the skin sore trial: exploring a better treatment option
TRANSCRIPT
The Skin Sore Trial: Exploring a better treatment option for
impetigo in Indigenous children living in remote Australia
Dr Asha Bowen
BA, DCH, MBBS
Thesis submitted for the degree of
Doctor of Philosophy
December 2014
Menzies School of Health Research
Charles Darwin University
Darwin, NT, Australia
DECLARATION
I hereby declare that the work herein now submitted as a thesis for the degree of
Doctor of Philosophy of the Charles Darwin University is the result of my own
investigations, and all references to the ideas and work of other researchers have
been specifically acknowledged. I hereby certify that the work embodied in this
thesis has not already been accepted in substance for any degree, and is not being
currently submitted in candidature for any other degree.
I give consent to this copy of my thesis, when deposited in the University
Library, being made available for loan and photocopying, and online via the
University’s Open Access repository eSpace.
Dr Asha Bowen
PhD Scholar
Menzies School of Health Research
December 2014
iii
ivxlcdm
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ABSTRACT
Impetigo is prevalent among Australian Indigenous children living in the
Northern Territory. The cause of this endemic skin infection is multifactorial
including household overcrowding, poverty, endemic scabies and fungal skin
infections, tropical climate, insect bites and minor trauma. Although impetigo is
widespread, affecting up to 50% of children in a community at one time, care
seeking for treatment of impetigo does not reflect this burden accurately,
possibly due to awareness of the painful injection prescribed. A better treatment
is needed.
This thesis presents the first randomised controlled trial conducted amongst
remote living Indigenous Australian children on the treatment of extensive
impetigo, and one of the largest impetigo trials worldwide. Methodological gaps
were addressed, with studies published on the susceptibility of Streptococcus
pyogenes to trimethoprim-sulphamethoxazole (SXT), the transport of impetigo
swabs in remote settings and photography protocols for capturing and scoring
sores. The principal findings are:
1. Either of two short courses of SXT are non-inferior to standard treatment of
impetigo with intramuscular benzathine penicillin G (BPG) – published in The
Lancet;
2. S. pyogenes remains the key driver of impetigo in our region; and
3. S. pyogenes is susceptible in vitro to SXT, and SXT is effective in vivo for the
treatment of skin infections caused by S. pyogenes.
These findings will inform the management of children with impetigo in remote
Indigenous communities and have already been incorporated into treatment
algorithms and guidelines regionally and nationally. This provides a simple,
short-course antibiotic regimen that is palatable, inexpensive and has a low side-
effect profile, as an alternative to an injection of intramuscular penicillin, which
has been the standard of care for more than 20 years. Ongoing surveillance will
v
be needed to understand how this new regimen is utilised and whether
widespread use for a common condition will alter antibiotic resistance profiles.
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TABLE OF CONTENTS
Declaration iii
Abstract v
Table of Contents vii
Acknowledgements xiii
Dedication xix
Publications xxi
Presentations xxiii
List of Figures xxvii
List of Tables xxix
Abbreviations xxxiii
Chapter 1: Introduction 1
1.1 Thesis overview 1
1.2 A brief overview of the Northern Territory, Australia 2
1.3 What is impetigo? 3
1.4 Why is understanding impetigo important? 7
1.5 How common is impetigo? 8
1.6 Natural history of impetigo resolution 8
1.7 How is impetigo treated? 10
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1.8 A randomised controlled trial for extensive impetigo in
Indigenous children
13
1.9 Ethics 14
1.10 Summary of Introduction 14
Chapter 2: The global epidemiology of impetigo 15
2.1 Summary 15
2.2 Statement of contribution to jointly authored work 15
2.3 Introduction 16
2.4 Methods 18
2.5 Results 21
2.6 Discussion 32
2.7 Conclusions 36
Chapter 3: Is Streptococcus pyogenes susceptible to
trimethoprim-sulphamethoxazole?
37
3.1 Chapter overview 37
3.2 Statement of contribution to jointly authored work 38
3.3 Journal article: Is Streptococcus pyogenes resistant or
susceptible to trimethoprim-sulphamethoxazole? Journal
of Clinical Microbiology, 50: 4067 – 72.
40
viii
Chapter 4: The challenges of collecting and transporting
impetigo swabs in a tropical environment
47
4.1 Chapter overview 47
4.2 Statement of contribution to jointly authored work 48
4.3 Journal article: Comparison of three methods for the
recovery of skin pathogens from impetigo swabs collected
in a remote community of the Northern Territory,
Australia. Transactions of the Royal Society of Tropical
Medicine and Hygiene, 107:384-9.
49
Chapter 5: Developing a methodology for capturing and scoring
standardised digital images of impetigo
57
5.1 Chapter overview 57
5.2 Statement of contribution to jointly authored work 58
5.3 Journal article: Standardising and assessing digital
images for use in clinical trials: A practical, reproducible
method that blinds the assessor to treatment allocation,
PLOS One: e110395
58
Chapter 6: The results of the Skin Sore Trial 69
6.1 Chapter overview 69
6.2 Statement of contribution to jointly authored work 70
6.3 Journal article: Short course oral cotrimoxazole versus
intramuscular benzathine benzylpenicillin for impetigo in
a highly endemic region: an open label, randomised,
71
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controlled, non-inferiority trial, The Lancet, 384: 2132-
2140.
Chapter 7: The microbiology of impetigo in Indigenous
children: associations between Streptococcus pyogenes,
Staphylococcus aureus, scabies and nasal carriage
81
7.1 Chapter overview 81
7.2 Statement of contribution to jointly authored work 82
7.3 Submitted journal article: The microbiology of
impetigo in Indigenous children: associations between
Streptococcus pyogenes, Staphylococcus aureus, scabies
and nasal carriage.
82
Chapter 8: Conclusions 95
8.1 Chapter overview 95
8.2 Discussion of main findings 97
8.3 Study limitations 103
8.4 Future work 105
8.5 Final conclusions 109
Bibliography 111
Appendices 147
Appendix 1 149
x
Appendix 2 153
Appendix 3 157
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ACKNOWLEDGEMENTS
There are many people to acknowledge for their support throughout the four
years of work that has gone into this thesis. Above all, I thank my Lord and
Saviour, Jesus Christ, through whom I am strengthened each day to accomplish
what he has called me to do (Philippians 4:13).
It would not have been possible without the steadfast love and support of my
husband Darren Westphal. He has been a constant source of encouragement, day
in and day out, even when it all seemed impossible. He has been by my side
throughout this PhD and has been a great sounding board for my thoughts as they
developed. His skills as an amateur photographer and involvement in health
research guided me through the early days of developing a method for
photographing digital images. He has kept me focused on the bigger world
outside my thesis, encouraged me to present my work at international
conferences so that he could come along too and kept our family functioning at
peak times of my thesis. Our children Zachary and Eliana, who have both been
born during my candidature, have grounded me when I felt overwhelmed. They
have kept me laughing, kept me busy and are really looking forward to when
Mummy no longer needs to work all the time! My family both in Australia and
the USA have taken care of my children to give me time to write, encouraged me
even when they weren’t too sure what I was talking about and listened along the
way to all of my challenges. My mum, Narelle Bowen, has been willing on
several occasions to come and care for my family, to give me the opportunity to
write, even arriving from the other side of the country with less than 24 hours
notice when I needed her.
My supervisors have taught me the art and science of research, motivated me
when paving a path forward seemed impossible and corrected endless drafts of
papers. To each of them I offer my sincere thanks for taking on a novice
researcher to lead this trial. I would like to thank Jonathan Carapetis for his
unending belief in my ability to achieve goals that seemed insurmountable; an
always open door, telephone line or email inbox to discuss my questions and
address challenges; and his vision for the big picture. My involvement in
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research was inspired by Jonathan and would not have been possible without him
opening this doorway. Likewise, my regular meetings would not have occurred
without his excellent personal assistants Caroline Sheridan, Kristy Coulston and
Linda Lorimer. Adding Steven Tong as a co-supervisor in my second year has
been an enormous blessing. His wisdom has been an asset, his understanding of
the PhD journey and timeline important, and his research savvy inspiring. Steve
has sat with me on many occasions nutting out statistical analyses that seemed
impossible, suggesting creative ways forward with papers and has been available
for endless questions. Ross Andrews oversaw the trial management team and
answered many questions on epidemiology along the way. He was willing from
the outset to consider my input into changes in the study design. I have learned
and grown under their collective supervision and am inspired by their ongoing
commitment to answering questions that make a meaningful difference to the
health of Indigenous Australians.
Alongside my core supervisors sit the two other chief investigators on this trial.
Bart Currie has inspired me throughout. His enthusiasm, knowledge and clinical
acumen and his endless energy for high quality research to improve the quality of
care for Indigenous people makes him an outstanding clinician researcher, who
was never too busy for my questions. Bart has also been integral in translating
our findings into treatment guidelines in real time and has guided me through this
process. Malcolm McDonald left Darwin early in my candidature, but has
remained a supporter, encourager and most importantly, an excellent critic of my
written work. His ability to edit words out of a manuscript without losing any
meaning is a skill I hope I will continue to develop in my future work. These five
investigators envisioned this trial, pursued it undeterred through three NHMRC
funding rounds and welcomed me to the team. I am humbled by their collective
experience, inspired by their enthusiasm and motivated to continue to conduct
translational research with meaningful outcomes for Indigenous children.
My PhD is a single, large randomised controlled trial, conducted in very remote
communities of the Northern Territory. It is one of the largest impetigo trials ever
conducted, and the first in truly remote settings where the burden of disease is
the highest. It would not have been possible without the large trial team: the chief
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investigators (Jonathan, Steve, Ross, Bart and Malcolm), the project managers
(Irene O’Meara and her predecessor Tammy Fernandes) and the research team
(Jane Nelson, Melita McKinnon, Therese Kearns, Valerie Coomber, Christine
Francais, Colleen Mitchell, Dianne Halliday, Dev Tilikiratne, Neil Kerinaiua,
Lynette Johnson, Simone Baker, Doreen Jinggarrabarra, Duncan Wutumbul,
Roslyn Gundjirrjirr and Elvira Dhumabuy). Alongside this group were residents,
and medical and nursing students, who participated for a brief week or two as
research assistants to gain exposure to research in Indigenous communities. Jane,
TK, Melita and Margaret Landrigan were critical in the establishment of the trial
and oversaw the operations when the team was in the field. The laboratory
scientists both at Menzies (Rebecca Towers, Rachael Lilliebridge, Donna
Woltring, Jacklyn Ng, Wajahat Mahmood, Bianca Hayes and Vanessa Theobold)
and at Royal Darwin Hospital (Jann Hennesey, Greg Haran, Pam Smith, Luke
Tennent, Nicole McMahon, Gloria de Castro and Nu Nu Swe) were tireless in
their commitment to processing thousands of swabs for culture and antibiotic
susceptibility testing.
Irene O’Meara has been an outstanding project manager throughout the trial,
keeping track of trial progress, planning trips to communities and endlessly
recruiting staff for the study. Irene’s willingness to answer any question, clean
data, discuss results and make suggestions has been invaluable. I cannot thank
her enough for her drive, dedication and enthusiasm to see this trial through to
completion. Irene’s willingness to go the extra mile and her dedication to
Indigenous health is highly commendable. I know with certainty that the results
reported in this thesis would not have been possible without Irene’s commitment.
Irene is the face of the Skin Sore Trial in the remote communities. Children and
families were keen to know the results when they saw Irene arrive back in their
communities. In addition to numerous recruitment trips, she coordinated all the
feedback trips on which we had the exciting opportunity of sharing our findings
with families, teachers, clinic staff and community elders. Having Irene in charge
saw recruitment completed in the required time frame when even this seemed
impossible. Irene has become a good friend and is a dedicated project manager.
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Robyn Liddle, Melita McKinnon, Tegan Harris and Linda Walsh were all
involved in data management for the study. Robyn’s willingness to teach me
Microsoft Access and to answer numerous queries about the data structure was
admirable. Robyn was also invaluable in teasing out various challenges in
statistical analysis. Her willingness to build databases and modify these until
working smoothly for the photography review was fantastic. Without Robyn’s
expertise in this area, it would not have been possible to organise and score the
thousands of digital images collected over the course of the study. The members
of the Area9 team at Menzies throughout my PhD were also incredibly helpful in
this process, making sure that the technology for scoring digital images by
colleagues in Melbourne, Perth, Auckland, Adelaide or Brisbane worked
smoothly. They problem solved and developed solutions to make the process
work faster when this was also a challenge.
Kara Burns was integral in developing simple protocols for amateur
photographers that gave us sets of digital images of the same sore that could be
scored. I thank my paediatric colleagues who willingly looked at the paired
digital images. Tom Snelling, Andrew Steer, Claire MacVicar, Deena Parbhoo,
Rebecca Cresp, Sarah Cherian, Sarah Martin and Ruth Lennox were all willing
and available when I needed their assistance.
Mark Chatfield’s assistance with statistical analysis for the study was invaluable.
His advice to the chief investigators on various important statistical decisions
strengthened the study. He also guided us through the process of engaging a data
safety and monitoring board. I thank Jim Buttery who chaired this group along
with Keith Edwards, Stephen Lambert and Peter O’Rourke for their commitment
to the study.
During my training to become an infectious diseases specialist, I experienced a
brief period of training in a microbiology laboratory. Whilst not being a
microbiologist, my PhD candidature has extended my knowledge and skills in
microbiology. It would not have been possible to do so without the willingness of
microbiology colleagues to share their insights and to answer my questions.
These included Rob Baird, Malcolm McDonald, Peter Ward, Phil Giffard, Steven
Tong and Jann Hennesey. To the scientists at Menzies and Royal Darwin
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Hospital who have conducted experiments, processed thousands of specimens
and provided a robust microbiological data set, I am truly grateful.
Sharing office space with other PhD students has been a privilege and
encouragement along the way. Many a problem felt less challenging after
discussion with peers, at various stages, on the same journey. The broader higher
degree community at Menzies has also been invaluable with their willingness to
stop for a chat in the tea room to commiserate, encourage or congratulate! The
education office of Caroline Sheridan and Catherine Richardson has helped me
keep the funding bodies and university reports timely. The finance department
was also integral in administering my scholarships.
Menzies has been a welcoming and productive environment in which to begin
my research career. Whilst not being my immediate supervisors, I have been
mentored and supported by Anne Chang and Peter Morris. Their experience in
conducting randomised trials, and their passion for doing excellent research that
makes a difference for Indigenous children, is inspiring. My colleagues in the
Infectious Diseases Department of Royal Darwin Hospital should also be
thanked. This group of world-class clinician researchers encourage and support
junior colleagues, and are in turn able to make ground-breaking discoveries. This
would not be possible without the extremely hard work of the research
administration and ethics teams. Christina Spargo and Maria Scarlett deserve
special mention for their assistance throughout my candidature. Thanks must also
go to the Communications team at Menzies who were instrumental in allowing
our research findings to gain media attention. Richmond Hodgson, Lucy Barnard
and Claire Addinsall were dedicated and their efforts led to local and national
media reports on the day our Lancet paper was released. I was keen for our
findings to be highlighted in the Indigenous media. Lucy coordinated a number
of interviews to ensure our research was communicated in plain language to
those who may one day need to be treated. I thank my mum, Narelle Bowen and
Sue Dibbs for assistance with editing my thesis.
xvii
It would not have been possible to enrol in a PhD without financial support. The
funding from the National Health and Medical Research Council, Australian
Academy of Sciences and Menzies School of Health Research enabled me to
train as a researcher and to present our findings at regional, national and
international meetings.
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DEDICATION
I dedicate this thesis to the participants and their families throughout the
Northern Territory who freely consented to join the Skin Sore Trial in the hope
that a better treatment for skin sores would be found. It is my hope that an
injection will no longer be a deterrent to seeking effective treatment for skin
sores, and one day we will report that Indigenous children have the same burden
of impetigo as their non-Indigenous counterparts.
To my children, Zachary and Eliana, I hope that one day you might understand
the importance of my work in striving to make a difference for Indigenous
children, and that you may live in a world where studies like this are no longer
needed.
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PUBLICATIONS
Bowen AC, Lilliebridge RA, Tong SY, Baird RW, Ward P, McDonald MI,
Currie BJ, Carapetis JR. Is Streptococcus pyogenes resistant or susceptible to
trimethoprim-sulfamethoxazole? Journal of Clinical Microbiology. 2012; 50
(12): 4067 – 4072.
Bowen AC, Tong SY, Carapetis JR. Reply to “Susceptibility to Streptococcus
pyogenes to trimethoprim-sulfamethoxazole”. Journal of Clinical Microbiology.
2013; 51( 4): 1351.
Bowen AC, Tong SY, Chatfield MD, Andrews RM, Carapetis JR. Comparison
of three methods for the recovery of skin pathogens from impetigo swabs
collected in a remote community of Northern Territory, Australia. Transactions
of the Royal Society of Tropical Medicine and Hygiene. 2013; 107 (6): 384 –
389.
Bowen AC, Tong SY, Andrews RM, et al. Short-course oral co-trimoxazole
versus intramuscular benzathine benzylpenicillin for impetigo in a highly
endemic region: an open-label, randomised, controlled non-inferiority trial. The
Lancet 2014, 384 (9960) 2132 - 2140.
Bowen AC, Burns K, Tong SYC, Andrews RM, Liddle R, O’Meara IM,
Westphal DW, Carapetis JR. Standardising and assessing digital images for use
in clinical trials: a practical, reproducible method that blinds the assessor to
treatment allocation. PLoS One. 2014, Nov 6; 9(11): e110395
Submitted Manuscripts under review
Bowen AC, Tong SYC, Chatfield MD, Carapetis JR. The microbiology of
impetigo in Indigenous children: associations of Streptococcus pyogenes,
Staphylococcus aureus, scabies and nasal carriage. Submitted to BMC
Microbiology, under review.
xxi
Manuscripts in development
Bowen AC, Tong SYC, Mahe A, Hay R, Steer AC, Andrews RM, Carapetis JR.
The global burden of impetigo: A systematic review.
Tasani M, Tong SYC, Holt D, Currie BJ, Carapetis JR, Bowen AC. Does the
presence of scabies impact treatment efficacy? Further analysis of the Skin Sore
Trial results
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PRESENTATIONS
International Conferences
Bowen AC, Tong SYC, McDonald MI, Carapetis JR. In vitro and in vivo
susceptibility of Streptococcus pyogenes to trimethoprim/sulphamethoxazole in
Northern Australia. European Congress of Clinical Microbiology and Infectious
Diseases, Barcelona, SPAIN, 10 – 13 May 2014 (Oral and ePoster presentation)
Bowen AC, Tong SYC, Carapetis JR. Epidemiology of impetigo in Indigenous
children in remote northern Australia: results from a randomised controlled trial.
Annual Meeting of the European Society of Paediatric Infectious Diseases,
Dublin, IRELAND, 6 – 9 May 2014 (Oral and poster presentation)
Bowen AC, Tong SYC, Andrews RM, O’Meara IM, Chatfield MD, McDonald
MI, Currie BJ, Carapetis JR. Short course oral trimethoprim-sulphamethoxazole
is as effective as intramuscular benzathine penicillin G for impetigo: Results of a
non-inferiority, randomised controlled trial. Australasian Society of Infectious
Diseases Conference, Adelaide, AUSTRALIA, 26 – 29 March 2014 (oral
presentation)
Bowen AC. The infectious disease burden of Australia’s Indigenous children
living in the Northern Territory. World Congress of the World Society of
Paediatric Infectious Diseases, Cape Town, SOUTH AFRICA, 19 - 22
November 2013 (invited speaker)
Bowen AC, Tong SYC, Andrews RM, O’Meara IM, Chatfield MD, McDonald
MI, Currie BJ, Carapetis JR. Trimethoprim-sulphamethoxazole is non-inferior to
benzathine penicillin G for the treatment of impetigo: Results of a randomised
controlled trial. World Congress of the World Society of Paediatric Infectious
Diseases, Cape Town, SOUTH AFRICA, 19 - 22 November 2013. (Poster)
Bowen AC, Tong SYC, Carapetis JR. The microbiology of impetigo in
Indigenous children of tropical Australia. World Congress of the World Society
of Paediatric Infectious Diseases. Cape Town, SOUTH AFRICA, 19 - 22
November 2013 (poster)
xxiii
Bowen AC, Lilliebridge RA, Tong SYC, Baird R, Ward P, Currie BJ, McDonald
MI, Andrews RM, Carapetis JR. Are Group A streptococci susceptible or
resistant to trimethoprim-sulphamethoxazole? World Congress of the World
Society of Paediatric Infectious Diseases, Melbourne, AUSTRALIA November
2011. (Poster)
Bowen AC, Tong SYC, Lilliebridge RA, Andrews RM, Carapetis JR. A practical
and effective method of transporting pyoderma and nose swabs for recovery of
Streptococcus pyogenes and Staphylococcus aureus in remote areas. Lancefield
Symposium XII, Palermo, ITALY 4 – 8 September 2011. (Poster)
Bowen AC, Lilliebridge RA, Tong SYC, Baird R, Ward P, Currie BJ, McDonald
MI, Andrews RM, Carapetis JR. Are Group A streptococci susceptible or
resistant to trimethoprim-sulphamethoxazole? Lancefield Symposium XII,
Palermo, ITALY 4 – 8 September 2011. (Poster)
Oral presentations at local meetings
Bowen AC, Tong SYC, Andrews RM, O’Meara IM, Chatfield MD, McDonald
MI, Currie BJ, Carapetis JR. The Skin Sore Trial. Child and Adolescent Health
Research Symposium 2014, Perth, WA, 22 – 23 October 2014.
Bowen AC. The infectious diseases burden of remote Indigenous children. The
Skin Sore Trial. Child and Adolescent Health Research Symposium 2014, Perth,
WA, 22 – 23 October 2014.
Bowen AC, Tong SYC, Andrews RM, O’Meara IM, Chatfield MD, McDonald
MI, Currie BJ, Carapetis JR. The Skin Sore Trial: results of a non-inferiority
randomised controlled trial on treatment of impetigo in remote Indigenous
children. Wesfarmers Infectious Diseases Seminar, Telethon Kids Institute,
Perth, WA, 3 September 2014.
Bowen AC, Tong SYC, Andrews RM, O’Meara IM, Chatfield MD, McDonald
MI, Currie BJ, Carapetis JR. Is cotrimoxazole a GAS drug? Results of the Skin
Sore Trial. Western Australian Infectious Diseases Evening Meeting
(WAIDEM), Perth, WA, 12 August 2014.
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Bowen AC, Tong SYC, Andrews RM, O’Meara IM, Chatfield MD, McDonald
MI, Currie BJ, Carapetis JR. The Skin Sore Trial. General Paediatric Research
Meeting, Princess Margaret Hospital, Perth, WA, 31 July 2014.
Bowen AC, Tong SYC, Andrews RM, O’Meara IM, Chatfield MD, McDonald
MI, Currie BJ, Carapetis JR. No more pus: a new paradigm for skin sores in the
Northern Territory. Royal Darwin Hospital Grand Rounds, Darwin, NT, 7 April
2014.
Bowen AC, Tong SYC, Andrews RM, O’Meara IM, Chatfield MD, McDonald
MI, Currie BJ, Carapetis JR. Short course oral trimethoprim-sulphamethoxazole
is as effective as intramuscular benzathine penicillin G for impetigo: Results of a
non-inferiority, randomised controlled trial. Menzies Seminar, Darwin, NT, 28
January 2014.
Bowen AC, Tong SYC, Andrews RM, O’Meara IM, Chatfield MD, McDonald
MI, Currie BJ, Carapetis JR. Trimethoprim-sulphamethoxazole is non-inferior to
benzathine penicillin G for the treatment of impetigo: Results of a Randomised
Controlled Trial. Northern Territory Royal Australasian College of Physicians,
Alice Springs, NT, 9 November 2013.
Bowen AC, Tong SYC, Andrews RM, O’Meara IM, Chatfield MD, McDonald
MI, Currie BJ, Carapetis JR. Trimethoprim-sulphamethoxazole is non-inferior to
benzathine penicillin G for the treatment of impetigo: Results of a Randomised
Controlled Trial. Paediatric Grand Rounds, Royal Darwin Hospital, Darwin, NT,
1 November 2013.
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LIST OF FIGURES
Chapter 1 1
Figure 1: Map of Australia, highlighting the Northern
Territory in red
2
Figure 2: The visual appearance of healing sores with
treatment
10
Chapter 2 15
Figure 1: Flowchart of systematic review according to
PRISMA statement
22
Figure 2: Map of countries with available population-
based studies of impetigo prevalence, highlighted and
colour coded by median prevalence of impetigo
25
Box: Applying prevalence estimates for impetigo to
remote living Australian Indigenous children
26
Chapter 4 47
Figure 1: Flow diagram of the collection of swabs from
participants with impetigo in study arms comparing the dry
or liquid Amies techniques with the STGGB technique
52
Chapter 5 57
Figure 1: The Panasonic DMC-TZ8 camera demonstrating
zoom and camera settings as indicated in figure 5
60
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Figure 2: Use of a scale to define the upper and lower
boundaries of image in the landscape position
61
Figure 3: An example of the participant identification card
described in table 1
61
Figure 4: Capturing the image using the study camera,
grey background, and grey scale in a remote context
61
Figure 5: The camera settings and icons as used in the
protocol
63
Figure 6: Cartoon demonstrating the photographer, camera
and sore were in the same plane to optimise reproducibility
of captured images
64
Figure 7: Database form used for Quality Control check by
medical photographer
64
Figure 8: This shows the database format utilised for
scoring digital images pairs. Image A was taken on day 0
and image B on day 7
65
Chapter 6 69
Figure 1: Digital images and scoring algorithm 74
Figure 2: Trial profile 75
Figure 3: Comparison of treatment success between co-
trimoxazole and benzathine benzylpenicillin for various
analyses
77
Figure 4: Microbiological clearance by treatment group 77
xxviii
LIST OF TABLES
Chapter 1 1
Table 1: Summary of key demographic differences
between the Indigenous and overall population of the
Northern Territory, Australia
3
Table 2: The CARPA manual and history of
recommendations for treatment of impetigo in the NT
12
Chapter 2 15
Table 1: Number of studies of impetigo prevalence by
decade, country and region
23
Table 2: Summary statistics of available studies by age
grouping
24
Table 3: Estimates of children with impetigo by regions of
the world with available data*
27
Table 4: Classification of studies by region and World
Bank Development Indicator in 2005
28
Table 5: Median prevalence of impetigo in childhood and
overall, categorised by the World Development Index
29
Table 6: Variability in median impetigo prevalence by
urban and rural study locations
31
Table 7: Regional variation in the use of pyoderma or
impetigo to describe bacterial skin infections
32
xxix
Chapter 3 37
Table 1: Antibiotic susceptibility testing methods and agar
used
41
Table 2: SXT Etest susceptibility results for study 1 42
Table 3: Difference in MIC in study 1a 42
Table 4: SXT Etest susceptibility results for study 2 42
Table 5: Difference in MIC in study 2a 42
Table 6: Summary of published results of S. pyogenes in
vitro susceptibility to SXT
43
Chapter 4 47
Table 1: Streptococcus pyogenes or Staphylococcus aureus
recovery from simultaneously collected swabs from
impetigo lesions, comparing the dry or liquid Amies
techniques versus the STGGB technique
52
Table 2: Overall detection rates of Streptococcus pyogenes
or Staphylococcus aureus from simultaneously collected
swabs from impetigo lesions
53
Chapter 5 57
Table 1: Study equipment chosen including the required
features, advantages and alternatives available or
recommended in the literature
62
Table 2: Quick list for capturing standardised photographs 63
xxx
Table 3: Definitions used for the quality control
assessment of digital images
65
Table 4: Definitions used for final outcome scoring of
digital images of impetigo
66
Chapter 6 69
Table 1: Baseline characteristics of all participants in the
trial at the first visit according to study group
76
Table 2: Modified intention-to-treat outcomes by treatment
group (unless stated)
77
Table 3: Adverse events associated with receipt of either
study drug
78
Chapter 7 81
Table 1: Results from logistic regression models to assess
associations between impetigo pathogens and age, sex,
severity, presence of scabies and region
87
Table 2: Identification of Staphylococcus aureus from any
impetigo lesion and the anterior nares for all children with
at least one skin and nose swab available (n=504 children)
90
Appendices 147
Appendix 1: Table Overall and childhood pyoderma and
scabies prevalence from 89 studies synthesised in Chapter
2 systematic review
149
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ABBREVIATIONS
APP As per protocol
ARF Acute rheumatic fever
AST Antibacterial susceptibility testing
BHS beta-haemolytic streptococci
BPG Benzathine penicillin G
CARPA Central Australian Rural Practitioners Association
CI Confidence Interval
CLSI Clinical and Laboratory Standards Institute
CNA Collistin and nalidixic acid
CRF Case report form
CTX cotrimoxazole (or trimethoprim-sulfamethoxazole)
DSMB Data Safety and Monitoring Board
EUCAST European Committee on Antimicrobial Susceptibility Testing
GAS Group A streptococcus (Streptococcus pyogenes)
HREC Human Research Ethics Committee
IDSA Infectious Diseases Society of America
IQR Interquartile range
ITT Intention to treat
JPEG Joint Photographic Experts Group
MHA Mueller Hinton agar
xxxiii
MHBA Mueller Hinton agar with horse blood
MHF Mueller Hinton agar containing defibrinated horse blood and
20mg/liter beta NAD
MHLHBA Mueller Hinton agar with lysed horse blood
MHS Mueller Hinton agar with sheep blood
MIC Minimum Inhibitory Concentration
mITT modified Intention to treat
mMRSA multidrug resistant MRSA
MRSA Methicillin resistant Staphylococcus aureus
MSSA Methicillin susceptible Staphylococcus aureus
NHMRC National Health and Medical Research Council
nmMRSA non multidrug resistant MRSA
NT Northern Territory
OR Odds Ratio
PhD Doctor of Philosophy
QC Quality Control
RCT Randomised Controlled Trial
SMGGB Skim milk tryptone glucose glycerol broth
SMZ sulfamethoxazole
SOP Standard Operating Procedure
SSTI Skin and soft tissue infections
STGGB Skim milk tryptone glucose glycerol broth
xxxiv
SXT Trimethoprim/sulphamethoxazole
TMP trimethoprim
WGS Whole Genome sequencing
WHO World Health Organization
xxxv
xxxvi
CHAPTER 1INTRODUCTION
1.1 THESIS OVERVIEW
This chapter will introduce the main themes of the thesis. To understand the
context of this thesis, it will be important to know about both the study setting
and impetigo: what it is, how it occurs, what treatments are available and what is
known about the natural history of impetigo resolution. This will pave the way
for Chapter 2, a systematic review of population-based studies on the prevalence
of impetigo to define the global burden. Methodology chapters will follow,
defining methods for transporting impetigo swabs in remote contexts (Chapter
3), testing for antibiotic susceptibility of Streptococcus pyogenes to
trimethoprim/sulphamethoxazole, an antibiotic which has long been thought to
be ineffective against this organism (Chapter 4) and capturing digital images for
measuring the outcome of a randomised controlled trial (Chapter 5). The results
of the major study will then be presented in Chapter 6, followed by the
microbiology of impetigo in Indigenous children of Northern Australia in
Chapter 7. Chapter 8 will conclude the thesis with future directions for research
translation, and highlight the need for ongoing studies to dernormalise skin
disease in remote Indigenous children of Australia.
1
0123456789
1.2 A BRIEF OVERVIEW OF THE NORTHERN TERRITORY, AUSTRALIA
Figure 1: Map of Australia highlighting the Northern Territory in red.
Source: www.virtualoceania.net/australia/nt_map.png (accessed 19/11/14).
All the work reported in this thesis has been undertaken in the Northern Territory
(NT), a federal territory of Australia (Figure 1). This brief orientation is provided
to place the work in context. The NT includes about one-sixth of the Australian
continent, with an area of 1.35 million square kilometres
(www.ga.gov.au/scientific-topics/geographic-information/dimensions/area-of-
australia-states-and-territories, accessed 17/11/14). The NT is divided into two
regions, the ‘Top End’ where a tropical climate prevails and ‘Central Australia’
with a semi-arid climate. The NT is vast, remote and sparsely populated. It has
five major centres, of which Darwin is the largest, and more than 100 remote
communities. At the most recent census (2011), the NT population was 211,945
of whom 56,776 (27%) were Indigenous (Aboriginal and Torres Strait Islanders)
(www.censusdata.abs.gov.au, accessed 15/11/14). Table 1 provides a limited
overview of the social deprivation disproportionately suffered by the NT
Indigenous population and consequent health impacts. Nurses and Aboriginal
Health Workers, under the guidance of medical practitioners, provide the bulk of
primary health care in remote community clinics. The CARPA standard
treatment manual is widely used as a clinic manual for the delivery of primary
health care (CARPA, 2014).
2
Table 1: Summary of key demographic differences between the Indigenous and overall population of the Northern Territory, Australia
Overall NT residents Indigenous NT residents
Median age 31 years 23 years
Proportion aged < 15 years 23.2% 33.2%
Median household income per week $1, 674 $1,099
Median number of people per household 2.9 4.2
Average number of people per bedroom 1.0 1.7
Unemployment rate 4.3% 13.7%
% Resident remote (outside Darwin) 50% 90%
Life expectancy at birth (2009)* Male 75.7y
Female 81.2y
Male 61.5y
Female 69.2y
Sources: 2011 census data available at www.abs.gov.au, accessed 15/11/14,
*www.health.gov.au/internet/publications/publishing.nsf/Content/oatish-hpf-2012-
toc~tier1~life-exp-wellb~119, accessed 15/11/14.
1.3 WHAT IS IMPETIGO?
Impetigo contagiosa, also known commonly as skin sores, school sores or
shortened to impetigo, is a non-bullous infection of the superficial layers of the
epidermis. (Hay et al., 2006) It was first described in 1864. (Fox, 1864)
Pyoderma is a term often used interchangeably with impetigo, but pyoderma
encompasses a broader definition of superficial bacterial skin infection that
includes bullous and non-bullous impetigo, ecthyma, folliculitis, cellulitis and,
sometimes, tropical ulcers. (Mahe, 2005) Whilst impetigo is a superficial
infection of the epidermis, ecthyma is a deeper infection that extends into the
dermis. (Hay et al., 2006) The distinction between these two conditions in terms
of clinical recognition and treatment recommendations at a primary care level is
poorly understood and as such they are often discussed together, both for
microbiology and treatment algorithms. (Stevens et al., 2014, Mahe et al., 2005b,
3
Steer et al., 2009) This thesis will predominantly use the terms impetigo or skin
sores to describe this bacterial skin infection.
1.3.1 Microbiology of impetigo
The gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes
commonly cause impetigo. These bacteria may be found independently or in a
mixed growth, with the relative proportions varying over time, region and
climate. (Koning et al., 2012) Previous studies identified early colonisation of the
skin with S. pyogenes resulting in the development of sores in the ensuing days
through minor breaches in skin integrity. (Ferrieri et al., 1972, Dajani et al.,
1972, Dajani et al., 1973) These early studies reported that impetiginous lesions
were later colonised with S. aureus (Ferrieri et al., 1972, Dillon, 1970) and as
such both pathogens were implicated at different time points in the development
of impetigo, with S. pyogenes causing the infection and S. aureus complicating it.
In more recent decades, S. aureus has assumed prominence in the global
microbiology of skin and soft tissue infections, raising treatment challenges, as
this organism has developed resistance mechanisms against the commonly used
antibiotics for skin infections. (Moran et al., 2013, Tong et al., 2008) The
microbiology of impetigo in the Northern Territory also reflected these trends,
with increasing prevalence of methicillin-resistant S. aureus (MRSA) as a
co-pathogen with S. pyogenes, seen in swabs taken from impetigo lesions
throughout the Northern Territory (McDonald et al., 2006) and in hospital
studies. (Tong et al., 2009) Based on this changing microbiology, it was
uncertain whether benzathine penicillin G remained the most effective treatment
for impetigo in our context.
1.3.2 Antecedents of impetigo
Intact skin is usually resistant to infection with S. aureus or S. pyogenes
(Tunnessen, 1985). Intact skin may be disrupted by trauma, abrasions, insect
bites, scratching, eczema or another dermatosis (e.g., scabies infestation, tinea,
pediculosis or varicella zoster). Individuals in contact with others who have a
high burden of skin infections may also be colonised, (Ferrieri et al., 1972)
followed by the development of impetiginous lesions at the site of skin
4
disruption. (Currie and Carapetis, 2000, Wannamaker, 1970) Skin pathogens are
highly transmissible (Ferrieri et al., 1972) (skin-to-skin and skin-to-fomite) and
result in endemic infections where household overcrowding is extreme, (Harris et
al., 1992) with epidemic peaks as new bacterial strains are introduced. (Dajani et
al., 1973)
Skin on any part of the body can be involved. However, due to trauma and insect
bites being the major risk factors in tropical settings, (Leng and Chay, 1982,
Kottenhahn and Heck, 1994, Taplin et al., 1973, Allen and Taplin, 1974) lesions
are most common on the lower limbs, followed by the upper limbs (Mahe, 2005)
and scalp when superinfected pediculosis occurs. (Jinadu, 1985) The predilection
for the limbs in tropical contexts may also be due to the wearing of minimal
clothes in the heat and humidity; as such, little protection is offered against
abrasions and minor trauma. (Belcher et al., 1977) In other populations, lesions
on the face, around the nose and mouth are more common. (Koning et al., 2012,
Kiriakis et al., 2012)
Impetigo is endemic in hot and humid climates, (Taplin et al., 1973) particularly
if there is a lack of ready access to water for cleaning. (Cairncross and Cliff,
1987, Bhavsar and Mehta, 1985, Taplin et al., 1973) Household overcrowding is
a suitable environment for transmission of skin pathogens and is more commonly
found in regions where poverty prevails. (Harris et al., 1992, Accorsi et al., 2009)
Impetigo is a disease of poverty, with burden increasing as socio-economic status
decreases. (Masawe et al., 1975) Impetigo also perpetuates where understanding
and recognition of the disease as a health priority is poor. (Mahe et al., 2005a,
Hay et al., 1994) Where access to dermatologists is limited, primary health care
providers may not recognise this (or other) skin diseases and few studies are
available to guide effective treatment decisions in these contexts. The integrated
management of childhood illness (IMCI) has been developed as an approach to
the recognition and treatment of impetigo and is widely used in Fiji, (Steer et al.,
2009) but in other regions where impetigo is common, treatment algorithms are
less well utilised. (Figueroa et al., 1998, Mahe et al., 1995) Prioritisation of
community dermatology is still desperately needed. (Ryan, 2008)
5
Finally, the normalisation of sores and poor skin health has been raised as a
possible explanation for ongoing disease burden. When the majority of children
in a community have sores at any one time, and these are recognised to
eventually resolve over weeks to months, sores may become an accepted norm in
childhood. This normalisation of sores can result in a failure to seek health care,
(Dogra and Kumar, 2003) and a failure of health-care practitioners to
appropriately treat for impetigo when the child presents with other complaints.
On the other hand, skin disease has been found to be the second most common
reason for health care seeking in childhood in developing countries. (Estrada
Castanon et al., 1992, Figueroa et al., 1996, Walker et al., 2008) This
contradictory information makes estimating the true burden of skin disease,
including impetigo, difficult. Focusing on episodes of health care sought
(through audits of clinic attendance) rather than population prevalence may result
in under-reporting of the true burden of impetigo.
1.3.3 Identification of impetigo
Skin sores are identified by the classic characteristics of purulence, erythema and
golden crusting, which progresses to form a thick scab. (Wannamaker, 1970)
Sores are often painful, itchy and unsightly. Children are aware of sores due to
pain and pruritus, but may not complain excessively. (Dogra and Kumar, 2003)
Training in the recognition of skin lesions has been identified as a priority for the
provision of primary health care in tropical contexts, where impetigo and other
skin diseases are common. (Mahe et al., 2005a, Mahe, 2005) Identification of the
correct diagnosis is a priority to ensure the most appropriate treatment is offered.
For the Skin Sore Trial, research nurses were trained in skin disease recognition
using a clinical diagnosis manual developed by our team in earlier work. This
diagnostic and treatment manual includes high quality images of dermatological
conditions and is widely used throughout the region for education and training of
health-care workers.
http://www.menzies.edu.au/icm_docs/162092RecognisingandTreatingSkin
Conditions.pdf, accessed 17/11/14.
6
1.4 WHY IS UNDERSTANDING IMPETIGO IMPORTANT?
While skin sores are rarely fatal, impetigo lesions cause discomfort,
inflammation and missed school days. (Hay et al., 1994) In addition, the bacteria
are highly transmissible, with early studies showing infection of all other family
members with streptococcal pyoderma occurring at a mean of 21 days from the
index case. (Ferrieri et al., 1972) More recently in the NT, secondary S. pyogenes
transmission within the household was detected in 19% of occupants. (McDonald
et al., 2008) Likewise S. aureus, particularly MRSA, colonisation of family
members of index cases with skin infections is high. (Miller et al., 2012, Fritz et
al., 2012) The break in the skin is the entry point for bacteria that may lead to
both severe infectious and post-infectious sequelae. Skin sores have been
reported as a risk factor in the development of S. aureus (Skull et al., 1999) and
S. pyogenes (Carapetis et al., 1999) bacteraemia, resulting in hospitalisation.
(Engelman et al., 2014) Acute post-streptococcal glomerulonephritis outbreaks
occur regularly where skin sores are endemic (Marshall et al., 2011) and
contribute to a high burden of chronic kidney disease. (Hoy et al., 2012, White et
al., 2001) Finally, the burden of acute rheumatic fever in Indigenous Australians
is amongst the highest reported in the world, (Parnaby and Carapetis, 2010,
Seckeler and Hoke, 2011) with consequent morbidity and mortality. (Lawrence
et al., 2013) This has long been hypothesised to arise from skin infection rather
than pharyngitis, as is seen elsewhere in the world. (McDonald et al., 2004)
These serious sequelae of impetigo are not reported in this thesis, as the design of
the randomised controlled trial and sample size needed were not sufficient to also
document these much rarer events. However, the importance of treating impetigo
stems from decreasing disease burden, reducing interpersonal transmission and
hence reducing these more serious consequences.
7
1.5 HOW COMMON IS IMPETIGO?
1.5.1 Global burden
A systematic review of population-based studies reported in Chapter 2 confirms
the global burden remains high. This updates previous literature that incorporated
impetigo into other GAS diseases (Carapetis et al., 2005) or referenced impetigo
as one of many skin conditions worthy of attention. (Mahe, 2005, Vos et al.,
2012, Hay et al., 2014) The included studies will describe the prevalence
predominantly in resource-poor contexts. Due to a paucity of population-based
prevalence studies of impetigo in developed countries, estimates from general
practitioner surveys (Koning et al., 2006, Rortveit et al., 2011, Shallcross et al.,
2013) confirm that impetigo is also a common bacterial skin infection in
childhood in industrialised settings.
1.5.2 Indigenous children of the NT
There are ten studies included in the systematic review from Australia, all
reporting impetigo prevalence in remote Indigenous children from northern
Australia. Seven are studies from the NT, two from Queensland and one from
Western Australia. There were no population-based, prevalence studies available
for non-Indigenous children of Australia.
Of 4026 children assessed in the seven studies from the NT, the median
prevalence of impetigo was 45.7% (inter quartile range, IQR 2.6% – 69.4%).
These studies were published between 1992 and 2009, and reflect an ongoing
high burden of disease. An estimate of prevalence from the Skin Sore Trial based
on episodes eligible for inclusion is similarly high at 870/1715 (50.7%).
Research assistants in our trial were seeking to recruit participants with impetigo,
which may result in over-estimation, but it does confirm the burden remains high
throughout the NT.
1.6 NATURAL HISTORY OF IMPETIGO RESOLUTION
Few studies of the natural history of untreated impetigo, documenting the process
and time to resolution, are available. Understanding the natural history of
8
impetigo resolution is critical in evaluating the end point of treatment studies.
Could the changes seen in the appearance of a sore be due to natural resolution
alone or is there a treatment effect apparent?
Current knowledge of the natural history of impetigo emerged from observations
during outbreaks of impetigo associated with acute post-streptococcal
glomerulonephritis at the Red Lake reservation in Minnesota, USA in the 1960s.
(Dajani et al., 1973, Ferrieri et al., 1972, Dajani et al., 1972) Professor Dajani
was available for correspondence via email during my candidature, to discuss the
pathogenesis and healing of impetigo lesions. Having him available to answer
my questions expanded my understanding of his observations in children and
hamster studies. (Dajani and Wannamaker, 1970) This was critical in developing
definitions to formalise objective assessments of the digital images used in our
trial, as studies of impetigo resolution have not been ongoing.
Impetigo usually begins with a 1–2cm erythematous macule that rapidly becomes
a vesicle or pustule. (Dajani and Wannamaker, 1970) The vesicle ruptures in the
first 24–48 hours, leaving a thin crust overlying the erosion. (Dajani and
Wannamaker, 1970) Erythema becomes more marked in the initial few days due
to underlying neovascularisation, then gradually resolves. Concurrent with
erythema, the thin crust thickens over several days and as healing progresses, the
crust reduces in size. (Dajani et al., 1973) As the crust dries and contracts, there
may be evidence of traction around the edges of the crust and peeling of the
surrounding layers of skin. Peeling skin is due to the presence of bacterial
proteases that break down the damaged extracellular matrix, providing the
opportunity for regeneration. Eventually the lesion becomes flat and dry with
peeling around the edge, followed by a slight hypo- or hyper-pigmentation of the
skin. This usually resolves with time, and scarring is reportedly uncommon with
impetigo (Dajani and Wannamaker, 1970) but more common with the deeper
lesion ecthyma. Untreated, impetigo lesions can remain unresolved for 30 days.
(Dillon, 1970)
Hamster models showed resolution of impetigo by 9–11 days from the
introduction of bacteria. (Dajani et al., 1971) This is faster than reported in
humans. (Dillon, 1970) The hamster model, while useful to understand
9
pathogenesis, may be a less reliable predictor of the time to resolution in human
impetigo, due to the absence of ongoing trauma in an animal laboratory,
compared to the real-world experience of children. Beyond this, the natural
history of impetigo without treatment remains poorly understood, as few studies
have focused on this aspect; treatment is usually recommended and when treated
outside of intervention studies, follow-up to determine outcome is rarely
included in the clinical algorithm.
Figure 2: The visual appearance of healing sores with treatment.
Note: This series of photographs obtained from a participant in the Skin Sore Trial on
days 0 (A), 2 (B), and 7 (C) highlight some of the features of sore healing described
above. The erythematous macule, pustule and development of thin crusts can be seen in
image A. Thickening of the crusts, traction at the edges of the sore and peeling skin are
evident in image B. Hypopigmentation can be seen in image C.
1.7 HOW IS IMPETIGO TREATED?
1.7.1 What is the evidence base for treatment of impetigo?
In developed country settings, impetigo is a common cause for childhood
presentations to the general practitioner. (Shallcross et al., 2013) Lesions are
often around the mouth and nose, of small diameter (0.5–1cm) and discrete.
(Sladden and Johnston, 2004, Hartman-Adams et al., 2014) The Cochrane review
on treatment of impetigo (Koning et al., 2012) provides strong evidence for the
topical treatment of limited impetigo. These findings have been incorporated into
guidelines, which are accessed throughout the world. (Stevens et al., 2014)
However, of the 68 included trials, only one includes children with severe
C B A
10
impetigo and that was conducted in a tropical, resource-poor context. (Faye et al.,
2007) The remainder studied limited impetigo and were conducted in hospital
outpatient departments or general practices, predominantly in developed
countries. (Koning et al., 2012) Therefore, while topical therapy with fusidic acid
or mupirocin is recommended for limited impetigo based on the synthesis of high
quality randomised controlled trials, (Koning et al., 2012, Shim et al., 2014) a
major evidence gap remains for the treatment of extensive impetigo.
In regions where impetigo is endemic, lesions are widespread and several body
areas are affected at the same time, topical therapies are impractical and
expensive. Neither guidelines from the Infectious Diseases Society of America,
(Stevens et al., 2014) nor Medecins Sans Frontiers in conjunction with the World
Health Organization, (Broek et al., 2013) recommend topical therapy for
extensive or endemic impetigo. Both rely on expert opinion to support treatment
with oral antibiotics that are active against S. pyogenes and methicillin-
susceptible S. aureus for extensive disease. In addition, the widespread use of
topical therapies leads to the rapid emergence of antimicrobial resistance. (Udo et
al., 1994) There remains an evidence gap for the most effective treatment for
impetigo where the burden is highest (tropical, resource-poor contexts).
1.7.2 How is impetigo treated in Indigenous children in the Northern Territory?
The treatment of impetigo in the NT has historically included a single dose of
intramuscular benzathine penicillin G (Table 2). This regimen was recommended
in the second edition of the CARPA manual in 1994, (CARPA, 1994) at the time
intramuscular benzathine penicillin G (LA-Bicillin®) was first registered for use
in Australia. (Currie, 2006) The inclusion of benzathine penicillin G in the NT
treatment algorithms was based on non-randomised studies conducted in the
USA (Esterly and Markowitz, 1970, Dillon, 1970) in children with extensive
impetigo. Cure rates of between 58% and 100% were achieved compared with
20% to 39% for placebo.
11
Table 2: The CARPA manual and history of recommendations for treatment of impetigo in the NT
Edition, date
When to treat?
Treatment Guidelines Notes
1st, 1992
Not stated Povidine iodine topically to clean sores Treat scabies when impetigo resolved Bicillin AP intramuscular
If no improvement in 48–72 hours, flucloxacillin (with probenicid) or erythromycin for 5–10 days
2nd, 1994
Not stated Povidine iodine topically to clean sores Treat scabies when impetigo resolved
Bicillin AP or Bicillin LA intramuscular
3rd, 1997
>6 infected lesions or the lesions appear severe
Povidine iodine topically to clean sores Give advice about scabies treatment
Bicillin LA intramuscular
If no improvement in 48–72 hours, flucloxacillin (with probenicid)
Roxithromycin if penicillin allergic
4th, 2003
>6 infected sores or the sores look severe
Povidine iodine topically to clean sores Treat impetigo and scabies together
Bicillin LA intramuscular Do not use topical mupirocin as resistance develops rapidlyRoxithromycin for 10 days if penicillin
allergic
5th, 2009
“Clearly infected sores”
Clean sores with soap and water, sponge off crusts
Check for scabies and treat together
Benzathine penicillin intramuscular or amoxicillin for 10 days if injection not possible
Do not use topical mupirocin as resistance develops rapidly
Trimethoprim-‐sulphamethoxazole for 5 days if penicillin allergic
Follow up. If not improving, flucloxacillin for 10 days or trimethoprim-‐sulphamethoxazole for 5 days
Due to the pain of the injection, intermittent BPG stock-outs (Currie, 2006) and
uncertainty about the overall number of sores needing treatment, the threshold
12
for treatment in the NT has varied. In the early CARPA editions, no guidance
was provided on when to treat; whereas in the middle editions, more than six
purulent or crusted sores were the treatment threshold, unless the “sores look
severe”. (Table 2) During the East Arnhem Healthy Skin Program (EAHSP,
2004–2006) when the guideline included the six sores threshold for prescribing
treatment, less than 10% of children qualifying for treatment were actually
receiving it. (Andrews et al., 2009) In 2009, the recommendations in CARPA for
treatment changed to “clearly infected sores”, due to concerns that counting the
number of sores was becoming the basis for treatment. (Andrews et al., 2009)
1.8 A RANDOMISED CONTROLLED TRIAL FOR EXTENSIVE IMPETIGO IN INDIGENOUS CHILDREN
Despite some of the highest reported impetigo rates reported in the world in
Indigenous children, (Carapetis et al., 2005) a formal evaluation of the efficacy
of BPG for impetigo had never been conducted in the NT. In addition, the rising
burden of MRSA in skin and soft tissue infections throughout the NT and
worldwide led us to hypothesise that an antibiotic active against MRSA was
needed. The suspicion that the painful needle was a deterrent to receiving
treatment was also important in considering a RCT.
BPG was well established as the standard of care for treatment of impetigo in the
NT (Table 2). A placebo has rarely been used in impetigo studies (Koning et al.,
2012) and it was not considered ethical in the context of high rates of
streptococcal and staphylococcal sequelae seen in children in the NT.
Clindamycin and trimethoprim-sulphamethoxazole were the available, affordable
antibiotics with activity against MRSA. The bitter, unpalatable taste of
clindamycin in syrup formulation, (Steele et al., 2006) resistance rates in
S. aureus at 25% (McDonald et al., 2006) and three times daily dosing, (Group,
2010) meant clindamycin was rejected. Conversely, trimethoprim-
sulphamethoxazole is palatable, has excellent skin penetration, (Krolicki et al.,
2004) resistance rates below 1% (McDonald et al., 2006), had demonstrated
efficacy in a pilot study, (Tong et al., 2010) and is one of the most widely used
antibiotics in the world.
13
Comparing a new treatment to a known standard or active control in a
randomised controlled trial, requires a non-inferiority design seeking to
determine that the new treatment is not worse than the current treatment, by an
acceptable amount. (Piaggio et al., 2012) A robust, measurable outcome is also
required.
1.9 ETHICS
This study was approved by the Human Research Ethics Committee of the
Northern Territory Department of Health and Menzies School of Health
Research (HREC09/08). Ethics approval was current throughout the period of the
study and concluded on 31 December 2013.
1.10 SUMMARY OF INTRODUCTION
Impetigo is common in childhood, with children living in resource-poor contexts
experiencing the highest burden. Despite this, few treatment studies to guide
evidence-based treatment algorithms are available for extensive impetigo.
Evidence for safe, palatable, simple, effective treatment regimens is needed. The
aim of this thesis is to find a better treatment option for impetigo amongst
Indigenous children living in remote communities of Australia, and add to the
limited available evidence for the treatment of extensive impetigo worldwide.
14
CHAPTER 2THE GLOBAL EPIDEMIOLOGY OF
IMPETIGO
2.1 SUMMARY
We conducted a comprehensive, systematic review of the population prevalence
of impetigo and the broader condition pyoderma. PubMed was systematically
searched for impetigo or pyoderma studies published between 1970 and 2014; 66
manuscripts relating to 89 studies met our inclusion criteria. From population-
based surveillance, 82 studies included data on children, giving a total of 145,028
children assessed for pyoderma or impetigo. Median childhood prevalence was
12.3% (IQR 4.2–19.4%). Fifty-eight (65%) of the studies were from low or low-
middle income countries, where median childhood prevalences were 8.4% (IQR
4.2–16.1%) and 14.5% (IQR 8.3–20.9%) respectively. However, the highest
burden was seen in underprivileged children of high-income countries; median
prevalence 19.4%, (IQR 3.9–43.3%). Based on data from studies published since
2000 from low and low-middle income countries, we estimate the global
population of children suffering from impetigo at any one time to be in excess of
162 million, predominantly in tropical, resource-poor contexts. Impetigo is an
under-recognised, neglected tropical disease and in conjunction with scabies,
comprises a major dermatological concern in childhood, with potential lifelong
consequences of untreated infection.
2.2 STATEMENT OF CONTRIBUTION TO JOINTLY AUTHORED WORK
This chapter is in preparation for submission to a peer-reviewed journal and is
authored by myself, Antoine Mahe, Roderick Hay, Ross Andrews, Andrew Steer,
Steven Tong and Jonathan Carapetis. I designed this study based on previous
work by Antoine Mahe, Roderick Hay, Andrew Steer and Jonathan Carapetis, to
15
systematically describe the global burden of impetigo. I wrote the protocol,
conducted the systematic literature review and assessed all papers for inclusion
in the study. Two reviewers, including myself and one of the other co-authors,
who all did a fraction of the reviews, conducted all of the full text reviews. I
synthesised the results, performed the data analysis and have written this chapter.
2.3 INTRODUCTION
Impetigo is a common dermatosis of childhood. Recent estimates of the global
burden of impetigo are in the order of 111 million children from developing
countries (Carapetis et al., 2005) to 140 million (Vos et al., 2012, Hay et al.,
2014) people affected at any one time. However, these estimates were based on a
limited literature review of impetigo in the context of larger studies, have not
been recently updated and acknowledge that impetigo estimates are imprecise
due to the paucity of published literature from the highest prevalence contexts.
This is because impetigo is a disease of poverty, occurring in settings where
systematic collection of accurate disease burden data is a lower priority and most
available data arise from hospital records, which may under-represent the true
population prevalence of skin disease due to selection bias (Hogewoning et al.,
2013, Saw et al., 2001, Lawrence et al., 1979, Bechelli et al., 1981, Gibbs, 1996,
Bissek et al., 2012) and minimal health-care seeking for skin diseases in
resource-poor contexts. (Hay et al., 1994, Behl, 1979, Mahe, 2005) This study
will systematically collate, and hence update understanding of, the population
prevalence of impetigo in children globally from 1970 to 2014. More precise
estimates for impetigo by age group, global region, climate, levels of
urbanisation and development are needed to target intervention studies and
algorithmic management at the primary health care level.
The bacteria Staphylococcus aureus and Streptococcus pyogenes typically cause
superficial bacterial infections of the skin. (Brook et al., 1997) These superficial
skin infections are variously labelled. The term pyoderma is often used to
describe all superficial bacterial skin infections in tropical contexts and is
inclusive of impetigo, ecthyma and furunculosis. (Mahe et al., 2005b) Impetigo
(also known as skin or school sores) is a subset of pyoderma and has both
16
bullous and non-bullous forms. In addition, impetigo is often divided into
primary and secondary impetigo, depending on whether an underlying
dermatological (e.g. eczema) or alternative skin infection (e.g. scabies,
pediculosis or tinea) preceded the bacterial component of the condition. (Mahe,
2005) Primary impetigo is often preceded by minor trauma or insect bites.
(Mahe, 2005) In an attempt to understand the global burden of superficial
bacterial skin infection, this study includes reports on both pyoderma and
impetigo as the major forms of bacterial skin infection under investigation.
Impetigo is more than a nuisance condition. While mortality estimates for
impetigo are low, the frequency of person-to-person transmission in resource-
poor communities maintains a high burden of disease and affects well-being.
(Murgia et al., 2010) In addition, the infectious (staphylococcal and streptococcal
cellulitis, bacteraemia and deep tissue infections) and post-infectious
(glomerulonephritis, rheumatic fever) sequelae are well known and cause high
morbidity (Carapetis et al., 2005, Hoy et al., 2012, Marshall et al., 2011, Skull et
al., 1999) and variable mortality. (Jackson et al., 2011, Tibazarwa et al., 2008)
The relative cost to families of missed school days and procuring treatments that
may or may not work is also high. (Hay et al., 1994, Yamamah et al., 2012)
Skilled diagnosticians are few where the burden is highest. Community health
workers often have poor training in skin disease recognition. (Mahe et al., 2005a)
Few treatment studies have been conducted in high-burden, endemic settings to
guide the development of evidence-based algorithms. (Bowen et al., 2014, van
der Wouden and Koning, 2014)
The aim of this systematic review is to evaluate the prevalence of impetigo from
studies in the general population and to explore variations in the epidemiology of
impetigo. The number of Indigenous Australian children with impetigo in remote
communities will also be estimated.
17
2.4 METHODS
2.4.1 Search Strategy
The systematic review is reported according to PRISMA guidelines. (Moher et
al., 2009) References were identified through searches of PubMed for papers
published in English between January 1970 and September 2014, which reported
population-based studies of skin disorders, with specific reference to impetigo or
pyoderma prevalence. We hand-searched the bibliographies of retrieved papers,
for additional references. Relevant articles published between 1970 and 2005
were identified through searches by AM and RH (Mahe, 2005) and are
synthesised in this manuscript. We used the following search terms ["impetigo"
OR "pyoderma"] AND ["Africa" OR "Asia" OR “Latin America” OR “Pacific”
OR “Oceania” OR “North America” OR “Europe” OR “Russia” OR “China” OR
“India” OR “Developing Country” OR “tropical” OR “Indigenous”]. Duplicates
were removed from the search before titles were reviewed for relevance
(epidemiology, prevalence, impetigo and pyoderma). If insufficient detail was
available in the title, abstracts or entire articles were reviewed to determine
whether the study met pre-determined inclusion criteria.
2.4.2 Selection Criteria
Studies were included if they were population-based, prevalence studies, with
extractable data on children with pyoderma or impetigo. A physical examination
by a clinician was essential for inclusion. Wherever a term was used that inferred
a bacterial skin infection (pyoderma, impetigo or sores) and numerator and
denominator or proportion-affected data were available, these have been
reported. Outpatient dermatology clinic and hospital-based studies from
developing countries are rich sources of data on treatment-seeking behaviours for
skin diseases. However, due to the management of impetigo occurring
predominantly at primary care settings rather than tertiary referral dermatology
clinics, these datasets were excluded. Our aim was to present the available
baseline data derived from prevalence studies performed in the general
population (community or school surveys).
18
2.4.3 Reviewer Assessment
Papers meeting the inclusion criteria were sourced in full-text and data extracted
by two reviewers independently. All papers were assessed by myself. Each of the
other co-authors independently assessed a subset of the studies to confirm
inclusion criteria and extract data. Data fields extracted included date of study,
country, climate, rural/urban environment, study site (e.g. school, household),
study design, sampling method, population, age range, gender of participants,
qualifications of person conducting the screening, case definition, definition of
bacterial skin infection, number of participants, number with impetigo, childhood
and adult prevalence, location of lesions, microbiology and presence of scabies.
2.4.4 Definitions
Where the study date was not reported, the year of publication was used. The
sampling method was defined as exhaustive if ≥85% of available population
were surveyed and non-exhaustive if it was below 85%. Other options for
sampling method included convenience (non-random selection of the available
population), targeted (orphanages or institutions) and random selection of
participants. Details of the definition used for recording a bacterial skin infection
were collected and later categorised as pyoderma (if the text used this term or
indicated that impetigo, folliculitis, ecthyma, furunculosis, cellulitis or tropical
ulcers were included), impetigo (only impetigo or skin sores were studied) and
secondarily infected scabies (the primary focus of the study was scabies with a
secondary focus on bacterial skin infection). We have concentrated on reporting
impetigo prevalence in children. Adult data were also incorporated where
available. If children and adults were included in the study, but separate rates
were not provided, then the community wide prevalence of impetigo was
reported. It was not possible to ascertain from the studies how representative the
study sample was of the broader population.
Countries were categorised into regions according to the United Nations (UN)
Population Division (www.esa.un.org/wpp/excel-Data/country-
Classification.pdf, last accessed 10 November 2014). Childhood was defined as
children aged 0 to 15 years, to calculate population regional and global
19
prevalence. To assess the total population at risk of impetigo based on the
median prevalence estimate, population data from the UN Department of
Economic and Social Affairs Population Division for 2012 were used. These
were available online at www.esa.un.org/unpd/wpp/unpp/panel_indicators.htm,
last accessed 7 December 2014. Each country was also categorised according to
the World Development Index as at July 2005 (www.data.worldbank.org) as high
(>$US10,725 gross national income (GNI) per capita), upper middle ($US3,466–
$10,725 GNI per capita), lower middle ($US876–$3465 GNI per capita) or low
(≤$US 875 GNI per capita) income.
The Koppen Climate Classification System is the most widely used classification
of climates (Peel et al., 2007) and recognises five major climate systems. These
include tropical, arid, temperate, cold and polar. Where data on climate was not
included in the paper, this classification has been used to code the climate by
country and region. (Peel et al., 2007)
2.4.5 Statistical Analysis
The data are synthesized into a narrative summary. Statistical analysis was
performed using Stata13 (Statacorp, Texas, USA). Where prevalence estimates
have been combined to understand regional and global burden of impetigo, the
median prevalence has been used. In order to estimate regional and global burden
of impetigo, the median prevalence has been applied to the 2011 Australian
Census and 2012 United Nations population estimate for less developed
countries. To take into account the variability in study size, the metaregression
command in Stata was used to calculate the pooled prevalence. Impetigo and
pyoderma are both used commonly to report bacterial skin infections. To assess
for any variation in reporting of prevalence based on the chosen term, we
assessed the use of each term, and calculated a statistical difference between the
median prevalence using a chi squared statistic.
20
2.5 RESULTS
Figure 1 summarises the results of the search conducted. Of the 1007 titles
identified, 952 were from database searching and 55 from additional sources.
Two hundred and thirteen duplicate records were removed and 628 papers
excluded, mainly due to an absence of data on impetigo or because the studies
were conducted in dermatology clinics or hospital settings. The 128 remaining
records were subjected to full-text review; 38 papers were excluded with reasons
given in Figure 1 leaving 90 papers, each of which was assessed by two
reviewers. A further 24 were excluded due to insufficient data reported on
impetigo prevalence. The final dataset includes 66 papers reporting on 89 studies
(Figure 1, Table 1, Appendix 1) conducted over a 45-year period.
The studies were predominantly from Africa (30/89, 34%), Asia (20/89, 22%)
and Oceania (19/89, 21%) and represented populations from 31 countries (Table
1, Figure 2). Studies with data available by decade were: 1970s (28, 32%), 1980s
(15, 17%), 1990s (20, 23%), 2000s (23, 26%) and since 2010 (3, 3%).
Data on impetigo prevalence were available for 174,508 individuals of whom
145,028 were children. The study size varied, ranging from 31 to 19,775
participants per study. The median study size was 636 participants (inter-quartile
range [IQR] 305–1817), median prevalence 11.2% (IQR 4.2–19.4%) and pooled
prevalence 15.5% (95% CI 12.1–19.0%) (Table 3). Extractable prevalence data
were available on children in 82 (92%) of the studies. The median impetigo
prevalence in children was 12.3%, (IQR 4.2–19.3%) and pooled prevalence
16.6% (95% CI 12.7–20.5%). The median number of children in each study was
534 (IQR 258–1,729).
21
Figure 1: Flowchart of systematic review according to PRISMA statement
952 Records identified through database searching
55 additional records identified through other sources
794 records after duplicates removed
166 records screened 38 records excluded
128 full-‐text articles assessed for eligibility
38 full-‐text articles excluded Not in English (n=6) Not a community
prevalence study (n=14) Review paper (n=1)
Unable to access full text (n=6)
Microbiology study (n=2) No impetigo prevalence
data (n=9)
90 papers assessed by 2 reviewers for inclusion
66 papers (89 studies) included in quantitative
synthesis
24 papers excluded by 2 reviewers
Data quality insufficient for reporting prevalence data
(n=24)
Scre
enin
g Id
entif
icat
ion
Elig
ibili
ty
Incl
uded
22
Table 1: Number of studies of impetigo prevalence by decade, country and region.
Decade Number of
studies
available
Countries Regions
1970 -‐ 1979 28 Colombia, Ghana, Tanzania,
New Zealand, Brazil, India,
USA, Gambia, Panama
Latin America &
Caribbean, Africa, Asia,
Oceania, North America
1980 -‐ 1989 15 Pakistan, Solomon Islands,
Nigeria, Ethiopia, Vanuatu,
India, Fiji, Canada
Asia, Oceania, Africa,
North America
1990 -‐ 1999 20 Australia, Honduras, Mali,
Malaysia, Ethiopia, Tanzania,
Ecuador, Samoa, Taiwan,
Kenya, Solomon Islands
Oceania, Latin America
& Caribbean, Africa,
Asia
2000 -‐ 2009 23 Nepal, Australia, India, Fiji,
Tanzania, Nigeria, Timor Leste,
Turkey, Mali, Ghana, Gabon,
Rwanda, Egypt
Asia, Oceania, Africa
2010 -‐ 2014 3* Ethiopia, Cameroon, Tanzania Africa
* Two studies were published in 2010 and did not provide a year of data collection in themanuscript.
23
Table 2: Summary statistics of available studies by age grouping Total available
population
(n=89 studies)
Childhood
population
(n=82 studies)
Adult population
(n=11 studies)
Median (IQR)
prevalence of
impetigo
11.2%
(4.2–19.4%)
12.3%
(4.2–19.4%)
4.9%
(3.1–9.6%)
Pooled prevalence
(95% CI)
15.5%
(12.1–19.0%)
16.6%
(12.7–20.5%)
9.7%
(2.2– 7.2%)
Median (IQR)
number of
participants per
study
636
(305–1,817)
534
(258–1,729)
638
(264–1,645)
Population with
impetigo
23,759 19,811 2,427
Total population
studied
(denominator)
174,508 145,028 18,246
24
Figure 2: Map of countries with available population-based studies of impetigo prevalence, highlighted and colour coded by median prevalence of impetigo.
Coding of median prevalence 0–5% blue, 5.1–10% green, 10.1–15% yellow, 15.1–20% orange and >20% red. Source: Map from powerpointworldmaps.com (accessed 14.10.2014).
2.5.1 Impetigo prevalence in children by region
Reported impetigo prevalence in populations under investigation ranged from
0.2% to 90%. The highest median prevalence of childhood impetigo was
reported from Oceania, where from 19 studies, the median prevalence was 40.2%
(IQR 17.2–48.1%). Excluding studies from Australia (see box) and New
Zealand, of the eight remaining studies from Oceania, the median prevalence
remained high at 29.7% (IQR 14.7–42.0%). The median impetigo prevalence in
Africa was 7% (IQR 4.1–12.3%), Asia 7.3% (IQR 3–16.1%), resource poor
populations in North America 13.3% (IQR 2.1–19.4%) and Latin America and
the Caribbean 15.5% (IQR 12.2–20.8%). There was no data available for Europe
or China.
25
Box: Applying prevalence estimates for impetigo to remote living Australian
Indigenous children
There were 10 population prevalence studies available for Australia. All
represented data from children living in remote Indigenous communities of
northern Australia, with no studies available for non-Indigenous children. The
median prevalence reported from these studies was 44.5% (IQR 34.0–49.2%).
Four studies were conducted since 2000, with a median prevalence of 43.0%
(IQR 40.2–45.7%). We estimated the total number of remote Indigenous children
with impetigo at any one time by applying the median prevalence of 44.5% to the
remote living Indigenous population from the states of Western Australia,
Queensland and Northern Territory aged less than 15 years in the 2011
Australian Census (35,272). We estimate 15,696 Indigenous children are
suffering from impetigo at any one time. This is the first time that prevalence
estimates have been used to generate a total number at risk amongst Australian
Indigenous children. This will be important for local health care planning.
2.5.2 Burden of impetigo in low and low-middle income countries
The United Nations Department of Economic and Social Affairs population
division estimated the global population below 15 years of age resident in less
developed countries in the years 2000–2009 to be at least 1.6 billion children.
Utilising the median population prevalence of 9.9%, (IQR 4.1–16.6%) from the
studies conducted in developing economies (WDI2005 index of lower-middle or
low) since 2000, the estimated population at risk of impetigo at any one time is
more than 162 million children. Excluding China, where no studies were
available, reduces the estimate to 137 million children with impetigo in low and
26
low-middle income countries. Table 3 outlines the regional estimates of children
with impetigo at any one time using the available data.
Table 3: Estimates of children with impetigo by regions of the world with available data*
Region Population in 2012 under 15 years
Median impetigo prevalence in
children
Estimated number of children with
impetigo
Africa 424,072,000 7%
(IQR 4.1–12.3%)
29,685,040
Asia 1,060,076,000 7.3%
(IQR 3–16.1%)
77,385,548
Oceania* 3,653,000 29.7%
(IQR 14.7–42.0%)
1,084,941
Latin American & Caribbean
167,654,000 15.5%
(IQR 12.2–20.8%)
25,986,370
*Studies from Australia, New Zealand and North America excluded as all these studies were
conducted in small, impoverished populations within these countries that may not reflect the
overall burden of impetigo for the childhood population.
2.5.3 Childhood population prevalence by age group
Only 13 (15%) studies reported prevalence of impetigo according to age group.
Of those that did, the median prevalence in 0–4 year olds studied was 19% (IQR
15–31%), 5–9 year olds 19% (IQR 12–43%) and 10–14 year olds 10%
(IQR 7–28%).
2.5.4 Childhood impetigo and World Development Index
The majority of studies, 58/89 (65%) (Table 4), were from low or low-middle
income countries. The remainder were from middle income or resource-poor
populations within high-income countries. Table 5 summarises the median
prevalence estimates according to income level of the country, with the highest
27
estimates coming from underprivileged populations within high-income
countries.
Table 4: Classification of studies by region and World Bank Development Indicator in 2005
Region High income Upper Middle income
Low Middle Income
Low Income
Oceania Australia (10) New Zealand (1)
Fiji* (4) Vanuatu (1) Samoa (1)
Solomon Islands* (2)
Africa Gabon (1) Egypt* (2) Ghana* (3) Rwanda (1)
Cameroon* (1) Ethiopia (3) Nigeria* (2) Tanzania (9) Kenya (3) Mali (3)
The Gambia (2) Asia Taiwan (1) Malaysia* (2)
Turkey* (1) India* (12) Nepal (2)
Pakistan* (1) Timor-‐Leste* (1)
Caribbean & Latin
America
Panama* (2) Honduras* (1) Brazil (2)*
Colombia* (1) Ecuador*(1)
North America
Canada (6) USA (7)
TOTAL 25 6 13 45
*Category has shifted rather than remaining stable within period from 1987–2013. Source:
data.worldbank.org/data-catalog/world-development-indicators, accessed 12.11.2014.
28
Table 5: Median prevalence of impetigo in childhood and overall, categorised by the World Development Index. WDI and number of
studies
Median childhood
prevalence (IQR)
N= 82
Median overall prevalence
(IQR)
N=89
High income
(N= 25)
19.4%
(IQR 3.9–43.3%)
19.4%
(IQR 3.9–43.3%)
Middle income
(n=4 [childhood] OR
n=6 [overall])
9.9%
(1.8–18.6%)
9.9%
(IQR 2–15.1%)
Low-‐Middle
(N=12)
14.5%
(IQR 8.3–20.9%)
12.8%
(7.8–18.3%)
Low
(n= 41 [childhood] OR
n= 46 [overall])
8.4%
(IQR 4.2–16.1%)
7.9%
(4.3–16.1%)
2.5.5 Scabies in association with impetigo
Fifty-seven (64%) studies also reported data on the prevalence of scabies. The
median prevalence of scabies from all included studies was 3.3% (IQR 0.7–
12.9%). Twenty-seven (87%) countries had available data on scabies prevalence.
Scabies prevalence varied by region, with the highest median prevalence found
in Oceania (n=14) at 16% (IQR 4.9–25%). The median scabies prevalence in
Africa (n=28) was 2% (IQR 0.7–7%) and in Asia (n=11) was 3.4% (IQR 0.9–
11.9%). The median prevalence of scabies in studies from Latin America and the
Caribbean (n=3) was 3% (IQR 0.6–10%). Scabies prevalence also varied by
decade. In the 1970s (n=12), the median was 3.2% (IQR 1.4–13%), 1980s (n=8)
1.1% (IQR 0.4–1.8%), 1990s (n=14) 9.2% (IQR 4.9–17%), 2000s (n=20) 1.9%
(IQR 0.6–15.1%) and since 2010 (n=3) 1.4% (IQR 0.3–1.8%).
29
2.5.6 Microbiology of impetigo
Thirty-four (38%) of the studies also reported culture-based microbiology results,
predominantly on streptococcal infection (n=31). Only 11/89 (12%) studies
reported on the relative contributions of S. pyogenes and S. aureus from
microbiological culture of skin lesions. S. pyogenes was identified in a median of
74% (IQR 57–95%) of cultures and S. aureus in a median of 64% (IQR 53–80%)
of cultures.
2.5.7 Body distribution of impetigo
Data were reported on body distribution of impetigo in 23 studies. Of these,
21/23 (91%) reported the lower limbs as being the most common site. In 11
studies, the proportion of body regions affected was given. These were re-
classified as lower limbs, upper limbs and other [scalp, face, neck, torso]. The
respective medians for body region distributions (these were not mutually
exclusive) were lower limbs 58% (IQR 44–86%), upper limbs 18% (IQR 14–
54%) and other 38% (IQR 5–43%).
2.5.8 Impetigo in rural versus urban settings
Studies were classified broadly as rural (n=61), urban (n=15) or both (n=13).
Table 6 shows the variation in prevalence when the data were examined by this
factor, with a higher prevalence of impetigo reported from rural locations
compared to urban settings.
30
Table 6: Variability in median impetigo prevalence by urban and rural study locations
Median impetigo prevalence overall
Median impetigo prevalence in children
Rural N=61 studies N=55 studies
13.3%
(6.7–20.9%)
16.1%
(5.9–22.6%)
Urban N=15 studies N=14 studies
4.8%
(2–10%)
4.5%
(2–7.3%)
Both N=13 studies N=13 studies
5.8%
(2.4–13.3%)
5.8%
(2.4–13.3%)
2.5.9 Childhood impetigo by climatic region
Many studies included details on the climatic conditions of the study region. If
not available, these were coded using the Koppen classification (Peel et al.,
2007). The majority of studies were from tropical environments, 67/89 (75%).
The remaining studies were from cold 9/89 (10%), temperate 9/89 (10%) and
arid 4/89 (5%) climates. Median impetigo prevalence in childhood was 12.2%
(IQR 4.8–20.8%), 17.5% (IQR 8.2–42.8%), 15.8% (IQR 2.2–30.2%) and 3.9%
(IQR 1–13.3%) for tropical, arid, temperate and cold climates respectively.
2.5.10 Variability in sampling technique
There was variability in the sampling techniques employed. The most common
technique described was exhaustive sampling. Of the exhaustive population
surveillance studies (n=57/89, 64%), the median number of participants was 636
(IQR 305–2528) and median prevalence 10.8% (IQR 4.3–19.4%). The median
prevalence was 4% (IQR 2–16.6%) in the 7 studies where random sampling was
employed.
31
2.5.11 Impetigo or pyoderma: is the reporting consistent?
Twenty-six (29%) studies reported on impetigo or skin sores as the primary
definition under investigation. Of these, 25 have data available on the prevalence
of impetigo in children with the median prevalence of 13.3% (IQR 2.3–22.6%).
Pyoderma was reported in 62 (70%) studies with a median prevalence of 11.8%
(IQR 5.1–19%). There was no statistical difference in the median prevalence
reported based on these definitions, p=0.85. Both definitions were used
throughout all decades of the study. There was some variability in the use of
definition by region: studies from Africa, Asia and Latin America predominantly
reported on pyoderma, whereas studies from Oceania used either definition and
North American studies were more likely to report on impetigo (Table 7).
Table 7: Regional variation in the use of pyoderma or impetigo to describe bacterial skin infections
Region Pyoderma reported(%) Impetigo reported(%)
Africa (n=30) 27 (90%) 3 (10%)
Asia (n=18) 15 (79%) 4 (21%)
Oceania (n=19) 10 (53%) 9 (47%)
North America (n=13) 3/13 (23%) 10/13 (77%)
Latin America & Caribbean (n=7)
7/7 (100%) 0
2.6 DISCUSSION
This systematic review provides comprehensive data and confirms an ongoing,
high burden of impetigo in childhood, estimating that more than 162 million
children in low and low-middle income countries are affected at any one time.
Our study revises upwards the estimate of children impacted by impetigo at any
one time, using the same methodology but incorporating more studies, compared
to the previous estimate of 111 million children. (Carapetis et al., 2005) The
32
reported burden has remained high throughout the study period, with a median
prevalence of 12.3% (IQR 4.2–19.3%). Our estimate derived from 89 studies
over 45 years is higher than previously published estimates, which were between
5 and 10%. (Mahe, 2005) Impetigo is more than a benign, nuisance conditions
and these numbers demonstrate the priority concern impetigo is for public health.
Each decade since 1970 has seen 15–20 new studies published in the peer-
reviewed literature. Our search strategy represents a greater depth of coverage
and enriches our understanding of the global burden of impetigo. The reported
data is derived from prevalence studies performed in the general population, at
the community or school level, and provides a more complete understanding of
the global burden of impetigo than previous estimates. Each study is valuable in
describing the burden of disease for a local or regional population, but
collectively they tell a far more compelling story of an under-appreciated disease.
These studies are predominantly reported from contexts of poverty or tropical
regions.
The data include large regions of the globe over a 45-year interval, suggesting
the burden of impetigo remains relatively unchanged over this period. Only three
studies were available for the current decade, of which two were published in
2010 (Komba and Mgonda, 2010, Murgia et al., 2010) and likely represent data
collected during the previous decade. The remaining study reports data from
March 2010. (Bissek et al., 2012) As such, the most precise, recent estimates are
from the decade ending in 2009 and have shown no change in the global burden
of impetigo. In view of this, we combined the prevalence estimates from all
studies published since 2000 in order to estimate the current burden of impetigo.
The data describe a context where progress against control of impetigo appears to
be lacking. Including bacterial skin infections in the list of neglected tropical
diseases may be one option to refocus attention on the need for this to be a
priority target. In addition, the burden experienced by impoverished populations
within wealthy countries is high. This is in keeping with version 2.0 of the
neglected tropical diseases agenda that utilises ‘blue marble health’ as a phrase to
describe the importance of poverty in all countries as a key underlying factor of
neglected tropical diseases. (Hotez, 2013) The widespread burden of impetigo
33
has remained unchanged over more than four decades. By highlighting the global
burden, which has previously been estimated at >2% of the global population at
any one time, (Hay et al., 2014) an agenda for screening, treatment and further
work can crystallise. This will inform primary prevention of kidney and heart
disease and gram-positive bacterial sepsis in resource-poor contexts.
A limitation of this systematic combination of datasets is the variability in
clinical skills of those performing cutaneous disease surveillance. Studies
reported using expert dermatologists, dermatologists in training, paediatricians,
general medical officers, nursing staff and community health workers trained in
identification of skin conditions, to screen populations. This variability may have
resulted in under-reporting of impetigo. In addition, many of the studies were
focused on a specific dermatosis e.g., scabies or pediculosis, or conducted in the
context of nutrition or child health surveys. Reporting of bacterial infection was a
secondary endpoint. Surveys of skin disease were conducted for a variety of
reasons, including to demonstrate the substantial burden of disease and
significant unmet need. (Walker et al., 2008) Guidelines for skin surveillance
studies have recently been developed. (Kotloff, 2008) Only one study to date
reported using these in the study design, (Steer et al., 2009) but the availability of
these guidelines is likely to inform future surveillance.
The definitions and clustering of conditions used in the studies was variable, but
using the two dominant terms of impetigo and pyoderma, there was no statistical
difference in the median prevalence. This suggests that our inclusion of both
terms as a descriptor of bacterial skin infections is robust.
Our study broadens our knowledge of the global distribution of impetigo, by
including studies from countries that were not available in previous disease
burden estimates. (Carapetis et al., 2005, Vos et al., 2012) An improved
understanding of the global burden may also help to prioritise treatment studies
in contexts with the highest burden of impetigo. The Cochrane review on the
optimal treatment of impetigo (Koning et al., 2012) includes studies
predominantly from high-income countries and references only one study (out of
68) conducted in a similar setting to those reported in our study. Similarly,
Karimkhani et al. report bacterial skin infections are under-represented in
34
Cochrane systematic reviews when matched with corresponding disability-
adjusted life years. (Karimkhani et al., 2014)
Initially, we hoped to define the burden of impetigo in developing countries by
including studies from low and low-middle income countries using the World
Bank Development Index. However, very few studies were excluded on this
basis, and the study was broadened to include all countries. It is possible that
studies have been conducted in the regions of highest disease burden leading to
an over-estimate of the global burden. (Mahe, 2005) We think this is unlikely,
given the size, consistency and duration of the estimated burden of impetigo that
we have described. However, population-based prevalence studies from Europe,
East and South-East Asia, and more recently North America, are under-
represented. No studies were available in English from the low or low-middle
income countries of Eastern Europe, and this is a significant gap in our global
understanding of the burden of impetigo. Recently, the burden of post-
streptococcal sequelae in these countries has been reported and is amongst the
highest in the world, (Tibazarwa et al., 2008) suggesting that impetigo may also
be common here. Despite these gaps, our findings are consistent with the 2010
global burden of disease estimates for impetigo. (Vos et al., 2012, Hay et al.,
2014)
This study is limited by the exclusion of unpublished data or grey literature
searching. This limitation is more likely to restrict the analysis to developing
countries; none of the three studies from developed countries which have been
included in the global burden of skin diseases (Hay et al., 2014) were found
using the search methods specified, as these studies were all health-care based.
Overall, few studies were identified from high-income countries. It is possible
that the implementation of a skin disease population prevalence study is because
of a pre-specified expectation of high rates of disease. The absence of data may
represent lower disease burden, or may just be an unstudied population. In
addition, hospital or health care seeking studies were not included.
Our study confirms that the greatest burden of impetigo is in children, with
steady decreases in prevalence with increasing age. (Mahe, 2005, Belcher et al.,
1977) Despite our study reflecting predominantly impoverished settings,
35
increases in impetigo have also been reported in children in developed countries
attending general practices for care (Shallcross et al., 2013) and it causes a large
volume of health care consultations in all regions of the world. (Mohammedamin
et al., 2006, Razmjou et al., 2009, Koning et al., 2006) Our summary of data has
consistently shown that impetigo predominantly affects the lower limbs in
children in resource poor contexts and that both S. pyogenes and S. aureus are
present in swabbed lesions.
Within the 89 studies, there was variability in the prevalence of impetigo
reported. Tropical climate, insect bites, scabies, poverty, limited access to water
and household overcrowding are all reported factors contributing to the
development of impetigo (Currie and Carapetis, 2000) but data on these was not
systematically available in the 89 studies. We looked at the burden of impetigo
by age group, level of urbanisation and broad climatic region. The individual
studies reported prevalence across a range of additional contexts including
ethnicity, gender, disability and socio-economic status but these were outside the
scope of our review and it is unlikely they would be amenable to systematic
analysis given the small numbers of studies addressing sub-groups.
2.7 CONCLUSIONS
Prevalence throughout the study period was highest in Oceania in both resource-
poor countries, and underprivileged populations within high-income countries.
This synthesis of studies is important for regional and national targeting of
healthy skin interventions, as the burden of disease is large. At a global level, this
study revises our estimate upwards of the number of children affected with
impetigo at any one time from 111 million (Carapetis et al., 2005) to 162 million;
this finding alone should drive a comprehensive research agenda for the
detection, treatment and prevention of impetigo in resource-poor contexts. It is
also important to appreciate that as antibiotics are the backbone of current
treatment for impetigo, this disease burden may contribute to burgeoning
antibiotic resistance in the absence of evidence-based treatment algorithms.
(Tong et al., 2014) This combination of high prevalence and moderate morbidity
makes impetigo a high population health priority
36
CHAPTER 3IS STREPTOCOCCUS PYOGENES
SUSCEPTIBLE TO TRIMETHOPRIM-SULPHAMETHOXAZOLE?
3.1 CHAPTER OVERVIEW
This chapter challenges the conventional wisdom that Streptococcus pyogenes is
not susceptible to trimethoprim/sulphamethoxazole (SXT). Historically, in vitro
antibiotic susceptibility testing was confounded by the presence of thymidine, an
inhibitor of SXT. As such, S. pyogenes appeared resistant to SXT in many earlier
assays. The long standing susceptibility of S. pyogenes to penicillin obviated the
need for an alternative antibiotic for treatment of S. pyogenes infections, and
exploration of the in vivo efficacy of SXT was deemed unnecessary or not
considered.
S. pyogenes is a key pathogen in impetigo and is often found in conjunction with
Staphylococcus aureus. The rising prevalence of methicillin-resistant S. aureus
(MRSA) in remote communities of northern Australia (Tong et al., 2008) was
concerning. Both hospital (Tong et al., 2009) and community (McDonald et al.,
2006) studies identified MRSA rates above 20%. As such, treatment with
antibiotics effective against both pathogens may be required and the Skin Sore
Trial was designed to address this question. When the Skin Sore Trial was
designed, SXT susceptibility in community S. aureus strains was 100%.
(McDonald et al., 2006) Susceptibility of S. pyogenes was similarly high in
unpublished laboratory work.
The work in this chapter demonstrates the in vitro susceptibility of S. pyogenes to
SXT. It was a necessary step in paving the way for our understanding of the
results of the Skin Sore Trial. In addition, it alerts the global community dealing
with skin and soft tissue infections where both S. pyogenes and MRSA are
37
implicated, that a simple, oral antibiotic regimen may be effective depending on
the local antibiogram.
3.2 STATEMENT OF CONTRIBUTION TO JOINTLY AUTHORED WORK
I designed this study based on preliminary work done by Peter Ward and
Malcolm McDonald during the process of achieving funding for this trial. The
preliminary work was included as Study 1 in this paper; I then extended that
work in Study 2. I wrote the standard operating procedures (SOP) for the conduct
of Study 2 and incorporated the recently released European Committee on
Antibiotic Susceptibility testing (EUCAST) breakpoints for S. pyogenes and
SXT. Whilst I planned to assist with the laboratory work, delay in the arrival of
supplies meant that Rachael Lilliebridge conducted the susceptibility testing,
while I was on maternity leave. I oversaw the microbiology, cleaned the data and
analysed the results with the assistance of Steven Tong. I conducted the literature
review and wrote all drafts of the manuscript. All co-authors contributed to
revisions of the manuscript and approved the final version for publication.
3.3 JOURNAL ARTICLE (FOLLOWING PAGE)
Is Streptococcus pyogenes resistant or susceptible to trimethoprim-sulfamethoxazole?
APPENDIX 2
Gelfand MS, Cleveland KO, Ketterer DC. Susceptibility of Streptococcus
pyogenes to trimethoprim-sulfamethoxazole. Journal of Clinical Microbiology.
2013; 51(4): 1350.
38
Bowen AC, Tong SY, Carapetis JR. Reply to “Susceptibility of Streptococcus
pyogenes to trimethoprim-sulfamethoxazole.” Journal of Clinical Microbiology.
2013; 51(4): 1351.
39
Is Streptococcus pyogenes Resistant or Susceptible to Trimethoprim-Sulfamethoxazole?
Asha C. Bowen,a,b Rachael A. Lilliebridge,a Steven Y. C. Tong,a,b Robert W. Baird,b Peter Ward,d Malcolm I. McDonald,c
Bart J. Currie,a,b and Jonathan R. Carapetisa,e
Menzies School of Health Research, Charles Darwin University, Darwin, Northern Territory, Australiaa; Royal Darwin Hospital, Darwin, Northern Territory, Australiab; JamesCook University, Cairns, Queensland, Australiac; Microbiology Department, Austin Pathology, Austin Health, Victoria, Australiad; and Telethon Institute for Child HealthResearch, Centre for Child Health Research, University of Western Australia, Perth, Western Australiae
Streptococcus pyogenes is commonly believed to be resistant to trimethoprim-sulfamethoxazole (SXT), resulting in reservationsabout using SXT for skin and soft tissue infections (SSTI) where S. pyogenes is involved. S. pyogenes’ in vitro susceptibility toSXT depends on the medium’s thymidine content. Thymidine allows S. pyogenes to bypass the sulfur-mediated inhibition offolate metabolism and, historically, has resulted in apparently reduced susceptibility of S. pyogenes to sulfur antibacterials. Thelow thymidine concentration in Mueller-Hinton agar (MHA) is now regulated. We explored S. pyogenes susceptibility to SXT onvarious media. Using two sets of 100 clinical S. pyogenes isolates, we tested for susceptibility using SXT Etests on MHA contain-ing defibrinated horse blood and 20 mg/liter �-NAD (MHF), MHA with sheep blood (MHS), MHA alone, MHA with horse blood(MHBA), and MHA with lysed horse blood (MHLHBA). European Committee on Antibacterial Susceptibility Testing (EUCAST)breakpoints defined susceptibility (MIC, <1 mg/liter) and resistance (MIC, >2 mg/liter). In study 1, 99% of S. pyogenes isolateswere susceptible to SXT on MHA, MHBA, and MHLHBA, with geometric mean MICs of 0.04, 0.04, and 0.05 mg/liter, respec-tively. In study 2, all 100 S. pyogenes isolates were susceptible to SXT on MHF, MHS, MHA, and MHLHBA with geometric meanMICs of 0.07, 0.16, 0.07, and 0.09 mg/liter, respectively. This study confirms the in vitro susceptibility of S. pyogenes to SXT, pro-viding support for the use of SXT for SSTIs. A clinical trial using SXT for impetigo is ongoing.
Streptococcus pyogenes was one of the first bacterial infections tobe treated with sulfur antibacterials in the 1930s (16) and
proved to be clinically effective in the treatment and prophylaxisof S. pyogenes infections (10, 16, 23, 29). However, when sulfadi-azine, an early short-acting sulfur antibacterial, was used in massprophylaxis programs to prevent S. pyogenes tonsillitis and acuterheumatic fever (ARF) in military recruits in the 1940s, the clinicalefficacy of this antibacterial was limited due to the presumed de-velopment of resistance (13, 15, 27, 31, 42) among some strains.Initial antibacterial susceptibility testing (AST) of S. pyogenes tosulfur antibacterials using a broth dilution method demonstratedthat some strains were resistant (25, 53); however, AST was in itsinfancy and no standardized reference methods existed at thattime. This early experience resulted in the belief that trim-ethoprim-sulfamethoxazole (SXT) is ineffective against S. pyo-genes, and its use has been discouraged in clinical practice fordecades (35).
Subsequent antibacterial susceptibility experiments showedapparently reduced susceptibility of S. pyogenes (and other bacte-ria) (6, 40) to the sulfur antibacterials due to antagonism of theinhibition of folate metabolism. Harper and Cawston discoveredan inhibitory substance in 1945, eventually identified as thymi-dine, which was interfering with the ability of sulfur antibacterialsto kill the organism (25). Because the activity of SXT is determinedby the antibacterial’s ability to deprive an organism of folate co-enzymes (7), there is a direct relationship between the thymidinelevels in culture media and SXT resistance (11). High thymidinecontent in agar provides an exogenous substrate which can beused by an organism to maintain folate metabolism and henceappear resistant to SXT. In early studies, most culture media con-tained sufficient thymidine to antagonize the inhibitory effects of
sulfur drugs and hence produced resistant results when this classof antibacterials was tested (6).
Notably, lysed horse blood was found to contain the enzymethymidine phosphorylase, which neutralized thymidine (46) andovercame this effect (21, 25). No other mammalian blood con-tains thymidine phosphorylase (21). However, the addition oflysed horse blood was not recommended for AST, despite severalauthors (5, 6, 21, 54) advising supplementation with lysed horseblood for any medium used to test sulfur antibacterial susceptibil-ity if the thymidine concentration was above 0.03 �g/ml (5) (be-low which inhibition does not occur). In this context, the notionthat S. pyogenes was resistant to sulfur antibacterials perpetuated.
SXT was introduced in 1968 (3, 28, 43) and has since becomeone of the most widely used antibacterials in the world. However,recommendations against the use of sulfur antibacterials, includ-ing SXT, for S. pyogenes infections continue in the belief that theorganism is intrinsically resistant (33, 47, 51). Two studies (33, 51)have reported S. pyogenes uniformly resistant to SXT, but this wasprior to thymidine content standardization in Mueller-Hintonagar (MHA) and was on agar containing sheep blood. There havealso been reported clinical failures in the use of this agent in erad-icating S. pyogenes from nasopharyngeal carriage (30). However,other centers have demonstrated full in vitro susceptibility of S.
Received 19 August 2012 Returned for modification 10 September 2012Accepted 4 October 2012
Published ahead of print 10 October 2012
Address correspondence to Asha C. Bowen, [email protected].
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.02195-12
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pyogenes to SXT (14, 22, 36, 54). Since 2006, when the thymidinecontent of MHA became strictly regulated by the Clinical andLaboratory Standards Institute (CLSI) to maintain a low level ofthymidine and hence avoid inhibition (M6-A2 protocol) (9), ithas no longer been necessary to add lysed horse blood to the me-dium for AST. However, current methods do use agar supple-mented with mammalian blood.
Given the prevailing view that caution should be exercised inusing SXT for infections involving S. pyogenes and the paucity ofclinical data of SXT efficacy against S. pyogenes, we sought to con-firm or disprove the notion that S. pyogenes is resistant to SXT invitro on various antibacterial susceptibility testing media. Coin-fection of S. pyogenes with Staphylococcus aureus in skin and softtissue infections (SSTI) and the rising prevalence of methicillin-resistant S. aureus (MRSA) provide added stimulus to explore theutility of SXT in the treatment of these infections.
MATERIALS AND METHODSSwab collection and identification of S. pyogenes. Skin, throat, or noseswabs were collected using a rayon tipped cotton swab (Copan, InterpathServices, Melbourne, Australia). In study 1, swabs were plated on horseblood agar (HBA) (Oxoid, Basingstoke, United Kingdom) and HBA con-taining colistin and nalidixic acid (HBA � CNA) (Oxoid) within 48 h ofcollection. In study 2, swabs were stored in skim milk-tryptone-glucose-glycerol broth (STGGB) at �70°C prior to plating on the above-describedmedia. Incubation was at 37°C for 16 h in 5% CO2. �-Hemolytic colonieswere identified morphologically, and confirmation of S. pyogenes was withthe Lancefield streptococcal grouping test for group A (Oxoid). Isolateswere stored in glycerol at �70°C until subsequent replating for AST.
Selection of isolates. (i) Study 1. We began by exploring the issue witha preliminary study of 100 skin and throat isolates of S. pyogenes collectedfrom 3 remote Australian Aboriginal communities between 2003 and2005 during surveillance studies (39). We used an SXT Etest strip (bio-Mérieux, France) on 3 different agars: MHA, MHA supplemented withhorse blood (MHBA), and MHA supplemented with lysed horse blood(MHLHBA) (Oxoid). Interpretation was based on European Committeeon Antibacterial Susceptibility Testing (EUCAST) breakpoints (20) re-leased in 2010.
(ii) Study 2. The Skin Sore Trial is a randomized, controlled trial(RCT) comparing benzathine penicillin G (BPG) treatment of impetigo(the standard of care) with oral SXT. The first 100 S. pyogenes isolates fromskin and nasal swabs from participants in the Skin Sore Trial were used instudy 2. The participants were 3 months to 13 years old and were from 4remote Aboriginal communities of the Top End of the Northern Terri-tory, Australia, recruited in 2010. Swabs were collected from the anteriornares and at least 2 purulent or crusted sores from all children. Swabs werecollected from skin sores on day 0 (pretreatment), day 2 (midtreatment),and day 7 (completion of treatment). Consent for participation and col-lection of specimens was obtained from the guardian or parent of eachparticipant. The study was approved by the Northern Territory Top EndHuman Research Ethics Committee (HREC 09/08) and has been regis-tered with the Australian and New Zealand Clinical Trials Registry(ACTRN 12609000858291).
Antibacterial susceptibility testing methods and agar used. The twointernationally accredited, standardized methods for AST (Table 1) arethe CLSI (8) and EUCAST (19) methods. CLSI does not provide referencebreakpoints for S. pyogenes susceptibility to SXT and recommends the useof Mueller-Hinton agar (MHA) supplemented with 5% sheep blood fortesting the susceptibility of S. pyogenes to other antibacterials. In contrast,EUCAST has released breakpoints for the disk diffusion method and MICon appropriate media. The medium recommended for Streptococcusgroups A, B, C, and G is MHA supplemented with 5% defibrinated horseblood � 20 mg/liter �-NAD. This agar is commonly referred to as MHF(http://www.eucast.org, accessed 25 July 2012).
Two experimental agars were also used for susceptibility testing. MHAis routinely used in a number of other antibacterial susceptibility tests notinvolving �-hemolytic streptococci. MHLHBA was used to explore thehypothesis based on historical literature that the lysing of horse bloodreleases thymidine phosphorylase which breaks thymidine down to thy-mine.
Using the EUCAST breakpoints (20), with an MIC of �1 mg/liter assensitive and an MIC of �2 mg/liter as resistant, and an SXT Etest toperform antibacterial susceptibility, these studies compared the variousmedia that have been recommended for AST. The Etest interpretation isbased on the trimethoprim component of the trimethoprim-sulfame-thoxazole combination, in a ratio of 1:19. The EUCAST methodologyusing the Etest was chosen due to the availability of published breakpoints;however, due to the widespread use of CLSI, the medium upon which thisorganism is tested was also used and results obtained were referenced tothe EUCAST breakpoints.
Antibacterial susceptibility testing. Single colonies were isolatedfrom frozen stocks following overnight incubation on HBA in 5% CO2 at37°C. Susceptibility testing for SXT was performed using a 0.5 McFarlandsuspension to create a confluent lawn inoculum and then applying an SXTEtest as per the manufacturer’s instructions. Plates were read by 2 readersfollowing incubation in 5% CO2 at 35°C for 16 to 20 h. The MIC wasrecorded where the inhibition ellipse intersected the scale. Where a differ-ence in results of more than 2 gradations was noted between the 2 readers,a repeat test was performed with a fresh subculture of S. pyogenes. As SXTis a bacteriostatic antibacterial, this mode of action can alter the appear-ance of an MIC endpoint, resulting in hazy zones. Where haze was pres-ent, both the 80% and 100% points of ellipse intersection were recorded.A penicillin, erythromycin, and clindamycin disk diffusion test accordingto CLSI guidelines (8) was conducted concurrently on all 100 strains instudy 2.
Quality control. In study 2, for every 20 S. pyogenes clinical isolates, acontrol strain of S. pyogenes (ATCC 19615) was also tested on the 4 agarsand found to be susceptible. Quality control of the SXT Etests was per-formed throughout the study using Escherichia coli (ATCC 25922) onMHA and Streptococcus pneumoniae (ATCC 49619) on MHS. The refer-ence ranges for each organism were achieved, namely, 0.064 to 0.25 �g/mlfor E. coli and 0.125 to 1 �g/ml for S. pneumoniae.
STATA version 12.0 (STATAcorp, College Station, TX) was used todetermine the geometric means. The data were logarithmically trans-formed to a normal distribution, and paired t tests were used to determinethe difference between agars for studies 1 and 2.
TABLE 1 Antibiotic susceptibility testing methods and agar used
Method Study Agar used Abbreviation
CLSI (8) 2 Mueller-Hinton agarsupplementedwith 5% sheepblood
MHS
EUCAST (19) 2 Mueller-Hinton agarsupplementedwith 5%defibrinated horseblood � �-NAD
MHF
Experimental 1 and 2 Mueller-Hinton agar MHAExperimental 1 and 2 Mueller-Hinton agar
containing 5%lysed horse blood
MHLHBA
Experimental 1 Mueller-Hinton agarcontaining horseblood
MHBA
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RESULTSStudy 1. On MHBA and MHLHBA, S. pyogenes isolates from theskin (n � 36) and throat (n � 64) were uniformly susceptible toSXT (Table 2). Ninety-nine isolates tested on MHA were suscep-tible to SXT (Table 2). The single isolate which appeared resistanton MHA was susceptible on both MHBA and MHLHBA.
There was no statistically significant difference between thegeometric mean MIC measurements on MHA and MHBA. How-ever, isolates tested on both MHA and MHBA had lower MICsthan isolates tested on MHLHBA (Table 3).
Study 2. One hundred isolates of S. pyogenes from 43 childrenwere included in this analysis. S. pyogenes isolates utilized werefrom sores (n � 98) and the anterior nares (n � 2). The majorityof isolates (76%) were from day 0 (before antibacterial treatment);19% were from day 2, and 5% were from day 7. Sixty-four of theswabs from which S. pyogenes was identified also cultured S. au-reus. Of these, 14% were methicillin-resistant S. aureus (MRSA)and 86% were methicillin-susceptible S. aureus (MSSA).
All 100 isolates of S. pyogenes were susceptible to SXT on allagars by both readers (Table 4). Interrater reliability was excellent,with 96% of all MIC readings within �1 MIC gradation. In view ofthis, all analyses were done on results from reader 1. All 100 S.pyogenes isolates were also susceptible to penicillin, erythromycin,and clindamycin.
The geometric means were similar for MHA, MHBA, andMHLHBA (Table 4). MHS had a higher geometric mean MICthan the other media. This was statistically significant, with iso-lates tested on MHS having higher geometric mean MICs than thesame isolates tested on all other agars (Table 5). Despite the higherMICs, all isolates tested on MHS were still susceptible to SXT.There was no difference in MIC between isolates tested on MHAand those tested on MHF. As in study 1, isolates tested on MHAhad lower MICs than the same isolates tested on MHLHBA. Thiswas also found for isolates tested on MHF (Table 5).
Ongoing surveillance with in vitro susceptibility testing isneeded to monitor for changes in rates of SXT resistance withincreased use of SXT. To date, we have tested 910 S. pyogenesisolates cultured from impetigo and anterior nares of children
randomized in the Skin Sore Trial on MHF using an SXT Etestaccording to EUCAST guidelines. Only 8 (0.9%) have been foundto be resistant, with MICs of �2 mg/liter (unpublished data).These results are consistent with those reported from the EUCASTgroup.
DISCUSSION
Although SXT is no longer commonly recommended for treat-ment of respiratory tract infections, it remains one of the mostwidely used and cheapest antibacterials in the world and is animportant option for treatment of SSTI, where S. pyogenes and S.aureus are often copathogens (4, 12, 24, 34). In the era of risingMRSA prevalence, antibacterials that are active against both bac-teria are highly valued.
Impetigo is a significant therapeutic problem in remote com-munities in the Northern Territory of Australia (37, 49, 50), withcommunity-associated MRSA having become highly prevalent inthis region (50). Impetigo is also an endemic problem in manyless-developed countries (41, 45), and MRSA is likely to be on therise in these contexts also (50). In patients with MRSA and S.pyogenes coinfection, finding a single oral agent that is effective,affordable, and easy to use would be a significant advance. Peni-cillins and cephalosporins are no longer an option for MRSAtreatment. In the Northern Territory context, clindamycin is notan option, with up to 22% of MRSA isolates resistant (49), asidefrom its poor palatability in young children and the difficulties inmaintaining adherence to a thrice-daily regimen. Tetracyclinesare not recommended in children under 8 years of age (17), andlinezolid is currently too expensive for the empirical treatment ofsuch a common childhood condition. SXT, which is cheap,widely available, and well tolerated and requires only twice-daily dosing, is a potential single agent for treatment of bothMRSA and S. pyogenes infections. Several studies have con-
TABLE 2 SXT Etest susceptibility results for study 1
Medium
% of isolatesGeometricmean MIC(mg/liter) SD (log)
Susceptible (MIC �
1 mg/liter)Resistant (MIC �2 mg/liter)
MHBA 100 0 0.04 1.52MHA 99 1 0.04 2.2MHLHBA 100 0 0.05 1.78
TABLE 3 Difference in MIC in study 1a
Medium
Difference in MICb
MHA MHLHBA
MHBA MHBA lower by 7%(�9% to 20%)
MHBA lower by 25%*(13% to 35%)
MHA MHA lower by 20%*(3% to 33%)
a Pairwise comparisons of geometric mean MICs of various media.b 95% confidence level in parentheses. *, P � 0.05.
TABLE 4 SXT Etest susceptibility results for study 2
Medium
% of susceptibleisolates (MIC �
1 mg/liter)
Geometricmean MIC(mg/liter) SD (log)
MHF 100 0.07 2.05MHS 100 0.16 1.79MHA 100 0.07 2.03MHLHBA 100 0.09 2.3
TABLE 5 Difference in MIC in study 2a
Medium
Difference in MICb
MHF MHA MHLHBA
MHS MHS higher by128%*(107% to150%)
MHS higher by132%*(113% to153%)
MHS higher by92%* (70%to 116%)
MHF MHF higher by2% (�12%to 7%)
MHF lower by16%* (3% to27%)
MHA MHA lower by17%* (6% to27%)
a Pairwise comparisons of geometric mean MICs of various media.b 95% confidence level in parentheses. *, P � 0.05.
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firmed the ongoing susceptibility of S. aureus to SXT in thisregion of Australia (38, 49).
The breakpoints utilized for this study were defined byEUCAST using data collated from a wide range of sources on morethan 2,500 isolates of S. pyogenes tested for susceptibility to SXT usinga variety of methods (32). Of the 2,596 tests reported from multiplesources, geographical areas, and time periods, 2,559 were susceptibleto SXT of �1 mg/liter and 23 isolates were resistant (0.9%) (Euro-pean Committee on Antimicrobial Susceptibility Testing, data fromthe EUCAST MIC distribution website, http://mic.eucast.org, last ac-cessed 18 September 2012). On disk diffusion testing for S. pyogenes
using SXT disks in a ratio of 1:19, a zone size of �18 mm indicatessusceptibility and �15 mm indicates resistance. Using these break-points, a total of 358 tests were performed, with 5 confirmed resistantstrains (1.4%) (http://mic.eucast.org, last accessed 18 September2012). This can be contrasted with results found in U.S.-based liter-ature using the CLSI methods, where AST for S. pyogenes is per-formed on agar supplemented with defibrinated sheep blood andSXT is not routinely tested, as S. pyogenes strains are considered uni-versally resistant (51). Defibrinated sheep blood is utilized, as thehemolytic reactions of �-hemolytic streptococci on blood agar con-taining sheep blood are deemed “true” (1).
TABLE 6 Summary of published results of S. pyogenes in vitro susceptibility to SXTa
Publication Yr Country Method and medium used Findings Resistance
Yourassowsky et al. (54) 1974 Belgium AST determined by the agardilution method on Wellcotestagar supplemented with 5%lysed horse blood
59/59 strains of S. pyogenessusceptible to TMP,SMZ, and SXT
0%
Finland et al. (22) 1976 United States Plate dilution method on amodified thymidine-deficientMueller-Hinton mediumcontaining 5% laked blood
35/35 strains of S. pyogenessusceptible to SXT; 32/35 strains of S. pyogenessusceptible to SMZalone
0%
Darrell et al. (14) 1968 United Kingdom MIC determined by the platedilution method on diagnosticsensitivity agar containing 5%lysed horse blood
14/14 strains of S. pyogenessusceptible to TMP withMIC � 1 �g/ml
0%
Liebowitz et al. (36) 2003 South Africa MICs determined by the brothmicrodilution methodaccording NCCLS guidelines
66/66 S. pyogenes strainssusceptible to SXT
0%
Hartman and Hoes (26) 1949 United States Wilson’s method (53, 53a), asemi-solid medium with lowsulfonamide antagonist content
94/96 strains of S. pyogenessusceptible tosulfadiazine
1.9%
Schultz and Frank (44) 1958 United States Wilson’s method (53, 53a), asemi-solid medium with lowsulfonamide antagonist content
84/86 strains of S. pyogenessusceptible to sulfurantibiotics
2.3%
Eliopoulos andWennersten (18)
1997 United States MICs determined by agar dilutionmethods on Mueller-Hinton IIagar � 5% lysed horse blood orwith thymidine phosphorylaseat 0.2 IU/ml added
58/60 S. pyogenessusceptible to SXT
3.3%
Berger-Rabinowitz andDavies (2)
1970 Israel Wilson’s method (53, 53a), asemi-solid medium with lowsulfonamide antagonist content
849/890 S. pyogenes strainssusceptible tosulfadiazine
4.6%
Dhanda et al. (15a) 2011 India Kirby-Bauer disk-diffusion test asper CLSI guidelines; agar notspecified
24/26 S. pyogenes strainssusceptible to SXT
6.7%
Bushby (6) 1973 United States Disk diffusion using TMP/SMZdisks 1.25/23.75 �g on variousmedia
699/757 S. pyogenes strainssusceptible to SXT
7.7%
Lakshmy et al. (34a) 2011 India Kirby-Bauer disk-diffusion test asper CLSI guidelines. Agar notspecified
95/119 S. pyogenes strainssusceptible to SXT
21.8%
Dumre et al. (16a) 2009 Nepal Kirby-Bauer disk-diffusion test asper CLSI guidelines; agar notspecified
11/38 S. pyogenes strainssusceptible to SXT
71%
Traub and Leonhard (51) 1997 Germany Agar disk diffusion using NCCLScriteria on sheep blood MHA
0/63 strains of S. pyogenessusceptible to SXT
100%
Kaplan et al. (33) 1999 United States Etest performed on MHAcontaining 5% sheep blood
0/169 strains of S. pyogenessusceptible to SXT withMIC � 32 �g/ml
100%
a TMP, trimethoprim, SMZ, sulfamethoxazole.
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The in vitro results reported in the current study confirmingthe susceptibility of S. pyogenes to SXT suggest that treatment ofSSTI with SXT is worth considering. Our current RCT to assess thenoninferiority of SXT to the standard treatment with benzathinepenicillin G (BPG) for impetigo will provide the necessary clinicalevidence to inform guidelines. It is based on a pilot study of 13participants which indicated that both BPG and SXT were effica-cious in healing impetigo (48). There is one study published com-paring these agents for S. pyogenes infection in tonsillitis, whichreported a 70% treatment efficacy for SXT compared to 88% forpenicillin, a non-statistically significant difference (52).
The infrequent reports of susceptibility of S. pyogenes to SXTdemonstrate resistance rates ranging from 0% to 100% dependingon which medium and testing conditions are used (Table 6). Al-though the variation in results may relate to the particular strainsincluded or the local prescribing patterns of SXT, it is most likelyrelated to the methodology of testing. All of the studies reportinghigh resistance rates either used media known to have high con-centrations of thymidine or did not provide details of the mediumused. As standardization to ensure a low thymidine concentrationof Mueller-Hinton medium was introduced only in 2006, it islikely that, unless low-thymidine media were specified (2, 14, 18,22, 26, 44, 54), studies in publications prior to this may not havecontrolled for thymidine content.
Alongside this, S. pyogenes has remained 100% susceptible invitro to penicillin. Hence there has been no pressing need to un-derstand SXT susceptibility as an alternative antibacterial in thepublic health approach to treatment of S. pyogenes infections.However, this is changing in the context of rising MRSA rates forSSTI.
Our results show that testing of S. pyogenes for susceptibility toSXT on MHS gives a higher MIC than all of the other agars, al-though the organism remains in the susceptible range. This couldpossibly be due to the availability of thymidine or other inhibitorysubstances in this medium. However, thymidine concentrationsof the various media utilized were not assessed. Alternatively, theabsence of an enzyme to reduce the inhibition in sheep bloodcompared to horse blood may be the explanation.
The original paper describing the identification of the Harper-Cawston factor (25) as thymidine (21) reports an interesting ob-servation that our study has partially demonstrated. Only lysedhorse blood contains thymidine phosphorylase to convert thymi-dine to thymine and hence overcome the inhibition of folate me-tabolism that occurs in the presence of thymidine. No other mam-malian blood contains this enzyme, which is a possible reason forthe higher MICs reported on MHS than on those agars containinghorse blood. In the original paper, the presence of thymidine atconcentrations of 1.6 �g/ml was sufficient to completely preventinhibition by the drugs. The inclusion of lysed horse blood re-stored the inhibition. This has also been shown by Coll et al. (11).However, the MIC of S. pyogenes isolates tested on MHLHBA washigher than those of isolates tested on MHF (study 2), MHA(studies 1 and 2), or MHBA (study 1), which suggests other factorsat play.
A limitation of this study is the reliance upon a single methodfor susceptibility testing, the Etest, which is a commercially de-rived method. Further work using broth or agar dilution methodswould add to our understanding of the susceptibility of S. pyogenesto SXT. As shown in Table 6, when these additional methods havebeen assessed, the susceptibility of S. pyogenes ranges from 0%
(54) to 3.3% (18) resistant, similarly low resistances to those re-ported in this study.
Reading MICs for SXT can be challenging due to haze. In par-ticular, only faint growth of S. pyogenes was achieved on MHA(due to the absence of blood), and this made reading endpointsmore difficult. MHLHBA and MHF had problems similar to thoseof MHA with respect to haze. Despite this in study 2, the MICswere reproducible between readers, with a high level of interraterreliability within 1 MIC gradation.
Conclusions. The widespread belief that SXT is ineffective forS. pyogenes infections because of inherent antimicrobial resistanceis a fallacy due to technical limitations in laboratory methodology:namely, the use of media containing high concentrations of thy-midine, which inhibits the action of sulfur antibacterials. Whenmedia containing low concentrations of thymidine and/or highconcentrations of the enzyme thymidine phosphorylase are used,resistance rates are low in most cases, although this must be mon-itored over time and may vary with local epidemiology and anti-bacterial prescribing patterns. This study provides justification toproceed to clinical trials of SXT for S. pyogenes infections. Corrob-oration with clinical trial data may convince clinicians that SXTcan safely and appropriately be used for infections involving S.pyogenes. The Skin Sore Trial will answer the clinical applicabilityof this current in vitro study. In the era of rising MRSA prevalence,more clinical trials of SXT for treatment of SSTI, where S. pyogenesand S. aureus are frequently copathogens, are needed.
ACKNOWLEDGMENTS
The work of the research assistants in study 1 and the Skin Sore Trial team(Irene O’Meara, Jane Nelson, Tammy Fernandes, Melita McKinnon, Di-anne Halliday, Colleen Mitchell, Valerie Coomber, and Christine Fran-cais) in recruiting participants for study 2 has made this research possible.Study scientists who carried out this work include R.A.L., P.W., andVanya Hampton. Mark Chatfield provided assistance with the statisticalanalysis. We also acknowledge all of the participants and their familieswho have participated in the Skin Sore Trial and Rheumatic Heart Diseasestudies.
This work was supported through a National Health and Medical Re-search Council (NHMRC) Project Grant (545234) on which A.C.B.,S.Y.C.T., M.I.M., B.J.C., and J.R.C. are all investigators. A.C.B. is the re-cipient of a NHMRC scholarship for Ph.D. research (605845) as well as anAustralian Academy of Sciences Douglas and Lola Douglas scholarship.S.Y.C.T. is the recipient of a NHMRC Early Career Fellowship (605829).
We have no conflicts of interest to declare.
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CHAPTER 4THE CHALLENGES OF COLLECTING ANDTRANSPORTING IMPETIGO SWABS IN A
TROPICAL ENVIRONMENT
4.1 CHAPTER OVERVIEW
Being able to effectively collect and transport impetigo swabs in a tropical
environment has been critical to the work undertaken in my thesis. This chapter
describes the method developed and validated for the transportation of impetigo
swabs from very remote contexts for processing in a central microbiology
laboratory. It also succinctly describes the methods used to culture and identify
Staphylococcus aureus and Streptococcus pyogenes. This supplements the
methods for antibiotic susceptibility testing described in Chapter 3.
The primary objective of the Skin Sore Trial was to determine non-inferiority of
trimethoprim/sulphamethoxazole to benzathine penicillin G for the treatment of
impetigo. This objective was complemented by the secondary outcomes, which
were to determine the relative abundance of S. aureus and S. pyogenes at days 0,
2, and 7 of the study. To be confident in these secondary outcomes, it was
necessary to develop and test robust methods for collecting and transporting skin
swab specimens.
The remote communities involved in the study were up to 1500 kilometres away
from the laboratory and did not have daily flights connecting the community to
the laboratory. As such, transportation of swabs was by either road or air.
Previous research protocols for detecting S. pyogenes recommended either:
1) immediate plating of the collected swab in the community and transportation
within 24 hours for incubation in the laboratory or; 2) plating and incubation of
the swab within 48 hours of sampling. (Johnson, 1996, Kotloff, 2008) These
recommendations were developed for contexts similar to the remote
communities of the Northern Territory and had been previously utilised in
47
smaller studies. (McDonald et al., 2006, Carapetis et al., 1995) However, the
size and complexity of recruiting 663 participants over three years from multiple
remote communities with varying available transportation logistics resulted in
the need to develop and test an alternative solution. Transport media have been
recommended for this situation, although little data was available on freezing
such specimens.
An additional benefit of the method reported in this chapter was that swabs
could be processed in batches. This was important for the laboratory scientists
working on the study to manage workflow as more than 4500 swabs of impetigo
lesions and the anterior nares were collected and processed during the course of
the RCT. This methodology also permitted freezing of the swabs for later
molecular and metagenomic studies that have the potential to further our
understanding of the mechanisms of transmission of bacterial skin pathogens
within and between communities.
The transport medium validated in this study is cheap, easy to produce, effective
in transporting skin pathogens, can be frozen and may be utilised in research in
other remote contexts.
4.2 STATEMENT OF CONTRIBUTION TO JOINTLY AUTHORED WORK
Together with Steven Tong, I conceived this experiment, with oversight from
Jonathan Carapetis. I wrote the SOP for this experiment and oversaw the data
collection. I, Steven Tong and Mark Chatfield conducted the data analysis. I
performed the literature review and wrote all drafts of the manuscript. All co-
authors contributed to revisions of the manuscript and approved the final version
for publication. Steven Tong agreed to be the corresponding author while I was
on maternity leave.
48
4.3 JOURNAL ARTICLE (FOLLOWING PAGE)
Comparison of three methods for the recovery of skin pathogens from impetigo swabs collected in a remote community of the Northern Territory, Australia
49
56
CHAPTER 5DEVELOPING A METHODOLOGY FOR
CAPTURING AND SCORING STANDARDISED DIGITAL IMAGES OF
IMPETIGO
5.1 CHAPTER OVERVIEW
This chapter describes the novel methods needed for assessing and blinding the
primary end point within our RCT, which has the potential to be a standardised
approach for similar studies in the future.
Prior impetigo studies synthesized in the Cochrane review of impetigo treatment
(Koning et al., 2012) have predominantly been conducted in urban physician
offices or hospital outpatient settings. This allows more ready access to experts
who can immediately assess the participant for the primary outcome. Due to the
remoteness of the research locations in our trial, it was not feasible to transport
an expert to assess the outcome of every one of the 663 episodes of impetigo
under investigation. As such, building on the work of tele-dermatology, digital
images were determined to be the best method available to capture and evaluate
the primary endpoint of this trial. However, no published protocols for capturing
standardised digital images by amateur photographers were available.
As an open label RCT, we needed a blinded, robust, reproducible and defendable
primary endpoint. This chapter describes in detail the development and
evaluation of our approach. This was primarily work which I led. Expert
photographic input was sought from a professional photographer to guide this
process. Once standardised digital images of all enrolled sores from days 0, 2 and
7 were available, a method for scoring these images that minimised bias and
provided readily analysable data was needed. This chapter will also describe the
57
method developed for scoring outcomes using assessors blinded to treatment
allocation.
Having published our approach, these methods are now available for future
impetigo treatment studies, and could be implemented in either remote or office-
based research. It may be possible to compare the results from an expert who is
immediately able to objectively assess the process of sore healing with results
from other experts scoring the digital images distant from the participant. This
translation will add value to future studies of impetigo, and facilitate high quality
treatment studies where the burden of impetigo is the highest.
5.2 STATEMENT OF CONTRIBUTION TO JOINTLY AUTHORED WORK
I designed and wrote all the standard operating procedures for collecting digital
images in the Skin Sore Trial. Assistance was sought from Kara Burns, a medical
photographer, for expert advice. I developed the training packages for the
research assistants and oversaw the quality checks of all digital images. Ross
Andrews proposed the concept for scoring the digital images in a randomly
allocated order, which I then revised and developed into a feasible protocol. I
oversaw all aspects of digital image scoring. Robyn Liddle developed the
databases within which digital images were scored. I cleaned and analysed the
data, developed the quick lists and drafted all versions of the manuscript. All co-
authors contributed to revisions of the manuscript and approved the final version
for publication.
5.3 JOURNAL ARTICLE (FOLLOWING PAGE)
Standardising and assessing digital images for use in Clinical Trials: A practical, reproducible method that blinds the assessor to treatment allocation
58
Standardising and Assessing Digital Images for Use inClinical Trials: A Practical, Reproducible Method ThatBlinds the Assessor to Treatment AllocationAsha C. Bowen1,2,4,5*, Kara Burns2,3, Steven Y. C. Tong1,2, Ross M. Andrews1, Robyn Liddle1,
Irene M. O9Meara1, Darren W. Westphal1,4, Jonathan R. Carapetis4,5
1 Menzies School of Health Research, Charles Darwin University, Darwin, NT, Australia, 2 Royal Darwin Hospital, Darwin, NT, Australia, 3 School of Business, Queensland
University of Technology, Brisbane, QLD, Australia, 4 Telethon Kids Institute for Child Health Research, University of Western Australia, Perth, WA, Australia, 5 Princess
Margaret Hospital for Children, Perth, WA, Australia
Abstract
With the increasing availability of high quality digital cameras that are easily operated by the non-professionalphotographer, the utility of using digital images to assess endpoints in clinical research of skin lesions has growingacceptance. However, rigorous protocols and description of experiences for digital image collection and assessment are notreadily available, particularly for research conducted in remote settings. We describe the development and evaluation of aprotocol for digital image collection by the non-professional photographer in a remote setting research trial, together witha novel methodology for assessment of clinical outcomes by an expert panel blinded to treatment allocation.
Citation: Bowen AC, Burns K, Tong SYC, Andrews RM, Liddle R, et al. (2014) Standardising and Assessing Digital Images for Use in Clinical Trials: A Practical,Reproducible Method That Blinds the Assessor to Treatment Allocation. PLoS ONE 9(11): e110395. doi:10.1371/journal.pone.0110395
Editor: H. Peter Soyer, The University of Queensland, Australia
Received June 17, 2014; Accepted September 13, 2014; Published November 6, 2014
Copyright: � 2014 Bowen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and itssupporting information files.
Funding: This work was supported by a National Health and Medical Research Council (NHMRC) of Australia project grant (545234) for which AB, ST, RA and JCare all investigators and IMO is funded as the trial project manager. AB is the recipient of an NHMRC scholarship for PhD research (605845) and an AustralianAcademy of Sciences Douglas and Lola Douglas scholarship. ST is the recipient of a NHMRC Career Development Fellowship (1065736). The funders had no role instudy design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: [email protected]
Introduction
Telemedicine is increasing in popularity, particularly for
dermatologists and other specialities where a clinician is not
always onsite for direct patient care [1]. With the increasing
availability of cheap, simple, high quality digital cameras for the
non-professional photographer, often a clinician or auxiliary [2,3],
the appeal of utilising digital images to diagnose or monitor
treatment progress is growing. An extension of this application is
the use of digital images of cutaneous disease to assess outcomes
for intervention studies including randomised controlled trials
(RCTs) [4]. Advantages include: maintaining blinding of the
outcome assessor to treatment allocation; scoring digital images in
batches to improve work flow; and utilising expert outcome
assessors remote from the site of data collection. These advantages
have particular appeal for the conduct of research in remote
regions or for multicentre studies where standardisation of clinical
outcome measures is critical. However, published methodologies
to guide such use of digital images are lacking. In addition, unlike
hospital or clinic based health photography which is usually
performed in a dedicated setting with instruments and lighting
operated by a professional photographer [5,6], this ideal may not
be achievable in remote, field-based research. Therefore we aimed
to develop a method to facilitate the acquisition of consistent, high
quality digital images of superficial skin lesions in the context of
conducting a RCT in a remote setting. A medical photographer
(KB) guided this process [4,7]. The digital images were taken by
field research staff and allowed the assessment of outcomes in a
blinded manner.
The four characteristics of an excellent clinical photograph are
correct perspective, use of a scale aligned with the image frame,
even lighting and a neutral background [8]. To achieve these
characteristics, it is important to standardise the equipment,
camera settings, participant positioning and photography tech-
nique so that reproducible images are captured [9,10]. Once
images of satisfactory quality have been captured, consideration
must be given to storing these confidential images for future use.
Standardised protocols that address these priorities for collecting
digital images are not available in the peer-reviewed literature.
Impetigo trials are an example of cutaneous disease research
where digital images can improve the objectivity of outcome
assessment. Impetigo is a common, non-benign cutaneous
infection that mostly occurs in resource-limited contexts [11],
affecting .2% of the global population at any one time [12].
Impetigo also regularly affects school-aged children in industrial-
ised settings [13]. Treatment of impetigo is a public health priority
to prevent severe sequelae including streptococcal and staphylo-
coccal sepsis, focal invasive disease, post-streptococcal glomerulo-
nephritis (which is in turn linked with chronic renal failure) [14],
and a postulated causative link with rheumatic fever and
rheumatic heart disease [15,16]. These sequelae mainly occur in
PLOS ONE | www.plosone.org November 2014 | Volume 9 | Issue 11 | e1103951
59
resource-limited settings and are responsible for hundreds of
thousands of deaths each year globally [11].
The authors of a meta-analysis on interventions for impetigo
recommended the use of clear and objective outcome measures for
future impetigo research [17]. As the burden of impetigo is in
resource-limited contexts where ready access to clinicians for
immediate end-point assessment may not be feasible, digital image
end-points are appealing and objective. The only RCT from a
resource-limited context included in the meta-analysis reported the
use of photographs for outcome assessment. In this RCT,
successful treatment was defined as clinical cure or marked
improvement with an additional measure using photographs, but
they did not report the methodology of image collection or how
they determined the outcome based on this end-point [18]. Well-
defined, reproducible endpoints are needed and blinded outcomes
are the cornerstone of good clinical trial design [19].
In conducting a RCT on impetigo treatment in a remote
setting, we developed a standardised, reproducible method for
collecting images of skin sores at different time points [6].
Reviewers blinded to treatment allocation assessed the images
using a simple, reproducible, quick method that provided readily
analysable data. The image capture protocol and novel method-
ology for blinded assessment may be useful for other trials in
cutaneous disease research.
Method
EthicsThis study and all consent documentation were approved by the
Human Research Ethics Committee of the Northern Territory
Department of Health and Menzies School of Health Research
(HREC 09/08). Indigenous elders provided community consent
before recruitment commenced. The parent or legal guardian for
all participants provided written informed consent. The study was
explained by a local interpreter or by using a talking book in the
participant’s first language. Written consent was itemised for all
study procedures including the collection of digital images.
SettingWe conducted a non-inferiority RCT in 7 remote Indigenous
communities of the Northern Territory of Australia between 2009
and 2012 [20]. The research team was based in Darwin and
travelled via plane or road to the remote communities up to 1500
kilometres away. There were 663 episodes of impetigo (in 508
children) enrolled in the trial and each had either one or two sores
under investigation. Research assistants trained in the photogra-
phy protocol captured digital images of all trial participants’ sores
on days 0, 2 and 7. Images were stored electronically. A panel of
paediatricians specialising in the care of Indigenous Australian
children externally reviewed these digital images at a later date,
according to a standardised scoring system as reported below.
Details of the protocol for capturing digital images ofimpetigo
Equipment. All images were captured using standardised
equipment (table 1, figures 1–4).
Camera Settings. Due to data collection occurring simulta-
neously in more than one community, we purchased three
identical cameras. All study camera settings were programmed
by the study manager (figure 5) and checked against the standard
operating procedure (SOP) by the research assistants before each
use. Training in the programming of settings and operation of the
camera was conducted with each new research assistant and image
quality reviewed at the completion of most field work trips. A
quick list to summarise the process for image capture in the field
was developed (table 2).
Lighting Conditions. The main light source was the flash as
force flash was always on. This provided a standard light source for
all three cameras in all settings. In addition, preference was given
to capturing digital images outdoors in the shade to improve the
ability of the camera to focus on the subject and avoid distracting
shadows [10]. Direct sunlight was avoided to minimise the
potential of a stronger light source resulting in an over-exposed
image. For uniformity of conditions, when it was not possible to
photograph participants outdoors, maximal ambient light was
achieved by turning on all lights and opening any curtains or
doors. Night time photography was avoided by returning to
photograph the participant first thing the following day.
Positioning of study participant. As we were working with
children, prior to taking any photographs, the participants were
reminded to remain still and the carer was engaged in reassuring
the child. The participant was positioned comfortably in a chair or
on the floor with a neutral grey background beneath the limb or
site to be photographed (figures 4 and 6). Jewellery, clothing or
hair that might obscure the area of interest were removed or tied
back. Any dressings covering the lesion were also removed.
Once settings were rechecked, a 5 cm neutral grey scale was
placed in a vertical position, in the same plane as the sore, as close
as possible to the left of the sore without obscuring any edges of the
lesion (figures 2 and 6). The upper limit (0) of the scale was
positioned at the top of the frame and the lower limit (5) at the
bottom. This ensured all images were captured at the same scale so
that when paired images were reviewed the sores were comparable
and any reduction in size could be assessed as a measure of sore
healing.
Photography techniqueTo maximise sharpness by depth of field, the camera lens plane
was positioned parallel to the sore plane with the photographer
standing above the sore (figure 6). The sore was centred using the
white square corners at the centre of the camera screen. The
shutter was depressed halfway to focus the lesion prior to capturing
the image.
A minimum of three images of each sore were taken at each
time point, to ensure that at least one adequate image was
available for outcome assessment, a technique known as bracket-
ing [21]. The research assistant was instructed to check each image
for SOP conformity and to take additional photographs if a clear,
focussed image showing all details of the sore had not been
Figure 1. The Panasonic DMC-TZ8 camera demonstrating zoomand camera settings as indicated in figure 5. http://www.digitalcamerawarehouse.com.au/prod6699.htm, last accessed 28 Sep-tember 2014).doi:10.1371/journal.pone.0110395.g001
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obtained. Each photograph number was recorded in the respective
participant’s case report form (CRF).
Once all images had been captured, brief notes were made in
the participant’s CRF to describe the positioning of the participant
so that whenever possible the same position could be used for
future images. Further follow up images of the same sore were
required on day 2 and day 7 from enrolment. For consistency of
orientation, previous images of the same sore were checked on the
study camera before capturing the next image.
Image download and storageStandardisation of image storage is critical to the meticulous
utilisation of this method [9,22]. At the completion of each day,
the image files were downloaded from the camera memory card to
a password-protected laptop. The laptop files served as a data
backup, which was important given that study visits lasted between
two and three weeks and internet was not reliable enough in the
remote context to upload numerous large files every day. Upon
return of the team to the research centre in Darwin; all images
were downloaded from the camera to the main computer server
where daily backups occur. All images were taken and stored in
high quality.JPEG (Joint Photographic Experts Group) format for
convenience as our chosen camera did not shoot in an
uncompressed (‘raw’ or ‘ loss-less’) format. There are limitations
to using a compressed or ‘lossy’ format but the benefits of using a
compact camera and storage of images for comparison outweighed
these and is in line with other clinical studies [23]. Three copies of
each unmodified image were saved: one in the participant’s folder
labelled with participant number; one in the generic backup folder
labelled with the camera-generated photograph number as
recorded in the participant CRF; and one labelled with a
randomly generated number between 1 and 15 000. Only once
this had occurred were images deleted from the memory card.
These re-identifiable images are to be stored on a secure server for
up to 25 years, in keeping with ethical requirements for research in
children [24]. From the three available digital images of each sore,
the best quality image (key criteria were focus, exposure and
magnification) was selected for outcome assessments.
Quality control processAs all images were collected by amateur photographers, images
were regularly checked by the study doctor (AB) and feedback
provided if the image did not conform to the SOP. In addition, prior
to commencing primary outcome reviews, a quality control (QC)
check of all available digital images was performed mid-way
through the study (collected from the first 200 study participants) by
a medical photographer (KB) experienced in capturing digital
images of skin conditions. The QC was a priority in this study as the
method described had not been previously used or evaluated and we
were unsure whether digital image manipulation might be needed
for image scoring. Digital images can be manipulated to overcome
flaws in image capture [21], however this has limitations. Our apriori hypothesis was that digital image manipulation would not be
needed. To confirm this after recruitment of 200 participants, 1 300
images were scored as either adequate or unable to be interpreted
using the definitions in table 3. If ‘‘unable to be interpreted’’ was
chosen, the reviewer was instructed to provide reasons (figure 7).
Figure 2. Use of scale to define the upper and lower boundaries of image in the landscape position. This series of images of the samesore on days 0 (A), 2 (B) and 7 (C) utilise the scale well with the 0 at the top of the image and the 5 at the bottom, are clear and focussed anddemonstrate sore healing over time. Limitations include different availability of light as captured during different parts of the day using outdoor light.doi:10.1371/journal.pone.0110395.g002
Figure 3. An example of the participant identification carddescribed in table 1. This card contains participant number, date ofimage, study day, and whether it is sore A or B as up to two-thirds ofstudy participants had two sores enrolled in the study.doi:10.1371/journal.pone.0110395.g003
Figure 4. Capturing the image using the study camera, greybackground, and grey scale in a remote context. The individualsin this image have given written informed consent to publish thisimage.doi:10.1371/journal.pone.0110395.g004
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Methods for digital image assessmentWe developed a method for scoring digital image pairs that was
simple, limited bias, quick and afforded readily analysable data. As
the methodology was novel, a paper-based pilot was conducted
employing 13 clinicians and researchers to confirm usability prior
to building an automated database for scoring. In the pilot, 22
paired digital images from 10 participants of either day 0 and 2 (5
participants) or day 0 and 7 (6 participants) were reviewed in
random order (days 0/2 or 2/0 and 0/7 or 7/0) by the 13
reviewers. The initial definition of healing or improved included
both a visual description of the sore pair and a clinical decision as
to whether further antibiotic treatment was needed.
The primary outcome for the RCT was treatment success at day
7 according to paired digital image scoring. After the successful
pilot, the digital image pairs were organised in random order in a
purpose built database (figure 8) using the randomly assigned
number between 0 and 15 000 as the only identifying information.
Scoring of the digital image pairs was on non-standardised
computer screens at locations remote from the primary study site.
Scoring was by a group of eight paediatricians with expertise in
caring for Indigenous children with impetigo. Primary outcome
reviewers blinded to treatment allocation were provided pairs of
images from day 0 and 7 (or day 0 and 2) in random order. Each
reviewer was unaware of which image (A or B) was pre- or post-
treatment and was asked to decide if image A, compared to image
B, was healed, improved, the same, worse, or unable to be
determined using the definitions (table 4) and vice versa (i.e.,
image B compared to image A). To expedite this process, an auto-
fill was used in the database. For example, when image A was
scored as ‘‘worse’’, auto-fill made available the options of ‘‘healed’’
or ‘‘improved’’ only for the comparison of image B to image A.
Where ‘‘healed’’ or ‘‘improved’’ were selected for image A, auto-
fill completed the scoring with ‘‘worse’’ for image B. The use of
auto-fill made the scoring process as rapid as possible. Thus
reviewers were blinded to both treatment allocation and the
chronological order of sores. Every image pair was evaluated by
two independent reviewers from the panel of eight. Where
disagreements occurred, an expert panel of three determined the
final result by consensus.
Following un-blinding of the chronological order of sores and to
produce readily analysable results from the blinded scoring system,
if image B was the day 2 or 7 sore, treatment success was deemed
to have occurred if image B was healed or improved compared to
image A (day 0 sore). If image B was the day 0 sore, then success
Table 1. Study equipment chosen including the required features, advantages and alternatives available or recommended in theliterature.
Item Specific Choice Features Advantages Alternatives
Digital Camera Panasonic LUMIXDMC-TZ8 14.5 megapixeldigital camera (figure 1)
Macro to 3 cm from a 12Xzoom lens equivalent to a25–300 mm lens on an SLR
Inexpensive Digital single lens reflex (SLR)camera [32]
Built in flash Readily available
Rugged metal casing Automatic
Lithium ion rechargeablebattery
Pre-specified settingsto achieve uniform, reproducibleimages whilst using amateurphotographers
4GB memory card
Background Grey background(figure 4)
A small grey board positionedbehind the body part beingassessed
Transportable and light weight Green or grey surgical drapes[33]
Manoeuvrable
Non-reflective
Neutral
Scale [34–36] 5cm, non-reflectivegrey scale (figure 2)
Vertical scale Defined the upper and lowerboundaries of the photograph(figure 2)
Paper or commercially availablescale e.g. the ABFO scale [34]
Single use sticker Easily removed and discarded
Good infection control
Identification Card Cardboard handwrittencard (figure 3)
Pre-formatted to add individualrandomisation number,study visit day, sore number,date and time of image capture
Maintained study blinding
Photographed prior to impetigoimage capture to avoid inclusionof any identifying details inthe image for scoring by blindedreviewers[37]
When forgotten, identification card wasphotographed upside downimmediately after the series of soreswas captured [37]
doi:10.1371/journal.pone.0110395.t001
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Figure 5. The camera settings and icons as used in the protocol (these icons and settings are standard across other popular cameramodels). In addition the rationale is provided on why these settings were adopted.doi:10.1371/journal.pone.0110395.g005
Table 2. Quick List for capturing standardised photographs.
Step Action
1 Confirm all camera settings are correct (figure 5), participant, paperwork and previous image for orientation (if available)
2 Position participant in the shade: comfort, lesion exposed, neutral background, scale in the same plane as the lesion
3 Photograph participant ID (table 1) prior to capturing series to preserve blinding.
4 Position camera in same plane as sore (figure 6). Centre the sore and focus camera. Take minimum of 3 photos. Take additional photographs if none are clearand focussed.
5 Record photograph number and notes in participant’s file
6 Save digital images in secure location and delete from camera
This could be printed on a small card to be carried with the camera as a reminder to research assistants capturing images.doi:10.1371/journal.pone.0110395.t002
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was deemed to have occurred if image B was worse compared to
image A (day 2 or 7 sores) and image A was either healed or
improved (table 4).
Results
Overall image collectionOver the 3-year study, almost 10 000 digital images were
collected and stored by more than 20 research assistants who
collected data in 7 remote communities covering an area of 1.35
million km2. From all of the images collected, the best available
image of the bracketed set was selected for outcome assessment.
Approximately 3 300 digital images were required to determine
the primary and secondary outcomes of the RCT by analysing the
results of paired comparisons. The project manager (IO) reviewed
all images and selected the best available image for the paired
comparison. The best available image (determined by focus,
exposure and magnification) was most often the second or third
image captured. This was consistent by study visit day.
Of the 9 944 digital images collected, only 17 (0.17%) were not
available for assessment due to staff error. These errors were the
wrong site photographed on subsequent days to the original site
(n = 6), the photograph not saved or filed (n = 4) and the
photograph not taken due to staff error (n = 7).
Quality control checkFor the QC check, 1 300 digital images from the first 200
participants that conformed to the described methodology were
reviewed. 1 258 (96.8%) were deemed adequate using the
definitions provided. Of the 42 images (3.2%) deemed unable to
be interpreted there was some overlap in categorisation: 29 were
due to incorrect exposure (8 too light, 21 too dark), 16 were due to
lack of focus, 2 had incorrect magnification due to lack of focus
and 1 had incorrect magnification (Table S1). Results of the QC
review were reassuring and consequently digital image modifica-
tion was not required.
Figure 6. Cartoon demonstrating the photographer, cameraand sore were in the same plane to optimise reproducibility ofcaptured imagesdoi:10.1371/journal.pone.0110395.g006
Figure 7. Database form used for Quality Control check by medical photographer.doi:10.1371/journal.pone.0110395.g007
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Pilot for digital image scoringThirteen reviewers piloted the digital image scoring process. All
pilot reviewers agreed the process was quick and manageable with
image quality being adequate. Initially definitions for healing,
improved, same or worse included a 2-armed definition with a
description of both sore healing and a clinical judgement as to
whether treatment with further antibiotics was indicated. The
reviewers reported that the need for additional treatment was
difficult based on images alone and as the decision had no timely
clinical impact, we removed this decision from the definitions.
Digital image scoring resultsOutcome scorers reported 98.3% of digital images as able to be
interpreted using the quality codes shown in figure 8. Of these,
89.9% were adequate and 8.4% suboptimal but still able to be
interpreted (Table S2). The inter-rater reliability of digital image
scoring was moderate. When assessing for treatment success
(pooled healed and improved, table 4) versus treatment failure
(unchanged or worse), there was 86% agreement between
reviewers with a kappa score of 0.4. When assessing using the 5
Table 3. Definitions used for the quality control assessment of digital images.
Assessment Definition
Adequate The entire sore was seen in enough detail to determine the margin and most of the interior; AND the scale was seenin sufficient detail to determine the approximate size.
Unable to be interpreted The image of the sore could not be interpreted due to incorrect exposure (too light or too dark), focus (lack of focusor depth of field) or incorrect magnification.
doi:10.1371/journal.pone.0110395.t003
Figure 8. This shows the database format utilised for scoring digital image pairs. Image A was taken on day 0 and image B on day 7.Image B shows erythema and as such was scored as improved using the definitions in table 4.doi:10.1371/journal.pone.0110395.g008
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available definitions (figure 8, table 4) there was 64% agreement
between reviewers, with a kappa score of 0.3 (Table S3).
Discussion
This is the first description of a method for capturing and
scoring comparative digital images of skin lesions in clinical
research. The methods outlined were practical even in remote
contexts, robust, reproducible and simple enough for non-
professional photographers to consistently follow. Strengths of
the described methodology include the quality control check and
more than 98% of captured images being interpretable. The gold
standard for needing to retake orthodontic photographs for poor
quality was set at 90% [3] and our findings of adequacy were at
this level, but when ‘suboptimal but still able to be interpreted’ was
included exceeded this gold standard. In addition, the described
method was followed by more than 20 study staff in remote
contexts resulting in ,0.2% of images being unavailable for
assessment. Digital images were the only available form of
documentary evidence for this blinded, clinical trial so it was
essential to have a robust process. Our results support that the
process outlined works.
The adoption of a standard set of image settings (figure 5) and a
Quick List that guided training in the methodology (table 2)
facilitated a uniform set of images that did not require any digital
manipulation. Guidelines on the manipulation of digital images
specify that while ‘‘it is acceptable practice to adjust the overall
brightness and contrast of a whole image’’ [25] it is best practice if
a group of images are to be compared to each other, that the
processing of individual images should be identical. The question
of ‘‘what constitutes a ‘‘reasonable’’ adjustment of image settings
such as brightness and contrast, etc.’’ has become important for
publication in scientific journals and is now included in
instructions to authors [26]. For example, the instructions to
authors in the Journal of Cell Biology outline that, if manipulation
of a digital image is undertaken, these manipulations must not
obscure, eliminate, or misrepresent any information present in the
original [25]. Forensic guidelines also emphasise this rigorous
approach for reproducibility [27]. As the method for capturing
digital images reported above had not been previously validated
and the outcome was based on a comparison of image pairs, the
QC check by a professional medical photographer was an
important step in determining whether our images would meet
this industry standard. Based on these results, we did not permit
the use of photo-editing software to modify any of the images [27].
We recommend following a protocol such as ours that has been
subjected to rigorous QC checks for future skin disease research
which should largely obviate the need for any digital image
manipulation.
High staff turnover when working in remote settings [28]
resulted in frequent training and re-training sessions in capturing
digital images using the methods described. Educational Power-
Point slides were developed for this purpose and supplemented by
the quick reference guides developed (tables 1 and 2, figures 5 and
6). In addition, real time review of captured digital images with
feedback to the research assistants was useful. Despite the use of a
standard protocol and ongoing training, occasional human errors
did occur as described above.
A limitation when using digital images for endpoint assessment
is the inability of reviewers to make a clinical decision using the
additional senses of hearing (patient feedback on pain and
pruritus), touch (warmth, fluctuance) and smell, when provided
only with the image. For cutaneous diseases where the appearance
of a lesion is the primary determinant of outcome, this limitation
can be partly addressed with a robust protocol for capturing
reproducible, diagnostic images for outcome assessment. This
known limitation impacted upon the inter-rater reliability agree-
ment as physicians were asked as reviewers to use a novel
diagnostic modality to score outcomes. To overcome this, we used
a consensus panel of three to adjudicate any discrepant scoring.
The consensus panel discussed all image pairs until consensus was
reached. The possibility that the use of the project manager to
select the best available image introduced bias is a possible
limitation. However, the QC check by a professional photogra-
pher confirms that the perceived bias was minimal with high
quality images consistently being provided to reviewers.
We suggest that this protocol could also be adapted from the
research setting for use in clinical care. In settings where
specialised clinicians are not readily available, standardised digital
photography of cutaneous lesions could be used in telemedicine to
allow highly skilled clinicians to assist local health staff to manage
patients in remote locations.
A unique feature of this protocol for standardising the
comparison between image pairs of the same sore where the only
detectable difference was changes in the appearance of the lesion
[6], was the requirement for all research assistants to check the
orientation of the image on the study camera before capturing the
next image. Guidelines on doing this were provided. Previous
expert advice has been for image capture to be performed
consistently by the same photographer [21]. This was not possible
within the remote research environment and overall ,5% of
participants had all 3 days of images collected by the same person.
Nonetheless, .99% of image pairs were assessable for the primary
outcome. This finding adds to the photography literature.
Providing amateur photographers with simple instructions and
guidance for collecting digital images using standardised camera
settings results in digital images that are of a high quality and can
Table 4. Definitions used for final outcome scoring of digital images of impetigo.
Assessment Criteria Outcome
Healed Lesions no longer evident or flat, with no evidence ofcrusting, erythema or purulence,but possibly with evidence of hyper- or hypo-pigmentationwhere the original sore was located.
Success
Improved Lesions reduced in sore diameter and erythema; ANDprogression from blister tocrusting and flattening of the sore. Purulence not evident.
Same No appreciable change in diameter, erythema or purulenceof lesion.
Failure
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be assessed by blinded, independent reviewers for outcome
determination.
A non-inferiority RCT comparing two treatments of a common
condition such as impetigo requires rigorous, blinded, objective
endpoints for assessment. Here, we have described the method
developed based on the available evidence and expertise, for
capturing digital images. We report these here for use in
subsequent research trials. This method was simple, reproducible
and from the QC check provided 97% of images that were
adequate for assessment. Whilst other RCTs have used a digital
image of the skin as a primary outcome, such as in pyoderma
gangrenosum [29], wound healing [30], or pressure sores [31], this
is the first report of a standardised, reproducible protocol that has
been subjected to a rigorous QC assessment for an impetigo RCT.
Our results confirm the reproducibility of the simple resources
developed and published herein which will further enhance the
rigour of trials using a photographic end-point.
Conclusions
This is the first report of a standardised, reproducible protocol
that has been subjected to a rigorous QC assessment for research
involving impetigo and could be adapted for other skin disease
research. Our study confirms non-professional photographers are
able to capture high quality digital images of skin for this purpose.
We present a simple method for capturing high quality digital
images of skin sores in a RCT and the methods used to score
digital image pairs. Future trials for management of skin
conditions, particularly in remote contexts, may benefit from
adopting this protocol.
Supporting Information
Table S1 Results of quality control (QC) check. When the
QC check was adequate, all other fields were automatically
completed as not applicable. Where the QC check score was not
interpretable, the subsequent fields of exposure, focus and
magnification were provided for the professional photographer
to give reasons for the decision.
(XLSX)
Table S2 Results of the quality assessment conductedby the primary outcome reviewers of the trial. Results
reported for simplicity are the combined quality result, as if either
one of the images were suboptimal, the image pair decision was
difficult. There were 8 reviewers in the study and quality
assessments from all reviewers are included in this table.
(XLSX)
Table S3 Dataset used to calculate inter-rater reliabil-ity and the kappa scores provided. Reviewers were
numbered 5, 6, 7, 9, 10, 12, 14 and 16. When all reviewers
scored all image pairs, the number of the reviewers selected for the
calculation is listed in columns revA_num and revB_num.
(XLSX)
Acknowledgments
We acknowledge the participants and families who participated in this trial
in the hope of finding a better treatment option for impetigo. Jane Nelson,
Deb Taylor-Thomson, Bart Currie, Malcolm McDonald and Mark
Chatfield provided critical evaluation of the methodology as it was
developed.
Author Contributions
Conceived and designed the experiments: AB ST RA RL JC. Performed
the experiments: AB KB IO. Analyzed the data: AB ST. Wrote the paper:
AB KB ST RL IO DW JC. Reviewed and approved the final version of the
manuscript: AB KB ST RA RL IO DW JC. Built databases: RL.
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Standardising Digital Images in Clinical Trials
PLOS ONE | www.plosone.org November 2014 | Volume 9 | Issue 11 | e11039510
68
CHAPTER 6THE RESULTS OF THE SKIN SORE
TRIAL
6.1 CHAPTER OVERVIEW
This chapter presents the results of the Skin Sore Trial, one of the largest
impetigo trials ever performed and the first to be conducted in remote Aboriginal
communities, where the disease burden is the highest worldwide (Chapter 2).
The trial results, now published in The Lancet, have already been incorporated
into local treatment guidelines.
It was possible to conduct a high quality, randomised controlled trial in these
remote communities due to a number of factors. These included the support from
the staff at the government and non-government remote clinics where extensive
consultation with health clinic managers and staff occurred. In all the
communities, elders or traditional owners provided consent to conduct the trial.
This facilitated participation. Finally, the commitment and dedication of the
senior trial staff, who trained and led a diverse and often transient group of
research assistants in the remote communities, was essential.
We initially proposed to recruit children presenting to the clinic for treatment of
skin sores. However, it was quickly realised that the high activity and acuity of
the clinics and the limited number of children presenting for treatment of sores,
made this approach unlikely to be successful. Ultimately, only 5% of participants
came from clinic referrals. Importantly, teachers at the local schools recognised
the need for sores to be treated and invited us in. The interest, support and active
participation of the school in each community made recruitment feasible. More
than 65% of the study participants were recruited from school settings, with
another 25% being siblings of those recruited in the school.
69
Each study trip had complicated logistics to arrange, including: coordination with
the school (avoiding holiday intervals) and with the clinic; arranging access to
accommodation and transportation within the community (both of which are in
short supply); coordinating, training and employing study staff (who had limited
longevity due to the demands of the trial) and; arranging transport schedules for
microbiology specimens to arrive back for processing at the laboratory in
Darwin. In total, 33 study trips were completed over the course of three years.
The NT is vast and sparsely populated. More than 50,000 kilometres were
travelled by road and air by the study team during the course of the study.
Recruitment commenced on 26 November 2009 and concluded on 20 November
2012. The proportion of participants recruited per study year was: 2009 (1%),
2010 (11%), 2011 (40%) and 2012 (48%). Due to challenges of conducting
research in these settings and previous experience, we allowed for up to 10% loss
to follow up in calculating our sample size. Pleasingly, more than 96% of study
participants completed all visits and of these, 97% received all doses of the study
drug.
The key findings were that: treatment success was achieved in 85% of
participants in both groups; SXT was non-inferior to BPG for the treatment of
impetigo; impetigo was driven by S. pyogenes even in the presence of S. aureus
and; BPG had a concerning rate of adverse events when administered
intramuscularly. These findings are important for Indigenous children from
remote communities. An alternative to the intramuscular BPG injection is now
available that is evidence-based, palatable, simple for adherence purposes and
pain-free. This study adds to the available evidence for treatment of extensive
impetigo.
6.2 STATEMENT OF CONTRIBUTION TO JOINTLY AUTHORED WORK
The chief investigators designed and secured funding for the trial before I joined
as a PhD student. I critically revised and oversaw the protocol, participated in
data collection, oversaw the scientific direction of the study, wrote SOPs, and
cleaned and analysed all the data. I was assisted in data cleaning by Irene
70
O’Meara and Robyn Liddle. I led the statistical analysis with input from Mark
Chatfield and Steven Tong. I conducted the literature review and wrote all drafts
of the manuscript. All co-authors contributed to revisions of the manuscript and
approved the final version for publication.
6.3 JOURNAL ARTICLE (FOLLOWING PAGE)
Short course oral cotrimoxazole versus intramuscular benzathine benzylpenicillin for impetigo in a highly, endemic region: an open label, randomised, controlled, non-inferiority trial
APPENDIX 3
van der Wouden JC and Koning S. Treatment of impetigo in resource-limited
settings. Lancet 2014, August 26.
71
CHAPTER 7THE MICROBIOLOGY OF IMPETIGO IN
INDIGENOUS CHILDREN: ASSOCIATIONSBETWEEN STREPTOCOCCUS PYOGENES,STAPHYLOCOCCUS AUREUS, SCABIES,
AND NASAL CARRIAGE
7.1 CHAPTER OVERVIEW
This chapter broadens our understanding of the microbiology of impetigo. It
reinforces the ongoing dominance of Streptococcus pyogenes as a key pathogen
in impetigo in tropical contexts, which differs to the microbiology reported from
developed contexts. Staphylococcus aureus, and increasingly methicillin-
resistant S. aureus (MRSA), are reported from these contexts where the majority
of impetigo trials have been conducted. Few high-quality microbiology studies
are conducted where the burden is the highest and it is important to know, in
2014, that treatment directed against S. pyogenes for impetigo remains a priority.
This was assumed in the only other RCT conducted in a similar resource-limited
context where severe disease was also prevalent, but no microbiology specimens
were included in that study. (Faye et al 2009)
This chapter explores the associations of the microbiology with severe disease,
the presence of scabies, age groups, sex and region. There was no association
found between children who were in the severe strata of our study (>= 2 purulent
or crusted sores and > 5 overall body sores) and the detection of S. pyogenes,
S. aureus or both. This was surprising, as our initial hypothesis was that severe
sores might be more likely to be co-infected with both pathogens. The presence
of scabies was associated with detection of S. pyogenes. Whilst this finding was
not unexpected, this is the first study to confirm the association. Swabbing of the
anterior nares was included in the study design to assess for S. aureus nasal
81
carriage. We found that there was no association between the presence of
S. aureus in the anterior nares and detecting S. aureus in impetiginous lesions. In
fact, participants without S. aureus in their skin sores were more likely to
harbour S. aureus in the anterior nares. This suggests that nasal decolonisation
strategies are unlikely to be effective in reducing the burden of impetigo in
remote Indigenous communities.
The results of antibiotic susceptibility testing for both S. pyogenes and S. aureus
are reported in more detail in this chapter. Susceptibility to trimethoprim-
sulphamethoxazole was above 99% for both skin pathogens in this study.
However, increasing use of SXT may drive increasing resistance rates, and
ongoing surveillance is a priority. Whilst performing antibiotic susceptibility
testing of S. pyogenes to SXT is not routine in Australian microbiology
laboratories, the methods to do so are available and interest is growing in
incorporating this test.
7.2 STATEMENT OF CONTRIBUTION TO JOINTLY AUTHORED WORK
I wrote all of the standard operating procedures for the collection, transportation,
storage, culture and antibiotic susceptibility testing of isolates from swabs
collected during the trial. I oversaw the scientists working in the laboratories at
the Menzies School of Health Research and Royal Darwin Hospital. I did all the
statistical analysis with assistance from Steven Tong and Mark Chatfield. I
drafted all versions of this manuscript. All co-authors contributed to revisions of
the manuscript and approved the final version for publication.
7.3 SUBMITTED JOURNAL ARTICLE, UNDER REVIEW (FOLLOWING PAGE)
The microbiology of impetigo in Indigenous children: associations of
Streptococcus pyogenes, Staphylococcus aureus, scabies and nasal carriage.
BMC Microbiology, under review.
82
Authors: Bowen AC, Tong SYC, Chatfield MD, Carapetis JR
7.4 ABSTRACT
Background: Impetigo is caused by both Streptococcus pyogenes and
Staphylococcus aureus; the relative contributions of each have been reported to
fluctuate with time and region. While S. aureus is reportedly on the increase in
most industrialised settings, S. pyogenes is still thought to drive impetigo in
endemic, tropical regions. However, few studies have utilised high quality
microbiological culture methods to confirm this assumption. We report the
prevalence and antimicrobial resistance of impetigo pathogens recovered in a
randomised, controlled trial of impetigo treatment conducted in remote
Indigenous communities of northern Australia.
Results: From 508 children, we collected 872 swabs of sores and 504 swabs
from the anterior nares prior to commencement of antibiotic therapy. S. pyogenes
and S. aureus were identified together in 503/872 (58%) of sores; with an
additional 207/872 (24%) sores having S. pyogenes and 81/872 (9%) S. aureus,
in isolation. Skin sore swabs taken during episodes with a concurrent diagnosis
of scabies were more likely to culture S. pyogenes (OR 2.2, 95% CI 1.1–4.4,
p=0.03). Eighteen percent of children had nasal carriage of skin pathogens. There
was no association between the presence of S. aureus in the nose and skin.
Methicillin-resistance was detected in 15% of children who cultured S. aureus
from either a sore or their nose. There was no association found between the
severity of impetigo and the detection of a skin pathogen.
Conclusions: S. pyogenes remains the principal pathogen in tropical impetigo;
the relatively high contribution of S. aureus as a co-pathogen has also been
confirmed. Children with scabies were more likely to have S. pyogenes detected.
While clearance of S. pyogenes is the key determinant of treatment efficacy, co-
infection with S. aureus warrants consideration of treatment options that are
effective against both pathogens where impetigo is severe and prevalent.
Trial Registration: This trial is registered; ACTRN12609000858291.
83
7.5 BACKGROUND
Impetigo is an epidermal infection caused by Staphylococcus aureus and
Streptococcus pyogenes. It is common in Indigenous children of northern
Australia, with prevalence as high as 70% (Currie and Carapetis, 2000). The
reported relative abundance of S. aureus and S. pyogenes has varied over time
(Koning et al., 2012). In recent decades, S. aureus and increasingly, methicillin-
resistant S. aureus (MRSA), has been the dominant reported pathogen in
impetigo studies worldwide, most of which have taken place in temperate-
climate regions, usually in affluent countries (Geria and Schwartz, 2010) where
there is a low burden of disease. By contrast, in tropical regions, impetigo is far
more common, and carries the greatest burden of sequelae (Carapetis et al.,
2005). S. pyogenes is assumed to have remained the dominant pathogen (Parks et
al., 2012) but some reports are emerging on skin and soft tissue infections caused
by S. aureus (Abdel Fattah and Darwish, 2012, Alvarez-Uria and Reddy, 2012).
There is limited microbiological surveillance of causative impetigo pathogens
from high burden contexts and rates of antimicrobial resistance are often
unknown (Parks et al., 2012). Impetigo is strongly associated with scabies
infestation in tropical environments (Steer et al., 2009), but the influence of
scabies on the microbiology of impetigo has not previously been described. We
report here on the microbiology of impetigo in a high-burden setting, and explore
the associations of this microbiology with age, sex, region, severity, presence of
scabies and nasal carriage of skin pathogens. Our dataset derives from a large,
non-inferiority randomised controlled trial (RCT) comparing trimethoprim-
sulphamethoxazole (SXT) with benzathine penicillin G (BPG) for the treatment
of impetigo in Indigenous children (Bowen et al., 2014).
7.6 METHODS
Study Design
Indigenous children aged 3 months to 13 years were participants in the RCT.
Children were eligible to participate on more than one occasion if at least 90 days
had elapsed since their last involvement. As such, 508 children from 12 remote
84
communities of the Northern Territory were enrolled for 663 episodes of
impetigo; all analysis presented here has been restricted to a child’s first episode
only. Six communities (comprising 463/508 children) were located in the tropical
climatic region, commonly referred to as the ‘Top End’. The remaining
communities were in Central Australia where a desert climate prevails. Children
were stratified by impetigo severity. The severe stratum included children with
≥2 purulent or crusted sores and ≥5 overall body sores.
Swabbing, transportation and culture methods
Each child had swabs taken from one or two sores (according to whether the
episode was classified as mild or severe) prior to commencing antibiotics. A
swab of the anterior nares was obtained to determine carriage of impetigo
pathogens in the context of infection. Swabs were collected between
26 November 2009 and 20 November 2012. Rayon tipped cotton swabs (Copan,
Italy) were transported at 4°C in 1 mL of skim milk tryptone glucose glycogen
broth (STGGB) and frozen at −70°C within 5 days of collection. Swabs were
defrosted, vortexed and an aliquot plated on horse blood agar and incubated for
48 hours at 37°C (Bowen et al., 2013). S. aureus and S. pyogenes were identified
morphologically and confirmed with latex agglutination. All S. aureus isolates
were staphytect (Oxoid, UK) and deoxyribonuclease (DNase, BD Diagnostics,
USA) positive. S. pyogenes agglutinated with group A Lancefield antisera
(Oxoid).
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing for S. aureus was determined on the Vitek2
platform using 22359 VITEK AST-P612 cards (bioMerieux, France) with
Clinical and Laboratory and Standards Institute (CLSI, 2011) breakpoints utilised
(CLSI, 2011). MRSA was defined as any S. aureus with a positive cefoxitin
screen. Non-multidrug resistant MRSA (nmMRSA) was defined as MRSA
resistant to <3 additional non beta-lactam antibiotics (Tong et al., 2009).
Multidrug-resistant MRSA (mMRSA) was defined as MRSA that was resistant
to ≥3 non beta-lactam antibiotics (Tong et al., 2009).
85
We performed susceptibility testing for S. pyogenes with penicillin, erythromycin
and clindamycin discs using CLSI disc diffusion standards. SXT susceptibility
for S. pyogenes was determined with an E test® (bioMerieux) according to the
European committee on antimicrobial susceptibility testing (EUCAST) standards
(www.eucast.org, last accessed 15 November 2014). SXT susceptible strains had
a MIC ≤1mg/L and resistant isolates had a MIC >2mg/L.
Ethics statement
This study was approved by the Northern Territory Department of Health and
Menzies School of Health Research human research ethics committee (09/08).
Written informed consent was obtained from a child’s parent or guardian for all
study procedures.
Statistical analysis
Mixed-effects logistic regression (random effects accounting for correlated data
due to multiple sores for the children in the severe impetigo strata) using Stata 13
(Statacorp, Texas, USA) was performed to assess associations between the
growth of skin pathogens and the presence of scabies, severe impetigo, age, sex,
region and nasal carriage.
7.7 RESULTS
Baseline Microbiology Results
Sores
We obtained 872 swabs of sores from 508 children with untreated impetigo
(median age 7 years, interquartile range [IQR] 5–9 years) at baseline. Seventy-
two percent of children were in the severe impetigo stratum, all of whom had two
sores swabbed. An impetigo pathogen was identified in 488/508 (96%) of
children. From the 872 sores swabbed, S. aureus and S. pyogenes were identified
together in 503/872 (58%), S. pyogenes alone in 207/872 (24%), and S. aureus
alone in 81/872 (9%) of sores. Swabs from children in the severe impetigo
stratum were not more likely to detect one or both skin pathogens compared with
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swabs from children in the mild impetigo stratum (Table 1). S. aureus was less
likely to be detected in older children (OR 0.6, 95% CI 0. –0.9 for children ≥ 5
years, Wald test on 2 degrees of freedom p = 0.04) or in children from Central
Australia (OR 0.5, 95% CI 0.3–0.9, p=0.02). Children from Central Australia
were less likely to have sores co-infected with both S. aureus and S. pyogenes
than children from the Top End (OR 0.5, 95% CI 0.3–0.9, p=0.01)
Table 1: Results from logistic regression models to assess associations between impetigo pathogens and age, sex, severity, presence of scabies and region.
Variable S. pyogenes in sores S. aureus in sores Both S. aureus
and S. pyogenes
in sores
MRSA in sores
positive for S.
aureus
OR 95% CI OR 95% CI OR 95% CI OR 95% CI
Female 1.3 0.8–2.1 1.0 0.7–1.4 1.1 0.8–1.5 1.0 0.6–1.7
0–4 years 1 (ref) 1 (ref) 1 (ref) 1 (ref)
5–9 years 1.1 0.7–2.0 0.6 0.4–0.9 0.8 0.5–1.1 0.9 0.5–1.6
10–13 years 1.2 0.6–2.4 0.5 0.3–0.9 0.7 0.4–1.2 0.7 0.3–1.5
Severe strata 1.4 0.8–2.6 0.7 0.4–1.1 1.1 0.7–1.7 0.6 0.3–1.1
Scabies present 2.2 1.1–4.4 0.8 0.5–1.3 0.9 0.6–1.4 1.4 0.7–2.6
Central
Australia
1.2 0.5–2.8 0.5 0.3–0.9 0.5 0.3–0.9 1.1 0.4–2.7
Scabies
Scabies was diagnosed at baseline in 84/508 (17%) of children: by age group: 0-
4 years (28/136, 21%); 5–9 years (40/271, 15%) and 10–13 years (16/101, 16%).
Those with scabies present were more likely to also have S. pyogenes detected
from sores (OR 2.2, 95% CI 1.1–4.4, p = 0.03) (Table 1).
87
Other beta haemolytic streptococci
Seven hundred and fifty-four beta haemolytic streptococci were cultured from
skin and nose swabs of 508 children with impetigo. Of these, 740/754 (98%)
were S. pyogenes with 710 from skin swabs and 30 from the nose swabs. The
remaining beta haemolytic streptococci were group C (2 skin, 2 nose) and group
G (7 skin, 3 nose).
Antibiotic susceptibility
One isolate each of S. pyogenes and S. aureus was selected per child for
reporting of the antibiotic susceptibility profile. Where possible, isolates cultured
from skin sores were selected, with the remainder coming from swabs of the
anterior nares.
S. pyogenes
There were 455 children with at least one S. pyogenes isolate available for
antibiotic susceptibility assessment. All S. pyogenes isolates were susceptible to
penicillin and erythromycin. Clindamycin resistance was detected in 9/455 (2%)
S. pyogenes isolates. SXT resistance was detected in 4/455 (<1%) isolates at
baseline with a MIC >2mg/L. (Bowen et al., 2012) The median SXT MIC for S.
pyogenes was 0.094 (interquartile range: 0.094–0.125) mg/L.
S. aureus
There were 435 children with at least one S. aureus isolate available for
antibiotic susceptibility assessment. Methicillin-resistance in S. aureus was
detected in 65/435 (15%) isolates, all of which were nmMRSA. There was no
detection of mMRSA. The respective resistance rates for other antibiotics by
child were penicillin 413/435 (95%), SXT 3/435 (<1%), erythromycin 60/435
(14%) and fusidic acid 9/435 (2%). Inducible clindamycin resistance was
reported in 60/435 (14%), 86% of which were MSSA. All 3 SXT-resistant
isolates were nmMRSA. Other antibiotics included on the VITEK card for
S. aureus susceptibility testing all had resistance rates below 0.02%. For those
88
with S. aureus detected, the presence of MRSA did not reach significance for
sex, age group, severe strata, the presence of scabies or region (table 1).
Nasal carriage of skin pathogens
S. aureus
A nasal swab was taken at baseline for 504/508 (99%) of children. Before
treatment, 91/504 (18%) children had confirmed carriage of a skin pathogen in
the anterior nares. Both S. aureus and S. pyogenes were recovered from 16/91
(18%), S. pyogenes alone from 14/91 (15%) and S. aureus alone from 61/91
(67%). Of children with nasal carriage of S. aureus, 66/77 (86%) had MSSA and
11/77 (14%), MRSA. Nasal carriage of S. aureus and S. pyogenes was
respectively 77/504 (15%) and 30/504 (6%).
We investigated the association of nasal carriage of S. aureus and the presence of
S. aureus in impetigo lesions. There were 504 children with both skin and nose
swabs available (Table 2). Surprisingly, of the 410 children culturing S. aureus
on the skin, only 54 (13%) also harboured S. aureus in the nose. Based on the
antibiogram, of the 54 with S. aureus in both the nose and skin sores, four had
discordant S. aureus strains (i.e., MSSA at one site and MRSA at the other). Of
the 94 children without S. aureus on the skin, there were 23 (24%) children who
harboured S. aureus in the nose. Of 424 children harbouring S. pyogenes on the
skin, 28 (7%) had concurrent growth of S. pyogenes from the anterior nares.
There were 2 children with isolated growth of S. pyogenes from the anterior
nares. Extending a model of Table 2, nasal colonisation with S. aureus was
associated with less, not more, S. aureus on the skin (OR 0.6, 95% CI 0.4–1.0,
p = 0.04), suggesting a separate epidemiology of S. aureus at these two sites.
89
Table2: Identification of Staphylococcus aureus from any impetigo lesion and the anterior nares for all children with at least one skin and nose swab available (n=504 children)
Anterior Nares Total
Positive Negative
Impetigo At least one sore
positive
54 (13%) 356 (87%) 410 (100%)
Negative 23 (24%) 71 (76%) 94 (100%)
Total 77 (15%) 427 (85%) 504 (100%)
7.8 DISCUSSION
S. pyogenes remains the key impetigo pathogen in Indigenous children of remote
Australia. Co-infection with S. aureus is also highly prevalent. In addition, our
study extends understanding of the microbiology of impetigo where scabies is
endemic. Where scabies is present, we found that S. pyogenes is more likely to
be recovered from impetigo lesions. We also found no positive correlation
between nasal carriage of S. aureus and recovery of S. aureus from impetigo
lesions, and no association between the severity of impetigo and recovery of
either S. pyogenes, S. aureus or both.
The stronger association of S. pyogenes (than S. aureus) with scabies presence
concurs with earlier work that linked scabies outbreaks with subsequent
epidemics of post-streptococcal glomerulonephritis (Svartman et al., 1972) and
recommended treatment of scabies at a community level to reduce the
complications of streptococcal pyoderma (Taplin et al., 1991, Carapetis et al.,
1997). Scabies association with impetigo in urban Indigenous children was
similarly high to that found in our study at a rate of 25/111 (23%) (Valery et al.,
2008). No prior studies have reported on the association between scabies and
MRSA detection. While the rates of both were high, we were unable to detect a
significant association between these.
90
Both S. pyogenes and S. aureus have been reported as key impetigo pathogens,
however the reported relative contributions of each have fluctuated over time and
region. Previous microbiology studies of impetigo in both urban and remote
Australian Indigenous children have shown high rates of co-infection (Valery et
al., 2008, McDonald et al., 2006b). Valery et al detected co-infection with skin
pathogens in 54% of urban Indigenous children. Of those children who had both
impetigo and scabies, S. pyogenes was recovered from 82% and S. aureus from
77% (Valery et al., 2008). S. pyogenes remains an important skin pathogen in our
region, as has also been reported from Fiji (Steer et al., 2009, Jenney et al., 2014)
and supports the ongoing prescription of antibiotics active against S. pyogenes
for the effective treatment of impetigo in tropical regions. Results of our clinical
trial confirmed the importance of clearing S. pyogenes in order to achieve healing
of impetigo lesions (Bowen et al., 2014).
The isolated dominance of S. aureus and rising rates of MRSA reported
elsewhere in skin and soft tissue infections (Abdel Fattah and Darwish, 2012,
Iovino et al., 2011, Phakade et al., 2012) were not found in our study.
Methicillin-resistance was detected in 15% of S. aureus isolates. This is lower
than previously reported for our remote tropical context (McDonald et al., 2006a)
and lower than recent rates found in a 20 year survey of S. aureus in our region
(Tong et al., 2014) but consistent with a study of impetigo in urban Indigenous
children of northern Australia (Valery et al., 2008). We were uncertain whether
MRSA varies by age, as this had not previously been explored, but we found no
evidence that it does. The observation that MRSA rates were stable across age
groups suggests early colonisation of children in remote communities with
MRSA strains.
We found that children in the severe strata, who had more than five purulent or
crusted sores, were not more likely than children in the mild strata to be infected
with S. pyogenes and/or S. aureus. Although those stratified as mild, indicating
fewer overall sores, may have had a less severe phenotype for each individual
sore, the microbiology of these sores was no different. We thus provide robust
evidence to refute earlier observational data in patients with impetigo where the
91
association of co-infection with a more severe phenotype was suspected but not
confirmed (Barrow, 1955, Hughes and Wan, 1967).
Whilst throat carriage of S. pyogenes is commonly reported and is not thought to
be the reservoir for skin infection, the anterior nares are thought to harbour S.
pyogenes only rarely. (Masters et al., 1958) As such, throat swabs were not
included in our study design as we focused on the characterisation of nasal
carriage of S. aureus. However, our findings of nasal carriage of S. pyogenes in
7% of children provide external validation of results from military recruits where
nasal carriage of S. pyogenes was found in 8% of those with ecthyma (Wasserzug
et al., 2009). There was no correlation between S. aureus nasal carriage and the
recovery of S. aureus from impetigo lesions. Previous studies have concluded
that when impetigo predominantly affects the lower limbs, there is either an
absence of S. aureus nasal carriage or a different genotype (Barth, 1987). Our
data supports this observation in that 67% of impetigo episodes involved only the
lower limbs (Bowen et al., 2014). As has previously been shown in military
recruits (Ellis et al., 2007), nasal decolonisation is also unlikely to be a useful
strategy in reducing the burden of impetigo in our tropical, endemic setting.
One study from Ghana in the 1970s with a similar tropical climate found
impetigo to be dominated by Lancefield groups C and G streptococci (Belcher et
al., 1975). These findings were not reproduced in our study and have not been
confirmed in other published microbiology studies from our region (McDonald et
al., 2006b) or other tropical contexts (Lawrence et al., 1979). Non-group A
streptococci do not appear to play a significant role in the pathogenesis of
impetigo.
A strength of this study is the standardisation of procedures for screening,
swabbing and microbiological culture in the context of a clinical trial conducted
according to International Conference on Harmonisation Good Clinical Practice
guidelines. In addition, the large number of children recruited from 12 distinct
communities from two regions of the Northern Territory suggests that the
conclusions are rigorous. Genotypic assessment of the isolates was not conducted
to determine the correlation of skin and nose isolates. The inability to correlate
the molecular epidemiology with the phenotype is a limitation of this study.
92
While the cost of molecular typing overall has become more affordable, the large
number of isolates in this study made further molecular analysis cost prohibitive.
7.9 CONCLUSIONS
S. pyogenes remains a key pathogen in impetigo in tropical contexts, despite the
rise in S. aureus found in many industrialised and tropical settings. Our findings
are in keeping with those reported from Fiji (Steer et al., 2009) and confirm that
impetigo in tropical contexts continues to be driven by S. pyogenes. In the
absence of local microbiology for impetigo, treatment algorithms should remain
focused on the treatment of S. pyogenes. However, we have demonstrated that
co-infection with both S. pyogenes and S. aureus is likely. As such, consideration
of treatment of impetigo with an agent that is effective against both S. pyogenes
and S. aureus is important. We have also concluded that in the context of
impetigo, there is no association between nasal colonisation and skin infection
with S. aureus.
Competing Interests
The authors declare that they have no competing interests.
Authors Contributions
AB, ST and JC participated in the design of the study. AB analysed the data and
drafted the manuscript with assistance from ST and MC. All authors read and
approved the final version of the manuscript.
Acknowledgements
This work was supported through a National Health and Medical Research
Council of Australia (NHMRC) project grant (545234) on which AB, ST, and JC
are all investigators. The funder had no role in the design, collection, analysis or
interpretation of the data, writing of the manuscript or decision to submit the
manuscript for publication. AB is the recipient of a NHMRC scholarship for PhD
research (605845) as well as an Australian Academy of Sciences Douglas and
93
Lola Douglas scholarship. ST is the recipient of an NHMRC Career
Development Fellowship (1065736).
We acknowledge the children and families who contributed to this trial in the
hope of finding a better treatment option for their sores, and the staff of the
Menzies School of Health Research and Royal Darwin Hospital microbiology
laboratory who contributed to data collection, culturing and antibiotic
susceptibility testing of all isolates. We acknowledge the co-investigators on this
study, Bart Currie, Ross Andrews and Malcolm McDonald. Malcolm McDonald
and Rob Baird provided feedback on the manuscript in development.
94
CHAPTER 8CONCLUSIONS
8.1 CHAPTER OVERVIEW
In this final chapter, the overall findings of the thesis are presented, conclusions
drawn and recommendations for future research outlined. I have reviewed the
global prevalence of impetigo and pyoderma using available population-based
studies. This has extended our understanding of the prevalence of impetigo at a
community level and, once published, should enhance the global understanding
of this high burden, but relatively unstudied, problem in the world’s poor. The
review also highlighted the ongoing problem that, despite living in a high-
income, developed economy, Australia’s Indigenous children have the highest
published rates of impetigo in the world.
There has been a long-standing dogma that trimethoprim-sulphamethoxazole
(SXT) is not active or effective in the treatment of Streptococcus pyogenes
infections. It was therefore important to re-look at the microbiology from which
this dogma arose, and use standardised susceptibility testing to confirm in vitro
susceptibility of S. pyogenes to (SXT). Chapter 3 describes this work and
confirms that S. pyogenes isolates from the NT are susceptible to SXT. The
accompanying literature review in Chapter 3 outlines the variability worldwide in
susceptibility of S. pyogenes to SXT, ranging from 0 to 100%. Much of this
variability can be attributed to the presence of thymidine, in media used for
susceptibility testing, acting as an inhibitor to SXT and thus appearing resistant.
The standardisation of thymidine content at a very low level, or addition of lysed
horse blood to susceptibility testing media, are two strategies that have been used
to overcome this appearance of resistance. This work was needed to better
understand the results of our clinical trial, and to legitimise the use of SXT as an
effective agent in the treatment of impetigo. Likewise, ongoing monitoring of
S. pyogenes susceptibility to SXT is needed, as this antibiotic is more widely
prescribed for impetigo.
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In addition, we addressed methodological issues that were critical to the
successful operation of the Skin Sore Trial in a very remote context. We
compared a cheap, simple and effective transport medium that is likely to be
cost-effective and efficient in resource-limited contexts for future impetigo
research, to a known standard. Previous guidelines have recommended
immediate plating of swabs. (Kotloff, 2008) This approach has microbiological
merit, but is difficult to achieve in remote settings with long distances to the
laboratory. As such, the microbiological description of impetigo where the
burden is highest is under-reported. The ability to perform high-quality
microbiology studies by transporting stored swabs back to a central laboratory
for later processing builds our understanding of the local microbiology and
antibiogram in endemic settings and, thus, makes uncovering the molecular
epidemiology achievable.
Digital images are useful for capturing and storing an endpoint when it is not
feasible to transport an expert to the point of care. These rigorous, standardised
methods for scoring digital images will be useful in future impetigo and other
skin disease trials.
Pleasingly, our in vivo results confirmed the in vitro finding of S. pyogenes being
susceptible to SXT, with the demonstration of non-inferiority of SXT to BPG for
the treatment of impetigo. In addition, clearance of S. pyogenes was equivalent
between these two antibiotics, with penicillin having known excellent efficacy
against S. pyogenes. No isolate of S. pyogenes has ever been reported that is
resistant to penicillin. The demonstration of equivalent clearance with both
antibiotics upholds the efficacy of SXT for S. pyogenes.
Surprisingly, in the MRSA era, our study confirmed the historical observations
made over four decades ago that S. pyogenes is the dominant pathogen driving
impetigo and that S. aureus is in fact a co-coloniser. We have shown that
benzathine penicillin G (BPG), an antibiotic with limited S. aureus activity but
very active against S. pyogenes, was effective in the treatment of impetigo, even
in the presence of high rates of S. aureus including MRSA. As such, MRSA-
targeted treatment for impetigo is not essential, but using an MRSA-active
antibiotic such as SXT may have the additional benefit of reducing S. aureus and
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MRSA skin carriage in a region where the incidence of sequelae of both gram-
positive pathogens is high. The increased use of SXT for treatment of impetigo
may see a reduction in the consequences of both pathogens on the short- and
long-term morbidity in Indigenous children.
Many questions remain and future studies, described below, are underway that
will continue to illuminate our understanding of this ubiquitous problem of
Indigenous childhood. The ultimate goal is that sores are not ignored, and are
effectively treated, with the outcome of this being a dramatic decline in the
prevalence of impetigo such that inter-personal transmission is no longer self-
sustaining.
8.2 DISCUSSION OF MAIN FINDINGS
8.2.1 Trimethoprim/sulphamethoxazole is effective against Streptococcus pyogenes in vitro
The current recommendation by the Infectious Diseases Society of America
(IDSA) when using SXT for treatment of skin and soft tissue infections involving
both S. pyogenes and S. aureus, is to include a second agent that is active against
S. pyogenes e.g. penicillin, cephalexin or amoxicillin. (Stevens et al., 2014) Our
findings dispute the need for a second antibiotic and are important. The use of a
single antibiotic when appropriate, rather than combination therapy, is a key
principle in antimicrobial stewardship. (SHEA et al., 2012) It is also a critical
factor in adherence to an oral treatment regimen, where the simplest, shortest
regimen, that is directly associated with symptoms, has the best correlation with
adherence. (Perez-Gorricho and Ripoll, 2003)
8.2.2 Trimethoprim/sulphamethoxazole is non-inferior to benzathine penicillin G for impetigo
The work in Chapter 3 paved the way for a complete understanding of the results
of our trial outlined in Chapter 6. We concluded, using the robust digital image
methodology described in Chapter 5, that SXT is non-inferior to BPG for the
treatment of skin sores. In both treatment arms, treatment success was achieved
in 85% of participants, absolute difference 0.5% (95% CI -6.2 to +7.3%).
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(Bowen et al., 2014) We set the non-inferiority margin at 10%, to detect a
clinically relevant difference, if one existed. There were two SXT dosing arms in
the study, one treating children twice a day for three days (six doses) and the
other once a day for five days (five doses). The regimens, pooled together and
also analysed separately, both reached significance with respect to the non-
inferiority margin and as such either can be recommended with confidence.
This provides a palatable, effective, short course, non-painful alternative
treatment to intramuscular BPG for children with impetigo in remote Indigenous
communities. The challenge now is to see these findings implemented into
practice. As discussed in Chapter 1, the CARPA manual is well utilised
throughout our region as the standard treatment manual. The 6th edition has
recently been published (October 2014) and the results of our study have been
included. (CARPA, 2014) The results have also been included in the national
antibiotic reference, Therapeutic Guidelines: Antibiotic, released in November
2014 (Antibiotic Expert Group, 2014) and in treatment guidelines in the
Kimberley, the northern region of Western Australia with a large, remote,
Indigenous population. SXT now sits in regional and national guidelines as an
effective treatment for impetigo, based on the results of our trial.
Our study also confirmed, for the first time in our region, that BPG remains an
effective treatment for impetigo. This is important, as there are circumstances
where BPG will remain the preferred treatment for impetigo. Clinic nurses and
clinicians in the region are familiar with prescribing and administering BPG, and
the simplicity of a single dose without the need to address adherence, has appeal.
This was the feedback received when our results were presented in health-care
settings in remote communities. As such, BPG remains as first-line therapy in
both the guidelines mentioned above, with the introduction of SXT as an
alternate, evidence-based option. Future evaluation of the uptake of both
antibiotics will be needed to understand the full impact of our study on
community-based health care.
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8.2.3 Trimethoprim/sulphamethoxazole is effective against S. pyogenes in vivo
Supporting our findings of in vitro efficacy outlined in Chapter 3, are the in vivo
results reported in Chapter 6. As outlined above, SXT is an effective antibiotic in
the treatment of impetigo caused by S. pyogenes. To be confident of any results
reported from our trial (and allay concerns that adherence would be poor), all
doses of antibiotics were directly observed. More than 96% of children had all
doses of study drug observed. This strengthens our findings. Likewise, we have
shown that S. pyogenes is cleared from infected sores with either BPG or SXT.
Culture positive rates before treatment were above 85% for S. pyogenes and by
day 7, S. pyogenes could be detected in less than 7% of sores in any of the three
treatment arms. (Bowen et al., 2014) The clearance of S. pyogenes from impetigo
lesions with BPG treatment is well known from earlier studies. (Ferrieri et al.,
1973) The declining detection of S. pyogenes on days 2 and 7 with both
antibiotics, with no difference in the rate of decline between BPG and SXT,
provides supportive data to confirm the in vivo efficacy of SXT.
8.2.4 S. pyogenes remains the driver of impetigo even in the MRSA era
We have confirmed that impetigo is driven by S. pyogenes, with clearance of
S. pyogenes being the only factor associated with sore healing in a step-wise
backwards logistic regression (OR 5.2, 95% CI 1.8–14.1). However, S. aureus is
frequently found in association with S. pyogenes as reported in Chapter 7. Whilst
severe outcomes can ensue if S. aureus is untreated, the focus for the interruption
of transmission of impetigo is on S. pyogenes. Our trial was designed mindful of
the rising prevalence of MRSA in both the hospital and community in the NT.
Our results suggest the significance of MRSA, as an impetigo driver, is lower
than previously thought.
8.2.5 Trimethoprim/sulphamethoxazole has fewer side effects than benzathine penicillin G
Our study confirmed very few side effects from SXT. In the 343 children treated
with SXT, we documented one case of urticarial rash without mucosal
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involvement that resolved within 24 hours. Three older children were unable to
tolerate the large volumes of SXT syrup administered using weight-based dosing
(40ml per day). When switched to tablets, they continued in the trial without
further problems. One child had persistent vomiting and was discontinued. SXT
has been identified in the global literature as an antibiotic with a concerning side-
effect profile, (Goldman et al., 2013) and we were closely monitoring for these
events. We did not see any rash heralding the potentially life-threatening Stevens
Johnson syndrome. However the study was not powered to detect uncommon
side effects. Given the high prevalence of impetigo in remote communities in our
region, ongoing education about and monitoring for this rare, but life-
threatening, side effect will be needed.
The high rate of adverse events in the BPG arm of the trial was somewhat of a
surprise. Whilst anecdotal reports abound of the painful intramuscular injection
being a deterrent to treatment, the high rate of reported pain at 48 hours –
affecting one in three children randomised to the BPG arm – is concerning.
Likewise, treatment refusal by four children randomised to the BPG arm
confirms the impression that the painful needle is a deterrent to treatment. It is
possible that the 22 children who declined to participate in the trial did so for
similar reasons. Early experience of the painful intramuscular injection for sores
and a recurring need for treatment, with a median of three visits per year in
infants, (Kearns et al., 2013) in addition to the other childhood needles for
immunisation, might have conditioned children to these responses. By one year
of age, almost all infants in five remote communities had documented skin sores.
(Kearns et al., 2013) Having an oral regimen that circumvents the painful
injection is important to preserve intramuscular antibiotic injections for
circumstances where there is currently no alternative e.g. rheumatic heart disease
secondary prophylaxis. (Wyber, 2013)
The early studies that introduced BPG to the arena of treatment for impetigo
were in the context of acute post-streptococcal glomerulonephritis (APSGN)
outbreaks, where treatment that was easy to deliver and long acting was a
priority. (Ferrieri et al., 1974) APSGN epidemics are also problematic in the NT,
occurring every 5–7 years (Marshall et al., 2011). In the context of the high
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burden of impetigo in the NT, and a limited evidence base for the treatment of
extensive impetigo, treatment that was effective during and possibly effective
between APSGN epidemics was adopted. Our study has confirmed that BPG is
an effective antibiotic for the treatment of impetigo, and confirmed the anecdotal
reports that the injection is a painful limitation to the use of this antibiotic. Where
an alternative, effective antibiotic exists with fewer adverse events, we would
recommend it in preference with antimicrobial resistance surveillance in place.
However, there will remain circumstances where health care providers or parents
prefer to administer BPG, and our study has confirmed that this remains an
effective option.
8.2.6 Australian Indigenous children continue to have the highest reported rates of impetigo in the world
Chapter 2 summarises the prevalence of impetigo globally. We searched for
population-based studies from anywhere in the world, however all available
studies were conducted in resource-poor countries or contexts. This reinforces
the connection between poverty and high rates of impetigo in children. In this
context, it is unacceptable that Australian Indigenous children have the highest
reported prevalence of impetigo in the world. Australia is a high-income country
and must do more to reduce this burden of disease.
In the NT, epidemiological surveys of bacterial skin infection and scabies have
been occurring for more than 20 years, with no real change in the prevalence.
This imposes a considerable, ongoing burden on primary health care (Kearns et
al., 2013, McMeniman et al., 2011) and we have estimated almost 16,000
children are in need of treatment in remote communities at any one time. Geo-
political borders do not restrict impetigo in remote Indigenous children; the
burden of disease is equally high in Western Australia and Queensland. The next
steps will involve developing a coordinated strategy for recognising and treating
skin infections across these jurisdictions, with routine evaluations.
Clearly, effective treatment that is simple, palatable and has few adverse events
is needed in our region. Communicating these findings is well underway and
translation into regional guidelines has occurred. The responsibility for future
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research lies in continuing to evaluate both the prevalence and utilisation of this
new regimen, with the hope that this population will not be world leaders for
impetigo prevalence in the future.
8.2.7 Methodologies to take impetigo research to high burden contexts
Previous impetigo trials have mostly been conducted in physician offices or
hospital outpatient settings. (Koning et al., 2012) In order to conduct this trial in
the remote communities of Australia, several methods were developed and
published. This contribution to the global literature on the methodologies for
impetigo research reported in Chapters 4 and 5 will hopefully facilitate adoption
of treatment studies in similar contexts, where the burden of disease is the
highest. We intentionally published these papers in journals that are more likely
to come to the attention of researchers in these contexts; either open-access or
tropical medicine journals. In the digital era, experts are no longer needed at the
point of care for assessing end-points, and could easily be located in Darwin,
New York or Bamako to confirm findings of studies using digital images. We
have developed a method for collecting and scoring digital images. The next step
is to validate this method. As most impetigo trials to date have been conducted in
hospital outpatient or office-based settings where experts are readily available for
end-point assessment, it may be possible to validate this method in a future
impetigo trial. This would involve comparing the point-of-care expert assessment
with the remote review of digital images by a similarly qualified expert. All
reviewers would remain blinded to the prescribed treatment.
Likewise, the transport of skin swabs from remote contexts to a central
laboratory benefits from the use of a transport medium. We used one that was
familiar in our laboratory, and validated it against the commercially-available
Amies transport medium. The benefit of STGGB as a transport medium is the
ability to freeze for longer periods in order to facilitate batching of specimens
and preserve bacteria for future molecular work.
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8.3 STUDY LIMITATIONS
8.3.1 Antiseptic or topical body washes?
In designing our trial, serious consideration was given to having a non-antibiotic
arm, one that used only topical antiseptic. Partly because of concerns around the
safety of not treating bacterial skin infection with antibiotics, but mainly to keep
our sample size feasible and our findings simple and easily interpretable, this arm
was not incorporated into the study design. Hygiene is very important in
managing impetigo as was shown in Mozambique, where access to water
influenced the burden of disease. (Cairncross and Cliff, 1987) In addition, hand-
washing studies have shown a significant decline in impetigo using soap and
water. (Luby et al., 2002, Luby et al., 2005) However, it is still unclear whether
antiseptics or topical body washes would add considerable value as an adjunct to
hygiene measures and treatment, (Koning et al., 2012) but these are unlikely to
cause harm.
Neither were traditional, Indigenous methods for controlling skin disease
evaluated in our trial. Bush medicine is still practised and many grandmothers
and elders asked questions about these treatments when we visited their
communities. Incorporating both worldviews into a regional treatment strategy to
reduce the burden of skin disease will be an important step to consider in
engaging the community.
8.3.2 Placebo control
Our trial lacked a placebo arm. It was not considered ethical with the high
disease burden and complications of impetigo seen in the NT. Few placebo-
controlled trials for impetigo exist, (Koning et al., 2012) and we deemed that the
best design was a non-inferiority study, comparing the new treatment with the
standard of care.
8.3.3 Adherence concerns
There has always been uncertainty about adherence to oral antibiotics and other
prescribed medicines in Indigenous Australians. (Burns, 1992, Kemp K, 1994,
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McConnel, 2003) This is a challenge when prescribing treatment for any patient,
but has an added complexity for Indigenous patients, due to language and
worldview differences between prescriber and patient. (McConnel, 2003) The
known strategies for achieving treatment adherence such as prescribing a simple,
short regimen, temporally linked to the disease process, are the most likely to be
successful. (Perez-Gorricho and Ripoll, 2003) In childhood, palatability of syrup
formulations is of equal importance. (Liu et al., 2014) These principles guided
the selection of an oral regimen to compare with an intramuscular injection. As
such, both oral regimens trialled were simple, palatable, short and easy to
administer. In our study, directly observed treatment was incorporated to
reinforce the message that adherence is important and to ensure our results were
valid.
The once daily regimen has the benefit of being simple enough to administer via
a directly observed approach at the school or health clinic in the remote
community, if there are ongoing concerns about treatment adherence. This would
be possible with a five-day course that coincides with the standard working week
when both the clinic and school would be open, but may not be feasible or
acceptable to families. This highlights an avenue for future qualitative research to
understand whether uptake of these strategies is occurring and if not, what
obstacles need to be overcome.
SXT is stable at room temperature (20–25ºC), but temperatures well above this
occur in most remote communities of the NT on a daily basis and where access
to a refrigerator is not universal, concerns abound about the efficacy of oral
treatment. SXT was stored in air-conditioned environments monitored by the
research assistants throughout the trial, and as such further work is needed to
understand the impact this may have on treatment efficacy.
8.3.4 Poverty and overcrowding
Whilst poverty, racism, household overcrowding and social disadvantage exist, it
is unclear whether a simple treatment regimen will on its own improve skin
disease. These primordial factors need to be addressed in an ongoing program of
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reducing the gap in health outcomes between Indigenous and non-Indigenous
Australians.
8.3.5 Antibiotic resistance
Recent work in synthesizing the antibiotic resistance patterns of S. aureus from
swabs collected in remote communities across the NT has shown a concerning
trend. During the period under investigation (1993–2012), more than 20,000
S. aureus isolates were collected. The more recent years showed a doubling in
the SXT resistance, albeit from a very low baseline value. (Tong et al., 2014b)
This requires ongoing evaluation, and studies are being developed to validate
both pharmaceutical prescriptions and changes in resistance phenotypes as
guidelines incorporate the SXT regimen into the standard of care.
8.4 FUTURE WORK
8.4.1 Does the presence of scabies impact treatment efficacy?
In Chapter 7 we showed that the presence of scabies in children with impetigo
was associated with an increased likelihood of detecting S. pyogenes from sores.
As both regimens trialed are active against S. pyogenes, we hypothesised that
treatment efficacy when analysed by the presence or absence of scabies would be
similar. Preliminary analysis suggests the opposite, in that children with scabies
co-infection had fewer treatment successes scored than children without scabies.
Surprisingly, this also revealed that the absolute difference in treatment success
for impetigo-scabies with BPG was 18.4% (95% CI -1 to 38%), whereas with
SXT it was only 7% (-4 to 19%). The lower overall treatment success for
impetigo-scabies and smaller difference between groups with and without
scabies, treated with SXT (rather than BPG), warrants further investigation. It is
possible that SXT might have added benefits in the treatment of impetigo when
scabies is also present. Current animal models could be used to determine
whether activity of SXT against scabies might explain some of these findings.
(Holt and Fischer, 2013)
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8.4.2 Whole genome sequencing to detect resistance mechanisms
Antibiotic resistance monitoring is an essential component of further research to
understand the changes in microbiology that occur with increasing antibiotic
pressure. However, we currently do not know the resistance mechanisms for
either organism against SXT for isolates from our region. There are several
possible molecular techniques that can be used to determine resistance
mechanisms. Of these, we assessed whole genome sequencing (WGS) to be the
most cost effective and efficient. In addition, the bioinformatics computer
platform and collaborators with experience in detecting resistance mechanisms
are also based at Menzies. We plan to use WGS to illuminate the possible
genotypic mechanisms of SXT resistance in skin sore isolates that are
phenotypically resistant. All SXT-resistant S. pyogenes and S. aureus isolates
have been sub-cultured and DNA extracted, then submitted for WGS on the
Illumina HiSeq2000 platform by Macrogen in Korea. Resulting data will be used
to determine the mechanism of resistance in these isolates. This question has
importance, as it will impact on the likely selective pressure of introducing SXT
into routine treatment of skin sores in the NT, based on the results of our trial.
There were 8 (<0.5%) S. pyogenes isolates resistant to SXT detected in the
overall study. The S pyogenes resistance was identified using an SXT E test
according to the EUCAST methodology reported in Chapter 3. Factors that cause
trimethoprim resistance in S. pyogenes have recently been described in isolates
from India and Germany. (Bergmann et al., 2014) We will explore whether these
horizontally transferrable, trimethoprim-insensitive dihydrofolate reductase (dfr)
genes are also found in our SXT resistant S. pyogenes isolates using WGS.
Likewise, S. aureus resistance to SXT was at a low level in our study with only
24 (1%) S. aureus isolates resistant to SXT. The S. aureus resistance was
identified using VITEK2 platform, with CLSI breakpoints as reported in Chapter
7. The correlation between the resistance phenotype and genotype will guide
future assessments of antibiotic resistance.
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8.4.3 Understanding the dynamics of skin sore transmission using WGS
The burden of impetigo suggests transmission of impetigo pathogens is common
in Indigenous communities. However, little work has been conducted on defining
the transmission dynamics in an endemic setting. We hypothesised that
transmission within a household is more common than elsewhere in the
community, in an endemic setting. If so, interventions to reduce household
crowding are most likely to be effective in reducing the burden of impetigo in
remote Indigenous communities. To explore this hypothesis, we have selected 54
isolates of S. pyogenes and S. aureus from 11 household clusters within the same
community on the same recruitment trip in November 2011 to sequence. As
such, we hope to understand how closely related these strains are and to build on
this by developing methods for inferring transmission. These models are needed
to determine the most appropriate interventions to reduce transmission, without
conducting expensive interventional studies. This work on transmission
dynamics can be built on the wealth of isolates, epidemiological data points and
outcomes of studies conducted in remote NT communities by Menzies
researchers over the past 20 years.
8.4.4 The niche for Staphylococcus argenteus?
Researchers at Menzies School of Health Research have recently described a
new lineage of Staphylococcus in the Northern Territory of Australia. (Tong et
al., 2013, Tong et al., 2014a) In light of this, we hypothesised that S. argenteus
might be better adapted to skin infection rather than nasal colonisation. To
explore this, multi-locus sequence typing (MLST) was performed on paired
samples of S. aureus from the nose and skin of participants from the Skin Sore
Trial. This hypothesis might explain why S. argenteus was more likely to be
found in community rather than hospital samples in previous work. Preliminary
work showed the rate of S. argenteus in our study samples to be very low, with
no significant difference between skin and nasal specimens.
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8.4.5 Evaluations of uptake
Our study was designed to find a better treatment option for impetigo, based on
an assumption that pain may be a limitation in the acceptance of treatment for
skin sores. However, health-care workers in the NT express a strong preference
for the use of an injection for treatment of sores due to concerns about adherence.
In a recent education session with remote doctors, comments about the injection
ranged from “the mothers come wanting the needle as they don’t want to muck
around with syrup or tablets” to “some mums won’t come to the clinic because
they don’t want their kids to get the needle”. These assumptions have not been
explored with parents or children in remote communities. There is a gap in our
knowledge that affects the translation of this research into practice. To explore
the uptake of our findings, qualitative interviews and monitoring of pharmacy
dispensing records will be helpful. While children and parents do not decide the
prescribed treatment when they seek care, qualitative research will guide the
implementation of guidelines and development of policy from our results. Future
studies are needed to evaluate the acceptance of this new regimen by both
prescribers and families.
It was important for clinicians in the region to have data to confirm BPG is
effective for the treatment of impetigo, despite high rates of S. aureus and
MRSA. Concerns about non-adherence to a short course antibiotic may drive the
ongoing prescription of BPG. However, as was seen in our trial, children may
vote with their feet, and run away if and when the needle is mentioned. Oral SXT
now has a strong evidence base for its efficacy and can be recommended with
confidence.
8.4.6 Ongoing surveillance
The increased use of any antibiotic causes increasing selection pressure and may
drive resistance. Likewise, the introduction of a resistant clone could also
compromise this regimen. (Nickerson et al., 2011) Ongoing surveillance is
necessary, both to assess whether a more palatable treatment is able to reduce the
burden of impetigo and what happens to antibiotic resistance profiles when this is
widely prescribed. Surveillance for the development of antimicrobial resistance
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in S. aureus and S. pyogenes to SXT will occur through routine microbiological
channels, using both hospital and community pathology specimens throughout
the region.
Our findings have highlighted the efficacy of SXT in S. pyogenes skin infections,
have outlined the complex history that resulted in the myth being perpetuated and
highlighted an available methodology with breakpoints for antibiotic
susceptibility testing to facilitate ongoing surveillance for the emergence of SXT
resistance.
Included in this will be ongoing surveillance of the incidence and prevalence of
APSGN and RHD over the long term to see whether there is any impact on these
severe complications of impetigo.
8.4.7 The ‘denormalisation’ of sores: A coordinated approach for recognition and treatment of skin disease in remote Indigenous children throughout Australia
Skin sores are common, rarely deemed serious enough to seek or obtain
treatment, highly transmissible and hence found at epidemic levels in many
remote Indigenous communities. The current paradigm is of skin infection being
normal rather than the exception. A paradigm shift is needed to achieve the de-
normalisation of skin sores. The next steps will involve developing a coordinated
strategy across jurisdictions for recognising and treating skin infections that is
routinely evaluated.
8.5 FINAL CONCLUSIONS
The studies described in this thesis have contributed to our understanding of the
burden, microbiology and efficacy of treatment of extensive impetigo. This fills a
gap in the impetigo literature on evidence-based treatments for extensive
impetigo. Our finding that impetigo is driven by S. pyogenes concurs with the
only other trial on the treatment of extensive impetigo, which subjectively
concluded that S. pyogenes was important. The methodology for conducting
impetigo studies in remote circumstances where disease burden is often the
highest has also been reported. This will provide a framework for future studies
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on the treatment of impetigo for children who need it the most. As is evident in
the systematic review (Koning et al., 2012), there is no generally agreed upon
standard treatment for impetigo and as such, treatments that are appropriate for
different contexts should continue to be trialled, perhaps using some of the
methodologies we have reported.
Chapter 2 summarises the prevalence of impetigo in many resource-poor
communities throughout the world, and highlights the ongoing burden suffered
by remote Indigenous children of Australia. The association of impetigo with
poverty make these rates unacceptable in a high-income country like Australia.
Clearly, effective treatment that is simple, palatable and has few adverse events
is needed in our region. These findings have been communicated throughout the
region and translated into treatment guidelines. In support of this, ongoing health
education and health promotion activities are needed to maintain a focus on skin
disease. In conjunction, surveillance of changes in the prevalence, microbiology
and antibiotic resistance profile as utilisation of a new regimen increases are
needed. These studies are in development, with the hope that Indigenous children
will not be world leaders for impetigo prevalence in the future.
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APPENDICES
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148
APPENDIX 1
Table: Overall and childhood pyoderma and scabies prevalence from 89 studies synthesised in the Chapter 2 systematic review.
Overall Children (0—15 years)
Country Year Number studied
Number with
pyoderma
Pyoderma prevalence
(%) Number studied
Number with
pyoderma Pyoderma prevalence
Scabies prevalence
India 1967 488 49 10.0 488 49 10.0 NS
USA* 1969 39 16 41.0 39 16 41.0 NS
USA* 1970 444 235 52.9 444 235 52.9 NS
USA* 1970 31 6 19.4 31 6 19.4 NS
Colombia 1971 1,269 169 13.3 1,269 169 13.3 NS
USA* 1971 80 12 15.0 80 12 15.0 NS
Panama 1972 892 135 15.1 628 111 17.7 0.60%
Panama 1972 131 17 13.0 NS NS NS NS
India 1973 6,641 392 5.9 6,641 392 5.9 NS
Tanzania 1973 532 37 7.0 532 37 7.0 31%
New Zealand* 1974 453 6 1.3 453 6 1.3 0
India 1974 1,038 176 17.0 1,038 176 17.0 Common
Brazil 1974 9,955 1211 12.2 9,955 1,211 12.2 3%
Ghana 1975 3,770 731 19.4 NS NS NS NS
Tanzania 1975^ 794 38 4.8 794 38 4.8 12.90%
Tanzania 1975^ 305 22 7.2 305 22 7.2 0.70%
Tanzania 1975^ 120 8 6.7 120 8 6.7 3.30%
Tanzania 1975^ 636 60 9.4 636 60 9.4 31.60%
Brazil 1976 775 87 11.2 307 64 20.8 NS
India 1976 89 11 12.4 89 11 12.4 NS
India 1976 19,775 586 3.0 19,775 586 3.0 NS
Gambia 1976 974 87 8.9 332 58 17.5 2.50%
USA* 1976 593 79 13.3 593 79 13.3 NS
USA* 1976 434 63 14.5 434 63 14.5 NS
India 1977 1,890 79 4.2 1,890 79 4.2 8.20%
Gambia 1977 945 68 7.2 387 39 10.1 2.00%
USA* 1977 535 104 19.4 535 104 19.4 NS
India 1978 2,771 59 2.1 1,509 48 3.2 13%
Pakistan 1980 444 86 19.4 444 86 19.4 1.90%
India 1982 4,133 431 10.4 NS NS NS 0.12%
Nigeria 1983 2,407 87 3.6 2,407 87 3.6 NS
Fiji 1983^ 61 14 23.0 61 14 23.0 33%
Canada* 1984 258 2 0.8 258 2 0.8 NS
149
Overall Children (0—15 years)
Country Year Number studied
Number with
pyoderma
Pyoderma prevalence
(%) Number studied
Number with
pyoderma Pyoderma prevalence
Scabies prevalence
Canada* 1984 378 8 2.1 378 8 2.1 NS
Solomon Islands 1984 10,224 4,370 42.7 5,160 2678 51.9 1.30%
Canada* 1985 239 10 4.2 239 10 4.2 NS
Canada* 1985 202 2 1.0 202 2 1.0 NS
Canada* 1985 336 13 3.9 336 13 3.9 NS
Canada* 1985 334 2 0.6 334 2 0.6 NS
India 1986^ 3,697 39 1.1 3,697 39 1.1 0.50%
Indi 1988 666 107 16.1 666 107 16.1 0.90%
Vanuatu 1989 18,223 2133 11.7 9,569 1537 16.1 16%
Ethiopia 1989 1,842 417 22.6 1,842 417 22.6 1.70%
Australia* 1990 120 52 43.3 120 52 43.3 NS
Tanzania 1991 936 178 19.0 NS NS NS 5.98%
Honduras 1992 206 43 20.9 206 43 20.9 10%
Ethiopia 1992 112 6 5.4 112 6 5.4 17%
Australia* 1992^ 180 31 17.2 180 31 17.2 NS
Australia* 1993 583 198 34.0 583 198 34.0 NS
Mali 1993 1,817 224 12.3 1,817 224 12.3 4.30%
Kenya 1993 5,780 735 12.7 5,780 735 12.7 8.30%
Ecuador 1993 495 22 4.4 495 22 4.4 NS
Tanzania 1994 1,114 18 1.6 NS NS NS 4.50%
Australia* 1994 81 39 48.1 81 39 48.1 NS
Australia* 1994 126 62 49.2 62 43 69.4 28.80%
Kenya 1995 4,358 470 10.8 4,358 470 10.8 10.50%
Nepal 1995 458 76 16.6 458 76 16.6 NS
Australia* 1995 79 71 89.9 79 71 89.9 23%
Malaysia 1995^ 41 8 19.5 41 8 19.5 NS
Solomon Islands
1997 226 91 40.3 226 91 40.3 25%
Taiwan 1998 3,029 72 2.4 3,029 72 2.4 1.40%
Malaysia 1998^ 356 24 6.7 NS NS NS 11.90%
Samoa 1998^ 8,767 3,822 43.6 8,767 3822 43.6 4.90%
Kenya 1999 4,961 563 11.3 4,961 563 11.3 8.00%
Australia* 2000 217 49 22.6 217 49 22.6 35%
Australia* 2000 121 80 66.1 121 80 66.1 5.0%
India 2000 4,249 28 0.7 4,249 28 0.7 NS
Egypt 2001 636 99 15.6 636 99 15.6 0.50%
Egypt 2001 720 6 0.8 720 6 0.8 0.10%
India 2001 12,586 925 7.3 12,586 925 7.3 5%
Mali 2001 1,729 310 17.9 1,729 310 17.9 1.30%
150
Overall Children (0—15 years)
Country Year Number studied
Number with
pyoderma
Pyoderma prevalence
(%) Number studied
Number with
pyoderma Pyoderma prevalence
Scabies prevalence
Mali 2002 1,632 304 18.6 1,632 304 18.6 0.70%
Turkey 2002^ 785 16 2.0 785 16 2.0 2.16%
Tanzania 2003 820 69 8.4 820 69 8.4 1.50%
Fiji 2004 258 6 2.3 258 6 2.3 32.6%
Australia* 2004 582 266 45.7 582 266 45.7 16.10%
Ghana 2004 463 20 4.3 463 20 4.3 0.00%
Gabon 2005 454 7 1.5 454 7 1.5 0.70%
Fiji 2006 3,462 1259 36.4 3,462 1259 36.4 18.50%
Fiji 2006 451 60 13.3 451 60 13.3 14%
Timor Leste 2007 1,535 112 7.3 728 82 11.3 17%
Ghana 2007 1,394 81 5.8 1,394 81 5.8 0.10%
Rwanda 2007 2,528 32 1.3 2,528 32 1.3 0.04%
Nepal 2008^ 878 54 6.2 NS NS NS 3.40%
Nigeria 2009^ 1,415 14 1.0 1,415 14 1.0 0.60%
Australia* 2009^ 2,001 804 40.2 2,001 804 40.2 NS
Cameroon 2010 400 43 10.8 48 2 4.2 1.80%
Ethiopia 2010^ 1,104 29 2.6 1,104 29 2.6 0.30%
Tanzania 2010^ 420 17 4.0 393 16 4.1 1.40%
*Resource-poor populations within high-income OECD countries, ^reflectsyear of publication when year study was commenced is unknown.
151
152
APPENDIX 2
GELFAND MS, CLEVELAND KO, KETTERER DC. SUSCEPTIBILITY OF STREPTOCOCCUS PYOGENES TO TRIMETHOPRIM-SULFAMETHOXAZOLE. JOURNAL OF CLINICAL MICROBIOLOGY. 2013; 51(4): 1350.
BOWEN AC, TONG SY, CARAPETIS JR. REPLY TO “SUSCEPTIBILITY OF STREPTOCOCCUS PYOGENES TO TRIMETHOPRIM-SULFAMETHOXAZOLE.” JOURNAL OF CLINICAL MICROBIOLOGY. 2013; 51(4): 1351.
153
Susceptibility of Streptococcus pyogenes to Trimethoprim-Sulfamethoxazole
Michael S. Gelfand, Kerry O. Cleveland, Daniel C. Ketterer
Department of Medicine, Division of Infectious Diseases, University of Tennessee Health Science Center, Memphis, Tennessee, USA
With interest we read the recent paper by Bowen et al. in whichthey propose that detection of in vitro susceptibility of Strep-
tococcus pyogenes to trimethoprim-sulfamethoxazole (TMP-SMX) is enhanced by testing on media containing low concentra-tions of thymidine (1). Whether their data on in vitrosusceptibility can be extrapolated to the clinical use of TMP-SMXin clinical infections due to this organism is unclear.
Traditional teaching in infectious diseases and microbiologyhas suggested that S. pyogenes is largely resistant to TMP-SMX,and, in fact, the drug has been incorporated into selective mediafor isolation of S. pyogenes from throat cultures (2–4).
Recently we observed two patients with skin and soft tissueinfections due to S. pyogenes who were treated with oral TMP-SMX and suffered adverse outcomes.
The first patient, a 40-year-old previously healthy man, pre-sented with a hand wound related to an occupational cut. After thewound was cleaned, he was discharged on TMP-SMX (160 mg ofTMP component twice daily). He returned 48 h later with a handinfection that had progressed to necrotizing fasciitis. Wound andblood cultures grew S. pyogenes. Susceptibility testing with TMP-SMX was not done.
The second patient, a 38-year-old woman with multiple skinabscesses, was treated with surgical debridement and tigecycline(50 mg intravenous [i.v.] administration twice daily) for 3 daysfollowed by oral TMP-SMX (160 mg of TMP component twicedaily) for 6 days. A wound culture grew S. pyogenes (no suscepti-bility testing for TMP-SMX was performed) and methicillin-sen-sitive Staphylococcus aureus (MRSA) susceptible to TMP-SMX.She expired after presenting in septic shock 24 h after discharge.
In vitro susceptibility of S. pyogenes to TMP-SMX is less rele-vant than the clinical efficacy of this agent in treating infectionsdue to that organism. Thymidine may become available to infect-ing microorganisms from damaged host tissues and bacteria, al-lowing microorganisms to overcome the metabolic block (5).Clinical failures in the treatment of MRSA infections have beenattributed to this mechanism, and animal model data in MRSAinfections support this explanation (5).
Human sera and urine contain detectable concentrations ofthymidine and folates (6, 7). Even when an organism is demon-strated to have in vitro susceptibility to the drug, use of TMP-SMXhas been discouraged for enterococcal urinary tract infections be-cause of concern about exogenous thymidine and folates beingavailable to the infecting organism (7). As pointed out, TMP-SMX
has not proven to be an effective agent for treatment of strepto-coccal pharyngitis (1).
We urge caution in the use of TMP-SMX for S. pyogenes skinand soft tissue infections before the availability of human clinicalstudies. The study of impetigo being conducted by the authorsmay not resolve the question of clinical utility of TMP-SMX forthese infections, as spontaneous resolution of impetigo is not un-common (8). Data from animal models of infection would bedesirable in helping to resolve this question.
ACKNOWLEDGMENTS
We declare that we have no conflicts of interest.No financial support was received for this work.
REFERENCES1. Bowen AC, Lilliebridge RA, Tong SY, Baird RW, Ward P, McDonald MI,
Currie BJ, Carapetis JR. 2012. Is Streptococcus pyogenes resistant or sus-ceptible to trimethoprim-sulfamethoxazole? J. Clin. Microbiol. 50:4067–4072.
2. Bisno AL, Stevens DL. 2009. Streptococcus pyogenes, p 2593–2610. In Man-dell GL, Bennett JE, Dolin R (ed), Mandell, Douglas, and Bennett’s princi-ples and practice of infectious diseases, 7th ed, vol 2. Churchill LivingstoneElsevier, Philadelphia, PA.
3. Milatovic D. 1981. Comparison of five selective media for beta-haemolyticstreptococci. J. Clin. Pathol. 34:556 –558.
4. Mirrett S, Monahan JS, Reller LB. 1987. Comparative evaluation of me-dium and atmosphere of incubation for isolation of Streptococcus pyogenes.Diagn. Microbiol. Infect. Dis. 6:217–221.
5. Proctor RA. 2008. Role of folate antagonists in the treatment of methicillin-resistant Staphylcoccus aureus infection. Clin. Infect. Dis. 46:584 –593.
6. Holden L, Hoffbrand AV, Tattersall MH. 1980. Thymidine concentra-tions in human sera: variations in patients with leukaemia and megaloblas-tic anaemia. Eur. J. Cancer 16:115–121.
7. Wisell KT, Kahlmeter G, Giske CG. 2008. Trimethoprim and enterococciin urinary tract infections: new perspectives on an old issue. J. Antimicrob.Chemother. 62:35– 40.
8. Koning S, van der Sande R, Verhagen AP, van Suijlekom-Smit LW,Morris AD, Butler CC, Berger M, van der Wouden JC. 2012. Interven-tions for impetigo. Cochrane Database Syst. Rev. 1:CD003261. doi:10.1002/14651858.CD003261.pub3.
Address correspondence to Kerry O. Cleveland, [email protected].
For the author reply, see doi:10.1128/JCM.03373-12.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.03329-12
LETTER TO THE EDITOR
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Reply to “Susceptibility of Streptococcus pyogenes to Trimethoprim-Sulfamethoxazole”
Asha C. Bowen,a Steven Y. C. Tong,a Jonathan R. Carapetisb
Menzies School of Health Research, Darwin, Northern Territory, Australiaa; Telethon Institute for Child Health Research, Perth, Western Australia, Australiab
We thank Gelfand et al. (1) for their interest in our article onthe in vitro susceptibility of Streptococcus pyogenes to trim-
ethoprim-sulfamethoxazole (SXT) (2). We agree that the twocases outlined by Gelfand et al. act as cautionary points for clinicalpractice, as there are currently no good clinical trial data to sup-port the use of SXT for the treatment of S. pyogenes skin and softtissue infections (SSTI). Our aim in publishing the in vitro data isto challenge the misconception that all S. pyogenes isolates areinherently resistant to SXT and hence open the way for clinicaltrials to more precisely determine the role for SXT in the treat-ment of S. pyogenes infections.
In the two clinical scenarios presented by Gelfand et al., theabsence of SXT susceptibility data for the S. pyogenes isolates isconcerning. Other reasons for treatment failure that are not elu-cidated include incomplete adherence, poor absorption, and thereality that the conditions of both of these patients might havedeteriorated regardless of the antibacterial used. One wonderswhy the authors prescribed SXT for these SSTI despite their un-derstanding of the traditional teaching that S. pyogenes is largelyresistant to SXT. Perhaps it was due to the need for a simple,palatable, oral antibacterial regimen that would treat undifferen-tiated SSTI, in the era of highly methicillin-resistant Staphylococ-cus aureus (MRSA) prevalence. We are in agreement with Gelfandet al. that human clinical studies are urgently needed to respond tothis challenge.
The results of our clinical trial comparing oral SXT with intra-muscular benzathine penicillin for impetigo treatment will in-form treatment decisions and generate confidence in the use ofSXT for impetigo treatment in our region and possibly beyond. Todate, with recruitment of 660 participants completed, despite two-thirds of the participants presenting with severe impetigo, nonehave been hospitalized with invasive S. pyogenes infection due topresumed treatment failure. Although the numbers in our studyare not large enough to detect a trend for bacteremia or othercomplications of inadequate treatment, we might have seen clin-ical outcomes similar to those Gelfand et al. have experienced if
SXT really has no role in the treatment of impetigo. Spontaneousresolution of mild impetigo may occur; however, the natural his-tory of impetigo, particularly when severe, is neither completelyunderstood nor benign (3).
We agree that the role of thymidine and its availability in thecontext of damaged host tissues (4) are unknown with respectto the treatment of SSTI with SXT. There are now numerous trialsunder way that involve the use of SXT for SSTI (http://clinicaltrials.gov/), and the results are eagerly awaited to betterdefine the clinical utility of SXT in such settings.
Rather than claiming in vitro data being less relevant than clin-ical efficacy, we would argue that in vitro data should be used toinform the design of appropriate clinical trials to determine theclinical role for SXT, particularly in an era with a truncated anti-microbial pipeline and high rates of mixed SSTI due to MRSA andS. pyogenes.
REFERENCES1. Gelfand MS, Cleveland KO, Ketterer DC. 2013. Susceptibility of Strepto-
coccus pyogenes to trimethoprim-sulfamethoxazole. J. Clin. Microbiol. 51:1350.
2. Bowen AC, Lilliebridge RA, Tong SYC, Baird R, Ward P, MacDonald M,Currie B, Carapetis JR. 2012. Is Streptococcus pyogenes resistant or sus-ceptible to trimethoprim-sulfamethoxazole? J. Clin. Microbiol. 50:4067–4072.
3. Koning S, van der Sande R, Verhagen AP, van Suijlekom-Smit LW,Morris AD, Butler CC, Berger M, van der Wouden JC. 2012. Interven-tions for impetigo. Cochrane Database Syst. Rev. 1:CD003261. doi:10.1002/14651858.pub3.
4. Proctor RA. 2008. Role of folate antagonists in the treatment of methicillin-resistant Staphylococcus aureus infection. Clin. Infect. Dis. 46:584 –593.
Address correspondence to Asha C. Bowen, [email protected].
This is a response to a letter by Gelfand et al. (doi:10.1128/JCM.03329-12).
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.03373-12
AUTHOR REPLY
Journal of Clinical Microbiology p. 1351April 2013 Volume 51 Number 4 jcm.asm.org 1351
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APPENDIX 3
VAN DER WOUDEN J AND KONING S. TREATMENT OF IMPETIGO IN RESOURCE-LIMITED SETTINGS. LANCET 2014, AUGUST 26.
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Comment
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Treatment of impetigo in resource-limited settingsWith an estimated worldwide prevalence of more than 140 million cases in 2010, impetigo is one of the seven skin disorders ranking in the 50 most common causes of disease.1 After dermatitis or eczema and viral warts, impetigo is the third most common skin disorder in children, and the most common skin infection in young children presenting in general practice in western Europe.2,3 In tropical ar eas, impetigo can be endemic and aff ect a large proportion of the population.4,5 Several Lancet articles have reported on the treatment of impetigo in endemic populations, notably in soldiers in World Wars 1 and 2, including two controlled (although not randomised) clinical trials.6–8
Indigenous Australian people, like other resource-poor populations in tropical areas, have a raised risk of severe complications associated with impetigo, such as post-streptococcal glomerulonephritis, sepsis, and bone and joint infections.9 In The Lancet, Asha Bowen and colleagues10 present the results of their randomised, controlled non-inferiority trial in Indigenous children in remote communities in the Northern Territory of Australia, in which they compared a short course of oral co-trimoxazole to the standard treatment with intramuscular benzathine benzylpenicillin.10 Unlike most previous impetigo studies, more than 70% of the children had severe impetigo (fi ve or more sores), and the lower limbs were the main aff ected area.
508 patients were randomly assigned to a study treatment: 165 to benzathine benzylpenicillin, 175 to twice-daily co-trimoxazole for 3 days, and 168 to once-daily co-trimoxazole for 5 days. The trial’s primary outcome was treatment success at day 7, objectively assessed by comparison of photographs of the sores at day 0 and day 7 by assessors who were masked to treatment and to the order of the photographs. Treatment success was achieved in 133 (85%) of 156 children who received benzathine benzylpenicillin, and in 283 (85%) of 334 who received co-trimoxazole (absolute diff erence 0·5% [95% CI –6·2 to 7·3]), falling within the prespecifi ed margin of non-inferiority (±10%). Results for the two treatment schedules of co-trimoxazole were similar. The study also underlines the key pathogenic role of Streptococcus pyogenes in impetigo in remote communities in Australia, instead of Staphylococcus aureus as seen over past decades in moderate climates. Adverse event profi les diff ered
substantially between the treatment groups, with 30% of children in the benzylpenicillin group reporting injection-site pain, and one hospital admission due to an injection-site abscess, compared with vomiting and rash reported by less than 1% of children who received co-trimoxazole.
Bowen and colleagues’ study,10 with 508 randomised patients, is one of the largest impetigo studies done so far. Our Cochrane review11 included 68 randomised trials, with an average of only 82 patients per study, and only one of the included studies had randomised more than 500 patients with impetigo. Additionally, this new study is one of the few randomised impetigo trials done in a tropical setting for the benefi t of an underprivileged population. Adherence to the prescribed treatment regimens, and follow-up to day 7 with very low attrition, are also commendable.
Cure rates were compared with those in the study done in Mali by Faye and colleagues,12 because Mali is a country where impetigo is also endemic and severe. However, the prevalence of disease might be less important for cure rates than for the risk of reinfection and development of complications.
A question not addressed by Bowen and colleagues’ study10 is whether successful treatment of severe impetigo reduces the risk of developing severe complications such as glomerulonephritis and sepsis, as is often believed to be the case, since this reduction in development of secondary complications is an important motive for treatment.
In conclusion, we believe that Bowen and colleagues’ new study10 in a resource-poor area makes a valuable contribution to the body of evidence about treatment of an important but under-investigated disease. The option of simple, palatable, pain-free, practical treatment of severe impetigo with co-trimoxazole is a welcome result for Indigenous children living in remote communities in Australia, and provides clinicians and patients with a simple regimen for treatment of impetigo in tropical regions where S pyogenes is the main causative agent.
*Johannes C van der Wouden, Sander KoningDepartment of General Practice and Elderly Care Medicine, EMGO Institute for Health and Care Research, VU University Medical Center, 1007 MB, Amsterdam, Netherlands (JCvdW); and Department of General Practice, Erasmus MC, University Medical Center Rotterdam, Netherlands (SK)[email protected]
Published OnlineAugust 27, 2014http://dx.doi.org/10.1016/S0140-6736(14)61395-7
See Online/Articleshttp://dx.doi.org/10.1016/S0140-6736(14)60841-2
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Streptococcus pyogenes
www.thelancet.com Published online August 27, 2014 http://dx.doi.org/10.1016/S0140-6736(14)61395-7
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Comment
2 www.thelancet.com Published online August 27, 2014 http://dx.doi.org/10.1016/S0140-6736(14)61395-7
We declare no competing interests.
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2 Fleming DM, Cross KW, Barley MA. Recent changes in the prevalence of diseases presenting for health care. Br J Gen Pract 2005; 55: 589–95.
3 Mohammedamin RSA, van der Wouden JC, Koning S, et al. Increasing incidence of skin disorders in children? A comparison between 1987 and 2001. BMC Dermatol 2006; 6: 4.
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10 Bowen AC, Tong SYC, Andrews RM, et al. Short-course oral co-trimoxazole versus intramuscular benzathine benzylpenicillin for impetigo in a highly endemic region: an open-label, randomised, controlled, non-inferiority trial. Lancet 2014; published online Aug 27. http://dx.doi.org/10.1016/S0140-6736(14)60841-2.
11 Koning S, van der Sande R, Verhagen AP, et al. Interventions for impetigo. Cochrane Database Syst Rev 2012; 1: CD003261.
12 Faye O, Hay RJ, Diawara I, Mahé A. Oral amoxicillin vs. oral erythromycin in the treatment of pyoderma in Bamako, Mali: an open randomized trial. Int J Dermatol 2007; 46: 19–22.
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