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,
and Anna Norrby-Teglund

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
Soft Tissue Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Edoardo Saccenti and Mattias Svensson
12 Systems and Precision Medicine in Necrotizing
Soft Tissue Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
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-
cine in necrotizing soft tissue infections
1.1 Necrotizing Soft Tissue
Infections
Necrotizing soft tissue infections (NSTI) are rap-
idly spreading infections that may cause exten-
sive soft tissue or limb loss, multiorgan failure,
and are associated with a considerable fatality
rate. These life-threatening infections that are
caused by a variety of pathogens are among the
few infectious diseases that remain associated
with substantial mortality and amputation even
in young individuals with no underlying condi-
tion. It is undisputed that rapid diagnosis and
prompted intervention is directly related to sur-
vival, but diagnosis and management are difficult
due to heterogeneity in clinical presentation,
co-morbidities, and microbiological etiology. To
tackle these challenges an international and mul-
tidisciplinary consortium, INFECT (Improving
Outcome of Necrotizing Fasciitis: Elucidation of
Complex Host and Pathogen Signatures that Dic-
tate Severity of Tissue Infection), was formed
https://permedinfect.com/projects/infect/.
1.2 The INFECT-Project
1.2.1 Initiative and Consortium
The initiative to INFECT was taken in 2012 when
a consortium of 14 partners applied for EU
funding within the Seventh Framework
Programme Health and Innovation, under the
programme “Systems medicine: Applying
systems biology approaches for understanding
multifactorial human diseases.” INFECT was
granted EU funding and was sponsored from
January 2013 to June 2018. The overall goal of
INFECT was to reveal pathophysiological
mechanisms and disease signatures, as well as
identifying prognostic and diagnostic markers of
the multifactorial highly lethal NSTI. To enable
this multifaceted work the consortium was
designed to include industrial, clinical, and pre-
clinical partners with expertise in different
disciplines including infectious diseases, inten-
sive care, microbiology, cell biology, bacterial
pathogenesis, systems biology, computational
modelling and diagnostics. INFECT partners are
listed in Table 1.1.
1.2.2 Objectives and Work Plan
The specific objectives of INFECT were to:
1. Establish a patient cohort and collect samples
for a biobank collection
2. Analyze patient samples and clinical isolates
to pin-point key host and pathogen factors
involved in the onset and development of
infection
3. Identify and quantify disease signatures and
underlying networks that contribute to disease
outcome
4. Exploit identified disease traits for the
innovation of optimized diagnostic tools
5. Translate the advanced knowledge generated
into improved classification and management,
as well as novel therapeutics
To achieve the objectives, a comprehensive
and integrated knowledge of diagnostic features,
causative microbial agent, treatment strategies,
and pathogenic mechanisms (host and bacterial
disease traits and their underlying interaction net-
work) were required. Therefore, the work of
INFECT was structured around an integrated
systems biology approach in patients and differ-
ent clinically relevant experimental models. The
INFECT-project was set up to be multidisciplin-
ary and included clinical partners, academic
experimentalists and mathematical modelers,
one patient organization, as well as two small-
and medium-sized enterprises focusing on data
integration in systems and synthetic biology,
and development of diagnostic tools. The partners
contributed with unique expertise, infectious dis-
ease and intensive care clinicians, technical
platforms and/or model systems that together
provided the means to successfully conduct the
multifaceted research proposed (Table 1.1.). The
work included seven highly integrated scientific
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
obtained, and one management work package. A
central part of the INFECT-project was the pro-
spective enrollment of patients with NSTI and
creation of a clinical data registry, the collection
of biobank samples and the causative microbes,
which all formed the basis for the experimental
studies (Madsen et al. 2019). Further, INFECT
was designed to use an integrated systems biol-
ogy approach in patients and different clinically
relevant experimental infection models, including
both murine NSTI models (Chella Krishnan et al.
2016; Nookala et al. 2018) and a human
organotypic skin model (Mairpady Shambat
et al. 2016; Siemens et al. 2015, 2016). The
work-flow also included a comprehensive set of
analyses followed by integration of results in
advanced computational platforms, which
enabled generation of pathophysiological models
of the disease and advanced understanding of the
underlying mechanisms and host–pathogen
interactions. The results were translated into
novel diagnostic tests and improved patient man-
agement (Fig. 1.1).
1.2.3 Achievements
The INFECT-project has generated comprehen-
sive knowledge of diagnostic features, causative
microbial agents, treatment strategies, and patho-
genic mechanisms (host and bacterial disease
traits and their underlying interaction network)
through analyses of the INFECT patient cohort
and associated biobank. Also, INFECT has
proven the value of systems medicine approaches
in acute infectious diseases to achieve improved
diagnostics and therapeutics to improve patient
4 M. Svensson and A. Norrby-Teglund

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. . .
disease outcome. In this book, we have
summarized the scope and outcome of the
INFECT-project. The results have been compiled
into 12 chapters, each of which has a unique focus
ranging from patients’ and doctors’ experiences
of NSTI, to different treatment alternatives and
the use of systems medicine in NSTI. In short,
Chaps. 2 and 3 portray affected patients’ and
treating physicians’ experiences; Chaps. 4, 5,
and 6 communicate clinical characteristics,
microbial etiology, and the role of beta-hemolytic
streptococci; Chaps. 7 and 8 describe treatment
options with particular attention to antibiotics and
polyspecific intravenous immunoglobulin
(IVIG); Chaps. 9 and 10 depict pathogenic
mechanisms revealed from in vitro and in vivo
models of NSTI; and finally, Chaps. 11 and 12
illustrate the merits of system biology for
identifying NSTI biomarkers, and the use of
systems medicine as a tool for stratifying patients
with NSTI.
Below we summarize some key findings and
achievements of INFECT:
• Establishment of the world’s largest NSTI
patient cohort with extended clinical registry
and associated biobank.
• Four hundred and nine NSTI patients with
completed clinical registries (>2000
variables) were enrolled and associated
biobank samples (> 6000) and clinical bacte-
rial isolates collected.
• In Chaps. 3, 4, 5, and 6 advanced insight into
the clinical aspects of NSTIs providing the
basis for evidence-based guidelines for patient
management and care is given (Bruun et al.
2020; Madsen et al. 2019).
• In Chaps. 9, 10, 11, and 12 it is shown how the
systems medicine approach within INFECT
substantially advanced our understanding of
these life-threatening infections, including the
identification of novel pathogenic mechanisms
and specific host and bacterial disease traits
associated with disease outcome (Afzal et al.
2020; Chella Krishnan et al. 2016; Siemens
et al. 2015, 2016; Thanert et al. 2019).
• In Chap. 10 it is demonstrated how the patho-
physiology of NSTI is influenced both by the
5
causative microbe and by host factors (Chella
Krishnan et al. 2016; Siemens et al. 2016;
Thanert et al. 2019). This underscores the
need for patient stratification and implementa-
tion of tailored therapy/personalized medicine
in these infections, presented in Chap. 13.
• Development and testing in the clinical setting
of multiplex diagnostic tools for rapid patho-
gen identification and monitoring of disease
associated biomarkers.
• The novel understanding of the disease
mechanisms of these infections has resulted
in changed clinical practice related to antibi-
otic usage as well as use of immunomodula-
tory treatments (Bergsten et al. 2020; Madsen
et al. 2017) (Chaps. 7, 8, and 9).
• Fostering the new generation of clinical and
preclinical scientists within the field of
systems medicine in infectious diseases.
1.3 Future Perspectives
We foresee that the achievements of INFECT will
form a basis for future advances in the area of
implementing personalized medicine in infectious
diseases. This book volume is designed to present
the contemporary understanding of clinical
features, treatment, and pathogenic aspects in
NSTI, as well as serve as an overview of the
contributions to this field achieved through the
INFECT study. Readers will also be guided to
other references that should be consulted for fur-
ther information on NSTI pathogenesis, causative
agents, treatments as well as novel diagnostic and
therapeutic strategies in order to improve out-
come of NSTIs. The foundation that the results
of INFECT has laid will be further explored and
implemented in the field of personal medicine in
infectious diseases in the current multinational
projects PerMIT and PerAID, www.
permedinfect.com.
Acknowledgement Financial support: The work was
supported by the European Union Seventh Framework
Programme: (FP7/2007-2013) under the grant agreement
305340.
6 M. Svensson and A. Norrby-Teglund

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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

# Springer Nature Switzerland AG 2020 7

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_2
8 D. Cartledge et al.
on the 6th October 1999. My son had been taken
away from me in 5 days.
Lee was a healthy 23-year-old, with a lovely
quite naive personality. Martyn, my younger Son,
Lee’s brother lived in Sheffield Yorkshire.
Lee had an opportunity for work in Sheffield,
so he travelled there in readiness of the interview.
Approximately 3 days into staying over there
Lee felt cold and had symptoms of flu. I informed
Martyn to advise Lee to take two paracetamol
tablets every 4 h and to drink plenty of water. I
could not speak to Lee as he was not on the
phone. Lee went for his interview feeling very
poorly but managed to get through it and waited
for a few days in the hope that he would be called
upon to attend a second interview. Five days later
Emma, Martyn’s partner, phoned me to say Lee
had something more serious than flu and Lee was
seriously ill and as a result they had phoned for an
ambulance. I remember the daunting tones of
Martyn yelling in the background, he sounded
like a wild animal and was hysterical. I lived
over a 100 miles away and I was in shock and
disbelief, so I knew I was unable to drive all the
way to Sheffield even at a time like this. The
emergency crew arrived and tried their best to
apply emergency care to Lee but he was falling
into unconsciousness. Martyn looked on in disbe-
lief, why was Lee so very poorly. Martyn
explained later that Lee’s eyes were rolling also.
What a terrible experience for Martyn to witness.
I just wish I could have been there for them both.
The emergency team tried their very best to keep
Lee alive. Placed appropriate drains in him but
Lee was not reacting to any treatment. The ambu-
lance crew decided to take Lee to the nearest
hospital and kindly passed the telephone number
of the hospital. Martyn travelled in the ambulance
with Lee and the crew. I am sure the stress of this
for such a young man was truly horrific. The
ambulance crew also said to ask mum to allow
15 min for them to arrive at the hospital before
telephoning. I phoned the hospital in disbelief
how was it possible for flu like symptoms to
develop into Lee being so terribly poorly. What
was the problem? I phoned Hallamshire Hospital
and spoke to the Staff Nurse in A & E. I explained
the circumstances and she replied “that there was
no one with them by the name of Stephen Lee
Spark”. At that moment I really thought I was in a
terrible bad dream. But then the staff nurse
replied “Oh wait a minute he is in resuscitation”
I replied “No, I do not think this is possible”. The
Staff Nurse asked me to phone back 15 min later,
and suggested I had a sweet cup of tea and some-
thing to eat. By this time, I was in shock and was
fainting every time I stood on my feet. I desper-
ately tried to eat a slice of toast, it was difficult
and akin to chewing on a piece of leather. I had no
saliva in my mouth to swallow. Twenty minutes
later I phoned the hospital and spoke to the Staff
Nurse she replied “I am so sorry to have to tell
you that Lee has passed away. Would you like to
speak with Martyn?” we could only cry together.
I eventually said “Go back home. I will see you
there”. All this time I was on my own and I now
needed my husband (not Lee’s father) to be with
me. I explained the whole horrific story. It was 2 h
before he arrived home and I had packed a few
things for the long journey to Sheffield. I took a
bottle of water, a bucket to for me to vomit in as I
was in shock and an old dressing gown that my
mum gave me when I was younger to give me
comfort. We eventually arrived in Sheffield. Dur-
ing the journey I tried my best to make the deci-
sion of whether to see Lee’s body or go straight to
Martyn I decided that Martyn needed support he
had been through a terrible trauma. After all it
was better for me to remember Lee the last time I
saw him full of health and happiness.
We asked Martyn a few gentle questions as he
was crying all the time and spent a long time just
holding one another for comfort. We will never,
never forget that morning as long as we live. We
then awaited the post-mortem results and we were
told Lee had died of a Strep milleri infection—
from an abscess in his gum and that it had devel-
oped into Necrotising Fasciitis. His internal
organs had been eaten away. Martyn informed
us that when the ambulance crew were applying
resuscitation to Lee all his rotting flesh was com-
ing down his nose and out of his mouth. Still to
this day he remembers every finer detail and
especially the smell of the rotten flesh. Martyn
also referred to a dark purple like rash around
Lee’s neck. What on earth was this terrible dis-
ease all about, I phoned Public Health England
several days later wanting to find out more about
2 Necrotizing Soft Tissue Infections: Case Reports from the Patients Prospective
necrotising fasciitis and spoke with the Strep A
and Respiratory Department, they were truly
compassionate and informative. Over the next
few days I spoke with many Consultants and
medical staff all informing a little more about
this terrible disease and all apologised that they
could not provide a telephone number of a sup-
port group and all mentioned that there was a
need for a support group to offer that listening
ear and an understanding of compassion.
Therefore, I launched my Charity The Lee
Spark NF Foundation in January 2000. My first
survivor contacted me in May 2000. What a thrill
it was to talk to her. I desperately hung on to
every word she said. That way I had a better
understanding of what my dear son had been
going through.
Future Perspectives
The early diagnosis and treatment of necrotising
fasciitis must be taught at core level in order to
save lives and devastation within families. The
financial drain on the medical system is also, a
major factor, to the patient if they survive a life of
surgery, medicine and psychological problems.
Martyn strives everyday living through the
tragic circumstances of Lee’s death, my dear
sons. What has happened to our family? His
father never got over the shock of losing Lee
and passed away a few years later (Fig. 2.1).
2.2 Robert’s Experience
The best way to describe living with the effects of
necrotising fasciitis is to say it is a journey which
continuous long after the infection has gone. This
journey can often feel like a roller coaster ride
with its highs and lows. Looking back, I would
now say the first part of my journey was easy as I
knew little about what was going on. I certainly
had no idea of the seriousness of my condition for
the powerful drugs I was given pretty much kept
me in a deep sleep for almost a fortnight.
I was admitted to my local hospital with
expected appendicitis on New Year’s Eve 31st
December 1981 and operated on the following
day 1st January 1982, but it was not until almost
9
2 days later that the necrotising fasciitis appeared
making it more likely that the infection was post-
operative. In those days after treatment it was felt
that I would heal better at home, so I was released
on 27 January 1982; the day my eldest child was
born. On this day I remember being pushed
through the streets of the hospital complex in a
wheelchair by a nurse from the main building to
the maternity department to meet my new-born
son. Later that day as my wife was to remain in
the hospital for 7 days, I was released into the care
of my mother. My wife remembers a few days
before my release having a conversation with the
consultant about my future prospects, he
informed her that there was no reason why I
could not live a normal life, “but” I would possi-
bly require some corrective surgery in the future.
Never was a truer word spoken.
After being released from the hospital I took
the consultant at his word and virtually just got on
with my life. Taking my new parental responsi-
bilities seriously as I set about being a father for
the first time. With hindsight I now realise that
carrying a pram up three flights of stairs after
defeating necrotising fasciitis was not the
brightest idea I have ever had. Now as I reflect,
I also realise that perhaps I should have taken
more time off work and gradually eased myself
back into the workplace. It really was not the
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
vital organs unsupported and unprotected. As a
result of this I now have a loopy bowel and
internal bruising to the stomach plus I have also
suffered no fewer than 17 abdominal hernias, two
of which are irreparable and one is a ruptured
repair for which I am required to wear a support
belt. Wearing a hernia (stoma) belt has its
advantages for it eases the pain considerably but
it also has disadvantages for it restricts my move-
ment and can be almost intolerable in hot
conditions. After my first hernia operation I dis-
covered that I suffered from acute acid reflux and
was given a chalky medication to drink, this did a
great job of easing the reflux but also caused
constipation due to a build-up of chalk in my
bowel. After several endoscopy exams and a
10
Fig. 2.1 Martyn Spark
(left) Stephen Lee Spark
(right) deceased
failed colonoscopy, I was sent for a CT colonos-
copy which revealed I had loops in my bowel on
my damaged side hence the reason for the intes-
tine pain caused by the constipation. It may sound
foolish, but this has resulted in me having to re
learn how to and what to eat. For example, I
cannot eat anything from the onion family except
the green part of a spring onion or the green part
of a leek, I cannot eat pineapples, lemons, limes,
oranges I cannot take vinegar or eat pickles and
many other everyday foods. I also must think
about the kind of food I need to eat to help prevent
any recurrence of acute constipation, alas this
means eating out can be difficult if not in some
cases impossible. As I mentioned earlier, I also
suffer from internal bruising to my stomach, to
this day I have never been given a reason for this,
but I believe it is caused by my bowel pressing on
my stomach when it is full.
Recently I got rather fed up having to keep
pulling at my waist band and tightening my trou-
ser belt so I changed to using braces, the easing of
the discomfort around my stomach was almost
instant; the pain is still there but it is a much
D. Cartledge et al.
more manageable pain. I feel incredibly lucky to
have almost accidently discovered the difference
wearing braces instead of a belt can make. Pain
management is without a doubt the most difficult
thing I have had to learn on my journey, for the
pain around my stomach causes by internal
bruising is relentless. The only help I receive for
this are Codeine and Paracetamol, but I find the
use of heat also helps, sadly the aforementioned
hernias make exercising the area around my
wound sight difficult, so exercise does not help.
Three years ago, during a gastroscopy proce-
dure it was discovered that I had an ampullary
adenoma (pre-cancerous lesion) in the damaged
area of my abdomen. This remains benign, it is
almost as if this has taken up residence in the
space where I lost the muscle due to Necrotising
Fasciitis, therefore I see this as a continuation of
my NF journey. Journeying with the effects of NF
is a continual battle in trying to adapt to the
demands placed upon my damaged body. For
years I have struggled with pain causing me to
overcompensate putting more pressure on my
undamaged side which now has osteoarthritis. A
2 Necrotizing Soft Tissue Infections: Case Reports from the Patients Prospective
Fig. 2.2 Robert Wilkie
survivor of necrotising
fasciitis
coincidence perhaps? This brings me to what I
find to be the most difficult thing to live with post
Necrotizing Fasciitis, dealing with medical peo-
ple, for there seems to be a willingness to keep NF
quiet, why? Surely if I had been better informed, I
could have been better prepared and more able to
reduce the risks of further damage to an already
battle-scarred body.
Future Perspective
With necrotising fasciitis listening to a survivor I
believe is absolutely crucial yet to often I feel like
no one is listening and many look at me and think
here he comes again. Surely listening to any sur-
vivor is crucial to after care and pain management
(Fig. 2.2).
2.3 Frankie’s Story Written by His
Mother Lucy Dove
Frankie became unwell on Monday 8th April
2013 and he was only a 1 year of age. He had
flu like symptoms. I describe his early symptoms
as flu like symptoms because Frankie had a runny
nose and a temperature of 39  C. His older
11
brother, Kayne was unwell the day before with
pustules on his face, flu like symptoms and a pin
prick rash on his back and chest. An out of hours
GP referred Kayne to A and E, where I was told
that his blood results were consistent with a viral
infection and he was prescribed flucloxacillin.
The links between Kayne and Frankie’s
symptoms are very important.
In the late morning of Tuesday 9th April,
Frankie appeared to become very unwell with
similar pustules on his face and a red pinprick
rash all over his arms, chest and back. However,
unlike his brother, Frankie was screaming when I
moved him and seemed to be in a lot of pain.
At our local walk in centre I stripped Frankie’s
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
touching the area, so it was obviously very pain-
ful to him. He began hitting me and did not want
to be cuddled. Frankie was a very cuddly baby, so
this sent alarm bells ringing in my head! I
explained this to the nurse practitioner, and they
told me his temperature and other observations
were fine. (I told then he had been given medica-
tion for a high temperature). They were going to
12
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
must have a viral infection. I informed them of
Frankie’s history including how his brother had
been unwell. I stressed that I was not happy with
how much pain Frankie was in, the paracetamol
and ibuprofen were not working to ease his pain
and so I really was not happy to go home with
him. They then told me I could take him to A
and E.
I took Frankie straight to Children’s A and
E. He was a very tough little boy, and nothing
ever fazed him and so I knew that something had
been missed because Frankie was in absolute
agony. He could not be pacified and seemed to
be getting worse before my eyes. He would not
stand up and kept screaming.
When seen by a consultant I pointed out the
lump under the skin on his thigh. Back in the
waiting room Frankie kept deteriorating and
now he was howling in pain as if he had been
really bad injured. At this point I was becoming
increasingly frustrated. I felt as though my
concerns were not being understood. The only
way I could get him comfortable was to lay him
on my chest facing me. This was when I began
examining him myself. I am not medically trained
but nor was I paranoid first-time mum. I knew
something was wrong and I decided to check
everywhere on his body. I refused to accept this
was just a viral infection. I began checking his
D. Cartledge et al.
back. Om his left side approximately halfway up,
I pressed Frankie cried out in pain. I worked out
there was an area of approximately 2 in. by 2 in.
that was very painful for Frankie when I touched
it. I then noticed that it could in fact be seen from
the side as a swelling under his skin.
When another doctor examined Frankie, she
concluded that she believed he had a tissue infec-
tion. At roughly 9 pm a doctor took a blood
sample from Frankie’s foot. An Orthopaedic doc-
tor examined Frankie and concluded that Frankie
had a possible tissue infection.
In the early hours a doctor came into the room
and told me that a “marker” in Frankie’s bloods
was very high, but they decided not to give
antibiotics and await more tests in the morning.
I trusted that the doctors knew what was medi-
cally right for my son. During the Frankie kept
waking and projectile vomiting. He kept crying
on and off for hours. He was given codeine earlier
in the evening which had made him drowsy, but
he was still in a great deal of pain. I had a tough
night with him as I could not comfort him, and he
was in so much pain.
In the morning Frankie looked totally deliri-
ous. He could barely move. I noticed that the skin
on Frankie’s back was now red in parts (Fig. 2.3).
It looked like the swelling had spread up his back
and as you all can see his back appears to be
swollen now (but more obvious on his left side).
2 Necrotizing Soft Tissue Infections: Case Reports from the Patients Prospective
The left side now had red marks appearing on the
surface. The boil on his left thigh seemed to be
moving outwards as if spreading. Were the boil
had been was now looking dark blue like a bruise.
Unfortunately, I could not get a photo of his leg.
He would not let me touch anywhere on the left
side of his back or indeed his body. I remember
touching his left arm and him crying out in pain.
A group of doctors came into the room I told
them that the swelling on Frankie’s back looked
like it had spread, and I showed the doctors
Frankie’s left thigh. I was told Frankie was to
start antibiotics, that he needed an emergency
MRI scan. Frankie was given oral sedation and I
went down with him for his MRI scan which
lasted over 45 min. After the consultant told me
Frankie needed to be sent by ambulance to
another hospital and that he would need to go to
theatre to have the pus removed from under his
skin. I asked him what it was, and he replied;
“Necrotising fasciitis”. I had never heard of this
before and before I had had time to look it up on
google two doctors came into the room and began
trying to put cannulas into Frankie’s hands. It was
now just before 3 pm. The doctors started putting
what she said were antibiotics into Frankie’s can-
nula and pushing large syringes of saline into him
one after the other. This was when I became
worried something was really, seriously wrong.
Frankie was transferred to the other hospital by
ambulance. When we arrived, we were taken to
the resuscitation area where doctors, consultants
and different specialist surgeons began
introducing themselves. As they were about to
put Frankie onto a ventilator, we kissed him
goodbye never knowing if we would ever see
him alive again. A consultant plastic surgeon
came into the room and told us that Frankie was
very seriously ill and that his condition was life
threatening. She told us that until they got him
into theatre and tried to remove all the dead layers
of skin and tissue, they could not know how bad
it was.
Frankie was in surgery for almost 10 h and
when he came back the lead plastic surgeon and
PICI Consultants took us into a private room. We
were told that Frankie’s body had been exten-
sively damaged by the infection. The surgeon
13
had removed all the layers of skin and fascia
from almost his entire back, left side and quite a
lot of his left thigh and muscle had been removed.
We were told that they had not been able to
remove all the infection because Frankie was too
weak for any further surgery and that he was been
given strong antibiotics intravenously. The con-
sultant told us that they had done everything they
could at the point and that it was now up to
Frankie’s body to fight the infection with the
help of some broad-spectrum antibiotics. We
were asked to get everyone we needed up to the
hospital because they did not believe our baby
was going to survive the night. Not only did he
have NF but the deadly toxins in his body causes
sepsis and put his internal organs at great risk.
As parents we could not believe we were being
told that our son might not survive never mind the
fact that he had no skin covering his back and
sides. They allowed us in to see Frankie and it
was unbearable to see him the way he was.
Frankie was fully ventilated, in an induced coma
and swollen to about five times his normal size.
As parents we could hardly recognise our own
son. The only thing that made him Frankie was
his beautiful white curly hair.
We cannot fault anything the second hospital
for the care they gave to our son. Frankie by some
miracle made it to Friday 12th April when doctors
took him back down to surgery. We were told that
if the infection had spread then Frankie would be
made comfortable and allowed to pass away
peacefully. He was down in the theatre for five
and a half hours the surgeon had managed to
remove the dead tissue from deep behind his
shoulder blade and had been forced to cut deeper
down into the muscle in his thigh. They were
confident that they had removed everything and
that everything now looked free from infection.
We were informed that the bacteria that has
caused the NF to develop was called strep A,
which knowing this meant that they could adjust
the antibiotics he was on.
After Frankie’s second operation we felt more
positive but were consistently told that Frankie
still had a very long way to go because of the
extensive damage the NF had causes to his body.
Over the weekend Frankie remained in an
14
Fig. 2.4 Frankie’s first
dressing change on the
ward with me
induced coma in PICU and then on Monday 15th
April 2013 he underwent a further 5 h of surgery
were expert plastic surgeons who specialise in
burns patients, grafted healthy skin from his
tummy, chest, right calf, left shin and back of
his thigh onto his back and left leg. They were
amazed with how well everything went and fol-
lowing two dressing changes in theatre on Friday
19th April 2013 Frankie was stable enough to be
taken of his ventilator. He remained on high doses
of morphine and clindamycin as well as other
drugs, but he became well enough to be trans-
ferred onto the burn’s unit.
Fast forward 7 years and Frankie is now
8 years old. We stayed in hospital for 3 months
as he underwent painful dressing changes and
procedures. He wore tight pressure suits and suf-
fered break downs in his scars for 2 years after.
D. Cartledge et al.
He had recurrent infections and 1 year later had to
have his central back re-grafted. He went to the-
atre every 6 weeks for steroid injections. His
grafts became raised and sore. He had to take
gabapentin for a year which really affected his
behaviour. He takes regular pain killers the stron-
gest being oral morphine. He has low self-esteem
and struggles with his confidence, but thankfully
he is very active, and we encourage him to not let
his skin grafts hold him back. We cream him four
times a day. I have included picture of Frankie’s
skin grafts from 2013 and now in 2020 (Figs. 2.4,
2.5, 2.6, and 2.7). Frankie is an inspiration. As he
grows, he will need more skin grafts and
procedures. We are incredibly lucky that he sur-
vived but our only wish is that it was diagnosed
and treated sooner.
2 Necrotizing Soft Tissue Infections: Case Reports from the Patients Prospective 15

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
learnt from Frankie’s case.
Future Perspective
Doctors must pay close attention to any bacterial
infections that a person may have been
predisposed to when taking history as this could
help with a faster diagnosis. I would argue that
this is very important with young children or
those with a weaker immune system. The links
between Kayne and Frankie’s symptoms are very
important because Impetigo can be caused by the
16 D. Cartledge et al.

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
2 Necrotizing Soft Tissue Infections: Case Reports from the Patients Prospective
eyes were watery; to me they were classic
symptoms of a cold. Forty-eight hours later Zara
came out with chickenpox. Over the next 24 h,
more pox came out but Zara suddenly become
really unwell which I thought was very unusual as
children are usually ill before the “pox” appears.
Zara started vomiting and developed a tempera-
ture of 38  C which could be controlled by para-
cetamol. The next day her temperature was
increasing and had now reached 39  C but was
no longer responding to paracetamol. The
chickenpox was still coming through but they
were now coming through as green/black in col-
our and were appearing more like “scabs” as
opposed to blistered “pox” texture. Zara had
now become very ill, she was not eating or drink-
ing and was starting to cry whenever she was
touched.
There were two significant “pox” that started
to look very nasty, one was on Zaras’ abdomen,
the other on her sternum. The “pox” were now
appearing as big green and black scabs. The pox
on her abdomen had now developed a red ring
around it, this is what lead me to take her to our
local A&E department in Salford Manchester
UK. They put us in an isolation room in the
children’s department. Zara was now starting to
develop a red rash around the “pox” on her
sternum.
We found out that 2 weeks prior to our visit a
3 year old with similar symptoms had been sent
home and had sadly died of sepsis, I think that
that tragic event helped my daughter because that
meant the staff was extremely vigilante with her.
The doctors were really concerned as they did not
know why Zara was ill or what was causing
it. Zara was now screaming if anyone touched
her. Her body was now in so much pain it was
heart breaking to watch. The doctors then decided
to transfer Zara to the local children’s hospital.
We were transferred into an isolation bay as
Zara was obviously still infectious and no one had
a clue what was happening to her. The ring
around the “pox” on her abdomen was now get-
ting larger. Another concern was that redness had
now appeared at the “pox” on her sternum. The
redness that was around Zara’s neck appeared to
17
look like a necklace. As the day wore on Zara
screamed whenever anybody entered her room
because she knew that they were going to touch
her. Her screams were ear piercing because she
was in so much pain.
The redness started spreading to what can only
be described as a “capped sleeve cardigan”. As
the hours went by, redness has now appeared near
the “pox” on her abdomen, it now looked like she
was wearing a red “belt”. As time passed,
“capped sleeve cardigan” had now turned into a
red “bolero jacket” and the red “belt” was now a
red “pair of hot pants/boxer shorts”. During the
night it now looked as if she was wearing a long
sleeved red cardigan. The weirdest thing was that
whilst the redness was overtaking her body, she
had a “band” of normal skin around her midriff.
Her “boxer shorts” were now a pair of “jammers”,
the redness had now spread to her knees. Within
30 min the redness was now a red pair of tights.
Zara was now “vacant”, just starting at nothing.
You could have thrown her around the room and
she would not have cared, her eyes were glazed,
she was not even responding to me her Mummy.
In the early hours the hospital room was filled
with the Heads of Departments, Medical
Consultants, Immunology consultants, Plastic
Surgery Consultants, Anaesthetists and other
Doctors and Nurses. A decision was made that
Zara now needed emergency surgery. Over 4 h
later myself and my partner were finally told that
she was out of surgery, we made our way up to
Intensive Care were we were ushered into a side
room where a Surgeon introduced himself and
began to explain the he was head of Plastics and
the Burns Unit. He told us what had taken place in
surgery. He explained that our daughter had
contracted Necrotising Fasciitis, we had never
heard of it. He told us that it was a flesh eating
bug, he explained that Necrotising Fasciitis had
eaten her chest wall so they had to debride the
whole of her chest wall and that the next 24–48 h
were critical as the child mortality rate was 95%.
He also informed us that he had operated on the
“pox” that was on her abdomen as it had turned
into a large abscess, so he had cut the “pox” out
and had packed the wound. We were then led into
18
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
our little girl for the first time, she was uncon-
scious as she was intubated and looking like an
American Football player. She was heavily ban-
daged and sedated; it was such a shock.
Five days later, the surgeon needed to see if the
Necrotising Fasciitis had spread so operated on
Zara whilst being in her intensive Care room
where I observed him removing silver foil from
her chest as he had left the wound open. He
observed that it had not spread any further so
decided to close the wound. It looked so surreal.
He said he had only seen a handful of cases but
had never witnessed Necrotising Fasciitis in the
chest wall nor seen in at its earliest detection. Zara
was transferred to the Burns Unit after a week in
Intensive Care where she remained for 3 weeks.
Zara was eventually discharged home after
D. Cartledge et al.
1 month. We attended various clinics and Zara
also wore a pressure vest for a period of time.
Zara is now 12 years old and is in her first year
of high school. Her scar spans from shoulder to
shoulder and is the shape of a “Y”. We see the
consultant regularly who has been amazed at her
healing process. Zara’s scar has not blanched but
has stretched over time as she has grown. Her
Consultant has said that she can have corrective
surgery to neaten it if she wishes when she is fully
grown. He also thinks that she contracted it from
Anastazja having Scarlet Fever as both are caused
by the Strep A bacteria. He thinks that Zara must
have had the bacteria on her skin when she devel-
oped chickenpox and that it entered her body
when the “pox” erupted. He also thinks that the
nastiest “pox” on her sternum was the entry point
(Figs. 2.8, 2.9, 2.10, and 2.11).
2 Necrotizing Soft Tissue Infections: Case Reports from the Patients Prospective 19

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
20 D. Cartledge et al.

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

# Springer Nature Switzerland AG 2020 21

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_3
22
monomicrobial infections caused predominantly
by Streptococcus pyogenes (group A streptococ-
cus; GAS). GAS infection of non-extremity loca-
tion is associated with immunosuppression.
Extensive surgical debridement is essential for
survival. Anaerobic necrotizing soft tissue
infections may benefit from hyperbaric oxygen
therapy.
3.1 Introduction
Necrotizing soft tissue infections (NSTIs) are life-
threatening infections typically characterized by
rapid progression of disease and significant local
tissue destruction. However, the speed of devel-
opment and associated clinical features differ
markedly depending on the bacterial etiology
(Hakkarainen et al. 2014; Calandra et al. 2005;
Anaya and Dellinger 2007). The tissue destruc-
tion involves any of the layers of the soft tissue
compartment, extending from the epidermis to the
deep muscle fascia. The disease frequently
progresses below the outer surface, and in the
absence of overt skin changes the diagnosis can
be difficult to establish in the early phases. How-
ever, signs of systemic toxicity and pain often
develop that are disproportionate to the findings
of the skin examination (Calandra et al. 2005).
The diagnosis is surgical with the findings of
friability of the superficial fascia, dishwater-gray
exudate, and absence of pus (Stevens and Bryant
2017). Several descriptions and classifications of
NSTI have been proposed, including stratification
based on (1) anatomical localization (i.e.,
Fournier’s gangrene), (2) depth of infection (nec-
rotizing fasciitis, necrotizing myositis), (3) sever-
ity (Sequential Organ Failure Assessment Score
(SOFA), simplified acute physiology score II
(SAPS II), or (4) microbiological findings.
Classifications due to microbiological findings
and their clinical characteristics are delineated in
Chap. 5. Shortly summarized, type 1 infections
are polymicrobial, type 2 are monomicrobial,
type 3 is a monomicrobial infection with marine
pathogens including Vibrio vulnificus and
Aeromonas hydrophila, whereas in type 4 the
etiology is fungal (Stevens and Bryant 2017;
T. Nedrebø and S. Skrede
Sartelli et al. 2018; Morgan 2010). Type 3 NSTI
clinically presents more like type 2 NSTI
(Hakkarainen et al. 2014), and the use of unique
clinical types 3 and 4 is yet to be justified. How-
ever, differentiating type 1 and 2 would serve
practical purposes, in which tailored diagnostics
and therapy may be warranted.
Type 1 polymicrobial infections most often
affect older patients with comorbidities like dia-
betes, and the predominant anatomical
localizations are perineum and the abdomen.
Type 2 monomicrobial infections are predomi-
nantly caused by S. pyogenes (group A strepto-
coccus; GAS) (Bruun et al. 2020).
The patients presented in the following three
case reports were all included in the INFECT
study, an international, multicenter, prospective
cohort study of adult patients (Madsen et al.
2019). Four hundred and nine patients
undergoing at least one operation with confirmed
NSTI were included, of which 402 were admitted
to an Intensive Care Unit (ICU). The most appar-
ent initial symptoms and signs were skin bruising
(202 patients, 51%) and opioid-requiring pain
(172 patients, 41%) and systemic toxicity was
frequent; 50% had septic shock and 20% acute
kidney injury (Madsen et al. 2019).
Microbiological findings in these patients
revealed polymicrobial infection in 50% (202)
and monomicrobial infection in 44%, of which
GAS was found in 126 cases (70%) among these
patients. In 26 of the patients (6%), no microbes
were identified.
The median number of surgical interventions
was 4, and 13% had amputation of either an
extremity or penis. Most patients received beta-
lactam (96%) and clindamycin (98%) as antibi-
otic treatment.
3.2 Cases
3.2.1 Case 1—Polymicrobial
Infection/Anaerobic Infection
Originating in the Pelvic Region
This patient was a 48-year-old man with a
30 years lasting history of substance abuse,
mostly heroin administered intravenously by
3 Necrotizing Soft Tissue Infections: Case Reports, from the Clinician’s Perspectives
way of the femoral veins. He had HCV RNA PCR
positive chronic hepatitis C genotype 3a and had
previously been hospitalized several times due to
opioid overdosing. Moreover, he had prior hospi-
tal admissions for a traumatic fracture of the left
femur and for surgical treatment of the right
ankle. Known medication at presentation was
alprazolam and oxazepam.
Six days prior to admission and during a
period of drug-abuse, he fell on his left knee
resulting in a blunt minor trauma. In the following
days he developed local swelling, pain, and red-
ness of the knee. At admission he was dehydrated
and opioid intoxicated, with a blood pressure
(BP) of 91/57 mmHg, a pulse rate of 96/min,
and a body temperature of 36,3
C. Clinical bio-
chemistry and hematologic tests revealed a
C-reactive protein (CRP) of 339 mg/L, white
blood cell count (WBC) of 26,2  109/L, creati-
nine of 233 μM, and a Laboratory Risk Indicator
for Necrotizing Fasciitis (LRINEC) score of
8, according to Wong et al. (2004). The medial
side of the left knee was warm, had a swelling and
erythema (Fig. 3.1), and joint flexion of the knee
was impaired due to pain. Septic arthritis was
suspected, and antibiotic therapy was initiated
with a combination of intravenous ceftriaxone
and clindamycin.
Ultrasound showed fluid in the knee joint and
between the superficial muscle layers in the lower
parts of the thigh. X-ray revealed gas along the
entire femoral shaft all the way from patella to
trochanter major, and the patient was suspected to
have infection with gas-producing microbes
(Fig. 3.2). Under suspicion of septic arthritis and
Fig. 3.1 Swelling and erythema of the knee at admission
23
possibly NSTI, he was taken to surgery. First, the
plastic surgeons made incisions on the medial and
lateral part of the thigh from patella in proximal
direction. The incisions were extended until they
reached the level of the deep fascia, but only
edema was present. The orthopedic surgeons
first did an arthroscopy of the knee, draining
purulent fluid (Fig. 3.3), samples of which were
collected for microbiologic diagnostics. Subse-
quently, they made a midline incision over the
prepatellar bursa. Gas had been seen along femur
on the X-ray, and when they opened the deep
fascia lots of necrotic tissue was detected beneath.
The incision was prolonged to previous incisions
made by the plastic surgeons, revealing direct
communication between the prepatellar bursa
and the intermediary vastus muscle. Thereafter
the vastus lateralis muscle was split to give access
to deeper structures. Pockets of pus were found
all along the fascia up to the anterior inferior iliac
spine, and parts of the quadriceps-muscle had to
be removed due to necrosis (Fig. 3.4). The wound
was covered with surgical swabs and left open
pending a revision the following day, in accor-
dance with contemporary recommendations
(Rogers 2020). On the first revision, necrotic
parts of the vastus intermedius muscle had to be
removed, but there was no more evident pus.
Microbiologic cultures from the wound taken
during surgery demonstrated Prevotella disiens,
Fusobacterium gonidiaformans, Actinomyces sp.,
and Streptococcus dysgalactiae (SD), a beta-
hemolytic streptococcus often found in
co-culture with intestinal flora. In blood culture,
only SD was found. Samples from the fluid in the
24
Fig. 3.2 Gas seen in the soft tissue along femur
knee joint were culture negative. At first, he was
treated with ceftriaxone and clindamycin, but
later this was changed to a combination of
benzylpenicillin and clindamycin following the
susceptibility tests, where the microbes were
Fig. 3.3 Purulent fluid during knee-arthroscopy
T. Nedrebø and S. Skrede
found to be sensitive to penicillin and/or
clindamycin.
There were serial wound revisions, before the
wounds were surgically closed, starting at day
4 and finished by day 6. Two days after closing
3 Necrotizing Soft Tissue Infections: Case Reports, from the Clinician’s Perspectives
Fig. 3.4 Necrotic muscle
(parts of quadriceps)
of the wound he developed more pain, and a rise
in CRP and WBC occurred. The sutures were
removed, and lots of pus were found and the
wound was washed with saline. From day 0 he
received 4 daily treatments with hyperbaric oxy-
gen therapy (HBOT), 90 min of 2.4 ATA (14/90),
as he was suspected to have a myositis due to
anaerobic bacteria, which was confirmed during
the initial phase of his hospital stay. He had
several more revisions before the wound was
closed, partly with a skin autograft.
Discussion Polymicrobial NSTI is referred to as
Type 1 NSTI, and often associated with gas in
the tissue (Stevens and Bryant 2017), although
gas is also a possible clinical presentation in
beta-hemolytic streptococcal (BHS) cases. In
the INFECT study, 5 of 114 mono-GAS NSTI
had palpable gas (crepitus) in the tissue and
10 of 114 had gas visualized on radiology pre-
operatively (Trond Bruun, personal communica-
tion). In type 1, strictly anaerobic microbes
predominate (Madsen et al. 2019; Thanert et al.
2019), often accompanied by facultative anaero-
bic gram-negative bacilli and gram-positive
cocci. A typical patient with NSTI type 1 is
likely to have comorbidities like diabetes
mellitus, peripheral vascular disease, or intrave-
nous drug use (IVDU) (Sarani et al. 2009; Chen
et al. 2001), thereby in a state of qualitative or
quantitative immunosuppression. This particular
patient had a minor trauma to his knee 1 week
prior to admission. Aspiration of pus from the
25
knee joint during arthroscopy supported a clini-
cal suspicion of septic arthritis. NSTI was
actively sought for, but at initial surgery there
were normal findings down to the fascia both
laterally and medially on the extremity. How-
ever, the X-ray had shown gas along the femur,
so the idea of a deeply situated primary NSTI
caused by IVDU inoculation, from the femoral
vein downwards along the femoral shaft to the
knee structures, was not abandoned. Notwith-
standing, the local trauma of his knee may
have created an environment for growth and
subsequent spread of the bacteria. Mechanistic
studies addressing the pathogenic strategies and
complex dynamics of bacterial communities in
polymicrobial NSTIs are lacking (Thanert et al.
2019). However, many of the bacteria found in
polymicrobial NSTI are the same as in
monomicrobial NSTI (e.g., GAS), and thereby
elaborate many of the same exotoxins and
tissue-degrading enzymes (Shiroff et al. 2014;
Snall et al. 2016). SD is an emerging cause of
invasive disease in general (Oppegaard et al.
2016) and specifically an increasing etiology of
NSTI in our patient population, and it may cause
monobacterial type 2 NSTI (Bruun et al. 2013)
or equally frequent, it is identified as part of
polymicrobial etiology of NSTI (Bruun et al.
2020). In the INFECT study, blood cultures
were more commonly positive in NSTI caused
by GAS (56%) than in SD (30%). IVDU is a
risk factor also for SD bacteremia, as shown
elsewhere (Ruppen et al. 2017).
26
In our hospital we have noted an emerging
association of severe SD infections and genotype
stG62647, which has predominated in invasive
SD infection in our population since 2013
(Oppegaard et al. 2017). This sequence type was
also dominating in SD NSTI in the Scandinavian
patient cohort of the INFECT study.
In this patient, SD was identified along with a
number of obligate anaerobic species that are
rarely culture positive in blood culture compared
to rates of identification in deep tissue cultures
(Brook and Frazier 1995), essentially confirmed
by the INFECT consortium (Madsen et al. 2019)
and even more so, in a study using nucleic acid
based detection of microbes in tissue (Thanert
et al. 2019). These results are in support of the
historic opinion that the importance of obligate
anaerobic species may be underestimated in NSTI
(Brook and Frazier 1995).
In this patient case, time from presentation at
hospital to surgery was 18 h, mostly explained by
the patient’s non-compliance and refusal to
receive help, delaying the time to surgery. This
puts the patient at risk of poor outcome. Since the
upsurge of NSTI in the mid 1980s, it was soon
evident that there was an association of time to
surgery and risk for poor outcome (Boyer et al.
2009; Elliott et al. 1996; Kobayashi et al. 2011;
McHenry et al. 1995; Wong et al. 2003). Two
recent meta-analyses reported a significantly
lower mortality if the initial surgery was
performed within 12 h of presentation (Gelbard
et al. 2018; Nawijn et al. 2020). Gas in the tissue
is one of the clinical signs more often associated
with polymicrobial NSTI (Stevens and Bryant
2017). In this patient gas had been seen on the
initial X-ray and although no evident sign of
deeply situated NSTI was detected in the early
phases of surgery, this radiologic finding spurred
the surgeons to continue, leading them to explore
muscle and other soft tissue all the way into the
femoral shaft, a strategy leading to drainage of
deeply situated necrotic tissue and pus, most
likely improving the outcomes for the patient
substantially. That a septic arthritis caused by
SD appears secondary to a NSTI of an extremity
is an observation made on several previous
occasions in our hospital.
T. Nedrebø and S. Skrede
Undertreatment with antibiotics in early
phases of obscure NSTI is common (Marwick
et al. 2012). Our patient received empirical ther-
apy with broad (1) gram-negative and (2) gram-
positive coverage that included effect on
(3) Staphylococcus aureus that in our community
is methicillin susceptible in >99% of invasive
cases, (4) anaerobic species other than
Bacteroides (that frequently expresses
clindamycin resistance), and on (5) toxin produc-
ing species that might be involved in a NSTI that
most likely was of polymicrobial etiology
(Peetermans et al. 2020). When results on suscep-
tibility were available, penicillin sensitivity in SD
was confirmed, whereas all anaerobic species
were either susceptible to both clindamycin and
benzylpenicillin or one of these agents. In line
with treatment guidelines (Stevens et al. 2014),
therapy was narrowed accordingly. Temporary
worsening seen in our patient was evaluated to
be caused by too early closure of wounds.
In NSTI the role of some of the therapeutic
measures is controversial. Firstly, in our hospital
HBOT is offered sparingly to patients with severe
NSTI, organ failure, and evidence of obligate
anaerobic microbial etiology. According to local
guidelines for treatment of NSTI in our hospital,
this patient did receive HBOT, as 1/3 of our
patients in the INFECT cohort did (unpublished
result). Furthermore, this patient was not offered
intravenous polyspecific human immunoglobulin
G (IVIG), which has been restrictedly used in our
hospital. Until the INFECT study, orders for use
were evidence of invasive GAS infection, shock,
and unstable patient as described by Anaya and
Dellinger (Anaya and Dellinger 2007). Only two
patients in our local INFECT cohort fulfilled
these criteria (unpublished data). However, there
is increasing evidence that patients with
monobacillary GAS NSTI or septic shock benefit
on IVIG treatment with improved outcome (Parks
et al. 2018), recently supported also by data for
the streptococcal cohort of the INFECT study
(Bergsten et al. 2020; Bruun et al. 2020). In this
case, SD and obligate anaerobic microbes were
identified. There is to date only one single
completed randomized, blinded, placebo-
controlled trial of immunoglobulin G treatment
3 Necrotizing Soft Tissue Infections: Case Reports, from the Clinician’s Perspectives
for patients with NSTI (Madsen et al. 2017). This
study failed to show any effect on primary and
secondary outcomes. Most patients included had
polymicrobial infection. In an analysis of associ-
ation between outcome and anatomic site of
infection, there was a tendency in favor of
placebo in infections of the anogenital and
abdominal areas (Madsen et al. 2017), in which
obligate anaerobes are frequently involved,
different to BHS, that are rarely encountered
(Madsen et al. 2019). This would support that
IVIG may have little effect in the polymicrobial,
non-BHS infections. A case report on beneficial
use of IVIG in NSTI caused by SD has been
presented, the effect attributed to blocking of
streptokinase-mediated fibrinolysis, with
subsequent reduced bacterial spread (Andreoni
et al. 2018).
The first case presented herein is a type
1 NSTI, a patient with a polymicrobial infection
caused by co-occurrence of obligate anaerobic
microbes and SD, a beta-hemolytic streptococcus.
The disease course developed slowly, but there
was a need for very resource demanding handling
including HBOT. This case illustrates host risk
factors (IVDU), as well as major clinical learning
point; the anticipated primary focus of infection
was an intra-articular knee joint infection as it was
here infection first became visible. Different to
this, the primary disease was judged to be
advanced NSTI originating in the groin,
descending along the femoral shaft.
Fig. 3.5 Area of marked swelling and erythema at first admission
27
3.2.2 Case 2—GAS of the Upper
Extremity
This patient was a 48-year-old male with a history
of psoriatic disease, but he was independent of
medication. One week prior to hospitalization the
patient got a minor laceration, as he cut a finger of
the left hand on a paper edge. He treated the finger
with a topical antibiotic due to a possible infec-
tion, and the finger was fine for the next few days.
However, while cross-country skiing his left
elbow rapidly became erythematous and painful
within a few hours, and his temperature rose to
39.1
C. He was sent to the local community
hospital on suspicion of severe skin and soft
tissue infection. On admission his left elbow
was painful, and there was an area of localized,
well defined erythema, and swelling (Fig. 3.5).
There were no signs of injury or breach of skin
barriers. Vital signs included a BP of
134/82 mmHg, pulse 91, temperature 39
C,
respiratory rate (RR) 12. CRP was <5 mg/L,
WBC 9.7  109/L. LRINEC score was 0 and
blood cultures were collected. Initial diagnosis
was bursitis with possible cellulitis. Five hours
later the patient felt worse as the erythema was
spreading. Vital signs were recorded with BP
112/57, a clinical chemistry showed CRP
72 mg/L, WBC 14.4  109/L, and blood lactate
was elevated to 3.6 mM. Treatment with intrave-
nous cloxacillin was initiated, but his condition
deteriorated. NSTI was suspected and he was
transferred to our tertiary care hospital.
28
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
had decreased to 86/60, and treatment with vaso-
pressor to maintain MAP >65 mm Hg had been
initiated. The erythema and swelling had spread
in distal direction from the MCP joints to the
middle upper arm (Fig. 3.6). The swelling over
the olecranon bursa was punctured, and fluid was
aspirated and sent to microscopy and culture and
gram-positive microbes with streptococcal mor-
phology were detected. Cloxacillin was
discontinued, and treatment was changed to
clindamycin, benzylpenicillin, and gentamycin.
He went to immediate surgery under general
anesthesia. In the surgical theatre a large dorsal
incision from olecranon to the lower forearm was
made. Extensive amounts of dishwater-like fluid
was found together with necrotic subcutaneous
tissue. An area of 7  20 cm of the skin over
the elbow, equaling approximately 1.5% of skin
surface, had to be removed due to necrosis
(Fig. 3.7). The muscle fascia and the muscle
tissue looked vital with no necrosis. The wound
was left open, covered with surgical swabs. Sub-
sequently, he was postoperatively transferred to
the intensive care unit (ICU), where SAPS III
score 52, and SOFA score 8 were noted at
admission.
The next morning a second look was
performed. The muscle fascia on the lower fore-
arm was cut open, and the muscle tissue below
appeared to be vital. Then the surgeons explored
proximally towards the elbow. Still the fascia
looked and felt vital, but extensive amounts of
pus-like fluid emanated from deeper muscle-
compartments. Immediately after surgery the
patient’s condition got worse with septic shock
and respiratory failure. On examination the
T. Nedrebø and S. Skrede
margins of the infection with redness and edema
had spread up to the shoulder. Initial bacteriologi-
cal tissue and fluid cultures grew GAS, whereas
blood cultures from the first admission were cul-
ture negative. A third revision uncovered some
purulent fluid just below the shoulder, but the
fascia and muscle tissue were vital with no signs
of progression (Fig. 3.8). His condition improved,
and 2 days later he was extubated and in no need
of circulatory support.
The patient went through several further
revisions, but no more necrotic tissue or signs of
infection were found. On day 4 they started to
close the wound, but he had a skin defect which
had to be transplanted at a later stage. On day 5 he
was discharged from ICU, with a SOFA score 5.
Discussion
Case 2 is a representative patient of type 2 NSTI:
GAS infection in a previously healthy young
individual, affecting an extremity (Madsen et al.
2019). GAS is a gram-positive coccus capable of
causing a broad array of infections, most com-
monly pharyngitis and non-necrotizing soft tissue
infections (Stevens 2020). Invasive GAS
infections include NSTI, which has a significant
potential for local spread and aggressive clinical
course, as experienced by this young individual.
This patient started out with swelling and
edema of his elbow, which only 12 h later spread
distally to his MCP joint and proximally up to his
shoulder. One week prior he had a small cut in his
finger. Most likely the cut in his finger caused an
entry of the bacteria, that may have been dormant
or in a very isolated, minute focus of infection not
giving rise to any attention. This was followed by
a NSTI development, first in the elbow due to a
3 Necrotizing Soft Tissue Infections: Case Reports, from the Clinician’s Perspectives 29

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-
country skiing. NSTI has historically been
associated with penetrating trauma, but recently
blunt trauma has also been verified as an indepen-
dent risk factor of GAS NSTI development
(Bruun et al. 2020).
In retrospective studies of NSTIs, upper
extremity infections are perceived as uncommon
and lethal, as reviewed elsewhere (Uehara et al.
2014). Differently, we demonstrate that upper
extremity is as common focus as head-neck
NSTI, and even half that of anogenital-abdominal

Fig. 3.8 Surgery after second revision had to go below the shoulder
30
or lower extremity focus of NSTI, and they are
not more lethal (Madsen et al. 2019). In extremity
NSTI GAS predominates vastly (Bruun et al.
2020; Madsen et al. 2019). In this case blood
cultures from the first admission were culture
negative, as seen in 44% of GAS cases (Bruun
et al. 2020), underlining the importance of
collecting bacteriological tissue and fluid for rou-
tine culture. In the entire INFECT cohort 93% of
the cases were tissue culture positive (Madsen
et al. 2019).
Our patient went to surgery 24 h after presen-
tation of symptoms, 18 h after admission to hos-
pital, and by then he was clinically affected with
hypotension in need of vasopressor. This is some-
what later than the average GAS patient in our
cohort. We showed that GAS cases receive sur-
gery after a median of 16 h compared to non-GAS
cases, whom received it 2 h later (Bruun et al.
2020). Invasive GAS infection can cause toxic
shock syndrome (TSS), in our study reported in
65% (Bruun et al. 2020). Clinical criteria for TSS
include hypotension (systolic blood pressure
>90 mmHg) and multi-organ involvement
(Breiman et al. 1993). This patient met the clini-
cal criteria of TSS, and the diagnosis was con-
firmed with isolation of GAS from tissue biopsies
and bursa fluid. This massive invasion and degra-
dation of the soft tissue caused by GAS are due to
both the bacteria’s production of exotoxins and
the features of M-proteins, the latter being cell
surface molecules on the bacteria. They both
result in a massive pro-inflammatory cytokine
release and thereby causing septic shock. A
study has also shown that patients with GAS
NSTI exhibited a lack of specific antibodies
directed against the causative S. pyogenes strains
and the majority of their exotoxins during initial
stages of infection (Babbar et al. 2018; Norrby-
Teglund et al. 1994; Basma et al. 1999),
antibodies meant to prevent the spread of
infection.
Several in vitro and in vivo studies have
indicated that the protein synthesis inhibitor
clindamycin abates the toxin production in
GAS, and improves survival rates in NSTIs and
streptococcal TSS (Andreoni et al. 2017;
T. Nedrebø and S. Skrede
Carapetis et al. 2014; Linner et al. 2014; Mascini
et al. 2001). Upon confirmation of streptococcal
etiology our patient received a combination of
penicillin and clindamycin, as advocated in con-
temporary guidelines for the treatment of GAS
NSTIs (Stevens and Bryant 2017). However, he
initially received inadequate antibiotic therapy, as
evaluated retrospectively. In a prospectively
enrolled cohort of skin and soft tissue infection
cases, Marwick et al. reported inadequate empiric
therapy in 10/11 (91%) cases with NSTI,
highlighting the diagnostic challenges of
distinguishing NSTIs from cellulitis in the early
phase (Marwick et al. 2012). The LRINEC score
has been proposed as a clinical tool for
identifying patients in need of surgical treatment.
However, at the primary admission our patient
had a LRINEC score of 0/13, demonstrating the
shortcomings of this scoring system, reviewed
elsewhere (Fernando et al. 2019). In our hospital
its use has been discouraged following our studies
(Bruun et al. 2013; Bruun et al. 2020; Madsen
et al. 2019). The diagnosis of NSTI is established
by surgery, but frequently diagnosis is delayed
(Goh et al. 2014). In general, it may therefore be
advisable to encourage surgical exploration ear-
lier when facing suspected NSTI patients, which
means a certain ratio of negative explorative
procedures must be accepted. It is our experience
that we explore suspected NSTI in the range of
1,5:1 vs. confirmed non-NSTI (unpublished
results), resembling published results of 1,2:1
that has been presented by others (Howell et al.
2019).
Extensive surgery is main therapy in NSTI. In
this case the fascia looked vital, but below the
fascia extensive amounts of necrotic tissue were
detected and removed. Intraoperative findings of
muscle tissue involvement showed that GAS
spread across tissue borders, and not only along
the tissue planes. Surgically it is important to
consider exploring below the fascia, even in
cases where they appear to be vital. The extent
of the excisions should be down to healthy bleed-
ing tissue at all margins. Often the spread in the
subcutaneous layer is more extensive than the
skin changes suggest. This was the case here
3 Necrotizing Soft Tissue Infections: Case Reports, from the Clinician’s Perspectives
too, as the patient only few hours after second
surgery developed TSS and had to undergo a
second excision, in proximal direction to reach
just below the shoulder, confirming challenges in
infectious source control of this case. This case
demonstrates the severe systemic manifestations
including streptococcal TSS in a previously
healthy man, the significance of blunt versus
penetrating trauma, initial antibiotic undertreatment,
and impact of too early surgical closure.
3.2.3 Case 3—GAS
in an Immunocompromised
Patient
A 59-year-old woman was admitted to her local
hospital after 2 days of increasing pain in her right
buttocks and groin. In addition, foul-smelling
dark green vaginal discharge was noted. She pre-
viously had one cesarean section and more
recently, surgery for urinary incontinence
(tension-free vaginal tape (TVT)) a few years
before enrollment in the INFECT study. Due to
an active psoriatic arthritis, she received regular
treatment with methotrexate per oral 10 mg once
weekly and the tumor necrosis factor-alpha inhib-
itor, etanercept subcutaneously 50 mg once
weekly. On admission to the hospital BP was
Fig. 3.9 Swelling and discolored area of the thigh 15 h after first admission
31
115/51 mmHg, pulse rate 107, RR 20, WBC
4.6  109/L, and temperature 38,2
C. CRP was
275 mg/L, while a LRINEC score was calculated
retrospectively showing a low score of 4/13.
Upon examination, she had no erythema or
signs of infection on her buttocks or thigh. A
gynecologic examination did not reveal any sign
of infection, but a vaginal ultrasound gave suspi-
cion of retroperitoneal edema against the pelvic
floor.
A CT scan showed either solid fluid or a
phlegmon along the pelvic muscles from upper
part of the right thigh to 5 cm above the pubic
tubercle. Edema was present in gluteal, pelvic,
and adductor muscles.
A pigtail catheter was inserted prevesically, in
an attempt to drain some of the fluid. Culture from
blood, and this fluid later grew GAS. Subse-
quently her condition deteriorated only a few
hours later, and soon she was in need of
vasopressors (norepinephrine and dobutamine).
She was rapidly sent by helicopter to the regional
hospital where she arrived 15 h after first admit-
tance to hospital. At arrival she was awake, self-
breathing, BP 89/35 mmHg (on vasopressor),
pulse rate 127, and temperature 39,5
C. On
examination, a swelling and discolored area on
the back of her thigh was found (Fig. 3.9) and a
CRP value of 225 mg/L was noted.
32
Fig. 3.10 Fasciotomy of
the medial side of the thigh
at first surgery
She went immediately to surgery under gen-
eral anesthesia. An explorative laparotomy did
not reveal any intra-abdominal pathology, except
from localized retroperitoneal edema in the ingui-
nal opening on the right side. Then a fasciotomy
both laterally and medially on the thigh was
performed (Fig. 3.10). There was edema of the
subcutaneous fat and dishwater-like fluid under
the fascia (Fig. 3.11). Both the fascia and the
muscle were found viable.
The antibiotic regimen was changed to
meropenem and clindamycin, and she was trans-
ferred to the ICU. Her condition worsened the
next few hours, and a swelling close to the right
labium majus appeared. She was once again taken
to immediate surgery. An incision was made
between the labium minus and labium majus on
the right side, to uncover devitalized subcutane-
ous tissue. There was no bleeding, due to wide-
spread thrombosis of minor vessels. A digital
dissection down to the fascia of the inferior part
of urogenital diaphragm was made. Necrotic sub-
cutaneous tissue was removed, but the fasciae
were vital. The same procedure was done on the
T. Nedrebø and S. Skrede
left side, and an external vulvectomy was
indicated. The wound was left open with wet
surgical swabs.
A second look at the fasciotomy of the right
thigh revealed some necrotic skin and a small
necrotic area of adductor magnus muscle. During
the surgical procedure the patient received IVIG
according to the contemporary treatment proto-
col, as there was evidence of GAS etiology and
she was circulatory unstable and in a critical state.
Upon return to the ICU after the second sur-
gery, her condition was judged critical and unsta-
ble, supported by maximal SOFA score of
17, SAPS II score of 66, and SAPS III score of
86. Arterial blood gas lactate exceeded 8 mM,
two vasopressors (norepinephrine and epineph-
rine) were administered as a Cardiac Index of
<4 L/min was detected. High-flow (300 mL/
min) continuous veno-venous hemofiltration
(CVVH) was started. However, following the
radical surgery, improvement was noted, and
after a few hours only epinephrine infusion
could be discontinued. She stabilized, and at a
new surgical revision the same day only minor
3 Necrotizing Soft Tissue Infections: Case Reports, from the Clinician’s Perspectives 33

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

Fig. 3.12 Picture taken of medial fasciotomy after second revision

34
58% of the non-IC (Butterworth and Murphy
2006). The newly developed SIARI score (Site
other than the lower limb, Immunosuppression,
Age <60 years, Renal impairment (s-creatinine
141) and Inflammatory markers (CRP  150,
WBC 25)) take into account
immunosuppressed patients, and is a better tool
in diagnosing NSTI in these patients (Cribb et al.
2019). Validation of this score is needed, how-
ever. In the INFECT-study population, 126 of
406 NSTI cases (31%) had GAS NSTI, only
12 of these having co-pathogens identified.
NSTI involving the extremities were also pre-
dominant, found in over 80% of streptococcal
NSTI (Bruun et al. 2020). In a retrospective
cohort study, patients with immunodeficiency or
an abdominal infection, had low probability of
GAS infection (Urbina et al. 2019). This case
presents an immunocompromised patient with
monomicrobial GAS infection of the perineum
and secondary to this, to the lower extremity.
Time from onset of symptoms to presentation at
hospital was 2 days. Time from presentation at
hospital to first surgery was 18 h as she had to be
transported to our referral hospital. A delay of
first surgery beyond 12 h of presentation has
been shown to increase mortality (Nawijn et al.
2020; Gelbard et al. 2018). At first surgery, exci-
sion of perineum was not performed, and
fasciotomy of upper lower extremity revealed
some dishwater-like fluid and edema. The first
CT scan taken at admission in the local hospital
showed a possible phlegmone of the pelvic and
gluteal muscle all the way up to the pubic tuber-
cle, so a more extensive excision at first surgery
probably had revealed affection of the perineum
at an earlier stage. The goal of the initial surgery
is to gain control and prevent further spread of the
infection (Sartelli et al. 2018; Stevens and Bryant
2017). Shortly after the initial surgery, she devel-
oped TSS and the NSTI presented on the labium
majus as a swelling. The inflammatory response
had caused massive thrombosis and thereby
necrosis of the soft tissue. At second surgery, a
more extensive debridement with bilateral
vulvectomy was performed. Her clinical condi-
tion was critical, with septic shock, but the exten-
sive surgery stopped the spreading, and together
T. Nedrebø and S. Skrede
with antibiotics and IVIG her condition improved
substantially within few hours and then stabilized.
This third case is a monomicrobial GAS infec-
tion localized to the perineum, which was seen in
only 6/126 (5%) of GAS cases (Bruun et al. 2020;
Madsen et al. 2019). Through the INFECT study
we have learned that this clinical setting, with
monomicrobial NSTI at a very uncommon infec-
tion focus site, is attributable to immunosuppres-
sion. Moreover, polyvalent human
immunoglobulin G (IVIG) has previously been
restrictedly used in our hospital, Haukeland Uni-
versity Hospital in western Norway. The INFECT
study has generated support to IVIG treatment in
GAS cases, as in multivariate analysis any dose of
IVIG was associated with survival, as was the
outcome in this case (Bruun et al. 2020).
3.3 Future Perspectives
NSTIs are rare infections and most often case
reports and small retrospective cohorts are
presented. An insufficient progress has been
noted in patient handling, as few steps have
been taken to narrow the gap between basic
pathophysiological knowledge and clinical rou-
tine handling. The INFECT project is to our
knowledge one of the largest prospective studies
of patients with NSTIs, offering with more rigor
than ever before, associations between clinical
presentation, microbial etiologies and anatomical
location of NSTIs, with data suited to aid clinical
decision-making. Examples of use of data
generated in the INFECT study have been given
herein. Through prospective inclusion, updated
clinical categorization, and studies on host–
microbe interactions with translational studies of
pathogenesis, the INFECT study has been paving
the road towards development of personalized
medicine in NSTIs.
Acknowledgement The INFECT study was supported
by the European Union’s Framework Program 7 (grant
number FP7/2007 – 2013 305340. The authors thank
Oddvar Oppegaard, Eivind Rath, and Trond Bruun for
their contribution and review of the manuscript.
3 Necrotizing Soft Tissue Infections: Case Reports, from the Clinician’s Perspectives 35

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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

# Springer Nature Switzerland AG 2020 39

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_4
40
Keywords
Necrotizing soft-tissue infections · Necrotizing
fasciitis · Fournier’s gangrene · Clinical
presentation · Diagnostic aspects ·
Microbiology
Highlights
• NSTI is a heterogenous infectious disease that
can involve any part of the body, and can
present in various ways.
• No set of diagnostic criteria exists; the diagno-
sis is based on clinical presentation and surgi-
cal intervention.
• Common symptoms include bruising of the
skin and severe pain.
• Bacterial etiology differs according to the
affected body part; infections of the upper
and lower extremities are dominated by
monomicrobial group A streptococci, and
infections of the abdomen and anogenital
area by polymicrobial infections.
4.1 Introduction
Necrotizing soft-tissue infection (NSTI) is a fast
progressing infection that may affect any layer of
the soft tissue, and results in severe morbidity,
mortality, and affected quality of life
(Hakkarainen et al. 2014). It is not a common
disease, with incidence rates ranging from 1.8
per 100,000 inhabitants per year in Denmark
(Hedetoft et al., unpublished) to 15.5 in
Thailand (Khamnuan et al. 2015a). The nomen-
clature varies; the terms gas gangrene, phagedena
gangrenosa, and hospital gangrene were used in
the early 1800s (Loudon 1994), and in 1952, the
term necrotizing fasciitis was introduced by
Wilson (Wilson 1952)—the term still used in
the WHO international classification of diseases
(WHO 2017). The term NSTI encompasses the
different variations of the infection (Stevens and
Bryant 2017). A description of a condition similar
to NSTI was described already by Hippocrates
(Jones 1923), but the first description is com-
monly attributed to the surgeon Ben Wilson,
who suggested early recognition, prompt surgery,
M. B. Madsen et al.
penicillin, and support for abnormal physiology
(Wilson 1952).
In this chapter we will review the clinical
presentation of NSTI and different aspects regard-
ing diagnosis.
4.2 Clinical Presentation
NSTIs can affect any part of the body, and as a
consequence, the presentation of the disease
varies considerably. Many patients with NSTI
have experienced a breach of the skin, e.g. by
pre-existing ulcers, trauma, or surgery (McHenry
et al. 1995; Wong et al. 2003; Madsen et al.
2019). But, as documented in early descriptions,
NSTI can develop without previous penetrating
trauma (Wilson 1952; Meleney 1924), and many
patients present without any known preceding
event (Wong et al. 2003; Hua et al. 2015). There-
fore, pain of the infected area can be the only
initial symptom. The triad of swelling, erythema,
and pain may be present—the cardinal symptoms
of inflammation, thus not distinguishing NSTI
from other infections. Notably, in NSTI without
obvious portal of entry, cutaneous manifestations
are a late sign. Severe skin manifestations with
purple or black discoloration are seen in 32% of
patients, and skin bullae in 27% (Figs. 4.1 and
4.2) (Madsen et al. 2019).
Systemic signs can include fever, tachycardia,
hypotension, and altered mental state—all gen-
eral symptoms of infection, and the progression
of infection (sepsis) (Table 4.1). The initial
symptoms can be even more vague as “influ-
enza-like” symptoms such as nausea, vomiting,
and diarrhea (Bisno et al. 2000). Pain requiring
opioids is seen in 42% of patients, and although
hard to quantitate, severe pain, or pain out of
proportion with the immediate appearance should
raise a high index of suspicion of NSTI, in
patients with infection (Wong et al. 2003; Madsen
et al. 2019; Borschitz et al. 2015). However, pain
may be absent due to use of analgesics or
neuropathy.
Most patients are tachycardic and have hypo-
tension, and in a Scandinavian observational
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,
20% had acute kidney injury, and some
multiorgan impairment. This is not, in itself,
prognostic of NSTI but is a marker of disease
severity.
As no symptom or sign is pathognomonic for
NSTI, and as these vary considerably from patient
to patient, it is important not to exclude the diag-
nosis in absence of specific symptoms (Stevens
and Bryant 2017).
The time from suspicion of NSTI to surgery
has previously been associated with an increased
risk of death in unadjusted analyses of retrospec-
tive data (McHenry et al. 1995; Wong et al. 2003;
42 M. B. Madsen et al.

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
4 Necrotizing Soft-Tissue Infections: Clinical Features and Diagnostic Aspects
surgery is an important modifiable factor that can
affect patient outcome.
Suspicion of NSTI is often measured from a
time when the patient is already in the hospital, as
this is when symptoms are described. However, a
recent publication has shown that patients experi-
ence a broad variety of symptoms while at home,
even up to several days before they were admitted
to hospital and eventually diagnosed with NSTI
(Erichsen Andersson et al. 2018). These anteced-
ent symptoms were categorized into three groups,
depending on the duration of these; more than
7 days, 3–7 days, and 1–2 days. Some of the
symptoms were often not easily describable. Ini-
tial symptoms included “flu-like symptoms” and
by some depicted as: “My muscles were sore like
after exercise.” Symptoms that progressed
included malaise, shivering, fever, nausea, and
pain—including escalation of pain—and
described as “something was seriously wrong”
and “losing control.” The last stage of symptoms
included anxiety, confusion, dizziness, and
fainting. Patients and/or their family reported
signs as high fever, local swelling, red/blue skin,
vomiting or diarrhea, boil/abscess, chills or fever,
rapid heart rate, and local heat. These symptoms
and signs are important to consider as the failure
to recognize these may delay further diagnostics
and correct diagnosis.
4.2.1 Pre-Existing Conditions
In many studies, a variety of pre-existing
conditions are described, including adipositas,
diabetes, cardiovascular disease, chronic kidney
and liver disease, and other immunocom-
promising diseases (Boyer et al. 2009; Sudarsky
et al. 1987). It is reasonable to assume that the
presence of some comorbidities increases the risk
of NSTI, but due to the low incidence and high
variety of comorbidities, any association will be
difficult to show. In a systematic review including
nine studies with 1493 patients, diabetes was
present in 45% (range: 15%–71%) of patients,
and diabetes seems to be overrepresented in
patients with NSTI (Goh et al. 2014). Patients
with diabetes have a higher risk of developing
43
infections (Carey et al. 2018), but whether diabe-
tes is an independent risk factor for developing
NSTI is not clear. Patients with diabetes also have
a higher risk of organ-failure and death from
sepsis (Beck et al. 2016; Esper et al. 2009), but
is not clear whether the same is applicable to
NSTI patients. In a prospective cohort study, dia-
betes (type I and II) was included as one of
11 predefined variables in an associative analysis
for risk of 90-day mortality. Of the 409 patients,
24% had diabetes at the time of diagnosis. The
odds ratio for patients with diabetes was 1.23
(95% CI: 0.55–2.71), thus showing no increased
risk of death compared to non-diabetic NSTI
patients. On the contrary, in a retrospective
study using a national database including 4178
patients with NSTI, diabetes was associated with
a lower mortality rate (Al-Qurayshi et al. 2020).
This is in contrast to a retrospective cohort study
including 9871 NSTI-related deaths in the USA,
where diabetes was more commonly observed
(Arif et al. 2016). As severe peripheral vascular
disease has been associated with increased mor-
tality (Hua et al. 2015), this could be one of the
mechanisms of which presence of diabetes results
in increased mortality, but in the same cohort,
diabetes was not associated with increased mor-
tality. There seems to be a higher rate of
amputations in diabetic NSTI patients (Horn
et al. 2020), but the etiology behind is not clear.
Other chronic diseases have been associated
with poorer outcomes. From several retrospective
studies, hepatic disease seems to be a determinant
for increased risk of mortality, either as cirrhosis
(Khamnuan et al. 2015b; Huang et al. 2011) or
simply hepatic disease (Al-Qurayshi et al. 2020;
Chuang et al. 2019). The studies by Huang,
Khamnuan, and Chuang were conducted in
Thailand and Taiwan, and it is possible that both
difference in population (Sarin et al. 2020) and
microbiological etiology may interact. Similarly,
associations between heart disease and increased
risk of mortality have been found in retrospective
studies (Boyer et al. 2009; Khamnuan et al.
2015b; Anaya et al. 2005).
With the exception of the study by Horn and
colleagues, the above-mentioned studies are of
retrospective nature, of varying sample size, and
44
often the methodology—including definitions of
diseases and mortality—is of modest quality. But
even though these associations have not been
reproduced in data from NSTI cohorts collected
prospectively, it is reasonable to assume that the
presence of a chronic disease may result in a
poorer outcome, and increased focus should be
given to these patients.
4.2.2 Site Specific Manifestations
NSTIs can be located to any part of the body, with
approximately 1/6 of the infections located to the
head and neck area, 1/6 to the upper extremities,
1/3 to the abdomen and anogenital area, and 1/3
to the lower extremities (Madsen et al. 2019).
Depending on the localization of the infection,
more pathognomonic signs can be seen.
Periocular affection is rare with approximately
0.24 cases per million (Flavahan et al. 2014).
Signs as swelling, redness, and pus are common
(Würtz et al. 2020). Due to the localization, the
symptoms are probably recognized fast. Beta-
hemolytic streptococci are the most frequently
cultured pathogen, with Streptococcus pyogenes
(group A streptococcus; GAS) being the most
common of these.
Cervical NSTIs may evolve after a common
throat infection as pharyngitis or tonsillitis or a
dental infection (Skitarelic et al. 2003), and the
initial symptoms will relate to the underlying
cause, e.g. sore throat, dental pain, fever, and
general weakness (Fig. 4.2). The infection can
also present with trismus. Although simple throat
infections are common, the incidence of cervical
NSTI is estimated to two per one million
inhabitants per year (Wolf et al. 2010). The infec-
tion can spread among the fascial planes involv-
ing the neck, and in more pronounced cases the
infection can involve mediastinum (Gunaratne
et al. 2018). NSTI of the neck can be complicated
by suppurative thrombophlebitis of the internal
jugular vein, referred to as Lemierre’s syndrome
(Deganello et al. 2009). The most commonly
cultured bacteria are streptococci and
staphylococci, but also a broad range of anaerobic
bacteria including Prevotella sp.,
M. B. Madsen et al.
Peptostreptococcus sp., Bacteroides sp., and
Fusobacterium sp. are seen (Gunaratne et al.
2018), reflecting the microbes normally seen in
the oral cavity.
Affection of the extremities (Fig. 4.3) is
dominated by monomicrobial GAS infections,
with odds ratios for GAS in upper extremity
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
and G streptococci, most commonly Streptococ-
cus dysgalactiae (SD), are also seen. Amputation
of an extremity occurs in 22% of these patients,
and elevated lactate is associated with amputation
of an extremity (Fig. 4.4).
NSTI involving the abdomen or anogenital
area—the latter also termed Fournier’s gan-
grene—is associated with polymicrobial infection
(Fig. 4.5) (Madsen et al. 2019; Yilmazlar et al.
2017). The incidence rate in males is 1.6 per
100.000 (Sorensen et al. 2009). NSTI in this
region often results from anorectal- or urogenital
skin infections (Singh et al. 2016). As the tissue is
often more loosely bound, deep cavities can
develop, and may spread to the abdomen, thigh,
or genitalia.
4.2.3 Laboratory Tests
In general, the standard laboratory blood tests
show the same pattern as in other infectious
diseases (Moore et al. 2015). Serum C-reactive
protein (CRP) is commonly available, and is
markedly elevated, with a median of 284 mg/L
(IQR: 179–373) before initial surgery (Madsen
et al. 2019). Furthermore, CRP levels may be
higher in patients with NSTI compared to patients
with cellulitis (Simonart et al. 2001). Leukocytes
are equally elevated, but not as markedly, with a
median of 16.3 109/L (IQR: 10.6–22.4) (Madsen
et al. 2019), although higher in other cohorts
(Wong et al. 2003). Procalcitonin (Madsen et al.
2019) and sedimentation rate (Wong et al. 2003)
are also elevated. In general, patients have a met-
abolic acidosis, with a low pH, low standard base
excess and high lactate. The level of acidose is
similar to that in cohorts of patients with septic
4 Necrotizing Soft-Tissue Infections: Clinical Features and Diagnostic Aspects 45

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

shock (ARISE Investigators et al. 2014;
Investigators 2014; PRISM Investigators 2017),
but the lactate levels are substantially lower.
New biomarkers emerge in the attempt to find
easily obtainable markers of diagnosis, disease
severity, and death. Pentraxin-3 (PTX3) is a
pattern-recognition molecule and is part of the
innate immune system. It is elevated in patients
with NSTI compared to controls, and correlated
to shock, amputation, and 180-day mortality—
although the association was not statistically sig-
nificant when adjusted for age, sex, chronic dis-
ease, and SAPS II (Hansen et al. 2016). Soluble
urokinase-type plasminogen activator receptor
(supAR) levels were higher in NSTI patients
with septic shock compared to no shock, and in
survivors compared to non-survivors (Polzik et al.
2019). Elevated supAR levels at admission were
associated with increased 90-day mortality, but
after adjusting for age, sex, and SOFA score, the
result was not statistically significant. Neither
PTX3 nor supAR are readily available in standard
laboratories.
In a quest to develop a score for differentiating
NSTI from common infectious disease, a score
based on six standard laboratory parameters
(potassium, glucose, hemoglobin, CRP, white
blood cells, and lactate) was developed—the

Fig. 4.4 Massive debridement after infection originating from the lower extremity. Courtesy of INFECT
46
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
(LRINEC) (Wong et al. 2004). The score was
developed using a cohort of 314 patients with
NSTI from a single center in Singapore included
retrospectively and validated in another retro-
spectively included cohort of 140 patients. In
this initial work, a LRINEC score of 6 or above
had excellent positive and negative predictive
values. A number of studies have shown varying
ability of the LRINEC score to distinguish
between NSTI and non-NSTI, but almost all
were retrospective observational studies, and of
rather small size. In a recent meta-analysis, the
LRINEC score had poor sensitivity for diagnosis
of NSTI and moderate sensitivity (Fernando et al.
2019). This is supported by a prospective, single-
center observational study conducted in Taiwan,
where the LRINEC was evaluated (Hsiao et al.
2020). Eight hundred and twenty five patients
with cellulitis and 106 patients with NSTI were
included. At a cut-off of a LRINEC score of six or
above, the positive predictive value (PPV) was
25% (95% CI: 20%–30%) and the negative pre-
dictive (NPV) was 92% (95% CI: 91%–93%),
and the area under the receiver operating charac-
teristic (ROC) for accuracy was 0.696 (95% CI:
0.640–0.751). When interpreting the score it is
M. B. Madsen et al.
important to consider that patients with NSTI are
heterogenous, and the quality of the studies
varies. A priori, a clinician should raise suspicion
for the ability of score based solely on standard
laboratory values to discriminate between
advanced tissue destruction and a
non-necrotizing infection, and further diagnostics
and treatment should not be delayed awaiting the
result of the score. Others have tried to develop an
alternative score for discriminating between NSTI
and non-NSTI. The SIARI score was developed
using data obtained retrospectively at a single-
center in New Zealand from 2000 to 2010
(Cribb et al. 2019). The score is based on the
variables site other than lower limb, immunosup-
pression, age 60 years, renal impairment, and
inflammatory markers, and the cohort included
138 patients with NSTI and 142 with severe cel-
lulitis; these were divided into a development and
a validation cohort. The PPVs in the two cohorts
were 73% (95% CI: 65%–80%) and 74% (95%
CI: 66–80%), respectively, and the NPVs were
80% (95% CI: 71%–87%) and 81% (71–88%),
respectively. The concordance statistics (C-statis-
tic—comparable to ROC curve) were 0.83 and
0.85, respectively. As for the LRINEC score,
limitations apply, including the lack of clinical
4 Necrotizing Soft-Tissue Infections: Clinical Features and Diagnostic Aspects
data; data were from cohorts included at a single-
center; data were collected retrospectively; the
limited number of included patients and data
were collected over large period of time. The
clinician should always have a high suspicion
for NSTI, and never rule out the diagnosis when
clinically suspected, despite a score below a cer-
tain cut-off.
4.2.4 Microbiology
Determining the microbiological etiology is of
uttermost important to target the antibiotic ther-
apy. Whenever a severe infection is suspected,
relevant cultures should be obtained immediately.
When reviewing microbiological data, it is
important to keep a number of factors in mind,
as these can differ considerably and introduce
substantial bias. The samples sent for
microbiological testing should be taken from the
wound or in its vicinity; alternatively, from blood
sampling. The samples should have been
obtained within a reasonable time from the time
of diagnosis. Samples taken too soon before
(in patients hospitalized for reasons other than
NSTI) may not reflect the pathogenic bacteria,
and samples taken too long after may represent
secondary contamination. It is also worth noting
the method in which the microbiological agents
are derived. Traditionally, the sample is spread
out and grown on an agar plate. This method may
facilitate the growth of certain bacteria above
others. Newer methods, including PCR analysis
and shotgun metabolomics (Rodriguez et al.
2020; Thänert et al. 2019; Rudkjøbing et al.
2016), are more sensitive, but may also find spe-
cies that are not relevant with regard to the infec-
tion. A great advantage is the ability to provide
faster bacterial etiology. The microbiologic etiol-
ogy of NSTI is reviewed in Chap. 5.
Although presumably seldom performed,
material obtained for histopathology can provide
additional information for the clinician. Only few
studies exist, but from these we have data on how
the different layers of the soft tissue are affected,
with inflammation and necrosis affecting dermis
and subcutaneous fat, and in some cases
47
involvement of epidermis (Barker et al. 1987;
Umbert et al. 1989; Stamenkovic and Lew
1984). Furthermore, thrombosis of the vessels is
seen. Histopathological examination may aid in
diagnosing NSTI in cases where the clinical
findings during surgery are not in unison and the
degree of the inflammatory response in the tissue
may correlate to disease severity, as proposed by
Bakleh and colleagues (Bakleh et al. 2005). Gram
staining would also provide information on pres-
ent bacteria if found in the tissue. An added bonus
to routine histopathology sampling could be a
more precise definition of NSTI at tissue level,
rendering it possible to include more homogenous
patients in future NSTI cohorts.
4.2.5 Imaging
Several imaging options are available, including
plain radiographs, ultrasonography (US),
computerized tomography (CT), and magnetic
resonance imaging (MRI).
Plain radiographs are readily available at most
centers. It is possible to visualize gas in the soft
tissue in the absence of trauma (Tso and Singh
2018). However, this symptom is far from present
in all patients, and if present, is easily overlooked
compared to findings from a CT scan (Leichtle
et al. 2016).
US findings include fluid accumulation in the
fascial layer and thickening of the subcutaneous
layer (Yen et al. 2002). US is limited by the low
resolution of deeper structures, but has been
shown to discriminate NSTI of the limbs from
non-necrotizing fasciitis (Yen et al. 2002). As US
is widely accessible, it is probably not likely to
postpone surgical intervention (Whitson and
Mayo 2016; Barbic et al. 2017). However, the
findings specific to NSTI require experience to
detect, and in all circumstances, US should not be
used to rule out NSTI.
The resolution of CT scans has increased
tremendously over the past years and exceeds
that of plain radiography and US (Hayeri et al.
2016). Findings of NSTI on CT scans include
asymmetric thickening of the fascia and gas track-
ing along fascial planes (Wysoki et al. 1997)—
48
especially gas within fluid collections along
subfascial planes. In a meta-analysis of seven
studies, the presence of fascial gas on CT had a
sensitivity of 88.5% (95% CI: 55.5%–97.9%) and
a specificity of 93.3% (95% CI: 80.8%–97.9%)
(Fernando et al. 2019). Other findings include
subfascial fluid, subcutaneous edema, and
increased soft-tissue attenuation (Tso and Singh
2018), and if all these findings are present, the
sensitivity rises to 94.3% (95% CI: 81.2%–
98.5%) but at the expense of specificity, which
decreases to 76.6% (95% CI: 21.3%–97.5%)
(Fernando et al. 2019) The use of CT scan can
aid in discriminating NSTI from non-necrotizing
infections (Carbonetti et al. 2016). Furthermore,
the scan may help surgeons determine the extent
of soft-tissue involvement and help localize the
source of infection. In some centers, CT scans can
be done within minutes, and thus probably do not
delay surgery. In this context, this modality can
be of help.
MRI has been used for more than 20 years to
help discriminate NSTI from non-necrotizing
infections (Rahmouni et al. 1994), and is consid-
ered the gold-standard for soft-tissue infections.
MRI has excellent resolution for soft tissue. MRI
findings of NSTI include thickening of the deep
fascia with abnormal signal intensity, edema, and
hypointense signals due to gas within fascial
layers (Ali et al. 2014; Kim et al. 2011). Changes
in muscle signal can be observed due to adjacent
inflammation. Unfortunately, MRI has major
limitations: accessibility and duration of the
scan—both which are likely to delay surgical
intervention.
Overall, the different imaging modalities may
aid in discriminating patients with NSTI from
non-necrotizing infections, but they must not
delay surgical intervention (Sartelli et al. 2018).
4.3 Diagnostic Aspects
Any patient with a cutaneous infection with local
or systemic signs of severity should be suspected
of NSTI (Fig. 4.6). No set of diagnostic criteria
for NSTI exists; the diagnosis is merely clinical,
meaning that it is based on intraoperative
M. B. Madsen et al.
findings. These typically include extensive necro-
sis of the subcutaneous tissue and/or muscle tis-
sue, undermining of the skin, thrombosis of the
superficial veins, and deliquescent tissue. Acute
microscopy for the microbiological agent can be
helpful for choice of antibiotic treatment, but the
microbiological flora is not a determinant of
whether the infection is a NSTI or not. Although
initial surgery may be without signs of NSTI,
patients should be closely monitored, as some of
these patients may eventually be diagnosed with
NSTI (Howell et al. 2019). Snap frozen biopsies
for histology have been used, but it is uncertain
whether these contribute to faster diagnosis and
thereby shorter time to surgery. As mentioned
above, MRI can potentially discriminate between
NSTI and non-necrotizing infections, but due to
the length of the procedure and the availability of
MR-scanner, for many, this is not a feasible
option. Ultrasound is readily available in many
hospitals, and as NSTI has certain specific ultra-
sonic properties, US can aid the diagnostic proce-
dure. However, caution should be taken when
ruling out a necrotizing infection. Finally, the
LRINEC score does not seem sufficient in
distinguishing NSTI from non-necrotizing
infections.
4.4 Future Perspectives
NSTIs are serious infections with diagnostic
challenges. In order to improve outcome for
future patients, focus on certain diagnostics
aspects is key. First and foremost, knowledge of
the disease is of uttermost importance. The initial
step is often when the patient contacts the
healthcare system. The threshold for contact
varies considerably from patient to patient, and
as the initial symptoms sometimes are the same as
“simple infections,” it is difficult to minimize the
time from the patients experience symptoms to
healthcare system contact. Every doctor seeing
patients may encounter one with NSTI (Anaya
and Dellinger 2007), and should be able to raise
suspicion and quickly refer the patient to a hospi-
tal or department capable of further diagnostics
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
50
disease suspected. Although never shown, early
recognition is probably one of the most
timesaving steps and could potentially reduce
morbidity and mortality considerably. Unfortu-
nately, there are several case stories of patients
acknowledging that “something is wrong,” but
where the healthcare workers did not recognize
the initial symptoms and thus the seriousness of
the situation (Erichsen Andersson et al. 2018).
Due to the low incidence, continuous education
of healthcare workers is needed to maintain
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.
Distinguishing NSTI from other infections is
challenging, and we still do not have solid diag-
nostic tools. New biomarkers emerge, but due to
the heterogeneity of the NSTI, it is not likely that
a single biomarker will be diagnostic—but as part
of a diagnostic panel it may help in diagnosing
NSTI. New methods for microbiological testing
seem more promising. A prompt microbiological
diagnosis can result in faster and a targeted anti-
biotic therapy. The use of PCR is already avail-
able in some centers, and the microbiological
catalogue is expanding. Shotgun metabolomics
is another emerging diagnostic tool. If these
methods can be made available as point-of-care
tests, time to targeted therapy may be reduced.
There is still not sufficient data to show
whether US can aid in the diagnosis of NSTI.
As it is readily available bedside in most centers,
US has the potential to push forward the diagno-
sis of NSTI. A large observational study
confirming the existing US findings and compar-
ing them to those found in severe cellulitis is
highly welcome.
The diagnostic aspect of NSTI should focus on
the recognition of symptoms compatible with
NSTI, and developing local logistics to develop
quick access to further diagnostic work-up: clini-
cal examination, preferably by an infectious dis-
ease specialist or dermatologist, blood sampling
including blood for microbiology, quick access to
imaging and quick access to an operating room
(Urbina et al. 2020).
M. B. Madsen et al.
It is important that the quest to understand the
underlying pathophysiology of NSTI continues.
This can only be done by meticulously collecting
data on these patients, preferably from prospec-
tive, multicenter cohort studies. Only when this is
done we can design randomized clinical trials
aimed to test the effect of specific treatments.
Acknowledgement Financial support: The work was
supported by the European Union Seventh Framework
Programme: (FP7/2007-2013) under the grant agreement
305340 (INFECT project).
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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

# Springer Nature Switzerland AG 2020 53

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_5
54
pathogen is essential. However, the choice of
empirical therapy is frequently inadequate,
underlining the need for comprehensive and
contemporary knowledge on causative
pathogens and relevant antimicrobial resis-
tance patterns in NSTIs. Also, studies of the
pathogenic mechanisms in different NSTIs are
needed, to improve handling of patients
through developing patient stratification and
tailored therapies. We review the current
knowledge on microbial etiology and provide
detailed characterizations of the predominant
pathogens.
Keywords
Bacteroides fragilis · Monomicrobial ·
Necrotizing soft tissue infection ·
Polymicrobial · Staphylococcus aureus ·
Streptococcus dysgalactiae · Streptococcus
pyogenes · Type 1 infection · Type 2 infection
Highlights
• Verification of microbial etiology is of para-
mount importance in NSTI
• The INFECT study provided strong
associations of affected localization and
microbial etiology in NSTI
• Monomicrobial NSTI is most often caused by
Streptococcus pyogenes and has strong corre-
lation to an extremity localization
• New microbial etiologies of NSTI are
emerging due to aging of populations and cli-
mate change
5.1 Introduction
Necrotizing soft tissue infections (NSTIs) are het-
erogeneous, rare and severe infections with high
morbidity and mortality rates. Mainstay therapy is
infection source control achieved by radical sur-
gical treatment supported by prompt administra-
tion of antimicrobial therapy adjusted to the
microbial etiology (Stevens and Bryant 2017).
Medical microbiology plays an important role in
this multidisciplinary work, as it can contribute to
improved prognosis through identification of
microbial etiology and relevant resistance profiles
S. Skrede et al.
that can guide antibiotic therapy. Moreover, veri-
fication of the causative pathogen can offer prog-
nostic information (Anaya et al. 2005; Huang
et al. 2011; Madsen et al. 2019). Notably, empiri-
cal antibiotic treatment of NSTIs is inadequate in
approximately 90% of the patients (Marwick
et al. 2011). This highlights the significant knowl-
edge gap in microbial etiology in NSTIs, both on
a regional and global level. There are several
reasons for this. NSTIs are rare, and with inci-
dence rates in the range of 0.3 to 15/100,000/year
(Stevens and Bryant 2017; Peetermans et al.
2020) most hospitals consequently treat a low
number of patients. This potentially leads to
insufficiently developed competence in adequate
microbiologic diagnostic procedures. Since obli-
gate anaerobic microbes are frequently involved,
samples are particularly vulnerable to incorrect
sample acquisition or transportation, which likely
has contributed to the low rates of culture positive
samples presented in several studies (McHenry
et al. 1995; Boyer et al. 2009; Khamnuan et al.
2015). Furthermore, many NSTI cases present as
medical emergencies, and establishing a
microbiological diagnosis is a lower priority
than initiating urgent therapeutic measures.
Finally, data reporting in many studies offer low
level of precision, e.g. when only crude
microbiological data for a patient cohort is given
(Childers et al. 2002), or because of varying qual-
ity in reporting of discharge diagnosis (Louis
et al. 2019). Notwithstanding, in NSTI medical
microbiology holds great promise, as test results
are offered with speed adding value to our
present-day diagnostic methods. Also, medical
microbiology is a key scientific discipline in
development of rapid diagnostic tests speeding
up decision making and leading towards
personalized therapeutic measures to well
stratified patient cohorts among NSTI patients.
In the following, microbiologic features of NSTI
are delineated.
5.2 Contemporary Classification
of NSTI
Necrotizing soft tissue infections have been
documented since the fifth century
5 Microbiological Etiology of Necrotizing Soft Tissue Infections
B.C. Historically classification according to
anatomical location has been practiced, giving
rise to a multitude of clinical syndromes bearing
different eponyms. In the head and neck area
Lemierre’s syndrome is a disseminated necrotiz-
ing infection characterized by jugular suppurative
thrombophlebitis and evidence of disseminated
infection, including signs of septic embolization.
Fusobacterium necrophorum is frequently
involved. Ludwig’s angina has been applied to a
number of infections of the sublingual, submaxil-
lary, or submandibular space. It should be
restricted to rapidly spreading infections
originating in the floor of the mouth that spreads
bilaterally within the submandibular and sublin-
gual spaces, without abscess formation or lym-
phatic involvement. Polymicrobial etiology is
predominant, and Peptostreptococcus,
Prevotella, and Porphyromonas species, and
viridans streptococci are frequently implicated.
Fournier’s gangrene is a progressive
polymicrobial synergistic necrotizing infection
of the external genitals and perineum, where a
number of pathogens can be expected, including
obligate anaerobic species, Enterobacteriaceae
and Enterococcus spp. Meleney’s bacterial gan-
grene, or hemolytic streptococcal gangrene, is a
type 2 necrotizing fasciitis caused by Streptococ-
cus pyogenes (group A streptococcus; GAS).
Many of these eponyms arose prior to the estab-
lishment of diagnostic microbiology, and their
use has frequently led to low precision in descrip-
tion of etiologies in severe soft tissue infections. It
was noted already in the 1970s that there was an
insufficient attention on the bacteria involved in
the pathogenesis of NSTI. In a single center study
from San Francisco, Giuliano and coworkers
described bacterial etiologies in sixteen patients
with NSTI, of which there were nine drug users,
three postoperative cases, and three with a story
of minor trauma (Giuliano 1977). They collected
intraoperative samples and identified
18 pathogens at species level, as well as a signifi-
cant number at genus level. The study culminated
in a novel system for classification of NSTI based
on microbial etiology, where category type
1 grew anaerobic bacteria and facultative anaero-
bic bacteria, such as Enterobacteriaceae or
55
streptococci other than GAS. They noted that in
each case of a type 1 infection, at least one anaer-
obic organism was cultured in combination with a
facultative anaerobe. In category type 2 GAS was
isolated. The authors noted that in type 2 GAS
occurred together with Staphylococcal species,
but not anaerobic bacteria or Enterobacteriaceae.
This categorization has been widely adopted and
used in guidelines (Sartelli et al. 2018) and
reviews (Sarani et al. 2009; Hakkarainen et al.
2014; Lamb et al. 2015; Stevens and Bryant
2017).
The list of implicated microbes has gradually
been expanded, though, and amendment of
monomicrobial infections caused by marine
gram-negative microbes as a type 3 category, as
well as a fungal type 4 category has also been
suggested (Morgan 2010). The latter is not widely
accepted as the usefulness of expanding necrotiz-
ing soft tissue infection entities is questioned
(Stevens and Bryant 2017). In Table 5.1 a practi-
cal overview of microbes presented by category is
offered and microbial causes of NSTIs presented
in more detail below.
5.3 Establishing Microbial Etiology
of NSTIs
There is a substantial heterogeneity in the distri-
bution of microbial etiologies reported in NSTIs,
and a plethora of pathogens have been implicated.
Temporospatial differences account for some of
the observed discrepancies, but unfortunately a
high variability in microbiological methods, case
definitions, and study designs is the major culprit.
In a substantial fraction of publications on NSTI,
characteristics of a low numbers of patients with a
particular etiology or single cases are reported,
occasionally accompanied by reviews of a limited
number of similar cases, identified in retrospec-
tive cohorts. Moreover, case definitions of NSTI
are not widely accepted due to lack of adequate
clinical precision (Calandra et al. 2005), leading
to inclusion of resembling clinical entities such as
deep-seated abscesses, vascular occlusion, and
cellulitis. Lastly, the precision of microbiological
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-
ing soft tissue infections and pathogenicity
delimiting what microbes that cause NSTIs
(Eron et al. 2003; Stevens et al. 2005, 2014).
In order to deal with this, classification catego-
rization systems for NSTIs have been created
according to microbial etiologies, as outlined in
Sect. 5.2. In the recent INFECT study, the world’s
largest prospective study of NSTIs, the method
for classification of cases by microbes, in a sys-
tem meant to aid categorization and evaluation of
microbiological data, was described in the sup-
plementary material accompanying the publica-
tion on streptococcal NSTI (Bruun et al. 2020).
According to this routine, categorization was
based on routine laboratory culture and identifi-
cation, essentially phrased as follows.
5.3.1 Sample Sources
The categorization of etiology and the certainty of
the etiological diagnosis was based on type of
sample, time of sampling, e.g. in relation to sur-
gery, host factors, patient story taking primary
focus into account, and ability of the microbes
to cause severe soft tissue infection confirmed by
description of pathogenicity.
The certainty of etiology was categorized as
definite in cases fulfilling documentation in elec-
tronic patient files at the highest level of
microbiological identification. Probable etiology
was delimited to “Likely pathogens” reviewed
below, whereas possible etiology was based on
weak documentation or low likelihood of being
etiological cause, including situations where
sampling method, host factors, patient story, or
clinical evaluation lead to uncertainty of the
role of the microbe. Finally, unknown etiology
was defined as no identification of relevant
pathogen.

Firstly, sample source was reviewed to support

whether a microbe would be definite, probable, or
unlikely to be involved in causes of surgically
confirmed NSTI. By definition, reliable sources
were limited to blood cultures or deeply situated
soft tissue in otherwise sterile environment,
e.g. superficial and deep fascia, muscle, fat or
unidentified necrotic tissue. Unreliable sources
were superficial swab samples, including those
taken from chronic abscesses with fistula.
5.3.3 Categorization According
to Pathogenicity
The microbes were classified into primary
pathogens, defined as microbes that may cause
soft tissue infection in patients without known
risk factors. These include Streptococcus
pyogenes, Vibrio vulnificus, and Clostridium
perfringens.
5 Microbiological Etiology of Necrotizing Soft Tissue Infections
Secondary pathogens included microbes that
may cause soft tissue infection, including
polymicrobial as well as monomicrobial etiology,
in patients with risk factors. Typical examples
were “group C and G streptococci” (most often
Streptococcus dysgalactiae, though frequently
not identified to species level in routine
laboratories), Streptococcus agalactiae (group B
streptococcus; GBS), Streptococcus anginosus
group, Streptococcus pneumoniae, Staphylococ-
cus aureus, Haemophilus influenzae, Neisseria
meningitidis, Enterobacteriaceae, Pseudomonas
spp., Acinetobacter spp., Bacteroides spp.,
Prevotella spp., and Fusobacterium spp.
Polymicrobial pathogens were delimited to
possible causes of soft tissue infections as part
of a polymicrobial flora, but rarely of NSTI in the
absence of primary or secondary pathogens,
e.g. Enterococcus.
Commensals were defined as microbes that do
not cause severe soft tissue infection, but that may
be occasional markers of polymicrobial etiology
in cases where they are cultured together with
other bacteria, with typical examples being Bacil-
lus other than B. anthracis, Corynebacterium,
Micrococcus, and Coagulase negative
staphylococci.
5.3.4 Classification as Either
Monomicrobial or
Polymicrobial
Monomicrobial cases were classified as such if a
primary or secondary pathogen was detected in
monoculture, or if a polymicrobial pathogen was
found in monoculture in a patient with severe
immunodeficiency.
Cases where a primary pathogen was
identified together with commensals were
categorized among the monomicrobial cases.
Polymicrobial cases would then comprise the
remaining cases, and in this publication it was
stated that polymicrobial infections are easily
categorized as such, deemed by patient stories
(e.g. head neck, pelvic) and culture results
(Bruun et al. 2020, supplementary material).
57
5.4 Microbiological Etiologies
in NSTI
5.4.1 Streptococcus pyogenes
Streptococcus pyogenes (group A streptococcus;
GAS) is exclusively a human pathogen. It causes
a substantial global disease burden, ranging from
superficial skin infections and tonsillitis, to severe
invasive infections (Ralph and Carapetis 2013).
Importantly, it is the predominant etiologic agent
of monobacillary type 2 NSTI, comprising
10-40% of all cases (Elliott et al. 1996, 2000;
Kobayashi et al. 2011; Das et al. 2012; Madsen
et al. 2019). The microbiological characteristics
of GAS are briefly reviewed below. Due to its
importance, details on pathophysiology of strep-
tococcal NSTI are presented in Chap. 10, clinical
aspects in Chap. 6, and antimicrobial treatment in
Chap. 7.
GAS belongs to the family Streptococcaceae
of the phylum Firmicutes. It is a gram-positive,
facultative anaerobic, catalase negative, spherical
or ovoid bacterium with approximate diameter
0.6–1 μM. It is non-motile and does not form
spores. In the microscope it appears in pairs or
short chains in clinical specimens, whereas long
chains are formed when it is grown in rich culture
media. Its binominal family name Streptococcus
comes from Greek στρεπτóς (streptós) “twisted,”
“chain,” or “necklace” and κóκκoς (kókkos)
meaning “berry” or “grain.” Its species epithet
pyogenes comes from the combination of Greek
πύoς (pyos), “pus,” and γεννάειν, (genein) “to
beget,” referring to its ability to form pus. The
microbe requires complex, enriched media to
grow. On solid culture media colonies may
appear glossy, or mucoid, more or less,
depending on the hyaluronate capsular material
a strain produces. When grown on blood agar it
forms white to gray colonies
0.5 mm and it
displays complete (β) hemolysis through red
blood cell disruption caused by streptolysin. For
almost a century, β-hemolytic streptococci have
been classified by detection of their respective
cell wall carbohydrate antigens, in the system
created by Rebecca Lancefield (Lancefield 1933;
58
Lancefield and Hare 1935). Phenotypic testing is
imperfect, exemplified by that GAS is not exclu-
sively expressing Lancefield group A carbohy-
drate antigen, yet it is still widely used.
Contemporary methods used for bacterial identi-
fication include mass-spectrometry, matrix-
assisted laser desorption/ionization time of flight
mass spectrometry (MALDI-ToF MS), a rapid
and cost-effective method that offers sufficient
taxonomic resolution at species level in the case
of GAS.
The major virulence factor of GAS is
M-protein, reviewed in Chap. 10. Almost every
GAS isolate expresses an emm-gene, and based
on antigenic differences more than 200 emm-
types have been described. The emm-types
emm1, emm3, emm12, emm28, and emm89 are
the predominant genotypes implicated in invasive
GAS disease (Chelsom et al. 1994;
Monnickendam et al. 1997; Kaul et al. 1997;
Sharkawy et al. 2002) although regional
differences in genotypic distribution has been
noted (Gherardi et al. 2018). In the INFECT
study, GAS comprised 126/409 (31%) of all
NSTI cases and 75% of all β-hemolytic
streptococci. The predominant emm-types were
expectedly emm1, emm3, and emm28 (Bruun
et al. 2020). Similar findings were also reported
from a retrospective cohort of 70 patients with
β-hemolytic streptococcal NSTI in western
Norway, whereof 61/70 (87%) were caused by
GAS (Bruun et al. 2013).
GAS is equipped with several other virulence
factors, of which the extracellular superantigens
are key mediators of the immunopathogenesis of
invasive GAS (iGAS) (Commons et al. 2014), on
the one hand able to elicit the unbalanced inflam-
matory responses seen in iGAS, and on the other
to ensure nasopharyngeal carriage (Zeppa et al.
2017). The repertoire of virulence determinants
and pathogenic mechanisms of GAS NSTIs are
further delineated in Chap. 10.
The disease signature of GAS NSTIs
represents the hallmark of features commonly
associated with NSTIs; rapid progression and a
fulminant clinical course. Following the upsurge
of GAS NSTIs in the mid-1980s, this pathogen
has become synonymous with the “Flesh-eating
S. Skrede et al.
bacteria” in the public media (Lamb et al. 2015).
In the recent INFECT-study another distinct char-
acteristic of GAS NSTIs was noted. A strong
association of GAS etiology and NSTI of the
upper extremity (Fig. 5.1), with and odds risk
(OR) of 7.80 (95% CI 4.36–14.25, p < 0.001)
was observed (Madsen et al. 2019).
A similar observation was reported almost
25 years ago in a small patient cohort from North-
ern America (Haywood et al. 1999).
In the INFECT-study only 12/126 cases (9%)
involving GAS were polymicrobial, of which 2/3
were co-cultured with Staphylococcus aureus
(Bruun et al. 2020). This co-residency was
observed also by the inventors of the currently
used categorization system of NSTI according to
microbial etiologies (Giuliano 1977). The authors
concluded that these infections were type 2 GAS
infections, where staphylococcal species were
detected, whereas anaerobic bacteria or
Enterobacteriaceae were not.
Penicillin is the undisputable treatment of
choice for GAS. However, GAS isolates with
reduced susceptibility to beta-lactam antibiotics
have recently been reported, associated with
mutations in penicillin binding protein pbp2x
(Vannice et al. 2019; Musser et al. 2020). The
clinical relevance has yet to be determined, but
sustained attentiveness is definitely warranted.
Increasing resistance to clindamycin is of more
immediate concern (Smit et al. 2015). However,
large regional variations are noted, and in the
INFECT study in Scandinavia only a single
GAS isolate displayed reduced susceptibility to
clindamycin. Persistent infection over days and
even weeks has been noted in patient cases
(Siemens et al. 2016). In part this may be
attributed to functional antimicrobial resistance
caused by GAS biofilm formation, first
documented in invasive GAS through the
INFECT study (Siemens et al. 2016), which
may impact future treatment strategies. Relevant
antibiotic resistance in GAS infection is further
detailed in the section on β-hemolytic
streptococci in Chap. 7.
The lack of efficient GAS vaccines and recent
concerns on antimicrobial susceptibilities in both
GAS and Streptococcus dysgalactiae
5 Microbiological Etiology of Necrotizing Soft Tissue Infections
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
as microbial etiology of NSTI.
5.4.2 Streptococcus dysgalactiae
Streptococcus dysgalactiae (SD) is a β-hemolytic
streptococcus that displays genetic similarity to
GAS, indicating a common phylogenetic ancestry
of human origin. The Latin species epithet
dysgalactiae refers to “loss of milk production,”
a symptom of bovine mastitis. SD expresses cell
wall carbohydrate antigens of Lancefield group C
and group G, and is divided into the subspecies
S. dysgalactiae subsp. equisimilis and
S. dysgalactiae subsp. dysgalactiae. The latter is
exclusively a zoonotic pathogen and not further
discussed. SD is a gram-positive, facultative
anaerobic, bacterium that does not form spores
and is non-motile. It forms large colonies >0.5
mm and on blood agar it is predominantly
β-hemolytic. Today many laboratories identify
bacteria by MALDI-ToF MS, but old methods
for identification are still frequently in use. It
does still not offer sufficient taxonomic resolution
beyond the species level, and multilocus
sequence typing (MLST) remains the most reli-
able method for identification of SD at subspecies
59
level. Different systems derived from GAS typing
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
SD, and presently more than 100 emm-types
have been described (https://www.cdc.gov/
streplab/index.html). Several other virulence
factors have been identified in SD, and when
comparing SD and GAS, genes for about half of
well-characterized GAS virulence factor genes,
including adhesins and protein G that binds
circulating immunoglobulins, are detected in
SD. Also, SD produces toxins and secreted
enzymes including Streptolysin O (SLO), Strepto
lysin S (SLS), and Streptokinase (Shimomura
et al. 2011).
Historically, SD was judged of little impor-
tance as etiology of human disease. The last few
decades, however, it is increasingly recognized as
an important cause of invasive disease (Ikebe
et al. 2004), particularly in the adult population
(Takahashi et al. 2010; Oppegaard et al. 2015).
From the upsurge of streptococcal NSTI in the
1980s and onwards scattered reports on group G
and C streptococcal NSTI were published (Barker
et al. 1987; Sharma et al. 2002). Even though
expression of Lancefield group G or C carbohy-
drate antigens is not unique to SD, it is
60
conceivable that most cases of these groups G and
C streptococcal NSTI were caused by SD. Today
it is well established that SD is an emerging cause
of monobacillary as well as polymicrobial NSTI
(Bruun et al. 2013, 2020; Madsen et al. 2019).
In a retrospective cohort of 70 patients in
western Norway with β-hemolytic NSTI, SD
caused 9/70 cases (Bruun et al. 2013). In the
INFECT study SD comprised 27/409 (7%) com-
pared to GAS 126/409 (31%), confirming the
importance of SD in NSTI. Furthermore, in SD
stG62647 was the predominant emm type and
expressing speG, 47 % of cases were
polymicrobial, 30% were blood culture positive,
and antimicrobial resistance towards erythromy-
cin and clindamycin was 5% and 0, respectively
(Bruun et al. 2020). Informatively, positive cul-
ture of SD was strongly associated with infection
of the lower extremities (Fig. 5.1), with an OR
9.76 (95% CI 2.57–54.85, p ¼ 0.001) (Madsen
et al. 2019). The worldwide increase in numbers
of elderly people and the increasing resistance
towards key antibiotics warrants attention toward
SD as microbial etiology of NSTI, and increasing
incidence rates may be anticipated.
5.4.3 Staphylococcus aureus,
Including MRSA
Staphylococcus aureus is a gram-positive, facul-
tative anaerobic round shaped bacterium with
approximate diameter 1 μM. It is non-motile and
does not form spores. In the microscope it appears
singly, in pairs, tetrads, and irregular grapelike
clusters, hence it is binominal family name
(of Greek σταφυλóκoκκoς; staphyle “grape-clus-
ter” and kokkos “small berry”), and its Latin
species epithet aureus referring to the “golden”
appearance of colonies when cultured. When
grown on blood agar it displays β-hemolysis.
S. aureus expresses catalase converting hydrogen
peroxide (H2O2) to water and oxygen, detection
of which has been a longstanding method used to
distinguish it from enterococci and streptococci.
Detection of coagulase production has been an
important, although imperfect method to differen-
tiate it from other staphylococcal species, as not
S. Skrede et al.
all S. aureus strains are coagulase-positive. Cul-
ture and antibiotic susceptibility testing are diag-
nostic corner-stones but today molecular
techniques, mass spectrometry, and other
methods are important novel measures to aid
detection (e.g. in slowly growing phenotypes),
in noncultivable cases (e.g. in some tissue
samples), rapid species determination, and detec-
tion of relevant resistance. S. aureus is a major
cause of human diseases. Its success can be
attributed to several microbial traits. Firstly, it is
a capable colonizer of the skin and mucosa in the
upper respiratory tract, and up to 30% of the
human population are carriers. Moreover,
S. aureus produces an arsenal of virulence factors
enabling it to adhere and expand locally, spread
or cause host responses systemically, distant from
the origin of infection, reviewed elsewhere
(Shumba et al. 2019). Surface adhesins are com-
monly covalently bound to the bacterial cell wall
peptidoglycan and mediate adherence to matrix
proteins of target tissues. The microbe confers
virulence also by use of multiple extracellular
factors, i.e. enzymes, cytotoxins, and
superantigens. Furthermore, small RNAs are
staphylococcal virulence factors, involved in,
e.g. increased biofilm formation. Strains of
S. aureus exchange phages, such as the Panton–
Valentine leukocidin (PVL) toxin producing,
mobile phage frequently expressed by strains of
methicillin resistant S. aureus (MRSA). The prev-
alence of PVL is low in methicillin susceptible
S. aureus (MSSA), whereas PVL-genes are pres-
ent in most isolates of community acquired (CA-)
MRSA. Of concern, PVL-toxin producing MRSA
is associated with skin and soft tissue infection,
including myositis and NSTI (Saeed et al. 2018;
Thapaliya et al. 2015). Therefore, despite MRSA
is an infrequent cause of NSTI in general, reports
support attentiveness towards MRSA as micro-
bial etiology in certain geographical areas, as
documented in the USA and Korea (Miller et al.
2005; Lee et al. 2007; Kim et al. 2019). S. aureus
has developed antibiotic resistance to all the rele-
vant antibiotic classes. The majority of S. aureus
strains produce penicillinase, for which reason
penicillinase-resistant beta-lactam antibiotics
belonging to the isoxazole penicillins are
5 Microbiological Etiology of Necrotizing Soft Tissue Infections
preferred for treatment of MSSA. In MRSA
methicillin resistance is conferred by the mecA
gene, which codes for an altered penicillin-
binding protein 2A (pbp2A), leaving most beta-
lactams inactive.
The prevalence of S. aureus in clinical cohorts
of NSTI is difficult to assess, as reporting on
microbiological data from relevant cohorts in
general holds low precision. It is also difficult to
quantify the impact of S. aureus as well as its
relative distribution between type 1 and type
2 infections. In the INFECT study it is
categorized a secondary pathogen, as detailed
above (Madsen et al. 2019). However, it is well
described that it displays a tropism for skeletal
muscle and is an established cause of pyomyositis
(Hall et al. 1990). In a study on selected S. aureus
isolates obtained in the INFECT study neither
clonal relationship nor MRSA was detected, and
there was only a single PVL positive case (Baude
et al. 2019). Nevertheless, when compared to a
different collection of blood stream infection
isolates, isolates from NSTI cases showed
increased myocyte internalization and cytopatho-
genic effect, suggesting alternative pathophysio-
logic mechanisms yet to be revealed.
Notwithstanding its cytotoxicity in muscle tissue,
it was not until 2005 that S. aureus (i.e. MRSA)
was shown to be etiology of monomicrobial NSTI
(Miller et al. 2005).
Through data obtained in the INFECT study a
more detailed picture of the prevalence and puta-
tive role of S. aureus is given. Firstly, in this
Scandinavian cohort it was an uncommon micro-
bial etiology, found in 6% (26/409) of cases in the
entire cohort. There were 13/179 (7%)
monomicrobial S. aureus cases, of which
12 were cases of extremities; 7/12 of the upper
and 5/12 of the lower. NSTI located to the upper
extremities was associated with monomicrobial
S. aureus infection (Fig. 5.1) with an OR of
5.80 (95% CI 1.61–21.60, p ¼ 0.0184) (Madsen
et al. 2019). In INFECT, monomicrobial growth
of S. aureus was associated with diabetes mellitus
when compared to those with no diabetes with an
OR of 5.4 (95% CI 1.57–18.7, p ¼ 0.01) (manu-
script, Rosén et al. 2020). There were 13/204
(6%) polymicrobial S. aureus cases, of which
61
6 were detected in anogenital infections (Madsen
et al. 2019). Among the 13 polymicrobial cases,
S. aureus was co-cultured with GAS in 8 (Madsen
et al. 2019; Bruun et al. 2020), inferring that in
only 5 cases (2%) other pathogens than GAS
were detected. Given this, Staphylococcus
aureus-colonization and sampling error may be
confounders. This assumption is partly supported
by a study of 148 INFECT cases, using 16S
rRNA sequencing with transcriptional analysis
of host and microbe using dual RNA sequencing
of peroperatively collected, infected, deep tissue
samples (Thänert et al. 2019). In this study it was
demonstrated that S. aureus is not an associate of
complex polymicrobial communities causing
NSTI. Thus, the INFECT study adds novel data
supporting the view that there is still a need both
to question as well as explore a putative role for
PVL negative MSSA as an etiology of
non-purulent NSTI.
5.4.4 Clostridium perfringens
The genus Clostridium belongs to the phylum
Firmicutes and includes more than 200 known
species, of which most are not regularly
associated with human disease. Differently,
C. perfringens, C. septicum, C. novyi, and
C. sordellii are microbial etiologies of NSTI,
comprising around 10% of culture positive cases
in some patient cohorts (Anaya et al. 2005; Boyer
et al. 2009; Kobayashi et al. 2011). The vegeta-
tive cells of Clostridium spp. form pairs, short
chains or appear in clusters, and they are
rod-shaped, forming spores, reflected in the
name of the genus, derived from Greek κλωστήρ;
klōstēr, “spindle,” derived from klōthein “to
spin.” The Latin species epithet of
C. perfringens from Latin per “through” and
frango “burst” alludes to the microbe
disrespecting tissue borders in expanding gas
gangrene, whereas the species epithet of
C. septicum means “putrefactive.” Clostridium
spp. form endospores and have a strictly fermen-
tative type of metabolism. Most species are obli-
gate anaerobic, but C. perfringens is aerotolerant.
Whereas vegetative cells in obligate anaerobic
62
spp. are killed by exposure to O2, spores are able
to survive long periods of exposure to air. Infor-
mative reviews on clostridial diseases are offered,
e.g. by Onderdonk and Garrett (2010), or Stevens
and Bryant (2020).
C. perfringens produces several extracellular
toxins (Stevens and Bryant 2002). Alpha toxin is
a hemolytic toxin that stimulates platelet aggrega-
tion and upregulates transcription of adherence
molecules on endothelial and polymorphonuclear
(PMN) cells. It causes intravascular aggregates of
leukocytes, activated platelets, and fibrin, leading
to impaired tissue perfusion, contributing to the
expanding borders of tissue destruction seen in
clostridial gas gangrene. The absence of PMNs in
tissue is caused by sequestration within the adja-
cent vasculature. Theta toxin is another impor-
tant, pore-forming cytolysin that contributes to
pathogenesis by its effects on cells of the vascular
and immune systems, including cytotoxicity
towards PMN.
C. perfringens remains largely susceptible to
first-line antibiotics, including clindamycin and
tetracyclines, offering protein synthesis inhibi-
tion, as well as penicillin, but resistance towards
clindamycin and penicillin warrants attention.
Nowadays clostridial gas gangrene is increas-
ingly rare in developed countries, attributed,
e.g. to improved socioeconomic conditions and
sanitation (Sarani et al. 2009), whereas other clos-
tridial species are identified in sporadic cases. In
the INFECT study blood cultures grew clostridial
species in 11/409 (3%) of the cases (Madsen et al.
2019), demonstrating the relevance of the genus.
It is known that Clostridium septicum associated
with intestinal comorbidities is cause of infections
with multiple foci, however, in this study a statis-
tically significant association between positive
culture for Clostridium spp. and NSTI of the
lower limb was found (Fig. 5.1), with an odds
ratio of 9.47 (95% CI 1.85–92.85, p ¼ 0.01). The
increasing prevalence of colonic cancer as rele-
vant comorbidity may possibly affect future
impact of clostridial species in NSTI and calls
for attention.
S. Skrede et al.
5.4.5 Vibrio vulnificus
Vibrio vulnificus of the family Vibrionaceae is a
motile gram-negative halophilic, rod-shaped bac-
terium with functional flagella. It is a marine
organism present in the temperate areas of the
world where it is found in coastal areas, including
estuaries and brackish ponds holding an optimal
temperature of 18–26  C (Huang et al. 2016). It is
an environmental pathogen with three distinct
biotypes (Horseman and Surani 2011). It
produces neutralizing metabolites and radical
scavengers to avoid destruction in the gastric
acidic environment upon ingestion. Some
V. vulnificus strains express a polysaccharide cap-
sule that can protect the organism against phago-
cytosis. It produces endotoxin,
lipopolysaccharide (LPS), as well as a variety of
extracellular virulence factors such as the
exotoxins metalloprotease VvpE, the cytotoxic
hemolysin VvhA releasing iron thus enhancing
bacterial growth, and the multifunctional
autoprocessing repeats-in-toxins (MARTX)
toxin, involved in, e.g. bacterial dissemination
from the intestine by hematogenous spread. It
also spreads by contamination of preexisting
wounds. It rarely affects healthy individuals and
usually it causes only moderate disease (Horse-
man and Surani 2011). However, in high-risk
individuals, i.e. male gender, immunosuppres-
sion, viral hepatitis, hepatic cirrhosis, iron over-
load states, or chronic wounds of the lower limbs
(Chuang et al. 2019) the infection may spread
rapidly, producing severe soft tissue destruction
resembling that seen in gas gangrene. Usually it is
affecting lower limbs, where a prominent local
feature of infection is blister formation and
bronze discoloration, as well as sepsis. Today it
is a frequent microbial etiology particularly
reported from the Mexican Gulf area and, partic-
ularly, South East Asia (Huang et al. 2016; Choi
et al. 2020). In a retrospective study from Taiwan
on 121 culture positive cases of NSTI, 75 cases
(62%) were blood culture positive, 57% in
survivors, and 74% in non-survivors, a difference
5 Microbiological Etiology of Necrotizing Soft Tissue Infections
not reaching statistical significance (Chao et al.
2013). In a Korean study on association between
V. vulnificus DNA load in bacteremia and out-
come, the use of real-time PCR targeting the toxR
gene in serum demonstrated a positive association
between bacterial load and mortality, and a possi-
ble role for RT-PCR for prognostic purposes was
suggested (Kim et al. 2011). Although
V. vulnificus in general is susceptible to all rele-
vant antibiotics, its expanded distribution area
and detection of a carbapenemase in the closely
related and co-residing species V. cholerae
warrants systematic monitoring of its drug sus-
ceptibility (Bier et al. 2015).
In the INFECT study enrolling patients from
2013 to 2017, Vibrio vulnificus was cultured in
deep tissue culture in 3 cases only (0.8%), of
which one also grew in blood culture (Madsen
et al. 2019). Although still rare in Northern
Europe, it is conceivable that its present global
range will expand due to climate change and that
its prevalence even in areas such as Scandinavia
will increase.
5.4.6 Aeromonas hydrophila
Aeromonas hydrophila is primarily a fish and
amphibian pathogen, but it is associated with
cellulitis and as an opportunistic microbial cause
of NSTI, with risk factors resembling those seen
in Vibro-infection (Tsai et al. 2015). Aeromonas
hydrophila is a heterotropic, non-spore-forming
facultative anaerobic gram-negative, pleomorphic
rod-shaped bacterium with a monotrichous flagel-
lum ensuring motility. As Vibrio, it thrives in
aquatic environments, including sewage, fresh
and brackish water, and soil. It can multiply at
temperatures as low as 4  C. It produces several
factors believed to play roles in pathogenesis,
e.g. the cytotoxin aerolysin, lipase, and amylase.
Also, production of hemolysins is evident on
blood agar, where it produces β-hemolysis. Resis-
tance in Aeromonas is reviewed elsewhere
(Piotrowska and Popowska 2014). Aeromonas
species produce beta-lactamases of three different
classes, a class C cephalosporinase, a class D
penicillinase, and a class B metallo-beta-
63
lactamase (MBL), of which VIM and IMP are
found, leaving it resistant to penicillin, ampicillin,
and carboxypenicillins. Regional resistance
patterns have been described. Clinical studies
have demonstrated differences in antimicrobial
susceptibility between species. Most strains are
susceptible to trimethoprim-sulfamethoxazole
(TMP-SMX), second and third generation
cephalosporins, aminoglycosides, carbapenems,
chloramphenicol, and tetracyclines, although
resistance against TMP-SMX, cephalosporins,
aminoglycoside and, expectedly,
fluoroquinolones is described. In two indepen-
dent retrospective studies from Taiwan it was
the second most important gram-negative micro-
bial etiology of NSTI, constituting 10% (Hsiao
et al. 2008) and 23% of all cases, respectively
(Lee et al. 2011), only surpassed by Vibrio
vulnificus. There were no Aeromonas cases
among the 409 cases included in the INFECT
study (Madsen et al. 2019).
5.4.7 Polymicrobial Etiologies
Today most NSTIs with polymicrobial etiologies
are categorized as type 1 infections. In the origi-
nal publication when such a categorization was
first presented, some requirements were described
that need to be fulfilled to be a type 1 infection
(Giuliano 1977); At least two microbes must be
cultured and identified, of which there must be
one obligate anaerobic microbe. The second
microbe in type 1 infections can include faculta-
tive anaerobic species, such as staphylococcal
species, a number of streptococcal species or
Enterobacteriaceae. The authors questioned that
GAS is involved in genuine type 1 infections,
even in the presence of other pathogens, an opin-
ion supported by sequencing data obtained in
infected tissue samples of patients with NSTI
(Thänert et al. 2019). In this respect it should be
noted that in a retrospective study on 75 GAS
NSTI patients, additional pathogens were recov-
ered in 31% of cases (Lin et al. 2013). However,
no description of standards of sampling was
presented, and the published cases could possibly
include late post-operative contaminants.
64
Different to GAS, SD appears to be a candi-
date etiology for both type 1 and type 2 infections
(Madsen et al. 2019; Bruun et al. 2020). Catego-
rization of type 1 infections is supported by infor-
mation on head-neck focus, anogenital or
abdominal focus or post-operative infection, as
well as by supporting information on elevated
age, the many and frequent comorbidities, and
low functional status of patients (Stevens and
Bryant 2017).
Most NSTIs are polymicrobial infections,
reportedly comprising some 45–85% of all cases
(Giuliano 1977; McHenry et al. 1995; Elliott et al.
2000, 1996; Childers et al. 2002; Anaya et al.
2005). It has been advocated that in NSTI specific
combinations of bacterial species is neither
observed nor diagnostic (Anaya and Dellinger
2007), and it has even been concluded that no
specific combination of microbial species in
NSTI exists and that polymicrobial infections
are to be considered unpredictable regarding
composition (Rudkjøbing et al. 2016). Opposite,
in the study using dual RNA sequencing of
intraoperatively collected, infected, deep tissue
samples from the INFECT study it was
demonstrated strict correlations of co-occurrence
between species and concluded firmly that
polymicrobial communities in NSTIs are not ran-
domly associated. It was demonstrated that spe-
cies form highly interconnected networks.
Relationships were demonstrated, e.g. oral anaer-
obic species such as Fusobacterium,
Porphyromonas, Parvimonas, and Peptostrep-
tococcus co-reside, but not with Bacteroides. Fur-
thermore, when E. coli is associated with an
obligate anaerobic species in a polymicrobial
NSTI, this will be a member of the Bacteroides
family. Specific and sophisticated metabolic and
signaling interactions in these communities were
demonstrated (Thänert et al. 2019), by ways
reviewed elsewhere (Stacy et al. 2016). Based
on clinical studies, microbiological diagnostic
results and mechanistic studies of polymicrobial
infections, it may be that the role of obligate
anaerobic bacteria is underestimated (Brook and
Frazier 1995; Bjurlin et al. 2013).
In deep neck space infections as well as in
anogenital and abdominal NSTIs, anaerobic
microbes predominate (Brook 2012; Bjurlin
S. Skrede et al.
et al. 2013). Severe deep space head and neck
infections generally follow oral, dental, and pha-
ryngeal infections, involving the bacteria of the
original focus of infection. Obligate anaerobic
microbes that are recovered include Prevotella
spp., Porphyromonas spp., Fusobacterium spp.,
and Peptostreptococcus spp. However, they are
frequently accompanied by facultative anaerobic
species including viridans streptococci, and less
often also Staphylococcus aureus (Poeschl et al.
2010). Type 2 infection with GAS is also a possi-
bility in the head and neck area (Brook 2012)
although uncommon (Madsen et al. 2019).
The majority of anogenital infections are
polymicrobial, and the most frequently cultured
organisms are obligate anaerobic species,
Enterobacteriaceae and Enterococcus spp.
(Bjurlin et al. 2013; Czymek et al. 2013). In the
INFECT study polymicrobial etiology confirmed
by routine diagnostic culture methods of the
laboratories of study hospitals was strongly
associated with infection of the anogenital and
abdominal region (Fig. 5.1), with an OR of 8.01
(95% CI 4.95–13.15, p < 0.001) (Madsen et al.
2019). Expectedly, in the anogenital region
microbes with troublesome antimicrobial resis-
tance are identified, e.g. MRSA, ESBL-producing
E. coli and Klebsiella spp., as well as Pseudomo-
nas sp. (Chia and Crum-Cianflone 2018), but also
antimicrobial resistance in the Bacteroides
fragilis group may be a concern (Nagy et al.
2011).
5.4.8 Microbial Etiologies
Documented by
the INFECT Study
As detailed in this chapter on microbiology,
numerous cases series and surveillance studies
of NSTI have been conducted. Notwithstanding,
the documentation of microbial etiologies has
often been hampered with low quality of data. In
the INFECT study strict standard operating
procedures for obtaining microbiological samples
were developed, that were adhered to in 407/409
cases (99.5%). Microbial etiologies were
established by culture in 94% of the cases through
routine diagnostic culture methods of the
5 Microbiological Etiology of Necrotizing Soft Tissue Infections
laboratories of study hospitals , while in 26 cases
(6%), no microbe was identified (Madsen et al.
2019). In 144 (35%) of the cases there was a
positive blood culture and in 378 (92%) there
was a culture positive sample from a sterile tissue
site. Associations of anatomical NSTI locations,
microbial etiologies, and thereby NSTI categories
were documented with rigor. Figure 5.2 offers a
visualization of the detailed microbiological
results that were obtained. Polymicrobial
infections were found in 204 (50%) patients,
whereas 179 (44%) had a monomicrobial infec-
tion, in which GAS was found in 126 (31%).
Most polymicrobial infections were located to
the pelvic region (118/204, 58%) or confined to
the head-neck area (43/204, 21%) (Madsen et al.
2019). Anaerobes were frequently identified in
polymicrobial infections. Furthermore, in the
anogenital tissue samples analyzed by 16S
rRNA sequencing, a strong association was
detected between E. coli and Bacteroides sp.,
almost entirely (i.e. 80%) comprised by
B. fragilis. Differently, in head-neck infections
there were strong associations between oral
anaerobic pathogens, in particular among species
belonging to five genus; Prevotella spp.,
Porphyromonas spp., Parvimonas spp.,
Peptostreptococcus spp., and Fusobacterium
spp. (Thänert et al. 2019). The results implicate
that in anogenital infections there is a higher risk
of both clindamycin resistant Bacteroides sp. and
extended spectrum beta-lactamase producing
Enterobacteriaceae when compared and
contrasted to NSTI of other anatomical areas,
including head-neck space infections. Finally, in
the INFECT study a substantial diversity in
microbial etiology was documented. Altogether
more than 50 different bacterial species were
found to be implicated in NSTIs (Thänert et al.
2019).
5.4.9 Non-culture Based
Microbiological Methods
in NSTI
Arguably, standard microbiological culture may
fail to identify unusual or fastidious organisms
65
and they can misrepresent relative abundance of
species. Furthermore, culture dependent methods
are time consuming. Some of these problems can
be dealt with using culture-independent methods.
Recently, use of a multiplex PCR with primers
allowing simultaneous detection of GAS, GBS,
MRSA, Vibrio vulnificus, and Aeromonas
hydrophila was reportedly successful, and the
authors concluded that PCR of tissue specimens
may be a useful and rapid diagnostic method in
NSTI (Tsai et al. 2019). Implementation of next-
generation molecular sequencing in routine
diagnostics in NSTIs could give a more complete
picture of the etiology, but few such studies have
been undertaken. In a novel study using next-
generation 16S rRNA gene sequencing
(NGS16S) to determine how often cultivable
organisms in complex polymicrobial samples
are not reported by standard culture,
20 bronchoalveolar lavage samples were cultured
and identified by NGS16S analysis of DNA
extracted directly from samples or from washed
culture plates. Only 21% of identities were
reported by standard culture. However, 96% of
microbes were cultivable. This study also
demonstrated that by using direct NGS16S the
same predominant organism was detected in
50% of samples when compared to identification
by culture. When predominant organisms dif-
fered, NGS16S most often detected anaerobes
(Cummings et al. 2020). Zhao-Fleming et al.
(2019) conducted a study to identify microbial
etiologies in human NSTIs, challenging previous
culture based data. Using 16S rRNA sequencing
they identified genera commonly found in NSTIs
that included Prevotella, Bacteroides,
Peptoniphilus, Porphyromonas, and Enterococ-
cus. Associations of bacterial constituents and
patient outcomes were detected, and data were
generated to support a recommendation of
increasing anaerobe antibiotic coverage during
the treatment of NSTIs (Zhao-Fleming et al.
2019).
As part of the INFECT project tissue biopsies
obtained preoperatively from 148 patients with
NSTI were analyzed to explore pathophysiologi-
cal mechanisms of monobacillary NSTIs compar-
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.
5 Microbiological Etiology of Necrotizing Soft Tissue Infections
(Thänert et al. 2019). Microbial community
profiling using 16S rRNA sequencing was
integrated with transcriptional analysis of host
and microbe, using dual RNA sequencing
(RNA-seq). It was shown that NSTIs were either
dominated by a single bacterial pathogen, usually
GAS, or associated with a diverse spectrum of
polymicrobial communities. In polymicrobial
infections Prevotella spp., Porphyromonas spp.,
Parvimonas spp., Fusobacterium spp.,
Peptostreptococcus spp., and Bacteroides
sp. were frequently detected. The diversity and
composition of the bacterial communities were
highly dependent on the focus of the infection,
supporting culture based data for the entire
INFECT patient cohort (Madsen et al. 2019,
Fig. 5.2). Using 16S rRNA sequencing, Thänert
and coworkers also showed that the bacterial
diversity in NSTIs of the upper and lower
extremities was significantly lower than in
NSTIs localized at the head/neck or anogenital
region (Thänert et al. 2019). The highest fre-
quency of polymicrobial infections was seen in
anogenital infections, where Bacteroides was pre-
dominant. In head and neck infections oral genera
Prevotella, Porphyromonas, and Fusobacterium
dominated, lending support to the hypothesis that
there is a connection between bacterial commu-
nity composition in NSTIs and commensals
associated with natural ecological niches. It was
also demonstrated that in polymicrobial NSTI
bacterial communities are not randomly
associated (Thänert et al. 2019). These
communities appear attractive targets for devel-
opment of novel therapies.
Use of nucleic acid based identification
methods has proven important in studies on path-
ogenesis of both polymicrobial and
monomicrobial infections, and studies on GAS
NSTI pathogenesis in primates (Kachroo et al.
2020) and mice (Hirose et al. 2019) using RNA
sequencing have recently been published.
Chapter 10 in this book covers this subject. At
present there is no role for use of nucleic acid
based methods in rapid identification of microbial
etiologies of NSTIs in the emergency setting, but
advances are made that makes such a goal within
reach.
67
5.5 Future Perspectives
NSTI are severe infections frequently leading to
septic shock and multiorgan failure (Madsen et al.
2019). Treatment is highly specialized, multidis-
ciplinary and very resource demanding, usually
taking place in an intensive care unit. Patients are
at risk of poor outcomes, and NSTI mortality rates
in the range of 20–30% and extremity amputation
rates of 10–20% are reported (Peetermans et al.,
2020).
There is a need for improvement of these
results. Of several reasons, strengthening the
microbiological diagnostic handling of patients
with NSTI is of great importance to improve
patient outcomes. Firstly, in many severe
infections there is an association between chances
of survival and the identification of
microbiological etiology and its antimicrobial
resistance properties. Most NSTIs are
polymicrobial. In these etiologies there are
emerging risks of antimicrobial resistance,
including clindamycin resistant obligate anaero-
bic species and β-hemolytic streptococci, ESBL
producing Enterobacteriaceae or, infrequently,
MRSA. Identification will aid in selection of effi-
cient antimicrobial treatment.
Secondly, medical microbiological studies can
spur future invention of tools to support treatment
decisions (Rello et al., 2018). NSTIs are highly
heterogeneous infections reflected by the multi-
tude of etiologies reviewed herein. Any future
development of individualized therapeutic
strategies will rely on identification of microbio-
logic etiology. Only then patient stratification
necessary to develop therapies targeting the host
and microbe interplay can find place.
It is a challenge to conduct studies on NSTIs.
Most clinical studies in this field have been focus-
ing on patient characteristics, whereas studies
arguably have focused insufficiently on the
pathogens and their interaction with the host. In
NSTIs, the systems medicine approach is in an
early phase, but studies using translation
approaches in well-defined patient groups are
needed. In this respect, identification and studies
on the NSTI pathogens are crucial, underlining
68
the importance of microbiology both in every day
patient handling as well as in clinical studies of
NSTI, in order ultimately to create individualized
therapies.
Acknowledgment This work was supported by the
European Union Seventh Framework Programme:
(FP7/2007-2013) under the grant agreement 305340
(INFECT project), NordForsk (Project no. 90456,
PerAID), the Norwegian Research Council under the
frame of ERA PerMed (Project 2018-151, PerMIT) and
The Swedish Research Council Framework Grant in
infections and antibiotics (2014-8609-117204-47).
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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
Department of Medicine, Haukeland University Hospital,
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
# Springer Nature Switzerland AG 2020 73
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_6
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
as opposed to 48% of the SD cases and just 2 out
of 15 GBS cases. Staphylococcus aureus was
found together with GAS in 6% of the cases.
In this chapter we give an overview of clinical
aspects of NSTIs caused by GAS or SD in light of
the INFECT study. This cohort provides a unique
set of clinical data describing what characterizes
these infections. The pathogenesis of streptococ-
cal NSTIs is explored in Chap. 9.
6.2 Epidemiology and Risk Factors
6.2.1 Incidence
Estimates of incidence for streptococcal NSTIs
can be derived from studies of NSTIs in general
or from surveillance studies of invasive strepto-
coccal infection. Either way, the lack of a uniform
definition of NSTIs is a major reason for the great
uncertainty attached to estimates of incidence. A
few nation-wide studies have been performed
showing highly variable incidence numbers of
NSTIs overall ranging from below 1 to above
10 per 100,000 population (Mulla et al. 2007;
Das et al. 2011; Oud and Watkins 2015;
Audureau et al. 2017; Chuang et al. 2018; Psoinos
et al. 2013), but different methods were used.
With respect to studies of NSTIs reporting
microbiological data, most are retrospective
single-center case series. The proportion of GAS
infections has varied greatly, as described in
Chap. 5, ranging from below 10% to 50% or
more. From the Scandinavian countries, three
smaller single-center studies have reported beta-
hemolytic streptococci in 29–52% of the cultured
cases, predominantly GAS (Skovsen et al. 2010;
Brink et al. 2014; Nordqvist et al. 2015).
Data on NSTIs caused by GAS is also avail-
able from surveillance studies of invasive GAS
and SD disease. Estimated incidence rates of
GAS NF in large surveillance studies from
industrialized countries performed since 1992
have been in the range 0.08–0.60/100,000/year
(Kaul et al. 1997; O’Brien et al. 2002; Sharkawy
et al. 2002; Tyrrell et al. 2005a, 2018; Ekelund
et al. 2005a, b; Hollm-Delgado et al. 2005;
O’Loughlin et al. 2007; Darenberg et al. 2007;
75
O’Grady et al. 2007; Lamagni et al. 2008a). As
for invasive GAS disease in general, great tempo-
ral fluctuations have been observed. Striking local
peaks in incidence have been described
(Cartwright et al. 1995; Barnham et al. 1997;
Erdem et al. 2005). In Norway, the mean inci-
dence of GAS NF registered by national surveil-
lance for the period 2010–2014 was 0.3/100,000/
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
may illustrate different methods of inclusion and
possible underreporting in microbiological sur-
veillance studies. Internationally, significantly
higher rates of GAS NSTIs and other invasive
GAS disease have been observed in disadvanta-
geous populations (Le Hello et al. 2010; Rudolph
et al. 2016).
Several cases of NSTIs attributed to group C
and G streptococci, presumably mostly SD, have
been published since a report in Lancet in 1984
(Gaunt et al. 1984), but the disease is rare com-
pared to GAS NSTIs. Large single- and multicen-
ter studies of NSTIs indicate that GAS is found
around 5–20 times more often than SD
(Kulasegaran et al. 2015; Bair et al. 2009;
Huang et al. 2011; Cheng et al. 2012; Bruun
et al. 2013; Chen et al. 2011). A similar ratio
between NSTIs caused by GAS and SD is
suggested by population-based surveillance stud-
ies of invasive streptococcal disease (Ekelund
et al. 2005a; Rantala et al. 2009a). Sometimes,
SD is found as part of a polymicrobial etiology
(Kulasegaran et al. 2015; Cheng et al. 2012; Chen
et al. 2011). GBS is found just as infrequent as SD
in NSTIs, occasionally as a single pathogen but
often together with other bacteria (Kulasegaran
et al. 2015, Cheng et al. 2012, Chen et al. 2011).
The INFECT study was not designed to give
exact epidemiological data. All study centers are
referral hospitals for NSTIs that offer hyperbaric
oxygen therapy, and most enrolled patients were
transferred from other hospitals. Some categories
are probably often not referred. These include
those without a fair chance of survival as well as
low risk patients. In Denmark, however, treat-
ment of NSTIs is more centralized than in
Norway and Sweden, and most hospitals send
76
their NSTIs cases to Rigshospitalet, i.e. the study
hospital. During 51 months of inclusion, 65 cases
with NSTIs caused by GAS were included at
Rigshospitalet. In a population of 5.6 million
this corresponds to an incidence of 0.3 cases per
100,000 per year. Because not all cases are trans-
ferred, the true incidence must be somewhat
higher. This estimate is comparable to other
data, including surveillance data from Norway
(www.msis.no). In the INFECT study overall,
NSTIs caused by SD represented 7% of the
cases, corresponding to an incidence of 0.07 per
100,000 per year, suggesting that the contribution
of SD might be greater than previously reported.
This may be related to the increase of invasive SD
infection in general (Oppegaard et al. 2015).
6.2.2 Risk Factors
In an early study during the upsurge of invasive
GAS infections in the industrialized world,
Chelsom and co-workers found that seven of
13 cases with GAS NSTIs were previously
healthy (Chelsom et al. 1994). The ability of
GAS to cause NSTIs in otherwise healthy people
has later been confirmed by other studies (Kaul
et al. 1997; Hassell et al. 2004). In the INFECT
study, more than a third of the GAS cases was
without known comorbidity. However, several
studies of invasive GAS disease in general as
well as of GAS NSTIs have identified various
risk factors. A study using non-invasive skin
and soft tissue infections as a control group
identified high age, diabetes mellitus, liver cirrho-
sis, and gout as independent risk factors for
NSTIs (Lin et al. 2013). In another study, com-
paring GAS NSTIs with other invasive skin and
soft tissue infections caused by GAS, alcoholism
and diabetes were more frequently identified in
the NSTIs group, but absence of cancer was the
only predictor of NSTIs in the adjusted analysis
(Sharkawy et al. 2002). Therefore, it has been
unclear what risk factors that are associated
with NSTIs compared to other invasive skin and
soft tissue infections. However, previous studies
were only powered to detect large differences.
When compared to non-necrotizing streptococcal
T. Bruun et al.
cellulitis, streptococcal NSTIs of the INFECT
cohort were associated with a history of blunt
trauma, absence of chronic wound and skin dis-
ease, and a lower BMI (Bruun et al. 2020). The
study confirms previous reports suggesting that
blunt trauma may be of importance
(Monnickendam et al. 1997; Factor et al. 2003;
Nuwayhid et al. 2007) and that streptococcal
NSTIs in most cases do not have an obvious
portal of entry (Kaul et al. 1997; Simonart et al.
2001). More than half of the GAS and SD cases of
the INFECT cohort had no preceding event. Stud-
ies in children, however, have found that varicella
is an important risk factor (Laupland et al. 2000;
Tyrrell et al. 2005b; Minodier et al. 2009; Imohl
et al. 2011). Not many studies have directly com-
pared risk factors of GAS NSTIs with NSTIs
caused by other etiologies. However, two studies
suggest that diabetes mellitus and immunosup-
pression are more common in NSTIs without
GAS (Bair et al. 2009; Cheng et al. 2012). A
recent study confirmed that GAS etiology is
unlikely in NSTIs of immunosuppressed
individuals (Urbina et al. 2019). Moreover, stud-
ies of NSTIs of all etiologies in general report a
higher frequency of comorbidity compared to
studies of GAS NSTIs. Also in the INFECT
cohort, comorbidities were more common
among non-streptococcal than streptococcal
cases (Bruun et al. 2020), and also more common
in SD compared to GAS cases, malignancy in
particular (Table 6.1). Overall the INFECT
study found that possible predisposing factors
were frequent, particularly in NSTIs caused by
SD, but the ability of GAS to cause NSTIs in
previously healthy individuals was confirmed.
6.2.3 Molecular Epidemiology
Serological M protein typing and later genetic
typing based on the emm gene and different
house-keeping genes (MLST typing) have been
the main tools to follow the epidemiology and
microbial diversity of invasive and non-invasive
GAS and SD strains. More recently, comparative
analysis of full genome sequences (WGS) has
emerged as the most powerful tool to provide
6 Beta-Hemolytic Streptococci and Necrotizing Soft Tissue Infections 77

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
35
Number with emm type
30
25
20
15
10
GAS
Fig. 6.1 emm type distribution with rates of septic shock
and 90-day mortality in 82 GAS and 11 SD cases of
necrotizing soft-tissue infection. Forty-four GAS isolates
and 16 SD isolates were not available for analysis.
insight into phylogenetic relationships and diver-
sity of strains as well as evolution and
characteristics of epidemic strains (McMillan
et al. 2013). Certain emm types have been
associated with invasive infection and disease
severity. In Europe and North America emm1,
emm28, emm89, emm3, emm12, emm4, and
emm6 have predominated among invasive GAS
strains since year 2000 (Gherardi et al. 2018). The
upsurge of invasive GAS disease since the 1980s
has been associated with certain GAS strains with
increased fitness, a hypervirulent emm1 clone in
particular (Barnett et al. 2018). In the INFECT
study, emm typing, MLST typing as well as WGS
have been performed and demonstrate great
diversity regarding involved strains (Bruun et al.
2020). As in most surveillance studies of invasive
GAS infections, however, emm1, emm3, and
emm28 were predominant emm types in the
INFECT cohort (Fig. 6.1). The fact that more
than 70% of the isolates belonged to only three
emm types are in line with evidence that some
T. Bruun et al.
Septicshock Dead
SD
Abbreviations: GAS group A Streptococcus, Streptococ-
cus pyogenes, SD Streptococcus dysgalactiae. Reprinted
from Bruun et al. (2020), with permission
emm types have a greater ability to cause invasive
infections (Nelson et al. 2016; Luca-Harari et al.
2009). Even in SD there was an association to
strains previously associated with severe
infections, with a predominance of the emm type
stG62747 (Oppegaard et al. 2017). The INFECT
study could not identify any clear relation
between emm type and septic shock or mortality,
probably reflecting the magnitude of microbial
and host-related factors contributing to severity
and death.
6.3 Clinical Features and Diagnosis
The clinical features of NSTIs are primarily
known from studies of NSTIs in general (see
Chap. 4), but a few studies reporting clinical
details on GAS NSTIs have been performed.
Clinical data on SD, however, have been avail-
able only from case reports and a retrospective
study of streptococcal NSTIs that included nine
6 Beta-Hemolytic Streptococci and Necrotizing Soft Tissue Infections
cases caused by SD (Bruun et al. 2013). The latter
study found that these patients were older, had a
higher burden of comorbidities and had
anatomically more superficial disease than the
GAS patients. Previous studies have reported
that GAS NSTIs are located in the lower
extremities in more than half the cases (Kaul
et al. 1997; Bruun et al. 2013; Lin et al. 2013).
One of the hallmarks of GAS NSTIs is severe
systemic toxicity. In studies comparing GAS
NSTIs with other invasive skin and soft tissue
infections caused by GAS, hypotension has sig-
nificantly more often been observed in the NSTIs
group (Lamagni et al. 2008b; Kittang et al. 2010;
Sharkawy et al. 2002). The frequency of strepto-
coccal toxic shock syndrome (STSS) in GAS
NSTIs is reported to be in the range 40–68%, far
greater than in other invasive GAS disease
(Darenberg et al. 2007; Lamagni et al. 2008b;
Svensson et al. 2000; Kaul et al. 1997; Lin et al.
2013; Davies et al. 1996; Bruun et al. 2013). In
most studies of NSTIs in general, the overall
frequency of septic shock is lower (Kao et al.
2011; Huang et al. 2011; Das et al. 2012; Keung
et al. 2013). Accordingly, severe systemic toxic-
ity is thought to be more typical of type 2 NSTIs
than of type 1 disease. In a comparative study,
however, shock was not more frequent among the
GAS NSTIs cases (Urbina et al. 2019). In addi-
tion, some non-streptococcal types of NSTIs are
associated with an increased risk of shock, includ-
ing monomicrobial infections with Vibrio
vulnificus or Klebsiella pneumoniae (Cheng
et al. 2012; Klontz et al. 1988). Detailed data on
local symptoms and signs in streptococcal NSTIs
are scarce except for case reports which probably
tend to present the most severe cases. Some data
are available from larger retrospective series that
report unspecific skin changes and no obvious
portal of entry in most of the cases (Sharkawy
et al. 2002; Simonart et al. 2001). As for NSTIs in
general, bullae are found in a minority (Lin et al.
2013). The frequency of other classical NF signs
in GAS disease is uncertain.
In the INFECT study, septic shock was more
common in GAS disease compared to
polymicrobial non-streptococcal cases (Bruun
et al. 2020). Furthermore, septic shock was
related to higher age rather than younger, in
79
contrast to studies of invasive GAS where STSS
was associated with younger age (Hoge et al.
1993; Nelson et al. 2016; Lamagni et al. 2008b).
In both GAS and SD patients, more than 70%
of the infections were located in the extremities
(Table 6.1). Upper extremity infections were typ-
ically caused by GAS and not SD. Bacteremia
and septic shock were more frequent in GAS
cases compared to cases of SD and polymicrobial
non-streptococcal NSTIs. This is in accordance
with major differences in the pathogenesis of
streptococcal compared to polymicrobial
infections, as demonstrated in a molecular study
of cases from the INFECT cohort (Thanert et al.
2019). Systemic severity according to presence
of septic shock, SOFA score, and laboratory
abnormalities was not more pronounced in
monomicrobial GAS disease compared to cases
involving co-pathogens, suggesting that GAS is
the dominant pathogen also in the latter type of
infection. Severe pain was prevalent in both GAS
and SD cases, but over 50% of the patients did not
receive opioid drugs before their first surgery. At
surgery, the finger test, a pathognomonic sign of
tissue necrosis, was not always positive. Muscle
affection was registered in about half of the
patients, including 13 cases consistent with myo-
sitis only, i.e. without signs of destruction of
subcutaneous tissue. CRP was in most patients
highly elevated, but 28% had preoperative values
below 200 mg/L. A clearly elevated creatinine of
150 μM preoperatively was found in 56% of
patients. Laboratory Risk Indicator for Necrotiz-
ing Fasciitis (LRINEC) score, a laboratory based
diagnostic tool for necrotizing fasciitis, is calcu-
lated based on preoperative measurements. A
score below 6, previously associated with low
risk of NSTIs (Wong et al. 2004), was found in
15% of the GAS and SD cases overall,
demonstrating suboptimal sensitivity among the
streptococcal cases. This was the conclusion also
in a recent systematic review (Fernando et al.
2018). Clinical features and surgical findings are
still the cornerstones in the diagnostic work
up. Clinical characteristics may also be of great
use in making an etiological diagnosis. As
demonstrated in a recent retrospective study,
where abdominal location and immunodeficiency
were strongly predictive of non-streptococcal
80
etiology (Urbina et al. 2019), the INFECT study
has shown major clinical differences separating
streptococcal and polymicrobial NSTIs. Both the
strong association of streptococcal NSTIs and
extremity location (Madsen et al. 2019) and
other characteristics may give diagnostic clues
on probable etiology and important guidance for
personalized treatment.
6.4 Treatment and Prognosis
6.4.1 Treatment
Surgery is the main treatment modality in NSTIs
regardless of etiology. This is due to the local
aggressiveness of disease, ischemia, tissue necro-
sis, and the limitations of antibiotic treatment
(Stevens et al. 2014). Surgical intervention must
be performed as early as possible when NSTIs are
suspected, to confirm the diagnosis and to remove
devitalized, infectious tissue, improve circulation
and oxygen supply, thereby halting the progress
of disease. The demonstration of the clear associ-
ation between a delay in surgery and increased
mortality underscore the importance of early sur-
gery (Wong et al. 2003). Misdiagnosis at admis-
sion is the main reason for surgical delay (Goh
et al. 2014). Daily revisions are recommended as
long as there is a need for debridement (Stevens
et al. 2014). In the INFECT study, however,
patients undergoing surgery later than 24 h after
admission did not have increased mortality
among the GAS cases. This could be due to
rapid identification and initiation of appropriate
treatment for the most severely sick patients, as
suggested by earlier surgery among patients with
shock.
In the absence of clinical trials comparing the
efficacy of different antibiotic regimens, current
recommendations rely on trials in other severe
infections, studies of etiology and resistance,
and of pharmacokinetic and pharmacodynamic
considerations, as further described in Chap. 7.
Empiric broad-spectrum therapy covering both
aerobic and anaerobic agents is warranted until a
T. Bruun et al.
probable bacteriological diagnosis is established.
Local frequency of antibiotic resistance should be
taken into consideration. In streptococcal NSTIs
or STSS, clindamycin is recommended in addi-
tion to penicillin (Stevens et al. 2014; Peetermans
et al. 2019). Clindamycin is known to inhibit
toxin release and thereby cytokine production
(Mascini et al. 2001). Furthermore, clindamycin
is superior to β-lactam antibiotics in infections
with large amounts of bacteria in a stationary
growth phase, which often is the case with
NSTIs (Eagle 1952; Stevens et al. 1988). Both
animal experiments and observational studies
have demonstrated increased survival with
clindamycin (Linner et al. 2014; Stevens et al.
1988; Zimbelman et al. 1999; Mulla et al. 2003;
Carapetis et al. 2014). In the INFECT study,
receiving clindamycin before inclusion at the
study hospital was not associated with increased
survival. As with surgery, this could reflect rapid
identification and initiation of appropriate treat-
ment for those in greatest need of such treatment.
Intravenous immunoglobulin (IVIG) is used
by some centers in STSS or streptococcal NSTIs
in order to neutralize streptococcal toxins. A
recent meta-analysis of clindamycin-treated
STSS patients from one randomized and four
non-randomized studies found that IVIG was sig-
nificantly associated with increased survival
(Parks et al. 2018). IVIG-therapy was associated
with increased survival in streptococcal NSTIs
also in the INFECT study (Bruun et al. 2020).
Moreover, a study on plasma of INFECT patients
demonstrated a dose-related toxin inhibition,
providing a mechanistic correlate that also
supports a possible benefit of IVIG (Bergsten
et al. 2020). This should encourage renewed
efforts to conduct a sufficiently powered
randomized clinical trial in GAS NSTIs.
Use of hyperbaric oxygen therapy (HBOT) in
NSTIs is supported by some observational data,
but results are diverging (Peetermans et al. 2019).
In the INFECT study, the case-fatality rate was
lower in HBOT treated GAS NSTIs patients, but
the effect of HBOT is difficult to assess due to
selection bias. Randomized trials are needed to
6 Beta-Hemolytic Streptococci and Necrotizing Soft Tissue Infections
define the role of HBOT in the treatment of strep-
tococcal NSTIs (Peetermans et al. 2019).
6.4.2 Prognosis
Mortality estimates are highly variable in differ-
ent studies of NSTIs caused by GAS. A large
European surveillance study of 254 cases of
GAS NSTIs included in 2003–2004 registered a
case-fatality-rate (CFR) of 32%, but CFR varied
greatly between different countries (Lamagni
et al. 2008a). In Sweden it was 19% (Darenberg
et al. 2007). Other surveillance studies with more
than 50 cases have reported a CFR varying
between 20% and 66% (O’Loughlin et al. 2007;
Lepoutre et al. 2011; O’Brien et al. 2002; Kaul
et al. 1997; Sharkawy et al. 2002; Monnickendam
et al. 1997). Two single-center studies of GAS
NSTIs reported a CFR of 16% and 11%, respec-
tively (Lin et al. 2013; Bruun et al. 2013). The
CFR is considerably lower in other invasive skin
and soft tissue infections caused by GAS
(Sharkawy et al. 2002).
Factors associated with increased rate of death
in GAS NSTIs include high age, comorbidities,
hypotension/STSS, muscle involvement, and bac-
teremia (O’Brien et al. 2002; Kaul et al. 1997;
Sharkawy et al. 2002; Bruun et al. 2013). In
studies of NSTIs in general, also surgical delay
has shown a strong association with mortality
(Wong et al. 2003; Childers et al. 2002; Elliott
et al. 1996; McHenry et al. 1995; Wall et al.
2000).
Data on mortality and prognostic factors in
NSTIs caused by SD are scarce. Among case
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
CFR was 20% and 33%, respectively. In the same
study there was an association of CFR with age
and heart disease (Bruun et al. 2013). Studies
comparing invasive GAS and SD disease in gen-
eral indicate that death among SD cases is more
often related to age and comorbidity and more
rarely to septic shock, than in GAS infections
(Ekelund et al. 2005a; Rantala et al. 2009b).
81
In the INFECT cohort, the frequency of
amputations was 16% among GAS and 11%
among SD cases (Bruun et al. 2020). The
90-day CFR was 10% for GAS and significantly
lower compared to polymicrobial
non-streptococcal cases (20%). The study
identified several independent variables
associated with 90-day CFR, including increased
age (odds ratio (OR) 1.15, per year), male sex
(OR 5.09), septic shock (OR 1.96), and IVIG not
administered (OR 3.15). Comorbidity was, how-
ever, not associated with mortality.
Associations of mortality and virulence factors
have been studied in invasive GAS infections.
Certain emm types have been associated with
increased mortality in invasive GAS infections
(Olafsdottir et al. 2014), even in NSTIs (Luca-
Harari et al. 2009). This association may be due to
virulence gene profiles, including superantigens,
associated with emm types and not the M protein
itself (Lintges et al. 2010). For superantigen
genes, association studies are diverging
(Commons et al. 2014; Lintges et al. 2010;
Imohl et al. 2017), but in the hypervirulent
M1T1 clone, speA2 has definitely been essential
for increased virulence (Barnett et al. 2018). The
role and mechanisms of superantigens are further
elaborated in Chap. 9.
In the INFECT study, the virulence gene
profiles of GAS reflected the emm type distribu-
tion. A high proportion with superantigen genes
speA and speG corresponds to predominance of
emm1 and emm3 types. No clear association of
emm types or other virulence genes with severity
was found, but for most emm types and virulence
gene profiles the numbers were low.
6.5 Conclusions and future
perspectives
The high severity and other specific features pre-
viously reported in NSTIs caused by β-hemolytic
streptococci were outlined in detail by the
INFECT study. The high frequency of toxic
shock, ICU treatment, and death underscores the
severity of the disease. The INFECT study also
82
supports previous research identifying blunt
trauma as an important risk factor for streptococ-
cal NSTIs. The contribution of S. dysgalactiae to
the disease burden was also confirmed. The sub-
stantial clinical and bacterial diversity observed
underscores the multifactorial background of
streptococcal NSTIs. Improved clinical tools and
predictive biomarker profiles are needed to early
discriminate between NSTIs in the need of sur-
gery and other severe soft tissue infections. Other
important knowledge gaps remain in the field, not
the least with respect to effects of treatment. IVIG
is among several factors associated with increased
survival in the INFECT study and other observa-
tional studies, but randomized trials are needed.
The high severity of streptococcal NSTIs
mandates also intensified clinical research on
other drugs targeting the cytokine effects in
NSTIs. At the same time, research should still
focus on the identification of bacterial traits,
host–pathogen interactions, and pathogenic
mechanisms associated with different clinical
phenotypes, and in this way continue the work
of the INFECT project and other studies of strep-
tococcal pathogenesis. Thereby, new targets for
therapy can be identified and a new platform for
tailored therapy can be created.
Acknowledgement Financial support: The INFECT
study was supported by the European Union’s Framework
Programme 7 (grant number FP7/2007–2013 305340).
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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

# Springer Nature Switzerland AG 2020 87

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_7
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
are frequently associated with monomicrobial
NSTIs (Madsen et al. 2019; Kim et al. 2019;
Louis et al. 2019). The water exposure associated
pathogens Vibrio vulnificus and Aeromonas
hydrophila have traditionally been regarded as
rare causes of monomicrobial NSTI, predomi-
nantly affecting subjects with pre-existing
chronic liver conditions (Stevens and Bryant
2017; Peetermans et al. 2020). However, recently
Hsiao et al. reported Vibrio spp. and Aeromonas
spp. to be responsible for approximately 20% of
the NSTIs in Taiwan (Hsiao et al. 2020). Increas-
ing incidence has also been noted on the Ameri-
can continent, indicating that these pathogens are
an emerging threat in several geographic regions
(King et al. 2019; Dechet et al. 2008). Clostridial
myonecrosis is an infrequent NSTI manifestation
caused by Clostridium perfringens, although
other clostridial species are occasionally
implicated (Stevens et al. 2012).
Prevalence of clinically relevant antimicrobial
resistance in NSTIs exhibit substantial regional
variations, and knowledge on local epidemiology
is essential to tailor the antibiotic treatment.
Although studies on NSTIs detailing antimicro-
bial resistance profiles are scarce to non-existent,
susceptibility data for invasive disease in general
is available for several of the key pathogens.
7.2.2 Antimicrobial Resistance
in b-Hemolytic Streptococci
The β-hemolytic streptococcal species
S. pyogenes, S. agalactiae, and S. dysgalactiae
have all been implicated in NSTIs, although
S. pyogenes is by far the most frequent (Bruun
et al. 2020; Das et al. 2012). These pathogens
have historically been regarded as unequivocally
sensitive to penicillin, but some concerns have
arisen the past decades. In 2008 the first reports
of S. agalactiae exhibiting reduced susceptibility
to penicillin were published, revealing increased
minimum inhibitory concentrations (MICs) to
several β-lactam antibiotics related to point
mutations in the penicillin binding protein pbp2x
(Dahesh et al. 2008). More recently, similar
mutations and reduced penicillin susceptibility
89
have been detected in both S. dysgalactiae and
S. pyogenes (Fuursted et al. 2016; Vannice et al.
2019). Although these resistance phenotypes are
still exceedingly rare, the mutational basis for
resistance is highly analogous to that of
S. pneumonia, where penicillin resistance has
gradually become widespread. All BHS are still
fully susceptible to broad spectrum β-lactam
antibiotics (e.g. cephalosporins and
carbapenems), as well as vancomycin, linezolid,
and daptomycin, but these agents are rarely
indicated in monomicrobial BHS NSTIs (Farrell
et al. 2013). Penicillin remains the drug of choice
for treating BHS infections, but sustained epide-
miological attentiveness is definitely warranted to
monitor the antimicrobial resistance trends in
these streptococcal species.
A significant increase in resistance to erythro-
mycin and clindamycin in BHS has been noted
over the past two decades in several regions.
Annual national surveillance reports from the
United Kingdom (UK) have revealed the rates
of clindamycin resistance in S. dysgalactiae to
increase gradually from 6% in 2004 to 25% in
2017 (Public Health England 2018). In the United
States of America (US), the clindamycin resis-
tance rates for S. agalactiae currently
approximates 50%, causing the Centers for Dis-
ease Control to revise their infectious diseases
guidelines (CDCgov 2010). Even higher rates of
resistance in BHS have been reported from south
east Asia (Lu et al. 2017). Conversely, in a large
prospective study of NSTIs conducted in northern
Europe, the prevalence of clindamycin resistance
in BHS was below 5%, underscoring the impor-
tance of taking regional resistance epidemiology
into consideration (Bruun et al. 2020).
7.2.3 Antimicrobial Resistance
in S. aureus
Staphylococcus aureus is a potent human patho-
gen, capable of causing the entire spectrum of
skin and soft tissue infections, including occa-
sionally implicated in NSTIs. The penicillinase-
stable penicillins (e.g. oxacillin and historically
methicillin) have been the cornerstone in
90 O. Oppegaard and E. Rath

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
Staphylococcus aureus (MRSA) emerged in the
1960s due to the acquisition of a mobile genetic
element carrying the mecA-gene, encoding a
penicillin binding protein with low affinity to
virtually all β-lactam antibiotics, including
carbapenems (Diekema et al. 2019b; Lee et al.
2018). A gradual global dissemination ensued,
and by the turn of the century, MRSA had
become one of the major health concerns in infec-
tious diseases. In a retrospective study of NSTIs
during 2001–2006 in Texas, MRSA was
identified as the causative pathogen in 39% of
the cases (Lee et al. 2007). A similar situation
was described in a contemporary report on NSTIs
from Los Angeles (Miller et al. 2005). Moreover,
approximately half the invasive S. aureus-isolates
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).
Extensive prevention and control measures
were implemented in several countries to coun-
teract this development, including isolation
protocols, patient screening, and MRSA eradica-
tion therapy. Two decades later, this massive
effort appears to have been successful, and the
prevalence of MRSA is declining in both the US
and in Europe, dropping below 5% in northern
European countries (Duncan et al. 2019; eCDC
2019; NORM/NORM-VET 2018). Nevertheless,
it is still a force to be reckoned with in several
geographic areas, and empiric MRSA coverage is
warranted in high endemic regions. An overview
delineated in Table 7.1.
Unfortunately, MRSA are frequently harbor-
ing co-resistance to other antimicrobial agents,
particularly tetracyclines, aminoglycosides,
clindamycin, and fluoroquinolones (Diekema
et al. 2019a, b). Vancomycin, linezolid, and
daptomycin are the most potent drugs against
MRSA, and resistance to these antimicrobial
agents is extremely rare in S. aureus (Farrell
et al. 2013; Duncan et al. 2019). The novel fifth
generation cephalosporins, ceftaroline, and
ceftobiprole, are marketed for their anti-MRSA
activity. Alarmingly, a recent report from Europe
revealed 10% of the MRSA-isolates to be
co-resistant to ceftaroline, indicating that these
cephalosporins should not be used for empirical
treatment of MRSA (Farrell et al. 2013).
7.2.4 Antimicrobial Resistance
in Enterobacteriaceae
A wide range of enterobacterial species have
been implicated in NSTIs, predominantly in
polymicrobial infections (Madsen et al. 2019;
Kim et al. 2019; Louis et al. 2019). Escherichia
coli and Klebsiella spp. are by far the most
commonly encountered Enterobacteriaceae,
reflecting their dominance as a cause of invasive
disease in humans in general. Resistance to beta-
lactam antibiotics has unfortunately become
widespread in these pathogens, including the dis-
semination of extended spectrum beta-lactamase
7 Treatment of Necrotizing Soft Tissue Infections: Antibiotics
(ESBL) enzymes (eCDC 2019; Diekema et al.
2019a). There is a plethora of ESBL genes cur-
rently circulating and their classification has been
much debated. In E. coli and Klebsiella spp.,
beta-lactamases mediating resistance to third gen-
eration cephalosporins (herein abbreviated
ESBLceph) have become endemic in several geo-
graphic regions (Diekema et al. 2019a; eCDC
2019). Piperacillin/tazobactam is also frequently
inactivated by these enzymes, but not invariably
(Kim et al. 2019). In Europe, there is a distinct
geographic polarization of the ESBLceph preva-
lence, ranging from 3rd generation cephalosporin
resistance in <5% of Klebsiella isolates in the
northern regions, to 75% of the isolates in the
south-east (eCDC 2019). A similar gradient is
evident between US and Latin America, although
less markedly (Castanheira et al. 2019). An over-
view of the global burden of ESBLceph is
delineated in Table 7.1.
Co-resistance in isolates harboring ESBL
genes is substantial, and in Greece, 50% of the
Klebsiella pneumonia isolates display combined
resistance to third generation cephalosporins,
fluoroquinolones, and aminoglycosides (eCDC
2019). Alarmingly, isolates harboring ESBL
enzymes also inactivating carbapenems
(ESBLcarba) have emerged in South East Europe,
and poses a major health concern (eCDC 2019;
Castanheira et al. 2019). Treatment options in
these multiresistant organisms are severely lim-
ited, and rapid implementation of effective pre-
vention and control measures are necessary to
contain the epidemiological situation.
7.2.5 Antimicrobial Resistance
in Obligate Anaerobic Bacteria
Obligate anaerobic bacteria are almost invariably
present in polymicrobial NSTIs, and Clostridium
perfringens is also an established cause of
monomicrobial NSTI (Thanert et al. 2019;
Zhao-Fleming et al. 2019). Gram-negative obli-
gate anaerobes such as Bacteroides spp.,
Prevotella spp., and Fusobacterium spp. are pre-
dominantly detected in infections below the
91
diaphragm, whereas gram-positive anaerobic
peptostreptococcal species are often associated
with infections emanating from the head and
neck area (Gajdacs et al. 2017; Brook et al.
2013). All these pathogens are uniformly suscep-
tible to metronidazole, but resistance rates to
other antimicrobial agents vary depending on
the species. Nevertheless, resistance to
carbapenems and piperacillin-tazobactam is suffi-
ciently rare to enable both these agents to function
as empiric coverage for anaerobes in
polymicrobial NSTIs (Gajdacs et al. 2017). The
broad spectrum tetracycline analog tigecycline
has also demonstrated excellent in vitro and
in vivo activity against obligate anaerobic bacte-
ria (Brook et al. 2013).
Clindamycin was historically regarded as a
potent anaerobic therapeutic agent, but resistance
has unfortunately become widespread. The per-
centage of Bacteroides isolates resistant to
clindamycin has increased from 5 % to 35 % in
the US in the period 1990–2010, and a similar
development has been observed in Europe
(Gajdacs et al. 2017). Hence, it is no longer a
first line agent in the treatment of these pathogens.
Penicillin has retained its activity against most
gram-positive anaerobic bacteria, including
Clostridium spp. However, the majority of
gram-negative anaerobes are equipped with
broad spectrum beta-lactamases, rendering both
penicillins and most cephalosporins ineffective
(Brook et al. 2013).
7.3 Clinical Efficacy Data
for Selected Antibiotics in Soft
Tissue Infections
Numerous clinical studies on antimicrobial treat-
ment of non-necrotizing soft tissue infections
have been performed. Key findings and principles
could likely be transferred to the management of
NSTIs, and will be briefly reviewed.
Historic studies from the dawn of the antibiotic
era reported approximately 70% cure rates in
cellulitis patients not receiving antibiotics, com-
pared to eradication of the infections in 98 % of
92
those receiving penicillin (Spellberg et al. 2009).
Moreover, a dramatic improvement in cellulitis
mortality and morbidity rates was observed after
penicillin became available (Spellberg et al.
2009).
In the modern age, placebo-controlled trials in
cellulitis are unethical, and studies are predomi-
nantly designed to demonstrate non-inferiority to
existing therapeutic agents. This is practical for
demonstrating clinical efficacy in general, but is
less suited for determining the optimal antimicro-
bial treatment strategy. Indeed, in a metanalysis
of 43 randomized clinical trials comparing the
efficacy of antimicrobial treatment in complicated
skin and soft tissue infections, Brindle et al. could
not find any evidence to support the preference of
one antibiotic over another (Brindle et al. 2019).
The efficacy of linezolid versus vancomycin in
complicated skin and soft tissue infections has
recently been much debated. Although the initial
registration trials demonstrated non-inferiority, a
Cochrane meta-analysis revealed a significant
improved clinical cure rate for linezolid (Yue
et al. 2016). However, the authors summarize
that the effect is small, and the majority of the
studies were funded by the pharmaceutical com-
pany, increasing the risk of bias. In a recent study
comparing vancomycin and linezolid for treat-
ment of invasive MRSA infections, linezolid
was associated with an increased mortality in
severely ill patients with an acute physiology
and chronic health evaluation (APACHE) II
score above 14 (Burnham et al. 2016). This
could lend caution to its clinical use in more
severe skin and soft tissue infections, such as
NSTIs.
The past decade, several new antibiotics have
received approval for the treatment of compli-
cated skin and soft tissue infections, including
daptomycin, tigecycline, tedizolid and the 5th
generation cephalosporins ceftaroline and
ceftobiprole (Brindle et al. 2019; Tirupathi et al.
2019). However, none has been shown to outper-
form the pre-existing treatment options, and their
role in the treatment of these condition has not
been firmly established. Most contemporary
guidelines recommend to reserve these antibiotics
for salvage therapy.
O. Oppegaard and E. Rath
7.4 Pharmacokinetic
and Pharmacodynamic
Considerations in Treatment
of NSTIs
7.4.1 Pharmacokinetics
in the Critically Ill
Knowledge on pharmacokinetics and pharmaco-
dynamics of antimicrobial agents is primarily
derived from studies on healthy subjects. How-
ever, several pathophysiological changes occur in
the critically ill that have potential implications
for the therapeutic strategy in NSTIs.
Firstly, there is a high prevalence of acute
kidney failure in patients with severe infectious
disease manifestations, including NSTIs,
necessitating adjustment of the daily dosage for
several antibiotics (Das et al. 2012; Bruun et al.
2020). Some agents are associated with a substan-
tial nephrotoxicity, and are contraindicated in the
presence of overt kidney failure. This particularly
concerns the aminoglycosides and colistin, but
neither are frequently administered in the treat-
ment of NSTIs. More problematic is the relative
contraindication for vancomycin, which is often
the agent of choice for MRSA coverage in NSTIs.
The risk of Vancomycin induced nephrotoxicity
correlates to the serum trough levels, and elevated
serum concentration over time is associated with
a high risk of renal failure (Arnaud and Liborio
2020). Vancomycin could potentially be
administered to patients with acute renal failure
if necessary, as long as adequate dose adjustment
is performed and daily serum concentrations are
measured. However, most clinicians would con-
sider switching to alternative MRSA active
agents in the presence of severely reduced renal
function, such as linezolid or daptomycin. It
should be noted that risk of linezolid-associated
bone marrow suppression is increased in the pres-
ence of kidney failure, further complicating the
therapeutic strategy in this patient group
(Wu et al. 2006). Hepatic impairment is an infre-
quent complication in NSTIs, but dosage reduc-
tion is recommended for clindamycin,
daptomycin, and metronidazole in patients with
7 Treatment of Necrotizing Soft Tissue Infections: Antibiotics
advanced liver failure classified as Child Pugh
class C (Perianez-Parraga et al. 2012).
The fluid homeostasis is frequently imbal-
anced in the critically ill due to significant capil-
lary leak coupled with aggressive fluid
resuscitation therapy, leading to an increase in
the extracellular fluid space (Udy et al. 2013).
Furthermore, the protein binding of antimicrobial
drugs is often reduced secondary to
hypoalbuminemia, and these pathophysiological
changes have implications for the pharmacokinet-
ics of several antibiotics (Udy et al. 2013; Kiang
et al. 2014; Minichmayr et al. 2017). Increased
distribution volumes have been demonstrated for
aminoglycosides, beta-lactams, daptomycin, and
glycopeptides in critically ill patients, and for
vancomycin the changes in distribution volume
were found to correlate with disease severity
(Udy et al. 2013). Redistribution of the therapeu-
tic drug to the extracellular compartment implic-
itly reduces the serum concentration of these
antimicrobial agents, potentially increasing the
risk of incomplete clearance of bacteremia. This
interaction between pharmacokinetics and sever-
ity should be taken into consideration in the treat-
ment of NSTIs, and underlines the importance of
administering antibiotics in sufficiently high
doses to compensate for the increased distribution
volume. Furthermore, the use of a loading dose
should be considered, especially for
glycopeptides and daptomycin (Udy et al. 2013).
7.4.2 Tissue Distribution
of Antibiotics
Although bacteremia is detected in a substantial
proportion of NSTIs, the predominance of the
bacterial burden is usually confined to the periph-
eral tissues at the affected site (Stevens and
Bryant 2017). In order to target these bacteria,
the therapeutic agents need to extravasate and
disseminate into the extracellular space. The dis-
tribution of antibiotic drugs into the soft tissue
compartment is dependent on several pharmaco-
kinetic properties, including solubility, molecular
weight, and protein binding. Consequently, the
tissue distribution profile displays high variability
93
between different antimicrobial classes. In gen-
eral, lipophilic agents, such as tigecycline,
linezolid, and clindamycin, have a larger distribu-
tion volume and tissue penetration than hydro-
philic antibiotics (e.g. beta-lactams, vancomycin,
and daptomycin) (Barbour et al. 2010; Kiang
et al. 2014). Nevertheless, all above agents
achieve sufficient tissue concentrations to be
potentially clinically effective in the treatment of
skin and soft tissue infections, but increased dos-
ing might be indicated in the latter antimicrobial
group (Pea 2016). In a small experimental study
of tissue distribution in diabetic foot infections,
vancomycin penetration was found to be signifi-
cantly impaired in the presence of
microangiopathy (Skhirtladze et al. 2006). The
clinical implications of this has yet to be
investigated, but increased dosage in the presence
of diabetes microangiopathy might be warranted.
Small vessel occlusion is also part of the local
pathophysiology in NSTIs, lending further sup-
port to using high dosing of glycopeptides in the
treatment of these infections (Stevens and Bryant
2017).
Intracellular survival of pathogens in
macrophages has emerged as a potentially impor-
tant aspect of the pathogenesis in NSTI, particu-
larly for S. aureus and S. pyogenes (Baude
et al. 2019; Hertzen et al. 2012). Hence, the
penetration of antimicrobial agents to the intracel-
lular compartment has become of interest. In
line with tissue distribution discussed above,
lipophilic drugs attain the highest intracellular
concentrations, approaching a one to one ratio
with plasma concentrations for macrolides,
tigecycline, and fluoroquinolones (Barcia-Macay
et al. 2006). Conversely, daptomycin has one of
the smallest distribution volumes of all
antimicrobials, and is not suitable for treatment
of intracellular microbes (Barbour et al. 2010).
However, the intracellular concentration of the
antibiotics is only half the story. The internalized
bacteria often reside in the phagolysosomes, and
this highly acidic environment has been
demonstrated to significantly alter the activity of
several antimicrobial agents. Barcia-Macay et al.
investigated the effect of various antibiotics
against intracellular S. aureus, and revealed that
94
a change in pH from 7.3 to 5.0 lead to a 1024-fold
increase in the MIC value for azithromycin, and a
fourfold increase for fluoroquinolones (Barcia-
Macay et al. 2006). Interestingly, when prolonged
exposure (>24 h) of the antibiotics was
investigated, cloxacillin was one of the most
effective agents in eradicating intracellular
S. aureus in this experimental model. This
contrasts the current dogma that beta-lactam
antibiotics have poor intracellular activity. Con-
versely, clindamycin achieves high intracellular
concentrations, and appears to eradicate intracel-
lular S. pyogenes more efficiently than penicillin
(Kaplan et al. 2006). The optimal treatment for
intracellular S. aureus and S. pyogenes in NSTIs
has yet to be elucidated, but combining beta-
lactam antibiotics and clindamycin is likely bene-
ficial, and in line with contemporary
recommendations.
7.4.3 Toxin Inhibition
Bacterial secretion of cytotoxins and
superantigens is highly implicated in the patho-
genesis of streptococcal and staphylococcal
NSTIs, and is one of the key disease mechanisms
distinguishing NSTIs from non-necrotizing soft
tissue infections (Shumba et al. 2019; Stevens
and Bryant 2017). Hence, the abrogation of
toxin production remains an important therapeu-
tic aspect in the management of NSTIs. Adminis-
tration of cell wall disrupting antibiotics,
e.g. beta-lactams, could potentially increase the
release of exotoxins through bacterial lysis
(Coyle et al. 2003). Conversely, clindamycin
inhibits the bacterial protein synthesis, and has
been shown to significantly reduce the production
of several streptococcal virulence determinant
in vitro (Andreoni et al. 2017; Mascini et al.
2001). Recently, these findings were
substantiated in vivo in a study by Andreoni
et al., revealing significantly attenuated Streptoly-
sin O activity after administration of clindamycin,
both in a murine model and a human case of NSTI
(Andreoni et al. 2017). Moreover, several clinical
studies have supported the adjunctive use of
clindamycin in severe S. pyogenes infections,
O. Oppegaard and E. Rath
demonstrating improved clinical outcome (Linner
et al. 2014; Carapetis et al. 2014). However, these
findings should not be extrapolated to the entire
spectrum of S. pyogenes skin and soft tissue
infections. Clinical benefit from the addition of
clindamycin could not be detected in less severe
cases, but was significantly associated with
increased risk of developing diarrhea (Brindle
et al. 2017). Adjunctive clindamycin treatment
should thus be reserved for S. pyogenes
associated NSTIs or toxic shock syndrome.
Clindamycin also reduces the production of
superantigens in S. aureus significantly more
than oxacillin, both in vitro and in murine models
(Coyle, and Society of Infectious Diseases, P
2003). Hence, some advocate that the administra-
tion of clindamycin is indicated also in the treat-
ment of severe S. aureus infections. However,
clinical studies pertaining to the use of adjunctive
clindamycin treatment in staphylococcal NSTIs
has not been performed.
Linezolid inhibits the bacterial protein synthe-
sis in a similar manner as clindamycin, by binding
to the 50S ribosomal subunit. Coyle et al. found
linezolid to be equally effective as clindamycin in
the reduction of superantigen production in
S. pyogenes in vitro, and it has also been
demonstrated to have similar activity in
S. aureus (Coyle, and Society of Infectious
Diseases, P 2003; Coyle et al. 2003). The ratio-
nale for administering linezolid in NSTIs is not as
strong as for clindamycin, but could be warranted
in strains resistant to clindamycin or in cases with
known allergy.
Interestingly, Andreoni et al. recently reported
clindamycin treatment to reduce toxin production
and disease severity in NSTIs caused by
S. pyogenes, regardless of the clindamycin sus-
ceptibility status of the strain (Andreoni et al.
2017). Although clindamycin monotherapy treat-
ment of clindamycin-resistant S. pyogenes soft
tissue infections previously has been associated
with an unfavorable outcome in experimental
mouse models, it is conceivable that clindamycin
retains a role as an adjunctive treatment of NSTIs
even in the presence of clindamycin-resistant
BHS (Lewis et al. 2014).
7 Treatment of Necrotizing Soft Tissue Infections: Antibiotics
7.4.4 Eagle Effect and Tolerance
The presence of tolerance to antimicrobial agents,
defined as a >32-fold ratio of minimum bacteri-
cidal concentration (MBC) to minimum inhibi-
tory concentration (MIC), has been reported as a
phenomenon for several decades, particularly in
various streptococcal species (Rolston et al.
1984). For S. pyogenes, the presence of penicillin
tolerance has been reported for up to 29% clinical
isolates derived from streptococcal tonsillitis
(Conley et al. 2003). However, the lack of
standardized laboratory methodology and repro-
ducibility has been criticized, and clinical impli-
cation for this observation has been difficult to
ascertain.
A related growth dependent phenomenon was
discovered by Eagle in 1952 in a murine
S. pyogenes pyomyositis model (Eagle 1952).
He reported that the efficacy of penicillin
correlated to the size of the bacterial inoculum,
and the beta-lactam antibiotic lost its potency
when the bacterial burden increased beyond a
certain point. It has become known as the Eagle
effect, and is presumed to reflect the reduced
activity of penicillin on S. pyogenes reaching a
stationary plateau-phase in their growth curve.
Later, Stevens et al. reproduced the results, but
also demonstrated that clindamycin was not
affected by growth phase or inoculum size, and
significantly outperformed penicillin in the treat-
ment of experimental pyomyositis (Stevens et al.
1988). A high inoculum of bacteria is a hallmark
of NSTIs as compared to less severe SSTIs, and
the Eagle effect is presumed to be of significance
in the treatment of NSTIs. The superiority of
clindamycin in bacteria in stationary growth
phase, combined with its ability to abrogate
toxin production in S. pyogenes, forms the exper-
imental basis for the recommendations of adjunc-
tive clindamycin treatment in NSTIs caused by
this pathogen.
Notably, the Eagle effect as well as cytotoxin
production has also been verified in
S. dysgalactiae-infections, but the potency of
clindamycin in this setting has not been examined
(Lam and Bayer 1983; Siemens et al. 2015).
95
7.4.5 Treatment of Biofilm
The organization of microbial communities into a
biofilm has long been recognized as a phenotypic
trait in several clinical pathogens, including
S. aureus (Gebreyohannes et al. 2019). Recently,
biofilm was revealed to also be implicated in
NSTIs caused by S. pyogenes (Siemens et al.
2016). The encapsulation of the microbes by
rich extracellular matrix and the distinct layering
of bacteria in different metabolic states is the
hallmark biofilm formation, and has been shown
to significantly alter the efficacy of several
antibiotics (Fiedler et al. 2015; Gebreyohannes
et al. 2019; Conley et al. 2003). The beta-lactam
antibiotics inhibit bacterial growth though disrup-
tion of the cell wall synthesis. However, in the
biofilm community some bacteria enter an inac-
tive stationary phase, rendering the beta-lactams
ineffective. Conversely, antibacterial agents
targeting DNA (fluoroquinolones), RNA (rifam-
picin) or protein synthesis (clindamycin and
linezolid) appear to retain their activity also in
biofilm, and have been suggested as potential
therapeutic agents in biofilm-associated
infections (Zimmerli and Sendi 2017; Chai et al.
2016).
In S. pyogenes, biofilm formation has previ-
ously been linked to penicillin treatment failure in
tonsillitis (Conley et al. 2003). The presence of
S. pyogenes biofilm has been demonstrated in
crypts in excised tonsillar tissue, and biofilm
capacity and reduced susceptibility to beta-lactam
antibiotics has been verified in S. pyogenes
isolates obtained from recurrent tonsillitis cases
(Conley et al. 2003; Roberts et al. 2012). Treat-
ment with clindamycin appears to more effective
in eradication of difficult to treat S. pyogenes
tonsillitis cases, though at the expense of more
adverse effect (Brook and Hirokawa 1985).
How discovery of S. pyogenes biofilm in
NSTIs translates into clinical practice has yet to
be elucidated, but it likely strengthens the posi-
tion of adjunctive clindamycin in the treatment of
these infections.
96 O. Oppegaard and E. Rath

7.5 Antimicrobial Treatment subsequently de-escalate treatment based on cul-

of NSTIs
7.5.1 Treatment Strategy
In the absence of clinical trials pertaining to
the antimicrobial treatment of NSTIs, general
recommendations for therapeutic management
are predominantly based on expert opinion.
Knowledge extrapolated from experimental stud-
ies on the pharmacodynamics and pharmacoki-
netics of relevant antibiotics as delineated
above, combined with data from clinical studies
on non-necrotizing skin and soft tissue infections,
have often been used as guidance in the decision
process. Importantly, the antimicrobial treatment
needs to cover the etiologic spectrum of
pathogens associated with NSTI.
In general, two different strategies exist in the
empiric treatment of infectious diseases. In some
clinical conditions, including uncomplicated
skins and soft tissue infections, the initiation of
narrow spectrum antibiotics is advised, and treat-
ment escalation can be considered in the event
of therapeutic failure. Inversely, in more severe
disease manifestations it is often recommended
to administer broad spectrum antibiotics, and
ture and susceptibility data. Given the substantial
mortality and morbidity associated with NSTIs
and the importance of swift infection control, a
de-escalation strategy is advocated by most
guidelines, and empiric coverage of all pathogens
frequently associated with this condition is essen-
tial (Stevens et al. 2014; Sunderkotter et al. 2019).
However, the major geographic differences in
antibiotic susceptibility described previously,
highlights the need to adapt antimicrobial treat-
ment to the local epidemiology of bacterial resis-
tance. Moreover, regional differences in
etiological distribution merit considerations,
especially in areas where water exposure
associated microbes (i.e. Vibrio and Aeromonas
spp.) are reported to be more frequent (Hsiao et al.
2020). Hence, a globally valid guideline for treat-
ment of NSTIs is perhaps not feasible.
7.5.2 Empirical Treatment
National guidelines for the treatment of NSTIs
have been developed in several countries
(Table 7.2), though many simply refer to the
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
7 Treatment of Necrotizing Soft Tissue Infections: Antibiotics
Society of America (IDSA) or World Society of
Emergency Surgery/Surgical Infections Society
Europe (WSES/SIS-E) (Stevens et al. 2014;
Sartelli et al. 2018). The various guidelines are
largely overlapping, but demonstrate some
important differences, predominantly reflecting
the variations in antimicrobial resistance.
One of the major ambiguities is the
incorporation of ani-MRSA coverage in the
empiric treatment regime. Understandably,
guidelines originating from US (IDSA) and
from Asia (South Korean guideline), where the
proportion of methicillin resistance in S. aureus
approximates 50%, advocate to initiate empiric
treatment with an MRSA active antibiotic in
all NSTI-patients (Stevens et al. 2014; Kwak
et al. 2017). The inverse holds true in
recommendations from regions of low MRSA
endemicity, including central and northern
Europe (Sunderkotter et al. 2019; Urbina et al.
2019; Helsedirektoratet 2013).
Another important consideration is the selec-
tion of empiric treatment for Enterobacteriaceae.
Most guidelines suggest administration of either
piperacillin/tazobactam or meropenem, and leave
it to the treating physician to decide the appropri-
ate choice based on local resistance epidemiology
(Stevens et al. 2014; Kwak et al. 2017; Sartelli
et al. 2018; Sunderkotter et al. 2019). Both these
agents have excellent broad spectrum activity
against streptococci, staphylococci (excluding
methicillin-resistant strains), anaerobes, and
Enterobacteriaceae, but carbapenems are usually
necessary to treat gram-negative bacteria harbor-
ing ESBLceph. In general, piperacillin/
tazobactam should be regarded as the first line
of defense in areas with low prevalence of
ESBLceph, but meropenem is warranted in geo-
graphic regions where resistance to piperacillin/
tazobactam is high.
A third major strategic decision pertains to
the administration of a protein synthesis inhibitor
as part of the empiric treatment regime. The
German guidelines and the WSES/SIS-E
recommendations incorporate clindamycin in
their therapeutic algorithm for all patients
(Sunderkotter et al. 2019; Sartelli et al. 2018).
However, the prevalence of S. pyogenes in
97
NSTIs emanating from the abdominal or genito-
urinary region is very low (Madsen et al. 2019).
Accordingly, reserving the toxin inhibitors for
NSTIs confined to the extremities or head/neck
area is suggested in France (Urbina et al. 2019).
The IDSA guidelines have also omitted
clindamycin from their empiric management,
but recommend administering a protein synthesis
inhibitor to all cases of verified S. pyogenes and
Clostridial NSTIs (Stevens et al. 2014).
In Norway, a country with very low preva-
lence of both MRSA and ESBLceph, a narrow
spectrum approach has been recommended in
the national guidelines for empiric treatment of
NSTIs located to the extremities; a combination
of penicillin, gentamicin, and clindamycin
(Helsedirektoratet 2013). Inversely, the local
antimicrobial treatment recommendations in
the neighboring country Denmark, advise
meropenem and clindamycin as the treatment of
choice for all cases of NSTIs (Rigshospitalet
2020). The rates of antimicrobial resistance in
these two countries are comparable, but two dia-
metrically opposing strategies have been chosen.
Interestingly, in the prospective INFECT study
including patients from both these countries, no
significant difference in mortality was observed
(Madsen et al. 2019). However, randomized clin-
ical trials are needed to address the optimal treat-
ment strategy in NSTIs, and to examine the
feasibility of tailoring the antimicrobial therapy
to the anatomical location.
7.5.3 Pathogen Specific Treatment
If the causative pathogen is identified, the antimi-
crobial treatment can often be de-escalated. The
treatment recommendations for monomicrobial
NSTIs are fairly similar across continents
(Table 7.3).
The combination of penicillin and clindamycin
is uniformly recommended for the treatment of
S. pyogenes NSTIs, to counteract bacterial toxin
production, biofilm formation and the Eagle
effect (Stevens et al. 2014; Helsedirektoratet
2013). However, therapeutic consideration in the
event of clindamycin resistance are not discussed
98 O. Oppegaard and E. Rath

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
evidence for a clindamycin mediated attenuation
of toxin production in S. pyogenes even in
the presence of clindamycin resistance, the size
of the bacterial inoculum remained unchanged
(Andreoni et al. 2017). Thus, the clinical
implications have yet to be elucidated, and until
further studies have been performed, it is advis-
able to switch to linezolid in clindamycin-
resistant cases.
S. dysgalactiae is an emerging cause of NSTIs
(Bruun et al. 2020; Oppegaard et al. 2015).
Experimental data has demonstrated its capability
of cytotoxin production, biofilm formation as
well occurrence of the Eagle effect when exposed
to penicillin (Siemens et al. 2015, 2016; Lam and
Bayer 1983). There are no studies pertaining to
the adjunctive treatment of clindamycin in this
pathogen, but the addition of a toxin inhibitor
could be considered in monomicrobial
S. dysgalactiae NSTIs, especially in the event of
therapeutic failure.
Different weight has been given to recent stud-
ies indicating a possible superiority for linezolid
over vancomycin in SSTIs, especially for MRSA
associated cases (Yue et al. 2016). Both IDSA
guidelines and French recommendations for
NSTI, as well as IDSA guidelines for MRSA
infections, maintain vancomycin as the treatment
of choice in MRSA infections (Liu et al.
2011; Stevens et al. 2014; Urbina et al. 2019).
Differently, WSES/SIS-E and the German
recommendations prefer linezolid or even
daptomycin (Sartelli et al. 2018; Sunderkotter
7 Treatment of Necrotizing Soft Tissue Infections: Antibiotics
et al. 2019). The quality of the evidence favoring
linezolid is very low, and both alternatives are
likely to be equally successful. However,
avoiding vancomycin in overt renal failure,
and possibly in the presence of diabetic
microangiopathy is advisable. The role of novel
5th generation cephalosporins and tigecycline in
the treatment of MRSA NSTIs has not been
firmly established, but should be reserved for
cases were the first line options cannot be used.
Vibrio spp. and Aeromonas spp. are still rare
causes of NSTI in most parts of the world, and
few guidelines address specific treatment
recommendations for these pathogens. A combi-
nation of doxycycline and ceftriaxone is
advocated in the South Korean and IDSA
guidelines, although doxycycline plus ciprofloxa-
cin might be preferable for Aeromonas infections
(Stevens et al. 2014; Kwak et al. 2017). In South
East Asia Vibrio spp. has become the dominant
cause of monomicrobial NSTIs, implicated in
20% of the cases in a recent study from Taiwan
(Hsiao et al. 2020). Accordingly, the South
Korean guidelines advice empiric coverage for
Vibrio spp. in patients with chronic liver
conditions and recent sea water exposure (Kwak
et al. 2017).
7.5.4 Treatment Duration
Studies investigating the optimal treatment dura-
tion in NSTI are lacking, and considerable
variations in clinical practice has been observed.
In a retrospective study from the US comparing
the management and outcomes in NSTIs between
three different hospitals, the treatment duration
ranged from 7 to 28 days (Faraklas et al. 2016).
Significant variations in treatment length were
detected between the three sites, but no
differences in clinical outcome was evident.
However, the study was not designed to clarify
the appropriate treatment duration.
Several guidelines offer a pragmatic approach,
and suggest continuation of treatment until sur-
gery is no longer indicated, clinical improvement
has occurred, and fever has been absent the past
72 h (Stevens et al. 2014; Sartelli et al. 2018).
99
7.6 Conclusions
Necrotizing soft tissue infection is a life-
threatening clinical condition, and rapid adminis-
tration of potent antibiotics is essential. Empiric
antimicrobial treatment should cover the likely
culprit, and the therapeutic regimen should be
adapted to local resistance epidemiology. The
decision to broadly administer vancomycin or
linezolid depends on the endemicity of MRSA
in the geographic region. Similarly, in areas with
low prevalence of ESBLceph, piperacillin/
tazobactam should be regarded as first line of
defense, but meropenem might be warranted in
regions where resistance to piperacillin/
tazobactam is high. Coverage of Vibrio spp. and
Aeromonas spp. should not be forgotten in areas
where these pathogens are endemic, particularly
in patients with pre-existing chronic liver
conditions and recent sea water exposure. The
use of a protein synthesis inhibitor could be omit-
ted in the empiric treatment regime in NSTIs
emanating from the abdominal and genital areas,
but is definitely indicated in cases localized to the
extremities, and in verified S. pyogenes and Clos-
tridial NSTIs. Furthermore, it should be strongly
considered in monomicrobial cases caused by
S. dysgalactiae and S. aureus. Randomized clini-
cal trials investigating the optimal antimicrobial
treatment of NSTIs are needed.
Acknowledgment This work was supported by the
European Union Seventh Framework Programme:
(FP7/2007-2013) under the grant agreement 305340
(INFECT project), NordForsk (Project no. 90456,
PerAID), the Norwegian Research Council under the
frame of ERA PerMed (Project 2018-151, PerMIT) and
The Swedish Research Council Framework Grant in
infections and antibiotics (2014-8609-117204-47).
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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

# Springer Nature Switzerland AG 2020 105

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_8
106 M. B. Madsen et al.

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
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-
ably less of IG3 and IgG4. In addition, the
subclasses vary in function due to differences in
the Fc region. IgG1 and IgG3 have a stronger
binding to C1q and to Fc receptors (FcR), and
are common in response to protein antigens
(Vidarsson et al. 2014). In contrast, IgG2 domi-
nate responses to bacterial polysaccharides
(Ferrante et al. 1990; Siber et al. 1980), while
IgG4 to allergens (Vidarsson et al. 2014).
IgA is the second most abundant isotype in
serum, and the principal Ig isotype in all mucosal
secretions such as saliva, tears, and secretions
covering the respiratory, gastrointestinal, and uro-
genital tract (Woof and Kerr 2006). IgA therefore
represents an important first line of defense
towards inhaled or ingested pathogens,
neutralizing toxins and inhibiting colonization of
epithelial surfaces, while at the same time
tolerating commensals and innocuous antigens.
In mucosal secretions, IgA exists predominantly
in a dimeric form where the two IgA molecules
are linked together by a small polypeptide, called
J chain. The dimeric IgA also includes the
107
secretory component; a residual polypeptide of
the extracellular proteolytic fragment of the poly-
meric Ig receptor required for transport of dimeric
IgA into the secretions. The complex of dimeric
IgA, J chain and secretory component is referred
to as secretory IgA (sIgA). In serum, monomeric
IgA is more common. Human IgA exists in two
subclasses, IgA1 and IgA2, and IgA1 is the domi-
nant subclass in serum as well as in many muco-
sal secretions. However in colon, IgA2 is most
frequent (Brandtzaeg and Johansen 2005).
IgM is the first isotype to be produced in a
humoral immune response and it offers an impor-
tant early defense against infections. IgM exists in
a large pentameric form, displaying 10 antigen-
binding sites and consequently high avidity inter-
action with antigens. The pentameric structure
also supports an efficient complement activation
(Schroeder and Cavacini 2010).
Both IgD and IgE are found at very low levels
in serum. Despite this, IgE is highly potent and
predominates in the immune response to allergens
and helminths parasites.
108
8.3 IVIG Preparations
8.3.1 Manufacturing
and Composition
IVIG is prepared from pooled plasma from sev-
eral thousands of blood donors and the resulting
polyvalent Ig preparation is supplied either in a
liquid or a lyophilized form. As a human blood-
derived product, manufacturing is tightly
regulated starting with the selection and testing
of individual donors to ensure quality and safety
control (Medicines Agency E 2018; WHO 2020).
Important quality and safety aspects include
retained structural and functional characteristics
of the Ig molecules, no infectious agents, no
procoagulant activity and absence of agglutina-
tion of anti-A and anti-B hemagglutinins
(Radosevich and Burnouf 2010). To this end,
the manufacturing of IVIG include several
procedures to remove such factors that might be
present in the plasma pool, including not the least
multiple steps (e.g. solvent/detergent treatment,
pasteurization, and nanofiltration) to reduce the
risk of disease transmission including viruses and
prions (for further detail see Sect. 8.3.4).
There are many IVIG preparations available
on the market, most of which are composed of
IgG, but there are also preparations enriched in
IgM and IgA (Table 8.1). The IgG subclass dis-
tribution in the IVIG preparation is comparable to
that found in serum with IgG1 being most abun-
dant followed by IgG2, and only small amounts of
IgG3 and IgG4. The Ig molecules are polyvalent
and offers a broad spectrum of antibodies specific
for a variety of foreign antigens (e.g. viral, bacte-
rial, parasitic, etc.), but also antibodies to self-
antigens, such as towards cytokines (natural
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
Privigen >98% IgG
Endobulin >98% IgG
Polyglobin >97% IgG
Trimodulin 56% IgG; 23% IgM; 21% IgA
Pentaglobin 76% IgG; 12% IgM; 12% IgA
NSTI necrotizing soft tissue infections, STSS Streptococcal toxic shock syndrome, CAP community acquired pneumonia
M. B. Madsen et al.
autoantibodies) and antibodies (anti-idiotypic
antibodies). As a consequence of the plasma
pool originating from different donors as well as
variations in manufacturing procedures, IVIG
preparations will vary in their final Ig composi-
tion and their functionality. This is evident not
only between different IVIG preparations but also
between batches of the same IVIG preparation
(Dhainaut et al. 2013; Norrby-Teglund et al.
1998).
8.3.2 Mechanisms of Action
IVIG has several mechanisms of actions
attributed to the dual functionality of Ig
molecules: (1) as cell-surface receptors allowing
for cellular signaling and activation, and (2) as
soluble effector molecules binding and
neutralizing antigens. This results in a direct
targeting of disease-causing antigens or agents,
and numerous immunomodulatory functions that
have been implicated in the mechanistic action of
IVIG in infections, as well as in autoimmune and
acute inflammatory diseases (Gelfand 2012;
Schwab and Nimmerjahn 2013). In the subsection
below, we focus on the role of IVIG in acute
infectious diseases, such as sepsis, toxic shock
syndrome and necrotizing soft tissue infections
(NSTI) with special attention on how IVIG can
neutralize specific virulence factors. However,
also the potent immunomodulatory functions of
IVIG are of key importance as these conditions
are characterized by dysregulated host responses
(Johansson and Norrby-Teglund 2012; Norrby-
Teglund et al. 2003; Steinhagen et al. 2020).
The immunomodulatory effects of IVIG are
numerous and include both Fc- and
Study subjects Reference
NSTI Madsen et al. (2017)
STSS Darenberg et al. (2003)
Sepsis Werdan et al. (2007)
Severe CAP Welte et al. (2018)
Sepsis, septic shock Rodríguez et al. (2005)
8 Treatment of Necrotizing Soft Tissue Infections: IVIG
Fab-mediated effects (Schwab and Nimmerjahn
2013). Fab-dependent effects comprise (1) killing
of target cells through antibody-dependent
cytotoxicity, (2) blockade of cellular receptors
and consequently cell–cell interactions, (3) neu-
tralization of autoantigens (e.g. cytokines, FAS
or FAsL, sialic acid-binding Ig-like lectin
(SIGLECs) expressed on neutrophils and
eosinophils) and autoantibodies by anti-idiotypic
antibodies, and (4) scavenging of the
anaphylatoxins C3a and C5a. The Fc-dependent
effects comprise (1) blockade of activating
Fc-gamma-receptors, (2) saturation of neonatal
FcR (FcRn) resulting in an accelerated IgG catab-
olism, (3) expansion of regulatory T cell popula-
tion, (4) modulation of FcγR expression on innate
immune cells and B cells, and modulation of
dendritic cells. FcγRIIIB has been shown in sev-
eral studies to be involved in the immunomodula-
tory activities of IVIG. This inhibitory FcR is
either upregulated on myeloid cells or the number
of myeloid cells expressing this receptor is
increased following IVIG administration (Bruhns
et al. 2003; Kaneko et al. 2006; Samuelsson
et al. 2001). Many of these IVIG-mediated effects
result in immunosuppressive and anti-
inflammatory responses, which are likely to
have impact on hyperinflammatory conditions
like NSTI and toxic shock syndrome (Norrby-
Teglund et al. 2000). However, it should be
noted that the vast majority of studies
investigating IVIG-mediated immunomodulation
have been performed in autoimmune or inflam-
matory diseases.
With regard to IVIG mediating neutralization
of virulence factors through specific antibodies,
more data are available. In sepsis, studies have
reported that low Ig-levels are associated with
increased mortality (Bermejo-Martín et al. 2014;
Martin-Loeches et al. 2017). A similar association
between lack of protective humoral immunity and
severity of infection has been reported in GAS
infections, where patients with streptococcal toxic
shock syndrome or NSTI have lower levels of
protective antibodies to streptococcal virulence
factors than patients with less severe GAS
infections (Babbar et al. 2018; Eriksson et al.
1999; Norrby-Teglund et al. 1994). In light of
109
this, IVIG was explored as a source of such pro-
tective antibodies towards streptococcal virulence
factors. Studies revealed that IVIG contains
broad spectrum antibodies that efficiently neutral-
ize bacterial virulence factors shown to be key
contributors to severe GAS infection (Table 8.2).
This is not surprising, considering the abundance
of asymptomatic carriage (especially in children)
and frequency of mild streptococcal diseases
in the population (Carapetis et al. 2005). The
antibodies target both surface attached virulence
factors, including the classical virulence factor M
protein, resulting in increased opsonophagocytic
killing (Table 8.2 and Fig. 8.2). In a recent
study applying immunoprecipitation of GAS
cell wall extracts revealed a broad range of
surface proteins, e.g. M protein, C5a peptidase,
S. pyogenes cell envelope protease (SpyCEP),
phosphoglycerate kinase, that were recognized
by IVIG (Reglinski et al. 2015). Also, secreted
factors including superantigens, proteases,
DNases, and cytolytic toxins are inhibited by
IVIG, which leads to reduced pro-inflammatory
responses (Table 8.2).
Similarly, IVIG has been reported to contain
antibodies that neutralize the activity of S. aureus
virulence factors involved in toxic shock syn-
drome and NSTI, including superantigens and
cytolytic toxins such as Panton Valentine
leukocidin (PVL) and α-hemolysin (Table 8.2).
IVIG was tested in an in vivo model of strep-
tococcal toxic shock syndrome using HLA class
II transgenic mice with increased susceptibility
to GAS superantigens (Sriskandan et al. 2006).
The results revealed that IVIG treatment
resulted in improved bacterial clearance, neutrali-
zation of circulating superantigen, reduced
pro-inflammatory responses, and reduce illness
severity. Similarly, in an experimental murine
model of necrotizing fasciitis caused by GAS,
IVIG administration resulted in smaller lesions
and reduced levels of several important GAS
virulence factors, including the SLO, Sda1, and
SpyCEP (Tarnutzer et al. 2019). However, in this
study, no effect on bacterial load in treated and
non-treated mice, indicating that the clinical treat-
ment effect noted in the infected mice was linked
to neutralization of virulence factors.
110 M. B. Madsen et al.

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
necrotizing pneumonia caused by MRSA strains
in an in vivo rabbit model and in particular
antibodies towards the pore-forming toxins
α-hemolysin and PVL were implicated in the
protective effect (Diep et al. 2016). Also
Mairpady Shambat et al. reported that IVIG
neutralized the effect of α-hemolysin and PVL
thereby attenuating tissue pathology in a human
organotypic model of lung (Mairpady Shambat
et al. 2015).
One important aspect is whether all IVIG
preparations are equally efficient. Studies have
investigated this and reported that variation do
exist between different preparation in their effi-
cacy to neutralize different virulence factors
(Norrby-Teglund et al. 1998; Schrage et al.
2006; Wood et al. 2017). When different IVIG
preparations were tested for neutralization of
broad panel of streptococcal superantigens, sig-
nificant differences were demonstrated between
preparation but also towards different
superantigens (Schrage et al. 2006). The
superantigens SSA, Spe-A, Spe-C, and Spe-K/L
were more efficiently neutralized by IVIG as
compared to Spe-J, Spe-H, and SmeZ-2. Notably,
within the concentration range tested none of the
preparations could completely block the activity
of SPE-J. This has implication for the dosage of
IVIG used and clinical efficacy. However, in
studies where supernatants from GAS strains
have been used, even from Spe-J-positive strains,
the superantigenic activity has always been
completely blocked by IVIG (Norrby-Teglund
et al. 1998, 1996a, b; Emgård et al. 2019). Most
of the studies described above have utilized IgG
preparations. However, it should be mentioned
that in studies comparing the IgG versus IgM-
and IgA-enriched IVIG preparations differences
have been noted. IgM-enriched IVIG has been
reported to contain higher anti-LPS antibody
titers in the IgM fraction (Trautmann et al.
1998), as well as increased opsonic activity
towards the Gram-negative bacteria Pseudomo-
nas aeruginosa, E coli, and Klebsiella
pneumoniae, as compared to IVIG containing
8 Treatment of Necrotizing Soft Tissue Infections: IVIG 111

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-
ence was also reported in regard to neutralization
of mitogenic and cytokine-inducing activity of
one of the group A streptococcal superantigens,
i.e. Spe-A (Norrby-Teglund et al. 1998). Whether
these noted differences would translate into a
varying clinical efficacy has not yet been
explored.
8.3.3 Therapeutic Indications
IVIG is mostly used as substitution treatment or
for immunomodulatory purposes. According to
the U.S. Food and Drug Administration (FDA)
and the European Medicines Agency, IVIG is
approved for replacement therapy in primary
immunodeficiencies with impaired antibody pro-
duction and secondary immunodeficiencies with
112
recurrent infections or low IgG levels, as well as
for immunomodulatory effect in primary immune
thrombocytopenia, acute inflammatory demyelin-
ating polyneuropathy (Guillain–Barré syndrome),
Kawasaki disease, and Chronic inflammatory
demyelinating polyradiculoneuropathy
(Table 8.3 and Fig. 8.3) (European Medicines
Agency 2020). IVIG can also be used as prophy-
laxis against hepatitis A and measles. In
outbreaks of measles, IVIG is often
recommended for healthy, non-immune persons
(Arciuolo et al. 2017; Matysiak-Klose et al. 2018;
Tunis et al. 2018). There is also widely accepted
off-label use of IVIG in several additional
conditions, supported by positive results but in
small-scale trials. In short, there is some level of
evidence for the use of IVIG against rheumatoid
and neurological autoimmune/inflammatory
diseases (Ballow 2011; Viard et al. 1998; Li
et al. 2005; Ephrem et al. 2008; Perlmutter et al.
1999), viral infections (Elsterova et al. 2017;
Vanderven and Kent 2020), bacterial infections
(Kakoullis et al. 2018; Ohlsson and Lacy 2020),
hematological conditions (Chang et al. 2008;
Sultan et al. 1984), obstetric (Egerup et al.
2015), and pulmonological conditions (Wade
and Chang 2015). How common off-label usage
of IVIG is for these indications is currently
unknown by the authors of this work.
8.3.4 Safety
Immunoglobulin use was generally considered
safe until the early 1990s, where patients who
received immunoglobulins were infected with
hepatitis C virus (Chapel 1999). In this section
we will review the safety profile of
immunoglobulins for medical use, with emphasis
on use for patients with NSTI.
8.3.4.1 Donor Selection
and Manufacturing Process
Plasma donation varies slightly from whole blood
donation, as a different technique is used in order
to obtain more plasma and reinfuse the remaining
blood products in the patient. It is also possible to
extract plasma from a “whole blood donation,”
M. B. Madsen et al.
but this technique renders less plasma. Donation
practices vary between countries; in some places,
donation is based on a voluntary basis, and some
have systems where the donor is compensated
economically (Farrugia et al. 2015). Plasma
derived from the national blood services is sold
to the medicine industry. Almost all plasma
derived products are manufactured by the
industry.
Transmission of viral disease was previously a
concern to patients receiving IVIG (Siegel 2006),
but few cases of viral transmission have been
reported through time. Cold ethanol fractioning
was used as part of the manufacturing process,
and this removed most viruses. No reports of HIV
transmission exist, but in the nineties two series of
incidents of hepatitis C virus transmission were
published (Farrugia and Quinti 2014; Power et al.
1994; Bresee et al. 1996). Consequently, the FDA
demanded viral removal and deactivation to be
included in the preparation process. Today, most
donor blood is screened for syphilis, hepatitis B,
C, and E and HIV and human T-lymphotropic
virus (NHS 2019).
A variety of different methods are used to
prevent transmission of virus. In the quest to
manufacture Ig preparation, Cohn developed
cold ethanol fractioning, which separated plasma
proteins (Cohn et al. 1940). Addition of pepsin or
incubation at low pH also help eliminate anti-
complementary activity (Gordon 1987). This pro-
cess itself eliminates HIV, but not HCV (Siegel
2006), and was the only elimination process for
many years.
The main methods for removing or
inactivating virus are described in Table 8.4
8.3.4.2 Supply
Immunoglobulin is a scarce blood product.
Immunoglobulin use is increasing
(Sundhedstyrelsen 2016; Blood 2019), and the
indications for its use are expanding. Data from
Australia suggest that the majority of immuno-
globulin use is for acknowledged indications, and
the increase is partly due to variations in adminis-
tration frequency, dosage, and population, and
not as a consequence of off-label use (Blood
2019). However, a report from Canada suggests
8 Treatment of Necrotizing Soft Tissue Infections: IVIG
Table 8.3 Indications for treatment with IVIG
Disease
Substitution
Primary humoral
immunodeficiency
Secondary humoral
immunodeficiency
Hematopoietic stem cell
transplantation, allogenic
Immune modulation
Immune thrombocytopenia/
idiopathic thrombocytopenic
purpura
Kawasaki disease
Acute inflammatory
demyelinating
polyneuropathy/Guillain–
Barré syndrome, Chronic
inflammatory demyelinating
polyneuropathy, Multifocal
motor neuropathy
Passive immunity
Hepatitis A prophylaxis
Common off-label clinical practice
Measles
113
Indication Dosage Main mechanism
Congenital agammaglobulinemia and 0.4–0.8 g/kg Substitution of Ab
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
Blood or bone marrow disorders - 0.2–0.4 g/kg every Substitution of Ab
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
Hypogammaglobulinemia, earlier also 2 g/kg during Substitution of Ab,
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)
Life threatening bleeding due to ITP, 0.8–1 g/kg Fc-rec blockade,
prophylaxis during surgery one/twice during decrease auto-Ab
3 days, or 0.4 g/kg (Ballow 2011; Imbach
every day during et al. 1981)
2–5 days
First line of treatment with 2 g/kg once Decrease
acetylsalicylic acid pro-inflammatory
cytokines, neutralize
bacterial toxins
(Ballow 2011;
Eleftheriou et al.
2014)
First line of treatment 0.4 g/kg Decrease auto-Ab
daily during 5 days (Ballow 2011;
for AIDP, 2 g/kg Finsterer 2005)
during 2–5 days,
then 1 g/kg every
third week for
CIDP and MMN
Pre/post-exposure for patients with 0,0032 g/kg Passive immunity
immunodeficiency disorders intramuscularly (FASS 2020; Askling
once and Herzog 2019)
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)
114 M. B. Madsen et al.

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
polyclonal immunoglobulin and approved indications.
AIDP acute inflammatory demyelinating polyneuropathy,
AIDS acquired immune deficiency syndrome, CIDP
chronic inflammatory demyelinating polyneuropathy,
CLL chronic lymphocytic leukemia, GBS Guillain–Barré
syndrome, HSCT hematopoietic stem cell transplantation,
allogenic, ITP idiopathic thrombocytopenic purpura, IVIg
intravenous immunoglobulin, MM multiple myeloma,
MMN multifocal motor neuropathy, PID primary humoral
immunodeficiency, SCIg subcutaneous immunoglobulin,
SID secondary humoral immunodeficiency
8 Treatment of Necrotizing Soft Tissue Infections: IVIG
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-
ficiency is not possible in a number of
countries—even in countries with high levels of
voluntary blood donation—and they are reliable
on plasma bought commercially.
8.3.4.3 Batch and Preparation Variation
Different manufacturers use different
compositions of immunoglobulin (Siegel 2015).
Furthermore, as IVIG is extracted from human
plasma, the content and thus the neutralizing
properties are subject to variation, dependent on
the exposure of the donors (Shankar-Hari et al.
2012).
8.3.4.4 Adverse Reactions of IVIG
Infusion
It is difficult to get an overview of adverse
reactions as use is indicated for a wide variety of
diseases. Furthermore, dosage, number of
infusions, infusion rate and composition of the
product vary. Consequently, different studies
have focused on different aspects of safety
profiles.
According to the summary of product
characteristics, common adverse reactions
include chills, headache, dizziness, fever,
vomiting, allergic reactions, nausea, arthralgia,
low blood pressure, and moderate lower back
pain. Severe adverse reactions include hypersen-
sitivity, hemolytic anemia, aseptic meningitis
syndrome, thromboembolism, acute renal failure,
transfusion-related acute lung injury, and trans-
missible agents (FDA 2019; Pierce and Jain
2003).
115
Adverse effects can be divided into immediate,
delayed, and late. As for all medications, infusion
of intravenous immunoglobulin may result in an
inflammatory response resulting in headache,
flushing, tight chestedness, dyspnea, and circula-
tory collapse (anaphylactic reaction). Headache is
the most frequent adverse reaction, and although
symptoms such as headache, malaise, and myal-
gia occur in about 5% of patients, these often
abate with a reduction in infusion rate. Rash is
seen in approximately 6% of patients.
As immunoglobulin preparations contain
varying amounts of IgA, patients with total IgA
deficiency may develop anti-IgA antibodies, ulti-
mately resulting in anaphylactic reaction
(Nydegger and Sturzenegger 1999).
Acute renal failure has been described in
patients with known impairment of renal function
(Cantú et al. 1995). The proposed mechanism
might be the presence of sucrose, which is used
as a stabilizing agent, but this is merely specula-
tive (Nydegger and Sturzenegger 1999). Serum
viscosity may increase as a result of high IgG
concentrations, immune complex formation, and
immunoglobulin aggregates, and may cause
symptoms as headache and visual blurring, and
could result in impaired circulation in patients
with atherosclerosis or other conditions that result
in poor vascular tone. Aseptic meningitis is a
condition mimicking bacterial meningitis with
affection of the cerebrovascular fluid, but the
mechanism is not clear. In a study on patients
with neuromuscular diseases receiving IVIG,
6/54 (11%) developed severe symptoms with
acute, severe headache, stiffneckedness, lethargy,
fever, and photophobia (Sekul et al. 1994).
116
The definitions of adverse events, and the
collection and reporting of safety data are
not standardized. Overall, the trials assessing
effects of IVIG in general did not have the
statistical power to detect differences in adverse
events and serious adverse reactions, and the
clinical trials performed to support marketing
applications for IVIG had insufficient power to
detect adverse events (Pierce and Jain 2003).
Regarding IVIG use in patients with NSTI,
only two trials have systematically collected
data on adverse reactions of IVIG (Darenberg
et al. 2003; Madsen et al. 2017); for a review of
these trials, please see the section “Rationale for
the use of IVIG for NSTI” below. In trials of IVIG
for sepsis and septic shock, the reporting of
adverse events is scarce (Pildal and Gotzsche
2004). In one of the larger trials (n ¼ 653), a
total of 19 adverse events were reported in
17 patients; 13 events in 11 patients in the IVIG
group, and 6 events in 6 patients in the placebo
group (Werdan et al. 2007). The adverse events in
the IVIG group included exanthema, thrombosis,
cardiac failure and arrest, anaphylactic reaction,
intracerebral hemorrhage, overhydration, and
acute kidney injury.
In an observational study with the specific aim
of studying the safety profile of IVIG use in a
general ICU population, acute kidney injury
(AKI) (defined as a rise in creatinine 20%
within 28 days of IVIG admission) occurred in
117 of 145 (81%) patients, and 30 (21%) required
renal replacement therapy (Foster et al. 2010).
Although most patients had other factors that
may have contributed to renal insufficiency,
10% had no obvious concurrent renal insult
other than IVIG. The number of patients with
thromboembolism was 4 (3%). The risk of AKI
may be associated with pre-existing conditions
such as sepsis and hypovolemia, but also with
the specific product used (Siegel 2006).
Lastly, it is worth considering the fact that
critically ill patients receive many different
therapies. In generally, little is known about
drug interactions, and the addition of IVIG is
likewise poorly described.
M. B. Madsen et al.
8.4 Rationale for the Use of IVIG
for NSTI
8.4.1 Laboratory Data
Patients with severe GAS infections including
NSTI have been reported to have lower levels of
protective antibodies towards several important
GAS virulence factors including Spe-B and
superantigens, as compared to uncomplicated
cases such as GAS tonsillitis or erysipelas cases
(Babbar et al. 2018; Eriksson et al. 1999; Norrby-
Teglund et al. 1994). IVIG has been shown to
contain protective antibodies targeting GAS viru-
lence factors, which are efficiently transferred to
the patients upon IVIG administration as evident
by an increased neutralizing activity in plasma
collected post-IVIG therapy (Tarnutzer et al.
2019; Norrby-Teglund et al. 1996a, b; Basma
et al. 1998).
Important points for discussion are what
dosages of IVIG that are efficacious, and
concentration-levels achieved at the tissue site of
infection. There are, as of yet, no clinical IVIG
dosage studies performed on NSTI or toxic shock
syndrome patients. However, insight was gained
through analyses of plasma collected from
patients included in the INSTINCT and the
INFECT studies (Bergsten et al. 2020). The
plasma analyses revealed that a dose of 25 g of
IVIG was sufficient to yield neutralizing activity
towards GAS superantigens. However, the level
of inhibition varied between strains, and towards
some GAS strains the inhibition was only border-
line protective. Taken together with the report by
Bruun et al. (Bruun et al. 2020) on GAS NSTI
patients in the observational INFECT study,
which showed an association between patients
who had not received IVIG and 90-day mortality,
Bergsten et al. (Bergsten et al. 2020) proposed a
dosage regimen of: 0.5 g/kg bodyweight (or a
minimum of 25 g) on day 1, followed by fixed
doses of 25 g daily for 1–2 additional days. The
higher dose on day 1 of 25 g offered only border-
line protective towards some strains in combina-
tion with often high levels of toxins and bacterial
8 Treatment of Necrotizing Soft Tissue Infections: IVIG
load at this time point and at the tissue site.
However, as acknowledged by the authors this
treatment regimen, i.e. dosages and treatment
days, should be evaluated in future trials.
Bioavailability of IVIG in the deep necrotic
tissue has been questioned, and the possibility of
local applications of antibodies, as used for post-
exposure prophylaxis in rabies (CDC 2020), has
been raised. In an in vivo model of GAS NSTI,
intraperitoneal and subcutaneous administration
of IVIG was compared (Tarnutzer et al. 2019).
This study showed that the subcutaneous admin-
istration yielded 1.6 times higher IgG concentra-
tion in the skin, but the serum concentration was
half as high compared to intraperitoneal adminis-
tration. Subcutaneous injection also resulted in
reduced clinical severity (i.e. smaller lesion size)
as well as reduced SLO activity.
8.4.2 Clinical Studies
The proportion of patients receiving IVIG as part
of the treatment for NSTI, as well as approaches
to IVIG use varies. Typically, IVIG is considered
in NSTI patients where the bacterial etiology is
highly suspicious of GAS (Anaya and Dellinger
2007). In this setting, 25% of all intensive care
units participating in a survey reported to always
use IVIG (de Prost et al. 2015), and in an
Australian observational study, 27% of patients
received IVIG (Carapetis et al. 2014). Overall use
independent of bacterial etiology has been
described, with 58% of patients receiving IVIG
(Madsen et al. 2019).
8.4.2.1 Randomized Clinical Trials
Despite the promising effects from in vitro stud-
ies, only two trials have compared the effects of
IVIG with placebo in patients with NSTI. In
1999, Darenberg and colleagues aimed to evalu-
ate the efficacy and safety of IVIG as adjunctive
therapy in patients with streptococcal toxic shock
syndrome (STSS) in a randomized, double blind,
placebo-controlled trial (Darenberg et al. 2003).
Patients were randomly assigned to either IVIG
for three consecutive days (1 g/kg day one, 0.5 g/
kg day 2 and 3) or an equal volume of 1%
117
albumin. After inclusion of 21 patients, 10 receiv-
ing IVIG (of whom 6 had NSTI) and 11 receiving
placebo (of whom 7 had NSTI), the trial was
prematurely terminated due to slow inclusion,
after enrolling only 21 of the intended
120 patients. The primary endpoint was 28-day
mortality and there was no difference between the
two groups; 1 of 10 patients in the IVIG group
had died, and 4 of 11 patients in the placebo
group ( p ¼ 0.30). After administration of IVIG,
patient serum contained sufficient neutralizing
antibodies to suppress proliferation of
T-lymphocytes to below pretreatment levels,
whereas in the placebo group, no change in
neutralizing activity was seen. The mean time to
resolution of shock was 100 h for the IVIG group
and 122 h for the placebo group. The mean time
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
death, a total of 12 adverse events were observed,
none of which were found related to the study
drug. None of these secondary endpoints were
statistically significant.
The INSTINCT trial was a randomized, clini-
cal trial including 100 patients with NSTI
(Madsen et al. 2017). Patients with NSTI,
irrespective of etiology, were included after
admission to the intensive care. Patients were
randomized to receive IVIG 25 g/day for three
consecutive days or placebo. The primary out-
come was physical quality of life assessed by
the physical component summary (PCS) score
of the Short Form-36 questionnaire (Ware et al.
1992). No difference in the primary outcome
(mean PCS score 29 versus 28, mean difference
1 (95% CI 7 to 10)) or in secondary outcomes,
including mortality, serious adverse reactions
(SARs), organ failure, organ support, bleeding,
resolution of shock and days in hospital, was
observed. SARs included acute kidney injury,
allergic reactions, aseptic meningitis syndrome,
hemolytic anemia, thrombi, and transmittable
agents. In the preplanned subgroup analysis on
patients with presumed GAS infection, no differ-
ence was seen. Importantly, the distribution of
patients with GAS or S. aureus was uneven
between the two intervention groups. Some
118
limitations of the trial deserve mentioning; the
trial had reduced power, as patients who died
where given a PCS score of 0. Furthermore,
20 (40%) patients in the placebo group had
received one dose of IVIG prior to enrolment.
This might have diluted a potential effect. Lastly,
the trial was not powered to detect differences in
secondary outcomes, making it difficult to con-
clude whether IVIG use is safe in NSTI patients.
8.4.2.2 Systematic Reviews
and Meta-Analyses
A systematic review from 2018 included only one
RCT on IVIG for NSTI, and thus could not add
additional data (Hua et al. 2018). In a meta-
analysis from 2018 (Parks et al. 2018), evaluating
the effect of IVIG in patients with STSS, data
from four observational studies (Carapetis et al.
2014; Kaul et al. 1999; Adalat et al. 2014; Linnér
et al. 2014) and one RCT (Darenberg et al. 2003)
were included. Studies were included if patients
were identified prospectively, met the consensus
criteria of STSS and whose antibiotic treatment
included clindamycin. A total of 165 patients
were included in the analysis; 70 received IVIG
and 95 did not. IVIG use was associated with a
reduction in overall mortality rate from 33.7% to
15.7% (RR, 0.46; 95% confidence interval,
0.26–0.83; p ¼ 0.01). The analysis has
limitations, the main being that data from 147 of
the 165 patients included were from observational
studies, and thus no causative conclusions can be
drawn. The studies included used different
comparators, and for some of the studies, no
information on dosing or timing was given.
8.4.2.3 Observational Studies
In a retrospective observational study by Kadri
and colleagues, 4127 patients with necrotizing
fasciitis and shock from 121 centers were
identified based on diagnosis code (Kadri et al.
2017). Only 164 of these patients had received
IVIG. These were propensity-matched and risk-
adjusted with those who had not received IVIG.
The groups were similar regarding age,
diagnoses, comorbidities, day of IVIG dose and
destination of discharge of survivors. The primary
outcome was in-hospital mortality; the crude
M. B. Madsen et al.
mortality rate was 27.3% in the IVIG group and
23.6% in the non-IVIG group (adjusted OR 1.00,
95% CI: 0.55–1.83).
In a publication focusing on the subgroup of
patients with GAS NSTI (Bruun et al. 2020) from
the Scandinavian INFECT observational study
(Madsen et al. 2019), the administration of IVIG
was associated with decreased risk of mortality by
Lasso Regression analysis, but not by ordinary
logistic regression analysis. Due to the observa-
tional design, these data may support the hypoth-
esis that IVIG can have a beneficial effect in this
subgroup of patients, but no causal conclusions
can be drawn.
In an Australian surveillance programme of
invasive GAS infections from 2002 to 2004, the
mortality rate in patients treated with IVIG and
clindamycin was lower (1/14 (7%)) than in those
who were treated with clindamycin alone (7/39
(18%)), but this was not statistically significant
(Carapetis et al. 2014). In a similar Swedish sur-
veillance programme of patients with STSS, also
from 2002 to 2004, 67 patients were identified of
whom 23 received IVIG and 44 did not (Linnér
et al. 2014). At day 28, 3/23 (13%) in the IVIG
group, and 22/44 (50%) in the non-IVIG group,
had died (OR for death 0.18 (95% CI: 0.04–0.83),
adjusted for SAPS II and use of clindamycin).
The proportion of patients with necrotizing fasci-
itis was larger in the IVIG group; in a subgroup
analysis of patients with necrotizing fasciitis (n ¼
19), the OR for death was 0.17 (95% CI:
0.01–2.5). A third surveillance programme for
invasive GAS infections in Ontario identified
62 patients from 1992 to 2002; 35 (56%) patients
received IVIG (Mehta et al. 2006). No difference
in ICU/hospital-mortality by univariate analysis
was seen. Lastly, a prospective observational
study enrolled patients with STSS in Canada in
1992–1994 (Kaul et al. 1999). A total of
53 patients with STSS were included; OR for
death for the 21 patients receiving IVIG was
0.18 (95% CI: 0.03–1.02).
8.4.2.4 IVIG for Sepsis
Several trials on IVIG for patients with sepsis/
septic shock have been conducted, and systematic
reviews have shown potential beneficial effects
8 Treatment of Necrotizing Soft Tissue Infections: IVIG
on mortality (Soares et al. 2012; Turgeon et al.
2007), although results vary, depending on the
inclusion criteria (Alejandria et al. 2013). Besides
general concerns regarding IVIG use (Shankar-
Hari et al. 2018) (see section below), there are
several other concerns that need attention, in par-
ticular the quality of the included trials. Inclusion
criteria varied, as several definitions of sepsis
were used. Sepsis is a heterogenous disease, and
the patients response to infection may differ
according to the site infected (Shankar-Hari
et al. 2016; Burnham et al. 2017). Most of the
trials were of a relatively small sample size, rang-
ing from 24 to 682 patients. Whereas IVIG in
NSTI patients is believed to function through a
neutralizing effect of exotoxins and other bacte-
rial virulence factors, the mechanisms in sepsis is
more poorly understood, and this patient group is
even more heterogenous than NSTI patients.
Lastly, the trials were conducted more than
10 years ago—some of them more than 20 years
ago. In conclusion, there is no evidence to support
the use of immunoglobulin for treating patients
with sepsis or septic shock (Rhodes et al. 2017).
8.4.3 IVIG in NSTI, Unresolved Issues
The use of IVIG for patients with NSTI is a
complex intervention, with multiple aspects that
are unknown, or have not been addressed
adequately.
NSTI is not a well-defined disease, and NSTI
patients represent a heterogenic population. The
term encompasses many different variations of
severe soft tissue infections (Madsen et al.
2019). The varying nomenclature and classifica-
tion schemes also reflect this diversity; Necrotiz-
ing Fasciitis, describing an infection along the
fascia in the subcutis; Fournier’s gangrene,
describing a progressing soft tissue infection
originating from the anogentital area.; Type 1
and 2 necrotizing fasciitis, depending on a poly-
or monomicrobial etiology. It is likely, that the
pathophysiology varies depending on the affected
body-part, mechanism of bacterial entry and bac-
terial etiology (Madsen et al. 2019). Especially
the latter is of importance regarding IVIG
119
treatment. IVIG has been used as adjuvant ther-
apy for NSTI (de Prost et al. 2015), but not
necessarily accounting for bacterial etiology,
and the neutralizing abilities of IVIG have pri-
marily been shown in patients affected by NSTI
caused by GAS or S. aureus.
Questions on dosage, timing of administration,
and duration of treatment are still debated. Tradi-
tionally, dosing has ranged from 0.5 g/kg to 2 g/
kg (Table 8.5). As discussed previously, one dose
of 25 g seems to be sufficient to suppress toxin
activity in NSTI patients infected by GAS, and
after two doses, no toxin activity is seen
(Bergsten et al. 2020). However, this has not
been confirmed in a clinical setting, and opti-
mally, a randomized clinical trial on GAS NSTI
should be performed to assess whether this treat-
ment has a beneficial effect and no unacceptable
serious adverse reactions.
The optimal composition of immunoglobulins
is not known. IgG has many potential
mechanisms of action (Di Rosa et al. 2014), but
whether an additional effect of adding IgM is
achieved is not known. Furthermore, the
properties of the preparations are dependent on
the donors and thus immunomodulating
properties may vary.
Only few prospective studies have evaluated
the safety profile of IVIG in patients with NSTI.
Of concern is the severity of the disease and
polypharmacy in these patients, with the possibil-
ity of drug interactions (Kane-Gill et al. 2012).
8.4.4 Future Perspectives
From in vitro and in vivo studies it has been
demonstrated that IVIG has the potential to alle-
viate the course of NSTIs where GAS or
S. aureus is the causative agent. However, we
do not have data from randomized clinical trials
to show that this treatment is beneficial to patients
and, just as important, is safe to use in patients
that are critically ill. Furthermore, plasma is a
scarce product. A large, multi-center RCT
evaluating the effect of IVIG on mortality in
patients with GAS NSTI is therefore warranted.
120 M. B. Madsen et al.

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
supported by the European Union Seventh Framework
Programme: (FP7/2007–2013) under the grant agreement
305340 (INFECT project); the Swedish Governmental
Agency for Innovation Systems (VINNOVA) under the
frame of NordForsk (Project no. 90456, PerAID), and the
Swedish Research Council under the frame of ERA
PerMed (Project 2018-151, PerMIT).
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 Pathogenic Mechanisms
of Streptococcal Necrotizing Soft Tissue 9
Infections

Nikolai Siemens, Johanna Snäll, Mattias Svensson,

and Anna Norrby-Teglund

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

# Springer Nature Switzerland AG 2020 127

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_9
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
the INFECT study by Thänert and colleagues
(Thanert et al. 2019). 16S rRNA gene sequencing
revealed that the diversity of the bacterial
communities was highly dependent on
anatomical location of NSTIs. Upper and lower
extremities showed a Gini—Simpson diversity
index lower than 0.25 with significant detection
of streptococcal species. However, 16S rRNA
sequencing of tissue biopsies also revealed the
abundance of obligate anaerobes in NSTI cases
(Thanert et al. 2019; Zhao-Fleming et al. 2019).
9.2 Initial Steps of Tissue
Infections: Bacterial Adhesins
and Colonizing Factors
NSTIs can present with and without a defined
portal of entry (Stevens and Bryant 2017). In
approximately half of the cases the bacteria gain
entry to the deep tissue through superficial
lesions, penetrating trauma or breaches of the
skin (Morgan 2010). In the other 50% of cases,
NSTIs initiate deep in the soft tissue, sometimes
at sites of a non-penetrating trauma (Kaul et al.
1997; Stevens et al. 1989). It has been suggested
that transient bacteremia originating from the
nasopharynx, a potential reservoir for
streptococci, is the source of such infections
(Johansson et al. 2010; Stevens and Bryant
2017). However, reports providing such evidence
are lacking.
The ability of GAS to establish an infection
can be primarily attributed to the surface
anchored and associated adhesins. Among these,
the streptococcal M protein is the most abundant
and probably best characterized protein, which is
also used for emm-typing (Beall et al. 1996).
Since its discovery more than 200 different emm
serotypes have been described and nearly all GAS
isolates harbor an emm gene (Bessen et al. 2015).
In addition, many GAS strains encode emm-like
genes, mrp and enn (Bessen et al. 2015). Based on
the chromosomal arrangement of these three
genes, five distinct patterns, A to E, are described.
Emm pattern A-C isolates show a significant asso-
ciation with nasopharynx, while pattern D strains
are often isolated from skin infections (Bessen
129
2009). In contrast, emm pattern E isolates account
for almost equal fractions at both tissue sites
(Bessen et al. 2015). Epidemiological studies
revealed that severe disease manifestations,
including NSTIs and STSS, are predominantly
associated with emm1 and emm3 serotypes
(Bruun et al. 2020; Darenberg et al. 2007; Luca-
Harari et al. 2009; Stevens 1999).
The adhesion by GAS to epithelial surfaces is
a two-step process. The first step is a relatively
weak and unspecific and overcomes electrostatic
repulsion. It was suggested that lipoteichoic acid
(LTA) is a mediator of the first step of adhesion.
This interaction is reversible and not sufficient to
provide for tissue tropism (Courtney et al. 2002).
The second step of adhesion may then involve
classical and non-classical surface proteins of
GAS. These adhesins either bind directly to the
cell surface receptors or use extracellular host
matrix proteins or plasma proteins as a prime
target for specific interactions (Kreikemeyer
et al. 2004). In skin infections, M proteins can
mediate cell adhesion to keratinocytes directly via
CD46 binding (Okada et al. 1995) or indirectly by
binding human fibronectin as a target on host
epithelial cells (Courtney et al. 1986). In addition
to proteinaceous ligands, M proteins bind to
glycosaminoglycans and that interaction
promotes GAS attachment to human skin
fibroblasts (Frick et al. 2003b). There are other
classical and non-classical adhesins described for
GAS, including hyaluronic acid capsule that
binds to CD44 on keratinocytes, fibronectin-
binding proteins FbaA, F2, serum opacity factor
(SOF), Fbp54, ScpA, Lbp, and FbaB, collagen-
binding protein Cpa, vitronectin-binding protein,
galactose-binding protein, and plasminogen-
binding proteins Epf, GAPDH, enolase, and
phosphoglycerate kinase (PGK) (Rohde and
Cleary 2016). Hence, GAS express a variety of
surface factors that employ different mechanisms
contributing to bacterial adhesion. However,
(1) not all of them occur in all serotypes, (2) they
are expressed at different bacterial growth cycles,
and (3) many of them are host cell type specific
(Courtney et al. 2002). For example, M proteins
mediate adhesion to human keratinocytes, but not
to buccal cells (Okada et al. 1994).
130
On another note, the secreted exotoxins,
namely superantigens (SAgs), were recently
shown to be critical for colonization. The reports
showed that the SAgs elicited an inflammatory
response that promoted bacterial colonization and
establishment of infection (Kasper et al. 2014;
Zeppa et al. 2016, 2017). This provided new
insight into the function of this family of bacterial
exotoxins, most well known for their
pro-inflammatory activities and association with
the cytokine storm characterizing toxic shock
syndrome (detailed in separate subsections
below).
Once the infection is established GAS can
translocate to deeper tissue or initiate internaliza-
tion into the cells. Although GAS have long been
considered to be an extracellular pathogen, now it
is appreciated that GAS can internalize into
non-phagocytic cells even to an equal level as
classical intracellular pathogens (Lapenta et al.
1994). For example, M protein forms complexes
with fibronectin and interacts with α5β1 integrins.
Extensive integrin clustering triggers caveolae
aggregation to form invaginations that ingest the
bacteria without detectable actin polymerization.
In that way intracellular bacteria bypasses fusion
with lysosome and intracellular streptococci
reside within caveolin-1-contating endosomes,
the so-called caveosomes. Alternatively, M
protein-fibronectin interaction leads to the cyto-
skeletal rearrangement with actin accumulation
around the streptococci. This results in trafficking
of the bacteria inside the cells through early and
late endosomes, which fuse with lysosomes to
form phagolysosomes (Rohde and Cleary 2016).
The first mechanism directs GAS to the safer
caveosomal compartment, while the second
results in most of the bacteria being killed by
the host cells (Dombek et al. 1999). In line with
this, Thänert and colleagues have shown that
transcripts of genes encoding fibronectin-binding
proteins, which mediate adherence to the extra-
cellular matrix and invasion into human host
cells, are highly abundant in NSTI patient
biopsies (Thanert et al. 2019). Thus, the results
underline the importance of streptococcal surface
proteins in NSTIs and suggest that there are addi-
tional functions to be explored in future studies.
N. Siemens et al.
9.3 Secreted Molecules
Contributing to Host Evasion
and Tissue Pathology
9.3.1 Streptolysins
To breach dermal barriers and enable the bacteria
to disseminate through the tissue, GAS secrete
several pore-forming exotoxins, proteases, and
DNases (summarized in Table 9.1). Studies have
shown that secreted factors are highly
up-regulated during fulminant tissue infections
and play a crucial role in NSTIs (Johansson
et al. 2010; Olsen and Musser 2010; Thanert
et al. 2019). Pore-forming toxins are a class of
bacterial virulence factors that cause cytolysis,
disrupt membranes, and have immune-
modulatory functions. Nearly all GAS isolates
secrete two potent hemolysins, namely Streptoly-
sin S (SLS) and Streptolysin O (SLO) (Barnett
et al. 2015; Yoshino et al. 2010). SLO is a 57 kDa
oxygen sensitive, cholesterol-dependent cytoly-
sin. It disrupts cytoplasmic membrane integrity
of epithelial and endothelial cells, monocytes,
macrophages, and neutrophils, thereby inducing
cell death through different mechanisms, includ-
ing apoptosis, necrosis, and/or pyroptosis
(Chandrasekaran and Caparon 2016; Keyel et al.
2013; Timmer et al. 2009). For example, SLO
blocks the clathrin-dependent pathway for GAS
internalization and induces keratinocytes apopto-
sis through dysregulation of the calcium signaling
(Cywes Bentley et al. 2005). At sub-lytic
concentrations, SLO suppresses crucial neutro-
phil functions, including chemotaxis and forma-
tion of neutrophil extracellular taps (NETs)
(Uchiyama et al. 2015). Furthermore, SLO
impairs intracellular GAS clearance by
neutrophils and professional phagocytes. It
inhibits dendritic cell (DC) maturation by
suppressing the expression of major histocompat-
ibility complex (MHC) class II and
co-stimulatory molecules CD80 and CD83
(Cortes and Wessels 2009) and prevents
phagolysosome acidification in macrophages by
damaging the phagolysosome membrane
(Bastiat-Sempe et al. 2014). In murine models of
9 Pathogenic Mechanisms of Streptococcal Necrotizing Soft Tissue Infections 131

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
mutants lacking slo gene were less virulent as
compared to the parental strain (Zhu et al. 2017).
In contrast to SLO, SLS is a small
non-immunogenic peptide of 2.7 kDa, which is
encoded by the SLS-associated gene (sag) locus
consisting of nine genes (sagABCDEFGHI). The
sagA gene encodes the premature form of SLS,
while other genes encode proteins for post-
translational processing and export (Nizet et al.
2000). SLS targets mainly human red blood cells,
platelets, and leukocytes. It accumulates on cell
membranes of eukaryotic cells and induces pore
formation by a yet not fully characterized mecha-
nism (Barnett et al. 2015). Higashi and colleagues
suggested that SLS interacts with the major
erythrocytes anion exchange protein band
3, thereby inducing osmotic change, rapid Cl -
influx, and erythrocyte lysis (Higashi et al. 2016).
Whether similar mechanisms apply to other
human host cells needs to be proven. However,
experimental NSTI models have shown that SLS
facilitates translocation of GAS across epithelial
barriers through direct cleavage of junctional
proteins E-cadherin and occludin (Sumitomo
et al. 2011). Furthermore, SLS-dependent cyto-
toxicity to human keratinocytes is mediated
through activation of pro-inflammatory mediators
p38 MAPK and NFκB. Subcutaneous infections
of mice result in IL-1β production in a
SLS-dependent manner (Flaherty et al. 2018). A
recent study by Pinho-Ribeiro and colleagues has
shown that SLS is also responsible for activation
of pain producing sensory nociceptor neurons
(Fig. 9.1) (Pinho-Ribeiro et al. 2018). This is of
great interest as an out-of-proportion pain at early
stages of infections is one of the critical hallmark
symptoms of NSTIs. SLS-dependent activation of
nociceptor neurons results in a release of calcito-
nin gene-related peptide (CGRP) into infected
tissue, which in turn suppresses critical neutrophil
functions, including recruitment to the site of
infection, opsonophagocytosis, and killing of the
bacteria (Pinho-Ribeiro et al. 2018). These
actions allow the bacteria to spread in the tissue.
Once deeper skin layers are reached, direct cyto-
toxicity of SLS towards different cell
compartments provokes neutrophil influx
(Humar et al. 2002). In turn, SLS actively
destroys neutrophils which are recruited to the
site of infection (Humar et al. 2002). These events
potentially negatively contribute to the outcome
in patients: (1) early CGRP-mediated suppression
of influx resulting in a lack or reduced number of
neutrophils is an unfavorable prognostic sign in
GAS NSTIs (Bakleh et al. 2005) and (2) hyper-
132
Fig. 9.1 Immune evasion strategies by GAS. GAS
secrete a variety of virulence factors responsible for bacte-
rial spread in the tissue. Streptokinase (Ska) forms
complexes with plasminogen (Plg). These Ska-Plg
complexes degrade fibrin clots, LL-37, and all classes of
human histones. Streptococcal pyrogenic exotoxin B
(SpeB) is a cysteine protease with a broad spectrum of
human substrates. SpeB degrades all classes of
immunoglobulins, complement components (e.g., C3),
antimicrobial peptides (e.g., LL-37), extracellular matrix
proteins (ECM), and CXCL chemokines including
activation and SLS-dependent damage of
neutrophils at later time points result in aug-
mented tissue pathology through neutrophil-
derived factors (Johansson et al. 2009, 2010;
Johansson and Norrby-Teglund 2013; Nuwayhid
et al. 2007).
N. Siemens et al.
CXCL1-7. SpyCEP, a subtilisin-like protease, degrades
CXCL8 which results in impaired neutrophil recruitment
to the site of infection. SLS-dependent activation of noci-
ceptor neurons (N) results in a release of calcitonin gene-
related peptide (CGRP), which suppresses neutrophil
recruitment. Furthermore, GAS have developed strategies
to avoid phagocytic killing. They reside within
macrophages. However, certain environmental signals
trigger the egress out of the cells and bacterial spread
within the tissue. In addition, GAS form biofilms within
the tissue
9.3.2 Proteases and DNases
9.3.2.1 The Cysteine Protease SpeB
In addition to pore-forming toxins, streptococcal
proteases damage tissue barriers, inhibit immune
cell transmigration to the site of infection, and
9 Pathogenic Mechanisms of Streptococcal Necrotizing Soft Tissue Infections
impair directly or indirectly immune cell
functions. One of the first proteases identified in
GAS is the streptococcal pyrogenic exotoxin B
(SpeB) (Elliott 1945). SpeB gene is highly
conserved in GAS (Yu and Ferretti 1991) and it
encodes a zymogen of 40 kDa which is auto-
catalytically cleaved into a mature 28 kDa prote-
ase (Liu and Elliott 1965). SpeB has a broad
spectrum of host and bacterial substrates. It
removes GAS surface anchored proteins, includ-
ing M protein, fibronectin-binding proteins, and
C5a-peptidase, and hydrolyzes secreted proteins,
such as streptokinase (Ska), SLO, EndoS, and
superantigens (Nelson et al. 2011). At the host
site, SpeB degrades immunoglobulins and
components of the complement system
(Fig. 9.1) (Kuo et al. 2008). These events result
in impaired opsonophagocytosis and subsequent
failure in phagocytic clearance of the bacteria
(Collin et al. 2002). Moreover, SpeB degrades
antimicrobial peptides produced by the host,
including LL-37, which was shown to provide
protection against murine soft tissue infections
(Nizet et al. 2001). Neutrophil-derived LL-37 is
highly abundant in human biopsy specimens and
its expression is positively correlated to the bac-
terial load (Johansson et al. 2008). However, such
a positive correlation suggests that LL-37 is not
efficient in bacterial killing. Nyberg and
colleagues have shown that SpeB gets entrapped
by α2-macroglobulin-GRAB complex on bacte-
rial surface. As a consequence, SpeB is retained at
the bacterial surface and protects bacteria against
killing by LL-37 (Nyberg et al. 2004). Further-
more, SpeB degrades a wide range of
chemokines, including CXCL chemokines
CXCL1-7, CXCL10-14, CXCL16, and CCL20,
XCL1, and CX3CL1 (Egesten et al. 2009), and
cleaves pro-IL-1β into mature form (Kapur et al.
1993). LaRock and colleagues suggested that
IL-1β acts as an intracellular sensor of bacterial
proteolytic activity (Larock et al. 2016). GAS
have adapted different strategies, including
mutations abrogating SpeB expression to evade
detection by IL-1β and subsequent killing of the
pathogen (Sumby et al. 2006). Genes encoding
for CovR/S two component system (TCS) and
transcriptional stand-alone regulator RopB,
133
which directly or indirectly control SpeB expres-
sion, are such mutational hot-spots (Hollands
et al. 2008; Sumby et al. 2006). Although SpeB
shows such a broad spectrum of substrates, its
role in invasive diseases is still under debate.
The speB gene can be found in all types of GAS
isolates (Darenberg et al. 2007; Luca-Harari et al.
2009). It was shown that SpeB is readily detect-
able in sera and tissues of NSTI patients (Gubba
et al. 1998; Siemens et al. 2016). Some studies
demonstrate that GAS isolates from non-invasive
infections express higher amounts of SpeB and
subsequently show greater activity as compared
to invasive strains (Kansal et al. 2000). However,
other studies demonstrate the opposite. GAS
isolates from invasive infections produce high
amounts of SpeB and low anti-SpeB antibody
titers in patients were associated with fatal out-
come (Holm et al. 1992). Furthermore, the con-
troversy of SpeB contribution in NSTIs continues
in murine soft tissue infection models. Some
authors report that SpeB highly contributes to
disease severity, tissue damage, bacterial dissem-
ination, and mortality (Kuo et al. 1998; Lukomski
et al. 1997, 1998, 1999). Others demonstrated that
speB-deficient mutant strains are as virulent as
their parental wild type strains (Ashbaugh et al.
1998; Ashbaugh and Wessels 2001). However,
human tissue biopsies from NSTI patients are
strongly positive for SpeB and a mixture of
SpeB-positive and negative clones is usually
seen in patients (Johansson et al. 2008; Siemens
et al. 2016).
9.3.2.2 Streptokinase (Ska) and SpyCEP
In contrast to eliminated SpeB expression in nat-
ural GAS covR/S mutants, several other secreted
factors are up-regulated in the same genetic back-
ground. This includes SLO, SpyCEP, Sda1, and
Streptokinase (Ska) (Cole et al. 2011; Siemens
et al. 2015; Sumby et al. 2006; Walker et al.
2007). Ska is a non-enzymatic plasminogen
(Plg) activator. GAS cover their surface with Plg
via different surface proteins, which in turn
results in acquisition of endogenously produced
and secreted Ska (Chandrahas et al. 2015;
Siemens et al. 2011). The Ska-Plg complex
exposes the active site and proteolytically
134
converts surface-bound Plg to plasmin (Boxrud
et al. 2004). Plasmin is a broad-spectrum host
protease that is involved in fibrinolysis. In the
absence of SpeB, active plasmin accumulates on
bacterial surface, resulting in protease activity
that enables the bacteria to degrade tissue barriers
and facilitate invasive spread (Cole et al. 2006;
Sanderson-Smith et al. 2008). Furthermore the
Ska-Plg complex degrades LL-37 and all classes
of human histones and abrogates their anti-
bactericidal effects (Fig. 9.1) (Hollands et al.
2012; Nitzsche et al. 2016). Due to human host-
specificity of Ska, no differences between wild
type and ska-deficient GAS strains were seen in
murine models (Khil et al. 2003). In contrast, by
using humanized mice expressing human plas-
minogen it was shown that activation of host Plg
by Ska leads to accelerated clearance of host
fibrin, which plays a central mechanism in GAS
invasion and spread in tissues (Sun et al. 2004).
It is becoming more evident that antimicrobial
peptides including LL-37 act as immunomodula-
tory and chemotactic agents (Beisswenger and
Bals 2005). Furthermore, LL-37 influences bac-
terial virulence properties. It induces an invasive
phenotype of GAS by up-regulating gene expres-
sion of SpyCEP, among others (Gryllos et al.
2008). SpyCEP is a surface localized subtilisin-
like protease of 180 kDa, which is highly
expressed in vivo (Zinkernagel et al. 2008). It
cleaves human CXCL chemokines, including
CXCL1, CXCL2, CXCL3, CXCL5, CXCL6,
and CXCL8, and murine chemokines CXCL1
and CXCL2 (Fig. 9.1) (Hidalgo-Grass et al.
2004, 2006; Sumby et al. 2008; Zinkernagel
et al. 2008). Considering the role of CXCL8 in
human neutrophil chemotaxis to the infected tis-
sue, it is tempting to assume that lack of
neutrophils due to CXCL8 degradation will result
in failed bacterial clearance and severe tissue
pathology caused by bacterial toxins. Indeed, sev-
eral murine studies of soft tissue infections have
demonstrated a paucity of neutrophil influx
resulting from chemokine degradation by
SpyCEP (Edwards et al. 2005; Hidalgo-Grass
et al. 2004, 2006; Sumby et al. 2008; Zinkernagel
et al. 2008). However, infiltration of neutrophils
in superficial fascia and dermis is one of the
N. Siemens et al.
pathological criteria for diagnosis of NSTIs
(Stamenkovic and Lew 1984). Several studies
utilizing patient’s biopsy material have shown
that neutrophils represent one of the major
immune cell infiltrating populations in NSTIs
(Johansson et al. 2009, 2014; Siemens et al.
2016; Thulin et al. 2006). The infiltration of
neutrophils positively correlated with bacterial
load in the tissue (Thulin et al. 2006). These
facts suggest that the murine models are limited
in mimicking the complexity of human infection
setting, in which a plethora of chemotactic signals
exists.
9.3.2.3 The Immunoglobulin Degrading
Enzymes IdeS and EndoS
GAS have also developed strategies to evade
adaptive immunity at the site of infection. The
bacteria secrete two enzymes, namely immuno-
globulin degrading enzyme (IdeS) and
endoglycosidase (EndoS). IdeS hydrolyzes four
subclasses of IgG (Von Pawel-Rammingen et al.
2002). However, its role in NSTIs is not yet clear.
EndoS, which transcription is highly
up-regulated during NSTIs (Thanert et al.
2019), specifically cleaves the conserved N-gly-
can of IgG antibodies. A recent study
demonstrated that EndoS specifically alters IgG
Fc glycosylation in vivo. At the site of infection,
IgG glycan hydrolysis positively correlates with
bacterial load and EndoS secretion (Naegeli et al.
2019). These actions potentially result in failed
opsonophagocytosis and pathogen clearance.
The authors showed that ndoS-mutant is more
susceptible to phagocytic killing as compared to
its parental wild type strain. In contrast to tissue
findings, glycan hydrolysis in circulation was
only detected in patients with septic shock
(Naegeli et al. 2019).
9.3.2.4 Streptodornase 1(Sda1)
Sda1 is a secreted enzyme which is predomi-
nantly associated with emm1 GAS NSTI isolates.
It displays potent sequence-nonspecific nuclease
activity on DNA substrates (Aziz et al. 2004a). It
occurs during phase-shifts to hyper-virulent inva-
sive infection and is believed to play a role in
evasion of the host’s innate immune responses
9 Pathogenic Mechanisms of Streptococcal Necrotizing Soft Tissue Infections
(Aziz et al. 2004b; Walker et al. 2007). Sda1 is
involved in degradation of the DNA component
of chromatin-rich NETs (Walker et al. 2007), a
mechanism which provides a protective effect
against bacterial killing by neutrophils. Recently
it was also shown that Sda1-mediated degradation
of streptococcal DNA alters recognition of GAS
by Toll-like receptor (TLR)9 (Uchiyama et al.
2012), which preferentially senses bacterial and
viral DNA. DNA degradation by Sda1 prevented
GAS induced IFNα and TNFα secretion from
murine macrophages and contributed to bacterial
survival. Furthermore, IFNα and TNFα levels
were significantly decreased in wild type mice
infected with Sda1-positive GAS strain as com-
pared to TLR9-deficient mice (Uchiyama et al.
2012). In addition, Sda1 impaired plasmacytoid
(p)DC recruitment to the site of infection. Keller
and colleagues suggested that Sda1 interferes
with stabilization of the DNA in a HMGB1-
dependent manner, which may result in decreased
IFN-1 levels and subsequently lead to reduced
numbers of pDCs (Keller et al. 2019).
9.3.3 The Streptococcal Inhibitor
of Complement (SIC)
In addition to proteases and DNases, a limited
number of GAS M serotypes (emm1, emm12,
emm55, emm57) secrete a protein called strepto-
coccal inhibitor of complement (SIC). SIC is a
highly polymorphic 31 kDa negatively charged
protein which interferes with complement
functions (Akesson et al. 1996; Fernie-King
et al. 2001). Furthermore, SIC blocks the lytic
activity of antimicrobial peptides such as LL-37,
defensins, H-kininogens, and lysozyme (Fernie-
King et al. 2002, 2004; Frick et al. 2003a, 2011).
Recently it was shown that SIC also binds to
extracellular histones, a group of host-associated
damage-associated molecular patterns (DAMPs)
released during necrotizing tissue infections, and
neutralizes their antimicrobial activity. Such
SIC-histone aggregates are readily detectable in
biopsies of NSTI patients (Westman et al. 2018).
Although SIC is considered to be an anti-
inflammatory agent (Akesson et al. 2010), SIC
135
binding to histones boosted the release of a
broad range of pro-inflammatory cytokines and
chemokines (Westman et al. 2018). Such actions
can potentially further contribute to hyper-
inflammation in tissue environment.
9.4 Local and Systemic
Inflammation in NSTIs
9.4.1 Neutrophil Responses in NSTI
Despite the fact that GAS factors contribute to
tissue pathology, it is widely accepted that
immune cells and host-derived factors play a cru-
cial role in hyper-inflammatory and tissue
destructive processes (Herwald et al. 2004;
Johansson et al. 2009; Kahn et al. 2008). Several
immune cell populations are recruited to the site
of infection, including neutrophils, macrophages,
T cells, and dendritic cells (Norrby-Teglund et al.
2001). Neutrophil migration to the site of infec-
tion is a crucial step in host defense. Once
neutrophils have reached the infection site, three
distinct mechanisms to fight an infection can
occur: phagocytosis, degranulation, and forma-
tion of NETs (Kolaczkowska and Kubes 2013).
Critical tissue damaging components of all these
processes are granule effector molecules, includ-
ing proteolytic enzymes and antimicrobial
peptides (Borregaard et al. 2007). GAS have
been demonstrated to have a significant impact
on neutrophils, by inducing an apoptotic program
and triggering degranulation (Kobayashi et al.
2003; Snall et al. 2016; Soehnlein et al. 2008)
and both, surface associated as well as secreted
factors are potent inducers of neutrophil degranu-
lation (Snall et al. 2016; Uhlmann et al. 2016b).
Strong neutrophil influx in the tissue is a typical
characteristic for GAS NSTIs and there is a sig-
nificant correlation between bacterial load, neu-
trophil influx, presence of degranulation markers
heparin-binding protein (HBP) and resistin, and
tissue pathology and inflammation (Fig. 9.2)
(Johansson et al. 2008, 2009, 2014; Snall et al.
2016). Streptococcal M protein is one of the
major triggers of neutrophil activation. Soluble
M1 protein forms complexes with fibrinogen
136
Fig. 9.2 Immune cell mediated hyper-inflammatory
response in the tissue triggered by secreted streptococcal
virulence factors. Several GAS factors contribute to hyper-
inflammation of the tissue. For example, soluble M1 pro-
tein (sM1) forms complexes with fibrinogen, which in turn
activates neutrophils. Furthermore, PGK and streptolysins
S and O (SLS/SLO) trigger neutrophil activation. This
results in a release of granule effector molecules, including
resistin, heparin-binding protein (HBP), LL-37, and
myeloperoxidase, which augments tissue inflammation.
and binds β2-integrins on neutrophil surface. This
event results in a release of massive amounts of
the granule proteins, including HBP, which is
N. Siemens et al.
SLS and SLO induce through different mechanisms host
cell lysis resulting in a release of pro-inflammatory
cytokines and chemokines. In addition, SpeB cleaves
proIL-1β into mature IL-1β leading to pyroptosis of
macrophages. GAS also secrete superantigens (SAgs).
SAgs bind without cellular processing to α- and β-chains
of MHC class II molecules on antigen-presenting cells
(APC) and variable β-chains of T cell receptor. These
interactions result in a massive cytokine storm
solely responsible for vascular leakage (Gautam
et al. 2001; Herwald et al. 2004). NSTI patient
biopsies are highly positive for such M protein/
9 Pathogenic Mechanisms of Streptococcal Necrotizing Soft Tissue Infections
fibrinogen complexes (Herwald et al. 2004; Kahn
et al. 2008). Furthermore, streptococcal secreted
factors which are susceptible to SpeB degrada-
tion, e.g., PGK and SLO, are potent inducers of
neutrophil degranulation (Nilsson et al. 2006;
Uhlmann et al. 2016b). Nilsson and colleagues
observed that GAS supernatants containing high
levels of SLO activated neutrophils by
perforating these cells. As a consequence,
neutrophils secreted HBP, LL-37, α-defensins,
and neutrophil elastase (Fig. 9.2). Bacterial
supernatants with negligible levels of SLO failed
to induce degranulation (Nilsson et al. 2006).
Most likely, these effects were observed due to
the presence or absence of SpeB in bacterial
supernatants. Uhlmann et al. demonstrated that
high degranulation response was triggered exclu-
sively by supernatants from SpeB-negative
strains (Uhlmann et al. 2016b). Global proteome
analyses identified phosphoglycerate kinase
(PGK) as a stimulatory factor for neutrophils.
Further experiments confirmed that PGK is sus-
ceptible to SpeB degradation (Uhlmann et al.
2016b). The neutrophil response and subsequent
tissue hyper-inflammation can be further aug-
mented through the streptococcal vesicle-like
structures. It was shown that GAS respond to
sub-inhibitory concentrations of LL-37 by releas-
ing vesicles containing several virulence factors.
These vesicles are of pro-inflammatory nature and
elicit resistin and myeloperoxidase release from
neutrophils (Uhlmann et al. 2016a).
9.4.2 Macrophage Responses in NSTI
Several studies have shown that in addition to
neutrophils, macrophages are also predominantly
infiltrating highly infected tissue of NSTI patients
(Johansson and Norrby-Teglund 2013; Johansson
et al. 2010, 2014; Siemens et al. 2016; Thulin
et al. 2006). Macrophages are highly effective
professional phagocytes. They ingest bacteria by
recognition of GAS via surface pattern recogni-
tion receptors (e.g., TLRs), which in turn induces
remodeling of the actin cytoskeleton to form a
phagosome (Valderrama and Nizet 2018). The
phagosome interacts with endosomes and
137
lysosomes, leading to phagolysosome formation,
which would usually kill the pathogen. In vitro
studies have shown that GAS activate NFκB and
MAPK in a MyD88-dependent manner leading to
production of pro-inflammatory cytokines,
including TNF and IL-6 (Gratz et al. 2008). In
addition, an up-regulation of the maturation
markers such as CD80 and CD86 was noted.
Experimental mice models of GAS skin infection
demonstrated that impaired MyD88 signaling
results in diminished pro-inflammatory response,
including IL-12, IFNγ, and TNF production as
well as chemoattractants such as CXCL1 and
CCL2. This in turn leads to the impaired recruit-
ment of macrophages and neutrophils (Loof et al.
2010). In addition, TNF-deficient mice is highly
susceptible to GAS soft tissue infections due to
the defect in macrophage recruitment to the site of
infection (Mishalian et al. 2011). However, mice
studies are highly controversial. MAPK and
NFκB activation and MyD88-dependent cytokine
production are TLR-independent processes in
mice as compared to humans (Gratz et al. 2008).
Analyses of human NSTI biopsies revealed that
even if the biopsies were highly infiltrated by
neutrophils and macrophages, high amounts of
viable bacteria were readily detectable, even in
those patients with prolonged antibiotic therapy.
Viable bacteria were mainly residing within
macrophages and such biopsies were
characterized by lower inflammation level and
lower bacterial load (Thulin et al. 2006). As pre-
viously mentioned, bacteria are normally killed
by phagolysosomal degradation. However, GAS
have acquired specific mechanisms to avoid such
killing. These processes involve M1-protein-
dependent trafficking and impaired fusion of
phagosome with lysosome leading to a persistent
infection (Hertzen et al. 2010), adaptation to
acidic environment, and escape from the
phagolysosome (Fig. 9.1) (Bastiat-Sempe et al.
2014). GAS pore-forming toxin SLO and its
co-toxin NAD-glycohydrolase (NADase) medi-
ate GAS intracellular survival in macrophages.
The two toxins prevent phagolysosomal acidifi-
cation. SLO perforates the phagolysosomal mem-
brane and enables translocation of NADase into
the macrophage cytosol. Subsequently, NADase
138
augments SLO-mediated cytotoxicity by deplet-
ing cellular energy stores (Bastiat-Sempe et al.
2014). However, the bacteria do not only persist,
but also replicate within macrophages and egress
(Hertzen et al. 2010). Ihk/Irr TCS was identified
as a key regulatory system at early stages of
bacterial persistence within macrophages. Fur-
thermore, Kachroo and colleagues have shown
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
down-regulation of Ihk/Irr TCS were seen at late
stages when the bacteria were exiting the cells
(Hertzen et al. 2012). It was also suggested that
SpeB, which is regulated by the CovR/S TCS,
contributes to intracellular survival and bacterial
egress out of the cell (Thulin et al. 2006). LaRock
and colleagues demonstrated that SpeB cleaves
IL-1β, which is usually seen as a marker of canon-
ical and caspase-1 dependent inflammasome acti-
vation and subsequent pyroptosis of the cells
(Larock et al. 2016). However, the direct cleavage
by SpeB induces a non-canonical IL-1β activation
in macrophages (Fig. 9.2). Analyses of patient’s
tissue biopsies revealed that once IL-1β is
released it forms immuno-stimulatory complexes
with High Mobility Group Box 1 (HMGB1) pro-
tein (Johansson et al. 2014). HMGB1 is a ubiqui-
tously expressed nuclear protein that is released
through active secretion by innate immune cells
or through passive release by injured cells (Skin-
ner 2010). Extracellular HMGB1 has been
reported to act as an alarmin with ability to acti-
vate the immune system and elevated levels of
HMGB1 were found in patients diagnosed with
sepsis and/or septic shock (Sunden-Cullberg et al.
2005). In GAS tissue infections, HMGB1 expres-
sion correlated with disease severity and
macrophages were identified as a predominant
source of HMGB1 release (Johansson et al.
2014). A close proximity between HMGB1 and
neutrophils suggested also a chemotactic role of
this molecule in GAS NSTIs which was further
confirmed by in vitro experiments (Johansson
et al. 2014).
Despite the fact that macrophages contribute to
the highly pro-inflammatory state in the tissue,
there is evidence rising that GAS induce a
N. Siemens et al.
mixed activation of murine macrophages
(Goldmann et al. 2007). Transcriptomic analyses
revealed that GAS infections induce both, Th1
(classical) signatures characterized by induction
of IL-1 and IL-6 cytokines, as well as Th2 (alter-
native) responses, e.g., induction of IL-1 decoy
receptor and IL-10 (Goldmann et al. 2007). How-
ever, this phenomenon was observed in mice.
9.4.3 Superantigens
NSTIs are often complicated by STSS (Johansson
et al. 2010). Sepsis-3 criteria define sepsis as a
life-threatening organ dysfunction caused by a
dysregulated host response to an infection. STSS
is a subset of sepsis in which particularly pro-
found circulatory, cellular, and metabolic
abnormalities are associated with a greater risk
of mortality (Singer et al. 2016). STSS is a result
of an invasive GAS infection (Low 2013) and
superantigens (SAgs) are key mediators of a sys-
temic excessive inflammatory response
(Llewelyn and Cohen 2002). To date, 13 GAS
SAgs were identified, which include streptococ-
cal pyrogenic exotoxins (Spe) A, C, G-M, strep-
tococcal superantigen (SSA), and streptococcal
mitogenic exotoxin Z (SmeZ) (Commons et al.
2014) and the two recently identified
superantigens SpeQ and SpeR (Reglinski et al.
2019). SAgs bind intact to α- and/or β-chains of
the major histocompatibility complex (MHC)
class II molecules on antigen-presenting cells
(APCs) and to the variable β-chains of the T cell
receptor. In addition, SAgs can also bind a
co-stimulatory molecule CD28 and its ligand
CD86 (B7-2) (Arad et al. 2011; Levy et al.
2016). Once the MHC-peptide specificity of T
cells is bypassed, these interactions result in a
massive cytokine storm, which includes produc-
tion of TNF, IFN-γ, IL-1, IL-2, IL-6, CXCL8,
CCL2, and CCL3 (Fig. 9.2) (Chatila and Geha
1993). However, such responses may vary due to
specific HLA class II haplotypes which may
influence the severity of GAS infections. Kotb
and colleagues have shown that certain
haplotypes (e.g., DRB1*1501/DQB1*0602) are
of a protective nature resulting in attenuated
9 Pathogenic Mechanisms of Streptococcal Necrotizing Soft Tissue Infections
inflammatory responses to the streptococcal
SAgs. In contrast, other haplotypes (e.g.,
DRB1*14/DQB1*0503 and DRB1*07/
*
DQB1 0201) are associated with a risk to develop
severe systemic responses (Kotb et al. 2002). A
recent report identified that Mucosa-associated
invariant T (MAIT) cells are strongly activated
by SAgs and represent the major T cell source of
IFNγ and TNF in the early cytokine response
associated with STSS (Emgard et al. 2019). How-
ever, the majority of the SAg studies are confined
to the systemic effects and only a limited number
of studies investigated SAg-driven events at the
deep tissue site (Johansson et al. 2010; Norrby-
Teglund et al. 2001). Norrby-Teglund and
colleagues showed a massive in vivo production
of SAgs in biopsies of GAS NSTI patients
(Norrby-Teglund et al. 2001). The infected tissue
was characterized by an enormous infiltration
with monocytes, macrophages, and T cells,
which correlated with a typical SAg-driven exces-
sive inflammatory response and the severity of
infection (Norrby-Teglund et al. 2001). In addi-
tion, up-regulation of the homing receptors
CCR5, CD44, and cutaneous lymphocyte-
associated antigen correlated with the expression
of Th1 cytokines at the tissue site (Norrby-
Teglund et al. 2001). T cell derived Th1 cytokines
and predominantly IFN-γ induce production of
IL-1β by monocytes and macrophages
(Chizzolini et al. 1997). In line with this, it was
shown that IL-1β plays a crucial role in GAS
NSTIs. Mice lacking IL-1R are more susceptible
to GAS infections (Hsu et al. 2011). Furthermore,
it was reported that patients who received the
IL-1R antagonist Anakinra experience more fre-
quently invasive GAS diseases including NSTIs,
which results in a greater risk of mortality (Larock
et al. 2016). In addition, by utilizing BXD mice
Chella Krishnan and colleagues have shown that
IL-1β participates in modulating GAS NSTIs
(Chella Krishnan et al. 2016). The authors
demonstrated that IL-1β gene expression was sig-
nificantly up-regulated in tissue biopsies of GAS
NSTI susceptible mice strains as compared to
non-susceptible strains. This finding was further
verified in plasma samples and tissue biopsies
from GAS NSTI patients, as well as samples
139
from an in vitro experimental model of human
skin. High IL-1β mRNA levels were detected in
infected tissues. In addition, analyses of systemic
IL-1β revealed high levels in patient plasma col-
lected during an acute phase of GAS NSTIs.
These levels declined during the treatment
(Chella Krishnan et al. 2016). A study by Hansen
and colleagues underlined the importance of
IL-1β in NSTIs. IL-1β was determined as an
independent predictor for 30-day mortality in
NSTIs (Hansen et al. 2017). In addition, signifi-
cantly higher IL-6 and TNF plasma levels in
streptococcal infected patients were detected as
compared to patients infected with other
microbiological agents, which is consistent with
septic shock being more prevalent in streptococ-
cal NSTIs (Hansen et al. 2017). Comparative
RNAseq analyses of tissue specimens from strep-
tococcal and polymicrobial NSTIs identified a
core of common up-regulated host genes
encoding pro-inflammatory mediators, including
IL-6 and CXCL8, complement components,
alarmins, and proteolytic enzymes (Thanert et al.
2019). However, GAS NSTI biopsies were
characterized by a higher expression of genes
encoding interferon-inducible mediators, which
includes CXCL9, CXCL10, CXCL11, MX1, and
MX2. This finding was further verified in patient
plasma samples. CXCL9, CXCL10, and
CXCL11 chemokine plasma levels were higher
in GAS NSTI patients as compared to patients
with polymicrobial NSTIs (Thanert et al. 2019).
Thus, these chemokines might serve as potential
biomarkers for GAS NSTIs.
9.5 Biofilm and Complex
Modelling of GAS NSTIs
in the Human Tissue Setting
Biofilm formation is recognized as a virulence
trait of many chronic tissue infections, including
cystic fibrosis, burn wound infections, and
infected diabetic foot ulcers (Bjarnsholt et al.
2018). Although GAS biofilm studies were
performed in the past, until recently, GAS have
not been considered a major biofilm forming spe-
cies due to the lack of clinically relevant reports.
140
Bacteria can exist in two states, planktonic or
biofilm and it has become clear that it is a
dynamic process allowing the bacteria to fre-
quently flux between these states (Mcdougald
et al. 2011). The process of biofilm formation
can be summarized in four steps: (1) reversible
attachment to abiotic or biotic surfaces driven by
environmental signals (e.g., pH, temperature, and
nutrients availability), (2) irreversible attachment
with initial microcolony formation and develop-
ment of an extracellular matrix, (3) biofilm matu-
ration, and (4) dispersal (Kostakioti et al. 2013).
The first report providing evidence of GAS bio-
film in vivo was published by Akiyama and
colleagues (Akiyama et al. 2003). Confocal laser
scanning microscopy (CLSM) of impetigo skin
biopsies revealed concanavalin A positive GAS
microcolonies consistent with biofilm formation
(Akiyama et al. 2003). The second study
performed on tonsils from children with recurrent
GAS pharyngitis identified three dimensional
communities of GAS within reticulated crypts
(Roberts et al. 2012). However, both studies
were of descriptive nature and have not addressed
potential pathogenic effects of GAS biofilms
within the host. A more recent clinical study on
GAS NSTIs presented a patient with a persistent
infection over a period of 24 days. The surgeons
reported a thick layer of biofilm covering
fasciotomy wounds (Siemens et al. 2016).
Analyses of biopsies collected from different
anatomical locations of 31 GAS NSTI patients
revealed that in 32% of cases, aggregations con-
sistent with biofilm communities were evident.
CLSM and scanning electron microscopy (SEM)
analyses confirmed the presence of amorphous or
fibrous biofilm structures consisting of bacteria,
polysaccharides, lipids, and DNA. Biofilm-
positive biopsies were characterized by a higher
bacterial load. In addition, higher neutrophil
influx and elevated pro-inflammatory responses
were noted in biofilm-positive tissue (Siemens
et al. 2016).
Classical mechanistic studies on biofilm for-
mation are confined to coated and uncoated plas-
tic or glass surfaces. These studies showed that
(1) not all clinically relevant GAS serotypes form
biofilms (Lembke et al. 2006), (2) biofilm
N. Siemens et al.
forming abilities within and between the
serotypes may vary (Conley et al. 2003), and
(3) GAS biofilms are up to ten times more resis-
tant to antibiotics as compared to planktonic cells
(Ogawa et al. 2011). In addition, several GAS
virulence factors have been implicated in biofilm
development. Among these, surface localized
factors, including M proteins, pili, and LTA,
were associated with initial attachment of bacteria
to different surfaces. Lack or diminished abun-
dance of such factors resulted in reduced biofilm
formation (Fiedler et al. 2015). Furthermore, cap-
sule and SpeB expression were implicated in bio-
film formation. However, reports on these two
factors are contradicting. While some authors
report a positive correlation, others report a nega-
tive impact (Marks et al. 2014; Roberts et al.
2010). Therefore, a recent study underlines the
importance of studying biofilm in a physiologi-
cally relevant setting (Siemens et al. 2016).
Although not all tested GAS serotypes were able
to form biofilms on abiotic surfaces, all of them
readily formed biofilms in a tissue setting. Fur-
thermore, Nra regulator, which is encoded within
the FCT-region (fibronectin-binding, collagen-
binding, T-pilus) and controls a variety of surface
localized factors including pili was implicated in
this process. A nra-negative mutant lost its ability
to form biofilm. In contrast to previous findings,
no associations between SpeB, capsule, CovR/S,
M protein, and Mga regulator and biofilm forma-
tion were found (Siemens et al. 2016). In support
of the importance to study biofilms in in vivo
settings, Vajjala and colleagues have
demonstrated biofilm formation in a mice NSTI
model (Vajjala et al. 2019). Both streptolysins,
SLS and SLO, which were previously excluded
as biofilm-contributing factors, were implicated
in this process. While GAS wild type strain
formed biofilms across the entire fascia already
12 h post infection, GAS mutant lacking both
streptolysins did not. The authors demonstrated
that streptolysins induce significant up-regulation
of endoplasmic reticulum (ER) stress which in
turn activates the unfolded protein response caus-
ing a release of host factors that support biofilm
formation. Treatment of mice with pharmacolog-
ical inducer of ER stress restored the ability of
9 Pathogenic Mechanisms of Streptococcal Necrotizing Soft Tissue Infections
streptolysin-null mutant to form biofilm (Vajjala
et al. 2019). These two studies underline the
importance of studying NSTIs in a complex
setting since simplified experimental in vitro
approaches did not revealed biofilms as clinical
feature of NSTIs. Such findings have direct
implications for antibiotic efficacy in the tissue
setting and call for reevaluation of treatment
protocols in which biofilm is considered.
Although in vitro assays in monolayer cultures
and the use of mice as an in vivo model of choice
contribute greatly to our understanding of infec-
tious diseases, human tissue biopsies and human
3D-organotypic tissue models are the most valu-
able tools to study host-pathogen interactions. As
described above, human NSTI tissue biopsies
provided valuable insight in host-pathogen
interactions at the tissue site. However, NSTIs
are rare acute and devastating conditions, making
it difficult to obtain patients consent for, e.g.,
sampling infected tissue or blood. Therefore,
3D-organotypic models might serve as a useful
tool to study these infections in a more complex
setting. Tissue engineering approaches originated
in the field of regenerative medicine (Langer and
Vacanti 1993). In contrast to standard monolayer
cell cultures, tissue models much more closely
resemble the architecture, cellular composition,
and matrix complexity of the respective organ
(Fig. 9.3a). In recent years 3D-organotypic
models were successfully employed in a number
of studies of human diseases involving the tissue
setting (Svensson and Chen 2018). These studies
include, but are not limited to, GAS, group G
streptococcus or staphylococcal skin/soft tissue
infections in human skin tissue model setting
(Mairpady Shambat et al. 2016; Siemens et al.
2015, 2016). The use of such organotypic tissue
models suggests a great potential of this approach
in infectious diseases. Considering the fact that
GAS are exclusively human pathogens, it
becomes convincing to use a complex humanized
model system. The engineered tissue recapitulates
key anatomical and functional features, including
a dermal layer consisting of fibroblasts and
stratified epidermis with stratum corneum
(Fig. 9.3a). The tissue produces important struc-
tural frame work proteins as well as the tight
141
junction and adherence junction proteins
(Fig. 9.3a). It was shown that GAS are able to
initiate tissue infection in this model system
(Fig. 9.3b). While at 8 h post infection most of
the bacteria were predominantly found with the
stratum corneum, at later time points, bacteria
disseminated throughout the entire tissue and
formed biofilms (Fig. 9.3c) (Siemens et al.
2016). 3D tissue models are also suitable for
implanting and studying immune cells, including
dendritic cells, monocytes, macrophages, and
even peripheral blood mononuclear cells
(Mairpady Shambat et al. 2015; Nguyen Hoang
et al. 2012). Such studies highlight a significant
progress in the field of infectious diseases and
contribute to the three R principle (replacement,
reduction, refinement) of animal experimentation
(Russell 1995).
9.6 Future Perspectives
The pathogenesis of NSTIs is a multifactorial
process which involves both, bacterial and host-
derived factors. These infections are hyper-
inflammatory conditions and are characterized
by a massive immune cell infiltration and high
bacterial load. GAS have evolved various
immune evasion mechanisms, including intracel-
lular survival and replication within phagocytes,
egress out of the cells, and biofilm formation in
the tissue. All these processes contribute greatly
to the bacterial persistence and as a result, the
antibiotic therapy fails to clear the infection.
Future studies should aim to improve therapeutic
strategies targeting intracellular bacterial reser-
voir and biofilm formation. Furthermore, early
diagnosis of GAS NSTIs might improve the out-
come for the patients. Therefore, the results from
the INFECT study, including anatomical location
and IL-1β, CXCL9, CXCL10, and CXCL11 as
potential promising biomarker candidates, should
be validated in a bigger cohort which encompass
clinical samples of patients with different
infections and other critical illnesses. Early diag-
nosis and subsequent early treatment might
reduce local and systemic hyper-inflammation
and will most likely further improve the outcome.

Fig. 9.3 GAS infections of the 3D-organotypic skin tissue models. (a) Comparative
microscopic analyses of the human skin (left) and 3D-organotypic skin tissue model
(right). Hematoxylin and eosin staining of human skin and tissue model (upper panel)
and images from immuno-fluorescence analyses of key anatomical proteins (lower
142
panels) are shown. (b) CLSM analyses of GAS within different epidermal and dermal
compartments of the 3D-organotypic model after 24 h of infection. (c) Comparative
CLSM analyses of GAS biofilms on glass surfaces (right panel) and within the skin
tissue model (left panel)
N. Siemens et al.
9 Pathogenic Mechanisms of Streptococcal Necrotizing Soft Tissue Infections
Acknowledgments Financial support: The work was
supported by the European Union Seventh Framework
Programme (FP7/2007–2013) under the grant agreement
305340 (INFECT project), the Swedish Governmental
Agency for Innovation Systems (VINNOVA) under the
frame of NordForsk (Project no. 90456, PerAID), the
Swedish Research Council under the frame of ERA
PerMed (Project 2018-151, PerMIT), the German
Research Foundation (DFG; grant no. 407176682), and
the Federal Excellence Initiative of Mecklenburg Western
Pomerania and European Social Fund (ESF) grant
KoInfekt (ESF_14-BM-A55-0001_16).
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 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

# Springer Nature Switzerland AG 2020 151

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_10
152
discuss the versatility of the HLA-II transgenic
mouse model that allowed the biological vali-
dation of known genetic associations to GAS
NSTI. The combined utility of ARI-BXD and
HLA-II mice as complementary approaches
that offer clinically translatable insights into
pathomechanisms driven by complex traits
and host genetic context and novel means to
evaluate the in vivo efficiency of therapies to
improve outcomes of GAS NSTI are also
discussed.
Keywords
Streptococcus pyogenes · NSTI · ARI-BXD ·
IL-1β, HLA-II
Highlights
A systems biology approach using advanced
intercross-derived recombinant inbred BXD
(ARI-BXD) mouse models and an HLA-II trans-
genic mouse model to validate known genetic
associations and superantigen-mediated
responses reveals:
• A significant quantitative trait locus (QTL) on
Chromosome 2 with pathways involving IL1β
as key upstream regulator mediating severity
and survival in NSTI susceptible
ARI-BXD mice.
• Decreased survival was associated with a sig-
nificant loss in the frequency of myeloid-
derived suppressor cells (MDSCs) in NSTI
susceptible ARI-BXD strains, which was
reversed with clindamycin treatment.
• Induction of FoxP3-expressing T regulatory
phenotype by the HLA-II mice expressing
the protective allele in contrast to a Th1 phe-
notype with concomitant IFN-γ and IL-2 by
mice expressing the HLA-II allele associated
with neutral risk during GAS NSTI.
• Transcriptome profile of GAS-infected skin
from HLA-II mice reveals unique signaling
pathways that are amenable to treatment with
existing therapeutic approaches.
S. Nookala et al.
10.1 Introduction
Host genetic variations have been implicated as
significant modulators of clinical presentation and
disease severity in almost all infectious diseases
and have been reported on extensively. The com-
plex polymorphic and nonpolymorphic traits
impact host immune responses and confer suscep-
tibility to a specific infection while at the same
time resistance to another type of infection. The
host genetic background significantly influences
not only the onset of the disease but also treat-
ment outcomes (Skamene 1983; Kotb 2004;
Frodsham and Hill 2004; Sadikot et al. 2005;
Howard 2013; Asner et al. 2014; Patarčić et al.
2015; Çalişkan et al. 2015). Polymorphisms in
HLA class II haplotypes, cytokine genes, and
innate pathogen recognition receptors including
TLR4 are among the key genetic determinants
that affect host immune responses to invading
pathogens. However, the complete spectrum of
genetic determinants that influence susceptibility
or resistance to infections remains unknown. The
identification of these factors can be impactful in
understanding the pathogenesis and inform man-
agement strategies. Furthermore, there is a grow-
ing concern and risk to human health with the
increase in the emerging and reemerging infec-
tious diseases, as well as natural or accidental
biological threats (Fauci 2005). Therefore,
recognizing emerging and reemerging infections
and preparedness to biothreats through thorough
knowledge and understanding of the mechanisms
that govern the pathogenesis and associated host
immune responses are key for substantially
controlling disease outbreaks and the develop-
ment of treatment possibilities.
Despite significant variations in the microbial
factors that are mostly enough to cause morbidity
and mortality, host genetic factors have a major
impact on disease susceptibility and outcomes
(Hill 1999). Mouse models are cornerstones for
studying human diseases and are indispensable
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. . .
models as surrogates of human infections
progresses to preclinical investigations that can
be assessed in the same mouse models. Further,
the successful use of mouse models to study
human infections and test potential interventions
is because of the genetic homology between
mouse and human (Peltonen and McKusick
2001).
10.2 Host Genetics and Group A
Streptococcus Infections
Infections caused by group A streptococcus
(GAS, Streptococcus pyogenes) are an ideal
model to explore the effects of interindividual
variations on susceptibility or resistance. The
pathophysiology of infections caused by GAS is
heterogeneous. Some individuals experience only
mild skin infections or sore throat, while in
others, GAS infections can lead to severe, inva-
sive, life-threatening complications such as strep-
tococcal toxic shock syndrome (STSS) and
necrotizing fasciitis (NF). In about 50% of the
cases, NF, a subgroup of necrotizing soft-tissue
infection (NSTI) can be complicated by STSS.
(Stevens 2000; Carapetis et al. 2005; Ralph and
Carapetis 2012). Despite early surgical debride-
ment, an appropriate combination of penicillin
and clindamycin, and adjunct therapies, an over-
arching pro-inflammatory cytokine storm and
aggressive tissue destruction lead to significant
morbidity and lethal outcomes with mortality as
high as 25% (Reglinski and Sriskandan 2014;
Barnett et al. 2019). The global burden of inva-
sive GAS infections remains alarming with over
600,000 new cases each year and an estimated
160,000 deaths annually (Ermert et al. 2015; Sims
Sanyahumbi et al. 2016). Antibiotics remain
effective against GAS infections; however,
antibiotic-resistant strains have been observed
and present a potential threat to public health
(Gilmer et al. 2013; Andreoni et al. 2017). Due
to the extensive genomic diversity in GAS strains,
there has been limited progress in GAS vaccine
development. However, vaccine discovery efforts
have increased more recently, and several
candidates including peptide antigens have been
153
proposed for undergoing preclinical studies
(Azuar et al. 2019; Vekemans et al. 2019). GAS
produces a spectrum of potent virulence factors
that are either secreted or surface associated that
aid in their establishment, colonization, multipli-
cation and dissemination, and to avoid recogni-
tion by the host immune system, antimicrobials,
and antibiotics (Thulin et al. 2006; Siemens et al.
2016; Andreoni et al. 2017; Keller et al. 2018).
Potential post-streptococcal autoimmune
complications that arise from GAS mimics of
host proteins include rheumatic heart disease,
rheumatic heart fever, arthritis, glomerulonephri-
tis, and Sydenham’s chorea (Dubost et al. 2004;
Kim et al. 2004; Chandnani et al. 2015; Sims
Sanyahumbi et al. 2016).
Superantigens (SAgs) are the most important
among the virulence factors secreted by GAS
bacteria. SAgs, which are potent immune toxins,
simultaneously cross-link T-cell receptor (TCR)-
bearing Vβ regions and conserved sequences of
HLA class II expressed on antigen-presenting
cells (APCs), thereby inducing the activation of
both types of cells (Kotb 1998; Proft and Fraser
2003). The net result is the fulminant activation of
the immune system and the massive release of
pro-inflammatory mediators leading to septic
shock and multiorgan failure with high mortality
rates (Norrby-Teglund et al. 2000; Kotb et al.
2003). The Kotb research group, through their
large epidemiological studies, demonstrated a sig-
nificant association of certain HLA class II
haplotypes with susceptibility or resistance to
invasive GAS infections (Kotb et al. 2002,
2003). The haplotype DRB1*15/DQB1*06
(DR15/DQ6) conferred strong protection from
severe systemic disease (SSD), the haplotype
DRB1*14/DQB1*05 (DR14/DQ5) was
associated with predisposition to SSD, and other
haplotypes, e.g., DRB1*04/DQB1*0302
(DR4/DQ8), were neutral with respect to SSD
(Kotb et al. 2002). Kotb et al. validated their
findings in in vitro human cell cultures and
demonstrated that the inflammatory responses to
the M1T1 SAgs were much attenuated in the
protective allele compared to high-risk or alleles
associated with neutral outcomes of invasive
GAS infections (Kotb et al. 2002).
154
10.2.1 Systems Genetics Approaches
Using BXD Recombinant Inbred
Mouse Models
It is only logical that HLA-II molecules are key
determinants of pathological outcomes of GAS
STSS and combined NF/STSS because GAS
SAgs, pivotal mediators of systemic shock,
simultaneously cross-link HLA-II molecules
expressed on antigen-presenting cells (APCs)
and TCRVβ elements and elicit
pro-inflammatory cytokine storm leading to mul-
tiple organ damage and shock (Norrby-Teglund
et al. 2001; Proft et al. 2003). More recently
Giesbrecht et al. demonstrated that cross-linking
of the SAg streptococcal pyrogenic exotoxin A
(SpeA) to HLA-II alone can cause activation of
the APCs independent of the responding T cells
(Giesbrecht et al. 2019). However, in addition to
SAgs, GAS produces a plethora of other virulence
factors that might modulate the host immune
responses besides involving the highly polymor-
phic HLA class II alleles. It is likely that
polymorphisms in cytokine genes and innate
pathogen recognition receptors including TLR4
affect host innate immune responses and might
contribute to the severity and susceptibility to
GAS infections. However, the complete spectrum
of genetic determinants that influence susceptibil-
ity remains unknown. Identification of the key
host factors will significantly impact the under-
standing of mechanisms underlying pathogenesis
due to invasive infections caused by GAS,
improve therapeutics, and management strategies.
One way to accomplish is to perform a genome-
wide search for susceptibility loci in humans that
requires the involvement of affected family
members; while this may be possible, it is quite
impractical and might be confounded by other
variables.
The influence of genetic background on sus-
ceptibility or resistance to invasive GAS
infections has been reported using inbred mouse
lines, including BALB/c, C3H/HeN, CBA/J, and
C57BL/10 (Medina et al. 2001). While the use of
these inbred lines offered insights, they lack the
power to mirror the host responses as seen in
S. Nookala et al.
humans that are associated with complex poly-
genic traits due to their uniform background and
limited genetic pool. To address this limitation,
recombinant inbred (RI) mice, and particularly,
the BXD family of RI mice, a murine reference
population with high genetic diversity, was cre-
ated using a breeding design originally
constructed by Benjamin A. Taylor by crossing
a female C57BL/6J (B6 or B) and a male DBA/2J
(D2 or D) (BXD) and followed by at least 20 con-
secutive generations of sib-matings (Taylor et al.
1973) (Fig. 10.1). These BXD RI strains have
been used extensively to study variation in path-
ogenicity and uncover genes modulating lethality
(Benjamin et al. 1986; Melvold et al. 1990;
Watters et al. 2001; Chesler et al. 2003). While
there is extensive variation between each strain,
there is virtually no genetic variation between
individuals of one strain (Peirce et al. 2004).
Thus, each BXD strain resembles a large human
population of monozygotic twins. The most com-
pelling feature of the BXD RI panel is that the
genomes of the parental inbred strains (C57BL/6J
and DBA/2J) have been fully sequenced, and they
are a replicable, replenishable, and recapitulatable
resource to map genetic loci of phenotypic traits
as well as to study causal and mechanistic links of
pathogenesis, treatment, prevention, and assess-
ment of the countermeasures of global biothreats
(Waterston et al. 2002). To further improve the
power and precision, the breeding strategy of
BXD lines was modified, and accordingly, the
production of strains BXD43 through BXD102,
started in the late 1990s, was derived from an
advanced intercross (BXD-ARI) progeny that
have been bred following a schema that
incorporated roughly twice as many
recombinations between the parental genomes
B6 and D2 compared to the F2-derived BXDs
(BXD-RI) and the ability to explore one or more
quantitative trait loci (QTL) associated with a
given disease or complex trait (Darvasi 1998;
Williams et al. 2001; Chesler et al. 2003; Peirce
et al. 2004; Parker et al. 2012; Pandey and
Williams 2014).
10 Systems Genetics Approaches in Mouse Models of Group A Streptococcal. . .
Fig. 10.1 BXD strains are generated by crossing two
fully inbred strains (C57BL/6J [female] and DBA/2J
[male]), abbreviated as B6 and D2 or simply B and D,
respectively. The resulting F1 generation (B6D2F1 or F1)
is in turn crossed to produce F2 progeny. Systemic
inbreeding is then performed beginning with the F2 gen-
eration for over 20 generations (recombinant inbred, RI) or
after random mating for 8–14 generations of F2 before
10.2.2 ARI-BXD Mouse Models
of GAS NSTI
In order to mimic the genetic diversity as seen in
humans, the Kotb research group undertook a
systems genetics approach to study the effect of
multiple genes in modulating disease phenotypes
to invasive GAS infections. Prior to establishing
the BXD ARI mouse models of GAS NSTIs, the
distinct role of host genetics and sex differences
in driving susceptibility to GAS NSTIs were
demonstrated in subcutaneous infections of con-
ventional mouse strains C57BL/6J, DBA/2J, AJ,
and CD-1 (Krishnan et al. 2016). Krishnan et al.
reported that DBA/2J strain was more susceptible
than C57BL/6J, and female mice of DBA/2J
155
inbreeding (advanced intercross-derived recombinant
inbred, ARI) to allow random recombination of the
chromosomes and generate progressively inbred BXD
strains whose genomes exhibit unique B to D or D to B
recombinations that are stably fixed. ARI mice increase the
recombinations and diversity of the genetic pool (Peirce
et al. 2004; Abdeltawab et al. 2008)
strain were more resistant than the male DBA/2J
mice, suggesting the propensity of sex in addition
to host genetics in driving susceptibility to GAS
NSTIs (Krishnan et al. 2016). In order to identify
the candidate genes involved in mediating differ-
ential susceptibility to GAS NSTIs, the Kotb
research group capitalized on the BXD-ARI
mice and established a mouse model of GAS
NSTI (subcutaneous infections) with the hyper-
virulent M1T1 clinical isolate, GAS 5448 (origi-
nally isolated from a patient with STSS)
(Chatellier et al. 2000). Using a large panel of
33 BXD strains along with the parental strains
C57BL/6J and DBA/2J, and their F1 strains
(B6D2F1), the GAS NSTI associated QTLs
were identified through genome-wide linkage
156
Fig. 10.2 Swivel graph showing survival against NSTIs
caused by GAS 5448 within the 33 BXD (black bars), B6
(green bar), D2 (red bar), and their F1 (blue bar) strains is
expressed as mean values of corrected relative survival
indices (cRSIs). Data is rank-ordered with positive indices
scans by mapping quantitative phenotypic traits
to the BXD mouse genotype available from the
gene network (Chella Krishnan et al. 2016).
There were significant differences in the differen-
tial susceptibility to GAS NSTI among different
BXDs, their parental, and F1 strains with BXD40,
BXD101, and BXD64 at the susceptible end
while BXD87, BXD85, and BXD73 strains
were extremely resistant (Fig. 10.2). Several
genomic loci proved significantly associated
with survival, body weight change, and maxi-
mum lesion size due to GAS NSTI. The strongest
and highly significant QTL modulating mouse
survival against GAS NSTI mapped to mouse
Chromosome (Chr) 2 between 24.5 and 35 Mb.
The QTLs for body weight change kinetics
S. Nookala et al.
indicating increased survival and negative indices
indicating decreased survival. Error bars indicate SEM.
P-values were calculated by Generalized Linear Model
(GLM) using ordinary least squares (OLS) ANOVA.
Data from Chella Krishnan et al. (2016)
mapped to Chr 7 between 125 and 131 Mb,
while the QTL modulating maximum lesion area
was mapped to Chr 6 (131.6–141.8 Mb) and Chr
18 (49.5–56.3 Mb) (Fig. 10.3).
Aziz et al. established the GAS sepsis model
(intravenous infections) using the M1T1 clinical
isolate, GAS5448, using B6 and D2 parents and a
panel of 20 BXD strains, and reported the influ-
ence of genetics, sex, age, body weight, and inoc-
ulum titer on susceptibility and survival to GAS
sepsis (Aziz et al. 2007). Their study revealed that
bacteremia and dissemination to organs were
major predictors of severity and mortality due to
GAS sepsis, and preliminary mapping results
suggested two significant QTLs on Chr 2 that
needed to be verified and further narrowed down
10 Systems Genetics Approaches in Mouse Models of Group A Streptococcal. . .
Fig. 10.3 Genome-wide interval mapping for survival
(a), body weight change (b), and maximum lesion size
(c) revealed suggestive QTLs on mouse Chr 2, 7, and
using additional BXD strains. By analyzing dis-
ease phenotypes in the context of BXD mice
genotypes in a larger panel of BXD strains
(32 strains including the parental strains, B6 and
D2), Abdeltawab et al. not only confirmed the
findings by Aziz et al. (2007) of a highly signifi-
cant QTL to mouse Chr 2 modulating BXD
157
6 and 18, respectively, suggesting a likely role for genes
under these QTLs in modulating the respective responses
during GAS NSTI (data from Chella Krishnan et al. 2016)
mouse GAS sepsis survival, but fine-mapped
genes between 22 and 34 Mb and verified the
differential expression of transcripts of key
genes in uninfected or GAS-infected blood and
spleens from highly resistant or susceptible BXD
strains, at selected times post-infection
(Abdeltawab et al. 2008). Further, Abdeltawab
158
et al. reported a second less significant QTL that
was also mapped on the same chromosome
between 125 and 150 Mb, and a third QTL on
Chr X. The QTLs for bacteremia and bacterial
dissemination to spleen overlapped with those for
survival, with a slight difference in significance,
with interleukin-1 alpha and prostaglandin E
synthases pathways as key networks involved in
modulating GAS sepsis (Abdeltawab et al. 2008).
GAS sepsis and NSTI were independent stud-
ies with different routes of infection performed on
BXD-ARI mice yet pointed out that the genetic
susceptibility loci for survival in both cases was
mapped to QTL on Chr 2. About 125 genes were
significantly differentially expressed in the GAS
NSTI susceptible strains (BXD 40 and 64) com-
pared to uninfected controls. BXD strains that
were resistant to GAS NSTIs were identified to
be BXD 73 and BXD87. Parsing of the 125 dif-
ferentially expressed genes identified two high-
scoring networks that likely mediate susceptibil-
ity to GAS NSTIs in susceptible hosts. Particu-
larly, IL-1β was identified as the top upstream
regulator likely mediating pro-inflammatory
effects driving severity seen in susceptible hosts.
The differential expression level of IL-1β
transcripts was verified in GAS-infected skin tis-
sue from susceptible mice and validated in tissue
biopsies and plasma from GAS-infected patients
(GAS 2006, INFECT Consortium) and in
GAS-infected 3D reconstructed skin models
(Chella Krishnan et al. 2016).
10.2.3 Assessing Heterogeneity
of Treatment Effects in BXD
Models of GAS NSTI
Invasive GAS infections have complex etiologies
that vary depending on the host genetic back-
ground, environment, gender, and other as yet
unidentified complex traits. The heterogeneity in
the outcomes and the cause, therefore, have to be
investigated to determine the best course of treat-
ment and prevention. The BXD mice offer a
promising model system to provide different
information and insights into pathomechanisms
underlying GAS infections. Importantly, data
S. Nookala et al.
derived from studies on these mouse models are
comparable to real-world data from diverse
human populations. These models help tease out
the mechanistic insights into disease and to assess
the efficacy of potential therapeutics. For exam-
ple, preliminary findings by Nookala et al. (man-
uscript in preparation) show that the survival and
the frequency of CD45+, CD11b+ monocytes, and
LY6G+LY6C+ myeloid-derived suppressor cells
(MDSCs) are significantly lower in BXD40
(NSTI susceptible) compared to BXD87 (NSTI
resistant) during subcutaneous GAS infections.
However, this condition was reversed with
clindamycin treatment (unpublished findings,
Nookala et al., Fig. 10.4a, b). The accumulation
of MDSCs is crucial for the host control of
infections. MDSCs are a heterogeneous collec-
tion of monocytes and polymorphonuclear innate
immune cells and function as initial effector cells
to restrict infection burden and suppress the
responses of activated effector T and NK-cells
(Ost et al. 2016; Dorhoi and Plessis 2018). The
understanding of the induction and frequency of
MDSCs in GAS infections is just beginning to
evolve. It is an attractive possibility that the loss
or lack of induction of MDSCs could be one of
the mechanisms against survival in GAS NSTI
susceptible mice, which needs to be further
investigated.
10.3 HLA-II Mouse Models
of Invasive GAS Infections
Invasive infections caused by GAS is one of the
most complex human diseases affecting multiple
organs driven by complex interactions that neces-
sarily include HLA-II haplotype, immunological
background, GAS burden, and the variations in
the secreted and surface-bound virulence factors
of the GAS pathogen. SAgs are among the most
potent toxins secreted by GAS. While the BXD
mice models described above are invaluable tools
to study the effect of complex traits, conventional
inbred mice are naturally resistant to
SAg-mediated outcomes of GAS infections
(Miethke et al. 1992; Blank et al. 1997;
Sriskandan et al. 2001). These setbacks were
10 Systems Genetics Approaches in Mouse Models of Group A Streptococcal. . .
Fig. 10.4 Immunophenotype of myeloid-derived sup-
pressor cells assessed by flow cytometry in blood from
10 days post GAS-infected BXD mice either susceptible or
resistant to GAS NSTI. Data is represented as a
t-distributed stochastic neighbor embedding (t-SNE) plot
to aid the visualization of distribution and clustering of
indicated subsets in untreated or clindamycin-treated
overcome through the use of transgenic mice
expressing human plasminogen (Sun et al. 2004;
Cole et al. 2006) and HLA-II alleles associated
with differential susceptibility to invasive GAS
infections (Nooh et al. 2007). Several studies
have reported the use of HLA-II transgenic mice
models to mimic responses to streptococcal and
staphylococcal SAgs as seen in humans
(Unnikrishnan et al. 2002; Welcher et al. 2002;
DaSilva et al. 2002; Roy et al. 2005; Rajagopalan
et al. 2006; Mangalam et al. 2008).
The first evidence of the dominant and
overriding effect of HLA-II molecules to GAS
infection in vivo and SAg stimulations in vitro
was reported by Nooh et al. (2007). Nooh et al.
(2007) demonstrated that HLA-II mice
expressing the neutral risk haplotype, DRB1*04/
DQB1*0302 (DR4/DQ8), mounted significantly
159
groups. t-SNE plot was run on ~5000 live cells
discriminated by viability dye on singlets. Manually
gated populations were overlaid onto t-SNE plots (a).
Kaplan–Meier survival curves for the susceptible or resis-
tant BXD mice 10 days post GAS infection that were
either untreated or treated with clindamycin (b)
higher levels of the pro-inflammatory cytokines,
TNF-α and IFN-γ, in vivo and in vitro compared
to mice expressing DQ6, the DQ allele that is in
linkage disequilibrium with the DR15 allele
(DRB1*15/DQB1*06 (DR15/DQ6)). While stud-
ies by Nooh et al. (2007) used models of invasive
GAS sepsis, Nookala et al. recently reported the
use of HLA-II transgenic mice expressing either
DRB1*1501 (DR15) or DRB1*04/DQB1*0302
(DR4/DQ8) as mouse models of GAS NSTI
(Nookala et al. 2018). They demonstrated that
HLA-II transgenic mice expressing the protective
DR15 allele significantly induced surface GARP/
LAP expressing FoxP3+ T regulatory cells com-
pared to mice expressing HLA-II DR4/DQ8, a
haplotype associated with neutral risk for com-
bined NF/STSS, which skewed the responses
toward a Th1 type during in vivo infections with
160
Fig. 10.5 Proposed pathomechanisms underlying severe
or nonsevere outcomes of GAS revealed from HLA-II
transgenic mouse models of GAS NF/STSS. GAS
superantigens simultaneously crosslink HLA-II and dis-
tinct variable beta regions in the TCR (TCR-Vβ), thereby
eliciting robust levels of pro-inflammatory cytokines. Cell
activation driven by the pro-inflammatory cytokine–recep-
tor interactions induces phosphorylation of STAT
molecules mediating the translocation of phosphorylated
a GAS 2006 (clinical isolate from a patient with
combined NF/STSS, INFECT Consortium) and
in vitro stimulations to SmeZ with the concomi-
tant expression of IFN-γ and IL-2 (Nookala et al.
2018). The summary of various cytokines and
transcription factors that are significantly
modulated during SAg stimulations in vitro in
splenocytes from HLA-II mice expressing risk
(severe) or protective (nonsevere) alleles and
S. Nookala et al.
STAT molecules into the nucleus resulting in the activa-
tion of transcription factors. The net effect is the polariza-
tion of naïve T cells into distinct subsets (Th1/Th17 in case
of severe and Tregs in case of nonsevere). Data presented
is a summary of analysis from transcriptomics data from
in vitro and in vivo studies in HLA-II mouse models of
GAS NF/STSS. Red arrows indicate increased expression
of transcripts
in vivo during GAS NSTI is shown in Fig. 10.5.
Transcriptome analysis of GAS2006-infected
skin from HLA-II DR4/DQ8 (a haplotype
associated with neutral risk for combined
NF/STSS) mice offered a plethora of information,
specifically, clues for the existence of a combined
adipogenic, metabolic, and inflammatory axis in
GAS NSTI (Nookala et al. 2018). The top five
most significantly downregulated pathways
10 Systems Genetics Approaches in Mouse Models of Group A Streptococcal. . .
Fig. 10.6 Heat map showing top 10 hits for canonical
pathways with significant activated (red) and inhibited
(green) z-score (A) associated with the differentially
expressed genes in GAS-infected skin lesions from
HLA-II mice expressing DR4/DQ8, a haplotype
associated with neutral risk for combined GAS NF/STSS
compared to PBS. A negative z-score indicates a predicted
during GAS NSTI were oxidative phosphoryla-
tion, PPAR, TCA cycle II, PPARα/RXRα activa-
tion, and fatty acid β-oxidation 1, while the top
five most significantly upregulated canonical sig-
naling pathways were neuroinflammation,
TREM1, IL-6, acute phase response,
p38MAPK, and leukocyte extravasation
(Nookala et al. manuscript in preparation). The
top ten canonical pathways that are significantly
enriched in HLA-II mice expressing the
DR4/DQ8 haplotype are represented as a heat
map in Fig. 10.6.
Collectively, we provided strong evidence
through a combination of systems genetics using
a large panel of BXD mice and humanized mouse
models of invasive GAS infections that host
genetic variations contribute significantly through
distinct molecular events involved in mediating
susceptibility and clinical outcomes of invasive
GAS infections. The systems genetics approach
161
inactivation, while a positive z-score indicates a predicted
activation of the enriched pathway. These pathways
predicted by the Ingenuity Pathway Analysis based on
the gene expression changes seen in our dataset may be
responsible for the pathophysiology underlying GAS
NF/STSS
using the ARI-BXD model provided the first evi-
dence of the architecture of complex traits that
were crucial in the understanding and the identifi-
cation of genes, networks, and pathways underly-
ing invasive GAS infections. The reductionist
(HLA-II) approach in murine models of invasive
GAS infections has contributed to unraveling the
important role of HLA class II allelic
polymorphisms leading to selective polarization
into distinct Th1/Th17 or T-regulatory subsets
during GAS NSTI, and the resulting
pro-inflammatory or anti-inflammatory patho-
genic processes they drive through their complex
cross talk with the immune system. Although
polymorphisms in HLA-II alleles are critical
determinants of severity of GAS infections,
systems genetics is becoming an important
approach to understanding both the underlying
biology and the distinct genetic loci, possible
genes that modulate the particular traits, and the
162
mechanisms driven by complex traits in the
genome.
10.4 Future Perspectives
There is an urgent need to reevaluate host
responses to identify host-directed strategies for
interference with various signaling pathways
combined with standard antibiotic care to attenu-
ate severity and improve outcomes of GAS NSTI.
The identification of the interactions driven by the
HLA-II haplotype as well as the complex traits of
the genome is critical because therapeutic effec-
tiveness is influenced by pathogen and host-
related genetic and nongenetic factors. Over the
last few years, the concept of “theranostics,” a
convergence of therapy and diagnostics catered
to patients based on their individual genetic
characteristics (Degrauwe et al. 2019) is becom-
ing increasingly popular. Given the disease het-
erogeneity, it is imperative to test whether or not a
patient may benefit from therapy or vaccination.
Due to the key role of HLA-II allelic
polymorphisms in self and nonself-recognition,
it is only logical that disease outcomes and ther-
apy benefits show heterogeneity due to interindi-
vidual variation. Both BXD and HLA-II
transgenic mouse models are invaluable tools to
help uncover biomarkers, therapeutic targets, and
vaccine candidates and test their efficacy for safe
and personalized therapeutic and vaccination
strategies. Most importantly, data from these
studies will translate directly to humans, will
enable the genetic evaluation of outcomes of
infections, and also appears as a promising forth-
coming revolutionary approach to patient-tailored
bedside theranostics. Particularly important, yet
poorly understood, is the host genetics-dependent
onset and progression of metabolic dysfunctions
and post-streptococcal sequelae, including rheu-
matic heart disease, rheumatic fever, arthritis,
glomerulonephritis, and psychiatric conditions.
Through carefully designed studies, the
established murine models will facilitate the dis-
section of consequences due to long-term and
recurrent GAS infections and complex host–path-
ogen interactions. Treatment or vaccination
S. Nookala et al.
strategies can then be personalized depending on
the host genetic context that confer susceptibility
or protection from particular disease in as much
as customized interventions can produce more
effective results than generalized therapeutic
approaches.
Acknowledgements The work presented was supported
by the grants from the European Union (FP7/2012–2017)
under the grant agreement 305340 (MK), grants from the
Swedish Research Council under grant number 20150338
(MK), UND CoBRE Host–Pathogen Interactions Pilot
Award (SN), and UND Genomics Core funded through
grant support from the National Institutes of Health grants
P20GM104360 (SN) and by the National Institute of Gen-
eral Medical Sciences of the National Institutes of Health
under grant numbers P20GM103442, U54GM128729,
and P20GM113123. We thank John Lee, Graphic Design
Specialist, at Information Resources of the University of
North Dakota School of Medicine & Health Sciences, for
his assistance with the figure illustrations.
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research0046
 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

# Springer Nature Switzerland AG 2020 167

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_11
168
clinically relevant experimental models by use
of systems biology approaches. This chapter
describes the current state of biomarkers in
NSTI and how biomarkers are categorized.
We introduce the fundamentals of top-down
systems biology approaches including analysis
tools and we review the use of current methods
and systems biology approaches to biomarker
discover. Further, we discuss how different
“omics” signatures (gene expression, protein,
and metabolites) from NSTI patient samples
can be used to identify key host and pathogen
factors involved in the onset and development
of infection, as well as exploring associations
to disease outcomes.
Keywords
Epigenomics · Genomics · Network inference
and analysis · Mathematical modelling ·
Metabolomics · Microbiome · Multivariate
statistics · Proteomics · Top-down approaches
Highlights
• Comprehensive systems biology analyses and
interpretation of omics data are emerging as
valuable tools for biomarker discovery
in NSTI.
• The inflammatory proteins IL1β, IL-6,
CXCL9, CXCL10, and CXCL11, were
identified as potential biomarkers to discrimi-
nate S. pyogenes NSTI from NSTI caused by
other pathogens.
• Metabolite–metabolite association networks
analysis has proven able to define sets of
metabolites associated with biofilm formation
in NSTI.
• Metabolites in combination with multivariate
and machine learning modelling have the poten-
tial to be utilized as predictive markers in NSTI.
11.1 Introduction
In search for biomarkers in NSTI, analyses have
usually focused specifically on measuring certain
selected factors, such as cytokines. This approach
is, to some extent, distorted because it is based on
already existing knowledge that somehow
E. Saccenti and M. Svensson
indicates that these factors must be associated
with the disease. In this context, a systems
biological approach, used correctly, has the
greater opportunity to identify hitherto unknown
factors that may be associated with disease and
that may be used as biomarkers.
Systems biology is a holistic approach to
deciphering the complexity of biological systems
that starts from the understanding that the
networks that form the whole of living organisms
are more than the sum of their parts. The systems
biology approach to understand disease-related
biology has the potential to revolutionize our
understanding of the cellular pathways and gene
networks underlying the onset of disease, and the
mechanisms of treatment strategies that amelio-
rate disease phenotypes. In the post-genomic era,
a wealth of novel approaches for generating and
analyzing large, high-dimensional genomic,
transcriptomic, proteomic, and metabolomic data
sets from cohorts of normal and diseased
individuals can be used in combination with
systems biology approaches (Rosato et al. 2018).
In research related to infectious diseases, such as
NSTI, the data sets include information from both
pathogen- and host-oriented analyses that can be
used at a systems biology level. The emerging
field of systems biology attempts to harness com-
plex, multi-parameter systems by computationally
integrating gene-level data with molecular
pathways and networks to extract new biological
insight. Systems biology may combine and aug-
ment current strategies to biomarker discovery,
generating novel, experimentally testable
candidates.
A biomarker is a measurable indicator of some
biological state or condition, and in recent years
systems biology approaches have been applied to
screen and identify diagnostic, prognostic, predic-
tive, and predisposing biomarkers as well as
targets for prevention and treatment of disease
(Feala et al. 2013; Vafaee et al. 2018; Anvar
et al. 2018). There are several successful
examples of molecular biomarkers that are cur-
rently the clinical standard for diagnostic screen-
ing in several diseases, for example, in
myocardial infarction (Hajar 2016) and certain
cancers (Mehta et al. 2010; Goossens et al.
2015). The search for novel molecular biomarkers
11 Systems Biology and Biomarkers in Necrotizing Soft Tissue Infections
continues to be a major research drive in many
biomedical fields. A PubMed (https://pubmed.
ncbi.nlm.nih.gov/) query for NSTI and biomarker
(Biomarker AND (NSTI OR “necrotising soft
tissue infection” OR “necrotising fasciitis”) in
March 2020 returned only 39 hits (44 using the
spelling “necrotizing”) demonstrating how little
is known and how limited the research activity on
this topic is, despite an incidence ranging from
0.3 to 15.5 case per 100.000 population (Stevens
and Bryant 2017) and all-cause 90-day mortality
of 18% (Madsen et al. 2019). In NSTI biomarkers
for early detection and guidance of treatment may
improve outcome and help to reduce the length of
hospital stay and mortality. There are good
opportunities that systems biology approaches in
NSTI can identify biomarkers that could be used
as novel tools for rapid and early identification
and stratification of patients with NSTI upon
admission. In this book chapter, we review bio-
marker types and detection methods, as well as a
number of systems biology approaches poten-
tially useful to identify candidate biomarkers
in NSTI.
11.2 State of the Art: Existing
Biomarkers for NSTI
There are currently no approved biomarkers for
the diagnosis or prognosis in NSTI, and the
molecular mechanisms of host responses during
NSTI remain poorly understood. This lack of
understanding reflects the complex, multifactorial
nature of NSTI, which is believed to involve a
network of interweaving host and pathogen
molecular pathways that dictate disease onset
and progression.
Available information on NSTI biomarkers are
restricted to a few research studies that focused on
a limited number of selected factors or informa-
tion of factors included as components of rela-
tively inaccurate scoring systems, such as the
LRINEC (Laboratory Risk Indicator for Necrotiz-
ing Fasciitis). LRINEC is generated from six rou-
tinely performed laboratory tests including the
analyses of patients´ C-reactive protein, white
blood cell count (
10,000/μL), hemoglobin
(g/dL), sodium (mEq/L), creatinine, and glucose.
169
Although LRINEC scoring is not optimal, it has
been used to distinguish NSTI from less severe
soft tissue infections (Wong et al. 2004).
Recently, LRINEC score has also been
analyzed in relation to pro-inflammatory
cytokines, and this revealed no significant associ-
ation between the LRINEC score and analyzed
specific cytokine levels on admission (Hansen
et al. 2017a). However, the analyses of cytokines
revealed IL-6 to be associated with disease sever-
ity, and that IL-1β was strongly associated with
30-day mortality (Hansen et al. 2017b). Another
factor suggested to have biomarker potential in
NSTI is Pentraxin-3 (PTX3) a glycoprotein which
belongs to the pentraxin protein superfamily,
including acute phase proteins, C-reactive protein
(CRP), and serum amyloid protein (SAP). PTX3
is produced by vascular cells or inflammatory
cells and is released into the circulation, possibly
reflecting systemic inflammation. The PTX3 level
in patients with NSTI at time of admission was
associated with septic shock, amputation, and risk
of death in patients, but was not an independent
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
prognostic markers. In addition, microbiological
cultures of Clostridium or Vibrio are negatively
associated, while a microbiological culture of
GAS is positively associated with survival
in NSTI. Nevertheless, there is a great need for
robust early measurable biomarkers to improve
identification and management of NSTI.
11.3 Biomarker Type and Detection
Molecular biomarkers generally consist of
molecules measured in body fluids or in samples
from the affected tissue. Challenges facing bio-
marker research include the lack of standardized
methods for fast reliable detection. Biomarkers
can be divided into four categories: diagnostic,
prognostic, predictive, and predisposing/suscepti-
bility (Quezada et al. 2017; Simon 2011, 2014;
Drucker and Krapfenbauer 2013). The typical
pattern of biomarker discovery is shown in
Fig. 11.1.
170
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
Diagnostic biomarkers are used, e.g. to determine
the health status of a patient to ensure appropriate
management. In infectious diseases such as NSTI
and sepsis, immediate treatment is required,
necessitating quick, early, and accurate diagnosis
which can help decision making. A singular ideal
biomarker may not be identified, but rather an
alternative approach focusing to determine the
diagnostic relevancy of multiple biomarkers
when used in concert may be useful. The ongoing
efforts in the development of a multiplex point-
of-care testing kit, enabling quick and reliable
detection of serum biomarkers, may have great
potential for early diagnosis of acute bacterial
infections. Thänert et al. (2019) provided insight
into the pathophysiology of mono- and
polymicrobial NSTIs and protein multiplex anal-
ysis highlighted three potential biomarkers,
CXCL9, CXCL10, CXCL11, as these three
E. Saccenti and M. Svensson
chemokines displayed statistically significant
concentration differences between S. pyogenes
and polymicrobial NSTIs. In addition, this study
performed RNA-seq analyses on tissue biopsies
and this showed that genes with significantly
greater expression in polymicrobial compared to
monomicrobial NSTIs were those encoding extra
cellular matrix (ECM) components like collagen,
fibronectin, lumican, and connective tissue
growth factor. In addition, gene sequencing data
showed, in line with protein data, that genes
encoding interferon-inducible mediators such as
CXCL9, CXCL10, CXCL11 were expressed at
higher levels in monomicrobial NSTIs, in partic-
ular those caused by S. pyogenes.
11.3.2 Prognostic Biomarkers
A prognostic marker identifies outcome in
patients. Usually, these markers provide insight
11 Systems Biology and Biomarkers in Necrotizing Soft Tissue Infections
into unfavorable outcome, such as mortality. The
presence or absence of a prognostic marker can be
useful for the selection of patients for a specific
treatment but does not directly predict the
response to a treatment.
In NSTI plasma biomarkers representing the
early inflammatory response are thought to be
useful as prognostic markers of disease severity
and mortality. To determine the association of
admission biochemical markers, such as lactate,
to limb loss and mortality both univariate and
multivariable analyses have been used. For exam-
ple, arterial lactate was found to be associated
with both mortality (odds ratio [OR], 1.5; 95%
confidence interval [CI], 1.1 to 2.0; P-
value ¼ 0.009) and limb loss (OR, 1.3; 95% CI,
1.0 to 1.7; P-value ¼ 0.02), and lactate was
suggested to be used early on to guide aggressive
therapeutic interventions (Schwartz et al. 2013).
In addition, attempts have been made to associate
markers with ICU (Intensive Care Unit) scoring
systems including simplified acute physiology
score (SAPS) II (Le Gall et al. 1993) and sequen-
tial organ failure assessment (SOFA) scores
(Vincent et al. 1996), as well as the LRINEC
score (Wong et al. 2004), presence of septic
shock, microbial etiology, renal replacement ther-
apy, and amputation.
In a study by Hansen et al. it was demonstrated
that plasma levels of mannose-binding lectin and
ficolins, which can activate the complement path-
way, were significantly lower in patients with
NSTI than in controls (Hansen et al. 2016b).
Furthermore, this study revealed that a high base-
line ficolin-2 level indicated a 94% chance of
surviving the first 28 days after admission.
Another study by Hansen et al. reported on the
association between cytokine (IL-1β, IL-6, IL-10,
and TNF-α) levels and the LRINEC score, dis-
ease severity and mortality in NSTI patients
(Hansen et al. 2017b). Although no significant
association between the LRINEC score and cyto-
kine levels on admission was found, the authors
found that IL-6 was consistently associated with
disease severity and that high IL-1β (OR 3.86
[95% CI, 1.43–10.40], P-value ¼ 0.008) and
IL-10 (4.80 [1.67–13.78], P-value ¼ 0.004)
were associated with 30-day mortality. Also,
171
PTX3 may be an appropriate marker of severity
and prognosis of NSTI (Hansen et al. 2016a).
Although PTX3 was reported to perform even
better than the “classical” inflammatory markers
CRP and procalcitonin (PCT), its usefulness has
been a matter of debate (Honore and Spapen
2016).
11.3.3 Predictive Biomarkers
Predictive biomarkers aim to objectively evaluate
the outcome of a specific clinical intervention, or
the differential outcomes of two or more
interventions, including toxicity. Predictive and
prognostic markers often get confused because
many markers can be both predictive and prog-
nostic. A marker can be an unfavorable prognos-
tic factor but could predict favorably for response
to therapy, or vice versa. Furthermore, it is possi-
ble that markers can be prognostically unfavor-
able, but predict favorably for one therapy and
predict unfavorably for another therapy.
When searching PubMed with the joint medi-
cal subject headings “NSTI” and “predictive
markers,” most, if not all, publications indexed
are studies that have identified prognostic rather
than predictive markers. In a recent study Afzal
and coworkers (Afzal et al. 2020) performed
metabolomics analysis combined with advanced
bioinformatics analysis of metabolite–metabolite
association networks, indicating potential of
metabolites as predictive markers in NSTI
(described in more detail in Sect. 11.4.5).
In conclusion, there is very little information
on biomarkers that can be used to predict patient
who benefits from different clinical interventions.
Thus, this could potentially be an accelerating
research field which would have a significant
clinical impact.
11.3.4 Predisposing Biomarkers
The fourth category of biomarkers is used to
identify individuals with a predisposition or sus-
ceptibility to develop disease. In NSTI, studies
have revealed that allelic variations in human
172
leukocyte antigen (HLA) class II haplotypes
result in striking differences in severity and
outcomes of invasive GAS infections via the dif-
ferential presentations of GAS superantigens
(SAgs) by class II HLAs to host T-cell receptor
(TCR) Vβ elements (Kotb et al. 2002, 2003;
Norrby-Teglund et al. 2000; Norrby-Teglund
and Kotb 2000). In particular, specific HLA
class II haplotypes have been found to confer
strong protection from severe systemic disease,
whereas others increased the risk of severe dis-
ease; patients with the DRB1*1501/DQB1*0602
haplotype mounted significantly reduced
responses and were less likely to develop severe
systemic disease.
It is likely that in NSTI a complex interaction
of multiple host and microbial factors that do not
lend themselves to reduction into a simple for-
mula exist, and therefore more holistic
approaches need to be implemented to identify
biomarkers for diagnosis, prognosis, prediction,
or predisposition/susceptibility.
11.4 Biomarker Use
11.4.1 Genomic Biomarkers in NSTI
To identify genomic biomarkers DNA sequenc-
ing (host and pathogen) technologies, such as
whole-genome sequencing can be used. While
cancer research has reached the farthest using
whole-genome sequencing (Staaf et al. 2019), it
is difficult to find examples where DNA sequenc-
ing has been used to identify biomarkers in NSTI.
It is likely that NSTI, with its acute course, poses
other challenges than cancer and in NSTI perhaps
gene sequencing may be best used to identify
predisposing conditions. However, the relatively
high costs likely prevent whole-genome sequenc-
ing analyses from becoming routine on a large
scale. Instead, “exome” sequencing (targeted
sequencing of protein-coding regions) may
become a cost-effective way to provide access to
the entire transcribed genome of individuals. In
addition, genome-wide association (GWA) stud-
ies identifying risk loci are feeding into the clini-
cal use of DNA variants. This research has been
E. Saccenti and M. Svensson
made possible by technological developments
that have expanded the throughput and reduced
the cost of single nucleotide polymorphisms
(SNP)-based research and allowed SNP-based
techniques to identify DNA copy number varia-
tion. Nevertheless, to identify SNPs remains a
significant challenge, due to the difficulty of
separating these. One of the limitations of GWA
studies is that it captures only common SNPs,
which by themselves may only contribute a
small extent of risk to develop disease. NSTI
patients infected with the same bacterial strain
can develop very different manifestations and a
role of immunogenetics of the host in shaping the
outcome of invasive streptococcal infections can
be identified (Kotb et al. 2002). However, to
identify rare SNPs that contribute to develop
NSTI for a small percentage of patients, more
cost-effective technologies will be required.
Despite its disadvantages DNA sequencing may
become more important for patient care over the
next 5 years as the costs are reduced and
capabilities extended.
11.4.2 Transcriptomic Biomarkers
in NSTI
Mapping genotypes to phenotypes is one of the
long-standing challenges in biology and medi-
cine, and a powerful strategy for tackling this
problem is performing transcriptome analysis.
Transcriptomics entails the measurement and
analysis of mRNA using microarray or RNA
sequencing technologies. At the beginning
microarrays offered tremendous opportunities by
capturing parallel information about many more
mRNAs than quantitative PCR techniques
(RT-PCR), which mostly focus on single mRNA
analyses. However, like all molecular techniques
there are limitations, such as dependence on tem-
plate quality (Bustin and Nolan 2004).
Although microarrays have been highly pro-
ductive research tools and used in several current
prognostic and predictive tests, other emerging
technologies have replaced the microarrays.
Next-generation DNA sequencing technology
known as “RNA-seq” allows simultaneous
11 Systems Biology and Biomarkers in Necrotizing Soft Tissue Infections
analysis of all RNA molecules, including alterna-
tive splice variants, mRNAs, non-coding RNAs
(ncRNAs), and miRNAs. RNA-seq has
revolutionized the analysis of RNAs. In a recent
study Thänert et al. took advantage of the next-
generation sequencing tools to enable microbial
profiling using 16S rRNA sequencing with tran-
scriptional analysis of host and microbe using
dual RNA sequencing (RNA-seq) in tissue
biopsies from NSTI patients (Thänert et al.
2019). The results from this study demonstrated
that, despite the similar clinical presentation of
NSTIs, the pathophysiology of mono- and
polymicrobial etiology differs significantly and
that these differences can potentially be exploited
for diagnostic purposes.
The transcriptional analysis of infected tissue
indicated that the gene expression profile differed
significantly between monomicrobial streptococ-
cal infections and polymicrobial NSTI. Among
the genes with significantly greater expression in
polymicrobial compared to monomicrobial
NSTIs were those encoding ECM components
like collagen, fibronectin or lumican, as well as
connective tissue growth factor, proteins. On the
other hand, a set of genes encoding interferon-
inducible mediators such as CXCL9, CXCL10,
CXCL11, MX1, and MX2 as well as the
guanylate-binding GTP1 and GTP2 were most
prominently higher expressed in monomicrobial
NSTIs, in particular those caused by S. pyogenes.
Because of the many diverse cell types in our
body each of which express a unique
transcriptome, conventional bulk population
sequencing can provide only the average expres-
sion signal for an ensemble of cells. More
recently there has been an explosion of interest
in obtaining high-resolution views of single-cell
transcriptomics (scRNA-seq) heterogeneity.
Assessing the differences in gene expression
between individual cells has the potential to iden-
tify rare populations that cannot be detected from
an analysis of pooled cells. Due to issues with
cost and analysis challenges, the implementation
of scRNA-seq as a tool for biomarker identifica-
tion in NSTI may still be some years away and is
unlikely to give the complete picture.
173
11.4.3 Epigenomic Biomarkers in NSTI
Epigenetics can be defined as the field of inherit-
able changes in gene expression that are not
caused by alterations in DNA sequences. Epige-
netic mechanisms include DNA methylation, his-
tone modifications, and non-coding RNAs, and
epigenetic alterations have an increasingly clear
role in modulating inflammatory and other immu-
nological processes. Identification of epigenetic
alterations may provide important prognostic,
predictive markers. This is an exciting field that
is likely to grow in clinical importance.
There are no studies on epigenetics in NSTI,
while the often-associated condition sepsis has
been subjected to epigenetic analyses (Cross
et al. 2019). To date, mostly the epigenetic modi-
fication associated with various stages of sepsis
has been assessed. A focus has been to identify
mechanisms involved in endothelial dysfunction
during the hyperinflammatory response and those
underpinning aspects of immunosuppression.
11.4.4 Proteomic Biomarkers in NSTI
Large scale analysis of specific proteomes, also
known as proteomics, defines protein diversity
and understands its biological consequences.
Analysis of proteins as inflammatory markers in
diseases could provide important information
about disease severity and guide decision making.
In true proteomics analysis all proteins from a
sample of interest are usually extracted and
digested with one or several proteases (typically
trypsin alone or in combination with Lys-C
(Wiśniewski and Mann 2012) to generate a
defined set of peptides. The peptides obtained
are subsequently analyzed by liquid chromatog-
raphy coupled to mass spectrometry (LC-MS).
The two most common approaches are either
designed to achieve a deep coverage of the prote-
ome (shotgun MS) or to collect as much quantita-
tive information as possible for a defined set of
proteins/peptides (targeted MS) (Picotti and
Aebersold 2012). Protein content information
can also be obtained through targeted approaches,
174
including multiplex analyses made to identify a
set of predetermined proteins. Since the test
results are needed within hours in NSTI,
approaches will most realistically include
analyses of samples, such as plasma, that can be
obtained quickly. However, rapid assessment
based on proteomics and/or multiplex analyses
of NSTI patients to identify protein-based
biomarkers is challenging, as these are time con-
suming analyses. Nevertheless, multiplex
approaches have been used to analyze NSTI
plasma samples and has provided important
insight to potential diagnostic biomarkers
(Thänert et al. 2019; Hansen et al. 2017a).
11.4.5 Metabolomic Biomarkers
in NSTI
Metabolomics takes a special position among the
omics disciplines in the system top-down
approach as the metabolome of biological pro-
cesses, carrying imprints of genetic, epigenetic,
and environmental factors, provides the link
between the genotype and phenotype (Karakitsou
et al. 2019; Rosato et al. 2018; Dunn and Ellis
2005; Kell 2004). Since metabolites represent the
endpoint of many molecular pathways,
metabolomics which quantifies the metabolite
content of cells or tissues is in addition to have
diagnostic and prognostic value also potentially
useful for predicting treatment response.
Metabolomics has been identified as a novel tool
to discover targets for sepsis diagnosis and prog-
nosis, as well as to gain insight into pathogenic
disease mechanisms (Eckerle et al. 2017).
Recently, we used untargeted metabolomics
analyses of plasma from NSTI patients and
healthy controls to identify the metabolic
signatures and connectivity patterns among
metabolites associated with NSTI. Out of
97 metabolites detected, the abundance of
33 was significantly altered in NSTI patients.
Analysis of metabolitemetabolite association
networks identified 20 metabolites differently
connected between NSTI and controls. Testing
of a set of differently connected metabolites (orni-
thine, ribose, urea, and glucuronic acid) in in vitro
E. Saccenti and M. Svensson
biofilm formation assays revealed
metabolite-specific effects on both bacterial
growth and biofilm formation. A biofilm can be
considered as a potential complicating
microbiological feature of NSTI and, conse-
quently, emphasizes reconsideration of antibiotic
treatment protocols. This study identifies for the
first time an NSTI-specific metabolic signature
with implications for optimized diagnostics and
therapies. Thus, the finding of metabolites in
NSTI associated with biofilm formation
emphasizes the potential of metabolites as predic-
tive markers in NSTI.
11.4.6 Microbiomic Biomarkers in NSTI
Analyzing the microbiome at the system biology
level for comparison in diseases to that of healthy
subjects has become increasingly popular. Rapid
progress in the development of next-generation
sequencing (NGS) technologies in recent years
has provided many valuable insights into com-
plex biological systems, including diverse micro-
bial communities.
As far as NSTI is concerned, this field of
research is in its infancy, but it may prove helpful
in providing biomarkers which will give insights
into the pathological process behind disease evo-
lution and progression by determining specific
etiological factors.
11.4.7 From One-Category-Analyses
Towards Systems Biological
Approaches
The classical approach to biomarker discovery
usually entails the comparison of molecules or
group of molecules among two or more groups,
usually in a case/control scenario. In NSTI typical
comparison is monomicrobial vs polymicrobial
infection or patient outcomes (septic shock/no-
septic shock (Hansen et al. 2018), amputation/
no-amputation, or mortality (Hansen et al.
2018)) or some clinical score (low/high LRINEC)
(Hansen et al. 2017a). Statistical approaches are
usually simple: candidate biomarkers are mostly
11 Systems Biology and Biomarkers in Necrotizing Soft Tissue Infections
tested one at a time in a univariate fashion using
procedure like ANOVA or linear mixed models,
logistic regression, or Kaplan–Meier curves when
association with survival is of interest or by
correlating biomarker concentration with some
clinical outcome.
Most new biomarkers proposed in the litera-
ture never reach the clinic, often because of a lack
of reproducibility. In a meta-analysis of highly
cited articles announcing new biomarker
candidates for a variety of diseases, it was
shown that follow-up experiments with greater
statistical power generally fail to reproduce the
same effect size as the original studies (Ioannidis
and Panagiotou 2011; Feala et al. 2013). More-
over, in addition to the need for validation of such
candidates, integration of the information
provided by each biomarker is also needed for a
comprehensive representation of the disease pro-
cess. Here we argue that biomarker discovery
would be greatly enhanced by applying systems
biology principles such as the multi-scale integra-
tion of information and the analysis of dynamic
patterns with the help of computational tools.
11.5 Systems Biology
Systems biology is an interdisciplinary field in
which experimental and computational methods
concur with mathematical models to the analysis
and the understanding of biological phenomena.
In systems biology different types of molecular
knowledge are integrated and exploited thanks to
the synergistic use and combination of statistical
and mathematical model together with experi-
mental data (Bruggeman and Westerhoff 2007).
Systems biology embraces a system prospec-
tive as opposed to the reductionistic vision which
is common in the mainstream biology: it aims to
tackle biological problems by considering the
interaction among the different parts of the
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
175
biomolecular mechanisms underlying complex
biological processes (Ahn et al. 2006)
Systems biology relies on the use of a mathe-
matical model to integrate and understand data
that are acquired using a vast array of experimen-
tal approaches ranging from molecular techniques
to comprehensive measurement often
encompassing multiple layers of the omics cas-
cade such as transcriptomics, proteomics, and
metabolomics. Different types of models can be
used depending on the data available, the research
questions, and whether a deductive or an induc-
tive approach is followed.
The deductive approach, usually referred to as
Top-down approach (Oltvai and Barabási 2002),
aims to gain insights into a given phenomenon
starting from the system-wide data acquired using
omics technologies from which information is
extracted, usually using statistical and machine
learning methods in combination with network
inference and network analysis (Rosato et al.
2018; Ideker and Krogan 2012). The ultimate
goal is to describe the interactions among the
molecular constituents of the system (genes,
proteins, metabolites), possibly across different
conditions (Rosato et al. 2018, Ideker and Krogan
2012) to understand how these parts interact to
shape the system-wide behavior of the system.
On the other side, the inductive approach,
referred to as Bottom-up approach (Oltvai and
Barabási 2002), starts from a detailed molecular
and biochemical knowledge of certain biological
mechanisms and aims to create mathematical
models that can reproduce experimental data.
The overarching goal is to predict the system-
wide behavior by building a complete genome-
scale model to provide an integrative view of the
biological interactions occurring inside living
systems (Shahzad and Loor 2012). The iterative
cycle of systems biology is illustrated in Fig. 11.1
in relationship to the pipeline for biomarker
discovery.
176
11.6 Systems Biology Approaches
to Biomarker Discovery
Determining whether a given biomarker is causal
or reactive to NSTI (or, in general, to any disease)
is likely to provide information on the underlying
pathogenic process and ultimately impact its gen-
eral applicability (Villoslada and Baranzini
2012). However, standard approaches that are
based on just univariate analysis can fail, by
their own nature, to account for the interrelated-
ness existing among genes, proteins, and
metabolites that behave in an orchestrated way
for what concerns regulation, transcription, and
translation.
For instance, Edwards and co-workers
(Edwards et al. 2018) investigated S. pyogenes
infections including NSTI among others, using a
proteomic approach and identified several human
proteins associated with NSTI. Yet, data was
analyzed in a univariate fashion, missing the
potential of highlighting association patterns
among proteins that could have been uniquely
associated with one or more type of infections.
Systems biology approaches rely on the con-
cept of mutual interaction among the constituents
of a biological system and thus poses a great
emphasis on measuring, describing, analyzing,
and interpreting the relationships among molecu-
lar features (genes, proteins, metabolites...) rather
than considering them one at a time. In this light,
top-down systems biology approaches can be
considered multivariate in nature, that is, the anal-
ysis is performed taking into consideration the
relationships among the variables and not only
their mean levels (i.e. concentration and
abundances) (Saccenti et al. 2014a) as in the
standard analysis for single molecule biomarkers.
Relationships among genes, proteins, and
metabolites are quantified using association
measures which describe the similarity of the
concentration and abundance profiles of these
molecular features measured on a set of biological
replicate samples. Correlations (either the
Pearson’s or Spearman’s indexes) are commonly
used and biological and biochemical information
can be derived from both the strength and the sign
E. Saccenti and M. Svensson
of correlations; for instance, if analyzing
metabolites, a strong positive correlation can indi-
cate an equilibrium condition or enzyme domi-
nance, while strong negative correlation can
indicate the presence of a conserved moiety
(Camacho et al. 2005).
It is fundamental to understand that the
associations among variables can carry informa-
tion also when concentrations (and abundances)
do not carry relevant information, for instance, to
discriminate between the case and control groups.
This situation, so common in the biomarker dis-
covery setting, is better illustrated with a
simulated example. With reference to Fig. 11.2a,
consider two metabolites (or protein or any other
biomolecular quantity) A and B measured from
case patients and controls: under this simulation
there is no difference between the concentration
levels of A and B in the two groups. However, if
the case and control group are compared taking
ratio of the concentrations of A and B a clear
difference between case and controls is evident,
as shown in Fig. 11.2b (see figure caption for
more details) and it is clear that the information
discriminating between cases and controls relies
on both metabolites and their mutual relationship
This phenomenon is a consequence of
metabolites A and B being correlated as shown
in Fig. 11.2c: from biological point of view what
is observed here is that given a certain concentra-
tion of metabolite A, the concentration of metab-
olite B changes with class (Saccenti et al. 2014a).
This indicates that when the relation or interaction
between pairs of metabolites or other biomolecu-
lar features is thought to be important and to carry
information about the problem studied, the ratios
of metabolite concentration (or abundances)
could be considered instead of single metabolite
concentrations. Examples of this exist in the clin-
ical setting where the deviation from the normal
range of the ratios of metabolites or protein is
used as diagnostic biomarkers (Petersen et al.
2012): ornithine-δ-aminotransferase deficiency
in young children is diagnosed using the proline
to citrulline ratio (De Sain-Van Der Velden et al.
2012), while the ratio between blood phenylala-
nine and tyrosine concentrations is used to
11 Systems Biology and Biomarkers in Necrotizing Soft Tissue Infections
Fig. 11.2 Idealized experiment aiming to find biomarkers
discrimination between the case and control group based
on the measurement and analysis of two metabolites A and
B measure on n1 ¼ 75 cases and n2 ¼ 75 controls. (a)
Comparison of the concentrations of metabolites A and B
tested with 1-way ANOVA: there is no statically signifi-
cant difference at the α ¼ 0.05 level (P-value 0.32 and
identify heterozygous carriers of phenylketonuria
risk alleles (Hsia 1958).
Metabolite and protein ratios and variation
thereof have often direct biological relevance
and can provide mechanistic insights. In fact, if
two metabolites are connected within (or by) a
biochemical pathway, metabolite ratios approxi-
mate (under idealized steady state assumptions)
the reaction rates (Petersen et al. 2012). For
instance, considering the elementary biochemical
reaction where metabolite A is converted to B and
then degraded
k1 k2
A !B!
described by the differential equations
8
>
> d½A
< ¼ k1 ½A
dt
> Analysis
: d ½B ¼ þk1 ½A  k2 ½B
>
dt
where [A] and [B] are the concentrations of A and
B at time t. At steady state the ratio of the con-
centration of A and B gives an approximation of
the ratios of the rate constants for the two
reactions
177
0.98, respectively, for metabolite A and B. (b) Compari-
son of the ratios of the concentrations of metabolites A and
B: a discrimination between the case and control groups is
evident (1-way ANOVA P-value ¼ 2.7
1018). (c)
Scatter plot of the concentration profiles of metabolites A
and B
½A k 2
¼
½B k 1
and can be used as a proxy to characterize the
system. Since reaction constants are tightly
constrained under normal physiological
conditions, increased (or decreased) metabolite
ratios can be an indication of alteration in the
activity of an enzyme that catalyzes the reaction
or a change in flux distribution. For instance,
variation of the ratios between sphingolipids that
differ by two carbon moieties has been linked to a
modified beta-oxidation, while variation of
the ratios between different classes of
phospholipids has been proposed to describe
modified activity of enzymes in the phospholipid
pathways (Altmaier et al. 2008).
11.7 Networks and Network
When many metabolites or other biochemical and
biomolecular features are measured, their mutual
relationships can be conveniently represented
using networks. A (biological) network is a
graphical representation of objects (called
nodes) and their relationships described by links
178
or edges connecting different nodes. The exact
meaning of the nodes and edges, i.e. of the kind of
relationships they represent, depends on the con-
text. In gene regulatory networks the nodes repre-
sent genes and the edge represents the existence
of regulatory mechanism where the protein pro-
duced by a given gene regulates the expression of
the target gene (Emmert-Streib et al. 2014). In
networks describing protein–protein interaction
the edge represents the existence of a physical
interaction between two proteins (Jordán et al.
2012).
In the context of biomarker discovery, the
most relevant networks are probably the
so-called metabolite–metabolite association
networks, or correlation networks, where the
nodes are metabolites and the edges represent
the existence of associations between two
metabolites, quantified through the correlation of
their concentration profiles measured on
replicated samples.
The simplest approach to build metabolite–
metabolite association networks is first taking
the correlation rij among pairs of metabolites
i and j and then imposing a threshold on either
the magnitude of the correlation or on the statisti-
cal significant (or both), setting to 0 the correla-
tion that do not satisfy such conditions. For
instance
 conditions. This approach is called differential
r ij if jr ij j > θ and Pval < α
r ij ¼ ð11:1Þ
0 otherwise
where θ and α (statistical significance) are
parameters to be determined; a commonly
accepted value for θ is 0.6 which is usually
taken as a threshold to discriminate between low
and medium-high correlation (Camacho et al.
2005). Other methods, like the Probabilistic Con-
text Likelihood of Relatedness for Correlations
(PCLRC) (Saccenti et al. 2014b), dispose of the
necessity of a pre-defined threshold on the cor-
relation but are context dependent, and the sig-
nificance of the correlations is established with
respect to the correlation background observed
in the data.
E. Saccenti and M. Svensson
If the value of the correlation (or any other
association measure) is retained, the network is
said to be weighted as every edge connecting two
nodes describes the weight of the association. In
other cases, only the presence/absence of an asso-
ciation is retained, and in this case the network is
said to be unweighted

1 if jr ij j > θ and Pval < α
r ij ¼ ð11:2Þ
0 otherwise
The nodes in a network can be characterized
using functions that can be derived from the
patterns of its association: a common measure is
the node degree or connectivity. For unweighted
networks this is simply the number of its connec-
tion. For weighted networks a weighted connec-
tivity is defined as the sum of the absolute values
of the edges:
X
χi ¼ j r ij j ð11:3Þ
i>j
In the framework of biomarker discovery, the
patterns of correlations between metabolites and
their connectivity can be exploited by comparing
them across conditions, for instance, cases and
controls, to identify associations that are
disrupted or altered by pathophysiological
network analysis and has been applied in NSTI
research (Afzal et al. 2020) as well in other fields
like cardiovascular disease (Vignoli et al. 2020;
Saccenti et al. 2014b) and aging (Vignoli et al.
2018).
Afzal et al. (2020) combined classical univari-
ate and multivariate approaches together with
differential analysis of metabolite–metabolite cor-
relation networks which were built from metabo-
lite concentrations of blood plasma metabolites
measured using GC-MS from NSTI patients
(n ¼ 34) and healthy (noninfected) controls
(n ¼ 24) enrolled in the INFECT project (Madsen
et al. 2019). In this study Spearman’s rank corre-
lation was used as an index of metabolite associ-
ation and implemented a conservative selection
procedure involving resampling to obtain robust
11 Systems Biology and Biomarkers in Necrotizing Soft Tissue Infections
Fig. 11.3 (a) Absolute differences in metabolite connec-
tivity in networks from NSTI and healthy control subject
(see Eqs. (11.3) and (11.4)) against its statistical signifi-
cance (Benjamini–Hochberg corrected -log10(P-value)).
The horizontal lines represent the significance thresholds
at α ¼ 0.05 and 0.01 level. (b) Scatter plot of the
estimates of the correlation because of the small
sample size available. Metabolite association
networks were constructed for both NSTI and
healthy and for each of the 97 measured
metabolites they calculated the differential con-
nectivity Δχ i defined as
Δχ i ¼ χ NSTI
i  χ Control
i ð11:4Þ
where χ NSTI
i and χ Control
i are the connectivity for
the metabolite i in the association network spe-
cific for NSTI and healthy control, respectively.
The significance of the observed difference in
connectivity was assessed using a permutation
test. Figure 11.3a shows the results of differential
network analysis and Fig. 11.3b shows the metab-
olite connectivity in the NSTI specific network
against connectivity in the uninfected surgery
controls. The authors noted that several
metabolites (urea, ribose, ribitol, pseudo uridine
penta, malic acid) that were not connected in the
control network were highly connected in the
179
metabolite connectivity observed in the metabolite–metab-
olite correlation networks built from NSTI and nonin-
fected surgical controls. The colors are proportional to –
log10(P-value) and the marker size is proportional to the
difference in connectivity
NSTI network, becoming hub node. In network
biology a hub is a node that is densely connected,
i.e. it is associated with many other nodes.
Because of this, hub nodes are assumed to play
crucial biological roles.
The concept of hub was first introduced within
the analysis of yeast protein–protein interaction
networks and it was shown that highly connected
nodes are more likely to be essential for survival
(Jeong et al. 2001; Carter et al. 2004). Analysis of
hub nodes has been extended to metabolite–
metabolite association networks: comparing low
and high cardiovascular risk patients. Saccenti
et al. (2014b) identified conserved hub metabo-
lite, and differentially conserved hubs and linked
them to alteration of mitochondrial activity.
Given this, Afzal et al. speculated that
metabolites showing differential connectivity
could play a role in disease pathogenesis either
by affecting the host response or the bacterial
infection. A set of significantly altered
metabolites was selected and tested in vitro to
180
investigate potential influence on NSTI group A
streptococcal strain growth and biofilm
formation.
The selected metabolites (ornithine, ribose,
urea, and glucuronic acid) were supplemented to
chemically defined media and revealed
metabolite-specific effects on both bacterial
growth and biofilm formation.
11.8 Multivariate Biomarker
and Omics Signatures
A generalized class of biomarkers are the
so-called multivariate biomarkers which are a
generalization of the bivariate biomarker
described in Sect. 11.6. This kind of biomarkers
are sometimes denoted with the term signatures,
especially in the context on omics research where
gene, protein and metabolite expression,
abundances and/or concentration levels are
measured in a comprehensive fashion on
biological samples using high-throughput analyt-
ical techniques like RNA sequencing, mass spec-
trometry, or nuclear magnetic resonance
spectrometry. These experimental techniques are
multivariate in nature since hundreds to
thousands of molecular features are measured
simultaneously and such molecular signatures
are exploited through multivariate and machine
learning approaches.
Sung et al. (2012) defined operatively molecu-
lar signature as a set of biomolecular features
together with a pre-defined computational proce-
dure that applies those features to predict a phe-
notype of clinical interest on a previously unseen
patient sample, stressing the computational/statis-
tical nature of such signatures.
Signatures are defined using statistical
approaches in combination with machine learning
techniques. Principal component analysis (PCA)
(Hotelling 1931; Pearson 1901; Jolliffe 2002) is a
common starting point when looking for bio-
marker signatures and it is used to explore data
and to highlight if such a signature may exist.
PCA is data reduction technique that allows for
visualization and exploration of high-dimensional
data by reducing its dimensionality. This is
E. Saccenti and M. Svensson
accomplished by taking linear combinations of
the original variables that explain as much as
data variability as possible which allows to
visualize the data in a lower dimensional space.
Figure 11.4 shows the results of a PCA analysis
on the data from Afzal and colleagues (Afzal et al.
2020). A clear separation between the metabolite
profiles of NSTI patient and healthy controls is
evident, indicating the existence of a
metabolomics (in this case) signature as shown
in the PCA score plot in Fig. 11.4. Figure 11.5
shows the relative importance (i.e. the PCA
loadings) corresponding to the PCA score plot.
The loadings are measure of the contribution of
each variable to explain the variability observed
in the data, in this case the separation between
NSTI and controls. As it can be seen all variables
(only the first 40 are shown for simplicity) con-
tribute to the model, showing the multivariate
nature of this metabolomics signature
differentiating NSTI subjects from the controls.
It is interesting to note that among the most
important metabolites there are urea, ribose,
ribitol, pseudo uridine penta, malic acid, the
same metabolites that showed significant differ-
ential connectivity in the network analysis (see
Fig. 11.3). This is because PCA, like all multivar-
iate approaches, takes into account the correlation
among variables which are also reflected in the
network structure underlying the data, making
network analysis and multivariate statistics two
complementary approaches.
Omics signatures consisting of many biomo-
lecular features have been defined and routinely
applied in the clinical setting. For instance, the
MammaPrint test (Van ’T Veer et al. 2002) used
to determine the most appropriate chemotherapy
treatment in lymph node negative breast cancer
patients with either positive or negative ER,
consists of 70 gene expression signatures which
were found to correlate with clinical phenotype
(metastatic vs. non-metastatic) and validated on
independent patient cohorts (Van de Vijver et al.
2002). Given the particular nature of NSTI, which
is a fast spreading infection requiring extremely
fast diagnosis and treatment, this type of high-
dimensional genomics signatures may not of
immediate applicability in the clinical setting
11 Systems Biology and Biomarkers in Necrotizing Soft Tissue Infections 181

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
also Chap. 13, Sect. 11.3.2).
However, omics signature can be explored to
single out key players and provide mechanistic
information about disease onset and progression
and can generate hypotheses that can be further
test in vitro and or in vivo in animal models,
closing the iterative cycle typical of systems
biology approaches. In this respect the main
limitation of PCA based signatures is that (1) all
variables contribute to the model and (2) PCA
base signatures are exploratory in nature and do

Fig. 11.5 Loading plot of principal component analysis
of the 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). Only the first 40, most contributing
metabolites are shown. Urea, ribose, ribitol, pseudo uri-
dine penta, and malic acid are also found to be relevant
from network analysis, See Fig. 11.4 for the corresponding
score plot
182
not allow, per se, the prediction of the clinical
phenotype on previously unseen patient
samples.
Signatures that are easier to interpret can be
obtained using advanced explorative techniques
like sparse approaches (Camacho et al. 2017;
Saccenti et al. 2018; Camacho et al. 2020). The
existence of a predictive power of such signatures
can be investigated using chemometrics or
machine learning approaches like partial least
squares discriminant analysis (PLS-DA) (Wold
and Eriksson 2001) or some of its extensions
(Lê Cao et al. 2008; Bylesjö et al. 2006; Camacho
and Saccenti 2018), Random Forests (Breiman
2001), Support vector machines (Cortes and
Vapnik 1995), or other machine learning tools.
When properly implemented, these methods
can allow unbiased prediction of the clinical phe-
notype on previously unseen patient samples: this
is accomplished by implementing cross-
validation, which is a computational strategy
that implies removing a set of samples from the
data set, building a predictive model, and then
testing in an unbiased way the ability of the
model to predict the left out samples, mimicking,
in this way, validation on an external cohort of
samples. This procedure can be particularly rele-
vant in NSTI where validation cohorts are diffi-
cult, to obtain. Random Forest was used to define
a multivariate biomarker consisting of 11 blood
analytes attaining specificity and sensitivity
around 90% for the early detection of ovarian
cancer (Bertenshaw et al. 2008), while Support
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.
2005).
Predictive omics signatures have great poten-
tial in NSTI. As an example, we show here a
Random Forest classification model fit on the
data from (Afzal et al. 2020) with default
parameters and implemented by splitting the
data into a validation set (20% of the data) and a
training set (80%) and repeating the cross-
validation procedure 100 times to take into
account sampling variability results. On the train-
ing set the accuracy for the prediction of NSTI
and control samples is 0.981 (0.978, 0.982 95%
E. Saccenti and M. Svensson
CI), while on the validation set one get average
accuracy equal to 0.980 (0.975–0.995, 95% CI),
average sensitivity 0.954 (0.938–0.969, 95% CI),
and an average specificity of 1. The classification
results on the validation set are similar to those on
the training set and this indicates that, in princi-
ple, these results may be generalizable to samples
acquired on external validation cohorts.
Many classification tools also produce
measures of importance of the predictors used in
the model and this allows to single out which
variables are most contributing to the discrimina-
tion among the groups to gain understanding of
the underlying biological processes or identify
potential biomarkers. Figure 11.6 shows the vari-
able importance plot of the Random Forest dis-
criminant model previously described to
discriminate between NSTI and healthy controls.
It is interesting to note that the group of variables
selected as important is somehow different from
the one from the PCA analysis (see Fig. 11.5):
only ribose is present in the top five most impor-
tant variables, while urea, which is important in
the PCA model, is not important in the Random
Forest model. This should not be surprising, since
PCA and Random Forest have different aims and
exploit different characteristics of the data: PCA
is an unsupervised method, while Random Forest
is a supervised method that uses information
about sample class (i.e. whether they belong to
NSTI patient or to controls) to build a model able
to predict to which class an unknown sample may
belong.
11.9 Future Perspective
Technology advances and critical analysis of
molecular pathways have opened new horizons
for management of diseases, exploring therapeu-
tic solutions to each individual patient beyond the
one-size fits all practice. As the amount of infor-
mation available grows, there comes a stage when
there is too much information to manually inte-
grate reliably. This leads to the need for formal
mathematical or computational models of disease
that incorporate all this information. For this rea-
son, clinical, microbiological, and experimental
11 Systems Biology and Biomarkers in Necrotizing Soft Tissue Infections
Fig. 11.6 Variable importance plot of a Random Forest
discriminant model fit to the 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
data can be integrated using various types of
mathematical algorithms to provide decision sup-
port tools for clinicians. This can pave the way for
application of personalized medicine in infectious
diseases, such as NSTI, with the aims to achieve
the right diagnosis and right treatment for the
right patient at the right time at the right cost.
Multivariate statistics, machine learning, and
reverse engineering approaches will allow
pinpointing and mapping key nodes that can
reveal possible biomarker sets from heteroge-
neous (meta-)data both from the tested pathogens
and the hosts (including the various “omics,”
pathotyping, patient stratification data). These
top-down analyses will be iteratively combined
with bottom-up modelling of specific
sub-networks/biomarkers. By thoroughly apply-
ing an iterative “dry-wet” cycle centered around
the cross-linking of bottom-up, hypothesis-driven
modelling of selected pathways and/or signatures,
with top-down, unbiased systems biology
approaches biomarkers that may contribute to
disease onset and outcome can be identified. In
particular, biomarker discovery in NSTI will
benefit from the integration of heterogenous
data, such as patient data and metadata, data
from the tested pathogens and the murine models
183
et al. 2019). Data is from Afzal et al. (2020). Only the first
40, most contributing metabolites are shown. The model is
used to investigate the predictive capability of the
metabolomics signature discriminating NSTI patient
from controls
and the wealth of omics data that can be collected
from them.
Even if the vast heterogeneity and/or quality of
the data will not allow detailed mechanistic
modelling of specific pathways, these top-down,
unbiased approaches will at least enable to iden-
tify basic structures in the topology of the various
networks underlying the disease. This in itself is a
substantial contribution to the state of the art in
biomarker identification. Finally, it will be impor-
tant to validate identified signatures/biomarkers
for patient classification through the use of large
patient cohorts. Indeed, validation remains one of
the major bottlenecks for biomarker discovery.
To become a clinically approved test, a potential
biomarker should be confirmed and validated
using large cohorts and should be reproducible,
specific, and sensitive (Drucker and
Krapfenbauer 2013). Although not uncommon,
NSTI is still a rare condition whose
characteristics (difficult diagnosis, fast spread,
high mortality, among others) make the creation
of validation cohorts difficult. However, the
INFECT cohort and the methodologies and
strategies for data collection and analysis devel-
oped within the INFECT project (Madsen et al.
2018, 2019; Afzal et al. 2020) are a first and
184 E. Saccenti and M. Svensson

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
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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 (*)

Laboratory of Systems and Synthetic Biology,
Wageningen University and Research, Wageningen, The
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
Department of Medicine, Haukeland University Hospital,
Bergen, Norway
E. Saccenti
Laboratory of Systems and Synthetic Biology,
Wageningen University and Research, Wageningen, The
Netherlands

# Springer Nature Switzerland AG 2020 187

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_12
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
to discrete and unspecific initial symptoms, scarce
clinical findings and lack of biomarkers in early
stages of many NSTI.
The cornerstones in the treatment of NSTI are
early aggressive surgical debridement and
appropriate antimicrobial therapy. Frequently
there is a need for advanced supportive
measures. There is clearly an urgent need for
refined bedside diagnostics and novel, targeted
treatment options to improve outcome in these
patients. Because of the multifactorial nature of
NSTI and sepsis, it is becoming increasingly
clear that individualized approaches are required
to improve outcome of these patients. These
infections are characterized by dysfunctional,
highly varying hyper- to hypo-inflammatory,
host responses contributing to disease severity
(Goldstein et al. 2007; Sarani et al. 2009;
Peetermans et al. 2020). Thus, individualized
therapeutic strategies targeting both pathogen
as well as the host response have great potential,
but require patient stratification based on disease
signatures.
Data are becoming available showing the
power of omics in identification of disease
signatures (Davenport et al. 2016), as well as in
providing the basis for patient stratification
through large-scale analysis of clinical data
(Hardt et al., in preparation), but fail to acknowl-
edge the complexity of the host-pathogen
interactions and the patient’s individual
responses to infection and its progression. In
this chapter, we address these problems from a
systems medicine perspective, provide an over-
view on the increasingly more frequent use of
Big Data and Artificial Intelligence (AI) to
aggregate, and generate knowledge from
burgeoning clinical and biochemical information
directing towards personalized approaches.
Moreover, we address the challenges to manage
this information and summarize current efforts
to translate this information into actionable
knowledge through the development of robust
computer-aided clinical decision support
systems (CDSS) to tackle the serious challenges
in NSTI diagnosis, stratification, and optimized
tailored therapy.
189
12.2 State of the Art: Systems,
Precision and Personalized
Medicine in NSTI
Current medical science is largely conducted
under a reductionist paradigm, which involves
the notion that complex phenomena like
mechanisms underlying onset of infectious
diseases, interactions between host and pathogens
and disease progression may be better understood
by breaking them down into smaller, simpler
components (Ahn et al. 2006).
However, given the complexity of the
conditions under study and the myriad of underly-
ing factors, this often leads to biased focus and
oversimplification (i.e. by focusing only on a hand-
ful of major factors with the biggest effect, while
the sum of minor factors may be considerable) and
generalization (i.e. assuming that a common cause-
effect relationship applies equally in all cases).
Such simplifications and generalizations often
limit our ability to understand how multiple
variables interact with one another to create emer-
gent effects and hamper not only our understand-
ing of the disease, but more importantly, our
capability of delivering better treatments. There
is clearly a need to address health and disease
from a systems perspective, that is, one that
accounts for all factors and interactions. For
instance, Chap. 12 illustrated the application of
such a systems (biology) approach for the identi-
fication of potential biomarker sets in NSTI. This
entailed the uncovering of metabolite–metabolite
association networks and analysis and deploy-
ment of machine learning methods (Afzal et al.
2020). These findings would not have been pos-
sible with a traditional, reductionist approach of
trying to identify potential biomarkers by stan-
dard approaches that are based, e.g. on simple
univariate analysis of datasets, as these fail to
account for the interrelatedness existing among
genes, proteins, and metabolites that behave in an
orchestrated way for what concerns regulation,
transcription, and translation (Rosato et al. 2018).
190 V. A. P. Martins dos Santos et al.

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-

Building upon the uncovering of biological
mechanisms as above and going beyond that,
“Systems medicine” applies systems biology
approaches to medical research and medical prac-
tice. Its objective is to integrate a variety of
biological/medical data at all relevant levels of
cellular organization using the power of compu-
tational and mathematical modelling, to enable
understanding of the pathophysiological
mechanisms, prognosis, diagnosis, and treatment
of disease (Auffray et al. 2010). Systems Medi-
cine involves iterative and reciprocal feedback
between the clinical practice and the research
carried on with computational, statistical, and
mathematical multiscale analysis and modelling
at both the epidemiological and individual patient
level (www.easym.eu).
This new paradigm of Systems Science and
Medicine complements the traditional reduction-
ist approach (as exemplified in Fig. 12.1) and,
ideally, leads to the identification of mechanisms
related to disease pathophysiology, selection of
novel drug targets and biomarkers, patient strati-
fication, risk assessment, and optimized therapy.
As described in Chap. 1, the systems medicine
INFECT project (https://permedinfect.com/
projects/infect/) has generated comprehensive
knowledge of diagnostic features, causative
genic mechanisms (host and bacterial disease
traits and their underlying interaction networks)
by using the largest thus far patient cohort in the
world. Also, INFECT has proven the value of
systems medicine approaches in acute infectious
diseases to achieve improved diagnostics and
therapeutics to improve patient disease outcome.
In particular, the novel understanding of the dis-
ease mechanisms has resulted in changed clinical
practice related to antibiotic usage as well as use of
immunomodulatory treatments (Bergsten et al. 2020;
Madsen et al. 2018) (see also Chaps. 7, 8, and 9).
However, the insights gained also underscore
the need for patient stratification and implemen-
tation of tailored therapy. Despite considerable
progress, there are substantial hurdles to be over-
come related to the difficulties in accounting for
patient’s individual conditions, or intrinsic to
Systems Medicine itself.
12.2.2 Challenges in Systems Medicine
and How to Tackle Them
The recent transition to data-rich (i.e. molecular
characterization) applications, which followed
the omics revolution with the advent of high
throughput genomics, transcriptomics,
metabolomics, and all other omics disciplines,
12 Systems and Precision Medicine in Necrotizing Soft Tissue Infections
have enabled Systems Medicine approaches
(Noble 2008). However, this data deluge is at
the same time an opportunity and a challenge.
An opportunity because it allows patient
phenotyping at different levels in an unprece-
dented manner, and a challenge because of the
heterogeneity observed in both data and patients.
There is an inherent data heterogeneity which
arises when different data types and different data
resources are integrated. This poses challenges
for both data handling and analysis which can
be addressed by vertical integration, i.e. when
different data informs on the same biological
level, or by horizontal integration, i.e. when mul-
tiple datasets inform on the same biological level
(Berthold et al. 2010). Both cases can be handled,
using FAIR data approaches (Findable, Accessi-
ble, Interoperable, Reusable) and semantics
technologies (Berthold et al. 2010) but, as a mat-
ter of fact, there are other inherent sources of
variability, which poses even greater challenges
to Systems medicine approaches.
Humans exhibit a great phenotypic diversity,
which originates from the complex interplay of
genetic, epigenetic, and environmental factors,
that affects both disease manifestations and
responses to therapy (Assfalg et al. 2008; Bernini
et al. 2009). Nonetheless, intra-individual
variability is less than half the inter-individual
variability, making personalized medicine possi-
ble (Hughes et al. 2015; Gruden et al. 2012).
Under this light, statistical and computational
characterization of individual and inter-individual
variability is pivotal to the deployment of systems
and personalized medicine approaches, which
will allow both higher sensitivity and specificity
of personalized assays and substantial new
insights into health and disease.
Characterization of phenotypic diversity in
both health and disease and underlying biomolec-
ular mechanisms should be carried out at different
levels. Examples could be developing new statis-
tical and computational methods, to exploit the
wealth of information obtainable at all omics
levels, or by developing and deploying multiscale
approaches to model how processes occurring at
widely different scales integrate, results in the
phenotypic variability observed in humans.
191
Multiscale modelling is another way to
approach the problem of phenotypic variability.
This includes bridging molecular and physiologi-
cal processes, even taking place at very different
time and spatial scales (Meier-Schellersheim et al.
2009). This could facilitate a better understanding
of the biology of health and disease, allowing us
to tailor models to individual patients
(e.g. genome-scale metabolic model constrained
by patient-specific data).
A key challenge is to select measurements and
data collected at the small scales and combine
them into informative metrics to be transferred
to a higher level. This is the realm of meta-
modelling, which is the statistical approximations
or predictions of the relationships between the
various model components. Meta-modelling has
been proposed as an efficient solution to link
models obtained at different scales, to link
modelling results and measured data, and to iden-
tify the most important metrics determining sys-
tem functionality at the various levels (Tøndel
et al. 2012).
Among statistical approaches derived from
multivariate analysis, multi-way analysis has
been proposed to retain the block-wise structure
of temporal data originating from nonlinear
dynamic models used to describe the systems at
different levels (Tøndel et al. 2012). However,
other solutions can be hypothesized that involve
other component methods: some examples are
principal component regression (Jolliffe 1982)
or partial least square regression (Wold and
Eriksson 2001) that can be used to model the
complex relationships between input parameters
and model outputs of nonlinear dynamic models
likely with embedded procedure for variable
selection to reduce dimensionality and complex-
ity of the parameter space (Tøndel et al. 2012).
Furthermore, parameter characteristics, such as
distributional properties, covariance, and correla-
tion patterns, could be inferred by stochastics
methods designed to deconvolute correlative or
noisy patterns: since signals can be separated
from background biological variability in data-
driven inference framework, this could ultimately
lead to a better definition of phenotype
characteristics.
192
12.2.3 Precision and Personalized
Medicine
Personalized Medicine builds upon Systems
Medicine and is an emerging data-driven health
care approach that integrates phenotypic, geno-
typic, epigenetic, lifestyle, and environmental
factors unique to an individual. The goal of
personalized medicine is to facilitate diagnosis,
predict effective therapy, and avoid adverse
reactions specific for each patient (Nimmesgern
et al. 2017; Union 2015).
Precision medicine is the concept of tailoring
disease treatment and prevention to account for
differences in genetic, environmental, or even
lifestyle factors specific to groups of people
(Bresnick 2018). Precision medicine takes
genetic and biochemical information unique to a
group of patients and uses that information to
develop more specific and streamlined
medications or treatments. The goal is to ensure
that each medication or treatment is best suited to
treat the individual, resulting in decreased side
effects and increased effectiveness (Hulsen et al.
2019).
Although the terms precision medicine and
personalized medicine are sometimes used inter-
changeably, generally speaking, “precision medi-
cine” seeks to create treatments that are applicable
to groups of individuals who meet certain
characteristics, whereas “personalized medicine”
implies individualized treatments available for
every unique patient (Erikainen and Chan 2019).
Precision and personalized medicine are in
their infancy in infectious diseases, in particular
acute diseases such as sepsis and NSTI (Lazăr
et al. 2019). As the patient populations in severe
infections are highly heterogeneous due to host-
microbe interactions governed by different host
factors and pathogens driving unique pathogenic
mechanisms, personalized and precision medi-
cine approaches may prove crucial. It is
recognized that a dysregulated host response to
infection is directly linked to severity and out-
come of severe infections, such as sepsis and
NSTI. Furthermore, the response profiles/disease
signatures can be highly variable, ranging from
V. A. P. Martins dos Santos et al.
hyper- to hypo-responses with different mediators
involved (Hotchkiss and Karl 2003; Anaya et al.
2005; Huang et al. 2011; Thänert et al. 2019).
The identification of disease signatures of
value for patient classification studies in well-
defined patient cohorts has been undertaken,
exploring host responses, pathogen profiles, and
their association with disease outcomes (Chella
Krishnan et al. 2016; Thänert et al. 2019). How-
ever, as for all biomarkers (see Chap. 12), these
findings must be validated by use of other patient
cohorts, as stated in a landmark position paper by
the European Society of Clinical Microbiology
and Infectious Diseases (Rello et al. 2018).
Accordingly, to advance on precision medicine
of NSTI and sepsis, two current multinational
projects have been designed and implemented
(PerMIT and PerAID, www.permedinfect.com).
These projects build upon the unique resources
created in the INFECT project, including clinical
expertise, patient registry, biobank, multi-omics
data, identified candidate disease signatures, data
stewardship resources, and basic science experi-
mental model systems.
These resources are currently being used to
test data-driven working hypotheses through
advanced preclinical and clinical studies com-
bined with Big Data integration and information
technology solutions, to develop patient stratifi-
cation schemes allowing for individualized ther-
apy in NSTI. The identified pathogenic
mechanisms and biomarkers linked to particular
NSTI disease signatures and clinical outcome are
being further validated in sepsis patients, a condi-
tion that, just like NSTI, is defined by the host’s
response to an infection. A major strength of
these on-going studies is that they resort on
well-defined patient cohorts, allowing for robust
conclusions linking host response signatures and
pathogenic mechanisms to clinical outcomes
encouraging translation of these findings into
future patient handling measures.
Interestingly as well, these two synergistic
projects envision that some stratification and clin-
ical trial designs will be shared for NSTI and
sepsis, whereas others will target more strictly
defined patient subgroups. For example, sepsis
patients with an immunosuppressive response
12 Systems and Precision Medicine in Necrotizing Soft Tissue Infections
profile might be targeted for an immune boosting
therapy, whereas NSTI and sepsis patients with a
hyperinflammatory profile will benefit from a
suppressive agent.
Undoubtedly, such insights will allow for
effective tailored treatments and development of
new tools and concepts for future clinical trials.
Crucially, however, for this potential to be ful-
filled, some further challenges have to be tackled,
in particular with regard to the handling of infor-
mation and translation to actionable knowledge.
Indeed, a key challenge in the personalized/
precision medicine field is the vast amount of
fragmented clinical and experimental datasets
that need to be organized, harmonized, and
integrated in order to achieve clinically relevant
results and to guide preclinical and clinical
research. This is also essential to enable fruitful
Big Data analytics to yield meaningful insights,
as well as for development of Clinical Decision
Support Systems (CDSS) to assist effectively and
efficiently in bedside decision.
12.3 Big Data, Machine Learning
and Deep Learning in Systems
Medicine
12.3.1 Big Data Definitions
and Characteristics
Big Data and the development of techniques to
handle it have the potential of enhancing our
ability to probe and understand which parts of
the biological machinery underlying the normal
functioning of the organism that may be or
become dysfunctional, given the pathophysiology
of a condition (Hulsen et al. 2019).
Big Data are characterized by the so-called
four V’s (Schroeck et al. 2012), which stands
for volume, variety, velocity, and variability.
1. Volume refers to the size of data, where size
indicates the physical occupancy of data files.
In Big Data applications exabytes, zettabytes,
and even higher amounts of data need to be
handled simultaneously. For what concerns
193
systems medicine applications, the volume of
the data is likely to be much smaller.
2. Variety refers to the heterogenous nature and
sources of data. In the biomedical field the
different types of data that can be collected,
mined, and analyzed are virtually endless.
Data comes not only in form of health records,
clinical data, to which data from omics
measurements can be added but also as medi-
cal imaging, including X-rays, CT, or MRI, or
images recorded on tissue samples, e.g. tissue
biopsies. These different data sources need to
be handled and integrated to be properly
analyzed, taking into account there are
structured data (e.g. data stored in excel for-
mat) and unstructured data (like doctor’s
notes). This is usually one of the most chal-
lenging tasks.
3. Velocity refers to rate of data sampling and
acquisition. Standard clinical measurements
and omics data are usually static or acquired
at a (very) low sampling rates. Differently,
health monitoring systems, including, but not
limited to, smartphones, smart watches, smart
bracelets/wristbands, connected sensors, and
wearable devices, enable continuous monitor-
ing of patient data by sensing and transmitting
measurements such as heart rate, blood pres-
sure, body temperature, respiratory rate, chest
sounds, and electrocardiogram (Vitabile et al.
2019) at high frequency, creating a flow of
data that often needs to be processed “on-the-
fly.”
4. Variability concerns the quality of the data
acquired and their inconsistency. For biomed-
ical and healthcare applications, data quality is
a very critical aspect, because erroneous infor-
mation can lead to erroneous diagnosis and
treatment. The problem of obtaining quality
data is complex and cross-disciplinary. Over
the years, several organizations have
contributed to defining the quality of various
products and services and identifying ways of
measuring such quality (Brighi 2018).
Handling and management of data inconsis-
tency, such those introduced by missing data,
194
is of paramount importance for the exploita-
tion of Big Data.
Accordingly, Big Data are high-volume, high-
velocity and/or high-variety information assets
that demand cost-effective, innovative forms of
information processing that enable enhanced
insight, decision making, and process automa-
tion. This definition (www.gartner.com/it-glos
sary/big-data/), albeit stated in the context of
informatics, summarizes the challenges and the
gain that Big Data present and can offer to
Systems Medicine.
The ultimate goal of the collection and use of
Big Data in Systems Medicine is the possibility of
obtaining better description of both health and
disease profiles and to use them to build integra-
tive models that can be used to predict disease
onset and progression and to tailor better treat-
ment for each patient. Under this light, Big Data
approaches are fundamental for the development
of Precision medicine, which aim to integrate
phenotypic, genomic, epigenetic, and environ-
mental factors unique to an individual to facilitate
diagnosis, predict effective therapy, and avoid
adverse reactions specific for each patient. Thus,
precision medicine needs to operate on different
scales to gain other insights into health and disease,
utilizing and integrating data from cells, tissues,
organs, and ecosystems, e.g. those constituted by
microbial communities (Stacy et al. 2016).
12.3.2 AI in Systems Medicine
Artificial intelligence encompasses the use of
software and algorithms to emulate the human
cognition in the analysis of complex medical data.
AI is being successfully employed in several
medical fields. Models have been constructed that
enable to distinguish high-risk breast lesions
(HRL) diagnosed with image-guided needle
biopsy that require surgical excision from HRLs
that are at low risk for upgrade to cancer at sur-
gery (Bahl et al. 2018) or able to detect pneumo-
nia using chest X-rays with an accuracy level
exceeding practicing radiologists (Rajpurkar
V. A. P. Martins dos Santos et al.
et al. 2017). Using images, artificial intelligence
approaches have been used to describe the impact
of orthognathic treatment on facial attractiveness
and age appearance (Patcas et al. 2019). A semi-
nal study (Esteva et al. 2017) tested the perfor-
mance of AI to distinguish keratinocyte
carcinomas versus benign seborrheic keratoses
and malignant melanomas versus benign nevi,
training it on >15,000 biopsy-proven diagnostic
images, against 21 board-certified dermatologists,
and found AI to perform on par with all tested
experts across both comparisons, and other stud-
ies confirmed AI ability in identifying melanoma
from dermoscopic images with accuracy similar
to that of specialists (Phillips et al. 2019, 2020).
There are few applications of Big Data and AI
to systems medicine specific to NSTI. The man-
agement of NSTI is complex given that clinical
presentation is highly variable and range from
early sepsis with obvious skin involvement to
minimal cutaneous manifestations with a dispro-
portionate systemic response (Bosshardt et al.
1996). Classic signs like fever, diffuse crepitus,
and shock are late signs: once large blisters and
gangrene develop, the infectious process is
already at an advanced stage (Bosshardt et al.
1996). NSTI treatment must be aggressive and
rapid and essential elements of the treatment are
resuscitation, antimicrobial therapy, surgical
debridement, and supportive care (Anaya et al.
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).
Hyperbaric oxygen treatment can also be used
as adjunctive therapy when infections involve
anaerobic bacteria, specifically the clostridial spe-
cies (Bakker 2012).
For instance, AI could be applied to the analy-
sis of microbiological findings. Identification of
the etiological agents can assist infection control
measures and antimicrobial therapy decision
making, and may offer prognostic information
(Anaya et al. 2005; Huang et al. 2011; Madsen
et al. 2019). The practical applicability of AI
methods for the analysis of microbiology finding
to aid diagnostic testing has been postulated and
discussed for the image analysis including Gram
12 Systems and Precision Medicine in Necrotizing Soft Tissue Infections
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
also to NSTI microbiology (see also Fig. 12.2).
The role of radiologic imaging in diagnosis of
NSTI is debated (Leichtle et al. 2016; Fernando
et al. 2019) and several imaging options are avail-
able, such as plain radiographs, ultrasonography,
computerized tomography (CT), and magnetic
resonance imaging (MRI).
Although MRI images were previously not
used in Big Data context or analyzed with AI
methods, those could be easily applied to support
and aid clinical decision. For instance, supervised
algorithms could be trained on large datasets
containing MRI images of patients with NSTI
and related conditions, and these algorithms
could be applied instantly, contextually with
image acquisition and reconstruction, to provide
guided diagnosis that could be integrated with
other clinical information.
Kim et al. (2011) suggested that magnetic res-
onance imaging could be used to differentiate
between necrotizing and non-necrotizing fasciitis.
They compared MRI findings between the two
groups and found that patients with necrotizing
fasciitis had a significantly greater frequency of,
195
among others, thick (3 mm) abnormal fascial
signal intensity on fat-suppressed T2-weighted
images, low signal intensity in the deep fascia
on fat-suppressed T2-weighted images. CT scan-
ning has also been proposed (Hietbrink et al.
2016) but MRI scanning proves to have the
highest sensitivity and specificity (Hietbrink
et al. 2016).
Rakus-Andersson and Frey (Rakus-Andersson
and Frey 2016) trained a modified neural net-
work, to identify groups of NSTI patient with
good prognosis of recovering without HBO com-
pared to patients for which HBO could be benefi-
cial, in such a way as to support clinical decision
making. They used data from 13 patients admitted
to the Blekinge County City Hospital in
Karlskrona between 2006 and 2010. The input
data consisted of clinical data (non-disclosed)
and the results were satisfactory, with an accuracy
of 92%; however, these results were obtained on a
very limited sample size and have not been cross-
validated.
The use of Big omics data in the clinical
setting of NSTI to support diagnosis at the bed-
side is less immediate since these measurement
196
platforms are usually not available in hospital
settings: sample work-up, preparation, acquisi-
tion, data cleaning, and processing may require
several days, which is a major hindrance, given
the severity of NSTI in critically ill patients
(Peetermans et al. 2020). A notable exception
is the use of nuclear magnetic resonance, which
requires minimal sample preparation to measure
metabolomics profiles of blood and tissues
samples collected in the operating theater and
that can provide almost real-time information to
the surgeons and to clinicians. Such a setting has
been implemented at St. Mary Hospital in
London (Nicholson et al. 2012).
The advent of new technologies is enabling
real-time sequencing of large genomes and it is
now possible to perform without delay sequenc-
ing and analysis of patient genetic information.
Implementations of automated phenotyping and
interpretation of genome sequencing by bead-
based genome library preparation directly from
blood samples and sequencing of paired 100-nt
reads obtained 15.5 h and used for fast
population-scale, provisional diagnosis of
genetic diseases of infants in neonatal and pedi-
atric intensive care units, have been reported
(Clark et al. 2019). They reported a prospective
100% precision and a mean time saving of 22 h
on diagnosis which subsequently affected
treatment.
If Big omics data are less applicable for bed-
side decision support due to technical limitations,
they are essential when defining strategies for
patient stratifications and individualized therapy
in NSTI. Indeed, the lack of stratification
strategies (one of the cornerstones of precision
medicine) is one of the biggest bottlenecks in
NSTI management. Omics data from NSTI
subjects (and possibly controls), such as
transcriptomics, proteomics, lipidomics, and
metabolomics profiles measured on blood and
tissue biopsies, could be analyzed in relation to
pathogens and clinical parameters using multivar-
iate statistics, machine learning, and reverse engi-
neering approaches to identify subgroups of
patients demonstrating a survival benefit or favor-
able response to given treatments.
V. A. P. Martins dos Santos et al.
12.4 Information Management
12.4.1 Personalized and Precision
Medicine
A key challenge in the fields of personalized and
precision medicine is to organize, harmonize, and
integrate the vast amount of fragmented clinical
and experimental datasets, in order to achieve
clinically relevant results and to guide preclinical
and clinical research.
Typically, data of different sources such as
electronic health record (EHR) systems, clinical
research databases, or biomedical knowledge
representations like (i.e. ontologies) have to be
reviewed and prepared. Furthermore, an often
overlooked weakness is the use of patient samples
and/or omics data despite lack of linked clinical
(meta)data, which greatly reduces the usefulness
of the studies. Thus, information management is
of paramount importance for systems and
personalized medicine in research as well as clin-
ical practice. To tackle these challenges, a variety
of approaches have been suggested and
implemented in different settings. One such
model is a three-layer information technology
architecture coupled to a cyclic data management
approach, as proposed by Ganzinger and Knaup
(2017). The generic high-level architecture of
such a three-layer model entails:
1. Data representation,
2. Decision support, and
3. User interface.
As for data representation (layer 1), data and
knowledge from different sources have to be
prepared and made available for use in systems
medicine. This includes data harmonization,
transformation, and storage. In decision support
(layer 2), the data and knowledge from layer 1 are
processed by applying decision support
approaches or systems biology models (see also
Sect. 12.3 on Big Data). Systems medicine
applications should be designed to assist and not
replace human decisions. Consequently, the user
interface for such an application (layer 3) must be
12 Systems and Precision Medicine in Necrotizing Soft Tissue Infections 197

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
198
Data fusion and standardization is ensured
throughout the use of specialized data fusion
algorithms and well-functioning interactions
among participating institutions.
The use of a RDF data model to manage data
provenance and storage ensures that data comply
with the FAIR guidelines for data management.
Minimum information checklists are used to facili-
tate interpretation and reproducibility of results
ensuring the inclusion of the relevant meta-data.
All data types are (should be) to be represented in
ontologies so that they can be integrated and remain
interoperable as the types and size of data increase.
A systematic treatment of the data is ensured
by the use of ontologies devoted to clinical and
disease-related terms, such as Systematized
Nomenclature of Medicine-Clinical Terms
(SNOMED CT) (Cote 1986; Lussier et al.
1998). The structure of the underlying data
resources is assessed using tools such as
RDF2Graph (van Dam et al. 2015) to ensure
that newly integrated datasets readily fulfill the
quality standards and indicators.
Operation of the www.permedinfect.com plat-
form enables both handling of heterogeneous data
and the standardization of operating procedures,
data, and models, as well as their storage and
stewardship.
The structure and workflows comply with
those required by the European Union on data
management of research (European Commission
2016). The platform itself aligns with interna-
tional data stewardship infrastructure such as the
ESFRI ELIXIR (elixir-europe.org) or Nordic
e-Infrastructure Collaboration (NeIC) program
(https://neic.no/).
12.4.4 Laying the Basis
for Computer-Assisted Decision
Support
Any CDSS has to rely on a data platform (see, for
instance, the HUNT platform, www.ntnu.edu/
V. A. P. Martins dos Santos et al.
hunt/data which inform the PerAID and PerMIT
data projects), allowing different layers with dif-
ferent level of access and different levels of gran-
ularity at the data level.
At the first level, a close interaction between
the clinicians and support personnel exists. The
interaction on the electronic devices is
optimized using the state-of-the-art technology
to monitor and to optimize human-computer
interfaces.
The second level prepares anonymized
databases from level 1 using software that can
process various types of data such as patient
records, data collected in various forms, and
biobank data to a format that can be included in
the RDF databases through previous
standardization. This software is to be used within
the hospitals in a protected environment and only
the anonymized data will be transferred to the
second level. This data is then to be further
digitalized and abstracted to the third level that
will represent a statistical view on the
original data.
The first and second level are unique for a
particular clinical center, while data at the third
level will be pooled across centers. The three-
layered data structure helps to overcome the
main limitations of medical support systems,
which are usually not scalable and not
interconnected. Data are translated to standard
English vocabulary while keeping the original
granularity of the data and third layer data will
be shareable between different clinical sites.
This allows the CDSS to be scalable and
used in other studied and countries by account-
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
best practice management of the disease. The
CDSS will therefore combine the information
of all patients in the patient cohorts, thereby
allowing for interoperability and scalability of
the system.
12 Systems and Precision Medicine in Necrotizing Soft Tissue Infections
12.5 Clinical Decision Support
Systems for Soft Tissue
Infections
12.5.1 The Need to Enhance Medical
Decisions
The quantity and quality of clinical data are
expanding rapidly, including EHRs, disease
registries, patient surveys, and information
exchanges. Also, burgeoning amounts of data
are becoming available for each patient, as is the
increasing body of medical and basic sciences
evidence. Hence, clinicians need tools to help
them make rational decisions based on all these
information (Wasylewicz and Scheepers-Hoeks
2019).
Big Data and digitalization, however, does not
automatically mean better patient care. Several
studies have shown that only implementing an
EHR and computerized physician order entry
(CPOE) has rapidly decreased the incidence of
certain errors, introducing, however, many more
(Magrabi et al. 2016). Therefore, high-quality
clinical decision support is essential if healthcare
organizations are to achieve the full benefits of
EHR and CPOE.
In the current healthcare setting, healthcare
providers often do not know that certain patient
data are available in the EHR, do not always
know how to access these data, do not have the
time to search for them, or are not fully informed
on the most current medical insights when facing
a decision. It is said the healthcare providers often
drown in the midst of plenty (Mamlin and Tierney
2016; Frost and Sullivan 2015; Bresnick 2016).
Moreover, decisions by healthcare
professionals are often made in conjunction
with/as part of direct patient contact, ward rounds,
or multidisciplinary meetings. This means that
many decisions are made in a matter of seconds
or minutes. This way, their quality depends on the
healthcare provider having all patient parameters
and medical knowledge readily available at the
time of the decision. Consequently, current
decisions are still strongly confounded by experi-
ence and knowledge of the professional.
199
Computer technology and algorithms can assist
by generating case-specific advice for clinical
decision making. The systems used are usually
referred to as CDSS, and are thus intended to
improve health care delivery by enhancing medi-
cal decisions with targeted clinical knowledge,
patient information, and other molecular or health
information (Wasylewicz and Scheepers-Hoeks
2019).
12.5.2 What Are CDSS What Is
Their Use
A traditional CDSS is comprised of software
designed to be a direct aid to clinical decision
making, in which the characteristics of an indi-
vidual patient are matched to a computerized
clinical knowledge base. Patient-specific
assessments or recommendations are then
presented to the clinician for a decision (Sutton
et al. 2020). From a historical point of view,
medication-related CDSS has been used for a
long time and is still the most advanced (Garg
et al. 2005). CDSSs today are primarily used at
the point-of-care, for the clinician to combine
their knowledge with information or suggestions
provided by the CDSS. However, there are CDSS
being developed with the capability to leverage
data and observations otherwise unobtainable or
uninterpretable by humans (Wasylewicz and
Scheepers-Hoeks 2019).
Current CDSS often makes use of
web-applications or integration with EHR and
CPOE systems. They can be administered not
only through desktop, tablet, smartphone but
also through other devices such as biometric
monitoring and wearable health technology.
These devices may or may not produce outputs
directly on the device or be linked into EHR
databases (Dias and Paulo Silva Cunha 2018).
The scope of functions provided by CDSS is
vast, including diagnostics, alarm systems, dis-
ease management, prescription, drug control,
and much more. CDSS ranges from personal dig-
ital assistant applications customized by a single
clinician to multihospital mainframe-based sur-
veillance systems meant to assure care for
200
Fig. 12.3 Clinical algorithm for suspected fasciitis, as
suggested by Hietbrink and co-workers (Hietbrink et al.
2016). The algorithm is used for gate specialties in patients
with suspected necrotizing fasciitis. It consists of aware-
ness, early surgical exploration, and early initiation of
treatment. When incision biopsy is indicated, the patient
thousands of patients (Pusic and Ansermino
2004). They can manifest as computerized alerts
and reminders, computerized guidelines, order
sets, patient data reports, documentation
templates, and clinical workflow tools.
Regarding the nature of the interaction with
the clinician, CDSS may be categorized into those
that entail solicited information (e.g. a clinician
asking for specific advice for a given condition)
or unsolicited information (deliver information or
knowledge that beneficially can alter clinical
decision making (Pusic and Ansermino 2004)).
Such applications can be particular useful if
CDSS builds upon robust and flexible data inte-
gration and includes a wide variety of data ana-
lytics. Such data analytics, in particular those
based on AI, Machine Learning, and other
V. A. P. Martins dos Santos et al.
is transported to the operation room for further treatment.
Treatment and aftercare are multidisciplinary. Analysis of
frozen section, microbiological findings, and biopsy could
be supported by AI technologies. Figure and caption from
Hietbrink et al. (2016), Licensed under Creative Commons
modelling tools, can be essential for rapid diag-
nosis, stratification, and assistance on decision
regarding disease treatment and intervention
strategies to be applied.
The benefits of CDSS, possible pitfalls, and
evidence-based mitigation strategies to overcome
have been published recently by Sutton et al.
(2020) and discussed as well by Pusic and
Ansermino (2004) and (Wasylewicz and
Scheepers-Hoeks (2019).
12.5.3 CDSS in NSTI
Algorithmic procedures to handle NSTI patients
have been defined (Hietbrink et al. 2016;
Peetermans et al. 2020), see Fig. 12.3, but there
12 Systems and Precision Medicine in Necrotizing Soft Tissue Infections
are currently no CDSS or modules thereof dedi-
cated to NSTI. Such modules or built-in tools
would possibly be very useful for a range of
uses, including those that could be included in
CDSSs or applications routinely used by general
practitioners, as these are often the first point of
entry of potential patients, which are frequently
misdiagnosed due to lack of familiarity with the
disease (Goh et al. 2014).
Indeed, due to the fast progression of the dis-
ease, NSTI management requires fast decisions to
determine the most appropriate course of action.
Such decisions, or part of them, could be
performed or supported by trained algorithms,
e.g. aiding in the main diagnostic problem, that
is to differentiate a lesion requiring surgery from a
lesion for which conservative treatment will be
sufficient. Furthermore, tasks like selection of
antibiotic therapy and adjunctive therapy could
be performed or optimized by dedicated
algorithms, as suggested in the closely relating
field of sepsis management (Komorowski 2020).
Simple algorithmic procedures are already rou-
tinely applied in the management of NSTI. An
example of this is the LRINEC (Laboratory Risk
Indicator for Necrotizing Fasciitis) score which is
generated from six routinely performed labora-
tory tests including the analyses of patients´
C-reactive protein, white blood cell count, hemo-
globin, sodium, creatinine, glucose. Wong et al.
(2004) proposed a tool to distinguishing NSTI
from the other severe soft tissue infections. How-
ever, recently its performance was evaluated in a
prospective cohort study and data were displayed
discouraging its use (Hsiao et al. 2020). An older
study within the National Surgical Quality
Improvement Program (NSQIP, USA) was used
to determine data on the incidence, treatment, and
outcomes of NSTIs (Mills et al. 2010). Partly on
the basis thereof, a 30-day postoperative mortality
risk calculator for patients with NSTI was devel-
oped and validated using a cohort of 1392
identified NSTI cases, of which 42% were female,
median age was 55 years, and median body mass
index was 32 kg/m2 (Mills et al. 2010). Thirty-
day mortality was 13%. Seven independent
variables were identified that correlated with mor-
tality: age older than 60 years, functional status,
requiring dialysis, American Society of
201
Anesthesiologists class 4 or higher, emergent sur-
gery, septic shock, and low platelet count. The
receiver operating characteristic area was 0.85
(95% CI 0.82-0.87), reflecting a reasonably
strong prediction. Using bootstrap validation,
the optimism-corrected receiver operating charac-
teristic area was 0.83 (95% CI 0.81–0.86), which
was used to develop an interactive risk calculator
for future patients. Although not a CDSS, this
correlation was nevertheless useful for stratifica-
tion, according to the authors.
Another cohort study was reported (Hua et al.
2015) that included 109 patients with a confirmed
diagnosis of NSTI, a median follow-up of
274 days (range 2–6135 days) and of which
31 (28%) died. On multivariate analysis, indepen-
dent risk factors of mortality were age older than
75 years, multifocal NSTI, severe peripheral vas-
cular disease, hospital-acquired infection, severe
sepsis, and septic shock on hospital admission.
Although a retrospective cohort, which disallows
a precise record of the delay between diagnosis
and surgery, these analyses could help building
information to develop a true CDSS and help
clinicians stratify NSTI severity at clinical course
onset.
A triple diagnostic procedure has been pro-
posed to manage NSTI (Hietbrink et al. 2016)
which combines the analysis of microscopic
findings on tissue biopsies together with Gram
staining to assess the presence, gram staining,
characteristic arrangements, and morphology of
microorganisms and analysis of fresh frozen
sections to detect necrosis of the superficial fascia
with fibrinous thrombi of arteries and veins
located in the fascia. The algorithmic procedure
is described in Fig. 12.3, in which many steps
could be replaced or supported by AI- and Big
Data-informed decision.
12.5.4 Current and Future
Developments in Relation
to Dedicated CDSS for NSTI
Specific efforts to develop and deploy innovative
clinical support tools for patient stratification and
bedside decisions suitable for the emergency and
intensive care setting for NSTI are currently being
202 V. A. P. Martins dos Santos et al.

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-
tional research projects comprising teams from
hospitals, medical research, academia, and indus-
try (see permedinfect.com). One specific task is
the development of Machine Learning and AI
models for the prediction of different outcomes
in NSTI, such as risk of sepsis, septic shock, or
amputation, using a combination of clinical
parameters including SAPS and SOFA severity
scores and biomarkers that can be measured dur-
ing the acute stages (e.g. in the emergency room),
thereby enabling rapid bedside decisions.
The same data approach is being used to iden-
tify an optimized scoring of NSTI patients, to
overcome some of sensitivity issues identified
with the severity score LRINEC indicated above
(Hansen et al. 2017).
Development of automatic calculations of
such severity score is part of the basis for
applications offering personalized decision sup-
port. Considering that different hospitals have
their own sepsis alert system, such automatic
calculations linked to the patients charts and
sepsis alarm are considered as a means to achieve
a more rapid and optimized identification of
patients. This type of systems can help
prioritizing severe cases and thereby reduce the
clinical burden and efficient use of hospital beds.
An effective and efficient CDSS is of particu-
lar importance for the stratification of patients and
personalized therapy in NSTI. Several dedicated
modules are currently under development (www.
permedinfect.com) for: (1) the integration of a
variety of quantitative or qualitative models
(i.e. statistical models, algorithms, etc.) to enable
a CDSS to perform data analytics (see previous
chapter and section on AI and Big Data above);
(2) aggregation of reasoning processes from the
domain and inference capabilities (e.g. rule-base
and case-based systems) to handle the data/infor-
mation (i.e. clinical and experimental
parameters); and (3) user interfaces to match the
need of practitioners in the clinic.
The software architecture model for the NSTI-
dedicated CDSS, the representation of interoper-
able clinical knowledge, and inference engine are
12 Systems and Precision Medicine in Necrotizing Soft Tissue Infections 203

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
204
allow patient stratification and individualized
therapy in NSTI/sepsis. In addition to individual
patient data, the system provides information of
patient classification into groups with similar dis-
ease status and comparable clinical and biological
parameters, which are of great value for
continued research. A simplified representation
of the envisaged CDSS for NSTI is shown in
Fig. 12.4.
An example of an initial prototype for a front-
end app to be directly used by a clinical practi-
tioner is depicted in Figs. 12.5 and 12.6 (www.
lifeglimmer.com).
These modules are currently under develop-
ment and testing and it is expected that they will
be eventually implemented in the clinical prac-
tice, with potentially substantial benefits for the
patient and disease management.
12.6 Conclusion and Perspectives
The clinical and biochemical research progress
over the last decades have provided a burgeoning
body of information on the possible mechanisms
underlying NSTI, on the clinical manifestation of
this fast-developing disease, and on
individualized patient characteristics. The organi-
zation and translation of this information into
actionable knowledge requires concerted, multi-
disciplinary efforts and accessible computational
systems that assist decision making. Altogether,
the efforts, platforms, and variety of modules
herein described for systems and personalized
medicine in acute infectious diseases form the
basis for development of tools and concepts for
refined diagnosis, patient stratification, and
individualized treatment. Thereby, such
approaches hold great promise for accurate and
rapid diagnosing and improving outcome in
NSTI, as well as potential to increase cost-
efficacy, as it will promote optimized tailored
therapy.
Acknowledgments The work was supported by the
European Union Seventh Framework Programme:
(FP7/2007-2013) under the grant agreement 305340
(INFECT project); the Swedish Governmental Agency
V. A. P. Martins dos Santos et al.
for Innovation Systems (VINNOVA), Innovation Fund
Denmark and the Research Council of Norway under the
frame of NordForsk (Project no. 90456, PerAID), and the
Swedish Research Council, Innovation Fund Denmark, the
Research Council of Norway, the Netherlands
Organisation for Health Research and Development
(ZonMW), and DLR Federal Ministry of Education and
Research, under the frame of ERA PerMed (Project 2018-
151, PerMIT).
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