absorbing substances
in these systems has consistently improved the recovery of S. aureus and members
of Enterobacteriaceae, particularly from treated patients.

Blood Culture and CMBCS for Mycobacteria

Bacteremia due to rapidly growing mycobacteria (RGM) can be detected by blood

cultures, similar to other fastidious organisms [44, 45]. In our experience with 115
consecutive clinical RGM strains [44], 46 (40%) were isolated from blood cultures
using the BACTEC 9240 and/or the Isolator 10 system (Wampole Laboratories,
Princeton, NJ). These RGM typically grow in 3–5 days; these bacteremias are usu-
ally associated with use of intravascular catheter. Among the 46 blood RGM iso-
lates, Mycobacterium mucogenicum was the most common (24 of 46 or 52%),
followed by M. abscessus complex and M. fortuitum complex. RGM can be recog-
nized as beaded Gram-positive rods and Kinyoun stain-positive acid-fast bacilli.
In contrast to RGM, Mycobacterium avium and M. tuberculosis are two species
of many slowly growing mycobacteria. Blood culture has been useful to detect and
monitor M. avium bacteremia in patients with AIDS. M. avium bacteremia usually
occurs when the CD4+ cell count falls below 50/mm3 [46]. Circulating M. avium,
exclusively within monocytes, usually range in 10–103 colony-forming units (CFU)
per ml blood but can be as high as 106 CFU/ml [46]. A number of blood culture
systems have been used: the earlier BACTEC 13A radiometric system and Isolator
10 system and the more recent CMBCS, such as BACTEC 9240 with MYCO/F
LYTIC medium and BacT/Alert MB. Several studies have evaluated these systems,
for example, in a controlled comparison of the performance of these systems.
Crump et al. [47] found that these systems perform comparably well in detecting
10 X. Y. Han

M. avium bacteremia and other mycobacterial and fungal sepsis. In addition, the
two CMBCS detect M. avium bacteremia 2–3 days sooner than the earlier systems.
On average, an incubation of 14 days is required.
Blood cultures also are able to detect M. tuberculosis bacteremia [47]. M. tuber-
culosis bacteremia appears to be particularly common in AIDS patients in develop-
ing countries. For examples, in Tanzania, it is the most common organism of all
sepsis-causing microorganisms, accounting for 48% (57 of 118 patients) [48]. In
Thailand, it ranks the second (27 of 114 patients), following Cryptococcus neofor-
mans (30 of 114) and surpassing M. avium (24 of 114) [49]. In Brazil, it is also the
most common cause of mycobacterial sepsis [50]. Blood cultures are as sensitive as
bone marrow cultures for the detection of M. tuberculosis, and the role of this
method appears to be expanding [51]. M. tuberculosis bacteremia in patients with
AIDS is associated with a rapid and high mortality [52]. Developing countries
recently have evaluated the performance of several blood culture systems for detect-
ing M. tuberculosis bacteremia; these include the Isolator 10 system and the
BACTEC using MYCO/F LYTIC medium, the conventional BacT/Alert FA, and the
BacT/Alert MB [52–55]. Crump et al. [55] found that BacT/Alert MB detected M.
tuberculosis bacteremia more rapidly than did manual methods, the BACTEC with
MYCO/F LYTIC medium, and the Isolator 10 system. However, the mean time to
positivity exceeded 3 weeks. The mean colony-forming units (CFU) per milliliter
blood were found to be 30 CFU/ml in one study [55] and 8 CFU/ml in another study
[52]. Together, these studies may provide some assistance with the initiation of
timely empiric antibiotic coverage against tuberculosis in patients with AIDS in
these countries in order to reduce the immediate mortality once the patient is seen
at the hospital. These data may also impact public health policies and healthcare
priorities in their respective countries.

Concluding Remarks

In conclusion, automated blood cultures have become the diagnostic mainstay for
bloodstream infections. The systems are refined and capable to cultivate various
bacteria, fungi, and mycobacteria. The laboratories have seen improved efficiency
through automation and a 5-day culturing cycle (except those for mycobacteria).
With the vast majority of significant isolates being detected within the first 72 h of
culture, the timely care of patients is facilitated. Recent integration of MALDI-TOF
in the rapid identification of bacteria from positive culture broth has further short-
ened the turnaround time in the culture diagnosis of bloodstream infections.
Automated Blood Cultures 11

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Laboratory Automation in Clinical
Bacteriology

Antony Croxatto

Introduction

About 70% of medical decisions depend on laboratory results, which indicates that
diagnostic tests have a great impact on health care [1, 2]. However, most routine
diagnostic laboratories currently face a steady increase in sample numbers despite a
limited budget as well as personnel shortages; this dichotomy significantly impacts
quality. Thus, a number of diagnostic disciplines, including chemistry, hematology,
and molecular diagnostics, have implemented automated methods over the past sev-
eral decades; in contrast, diagnostic microbiology has been considered as too com-
plex and having an insufficient number of samples to justify the development of
automated solutions. Thus, conventional methods in bacteriology did not change
much over these several decades until the relatively recent introduction of advanced
approaches such as automated antibiotic susceptibility testing (AST), molecular
diagnostic microbiology, and matrix-assisted laser desorption/ionization time-of-­
flight mass spectrometry (MALDI-TOF). The introduction of these advanced tech-
nologies has greatly improved the productivity of the clinical microbiology
laboratory, but is not sufficient to deal with the continued increase in the volume of
samples. Fortunately, the development of advanced technologies such as MALDI-­
TOF together with the introduction of liquid-based transport devices as well as the
consolidation of clinical microbiology laboratories have finally triggered the devel-
opment of “total automated systems” for diagnostic bacteriology. Even though the
first automated modules for sample inoculation such as the Autostreaker were intro-
duced in the 1970s [3, 4], the demand for such automated solutions has only emerged
at the beginning of the twenty-first century. One particular company, Kiestra Lab

A. Croxatto (*)
Institute of Microbiology, Lausanne University Hospital and Lausanne University,
Lausanne, Switzerland
e-mail: Antony.croxatto@chuv.ch

© Springer Nature Switzerland AG 2018 15

Y.-W. Tang, C. W. Stratton (eds.), Advanced Techniques in Diagnostic
Microbiology, https://doi.org/10.1007/978-3-319-33900-9_2
16 A. Croxatto

Automation (the Netherlands), has developed and proposed automated incubators

since 2006. In 2008, Kiestra introduced the first automated line in bacteriology
called “total laboratory automation” (TLA); this TLA connected an agar plate sort-
ing and barcoding instrument with automated incubators and workbenches through
a bidirectional conveyor system. The same year, three other companies, Becton-­
Dickinson (Baltimore, USA), bioMérieux (Marcy l’Etoile, France), and Copan
(Brescia, Italy), commercialized their automated inoculation systems Innova, Previ-­
Isola, and WASP (Walk-Away Specimen Processer), respectively. Kiestra Lab
Automation launched a semiautomated sample processor (InoqulA) in 2009 and a
second generation that was fully automated in 2011. Eventually, Becton-Dickinson
(BD) acquired Kiestra and formed BD Kiestra in 2012, which allowed the improve-
ment of the TLA system by introducing updated inoculation and incubator systems,
while Copan commercialized the first WASPLab in 2012. Thus, today there are
several different automated systems for bacteriology, each with different techno-
logical architectures and workflows and each with their own advantages and disad-
vantages. Importantly, automated solutions represent a new revolution for diagnostic
bacteriology with the promise of significant impact for productivity and quality in
the clinical microbiology laboratory.

The Different Automated Systems

Currently, four major diagnostic bacteriology activities can be automated: (1) inoc-
ulation, (2) plate management with conveyor systems and robotic arms, (3) incuba-
tion, and (4) digital imaging which allows plate reading by telebacteriology (Fig. 1).
There are a number of commercially available different systems that address par-
tially or totally these four processes (Fig. 1). Thus, the automated systems for bac-
teriology are commonly classified in three levels of automation: Level 1, inoculation
only; Level 2, partial automation; and Level 3, complete automation (Fig. 1). The
first level only includes specimen processors (Fig. 2). Currently, there are four auto-
mated inoculation systems commercially available: (1) the Autoplak (NTE-SENER),
(2) the InoqulA (BD Kiestra), (3) the PreLUD (I2A diagnostics), and (4) the
WASP (Copan). The Previ-Isola (bioMérieux) is no longer commercially avail-
able but has been installed in many laboratories during the past decade. The sec-
ond level, partial automation, includes the Work Cell Automation (WCA; BD) and
WASPLab (Copan) and consists of an inoculation module connected to incubators
by a unidirectional conveyor system (Fig. 3). The third level, complete automa-
tion, only includes the TLA from BD and consists of an inoculation module, incu-
bators, and workbenches that are connected by a bidirectional conveyor system
(Fig. 3). The MAESTRO system from I2A, which belongs to the second level of
automation, is still in a research and development phase and will not be discussed.
This chapter will focus only on the current commercially available automated systems
by BD and Copan.
Laboratory Automation in Clinical Bacteriology 17

Automated Manual

Follow up
Inoculation Incubation Imaging Reading work

AUTOPLAK

Previ-Isola

WASP WASPLab / WCA

PRELUD MAESTRO

InoqulA TLA

Specimen Incubators Tele- Plates

processors Digital imaging bacteriology delivery

Level of automation

Inoculation Partial lab automation Complete lab automation

Fig. 1 Automated and manual laboratory activities. Different levels of automation are commer-
cialized: Inoculation only (AUTOPLAK, Previ-Isola, WASP, PreLUD, InoqulA), partial automa-
tion (WCA, WASPLab, MAESTRO), and total automation (TLA). (Adapted with permission from
Croxatto et al. [6])

Specimen Processors

The InoqulA and WASP specimen processors (also called inoculation systems) uti-
lize different technological approaches with different features and different work-
flows (Fig. 2 and Table 1).
The InoqulA is composed of a fully automated (FA) and semiautomated (SA)
module which are linked to the SorterA (media storage) and the BarcodA (barcode
labeling) modules (Fig. 2). The FA module can only process liquid-based samples
and uses a pipetting system that has the ability to inoculate 10–800 ul on agar plates,
on slides, and/or in enrichment broth. The FA element is equipped with two robotic
arms to manage sample containers and transportation, including decapping and
recapping during the sample inoculation process. The system is also equipped with
a vortex for sample agitation prior to pipetting. The SA module can be equipped
with a safety hood and is used to inoculate nonliquid samples or liquid samples with
insufficient homogenization or containing aggregates that can clog the pipetting
system. In the SA mode, samples are deposited on the agar, but the streaking is
automatically processed. The InoqulA is characterized by an innovative streaking
approach consisting of a rolling magnetic bead. The magnetic bead covered with
sample material streaks the microbes using different rolling movements such as
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