Necrotizing Soft Tissue Infections: Anna Norrby-Teglund Mattias Svensson Steinar Skrede Editors

Advances in Experimental Medicine and Biology 1294
Anna Norrby-Teglund
Mattias Svensson
Steinar Skrede Editors
Necrotizing
Soft Tissue
Infections
Clinical and Pathogenic Aspects
Advances in Experimental Medicine
and Biology
Volume 1294
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Anna Norrby-Teglund •
Mattias Svensson • Steinar Skrede
Editors
Necrotizing Soft Tissue

Infections
Clinical and Pathogenic Aspects
Editors
Anna Norrby-Teglund Mattias Svensson
Center for Infectious Medicine, Center for Infectious Medicine,
Department of Medicine Department of Medicine
Karolinska Institutet Karolinska Institutet
Huddinge, Sweden Huddinge, Sweden
Steinar Skrede
Department of Clinical Science
University of Bergen
Bergen, Norway
Department of Medicine
Haukeland University Hospital
Bergen, Norway
ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-3-030-57615-8 ISBN 978-3-030-57616-5 (eBook)
https://doi.org/10.1007/978-3-030-57616-5
# Springer Nature Switzerland AG 2020

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Preface
During the last four decades, there has been a worldwide upsurge of severe
skin and soft tissue infections. These infections are frequently associated with
severe complications such as septic shock and multi-organ failure, resulting in
need for intensive care and challenging rehabilitation. Unfortunately, in too
many cases the outcome is loss of life or limbs.
This book is devoted entirely to these severe and rapidly spreading necro-
tizing soft tissue infections. It includes 12 chapters ranging from case reports
written by relatives or survivors providing the patient perspective, as well as
clinical case reports written by clinicians caring for the patients. In addition,
the book contains chapters describing the current understanding of therapeutic
strategies, pathogenic mechanisms, and systems medicine approaches
allowing for new concepts for patient stratification. The work has its founda-
tion in a recently completed European Union funded project called INFECT,
which during the period 2013–2018 conducted comprehensive studies of
these heterogenous and complex life-threatening infections.
The project involved the establishment of the largest prospective patient
cohort to date, a collection of biobank samples that were further analyzed to
delineate key events underlying disease outcome and exploring their value for
improved diagnostics and therapeutics. Advanced systems biology analysis
and computational modeling were employed, and findings were validated in
clinically relevant experimental in vitro and in vivo tissue infection models.
To achieve its goals, the INFECT consortium gathered experts within the
fields of intensive care medicine, infectious diseases, microbiology, bacterial
pathogenesis, systems biology, and diagnostics, as well as the patient organi-
zation Lee Spark NF Foundation. A success factor of the INFECT project was
the integrated work requiring the close interaction between partners from
different disciplines and full utilization of all available expertise and resources
provided by the consortium partners (Fig. 1).
In this book, different aspects of necrotizing soft tissue infections are
summarized by INFECT partners, providing contemporary state-of-the-art
presentations, highlighting both advances made within INFECT and else-
where. The different chapters of this volume collectively demonstrate the
great heterogeneity of these infections and demonstrate the necessity for
tools to stratify patients for novel, personalized tailored therapies. Hence,
the book underlines the need for further actions toward the implementation
v
vi Preface
Fig. 1 INFCT consortium meeting in Stockholm 2017 attended by INFECT partners, i.e. Karolinska Institutet (coordi-
nator, Sweden), Rigshospitalet (Denmark), Karolinska University Hospital (Sweden), Blekinge County Hospital
(Sweden), Sahlgrenska University Hospital (Sweden), University of Bergen (Norway), Helmholtz Centre for Infection
Research (Germany), Wageningen University and Research (The Netherlands), University of Lyon (France),
LifeGlimmer GmbH (Germany), Tel Aviv University (Israel), Lee Spark NF Foundation (UK), University of North
Dakota (US), and Cube Dx (Austria)
of personalized medicine for therapeutic advances for patients with necrotiz-

ing soft tissue infections.
The INFECT consortium was supported by two scientific advisors:
Dr. Marina Morgan, consultant clinical microbiologist at Royal Devon and
Exeter hospital in the UK, and Prof. Matthias Reuss from Stuttgart Research
Center Systems Biology, who provided valuable scientific advice throughout
the entire project period. We also want to acknowledge the financial support
from the European Commission, and we are grateful to the invaluable guid-
ance we received from the Scientific Officer, Ms. Christina Kyriakopoulou.
Last but not least, we would like to express our sincere gratitude to patients
and relatives for their participation in the INFECT project.
Huddinge, Sweden Anna Norrby-Teglund

Huddinge, Sweden Mattias Svensson
Bergen, Norway Steinar Skrede
Contents
1 The INFECT-Project: An International

and Multidisciplinary Project on Necrotizing Soft Tissue
Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Mattias Svensson and Anna Norrby-Teglund
2 Necrotizing Soft Tissue Infections: Case Reports
from the Patients Prospective . . . . . . . . . . . . . . . . . . . . . . . . . 7
Doreen Cartledge, Lucy Dove, Emma Richardson,
and Robert Wilkie
3 Necrotizing Soft Tissue Infections: Case Reports,
from the Clinician’s Perspectives . . . . . . . . . . . . . . . . . . . . . . 21
Torbjørn Nedrebø and Steinar Skrede
4 Necrotizing Soft-Tissue Infections: Clinical Features
and Diagnostic Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Martin Bruun Madsen, Per Arnell, and Ole Hyldegaard
5 Microbiological Etiology of Necrotizing Soft Tissue
Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Steinar Skrede, Trond Bruun, Eivind Rath, and Oddvar
Oppegaard
6 Beta-Hemolytic Streptococci and Necrotizing Soft Tissue
Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Trond Bruun, Eivind Rath, Oddvar Oppegaard,
and Steinar Skrede
7 Treatment of Necrotizing Soft Tissue Infections:
Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Oddvar Oppegaard and Eivind Rath
8 Treatment of Necrotizing Soft Tissue Infections: IVIG . . . . . . 105
Martin Bruun Madsen, Helena Bergsten,
and Anna Norrby-Teglund
9 Pathogenic Mechanisms of Streptococcal Necrotizing
Soft Tissue Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Nikolai Siemens, Johanna Snäll, Mattias Svensson,
vii
viii Contents
10 Systems Genetics Approaches in Mouse Models

of Group A Streptococcal Necrotizing Soft-Tissue
Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Suba Nookala, Karthickeyan Chella Krishnan, Santhosh
Mukundan, and Malak Kotb
11 Systems Biology and Biomarkers in Necrotizing
Edoardo Saccenti and Mattias Svensson
12 Systems and Precision Medicine in Necrotizing
Vitor A. P. Martins dos Santos, Christopher Hardt, Steinar
Skrede, and Edoardo Saccenti
The INFECT-Project: An International
and Multidisciplinary Project 1
on Necrotizing Soft Tissue Infections
Mattias Svensson and Anna Norrby-Teglund
Contents
1.1 Necrotizing Soft Tissue Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 The INFECT-Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.1 Initiative and Consortium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.2 Objectives and Work Plan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.3 Achievements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Abstract Keywords
This book describes clinical and microbiologic Necrotizing soft tissue infections · Systems
aspects, pathogenesis, and diagnostics, related medicine · Pathogenesis
to the severe and rapidly spreading necrotizing
soft tissue infections. The work has its founda-
tion in a recently completed European Union
funded FP7-project called INFECT, which
during the period 2013–2018 focused on
utilizing a systems medicine approach to
increase our understanding of these heteroge- Highlights
nous and complex life-threatening infections. The work accomplished in the INFECT-project is
In this chapter, the aim and scope as well as summarized in book Chaps. 2, 3, 4, 5, 6, 7, 8, 9,
key achievements of the INFECT-project are 10, 11, and 12, and highlights from these are:
described.
• Advanced understanding of necrotizing soft
tissue infections and associated pathogenesis
• Clinical and experimental data of importance
for improved diagnostic and therapeutics
M. Svensson (*) · A. Norrby-Teglund
• Demonstrated the value of a systems medicine
Center for Infectious Medicine, Department of Medicine, approach in infectious diseases
Karolinska Institutet, Huddinge, Sweden
e-mail: mattias.svensson@ki.se
# Springer Nature Switzerland AG 2020 1

A. Norrby-Teglund et al. (eds.), Necrotizing Soft Tissue Infections, Advances in Experimental Medicine
and Biology 1294, https://doi.org/10.1007/978-3-030-57616-5_1
2 M. Svensson and A. Norrby-Teglund
• Scientific foundation for personalized medi- disciplines including infectious diseases, inten-
cine in necrotizing soft tissue infections sive care, microbiology, cell biology, bacterial
pathogenesis, systems biology, computational
1.1 Necrotizing Soft Tissue modelling and diagnostics. INFECT partners are
Infections listed in Table 1.1.
Necrotizing soft tissue infections (NSTI) are rap-

idly spreading infections that may cause exten- 1.2.2 Objectives and Work Plan
sive soft tissue or limb loss, multiorgan failure,
and are associated with a considerable fatality The specific objectives of INFECT were to:
rate. These life-threatening infections that are
caused by a variety of pathogens are among the 1. Establish a patient cohort and collect samples
few infectious diseases that remain associated for a biobank collection
with substantial mortality and amputation even 2. Analyze patient samples and clinical isolates
in young individuals with no underlying condi- to pin-point key host and pathogen factors
tion. It is undisputed that rapid diagnosis and involved in the onset and development of
prompted intervention is directly related to sur- infection
vival, but diagnosis and management are difficult 3. Identify and quantify disease signatures and
due to heterogeneity in clinical presentation, underlying networks that contribute to disease
co-morbidities, and microbiological etiology. To outcome
tackle these challenges an international and mul- 4. Exploit identified disease traits for the
tidisciplinary consortium, INFECT (Improving innovation of optimized diagnostic tools
Outcome of Necrotizing Fasciitis: Elucidation of 5. Translate the advanced knowledge generated
Complex Host and Pathogen Signatures that Dic- into improved classification and management,
tate Severity of Tissue Infection), was formed as well as novel therapeutics
https://permedinfect.com/projects/infect/.
To achieve the objectives, a comprehensive
and integrated knowledge of diagnostic features,
causative microbial agent, treatment strategies,
1.2 The INFECT-Project and pathogenic mechanisms (host and bacterial
disease traits and their underlying interaction net-
1.2.1 Initiative and Consortium work) were required. Therefore, the work of
INFECT was structured around an integrated
The initiative to INFECT was taken in 2012 when systems biology approach in patients and differ-
a consortium of 14 partners applied for EU ent clinically relevant experimental models. The
funding within the Seventh Framework INFECT-project was set up to be multidisciplin-
Programme Health and Innovation, under the ary and included clinical partners, academic
programme “Systems medicine: Applying experimentalists and mathematical modelers,
systems biology approaches for understanding one patient organization, as well as two small-
multifactorial human diseases.” INFECT was and medium-sized enterprises focusing on data
granted EU funding and was sponsored from integration in systems and synthetic biology,
January 2013 to June 2018. The overall goal of and development of diagnostic tools. The partners
INFECT was to reveal pathophysiological contributed with unique expertise, infectious dis-
mechanisms and disease signatures, as well as ease and intensive care clinicians, technical
identifying prognostic and diagnostic markers of platforms and/or model systems that together
the multifactorial highly lethal NSTI. To enable provided the means to successfully conduct the
this multifaceted work the consortium was multifaceted research proposed (Table 1.1.). The
designed to include industrial, clinical, and pre- work included seven highly integrated scientific
clinical partners with expertise in different work packages, one work package on
1 The INFECT-Project: An International and Multidisciplinary Project on. . . 3
Table 1.1 INFECT partners

Partner organization name Country Team leader Main role
Karolinska Institutet Sweden Anna Norrby- Coordinator, bacterial pathogenesis, experimental
Teglund human tissue model systems
Mattias Svensson
Rigshospitalet Denmark Ole Hyldegaard Patient cohort, biobank, therapeutics
Erik Jansen
Karolinska University Sweden Michael Nekludov Patient cohort, biobank, therapeutics
Hospital Folke Lind
Blekinge County Hospital Sweden Ylva Karlsson Patient cohort, biobank, therapeutics
Sahlgrenska University Sweden Per Arnell Patient cohort, biobank, therapeutics
Hospital Anders Rosén
University of Bergen Norway Steinar Skrede Patient cohort, biobank, therapeutics, microbiology
Trond Bruun
University of North USA Malak Kotb NSTI murine model, forward genetics
Dakota Suba Nookala
Helmholtz Center for Germany Dietmar Pieper Systems biology
Infection Research Singh Chhatwal
University of Lyon France Francois Microbiology, systems biology
Vandenesch
Wageningen University The Vitor Martins dos Systems biology, integration modeling
and Research Netherlands Santos
LifeGlimmer GmbH Germany Vitor Martins dos Systems biology, integration modeling, SME
Santos
Edoardo Saccenti
Tel Aviv University Israel Eytan Ruppin Systems biology, integration modeling
Raphy Zarecki
Cube Dx Austria Christoph Diagnostic tools, SME
Reschreiter
The Lee Spark NF UK Doreen Cartledge Patient organization
Foundation
dissemination and exploitation of the knowledge of the disease and advanced understanding of the
obtained, and one management work package. A underlying mechanisms and host–pathogen
central part of the INFECT-project was the pro- interactions. The results were translated into
spective enrollment of patients with NSTI and novel diagnostic tests and improved patient man-
creation of a clinical data registry, the collection agement (Fig. 1.1).
of biobank samples and the causative microbes,
which all formed the basis for the experimental
studies (Madsen et al. 2019). Further, INFECT 1.2.3 Achievements
was designed to use an integrated systems biol-
ogy approach in patients and different clinically The INFECT-project has generated comprehen-
relevant experimental infection models, including sive knowledge of diagnostic features, causative
both murine NSTI models (Chella Krishnan et al. microbial agents, treatment strategies, and patho-
2016; Nookala et al. 2018) and a human genic mechanisms (host and bacterial disease
organotypic skin model (Mairpady Shambat traits and their underlying interaction network)
et al. 2016; Siemens et al. 2015, 2016). The through analyses of the INFECT patient cohort
work-flow also included a comprehensive set of and associated biobank. Also, INFECT has
analyses followed by integration of results in proven the value of systems medicine approaches
advanced computational platforms, which in acute infectious diseases to achieve improved
enabled generation of pathophysiological models diagnostics and therapeutics to improve patient
Fig. 1.1 An overview of the INFECT consortium and activities, in which clinical challenges, identification of
pathophysiological mechanisms, and development of diagnostic tools were in focus
1 The INFECT-Project: An International and Multidisciplinary Project on. . . 5
disease outcome. In this book, we have causative microbe and by host factors (Chella
summarized the scope and outcome of the Krishnan et al. 2016; Siemens et al. 2016;
INFECT-project. The results have been compiled Thanert et al. 2019). This underscores the
into 12 chapters, each of which has a unique focus need for patient stratification and implementa-
ranging from patients’ and doctors’ experiences tion of tailored therapy/personalized medicine
of NSTI, to different treatment alternatives and in these infections, presented in Chap. 13.
the use of systems medicine in NSTI. In short, • Development and testing in the clinical setting
Chaps. 2 and 3 portray affected patients’ and of multiplex diagnostic tools for rapid patho-
treating physicians’ experiences; Chaps. 4, 5, gen identification and monitoring of disease
and 6 communicate clinical characteristics, associated biomarkers.
microbial etiology, and the role of beta-hemolytic • The novel understanding of the disease
streptococci; Chaps. 7 and 8 describe treatment mechanisms of these infections has resulted
options with particular attention to antibiotics and in changed clinical practice related to antibi-
polyspecific intravenous immunoglobulin otic usage as well as use of immunomodula-
(IVIG); Chaps. 9 and 10 depict pathogenic tory treatments (Bergsten et al. 2020; Madsen
mechanisms revealed from in vitro and in vivo et al. 2017) (Chaps. 7, 8, and 9).
models of NSTI; and finally, Chaps. 11 and 12 • Fostering the new generation of clinical and
illustrate the merits of system biology for preclinical scientists within the field of
identifying NSTI biomarkers, and the use of systems medicine in infectious diseases.
systems medicine as a tool for stratifying patients
with NSTI.
Below we summarize some key findings and
achievements of INFECT:
1.3 Future Perspectives
• Establishment of the world’s largest NSTI
We foresee that the achievements of INFECT will
patient cohort with extended clinical registry
form a basis for future advances in the area of
and associated biobank.
implementing personalized medicine in infectious
• Four hundred and nine NSTI patients with
diseases. This book volume is designed to present
completed clinical registries (>2000
the contemporary understanding of clinical
variables) were enrolled and associated
features, treatment, and pathogenic aspects in
biobank samples (> 6000) and clinical bacte-
NSTI, as well as serve as an overview of the
rial isolates collected.
contributions to this field achieved through the
• In Chaps. 3, 4, 5, and 6 advanced insight into
INFECT study. Readers will also be guided to
the clinical aspects of NSTIs providing the
other references that should be consulted for fur-
basis for evidence-based guidelines for patient
ther information on NSTI pathogenesis, causative
management and care is given (Bruun et al.
agents, treatments as well as novel diagnostic and
2020; Madsen et al. 2019).
therapeutic strategies in order to improve out-
• In Chaps. 9, 10, 11, and 12 it is shown how the
come of NSTIs. The foundation that the results
systems medicine approach within INFECT
of INFECT has laid will be further explored and
substantially advanced our understanding of
implemented in the field of personal medicine in
these life-threatening infections, including the
infectious diseases in the current multinational
identification of novel pathogenic mechanisms
projects PerMIT and PerAID, www.
and specific host and bacterial disease traits
permedinfect.com.
associated with disease outcome (Afzal et al.
2020; Chella Krishnan et al. 2016; Siemens
Acknowledgement Financial support: The work was
et al. 2015, 2016; Thanert et al. 2019). supported by the European Union Seventh Framework
• In Chap. 10 it is demonstrated how the patho- Programme: (FP7/2007-2013) under the grant agreement
physiology of NSTI is influenced both by the 305340.
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Necrotizing Soft Tissue Infections: Case
Reports from the Patients Prospective 2
Doreen Cartledge, Lucy Dove, Emma Richardson,
and Robert Wilkie
Contents
2.1 Stephen Lee Spark’s Story as Told by His Mum Doreen Cartledge . . . . . . . . . 7
2.2 Robert’s Experience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3 Frankie’s Story Written by His Mother Lucy Dove . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.4 Zara’s Story Written by Her Mum Emma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Abstract support them through further operations,

let alone mental scars. Parents show a strength
All who have contributed in writing this chap-
of support like no other and we hope that their
ter have been patients and parents that have
lives can be enhanced through the battle they
experienced an horrific life event. The horrific
have individually won let alone their family.
disease named necrotising fasciitis has
Robert’s story from a patient’s perspective is
affected our lives for ever. All four stories
quite different and you will read his courage
have explained how easily an everyday infec-
throughout. We continue to raise awareness
tion can develop incredibly quickly into a life-
through education.
threatening experience. Three stories are
expressed from the worn hearts of being a
Keywords
mother, fighting for their child every step of
the way. Knowing our children and how they Necrotizing soft tissue infections · Patient
react through pain and illness is felt in each experience
word, sentence, paragraph and even between
the lines. Dedicating our unmarkable love and
devotion for the child we carried for 9 months.
To see them suffer in illness is heart 2.1 Stephen Lee Spark’s Story
wrenching, but to experience this disease as Told by His Mum Doreen
necrotising fasciitis is something else. We Cartledge
must live through every day watching them
grow with their scars of debridement, and to We lost Stephen Lee Spark, Son, Brother,
Nephew, Cousin and Grandson to a horrific dis-
D. Cartledge (*) · L. Dove · E. Richardson · R. Wilkie
ease named necrotising fasciitis. The time is
Lancashire, UK important when being bereaved it was 9.45 am

8 D. Cartledge et al.
on the 6th October 1999. My son had been taken Spark”. At that moment I really thought I was in a
away from me in 5 days. terrible bad dream. But then the staff nurse
Lee was a healthy 23-year-old, with a lovely replied “Oh wait a minute he is in resuscitation”
quite naive personality. Martyn, my younger Son, I replied “No, I do not think this is possible”. The
Lee’s brother lived in Sheffield Yorkshire. Staff Nurse asked me to phone back 15 min later,
Lee had an opportunity for work in Sheffield, and suggested I had a sweet cup of tea and some-
so he travelled there in readiness of the interview. thing to eat. By this time, I was in shock and was
Approximately 3 days into staying over there fainting every time I stood on my feet. I desper-
Lee felt cold and had symptoms of flu. I informed ately tried to eat a slice of toast, it was difficult
Martyn to advise Lee to take two paracetamol and akin to chewing on a piece of leather. I had no
tablets every 4 h and to drink plenty of water. I saliva in my mouth to swallow. Twenty minutes
could not speak to Lee as he was not on the later I phoned the hospital and spoke to the Staff
phone. Lee went for his interview feeling very Nurse she replied “I am so sorry to have to tell
poorly but managed to get through it and waited you that Lee has passed away. Would you like to
for a few days in the hope that he would be called speak with Martyn?” we could only cry together.
upon to attend a second interview. Five days later I eventually said “Go back home. I will see you
Emma, Martyn’s partner, phoned me to say Lee there”. All this time I was on my own and I now
had something more serious than flu and Lee was needed my husband (not Lee’s father) to be with
seriously ill and as a result they had phoned for an me. I explained the whole horrific story. It was 2 h
ambulance. I remember the daunting tones of before he arrived home and I had packed a few
Martyn yelling in the background, he sounded things for the long journey to Sheffield. I took a
like a wild animal and was hysterical. I lived bottle of water, a bucket to for me to vomit in as I
over a 100 miles away and I was in shock and was in shock and an old dressing gown that my
disbelief, so I knew I was unable to drive all the mum gave me when I was younger to give me
way to Sheffield even at a time like this. The comfort. We eventually arrived in Sheffield. Dur-
emergency crew arrived and tried their best to ing the journey I tried my best to make the deci-
apply emergency care to Lee but he was falling sion of whether to see Lee’s body or go straight to
into unconsciousness. Martyn looked on in disbe- Martyn I decided that Martyn needed support he
lief, why was Lee so very poorly. Martyn had been through a terrible trauma. After all it
explained later that Lee’s eyes were rolling also. was better for me to remember Lee the last time I
What a terrible experience for Martyn to witness. saw him full of health and happiness.
I just wish I could have been there for them both. We asked Martyn a few gentle questions as he
The emergency team tried their very best to keep was crying all the time and spent a long time just
Lee alive. Placed appropriate drains in him but holding one another for comfort. We will never,
Lee was not reacting to any treatment. The ambu- never forget that morning as long as we live. We
lance crew decided to take Lee to the nearest then awaited the post-mortem results and we were
hospital and kindly passed the telephone number told Lee had died of a Strep milleri infection—
of the hospital. Martyn travelled in the ambulance from an abscess in his gum and that it had devel-
with Lee and the crew. I am sure the stress of this oped into Necrotising Fasciitis. His internal
for such a young man was truly horrific. The organs had been eaten away. Martyn informed
ambulance crew also said to ask mum to allow us that when the ambulance crew were applying
15 min for them to arrive at the hospital before resuscitation to Lee all his rotting flesh was com-
telephoning. I phoned the hospital in disbelief ing down his nose and out of his mouth. Still to
how was it possible for flu like symptoms to this day he remembers every finer detail and
develop into Lee being so terribly poorly. What especially the smell of the rotten flesh. Martyn
was the problem? I phoned Hallamshire Hospital also referred to a dark purple like rash around
and spoke to the Staff Nurse in A & E. I explained Lee’s neck. What on earth was this terrible dis-
the circumstances and she replied “that there was ease all about, I phoned Public Health England
no one with them by the name of Stephen Lee several days later wanting to find out more about
2 Necrotizing Soft Tissue Infections: Case Reports from the Patients Prospective 9
necrotising fasciitis and spoke with the Strep A 2 days later that the necrotising fasciitis appeared
and Respiratory Department, they were truly making it more likely that the infection was post-
compassionate and informative. Over the next operative. In those days after treatment it was felt
few days I spoke with many Consultants and that I would heal better at home, so I was released
medical staff all informing a little more about on 27 January 1982; the day my eldest child was
this terrible disease and all apologised that they born. On this day I remember being pushed
could not provide a telephone number of a sup- through the streets of the hospital complex in a
port group and all mentioned that there was a wheelchair by a nurse from the main building to
need for a support group to offer that listening the maternity department to meet my new-born
ear and an understanding of compassion. son. Later that day as my wife was to remain in
Therefore, I launched my Charity The Lee the hospital for 7 days, I was released into the care
Spark NF Foundation in January 2000. My first of my mother. My wife remembers a few days
survivor contacted me in May 2000. What a thrill before my release having a conversation with the
it was to talk to her. I desperately hung on to consultant about my future prospects, he
every word she said. That way I had a better informed her that there was no reason why I
understanding of what my dear son had been could not live a normal life, “but” I would possi-
going through. bly require some corrective surgery in the future.
Never was a truer word spoken.
Future Perspectives After being released from the hospital I took
The early diagnosis and treatment of necrotising the consultant at his word and virtually just got on
fasciitis must be taught at core level in order to with my life. Taking my new parental responsi-
save lives and devastation within families. The bilities seriously as I set about being a father for
financial drain on the medical system is also, a the first time. With hindsight I now realise that
major factor, to the patient if they survive a life of carrying a pram up three flights of stairs after
surgery, medicine and psychological problems. defeating necrotising fasciitis was not the
Martyn strives everyday living through the brightest idea I have ever had. Now as I reflect,
tragic circumstances of Lee’s death, my dear I also realise that perhaps I should have taken
sons. What has happened to our family? His more time off work and gradually eased myself
father never got over the shock of losing Lee back into the workplace. It really was not the
and passed away a few years later (Fig. 2.1). greatest idea I have ever had to go straight back
into such a physical job. After all, a huge area of
abdominal muscle had been lost forever leaving
2.2 Robert’s Experience vital organs unsupported and unprotected. As a
result of this I now have a loopy bowel and
The best way to describe living with the effects of internal bruising to the stomach plus I have also
necrotising fasciitis is to say it is a journey which suffered no fewer than 17 abdominal hernias, two
continuous long after the infection has gone. This of which are irreparable and one is a ruptured
journey can often feel like a roller coaster ride repair for which I am required to wear a support
with its highs and lows. Looking back, I would belt. Wearing a hernia (stoma) belt has its
now say the first part of my journey was easy as I advantages for it eases the pain considerably but
knew little about what was going on. I certainly it also has disadvantages for it restricts my move-
had no idea of the seriousness of my condition for ment and can be almost intolerable in hot
the powerful drugs I was given pretty much kept conditions. After my first hernia operation I dis-
me in a deep sleep for almost a fortnight. covered that I suffered from acute acid reflux and
I was admitted to my local hospital with was given a chalky medication to drink, this did a
expected appendicitis on New Year’s Eve 31st great job of easing the reflux but also caused
December 1981 and operated on the following constipation due to a build-up of chalk in my
day 1st January 1982, but it was not until almost bowel. After several endoscopy exams and a
Fig. 2.1 Martyn Spark

(left) Stephen Lee Spark
(right) deceased
failed colonoscopy, I was sent for a CT colonos- more manageable pain. I feel incredibly lucky to
copy which revealed I had loops in my bowel on have almost accidently discovered the difference
my damaged side hence the reason for the intes- wearing braces instead of a belt can make. Pain
tine pain caused by the constipation. It may sound management is without a doubt the most difficult
foolish, but this has resulted in me having to re thing I have had to learn on my journey, for the
learn how to and what to eat. For example, I pain around my stomach causes by internal
cannot eat anything from the onion family except bruising is relentless. The only help I receive for
the green part of a spring onion or the green part this are Codeine and Paracetamol, but I find the
of a leek, I cannot eat pineapples, lemons, limes, use of heat also helps, sadly the aforementioned
oranges I cannot take vinegar or eat pickles and hernias make exercising the area around my
many other everyday foods. I also must think wound sight difficult, so exercise does not help.
about the kind of food I need to eat to help prevent Three years ago, during a gastroscopy proce-
any recurrence of acute constipation, alas this dure it was discovered that I had an ampullary
means eating out can be difficult if not in some adenoma (pre-cancerous lesion) in the damaged
cases impossible. As I mentioned earlier, I also area of my abdomen. This remains benign, it is
suffer from internal bruising to my stomach, to almost as if this has taken up residence in the
this day I have never been given a reason for this, space where I lost the muscle due to Necrotising
but I believe it is caused by my bowel pressing on Fasciitis, therefore I see this as a continuation of
my stomach when it is full. my NF journey. Journeying with the effects of NF
Recently I got rather fed up having to keep is a continual battle in trying to adapt to the
pulling at my waist band and tightening my trou- demands placed upon my damaged body. For
ser belt so I changed to using braces, the easing of years I have struggled with pain causing me to
the discomfort around my stomach was almost overcompensate putting more pressure on my
instant; the pain is still there but it is a much undamaged side which now has osteoarthritis. A
Fig. 2.2 Robert Wilkie

survivor of necrotising
fasciitis
coincidence perhaps? This brings me to what I brother, Kayne was unwell the day before with
find to be the most difficult thing to live with post pustules on his face, flu like symptoms and a pin
Necrotizing Fasciitis, dealing with medical peo- prick rash on his back and chest. An out of hours
ple, for there seems to be a willingness to keep NF GP referred Kayne to A and E, where I was told
quiet, why? Surely if I had been better informed, I that his blood results were consistent with a viral
could have been better prepared and more able to infection and he was prescribed flucloxacillin.
reduce the risks of further damage to an already The links between Kayne and Frankie’s
battle-scarred body. symptoms are very important.
In the late morning of Tuesday 9th April,
Future Perspective Frankie appeared to become very unwell with
With necrotising fasciitis listening to a survivor I similar pustules on his face and a red pinprick
believe is absolutely crucial yet to often I feel like rash all over his arms, chest and back. However,
no one is listening and many look at me and think unlike his brother, Frankie was screaming when I
here he comes again. Surely listening to any sur- moved him and seemed to be in a lot of pain.
vivor is crucial to after care and pain management At our local walk in centre I stripped Frankie’s
(Fig. 2.2). clothes off and I could see that a boil type lump
had developed on his left thigh. I had not been
there when I got him ready and he did not like me
2.3 Frankie’s Story Written by His touching the area, so it was obviously very pain-
Mother Lucy Dove ful to him. He began hitting me and did not want
to be cuddled. Frankie was a very cuddly baby, so
Frankie became unwell on Monday 8th April this sent alarm bells ringing in my head! I
2013 and he was only a 1 year of age. He had explained this to the nurse practitioner, and they
flu like symptoms. I describe his early symptoms told me his temperature and other observations
as flu like symptoms because Frankie had a runny were fine. (I told then he had been given medica-
nose and a temperature of 39 C. His older tion for a high temperature). They were going to
Fig. 2.3 Frankie’s back

the morning after being
admitted. Once can clearly
see the swelling on his left
side and changes to the skin
colour on the surface
discharge him home as they said they believed he back. Om his left side approximately halfway up,
must have a viral infection. I informed them of I pressed Frankie cried out in pain. I worked out
Frankie’s history including how his brother had there was an area of approximately 2 in. by 2 in.
been unwell. I stressed that I was not happy with that was very painful for Frankie when I touched
how much pain Frankie was in, the paracetamol it. I then noticed that it could in fact be seen from
and ibuprofen were not working to ease his pain the side as a swelling under his skin.
and so I really was not happy to go home with When another doctor examined Frankie, she
him. They then told me I could take him to A concluded that she believed he had a tissue infec-
and E. tion. At roughly 9 pm a doctor took a blood
I took Frankie straight to Children’s A and sample from Frankie’s foot. An Orthopaedic doc-
E. He was a very tough little boy, and nothing tor examined Frankie and concluded that Frankie
ever fazed him and so I knew that something had had a possible tissue infection.
been missed because Frankie was in absolute In the early hours a doctor came into the room
agony. He could not be pacified and seemed to and told me that a “marker” in Frankie’s bloods
be getting worse before my eyes. He would not was very high, but they decided not to give
stand up and kept screaming. antibiotics and await more tests in the morning.
When seen by a consultant I pointed out the I trusted that the doctors knew what was medi-
lump under the skin on his thigh. Back in the cally right for my son. During the Frankie kept
waiting room Frankie kept deteriorating and waking and projectile vomiting. He kept crying
now he was howling in pain as if he had been on and off for hours. He was given codeine earlier
really bad injured. At this point I was becoming in the evening which had made him drowsy, but
increasingly frustrated. I felt as though my he was still in a great deal of pain. I had a tough
concerns were not being understood. The only night with him as I could not comfort him, and he
way I could get him comfortable was to lay him was in so much pain.
on my chest facing me. This was when I began In the morning Frankie looked totally deliri-
examining him myself. I am not medically trained ous. He could barely move. I noticed that the skin
but nor was I paranoid first-time mum. I knew on Frankie’s back was now red in parts (Fig. 2.3).
something was wrong and I decided to check It looked like the swelling had spread up his back
everywhere on his body. I refused to accept this and as you all can see his back appears to be
was just a viral infection. I began checking his swollen now (but more obvious on his left side).
The left side now had red marks appearing on the had removed all the layers of skin and fascia
surface. The boil on his left thigh seemed to be from almost his entire back, left side and quite a
moving outwards as if spreading. Were the boil lot of his left thigh and muscle had been removed.
had been was now looking dark blue like a bruise. We were told that they had not been able to
Unfortunately, I could not get a photo of his leg. remove all the infection because Frankie was too
He would not let me touch anywhere on the left weak for any further surgery and that he was been
side of his back or indeed his body. I remember given strong antibiotics intravenously. The con-
touching his left arm and him crying out in pain. sultant told us that they had done everything they
A group of doctors came into the room I told could at the point and that it was now up to
them that the swelling on Frankie’s back looked Frankie’s body to fight the infection with the
like it had spread, and I showed the doctors help of some broad-spectrum antibiotics. We
Frankie’s left thigh. I was told Frankie was to were asked to get everyone we needed up to the
start antibiotics, that he needed an emergency hospital because they did not believe our baby
MRI scan. Frankie was given oral sedation and I was going to survive the night. Not only did he
went down with him for his MRI scan which have NF but the deadly toxins in his body causes
lasted over 45 min. After the consultant told me sepsis and put his internal organs at great risk.
Frankie needed to be sent by ambulance to As parents we could not believe we were being
another hospital and that he would need to go to told that our son might not survive never mind the
theatre to have the pus removed from under his fact that he had no skin covering his back and
skin. I asked him what it was, and he replied; sides. They allowed us in to see Frankie and it
“Necrotising fasciitis”. I had never heard of this was unbearable to see him the way he was.
before and before I had had time to look it up on Frankie was fully ventilated, in an induced coma
google two doctors came into the room and began and swollen to about five times his normal size.
trying to put cannulas into Frankie’s hands. It was As parents we could hardly recognise our own
now just before 3 pm. The doctors started putting son. The only thing that made him Frankie was
what she said were antibiotics into Frankie’s can- his beautiful white curly hair.
nula and pushing large syringes of saline into him We cannot fault anything the second hospital
one after the other. This was when I became for the care they gave to our son. Frankie by some
worried something was really, seriously wrong. miracle made it to Friday 12th April when doctors
Frankie was transferred to the other hospital by took him back down to surgery. We were told that
ambulance. When we arrived, we were taken to if the infection had spread then Frankie would be
the resuscitation area where doctors, consultants made comfortable and allowed to pass away
and different specialist surgeons began peacefully. He was down in the theatre for five
introducing themselves. As they were about to and a half hours the surgeon had managed to
put Frankie onto a ventilator, we kissed him remove the dead tissue from deep behind his
goodbye never knowing if we would ever see shoulder blade and had been forced to cut deeper
him alive again. A consultant plastic surgeon down into the muscle in his thigh. They were
came into the room and told us that Frankie was confident that they had removed everything and
very seriously ill and that his condition was life that everything now looked free from infection.
threatening. She told us that until they got him We were informed that the bacteria that has
into theatre and tried to remove all the dead layers caused the NF to develop was called strep A,
of skin and tissue, they could not know how bad which knowing this meant that they could adjust
it was. the antibiotics he was on.
Frankie was in surgery for almost 10 h and After Frankie’s second operation we felt more
when he came back the lead plastic surgeon and positive but were consistently told that Frankie
PICI Consultants took us into a private room. We still had a very long way to go because of the
were told that Frankie’s body had been exten- extensive damage the NF had causes to his body.
sively damaged by the infection. The surgeon Over the weekend Frankie remained in an
Fig. 2.4 Frankie’s first

dressing change on the
ward with me
induced coma in PICU and then on Monday 15th He had recurrent infections and 1 year later had to
April 2013 he underwent a further 5 h of surgery have his central back re-grafted. He went to the-
were expert plastic surgeons who specialise in atre every 6 weeks for steroid injections. His
burns patients, grafted healthy skin from his grafts became raised and sore. He had to take
tummy, chest, right calf, left shin and back of gabapentin for a year which really affected his
his thigh onto his back and left leg. They were behaviour. He takes regular pain killers the stron-
amazed with how well everything went and fol- gest being oral morphine. He has low self-esteem
lowing two dressing changes in theatre on Friday and struggles with his confidence, but thankfully
19th April 2013 Frankie was stable enough to be he is very active, and we encourage him to not let
taken of his ventilator. He remained on high doses his skin grafts hold him back. We cream him four
of morphine and clindamycin as well as other times a day. I have included picture of Frankie’s
drugs, but he became well enough to be trans- skin grafts from 2013 and now in 2020 (Figs. 2.4,
ferred onto the burn’s unit. 2.5, 2.6, and 2.7). Frankie is an inspiration. As he
Fast forward 7 years and Frankie is now grows, he will need more skin grafts and
8 years old. We stayed in hospital for 3 months procedures. We are incredibly lucky that he sur-
as he underwent painful dressing changes and vived but our only wish is that it was diagnosed
procedures. He wore tight pressure suits and suf- and treated sooner.
fered break downs in his scars for 2 years after.
Fig. 2.5 Tissue loss on Frankie’s leg with skin graft. May 2013
Fig. 2.6 Frankie May 2013
I think there are four vital lessons that could be predisposed to when taking history as this could
learnt from Frankie’s case. help with a faster diagnosis. I would argue that
this is very important with young children or
Future Perspective those with a weaker immune system. The links
Doctors must pay close attention to any bacterial between Kayne and Frankie’s symptoms are very
infections that a person may have been important because Impetigo can be caused by the
Fig. 2.7 Frankie

January 2020
bacteria Streptococcus (most common) or Staph- Listen to parents! They see their child every
ylococcus (staph). Methicillin-resistant Staph day and they can be the best judge of their child’s
aureus (MRSA) is also becoming a common condition. If they are worried, it is for a reason.
cause. This could then help with early treatment This is vital to helping get an early diagnosis.
and prevent complications.
Although Frankie had similar symptoms to his
brother, Frankie was clearly suffering from a
2.4 Zara’s Story Written by Her
great deal of pain. Doctors need to be aware of
Mum Emma
this. His symptoms should not have caused the
level of pain he was in and this is an important
I have four children, two boys and two girls. My
indicator that something more serious is
daughter Anastazja was 4 years old when she was
developing.
diagnosed with Scarlet Fever, to be honest I
Any break in the skin or boil in conjunction
thought it was an old fashioned disease that had
with the symptoms and pain score should be
been eradicated. Two weeks later my other
taken seriously and treated with IV antibiotics
daughter Zara who was then 3 years old became
immediately without waiting for blood test
unwell with what seemed to be a cold, her nose
results.
was running but snotty, her cheeks were red, her
eyes were watery; to me they were classic look like a necklace. As the day wore on Zara
symptoms of a cold. Forty-eight hours later Zara screamed whenever anybody entered her room
came out with chickenpox. Over the next 24 h, because she knew that they were going to touch
more pox came out but Zara suddenly become her. Her screams were ear piercing because she
really unwell which I thought was very unusual as was in so much pain.
children are usually ill before the “pox” appears. The redness started spreading to what can only
Zara started vomiting and developed a tempera- be described as a “capped sleeve cardigan”. As
ture of 38 C which could be controlled by para- the hours went by, redness has now appeared near
cetamol. The next day her temperature was the “pox” on her abdomen, it now looked like she
increasing and had now reached 39 C but was was wearing a red “belt”. As time passed,
no longer responding to paracetamol. The “capped sleeve cardigan” had now turned into a
chickenpox was still coming through but they red “bolero jacket” and the red “belt” was now a
were now coming through as green/black in col- red “pair of hot pants/boxer shorts”. During the
our and were appearing more like “scabs” as night it now looked as if she was wearing a long
opposed to blistered “pox” texture. Zara had sleeved red cardigan. The weirdest thing was that
now become very ill, she was not eating or drink- whilst the redness was overtaking her body, she
ing and was starting to cry whenever she was had a “band” of normal skin around her midriff.
touched. Her “boxer shorts” were now a pair of “jammers”,
There were two significant “pox” that started the redness had now spread to her knees. Within
to look very nasty, one was on Zaras’ abdomen, 30 min the redness was now a red pair of tights.
the other on her sternum. The “pox” were now Zara was now “vacant”, just starting at nothing.
appearing as big green and black scabs. The pox You could have thrown her around the room and
on her abdomen had now developed a red ring she would not have cared, her eyes were glazed,
around it, this is what lead me to take her to our she was not even responding to me her Mummy.
local A&E department in Salford Manchester In the early hours the hospital room was filled
UK. They put us in an isolation room in the with the Heads of Departments, Medical
children’s department. Zara was now starting to Consultants, Immunology consultants, Plastic
develop a red rash around the “pox” on her Surgery Consultants, Anaesthetists and other
sternum. Doctors and Nurses. A decision was made that
We found out that 2 weeks prior to our visit a Zara now needed emergency surgery. Over 4 h
3 year old with similar symptoms had been sent later myself and my partner were finally told that
home and had sadly died of sepsis, I think that she was out of surgery, we made our way up to
that tragic event helped my daughter because that Intensive Care were we were ushered into a side
meant the staff was extremely vigilante with her. room where a Surgeon introduced himself and
The doctors were really concerned as they did not began to explain the he was head of Plastics and
know why Zara was ill or what was causing the Burns Unit. He told us what had taken place in
it. Zara was now screaming if anyone touched surgery. He explained that our daughter had
her. Her body was now in so much pain it was contracted Necrotising Fasciitis, we had never
heart breaking to watch. The doctors then decided heard of it. He told us that it was a flesh eating
to transfer Zara to the local children’s hospital. bug, he explained that Necrotising Fasciitis had
We were transferred into an isolation bay as eaten her chest wall so they had to debride the
Zara was obviously still infectious and no one had whole of her chest wall and that the next 24–48 h
a clue what was happening to her. The ring were critical as the child mortality rate was 95%.
around the “pox” on her abdomen was now get- He also informed us that he had operated on the
ting larger. Another concern was that redness had “pox” that was on her abdomen as it had turned
now appeared at the “pox” on her sternum. The into a large abscess, so he had cut the “pox” out
redness that was around Zara’s neck appeared to and had packed the wound. We were then led into
Fig. 2.8 Zara had given

up, her eyes were empty,
she was no longer
screaming whenever she
was touched. This is the
moment they decided she
needed emergency surgery
the Intensive Care isolation room where we saw 1 month. We attended various clinics and Zara
our little girl for the first time, she was uncon- also wore a pressure vest for a period of time.
scious as she was intubated and looking like an Zara is now 12 years old and is in her first year
American Football player. She was heavily ban- of high school. Her scar spans from shoulder to
daged and sedated; it was such a shock. shoulder and is the shape of a “Y”. We see the
Five days later, the surgeon needed to see if the consultant regularly who has been amazed at her
Necrotising Fasciitis had spread so operated on healing process. Zara’s scar has not blanched but
Zara whilst being in her intensive Care room has stretched over time as she has grown. Her
where I observed him removing silver foil from Consultant has said that she can have corrective
her chest as he had left the wound open. He surgery to neaten it if she wishes when she is fully
observed that it had not spread any further so grown. He also thinks that she contracted it from
decided to close the wound. It looked so surreal. Anastazja having Scarlet Fever as both are caused
He said he had only seen a handful of cases but by the Strep A bacteria. He thinks that Zara must
had never witnessed Necrotising Fasciitis in the have had the bacteria on her skin when she devel-
chest wall nor seen in at its earliest detection. Zara oped chickenpox and that it entered her body
was transferred to the Burns Unit after a week in when the “pox” erupted. He also thinks that the
Intensive Care where she remained for 3 weeks. nastiest “pox” on her sternum was the entry point
Zara was eventually discharged home after (Figs. 2.8, 2.9, 2.10, and 2.11).
Fig. 2.9 This was my first image of my 3 year old after surgery. No words could have prepared me. Unconscious and
intubated. I could not comprehend or believe this was my little girl
Fig. 2.10 Seventy-two hours after Zara’s surgery they needed to perform more surgery to ensure that necrotising
fasciitis had not spread and to remove the silver mesh they had placed into her
Fig. 2.11 We finally had

our little girl back, smiling
and laughing. No one
would believe the trauma
Zara had been through and
survived. Our little miracle
Necrotizing Soft Tissue Infections: Case
Reports, from the Clinician’s 3
Perspectives
Torbjørn Nedrebø and Steinar Skrede
Contents
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2 Cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2.1 Case 1—Polymicrobial Infection/Anaerobic Infection Originating in the Pelvic
Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.2.2 Case 2—GAS of the Upper Extremity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2.3 Case 3—GAS in an Immunocompromised Patient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.3 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Abstract chapter, we present three cases from the

INFECT-study population. This study was an
Necrotizing soft tissue infections (NSTI) are
international, multicenter, prospective cohort
rapidly spreading and life-threatening
study of adult patients with NSTI. We describe
infections of skin and soft tissue. Essentially
the clinical presentations, pre-, peri-, and post-
there are two types of NSTI, based on the
operative clinical findings, microbiology, and
invasive microorganisms. The speed of devel-
treatment in cases of monobacillary Streptococ-
opment and associated clinical features differ
cus pyogenes necrotizing soft tissue infections
markedly depending on the bacterial etiology.
NSTI, polymicrobial infection, and an unusual
Early recognition, extensive surgical debride-
presentation of pelvic monobacillary S. pyogenes
ment, and appropriate antimicrobials are piv-
infection in an immunocompromised patient.
otal for successful management. In this
Keywords
T. Nedrebø (*) NSTI · Streptococcus pyogenes ·
Department of Anaesthesia, Haraldsplass Deaconess
Hospital, Bergen, Norway Polymicrobial infections ·
e-mail: torbjorn.nedrebo@ihelse.net Immunosuppression · Toxic shock syndrome
S. Skrede
Department of Clinical Science, University of Bergen, Highlights
Bergen, Norway Necrotizing soft tissue infections located to the
Department of Medicine, Haukeland University Hospital, extremities are most commonly associated with
Bergen, Norway

22 T. Nedrebø and S. Skrede
monomicrobial infections caused predominantly Sartelli et al. 2018; Morgan 2010). Type 3 NSTI
by Streptococcus pyogenes (group A streptococ- clinically presents more like type 2 NSTI
cus; GAS). GAS infection of non-extremity loca- (Hakkarainen et al. 2014), and the use of unique
tion is associated with immunosuppression. clinical types 3 and 4 is yet to be justified. How-
Extensive surgical debridement is essential for ever, differentiating type 1 and 2 would serve
survival. Anaerobic necrotizing soft tissue practical purposes, in which tailored diagnostics
infections may benefit from hyperbaric oxygen and therapy may be warranted.
therapy. Type 1 polymicrobial infections most often
affect older patients with comorbidities like dia-
betes, and the predominant anatomical
3.1 Introduction localizations are perineum and the abdomen.
Type 2 monomicrobial infections are predomi-
Necrotizing soft tissue infections (NSTIs) are life- nantly caused by S. pyogenes (group A strepto-
threatening infections typically characterized by coccus; GAS) (Bruun et al. 2020).
rapid progression of disease and significant local The patients presented in the following three
tissue destruction. However, the speed of devel- case reports were all included in the INFECT
opment and associated clinical features differ study, an international, multicenter, prospective
markedly depending on the bacterial etiology cohort study of adult patients (Madsen et al.
(Hakkarainen et al. 2014; Calandra et al. 2005; 2019). Four hundred and nine patients
Anaya and Dellinger 2007). The tissue destruc- undergoing at least one operation with confirmed
tion involves any of the layers of the soft tissue NSTI were included, of which 402 were admitted
compartment, extending from the epidermis to the to an Intensive Care Unit (ICU). The most appar-
deep muscle fascia. The disease frequently ent initial symptoms and signs were skin bruising
progresses below the outer surface, and in the (202 patients, 51%) and opioid-requiring pain
absence of overt skin changes the diagnosis can (172 patients, 41%) and systemic toxicity was
be difficult to establish in the early phases. How- frequent; 50% had septic shock and 20% acute
ever, signs of systemic toxicity and pain often kidney injury (Madsen et al. 2019).
develop that are disproportionate to the findings Microbiological findings in these patients
of the skin examination (Calandra et al. 2005). revealed polymicrobial infection in 50% (202)
The diagnosis is surgical with the findings of and monomicrobial infection in 44%, of which
friability of the superficial fascia, dishwater-gray GAS was found in 126 cases (70%) among these
exudate, and absence of pus (Stevens and Bryant patients. In 26 of the patients (6%), no microbes
2017). Several descriptions and classifications of were identified.
NSTI have been proposed, including stratification The median number of surgical interventions
based on (1) anatomical localization (i.e., was 4, and 13% had amputation of either an
Fournier’s gangrene), (2) depth of infection (nec- extremity or penis. Most patients received beta-
rotizing fasciitis, necrotizing myositis), (3) sever- lactam (96%) and clindamycin (98%) as antibi-
ity (Sequential Organ Failure Assessment Score otic treatment.
(SOFA), simplified acute physiology score II
(SAPS II), or (4) microbiological findings.
Classifications due to microbiological findings 3.2 Cases
and their clinical characteristics are delineated in
Chap. 5. Shortly summarized, type 1 infections 3.2.1 Case 1—Polymicrobial
are polymicrobial, type 2 are monomicrobial, Infection/Anaerobic Infection
type 3 is a monomicrobial infection with marine Originating in the Pelvic Region
pathogens including Vibrio vulnificus and
Aeromonas hydrophila, whereas in type 4 the This patient was a 48-year-old man with a
etiology is fungal (Stevens and Bryant 2017; 30 years lasting history of substance abuse,
mostly heroin administered intravenously by
3 Necrotizing Soft Tissue Infections: Case Reports, from the Clinician’s Perspectives 23
way of the femoral veins. He had HCV RNA PCR possibly NSTI, he was taken to surgery. First, the
positive chronic hepatitis C genotype 3a and had plastic surgeons made incisions on the medial and
previously been hospitalized several times due to lateral part of the thigh from patella in proximal
opioid overdosing. Moreover, he had prior hospi- direction. The incisions were extended until they
tal admissions for a traumatic fracture of the left reached the level of the deep fascia, but only
femur and for surgical treatment of the right edema was present. The orthopedic surgeons
ankle. Known medication at presentation was first did an arthroscopy of the knee, draining
alprazolam and oxazepam. purulent fluid (Fig. 3.3), samples of which were
Six days prior to admission and during a collected for microbiologic diagnostics. Subse-
period of drug-abuse, he fell on his left knee quently, they made a midline incision over the
resulting in a blunt minor trauma. In the following prepatellar bursa. Gas had been seen along femur
days he developed local swelling, pain, and red- on the X-ray, and when they opened the deep
ness of the knee. At admission he was dehydrated fascia lots of necrotic tissue was detected beneath.
and opioid intoxicated, with a blood pressure The incision was prolonged to previous incisions
(BP) of 91/57 mmHg, a pulse rate of 96/min, made by the plastic surgeons, revealing direct
and a body temperature of 36,3 C. Clinical bio- communication between the prepatellar bursa
chemistry and hematologic tests revealed a and the intermediary vastus muscle. Thereafter
C-reactive protein (CRP) of 339 mg/L, white the vastus lateralis muscle was split to give access
blood cell count (WBC) of 26,2 109/L, creati- to deeper structures. Pockets of pus were found
nine of 233 μM, and a Laboratory Risk Indicator all along the fascia up to the anterior inferior iliac
for Necrotizing Fasciitis (LRINEC) score of spine, and parts of the quadriceps-muscle had to
8, according to Wong et al. (2004). The medial be removed due to necrosis (Fig. 3.4). The wound
side of the left knee was warm, had a swelling and was covered with surgical swabs and left open
erythema (Fig. 3.1), and joint flexion of the knee pending a revision the following day, in accor-
was impaired due to pain. Septic arthritis was dance with contemporary recommendations
suspected, and antibiotic therapy was initiated (Rogers 2020). On the first revision, necrotic
with a combination of intravenous ceftriaxone parts of the vastus intermedius muscle had to be
and clindamycin. removed, but there was no more evident pus.
Ultrasound showed fluid in the knee joint and Microbiologic cultures from the wound taken
between the superficial muscle layers in the lower during surgery demonstrated Prevotella disiens,
parts of the thigh. X-ray revealed gas along the Fusobacterium gonidiaformans, Actinomyces sp.,
entire femoral shaft all the way from patella to and Streptococcus dysgalactiae (SD), a beta-
trochanter major, and the patient was suspected to hemolytic streptococcus often found in
have infection with gas-producing microbes co-culture with intestinal flora. In blood culture,
(Fig. 3.2). Under suspicion of septic arthritis and only SD was found. Samples from the fluid in the
Fig. 3.1 Swelling and erythema of the knee at admission

Fig. 3.2 Gas seen in the soft tissue along femur
knee joint were culture negative. At first, he was found to be sensitive to penicillin and/or
treated with ceftriaxone and clindamycin, but clindamycin.
later this was changed to a combination of There were serial wound revisions, before the
benzylpenicillin and clindamycin following the wounds were surgically closed, starting at day
susceptibility tests, where the microbes were 4 and finished by day 6. Two days after closing
Fig. 3.3 Purulent fluid during knee-arthroscopy

Fig. 3.4 Necrotic muscle

(parts of quadriceps)
of the wound he developed more pain, and a rise knee joint during arthroscopy supported a clini-
in CRP and WBC occurred. The sutures were cal suspicion of septic arthritis. NSTI was
removed, and lots of pus were found and the actively sought for, but at initial surgery there
wound was washed with saline. From day 0 he were normal findings down to the fascia both
received 4 daily treatments with hyperbaric oxy- laterally and medially on the extremity. How-
gen therapy (HBOT), 90 min of 2.4 ATA (14/90), ever, the X-ray had shown gas along the femur,
as he was suspected to have a myositis due to so the idea of a deeply situated primary NSTI
anaerobic bacteria, which was confirmed during caused by IVDU inoculation, from the femoral
the initial phase of his hospital stay. He had vein downwards along the femoral shaft to the
several more revisions before the wound was knee structures, was not abandoned. Notwith-
closed, partly with a skin autograft. standing, the local trauma of his knee may
have created an environment for growth and
Discussion Polymicrobial NSTI is referred to as subsequent spread of the bacteria. Mechanistic
Type 1 NSTI, and often associated with gas in studies addressing the pathogenic strategies and
the tissue (Stevens and Bryant 2017), although complex dynamics of bacterial communities in
gas is also a possible clinical presentation in polymicrobial NSTIs are lacking (Thanert et al.
beta-hemolytic streptococcal (BHS) cases. In 2019). However, many of the bacteria found in
the INFECT study, 5 of 114 mono-GAS NSTI polymicrobial NSTI are the same as in
had palpable gas (crepitus) in the tissue and monomicrobial NSTI (e.g., GAS), and thereby
10 of 114 had gas visualized on radiology pre- elaborate many of the same exotoxins and
operatively (Trond Bruun, personal communica- tissue-degrading enzymes (Shiroff et al. 2014;
tion). In type 1, strictly anaerobic microbes Snall et al. 2016). SD is an emerging cause of
predominate (Madsen et al. 2019; Thanert et al. invasive disease in general (Oppegaard et al.
2019), often accompanied by facultative anaero- 2016) and specifically an increasing etiology of
bic gram-negative bacilli and gram-positive NSTI in our patient population, and it may cause
cocci. A typical patient with NSTI type 1 is monobacterial type 2 NSTI (Bruun et al. 2013)
likely to have comorbidities like diabetes or equally frequent, it is identified as part of
mellitus, peripheral vascular disease, or intrave- polymicrobial etiology of NSTI (Bruun et al.
nous drug use (IVDU) (Sarani et al. 2009; Chen 2020). In the INFECT study, blood cultures
et al. 2001), thereby in a state of qualitative or were more commonly positive in NSTI caused
quantitative immunosuppression. This particular by GAS (56%) than in SD (30%). IVDU is a
patient had a minor trauma to his knee 1 week risk factor also for SD bacteremia, as shown
prior to admission. Aspiration of pus from the elsewhere (Ruppen et al. 2017).
In our hospital we have noted an emerging Undertreatment with antibiotics in early

association of severe SD infections and genotype phases of obscure NSTI is common (Marwick
stG62647, which has predominated in invasive et al. 2012). Our patient received empirical ther-
SD infection in our population since 2013 apy with broad (1) gram-negative and (2) gram-
(Oppegaard et al. 2017). This sequence type was positive coverage that included effect on
also dominating in SD NSTI in the Scandinavian (3) Staphylococcus aureus that in our community
patient cohort of the INFECT study. is methicillin susceptible in >99% of invasive
In this patient, SD was identified along with a cases, (4) anaerobic species other than
number of obligate anaerobic species that are Bacteroides (that frequently expresses
rarely culture positive in blood culture compared clindamycin resistance), and on (5) toxin produc-
to rates of identification in deep tissue cultures ing species that might be involved in a NSTI that
(Brook and Frazier 1995), essentially confirmed most likely was of polymicrobial etiology
by the INFECT consortium (Madsen et al. 2019) (Peetermans et al. 2020). When results on suscep-
and even more so, in a study using nucleic acid tibility were available, penicillin sensitivity in SD
based detection of microbes in tissue (Thanert was confirmed, whereas all anaerobic species
et al. 2019). These results are in support of the were either susceptible to both clindamycin and
historic opinion that the importance of obligate benzylpenicillin or one of these agents. In line
anaerobic species may be underestimated in NSTI with treatment guidelines (Stevens et al. 2014),
(Brook and Frazier 1995). therapy was narrowed accordingly. Temporary
In this patient case, time from presentation at worsening seen in our patient was evaluated to
hospital to surgery was 18 h, mostly explained by be caused by too early closure of wounds.
the patient’s non-compliance and refusal to In NSTI the role of some of the therapeutic
receive help, delaying the time to surgery. This measures is controversial. Firstly, in our hospital
puts the patient at risk of poor outcome. Since the HBOT is offered sparingly to patients with severe
upsurge of NSTI in the mid 1980s, it was soon NSTI, organ failure, and evidence of obligate
evident that there was an association of time to anaerobic microbial etiology. According to local
surgery and risk for poor outcome (Boyer et al. guidelines for treatment of NSTI in our hospital,
2009; Elliott et al. 1996; Kobayashi et al. 2011; this patient did receive HBOT, as 1/3 of our
McHenry et al. 1995; Wong et al. 2003). Two patients in the INFECT cohort did (unpublished
recent meta-analyses reported a significantly result). Furthermore, this patient was not offered
lower mortality if the initial surgery was intravenous polyspecific human immunoglobulin
performed within 12 h of presentation (Gelbard G (IVIG), which has been restrictedly used in our
et al. 2018; Nawijn et al. 2020). Gas in the tissue hospital. Until the INFECT study, orders for use
is one of the clinical signs more often associated were evidence of invasive GAS infection, shock,
with polymicrobial NSTI (Stevens and Bryant and unstable patient as described by Anaya and
2017). In this patient gas had been seen on the Dellinger (Anaya and Dellinger 2007). Only two
initial X-ray and although no evident sign of patients in our local INFECT cohort fulfilled
deeply situated NSTI was detected in the early these criteria (unpublished data). However, there
phases of surgery, this radiologic finding spurred is increasing evidence that patients with
the surgeons to continue, leading them to explore monobacillary GAS NSTI or septic shock benefit
muscle and other soft tissue all the way into the on IVIG treatment with improved outcome (Parks
femoral shaft, a strategy leading to drainage of et al. 2018), recently supported also by data for
deeply situated necrotic tissue and pus, most the streptococcal cohort of the INFECT study
likely improving the outcomes for the patient (Bergsten et al. 2020; Bruun et al. 2020). In this
substantially. That a septic arthritis caused by case, SD and obligate anaerobic microbes were
SD appears secondary to a NSTI of an extremity identified. There is to date only one single
is an observation made on several previous completed randomized, blinded, placebo-
occasions in our hospital. controlled trial of immunoglobulin G treatment
for patients with NSTI (Madsen et al. 2017). This 3.2.2 Case 2—GAS of the Upper
study failed to show any effect on primary and Extremity
secondary outcomes. Most patients included had
polymicrobial infection. In an analysis of associ- This patient was a 48-year-old male with a history
ation between outcome and anatomic site of of psoriatic disease, but he was independent of
infection, there was a tendency in favor of medication. One week prior to hospitalization the
placebo in infections of the anogenital and patient got a minor laceration, as he cut a finger of
abdominal areas (Madsen et al. 2017), in which the left hand on a paper edge. He treated the finger
obligate anaerobes are frequently involved, with a topical antibiotic due to a possible infec-
different to BHS, that are rarely encountered tion, and the finger was fine for the next few days.
(Madsen et al. 2019). This would support that However, while cross-country skiing his left
IVIG may have little effect in the polymicrobial, elbow rapidly became erythematous and painful
non-BHS infections. A case report on beneficial within a few hours, and his temperature rose to
use of IVIG in NSTI caused by SD has been 39.1 C. He was sent to the local community
presented, the effect attributed to blocking of hospital on suspicion of severe skin and soft
streptokinase-mediated fibrinolysis, with tissue infection. On admission his left elbow
subsequent reduced bacterial spread (Andreoni was painful, and there was an area of localized,
et al. 2018). well defined erythema, and swelling (Fig. 3.5).
The first case presented herein is a type There were no signs of injury or breach of skin
1 NSTI, a patient with a polymicrobial infection barriers. Vital signs included a BP of
caused by co-occurrence of obligate anaerobic 134/82 mmHg, pulse 91, temperature 39 C,
microbes and SD, a beta-hemolytic streptococcus. respiratory rate (RR) 12. CRP was <5 mg/L,
The disease course developed slowly, but there WBC 9.7 109/L. LRINEC score was 0 and
was a need for very resource demanding handling blood cultures were collected. Initial diagnosis
including HBOT. This case illustrates host risk was bursitis with possible cellulitis. Five hours
factors (IVDU), as well as major clinical learning later the patient felt worse as the erythema was
point; the anticipated primary focus of infection spreading. Vital signs were recorded with BP
was an intra-articular knee joint infection as it was 112/57, a clinical chemistry showed CRP
here infection first became visible. Different to 72 mg/L, WBC 14.4 109/L, and blood lactate
this, the primary disease was judged to be was elevated to 3.6 mM. Treatment with intrave-
advanced NSTI originating in the groin, nous cloxacillin was initiated, but his condition
descending along the femoral shaft. deteriorated. NSTI was suspected and he was
transferred to our tertiary care hospital.
Fig. 3.5 Area of marked swelling and erythema at first admission

Fig. 3.6 Spread of

erythema and swelling
down to MCP joints.
Picture taken 12 h after first
admission
At arrival 12 h after first hospitalization, BP margins of the infection with redness and edema
had decreased to 86/60, and treatment with vaso- had spread up to the shoulder. Initial bacteriologi-
pressor to maintain MAP >65 mm Hg had been cal tissue and fluid cultures grew GAS, whereas
initiated. The erythema and swelling had spread blood cultures from the first admission were cul-
in distal direction from the MCP joints to the ture negative. A third revision uncovered some
middle upper arm (Fig. 3.6). The swelling over purulent fluid just below the shoulder, but the
the olecranon bursa was punctured, and fluid was fascia and muscle tissue were vital with no signs
aspirated and sent to microscopy and culture and of progression (Fig. 3.8). His condition improved,
gram-positive microbes with streptococcal mor- and 2 days later he was extubated and in no need
phology were detected. Cloxacillin was of circulatory support.
discontinued, and treatment was changed to The patient went through several further
clindamycin, benzylpenicillin, and gentamycin. revisions, but no more necrotic tissue or signs of
He went to immediate surgery under general infection were found. On day 4 they started to
anesthesia. In the surgical theatre a large dorsal close the wound, but he had a skin defect which
incision from olecranon to the lower forearm was had to be transplanted at a later stage. On day 5 he
made. Extensive amounts of dishwater-like fluid was discharged from ICU, with a SOFA score 5.
was found together with necrotic subcutaneous
tissue. An area of 7 20 cm of the skin over Discussion
the elbow, equaling approximately 1.5% of skin Case 2 is a representative patient of type 2 NSTI:
surface, had to be removed due to necrosis GAS infection in a previously healthy young
(Fig. 3.7). The muscle fascia and the muscle individual, affecting an extremity (Madsen et al.
tissue looked vital with no necrosis. The wound 2019). GAS is a gram-positive coccus capable of
was left open, covered with surgical swabs. Sub- causing a broad array of infections, most com-
sequently, he was postoperatively transferred to monly pharyngitis and non-necrotizing soft tissue
the intensive care unit (ICU), where SAPS III infections (Stevens 2020). Invasive GAS
score 52, and SOFA score 8 were noted at infections include NSTI, which has a significant
admission. potential for local spread and aggressive clinical
The next morning a second look was course, as experienced by this young individual.
performed. The muscle fascia on the lower fore- This patient started out with swelling and
arm was cut open, and the muscle tissue below edema of his elbow, which only 12 h later spread
appeared to be vital. Then the surgeons explored distally to his MCP joint and proximally up to his
proximally towards the elbow. Still the fascia shoulder. One week prior he had a small cut in his
looked and felt vital, but extensive amounts of finger. Most likely the cut in his finger caused an
pus-like fluid emanated from deeper muscle- entry of the bacteria, that may have been dormant
compartments. Immediately after surgery the or in a very isolated, minute focus of infection not
patient’s condition got worse with septic shock giving rise to any attention. This was followed by
and respiratory failure. On examination the a NSTI development, first in the elbow due to a
Fig. 3.7 First surgery over the elbow and forearm. 7 * 20 cm of the skin removed due to necrosis
possible minor tissue trauma caused by the cross- In retrospective studies of NSTIs, upper
country skiing. NSTI has historically been extremity infections are perceived as uncommon
associated with penetrating trauma, but recently and lethal, as reviewed elsewhere (Uehara et al.
blunt trauma has also been verified as an indepen- 2014). Differently, we demonstrate that upper
dent risk factor of GAS NSTI development extremity is as common focus as head-neck
(Bruun et al. 2020). NSTI, and even half that of anogenital-abdominal
Fig. 3.8 Surgery after second revision had to go below the shoulder
or lower extremity focus of NSTI, and they are Carapetis et al. 2014; Linner et al. 2014; Mascini
not more lethal (Madsen et al. 2019). In extremity et al. 2001). Upon confirmation of streptococcal
NSTI GAS predominates vastly (Bruun et al. etiology our patient received a combination of
2020; Madsen et al. 2019). In this case blood penicillin and clindamycin, as advocated in con-
cultures from the first admission were culture temporary guidelines for the treatment of GAS
negative, as seen in 44% of GAS cases (Bruun NSTIs (Stevens and Bryant 2017). However, he
et al. 2020), underlining the importance of initially received inadequate antibiotic therapy, as
collecting bacteriological tissue and fluid for rou- evaluated retrospectively. In a prospectively
tine culture. In the entire INFECT cohort 93% of enrolled cohort of skin and soft tissue infection
the cases were tissue culture positive (Madsen cases, Marwick et al. reported inadequate empiric
et al. 2019). therapy in 10/11 (91%) cases with NSTI,
Our patient went to surgery 24 h after presen- highlighting the diagnostic challenges of
tation of symptoms, 18 h after admission to hos- distinguishing NSTIs from cellulitis in the early
pital, and by then he was clinically affected with phase (Marwick et al. 2012). The LRINEC score
hypotension in need of vasopressor. This is some- has been proposed as a clinical tool for
what later than the average GAS patient in our identifying patients in need of surgical treatment.
cohort. We showed that GAS cases receive sur- However, at the primary admission our patient
gery after a median of 16 h compared to non-GAS had a LRINEC score of 0/13, demonstrating the
cases, whom received it 2 h later (Bruun et al. shortcomings of this scoring system, reviewed
2020). Invasive GAS infection can cause toxic elsewhere (Fernando et al. 2019). In our hospital
shock syndrome (TSS), in our study reported in its use has been discouraged following our studies
65% (Bruun et al. 2020). Clinical criteria for TSS (Bruun et al. 2013; Bruun et al. 2020; Madsen
include hypotension (systolic blood pressure et al. 2019). The diagnosis of NSTI is established
>90 mmHg) and multi-organ involvement by surgery, but frequently diagnosis is delayed
(Breiman et al. 1993). This patient met the clini- (Goh et al. 2014). In general, it may therefore be
cal criteria of TSS, and the diagnosis was con- advisable to encourage surgical exploration ear-
firmed with isolation of GAS from tissue biopsies lier when facing suspected NSTI patients, which
and bursa fluid. This massive invasion and degra- means a certain ratio of negative explorative
dation of the soft tissue caused by GAS are due to procedures must be accepted. It is our experience
both the bacteria’s production of exotoxins and that we explore suspected NSTI in the range of
the features of M-proteins, the latter being cell 1,5:1 vs. confirmed non-NSTI (unpublished
surface molecules on the bacteria. They both results), resembling published results of 1,2:1
result in a massive pro-inflammatory cytokine that has been presented by others (Howell et al.
release and thereby causing septic shock. A 2019).
study has also shown that patients with GAS Extensive surgery is main therapy in NSTI. In
NSTI exhibited a lack of specific antibodies this case the fascia looked vital, but below the
directed against the causative S. pyogenes strains fascia extensive amounts of necrotic tissue were
and the majority of their exotoxins during initial detected and removed. Intraoperative findings of
stages of infection (Babbar et al. 2018; Norrby- muscle tissue involvement showed that GAS
Teglund et al. 1994; Basma et al. 1999), spread across tissue borders, and not only along
antibodies meant to prevent the spread of the tissue planes. Surgically it is important to
infection. consider exploring below the fascia, even in
Several in vitro and in vivo studies have cases where they appear to be vital. The extent
indicated that the protein synthesis inhibitor of the excisions should be down to healthy bleed-
clindamycin abates the toxin production in ing tissue at all margins. Often the spread in the
GAS, and improves survival rates in NSTIs and subcutaneous layer is more extensive than the
streptococcal TSS (Andreoni et al. 2017; skin changes suggest. This was the case here
too, as the patient only few hours after second 115/51 mmHg, pulse rate 107, RR 20, WBC
surgery developed TSS and had to undergo a 4.6 109/L, and temperature 38,2 C. CRP was
second excision, in proximal direction to reach 275 mg/L, while a LRINEC score was calculated
just below the shoulder, confirming challenges in retrospectively showing a low score of 4/13.
infectious source control of this case. This case Upon examination, she had no erythema or
demonstrates the severe systemic manifestations signs of infection on her buttocks or thigh. A
including streptococcal TSS in a previously gynecologic examination did not reveal any sign
healthy man, the significance of blunt versus of infection, but a vaginal ultrasound gave suspi-
penetrating trauma, initial antibiotic undertreatment, cion of retroperitoneal edema against the pelvic
and impact of too early surgical closure. floor.
A CT scan showed either solid fluid or a
phlegmon along the pelvic muscles from upper
3.2.3 Case 3—GAS part of the right thigh to 5 cm above the pubic
in an Immunocompromised tubercle. Edema was present in gluteal, pelvic,
Patient and adductor muscles.
A pigtail catheter was inserted prevesically, in
A 59-year-old woman was admitted to her local an attempt to drain some of the fluid. Culture from
hospital after 2 days of increasing pain in her right blood, and this fluid later grew GAS. Subse-
buttocks and groin. In addition, foul-smelling quently her condition deteriorated only a few
dark green vaginal discharge was noted. She pre- hours later, and soon she was in need of
viously had one cesarean section and more vasopressors (norepinephrine and dobutamine).
recently, surgery for urinary incontinence She was rapidly sent by helicopter to the regional
(tension-free vaginal tape (TVT)) a few years hospital where she arrived 15 h after first admit-
before enrollment in the INFECT study. Due to tance to hospital. At arrival she was awake, self-
an active psoriatic arthritis, she received regular breathing, BP 89/35 mmHg (on vasopressor),
treatment with methotrexate per oral 10 mg once pulse rate 127, and temperature 39,5 C. On
weekly and the tumor necrosis factor-alpha inhib- examination, a swelling and discolored area on
itor, etanercept subcutaneously 50 mg once the back of her thigh was found (Fig. 3.9) and a
weekly. On admission to the hospital BP was CRP value of 225 mg/L was noted.
Fig. 3.9 Swelling and discolored area of the thigh 15 h after first admission
Fig. 3.10 Fasciotomy of

the medial side of the thigh
at first surgery
She went immediately to surgery under gen- left side, and an external vulvectomy was
eral anesthesia. An explorative laparotomy did indicated. The wound was left open with wet
not reveal any intra-abdominal pathology, except surgical swabs.
from localized retroperitoneal edema in the ingui- A second look at the fasciotomy of the right
nal opening on the right side. Then a fasciotomy thigh revealed some necrotic skin and a small
both laterally and medially on the thigh was necrotic area of adductor magnus muscle. During
performed (Fig. 3.10). There was edema of the the surgical procedure the patient received IVIG
subcutaneous fat and dishwater-like fluid under according to the contemporary treatment proto-
the fascia (Fig. 3.11). Both the fascia and the col, as there was evidence of GAS etiology and
muscle were found viable. she was circulatory unstable and in a critical state.
The antibiotic regimen was changed to Upon return to the ICU after the second sur-
meropenem and clindamycin, and she was trans- gery, her condition was judged critical and unsta-
ferred to the ICU. Her condition worsened the ble, supported by maximal SOFA score of
next few hours, and a swelling close to the right 17, SAPS II score of 66, and SAPS III score of
labium majus appeared. She was once again taken 86. Arterial blood gas lactate exceeded 8 mM,
to immediate surgery. An incision was made two vasopressors (norepinephrine and epineph-
between the labium minus and labium majus on rine) were administered as a Cardiac Index of
the right side, to uncover devitalized subcutane- <4 L/min was detected. High-flow (300 mL/
ous tissue. There was no bleeding, due to wide- min) continuous veno-venous hemofiltration
spread thrombosis of minor vessels. A digital (CVVH) was started. However, following the
dissection down to the fascia of the inferior part radical surgery, improvement was noted, and
of urogenital diaphragm was made. Necrotic sub- after a few hours only epinephrine infusion
cutaneous tissue was removed, but the fasciae could be discontinued. She stabilized, and at a
were vital. The same procedure was done on the new surgical revision the same day only minor
Fig. 3.11 Edema and dishwater-like fluid under the fascia
necrotic muscle of the adductor muscle was pres- Discussion

ent (Fig. 3.12). The inner genitals were not NSTI in immunocompromised (IC) patients has
affected by the infection and were intact. She been found to have a two times higher mortality
had several revisions the following days and sub- than non-immunocompromised and can be clini-
sequently moved to the National Burn Center, cally challenging as their symptoms may not
located in our hospital, offering expertise in present in the same manner (Keung et al. 2013).
handling of large wounds and need for recon- In the same study, 59% of the wound cultures
struction surgery. from first surgical debridement were found to be
On day 8, reconstructive surgery of the area polymicrobial, with lower extremities involved in
around vulva started with graft from the gracilis 54%, perineum 40%, and trunk 37%. In a retro-
muscle. After 5 weeks she was transferred to the spective review of children with NSTI, 75% of
local hospital for rehabilitation. the IC had polymicrobial infections compared to
Fig. 3.12 Picture taken of medial fasciotomy after second revision

58% of the non-IC (Butterworth and Murphy with antibiotics and IVIG her condition improved
2006). The newly developed SIARI score (Site substantially within few hours and then stabilized.
other than the lower limb, Immunosuppression, This third case is a monomicrobial GAS infec-
Age <60 years, Renal impairment (s-creatinine tion localized to the perineum, which was seen in
141) and Inflammatory markers (CRP 150, only 6/126 (5%) of GAS cases (Bruun et al. 2020;
WBC 25)) take into account Madsen et al. 2019). Through the INFECT study
immunosuppressed patients, and is a better tool we have learned that this clinical setting, with
in diagnosing NSTI in these patients (Cribb et al. monomicrobial NSTI at a very uncommon infec-
2019). Validation of this score is needed, how- tion focus site, is attributable to immunosuppres-
ever. In the INFECT-study population, 126 of sion. Moreover, polyvalent human
406 NSTI cases (31%) had GAS NSTI, only immunoglobulin G (IVIG) has previously been
12 of these having co-pathogens identified. restrictedly used in our hospital, Haukeland Uni-
NSTI involving the extremities were also pre- versity Hospital in western Norway. The INFECT
dominant, found in over 80% of streptococcal study has generated support to IVIG treatment in
NSTI (Bruun et al. 2020). In a retrospective GAS cases, as in multivariate analysis any dose of
cohort study, patients with immunodeficiency or IVIG was associated with survival, as was the
an abdominal infection, had low probability of outcome in this case (Bruun et al. 2020).
GAS infection (Urbina et al. 2019). This case
presents an immunocompromised patient with
monomicrobial GAS infection of the perineum
3.3 Future Perspectives
and secondary to this, to the lower extremity.
Time from onset of symptoms to presentation at
NSTIs are rare infections and most often case
hospital was 2 days. Time from presentation at
reports and small retrospective cohorts are
hospital to first surgery was 18 h as she had to be
presented. An insufficient progress has been
transported to our referral hospital. A delay of
noted in patient handling, as few steps have
first surgery beyond 12 h of presentation has
been taken to narrow the gap between basic
been shown to increase mortality (Nawijn et al.
pathophysiological knowledge and clinical rou-
2020; Gelbard et al. 2018). At first surgery, exci-
tine handling. The INFECT project is to our
sion of perineum was not performed, and
knowledge one of the largest prospective studies
fasciotomy of upper lower extremity revealed
of patients with NSTIs, offering with more rigor
some dishwater-like fluid and edema. The first
than ever before, associations between clinical
CT scan taken at admission in the local hospital
presentation, microbial etiologies and anatomical
showed a possible phlegmone of the pelvic and
location of NSTIs, with data suited to aid clinical
gluteal muscle all the way up to the pubic tuber-
decision-making. Examples of use of data
cle, so a more extensive excision at first surgery
generated in the INFECT study have been given
probably had revealed affection of the perineum
herein. Through prospective inclusion, updated
at an earlier stage. The goal of the initial surgery
clinical categorization, and studies on host–
is to gain control and prevent further spread of the
microbe interactions with translational studies of
infection (Sartelli et al. 2018; Stevens and Bryant
pathogenesis, the INFECT study has been paving
2017). Shortly after the initial surgery, she devel-
the road towards development of personalized
oped TSS and the NSTI presented on the labium
medicine in NSTIs.
majus as a swelling. The inflammatory response
had caused massive thrombosis and thereby
Acknowledgement The INFECT study was supported
necrosis of the soft tissue. At second surgery, a by the European Union’s Framework Program 7 (grant
more extensive debridement with bilateral number FP7/2007 – 2013 305340. The authors thank
vulvectomy was performed. Her clinical condi- Oddvar Oppegaard, Eivind Rath, and Trond Bruun for
tion was critical, with septic shock, but the exten- their contribution and review of the manuscript.
sive surgery stopped the spreading, and together
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Necrotizing Soft-Tissue Infections:
Clinical Features and Diagnostic Aspects 4
Martin Bruun Madsen, Per Arnell, and Ole Hyldegaard
Contents
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.2 Clinical Presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.2.1 Pre-Existing Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.2.2 Site Specific Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.2.3 Laboratory Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.2.4 Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4.2.5 Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4.3 Diagnostic Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.4 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Abstract and deliquescent tissue can be seen. Patients

can present with vague symptoms, and
The term necrotizing soft-tissue infection
approximately half of patients experience
(NSTI) encompasses a heterogenous group of
severe pain. The clinical presentation and
patients with necrotizing infections, involving
microbiological etiology vary according to
any body part. NSTI is diagnosed by surgical
affected body site, with NSTI located to the
exploration, where necrosis of the subcutane-
extremities being dominated by
ous tissue and/or muscle tissue, undermining
monomicrobial group A streptococcal
of the skin, thrombosis of the superficial veins,
infections, and NSTI located to the anogenital
area dominated by polymicrobial infections.
M. B. Madsen (*)
Department of Intensive Care, Copenhagen University No set of diagnostic criteria exists, and suspi-
Hospital, Rigshospitalet, Copenhagen, Denmark cion of the diagnosis should come from careful
e-mail: martin.bruun.madsen.01@regionh.dk clinical examination and signs of local or sys-
P. Arnell temic severity. Laboratory blood values show
Department of Anaesthesia and Intensive Care, no distinct pattern but resemble those of sep-
Sahlgrenska University Hospital, Gothenburg, Sweden sis. Imaging can aid the diagnostic process but
O. Hyldegaard must not delay surgical intervention.
Department of Anaesthesia, Centre of Head and
Orthopaedics, Copenhagen University Hospital,
Rigshospitalet, Copenhagen, Denmark

40 M. B. Madsen et al.
Keywords penicillin, and support for abnormal physiology

(Wilson 1952).
Necrotizing soft-tissue infections · Necrotizing In this chapter we will review the clinical
fasciitis · Fournier’s gangrene · Clinical presentation of NSTI and different aspects regard-
presentation · Diagnostic aspects · ing diagnosis.
Microbiology
Highlights 4.2 Clinical Presentation

• NSTI is a heterogenous infectious disease that
can involve any part of the body, and can NSTIs can affect any part of the body, and as a
present in various ways. consequence, the presentation of the disease
• No set of diagnostic criteria exists; the diagno- varies considerably. Many patients with NSTI
sis is based on clinical presentation and surgi- have experienced a breach of the skin, e.g. by
cal intervention. pre-existing ulcers, trauma, or surgery (McHenry
• Common symptoms include bruising of the et al. 1995; Wong et al. 2003; Madsen et al.
skin and severe pain. 2019). But, as documented in early descriptions,
• Bacterial etiology differs according to the NSTI can develop without previous penetrating
affected body part; infections of the upper trauma (Wilson 1952; Meleney 1924), and many
and lower extremities are dominated by patients present without any known preceding
monomicrobial group A streptococci, and event (Wong et al. 2003; Hua et al. 2015). There-
infections of the abdomen and anogenital fore, pain of the infected area can be the only
area by polymicrobial infections. initial symptom. The triad of swelling, erythema,
and pain may be present—the cardinal symptoms
of inflammation, thus not distinguishing NSTI
4.1 Introduction from other infections. Notably, in NSTI without
obvious portal of entry, cutaneous manifestations
Necrotizing soft-tissue infection (NSTI) is a fast are a late sign. Severe skin manifestations with
progressing infection that may affect any layer of purple or black discoloration are seen in 32% of
the soft tissue, and results in severe morbidity, patients, and skin bullae in 27% (Figs. 4.1 and
mortality, and affected quality of life 4.2) (Madsen et al. 2019).
(Hakkarainen et al. 2014). It is not a common Systemic signs can include fever, tachycardia,
disease, with incidence rates ranging from 1.8 hypotension, and altered mental state—all gen-
per 100,000 inhabitants per year in Denmark eral symptoms of infection, and the progression
(Hedetoft et al., unpublished) to 15.5 in of infection (sepsis) (Table 4.1). The initial
Thailand (Khamnuan et al. 2015a). The nomen- symptoms can be even more vague as “influ-
clature varies; the terms gas gangrene, phagedena enza-like” symptoms such as nausea, vomiting,
gangrenosa, and hospital gangrene were used in and diarrhea (Bisno et al. 2000). Pain requiring
the early 1800s (Loudon 1994), and in 1952, the opioids is seen in 42% of patients, and although
term necrotizing fasciitis was introduced by hard to quantitate, severe pain, or pain out of
Wilson (Wilson 1952)—the term still used in proportion with the immediate appearance should
the WHO international classification of diseases raise a high index of suspicion of NSTI, in
(WHO 2017). The term NSTI encompasses the patients with infection (Wong et al. 2003; Madsen
different variations of the infection (Stevens and et al. 2019; Borschitz et al. 2015). However, pain
Bryant 2017). A description of a condition similar may be absent due to use of analgesics or
to NSTI was described already by Hippocrates neuropathy.
(Jones 1923), but the first description is com- Most patients are tachycardic and have hypo-
monly attributed to the surgeon Ben Wilson, tension, and in a Scandinavian observational
who suggested early recognition, prompt surgery, study, half of the patients fulfilled the criteria for
4 Necrotizing Soft-Tissue Infections: Clinical Features and Diagnostic Aspects 41
Fig. 4.1 Presentation of

NSTI before surgery. (a)
The skin is only slightly
red. Note the bullae. (b)
After initial surgery. The
infection has undermined
the skin to a much larger
extent than seen in picture
(a). Courtesy of INFECT
septic shock (Madsen et al. 2019). Furthermore, to patient, it is important not to exclude the diag-
20% had acute kidney injury, and some nosis in absence of specific symptoms (Stevens
multiorgan impairment. This is not, in itself, and Bryant 2017).
prognostic of NSTI but is a marker of disease The time from suspicion of NSTI to surgery
severity. has previously been associated with an increased
As no symptom or sign is pathognomonic for risk of death in unadjusted analyses of retrospec-
NSTI, and as these vary considerably from patient tive data (McHenry et al. 1995; Wong et al. 2003;
Fig. 4.2 NSTI before

surgery, originating from
the neck, spreading to the
thoracic region. Courtesy of
INFECT
Stevens et al. 2014; Boyer et al. 2009; Lille et al. Despite methodological issues regarding these
1996; Bucca et al. 2013; Hadeed et al. 2016). analyses, it is reasonable to assume that time to
Table 4.1 Symptoms and findings related to organ systems

Organ system Comment
Central nervous system
Altered mental state
Pain
Circulatory system
Tachycardia (beats/min) 107 (93–127)
Hypotension
Elevated lactate (mmol/L) 2.1 (1.2–4.4)
Renal functioning
Elevated creatinine (μmol/L) 122 (83–200)
Elevate carbamide (mmol/L) 10.0 (5.9–15.0)
Skin
Erythema/bruising Seen in app. 50% of patients
Purple/black discoloration Seen in 32% of patients. A late sign when there is no portal of entry
Skin bullae Seen in 27% of patients
Crepitus Seen in 14% of patients
Coagulation
Low platelets (109/L) 171 (111–267)
Elevated INR 1.4 (1.2–1.6)
Systemic
Metabolic acidosis pH 7.30 (7.23–7.36)
Bicarbonate 20.0 (16.9–22.8)
Fever 15% of patients had temperature above 39 degrees Celsius
Elevated C-reactive protein (mg/L) 284 (179–373)
Elevated white blood cells (109/L) 16.3 (10.6–22.4)
Elevated procalcitonin (μg/L) 8.46 (1.66–33.40)
Values are median (IQR). Data from (Madsen et al. 2019)
For creatinine, white blood cells, C-reactive protein, and lactate, values are from before initial surgery. For the remaining
values, data are from the first 24 h in the ICU following surgery
surgery is an important modifiable factor that can infections (Carey et al. 2018), but whether diabe-
affect patient outcome. tes is an independent risk factor for developing
Suspicion of NSTI is often measured from a NSTI is not clear. Patients with diabetes also have
time when the patient is already in the hospital, as a higher risk of organ-failure and death from
this is when symptoms are described. However, a sepsis (Beck et al. 2016; Esper et al. 2009), but
recent publication has shown that patients experi- is not clear whether the same is applicable to
ence a broad variety of symptoms while at home, NSTI patients. In a prospective cohort study, dia-
even up to several days before they were admitted betes (type I and II) was included as one of
to hospital and eventually diagnosed with NSTI 11 predefined variables in an associative analysis
(Erichsen Andersson et al. 2018). These anteced- for risk of 90-day mortality. Of the 409 patients,
ent symptoms were categorized into three groups, 24% had diabetes at the time of diagnosis. The
depending on the duration of these; more than odds ratio for patients with diabetes was 1.23
7 days, 3–7 days, and 1–2 days. Some of the (95% CI: 0.55–2.71), thus showing no increased
symptoms were often not easily describable. Ini- risk of death compared to non-diabetic NSTI
tial symptoms included “flu-like symptoms” and patients. On the contrary, in a retrospective
by some depicted as: “My muscles were sore like study using a national database including 4178
after exercise.” Symptoms that progressed patients with NSTI, diabetes was associated with
included malaise, shivering, fever, nausea, and a lower mortality rate (Al-Qurayshi et al. 2020).
pain—including escalation of pain—and This is in contrast to a retrospective cohort study
described as “something was seriously wrong” including 9871 NSTI-related deaths in the USA,
and “losing control.” The last stage of symptoms where diabetes was more commonly observed
included anxiety, confusion, dizziness, and (Arif et al. 2016). As severe peripheral vascular
fainting. Patients and/or their family reported disease has been associated with increased mor-
signs as high fever, local swelling, red/blue skin, tality (Hua et al. 2015), this could be one of the
vomiting or diarrhea, boil/abscess, chills or fever, mechanisms of which presence of diabetes results
rapid heart rate, and local heat. These symptoms in increased mortality, but in the same cohort,
and signs are important to consider as the failure diabetes was not associated with increased mor-
to recognize these may delay further diagnostics tality. There seems to be a higher rate of
and correct diagnosis. amputations in diabetic NSTI patients (Horn
et al. 2020), but the etiology behind is not clear.
Other chronic diseases have been associated
4.2.1 Pre-Existing Conditions with poorer outcomes. From several retrospective
studies, hepatic disease seems to be a determinant
In many studies, a variety of pre-existing for increased risk of mortality, either as cirrhosis
conditions are described, including adipositas, (Khamnuan et al. 2015b; Huang et al. 2011) or
diabetes, cardiovascular disease, chronic kidney simply hepatic disease (Al-Qurayshi et al. 2020;
and liver disease, and other immunocom- Chuang et al. 2019). The studies by Huang,
promising diseases (Boyer et al. 2009; Sudarsky Khamnuan, and Chuang were conducted in
et al. 1987). It is reasonable to assume that the Thailand and Taiwan, and it is possible that both
presence of some comorbidities increases the risk difference in population (Sarin et al. 2020) and
of NSTI, but due to the low incidence and high microbiological etiology may interact. Similarly,
variety of comorbidities, any association will be associations between heart disease and increased
difficult to show. In a systematic review including risk of mortality have been found in retrospective
nine studies with 1493 patients, diabetes was studies (Boyer et al. 2009; Khamnuan et al.
present in 45% (range: 15%–71%) of patients, 2015b; Anaya et al. 2005).
and diabetes seems to be overrepresented in With the exception of the study by Horn and
patients with NSTI (Goh et al. 2014). Patients colleagues, the above-mentioned studies are of
with diabetes have a higher risk of developing retrospective nature, of varying sample size, and
often the methodology—including definitions of Peptostreptococcus sp., Bacteroides sp., and

diseases and mortality—is of modest quality. But Fusobacterium sp. are seen (Gunaratne et al.
even though these associations have not been 2018), reflecting the microbes normally seen in
reproduced in data from NSTI cohorts collected the oral cavity.
prospectively, it is reasonable to assume that the Affection of the extremities (Fig. 4.3) is
presence of a chronic disease may result in a dominated by monomicrobial GAS infections,
poorer outcome, and increased focus should be with odds ratios for GAS in upper extremity
given to these patients. NSTI being 7.80, 95%CI 4.36–14.25, and for
lower extremity 1.91, 95%CI 1.18–3.09 (Madsen
et al. 2019). Staphylococcus aureus and group C
4.2.2 Site Specific Manifestations and G streptococci, most commonly Streptococ-
cus dysgalactiae (SD), are also seen. Amputation
NSTIs can be located to any part of the body, with of an extremity occurs in 22% of these patients,
approximately 1/6 of the infections located to the and elevated lactate is associated with amputation
head and neck area, 1/6 to the upper extremities, of an extremity (Fig. 4.4).
1/3 to the abdomen and anogenital area, and 1/3 NSTI involving the abdomen or anogenital
to the lower extremities (Madsen et al. 2019). area—the latter also termed Fournier’s gan-
Depending on the localization of the infection, grene—is associated with polymicrobial infection
more pathognomonic signs can be seen. (Fig. 4.5) (Madsen et al. 2019; Yilmazlar et al.
Periocular affection is rare with approximately 2017). The incidence rate in males is 1.6 per
0.24 cases per million (Flavahan et al. 2014). 100.000 (Sorensen et al. 2009). NSTI in this
Signs as swelling, redness, and pus are common region often results from anorectal- or urogenital
(Würtz et al. 2020). Due to the localization, the skin infections (Singh et al. 2016). As the tissue is
symptoms are probably recognized fast. Beta- often more loosely bound, deep cavities can
hemolytic streptococci are the most frequently develop, and may spread to the abdomen, thigh,
cultured pathogen, with Streptococcus pyogenes or genitalia.
(group A streptococcus; GAS) being the most
common of these.
Cervical NSTIs may evolve after a common 4.2.3 Laboratory Tests
throat infection as pharyngitis or tonsillitis or a
dental infection (Skitarelic et al. 2003), and the In general, the standard laboratory blood tests
initial symptoms will relate to the underlying show the same pattern as in other infectious
cause, e.g. sore throat, dental pain, fever, and diseases (Moore et al. 2015). Serum C-reactive
general weakness (Fig. 4.2). The infection can protein (CRP) is commonly available, and is
also present with trismus. Although simple throat markedly elevated, with a median of 284 mg/L
infections are common, the incidence of cervical (IQR: 179–373) before initial surgery (Madsen
NSTI is estimated to two per one million et al. 2019). Furthermore, CRP levels may be
inhabitants per year (Wolf et al. 2010). The infec- higher in patients with NSTI compared to patients
tion can spread among the fascial planes involv- with cellulitis (Simonart et al. 2001). Leukocytes
ing the neck, and in more pronounced cases the are equally elevated, but not as markedly, with a
infection can involve mediastinum (Gunaratne median of 16.3 109/L (IQR: 10.6–22.4) (Madsen
et al. 2018). NSTI of the neck can be complicated et al. 2019), although higher in other cohorts
by suppurative thrombophlebitis of the internal (Wong et al. 2003). Procalcitonin (Madsen et al.
jugular vein, referred to as Lemierre’s syndrome 2019) and sedimentation rate (Wong et al. 2003)
(Deganello et al. 2009). The most commonly are also elevated. In general, patients have a met-
cultured bacteria are streptococci and abolic acidosis, with a low pH, low standard base
staphylococci, but also a broad range of anaerobic excess and high lactate. The level of acidose is
bacteria including Prevotella sp., similar to that in cohorts of patients with septic
Fig. 4.3 Infection of the lower leg. Courtesy of INFECT
shock (ARISE Investigators et al. 2014; (supAR) levels were higher in NSTI patients
Investigators 2014; PRISM Investigators 2017), with septic shock compared to no shock, and in
but the lactate levels are substantially lower. survivors compared to non-survivors (Polzik et al.
New biomarkers emerge in the attempt to find 2019). Elevated supAR levels at admission were
easily obtainable markers of diagnosis, disease associated with increased 90-day mortality, but
severity, and death. Pentraxin-3 (PTX3) is a after adjusting for age, sex, and SOFA score, the
pattern-recognition molecule and is part of the result was not statistically significant. Neither
innate immune system. It is elevated in patients PTX3 nor supAR are readily available in standard
with NSTI compared to controls, and correlated laboratories.
to shock, amputation, and 180-day mortality— In a quest to develop a score for differentiating
although the association was not statistically sig- NSTI from common infectious disease, a score
nificant when adjusted for age, sex, chronic dis- based on six standard laboratory parameters
ease, and SAPS II (Hansen et al. 2016). Soluble (potassium, glucose, hemoglobin, CRP, white
urokinase-type plasminogen activator receptor blood cells, and lactate) was developed—the
Fig. 4.4 Massive debridement after infection originating from the lower extremity. Courtesy of INFECT
Fig. 4.5
(a)
Infection of
the scrotum
as seen after
initial
surgery. (b)
Picture of
Initial
debridement of widespread
infection (different patient
from a). Courtesy of
INFECT
laboratory risk indicator for necrotizing fasciitis important to consider that patients with NSTI are
(LRINEC) (Wong et al. 2004). The score was heterogenous, and the quality of the studies
developed using a cohort of 314 patients with varies. A priori, a clinician should raise suspicion
NSTI from a single center in Singapore included for the ability of score based solely on standard
retrospectively and validated in another retro- laboratory values to discriminate between
spectively included cohort of 140 patients. In advanced tissue destruction and a
this initial work, a LRINEC score of 6 or above non-necrotizing infection, and further diagnostics
had excellent positive and negative predictive and treatment should not be delayed awaiting the
values. A number of studies have shown varying result of the score. Others have tried to develop an
ability of the LRINEC score to distinguish alternative score for discriminating between NSTI
between NSTI and non-NSTI, but almost all and non-NSTI. The SIARI score was developed
were retrospective observational studies, and of using data obtained retrospectively at a single-
rather small size. In a recent meta-analysis, the center in New Zealand from 2000 to 2010
LRINEC score had poor sensitivity for diagnosis (Cribb et al. 2019). The score is based on the
of NSTI and moderate sensitivity (Fernando et al. variables site other than lower limb, immunosup-
2019). This is supported by a prospective, single- pression, age 60 years, renal impairment, and
center observational study conducted in Taiwan, inflammatory markers, and the cohort included
where the LRINEC was evaluated (Hsiao et al. 138 patients with NSTI and 142 with severe cel-
2020). Eight hundred and twenty five patients lulitis; these were divided into a development and
with cellulitis and 106 patients with NSTI were a validation cohort. The PPVs in the two cohorts
included. At a cut-off of a LRINEC score of six or were 73% (95% CI: 65%–80%) and 74% (95%
above, the positive predictive value (PPV) was CI: 66–80%), respectively, and the NPVs were
25% (95% CI: 20%–30%) and the negative pre- 80% (95% CI: 71%–87%) and 81% (71–88%),
dictive (NPV) was 92% (95% CI: 91%–93%), respectively. The concordance statistics (C-statis-
and the area under the receiver operating charac- tic—comparable to ROC curve) were 0.83 and
teristic (ROC) for accuracy was 0.696 (95% CI: 0.85, respectively. As for the LRINEC score,
0.640–0.751). When interpreting the score it is limitations apply, including the lack of clinical
data; data were from cohorts included at a single- involvement of epidermis (Barker et al. 1987;
center; data were collected retrospectively; the Umbert et al. 1989; Stamenkovic and Lew
limited number of included patients and data 1984). Furthermore, thrombosis of the vessels is
were collected over large period of time. The seen. Histopathological examination may aid in
clinician should always have a high suspicion diagnosing NSTI in cases where the clinical
for NSTI, and never rule out the diagnosis when findings during surgery are not in unison and the
clinically suspected, despite a score below a cer- degree of the inflammatory response in the tissue
tain cut-off. may correlate to disease severity, as proposed by
Bakleh and colleagues (Bakleh et al. 2005). Gram
staining would also provide information on pres-
4.2.4 Microbiology ent bacteria if found in the tissue. An added bonus
to routine histopathology sampling could be a
Determining the microbiological etiology is of more precise definition of NSTI at tissue level,
uttermost important to target the antibiotic ther- rendering it possible to include more homogenous
apy. Whenever a severe infection is suspected, patients in future NSTI cohorts.
relevant cultures should be obtained immediately.
When reviewing microbiological data, it is
important to keep a number of factors in mind, 4.2.5 Imaging
as these can differ considerably and introduce
substantial bias. The samples sent for Several imaging options are available, including
microbiological testing should be taken from the plain radiographs, ultrasonography (US),
wound or in its vicinity; alternatively, from blood computerized tomography (CT), and magnetic
sampling. The samples should have been resonance imaging (MRI).
obtained within a reasonable time from the time Plain radiographs are readily available at most
of diagnosis. Samples taken too soon before centers. It is possible to visualize gas in the soft
(in patients hospitalized for reasons other than tissue in the absence of trauma (Tso and Singh
NSTI) may not reflect the pathogenic bacteria, 2018). However, this symptom is far from present
and samples taken too long after may represent in all patients, and if present, is easily overlooked
secondary contamination. It is also worth noting compared to findings from a CT scan (Leichtle
the method in which the microbiological agents et al. 2016).
are derived. Traditionally, the sample is spread US findings include fluid accumulation in the
out and grown on an agar plate. This method may fascial layer and thickening of the subcutaneous
facilitate the growth of certain bacteria above layer (Yen et al. 2002). US is limited by the low
others. Newer methods, including PCR analysis resolution of deeper structures, but has been
and shotgun metabolomics (Rodriguez et al. shown to discriminate NSTI of the limbs from
2020; Thänert et al. 2019; Rudkjøbing et al. non-necrotizing fasciitis (Yen et al. 2002). As US
2016), are more sensitive, but may also find spe- is widely accessible, it is probably not likely to
cies that are not relevant with regard to the infec- postpone surgical intervention (Whitson and
tion. A great advantage is the ability to provide Mayo 2016; Barbic et al. 2017). However, the
faster bacterial etiology. The microbiologic etiol- findings specific to NSTI require experience to
ogy of NSTI is reviewed in Chap. 5. detect, and in all circumstances, US should not be
Although presumably seldom performed, used to rule out NSTI.
material obtained for histopathology can provide The resolution of CT scans has increased
additional information for the clinician. Only few tremendously over the past years and exceeds
studies exist, but from these we have data on how that of plain radiography and US (Hayeri et al.
the different layers of the soft tissue are affected, 2016). Findings of NSTI on CT scans include
with inflammation and necrosis affecting dermis asymmetric thickening of the fascia and gas track-
and subcutaneous fat, and in some cases ing along fascial planes (Wysoki et al. 1997)—
especially gas within fluid collections along findings. These typically include extensive necro-
subfascial planes. In a meta-analysis of seven sis of the subcutaneous tissue and/or muscle tis-
studies, the presence of fascial gas on CT had a sue, undermining of the skin, thrombosis of the
sensitivity of 88.5% (95% CI: 55.5%–97.9%) and superficial veins, and deliquescent tissue. Acute
a specificity of 93.3% (95% CI: 80.8%–97.9%) microscopy for the microbiological agent can be
(Fernando et al. 2019). Other findings include helpful for choice of antibiotic treatment, but the
subfascial fluid, subcutaneous edema, and microbiological flora is not a determinant of
increased soft-tissue attenuation (Tso and Singh whether the infection is a NSTI or not. Although
2018), and if all these findings are present, the initial surgery may be without signs of NSTI,
sensitivity rises to 94.3% (95% CI: 81.2%– patients should be closely monitored, as some of
98.5%) but at the expense of specificity, which these patients may eventually be diagnosed with
decreases to 76.6% (95% CI: 21.3%–97.5%) NSTI (Howell et al. 2019). Snap frozen biopsies
(Fernando et al. 2019) The use of CT scan can for histology have been used, but it is uncertain
aid in discriminating NSTI from non-necrotizing whether these contribute to faster diagnosis and
infections (Carbonetti et al. 2016). Furthermore, thereby shorter time to surgery. As mentioned
the scan may help surgeons determine the extent above, MRI can potentially discriminate between
of soft-tissue involvement and help localize the NSTI and non-necrotizing infections, but due to
source of infection. In some centers, CT scans can the length of the procedure and the availability of
be done within minutes, and thus probably do not MR-scanner, for many, this is not a feasible
delay surgery. In this context, this modality can option. Ultrasound is readily available in many
be of help. hospitals, and as NSTI has certain specific ultra-
MRI has been used for more than 20 years to sonic properties, US can aid the diagnostic proce-
help discriminate NSTI from non-necrotizing dure. However, caution should be taken when
infections (Rahmouni et al. 1994), and is consid- ruling out a necrotizing infection. Finally, the
ered the gold-standard for soft-tissue infections. LRINEC score does not seem sufficient in
MRI has excellent resolution for soft tissue. MRI distinguishing NSTI from non-necrotizing
findings of NSTI include thickening of the deep infections.
fascia with abnormal signal intensity, edema, and
hypointense signals due to gas within fascial
layers (Ali et al. 2014; Kim et al. 2011). Changes 4.4 Future Perspectives
in muscle signal can be observed due to adjacent
inflammation. Unfortunately, MRI has major NSTIs are serious infections with diagnostic
limitations: accessibility and duration of the challenges. In order to improve outcome for
scan—both which are likely to delay surgical future patients, focus on certain diagnostics
intervention. aspects is key. First and foremost, knowledge of
Overall, the different imaging modalities may the disease is of uttermost importance. The initial
aid in discriminating patients with NSTI from step is often when the patient contacts the
non-necrotizing infections, but they must not healthcare system. The threshold for contact
delay surgical intervention (Sartelli et al. 2018). varies considerably from patient to patient, and
as the initial symptoms sometimes are the same as
“simple infections,” it is difficult to minimize the
4.3 Diagnostic Aspects time from the patients experience symptoms to
healthcare system contact. Every doctor seeing
Any patient with a cutaneous infection with local patients may encounter one with NSTI (Anaya
or systemic signs of severity should be suspected and Dellinger 2007), and should be able to raise
of NSTI (Fig. 4.6). No set of diagnostic criteria suspicion and quickly refer the patient to a hospi-
for NSTI exists; the diagnosis is merely clinical, tal or department capable of further diagnostics
meaning that it is based on intraoperative and surgery, emphasizing the severity of the
4
Obtain cultures for microbiology

Signs of NSTI? Initiate broad spectrum antibiotics
Contact members of multidisciplinary team according to local protocols

• Surgeon: immediate evaluation and preparation for emergency surgery
• Anaesthesiologist: Preparation for surgery / optimisation of patient
• Intensivist: optimisation of patient / plan ICU admittance after surgery
Consider
Early symptoms • Imaging – but should not delay surgery when NSTI is suspected
• Pain, nausea vomiting
• Redness, swelling
Late symptoms
• Pain out of proportion
• Altered consciousness
• Cyanosis Close observation
Emergency surgery Further diagnostics
• Crepitus
• Bullae Consider ICU
• Necrosis
Necrotizing Soft-Tissue Infections: Clinical Features and Diagnostic Aspects
Systemic symptoms
• Fever
• Tachycardia
• Shock ICU
• Elevated acute phase reactants (see table 1)
Fig. 4.6 The purpose of the flow chart is to give an overview of the most important diagnostic, logistic, and treatment aspects. It is important to consider that no combination of
symptoms and sign is pathognomonic for NSTI. Suspicion of NSTI should have a low threshold. This chapter does not cover treatment of NSTI. Adapted from (Urbina et al. 2020)
49
disease suspected. Although never shown, early It is important that the quest to understand the
recognition is probably one of the most underlying pathophysiology of NSTI continues.
timesaving steps and could potentially reduce This can only be done by meticulously collecting
morbidity and mortality considerably. Unfortu- data on these patients, preferably from prospec-
nately, there are several case stories of patients tive, multicenter cohort studies. Only when this is
acknowledging that “something is wrong,” but done we can design randomized clinical trials
where the healthcare workers did not recognize aimed to test the effect of specific treatments.
the initial symptoms and thus the seriousness of
the situation (Erichsen Andersson et al. 2018). Acknowledgement Financial support: The work was
Due to the low incidence, continuous education supported by the European Union Seventh Framework
Programme: (FP7/2007-2013) under the grant agreement
of healthcare workers is needed to maintain
305340 (INFECT project).
awareness of the disease. This is presumably
even more relevant in settings where NSTI is
not routinely encountered and focus should be
on signs of severity. References
Distinguishing NSTI from other infections is
challenging, and we still do not have solid diag- Ali SZ, Srinivasan S, Peh WCG (2014) MRI in necrotizing
fasciitis of the extremities. Br J Radiol 87:20130560
nostic tools. New biomarkers emerge, but due to Al-Qurayshi Z, Nichols RL, Killackey MT, Kandil E
the heterogeneity of the NSTI, it is not likely that (2020) Mortality risk in necrotizing fasciitis: national
a single biomarker will be diagnostic—but as part prevalence, trend, and burden. Surg Infect 21:
of a diagnostic panel it may help in diagnosing sur.2019.277
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NSTI. New methods for microbiological testing infection: diagnosis and management. Clin Infect Dis
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biotic therapy. The use of PCR is already avail- Predictors of mortality and limb loss in necrotizing
soft tissue infections. Arch Surg 140:151–157
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Peake SL et al (2014) Goal-directed resuscitation for
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There is still not sufficient data to show 371:1496–1506
whether US can aid in the diagnosis of NSTI. Bakleh M, Wold LE, Mandrekar JN et al (2005) Correla-
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Microbiological Etiology of Necrotizing
Soft Tissue Infections 5
Steinar Skrede, Trond Bruun, Eivind Rath,
and Oddvar Oppegaard
Contents
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5.2 Contemporary Classification of NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5.3 Establishing Microbial Etiology of NSTIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
5.3.1 Sample Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
5.3.2 Quality of Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
5.3.3 Categorization According to Pathogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
5.3.4 Classification as Either Monomicrobial or Polymicrobial . . . . . . . . . . . . . . . . . . . . . . 57
5.4 Microbiological Etiologies in NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.4.1 Streptococcus pyogenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5.4.2 Streptococcus dysgalactiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5.4.3 Staphylococcus aureus, Including MRSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.4.4 Clostridium perfringens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.4.5 Vibrio vulnificus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
5.4.6 Aeromonas hydrophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.4.7 Polymicrobial Etiologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.4.8 Microbial Etiologies Documented by the INFECT Study . . . . . . . . . . . . . . . . . . . . . . 64
5.4.9 Non-culture Based Microbiological Methods in NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . 65
5.5 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Abstract
S. Skrede (*) · T. Bruun
Department of Clinical Science, University of Bergen, Necrotizing soft tissue infections (NSTIs) are
Bergen, Norway severe clinical conditions requiring swift ther-
Department of Medicine, Haukeland University Hospital, apeutic intervention, including surgical
Bergen, Norway removal of infected tissue and administration
e-mail: Steinar.skrede@helse-bergen.no
of potent antibiotics. There is wide diversity in
E. Rath · O. Oppegaard the microbial etiologic agents, and tailoring
Department of Medicine, Haukeland University Hospital,
Bergen, Norway the antibiotic treatment to the offending

54 S. Skrede et al.
pathogen is essential. However, the choice of that can guide antibiotic therapy. Moreover, veri-
empirical therapy is frequently inadequate, fication of the causative pathogen can offer prog-
underlining the need for comprehensive and nostic information (Anaya et al. 2005; Huang
contemporary knowledge on causative et al. 2011; Madsen et al. 2019). Notably, empiri-
pathogens and relevant antimicrobial resis- cal antibiotic treatment of NSTIs is inadequate in
tance patterns in NSTIs. Also, studies of the approximately 90% of the patients (Marwick
pathogenic mechanisms in different NSTIs are et al. 2011). This highlights the significant knowl-
needed, to improve handling of patients edge gap in microbial etiology in NSTIs, both on
through developing patient stratification and a regional and global level. There are several
tailored therapies. We review the current reasons for this. NSTIs are rare, and with inci-
knowledge on microbial etiology and provide dence rates in the range of 0.3 to 15/100,000/year
detailed characterizations of the predominant (Stevens and Bryant 2017; Peetermans et al.
pathogens. 2020) most hospitals consequently treat a low
number of patients. This potentially leads to
Keywords insufficiently developed competence in adequate
microbiologic diagnostic procedures. Since obli-
Bacteroides fragilis · Monomicrobial ·
gate anaerobic microbes are frequently involved,
Necrotizing soft tissue infection ·
samples are particularly vulnerable to incorrect
Polymicrobial · Staphylococcus aureus ·
sample acquisition or transportation, which likely
Streptococcus dysgalactiae · Streptococcus
has contributed to the low rates of culture positive
pyogenes · Type 1 infection · Type 2 infection
samples presented in several studies (McHenry
et al. 1995; Boyer et al. 2009; Khamnuan et al.
Highlights
2015). Furthermore, many NSTI cases present as
• Verification of microbial etiology is of para-
medical emergencies, and establishing a
mount importance in NSTI
microbiological diagnosis is a lower priority
• The INFECT study provided strong
than initiating urgent therapeutic measures.
associations of affected localization and
Finally, data reporting in many studies offer low
microbial etiology in NSTI
level of precision, e.g. when only crude
• Monomicrobial NSTI is most often caused by
microbiological data for a patient cohort is given
Streptococcus pyogenes and has strong corre-
(Childers et al. 2002), or because of varying qual-
lation to an extremity localization
ity in reporting of discharge diagnosis (Louis
• New microbial etiologies of NSTI are
et al. 2019). Notwithstanding, in NSTI medical
emerging due to aging of populations and cli-
microbiology holds great promise, as test results
mate change
are offered with speed adding value to our
present-day diagnostic methods. Also, medical
microbiology is a key scientific discipline in
5.1 Introduction
development of rapid diagnostic tests speeding
up decision making and leading towards
Necrotizing soft tissue infections (NSTIs) are het-
personalized therapeutic measures to well
erogeneous, rare and severe infections with high
stratified patient cohorts among NSTI patients.
morbidity and mortality rates. Mainstay therapy is
In the following, microbiologic features of NSTI
infection source control achieved by radical sur-
are delineated.
gical treatment supported by prompt administra-
tion of antimicrobial therapy adjusted to the
microbial etiology (Stevens and Bryant 2017).
5.2 Contemporary Classification
Medical microbiology plays an important role in
of NSTI
this multidisciplinary work, as it can contribute to
improved prognosis through identification of
Necrotizing soft tissue infections have been
microbial etiology and relevant resistance profiles
documented since the fifth century
5 Microbiological Etiology of Necrotizing Soft Tissue Infections 55
B.C. Historically classification according to streptococci other than GAS. They noted that in
anatomical location has been practiced, giving each case of a type 1 infection, at least one anaer-
rise to a multitude of clinical syndromes bearing obic organism was cultured in combination with a
different eponyms. In the head and neck area facultative anaerobe. In category type 2 GAS was
Lemierre’s syndrome is a disseminated necrotiz- isolated. The authors noted that in type 2 GAS
ing infection characterized by jugular suppurative occurred together with Staphylococcal species,
thrombophlebitis and evidence of disseminated but not anaerobic bacteria or Enterobacteriaceae.
infection, including signs of septic embolization. This categorization has been widely adopted and
Fusobacterium necrophorum is frequently used in guidelines (Sartelli et al. 2018) and
involved. Ludwig’s angina has been applied to a reviews (Sarani et al. 2009; Hakkarainen et al.
number of infections of the sublingual, submaxil- 2014; Lamb et al. 2015; Stevens and Bryant
lary, or submandibular space. It should be 2017).
restricted to rapidly spreading infections The list of implicated microbes has gradually
originating in the floor of the mouth that spreads been expanded, though, and amendment of
bilaterally within the submandibular and sublin- monomicrobial infections caused by marine
gual spaces, without abscess formation or lym- gram-negative microbes as a type 3 category, as
phatic involvement. Polymicrobial etiology is well as a fungal type 4 category has also been
predominant, and Peptostreptococcus, suggested (Morgan 2010). The latter is not widely
Prevotella, and Porphyromonas species, and accepted as the usefulness of expanding necrotiz-
viridans streptococci are frequently implicated. ing soft tissue infection entities is questioned
Fournier’s gangrene is a progressive (Stevens and Bryant 2017). In Table 5.1 a practi-
polymicrobial synergistic necrotizing infection cal overview of microbes presented by category is
of the external genitals and perineum, where a offered and microbial causes of NSTIs presented
number of pathogens can be expected, including in more detail below.
obligate anaerobic species, Enterobacteriaceae
and Enterococcus spp. Meleney’s bacterial gan-
grene, or hemolytic streptococcal gangrene, is a 5.3 Establishing Microbial Etiology
type 2 necrotizing fasciitis caused by Streptococ- of NSTIs
cus pyogenes (group A streptococcus; GAS).
Many of these eponyms arose prior to the estab- There is a substantial heterogeneity in the distri-
lishment of diagnostic microbiology, and their bution of microbial etiologies reported in NSTIs,
use has frequently led to low precision in descrip- and a plethora of pathogens have been implicated.
tion of etiologies in severe soft tissue infections. It Temporospatial differences account for some of
was noted already in the 1970s that there was an the observed discrepancies, but unfortunately a
insufficient attention on the bacteria involved in high variability in microbiological methods, case
the pathogenesis of NSTI. In a single center study definitions, and study designs is the major culprit.
from San Francisco, Giuliano and coworkers In a substantial fraction of publications on NSTI,
described bacterial etiologies in sixteen patients characteristics of a low numbers of patients with a
with NSTI, of which there were nine drug users, particular etiology or single cases are reported,
three postoperative cases, and three with a story occasionally accompanied by reviews of a limited
of minor trauma (Giuliano 1977). They collected number of similar cases, identified in retrospec-
intraoperative samples and identified tive cohorts. Moreover, case definitions of NSTI
18 pathogens at species level, as well as a signifi- are not widely accepted due to lack of adequate
cant number at genus level. The study culminated clinical precision (Calandra et al. 2005), leading
in a novel system for classification of NSTI based to inclusion of resembling clinical entities such as
on microbial etiology, where category type deep-seated abscesses, vascular occlusion, and
1 grew anaerobic bacteria and facultative anaero- cellulitis. Lastly, the precision of microbiological
bic bacteria, such as Enterobacteriaceae or sampling is often not detailed, and the influence
56 S. Skrede et al.
Table 5.1 Categorization of necrotizing soft tissue infections by microbiological etiology

Category Type 1 Type 2 Type 3 Type 4
Etiology Polymicrobial mixed aerobic/ Monomicrobial Gram-negative marine Fungal
anaerobic organisms
Species Staphylococcus aureus Streptococcus Vibrio vulnificus Candida
Viridans streptococci pyogenes Aeromonas hydrophila spp.
Streptococcus dysgalactiae Streptococcus Shewanella spp. Zygomycetes
Enterococcus spp. dysgalactiae
E. coli/Klebsiella CA-MRSAa
Bacteroides spp.
Prevotella spp.
Fusobacterium spp.
a
CA-MRSA community acquired methicillin resistant Staphylococcus aureus
of contaminants on the etiologic distribution is 5.3.2 Quality of Identification

thus difficult to evaluate. Consequently, there is
still no international consensus definition regard- The categorization of etiology and the certainty of
ing soft tissue infections and pathogenicity the etiological diagnosis was based on type of
delimiting what microbes that cause NSTIs sample, time of sampling, e.g. in relation to sur-
(Eron et al. 2003; Stevens et al. 2005, 2014). gery, host factors, patient story taking primary
In order to deal with this, classification catego- focus into account, and ability of the microbes
rization systems for NSTIs have been created to cause severe soft tissue infection confirmed by
according to microbial etiologies, as outlined in description of pathogenicity.
Sect. 5.2. In the recent INFECT study, the world’s The certainty of etiology was categorized as
largest prospective study of NSTIs, the method definite in cases fulfilling documentation in elec-
for classification of cases by microbes, in a sys- tronic patient files at the highest level of
tem meant to aid categorization and evaluation of microbiological identification. Probable etiology
microbiological data, was described in the sup- was delimited to “Likely pathogens” reviewed
plementary material accompanying the publica- below, whereas possible etiology was based on
tion on streptococcal NSTI (Bruun et al. 2020). weak documentation or low likelihood of being
According to this routine, categorization was etiological cause, including situations where
based on routine laboratory culture and identifi- sampling method, host factors, patient story, or
cation, essentially phrased as follows. clinical evaluation lead to uncertainty of the
role of the microbe. Finally, unknown etiology
was defined as no identification of relevant
pathogen.
5.3.1 Sample Sources
Firstly, sample source was reviewed to support

whether a microbe would be definite, probable, or 5.3.3 Categorization According
unlikely to be involved in causes of surgically to Pathogenicity
confirmed NSTI. By definition, reliable sources
were limited to blood cultures or deeply situated The microbes were classified into primary
soft tissue in otherwise sterile environment, pathogens, defined as microbes that may cause
e.g. superficial and deep fascia, muscle, fat or soft tissue infection in patients without known
unidentified necrotic tissue. Unreliable sources risk factors. These include Streptococcus
were superficial swab samples, including those pyogenes, Vibrio vulnificus, and Clostridium
taken from chronic abscesses with fistula. perfringens.
Secondary pathogens included microbes that 5.4 Microbiological Etiologies

may cause soft tissue infection, including in NSTI
polymicrobial as well as monomicrobial etiology,
in patients with risk factors. Typical examples 5.4.1 Streptococcus pyogenes
were “group C and G streptococci” (most often
Streptococcus dysgalactiae, though frequently Streptococcus pyogenes (group A streptococcus;
not identified to species level in routine GAS) is exclusively a human pathogen. It causes
laboratories), Streptococcus agalactiae (group B a substantial global disease burden, ranging from
streptococcus; GBS), Streptococcus anginosus superficial skin infections and tonsillitis, to severe
group, Streptococcus pneumoniae, Staphylococ- invasive infections (Ralph and Carapetis 2013).
cus aureus, Haemophilus influenzae, Neisseria Importantly, it is the predominant etiologic agent
meningitidis, Enterobacteriaceae, Pseudomonas of monobacillary type 2 NSTI, comprising
spp., Acinetobacter spp., Bacteroides spp., 10-40% of all cases (Elliott et al. 1996, 2000;
Prevotella spp., and Fusobacterium spp. Kobayashi et al. 2011; Das et al. 2012; Madsen
Polymicrobial pathogens were delimited to et al. 2019). The microbiological characteristics
possible causes of soft tissue infections as part of GAS are briefly reviewed below. Due to its
of a polymicrobial flora, but rarely of NSTI in the importance, details on pathophysiology of strep-
absence of primary or secondary pathogens, tococcal NSTI are presented in Chap. 10, clinical
e.g. Enterococcus. aspects in Chap. 6, and antimicrobial treatment in
Commensals were defined as microbes that do Chap. 7.
not cause severe soft tissue infection, but that may GAS belongs to the family Streptococcaceae
be occasional markers of polymicrobial etiology of the phylum Firmicutes. It is a gram-positive,
in cases where they are cultured together with facultative anaerobic, catalase negative, spherical
other bacteria, with typical examples being Bacil- or ovoid bacterium with approximate diameter
lus other than B. anthracis, Corynebacterium, 0.6–1 μM. It is non-motile and does not form
Micrococcus, and Coagulase negative spores. In the microscope it appears in pairs or
staphylococci. short chains in clinical specimens, whereas long
chains are formed when it is grown in rich culture
media. Its binominal family name Streptococcus
5.3.4 Classification as Either comes from Greek στρεπτóς (streptós) “twisted,”
Monomicrobial or “chain,” or “necklace” and κóκκoς (kókkos)
Polymicrobial meaning “berry” or “grain.” Its species epithet
pyogenes comes from the combination of Greek
Monomicrobial cases were classified as such if a πύoς (pyos), “pus,” and γεννάειν, (genein) “to
primary or secondary pathogen was detected in beget,” referring to its ability to form pus. The
monoculture, or if a polymicrobial pathogen was microbe requires complex, enriched media to
found in monoculture in a patient with severe grow. On solid culture media colonies may
immunodeficiency. appear glossy, or mucoid, more or less,
Cases where a primary pathogen was depending on the hyaluronate capsular material
identified together with commensals were a strain produces. When grown on blood agar it
categorized among the monomicrobial cases. forms white to gray colonies 0.5 mm and it
Polymicrobial cases would then comprise the displays complete (β) hemolysis through red
remaining cases, and in this publication it was blood cell disruption caused by streptolysin. For
stated that polymicrobial infections are easily almost a century, β-hemolytic streptococci have
categorized as such, deemed by patient stories been classified by detection of their respective
(e.g. head neck, pelvic) and culture results cell wall carbohydrate antigens, in the system
(Bruun et al. 2020, supplementary material). created by Rebecca Lancefield (Lancefield 1933;
58 S. Skrede et al.
Lancefield and Hare 1935). Phenotypic testing is bacteria” in the public media (Lamb et al. 2015).
imperfect, exemplified by that GAS is not exclu- In the recent INFECT-study another distinct char-
sively expressing Lancefield group A carbohy- acteristic of GAS NSTIs was noted. A strong
drate antigen, yet it is still widely used. association of GAS etiology and NSTI of the
Contemporary methods used for bacterial identi- upper extremity (Fig. 5.1), with and odds risk
fication include mass-spectrometry, matrix- (OR) of 7.80 (95% CI 4.36–14.25, p < 0.001)
assisted laser desorption/ionization time of flight was observed (Madsen et al. 2019).
mass spectrometry (MALDI-ToF MS), a rapid A similar observation was reported almost
and cost-effective method that offers sufficient 25 years ago in a small patient cohort from North-
taxonomic resolution at species level in the case ern America (Haywood et al. 1999).
of GAS. In the INFECT-study only 12/126 cases (9%)
The major virulence factor of GAS is involving GAS were polymicrobial, of which 2/3
M-protein, reviewed in Chap. 10. Almost every were co-cultured with Staphylococcus aureus
GAS isolate expresses an emm-gene, and based (Bruun et al. 2020). This co-residency was
on antigenic differences more than 200 emm- observed also by the inventors of the currently
types have been described. The emm-types used categorization system of NSTI according to
emm1, emm3, emm12, emm28, and emm89 are microbial etiologies (Giuliano 1977). The authors
the predominant genotypes implicated in invasive concluded that these infections were type 2 GAS
GAS disease (Chelsom et al. 1994; infections, where staphylococcal species were
Monnickendam et al. 1997; Kaul et al. 1997; detected, whereas anaerobic bacteria or
Sharkawy et al. 2002) although regional Enterobacteriaceae were not.
differences in genotypic distribution has been Penicillin is the undisputable treatment of
noted (Gherardi et al. 2018). In the INFECT choice for GAS. However, GAS isolates with
study, GAS comprised 126/409 (31%) of all reduced susceptibility to beta-lactam antibiotics
NSTI cases and 75% of all β-hemolytic have recently been reported, associated with
streptococci. The predominant emm-types were mutations in penicillin binding protein pbp2x
expectedly emm1, emm3, and emm28 (Bruun (Vannice et al. 2019; Musser et al. 2020). The
et al. 2020). Similar findings were also reported clinical relevance has yet to be determined, but
from a retrospective cohort of 70 patients with sustained attentiveness is definitely warranted.
β-hemolytic streptococcal NSTI in western Increasing resistance to clindamycin is of more
Norway, whereof 61/70 (87%) were caused by immediate concern (Smit et al. 2015). However,
GAS (Bruun et al. 2013). large regional variations are noted, and in the
GAS is equipped with several other virulence INFECT study in Scandinavia only a single
factors, of which the extracellular superantigens GAS isolate displayed reduced susceptibility to
are key mediators of the immunopathogenesis of clindamycin. Persistent infection over days and
invasive GAS (iGAS) (Commons et al. 2014), on even weeks has been noted in patient cases
the one hand able to elicit the unbalanced inflam- (Siemens et al. 2016). In part this may be
matory responses seen in iGAS, and on the other attributed to functional antimicrobial resistance
to ensure nasopharyngeal carriage (Zeppa et al. caused by GAS biofilm formation, first
2017). The repertoire of virulence determinants documented in invasive GAS through the
and pathogenic mechanisms of GAS NSTIs are INFECT study (Siemens et al. 2016), which
further delineated in Chap. 10. may impact future treatment strategies. Relevant
The disease signature of GAS NSTIs antibiotic resistance in GAS infection is further
represents the hallmark of features commonly detailed in the section on β-hemolytic
associated with NSTIs; rapid progression and a streptococci in Chap. 7.
fulminant clinical course. Following the upsurge The lack of efficient GAS vaccines and recent
of GAS NSTIs in the mid-1980s, this pathogen concerns on antimicrobial susceptibilities in both
has become synonymous with the “Flesh-eating GAS and Streptococcus dysgalactiae
Fig. 5.1 Associations between affected body part and microbiological findings. GAS Streptococcus pyogenes, GCS/
GGS 27 S. dysgalactiae, 1 S. equi sp. zooepidemicus. Inspired by Madsen (Madsen 2018), with permission
(SD) warrants continuous attention towards GAS level. Different systems derived from GAS typing
as microbial etiology of NSTI. have been used for typing of SD, most often by
emm-typing. The emm-gene encodes the
M-protein, that is a virulence factor present in
5.4.2 Streptococcus dysgalactiae SD, and presently more than 100 emm-types
have been described (https://www.cdc.gov/
Streptococcus dysgalactiae (SD) is a β-hemolytic streplab/index.html). Several other virulence
streptococcus that displays genetic similarity to factors have been identified in SD, and when
GAS, indicating a common phylogenetic ancestry comparing SD and GAS, genes for about half of
of human origin. The Latin species epithet well-characterized GAS virulence factor genes,
dysgalactiae refers to “loss of milk production,” including adhesins and protein G that binds
a symptom of bovine mastitis. SD expresses cell circulating immunoglobulins, are detected in
wall carbohydrate antigens of Lancefield group C SD. Also, SD produces toxins and secreted
and group G, and is divided into the subspecies enzymes including Streptolysin O (SLO), Strepto
S. dysgalactiae subsp. equisimilis and lysin S (SLS), and Streptokinase (Shimomura
S. dysgalactiae subsp. dysgalactiae. The latter is et al. 2011).
exclusively a zoonotic pathogen and not further Historically, SD was judged of little impor-
discussed. SD is a gram-positive, facultative tance as etiology of human disease. The last few
anaerobic, bacterium that does not form spores decades, however, it is increasingly recognized as
and is non-motile. It forms large colonies >0.5 an important cause of invasive disease (Ikebe
mm and on blood agar it is predominantly et al. 2004), particularly in the adult population
β-hemolytic. Today many laboratories identify (Takahashi et al. 2010; Oppegaard et al. 2015).
bacteria by MALDI-ToF MS, but old methods From the upsurge of streptococcal NSTI in the
for identification are still frequently in use. It 1980s and onwards scattered reports on group G
does still not offer sufficient taxonomic resolution and C streptococcal NSTI were published (Barker
beyond the species level, and multilocus et al. 1987; Sharma et al. 2002). Even though
sequence typing (MLST) remains the most reli- expression of Lancefield group G or C carbohy-
able method for identification of SD at subspecies drate antigens is not unique to SD, it is
60 S. Skrede et al.
conceivable that most cases of these groups G and all S. aureus strains are coagulase-positive. Cul-
C streptococcal NSTI were caused by SD. Today ture and antibiotic susceptibility testing are diag-
it is well established that SD is an emerging cause nostic corner-stones but today molecular
of monobacillary as well as polymicrobial NSTI techniques, mass spectrometry, and other
(Bruun et al. 2013, 2020; Madsen et al. 2019). methods are important novel measures to aid
In a retrospective cohort of 70 patients in detection (e.g. in slowly growing phenotypes),
western Norway with β-hemolytic NSTI, SD in noncultivable cases (e.g. in some tissue
caused 9/70 cases (Bruun et al. 2013). In the samples), rapid species determination, and detec-
INFECT study SD comprised 27/409 (7%) com- tion of relevant resistance. S. aureus is a major
pared to GAS 126/409 (31%), confirming the cause of human diseases. Its success can be
importance of SD in NSTI. Furthermore, in SD attributed to several microbial traits. Firstly, it is
stG62647 was the predominant emm type and a capable colonizer of the skin and mucosa in the
expressing speG, 47 % of cases were upper respiratory tract, and up to 30% of the
polymicrobial, 30% were blood culture positive, human population are carriers. Moreover,
and antimicrobial resistance towards erythromy- S. aureus produces an arsenal of virulence factors
cin and clindamycin was 5% and 0, respectively enabling it to adhere and expand locally, spread
(Bruun et al. 2020). Informatively, positive cul- or cause host responses systemically, distant from
ture of SD was strongly associated with infection the origin of infection, reviewed elsewhere
of the lower extremities (Fig. 5.1), with an OR (Shumba et al. 2019). Surface adhesins are com-
9.76 (95% CI 2.57–54.85, p ¼ 0.001) (Madsen monly covalently bound to the bacterial cell wall
et al. 2019). The worldwide increase in numbers peptidoglycan and mediate adherence to matrix
of elderly people and the increasing resistance proteins of target tissues. The microbe confers
towards key antibiotics warrants attention toward virulence also by use of multiple extracellular
SD as microbial etiology of NSTI, and increasing factors, i.e. enzymes, cytotoxins, and
incidence rates may be anticipated. superantigens. Furthermore, small RNAs are
staphylococcal virulence factors, involved in,
e.g. increased biofilm formation. Strains of
5.4.3 Staphylococcus aureus, S. aureus exchange phages, such as the Panton–
Including MRSA Valentine leukocidin (PVL) toxin producing,
mobile phage frequently expressed by strains of
Staphylococcus aureus is a gram-positive, facul- methicillin resistant S. aureus (MRSA). The prev-
tative anaerobic round shaped bacterium with alence of PVL is low in methicillin susceptible
approximate diameter 1 μM. It is non-motile and S. aureus (MSSA), whereas PVL-genes are pres-
does not form spores. In the microscope it appears ent in most isolates of community acquired (CA-)
singly, in pairs, tetrads, and irregular grapelike MRSA. Of concern, PVL-toxin producing MRSA
clusters, hence it is binominal family name is associated with skin and soft tissue infection,
(of Greek σταφυλóκoκκoς; staphyle “grape-clus- including myositis and NSTI (Saeed et al. 2018;
ter” and kokkos “small berry”), and its Latin Thapaliya et al. 2015). Therefore, despite MRSA
species epithet aureus referring to the “golden” is an infrequent cause of NSTI in general, reports
appearance of colonies when cultured. When support attentiveness towards MRSA as micro-
grown on blood agar it displays β-hemolysis. bial etiology in certain geographical areas, as
S. aureus expresses catalase converting hydrogen documented in the USA and Korea (Miller et al.
peroxide (H2O2) to water and oxygen, detection 2005; Lee et al. 2007; Kim et al. 2019). S. aureus
of which has been a longstanding method used to has developed antibiotic resistance to all the rele-
distinguish it from enterococci and streptococci. vant antibiotic classes. The majority of S. aureus
Detection of coagulase production has been an strains produce penicillinase, for which reason
important, although imperfect method to differen- penicillinase-resistant beta-lactam antibiotics
tiate it from other staphylococcal species, as not belonging to the isoxazole penicillins are
preferred for treatment of MSSA. In MRSA 6 were detected in anogenital infections (Madsen
methicillin resistance is conferred by the mecA et al. 2019). Among the 13 polymicrobial cases,
gene, which codes for an altered penicillin- S. aureus was co-cultured with GAS in 8 (Madsen
binding protein 2A (pbp2A), leaving most beta- et al. 2019; Bruun et al. 2020), inferring that in
lactams inactive. only 5 cases (2%) other pathogens than GAS
The prevalence of S. aureus in clinical cohorts were detected. Given this, Staphylococcus
of NSTI is difficult to assess, as reporting on aureus-colonization and sampling error may be
microbiological data from relevant cohorts in confounders. This assumption is partly supported
general holds low precision. It is also difficult to by a study of 148 INFECT cases, using 16S
quantify the impact of S. aureus as well as its rRNA sequencing with transcriptional analysis
relative distribution between type 1 and type of host and microbe using dual RNA sequencing
2 infections. In the INFECT study it is of peroperatively collected, infected, deep tissue
categorized a secondary pathogen, as detailed samples (Thänert et al. 2019). In this study it was
above (Madsen et al. 2019). However, it is well demonstrated that S. aureus is not an associate of
described that it displays a tropism for skeletal complex polymicrobial communities causing
muscle and is an established cause of pyomyositis NSTI. Thus, the INFECT study adds novel data
(Hall et al. 1990). In a study on selected S. aureus supporting the view that there is still a need both
isolates obtained in the INFECT study neither to question as well as explore a putative role for
clonal relationship nor MRSA was detected, and PVL negative MSSA as an etiology of
there was only a single PVL positive case (Baude non-purulent NSTI.
et al. 2019). Nevertheless, when compared to a
different collection of blood stream infection
isolates, isolates from NSTI cases showed 5.4.4 Clostridium perfringens
increased myocyte internalization and cytopatho-
genic effect, suggesting alternative pathophysio- The genus Clostridium belongs to the phylum
logic mechanisms yet to be revealed. Firmicutes and includes more than 200 known
Notwithstanding its cytotoxicity in muscle tissue, species, of which most are not regularly
it was not until 2005 that S. aureus (i.e. MRSA) associated with human disease. Differently,
was shown to be etiology of monomicrobial NSTI C. perfringens, C. septicum, C. novyi, and
(Miller et al. 2005). C. sordellii are microbial etiologies of NSTI,
Through data obtained in the INFECT study a comprising around 10% of culture positive cases
more detailed picture of the prevalence and puta- in some patient cohorts (Anaya et al. 2005; Boyer
tive role of S. aureus is given. Firstly, in this et al. 2009; Kobayashi et al. 2011). The vegeta-
Scandinavian cohort it was an uncommon micro- tive cells of Clostridium spp. form pairs, short
bial etiology, found in 6% (26/409) of cases in the chains or appear in clusters, and they are
entire cohort. There were 13/179 (7%) rod-shaped, forming spores, reflected in the
monomicrobial S. aureus cases, of which name of the genus, derived from Greek κλωστήρ;
12 were cases of extremities; 7/12 of the upper klōstēr, “spindle,” derived from klōthein “to
and 5/12 of the lower. NSTI located to the upper spin.” The Latin species epithet of
extremities was associated with monomicrobial C. perfringens from Latin per “through” and
S. aureus infection (Fig. 5.1) with an OR of frango “burst” alludes to the microbe
5.80 (95% CI 1.61–21.60, p ¼ 0.0184) (Madsen disrespecting tissue borders in expanding gas
et al. 2019). In INFECT, monomicrobial growth gangrene, whereas the species epithet of
of S. aureus was associated with diabetes mellitus C. septicum means “putrefactive.” Clostridium
when compared to those with no diabetes with an spp. form endospores and have a strictly fermen-
OR of 5.4 (95% CI 1.57–18.7, p ¼ 0.01) (manu- tative type of metabolism. Most species are obli-
script, Rosén et al. 2020). There were 13/204 gate anaerobic, but C. perfringens is aerotolerant.
(6%) polymicrobial S. aureus cases, of which Whereas vegetative cells in obligate anaerobic
62 S. Skrede et al.
spp. are killed by exposure to O2, spores are able 5.4.5 Vibrio vulnificus
to survive long periods of exposure to air. Infor-
mative reviews on clostridial diseases are offered, Vibrio vulnificus of the family Vibrionaceae is a
e.g. by Onderdonk and Garrett (2010), or Stevens motile gram-negative halophilic, rod-shaped bac-
and Bryant (2020). terium with functional flagella. It is a marine
C. perfringens produces several extracellular organism present in the temperate areas of the
toxins (Stevens and Bryant 2002). Alpha toxin is world where it is found in coastal areas, including
a hemolytic toxin that stimulates platelet aggrega- estuaries and brackish ponds holding an optimal
tion and upregulates transcription of adherence temperature of 18–26 C (Huang et al. 2016). It is
molecules on endothelial and polymorphonuclear an environmental pathogen with three distinct
(PMN) cells. It causes intravascular aggregates of biotypes (Horseman and Surani 2011). It
leukocytes, activated platelets, and fibrin, leading produces neutralizing metabolites and radical
to impaired tissue perfusion, contributing to the scavengers to avoid destruction in the gastric
expanding borders of tissue destruction seen in acidic environment upon ingestion. Some
clostridial gas gangrene. The absence of PMNs in V. vulnificus strains express a polysaccharide cap-
tissue is caused by sequestration within the adja- sule that can protect the organism against phago-
cent vasculature. Theta toxin is another impor- cytosis. It produces endotoxin,
tant, pore-forming cytolysin that contributes to lipopolysaccharide (LPS), as well as a variety of
pathogenesis by its effects on cells of the vascular extracellular virulence factors such as the
and immune systems, including cytotoxicity exotoxins metalloprotease VvpE, the cytotoxic
towards PMN. hemolysin VvhA releasing iron thus enhancing
C. perfringens remains largely susceptible to bacterial growth, and the multifunctional
first-line antibiotics, including clindamycin and autoprocessing repeats-in-toxins (MARTX)
tetracyclines, offering protein synthesis inhibi- toxin, involved in, e.g. bacterial dissemination
tion, as well as penicillin, but resistance towards from the intestine by hematogenous spread. It
clindamycin and penicillin warrants attention. also spreads by contamination of preexisting
Nowadays clostridial gas gangrene is increas- wounds. It rarely affects healthy individuals and
ingly rare in developed countries, attributed, usually it causes only moderate disease (Horse-
e.g. to improved socioeconomic conditions and man and Surani 2011). However, in high-risk
sanitation (Sarani et al. 2009), whereas other clos- individuals, i.e. male gender, immunosuppres-
tridial species are identified in sporadic cases. In sion, viral hepatitis, hepatic cirrhosis, iron over-
the INFECT study blood cultures grew clostridial load states, or chronic wounds of the lower limbs
species in 11/409 (3%) of the cases (Madsen et al. (Chuang et al. 2019) the infection may spread
2019), demonstrating the relevance of the genus. rapidly, producing severe soft tissue destruction
It is known that Clostridium septicum associated resembling that seen in gas gangrene. Usually it is
with intestinal comorbidities is cause of infections affecting lower limbs, where a prominent local
with multiple foci, however, in this study a statis- feature of infection is blister formation and
tically significant association between positive bronze discoloration, as well as sepsis. Today it
culture for Clostridium spp. and NSTI of the is a frequent microbial etiology particularly
lower limb was found (Fig. 5.1), with an odds reported from the Mexican Gulf area and, partic-
ratio of 9.47 (95% CI 1.85–92.85, p ¼ 0.01). The ularly, South East Asia (Huang et al. 2016; Choi
increasing prevalence of colonic cancer as rele- et al. 2020). In a retrospective study from Taiwan
vant comorbidity may possibly affect future on 121 culture positive cases of NSTI, 75 cases
impact of clostridial species in NSTI and calls (62%) were blood culture positive, 57% in
for attention. survivors, and 74% in non-survivors, a difference
not reaching statistical significance (Chao et al. lactamase (MBL), of which VIM and IMP are
2013). In a Korean study on association between found, leaving it resistant to penicillin, ampicillin,
V. vulnificus DNA load in bacteremia and out- and carboxypenicillins. Regional resistance
come, the use of real-time PCR targeting the toxR patterns have been described. Clinical studies
gene in serum demonstrated a positive association have demonstrated differences in antimicrobial
between bacterial load and mortality, and a possi- susceptibility between species. Most strains are
ble role for RT-PCR for prognostic purposes was susceptible to trimethoprim-sulfamethoxazole
suggested (Kim et al. 2011). Although (TMP-SMX), second and third generation
V. vulnificus in general is susceptible to all rele- cephalosporins, aminoglycosides, carbapenems,
vant antibiotics, its expanded distribution area chloramphenicol, and tetracyclines, although
and detection of a carbapenemase in the closely resistance against TMP-SMX, cephalosporins,
related and co-residing species V. cholerae aminoglycoside and, expectedly,
warrants systematic monitoring of its drug sus- fluoroquinolones is described. In two indepen-
ceptibility (Bier et al. 2015). dent retrospective studies from Taiwan it was
In the INFECT study enrolling patients from the second most important gram-negative micro-
2013 to 2017, Vibrio vulnificus was cultured in bial etiology of NSTI, constituting 10% (Hsiao
deep tissue culture in 3 cases only (0.8%), of et al. 2008) and 23% of all cases, respectively
which one also grew in blood culture (Madsen (Lee et al. 2011), only surpassed by Vibrio
et al. 2019). Although still rare in Northern vulnificus. There were no Aeromonas cases
Europe, it is conceivable that its present global among the 409 cases included in the INFECT
range will expand due to climate change and that study (Madsen et al. 2019).
its prevalence even in areas such as Scandinavia
will increase.
5.4.7 Polymicrobial Etiologies
5.4.6 Aeromonas hydrophila Today most NSTIs with polymicrobial etiologies

are categorized as type 1 infections. In the origi-
Aeromonas hydrophila is primarily a fish and nal publication when such a categorization was
amphibian pathogen, but it is associated with first presented, some requirements were described
cellulitis and as an opportunistic microbial cause that need to be fulfilled to be a type 1 infection
of NSTI, with risk factors resembling those seen (Giuliano 1977); At least two microbes must be
in Vibro-infection (Tsai et al. 2015). Aeromonas cultured and identified, of which there must be
hydrophila is a heterotropic, non-spore-forming one obligate anaerobic microbe. The second
facultative anaerobic gram-negative, pleomorphic microbe in type 1 infections can include faculta-
rod-shaped bacterium with a monotrichous flagel- tive anaerobic species, such as staphylococcal
lum ensuring motility. As Vibrio, it thrives in species, a number of streptococcal species or
aquatic environments, including sewage, fresh Enterobacteriaceae. The authors questioned that
and brackish water, and soil. It can multiply at GAS is involved in genuine type 1 infections,
temperatures as low as 4 C. It produces several even in the presence of other pathogens, an opin-
factors believed to play roles in pathogenesis, ion supported by sequencing data obtained in
e.g. the cytotoxin aerolysin, lipase, and amylase. infected tissue samples of patients with NSTI
Also, production of hemolysins is evident on (Thänert et al. 2019). In this respect it should be
blood agar, where it produces β-hemolysis. Resis- noted that in a retrospective study on 75 GAS
tance in Aeromonas is reviewed elsewhere NSTI patients, additional pathogens were recov-
(Piotrowska and Popowska 2014). Aeromonas ered in 31% of cases (Lin et al. 2013). However,
species produce beta-lactamases of three different no description of standards of sampling was
classes, a class C cephalosporinase, a class D presented, and the published cases could possibly
penicillinase, and a class B metallo-beta- include late post-operative contaminants.
64 S. Skrede et al.
Different to GAS, SD appears to be a candi- et al. 2013). Severe deep space head and neck
date etiology for both type 1 and type 2 infections infections generally follow oral, dental, and pha-
(Madsen et al. 2019; Bruun et al. 2020). Catego- ryngeal infections, involving the bacteria of the
rization of type 1 infections is supported by infor- original focus of infection. Obligate anaerobic
mation on head-neck focus, anogenital or microbes that are recovered include Prevotella
abdominal focus or post-operative infection, as spp., Porphyromonas spp., Fusobacterium spp.,
well as by supporting information on elevated and Peptostreptococcus spp. However, they are
age, the many and frequent comorbidities, and frequently accompanied by facultative anaerobic
low functional status of patients (Stevens and species including viridans streptococci, and less
Bryant 2017). often also Staphylococcus aureus (Poeschl et al.
Most NSTIs are polymicrobial infections, 2010). Type 2 infection with GAS is also a possi-
reportedly comprising some 45–85% of all cases bility in the head and neck area (Brook 2012)
(Giuliano 1977; McHenry et al. 1995; Elliott et al. although uncommon (Madsen et al. 2019).
2000, 1996; Childers et al. 2002; Anaya et al. The majority of anogenital infections are
2005). It has been advocated that in NSTI specific polymicrobial, and the most frequently cultured
combinations of bacterial species is neither organisms are obligate anaerobic species,
observed nor diagnostic (Anaya and Dellinger Enterobacteriaceae and Enterococcus spp.
2007), and it has even been concluded that no (Bjurlin et al. 2013; Czymek et al. 2013). In the
specific combination of microbial species in INFECT study polymicrobial etiology confirmed
NSTI exists and that polymicrobial infections by routine diagnostic culture methods of the
are to be considered unpredictable regarding laboratories of study hospitals was strongly
composition (Rudkjøbing et al. 2016). Opposite, associated with infection of the anogenital and
in the study using dual RNA sequencing of abdominal region (Fig. 5.1), with an OR of 8.01
intraoperatively collected, infected, deep tissue (95% CI 4.95–13.15, p < 0.001) (Madsen et al.
samples from the INFECT study it was 2019). Expectedly, in the anogenital region
demonstrated strict correlations of co-occurrence microbes with troublesome antimicrobial resis-
between species and concluded firmly that tance are identified, e.g. MRSA, ESBL-producing
polymicrobial communities in NSTIs are not ran- E. coli and Klebsiella spp., as well as Pseudomo-
domly associated. It was demonstrated that spe- nas sp. (Chia and Crum-Cianflone 2018), but also
cies form highly interconnected networks. antimicrobial resistance in the Bacteroides
Relationships were demonstrated, e.g. oral anaer- fragilis group may be a concern (Nagy et al.
obic species such as Fusobacterium, 2011).
Porphyromonas, Parvimonas, and Peptostrep-
tococcus co-reside, but not with Bacteroides. Fur-
thermore, when E. coli is associated with an 5.4.8 Microbial Etiologies
obligate anaerobic species in a polymicrobial Documented by
NSTI, this will be a member of the Bacteroides the INFECT Study
family. Specific and sophisticated metabolic and
signaling interactions in these communities were As detailed in this chapter on microbiology,
demonstrated (Thänert et al. 2019), by ways numerous cases series and surveillance studies
reviewed elsewhere (Stacy et al. 2016). Based of NSTI have been conducted. Notwithstanding,
on clinical studies, microbiological diagnostic the documentation of microbial etiologies has
results and mechanistic studies of polymicrobial often been hampered with low quality of data. In
infections, it may be that the role of obligate the INFECT study strict standard operating
anaerobic bacteria is underestimated (Brook and procedures for obtaining microbiological samples
Frazier 1995; Bjurlin et al. 2013). were developed, that were adhered to in 407/409
In deep neck space infections as well as in cases (99.5%). Microbial etiologies were
anogenital and abdominal NSTIs, anaerobic established by culture in 94% of the cases through
microbes predominate (Brook 2012; Bjurlin routine diagnostic culture methods of the
laboratories of study hospitals , while in 26 cases and they can misrepresent relative abundance of
(6%), no microbe was identified (Madsen et al. species. Furthermore, culture dependent methods
2019). In 144 (35%) of the cases there was a are time consuming. Some of these problems can
positive blood culture and in 378 (92%) there be dealt with using culture-independent methods.
was a culture positive sample from a sterile tissue Recently, use of a multiplex PCR with primers
site. Associations of anatomical NSTI locations, allowing simultaneous detection of GAS, GBS,
microbial etiologies, and thereby NSTI categories MRSA, Vibrio vulnificus, and Aeromonas
were documented with rigor. Figure 5.2 offers a hydrophila was reportedly successful, and the
visualization of the detailed microbiological authors concluded that PCR of tissue specimens
results that were obtained. Polymicrobial may be a useful and rapid diagnostic method in
infections were found in 204 (50%) patients, NSTI (Tsai et al. 2019). Implementation of next-
whereas 179 (44%) had a monomicrobial infec- generation molecular sequencing in routine
tion, in which GAS was found in 126 (31%). diagnostics in NSTIs could give a more complete
Most polymicrobial infections were located to picture of the etiology, but few such studies have
the pelvic region (118/204, 58%) or confined to been undertaken. In a novel study using next-
the head-neck area (43/204, 21%) (Madsen et al. generation 16S rRNA gene sequencing
2019). Anaerobes were frequently identified in (NGS16S) to determine how often cultivable
polymicrobial infections. Furthermore, in the organisms in complex polymicrobial samples
anogenital tissue samples analyzed by 16S are not reported by standard culture,
rRNA sequencing, a strong association was 20 bronchoalveolar lavage samples were cultured
detected between E. coli and Bacteroides sp., and identified by NGS16S analysis of DNA
almost entirely (i.e. 80%) comprised by extracted directly from samples or from washed
B. fragilis. Differently, in head-neck infections culture plates. Only 21% of identities were
there were strong associations between oral reported by standard culture. However, 96% of
anaerobic pathogens, in particular among species microbes were cultivable. This study also
belonging to five genus; Prevotella spp., demonstrated that by using direct NGS16S the
Porphyromonas spp., Parvimonas spp., same predominant organism was detected in
Peptostreptococcus spp., and Fusobacterium 50% of samples when compared to identification
spp. (Thänert et al. 2019). The results implicate by culture. When predominant organisms dif-
that in anogenital infections there is a higher risk fered, NGS16S most often detected anaerobes
of both clindamycin resistant Bacteroides sp. and (Cummings et al. 2020). Zhao-Fleming et al.
extended spectrum beta-lactamase producing (2019) conducted a study to identify microbial
Enterobacteriaceae when compared and etiologies in human NSTIs, challenging previous
contrasted to NSTI of other anatomical areas, culture based data. Using 16S rRNA sequencing
including head-neck space infections. Finally, in they identified genera commonly found in NSTIs
the INFECT study a substantial diversity in that included Prevotella, Bacteroides,
microbial etiology was documented. Altogether Peptoniphilus, Porphyromonas, and Enterococ-
more than 50 different bacterial species were cus. Associations of bacterial constituents and
found to be implicated in NSTIs (Thänert et al. patient outcomes were detected, and data were
2019). generated to support a recommendation of
increasing anaerobe antibiotic coverage during
the treatment of NSTIs (Zhao-Fleming et al.
5.4.9 Non-culture Based 2019).
Microbiological Methods As part of the INFECT project tissue biopsies
in NSTI obtained preoperatively from 148 patients with
NSTI were analyzed to explore pathophysiologi-
Arguably, standard microbiological culture may cal mechanisms of monobacillary NSTIs compar-
fail to identify unusual or fastidious organisms ing and contrasting polymicrobial infections
66
Fig. 5.2 Most monomicrobial infections are caused by GAS and associated with infections of the extremities. Most polymicrobial infections are associated with the oral or the
anogenital orifices, anaerobes are frequently detected, whereas GAS is infrequently retrieved in these infections. Reprinted from Madsen et al. (2019), with permission
S. Skrede et al.
(Thänert et al. 2019). Microbial community 5.5 Future Perspectives

profiling using 16S rRNA sequencing was
integrated with transcriptional analysis of host NSTI are severe infections frequently leading to
and microbe, using dual RNA sequencing septic shock and multiorgan failure (Madsen et al.
(RNA-seq). It was shown that NSTIs were either 2019). Treatment is highly specialized, multidis-
dominated by a single bacterial pathogen, usually ciplinary and very resource demanding, usually
GAS, or associated with a diverse spectrum of taking place in an intensive care unit. Patients are
polymicrobial communities. In polymicrobial at risk of poor outcomes, and NSTI mortality rates
infections Prevotella spp., Porphyromonas spp., in the range of 20–30% and extremity amputation
Parvimonas spp., Fusobacterium spp., rates of 10–20% are reported (Peetermans et al.,
Peptostreptococcus spp., and Bacteroides 2020).
sp. were frequently detected. The diversity and There is a need for improvement of these
composition of the bacterial communities were results. Of several reasons, strengthening the
highly dependent on the focus of the infection, microbiological diagnostic handling of patients
supporting culture based data for the entire with NSTI is of great importance to improve
INFECT patient cohort (Madsen et al. 2019, patient outcomes. Firstly, in many severe
Fig. 5.2). Using 16S rRNA sequencing, Thänert infections there is an association between chances
and coworkers also showed that the bacterial of survival and the identification of
diversity in NSTIs of the upper and lower microbiological etiology and its antimicrobial
extremities was significantly lower than in resistance properties. Most NSTIs are
NSTIs localized at the head/neck or anogenital polymicrobial. In these etiologies there are
region (Thänert et al. 2019). The highest fre- emerging risks of antimicrobial resistance,
quency of polymicrobial infections was seen in including clindamycin resistant obligate anaero-
anogenital infections, where Bacteroides was pre- bic species and β-hemolytic streptococci, ESBL
dominant. In head and neck infections oral genera producing Enterobacteriaceae or, infrequently,
Prevotella, Porphyromonas, and Fusobacterium MRSA. Identification will aid in selection of effi-
dominated, lending support to the hypothesis that cient antimicrobial treatment.
there is a connection between bacterial commu- Secondly, medical microbiological studies can
nity composition in NSTIs and commensals spur future invention of tools to support treatment
associated with natural ecological niches. It was decisions (Rello et al., 2018). NSTIs are highly
also demonstrated that in polymicrobial NSTI heterogeneous infections reflected by the multi-
bacterial communities are not randomly tude of etiologies reviewed herein. Any future
associated (Thänert et al. 2019). These development of individualized therapeutic
communities appear attractive targets for devel- strategies will rely on identification of microbio-
opment of novel therapies. logic etiology. Only then patient stratification
Use of nucleic acid based identification necessary to develop therapies targeting the host
methods has proven important in studies on path- and microbe interplay can find place.
ogenesis of both polymicrobial and It is a challenge to conduct studies on NSTIs.
monomicrobial infections, and studies on GAS Most clinical studies in this field have been focus-
NSTI pathogenesis in primates (Kachroo et al. ing on patient characteristics, whereas studies
2020) and mice (Hirose et al. 2019) using RNA arguably have focused insufficiently on the
sequencing have recently been published. pathogens and their interaction with the host. In
Chapter 10 in this book covers this subject. At NSTIs, the systems medicine approach is in an
present there is no role for use of nucleic acid early phase, but studies using translation
based methods in rapid identification of microbial approaches in well-defined patient groups are
etiologies of NSTIs in the emergency setting, but needed. In this respect, identification and studies
advances are made that makes such a goal within on the NSTI pathogens are crucial, underlining
reach.
68 S. Skrede et al.
the importance of microbiology both in every day Skrede S (2013) Necrotizing soft tissue infections
patient handling as well as in clinical studies of caused by Streptococcus pyogenes and Streptococcus
dysgalactiae subsp. equisimilis of groups C and G in
NSTI, in order ultimately to create individualized western Norway. Clin Microbiol Infect 19:E545–E550
therapies. Bruun T, Rath E, Madsen MB, Oppegaard O,
Acknowledgment This work was supported by the Bergey F, Itzek A, Hyldegaard O, Norrby-Teglund A,
European Union Seventh Framework Programme: Skrede S, Infect Study Group (2020) Risk factors and
(FP7/2007-2013) under the grant agreement 305340 predictors of mortality in streptococcal necrotizing
(INFECT project), NordForsk (Project no. 90456, soft-tissue infections: a Multicenter prospective study.
PerAID), the Norwegian Research Council under the Clin Infect Dis. https://doi.org/10.1093/cid/ciaa027
frame of ERA PerMed (Project 2018-151, PerMIT) and Calandra T, Cohen J, International Sepsis Forum Defini-
The Swedish Research Council Framework Grant in tion of Infection in the ICU Consensus Conference
infections and antibiotics (2014-8609-117204-47). (2005) The international sepsis forum consensus con-
ference on definitions of infection in the intensive care
unit. Crit Care Med 33:1538–1548
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and soft-tissue infections. Clin Infect Dis Dissanaike S, Rumbaugh K (2019) Obligate anaerobes
41:1373-1406. Erratum in: Clin Infect Dis are abundant in human necrotizing soft tissue infection
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toxins in the pathogenesis of gas gangrene. Clin Infect Zeppa JJ, Kasper KJ, Mohorovic I, Mazzuca DM,
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Beta-Hemolytic Streptococci
and Necrotizing Soft Tissue Infections 6
Trond Bruun, Eivind Rath, Oddvar Oppegaard,
and Steinar Skrede
Contents
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
6.2 Epidemiology and Risk Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
6.2.1 Incidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
6.2.2 Risk Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
6.2.3 Molecular Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
6.3 Clinical Features and Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
6.4 Treatment and Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
6.4.1 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
6.4.2 Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
6.5 Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Abstract cases were caused by GAS (31%) or SD (7%).

Risk factors of streptococcal NSTIs compared
β-hemolytic streptococci are major causes of
to streptococcal cellulitis have previously been
necrotizing soft tissue infections (NSTIs),
largely unknown. The INFECT study con-
Streptococcus pyogenes (group A streptococ-
firmed blunt trauma as an important risk factor.
cus; GAS) in particular. NSTIs caused by
In addition, absence of pre-existing skin
Streptococcus dysgalactiae (SD) have also
lesions and a lower BMI were associated
been reported. In the INFECT cohort of
with NSTIs. The study also confirmed that
409 NSTIs patients, more than a third of the
septic shock is more frequent in GAS cases
than in other types of NSTIs. Septic shock was
T. Bruun (*) · S. Skrede also among several predictors of mortality.
Department of Clinical Science, University of Bergen,
The role of intravenous immunoglobulin
Bergen, Norway
(IVIG) in streptococcal NSTIs has been
unclear. In the INFECT cohort, IVIG treat-
Bergen, Norway
e-mail: trond.bruun@helse-bergen.no ment was associated with increased survival.
As in other studies, a significant microbial
E. Rath · O. Oppegaard
Department of Medicine, Haukeland University Hospital, diversity was observed, but with predomi-
Bergen, Norway nance of a few emm types. Overall, the
74 T. Bruun et al.
INFECT study gives a comprehensive and was observed (Stevens et al. 1989; Hoge et al.
contemporary picture of the clinical 1993; Martin and Hoiby 1990). Among the most
characteristics and the microbes involved in severe manifestations of invasive GAS infection
streptococcal NSTIs. The reported severity of encountered were necrotizing fasciitis (NF) and
disease underscores the need for new efforts other necrotizing soft tissue infections (NSTIs),
aimed at identifying novel diagnostic including rare cases of necrotizing myositis. Case
measures and improved treatment. series and surveillance studies at that time
described high rates of septic shock, need for
Keywords intensive care, and death (Stevens et al. 1989;
Chelsom et al. 1994; Kaul et al. 1997; Sharkawy
NSTIs · Necrotizing fasciitis · Streptococcus
et al. 2002; Hoge et al. 1993). Notably, a signifi-
pyogenes · Group A streptococcus ·
cant proportion of the cases were previously
Streptococcus dysgalactiae
healthy individuals. Later, surveillance studies on
invasive GAS disease have confirmed such
Highlights
characteristics (Lamagni et al. 2008a; Nelson
• The INFECT cohort of 409 cases with NSTIs
et al. 2016).
confirmed that Streptococcus pyogenes (GAS)
Even other streptococci have the ability to
and Streptococcus dysgalactiae (SD) are
cause NSTIs. Beta-hemolytic group C and G
major agents in NSTIs.
streptococci of human origin mainly belong to
• When comparing to non-necrotizing strepto-
the species Streptococcus dysgalactiae (SD) of
coccal cellulitis, streptococcal NSTIs were
which almost all belong to the subspecies Strep-
associated with blunt trauma, absence of
tococcus dysgalactiae subsp. equisimilis (SDSE).
pre-existing skin lesions, and a lower BMI.
The term SD is herein used instead of group C or
• Septic shock was significantly more frequent
G streptococci or SDSE. SD is phylogenetically
in GAS compared to SD and polymicrobial
closely related to GAS. Several recent studies
NSTIs.
suggest that invasive infections with this species
• Among the GAS cases, several factors were
are increasing (Oppegaard et al. 2015; Schwartz
associated with mortality, including age, septic
et al. 2014; Couture-Cossette et al. 2018; Fujiya
shock, and no administration of intravenous
et al. 2019). Reports even describe the ability of
immunoglobulin.
SD to cause NSTIs (Ekelund et al. 2005a; Bruun
et al. 2013). NSTIs caused by group B streptococ-
cus (Streptococcus agalactiae; GBS), a more dis-
6.1 Introduction
tant relative of GAS, have also been reported, but
most such cases have been polymicrobial
Streptococcus pyogenes (group A streptococcus;
(Kulasegaran et al. 2015; Cheng et al. 2012;
GAS) is perhaps the human pathogen causing the
Chen et al. 2011).
most diverse spectrum of infections, ranging from
Apart from two retrospective single-center
mild impetigo or pharyngitis to immunological
studies, contemporary detailed data on strepto-
sequelae and life-threatening streptococcal toxic
coccal NSTIs have been lacking (Bruun et al.
shock syndrome (Bryant and Stevens 2014).
2013; Lin et al. 2013). The INFECT cohort, how-
Globally, it is estimated that more than half a
ever, includes the largest clinical cohort of strep-
million people die each year due to diseases
tococcal NSTIs cases to date (Madsen et al. 2019;
caused by GAS (Sims Sanyahumbi et al. 2016).
Bruun et al. 2020). Among 409 NSTIs patients,
Developing countries suffer the great majority of
there were 126 with GAS and 27 with SD, in total
severe disease and deaths caused by GAS, mainly
37% of the cohort. In addition, one case with
due to rheumatic heart disease. However, also the
S. equi and 15 cases with S. agalactiae (Group
industrialized world is affected, and in the 1980s
B streptococcus; GBS) were identified. Ninety
and 1990s an increase of invasive GAS infections
percent of the GAS cases were monomicrobial,
6 Beta-Hemolytic Streptococci and Necrotizing Soft Tissue Infections 75
as opposed to 48% of the SD cases and just 2 out O’Grady et al. 2007; Lamagni et al. 2008a). As
of 15 GBS cases. Staphylococcus aureus was for invasive GAS disease in general, great tempo-
found together with GAS in 6% of the cases. ral fluctuations have been observed. Striking local
In this chapter we give an overview of clinical peaks in incidence have been described
aspects of NSTIs caused by GAS or SD in light of (Cartwright et al. 1995; Barnham et al. 1997;
the INFECT study. This cohort provides a unique Erdem et al. 2005). In Norway, the mean inci-
set of clinical data describing what characterizes dence of GAS NF registered by national surveil-
these infections. The pathogenesis of streptococ- lance for the period 2010–2014 was 0.3/100,000/
cal NSTIs is explored in Chap. 9. year (Naseer et al. 2016). In contrast, a study from
western Norway found an incidence of 1.2 per
100,000 (Bruun et al. 2013). This great difference
6.2 Epidemiology and Risk Factors may illustrate different methods of inclusion and
possible underreporting in microbiological sur-
6.2.1 Incidence veillance studies. Internationally, significantly
higher rates of GAS NSTIs and other invasive
Estimates of incidence for streptococcal NSTIs GAS disease have been observed in disadvanta-
can be derived from studies of NSTIs in general geous populations (Le Hello et al. 2010; Rudolph
or from surveillance studies of invasive strepto- et al. 2016).
coccal infection. Either way, the lack of a uniform Several cases of NSTIs attributed to group C
definition of NSTIs is a major reason for the great and G streptococci, presumably mostly SD, have
uncertainty attached to estimates of incidence. A been published since a report in Lancet in 1984
few nation-wide studies have been performed (Gaunt et al. 1984), but the disease is rare com-
showing highly variable incidence numbers of pared to GAS NSTIs. Large single- and multicen-
NSTIs overall ranging from below 1 to above ter studies of NSTIs indicate that GAS is found
10 per 100,000 population (Mulla et al. 2007; around 5–20 times more often than SD
Das et al. 2011; Oud and Watkins 2015; (Kulasegaran et al. 2015; Bair et al. 2009;
Audureau et al. 2017; Chuang et al. 2018; Psoinos Huang et al. 2011; Cheng et al. 2012; Bruun
et al. 2013), but different methods were used. et al. 2013; Chen et al. 2011). A similar ratio
With respect to studies of NSTIs reporting between NSTIs caused by GAS and SD is
microbiological data, most are retrospective suggested by population-based surveillance stud-
single-center case series. The proportion of GAS ies of invasive streptococcal disease (Ekelund
infections has varied greatly, as described in et al. 2005a; Rantala et al. 2009a). Sometimes,
Chap. 5, ranging from below 10% to 50% or SD is found as part of a polymicrobial etiology
more. From the Scandinavian countries, three (Kulasegaran et al. 2015; Cheng et al. 2012; Chen
smaller single-center studies have reported beta- et al. 2011). GBS is found just as infrequent as SD
hemolytic streptococci in 29–52% of the cultured in NSTIs, occasionally as a single pathogen but
cases, predominantly GAS (Skovsen et al. 2010; often together with other bacteria (Kulasegaran
Brink et al. 2014; Nordqvist et al. 2015). et al. 2015, Cheng et al. 2012, Chen et al. 2011).
Data on NSTIs caused by GAS is also avail- The INFECT study was not designed to give
able from surveillance studies of invasive GAS exact epidemiological data. All study centers are
and SD disease. Estimated incidence rates of referral hospitals for NSTIs that offer hyperbaric
GAS NF in large surveillance studies from oxygen therapy, and most enrolled patients were
industrialized countries performed since 1992 transferred from other hospitals. Some categories
have been in the range 0.08–0.60/100,000/year are probably often not referred. These include
(Kaul et al. 1997; O’Brien et al. 2002; Sharkawy those without a fair chance of survival as well as
et al. 2002; Tyrrell et al. 2005a, 2018; Ekelund low risk patients. In Denmark, however, treat-
et al. 2005a, b; Hollm-Delgado et al. 2005; ment of NSTIs is more centralized than in
O’Loughlin et al. 2007; Darenberg et al. 2007; Norway and Sweden, and most hospitals send
76 T. Bruun et al.
their NSTIs cases to Rigshospitalet, i.e. the study cellulitis, streptococcal NSTIs of the INFECT
hospital. During 51 months of inclusion, 65 cases cohort were associated with a history of blunt
with NSTIs caused by GAS were included at trauma, absence of chronic wound and skin dis-
Rigshospitalet. In a population of 5.6 million ease, and a lower BMI (Bruun et al. 2020). The
this corresponds to an incidence of 0.3 cases per study confirms previous reports suggesting that
100,000 per year. Because not all cases are trans- blunt trauma may be of importance
ferred, the true incidence must be somewhat (Monnickendam et al. 1997; Factor et al. 2003;
higher. This estimate is comparable to other Nuwayhid et al. 2007) and that streptococcal
data, including surveillance data from Norway NSTIs in most cases do not have an obvious
(www.msis.no). In the INFECT study overall, portal of entry (Kaul et al. 1997; Simonart et al.
NSTIs caused by SD represented 7% of the 2001). More than half of the GAS and SD cases of
cases, corresponding to an incidence of 0.07 per the INFECT cohort had no preceding event. Stud-
100,000 per year, suggesting that the contribution ies in children, however, have found that varicella
of SD might be greater than previously reported. is an important risk factor (Laupland et al. 2000;
This may be related to the increase of invasive SD Tyrrell et al. 2005b; Minodier et al. 2009; Imohl
infection in general (Oppegaard et al. 2015). et al. 2011). Not many studies have directly com-
pared risk factors of GAS NSTIs with NSTIs
caused by other etiologies. However, two studies
6.2.2 Risk Factors suggest that diabetes mellitus and immunosup-
pression are more common in NSTIs without
In an early study during the upsurge of invasive GAS (Bair et al. 2009; Cheng et al. 2012). A
GAS infections in the industrialized world, recent study confirmed that GAS etiology is
Chelsom and co-workers found that seven of unlikely in NSTIs of immunosuppressed
13 cases with GAS NSTIs were previously individuals (Urbina et al. 2019). Moreover, stud-
healthy (Chelsom et al. 1994). The ability of ies of NSTIs of all etiologies in general report a
GAS to cause NSTIs in otherwise healthy people higher frequency of comorbidity compared to
has later been confirmed by other studies (Kaul studies of GAS NSTIs. Also in the INFECT
et al. 1997; Hassell et al. 2004). In the INFECT cohort, comorbidities were more common
study, more than a third of the GAS cases was among non-streptococcal than streptococcal
without known comorbidity. However, several cases (Bruun et al. 2020), and also more common
studies of invasive GAS disease in general as in SD compared to GAS cases, malignancy in
well as of GAS NSTIs have identified various particular (Table 6.1). Overall the INFECT
risk factors. A study using non-invasive skin study found that possible predisposing factors
and soft tissue infections as a control group were frequent, particularly in NSTIs caused by
identified high age, diabetes mellitus, liver cirrho- SD, but the ability of GAS to cause NSTIs in
sis, and gout as independent risk factors for previously healthy individuals was confirmed.
NSTIs (Lin et al. 2013). In another study, com-
paring GAS NSTIs with other invasive skin and
soft tissue infections caused by GAS, alcoholism 6.2.3 Molecular Epidemiology
and diabetes were more frequently identified in
the NSTIs group, but absence of cancer was the Serological M protein typing and later genetic
only predictor of NSTIs in the adjusted analysis typing based on the emm gene and different
(Sharkawy et al. 2002). Therefore, it has been house-keeping genes (MLST typing) have been
unclear what risk factors that are associated the main tools to follow the epidemiology and
with NSTIs compared to other invasive skin and microbial diversity of invasive and non-invasive
soft tissue infections. However, previous studies GAS and SD strains. More recently, comparative
were only powered to detect large differences. analysis of full genome sequences (WGS) has
When compared to non-necrotizing streptococcal emerged as the most powerful tool to provide
Table 6.1 Clinical and biochemical findings in NSTIs caused by GAS or SD

GAS (N ¼ 126) SD (N ¼ 27) P value
Site of infection
Head/neck including intrathoracic space 14/126 (11) 3/27 (11) 1.000
Upper extremities including thoracic 50/126 (40) 2/27 (7) 0.001
involvement
Abdomen and anogenital area 9/126 (7) 5/27 (19) 0.075
Lower extremities 53/126 (42) 17/27 (63) 0.048
Preoperative symptoms/signs
Pain treated with opioids 55/122 (45) 10/24 (42) 0.758
Skin bullae 47/124 (38) 6/26 (23) 0.150
Purple/black skin discoloration 47/125 (38) 12/26 (46) 0.416
Skin bruising 78/124 (63) 15/25 (60) 0.785
Skin anesthesia 6/94 (6) 1/23 (4) 1.000
Preoperative biochemistrya
CRP (mg/L)b 295 (193–370) 261 (148–339) 0.131
Leukocytes (109/L)c 14.2 (8.2–22.2) 16.9 (11.2–23.7) 0.393
Creatinine (μM)d 178 (109–266) 103 (73–181) 0.001
Other biochemistry
Thrombocytes (109/L)e 143 (89–202) 198 (100–284) 0.081
Bilirubin (μM)f 19 (11–36) 17 (10–28) 0.496
Severity
Bacteremia 69/126 (56) 8/27 (30) 0.014
Septic shockg 82/126 (65) 11/27 (41) 0.019
SAPS II (0–163)h 44 (34–57) 44 (36–58) 0.758
SOFA score day 1 (0–20)i 9 (7–12) 8 (6–11) 0.129
LRINEC score 6j 90/104 (87) 19/24 (79) 0.352
Surgical findingsk
Subcutaneous tissue dissolved or deliquescent 103/126 (82) 23/27 (85) 0.787
Subcutaneous greyish discolouration 96/126 (76) 19/27 (70) 0.525
Dishwater pusl 81/126 (64) 14/27 (52) 0.227
Muscle affected 83/126 (66) 14/27 (52) 0.170
Finger test positivem 63/126 (50) 11/27 (41) 0.382
Maximum skin defect (%)n 5 (2–12) 4 (1–8) 0.232
Adapted from Bruun et al. (2020), with addition of site of infection and surgical findings. Data are presented as median
(inter-quartile range) or No./No. evaluated (%). Boldface indicates statistical significance (P < 0.05)
NSTIs necrotizing soft tissue infection, GAS group A streptococcus/S. pyogenes, SD Streptococcus dysgalactiae, SAPS
simplified acute physiology score, SOFA sequential organ failure assessment, LRINEC Laboratory risk indicator for
necrotizing fasciitis
a
Highest values observed
b
Data missing for five patients
c
Data missing for seven patients
d
Data missing for nine patients
e
Lowest value day 1 in the intensive care unit/high-dependency unit. Data were missing for six patients
f
Highest value day 1 in the intensive care unit/high-dependency unit. Data were missing for 12 patients
g
Defined by use of vasopressor or inotropic agents and lactate >2 mmol/L
h
SAPS II is calculated the first 24 h in the intensive care unit/high-dependency unit from 17 variables; scores range from
0 to 163, with higher scores indicating more severe disease. Data were missing for 16 patients
i
The SOFA score includes subscores ranging from 0 to 4 for each of five components (circulation, lungs, liver, kidneys,
and coagulation). Aggregated scores range from 0 to 20, with higher scores indicating more severe organ failure. The
scoring was modified because cerebral failure was not assessed. Data were missing for three patients
j
Data were missing for 25 patients
k
At primary hospital or during first 7 days of stay at intensive care unit or high-dependency unit at specialized hospital
l
‘Dishwater’ colored fluid
m
Tissues dissect with minimal resistance when sweeping a finger rapidly at the level of the fascia
n
Data was missing for one patient. Estimated by the rule of nines
78 T. Bruun et al.
35
Number with emm type Septicshock Dead
30
25
20
15
10
GAS SD
Fig. 6.1 emm type distribution with rates of septic shock Abbreviations: GAS group A Streptococcus, Streptococ-
and 90-day mortality in 82 GAS and 11 SD cases of cus pyogenes, SD Streptococcus dysgalactiae. Reprinted
necrotizing soft-tissue infection. Forty-four GAS isolates from Bruun et al. (2020), with permission
and 16 SD isolates were not available for analysis.
insight into phylogenetic relationships and diver- emm types have a greater ability to cause invasive
sity of strains as well as evolution and infections (Nelson et al. 2016; Luca-Harari et al.
characteristics of epidemic strains (McMillan 2009). Even in SD there was an association to
et al. 2013). Certain emm types have been strains previously associated with severe
associated with invasive infection and disease infections, with a predominance of the emm type
severity. In Europe and North America emm1, stG62747 (Oppegaard et al. 2017). The INFECT
emm28, emm89, emm3, emm12, emm4, and study could not identify any clear relation
emm6 have predominated among invasive GAS between emm type and septic shock or mortality,
strains since year 2000 (Gherardi et al. 2018). The probably reflecting the magnitude of microbial
upsurge of invasive GAS disease since the 1980s and host-related factors contributing to severity
has been associated with certain GAS strains with and death.
increased fitness, a hypervirulent emm1 clone in
particular (Barnett et al. 2018). In the INFECT
study, emm typing, MLST typing as well as WGS 6.3 Clinical Features and Diagnosis
have been performed and demonstrate great
diversity regarding involved strains (Bruun et al. The clinical features of NSTIs are primarily
2020). As in most surveillance studies of invasive known from studies of NSTIs in general (see
GAS infections, however, emm1, emm3, and Chap. 4), but a few studies reporting clinical
emm28 were predominant emm types in the details on GAS NSTIs have been performed.
INFECT cohort (Fig. 6.1). The fact that more Clinical data on SD, however, have been avail-
than 70% of the isolates belonged to only three able only from case reports and a retrospective
emm types are in line with evidence that some study of streptococcal NSTIs that included nine
cases caused by SD (Bruun et al. 2013). The latter contrast to studies of invasive GAS where STSS
study found that these patients were older, had a was associated with younger age (Hoge et al.
higher burden of comorbidities and had 1993; Nelson et al. 2016; Lamagni et al. 2008b).
anatomically more superficial disease than the In both GAS and SD patients, more than 70%
GAS patients. Previous studies have reported of the infections were located in the extremities
that GAS NSTIs are located in the lower (Table 6.1). Upper extremity infections were typ-
extremities in more than half the cases (Kaul ically caused by GAS and not SD. Bacteremia
et al. 1997; Bruun et al. 2013; Lin et al. 2013). and septic shock were more frequent in GAS
One of the hallmarks of GAS NSTIs is severe cases compared to cases of SD and polymicrobial
systemic toxicity. In studies comparing GAS non-streptococcal NSTIs. This is in accordance
NSTIs with other invasive skin and soft tissue with major differences in the pathogenesis of
infections caused by GAS, hypotension has sig- streptococcal compared to polymicrobial
nificantly more often been observed in the NSTIs infections, as demonstrated in a molecular study
group (Lamagni et al. 2008b; Kittang et al. 2010; of cases from the INFECT cohort (Thanert et al.
Sharkawy et al. 2002). The frequency of strepto- 2019). Systemic severity according to presence
coccal toxic shock syndrome (STSS) in GAS of septic shock, SOFA score, and laboratory
NSTIs is reported to be in the range 40–68%, far abnormalities was not more pronounced in
greater than in other invasive GAS disease monomicrobial GAS disease compared to cases
(Darenberg et al. 2007; Lamagni et al. 2008b; involving co-pathogens, suggesting that GAS is
Svensson et al. 2000; Kaul et al. 1997; Lin et al. the dominant pathogen also in the latter type of
2013; Davies et al. 1996; Bruun et al. 2013). In infection. Severe pain was prevalent in both GAS
most studies of NSTIs in general, the overall and SD cases, but over 50% of the patients did not
frequency of septic shock is lower (Kao et al. receive opioid drugs before their first surgery. At
2011; Huang et al. 2011; Das et al. 2012; Keung surgery, the finger test, a pathognomonic sign of
et al. 2013). Accordingly, severe systemic toxic- tissue necrosis, was not always positive. Muscle
ity is thought to be more typical of type 2 NSTIs affection was registered in about half of the
than of type 1 disease. In a comparative study, patients, including 13 cases consistent with myo-
however, shock was not more frequent among the sitis only, i.e. without signs of destruction of
GAS NSTIs cases (Urbina et al. 2019). In addi- subcutaneous tissue. CRP was in most patients
tion, some non-streptococcal types of NSTIs are highly elevated, but 28% had preoperative values
associated with an increased risk of shock, includ- below 200 mg/L. A clearly elevated creatinine of
ing monomicrobial infections with Vibrio 150 μM preoperatively was found in 56% of
vulnificus or Klebsiella pneumoniae (Cheng patients. Laboratory Risk Indicator for Necrotiz-
et al. 2012; Klontz et al. 1988). Detailed data on ing Fasciitis (LRINEC) score, a laboratory based
local symptoms and signs in streptococcal NSTIs diagnostic tool for necrotizing fasciitis, is calcu-
are scarce except for case reports which probably lated based on preoperative measurements. A
tend to present the most severe cases. Some data score below 6, previously associated with low
are available from larger retrospective series that risk of NSTIs (Wong et al. 2004), was found in
report unspecific skin changes and no obvious 15% of the GAS and SD cases overall,
portal of entry in most of the cases (Sharkawy demonstrating suboptimal sensitivity among the
et al. 2002; Simonart et al. 2001). As for NSTIs in streptococcal cases. This was the conclusion also
general, bullae are found in a minority (Lin et al. in a recent systematic review (Fernando et al.
2013). The frequency of other classical NF signs 2018). Clinical features and surgical findings are
in GAS disease is uncertain. still the cornerstones in the diagnostic work
In the INFECT study, septic shock was more up. Clinical characteristics may also be of great
common in GAS disease compared to use in making an etiological diagnosis. As
polymicrobial non-streptococcal cases (Bruun demonstrated in a recent retrospective study,
et al. 2020). Furthermore, septic shock was where abdominal location and immunodeficiency
related to higher age rather than younger, in were strongly predictive of non-streptococcal
80 T. Bruun et al.
etiology (Urbina et al. 2019), the INFECT study probable bacteriological diagnosis is established.
has shown major clinical differences separating Local frequency of antibiotic resistance should be
streptococcal and polymicrobial NSTIs. Both the taken into consideration. In streptococcal NSTIs
strong association of streptococcal NSTIs and or STSS, clindamycin is recommended in addi-
extremity location (Madsen et al. 2019) and tion to penicillin (Stevens et al. 2014; Peetermans
other characteristics may give diagnostic clues et al. 2019). Clindamycin is known to inhibit
on probable etiology and important guidance for toxin release and thereby cytokine production
personalized treatment. (Mascini et al. 2001). Furthermore, clindamycin
is superior to β-lactam antibiotics in infections
with large amounts of bacteria in a stationary
6.4 Treatment and Prognosis growth phase, which often is the case with
NSTIs (Eagle 1952; Stevens et al. 1988). Both
6.4.1 Treatment animal experiments and observational studies
have demonstrated increased survival with
Surgery is the main treatment modality in NSTIs clindamycin (Linner et al. 2014; Stevens et al.
regardless of etiology. This is due to the local 1988; Zimbelman et al. 1999; Mulla et al. 2003;
aggressiveness of disease, ischemia, tissue necro- Carapetis et al. 2014). In the INFECT study,
sis, and the limitations of antibiotic treatment receiving clindamycin before inclusion at the
(Stevens et al. 2014). Surgical intervention must study hospital was not associated with increased
be performed as early as possible when NSTIs are survival. As with surgery, this could reflect rapid
suspected, to confirm the diagnosis and to remove identification and initiation of appropriate treat-
devitalized, infectious tissue, improve circulation ment for those in greatest need of such treatment.
and oxygen supply, thereby halting the progress Intravenous immunoglobulin (IVIG) is used
of disease. The demonstration of the clear associ- by some centers in STSS or streptococcal NSTIs
ation between a delay in surgery and increased in order to neutralize streptococcal toxins. A
mortality underscore the importance of early sur- recent meta-analysis of clindamycin-treated
gery (Wong et al. 2003). Misdiagnosis at admis- STSS patients from one randomized and four
sion is the main reason for surgical delay (Goh non-randomized studies found that IVIG was sig-
et al. 2014). Daily revisions are recommended as nificantly associated with increased survival
long as there is a need for debridement (Stevens (Parks et al. 2018). IVIG-therapy was associated
et al. 2014). In the INFECT study, however, with increased survival in streptococcal NSTIs
patients undergoing surgery later than 24 h after also in the INFECT study (Bruun et al. 2020).
admission did not have increased mortality Moreover, a study on plasma of INFECT patients
among the GAS cases. This could be due to demonstrated a dose-related toxin inhibition,
rapid identification and initiation of appropriate providing a mechanistic correlate that also
treatment for the most severely sick patients, as supports a possible benefit of IVIG (Bergsten
suggested by earlier surgery among patients with et al. 2020). This should encourage renewed
shock. efforts to conduct a sufficiently powered
In the absence of clinical trials comparing the randomized clinical trial in GAS NSTIs.
efficacy of different antibiotic regimens, current Use of hyperbaric oxygen therapy (HBOT) in
recommendations rely on trials in other severe NSTIs is supported by some observational data,
infections, studies of etiology and resistance, but results are diverging (Peetermans et al. 2019).
and of pharmacokinetic and pharmacodynamic In the INFECT study, the case-fatality rate was
considerations, as further described in Chap. 7. lower in HBOT treated GAS NSTIs patients, but
Empiric broad-spectrum therapy covering both the effect of HBOT is difficult to assess due to
aerobic and anaerobic agents is warranted until a selection bias. Randomized trials are needed to
define the role of HBOT in the treatment of strep- In the INFECT cohort, the frequency of
tococcal NSTIs (Peetermans et al. 2019). amputations was 16% among GAS and 11%
among SD cases (Bruun et al. 2020). The
90-day CFR was 10% for GAS and significantly
6.4.2 Prognosis lower compared to polymicrobial
non-streptococcal cases (20%). The study
Mortality estimates are highly variable in differ- identified several independent variables
ent studies of NSTIs caused by GAS. A large associated with 90-day CFR, including increased
European surveillance study of 254 cases of age (odds ratio (OR) 1.15, per year), male sex
GAS NSTIs included in 2003–2004 registered a (OR 5.09), septic shock (OR 1.96), and IVIG not
case-fatality-rate (CFR) of 32%, but CFR varied administered (OR 3.15). Comorbidity was, how-
greatly between different countries (Lamagni ever, not associated with mortality.
et al. 2008a). In Sweden it was 19% (Darenberg Associations of mortality and virulence factors
et al. 2007). Other surveillance studies with more have been studied in invasive GAS infections.
than 50 cases have reported a CFR varying Certain emm types have been associated with
between 20% and 66% (O’Loughlin et al. 2007; increased mortality in invasive GAS infections
Lepoutre et al. 2011; O’Brien et al. 2002; Kaul (Olafsdottir et al. 2014), even in NSTIs (Luca-
et al. 1997; Sharkawy et al. 2002; Monnickendam Harari et al. 2009). This association may be due to
et al. 1997). Two single-center studies of GAS virulence gene profiles, including superantigens,
NSTIs reported a CFR of 16% and 11%, respec- associated with emm types and not the M protein
tively (Lin et al. 2013; Bruun et al. 2013). The itself (Lintges et al. 2010). For superantigen
CFR is considerably lower in other invasive skin genes, association studies are diverging
and soft tissue infections caused by GAS (Commons et al. 2014; Lintges et al. 2010;
(Sharkawy et al. 2002). Imohl et al. 2017), but in the hypervirulent
Factors associated with increased rate of death M1T1 clone, speA2 has definitely been essential
in GAS NSTIs include high age, comorbidities, for increased virulence (Barnett et al. 2018). The
hypotension/STSS, muscle involvement, and bac- role and mechanisms of superantigens are further
teremia (O’Brien et al. 2002; Kaul et al. 1997; elaborated in Chap. 9.
Sharkawy et al. 2002; Bruun et al. 2013). In In the INFECT study, the virulence gene
studies of NSTIs in general, also surgical delay profiles of GAS reflected the emm type distribu-
has shown a strong association with mortality tion. A high proportion with superantigen genes
(Wong et al. 2003; Childers et al. 2002; Elliott speA and speG corresponds to predominance of
et al. 1996; McHenry et al. 1995; Wall et al. emm1 and emm3 types. No clear association of
2000). emm types or other virulence genes with severity
Data on mortality and prognostic factors in was found, but for most emm types and virulence
NSTIs caused by SD are scarce. Among case gene profiles the numbers were low.
reports, CFR is high, but the most severe cases
may be overrepresented. In a retrospective study
of 70 GAS and 9 SD NSTIs cases, the 90-day 6.5 Conclusions and future
CFR was 20% and 33%, respectively. In the same perspectives
study there was an association of CFR with age
and heart disease (Bruun et al. 2013). Studies The high severity and other specific features pre-
comparing invasive GAS and SD disease in gen- viously reported in NSTIs caused by β-hemolytic
eral indicate that death among SD cases is more streptococci were outlined in detail by the
often related to age and comorbidity and more INFECT study. The high frequency of toxic
rarely to septic shock, than in GAS infections shock, ICU treatment, and death underscores the
(Ekelund et al. 2005a; Rantala et al. 2009b). severity of the disease. The INFECT study also
82 T. Bruun et al.
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Treatment of Necrotizing Soft Tissue
Infections: Antibiotics 7
Oddvar Oppegaard and Eivind Rath
Contents
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
7.2 Antimicrobial Resistance in Necrotizing Soft Tissue Infections . . . . . . . . . . . . 88
7.2.1 Brief Overview of the Etiology of NSTIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
7.2.2 Antimicrobial Resistance in β-Hemolytic Streptococci . . . . . . . . . . . . . . . . . . . . . . . . . 89
7.2.3 Antimicrobial Resistance in S. aureus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
7.2.4 Antimicrobial Resistance in Enterobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
7.2.5 Antimicrobial Resistance in Obligate Anaerobic Bacteria . . . . . . . . . . . . . . . . . . . . . . 91
7.3 Clinical Efficacy Data for Selected Antibiotics in Soft Tissue Infections . . 91
7.4 Pharmacokinetic and Pharmacodynamic Considerations in Treatment
of NSTIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
7.4.1 Pharmacokinetics in the Critically Ill . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
7.4.2 Tissue Distribution of Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7.4.3 Toxin Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
7.4.4 Eagle Effect and Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
7.4.5 Treatment of Biofilm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
7.5 Antimicrobial Treatment of NSTIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
7.5.1 Treatment Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
7.5.2 Empirical Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
7.5.3 Pathogen Specific Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
7.5.4 Treatment Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
7.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Abstract administration of potent antimicrobial agents

is pivotal, but inadequate empirical therapy is
Necrotizing soft tissue infections (NSTIs) are
unfortunately common. Optimization of the
severe, life-threatening infections, and early
antibiotic treatment strategy in NSTIs requires
therapeutic intervention is essential. Prompt
consideration of local epidemiology of causa-
O. Oppegaard (*) · E. Rath
tive pathogens and antimicrobial resistance
Department of Medicine, Haukeland University Hospital, patterns, knowledge on common pathogenetic
Bergen, Norway mechanisms in NSTIs, and adaptations to
e-mail: Oddvar.oppegaard@helse-bergen.no

88 O. Oppegaard and E. Rath
pharmacokinetic and pharmacodynamic phys- and constitutes the second pillar in the clinical
iological changes in critically ill patients. In management of NSTIs. Delayed administration
the present article we address all these issues, of antibiotics in sepsis is associated with an unfa-
as well as review and compare contemporary vorable outcome, with a linear increase in the risk
guidelines for antimicrobial treatment of of mortality for each hour of delay (Ferrer et al.
NSTIs from around the world. 2014).
On a similar note, severe infections caused by
Keywords drug-resistant microbes are also associated with
increased mortality, highlighting the importance
Necrotizing soft tissue infections · Skin and
of selecting empiric treatment regimens covering
soft tissue infections · Necrotizing fasciitis ·
the likely pathogens and resistance phenotypes
Resistance · Antimicrobial treatment ·
associated with the disease entity (Melzer and
Guidelines
Petersen 2007; van Hal et al. 2012).
In NSTIs, though, there is a paucity of suscep-
Highlights
tibility data from epidemiological studies, and
• Early administration of potent and adequate
clinical trials pertaining to the antimicrobial treat-
antimicrobial treatment is paramount.
ment of NSTIs have never been performed.
• Empirical treatment of necrotizing soft tissue
Existing knowledge is thus predominantly based
infections should be tailored to local antimi-
on extrapolation of data from non-necrotizing soft
crobial resistance epidemiology, particularly
tissue infections. However, there are important
the prevalence of methicillin-resistant Staphy-
differences between these two clinical entities
lococcus aureus and Enterobacteriaceae har-
regarding the specter of causative pathogens, bac-
boring extended spectrum beta-lactamases.
terial resistance phenotypes and properties, antimi-
• Antimicrobial therapy should be de-escalated
crobial pharmacokinetics and pharmacodynamics
and targeted as soon as the microbial etiology
in the host, and clinical treatment strategies
is identified.
(Stevens and Bryant 2017; Stevens et al. 2014).
• Toxin-inhibitor treatment is strongly
In the present chapter we will address these
suggested in the empirical treatment of
issues and review the data forming the basis for
NSTIs confined to the extremities, and in
contemporary guidelines on the antimicrobial
verified Streptococcus pyogenes or Clostrid-
treatment of NSTIs.
ium spp. NSTIs.
• Vibrio vulnificus is an endemic cause of NSTI
in East Asia, but is also an emerging pathogen
7.2 Antimicrobial Resistance
in other continents.
in Necrotizing Soft Tissue
Infections
7.1 Introduction
7.2.1 Brief Overview of the Etiology
of NSTIs
Necrotizing soft tissue infection (NSTI) is a
severe disease manifestation requiring rapid clin-
The distribution of causal pathogens in NSTIs
ical intervention to avoid a fatal outcome or
vary widely in epidemiologic reports, partly
amputation (Stevens and Bryant 2017; Wong
related to their temporal and geographic context.
et al. 2003). Swift surgical removal of infected
Nevertheless, some etiologic features appear to be
tissue has been demonstrated to be essential, and
relatively conserved; polymicrobial cases usually
prolonged time to first incision is an independent
accounts for approximately 50% of the cases,
risk factor for mortality (Wong et al. 2003).
often consisting of a mixture of Enterobac-
Administration of potent and adequate antimicro-
teriaceae and anaerobic bacteria, whereas
bial treatment is also of immediate importance,
β-hemolytic streptococci (BHS) and S. aureus
7 Treatment of Necrotizing Soft Tissue Infections: Antibiotics 89
are frequently associated with monomicrobial have been detected in both S. dysgalactiae and
NSTIs (Madsen et al. 2019; Kim et al. 2019; S. pyogenes (Fuursted et al. 2016; Vannice et al.
Louis et al. 2019). The water exposure associated 2019). Although these resistance phenotypes are
pathogens Vibrio vulnificus and Aeromonas still exceedingly rare, the mutational basis for
hydrophila have traditionally been regarded as resistance is highly analogous to that of
rare causes of monomicrobial NSTI, predomi- S. pneumonia, where penicillin resistance has
nantly affecting subjects with pre-existing gradually become widespread. All BHS are still
chronic liver conditions (Stevens and Bryant fully susceptible to broad spectrum β-lactam
2017; Peetermans et al. 2020). However, recently antibiotics (e.g. cephalosporins and
Hsiao et al. reported Vibrio spp. and Aeromonas carbapenems), as well as vancomycin, linezolid,
spp. to be responsible for approximately 20% of and daptomycin, but these agents are rarely
the NSTIs in Taiwan (Hsiao et al. 2020). Increas- indicated in monomicrobial BHS NSTIs (Farrell
ing incidence has also been noted on the Ameri- et al. 2013). Penicillin remains the drug of choice
can continent, indicating that these pathogens are for treating BHS infections, but sustained epide-
an emerging threat in several geographic regions miological attentiveness is definitely warranted to
(King et al. 2019; Dechet et al. 2008). Clostridial monitor the antimicrobial resistance trends in
myonecrosis is an infrequent NSTI manifestation these streptococcal species.
caused by Clostridium perfringens, although A significant increase in resistance to erythro-
other clostridial species are occasionally mycin and clindamycin in BHS has been noted
implicated (Stevens et al. 2012). over the past two decades in several regions.
Prevalence of clinically relevant antimicrobial Annual national surveillance reports from the
resistance in NSTIs exhibit substantial regional United Kingdom (UK) have revealed the rates
variations, and knowledge on local epidemiology of clindamycin resistance in S. dysgalactiae to
is essential to tailor the antibiotic treatment. increase gradually from 6% in 2004 to 25% in
Although studies on NSTIs detailing antimicro- 2017 (Public Health England 2018). In the United
bial resistance profiles are scarce to non-existent, States of America (US), the clindamycin resis-
susceptibility data for invasive disease in general tance rates for S. agalactiae currently
is available for several of the key pathogens. approximates 50%, causing the Centers for Dis-
ease Control to revise their infectious diseases
guidelines (CDCgov 2010). Even higher rates of
7.2.2 Antimicrobial Resistance resistance in BHS have been reported from south
in b-Hemolytic Streptococci east Asia (Lu et al. 2017). Conversely, in a large
prospective study of NSTIs conducted in northern
The β-hemolytic streptococcal species Europe, the prevalence of clindamycin resistance
S. pyogenes, S. agalactiae, and S. dysgalactiae in BHS was below 5%, underscoring the impor-
have all been implicated in NSTIs, although tance of taking regional resistance epidemiology
S. pyogenes is by far the most frequent (Bruun into consideration (Bruun et al. 2020).
et al. 2020; Das et al. 2012). These pathogens
have historically been regarded as unequivocally
sensitive to penicillin, but some concerns have 7.2.3 Antimicrobial Resistance
arisen the past decades. In 2008 the first reports in S. aureus
of S. agalactiae exhibiting reduced susceptibility
to penicillin were published, revealing increased Staphylococcus aureus is a potent human patho-
minimum inhibitory concentrations (MICs) to gen, capable of causing the entire spectrum of
several β-lactam antibiotics related to point skin and soft tissue infections, including occa-
mutations in the penicillin binding protein pbp2x sionally implicated in NSTIs. The penicillinase-
(Dahesh et al. 2008). More recently, similar stable penicillins (e.g. oxacillin and historically
mutations and reduced penicillin susceptibility methicillin) have been the cornerstone in
Table 7.1 Global burden of antimicrobial resistance for selected pathogens

Year S. aureus MRSA (%) E. coli ESBLceph (%) K. pneumonia ESBLceph (%)
Nordic countries 2018 0–2 0–8 0–8
Central Europe 2018 5–10 10–15 10–20
South East Europe 2018 30–40 20–30 50–75
United States 2016 45 12 13
South America 2016 – 26 35
Japan 2018 46 27 10
MRSA methicillin-resistant Staphylococcus aureus, ESBLceph extended spectrum beta-lactamase resistant to third gener-
ation cephalosporins. Nordic countries include Norway, Sweden, Denmark, Iceland and Finland. Central Europe refers
herein to the United Kingdom and Germany. South East Europe refers in this context to Italy, Greece, Romania and
Bulgaria. Adapted from references (Castanheira et al. 2019; eCDC 2019; JANIS 2018)
antimicrobial treatment of infections involving of the contemporary burden of MRSA is

this microbe. However, methicillin-resistant delineated in Table 7.1.
Staphylococcus aureus (MRSA) emerged in the Unfortunately, MRSA are frequently harbor-
1960s due to the acquisition of a mobile genetic ing co-resistance to other antimicrobial agents,
element carrying the mecA-gene, encoding a particularly tetracyclines, aminoglycosides,
penicillin binding protein with low affinity to clindamycin, and fluoroquinolones (Diekema
virtually all β-lactam antibiotics, including et al. 2019a, b). Vancomycin, linezolid, and
carbapenems (Diekema et al. 2019b; Lee et al. daptomycin are the most potent drugs against
2018). A gradual global dissemination ensued, MRSA, and resistance to these antimicrobial
and by the turn of the century, MRSA had agents is extremely rare in S. aureus (Farrell
become one of the major health concerns in infec- et al. 2013; Duncan et al. 2019). The novel fifth
tious diseases. In a retrospective study of NSTIs generation cephalosporins, ceftaroline, and
during 2001–2006 in Texas, MRSA was ceftobiprole, are marketed for their anti-MRSA
identified as the causative pathogen in 39% of activity. Alarmingly, a recent report from Europe
the cases (Lee et al. 2007). A similar situation revealed 10% of the MRSA-isolates to be
was described in a contemporary report on NSTIs co-resistant to ceftaroline, indicating that these
from Los Angeles (Miller et al. 2005). Moreover, cephalosporins should not be used for empirical
approximately half the invasive S. aureus-isolates treatment of MRSA (Farrell et al. 2013).
were found to be methicillin-resistant in the UK,
southern Europe, the US, and South East Asia
during this period (Diekema et al. 2019a, 2019b). 7.2.4 Antimicrobial Resistance
Extensive prevention and control measures in Enterobacteriaceae
were implemented in several countries to coun-
teract this development, including isolation A wide range of enterobacterial species have
protocols, patient screening, and MRSA eradica- been implicated in NSTIs, predominantly in
tion therapy. Two decades later, this massive polymicrobial infections (Madsen et al. 2019;
effort appears to have been successful, and the Kim et al. 2019; Louis et al. 2019). Escherichia
prevalence of MRSA is declining in both the US coli and Klebsiella spp. are by far the most
and in Europe, dropping below 5% in northern commonly encountered Enterobacteriaceae,
European countries (Duncan et al. 2019; eCDC reflecting their dominance as a cause of invasive
2019; NORM/NORM-VET 2018). Nevertheless, disease in humans in general. Resistance to beta-
it is still a force to be reckoned with in several lactam antibiotics has unfortunately become
geographic areas, and empiric MRSA coverage is widespread in these pathogens, including the dis-
warranted in high endemic regions. An overview semination of extended spectrum beta-lactamase
(ESBL) enzymes (eCDC 2019; Diekema et al. diaphragm, whereas gram-positive anaerobic
2019a). There is a plethora of ESBL genes cur- peptostreptococcal species are often associated
rently circulating and their classification has been with infections emanating from the head and
much debated. In E. coli and Klebsiella spp., neck area (Gajdacs et al. 2017; Brook et al.
beta-lactamases mediating resistance to third gen- 2013). All these pathogens are uniformly suscep-
eration cephalosporins (herein abbreviated tible to metronidazole, but resistance rates to
ESBLceph) have become endemic in several geo- other antimicrobial agents vary depending on
graphic regions (Diekema et al. 2019a; eCDC the species. Nevertheless, resistance to
2019). Piperacillin/tazobactam is also frequently carbapenems and piperacillin-tazobactam is suffi-
inactivated by these enzymes, but not invariably ciently rare to enable both these agents to function
(Kim et al. 2019). In Europe, there is a distinct as empiric coverage for anaerobes in
geographic polarization of the ESBLceph preva- polymicrobial NSTIs (Gajdacs et al. 2017). The
lence, ranging from 3rd generation cephalosporin broad spectrum tetracycline analog tigecycline
resistance in <5% of Klebsiella isolates in the has also demonstrated excellent in vitro and
northern regions, to 75% of the isolates in the in vivo activity against obligate anaerobic bacte-
south-east (eCDC 2019). A similar gradient is ria (Brook et al. 2013).
evident between US and Latin America, although Clindamycin was historically regarded as a
less markedly (Castanheira et al. 2019). An over- potent anaerobic therapeutic agent, but resistance
view of the global burden of ESBLceph is has unfortunately become widespread. The per-
delineated in Table 7.1. centage of Bacteroides isolates resistant to
Co-resistance in isolates harboring ESBL clindamycin has increased from 5 % to 35 % in
genes is substantial, and in Greece, 50% of the the US in the period 1990–2010, and a similar
Klebsiella pneumonia isolates display combined development has been observed in Europe
resistance to third generation cephalosporins, (Gajdacs et al. 2017). Hence, it is no longer a
fluoroquinolones, and aminoglycosides (eCDC first line agent in the treatment of these pathogens.
2019). Alarmingly, isolates harboring ESBL Penicillin has retained its activity against most
enzymes also inactivating carbapenems gram-positive anaerobic bacteria, including
(ESBLcarba) have emerged in South East Europe, Clostridium spp. However, the majority of
and poses a major health concern (eCDC 2019; gram-negative anaerobes are equipped with
Castanheira et al. 2019). Treatment options in broad spectrum beta-lactamases, rendering both
these multiresistant organisms are severely lim- penicillins and most cephalosporins ineffective
ited, and rapid implementation of effective pre- (Brook et al. 2013).
vention and control measures are necessary to
contain the epidemiological situation.
7.3 Clinical Efficacy Data
for Selected Antibiotics in Soft
7.2.5 Antimicrobial Resistance Tissue Infections
in Obligate Anaerobic Bacteria
Numerous clinical studies on antimicrobial treat-
Obligate anaerobic bacteria are almost invariably ment of non-necrotizing soft tissue infections
present in polymicrobial NSTIs, and Clostridium have been performed. Key findings and principles
perfringens is also an established cause of could likely be transferred to the management of
monomicrobial NSTI (Thanert et al. 2019; NSTIs, and will be briefly reviewed.
Zhao-Fleming et al. 2019). Gram-negative obli- Historic studies from the dawn of the antibiotic
gate anaerobes such as Bacteroides spp., era reported approximately 70% cure rates in
Prevotella spp., and Fusobacterium spp. are pre- cellulitis patients not receiving antibiotics, com-
dominantly detected in infections below the pared to eradication of the infections in 98 % of
those receiving penicillin (Spellberg et al. 2009). 7.4 Pharmacokinetic

Moreover, a dramatic improvement in cellulitis and Pharmacodynamic
mortality and morbidity rates was observed after Considerations in Treatment
penicillin became available (Spellberg et al. of NSTIs
2009).
In the modern age, placebo-controlled trials in 7.4.1 Pharmacokinetics
cellulitis are unethical, and studies are predomi- in the Critically Ill
nantly designed to demonstrate non-inferiority to
existing therapeutic agents. This is practical for Knowledge on pharmacokinetics and pharmaco-
demonstrating clinical efficacy in general, but is dynamics of antimicrobial agents is primarily
less suited for determining the optimal antimicro- derived from studies on healthy subjects. How-
bial treatment strategy. Indeed, in a metanalysis ever, several pathophysiological changes occur in
of 43 randomized clinical trials comparing the the critically ill that have potential implications
efficacy of antimicrobial treatment in complicated for the therapeutic strategy in NSTIs.
skin and soft tissue infections, Brindle et al. could Firstly, there is a high prevalence of acute
not find any evidence to support the preference of kidney failure in patients with severe infectious
one antibiotic over another (Brindle et al. 2019). disease manifestations, including NSTIs,
The efficacy of linezolid versus vancomycin in necessitating adjustment of the daily dosage for
complicated skin and soft tissue infections has several antibiotics (Das et al. 2012; Bruun et al.
recently been much debated. Although the initial 2020). Some agents are associated with a substan-
registration trials demonstrated non-inferiority, a tial nephrotoxicity, and are contraindicated in the
Cochrane meta-analysis revealed a significant presence of overt kidney failure. This particularly
improved clinical cure rate for linezolid (Yue concerns the aminoglycosides and colistin, but
et al. 2016). However, the authors summarize neither are frequently administered in the treat-
that the effect is small, and the majority of the ment of NSTIs. More problematic is the relative
studies were funded by the pharmaceutical com- contraindication for vancomycin, which is often
pany, increasing the risk of bias. In a recent study the agent of choice for MRSA coverage in NSTIs.
comparing vancomycin and linezolid for treat- The risk of Vancomycin induced nephrotoxicity
ment of invasive MRSA infections, linezolid correlates to the serum trough levels, and elevated
was associated with an increased mortality in serum concentration over time is associated with
severely ill patients with an acute physiology a high risk of renal failure (Arnaud and Liborio
and chronic health evaluation (APACHE) II 2020). Vancomycin could potentially be
score above 14 (Burnham et al. 2016). This administered to patients with acute renal failure
could lend caution to its clinical use in more if necessary, as long as adequate dose adjustment
severe skin and soft tissue infections, such as is performed and daily serum concentrations are
NSTIs. measured. However, most clinicians would con-
The past decade, several new antibiotics have sider switching to alternative MRSA active
received approval for the treatment of compli- agents in the presence of severely reduced renal
cated skin and soft tissue infections, including function, such as linezolid or daptomycin. It
daptomycin, tigecycline, tedizolid and the 5th should be noted that risk of linezolid-associated
generation cephalosporins ceftaroline and bone marrow suppression is increased in the pres-
ceftobiprole (Brindle et al. 2019; Tirupathi et al. ence of kidney failure, further complicating the
2019). However, none has been shown to outper- therapeutic strategy in this patient group
form the pre-existing treatment options, and their (Wu et al. 2006). Hepatic impairment is an infre-
role in the treatment of these condition has not quent complication in NSTIs, but dosage reduc-
been firmly established. Most contemporary tion is recommended for clindamycin,
guidelines recommend to reserve these antibiotics daptomycin, and metronidazole in patients with
for salvage therapy.
advanced liver failure classified as Child Pugh between different antimicrobial classes. In gen-
class C (Perianez-Parraga et al. 2012). eral, lipophilic agents, such as tigecycline,
The fluid homeostasis is frequently imbal- linezolid, and clindamycin, have a larger distribu-
anced in the critically ill due to significant capil- tion volume and tissue penetration than hydro-
lary leak coupled with aggressive fluid philic antibiotics (e.g. beta-lactams, vancomycin,
resuscitation therapy, leading to an increase in and daptomycin) (Barbour et al. 2010; Kiang
the extracellular fluid space (Udy et al. 2013). et al. 2014). Nevertheless, all above agents
Furthermore, the protein binding of antimicrobial achieve sufficient tissue concentrations to be
drugs is often reduced secondary to potentially clinically effective in the treatment of
hypoalbuminemia, and these pathophysiological skin and soft tissue infections, but increased dos-
changes have implications for the pharmacokinet- ing might be indicated in the latter antimicrobial
ics of several antibiotics (Udy et al. 2013; Kiang group (Pea 2016). In a small experimental study
et al. 2014; Minichmayr et al. 2017). Increased of tissue distribution in diabetic foot infections,
distribution volumes have been demonstrated for vancomycin penetration was found to be signifi-
aminoglycosides, beta-lactams, daptomycin, and cantly impaired in the presence of
glycopeptides in critically ill patients, and for microangiopathy (Skhirtladze et al. 2006). The
vancomycin the changes in distribution volume clinical implications of this has yet to be
were found to correlate with disease severity investigated, but increased dosage in the presence
(Udy et al. 2013). Redistribution of the therapeu- of diabetes microangiopathy might be warranted.
tic drug to the extracellular compartment implic- Small vessel occlusion is also part of the local
itly reduces the serum concentration of these pathophysiology in NSTIs, lending further sup-
antimicrobial agents, potentially increasing the port to using high dosing of glycopeptides in the
risk of incomplete clearance of bacteremia. This treatment of these infections (Stevens and Bryant
interaction between pharmacokinetics and sever- 2017).
ity should be taken into consideration in the treat- Intracellular survival of pathogens in
ment of NSTIs, and underlines the importance of macrophages has emerged as a potentially impor-
administering antibiotics in sufficiently high tant aspect of the pathogenesis in NSTI, particu-
doses to compensate for the increased distribution larly for S. aureus and S. pyogenes (Baude
volume. Furthermore, the use of a loading dose et al. 2019; Hertzen et al. 2012). Hence, the
should be considered, especially for penetration of antimicrobial agents to the intracel-
glycopeptides and daptomycin (Udy et al. 2013). lular compartment has become of interest. In
line with tissue distribution discussed above,
lipophilic drugs attain the highest intracellular
7.4.2 Tissue Distribution concentrations, approaching a one to one ratio
of Antibiotics with plasma concentrations for macrolides,
tigecycline, and fluoroquinolones (Barcia-Macay
Although bacteremia is detected in a substantial et al. 2006). Conversely, daptomycin has one of
proportion of NSTIs, the predominance of the the smallest distribution volumes of all
bacterial burden is usually confined to the periph- antimicrobials, and is not suitable for treatment
eral tissues at the affected site (Stevens and of intracellular microbes (Barbour et al. 2010).
Bryant 2017). In order to target these bacteria, However, the intracellular concentration of the
the therapeutic agents need to extravasate and antibiotics is only half the story. The internalized
disseminate into the extracellular space. The dis- bacteria often reside in the phagolysosomes, and
tribution of antibiotic drugs into the soft tissue this highly acidic environment has been
compartment is dependent on several pharmaco- demonstrated to significantly alter the activity of
kinetic properties, including solubility, molecular several antimicrobial agents. Barcia-Macay et al.
weight, and protein binding. Consequently, the investigated the effect of various antibiotics
tissue distribution profile displays high variability against intracellular S. aureus, and revealed that
a change in pH from 7.3 to 5.0 lead to a 1024-fold demonstrating improved clinical outcome (Linner
increase in the MIC value for azithromycin, and a et al. 2014; Carapetis et al. 2014). However, these
fourfold increase for fluoroquinolones (Barcia- findings should not be extrapolated to the entire
Macay et al. 2006). Interestingly, when prolonged spectrum of S. pyogenes skin and soft tissue
exposure (>24 h) of the antibiotics was infections. Clinical benefit from the addition of
investigated, cloxacillin was one of the most clindamycin could not be detected in less severe
effective agents in eradicating intracellular cases, but was significantly associated with
S. aureus in this experimental model. This increased risk of developing diarrhea (Brindle
contrasts the current dogma that beta-lactam et al. 2017). Adjunctive clindamycin treatment
antibiotics have poor intracellular activity. Con- should thus be reserved for S. pyogenes
versely, clindamycin achieves high intracellular associated NSTIs or toxic shock syndrome.
concentrations, and appears to eradicate intracel- Clindamycin also reduces the production of
lular S. pyogenes more efficiently than penicillin superantigens in S. aureus significantly more
(Kaplan et al. 2006). The optimal treatment for than oxacillin, both in vitro and in murine models
intracellular S. aureus and S. pyogenes in NSTIs (Coyle, and Society of Infectious Diseases, P
has yet to be elucidated, but combining beta- 2003). Hence, some advocate that the administra-
lactam antibiotics and clindamycin is likely bene- tion of clindamycin is indicated also in the treat-
ficial, and in line with contemporary ment of severe S. aureus infections. However,
recommendations. clinical studies pertaining to the use of adjunctive
clindamycin treatment in staphylococcal NSTIs
has not been performed.
7.4.3 Toxin Inhibition Linezolid inhibits the bacterial protein synthe-
sis in a similar manner as clindamycin, by binding
Bacterial secretion of cytotoxins and to the 50S ribosomal subunit. Coyle et al. found
superantigens is highly implicated in the patho- linezolid to be equally effective as clindamycin in
genesis of streptococcal and staphylococcal the reduction of superantigen production in
NSTIs, and is one of the key disease mechanisms S. pyogenes in vitro, and it has also been
distinguishing NSTIs from non-necrotizing soft demonstrated to have similar activity in
tissue infections (Shumba et al. 2019; Stevens S. aureus (Coyle, and Society of Infectious
and Bryant 2017). Hence, the abrogation of Diseases, P 2003; Coyle et al. 2003). The ratio-
toxin production remains an important therapeu- nale for administering linezolid in NSTIs is not as
tic aspect in the management of NSTIs. Adminis- strong as for clindamycin, but could be warranted
tration of cell wall disrupting antibiotics, in strains resistant to clindamycin or in cases with
e.g. beta-lactams, could potentially increase the known allergy.
release of exotoxins through bacterial lysis Interestingly, Andreoni et al. recently reported
(Coyle et al. 2003). Conversely, clindamycin clindamycin treatment to reduce toxin production
inhibits the bacterial protein synthesis, and has and disease severity in NSTIs caused by
been shown to significantly reduce the production S. pyogenes, regardless of the clindamycin sus-
of several streptococcal virulence determinant ceptibility status of the strain (Andreoni et al.
in vitro (Andreoni et al. 2017; Mascini et al. 2017). Although clindamycin monotherapy treat-
2001). Recently, these findings were ment of clindamycin-resistant S. pyogenes soft
substantiated in vivo in a study by Andreoni tissue infections previously has been associated
et al., revealing significantly attenuated Streptoly- with an unfavorable outcome in experimental
sin O activity after administration of clindamycin, mouse models, it is conceivable that clindamycin
both in a murine model and a human case of NSTI retains a role as an adjunctive treatment of NSTIs
(Andreoni et al. 2017). Moreover, several clinical even in the presence of clindamycin-resistant
studies have supported the adjunctive use of BHS (Lewis et al. 2014).
clindamycin in severe S. pyogenes infections,
7.4.4 Eagle Effect and Tolerance 7.4.5 Treatment of Biofilm
The presence of tolerance to antimicrobial agents, The organization of microbial communities into a
defined as a >32-fold ratio of minimum bacteri- biofilm has long been recognized as a phenotypic
cidal concentration (MBC) to minimum inhibi- trait in several clinical pathogens, including
tory concentration (MIC), has been reported as a S. aureus (Gebreyohannes et al. 2019). Recently,
phenomenon for several decades, particularly in biofilm was revealed to also be implicated in
various streptococcal species (Rolston et al. NSTIs caused by S. pyogenes (Siemens et al.
1984). For S. pyogenes, the presence of penicillin 2016). The encapsulation of the microbes by
tolerance has been reported for up to 29% clinical rich extracellular matrix and the distinct layering
isolates derived from streptococcal tonsillitis of bacteria in different metabolic states is the
(Conley et al. 2003). However, the lack of hallmark biofilm formation, and has been shown
standardized laboratory methodology and repro- to significantly alter the efficacy of several
ducibility has been criticized, and clinical impli- antibiotics (Fiedler et al. 2015; Gebreyohannes
cation for this observation has been difficult to et al. 2019; Conley et al. 2003). The beta-lactam
ascertain. antibiotics inhibit bacterial growth though disrup-
A related growth dependent phenomenon was tion of the cell wall synthesis. However, in the
discovered by Eagle in 1952 in a murine biofilm community some bacteria enter an inac-
S. pyogenes pyomyositis model (Eagle 1952). tive stationary phase, rendering the beta-lactams
He reported that the efficacy of penicillin ineffective. Conversely, antibacterial agents
correlated to the size of the bacterial inoculum, targeting DNA (fluoroquinolones), RNA (rifam-
and the beta-lactam antibiotic lost its potency picin) or protein synthesis (clindamycin and
when the bacterial burden increased beyond a linezolid) appear to retain their activity also in
certain point. It has become known as the Eagle biofilm, and have been suggested as potential
effect, and is presumed to reflect the reduced therapeutic agents in biofilm-associated
activity of penicillin on S. pyogenes reaching a infections (Zimmerli and Sendi 2017; Chai et al.
stationary plateau-phase in their growth curve. 2016).
Later, Stevens et al. reproduced the results, but In S. pyogenes, biofilm formation has previ-
also demonstrated that clindamycin was not ously been linked to penicillin treatment failure in
affected by growth phase or inoculum size, and tonsillitis (Conley et al. 2003). The presence of
significantly outperformed penicillin in the treat- S. pyogenes biofilm has been demonstrated in
ment of experimental pyomyositis (Stevens et al. crypts in excised tonsillar tissue, and biofilm
1988). A high inoculum of bacteria is a hallmark capacity and reduced susceptibility to beta-lactam
of NSTIs as compared to less severe SSTIs, and antibiotics has been verified in S. pyogenes
the Eagle effect is presumed to be of significance isolates obtained from recurrent tonsillitis cases
in the treatment of NSTIs. The superiority of (Conley et al. 2003; Roberts et al. 2012). Treat-
clindamycin in bacteria in stationary growth ment with clindamycin appears to more effective
phase, combined with its ability to abrogate in eradication of difficult to treat S. pyogenes
toxin production in S. pyogenes, forms the exper- tonsillitis cases, though at the expense of more
imental basis for the recommendations of adjunc- adverse effect (Brook and Hirokawa 1985).
tive clindamycin treatment in NSTIs caused by How discovery of S. pyogenes biofilm in
this pathogen. NSTIs translates into clinical practice has yet to
Notably, the Eagle effect as well as cytotoxin be elucidated, but it likely strengthens the posi-
production has also been verified in tion of adjunctive clindamycin in the treatment of
S. dysgalactiae-infections, but the potency of these infections.
clindamycin in this setting has not been examined
(Lam and Bayer 1983; Siemens et al. 2015).
7.5 Antimicrobial Treatment subsequently de-escalate treatment based on cul-

of NSTIs ture and susceptibility data. Given the substantial
mortality and morbidity associated with NSTIs
7.5.1 Treatment Strategy and the importance of swift infection control, a
de-escalation strategy is advocated by most
In the absence of clinical trials pertaining to guidelines, and empiric coverage of all pathogens
the antimicrobial treatment of NSTIs, general frequently associated with this condition is essen-
recommendations for therapeutic management tial (Stevens et al. 2014; Sunderkotter et al. 2019).
are predominantly based on expert opinion. However, the major geographic differences in
Knowledge extrapolated from experimental stud- antibiotic susceptibility described previously,
ies on the pharmacodynamics and pharmacoki- highlights the need to adapt antimicrobial treat-
netics of relevant antibiotics as delineated ment to the local epidemiology of bacterial resis-
above, combined with data from clinical studies tance. Moreover, regional differences in
on non-necrotizing skin and soft tissue infections, etiological distribution merit considerations,
have often been used as guidance in the decision especially in areas where water exposure
process. Importantly, the antimicrobial treatment associated microbes (i.e. Vibrio and Aeromonas
needs to cover the etiologic spectrum of spp.) are reported to be more frequent (Hsiao et al.
pathogens associated with NSTI. 2020). Hence, a globally valid guideline for treat-
In general, two different strategies exist in the ment of NSTIs is perhaps not feasible.
empiric treatment of infectious diseases. In some
clinical conditions, including uncomplicated
skins and soft tissue infections, the initiation of 7.5.2 Empirical Treatment
narrow spectrum antibiotics is advised, and treat-
ment escalation can be considered in the event National guidelines for the treatment of NSTIs
of therapeutic failure. Inversely, in more severe have been developed in several countries
disease manifestations it is often recommended (Table 7.2), though many simply refer to the
to administer broad spectrum antibiotics, and recommendations from the Infectious Diseases
Table 7.2 Overview of contemporary guideline recommendations for empirical treatment of NSTIs
IDSA 2014 WSES/SIS-E Germany 2018 France 2019 Norway 2013 South Korea
(Stevens et al. 2018 (Sartelli (Sunderkotter (Urbina et al. (Helsedirektoratet 2017 (Kwak
2014) et al. 2018) et al. 2019) 2019) 2013) et al. 2017)
Standard Pip/tazo Pip/tazo Pip/tazo Pip/tazoa Penicillinb Pip/tazo
or or or and or
Meropenem Meropenem Meropenem Gentamicin Meropenem
or or or
3. gen ceph 3. gen ceph and 3. gen ceph and
and metro metro metro
Plus Vancomycin Linezolid NA NA NA Vancomycin
MRSA or or or
coverage Linezolid Daptomycin Linezolid
Plus NA Clindamycin Clindamycin NA Clindamycin NA
toxin
inhibit
NSTIs necrotizing soft tissue infections, IDSA Infectious Diseases Society of America, WSES/SIS-E World Society of
Emergency Surgery/ Surgical Infection Society—Europe, pip/tazo, piperacillin/tazobactam, 3. gen ceph, third generation
cephalosporin (e.g. cefotaxime or ceftriaxone), metro metronidazole, MRSA methicillin-resistant Staphylococcus aureus,
toxin inhibit toxin inhibitor, NA not advised as empirical treatment
a
The French guideline recommends to use meropenem or vancomycin in the presence of risk factors for ESBLceph or
MRSA, respectively
b
The Norwegian guideline recommends using third generation cephalosporin and metronidazole, or even meropenem, in
NSTIs emanating from the abdominal or genitourinary region
Society of America (IDSA) or World Society of NSTIs emanating from the abdominal or genito-
Emergency Surgery/Surgical Infections Society urinary region is very low (Madsen et al. 2019).
Europe (WSES/SIS-E) (Stevens et al. 2014; Accordingly, reserving the toxin inhibitors for
Sartelli et al. 2018). The various guidelines are NSTIs confined to the extremities or head/neck
largely overlapping, but demonstrate some area is suggested in France (Urbina et al. 2019).
important differences, predominantly reflecting The IDSA guidelines have also omitted
the variations in antimicrobial resistance. clindamycin from their empiric management,
One of the major ambiguities is the but recommend administering a protein synthesis
incorporation of ani-MRSA coverage in the inhibitor to all cases of verified S. pyogenes and
empiric treatment regime. Understandably, Clostridial NSTIs (Stevens et al. 2014).
guidelines originating from US (IDSA) and In Norway, a country with very low preva-
from Asia (South Korean guideline), where the lence of both MRSA and ESBLceph, a narrow
proportion of methicillin resistance in S. aureus spectrum approach has been recommended in
approximates 50%, advocate to initiate empiric the national guidelines for empiric treatment of
treatment with an MRSA active antibiotic in NSTIs located to the extremities; a combination
all NSTI-patients (Stevens et al. 2014; Kwak of penicillin, gentamicin, and clindamycin
et al. 2017). The inverse holds true in (Helsedirektoratet 2013). Inversely, the local
recommendations from regions of low MRSA antimicrobial treatment recommendations in
endemicity, including central and northern the neighboring country Denmark, advise
Europe (Sunderkotter et al. 2019; Urbina et al. meropenem and clindamycin as the treatment of
2019; Helsedirektoratet 2013). choice for all cases of NSTIs (Rigshospitalet
Another important consideration is the selec- 2020). The rates of antimicrobial resistance in
tion of empiric treatment for Enterobacteriaceae. these two countries are comparable, but two dia-
Most guidelines suggest administration of either metrically opposing strategies have been chosen.
piperacillin/tazobactam or meropenem, and leave Interestingly, in the prospective INFECT study
it to the treating physician to decide the appropri- including patients from both these countries, no
ate choice based on local resistance epidemiology significant difference in mortality was observed
(Stevens et al. 2014; Kwak et al. 2017; Sartelli (Madsen et al. 2019). However, randomized clin-
et al. 2018; Sunderkotter et al. 2019). Both these ical trials are needed to address the optimal treat-
agents have excellent broad spectrum activity ment strategy in NSTIs, and to examine the
against streptococci, staphylococci (excluding feasibility of tailoring the antimicrobial therapy
methicillin-resistant strains), anaerobes, and to the anatomical location.
Enterobacteriaceae, but carbapenems are usually
necessary to treat gram-negative bacteria harbor-
ing ESBLceph. In general, piperacillin/ 7.5.3 Pathogen Specific Treatment
tazobactam should be regarded as the first line
of defense in areas with low prevalence of If the causative pathogen is identified, the antimi-
ESBLceph, but meropenem is warranted in geo- crobial treatment can often be de-escalated. The
graphic regions where resistance to piperacillin/ treatment recommendations for monomicrobial
tazobactam is high. NSTIs are fairly similar across continents
A third major strategic decision pertains to (Table 7.3).
the administration of a protein synthesis inhibitor The combination of penicillin and clindamycin
as part of the empiric treatment regime. The is uniformly recommended for the treatment of
German guidelines and the WSES/SIS-E S. pyogenes NSTIs, to counteract bacterial toxin
recommendations incorporate clindamycin in production, biofilm formation and the Eagle
their therapeutic algorithm for all patients effect (Stevens et al. 2014; Helsedirektoratet
(Sunderkotter et al. 2019; Sartelli et al. 2018). 2013). However, therapeutic consideration in the
However, the prevalence of S. pyogenes in event of clindamycin resistance are not discussed
Table 7.3 Overview of contemporary guideline recommendations for treatment of NSTI with known etiology
IDSA 2014 WSES/SIS-E 2018 Germany 2018 France 2019 Norway 2013
(Stevens et al. (Sartelli et al. (Sunderkotter et al. (Urbina et al. (Helsedirektoratet
2014) 2018) 2019) 2019) 2013)
S. pyogenes Penicillin NA NA NA Penicillin
and and
Clindamycin Clindamycin
S. aureus Oxacillin NA NA NA NA
MSSA
S. aureus Vancomycin Linezolid Linezolid Vancomycin NA
MRSA or or or
Linezolid Daptomycin Daptomycin
Polymicrobial Pip/tazo Pip/tazo Pip/tazo Pip/tazoa 3. gen ceph and
infections or or or metro
Meropenem Meropenem Meropenem or
or Meropenem
3. gen ceph and
metro
V. vulnificus Doxycycline NA NA NA NA
and
Ceftriaxone
A. hydrophilia Doxycycline NA NA NA NA
and
Ceftriaxone
C. perfringens Penicillin NA NA NA Penicillin
and and
Clindamycin Clindamycin
NSTIs necrotizing soft tissue infections, IDSA Infectious Diseases Society of America, WSES/SIS-E World Society of
Emergency Surgery/Surgical Infection Society—Europe, pip/tazo, piperacillin/tazobactam, 3. gen ceph, third generation
cephalosporin (e.g. cefotaxime or ceftriaxone), metro metronidazole, MSSA methicillin susceptible Staphylococcus
aureus, MRSA methicillin-resistant Staphylococcus aureus, NA not addressed
a
The French guideline recommends to use meropenem or vancomycin in the presence of risk factors for ESBLceph or
MRSA, respectively
in any guidelines. Although Andreoni et al. found the adjunctive treatment of clindamycin in this
evidence for a clindamycin mediated attenuation pathogen, but the addition of a toxin inhibitor
of toxin production in S. pyogenes even in could be considered in monomicrobial
the presence of clindamycin resistance, the size S. dysgalactiae NSTIs, especially in the event of
of the bacterial inoculum remained unchanged therapeutic failure.
(Andreoni et al. 2017). Thus, the clinical Different weight has been given to recent stud-
implications have yet to be elucidated, and until ies indicating a possible superiority for linezolid
further studies have been performed, it is advis- over vancomycin in SSTIs, especially for MRSA
able to switch to linezolid in clindamycin- associated cases (Yue et al. 2016). Both IDSA
resistant cases. guidelines and French recommendations for
S. dysgalactiae is an emerging cause of NSTIs NSTI, as well as IDSA guidelines for MRSA
(Bruun et al. 2020; Oppegaard et al. 2015). infections, maintain vancomycin as the treatment
Experimental data has demonstrated its capability of choice in MRSA infections (Liu et al.
of cytotoxin production, biofilm formation as 2011; Stevens et al. 2014; Urbina et al. 2019).
well occurrence of the Eagle effect when exposed Differently, WSES/SIS-E and the German
to penicillin (Siemens et al. 2015, 2016; Lam and recommendations prefer linezolid or even
Bayer 1983). There are no studies pertaining to daptomycin (Sartelli et al. 2018; Sunderkotter
et al. 2019). The quality of the evidence favoring 7.6 Conclusions

linezolid is very low, and both alternatives are
likely to be equally successful. However, Necrotizing soft tissue infection is a life-
avoiding vancomycin in overt renal failure, threatening clinical condition, and rapid adminis-
and possibly in the presence of diabetic tration of potent antibiotics is essential. Empiric
microangiopathy is advisable. The role of novel antimicrobial treatment should cover the likely
5th generation cephalosporins and tigecycline in culprit, and the therapeutic regimen should be
the treatment of MRSA NSTIs has not been adapted to local resistance epidemiology. The
firmly established, but should be reserved for decision to broadly administer vancomycin or
cases were the first line options cannot be used. linezolid depends on the endemicity of MRSA
Vibrio spp. and Aeromonas spp. are still rare in the geographic region. Similarly, in areas with
causes of NSTI in most parts of the world, and low prevalence of ESBLceph, piperacillin/
few guidelines address specific treatment tazobactam should be regarded as first line of
recommendations for these pathogens. A combi- defense, but meropenem might be warranted in
nation of doxycycline and ceftriaxone is regions where resistance to piperacillin/
advocated in the South Korean and IDSA tazobactam is high. Coverage of Vibrio spp. and
guidelines, although doxycycline plus ciprofloxa- Aeromonas spp. should not be forgotten in areas
cin might be preferable for Aeromonas infections where these pathogens are endemic, particularly
(Stevens et al. 2014; Kwak et al. 2017). In South in patients with pre-existing chronic liver
East Asia Vibrio spp. has become the dominant conditions and recent sea water exposure. The
cause of monomicrobial NSTIs, implicated in use of a protein synthesis inhibitor could be omit-
20% of the cases in a recent study from Taiwan ted in the empiric treatment regime in NSTIs
(Hsiao et al. 2020). Accordingly, the South emanating from the abdominal and genital areas,
Korean guidelines advice empiric coverage for but is definitely indicated in cases localized to the
Vibrio spp. in patients with chronic liver extremities, and in verified S. pyogenes and Clos-
conditions and recent sea water exposure (Kwak tridial NSTIs. Furthermore, it should be strongly
et al. 2017). considered in monomicrobial cases caused by
S. dysgalactiae and S. aureus. Randomized clini-
cal trials investigating the optimal antimicrobial
7.5.4 Treatment Duration treatment of NSTIs are needed.
Studies investigating the optimal treatment dura- Acknowledgment This work was supported by the
tion in NSTI are lacking, and considerable European Union Seventh Framework Programme:
variations in clinical practice has been observed. (FP7/2007-2013) under the grant agreement 305340
(INFECT project), NordForsk (Project no. 90456,
In a retrospective study from the US comparing PerAID), the Norwegian Research Council under the
the management and outcomes in NSTIs between frame of ERA PerMed (Project 2018-151, PerMIT) and
three different hospitals, the treatment duration The Swedish Research Council Framework Grant in
ranged from 7 to 28 days (Faraklas et al. 2016). infections and antibiotics (2014-8609-117204-47).
Significant variations in treatment length were
detected between the three sites, but no
differences in clinical outcome was evident. References
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Treatment of Necrotizing Soft Tissue
Infections: IVIG 8
Martin Bruun Madsen, Helena Bergsten, and Anna Norrby-
Teglund
Contents
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
8.2 Immunoglobulins: Structure and Function of Different Ig Isotypes . . . . . . 106
8.3 IVIG Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
8.3.1 Manufacturing and Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
8.3.2 Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
8.3.3 Therapeutic Indications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
8.3.4 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
8.4 Rationale for the Use of IVIG for NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
8.4.1 Laboratory Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
8.4.2 Clinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
8.4.3 IVIG in NSTI, Unresolved Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
8.4.4 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Abstract septic shock, and necrotizing soft tissue

infections. IVIG has been used for patients
Immunoglobulins are key effector molecules
with severe invasive group A streptococcal
in the humoral immune response. Intravenous
infection since the early nineties and off-label
polyspecific immunoglobulin (IVIG) is a prep-
use of IVIG for necrotizing soft tissue
aration of polyclonal serum immunoglobulins,
infections is common. It is also used for a
typically IgG, from thousands of donors. It has
variety of autoimmune, inflammatory, and
been used as adjunctive therapy in critically ill
immunodeficiency diseases. A meta-analysis
patients with severe infections, i.e. sepsis,
of the clinical studies available for IVIG use
in group A streptococcal toxic shock syn-
M. B. Madsen drome indicates a survival benefit. A blinded,
Department of Intensive Care, Copenhagen University placebo-controlled clinical trial (INSTINCT)
Hospital, Rigshospitalet, Copenhagen, Denmark
assessed the effect of IVIG in 100 intensive
H. Bergsten · A. Norrby-Teglund (*) care unit patients with necrotizing soft tissue
Center for Infectious Medicine, Department of Medicine
Huddinge, Karolinska Institutet, Karolinska University infections, including all bacterial etiologies.
Hospital, Huddinge, Sweden The study did not demonstrate any effect on
e-mail: Anna.norrby-teglund@ki.se

self-reported physical functioning at 6 months. into different groups (heavy (IgM), regular (IgA,
In this chapter, we review the mechanisms of IgE, IgD, and IgG), and light (light chain dimers)
action of IVIG and the clinical studies that are (Gordon 1987). The product was used to prevent
available for necrotizing soft tissue infections measles and hepatitis, and for the treatment of
as well as severe group A streptococcal sepsis due to pneumococci in pediatric patients
infections. with agammaglobulinemia (Bruton 1952).
Immunoglobulins have also been used for a vari-
Keywords ety of autoimmune and inflammatory diseases.
The first reports of its use in patients with toxic
Intravenous immunoglobulin · Streptococcus
shock syndrome caused by group A streptococcus
pyogenes Group A streptococcus ·
(GAS) were published in 1992 (Barry et al. 1992;
Superantigen neutralization · Necrotizing soft
Stegmayr et al. 1992).
tissue infections
In this chapter we will review the structure and
function of immunoglobulins and discuss the
Highlights
indications of intravenous immunoglobulins with
• IVIG can neutralize virulence factors
emphasis on necrotizing soft tissue infections.
expressed by group A streptococcus and
Staphylococcus aureus.
• Epidemiologic studies of IVIG use in strepto-
8.2 Immunoglobulins: Structure
coccal toxic shock syndrome demonstrate a
and Function of Different Ig
survival benefit in clindamycin treated
Isotypes
patients.
• Only one RCT of IVIG versus placebo has
Immunoglobulins (Ig) are heterodimeric
been conducted in patients with necrotizing
Y-shaped glycoproteins, highly abundant in
soft tissue infection, and no difference in phys-
plasma with concentrations of 7–12 g/L. The Ig
ical quality of life between the two groups was
molecules are composed of two identical heavy
demonstrated.
polypeptide chains and two identical light chains
bridged together by disulphide bonds (Schroeder
and Cavacini 2010). Each chain contains an NH2-
8.1 Introduction
terminal variable region and a COOH-terminal
constant (Fc) region (Fig. 8.1). Functionally, the
Immunoglobulins, also known as antibodies, are
molecules are separated by a hinge region into a
glycoproteins secreted by plasma cells, and are an
variable, antigen binding, Fab part, and a constant
important part of the immune system where they
Fc-part that mediate immunomodulatory effector
help neutralize pathogens. Immunoglobulins for
function through complement interaction and
medical use are made from human serum and are
binding to cellular Fc receptors (Schroeder and
used for a variety of indications. Already in 1890,
Cavacini 2010) (Fig. 8.1). The Fc region of the
von Behring and Kitasato reported of an agent in
heavy chain defines the five classes (isotypes) of
the blood that could neutralize diphtheria toxin—
Ig: IgA, IgD, IgE, IgG, and IgM (Black 1997).
“Antikörper,” and proposed that this agent in
IgG exists in four subclasses IgG1-IgG4, and IgA
serum could react against foreign antigens
in two subclasses IgA1 and IgA2. The isotypes
(Schroeder and Cavacini 2010). In the 1930s,
and Ig subclasses differ with respect to abun-
clinical studies by McKhann showed that placen-
dance, location, and effector function.
tal extracts could prevent measles (Robinson and
IgG is the most abundant Ig isotype in serum
McKhann 1935), and by 1939, electrophoresis
and provides the majority of pathogen-specific
and later cold fractioning made it possible for
antibodies involved in humoral immunity
serum to be separated into albumin and globulin
(Vidarsson et al. 2014). The four IgG subclasses
fractions, and immunoglobulins to be separated
vary greatly in abundance with IgG1 being most
8 Treatment of Necrotizing Soft Tissue Infections: IVIG 107
Fig. 8.1 Schematic model

of the immunoglobulin
structure. Fab fragment
antigen binding, Fc
fragment constant, VL
variable region of the light
chain, CL constant region
of the light chain, VH
variable region of the heavy
chain, CH constant region
of the heavy chain,
SS disulfide
abundant (60%) followed by IgG2 and consider- secretory component; a residual polypeptide of
ably less of IG3 and IgG4. In addition, the the extracellular proteolytic fragment of the poly-
subclasses vary in function due to differences in meric Ig receptor required for transport of dimeric
the Fc region. IgG1 and IgG3 have a stronger IgA into the secretions. The complex of dimeric
binding to C1q and to Fc receptors (FcR), and IgA, J chain and secretory component is referred
are common in response to protein antigens to as secretory IgA (sIgA). In serum, monomeric
(Vidarsson et al. 2014). In contrast, IgG2 domi- IgA is more common. Human IgA exists in two
nate responses to bacterial polysaccharides subclasses, IgA1 and IgA2, and IgA1 is the domi-
(Ferrante et al. 1990; Siber et al. 1980), while nant subclass in serum as well as in many muco-
IgG4 to allergens (Vidarsson et al. 2014). sal secretions. However in colon, IgA2 is most
IgA is the second most abundant isotype in frequent (Brandtzaeg and Johansen 2005).
serum, and the principal Ig isotype in all mucosal IgM is the first isotype to be produced in a
secretions such as saliva, tears, and secretions humoral immune response and it offers an impor-
covering the respiratory, gastrointestinal, and uro- tant early defense against infections. IgM exists in
genital tract (Woof and Kerr 2006). IgA therefore a large pentameric form, displaying 10 antigen-
represents an important first line of defense binding sites and consequently high avidity inter-
towards inhaled or ingested pathogens, action with antigens. The pentameric structure
neutralizing toxins and inhibiting colonization of also supports an efficient complement activation
epithelial surfaces, while at the same time (Schroeder and Cavacini 2010).
tolerating commensals and innocuous antigens. Both IgD and IgE are found at very low levels
In mucosal secretions, IgA exists predominantly in serum. Despite this, IgE is highly potent and
in a dimeric form where the two IgA molecules predominates in the immune response to allergens
are linked together by a small polypeptide, called and helminths parasites.
J chain. The dimeric IgA also includes the
8.3 IVIG Preparations autoantibodies) and antibodies (anti-idiotypic

antibodies). As a consequence of the plasma
8.3.1 Manufacturing pool originating from different donors as well as
and Composition variations in manufacturing procedures, IVIG
preparations will vary in their final Ig composi-
IVIG is prepared from pooled plasma from sev- tion and their functionality. This is evident not
eral thousands of blood donors and the resulting only between different IVIG preparations but also
polyvalent Ig preparation is supplied either in a between batches of the same IVIG preparation
liquid or a lyophilized form. As a human blood- (Dhainaut et al. 2013; Norrby-Teglund et al.
derived product, manufacturing is tightly 1998).
regulated starting with the selection and testing
of individual donors to ensure quality and safety
control (Medicines Agency E 2018; WHO 2020). 8.3.2 Mechanisms of Action
Important quality and safety aspects include
retained structural and functional characteristics IVIG has several mechanisms of actions
of the Ig molecules, no infectious agents, no attributed to the dual functionality of Ig
procoagulant activity and absence of agglutina- molecules: (1) as cell-surface receptors allowing
tion of anti-A and anti-B hemagglutinins for cellular signaling and activation, and (2) as
(Radosevich and Burnouf 2010). To this end, soluble effector molecules binding and
the manufacturing of IVIG include several neutralizing antigens. This results in a direct
procedures to remove such factors that might be targeting of disease-causing antigens or agents,
present in the plasma pool, including not the least and numerous immunomodulatory functions that
multiple steps (e.g. solvent/detergent treatment, have been implicated in the mechanistic action of
pasteurization, and nanofiltration) to reduce the IVIG in infections, as well as in autoimmune and
risk of disease transmission including viruses and acute inflammatory diseases (Gelfand 2012;
prions (for further detail see Sect. 8.3.4). Schwab and Nimmerjahn 2013). In the subsection
There are many IVIG preparations available below, we focus on the role of IVIG in acute
on the market, most of which are composed of infectious diseases, such as sepsis, toxic shock
IgG, but there are also preparations enriched in syndrome and necrotizing soft tissue infections
IgM and IgA (Table 8.1). The IgG subclass dis- (NSTI) with special attention on how IVIG can
tribution in the IVIG preparation is comparable to neutralize specific virulence factors. However,
that found in serum with IgG1 being most abun- also the potent immunomodulatory functions of
dant followed by IgG2, and only small amounts of IVIG are of key importance as these conditions
IgG3 and IgG4. The Ig molecules are polyvalent are characterized by dysregulated host responses
and offers a broad spectrum of antibodies specific (Johansson and Norrby-Teglund 2012; Norrby-
for a variety of foreign antigens (e.g. viral, bacte- Teglund et al. 2003; Steinhagen et al. 2020).
rial, parasitic, etc.), but also antibodies to self- The immunomodulatory effects of IVIG are
antigens, such as towards cytokines (natural numerous and include both Fc- and
Table 8.1 Different types of IVIG preparations used in randomized placebo-controlled trials of IVIG as adjunctive
therapy for severe acute bacterial infections
Commercial name Ig content Study subjects Reference
Privigen >98% IgG NSTI Madsen et al. (2017)
Endobulin >98% IgG STSS Darenberg et al. (2003)
Polyglobin >97% IgG Sepsis Werdan et al. (2007)
Trimodulin 56% IgG; 23% IgM; 21% IgA Severe CAP Welte et al. (2018)
Pentaglobin 76% IgG; 12% IgM; 12% IgA Sepsis, septic shock Rodríguez et al. (2005)
NSTI necrotizing soft tissue infections, STSS Streptococcal toxic shock syndrome, CAP community acquired pneumonia
Fab-mediated effects (Schwab and Nimmerjahn this, IVIG was explored as a source of such pro-
2013). Fab-dependent effects comprise (1) killing tective antibodies towards streptococcal virulence
of target cells through antibody-dependent factors. Studies revealed that IVIG contains
cytotoxicity, (2) blockade of cellular receptors broad spectrum antibodies that efficiently neutral-
and consequently cell–cell interactions, (3) neu- ize bacterial virulence factors shown to be key
tralization of autoantigens (e.g. cytokines, FAS contributors to severe GAS infection (Table 8.2).
or FAsL, sialic acid-binding Ig-like lectin This is not surprising, considering the abundance
(SIGLECs) expressed on neutrophils and of asymptomatic carriage (especially in children)
eosinophils) and autoantibodies by anti-idiotypic and frequency of mild streptococcal diseases
antibodies, and (4) scavenging of the in the population (Carapetis et al. 2005). The
anaphylatoxins C3a and C5a. The Fc-dependent antibodies target both surface attached virulence
effects comprise (1) blockade of activating factors, including the classical virulence factor M
Fc-gamma-receptors, (2) saturation of neonatal protein, resulting in increased opsonophagocytic
FcR (FcRn) resulting in an accelerated IgG catab- killing (Table 8.2 and Fig. 8.2). In a recent
olism, (3) expansion of regulatory T cell popula- study applying immunoprecipitation of GAS
tion, (4) modulation of FcγR expression on innate cell wall extracts revealed a broad range of
immune cells and B cells, and modulation of surface proteins, e.g. M protein, C5a peptidase,
dendritic cells. FcγRIIIB has been shown in sev- S. pyogenes cell envelope protease (SpyCEP),
eral studies to be involved in the immunomodula- phosphoglycerate kinase, that were recognized
tory activities of IVIG. This inhibitory FcR is by IVIG (Reglinski et al. 2015). Also, secreted
either upregulated on myeloid cells or the number factors including superantigens, proteases,
of myeloid cells expressing this receptor is DNases, and cytolytic toxins are inhibited by
increased following IVIG administration (Bruhns IVIG, which leads to reduced pro-inflammatory
et al. 2003; Kaneko et al. 2006; Samuelsson responses (Table 8.2).
et al. 2001). Many of these IVIG-mediated effects Similarly, IVIG has been reported to contain
result in immunosuppressive and anti- antibodies that neutralize the activity of S. aureus
inflammatory responses, which are likely to virulence factors involved in toxic shock syn-
have impact on hyperinflammatory conditions drome and NSTI, including superantigens and
like NSTI and toxic shock syndrome (Norrby- cytolytic toxins such as Panton Valentine
Teglund et al. 2000). However, it should be leukocidin (PVL) and α-hemolysin (Table 8.2).
noted that the vast majority of studies IVIG was tested in an in vivo model of strep-
investigating IVIG-mediated immunomodulation tococcal toxic shock syndrome using HLA class
have been performed in autoimmune or inflam- II transgenic mice with increased susceptibility
matory diseases. to GAS superantigens (Sriskandan et al. 2006).
With regard to IVIG mediating neutralization The results revealed that IVIG treatment
of virulence factors through specific antibodies, resulted in improved bacterial clearance, neutrali-
more data are available. In sepsis, studies have zation of circulating superantigen, reduced
reported that low Ig-levels are associated with pro-inflammatory responses, and reduce illness
increased mortality (Bermejo-Martín et al. 2014; severity. Similarly, in an experimental murine
Martin-Loeches et al. 2017). A similar association model of necrotizing fasciitis caused by GAS,
between lack of protective humoral immunity and IVIG administration resulted in smaller lesions
severity of infection has been reported in GAS and reduced levels of several important GAS
infections, where patients with streptococcal toxic virulence factors, including the SLO, Sda1, and
shock syndrome or NSTI have lower levels of SpyCEP (Tarnutzer et al. 2019). However, in this
protective antibodies to streptococcal virulence study, no effect on bacterial load in treated and
factors than patients with less severe GAS non-treated mice, indicating that the clinical treat-
infections (Babbar et al. 2018; Eriksson et al. ment effect noted in the infected mice was linked
1999; Norrby-Teglund et al. 1994). In light of to neutralization of virulence factors.
Table 8.2 Neutralizing antibodies in IVIG that target streptococcal and staphylococcal virulence factors
Virulence factors IVIG effect References
Group A streptococcus
M proteins Opsonophagocytic killing Reglinski et al. (2015), Basma et al. (1998)
Serine protease Inhibition of IL8 cleaving Tarnutzer et al. (2019)
SpyCEP activity
Superantigens Neutralization of immune Sriskandan et al. (2006), Schrage et al. (2006), Norrby-
stimulation Teglund et al. (1996a)
Streptolysin O Inhibition of hemolytic activity Tarnutzer et al. (2019)
(SLO)
The DNase Sda1 Inhibition of DNase activity Tarnutzer et al. (2019)
SDSE
Streptokinase Inhibition of Ska-mediated Andreoni et al. (2018)
(Ska) fibrinolysis
Staphylococcus aureus
Cell wall proteins Opsonophagocytic killing Reglinski and Sriskandan (2019)
Superantigens Neutralization of immune Darenberg et al. (2004), Darville et al. (1997), Takei et al.
stimulation (1993)
LukAB (LukGH) Inhibition of cytotoxic activity Wood et al. (2017)
PVL Inhibition of cytotoxic activity Diep et al. (2016), Mairpady Shambat et al. (2015),
Gauduchon et al. (2004)
α-hemolysin Inhibition of cytotoxic activity Diep et al. (2016), Mairpady Shambat et al. (2015), Farag et al.
(2013)
SDSE Streptococcus dysgalactiae subspecies equisimilis; Sda Streptodornase 1, SLO Streptolysin O, SpyCEP
S. pyogenes cell envelope protease, Luk Leukocidin, PVL Panton Valentine Leukocidin
IVIG was also shown to be protective against superantigens SSA, Spe-A, Spe-C, and Spe-K/L
necrotizing pneumonia caused by MRSA strains were more efficiently neutralized by IVIG as
in an in vivo rabbit model and in particular compared to Spe-J, Spe-H, and SmeZ-2. Notably,
antibodies towards the pore-forming toxins within the concentration range tested none of the
α-hemolysin and PVL were implicated in the preparations could completely block the activity
protective effect (Diep et al. 2016). Also of SPE-J. This has implication for the dosage of
Mairpady Shambat et al. reported that IVIG IVIG used and clinical efficacy. However, in
neutralized the effect of α-hemolysin and PVL studies where supernatants from GAS strains
thereby attenuating tissue pathology in a human have been used, even from Spe-J-positive strains,
organotypic model of lung (Mairpady Shambat the superantigenic activity has always been
et al. 2015). completely blocked by IVIG (Norrby-Teglund
One important aspect is whether all IVIG et al. 1998, 1996a, b; Emgård et al. 2019). Most
preparations are equally efficient. Studies have of the studies described above have utilized IgG
investigated this and reported that variation do preparations. However, it should be mentioned
exist between different preparation in their effi- that in studies comparing the IgG versus IgM-
cacy to neutralize different virulence factors and IgA-enriched IVIG preparations differences
(Norrby-Teglund et al. 1998; Schrage et al. have been noted. IgM-enriched IVIG has been
2006; Wood et al. 2017). When different IVIG reported to contain higher anti-LPS antibody
preparations were tested for neutralization of titers in the IgM fraction (Trautmann et al.
broad panel of streptococcal superantigens, sig- 1998), as well as increased opsonic activity
nificant differences were demonstrated between towards the Gram-negative bacteria Pseudomo-
preparation but also towards different nas aeruginosa, E coli, and Klebsiella
superantigens (Schrage et al. 2006). The pneumoniae, as compared to IVIG containing
Fig. 8.2 Mechanisms of action of IVIG in severe invasive group A streptococcal infections. SAg superantigen, SLO
streptolysin, SdaI streptodornase I, APC antigen presenting cell
only IgG (Garbett et al. 1989). A similar differ- 8.3.3 Therapeutic Indications
ence was also reported in regard to neutralization
of mitogenic and cytokine-inducing activity of IVIG is mostly used as substitution treatment or
one of the group A streptococcal superantigens, for immunomodulatory purposes. According to
i.e. Spe-A (Norrby-Teglund et al. 1998). Whether the U.S. Food and Drug Administration (FDA)
these noted differences would translate into a and the European Medicines Agency, IVIG is
varying clinical efficacy has not yet been approved for replacement therapy in primary
explored. immunodeficiencies with impaired antibody pro-
duction and secondary immunodeficiencies with
recurrent infections or low IgG levels, as well as but this technique renders less plasma. Donation
for immunomodulatory effect in primary immune practices vary between countries; in some places,
thrombocytopenia, acute inflammatory demyelin- donation is based on a voluntary basis, and some
ating polyneuropathy (Guillain–Barré syndrome), have systems where the donor is compensated
Kawasaki disease, and Chronic inflammatory economically (Farrugia et al. 2015). Plasma
demyelinating polyradiculoneuropathy derived from the national blood services is sold
(Table 8.3 and Fig. 8.3) (European Medicines to the medicine industry. Almost all plasma
Agency 2020). IVIG can also be used as prophy- derived products are manufactured by the
laxis against hepatitis A and measles. In industry.
outbreaks of measles, IVIG is often Transmission of viral disease was previously a
recommended for healthy, non-immune persons concern to patients receiving IVIG (Siegel 2006),
(Arciuolo et al. 2017; Matysiak-Klose et al. 2018; but few cases of viral transmission have been
Tunis et al. 2018). There is also widely accepted reported through time. Cold ethanol fractioning
off-label use of IVIG in several additional was used as part of the manufacturing process,
conditions, supported by positive results but in and this removed most viruses. No reports of HIV
small-scale trials. In short, there is some level of transmission exist, but in the nineties two series of
evidence for the use of IVIG against rheumatoid incidents of hepatitis C virus transmission were
and neurological autoimmune/inflammatory published (Farrugia and Quinti 2014; Power et al.
diseases (Ballow 2011; Viard et al. 1998; Li 1994; Bresee et al. 1996). Consequently, the FDA
et al. 2005; Ephrem et al. 2008; Perlmutter et al. demanded viral removal and deactivation to be
1999), viral infections (Elsterova et al. 2017; included in the preparation process. Today, most
Vanderven and Kent 2020), bacterial infections donor blood is screened for syphilis, hepatitis B,
(Kakoullis et al. 2018; Ohlsson and Lacy 2020), C, and E and HIV and human T-lymphotropic
hematological conditions (Chang et al. 2008; virus (NHS 2019).
Sultan et al. 1984), obstetric (Egerup et al. A variety of different methods are used to
2015), and pulmonological conditions (Wade prevent transmission of virus. In the quest to
and Chang 2015). How common off-label usage manufacture Ig preparation, Cohn developed
of IVIG is for these indications is currently cold ethanol fractioning, which separated plasma
unknown by the authors of this work. proteins (Cohn et al. 1940). Addition of pepsin or
incubation at low pH also help eliminate anti-
complementary activity (Gordon 1987). This pro-
8.3.4 Safety cess itself eliminates HIV, but not HCV (Siegel
2006), and was the only elimination process for
Immunoglobulin use was generally considered many years.
safe until the early 1990s, where patients who The main methods for removing or
received immunoglobulins were infected with inactivating virus are described in Table 8.4
hepatitis C virus (Chapel 1999). In this section
we will review the safety profile of 8.3.4.2 Supply
immunoglobulins for medical use, with emphasis Immunoglobulin is a scarce blood product.
on use for patients with NSTI. Immunoglobulin use is increasing
(Sundhedstyrelsen 2016; Blood 2019), and the
8.3.4.1 Donor Selection indications for its use are expanding. Data from
and Manufacturing Process Australia suggest that the majority of immuno-
Plasma donation varies slightly from whole blood globulin use is for acknowledged indications, and
donation, as a different technique is used in order the increase is partly due to variations in adminis-
to obtain more plasma and reinfuse the remaining tration frequency, dosage, and population, and
blood products in the patient. It is also possible to not as a consequence of off-label use (Blood
extract plasma from a “whole blood donation,” 2019). However, a report from Canada suggests
Table 8.3 Indications for treatment with IVIG

Disease Indication Dosage Main mechanism
Substitution
Primary humoral Congenital agammaglobulinemia and 0.4–0.8 g/kg Substitution of Ab
immunodeficiency hypogammaglobulinemia, common initially, then (Ammann et al. 1982;
variable immunodeficiency, severe 0.2–0.8 g/kg every Oates et al. 1991)
combined immunodeficiency, 3–4 weeks
Wiskott–Aldrich syndrome
Secondary humoral Blood or bone marrow disorders - 0.2–0.4 g/kg every Substitution of Ab
immunodeficiency especially chronic lymphocytic 3–4 weeks (Raanani et al. 2009)
leukemia (CLL) or multiple myeloma
(MM) with hypogammaglobulinemia
and recurrent bacterial infections,
congenital acquired
immunodeficiency syndrome (AIDS)
with recurrent infections, in rare cases:
patients treated with
immunosuppressant/'biological' drugs
or undergoing chemotherapy,
transcobalamin deficiency, gut
lymphangiectasia
Hematopoietic stem cell Hypogammaglobulinemia, earlier also 2 g/kg during Substitution of Ab,
transplantation, allogenic prophylaxis at transplant surgery and 4 days, then infection prophylaxis,
treatment of graft versus host disease 0.2–0.4 g/kg every decrease donor
3–4 weeks specific Ab (NIH
1990; Jamil and
Mineishi 2015)
Immune modulation
Immune thrombocytopenia/ Life threatening bleeding due to ITP, 0.8–1 g/kg Fc-rec blockade,
idiopathic thrombocytopenic prophylaxis during surgery one/twice during decrease auto-Ab
purpura 3 days, or 0.4 g/kg (Ballow 2011; Imbach
every day during et al. 1981)
2–5 days
Kawasaki disease First line of treatment with 2 g/kg once Decrease
acetylsalicylic acid pro-inflammatory
cytokines, neutralize
bacterial toxins
(Ballow 2011;
Eleftheriou et al.
2014)
Acute inflammatory First line of treatment 0.4 g/kg Decrease auto-Ab
demyelinating daily during 5 days (Ballow 2011;
polyneuropathy/Guillain– for AIDP, 2 g/kg Finsterer 2005)
Barré syndrome, Chronic during 2–5 days,
inflammatory demyelinating then 1 g/kg every
polyneuropathy, Multifocal third week for
motor neuropathy CIDP and MMN
Passive immunity
Hepatitis A prophylaxis Pre/post-exposure for patients with 0,0032 g/kg Passive immunity
immunodeficiency disorders intramuscularly (FASS 2020; Askling
once and Herzog 2019)
Common off-label clinical practice
Measles Post-exposure for non-immune 0.4 g/kg within Passive immunity
persons 72 h (Arciuolo et al. 2017;
Matysiak-Klose et al.
2018; Tunis et al.
2018)
(continued)
Table 8.3 (continued)

Disease Indication Dosage Main mechanism
Myasthenia gravis Second line of treatment—severe 2 g/kg during Decrease auto-Ab
symptoms, myasthenic crisis 5 days, then 0.4 (Ballow 2011; García-
g/kg every month Carrasco et al. 2007;
Zinman et al. 2007;
Barth et al. 2011)
Streptococcal toxic shock Adjunctive treatment 0.5–2 g/kg initially, Neutralize bacterial
syndrome followed by lower toxins, opsonization of
doses bacteria (Parks et al.
2018; Linnér et al.
2014)
Necrotizing soft tissue Streptococcal or staphylococcal 0.5–2 g/kg initially, Neutralize bacterial
infections /fasciitis etiology, adjunctive treatment followed by lower toxins, opsonization of
doses bacteria (Madsen et al.
2017; Bergsten et al.
2020)
The dosages are given intravenously unless otherwise specified. Ab indicate antibodies. Subcutaneous alternatives are
available for primary immunodeficiencies and chronic inflammatory demyelinating polyneuropathy
Fig. 8.3 FDA-registered compounds with human normal syndrome, HSCT hematopoietic stem cell transplantation,
polyclonal immunoglobulin and approved indications. allogenic, ITP idiopathic thrombocytopenic purpura, IVIg
AIDP acute inflammatory demyelinating polyneuropathy, intravenous immunoglobulin, MM multiple myeloma,
AIDS acquired immune deficiency syndrome, CIDP MMN multifocal motor neuropathy, PID primary humoral
chronic inflammatory demyelinating polyneuropathy, immunodeficiency, SCIg subcutaneous immunoglobulin,
CLL chronic lymphocytic leukemia, GBS Guillain–Barré SID secondary humoral immunodeficiency
Table 8.4 Methods for removing or inactivating of virus

Cold ethanol fractioning (Cohn Involves increasing ethanol fraction, decreasing temperature and decreasing pH
process)
Octanoic/caprylic acid Addition of caprylic acid will make non-immunoglobulin proteins precipitate
fractioning
Filtration (Ultra-, dia- and A membrane separation process, where components are removed based on their
nanofiltration) solvated size and structure
Anion exchange chromatography Separation of molecules based on their net surface charge
Pasteurization Heating in solution
Low pH incubation
the opposite (Foster et al. 2010). Plasma-self-suf- Adverse effects can be divided into immediate,
ficiency is not possible in a number of delayed, and late. As for all medications, infusion
countries—even in countries with high levels of of intravenous immunoglobulin may result in an
voluntary blood donation—and they are reliable inflammatory response resulting in headache,
on plasma bought commercially. flushing, tight chestedness, dyspnea, and circula-
tory collapse (anaphylactic reaction). Headache is
the most frequent adverse reaction, and although
8.3.4.3 Batch and Preparation Variation
symptoms such as headache, malaise, and myal-
Different manufacturers use different
gia occur in about 5% of patients, these often
compositions of immunoglobulin (Siegel 2015).
abate with a reduction in infusion rate. Rash is
Furthermore, as IVIG is extracted from human
seen in approximately 6% of patients.
plasma, the content and thus the neutralizing
As immunoglobulin preparations contain
properties are subject to variation, dependent on
varying amounts of IgA, patients with total IgA
the exposure of the donors (Shankar-Hari et al.
deficiency may develop anti-IgA antibodies, ulti-
2012).
mately resulting in anaphylactic reaction
(Nydegger and Sturzenegger 1999).
8.3.4.4 Adverse Reactions of IVIG Acute renal failure has been described in
Infusion patients with known impairment of renal function
It is difficult to get an overview of adverse (Cantú et al. 1995). The proposed mechanism
reactions as use is indicated for a wide variety of might be the presence of sucrose, which is used
diseases. Furthermore, dosage, number of as a stabilizing agent, but this is merely specula-
infusions, infusion rate and composition of the tive (Nydegger and Sturzenegger 1999). Serum
product vary. Consequently, different studies viscosity may increase as a result of high IgG
have focused on different aspects of safety concentrations, immune complex formation, and
profiles. immunoglobulin aggregates, and may cause
According to the summary of product symptoms as headache and visual blurring, and
characteristics, common adverse reactions could result in impaired circulation in patients
include chills, headache, dizziness, fever, with atherosclerosis or other conditions that result
vomiting, allergic reactions, nausea, arthralgia, in poor vascular tone. Aseptic meningitis is a
low blood pressure, and moderate lower back condition mimicking bacterial meningitis with
pain. Severe adverse reactions include hypersen- affection of the cerebrovascular fluid, but the
sitivity, hemolytic anemia, aseptic meningitis mechanism is not clear. In a study on patients
syndrome, thromboembolism, acute renal failure, with neuromuscular diseases receiving IVIG,
transfusion-related acute lung injury, and trans- 6/54 (11%) developed severe symptoms with
missible agents (FDA 2019; Pierce and Jain acute, severe headache, stiffneckedness, lethargy,
2003). fever, and photophobia (Sekul et al. 1994).
The definitions of adverse events, and the 8.4 Rationale for the Use of IVIG
collection and reporting of safety data are for NSTI
not standardized. Overall, the trials assessing
effects of IVIG in general did not have the 8.4.1 Laboratory Data
statistical power to detect differences in adverse
events and serious adverse reactions, and the Patients with severe GAS infections including
clinical trials performed to support marketing NSTI have been reported to have lower levels of
applications for IVIG had insufficient power to protective antibodies towards several important
detect adverse events (Pierce and Jain 2003). GAS virulence factors including Spe-B and
Regarding IVIG use in patients with NSTI, superantigens, as compared to uncomplicated
only two trials have systematically collected cases such as GAS tonsillitis or erysipelas cases
data on adverse reactions of IVIG (Darenberg (Babbar et al. 2018; Eriksson et al. 1999; Norrby-
et al. 2003; Madsen et al. 2017); for a review of Teglund et al. 1994). IVIG has been shown to
these trials, please see the section “Rationale for contain protective antibodies targeting GAS viru-
the use of IVIG for NSTI” below. In trials of IVIG lence factors, which are efficiently transferred to
for sepsis and septic shock, the reporting of the patients upon IVIG administration as evident
adverse events is scarce (Pildal and Gotzsche by an increased neutralizing activity in plasma
2004). In one of the larger trials (n ¼ 653), a collected post-IVIG therapy (Tarnutzer et al.
total of 19 adverse events were reported in 2019; Norrby-Teglund et al. 1996a, b; Basma
17 patients; 13 events in 11 patients in the IVIG et al. 1998).
group, and 6 events in 6 patients in the placebo Important points for discussion are what
group (Werdan et al. 2007). The adverse events in dosages of IVIG that are efficacious, and
the IVIG group included exanthema, thrombosis, concentration-levels achieved at the tissue site of
cardiac failure and arrest, anaphylactic reaction, infection. There are, as of yet, no clinical IVIG
intracerebral hemorrhage, overhydration, and dosage studies performed on NSTI or toxic shock
acute kidney injury. syndrome patients. However, insight was gained
In an observational study with the specific aim through analyses of plasma collected from
of studying the safety profile of IVIG use in a patients included in the INSTINCT and the
general ICU population, acute kidney injury INFECT studies (Bergsten et al. 2020). The
(AKI) (defined as a rise in creatinine 20% plasma analyses revealed that a dose of 25 g of
within 28 days of IVIG admission) occurred in IVIG was sufficient to yield neutralizing activity
117 of 145 (81%) patients, and 30 (21%) required towards GAS superantigens. However, the level
renal replacement therapy (Foster et al. 2010). of inhibition varied between strains, and towards
Although most patients had other factors that some GAS strains the inhibition was only border-
may have contributed to renal insufficiency, line protective. Taken together with the report by
10% had no obvious concurrent renal insult Bruun et al. (Bruun et al. 2020) on GAS NSTI
other than IVIG. The number of patients with patients in the observational INFECT study,
thromboembolism was 4 (3%). The risk of AKI which showed an association between patients
may be associated with pre-existing conditions who had not received IVIG and 90-day mortality,
such as sepsis and hypovolemia, but also with Bergsten et al. (Bergsten et al. 2020) proposed a
the specific product used (Siegel 2006). dosage regimen of: 0.5 g/kg bodyweight (or a
Lastly, it is worth considering the fact that minimum of 25 g) on day 1, followed by fixed
critically ill patients receive many different doses of 25 g daily for 1–2 additional days. The
therapies. In generally, little is known about higher dose on day 1 of 25 g offered only border-
drug interactions, and the addition of IVIG is line protective towards some strains in combina-
likewise poorly described. tion with often high levels of toxins and bacterial
load at this time point and at the tissue site. albumin. After inclusion of 21 patients, 10 receiv-
However, as acknowledged by the authors this ing IVIG (of whom 6 had NSTI) and 11 receiving
treatment regimen, i.e. dosages and treatment placebo (of whom 7 had NSTI), the trial was
days, should be evaluated in future trials. prematurely terminated due to slow inclusion,
Bioavailability of IVIG in the deep necrotic after enrolling only 21 of the intended
tissue has been questioned, and the possibility of 120 patients. The primary endpoint was 28-day
local applications of antibodies, as used for post- mortality and there was no difference between the
exposure prophylaxis in rabies (CDC 2020), has two groups; 1 of 10 patients in the IVIG group
been raised. In an in vivo model of GAS NSTI, had died, and 4 of 11 patients in the placebo
intraperitoneal and subcutaneous administration group ( p ¼ 0.30). After administration of IVIG,
of IVIG was compared (Tarnutzer et al. 2019). patient serum contained sufficient neutralizing
This study showed that the subcutaneous admin- antibodies to suppress proliferation of
istration yielded 1.6 times higher IgG concentra- T-lymphocytes to below pretreatment levels,
tion in the skin, but the serum concentration was whereas in the placebo group, no change in
half as high compared to intraperitoneal adminis- neutralizing activity was seen. The mean time to
tration. Subcutaneous injection also resulted in resolution of shock was 100 h for the IVIG group
reduced clinical severity (i.e. smaller lesion size) and 122 h for the placebo group. The mean time
as well as reduced SLO activity. to no further progression of necrotizing fasciitis
or cellulitis was 69 h for the IVIG group, com-
pared with 36 h for the placebo group. Besides
8.4.2 Clinical Studies death, a total of 12 adverse events were observed,
none of which were found related to the study
The proportion of patients receiving IVIG as part drug. None of these secondary endpoints were
of the treatment for NSTI, as well as approaches statistically significant.
to IVIG use varies. Typically, IVIG is considered The INSTINCT trial was a randomized, clini-
in NSTI patients where the bacterial etiology is cal trial including 100 patients with NSTI
highly suspicious of GAS (Anaya and Dellinger (Madsen et al. 2017). Patients with NSTI,
2007). In this setting, 25% of all intensive care irrespective of etiology, were included after
units participating in a survey reported to always admission to the intensive care. Patients were
use IVIG (de Prost et al. 2015), and in an randomized to receive IVIG 25 g/day for three
Australian observational study, 27% of patients consecutive days or placebo. The primary out-
received IVIG (Carapetis et al. 2014). Overall use come was physical quality of life assessed by
independent of bacterial etiology has been the physical component summary (PCS) score
described, with 58% of patients receiving IVIG of the Short Form-36 questionnaire (Ware et al.
(Madsen et al. 2019). 1992). No difference in the primary outcome
(mean PCS score 29 versus 28, mean difference
8.4.2.1 Randomized Clinical Trials 1 (95% CI 7 to 10)) or in secondary outcomes,
Despite the promising effects from in vitro stud- including mortality, serious adverse reactions
ies, only two trials have compared the effects of (SARs), organ failure, organ support, bleeding,
IVIG with placebo in patients with NSTI. In resolution of shock and days in hospital, was
1999, Darenberg and colleagues aimed to evalu- observed. SARs included acute kidney injury,
ate the efficacy and safety of IVIG as adjunctive allergic reactions, aseptic meningitis syndrome,
therapy in patients with streptococcal toxic shock hemolytic anemia, thrombi, and transmittable
syndrome (STSS) in a randomized, double blind, agents. In the preplanned subgroup analysis on
placebo-controlled trial (Darenberg et al. 2003). patients with presumed GAS infection, no differ-
Patients were randomly assigned to either IVIG ence was seen. Importantly, the distribution of
for three consecutive days (1 g/kg day one, 0.5 g/ patients with GAS or S. aureus was uneven
kg day 2 and 3) or an equal volume of 1% between the two intervention groups. Some
limitations of the trial deserve mentioning; the mortality rate was 27.3% in the IVIG group and
trial had reduced power, as patients who died 23.6% in the non-IVIG group (adjusted OR 1.00,
where given a PCS score of 0. Furthermore, 95% CI: 0.55–1.83).
20 (40%) patients in the placebo group had In a publication focusing on the subgroup of
received one dose of IVIG prior to enrolment. patients with GAS NSTI (Bruun et al. 2020) from
This might have diluted a potential effect. Lastly, the Scandinavian INFECT observational study
the trial was not powered to detect differences in (Madsen et al. 2019), the administration of IVIG
secondary outcomes, making it difficult to con- was associated with decreased risk of mortality by
clude whether IVIG use is safe in NSTI patients. Lasso Regression analysis, but not by ordinary
logistic regression analysis. Due to the observa-
8.4.2.2 Systematic Reviews tional design, these data may support the hypoth-
and Meta-Analyses esis that IVIG can have a beneficial effect in this
A systematic review from 2018 included only one subgroup of patients, but no causal conclusions
RCT on IVIG for NSTI, and thus could not add can be drawn.
additional data (Hua et al. 2018). In a meta- In an Australian surveillance programme of
analysis from 2018 (Parks et al. 2018), evaluating invasive GAS infections from 2002 to 2004, the
the effect of IVIG in patients with STSS, data mortality rate in patients treated with IVIG and
from four observational studies (Carapetis et al. clindamycin was lower (1/14 (7%)) than in those
2014; Kaul et al. 1999; Adalat et al. 2014; Linnér who were treated with clindamycin alone (7/39
et al. 2014) and one RCT (Darenberg et al. 2003) (18%)), but this was not statistically significant
were included. Studies were included if patients (Carapetis et al. 2014). In a similar Swedish sur-
were identified prospectively, met the consensus veillance programme of patients with STSS, also
criteria of STSS and whose antibiotic treatment from 2002 to 2004, 67 patients were identified of
included clindamycin. A total of 165 patients whom 23 received IVIG and 44 did not (Linnér
were included in the analysis; 70 received IVIG et al. 2014). At day 28, 3/23 (13%) in the IVIG
and 95 did not. IVIG use was associated with a group, and 22/44 (50%) in the non-IVIG group,
reduction in overall mortality rate from 33.7% to had died (OR for death 0.18 (95% CI: 0.04–0.83),
15.7% (RR, 0.46; 95% confidence interval, adjusted for SAPS II and use of clindamycin).
0.26–0.83; p ¼ 0.01). The analysis has The proportion of patients with necrotizing fasci-
limitations, the main being that data from 147 of itis was larger in the IVIG group; in a subgroup
the 165 patients included were from observational analysis of patients with necrotizing fasciitis (n ¼
studies, and thus no causative conclusions can be 19), the OR for death was 0.17 (95% CI:
drawn. The studies included used different 0.01–2.5). A third surveillance programme for
comparators, and for some of the studies, no invasive GAS infections in Ontario identified
information on dosing or timing was given. 62 patients from 1992 to 2002; 35 (56%) patients
received IVIG (Mehta et al. 2006). No difference
8.4.2.3 Observational Studies in ICU/hospital-mortality by univariate analysis
In a retrospective observational study by Kadri was seen. Lastly, a prospective observational
and colleagues, 4127 patients with necrotizing study enrolled patients with STSS in Canada in
fasciitis and shock from 121 centers were 1992–1994 (Kaul et al. 1999). A total of
identified based on diagnosis code (Kadri et al. 53 patients with STSS were included; OR for
2017). Only 164 of these patients had received death for the 21 patients receiving IVIG was
IVIG. These were propensity-matched and risk- 0.18 (95% CI: 0.03–1.02).
adjusted with those who had not received IVIG.
The groups were similar regarding age, 8.4.2.4 IVIG for Sepsis
diagnoses, comorbidities, day of IVIG dose and Several trials on IVIG for patients with sepsis/
destination of discharge of survivors. The primary septic shock have been conducted, and systematic
outcome was in-hospital mortality; the crude reviews have shown potential beneficial effects
on mortality (Soares et al. 2012; Turgeon et al. treatment. IVIG has been used as adjuvant ther-
2007), although results vary, depending on the apy for NSTI (de Prost et al. 2015), but not
inclusion criteria (Alejandria et al. 2013). Besides necessarily accounting for bacterial etiology,
general concerns regarding IVIG use (Shankar- and the neutralizing abilities of IVIG have pri-
Hari et al. 2018) (see section below), there are marily been shown in patients affected by NSTI
several other concerns that need attention, in par- caused by GAS or S. aureus.
ticular the quality of the included trials. Inclusion Questions on dosage, timing of administration,
criteria varied, as several definitions of sepsis and duration of treatment are still debated. Tradi-
were used. Sepsis is a heterogenous disease, and tionally, dosing has ranged from 0.5 g/kg to 2 g/
the patients response to infection may differ kg (Table 8.5). As discussed previously, one dose
according to the site infected (Shankar-Hari of 25 g seems to be sufficient to suppress toxin
et al. 2016; Burnham et al. 2017). Most of the activity in NSTI patients infected by GAS, and
trials were of a relatively small sample size, rang- after two doses, no toxin activity is seen
ing from 24 to 682 patients. Whereas IVIG in (Bergsten et al. 2020). However, this has not
NSTI patients is believed to function through a been confirmed in a clinical setting, and opti-
neutralizing effect of exotoxins and other bacte- mally, a randomized clinical trial on GAS NSTI
rial virulence factors, the mechanisms in sepsis is should be performed to assess whether this treat-
more poorly understood, and this patient group is ment has a beneficial effect and no unacceptable
even more heterogenous than NSTI patients. serious adverse reactions.
Lastly, the trials were conducted more than The optimal composition of immunoglobulins
10 years ago—some of them more than 20 years is not known. IgG has many potential
ago. In conclusion, there is no evidence to support mechanisms of action (Di Rosa et al. 2014), but
the use of immunoglobulin for treating patients whether an additional effect of adding IgM is
with sepsis or septic shock (Rhodes et al. 2017). achieved is not known. Furthermore, the
properties of the preparations are dependent on
the donors and thus immunomodulating
8.4.3 IVIG in NSTI, Unresolved Issues properties may vary.
Only few prospective studies have evaluated
The use of IVIG for patients with NSTI is a the safety profile of IVIG in patients with NSTI.
complex intervention, with multiple aspects that Of concern is the severity of the disease and
are unknown, or have not been addressed polypharmacy in these patients, with the possibil-
adequately. ity of drug interactions (Kane-Gill et al. 2012).
NSTI is not a well-defined disease, and NSTI
patients represent a heterogenic population. The
term encompasses many different variations of
8.4.4 Future Perspectives
severe soft tissue infections (Madsen et al.
2019). The varying nomenclature and classifica-
From in vitro and in vivo studies it has been
tion schemes also reflect this diversity; Necrotiz-
demonstrated that IVIG has the potential to alle-
ing Fasciitis, describing an infection along the
viate the course of NSTIs where GAS or
fascia in the subcutis; Fournier’s gangrene,
S. aureus is the causative agent. However, we
describing a progressing soft tissue infection
do not have data from randomized clinical trials
originating from the anogentital area.; Type 1
to show that this treatment is beneficial to patients
and 2 necrotizing fasciitis, depending on a poly-
and, just as important, is safe to use in patients
or monomicrobial etiology. It is likely, that the
that are critically ill. Furthermore, plasma is a
pathophysiology varies depending on the affected
scarce product. A large, multi-center RCT
body-part, mechanism of bacterial entry and bac-
evaluating the effect of IVIG on mortality in
terial etiology (Madsen et al. 2019). Especially
patients with GAS NSTI is therefore warranted.
the latter is of importance regarding IVIG
Table 8.5 Doses of IVIG used in patients with severe invasive infections, including necrotizing soft tissue infections
and toxic shock syndrome
Number of included
patients (number Initial IVIG Repeat Number of
Study Patient cohort receiving IVIG) dosage dosages treatment days
Kadri et al. (2017) Debrided NF with 4127 (164) 1 g/kg n/a 1b
shock (0.66–1)a
Madsen et al. NSTI admitted to the 100 (50) 25 g 25 g 2–3
(2017) ICU
Adalat et al. (2014) Streptococcal and 49 (10) n/a n/a n/a
staphylococcal TSS
Carapetis et al. Severe invasive GAS 84 (14) n/a n/a n/a
(2014) infections
Linnér et al. (2014) STSS 67 (23) 0.5 g/kg 0.5 g/kg 1–6
Mehta et al. (2006) Invasive GAS 62(45) 1.61 0.75 n/a
infections 0.72 g/kgc 0.52 g/kgc
Norrby-Teglund Severe GAS soft tissue 7 (7) 2 g/kg n/a 1–3
et al. (2005) infections
Arnholm et al. Invasive GAS 41 (11) 50 g (n ¼ 7) n/a 2-3
(2004) infections 80 g (n ¼ 1)
Darenberg et al. STSS 21 (10) 1 g/kg 0.5 mg/kg 3
(2003) day 2 and 3
Haywood et al. Streptococcal NF 20 (16) 0.001–0.002 g/ n/a n/a
(1999) kg
Kaul et al. (1999) STSS 53 (21) 0.2–2 g/kgd n/a Up to 5
Case-reports not included. NSTI necrotizing soft tissue infection, NF necrotizing fasciitis, ICU intensive care unit, TSS
toxic shock syndrome, GAS group A streptococcal infections, STSS streptococcal toxic shock syndrome
a
Median dose (IQR)
b
Median number of treatment days
c
Mean dosage
d
The median cumulative dose was 2 g/kg (range 0.2–3.6 g/kg)
Acknowledgement Financial support: The work was Anaya DA, Dellinger EP (2007) Necrotizing soft-tissue
supported by the European Union Seventh Framework infection: diagnosis and management. Clin Infect Dis
Programme: (FP7/2007–2013) under the grant agreement 44:705–710
305340 (INFECT project); the Swedish Governmental Andreoni F, Ugolini F, Keller N et al (2018) Immunoglob-
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Swedish Research Council under the frame of ERA rotizing fasciitis. J Infect Dis 217:270–279
PerMed (Project 2018-151, PerMIT). Arciuolo RJ, Jablonski RR, Zucker JR, Rosen JB (2017)
Effectiveness of measles vaccination and immune
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Pathogenic Mechanisms
of Streptococcal Necrotizing Soft Tissue 9
Infections
Nikolai Siemens, Johanna Snäll, Mattias Svensson,

Contents
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
9.2 Initial Steps of Tissue Infections: Bacterial Adhesins and Colonizing
Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
9.3 Secreted Molecules Contributing to Host Evasion and Tissue Pathology 130
9.3.1 Streptolysins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
9.3.2 Proteases and DNases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
9.3.3 The Streptococcal Inhibitor of Complement (SIC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
9.4 Local and Systemic Inflammation in NSTIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
9.4.1 Neutrophil Responses in NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
9.4.2 Macrophage Responses in NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
9.4.3 Superantigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
9.5 Biofilm and Complex Modelling of GAS NSTIs in the Human Tissue
Setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
9.6 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Abstract S. dysgalactiae subsp. equisimilis (SDSE,

most group G and C streptococcus), are the
Necrotizing skin and soft tissue infections
main causative agents of monomicrobial
(NSTIs) are severe life-threatening and rapidly
NSTIs and certain types, such as emm1 and
progressing infections. Beta-hemolytic
emm3, are over-represented in NSTI cases. An
streptococci, particularly S. pyogenes (group
arsenal of bacterial virulence factors contribute
A streptococci (GAS)) but also
to disease pathogenesis, which is a complex
and multifactorial process. In this chapter, we
N. Siemens (*) summarize data that have provided mechanis-
Department of Molecular Genetics and Infection Biology, tic and immuno-pathologic insight into host-
University of Greifswald, Greifswald, Germany pathogens interactions that contribute to tissue
e-mail: nikolai.siemens@uni-greifswald.de
pathology in streptococcal NSTIs. The role of
J. Snäll · M. Svensson · A. Norrby-Teglund streptococcal surface associated and secreted
Department of Medicine, Center for Infectious Medicine,
Karolinska Institutet, Huddinge, Sweden
factors contributing to the hyper-inflammatory

128 N. Siemens et al.
state and immune evasion, bacterial load in the patients, are monomicrobial infections, mostly due
tissue and persistence strategies, including to Gram-positive microorganisms (Harbrecht and
intracellular survival and biofilm formation, as Nash 2016; Morgan 2010). Among these, Strepto-
well as strategies to mimic NSTIs in vitro are coccus pyogenes (group A streptococcus; GAS)
discussed. remains the most common pathogen (Bruun et al.
2020; Madsen et al. 2019). Compared to type 1
Keywords NSTI, type 2 infections are more likely to occur in
younger patients often without underlying illnesses
Streptococcus · Necrotizing soft tissue
(Stevens and Bryant 2017).
infections · Pathogenesis · Neutrophils ·
It is estimated that more than 18 million people
Macrophages
worldwide develop invasive streptococcal
diseases annually (Sims Sanyahumbi et al.
Highlights
2016). Several prospective population-based
Necrotizing soft tissue infections (NSTIs) located
studies have reported skin and soft tissue as the
to the extremities are associated with
dominating foci of invasive streptococcal
monomicrobial infections caused predominantly
infections (Davies et al. 1996; Lamagni et al.
by group A streptococcus (GAS). Current data
2008; Moses et al. 2002). Depending on the geo-
indicate that the pathogenesis of GAS NSTIs is
graphic region and varying between years, the
associated with a variety of bacterial virulence
annual incidence rate is ranging from 1.4 to 4.7
factors, such as superantigens, cytolysins, and
severe cases per 100,000 populations (Kaul et al.
Sda1. IL-1β, CXCL9, CXCL10, and CXCL11
1997; Lamagni et al. 2008; Naseer et al. 2016;
have been implicated as potential biomarkers for
Nelson et al. 2016; Stevens et al. 1989). A Cana-
GAS NSTIs. Within the tissue, GAS have
dian prospective study on invasive GAS
evolved different strategies to promote bacterial
infections linked 48% of all cases to soft tissue
persistence and antibiotic resistance strategies.
infections and 24% met the criteria for NSTIs.
These include (1) residing within macrophages
Among these patients, 47% also presented with
and (2) biofilm formation. 3D-organotypic skin
streptococcal toxic shock syndrome (STSS).
tissue models recapitulate key anatomical and
NSTI patients who also developed STSS had a
functional features of the skin and their use as
mortality of 67%, as compared to only 4.9% of
infection model systems has provided novel
patients who did not (Kaul et al. 1997). Recently
insights into the pathogenesis of GAS NSTIs.
an international multicenter, prospective cohort
study, called INFECT, with the aim of studying
the clinical profile of NSTI patients was
9.1 Introduction
performed in Scandinavia (Madsen et al. 2018).
The INFECT study is the largest prospective
Necrotizing soft tissue infections (NSTIs) are rare,
cohort study in patients with NSTIs to date. In
severe, and rapidly progressing infections of the
total, 409 patients were included. 179 (44%) had
skin and soft tissue that account for significant
a monomicrobial infection, and GAS was pre-
morbidity and mortality (Anaya et al. 2005;
dominant bacterial species (126 [31%]). Among
Madsen et al. 2019; Morgan 2010). NSTIs can
GAS NSTI patients, 65% presented with STSS.
occur after traumatic injuries, including minor
All-cause mortality was 14% at day 30 and 18%
breaches of the skin or mucosa and
at day 90. However, GAS NSTIs were associated
non-penetrating injuries of the soft tissue (Stevens
with lower mortality rates (10%). Interestingly,
and Bryant 2017). Based on the invading
NSTIs located to the extremities were associated
microorganisms, NSTIs are commonly classified
with monomicrobial infections caused predomi-
into two types. Type 1 NSTIs are of polymicrobial
nantly by GAS (Madsen et al. 2019). Such
nature and are affecting around 70–80% of patients.
anatomical preference by GAS was also seen in
Type 2 NSTIs, affecting around 20–30% of
a study of a selected cohort of 148 patients from
9 Pathogenic Mechanisms of Streptococcal Necrotizing Soft Tissue Infections 129
the INFECT study by Thänert and colleagues 2009). In contrast, emm pattern E isolates account
(Thanert et al. 2019). 16S rRNA gene sequencing for almost equal fractions at both tissue sites
revealed that the diversity of the bacterial (Bessen et al. 2015). Epidemiological studies
communities was highly dependent on revealed that severe disease manifestations,
anatomical location of NSTIs. Upper and lower including NSTIs and STSS, are predominantly
extremities showed a Gini—Simpson diversity associated with emm1 and emm3 serotypes
index lower than 0.25 with significant detection (Bruun et al. 2020; Darenberg et al. 2007; Luca-
of streptococcal species. However, 16S rRNA Harari et al. 2009; Stevens 1999).
sequencing of tissue biopsies also revealed the The adhesion by GAS to epithelial surfaces is
abundance of obligate anaerobes in NSTI cases a two-step process. The first step is a relatively
(Thanert et al. 2019; Zhao-Fleming et al. 2019). weak and unspecific and overcomes electrostatic
repulsion. It was suggested that lipoteichoic acid
(LTA) is a mediator of the first step of adhesion.
9.2 Initial Steps of Tissue This interaction is reversible and not sufficient to
Infections: Bacterial Adhesins provide for tissue tropism (Courtney et al. 2002).
and Colonizing Factors The second step of adhesion may then involve
classical and non-classical surface proteins of
NSTIs can present with and without a defined GAS. These adhesins either bind directly to the
portal of entry (Stevens and Bryant 2017). In cell surface receptors or use extracellular host
approximately half of the cases the bacteria gain matrix proteins or plasma proteins as a prime
entry to the deep tissue through superficial target for specific interactions (Kreikemeyer
lesions, penetrating trauma or breaches of the et al. 2004). In skin infections, M proteins can
skin (Morgan 2010). In the other 50% of cases, mediate cell adhesion to keratinocytes directly via
NSTIs initiate deep in the soft tissue, sometimes CD46 binding (Okada et al. 1995) or indirectly by
at sites of a non-penetrating trauma (Kaul et al. binding human fibronectin as a target on host
1997; Stevens et al. 1989). It has been suggested epithelial cells (Courtney et al. 1986). In addition
that transient bacteremia originating from the to proteinaceous ligands, M proteins bind to
nasopharynx, a potential reservoir for glycosaminoglycans and that interaction
streptococci, is the source of such infections promotes GAS attachment to human skin
(Johansson et al. 2010; Stevens and Bryant fibroblasts (Frick et al. 2003b). There are other
2017). However, reports providing such evidence classical and non-classical adhesins described for
are lacking. GAS, including hyaluronic acid capsule that
The ability of GAS to establish an infection binds to CD44 on keratinocytes, fibronectin-
can be primarily attributed to the surface binding proteins FbaA, F2, serum opacity factor
anchored and associated adhesins. Among these, (SOF), Fbp54, ScpA, Lbp, and FbaB, collagen-
the streptococcal M protein is the most abundant binding protein Cpa, vitronectin-binding protein,
and probably best characterized protein, which is galactose-binding protein, and plasminogen-
also used for emm-typing (Beall et al. 1996). binding proteins Epf, GAPDH, enolase, and
Since its discovery more than 200 different emm phosphoglycerate kinase (PGK) (Rohde and
serotypes have been described and nearly all GAS Cleary 2016). Hence, GAS express a variety of
isolates harbor an emm gene (Bessen et al. 2015). surface factors that employ different mechanisms
In addition, many GAS strains encode emm-like contributing to bacterial adhesion. However,
genes, mrp and enn (Bessen et al. 2015). Based on (1) not all of them occur in all serotypes, (2) they
the chromosomal arrangement of these three are expressed at different bacterial growth cycles,
genes, five distinct patterns, A to E, are described. and (3) many of them are host cell type specific
Emm pattern A-C isolates show a significant asso- (Courtney et al. 2002). For example, M proteins
ciation with nasopharynx, while pattern D strains mediate adhesion to human keratinocytes, but not
are often isolated from skin infections (Bessen to buccal cells (Okada et al. 1994).
On another note, the secreted exotoxins, 9.3 Secreted Molecules

namely superantigens (SAgs), were recently Contributing to Host Evasion
shown to be critical for colonization. The reports and Tissue Pathology
showed that the SAgs elicited an inflammatory
response that promoted bacterial colonization and 9.3.1 Streptolysins
establishment of infection (Kasper et al. 2014;
Zeppa et al. 2016, 2017). This provided new To breach dermal barriers and enable the bacteria
insight into the function of this family of bacterial to disseminate through the tissue, GAS secrete
exotoxins, most well known for their several pore-forming exotoxins, proteases, and
pro-inflammatory activities and association with DNases (summarized in Table 9.1). Studies have
the cytokine storm characterizing toxic shock shown that secreted factors are highly
syndrome (detailed in separate subsections up-regulated during fulminant tissue infections
below). and play a crucial role in NSTIs (Johansson
Once the infection is established GAS can et al. 2010; Olsen and Musser 2010; Thanert
translocate to deeper tissue or initiate internaliza- et al. 2019). Pore-forming toxins are a class of
tion into the cells. Although GAS have long been bacterial virulence factors that cause cytolysis,
considered to be an extracellular pathogen, now it disrupt membranes, and have immune-
is appreciated that GAS can internalize into modulatory functions. Nearly all GAS isolates
non-phagocytic cells even to an equal level as secrete two potent hemolysins, namely Streptoly-
classical intracellular pathogens (Lapenta et al. sin S (SLS) and Streptolysin O (SLO) (Barnett
1994). For example, M protein forms complexes et al. 2015; Yoshino et al. 2010). SLO is a 57 kDa
with fibronectin and interacts with α5β1 integrins. oxygen sensitive, cholesterol-dependent cytoly-
Extensive integrin clustering triggers caveolae sin. It disrupts cytoplasmic membrane integrity
aggregation to form invaginations that ingest the of epithelial and endothelial cells, monocytes,
bacteria without detectable actin polymerization. macrophages, and neutrophils, thereby inducing
In that way intracellular bacteria bypasses fusion cell death through different mechanisms, includ-
with lysosome and intracellular streptococci ing apoptosis, necrosis, and/or pyroptosis
reside within caveolin-1-contating endosomes, (Chandrasekaran and Caparon 2016; Keyel et al.
the so-called caveosomes. Alternatively, M 2013; Timmer et al. 2009). For example, SLO
protein-fibronectin interaction leads to the cyto- blocks the clathrin-dependent pathway for GAS
skeletal rearrangement with actin accumulation internalization and induces keratinocytes apopto-
around the streptococci. This results in trafficking sis through dysregulation of the calcium signaling
of the bacteria inside the cells through early and (Cywes Bentley et al. 2005). At sub-lytic
late endosomes, which fuse with lysosomes to concentrations, SLO suppresses crucial neutro-
form phagolysosomes (Rohde and Cleary 2016). phil functions, including chemotaxis and forma-
The first mechanism directs GAS to the safer tion of neutrophil extracellular taps (NETs)
caveosomal compartment, while the second (Uchiyama et al. 2015). Furthermore, SLO
results in most of the bacteria being killed by impairs intracellular GAS clearance by
the host cells (Dombek et al. 1999). In line with neutrophils and professional phagocytes. It
this, Thänert and colleagues have shown that inhibits dendritic cell (DC) maturation by
transcripts of genes encoding fibronectin-binding suppressing the expression of major histocompat-
proteins, which mediate adherence to the extra- ibility complex (MHC) class II and
cellular matrix and invasion into human host co-stimulatory molecules CD80 and CD83
cells, are highly abundant in NSTI patient (Cortes and Wessels 2009) and prevents
biopsies (Thanert et al. 2019). Thus, the results phagolysosome acidification in macrophages by
underline the importance of streptococcal surface damaging the phagolysosome membrane
proteins in NSTIs and suggest that there are addi- (Bastiat-Sempe et al. 2014). In murine models of
tional functions to be explored in future studies.
Table 9.1 GAS secreted virulence factors

GAS
factor Main immunological functions Key references
SLO Cytolysis, suppression of neutrophil chemotaxis Cywes Bentley et al. (2005),
Uchiyama et al. (2015)
SLS Cytolysis, activation of pain producing sensory nociceptor neurons Nizet et al. (2000), Pinho-Ribeiro
et al. (2018)
SpeB Hydrolysis of a broad spectrum of host and bacterial substrates Nelson et al. (2011), Larock et al.
leading to an impaired recognition and killing of GAS (2016)
Ska Systemic spread through plasminogen activation and fibrinolysis Boxrud et al. (2004), Nitzsche et al.
(2016)
SpyCEP Chemokine degradation resulting in an impaired chemotaxis Zinkernagel et al. (2008)
EndoS/ Immunoglobulin degradation, impaired opsonization Collin et al. (2002), Naegeli et al.
IdeS (2019)
Sda1 DNA degradation leading to an impaired bacterial recognition and Keller et al. (2019), Uchiyama et al.
killing by phagocytes (2012)
SIC Interferes with complement functions and blocks lytic activities of Westman et al. (2018)
antimicrobial peptides
SAgs Cytokine storm through excessive T cell and APC activation, Kasper et al. (2014), Llewelyn and
colonization Cohen (2002), Levy et al. (2016)
soft tissue infections, it was shown that GAS p38 MAPK and NFκB. Subcutaneous infections
mutants lacking slo gene were less virulent as of mice result in IL-1β production in a
compared to the parental strain (Zhu et al. 2017). SLS-dependent manner (Flaherty et al. 2018). A
In contrast to SLO, SLS is a small recent study by Pinho-Ribeiro and colleagues has
non-immunogenic peptide of 2.7 kDa, which is shown that SLS is also responsible for activation
encoded by the SLS-associated gene (sag) locus of pain producing sensory nociceptor neurons
consisting of nine genes (sagABCDEFGHI). The (Fig. 9.1) (Pinho-Ribeiro et al. 2018). This is of
sagA gene encodes the premature form of SLS, great interest as an out-of-proportion pain at early
while other genes encode proteins for post- stages of infections is one of the critical hallmark
translational processing and export (Nizet et al. symptoms of NSTIs. SLS-dependent activation of
2000). SLS targets mainly human red blood cells, nociceptor neurons results in a release of calcito-
platelets, and leukocytes. It accumulates on cell nin gene-related peptide (CGRP) into infected
membranes of eukaryotic cells and induces pore tissue, which in turn suppresses critical neutrophil
formation by a yet not fully characterized mecha- functions, including recruitment to the site of
nism (Barnett et al. 2015). Higashi and colleagues infection, opsonophagocytosis, and killing of the
suggested that SLS interacts with the major bacteria (Pinho-Ribeiro et al. 2018). These
erythrocytes anion exchange protein band actions allow the bacteria to spread in the tissue.
3, thereby inducing osmotic change, rapid Cl - Once deeper skin layers are reached, direct cyto-
influx, and erythrocyte lysis (Higashi et al. 2016). toxicity of SLS towards different cell
Whether similar mechanisms apply to other compartments provokes neutrophil influx
human host cells needs to be proven. However, (Humar et al. 2002). In turn, SLS actively
experimental NSTI models have shown that SLS destroys neutrophils which are recruited to the
facilitates translocation of GAS across epithelial site of infection (Humar et al. 2002). These events
barriers through direct cleavage of junctional potentially negatively contribute to the outcome
proteins E-cadherin and occludin (Sumitomo in patients: (1) early CGRP-mediated suppression
et al. 2011). Furthermore, SLS-dependent cyto- of influx resulting in a lack or reduced number of
toxicity to human keratinocytes is mediated neutrophils is an unfavorable prognostic sign in
through activation of pro-inflammatory mediators GAS NSTIs (Bakleh et al. 2005) and (2) hyper-
Fig. 9.1 Immune evasion strategies by GAS. GAS CXCL1-7. SpyCEP, a subtilisin-like protease, degrades
secrete a variety of virulence factors responsible for bacte- CXCL8 which results in impaired neutrophil recruitment
rial spread in the tissue. Streptokinase (Ska) forms to the site of infection. SLS-dependent activation of noci-
complexes with plasminogen (Plg). These Ska-Plg ceptor neurons (N) results in a release of calcitonin gene-
complexes degrade fibrin clots, LL-37, and all classes of related peptide (CGRP), which suppresses neutrophil
human histones. Streptococcal pyrogenic exotoxin B recruitment. Furthermore, GAS have developed strategies
(SpeB) is a cysteine protease with a broad spectrum of to avoid phagocytic killing. They reside within
human substrates. SpeB degrades all classes of macrophages. However, certain environmental signals
immunoglobulins, complement components (e.g., C3), trigger the egress out of the cells and bacterial spread
antimicrobial peptides (e.g., LL-37), extracellular matrix within the tissue. In addition, GAS form biofilms within
proteins (ECM), and CXCL chemokines including the tissue
activation and SLS-dependent damage of 9.3.2 Proteases and DNases

neutrophils at later time points result in aug-
mented tissue pathology through neutrophil- 9.3.2.1 The Cysteine Protease SpeB
derived factors (Johansson et al. 2009, 2010; In addition to pore-forming toxins, streptococcal
Johansson and Norrby-Teglund 2013; Nuwayhid proteases damage tissue barriers, inhibit immune
et al. 2007). cell transmigration to the site of infection, and
impair directly or indirectly immune cell which directly or indirectly control SpeB expres-
functions. One of the first proteases identified in sion, are such mutational hot-spots (Hollands
GAS is the streptococcal pyrogenic exotoxin B et al. 2008; Sumby et al. 2006). Although SpeB
(SpeB) (Elliott 1945). SpeB gene is highly shows such a broad spectrum of substrates, its
conserved in GAS (Yu and Ferretti 1991) and it role in invasive diseases is still under debate.
encodes a zymogen of 40 kDa which is auto- The speB gene can be found in all types of GAS
catalytically cleaved into a mature 28 kDa prote- isolates (Darenberg et al. 2007; Luca-Harari et al.
ase (Liu and Elliott 1965). SpeB has a broad 2009). It was shown that SpeB is readily detect-
spectrum of host and bacterial substrates. It able in sera and tissues of NSTI patients (Gubba
removes GAS surface anchored proteins, includ- et al. 1998; Siemens et al. 2016). Some studies
ing M protein, fibronectin-binding proteins, and demonstrate that GAS isolates from non-invasive
C5a-peptidase, and hydrolyzes secreted proteins, infections express higher amounts of SpeB and
such as streptokinase (Ska), SLO, EndoS, and subsequently show greater activity as compared
superantigens (Nelson et al. 2011). At the host to invasive strains (Kansal et al. 2000). However,
site, SpeB degrades immunoglobulins and other studies demonstrate the opposite. GAS
components of the complement system isolates from invasive infections produce high
(Fig. 9.1) (Kuo et al. 2008). These events result amounts of SpeB and low anti-SpeB antibody
in impaired opsonophagocytosis and subsequent titers in patients were associated with fatal out-
failure in phagocytic clearance of the bacteria come (Holm et al. 1992). Furthermore, the con-
(Collin et al. 2002). Moreover, SpeB degrades troversy of SpeB contribution in NSTIs continues
antimicrobial peptides produced by the host, in murine soft tissue infection models. Some
including LL-37, which was shown to provide authors report that SpeB highly contributes to
protection against murine soft tissue infections disease severity, tissue damage, bacterial dissem-
(Nizet et al. 2001). Neutrophil-derived LL-37 is ination, and mortality (Kuo et al. 1998; Lukomski
highly abundant in human biopsy specimens and et al. 1997, 1998, 1999). Others demonstrated that
its expression is positively correlated to the bac- speB-deficient mutant strains are as virulent as
terial load (Johansson et al. 2008). However, such their parental wild type strains (Ashbaugh et al.
a positive correlation suggests that LL-37 is not 1998; Ashbaugh and Wessels 2001). However,
efficient in bacterial killing. Nyberg and human tissue biopsies from NSTI patients are
colleagues have shown that SpeB gets entrapped strongly positive for SpeB and a mixture of
by α2-macroglobulin-GRAB complex on bacte- SpeB-positive and negative clones is usually
rial surface. As a consequence, SpeB is retained at seen in patients (Johansson et al. 2008; Siemens
the bacterial surface and protects bacteria against et al. 2016).
killing by LL-37 (Nyberg et al. 2004). Further-
more, SpeB degrades a wide range of 9.3.2.2 Streptokinase (Ska) and SpyCEP
chemokines, including CXCL chemokines In contrast to eliminated SpeB expression in nat-
CXCL1-7, CXCL10-14, CXCL16, and CCL20, ural GAS covR/S mutants, several other secreted
XCL1, and CX3CL1 (Egesten et al. 2009), and factors are up-regulated in the same genetic back-
cleaves pro-IL-1β into mature form (Kapur et al. ground. This includes SLO, SpyCEP, Sda1, and
1993). LaRock and colleagues suggested that Streptokinase (Ska) (Cole et al. 2011; Siemens
IL-1β acts as an intracellular sensor of bacterial et al. 2015; Sumby et al. 2006; Walker et al.
proteolytic activity (Larock et al. 2016). GAS 2007). Ska is a non-enzymatic plasminogen
have adapted different strategies, including (Plg) activator. GAS cover their surface with Plg
mutations abrogating SpeB expression to evade via different surface proteins, which in turn
detection by IL-1β and subsequent killing of the results in acquisition of endogenously produced
pathogen (Sumby et al. 2006). Genes encoding and secreted Ska (Chandrahas et al. 2015;
for CovR/S two component system (TCS) and Siemens et al. 2011). The Ska-Plg complex
transcriptional stand-alone regulator RopB, exposes the active site and proteolytically
converts surface-bound Plg to plasmin (Boxrud pathological criteria for diagnosis of NSTIs
et al. 2004). Plasmin is a broad-spectrum host (Stamenkovic and Lew 1984). Several studies
protease that is involved in fibrinolysis. In the utilizing patient’s biopsy material have shown
absence of SpeB, active plasmin accumulates on that neutrophils represent one of the major
bacterial surface, resulting in protease activity immune cell infiltrating populations in NSTIs
that enables the bacteria to degrade tissue barriers (Johansson et al. 2009, 2014; Siemens et al.
and facilitate invasive spread (Cole et al. 2006; 2016; Thulin et al. 2006). The infiltration of
Sanderson-Smith et al. 2008). Furthermore the neutrophils positively correlated with bacterial
Ska-Plg complex degrades LL-37 and all classes load in the tissue (Thulin et al. 2006). These
of human histones and abrogates their anti- facts suggest that the murine models are limited
bactericidal effects (Fig. 9.1) (Hollands et al. in mimicking the complexity of human infection
2012; Nitzsche et al. 2016). Due to human host- setting, in which a plethora of chemotactic signals
specificity of Ska, no differences between wild exists.
type and ska-deficient GAS strains were seen in
murine models (Khil et al. 2003). In contrast, by 9.3.2.3 The Immunoglobulin Degrading
using humanized mice expressing human plas- Enzymes IdeS and EndoS
minogen it was shown that activation of host Plg GAS have also developed strategies to evade
by Ska leads to accelerated clearance of host adaptive immunity at the site of infection. The
fibrin, which plays a central mechanism in GAS bacteria secrete two enzymes, namely immuno-
invasion and spread in tissues (Sun et al. 2004). globulin degrading enzyme (IdeS) and
It is becoming more evident that antimicrobial endoglycosidase (EndoS). IdeS hydrolyzes four
peptides including LL-37 act as immunomodula- subclasses of IgG (Von Pawel-Rammingen et al.
tory and chemotactic agents (Beisswenger and 2002). However, its role in NSTIs is not yet clear.
Bals 2005). Furthermore, LL-37 influences bac- EndoS, which transcription is highly
terial virulence properties. It induces an invasive up-regulated during NSTIs (Thanert et al.
phenotype of GAS by up-regulating gene expres- 2019), specifically cleaves the conserved N-gly-
sion of SpyCEP, among others (Gryllos et al. can of IgG antibodies. A recent study
2008). SpyCEP is a surface localized subtilisin- demonstrated that EndoS specifically alters IgG
like protease of 180 kDa, which is highly Fc glycosylation in vivo. At the site of infection,
expressed in vivo (Zinkernagel et al. 2008). It IgG glycan hydrolysis positively correlates with
cleaves human CXCL chemokines, including bacterial load and EndoS secretion (Naegeli et al.
CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, 2019). These actions potentially result in failed
and CXCL8, and murine chemokines CXCL1 opsonophagocytosis and pathogen clearance.
and CXCL2 (Fig. 9.1) (Hidalgo-Grass et al. The authors showed that ndoS-mutant is more
2004, 2006; Sumby et al. 2008; Zinkernagel susceptible to phagocytic killing as compared to
et al. 2008). Considering the role of CXCL8 in its parental wild type strain. In contrast to tissue
human neutrophil chemotaxis to the infected tis- findings, glycan hydrolysis in circulation was
sue, it is tempting to assume that lack of only detected in patients with septic shock
neutrophils due to CXCL8 degradation will result (Naegeli et al. 2019).
in failed bacterial clearance and severe tissue
pathology caused by bacterial toxins. Indeed, sev- 9.3.2.4 Streptodornase 1(Sda1)
eral murine studies of soft tissue infections have Sda1 is a secreted enzyme which is predomi-
demonstrated a paucity of neutrophil influx nantly associated with emm1 GAS NSTI isolates.
resulting from chemokine degradation by It displays potent sequence-nonspecific nuclease
SpyCEP (Edwards et al. 2005; Hidalgo-Grass activity on DNA substrates (Aziz et al. 2004a). It
et al. 2004, 2006; Sumby et al. 2008; Zinkernagel occurs during phase-shifts to hyper-virulent inva-
et al. 2008). However, infiltration of neutrophils sive infection and is believed to play a role in
in superficial fascia and dermis is one of the evasion of the host’s innate immune responses
(Aziz et al. 2004b; Walker et al. 2007). Sda1 is binding to histones boosted the release of a
involved in degradation of the DNA component broad range of pro-inflammatory cytokines and
of chromatin-rich NETs (Walker et al. 2007), a chemokines (Westman et al. 2018). Such actions
mechanism which provides a protective effect can potentially further contribute to hyper-
against bacterial killing by neutrophils. Recently inflammation in tissue environment.
it was also shown that Sda1-mediated degradation
of streptococcal DNA alters recognition of GAS
by Toll-like receptor (TLR)9 (Uchiyama et al. 9.4 Local and Systemic
2012), which preferentially senses bacterial and Inflammation in NSTIs
viral DNA. DNA degradation by Sda1 prevented
GAS induced IFNα and TNFα secretion from 9.4.1 Neutrophil Responses in NSTI
murine macrophages and contributed to bacterial
survival. Furthermore, IFNα and TNFα levels Despite the fact that GAS factors contribute to
were significantly decreased in wild type mice tissue pathology, it is widely accepted that
infected with Sda1-positive GAS strain as com- immune cells and host-derived factors play a cru-
pared to TLR9-deficient mice (Uchiyama et al. cial role in hyper-inflammatory and tissue
2012). In addition, Sda1 impaired plasmacytoid destructive processes (Herwald et al. 2004;
(p)DC recruitment to the site of infection. Keller Johansson et al. 2009; Kahn et al. 2008). Several
and colleagues suggested that Sda1 interferes immune cell populations are recruited to the site
with stabilization of the DNA in a HMGB1- of infection, including neutrophils, macrophages,
dependent manner, which may result in decreased T cells, and dendritic cells (Norrby-Teglund et al.
IFN-1 levels and subsequently lead to reduced 2001). Neutrophil migration to the site of infec-
numbers of pDCs (Keller et al. 2019). tion is a crucial step in host defense. Once
neutrophils have reached the infection site, three
distinct mechanisms to fight an infection can
9.3.3 The Streptococcal Inhibitor occur: phagocytosis, degranulation, and forma-
of Complement (SIC) tion of NETs (Kolaczkowska and Kubes 2013).
Critical tissue damaging components of all these
In addition to proteases and DNases, a limited processes are granule effector molecules, includ-
number of GAS M serotypes (emm1, emm12, ing proteolytic enzymes and antimicrobial
emm55, emm57) secrete a protein called strepto- peptides (Borregaard et al. 2007). GAS have
coccal inhibitor of complement (SIC). SIC is a been demonstrated to have a significant impact
highly polymorphic 31 kDa negatively charged on neutrophils, by inducing an apoptotic program
protein which interferes with complement and triggering degranulation (Kobayashi et al.
functions (Akesson et al. 1996; Fernie-King 2003; Snall et al. 2016; Soehnlein et al. 2008)
et al. 2001). Furthermore, SIC blocks the lytic and both, surface associated as well as secreted
activity of antimicrobial peptides such as LL-37, factors are potent inducers of neutrophil degranu-
defensins, H-kininogens, and lysozyme (Fernie- lation (Snall et al. 2016; Uhlmann et al. 2016b).
King et al. 2002, 2004; Frick et al. 2003a, 2011). Strong neutrophil influx in the tissue is a typical
Recently it was shown that SIC also binds to characteristic for GAS NSTIs and there is a sig-
extracellular histones, a group of host-associated nificant correlation between bacterial load, neu-
damage-associated molecular patterns (DAMPs) trophil influx, presence of degranulation markers
released during necrotizing tissue infections, and heparin-binding protein (HBP) and resistin, and
neutralizes their antimicrobial activity. Such tissue pathology and inflammation (Fig. 9.2)
SIC-histone aggregates are readily detectable in (Johansson et al. 2008, 2009, 2014; Snall et al.
biopsies of NSTI patients (Westman et al. 2018). 2016). Streptococcal M protein is one of the
Although SIC is considered to be an anti- major triggers of neutrophil activation. Soluble
inflammatory agent (Akesson et al. 2010), SIC M1 protein forms complexes with fibrinogen
Fig. 9.2 Immune cell mediated hyper-inflammatory SLS and SLO induce through different mechanisms host
response in the tissue triggered by secreted streptococcal cell lysis resulting in a release of pro-inflammatory
virulence factors. Several GAS factors contribute to hyper- cytokines and chemokines. In addition, SpeB cleaves
inflammation of the tissue. For example, soluble M1 pro- proIL-1β into mature IL-1β leading to pyroptosis of
tein (sM1) forms complexes with fibrinogen, which in turn macrophages. GAS also secrete superantigens (SAgs).
activates neutrophils. Furthermore, PGK and streptolysins SAgs bind without cellular processing to α- and β-chains
S and O (SLS/SLO) trigger neutrophil activation. This of MHC class II molecules on antigen-presenting cells
results in a release of granule effector molecules, including (APC) and variable β-chains of T cell receptor. These
resistin, heparin-binding protein (HBP), LL-37, and interactions result in a massive cytokine storm
myeloperoxidase, which augments tissue inflammation.
and binds β2-integrins on neutrophil surface. This solely responsible for vascular leakage (Gautam
event results in a release of massive amounts of et al. 2001; Herwald et al. 2004). NSTI patient
the granule proteins, including HBP, which is biopsies are highly positive for such M protein/
fibrinogen complexes (Herwald et al. 2004; Kahn lysosomes, leading to phagolysosome formation,
et al. 2008). Furthermore, streptococcal secreted which would usually kill the pathogen. In vitro
factors which are susceptible to SpeB degrada- studies have shown that GAS activate NFκB and
tion, e.g., PGK and SLO, are potent inducers of MAPK in a MyD88-dependent manner leading to
neutrophil degranulation (Nilsson et al. 2006; production of pro-inflammatory cytokines,
Uhlmann et al. 2016b). Nilsson and colleagues including TNF and IL-6 (Gratz et al. 2008). In
observed that GAS supernatants containing high addition, an up-regulation of the maturation
levels of SLO activated neutrophils by markers such as CD80 and CD86 was noted.
perforating these cells. As a consequence, Experimental mice models of GAS skin infection
neutrophils secreted HBP, LL-37, α-defensins, demonstrated that impaired MyD88 signaling
and neutrophil elastase (Fig. 9.2). Bacterial results in diminished pro-inflammatory response,
supernatants with negligible levels of SLO failed including IL-12, IFNγ, and TNF production as
to induce degranulation (Nilsson et al. 2006). well as chemoattractants such as CXCL1 and
Most likely, these effects were observed due to CCL2. This in turn leads to the impaired recruit-
the presence or absence of SpeB in bacterial ment of macrophages and neutrophils (Loof et al.
supernatants. Uhlmann et al. demonstrated that 2010). In addition, TNF-deficient mice is highly
high degranulation response was triggered exclu- susceptible to GAS soft tissue infections due to
sively by supernatants from SpeB-negative the defect in macrophage recruitment to the site of
strains (Uhlmann et al. 2016b). Global proteome infection (Mishalian et al. 2011). However, mice
analyses identified phosphoglycerate kinase studies are highly controversial. MAPK and
(PGK) as a stimulatory factor for neutrophils. NFκB activation and MyD88-dependent cytokine
Further experiments confirmed that PGK is sus- production are TLR-independent processes in
ceptible to SpeB degradation (Uhlmann et al. mice as compared to humans (Gratz et al. 2008).
2016b). The neutrophil response and subsequent Analyses of human NSTI biopsies revealed that
tissue hyper-inflammation can be further aug- even if the biopsies were highly infiltrated by
mented through the streptococcal vesicle-like neutrophils and macrophages, high amounts of
structures. It was shown that GAS respond to viable bacteria were readily detectable, even in
sub-inhibitory concentrations of LL-37 by releas- those patients with prolonged antibiotic therapy.
ing vesicles containing several virulence factors. Viable bacteria were mainly residing within
These vesicles are of pro-inflammatory nature and macrophages and such biopsies were
elicit resistin and myeloperoxidase release from characterized by lower inflammation level and
neutrophils (Uhlmann et al. 2016a). lower bacterial load (Thulin et al. 2006). As pre-
viously mentioned, bacteria are normally killed
by phagolysosomal degradation. However, GAS
9.4.2 Macrophage Responses in NSTI have acquired specific mechanisms to avoid such
killing. These processes involve M1-protein-
Several studies have shown that in addition to dependent trafficking and impaired fusion of
neutrophils, macrophages are also predominantly phagosome with lysosome leading to a persistent
infiltrating highly infected tissue of NSTI patients infection (Hertzen et al. 2010), adaptation to
(Johansson and Norrby-Teglund 2013; Johansson acidic environment, and escape from the
et al. 2010, 2014; Siemens et al. 2016; Thulin phagolysosome (Fig. 9.1) (Bastiat-Sempe et al.
et al. 2006). Macrophages are highly effective 2014). GAS pore-forming toxin SLO and its
professional phagocytes. They ingest bacteria by co-toxin NAD-glycohydrolase (NADase) medi-
recognition of GAS via surface pattern recogni- ate GAS intracellular survival in macrophages.
tion receptors (e.g., TLRs), which in turn induces The two toxins prevent phagolysosomal acidifi-
remodeling of the actin cytoskeleton to form a cation. SLO perforates the phagolysosomal mem-
phagosome (Valderrama and Nizet 2018). The brane and enables translocation of NADase into
phagosome interacts with endosomes and the macrophage cytosol. Subsequently, NADase
augments SLO-mediated cytotoxicity by deplet- mixed activation of murine macrophages

ing cellular energy stores (Bastiat-Sempe et al. (Goldmann et al. 2007). Transcriptomic analyses
2014). However, the bacteria do not only persist, revealed that GAS infections induce both, Th1
but also replicate within macrophages and egress (classical) signatures characterized by induction
(Hertzen et al. 2010). Ihk/Irr TCS was identified of IL-1 and IL-6 cytokines, as well as Th2 (alter-
as a key regulatory system at early stages of native) responses, e.g., induction of IL-1 decoy
bacterial persistence within macrophages. Fur- receptor and IL-10 (Goldmann et al. 2007). How-
thermore, Kachroo and colleagues have shown ever, this phenomenon was observed in mice.
that Ihk/Irr TCS is highly up-regulated in a necro-
tizing myositis model in primates (Kachroo et al.
2020). In contrast, up-regulation of CovR/S and 9.4.3 Superantigens
down-regulation of Ihk/Irr TCS were seen at late
stages when the bacteria were exiting the cells NSTIs are often complicated by STSS (Johansson
(Hertzen et al. 2012). It was also suggested that et al. 2010). Sepsis-3 criteria define sepsis as a
SpeB, which is regulated by the CovR/S TCS, life-threatening organ dysfunction caused by a
contributes to intracellular survival and bacterial dysregulated host response to an infection. STSS
egress out of the cell (Thulin et al. 2006). LaRock is a subset of sepsis in which particularly pro-
and colleagues demonstrated that SpeB cleaves found circulatory, cellular, and metabolic
IL-1β, which is usually seen as a marker of canon- abnormalities are associated with a greater risk
ical and caspase-1 dependent inflammasome acti- of mortality (Singer et al. 2016). STSS is a result
vation and subsequent pyroptosis of the cells of an invasive GAS infection (Low 2013) and
(Larock et al. 2016). However, the direct cleavage superantigens (SAgs) are key mediators of a sys-
by SpeB induces a non-canonical IL-1β activation temic excessive inflammatory response
in macrophages (Fig. 9.2). Analyses of patient’s (Llewelyn and Cohen 2002). To date, 13 GAS
tissue biopsies revealed that once IL-1β is SAgs were identified, which include streptococ-
released it forms immuno-stimulatory complexes cal pyrogenic exotoxins (Spe) A, C, G-M, strep-
with High Mobility Group Box 1 (HMGB1) pro- tococcal superantigen (SSA), and streptococcal
tein (Johansson et al. 2014). HMGB1 is a ubiqui- mitogenic exotoxin Z (SmeZ) (Commons et al.
tously expressed nuclear protein that is released 2014) and the two recently identified
through active secretion by innate immune cells superantigens SpeQ and SpeR (Reglinski et al.
or through passive release by injured cells (Skin- 2019). SAgs bind intact to α- and/or β-chains of
ner 2010). Extracellular HMGB1 has been the major histocompatibility complex (MHC)
reported to act as an alarmin with ability to acti- class II molecules on antigen-presenting cells
vate the immune system and elevated levels of (APCs) and to the variable β-chains of the T cell
HMGB1 were found in patients diagnosed with receptor. In addition, SAgs can also bind a
sepsis and/or septic shock (Sunden-Cullberg et al. co-stimulatory molecule CD28 and its ligand
2005). In GAS tissue infections, HMGB1 expres- CD86 (B7-2) (Arad et al. 2011; Levy et al.
sion correlated with disease severity and 2016). Once the MHC-peptide specificity of T
macrophages were identified as a predominant cells is bypassed, these interactions result in a
source of HMGB1 release (Johansson et al. massive cytokine storm, which includes produc-
2014). A close proximity between HMGB1 and tion of TNF, IFN-γ, IL-1, IL-2, IL-6, CXCL8,
neutrophils suggested also a chemotactic role of CCL2, and CCL3 (Fig. 9.2) (Chatila and Geha
this molecule in GAS NSTIs which was further 1993). However, such responses may vary due to
confirmed by in vitro experiments (Johansson specific HLA class II haplotypes which may
et al. 2014). influence the severity of GAS infections. Kotb
Despite the fact that macrophages contribute to and colleagues have shown that certain
the highly pro-inflammatory state in the tissue, haplotypes (e.g., DRB1*1501/DQB1*0602) are
there is evidence rising that GAS induce a of a protective nature resulting in attenuated
inflammatory responses to the streptococcal from an in vitro experimental model of human

SAgs. In contrast, other haplotypes (e.g., skin. High IL-1β mRNA levels were detected in
DRB1*14/DQB1*0503 and DRB1*07/ infected tissues. In addition, analyses of systemic
*
DQB1 0201) are associated with a risk to develop IL-1β revealed high levels in patient plasma col-
severe systemic responses (Kotb et al. 2002). A lected during an acute phase of GAS NSTIs.
recent report identified that Mucosa-associated These levels declined during the treatment
invariant T (MAIT) cells are strongly activated (Chella Krishnan et al. 2016). A study by Hansen
by SAgs and represent the major T cell source of and colleagues underlined the importance of
IFNγ and TNF in the early cytokine response IL-1β in NSTIs. IL-1β was determined as an
associated with STSS (Emgard et al. 2019). How- independent predictor for 30-day mortality in
ever, the majority of the SAg studies are confined NSTIs (Hansen et al. 2017). In addition, signifi-
to the systemic effects and only a limited number cantly higher IL-6 and TNF plasma levels in
of studies investigated SAg-driven events at the streptococcal infected patients were detected as
deep tissue site (Johansson et al. 2010; Norrby- compared to patients infected with other
Teglund et al. 2001). Norrby-Teglund and microbiological agents, which is consistent with
colleagues showed a massive in vivo production septic shock being more prevalent in streptococ-
of SAgs in biopsies of GAS NSTI patients cal NSTIs (Hansen et al. 2017). Comparative
(Norrby-Teglund et al. 2001). The infected tissue RNAseq analyses of tissue specimens from strep-
was characterized by an enormous infiltration tococcal and polymicrobial NSTIs identified a
with monocytes, macrophages, and T cells, core of common up-regulated host genes
which correlated with a typical SAg-driven exces- encoding pro-inflammatory mediators, including
sive inflammatory response and the severity of IL-6 and CXCL8, complement components,
infection (Norrby-Teglund et al. 2001). In addi- alarmins, and proteolytic enzymes (Thanert et al.
tion, up-regulation of the homing receptors 2019). However, GAS NSTI biopsies were
CCR5, CD44, and cutaneous lymphocyte- characterized by a higher expression of genes
associated antigen correlated with the expression encoding interferon-inducible mediators, which
of Th1 cytokines at the tissue site (Norrby- includes CXCL9, CXCL10, CXCL11, MX1, and
Teglund et al. 2001). T cell derived Th1 cytokines MX2. This finding was further verified in patient
and predominantly IFN-γ induce production of plasma samples. CXCL9, CXCL10, and
IL-1β by monocytes and macrophages CXCL11 chemokine plasma levels were higher
(Chizzolini et al. 1997). In line with this, it was in GAS NSTI patients as compared to patients
shown that IL-1β plays a crucial role in GAS with polymicrobial NSTIs (Thanert et al. 2019).
NSTIs. Mice lacking IL-1R are more susceptible Thus, these chemokines might serve as potential
to GAS infections (Hsu et al. 2011). Furthermore, biomarkers for GAS NSTIs.
it was reported that patients who received the
IL-1R antagonist Anakinra experience more fre-
quently invasive GAS diseases including NSTIs, 9.5 Biofilm and Complex
which results in a greater risk of mortality (Larock Modelling of GAS NSTIs
et al. 2016). In addition, by utilizing BXD mice in the Human Tissue Setting
Chella Krishnan and colleagues have shown that
IL-1β participates in modulating GAS NSTIs Biofilm formation is recognized as a virulence
(Chella Krishnan et al. 2016). The authors trait of many chronic tissue infections, including
demonstrated that IL-1β gene expression was sig- cystic fibrosis, burn wound infections, and
nificantly up-regulated in tissue biopsies of GAS infected diabetic foot ulcers (Bjarnsholt et al.
NSTI susceptible mice strains as compared to 2018). Although GAS biofilm studies were
non-susceptible strains. This finding was further performed in the past, until recently, GAS have
verified in plasma samples and tissue biopsies not been considered a major biofilm forming spe-
from GAS NSTI patients, as well as samples cies due to the lack of clinically relevant reports.
Bacteria can exist in two states, planktonic or forming abilities within and between the
biofilm and it has become clear that it is a serotypes may vary (Conley et al. 2003), and
dynamic process allowing the bacteria to fre- (3) GAS biofilms are up to ten times more resis-
quently flux between these states (Mcdougald tant to antibiotics as compared to planktonic cells
et al. 2011). The process of biofilm formation (Ogawa et al. 2011). In addition, several GAS
can be summarized in four steps: (1) reversible virulence factors have been implicated in biofilm
attachment to abiotic or biotic surfaces driven by development. Among these, surface localized
environmental signals (e.g., pH, temperature, and factors, including M proteins, pili, and LTA,
nutrients availability), (2) irreversible attachment were associated with initial attachment of bacteria
with initial microcolony formation and develop- to different surfaces. Lack or diminished abun-
ment of an extracellular matrix, (3) biofilm matu- dance of such factors resulted in reduced biofilm
ration, and (4) dispersal (Kostakioti et al. 2013). formation (Fiedler et al. 2015). Furthermore, cap-
The first report providing evidence of GAS bio- sule and SpeB expression were implicated in bio-
film in vivo was published by Akiyama and film formation. However, reports on these two
colleagues (Akiyama et al. 2003). Confocal laser factors are contradicting. While some authors
scanning microscopy (CLSM) of impetigo skin report a positive correlation, others report a nega-
biopsies revealed concanavalin A positive GAS tive impact (Marks et al. 2014; Roberts et al.
microcolonies consistent with biofilm formation 2010). Therefore, a recent study underlines the
(Akiyama et al. 2003). The second study importance of studying biofilm in a physiologi-
performed on tonsils from children with recurrent cally relevant setting (Siemens et al. 2016).
GAS pharyngitis identified three dimensional Although not all tested GAS serotypes were able
communities of GAS within reticulated crypts to form biofilms on abiotic surfaces, all of them
(Roberts et al. 2012). However, both studies readily formed biofilms in a tissue setting. Fur-
were of descriptive nature and have not addressed thermore, Nra regulator, which is encoded within
potential pathogenic effects of GAS biofilms the FCT-region (fibronectin-binding, collagen-
within the host. A more recent clinical study on binding, T-pilus) and controls a variety of surface
GAS NSTIs presented a patient with a persistent localized factors including pili was implicated in
infection over a period of 24 days. The surgeons this process. A nra-negative mutant lost its ability
reported a thick layer of biofilm covering to form biofilm. In contrast to previous findings,
fasciotomy wounds (Siemens et al. 2016). no associations between SpeB, capsule, CovR/S,
Analyses of biopsies collected from different M protein, and Mga regulator and biofilm forma-
anatomical locations of 31 GAS NSTI patients tion were found (Siemens et al. 2016). In support
revealed that in 32% of cases, aggregations con- of the importance to study biofilms in in vivo
sistent with biofilm communities were evident. settings, Vajjala and colleagues have
CLSM and scanning electron microscopy (SEM) demonstrated biofilm formation in a mice NSTI
analyses confirmed the presence of amorphous or model (Vajjala et al. 2019). Both streptolysins,
fibrous biofilm structures consisting of bacteria, SLS and SLO, which were previously excluded
polysaccharides, lipids, and DNA. Biofilm- as biofilm-contributing factors, were implicated
positive biopsies were characterized by a higher in this process. While GAS wild type strain
bacterial load. In addition, higher neutrophil formed biofilms across the entire fascia already
influx and elevated pro-inflammatory responses 12 h post infection, GAS mutant lacking both
were noted in biofilm-positive tissue (Siemens streptolysins did not. The authors demonstrated
et al. 2016). that streptolysins induce significant up-regulation
Classical mechanistic studies on biofilm for- of endoplasmic reticulum (ER) stress which in
mation are confined to coated and uncoated plas- turn activates the unfolded protein response caus-
tic or glass surfaces. These studies showed that ing a release of host factors that support biofilm
(1) not all clinically relevant GAS serotypes form formation. Treatment of mice with pharmacolog-
biofilms (Lembke et al. 2006), (2) biofilm ical inducer of ER stress restored the ability of
streptolysin-null mutant to form biofilm (Vajjala junction and adherence junction proteins
et al. 2019). These two studies underline the (Fig. 9.3a). It was shown that GAS are able to
importance of studying NSTIs in a complex initiate tissue infection in this model system
setting since simplified experimental in vitro (Fig. 9.3b). While at 8 h post infection most of
approaches did not revealed biofilms as clinical the bacteria were predominantly found with the
feature of NSTIs. Such findings have direct stratum corneum, at later time points, bacteria
implications for antibiotic efficacy in the tissue disseminated throughout the entire tissue and
setting and call for reevaluation of treatment formed biofilms (Fig. 9.3c) (Siemens et al.
protocols in which biofilm is considered. 2016). 3D tissue models are also suitable for
Although in vitro assays in monolayer cultures implanting and studying immune cells, including
and the use of mice as an in vivo model of choice dendritic cells, monocytes, macrophages, and
contribute greatly to our understanding of infec- even peripheral blood mononuclear cells
tious diseases, human tissue biopsies and human (Mairpady Shambat et al. 2015; Nguyen Hoang
3D-organotypic tissue models are the most valu- et al. 2012). Such studies highlight a significant
able tools to study host-pathogen interactions. As progress in the field of infectious diseases and
described above, human NSTI tissue biopsies contribute to the three R principle (replacement,
provided valuable insight in host-pathogen reduction, refinement) of animal experimentation
interactions at the tissue site. However, NSTIs (Russell 1995).
are rare acute and devastating conditions, making
it difficult to obtain patients consent for, e.g.,
sampling infected tissue or blood. Therefore, 9.6 Future Perspectives
3D-organotypic models might serve as a useful
tool to study these infections in a more complex The pathogenesis of NSTIs is a multifactorial
setting. Tissue engineering approaches originated process which involves both, bacterial and host-
in the field of regenerative medicine (Langer and derived factors. These infections are hyper-
Vacanti 1993). In contrast to standard monolayer inflammatory conditions and are characterized
cell cultures, tissue models much more closely by a massive immune cell infiltration and high
resemble the architecture, cellular composition, bacterial load. GAS have evolved various
and matrix complexity of the respective organ immune evasion mechanisms, including intracel-
(Fig. 9.3a). In recent years 3D-organotypic lular survival and replication within phagocytes,
models were successfully employed in a number egress out of the cells, and biofilm formation in
of studies of human diseases involving the tissue the tissue. All these processes contribute greatly
setting (Svensson and Chen 2018). These studies to the bacterial persistence and as a result, the
include, but are not limited to, GAS, group G antibiotic therapy fails to clear the infection.
streptococcus or staphylococcal skin/soft tissue Future studies should aim to improve therapeutic
infections in human skin tissue model setting strategies targeting intracellular bacterial reser-
(Mairpady Shambat et al. 2016; Siemens et al. voir and biofilm formation. Furthermore, early
2015, 2016). The use of such organotypic tissue diagnosis of GAS NSTIs might improve the out-
models suggests a great potential of this approach come for the patients. Therefore, the results from
in infectious diseases. Considering the fact that the INFECT study, including anatomical location
GAS are exclusively human pathogens, it and IL-1β, CXCL9, CXCL10, and CXCL11 as
becomes convincing to use a complex humanized potential promising biomarker candidates, should
model system. The engineered tissue recapitulates be validated in a bigger cohort which encompass
key anatomical and functional features, including clinical samples of patients with different
a dermal layer consisting of fibroblasts and infections and other critical illnesses. Early diag-
stratified epidermis with stratum corneum nosis and subsequent early treatment might
(Fig. 9.3a). The tissue produces important struc- reduce local and systemic hyper-inflammation
tural frame work proteins as well as the tight and will most likely further improve the outcome.
142
Fig. 9.3 GAS infections of the 3D-organotypic skin tissue models. (a) Comparative panels) are shown. (b) CLSM analyses of GAS within different epidermal and dermal
microscopic analyses of the human skin (left) and 3D-organotypic skin tissue model compartments of the 3D-organotypic model after 24 h of infection. (c) Comparative
(right). Hematoxylin and eosin staining of human skin and tissue model (upper panel) CLSM analyses of GAS biofilms on glass surfaces (right panel) and within the skin
and images from immuno-fluorescence analyses of key anatomical proteins (lower tissue model (left panel)
N. Siemens et al.
Acknowledgments Financial support: The work was Bakleh M, Wold LE, Mandrekar JN, Harmsen WS,
supported by the European Union Seventh Framework Dimashkieh HH, Baddour LM (2005) Correlation of
Programme (FP7/2007–2013) under the grant agreement histopathologic findings with clinical outcome in nec-
305340 (INFECT project), the Swedish Governmental rotizing fasciitis. Clin Infect Dis 40:410–414
Agency for Innovation Systems (VINNOVA) under the Barnett TC, Cole JN, Rivera-Hernandez T,
frame of NordForsk (Project no. 90456, PerAID), the Henningham A, Paton JC, Nizet V, Walker MJ
Swedish Research Council under the frame of ERA (2015) Streptococcal toxins: role in pathogenesis and
PerMed (Project 2018-151, PerMIT), the German disease. Cell Microbiol 17:1721–1741
Research Foundation (DFG; grant no. 407176682), and Bastiat-Sempe B, Love JF, Lomayesva N, Wessels MR
the Federal Excellence Initiative of Mecklenburg Western (2014) Streptolysin O and NAD-glycohydrolase pre-
Pomerania and European Social Fund (ESF) grant vent phagolysosome acidification and promote group
KoInfekt (ESF_14-BM-A55-0001_16). A Streptococcus survival in macrophages. MBio 5:
e01690–e01614
Beall B, Facklam R, Thompson T (1996) Sequencing
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187:605–613
Systems Genetics Approaches in Mouse
Models of Group A Streptococcal 10
Necrotizing Soft-Tissue Infections
Suba Nookala, Karthickeyan Chella Krishnan,

Santhosh Mukundan, and Malak Kotb
Contents
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
10.2 Host Genetics and Group A Streptococcus Infections . . . . . . . . . . . . . . . . . . . . 153
10.2.1 Systems Genetics Approaches Using BXD Recombinant Inbred Mouse
Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
10.2.2 ARI-BXD Mouse Models of GAS NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
10.2.3 Assessing Heterogeneity of Treatment Effects in BXD Models of GAS NSTI 158
10.3 HLA-II Mouse Models of Invasive GAS Infections . . . . . . . . . . . . . . . . . . . . . . . 158
10.4 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
Abstract and C57BL/10 offered significant insights,

the human specificity and the interindividual
Mouse models are invaluable resources for
variations on susceptibility or resistance to
studying the pathogenesis and preclinical eval-
GAS infections limit their ability to mirror
uation of therapeutics and vaccines against
responses as seen in humans. In this chapter,
many human pathogens. Infections caused by
we discuss the advanced recombinant inbred
group A streptococcus (GAS, Streptococcus
(ARI) BXD mouse model that mimics the
pyogenes) are heterogeneous ranging from
genetic diversity as seen in humans and
mild pharyngitis to severe invasive necrotizing
underpins the feasibility to map multiple
fasciitis, a subgroup of necrotizing soft-tissue
genes (genetic loci) modulating GAS NSTI.
infections (NSTIs). While several strains of
GAS produces a myriad of virulence factors,
mice including BALB/c, C3H/HeN, CBA/J,
including superantigens (SAg). Superantigens
S. Nookala (*) · S. Mukundan · M. Kotb are potent immune toxins that activate T cells
Department of Biomedical Sciences, School of Medicine by cross-linking T cell receptors with human
and Health Sciences, University of North Dakota, Grand leukocyte antigen class-II (HLA-II) molecules
Forks, ND, USA expressed on antigen-presenting cells. This
e-mail: suba.nookala@und.edu
leads to a pro-inflammatory cytokine storm
K. C. Krishnan and the subsequent multiple organ damage
Department of Medicine/Division of Cardiology, David
Geffen School of Medicine, University of California, Los and shock. Inbred mice are innately refractive
Angeles, CA, USA to SAg-mediated responses. In this chapter, we

152 S. Nookala et al.
discuss the versatility of the HLA-II transgenic 10.1 Introduction

mouse model that allowed the biological vali-
dation of known genetic associations to GAS Host genetic variations have been implicated as
NSTI. The combined utility of ARI-BXD and significant modulators of clinical presentation and
HLA-II mice as complementary approaches disease severity in almost all infectious diseases
that offer clinically translatable insights into and have been reported on extensively. The com-
pathomechanisms driven by complex traits plex polymorphic and nonpolymorphic traits
and host genetic context and novel means to impact host immune responses and confer suscep-
evaluate the in vivo efficiency of therapies to tibility to a specific infection while at the same
improve outcomes of GAS NSTI are also time resistance to another type of infection. The
discussed. host genetic background significantly influences
not only the onset of the disease but also treat-
Keywords ment outcomes (Skamene 1983; Kotb 2004;
Streptococcus pyogenes · NSTI · ARI-BXD · Frodsham and Hill 2004; Sadikot et al. 2005;
IL-1β, HLA-II Howard 2013; Asner et al. 2014; Patarčić et al.
2015; Çalişkan et al. 2015). Polymorphisms in
Highlights HLA class II haplotypes, cytokine genes, and
A systems biology approach using advanced innate pathogen recognition receptors including
intercross-derived recombinant inbred BXD TLR4 are among the key genetic determinants
(ARI-BXD) mouse models and an HLA-II trans- that affect host immune responses to invading
genic mouse model to validate known genetic pathogens. However, the complete spectrum of
associations and superantigen-mediated genetic determinants that influence susceptibility
responses reveals: or resistance to infections remains unknown. The
identification of these factors can be impactful in
• A significant quantitative trait locus (QTL) on understanding the pathogenesis and inform man-
Chromosome 2 with pathways involving IL1β agement strategies. Furthermore, there is a grow-
as key upstream regulator mediating severity ing concern and risk to human health with the
and survival in NSTI susceptible increase in the emerging and reemerging infec-
ARI-BXD mice. tious diseases, as well as natural or accidental
• Decreased survival was associated with a sig- biological threats (Fauci 2005). Therefore,
nificant loss in the frequency of myeloid- recognizing emerging and reemerging infections
derived suppressor cells (MDSCs) in NSTI and preparedness to biothreats through thorough
susceptible ARI-BXD strains, which was knowledge and understanding of the mechanisms
reversed with clindamycin treatment. that govern the pathogenesis and associated host
• Induction of FoxP3-expressing T regulatory immune responses are key for substantially
phenotype by the HLA-II mice expressing controlling disease outbreaks and the develop-
the protective allele in contrast to a Th1 phe- ment of treatment possibilities.
notype with concomitant IFN-γ and IL-2 by Despite significant variations in the microbial
mice expressing the HLA-II allele associated factors that are mostly enough to cause morbidity
with neutral risk during GAS NSTI. and mortality, host genetic factors have a major
• Transcriptome profile of GAS-infected skin impact on disease susceptibility and outcomes
from HLA-II mice reveals unique signaling (Hill 1999). Mouse models are cornerstones for
pathways that are amenable to treatment with studying human diseases and are indispensable
existing therapeutic approaches. for the prediction of outcomes of infections and
diseases including genetic abnormalities,
neurodegeneration, and cancer (Bedell et al.
1997). The knowledge gained from mouse
10 Systems Genetics Approaches in Mouse Models of Group A Streptococcal. . . 153
models as surrogates of human infections proposed for undergoing preclinical studies

progresses to preclinical investigations that can (Azuar et al. 2019; Vekemans et al. 2019). GAS
be assessed in the same mouse models. Further, produces a spectrum of potent virulence factors
the successful use of mouse models to study that are either secreted or surface associated that
human infections and test potential interventions aid in their establishment, colonization, multipli-
is because of the genetic homology between cation and dissemination, and to avoid recogni-
mouse and human (Peltonen and McKusick tion by the host immune system, antimicrobials,
2001). and antibiotics (Thulin et al. 2006; Siemens et al.
2016; Andreoni et al. 2017; Keller et al. 2018).
Potential post-streptococcal autoimmune
10.2 Host Genetics and Group A complications that arise from GAS mimics of
Streptococcus Infections host proteins include rheumatic heart disease,
rheumatic heart fever, arthritis, glomerulonephri-
Infections caused by group A streptococcus tis, and Sydenham’s chorea (Dubost et al. 2004;
(GAS, Streptococcus pyogenes) are an ideal Kim et al. 2004; Chandnani et al. 2015; Sims
model to explore the effects of interindividual Sanyahumbi et al. 2016).
variations on susceptibility or resistance. The Superantigens (SAgs) are the most important
pathophysiology of infections caused by GAS is among the virulence factors secreted by GAS
heterogeneous. Some individuals experience only bacteria. SAgs, which are potent immune toxins,
mild skin infections or sore throat, while in simultaneously cross-link T-cell receptor (TCR)-
others, GAS infections can lead to severe, inva- bearing Vβ regions and conserved sequences of
sive, life-threatening complications such as strep- HLA class II expressed on antigen-presenting
tococcal toxic shock syndrome (STSS) and cells (APCs), thereby inducing the activation of
necrotizing fasciitis (NF). In about 50% of the both types of cells (Kotb 1998; Proft and Fraser
cases, NF, a subgroup of necrotizing soft-tissue 2003). The net result is the fulminant activation of
infection (NSTI) can be complicated by STSS. the immune system and the massive release of
(Stevens 2000; Carapetis et al. 2005; Ralph and pro-inflammatory mediators leading to septic
Carapetis 2012). Despite early surgical debride- shock and multiorgan failure with high mortality
ment, an appropriate combination of penicillin rates (Norrby-Teglund et al. 2000; Kotb et al.
and clindamycin, and adjunct therapies, an over- 2003). The Kotb research group, through their
arching pro-inflammatory cytokine storm and large epidemiological studies, demonstrated a sig-
aggressive tissue destruction lead to significant nificant association of certain HLA class II
morbidity and lethal outcomes with mortality as haplotypes with susceptibility or resistance to
high as 25% (Reglinski and Sriskandan 2014; invasive GAS infections (Kotb et al. 2002,
Barnett et al. 2019). The global burden of inva- 2003). The haplotype DRB1*15/DQB1*06
sive GAS infections remains alarming with over (DR15/DQ6) conferred strong protection from
600,000 new cases each year and an estimated severe systemic disease (SSD), the haplotype
160,000 deaths annually (Ermert et al. 2015; Sims DRB1*14/DQB1*05 (DR14/DQ5) was
Sanyahumbi et al. 2016). Antibiotics remain associated with predisposition to SSD, and other
effective against GAS infections; however, haplotypes, e.g., DRB1*04/DQB1*0302
antibiotic-resistant strains have been observed (DR4/DQ8), were neutral with respect to SSD
and present a potential threat to public health (Kotb et al. 2002). Kotb et al. validated their
(Gilmer et al. 2013; Andreoni et al. 2017). Due findings in in vitro human cell cultures and
to the extensive genomic diversity in GAS strains, demonstrated that the inflammatory responses to
there has been limited progress in GAS vaccine the M1T1 SAgs were much attenuated in the
development. However, vaccine discovery efforts protective allele compared to high-risk or alleles
have increased more recently, and several associated with neutral outcomes of invasive
candidates including peptide antigens have been GAS infections (Kotb et al. 2002).
10.2.1 Systems Genetics Approaches humans that are associated with complex poly-
Using BXD Recombinant Inbred genic traits due to their uniform background and
Mouse Models limited genetic pool. To address this limitation,
recombinant inbred (RI) mice, and particularly,
It is only logical that HLA-II molecules are key the BXD family of RI mice, a murine reference
determinants of pathological outcomes of GAS population with high genetic diversity, was cre-
STSS and combined NF/STSS because GAS ated using a breeding design originally
SAgs, pivotal mediators of systemic shock, constructed by Benjamin A. Taylor by crossing
simultaneously cross-link HLA-II molecules a female C57BL/6J (B6 or B) and a male DBA/2J
expressed on antigen-presenting cells (APCs) (D2 or D) (BXD) and followed by at least 20 con-
and TCRVβ elements and elicit secutive generations of sib-matings (Taylor et al.
pro-inflammatory cytokine storm leading to mul- 1973) (Fig. 10.1). These BXD RI strains have
tiple organ damage and shock (Norrby-Teglund been used extensively to study variation in path-
et al. 2001; Proft et al. 2003). More recently ogenicity and uncover genes modulating lethality
Giesbrecht et al. demonstrated that cross-linking (Benjamin et al. 1986; Melvold et al. 1990;
of the SAg streptococcal pyrogenic exotoxin A Watters et al. 2001; Chesler et al. 2003). While
(SpeA) to HLA-II alone can cause activation of there is extensive variation between each strain,
the APCs independent of the responding T cells there is virtually no genetic variation between
(Giesbrecht et al. 2019). However, in addition to individuals of one strain (Peirce et al. 2004).
SAgs, GAS produces a plethora of other virulence Thus, each BXD strain resembles a large human
factors that might modulate the host immune population of monozygotic twins. The most com-
responses besides involving the highly polymor- pelling feature of the BXD RI panel is that the
phic HLA class II alleles. It is likely that genomes of the parental inbred strains (C57BL/6J
polymorphisms in cytokine genes and innate and DBA/2J) have been fully sequenced, and they
pathogen recognition receptors including TLR4 are a replicable, replenishable, and recapitulatable
affect host innate immune responses and might resource to map genetic loci of phenotypic traits
contribute to the severity and susceptibility to as well as to study causal and mechanistic links of
GAS infections. However, the complete spectrum pathogenesis, treatment, prevention, and assess-
of genetic determinants that influence susceptibil- ment of the countermeasures of global biothreats
ity remains unknown. Identification of the key (Waterston et al. 2002). To further improve the
host factors will significantly impact the under- power and precision, the breeding strategy of
standing of mechanisms underlying pathogenesis BXD lines was modified, and accordingly, the
due to invasive infections caused by GAS, production of strains BXD43 through BXD102,
improve therapeutics, and management strategies. started in the late 1990s, was derived from an
One way to accomplish is to perform a genome- advanced intercross (BXD-ARI) progeny that
wide search for susceptibility loci in humans that have been bred following a schema that
requires the involvement of affected family incorporated roughly twice as many
members; while this may be possible, it is quite recombinations between the parental genomes
impractical and might be confounded by other B6 and D2 compared to the F2-derived BXDs
variables. (BXD-RI) and the ability to explore one or more
The influence of genetic background on sus- quantitative trait loci (QTL) associated with a
ceptibility or resistance to invasive GAS given disease or complex trait (Darvasi 1998;
infections has been reported using inbred mouse Williams et al. 2001; Chesler et al. 2003; Peirce
lines, including BALB/c, C3H/HeN, CBA/J, and et al. 2004; Parker et al. 2012; Pandey and
C57BL/10 (Medina et al. 2001). While the use of Williams 2014).
these inbred lines offered insights, they lack the
power to mirror the host responses as seen in
Fig. 10.1 BXD strains are generated by crossing two inbreeding (advanced intercross-derived recombinant
fully inbred strains (C57BL/6J [female] and DBA/2J inbred, ARI) to allow random recombination of the
[male]), abbreviated as B6 and D2 or simply B and D, chromosomes and generate progressively inbred BXD
respectively. The resulting F1 generation (B6D2F1 or F1) strains whose genomes exhibit unique B to D or D to B
is in turn crossed to produce F2 progeny. Systemic recombinations that are stably fixed. ARI mice increase the
inbreeding is then performed beginning with the F2 gen- recombinations and diversity of the genetic pool (Peirce
eration for over 20 generations (recombinant inbred, RI) or et al. 2004; Abdeltawab et al. 2008)
after random mating for 8–14 generations of F2 before
10.2.2 ARI-BXD Mouse Models strain were more resistant than the male DBA/2J
of GAS NSTI mice, suggesting the propensity of sex in addition
to host genetics in driving susceptibility to GAS
In order to mimic the genetic diversity as seen in NSTIs (Krishnan et al. 2016). In order to identify
humans, the Kotb research group undertook a the candidate genes involved in mediating differ-
systems genetics approach to study the effect of ential susceptibility to GAS NSTIs, the Kotb
multiple genes in modulating disease phenotypes research group capitalized on the BXD-ARI
to invasive GAS infections. Prior to establishing mice and established a mouse model of GAS
the BXD ARI mouse models of GAS NSTIs, the NSTI (subcutaneous infections) with the hyper-
distinct role of host genetics and sex differences virulent M1T1 clinical isolate, GAS 5448 (origi-
in driving susceptibility to GAS NSTIs were nally isolated from a patient with STSS)
demonstrated in subcutaneous infections of con- (Chatellier et al. 2000). Using a large panel of
ventional mouse strains C57BL/6J, DBA/2J, AJ, 33 BXD strains along with the parental strains
and CD-1 (Krishnan et al. 2016). Krishnan et al. C57BL/6J and DBA/2J, and their F1 strains
reported that DBA/2J strain was more susceptible (B6D2F1), the GAS NSTI associated QTLs
than C57BL/6J, and female mice of DBA/2J were identified through genome-wide linkage
Fig. 10.2 Swivel graph showing survival against NSTIs indicating increased survival and negative indices
caused by GAS 5448 within the 33 BXD (black bars), B6 indicating decreased survival. Error bars indicate SEM.
(green bar), D2 (red bar), and their F1 (blue bar) strains is P-values were calculated by Generalized Linear Model
expressed as mean values of corrected relative survival (GLM) using ordinary least squares (OLS) ANOVA.
indices (cRSIs). Data is rank-ordered with positive indices Data from Chella Krishnan et al. (2016)
scans by mapping quantitative phenotypic traits mapped to Chr 7 between 125 and 131 Mb,
to the BXD mouse genotype available from the while the QTL modulating maximum lesion area
gene network (Chella Krishnan et al. 2016). was mapped to Chr 6 (131.6–141.8 Mb) and Chr
There were significant differences in the differen- 18 (49.5–56.3 Mb) (Fig. 10.3).
tial susceptibility to GAS NSTI among different Aziz et al. established the GAS sepsis model
BXDs, their parental, and F1 strains with BXD40, (intravenous infections) using the M1T1 clinical
BXD101, and BXD64 at the susceptible end isolate, GAS5448, using B6 and D2 parents and a
while BXD87, BXD85, and BXD73 strains panel of 20 BXD strains, and reported the influ-
were extremely resistant (Fig. 10.2). Several ence of genetics, sex, age, body weight, and inoc-
genomic loci proved significantly associated ulum titer on susceptibility and survival to GAS
with survival, body weight change, and maxi- sepsis (Aziz et al. 2007). Their study revealed that
mum lesion size due to GAS NSTI. The strongest bacteremia and dissemination to organs were
and highly significant QTL modulating mouse major predictors of severity and mortality due to
survival against GAS NSTI mapped to mouse GAS sepsis, and preliminary mapping results
Chromosome (Chr) 2 between 24.5 and 35 Mb. suggested two significant QTLs on Chr 2 that
The QTLs for body weight change kinetics needed to be verified and further narrowed down
Fig. 10.3 Genome-wide interval mapping for survival 6 and 18, respectively, suggesting a likely role for genes
(a), body weight change (b), and maximum lesion size under these QTLs in modulating the respective responses
(c) revealed suggestive QTLs on mouse Chr 2, 7, and during GAS NSTI (data from Chella Krishnan et al. 2016)
using additional BXD strains. By analyzing dis- mouse GAS sepsis survival, but fine-mapped
ease phenotypes in the context of BXD mice genes between 22 and 34 Mb and verified the
genotypes in a larger panel of BXD strains differential expression of transcripts of key
(32 strains including the parental strains, B6 and genes in uninfected or GAS-infected blood and
D2), Abdeltawab et al. not only confirmed the spleens from highly resistant or susceptible BXD
findings by Aziz et al. (2007) of a highly signifi- strains, at selected times post-infection
cant QTL to mouse Chr 2 modulating BXD (Abdeltawab et al. 2008). Further, Abdeltawab
et al. reported a second less significant QTL that derived from studies on these mouse models are
was also mapped on the same chromosome comparable to real-world data from diverse
between 125 and 150 Mb, and a third QTL on human populations. These models help tease out
Chr X. The QTLs for bacteremia and bacterial the mechanistic insights into disease and to assess
dissemination to spleen overlapped with those for the efficacy of potential therapeutics. For exam-
survival, with a slight difference in significance, ple, preliminary findings by Nookala et al. (man-
with interleukin-1 alpha and prostaglandin E uscript in preparation) show that the survival and
synthases pathways as key networks involved in the frequency of CD45+, CD11b+ monocytes, and
modulating GAS sepsis (Abdeltawab et al. 2008). LY6G+LY6C+ myeloid-derived suppressor cells
GAS sepsis and NSTI were independent stud- (MDSCs) are significantly lower in BXD40
ies with different routes of infection performed on (NSTI susceptible) compared to BXD87 (NSTI
BXD-ARI mice yet pointed out that the genetic resistant) during subcutaneous GAS infections.
susceptibility loci for survival in both cases was However, this condition was reversed with
mapped to QTL on Chr 2. About 125 genes were clindamycin treatment (unpublished findings,
significantly differentially expressed in the GAS Nookala et al., Fig. 10.4a, b). The accumulation
NSTI susceptible strains (BXD 40 and 64) com- of MDSCs is crucial for the host control of
pared to uninfected controls. BXD strains that infections. MDSCs are a heterogeneous collec-
were resistant to GAS NSTIs were identified to tion of monocytes and polymorphonuclear innate
be BXD 73 and BXD87. Parsing of the 125 dif- immune cells and function as initial effector cells
ferentially expressed genes identified two high- to restrict infection burden and suppress the
scoring networks that likely mediate susceptibil- responses of activated effector T and NK-cells
ity to GAS NSTIs in susceptible hosts. Particu- (Ost et al. 2016; Dorhoi and Plessis 2018). The
larly, IL-1β was identified as the top upstream understanding of the induction and frequency of
regulator likely mediating pro-inflammatory MDSCs in GAS infections is just beginning to
effects driving severity seen in susceptible hosts. evolve. It is an attractive possibility that the loss
The differential expression level of IL-1β or lack of induction of MDSCs could be one of
transcripts was verified in GAS-infected skin tis- the mechanisms against survival in GAS NSTI
sue from susceptible mice and validated in tissue susceptible mice, which needs to be further
biopsies and plasma from GAS-infected patients investigated.
(GAS 2006, INFECT Consortium) and in
GAS-infected 3D reconstructed skin models
(Chella Krishnan et al. 2016). 10.3 HLA-II Mouse Models
of Invasive GAS Infections
10.2.3 Assessing Heterogeneity Invasive infections caused by GAS is one of the

of Treatment Effects in BXD most complex human diseases affecting multiple
Models of GAS NSTI organs driven by complex interactions that neces-
sarily include HLA-II haplotype, immunological
Invasive GAS infections have complex etiologies background, GAS burden, and the variations in
that vary depending on the host genetic back- the secreted and surface-bound virulence factors
ground, environment, gender, and other as yet of the GAS pathogen. SAgs are among the most
unidentified complex traits. The heterogeneity in potent toxins secreted by GAS. While the BXD
the outcomes and the cause, therefore, have to be mice models described above are invaluable tools
investigated to determine the best course of treat- to study the effect of complex traits, conventional
ment and prevention. The BXD mice offer a inbred mice are naturally resistant to
promising model system to provide different SAg-mediated outcomes of GAS infections
information and insights into pathomechanisms (Miethke et al. 1992; Blank et al. 1997;
underlying GAS infections. Importantly, data Sriskandan et al. 2001). These setbacks were
Fig. 10.4 Immunophenotype of myeloid-derived sup- groups. t-SNE plot was run on ~5000 live cells
pressor cells assessed by flow cytometry in blood from discriminated by viability dye on singlets. Manually
10 days post GAS-infected BXD mice either susceptible or gated populations were overlaid onto t-SNE plots (a).
resistant to GAS NSTI. Data is represented as a Kaplan–Meier survival curves for the susceptible or resis-
t-distributed stochastic neighbor embedding (t-SNE) plot tant BXD mice 10 days post GAS infection that were
to aid the visualization of distribution and clustering of either untreated or treated with clindamycin (b)
indicated subsets in untreated or clindamycin-treated
overcome through the use of transgenic mice higher levels of the pro-inflammatory cytokines,
expressing human plasminogen (Sun et al. 2004; TNF-α and IFN-γ, in vivo and in vitro compared
Cole et al. 2006) and HLA-II alleles associated to mice expressing DQ6, the DQ allele that is in
with differential susceptibility to invasive GAS linkage disequilibrium with the DR15 allele
infections (Nooh et al. 2007). Several studies (DRB1*15/DQB1*06 (DR15/DQ6)). While stud-
have reported the use of HLA-II transgenic mice ies by Nooh et al. (2007) used models of invasive
models to mimic responses to streptococcal and GAS sepsis, Nookala et al. recently reported the
staphylococcal SAgs as seen in humans use of HLA-II transgenic mice expressing either
(Unnikrishnan et al. 2002; Welcher et al. 2002; DRB1*1501 (DR15) or DRB1*04/DQB1*0302
DaSilva et al. 2002; Roy et al. 2005; Rajagopalan (DR4/DQ8) as mouse models of GAS NSTI
et al. 2006; Mangalam et al. 2008). (Nookala et al. 2018). They demonstrated that
The first evidence of the dominant and HLA-II transgenic mice expressing the protective
overriding effect of HLA-II molecules to GAS DR15 allele significantly induced surface GARP/
infection in vivo and SAg stimulations in vitro LAP expressing FoxP3+ T regulatory cells com-
was reported by Nooh et al. (2007). Nooh et al. pared to mice expressing HLA-II DR4/DQ8, a
(2007) demonstrated that HLA-II mice haplotype associated with neutral risk for com-
expressing the neutral risk haplotype, DRB1*04/ bined NF/STSS, which skewed the responses
DQB1*0302 (DR4/DQ8), mounted significantly toward a Th1 type during in vivo infections with
Fig. 10.5 Proposed pathomechanisms underlying severe STAT molecules into the nucleus resulting in the activa-
or nonsevere outcomes of GAS revealed from HLA-II tion of transcription factors. The net effect is the polariza-
transgenic mouse models of GAS NF/STSS. GAS tion of naïve T cells into distinct subsets (Th1/Th17 in case
superantigens simultaneously crosslink HLA-II and dis- of severe and Tregs in case of nonsevere). Data presented
tinct variable beta regions in the TCR (TCR-Vβ), thereby is a summary of analysis from transcriptomics data from
eliciting robust levels of pro-inflammatory cytokines. Cell in vitro and in vivo studies in HLA-II mouse models of
activation driven by the pro-inflammatory cytokine–recep- GAS NF/STSS. Red arrows indicate increased expression
tor interactions induces phosphorylation of STAT of transcripts
molecules mediating the translocation of phosphorylated
a GAS 2006 (clinical isolate from a patient with in vivo during GAS NSTI is shown in Fig. 10.5.
combined NF/STSS, INFECT Consortium) and Transcriptome analysis of GAS2006-infected
in vitro stimulations to SmeZ with the concomi- skin from HLA-II DR4/DQ8 (a haplotype
tant expression of IFN-γ and IL-2 (Nookala et al. associated with neutral risk for combined
2018). The summary of various cytokines and NF/STSS) mice offered a plethora of information,
transcription factors that are significantly specifically, clues for the existence of a combined
modulated during SAg stimulations in vitro in adipogenic, metabolic, and inflammatory axis in
splenocytes from HLA-II mice expressing risk GAS NSTI (Nookala et al. 2018). The top five
(severe) or protective (nonsevere) alleles and most significantly downregulated pathways
Fig. 10.6 Heat map showing top 10 hits for canonical inactivation, while a positive z-score indicates a predicted
pathways with significant activated (red) and inhibited activation of the enriched pathway. These pathways
(green) z-score (A) associated with the differentially predicted by the Ingenuity Pathway Analysis based on
expressed genes in GAS-infected skin lesions from the gene expression changes seen in our dataset may be
HLA-II mice expressing DR4/DQ8, a haplotype responsible for the pathophysiology underlying GAS
associated with neutral risk for combined GAS NF/STSS NF/STSS
compared to PBS. A negative z-score indicates a predicted
during GAS NSTI were oxidative phosphoryla- using the ARI-BXD model provided the first evi-
tion, PPAR, TCA cycle II, PPARα/RXRα activa- dence of the architecture of complex traits that
tion, and fatty acid β-oxidation 1, while the top were crucial in the understanding and the identifi-
five most significantly upregulated canonical sig- cation of genes, networks, and pathways underly-
naling pathways were neuroinflammation, ing invasive GAS infections. The reductionist
TREM1, IL-6, acute phase response, (HLA-II) approach in murine models of invasive
p38MAPK, and leukocyte extravasation GAS infections has contributed to unraveling the
(Nookala et al. manuscript in preparation). The important role of HLA class II allelic
top ten canonical pathways that are significantly polymorphisms leading to selective polarization
enriched in HLA-II mice expressing the into distinct Th1/Th17 or T-regulatory subsets
DR4/DQ8 haplotype are represented as a heat during GAS NSTI, and the resulting
map in Fig. 10.6. pro-inflammatory or anti-inflammatory patho-
Collectively, we provided strong evidence genic processes they drive through their complex
through a combination of systems genetics using cross talk with the immune system. Although
a large panel of BXD mice and humanized mouse polymorphisms in HLA-II alleles are critical
models of invasive GAS infections that host determinants of severity of GAS infections,
genetic variations contribute significantly through systems genetics is becoming an important
distinct molecular events involved in mediating approach to understanding both the underlying
susceptibility and clinical outcomes of invasive biology and the distinct genetic loci, possible
GAS infections. The systems genetics approach genes that modulate the particular traits, and the
mechanisms driven by complex traits in the strategies can then be personalized depending on
genome. the host genetic context that confer susceptibility
or protection from particular disease in as much
as customized interventions can produce more
10.4 Future Perspectives effective results than generalized therapeutic
approaches.
There is an urgent need to reevaluate host
responses to identify host-directed strategies for Acknowledgements The work presented was supported
interference with various signaling pathways by the grants from the European Union (FP7/2012–2017)
under the grant agreement 305340 (MK), grants from the
combined with standard antibiotic care to attenu- Swedish Research Council under grant number 20150338
ate severity and improve outcomes of GAS NSTI. (MK), UND CoBRE Host–Pathogen Interactions Pilot
The identification of the interactions driven by the Award (SN), and UND Genomics Core funded through
HLA-II haplotype as well as the complex traits of grant support from the National Institutes of Health grants
P20GM104360 (SN) and by the National Institute of Gen-
the genome is critical because therapeutic effec-
eral Medical Sciences of the National Institutes of Health
tiveness is influenced by pathogen and host- under grant numbers P20GM103442, U54GM128729,
related genetic and nongenetic factors. Over the and P20GM113123. We thank John Lee, Graphic Design
last few years, the concept of “theranostics,” a Specialist, at Information Resources of the University of
North Dakota School of Medicine & Health Sciences, for
convergence of therapy and diagnostics catered
his assistance with the figure illustrations.
to patients based on their individual genetic
characteristics (Degrauwe et al. 2019) is becom-
ing increasingly popular. Given the disease het-
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Systems Biology and Biomarkers
in Necrotizing Soft Tissue Infections 11
Edoardo Saccenti and Mattias Svensson
Contents
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
11.2 State of the Art: Existing Biomarkers for NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . 169
11.3 Biomarker Type and Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
11.3.1 Diagnostic Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
11.3.2 Prognostic Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
11.3.3 Predictive Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
11.3.4 Predisposing Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
11.4 Biomarker Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
11.4.1 Genomic Biomarkers in NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
11.4.2 Transcriptomic Biomarkers in NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
11.4.3 Epigenomic Biomarkers in NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
11.4.4 Proteomic Biomarkers in NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
11.4.5 Metabolomic Biomarkers in NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
11.4.6 Microbiomic Biomarkers in NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
11.4.7 From One-Category-Analyses Towards Systems Biological Approaches . . . 174
11.5 Systems Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
11.6 Systems Biology Approaches to Biomarker Discovery . . . . . . . . . . . . . . . . . . . 176
11.7 Networks and Network Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
11.8 Multivariate Biomarker and Omics Signatures . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
11.9 Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
E. Saccenti (*) Abstract

Laboratory of Systems and Synthetic Biology,
In necrotizing soft tissue infection (NSTI)
Wageningen University and Research, Wageningen, The
Netherlands there is a need to identify biomarker sets that
e-mail: edoardo.saccenti@wur.nl can be used for diagnosis and disease manage-
M. Svensson ment. The INFECT study was designed to
Center for Infectious Medicine, Department of Medicine, obtain such insights through the integration
ANA Futura, Karolinska Institutet, Karolinska University of patient data and results from different
Hospital, Huddinge, Sweden

168 E. Saccenti and M. Svensson
clinically relevant experimental models by use indicates that these factors must be associated
of systems biology approaches. This chapter with the disease. In this context, a systems
describes the current state of biomarkers in biological approach, used correctly, has the
NSTI and how biomarkers are categorized. greater opportunity to identify hitherto unknown
We introduce the fundamentals of top-down factors that may be associated with disease and
systems biology approaches including analysis that may be used as biomarkers.
tools and we review the use of current methods Systems biology is a holistic approach to
and systems biology approaches to biomarker deciphering the complexity of biological systems
discover. Further, we discuss how different that starts from the understanding that the
“omics” signatures (gene expression, protein, networks that form the whole of living organisms
and metabolites) from NSTI patient samples are more than the sum of their parts. The systems
can be used to identify key host and pathogen biology approach to understand disease-related
factors involved in the onset and development biology has the potential to revolutionize our
of infection, as well as exploring associations understanding of the cellular pathways and gene
to disease outcomes. networks underlying the onset of disease, and the
mechanisms of treatment strategies that amelio-
Keywords rate disease phenotypes. In the post-genomic era,
a wealth of novel approaches for generating and
Epigenomics · Genomics · Network inference
analyzing large, high-dimensional genomic,
and analysis · Mathematical modelling ·
transcriptomic, proteomic, and metabolomic data
Metabolomics · Microbiome · Multivariate
sets from cohorts of normal and diseased
statistics · Proteomics · Top-down approaches
individuals can be used in combination with
systems biology approaches (Rosato et al. 2018).
Highlights
In research related to infectious diseases, such as
• Comprehensive systems biology analyses and
NSTI, the data sets include information from both
interpretation of omics data are emerging as
pathogen- and host-oriented analyses that can be
valuable tools for biomarker discovery
used at a systems biology level. The emerging
in NSTI.
field of systems biology attempts to harness com-
• The inflammatory proteins IL1β, IL-6,
plex, multi-parameter systems by computationally
CXCL9, CXCL10, and CXCL11, were
integrating gene-level data with molecular
identified as potential biomarkers to discrimi-
pathways and networks to extract new biological
nate S. pyogenes NSTI from NSTI caused by
insight. Systems biology may combine and aug-
other pathogens.
ment current strategies to biomarker discovery,
• Metabolite–metabolite association networks
generating novel, experimentally testable
analysis has proven able to define sets of
candidates.
metabolites associated with biofilm formation
A biomarker is a measurable indicator of some
in NSTI.
biological state or condition, and in recent years
• Metabolites in combination with multivariate
systems biology approaches have been applied to
and machine learning modelling have the poten-
screen and identify diagnostic, prognostic, predic-
tial to be utilized as predictive markers in NSTI.
tive, and predisposing biomarkers as well as
targets for prevention and treatment of disease
(Feala et al. 2013; Vafaee et al. 2018; Anvar
11.1 Introduction
et al. 2018). There are several successful
examples of molecular biomarkers that are cur-
In search for biomarkers in NSTI, analyses have
rently the clinical standard for diagnostic screen-
usually focused specifically on measuring certain
ing in several diseases, for example, in
selected factors, such as cytokines. This approach
myocardial infarction (Hajar 2016) and certain
is, to some extent, distorted because it is based on
cancers (Mehta et al. 2010; Goossens et al.
already existing knowledge that somehow
2015). The search for novel molecular biomarkers
11 Systems Biology and Biomarkers in Necrotizing Soft Tissue Infections 169
continues to be a major research drive in many Although LRINEC scoring is not optimal, it has
biomedical fields. A PubMed (https://pubmed. been used to distinguish NSTI from less severe
ncbi.nlm.nih.gov/) query for NSTI and biomarker soft tissue infections (Wong et al. 2004).
(Biomarker AND (NSTI OR “necrotising soft Recently, LRINEC score has also been
tissue infection” OR “necrotising fasciitis”) in analyzed in relation to pro-inflammatory
March 2020 returned only 39 hits (44 using the cytokines, and this revealed no significant associ-
spelling “necrotizing”) demonstrating how little ation between the LRINEC score and analyzed
is known and how limited the research activity on specific cytokine levels on admission (Hansen
this topic is, despite an incidence ranging from et al. 2017a). However, the analyses of cytokines
0.3 to 15.5 case per 100.000 population (Stevens revealed IL-6 to be associated with disease sever-
and Bryant 2017) and all-cause 90-day mortality ity, and that IL-1β was strongly associated with
of 18% (Madsen et al. 2019). In NSTI biomarkers 30-day mortality (Hansen et al. 2017b). Another
for early detection and guidance of treatment may factor suggested to have biomarker potential in
improve outcome and help to reduce the length of NSTI is Pentraxin-3 (PTX3) a glycoprotein which
hospital stay and mortality. There are good belongs to the pentraxin protein superfamily,
opportunities that systems biology approaches in including acute phase proteins, C-reactive protein
NSTI can identify biomarkers that could be used (CRP), and serum amyloid protein (SAP). PTX3
as novel tools for rapid and early identification is produced by vascular cells or inflammatory
and stratification of patients with NSTI upon cells and is released into the circulation, possibly
admission. In this book chapter, we review bio- reflecting systemic inflammation. The PTX3 level
marker types and detection methods, as well as a in patients with NSTI at time of admission was
number of systems biology approaches poten- associated with septic shock, amputation, and risk
tially useful to identify candidate biomarkers of death in patients, but was not an independent
in NSTI. predictor of 180-day mortality in this patient
group (Hansen et al. 2016a). Levels of s-lactate
>6 mM and creatinine >144 mM are considered
11.2 State of the Art: Existing prognostic markers. In addition, microbiological
Biomarkers for NSTI cultures of Clostridium or Vibrio are negatively
associated, while a microbiological culture of
There are currently no approved biomarkers for GAS is positively associated with survival
the diagnosis or prognosis in NSTI, and the in NSTI. Nevertheless, there is a great need for
molecular mechanisms of host responses during robust early measurable biomarkers to improve
NSTI remain poorly understood. This lack of identification and management of NSTI.
understanding reflects the complex, multifactorial
nature of NSTI, which is believed to involve a
network of interweaving host and pathogen
11.3 Biomarker Type and Detection
molecular pathways that dictate disease onset
and progression.
Molecular biomarkers generally consist of
Available information on NSTI biomarkers are
molecules measured in body fluids or in samples
restricted to a few research studies that focused on
from the affected tissue. Challenges facing bio-
a limited number of selected factors or informa-
marker research include the lack of standardized
tion of factors included as components of rela-
methods for fast reliable detection. Biomarkers
tively inaccurate scoring systems, such as the
can be divided into four categories: diagnostic,
LRINEC (Laboratory Risk Indicator for Necrotiz-
prognostic, predictive, and predisposing/suscepti-
ing Fasciitis). LRINEC is generated from six rou-
bility (Quezada et al. 2017; Simon 2011, 2014;
tinely performed laboratory tests including the
Drucker and Krapfenbauer 2013). The typical
analyses of patients´ C-reactive protein, white
pattern of biomarker discovery is shown in
blood cell count (10,000/μL), hemoglobin
Fig. 11.1.
(g/dL), sodium (mEq/L), creatinine, and glucose.
Fig. 11.1 Relationship between the classical biomarker discovery pipeline and the systems biology cycle. Systems
biology can play a pivotal role in both discovery and analytical validations steps. Figure adapted from Del Campo et al.
(2015) and Rosato et al. (2018)
11.3.1 Diagnostic Biomarkers chemokines displayed statistically significant

concentration differences between S. pyogenes
Diagnostic biomarkers are used, e.g. to determine and polymicrobial NSTIs. In addition, this study
the health status of a patient to ensure appropriate performed RNA-seq analyses on tissue biopsies
management. In infectious diseases such as NSTI and this showed that genes with significantly
and sepsis, immediate treatment is required, greater expression in polymicrobial compared to
necessitating quick, early, and accurate diagnosis monomicrobial NSTIs were those encoding extra
which can help decision making. A singular ideal cellular matrix (ECM) components like collagen,
biomarker may not be identified, but rather an fibronectin, lumican, and connective tissue
alternative approach focusing to determine the growth factor. In addition, gene sequencing data
diagnostic relevancy of multiple biomarkers showed, in line with protein data, that genes
when used in concert may be useful. The ongoing encoding interferon-inducible mediators such as
efforts in the development of a multiplex point- CXCL9, CXCL10, CXCL11 were expressed at
of-care testing kit, enabling quick and reliable higher levels in monomicrobial NSTIs, in partic-
detection of serum biomarkers, may have great ular those caused by S. pyogenes.
potential for early diagnosis of acute bacterial
infections. Thänert et al. (2019) provided insight
into the pathophysiology of mono- and
11.3.2 Prognostic Biomarkers
polymicrobial NSTIs and protein multiplex anal-
ysis highlighted three potential biomarkers,
A prognostic marker identifies outcome in
CXCL9, CXCL10, CXCL11, as these three
patients. Usually, these markers provide insight
into unfavorable outcome, such as mortality. The PTX3 may be an appropriate marker of severity
presence or absence of a prognostic marker can be and prognosis of NSTI (Hansen et al. 2016a).
useful for the selection of patients for a specific Although PTX3 was reported to perform even
treatment but does not directly predict the better than the “classical” inflammatory markers
response to a treatment. CRP and procalcitonin (PCT), its usefulness has
In NSTI plasma biomarkers representing the been a matter of debate (Honore and Spapen
early inflammatory response are thought to be 2016).
useful as prognostic markers of disease severity
and mortality. To determine the association of
admission biochemical markers, such as lactate, 11.3.3 Predictive Biomarkers
to limb loss and mortality both univariate and
multivariable analyses have been used. For exam- Predictive biomarkers aim to objectively evaluate
ple, arterial lactate was found to be associated the outcome of a specific clinical intervention, or
with both mortality (odds ratio [OR], 1.5; 95% the differential outcomes of two or more
confidence interval [CI], 1.1 to 2.0; P- interventions, including toxicity. Predictive and
value ¼ 0.009) and limb loss (OR, 1.3; 95% CI, prognostic markers often get confused because
1.0 to 1.7; P-value ¼ 0.02), and lactate was many markers can be both predictive and prog-
suggested to be used early on to guide aggressive nostic. A marker can be an unfavorable prognos-
therapeutic interventions (Schwartz et al. 2013). tic factor but could predict favorably for response
In addition, attempts have been made to associate to therapy, or vice versa. Furthermore, it is possi-
markers with ICU (Intensive Care Unit) scoring ble that markers can be prognostically unfavor-
systems including simplified acute physiology able, but predict favorably for one therapy and
score (SAPS) II (Le Gall et al. 1993) and sequen- predict unfavorably for another therapy.
tial organ failure assessment (SOFA) scores When searching PubMed with the joint medi-
(Vincent et al. 1996), as well as the LRINEC cal subject headings “NSTI” and “predictive
score (Wong et al. 2004), presence of septic markers,” most, if not all, publications indexed
shock, microbial etiology, renal replacement ther- are studies that have identified prognostic rather
apy, and amputation. than predictive markers. In a recent study Afzal
In a study by Hansen et al. it was demonstrated and coworkers (Afzal et al. 2020) performed
that plasma levels of mannose-binding lectin and metabolomics analysis combined with advanced
ficolins, which can activate the complement path- bioinformatics analysis of metabolite–metabolite
way, were significantly lower in patients with association networks, indicating potential of
NSTI than in controls (Hansen et al. 2016b). metabolites as predictive markers in NSTI
Furthermore, this study revealed that a high base- (described in more detail in Sect. 11.4.5).
line ficolin-2 level indicated a 94% chance of In conclusion, there is very little information
surviving the first 28 days after admission. on biomarkers that can be used to predict patient
Another study by Hansen et al. reported on the who benefits from different clinical interventions.
association between cytokine (IL-1β, IL-6, IL-10, Thus, this could potentially be an accelerating
and TNF-α) levels and the LRINEC score, dis- research field which would have a significant
ease severity and mortality in NSTI patients clinical impact.
(Hansen et al. 2017b). Although no significant
association between the LRINEC score and cyto-
kine levels on admission was found, the authors 11.3.4 Predisposing Biomarkers
found that IL-6 was consistently associated with
disease severity and that high IL-1β (OR 3.86 The fourth category of biomarkers is used to
[95% CI, 1.43–10.40], P-value ¼ 0.008) and identify individuals with a predisposition or sus-
IL-10 (4.80 [1.67–13.78], P-value ¼ 0.004) ceptibility to develop disease. In NSTI, studies
were associated with 30-day mortality. Also, have revealed that allelic variations in human
leukocyte antigen (HLA) class II haplotypes made possible by technological developments

result in striking differences in severity and that have expanded the throughput and reduced
outcomes of invasive GAS infections via the dif- the cost of single nucleotide polymorphisms
ferential presentations of GAS superantigens (SNP)-based research and allowed SNP-based
(SAgs) by class II HLAs to host T-cell receptor techniques to identify DNA copy number varia-
(TCR) Vβ elements (Kotb et al. 2002, 2003; tion. Nevertheless, to identify SNPs remains a
Norrby-Teglund et al. 2000; Norrby-Teglund significant challenge, due to the difficulty of
and Kotb 2000). In particular, specific HLA separating these. One of the limitations of GWA
class II haplotypes have been found to confer studies is that it captures only common SNPs,
strong protection from severe systemic disease, which by themselves may only contribute a
whereas others increased the risk of severe dis- small extent of risk to develop disease. NSTI
ease; patients with the DRB1*1501/DQB1*0602 patients infected with the same bacterial strain
haplotype mounted significantly reduced can develop very different manifestations and a
responses and were less likely to develop severe role of immunogenetics of the host in shaping the
systemic disease. outcome of invasive streptococcal infections can
It is likely that in NSTI a complex interaction be identified (Kotb et al. 2002). However, to
of multiple host and microbial factors that do not identify rare SNPs that contribute to develop
lend themselves to reduction into a simple for- NSTI for a small percentage of patients, more
mula exist, and therefore more holistic cost-effective technologies will be required.
approaches need to be implemented to identify Despite its disadvantages DNA sequencing may
biomarkers for diagnosis, prognosis, prediction, become more important for patient care over the
or predisposition/susceptibility. next 5 years as the costs are reduced and
capabilities extended.
11.4 Biomarker Use

11.4.2 Transcriptomic Biomarkers
11.4.1 Genomic Biomarkers in NSTI in NSTI
To identify genomic biomarkers DNA sequenc- Mapping genotypes to phenotypes is one of the
ing (host and pathogen) technologies, such as long-standing challenges in biology and medi-
whole-genome sequencing can be used. While cine, and a powerful strategy for tackling this
cancer research has reached the farthest using problem is performing transcriptome analysis.
whole-genome sequencing (Staaf et al. 2019), it Transcriptomics entails the measurement and
is difficult to find examples where DNA sequenc- analysis of mRNA using microarray or RNA
ing has been used to identify biomarkers in NSTI. sequencing technologies. At the beginning
It is likely that NSTI, with its acute course, poses microarrays offered tremendous opportunities by
other challenges than cancer and in NSTI perhaps capturing parallel information about many more
gene sequencing may be best used to identify mRNAs than quantitative PCR techniques
predisposing conditions. However, the relatively (RT-PCR), which mostly focus on single mRNA
high costs likely prevent whole-genome sequenc- analyses. However, like all molecular techniques
ing analyses from becoming routine on a large there are limitations, such as dependence on tem-
scale. Instead, “exome” sequencing (targeted plate quality (Bustin and Nolan 2004).
sequencing of protein-coding regions) may Although microarrays have been highly pro-
become a cost-effective way to provide access to ductive research tools and used in several current
the entire transcribed genome of individuals. In prognostic and predictive tests, other emerging
addition, genome-wide association (GWA) stud- technologies have replaced the microarrays.
ies identifying risk loci are feeding into the clini- Next-generation DNA sequencing technology
cal use of DNA variants. This research has been known as “RNA-seq” allows simultaneous
analysis of all RNA molecules, including alterna- 11.4.3 Epigenomic Biomarkers in NSTI
tive splice variants, mRNAs, non-coding RNAs
(ncRNAs), and miRNAs. RNA-seq has Epigenetics can be defined as the field of inherit-
revolutionized the analysis of RNAs. In a recent able changes in gene expression that are not
study Thänert et al. took advantage of the next- caused by alterations in DNA sequences. Epige-
generation sequencing tools to enable microbial netic mechanisms include DNA methylation, his-
profiling using 16S rRNA sequencing with tran- tone modifications, and non-coding RNAs, and
scriptional analysis of host and microbe using epigenetic alterations have an increasingly clear
dual RNA sequencing (RNA-seq) in tissue role in modulating inflammatory and other immu-
biopsies from NSTI patients (Thänert et al. nological processes. Identification of epigenetic
2019). The results from this study demonstrated alterations may provide important prognostic,
that, despite the similar clinical presentation of predictive markers. This is an exciting field that
NSTIs, the pathophysiology of mono- and is likely to grow in clinical importance.
polymicrobial etiology differs significantly and There are no studies on epigenetics in NSTI,
that these differences can potentially be exploited while the often-associated condition sepsis has
for diagnostic purposes. been subjected to epigenetic analyses (Cross
The transcriptional analysis of infected tissue et al. 2019). To date, mostly the epigenetic modi-
indicated that the gene expression profile differed fication associated with various stages of sepsis
significantly between monomicrobial streptococ- has been assessed. A focus has been to identify
cal infections and polymicrobial NSTI. Among mechanisms involved in endothelial dysfunction
the genes with significantly greater expression in during the hyperinflammatory response and those
polymicrobial compared to monomicrobial underpinning aspects of immunosuppression.
NSTIs were those encoding ECM components
like collagen, fibronectin or lumican, as well as
connective tissue growth factor, proteins. On the
11.4.4 Proteomic Biomarkers in NSTI
other hand, a set of genes encoding interferon-
inducible mediators such as CXCL9, CXCL10,
Large scale analysis of specific proteomes, also
CXCL11, MX1, and MX2 as well as the
known as proteomics, defines protein diversity
guanylate-binding GTP1 and GTP2 were most
and understands its biological consequences.
prominently higher expressed in monomicrobial
Analysis of proteins as inflammatory markers in
NSTIs, in particular those caused by S. pyogenes.
diseases could provide important information
Because of the many diverse cell types in our
about disease severity and guide decision making.
body each of which express a unique
In true proteomics analysis all proteins from a
transcriptome, conventional bulk population
sample of interest are usually extracted and
sequencing can provide only the average expres-
digested with one or several proteases (typically
sion signal for an ensemble of cells. More
trypsin alone or in combination with Lys-C
recently there has been an explosion of interest
(Wiśniewski and Mann 2012) to generate a
in obtaining high-resolution views of single-cell
defined set of peptides. The peptides obtained
transcriptomics (scRNA-seq) heterogeneity.
are subsequently analyzed by liquid chromatog-
Assessing the differences in gene expression
raphy coupled to mass spectrometry (LC-MS).
between individual cells has the potential to iden-
The two most common approaches are either
tify rare populations that cannot be detected from
designed to achieve a deep coverage of the prote-
an analysis of pooled cells. Due to issues with
ome (shotgun MS) or to collect as much quantita-
cost and analysis challenges, the implementation
tive information as possible for a defined set of
of scRNA-seq as a tool for biomarker identifica-
proteins/peptides (targeted MS) (Picotti and
tion in NSTI may still be some years away and is
Aebersold 2012). Protein content information
unlikely to give the complete picture.
can also be obtained through targeted approaches,
including multiplex analyses made to identify a biofilm formation assays revealed

set of predetermined proteins. Since the test metabolite-specific effects on both bacterial
results are needed within hours in NSTI, growth and biofilm formation. A biofilm can be
approaches will most realistically include considered as a potential complicating
analyses of samples, such as plasma, that can be microbiological feature of NSTI and, conse-
obtained quickly. However, rapid assessment quently, emphasizes reconsideration of antibiotic
based on proteomics and/or multiplex analyses treatment protocols. This study identifies for the
of NSTI patients to identify protein-based first time an NSTI-specific metabolic signature
biomarkers is challenging, as these are time con- with implications for optimized diagnostics and
suming analyses. Nevertheless, multiplex therapies. Thus, the finding of metabolites in
approaches have been used to analyze NSTI NSTI associated with biofilm formation
plasma samples and has provided important emphasizes the potential of metabolites as predic-
insight to potential diagnostic biomarkers tive markers in NSTI.
(Thänert et al. 2019; Hansen et al. 2017a).
11.4.6 Microbiomic Biomarkers in NSTI

11.4.5 Metabolomic Biomarkers
in NSTI Analyzing the microbiome at the system biology
level for comparison in diseases to that of healthy
Metabolomics takes a special position among the subjects has become increasingly popular. Rapid
omics disciplines in the system top-down progress in the development of next-generation
approach as the metabolome of biological pro- sequencing (NGS) technologies in recent years
cesses, carrying imprints of genetic, epigenetic, has provided many valuable insights into com-
and environmental factors, provides the link plex biological systems, including diverse micro-
between the genotype and phenotype (Karakitsou bial communities.
et al. 2019; Rosato et al. 2018; Dunn and Ellis As far as NSTI is concerned, this field of
2005; Kell 2004). Since metabolites represent the research is in its infancy, but it may prove helpful
endpoint of many molecular pathways, in providing biomarkers which will give insights
metabolomics which quantifies the metabolite into the pathological process behind disease evo-
content of cells or tissues is in addition to have lution and progression by determining specific
diagnostic and prognostic value also potentially etiological factors.
useful for predicting treatment response.
Metabolomics has been identified as a novel tool
to discover targets for sepsis diagnosis and prog- 11.4.7 From One-Category-Analyses
nosis, as well as to gain insight into pathogenic Towards Systems Biological
disease mechanisms (Eckerle et al. 2017). Approaches
Recently, we used untargeted metabolomics
analyses of plasma from NSTI patients and The classical approach to biomarker discovery
healthy controls to identify the metabolic usually entails the comparison of molecules or
signatures and connectivity patterns among group of molecules among two or more groups,
metabolites associated with NSTI. Out of usually in a case/control scenario. In NSTI typical
97 metabolites detected, the abundance of comparison is monomicrobial vs polymicrobial
33 was significantly altered in NSTI patients. infection or patient outcomes (septic shock/no-
Analysis of metabolitemetabolite association septic shock (Hansen et al. 2018), amputation/
networks identified 20 metabolites differently no-amputation, or mortality (Hansen et al.
connected between NSTI and controls. Testing 2018)) or some clinical score (low/high LRINEC)
of a set of differently connected metabolites (orni- (Hansen et al. 2017a). Statistical approaches are
thine, ribose, urea, and glucuronic acid) in in vitro usually simple: candidate biomarkers are mostly
tested one at a time in a univariate fashion using biomolecular mechanisms underlying complex
procedure like ANOVA or linear mixed models, biological processes (Ahn et al. 2006)
logistic regression, or Kaplan–Meier curves when Systems biology relies on the use of a mathe-
association with survival is of interest or by matical model to integrate and understand data
correlating biomarker concentration with some that are acquired using a vast array of experimen-
clinical outcome. tal approaches ranging from molecular techniques
Most new biomarkers proposed in the litera- to comprehensive measurement often
ture never reach the clinic, often because of a lack encompassing multiple layers of the omics cas-
of reproducibility. In a meta-analysis of highly cade such as transcriptomics, proteomics, and
cited articles announcing new biomarker metabolomics. Different types of models can be
candidates for a variety of diseases, it was used depending on the data available, the research
shown that follow-up experiments with greater questions, and whether a deductive or an induc-
statistical power generally fail to reproduce the tive approach is followed.
same effect size as the original studies (Ioannidis The deductive approach, usually referred to as
and Panagiotou 2011; Feala et al. 2013). More- Top-down approach (Oltvai and Barabási 2002),
over, in addition to the need for validation of such aims to gain insights into a given phenomenon
candidates, integration of the information starting from the system-wide data acquired using
provided by each biomarker is also needed for a omics technologies from which information is
comprehensive representation of the disease pro- extracted, usually using statistical and machine
cess. Here we argue that biomarker discovery learning methods in combination with network
would be greatly enhanced by applying systems inference and network analysis (Rosato et al.
biology principles such as the multi-scale integra- 2018; Ideker and Krogan 2012). The ultimate
tion of information and the analysis of dynamic goal is to describe the interactions among the
patterns with the help of computational tools. molecular constituents of the system (genes,
proteins, metabolites), possibly across different
conditions (Rosato et al. 2018, Ideker and Krogan
11.5 Systems Biology 2012) to understand how these parts interact to
shape the system-wide behavior of the system.
Systems biology is an interdisciplinary field in On the other side, the inductive approach,
which experimental and computational methods referred to as Bottom-up approach (Oltvai and
concur with mathematical models to the analysis Barabási 2002), starts from a detailed molecular
and the understanding of biological phenomena. and biochemical knowledge of certain biological
In systems biology different types of molecular mechanisms and aims to create mathematical
knowledge are integrated and exploited thanks to models that can reproduce experimental data.
the synergistic use and combination of statistical The overarching goal is to predict the system-
and mathematical model together with experi- wide behavior by building a complete genome-
mental data (Bruggeman and Westerhoff 2007). scale model to provide an integrative view of the
Systems biology embraces a system prospec- biological interactions occurring inside living
tive as opposed to the reductionistic vision which systems (Shahzad and Loor 2012). The iterative
is common in the mainstream biology: it aims to cycle of systems biology is illustrated in Fig. 11.1
tackle biological problems by considering the in relationship to the pipeline for biomarker
interaction among the different parts of the discovery.
systems rather than subdividing it into indepen-
dent sub-problems. In biological systems the
interactions between components dominate the
component themselves in shaping the system-
wide behavior and for this reason the reductionist
approach is less effective to understand the
11.6 Systems Biology Approaches of correlations; for instance, if analyzing

to Biomarker Discovery metabolites, a strong positive correlation can indi-
cate an equilibrium condition or enzyme domi-
Determining whether a given biomarker is causal nance, while strong negative correlation can
or reactive to NSTI (or, in general, to any disease) indicate the presence of a conserved moiety
is likely to provide information on the underlying (Camacho et al. 2005).
pathogenic process and ultimately impact its gen- It is fundamental to understand that the
eral applicability (Villoslada and Baranzini associations among variables can carry informa-
2012). However, standard approaches that are tion also when concentrations (and abundances)
based on just univariate analysis can fail, by do not carry relevant information, for instance, to
their own nature, to account for the interrelated- discriminate between the case and control groups.
ness existing among genes, proteins, and This situation, so common in the biomarker dis-
metabolites that behave in an orchestrated way covery setting, is better illustrated with a
for what concerns regulation, transcription, and simulated example. With reference to Fig. 11.2a,
translation. consider two metabolites (or protein or any other
For instance, Edwards and co-workers biomolecular quantity) A and B measured from
(Edwards et al. 2018) investigated S. pyogenes case patients and controls: under this simulation
infections including NSTI among others, using a there is no difference between the concentration
proteomic approach and identified several human levels of A and B in the two groups. However, if
proteins associated with NSTI. Yet, data was the case and control group are compared taking
analyzed in a univariate fashion, missing the ratio of the concentrations of A and B a clear
potential of highlighting association patterns difference between case and controls is evident,
among proteins that could have been uniquely as shown in Fig. 11.2b (see figure caption for
associated with one or more type of infections. more details) and it is clear that the information
Systems biology approaches rely on the con- discriminating between cases and controls relies
cept of mutual interaction among the constituents on both metabolites and their mutual relationship
of a biological system and thus poses a great This phenomenon is a consequence of
emphasis on measuring, describing, analyzing, metabolites A and B being correlated as shown
and interpreting the relationships among molecu- in Fig. 11.2c: from biological point of view what
lar features (genes, proteins, metabolites...) rather is observed here is that given a certain concentra-
than considering them one at a time. In this light, tion of metabolite A, the concentration of metab-
top-down systems biology approaches can be olite B changes with class (Saccenti et al. 2014a).
considered multivariate in nature, that is, the anal- This indicates that when the relation or interaction
ysis is performed taking into consideration the between pairs of metabolites or other biomolecu-
relationships among the variables and not only lar features is thought to be important and to carry
their mean levels (i.e. concentration and information about the problem studied, the ratios
abundances) (Saccenti et al. 2014a) as in the of metabolite concentration (or abundances)
standard analysis for single molecule biomarkers. could be considered instead of single metabolite
Relationships among genes, proteins, and concentrations. Examples of this exist in the clin-
metabolites are quantified using association ical setting where the deviation from the normal
measures which describe the similarity of the range of the ratios of metabolites or protein is
concentration and abundance profiles of these used as diagnostic biomarkers (Petersen et al.
molecular features measured on a set of biological 2012): ornithine-δ-aminotransferase deficiency
replicate samples. Correlations (either the in young children is diagnosed using the proline
Pearson’s or Spearman’s indexes) are commonly to citrulline ratio (De Sain-Van Der Velden et al.
used and biological and biochemical information 2012), while the ratio between blood phenylala-
can be derived from both the strength and the sign nine and tyrosine concentrations is used to
Fig. 11.2 Idealized experiment aiming to find biomarkers 0.98, respectively, for metabolite A and B. (b) Compari-
discrimination between the case and control group based son of the ratios of the concentrations of metabolites A and
on the measurement and analysis of two metabolites A and B: a discrimination between the case and control groups is
B measure on n1 ¼ 75 cases and n2 ¼ 75 controls. (a) evident (1-way ANOVA P-value ¼ 2.7 1018). (c)
Comparison of the concentrations of metabolites A and B Scatter plot of the concentration profiles of metabolites A
tested with 1-way ANOVA: there is no statically signifi- and B
cant difference at the α ¼ 0.05 level (P-value 0.32 and
identify heterozygous carriers of phenylketonuria ½A k 2

¼
risk alleles (Hsia 1958). ½B k 1
Metabolite and protein ratios and variation
thereof have often direct biological relevance and can be used as a proxy to characterize the
and can provide mechanistic insights. In fact, if system. Since reaction constants are tightly
two metabolites are connected within (or by) a constrained under normal physiological
biochemical pathway, metabolite ratios approxi- conditions, increased (or decreased) metabolite
mate (under idealized steady state assumptions) ratios can be an indication of alteration in the
the reaction rates (Petersen et al. 2012). For activity of an enzyme that catalyzes the reaction
instance, considering the elementary biochemical or a change in flux distribution. For instance,
reaction where metabolite A is converted to B and variation of the ratios between sphingolipids that
then degraded differ by two carbon moieties has been linked to a
modified beta-oxidation, while variation of
the ratios between different classes of
k1 k2
A !B! phospholipids has been proposed to describe
modified activity of enzymes in the phospholipid
described by the differential equations pathways (Altmaier et al. 2008).
8
>
> d½A
< ¼ k1 ½A 11.7 Networks and Network
dt
> Analysis
: d ½B ¼ þk1 ½A k2 ½B
>
dt
When many metabolites or other biochemical and
where [A] and [B] are the concentrations of A and biomolecular features are measured, their mutual
B at time t. At steady state the ratio of the con- relationships can be conveniently represented
centration of A and B gives an approximation of using networks. A (biological) network is a
the ratios of the rate constants for the two graphical representation of objects (called
reactions nodes) and their relationships described by links
or edges connecting different nodes. The exact If the value of the correlation (or any other
meaning of the nodes and edges, i.e. of the kind of association measure) is retained, the network is
relationships they represent, depends on the con- said to be weighted as every edge connecting two
text. In gene regulatory networks the nodes repre- nodes describes the weight of the association. In
sent genes and the edge represents the existence other cases, only the presence/absence of an asso-
of regulatory mechanism where the protein pro- ciation is retained, and in this case the network is
duced by a given gene regulates the expression of said to be unweighted
the target gene (Emmert-Streib et al. 2014). In
networks describing protein–protein interaction
1 if jr ij j > θ and Pval < α
the edge represents the existence of a physical r ij ¼ ð11:2Þ
interaction between two proteins (Jordán et al. 0 otherwise
2012).
The nodes in a network can be characterized
In the context of biomarker discovery, the
using functions that can be derived from the
most relevant networks are probably the
patterns of its association: a common measure is
so-called metabolite–metabolite association
the node degree or connectivity. For unweighted
networks, or correlation networks, where the
networks this is simply the number of its connec-
nodes are metabolites and the edges represent
tion. For weighted networks a weighted connec-
the existence of associations between two
tivity is defined as the sum of the absolute values
metabolites, quantified through the correlation of
of the edges:
their concentration profiles measured on
replicated samples.
X
The simplest approach to build metabolite– χi ¼ j r ij j ð11:3Þ
metabolite association networks is first taking i>j
the correlation rij among pairs of metabolites
i and j and then imposing a threshold on either In the framework of biomarker discovery, the
the magnitude of the correlation or on the statisti- patterns of correlations between metabolites and
cal significant (or both), setting to 0 the correla- their connectivity can be exploited by comparing
tion that do not satisfy such conditions. For them across conditions, for instance, cases and
instance controls, to identify associations that are
disrupted or altered by pathophysiological
conditions. This approach is called differential
r ij if jr ij j > θ and Pval < α network analysis and has been applied in NSTI
r ij ¼ ð11:1Þ
0 otherwise research (Afzal et al. 2020) as well in other fields
like cardiovascular disease (Vignoli et al. 2020;
where θ and α (statistical significance) are Saccenti et al. 2014b) and aging (Vignoli et al.
parameters to be determined; a commonly 2018).
accepted value for θ is 0.6 which is usually Afzal et al. (2020) combined classical univari-
taken as a threshold to discriminate between low ate and multivariate approaches together with
and medium-high correlation (Camacho et al. differential analysis of metabolite–metabolite cor-
2005). Other methods, like the Probabilistic Con- relation networks which were built from metabo-
text Likelihood of Relatedness for Correlations lite concentrations of blood plasma metabolites
(PCLRC) (Saccenti et al. 2014b), dispose of the measured using GC-MS from NSTI patients
necessity of a pre-defined threshold on the cor- (n ¼ 34) and healthy (noninfected) controls
relation but are context dependent, and the sig- (n ¼ 24) enrolled in the INFECT project (Madsen
nificance of the correlations is established with et al. 2019). In this study Spearman’s rank corre-
respect to the correlation background observed lation was used as an index of metabolite associ-
in the data. ation and implemented a conservative selection
procedure involving resampling to obtain robust
Fig. 11.3 (a) Absolute differences in metabolite connec- metabolite connectivity observed in the metabolite–metab-
tivity in networks from NSTI and healthy control subject olite correlation networks built from NSTI and nonin-
(see Eqs. (11.3) and (11.4)) against its statistical signifi- fected surgical controls. The colors are proportional to –
cance (Benjamini–Hochberg corrected -log10(P-value)). log10(P-value) and the marker size is proportional to the
The horizontal lines represent the significance thresholds difference in connectivity
at α ¼ 0.05 and 0.01 level. (b) Scatter plot of the
estimates of the correlation because of the small NSTI network, becoming hub node. In network
sample size available. Metabolite association biology a hub is a node that is densely connected,
networks were constructed for both NSTI and i.e. it is associated with many other nodes.
healthy and for each of the 97 measured Because of this, hub nodes are assumed to play
metabolites they calculated the differential con- crucial biological roles.
nectivity Δχ i defined as The concept of hub was first introduced within
the analysis of yeast protein–protein interaction
networks and it was shown that highly connected
Δχ i ¼ χ NSTI
i χ Control
i ð11:4Þ nodes are more likely to be essential for survival
(Jeong et al. 2001; Carter et al. 2004). Analysis of
where χ NSTI
i and χ Control
i are the connectivity for
hub nodes has been extended to metabolite–
the metabolite i in the association network spe-
metabolite association networks: comparing low
cific for NSTI and healthy control, respectively.
and high cardiovascular risk patients. Saccenti
The significance of the observed difference in
et al. (2014b) identified conserved hub metabo-
connectivity was assessed using a permutation
lite, and differentially conserved hubs and linked
test. Figure 11.3a shows the results of differential
them to alteration of mitochondrial activity.
network analysis and Fig. 11.3b shows the metab-
Given this, Afzal et al. speculated that
olite connectivity in the NSTI specific network
metabolites showing differential connectivity
against connectivity in the uninfected surgery
could play a role in disease pathogenesis either
controls. The authors noted that several
by affecting the host response or the bacterial
metabolites (urea, ribose, ribitol, pseudo uridine
infection. A set of significantly altered
penta, malic acid) that were not connected in the
metabolites was selected and tested in vitro to
control network were highly connected in the
investigate potential influence on NSTI group A accomplished by taking linear combinations of

streptococcal strain growth and biofilm the original variables that explain as much as
formation. data variability as possible which allows to
The selected metabolites (ornithine, ribose, visualize the data in a lower dimensional space.
urea, and glucuronic acid) were supplemented to Figure 11.4 shows the results of a PCA analysis
chemically defined media and revealed on the data from Afzal and colleagues (Afzal et al.
metabolite-specific effects on both bacterial 2020). A clear separation between the metabolite
growth and biofilm formation. profiles of NSTI patient and healthy controls is
evident, indicating the existence of a
metabolomics (in this case) signature as shown
11.8 Multivariate Biomarker in the PCA score plot in Fig. 11.4. Figure 11.5
and Omics Signatures shows the relative importance (i.e. the PCA
loadings) corresponding to the PCA score plot.
A generalized class of biomarkers are the The loadings are measure of the contribution of
so-called multivariate biomarkers which are a each variable to explain the variability observed
generalization of the bivariate biomarker in the data, in this case the separation between
described in Sect. 11.6. This kind of biomarkers NSTI and controls. As it can be seen all variables
are sometimes denoted with the term signatures, (only the first 40 are shown for simplicity) con-
especially in the context on omics research where tribute to the model, showing the multivariate
gene, protein and metabolite expression, nature of this metabolomics signature
abundances and/or concentration levels are differentiating NSTI subjects from the controls.
measured in a comprehensive fashion on It is interesting to note that among the most
biological samples using high-throughput analyt- important metabolites there are urea, ribose,
ical techniques like RNA sequencing, mass spec- ribitol, pseudo uridine penta, malic acid, the
trometry, or nuclear magnetic resonance same metabolites that showed significant differ-
spectrometry. These experimental techniques are ential connectivity in the network analysis (see
multivariate in nature since hundreds to Fig. 11.3). This is because PCA, like all multivar-
thousands of molecular features are measured iate approaches, takes into account the correlation
simultaneously and such molecular signatures among variables which are also reflected in the
are exploited through multivariate and machine network structure underlying the data, making
learning approaches. network analysis and multivariate statistics two
Sung et al. (2012) defined operatively molecu- complementary approaches.
lar signature as a set of biomolecular features Omics signatures consisting of many biomo-
together with a pre-defined computational proce- lecular features have been defined and routinely
dure that applies those features to predict a phe- applied in the clinical setting. For instance, the
notype of clinical interest on a previously unseen MammaPrint test (Van ’T Veer et al. 2002) used
patient sample, stressing the computational/statis- to determine the most appropriate chemotherapy
tical nature of such signatures. treatment in lymph node negative breast cancer
Signatures are defined using statistical patients with either positive or negative ER,
approaches in combination with machine learning consists of 70 gene expression signatures which
techniques. Principal component analysis (PCA) were found to correlate with clinical phenotype
(Hotelling 1931; Pearson 1901; Jolliffe 2002) is a (metastatic vs. non-metastatic) and validated on
common starting point when looking for bio- independent patient cohorts (Van de Vijver et al.
marker signatures and it is used to explore data 2002). Given the particular nature of NSTI, which
and to highlight if such a signature may exist. is a fast spreading infection requiring extremely
PCA is data reduction technique that allows for fast diagnosis and treatment, this type of high-
visualization and exploration of high-dimensional dimensional genomics signatures may not of
data by reducing its dimensionality. This is immediate applicability in the clinical setting
Fig. 11.4 Score plot of

principal component
analysis of concentration
profiles of 97 metabolites
measured on using GC-MS
from NSTI patients
(n ¼ 34) and healthy
(surgery, noninfected)
controls (n ¼ 24) enrolled
in the INFECT project
(Madsen et al. 2019). Data
is from (Afzal et al. 2020).
A clear separation between
NSTI patient and controls is
evident, indicating the
existence of a multivariate
metabolomics signature
specific to NSTI. See
Fig. YY for the
corresponding loading plot
although some applications are emerging (See test in vitro and or in vivo in animal models,
also Chap. 13, Sect. 11.3.2). closing the iterative cycle typical of systems
However, omics signature can be explored to biology approaches. In this respect the main
single out key players and provide mechanistic limitation of PCA based signatures is that (1) all
information about disease onset and progression variables contribute to the model and (2) PCA
and can generate hypotheses that can be further base signatures are exploratory in nature and do
Fig. 11.5 Loading plot of principal component analysis et al. 2020). Only the first 40, most contributing
of the concentration profiles of 97 metabolites measured metabolites are shown. Urea, ribose, ribitol, pseudo uri-
on using GC-MS from NSTI patients (n ¼ 34) and healthy dine penta, and malic acid are also found to be relevant
(surgery, noninfected) controls (n ¼ 24) enrolled in the from network analysis, See Fig. 11.4 for the corresponding
INFECT project (Madsen et al. 2019). Data is from (Afzal score plot
not allow, per se, the prediction of the clinical CI), while on the validation set one get average
phenotype on previously unseen patient accuracy equal to 0.980 (0.975–0.995, 95% CI),
samples. average sensitivity 0.954 (0.938–0.969, 95% CI),
Signatures that are easier to interpret can be and an average specificity of 1. The classification
obtained using advanced explorative techniques results on the validation set are similar to those on
like sparse approaches (Camacho et al. 2017; the training set and this indicates that, in princi-
Saccenti et al. 2018; Camacho et al. 2020). The ple, these results may be generalizable to samples
existence of a predictive power of such signatures acquired on external validation cohorts.
can be investigated using chemometrics or Many classification tools also produce
machine learning approaches like partial least measures of importance of the predictors used in
squares discriminant analysis (PLS-DA) (Wold the model and this allows to single out which
and Eriksson 2001) or some of its extensions variables are most contributing to the discrimina-
(Lê Cao et al. 2008; Bylesjö et al. 2006; Camacho tion among the groups to gain understanding of
and Saccenti 2018), Random Forests (Breiman the underlying biological processes or identify
2001), Support vector machines (Cortes and potential biomarkers. Figure 11.6 shows the vari-
Vapnik 1995), or other machine learning tools. able importance plot of the Random Forest dis-
When properly implemented, these methods criminant model previously described to
can allow unbiased prediction of the clinical phe- discriminate between NSTI and healthy controls.
notype on previously unseen patient samples: this It is interesting to note that the group of variables
is accomplished by implementing cross- selected as important is somehow different from
validation, which is a computational strategy the one from the PCA analysis (see Fig. 11.5):
that implies removing a set of samples from the only ribose is present in the top five most impor-
data set, building a predictive model, and then tant variables, while urea, which is important in
testing in an unbiased way the ability of the the PCA model, is not important in the Random
model to predict the left out samples, mimicking, Forest model. This should not be surprising, since
in this way, validation on an external cohort of PCA and Random Forest have different aims and
samples. This procedure can be particularly rele- exploit different characteristics of the data: PCA
vant in NSTI where validation cohorts are diffi- is an unsupervised method, while Random Forest
cult, to obtain. Random Forest was used to define is a supervised method that uses information
a multivariate biomarker consisting of 11 blood about sample class (i.e. whether they belong to
analytes attaining specificity and sensitivity NSTI patient or to controls) to build a model able
around 90% for the early detection of ovarian to predict to which class an unknown sample may
cancer (Bertenshaw et al. 2008), while Support belong.
vector machine has been used to derive a panel of
5 markers able to attaining a prediction sensitivity
of 95.3% and a specificity of 99.4% (Mor et al. 11.9 Future Perspective
2005).
Predictive omics signatures have great poten- Technology advances and critical analysis of
tial in NSTI. As an example, we show here a molecular pathways have opened new horizons
Random Forest classification model fit on the for management of diseases, exploring therapeu-
data from (Afzal et al. 2020) with default tic solutions to each individual patient beyond the
parameters and implemented by splitting the one-size fits all practice. As the amount of infor-
data into a validation set (20% of the data) and a mation available grows, there comes a stage when
training set (80%) and repeating the cross- there is too much information to manually inte-
validation procedure 100 times to take into grate reliably. This leads to the need for formal
account sampling variability results. On the train- mathematical or computational models of disease
ing set the accuracy for the prediction of NSTI that incorporate all this information. For this rea-
and control samples is 0.981 (0.978, 0.982 95% son, clinical, microbiological, and experimental
Fig. 11.6 Variable importance plot of a Random Forest et al. 2019). Data is from Afzal et al. (2020). Only the first
discriminant model fit to the concentration profiles of 40, most contributing metabolites are shown. The model is
97 metabolites measured on using GC-MS from NSTI used to investigate the predictive capability of the
patients (n ¼ 34) and healthy (surgery, noninfected) metabolomics signature discriminating NSTI patient
controls (n ¼ 24) enrolled in the INFECT project (Madsen from controls
data can be integrated using various types of and the wealth of omics data that can be collected
mathematical algorithms to provide decision sup- from them.
port tools for clinicians. This can pave the way for Even if the vast heterogeneity and/or quality of
application of personalized medicine in infectious the data will not allow detailed mechanistic
diseases, such as NSTI, with the aims to achieve modelling of specific pathways, these top-down,
the right diagnosis and right treatment for the unbiased approaches will at least enable to iden-
right patient at the right time at the right cost. tify basic structures in the topology of the various
Multivariate statistics, machine learning, and networks underlying the disease. This in itself is a
reverse engineering approaches will allow substantial contribution to the state of the art in
pinpointing and mapping key nodes that can biomarker identification. Finally, it will be impor-
reveal possible biomarker sets from heteroge- tant to validate identified signatures/biomarkers
neous (meta-)data both from the tested pathogens for patient classification through the use of large
and the hosts (including the various “omics,” patient cohorts. Indeed, validation remains one of
pathotyping, patient stratification data). These the major bottlenecks for biomarker discovery.
top-down analyses will be iteratively combined To become a clinically approved test, a potential
with bottom-up modelling of specific biomarker should be confirmed and validated
sub-networks/biomarkers. By thoroughly apply- using large cohorts and should be reproducible,
ing an iterative “dry-wet” cycle centered around specific, and sensitive (Drucker and
the cross-linking of bottom-up, hypothesis-driven Krapfenbauer 2013). Although not uncommon,
modelling of selected pathways and/or signatures, NSTI is still a rare condition whose
with top-down, unbiased systems biology characteristics (difficult diagnosis, fast spread,
approaches biomarkers that may contribute to high mortality, among others) make the creation
disease onset and outcome can be identified. In of validation cohorts difficult. However, the
particular, biomarker discovery in NSTI will INFECT cohort and the methodologies and
benefit from the integration of heterogenous strategies for data collection and analysis devel-
data, such as patient data and metadata, data oped within the INFECT project (Madsen et al.
from the tested pathogens and the murine models 2018, 2019; Afzal et al. 2020) are a first and
promising step towards the definition of clinically Camacho J, Rodríguez-GÓMEZ RA, Saccenti E (2017)
relevant biomarker in NSTI. Group-wise principal component analysis for explor-
atory data analysis. J Comput Graph Stat 26:501–512
Camacho J, Smilde AK, Saccenti E, Westerhuis JA (2020)
Acknowledgement Financial support: The work was All sparse PCA models are wrong, but some are useful.
supported by the European Union Seventh Framework Part I: computation of scores, residuals and explained
Programme: (FP7/2007-2013) under the grant agreement variance. Chemom Intel Lab Syst 196:103907
305340 (INFECT project); the Swedish Governmental Carter S, Brechbuhler C, Griffin M, Bond A (2004) Gene
Agency for Innovation Systems (VINNOVA) under the co-expression network topology provides a framework
frame of NordForsk (Project no. 90456, PerAID), and the for molecular characterization of cellular state. Bioin-
Swedish Research Council and The Netherlands Organi- formatics 20:2242–2250
zation for Health Research and Development (ZonMv) Cortes C, Vapnik V (1995) Support-vector networks.
under the frame of ERA PerMed (Project 2018-151, Mach Learn 20:273–297
PerMIT). Cross D, Drury RE, Hill JL, Pollard AJ (2019) Epigenetics
in sepsis: understanding its role in endothelial dysfunc-
tion, immunosuppression and potential therapeutics.
Front Immunol 10:1363
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Systems and Precision Medicine
in Necrotizing Soft Tissue Infections 12
Vitor A. P. Martins dos Santos, Christopher Hardt,
Steinar Skrede, and Edoardo Saccenti
Contents
12.1 Introduction: The Case for a Systems and Personalized Approach
to NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
12.2 State of the Art: Systems, Precision and Personalized Medicine in NSTI 189
12.2.1 Systems Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
12.2.2 Challenges in Systems Medicine and How to Tackle Them . . . . . . . . . . . . . . . . . 190
12.2.3 Precision and Personalized Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
12.3 Big Data, Machine Learning and Deep Learning in Systems Medicine 193
12.3.1 Big Data Definitions and Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
12.3.2 AI in Systems Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
12.4 Information Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
12.4.1 Personalized and Precision Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
12.4.2 The Case of NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
12.4.3 Integrating Heterogeneous Data with FAIR Principles . . . . . . . . . . . . . . . . . . . . . . 197
12.4.4 Laying the Basis for Computer-Assisted Decision Support . . . . . . . . . . . . . . . . . . 198
V. A. P. Martins dos Santos (*)

Netherlands
Lifeglimmer GmbH, Berlin, Germany
e-mail: vitor.martinsdossantos@wur.nl
C. Hardt
Lifeglimmer GmbH, Berlin, Germany
S. Skrede
Department of Clinical Science, University of Bergen,
Bergen, Norway
Bergen, Norway
E. Saccenti
Netherlands

188 V. A. P. Martins dos Santos et al.
12.5 Clinical Decision Support Systems for Soft Tissue Infections . . . . . . . . . . . . . .199
12.5.1 The Need to Enhance Medical Decisions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .199
12.5.2 What Are CDSS What Is Their Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .199
12.5.3 CDSS in NSTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .200
12.5.4 Current and Future Developments in Relation to Dedicated CDSS for NSTI . 201
12.6 Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .204
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .204
Abstract Information management · Personalized

medicine · Semantic technologies
Necrotizing soft tissue infections (NSTI) are
multifactorial and characterized by dysfunc-
Highlights
tional, time dependent, highly varying hyper-
• Systems, precision and personalized medicine
to hypo-inflammatory host responses
approaches are methods to facilitate and
contributing to disease severity. Furthermore,
improve contemporary diagnosis, accurate
host-pathogen interactions are diverse and dif-
stratification, and optimized tailored therapy
ficult to identify and characterize, due to the
in NSTI.
many different disease endotypes. There is a
• Big Data analytics and artificial intelligence
need for both refined bedside diagnostics as
are increasingly allowing to uncover disease
well as novel targeted treatment options to
mechanisms, giving a basis for patient
improve outcome in NSTI. In order to achieve
stratification.
clinically relevant results and to guide preclin-
• We have established a prototype of an
ical and clinical research the vast amount of
advanced platform for personalized medicine
fragmented clinical and experimental datasets,
in NSTI according to FAIR principles.
which often include omics data at different
• We have developed the basis for a clinical
levels (transcriptomics, proteomics,
decision support system in NSTI.
metabolomics, etc.), need to be organized,
harmonized, integrated, and analyzed taking
into account the Big Data nature of these
datasets. In this chapter, we address these
12.1 Introduction: The Case
matters from a systems perspective and yet
for a Systems and Personalized
personalized approach. The chapter provides
Approach to NSTI
an overview on the increasingly more frequent
NSTI are complex multifactorial diseases that can
use of Big Data and Artificial Intelligence
be caused by a variety of microbes. They are
(AI) to aggregate and generate knowledge
frequently complicated by septic shock and
from burgeoning clinical and biochemical
multi-organ failure. Despite modern medicine,
information, addresses the challenges to man-
the mortality is high, often exceeding 25%, and
age this information, and summarizes current
amputation is required in up to 15% of the cases
efforts to develop robust computer-aided clini-
(Peetermans et al. 2020). Most patients affected
cal decision support systems so to tackle the
are individuals with co-morbidities,
serious challenges in NSTI diagnosis, stratifi-
e.g. cardiovascular diseases and diabetes mellitus,
cation, and optimized tailored therapy.
but some patients are young immunocompetent
individuals. The fulminant, often rapid course of
Keywords
these invasive infections (Kittang et al. 2010),
Artificial intelligence · Big data · Clinical demands early diagnosis and immediate interven-
decision support systems · Deep learning · tion. However, misdiagnosis and subsequent
doctor’s delay is frequent (Goh et al. 2014), due
12 Systems and Precision Medicine in Necrotizing Soft Tissue Infections 189
to discrete and unspecific initial symptoms, scarce 12.2 State of the Art: Systems,
clinical findings and lack of biomarkers in early Precision and Personalized
stages of many NSTI. Medicine in NSTI
The cornerstones in the treatment of NSTI are
early aggressive surgical debridement and Current medical science is largely conducted
appropriate antimicrobial therapy. Frequently under a reductionist paradigm, which involves
there is a need for advanced supportive the notion that complex phenomena like
measures. There is clearly an urgent need for mechanisms underlying onset of infectious
refined bedside diagnostics and novel, targeted diseases, interactions between host and pathogens
treatment options to improve outcome in these and disease progression may be better understood
patients. Because of the multifactorial nature of by breaking them down into smaller, simpler
NSTI and sepsis, it is becoming increasingly components (Ahn et al. 2006).
clear that individualized approaches are required However, given the complexity of the
to improve outcome of these patients. These conditions under study and the myriad of underly-
infections are characterized by dysfunctional, ing factors, this often leads to biased focus and
highly varying hyper- to hypo-inflammatory, oversimplification (i.e. by focusing only on a hand-
host responses contributing to disease severity ful of major factors with the biggest effect, while
(Goldstein et al. 2007; Sarani et al. 2009; the sum of minor factors may be considerable) and
Peetermans et al. 2020). Thus, individualized generalization (i.e. assuming that a common cause-
therapeutic strategies targeting both pathogen effect relationship applies equally in all cases).
as well as the host response have great potential, Such simplifications and generalizations often
but require patient stratification based on disease limit our ability to understand how multiple
signatures. variables interact with one another to create emer-
Data are becoming available showing the gent effects and hamper not only our understand-
power of omics in identification of disease ing of the disease, but more importantly, our
signatures (Davenport et al. 2016), as well as in capability of delivering better treatments. There
providing the basis for patient stratification is clearly a need to address health and disease
through large-scale analysis of clinical data from a systems perspective, that is, one that
(Hardt et al., in preparation), but fail to acknowl- accounts for all factors and interactions. For
edge the complexity of the host-pathogen instance, Chap. 12 illustrated the application of
interactions and the patient’s individual such a systems (biology) approach for the identi-
responses to infection and its progression. In fication of potential biomarker sets in NSTI. This
this chapter, we address these problems from a entailed the uncovering of metabolite–metabolite
systems medicine perspective, provide an over- association networks and analysis and deploy-
view on the increasingly more frequent use of ment of machine learning methods (Afzal et al.
Big Data and Artificial Intelligence (AI) to 2020). These findings would not have been pos-
aggregate, and generate knowledge from sible with a traditional, reductionist approach of
burgeoning clinical and biochemical information trying to identify potential biomarkers by stan-
directing towards personalized approaches. dard approaches that are based, e.g. on simple
Moreover, we address the challenges to manage univariate analysis of datasets, as these fail to
this information and summarize current efforts account for the interrelatedness existing among
to translate this information into actionable genes, proteins, and metabolites that behave in an
knowledge through the development of robust orchestrated way for what concerns regulation,
computer-aided clinical decision support transcription, and translation (Rosato et al. 2018).
systems (CDSS) to tackle the serious challenges
in NSTI diagnosis, stratification, and optimized
tailored therapy.
Fig. 12.1 Schematic

illustration of the core
differences between
reductionism and systems
science, when analyzing the
properties of a system
(Tillmann et al. 2015).
Licensed under Creative
Commons
12.2.1 Systems Medicine microbial agents, treatment strategies, and patho-

genic mechanisms (host and bacterial disease
Building upon the uncovering of biological traits and their underlying interaction networks)
mechanisms as above and going beyond that, by using the largest thus far patient cohort in the
“Systems medicine” applies systems biology world. Also, INFECT has proven the value of
approaches to medical research and medical prac- systems medicine approaches in acute infectious
tice. Its objective is to integrate a variety of diseases to achieve improved diagnostics and
biological/medical data at all relevant levels of therapeutics to improve patient disease outcome.
cellular organization using the power of compu- In particular, the novel understanding of the dis-
tational and mathematical modelling, to enable ease mechanisms has resulted in changed clinical
understanding of the pathophysiological practice related to antibiotic usage as well as use of
mechanisms, prognosis, diagnosis, and treatment immunomodulatory treatments (Bergsten et al. 2020;
of disease (Auffray et al. 2010). Systems Medi- Madsen et al. 2018) (see also Chaps. 7, 8, and 9).
cine involves iterative and reciprocal feedback However, the insights gained also underscore
between the clinical practice and the research the need for patient stratification and implemen-
carried on with computational, statistical, and tation of tailored therapy. Despite considerable
mathematical multiscale analysis and modelling progress, there are substantial hurdles to be over-
at both the epidemiological and individual patient come related to the difficulties in accounting for
level (www.easym.eu). patient’s individual conditions, or intrinsic to
This new paradigm of Systems Science and Systems Medicine itself.
Medicine complements the traditional reduction-
ist approach (as exemplified in Fig. 12.1) and,
ideally, leads to the identification of mechanisms
12.2.2 Challenges in Systems Medicine
related to disease pathophysiology, selection of
and How to Tackle Them
novel drug targets and biomarkers, patient strati-
fication, risk assessment, and optimized therapy.
The recent transition to data-rich (i.e. molecular
As described in Chap. 1, the systems medicine
characterization) applications, which followed
INFECT project (https://permedinfect.com/
the omics revolution with the advent of high
projects/infect/) has generated comprehensive
throughput genomics, transcriptomics,
knowledge of diagnostic features, causative
metabolomics, and all other omics disciplines,
have enabled Systems Medicine approaches Multiscale modelling is another way to

(Noble 2008). However, this data deluge is at approach the problem of phenotypic variability.
the same time an opportunity and a challenge. This includes bridging molecular and physiologi-
An opportunity because it allows patient cal processes, even taking place at very different
phenotyping at different levels in an unprece- time and spatial scales (Meier-Schellersheim et al.
dented manner, and a challenge because of the 2009). This could facilitate a better understanding
heterogeneity observed in both data and patients. of the biology of health and disease, allowing us
There is an inherent data heterogeneity which to tailor models to individual patients
arises when different data types and different data (e.g. genome-scale metabolic model constrained
resources are integrated. This poses challenges by patient-specific data).
for both data handling and analysis which can A key challenge is to select measurements and
be addressed by vertical integration, i.e. when data collected at the small scales and combine
different data informs on the same biological them into informative metrics to be transferred
level, or by horizontal integration, i.e. when mul- to a higher level. This is the realm of meta-
tiple datasets inform on the same biological level modelling, which is the statistical approximations
(Berthold et al. 2010). Both cases can be handled, or predictions of the relationships between the
using FAIR data approaches (Findable, Accessi- various model components. Meta-modelling has
ble, Interoperable, Reusable) and semantics been proposed as an efficient solution to link
technologies (Berthold et al. 2010) but, as a mat- models obtained at different scales, to link
ter of fact, there are other inherent sources of modelling results and measured data, and to iden-
variability, which poses even greater challenges tify the most important metrics determining sys-
to Systems medicine approaches. tem functionality at the various levels (Tøndel
Humans exhibit a great phenotypic diversity, et al. 2012).
which originates from the complex interplay of Among statistical approaches derived from
genetic, epigenetic, and environmental factors, multivariate analysis, multi-way analysis has
that affects both disease manifestations and been proposed to retain the block-wise structure
responses to therapy (Assfalg et al. 2008; Bernini of temporal data originating from nonlinear
et al. 2009). Nonetheless, intra-individual dynamic models used to describe the systems at
variability is less than half the inter-individual different levels (Tøndel et al. 2012). However,
variability, making personalized medicine possi- other solutions can be hypothesized that involve
ble (Hughes et al. 2015; Gruden et al. 2012). other component methods: some examples are
Under this light, statistical and computational principal component regression (Jolliffe 1982)
characterization of individual and inter-individual or partial least square regression (Wold and
variability is pivotal to the deployment of systems Eriksson 2001) that can be used to model the
and personalized medicine approaches, which complex relationships between input parameters
will allow both higher sensitivity and specificity and model outputs of nonlinear dynamic models
of personalized assays and substantial new likely with embedded procedure for variable
insights into health and disease. selection to reduce dimensionality and complex-
Characterization of phenotypic diversity in ity of the parameter space (Tøndel et al. 2012).
both health and disease and underlying biomolec- Furthermore, parameter characteristics, such as
ular mechanisms should be carried out at different distributional properties, covariance, and correla-
levels. Examples could be developing new statis- tion patterns, could be inferred by stochastics
tical and computational methods, to exploit the methods designed to deconvolute correlative or
wealth of information obtainable at all omics noisy patterns: since signals can be separated
levels, or by developing and deploying multiscale from background biological variability in data-
approaches to model how processes occurring at driven inference framework, this could ultimately
widely different scales integrate, results in the lead to a better definition of phenotype
phenotypic variability observed in humans. characteristics.
12.2.3 Precision and Personalized hyper- to hypo-responses with different mediators

Medicine involved (Hotchkiss and Karl 2003; Anaya et al.
2005; Huang et al. 2011; Thänert et al. 2019).
Personalized Medicine builds upon Systems The identification of disease signatures of
Medicine and is an emerging data-driven health value for patient classification studies in well-
care approach that integrates phenotypic, geno- defined patient cohorts has been undertaken,
typic, epigenetic, lifestyle, and environmental exploring host responses, pathogen profiles, and
factors unique to an individual. The goal of their association with disease outcomes (Chella
personalized medicine is to facilitate diagnosis, Krishnan et al. 2016; Thänert et al. 2019). How-
predict effective therapy, and avoid adverse ever, as for all biomarkers (see Chap. 12), these
reactions specific for each patient (Nimmesgern findings must be validated by use of other patient
et al. 2017; Union 2015). cohorts, as stated in a landmark position paper by
Precision medicine is the concept of tailoring the European Society of Clinical Microbiology
disease treatment and prevention to account for and Infectious Diseases (Rello et al. 2018).
differences in genetic, environmental, or even Accordingly, to advance on precision medicine
lifestyle factors specific to groups of people of NSTI and sepsis, two current multinational
(Bresnick 2018). Precision medicine takes projects have been designed and implemented
genetic and biochemical information unique to a (PerMIT and PerAID, www.permedinfect.com).
group of patients and uses that information to These projects build upon the unique resources
develop more specific and streamlined created in the INFECT project, including clinical
medications or treatments. The goal is to ensure expertise, patient registry, biobank, multi-omics
that each medication or treatment is best suited to data, identified candidate disease signatures, data
treat the individual, resulting in decreased side stewardship resources, and basic science experi-
effects and increased effectiveness (Hulsen et al. mental model systems.
2019). These resources are currently being used to
Although the terms precision medicine and test data-driven working hypotheses through
personalized medicine are sometimes used inter- advanced preclinical and clinical studies com-
changeably, generally speaking, “precision medi- bined with Big Data integration and information
cine” seeks to create treatments that are applicable technology solutions, to develop patient stratifi-
to groups of individuals who meet certain cation schemes allowing for individualized ther-
characteristics, whereas “personalized medicine” apy in NSTI. The identified pathogenic
implies individualized treatments available for mechanisms and biomarkers linked to particular
every unique patient (Erikainen and Chan 2019). NSTI disease signatures and clinical outcome are
Precision and personalized medicine are in being further validated in sepsis patients, a condi-
their infancy in infectious diseases, in particular tion that, just like NSTI, is defined by the host’s
acute diseases such as sepsis and NSTI (Lazăr response to an infection. A major strength of
et al. 2019). As the patient populations in severe these on-going studies is that they resort on
infections are highly heterogeneous due to host- well-defined patient cohorts, allowing for robust
microbe interactions governed by different host conclusions linking host response signatures and
factors and pathogens driving unique pathogenic pathogenic mechanisms to clinical outcomes
mechanisms, personalized and precision medi- encouraging translation of these findings into
cine approaches may prove crucial. It is future patient handling measures.
recognized that a dysregulated host response to Interestingly as well, these two synergistic
infection is directly linked to severity and out- projects envision that some stratification and clin-
come of severe infections, such as sepsis and ical trial designs will be shared for NSTI and
NSTI. Furthermore, the response profiles/disease sepsis, whereas others will target more strictly
signatures can be highly variable, ranging from defined patient subgroups. For example, sepsis
patients with an immunosuppressive response
profile might be targeted for an immune boosting systems medicine applications, the volume of
therapy, whereas NSTI and sepsis patients with a the data is likely to be much smaller.
hyperinflammatory profile will benefit from a 2. Variety refers to the heterogenous nature and
suppressive agent. sources of data. In the biomedical field the
Undoubtedly, such insights will allow for different types of data that can be collected,
effective tailored treatments and development of mined, and analyzed are virtually endless.
new tools and concepts for future clinical trials. Data comes not only in form of health records,
Crucially, however, for this potential to be ful- clinical data, to which data from omics
filled, some further challenges have to be tackled, measurements can be added but also as medi-
in particular with regard to the handling of infor- cal imaging, including X-rays, CT, or MRI, or
mation and translation to actionable knowledge. images recorded on tissue samples, e.g. tissue
Indeed, a key challenge in the personalized/ biopsies. These different data sources need to
precision medicine field is the vast amount of be handled and integrated to be properly
fragmented clinical and experimental datasets analyzed, taking into account there are
that need to be organized, harmonized, and structured data (e.g. data stored in excel for-
integrated in order to achieve clinically relevant mat) and unstructured data (like doctor’s
results and to guide preclinical and clinical notes). This is usually one of the most chal-
research. This is also essential to enable fruitful lenging tasks.
Big Data analytics to yield meaningful insights, 3. Velocity refers to rate of data sampling and
as well as for development of Clinical Decision acquisition. Standard clinical measurements
Support Systems (CDSS) to assist effectively and and omics data are usually static or acquired
efficiently in bedside decision. at a (very) low sampling rates. Differently,
health monitoring systems, including, but not
limited to, smartphones, smart watches, smart
12.3 Big Data, Machine Learning bracelets/wristbands, connected sensors, and
and Deep Learning in Systems wearable devices, enable continuous monitor-
Medicine ing of patient data by sensing and transmitting
measurements such as heart rate, blood pres-
12.3.1 Big Data Definitions sure, body temperature, respiratory rate, chest
and Characteristics sounds, and electrocardiogram (Vitabile et al.
2019) at high frequency, creating a flow of
Big Data and the development of techniques to data that often needs to be processed “on-the-
handle it have the potential of enhancing our fly.”
ability to probe and understand which parts of 4. Variability concerns the quality of the data
the biological machinery underlying the normal acquired and their inconsistency. For biomed-
functioning of the organism that may be or ical and healthcare applications, data quality is
become dysfunctional, given the pathophysiology a very critical aspect, because erroneous infor-
of a condition (Hulsen et al. 2019). mation can lead to erroneous diagnosis and
Big Data are characterized by the so-called treatment. The problem of obtaining quality
four V’s (Schroeck et al. 2012), which stands data is complex and cross-disciplinary. Over
for volume, variety, velocity, and variability. the years, several organizations have
contributed to defining the quality of various
1. Volume refers to the size of data, where size products and services and identifying ways of
indicates the physical occupancy of data files. measuring such quality (Brighi 2018).
In Big Data applications exabytes, zettabytes, Handling and management of data inconsis-
and even higher amounts of data need to be tency, such those introduced by missing data,
handled simultaneously. For what concerns
is of paramount importance for the exploita- et al. 2017). Using images, artificial intelligence
tion of Big Data. approaches have been used to describe the impact
of orthognathic treatment on facial attractiveness
Accordingly, Big Data are high-volume, high- and age appearance (Patcas et al. 2019). A semi-
velocity and/or high-variety information assets nal study (Esteva et al. 2017) tested the perfor-
that demand cost-effective, innovative forms of mance of AI to distinguish keratinocyte
information processing that enable enhanced carcinomas versus benign seborrheic keratoses
insight, decision making, and process automa- and malignant melanomas versus benign nevi,
tion. This definition (www.gartner.com/it-glos training it on >15,000 biopsy-proven diagnostic
sary/big-data/), albeit stated in the context of images, against 21 board-certified dermatologists,
informatics, summarizes the challenges and the and found AI to perform on par with all tested
gain that Big Data present and can offer to experts across both comparisons, and other stud-
Systems Medicine. ies confirmed AI ability in identifying melanoma
The ultimate goal of the collection and use of from dermoscopic images with accuracy similar
Big Data in Systems Medicine is the possibility of to that of specialists (Phillips et al. 2019, 2020).
obtaining better description of both health and There are few applications of Big Data and AI
disease profiles and to use them to build integra- to systems medicine specific to NSTI. The man-
tive models that can be used to predict disease agement of NSTI is complex given that clinical
onset and progression and to tailor better treat- presentation is highly variable and range from
ment for each patient. Under this light, Big Data early sepsis with obvious skin involvement to
approaches are fundamental for the development minimal cutaneous manifestations with a dispro-
of Precision medicine, which aim to integrate portionate systemic response (Bosshardt et al.
phenotypic, genomic, epigenetic, and environ- 1996). Classic signs like fever, diffuse crepitus,
mental factors unique to an individual to facilitate and shock are late signs: once large blisters and
diagnosis, predict effective therapy, and avoid gangrene develop, the infectious process is
adverse reactions specific for each patient. Thus, already at an advanced stage (Bosshardt et al.
precision medicine needs to operate on different 1996). NSTI treatment must be aggressive and
scales to gain other insights into health and disease, rapid and essential elements of the treatment are
utilizing and integrating data from cells, tissues, resuscitation, antimicrobial therapy, surgical
organs, and ecosystems, e.g. those constituted by debridement, and supportive care (Anaya et al.
microbial communities (Stacy et al. 2016). 2005; Morgan 2010; Hakkarainen et al. 2014;
Stevens and Bryant 2017), and constant monitor-
ing is required to achieve fluid, electrolyte, and
hemodynamic stability (Bosshardt et al. 1996).
12.3.2 AI in Systems Medicine
Hyperbaric oxygen treatment can also be used
as adjunctive therapy when infections involve
Artificial intelligence encompasses the use of
anaerobic bacteria, specifically the clostridial spe-
software and algorithms to emulate the human
cies (Bakker 2012).
cognition in the analysis of complex medical data.
For instance, AI could be applied to the analy-
AI is being successfully employed in several
sis of microbiological findings. Identification of
medical fields. Models have been constructed that
the etiological agents can assist infection control
enable to distinguish high-risk breast lesions
measures and antimicrobial therapy decision
(HRL) diagnosed with image-guided needle
making, and may offer prognostic information
biopsy that require surgical excision from HRLs
(Anaya et al. 2005; Huang et al. 2011; Madsen
that are at low risk for upgrade to cancer at sur-
et al. 2019). The practical applicability of AI
gery (Bahl et al. 2018) or able to detect pneumo-
methods for the analysis of microbiology finding
nia using chest X-rays with an accuracy level
to aid diagnostic testing has been postulated and
exceeding practicing radiologists (Rajpurkar
discussed for the image analysis including Gram
Fig. 12.2 Information management model for systems and personalized medicine. Figure from (Ganzinger and Knaup
2017), Licensed under Creative Commons
stains (Smith et al. 2020) which could be applied among others, thick (3 mm) abnormal fascial
also to NSTI microbiology (see also Fig. 12.2). signal intensity on fat-suppressed T2-weighted
The role of radiologic imaging in diagnosis of images, low signal intensity in the deep fascia
NSTI is debated (Leichtle et al. 2016; Fernando on fat-suppressed T2-weighted images. CT scan-
et al. 2019) and several imaging options are avail- ning has also been proposed (Hietbrink et al.
able, such as plain radiographs, ultrasonography, 2016) but MRI scanning proves to have the
computerized tomography (CT), and magnetic highest sensitivity and specificity (Hietbrink
resonance imaging (MRI). et al. 2016).
Although MRI images were previously not Rakus-Andersson and Frey (Rakus-Andersson
used in Big Data context or analyzed with AI and Frey 2016) trained a modified neural net-
methods, those could be easily applied to support work, to identify groups of NSTI patient with
and aid clinical decision. For instance, supervised good prognosis of recovering without HBO com-
algorithms could be trained on large datasets pared to patients for which HBO could be benefi-
containing MRI images of patients with NSTI cial, in such a way as to support clinical decision
and related conditions, and these algorithms making. They used data from 13 patients admitted
could be applied instantly, contextually with to the Blekinge County City Hospital in
image acquisition and reconstruction, to provide Karlskrona between 2006 and 2010. The input
guided diagnosis that could be integrated with data consisted of clinical data (non-disclosed)
other clinical information. and the results were satisfactory, with an accuracy
Kim et al. (2011) suggested that magnetic res- of 92%; however, these results were obtained on a
onance imaging could be used to differentiate very limited sample size and have not been cross-
between necrotizing and non-necrotizing fasciitis. validated.
They compared MRI findings between the two The use of Big omics data in the clinical
groups and found that patients with necrotizing setting of NSTI to support diagnosis at the bed-
fasciitis had a significantly greater frequency of, side is less immediate since these measurement
platforms are usually not available in hospital 12.4 Information Management

settings: sample work-up, preparation, acquisi-
tion, data cleaning, and processing may require 12.4.1 Personalized and Precision
several days, which is a major hindrance, given Medicine
the severity of NSTI in critically ill patients
(Peetermans et al. 2020). A notable exception A key challenge in the fields of personalized and
is the use of nuclear magnetic resonance, which precision medicine is to organize, harmonize, and
requires minimal sample preparation to measure integrate the vast amount of fragmented clinical
metabolomics profiles of blood and tissues and experimental datasets, in order to achieve
samples collected in the operating theater and clinically relevant results and to guide preclinical
that can provide almost real-time information to and clinical research.
the surgeons and to clinicians. Such a setting has Typically, data of different sources such as
been implemented at St. Mary Hospital in electronic health record (EHR) systems, clinical
London (Nicholson et al. 2012). research databases, or biomedical knowledge
The advent of new technologies is enabling representations like (i.e. ontologies) have to be
real-time sequencing of large genomes and it is reviewed and prepared. Furthermore, an often
now possible to perform without delay sequenc- overlooked weakness is the use of patient samples
ing and analysis of patient genetic information. and/or omics data despite lack of linked clinical
Implementations of automated phenotyping and (meta)data, which greatly reduces the usefulness
interpretation of genome sequencing by bead- of the studies. Thus, information management is
based genome library preparation directly from of paramount importance for systems and
blood samples and sequencing of paired 100-nt personalized medicine in research as well as clin-
reads obtained 15.5 h and used for fast ical practice. To tackle these challenges, a variety
population-scale, provisional diagnosis of of approaches have been suggested and
genetic diseases of infants in neonatal and pedi- implemented in different settings. One such
atric intensive care units, have been reported model is a three-layer information technology
(Clark et al. 2019). They reported a prospective architecture coupled to a cyclic data management
100% precision and a mean time saving of 22 h approach, as proposed by Ganzinger and Knaup
on diagnosis which subsequently affected (2017). The generic high-level architecture of
treatment. such a three-layer model entails:
If Big omics data are less applicable for bed-
side decision support due to technical limitations, 1. Data representation,
they are essential when defining strategies for 2. Decision support, and
patient stratifications and individualized therapy 3. User interface.
in NSTI. Indeed, the lack of stratification
strategies (one of the cornerstones of precision As for data representation (layer 1), data and
medicine) is one of the biggest bottlenecks in knowledge from different sources have to be
NSTI management. Omics data from NSTI prepared and made available for use in systems
subjects (and possibly controls), such as medicine. This includes data harmonization,
transcriptomics, proteomics, lipidomics, and transformation, and storage. In decision support
metabolomics profiles measured on blood and (layer 2), the data and knowledge from layer 1 are
tissue biopsies, could be analyzed in relation to processed by applying decision support
pathogens and clinical parameters using multivar- approaches or systems biology models (see also
iate statistics, machine learning, and reverse engi- Sect. 12.3 on Big Data). Systems medicine
neering approaches to identify subgroups of applications should be designed to assist and not
patients demonstrating a survival benefit or favor- replace human decisions. Consequently, the user
able response to given treatments. interface for such an application (layer 3) must be
carefully designed to support well-informed, platform. This required previous ethical approvals
reproducible, clinical decisions in an appropriate and written informed consent from all patients
time frame. The core concept of this model is the [and to be updated and amended as need arises].
knowledge base, which contains patient and It required as well full compliance on Data &
disease-related data as well as formally Ethics governance, Good Clinical Practice
represented knowledge including a variety of guidelines, and European General Data Protec-
omics and biomedical data. The Decision Support tion Regulations (GDPR) (Regulation 2016).
is suggested as case-based and rule-based The data platform developed includes systems to
components (Ganzinger and Knaup 2017), see handle data in separated data domains and stores
also Sect. 12.5 below pertaining Decision Sup- to protect data ownership and privacy
port. This model is shown schematically in requirements.
Fig. 12.2.
The complexity of the data management pro-
cess depends on the level of heterogeneity preva-
12.4.3 Integrating Heterogeneous
lent in the data sources. To achieve sufficient case
Data with FAIR Principles
numbers it is often necessary to combine data on
the same entity types from different sources. For
A key feature and goal of such multinational,
example, in the multi-center approach hospitals
multi-center projects like INFECT, PERAID,
decide to collaborate and share clinical data on a
and PerMIT is to ensure that all data resources
specific disease area to build a joint systems med-
(both institutions’ own and public) are properly
icine application with a higher number of cases
integrated into a common framework. A
and therefore greater statistical power. In most
distributed data lookup and retrieval service
cases, clinical documentation will not be based
allow users to select relevant datasets for inclu-
on identical specifications. Thus, in a
sion in analysis based on not only matching meta-
harmonization step data definitions have to be
data but importantly clinical and biological rele-
evaluated for each attribute, both on a syntactic
vance and this adds a vital level of quality control
and semantic level.
ensuring that only clinically relevant datasets are
included. These encompass also demographics,
clinical, and treatment aspects.
12.4.2 The Case of NSTI
Strict clinical case definition criteria, as well as
source of infection and severity scores such as
In the context of systems and personalized medi-
simplified acute physiology score (SAPS) and
cine in NSTI, the most comprehensive endeavor
sequential organ failure assessment (SOFA) are
thus far is that being undertaken under the scope
used for precise patient classifications, whereas
of a series of national and international research
microbiological results will be documented and
programs comprising teams from hospitals, med-
allow for stratification according to etiology and
ical research, academia, and industry (see Chap. 1
virulence properties. A key component is the
and www.permedinfect.com). These programs
inclusion of heterogeneous omics and biochemi-
have built a platform for personalized medicine
cal datasets, which are ultimately essential for
in acute infectious diseases with focus on NSTI
establishing the mechanisms underlying clinical
and sepsis that form the basis for development of
conditions. The carefully curated studies/datasets
tools and concepts for refined diagnosis, patient
are integrated with a minimal data model for
stratification, and individualized treatment.
meta-data exposure using a Resource Description
The various participating hospitals have made
Framework (RDF) model (Lassila and Swick
substantial standardization efforts, so that clinical
1998), which is further empowered by using
partners provide patient cohorts, including both
distributed search and indexing technologies
clinical registries and associated biobanks, which
such as Apache Lucene (Białecki et al. 2012).
were used to populate a common information
Data fusion and standardization is ensured hunt/data which inform the PerAID and PerMIT
throughout the use of specialized data fusion data projects), allowing different layers with dif-
algorithms and well-functioning interactions ferent level of access and different levels of gran-
among participating institutions. ularity at the data level.
The use of a RDF data model to manage data At the first level, a close interaction between
provenance and storage ensures that data comply the clinicians and support personnel exists. The
with the FAIR guidelines for data management. interaction on the electronic devices is
Minimum information checklists are used to facili- optimized using the state-of-the-art technology
tate interpretation and reproducibility of results to monitor and to optimize human-computer
ensuring the inclusion of the relevant meta-data. interfaces.
All data types are (should be) to be represented in The second level prepares anonymized
ontologies so that they can be integrated and remain databases from level 1 using software that can
interoperable as the types and size of data increase. process various types of data such as patient
A systematic treatment of the data is ensured records, data collected in various forms, and
by the use of ontologies devoted to clinical and biobank data to a format that can be included in
disease-related terms, such as Systematized the RDF databases through previous
Nomenclature of Medicine-Clinical Terms standardization. This software is to be used within
(SNOMED CT) (Cote 1986; Lussier et al. the hospitals in a protected environment and only
1998). The structure of the underlying data the anonymized data will be transferred to the
resources is assessed using tools such as second level. This data is then to be further
RDF2Graph (van Dam et al. 2015) to ensure digitalized and abstracted to the third level that
that newly integrated datasets readily fulfill the will represent a statistical view on the
quality standards and indicators. original data.
Operation of the www.permedinfect.com plat- The first and second level are unique for a
form enables both handling of heterogeneous data particular clinical center, while data at the third
and the standardization of operating procedures, level will be pooled across centers. The three-
data, and models, as well as their storage and layered data structure helps to overcome the
stewardship. main limitations of medical support systems,
The structure and workflows comply with which are usually not scalable and not
those required by the European Union on data interconnected. Data are translated to standard
management of research (European Commission English vocabulary while keeping the original
2016). The platform itself aligns with interna- granularity of the data and third layer data will
tional data stewardship infrastructure such as the be shareable between different clinical sites.
ESFRI ELIXIR (elixir-europe.org) or Nordic This allows the CDSS to be scalable and
e-Infrastructure Collaboration (NeIC) program used in other studied and countries by account-
(https://neic.no/). ing for local language requirements (see Sect.
12.5). Then the third layer, containing highly
processed data, can be made public and used to
predict treatment outcomes and contributing to
12.4.4 Laying the Basis
best practice management of the disease. The
for Computer-Assisted Decision
CDSS will therefore combine the information
Support
of all patients in the patient cohorts, thereby
allowing for interoperability and scalability of
Any CDSS has to rely on a data platform (see, for
the system.
instance, the HUNT platform, www.ntnu.edu/
12.5 Clinical Decision Support Computer technology and algorithms can assist
Systems for Soft Tissue by generating case-specific advice for clinical
Infections decision making. The systems used are usually
referred to as CDSS, and are thus intended to
12.5.1 The Need to Enhance Medical improve health care delivery by enhancing medi-
Decisions cal decisions with targeted clinical knowledge,
patient information, and other molecular or health
The quantity and quality of clinical data are information (Wasylewicz and Scheepers-Hoeks
expanding rapidly, including EHRs, disease 2019).
registries, patient surveys, and information
exchanges. Also, burgeoning amounts of data
are becoming available for each patient, as is the 12.5.2 What Are CDSS What Is
increasing body of medical and basic sciences Their Use
evidence. Hence, clinicians need tools to help
them make rational decisions based on all these A traditional CDSS is comprised of software
information (Wasylewicz and Scheepers-Hoeks designed to be a direct aid to clinical decision
2019). making, in which the characteristics of an indi-
Big Data and digitalization, however, does not vidual patient are matched to a computerized
automatically mean better patient care. Several clinical knowledge base. Patient-specific
studies have shown that only implementing an assessments or recommendations are then
EHR and computerized physician order entry presented to the clinician for a decision (Sutton
(CPOE) has rapidly decreased the incidence of et al. 2020). From a historical point of view,
certain errors, introducing, however, many more medication-related CDSS has been used for a
(Magrabi et al. 2016). Therefore, high-quality long time and is still the most advanced (Garg
clinical decision support is essential if healthcare et al. 2005). CDSSs today are primarily used at
organizations are to achieve the full benefits of the point-of-care, for the clinician to combine
EHR and CPOE. their knowledge with information or suggestions
In the current healthcare setting, healthcare provided by the CDSS. However, there are CDSS
providers often do not know that certain patient being developed with the capability to leverage
data are available in the EHR, do not always data and observations otherwise unobtainable or
know how to access these data, do not have the uninterpretable by humans (Wasylewicz and
time to search for them, or are not fully informed Scheepers-Hoeks 2019).
on the most current medical insights when facing Current CDSS often makes use of
a decision. It is said the healthcare providers often web-applications or integration with EHR and
drown in the midst of plenty (Mamlin and Tierney CPOE systems. They can be administered not
2016; Frost and Sullivan 2015; Bresnick 2016). only through desktop, tablet, smartphone but
Moreover, decisions by healthcare also through other devices such as biometric
professionals are often made in conjunction monitoring and wearable health technology.
with/as part of direct patient contact, ward rounds, These devices may or may not produce outputs
or multidisciplinary meetings. This means that directly on the device or be linked into EHR
many decisions are made in a matter of seconds databases (Dias and Paulo Silva Cunha 2018).
or minutes. This way, their quality depends on the The scope of functions provided by CDSS is
healthcare provider having all patient parameters vast, including diagnostics, alarm systems, dis-
and medical knowledge readily available at the ease management, prescription, drug control,
time of the decision. Consequently, current and much more. CDSS ranges from personal dig-
decisions are still strongly confounded by experi- ital assistant applications customized by a single
ence and knowledge of the professional. clinician to multihospital mainframe-based sur-
veillance systems meant to assure care for
Fig. 12.3 Clinical algorithm for suspected fasciitis, as is transported to the operation room for further treatment.
suggested by Hietbrink and co-workers (Hietbrink et al. Treatment and aftercare are multidisciplinary. Analysis of
2016). The algorithm is used for gate specialties in patients frozen section, microbiological findings, and biopsy could
with suspected necrotizing fasciitis. It consists of aware- be supported by AI technologies. Figure and caption from
ness, early surgical exploration, and early initiation of Hietbrink et al. (2016), Licensed under Creative Commons
treatment. When incision biopsy is indicated, the patient
thousands of patients (Pusic and Ansermino modelling tools, can be essential for rapid diag-
2004). They can manifest as computerized alerts nosis, stratification, and assistance on decision
and reminders, computerized guidelines, order regarding disease treatment and intervention
sets, patient data reports, documentation strategies to be applied.
templates, and clinical workflow tools. The benefits of CDSS, possible pitfalls, and
Regarding the nature of the interaction with evidence-based mitigation strategies to overcome
the clinician, CDSS may be categorized into those have been published recently by Sutton et al.
that entail solicited information (e.g. a clinician (2020) and discussed as well by Pusic and
asking for specific advice for a given condition) Ansermino (2004) and (Wasylewicz and
or unsolicited information (deliver information or Scheepers-Hoeks (2019).
knowledge that beneficially can alter clinical
decision making (Pusic and Ansermino 2004)).
Such applications can be particular useful if
12.5.3 CDSS in NSTI
CDSS builds upon robust and flexible data inte-
gration and includes a wide variety of data ana-
Algorithmic procedures to handle NSTI patients
lytics. Such data analytics, in particular those
have been defined (Hietbrink et al. 2016;
based on AI, Machine Learning, and other
Peetermans et al. 2020), see Fig. 12.3, but there
are currently no CDSS or modules thereof dedi- Anesthesiologists class 4 or higher, emergent sur-
cated to NSTI. Such modules or built-in tools gery, septic shock, and low platelet count. The
would possibly be very useful for a range of receiver operating characteristic area was 0.85
uses, including those that could be included in (95% CI 0.82-0.87), reflecting a reasonably
CDSSs or applications routinely used by general strong prediction. Using bootstrap validation,
practitioners, as these are often the first point of the optimism-corrected receiver operating charac-
entry of potential patients, which are frequently teristic area was 0.83 (95% CI 0.81–0.86), which
misdiagnosed due to lack of familiarity with the was used to develop an interactive risk calculator
disease (Goh et al. 2014). for future patients. Although not a CDSS, this
Indeed, due to the fast progression of the dis- correlation was nevertheless useful for stratifica-
ease, NSTI management requires fast decisions to tion, according to the authors.
determine the most appropriate course of action. Another cohort study was reported (Hua et al.
Such decisions, or part of them, could be 2015) that included 109 patients with a confirmed
performed or supported by trained algorithms, diagnosis of NSTI, a median follow-up of
e.g. aiding in the main diagnostic problem, that 274 days (range 2–6135 days) and of which
is to differentiate a lesion requiring surgery from a 31 (28%) died. On multivariate analysis, indepen-
lesion for which conservative treatment will be dent risk factors of mortality were age older than
sufficient. Furthermore, tasks like selection of 75 years, multifocal NSTI, severe peripheral vas-
antibiotic therapy and adjunctive therapy could cular disease, hospital-acquired infection, severe
be performed or optimized by dedicated sepsis, and septic shock on hospital admission.
algorithms, as suggested in the closely relating Although a retrospective cohort, which disallows
field of sepsis management (Komorowski 2020). a precise record of the delay between diagnosis
Simple algorithmic procedures are already rou- and surgery, these analyses could help building
tinely applied in the management of NSTI. An information to develop a true CDSS and help
example of this is the LRINEC (Laboratory Risk clinicians stratify NSTI severity at clinical course
Indicator for Necrotizing Fasciitis) score which is onset.
generated from six routinely performed labora- A triple diagnostic procedure has been pro-
tory tests including the analyses of patients´ posed to manage NSTI (Hietbrink et al. 2016)
C-reactive protein, white blood cell count, hemo- which combines the analysis of microscopic
globin, sodium, creatinine, glucose. Wong et al. findings on tissue biopsies together with Gram
(2004) proposed a tool to distinguishing NSTI staining to assess the presence, gram staining,
from the other severe soft tissue infections. How- characteristic arrangements, and morphology of
ever, recently its performance was evaluated in a microorganisms and analysis of fresh frozen
prospective cohort study and data were displayed sections to detect necrosis of the superficial fascia
discouraging its use (Hsiao et al. 2020). An older with fibrinous thrombi of arteries and veins
study within the National Surgical Quality located in the fascia. The algorithmic procedure
Improvement Program (NSQIP, USA) was used is described in Fig. 12.3, in which many steps
to determine data on the incidence, treatment, and could be replaced or supported by AI- and Big
outcomes of NSTIs (Mills et al. 2010). Partly on Data-informed decision.
the basis thereof, a 30-day postoperative mortality
risk calculator for patients with NSTI was devel-
oped and validated using a cohort of 1392 12.5.4 Current and Future
identified NSTI cases, of which 42% were female, Developments in Relation
median age was 55 years, and median body mass to Dedicated CDSS for NSTI
index was 32 kg/m2 (Mills et al. 2010). Thirty-
day mortality was 13%. Seven independent Specific efforts to develop and deploy innovative
variables were identified that correlated with mor- clinical support tools for patient stratification and
tality: age older than 60 years, functional status, bedside decisions suitable for the emergency and
requiring dialysis, American Society of intensive care setting for NSTI are currently being
E
e.g xtern
. lit al
MedTrender era dat
tur a,
e ML models
(+ others?) CDSS running on
Patient Clinical Nordic platform
PerMIT /
PerAID Toolbox
Genetic Automated scoring
Database
Consider overlap!
Mortality prediction
Severity assessment
...
INFECT cohort Metabolomic
+
New data from Health records?
Findable
PerMIT / Accessible
PerAID Questionnaires?
Interoperable
Reusable Expert
Interface
knowledge
Other? (App)
Personalised Improved
(pre-emptive) decision-
treatment making
Fig. 12.4 Simplified representation of a CDSS for NSTI under development in the scope of the projects PerAID and
PerMIT (Figure reproduced from permedinfect.com, author’s copyright)
undertaken in a series of national and interna- sepsis alarm are considered as a means to achieve
tional research projects comprising teams from a more rapid and optimized identification of
hospitals, medical research, academia, and indus- patients. This type of systems can help
try (see permedinfect.com). One specific task is prioritizing severe cases and thereby reduce the
the development of Machine Learning and AI clinical burden and efficient use of hospital beds.
models for the prediction of different outcomes An effective and efficient CDSS is of particu-
in NSTI, such as risk of sepsis, septic shock, or lar importance for the stratification of patients and
amputation, using a combination of clinical personalized therapy in NSTI. Several dedicated
parameters including SAPS and SOFA severity modules are currently under development (www.
scores and biomarkers that can be measured dur- permedinfect.com) for: (1) the integration of a
ing the acute stages (e.g. in the emergency room), variety of quantitative or qualitative models
thereby enabling rapid bedside decisions. (i.e. statistical models, algorithms, etc.) to enable
The same data approach is being used to iden- a CDSS to perform data analytics (see previous
tify an optimized scoring of NSTI patients, to chapter and section on AI and Big Data above);
overcome some of sensitivity issues identified (2) aggregation of reasoning processes from the
with the severity score LRINEC indicated above domain and inference capabilities (e.g. rule-base
(Hansen et al. 2017). and case-based systems) to handle the data/infor-
Development of automatic calculations of mation (i.e. clinical and experimental
such severity score is part of the basis for parameters); and (3) user interfaces to match the
applications offering personalized decision sup- need of practitioners in the clinic.
port. Considering that different hospitals have The software architecture model for the NSTI-
their own sepsis alert system, such automatic dedicated CDSS, the representation of interoper-
calculations linked to the patients charts and able clinical knowledge, and inference engine are
PatientDB
te
Tr
la
ea
cu
t
al
C
Predict Mine
Fig. 12.5 Representation of a simple mobile app for use databases, calculating specific scores, predicting outcomes
by clinicians as a front-end of a CDSS for NSTI and generally supports the clinician for decisions
(LifeGlimmer GmbH). It enables data mining, querying
Example with 2 variables

predicting “death after 1 yea
ar”
Enter data Predict
Fig. 12.6 Example of the use of the app as support to (e.g. 90 days mortality) of NSTI patients with high accu-
beside decision. Based on over 2000 current clinical racy, supporting thereby patient stratification for
parameters and by using Machine Learning algorithms differentiated treatment (LifeGlimmer GmbH)
(tested in the INFECT patient cohort), it predicts severity
being designed to form a base for a CDSS frame- validation cycles using enterprise-grade soft-
work of wider applicability. The CDSS ware-engineering methodologies and
functionalities are being iteratively developed technologies. The CDSS prepares views for the
through requirement-adjustment-development- clinicians and supports personnel with data that
allow patient stratification and individualized for Innovation Systems (VINNOVA), Innovation Fund
therapy in NSTI/sepsis. In addition to individual Denmark and the Research Council of Norway under the
frame of NordForsk (Project no. 90456, PerAID), and the
patient data, the system provides information of Swedish Research Council, Innovation Fund Denmark, the
patient classification into groups with similar dis- Research Council of Norway, the Netherlands
ease status and comparable clinical and biological Organisation for Health Research and Development
parameters, which are of great value for (ZonMW), and DLR Federal Ministry of Education and
Research, under the frame of ERA PerMed (Project 2018-
continued research. A simplified representation 151, PerMIT).
of the envisaged CDSS for NSTI is shown in
Fig. 12.4.
An example of an initial prototype for a front-
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Advances in Experimental Medicine and Biology 1294

More information about this series at http://www.springer.com/series/5584

Necrotizing Soft Tissue

ISSN 0065-2598 ISSN 2214-8019 (electronic)

# Springer Nature Switzerland AG 2020

of personalized medicine for therapeutic advances for patients with necrotiz-

Huddinge, Sweden Anna Norrby-Teglund

1 The INFECT-Project: An International

10 Systems Genetics Approaches in Mouse Models

Mattias Svensson and Anna Norrby-Teglund

# Springer Nature Switzerland AG 2020 1

Necrotizing soft tissue infections (NSTI) are rap-

Table 1.1 INFECT partners

References Dos Santos VAP, INFECT Study Group, Norrby-

Abstract support them through further operations,

# Springer Nature Switzerland AG 2020 7

Fig. 2.1 Martyn Spark

Fig. 2.2 Robert Wilkie

Fig. 2.3 Frankie’s back

Fig. 2.4 Frankie’s ﬁrst

Fig. 2.6 Frankie May 2013

Fig. 2.7 Frankie

Fig. 2.8 Zara had given

Fig. 2.11 We ﬁnally had

Torbjørn Nedrebø and Steinar Skrede

Abstract chapter, we present three cases from the

# Springer Nature Switzerland AG 2020 21

Fig. 3.1 Swelling and erythema of the knee at admission

Fig. 3.2 Gas seen in the soft tissue along femur

Fig. 3.3 Purulent ﬂuid during knee-arthroscopy

Fig. 3.4 Necrotic muscle

In our hospital we have noted an emerging Undertreatment with antibiotics in early

Fig. 3.5 Area of marked swelling and erythema at ﬁrst admission

Fig. 3.6 Spread of

Fig. 3.10 Fasciotomy of

Fig. 3.11 Edema and dishwater-like ﬂuid under the fascia

necrotic muscle of the adductor muscle was pres- Discussion

Fig. 3.12 Picture taken of medial fasciotomy after second revision

References Bruun T, Rath E, Bruun Madsen M, Oppegaard O,

Abstract and deliquescent tissue can be seen. Patients

# Springer Nature Switzerland AG 2020 39

Keywords penicillin, and support for abnormal physiology

Highlights 4.2 Clinical Presentation

Fig. 4.1 Presentation of

Fig. 4.2 NSTI before

Table 4.1 Symptoms and findings related to organ systems

often the methodology—including deﬁnitions of Peptostreptococcus sp., Bacteroides sp., and

Fig. 4.3 Infection of the lower leg. Courtesy of INFECT

Obtain cultures for microbiology

Contact members of multidisciplinary team according to local protocols

# Springer Nature Switzerland AG 2020 53

Table 5.1 Categorization of necrotizing soft tissue infections by microbiological etiology

of contaminants on the etiologic distribution is 5.3.2 Quality of Identification

Firstly, sample source was reviewed to support

Secondary pathogens included microbes that 5.4 Microbiological Etiologies

5.4.6 Aeromonas hydrophila Today most NSTIs with polymicrobial etiologies

(Thänert et al. 2019). Microbial community 5.5 Future Perspectives

subsp. equisimilis strain causing streptococcal toxic 26, 2020 https://www.uptodate.com/contents/clostrid

Abstract cases were caused by GAS (31%) or SD (7%).