Edited by

Stuart H. Ralston Ian D. Penman Mark W. J. Strachan Richa rd P. Hobson

Ill

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Principles and Practice of

Medicine

Sir Stanley Davidson (1894–1981)

This famous textbook was the brainchild of one of the great Professors of Medicine of the 20th century. Stanley Davidson was born in

Sri Lanka and began his medical undergraduate training at Trinity College, Cam bridge; this was interrupted by World War I and later

resumed in Edinburgh. He was seriously wounded in battle, and the carnage and shocking wa ste of young life that he encountered at that

time had a profound effect on his subsequent attitudes and values.

In 1930 Stanley Davidson was appointed Professor of Medicine at the University of A berdeen, one of the rst full-time Chairs of

Medicine anywhere and the rst in Scotland. In 1938 he took up the Chair of Medicine at Ed inburgh and was to remain in this post until

retirement in 1959. He was a renowned educator and a particularly gifted teacher at t he bedside, where he taught that everything had to

be questioned and explained. He himself gave most of the systematic lectures in Medicin e, which were made available as typewritten

notes that emphasised the essentials and far surpassed any textbook available at the time.

Principles and Practice of Medicine was conceived in the late 1940s with its origins in those lecture notes . The first edition, published in

1952, was a masterpiece of clarity and uniformity of style. It was of modest size and pr ice, but sufciently comprehensive and up to date

to provide students with the main elements of sound medical practice. Although the form at and presentation have seen many changes in

22 subsequent editions, Sir Stanley’s original vision and objectives remain. More than hal f a century after its rst publication, his book

continues to inform and educate students, doctors and health professionals all over the wor ld.

Principles and Practice of

23rd Edition

Edited by

Stuart H Ralston

MD, FRCP, FMedSci, FRSE, FFPM(Hon)

Arthritis Research UK Professor of Rheumatology,

University of Edinburgh; Honorary Consultant Rheumatologist,

Western General Hospital, Edinburgh, UK

Ian D Penman

BSc(Hons), MD, FRCPE

Consultant Gastroenterologist,

Royal Inrmary of Edinburgh;

Honorary Senior Lecturer, University of Edinburgh, UK

Mark WJ Strachan

BSc(Hons), MD, FRCPE

Consultant Endocrinologist,

Metabolic Unit, Western General Hospital, Edinburgh;

Honorary Professor, University of Edinburgh, UK

Richard P Hobson

LLM, PhD, MRCP(UK), FRCPath

Consultant Microbiologist,

Harrogate and District NHS Foundation Trust;

Honorary Senior Lecturer, University of Leeds, UK

Illustrations by Robert Britton

Illustrations and boxes in Chapter 8 © Julian White.

No part of this publication may be reproduced or transmitted in any form or by any me ans, electronic or mechanical, including

photocopying, recording, or any information storage and retrieval system, without perm ission in writing from the publisher. Details on

how to seek permission, further information about the publisher’s permissions policies a nd our arrangements with organizations such as

the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions.

This book and the individual contributions contained in it are protected under copyright by the publisher (other than as may be noted

herein).

First edition 1952

Second edition 1954

Third edition 1956

Fourth edition 1958

Fifth edition 1960

Sixth edition 1962

Seventh edition 1964

Eighth edition 1966

ISBN 978-0-7020-7028-0 International ISBN 978-0-7020-7027-3 Ninth edition 1 968

Tenth edition 1971

Eleventh edition 1974 Twelfth edition 1977 Thirteenth edition 1981 Fourteenth edition 1 984 Fifteenth edition 1987 Sixteenth edition

1991

Notices

Seventeenth edition 1995 Eighteenth edition 1999 Nineteenth edition 2002 Twentieth ed ition 2006

Twenty-rst edition 2010 Twenty-second edition 2014 Twenty-third edition 2018

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information,

methods, compounds or experiments described herein. Because of rapid advances in th e medical sciences, in particular, independent

verication of diagnoses and drug dosages should be made. To the fullest extent of th e law, no responsibility is assumed by Elsevier,

authors, editors or contributors for any injury and/or damage to persons or property as a matter of products liability, negligence or

otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

The

publisher’s

policy is to use

paper manufactured from sustainable forests

Content Strategist: Laurence Hunter Content Development Specialist: Wendy Le e Content Coordinator: Susan Jansons

Printed in China Project Manager: Louisa Talbott Last digit is the p rint number: 9 8 7 6 5 4 3 2 1 Designer: Miles Hitchen

Contents

Preface ix Contributors xi International Advisory Board xv Acknowledgements xvii Introduction xix

PART 1 FUNDAMENTALS OF MEDICINE 1

1. Clinical decision-making 1

N Cooper, AL Cracknell

2. Clinical therapeutics and good prescribing 13

SRJ Maxwell

3. Clinical genetics 37

K Tatton-Brown, DR FitzPatrick

4. Clinical immunology 61

SE Marshall, SL Johnston

5. Population health and epidemiology 91

H Campbell, DA McAllister

6. Principles of infectious disease 99

JAT Sandoe, DH Dockrell

PART 2 EMERGENCY AND CRITICAL CARE MEDICINE 131

7. Poisoning 131

SHL Thomas

8. Envenomation 151

J White

9. Environmental medicine 163

M Byers

10. Acute medicine and critical illness 173

VR Tallentire, MJ MacMahon

vi • CONTENTS

PART 3 CLINICAL MEDICINE 215

11. Infectious disease 215

DH Dockrell, S Sundar, BJ Angus

12. HIV infection and AIDS 305

G Maartens

13. Sexually transmitted infections 329

GR Scott

14. Clinical biochemistry and metabolic medicine 345

A Mather, L Burnett, DR Sullivan, P Stewart

15. Nephrology and urology 381

B Conway, PJ Phelan, GD Stewart

16. Cardiology 441

DE Newby, NR Grubb

17. Respiratory medicine 545

PT Reid, JA Innes

18. Endocrinology 629

MWJ Strachan, JDC Newell-Price

19. Nutritional factors in disease 691

AG Shand, JPH Wilding

20. Diabetes mellitus 719

ER Pearson, RJ McCrimmon

21. Gastroenterology 763

E El-Omar, MH McLean

22. Hepatology 845

QM Anstee, DEJ Jones

23. Haematology and transfusion medicine 911

HG Watson, DJ Culligan, LM Manson

24. Rheumatology and bone disease 981

GPR Clunie, SH Ralston

25. Neurology 1061

JP Leach, RJ Davenport

26. Stroke medicine 1147

P Langhorne

27. Medical ophthalmology 1163

J Olson

28. Medical psychiatry 1179

RM Steel, SM Lawrie

29. Dermatology 1209

SH Ibbotson

30. Maternal medicine 1269

L Mackillop, FEM Neuberger

CONTENTS • vii

31. Adolescent and transition medicine 1287

R Mann

32. Ageing and disease 1301

MD Witham

33. Oncology 1313

GG Dark

34. Pain and palliative care 1337

LA Colvin, M Fallon

35. Laboratory reference ranges 1357

SJ Jenks

Index 1365

Well over two million copies of Davidson’s Principles and Practice of Medicine have been sold s ince it was rst published in 1952. Now

in its 23rd Edition, Davidson’s is regarded as a ‘must-have’ textbook for thousand s of medical students, doctors and health professionals

across the world, describing the pathophysiology and clinical features of the m ost important conditions encountered in the major

specialties of adult medicine and explaining how to investigate, diagnose and manage the m. The book is the winner of numerous prizes

and awards and has been translated into many languages. Taking its origins from Sir St anley Davidson’s much-admired lecture notes, the

book has endured because it continues to keep pace with how modern medicine is taught and to provide a wealth of information in an

easy-to-read, concise and beautifully illustrated format.

Davidson’s strives to ensure that readers can not only recognise the clinical features of a disease but also understand the underlying

causes. To achieve this, each chapter begins with a summary of the relevant p re-clinical science, linking pathophysiology with clinical

presentation and treatment so that students can use the book from the outset of their medical studies right through to their nal

examinations and beyond.

The regular introduction of new authors and editors is important for maintaining freshn ess. On this occasion, Professor Mark Strachan

and Dr Richard Hobson have come on board as editors, and 26 new authors have join ed our existing contributors to make up an

outstanding team of authorities in their respective elds. As well as recruiting auth ors from around the globe, particularly for topics such

as infectious diseases, HIV and envenomation, we welcome members from 17 coun tries on to our International Advisory Board. These

leading experts provide detailed comments that are crucial to our revision of each new e dition. A particularly important aspect in

planning the revision is for the editors to meet students and faculty in medical school s in those countries where the book is most widely

read, so that we can respond to the feedback of our global readership and the ir tutors. We use this feedback, along with the information

we gather via detailed student reviews and surveys, to craft each edition. The auth ors, editors and publishing team aim to ensure that

readers all over the world are best served by a book that integrates medical science with clinical medicine to convey key knowledge and

practical advice in an accessible and readable format. The amount of detail is tailored to t he needs of medical students working towards

their final examinations, as well as candidates preparing for Membership of the Royal C olleges of Physicians (MRCP) or its equivalent.

With this new edition we have introduced several changes in both structure an d content. The opening six chapters provide an account of

the principles of genetics, immunology, infectious diseases and population health, along w ith a discussion of

Preface

the core principles behind clinical decision-making and good prescribing. Subsequent c hapters discuss medical emergencies in

poisoning, envenomation and environmental medicine, while a new chapter explores co mmon presentations in acute medicine, as well as

the recognition and management of the critically ill. The disease-specic chapters that fol low cover the major medical specialties, each

one thoroughly revised and updated to ensure that readers have access to the ‘cutting e dge’ of medical knowledge and practice. Two new

chapters on maternal and adolescent/transition medicine now complement the one on ageing a nd disease, addressing particular problems

encountered at key stages of patients’ lives. Medical ophthalmology is also now included as a direct response to readers’ requests.

The innovations introduced in recent editions have been maintained and, in many cases, developed. The highly popular ‘Clinical

Examination’ overviews have been extended to the biochemistry, nutrition and dermato logy chapters. The ‘Presenting Problems’

sections continue to provide an invaluable overview of the most common presentations in each disease area. The ‘Emergency’ and

‘Practice Point’ boxes have been retained along with the ‘In Old Age’, ‘In Pregnancy’ and ‘In Adolescence’ boxes, which emphasise key

practical points in the presentation and management of the elderly, women with medical disord ers who are pregnant or planning

pregnancy, and teenagers transitioning between paediatric and adult services.

Education is achieved by assimilating information from many sources and reader s of this book can enhance their learning experience by

using several complementary resources. We are delighted to have a new self-testing companion book entitled Davidson’s Assessment in

Medicine, containing over 1250 multiple choice questions specically tailored to the contents of Davidson’s. The long-standing

association of Davidson’s with its sister books, Macleod’s Clinical Examination (now in its 14th Edition) and Principles and Practice of

Surgery (7th Edition), still holds good. Our ‘family’ has also expanded with the pu blication of Davidson’s Essentials of Medicine, a long-

requested pocket-sized version of the main text; Davidson’s 100 Clinical Cases, which contains scenarios directly based on our

‘Presenting Problems’; and Macleod’s Clinical Diagnosis, which d escribes a systematic approach to the differential diagnosis of

symptoms and signs. We congratulate the editors and authors of these books for contin uing the tradition of easily digested and expertly

illustrated texts.

We all take immense pride in continuing the great tradition first established by Sir Stan ley Davidson and in producing an outstanding

book for the next generation of doctors.

SHR, IDP, MWJS, RPH Edinburgh 2018

Brian J Angus BSc(Hons), DTM&H, FRCP, MD, FFTM(Glas)

Associate Professor, Nufeld Department of Medicine, University of Oxford, UK

Quentin M Anstee BSc(Hons), PhD, FRCP

Professor of Experimental Hepatology, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne; Honorary Consultant

Hepatologist, Freeman Hospital, Newcastle upon Tyne, UK

Leslie Burnett MBBS, PhD, FRCPA, FHGSA

Chief Medical Ofcer, Genome.One, Garvan Institute of Medical Research, Sydn ey; Conjoint Professor, St Vincent’s Clinical School,

UNSW; Honorary Professor in Pathology and Genetic Medicine, Sydney Med ical School, University of Sydney, Australia

Mark Byers OBE, FRCGP, FFSEM, FIMC, MRCEM Consultant in Pre-Hospi tal Emergency Medicine, Institute of Pre-Hospital

Care, London, UK

Harry Campbell MD, FRCPE, FFPH, FRSE

Professor of Genetic Epidemiology and Public Health, Centre for Global Health Research, U sher Institute of Population Health Sciences

and Informatics, University of Edinburgh, UK

Gavin PR Clunie BSc, MD, FRCP

Consultant Rheumatologist and Metabolic Bone Physician, Cambridge University Hospitals NHS Foundation Trust, Addenbrooke’s

Hospital, Cambridge, UK

Lesley A Colvin BSc, FRCA, PhD, FRCPE, FFPMRCA Consultant, Department of Anaesthesia, Critical Care and Pain Medicine,

Western General Hospital, Edinburgh; Honorary Professor in Anaesthesia and Pain Me dicine, University of Edinburgh, UK

Bryan Conway MB, MRCP, PhD

Senior Lecturer, Centre for Cardiovascular Science, University of Edinburgh; H onorary Consultant Nephrologist, Royal Inrmary of

Edinburgh, UK

Nicola Cooper FAcadMEd, FRCPE, FRACP

Consultant Physician, Derby Teaching Hospitals NHS Foundation Trust, Derby; Honorary Cl inical Associate Professor, Division of

Medical Sciences and Graduate Entry Medicine, University of Nottingham, U K

Contributors

Alison L Cracknell FRCP

Consultant, Medicine for Older People, Leeds Teaching Hospitals NHS Trust, Leeds; Honorary Clinical Associate Professor, University

of Leeds, UK

Dominic J Culligan BSc, MD, FRCP, FRCPath

Consultant Haematologist, Aberdeen Royal Inrmary; Honorary Senior Lecturer, University of Aberdeen, UK

Graham G Dark FRCP, FHEA

Senior Lecturer in Medical Oncology and Cancer Education, Newcastle University, Ne wcastle upon Tyne, UK

Richard J Davenport DM, FRCPE, BMedSci

Consultant Neurologist, Royal Inrmary of Edinburgh and Western General Hospital, Edinbur gh; Honorary Senior Lecturer, University

of Edinburgh, UK

David H Dockrell MD, FRCPI, FRCPG, FACP

Professor of Infection Medicine, Medical Research Council/ University of Edinburgh C entre for Inammation Research, University of

Edinburgh, UK

Emad El-Omar BSc(Hons), MD(Hons), FRCPE, FRSE, FRACP

Professor of Medicine, St George and Sutherland Clinical School, University of New South Wale s, Sydney, Australia

Marie Fallon MD, FRCP

St Columba’s Hospice Chair of Palliative Medicine, University of Edinburgh, UK

David R FitzPatrick MD, FMedSci

Professor, Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, UK

Neil R Grubb MD, FRCP

Consultant in Cardiology, Royal Inrmary of Edinburgh; Honorary Senior Le cturer in Cardiovascular Sciences, University of Edinburgh,

Sally H Ibbotson BSc(Hons), MD, FRCPE

Professor of Photodermatology, University of Dundee; Honorary Consultant Derma tologist and Head of Photobiology Unit, Ninewells

Hospital and Medical School, Dundee, UK xii • CONTRIBUTORS

J Alastair Innes BSc, PhD, FRCPE

Consultant, Respiratory Unit, Western General Hospital, Edinburgh; Honorary Reader in Resp iratory Medicine, University of Edinburgh,

Sara J Jenks BSc(Hons), MRCP, FRCPath Consultant in Metabolic Medicine, D epartment of Clinical Biochemistry, Royal Inrmary

of Edinburgh, UK

Sarah L Johnston FCRP, FRCPath

Consultant Immunologist, Department of Immunology and Immunogenetics, N orth Bristol NHS Trust, Bristol, UK

Simon RJ Maxwell BSc, MD, PhD, FRCP, FRCPE, FBPhS, FHEA

Professor of Student Learning – Clinical Pharmacology and Prescribing, and Medical Director, UK Prescribing Safety Assessment,

Clinical Pharmacology Unit, University of Edinburgh, UK

David A McAllister MSc, MD, MRCP, MFPH

Wellcome Trust Intermediate Clinical Fellow and Beit Fellow, Senior Clinical Lecturer in Epidem iology, and Honorary Consultant in

Public Health Medicine, University of Glasgow, UK

David EJ Jones MA, BM, PhD, FRCP

Professor of Liver Immunology, Institute of Cellular Medicine, Newcastle Univ ersity, Newcastle upon Tyne; Consultant Hepatologist,

Freeman Hospital, Newcastle upon Tyne, UK

Rory J McCrimmon MD, FRCPE

Professor of Experimental Diabetes and Metabolism, University of Dundee, UK

Peter Langhorne PhD, FRCPG, FRCPI

Professor of Stroke Care, Institute of Cardiovascular and Medical Sciences, University of Gl asgow, UK

Mairi H McLean BSc(Hons), MRCP, PhD

Senior Clinical Lecturer in Gastroenterology, School of Medicine, Medical Scienc es and Nutrition, University of Aberdeen; Honorary

Consultant Gastroenterologist, Aberdeen Royal Inrmary, UK

Stephen M Lawrie MD(Hons), FRCPsych, FRCPE(Hon) Professor of Psychiatry, U niversity of Edinburgh, UK

John Paul Leach MD, FRCPG, FRCPE

Consultant Neurologist, Institute of Neuroscience, Queen Elizabeth University Hospital, Glasg ow; Head of Undergraduate Medicine and

Honorary Associate Clinical Professor, University of Glasgow, UK

Gary Maartens MBChB, FCP(SA), MMed

Professor of Clinical Pharmacology, University of Cape Town, South Africa

Lucy Mackillop MA(Oxon), FRCP

Consultant Obstetric Physician, Oxford University Hospitals NHS Foundation Trust, Oxfor d; Honorary Senior Clinical Lecturer,

Nufeld Department of Obstetrics and Gynaecology, University of Oxford, UK

Francesca EM Neuberger MRCP(UK)

Consultant Physician in Acute Medicine and Obstetric Medicine, Southmead Hospital, Bristo l, UK

David E Newby BA, BSc(Hons), PhD, BM DM DSc, FMedSci, FRSE, FESC, FA CC

British Heart Foundation John Wheatley Professor of Cardiology, British Heart Foundatio n Centre for Cardiovascular Science,

University of Edinburgh, UK

John DC Newell-Price MA, PhD, FRCP

Professor of Endocrinology and Consultant Endocrinologist, Department of Oncology and M etabolism, University of Shefeld, UK

John Olson MD, FRPCE, FRCOphth

Consultant Ophthalmic Physician, Aberdeen Royal Inrmary; Honorary Reader, Universi ty of Aberdeen, UK

Michael J MacMahon FRCA, FICM, EDIC Consultant in Anaesthesia and Intensive Ca re, Victoria Hospital, Kirkcaldy, UK

Ewan R Pearson MA, PhD, FRCPE

Professor of Diabetic Medicine, University of Dundee, UK

Rebecca Mann BMedSci, MRCP, FRCPCh Consultant Paediatrician, Taunton and Somerset NHS Foundation Trust, Taunton, UK

Paul J Phelan MD, FRCPE

Consultant Nephrologist and Renal Transplant Physician, Royal Inrmary of Edin burgh; Honorary Senior Lecturer, University of

Edinburgh, UK

Lynn M Manson MD, FRCPE, FRCPath

Consultant Haematologist, Scottish National Blood Transfusion Service, Department of Tran sfusion Medicine, Royal Inrmary of

Edinburgh, UK

Sara E Marshall FRCP, FRCPath, PhD

Professor of Clinical Immunology, Medical Research Institute, University of Dundee, UK

Amanda Mather MBBS, FRACP, PhD

Consultant Nephrologist, Department of Renal Medicine, Royal North Shore Hospital, Sydne y; Conjoint Senior Lecturer, Faculty of

Medicine, University of Sydney, Australia

Stuart H Ralston MD, FRCP, FMedSci, FRSE, FFPM(Hon)

Arthritis Research UK Professor of Rheumatology, University of Edinburgh; Honorary Co nsultant Rheumatologist, Western General

Hospital, Edinburgh, UK

Peter T Reid MD, FRCPE

Consultant Physician, Respiratory Medicine, Lothian University Hospitals, Edinbu rgh, UK

Jonathan AT Sandoe PhD, FRCPath

Associate Clinical Professor, University of Leeds; Consultant Microbiologist, Leeds Te aching Hospitals NHS Trust, UK

CONTRIBUTORS • xiii

Gordon R Scott BSc, FRCP

Consultant in Genitourinary Medicine, Chalmers Sexual Health Centre, Edinburgh, UK

Shyam Sundar MD, FRCP(London), FAMS, FNASc, FASc, FNA

Professor of Medicine, Institute of Medical Sciences, Banaras Hindu University , Varanasi, India

Alan G Shand MD, FRCPE

Consultant Gastroenterologist, Western General Hospital, Edinburgh, UK

Victoria R Tallentire BSc(Hons), MD, FRCPE

Consultant Physician, Western General Hospital, Edinburgh; Honorary Clinical Senior Lecturer, U niversity of Edinburgh, UK

Robby M Steel MA, MD, FRCPsych

Consultant Liaison Psychiatrist, Department of Psychological Medicine, Royal Inrmary of Edin burgh; Honorary (Clinical) Senior

Lecturer, Department of Psychiatry, University of Edinburgh, UK

Katrina Tatton-Brown BA, MD, FRCP

Consultant in Clinical Genetics, South West Thames Regional Genetics Service, St G eorge’s Universities NHS Foundation Trust,

London; Reader in Clinical Genetics and Genomic Education, St George’s University, Lon don, UK

Grant D Stewart BSc(Hons), FRCSEd(Urol), PhD University Lecturer in Uro logical Surgery, Academic Urology Group, University

of Cambridge; Honorary Consultant Urological Surgeon, Department of Urology, Addenbroo ke’s Hospital, Cambridge; Honorary Senior

Clinical Lecturer, University of Edinburgh, UK

Simon HL Thomas MD, FRCP, FRCPE

Professor of Clinical Pharmacology and Therapeutics, Medical Toxicology Centre , Newcastle University, Newcastle upon Tyne, UK

Peter Stewart MBBS, FRACP, FRCPA, MBA Associate Professor in Chemical Pathology, University of Sydney; Ar ea Director of

Clinical Biochemistry and Head of the Biochemistry Department, Royal Prince Alfred and Liverpool Hospitals, Sydney, Australia

Henry G Watson MD, FRCPE, FRCPath

Consultant Haematologist, Aberdeen Royal Inrmary; Honorary Professor of Me dicine, University of Aberdeen, UK

Julian White MB, BS, MD, FACTM

Head of Toxinology, Women’s and Children’s Hospital, North Adelaide; Professor, U niversity of Adelaide, Australia

Mark WJ Strachan BSc(Hons), MD, FRCPE

Consultant Endocrinologist, Metabolic Unit, Western General Hospital, Edinbu rgh; Honorary Professor, University of Edinburgh, UK

John PH Wilding DM, FRCP

Professor of Medicine, Obesity and Endocrinology, University of Liverpool , UK

David R Sullivan MBBS, FRACP, FRCPA, FCSANZ Clinical Associate Professor, Fac ulty of Medicine, University of Sydney;

Physician and Chemical Pathologist, Department of Chemical Pathology, Royal Prince Alfred Hospital, Sydney, Australia

Miles D Witham PhD, FRCPE

Clinical Reader in Ageing and Health, University of Dundee, UK

International Advisory Board

Amitesh Aggarwal

Associate Professor, Department of Medicine, University College of Medical Sciences and G TB Hospital, Delhi, India

AG Frauman

Professor of Clinical Pharmacology and Therapeutics, University of Melbourne, Australia

Ragavendra Bhat

Professor of Internal Medicine, Ras Al Khaimah Medical and Health Sciences Unive rsity, Ras Al Khaimah, United Arab Emirates

Sujoy Ghosh

Associate Professor, Department of Endocrinology, Institute of Post Graduate Medical Ed ucation and Research, Kolkata, India

Matthew A Brown

Professor and Director of Genomics, Queensland University of Technology, Brisbane, Aus tralia

Khalid I Bzeizi

Senior Consultant and Head of Hepatology, Prince Sultan Military Medical Ci ty, Riyadh, Saudi Arabia

Arnold Cohen

Clinical Professor of Medicine, Elson S. Floyd College of Medicine at Washington State Univers ity, Spokane, Washington; Associate

Clinical Professor, University of Washington School of Medicine; Gastroenterologis t, Spokane Digestive Disease Center, Washington,

USA

Hadi A Goubran

Haematologist, Saskatoon Cancer Centre and Adjunct Professor, College of Medicine, University of Saskatchewan, Canada; Professor of

Medicine and Haematology (Sabbatical), Cairo University, Egypt

Rajiva Gupta

Director and Head, Rheumatology and Clinical Immunology, Medanta – The M edicity, Gurgaon, India

Saroj Jayasinghe

Chair Professor of Medicine, Faculty of Medicine, University of Colombo; Honorary Co nsultant Physician, National Hospital of Sri

Lanka, Colombo, Sri Lanka

MK Daga

Director Professor of Medicine, and In-Charge Intensive Care Unit and Center for Occu pational and Environment Medicine, Maulana

Azad Medical College, New Delhi, India

AL Kakrani

Professor and Head, Department of Medicine, Dr DY Patil Medical College, Ho spital and Research Centre; Dean, Faculty of Medicine,

Dr DY Patil Vidyapeeth Deemed University, Pimpri, Pune, India

D Dalus

Professor and Head, Department of Internal Medicine, Medical College and Hospital, Triv andrum, India

Sydney C D’Souza

Professor of Medicine, AJ Institute of Medical Science, Mangalore; Former Hea d, Department of Medicine, Kasturba Medical College

Mangalore, Manipal University, India

Tarun Kumar Dutta

Professor of Medicine and Clinical Hematology, Mahatma Gandhi Medical College and Rese arch Institute, Puducherry, India

M Abul Faiz

Professor of Medicine (Retired), Sir Salimullah Medical College, Mitford, Dhaka, Banglad esh

Vasantha Kamath

Senior Professor, Department of Internal Medicine, MVJ Medical College and Research Hospital, Bengaluru, Karnataka, India

Piotr Kuna

Professor of Medicine; Chairman, 2nd Department of Medicine; Head of Division of Internal M edicine, Asthma and Allergy, Medical

University of Lodz, Poland

Pravin Manga

Emeritus Professor of Medicine, Department of Internal Medicine, University of Witwatersran d, Johannesburg, South Africa

xvi • INTERNATIONAL ADVISORY BOARD

Ammar F Mubaidin

Professor of Neurology, Jordan University Hospital, Khalidi Medical Center, Amman, Jordan

Nirmalendu Sarkar

Professor and Head (Retired), Department of Medicine, Institute of Post Graduate Medica l Education and Research and SSKM Hospital,

Kolkata, India

Milind Nadkar

Professor of Medicine and Chief of Rheumatology and Emergency Medicine, Seth GS Medic al College and KEM Hospital, Mumbai,

India

KR Sethuraman

Vice-Chancellor, Sri Balaji Vidyapeeth University, Pondicherry, India

Matthew Ng

Honorary Clinical Professor, University of Hong Kong, Tung Wah Hospital, Hong Kong

Moffat Nyirenda

Professor of Medicine (Global Non-Communicable Diseases), London School of Hygiene and Tropical Medicine, UK; Honorary

Professor of Research, College of Medicine, University of Malawi

Tommy Olsson

Professor of Medicine, Department of Medicine, Umeå University Hospital, Sweden

Ami Prakashvir Parikh

Consultant Physician, Sheth VS General Hospital, Ellisbridge, Ahmedabad; Professor of Med icine and Head of Department of Medicine,

NHL Municipal Medical College, Ahmedabad, India

Surendra K Sharma

Adjunct Professor, Department of Molecular Medicine, Jamia Hamdard Institute of Mole cular Medicine, Hamdard University, Delhi;

Director of Research and Adjunct Professor, Departments of General Medicine and Pulmo nary Medicine, JNMC, Datta Meghe Institute

of Medical Sciences, Sawangi (M), Wardha Maharashtra, India

Ibrahim Sherif

Emeritus Professor of Medicine, Tripoli University; Consultant Endocrinologist, Alaa Clinic, Trip oli, Libya

Ian J Simpson

Emeritus Professor of Medicine, Faculty of Medical and Health Sciences, University of Auc kland, New Zealand

SG Siva Chidambaram

Professor of Medicine, Dean/SPL Ofcer, Perambalur Government Medical Colleg e and GHQ, Perambalur, India

Medha Y Rao

Professor of Internal Medicine, Principal and Dean, MS Ramaiah Medical College, Bangalore, In dia

Arvind K Vaish

Professor and Head, Department of Medicine, Hind Institute of Medical Sciences, Lucknow; Ex-Professor and Head of Medicine, KG

Medical University, Lucknow, India

NR Rau

Consultant Physician, Anugraha Medical Centre and Adarsha Hospital, Udupi, Karna taka; Former Professor and Head, Department of

Medicine, Kasturba Medical College, Manipal University, Karnataka, India

Josanne Vassallo

Professor of Medicine, Faculty of Medicine and Surgery, University of Malt a; Consultant Endocrinologist, Division of Endocrinology,

Mater Dei Hospital, Msida, Malta

Acknowledgements

Following the publication of the 22nd edition of Davidson’s, Professor Brian W alker and Dr Nicki Colledge retired as editors. We would

like to express our gratitude for the immense contribution they both made to the continuing success of this textbook.

The current editors would like to acknowledge and offer grateful thanks for the input o f all previous editions’ contributors, without

whom this new edition would not have been possible. In particular we are indebted to th ose former authors who step down with the

arrival of this new edition. They include Assistant Professor Albiruni Ryan Abdul-Raza k, Professor Andrew Bradbury, Dr Jenny Craig,

Professor Allan Cumming, Dr Robert Dawe, Emeritus Professor Michael Field, D r Jane Goddard, Professor Philip Hanlon, Dr Charlie

Lees, Dr Helen Macdonald, Professor Iain McInnes, Dr Graham Nimmo, Dr Simon Noble, Dr David Oxenham, Professor Jonathan

Seckl, Professor Michael Sharpe, Professor Neil Turner, Dr Simon Walker and Profes sor Timothy Walsh.

We are grateful to members of the International Advisory Board, all of whom provide d detailed suggestions that have improved the

book. Several members have now retired from the Board and we are grateful for their support during the preparation of previous

editions. They include Professor OC Abraham, Professor Tofayel Ahmed, Professor S amar Banerjee, Professor Tapas Das, Professor

Tsuguya Fukui, Professor Saman Gunatilake, Professor Wasim Jafri, Professor Saralad evi Naicker, Professor Nardeep Naithani,

Professor Prem Pais, Professor A Ramachandran, the late Professor (Mrs) Harsh a R Salkar and Professor Subhash Varma.

Detailed chapter reviews were commissioned to help plan this new edition and we are g rateful to all those who assisted, including

Professor Rustam Al-Shahi, Dr David Enoch, Dr Colin Forfar, Dr Richard Herriot and Dr Robert Lindsay.

The Editors and Publisher would like to thank all those who have provided valuab le feedback on this textbook and whose comments

have helped shape this new edition. We would particularly like to extend our thanks to the many readers who contact us with suggestions

for improvements. This input has been invaluable and is much appreciated; we reg ret the names are too numerous to mention

individually.

As part of the Publisher’s review, 360 readers from over 200 universities and colleges around the world supplied many innovative ideas

on how to enhance the book. We are deeply indebted to the following for their enthusiastic support and we trust we have listed all those

who contributed; we apologise if any names have been accidentally omitted. R abab Hasan Abbasi, Ayesha Abdul Aziz, Ahmed Al

Abdullah, Osama Abuzagaia, Humaira Achakzai, Sajan Acharya, Anurag Adhikari, E sha Agarwal, Avin Aggarwal, Dorothy Agrapidis,

Sakir Ahmed, Iman Akhtar, Muhammad Faizan Ali, Syeda A Rfa Ali, M Amogh, Rampr asath Anbazhagan, Anju George C, Vamseedhar

Annam, Muhammad Ehtisham Ansar, David C Antoney, Hina Arif, Sriharsha Athota, Muhamm ed Ali Azher, Bilal Al Azzawi, Janak

Bahirwani, Devyani Bahl, Mohammed Naseem Baig, Deepak Kumar Bandari, Sag nik Banerjee, Tapastanu Banerjee, Kieran Bannerman,

Emily Bart, Suranjana Basak, Saravana Bavan, Mark Beeston, Andrew Bever stock, Jeetendra Bhandari, Navin Bhatt, Soumyadeep

Bhaumik, Rajrsh Bhawani, Kriti Bhayana, Praveen Bhugra, Amit Bisht, Rahul Bisht, Ru dradeep Biswas, Tamoghna Biswas, Moira

Bradfield, S Charles Bronson, Rosie Jane Campbell, Anup Chalise, Subhankar Chatter jee, Chiranjivi Chaudhari, Lok Bandhu

Chaudhary, Muhammad Hamid Chaudhary, Umar Iftikhar Chaudhry, Himshree Gau rang Chhaya, Smitha Chirayil, Alexandra Choa,

Rahul Choudhary, Guy Conlon, Gabriel MetcalfCuenca, Jack Cuningham, Gopal Dab ade, Saraswata Das, Rahul Dev, Muinul Islam

Dewan, Sree Divya, Muhammad Dizayee, Lucy Drummond, Simon Dryden, Fiona Dr ysdale, Kaitlin Duell, Gemma Dwyer, Md Khader

Faheem N, Mahedi Hasan Faisal, Ali Mokhtari Farivar, Mohammed Omar Farooq, Ten Fu Fatt, Muhammad Zubair Fazal, Rebecca

Ferris, Rebecca Fisher, Kartik Nimish Gandhi, Vibhav Gandhi, James Gao, Ella Gardne r, Ankit Kumar Garg, Vibhuti Garg, Vishal Garg,

Rakesh Garlapati, Partha Sarathi Ghosh, Prattay Ghosh, Muhammad Umer Gi ll, Madelaine Gimzewska, Nikolaos D Giotis, Evan Goh,

Iain Gow, Bharatiram Guduri, Aantriksha Gupta, Shiwam Kumar Gupta, Michael Hagarty, M d Rashid Haider, Iqra Haq, Abdalla A

Hassan, Fatima Hemani, Bianca Honnekeri, Prerana Huddar, Sandip Hulke, Mohamme d Laique Hussain, Syahir Ibrahim, Victor

Ilubaera, Sushrut Ingawale, Aroobah Iqbal, Tooba Irshad, Nagib Ul-Islam, Sehra Jabeen, Jeev an Jacob, Samreen Jaffar, Ankita Jain,

Anukriti Jain, Ghazfa Jamil, Karan Jatwani, Surani Dilhani Jayawickrema, Per-Kristian Jensen, Love Kapil, Ishwor Karki, Ewan D

Kennedy, Amit Keshri, Haider Ali Khalid, Ali Khaliq, Hina Khan, Md Taha Ali Khan, Raazia Kh an, Priya Khetarpal, Robert Kimmitt,

Navneet Kishore, Narendranath Reddy Konda, Kirsten Kramers, Ajay Kumar, Gaura v Kumar, Karun Kumar, Mudit Kumar, Sathish

Kumar A, Sonu Kumar, Ramasamy Shakthi Kumaran, Joachim Langhans, Keith Leun g, Ang Li, Lai Nai Lim (Jeremiah), Marlene Da

Vitoria Lobo, Bo Løfgren, Sham Kumar Lulla, Apurva Lunia, Arslan Luqman, Faizan L uqman, Mithilesh Chandra Malviya, Sudhir

Mane, Sachin Mangal, G. Manju, Adupa Manupranay, Ussama Maqbool, Za hid Marwat, Ronny John Mathew, Ross Mclaren, Lucy

Mcnally, Varshil Mehta, Kamran Younis Memon, Nisha Menon, Varun Menon P, Jessica Mills, Asim Abid Minhas, xviii •

ACKNOWLEDGEMENTS

Mohd Nasir Mohiuddin, Matshidiso Mokgwathi, Kristin Monk, Joseph Raja Mony, Sudeb Mu kherjee, Sadaf Nadeem, Huda Naim, B

Abhilash Nair, Ramya Narasimhan, Rahul Navani, Ayesha Nazir, Prakhar Nigam, Srip riya Nopany, Prakash Raj Oli, Chieh Yin Ooi,

Muhammad Osama, Kate O’Sullivan, Hajer Oun, Ashwin Pachat, Akshay Pa chyala, Kannan Padma, Gregory Page, Vishal Krishna Pai,

Dhiman Pal, Vidit Panchal, Madhav Pandey, Ambikapathi Panneerselvam, Rakesh S ingh Panwar, Prakash Parida, Ashwin Singh Parihar,

Kunal Parwani, Riya Patel, Himanshu Pathak, Prathamesh Pathrikar, Samanvith Patlori, G ary Paul, Harris Penman, Lydia B Peters,

Kausthubha Puttalingaiah, Muneeb Qureshi, Sidra Qureshi, Varun Venkat Ragha van MS, Raghavendra MS, Abdur Rahaman, Ajmal

Rahman Km, SM Tajdit Rahman, Ankit Raj, Solai Raj, Namita Raja, Revathi Rajagopal, Aswathy Raju B, Nagarajan Raju, Al-Amin

Hossain Rakib, Abhiraj Ramchandani, Varun Ramchandani, Viviktha Ramesh, Jai Ran jan, Ganga Raghava Rao, Gomathi Ravula,

Aneeqa Abdul Rehman, Neeha Abdul Rehman, Varun Bhaktarahalli Renukappa, Rosali nd Revans, Madina Riaz, Lachlan Rodd, Jan

Ross, Pritam Roy, Jazeel Sa, Iqra Saeed, Israel Sariyu, Partha Saha, Mohammed Sulaiman Sait J, Ribha Salman, Souvik Samaddar,

Juliane Rf Sanner, Abhinav Sathe, Smarter Sawant, Jeff Schwartz, Somanshi Sehgal, Arbind Shah, Basit Shah, Rakesh Kumar Shah,

Mohith Shamdas, Abhishek Sharma, Anmol Sharma, Deepak Sharma, Pawan Kumar Sharma, Nisha Sharoff, Alsba Sheikh, Haris

Sheikh, Nujood Al Shirawi, William Shomali, Pratima Shrestha, Suhana Shresth a, Ajay Shukla, Prithiv Siddharth, Arpit Sikri, Ankita

Singh, Avinash Singh, Deepali Singh, Jeevika Singh, Prince Singh, Rajshree Singh, Shr uti Singh, Vikas Singh, Yogita Singh, Ayush

Keshav Singhal, Dattatreya Sitaram, Brooke Smith, Yeshwanth Sonnathi, Soundarya So undararajan, Nicola Stuber, Maleeha Subhan,

Udayasree Sagar Surampally, Monika Surana, Jason Suresh, Salman Ali Syed, Moham mad Hasnain Tahir, Syeda Sara Tamkanath,

Areeba Tariq, Saipriya Tatineni, Ghias Un Nabi Tayyab, Arul Qubert Thaya, J olley Thomas, Stephanie Tristram, Neelanchal Trivedi,

Vaibhav Trivedi, Kriti Upadhyay, Sanjaya Kumar Upadhyaya, Rukhsar Vankani, Rajk umar Krishnan Vasanthi, D Vijayaraju, R

Vinayak, Neelakantan Viswanathan, Joarder Wakil, Syed Hamza Bin Waqar, Waiz A Wasey, William Wasif, Kiran Wasti, Donald

Waters, Katherine Weir, Clinton White, Aalok Yadav, Loong Yee Yew, Shahwar You suf, Amal Yusof and Hassam Zulqar.

The authors of Chapter 20 would like to thank Dr Drew Henderson, who reviewed the ‘Diabetic nephropathy’ section.

Two short sections in Chapter 3 on array comparative genomic hybridisation and sin gle-molecule sequencing are adapted from Dr K

Tatton-Brown’s Massive Open Online Course for FutureLearn. We would like to than k the Open University and St George’s, University

of London, for permission to use this material.

We are especially grateful to all those working for Elsevier, in particular Laurence H unter, Wendy Lee and Robert Britton, for their

endless support and expertise in the shaping, collation and illustration of this e dition.

SHR, IDP, MWJS, RPH Edinburgh 2018 The opening six chapters of the book, makin g up Part 1 on ‘Fundamentals of Medicine’,

provide an account of the principles of genetics, immunology, infectious diseases and p opulation health, along with a discussion of the

core principles behind clinical decision-making and good prescribing. Subsequent chap ters in Part 2, ‘Emergency and Critical Care

Medicine’, discuss medical emergencies in poisoning, envenomation and enviro nmental medicine, while a new chapter explores

common presentations in acute medicine, as well as the recognition and managem ent of the critically ill. The third part, ‘Clinical

Medicine’, is devoted to the major medical specialties. Each chapter has been writt en by experts in the eld to provide the level of detail

expected of trainees in their discipline. To maintain the book’s virtue of being concise, care has been taken to avoid unnecessary

duplication between chapters.

The system-based chapters in Part 3 follow a standard format, beginning with an overv iew of the relevant aspects of clinical examination,

followed by an account of functional anatomy, physiology and investigations, then the common presentations of disease, and details of

the individual diseases and treatments relevant to that system. In chapters that descri be the immunological, cellular and molecular basis

of disease, this problem-based approach brings the close links between moder n medical science and clinical practice into sharp focus.

The methods used to present information are described below. Clinical examination overview s

Introduction

and a plan of investigation for patients who present with particular symptoms or signs. I n Davidson’s this is addressed by incorporating a

‘Presenting Problems’ section into all relevant chapters. Nearly 250 presentations are inc luded, which represent the most common

reasons for referral to each medical specialty.

Boxes

Boxes are a popular way of presenting information and are particularly useful for rev ision. They are classied by the type of information

they contain, using specic symbols.

General Information These include causes, clinical features, investigations, tre atments and other useful information.

Practice Point

There are many practical skills that students and doctors must master. These vary f rom inserting a nasogastric tube to reading an ECG or

X-ray, or interpreting investigations such as arterial blood gases or thyroid function te sts. ‘Practice Point’ boxes provide straightforward

guidance on how these and many other skills can be acquired and applied.

The value of good clinical skills is highlighted by a two-page overview of the important elements of the clinical examination at the

beginning of most chapters. The left-hand page includes a mannikin to illustrate k ey steps in examination of the relevant system,

beginning with simple observations and progressing in a logical sequence around the bo dy. The right-hand page expands on selected

themes and includes tips on examination technique and interpretation of physical sig ns. These overviews are intended to act as an aide-

mémoire and not as a replacement for a detailed text on clinical examination, as provided in our sister title, Macleod’s Clinical

Examination.

Presenting problems

Medical students and junior doctors must not only assimilate a great many facts about var ious disorders but also develop an analytical

approach to formulating a differential diagnosis

Emergency These boxes describe the management of many of the most common emerg encies in medicine.

In Old Age

In many countries, older people comprise 20% of the population and are the chief use rs of health care. While they contract the same

diseases as those who are younger, there are often important differences in the way the y present and how they are best managed. Chapter

32, ‘Ageing and disease’, concentrates on the principles of managing the frailest gro up who suffer from multiple comorbidity and

disability, and who tend to present with non-specific problems such as falls or delirium. Many older people, though, also suffer from

specic single-organ pathology. ‘In Old Age’ boxes are thus included in each chapter and describe common presentations, implications

of physiological changes of ageing, effects of age on investigations, problems of xx • INTRODUCTION

treatment in old age, and the benets and risks of intervention in older people.

noradrenaline. British spellings have been retained for drug classes and groups (e.g. am phetamines not amfetamines).

In Pregnancy

Units of measurement

Many conditions are different in the context of pregnancy, while some arise only durin g or shortly after pregnancy. Particular care must

be taken with investigations (for example, to avoid radiation exposure to the fetus) and tr eatment (to avoid the use of drugs that harm the

fetus). These issues are highlighted by ‘In Pregnancy’ boxes distributed throughout the b ook, which complement the new chapter on

maternal medicine.

In Adolescence

Although paediatric medicine is not covered in Davidson’s, many chronic disorders beg in in childhood and adult physicians often

contribute to multidisciplinary teams that manage young patients ‘in transition’ between p aediatric and adult health-care services. This

group of patients often presents a particular challenge, due to the physiological and psy chological changes that occur in adolescence and

which can have a major impact on the disease and its management. Adolescents can be encouraged to take over responsibility from their

parents/carers in managing their disease, but are naturally rebellious and often struggle to adhere to the impositions of chronic treatment.

To highlight these issues, we have introduced a new chapter on adolescent and transition m edicine to accompany the ‘In Adolescence’

boxes that appear in relevant chapters.

Terminology

The recommended International Non-proprietary Names (INNs) are used for all drugs, with the exception of adrenaline and The

International System of Units (SI units) is the recommended means of presentation for laboratory data and has been used throughout

Davidson’s. We recognise, though, that many laboratories around the world contin ue to provide data in non-SI units, so these have been

included in the text for the commonly measured analytes. Both SI and non-SI u nits are also given in Chapter 35, which describes the

reference ranges used in Edinburgh’s laboratories. It is important to appreciate that the se reference ranges may vary from those used in

other laboratories.

Finding what you are looking for

A contents list is given on the opening page of each chapter. In addition, the book contains numerous cross-references to help readers

nd their way around, along with an extensive index. A list of up-to-date reviews and usef ul websites with links to management

guidelines appears at the end of each chapter.

Giving us your feedback

The Editors and Publisher hope that you will nd this edition of Davidson’s informative and easy to use. We would be delighted to hear

from you if you have any comments or suggestions to make for future editions o f the book. Please contact us by e-mail at: davidson.

feedback@elsevier.com. All comments received will be much appreciated a nd will be considered by the editorial team.

N Cooper

AL Cracknell 1

Clinical decision-making

Introduction 2
The problem of diagnostic error 2 Clinical reasoning: denitions 2  Clinical skills and decision-making 3
Use and interpretation of diagnostic tests 3 Normal values 3
Factors other than disease that inuence test results 4 Operating chara cteristics 4
Sensitivity and specicity 4
Prevalence of disease 5
Dealing with uncertainty 5
Cognitive biases 6
Type 1 and type 2 thinking 7
Common cognitive biases in medicine 7
Human factors 9
Reducing errors in clinical decision-making 9 Cognitive debiasing strategies 9
Using clinical prediction rules and other decision aids 10 Effective team communication 10
Patient-centred evidence-based medicine and shared decision-making 10
Clinical decision-making: putting it all together 10 Answers to problems 12
Introduction
A great deal of knowledge and skill is required to practise as a doctor. Physicians in the  21st century need to have a comprehensive
knowledge of basic and clinical sciences, have good communication skills, be able to p erform procedures, work effectively in a team and
demonstrate professional and ethical behaviour. But how doctors think, reason and ma ke decisions is arguably their most critical skill.
Knowledge is necessary, but not sufcient on its own for good performance and safe  care. This chapter describes the principles of
clinical decision-making, or clinical reasoning.
The problem of diagnostic error
It is estimated that diagnosis is wrong 10–15% of the time in specialties such as e mergency medicine, internal medicine and general
practice. Diagnostic error is associated with greater morbidity than other types of medic al error, and the majority is considered to be
preventable. For every diagnostic error there are a number of root causes. Studies of m isdiagnosis assign three main categories, shown in
Box 1.1; however, errors in clinical reasoning play a signicant role in the majority of  diagnostic adverse events.
1.1 Root causes of diagnostic error in studies Error category No fault
System error
Human cognitive error Examples
Unusual presentation of a disease Missing information
Inadequate diagnostic support
Results not available
Error-prone processes
Poor supervision of inexperienced staff Poor team communication
Inadequate data-gathering
Errors in reasoning
‘Clinical reasoning’ describes the thinking and decision-making processes associated with clinical  practice. It is a clinician’s ability to
make decisions (often with others) based on all the available clinical information, starting  with the history and physical examination. Our
understanding of clinical reasoning derives from the elds of education, cognitive psyc hology and studies of expertise.
Figure 1.1 shows the different elements involved in clinical reasoning. Good clinic al skills are fundamental, followed by understanding
how to use and interpret diagnostic tests. Other essential elements include an understandin g of cognitive biases and human factors, and
the ability to think about one’s own thinking (which is explained in more detail later). Ot her key elements of clinical reasoning include
patient-centred evidencebased medicine (EBM) and shared decision-making with patien ts and/or carers.
Adapted from Graber M, Gordon R, Franklin N. Reducing diagnostic errors in med icine: what’s the goal? Acad Med 2002; 77:981–992.
Clinical skills (history and physical
examination)
1.2 Reasons for errors in clinical reasoning
Shared decision-making
Source of error Knowledge gaps
Misinterpretation of diagnostic tests
Cognitive biases Examples
Telling a patient she cannot have biliary
colic because she has had her gallbladder removed – gallstones can form in the b ile ducts in patients who have had a 
cholecystectomy

Deciding a patient has not had a stroke because his brain scan is normal – computed tomography and even magnetic resonance imaging,

especially when performed early, may not identify an infarct Accepting a diag nosis handed over to you without question (the ‘framing

effect’) instead of asking yourself ‘What is the evidence that supports this diagnosis?’

Diagnostic error has been dened as ‘a situation in which the clinician has all the in formation necessary to make the diagnosis but then

makes the wrong diagnosis’. Why does this happen? Studies reveal three main rea sons:

• knowledge gaps

• misinterpretation of diagnostic tests

• cognitive biases.

Examples of errors in these three categories are shown in Box 1.2.

Clearly, clinical knowledge is required for sound clinical reasoning, and an incomplete knowledge base or inadequate experience can

lead to diagnostic error. However, this chapter focuses on other elements of cli nical reasoning: namely, the interpretation of diagnostic

tests, cognitive biases and human factors.

Clinical reasoning: denitions

Clinical reasoning

Using and interpreting diagnostic

tests

Patient-centred Understanding

EBM

cognitive biases and human factors

Thinking

about

thinking

Fig. 1.1 Elements of clinical reasoning. (EBM = evidence-based medicine)

Use and interpretation of diagnostic tests • 3

Clinical skills and decision-making

Even with major advances in medical technology, the history remains the most importan t part of the clinical decision-making process.

Studies show that physicians make a diagnosis in 70–90% of cases from the history alon e. It is important to remember that a good history

is gathered not only from the patient but also, if necessary (and with consent if required ), from all available sources: for example,

paramedic and emergency department notes, eye-witnesses, relatives and/or carers.

Clinicians need to be aware of the diagnostic usefulness of clinical features in the history and examination. For example, students are

taught that meningitis presents with the following features:

• headache

• fever

• meningism (photophobia, nuchal rigidity).

However, the frequency with which patients present with certain features and the diagn ostic weight of each feature are important in

clinical reasoning. For example, many patients with meningitis do not have classical signs of meningeal irritation (Kernig’s sign,

Brudzinski’s sign and nuchal rigidity). In one prospective study, they had likelihood ratios of around 1, meaning they carried little

diagnostic weight (Fig. 1.2).

Likelihood ratios (LR) are clinical diagnostic weights. An LR of greater than 1 increases t he probability of disease (the higher the

Infinity

LR 10

Change in probability of disease

+ 45%

value, the greater the probability). Similarly, an LR of less than 1

1decreases the probability of disease. LRs are developed against

a diagnostic standard (e.g. in the case of meningitis, lumbar puncture results), so do not exist for all clinical ndings. LRs illustrate how

an individual clinical nding changes the probability of a disease. For example, in a pers on presenting with headache and fever, the

clinical nding of nuchal rigidity (neck stiffness) may carry little weight in dec iding whether to perform a lumbar puncture because LRs

do not determine the prior probability of disease; they reect only how a single clinical nding cha nges it. Clinicians have to take all the

available information from the history and physical examination into account. If the overall clinical probability is high to begin with, a

clinical nding with an LR of around 1 does not change this.

‘Evidence-based history and examination’ is a term used to describe how clinicians inco rporate knowledge about the prevalence and

diagnostic weight of clinical ndings into their history and physical examination. This is importa nt because an estimate of clinical

probability is vital in decision-making and the interpretation of diagnostic tests.

Use and interpretation of

diagnostic tests

There is no such thing as a perfect diagnostic test. Test results give us test probabilities, no t real probabilities. Test results have to be

interpreted because they are affected by the following:

• how ‘normal’ is dened

• factors other than disease

• operating characteristics

• sensitivity and specicity

• prevalence of disease in the population.

Normal values

Increase 5

probability

+ 30%

2 + 15%

No change 1 Kernig’s sign

Brudzinski’s sign

Nuchal rigidity 0.5

No change

– 15%

Decrease

0.2

probability

– 30%

0.1 – 45%

Zero Fig. 1.2 Likelihood ratio (LR) of Kernig’s sign, Brudzinski’s sign and nuchal rigidity in th e clinical diagnosis of meningitis.

probability of finding in patients withdiseaseLR =

probability of finding in patientswithout disease LRs are also used for diagnostic tests; here a

physical examination nding can be considered a diagnostic test. Data from Tho mas KE, Hasbun R, Jekel J, Quagliarello VJ. The

diagnostic accuracy of Kernig’s sign, Brudzinski’s sign, and nuchal rigidity in adults with suspec ted meningitis. Clin Infect Dis 2002;

35:46–52.

Most tests provide quantitative results (i.e. a value on a continuous numerical scale). In o rder to classify quantitative results as normal or

abnormal, it is necessary to dene a cut-off point. Many quantitative measurements in p opulations have a Gaussian or ‘normal’

distribution. By convention, the normal range is dened as those values that en compass 95% of the population, or 2 standard deviations

above and below the mean. This means that 2.5% of the normal population will have va lues above, and 2.5% will have values below the

normal range. For this reason, it is more appropriate to talk about the ‘reference ran ge’ rather than the ‘normal range’ (Fig. 1.3).

Test results in abnormal populations also have a Gaussian distribution, with a different mean an d standard deviation. In some diseases

there is no overlap between results from the abnormal and normal population. Howeve r, in many diseases there is overlap; in these

circumstances, the greater the difference between the test result and the limits of the reference ran ge, the higher the chance that the

person has a disease.

However, there are also situations in medicine when ‘normal’ is abnormal and ‘ab normal’ is normal. For example, a normal PaCO

the context of a severe asthma attack is abnormal and means the patient has life-threaten ing asthma. A low ferritin in a young

menstruating woman is not considered to be a disease at all. Normal, to some exten t, is therefore arbitrary. Normal

population

1.3 Examples of factors other than disease that inuence test results

Abnormal populations Factor Age

Ethnicity

Mean Mean Mean Value – 2SD + 2SD

‘Reference range’

Fig. 1.3 Normal distribution and reference range. For many tests, the frequency distrib ution of results in the normal healthy population

(red line) is a symmetrical bell-shaped curve. The mean ± 2 standard deviations (SD) encompass es 95% of the normal population and

usually denes the ‘reference range’; 2.5% of the normal population have values abov e, and 2.5% below, this range (shaded areas). For

some diseases (blue line), test results overlap with the normal population or even with the refe rence range. For other diseases (green line),

tests may be more reliable because there is no overlap between the normal and abnor mal population.

Pregnancy

Sex

Spurious (in vitro) results Examples

Creatinine is lower in old age (due to relatively lower muscle mass) – an older person can have a signicantly reduced eGFR rate with a

‘normal’ creatinine

Healthy people of African ancestry have lower white cell counts

Several tests are affected by late pregnancy, due to the effects of a growing f etus, including:

Reduced urea and creatinine (haemodilution) Iron deciency anaemia (increased dema nd) Increased alkaline phosphatase (produced by

the placenta)

Raised D-dimer (physiological changes in the coagulation system)

Mild respiratory alkalosis (physiological maternal hyperventilation)

ECG changes (tachycardia, left axis deviation)

Males and females have different reference ranges for many tests, e.g. haemoglobin

A spurious high potassium is seen in haemolysis and in thrombocytosis (‘pseudohyperkalaemia’)

(ECG = electrocardiogram; eGFR = estimated glomerular ltration rate, a better estimate of r enal function than creatinine)

Factors other than disease that inuence test results

A number of factors other than disease inuence test results:

• age

• ethnicity

• pregnancy

• sex

• spurious (in vitro) results.

Box 1.3 gives some examples.

Operating characteristics

Tests are also subject to operating characteristics. This refers to the way the test is performed. P atients need to be able to comply fully

with some tests, such as spirometry (p. 569), and if they cannot, then the test result will b e affected. Some tests are very dependent on the

skill of the operator and are also affected by the patient’s body habitus and c linical state; ultrasound of the heart and abdomen are

examples. A common mistake is when doctors refer to a test result as ‘no abnormality detecte d’ when, in fact, the report describes a

technically difcult and incomplete scan that should more accurately be described as ‘non- diagnostic’.

Some conditions are paroxysmal. For example, around half of patients with epilepsy ha ve a normal standard electroencephalogram

(EEG). A normal EEG therefore does not exclude epilepsy. On the other hand, around 1 0% of patients who do not have epilepsy have

epileptiform discharges on their EEG. This is referred to as an ‘incidental nding’. I ncidental findings are common in medicine, and are

increasing in incidence with the greater availability of more sensitive tests. Test results should alwa ys be interpreted in the light of the

patient’s history and physical examination.

Sensitivity and specicity

Diagnostic tests have characteristics termed ‘sensitivity’ and ‘specicity’. Sensitivity is the ability to detect true positives; specicity is

the ability to detect true negatives. Even a very good test, with 95% sensitivity, will miss 1 in 20 people with the disease. Every test

therefore has ‘false positives’ and ‘false negatives’ (Box 1.4).

A very sensitive test will detect most disease but generate abnormal ndings in healthy p eople. A negative result will therefore reliably

exclude disease but a positive result does not mean the disease is present – it means furth er evaluation is required. On the other hand, a

very specic test may miss signicant pathology but is likely to establish the diagnosis bey ond doubt when the result is positive. All tests

differ in their sensitivity and specicity, and clinicians require a working knowledge of th e tests they use in this respect.

In choosing how a test is used to guide decision-making there is a trade-off between se nsitivity versus specicity. For example, dening

an exercise electrocardiogram (p. 449) as abnormal if there is at least 0.5 mm of ST depression would ensure that very few cases of

coronary artery disease are missed but would generate many false-positive results (high sensitivity, low specicity). On the other hand, a

cut-off point of 2.0 mm of ST depression would detect most cases of important corona ry artery disease with far fewer false positives.

This trade-off is illustrated by the receiver operating characteristic curve of the test ( Fig. 1.4).

An extremely important concept is this: the probability that a person has a disease depends on t he pre-test probability, and the sensitivity

and specicity of the test. For example, imagine that an elderly lady has fallen and hurt h er left hip. On examination,

Dealing with uncertainty • 5

1.4 Sensitivity and specicity Disease No disease Positive testAB (True positive) (False positive ) Negative testCD (False negative)

(True negative) Sensitivity = A/(A+C) × 100

Specicity = D/(D+B) × 100

1.0

0.8

0.6

0.4

0.2

0.01.0 0.8 0.6 0.4 0.2 0

Specificity

Fig. 1.4 Receiver operating characteristic graph illustrating the trade-off between se nsitivity and specicity for a given test. The curve is

generated by ‘adjusting’ the cut-off values dening normal and abnormal results, calculati ng the effect on sensitivity and specicity and

then plotting these against each other. The closer the curve lies to the top left-hand corner, th e more useful the test. The red line

illustrates a test with useful discriminant value and the green line illustrates a less useful, poorly discriminant test.

the hip is extremely painful to move and she cannot stand. However, her hip X-rays ar e normal. Does she have a fracture? The sensitivity

of plain X-rays of the hip performed in the emergency department for suspected hip f racture is around 95%. A small percentage of

fractures are therefore missed. If our patient has (or is at risk of) osteoporosis, has severe pain on hip movement and cannot bear weight

on the affected side, then the clinical probability of hip fracture is high. If, on the o ther hand, she is unlikely to have osteoporosis, has no

pain on hip movement and is able to bear weight, then the clinical probability of hip fra cture is low.

Doctors are continually making judgements about whether something is true, given that something else is true. This is known as

‘conditional probability’. Bayes’ Theorem (named after English clergyman Thomas Ba yes, 1702–1761) is a mathematical way to

describe the post-test probability of a disease by combining pre-test probability, sensitivity a nd specicity. In clinical practice, doctors

are not able to make complex mathematical calculations for every decision they make. In practical terms, the answer to the question of

whether there is a fracture is that in a high-probability patient a normal test result does no t exclude the condition, but in a low-probability

patient it makes it very unlikely. This principle is illustrated in Figure 1.5.

Sox and colleagues (see ‘ Further information’) state a fundamental assertion, which they describe as a profound and subtle principle of

clinical medicine: the interpretation of

new information depends on what you believed beforehand. In 1

other words, the interpretation of a test result

depends on the probability of disease before the test.

Prevalence of disease

Consider this problem that was posed to a group of Harvard doctors: if a test to dete ct a disease whose prevalence is 1 : 1000 has a false-

positive rate of 5%, what is the chance that a person found to have a positive result actu ally has the disease, assuming you know nothing

about the person’s symptoms and signs? Take a moment to work this out. In this proble m, we have removed clinical probability and are

only considering prevalence. The answer is at the end of the chapter.

Predictive values combine sensitivity, specicity and prevalence. Sensitivity and specicity are characteristics of the test; the population

does not change this. However, as doctors, we are interested in the question, ‘What is th e probability that a person with a positive test

actually has the disease?’ This is illustrated in Box 1.5.

Post-test probability and predictive values are different. Posttest probability is the probab ility of a disease after taking into account new

information from a test result. Bayes’ Theorem can be used to calculate post-test probab ility for a patient in any population. The pre-test

probability of disease is decided by the doctor; it is a judgement based on infor mation gathered prior to ordering the test. Predictive value

is the proportion of patients with a test result who have the disease (or no disease) a nd is calculated from a table of results in a specic

population (see Box 1.5). It is not possible to transfer this value to a different populatio n. This is important to realise because published

information about the performance of diagnostic tests may not apply to different po pulations.

In deciding the pre-test probability of disease, clinicians often neglect to take pre valence into account and this distorts their estimate of

probability. To estimate the probability of disease in a patient more accurately, clinicians should a nchor on the prevalence of disease in

the subgroup to which the patient belongs and then adjust to take the individual factors i nto account.

1.5 Predictive values: ‘What is the probability that a person with a positive test act ually has the disease?’ Disease No disease

Positive testAB (True positive) (False positive)

Negative test CD (False negative) (True negative) Positive predictive value = A/(A+B) × 100

Negative predictive value

D/(D

100

Dealing with uncertainty

Clinical ndings are imperfect and diagnostic tests are imperfect. It is important to recogn ise that clinicians frequently deal with

uncertainty. By expressing uncertainty as probability, new information from diagnostic tests can be incorporated more accurately.

However, subjective estimates of probability can sometimes be unreliable. As th e section on cognitive biases will demonstrate (see

below), intuition can be a source of error.

% probability of having the disease

0 1020304050 60 70 80 90 100

90% chance of having the disease before the test is done

Patient A

34.6% chance of having the disease if the

test is negative

98.3% chance of having the disease if the

test is positive

50% chance of having the disease before the test is done

Patient B

5.6% chance of having the disease if the

test is negative

86.4% chance of having the disease if the test is positive

0 102030405060708090 100 Fig. 1.5 The interpretation of a test result depends on the pro bability of the disease before the test is carried

out. In the example shown, the test

being carried out has a sensitivity of 95% and a specicity of 85%. Patient A has very c haracteristic clinical ndings, which make the

pre-test probability of the condition for which the test is being used very high – estimated as 90% . Patient B has more equivocal ndings,

such that the pre-test probability is estimated as only 50%. If the result in Patien t A is negative, there is still a signicant chance that he

has the condition for which he is being tested; in Patient B, however, a negative result makes the diagnosis very unlikely.

Knowing the patient’s true state is often unnecessary in clinical decision-making. Sox an d colleagues (see ‘Further information’) argue

that there is a difference between knowing that a disease is present and acting as if it w ere present. The requirement for diagnostic

certainty depends on the penalty for being wrong. Different situations require differen t levels of certainty before starting treatment. How

we communicate uncertainty to patients will be discussed later in this chapter (p. 10).

The treatment threshold combines factors such as the risks of the test, and the risks vers us benets of treatment. The point at which the

factors are all evenly weighed is the threshold. If a test or treatment for a disease is effe ctive and low-risk (e.g. giving antibiotics for a

suspected urinary tract infection), then there is a lower threshold for going ahead. On the other hand, if a test or treatment is less effective

or high-risk (e.g. starting chemotherapy for a malignant brain tumour), then greater co ndence is required in the clinical diagnosis and

potential benets of treatment rst. In principle, if a diagnostic test will not change the manage ment of the patient, then careful

consideration should be given to whether it is necessary to do the test at all.

In summary, test results shift our thinking, but rarely give a ‘yes’ or a ‘no’ answer in term s of a diagnosis. Sometimes tests shift the

probability of disease by less than we realise. Pre-test probability is key, and this is derived from the history and physical examination,

combined with a sound knowledge of medicine and an understanding of the prevalenc e of disease in the particular care setting or the

population to which the patient belongs.

Cognitive biases

Advances in cognitive psychology in recent decades have demonstrated that human thin king and decision-making are prone to error.

Cognitive biases are subconscious errors that lead to inaccurate judgement and illogical i nterpretation of information. They are prevalent

in everyday life; as the famous saying goes, ‘to err is human.’

Take a few moments to look at this simple puzzle. Do not try to solve it mathematically but liste n to your intuition: A bat and ball cost

£1.10.

The bat costs £1 more than the ball.

How much does the ball cost?

The answer is at the end of the chapter. Most people get the answer to this puzzle wron g. Two things are going on: one is that humans

have two distinct types of processes when it comes to thinking and decision-making – te rmed ‘type 1’ and ‘type 2’ thinking. The other is

that the human brain is wired to jump to conclusions sometimes or to miss things that are obvio us. British psychologist and patient safety

pioneer James

Cognitive biases • 7

Experience

Context

Ambient conditions

Cognitive biases more likely

Recognised Type 1 processes

Clinical

presentation

Rational override Irrational override

Working diagnosis

Not

recognised Type 2 processes

Education Training Logical competence Fig. 1.6 The interplay between type 1 and

type 2 thinking in the diagnostic process. Adapted from Croskerry P. A universal mode l of diagnostic reasoning. Acad Med 2009;

84:1022–1028.

Reason said that, ‘Our propensity for certain types of error is the price we pay for the brain’s re markable ability to think and act

intuitively – to sift quickly through the sensory information that constantly bombards us with out wasting time trying to work through

every situation anew.’ This property of human thinking is highly relevant to clinical decision-mak ing.

1.6 Type 1 and type 2 thinking

Type 1 and type 2 thinking

Studies of cognitive psychology and functional magnetic resonance imaging demonstrat e two distinct types of processes when it comes

to decision-making: intuitive (type 1) and analytical (type 2). This has been termed ‘dua l process theory’. Box 1.6 explains this in more

detail.

Psychologists estimate that we spend 95% of our daily lives engaged in type 1 thinking – the intuitive, fast, subconscious mode of

decision-making. Imagine driving a car, for example; it would be impossible to functio n efciently if every decision and movement were

as deliberate, conscious, slow and effortful as in our rst driving lesson. With experience, co mplex procedures become automatic, fast

and effortless. The same applies to medical practice. There is evidence that expert decisio n-making is well served by intuitive thinking.

The problem is that although intuitive processing is highly efcient in many circums tances, in others it is prone to error.

Clinicians use both type 1 and type 2 thinking, and both types are important in clinical de cision-making. When encountering a problem

that is familiar, clinicians employ pattern recognition and reach a working diagnosis or d ifferential diagnosis quickly (type 1 thinking).

When encountering a problem that is more complicated, they use a slower, systematic ap proach (type 2 thinking). Both types of thinking

interplay – they are not mutually exclusive in the diagnostic process. Figure 1.6 illustra tes the interplay between type 1 and type 2

thinking in clinical practice.

Errors can occur in both type 1 and type 2 thinking; for example, people can apply the wrong rules or make errors in their application

while using type 2 thinking. However, it has been argued that the common cognitive bia ses encountered in medicine tend to occur when

clinicians are engaged in type 1 thinking.

For example, imagine being asked to see a young woman who is drowsy. She is hande d over to you as a ‘probable overdose’ because she

has a history of depression and a packet of painkillers Type 1

Intuitive, heuristic (pattern recognition) Automatic, subconscious

Fast, effortless

Low/variable reliability

Vulnerable to error

Highly affected by context

High emotional involvement

Low scientic rigour

Type 2

Analytical, systematic

Deliberate, conscious

Slow, effortful

High/consistent reliability Less prone to error

Less affected by context Low emotional involvement High scientic rigour

was found beside her at home. Her observations show she has a Glasgow Coma Scale score of 10/15, heart rate 100 beats/ min, blood

pressure 100/60 mmHg, respiratory rate 14 breaths/ min, oxygen saturations 98% on a ir and temperature 37.5°C. Already your mind has

reached a working diagnosis. It ts a pattern (type 1 thinking). You think she has taken an overdose. At this point you can stop to think

about your thinking (rational override in Fig. 1.6): ‘What is the evidence for this diagnosis? W hat else could it be?’

On the other hand, imagine being asked to assess a patient who has been admi tted with syncope. There are several different causes of

syncope and a systematic approach is required to reach a diagnosis (type 2 thinking). H owever, you recently heard about a case of

syncope due to a leaking abdominal aortic aneurysm. At the end of your assessment, fo llowing evidence-based guidelines, it is clear the

patient can be discharged. Despite this, you decide to observe the patient overnight ‘just in case’ (irrational override in Fig. 1.6). In this

example, your intuition is actually availability bias (when things are at the forefront o f your mind), which has signicantly distorted your

estimate of probability.

Common cognitive biases in medicine

Figure 1.7 illustrates the common cognitive biases prevalent in medical practice. Biases often work together; for example, in

Anchoring

The common human tendency to rely too heavily on the first piece of information offer ed (the

‘anchor’) when making decisions

Diagnostic momentum

Once a diagnostic label has been attached to a patient (by the patient or other health-care professionals), it can gather momentum with

each review, leading others to exclude other possibilities in their thinking

Premature closure

The tendency to close the decisionmaking process prematurely and accept a diagnosis b efore it, and other possibilities, have been fully

explored

Ascertainment bias

We sometimes see what we expect to see (‘self-fulfilling prophecy’). For example, a

frequent self-harmer attends the emergency department with drowsiness; everyone assu mes he has taken another overdose and misses a

brain injury

Framing effect

How a case is presented – for example, in handover – can

generate bias in the listener. This can be mitigated by always having ‘healthy scepticism’ about other people’s diagnoses

Psych-out error

Psychiatric patients who present with medical problems are underassessed, under-exam ined and under-investigated because problems

are presumed to be due to, or exacerbated by, their

psychiatric condition

Availability bias

Things may be at the forefront of your mind because you have seen several cases rece ntly or have been studying that condition in

particular. For example, when one of the authors worked in an epilepsy clinic, all black outs were possible seizures

Hindsight bias

Knowing the outcome may

profoundly influence the perception of past events and decision-making, preventing a realistic appraisal of what actually occurred – a

major problem in learning from

diagnostic error

Search satisficing

We may stop searching because we have found something that fits or is convenient, ins tead of

systematically looking for the best alternative, which involves more effort

Base rate neglect

The tendency to ignore the prevalence of a disease, which then distorts Bayesian reason ing. In some cases, clinicians do this deliberately

in order to rule out an unlikely but worst-case scenario

Omission bias

The tendency towards inaction, rooted in the principle of ‘first do no harm.’ Events tha t occur through natural progression of disease are

more acceptable than those that may be attributed directly to the action of the health-care team

Triage-cueing

Triage ensures patients are sent to the right department. However, this leads to ‘geograp hy is destiny’. For example, a diabetic

ketoacidosis patient with abdominal pain and vomiting is sent to surgery. The wrong loc ation (surgical ward) stops people thinking

about

medical causes of abdominal pain and vomiting

Commission bias

The tendency towards action rather than inaction, on the assumption that good can com e only from doing something (rather than

‘watching and waiting’)

Overconfidence bias

The tendency to believe we know more than we actually do, placing too much faith in o pinion instead of gathered evidence

Unpacking principle

Failure to ‘unpack’ all the available information may mean things are missed. For examp le, if a thorough history is not obtained from

either the patient or carers (a common problem in geriatric medicine), diagnostic possibi lities may be discounted

Confirmation bias

The tendency to look for

confirming evidence to support a theory rather than looking for disconfirming evidenc e to refute it, even if the latter is clearly present.

Confirmation bias is common when a patient has been seen first by another doctor

Posterior probability

Our estimate of the likelihood of disease may be unduly influenced by what has gone o n before for a particular patient. For example, a

patient who has been extensively investigated for headaches

presents with a severe headache, and serious causes are discounted

Visceral bias

The influence of either negative or positive feelings towards patients, which can affect o ur decisionmaking

Fig. 1.7 Common cognitive biases in medicine. Adapted from Croskerry P. Achieving qual ity in clinical decision-making: cognitive

strategies and detection of bias. Acad Emerg Med 2002; 9:1184–1204.

Reducing errors in clinical decision-making • 9

overcondence bias (the tendency to believe we know more than we actually do), too much faith is placed in opinion instead of gathered

evidence. This bias can be augmented by the availability bias and nally by commission bias (the tendency towards action rather than

inaction) – sometimes with disastrous results.

The mark of a well-calibrated thinker is the ability to recognise what mode of thinking is being employed and to anticipate and recognise

situations in which cognitive biases and errors are more likely to occur.

• adopting ‘cognitive debiasing strategies’ 1

• using clinical prediction rules and other decision aids

• engaging in effective team communication.

Cognitive debiasing strategies

There are some simple and established techniques that can be used to avoid cognitive bia ses and errors in clinical decision-making.

Human factors

‘Human factors’ is the science of the limitations of human performance, and how techn ology, the work environment and team

communication can adapt for this to reduce diagnostic and other types of error. An alysis of serious adverse events in clinical practice

shows that human factors and poor team communication play a signicant role when thi ngs go wrong.

Research shows that many errors are beyond an individual’s conscious control and are precipita ted by many factors. The discipline of

human factors seeks to understand interactions between:

• people and tasks or technology

• people and their work environment

• people in a team.

An understanding of these interactions makes it easier for health-care professionals, wh o are committed to ‘rst do no harm,’ to work in

the safest way possible. For example, performance is adversely affected by factors such as poorly designed processes and equipment,

frequent interruptions and fatigue. The areas of the brain required for type 2 processing are most affected by things like fatigue and

cognitive overload, and the brain reverts to type 1 processing to conserve cognitive energy. Figure 1.8 illustrates some of the internal and

external factors that affect human judgement and decision-making.

Various experiments demonstrate that we focus our attention to lter out distractions. This is adva ntageous in many situations, but in

focusing on what we are trying to see we may not notice the unexpected. In a team con text, what is obvious to one person may be

completely missed by someone else. Safe and effective team communication th erefore requires us never to assume, and to verbalise

things, even though they may seem obvious.

History and physical examination

Taking a history and performing a physical examination may seem obvious, but these a re sometimes carried out inadequately. This is the

‘unpacking principle’: failure to unpack all the available information means things can b e missed and lead to error.

Problem lists and differential diagnosis

Once all the available data from history, physical examination and (sometimes) initial test results a re available, these need to be

synthesised into a problem list. The ability to identify key clinical data and create a proble m list is a key step in clinical reasoning. Some

problems (e.g. low serum potassium) require action but not necessarily a differential diagno sis. Other problems (e.g. vomiting) require a

differential diagnosis. The process of generating a problem list ensures nothing is missed. The pr ocess of generating a differential

diagnosis works against anchoring on a particular diagnosis too early, thereby avoiding search satisficing and premature closure (see Fig.

1.7).

Mnemonics and checklists

These are used frequently in medicine in order to reduce reliance on fallible human me mory. ABCDE (airway, breathing, circulation,

disability, exposure/examination) is probably the most successful checklist in medicine, u sed during the assessment and treatment of

critically ill patients (ABCDE is sometimes prexed with ‘C’ for ‘control of any obvious problem’; see p. 188). Checklists ensure that

important issues have been considered and completed, especially under conditions of co mplexity, stress or fatigue.

Reducing errors in clinical decision-making

Knowledge and experience do not eliminate errors. Instead, there are a number of wa ys in which we can act to reduce errors in clinical

decision-making. Examples are:

Red ags and ROWS (‘rule out worst case scenario’)

These are strategies that force doctors to consider serious diseases that can present with common symptoms. Red ags in back pain are

listed in Box 24.19 (p. 996). Considering and investigating for possible pulmona ry embolism in patients who

Internal factors Knowledge Training

Beliefs and values Emotions

Sleep/fatigue Stress

Physical illness Personality type Type 1 thinking/ conservation of cognitive effort Cognitiv e and affective biases

Error External factors Interruptions Cognitive overload Time pressure Am bient conditions Insufficient data Team factors Patient

factors Poor feedback Fig. 1.8 Factors that affect our judgement and decision-making. Type 1 thinking = f ast, intuitive,

subconscious, low-effort.

present with pleuritic chest pain and breathlessness is a common example of ruling out a worst-case scenario, as pulmonary embolism

can be fatal if missed. Red ags and ROWS help to avoid cognitive biases such as the ‘fr aming effect’ and ‘premature closure’.

Newer strategies to avoid cognitive biases and errors in decisionmaking are emerging. T hese involve explicit training in clinical

reasoning and human factors. In theory, if doctors are aware of the science of human thinking and decision-making, then they are more

able to think about their thinking, understand situations in which their decision-making m ay be affected, and take steps to mitigate this.

Assessment

Using clinical prediction rules and other decision aids

A clinical prediction rule is a statistical model of the diagnostic process. When clinical p rediction rules are matched against the opinion

of experts, the model usually outperforms the experts, because it is applied consistently in each case. However, it is important that

clinical prediction rules are used correctly – that is, applied to the patient popula tion that was used to create the rule. Clinical prediction

rules force a scientic assessment of the patient’s symptoms, signs and other dat a to develop a numerical probability of a disease or an

outcome. They help clinicians to estimate probability more accurately.

A good example of a clinical prediction rule to estimate pre-test probability is the Wells s core in suspected deep vein thrombosis (see

Box 10.15, p. 187). Other commonly used clinical prediction rules predict outco mes and therefore guide the management plan. These

include the GRACE score in acute coronary syndromes (see Fig. 16.62, p. 494) and the CURB-65 score in communityacquired

pneumonia (see Fig. 17.32, p. 583).

Effective team communication

Effective team communication and proper handovers are vital for safe clinical care. Th e SBAR system of communication has been

recommended by the UK’s Patient Safety First campaign. It is a structured way to com municate about a patient with another health-care

professional (e.g. during handover or when making a referral) and increases the amount of relevant information being communicated in a

shorter time. It is illustrated in Box 1.7.

In increasingly complex health-care systems, patients are looked after by a wide variety of professionals, each of whom has access to

important information required to make clinical decisions. Strict hierarchies are hazardou s to patient safety if certain members of the

team are not able to speak up.

Patient-centred evidence-based medicine and shared decision-making

1.7 The SBAR system of communicating

R ecommendation Example (a telephone call to the Intensive Care team)

I am [name] calling from [place] about a patient with a NEWS of 10.

[Patient’s name], 30-year-old woman, no past medical history, was admitted last night with community-acquired pneumonia. Since then

her oxygen requirements have been steadily

increasing.

Her vital signs are: blood pressure 115/60 mmHg, heart rate 120 beats/min, temper ature 38°C, respiratory rate 32 breaths/min, oxygen

saturations 89% on 15 L via reservoir bag mask. An arterial blood gas shows pH 7.3 ( H

50 nmol/L), PaCO

4.0 kPa (30 mmHg), PaO

7 kPa (52.5 mmHg), standard bicarbonate 14 mmol/L.

Chest X-ray shows extensive right lower zone consolidation.

Please can you come and see her as soon as possible? I think she needs admission to Intensive Care.

(NEWS = National Early Warning Score; a patient with normal vital signs scores 0 )

From Royal College of Physicians of London. National Early Warning Score: standa rdising the assessment of illness severity in the

NHS. Report of a working party. RCP, July 2012; www.rcplondon.ac.uk/projects/outputs/nation al-earlywarning-score-news (accessed

March 2016).

with dual antiplatelet therapy and low-molecular-weight heparin as recommended in clin ical guidelines?

As this chapter has described, clinicians frequently deal with uncertainty/prob ability. Clinicians need to be able to explain risks and

benets of treatment in an accurate and understandable way. Providing the relevant statist ics is seldom sufcient to guide decision-

making because a patient’s perception of risk may be inuenced by irrational factors as well as individual values.

Research evidence provides statistics but these can be confusing. Terms such as ‘comm on’ and ‘rare’ are nebulous. Whenever possible,

clinicians should quote numerical information using consistent denominators (e.g. ‘90 o ut of 100 patients who have this operation feel

much better, 1 will die during the operation and 2 will suffer a stroke’). Visual aids can be used to present complex statistical information

(Fig. 1.9).

How uncertainty is conveyed to patients is important. Many studies demonstrate a correlation between effective clinician– patient

communication and improved health outcomes. If patients feel they have been listened to and understand the problem and proposed

treatment plan, they are more likely to follow the plan and less likely to re-attend.

‘Patient-centred evidence-based medicine’ refers to the application of best-available res earch evidence while taking individual patient

factors into account; these include both clinical and non-clinical factors (e.g. the patient’ s social circumstances, values and wishes). For

example, a 95-year-old man with dementia and a recent gastrointestinal bleed is admitted with an inferior myocardial infarction. He is

clinically well. Should he be treated SBAR

Situation

Background

Clinical decision-making: putting it all together

The following is a practical example that brings together many of the concepts outlin ed in this chapter:

A 25-year-old woman presents with right-sided pleuritic chest pain and breathlessness. She reports that she had an upper

Clinical decision-making: putting it all together • 11

Feel better

No difference

Stroke

Dead

<500 ng/mL). A normal chest X-ray is a common nding in 1

pulmonary embolism. Several studies have shown that the D-dimer assay has at

least 95% sensitivity in acute pulmonary embolism but it has a low specicity. A very sensitive test will detect most disease but generate

abnormal ndings in healthy people. On the other hand, a negative result virtually, but not completely, excl udes the disease. It is

important at this point to realise that a raised D-dimer result does not mean this patient has a pulmonary embolism; it just means that we

have not been able to exclude it. Since pulmonary embolism is a potentially fatal con dition we need to rule out the worst-case scenario

(ROWS), and the next step is therefore to arrange further imaging. What kind of imagin g depends on individual patient characteristics

and what is available.

Fig. 1.9 Visual portrayal of benets and risks. The image refers to an operation that is expected to relieve symptoms in 90% of patients,

but cause stroke in 2% and death in 1%. From Edwards A, Elwyn G, Mulley A. Explaining risk s: turning numerical data into meaningful

pictures. BMJ 2002; 324:827–830, reproduced with permission from the BMJ Pu blishing Group.

respiratory tract infection a week ago and was almost back to normal when the symptoms started. The patient has no past medical history

and no family history, and her only medication is the combined oral contraceptive pill. On ex amination, her vital signs are normal

(respiratory rate 19 breaths/min, oxygen saturations 98% on air, blood pressure 115/60 m mHg, heart rate 90 beats/min, temperature

37.5°C) and the physical examination is also normal. You have been asked to assess he r for the possibility of a pulmonary embolism.

(More information on pulmonary embolism can be found on page 619.)

Treatment threshold

The treatment threshold combines factors such as the risks of the test, and the risks vers us benets of treatment. A CT pulmonary

angiogram (CTPA) could be requested for this patient, although in some circumstances ventilation–perfusion single-photon emission

computed tomography (V/Q SPECT, p. 620) may be a more suitable alternative. However , what if the scan cannot be performed until the

next day? Because pulmonary embolism is potentially fatal and the risks of treatment in this case are low, the patient should be started on

treatment while awaiting the scan.

Post-test probability

The patient’s scan result is subsequently reported as ‘no pulmonary embolism’. Combin ed with the low pre-test probability, this scan

result reliably excludes pulmonary embolism.

Evidence-based history and examination

Information from the history and physical examination is vital in deciding whether this could be a pulmonary embolism. Pleurisy and

breathlessness are common presenting features of this disease but are also common pre senting features in other diseases. There is

nothing in the history to suggest an alternative diagnosis (e.g. high fever, productive co ugh, recent chest trauma). The patient’s vital signs

are normal, as is the physical examination. However, the only feature in the history and examination that has a negative likelihood ratio

in the diagnosis of pulmonary embolism is a heart rate of less than 90 beats/min. In othe r words, the normal physical examination

ndings (including normal oxygen saturations) carry very little diagnostic weight .

Deciding pre-test probability

The prevalence of pulmonary embolism in 25-year-old women is low. We anchor o n this prevalence and then adjust for individual

patient factors. This patient has no major risk factors for pulmonary embolism. To assist our estimate of pre-test probability, we could

use a clinical prediction rule: in this case, the modied Wells score for pulmonar y embolism, which would give a score of 3 (low

probability – answering yes only to the criterion ‘PE is the number one diagnos is, an alternative is less likely’).

Interpreting test results

Imagine the patient went on to have a normal chest X-ray and blood results, apart from a raised D-dimer of 900 (normal

Cognitive biases

Imagine during this case that the patient had been handed over to you as ‘nothing wron g – probably a pulled muscle’. Cognitive biases

(subconscious tendencies to respond in a certain way) would come into play, such as th e ‘framing effect’, ‘conrmation bias’ and ‘search

satiscing’. The normal clinical examination might conrm the diagnosis of musculoskeletal pain in your mind, despite the examination

being entirely consistent with pulmonary embolism and despite the lack of history and examination ndings (e.g. chest wall tenderness)

to support the diagnosis of musculoskeletal chest pain.

Human factors

Imagine that, after you have seen the patient, a nurse hands you some blood forms and asks you what tests you would like to request on

‘this lady’. You request blood tests including a D-dimer on the wrong patient. Luckily, this error is intercepted.

Reducing cognitive error

The diagnosis of pulmonary embolism can be difcult. Clinical prediction rules (e.g. modied We lls score), guidelines (e.g. from the

UK’s National Institute for Health and Care Excellence, or NICE) and decision aids (e.g . simplied pulmonary embolism severity index,

or PESI) are frequently used in combination with the doctor’s opinion, derived from information gathered in the history and physical

examination.

Person-centred EBM and information given to patient

The patient is treated according to evidence-based guidelines that apply to her particular situat ion. Tests alone do not make a diagnosis

and at the end of this process the patient is told that the combination of history, examination a nd test results mean she is extremely

unlikely to have a pulmonary embolism. Viral pleurisy is offered as an alternative diagnosis and sh e is reassured that her symptoms are

expected to settle over the coming days with analgesia. She is advised to re-present to h ospital if her symptoms suddenly get worse.

Answers to problems

Harvard problem (p. 5)

Almost half of doctors surveyed said 95%, but they neglected to take prevalence into a ccount. If 1000 people are tested, there will be 51

positive results: 50 false positives and 1 true positive. The chance that a person found to have a positive result actually has the disease is

1/51 or 2%.

Bat and ball problem (p. 6)

This puzzle is from the book, Thinking, Fast and Slow, by Nobel laureate Daniel Kahneman (see ‘ Further information’). He writes, ‘A

number came to your mind. The number, of course, is 10p. The distinctive mark of this easy puzzle is that it evokes an answer that is

intuitive, appealing – and wrong. Do the math, and you will see.’ The correct answer is 5p.

Further information

Books and journal articles

Cooper N, Frain J (eds). ABC of clinical reasoning. Oxford: Wiley–Blackwell; 2016.

Kahneman D. Thinking, fast and slow. Harmondsworth: Penguin; 2012.

McGee S. Evidence-based physical diagnosis, 3rd edn. Philadelphia: Saunders; 2012.

Scott IA. Errors in clinical reasoning: causes and remedial strategies. BMJ 2009; 338: b186.

Sox H, Higgins MC, Owens DK. Medical decision making, 2nd edn. Chichester: Wiley–Bl ackwell; 2013.

Trowbridge RL, Rencic JJ, Durning SJ. Teaching clinical reasoning. Philadelphia: Am erican College of Physicians; 2015.

Vincent C. Patient safety. Edinburgh: Churchill Livingstone; 2006.

Websites

chfg.org UK Clinical Human Factors Group.

clinical-reasoning.org Clinical reasoning resources.

creme.org.uk UK Clinical Reasoning in Medical Education group. improvediagnosis.org Society to Improve Diagnosis in Medicine.

vassarstats.net/index.html Suite of calculators for statistical

computation (Calculator 2 is a calculator for predictive values and likelihood ratios).

SRJ Maxwell

Clinical therapeutics and good prescribing

Drug regulation and management 26

Drug development and marketing 26

Managing the use of medicines 27

Prescribing in practice 28

Decision-making in prescribing 28

Prescribing in special circumstances 31

Writing prescriptions 33

Monitoring drug therapy 34

Principles of clinical pharmacology 14 Pharmacodynamics 14

Pharmacokinetics 17

Inter-individual variation in drug responses 19 Adverse outcomes of drug therapy 21 Adverse drug reactions 21

Drug interactions 23

Medication errors 24

Prescribing medicines is the major tool used by doctors to restore or preserve the health of patients. Medicines contain drugs (the specic

chemical substances with pharmacological effects), either alone or in combination with ad ditional drugs, in a formulation mixed with

other ingredients. The benecial effects of medicines must be weighed against their cost and potential adverse drug reactions and

interactions. The latter two factors are sometimes caused by injudicious prescribing decisions and by prescribing errors. The modern

prescriber must meet the challenges posed by the increasing number of drugs and formulations a vailable and of indications for

prescribing them, and the greater complexity of treatment regimens followed by individ ual patients (‘polypharmacy’, a particular

challenge in the ageing population). The purpose of this chapter is to elaborate on the princi ples and practice that underpin good

prescribing (Box 2.1).

Dosage

Pharmacokinetics regimen

‘what the body does to a drug’ Monitoring

Measure plasma drug

Time

concentration Plasma

concentration

Concentration at the site of action Pharmacodynamics

Concentration

‘what a drug does to the body’

Monitoring Measure clinical effects

2.1 Steps in good prescribing

Pharmacological effects

• Make a diagnosis

• Consider factors that might inuence the patient’s response to therapy (age, con comitant drug therapy, renal and liver function etc.)

• Establish the therapeutic goal*

• Choose the therapeutic approach*

• Choose the drug and its formulation (the ‘medicine’)

• Choose the dose, route and frequency

• Choose the duration of therapy

• Write an unambiguous prescription (or ‘medication order’)

• Inform the patient about the treatment and its likely effects

• Monitor treatment effects, both benecial and harmful

• Review/alter the prescription

*These steps in particular take the patient’s views into consideration to establish a th erapeutic partnership (shared decision-making to

achieve ‘concordance’).

Principles of clinical pharmacology

Prescribers need to understand what the drug does to the body (pharmacodynamics) a nd what the body does to the drug

(pharmacokinetics) (Fig. 2.1). Although this chapter is focused on the most common dru gs, which are synthetic small molecules, the

same principles apply to the increasingly numerous ‘biological’ therapies (sometimes ab breviated to ‘biologics’) now in use, which

include peptides, proteins, enzymes and monoclonal antibodies (see Box 4.2, p. 65).

Pharmacodynamics

Drug targets and mechanisms of action

Modern drugs are usually discovered by screening compounds for activity either to stimula te or to block the function of a specic

molecular target, which is predicted to have a benecial effect in a particular diseas e (Box 2.2). Other drugs have useful but less selective

chemical properties, such as chelators (e.g. for treatment of iron or copper overload), o smotic agents (used as diuretics in cerebral

oedema) or general anaesthetics (that alter the biophysical properties of lipid membranes). The following characteristics of the interaction

of drugs with receptors illustrate some of the important determinants of the effects of dr ugs:

• Afnity describes the propensity for a drug to bind to a

receptor and is related to the ‘molecular t’ and the Fig. 2.1 Pharmacokinetics and pharmacod ynamics.

strength of the chemical bond. Some drug–receptor interactions are irreversible, eith er because the afnity is so strong or because the

drug modies the structure of its molecular target.

• Selectivity describes the propensity for a drug to bind to one target ra ther than another. Selectivity is a relative term, not to be confused

with absolute specicity. It is common for drugs targeted at a particular subtype of receptor to exhibit some effect at other subtypes. For

example, β-adrenoceptors can be subtyped on the basis of their responsiveness to th e endogenous agonist noradrenaline (norepinephrine):

the concentration of noradrenaline required to cause bronchodilatation (via β

-adrenoceptors) is ten times higher than that required to

cause tachycardia (via

β1

-adrenoceptors). ‘Cardioselective’ β-blockers have anti-anginal effects on the heart (β

) but may still cause

bronchospasm in the lung (

β2

) and are contraindicated for asthmatic patients.

• Agonists bind to a receptor to produce a conformational change that is coupled to a biological response. As agonist concentration

increases, so does the proportion of receptors occupied, and hence the biolo gical effect. Partial agonists activate the receptor but cannot

produce a maximal signalling effect equivalent to that of a full agonist, even when a ll available receptors are occupied.

• Antagonists bind to a receptor but do not produce the conformational ch ange that initiates an intracellular signal. A competitive

antagonist competes with endogenous ligands to occupy receptor-binding sites, w ith the resulting antagonism depending on the relative

afnities and concentrations of drug and ligand. Non-competitive antagonists inhibit the effect of an agonist by mechanisms other than

direct competition for receptor binding with the agonist (e.g. by affecting post-r eceptor signalling).

Dose–response relationships

Plotting the logarithm of drug dose against drug response typically produces a sigmoida l dose–response curve (Fig. 2.2). Progressive

increases in drug dose (which, for most drugs, is proportional to the plasma drug conce ntration) produce increasing

2.2 Examples of target molecules for drugs

Drug target

Receptors

Channel-linked receptors

G-protein-coupled receptors (GPCRs)

Description

Ligand binding controls a linked ion channel, known as ‘ligand-gated’ (in contrast to ‘voltage- gated’ channels that respond to changes in

membrane potential)

Ligand binding affects one of a family of ‘G-proteins’ that mediate signal transduction eith er by activating intracellular enzymes (such as

adenylate or guanylate cyclase, producing cyclic AMP or GMP, respectively) or by controllin g ion channels

Kinase-linked receptors

Transcription factor receptors Ligand binding activates an intracellular protein kinase th at triggers a cascade of phosphorylation reactions

Intracellular and also known as ‘nuclear receptors’; ligand binding promotes or inhibits gene tr anscription and hence synthesis of new

proteins

Examples 2

Nicotinic acetylcholine receptor

GABA receptor

Sulphonylurea receptor

Muscarinic acetylcholine receptor

-adrenoceptors

Dopamine receptors

5-Hydroxytryptamine (5-HT,

serotonin) receptors

Opioid receptors

Insulin receptor

Cytokine receptors

Steroid receptors

Thyroid hormone receptors

Vitamin D receptors

Retinoid receptors

PPARγ and α receptors

Other targets

Voltage-gated ion channels

Enzymes Mediate electrical signalling in excitable tissues (muscle and nervous syst em)

Catalyse biochemical reactions. Drugs interfere with binding of substrate to the active site or of co-factors

Transporter proteins Carry ions or molecules across cell membranes

Cytokines and other signalling molecules Cell surface antigens Small proteins that are important in cell signalling (autocrine, paracrine

and endocrine), especially affecting the immune response Block the recognition of cell surface molecules that modulate cellular

responses

channels

Cyclo-oxygenase

ACE

Xanthine oxidase

5-HT re-uptake transporter

ATPase

Tumour necrosis factors

Interleukins

Cluster of differentiation molecules (e.g. CD20, CD80)

(ACE = angiotensin-converting enzyme; AMP = adenosine monophosphate; ATPase = adenosine triphosphatase; GABA = γ-

aminobutyric acid; GMP = guanosine monophosphate; PPAR = peroxisome prolifer ator-activated receptor)

Hypersusceptibility Side-effects

100

Beneficial

Emax effect

= 0.1 mg

Toxic effects

Adverse effect

= 100 mg

60 Therapeutic index 100/0.1 = 1000

0.0001 0.001 0.01 0.1 1 10 100 1000 Drug dose (mg)

Fig. 2.2 Dose–response curve. The green curve represents the benecial effect of the drug . The maximum response on the curve is the

max

and the dose (or concentration) producing half this value (E

max

/2) is the ED

(or EC

). The red curve illustrates the dose–

response relationship for the most important adverse effect of this drug. This occu rs at much higher doses; the ratio between the ED

for

the adverse effect and that for the benecial effect is the ‘therapeutic index’, which indicates how much margin there is for prescribers

when choosing a dose that will provide benecial effects without also causing this ad verse effect. Adverse effects that occur at doses

above the therapeutic range are normally called ‘toxic effects’, while those occurring w ithin the therapeutic range are ‘side-effects’ and

those below it are ‘hyper-susceptibility effects’.

response but only within a relatively narrow range of dose; further increases in dose b eyond this range produce little extra effect. The

following characteristics of the drug response are useful in comparing different dr ugs:

• Efcacy describes the extent to which a drug can produce

a target-specic response when all available receptors or binding sites are occupied (i.e. E

max

on the dose–response curve). A full

agonist can produce the maximum response of which the receptor is capable, while a partial agonist at the same receptor will have lower

efcacy. Therapeutic efcacy describes the effect of the drug on a desired bi ological endpoint and can be used to compare drugs that act

via different pharmacological mechanisms (e.g. loop diuretics induce a greater diuresis than thiazide diuretics and therefore have greater

therapeutic efcacy).

• Potency describes the amount of drug required for a given response. More potent drugs p roduce biological effects at lower doses, so

they have a lower ED

. A less potent drug can still have an equivalent efcacy if it is given in higher doses.

The dose–response relationship varies between patients because of variations in the man y determinants of pharmacokinetics and

pharmacodynamics. In clinical practice, the prescriber is unable to construct a dose–res ponse curve for each individual patient.

Therefore, most drugs are licensed for use within a recommended range of doses that is expected to reach close to the top of the dose–

response curve for most patients. However, it is sometimes possible to achieve the desire d therapeutic efcacy at doses towards the

lower end of, or even below, the recommended range.

Therapeutic index

The adverse effects of drugs are often dose-related in a similar way to the ben ecial effects, although the dose–response curve for these

adverse effects is normally shifted to the right (Fig. 2.2). The ratio of the ED

for therapeutic efcacy and for a major adverse effect is

known as the ‘therapeutic index’. In reality, drugs have multiple potential adverse effec ts, but the concept of therapeutic index is usually

based on adverse effects that might require dose reduction or discontinuation. For most dr ugs, the therapeutic index is greater than 100

but there are some notable exceptions with therapeutic indices of less than 10 (e.g. digo xin, warfarin, insulin, phenytoin, opioids). The

doses of such drugs have to be titrated carefully for individual patients to maximis e benets but avoid adverse effects.

Desensitisation and withdrawal effects

Desensitisation refers to the common situation in which the biological response to a drug diminishes when it is given continuously or

repeatedly. It may be possible to restore the response by increasing the dose of the dru g but, in some cases, the tissues may become

completely refractory to its effect.

• Tachyphylaxis describes desensitisation that occurs very

rapidly, sometimes with the initial dose. This rapid loss of response implies depletio n of chemicals that may be necessary for the

pharmacological actions of the drug (e.g. a stored neurotransmitter released from a nerve termin al) or receptor phosphorylation.

• Tolerance describes a more gradual loss of response to a drug that occurs over days or weeks. This slower change implies changes in

receptor numbers or the development of counter-regulatory physiological changes th at offset the actions of the drug (e.g. accumulation of

salt and water in response to vasodilator therapy).

• Drug resistance is a term normally reserved for describing the loss of eff ectiveness of an antimicrobial (p. 116) or cancer chemotherapy

drug.

• In addition to these pharmacodynamic causes of desensitisation, reduced respons e may be the

consequence of lower plasma and tissue drug concentrations as a result of alte red pharmacokinetics (see below).

When drugs induce chemical, hormonal and physiological changes that offset their actio ns, discontinuation may allow these changes to

cause ‘rebound’ withdrawal effects (Box 2.3).

2.3 Examples of drugs associated with withdrawal effects

Drug

Alcohol Symptoms

Anxiety, panic, paranoid delusions, visual and auditory hallucinations

Barbiturates, benzodiazepines Similar to alcohol Signs

Agitation, restlessness,

delirium, tremor, tachycardia, ataxia, disorientation, seizures

Similar to alcohol

Glucocorticoids Weakness, fatigue, decreased appetite, weight loss, nausea, vomiting, diarrhoea, abdomi nal pain Hypotension,

hypoglycaemia

Opioids

Selective serotonin re-uptake inhibitors (SSRIs)

Rhinorrhoea, sneezing, yawning, lacrimation, abdominal and leg

cramping, nausea, vomiting, diarrhoea

Dizziness, sweating, nausea, insomnia, tremor, delirium, nightmares

Dilated pupils Treatment

Treat immediate withdrawal syndrome with benzodiazepines

Transfer to long-acting

benzodiazepine then gradually reduce dosage

Prolonged therapy suppresses the hypothalamic–pituitary–adrenal axis and caus es adrenal insufciency requiring glucocorticoid

replacement. Withdrawal should be gradual after prolonged therapy (p. 670)

Transfer addicts to long-acting agonist methadone

Tremor Reduce SSRIs slowly to avoid withdrawal effects

Pharmacokinetics

Parenteral administration

These routes avoid absorption via the gastrointestinal tract and

Understanding ‘what the body does to the drug’ ( Fig. 2.3) is

first-pass metabolism in the liver:

2 extremely important for prescribers because

this forms the basis • Intravenous (IV). The IV route enables all of a dose to en ter on which the optimal route of administration and dose

regimen the systemic circulation reliably, without any concerns are chosen and explains the majority of in ter-individual variation about

absorption or rst-pass metabolism (i.e. the dose is in the response to drug the rapy. 100% bioavailable), and rapidly achieve a high

plasma

Drug absorption and routes of administration

concentration. It is ideal for very ill patients when a rapid, certain effect is critical to outcome (e.g. benzathine Absorption is the process

by which drug molecules gain access

benzylpenicillin for meningococcal meningitis).

• Intramuscular (IM). IM administration is easier to achieve

to the blood stream. The rate and extent of drug absorption

than the IV route (e.g. adrenaline

(epinephrine) for acute depend on the route of administration (Fig. 2.3). anaphylaxis) but absorption is less pre dictable and

Enteral administration depends on muscle blood ow.

These routes involve administration via the gastrointestinal

• Subcutaneous (SC). The SC route is ideal for drugs that

tract:

have to be administered

parenterally because of low oral

• Oral. This is the most common route of administration

bioavailability, are absorbed well from subcutaneous fat,

because it is simple, convenient and

readily used by

and might ideally be injected by patients themselves (e.g. patients to self-administer t heir medicines. Absorption after

insulin, heparin).

• Transdermal. A transdermal patch can enable a drug to be

an oral dose is a complex process that depends on the absorbed through the skin and into the

circulation (e.g. drug being swallowed, surviving exposure to gastric acid, oestrogens, nicotine, nitrates).avoiding unacceptable food binding, being absorbed

across the small bowel mucosa into the portal venous

Other routes of administrationsystem, and surviving metabolism by gut wall or liver

enzymes (‘rst-pass metabolism’). As a consequence, • Topical application of a drug involves direct administration absorption is

frequently incomplete following oral to the site of action (e.g. skin, eye, ear). This h as the administration. The term ‘bioavailability’

describes the advantage of achieving sufcient concentration at this site proportio n of the dose that reaches the systemic while

minimising systemic exposure and the risk of circulation intact. adverse effects elsewhere.

• Buccal, intranasal and sublingual (SL). These routes • Inhaled (INH) administr ation allows drugs to be delivered have the advantage of

enabling rapid absorption

into the systemic circulation without the uncertainties

associated with oral administration (e.g. organic nitrates

for angina pectoris, triptans for migraine, opioid

analgesics).

• Rectal (PR). The rectal mucosa is occasionally used

as a site of drug administration when the oral route is

compromised because of nausea and vomiting or

unconsciousness (e.g. diazepam in status epilepticus).

directly to a target in the respiratory tree, usually the small airways (e.g. salbutamol, beclometa sone). However, a signicant proportion

of the inhaled dose may be absorbed from the lung or is swallowed and can reach t he systemic circulation. The most common mode of

delivery is the metered-dose inhaler but its success depends on some degree of manual dexterity and timing (see Fig. 17.23, p. 571).

Patients who nd these difcult may use a ‘spacer’ device to improve drug delivery. A special m ode

Oral

Mouth

Stomach

Buccal

Circulating Parenteral

plasma Metabolism

Intestinal wall enzymes

itialflu

Liver enzymes e

r s t id

Small intestine

Liver

Intracellular fluid

Kidney

Large intestine Portal

venous system

Excretion in urine

Rectum Rectal Excretion in faeces

Fig. 2.3 Pharmacokinetics summary. Most drugs are taken orally, are absorbed from the intestinal lumen and enter the portal venous

system to be conveyed to the liver, where they may be subject to rst-pass meta bolism and/or excretion in bile. Active drugs then enter

the systemic circulation, from which they may diffuse (or sometimes be activ ely transported) in and out of the interstitial and

intracellular uid

compartments. Drug that remains in circulating plasma is subject to liver metabolism a nd renal excretion. Drugs excreted in bile may be

reabsorbed, creating an enterohepatic circulation. First-pass metabolism in the liver is avoide d if drugs are administered via the buccal or

rectal mucosa, or parenterally (e.g. by intravenous injection).

of inhaled delivery is via a nebulised solution created by using pressurised oxygen or ai r to break up solutions and suspensions into small

aerosol droplets that can be directly inhaled from the mouthpiece of the device.

Dose

Drug distribution

Distribution is the process by which drug molecules transfer into and out of the blood s tream. This is inuenced by the drug’s molecular

size and lipid solubility, the extent to which it binds to proteins in plasma, its suscepti bility to drug transporters expressed on cell

surfaces, and its binding to its molecular target and to other cellular proteins (which can be irreversible). Most drugs diffuse passively

across capillary walls down a concentration gradient into the interstitial uid until the con centration of free drug molecules in the

interstitial uid is equal to that in the plasma. As drug molecules in the blood are removed by metabolism or excretion, the plasma

concentration falls, drug molecules diffuse back from the tissue compartment into the bl ood and eventually all will be eliminated. Note

that this reverse movement of drug away from the tissues will be prevented if further drug dos es are administered and absorbed into the

plasma.

Volume of distribution

The apparent volume of distribution (V

) is the volume into which

a drug appears to have distributed following intravenous injection. It is calculated from the equation

= DC

where D is the amount of drug given and C

is the initial plasma concentration (Fig. 2.4A). Drugs that are highly bound to plasma

proteins may have a V

below 10 L (e.g. warfarin, aspirin), while those that diffuse into the interstitial uid but do not enter cells

because they have low lipid solubility may have a V

between 10 and 30 L (e.g. gentamicin, amoxicillin). It is an ‘apparent’ volume

because those drugs that are lipid-soluble and highly tissue-bound may have a V

of greater than 100 L (e.g. digoxin, amitriptyline).

Drugs with a larger V

have longer half-lives (see below), take longer to reach steady state on repeated administra tion and are eliminated

more slowly from the body following discontinuation.

Drug elimination

Drug metabolism

Metabolism is the process by which drugs are chemically altered from a lipid-soluble fo rm suitable for absorption and distribution to a

more water-soluble form that is necessary for excretion. Some drugs, known as ‘pr odrugs’, are inactive in the form in which they are

administered but are converted to an active metabolite in vivo.

Phase I metabolism involves oxidation, reduction or hydrolysis to make drug molecules suitable for phase II reactions or for excretion.

Oxidation is by far the most common form of phase I reaction and chiefly involve s members of the cytochrome P450 family of

membrane-bound enzymes in the endoplasmic reticulum of hepatocytes.

Phase II metabolism involves combining phase I metabolites with an endogeno us substrate to form an inactive conjugate that is much

more water-soluble. Reactions include glucuronidation, sulphation, acetylation, methylation an d conjugation with glutathione. This is

necessary to enable renal excretion, because lipid-soluble metabolites will simply diffuse back into the body after glomerular ltration (p.

349).

A constant fraction of drug is cleared in unit time

1/2

= 8 hours

6 121824 B

Time (hours)

Dose Dose Dose Dose Dose Dose

Loading dose

Adverse effects Therapeutic range

Subtherapeutic

1/2

= 30 hours Dose interval = 24 hours 123456 Time (days)

Fig. 2.4 Drug concentrations in plasma following single and multiple drug dosing. A In this example of rst-order kinetics following a

single intravenous dose, the time period required for the plasma drug concentration to h alve (half-life, t

1/2

) remains constant throughout

the elimination process. B After multiple dosing, the plasma drug concentration rises if each dose is administered before the previous

dose has been entirely cleared. In this example, the drug’s half-life is 30 hours, so that with daily dosing the peak, average and trough

concentrations steadily increase as drug accumulates in the body (black line). Steady state is reached after approximately 5 half-lives,

when the rate of elimination (the product of concentration and clearance) is equal to the rate of drug absorption (the product of rate of

administration and bioavailability). The long half-life in this example means that it takes 6 days for steady state to be achieved and, for

most of the rst 3 days of treatment, plasma drug concentrations are below the therapeutic range . This problem can be overcome if a

larger loading dose (red line) is used to achieve steady-state drug concentrations more rapidly .

Drug excretion

Excretion is the process by which drugs and their metabolites are removed from the bo dy.

Renal excretion is the usual route of elimination for drugs or their metabolites that are of low molecular weight and sufciently water-

soluble to avoid reabsorption from the renal tubule. Drugs bound to plasma proteins ar e not ltered by the glomeruli. The pH of the urine

is more acidic than that of plasma, so that some drugs (e.g. salicylates) become un-ionise d and tend to be reabsorbed. Alkalination of the

urine can hasten excretion (e.g. after a salicylate overdose; p. 138). For some drugs, active secretion into the proximal tubule lumen,

rather than glomerular filtration, is the predominant mechanism of excretion (e.g. metho trexate, penicillin).

Faecal excretion is the predominant route of elimination for drugs with high molecula r weight, including those that are excreted in the

bile after conjugation with glucuronide in the liver, and any drugs that are not absorbed after enteral administration. Molecules of drug or

metabolite that are excreted in the bile enter the small intestine, where they may, if they ar e sufciently lipid-soluble, be reabsorbed

through the gut wall and return to the liver via the portal vein (see Fig. 2.3). This recycling between the liver, bile, gut and portal vein is

known as ‘enterohepatic circulation’ and can signicantly prolong the residence of drugs in the body.

Elimination kinetics

The net removal of drug from the circulation results from a combination of drug metab olism and excretion, and is usually described as

‘clearance’, i.e. the volume of plasma that is completely cleared of drug per unit time.

For most drugs, elimination is a high-capacity process that does not become saturated, ev en at high dosage. The rate of elimination is

therefore directly proportional to the drug concentration because of the ‘law of mass a ction’, whereby higher drug concentrations will

drive faster metabolic reactions and support higher renal filtration rates. This results in ‘ rst-order’ kinetics, when a constant fraction of

the drug remaining in the circulation is eliminated in a given time and the decline in conc entration over time is exponential (Fig. 2.4A).

This elimination can be described by the drug’s half-life (t

1/2

), i.e. the time taken for the plasma drug concentration to halve, which

remains constant throughout the period of drug elimination. The signicance of this phenomenon for prescribers is that the effect of

increasing doses on plasma concentration is predictable – a doubled dose lead s to a doubled concentration at all time points.

For a few drugs in common use (e.g. phenytoin, alcohol), elimination capacity is excee ded (saturated) within the usual dose range. This

is called ‘zero-order’ kinetics. Its signicance for prescribers is that, if the rate of administ ration exceeds the maximum rate of

elimination, the drug will accumulate progressively, leading to serious toxicity.

Repeated dose regimens

The goal of therapy is usually to maintain drug concentrations within the therapeutic range (see Fig. 2.2) over several days (e.g.

antibiotics) or even for months or years (e.g. antihypertensives, lipid-lowering drugs, thyroid hormone replacement therapy). This goal is

rarely achieved with single doses, so prescribers have to plan a regimen of repeate d doses. This involves choosing the size of each

individual dose and the frequency of dose administration.

As illustrated in Figure 2.4B, the time taken to reach drug concentrations within the therapeutic range depends on the half-life of the

drug. Typically, with doses administered regularly, it takes approximately 5 half-lives to reach a ‘steady state’ in which the rate of drug

elimination is equal to the rate of drug administration. This applies when starting n ew drugs and when adjusting doses of current drugs.

With appropriate dose selection, steady-state drug concentrations will be maintained with in the therapeutic range. This is important for

prescribers because it means that the effects of a new prescription, or dose titra tion, for a drug with a long half-life (e.g. digoxin – 36

hours) may not be

known for a few days. In contrast, drugs with a very short half-life 2

(e.g. dobutamine – 2 minutes) have to be given continuously by infusion but

reach a new steady state within minutes.

For drugs with a long half-life, if it is unacceptable to wait for 5 half-lives unti l concentrations within the therapeutic range are achieved,

then an initial ‘loading dose’ can be given that is much larger than the maintenance dose and equivalent to the amount of drug required in

the body at steady state. This achieves a peak plasma concentration close to the pla teau concentration, which can then be maintained by

successive maintenance doses.

‘Steady state’ actually involves uctuations in drug concentrations, with peaks just after administration followed by troughs just prior to

the next administration. The manufacturers of medicines recommend dosing regimens th at predict that, for most patients, these

oscillations result in troughs within the therapeutic range and peaks that are not high eno ugh to cause adverse effects. The optimal dose

interval is a compromise between convenience for the patient and a constant level of dr ug exposure. More frequent administration (e.g.

25 mg 4 times daily) achieves a smoother plasma concentration prole than 100 mg once daily but is much more difcult for patients to

sustain. A solution to this need for compromise in dosing frequency for drugs with half -lives of less than 24 hours is the use of

‘modied-release’ formulations. These allow drugs to be absorbed more slowly from th e gastrointestinal tract and reduce the oscillation

in plasma drug concentration prole, which is especially important for drugs with a low therapeutic index (e.g. levodopa).

Inter-individual variation in drug responses

Prescribers have numerous sources of guidance about how to use drugs appropria tely (e.g. dose, route, frequency, duration) for many

conditions. However, this advice is based on average dose–response data derived f rom observations in many individuals. When applying

this information to an individual patient, prescribers must take account of inter-individua l variability in response. Some of this variability

is predictable and good prescribers are able to anticipate it and adjust their prescriptions according ly to maximise the chances of benet

and minimise harm. Inter-individual variation in responses also mandates that effects of treatment should be monitored (p. 34).

Some inter-individual variation in drug response is accounted for by differences in p harmacodynamics. For example, the benecial

natriuresis produced by the loop diuretic furosemide is often signicantly reduced at a given dose in patients with renal impairment,

while delirium caused by opioid analgesics is more likely in the elderly. Differences in p harmacokinetics more commonly account for

different drug responses, however. Examples of factors inuencing the absorp tion, metabolism and excretion of drugs are shown in Box

2.4.

It is hoped that a signicant proportion of the inter-individual variation in drug responses can be explained by studying genetic

differences in single genes (‘pharmacogenetics’; Box 2.5) or the effects of multip le gene variants (‘pharmacogenomics’). The aim is to

identify those patients most likely to benet from specic treatments and those m ost susceptible to adverse effects. In this way, it may be

possible to select drugs and dose regimens for individual patients to maximise the be net-to-hazard ratio (‘personalised medicine’).

2.4 Patient-specic factors that inuence pharmacokinetics Age

• Drug metabolism is low in the fetus and newborn, may be enhanced in young chi ldren, and becomes less effective with age

• Drug excretion falls with the age-related decline in renal function

Sex

• Women have a greater proportion of body fat than men, increasing the volume of distribution and half-life of lipid-soluble drugs

Body weight

• Obesity increases volume of distribution and half-life of lipid-soluble drugs

• Patients with higher lean body mass have larger body compartments into which drugs are distributed and may require higher doses

Liver function

• Metabolism of most drugs depends on several cytochrome P450 enzymes that are imp aired in patients with advanced liver disease

• Hypoalbuminaemia inuences the distribution of drugs that are highly protein-bound

Kidney function

• Renal disease and the decline in renal function with ageing may lead to drug acc umulation

Gastrointestinal function

• Small intestinal absorption of oral drugs may be delayed by reduced gastric mo tility

• Absorptive capacity of the intestinal mucosa may be reduced in disease (e.g. Crohn ’s or coeliac disease) or after surgical resection

Food

• Food in the stomach delays gastric emptying and reduces the rate (but not usually the extent) of drug absorption

• Some food constituents bind to certain drugs and prevent their absorption

Smoking

• Tar in tobacco smoke stimulates the oxidation of certain drugs

Alcohol

• Regular alcohol consumption stimulates liver enzyme synthesis, while binge drinking may temporarily inhibit drug metabolism

Drugs

• Drug–drug interactions cause marked variation in pharmacokinetics (see Box 2.11)

2.5 Examples of pharmacogenetic variations that inuence drug response

Genetic variant

Pharmacokinetic

Aldehyde dehydrogenase-2 deciency

Drug affected Clinical outcome

Ethanol

Acetylation Isoniazid, hydralazine, procainamide

Oxidation (CYP2D6) Nortriptyline Codeine

Oxidation (CYP2C18) Proguanil

Oxidation (CYP2C9) Oxidation (CYP2C19) Warfarin

Clopidogrel

Sulphoxidation

Human leucocyte antigen (HLA)-B*1502 Penicillamine Carbamazepine

Pseudocholinesterase deciency Suxamethonium (succinylcholine)

Elevated blood acetaldehyde causes facial ushing and increased heart rate in ~5 0% of Japanese, Chinese and other Asian populations

Increased responses in slow acetylators, up to 50% of some populations

Increased risk of toxicity in poor metabolisers

Reduced responses with slower conversion of codeine to more active morphine in poor metabolisers, 10% of European populations

Increased risk of toxicity in ultra-fast metabolisers, 3% of Europeans but 40% of Nort h Africans

Reduced efcacy with slower conversion to active cycloguanil in poor metabolisers

Polymorphisms known to inuence dosages

Reduced enzymatic activation results in reduced antiplatelet effect

Increased risk of toxicity in poor metabolisers

Increased risk of serious dermatological reactions (e.g. Stevens–Johnson syndrome) for 1 in 2000 in Caucasian populations (much higher

in some Asian countries)

Decreased drug inactivation leads to prolonged paralysis and sometimes persistent apnoea requir ing mechanical ventilation until the drug

can be eliminated by alternate pathways; occurs in 1 in 1500 people

Pharmacodynamic

Glucose-6-phosphate dehydrogenase (G6PD) deciency

Acute intermittent porphyria

SLC01B1 polymorphism

HLA-B*5701 polymorphism

HLA-B*5801 polymorphism

HLA-B*1502 polymorphism

Hepatic nuclear factor 1 alpha (HNF1A) polymorphism

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells Oxida nt drugs, including antimalarials (e.g. chloroquine,

primaquine)

Enzyme-inducing drugs

Statins

Abacavir

Allopurinol

Carbamazepine

Sulphonylureas

Risk of haemolysis in G6PD deciency

Increased risk of an acute attack

Increased risk of rhabdomyolysis

Increased risk of skin hypersensitivity reactions

Increased risk of rashes in Han Chinese

Increased risk of skin hypersensitivity reactions in Han Chinese Increased sensitivity to the blood glucose-lowering effects

Trastuzumab Increased sensitivity to the inhibitory effects on growth and division of the target can cer cells

Adverse outcomes of drug therapy

2.6 How to take a drug history

The decision to prescribe a drug always involves a judgement of the balance betw een therapeutic benets and risk of an adverse

outcome. Both prescribers and patients tend to be more focused on the former but a tru ly informed decision requires consideration of

both.

Adverse drug reactions

Some important denitions for the adverse effects of drugs are:

• Adverse event. A harmful event that occurs while a patient is taking a drug , irrespective of whether the drug is suspected of being the

cause.

• Adverse drug reaction (ADR). An unwanted or harmful reaction that is experienced following the administration of a drug or

combination of drugs under normal conditions of use and is suspected to be related to the drug. A n ADR will usually require the drug to

be discontinued or the dose reduced.

• Side-effect. Any effect caused by a drug other than the intended therapeutic effec t, whether benecial, neutral or harmful. The term

‘side-effect’ is often used interchangeably with ‘ADR’, although the former usually impl ies an ADR that occurs during exposure to

normal therapeutic drug concentrations (e.g. vasodilator-induced ankle oedema).

• Hypersensitivity reaction. An ADR that occurs as a result of an immunological r eaction and often at exposure to subtherapeutic drug

concentrations. Some of these reactions are immediate and result from the interaction of drug antigens with immunoglobulin E (IgE) on

mast cells and basophils, which causes a release of vasoactive biomolecules (e.g. penicill in-related anaphylaxis). ‘Anaphylactoid’

reactions present similarly but occur through a direct non-immune-mediated rele ase of the same mediators or result from direct

complement activation (p. 75). Hypersensitivity reactions may occur via other mechanisms such as ant ibody-dependent (IgM or IgG),

immune complex-mediated or cell-mediated pathways.

• Drug toxicity. Adverse effects of a drug that occur because the dose or pla sma concentration has risen above the therapeutic range,

either unintentionally or intentionally (drug overdose; see Fig. 2.2 and p. 137).

• Drug abuse. The misuse of recreational or therapeutic drugs that may lead to addiction or dependence, serious physiological injury

(such as liver damage), psychological harm (abnormal behaviour patterns, hallucinations, memory loss) or death (p. 1184).

Information from the patient (or carer) 2 Use language that patients will understand (e.g. ‘medic ines’ rather than ‘drugs’, which may be

mistaken for drugs of abuse) while

gathering the following information:

• Current prescribed drugs, including formulations (e.g. modiedrelease tablets ), doses, routes of administration, frequency and timing,

duration of treatment

• Other medications that are often forgotten (e.g. contraceptives, over-the-counter dru gs, herbal remedies, vitamins)

• Drugs that have been taken in the recent past and reasons for stopp ing them

• Previous drug hypersensitivity reactions, their nature and time course (e.g. rash, anaphylaxis)

• Previous ADRs, their nature and time course (e.g. ankle oedema with amlodipine)

• Adherence to therapy (e.g. ‘Are you taking your medication

regularly?’)

Information from GP medical records and/or pharmacist

• Up-to-date list of medications

• Previous ADRs

• Last order dates for each medication

Inspection of medicines

• Drugs and their containers (e.g. blister packs, bottles, vials) should be ins pected for name, dosage, and the number of dosage forms

taken since dispensed

(ADR = adverse drug reaction)

2.7 Risk factors for adverse drug reactions

Patient factors

• Elderly age (e.g. low physiological reserve)

• Gender (e.g. ACE inhibitor-induced cough in women)

• Polypharmacy (e.g. drug interactions)

• Genetic predisposition (see Box 2.5)

• Hypersensitivity/allergy (e.g.

-lactam antibiotics)

• Diseases altering pharmacokinetics (e.g. hepatic or renal impairment) or pharmacody namic responses (e.g. bladder instability)

• Adherence problems (e.g. cognitive impairment)

Drug factors

• Steep dose–response curve (e.g. insulin)

• Low therapeutic index (e.g. digoxin, cytotoxic drugs)

Prescriber factors

• Inadequate understanding of principles of clinical pharmacology

• Inadequate knowledge of the patient

• Inadequate knowledge of the prescribed drug

• Inadequate instructions and warnings provided to patients

• Inadequate monitoring arrangements planned

Prevalence of ADRs

ADRs are a common cause of illness, accounting in the UK for approximately 3% of c onsultations in primary care and 7% of emergency

admissions to hospital, and affecting around 15% of hospital inpatients. Many ‘disease’ presentations are eventually attributed to ADRs,

emphasising the importance of always taking a careful drug history (Box 2.6 ). Factors accounting for the rising prevalence of ADRs are

the increasing age of patients, polypharmacy (higher risk of drug interactions), increas ing

(ACE

angiotensin-converting enzyme)

availability of over-the-counter medicines, increasing use of herbal or traditional medici nes, and the increase in medicines available via

the Internet that can be purchased without a prescription from a health-care pro fessional. Risk factors for ADRs are shown in Box 2.7.

ADRs are important because they reduce quality of life for patients, reduce adherence to and therefore efcacy of benecial treatments,

cause diagnostic confusion, undermine the condence of patients in their health-care pr ofessional(s) and consume health-care resources.

Retrospective analysis of ADRs has shown that more than half could have been avoided if the prescriber had taken more care in

anticipating the potential hazards of drug therapy. For example, non-steroidal anti-inflam matory drug (NSAID) use accounts for many

thousands of emergency admissions, gastrointestinal bleeding episodes and a signicant number of deaths. In many cases, the patients

are at increased risk due to their age, interacting drugs (e.g. aspirin, warfarin) or a past history of peptic u lcer disease. Drugs that

commonly cause ADRs are listed in Box 2.8.

Prescribers and their patients ideally want to know the frequency with which ADRs occ ur for a specic drug. Although this may be well

characterised for more common ADRs observed in clinical trials, it is less clear for rare ly reported ADRs when the total numbers of

reactions and patients exposed are not known. The words used to describe frequency can be misinterpreted by

2.8 Drugs that are common causes of adverse drug reactions Drug or drug class

ACE inhibitors

(e.g. lisinopril)

Antibiotics

(e.g. amoxicillin)

Anticoagulants

(e.g. warfarin, heparin) Antipsychotics

(e.g. haloperidol)

Aspirin

Benzodiazepines

(e.g. diazepam)

β-blockers

(e.g. atenolol)

Calcium channel blockers (e.g. amlodipine)

Digoxin

Diuretics

(e.g. furosemide, bendroumethiazide)

Insulin

NSAIDs

(e.g. ibuprofen)

Opioid analgesics (e.g. morphine)

Common adverse drug reactions Renal impairment

Hyperkalaemia

Nausea

Diarrhoea

Bleeding

Falls

Sedation

Delirium

Gastrotoxicity (dyspepsia, gastrointestinal bleeding)

Drowsiness

Falls

Cold peripheries

Bradycardia

Ankle oedema

patients but widely accepted meanings include: very common (10% or more), com mon (1–10%), uncommon (0.1–1%), rare (0.01–0.1%)

and very rare (0.01% or less).

Classication of ADRs

ADRs have traditionally been classied into two major groups:

• Type A (‘augmented’) ADRs. These are predictable from

the known pharmacodynamic effects of the drug and are dose-dependent, co mmon (detected early in drug development) and usually

mild. Examples include constipation caused by opioids, hypotension caused by antihypertensives and deh ydration caused by diuretics.

• Type B (‘bizarre’) ADRs. These are not predictable, are not obviously d ose-dependent in the therapeutic range, are rare (remaining

undiscovered until the drug is marketed) and often severe. Patients who experience ty pe B reactions are generally ‘hyper-susceptible’

because of unpredictable immunological or genetic factors (e.g. anaphylaxis caused by pe nicillin, peripheral neuropathy caused by

isoniazid in poor acetylators).

This simple classification has shortcomings, and a more detailed classication based on d ose (see Fig. 2.2), timing and susceptibility

(DoTS) is now used by those analysing ADRs in greater depth (Box 2.9). The A B classication can be extended as a reminder of some

other types of ADR:

• Type C (‘chronic/continuous’) ADRs. These occur only

after prolonged continuous exposure to a drug. Examples include osteoporosis caused by glucocorticoids, retinopathy caused by

chloroquine, and tardive dyskinesia caused by phenothiazines.

• Type D (‘delayed’) ADRs. These are delayed until long after drug exposure, m aking diagnosis difcult. Examples include malignancies

that may emerge after

immunosuppressive treatment post-transplantation (e.g. azathioprine, tacrolimus) a nd vaginal cancer occurring many years after

exposure to diethylstilboestrol.

• Type E (‘end-of-treatment’) ADRs. These occur after abrupt drug withdra wal (see Box 2.3).

A teratogen is a drug with the potential to affect the development of the fetus in the rst 10 weeks of intrauterine life (e.g. phenytoin,

warfarin). The thalidomide disaster in the early 1960s highlighted the risk of teratogenic ity and led to mandatory testing of all new drugs.

Congenital defects in a live infant or aborted fetus should

Nausea and anorexia

Bradycardia

Dehydration

Electrolyte disturbance

(hypokalaemia, hyponatraemia) Hypotension

Renal impairment

Hypoglycaemia

Gastrotoxicity (dyspepsia, gastrointestinal bleeding) Renal impairment

Nausea and vomiting

Delirium

Constipation

2.9 DoTS classication of adverse drug reactions Category

Dose

Below therapeutic dose

In the therapeutic dose range At high doses

Timing

With the rst dose

Early stages of treatment On stopping treatment

Signicantly delayed

Susceptibility Example

Anaphylaxis with penicillin Nausea with morphine

Hepatotoxicity with paracetamol

Anaphylaxis with penicillin

Hyponatraemia with diuretics

Benzodiazepine withdrawal syndrome Clear-cell cancer with

diethylstilboestrol

See patient factors in Box 2.7

(ACE

angiotensin-converting enzyme; NSAID

non-steroidal anti-inammatory drug)

(INR = international normalised ratio)

provoke suspicion of an ADR and a careful exploration of drug exposures (including s elf-medication and herbal remedies).

Detecting ADRs – pharmacovigilance

Type A ADRs become apparent early in the development of a new drug. By the time a n ew drug is licensed and launched on to a

possible worldwide market, however, a relatively small number of patients (just several hundre d) may have been exposed to it, meaning

that rarer but potentially serious type B ADRs may remain undiscovered. Pharmacovigi lance is the process of detecting (‘signal

generation’) and evaluating ADRs in order to help prescribers and patients to be better informed about the risks of drug therapy. Drug

regulatory agencies may respond to this information by placing restrictions on the licensed indic ations, reducing the recommended dose

range, adding special warnings and precautions for prescribers in the product literature , writing to all health-care professionals or

withdrawing the product from the market.

Voluntary reporting systems allow health-care professionals and patients to report suspe cted ADRs to the regulatory authorities. A good

example is the ‘Yellow Card’ scheme that was set up in the UK in response to th e thalidomide tragedy. Reports are analysed to assess the

likelihood that they represent a true ADR (Box 2.10). Although voluntary repo rting is a continuously operating and effective early-

warning system for previously unrecognised rare ADRs, its weaknesses include low re porting rates (only 3% of all ADRs and 10% of

serious ADRs are ever reported), an inability to quantify risk (because the ratio of ADRs to prescriptions is unknown), and the inuence

of prescriber awareness on likelihood of reporting (reporting rates rise rapidly following pu blicity about potential ADRs).

More systematic approaches to collecting information on ADRs include ‘prescription ev ent monitoring’, in which a sample

2.10 TREND analysis of suspected adverse drug reactions

of prescribers of a particular drug are issued with questionnaires concerning the clinica l outcome for their patients, and the collection of

population statistics. Many health-care systems 2

routinely collect patient-identiable data on prescriptions (a

surrogate marker of exposure to a drug), health-care events (e.g. hospitalisation, opera tions, new clinical diagnoses) and other clinical

data (e.g. haematology, biochemistry). As these records are linked, with appropriate safe guards for condentiality and data protection,

they are providing a much more powerful mechanism for assessing both the harms and benets of drugs.

All prescribers will inevitably see patients experiencing ADRs caused by prescriptions w ritten by themselves or their colleagues. It is

important that these are recognised early. In addition to the features in Box 2.10, features that should raise suspicion of an ADR and the

need to respond (by drug withdrawal, dosage reduction or reporting to the regulatory authorities) include:

• concern expressed by a patient that a drug has

harmed them

• abnormal clinical measurements (e.g. blood pressure,

temperature, pulse, blood glucose and weight) or

laboratory results (e.g. abnormal liver or renal function, low

haemoglobin or white cell count) while on drug therapy

• new therapy started that could be in response to an ADR

(e.g. omeprazole, allopurinol, naloxone)

• the presence of risk factors for ADRs (see Box 2.7).

Drug interactions

A drug interaction has occurred when the administration of one drug increases or decreases the benecial or adverse responses to another

drug. Although the number of potential interacting drug combinations is very large, on ly a small number are common in clinical practice.

Important drug interactions are most likely to occur when the affected drug has a low therapeut ic index, steep dose–response curve, high

rst-pass or saturable metabolism, or a single mechanism of elimination.

Factor

Temporal relationship

Re-challenge

E xclusion Key question

What is the time

interval between the start of drug therapy and the reaction?

What happens when the patient is

re-challenged with the drug?

Have concomitant drugs and other

non-drug causes been excluded?

Novelty Has the reaction been reported before?

De-challenge Does the reaction

improve when the drug is withdrawn or the dose is reduced? Comment

Most ADRs occur soon after starting treatment and

within hours in the case of anaphylactic reactions

Re-challenge is rarely

possible because of the need to avoid exposing patients to unnecessary risk ADR i s a diagnosis of

exclusion following clinical assessment and relevant investigations for non-drug causes

The suspected ADR may already be recognised and mentioned in the SPC

approved by the regulatory authorities

Most, but not all, ADRs improve on drug

withdrawal, although

recovery may be slow

(SPC = summary of product characteristics)

Mechanisms of drug interactions

Pharmacodynamic interactions occur when two drugs produce additive, synergistic or antagonistic effects at the same drug target (e.g.

receptor, enzyme) or physiological system (e.g. electrolyte excretion, heart rate). These are the most common interactions in clinical

practice and some important examples are given in Box 2.11.

Pharmacokinetic interactions occur when the administration of a second drug alters the co ncentration of the rst at its site of action.

There are numerous potential mechanisms:

• Absorption interactions. Drugs that either delay (e.g.

anticholinergic drugs) or enhance (e.g. prokinetic drugs) gastric emptying inu ence the rate of rise in plasma concentration of other drugs

but not the total amount of drug absorbed. Drugs that bind to form insoluble comple xes or chelates (e.g. aluminium-containing antacids

binding with ciprooxacin) can reduce drug absorption.

• Distribution interactions. Co-administration of drugs that compete for pro tein binding in plasma (e.g. phenytoin and diazepam) can

increase the unbound drug concentration, but the effect is usually short-lived due to increase d elimination and hence restoration of the

pre-interaction equilibrium.

2.11 Common drug interactions Mechanism

Pharmaceutical* Chemical reaction Object drug Precipitant drug Result

Sodium

bicarbonate Calcium gluconate Precipitation of insoluble calcium carbonate

Pharmacokinetic Reduced absorption Tetracyclines

Reduced protein binding Phenytoin

Calcium, aluminium, and magnesium salts

Aspirin

Reduced tetracycline absorption

Increased unbound and reduced total phenytoin plasma concentration

Reduced metabolism: CYP3A4

Amiodarone Grapefruit juice

CYP2C19

CYP2D6

Xanthine oxidase

Monoamine oxidase

Increased metabolism (enzyme induction)

Reduced renal elimination Warfarin

Phenytoin

Clozapine

Azathioprine

Catecholamines Ciclosporin

Clarithromycin

Omeprazole

Paroxetine

Allopurinol

Monoamine oxidase inhibitors St John’s wort

Cardiac arrhythmias because of prolonged QT interval (p. 476)

Enhanced anticoagulation

Phenytoin toxicity

Clozapine toxicity

Azathioprine toxicity

Hypertensive crisis due to monoamine toxicity Loss of immunosuppression

Lithium

Methotrexate Diuretics NSAIDs Lithium toxicity

Methotrexate toxicity

Pharmacodynamic

Direct antagonism at same receptor

Direct potentiation in same organ system

Indirect potentiation by actions in different organ systems

Opioids

Salbutamol

Benzodiazepines ACE inhibitors Digoxin

Warfarin

Naloxone

Atenolol

Alcohol

NSAIDs

Diuretics

Aspirin, NSAIDs

Diuretics ACE inhibitors

*Pharmaceutical interactions are related to the formulation of the drugs and occur befo re drug absorption. (ACE = angiotensin-

converting enzyme; NSAID = non-steroidal anti-inammatory drug)

Reversal of opioid effects used therapeutically Inhibits bronchodilator effect

Increased sedation

Increased risk of renal impairment

Digoxin toxicity enhanced because of hypokalaemia Increased risk of bleeding becaus e of gastrotoxicity and antiplatelet effects

Blood pressure reduction (may be therapeutically advantageous) because of the i ncreased activity of the renin–angiotensin system in

response to diuresis

• Metabolism interactions. Many drugs rely on metabolism by different isoenzymes of cytochrome P450 (CYP) in the liver. CYP

enzyme inducers (e.g. phenytoin, rifampicin) generally reduce plasma concentrations of ot her drugs, although they may enhance

activation of prodrugs. CYP enzyme inhibitors (e.g. clarithromycin, cimetidine, grapefruit juice) have the opposite effect. Enzyme

induction effects usually take a few days to manifest because of the need to synthesise new CY P enzyme, in contrast to the rapid effects

of enzyme inhibition.

• Excretion interactions. These primarily affect renal excretion. For ex ample, drug-induced reduction in glomerular ltration rate (e.g.

diuretic-induced dehydration, angiotensin-converting enzyme (ACE) inhibitors, NSAIDs) can reduce the clearance and increase the

plasma concentration of many drugs, including some with a low therapeutic index (e.g. digoxin , lithium, aminoglycoside antibiotics).

Less commonly, interactions may be due to competition for a common tubular organic anion tr ansporter (e.g. methotrexate excretion may

be inhibited by competition with NSAIDs).

Avoiding drug interactions

Drug interactions are increasing as patients are prescribed more medicines (polypharma cy). Prescribers can avoid the adverse

consequences of drug–drug interactions by taking a careful drug history (see Box 2.6) before prescribing additional drugs, only

prescribing for clear indications, and taking special care when prescribing drugs with a narrow therapeutic index (e.g. warfarin). When

prescribing an interacting drug is unavoidable, good prescribers will seek fur ther information and anticipate the potential risk. This will

allow them to provide special warnings for the patient and arrange for monitoring, eithe r of the clinical effects (e.g. coagulation tests for

warfarin) or of plasma concentration (e.g. digoxin).

Medication errors

A medication error is any preventable event that may lead to inappropriate medication use or patient harm whi le the medication is in the

control of the health-care professional or patient. Errors may occur in prescribing, dispen sing, preparing solutions, administration or

monitoring. Many ADRs are considered in retrospect to have been ‘avoidable’ with mo re care or forethought; in other words, an adverse

event considered by one prescriber to be an unfortunate ADR might be considered b y another to be a prescribing error.

Medication errors are very common. Several thousand medication orders are dispensed and administered each day in a medium-sized

hospital. Recent UK studies suggest that

2.12 Hospital prescribing errors

Error type

Omission on admission

Underdose

Overdose

Strength/dose missing

Omission on discharge

Administration times incorrect/missing Duplication

Product or formulation not specied

Incorrect formulation

No maximum dose

Unintentional prescribing

No signature

Clinical contraindication

Incorrect route

No indication

Intravenous instructions incorrect/missing Drug not prescribed but indicated

Drug continued for longer than needed Route of administration missing

Start date incorrect/missing

Risk of drug interaction

Controlled drug requirements incorrect/missing Daily dose divided incorrectly

Signicant allergy

Drug continued in spite of adverse effects Premature discontinuation

Failure to respond to out-of-range drug level

Approximate % of total

< 0.5

Systems factors 2

• Working hours of prescribers (and others)

• Patient throughput

• Professional support and supervision by colleagues

• Availability of information (medical records)

• Design of prescription forms

• Distractions

• Availability of decision support

• Checking routines (e.g. clinical pharmacy)

• Reporting and reviewing of incidents

Prescriber factors

Knowledge

• Clinical pharmacology principles

• Drugs in common use

• Therapeutic problems commonly encountered

• Knowledge of workplace systems

Skills

• Taking a good drug history

• Obtaining information to support prescribing

• Communicating with patients

• Numeracy and calculations

• Prescription writing

Attitudes

• Coping with risk and uncertainty

• Monitoring of prescribing

• Checking routines

Unintended action

Slip

Prescription not as intended Prescriber unaware

Correct plan known but not executed (Causes include workload, time

pressures, distractions)

Lapse

Prescription incomplete or forgotten

Prescriber may remember

7–9% of hospital prescriptions contain an error, and most are written by junior doc tors. Common prescribing errors in hospitals include

omission of medicines (especially failure to prescribe regular medicines at the point of a dmission or discharge, i.e. ‘medicines

reconciliation’), dosing errors, unintentional prescribing and poor use of documentatio n (Box 2.12).

Most prescription errors result from a combination of failures by the individual prescriber and th e health-service systems in which they

work (Box 2.13). Health-care organisations increasingly encourage reporting of er rors within a ‘no-blame culture’ so that they can be

subject to ‘root cause analysis’ using human error theory (Fig. 2.5). Prevention is targeted at the factors in Box 2.13, and can be

supported by prescribers communicating and cross-checking with colleagues (e. g. when calculating doses adjusted for body weight, or

planning appropriate monitoring after drug administration), and by health-care syste ms providing clinical pharmacist support (e.g. for

checking the patient’s previous medications and current prescriptions) and electronic pr escribing (which avoids errors due to illegibility

or serious dosing mistakes, and may be combined with a clinical decision support system to take account of patient characteristics and

drug history, and provide warnings of potential contraindications and drug interactions).

2.13 Causes of prescribing errors

Planned

Correct action

action

Prescribing

Intended outcome

Intended action

Mistake

Prescription as intended but written based on

the wrong principles or lack of knowledge Prescriber unaware

Wrong plan selected (Causes include poor training and

lack of experience)

Violation

Deliberate deviations from standard practice Prescriber aware

Fig. 2.5 Human error theory. Unintended errors may occur because the prescriber fails to c omplete the prescription correctly (a slip; e.g.

writes the dose in ‘mg’ not ‘micrograms’) or forgets part of the action that is important for succ ess (a lapse; e.g. forgets to co-prescribe

folic acid with methotrexate); prevention requires the system to provide appropriate checking rout ines. Intended errors occur when the

prescriber acts incorrectly due to lack of knowledge (a mistake; e.g. prescribes ate nolol for a patient with known severe asthma because

of ignorance about the contraindication); prevention must focus on training the prescriber.

Responding when an error is discovered

All prescribers will make errors. When they do, their rst duty is to protect the p atient’s safety. This will involve a clinical review and

the taking of any steps that will reduce harm (e.g. remedial treatment, monitorin g, recording the event in the notes, informing

colleagues). Patients should be informed if they have been exposed to potential harm. For err ors that do not reach the patient, it is the

prescriber’s duty to report them, so that others can learn from the experience an d take the opportunity to reect on how a similar incident

might be avoided in the future.

Drug regulation and management

Given the powerful benecial and potentially adverse effects of drugs, the production and use of medicines are strictly regulated (e.g. by

the Food and Drug Administration in the USA, Medicines and Healthcare Products Regula tory Agency in the UK, and Central Drugs

Standard Control Organisation in India). Regulators are responsible for licensing med icines, monitoring their safety (pharmacovigilance;

p. 23), approving clinical trials, and inspecting and maintaining standards of drug developm ent and manufacture.

In addition, because of the high costs of drugs and their adverse effects, health-care se rvices must prioritise their use in light of the

evidence of their benet and harm, a process referred to as ‘medicines management’.

2.14 Clinical development of new drugs

Phase I

• Healthy volunteers (20–80)

• These involve initial single-dose, ‘rst-into-man’ studies, followed by

repeated-dose studies. They aim to establish the basic pharmacokinetic and pharma codynamic properties, and short-term safety

• Duration: 6–12 months

Phase II

• Patients (100–200)

• These investigate clinical effectiveness (‘proof of concept’), safety and dose–r esponse relationship, often with a surrogate clinical

endpoint, in the target patient group to determine the optimal dosing regimen for large r conrmatory studies

• Duration: 1–2 years

Phase III

• Patients (100s–1000s)

• These are large, expensive clinical trials that conrm safety and efc acy in the target patient population, using relevant clinical

endpoints. They may be placebo-controlled studies or comparisons with other active comp ounds

• Duration: 1–2 years

Phase IV

• Patients (100s–1000s)

• These are undertaken after the medicine has been marketed for its rst in dication to evaluate new indications, new doses or

formulations, long-term safety or cost-effectiveness

Drug development and marketing

Naturally occurring products have been used to treat illnesses for thousands of years and some remain in common use today. Examples

include morphine from the opium poppy (Papaver somniferum), digitalis from the fo xglove (Digitalis purpurea), curare from the bark of

a variety of species of South American trees, and quinine from the bark of the Cin chona species. Although plants and animals remain a

source of discovery, the majority of new drugs come from drug discovery progr ammes that aim to identify small-molecule compounds

with specic interactions with a molecular target that will induce a predicted biolog ical effect.

The usual pathway for development of these small molecules includes: identifyin g a plausible molecular target by investigating

pathways in disease; screening a large library of compounds for those that interact with th e molecular target in vitro; conducting

extensive medicinal chemistry to optimise the properties of lead compounds; testing efcacy and toxicity of these compounds in vitro

and in animals; and undertaking a clinical development programme (Box 2.14) . This process typically takes longer than 10 years and

may cost up to US$1 billion. Manufacturers have a dened period of exclusive marketing of t he drug while it remains protected by an

original patent, typically 10–15 years, during which time they must recoup the costs of developing the drug. Meanwhile, competitor

companies will often produce similar ‘me too’ drugs of the same class. Once th e drug’s patent has expired, ‘generic’ manufacturers may

step in to produce cheaper formulations of the drug. Paradoxically, if a generic drug is pr oduced by only one manufacturer, the price may

actually rise, sometimes substantially. Newer ‘biological’ products are based on la rge molecules (e.g. human recombinant antibodies)

derived from complex manufacturing processes involving specic cell lines, molecular c loning and purication processes. After the

patent for the originator product expires, other manufacturers may develop simi lar products (‘biosimilars’) that share similar

pharmacological actions but are not completely identical. ‘Biosimilars’ are considered dis tinct from ‘generic’ medications, as complex

biological molecules are more susceptible to differences in manufacturing processes tha n conventional small-molecule-type

pharmaceuticals.

The number of new drugs produced by the pharmaceutical industry has declined in r ecent years. The traditional approach of targeting

membrane-bound receptors and enzymes with small molecules (see Box 2.2) is n ow giving way to new targets, such as complex second-

messenger systems, cytokines, nucleic acids and cellular networks. These require novel therapeutic agents, which present new challenges

for ‘translational medicine’, the discipline of converting scientic discoveries into a useful medicin e with a well-dened benet–risk

prole (Box 2.15).

Licensing new medicines

New drugs are given a ‘market authorisation’, based on the evidence of quality, safety and efficacy presented by the manufacturer. The

regulator not only will approve the drug but also will take great care to ensure that the a ccompanying information reects the evidence

that has been presented. The summary of product characteristics (SPC), or ‘lab el’, provides detailed information about indications,

dosage, adverse effects, warnings, monitoring and so on. If approved, drugs can be made available with different levels of restriction:

• Controlled drug (CD). These drugs are subject to strict

legal controls on supply and possession, usually due to their abuse potential (e.g. opioid analgesic s).

Drug regulation and management • 27

2.15 Novel therapeutic alternatives to conventional small-molecule drugs Approaches

Monoclonal antibodies

Targeting of receptors or other molecules with relatively specic antibodies

Therapeutic indications

Cancer

Chronic inammatory diseases (e.g. rheumatoid arthritis, inammatory bowel disease)

Challenges 2

Selectivity of action

Complex manufacturing process

Small interfering RNA (siRNA)

Inhibition of gene expression

Gene therapy

Delivery of modied genes that supplement or alter host DNA

Macular degeneration Delivery to target

Cystic brosis

Cancer

Cardiovascular disease Delivery to target

Adverse effects of delivery vector (e.g. virus)

Stem cell therapy

Stem cells differentiate and replace damaged host cells

Parkinson’s disease Spinal cord injury

Ischaemic heart disease Delivery to target

Immunological compatibility Long-term effects unknown

• Prescription-only medicine (PoM). These are available only from a pharmacis t and can be supplied only if prescribed by an appropriate

practitioner.

• Pharmacy (P). These are available only from a pharmacist but can be supplied with out a prescription.

• General sales list (GSL). These medicines may be bought ‘over the counter’ ( OTC) from any shop and without a prescription.

Although the regulators take great care to agree the exact indications for prescribing a medicine , based on the evidence provided by the

manufacturer, there are some circumstances in which prescribers may direct its use outside the te rms stated in the SPC (‘off-label’

prescribing). Common situations where this might occur include prescribing outside the appr oved age group (e.g. prescribing for

children) or using an alternative formulation (e.g. administering a medicine provid ed in a solid form as an oral solution). Other important

examples might include prescribing for an indication for which there are no app roved medicines or where all of the approved medicines

have caused unacceptable adverse effects. Occasionally, medicines may be prescribed wh en there is no marketing authorisation in the

country of use. Examples include when a medicine licensed in another count ry is imported for use for an individual patient (‘unlicensed

import’) or when a patient requires a specic preparation of a medicine to be manufactur ed (‘unlicensed special’). When prescribing is

‘off-label’ or ‘unlicensed’, there is an increased requirement for prescribers to be able to justify their actions and to inform and agree the

decision with the patient.

Drug marketing

The marketing activities of the pharmaceutical industry are well resourced and are important in the process of recouping the massive

costs of drug development. In some countries, such as the USA, it is possible to promote a n ew drug by direct-toconsumer advertising,

although this is illegal in the countries of the European Union. A major focus is on promotion to prescribers via educational events,

sponsorship of meetings, advertisements in journals, involvement with opinion leaders, a nd direct contact by company representatives.

Such largesse has the potential to cause signicant conicts of interest and might te mpt prescribers to favour one drug over another, even

in the face of evidence on effectiveness or cost-effectiveness.

Managing the use of medicines

Many medicines meet the three key regulatory requirements of quality, safety and efc acy. Although prescribers are legally entitled to

prescribe any of them, it is desirable to limit the choice so that treatments for specic dise ases can be focused on the most effective and

cost-effective options, prescribers (and patients) gain familiarity with a smaller n umber of medicines, and pharmacies can concentrate

stocks on them.

The process of ensuring optimal use of available medicines is known as ‘medicines manage ment’ or ‘quality use of medicines’. It

involves careful evaluation of the evidence of benefit and harm from using the medicin e, an assessment of cost-effectiveness, and

support for processes to implement the resulting recommendations. These activities usually involve both national (e.g. National Institute

for Health and Care Excellence (NICE) in the UK) and local organisations (e.g. drug and therapeutics committees).

Evaluating evidence

The principles of evidence-based medicine are described on page 10. Drugs are often evalua ted in high-quality randomised controlled

trials, the results of which can be considered by systematic review (Fig. 2.6). Ideally, data are available n ot only for comparison with

placebo but also for ‘head-to-head’ comparison with alternative therapies. Trials are co nducted in selected patient populations, however,

and are not representative of every clinical scenario, so extrapolation to individual patien ts is not always straightforward. Other subtle

bias may be introduced because of the sources of funding (e.g. pharmaceutical industry) and the interests of the investigators in being

involved in research that has a ‘positive’ impact. These biases may be manifest in the way the trials are conducted or in how they are

interpreted or reported. A common example of the latter is the difference between relative a nd absolute risk of clinical events reported in

prevention trials. If a clinical event is encountered in the placebo arm at a rate of 1 in 50 patie nts (2%) but only 1 in 100 patients (1%) in

the active treatment arm, then the impact of treatment can be described as either a 50% rela tive risk reduction or 1% absolute risk

reduction. Although the former sounds more impressive, it is the latter that is of more impor tance to the Odds ratio

0.1 0.2 0.5 1 2 5 10

Favours treatment Favours placebo Fig. 2.6 Systematic review of the evid ence from randomised controlled clinical trials. This forest plot

shows the effect of warfarin compared with placebo on the likelihood of stroke in patients with atrial fibrillation in ve randomised

controlled trials that passed the quality criteria required for inclusion in a meta-analysis. For eac h trial, the purple box is proportionate to

the number of participants. The tick marks show the mean odds ratio and the black line s indicate its 95% condence intervals. Note that

not all the trials showed statistically signicant effects (i.e. the condence intervals c ross 1.0). However, the meta-analysis, represented

by the black diamond, conrms a highly signicant statistical benet. The overall odds ra tio is approximately 0.4, indicating a mean 60%

risk reduction with warfarin treatment in patients with the characteristics of the participants in these trials.

individual patient. It means that the number of patients that needed to be treated (NNT) f or 1 to benet (compared to placebo) was 100.

This illustrates how large clinical trials of new medicines can produce highly statistical ly signicant and impressive relative risk

reductions and still predict a very modest clinical impact.

Evaluating cost-effectiveness

New drugs often represent an incremental improvement over the current standard of care but a re usually more expensive. Health-care

budgets are limited in every country and so it is impossible to fund all new medicines. Th is means that very difcult nancial decisions

have to be taken with due regard to the principles of ethical justice. The main approach taken is cost-effectiveness analysis (CEA), where

a comparison is made between the relative costs and outcomes of different courses of action. CEA is usually expressed as a ratio where

the denominator is a gain in health and the numerator is the cost associated with the health gain. A major challenge is to compare the

value of interventions for different clinical outcomes. One method is to calculate the quality -adjusted life years (QALYs) gained if the

new drug is used rather than standard treatment. This analysis involves estimating the ‘utility’ of various health states between 1 (perfect

health) and 0 (dead). If the additional costs and any savings are known, then it is possible to derive the incremental cost-effectiveness

ratio (ICER) in terms of cost/QALY. These principles are exemplied in Box 2.16 . There are, however, inherent weaknesses in this kind

of analysis: it usually depends on modelling future outcomes well beyond the dura tion of the clinical trial data that are available; it

assumes that QALYs gained at all ages are of equivalent value; and the appropriate standard ca re against which the new drug should be

compared is often uncertain.

These pharmacoeconomic assessments are challenging and resource-intensive, and are undertaken at national level in most countries,

e.g. in the UK by NICE.

2.16 Cost-effectiveness analysis

A clinical trial lasting 2 years compares two interventions for the treatment of co lon cancer:

• Treatment A: standard treatment, cost £1000/year, oral therapy

• Treatment B: new treatment, cost £6000/year, monthly intravenous

infusions, often followed by a week of nausea.

The new treatment (B) signicantly increases the average time to progression (18 mo nths versus 12 months) and reduces overall

mortality (40% versus 60%). The health economist models the survival curves f rom the trial in order to undertake a cost–utility analysis

and concludes that:

• Intervention A: allows an average patient to live for 2 extra years at

a utility 0.7

1.4 QALYs (cost £2000)

• Intervention B: allows an average patient to live for 3 extra years at

a utility 0.6

1.8 QALYs (cost £18 000).

The health economists conclude that treatment B provides an extra 0.4 QALYs at an extra cost of £16 000, meaning that the ICER

£40

000/QALY. They recommend that the new treatment should not be funded on the basis that their threshold for cost acceptability is £30

000/QALY.

(ICER = incremental cost-effectiveness ratio; QALY = quality-adjusted life year)

Implementing recommendations

Many recommendations about drug therapy are included in clinical guidelines written b y an expert group after systematic review of the

evidence. Guidelines provide recommendations rather than obligations for prescribers and are h elpful in promoting more consistent and

higher-quality prescribing. They are often written without concern for cost-effectivenes s, however, and may be limited by the quality of

available evidence. Guidelines cannot anticipate the extent of the variation between individual pa tients who may, for example, have

unexpected contraindications to recommended drugs or choose different priorities for treatmen t. When deviating from respected national

guidance, prescribers should be able to justify their practice.

Additional recommendations for prescribing are often implemented locally or imposed b y bodies responsible for paying for health care.

Most health-care units have a drug and therapeutics committee (or equivalent) co mprised of senior and junior medical staff, pharmacists

and nurses, as well as managers (because of the implications of the committee’s work for governance and resources). This group

typically develops local prescribing policy and guidelines, maintains a local drug formulary a nd evaluates requests to use new drugs. The

local formulary contains a more limited list than any national formulary (e.g. B ritish National Formulary) because the latter lists all

licensed medicines that can be prescribed legally, while the former contains only those th at the health-care organisation has approved for

local use. The local committee may also be involved, with local specialists, in pro viding explicit protocols for management of clinical

scenarios.

Prescribing in practice

Decision-making in prescribing

Prescribing should be based on a rational approach to a series of challenges (see Box 2.1).

Making a diagnosis

Ideally, prescribing should be based on a conrmed diagnosis but, in reality, many prescriptions are based on the balance of probability,

taking into account the differential diagnosis (e.g. proton pump inhibitors for post-pran dial retrosternal discomfort).

Establishing the therapeutic goal

The goals of treatment are usually clear, particularly when relieving symptoms (e.g. pain , nausea, constipation). Sometimes the goal is

less obvious to the patient, especially when aiming to prevent future events (e.g. ACE inhibitors to prevent hospitalisation and extend life

in chronic heart failure). Prescribers should be clear about the therapeutic goal against whic h they will judge success or failure of

treatment. It is also important to establish that the value placed on this goal by the prescriber is shared by the patient (concordance).

Choosing the therapeutic approach

For many clinical problems, drug therapy is not absolutely mandated. Having taken the potential benets and harms into account,

prescribers must consider whether drug therapy is in the patient’s interest and is preferred to no treatment or one of a range of

alternatives (e.g. physiotherapy, psychotherapy, surgery). Assessing the balance of be net and harm is often complicated and depends on

various features associated with the patient, disease and drug (Box 2.17).

Choosing a drug

For most common clinical indications (e.g. type 2 diabetes, depression), more than one d rug is available, often from more than one drug

class. Although prescribers often have guidance about which represents the rational choice for the average patient, they still need to

consider whether this is the optimal choice for the individual patient. Certain factors may inuence the choice of drug:

Absorption

Patients may nd some formulations easier to swallow than others or may be vomiting an d require a drug available for parenteral

administration.

Distribution

Distribution of a drug to a particular tissue sometimes dictates

choice (e.g. tetracyclines and rifampicin are concentrated in the bile, and lincomyc in and clindamycin in bones).

Metabolism

Drugs that are extensively metabolised should be avoided in severe liver disease (e.g. o pioid analgesics).

Excretion

Drugs that depend on renal excretion for elimination (e.g. digoxin, aminoglycoside anti biotics) should be avoided in patients with

impaired renal function if suitable alternatives exist.

Efcacy

Prescribers normally choose drugs with the greatest efcacy in

achieving the goals of therapy (e.g. proton pump inhibitors rather than H

-receptor antagonists). It may be appropriate, however, to

compromise on efcacy if other drugs are more convenient, safer to use or less expensive.

Avoiding adverse effects

Prescribers should be wary of choosing drugs that are more likely to cause adverse eff ects (e.g. cephalosporins rather than alternatives

for patients allergic to penicillin) or worsen coexisting conditions (e.g. β-blockers as trea tment for angina in patients with asthma).

Features of the disease

This is most obvious when choosing antibiotic therapy, which should be based o n the known or suspected sensitivity of the infective

organism (p. 116).

Severity of disease

The choice of drug should be appropriate to disease severity (e.g. paracetamol for mild pain, morphine for severe pain). Coexisting

disease

This may be either an indication or a contraindication to therapy. Hypertensive patients m ight be prescribed a

-blocker if they also have

left ventricular impairment but not if they have asthma.

Avoiding adverse drug interactions

Prescribers should avoid giving combinations of drugs that might interact, either directly or indirectly (see Box 2.11).

Patient adherence to therapy

Prescribers should choose drugs with a simple dosing schedule

or easier administration (e.g. the ACE inhibitor lisinopril once daily rather than captopril 3 times daily for hypertension).

Cost

Prescribers should choose the cheaper drug (e.g. a generic

or biosimilar) if two drugs are of equal efficacy and safety. Even if cost is not a concer n for the individual patient, it is important to

remember that unnecessary expenditure will ultimately limit choices for other prescriber s and patients. Sometimes a more costly drug

may be appropriate (e.g. if it yields improved adherence).

Genetic factors

There are already a small number of examples where genotype inuences the choic e of drug therapy (see Box 2.5).

2.17 Factors to consider when balancing benets and harms of drug therapy

• Seriousness of the disease or symptom

• Efcacy of the drug

• Seriousness of potential adverse effects

• Likelihood of adverse effects

• Efcacy of alternative drugs or non-drug therapies

• Safety of alternative drugs or non-drug therapies

Choosing a dosage regimen

Prescribers have to choose a dose, route and frequency of administration (dosage regi men) to achieve a steady-state drug concentration

that provides sufcient exposure of the target tissue without producing toxic effects. Ma nufacturers draw up dosage recommendations

based on average observations in many patients but the optimal regimen that will maximi se the benet to harm ratio for an individual

patient is never certain. Rational prescribing involves treating each prescription as an exper iment and gathering sufcient information to

amend it if necessary. There are some general principles that should be followed, as describe d below.

Dose titration

Prescribers should generally start with a low dose and titrate

this slowly upwards as necessary. This cautious approach is particularly important if the patient is likely to be more sensitive to adverse

pharmacodynamic effects (e.g. delirium or postural hypotension in the elderly) or have al tered pharmacokinetic handling (e.g. renal or

hepatic impairment), and when using drugs with a low therapeutic index (e.g. benzodia zepines, lithium, digoxin). There are some

exceptions, however. Some drugs must achieve therapeutic concentration quickly be cause of the clinical circumstance (e.g. antibiotics,

glucocorticoids, carbimazole). When early effect is important but there may be a delay in achievin g steady state because of a drug’s long

half-life (e.g. digoxin, warfarin, amiodarone), an initial loading dose is given prior to establishing the appropriate maintenance dose (see

Fig. 2.4).

If adverse effects occur, the dose should be reduced or an alternative drug prescribed; in s ome cases, a lower dose may sufce if it can be

combined with another synergistic drug (e.g. the immunosuppressant azathioprine reduces gluco corticoid requirements in patients with

inflammatory disease). It is important to remember that the shape of the dose–response curve (s ee Fig. 2.2) means that higher doses may

produce little added therapeutic effect and might increase the chances of toxicity.

Route

There are many reasons for choosing a particular route of administration (Box 2.18 ).

Frequency

Frequency of doses is usually dictated by a manufacturer’s

recommendation. Less frequent doses are more convenient for patients but result in gre ater uctuation between peaks and troughs in drug

concentration (see Fig. 2.4). This is relevant if the peaks are associated with adverse effe cts (e.g. dizziness with antihypertensives) or the

troughs are associated with troublesome loss of effect (e.g. anti-Parkinsonian drugs). T hese problems can be tackled either by splitting

the dose or by employing a modied-release formulation, if available.

Timing

For many drugs the time of administration is unimportant. There

are occasionally pharmacokinetic or therapeutic reasons, however, for giving drugs at particular times (Box 2.19).

Formulation

For some drugs there is a choice of formulation, some for use by different routes. Som e are easier to ingest, particularly by children (e.g.

elixirs). The formulation is important when writing repeat prescriptions for drugs with a low th erapeutic index that come in different

formulations (e.g. lithium, phenytoin, theophylline). Even if the prescribed dos e remains constant, an alternative formulation may differ

in its absorption and bioavailability, and hence plasma drug concentration. The se are examples of the small number of drugs that should

be prescribed by specic brand name rather than ‘generic’ international non-proprieta ry name (INN).

2.18 Factors inuencing the route of drug administration

Reason

Only one route possible

Patient adherence

Poor absorption

Rapid action

Vomiting

Avoidance of rst-pass metabolism

Certainty of effect

Direct access to the site of action (avoiding

unnecessary systemic exposure)

Ease of access

Comfort Example

Dobutamine (IV)

Gliclazide (oral)

Phenothiazines and thioxanthenes (2 weekly IM depot injections rather than daily tablets, in schizophrenia) Furosemide (IV rather than

oral, in severe heart failure)

Haloperidol (IM rather than oral, in acute behavioural disturbance)

Phenothiazines (PR or buccal rather than oral, in nausea)

Glyceryl trinitrate (SL, in angina pectoris)

Amoxicillin (IV rather than oral, in acute chest infection)

Bronchodilators (INH rather than oral, in asthma)

Local application of drugs to skin, eyes etc.

Diazepam (PR, if IV access is difcult in status epilepticus)

Adrenaline (epinephrine) (IM, if IV access is difcult in acute anaphylaxis) Mor phine (SC rather than IV in terminal care)

(IM

intramuscular; INH

by inhalation; IV

intravenous; PR

per rectum; SC = subcutaneous; SL = sublingual)

Duration

Some drugs require a single dose (e.g. thrombolysis post

myocardial infarction), while for others the duration of the course of treatment is certain at the outset (e.g. antibiotics). For most, the

duration will be largely at the prescriber’s discretion and will depend on response and disea se progression (e.g. analgesics,

antidepressants). For many, the treatment will be long-term (e.g. insulin, antihypertensiv es, levothyroxine).

Involving the patient

Patients should, whenever possible, be engaged in making choices about drug therapy. Their beliefs and expectations affect the goals of

therapy and help in judging the acceptable benet/ harm balance when selecting treatme nts. Very often, patients may wish to defer to the

professional expertise of the prescriber. Nevertheless, they play key roles in adherence to therapy and in monitoring treatment, not least

by providing early warning of adverse events. It is important for them to be provide d with the necessary information to understand the

choice that has been made, what to expect from the treatment, and any measurements that m ust be undertaken (Box 2.20).

A major drive to include patients has been the recognition that up to half of the drug do ses for chronic preventative therapy are not taken.

This is often termed ‘non-compliance’ but is more appropriately called ‘non-adherenc e’, to reect a less paternalistic view of the doctor–

patient relationship; it may or may not be intentional. Non-adherence to the dose regimen red uces the likelihood of benets to the patient

and can be costly in terms

2.19 Factors inuencing the timing of drug therapy

Drug

Diuretics (e.g. furosemide)

Statins (e.g. simvastatin)

Antidepressants (e.g. amitriptyline) Salbutamol

Glyceryl trinitrate

Paracetamol

Regular nitrate therapy (e.g. isosorbide mononitrate)

Aspirin

Alendronate

Tetracyclines

Hypnotics (e.g. temazepam)

Antihypertensive drugs (e.g. amlodipine) Recommended timing Once in the morning O nce at night

Once at night

Before exercise

When required

Reasons 2 Night-time diuresis undesirable

HMG CoA reductase activity is greater at night

Allows adverse effects to occur during sleep

Reduces symptoms in exercise-induced asthma

Relief of acute symptoms only

Eccentric dosing regimen (e.g. twice daily at 8 a.m. and 2 p.m.)

With food

Once in the morning before breakfast, sitting upright

2 hours before or after food or antacids

Once at night

Once in the morning

Reduces development of nitrate tolerance by allowing drug-free period each night

Minimises gastrotoxic effects

Minimises risk of oesophageal irritation

Divalent and trivalent cations chelate tetracyclines, preventing absorption

Maximises therapeutic effect and minimises daytime sedation Blood pressure is higher d uring the daytime

(HMG CoA

3-hydroxy-3-methylglutaryl-coenzyme A)

2.20 What patients need to know about their medicines*

Comment

Reinforces the goals of therapy Knowledge

The reason for taking the medicine How the medicine works

How to take the medicine

What benets to expect

What adverse effects might occur

Precautions that improve safety

When to return for review May be important for the effectiveness (e.g. inhaled salbuta mol in asthma) and safety (e.g. alendronate for

osteoporosis) of treatment

May help to support adherence or prompt review because of treatment failure

Discuss common and mild effects that may be transient and might not require discontinu ation Mention rare but serious effects that might

inuence the patient’s consent

Explain symptoms to report that might allow serious adverse effects to be averted, moni toring that will be required and potentially

important drug–drug interactions

This will be important to enable monitoring

*Many medicines are provided with patient information leaets, which the patient should be encouraged to read.

of wasted medicines and unnecessary health-care episodes. An important reason may b e lack of concordance with the prescriber about

the goals of treatment. A more open and shared decisionmaking process might resolve any misunderstandings at the outset and foster

improved adherence, as well as improved satisfaction with health-care services and con dence in prescribers. Fully engaging patients in

shared decision-making is sometimes constrained by various factors, such as limited con sultation time and challenges in communicating

complex numerical data.

Writing the prescription

The culmination of the planning described above is writing an accurate and legible prescription so that the drug will be dispensed and

administered as planned (see ‘Writing prescriptions’ below).

Monitoring treatment effects

Rational prescribing involves monitoring for the benecial and adverse effects of tr eatment so that the balance remains in favour of a

positive outcome (see ‘Monitoring drug therapy’ below).

Stopping drug therapy

It is also important to review long-term treatment at regular intervals to assess whether co ntinued treatment is required. Elderly patients

are keen to reduce their medication burden and are often prepared to compromise on the original goals of long-term preventative therapy

to achieve this.

Prescribing in special circumstances Prescribing for patients with renal disease

Patients with renal impairment are readily identied by having a low estimated glomerular ltration rate (eGFR < 60 mL/min) based on

their serum creatinine, age, sex and ethnic group (p. 386). This group includes a large proportion of elderly patients. If a drug (or its

active metabolites) is eliminated predominantly by the kidneys, it will tend to accumulate a nd so the maintenance dose must be reduced.

For some drugs, renal impairment makes patients more sensitive to their adverse pharma codynamic effects.

2.21 Some drugs that require extra caution in patients with renal or hepatic disease

Kidney disease Liver disease Pharmacodynamic effects enhanced

ACE inhibitors and ARBs (renal impairment, hyperkalaemia) Metformin (lactic aci dosis) Spironolactone (hyperkalaemia) NSAIDs

(impaired renal function) Sulphonylureas (hypoglycaemia) Insulin (hypoglycaemia)

Warfarin (increased

anticoagulation because of reduced clotting factor synthesis) Metformin (lactic acidosis)

Chloramphenicol (bone marrow suppression)

NSAIDs (gastrointestinal

bleeding, uid retention)

Sulphonylureas (hypoglycaemia) Benzodiazepines (coma)

Pharmacokinetic handling altered (reduced clearance) Aminoglycosides (e.g. gentamicin) Vancomycin

Digoxin

Lithium

Other antibiotics (e.g.

ciprooxacin)

Atenolol

Allopurinol

Cephalosporins

Methotrexate

Opioids (e.g. morphine)

Phenytoin

Rifampicin

Propranolol

Warfarin

Diazepam

Lidocaine

Opioids (e.g. morphine)

(ACE

angiotensin-converting enzyme; ARB

angiotensin receptor blocker; NSAID = non-steroidal anti-inammatory drug)

2.22 Prescribing in old age

• Reduced drug elimination: partly due to impaired renal function.

• Increased sensitivity to drug effects: notably in the brain (leading to sedation or delirium) and as a result of comorbidities.

• More drug interactions: largely as a result of polypharmacy.

• Lower starting doses and slower dose titration: often required, with careful monitoring of drug effects.

• Drug adherence: may be poor because of cognitive impairment, difculty swallow ing (dry mouth) and complex polypharmacy

regimens. Supplying medicines in pill organisers (e.g. dosette boxes or calendar blister packs), providing automatic reminders, and

regularly reviewing and simplifying the drug regimen can help.

• Some drugs that require extra caution, and their mechanisms:

Digoxin : increased sensitivity of Na

pump; hypokalaemia due to diuretics; renal impairment favours accumulation → increased risk

of toxicity.

Antihypertensive drugs: reduced baroreceptor function → increased risk of postural h ypotension.

Antidepressants, hypnotics, sedatives, tranquillisers: increased sensitivity of the brain; reduce d metabolism → increased risk of toxicity.

Warfarin: increased tendency to falls and injury and to bleeding from intra- and extracra nial sites; increased sensitivity to inhibition of

clotting factor synthesis → increased risk of bleeding.

Clomethiazole, lidocaine, nifedipine, phenobarbital, propranolol, theophylline: metabolism re duced

→

increased risk of toxicity. Non-

steroidal anti-inammatory drugs: poor renal function → increased risk of renal impair ment; susceptibility to gastrotoxicity → increased

risk of upper gastrointestinal bleeding.

Examples of drugs that require extra caution in patients with renal disease are listed in Box 2.21 .

Prescribing for patients with hepatic disease

The liver has a large capacity for drug metabolism and hepatic insufciency ha s to be advanced before drug dosages need to be modied.

Patients who may have impaired metabolism include those with jaundice, ascites, hypoalb uminaemia, malnutrition or encephalopathy.

Hepatic drug clearance may also be reduced in acute hepatitis, in hepatic congestion due to car diac failure, and in the presence of

intrahepatic arteriovenous shunting (e.g. in hepatic cirrhosis). There are no good tests o f hepatic drugmetabolising capacity or of biliary

excretion, so dosage should be guided by the therapeutic response and careful mo nitoring for adverse effects. The presence of liver

disease also increases the susceptibility to adverse pharmacological effects of drugs. Some drugs th at require extra caution in patients

with hepatic disease are listed in Box 2.21.

Prescribing for elderly patients

The issues around prescribing in the elderly are discussed in Box 2.22.

Prescribing for women who are pregnant or breastfeeding

Prescribing in pregnancy should be avoided if possible to minimise the risk of adverse e ffects in the fetus. Drug therapy in pregnancy

may, however, be required either for a pre-existing problem (e.g. epilepsy, asthma, hy pothyroidism) or for problems that arise during

pregnancy (e.g. morning sickness, anaemia, prevention of neural tube defects, gestational diabetes, hypertension). About 35% of women

take drug therapy at least once during

2.23 Prescribing in pregnancy

• Teratogenesis: a potential risk, especially when drugs are taken between 2 and 8 weeks of gestation (4–10 weeks from last menstrual

period). Common teratogens include retinoids (e.g. isotretinoin), cytotoxic drugs, ang iotensin-converting enzyme inhibitors,

antiepileptics and warfarin. If there is inadvertent exposure, then the timing of co nception should be established, counselling given and

investigations undertaken for fetal abnormalities.

• Adverse fetal effects in late gestation: e.g. tetracyclines may stain growing teeth and b ones; sulphonamides displace fetal bilirubin from

plasma proteins, potentially causing kernicterus; opioids given during delivery may be associated w ith respiratory depression in the

neonate.

• Altered maternal pharmacokinetics: extracellular uid volume and V

increase. Plasma albumin falls but other binding globulins (e.g.

for thyroid and steroid hormones) increase. Glomerular ltration increases by approxim ately 70%, enhancing renal clearance. Placental

metabolism contributes to increased clearance, e.g. of levothyroxine and glucocorticoids. Th e overall effect is a fall in plasma levels of

many drugs.

• In practice:

Avoid any drugs unless the risk:benet analysis is in favour of treating (usually th e mother).

Use drugs for which there is some record of safety in humans. Use the lowest dose for the shortest time possible.

Choose the least harmful drug if alternatives are available.

pregnancy and 6% take drug therapy during the rst trimester (excluding iron, folic acid and vitamins). The most commonly used drugs

are simple analgesics, antibacterial drugs and antacids. Some considerations when pr escribing in pregnancy are listed in Box 2.23.

Drugs that are excreted in breast milk may cause adverse effects in the baby. Prescribe rs should always consult the summary of product

characteristics for each drug or a reliable formulary when treating a pregnant woman or breastfeeding mother.

Writing prescriptions

A prescription is a means by which a prescriber communicates the intended plan of tre atment to the pharmacist who dispenses a

medicine and to a nurse or patient who administers it. It should be precise, accurate, clea r and legible. The two main kinds of

prescription are those written, dispensed and administered in hospital and those written in p rimary care (in the UK by a GP), dispensed at

a community pharmacy and self-administered by the patient. The information supplied m ust include:

• the date

• the identication details of the patient

• the name of the drug

• the formulation

• the dose

• the frequency of administration

• the route and method of administration

• the amount to be supplied (primary care only)

• instructions for labelling (primary care only)

• the prescriber’s signature.

2.24 High-risk prescribing moments

• Trying to amend an active prescription (e.g. altering the dose/ 2 timing) – always avoid and start again

• Writing up drugs in the immediate presence of more than one prescription chart or set of notes – avoid

• Allowing one’s attention to be diverted in the middle of completing a p rescription – avoid

• Prescribing ‘high-risk’ drugs (e.g. anticoagulants, opioids, insulin, sedatives) – a sk for help if necessary

• Prescribing parenteral drugs – take care

• Rushing prescribing (e.g. in the midst of a busy ward round)

– avoid

• Prescribing unfamiliar drugs – consult the formulary and ask for help if necessary

• Transcribing multiple prescriptions from an expired chart to a new one – take care to review the rationale for each medicine

• Writing prescriptions based on information from another source such as a ref erral letter (the list may contain errors and some of the

medicines may be the cause of the patient’s illness) – review the justication for each as if it is a new prescription

• Writing up ‘to take out’ drugs (because these will become the patient’s regular medi cation for the immediate future) – take care and

seek advice if necessary

• Calculating drug doses – ask a colleague to perform an

independent calculation or use approved electronic dose

calculators

• Prescribing sound-alike or look-alike drugs (e.g. chlorphenamine an d chlorpromazine) – take care

Prescribing in hospital

Although GP prescribing is increasingly electronic, most hospital prescribing continues t o be based around the prescription and

administration record (the ‘drug chart’) (Fig. 2.7). A variety of charts are in u se and prescribers must familiarise themselves with the

local version. Most contain the following sections:

• Basic patient information: will usually include name, age,

date of birth, hospital number and address. These details are often ‘lled in’ us ing a sticky addressograph label but this increases the risk

of serious error.

• Previous adverse reactions/allergies: communicates important patient safety inform ation based on a careful drug history and/or the

medical record.

• Other medicines charts: notes any other hospital prescription documents that contain current prescriptions being received by the patient

(e.g. anticoagulants, insulin, oxygen, uids).

• Once-only medications: for prescribing medicines to be used infrequently, such as single-dose prophylactic antibiotics and other pre-

operative medications.

• Regular medications: for prescribing medicines to be taken for a number of days or continuously, such as a course of antibiotics,

antihypertensive drugs and so on.

• ‘As required’ medications: for prescribing for symptomatic relief, usually to be adm inistered at the discretion of the nursing staff (e.g.

antiemetics, analgesics).

Prescribers should be aware of the risks of prescription error (Box 2.24 and see Box 2.13 ), ensure they have considered the rational basis

for their prescribing decision described above, and then follow the guidance illustrated in Figure 2.7 in order to write the prescription. It

is a basic principle that a prescription will be followed by a judgement as to its succes s or failure and any appropriate changes made (e.g.

altered dosage, discontinuation or substitution).

Hospital discharge (‘to take out’) medicines

Most patients will be prescribed a short course of their medicines at discharge. This pres cription is particularly important because it

usually informs future therapy at the point of transfer of prescribing responsibility to pr imary care. Great care is required to ensure that

this list is accurate. It is particularly important to ensure that any hospital medicines that s hould be stopped are not included and that

those intended to be administered for a short duration only are clearly identied. It is al so important for any signicant ADRs

experienced in hospital to be recorded and any specic monitoring or review ident ied.

Prescribing in primary care

Most of the considerations above are equally applicable to primary care (GP) prescripti ons. In many health-care systems, community

prescribing is electronic, making issues of legibility irrelevant and often providing b asic decision support to limit the range of doses that

can be written and highlight potential drug interactions. Important additional issues more relevan t to GP prescribing are:

• Formulation. The prescription needs to carry information

about the formulation for the dispensing pharmacist (e.g. tablets or oral suspensio n).

• Amount to be supplied. In the hospital the pharmacist will organise this. Elsewhere it mus t be specied either as the precise number of

tablets or as the duration of treatment. Creams and ointments should be specied in gr ams and lotions in mL.

• Controlled drugs. Prescriptions for ‘controlled’ drugs (e.g. opi oid analgesics, with potential for drug abuse) are subject to additional

legal requirements. In the UK, they

A PRESCRIPTION AND ADMINISTRATION RECORD

Hospital/Ward: Weight: If rewritten, date:

Standard Chart

Consultant: Name of patient: Height: Hospital number:

D.O.B.: DISCHARGE PRESCRIPTION Date completed:–

(Attach printed label here) Completed by:–

OTHER MEDICINES CHARTS PREVIOUS ADVERSE REACTIONS

(This must be completed before prescribing on this chart)

Date Type of chart Medicine Description of reaction Completed by Date

CODES FOR NON-ADMINISTRATION OF PRESCRIBED MEDICINE If a dose is not administered as prescribed, intial and enter a code in

the column with a circle drawn round the code according to the reason as shown below. Inform the responsible doctor of the appropriate timescale.

1. Patient refuses

2. Patient not present

3. Medicines not available – CHECK ORDERED

4. Asleep/drowsy

5. Administration route not available – CHECK FOR ALTERNATIVE 6. Vomiting/nausea

7. Time varied on doctor’s instructions 8. Once-only/as-required medicine given 9. Dose withheld on doc tor’s instructions 10. Possible adverse reaction/side-effect

ONCE-ONLY MEDICINES

Date

Time

Medicine (approved name)

Dose

Route

Prescriber – sign and print Time Given given by

Fig. 2.7 Example of a hospital prescription and administration record (‘drug chart’). A Front page. The correct identication of the

patient is critical to reducing the risk of an administration error. This page also clearly identi es other prescriptions charts in use and

previous adverse reactions to drugs to minimise the risk of repeated exposure. Note also the codes employed by the nursing staff to

indicate reasons why drugs may not have been administered. The patient’s name and d ate of birth should be written on each page of the

chart. The patient’s weight and height may be required to calculate safe doses for m any drugs with narrow therapeutic indices. B ‘Once-

only medicines’. This area is used for prescribing medicines that are unlikely to be repeated on a regular basis. Note that the prescriber

has written the names of all drugs legibly in block capitals. The generic international non-proprietary name (INN) should be used in

preference to the brand name (e.g. write ‘SIMVASTATIN’, not ‘ZOCOR’). The only exceptions are when variation occurs in the

properties of alternative branded formulations (e.g. modied-release preparations of dr ugs such as lithium, theophylline, phenytoin and

nifedipine) or when the drug is a combination product with no generic name (e.g. Klio vance). The only acceptable abbreviations for

drug dose units are ‘g’ and ‘mg’. ‘Units’ (e.g. of insulin or heparin) and ‘micrograms’ m ust always be written in full, never as ‘U’ or ‘

g’

(nor ‘mcg’, nor ‘ug’). For liquid preparations write the dose in mg; ‘mL’ can b e written only for a combination product (e.g. Gaviscon

liquid) or if the strength is not expressed in weight (e.g. adrenaline (epinephrine) 1 in 1000). Use numbers/gures (e.g. 1 or ‘one’) to

denote use of a sachet/enema but avoid prescribing numbers of tablets without specifying their str ength. Always include the dose of

inhaled drugs in addition to stating numbers of ‘puffs’, as strengths can vary. Widely a ccepted abbreviations for route of administration

are: intravenous – ‘IV’; intramuscular – ‘IM’; subcutaneous – ‘SC’; sublingual – ‘SL’; per rectum – ‘PR’; per vaginam – ‘PV’;

nasogastric – ‘NG’; inhaled – ‘INH’; and topical – ‘TOP’. ‘ORAL’ is preferred to per oram – ‘PO’. Care should be taken in specifying

‘RIGHT’ or ‘LEFT’ for eye and ear drops. The prescriber should sign and print their name clearly so that they can be identied by

colleagues. The prescription should be dated and have an administration time. The nurse who administered the prescription has signed to

conrm that the dose has been administered.

must contain the address of the patient and prescriber (not necessary on most hospital fo rms), the form and the strength of the

preparation, and the total quantity of the preparation/number of dose units in both words and figures.

• ‘Repeat prescriptions’. A large proportion of GP prescribing involves ‘repeat pre scriptions’ for chronic medication. These are often

generated automatically, although the prescriber remains responsible for regular revie w and for ensuring that the benet-to-harm ratio

remains favourable.

Monitoring drug therapy

Prescribers should measure the effects of the drug, both benecial and harmful, to info rm decisions about dose titration (up or down),

discontinuation or substitution of treatment. Monitoring can be achieved subjectively by asking t he patient about symptoms or, more

objectively, by measuring a clinical effect. Alternatively, if the pharmacodynamic effect s of the drug are difcult to assess, the plasma

drug concentration may be measured, on the basis that it will be closely related to the effect of the drug (see Fig. 2.2).

Dose Date

REGULAR MEDICINES

Time

Drug (approved name) 6

Route

Prescriber– sign and print

Notes

12 Start date

Pharmacy

22 Drug (approved name)

Dose

Route

Prescriber– sign and print

Notes

12 Start date

Pharmacy

Drug (approved name)

Dose

22 6

Route

Prescriber– sign and print

Notes

12 Start date

Pharmacy

AS-REQUIRED THERAPY

Drug (approved name) Date Time Dose and frequency Route Dose Initials Prescriber– sign and print Star t date Date Time Indication/notes Pharmacy Dose Initials Drug

(approved name) Date Time Dose and frequency Route Dose Initials Prescriber– sign and print Start date Date Time Indication/notes Pharmacy Dose Initials

Fig. 2.7, cont’d C ‘Regular medicines’. This area is used for prescribin g medicines that are going to be given regularly. In addition to

the name, dose and route, a frequency of administration is required for each medicine. Widely accepted Latin abbrev iations for dose

frequency are: once daily – ‘OD’; twice daily – ‘BD’; 3 times daily – ‘TDS’; 4 times daily – ‘QDS’; as required – ‘PRN’; in the morning

– ‘OM’ (omni mane); at night – ‘ON’ (omni nocte); and immediately – ‘stat’. The hospital chart usually requires specic times to be

identied for regular medicines that coincide with nursing drug rounds and thes e can be circled. If treatment is for a known time period,

cross off subsequent days when the medicine is not required. The ‘notes’ box can be used to communicate additional important

information (e.g. whether a medicine should be taken with food, type of inhaler device used, and anything else that the drug dispenser

should know). State here the times for peak/trough plasma levels for drugs requiring th erapeutic monitoring. Prescriptions should be

discontinued by drawing a vertical line at the point of discontinuation, horizontal lines thro ugh the remaining days on the chart, and

diagonal lines through the drug details and administration boxes. This action should be si gned and dated and a supplementary note

written to explain it (e.g. describing any adverse effect). In this example, amlodipin e has been discontinued because of ankle oedema.

There is room for the ward pharmacist to sign to indicate that the prescription has been rev iewed and that a supply of the medicine is

available. The administration boxes allow the nurse to sign to conrm that the dose has been given. Note that these boxes also allow for

recording of reasons for non-administration (in this example ‘2’ indicates that the patien t was not present on the ward at the time) and

the prevention of future administration by placing an ‘X’ in the box. D ‘As-req uired medicines’. These prescriptions leave the

administration of the drug to the discretion of the nursing staff. The prescription must de scribe clearly the indication, frequency, minimal

time interval between doses, and maximum dose in any 24-hour period (in this case, the maximum daily dose of paracetamol is 4 g).

Clinical and surrogate endpoints

Ideally, clinical endpoints are measured directly and the drug dosage titrated to achieve the therapeutic goal and avoid toxicity (e.g.

control of ventricular rate in a patient with atrial brillation). Sometimes this is impractica l because the clinical endpoint is a future event

(e.g. prevention of myocardial infarction by statins or resolution of a chest infection wi th antibiotics); in these circumstances, it may be

possible to select a ‘surrogate’ endpoint that will predict success or failure. This may be an interme diate step in the pathophysiological

process (e.g. serum cholesterol as a surrogate for risk of myocardial infarction) or a

2.25 Drugs commonly monitored by plasma drug concentration Drug

Digoxin

Gentamicin

Levothyroxine Lithium

Phenytoin

Theophylline (oral)

Vancomycin Half-life (hrs)* Comment

36 Steady state takes several days to achieve. Samples should be taken 6 hrs post dose. Measurement is useful to conrm the clinical

impression of toxicity or non-adherence but clinical effectiveness is better assessed by ventricular heart rate. Risk of toxicity increases

progressively at concentrations > 1.5 μg/L, and is likely at concentrations

3.0

g/L (toxicity is more likely in the presence of

hypokalaemia)

2 Measure pre-dose trough concentration (should be

g/mL) to ensure that accumulation (and the risk of nephrotoxicity and

ototoxicity) is avoided; see Fig. 6.18 (p. 122)

>120 Steady state may take up to 6 weeks to achieve (p. 640)

24 Steady state takes several days to achieve. Samples should be taken 12 hrs post dose . Target range 0.4–1 mmol/L

24 Measure pre-dose trough concentration (should be 10–20 mg/L) to ensure that acc umulation is avoided. Good correlation between

concentration and toxicity. Concentration may be misleading in the presence of hypoalbuminaemia

6 Steady state takes 2–3 days to achieve. Samples should be taken 6 hrs post dose. Targ et concentration is 10–20 mg/L but its

relationship with bronchodilator effect and adverse effects is variable

6 Measure pre-dose trough concentration (should be 10–15 mg/L) to ensure clinical ef cacy and that accumulation and the risk of

nephrotoxicity are avoided (p. 123)

*Half-lives vary considerably with different formulations and between patients.

measurement that follows the pathophysiology, even if it is not a key factor in its progre ssion (e.g. serum C-reactive protein as a

surrogate for resolution of inammation in chest infection). Such surrogates are sometimes termed ‘biomarkers’.

Plasma drug concentration

The following criteria must be met to justify routine monitoring by plasma drug concentrati on:

• Clinical endpoints and other pharmacodynamic (surrogate)

effects are difcult to monitor.

• The relationship between plasma concentration and clinical

effects is predictable.

• The therapeutic index is low. For drugs with a high

therapeutic index, any variability in plasma concentrations

is likely to be irrelevant clinically.

Some examples of drugs that full these criteria are listed in Box 2.25.

Measurement of plasma concentration may be useful in planning adjustments of drug d ose and frequency of administration; to explain an

inadequate therapeutic response (by identifying subtherapeutic concentration or incomplete adherence); to establish whether a suspected

ADR is likely to be caused by the drug; and to assess and avoid potential drug interactions.

Timing of samples in relation to doses

The concentration of drug rises and falls during the dosage

interval (see Fig. 2.4B). Measurements made during the initial absorption and distribution phases are unpredictable because of the

rapidly changing concentration, so samples are usually taken at the end of the dosage interval ( a ‘trough’ or ‘pre-dose’ concentration).

This measurement is normally made in steady state, which usually takes ve half-lives to achieve after the drug is introduced or the dose

changed (unless a loading dose has been given).

Interpreting the result

A target range is provided for many drugs, based on average thresholds for thera peutic benet and toxicity. Inter-individual variability

means that these can be used only as a guide. For instance, in a patient who describe s symptoms that could be consistent with toxicity but

has a drug concentration in the top half of the target range, toxic effects should still be s uspected. Another important consideration is that

some drugs are heavily protein-bound (e.g. phenytoin) but only the unbound drug is p harmacologically active. Patients with

hypoalbuminaemia may therefore have a therapeutic or even toxic concentration of un bound drug, despite a low ‘total’ concentration.

Further information

Websites

bnf.org The British National Formulary (BNF) is a key refere nce resource for UK NHS prescribers, with a list of licensed drugs,

chapters on prescribing in renal failure, liver disease, pregnancy and during breastfeeding, and a ppendices on drug interactions.

cochrane.org The Cochrane Collaboration is a leading international body that provi des evidence-based reviews (around 7000 so far).

evidence.nhs.uk NHS Evidence provides a wide range of health information relevan t to delivering quality patient care.

icp.org.nz The Interactive Clinical Pharmacology site is designed to increase understan ding of principles in clinical pharmacology.

medicines.org.uk/emc/ The electronic Medicines Compendium (eMC) co ntains up-to-date, easily accessible information about medicines

licensed by the UK Medicines and Healthcare Products Regulatory Agency (M HRA) and the European Medicines Agency (EMA).

nice.org.uk The UK National Institute for Health and Care Excellence makes recomm endations to the UK NHS on new and existing

medicines, treatments and procedures.

who.int/medicines/en/ The World Health Organisation Essential Medicines and Pharmac eutical Policies.

K Tatton-Brown

DR FitzPatrick 3

Clinical genetics

The fundamental principles of genomics 38

The packaging of genes: DNA, chromatin and chromosomes 38 Fro m DNA to protein 38

Non-coding RNA 40

Cell division, differentiation and migration 40

Cell death, apoptosis and senescence 41

Genomics, health and disease 42

Classes of genetic variant 42

Consequences of genomic variation 44

Normal genomic variation 45

Constitutional genetic disease 46

Somatic genetic disease 50

Interrogating the genome: the changing landscape of genomic technologies 51

Looking at chromosomes 51

Looking at genes 52

Genomics and clinical practice 56

Genomics and health care 56

Treatment of genetic disease 58

Ethics in a genomic age 59

We have entered a genomic era. Powerful new technologies are driving forward trans formational change in health care. Genetic

sequencing has evolved from the targeted sequencing of a single gene to the parallel s equencing of multiple genes. In addition to

improving the chances of identifying a genetic cause of rare diseases, these technologie s are increasingly directing therapies and, in the

future, are likely to be used in the diagnosis and prevention of common diseases such a s diabetes. In this chapter we explore the

fundamentals of genomics, the basic principles underlying these new genomic technologie s and how the data generated can be applied

safely for patient benet. We will review the use of genomic technology across a breadt h of medical specialties, including obstetrics,

paediatrics, oncology and infectious disease, and consider how health care is likely to be transformed by technology over the coming

decade. Finally, we will consider the ethical impact that these technologies are likely to have , both for the individual and for their wider

family. Normal female karyotype

Chromosome

Chromatin

The fundamental principles of genomics

Nucleosome Histones

The packaging of genes: DNA, chromatin and chromosomes

Genes are functional units encoded in double-stranded deoxyribonucleic acid (DNA), packaged as chromosomes and located in the

nucleus of the cell: a membrane-bound compartment found in all cells except erythrocy tes and platelets (Fig. 3.1). DNA consists of a

linear sequence of just four bases: adenine (A,) cytosine (C), thymine (T) and guanine (G .) It forms a ‘double helix’, a twisted ladder-like

structure formed from two complementary strands of DNA joined by hydrogen bond s between bases on the opposite strand that can form

only between a C and a G base and an A and a T base. It is this feature of DNA that enables faithful DNA replication and is the basis for

many of the technologies designed to interrogate the genome: when the DNA double helix ‘un zips’, one strand can act as a template for

the creation of an identical strand.

A single copy of the human genome comprises approximately 3.1 billion base pairs of DNA, wound around proteins called histones. The

unit consisting of 147 base pairs wrapped around four different histone proteins is calle d the nucleosome. Sequences of nucleosomes

(resembling a string of beads) are wound and packaged to form chromatin: tightly woun d, densely packed chromatin is called

heterochromatin and open, less tightly wound chromatin is called euchromatin.

The chromatin is nally packaged into the chromosomes. Humans are diploid organisms : the nucleus contains two copies of the genome,

visible microscopically as 23 chromosome pairs (known as the karyotype). Chromosom es 1 through to 22 are known as the autosomes

and consist of identical chromosomal pairs. The 23rd ‘pair’ of chromosomes are the two sex chromosomes: females have two X

chromosomes and males an X and Y chromosome. A normal female karyotype is there fore written as 46,XX and a normal male is

46,XY.

From DNA to protein

Genes are functional elements on the chromosome that are capable of transmitting infor mation from the DNA template

A T G A C GG A T

T A C T G CC T A

DNA helix

Fig. 3.1 The packaging of DNA, genes and chromosomes. From bottom to top: th e double helix and the complementary DNA bases;

chromatin; and a normal female chromosome pattern – the karyotype.

via the production of messenger ribonucleic acid (mRNA) to the production of protein s. The human genome contains over 20 000 genes,

although many of these are inactive or silenced in different cell types, reecting the variable gene expression responsible for cell-specic

characteristics. The central dogma is the pathway describing the basic steps of protein produc tion: transcription, splicing, translation and

protein modication (Fig. 3.2). Although this is now recognised as an over-simplication (contrary to this linear relationship, a single

gene will often encode many different proteins), it remains a useful starting point to exp lore protein production.

Transcription: DNA to messenger RNA

Transcription describes the production of ribonucleic acid (RNA) from the DNA template. For transcription to commence, an enzyme

called RNA polymerase binds to a segment of DNA at the start of the gene: the promote r. Once bound, RNA polymerase moves along

one strand of DNA, producing an RNA molecule complementary to the DNA template. In prote in-coding genes this is known as

messenger RNA (mRNA). A DNA sequence close to the end of the gene, called the p olyadenylation signal, acts as a signal for

termination of the RNA transcript (Fig. 3.3).

DNA

5’CGATTC3’ 3’GCTAAG5’ Transcription

Nucleus

Nuclear

membrane

Gene A

RNA 5’CGAUUC3’

Translation Nucleolus

Gene B

Protein N_ArgPhe_C Enhancer

Fig. 3.2 The central dogma of protein production. Double-stranded DNA as a template for single-s tranded RNA, which codes for the

production of a peptide chain of amino acids. Each of these chains has an orientatio n. For DNA and RNA, this is 5

′

to 3

′

. For peptides,

this is N-terminus to C-terminus.

RNA differs from DNA in three main ways:

• RNA is single-stranded.

• The sugar residue within the nucleotide is ribose, rather

than deoxyribose.

• It contains uracil (U) in place of thymine (T).

The activity of RNA polymerase is regulated by transcription factors. These p roteins bind to specic DNA sequences at the promoter or

to enhancer elements that may be many thousands of base pairs away from the promot er; a loop in the chromosomal DNA brings the

enhancer close to the promoter, enabling the bound proteins to interact. The huma n genome encodes more than 1200 different

transcription factors. Mutations within transcription factors, promoters and enhancers ca n cause disease. For example, the blood disorder

alpha-thalassaemia is usually caused by gene deletions (see p. 954 and Box 3.4). However, it can also result from a mutation in an

enhancer located more than 100 000 base pairs (bp) from the α-globin gene pro moter, leading to greatly reduced transcription.

Gene activity, or expression, is inuenced by a number of complex interacting factors, including the accessibility of the gene promoter to

transcription factors. DNA can be modied by the addition of a methyl group to cytosine molecu les (methylation). If DNA methylation

occurs in promoter regions, transcription is silenced, as methyl cytosines are usually not available for transcription factor binding. A

second mechanism determining promoter accessibility is the structural conguration of chroma tin. In open chromatin, called

euchromatin, gene promoters are accessible to RNA polymerase and transcription factors; therefore it is transcriptionally active. This

contrasts with heterochromatin, which is densely packed and transcriptionally silent. The chromatin conguration is determined by

modications (such as methylation or acetylation) of specic amino acid residues of histone pro tein tails.

Modications of DNA and histones are termed epigenetic (‘epi-’ meaning ‘above’ the genome), as they do not alter the primary

sequence of the DNA code but have biological signicance in chromosomal function. A bnormal epigenetic changes are increasingly

recognised as important events in the progression of cancer, allowing expression of norma lly silenced genes that result in cancer cell de-

differentiation and proliferation. They also afford therapeutic targets. For instance, the h istone deacetylase inhibitor vorinostat has been

successfully used to treat cutaneous T-cell lymphoma, due to the re-expression of gene s that had

Active gene

Gene C Transcription

factors

Promoter RNA

polymerase II Sense strand

5' 3' RNA

Exon 1 Exon 2 Exon 3 3' Intron 1 Intron 2

Transcription

Primary RNA

Exon 1 Exon 2 Exon 3 transcript

5'UTR

3'UTR

Splicing cap AAAAA Spliceosome PolyA tail

Messenger RNA

cap(mRNA)

AAAAA

Nuclear pore

RNA export to cytoplasm

Nuclear

membrane

tRNAs

Messenger RNA

cap(mRNA)

Ribosome Translation

AAAAA

Protein product N-term C-term

Fig. 3.3 RNA synthesis and its translation into protein. Gene transcription involves binding o f RNA polymerase II to the promoter of

genes being transcribed with other proteins (transcription factors) that regulate the transcription rate. T he primary RNA transcript is a

copy of the whole gene and includes both introns and exons, but the introns are removed w ithin the nucleus by splicing and the exons are

joined to form the messenger RNA (mRNA). Prior to export from the nucleu s, a methylated guanosine nucleotide is added to the 5

′

end

of the RNA (‘cap’) and a string of adenine nucleotides is added to the 3′ (‘polyA tail’). This protects the RNA from degradation and

facilitates transport into the cytoplasm. In the cytoplasm, the mRNA binds to ribos omes and forms a template for protein production.

(tRNA

transfer RNA; UTR

untranslated region)

previously been silenced in the tumour. These genes encode transcription factors that pro mote T-cell differentiation as opposed to

proliferation, thereby causing tumour regression.

RNA splicing, editing and degradation

Transcription produces an RNA molecule that is a copy of the whole gene, termed the primary or nascent transcript. This nascent

transcript then undergoes splicing, whereby regions not required to make protein (the i ntronic regions) are removed while those

segments that are necessary for protein production (the exonic regions) are retained and rejoin ed.

Splicing is a highly regulated process that is carried out by a multimeric protein comple x called the spliceosome. Following splicing, the

mRNA molecule is exported from the nucleus and used as a template for protein synth esis. Many genes produce more than one form of

mRNA (and thus protein) by a process termed alternative splicing, in which different c ombinations of exons are joined together.

Different proteins from the same gene can have entirely distinct functions. For example , in thyroid C cells the calcitonin gene produces

mRNA encoding the osteoclast inhibitor calcitonin (p. 634), but in neurons the same gene p roduces an mRNA with a different

complement of exons via alternative splicing that encodes a neurotransmitter, calcitonin-gene-relate d peptide (p. 772).

into the cytoplasm or packaged into vesicles for secretion. The clinical importance of po st-translational modication of proteins is shown

by the severe developmental, neurological, haemostatic and soft tissue abnormalities that are asso ciated with the many different

congenital disorders of glycosylation. Post-translational modications can also be disrupt ed by the synthesis of proteins with abnormal

amino acid sequences. For example, the most common mutation in cystic brosis (ΔF508) results in an abnormal protein that cannot be

exported from the ER and Golgi (see Box 3.4).

Non-coding RNA

Approximately 4500 genes in humans encode non-coding RNAs (ncRNA) rather than proteins. There are various categories of ncRNA,

including transfer RNA (tRNA), ribosomal RNA (rRNA), ribozymes and microRNA (miRN A). The miRNAs, which number over 1000,

have a role in post-translational gene expression: they bind to mRNAs, typically in the 3

′

UTR, promoting target mRNA degradation and

gene silencing. Together, miRNAs affect over half of all human genes and have impor tant roles in normal development, cancer and

common degenerative disorders. This is the subject of considerable research interest at p resent.

Translation and protein production

Following splicing, the segment of mRNA containing the code that directs synthesis of a protein product is called the open reading frame

(ORF). The inclusion of a particular amino acid in the protein is specied by a codon c omposed of three contiguous bases. There are 64

different codons with some redundancy in the system: 61 codons encode one of the 20 amino acids, and the remaining three codons –

UAA, UAG and UGA (known as stop codons) – cause termination of the growing polype ptide chain. ORFs in humans most commonly

start with the amino acid methionine. All mRNA molecules have domains before and after the ORF calle d the 5′ untranslated region

(UTR) and 3′UTR, respectively. The start of the 5′UTR contains a cap structure that pr otects mRNA from enzymatic degradation, and

other elements within the 5′UTR are required for efcient translation. The 3′UTR also conta ins elements that regulate efciency of

translation and mRNA stability, including a stretch of adenine bases known as a polyA tail (see Fig. 3.3).

The mRNAs then leave the nucleus via nuclear pores and associate with ribosomes, the sites of protein production (see Fig. 3.3). Each

ribosome consists of two subunits (40S and 60S), which comprise non-coding rRNA m olecules (see Fig. 3.9, p. 50) complexed with

proteins. During translation, a different RNA molecule known as transfer RNA (tRNA ) binds to the ribosome. The tRNAs deliver amino

acids to the ribosome so that the newly synthesised protein can be assembled in a stepwis e fashion. Individual tRNA molecules bind a

specic amino acid and ‘read’ the mRNA ORF via an ‘anticodon’ of three nucleotides that is complementary to the codon in mRNA (see

Fig. 3.3). A proportion of ribosomes is bound to the membrane of the endoplasmic reticulum (ER), a complex tubular structure that

surrounds the nucleus.

Proteins synthesised on these ribosomes are translocated into the lumen of the ER, wher e they undergo folding and processing. From

here, the protein may be transferred to the Golgi apparatus, where it undergoes post-tr anslational modications, such as glycosylation

(covalent attachment of sugar moieties), to form the mature protein that can be exported

Cell division, differentiation and migration

In normal tissues, molecules such as hormones, growth factors and cytokines p rovide the signal to activate the cell cycle: a controlled

programme of biochemical events that culminates in cell division. In all cells of the body , except the gametes (the sperm and egg cells,

also known as the germ line), mitosis completes cell division, resulting in two diploid daughter c ells. In contrast, the sperm and eggs

cells complete cell division with meiosis, resulting in four haploid daughter cells ( Fig. 3.4).

The stages of cell division in the non-germ-line, somatic cells are shown below:

• Cells not committed to mitosis are said to be in G

• Cells committed to mitosis must go through the

preparatory phase of interphase consisting of G

, S and G

• G

(rst gap): synthesis of the cellular components

necessary to complete cell division

• S (synthesis): DNA replication producing identical

copies of each chromosome called the sister

chromatids

• G

(second gap): repair of any errors in the replicated

DNA before proceeding to mitosis.

• Mitosis (M) consists of four phases:

• Prophase: the chromosomes condense and become

visible, the centrioles move to opposite ends of the cell

and the nuclear membrane disappears.

• Metaphase: the centrioles complete their migration to

opposite ends of the cell and the chromosomes –

consisting of two identical sister chromatids – line up at

the equator of the cell.

• Anaphase: spindle bres attach to the chromosome

and pull the sister chromatids apart.

• Telophase: the chromosomes decondense, the nuclear

membrane reforms and two daughter cells – each with

46 chromosomes – are formed.

The progression from one phase to the next is tightly controlled by cell-cycle checkpoin ts. For example, the checkpoint between

Father Mother

Individual chromosome pair (homologues)

DNA replication

Sister chromatids

Homologous pairing

Swapping of

genetic material between homologues: Recombination

Meiotic cell divisions

Non-disjunction of chromosomes is a common error in human meiosis, resulting in triso my of individual chromosomes or uniparental

disomy (both chromosomes from single parent)

1st

polar bodies

Egg 2nd

polar body

Sperm Fig. 3.4 Meiosis and gametogenesis: the main chromosomal stages

of meiosis in both males and females. A single homologous pair of chromosomes is repres ented in different colours. The nal step is the

production of haploid germ cells. Each round of meiosis in the male results in four spe rm cells; in the female, however, only one egg cell

is produced, as the other divisions are sequestered on the periphery of the mature egg as peripheral polar bodies.

and mitosis ensures that all damaged DNA is repaired prior to segregation of the chromosomes. Failure of these control processes is a

crucial driver in the pathogenesis of cancer, as discussed on page 1316.

• Meiosis is a special, gamete-specic, form of cell division

( Fig. 3.4). Like mitosis, meiosis consists of four phases (prophase, metaphase, anaphase and teloph ase) but differs from mitosis in the

following ways:

• It consists of two separate cell divisions known as

meiosis I and meiosis II.

• It reduces the chromosome number from the 3

diploid to the haploid number via a tetraploid

stage, i.e. from 46 to 92 (MI S) to 46 (MI M) to

23 (MII M) chromosomes, so that when a sperm cell fertilises the egg, the resu lting zygote will return to a diploid, 46, chromosome

complement. This reduction to the haploid number occurs at the end of meiosis II.

• The 92 chromosome stage consists of 23 homologous pairs of sister chromatids, whi ch then swap genetic material, a process known as

recombination. This occurs at the end of MI prophase and ensures that t he chromosome that a parent passes to his or her offspring is a

mix of the chromosomes that the

parent inherited from his or her own mother

and father.

The individual steps in meiotic cell division are similar in males and females. However, the timin g of the cell divisions is very different.

In females, meiosis begins in fetal life but does not complete until after ovulation. A sing le meiotic cell division can thus take more than

40 years to complete. As women become older, the separation of chromosomes at meiosis II b ecomes less efcient. That is why the risk

of trisomies (p. 44) due to non-disjunction grows greater with increasing maternal age. In males, meiotic division does not begin until

puberty and continues throughout life. In the testes, both meiotic divisions are completed in a matter of days.

Cell death, apoptosis and senescence

With the exception of stem cells, human cells have only a limited capacity for cell division . The Hayick limit is the number of divisions

a cell population can go through in culture before division stops and enters a state know n as senescence. This ‘biological clock’ is of

great interest in the study of the normal ageing process. Rare human diseases associated with premature ageing, called progeric

syndromes, have been very helpful in identifying the importance of DNA repair mech anisms in senescence (p. 1034). For example, in

Werner’s syndrome, a DNA helicase (an enzyme that separates the two DNA stra nds) is mutated, leading to failure of DNA repair and

premature ageing. A distinct mechanism of cell death is seen in apoptosis, or programme d cell death.

Apoptosis is an active process that occurs in normal tissues and plays an important r ole in development, tissue remodelling and the

immune response. The signal that triggers apoptosis is specic to each tissue or cell type. This s ignal activates enzymes, called caspases,

which actively destroy cellular components, including chromosomal DNA. This degrad ation results in cell death, but the cellular corpse

contains characteristic vesicles called apoptotic bodies. The corpse is then recognised and removed by phagocytic cells of the immune

system, such as macrophages, in a manner that does not provoke an inammatory resp onse.

A third mechanism of cell death is necrosis. This is a pathological process in which the c ellular environment loses one or more of the

components necessary for cell viability. Hypoxia is probably the most common cause of necrosis.

Genomics, health and disease

Classes of genetic variant

There are many different classes of variation in the human genome, categorised by the size of the DNA segment involved and/or by the

mechanism giving rise to the variation. mutation (Box 3.1 and Fig. 3.5). If a multiple of three nucleotides is involved, this is in-frame. If

an indel change affects one or two nucleotides within the ORF of a protein-coding gen e, this can have serious consequences because the

triple nucleotide sequence of the codons is disrupted, resulting in a frameshift mutation. The effec t on the gene is typically severe

because the amino acid sequence is totally disrupted.

Nucleotide substitutions

The substitution of one nucleotide for another is the most common type of genomic var iation. Depending on their frequency and

functional consequences, these changes are known as point mutations or single nucleotide poly morphisms (SNPs). They occur by

misincorporation of a nucleotide during DNA synthesis or by chemical modica tion of the base. When these substitutions occur within

ORFs of a protein-coding gene, they are further classied into:

• synonymous – resulting in a change in the codon without

altering the amino acid

• non-synonymous (also known as a missense mutation) –

resulting in a change in the codon and the encoded amino

acid

• stop gain (or nonsense mutation) – introducing a

premature stop codon and resulting in truncation of the

protein

• splicing – taking place at splice sites that most frequently

occur at the junction between an intron and an exon.

These different types of mutation are illustrated in Box 3.1 and examples are sho wn in Figures 3.5 and 3.6.

Insertions and deletions

One or more nucleotides may be inserted or lost in a DNA sequence, resulting in an ins ertion/deletion (indel) polymorphism or

Stop gain (also called a nonsense mutation) Causing the generation of a premature stop codon

Indel

Where bases are either inserted or deleted; disruption of the reading fram e is dependent on the number of bases inserted or deleted

THE CAT

where the F of FAT is replaced by a C generating a premature stop codon

THE FAT FOX WAS ILL ILL COS SHE ATE THE OLD CAT

where the insertion of three bases results in maintenance of the reading frame THE FAT FO X WAW ASI LLC OSS HEA TET HEO

LDC AT

where the insertion of two bases results in disruption of the reading frame

A Normal

B Silent polymorphism (no amino acid change)

C Missense mutation causing Lys–Gln amino acid change

D ‘G’ insertion causing frameshift mutation

3.1 Classes of genetics variant

The classes of genetic variant can be illustrated using the sentence ‘THE FAT FOX WA S ILL COS SHE ATE THE OLD CAT’

Synonymous

Silent polymorphism with no amino acid change

Non-synonymous Causing an amino acid change

THE FAT FOX WAS ILL COS SHE ATE THE OLD KAT

where the C is replaced with a K but the meaning remains the same

THE FAT BOX WAS ILL COS SHE ATE THE OLD CAT

where the F of FOX is replaced by a B and the original meaning of the sentence is lost

Nonsense mutation causing premature termination codon

Fig. 3.5 Different types of mutation affecting coding exons. A Normal sequence. B A synonymous nucleotide substitution changing the

third base of a codon; the resulting amino acid sequence is unchanged. C A missense mutation in which the nucleotide substitution

results in a change in a single amino acid from the normal sequence (AAG) encoding lysine to glutamine (CAG). D Insertion of a G

residue (boxed) causes a frameshift mutation, completely altering the amino acid sequence do wnstream. This usually results in a loss-of-

function mutation. E A nonsense mutation resulting in a single nucleotide chan ge from a lysine codon (AAG) to a premature stop codon

(TAG).

A Normal

Splice donor site Splice acceptor site

Exon Intron Exon

Intron removed by 3 splicing

B Splice site mutation

Exon Intron Exon

mRNA ‘reads through’ intron

Abnormal protein with

premature stop codon

Fig. 3.6 Splice site mutations. A The normal sequence is shown, illustrating two exons, and intervening intron (blue) with splice donor

(AG) and splice acceptor sites (GT) underlined. Normally, the intron is removed by splicin g to give the mature messenger RNA that

encodes the protein. B In a splice site mutation the donor site is mutated. As a result, sp licing no longer occurs, leading to read-through

of the mRNA into the intron, which contains a premature termination codon downstre am of the mutation.

3.2 Diseases associated with triplet and other repeat expansions* No . of repeats Normal Mutant Repeat

Coding repeat expansion

Huntington’s disease [CAG]

Spinocerebellar ataxia (type 1) [CAG]

Spinocerebellar ataxia (types 2, [CAG]

3, 6, 7)

Dentatorubral-pallidoluysian [CAG]

atrophy

Machado–Joseph disease [CAG]

Spinobulbar muscular atrophy [CAG]

Non-coding repeat expansion

Myotonic dystrophy [CTG]

Friedreich’s ataxia [GAA]

Progressive myoclonic epilepsy [CCCCGCCCCGCG]

4–8

Fragile X mental retardation [CGG]

Fragile site mental retardation 2 [GCC]

(FRAXE)

Gene Gene location Inheritance

6–34

6–39 > 40

Various Various Huntingtin Ataxin

Various

4p16

6p22–23 Various

AD AD AD

7–25 > 49 Atrophin 12p12–13 AD

12–40 > 67

11–34

MJD

Androgen receptor 14q32

Xq11–12 AD

XL recessive

5–37 > 50

7–22 > 200

2–3

5–52 > 200

6–35 > 200 DMPK-3′UTR

Frataxin-intronic Cystatin B-5

′

UTR FMR1–5′UTR

FMR2

19q13 9q13

21q

Xq27

Xq28

XL dominant

XL, probably recessive

*The triplet repeat diseases fall into two major groups: those with disease stemming from expansion of [CAG]

repeats in coding DNA,

resulting in multiple adjacent glutamine residues (polyglutamine tracts), and those with n on-coding repeats. The latter tend to be longer.

Unaffected parents usually display ‘pre-mutation’ allele lengths that are just abo ve the normal range. (AD/AR

autosomal

dominant/recessive; UTR

untranslated region; XL

X-linked)

Simple tandem repeat mutations

Variations in the length of simple tandem repeats of DNA are thought to arise as the result of s lippage of DNA during meiosis and are

termed microsatellite (small) or minisatellite (larger) repeats. These repeats are unstable a nd can expand or contract in different

generations. This instability is proportional to the size of the original repeat, in that longer repeats tend to be more unstable. Many

microsatellites and minisatellites occur in introns or in chromosomal regions between genes and ha ve no obvious adverse effects.

However, some genetic diseases are caused by microsatellite repeats that result in dup lication of amino acids within the affected gene

product or affect gene expression (Box 3.2).

Copy number variations

Variation in the number of copies of an individual segment of the genome from the usual dip loid (two copies) content can be categorised

by the size of the segment involved. Rarely, individuals may gain (trisomy) or lose (mo nosomy) a whole chromosome. Such numerical

chromosome anomalies most commonly occur by a process known as non-disjunction, where pairs of homologous chromosomes do not

separate at meiosis II (p. 40). Common trisomies include Down’s syndrome (triso my 21), Edward’s syndrome (trisomy 18) and Patau’s

syndrome (trisomy 13). Monosomy of the autosomes (present in all the cells, as oppose d to in a mosaic distribution) does not occur but

Turner’s syndrome, in which there is monosomy for the X chromosome, affects approx imately 1 in 2500 live births (Box 3.3).

Large insertions or deletions of chromosomal DNA also occur and are usually associate d with a learning disability and/or congenital

malformations. Such structural chromosomal anomalies usually arise as the result of one of two different processes:

• non-homologous end-joining

• non-allelic homologous recombination.

Random double-stranded breaks in DNA are a necessary process in meiotic recombina tion and also occur during mitosis at a predictable

rate. The rate of these breaks is dramatically increased by exposure to ionising radiation. Wh en such breaks take place, they are usually

repaired accurately by DNA repair mechanisms within the cell. However, in a proport ion of breaks, segments of DNA that are not

normally contiguous will be joined (‘non-homologous end-joining’). If the joined frag ments are from different chromosomes, this results

in a translocation. If they are from the same chromosome, this will result in either inv ersion, duplication or deletion of a chromosomal

fragment (Fig. 3.7). Large insertions and deletions may be cytogenetically visible as chromosomal dele tions or duplications. If the

anomalies are too small to be detected by microscopy, they are termed microdeletions and mi croduplications. Many microdeletion

syndromes have been described and most result from nonallelic homologous recombin ation between repeats of highly similar DNA

sequences, which leads to recurrent chromosome anomalies – and clinical syndromes – occurring in unrelated individuals (Fig. 3.7 and

Box 3.3).

Consequences of genomic variation

The consequence of an individual mutation depends on many factors, includin g the mutation type, the nature of the gene product and the

position of the variant in the protein. Mutations can have profound or subtle effects on gene and cell function. Variations that have

profound effects are responsible for ‘classical’ genetic diseases, whereas those with subtle effects may contribute to the pathogenesis of

common disease where there is a genetic component, such as diabetes.

• Neutral variants have no effect on quality or type of protein

produced.

• Loss-of-function mutations result in loss or reduction in the

normal protein function. Whole-gene deletions are the

archetypal loss-of-function variants but stop-gain or indel

mutations (early in the ORF), missense mutations affecting

a critical domain and splice-site mutations can also result

in loss of protein function.

3.3 Chromosome and contiguous gene disorders

Disease Locus Incidence Clinical features Numerical chromosomal abnormalities

Down’s syndrome (trisomy 21)

Edwards’ syndrome (trisomy 18)

Patau’s syndrome (trisomy 13)

Klinefelter’s syndrome

XYY

Triple X syndrome Turner’s syndrome

the aorta, primary amenorrhoea (p. 659) Recurrent deletions, microdeletions and contigu ous gene defects

47,XY,

21 or 47,XX

21 1 in 800 Characteristic facies, IQ usually

50, congenital heart disease, reduced life expectancy

47,XY,

18 or 47,XX,

18 1 in 6000 Early lethality, characteristic skull and facies, frequent malformations of heart, kid ney and other

organs

47,XY,+13 or 47, XX,+13 1 in Early lethality, cleft lip and palate, polydactyly, small h ead, 15 000 frequent congenital heart disease

47,XXY 1 in 1000 Phenotypic male, infertility, gynaecomastia, small testes (p. 660)

47,XYY 1 in 1000 Usually asymptomatic, some impulse control problems

47,XXX 1 in 1000 Usually asymptomatic, may have reduced IQ

45,X 1 in 5000 Phenotypic female, short stature, webbed neck, coarctation of

Di George/velocardiofacial syndrome 22q11.2

Prader–Willi syndrome 15q11–q13

Angelman’s syndrome 15q11–q13

Williams’ syndrome 7q11.23

Smith–Magenis syndrome 17p11.2 1 in 4000 Cardiac outow tract defects, distinctive f acial appearance, thymic hypoplasia, cleft palate

and hypocalcaemia. Major gene seems to be TBX1 (cardiac defects and cleft palate)

1 in Distinctive facial appearance, hyperphagia, small hands and

15 000 feet, distinct behavioural phenotype. Imprinted region, deletions on patern al allele in 70% of cases

1 in Distinctive facial appearance, absent speech,

15 000 electroencephalogram (EEG) abnormality, characteristic gait. Imprinted region, deletio ns on maternal allele encompassing

UBE3A

1 in Distinctive facial appearance, supravalvular aortic stenosis,

10 000 learning disability and infantile hypercalcaemia. Major gene for supravalvular aortic steno sis is elastin

1 in Distinctive facial appearance and behavioural phenotype,

25 000 self-injury and rapid eye movement (REM) sleep abnormalities. Major gene seems to be RAI1

A How structural chromosomal anomalies are described

Chromosome Chromosome

9 14

Deletions Duplications Interstitial Terminal Tandem Inverted 23

Reciprocal Robertsonian Inversions

translocation translocation

(st)

13 (sa)

pNNANANANA 3

Cen

11.2

Cen

21.1

21.3 21 q

21.2 q 23

24.2

34.2

32.2

Metacentric Acrocentric

B Mechanism underlying recurrent deletions and duplication: non-allelic homologous re combination

Normal pairing

2 Recombination

Abnormal pairing between DNA repeats 1

Deletion Duplication DNA repeat

Maternal chromosome Paternal chromosome

1 1 2

1 2

Fig. 3.7 Chromosomal analysis and structural chromosomal disorders. A Human chrom osomes can be classed as metacentric if the

centromere is near the middle, or acrocentric if the centromere is at the end. Th e bands of each chromosome are given a number, starting

at the centromere and working out along the short (p) arm and long (q) arm. Transloca tions and inversions are balanced structural

chromosome anomalies where no genetic material is missing but it is in the wrong order. Translocations can be divided into reciprocal

(direct swap of chromosomal material between nonhomologous chromosomes) and Ro bertsonian (fusion of acrocentric chromosomes).

Deletions and duplications can also occur due to non-allelic homologous recombinati on (illustrated in part B). Deletions are classied as

interstitial if they lie within a chromosome, and terminal if the terminal region of the chromosome is affected. Duplications can be either

in tandem (where the duplicated fragment is inserted next to the region that is duplicated and orientated in the correct direction) or

inverted (where the duplicated fragment is in the wrong direction). (N = normal; A = a bnormal) B A common error of meiotic

recombination, known as non-allelic homologous recombination, can occur (right pane l), resulting in a deletion on one chromosome and

a duplication in the homologous chromosome. The error is induced by tandem repeats in the DNA sequences (green), which can

misalign and bind to each other, thereby ‘fooling’ the DNA into thinking the pairing prior to r ecombination is correct.

• Gain-of-function mutations result in a gain of protein function. They are typically non-synonymous mutations that alter the protein

structure, leading to activation/ alteration of its normal function through causing either an interaction with a novel substrate or a change in

its normal function.

• Dominant negative mutations are the result of nonsynonymous mutations or in-fr ame deletions/duplications but may also, less

frequently, be caused by triplet repeat expansion mutations. Dominant negativ e mutations are heterozygous changes that result in the

production of an abnormal protein that interferes with the normal functioning of the wild-type protein.

Normal genomic variation

We each have 5–50 million variants in our genome, occurring approximately every 300 b ases. These variants are mostly polymorphisms,

arising in more than 1% of the population; they have no or subtle effects on gene and c ell function, and are not associated with a high

risk of disease. Polymorphisms can occur within exons, introns or the intergenic regi ons that comprise 98–99% of the human genome.

Each of the classes of genetic variant discussed on page 42 is present in the genome as a common polymorphism. However, the most

frequent is the single nucleotide polymorphism, or SNP (pronounced ‘snip’), describing the subs titution of a single base.

Polymorphisms and common disease

The protective and detrimental polymorphisms associated with common disease have be en identied primarily through genome-wide

association studies (GWAS, p. 56) and are the basis for many direct-to-consumer tests that pu rport to determine individual risk proles

for common diseases or traits such as cardiovascular disease, diabetes and even male-p attern baldness! An example is the polymorphism

in the gene SLC2A9 that not only explains a signicant proportion of the normal population varia tion in serum urate concentration but

also predisposes ‘high-risk’ allele carriers to the development of gout. However, the curren t reality is that, until we have a more

comprehensive understanding of the full genomic landscape and knowledge of the comple te set of detrimental and protective

polymorphisms, we cannot accurately assess risk.

Evolutionary selection

Genetic variants play an important role in evolutionary selection, with advantageous varia nts resulting in positive selection via improved

reproductive tness, and variations that decrease reproductive fitness becoming exclude d through evolution. Given this simple paradigm,

it would be tempting to assume that common mutations are all advantageous and all rare mutations are pathogenic. Unfortunately, it is

often difcult to classify any common mutation as either advantageous or deleterious – or, indeed, neutral. Mutations that are

advantageous in early life and thus enhance reproductive tness may be deleterious in l ater life. There may be mutations that are

advantageous for survival in particular conditions (e.g. famine or pandemic) that may be disadvantageous in more benign circumstances

by causing a predisposition to obesity or autoimmune disorders.

Constitutional genetic disease

Familial genetic disease is caused by constitutional mutations, which are inherited thro ugh the germ line. However, different mutations

in the same gene can have different consequences, depending on the genetic mechanism unde rlying that disease. About 1% of the human

population carries constitutional mutations that cause disease.

Constructing a family tree

The family tree – or pedigree – is a fundamental tool of the clinical geneticist, who will r outinely take a three-generation family history,

on both sides of the family, enquiring about details of all medical conditions in family members, consanguinity, dates of birth and death,

and any history of pregnancy loss or infant death. The basic symbols and nomenclature used in drawing a pedigree are shown in Figure

3.8.

Patterns of disease inheritance

Autosomal dominant inheritance

Take some time to draw out the following pedigree:

Anne is referred to Clinical Genetics to discuss her personal history of colon cancer (she was diagnosed at the age of 46 years) and

family history of colon/endometrial cancer: her mother was diagnosed with en dometrial cancer at the age of 60 years and her cousin

through her healthy maternal aunt was diagnosed with colon cancer in her fties. Both her mate rnal grandmother and grandfather died of

‘old age’. There is no family history of note on her father’s side of the family. H e has one brother and both his parents died of old age, in

their eighties. Anne has two healthy daughters, aged 12 and 14 years, and a healthy fu ll sister.

This family history is typical of an autosomal dominant condition (Fig. 3.8): in this case, a colon/endometrial cancer s usceptibility

syndrome known as Lynch’s syndrome, associated with disruption of one of the misma tch repair genes: MSH2, MSH6, MLH1 and PMS2

(see p. 830 and Box 3.11, p. 57).

Male

Female

Dominant inheritance Recessive inheritance

Unknown sex

1 2

Deceased

individual

(with age at death)

d. 50 y III

Partners

1 23 4

II 1 234 III 23 4 5 6

Separated

12 3 4

Consanguinity

Transmission to 50% of offspring independent of gender

Monozygotic twins

1 23 4 5 6

IV 367

V 567 8 Consanguinity

Dizygotic twins X-linked recessive inheritance Stillbirth

(with gestation)

SB SB

30 wk 39 wk

1 2

Miscarriage

(with gestation)

16 wk

1 23 4

Mitochondrial DNA disorder

1 2 II 1 23 4 Termination III 1 23 4 5 6

III 1 23 4 5 6

Clinically affected IV

12 3 4

Clinically affected,

several diagnoses

Affected males related through

Carrier

unaffected females Positive pre

symptomatic test

IV 12 3 4 Both sexes affected but only inherited through female meiosis

Fig. 3.8 Drawing a pedigree and patterns of inheritance. A The main symbols used to represent pedigrees in diagrammatic form. B The

main modes of disease inheritance (see text for details).

Features of an autosomal dominant pedigree include:

• There are affected individuals in each generation (unless the mutation has arisen de novo, i.e. for the rst time in an affected

individual). However, variable penetrance and expressivity can inuence the n umber of affected individuals and the severity of disease in

each generation. Penetrance is dened as the proportion of individuals bea ring a mutated allele who develop the disease phenotype. The

mutation is said to be fully penetrant if all individuals who inherit a mutation develop the disea se. Expressivity describes the level of

severity of each aspect of the disease phenotype.

• Males and females are usually affected in roughly equal numbers (unless the clinical presentation of the condition is gender-specic,

such as an inherited susceptibility to breast and/or ovarian cancer).

The offspring risk for an individual affected with an 3 autosomal dominant condition is 1 in 2 (or 50%). This offspring risk is true for

each pregnancy, since half the affected individual gametes (sperm or egg cells) will con tain the affected chromosome/gene and half will

contain the normal chromosome/gene.

There is a long list of autosomal dominant conditions, some of which are shown in Box 3.4.

3.4 Genetic conditions dealt with by clinicians in other specialties

Name of condition

Autosomal dominant conditions

Autosomal dominant polycystic kidney disease (ADPKD)

Tuberous sclerosis

Marfan’s syndrome

Long QT syndrome

Brugada’s syndrome Neurobromatosis type 1

Neurobromatosis type 2

Hereditary spherocytosis

Vascular Ehlers–Danlos syndrome (EDS type 4) Hereditary haemorrhagic telangiectas ia

Osteogenesis imperfecta

Charcot–Marie–Tooth disease

Hereditary neuropathy with liability to pressure palsies Autosomal recessive con ditions

Familial Mediterranean fever

Mevalonic aciduria (mevalonate kinase deciency) Autosomal recessive polycystic kidn ey disease (ARPKD) Kartagener’s syndrome

(primary ciliary dyskinesia) Cystic brosis

Pendred’s syndrome

Congenital adrenal hyperplasia-21 hydroxylase deciency

Haemochromatosis

Wilson’s disease

Alpha

-antitrypsin deciency

Gilbert’s syndrome

Benign recurrent intrahepatic cholestasis Alpha-thalassaemia

Beta-thalassaemia

Sickle cell disease

Spinal muscular atrophy X-linked conditions Alport’s syndrome

Primary agammaglobulinaemia Haemophilia A (factor VIII deciency) Haemophilia B (factor IX deciency) Duchenne muscular

dystrophy Gene Reference

PKD1 (85%), PKD2 (15%) p. 405 Box 15.28, p. 415

TSC1 p. 1264

TSC2 p. 1264

FBN1 p. 508

KCNQ1 p. 476

SCN5A p. 477

NF1 p. 1131 Box 25.77, p. 1132

NF2 p. 1131 Box 25.77, p. 1132

ANK1 p. 947

COL3A1 p. 970

ENG, ALK1, GDF2 p. 970

COL1A1, COL1A2 p. 1055

PMP22, MPZ, GJB1 p. 1140

PMP22

MEFV p. 81 MVK p. 81 PKHD1 Box 15.28, p. 415 DNAI1 Box 17.30, p. 578 CFTR1 p. 580

Box 17.30 , p. 578 p. 842 SLC26A4 p. 650

CYP21A p. 676 Box 18.27, p. 658

HFE p. 895

ATP7B p. 896

SERPINA1 p. 897

UGT1A1 p. 897

ATP8B1 p. 902

HBA1, HBA2 p. 951 p. 954

HBB p. 951 p. 953

HBB p. 951

SMN1 p. 1117

COL4A5 Box 15.28, p. 415 p. 403

BTK p. 78

F8 p. 971

F9 p. 973

DMD p. 1143 and Box 25.91

Autosomal recessive inheritance

As above, take some time to draw a pedigree representing the following:

Mr and Mrs Kent, a non-consanguineous couple, are referred because their son, Jamie, had severe neonatal liver disease. Included among

the many investigations that the paediatric hepatologist undertook was testing for α

-antitrypsin deciency (Box 3.5). Jamie was shown

to have the PiZZ phenotype. Testing confirmed both parents as carriers with PiMZ phen otypes. In the family, Jamie has an older sister

who has no medical problems. Mr Kent is one of four children with two brothers and a sister and Mrs Kent has a younger brother. Both

sets of grandparents are alive and well. There is no family history of α

-antitrypsin deciency.

This family history is characteristic of an autosomal recessive disorder (Fig. 3.8), where bot h alleles of a gene must be mutated before the

disease is manifest in an individual; an affected individual inherits one mutant allele from each of their parents, who are therefore healthy

carriers for the condition. An autosomal recessive condition might be suspected in a fam ily where:

• Males and females are affected in roughly equal

proportions.

• Parents are blood related; this is known as consanguinity.

Where there is consanguinity, the mutations are usually

homozygous, i.e. the same mutant allele is inherited from

both parents.

3.5 Alpha

-antitrypsin deciency

Inheritance pattern

• Autosomal recessive

Genetic cause

• Two common mutations in the SERPINA1 gene: p.Glu342Lys and

p.Glu264Val

Prevalence

• 1 in 1500–3000 of European ancestry

Clinical presentation

• Variable presentation from neonatal period through to adulthood

• Neonatal period: prolonged jaundice with conjugated

hyperbilirubinaemia or (rarely) liver disease

• Adulthood: pulmonary emphysema and/or cirrhosis. Rarely, the skin disease, pann iculitis, develops

Disease mechanism

• SERPINA1 encodes α

-antitrypsin, which protects the body from the effects of neutrophil elastase. Th e symptoms of

α1

-antitrypsin

deciency result from the effects of this enzyme attacking normal tissue

Disease variants

• M variant: if an individual has normal SERPINA1 genes and produ ces normal levels of

α1

-antitrypsin, they are said to have an M

variant

• S variant: p.Glu264Val mutation results in

α1

-antitrypsin levels reduced to about 40% of normal

• Z variant: p.Glu342Lys mutation results in very little

α1

-antitrypsin

• PiZZ: individuals who are homozygous for the p.Glu342Lys mutation are likely t o have

α1

-antitrypsin deciency and the associated

symptoms

• PiZS: individuals who are compound heterozygous for p.Glu342Lys and p. Glu264Val are likely to be affected, especially if they

smoke, but usually to a milder degree

• Individuals within one sibship in one generation are affected and so the condition can appear to have arisen ‘out of the blue’.

Approximately 1 in 4 children born to carriers of an autosomal recessive condition will be affected. The offspring risk for carrier parents

is therefore 25% and the chances of an unaffected child, with an affected sibling, bein g a carrier is 2/3.

Examples of some autosomal recessive conditions, discussed elsewhere in this book, are shown in Box 3.4.

X-linked inheritance

The following is an exemplar of an X-linked recessive pedigree (Fig. 3.8):

Edward has a diagnosis of Duchenne muscular dystrophy (DMD, Box 3.6). H is parents had suspected the diagnosis when he was 3 years

old because he was not yet walking and there was a family history of DMD: Edward’s maternal uncle had been affected and died at the

age of 24 years. Edward’s mother has no additional siblings. After Edward demonstrated a very high creatinine kinase level, the

paediatrician also requested genetic testing, which identied a deletion of exons 2– 8 of the dystrophin gene. Edward has a younger,

healthy sister and grandparents on both sides of the family are well, although the maternal grandm other has recently developed a

cardiomyopathy. Edward’s father has an older sister and an older brother who are bo th well.

Genetic diseases caused by mutations on the X chromosome have specic characteristics :

• X-linked diseases are mostly recessive and restricted to

males who carry the mutant allele. This is because males

3.6 Duchenne muscular dystrophy*

Inheritance pattern

• X-linked recessive

Genetic cause

• Mutations or deletions encompassing/within the DMD (dystrophin)

gene located at Xp21

Prevalence

• 1 in 3000–4000 live male births

Clinical presentation

• Delayed motor milestones

• Speech delay

• Grossly elevated creatine kinase (CK) levels (in the thousands)

• Ambulation is usually lost between the ages of 7 and 13 years

• Lifespan is reduced with a mean age of death, usually from respiratory failure, in the mid-twenties

• Cardiomyopathy affects almost all boys with Duchenne muscular dystro phy and some female carriers

Disease mechanism

• DMD encodes dystrophin, a major structural component of muscle

• Dystrophin links the internal cytoskeleton to the extracellular matrix Disease va riants

• Becker muscular dystrophy, although a separate disease, is also

caused by mutations in the dystrophin gene

• In Duchenne muscular dystrophy, there is no dystrophin protein,

whereas in Becker muscular dystrophy there is a reduction in the

amount or alteration in the size of the dystrophin protein

*See also page 1143.

have only one X chromosome, whereas females have two (see Fig. 3.1). However, occasi onally, female carriers may exhibit signs of an

X-linked disease due to a phenomenon called skewed X-inactivation. All female embryos, at about 100 cells in size, stably inactivate one

of their two X chromosomes in each cell. Where this inactivation is random, approximately 50% of the cells will express the genes from

one X chromosome and 50% of cells will express genes from the other. Where there i s a mutant gene, there is often skewing away from

the associated X chromosome, resulting in an unaffected female carrier. H owever, if, by chance, there is a disproportionate inactivation

of the normal X chromosome with skewing towards the mutant allele, then an affected female carrier may be affected (albeit more

mildly than males).

• The gene can be transmitted from female carriers to their sons: in families with an X-link ed recessive condition, there are often a

number of affected males related through unaffected females.

• Affected males cannot transmit the condition to their sons (but all their daughters wou ld be carriers).

The risk of a female carrier having an affected child is 25% or half of her m ale offspring.

Mitochondrial inheritance

The mitochondrion is the main site of energy production within the cell. Mitochondria ar ose during evolution via the symbiotic

association with an intracellular bacterium. They have a distinctive structure with function ally distinct inner and outer membranes.

Mitochondria produce energy in the form of adenosine triphosphate (ATP). ATP is mo stly derived from the metabolism of glucose and

fat (Fig. 3.9). Glucose cannot enter mitochondria directly but is rst metabolised to pyruvate via gly colysis. Pyruvate is then imported

into the mitochondrion and metabolised to acetyl-co-enzyme A (acetyl-CoA). F atty acids are transported into the mitochondria following

conjugation with carnitine and are sequentially catabolised by a process called β-oxidatio n to produce acetyl-CoA. The acetyl-CoA from

both pyruvate and fatty acid oxidation is used in the citric acid (Krebs) cycle – a series o f enzymatic reactions that produces CO

, the

reduced form of nicotinamide adenine dinucleotide (NADH) and the reduced form of avine adenine dinucleotide (FADH

). Both

NADH and FADH

then donate electrons to the respiratory chain. Here these elections are transferred via a complex series of reactions,

resulting in the formation of a proton gradient across the inner mitochondrial 3

membrane. The gradient is used by an inner mitochondrial

membrane protein, ATP synthase, to produce ATP, which is then transported to other p arts of the cell. Dephosphorylation of ATP is used

to produce the energy required for many cellular processes.

Each mitochondrion contains 2–10 copies of a 16-kilobase (kB) double-stranded circular D NA molecule (mtRNA). This mtDNA

contains 13 protein-coding genes, all involved in the respiratory chain, and the ncRNA genes required for protein synthesis within the

mitochondria (Fig. 3.9). The mutational rate of mtDNA is relatively high due to the lac k of protection by chromatin. Several mtDNA

diseases characterised by defects in ATP production have been described. Mitochond ria are most numerous in cells with high metabolic

demands, such as muscle, retina and the basal ganglia, and these tissues tend to be the ones most severely affected in mitochondrial

diseases (Box 3.7). There are many other mitochondrial diseases that are c aused by mutations in nuclear genes, which encode proteins

that are then imported into the mitochondrion and are critical for energy production, e.g. most fo rms of Leigh’s syndrome (although

Leigh’s syndrome may also be caused by a mitochondrial gene mutation).

The inheritance of mtDNA disorders is characterised by transmission from females, but males and females generally are equally affected

(see Fig. 3.8). Unlike the other inheritance patterns mentioned above, mitochondrial inheritanc e has nothing to do with meiosis but

reects the fact that mitochondrial DNA is transmitted by oöcytes: sperm do not contrib ute mitochondria to the zygote. Mitochondrial

disorders tend to be variable in penetrance and expressivity within families, and this is m ostly accounted for by the fact that only a

proportion of multiple mtDNA molecules within mitochondria contain the causal mutation (the degree of mtDNA heteroplasmy).

Imprinting

Several chromosomal regions (loci) have been identied where gene express ion is inherited in a parent-of-origin-specic manner;

3.7 The structure of the respiratory chain complexes and the diseases associated wi th their dysfunction Complex Enzyme nDNA

subunits

mtDNA subunits

Diseases

I NADH dehydrogenase 38 7 MELAS, MERRF bilateral striatal necrosis, LHON, myo pathy and exercise intolerance, Parkinsonism,

Leigh’s syndrome, exercise myoglobinuria, leucodystrophy/myoclonic epilepsy

II Succinate dehydrogenase 4 0 Phaeochromocytoma, Leigh’s syndrome III Cytochro me bc

complex 10 1 Parkinsonism/MELAS,

cardiomyopathy, myopathy, exercise myoglobinuria, Leigh’s syndrome

IV Cytochrome c oxidase 10 3 Sideroblastic anaemia, myoclonic ataxia, deafness, myo pathy, MELAS, MERRF mitochondrial

encephalomyopathy, motor neuron disease-like, exercise myoglobinuria, Leigh’s synd rome

V ATP synthase 14 2 Leigh’s syndrome, NARP, bilateral striatal necrosis

nDNA subunits.

mtDNA subunits

number of different protein subunits in each complex that are encoded in the nDNA a nd mtDNA,

respectively. (ATP = adenosine triphosphate; LHON = Leber hereditary optic neuropa thy; MELAS = myopathy, encephalopathy, lactic

acidosis and stroke-like episodes; MERRF = myoclonic epilepsy and ragged red bres; mtDNA

mitochondrial DNA; NADH

the

reduced form of nicotinamide adenine dinucleotide; NARP

neuropathy, ataxia and retinitis pigmentosa; nDNA = nuclear DNA)

Inner AB

strand membrane

Outer

membrane

Cristae

Matrix 22 tRNAs

trand

NADH dehydrogenase 7

subunits

Cytochrome B/C oxidase 4 subunits

2 ribosomal RNA subunits 2 ATP synthase subunits Intragenic DNA

FFA

Glucose Carnitine

Pyruvate

Lactate

Outer

membrane

CPT I

CPT II H

Cyt

2e Q

III

Inner

membrane

PDH Carnitine-FA ester Carnitine

FFA Acetyl-CoA NADH

ATP synthase

NAD

FADH

FAD

ADP + Pi

ATP

Fatty acid β

-oxidation cycle

Citric acid

(Krebs) FADH

2cycle

FADH

NADH

Fig. 3.9 Mitochondria. A Mitochondrial structure. There is a smooth outer membra ne surrounding a convoluted inner membrane, which

has inward projections called cristae. The membranes create two compartments: th e inter-membrane compartment, which plays a crucial

role in the electron transport chain, and the inner compartment (or matrix), which contains mit ochondrial DNA and the enzymes

responsible for the citric acid (Krebs) cycle and the fatty acid

-oxidation cycle. B Mitochondrial DNA. The mitochondrion contains

several copies of a circular double-stranded DNA molecule, which has a non- coding region, and a coding region that encodes the genes

responsible for energy production, mitochondrial transfer RNA (tRNA) molecules and mitochon drial ribosomal RNA (rRNA)

molecules. (ATP

adenosine triphosphate; NADH

the reduced form of nicotinamide adenine dinucleotide) C Mitochondrial energ y

production. Fatty acids enter the mitochondrion conjugated to carnitine by carnitine-palm ityl transferase type 1 (CPT I) and, once inside

the matrix, are unconjugated by CPT II to release free fatty acids (FFA). These are bro ken down by the

-oxidation cycle to produce

acetyl-co-enzyme A (acetyl-CoA). Pyruvate can enter the mitochondrion directly and i s metabolised by pyruvate dehydrogenase (PDH)

to produce acetyl-CoA. The acetyl-CoA enters the Krebs cycle, leading to the produc tion of NADH and avine adenine dinucleotide

(reduced form) (FADH

), which are used by proteins in the electron transport chain to generate a hydrogen i on gradient across the inter-

membrane compartment. Reduction of NADH and FADH

by proteins I and II, respectively, releases electrons (e), and the energy

released is used to pump protons into the inter-membrane compartment. Coenzyme Q

/ubiquinone (Q) is an intensely hydrophobic

electron carrier that is mobile within the inner membrane. As electrons are exchanged b etween proteins in the chain, more protons are

pumped across the membrane, until the electrons reach complex IV (cytochrome oxida se), which uses the energy to reduce oxygen to

water. The hydrogen ion gradient is used to produce ATP by the enzyme ATP synthas e, which consists of a proton channel and catalytic

sites for the synthesis of ATP from ADP. When the channel opens, hydrogen ions ente r the matrix down the concentration gradient, and

energy is released that is used to make ATP.

these are called imprinted loci. Within these loci the paternally inherited gene may be active whi le the maternally inherited may be

silenced, or vice versa. Mutations within imprinted loci lead to an unusual patte rn of inheritance where the phenotype is manifest only if

inherited from the parent who contributes the transcriptionally active allele. Ex amples of imprinting disorders are given in Box 3.8.

Somatic genetic disease

Somatic mutations are not inherited but instead occur during post-zygotic mitotic cell divisions at any point from embryonic

development to late adult life. An example of this phenomenon is polyostotic brous dyspl asia (McCune–Albright syndrome), in

3.8 Imprinting disorders Disorder Locus Beckwith–Wiedemann 1 1p15 syndrome

Prader–Willi syndrome 15q11–q13

Angelman’s syndrome (AS) 15q11–q13

Pseudohypoparathyroidism 20q13 (p. 664)

Genes Notes

CDKN1C, IGF2, Increased growth, macroglossia, hemihypertrophy, abdominal wall defects, ear 3

H19 lobe pits/creases and increased

susceptibility to developing childhood tumours

SNRPN, Necdin Obesity, hypogonadism and learning disability. Lack of paternal contr ibution (due and others to deletion of paternal

15q11–q13, or inheritance of both chromosome 15q11–q13 regions from the m other)

UBE3A Severe mental retardation, ataxia, epilepsy and inappropriate laughing bouts. D ue to loss-of-function mutations in the maternal

UBE3A gene. The neurological phenotype results because most tissues expre ss both maternal and paternal alleles of UBE3A, whereas

the brain expresses predominantly the maternal allele

GNAS1 Inheritance of the mutation from the mother results in hypocalcaemia, hyperphos phataemia, raised parathyroid hormone (PTH)

levels, ectopic calcication, obesity, delayed puberty and shortened 4th and 5th metacarpals (the syndrome known as Albright’s

hereditary osteodystrophy, AHO). When the mutation is inherited from the father, PTH, cal cium and phosphate levels are normal but the

other features are present (pseudopseudohypoparathyroidism, p. 664). These differ ences are due to the fact that, in the kidney (the main

target organ through which PTH regulates serum calcium and phosphate), the paternal allele is silen ced and the maternal allele is

expressed, whereas both alleles are expressed in other tissues.

which a somatic mutation in the G

alpha gene causes constitutive activation of downstream signalling, resulting in focal lesi ons in the

skeleton and endocrine dysfunction (p. 1055).

The most important example of human disease caused by somatic mutations is cance r (see Ch. 33). Here, ‘driver’ mutations occur within

genes that are involved in regulating cell division or apoptosis, resulting in abnormal cell g rowth and tumour formation. The two general

categories of cancer-causing mutation are gain-of-function mutations in growth-promo ting genes (oncogenes) and loss-of-function

mutations in growth-suppressing genes (tumour suppressor genes). Whichever mecha nism is acting, most tumours require an initiating

mutation in a single cell that can then escape from normal growth controls. This cell replicates more frequently or fails to undergo

programmed death, resulting in clonal expansion. As the size of the clone increases, on e or more cells may acquire additional mutations

that confer a further growth advantage, leading to proliferation of these subclones, which m ay ultimately result in aggressive metastatic

cancer. The cell’s complex self-regulating machinery means that more than one mutation is usually required to produce a malignant

tumour (see Fig. 33.3, p. 1318). For example, if a mutation results in activation of a growth f actor gene or receptor, then that cell will

replicate more frequently as a result of autocrine stimulation. However, this mutant cell will still b e subject to normal cell-cycle

checkpoints to promote DNA integrity in its progeny. If additional mutations in the same cell resul t in defective cell-cycle checkpoints,

however, it will rapidly accumulate further mutations, which may allow completely unre gulated growth and/ or separation from its

matrix and cellular attachments and/or resistance to apoptosis. As cell growth becomes i ncreasingly dysregulated, cells de-differentiate,

lose their response to normal tissue environment and cease to ensure appropriate mitotic chromosomal segregation. These processes

combine to generate the classical malignant characteristics of disorganised growth, variable levels of differentiation, and numerical and

structural chromosome abnormalities. An increase in somatic mutation rate can occur on exposure to external mutagens, such as

ultraviolet light or cigarette smoke, or if the cell has defects in DNA repair systems . Cancer is thus a disease that affects the fundamental

processes of molecular and cell biology.

Interrogating the genome: the changing landscape of genomic technologies

Looking at chromosomes

The analysis of metaphase chromosomes by light microscopy was the mainstay of clin ical cytogenetic analysis for decades, the aim

being to detect gain or loss of whole chromosomes (aneuploidy) or large chromosoma l segments (> 4 million bp). More recently,

genome-wide microarrays (array comparative genomic hybridisation or array CGH) h ave replaced chromosome analysis, allowing rapid

and precise detection of segmental gain or loss of DNA throughout the genome (see Box 3.3). Microarrays consist of grids of multiple

wells containing short DNA sequences (reference DNA) that are complementary to known sequences in the genome. Patient and

reference DNA are each labelled with a coloured uorescent dye (generally, patient DNA is labelled with a green uorescent dye and

reference DNA with a red uorescent dye) and added to the microarray grid. W here there is an equal quantity of patient and reference

DNA bound to the spot, this results in yellow uorescence. Where there is too much patient DNA (representing a duplication of a

chromosome region), the spot will be greener; it will be more red (appears orange) wh ere there is 2 : 1 ratio of the control:patient DNA

(representing heterozygous deletion of a chromosome region; Fig. 3.10).

Array CGH and other array-based approaches can detect small chromosomal deletions and duplications. They are also generally more

sensitive than conventional karyotyping at detecting mosaicism (where there are two or more populations of cells, derived from a single

fertilised egg, with different genotypes).

CGH

Patient Normal control DNA DNA Label DNA

with different

fluorescent

dyes

Mix equimolar

amounts of

labelled DNA

Apply DNA mix to glass slide with

high-density array of different DNA

probes with known location in the human genome

Patient/control ratio = 0.5:1 → deletion of patient DNA

Patient/control ratio = 1.5:1 → duplication of patient DNA

Patient/control ratio = 1:1

→ normal

Fig. 3.10 Detection of chromosome abnormalities by comparative genomic hybridisation (C GH). Deletions and duplications are detected

by looking for deviation from the 1 : 1 ratio of patient and control DNA in a microar ray. Ratios in excess of 1 indicate duplications,

whereas ratios below 1 indicate deletions.

However, array-based approaches will not detect balanced chromosome rearrangemen ts where there is no loss or gain of

genes/chromosome material, such as balanced reciprocal translocations, or a global incr ease in copy number, such as triploidy.

The widespread use of array-based approaches has brought a number of challenge s for clinical interpretation, including the identication

of copy number variants (CNVs) of uncertain clinical signicance, CNVs of variable p enetrance and incidental ndings. A CNV of

uncertain clinical signicance describes a loss or gain of chromosome material where there are insufcient data to conclude whether or

not it is associated with a learning disability and/or medical problems. While this uncertain ty can be difcult to prepare families for and

can be associated with considerable anxiety, it is likely that there will be greater clarity in the future as we generate larger CNV datasets.

A CNV of variable penetrance, also known as a neurosusceptibility locus, describes a c hromosome deletion or duplication associated

with a lower threshold for manifesting a learning disability or autistic spectrum dis order. CNVs of variable penetrance are therefore

identied at greater frequencies among individuals with a learning disability and/or autist ic spectrum disorder than in the general

population. The current understanding is that additional modifying factors (genetic, env ironmental or stochastic) must inuence the

phenotypic expression of these neurosusceptibility loci.

Finally, an incidental CNV nding describes a deletion or duplication encompassing a ge ne or genes that are causative of a phenotype or

risk unrelated to the presenting complaint. For instance, if, through the array CGH inve stigation for an intellectual disability, a deletion

encompassing the BRCA1 gene were identied, this would be considered an incidental nding .

Looking at genes

Gene amplication: polymerase

chain reaction

The polymerase chain reaction (PCR) is a fundamental laboratory technique that ampli es targeted sections of the human genome for

further analyses – most commonly, DNA sequencing. The method utilises therm al cycling: repeated cycles of heating and cooling allow

the initial separation of double-stranded DNA into two single strands (known as denaturation), each of which serves as a template during

the subsequent replication step, guided by primers designed to anneal to a specic genomic r egion. This cycle of heating/cooling and

denaturation/replication is repeated many times, resulting in the exponential amplication of DNA between primer sites (Fig. 3.11).

Gene sequencing

In the mid-1970s, a scientist called Fred Sanger pioneered a DNA sequencing techniqu e (‘Sanger sequencing’) that determined the

precise order and nucleotide type (thymine, cytosine, adenine and guanine) in a molec ule of DNA. Modern Sanger sequencing uses

uorescently labelled, chain-terminating nucleotides that are sequentially incorpo rated into the newly synthesised DNA, generating

multiple DNA chains of differing lengths. These DNA chains are subject to capillary elec trophoresis, which separates them by size,

allowing the fragments to be ‘read’ by a laser and producing a sequence chromatogra m that corresponds to the target sequence (Fig.

3.12). Although transformative, Sanger sequencing was difcult and costly to scale, as exemplied by the Human Genome Project, which

took 12 years to sequence the entire human genome at a cost approaching 3 billion dollars. Recently, DNA sequencing has been

transformed again by a group of technologies collectively known as ‘next-generation sequencing’ (NGS; Fig. 3.13). This refers to a

family of postSanger sequencing technologies that utilise the same ve basic principles:

• Library preparation: DNA samples are fragmented (by

enzyme cleavage or ultrasound) and then modied with a custom adapter sequence .

• Amplication: the library fragment is amplied to produce DNA clusters, each originatin g from a single DNA fragment. Each cluster

will act as a single sequencing reaction.

• Capture: if an entire genome is being sequenced, this step will not be included. The ca pture step is required if targeted resequencing is

necessary, such as for a panel gene test or an exome (Box 3.9).

• Sequencing: each DNA cluster is simultaneously sequenced and the data from ea ch captured; this is known as a ‘read’ and is usually

between 50 and 300 bases long sequenced (see Box 3.10 for a detailed description of the three most c ommonly used sequencing

methods: synthesis, ligation and ion semiconductor sequencing).

• Alignment and variant identication: specialised software analyses read sequ ences and compares the data to a reference template. This

is known as ‘alignment’ or ‘mapping’ and, although there are 3 billion bases in the

DNA

Polymerase

Primers

sample

+ dNTPs

Heat 95°C

DNA strands separate Fig. 3.11 The polymerase chain reaction (PCR). PCR involves adding a tiny amount of the patient’s DNA to a

reaction containing primers (short oligonucleotides 18–21 bp in length, which bind to the D NA anking the region of interest) and

deoxynucleotide phosphates (dATP, dCTP, dGTP,

dTTP), which are used to synthesise new DNA and a heat-stable

3polymerase. The reaction mix is rst heated to 95°C, which causes the double-

stranded DNA molecules to separate. The reaction is then cooled to 50–60°C, which allo ws the primers to bind to the target DNA. The

reaction is then heated to 72°C, at which point the polymerase starts making new DNA strands. These cycle s are repeated 20–30 times,

resulting in exponential amplication of the DNA fragment between the primer sites. The resultin g PCR products can then be used for

further analysis – most commonly, DNA sequencing (see Fig. 3.12).

Cool ~60°C

Cycle Primers bind no. 1 to DNA

DNA replicated Heat ~72°C

Heat 95°C

DNA strands separate

Primers

Cycle

bind to no. 2

DNA

DNA replicated Cool ~60°C

Heat ~72°C

0 5 Repeat cycles 20 – 30 times

10 15 20 25 30 PCR cycles

Exponential amplification of DNA between primer sites human genome, allows the rem arkably accurate

determination of the genomic origin where a read consists of 25 nucleotides or more. Varia nts are identied as differences between the

read and the reference genome. For instance, if there is a different nucleotide in half the reads at a given position compared to the

reference genome, this is likely to represent a heterozygous base substitution. The numb er of reads that align at a given point is called the

‘depth’ or ‘coverage’. The higher the read depth, the more accurate the variant call. How ever, in general, a depth of 30 or more reads is

generally accepted as producing diagnostic-grade results.

Rather than sequencing only one small section of DNA at a time, NGS allows the analysis o f many hundreds of thousands of DNA

strands in a single experiment and so is also commonly referred to as multiple parallel seq uencing technology. Today’s NGS machines

can sequence the entire human genome in a single day at a cost approaching 1000 US dollars.

NGS capture

Although we now have the capability to sequence the entire

genome in a single experiment, whole-genome sequencing is not always the optimal use of NGS. NGS capture refers to the ‘pull-down’

of a targeted region of the genome and may constitute several to several hundred gene s associated with a given phenotype (a gene panel),

the exons of all known coding genes (an exome), or the exons of all coding genes known to be associated with disease (a clinical exome).

Each of these targeted resequencing approaches is associated with a number of advantages and disadvantages (see Box 3.9). In order for

NGS to be used for optimal patient benet, it is essential for the clinician to have a good un derstanding of which test is the best one to

request in any given clinical presentation.

Challenges of NGS technologies

Genomic technologies have the potential to transform the way that we practise medici ne, and ever faster and cheaper DNA sequencing

offers increasing opportunities to prevent, diagnose and treat disease. However , genomic technologies are not without their challenges:

for instance, storing the enormous quantities of data generated by NGS. While the A, C, T a nd G of our genomic code could be stored on

the memory of a smartphone, huge computers, able to store several petabytes of data (w here 1 petabyte is 1 million gigabytes of data),

are required to store the information needed to generate each individual’s genome.

Even if we can store and handle these huge datasets successfully, we then need to be ab le to sift through the millions of

DNA

Polymerase

Primers

sample

+ ddNTPs

PCR

Key

ddATP

ddGTP

ddCTP

ddTTP

New DNA molecules terminated by incorporation of ddNTP

Capillary

electrophoresis Largest fragments migrate slowest

DNA sequence chromatogram Smallest fastest

Laser

fluorescence detector

Fragments detected by laser fluorescence Fig. 3.12 Sanger sequencing of DNA, whi ch is very widely used in DNA diagnostics. This is

performed using PCR-amplied fragments of DNA

corresponding to the gene of interest. The sequencing reaction is carried out with a com bination dNTP and uorescently labelled di-

deoxy-dNTP (ddATP, ddTTP, ddCTP and ddGTP), which become incorporated into the newly synthesised DNA, causing termination of

the chain at that point. The reaction products are then subject to capillary electrophore sis and the different-sized fragments are detected

by a laser, producing a sequence chromatogram that corresponds to the target DNA seq uence.

3.9 The advantages and disadvantages of whole-genome sequencing, whole-exome sequencing and gene panels

Test

Whole-genome sequencing (WGS)

Whole-exome

sequencing (WES)

Gene panels Advantages

The most comprehensive analysis of the genome available

More even coverage of genes, allowing better identication of dosage abnormalities

Will potentially detect all gene mutations, including intronic mutations

Cheaper than whole-genome sequencing

Analysis is not restricted to only those genes known to cause a given condition

Fewer variants detected than in WGS and so easier interpretation

Deeper sequencing than WGS increases sensitivity and detection of mosaicism

Cost-effective

Very deep sequencing, increasing the chances of mosaicism being detected

Fewer variants detected and so data easier to interpret As analysis is restricted to known genes, the likelihood of a variant being

pathogenic is greatly increased Disadvantages

More expensive to generate and store

Will detect millions of variants in non-coding DNA, which can be very difcult to interpre t

Associated with a greater risk of identifying incidental ndings Shallow sequencing (few reads per gene) and so less sensitive and less

able to detect mosaicism

Less even coverage of the genome and so dosage abnormalities are more difcult to detect

Less comprehensive analysis (1–2% of the genome) than WGS Increased risk of iden tifying incidental ndings over targeted gene

sequencing

Will only detect variation in genes known to cause a given condition

Difcult to add new genes to the panel as they are discovered

normal variants to identify the single (or, rarely, several) pathogenic, disease-causing m utation. While this can, to an extent, be achieved

through the application of complex algorithms, these take time and considerable expertis e to develop and are not infallible.

Furthermore, even after these data have been sifted by bioinformaticians, it is highly like ly that clinicians will be left with some variants

for which there are insufcient data to enable their denitive categorisation as either path ogenic or non-pathogenic. This may be because

we simply do not know enough about the gene, because the particular variant has not p reviously been reported and/or it is identied in an

unaffected parent. These variants must be interpreted with caution and, more u sually, their interpretation will require input from a

genetics expert in the context of the clinical presentation, where an ‘innocent until proven gu ilty’ approach is often adopted.

Finally, if we are to interrogate the entire genome or even the exome, it is for eseeable that we will routinely identify ‘incidental’ or

secondary ndings – in other words, ndings not related to the initial diagnostic q uestion. The UK has so far advocated a conservative

approach to incidental ndings.

Uses of NGS

NGS is now frequently used, within diagnostic laboratories, to identify base subst itutions and indels (although the latter were

1 Library preparation 3.10 Next-generation sequencing methods Ge nomic DNA Fragmentation

2 Cluster amplification

Adapter ligation

Flow cell

Amplification

3 Sequencing G A T C

4 Alignment and variant interpretation Reference genome C CGATATCTAGCTTA ATATCTAGC Reads

CG TAGC

TATCTAGC CCG TAGCTAGCTTA

Fig. 3.13 Sequencing by synthesis as used in the Illumina system. (1) Library preparation: D NA is fragmented and specialist adapters are

ligated to the fragmented ends. (2) Cluster amplication: the library is loaded to a ow ce ll and the adapters hybridise to the ow-cell

surface. Each bound fragment is hybridised. (3) Sequencing. (4) Alignment and variant interpr etation: reads are aligned to a reference

sequence using complex software and differences between reference and case genomes are identi ed.

initially problematic). The current NGS challenge is to detect large deletions or duplicatio ns spanning several hundreds or thousands of

bases and therefore exceeding any single read. Increasingly, however, this dosage ana lysis is being achieved using sophisticated

computational methods, negating the need for more traditional technologies such as arra y CGH. Additional potential uses of NGS

include detection of balanced and unbalanced translocations and mosaicism: NGS h as proved remarkably sensitive at detecting the latter

when there is high read coverage for a given region. Of note, however, Sequ encing by synthesis (Fig. 3.13)

• The most frequently used NGS method 3

• Used in Illumina systems (commonly used in diagnostic laboratories)

• Uses uorescently labelled, terminator nucleotides that are

sequentially incorporated into a growing DNA chain

• Library DNA samples (fragmented DNA anked by DNA adapter s equences) are anchored to a ow cell by hybridisation of the DNA

adapter sequence to probes on the ow-cell surface

• Amplication occurs by washing the ow cell in a mixture

containing all four uorescently labelled terminator nucleotides: A, C, T and G

• Once the nucleotide, complementary to the rst base of the DNA template, is in corporated, no further nucleotides can be added until

the mixture is washed away

• The nucleotide terminator is shed and the newly incorporated

nucleotide reverts to a regular, non-uorescent nucleotide that can be extended

• The process is then repeated with the incorporation of a second base etc.

• Sequencing by synthesis is therefore space- and time-dependent: a sensor will detect the order of uorescent emissions for each spot on

the plate (representing the cluster) and determine the sequence for that read

Sequencing by ligation

• Used in SOLiD systems

• Uses DNA ligase rather than DNA polymerase (as is used in

sequencing by synthesis) and short oligonucleotides (as opposed to single nucleotides)

• Library DNA samples are washed in a mixture containing

oligonucleotide probes representing 4–16 dinucleotide sequences. Only one nucleotide in the probe is uorescently labelled

• The complementary oligo probes will hybridise, using DNA ligase, to the target se quence, initially at a primer annealed to the anchor

site and then progressively along the DNA strand

• After incorporation of each probe, uorescence is measured and the dye is cleaved off

• Eventually, a new strand is synthesised (composed of a series of the olig o probes)

• A new strand is then synthesised but is offset by one nucleotide

• The process is repeated a number of times (5 rounds in the SOLiD system), providing overlapping templates that are analysed and a

composite of the target sequence determined

Ion semiconductor sequencing

• When a nucleotide is incorporated into a growing DNA strand, a hydrogen ion is rele ased that can be detected by an alteration in the pH

of the solution. This hydrogen ion release forms the basis of ion semiconductor sequencing

• Each amplied DNA cluster is located above a semiconductor transistor, c apable of detecting differences in the pH of the solution

• The DNA cluster is washed in a mixture containing only one type of nucle otide

• If the correct nucleotide, complementary to the next base on the DNA template , is in the mixture and incorporated, a hydrogen ion is

released and detected

• If a homopolymer (sequence of two or more identical nucleotides) is pr esent, this will be detected as a decrease in pH proportionate to

the number of identical nucleotides in the sequence

NGS is still not able to interrogate the epigenome (and so will not identify cond itions caused by a disruption of imprinting, such as

Beckwith–Wiedemann, Silver–Russell, Angelman’s and Prader–Willi syndromes) and w ill not detect triplet repeat expansions such as

those that cause Huntington’s disease, myotonic dystrophy and fragile X syndrome (se e Boxes 3.8 and 3.2).

Third-generation sequencing

Increasingly, third-generation or single-molecule sequencing is entering the diagnostic ar ena. As with next- or second-generation

sequencing, a number of different platforms are commercially available. One of the m ost successful is SMRT technology (single-

molecule sequencing in real time), developed by Pacic Biosciences. This system utilis es a single-stranded DNA molecule (as compared

to the amplied clusters used in NGS), which acts as a template for the sequential incorp oration, using a polymerase, of uorescently

labelled nucleotides. As each complementary nucleotide is added, the uorescence (an d therefore the identity of the nucleotide) is

recorded before it is removed and another nucleotide is added.

A key advantage of third-generation sequencing is the long length of the read it generates: in th e region of 10–15 kilobases. It is also

cheaper than NGS, as fewer reagents are required. Given these inherent advantages, t hird-generation sequencing is likely to supersede

NGS in the near future. Given the confusion surrounding the terminology of NGS and third-g eneration sequencing, these technologies

are increasingly referred to as ‘massively parallel sequencing’.

Genomics and clinical practice

Genomics and health care

Genomics in rare

neurodevelopmental disorders

Although, by denition, the diagnosis of a rare disorder is made infrequently, r are diseases, when considered together, affect about 3

million people in the UK, the majority of whom are children. NGS has transformed the ability to diagnose individuals affected by a rare

disease. Whereas previously, when we were restricted to the sequential analysis of sin gle genes, a clinician would need to make a clinical

diagnosis in order to target testing, NGS allows the interrogation of multiple genes in a si ngle experiment. This might be done through a

gene panel, a clinical exome or an exome (see Box 3.9 and p. 53), and has increase d the diagnostic yield in neurodevelopmental

disorders to approximately 30%. Not only does the identication of the genetic cause of a rare disorder potentially provide families with

answers, prognostic information and the opportunity to meet and derive support from other a ffected families but also it can provide

valuable information for those couples planning further children and wishing to consider prena tal testing in the future.

Genomics and common disease

Most common disorders are determined by interactions between a number of genes an d the environment. In this situation, the genetic

contribution to disease is termed polygenic. Until recently, very little progress had been made in identifying the genetic variants that

predispose to common disorders, but this has been changed by the advent of genome-w ide association studies. A GWAS typically

involves genotyping many (> 500 000) genetic markers (SNPs) spread acro ss the genome in a large group of individuals with the disease

and in controls. By comparing the SNP genotypes in cases and controls, it is pos sible to identify regions of the genome, and therefore

genes, more strongly associated with a given SNP prole and therefore more likely to contribute to the disease under study.

Genomics and obstetrics

Prenatal genetic testing may be performed where a pregnancy is considered at increased r isk of being affected with a genetic condition,

either because of the ultrasound/biochemical screening results or because of the family history. While invasive tests, such as

amniocentesis and chorionic villus sampling, have been the mainstay of prenata l diagnosis for many years, they are increasingly being

superseded by non-invasive testing of cell-free fetal DNA (cffDNA), originating from placental trophoblasts and detectable in the

maternal circulation from 4–5 weeks’ gestation; it is present in sufcient quantities for tes ting by 9 weeks.

• Non-invasive prenatal testing (NIPT): the sequencing and

quantication, using NGS, of cffDNA chromosome-specic DNA sequences to identify trisomy 13, 18 or 21. The accuracy of NIPT in

detecting pregnancy-specic aneuploidy approaches 98%. A false-negative result can occur when there is too little cffDNA (possibly

due to early gestation or high maternal body mass index) or when aneuploidy has arisen later in deve lopment and is conned to the

embryo and not represented in the placenta. False positives can occur with conned plac ental mosaicism (describing aneuploidy in the

placenta, not the fetus) or with an alternative cause of aneuploidy in the maternal circulation, such a cell-free tumour DNA.

• Non-invasive prenatal diagnosis (NIPD): the identication of a fetal single-gene defect t hat either has been paternally inherited or has

arisen de novo and so is not identiable in the maternal genome. Examples of conditions that are currently amenable to NIPD include

achondroplasia and the craniosynostoses. Increasingly, however, NIPD is being used for auto somal recessive conditions such as cystic

fibrosis, where parents are carriers for different mutations. The free fetal DNA is tested to see w hether the paternal mutation is identied

and, if absent, the fetus is not affected. If the paternal mutation is identied, however , a denitive invasive test is required to determine

whether the maternal mutation has also been inherited and the fetus is affected.

Where a genetic diagnosis is known in a family, a couple may opt to undertake pre-imp lantation genetic diagnosis (PGD). PGD is used

as an adjunct to in vitro fertilisation and involves the genetic testing of a single cell from a developing embryo, prior to implantation.

Genomics and oncology

Until recently, individuals were stratied to genetic testing if they presented with a pers onal and/or family history suggestive of an

inherited cancer predisposition syndrome (Box 3.11). Relevant clinical inf ormation included the age of cancer diagnosis and

number/type of tumours. For example, the diagnosis of bilateral breast cancer in a woman in her thirties with a mother who had ovarian

cancer in her forties is suggestive of BRCA1/2-associated familial breast/ovarian cancer. In ma ny familial cancer syndromes, somatic

mutations act together with an inherited mutation to cause specic cancers (p. 50). Famili al cancer syndromes may be due to germ-line

loss-of-function mutations in tumour suppressor genes encoding DNA repair enzymes or proto-oncogenes. At the cellular level, loss of

one copy of a tumour suppressor

Genomics and clinical practice • 57

3.11 Inherited cancer predisposition syndromes Syndrome name Gene Associate d cancers

Birt–Hogg–Dubé syndrome FLCN Renal tumour (oncocytoma, chromophobe (and mixed), r enal cell carcinoma) Additional clinical

features

Fibrofolliculoma 3

Trichodiscoma

Pulmonary cysts

Breast/ovarian hereditary BRCA1 Breast carcinoma susceptibility BRCA2 Ovarian carci noma Pancreatic carcinoma

Prostate carcinoma

Cowden’s syndrome PTEN Breast carcinoma Thyroid carcinoma Endometrial carcinom a

Gorlin’s syndrome/basal PTCH1 Basal cell carcinoma cell naevus syndrome Me dulloblastoma

Li–Fraumeni syndrome TP53 Sarcoma (e.g. osteosarcoma, chondrosarcoma, rhabdomyosar coma)

Breast carcinoma

Brain cancer (esp. glioblastoma) Adrenocortical carcinoma

Brain

Lynch’s syndrome/ MLH1 Colorectal carcinoma (majority right-sided) hereditary non- polyposis MSH2 Endometrial carcinoma

colon cancer MSH6 Gastric carcinoma

PMS2 Cholangiocarcinoma

Ovarian carcinoma (esp. mucinous) Multiple endocrine MEN1 Parathyroid tumour

neoplasia 1 Endocrine pancreatic tumour

Anterior pituitary tumour

Multiple endocrine RET Medullary thyroid tumour

neoplasia 2 and 3 (also Phaeochromocytoma

known as 2a and 2b, Parathyroid tumour

respectively)

Polyposis, familial APC Colorectal adenocarcinoma (FAP is characterised adenomatous (FAP) by thousands of polyps from the second

decade; without colectomy, malignant transformation of at least one of these poly ps is inevitable)

Duodenal carcinoma Hepatoblastoma

Polyposis, MYH-associated MYH (MUTYH) Colorectal adenocarcinoma Duodenal ad enocarcinoma

Retinoblastoma, familial RB1 Retinoblastoma Osteosarcoma Macrocephaly

Intellectual disability/autistic spectrum disorder Trichilemmoma

Acral keratosis

Papillomatous papule

Thyroid cyst

Lipoma

Haemangioma

Intestinal hamartoma

Odontogenic keratocyst

Palmar or plantar pits

Falx calcication

Rib abnormalities (e.g. bid, fused or missing ribs)

Macrocephaly

Cleft lip/palate

Lipoma

Facial angiobroma

Desmoid tumour

Congenital hypertrophy of the retinal pigment epithelium (CHRPE)

gene does not have any functional consequences, as the cell is protected by the rema ining normal copy. However, a somatic mutation

affecting the normal allele is likely to occur in one cell at some point during life, resul ting in complete loss of tumour suppressor activity

and a tumour developing by clonal expansion of that cell. This two-hit mechanism (one inherited, one somatic) for cancer development is

known as the Knudson hypothesis. It explains why tumours may not develop for many years (or ever) in some members of these cancer-

prone families. In DNA repair diseases, the inherited mutations increase the somatic mutation rate. Autosomal dominant mutations in

genes encoding components of specic DNA repair systems are relatively common cau ses of familial colon cancer and breast cancer

(e.g. BRCA1).

Increasingly, genetics is moving into the mainstream, becoming integrated into routine o ncological care as new gene-specic treatments

are introduced. Testing for a genetic predisposition to cancer is therefore moving from the dom ain of clinical genetics, where it has

informed diagnosis, cascade treatment and screening/prophylactic management, to oncolo gy, where it is informing the immediate

management of the patient following cancer diagnosis. This is exemplied by BRCA1 and BRCA2 (BRCA1/2)-related breast cancer.

Previously, women with a mutation in either the BRCA1 or BRCA2 gene would have received sim ilar rst-line chemotherapy to women

with a sporadic breast cancer without a known genetic association. More recently, it has been shown that BRCA1/2 mutation-positive

tumours are sensitive to poly ADP ribose polymerase (PARP) inhibitors. PARP inhibitors block th e single-strand break-repair pathway.

In a BRCA1/2 mutation-positive tumour – with compromised double-strand break repair – the additional loss of the singlestrand break-

repair pathway will drive the cell towards apoptosis. Indeed, PARP inhibitors have been shown to be so effective at destroying BRCA1/2

mutation-positive tumour cells, and with such minimal side-effects, that BRCA1/2 gene testing is increasingly determining patient

management. It is likely, with a growing understanding of the genomic architecture of tu mours, increasing accessibility of NGS and an

expanding portfolio of gene-directed therapies, that testing for many of the other inhe rited cancer susceptibility genes will, in time, move

into the mainstream.

Genomics in infectious disease

NGS technologies are also transforming infectious disease. Given that a microbial genom e can be sequenced within a single day at a

current cost of less than 100 US dollars, microbiologists are able to identify a causative m icroorganism and target effective treatment

rapidly and accurately. Moreover, microbial genome sequencing enables the effective s urveillance of infections to reduce and prevent

transmission. Finally, an understanding of the microbial genome will drive the development of vaccines and antibiotics, essential in an

era characterised by increasing microbial resistance to established antibiotic age nts.

Treatment of genetic disease

Pharmacogenomics

Pharmacogenomics is the science of dissecting the genetic determinants of drug kinetics and effects using information from the human

genome. For more than 50 years, it has been appreciated that polymorphic mutations within ge nes can affect individual responses to

some drugs, such as loss-of-function mutations in CYP2D6 that cause hyper sensitivity to debrisoquine, an adrenergic-blocking

medication formerly used for the treatment of hypertension, in 3% of the population. T his gene is part of a large family of highly

polymorphic genes encoding cytochrome P450 proteins, mostly expressed in the liver, which determine the metabolism of a host of

specic drugs. Polymorphisms in the CYP2D6 gene also determine codeine activ ation, while those in the CYP2C9 gene affect warfarin

inactivation. Polymorphisms in these and other drug metabolic genes determine the persistence of drugs and, therefore, should provide

information about dosages and toxicity. With the increasing use of NGS, genetic testing for assessment of drug response is seldom

employed routinely, but in the future it may be possible to predict the best specic drugs and dosages for individual patients based on

genetic proling: so-called ‘personalised medicine’. An example is the enzyme thiopurine methylt ransferase (TPMT), which catabolises

azathioprine, a drug that is used in the treatment of autoimmune diseases and in cancer c hemotherapy. Genetic screening for polymorphic

variants of TPMT can be useful in identifying patients who have increased sensitivity to the effects of azathioprine and who can be

treated with lower doses than normal.

Gene therapy and genome editing

Replacing or repairing mutated genes (gene therapy) is challenging in humans. Retrovi ral-mediated ex vivo replacement of the defective

gene in bone marrow cells for the treatment of severe combined immune deciency syn drome (p. 79) has been successful. The major

problems with clinical use of virally delivered gene therapy have been oncogenic integration o f the exogenous DNA into the genome and

severe immune response to the virus.

Other therapies for genetic disease include PTC124, a compound that can ‘force’ cells to read through a mutation that results in a

premature termination codon in an ORF with the aim of producing a near-normal prote in product. This therapeutic approach could be

applied to any genetic disease caused by nonsense mutations.

The most exciting development in genetics for a generation has been the discovery of a ccurate, efcient and specic techniques to enable

editing of the genome in cells and organisms. This technology is known as CRISPR/C as9 (clustered regularly interspaced short

palindromic repeats and CRISPR-associated) genome editing. It is likely that ex vivo corre ction of genetic disease will become

commonplace over the next few years. In vivo correction is not yet possible and will take m uch longer to become part of clinical

practice.

Induced pluripotent stem cells and

regenerative medicine

Adult stem-cell therapy has been in wide use for decades in the form of bon e marrow transplantation. The identication of adult stem

cells for other tissues, coupled with the ability to purify and maintain such cells in vitro, n ow offers exciting therapeutic potential for

other diseases. It was recently discovered that many different adult cell types can be tran s-differentiated to form cells (induced

pluripotent stem cells or iPS cells) with almost all the characteristics of embryonal stem cel ls derived from the early blastocyst. In

mammalian model species, such cells can be taken and used to regenerate differentiate d tissue cells, such as in heart and brain. They have

great potential both for the development of tissue models of human disease and for regenerative medicin e.

Pathway medicine

The ability to manipulate pathways that have been altered in genetic disease has tremendo us therapeutic potential for Mendelian disease,

but a rm understanding of both disease pathogenesis and drug action at a bioche mical level is required. An exciting example has been

the discovery that the vascular pathology associated with Marfan’s syndrome is due to the defecti ve brillin molecules causing up-

regulation of transforming growth factor (TGF)β signalling in the vessel wall. Losartan is an antihypertensive drug that is marketed as an

angiotensin II receptor antagonist. However, it also acts as a partial antagonist of TGFβ signallin g and is effective in preventing aortic

dilatation in a mouse model of Marfan’s syndrome, showing promising effects in early human clinical trials.

Further information • 59

Ethics in a genomic age

As genomic technology is increasingly moving into mainstream clinical practice, it is essen tial for clinicians from all specialties to

appreciate the complexities of genetic testing and consider whether genetic testing is the right th ing to do in a given clinical scenario. To

exemplify the ethical considerations associated with genetic testing, it may be helpful to th ink about them in the context of a clinical

scenario. As you read the scenario, try to think what counselling/ethical issues might arise.

A 32-year-old woman is referred to discuss BRCA2 testing; she is currently pregnant with her second child (she already has a 2-year-old

daughter) and has an identical twin sister. Her mother, a healthy 65-year-old with Ash kenazi Jewish ancestry, participated in direct-to-

consumer testing (DCT) for ‘a bit of fun’ and a BRCA2 mutation – common in the Ashkenazi Jew ish population – was identied. There

is no signicant cancer family history of note.

Consider the following issues:

• Pre-symptomatic/predictive testing: this describes testing for a known famil ial gene mutation in an unaffected individual (compared

with diagnostic testing, where genetic testing is undertaken in an affected individual). A lthough this could be considered for the

unaffected patient, in the current scenario any testing would also have implications for her identical twin sister. This needs to be fully

explored with the patient and her sister prior to testing. There is also the potential iss ue of predictive testing in the patient’s first child. A

fundamental tenet in clinical genetics is that predictive genetic testing should be avoided in childhoo d for adult-onset conditions. This is

because, if no benet to the patient is accrued through childhood testing, it is better to retain the child ’s right to decide for herself, when

she is old enough, whether she wishes to participate in genetic testing or not.

• Prenatal testing: the principles behind predictive genetic testing in childhood can be extended to prenatal testing, i.e. if a pregnancy is

being continued, a baby should not be tested for an adult-onset condition that cannot be prevented or treated in childhood. However,

prenatal testing itself is hugely controversial and there is much debate as to how severe a condition should b e to justify prenatal

diagnosis, which would determine ongoing pregnancy decisions.

• DCT: while DCT can be interesting and empowering for individuals wishing to nd out more about their genetic backgrounds, it also

has several drawbacks. Perhaps the main one is that, unlike face-to-face genetic counsellin g (which usually precedes any genetic testing,

certainly where there are serious health implications for the individual and their family, su ch as is associated with BRCA1/2 mutations),

DCT is undertaken in isolation with no direct access to professional support. Fu rthermore, in addition to some (common) single-gene

mutations, such as the founder BRCA1/2 mutations frequently identied in

the Ashkenazi Jewish population and discussed in this 3

example, current DCT packages utilise a series of SNPs to determine an overall risk p role;

they evaluate the number of detrimental and protective SNPs for a given disease. However, g iven that only a minority of the risk SNPs

have so far been characterised, this is often inaccurate.

Individuals may be falsely reassured that they are not at increased risk of a ge netic condition despite a family history suggesting

otherwise, resulting in inadequate surveillance and/or management.

The ethical considerations listed in this clinical scenario give just a avour of some of the issues frequently encountered in clinical

genetics. They are not meant to be an exhaustive summary and whole textbooks and meetings are devoted to the discussion of hugely

complex ethical issues in genetics. However, a guiding principle is that, although each counse lling situation will be unique with specic

communication and ethical challenges, a genetic result is permanent and has implic ations for the whole family, not just the individual.

Where possible, therefore, an informed decision regarding genetic testing should be tak en by a competent adult following counselling by

an experienced and appropriately trained clinician.

Further information

Books and journal articles

Alberts B, Bray D, Hopkin K et al. Essential cell biology, 4th edn. New York: Garland Sc ience; 2013.

Firth H, Hurst JA. Oxford desk reference: clinical genetics. Oxford: Oxford University Press; 2 005.

Read A, Donnai D. New clinical genetics, 2nd edn. Banbury: Scion; 2010.

Strachan T, Read A. Human molecular genetics, 4th edn. New York: Garlan d Science; 2010.

Websites

bsgm.org.uk British Society for Genetic Medicine; has a report on genetic testing of child ren.

decipher.sanger.ac.uk Excellent, comprehensive genomic database.

ensembl.org Annotated genome databases from multiple organisms.

futurelearn.com/courses/the-genomics-era Has a Massive Open Online Course on genomics, for which one of the authors of the current

chapter is the lead educator.

genome.ucsc.edu Excellent source of genomic information.

ncbi.nlm.nih.gov Online Mendelian Inheritance in Man (OMIM).

ncbi.nlm.nih.gov/books/NBK1116/ Gene Reviews: excellent US-based so urce of information about many rare genetic diseases.

orpha.net/consor/cgi-bin/index.php Orphanet: European-based database on rare disease.

Functional anatomy and physiology 62 The innate immune system 62

The adaptive immune system 67

The inammatory response 70

Acute inammation 70

Chronic inammation 71

Laboratory features of inammation 71

Presenting problems in immune disorders 73 Recurrent infections 73

Intermittent fever 74

Anaphylaxis 75

Immune deciency 77

Primary phagocyte deciencies 77

Complement pathway deciencies 78

Primary antibody deciencies 78

Primary T-lymphocyte deciencies 79

Secondary immune deciencies 80

SE Marshall

SL Johnston

Clinical immunology

Periodic fever syndromes 81

Amyloidosis 81

Autoimmune disease 81

Allergy 84

Angioedema 87

Transplantation and graft rejection 88

Transplant rejection 88

Complications of transplant immunosuppression 89

Organ donation 90

Tumour immunology 90

The immune system has evolved to identify and destroy pathogens while minimising dam age to host tissue. Despite the ancient

observation that recovery from an infectious disease frequently results in protec tion against that condition, the existence of the immune

system as a functional entity was not recognised until the end of the 19th century. More recently, it has become clear that the immune

system not only protects against infection but also regulates tissue repair following injury, an d when dysregulated, governs the responses

that can lead to autoimmune and auto-inammatory diseases. Dysfunction or deciency of the immune response can lead to a wide

variety of diseases that may potentially involve every organ system in the body.

The aim of this chapter is to provide a general understanding of the immune system, ho w it contributes to human disease and how

manipulation of the immune system can be put to therapeutic use. A review of the key c omponents of the immune response is followed

by sections that illustrate the clinical presentation of the most common forms of immune d ysfunction: immune deciency, inammation,

autoimmunity and allergy. More detailed discussion of individual conditions can be foun d in the relevant organ-specic chapters of this

book.

Functional anatomy and physiology

The immune system consists of an intricately linked network of lymphoid organs, cells and p roteins that are strategically placed to

protect against infection (Fig. 4.1). Immune defences are normally categorised into the inna te immune response, which provides

immediate protection against an invading pathogen, and the adaptive or acquired immun e response, which takes more time to develop but

confers exquisite specicity and long-lasting protection.

The innate immune system

Innate defences against infection include anatomical barriers, phagocytic cells, soluble m olecules such as complement and acute phase

proteins, and natural killer cells. The innate immune system recognises generic microbial structures present on non-mammalian tissue and

can be mobilised within minutes. A specic stimulus will elicit essentially identical response s in different individuals, in contrast with

adaptive antibody and T-cell responses, which vary greatly between individuals.

Physical barriers

The tightly packed keratinised cells of the skin physically limit colonisation by micro organisms. The hydrophobic oils that are secreted

by sebaceous glands further repel water and microorganisms, and microbial growth is inhibited by the skin’s low pH and low oxygen

tension. Sweat also contains lysozyme, an enzyme that destroys the structural in tegrity of bacterial cell walls; ammonia, which has

antibacterial properties; and several

Cortex B cells in primary lymphoid follicles

Germinal centre

Proliferating B cells after antigen exposure

Paracortex T cells Dendritic cells

Medulla Plasma cells Sinuses with

macrophages

Blood vessels Efferent lymph Afferent lymph Capsule

Adenoids Lymph nodes Tonsils

Thymus

Thoracic duct

Liver

Spleen

Peyer’s patches in small intestine Lymph node section

B lymphocyte T lymphocyte Appendix Bone marrow Neutrophil Natural killer cell

Lymphatics Monocyte Macrophage Eosinophil

Antigen-presenting cell Basophil Mast cell

Cells of the adaptive immune system

Fig. 4.1 Anatomy of the immune system.

Cells of the innate immune system

antimicrobial peptides such as defensins. Similarly, the mucous membranes of the respiratory, g astrointestinal and genitourinary tracts

provide a physical barrier to infection. Secreted mucus traps invading pathogens, and immunoglobulin A (IgA), generated by the

adaptive immune system, prevents bacteria and viruses attaching to and penetrating epithel ial cells. As in the skin, lysozyme and

antimicrobial peptides within mucosal membranes directly kill invading pathogens, and la ctoferrin acts to starve invading bacteria of

iron. Within the respiratory tract, cilia directly trap pathogens and contribute to removal of mucus, assisted by physical manœuvres such

as sneezing and coughing. In the gastrointestinal tract, hydrochloric acid and saliv ary amylase chemically destroy bacteria, while normal

peristalsis and induced vomiting or diarrhoea assist clearance of invading organisms.

The microbiome, which is made up of endogenous commensal bacteria, provides an ad ditional constitutive defence against infection.

Approximately 10

bacteria normally reside at epithelial surfaces in symbiosis with the human host (p. 102). They compete with

pathogenic microorganisms for scarce resources, including space and nutrien ts, and produce fatty acids and bactericidins that inhibit the

growth of many pathogens. In addition, recent research has demonstrated that com mensal bacteria help to shape the immune response by

inducing specic regulatory T cells within the intestine. Eradication of the normal ora with br oad-spectrum antibiotics commonly

results in opportunistic infection by organisms such as Clostridium difcile, which rapidly c olonise an undefended ecological niche.

These constitutive barriers are highly effective, but if external defences are bre ached by a wound or pathogenic organism, the specic

soluble proteins and cells of the innate immune system are activated.

Phagocytes

Phagocytes (‘eating cells’) are specialised cells that ingest and kill microorganisms , scavenge cellular and infectious debris, and 4

produce

inammatory molecules that regulate other components

of the immune system. They include neutrophils, monocytes and macrophages, and are particularly important for defence against

bacterial and fungal infections. Phagocytes express a wide range of surface receptor s, including pattern recognition receptors (PRRs),

which recognise pathogen-associated molecular patterns (PAMPs) on invading microor ganisms, allowing their identication. The PRRs

include Toll-like receptors, nucleotide oligomerisation domain (NOD) protein-like receptors and mannose receptors, whereas the

PAMPs they recognise are molecular motifs not present on mammalian cells, including b acterial cell wall components, bacterial DNA

and viral double-stranded RNA. While phagocytes can recognise microorganisms thro ugh PRRs alone, engulfment of microorganisms is

greatly enhanced by opsonisation. Opsonins include acute phase proteins produced by the liver, such as C-reactive protein and

complement. Antibodies generated by the adaptive immune system also act as opsonins. They bind both to the pathogen and to

phagocyte receptors, acting as a bridge between the two to facilitate phagocytosis (Fig. 4.2). This is followe d by intracellular pathogen

destruction and downstream activation of pro-inammatory genes, resulting in the generati on of pro-inammatory cytokines as discussed

below.

Microbes C3b

Antibody Lipopolysaccharide

C-reactive protein Toll-like receptors Bacterial DNA Fc

receptor Bacterial RNA NOD-like

C3b

receptors receptor

NFκB Peptidoglycans Crystals

Lysosome

ResponseNFκB genes

Phagocytic cell

Pro-inflammatory gene expression

Fig. 4.2 Phagocytosis and opsonisation. Phagocytosis of microbes can be augmented by sev eral opsonins, such as C-reactive protein,

antibodies and complement fragments like C3b, which enhance the ability of phago cytic cells to engulf microorganisms and destroy

them. Phagocytes also recognise components of microbes, such as lipopolysacchari de, peptidoglycans, DNA and RNA, collectively as

pathogen-associated molecular patterns (PAMPs). These activate pattern recogn ition receptors (PRRs), such as Toll-like receptors and

nucleotide oligomerisation domain (NOD)-like receptors, which promote inammatory gene expr ession through the nuclear factor kappa

beta (NFκB) pathway. Uric acid and other crystals can also promote inammation throu gh the NOD pathway.

Neutrophils

Neutrophils, also known as polymorphonuclear leucocytes, are derived from the bone marrow and circulate freely in the blood. They are

short-lived cells with a half-life of 6 hours, and are produced at the rate of 10

cells daily. Their functions are to kill microorganisms, to

facilitate rapid transit of cells through tissues, and to amplify the immune response non-sp ecically. These functions are mediated by

enzymes contained in granules, which also provide an intracellular milieu for the killing and degradation of microorganisms.

Two main types of granule are recognised: primary or azurophil granules, and the mor e numerous secondary or specic granules.

Primary granules contain myeloperoxidase and other enzymes important for intracell ular killing and digestion of ingested microbes.

Secondary granules are smaller and contain lysozyme, collagenase and lactoferrin, whi ch can be released into the extracellular space.

Enzyme production is increased in response to infection, which is reected by more inte nse granule staining on microscopy, known as

‘toxic granulation’.

Changes in damaged or infected cells trigger local production of inflammatory molecule s and cytokines. These cytokines stimulate the

production and maturation of neutrophils in the bone marrow, and their release into th e circulation. Neutrophils are recruited to specic

sites of infection by chemotactic agents, such as interleukin 8 (IL-8), and by activation o f local endothelium. Up-regulation of cellular

adhesion molecules on neutrophils and the endothelium also facilitates neutrophil m igration. The transit of neutrophils through the blood

stream is responsible for the rise in neutrophil count that occurs in early infection. Once present within infected tissue, activated

neutrophils seek out and engulf invading microorganisms. These are initially enclo sed within membrane-bound vesicles, which fuse with

cytoplasmic granules to form the phagolysosome. Within this protected compartment, ki lling of the organism occurs through a

combination of oxidative and non-oxidative killing. Oxidative killing, also known as the respiratory burst, is mediated by the

nicotinamide adenine dinucleotide phosphate (NADPH)–oxidase enzyme complex, whi ch converts oxygen into reactive oxygen species

such as hydrogen peroxide and superoxide that are lethal to microorganisms. The myel operoxidase enzyme within neutrophils produces

hypochlorous acid, which is a powerful oxidant and antimicrobial agent. Non-oxidative (oxygen-independent) killing occurs through the

release of bactericidal enzymes into the phagolysosome. Each enzyme has a distinct antim icrobial spectrum, providing broad coverage

against bacteria and fungi.

An additional, recently identied form of neutrophil-mediated killing is neutrophil extracellular tra p (NET) formation. Activated

neutrophils can release chromatin with granule proteins such as elastase to form an e xtracellular matrix that binds to microbial proteins.

This can immobilise or kill microorganisms without requiring phagocytosis. The process of phagocytosis and NET formation (NETosis)

depletes neutrophil glycogen reserves and is followed by neutrophil death. As the cells die, their contents are released and lysosomal

enzymes degrade collagen and other components of the interstitium, causing liquefactio n of closely adjacent tissue. The accumulation of

dead and dying neutrophils results in the formation of pus, which, if extensive, may lead to abscess formation.

Monocytes and macrophages

Monocytes are the precursors of tissue macrophages. They are produced in the bone marr ow and enter the circulation, where they

4.1 Functions of macrophages Amplication of the inammatory response

• Stimulate the acute phase response (through production of IL-1 and IL-6)

• Activate vascular endothelium (IL-1, TNF

)

• Stimulate neutrophil maturation and chemotaxis (IL-1, IL-8)

• Stimulate monocyte chemotaxis

Killing of microorganisms

• Phagocytosis

• Microbial killing through oxidative and non-oxidative mechanisms

Clearance, resolution and repair

• Scavenging of necrotic and apoptotic cells

• Clearance of toxins and other inorganic debris

• Tissue remodelling (elastase, collagenase, matrix proteins)

• Down-regulation of inammatory cytokines

• Wound healing and scar formation (IL-1, platelet-derived growth factor, broblast gr owth factor)

Link between innate and adaptive immune systems

• Activate T cells by presenting antigen in a recognisable form

• T cell-derived cytokines increase phagocytosis and microbicidal activity of macro phages in a positive feedback loop

(IL = interleukin; TNF = tumour necrosis factor)

constitute about 5% of leucocytes. From the blood stream they migrate to perip heral tissues, where they differentiate into tissue

macrophages and reside for long periods. Specialised populations of tissue macrophage s include Kupffer cells in the liver, alveolar

macrophages in the lung, mesangial cells in the kidney, and microglial cells in the brain. Macrophages, like neutrophils, are capable of

phagocytosis and killing of microorganisms but also play an important role in the amp lication and regulation of the inammatory

response (Box 4.1). They are particularly important in tissue surveillance and constantly survey their immediate surroundings for signs of

tissue damage or invading organisms.

Dendritic cells

Dendritic cells are specialised antigen-presenting cells that are present in tissues in contact with the external environment, such as the

skin and mucosal membranes. They can also be found in an immature state in the blood . They sample the environment for foreign

particles and, once activated, carry microbial antigens to regional lymph nodes, where t hey interact with T cells and B cells to initiate and

shape the adaptive immune response.

Cytokines

Cytokines are signalling proteins produced by cells of the immune system and a variety of other cell types. More than 100 have been

identied. Cytokines have complex and overlapping roles in cellular communication and regulation of the immune response. Subtle

differences in cytokine production, particularly at the initiation of an immune response, can have a major impact on outcome. Cytokines

bind to specic receptors on target cells and activate downstream intracellular signalling p athways, ultimately leading to changes in gene

transcription and cellular function. Two important signalling pathways are illustrated in Figure 4.3. The nuclear factor kappa B (NFκB)

pathway is activated by tumour necrosis factor (TNF), by other members of the TNF su perfamily such as receptor activator of nuclear

kappa B ligand IL-6

IL-2 Cytokines

IFNγTNF

Cytokine receptor

TNF receptor TRAF JAK JAK JAK inhibitor P P

STAT

IKKκ P P

IKK

STAT

B NFκB

P I

κ B

Response

STAT

genes PP

STAT

Response genes

DNA

Fig. 4.3 Cytokines signalling pathways and 4

the immune response. Cytokines regulate the immune response through binding to specic

receptors that activate a variety of intracellular signalling pathways, two of which are shown. Me mbers of the tumour necrosis factor

(TNF) superfamily and the Toll-like receptors and

NOD-like receptors (Fig. 4.2) signal through the nuclear factor kappa B (NFκB) pathw ay. Several other cytokines, including interleukin-

2 (IL-2), IL-6 and interferons, employ the Janus kinase/ signal transducer and activator of transcription (JAK-STAT) pathway to regulate

cellular

function (see text for more details). (I

inhibitor of kappa B; IKK = I kappa B kinase; P

phosphorylation of the signalling protein; TRAF = tumour necrosis factor rece ptor

associated factor)

4.2 Important cytokines in the regulation of the immune response

Cytokine

Interferon-alpha (IFNα)

Interferon-gamma (IFN

)

Tumour necrosis factor alpha

(TNF

)

Macrophages, NK cells and others, including T cells

Interleukin-1 (IL-1)

Interleukin-2

(IL-2)

Interleukin-4

(IL-4)

Interleukin-6

(IL-6)

CD4

T cells

CD4

T cells

Monocytes and macrophages

Interleukin-12 (IL-12)

Interleukin-17 (IL-17)

Monocytes and

macrophages

Th17 cells (T helper), NK cells, NK-T cells

Interleukin-22 (IL-22)

Source

T cells and

macrophages

T cells and NK cells

Macrophages and neutrophils

Th17 cells Stimulates proliferation and differentiation of antigenspecic T lymphocytes

Stimulates maturation of B and T cells, and production of IgE antibody

Stimulates neutrophil recruitment, fever, and T-cell and macrophage activation as part of t he inammatory response, stimulates

maturation of B cells into plasma cells Stimulates IFNγ and TNFα release by T cells

Activates NK cells

Pro-inammatory cytokine

Involved in mucosal immunity and control of extracellular pathogens, synergy with IL-1 and T NF

Induction of epithelial cell proliferation and antimicrobial proteins in keratinocytes

(IgE = immunoglobulin E; NK = natural killer)

Actions

Antiviral activity

Activates NK cells, CD8

T cells and macrophages

Increases antimicrobial activity of macrophages Regulates cytokine production by T cel ls and macrophages

Pro-inammatory

Increases expression of other cytokines and adhesion molecules

Causes apoptosis of some target cells

Directly cytotoxic

Stimulates neutrophil recruitment, fever, and T-cell and macrophage activation as part of t he inammatory response Biologic therapies

Recombinant IFN

used in hepatitis C and some malignancies

Used in chronic granulomatous disease

TNF

inhibitors used in rheumatoid arthritis, inammatory bowel disease, psoriasis and many other

inammatory conditions

IL-1 inhibitors used in systemic juvenile rheumatoid arthritis, periodic fever syndromes and acute gout

Antibodies to IL-4 receptor used in severe atopic dermatitis

Antibodies to IL-6 receptor used in rheumatoid arthritis

Antibody to p40 subunit of IL-12 used in psoriasis and psoriatic arthritis

Antibody to IL-17 used in psoriasis, psoriatic arthritis and ankylosing spondylitis

(RANKL; p. 985), and by the Toll-like receptors and NOD-like receptor s (see Fig. 4.2). In the case of TNF superfamily members,

receptor binding causes the inhibitor of kappa B kinase (IKK) complex of three proteins to be recruited to the receptor by binding TNF

receptor-associated proteins (TRAF). This activates IKK, which in turn leads to ph osphorylation of the inhibitor of nuclear factor kappa

B protein (IκB), causing it to be degraded, and allowing NFκB to translocate to the nucleus and activate gene transcription. The Janus

kinase/signal transducers and activators of transcription (JAK-STAT) pathway is involv ed in transducing signals downstream of many

cytokine receptors, including those for IL-2, IL-6 and interferon-gamma (IFNγ ). On receptor binding, JAK proteins are recruited to the

intracellular portion of the receptor and are phosphorylated. These in turn phosphorylate ST AT proteins, which translocate to the nucleus

and activate gene transcription, altering cellular function. The function and diseas e associations of several important cytokines are shown

in Box 4.2. Cytokine inhibitors are now routinely used in the treatment of autoimmune dis eases, most of which are monoclonal

antibodies to cytokines or their receptors. In addition, small-molecule inhibitors have bee n developed that inhibit the intracellular

signalling pathways used by cytokines. These include the Janus kinase inhibitors tofacitin ib and baracitinib, which are used in

rheumatoid arthritis (p. 1026), and the tyrosine kinase inhibitor imatinib, which is used in ch ronic myeloid leukaemia (p. 959).

Classical

pathway

Antibody–antigen complexes

Lectin Alternate pathway pathway Mannose

binding lectin

C1inh C4

Direct

activation

Smooth muscle contraction Activation of cells Vascular permeability

C3a C3b

C5a

Integrins

Integrins are transmembrane proteins that play important roles in cell–cell and cell–matrix int eractions. They mediate attachment of the

cell to the extracellular matrix, signal transduction and cell migration. Their role in autoimm une disease has been extensively studied.

Targeted therapy with a recombinant humanised antiα4 integrin antibody, natalizuma b, is an effective treatment for multiple sclerosis,

which works by preventing immune cells from traversing the vascular endothelium and entering the central nervous system (p. 1109).

Complement

The complement system comprises a group of more than 20 tightly regulated, functionally lin ked proteins that act to promote

inammation and eliminate invading pathogens. Complement proteins are produced in th e liver and are present in inactive form in the

circulation. When the complement system is activated, it sets in motion a rapidly amp lied biological cascade analogous to the

coagulation cascade (p. 918).

There are three mechanisms by which the complement cascade can be activated ( Fig. 4.4):

• The alternate pathway is triggered directly by binding of

C3 to bacterial cell-wall components, such as

lipopolysaccharide of Gram-negative bacteria and teichoic acid of Gram-positive bacteria.

• The classical pathway is initiated when two or more IgM or IgG antibody molecules bind to antigen. The associated conformational

change exposes binding sites on the antibodies for the rst protein in the classical p athway, C1, which is a multiheaded molecule that can

bind up to six antibody molecules. Once two or more ‘heads’ of a C1 mo lecule are bound to antibody, the classical cascade is triggered.

An important inhibitor of the classical pathway is C1 inhibitor (C1inh), as illustra ted in Figure 4.4.

• The lectin pathway is activated by the direct binding of mannose-binding le ctin to microbial cell surface carbohydrates. This mimics

the binding of C1 to immune complexes and directly stimulates the classical pathway, bypass ing the need for immune complex

formation.

Activation of complement by any of these pathways results in activation of C3. T his in turn activates the nal common pathway, in which

the complement proteins C5–C9 assemble to form the membrane attack complex (MAC). This can puncture the cell wall, leading to

osmotic lysis of target cells. This step is particularly C5

Opsonisation of bacteria C5b

Membrane C7

attack

complex

(MAC)

Lysis of bacteria

Fig. 4.4 The complement pathway. The classical pathway is activated by binding of antigen–antibody complexes to C1 but is blocked by

C1 inhibitor (C1inh), whereas mannose-binding lectins, which are macr omolecules that bind to various microorganisms, activate the

pathway by binding C4. Bacteria can directly activate the pathway through C3, wh ich plays a pivotal role in complement activation

through all three pathways.

important in the defence against encapsulated bacteria such as Neisseria spp. and Haemophilus inu enzae.

Complement fragments generated by activation of the cascade can also act as opsonins, rendering microorganisms more susceptible to

phagocytosis by macrophages and neutrophils (see Fig. 4.2). In addition, they are che motactic agents, promoting leucocyte trafcking to

sites of inammation. Some fragments act as anaphylotoxins, binding to complement rec eptors on mast cells and triggering release of

histamine, which increases vascular permeability. The products of complement activation also help to target immune complexes to

antigen-presenting cells, providing a link between the innate and the acquired immune systems. Finally, activated complement products

dissolve the immune complexes that triggered the cascade, minimising bystander damage to surrounding tissues.

A monoclonal antibody directed against the central complement molecule C5, eculizuma b, has been developed for therapeutic use in

paroxysmal nocturnal haemoglobinuria and atypical haemolytic uraemic syndromes (p. 408 ). Invasive infection, including

meningococcal sepsis, has been reported with eculizumab therapy, highlighting the impo rtance of the complement system in preventing

such infections.

Mast cells and basophils

Mast cells and basophils are bone marrow-derived cells that play a central role in allergi c disorders. Mast cells reside predominantly in

tissues exposed to the external environment, such as the skin and gut, while basophils cir culate in peripheral blood and are recruited into

tissues in response to inammation. Both contain large cytoplasmic granules that enclose vasoactive substances such as histamine (see

Fig. 4.14). Mast cells and basophils express IgE receptors on their cell surface, which b ind IgE antibody. On encounter with specic

antigen, the cell is triggered to release histamine and other mediators present within the g ranules and to synthesise additional mediators,

including leukotrienes, prostaglandins and cytokines. An inammatory cascade is initiated that inc reases local blood ow and vascular

permeability, stimulates smooth muscle contraction, and increases secretion at mucosal su rfaces.

Natural killer cells

Natural killer (NK) cells are large granular lymphocytes that play a major role in defenc e against tumours and viruses. They exhibit

features of both the adaptive and the innate immune systems in that they are morph ologically similar to lymphocytes and recognise

similar ligands, but they are not antigen-specic and cannot generate immunological memory. NK cells express a variety of cell surface

receptors, some of which are stimulatory and others inhibitory. The effects of inhibitory r eceptors normally predominate. These

recognise human leucocyte antigen (HLA) molecules that are expressed on nor mal nucleated cells, preventing NK cell-mediated attack,

whereas the stimulatory receptors recognise molecules that are expressed primarily whe n cells are damaged. This allows NK cells to

remain tolerant to healthy cells but not to damaged ones. When cells become infected by viruses or undergo malignant change, expression

of HLA class I molecules on the cell surface can be down-regulated. This is an important mec hanism by which these cells then evade

adaptive T-lymphocyte responses. In this circumstance, however, NK cell defences bec omes important, as down-regulation of HLA class

I abrogates the inhibitory signals that normally prevent NK activation. The net result is NK attac k on the abnormal target cell. NK cells

can also be activated by binding of antigen–antibody complexes to surface receptors. This physically links the NK cell to its target in a

manner analogous to opsonisation and is known as antibody-dependent cellular cytotox icity (ADCC).

Activated NK cells can kill their targets in various ways. They secrete pore-forming protein s such as perforin into the membrane of the

target cell, and proteolytic enzymes called granzymes into the target cell, which cause ap optosis. In addition, NK cells produce a variety

of cytokines such as TNFα and IFNγ, which have direct antiviral and anti-tumour e ffects.

The adaptive immune system

If the innate immune system fails to provide effective protection against an invad ing pathogen, the adaptive immune system is mobilised

(see Fig. 4.1). This has three key characteristics:

• It has exquisite specicity and can discriminate between

very small differences in molecular structure.

• It is highly adaptive and can respond to an almost

unlimited number of molecules.

• It possesses immunological memory, and changes

consequent to initial activation by an antigen allow a more

effective immune response on subsequent encounters.

There are two major arms of the adaptive immune response. Humoral immun ity involves the production of antibodies by B lymphocytes,

and cellular immunity involves the activation of T lymphocytes, which synthesise and re lease cytokines that affect other cells, as well as

directly killing target cells. These interact closely with each other and with the components of the innate immune system to maximise

effectiveness of the immune response.

Lymphoid organs

The primary lymphoid organs are involved in lymphocyte development. They include t he bone marrow, where T and B lymphocytes

differentiate from haematopoietic stem cells (p. 914) and where B lymphocytes also ma ture, and the thymus, the site

of T-cell maturation (see Fig. 4.1). After maturation, lymphocytes 4

migrate to the secondary lymphoid organs. These include the spleen, lymph

nodes and mucosa-associated lymphoid tissue. These trap and concentrate foreign subs tances and are the major sites of interaction

between naïve lymphocytes and microorganisms.

The thymus

The thymus is a bi-lobed structure in the anterior mediastinum, and is organised into cortical and medullary areas. The cortex is densely

populated with immature T cells, which migrate to the medulla to undergo selection and mat uration. The thymus is most active in the

fetal and neonatal period, and involutes after puberty. Failure of thymic development is associated with profound T-cell immune

deciency (p. 79) but surgical removal of the thymus in childhood (usually during major cardiac surgery) is not associated with

signicant immune dysfunction.

The spleen

The spleen is the largest of the secondary lymphoid organs. It is highly effective at lter ing blood and is an important site of

phagocytosis of senescent erythrocytes, bacteria, immune complexes and other debris, an d of antibody synthesis. It is important for

defence against encapsulated bacteria, and asplenic individuals are at risk of overwhelm ing Streptococcus pneumoniae and H. inuenzae

infection (see Box 4.5).

Lymph nodes

These are positioned to maximise exposure to lymph draining from sites of external contact, a nd are highly organised (Fig. 4.1)

• The cortex contains primary lymphoid follicles, which are

the site of B-lymphocyte interactions. When B cells encounter antigen, they undergo intense proliferation, forming germinal centres.

• The paracortex is rich in T lymphocytes and dendritic cells.

• The medulla is the major site of antibody-secreting plasma cells.

• Within the medulla there are many sinuses, which contain large numbers of macrophages.

Mucosa-associated lymphoid tissue

Mucosa-associated lymphoid tissue (MALT) consists of diffusely distributed lymphoid c ells and follicles present along mucosal surfaces.

It has a similar function to the more organised, encapsulated lymph nodes. They include the tonsils, adenoids and Peyer’s patches in the

small intestine.

Lymphatics

Lymphoid tissue is connected by a network of lymphatics, with three major functions: it provides access to lymph nodes, returns

interstitial uid to the venous system, and transports fat from the small intestine to the blo od stream (see Fig. 14.13, p. 372). The

lymphatics begin as blind-ending capillaries, which come together to form lymphatic ducts , entering and leaving regional lymph nodes as

afferent and efferent ducts, respectively. They eventually coalesce and drain into the thoracic d uct and left subclavian vein. Lymphatics

may be either deep or supercial, and follow the distribution of major blood vessels.

Humoral immunity

Humoral immunity is mediated by B lymphocytes, which differentiate from haematopoie tic stem cells in the bone marrow. Their major

functions are to produce antibody and interact with T cells, but they are also involved in antigen presentation. Mature B lymphocytes can

be found in the bone marrow, lymphoid tissue, spleen and, to a lesser extent, the blood stream. They

T-helper cell

Immunoglobulin receptor

Antigen

CD40L

IL-4

IL-5

CD40

TCR

HLA

B cell

B-cell activation

Antibodies

Clonal

expansion

express a unique immunoglobulin receptor on their cell surface, the B-cell receptor, whic h binds to soluble antigen targets (Fig. 4.5).

Encounters with antigen usually occur within lymph nodes. If provided with approp riate cytokines and other signals from nearby T

lymphocytes, antigen-specic B cells respond by rapidly proliferating in a process know n as clonal expansion (Fig. 4.5). This is

accompanied by a highly complex series of genetic rearrangements known as somatic hy permutation, which generates B-cell populations

that express receptors with greater afnity for antigen than the original. These cells diffe rentiate into either long-lived memory cells,

which reside in the lymph nodes, or plasma cells, which produce antibody. Memory cells allow produc tion of a more rapid and more

effective response on subsequent exposure to that pathogen.

Immunoglobulins

Immunoglobulins (Ig) play a central role in humoral immunity. They are soluble protein s produced by plasma cells and are made up of

two heavy and two light chains (Fig. 4.6). The heavy chain determines the antibody class or isotype, such as IgG, IgA, IgM, IgE or IgD.

Subclasses of IgG and IgA also occur. The antigen is recognised by the antigen-binding re gions (F

) of both heavy and light chains,

while the consequences of antibody binding are determined by the constant region of the heavy chain (F

) (Box 4.3). Antibodies have

several functions.

Memory B cells Plasma cells

Fig. 4.5 B-cell activation. Activation of B cells is initiated through binding of an antige n with the immunoglobulin receptor on the cell

surface. For activation to proceed, an interaction with T-helper cells is also required, pro viding additional signals through binding of

CD40 ligand (CD40L) to CD40; an interaction between the T-cell receptor (TCR) and processed a ntigenic peptides presented by human

leucocyte antigen (HLA) molecules on the B-cell surface; and cytokines released by the T-helper cells. Fully activated B cells undergo

clonal expansion with differentiation towards plasma cells that produce antibody. Fo llowing activation, memory cells are generated that

allow rapid antibody responses when the same Light chain Variable

region (F

)

Heavy chain Constant region (F

)

antigen is encountered on a second occasion. (CD = cluster of differentiation; IL

interleukin)

Fig. 4.6 The structure of an immunoglobulin (antibody) molecule. The variable region is re sponsible for antigen binding, whereas the

constant region can interact with immunoglobulin receptors expressed on immune cells .

4.3 Classes and properties of antibody Concentration Antibody in adult serum

IgG 6.0–16.0 g/L Complement

activation* Opsonisation

IgG1

+++

IgG1

IgG2 + IgG3 ++ IgG3

+++

Presence in external secretions

IgA 1.5–4.0 g/L – – ++++

IgM 0.5–2.0 g/L IgE 0.003–0.04 g/L ++++

–

– – + –

IgD Not detected – –

– Other properties

Four subclasses: IgG1, IgG2, IgG3, IgG4

Distributed equally between blood and extracellular uid, and transported across placen ta

IgG2 is particularly important in defence against polysaccharides antigens

Two subclasses: IgA1, IgA2

Highly effective at neutralising toxins

Particularly important at mucosal surfaces

Highly effective at agglutinating pathogens

Majority of IgE is bound to mast cells, basophils and eosinophils

Important in allergic disease and defence against parasite infection

Function in B-cell development

*Activation of the classical pathway, also called ‘complement xation’.

They facilitate phagocytosis by acting as opsonins (see Fig. 4.2) and facilitate c ell killing by cytotoxic cells, particularly NK cells by

antibody-dependent cellular cytotoxicity. Binding of antibodies to antigen can trigger ac tivation of the classical complement pathway

(see Fig. 4.4). In addition, antibodies can directly neutralise the biological activity of their antigen target. This is a particularly important

feature of IgA antibodies, which act predominantly at mucosal surfaces.

The humoral immune response is characterised by immunological memory, in which the antibody response to successive exposures to an

antigen is qualitatively and quantitatively improved from the rst exposure. When a prev iously unstimulated or ‘naïve’ B lymphocyte is

activated by antigen, the rst antibody to be produced is IgM, which appears in th e serum after 5–10 days. Depending on additional

stimuli provided by T lymphocytes, other antibody classes (IgG, IgA and IgE) are prod uced 1–2 weeks later. If the memory B cell is

subsequently re-exposed to the same antigen, the lag time between exposure and production of antibody is decreased to 2–3 days, the

amount of antibody produced is increased, and the response is dominated by IgG antib odies of high afnity. Furthermore, in contrast to

the initial antibody response, secondary antibody responses do not require additio nal input from T lymphocytes. This allows the rapid

generation of highly specic responses on re-exposure to a pathogen and is an important mechanism in vaccine efcacy. 4

Cellular immunity

Cellular immunity is mediated by T lymphocytes, which play important roles in defence against viruses, fungi and intracellular bacteria.

They also play an important immunoregulatory role, by orchestrating and regulating the respo nses of other components of the immune

system. T-lymphocyte precursors differentiate from haematopoietic stem cells in the bon e marrow and are exported to the thymus when

they are still immature (see Fig. 4.1). Individual T cells express a unique receptor that is highly

Antigen-presenting cell

CD80 CD86

IL-2 HLA Antigenic peptide T-cell receptor

CD28

CTLA4

PDL1

PD1

T-cell activation

Cytokines

Fas ligand

TNFα, IFNγ

Function

Direct cell killing Th2 cells

Cytokines

IL-4, IL-5, IL-10,

IL-13

Function

B-cell activation

Eosinophil activation

CD8

T cells

Memory T cells CD4

T cells

Memory T cells Th1 cells

Th17 cells

Cytokines

TNFα, IFNγ, IL2 Function

Pro-inflammatory

Cytokines

IL-17

Function

Mucosal immunity Pro-inflammatory

Regulatory T cells

Cytokines

IL-10, TGF

Function

Anti-inflammatory

Fig. 4.7 T-cell activation. Activation of T cells is initiated when an antigenic peptide bound to a human leucocyte antigen (HLA)

molecule on antigenpresenting cells interacts with the T-cell receptor expressed by T lym phocytes. Additional signals are required for T-

cell activation, however. These include binding of the co-stimulatory molecules CD80 and CD8 6 with CD28 on the T cell, and

interleukin 2 (IL-2), which is produced in an autocrine manner by T cells that are undergoing activation. Other molecules are present that

can inhibit T-cell activation, however, including cytotoxic T-lymphocyte-associated protein 4 (CTLA4), which competes with CD28 for

binding to CD80 and CD86; and PD1, which, by binding PDL1, is also inhibitory. Follo wing activation, T cells proliferate and,

depending on their subtype, have various functions with distinct patterns of cytokine pr oduction, as indicated. Memory cells are also

generated that can mount a rapid immune response on encountering the same antigen. ( CD = cluster of differentiation; CD40L = CD40

ligand; IFNγ = interferon-gamma; IL = interleukin; PD1 = programmed cell death 1; PD L1 = programmed death ligand 1; TGFβ =

transforming growth factor beta; TNFα = tumour necrosis factor alpha)

specic for a single antigen. Within the thymus T cells undergo a process of stringent selection to ensure that autoreactive cells are

destroyed. Mature T lymphocytes leave the thymus and expand to populate other organ s of the immune system. It has been estimated that

an individual possesses 10

–10

T-cell clones, each with a unique T-cell receptor, ensuring at least partial coverage for any antigen

encountered.

Unlike B cells, T cells cannot recognise intact protein antigens in their native form. Instea d, the protein must be broken down into

component peptides by antigen-presenting cells for presentation to T lymphocytes in ass ociation with HLA molecules on the antigen-

presenting cell surface. This process is known as antigen processing and presentation, a nd it is the complex of peptide and HLA together

that is recognised by individual T cells (Fig. 4.7). The structure of HLA molecules varies widely between individuals. Since each HLA

molecule has the capacity to present a subtly different peptide repertoire to T lymp hocytes, this ensures enormous diversity in recognition

of antigens by the T-cell population. All nucleated cells have the capacity to process and p resent antigens, but cells with specialised

antigenpresenting functions include dendritic cells, macrophages and B lymphocytes. Th ese carry additional co-stimulatory molecules,

such as CD80 and CD86, providing the necessary ‘second signal’ for full T-cell activa tion.

T lymphocytes can be divided into two subgroups on the basis of function and recogni tion of HLA molecules. These are designated

CD4

and CD8

T cells, according to the ‘cluster of differentiation’ (CD) antigen number of key proteins exp ressed on their cell surface.

CD8

T lymphocytes

These cells recognise antigenic peptides in association with HLA class I molecules (HLA -A, HLA-B, HLA-C). They kill infected cells

directly through the production of pore-forming molecules such as perforin and re lease of digesting enzymes triggering apoptosis of the

target cell, and are particularly important in defence against viral infection.

CD4

T lymphocytes

These cells recognise peptides presented on HLA class II molecules (HLA-DR, HLA-D P and HLA-DQ) and have mainly

immunoregulatory functions. They produce cytokines and provide co-stimulatory signa ls that support the activation of CD8

lymphocytes and assist the production of mature antibody by B cells. In addition, their close interaction with phagocytes determines

cytokine production by both cell types. CD4

lymphocytes can be further subdivided into subsets on the basis of the cytokines they

produce:

• Th1 (T helper) cells typically produce IL-2, IFNγ and

TNFα, and support the development of delayed-type hypersensitivity responses ( p. 83).

• Th2 cells typically produce IL-4, IL-5, IL-10 and IL-13, and promote allergic respo nses (p. 84).

• T-regulatory cells (T regs) are a further subset of specialised CD4

lymphocytes that are important in actively suppressing activation of

other cells and preventing autoimmune disease.

• Th17 cells are pro-inammatory cells dened by their production of IL-17. T hey are related to regulatory T cells, and play a role in

immune defence at mucosal surfaces T-cell activation is regulated by a balance between co-stimulatory

molecules, the second signal required for activation, and inhibitory molecules that down -regulate T-cell activity. One such inhibitory

molecule, CTLA4, has been harnessed therapeutically in the form of abatacept, which i s a fusion protein comprised of the Fc fragment of

immunoglobulin linked to CTLA4. This is used to inhibit T-cell activation in rheumatoid arthr itis and solid organ transplantation.

The inammatory response

Inammation is the response of tissues to injury or infection, and is necessary for norm al repair and healing. This section focuses on the

general principles of the inammatory response and its multisystem manifestations. The role o f inammation in specic diseases is

discussed in many other chapters of this book.

Acute inammation

Acute inammation is the result of rapid and complex interplay between the cells and soluble m olecules of the innate immune system.

The classical external signs include heat, redness, pain and swelling (F ig. 4.8). The inammatory process is initiated by local tissue injury

or infection. Damaged epithelial cells produce cytokines and antimicrobial peptide s, causing early inltration of phagocytic cells.

Production of leukotrienes, prostaglandins, histamine, kinins, anaphylotoxins and indu cible nitric oxide synthase also occurs within

inamed tissue. These mediators cause vasodilatation and increased vascular pe rmeability, causing trafcking of uid and cells into the

affected tissue. In addition, pro-inammatory cytokines, such as IL-1, TNFα and IL-6 produced at the site of injury, are released

systemically and act on the hypothalamus to cause fever, and on the liver to stim ulate production of acute phase proteins.

The acute phase response

The acute phase response refers to the production of a variety of proteins by the liver in response to inflammatory stimuli. These proteins

have a wide range of activities. Circulating levels of C-reactive protein (CRP) and serum amyloid A may be increased 1000-fold,

contributing to host defence and stimulating repair and regeneration. Fibrinogen plays a n essential role in wound healing, and α

antitrypsin and

α1

-antichymotrypsin control the pro-inammatory cascade by neutralising the enzymes pr oduced by activated

neutrophils, preventing widespread tissue destruction. In addition, antioxidants such as h aptoglobin and manganese superoxide dismutase

scavenge for oxygen free radicals, while increased levels of iron-binding proteins such as ferritin and lactoferrin decrease the iron

available for uptake by bacteria (p. 941). Immunoglobulins are not acute phase prot eins but are often increased in chronic inammation.

Septic shock

Septic shock is the clinical manifestation of overwhelming inammation (p. 196). It is characterised by ex cessive production of pro-

inflammatory cytokines by macrophages, causing hypotension, hypovolaemia and tissu e oedema. In addition, uncontrolled neutrophil

activation causes release of proteases and oxygen free radicals within blood vessels, da maging the vascular endothelium and further

increasing capillary permeability. Direct activation of the coagulation pathway combines with en dothelial cell disruption to form clots

within the damaged vessels. The

The inflammatory response • 71

Headache Delirium Anorexia

Low blood pressure

Liver: ↑

Synthesis of acute phase proteins Flushing

↑Respiratory rate

↑

Heart rate, flow murmur

Adrenal release of glucocorticoids and catecholamines

Release of insulin from pancreas

Enlarged draining lymph nodes

Bone marrow:

↑Production and mobilisation of neutrophils

Skin rupture Local infection Bacteria

Tissue damage Ascending lymphangitis Inflammatory mediators and cytokines

Local cellulitis Pain Redness Swelling Nail

Hypothalamus:

Change in temperature set point

Fever

Sweating

Neuro-endocrine and autonomic stress responses

Vasodilatation ↑Local vascular permeability

Neutrophils +

Macrophages Phagocytosis

Cytokine production Vasodilatation

↑

Local vascular

permeability

↑

Leucocyte influx Fig. 4.8 Clinical features of acute inammation. In this example, the re sponse is to a penetrating injury and infection

of the foot.

clinical consequences include cardiovascular collapse, acute respiratory distress syndrom e, disseminated intravascular coagulation,

multi-organ failure and often death. Septic shock most frequently results from infection w ith Gram-negative bacteria, because

lipopolysaccharide produced by these organisms is particularly effective at activating the inammatory cascade. Early recognition and

appropriate early intervention can improve patient outcome (p. 196).

Resolution of inammation

Resolution of an inammatory response is crucial for normal healing. This involves activ e down-modulation of inammatory stimuli and

repair of bystander damage to local tissues. Extravasated neutrophils undergo apoptosis and are phagocytosed by macrophages, along

with the remains of microorganisms. Macrophages also synthesise collagenase and elast ase, which break down local connective tissue

and aid in the removal of debris. Normal tissue homeostasis is also associated with rever sion of parenchymal cells to a non-inammatory

phenotype. Macrophage-derived cytokines, including transforming growth factor-beta (TGFβ) and platelet-derived growth factor,

stimulate fibroblasts and promote the synthesis of new collagen, while angiogenic factor s stimulate new vessel formation.

Chronic inammation

In most instances, the development of an active immune response results in clearance an d control of the inammatory stimulus and

resolution of tissue damage. Failure of this process may result in chronic inammation, with signicant associated bystander damage,

known as hypersensitivity responses. Persistence of microorganisms can result in ongoi ng accumulation of neutrophils, macrophages and

activated T lymphocytes within the lesion. If this is associated with local deposition of br ous tissue, a granuloma may form.

Granulomas are characteristic of tuberculosis and leprosy (Hansen’s disease), in wh ich the microorganism is protected by a robust cell

wall that shields it from killing, despite phagocytosis.

Laboratory features of inammation

Inammation is associated with changes in many laboratory investigations. Leucocytosis is common, and reects the transit of activated

neutrophils and monocytes to the site of infection. The platelet count may also be increased. The most widely used laboratory measure of

acute inammation is CRP. Circulating levels of many other acute phase reactants, in cluding brinogen, ferritin and complement

components, are also increased in response to acute inammation, while albumin levels a re reduced. Chronic inammation is frequently

associated with a normocytic normochromic anaemia (p. 943).

C-reactive protein

C-reactive protein (CRP) is an acute phase reactant synthesised by the liver, which op sonises invading pathogens. Circulating

concentrations of CRP increase within 6 hours of the start of an inammatory stimulus. Serum c oncentrations of CRP provide a direct

biomarker of acute inammation and, because the serum half-life of CRP is 18 hours, l evels fall promptly once the inammatory

stimulus is removed. Sequential measurements are useful in monitoring disease activity (Box 4 .4). For reasons that remain unclear, some

diseases are associated with only minor elevations of CRP despite unequivocal evidence of active inflammation. These include systemic

lupus erythematosus (SLE), systemic sclerosis, ulcerative colitis and leukaemia. An important practi cal point is that if the CRP is raised

in these conditions, it suggests intercurrent infection rather than disease activity. Since the CRP is a more sensitive early indicator of the

acute phase response, it is generally used in preference to the erythrocyte sedimentation rate (ESR). If both ESR and CRP are used, any

discrepancy should be resolved by assessing the individual determinants of the ESR, wh ich are discussed below.

Erythrocyte sedimentation rate

The ESR is an indirect measure of inammation. It measures how fast erythrocytes fa ll through plasma, which is determined by the

composition of plasma proteins and the morphology of circulating erythrocytes. These factors govern the propensity of red cells to

aggregate, the major determinant of the ESR. Erythrocytes are inherently negatively cha rged, which prevents them from clumping

together in the blood stream. Since plasma proteins are positively charged, an increase in pla sma protein concentrations neutralises the

negative charge of erythrocytes, overcoming their inherent repulsive forces and causing them aggregate, resulting in rouleaux formation.

Rouleaux have a higher mass-to-surface area ratio than single red cells, and therefore sediment faster. The most common reason for an

increased ESR is an acute phase response, which causes an increase in the concentration of acu te phase proteins, including CRP.

However, other conditions that do not affect acute phase proteins may alter the compos ition and concentration of other plasma protein

(Box 4.4). For example, immunoglobulins comprise a signicant proportion of plasma proteins but do not participate in the acute phase

response. Thus any condition that causes an increase in serum immunoglobulins will inc rease the ESR without a corresponding increase

in CRP. In addition, abnormal red cell morphology can make rouleaux formation impossible. For these reasons, an inappropriately low

ESR occurs in spherocytosis and sickle-cell anaemia.

Plasma viscosity

Plasma viscosity is another surrogate measure of plasma protein concentration. Like the ESR, it is affected by the concentration of large

plasma proteins, including brinogen and immunoglobulins. It is not affected by proper ties of erythrocytes and is generally considered to

be more reliable than the ESR as a marker of inammation.

4.4 Conditions commonly associated with abnormal C-reactive protein (CRP) and /or erythrocyte sedimentation rate (ESR)

Condition

Acute bacterial, fungal or viral infection

Consequence

Stimulates acute phase response

Necrotising bacterial infection

Chronic bacterial or fungal infection Localised abscess, bacterial

endocarditis or tuberculosis

Acute inammatory diseases Crohn’s disease, polymyalgia rheumatica, inammatory arthritis

Systemic lupus erythematosus, Sjögren’s syndrome, ulcerative colitis

Multiple myeloma

Pregnancy, old age, end-stage renal disease

Stimulates profound acute

inammatory response

Stimulates acute and chronic

inammatory response with polyclonal increase in immunoglobulins,

as well as increased acute phase proteins

Stimulates acute phase response Effect on CRP

Effect on ESR

Increased (range 50–150 mg/L; in severe infections may be > 300 mg/L)

Greatly increased (may be

300 mg/L)

Increased (range 50–150 mg/L) Increased

Increased

disproportionately to CRP

Increased (range 50–150 mg/L) Increased

Chronic inammatory response Normal Increased

Monoclonal increase in serum immunoglobulin without acute inammation

Increased brinogen

Normal Increased

Normal Moderately increased

Reference range < 10 mg/L.

Reference range: adult males <10 mm/hr, adult females < 20 mm/hr.

Presenting problems in immune disorders

Recurrent infections

Infections can occur in otherwise healthy individuals but recurrent infection raises susp icion of an immune deciency. Depending on the

component of the immune system affected, the infections may involve bacteria, viruses, fungi or protozoa, as summarised in Box 4.5. T-

cell deciencies can involve pathogens from all groups.

Aetiology

Infections secondary to immune deciency occur because of defects in the numbe r or function of phagocytes, B cells, T cells or

complement, as described later in this chapter.

Clinical assessment

Clinical features that may indicate immune deciency are listed in Box 4.6. Frequent or severe infections, or ones caused b y unusual

organisms or at unusual sites are typical of immune deciency.

Investigations

Initial investigations should include full blood count and white cell differential, CRP, renal and live r function tests, urine dipstick, serum

immunoglobulins with protein electrophoresis, and HIV testing. Additional microbiolo gical tests, virology and imaging are required to

identify the causal organism and localise the site of infection, as outlined in Box 4 .7. If primary immune deciency is suspected on the

basis of initial investigations, more specialised tests should be considered, as summarised in Box 4.8 .

Management

If an immune deciency is suspected but has not yet been formally characterised, patien ts should not receive live vaccines because of the

risk of vaccine-induced disease. Further management depends on the underlying cause and details are provided later.

4.6 Warning signs of primary immune deciency* In children

≥4 new ear infections within 1 year

≥

2 serious sinus infections within 1 year

≥

2 months on antibiotics with little effect

≥2 pneumonias within 1 year

Failure of an infant to

gain weight or grow

normally

Recurrent deep skin or organ abscesses

Persistent thrush in mouth or elsewhere on skin after infancy

Need for intravenous antibiotics to clear infections

≥ 2 deep-seated infections such as sepsis, meningitis or cellulitis

A family history of

primary immune

deciency

In adults

≥2 new ear infections within

1 year

≥2 new sinus infections 4

within 1 year, in the absence

of allergy

Recurrent viral infections

≥1 pneumonia per year for more than 1 year

Chronic diarrhoea with weight loss

Recurrent deep skin or organ abscesses

Persistent thrush or fungal infection on skin or elsewhere

Recurrent need for

intravenous antibiotics to clear infections

Infection with atypical mycobacteria

A family history of primary immune deciency

*The presence of two or more of the above features may indicate the presenc e of an underlying primary immunodeciency.

4.5 Immune deciencies and common patterns of infection

Phagocyte deciency Bacteria

Staphylococcus aureus Pseudomonas aeruginosa Serratia marcescens

Burkholderia cenocepacia Nocardia

Mycobacterium tuberculosis Atypical mycobacteria

Fungi

Candida spp.

Aspergillus spp.

Complement deciency Antibody deciency T-lymphocyte deciency

Neisseria meningitidis Neisseria gonorrhoeae Haemophilus inuenzae Streptococcus pn eumoniae Haemophilus inuenzae Streptococcus

pneumoniae Staphylococcus aureus Mycobacterium tuberculosis Atypical mycobacteria

––

Viruses – –

Protozoa Giardia lamblia

– Candida spp.

Aspergillus spp.

Pneumocystis jirovecii

Cytomegalovirus (CMV) Enteroviruses

Epstein–Barr virus (EBV) Herpes zoster virus

Human papillomavirus Human herpesvirus 8 Toxoplasma gondii

Cryptosporidia

4.7 Initial investigations in suspected immune deciency

Test

Full blood count

Acute phase reactants

Serum immunoglobulins

Serum protein electrophoresis Value

Full white cell differential

Help determine presence of active infection Detection of antibody deciency

Detection of paraprotein

Comment

May dene pathway for further investigation

May be the cause of immune paresis; paraprotein should be excluded prior to diagn osis of primary antibody deciency

Serum free light chains/Bence Jones proteins Human immunodeciency virus (HIV) te st

Imaging according to history and examination findings

Detection of paraprotein

To exclude HIV as cause of secondary immune deciency

Detection of active infection/end-organ damage

May support treatment decisions, e.g. if there is evidence of bronchiectasis

4.8 Specialist investigations in suspected immune deciency

Test

Complement

(C3/C4/CH50/AP50) Test vaccination

Neutrophil function

Lymphocyte immunophenotyping (by ow cytometry)

Lymphocyte proliferation

Cytokine production

Genetic testing Investigation of recurrent invasive bacterial and fungal infection, especially with catalase-positive organisms

Investigation of leucocyte adhesion deciency

Determination of specic lymphocyte subsets, T cell, B cell, NK cell

Determination of lymphocyte proliferation in response to mitogenic stimulation

To determine T-cell immune function in response to antigen stimulation; limited availability, not ro utine Under specialist supervision

when specic primary immune deciency suspected Comment

Inherited complement deciency likely to give low/ absent results on functional assays

Helpful in patients with borderline low or normal immunoglobulins but conrmed recurrent infection Respiratory burst low/absent in

chronic

granulomatous disease

Leucocytosis with absent CD11a, b, c expression

May dene specic primary immune deciency, e.g. absent B cells in X-linked agamm aglobulinaemia

Poor responses seen in certain T-cell immune deciencies

Can be helpful, for example, in investigation of atypical mycobacterial infection

May conrm genetic cause, with implications for family members and future antena tal testing

(NK = natural killer)

Intermittent fever

Value

Investigation of recurrent pyogenic bacterial infection

Determination of functional humoral immune response

Intermittent fever has a wide differential diagnosis, including recurrent infection, malign ancy and certain rheumatic disorders, such as

Still’s disease, vasculitis and SLE (pp. 1040 and 1034), but a familial fever syndrome is a potential cause.

Aetiology

Familial fever syndromes are genetic disorders caused by mutations in genes responsibl e for regulating the inammatory response. The

symptoms are caused by activation of intracellular signalling pathways involved in the re gulation of inammation, with over-production

of pro-inammatory cytokines such as IL-1.

Clinical assessment

A full clinical history and physical examination should be performed, paying attention to the patient’s ethnic background and any family

history of a similar disorder. If this assessment shows no evidence of underlying infection, malig nancy or a rheumatic disorder and there

is a positive family history and early age at onset, then the likelihood of a familial fever s yndrome is increased.

Investigations Blood should be taken for a full blood count, measurement

of ESR and CRP, and assessment of renal and liver function. Serum ferritin should be ch ecked, as very high levels support the diagnosis

of Still’s disease. Blood and urine cultures should also be performed, along with a n autoimmune screen that includes measurement of

antinuclear antibodies and consideration of antineutrophil cytoplasmic antibodies to chec k for evidence of SLE or vasculitis, respectively.

Imaging may be required to exclude occult infection. If these investigations provide no evidence of infection or another cause, then

genetic analysis should be considered to conrm the diagnosis of a familial fever sy ndrome (p. 81). Negative genetic testing does not,

however, entirely exclude a periodic fever syndrome.

Management

Symptomatic management with non-steroidal anti-inammatory drugs (NSAIDs) should be initiated, pending the results of

investigations. If the response to NSAIDs is inadequate, glucocorticoids can be tried, pr ovided that infection has been excluded. If a

familial fever syndrome is conrmed, then denitive therapy should be initiated, depend ing on the underlying diagnosis (p. 81).

Anaphylaxis

Anaphylaxis is a potentially life-threatening, systemic allergic reaction characterised by c irculatory collapse, bronchospasm, laryngeal

stridor, often associated with angioedema, and urticaria. The risk of death is increased in patients with pre-existing asthma, particularly if

this is poorly controlled, and in situations where treatment with adrenaline (epinephrine) is de layed.

Aetiology

Anaphylaxis occurs when an allergen binds to and cross-links

membrane-bound IgE on mast cells in a susceptible individual, causing release of h istamine, tryptase and other vasoactive mediators

from mast cells. These mediators have a variety of effects, including vasodilatation, increased capillary permeability

4.9 Clinical features of mast cell degranulation Mediator Biological effects Pre-formed and stored within granules

Histamine

Tryptase

Eosinophil chemotactic factor Neutrophil chemotactic factor Vasodilatation, chemotaxis, bronchoconstriction, increased capillary

permeability and increased mucus secretion Bronchoconstriction, activates complement C3

Eosinophil chemotaxis

Neutrophil chemotaxis

Synthesised on activation of mast cells Leukotrienes

Prostaglandins

Thromboxanes

Platelet-activating factor Increase vascular permeability, chemotaxis, mucus secretion and smooth muscle contraction

Bronchoconstriction, platelet aggregation and vasodilatation Bronchoconstrictio n

Bronchoconstriction, chemotaxis of eosinophils and neutrophils • No c ause is identied in 20% of patients with anaphylaxis

Feeling of impending doom, loss of consciousness

Conjunctival injection

Flushing

Sweating

Wheeze,

bronchoconstriction

Angioedema

of lips and mucous membrane

Laryngeal obstruction Stridor

Hypotension Wasp sting

Urticaria leading to hypotension, and bronchoconstriction, as summarised in Box 4.9. It can be difficult to disting uish IgE-mediated

anaphylaxis clinically from non-specic degranulation of mast cells on exposu re to drugs, chemicals or other triggers where IgE is not

involved, previously known as anaphylactoid reactions. Common triggers are shown in Box 4.10.

Clinical assessment

The clinical features of anaphylaxis and ‘anaphylactoid’ reactions are indistinguishable and are summarised in Figure 4.9. Several other

conditions can mimic anaphylaxis and these are listed in Box 4.11.

It is important to assess the severity of the reaction, and the time between allergen exposure a nd onset of symptoms provides

Cardiac

arrhythmias

Itching of

palms, soles of feet and genitalia

4.10 Common causes of systemic allergic reactions Anaphylaxis: IgE-m ediated mast cell degranulation

Foods

• Peanuts

• Tree nuts

• Fish and shellsh Insect stings

• Bee venom

• Milk

• Eggs

• Soy products

• Wasp venom Chemicals, drugs and other foreign proteins

• Intravenous anaesthetic agents • Penicillin and other ant ibiotics

(suxamethonium) • Latex

Anaphylactoid: non-lgE-mediated mast cell degranulation

Drugs

• Aspirin and non-steroidal • Opiates

anti-inammatory drugs • Radiocontrast media (NSAIDs)

Physical

• Exercise • Cold

Idiopathic

Abdominal pain

Diarrhoea Fig. 4.9 Clinical manifestations of anaphylaxis. In this example, the response is to an insect sting containing venom to which

the patient is allergic. This causes release of histamine and other vasoactive mediators, whic h cause the characteristic features of

anaphylaxis that are illustrated.

4.11 Differential diagnosis of anaphylaxis

Causes of hypotension

• Vasovagal syncope

• Cardiac arrhythmia

Causes of respiratory distress

• Status asthmaticus

Causes of laryngeal obstruction

• C1 inhibitor deciency

Causes of generalised ushing

• Systemic mastocytosis

• Carcinoid syndrome

• Cardiogenic shock

• Pulmonary embolus

• Idiopathic angioedema

• Phaeochromocytoma

4.12 Emergency management of anaphylaxis Treatment

Prevent further contact with allergen

Ensure airway patency

Administer adrenaline (epinephrine)

promptly:

0.3–1.0 mL 1 : 1000 solution IM in adults

Repeat at 5–10-min intervals if initial response is inadequate

Administer antihistamines:

Chlorphenamine 10 mg IM or slow IV injection

Administer glucocorticoids:

Hydrocortisone 200 mg IV

Provide supportive treatment: Nebulised β

-agonists

IV uids

Oxygen

(IM = intramuscular; IV = intravenous)

Comment

Prevents ongoing mast cell activation

Prevents hypoxia

Intramuscular route important because of peripheral

vasoconstriction

Acts within minutes

Increases blood pressure Reverses bronchospasm

Blocks effect of histamine on target cells

Reduces cytokine release Prevents rebound symptoms in severe cases

Reverses bronchospasm Restores plasma volume Reverses hypoxia

a guide. Enquiry should be made about potential triggers. If none is immediately obviou s, a detailed history of the previous 24 hours

may be helpful. The most common triggers of anaphylaxis are foods, latex, insect ven om and drugs (see Box 4.10). A history of previous

local allergic responses to the offending agent is common. The route of allergen exposu re may inuence the principal clinical features of

a reaction; for example, if an allergen is inhaled, the major symptom is frequently whee zing. Features of anaphylaxis may overlap with

the direct toxic effects of drugs and venoms (Chs 7 and 8). Potentiating factors, such as exercise o r alcohol, can lower the threshold for

an anaphylactic event. It is important to identify precipitating factors so that appropriate avoidance m easures may be taken in the longer

term.

Investigations

Measurement of serum mast cell tryptase concentrations is useful to conrm the diagnos is but cannot distinguish between anaphylaxis

and non-IgE-mediated anaphylactoid reactions. Specic IgE tests may be useful in con rming hypersensitivity and may be preferable to

skin-prick tests when investigating patients with a history of anaphylaxis.

4.13 How to prescribe self-injectable

adrenaline (epinephrine)

Prescription (normally initiated by an immunologist or allergist)

• Specify the brand of autoinjector, as they have different triggering m echanisms

• Prescribe two devices

Indications

• Anaphylaxis to allergens that are difcult to avoid:

Insect venom

Foods

• Idiopathic anaphylactic reactions

• History of severe localised reactions with high risk of future

anaphylaxis:

Reaction to trace allergen

Likely repeated exposure to allergen

• History of severe localised reactions with high risk of adverse outcome: Poor ly controlled asthma

Lack of access to emergency care

Patient and family education

• Know when and how to use the device

• Carry the device at all times

• Seek medical assistance immediately after use

• Wear an alert bracelet or necklace

• Include the school in education for young patients (see ‘Furth er information’)

Other considerations

• Caution with β-blockers in anaphylactic patients as they may increase the seve rity of an anaphylactic reaction and reduce the response

to adrenaline (epinephrine)

4.14 Allergy in adolescence

• Resolution of childhood allergy: most children affected by allergy to milk , egg, soybean or wheat will grow out of their food allergies

by adolescence but allergies to peanuts, tree nuts, sh and shellsh are frequently life-long.

• Risk-taking behaviour and fatal anaphylaxis: serious allergy is increasingly commo n in adolescents and this is the highest risk group for

fatal, food-induced anaphylaxis. This is associated with increased risk-taking behaviour, and food-allergic teenagers are more likely than

adults to eat unsafe foods, deny reaction symptoms and delay emergency treatment.

• Emotional impact of food allergies: some adolescents may neglect to carry a prescribed adrenalin autoinjector because of the associated

nuisance and/or stigma. Surveys of food-allergic teens reveal that many take risks because they feel socially isolated by their allergy.

Management

The principles of management of the acute event are summarised in Box 4 .12. Individuals who have recovered from an anaphylactic

event should be referred for specialist assessment. The aim is to identify the trigg er factor, to educate the patient regarding avoidance

and management of subsequent episodes, and to establish whether specic treatmen t, such as immunotherapy, is indicated. If the trigger

factor cannot be identied or avoided, recurrence is common. Patients who have previously ex perienced an anaphylactic event should be

prescribed self-injectable adrenaline (epinephrine) and they and their families or carers should be instructed in its use (Box 4.13). The use

of a MedicAlert (or similar) bracelet will increase the likelihood of the injector being adm inistered in an emergency. Allergy in

adolescence requires additional consideration and management, as set out in B ox 4.14.

Immune deciency

The consequences of immune deciency include recurrent infection, autoimmunity as a result of immune dysregulation, and increased

susceptibility to malignancy, especially malignancy driven by viral infections such as Eps tein–Barr virus. Immune deciency may arise

through intrinsic defects in immune function but is much more commonly due to second ary causes, including infection, drug therapy,

malignancy and ageing. This section gives an overview of primary immune deciencies . More than a hundred such deciencies have

been described, most of which are genetically determined and present in childhood or a dolescence. The presentation of immune

deciency depends on the component of the immune system that is defective (see Box 4.5 ). There is considerable overlap and

redundancy in the immune network, however, and some diseases do not fall easily into this classication.

Primary phagocyte deciencies

Primary phagocyte deciencies typically present with recurrent bacterial and fungal in fections, which may involve unusual sites.

Affected patients require aggressive management of infections, including intravenous antibiotics a nd surgical drainage of abscesses, and

long-term prophylaxis with antibacterial and antifungal agents. The most important exam ples are illustrated in Figure 4.10 and discussed

below.

Chronic granulomatous disease

This is caused by mutations in genes that encode NADPH oxidase enzymes, which resu lts in failure of oxidative killing. The defect leads

to susceptibility to catalase-positive organisms 4

such as Staphylococcus aureus, Burkholderia cenocepacia and

Aspergillus . Intracellular killing of mycobacteria in macrophages is also impaired. Infe ctions most commonly involve the lungs, lymph

nodes, soft tissues, bone, skin and urinary tract, and are characterised histologically by granuloma formation. Most cases are X-linked (p.

48).

Leucocyte adhesion deciencies

These very rare disorders of phagocyte migration occur because

of failure to express adhesion molecules on the surface of leucocytes, resulting in their inability to exit the blood stream. The most

common cause is loss-of-function mutations affecting the ITGB2 gene, which encodes the integrin

-2 chain, a component of the

adhesion molecule LFA1. They are characterised by recurrent bacterial infections but s ites of infection lack evidence of neutrophil

inltration, such as pus formation. Peripheral blood neutrophil counts may be very high during acute infection because of the failure of

mobilised neutrophils to exit blood vessels. Specialised tests show reduced or absent exp ression of adhesion molecules on neutrophils.

Normal

LFA1

IL-23

IL-12

ICAM1

IFNγ

IL-23

IL-12

IFNγ

Neutrophils traverse endothelium through binding of LFA1 to ICAM1 Cytokines activa te macrophages Destruction of microorganisms

through NADPH oxidase-mediated killing

Primary phagocyte deficiency

Leucocyte adhesion deficiency Cytokine defects IL-12

IL-23

IFN

Chronic granulomatous disease

Neutrophils cannot traverse endothelium due to defects in ITGB2, a component of LFA 1

Phagocytes cannot be activated due to defects in cytokines or their receptors Microorga nisms cannot be destroyed in lysosomes due

to NADPH oxidase deficiency

Fig. 4.10 Normal phagocyte function and mechanisms of primary phagocyte deciency. U nder normal circumstances, neutrophils

traverse the endothelium to enter tissues by the cell surface molecule lymphocyte function- associated antigen 1 (LFA1), which binds to

intercellular adhesion molecule 1 (ICAM1) on endothelium. In order for macro phages to engulf and kill microorganisms, they need to be

activated by cytokines and also require nicotinamide adenine dinucleotide phosphate (NA DPH) oxidase to generate free radicals.

Primary phagocyte deciencies can occur as the result of leucocytes being unable to trav erse endothelium due to defects in LFA1,

because of mutations in cytokines or their receptors, or because of defects in NADP H oxidase. (IFNγ = interferon-gamma; IL =

interleukin)

Defects in cytokines and cytokine receptors

Mutations of the genes encoding cytokines such as IFNγ,

IL-12, IL-23 or their receptors result in failure of intracellular killing by macrophages, and affected individuals are particularly

susceptible to mycobacterial infections.

Complement pathway deciencies

Loss-of-function mutations have been identied in almost all the complement pathway p roteins (see Fig. 4.4). While most complement

deciencies are rare, mannose-binding lectin deciency is common and affects about 5% of the northern European population, many of

whom are asymptomatic (see below).

Clinical features

Patients with deciency in complement proteins can present in

different ways. In some cases, the presenting feature is recurrent infection with encaps ulated bacteria, particularly Neisser ia spp.,

reecting the importance of the membrane attack complex in defence against these o rganisms. However, genetic deciencies of the

classical complement pathway (C1, C2 and C4) also present with an increased risk of a utoimmune disease, particularly SLE (p. 1034).

Individuals with mannose-binding lectin deciency have an increased incidence of bact erial infections if subjected to an additional cause

of immune compromise, such as premature birth or chemotherapy. The signicance of this condition has been debated, however, since

population studies have shown no overall increase in infectious disease or mortality in patients with this disorder. Deciency of the

regulatory protein Cl inhibitor is not associated with recurrent infection but causes recur rent angioedema (p. 87).

Investigations

Screening for complement deciencies usually involves specialised functional tests of co mplement-mediated haemolysis. These are

known as the CH50 (classical haemolytic pathway 50) and AP50 (alternative pat hway 50) tests. If abnormal, haemolytic tests are

followed by measurement of individual complement components.

Management

Patients with complement deciencies should be vaccinated with meningococcal, pneumo coccal and H. inuenzae B vaccines to boost

their adaptive immune responses. Lifelong prophylactic penicillin to prevent meningococ cal infection is recommended, as is early access

to acute medical assessment in the event of infection. Patients should also carry a M edicAlert or similar. At-risk family members should

be screened for complement deciencies with functional complement assays. The manag ement of C1 esterase deciency is discussed

elsewhere.

Primary antibody deciencies

Primary antibody deciencies occur as the result of abnormalities in B-cell function, as s ummarised in Figure 4.11. They are

characterised by recurrent bacterial infections, particularly of the respiratory an d gastrointestinal tract. The most common causative

organisms are encapsulated bacteria such as Streptococcus pneumoniae and H. inuen zae. These disorders usually present in infancy,

when the protective benet of placental transfer of maternal immunoglobulin has w aned. The most important causes are discussed in

more detail below.

X-linked agammaglobulinaemia

This rare X-linked disorder ( p. 48) is caused by mutations in the BTK gene, which encodes Bruton ty rosine kinase, a signalling protein

that is required for B-cell development. Affected males present with severe bacterial infections d uring infancy. There is a marked

reduction in B-cell numbers and immunoglobulin levels are low or undetectable. Manag ement is with immunoglobulin replacement

therapy and antibiotics to treat infections.

Selective IgA deciency

This is the most common primary antibody deciency, affecting

1 : 600 northern Europeans. Although IgA deciency is usually asymptomatic with no clinical sequelae, about 30% of individuals

experience recurrent mild respiratory and gastrointestinal infections. The diagnosis can be conrmed by measurement of IgA levels,

which are low or undetectable (<0.05 g/L). In some

Failure of production of IgG antibodies: Common variable immune deficiency Specific antibody deficiency

IgG

Plasma cells

Immature

IgM-producing

B cells B cells

IgE

Failure of B-cell maturation: X-linked agammaglobulinaemia IgA Lymphoid progenitor s

Failure of lymphocyte precursors: Severe combined immune deficiency Failure of IgA production: Selective IgA deficiency

Stem cells

Bone marrow

Fig. 4.11 B lymphocytes and primary antibody deciencies (green boxes). (Ig = immunog lobulin)

4.15 Investigation of primary antibody deciencies

Selective IgA

deciency

Common variable immune deciency Specic antibody deciency

Circulating lymphocyte

Serum immunoglobulin (Ig) concentrations numbers IgM IgG IgA IgE B cells Norma l Often elevated Absent Normal Normal T cells

Normal

Normal or low Low Low or absent Low or absent Variable Variable Test immunisation

Not applicable* 4

No antibody response

Normal Normal Normal Normal Normal Normal No antibody response to polysacchar ide antigens

*Test immunisation is not usually performed in IgA deciency but some patients may ha ve impaired responses.

patients, there is a compensatory increase in serum IgG levels. Specic treatment is generall y not required.

Common variable immune deciency

Common variable immune deciency (CVID) is characterised by

low serum IgG levels and failure to make antibody responses to exogenous pathogens. It is a heterogeneous adult-onset primary immune

deciency of unknown cause. The presentation is with recurrent infections, and bronc hiectasis is a recognised complication.

Paradoxically, antibody-mediated autoimmune diseases, such as idiopathic thrombocytop enic purpura and autoimmune haemolytic

anaemia, are common in CVID. It is also associated with an increased risk of malignanc y, particularly lymphoproliferative disease.

Functional IgG antibody deciency

This is a poorly characterised condition resulting in defective

antibody responses to polysaccharide antigens. Some patients are also decient in the antibody subclasses IgG2 and IgG4, and this

condition was previously called IgG subclass deciency. There is overlap between spec ic antibody deciency, IgA deciency and

CVID, and some patients may progress to a more global antibody deciency over time.

Investigations

Serum immunoglobulins ( Box 4.15) should be measured in conjunction with p rotein and urine electrophoresis to exclude secondary

causes of hypogammaglobulinaemia, and B- and T-lymphocyte subsets should be meas ured. Specic antibody responses to known

pathogens should be assessed by measuring IgG antibodies against tetanus, H . inuenzae and S. pneumoniae (most patients will have

been exposed to these antigens through infection or immunisation). If specic antibody levels are low, immunisation with the appropriate

killed vaccine should be followed by repeat antibody measurement 6–8 weeks later; fai lure to mount a response indicates a signicant

defect in antibody production. These functional tests have generally superseded IgG su bclass quantitation.

Management

Patients with antibody deciencies generally require aggressive treatment of infect ions and prophylactic antibiotics may be indicated. An

exception is deciency of IgA, which usually does not require treatment. The mainstay of treatment in most patients with antibody

deciency is immunoglobulin replacement therapy. This is derived from plasma from hu ndreds of donors and contains IgG antibodies to

a wide variety of common organisms. Replacement immunoglobulin may be administere d either intravenously or subcutaneously, with

the aim of maintaining trough IgG levels (the IgG level just prior to an infusion) within the normal range. This has been shown to

minimise progression of end-organ damage and improve clinical outcome. Treatment m ay be self-administered and is life-long. Benets

of immunisation are limited because of the defect in IgG antibody production, and as w ith all primary immune deciencies, live vaccines

should be avoided.

Primary T-lymphocyte deciencies

These are a group of diseases characterised by recurrent viral, protozoal and fungal infe ctions (see Box 4.5). Many T-cell deciencies are

also associated with defective antibody production because of the importance of T cells in providing help for B cells. These disorders

generally present in childhood. Several causes of T-cell deciency are recognised . These are summarised in Figure 4.12 and discussed in

more detail below.

DiGeorge syndrome

This results from failure of development of the third and fourth pharyngeal pouches, and is usually caused by a deletion of chromosome

22q11. The immune deciency is accounted for by failure of thymic development; how ever, the immune deciency can be very

heterogeneous. Affected patients tend to have very low numbers of circulating T cells despite normal development in the bone marrow. It

is associated with multiple developmental anomalies, including congenital heart disease, h ypoparathyroidism, tracheo-oesophageal

stulae, cleft lip and palate.

Bare lymphocyte syndromes

These rare disorders are caused by mutations in a variety of genes that regulate express ion of HLA molecules or their transport to the cell

surface. If HLA class I molecules are affected, CD8

lymphocytes fail to develop normally, while absent expression of HLA class I I

molecules affects CD4

lymphocyte maturation. In addition to recurrent infections, failure to express HLA class I is associated with

systemic vasculitis caused by uncontrolled activation of NK cells.

Severe combined immune deciency

Severe combined immune deciency (SCID) results from mutations

in a number of genes that regulate lymphocyte development, with failure of T-ce ll maturation, with or without accompanying B- and NK-

cell maturation. The most common cause is X-linked SCID, resulting from loss-of-func tion mutations in the interleukin-2 receptor

gamma (IL2RG) gene. The gene product is a component of several interleukin recep tors, including those for IL-2, IL-7 and IL-15, which

are absolutely required for T-cell and NK development. This results in T-cell-negative, NK-cell-negative, Failure of thymic development:

DiGeorge syndrome Failure of expression of HLA molecules: Bare lymphocyte syndro mes

Lymphoid progenitors

Failure of lymphocyte

precursors:

Severe combined

immune deficiency

Proliferation and

maturation of thymocytes Thymus

Export of mature T lymphocytes to periphery

Failure of apoptosis:

Stem

Autoimmune lymphoproliferative

cells

syndromes

Bone

marrow Failure of cytokine production: Cytokine deficiencies

Apoptotic cell death T-lymphocyte activation and effector function

Fig. 4.12 T-lymphocyte function and dysfunction (green boxes). (HLA = human leucocyt e antigen)

B-cell-positive SCID. Another cause is deciency of the enzyme adenosine deaminase (ADA ), which causes lymphocyte death due to

accumulation of toxic purine metabolites intracellularly, resulting in T-cell-negative, B-ce ll-negative and NK-cell-negative SCID.

The absence of an effective adaptive immune response causes recurrent bacterial, fung al and viral infections soon after birth. Bone

marrow transplantation (BMT; p. 936) is the treatment option of first choice. Gene therapy has been approved for treatment of ADA

deciency when there is no suitable donor for BMT and is under investigation for a nu mber of other causes of SCID.

Investigations

The principal tests for T-lymphocyte deciencies are a total lymphocyte count and quan titation of individual lymphocyte subpopulations.

Serum immunoglobulins should also be measured. Second-line, functional tests of T-cel l activation and proliferation may be indicated.

Patients in whom T-lymphocyte deciencies are suspected should be tested for HIV infec tion (p. 310).

Management

Patients with T-cell deciencies should be considered for antiPneumocystis and antifungal pr ophylaxis, and require aggressive

management of infections when they occur. Immunoglobulin replacement is indicated f or associated defective antibody production.

Stem cell transplantation (p. 936) or gene therapy may be appropriate in some disorders. Where a family history is known and antenatal

testing conrms a specic defect, stem cell therapy prior to recurrent invasive infection c an improve outcome.

Autoimmune lymphoproliferative syndrome

This rare disorder is caused by failure of normal lymphocyte apoptosis, most commonly due to mutations in the FAS gene, which

encodes Fas, a signalling protein that regulates programmed cell death in lymphocytes. T his results in massive accumulation of

autoreactive T cells, which cause autoimmune-mediated anaemia, thrombocytopenia and neutropenia. Other features include

lymphadenopathy, splenomegaly and a variety of other autoimmune diseases. Susceptibility to infection is increased because of the

neutropenia.

Secondary immune deciencies

Secondary immune deciencies are much more common than primary immune de ciencies and occur when the immune system is

compromised by external factors (Box 4.16). Common causes include infections, such as HIV and measles, and cytotoxic

4.16 Causes of secondary immune deciency

Physiological

• Ageing

• Prematurity

Infection

• HIV infection

• Measles

Iatrogenic

• Immunosuppressive therapy

• Antineoplastic agents

• Glucocorticoids

Malignancy

• B-cell malignancies including

leukaemia, lymphoma and myeloma

• Pregnancy

• Mycobacterial infection

• Stem cell transplantation

• Radiation injury

• Antiepileptic agents

• Solid tumours

• Thymoma

Biochemical and nutritional disorders

• Malnutrition

• Renal insufciency/dialysis

• Diabetes mellitus

Other conditions

• Burns

• Specic mineral deciencies (iron, zinc)

• Asplenia/hyposplenism

4.17 Immune senescence

• T-cell responses: decline, with reduced delayed-type hypersensitivity responses.

• Antibody production: decreased for many exogenous antigens. Although auto antibodies are frequently detected, autoimmune disease is

less common.

• Response to vaccination: reduced; 30% of healthy older people may not develo p protective immunity after inuenza vaccination.

• Allergic disorders and transplant rejection: less common.

• Susceptibility to infection: increased; community-acquired pneumonia b y threefold and urinary tract infection by 20-fold. Latent

infections, including tuberculosis and herpes zoster, may be reactivated.

• Manifestations of inammation: may be absent, with lack of pyrexia or leuc ocytosis.

• Secondary immune deciency: common.

and immunosuppressive drugs, particularly those used in the management of transplantation, autoimmunity and cancer. Physiological

immune deciency occurs at the extremes of life; the decline of the immune response in the elderly is known as immune senescence

(Box 4.17). Management of secondary immune deciency is described in the relevant chapters on infectious diseases (Ch. 11), HIV (Ch.

12), haematological disorders (Ch. 23) and oncology (Ch. 33).

Periodic fever syndromes

These rare disorders are characterised by recurrent episodes of fever and organ inammati on, associated with an elevated acute phase

response (p. 74).

Familial Mediterranean fever

Familial Mediterranean fever (FMF) is the most common of the familial periodic fevers, pr edominantly affecting Mediterranean people,

including Arabs, Turks, Sephardic Jews and Armenians. It results from mutations of th e MEFV gene, which encodes a protein called

pyrin that regulates neutrophil-mediated inammation by indirectly suppressing the production o f IL-1. FMF is characterised by

recurrent painful attacks of fever associated with peritonitis, pleuritis and arthritis, which last for a few hours to 4 days and are associated

with markedly increased CRP levels. Symptoms resolve completely between episodes. Mo st individuals have their rst attack before the

age of 20. The major complication of FMF is AA amyloidosis (see below). Colchicine sign icantly reduces the number of febrile

episodes in 90% of patients but is ineffective during acute attacks.

Mevalonic aciduria (mevalonate

kinase deciency)

Mevalonate kinase deciency, previously known as hyper-IgD syndrome, is an autoso mal recessive disorder that causes recurrent attacks

of fever, abdominal pain, diarrhoea, lymphadenopathy, arthralgia, skin lesions and aphtho us ulceration. Most patients are from Western

Europe, particularly the Netherlands and northern France. It is caused by loss-of-func tion mutations in the gene encoding mevalonate

kinase, which is involved in the metabolism of cholesterol. It remains unclear w hy this causes an inammatory periodic fever. Serum

IgD and IgA levels may be persistently elevated, and CRP levels are increased dur ing acute attacks. Standard anti-inammatory drugs,

including colchicine and glucocorticoids, are ineffective in suppressing the attacks but IL-1 inhibit ors, such as anakinra, and TNF

inhibitors, such as etanercept, may improve symptoms and can induce complete r emission in some patients.

TNF receptor-associated periodic syndrome

TNF receptor-associated periodic syndrome (TRAPS) also known as Hibernian fever, is an autosomal dominant syndrome caused by

mutations in the TNFRSF1A gene. The presentation is with recurrent attacks of fever, arthralgia, myalgia, serositis and rashes. Attacks

may be prolonged for 1 week or more. During a typical attack, laboratory ndings inc lude neutrophilia, increased CRP and elevated IgA

levels. The diagnosis can be conrmed by low serum levels of the soluble type 1 TNF receptor and by mutation screening of the

TNFRSF1A gene. As in FMF, the major complication is amyloidosis, and regular screening for proteinuria is advised. Acute episodes

respond to systemic glucocorticoids. Therapy with IL-1 inhibitors, such as anakinra, ca n be effective in preventing attacks.

Amyloidosis

Amyloidosis is the name given to a group of acquired and hereditary disorders charact erised by the extracellular deposition of insoluble

proteins.

Pathophysiology

Amyloidosis is caused by deposits consisting of fibrils of the specic protein involved, lin ked to glycosaminoglycans, proteoglycans and

serum amyloid P. Protein accumulation may be localised or systemic, and the clinical manifest ations depend on the organ(s) affected.

Amyloid diseases are classied by the aetiology and type of protein deposited (Box 4.18 ). Clinical features

The clinical presentation may be with nephrotic syndrome (p. 395), cardiomyopathy (p. 538) or peripheral neuropat hy (p. 1138).

Amyloidosis should always be considered as a potential diagnosis in patients with these d isorders when the cause is unclear.

Investigations

The diagnosis is established by biopsy, which may be of an affected organ, rectum or s ubcutaneous fat. The pathognomonic histological

feature is apple-green birefringence of amyloid deposits when stained with Congo red dye and viewed under polarised light.

Immunohistochemical staining can identify the type of amyloid bril present. Quantitative scintigraphy with radiolabelled serum amyloid

P is a valuable tool in determining the overall load and distribution of amyloid deposits.

Management

The aims of treatment are to support the function of affected organs and, in acquired amyloid osis, to prevent further amyloid deposition

through treatment of the primary cause. When the latter is possible, regression of existin g amyloid deposits may occur.

Autoimmune disease

Autoimmunity can be dened as the presence of immune responses against self-tissue. T his may be a harmless phenomenon, identied

4.18 Causes of amyloidosis

Disorder Pathological basis Acquired systemic amyloidosis

Reactive (AA) amyloidosis (p. 81)

Increased production of serum amyloid A as part of prolonged or recurrent acute inammato ry response

Light chain amyloidosis (AL) Increased production of monoclonal

light chain

Chronic infection (tuberculosis, bronchiectasis, chronic abscess, osteomyelitis)

Chronic inammatory diseases (untreated rheumatoid arthritis, familial Mediterranean fever) Mo noclonal gammopathies, including

myeloma, benign gammopathies and plasmacytoma

Dialysis-associated (A

β2

M) amyloidosis

Accumulation of circulating

-microglobulin due to failure of renal catabolism in kidney failure

Renal dialysis

Senile systemic amyloidosis Normal transthyretin protein deposited in tissues

Age

70 years

90% of patients present with

non-selective proteinuria or nephrotic syndrome

Restrictive cardiomyopathy, peripheral and autonomic neuropathy, carpal tunn el syndrome, proteinuria,

spontaneous purpura, amyloid nodules and plaques

Macroglossia occurs rarely but is pathognomonic

Prognosis is poor

Carpal tunnel syndrome, chronic arthropathy and pathological fractures se condary to amyloid bone cyst formation

Manifestations occur 5–10 years after the start of dialysis

Feature of normal ageing (affects >90% of 90-year-olds)

Usually asymptomatic

Hereditary systemic amyloidosis

>20 forms of hereditary Production of protein with an abnormal systemic amyloidosis struc ture that predisposes to amyloid

fibril formation. Most commonly due to mutations in transthyretin gene

only by the presence of low-titre autoantibodies or autoreactive T cells. However, if th ese responses cause signicant organ damage,

autoimmune diseases occur. These are a major cause of chronic morbidity and disability, affectin g up to 1 in 30 adults at some point

during life.

Pathophysiology

Autoimmune diseases result from the failure of immune tolerance, the process by which the immune system recognises and accepts self-

tissue. Central immune tolerance occurs during lymphocyte development, when T and B lymphocytes that recognise self-antigens are

eliminated before they develop into fully immunocompetent cells. This process is most active in f etal life but continues throughout life as

immature lymphocytes are generated. Some autoreactive cells inevitably evad e deletion and escape into the circulation, however, and are

controlled through peripheral tolerance mechanisms. Peripheral immune toleran ce mechanisms include the suppression of autoreactive

cells by regulatory T cells; the generation of functional hyporesponsiveness (anergy) in lymp hocytes that encounter antigen in the

absence of the co-stimulatory signals that accompany inammation; and cell death by ap optosis. Autoimmune diseases develop when

selfreactive lymphocytes escape from these tolerance mechanisms.

Multiple genetic and environmental factors contribute to the development of autoimmun e disease. Autoimmune diseases are much more

common in women than in men, for reasons that remain unclear. Many are as sociated with genetic variations in the HLA loci, reecting

the importance of HLA genes in shaping lymphocyte responses. Other important susceptibili ty genes include Predisposing conditions

Autosomal dominant inheritance Other features

Peripheral and autonomic neuropathy, cardiomyopathy

Renal involvement unusual

10% of gene carriers are

asymptomatic throughout life

4.19 Association of specic gene polymorphisms with autoimmune diseases Gene

HLA

complex

PTPN22

Function

Key determinants of antigen presentation to T cells

Regulation of T- and B-cell receptor signalling

CTLA4

IL23R

Important co-stimulatory molecule that transmits inhibitory signals to T cells

Cytokine-mediated

control of T cells

TNFRSF1A

ATG5

Control of tumour

necrosis factor network

Autophagy

Diseases

Most autoimmune diseases

Rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus

Rheumatoid arthritis, type 1 diabetes

Inammatory bowel disease, psoriasis, ankylosing spondylitis

Multiple sclerosis

Systemic lupus erythematosus

those determining cytokine activity, co-stimulation (the expression of second signals req uired for full T-cell activation; see Fig. 4.7) and

cell death. Many of the same gene variants underlie multiple autoimmune disorders, re ecting their common pathogenesis (Box 4.19).

Even though some of these associations are the strongest that have been identied in com plex genetic diseases, they have very limited

predictive value and are generally not useful in determining management of individual p atients. Several environmental factors may be

associated with autoimmunity in genetically predisposed individuals, including infection, cigare tte smoking and hormone levels. The

most widely studied of these is infection, as occurs in acute rheumatic fever following st reptococcal infection or reactive arthritis

following bacterial infection. Several mechanisms have been invoked to explain the aut oimmunity that occurs after an infectious trigger.

These include crossreactivity between proteins expressed by the pathogen and the host ( molecular mimicry), such as Guillain–Barré

syndrome and Campylobacter infection (p. 1140); release of sequestered antigens fr om tissues that are damaged during infections that

are not usually visible to the immune system; and production of inammatory cytokin es that overwhelm the normal control mechanisms

that prevent bystander damage. Occasionally, autoimmune disease may be an adverse e ffect of drug treatment. For example, metabolic

products of the anaesthetic agent halothane can bind to liver enzymes, resulting in a struct urally novel protein that is recognised as a

foreign antigen by the immune system. This can provoke the development of au toantibodies and activated T cells, which can cause

hepatic necrosis.

Clinical features

The clinical presentation of autoimmune disease is highly variable. Autoimmune diseases can be classied by organ involvement or by

the predominant mechanism responsible for tissue damage. The Gell and Coombs c lassication of hypersensitivity is the most widely

used, and distinguishes four types of immune response that result in tissue damage (Box 4.20 ).

• Type I hypersensitivity is relevant in allergy but is not

associated with autoimmune disease.

• Type II hypersensitivity causes injury to a single tissue or

organ and is mediated by specic autoantibodies.

• Type III hypersensitivity results from deposition of immune

complexes, which initiates activation of the classical

complement cascade, as well as recruitment and

activation of phagocytes and CD4

lymphocytes. The site

of immune complex deposition is determined by the

relative amount of antibody, size of the immune

complexes, nature of the antigen and local

haemodynamics. Generalised deposition of immune

complexes gives rise to systemic diseases such as SLE.

• Type IV hypersensitivity is mediated by activated T cells

and macrophages, which together cause tissue damage.

Investigations

Autoantibodies

Many autoantibodies have been identied and are used in the

diagnosis and monitoring of autoimmune diseases, as discussed elsewhere in this book. Antibodies can be quantied either by titre (the

maximum dilution of the serum at which the antibody 4

can be detected) or by concentration in standardised units using

an enzyme-linked immunosorbent assay (ELISA) in which the antigen is used to coat micr otitre plates to which the patient’s serum is

added (Fig. 4.13A). Qualitative tests are also employed for antinuclear antibo dies in which the pattern of nuclear staining is recorded

(Fig. 4.13B).

Antibodies Detection of

bind to target bound antibody Quantitate on plate reader

Wash Wash

Target antigen Target antigen

Nucleolar

Homogenous

Speckled

Fig. 4.13 Autoantibody testing. A Measurement of antibody levels by

enzyme-linked immunosorbent assay (ELISA). The antigen of interest is used to coat m icrotitre plates to which patient serum is added. If

autoantibodies are present, these bind to the target antigen on the microtitre plate. The amou nt of bound antibody is quantitated by

adding a secondary antibody linked to an enzyme that converts a colourless subs trate to a coloured one, which can be detected by a plate

reader. B Qualitative analysis of autoantibodies by patterns of nuclear staining. In this assay, patient serum is added to cultured cells and

a secondary antibody is added with a uorescent label to detect any bound antibody. If an tinuclear antibodies are present, they are

detected as bright green staining. Different antinuclear antibody patterns may be seen in diffe rent types of connective tissue disease (Ch.

24). B (Nucleolar and Homogenous), Courtesy of Juliet Dunphy, Biomedical Scientist, Royal United Hospital Bath, previously of Bath

Institute of Rheumatic Diseases, UK; (Speckled), Courtesy of Mr Richard Brown, Cl inical Scientist in Immunology, Southwest

Pathology Services, UK.

4.20 Gell and Coombs classication of hypersensitivity diseases

Type

Type I

Immediate hypersensitivity Type II

Antibody-mediated

Type III

Immune complex-mediated

Type IV

Delayed type Mechanism

IgE-mediated mast cell degranulation Example of disease in

response to exogenous agent

Allergic disease

Example of autoimmune disease None described

Binding of cytotoxic IgG or IgM antibodies to antigens on cell surface causes cell killing

IgG or IgM antibodies bind soluble antigen to form immune complexes that trigger classi cal complement pathway activation

Activated T cells, and phagocytes

ABO blood transfusion reaction Hyperacute transplant rejection

Serum sickness Farmer’s lung Autoimmune haemolytic anaemia Idiopathic thrombocyto penic purpura Goodpasture’s disease

Systemic lupus erythematosus Cryoglobulinaemia

Acute cellular transplant rejection Nickel hypersensitivity

Type 1 diabetes

Hashimoto’s thyroiditis

4.21 Classication of cryoglobulins

Immunoglobulin (Ig) isotype and specicity

Prevalence

Disease association Type I

Isolated monoclonal IgM paraprotein with no particular specicity

25%

Lymphoproliferative disease, especially Waldenström macroglobulinaemia (p. 966) Type I I

Immune complexes formed by monoclonal IgM paraprotein directed towards constant re gion of IgG

25%

Infection, particularly hepatitis C;

lymphoproliferative disease

Symptoms Hyperviscosity:

Raynaud’s phenomenon

Acrocyanosis

Retinal vessel occlusion

Arterial and venous thrombosis

Protein electrophoresis Rheumatoid factor Complement

Serum viscosity

Monoclonal IgM paraprotein Negative

Usually normal

Raised

Small-vessel vasculitis:

Purpuric rash

Arthralgia

Neuropathy

Cutaneous ulceration, hepatosplenomegaly, glomerulonephritis, Raynaud’s phenomeno n

Monoclonal IgM paraprotein

Strongly positive

Decreased C4

Normal Type III

Immune complexes formed by

polyclonal IgM or IgG directed towards constant region of IgG

50%

Infection, particularly hepatitis C; autoimmune disease, including

rheumatoid arthritis and systemic lupus erythematosus

Small-vessel vasculitis:

Purpuric rash, arthralgia

Cutaneous ulceration

Hepatosplenomegaly,

glomerulonephritis

Raynaud’s phenomenon

No monoclonal paraprotein

Strongly positive

Decreased C4

Normal

Complement Measurement of complement components can be useful in the

evaluation of immune complex-mediated diseases. Classical complement pathway activati on leads to a decrease in circulating C4 levels

and is often also associated with decreased C3 levels. Serial measurement of C3 and C4 is a usef ul surrogate measure of disease activity

in conditions such as SLE.

Cryoglobulins

Cryoglobulins are antibodies directed against other

immunoglobulins, forming immune complexes that precipitate in the cold. They can lead to type III hypersensitivity reactions, with

typical clinical manifestations including purpuric rash, often of the lower extremities, arthralgia and peripheral neuropathy.

Cryoglobulins are classied into three types, depending on the properties of the immunoglob ulin involved (Box 4.21). Testing for

cryoglobulins requires the transport of a serum specimen to the laboratory at 37°C. Cryog lobulins should not be confused with cold

agglutinins; the latter are autoantibodies specically directed against the I/i antigen on the surface o f red cells, which can cause

intravascular haemolysis in the cold (p. 950).

Management

The management of autoimmune disease depends on the organ system involved and fu rther details are provided elsewhere in this book.

In general, treatment of autoimmune diseases involves the use of glucocorticoids and im munosuppressive agents, which are increasingly

used in combination with biologic agents targeting disease-specic cytokines and their re ceptors. Not all conditions require immune

suppression, however. For example, the management of coeliac disease involves di etary gluten withdrawal, while autoimmune

hypothyroidism requires appropriate thyroxine supplementation.

Allergy

Allergic diseases are a common and increasing cause of illness, affecting between 1 5% and 20% of the population at some time. They

comprise a range of disorders from mild to life-threatening and affect many o rgans. Atopy is the tendency to produce an exaggerated IgE

immune response to otherwise harmless environmental substances, while an allergic disease can be dened as the clinical manifestation

of this inappropriate IgE immune response.

Pathophysiology

The immune system does not normally respond to the many environmental substances to whic h it is exposed on a daily basis. In allergic

individuals, however, an initial exposure to a normally harmless exogenous s ubstance (known as an allergen) triggers the production of

specic IgE antibodies by activated B cells. These bind to high-afnity IgE receptors on the su rface of mast cells, a step that is not itself

associated with clinical sequelae. However, re-exposure to the allergen binds to and cross-l inks membrane-bound IgE, which activates

the mast cells, releasing a variety of vasoactive mediators (the early phase respons e; Fig. 4.14 and see Box 4.9). This type I

hypersensitivity reaction forms the basis of an allergic reaction, which can rang e from sneezing and rhinorrhoea to anaphylaxis (Box

4.22). In some individuals, the early phase response is followed by persistent activation o f mast cells, manifest by ongoing swelling and

local inammation. This is known as the late phase reaction and is mediated by mast cell metabolites, basophils, eosinophils and

macrophages. Long-standing or recurrent allergic inammation may give rise to a ch ronic inammatory response characterised by a

complex infiltrate of macrophages, eosinophils and T lymphocytes, in addition to mast c ells and basophils. Once this has been

established, inhibition of mast cell mediators with antihistamines is clinically ineffective in i solation. Mast cell activation may also be

non-specically triggered through other signals, such as neuropeptides, anaphylotoxins and bacte rial peptides.

The increasing incidence of allergic diseases is largely unexplained but one widely held theory is the ‘hygiene hypothesis’. This proposes

that infections in early life are critically important in maturation of the immune response and bias the immune system against the

development of allergies; the high prevalence

Allergy • 85

A B C Allergen

Mast cell

B BB B

B B B

T and B cells IgE antibody

IgE receptor

Histamine, tryptase and vasoactive peptides

Fig. 4.14 Type I (immediate) hypersensitivity response. A After an encounter with allergen , B cells produce immunoglobulin E (IgE)

antibody against the allergen. B Specic IgE antibodies bind to circulating mast cells via high-afnity IgE cell surface receptors. C On

re-encounter with allergen, the allergen binds to the IgE antibody-coated mast cel ls. This cross-linking of the IgE triggers mast cell

activation with release of vasoactive mediators (see Box 4.9).

4.22 Clinical manifestations of allergy Dermatological

• Urticaria

• Atopic eczema if chronic

Respiratory

• Asthma

Ophthalmological

• Allergic conjunctivitis Gastrointestinal

• Food allergy

Other

• Anaphylaxis

• Drug allergy

• Allergic contact eczema

• Angioedema

• Atopic rhinitis

• Allergy to insect venom

of allergic disease is the penalty for the decreased exposure to infection that has resulted from improvements in sanitation and health

care. Genetic factors also contribute strongly to the development of allergic disease s. A positive family history is common in patients

with allergy, and genetic association studies have identied a wide variety of predisposin g variants in genes controlling innate immune

responses, cytokine production, IgE levels and the ability of the epithelial barrie r to protect against environmental agents. The expression

of a genetic predisposition is complex; it is governed by environmental factors, such as pollutants and cigarette smoke, and the incidence

of bacterial and viral infection.

Clinical features

Common presentations of allergic disease are shown in Box 4.22. Those that affe ct the respiratory system and skin are discussed in more

detail in Chapters 17 and 29, respectively. Here we focus on general principles of the app roach to the allergic patient, some specic

allergies and anaphylaxis.

Insect venom allergy

Local non-IgE-mediated reactions to insect stings are common

and may cause extensive swelling around the site lasting up to 7 days. These usually d o not require specic treatment. Toxic reactions to

venom after multiple (50–100) simultaneous stings may mimic anaphylaxis. In addition, exposure to large amounts of insect venom

frequently stimulates the production of IgE antibodies, and thus may be followed by alle rgic reactions to single stings. Allergic IgE-

mediated reactions vary from mild to life-threatening. Antigen-specic immuno therapy (desensitisation; see below) with bee or wasp

venom can reduce the incidence of recurrent anaphylaxis from 50–60% to approximatel y 10% but requires up to 5 years of treatment.

Peanut allergy

Peanut allergy is the most common food-related allergy. More than 50% of patients present be fore the age of 3 years and some

individuals react to their rst known exposure to peanuts, thought to result from sensitisation to arachis oil in topical creams. Peanuts are

ubiquitous in the Western diet, and every year up to 25% of peanut-allergic individu als experience a reaction as a result of inadvertent

exposure.

Birch oral allergy syndrome

This syndrome is characterised by the combination of birch pollen

hay fever and local oral symptoms, including itch and angioedema, after contact with ce rtain raw fruits, raw vegetables and nuts. Cooked

fruits and vegetables are tolerated without difculty. It is due to shared or cross-reactive allergens that are destroyed by cooking or

digestion, and can be conrmed by skin prick testing using fresh fruit. Severe allergic reactions are unusual.

Diagnosis

When assessing a patient with a complaint of allergy, it is important

to identify what the patient means by the term, as up to 20% of the UK population descr ibe themselves as having a food allergy; in fact,

less than 1% have true allergy, as dened by an IgE-mediated hypersensitivity rea ction conrmed on double-blind challenge. The nature

of the symptoms should be established and specic triggers identied, along with the predictabilit y of a reaction, and the time lag

between exposure to a potential allergen and onset of symptoms. An allergic reaction us ually occurs within minutes of exposure and

provokes predictable, reproducible symptoms such as angioedema, urticaria and wheez ing. Specic enquiry should be made about other

allergic symptoms, past and present, and about a family history of allergic disease. Poten tial allergens in the home and workplace should

be identied. A detailed drug history should always be taken, including details of adherence t o medication, possible adverse effects and

the use of over-the-counter or complementary therapies.

Investigations

Skin-prick tests

Skin-prick testing is a key investigation in the assessment

of patients suspected of having allergy. A droplet of diluted standardised allergen is plac ed on the forearm and the skin is supercially

punctured through the droplet with a sterile lancet. Positive and negative control material must be included in the assessment. After 15

minutes, a positive response is indicated by a local weal and are response 2 m m or more larger than the negative control. A major

advantage of skin-prick testing is that patient can clearly see the results, which may be useful in gaining adherence to avoidance

measures. Disadvantages include the remote risk of a severe allergic reaction, so resusc itation facilities should be available. Results are

unreliable in patients with extensive skin disease. Antihistamines inhibit the magnitude of th e response and should be discontinued for at

least 3 days before testing; low-dose glucocorticoids do not influence test result s. A number of other prescribed medicines can also lead

to false-negative results, including amitriptyline and risperidone.

Specic IgE tests

An alternative to skin-prick testing is the quantitation of IgE directed

against the suspected allergen. The sensitivity and specicity of specic IgE tests (previously known as radioallergosorbent tests, RAST)

are lower than those of skin-prick tests. However, IgE tests may be very useful if skin testing is in appropriate, such as in patients taking

antihistamines or those with severe skin disease or dermatographism. They can also be used to test for cross-reactivity – for example,

with multiple insect venoms, where component-resolved diagnostics, using recombinant allergens, is increasingly used rather than crude

allergen extract. Specic IgE tests can also be used post-mortem to identify allergens responsible for lethal a naphylaxis.

Supervised exposure to allergen

Tests involving supervised exposure to an allergen (allergen challenge) are usually per formed in specialist centres on carefully selected

patients, and include bronchial provocation testing, nasal challenge, and food or drug c hallenge. These may be particularly useful in the

investigation of occupational asthma or food allergy. Patients can be considered for challeng e testing when skin tests and/or IgE tests are

negative, as they can be helpful in ruling out allergic disease.

Mast cell tryptase

Measurement of serum mast cell tryptase is extremely useful in investigating a possible anaphyla ctic event. Ideally, measurements

should be made at the time of the reaction following appropriate resuscitation, and 3 ho urs and 24 hours later. The basis of the test is the

fact that circulating levels of mast cell degranulation products rise dramatically to peak 1–2 hours after a systemic allergic reaction.

Tryptase is the most stable of these and is easily measured in serum.

Serum total IgE

Serum total IgE measurements are not routinely indicated in the investigation of allergic d isease, other than to aid in the interpretation of

specic IgE results, as false-positive specic IgEs are common in patients with atopy, wh o often have a high total IgE level. Although

atopy is the most common cause of an elevated total IgE in developed countries, t here are many other causes, including parasitic and

helminth infections (pp. 299 and 288), lymphoma (p. 961), drug reactions and eosinophilic granulomatosis with po lyangiitis (previously

known as Churg–Strauss vasculitis; p. 1043). Normal total IgE levels do not exclude allergic disease.

Eosinophilia

Peripheral blood eosinophilia is common in atopic individuals but

lacks specicity. Eosinophilia of more than 20% or an absolute eosinophil count over 1.5

/L should initiate a search for a non-atopic

cause, such as eosinophilic granulomatosis with polyangiitis or parasitic infection ( p. 928).

Management

Several approaches can be deployed in the management of allergic individuals, as discu ssed below.

Avoidance of the allergen

This is indicated in all cases and should be rigorously attempted, with the advice of speci alist dietitians and occupational physicians if

necessary.

Antihistamines

Antihistamines are useful in the management of allergy as they

inhibit the effects of histamine on tissue H

receptors. Long-acting, non-sedating preparations are particularly useful for prophyla xis.

Glucocorticoids

These are highly effective in allergic disease, and if used topically, adverse effects can b e minimised.

Sodium cromoglicate

Sodium cromoglicate stabilises the mast cell membrane, inhibiting

release of vasoactive mediators. It is effective as a prophylactic agent in asthma and a llergic rhinitis but has no role in management of

acute attacks. It is poorly absorbed and therefore ineffective in the management of food a llergies.

Antigen-specic immunotherapy

This involves the sequential administration of increasing doses of allergen extract over a prolonged period of time. The mechanism of

action is not fully understood but it is highly effective in the prevention of insect venom ana phylaxis and of allergic rhinitis secondary to

grass pollen. The traditional route of administration is by subcutaneous injection, which carries a risk of anaphylaxis and should be

performed only in specialised centres. Sublingual immunotherapy is also increasingly used. Clinical studies to date do not support the use

of allergen immunotherapy for food hypersensitivity, although this is an area of active i nvestigation.

Omalizumab

Omalizumab is a monoclonal antibody directed against IgE;

it inhibits the binding of IgE to mast cells and basophils. It is licensed for treatm ent of refractory chronic spontaneous urticaria and also

for severe persistent allergic asthma that has failed to respond to standard therapy ( p. 572). The dose and frequency are determined by

baseline IgE (measured before the start of treatment) and body weight. It is under investigatio n for allergic rhinitis but not yet approved

for this indication.

Adrenaline (epinephrine)

Adrenaline given by injection in the form of a pre-loaded self

injectable device can be life-saving in the acute management of anaphylaxis (see Box 4.12 ).

Angioedema • 87

Angioedema

Angioedema is an episodic, localised, non-pitting swelling of submucous or subcutaneo us tissues.

Pathophysiology

The causes of angioedema are summarised in Box 4.23. It may

be a manifestation of allergy or non-allergic degranulation of mast cells in response to d rugs and toxins. In these conditions the main

cause is mast cell degranulation with release of histamine and other vasoactive m ediators. In hereditary angioedema, the cause is C1

inhibitor deciency, which causes increased local release of bradykinin. Angiotensin-co nverting enzyme (ACE) inhibitor-induced

angioedema also occurs as the result of increased bradykinin levels due to inhibition of its breakdown.

Clinical features

Angioedema is characterised by soft-tissue swelling that most frequently affects the face (Fig. 4.15) but can also affect the extremities

and genitalia. Involvement of the larynx or tongue may cause life-threatening respiratory tract obstruction, and oedema of the intestinal

mucosa may cause abdominal pain and distension.

Investigations

Differentiating the mechanism of angioedema is important in

determining the most appropriate treatment. A clinical history of allergy or drug exposur e can give clues to the underlying diagnosis. If

no obvious trigger can be identied, measurement of complement C4 is useful in differentia ting hereditary and acquired angioedema

from other causes. If C4 levels are low, further investigations should be initiated to look for evidence of C1 inhibitor deciency.

Management 4

Management depends on the underlying cause. Angioedema associated with allergen exposure generally responds to

antihistamines and glucocorticoids. Following acute management of angioedema second ary to drug therapy, drug withdrawal

Fig. 4.15 Angioedema. This young man has hereditary angioedema. A Normal ap pearance. B During an acute attack. From Helbert M.

Flesh and bones of immunology. Edinburgh: Churchill Livingstone, Elsevier Ltd; 2006.

4.23 Types of angioedema

Pathogenesis Allergic reaction to specic trigger

IgE-mediated degradation of mast cells

Idiopathic angioedema Non-IgE-mediated degranulation of mast cells

Key mediator Prevalence Histamine Common Histamine Common

Clinical features Usually associated with urticaria

History of other allergies common

Follows exposure to specic allergen, in food, animal dander or insect v enom

Investigations Specic IgE tests or skin-prick tests

Treatment

Associated

drug reactions Allergen avoidance Antihistamines

Specic drug allergies Usually associated with urticaria May be triggered by physical stimuli such as heat, pressure or exercise

Dermatographism common Occasionally associated with underlying infection or thyroid disease

Specic IgE tests and skin-prick tests often negative

Hypothyroidism should be excluded

Antihistamines are mainstay of treatment and prophylaxis

NSAIDs

Opioids, radiocontrast media Hereditary angioedema C1 inhibitor deciency, with resulting increased local bradykinin concentration

Bradykinin

Rare autosomal dominant disorder

Not associated with urticaria or other features of allergy Does not cause anaphylaxis May caus e life-threatening respiratory tract

obstruction Can cause severe

abdominal pain

Complement C4 (invariably low in acute attacks)

C1 inhibitor levels

ACE-inhibitor associated angioedema

Inhibition of breakdown of bradykinin

Bradykinin

0.1–0.2% of patients treated with ACE inhibitors

Not associated with urticaria Does not cause anaphylaxis Usually affects the head and neck, and may cause

life-threatening respiratory tract obstruction

Can occur years after the start of treatment

No specic investigations

Unresponsive to

antihistamines

Anabolic steroids

C1 inhibitor concentrate or icatibant for acute attacks ACE inhibitor should be discontinued

ARBs should be avoided if possible unless there is a strong indication

ACE inhibitors, ARBs

(ACE = angiotensin-converting enzyme; ARBs = angiotensin II receptor blockers; NSA IDs = non-steroidal anti-inammatory drugs)

should prevent further attacks, although ACE inhibitor-induced angioedema can continue for a li mited period post drug withdrawal.

Management of angioedema associated with C1 inhibitor deciency is discussed below.

Hereditary angioedema

Hereditary angioedema (HAE), also known as inherited C1 inhibitor deciency, is an a utosomal dominant disorder caused by decreased

production or activity of C1 inhibitor protein. This complement regulatory protein inhibits spont aneous activation of the classical

complement pathway (see Fig. 4.4). It also acts as an inhibitor of the kinin cascade, activation of which increases local bradykinin levels,

giving rise to local pain and swelling.

Clinical features

The angioedema in HAE may be spontaneous or triggered by local trauma or infection. M ultiple parts of the body may be involved,

especially the face, extremities, upper airway and gastrointestinal tract. Oedema of the in testinal wall causes severe abdominal pain and

many patients with undiagnosed HAE undergo exploratory laparotomy. The most impo rtant complication is laryngeal obstruction, often

associated with minor dental procedures, which can be fatal. Episodes of angioedema a re self-limiting and usually resolve within 48

hours. Patients with HAE generally present in adolescence but may go undiagnosed for many years. A family history can be identied in

80% of cases. HAE is not associated with allergic diseases and is specically not associated wit h urticaria.

Investigations

Acute episodes are accompanied by low C4 levels; a low C4 during an episode of angioede ma should therefore trigger further

investigation. The diagnosis can be conrmed by measurement of C1 inhibitor levels and fun ction.

Management

Severe acute attacks should be treated with puried C1 inhibitor concentrate or the brad ykinin receptor antagonist icatibant. Anabolic

steroids, such as danazol, can be used to prevent attacks and act by increasing endogen ous production of complement proteins.

Tranexamic acid can be helpful as prophylaxis in some patients. Patients can be taught to self-administer therapy and should be advised

to carry a MedicAlert or similar.

Acquired C1 inhibitor deciency

This rare disorder is clinically indistinguishable from HAE but presents in late adulthood . It is associated with autoimmune and

lymphoproliferative diseases. Most cases are due to the

4.24 Immunological diseases in pregnancy

Allergic disease

• Maternal dietary restrictions during pregnancy or lactation: current evidence does n ot support these for prevention of allergic disease.

• Breastfeeding for at least 4 months: prevents or delays the occurrence of atopic dermatitis, cow’s milk allergy and wheezing in early

childhood, as compared with feeding formula milk containing intact cow’s milk protein.

Autoimmune disease

• Suppressed T-cell-mediated immune responses in pregnancy: may suddenly reactivate post-partum. Some autoimmune diseases may

improve during pregnancy but are immediately after delivery. Systemic lupus erythemato sus (SLE) is an exception, however, as it is

prone to exacerbation in pregnancy or the puerperium.

• Passive transfer of maternal antibodies: can mediate autoimmune disease in the fetus and newborn, including SLE, Graves’ disease and

myasthenia gravis.

• Antiphospholipid syndrome (p. 977): an important cause of fetal loss, intrauterine growth restriction and pre-eclampsia.

• HIV in pregnancy: see p. 326.

development of autoantibodies to C1 inhibitor, but the condition can also be caused by autoantibodies that activate C1. Treatment of the

underlying disorder may induce remission of angioedema. As with HAE, a low C4 is s een during acute episodes.

Transplantation and graft rejection

Transplantation provides the opportunity for denitive treatment of end-stage organ dis ease. The major complications are graft rejection,

drug toxicity and infection consequent to immunosuppression. Transplant survival conti nues to improve, as a result of the introduction of

less toxic immunosuppressive agents and increased understanding of the processes o f transplant rejection. Stem cell transplantation and

its complications are discussed on page 936.

Transplant rejection

Solid organ transplantation inevitably stimulates an aggressive immune respons e by the recipient, unless the transplant is between

monozygotic twins. The type and severity of the rejection response is determined by the genetic disparity between the donor and

recipient, the immune status of the host and the nature of the tissue transplanted (Box 4.25 ). The most important genetic determinant

4.25 Classication of transplant rejection

Type

Hyperacute rejection Time Pathological ndings Minutes to hours Thrombosis, necrosis

Acute cellular rejection 5–30 days Cellular inltration

Acute vascular rejection Chronic allograft failure 5–30 days Vasculitis

>30 days Fibrosis, scarring Mechanism

Pre-formed antibody to donor antigens results in complement activation (type II hyperse nsitivity)

CD4

and CD8

T cells (type IV hypersensitivity)

Antibody and complement activation Immune and non-immune

mechanisms

Treatment

None – irreversible graft loss

Increase immunosuppression

Increase immunosuppression Minimise drug toxicity, control hypertension and hype rlipidaemia

Transplantation and graft rejection • 89

is the difference between donor and recipient HLA proteins (p. 67). The extensive pol ymorphism of these proteins means that donor

HLA antigens are almost invariably recognised as foreign by the recipient immune sy stem, unless an active attempt has been made to

minimise incompatibility.

• Hyperacute rejection results in rapid and irreversible

destruction of the graft ( Box 4.25). It is mediated by pre-existing recipient antibodies against do nor HLA antigens, which arise as a

result of previous exposure through transplantation, blood transfusion or pregn ancy. It is very rarely seen in clinical practice, as the use

of screening for anti-HLA antibodies and pre-transplant cross-matching ensures the pr ior identication of recipient–donor

incompatibility.

• Acute cellular rejection is the most common form of graft rejection. It is m ediated by activated T lymphocytes and results in

deterioration in graft function. If allowed to progress, it may cause fever, pain and tenderness over the graft. It is usually amenable to

increased

immunosuppressive therapy.

• Acute vascular rejection is mediated by antibody formed

de novo after transplantation. It is more curtailed than the

hyperacute response because of the use of intercurrent

immunosuppression but it is also associated with reduced

graft survival. Aggressive immunosuppressive therapy is

indicated and physical removal of antibody through

plasmapheresis may be indicated in severe causes. Not all

post-transplant anti-donor antibodies cause graft damage;

their consequences are determined by specicity and

ability to trigger other immune components, such as the

complement cascade.

• Chronic allograft failure, also known as chronic rejection,

is a major cause of graft loss. It is associated with

proliferation of transplant vascular smooth muscle,

interstitial brosis and scarring. The pathogenesis is poorly

understood but contributing factors include immunological

damage caused by subacute rejection, hypertension,

hyperlipidaemia and chronic drug toxicity.

Investigations

Pre-transplantation testing

HLA typing determines an individual’s HLA polymorphisms and

facilitates donor–recipient matching. Potential transplant recipients are also screened for the presence of anti-HLA antibodies. The

recipient is excluded from receiving a transplant that carries 4

these alleles.

Donor–recipient cross-matching is a functional assay that directly tests whether serum fr om a recipient (which potentially contains anti-

donor antibodies) is able to bind and/or kill donor lymphocytes. It is specic to a prospec tive donor–recipient pair and is done

immediately prior to transplantation. A positive cross-match is a contraindication to transp lantation because of the risk of hyperacute

rejection.

Post-transplant biopsy: C4d staining

C4d is a fragment of the complement protein C4 (see Fig. 4.4).

Deposition of C4d in graft capillaries indicates local activation of the classical complement pathw ay and provides evidence of antibody-

mediated damage. This is useful in the early diagnosis of vascular rejection.

Complications of transplant immunosuppression

Transplant recipients require indenite treatment with immunosuppressive agents. In gen eral, two or more immunosuppressive drugs are

used in synergistic combination in order to minimise adverse effects (Box 4.26 ). The major complications of long-term

immunosuppression are infection and malignancy. The risk of some opportunistic infect ions may be minimised through the use of

prophylactic medication, such as ganciclovir for cytomegalovirus prophylaxis and trime thoprim–sulfamethoxazole for Pneumocystis

prophylaxis. Immunisation with killed vaccines is appropriate, although the immune resp onse may be curtailed. Live vaccines should not

be given.

4.26 Immunosuppressive drugs used in transplantation

Drug

Anti-proliferative agents

Azathioprine, mycophenolate mofetil

Calcineurin inhibitors

Ciclosporin, tacrolimus

Mechanism of action Inhibit lymphocyte proliferation by blocking DNA synthesis May be directly cytotoxic at high doses

Inhibit T-cell signalling; prevent lymphocyte activation; block cytokine transcription

Glucocorticoids

Anti-thymocyte globulin (ATG)

Basiliximab

Belatacept Decrease phagocytosis and release of proteolytic enzymes; decrease lymph ocyte activation and proliferation; decrease

cytokine production; decrease antibody production Antibodies to cell surface proteins d eplete or block T cells

Monoclonal antibody directed against CD25 (IL-2Rα chain), expressed on activated T cells

Selectively inhibits T-cell activation through blockade of CTLA4

Major adverse effects

Increased susceptibility to infection Leucopenia

Hepatotoxicity

Increased susceptibility to infection Hypertension

Nephrotoxicity

Diabetogenic (especially tacrolimus) Gingival hypertrophy, hirsutism (ciclosporin) Incr eased susceptibility to infection Multiple other

complications (p. 670)

Profound non-specic immunosuppression Increased susceptibility to infection

Increased susceptibility to infection

Gastrointestinal side-effects

Increased susceptibility to infection and malignancy Gastrointestinal side-effects

Hypertension

Anaemia/leucopenia

The increased risk of malignancy arises because T-cell suppression results in failure to c ontrol viral infections associated with malignant

transformation. Virus-associated tumours include lymphoma (associated with Epstein–B arr virus), Kaposi’s sarcoma (associated with

human herpesvirus 8) and skin tumours (associated with human papillomavirus). Immu nosuppression is also linked with a small increase

in the incidence of common cancers not associated with viral infection (such a s lung, breast and colon cancer), reecting the importance

of T cells in anticancer surveillance.

Organ donation

The major problem in transplantation is the shortage of organ donors. Cadaveric organ donors are usually previously healthy individuals

who experience brainstem death (p. 211), frequently as a result of road trafc acci dents or cerebrovascular events. Even if organs were

obtained from all potential cadaveric donors, though, their numbers would be insufcie nt to meet current needs. An alternative is the use

of living donors. Altruistic living donation, usually from close relatives, is widely used in renal transplantation. Living organ donation is

inevitably associated with some risk to the donor and it is highly regulated to ensure app ropriate appreciation of the risks involved.

Because of concerns about coercion and exploitation, non-altruistic organ donation (th e sale of organs) is illegal in most countries.

Tumour immunology

Surveillance by the immune system is critically important in monitoring and removing da maged and mutated cells as they arise. The

ability of the immune system to kill cancer cells effectively is inuenced by tumour immu nogenicity and specicity. Many cancer

antigens are poorly expressed and specic antigens can mutate, either spontaneously or in response to treatment, which can result in

evasion of immune responses. In addition, the inhibitory pathways that are used to maintain self-to lerance and limit collateral tissue

damage during antimicrobial immune responses can be co-opted by cancerous cells to eva de immune destruction. Recognition and

understanding of these immune checkpoint pathways has led to the development of a numb er of new treatments for cancers that are

otherwise refractory to treatment. For example, antibodies to CTLA4, a co-stimu latory molecule normally involved in down-regulation

of immune responses, have been licensed for refractory melanoma, and antibodies to P D1 (programmed cell death protein 1) are used in

melanoma, non-small-cell lung cancer and renal cell carcinoma. Potential risks inclu de the development of autoimmunity, reecting the

importance of these pathways in the control of self-tolerance.

Further information

allergy.org.au An Australasian site providing information on allergy, asthma and immune diseases.

allergyuk.org UK site for patients and health-care professionals.

anaphylaxis.org.uk Provides information and support for patients with severe allerg ies.

info4pi.org A US site managed by the non-prot Jeffrey Modell Fo undation, which provides extensive information about primary

immune deciencies.

niaid.nih.gov National Institute of Allergy and Infectious Diseases: provides useful information on a variety of allergic diseases, immune

deciency syndromes and autoimmune diseases.

H Campbell

DA McAllister 5

Population health and epidemiology

Global burden of disease and underlying risk factors 92 Life expectancy 92

Global causes of death and disability 92

Risk factors underlying disease 93

Social determinants of health 93

The hierarchy of systems – from molecules to ecologies 93 The life course 93

Preventive medicine 93

Principles of screening 94

Epidemiology 95

Understanding causes and effect 95

Health data/informatics 97

The UK Faculty of Public Health denes public health as ‘the science and art of promoting a nd protecting health and well-being,

preventing ill-health and prolonging life through the organised efforts of society’. This de nition recognises that there is a collective

responsibility for the health of the population that requires partnerships between govern ment, health services and others to promote and

protect health and prevent disease. Population health has been dened as ‘the he alth outcomes of a group of individuals, including the

distribution of such outcomes within the group’. Medical doctors can play a role in all th ese efforts to improve health both through their

clinical work and through their support of broader actions to improve public health.

Global burden of disease and underlying risk factors

5.1 Global premature mortality: top 15 ranked causes, 2015

1,2

1. Ischaemic heart disease (4)

2. Cerebrovascular disease (5)

3. Lower respiratory infections (1)

4. Neonatal preterm birth complications (2)

5. Diarrhoeal diseases (3)

6. Neonatal encephalopathy (6)

7. HIV/AIDS (29)

8. Road injuries (10)

9. Malaria (7)

10. Chronic obstructive pulmonary disease (12) 11. Congenital anomalies (9)

12. Tuberculosis (11)

13. Lung cancer

(20)

14. Self-harm (16)

15. Diabetes (

30)

By Years of Life Lost (YLL).

Rank in 1990 is shown in brackets.

‘All cancers combined’ would rank in the top three causes.

The Global Burden of Disease (GBD) exercise was initiated by

the World Bank in 1992, with rst estimates appearing in 1993.

Regular updated gures have been published since, together with

projections of future disease burden. The aim was to produce

reliable and internally consistent estimates of disease burden

for all diseases and injuries, and to assess their physiological,

behavioural and social risk factors, so that this information

could be made available to health workers, researchers and

policy-makers.

The GBD exercise adopted the metric ‘disability adjusted

life year’ (DALY) to describe population health. This combines

information about premature mortality in a population (measured

as Years of Life Lost from an ‘expected’ life expectancy) and years

of life lived with disability (Years of Life lived with Disability, which

is weighted by a severity factor). The International Classication

of Disease (ICD) rules, which assign one cause to each death,

are followed. All estimates are presented by age and sex groups

and by regions of the world. Many countries now also report

their own national burden of disease data.

5.2 Global disability: top 15 ranked causes, 2015

1. Lower back and neck pain (1)

2. Sense organ diseases (3)

3. Depressive disorders (4)

4. Iron deciency anaemia (2)

5. Skin diseases (5)

6. Diabetes (9)

7. Migraine (6)

8. Other musculoskeletal conditions

(7)

9. Anxiety disorders (8)

10. Oral disorders (11)

11. Asthma (10)

12. Schizophrenia (13)

13. Osteoarthritis (19)

14. Chronic obstructive pulmonary disease (14) 15. Falls (12)

Life expectancy

Global life expectancy at birth increased from 61.7 years in 1980 to 71.8 years in 2015 , an increase of 0.29 years per calendar year. This

change is due to a substantial fall in child mortality (mainly caused by common infections), pa rtly offset by rises in mortality from adult

conditions such as diabetes and chronic kidney disease. Some areas have not sho wn these increases in life expectancy in men, often due

to war and interpersonal violence.

Global causes of death and disability

Box 5.1 shows a ranked list of the major causes of global premature deaths in 2 015. Communicable, maternal, neonatal and nutritional

causes accounted for about one-quarter of deaths worldwide, down from about one-t hird in 1990. In contrast, deaths from non-

communicable diseases are increasing in importance and now account for about two-thir ds of all deaths globally, including about 13

million from ischaemic heart disease and stroke, and about 8 million from cancer. The a ge-standardised death rates for most diseases

globally are falling. However, despite this, the numbers of deaths from many diseases a re rising due to global population growth and the

change in age structure of

By Years of Life lived with Disability (YLD).

Rank in 1990 is shown in brackets.

Not otherwise classied as specic conditions such

as osteoarthritis.

the population to older ages, and this is placing an increasing burden on health systems. For a f ew conditions (e.g. HIV/AIDS, diabetes

mellitus and chronic kidney disease), age-standardised death rates continue to rise. With in this overall pattern, signicant regional

variations exist: for example, communicable, maternal, neonatal and nutritiona l causes still account for about two-thirds of premature

mortality in sub-Saharan Africa.

GBD also provides estimates of disability from disease ( Box 5.2). This has raised awarene ss of the importance of conditions like

depression, low back and neck pain, and asthma, which account for a relatively lar ge disease burden but relatively few deaths. This, in

turn, has resulted in greater health policy priority being given to these conditio ns. Since the policy focus in national health systems is

increasingly on keeping people healthy rather than only on reducing premature d eaths, it is important to have measures of these health

outcomes.

It is also essential to recognise that, although these estimates represent the best overall picture of burden of disease, they are based on

imperfect data. Nevertheless, the quality of data underlying the estimates and the modelling pro cesses are

Social determinants of health • 93

5.3 Global risk factors: top 15 ranked causes, 2015

1–3

1. High blood pressure (3)

2. Smoking/second-hand smoke exposure (5)

3. High fasting blood glucose (10)

4. High body mass index (13)

5. Childhood underweight (1)

6. Ambient particulate matter pollution (6)

7. High total cholesterol (12)

8. Household air pollution (4)

9. Alcohol use (11)

10. High sodium intake (14)

11. Low wholegrain intake (15)

12. Unsafe sex (20)

13. Low fruit intake (16)

14. Unsafe water (2)

15. Low glomerular ltration rate (21)

By percentage of burden of disease they cause.

Rank in 1990 is shown in brackets.

All dietary risk factors and physical inactivity

combined accounted for

10% of global burden of disease. Low physical activity was ranked 21, iron deciency 16 and suboptimal breastfeeding 22 in 2015.

improving over time and provide an increasingly robust basis for evidence-based health plan ning and priority setting.

G l o b

al ecosys

tem

vironme

ntE n

econ

o m munit

y t y

i festy

i 5

People

Age, sex and

hereditary factors

Macr

o-economy

ces

global for

politics, cultur

Fig. 5.1 Hierarchy of systems that inuence population health. Adapted from an original mo del by Whitehead M, Dahlgren G. What can

be done about inequalities in health? Lancet 1991; 338:1059–1063.

Risk factors underlying disease

Box 5.3 shows a ranked list of the main risk factors that underlay GBD in 2015 and how this ranking has changed in recent years.

Social determinants of health

Health emerges from a highly complex interaction between a person’s genetic bac kground and environmental factors (aspects of the

physical, biological (microbes), built and social environments, and also distant inuences such as the global ecosystem; Fig. 5.1).

The hierarchy of systems – from molecules to ecologies

5.4 ‘Hierarchy of systems’ applied to ischaemic heart disease

Level in the hierarchy

Molecular

Cellular

Tissue

Organ

System

Person

Family

Community Population Society

Ecology

Example of effect

ApoB mutation causing hypercholesterolaemia Macrophage foam cells accumulate in ve ssel wall Atheroma and thrombosis of coronary

artery Ischaemia and infarction of myocardium Cardiac failure

Limited exercise capacity, impact on employment Passive smoking, diet

Shops and leisure opportunities

Prevalence of obesity

Policies on smoking, screening for risk factors

Agriculture inuencing fat content in diet Inuences on health exist at many levels and e xtend beyond the individual to include the

family, community, population and ecology. Box 5.4 shows an example o f this for determinants of coronary heart disease and

demonstrates the importance of considering not only the disease process in a patient but als o its context. Health care is not the only

determinant – and is usually not the major determinant – of health status in the population. The concept of ‘global health’ recognises the

global dimension of health problems, whether these be emerging or pandemic infec tions or global economic inuences on health. to

higher risk of hypertension and type 2 diabetes in young adults, and of cardiovascular disease in middle age. It has been suggested that

under-nutrition during middle to late gestation permanently ‘programs’ cardiovascular and m etabolic responses. This ‘life course’

perspective highlights the cumulative effect on health of exposures to illness, adve rse environmental conditions and behaviours that

damage health.

The life course Preventive medicine

The determinants of health operate over the whole lifespan. Values and behaviours acq uired during childhood and adolescence have a

profound inuence on educational outcomes, job prospects and risk of disease. These c an have a strong inuence, for example, on

whether a young person takes up damaging behaviour like smoking, risky sexual activity and drug misuse. Inuences on health can

operate even before birth. Low birth weight can lead The complexity of interactions betwe en physical, social and economic determinants

of health means successful prevention is often difcult. Moreover, the life-course perspective illustrates that it may be necessary to

intervene early in life or even before birth, to prevent important disease later. Successful prevention is likely to require many

interventions across the life course and at several levels in the hierarchy of systems. The examples below illustrate this.

Alcohol

Alcohol use is an increasingly important risk factor underlying GBD (see Box 5.3 ). Reasons for increasing rates of alcohol-related harm

vary by place and time but include the falling price of alcohol (in real terms), increased availability and cultural change fostering higher

levels of consumption. Public, professional and governmental concern has now led to a minimu m price being charged for a unit of

alcohol, tightening of licensing regulations and curtailment of some promotional activity in many countries. However, even more

aggressive public health measures will be needed to reverse the levels of harm in the po pulation. The approach for individual patients

suffering adverse effects of alcohol is described elsewhere (e.g. pp. 1184 and 880) .

Smoking

Smoking is one of the top three risk factors underlying GBD (see B ox 5.3). It is responsible for a substantial majority of cases of chronic

obstructive pulmonary disease (COPD) and lung cancer (pp. 573 and 598), and most smokers d ie either from these or from ischaemic

heart disease. Smoking also causes cancers of the upper respiratory and gastrointestinal tracts, pancreas, bladder and kidney, and

increases risks of peripheral vascular disease, stroke and peptic ulceration. Maternal smokin g is an important cause of fetal growth

retardation. Moreover, there is evidence that passive (‘second-hand’) smoking has ad verse effects on cardiovascular and respiratory

health.

The decline in smoking in many high-income countries has been achieved not only by warni ng people of the health risks but also by

increasing taxation of tobacco, banning advertising, legislating against smoking in pu blic places and giving support for smoking

cessation to maintain this decline. However, smoking rates remain high in many poore r areas and are increasing among young women. In

many developing countries, tobacco companies have found new markets and rates ar e rising.

A complex hierarchy of systems interacts to cause smokers to initiate and maintain their habit. A t the molecular and cellular levels,

nicotine acts on the nervous system to create dependence and maintain the smoking hab it. There are also strong inuences at the

personal and social level, such as young female smokers being motivated to ‘stay thin’ o r ‘look cool’ and peer pressure. Other important

inuences include cigarette advertising, with the advertising budget of the tobacco indus try being much greater than that of health

services. Strategies to help individuals stop smoking (such as nicotine replacement therapy, a nti-smoking advice and behavioural

support) are cost-effective and form an important part of the overall strategy.

Obesity

Obesity is an increasingly important risk factor underlying GBD (see Box 5.3 ). The weight distribution of almost the whole population is

shifting upwards: the slim are becoming less slim while the fat are getting fatter (p. 698). In the UK, this translates into a 1 kg increase in

weight per adult per year (on average over the adult population). The current obe sity epidemic cannot be explained simply by individual

behaviour and poor choice but also requires an understanding of the obesogenic envir onment that encourages people to eat more and

exercise less. This includes the availability of cheap and heavily marketed energy-rich f oods, the increase in labour-saving devices (e.g.

lifts and remote controls) and the rise in passive transport (cars as opposed to walki ng, cycling, or walking to public transport hubs). To

combat the health impact of obesity, therefore, we not only need to help those who are already obese but also develop strategies that

impact on the whole population and reverse the obesogenic environment.

Poverty and afuence

The adverse health and social consequences of poverty are well documented: high birth rates, high death rates and short life expectancy.

Typically, with industrialisation, the pattern changes: low birth rates, low death rates and longer life expectancy. Instead of infections,

chronic conditions such as heart disease dominate in an older population. Adverse health cons equences of excessive afuence are also

becoming apparent. Despite experiencing sustained economic growth for the last 50 ye ars, people in many industrialised countries are

not growing any happier and the litany of socioeconomic problems – crime, congestion , inequality – persists.

Many countries are now experiencing a ‘double burden’. They have large population s still living in poverty who are suffering from

problems such as diarrhoea and malnutrition, alongside afuent populations (often in cities) who suffer from chronic illness such as

diabetes and heart disease.

Atmospheric pollution

Emissions from industry, power plants and motor vehicles of sulphur oxides, nitrogen o xides, respirable particles and metals are severely

polluting cities and towns in Asia, Africa, Latin America and Eastern Europe. Burning of fossil and biomass fuels, with production of

short-lived carbon pollutants (SLCPs – methane, ozone, black carbon and hydrofl uorocarbons), contributes to increased death rates from

respiratory and cardiovascular disease in vulnerable adults, such as those with establishe d respiratory disease and the elderly, while

children experience an increase in bronchitic symptoms. Developing countries a lso suffer high rates of respiratory disease as a result of

indoor pollution caused mainly by heating and cooking using solid biomass fuels.

Climate change and global warming

Climate change is arguably the world’s most important environmental health issue. A co mbination of habitat destruction and increased

production of carbon dioxide and SLCPs, caused primarily by human activity, seems to be the main cause. The temperature of the globe

is rising, and if current trends continue, warming by 4°C is predicted by 2050. The clim ate is being affected, putting millions of people at

risk of rising sea levels, ooding, droughts and failed crops These have already claimed millio ns of lives during the past 20 years and

have adversely affected the lives of many more. The economic costs of property damage and the impact on agriculture, food supplies and

prosperity have also been substantial. Global warming will also include changes in the g eographical range of some vector-borne

infectious diseases. Currently, politicians cannot agree an effective framework of actio ns to tackle the problem, but reducing emissions of

and SLCPs is essential.

Principles of screening

Screening is the application of a test to a large number of asymptomatic people with the aim of reducing morbidity or mortality from a

disease. The World Health Organisation (WHO)

Epidemiology • 95

has identied a set of (‘Wilson and Jungner’) criteria to guide health systems in deciding when it is appropriate to implement screening

programmes. The essential criteria are:

• Is the disease an important public health problem?

• Is there a suitable screening test available?

• Is there a recognisable latent or early stage?

• Is there effective treatment for the disease at this stage

that improves prognosis?

A suitable screening test is one that is cheap, acceptable, easy to perform and safe, a nd gives a valid result in terms of sensitivity and

specicity (p. 4). Screening programmes should always be evaluated in trials so that r obust evidence is provided in favour of their

adoption. These evaluations are prone to several biases – self-selection bias, lead-time b ias and length bias – and these need to be

accounted for in the analysis. Examples of large-scale programmes in the UK include b reast, colorectal and cervical cancer national

screening programmes and a number of screening tests carried out in pregnancy and in the newborn, such as the:

• diabetic eye screening programme

• fetal anomaly screening programme

• infectious diseases in pregnancy screening programme

• newborn and infant physical examination screening

programme

• newborn blood spot screening programme

• newborn hearing screening programme

• sickle-cell and thalassaemia screening programme. These are illustrated in Figure 5.2 .

Problems with screening include:

• over-diagnosis (of a disease that would not have come to attention on it s own or would not have led to death)

• false reassurance

• diversion of resources from investments that could control the disease more cost-effecti vely.

An example of these problems is the use of prostate-specic

antigen (PSA) testing as a screening test for the diagnosis of

prostate cancer (p. 438).

5.5 Calculation of risk using descriptive epidemiology Prevalence

• The ratio of the number of people with a longer-term disease or condition, at a spec ied time, to the number of people in the

population

Incidence

• The number of events (new cases or episodes) occurring in the 5 p opulation at risk during a dened period of time

Attributable risk

• The difference between the risk (or incidence) of disease in exposed and no n-exposed populations

Attributable fraction

• The ratio of the attributable risk to the incidence

Relative risk

• The ratio of the risk (or incidence) in the exposed population to the risk (or i ncidence) in the non-exposed population

A similar measure is the cumulative incidence or risk, which is the number of new cases a s a proportion of the total people at risk at the

beginning of the exposure time. If, in the example above, the same 1000 people w ere observed for a year (i.e. with no one joining or

leaving the group), then the 1-year risk is 10% (100/1000). The time period shoul d always be specied.

These rates and proportions are used to describe how diseases (and risk factors) vary according to time, person and place. Temporal

variation may occur seasonally (e.g. malaria occurs in the wet season but not t he dry) or as longer-term ‘secular’ trends (e.g. malaria may

re-emerge due to drug resistance). Person comparisons include age, sex, socioeconom ic status, employment, and lifestyle characteristics.

Place comparisons include the local environment (e.g. urban versus rural) and internati onal comparisons.

Understanding causes and effect Epidemiology

Epidemiologists study disease in free-living humans, seeking to describe patterns of hea lth and disease and to understand how different

exposures cause or prevent disease (Box 5.5).

Chronic diseases and risk factors (e.g. smoking, obesity etc.) are often described in terms of their prevalence. A prevalence is simply a

proportion: e.g. the prevalence of diabetes in people aged 80 and older in dev eloped countries is around 10%.

Events such as deaths, hospitalisations and rst occurrences of a disease are describ ed using incidence rates: e.g. if there are 100 new

cases of a disease in a single year in a population of 1000, the incidence rate is 105 per 1000 per son-years, not 100, because of the effect

of ‘person-time’. Person-time is the sum of the total ‘exposed’ time for the populatio n and in this example is 950 person-years. The

reason person-time is less than 1000 is that 100 people experienced the event. The se 100 people are assumed to have had an event, on

average, halfway through the time period, removing 100 × 0.5 person-years from the exposure time (as it is not possible to have a rst

occurrence of a disease twice). Hence, the incidence per 1000 person-years is 105, no t 100.

Epidemiological research complements that based on animal, cell and tissue models, the  ndings of which do not always translate to

humans. For example, only a minority of drug discoveries from laboratory research ar e effective when tested in people.

However, differentiating causes from mere non-causal associations is a considerable ch allenge for epidemiology. This is because while

laboratory researchers can directly manipulate conditions to isolate and understand caus es, such approaches are impossible in free-living

populations. Epidemiologists have developed a different approach, based around a num ber of study designs (Box 5.6). Of these, the

clinical trial is closest to the laboratory experiment. An early example of a clinical trial is s hown in Figure 5.3, along with ‘effect

measures’, which are used to quantify the difference in rates and risks.

In clinical trials, patients are usually allocated randomly to treatments so that, on average, groups are similar, apart from the intervention

of interest. Nevertheless, for any particular trial, especially a small trial, the laws of prob ability mean that differences can and do occur

by chance. Poorly designed or executed trials can also limit comparability between groups. A llocation may not be truly random (e.g.

because of inadequate concealment of the randomisation sequence), and there may

Pre-conception

Commence folic acid

Pregnancy Blood for sickle cell and thalassaemia

Blood for syphilis, hepatitis B and HIV as early as possible, or at any stage of the pregn ancy, including labour

Blood for haemoglobin,

group, Rhesus and

antibodies as early as Blood for T21,possible, or as soon T18 and T13

as a woman arrives for

(combined test)care, including labour

Blood for T21 (quadruple test) Reoffer screening for

infectious diseases if

initially declined

Repeat haemoglobin and antibodies

Newborn

Newborn physical examination by 72 hours

Hepatitis B vaccination ± immunoglobulin within 24 hours Newborn hearing screen

Infant physical examination at 6–8 weeks Women with type 1 or type 2 diabetes are of fered diabetic eye (DE) screening annually.

Week

In pregnancy women with

type 1 or type 2 diabetes are

offered a DE screen when

they first present for care

Early pregnancy scan to

12 support T21, T18 and13 T13 screening14

18 Detailed ultrasound19 scan for structural20

abnormalities, including

21 T18 and T1322

32 Give and discuss

33 newborn screening

34 information

Birth

Newborn blood spot screens +1

(ideally on day 5) for

sickle cell disease (SCD), +2

cystic fibrosis (CF),

congenital

hypothyroidism (CHT)

and inherited metabolic +4 diseases (PKU, MCADD,

MSUD, IVA, GA1 and HCU). +5 NB: babies who missed the

screen can be tested up to +6

1 year (except CF offered up to 8 weeks)

Follow-up DE screen for women with type 1 or 2 diabetes found to have diabetic retin opathy

Further DE screen for women with type 1 or 2 diabetes

Key T21, T18, T13 and fetal anomaly ultrasound

Sickle cell and

thalassaemia

Newborn and infant

physical examination

Newborn hearing

Give screening information as soon as possible

Diabetic eye

Newborn blood spot

Infectious diseases in pregnancy

Fig. 5.2 UK NHS Pregnancy and Newborn Screening Programmes: optimum times for test ing. (GA1 = glutaric aciduria type 1; HCU =

homocystinuria; IVA

isovaleric acidaemia; MCADD

medium-chain acyl-CoA dehydrogenase deciency; MSUD

maple syrup urine

disease; PKU = phenylketonuria; T13, 18, 21 = trisomy 13, 18 and 21) Based on Version 8.1, March 2016, Gateway ref: 2014696, Public

Health England.

Health data/informatics • 97

5.6 Epidemiological study designs Design

Clinical trial

Cohort

Case–control

Cross-sectional Description

Enrols a sample from a population and compares outcomes after randomly allocating patients to an intervention

Enrols a sample from a population and compares outcomes according to exposures

Enrols cases with an outcome of interest and controls without that outcome and compare s exposures between the groups

Enrols a cross-section (sample) of people from the population of interest; obtains da ta on exposures and outcomes

Example

Medical Research Council (MRC) Streptomycin Trial –

demonstrated effectiveness of streptomycin in tuberculosis

Framingham Study – identied risk factors for cardiovascular

disease

Doll R, Hill AB. Smoking and carcinoma of the lung. British Medical 5 Journal 195 0 – demonstrated that smoking caused lung cancer

World Health Organisation Demographic and Health Survey – captures risk factor data in a uniform way across many countries

Enrolled 107 patients with tuberculosis

Random allocation

Streptomycin 55 patients

Bed rest 52 patients or more often practical, considerations. Epidemiologis ts therefore seek to minimise bias and confounding by good

study analysis and design. They subsequently make causal inferences by balancing the pro bability that an observed association has been

caused by chance, bias and/or confounding against the alternative probability that the re lationship is causal. This weighing-up requires an

understanding of the frequency and importance of different sources of bias and confounding, as well as the scientic rationale of the

putative causal relationship. It was this approach, collectively and over a number of yea rs, that settled the fact that smoking causes lung

cancer and, subsequently, heart disease.

Follow-up and count deaths

Events 4 Events 15 Risk 7.3% Risk 28.8% Odds 0.068 Odds 0.224

Effect measures

Risk ratio (relative risk, RR) Odds ratio (OR) Absolute risk reduction (ARR) Relative r isk reduction (RRR) Number needed to treat to

prevent one death (NNT= 1/ARR) 0.25

0.30

21.6%

74.8%

4.6

Fig. 5.3 An example of a clinical trial: streptomycin versus bed rest in tuberculosis. Both prevalences and risks are, in fact, proportions,

and are therefore frequently expressed as odds. The reasons for doing so are beyond the s cope of this text.

be systematic differences (biases) in the way people allocated to different groups are treated or studied.

Such biases also occur in observational epidemiological study designs, such as cohort, c ase–control and cross-sectional studies (Box 5.6).

These designs are also much more subject to the problem of confounding than are rand omised trials.

Confounding is where the relationship between an exposure and outcome of interest is confu sed by the presence of some other causal

factor. For example, coffee consumption may be associated with lung cancer becau se smoking is more common among coffee-drinkers.

Here, smoking is said to confound the association between coffee and lung cancer.

Despite these limitations, for most causes of diseases, randomised controlled trials are no t feasible because of ethical,

Health data/informatics

As patients pass through health and social care systems, data are recorded concerning t heir family background, lifestyle and disease

states, which is of potential interest to healthcare organisations seeking to deliver services , policy-makers concerned with improving

health, scientic researchers trying to understand health, and also pharmaceutical and other co mmercial organisations aiming to identify

markets.

There is a long tradition of maintaining health information systems. In most countries, re gistration of births and deaths is required by law,

and in the majority, the cause of death is also recorded (Fig. 5.4). There are many challenge s in ensuring such data are useful, especially

for comparisons across time and place:

• A system of standard terminologies is needed, such as the

WHO International Classication of Diseases (ICD-10), which provides a list of diagnostic codes attempting to cover every diagnostic

entity.

• These terms must be understood to refer to the same, or at least similar, diseases in different places.

• Access to diagnostic skill and facilities is required.

• Standard protocols for assigning clinical diagnoses to ICD-10 codes are nee ded

• Robust quality control processes are needed to maintain some level of data completenes s and accuracy. Many countries employ similar

systems for hospitalisations, to

allow recovery of health-care utilisation costs or to manage and

plan services. Similar data are rarely collected for communitybased health care, nor are detailed data on health-care processes

generally included in national data systems. Consequently, there

has been considerable interest in using data from information

technology systems used to deliver care, such as electronic

INTERNATIONAL FORM OF MEDICAL CERTIFICATE OF CAUSE OF DEATH Cause of death

Disease or condition directly leading to death*

(a)

I21.9

due to (or as a consequence of)

Antecedent causes

Morbid conditions, if any, giving rise to the above cause, stating the underlying

condition last

(b)

E78.0

due to (or as a consequence of)

(c)

due to (or as a consequence of)

(d)

Other significant conditions contributing to the death, but not related to the disease or co ndition causing it

J47

*This does not mean the mode of dying, e.g. heart failure, respiratory failure. It means the disease, injury, or complication that caused

death.

Approximate interval between onset and death

Fig. 5.4 Completed death certicate. International Classication of Diseases 10 (ICD-10) co des are appended in red. WHO ICD-10, vol.

2; 1990. Available at https://commons.m.wikimedia.org/wiki/File:International_form_of_me dical_certicate_of_cause_of_death.png.

patient records, drug-dispensing databases, radiological software and clinical laboratory information systems.

Data from such systems are, of course, much less structured than those obtained from vital r egistrations. Moreover, the completeness of

such data depends greatly on local patterns of health-care utilisation, as well as how clinicians an d others use information technology

systems within different settings. As such, deriving useful, unbiased information from such data is a considerable challenge.

Much of the discipline of health informatics is concerned with addressing this challenge . One approach has been to develop

comprehensive standard classication systems such as SNOMED-CT, ‘a standardised, m ultilingual vocabulary of terms relating to the

care of the individual’, which has been designed for electronic health-care records.

An alternative has been to use statistical methods such as natural language processing to derive in formation automatically from free text

(such as culling diagnoses from radiological reports), or to employ ‘machine learning’, in which software algorithms are applied to data

in order to derive useful insights. Such approaches are suited to large, messy data wher e the costs of systematisation would be

prohibitive. It is likely that such innovations will, over the coming years, provide useful information to complement that obtained from

more traditional health information systems.

Further information

Books and journal articles

GBD 2015 Disease and Injury Incidence and Prevalence Collaborators. Global, regiona l, and national incidence, prevalence, and years

lived with disability for 310 diseases and injuries, 1990–2015: a systematic an alysis for the Global Burden of Disease Study 2015.

Lancet 2016; 388:1545–1602.

GBD 2015 Mortality and Causes of Death Collaborators. Global, regional, and national life expectancy, and cause-specic mortality for

249 causes of death, 1980–2015: a systematic analysis for the Global Burden of Di sease Study 2015. Lancet 2016; 388:1459–1544.

GBD 2015 Risk Factors Collaborators. Global, regional, and national comparativ e risk assessment of 79 behavioural, environmental and

occupational, and metabolic risks or clusters of risks, 1990–2015: a systematic analysis for th e Global Burden of Disease Study 2015.

Lancet 2016; 388:1659–1724.

Kindig D, Stoddart G. What is population health? Am J Public Health 2003; 93:380–383.

Websites

fph.org.uk UK Faculty of Public Health: What is public health?

gov.uk UK Government: population screening programmes.

JAT Sandoe

DH Dockrell

Principles of infectious disease

Infectious agents 100

Normal microbial ora 102

Host–pathogen interactions 104 Investigation of infection 105

Direct detection of pathogens 105 Culture 106

Indirect detection of pathogens 106 Antimicrobial susceptibility testing 109 Epidemiology of infection 110 Infection prevention and

control 111 Health care-associated infection 111 Outbreaks of infection 114

Immunisation 114

Antimicrobial stewardship 115 Treatment of infectious diseases 116 Principles of antimicrobial ther apy 116 Antibacterial agents 120

Antimycobacterial agents 125

Antifungal agents 125

Antiviral agents 126

Antiparasitic agents 128

‘Infection’ in its strict sense describes the situation where microorganisms or other infec tious agents become established in the host

organism’s cells or tissues, replicate, cause harm and induce a host response. If a micro organism survives and replicates on a mucosal

surface without causing harm or illness, the host is said to be ‘colonised’ by that organism . If a microorganism survives and lies dormant

after invading host cells or tissues, infection is said to be ‘latent’. When the infectious agent , or the host response to it, is sufcient to

cause illness or harm, then the process is termed an ‘infectious disease’. Most pathogens (infe ctious agents that can cause disease) are

microorganisms but some are multicellular organisms. The manifestations of disease may aid pathogen dissemination (e.g. diarrhoea).

The term ‘infection’ is often used interchangeably with ‘infectious disease’ but not all in fections are ‘infectious’, i.e. transmissible from

person to person. Infectious diseases transmitted between hosts are called communicable dise ases, whereas those caused by organisms

that are already colonising the host are described as endogenous. The distinction is blurred in some situations, including health care-

associated infections such as meticillinresistant Staphylococcus aureus (MRSA) or Clostridiu m difcile infection (CDI), in which

colonisation precedes infection but the colonising bacteria may have been recently tra nsmitted between patients. The chain of infection

(Fig. 6.1) describes six essential elements for communicable disease transmission.

Despite dramatic advances in hygiene, immunisation and antimicrobial therapy, infectiou s agents still cause a massive burden of disease

worldwide. Key challenges remain in

Infectious agent Reservoir

Susceptible host

Exit tackling infection in resource-poor countries. Microorganisms are continua lly mutating and evolving; the emergence of new

infectious agents and antimicrobial-resistant microorganisms is therefore inevi table. This chapter describes the biological and

epidemiological principles of infectious diseases and the general approach to their prevention, dia gnosis and treatment. Specic

infectious diseases are described in Chapters 11–13 and many of the organ-based chapters.

Infectious agents

The concept of an infectious agent was established by Robert Koch in the 19th century (Box 6. 1). Although fulfilment of ‘Koch’s

postulates’ became the standard for the denition of an infectious agent, they d o not apply to uncultivable organisms (e.g.

Mycobacterium leprae, Tropheryma whipplei ) or members of the normal human ora (e.g. Escherichia col i, Candida spp.). The

following groups of infectious agents are now recognised.

Viruses

Viruses are incapable of independent replication. Instead, they subvert host cellular proces ses to ensure synthesis of their nucleic acids

and proteins. Viruses’ genetic material (the genome) consists of single- or double-stran ded DNA or RNA. Retroviruses transcribe their

RNA into DNA in the host cell by reverse transcription. An antigenically unique protein coat (cap sid) encloses the genome, and together

these form the nucleocapsid. In many viruses, the nucleocapsid is packaged wit hin a lipid envelope. Enveloped viruses are less able to

survive in the environment and are spread by respiratory, sexual or blood-borne route s, including arthropod-based transmission. Non-

enveloped viruses survive better in the environment and are predominantly transmitted by faecal–oral or, less often, respiratory routes. A

generic virus life cycle is shown in Figure 6.2. A virus that infects a bacterium is a bacteriophage (phage).

Prokaryotes: bacteria (including mycobacteria and actinomycetes)

Prokaryotic cells are capable of synthesising their own proteins and nucleic acids, and are able to reproduce autonomously, although

they lack a nucleus. The bacterial cell membrane is bounded by a peptidoglycan cell wall, w hich is thick (20–80 nm) in Gram-positive

organisms and thin (5–10 nm) in Gram-negative ones. The Gram-negative cell wall is s urrounded by an outer membrane containing

lipopolysaccharide. Genetic information is contained within a chromosome but bacteria may also contain rings of extra-chromosomal

DNA, known as plasmids, which can be transferred between organisms, without cells h aving to divide. Bacteria may be embedded in a

polysaccharide capsule,

Entry

Transmission

Fig. 6.1 Chain of infection. The infectious agent is the organism that causes the dise ase. The reservoir is the place where the population

of an infectious agent is maintained. The portal of exit is the point from which the infectious agent leaves the reservoir. Transmission is

the process by which the infectious agent is transferred from the reservoir to the human host, either directly or via a vector or fomite.

The portal of entry is the body site that is rst accessed by the infectious agent. Finally, in order for disease to ensue, the person to whom

the infectious agent is transmitted must be a susceptible host.

6.1 Denition of an infectious agent –

Koch’s postulates

1. The same organism must be present in every case of the disease

2. The organism must be isolated from the diseased host and grown in pure cultu re

3. The isolate must cause the disease, when inoculated into a healthy, susceptible animal

4. The organism must be re-isolated from the inoculated, diseased animal

Infectious agents • 101

Interaction between host receptor molecule and virus ligand (determin es host-specificity of the virus) Adsorption 1

Lipid envelope

Capsid Nucleic acid

Release 6 Complete virus particles are released by budding of host cell membrane (shown here) or disintegration of host cell

Assembly 5 Assembly of virus components is mediated by host and/or viral enzymes Host cell

4 Synthesis Nucleic acid and protein synthesis is mediated by host and/or viral enzymes. This takes place in nucleus or cytoplasm,

depending on the specific virus Virus

2 Penetration

Receptor-mediated endocytosis

or, in some enveloped viruses,

membrane fusion (shown here)

Uncoating

Nucleic acid is liberated from the phagosome (if endocytosed)

and/or capsid by complex

enzymatic and/or receptor-mediated processes

Fig. 6.2 A generic virus life cycle. Life cycle components common to most viruses are host cell attachment and penetration, virus

uncoating, nucleic acid and protein synthesis, virus assembly and release. Virus release is ac hieved either by budding, as illustrated, or by

lysis of the cell membrane. Life cycles vary between viruses.

6.2 How bacteria are identied Gram stain reaction (see Fig. 6.3)

• Gram-positive (thick peptidoglycan layer), Gram-negative (thin peptid oglycan) or unstainable

Microscopic morphology

• Cocci (round cells) or bacilli (elongated cells)

• Presence or absence of capsule

Cell association

• Association in clusters, chains or pairs

Colonial characteristics

• Colony size, shape or colour

• Effect on culture media (e.g. β-haemolysis of blood agar in haemolytic strepto cocci; see Fig. 6.4)

Atmospheric requirements

• Strictly aerobic (requires O

), strictly anaerobic (requires absence of O

), facultatively aerobic (grows with or without O

) or

microaerophilic (requires reduced O

)

Biochemical reactions

• Expression of enzymes (oxidase, catalase, coagulase)

• Ability to ferment or hydrolyse various biochemical substrates Motility

• Motile or non-motile

Antibiotic susceptibility

• Identies organisms with invariable susceptibility (e.g. to optochin in

Streptococcus pneumoniae or metronidazole in obligate anaerobes) Matrix-assisted laser desorption/ionisation time-of-ight mass

spectrometry (MALDI-TOF-MS)

• A rapid technique that identies bacteria and some fungi from their spec ic molecular composition

Sequencing bacterial 16s ribosomal RNA gene

• A highly specic test for identication of organisms in pure culture and in samples from normally sterile sites

Whole-genome sequencing

• Although not yet in routine use, whole-genome sequencing (WGS) offers the potential to provide rapid and simultaneous identication,

sensitivity testing and typing of organisms from pure culture and/or directly from clinical sa mples. As such, WGS is likely to replace

many of the technologies described above over the next few years (p . 58)

and motile bacteria are equipped with agella. Although many prokaryotes are capable o f independent existence, some (e.g. Chlamydia

trachomatis, Coxiella burnetii ) are obligate intracellular organisms. Bacteria that can grow in articial culture media are classied and

identied using a range of characteristics (Box 6.2); examples are shown i n Figures 6.3 and 6.4.

Eukaryotes: fungi, protozoa and helminths

Eukaryotic cells contain membrane-bound organelles, including nuclei, mitochondria and Golgi apparatus. Pathogenic eukaryotes are

unicellular (e.g. fungi, protozoa) or complex multicellular organisms (e.g. nematodes, tr ematodes and cestodes, p. 288).

Gram stain

Gram-positive

cocci

Colony morphology (e.g. haemolysis), Gram stain appearance, agglutination reactions, coagulase test, catalase

Gram-positive bacilli Gram-negative cocci Gram-negative bacilli

Colony morphology, growth characteristics (e.g. growth in anaerobic atmosphere),

Gram stain appearance, MALDI-TOF-MS identification

Colony morphology, growth characteristics, oxidase reaction, sugar

fermentation/MALDI-TOF-MS identification

Colony morphology, growth characteristics, lactose fermentation, oxidase reaction, MA LDI-TOF-MS identification

Gram-positive cocci–clusters Examples

Staphylococcus

aureus

Coagulase-negative

staphylococci

Gram-positive cocci–chains

Examples

Oral streptococci

Streptococcus

pneumoniae (often

pairs)

Beta-haemolytic streptococci

Enterococci (short chains)

Examples

Actinomycetes

Arcanobacterium haemolyticum

Bacillus spp.

Corynebacterium diphtheriae Lactobacillus spp.

Listeria monocytogenes Nocardia spp.

Clostridium spp.

Examples

Neisseria meningitidis Neisseria gonorrhoeae Moraxella catarrhalis

Examples

Escherichia coli

Klebsiella pneumoniae Proteus spp.

Enterobacter spp.

Serratia spp.

Salmonella spp.

Shigella spp.

Yersinia spp.

Vibrio spp.

Pseudomonas aeruginosa

Fig. 6.3 Flow chart for bacterial identication, including Gram lm appearances on light mic roscopy (×100). (MALDI-TOF-MS =

matrix-assisted laser desorption/ionisation time-of-ight mass spectroscopy)

Fig. 6.4 Beta-haemolytic streptococci (A) and alpha-haemolytic streptococci (B) spread on eac h half of a blood agar plate (backlit). This

image is half life size.

0.5. Beta-haemolysis renders the agar transparent around the colonies (A) and a lpha-haemolysis imparts a green

tinge to the agar (B).

Fungi exist as either moulds (lamentous fungi) or yeasts.

Dimorphic fungi exist in either form, depending on environmental conditions (see Fig. 11.59, p. 300). T he fungal plasma membrane

differs from the human cell membrane in that it contains the sterol, ergosterol. Fungi hav e a cell wall made up of polysaccharides, chitin

and mannoproteins. In most fungi, the main structural component of the cell wall is β-1, 3-D-glucan, a glucose polymer. These

differences from mammalian cells are important because they offer useful therapeutic targ ets.

Protozoa and helminths are often referred to as parasites. Many parasites have complex multi-stage life cycles, which involve animal

and/or plant hosts in addition to humans.

Prions

Although prions are transmissible and have some of the characteristics of infectious age nts, they are not microorganisms and are not

diagnosed in microbiology laboratories. Prions are covered on page 250.

Normal microbial ora

The human body is colonised by large numbers of microorganisms (collectively termed the human microbiota). These colonising

Normal microbial flora • 103

Scalp

As for skin

Nares

Staph. aureus

Coagulase-negative staphylococci

Oral cavity

Oral streptococci (α-haemolytic)

Anaerobic Gram-positive bacilli

(including Actinomyces spp.)

Anaerobic Gram-negative bacilli

Prevotella spp.

Fusobacterium spp.

Candida spp.

Skin

Coagulase-negative staphylococci

Staph. aureus

Corynebacterium spp.

Propionibacterium spp.

Malassezia spp.

Hands

Resident: as for skin

Transient: skin flora (including

meticillin-resistant and other

Staph. aureus), bowel flora

(including Clostridium difficile,

Candida spp. and Enterobacteriaceae)

Vagina

Lactobacillus spp.

Staph. aureus

Candida spp.

Enterobacteriaceae

Strep. agalactiae (group B)

Perineum As for skin As for large bowel

Pharynx

Haemophilus spp.

Moraxella catarrhalis

Neisseria spp. (including N. meningitidis) Staph. aureus

Strep. pneumoniae

Strep. pyogenes (group A)

Oral streptococci (

-haemolytic)

Small bowel

Distally, progressively increasing

numbers of large bowel bacteria

Candida spp.

Large bowel

Enterobacteriaceae

Escherichia coli

Klebsiella spp.

Enterobacter spp.

Proteus spp.

Enterococci

E. faecalis

E. faecium

Streptococcus anginosus group Strep. anginosus

Strep. intermedius

Strep. constellatus

Anaerobic Gram-positive bacilli Clostridium spp.

Anaerobic Gram-negative bacilli Bacteroides spp.

Prevotella spp.

Candida spp.

Fig. 6.5 Human non-sterile sites and normal ora in health.

bacteria, also referred to as the ‘normal ora’, are able to survive Physical barriers, inc luding the skin, lining of the gastrointestinal and

replicate on skin and mucosal surfaces. The gastrointestinal tract and other mucous mem branes, maintain sterility of the tract and the

mouth are the two most heavily colonised sites in submucosal tissues, blood stream and perito neal and pleural the body and their

microbiota are distinct, in both composition cavities, for example.

and function. Knowledge of non-sterile body sites and their normal The normal ora c ontribute to endogenous disease mainly flora is

required to inform microbiological sampling strategies by translocation to a sterile site bu t excessive growth at the and interpret culture

results (Fig. 6.5). ‘normal’ site (overgrowth) can also cause disease. Overgrowth

The microbiome is the total burden of microorganisms, their is exemplied by denta l caries, vaginal thrush and ‘blind loop’ genes and

their environmental interactions, and is now recognised syndrome (p. 808). Translocation r esults from spread along a to have a profound

inuence over human health and disease. surface or penetration though a colonised surface, e.g. urinary Maintenance of the normal flora

is beneficial to health. For tract infection caused by perineal/enteric ora, and surgical sit e example, lower gastrointestinal tract bacteria

synthesise and infections, particularly of prosthetic materials, caused by skin ora excre te vitamins (e.g. vitamins K and B

);

colonisation with such as staphylococci. Normal ora also contribute to disease normal ora conf ers ‘colonisation resistance’ to infection

with by cross-infection, in which organisms that are colonising one pathogenic organisms by altering the lo cal environment (e.g.

individual cause disease when transferred to another, more lowering pH), producing a ntibacterial agents (e.g. bacteriocins susceptible,

individual.

(small antimicrobial peptides/proteins), fatty acids and metabolic The importance of limitin g perturbations of the microbiota by waste

products), and inducing host antibodies that cross-react antimicrobial therapy is increasin gly recognised. Probiotics are with pathogenic

organisms. microbes or mixtures of microbes that are given to a patient to

Conversely, normally sterile body sites must be kept sterile. The prevent or treat infectio n and are intended to restore a benecial

mucociliary escalator transports environmental material deposited prole of microbiota. A lthough probiotics have been used in a in the

respiratory tract to the nasopharynx. The urethral sphincter number of settings, whethe r they have demonstrable clinical prevents ow

from the non-sterile urethra to the sterile bladder. benets remains a subject of debate.

Host–pathogen interactions

‘Pathogenicity’ is the capability of an organism to cause disease and ‘virulence’ is th e extent to which a pathogen is able to cause

disease. Pathogens produce proteins and other factors, termed virulence factors, w hich contribute to disease.

• Primary pathogens cause disease in a proportion of

individuals to whom they are exposed, regardless of the host’s immunological s tatus.

• Opportunistic pathogens cause disease only in individuals wh ose host defences are compromised, e.g. by an intravascular catheter, or

when the immune system is compromised, by genetic susceptibility or

immunosuppressive therapy.

Characteristics of successful pathogens

Successful pathogens have a number of attributes. They compete with host cells and co lonising ora by various methods, including

sequestration of nutrients and production of bacteriocins. Motility enables pathogens to reach their site of infection, often sterile sites that

colonising bacteria do not reach, such as the distal airway. Many microorganisms, includ ing viruses, use ‘adhesins’ to attach to host cells

initially. Some pathogens can invade through tissues. Many bacterial and fungal infectio ns form ‘biolms’. After initial adhesion to a

host surface, bacteria multiply in biolms to form complex three-dimensional structures surround ed by a matrix of host and bacterial

products that afford protection to the colony and limit the effectiveness of antimicrobials. Biolm s forming on man-made medical

devices such as vascular catheters or grafts can be particularly difcult to treat.

Pathogens may produce toxins, microbial molecules that cause adverse effects on host c ells, either at the site of infection, or remotely

following carriage through the blood stream. Endotoxin is the lipid component of Gram -negative bacterial outer membrane

lipopolysaccharide. It is released when bacterial cells are damaged and has generalised inammatory effects. Exotoxins are proteins

released by living bacteria, which often have specic effects on target organs (Box 6.3 ).

Intracellular pathogens, including viruses, bacteria (e.g. Salmonella spp., Listeria monocytogenes and Mycobacterium tuberculosis),

parasites (e.g. Leishmania spp.) and fungi (e.g.

6.3 Exotoxin-mediated bacterial diseases

Disease

Antibiotic-associated diarrhoea/ pseudomembranous colitis Botulism

Cholera

Diphtheria

Haemolytic uraemic syndrome

Necrotising pneumonia Tetanus

Toxic shock syndrome Organism

Clostridium difcile (p. 230)

Clostridium botulinum (p. 1126) Vibrio cholerae (p. 264)

Corynebacterium diphtheriae (p. 265)

Enterohaemorrhagic Escherichia coli (E. coli O157 and other strains) (p. 263)

Staphylococcus aureus (p. 250) Clostridium tetani (p. 1125)

Staphylococcus aureus (p. 252) Streptococcus pyogenes (p. 253) Histoplasma capsulatum), are able to surv ive in intracellular

environments, including after phagocytosis by macrophages. Pathogenic bacteria expre ss different genes, depending on environmental

stress (pH, iron starvation, O

starvation etc.) and anatomical location.

Genetic diversity enhances the pathogenic capacity of bacteria. Some virulence factor g enes are found on plasmids or in phages and are

exchanged between different strains or species. The ability to acquire genes from the gene pool of all strains of the species (the ‘bacterial

supragenome’) increases diversity and the potential for pathogenicity. Viruses exploit th eir rapid reproduction and potential to exchange

nucleic acid with host cells to enhance diversity. Once a strain acquires a particularly effective combination of virulence genes, it may

become an epidemic strain, accounting for a large subset of infections in a par ticular region. This phenomenon accounts for inuenza

pandemics (see Box 6.10).

The host response

Innate and adaptive immune and inammatory responses, which humans use to control the normal ora and respond to pathogens, are

reviewed in Chapter 4.

Pathogenesis of infectious disease

The harmful manifestations of infection are determined by a combination of the virulen ce of the organism and the host response to

infection. Despite the obvious benets of an intact host response, an excessive response is undesirable. Cytokines and antimicrobial

factors contribute to tissue injury at the site of infection, and an excessive inammatory response may lead to hypotension and organ

dysfunction (p. 196). The contribution of the immune response to disease ma nifestations is exemplied by the immune reconstitution

inammatory syndrome (IRIS). This is seen, for example, in human immunodeciency viru s (HIV) infection, post-transplantation

neutropenia or tuberculosis (which causes suppression of T-cell function): there is a parado xical worsening of the clinical condition as

the immune dysfunction is corrected, caused by an exuberant but dysregulated inammatory response.

The febrile response

Thermoregulation is altered in infectious disease, which may cause both hyperthermia ( fever) and hypothermia. Fever is mediated mainly

by ‘pyrogenic cytokines’ (e.g. interleukins IL-1 and IL-6, and tumour necrosis f actor alpha (TNFα)), which are released in response to

various immunological stimuli including activation of pattern recognition receptors (PRRs) by microbial pyrogens (e.g.

lipopolysaccharide) and factors released by injured cells. Their ultimate effect is to induce the synthesis of prostaglandin E

, which binds

to specic receptors in the preoptic nucleus of the hypothalamus (thermoregulatory centre), cau sing the core temperature to rise.

Rigors are a clinical symptom (or sign if they are witnessed) characterised by feeling very cold (‘chills’) and uncontrollable shivering,

usually followed by fever and sweating. Rigors occur when the thermoregulatory cen tre attempts to correct a core temperature to a

higher level by stimulating skeletal muscle activity and shaking.

There are data to support the hypothesis that raised body temperature interferes with t he replication and/or virulence of pathogens. The

mechanisms and possible protective role of infection-driven hypothermia, however, are poorly understood, and require further study.

Investigation of infection

The aims of investigating a patient with suspected infection are to conrm the presence o f infection, identify the specic pathogen(s) and

identify its susceptibility to specic antimicrobial agents in order to optimise therapy. The presence of infection may be suggested by

identifying proteins that are produced in response to pathogens as part of the innate imm une and acute phase responses (p. 70). Pathogens

may be detected directly (e.g. by culturing a normally sterile body site) or their prese nce may be inferred by identifying the host response

to the organism, (‘indirect detection’, Box 6.4). Careful sampling increases the likelihood of diag nosis (Box 6.5). Culture results must be

interpreted in the context of the normal ora at the sampled site (see Fig. 6.5). The extent to wh ich a microbiological test result supports

or excludes a particular diagnosis depends on its statistical performance (e.g. sensitivity, specicity , positive and negative predictive

value, p. 4). Sensitivity and specicity vary according to the time betw een infection and testing, and positive and negative predictive

values depend on the prevalence of the condition in the test population. The complexity of test interpretation is illustrated in Figure 6.8

below, which shows the ‘windows of opportunity’ afforded by various testing methods. Given this complexity, effective communication

between the clinician and the microbiologist is vital to ensure accurate test interpretation.

Direct detection of pathogens

Some direct detection methods provide rapid results and enable detection of organisms t hat cannot be grown easily on articial culture

media, such as Chlamydia spp.; they can also provide information on antimicrobial se nsitivity, e.g. Mycobacterium tuberculosis.

Detection of whole organisms

Whole organisms are detected by examination of biological uids or tissue using a micro scope.

• Bright eld microscopy (in which the test sample is

interposed between the light source and the objective lens) uses stains to enhance visual contrast between the

6.4 Tests used to diagnose infection

Non-specic markers of inammation/infection

• e.g. White cell count in blood sample (WCC), plasma C-reactive protein (C RP), procalcitonin, serum lactate, cell counts in urine or

cerebrospinal uid (CSF), CSF protein and glucose

Direct detection of organisms or organism components

• Microscopy

• Detection of organism components (e.g. antigen, toxin)

• Nucleic acid amplication (e.g. polymerase chain reaction)

Culture of organisms

•

Antimicrobial susceptibility testing

Tests of the host’s specic immune response

• Antibody detection

• Interferon-gamma release assays (IGRA)

organism and its background. Examples include Gram staining of bacteria and Zie hl–Neelsen or auramine staining of acid- and alcohol-

fast bacilli (AAFB) in tuberculosis (the latter requires an ultraviolet light source). In

histopathological examination of tissue samples, multiple stains are used to demons trate not only the presence of microorganisms but also

features of disease pathology.

• Dark eld microscopy (in which light is scattered to make organisms app ear bright on a dark background) is used, for example, to

examine genital chancre uid in suspected syphilis. 6

• Electron microscopy may be used to examine stool and vesicle uid to detect enteric and herpesviruses,

respectively, but its use has largely been supplanted by nucleic acid detection (see below).

• Flow cytometry can be used to analyse liquid samples (e.g. urine) for t he presence of particles based on

properties such as size, impedance and light scatter. This technique can detect bacteria but m ay misidentify other particles as bacteria too.

6.5 How to provide samples for

microbiological sampling

Communicate with the laboratory

• Discuss samples that require processing urgently or that may contain hazardo us or unusual pathogens with laboratory staff before

collection

• Communication is key to optimising microbiological diagnosis. If there is dou bt about any aspect of sampling, it is far better to discuss

it with laboratory staff beforehand than to risk diagnostic delay by inappropriate samplin g or sample handling

Take samples based on a clinical diagnosis

• Sampling in the absence of clinical evidence of infection is rarely appropriate (e .g. collecting urine, or sputum for culture)

Use the correct container

• Certain tests (e.g. nucleic acid and antigen detection tests) require proprietary sample co llection equipment

Follow sample collection procedures

• Failure to follow sample collection instructions precisely can result in false- positive (e.g. contamination of blood culture samples) or

false-negative (e.g. collection of insufcient blood for culture) results

Label sample and request form correctly

• Label sample containers and request forms according to local polic ies, with demographic identiers, specimen type and time/date

collected

• Include clinical details on request forms

• Identify samples carrying a high risk of infection (e.g. blood liable to conta in a blood-borne virus) with a hazard label

Use appropriate packaging

• Close sample containers tightly and package securely (usually in sealed pla stic bags)

• Attach request forms to samples but not in the same compartment (to avoid con tamination, should leakage occur)

Manage storage and transport

• Transport samples to the microbiology laboratory quickly

• If pre-transport storage is required, conditions (e.g. refrigeration, incubation, storag e at room temperature) vary with sample type

• Notify the receiving laboratory prior to arrival of unusual or urgent samples, to ensure timely processing

Detection of components of organisms

Components of microorganisms detected for diagnostic purposes include nucleic acids, cell wall molecules, toxins and other antigens.

Commonly used examples include Legionella pneumophila serogroup 1 antigen in urine and cryptococcal polysaccharide antigen in

cerebrospinal uid (CSF). Most antigen detection methods are based on in vitro binding of specic antigen/antibody and are described

below. Other methods may be used, such as tissue culture cytotoxicity assay for C. difcile tox in. In toxin-mediated disease, detection of

toxin may be of greater relevance than identication of the organism itself (e.g. stool C. dif cile toxin).

Nucleic acid amplication tests

In a nucleic acid amplication test (NAAT), specic sequences of microbial DNA and RNA are identied using a nucleic acid primer

that is amplied exponentially by enzymes to generate multiple copies of a targe t nucleotide sequence. The most commonly used

amplication method is the polymerase chain reaction (PCR; see Fig. 3.11, p. 53). Revers e transcription (RT) PCR is used to detect RNA

from RNA viruses (e.g. hepatitis C virus and HIV-1). The use of uorescent labels in the reacti on enables ‘real-time’ detection of

amplied DNA; quantication is based on the principle that the time taken to reach the det ection threshold is proportional to the initial

number of copies of the target nucleic acid sequence. In multiplex PCR, multiple prim er pairs are used to enable detection of several

different organisms at once.

Determination of nucleotide sequences in a target gene(s) can be used to assign microo rganisms to specic strains, which may be relevant

to treatment and/or prognosis (e.g. in hepatitis C infection, p. 877). Genes that are relevant to pathogenicity (such as toxin genes) or

antimicrobial resistance can also be detected; for example, the mecA gene is used to screen for MRSA.

NAATs are the most sensitive direct detection methods and are also relatively rapid. The y are used widely in virology, where the

possibility of false-positive results from colonising or contaminating organisms is rem ote, and are applied to blood, respiratory samples,

stool and urine. In bacteriology, PCR is used to examine CSF, blood, tissue and genital s amples, and multiplex PCR is being developed

for use in faeces. PCR is particularly helpful for microorganisms that cannot be readi ly cultured, e.g. Tropheryma whipplei, and is being

used increasingly in mycology and parasitology.

Culture

Microorganisms may be both detected and further characterised by culture from clinica l samples (e.g. tissue, swabs and body fluids).

• Ex vivo culture (tissue or cell culture) was widely used in

the isolation of viruses but has been largely supplanted by NAAT.

• In vitro culture (in articial culture media) of bacteria and fungi is use d to conrm the presence of pathogens, allow identication, test

antimicrobial susceptibility and subtype the organism for epidemiological purposes.

Culture has its limitations: results are not immediate, even for organisms that are easy to g row, and negative cultures rarely exclude

infection. Organisms such as Mycobacterium tuberculosis are slow-growing, typically taking at least 2 weeks, even in rapid-culture

systems. Certain organisms, such as Mycobacterium leprae and Tropheryma whipplei, cannot be cultivated on articial media, and others

(e.g. Chlamydia spp. and viruses) grow only in culture systems, which are slow and labour-intensive.

Blood culture

The terms ‘bacteraemia’ and ‘fungaemia’ describe the presence of bacteria and fungi in the blood. ‘Blood-stream infection’ (p. 225) is

the association of bacteraemia/fungaemia with clinical evidence of infection. The presen ce of bacteraemia/fungaemia can be determined

by inoculating a liquid culture medium with freshly drawn blood, which is then incubated in a system that monitors it constantly for

growth of microorganisms (e.g. by detecting products of microbial respiration using u orescence; Fig. 6.6). If growth is detected,

organisms are identied and sensitivity testing is performed. Traditionally, identication h as been achieved by Gram stain appearance

and biochemical reactions. However, matrix-assisted laser desorption/ionisation time-of-ight mass spectroscopy (MALDI-TOF-MS;

see Box 6.2) is being used increasingly to identify organisms. MALDI-TOF-MS produces a pr ole of proteins of different sizes from the

target microorganism and uses databases of such proles to identify the organism (Fig. 6.7). It is rapid and accurate. Tak ing multiple

blood samples for culture at different times allows differentiation of transient (one or two positiv e samples) and persistent (majority are

positive) bacteraemia. This can be clinically important in the identication of the source of infection (p. 530).

Indirect detection of pathogens

Tests may be used to detect the host’s immune (antibody) response to a specific microor ganism, and can enable the diagnosis of infection

with organisms that are difcult to detect by other methods or are no longer present i n the host. The term ‘serology’ describes tests

carried out on serum and includes both antigen (direct) and antibody (indirect) detection.

Antibody detection

Organism-specic antibody detection is applied mainly to blood (Fig. 6.8). Results are typically ex pressed as titres: that is, the reciprocal

of the highest dilution of the serum at which antibody is detectable (for example, detection at se rum dilution of 1 : 64 gives a titre of 64).

‘Seroconversion’ is defined as either a change from negative to positive detection or a fourfold rise in titre between acute and

convalescent serum samples. An acute sample is usually taken during the rst week of disease an d the convalescent sample 2–4 weeks

later. Earlier diagnosis can be achieved by detection of immunoglobulin M (Ig M) antibodies, which are produced early in infection (p.

68). A limitation of these tests is that antibody production requires a fully functional host im mune system, so there may be false-negative

results in immunocompromised patients. Also, other than in chronic infections and with I gM detection, antibody tests usually provide a

retrospective diagnosis.

Antibody detection methods are described below (antigen detection methods are also de scribed here as they share similar methodology).

Enzyme-linked immunosorbent assay

The principles of the enzyme-linked immunosorbent assay (ELISA, EIA) are illustrated in Figure 6.9. These assays rely on linking

1 Patient sampling

Contamination minimised by aseptic technique. Maximise sensitivity by sampling correct volume

2 Sample handling 3 Specimen transport

Department of Microbiology

Follow local instructions for safety, labelling, and numbers of samples and bottles requir ed Transport samples to laboratory as quickly as

possible. Follow manufacturer’s

instructions for the blood culture system

6used if temporary storage is required

4 Incubation 5 Growth detection

Incubate at 35–37°C for 5–7 days. Microbial growth is usually detected by constant au tomatic monitoring of CO

. If no growth,

specimen is negative and discarded

Time to positivity (TTP) is usually 12–24 hrs in significant bacteraemia, but may be shor ter in overwhelming sepsis or longer with

fastidious organisms (e.g. Brucella spp.)

6 Preliminary results*

A Gram film of the blood culture medium is examined and results are communicated immediately to the clinician to guide antibiotic

therapy

Preliminary

susceptibility results are communicated to the clinician

7 Incubation

9 Definitive results

8 Culture results*

Further overnight incubation is often required for definitive identification of organisms (by biochemical testing) and additional

susceptibility testing; identification by MALDI-TOF MS (Fig. 6.7) is more rapid

10 Reporting A final summary is released when all testing is complete. For

clinical care, communication of interim results (Gram film, preliminary identification and susceptibility) is usually more important than

the final report. Effective clinical–laboratory communication is vital

A small amount of the medium is incubated on a range of culture media. Preliminary sus ceptibility testing may be carried out

Overnight incubation required

Urgent communication required

Fig. 6.6 An overview of the processing of blood cultures. *In laboratories equipped with M ALDI-TOF-MS (p. 106), rapid denitive

organism identication may be achieved at stage 6 and/or stage 8.

an antibody with an enzyme that generates a colour change on exposure to a chrom ogenic substrate. Various congurations allow

detection of antigens or specic subclasses of immunoglobulin (e.g. IgG, IgM, IgA). ELISA may also be adapted to detect PCR products,

using immobilised oligonucleotide hybridisation probes and various detection systems.

Immunoblot (Western blot)

Microbial proteins are separated according to molecular weight by polyacrylamide gel electrop horesis (PAGE) and transferred (blotted)

on to a nitrocellulose membrane, which is incubated with patient serum. Binding of spe cic antibody is detected with an enzyme–anti-

immunoglobulin conjugate similar to that used in ELISA, and specicity is conr med by its location on the membrane. Immunoblotting

is a highly specic test, which may be used to conrm the results of less specic tes ts such as ELISA (e.g. in Lyme disease, p. 255).

Immunouorescence assays

Indirect immunouorescence assays (IFAs) detect antibodies by incubating a serum sam ple with immobilised antigen (e.g. cells known

to be infected with virus on a glass slide); any virus-specic antibody present in the seru m binds to antigen and is then detected using a

uorescent-labelled anti-human immunoglobulin (‘secondary’ antibody). Fluorescence is visualised using a microscope. This method

can also detect organisms in clinical samples (usually tissue or centrifuged cells) using a speci c antibody in place of immobilised

antigen to achieve capture.

Complement xation test

In a complement xation test (CFT), patient serum is heat-treated to inactivate complemen t and mixed with the test antigen. Any specic

antibody in the serum will complex with the antigen. Complement is then added to the re action. If antigen–antibody complexes are

present, the complement will be ‘xed’ (consumed). Sheep erythrocytes, coated with an anti-erythrocyte antibody, are added. The degree

of erythrocyte lysis reects the remaining complement and is inversely proportional to th e quantity of the specic antigen–antibody

complex present.

Agglutination tests

When antigens are present on the surface of particles (e.g.

cells, latex particles or microorganisms) and cross-linked with antibodies, visible clum ping (or ‘agglutination’) occurs.

• In direct agglutination, patient serum is added to a

suspension of organisms that express the test antigen.

Detector

Lighter Heavier

m/z

Mass spectrum

Separation region (electric field-free)

Flight tube

Laser

Sample plate

Voltage grid

Fig. 6.7 The workings of matrix-assisted laser desorption/ionisation

time-of-ight mass spectrometry (MALDI-TOF MS). Adapted from Sobin K, Hameer D, Ruparel T. Digital genotyping using molecular

afnity and mass spectrometry. Nature Rev Genet 2003; 4:1001–1008. For example, in the Weil–Felix test, if a patient’s serum contains

antibodies to rickettsial species they cause agglutination when Proteus spp. surface (O) ant igens are added because the antibodies cross-

react with the Proteus antigens. The test lacks sensitivity and specicity but is still used to diagnose rickettsial infection in resource-

limited settings. The Widal test reaction uses a suspension of Salmonella typhi and S. p aratyphi ‘A’ and ‘B’, treated to retain only ‘O’ and

‘H’ antigens. These antigens are kept to detect corresponding antibodies in serum from a patient suspected of having typhoid fever. The

test is not specic but is still used in some parts of the world.

• In indirect (passive) agglutination, specic antigen is attached to the surface of carrier particles, which agglutinate when incubated with

patient samples that contain specic antibodies.

• In reverse passive agglutination (an antigen detection test), the carrier particle is co ated with antibody rather than antigen.

Other tests

Immunodiffusion involves antibodies and antigen migrating through gels, with or witho ut the assistance of electrophoresis, and forming

insoluble complexes where they meet. The complexes are seen on staining as ‘precipi tin bands’. Immunodiffusion is used in the

diagnosis of dimorphic fungi (p. 300) and some forms of aspergillosis (p. 596).

Immunochromatography is used to detect antigen. The system consists of a porous test s trip (e.g. a nitrocellulose membrane), at one end

of which there is target-specic antibody, complexed with coloured microparticles. Furt her specic antibody is immobilised in a

transverse narrow line some distance along the strip. Test material (e.g. blood or urine) is ad ded to the antibody–particle complexes,

which then migrate along the strip by capillary action. If these are complexed with antig en, they will be immobilised by the specic

antibody and visualised as a transverse line across the strip. If the test is negative, the antibo dy–particle complexes will bind to a line of

immobilised anti-immunoglobulin antibody placed further along the strip, which acts as a negative control. Immunochromatographic

tests are rapid and relatively cheap to perform, and are appropriate for point-of-care testing, e.g. in HIV 1 and malaria (p. 276).

Acute sample Convalescent sample

Antibody detection: IgM

Nucleic acid (NA)

Antibody detection: IgG (seroconversion)

detection

Antigen (Ag)

detection

Antibody detection: IgG (fourfold rise in titre)

IgM

NA IgG

Ag Limit

of detection Fig. 6.8 Detection of antigen, nucleic acid and antibody in infectious disease. The acut e sample is usually taken during the

rst week of illness, and the convalescent sample 2–4 weeks later. Detection limits and dura tion of detectability vary between tests and

diseases, although in most diseases immunoglobulin M (IgM) is detectable within the  rst 1–2 weeks.

A B C D

Antibody detection Antibody capture Competitive antibody Double antibody sandwich ELISA ELISA detection ELISA ELISA (for

antigen detection) Patient Ab

Antibody–enzyme

conjugate

Ig subclass-specific Ab

Specific Ag

Chromogenic substrate

6Ab specific to Ag from

the disease-causing

organism

Fig. 6.9 Antibody (Ab) and antigen (Ag) detection by enzyme-linked immunosorbent assa y (ELISA). This can be congured in various

ways. A Patient Ab binds to immobilised specic Ag and is detected by addi tion of anti-immunoglobulin–enzyme conjugate and

chromogenic substrate. B Patient Ab binds to immobilised Ig subclass-specic Ab and is detected by addition of specic Ag, followed

by antibody–enzyme conjugate and chromogenic substrate. C Patient Ab and antib ody–enzyme conjugate bind to immobilised specic

Ag. Magnitude of colour change reaction is inversely proportional to concentration of p atient Ab. D Patient Ag binds to immobilised Ab

and is detected by addition of antibody–enzyme conjugate and chromogenic sub strate. In A, the conjugate Ab is specic for human

immunoglobulin. In B–D, it is specic for Ag from the disease-causing organism.

Antibody-independent specic

immunological tests

The interferon-gamma release assay (IGRA) is being used increasingly to diagnose late nt tuberculosis infection (LTBI). The principle

behind IGRA is discussed on page 594. IGRA cannot distinguish between l atent and active tuberculosis infection and is therefore

appropriate for use only in countries where the background incidence of tuberculo sis is low.

A B

F C

E D

Antimicrobial susceptibility testing

If growth of microorganisms in culture is inhibited by the addition of an antimicrobial ag ent, the organism is considered to be susceptible

to that antimicrobial. Bacteriostatic agents cause reversible inhibition of growth and bacte ricidal agents cause cell death; the terms

fungistatic/fungicidal are equivalent for antifungal agents, and virustatic/virucidal for an tiviral agents. The lowest concentration of

antimicrobial agent at which growth is inhibited is the minimum inhibitory concentration ( MIC), and the lowest concentration that

causes cell death is the minimum bactericidal concentration (MBC). If the MIC is le ss than or equal to a predetermined breakpoint

threshold, the organism is considered susceptible, and if the MIC is greater tha n the breakpoint, it is resistant.

Breakpoints are determined for each antimicrobial agent from a combination of pharmacokinetic (p. 17) and clinical data. The

relationship between in vitro antimicrobial susceptibility and clinical response is complex, as response also depends on immune status,

pharmacokinetic variability (p. 17), comorbidities that may inuence pharmaco kinetics or pharmacodynamics, and antibiotic dosing, as

well as MIC/MBC. Thus, although treating a patient according to the results of susceptibility testin g increases the likelihood of recovery,

it does not guarantee therapeutic success.

Susceptibility testing is often carried out by disc diffusion (Fig. 6.10). Antibiotic-impregn ated lter paper discs are placed on agar plates

containing bacteria; antibiotic diffuses into the agar, resulting in a concentration gradien t centred on the disc. Bacteria are unable to grow

where the antibiotic concentration exceeds the MIC, which may therefore be inferred from the size of the zone of inhibition. The MIC is

commonly measured in diagnostic laboratories using ‘diffusion strips’.

A B

F C

E D

Zone of Zone of inhibition inhibition 5

Fig. 6.10 Antimicrobial susceptibility testing by disc diffusion (panels 1-4) and minimum inhib itory concentration (MIC, panel 5). 1. The

test organism is spread over the surface of an agar plate. 2. Antimicrobial-impregnated disc s (A–F) are placed on the surface and the

plate is incubated (e.g. overnight). 3–4. After incubation, zones of growth inhibition may be seen. The organism is considered susceptible

if the diameter of the zone of inhibition exceeds a pre-determined threshold. 5. I n a ‘diffusion strip’ test, the strip is impregnated with

antimicrobial at a concentration gradient that decreases steadily from top to bottom. The system is d esigned so that the MIC value is the

point at which the ellipse cuts a scale on the strip (arrow). 4, Kindly supplied by Charlotte Symes.

Epidemiology of infection

The communicability of infectious disease means that, once a clinician has diagnosed an infectious disease, potential exposure of other

patients must also be considered. The patient may require separation from other patients (‘isolation’), or an outbreak of disease may need

to be investigated in the community (Ch. 5). The approach will be specic to the microorganism involved (Chs 11–13) but the principles

are outlined below.

Geographical and temporal patterns of infection

Endemic disease

Endemic disease has a constant presence within a given geographical area or population . The infectious agent may have a reservoir,

vector or intermediate host that is geographically restricted, or may itself have restrictive environm ental requirements (e.g. temperature

range, humidity). The population affected may be geographically isolated or the disease may be limited to unvaccinated populations.

Factors that alter geographical restriction include:

• expansion of an animal reservoir (e.g. Lyme disease from

reforestation)

• vector escape (e.g. airport malaria)

• extension of host range (e.g. schistosomiasis from dam

construction)

• human migration (e.g. carbapenemase-producing

Klebsiella pneumoniae)

• public health service breakdown (e.g. diphtheria in

unvaccinated areas)

• climate change (e.g. dengue virus and Rift Valley fever).

Emerging and re-emerging disease

An emerging infectious disease is one that has newly appeared in

a population, or has been known for some time but is increasing in incidence or geogra phical range. If the disease was previously known

and thought to have been controlled or eradicated, it is considered to be re-emerging. Man y emerging diseases are caused by organisms

that infect animals and have undergone adaptations that enable them to infect humans . This is exemplied by HIV-1, which is believed to

have originated in higher primates in Africa. The geographical pattern of some recent e merging and re-emerging infections is shown in

Figure 6.11.

Reservoirs of infection

The US Centers for Disease Control (CDC) dene a reservoir of infection as any pe rson, other living organism, environment or

combination of these in which the infectious agent lives and replicates and on which the in fectious agent is dependent for its survival.

The infectious agent is transmitted from this reservoir to a susceptible host.

Human reservoirs

Both colonised individuals and those with infection can act as reservoirs, e.g. with Staph. aureus (including MRSA), Strep. pyogenes and

C. difficile. For infected humans to act as reservoirs, the infections caused mu st be long-lasting in at least a proportion of those affected,

to enable onward transmission (e.g. tuberculosis, sexually transmitted infections). Hum ans are the only reservoir for some infections (e.g.

measles).

Animal reservoirs

The World Health Organization (WHO) denes a zoonosis as ‘a disease or infect ion that is naturally transmissible from vertebrate

animals to humans’. Infected animals may be asymptomatic. Zoonotic agents may be transmitted via any of the routes described below.

Primary infection with zoonoses may be transmitted onward between humans, causing secondary disease (e.g. Q fever, brucellosis,

Ebola).

Environmental reservoirs

Many infective pathogens are acquired from an environmental source. However, some of these are maintained in human or animal

reservoirs, with the environment acting only as a conduit for infection.

MDR-TB

Cryptococcus CPE gattii

Cholera

Chikungunya virus Cyclospora

Zika virus Ebola virus disease

Cholera

Chikungunya Cholera

virus

Anthrax Zika virus

MERS-Co-V

Cryptococcus gattii

XDR-TB

Fig. 6.11 Geographical locations of some infectious disease outbreaks, with examples of em erging and re-emerging diseases. (CPE =

carbapenemase-producing Enterobacteriaceae; MDR-TB = multidrug-resistant tubercu losis; MERS-Co-V = Middle East respiratory

syndrome coronavirus; XDR-TB = extensively drug-resistant tuberculosis)

6.6 Incubation periods of important infections

6.7 Periods of infectivity in common childhood infectious diseases

Infection

Short incubation periods Anthrax, cutaneous

Anthrax, inhalational

Bacillary dysentery

Cholera

Dengue haemorrhagic fever

Diphtheria

Gonorrhoea

Inuenza

Meningococcaemia

Norovirus

SARS coronavirus

Scarlet fever

Intermediate incubation periods Amoebiasis

Brucellosis

Chickenpox

Lassa fever

Malaria

Measles

Mumps

Poliomyelitis

Psittacosis

Rubella

Typhoid

Whooping cough

Incubation period

9 hrs to 2 weeks

2 days

1–6 days

2 hrs to 5 days

3–14 days

1–10 days

2–10 days

1–3 days

2–10 days

1–3 days

2–7 days

2–4 days

1–4 weeks

5–30 days

11–20 days

3–21 days

10–15 days

6–19 days

15–24 days

3–35 days

1–4 weeks

15–20 days

5–31 days

5–21 days

Long incubation periods

Hepatitis A

Hepatitis B

Leishmaniasis, cutaneous

Leishmaniasis, visceral

Leprosy (Hansen’s disease)

Rabies

Trypanosoma brucei gambiense infection

Tuberculosis

3–7 weeks

6 weeks to 6 months Weeks to months Months to years

5–20 years

2–8 weeks

Months to years

1–12 months

Incubation periods are approximate and may differ from local or national guidance.

Longer incubation periods have been reported.

WHO.

Health Protection Agency (now Health Protection England).

Richardson M, Elliman D, Maguire H, et al. Pediatr Infect Dis J

2001; 20:380–388.

Centers for Disease Control, USA.

(SARS

severe acute respiratory syndrome)

Transmission of infection

Communicable diseases may be transmitted by one or more of the following routes:

• Respiratory route: inhalation.

• Faecal–oral route: ingestion of material originating from

faeces.

• Sexually transmitted infections: direct contact between

mucous membranes.

• Blood-borne infections: direct inoculation of blood or body

fluids.

• Direct contact: very few organisms are capable of causing

infection by direct contact with intact skin. Most infection

by this route requires contact with damaged skin (e.g.

surgical wound).

• Via a vector or fomite: the vector/fomite bridges the gap

between the infected host or reservoir and the uninfected

host. Vectors are animate, and include mosquitoes in

malaria, dengue and Zika virus infection, eas in plague Disease

Chickenpox

Measles

Mumps

Rubella

Scarlet fever

Whooping cough

Infectious period

From 4 days before until 5 days after

appearance of the rash (transmission before 48 hrs prior to the onset of rash is rare)

From 4 days before onset to 4 days after

onset of the rash

From 2–3 days before to 5 days after

disease onset

6 From 10 days before until 15 days after the onset of the rash, but most infectious during prodromal illness

Unknown

6,7

From Richardson M, Elliman D, Maguire H, et al. Pediatr Infect Dis J 2001;

20:380–388.

Centers for Disease Control, USA; cdc.gov/measles/hcp/.

Bennett JE, Dolin R, Blaser MJ. Mandell, Douglas and

Bennett’s Principles and practice of infectious diseases, 8th edn. Philadelphia: Elsevier ; 2015.

4–6

Exclude from contact with non-immune

and immunocompromised people for 5 days from

onset of rash,

onset of parotitis, or

start of antibiotic treatment.

Exclude for

3 weeks if untreated.

Durations are approximate and vary between information sources, and these rec ommendations may differ from local or national

guidance.

and humans in MRSA. Fomites are inanimate objects such as door handles, water taps and ult rasound probes, which are particularly

associated with health care-associated infection (HCAI).

The likelihood of infection following transmission of a pathogen depends on organism factors (virulence, p. 104) and host susceptibility.

The incubation period is the time between exposure and development of symptoms, and the period of infectivity is the period after

exposure during which the patient is infectious to others. Knowledge of incubation periods an d of periods of infectivity is important in

controlling the spread of disease, although for many diseases these estimates are impre cise (Boxes 6.6 and 6.7).

Deliberate release

Deliberate release of pathogens with the intention of causing disease is known as biologic al warfare or bioterrorism, depending on the

scale and context. Deliberate release incidents have included a 750-person outbreak of Salmonella typhimurium caused by contamination

of salads in 1984 (Oregon, USA) and 22 cases of anthrax (ve fatal) from t he mailing of nely powdered (weaponised) anthrax spores in

2001 (New Jersey, USA). Diseases with high potential for deliberate release include an thrax, plague, tularaemia, smallpox and botulism

(through toxin release).

Infection prevention and control

Infection prevention and control (IPC) describes the measures applied to pop ulations with the aim of breaking the chain of infection (see

Fig. 6.1, p. 100).

Health care-associated infection

The risk of developing infection following admission to a health-care facility (health car e-associated infection, HCAI) in

External ventricular drain and ventriculoperitoneal shunt infection C oagulase-negative staphylococci

Staphylococcus aureus

Diphtheroids

Pseudomonas aeruginosa

Temporary central venous

catheter infection

Staphylococcus aureus (incl. MRSA)

Coagulase-negative staphylococci

Coliforms

Candida

Cuffed/tunnelled central venous catheter infection

Coagulase-negative staphylococci

Staphylococcus aureus (incl. MRSA)

Coliforms

Candida

Pseudomonas spp.

Enterococcus spp.

Surgical site infection

Staphylococcus aureus

Beta-haemolytic streptococci Coliforms

Anaerobes

Breast implant infection

Staphylococcus aureus

Coagulase-negative staphylococci

Peritoneal dialysis-related peritonitis Staphylococcus aureus

Coagulase-negative staphylococci Coliforms

Pseudomonas spp.

Prosthetic joint infection

Coagulase-negative staphylococci Staphylococcus aureus

Streptococci

Coliforms

Propionibacterium acnes

Fig. 6.12 Commonly encountered health care-associated infections (HCAIs) and the factor s that predispose to them.

6.8 Measures used in infection prevention and control (IPC) Institut ions

• Handling, storage and disposal of clinical waste

• Containment and safe removal of spilled blood and body uids

• Cleanliness of environment and medical equipment

• Specialised ventilation (e.g. laminar ow, air ltration, controlled press ure gradients)

• Sterilisation and disinfection of instruments and equipment

• Food hygiene

• Laundry management

Health-care staff

• Education

• Hand hygiene, including hand-washing (see Fig. 6.13)

• Sharps management and disposal

• Use of personal protective equipment (masks, sterile and non-sterile g loves, gowns and aprons)

• Screening health workers for disease (e.g. tuberculosis, hepatitis B virus, M RSA)

• Immunisation and post-exposure prophylaxis

Clinical practice

• Antibiotic stewardship (p. 115)

• Aseptic technique

• Perioperative antimicrobial prophylaxis

• Screening patients for colonisation or infection (e.g. MRSA, GRE, CPE)

Response to infections

• Surveillance to detect alert organism (see text) outbreaks and antimicrob ial resistance

• Antibiotic chemoprophylaxis in infectious disease contacts, if indicated (see Box 6.18)

• Isolation (see Box 6.9)

• Reservoir control

• Vector control

(CPE = carbapenemase-producing Enterobacteriaceae; GRE = glycopeptide-resistant e nterococci; MRSA = meticillin-resistant

Staphylococcus aureus )

the developed world is about 10%. Many nosocomial bacterial infections are caused by o rganisms that are resistant to numerous

antibiotics (multi-resistant bacteria), including MRSA, extendedspectrum β-lactamases (E SBLs) and carbapenemase-producing

Enterobacteriaceae (CPE), and glycopeptide-resistant enterococci (GRE). Other infectio ns of particular concern in hospitals include C.

difcile (p. 264) and norovirus (p. 249). Some examples are shown in Figure 6.12.

IPC measures are described in Box 6.8. The most important is maintenance of good hand hygie ne (Fig. 6.13). Hand

Wash hands only when visibly soiled! Otherwise use handrub! Duration of the entire procedure: 40–60 sec.

12 3 4 56

Wet hands with water using elbow-operated or nontouch taps (if available)

Apply enough soap to cover Rub hands palm to palm all hand surfaces

Right palm over left dorsum with interlaced fingers and vice versa

Palm to palm with fingers interlaced

789 10 11

Backs of fingers to

opposing palms with

fingers interlaced 6 12

Rotational rubbing of left thumb clasped in right palm and vice versa Rotational rubbing , Rinse hands with water backwards and

forwards

with clasped fingers of

right hand in left palm

and vice versa

Dry thoroughly with a single-use towel If hand-operated taps have been used, use tow el to turn off tap

...and your hands are clean

Fig. 6.13 Hand-washing. Good hand hygiene, whether with soap/water or alcohol handru b, includes areas that are often missed, such as

ngertips, web spaces, palmar creases and the backs of hands. Adapted from the ‘How to Handwash’ URL:

6.9 Types of isolation precaution

Airborne transmission

Precautions

Negative pressure room with air exhausted externally or ltered

N95 masks or personal respiratiors for staff; avoid using non-immune staff

Contact transmission Droplet transmission

Private room preferred (otherwise, inter-patient spacing ≥ 1 m)

Gloves and gown for staff in contact with patient or contaminated areas

Infections managed with these precautions Measles

Tuberculosis, pulmonary or laryngeal, conrmed or suspected

Enteroviral infections in young children (diapered or incontinent)

Norovirus

C. difcile infection

Multidrug-resistant organisms (e.g. MRSA, ESBL, GRE, VRSA, penicillin-resistant Strep . pneumoniae)

Parainuenza in infants and

young children Rotavirus

RSV in infants, children and immunocompromised Viral conjunctivitis, acute

Private room preferred (otherwise, inter-patient spacing ≥ 1 m)

Surgical masks for staff in close contact with patient

Diphtheria, pharyngeal

Haemophilus inuenzae type B infection

Herpes simplex virus, disseminated or severe Inuenza

Meningococcal infection

Mumps

Mycoplasma pneumoniae

Parvovirus (erythrovirus) B19 (erythema infectiosum, fifth disease)

Pertussis

Plague, pneumonic/bubonic

Rubella

Strep. pyogenes (group A), pharyngeal

Infections managed with multiple precautions

Smallpox, monkeypox, VZV (chickenpox or disseminated disease)

Adenovirus pneumonia SARS, viral haemorrhagic fever

Recommendations based on 2007 CDC guideline for isolation precautions. May differ from local or national recommendations.

Not a

CDC recommendation.

Subject to local risk assessment.

Or in any immunocompromised patient until possibility of disseminated

infection excluded. (ESBL = extended-spectrum β-lactamase; GRE = glycopeptide-resis tant enterococci; MRSA = meticillin-resistant

Staph. aureus; RSV = respiratory syncytial virus; SARS = severe acute respiratory synd rome; VRSA = vancomycin-resistant Staph.

aureus; VZV = varicella zoster virus)

decontamination (e.g. using alcohol gel or washing) is mandatory before and after eve ry patient contact. Decontamination with alcohol

gel is usually adequate but hand-washing (with hot water, liquid soap and complete dry ing) is required after any procedure that involves

more than casual physical contact, or if hands are visibly soiled. In situations where the pr evalence of C. difcile is high (e.g. a local

outbreak), alcohol gel decontamination between patient contacts is inadequate as it does not kill C. difcile spores, and hands must be

washed.

Some infections necessitate additional measures to prevent cross-infection (Box 6.9 ). To minimise risk of infection, invasive procedures

must be performed using strict aseptic technique.

Outbreaks of infection

Descriptive terms are dened in Box 6.10. Conrmation of an infectious disease outbreak usually requires evidence from ‘typing’ that

the causal organisms have identical phenotypic and/or genotypic characteristics. If this is found not to be the case, the term pseudo-

outbreak is used. When an outbreak of infection is suspected, a case denition is agreed. The number of cases that meet the case

denition is then assessed by case-nding, using methods ranging from administratio n of questionnaires to national reporting systems.

Case-nding usually includes microbiological testing, at least in the early stages of an out break. Temporal changes in cases are noted in

order to plot an outbreak curve, and demographic details are collected to identify possib le sources of infection. A case–control study, in

which recent activities (potential exposures) of affected ‘cases’ are compared to those of unaffected ‘controls’, may be undertaken to

establish the outbreak source, and measures are taken to manage the outbreak and control its spread. Good communication between

relevant personnel during and after the outbreak is important to inform practic e in future outbreaks.

Surveillance ensures that disease outbreaks are either prevented or identied early. In h ospitals, staff are made aware of the isolation of

alert organisms, which have the propensity to cause outbreaks, and alert conditions, which are lik ely to be caused by such organisms.

Analogous systems are used nationally; many countries publish lists of organisms and di seases, which, if detected (or suspected), must

be reported to public health authorities (reportable or notiable diseases). Reasons for a disease to be classied as reportable are shown in

Box 6.11.

6.11 Reasons for including an infectious disease on a regional/national list of reporta ble diseases* Examples

Inuenza, Salmonella, tuberculosis

Reason for inclusion

Endemic/local disease with the potential to spread and/or cause outbreaks

Imported disease with the

propensity to spread and/or cause outbreaks

Evidence of a possible breakdown in health protection/public health functions

Evidence of a possible breakdown in food safety practices

Evidence of a possible failure of a vaccination programme

Disease with the potential to be a novel or increasing threat to human health

Evidence of expansion of the range of a reservoir/vector

Evidence of possible deliberate release

Typhoid, cholera (depending on local epidemiology)

Legionella, Cryptosporidium

Botulism, verotoxigenic E. coli

Measles, poliomyelitis, pertussis

SARS, MERS-CoV, multi-resistant bacteria

Lyme disease, rabies, West Nile encephalitis

Anthrax, tularaemia, plague, smallpox, botulism

*Given the different geographical ranges of individual diseases and wide national variation s in public health services, vaccination

programmes and availability of resources, reporting regulations vary between regions, states and countries. Many diseases are reportable

for more than one reason. (MERS-CoV = Middle East respiratory syndrome coronavi rus; SARS = severe acute respiratory syndrome)

6.10 Terminology in outbreaks of infection Term Denition

Classication of related cases of infectious disease * Cluster An aggrega tion of cases of a disease that are closely grouped in time and

place, and may or may not exceed the expected number

Epidemic The occurrence of more cases of disease than expected in a given area or amon g a specic group of people over a particular

period of time

Outbreak Synonymous with epidemic. Alternatively, a localised, as opposed t o generalised, epidemic

Pandemic An epidemic occurring over a very wide area (several countries or continents) and usually affecting a large proportion of the

population

Classication of affected patients (cases)

Index case

Primary cases

Secondary cases

Types of outbreak

Common source

outbreak

Point source outbreak

Person-to

person spread The rst case identied in an outbreak

Cases acquired from a specic source of infection Cases acquired from primary cases

Exposure to a common source of infection (e.g. water-cooling tower, medical staff member shedding MRSA). New primary cases will

arise until the source is no longer present

Exposure to a single source of infection at a specic point in time (e.g. contamin ated food at a party). Primary cases will develop disease

synchronously

Outbreak with both primary and secondary cases. May complicate point source or common source outbreak

*Adapted from cdc.gov. (MRSA

meticillin-resistant Staphylococcus aureus )

Immunisation

Immunisation may be passive or active. Passive immunisation is achieved by administe ring antibodies targeting a specic pathogen.

Antibodies are obtained from blood, so confer some of the risks associated with blood products (p. 933). The protection afforded by

passive immunisation is immediate but of short duration (a few weeks or months); it is us ed to prevent or attenuate infection before or

after exposure (Box 6.12).

Vaccination

Active immunisation is achieved by vaccination with whole organisms or organism comp onents (Box 6.13).

Types of vaccine

Whole-cell vaccines consist of live or inactivated (killed) microorganisms. Component v accines contain only extracted or synthesised

components of microorganisms (e.g. polysaccharides or proteins). Live vaccines conta in organisms with attenuated (reduced) virulence,

which cause only mild symptoms but induce T-lymphocyte and humoral responses ( p. 68) and are therefore more immunogenic than

inactivated whole-cell vaccines. The use of live vaccines in immunocompromised individuals is not generally recommended, but they

may be used by specialists following a risk/benet assessment.

Component vaccines consisting only of polysaccharides, such as the pneumococcal pol ysaccharide vaccine (PPV), are poor activators of

T lymphocytes and produce a short-lived antibody response without long-lasting memo ry. Conjugation of polysaccharide to a protein, as

in the Haemophilus inuenzae type B (Hib) vaccine and the protein conjugate pneumoco ccal

Antimicrobial stewardship • 115

6.12 Indications for post-exposure prophylaxis with immunoglobulins

Human normal immunoglobulin (pooled immunoglobulin)

• Hepatitis A (unvaccinated contacts*)

• Measles (exposed child with heart or lung disease)

Human specic immunoglobulin

• Hepatitis B (sexual partners, inoculation injuries, infants born to infected mothers)

• Tetanus (high-risk wounds or incomplete or unknown immunisation status)

• Rabies

• Chickenpox (immunosuppressed children and adults, pregnant women )

*Active immunisation is preferred if contact is with a patient who is within 1 week of onset o f jaundice.

6.14 Guidelines for vaccination against

infectious disease

• The principal contraindication to inactivated vaccines is an

anaphylactic reaction to a previous dose or a vaccine component

• Live vaccines should not be given during an acute infection, to pregnant w omen or to the immunosuppressed, unless the

immunosuppression is mild and the benets outweigh the risks

• If two live vaccines are required, they should be given either

simultaneously in opposite arms or 4 weeks apart

• Live vaccines should not be given for 3 months after an injection of

human normal immunoglobulin (HNI) 6

• HNI should not be given for 2 weeks after a live vaccine

• Hay fever, asthma, eczema, sickle-cell disease, topical

glucocorticoid therapy, antibiotic therapy, prematurity and chronic heart and lung diseases, in cluding tuberculosis, are not

contraindications to vaccination

6.13 Vaccines in current clinical use Live attenuated vaccines

• Measles, mumps, rubella (MMR)

• Oral poliomyelitis (OPV, not used in UK)

• Rotavirus

• Tuberculosis (bacille Calmette–Guérin, BCG)

• Typhoid (oral typhoid vaccine)

• Varicella zoster virus

Inactivated (killed) whole-cell vaccines

• Cholera

• Hepatitis A

• Inuenza

• Poliomyelitis (inactivated polio virus, IPV)

• Rabies

Component vaccines

• Anthrax (adsorbed extracted antigens)

• Diphtheria (adsorbed toxoid)

• Hepatitis B (adsorbed recombinant hepatitis B surface antigen,

HBsAg)

• Haemophilus inuenzae type B (conjugated capsular polysaccharide)

• Human papillomavirus (recombinant capsid proteins)

• Meningococcal, quadrivalent A, C, Y, W135 (conjugated capsular

polysaccharide)

• Meningococcal, serogroup C (conjugated capsular polysaccharide)

• Pertussis (adsorbed extracted antigens)

• Pneumococcal conjugate (PCV; conjugated capsular polysaccharide,

13 serotypes)

• Pneumococcal polysaccharide (PPV; puried capsular

polysaccharide, 23 serotypes)

• Tetanus (adsorbed toxoid)

• Typhoid (puried Vi capsular polysaccharide)

vaccinated to curtail further spread. Vaccination is aimed mainly at preventing infectious dise ase. However, vaccination against human

papillomavirus (HPV) was introduced to prevent cervical and other cancers that co mplicate HPV infection. Vaccination guidelines for

individuals are shown in Box 6.14.

Vaccination becomes successful once the number of susceptible hosts in a population fa lls below the level required to sustain continued

transmission of the target organism (herd immunity). Naturally acquired smallpox was declared to have been eradicated worldwide in

1980 through mass vaccination. In 1988, the WHO resolved to eradicate poliomyelitis b y vaccination; the number of cases worldwide

has since fallen from approximately 350 000 per annum to 74 in 2015. Recommended vaccination schedules vary between countries. In

addition to standard vaccination schedules, catch-up schedules are specied for individu als who join vaccination programmes later than

the recommended age.

Antimicrobial stewardship

Antimicrobial stewardship (AMS) refers to the systems and processes applied to a popu lation to optimise the use of antimicrobial agents.

The populations referred to here may be a nation, region, hospital, or a unit within a he alth-care organisation (e.g. ward or clinic). AMS

aims to improve patient outcomes and reduce antimicrobial resistance (AMR). IPC and AMS compleme nt each other (Fig. 6.14).

Elements of AMS include treatment guidelines, antimicrobial formularies and ward rounds by infection specialists.

vaccine (PCV), activates T lymphocytes, which results in a sustained response and immu nological memory. Toxoids are bacterial toxins

that have been modied to reduce toxicity but maintain antigenicity. Vaccine re sponse can be improved by co-administration with mildly

pro-inammatory adjuvants, such as aluminium hydroxide.

Use of vaccines

Vaccination may be applied to entire populations or to subpopulations at specic risk thr ough travel, occupation or other activities. In

ring vaccination, the population immediately surrounding a case or outbreak of infectious dise ase is Effective antimicrobial stewardship

reduces

health care-associated infections

Effective infection control reduces the need

for antimicrobials

Antimicrobial stewardship

Infection prevention and control

Fig. 6.14 The relationship between infection prevention and control (IPC) and antimicrobial stew ardship (AMS).

Treatment of infectious diseases

Key components of treating infection are:

• optimising antimicrobial therapy while minimising selection for antimicrob ial resistance and the impact on commensal flora

• addressing predisposing factors, e.g. glycaemic control in diabetes mellitus; viral load control in HIV-1-associated opportunistic

infection

• considering adjuvant therapy, e.g. removal of an infected medical device or n ecrotic tissue

• managing complications, e.g. severe sepsis (systemic inammatory response syn drome, or SIRS, p. 196) and acute kidney injury (p.

411).

For communicable disease, treatment must also take into

account contacts of the infected patient, and may include

IPC interventions such as isolation, antimicrobial prophylaxis,

vaccination and contact tracing.

Principles of antimicrobial therapy

In some situations (e.g. pneumonia) it is important to start appropriate antimicrobial thera py promptly, whereas in others prior

conrmation of the diagnosis and pathogen is preferred. The principles underlying the choice of antimicrobial agent(s) are discussed

below. The WHO ‘World Antibiotic Awareness Week’ campaign is a yearly event aim ed at highlighting the importance of prudent

antimicrobial prescribing (see ‘Further information’).

Antimicrobial action and spectrum

Antimicrobial agents may kill or inhibit microorganisms by targeting essential and non-es sential cellular processes, respectively. The

range, or spectrum, of microorganisms that is killed or inhibited by a particular anti microbial agent needs consideration when selecting

therapy. Mechanisms of action of the major classes of antibacterial agent are listed in Box 6. 15 and appropriate agents for some common

infecting organisms are shown in Box 6.16. In severe infections and/ or immunocomp romised patients, it is customary to use bactericidal

agents in preference to bacteriostatic agents.

Empiric versus targeted therapy

Empiric antimicrobial therapy is selected to treat a suspected infection (e.g. meningitis) before the microbiological cause is known.

Targeted or ‘directed’ therapy can be prescribed when the pathogen(s) is known . Empirical antimicrobial regimens need to have activity

against the range of pathogens that could be causing the infection in question; because bro ad-spectrum agents affect many more bacteria

than needed, they select for antimicrobial resistance. ‘Start Smart – Then Focus’ (Fig. 6.1 5) describes the principle of converting from

empiric therapy to narrow-spectrum targeted therapy. Optimum empiric therapy depen ds on the site of infection, patient characteristics

and local antimicrobial resistance patterns. National or local guidelines are often used to inform antimicrobial prescribing decisions.

Combination therapy

It is sometimes appropriate to combine antimicrobial agents:

• when there is a need to increase clinical effectiveness (e.g. biolm infections)

6.15 Target and mechanism of action of common antibacterial agents

Aminoglycosides, chloramphenicol, macrolides,

lincosamides, oxazolidinones

• Inhibition of bacterial protein synthesis by binding to subunits of bacterial ribosomes

Tetracyclines

• Inhibition of protein synthesis by preventing transfer RNA binding to ribosomes

Beta-lactams

• Inhibition of cell wall peptidoglycan synthesis by competitive inhibition of transpeptidases (‘penicillin-binding proteins’)

Cyclic lipopeptide (daptomycin)

• Insertion of lipophilic tail into plasma membrane causing depolarisation and cell death

Glycopeptides

• Inhibition of cell wall peptidoglycan synthesis by forming complexes with D-ala nine residues on peptidoglycan precursors

Nitroimidazoles

• The reduced form of the drug causes strand breaks in DNA

Quinolones

• Inhibition of DNA replication by binding to DNA topoisomerases (DNA gy rase and topoisomerase IV), preventing supercoiling and

uncoiling of DNA

Rifamycins

• Inhibition of DNA synthesis by inhibiting DNA-dependent RNA polymerase

Sulphonamides and trimethoprim

• Inhibition of folate synthesis by dihydropteroate synthase (sulphonamid es) and dihydrofolate reductase (trimethoprim) inhibition

• when no single agent’s spectrum covers all potential pathogens (e.g. polymicrobial infection)

• when there is a need to reduce development of antimicrobial resista nce in the target pathogen, as the organism would need to develop

resistance to multiple agents simultaneously (e.g. antituberculous chemotherapy, p. 592; antiretroviral therapy (ART), p. 324).

Antimicrobial resistance

Microorganisms have evolved in the presence of naturally occurring antibiotics and ha ve therefore developed resistance mechanisms

(categorised in Fig. 6.16) to all classes of antimicrobial agent (antibiotics and their derivatives ). Intrinsic resistance is an innate property

of a microorganism, whereas acquired resistance arises by spontaneous mutation or ho rizontal transfer of genetic material from another

organism (e.g. via a plasmid, p. 100). Plasmids often encode resistance to multiple antibiotics.

The mecA gene encodes a penicillin-binding protein, which has a low afnity for penicillins and therefore confers resistance to β-lactam

antibiotics in staphylococci. Extended-spectrum β-lactamases (ESBLs) are frequently en coded on plasmids, which are transferred

relatively easily between bacteria, including Enterobacteriaceae. Plasmid-encoded carba penemases have been detected in strains of

Klebsiella pneumoniae (e.g. New Delhi metalloβ-lactamase 1, NDM-1). Strains of MRSA have be en described that also have reduced

susceptibility to glycopeptides through the development of a relatively impermeable cell w all.

6.16 Antimicrobial options for common infecting bacteria Organis m

Gram-positive organisms Enterococcus faecalis

Enterococcus faecium

Glycopeptide-resistant enterococci MRSA

Staphylococcus aureus

Streptococcus pyogenes

Streptococcus pneumoniae

Gram-negative organisms