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The Atlas of Sexually Transmitted Diseases and AIDS, 4th Edition, by Drs. Stephen A. Morse, King K. Holmes, Adele A. Moreland, MD, and Ronald C. Ballard, provides you with an exclusive gallery of STD and AIDS images so you can better diagnose and treat these diseases. Approximately 1,100 unique images – most in full color and 30% new to this edition – depict the clinical signs associated with each type of infection. You’ll also find expert guidance on new vaccines, screening techniques, treatment guidelines, and best practices in the field.

  • Get expert advice on the tests available to reach a definitive diagnosis and review therapeutic options, treatment guidelines, prevention strategies, and management of complications.
  • Access appendices on the selection and evaluation of diagnostic tests, quality control, and test technologies.
  • Effectively diagnose all types of STDs and HIV/AIDS with approximately 1,100 images—most in full color and more than 30% new to this edition―that depict the epidemiology as well as the clinical manifestations of these diseases.
  • Effectively utilize new vaccines for HPV and Hepatitis B, new screening tests for Chlamydia, new drugs under development, new treatment guidelines and best practices in HIV screening, and much more.



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Date de parution 17 septembre 2010
Nombre de lectures 5
EAN13 9780702047640
Langue English
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Atlas of Sexually Transmitted Diseases and AIDS
Fourth Edition

Stephen A. Morse, MSPH, PhD
Associate Director for Environmental Microbiology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, USA

Ronald C. Ballard, PhD
Chief, Laboratory Reference and Research Branch, Division of STD Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention, Centers for Disease Control and Prevention Atlanta, USA

King K. Holmes, MD, PhD
William H. Foege Chair and Professor, Department of Global Health, Schools of Medicine and Public Health, Director, Center for AIDS and STD, University of Washington, Seattle, USA

Adele A. Moreland, MD, FAAD
Senior Staff, Department of Dermatology, Lahey Clinic Medical Center, Burlington, Massachusetts USA
Front Matter

Atlas of Sexually Transmitted Diseases and AIDS
Stephen A. Morse MSPH, PhD
Associate Director for Environmental Microbiology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, USA
Ronald C. Ballard PhD
Chief, Laboratory Reference and Research Branch, Division of STD Prevention, National Center for HIV/AIDS, Viral Hepatitis,STD and TB Prevention,Centers for Disease Control and Prevention Atlanta, USA
King K. Holmes MD, PhD
William H. Foege Chair and Professor, Department of Global Health, Schools of Medicine and Public Health, Director, Center for AIDS and STD, University of Washington, Seattle, USA
Adele A. Moreland MD, FAAD
Senior Staff, Department of Dermatology, Lahey Clinic Medical Center, Burlington, Massachusetts USA

Commissioning Editor:  Sue Hodgson
Development Editor:  Nani Clansey
Editorial Assistant:  Poppy Garraway/Rachael Harrison
Project Manager:  Sruthi Viswam
Design:  Charles Gray
Illustration Manager:  Bruce Hogarth
Illustrator:  Maurice Murphy/Antbits
Marketing Manager(s) (UK/USA):  Helena Mutak

An imprint of Elsevier Limited
© 2010, Elsevier Limited. All rights reserved.
First edition 1990
Second edition 1996
Third edition 2003
The right of Stephen A. Morse, Ronald C. Ballard, King K. Holmes and Adele A. Moreland to be identified as editors of this work has been asserted by them in accordance with the Copyright, Designs and Patents Act 1988.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: .

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.
With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability 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.
ISBN-13: 9780702040603
British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library
Atlas of sexually transmitted diseases and AIDS.—4th ed.
1. Sexually transmitted diseases–Atlases. 2. AIDS (Disease)–Atlases.
I. Morse, Stephen A.
Library of Congress Cataloging in Publication Data
A catalog record for this book is available from the Library of Congress
Printed in China
Last digit is the print number: 9 8 7 6 5 4 3 2 1

Ronald C. Ballard, PhD, Chief, Laboratory Reference and Research Branch National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention Centers for Disease Control and Prevention Atlanta, USA

Consuelo M. Beck-Sague, MD, FAAP, FASM, Assistant Professor, Department of Epidemiology and Public Health, University of Miami School of Medicine Miami, USA

Francis Bowden, MBBS, MD, FRACP, FAChSHM, Professor of Medicine, Infectious Diseases and Sexual Health Physician Australian National University The Canberra Hospital Canberra, Australia

William A. Bower, MD, FIDSA, Commander, U.S. Public Health Service Office of Blood, Organ, and other Tissue Safety Division of Healthcare Quality Promotion, National Center for Emerging and Zoonotic Infectious Diseases Centers for Disease Control and Prevention Atlanta, USA

Cheng-Yen Chen, PhD, Microbiologist, Laboratory Reference and Research Branch Division of STD Prevention National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention Centers for Disease Control and Prevention Atlanta, USA

Robert W. Coombs, MD, PhD, Professor of Laboratory Medicine and Medicine Department of Laboratory Medicine Harborview Medical Center University of Washington Seattle, USA

David L. Cox, PhD, Microbiologist, Laboratory Reference and Research Branch Division of STD Prevention National Center for HIV/AIDS, Viral Hepatitis STD and TB Prevention Centers for Disease Control and Prevention Atlanta, USA

Shireesha Dhanireddy, MD, Assistant Professor of Medicine Division of Allergy and Infectious Diseases University of Washington Harborview Medical Center Seattle, USA

Kenneth L. Dominguez, MD, MPH, Medical Microbiologist, Commander, USPHS National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention Centers for Disease Control and Prevention Atlanta, USA

John M. Douglas, Jr, MD, Senior Medical Advisor National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention Centers for Disease Control and Prevention Atlanta, USA

Kevin A. Fenton, MD, PhD, FFPH, Director, National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention Centers for Disease Control and Prevention Atlanta, USA

Charlotte A. Gaydos, MS, MPH, DrPH, Professor of Medicine Department of Medicine Division of Infectious Diseases Johns Hopkins Medical Institutions Baltimore, USA

Robert D. Harrington, MD, Professor, Allergy & Infectious Diseases, Medicine Harborview Medical Center University of Washington Seattle, USA

Sharon L. Hillier, PhD, Professor of Microbiology Department of Obstetrics, Gynecology & Reproductive Sciences University of Pittsburgh School of Medicine Pittsburgh, USA

Dale Hu, MD, MPH, Epidemiologist, Division of Viral Hepatitis National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention Centers for Disease Control and Prevention Atlanta, USA

Catherine A. Ison, PhD, FRCPath, Professor Director of Sexually Transmitted Bacteria Reference Laboratory Health Protection Agency Centre for Infections Visiting Professor of Investigative Science and Infectious Disease Epidemiology Imperial College London Sexually Transmitted Bacteria Reference Laboratory Health Protection Agency Centre for Infections London, UK

Jørgen S. Jensen, MD, PhD, DMedSci, Mycoplasma Laboratory Statens Serum Institut Copenhagen, Denmark

Christine Johnston, MD, MPH, Acting Instructor of Medicine Division of Infectious Diseases University of Washington Virology Research Clinic Seattle, USA

Peter K. Kohl, FRCP Lon, Professor of Dermatology, Director Department of Dermatology and Venereology Vivantes Klinikum Neukölln Academic Teaching Hospital of Charite University Medicine Berlin Berlin, Germany

Helen H. Lee, PhD, Associate Professor of Medical Biotechnology Department of Haematology University of Cambridge Cambridge, UK

David Lewis, FRCP(UK), PhD, DTM&H, Head of the Sexually Transmitted Infections Reference Centre National Institute for Communicable Diseases, Sandringham, South Africa Honorary Associate Professor, Department of Internal Medicine University of the Witwatersrand, South Africa Honorary Associate Professor, Division of Medical Microbiology University of Cape Town, South Africa

Lourdes Mahilum-Tapay, PhD, Research Associate Department of Haematology University of Cambridge Cambridge, UK

Jeanne M. Marrazzo, MD, MPH, Associate Professor, Medicine/Division of Allergy and Infectious Diseases Medical Director, Seattle STD/HIV Prevention Training Center University of Washington Seattle, USA

Adele A. Moreland, MD, FAAD, Senior Staff, Department of Dermatology Lahey Clinic Medical Center Burlington, Massachusetts USA

Stephen A. Morse, MSPH, PhD, Associate Director for Environmental Microbiology National Center for Emerging and Zoonotic Infectious Diseases Centers for Disease Control and Prevention Atlanta, USA

Rhoda A. Morrow, PhD, Professor Department of Laboratory Medicine University of Washington, Seattle, WA Affiliate Investigator, Clinical Research Fred Hutchinson Cancer Research Center Seattle, USA

Francis J. Ndowa, MB, ChB, Dip Derm, Dip GU Med, Medical Officer Department of Reproductive Health and Research World Health Organisation Geneva, Switzerland

Winnie W. Ooi, MD, DMD, MPH, Assistant Professor of Medicine Department of Infectious Diseases Tufts University School of Medicine Lahey Clinic Medical Center Burlington, USA

John R. Papp, PhD, Microbiologist Laboratory Reference and Research Branch, Division of STD Prevention National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention Centers for Disease Control and Prevention Atlanta, USA

Jorma Paavonen, MD, Professor of Obstetrics and Gynecology Department of Obstetrics and Gynecology University of Helsinki Helsinki, Finland

Alan Pillay, PhD, Microbiologist Laboratory Reference and Research Branch Division of STD Prevention National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention Centers for Disease Control and Prevention Atlanta, USA

Angela J. Robinson, MBBS, FRCP, Consultant in Sexual Health Department of Genitourinary Medicine Mortimer Market Centre London, UK

Jack D. Sobel, MD, Professor of Internal Medicine Chief, Division of Infectious Diseases Wayne State University School of Medicine Harper University Hospital Detroit, USA

Stanley Spinola, MD, Director, Division of Infectious Diseases, David H. Jacobs Professor of Infectious Diseases, Professor of Medicine, Mircrobiology and Immunology, Pathology and Laboratory Medicine Indiana University School of Medicine Indianapolis, USA

David Taylor-Robinson, PhD, Emeritus Professor of Genitourinary Microbiology and Medicine Division of Medicine Imperial College of Science, Technology and Medicine St Mary’s Hospital London, UK

Magnus Unemo, PhD, Associate Professor of Clinical Microbiology National Reference Laboratory for Pathogenic Neisseria Department of Clinical Microbiology Orebro University Hospital Orebro, Sweden

Elizabeth Unger, PhD, MD, Chronic Viral Diseases Branch Division of High-Consequence Pathogens and Pathology National Center for Emerging and Zoonotic Infectious Diseases Centers for Disease Control and Prevention Atlanta, USA

Anna Wald, MD, MPH, Professor, Medicine/Allergy & Infectious Diseases; Epidemiology Virology Research Clinic University of Washington Seattle, USA

John Ward, PhD, Director, Division of Viral Hepatitis National Center for HIV/AIDS, Viral Hepatitis, STD and TB PreventionCenters for Disease Control and Prevention Atlanta, USA

Harold Wiesenfeld, MD, CM, Associate Professor, Department of Obstetrics, Gynecology and Reproductive Sciences University of Pittsburgh School of Medicine Pittsburgh, USA
Experience alone does not produce an expert clinician or laboratory scientist. The expert must be able to distinguish the relevant from the irrelevant in the face of new knowledge and emerging technologies in the clinical and laboratory disciplines. The bewildered beginner needs a practical synthesis of the essential facts, the classical together with atypical clinical manifestations of disease and application of the most appropriate clinical techniques, laboratory tests and therapies. This fourth edition of the Atlas of Sexually Transmitted Diseases and AIDS again presents an enormous amount of well-illustrated, up-to-date and practical material on the most common (and several uncommon) sexually transmitted infections. The first chapter reviews genital anatomy and examination, and includes dermatological conditions which may be confused with STDs. The chapters following cover the etiological agents, epidemiology, clinical manifestations, laboratory diagnosis and current treatment recommendations for sexually transmitted infections, including opportunistic infections associated with AIDS.
We thank each of the authors of this edition of the Atlas, who were selected on the basis of their outstanding reputations as clinicians, researchers, and teachers. It is gratifying that they have been able to combine succinct text with a wealth of photographs and helpful illustrations to distill essential, practical information into a uniquely accessible format. Specialists and generalists alike can literally complete the book in a day and, like the editors, will learn dozens of important new findings and approaches (while relearning many old ones!). They will find this a very useful resource to return to, again and again, while teachers in the field will be able to use the illustrations in electronic form to enhance their presentations. The format will also aid the student or trainee to understand and assimilate new information more rapidly, because it is so richly illustrated and so clearly presented.
We believe that the Atlas will provide an additional perspective on the subject to both novice and expert, both as a quick reference and as an additional, pictorial resource when studying for board examinations or applying new, innovative diagnostic tests.

Stephen A. Morse

Ronald C. Ballard

King K. Holmes

Adele A. Moreland
We would like to recognize and express deep gratitude to the late Sumner E (Sam) Thompson, MD and Sidney Olansky, MD for their inspiration, teaching, clinical expertise and hard work. Dr Thompson inspired us to tackle this project and was a co-editor of the first edition of this Atlas of Sexually Transmitted Diseases. His clinical vision inspired physicians, medical students and clinicians at Emory University and its affiliates. Dr Sidney Olansky transmitted his vast knowledge and love of dermatology and syphilology to all those he taught. His long associations with the Fulton County Health Department in conjunction with his teaching in the Dermatology Department at Emory University stimulated remarkable interest in STDs at Emory and the inspiration to produce the first edition of this book.
We would like to thank Sue Hodgson and Nani Clansey at Elsevier Publishing for their help and enthusiasm in tackling this project. We would also like to express our thanks to the authors of previous editions for their contributions, which were responsible for the success of earlier editions of the Atlas. We would also like to acknowledge the contributions made by Samuel K Sarafian, PhD (d1996) Sandra Larsen, PhD (d2001) and Walter Stamm, MD (d2009) for their contributions to previous editions of the Atlas and to the fields of STDs and HIV/AIDS.

Stephen A. Morse

Ronald C. Ballard

King K. Holmes

Adele A. Moreland
Table of Contents
Front Matter
Chapter 1: Genital and Dermatologic Examination
Chapter 2: Gonorrhea
Chapter 3: Infections Caused by Chlamydia Trachomatis
Chapter 4: Genital Mycoplasmas
Chapter 5: Vaginal Infections
Chapter 6: Pelvic Inflammatory Disease
Chapter 7: Syphilis
Chapter 8: Chancroid
Chapter 9: Donovanosis
Chapter 10: Genital Herpes
Chapter 11: Genital Human Papillomavirus Infections
Chapter 12: Viral Hepatitis
Chapter 13: Human Immunodeficiency Virus Infection and the Acquired Immunodeficiency Syndrome: Epidemiology
Chapter 14: Human Immunodeficiency Virus Infection and the Acquired Immunodeficiency Syndrome: Viral Pathogenesis, Laboratory Diagnosis and Monitoring
Chapter 15: Human Immunodeficiency Virus Infection and the Acquired Immunodeficiency Syndrome: Diagnosis and Management
Chapter 16: Sexually Transmissible Infections in Infants, Children and Adolescents
Chapter 17: Infestations
Chapter 18: Syndromic Management
The Laboratory Diagnosis of STDs – Principles, Selection, and Evaluation of Diagnostic Tests
Media, Stains, Reagents and Test Procedures
1 Genital and Dermatologic Examination

A. Moreland, P. Kohl

Skin changes (cutaneous disorders) of the genital skin may be assumed either by patient or physician to be of a sexually transmitted nature because of their location. This chapter will review genital anatomy and examination, and general principles of dermatologic examination. The latter will include common cutaneous disorders of the genitalia that are not sexually transmitted, but that may be seen in a sexually transmitted disease (STD) clinic or be mistaken for a STD.

Genital Anatomy and Examination
The examination of both the male and female genital region should begin below the umbilicus at the mons pubis. It is generally unsatisfactory to try to evaluate a partially clothed patient, because important ancillary findings (e.g. lymph nodes) may be missed. The patient should be undressed below the waist and gowned or draped; the gloved physician then exposes the lower abdomen, buttocks and genitalia in a systematic manner. Exam table stirrups afford better visualization of the genitalia and perianal area in either sex, and should be used if available. Inspect the inguinal folds, noting erythema or scaling and palpating for nodes. Examine pubic hair for nits, lice, papules of molluscum contagiosum, folliculitis, human papillomavirus (HPV), or scabies burrows. Other skin lesions such as blisters (herpesvirus, HSV) or scaly plaques (tinea, syphilis) should be noted, as well as ulcerations anywhere on the genitalia, abdomen or buttocks. At the inferior midline near the male penis or female clitoris, the hair becomes more sparse.

Male Genital Examination

Penis and scrotum
Male genital anatomy is shown in Fig. 1.1 . Deep pigmentation is usual on the shaft of the penis and the hair is almost absent. A few minute yellowish papules may be seen ( Fig. 1.2 ). These are pilosebaceous units (sometimes a vestigial hair and its associated oil gland). Sweat glands are also present on the base, shaft and glans of the penis.

Fig. 1.1 Male genitals.

Fig. 1.2 Sebaceous glands. Penile sebaceous glands appear as yellowish papules on shaft of penis and may also be present on the scrotum. These may be quite prominent on some individuals.
The redundant prepuce (foreskin) projects over the glans where sebaceous glands (of Tyson) secrete a keratinous material called smegma which may accumulate between the prepuce and glans in an uncircumcised male. 1 The inner surface of the prepuce has a moist appearance, much like a mucous membrane. Scattered sebaceous glands empty directly to the surface of the glans and are not associated with hair follicles.
The firm raised ridge encircling the shaft of the penis near the distal tip is termed the corona of the penis. The coronal sulcus, a slight depression, lies proximal to the corona. The tip distal to the corona is termed the glans penis. A varying number of smooth or slightly pebbly flesh-colored papules in one or two orderly rows may rim some or all of the corona; these are a normal variant called ‘pearly penile papules’. They may be mistaken for condylomata (HPV) but histologically are angiofibromas ( Fig. 1.3 ). 2 The urethral meatus is usually located on the posterior or undersurface of the glans and should be carefully examined for discharge, ulcers, or growths such as condylomata.

Fig. 1.3 ‘Pearly penile papules.’ A normal variant, these tiny papules are sometimes mistaken for condylomata.
The skin of the scrotum is thin and more deeply pigmented than the surrounding skin. Scrotal skin is closely adherent to the underlying dartos muscle, which gives it a rugose wrinkled appearance with contraction of the muscle (e.g. at rest in younger individuals, and in the cold at all ages). The scrotum has numerous pilosebaceous, eccrine and apocrine glands. Hair is sparse and coarse. 3
Gentle palpation of the testes, spermatic cord and epididymis within the scrotal sac will reveal any tenderness or masses which may indicate infection. The scrotum should be raised to examine the perineal skin between the scrotum and the anus. Sparse, coarse hair covers the skin up to the anal mucosa and sweat and sebaceous glands are present.

Anus and rectum
The folds of the anus are hairless and should be examined for hemorrhoids, fissures, ulcers, erosions, and growths. By gentle pressure with a gloved finger the rectal mucosa can be palpated for tenderness, ulcers, discharge, or masses beyond the anal sphincter.

Female Genital Examination

The vulva
The female genitalia are shown in Fig. 1.4 . The commonest diagnostic error in evaluation of the vulva is failure to systematically examine the area. Patients may be unaware of proper terminology and anatomy and refer to all areas of the female genital anatomy as vaginal. The vulva is the area between the mons pubis and the perineum bounded by the inguinal folds. For all genital complaints the examination routinely proceeds in the same way from the labia majora inward to assure recognition of the range of normal anatomic variations. During the examination, the physician can reassure the patient by verbalizing normal findings, and an office hand-mirror may help communication between patient and physician regarding specific areas of concern.

Fig. 1.4 Female genitalia.
Courtesy of Stevens A, Lowe J, Histology. London, Mosby, 1992.

The labia majora
The plump paired labia majora fuse anteriorly at the mons pubis and posteriorly merge with the perineal area. They are bounded laterally by the intertriginous folds and are covered laterally by coarse hair. Sweat and sebaceous glands are present so the medial surfaces may be slightly moist. Abnormal findings or signs of infection may be difficult to see because of the pubic hair. 4

The labia minora
In addition to the pigmentation of the outer labia minora, there are two other normal findings which can confuse the untrained examiner. First are the normal smooth yellowish ‘pebbly’ papules that are most numerous at the outer edges of the labia minora. These are sebaceous glands and normally occur along the outer minora and inner majora ( Fig. 1.5 ). Under the clitoral hood, sebaceous secretions called smegma may accumulate. The sebaceous glands usually stop about halfway along the surface of the inner minora, and this line (Hart’s line) demarcates mucosal from squamous epithelium (see Fig. 1.4 ). 5 Patients sometimes mistake these papules for vesicles or pustules and become concerned. In some cases, the sebaceous glands give rise to inclusion cysts; small ones are called milia and larger ones may develop into epidermoid cysts. If they are not symptomatic, they can be ignored.

Fig. 1.5 Normal vulva with finely textured papular sebaceous glands on the inner labia majora and labia minora. In this case, glands are confluent over the outer minora, producing an almost white appearance. By contrast, the vestibular mucosa inside Hart’s line appears red, although this is normal coloration. There is much individual variation in the size and distribution of genital sebaceous glands, but in general, they decrease in number with age.
The second normal variant is the presence of small, often asymptomatic, cutaneous papillae on the inner labia minora, especially at the posterior vaginal introitus ( Fig. 1.6 ). Longer papillae in the posterior introitus have been described as a normal variant and a rough, papillomatous labial mucosal surface often becomes more prominent in inflammatory conditions or lichenification. The clinician should be careful not to overdiagnose benign papillomatosis as ‘subclinical HPV’. Condylomatous HPV lesions in the posterior introitus are typically plaque-like and are relatively easy to differentiate from the delicate stalk-like papillae. 6 , 7

Fig. 1.6 Normal vulva with prominent vestibular papillae on the mucosa inside Hart’s line and at the posterior fourchette. Although they are frequently mistaken for condylomata, biopsied papillae are typically negative for human papillomavirus (HPV). HPV lesions are usually more keratotic and less translucent than papillae; the latter are often symmetrical and/or linear on both sides of the vulva, unlike condylomata. No treatment is necessary.
Some investigations of vulval mucosal papillae initially implicated HPV infection, especially when magnified inspection of papillomatous epithelium revealed an associated mosaic or punctate vascular pattern with capillaries extending into individual papillae. Since there might not have been either a history of previous infection nor obvious condylomata acuminata, it was suggested that papillomatosis represented ‘subclinical’ HPV infection. Research studies using polymerase chain reaction (PCR) technology, Southern blot or other molecular biologic techniques have made this assumption valid. 8 , 9

The vulvar vestibule
The vestibule is the inner portion of the vulva extending from Hart’s line on the labia minora inward to the hymenal ring. Within the vestibule are located the urethral meatus and the openings of Skene’s and Bartholin’s glands ( Fig. 1.4 ). Smaller minor mucous glands are found throughout the vestibule, mostly in the posterior fourchette and in the groove at the base of the hymenal ring, where they may be seen as tiny pit-like openings. Vulvar vestibulitis should be suspected by the patient’s complaint of significant and persistent entry dyspareunia and discomfort at the opening of the vagina. The diagnosis is made by finding erythema and point tenderness upon palpation of the gland orifice with a cotton-tipped applicator. 10
Visible changes (plaques, scarring, thickening) should be biopsied, preferably in the thickest portion of a lesion. Acetowhitening (application of vinegar or 3–5% acetic acid for 1–2 minutes) can be used to highlight thickened areas. If HPV infection is found on the vulva, colposcopy of the vagina and cervix is recommended; if on the anus, proctoscopy. Biopsies should be performed on any diagnostically questionable areas, especially if intraepithelial neoplasia is suspected.

The vagina and cervix
Before inserting a speculum into the vagina, gentle pressure with two fingers on the posterior fourchette relaxes the muscles at the vaginal opening. To view the vagina, a warm speculum of proper size, moistened with water, should be inserted with the blades closed and positioned obliquely. The blades are then slipped horizontally and opened slowly.
The moist vaginal mucosal lining is erythematous and has a slightly irregular surface. Numerous transverse and longitudinal folds give the vaginal canal a rugose appearance. The cervix appears at the end of the vaginal vault as a firm, smooth, somewhat circular or dome-shaped mass with a central concavity, the os ( Figs 1.7 – 1.10 ), which is the entrance to the endocervical canal. Notations of vaginal discharges, lesions, or ulcers, and cervical mucosal abnormalities, ectropion, or lacerations should be made. Before the speculum is removed, samples can be obtained for cytology, cultures, and other diagnostic tests, including direct microscopy of vaginal and cervical secretions.

Fig. 1.7 Normal cervix. The squamocolumnar junction is seen and also the lower part of the endocervical canal. (See also Fig. 3.17 .)
Courtesy of Mr Peter Greenhouse.

Fig. 1.8 Stratified squamous epithelium covers the ectocervix. Like those of the vagina, the cells are rich in glycogen during the period of sexual maturity.
Courtesy of Stevens A, Lowe J, Histology. London, Mosby, 1992.

Fig. 1.9 Photomicrograph of the endocervical canal. (Left) The endocervical canal is lined by a single layer of tall columnar mucus-secreting epithelium. (Right) Numerous deep invaginations of the mucus-secreting epithelium extend into the cervical stroma and greatly increase the surface for mucus production.
Courtesy of Stevens A, Lowe J, Histology. London, Mosby, 1992.

Fig. 1.10 Colposcopy. Ectopy showing early squamous metaplasia.
Courtesy of Mr Peter Greenhouse.

Bimanual examination
The middle and index fingers of one hand should be inserted along the posterior vaginal wall after the speculum is withdrawn. The cervix is then lifted toward the abdominal wall and the opposite hand presses down to palpate the uterus, which can be gently moved to determine presence of tenderness or tumors. The ovaries and Fallopian tubes (adnexa) are found laterally or posterolaterally to the uterus. Bimanual palpation will usually ascertain tenderness and presence of masses. A clean glove should be used for the rectovaginal examination. The examining finger is gently placed into the anal opening and when the sphincter is relaxed the examination can comfortably proceed. The index finger is placed into the vagina and the middle finger into the rectum to palpate the posterior uterine and vaginal structures. Rectal hemorrhoids, polyps, and tumors can be observed and noted.

Principles of Dermatologic Examination of the Genitalia
Although it is traditional to classify and discuss infectious diseases and conditions with respect to etiology, this approach has significant shortcomings when applied to cutaneous disorders. A 3 mm papule, for example, can be a congenital nevus, a benign or malignant neoplasm, or the result of infection with a bacteria, a virus, or a fungus. Recognizing dermatologic diseases is facilitated by searching for primary lesions, which provide a starting point for the development of a differential diagnosis. Primary lesions are fresh, fully developed lesions, which have not dried, crusted, eroded, been scratched, or become secondarily infected. Texture, size, pigmentation, or other identifiable characteristics must be noted. Finding and describing the primary lesion morphology should then be followed by observation of the distribution and configuration of the lesions on the skin. The patient should be asked whether similar lesions or other cutaneous changes are present on other areas of the body. The cutaneous examination should be expanded as necessary to help categorize and identify the extent of the disorder.
With these principles in mind and for the purpose of this discussion, the genital dermatoses which are not sexually transmitted will be grouped into seven different morphologic categories. Six of these are defined by the primary lesion, such as pustules, pigmentary disorders, and dermatitis. The seventh category, erosions and ulcers, deals with the so-called ‘minus’ lesions, wherein the original morphology has been altered by the loss of superficial epidermis (erosion) or of the entire skin surface itself (ulcer). This latter category may be difficult to assess, for normal morphologic clues to differential diagnosis frequently are absent.
The final category is itching, defined by the symptom. This section will discuss evaluation of pruritus ani, scrota, and vulvae.

The term dermatitis simply means inflammation of the skin. The Greek root eczema, which means ‘boiling over or out,’ is remarkably descriptive of the oozing, wet appearance of dermatitic skin. Eczemas characteristically are pruritic. The patient complains of itching, and scratch marks (excoriations) may be seen on the skin surface. Dermatitis typically changes its appearance over time. The first sign simply may be erythema, which is followed by a pebbly appearance to the skin surface that rapidly evolves into small blisters which may ooze and crust ( Fig. 1.11 ). As dermatitis evolves, the skin becomes thickened, leathery, and often scaly, with increased skin markings. These findings are the hallmark of lichenification ( Fig. 1.12A and 1.12B ) and are even more important than scaling in making this diagnosis. The patient’s rubbing or scratching of the initial condition will increase the likelihood of lichenification, which persists long after the original insult has been removed. Acute dermatitis, then, is seen as a plaque that is erythematous, edematous, and oozing; chronic dermatitis is a plaque that may be purplish, hyperpigmented, and lichenified. In the latter case, the patient is said to have lichen simplex chronicus, a descriptive term that indicates only that the patient has a plaque of thickened skin that has been rubbed or scratched. Any area of the body may be involved ( Fig. 1.13 ), but genital skin is a common area of involvement ( Figs 1.14 , 1.15A, and 1.15B ). Some underlying skin conditions, such as atopic dermatitis, may make it more likely that the patient will develop areas of lichen simplex chronicus. In other cases, the skin reaction is due to something that has come in contact with the epidermis. The offending substance may be an irritant such as urine or a true allergen. Neomycin and benzocaine are relatively common allergens found in non-prescription topical medications. These medications may be self-prescribed by patients or prescribed by physicians to treat both pruritus and any type of irritation, abrasion, or ulcer. 11

Fig. 1.11 Allergic contact dermatitis of the penis due to spermicidal jelly. Note the typical appearance of microvesicles on the glans penis. This patient complained of pruritus and rash developing approximately 2 days after use of the product.

Fig. 1.12A Lichenification of intertriginous skin as a result of chronic rubbing and scratching with an ulcer due to scratching (excoriation).

Fig.1.12B Thickened skin markings and excoriations in lichen simplex of the scrotum.

Fig. 1.13 Lichen simplex chronicus on the foot. The hallmark of this diagnosis is the leathery appearance of the skin.

Fig. 1.14 Lichen simplex chronicus of the scrotum. The accentuation of normal skin markings is shown clearly. The inguinal area is hyperpigmented—a milder sign of continuous rubbing.

Fig. 1.15A Vulvar lichen simplex chronicus. The extensiveness of the area involved suggests that pruritus has been present for several months or more. The skin is lichenified, scaly, and in some areas hyperpigmented.

Fig. 1.15B Chronic scratching caused the lichenification on the mons and the hyperpigmentation on the thighs. Mild edema and erythema is still visible on the labiae in this resolving dermatitis.

Papulosquamous disorders
The papulosquamous dermatoses, as the name implies, are characterized by papules and plaques that typically have a scaly surface. While a plaque of lichen simplex chronicus might fit this description, it should be noted that lichenification is secondary to rubbing and scratching of the affected skin. The papulosquamous dermatoses, on the other hand, begin with a scaly papule as the primary lesion. Of all dermatologic disorders, probably the most commonly encountered are those in the papulosquamous category, and it is important for the clinician to develop a logical approach to the differential diagnosis of these problems ( Table 1.1 ).

Table 1.1 Differential diagnosis of common papulosquamous dermatoses.
The acute onset of a pruritic annular lesion anywhere on the body, especially in intertriginous areas, should raise the suspicion of a dermatophyte ( Fig. 1.16 ) infection. Scraping a bit of scale from the border of a lesion and examining it under 10–20% potassium hydroxide (KOH) solution will allow the visualization of fungal hyphae ( Figs 1.17 – 1.20 ). Dermatophyte infections are more common in men than in women, but the latter are more likely to develop candidal infections, usually as a result of spread from the vagina (see section on pustular disorders, pp. 11 – 16 ). Griseofulvin is effective only for dermatophytes and nystatin only for Candida , but the imidazole antifungals are effective treatment for both dermatophyte and candidal infections. 12 , 13 Annular lesions also may occur in secondary syphilis, but syphilis only rarely itches, and no hyphae can be seen on KOH and, of course, the serology is positive.

Fig. 1.16 Tinea cruris. Erythema and scaling associated with pruritus are typical features of a dermatophyte infection. Scrapings for potassium hydroxide (KOH) and fungal cultures should be taken from the leading edge of the involved skin, even though scaling there may be minimal.

Fig. 1.17

Fig. 1.18

Fig. 1.19

Figs 1.17–1.20 Examination of skin scraping for fungal infection with potassium hydroxide (KOH) solution.
Fig. 1.17 (top left) Equipment needed for KOH examination. Curved scalpel blades, glass microscope slides, 10 to 20% KOH, glass coverslips, heat source, and microscope are shown.
Fig. 1.18 (top right) A curved scalpel blade allows gentle scraping of the skin with minimal trauma. The scale should be collected directly onto a glass slide and the coverslip applied.
Fig. 1.19 (lower left) KOH applied by dropper to the edge of the covered specimen allows it to penetrate under the coverslip by capillary action. The slide is gently warmed, without boiling, to allow clearing of the specimen. The alcohol lamp produces a cleaner flame than do matches.
Fig. 1.20 (lower right) Microscopic view of branched hyphae among cleared keratinocytes as they appear in a positive KOH preparation.
Psoriasis is another commonly encountered papulosquamous disorder with a distinct familial association, even though many patients are unaware of family members with psoriasis. Typically seen as thick, red plaques with adherent white scales, psoriasis occurs most commonly on the arms ( Fig. 1.21A ), knees, elbows, trunk, and sacrum, as well as the scalp ( Fig. 1.21B ). Genital lesions, however, are apt to have little, if any, scale, and may be seen simply as persistent intertriginous erythema ( Figs 1.22 , 1.23A, and 1.23B ) usually with sharply demarcated borders which helps to differentiate it from other intertriginous conditions. Scaly plaques may be seen on the penis ( Fig. 1.23C ) and scrotum as well as on the pubic area, and in some cases may closely resemble the papulosquamous form of secondary syphilis, with minimal involvement of the rest of the body. Fingernail pitting may lead one to suspect the diagnosis of psoriasis in a persistent genital papulosquamous disorder. A mild topical corticosteroid (hydrocortisone 1%) generally is effective in treating genital psoriasis. Topical pimecrolemus and tacrolimus may also be used, though this use is currently off-label in the United States. 14 The use of strong fluorinated steroids on genital skin may lead to the development of cutaneous atrophy or striae, which are permanent and unsightly.

Fig. 1.21A Psoriasis. Thick reddish plaques with an adherent thick white scale are typical on non-genital skin such as the arms, elbows, knees, and scalp.

Fig. 1.21B Psoriasis of the scalp with well demarcated pink plaques and white scale behind the ear in the patient seen in Fig 1.23 with genital psoriasis.

Fig. 1.22 Psoriasis of the vulva. The typical thick scale seen here is sometimes absent when psoriasis occurs on the genital skin, leaving erythematous patches and plaques with a more macerated and moist scale, or with no scale at all.

Fig. 1.23C Psoriasis of the penis. The typical intense erythema of psoriatic plaques, but with complete lack of scale, is seen in this largely intertriginous plaque of psoriasis under the foreskin.

Fig. 1.23A Psoriasis of the penis. Scaling is present in this case of non-intertriginous penile psoriasis.

Fig. 1.23B Psoriasis of the natal cleft. Intertriginous plaque lacks thick scale.
Seborrheic dermatitis usually is seen as scaling in the hairy areas of the body, with a more or less prominent erythematous and papular component. Most commonly diagnosed in the scalp as ‘dandruff’, seborrheic dermatitis also affects the eyebrows, nasolabial folds ( Fig. 1.24 ), axillae, central chest, and genital region. Women usually experience only mild erythema and scaling on the mons pubis ( Figs 1.25 and 1.26 ), but men may have erythematous plaques on the penis ( Fig. 1.27 ) that are difficult to differentiate from psoriasis or secondary syphilis. Treatment with mild corticosteroids is effective in this condition, as well.

Fig. 1.24 Typical seborrheic dermatitis in the nasolabial crease. Erythema with mild scaling is seen.

Figs. 1.25

Figs. 1.25 (left) and 1.26 (right) Seborrheic dermatitis of the vulva may present as pruritus of the vulva or mons. The skin appears red or slightly pigmented and thick ‘dandruff’ scales may be seen.
Courtesy of du Vivier A, Atlas of Clinical Dermatology. New York, Gower Medical Publishing, 1986.

Fig. 1.27 Seborrheic dermatitis on the penis. Erythematous changes are sometimes difficult to differentiate from psoriasis, and may resolve with a mottled hypopigmentation.
Lichen planus (classically described as purple, pruritic, polygonal papules and plaques) is not as scaly as are the above disorders. The typical areas of involvement are the flexor surfaces of the wrists, the trunk ( Fig. 1.28 ), and the anterior shins. An examination of the tongue ( Fig. 1.29 ) or buccal mucosa ( Fig. 1.30 ) may show a lacy white pattern and sometimes erosions difficult to distinguish from Candida or thrush. Genital mucosal surfaces may exhibit several types of lesions including a similar lacy white pattern, and scaly or smooth violaceous papules ( Figs 1.31 and 1.32 ). Treatment of lichen planus is symptomatic, with corticosteroids and, if necessary, antipruritic agents. Lichen sclerosus and the so-called mixed dystrophies (see below) may be responsible for thickened, scaly plaques appearing on the genitalia. Although these disorders are not always pruritic, itching may occur primarily or may be secondary to medications that have been applied to the affected area. A biopsy may be necessary to differentiate lichenified dermatitis from a primarily papulosquamous disorder.

Fig. 1.28 Lichen planus on the trunk. Typical violaceous flat-topped papules are seen, some with angular borders and adherent scale in the form of Wickham’s striae.

Fig. 1.29 Oral lichen planus of the tongue. Whitish plaques are seen centrally.
Courtesy of Emory University School of Dentistry.

Fig. 1.30 Oral lichen planus. Thin whitish linear streaks or Wickham’s striae are seen on the buccal mucosa. This is not symptomatic unless it is erosive.
Courtesy of Emory University School of Dentistry.

Fig. 1.31 Flesh-colored papules of lichen planus have a lacy white surface and assume an annular configuration.
Courtesy of du Vivier A, Atlas of Clinical Dermatology. New York, Gower Medical Publishing, 1986.

Fig. 1.32 These papules on the glans are scalier and more extensive than those in Fig. 1.31 .
Courtesy of du Vivier A, Atlas of Clinical Dermatology. New York, Gower Medical Publishing, 1986.

Pigmentary disorders

A black macule on the genitalia is an obvious lesion of concern, for it is important to rule out malignant melanoma as a diagnostic possibility. 15 , 16 Typically, however, a melanoma ( Fig. 1.33 ) is a single lesion with an irregular ‘notched’ border with variable hyperpigmentation, which also may show areas of depigmentation within the larger macule. This malignant change should be distinguished from that of freckle or lentigo ( Fig. 1.34A and 1.34B )—benign macules having regular borders and smooth pigmentation. Diffuse hyperpigmentation as a result of chronic inflammation, postinflammatory hyperpigmentation, also can occur as multiple macules, giving a ‘spotty’ appearance to genital skin, especially around the vaginal introitus ( Fig. 1.35 ).

Fig. 1.33 Malignant melanoma. Note the asymmetry, irregular contours, and variable pigmentation that are the hallmarks of this malignancy.

Fig. 1.34A Lentigines of the vulva. Multiple dark macules or freckles on the labia minora and vaginal introitus may appear as a result of previous inflammation, but single lesions should be evaluated carefully to rule out the possibility of malignant melanoma.

Fig. 1.34B Penile lentigo. Sharp borders, regular pigmentation and symmetry characterize a benign lentigo.

Fig. 1.35 Postinflammatory hyperpigmentation of the vulva. The spotty hyperpigmentation on the right labium majus and left labium minus seen in association with a more diffuse hypopigmentation around the clitoris and the lower introitus.
Another form of diffuse hyperpigmentation, but with a thickened velvety appearance to the skin, is that of acanthosis nigricans. This pigmentary change may be seen around the neck ( Fig. 1.36 ), genitalia ( Fig. 1.37 ), and the axillae of genetically predisposed obese individuals or in some patients with endocrine abnormalities. This ‘benign’ or pseudoacanthosis nigricans cannot be distinguished clinically or histologically from the form that is associated with internal malignancy, usually a gastric adenocarcinoma. Thus a thorough evaluation for malignancy should be made in patients who present with new-onset acanthosis nigricans. Unfortunately, the malignancy may be well established by the time the cutaneous changes are seen. 17

Fig. 1.36 Pseudoacanthosis nigricans of the neck. The finely papillated surface of the skin gives it a velvety appearance. This feature, in combination with hyperpigmentation, is the cardinal sign of acanthosis nigricans of any etiology.

Fig. 1.37 Acanthosis nigricans of the vulva. This patient, who has extensive involvement of all intertriginous skin and of the hands and mouth, was found to have gastric adenocarcinoma—the most common cancer associated with this disorder. The acute onset of thick, velvety intertriginous plaques, hyperpigmented or not, should prompt a thorough evaluation for internal malignancy.

By far the most common color change on the genitalia is the loss of pigment in the form of vitiligo. This pigment loss is quite remarkable in persons with dark complexions and may be overlooked entirely in fair-skinned people. Characteristically symmetric in distribution, it may be seen as white patches on the glans penis ( Fig. 1.38 ) or as a ‘keyhole’ pattern around the vagina ( Fig. 1.39 ) and anus. When it occurs on other areas of the body, it also is often periorificial, around the mouth, eyes ( Fig. 1.40 ), and nares. Vitiligo also may develop distally over the fingers and toes, again, in a typically symmetric pattern. Asymmetric vitiligo is unusual but does occur, often in a dermatomal distribution. Some vitiligo patients have autoimmune thyroid disorders or diabetes, but many have no systemic abnormalities. 18 , 19 Treatment should be directed to a dermatologist, but spontaneous repigmentation has been known to occur. Postinflammatory hypopigmentation may be seen after an episode of primary or secondary syphilis ( Fig. 1.41 ), any form of genital ulcer, a dermatophyte infection, or chronic dermatitis or intertrigo ( Figs 1.42A, 1.42B , and 1.43 ).

Fig. 1.38 Vitiligo of the glans penis. This is a relatively common condition, which, although asymptomatic, may be a source of great anxiety for the patient.

Fig. 1.39 Vitiligo of the vulva. This photograph shows the typical symmetric loss of pigmentation from the periorificial skin. Notice that the epidermis is quite normal in appearance. There is no sign of the atrophy usually associated with lichen sclerosus, which also may be hypopigmented.

Fig. 1.40 Periorificial facial vitiligo. The patchy symmetric loss of pigmentation in vitiligo may be localized to one area of the body or it may involve many sites. In this patient, the vitiligo was confined to the face and extremities.

Fig. 1.41 Postinflammatory hypopigmentation and hyperpigmentation of the penis. This finding may be caused by a balanitis or a syphilitic chancre may have been the cause of the spotty pigmentation of the glans that appeared months prior to the development of a generalized papular eruption. This generalized eruption proved to be a manifestation of secondary syphilis.

Fig. 1.42A Postinflammatory hypopigmentation of the foreskin and corona of the penis. Seborrheic dermatitis caused the pigment changes in this patient, who visited the STD clinic for this problem.

Fig. 1.42B Vulvar hypopigmentationn associated with long term hydrocortisone use in lichen simplex chronicus.

Fig. 1.43 Postinflammatory hypopigmentation and hyperpigmentation of chronic intertrigo. Pigment variations may be seen as inflammatory cutaneous conditions flare and resolve.

Itching or burning may be the presenting symptom in lichen sclerosus (lichen sclerosus et atrophicus). Occurring more commonly on female genitalia, this condition is seen clinically as depigmentation of the skin ( Fig. 1.44A ). The atrophic epidermis shows fine ‘cigarette paper’ wrinkling, while the sclerotic or thickened dermis obscures normal capillary filling, giving a white appearance to the skin. Severe cases may result in complete resorption of the labia minora, and vulvar adhesions are not uncommon ( Fig. 1.44B and 1.44C ).

Fig. 1.44A Lichen sclerosus of the vulva. Thinning and atrophy of epidermal skin are seen with loss of architecture of the labia minora, including adhesion formation at the posterior introitus. Note the presence of erosions and petechiae secondary to mild trauma of the fragile skin.

Fig. 1.44B Lichen sclerosus of the vulva. Atrophy, edema and pigmentary changes in this painful chronic case of lichen sclerosus.

Fig. 1.44C Perianal lichen sclerosus. Perianal hypopigmentation and telangiectasia in patient.
The etiology of this condition is unknown, and occasionally it may be seen in young girls, in some cases resolving at puberty. Symptoms vary in such cases of lichen sclerosus, ranging from the patient’s complete unawareness of the problem to severe itching and burning. The thinned epidermis is extremely friable, and petechiae or purpura may be seen as a result of scratching. 20 When seen in the male, lichen sclerosus may cause the glans penis to have an extremely white, scarred-down appearance known as balanitis xerotica obliterans ( Figs 1.45A , 45B ). As with lichen sclerosus in the female, balanitis xerotica may respond to topical treatment with glucocorticoids.

Fig. 1.45A Balanitis xerotica obliterans. Lichen sclerosus on the glans penis exhibits white atrophic patches similar to those seen on the vulva. Meatal stenosis may occur.

Fig. 1.45B Absolute phimosis caused by lichen sclerosus of the foreskin. This finding needs surgical intervention.
In some cases, lichen sclerosus may develop discrete areas of thickened hyperkeratotic stratum corneum ( Fig. 1.46 ). Several biopsies should be taken from different areas of thickened dystrophic skin to rule out the possibility of vulvar intraepithelial neoplasia (VIN). 21 Atrophic vaginitis may be seen in the postmenopausal woman, though cutaneous changes may consist only of mild thinning and loss of subcutaneous substance. 22

Fig. 1.46 (A) Early changes in lichen sclerosus. White thickened areas of vulva caused by lichen sclerosus. (B) More advanced vulvar lichen sclerosis showing hypertrophic plaques, edema, loss of normal architecture, introital narrowing and perineal involvement. This patient has biopsy-proven lichen sclerosus with areas of cutaneous hyperplasia. There was no evidence of malignancy.

Most physicians regard the presence of pustules on the skin as prima facie evidence of infection. In most cases this is true, and infection certainly should be ruled out when pus-containing papules are seen. The presence of pus generally implies infection; however, there are certain cutaneous conditions that are characterized by the presence of aggregates of white cells that are sterile to culture for bacteria, fungi, or viruses. In the following section, we will discuss the pustular conditions of the genitalia ( Tables 1.2 and 1.3 ).
Table 1.2 Infectious pustular conditions occurring on the genitalia. Condition Findings Treatment Candidiasis Most common, itches and burns. Imidazole or azole creams   Intense erythema; often edema, satellite lesions. Oral ketoconazole or fluconazole in resistant cases—short courses. Tinea Serpiginous ‘active’ border, itchy, relatively unusual in women. Topical imidazole or azole or oral antifungals. Impetigo Usually secondary to pruritic or irritant dermatitis, excoriations, Topical antibacterial scrubs, topical or oral antibiotics. Folliculitis Pustules at base of hairs, Benzoyl peroxide or mupuricin, topical and oral anatibiotics if necessary according to cultures and sensitivities. Furunculosis Painful, deep-seated nodules may be topped by pustules; may suppurate; recurrent lesions may indicate transmission by close contact. Early treatment with mupuricin or oral antibiotics may abort early lesions and prevent suppuration. Herpes simplex WBCs in old intact vesicles may cause lesions to look pustular. Antivirals or topical antibiotics in mild cases. (See Chapter 10 , Herpes.) Syphilis Scattered scaly pustules may be seen in secondary syphilis. Penicillin: see treatment schedule in Chapter 7 .
Table 1.3 Non-infectious pustular conditions occurring on the genitalia. Condition Findings Treatment Pseudofolliculitis Ingrown hairs indicate mechanical trauma. Stop shaving or use benzoyl peroxide, wash prior to shaving. Acneiform rashes Withdrawal of potent topical steroids; contact with oils. Wean off hydrocortisones. Eliminate work-related industrial hydrocarbon exposure. Hidradenitis suppurativa Chronic acneiform condition with sinus tracts and scarring. Minocin, 100 mg po daily. Surgical excision of affected area. Pustular psoriasis Often associated with arthritis. Systemic or phototherapy. Refer to dermatologist. Pemphigus (acquired P. vulgaris or Chronic Familial Pemphigus) Erosions, pustules or bullae Refer to dermatologist
While the presence of pus generally implies infection, this finding is not specific. Just as there are non-pyogenic infections, there are certain pustular skin conditions not at all associated with infectious organisms. Gram stains of pustule contents should be examined for bacteria and Gram-positive budding yeast forms; KOH of the pustule roof may reveal fungal hyphae; and bacterial cultures should be done on material from cleaned, intact lesions. If lesion morphology suggests herpes, Tzanck smears and viral cultures should also be performed; dark-field examination should be done if syphilis is suspected.

Infectious pustules
One of the most common causes of genital pustules, especially with inflammation, is cutaneous candidiasis or monilia ( Fig. 1.47 ). Skin lesions generally are seen in conjunction with a candidal vaginitis in the female ( Fig. 1.48A ). Males may also harbor the organism ( Candida albicans ) in the inguinal or gluteal folds ( Fig. 1.48B ) on the scrotum and, especially if uncircumcised, on the penis ( Fig. 1.49 ). Factors predisposing to cutaneous candidiasis include immunosuppression, diabetes mellitus, and the administration of systemic antibiotics. 22 While candidal pseudohyphae sometimes may be seen on KOH examination of material from superficial intertriginous erosions, the better diagnostic tests for this organism are a Gram or PAS stain of material from a pustule. The typical budding yeast forms are Gram-positive and somewhat larger than lymphocytes ( Fig. 1.50 ).Topical treatment with antifungal creams is usually successful. 23

Fig. 1.47 Candida infection showing the intense inflammation with satellite pustules. Note that the pustules are superficial and not located at the base of hairs.

Fig. 1.48A Candida vulvovaginitis. Intense erythema and edema appear around the introitus, perineum, and perianal areas. The discrete erythematous macules at the active borders are resolving pustules.

Fig. 1.48B Candida intertrigo. Confluent erythema, scaling and satellite pustules at the periphery of the involved area in pannicular and inguinal folds.

Fig. 1.49 Candida balanitis showing edema and erythema in a diabetic. This is seen most commonly in uncircumcised males. Candidiasis should be considered a sexually transmissible disease, treatment with topical antifungal creams may be adequate but systemic antifungals may at times be necessary.

Fig. 1.50 PAS stain of budding yeast and pseudohyphae seen in Candida albicans .
Acute inflammatory tinea infections may have a vesiculopustular scaly border ( Figs 1.51A and 1.51B ). KOH of blister or pustule roof will demonstrate fungal hyphae. In the presence of a chronic intertrigo, foci of dermatophyte infections may remain deep in follicles, which can occasionally become nodular (Majocchi’s granuloma). The diagnosis of fungal folliculitis should be considered when the patient fails to respond to systemic antibiotics.

Fig. 1.51A Tinea corporis showing vesicles and pustules at the active advancing edge of a typical scaly plaque.

Fig. 1.51B (right) Tinea corporis of buttocks and perineum. Erythemetous serpiginous plaques with well-defined borders and scale are classic signs of tinea infection.
Discrete, scattered pustules in hairy areas of the body generally are caused by Staphylococcus aureus and less frequently, Streptococcus pyogenes . Superficial infections usually respond well to topical antibiotics such as mupuricin 2%. Oral antibiotics should be used conservatively and cultures and sensitivities used to guide therapy as methicillin reisitant strains of bacteria may be present. 24
In susceptible individuals, folliculitis may develop into a larger cutaneous abscess called a carbuncle or a furuncle ( Fig. 1.52 ). Typically caused by Staphylococcus aureus , early lesions will respond to systemic antibiotics. Most later lesions benefit from application of warm compresses until spontaneous rupture of the abscess occurs, but fully developed walled-off abscesses may require incision and drainage. Recurrent furunculosis does not necessarily imply that a patient has an immune deficiency. Pustules also may be seen in mixed bacterial impetigo ( Fig. 1.53 ), an extremely common skin infection that may be the result of secondary bacterial colonization of a pre-existing dermatitis. 25

Fig. 1.52 Carbuncle on the upper thigh. A thick crust with surrounding erythema and tenderness is characteristic.

Fig. 1.53 Impetigo. Pustules, pus-filled bullae, crusts, and erosions are all present in this superficial bacterial skin infection, which may be localized to the groin in sexually active patients.
While the umbilicated papules of molluscum contagiosum ( Figs 1.54 and 1.55 ) are not actually pustules, the initial appearance of these lesions may mislead the patient and physician. Since usually they are pale or flesh-colored, they can give the appearance of pustules; however, they are actually rather sturdy papules, which may persist for many weeks. The central dell or umbilication is characteristic of the viral etiology of these lesions, which are caused by a pox virus. Therapy is directed toward destruction of the lesion, with curettage or blistering agents applied to the lesions.

Figs 1.54

Figs 1.54 (left) and 1.55 (right) Molluscum contagiosum. Flesh-colored papules of molluscum may be distinguished by their umbilicated centers. The papules contain a white cheesy substance, which may be stained for the presence of viral inclusion bodies.
Courtesy of du Vivier A, Atlas of Clinical Dermatology. New York, Gower Medical Publishing, 1986.

Non-infectious pustules
In most clinical situations, the presence of pus implies infection, and it is entirely appropriate to obtain bacterial, fungal, and/or viral cultures in this setting. There are certain dermatologic conditions, however, in which pustules or the accumulation of white cells in the epidermis is initiated by stimuli other than bacterial infection.
While bacterial superinfection can play an important part in hidradenitis suppurativa, also termed acne inversa ( Figs 1.56A and 1.56B ), the mechanism of this severe acneiform eruption in the groin and/or axillae ( Fig. 1.57 ) is related to occlusion of hair follicles and retention of follicular contents, resulting in an inflammatory process that includes hair follicles and sweat glands. Secondary bacterial infection is common. The presence of multiple papules, pustules, cysts, and sinus tracts is the cutaneous constellation common to cystic acne, hidradenitis, and dissecting folliculitis of the scalp, which may occur together. In some chronic cases, keloid formation may be the most prominent feature of a ‘burned-out’ case of hidradenitis. Antibiotic therapy can be helpful in acute flares of this disease, and resistance to tetracycline or erythromycin should raise the suspicion of superinfection with Gram-negative organisms. Surgical excision and grafting remains the treatment of choice for recalcitrant cases, although the retinoids have shown some promise in the treatment of this distressing condition. 26 , 27

Figs 1.56

Figs 1.56A (left) and 1.56B (right) Hidradenitis suppurativa of the vulva. Indolent painful pustules and nodules are associated with this chronic disorder. Sinuses and scars result.
Courtesy of du Vivier A, Atlas of Clinical Dermatology. New York, Gower Medical Publishing, 1986.

Fig. 1.57 Axillary hidradenitis suppurativa. Draining nodules, sinus tracts and scars as well as postinflammatory hyperpigmentation typify hidradenitis suppurativa.
Pustular psoriasis ( Fig. 1.58 ) may begin as groups of sterile pustules in intertriginous areas. These rapidly enlarge and spread across the trunk and extremities in waves that coalesce, forming ‘lakes’ of pus in the superficial epidermis. This severe form of psoriasis is associated with high fevers and malaise. It occurs primarily in patients already diagnosed with psoriasis and is seen sometimes as a result of systemic steroid therapy. Acute episodes may be difficult to manage, and generally respond best to systemic therapy with antimetabolites such as methotrexate or cyclosporin or with systemic retinoids. 28

Fig. 1.58 Pustular psoriasis. Typical clusters of pustules arise in intertriginous areas and spread outward, forming ‘lakes’ of pus at the periphery of the eruption. Patients are febrile and ill, though the pustules are sterile. This form of psoriasis is relatively rare but may be precipitated by systemic corticosteroid therapy.
Reactive arthritis, formerly known as Reiter’s syndrome, is an uncommon condition in which urethritis and arthritis may be associated with psoriasis-like lesions on the skin, including an inflammatory condition of the penis or vulva known as circinate balanitis or vulvitis. 29 , 30 The urethritis and involvement of genital mucosa make the STD clinic a likely setting in which to diagnose this disease. The circinate balanitis ( Figs 1.59A, 1.59B ) may appear as non-scaly erythematous plaques, or the eruption may be more pustular, crusted, and scaly. On non-genital skin, it is very similar in appearance to pustular psoriasis ( Fig. 1.60 ). Arthritis is also a typical feature, and conjunctivitis also may be seen. Patients with reactive arthritis usually have histocompatibility antigen HLA-B27, 31 with a high risk of developing ankylosing spondylitis. A link to infections caused by Chlamydia trachomatis has been postulated. Fortunately, skin lesions often respond to low-potency topical corticosteroids. The arthritis may be more difficult to treat and can be disabling. 32

Fig. 1.59A and B Balanitis circinata. Reiter’s disease presented in this patient with an erythematous circinate eruption on the glans penis resembling psoriasis. This disorder is associated with HLA-B27, and symptoms of arthritis are extremely common.

Fig. 1.60 Reactive arthritis. Scaly papules cover the instep and heel. Palmar and plantar involvement is termed keratoderma blenorrhagica.
Benign familial pemphigus (Hailey–Hailey disease) frequently presents as pustules and erosions in intertriginous areas ( Fig. 1.61 ), but this inherited disorder can easily become widespread or superinfected with Candida or bacteria, which may obscure the initial diagnosis. The familial occurrence and chronicity, as well as a typical histologic picture, make diagnosis relatively easy, although the varied spectrum of lesions from hyperkeratotic papules to erosions may mislead the clinician who looks for fluid-filled vesicles in this so-called ‘bullous disease’. Treatment must include appropriate antibiotics or antifungals and, if necessary, corticosteroids, retinoids, antimetabolites or surgery. 33

Fig. 1.61 Erosive lesions of benign familial pemphigus (Hailey–Hailey disease) on the scrotum and groin. Traumatic loss of the blister roof in an intertriginous area may cause an otherwise typical bullous disease to appear as multiple erosions.

Nodules and tumors
Epidermoid cysts are firm, yellow, subcutaneous nodules that may occur singly or, in some cases, prolifically over the vulva or scrotum ( Fig. 1.62 ). Treatment usually is sought when the cysts rupture or become secondarily infected. Although cutaneous crusting and erosion may be present over the cyst, the nodular nature of the lesion is unlike other sexually transmitted genital ulcers, which are more superficial. Antistaphylococcal antibiotics and sitz baths usually resolve the secondary infection and, if necessary, the cyst may later be removed. In most cases, the patient is aware of the diagnosis, although with multiple lesions one also should consider the possibility of steatocystoma multiplex. The latter cysts extrude a clear to yellowish gel-like material when punctured, and often appear on the face, neck, upper trunk, and axillae as well. This condition is a hereditary disorder, primarily of cosmetic concern.

Fig. 1.62 Epidermoid cysts of the scrotum. Generally asymptomatic, these lesions occasionally may rupture and cause discomfort to the patient. They should be differentiated from steatocystoma multiplex, which contain a gel-like material rather than the thick, yellow, sebaceous substance typical of the epidermoid cyst.
Fox–Fordyce disease is characterized by aggregations of tiny 1–2 mm papules in the groin or axilla. This hereditary condition affects the apocrine sweat ducts and is much more common in women than in men. The most common complaint is severe pruritus, which may respond to systemic estrogen therapy. 34
Keloids ( Fig. 1.63 ) are irregular, often linear, firm nodules, seen most often in patients with recurrent episodes of folliculitis or hidradenitis. They should be differentiated from epidermoid cysts, for they will often respond to intralesional steroid therapy, and excision may worsen the condition.

Fig. 1.63 Vulvar keloids. Thickened, linear nodular scars are present on both labia majora. Inciting factors in susceptible individuals include any inflammation or infectious or traumatic insult to skin.
Seborrheic keratoses are elevated ‘stuck-on’ growths that may be pigmented or flesh-colored, which most often appear on the trunk ( Fig. 1.64A ) but may occur on the genitalia ( Fig. 1.64B ). These warty growths are quite benign, and similar lesions are usually found elsewhere on the body. They require removal only if they occur in areas where friction from clothing causes irritation. In intertriginous areas, seborrheic keratoses or even simple acrochordons (skin tags) may, with time, become pedunculated and prominent ( Fig. 1.65 ). When one of these lesions becomes twisted on its stalk the entire lesion may infarct, becoming black and alarming the patient.

Fig. 1.64A Seborrheic keratosis. This thickened, warty lesion has a typical ‘stuck-on’ appearance. Similar lesions may be found elsewhere on the trunk.

Fig. 1.64B Penile seborrheic keratosis. Recent growth and the dark color concerned the patient. Biopsy confirmed the benign nature of the lesions.

Fig. 1.65 (A) Acrochordons. ‘Skin tags’ are often found in intertriginous areas. They usually are asymptomatic unless traumatized. (B) Huge gluteal fibroma.
Hyperkeratotic or ulcerated lesions that are asymmetrically located on the genitalia should be evaluated carefully for the possibility of squamous cell carcinoma ( Figs 1.66A, 1.66B , and 1.67 ). The lesions may be asymptomatic, and patients may be unaware of them or deny the chronicity of the problem. Biopsy is recommended for suspicious lesions, and multiple biopsies should be taken of all suspicious areas. In some forms of squamous cell carcinoma, such as Bowen’s disease of the vulva and Bowenoid papulosis, the presence of certain human papillomaviruses (HPV 16 and 18) has been reported. Evaluation of the patient with a suspicious lesion should include palpation of regional lymph nodes. It may be appropriate to refer the patient directly to a specialist for evaluation and biopsy, although physicians should be aware that apprehension may make the patient reluctant to seek appropriate and timely health care. For this reason it may be expeditious to perform a biopsy on the first visit so that the correct diagnosis may be made ( Fig. 1.68 ).

Fig. 1.66A Squamous cell carcinoma of the penis. This large chronic ulcer had been present for over a year. Patients may delay consultation with a physician because they are afraid that a malignancy will be diagnosed. Biopsy proved squamous cell carcinoma.

Fig. 1.66B Carcinoma in situ of penis. White plaques representing residual carcinoma in situ and scarring after surgery for squamous cell carcinoma.

Fig. 1.67 Carcinoma in situ of the vulva. Note the asymmetric, rough, whitish, eroded, thickened appearance of this malignancy on the labium.

Fig. 1.68 Needle shave technique for skin biopsy.
(A) Using a small (27–30 gauge) needle, a wheal is formed under and around the lesion with local anesthetic (generally less than 1 mL of 1% xylocaine with epinephrine.
(B) The needle is inserted just proximal to the lesion, advanced just under it, and exited just distal to it.
(C) A small scalpel blade is inserted under the point of the needle, with the back of the blade actually touching the distal needle shaft. The biopsy incision is begun slightly distal to the exit point of the needle, and is directed toward the hub for maximum control.
(D) The angle of the blade should be determined before beginning the incision—a shallow angle for a superficial incision and a wider angle for a deeper specimen.
(E) The incision is begun and ended slightly beyond the needle entrance and exit. The skin is lifted gently with the needle, as the blade slices underneath. The biopsy specimen should be impaled neatly on the needle for ease of handling. The biopsy specimen may be left on the needle and set aside briefly until bleeding is stopped, or it may be placed directly into fixative.
(F) To remove the biopsy specimen from the needle, use the back of the scalpel blade to slide the specimen off into the bottle of fixative.

Erosions and ulcers
An erosion is defined as the loss of epidermis, while an ulcer extends through the epidermis into the dermis. The lack of a primary lesion makes evaluation of erosions and ulcers extremely difficult for most physicians, and biopsies rarely are helpful unless taken from the edge of a fresh lesion. Infectious ulcers will be covered in other chapters, so this discussion will be limited to non-infectious genital erosions and ulcers.

Bullous diseases
The fragility of a blister roof in an intertriginous area makes erosions the most common presentation of the bullous diseases, which classically appear as blisters elsewhere on the skin. Erythema multiforme (EM) typically appears as ‘target’ or ‘bull’s-eye’ lesions on the extremities ( Fig. 1.69 ). Involvement of the oral mucosa ( Fig. 1.70A ), palms, soles, and glans penis ( Fig. 1.70B ) is seen most often in the bullous form called the Stevens–Johnson syndrome. EM often is associated with ingestion of drugs or a preceding HSV infection; however, other infections such as mycoplasmal pneumonia, or other viral or bacterial infections may be associated with the occurrence of this disorder. 35 , 36 Recurrent episodes are not uncommon and may be limited to mucous membranes such as the mouth and genitalia. It is important to ask the patient whether or not there has been an episode of HSV preceding the outbreak of EM, for control of HSV recurrences with acyclovir may lead to control of EM as well. 37 , 38

Fig. 1.69 Erythema multiforme (EM) on the arms. The concentric shape (‘target’ lesions) and presence of bullae are helpful clues to the recognition of this skin disorder, in which many different morphologic types of lesions may be present.

Fig. 1.70A Stevens–Johnson syndrome. This patient’s lips and conjunctivae exhibit painful erosions and crusting. There were multiple tender erythematous plaques on the palms and soles.

Fig. 1.70B Penile erosion in erythema multiforme. These painful shallow erosions developed after a herpesvirus infection of the mouth—a relatively common association.

Fig. 1.71A Vulvar erosions in mucous membrane pemphigoid. Persistent painful vulvar erosions and oral involvement were the only manifestations in this patient.

Fig. 1.71B Painful erythemetous erosions of the gingiva present in the patient in Fig. 1.71 are typical of the desquamative gingivitis of mucous membrane pemphigoid.

Fig. 1.71C Mucous membrane pemphigoid. Two new painful oral erosions on the left tonsilar pillar in a patient with scalp and trunk involvement.

Fig. 1.71D Pemphigus vulgaris. Oral erosions are common in pemphigus vulgaris. Courtesy of Dr. Patrick W. Edmunds, Private Practice of Oral and Maxillofacial Surgery.

Fig. 1.71E Penile erosions in pemphigus vulgaris. Genital lesions are less common than in mucous membrane pemphigoid. Courtesy of Dr. Patrick W. Edmunds, Private Practice of Oral and Maxillofacial Surgery.

Fig. 1.71F Conjunctival erythema in pemphigus vulgaris. Courtesy of Dr. Patrick W. Edmunds, Private Practice of Oral and Maxillofacial Surgery.

Fig. 1.71G Indirect immunofluorecence positive to IgG in pemphigus vulgaris patient in 1.71D, E, and F.
As mentioned previously in the section on pustular dermatoses, benign familial pemphigus is most commonly seen as localized erosions in the groin. Chronic erosions of the vulvar mucosa may be seen in mucous membrane (cicatricial) pemphigoid, an uncommon blistering disorder of the mucosae. The disease most often presents with erosions of oral or conjunctival mucosae, desquamative gingivitis or conjunctivitis. Genital involvement is less common and may affect the vulva, penis, or anal mucosae. The scalp, face, neck, nasopharynx and esophagous may also be affected ( Fig. 1.71A, B, and C ). Chronic disease can cause scarring, strictures, blindness, loss of teeth and life-threatening laryngeal strictures. Pemphigus vulgaris also may present with similar ulcers or erosions ( Fig. 1.71D, E, F, and G ). Chronic pemphigus may even result in somewhat heaped-up, friable papules (pemphigus vegetans). Diagnosis and treatment of these bullous diseases is difficult and referral to a dermatologist familiar with the management of these painful, chronic and sometimes life-threatening disorders is preferable. 39

Ulcerative dermatoses
Ulcerative forms of dermatoses may occur when a dermatologic condition makes the skin exceptionally fragile and easily traumatized. When these conditions occur in the genitalia, their presentation may be obscured by their erosive appearance.
Seen most commonly on the glans penis or hands, the fixed drug eruption ( Fig. 1.72 ) has been linked with tetracycline therapy, phenolphthalein, that is found in certain laxatives, and several other drugs ( Table 1.4 ). 40 , 41 Typically appearing as hyperpigmented round macules on the skin, acute lesions may be eczematous, bullous, or erosive in appearance. The appearance of genital lesions in a patient who is being treated with tetracycline for an STD may cause that patient to believe that he or she is experiencing a relapse of the disease or has another STD (see Chapter 7 , Differential Diagnosis).

Fig. 1.72 Fixed drug eruption of the penis. Lesions may appear elsewhere on the body as hyperpigmented round macules or bullae that flare with re-administration of the offending drug.
Table 1.4 Drugs commonly causing fixed drug eruptions.
Non-steroidal anti-inflammatories
Quinine and derivatives
Lichen planus was discussed under its most typical presentation as a papulosquamous disorder, but ulcerative forms of this disorder do occur on mucous membranes ( Figs 1.73 and 1.74 ) and can be extremely difficult to manage. Vulvovaginal erosions may be extensive, and their chronicity may cause the physician to consider the possibility of malignancy. Superinfection with Candida may also occur in this disorder, and should be considered and treated, if present. Treatment for ulcerative lichen planus generally is symptomatic, and topical steroids may be necessary.

Fig. 1.73 Lichen Planus. Buccal mucosa with thin whitish linear streaks.

Fig. 1.74 Lichen Planus. White striae are seen on buccal mucosa.
Courtesy of Emory University School of Dentistry.
Lichen sclerosus was discussed under the category of atrophy ( pp. 9 – 10 ), but the extreme friability of the epidermis in this condition makes the presence of erosions, petechiae, and purpura a common occurrence. The patient should be examined carefully for the typical white atrophic epidermis occurring symmetrically around the rectum and perineum.
Cutaneous trauma is an often overlooked source of genital ulceration. A relatively innocuous dermatitis on the genitalia may be extremely pruritic and bothersome, and may result in the patient traumatizing the skin during bouts of itching and scratching. Erosions can be deep and severe ( Fig. 1.75 ), and secondary infection may make evaluation difficult. Questioning the patient about his underlying symptoms will frequently evoke an admission of intractable pruritus, and therapy should be directed toward alleviation of symptoms. Trauma induced by the patient’s sexual partner also should be considered, especially following oral sex. Human bites ( Fig. 1.76 ) are notoriously infectious, and cultures may be necessary to determine appropriate broad-spectrum antibiotic therapy. The presence of symmetric bruises or cuts encircling the penis should lead the physician to suspect cutaneous trauma as a likely etiology; this becomes especially important in the evaluation of children for possible sexual abuse.

Fig. 1.75 Traumatic ulcer of the penis. The sharply angled borders of this lesion are a clue to its traumatic rather than infectious etiology.

Fig. 1.76 Ulcer secondary to human bite of the penile shaft. Secondary infection is a common sequela of human bite wounds and cultures may be necessary for appropriate antibiotic therapy.

Systemic diseases
Systemic diseases also may lead to secondary genital ulcers. Behçet’s disease is a multisystem disorder that may present with skin involvement in a majority of cases. In the full-blown syndrome, oral and genital ulcerations are present ( Figs 1.77 – 1.79 ), as well as a pustular eruption, which may involve the genitals. A spectrum of ocular involvement includes conjunctivitis, photophobia, uveitis, and optic neuritis. Central nervous system changes are variable and can be severe, thus frequently dominating the clinical picture. Fever, arthralgias, and cardiac or pulmonary involvement also may be present. The mucosal ulcerations are non-specific, and more common causes should be excluded before a diagnosis of Behçet’s is made on the basis of oral and genital ulcers alone. 42 , 43

Figs 1.77

Figs 1.78

Figs 1.77–1.79 Behçet’s disease. Non-specific painful recurrent ulcers of the oral and genital mucosa were the presenting complaints in these young patients in whom Behçet’s disease was diagnosed. Fig. 1.79 shows scrotal ulcer caused by Behçet’s disease.
Pyoderma gangrenosum ( Fig. 1.80 ) is a shaggy, painful, ‘dirty’ looking ulcer with a bluish overhanging border. The name reflects the exceptionally infectious appearance of this actually non-infectious ulcer. It is seen most commonly in patients with inflammatory bowel disease, but also may be seen with multiple myeloma or other hematologic or immunologic disorders. 44 , 45

Fig. 1.80 Pyoderma gangrenosum. Multiple deep necrotic ulcers with dusky overhanging margins are characteristic of pyoderma gangrenosum. These lesions may be seen in patients with various systemic diseases.
Although pyoderma gangrenosum may be seen with gastrointestinal disease, cutaneous Crohn’s disease classically presents long ‘knife-cut’ ulcers along the intertriginous groin folds. Flares of these cutaneous lesions often parallel the course of the gastrointestinal disease, and control of one often will lead to control of the other. 46 , 47
Asymmetric ulcers of the genitalia that do not heal with appropriate therapy should be biopsied to rule out carcinoma. Biopsies should be multiple and taken from the thickest part of a lesion and the edge of an ulcer. Squamous cell carcinoma was discussed under nodules and tumors ( pp. 16 – 18 ). Extramammary Paget’s disease, an uncommon disorder frequently associated with an underlying adenocarcinoma, presents as a chronic eczematous plaque. Pruritus or pain may be present. Hallmarks of this diagnosis are its chronicity, asymmetry, and lack of response to topical therapy. The vulva is one of the most common sites for extramammary Paget’s disease, but it also occurs on the penis, scrotum and perianal region. Biopsy is essential for diagnosis. 48 , 49 , 50

Itching (pruritus ani, scroti, vulvae)
Acute-onset perineal itching or burning should take the physician through a standard differential diagnosis, including Candida infection, irritant and contact dermatitis, urinary tract infection, hemorrhoids, pinworms, and condylomata. It is a different challenge to evaluate chronic cutaneous symptomatology, and this problem is not within the scope of this text. However, a few points should be made: lichen simplex chronicus (LSC) (see earlier section on dermatitis/eczema , pp. 5–7) is thickening of the skin in response to chronic scratching, and several underlying causes of pruritus must be considered. On the genitalia, maceration and intertriginous rubbing contribute to flares and continuation of symptoms, and infections are particularly likely to initiate itching. Cultures should be done for yeast and fungus as tinea and Candida are the most common offenders, and may be primary or secondary to the process. Vigorous scratching or eczematous change disturbs the skin barrier, allowing the development of secondary bacterial infection. In many cases of LSC, the initiating cause cannot be identified; the patient should be reassured that this is probably of no consequence, because the problem is now only the secondary change which has developed as a result of scratching.
In childhood, genital pruritus is often the result of irritant dermatitis, although STDs are an obvious consideration. Young girls may have fecal contamination of the vulva from careless hygiene, or conversely may irritate the skin from vigorous scrubbing or washing with soap. Pinworms are more common in childhood and typically involve the anus, but may also be seen at the vaginal opening. Vaginal or rectal discharge in childhood should be evaluated for evidence of possible sexual abuse, and genital lesions should be examined carefully. Lichen sclerosus (see below) can occur in childhood, and traumatic-appearing purpuric lesions are typical with the fragile epithelium of this cutaneous condition.
The symptomatic patient should be asked about pre-existing dermatoses (including oral mucosal lesions), Candida , condylomata, methods of cleansing, and the use of topical and systemic medications. Previous treatments should be explored, especially if they resulted in a clearly allergic response (vesicles or erosions lasting for 2 weeks) rather than local irritation (stinging and burning on application). The patient should be asked specifically about risk factors: for example, the Candida -prone patient may receive frequent rounds of antibiotics for sinusitis, urinary tract infections, or acne; steroids or other immunosuppressants may be prescribed for a variety of disorders. Estrogen deficiency may be important if the patient is perimenopausal. On the genitalia, erythematous papules and pustules may develop as a complication of topical steroid use, as well as with cutaneous infections.
While some patients will describe elements of itching and burning, the two conditions can usually be differentiated on physical examination. Cutaneous changes of lichenification (leathery thickening) or excoriation (scratch marks) are more typical of pruritus, because the patient with burning skin rarely rubs or scratches the affected area. Without evidence of scratching, the patient with cutaneous burning or dysesthesia may appear to have a normal examination. 51, 52, 53


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2 Gonorrhea

C.A. Ison, D.A. Lewis

The clinical syndrome of gonorrhea was described by biblical authors, but the etiologic agent, Neisseria gonorrhoeae , was not described until 1879, when Albert Neisser observed the organism in smears of purulent exudates from urethritis, cervicitis, and ophthalmia neonatorum. N. gonorrhoeae colonizes and infects primarily the mucosa of the lower anogenital tract, the oropharynx and the conjunctivae and occasionally ascends to colonize and infect the normally sterile upper genital tract or invades the blood to cause disseminated infection. If innappropriately treated, or left untreated, uncomplicated gonorrhea may lead to epididymitis in men and pelvic inflammatory disease (PID) in women. In addition, epidemiological and biological studies provide strong evidence that gonococcal infections facilitate HIV transmission, hence effective treatment of gonorrhea should remain an important part of HIV prevention strategies.
The genus Neisseria includes the pathogenic species N. gonorrhoeae and N. meningitidis , as well as species that are normal flora of the oropharynx and nasopharynx. The cell envelope of N. gonorrhoeae consists of a cytoplasmic membrane, a thin peptidoglycan layer and an outer membrane. Colonization of the mucosal surface by the organism occurs by attachment to the epithelial cell surface, which is mediated by pili and opacity proteins, followed by internalization and transcytosis, mediated by opacity proteins and porins, to establish an infection in the subepithelial space. An immune response is elicited resulting in a polymorphonuclear infiltrate containing intracellular gonococci. Sialylation of the lipo-oligosaccharide results in resistance to the bactericidal action of serum, an attribute necessary for dissemination into the blood to occur.
N. gonorrhoeae is always considered a pathogen that requires treatment and is not considered part of the normal flora. However, strains of commensal Neisseria spp. may occasionally be isolated in clinical specimens from anogenital sites and observed intracellularly in polymorphonuclear leukocytes, and are morphologically indistinguishable from the pathogenic Neisseria e. Thus, accurate laboratory identification of the gonococcus is essential because of the social and medicolegal consequences of misidentifying strains of non-pathogenic Neisseria spp. as N. gonorrhoeae .
Because of the fastidious growth requirements of N. gonorrhoeae , it was difficult to culture the organism until the development of chocolatized blood agar supplemented with growth factors. In the 1960s, the development of selective media containing antimicrobial and antifungal agents (such as Thayer–Martin medium), which enhanced the isolation of the gonococcus by inhibiting not only Gram-positive bacteria but also the closely related Neisseria spp., further simplified the laboratory diagnosis of gonorrhea. Rapid biochemical and serologic tests are available, allowing identification of the gonococcus within a few hours of its isolation. More recently, nucleic acid amplification technology has shed new light on the extent of asymptomatic gonococcal infection. All of these innovations have advanced our knowledge of this common pathogen, its epidemiology and its clinical manifestations.

Gonorrhea is a disease of worldwide importance ( Fig 2.1 ). In the USA, between 1975 and 1997, the national gonorrhea rate declined 74% following implementation of the national gonorrhea control program in the mid-1970s ( Fig. 2.2 ). For the past 10 years, the gonorrhea rate per 100 000 population has reached a plateau and in the past few years has even increased slightly ( Fig. 2.2 ). These data probably reflect the fact that the extensive screening programs for asymptomatic gonorrhea infections in women attending prenatal, family planning, sexually transmitted disease clinics, and other clinics have achieved maximal effect in shortening the average duration of gonococcal infection in that country’s population. Further reductions in annual infection rates will require extending female screening programs to unconventional sites such as schools and including men, both heterosexuals and men-who-have-sex-with-men (MSM), in the search for asymptomatic cases. In the UK the decline in the number of cases of gonorrhea seen in the 1980s and early 1990s has now been reversed with the diagnoses of heterosexually-acquired gonorrhea reaching a peak in 2002, and diagnoses of gonorrhoea among MSM peaking in 2006 ( Fig. 2.3 ). The UK has also observed a 2.4-fold increase in the number of cases receiving epidemiological treatment of suspected gonorrhea from 1999 to 2008 ( Fig 2.4 ).

Fig. 2.1 Estimated new cases of gonorrhea.
(In millions, 1999, courtesy WHO 2000.)

Fig. 2.2 Reported cases of gonorrhea in the USA, 1970–2007.

Fig. 2.3 Diagnoses of uncomplicated gonorrhea by gender and sexual orientation: United Kingdom 1999–2008.

Fig. 2.4 Diagnoses of epidemiological treatment for gonorrhea by gender and sexual orientation: United Kingdom 1999–2008.
Gonorrhea is transmitted almost exclusively by sexual contact. Persons under 25 years of age who have unprotected sexual intercourse with multiple sexual partners are at highest risk. Rates of clinical gonococcal infection are higher in men, and in particular among MSM. Overall, infection prevalence is higher in minority and inner-city populations. As noted above, gonorrhea is often acquired from a sexual partner who is either asymptomatic or who has only minimal symptoms. Transmission efficiency (a measure of transmission through one sexual exposure) is estimated to be 50–60% from an infected man to an uninfected woman and 20% from an infected woman to an uninfected man. More than 90% of men with urethral gonorrhea will develop symptoms within 5 days of infection. Most men with symptomatic urethritis will seek health care because of the relative severity of the symptoms. Infections at other anatomic sites in men, such as pharyngeal and anorectal infections in MSM, and infections in women are far less likely to produce early symptoms and, therefore, are less likely to be diagnosed and treated.
The rationale for public health measures, such as screening and contact tracing, is to identify and treat patients with asymptomatic or minimally symptomatic infections, thus shortening the duration of infection and preventing further transmission of the disease. Additionally, because all women with lower genital tract gonococcal infection regardless of the presence or absence of symptoms are at risk for PID, the early identification and treatment of infected women is important.
The epidemiology of gonorrhoea has been studied extensively using phenotypic methods, such as auxotyping ( Fig. 2.5 ) and serotyping, as well as by genotyping, such as opa-typing ( Fig 2.6 ) and NG-MAST ( Fig 2.7 ), to monitor temporal changes and movement of antibiotic-resistant strains as well as the complexity of sexual networks. Phenotyping has been useful in identifying potential clusters but isolates of the same phenotype may not necessarily reflect the same genotype. Genotyping methodologies are more discriminatory than phenotypic methods, and will be discussed in more detail below.

Fig. 2.5 Chemically defined media used for auxotyping: complete medium, without proline and without arginine. Absence of growth indicates requirement for the specific amino acid.

Fig. 2.6 Polyacrylamide gel showing different opa-types of N. gonorrhoeae . Molecular weight markers in lanes 1, 9, 16 and 22, all other tracks different strains of gonococci.

Fig. 2.7 NG-MAST sequence trace for tbpB 26 and tppB 16. Yellow bars indicate sequence differences.

Antimicrobial Resistance
The evolution of antimicrobial resistance in N. gonorrhoeae has, in both developed and developing countries, 1 potential negative implications for the control of gonorrhea. Resistance to a number of therapeutic agents, e.g. sulphonamides, penicillins, spectinomycin, quinolones and most recently oral cephalosporins, has been observed historically, and for each agent, the observed resistance has generally developed within a few years of its introduction. Resistance may be either chromosomally-mediated or plasmid-mediated ( Table 2.1 ). Chromosomal mutations are the most frequent mutations observed, and may alter the affinity of the drug for its target, increase efflux of drug from the bacterial cell, or decrease permeability of gonococci. Strains with chromosomal resistance to penicillins, tetracyclines, macrolides and quinolones, as well as decreased susceptibility to cephalosporins, have been identified in many parts of the world. Sporadic high-level chromosomal resistance to spectinomycin has also been reported, particularly when it was used as a first-line therapy in the 1970s in Korea.
Table 2.1 Types of antimicrobial resistance in N. gonorrhoeae . Type of resistance Antimicrobial agent Chromosomal (genes located on the chromosome) Sulphonamides Penicillin   Tetracycline   Spectinomycin   Macrolides   Fluoroquinolones   Cephalosporins Plasmid-mediated (genes located on plasmids) Penicillin (PPNG) Tetracycline (TRNG)
PPNG = penicillinase-producing N. gonorrhoeae ; TRNG = tetracycline-resistant N. gonorrhoeae
Penicillinase-producing N. gonorrhoeae (PPNG) strains, which inactivate penicillins and other β-lactams, were first described in 1976. Several β-lactamase plasmids have been identified in PPNG strains, including the 3.2 MDa ‘African’, 4.4 MDa ‘Asian’, 3.05 MDa ‘Toronto’, 4.0 MDa ‘Nimes’ and the 6.5 MDa New Zealand plasmids. The 2.9 MDa ‘Rio’ plasmid has been found to be identical to the Toronto plasmid. All of these encode for the TEM-1-type β-lactamase but differ in size due to deletions in the non-functional part of the plasmid. They require the presence of the larger 24.5 MDa conjugative plasmid for dissemination to other gonococci. The highest prevalence of PPNG strains is found in parts of Africa and Asia, but they have been endemic in the USA and Europe since 1981. PPNG exhibit high levels of resistance that make the penicillins inappropriate agents for gonococcal therapy.
Plasmid-mediated, high-level resistance to tetracycline was reported in N. gonorrhoeae (TRNG) in 1985. It has resulted from the insertion of the TetM determinant into the 24.5 MDa conjugative plasmid resulting in a plasmid of 25.2 MDa. Based on restriction analysis, two TetM plasmids have been identified in N. gonorrhoeae , which have been designated ‘US’ and ‘Dutch’. These plasmids can mobilize themselves between both gonococcal isolates and different species and have been reported in N. meningitidis and Kingella denitrificans . They also have the ability to mobilize β-lactamase plasmids and isolates with plasmid-mediated resistance to penicillin and tetracycline (PPNG/TRNG) are commonly found. Like the penicillins, tetracyclines are inappropriate sole therapies for gonococcal infections.
Decreased susceptibility and high-level chromosomal resistance to the fluoroquinolones (e.g. ciprofloxacin, ofloxacin), acquired through the acquisition of point mutations in the DNA gyrase ( gyr A) and topoisomerase IV ( par C) genes, are now widespread and have resulted in many regions of the world abandoning this antibiotic class as first-line therapies for presumptive or confirmed gonorrhea. It seems probable that strains exhibiting high-levels of resistance also have changes in membrane permeability that may be associated with an efflux system.
Most countries in the world where fluoroquinolones are no longer effective have now turned to use of cephalosporins, administered either orally (e.g. cefixime or cefpodoxime) or parenterally (e.g. ceftriaxone). Resistance to oral cephalosporins has recently been described in Japan 1 which appears, in the main part, to be due to the acquisition of mosaic pen A genes as the result of genetic exchange of DNA between N. gonorrhoeae and commensal Neisseria spp. 2 The strains showing decreased susceptibility and resistance to oral cephalosporins remain susceptible to intramuscular ceftriaxone. However, a drift in the minimum inhibitory concentration (MIC) upwards for both oral cephalosporins and ceftriaxone has been described in longitudinal surveillance programs in both the USA and the UK, raising concerns about the future viability of cephalosporins in the treatment of gonorrhea. Azithromycin may also be used in the management of gonorrhea, although a single 2 g oral dose is required for optimal efficacy. Whilst useful in the management of individual cases, concerns exist over recommending any macrolide as first line therapy for gonorrhea, given the ease with which macrolide resistance may develop in N. gonorrhoeae . Isolates with decreased susceptibility to azithromycin were identified by the Gonococcal Isolate Surveillance Project (GISP) in 1999 and isolates with very high MICs were recently reported in the United Kingdom. 3
The continued emergence of resistance to antimicrobial agents used for the treatment of gonorrhea 1 , 4 is a concern and requires effective surveillance programs to monitor susceptibility patterns, detect drifts in susceptibility and emergence of resistance. There are established surveillance programs in the USA, Canada, Australia and The Netherlands which have produced valuable temporal data in these individual countries. The Gonococcal Antimicrobial Susceptibility Program (GASP) was established to provide a global surveillance network. GASP is co-ordinated by the World Health Organization and the aim was to create a series of networks based on WHO regions. Important elements of GASP are that data should be comparable between laboratories and this requires the development of training and quality assurance programs. Currently GASP is most active in the Americas and the Caribbean, the western Pacific region and the southeast Asian region although new GASP initiatives are now underway in Africa, the European Union and Eastern Europe. The GASP team is currently working on definitions for multi-drug (MDR) and extensively drug-resistant (XDR) N. gonorrhoeae .

Clinical Manifestations
In the majority of cases, gonococcal infections are limited to mucosal surfaces. 5 Infection occurs in areas of columnar epithelium including the cervix, urethra, rectum, pharynx, and conjunctiva ( Table 2.2 ). Squamous epithelium is not susceptible to infection by the gonococcus. However, the prepubertal vaginal epithelium which has not been keratinized under the influence of estrogen, may be infected. Hence, gonorrhea may present in a young girl as a vulvovaginitis. In mucosal infections, there is usually a brisk, local neutrophilic response manifested clinically as a purulent discharge.
Table 2.2 Clinical manifestations of gonococcal infections. Category Uncomplicated Gonorrhea Urethra Symptomatic Scant, clear discharge     Copious purulent discharge   Asymptomatic   Cervix Symptomatic Red, friable cervical os     Purulent discharge from os     Salpingitis     Bilateral or unilateral lower abdominal tenderness   Asymptomatic   Rectum Symptomatic Copious, purulent discharge     Burning/stinging pain     Tenesmus     Blood in stools   Asymptomatic   Pharynx Symptomatic Mild pharyngitis     Mild sore throat     Erythema   Asymptomatic   Conjunctiva Symptomatic Copious purulent discharge     Keratitis and corneal ulceration; perforation, extrusion of lens     Scarring; opacification of lens     Blindness Category Complicated Gonorrhea   Male complications Penile edema   Tyson’s glands abscess   Cowper’s glands abscess   Seminal vesiculitis   Epididymitis Female complications Endometritis   Salpingitis   Tubo-ovarian abscess   Ectopic pregnancy   Infertility Disseminated gonococcal infection (DGI) Bacteremia   Fever   Skin lesions: macular, erythematous, pustular, necrotic, hemorrhagic   Tenosynovitis   Joints; septic arthritis   Endocarditis   Meningitis
In women, untreated cervical infection may lead to endometritis and salpingitis, a sign–symptom complex more commonly known as PID (see also Chapter 6 ). It has been estimated that in approximately 1–3% of female patients with mucosal infection, hematogenous spread occurs, causing disseminated gonococcal infection (DGI). However, the risk may be much lower in populations with a low prevalence of the gonococcal auxotypes that have been shown to be associated with dissemination.

The most common symptom of gonorrhea in men is urethral discharge that may range from a scanty, clear, or cloudy fluid to one that is copious and purulent ( Figs 2.8 and 2.9 ). Dysuria is usually present and the meatus may be inflamed. However, men with asymptomatic urethritis may be an important reservoir for transmission. Although most men with gonorrhea develop symptoms, those who ignore their symptoms or have asymptomatic infection are at increased risk of developing complications (see Table 2.2 ).

Fig. 2.8 Symptomatic gonococcal urethritis. Scanty urethral discharge obtained after urethral stripping.

Fig. 2.9 Symptomatic gonococcal urethritis. Copious spontaneous urethral discharge.
Endocervical infection is the most common type of uncomplicated gonorrhea in women ( Figs 2.10 and 2.11 ). At least one-half of infected women are asymptomatic or have symptoms that are mild to non-specific. Cervical infections may be accompanied by vaginal discharge, abnormal vaginal bleeding, or dysuria. Local complications include abscesses in Bartholin’s and Skene’s glands ( Fig. 2.12 ). Asymptomatic infections are found most often in women who are screened for gonorrhea in routine gynecologic examinations or who are seen as sexual contacts of men with gonorrhea. On examination, the cervical os may be erythematous and friable, with a purulent exudate ( Figs 2.11 and 2.13 ), or may be normal.

Fig. 2.10 Endocervical gonorrhea. A small amount of purulent discharge is visible in the endocervical canal.

Fig. 2.11 Signs of endocervical gonorrhea: cervical edema and erythema as well as discharge.

Fig. 2.12 Urethral gonorrhea in the female. Purulent discharge is visible, with involvement of Bartholin’s gland.

Fig. 2.13 Gonococcal cervicitis with mucoid discharge and marked cervical erythema and edema. This is indistinguishable clinically from chlamydial cervicitis.
Anorectal infections, which occur in 30% of women with cervical gonorrhea, probably represent secondary colonization from a primary cervical infection and are symptomatic in less than 5% of women. Infections in MSM, however, result from both penile-anal and oro-anal intercourse and are more often symptomatic (18–34%). Symptoms and signs range from mild burning on defecation to itching to severe tenesmus, and from mucopurulent discharge to frank blood in the stools.
Pharyngeal ‘infections’ are diagnosed most often in women and MSM with a history of fellatio through screening. Over 90% of pharyngeal infections are asymptomatic and there has never been a convincing demonstration of a relationship between pharyngeal infection/colonization and the signs and symptoms of a sore throat or tonsillitis.
Ocular infections occur in newborns who are exposed to infected secretions in the birth canal of an infected mother ( Fig. 2.14 ). Occasionally, keratoconjunctivitis is seen in adults through self-inoculation ( Fig. 2.15 ). Conjunctival infection, tearing, and lid edema occur early, followed rapidly by the appearance of a frankly purulent exudate. Prompt diagnosis and treatment are important because corneal scarring or perforation may result ( Fig. 2.16 ).

Fig. 2.14 Gonococcal ophthalmia neonatorum. Lid edema, erythema, and marked purulent discharge are seen. The Gram-stained smear was loaded with Gram-negative diplococci within neutrophils.

Fig. 2.15 Early gonococcal ophthalmia in an adult showing marked chemosis and tearing with no discharge.

Fig. 2.16 Corneal clouding following gonococcal ophthalmia in an adult.

Disseminated gonococcal infection
Disseminated gonococcal infection (DGI) is the result of gonococcal bacteremia and occurs more frequently in women than men. The sources of infection are primarily asymptomatic infections of the pharynx, urethra, or cervix. In describing this disease, it is useful to divide patients into two groups: those with suppurative arthritis and those without. The term tenosynovitis-dermatitis syndrome often is applied to the latter group of patients, because the majority present with, or give a history of, one or both of tenosynovitis or skin rash. Only a minority of patients with suppurative gonococcal arthritis have tenosynovitis and/or skin lesions at the time of presentation. It is generally thought that the tenosynovitis-dermatitis syndrome represents the initial stage which then progresses to a frank septic arthritis.
Patients with the tenosynovitis-dermatitis syndrome may be febrile. The majority will have skin lesions which begin as small erythematous maculopapular petechial lesions. These lesions usually develop a central pustule which may then progress to a lesion with central necrosis ( Figs 2.17 – 2.19 ). Early on the lesions of meningococcemia and DGI may be indistinguishable though the former usually progress to confluent petechial and then hemorrhagic lesions. Patients with DGI usually have less than 20 lesions, whereas patients with meningococcemia usually have many more. DGI lesions are located peripherally, with the wrists and ankles being the most common locations. Polyarthralgia is a common presenting symptom of DGI regardless of the clinical classification of the patient. Tenosynovitis is characterized as erythema, swelling, and direct tenderness upon palpation of the affected tendon group ( Fig. 2.20 ). It is found most commonly around the wrist and dorsum of the hand, and less often at the ankle, including the Achilles tendon, and the dorsum of the foot. In contrast to those with the tenosynovitis-dermatitis syndrome, most patients with gonococcal suppurative arthritis are afebrile. The arthritis typically affects the wrists, the small joints of the hands and the knees. The majority of cases will give a history of migratory polyathralgia and tenosynovitis may be present. About one third have skin lesions as described for tenosynovitis-dermatitis syndrome. Left untreated, many cases of DGI eventually resolve without specific therapy, but a significant proportion will suffer serious morbidity including endocarditis, meningitis, and osteomyelitis. Since the advent of antibiotics, these complications have been observed only rarely.

Fig. 2.17 Skin lesions of disseminated gonococcal infection. Papular and pustular lesions on the foot.

Fig. 2.18 Skin lesions of disseminated gonococcal infection. Small painful midpalmar lesion on an erythematous base.

Fig. 2.19 Skin lesions of disseminated gonococcal infection. Classic large lesions with a necrotic, grayish central lesion on an erythematous base.

Fig. 2.20 Disseminated gonococcal infection. Tenosynovitis of the dorsal foot.
(Courtesy of Dr Charles V. Sanders, LSU Medical School and Williams & Williams Publishers.)
The diagnostic method of choice is blood culture although all suspected cases should be screened for N. gonorrhoeae infection at pharyngeal and anorectal sites, as well as at urethral and (in women) endocervical sites, prior to commencing antibiotics as asymptomatic gonococcal colonization is often present. Gonococci may occasionally be detected by microscopy and culture from pus expressed from broken skin lesions ( Fig. 2.21 ). Blood cultures are most likely to be positive among patients with the tenosynovitis-dermatitis syndrome and are rarely positive when taken from septic arthritis cases. Aspiration of pus from joints of septic arthritis patients should undergo full microbiological investigation, including microscopy and culture for N. gonorrhoeae . In one-half of DGI cases, gonococci cannot be isolated from blood, CSF, or synovial fluid, even with the best laboratory techniques. 6

Fig. 2.21 Gram-negative diplococci visible in one neutrophil in a smear of a pustular skin lesion from a patient with disseminated gonococcal infection. Meningococci cannot be distinguished from gonococci with this method.
DGI must be distinguished from Reiter’s syndrome, meningococcemia, acute rheumatoid arthritis, other forms of septic arthritis, and the immune complex-mediated arthritides caused by hepatitis B virus and HIV ( Box 2.1 and Table 2.3 ). In the absence of positive blood or synovial fluid cultures, a presumptive diagnosis of DGI can be made according to Box 2.2 .

Box 2.1 Differential diagnosis of disseminated gonococcal infection.

Dermatitis-tenosynovitis syndrome
Staphylococcal sepsis or endocarditis
Other bacterial septicemias (rare)
HIV infection: Acute thrombocytopenia and arthritis
Hepatitis B prodrome
Acute Reiter’s syndrome
Juvenile rheumatoid arthritis
Lyme disease
Table 2.3 Differential diagnosis of monarticular arthritis. Infectious Non-infectious Bacterial Gout, pseudogout * Adults Neisseria gonorrhoeae * Rheumatoid arthritis (especially juvenile rheumatoid arthritis) Staphylococcus aureus Trauma Streptococcus pneumoniae Tumors Other Streptococcus spp. Hemarthrosis Children Osteochondritis Staphylococcus aureus Psoriatic arthritis Streptococcus pneumoniae Pigmented/villonodular synovitis Other Streptococcus spp.   Haemophilus influenzae   Gram-negative rods   Tuberculosis   Fungal  
* Most common.

Box 2.2 Criteria supporting a clinical diagnosis of disseminated gonococcal infection (DGI).

Gonococci are demonstrated in synovial fluid, blood, cerebrospinal fluid, or skin lesions by culture
Observation of diplococci in Gram- or methylene blue-stained smear
Clinical diagnosis of DGI may be based on two of the following three criteria:
Isolation of gonococci from urogenital, rectal, pharyngeal, or conjunctival sites of the patient or the patient’s sexual partner(s)
Infection is manifested as pustular, hemorrhagic, or necrotic skin lesions distributed on the extremities
Patient responds rapidly to appropriate antimicrobial therapy

Gonococcal pelvic inflammatory disease (PID)
As is the case with chlamydia, the gonococcus may ascend from the endocervical canal through the endometrium to the fallopian tubes and ultimately to the pelvic peritoneum ( Fig. 2.22 ), resulting in endometritis, salpingitis, and finally peritonitis (see also Chapter 6 ). Patients may report pelvic and abdominal pain, fever, and chills. The proportion of PID cases caused by N. gonorrhoeae , based on the recovery of the organism from laparoscopic specimens, varies from 8 to 70% depending on geographic location. The proportion of women with cervical gonococcal infection who will develop upper tract disease is uncertain. PID is the most common and costly consequence of gonorrhea, and recurrent episodes of PID are common. Initial PID infections are more likely to be gonococcal or chlamydial, while other bacteria are isolated more frequently from recurrent episodes ( Table 2.4 ). The consequences of PID include an increased probability of infertility (tubal factor infertility), ectopic pregnancy, and chronic pelvic pain.

Fig. 2.22 The evolution of gonococcal pelvic inflammatory disease.
Table 2.4 Etiologic agents of primary and recurrent pelvic inflammatory disease. Neisseria gonorrhoeae Bacteroides spp. Chlamydia trachomatis Peptococcus spp. Group B Streptococci Peptostreptococcus spp. Escherichia coli and other enterobacteria Genital mycoplasmas Gardnerella vaginalis Actinomyces israelii Haemophilus spp. (only with IUD-associated disease) Fusobacterium spp.  
The clinical diagnosis of PID is imprecise. The disease may present with lower abdominal pain, fever, chills, abnormally painful menses and metromenorrhagia. Adnexal, fundal, and cervical motion tenderness are found on examination. Acute pyogenic complications of PID include tubo-ovarian abscesses, pelvic peritonitis (often mimicking appendicitis), or the Fitz–Hugh–Curtis syndrome, which is an inflammation of Glisson’s capsule of the liver.
Laparoscopy is the best method for confirming the clinical diagnosis; however, especially in early PID, the inflammation may not have extended to the tubal surface, and the peritoneal mucosa may appear normal despite inflammation within the tubes ( Figs 2.23 and 2.24 ). Endometrial biopsy represents a less invasive alternative to laparoscopic examination. Abnormalities are seen in as many as 90% of endometrial biopsies from women with laparoscopic evidence of salpingitis. Additionally, as many as one-third of women with examination findings suggestive of PID and negative laparoscopic findings will have histopathologic findings that suggest endometritis. Endocervical specimens for N. gonorrhoeae and C. trachomatis testing are recommended for all women with suspected PID. Male partners of women with confirmed gonococcal PID should be investigated, treated and managed accordingly.

Fig. 2.23 Normal laparoscopic view of the female genital tract: view from above the dome of the uterus.

Fig. 2.24 Uterus and fallopian tubes of a patient with advanced recurrent pelvic inflammatory disease showing so-called ‘retort’ tubes.

Laboratory Tests
The laboratory diagnosis of gonorrhea has undergone considerable change in recent years. The presumptive diagnosis by examination of a Gram-stained smear for the presence of intracellular Gram-negative cocci remains the mainstay in many clinical settings, particularly for patients with the signs and symptoms of gonorrhea. However, isolation of N. gonorrhoeae , historically the gold standard, has been replaced in many settings by molecular detection using nucleic acid amplification tests (NAATs). Culture is still required to provide a viable organism for susceptibility testing to monitor the emergence of resistance to current therapies but NAATs offer detection using highly sensitive and specific tests which are more tolerant of delays or inadequacies in specimen collection or transportation to the laboratory. It must be remembered that no NAAT is 100% sensitive and specific and although the commercially available kits will detect both N. gonorrhoeae and Chlamydia trachomatis , often simultaneously, the genetic targets for N. gonorrhoeae are not as robust as for C. trachomatis because of the extraordinary ability of the gonococcus for genetic recombination and exchange.

Presumptive laboratory diagnosis
Microscopic examination of a Gram-stained smear from the urethra and cervix are considered positive for the presumptive diagnosis of gonorrhea if Gram-negative diplococci are observed intracellularly in polymorphonuclear leukocytes ( Figs 2.25 and 2.26 ). A positive smear is 95% sensitive in men with symptomatic urethritis but is less sensitive (30–50%) in cervical specimens from women and in asymptomatic men. This probably reflects the greater number of asymptomatic infections in women and the lower numbers of organisms present in an asymptomatic infection. It is no longer common practice to examine rectal smears as the sensitivity is likely to be less (40–60%) in blindly obtained rectal specimens, due primarily to the large number of normal flora present in the gut, many of which may also be Gram-negative cocci. A rectal smear is most useful if taken with a proctoscope from patients with evidence of infection. Diagnosis of pharyngeal infection cannot be based on the Gram stain because of the frequent presence of other Neisseria and related spp. in the oropharynx. In some parts of the world, smears are stained with methylene blue which is simple and rapid and particularly useful where laboratory facilities are not well developed ( Fig. 2.27 ). It does give the classical picture of intracellular diplococci but lacks specificity; if resources and facilities are available, any smears showing intracellular diplococci should be stained by Gram’s method as confirmation of the presumptive diagnosis.

Fig. 2.25 Gram-stained smear of urethral exudate from a male showing a sheet of neutrophils (PMNs) and many Gram-negative diplococci within PMNs. This finding is sufficient for a presumptive diagnosis of gonorrhea in the male.

Fig. 2.26 Gram-stained smear of endocervical exudate showing scattered neutrophils and squamous epithelial cells. Gram-negative diplococci are present in one neutrophil. This finding supports a presumptive diagnosis of gonorrhea in the female, but should be confirmed by culture.

Fig. 2.27 Methylene blue-stained smear of urethral exudate from a man showing neutrophils and intracellular organisms.

Culture for N. Gonorrhoeae

Specimen collection and transportation
When culture is to be used for diagnosis, specimens should always be collected before treatment is administered. N. gonorrhoeae is most successfully isolated when the specimen is inoculated onto media directly from the patient and incubated at 35–36.5°C in a CO 2 -enriched atmosphere immediately after collection. Where possible, incubation should be in an appropriate incubator but if this is not available it is important to place the media into the presence of a CO 2 -enriched atmosphere as soon as possible. Thus, streaked plates should be immediately placed in a CO 2 candle-extinction jar held at room temperature until they can be transported to the laboratory incubator. Specimens may be kept at room temperature for up to 5 hours without loss of viability. For small numbers of specimens CO 2 envelopes designed for individual plates are commercially available. Collection of specimens using Amies’ or Stuart’s medium is an acceptable alternative and retrieval is best if delivered to the laboratory within 4–8 hours, although acceptable recovery can be achieved after up to 48 hours if stored correctly.
Specimens for culture may be collected from the urethra, cervix, rectum, and pharynx as well as from the endometrium, fallopian tubes, joint fluid or blood, if PID or DGI is suspected. The precise choice of anatomic sites from which to collect specimens is made on the basis of the patient’s potential for sexual exposure and presenting symptoms. Specimens are collected with cotton, polyester, or calcium alginate swabs or plastic loops. Normally, only urethral specimens are collected from heterosexual men, while urethral, rectal, and pharyngeal specimens are collected from homosexual men. Cervical and rectal specimens are routinely collected from women; specimens may also be collected from the urethra and from Bartholin’s and Skene’s glands when appropriate. In men, N. gonorrhoeae may also be isolated from the culture of urine sediment, but this method has a low sensitivity and is time consuming to perform. Blood cultures should be collected from patients with suspected disseminated infection. In patients with septic arthritis, synovial fluid should be cultured, although it is not uncommon for cultures from these sites to be negative and molecular detection may be helpful.
If specimens must be transported a significant distance from the clinical facility to the laboratory, they should be inoculated onto an isolation medium (Jembec or Transgrow) and incubated overnight before shipment. The transport medium should be shipped by courier or an express delivery service to insure delivery of the inoculated medium within 24–48 hours.

Isolation methods
Specimens should be inoculated onto selective media such as Thayer–Martin (TM) medium, modified Thayer–Martin medium (MTM), Martin–Lewis (ML) medium, New York City (NYC) medium, or GC–Lect (GC–L) medium, which are composed of GC base or equivalent media supplemented with growth factors and antimicrobial and antifungal agents ( Table 2.5 ); some media contain hemoglobin. The addition of the selective agents allows the colonies of N. gonorrhoeae to grow and bacteria found in the normal flora to be inhibited ( Fig. 2.28 ). If the specimen is obtained from a site that is normally sterile (e.g. blood, synovial fluid, conjunctiva), it may be inoculated on a non-selective medium such as chocolate agar. It should be remembered that other Neisseria and related spp. may grow on non-selective media and therefore confirmation by identification of N. gonorrhoeae should be undertaken. Vancomycin-susceptible strains of N. gonorrhoeae occasionally occur and may not grow on selective media containing 4 µg vancomycin/mL, which is routinely used for the isolation of the gonococcus. Selective media have been modified to contain 2 µg or 3 µg vancomycin/mL in order to overcome this problem. Lincomycin has also been used as an alternative but is less inhibitory than vancomycin and overgrowth, particularly from rectal specimens, can occur. Vancomycin-susceptible gonococci should be suspected in a community when false-negative cultures are obtained, as evidenced by a discrepancy between Gram-stain positivity and culture-positivity rates for urethral gonorrhea in men; these should agree for at least 95% of cases. In an ideal situation both selective and non-selective media ( Fig. 2.29 ) would be used, however in most instances resources are only available for a single medium which should contain selective agents.
Table 2.5 Antimicrobial agents used in selective media for isolation of N. gonorrhoeae. Selective agent Concentrations used Organisms inhibited Vancomycin 2–4 mg/L Gram-positive bacteria Lincomycin 1 mg/L Gram-positive bacteria Colistin 7.5 mg/L Gram-negative bacteria     (other than Neisseria ) Trimethoprim 5 mg/L Gram-negative bacteria     (other than Neisseria ) Nystatin 12,500 IU/L Candida spp. Amphotericin 1–1.5 mg/L Candida spp.

Fig. 2.28 Comparison of growth from endocervical swab on selective (left) and non-selective medium (right).

Fig. 2.29 Comparison of growth of a pure growth of N. gonorrhoeae on selective (left) and non-selective medium (right) demonstrating growth retardation by the selective agents.
The specimen should be inoculated over the entire surface of the plate in a ‘Z’ pattern, followed by streak inoculation of the plate ( Figs 2.30 – 2.31 ). This inoculation technique yields isolated colonies that can be more easily processed, particularly in pharyngeal specimens, from which strains of N. meningitidis , N. lactamica , and K. denitrificans may also be isolated.

Fig. 2.30 ‘Z’ streak method of inoculation to obtain isolated colonies for the identification of Neisseria .

Fig. 2.31 Cross-streaking of ‘Z’ inoculated plate to insure separation of colonies.
Inoculated plates should be incubated immediately at 35–36.5°C in a CO 2 -enriched, humid atmosphere. Gonococci require CO 2 for primary isolation; the supplemental CO 2 can be provided in a CO 2 incubator, a container with a CO 2 -generating tablet, or a candle-extinction jar with white, unscented, non-toxic candles.

Identification of N. Gonorrhoeae

Presumptive identification
Culture plates are examined for growth after incubation for 24 hours; those that show no growth at this time are reincubated for 24–48 hours before being discarded and before a report of negative for N. gonorrhoeae is issued. Translucent, non-pigmented-to-brownish colonies measuring 0.5–1.0 mm in diameter on isolation media should be further characterized ( Fig. 2.32 ). Representative colonies are Gram stained and examined for oxidase production. Smears prepared from suspect colonies should be examined microscopically for the presence of Gram-negative diplococci. Cells of Neisseria spp. occur as diplococci composed of kidney-shaped cells (0.8 µm–0.6 µm) with adjacent sides flattened (see Fig. 2.25 ). Oxidase is detected either by placing a drop of oxidase reagent (tetramethyl-paraphenylenediamine-dihydrochloride) on a few representative colonies ( Fig. 2.33 ) or by rubbing representative colonies on filter paper moistened with oxidase reagent with a platinum or plastic loop ( Fig 2.34 ). In a positive test, the colonies will turn purple within 10 seconds. The oxidase reagent should not be placed on all suspect colonies. If few suspect colonies are available, they must be subcultured to chocolate agar immediately after the application of the oxidase reagent because of the toxicity of the reagent to the cells. Thin smears of suspect colonies are Gram stained as described above. If the suspect colonies are oxidase-positive, Gram-negative diplococci, a report of presumptive N. gonorrhoeae may be made for cervical, urethral, or rectal specimens.

Fig. 2.32 Inoculated plate after 24 hours of incubation.

Fig. 2.33 Positive oxidase reaction on culture of N. gonorrhoeae .

Fig. 2.34 Oxidase strip showing a negative reaction (yellow) indicating not N. gonorrhoeae and a positive reaction (purple) indicating presumptive N. gonorrhoeae .

Confirmation of identification
Ideally, when culture is performed, all isolates should be confirmed as N. gonorrhoeae , although this is not always possible when adequate laboratory facilities and resources are not available. Certainly, pharyngeal isolates must be confirmed because other Neisseria and related spp. may frequently be isolated on gonococcal selective media. The aim of identification tests is to distinguish between N. gonorrhoeae and other species of Neisseria , particularly N. meningitidis and N. lactamica . 7 Traditonally, N. gonorrhoeae is characterized by its ability to produce acid from glucose but not from maltose, lactose and sucrose after incubation at 35–36.5°C without CO 2 for 24–48 hours ( Figs 2.35 and 2.36 ) and, from growth on a selective isolation medium, this may be sufficient. However, many commensal Neisseria grow on non-selective media and these may produce acid from glucose and therefore additional tests may be required such as reduction of nitrate and the production of polysaccharide. 7

Fig. 2.35 Acid production from carbohydrates in Cystine Trypticase Agar (CTA) medium. Tubes from left to right are CTA base medium containing no carbohydrate, CTA medium containing 1% glucose and CTA medium containing 1% maltose. The organism is N. gonorrhoeae .

Fig. 2.36 Acid production from carbohydrates in CTA medium (see Fig. 2.32 for further details). The organism is N. meningitidis .
It is now more usual to use rapid identification kits ( Fig 2.37 and 2.38 ) which combine the detection of acid production from carbohydrates with that of preformed enzymes such as prolyliminopeptidase which is produced by N. gonorrhoeae , β-glutamyl aminopeptidase produced by N. meningitidis , and β-galactosidase produced by N. lactamica ; strains of Branlamella catarrhalis produce none of these enzymes. A biochemical profile is obtained from these kits usually after 2–4 hours’ incubation and this gives a precentage likelihood of the identification. A pure growth of the organism to be identified is required and therefore an overnight subculture is usually required before identification can take place. The use of tests which only detect the presence of aminopeptidases should be discouraged as N. gonorrhoeae that lack the ability to produce prolyliminopeptidase have been reported in many countries in recent years. 8

Fig. 2.37 API NH identification kit in incubation chamber demonstrating the profile of N. gonorrhoeae .

Fig. 2.38 Rapid NH identification kit demonstrating the profile of N. gonorrhoeae .

Serologic tests
There are currently no available tests that permit the detection of gonococcal antibodies in serum. Serologic tests for the identification of N. gonorrhoeae in primary cultures are commercially available as immunofluorescence (FA), coagglutination or latex tests. Thin smears may be stained with a monoclonal FA reagent ( Fig. 2.39 ) to confirm the identification of N. gonorrhoeae . Cross-reaction with non-gonococcal species has not been observed with monoclonal reagents and reagents containing polyclonal antibodies are no longer commercially available. Occasionally, some gonococcal strains do not react in this test (false negative).

Fig. 2.39 Monoclonal FA stain.
Coagglutination tests consist of cocktails of monoclonal antibodies directed toward the major gonococcal porin, Por, which have been adsorbed to protein A-producing Staphylococcus aureus cells. Suspect colonies are suspended in buffer or saline and heated in a boiling-water bath. A drop of the cooled suspension is mixed with a drop each of the antigonococcal reagents and a negative control. After rotation for 1–2 minutes, the reactions are interpreted. If a suspension gives a positive reaction with the antigonococcal reagent and a negative reaction with the control, the isolate is identified as N. gonorrhoeae and the use of the two reagents also gives the serogroup WI or WII/III ( Fig. 2.40 ). Cross-reactions between non-gonococcal Neisseria and related spp. is rare ( Fig. 2.41 ) although gonococcal strains that do not react with the reagents do occur. Reagents that are based on latex particles do exist but have little advantage over the co-agglutination reagents.

Fig. 2.40 Reaction of two strains (A, wells 1 and 2 and B, wells 5 and 6) of N. gonorrhoeae with Phadebact coagglutination reagents WI (wells 1 and 5) and WII/III (wells 2 and 6).

Fig. 2.41 Reactions of N. gonorrhoeae , N. meningitidis , and related spp. in the Phadebact coagglutination test:
1 N. gonorrhoeae .
2 Negative control.
3 N. meningitidis .
4 N. cinerea.
5 N. lactamica .
6 Negative control.
The approach to identification of N. gonorrhoeae will depend on the number and source of isolates obtained in any laboratory but careful consideration should be taken to the limitations of the tests available 9 and where possible it is advisable to use more than one test, for instance a biochemical kit and immunological reagent together. The identification of organisms isolated from sexual or child abuse cases is always difficult and the use of multiple tests is essential.

Isolates of N. gonorrhoeae must be subcultured every 24–48 hours or suspended in a solution of trypticase soy broth containing 15% glycerol and frozen at −70°C. Strains cannot be stored at −20°C for long periods but can be stored at this temperature for a short time (approximately 2 months).

Molecular Detection of N. Gonorrhoeae
Molecular diagnosis of gonorrhea is available using commercially available dual kits that detect C. trachomatis and N. gonorrhoeae by different methods of nucleic acid amplification; polymerase chain reaction (PCR, Roche Amplicor), strand displacement amplification (SDA; BD Probe Tec), transcriptional mediated amplification (TMA; Gen Probe) and real-time PCR (Abbott). A number of in-house assays have also been reported and two of the most useful of these detect the pseudo por A gene and the opa gene 10 and are particularly useful as confirmatory assays. The use of molecular methods for the diagnosis of gonorrhea has increased markedly in the last five years and this has been driven by the widespread use of NAATs for chlamydia screening, the opportunity to detect two sexually transmitted infections for little or no extra cost, together with a vast improvement in the sensitivity and specificity of these tests. The high sensitivity allows the use of non-invasive specimens such as urine or self-taken vaginal swabs and hence enables patients to be screened in many different settings, although there is evidence that urines are not the optimal specimen for women. 11 The increased specificity allows use with extragenital specimens, although these kits are not approved for use with rectal and pharyngeal specimens, there is increasing evidence that molecular detection is becoming the method of choice for these sites as culture is shown to have a low sensitivity. 12 However, it is recommended NAATs should only be used with specimens from individuals at risk of infection and that all positive results should be confirmed using an alternative target to avoid misdiagnosis due to cross-reactions with other Neisseria spp., especially with pharyngeal specimens.
While NAATs offer many advantages for the diagnosis of gonorrhea their use will be affected by the prevalence of infection in the population being tested. In low prevalence populations the positive predicitive value (PPV) will be unacceptably low unless a supplementary test is used to confirm using a different genetic target. For example: in a population with a 1% prevalence using a test with sensitivity of 98% and specificity of 99% the PPV is 50% and the negative predictive value (NPV) is 100%. If the positive specimens are tested using a supplementary test of equal sensitivity and specificity (98% and 99% respectively) then the prevalence becomes 50% and the PPV is 99% and the NPV is 98%. It is generally recommended that the algorithm for testing should produce a PPV of >90% to limit misdiagnosis. 10

Antimicrobial Susceptibility Testing

β-Lactamase tests
All gonococcal isolates should be tested for β-lactamase, which can be detected in colonies on primary isolation medium ( Fig 2.42 ) or after subculture and as part of the identification profile ( Fig 2.43 ). Several tests for β-lactamase are available commercially; these include chromogenic cephalosporin ( Fig. 2.44 ), acidometric, and iodometric tests. The chromogenic cephalosporin (nitrocefin) tests are preferred because the substrate is stable and the reactions are specific and highly sensitive for β-lactamase. The specificity and sensitivity of the acidometric and iodometric tests may be affected by several factors, including incorrect storage of the product, which may result in non-specific hydrolysis of the substrate. β-lactamase-positive and β-lactamase-negative strains should be included as controls with each batch of clinical isolates.

Fig. 2.42 Chromogenic cephalosporin, nitrocefin, directly on a gonococcal culture showing the colour change to pink for a positive reaction (left) and no change for a negative reaction (right) for β-lactamase production.

Fig. 2.43 Comparison of β-lactamase production (*) as detected by API NH (well) for a positive (top) and negative (bottom) strain of N. gonorrhoeae .

Fig. 2.44 Reactions of β-lactamase-positive (left) and negative (right) strains of N. gonorrhoeae in a nitrocefin test.

Determination of antimicrobial susceptibility

Disk-diffusion susceptibility testing
Disk-diffusion susceptibility testing is most frequently used to measure the antimicrobial resistance of isolates of N. gonorrhoeae for patient management whereas determination of the minimal inhibitory concentration (MIC) is more appropriate for surveillance programs. The methodology varies in different parts of the world and can be performed on both blood-containing media ( Fig 2.45 and 2.46 ). or on GC agar base supplemented with IsoVitaleX or an equivalent and to the use of either high- or low-dose discs. 13

Fig. 2.45 Disc susceptibility test for N. gonorrhoeae using blood-containing medium.

Fig. 2.46 Disc susceptibility test for N. gonorrhoeae using GC agar medium.

Agar-dilution susceptibility testing
Agar-dilution susceptibility testing is the reference method for measuring the antimicrobial susceptibilities of strains of N. gonorrhoeae . Resistance to antimicrobial agents is measured as the MIC of the agent that inhibits growth of an isolate. Determination of susceptibilities to antimicrobial agents used for therapy should be tested such as the third generation cephalosporins, cefixime and ceftriaxone although other agents, such as ceftobutin, are used in different parts of the world. The methodology for susceptibility testing of N. gonorrhoeae can vary between countries, differing primarily in the base medium used; GC agar base (CLSI, North America), Isosensitest (Australia) and Diagnostic Sensitivity Agar (Europe). For therapeutic antimicrobial agents this has historically been most important for the concentration of penicillin considered resistant; which can differ from ≥2 mg/L (GC base agar) and ≥1 mg/L (Isosensitest and DST agar). Other antimicrobials agents are affected less but this should be remembered when comparing different methodologies.
Susceptibility testing is performed on the base medium of choice containing 1% (vol/vol) IsoVitaleX or an equivalent supplement; antimicrobial agents are incorporated into the base medium in serial twofold dilutions. Isolates to be tested are grown overnight on chocolate agar and suspended in Mueller–Hinton broth (or equivalent) to an optical density equivalent to a 0.5 McFarland standard, containing approximately 10 8 colony-forming units (CFU)/mL. The suspensions are diluted 1 : 10 in Mueller–Hinton broth, and 10 4  CFU/mL are inoculated onto the surface of the antibiotic-containing media and an antibiotic-free control medium with a Steer’s replicator, multipoint inoculator or a calibrated loop. Plates are incubated at 35–36°C in a CO 2 -enriched atmosphere for 24 hours and then examined for growth. The MIC of the antimicrobial agent for an isolate is the lowest concentration that inhibits its growth ( Fig. 2.47 ). A modification of the full MIC is the use of breakpoints which uses a similar method but with medium containing one or two concentrations of antibiotic that can be used to categorize isolates as being resistant or having decreased susceptibility. This is useful for screening isolates, particularly for high-level resistance.

Fig. 2.47 Susceptibility of N. gonorrhoeae to penicillin determined by the agar dilution method. Medium containing 0.5 µg/mL penicillin (Left) showing inhibition of some strains compared to the control medium without antibiotic (Right).
An alternative method for determining the MIC is the use of the Etest. This uses a strip, impregnated with antibiotic at different concentrations which is placed on a previously seeded agar plate which results in the antibiotic immediately being released into the medium. After overnight incubation the MIC is determined where the elipse crosses the strip ( Fig 2.48 ). This is a very useful method for testing single strains but is also useful for confirming MICs determined by the agar dilution method.

Fig. 2.48 Susceptibility of N. gonorrhoeae to penicillin using the Etest. MIC is determined at the point the elipse crosses the strip (0.06 mg/L).

Interpretation of antimicrobial susceptibility test results
An antimicrobial susceptibility result determined in the laboratory is only a measure of the in vitro susceptibility of an isolate. A patient may have a positive test-of-cure culture for a variety of reasons:
1 Failure of therapy because of infection with a resistant isolate
2 Failure of therapy even when the patient is infected with a strain that is susceptible by in vitro measurements because of non-compliance with treatment.
3 Reinfection.
Thus, antimicrobial susceptibilities must be used as an adjunct to, but cannot be substituted for, clinical findings. The aim of susceptibility testing is to correlate with treatment outcome ( Table 2.6 ).
Table 2.6 Correlates of disk-diffusion susceptibility results with clinical outcome. SUSCEPTIBLE Less than 5% likelihood of treatment failure INTERMEDIATE Predictable failure rates of 5 to 15% if the patient is   treated with the tested antibiotic in the standard dosage   (in most cases of intermediate susceptibility, a higher dose   or prolonged therapy results in greater than 95% cure   rates) RESISTANT May be associated with treatment failure rates of greater   than 15%

Antimicrobial therapy
In response to the continued emergence of resistant strains in the USA, the current CDC STD Treatment Guidelines 14 recommend treatment of all gonococcal infections presumptively with antibiotic regimens effective against strains resistant to penicillin, quinolones and/or tetracycline. First line agents recommeded by CDC are either imtramuscular ceftriaxone (125 mg) or oral cefixime (400 mg), although availability of the latter has been a problem in the USA in recent years. Spectinomycin, 2 g intramuscularly, is a useful regimen for the treatment of patients who have severe allergic reactions to penicillins (e.g. bronchospasm, anaphylaxis) in whom cephalosporins would be contraindicated due to the 10% risk of cross-allergy in penicillin-allergic patients. The World Health Organization’s (WHO) treatment guidelines 15 are very similar to former CDC treatment guidelines and are currently being updated. Similar to the CDC guidelines, the UK National Guidelines 16 and European guidelines 17 recommend either a single dose cephalosporin (ceftriaxone or cefixime), although the ceftriaxone is used at the higher 250 mg dose, or spectinomycin as first-line therapy. It should be noted that treatment recommendations for complicated gonococcal infections ( Table 2.7 ) are based on expert opinion, not on therapeutic trials.
Table 2.7 Recommended therapy for patients with complicated gonococcal infections. Syndrome Recommended therapy Disseminated gonococcal infection Ceftriaxone * : 1 g, i.m. or i.v., every 24 hoursAll regimens should be continued for 24–48 hours after improvement begins; therapy may then be switched to cefixime 400 mg p.o. b.i.d. or ciprofloxacin 500 mg p.o. b.i.d. (if isolate is proven to be susceptible) to complete 7 days of therapy Meningitis/endocarditis Consultation with an expert is vital. Ceftriaxone 1–2 g i.v. every 12 hours. Patients with meningitis should be treated for 10–14 days; those with endocarditis should be treated for at least 1 month Ophthalmia   Neonatal Ceftriaxone: 25–50 mg/kg body weight, i.m. or i.v., in a single dose, not to exceed 125 mg Adult Ceftriaxone: 1 g i.m., 1 dose †
* Or cefotaxime 1 g i.v. every 8 hours, or ceftizoxime 1 g i.v. every 8 hours, or spectinomycin 2 g i.m. every 12 hours.
† Infected eye should be lavaged with saline solution.
Although not listed as a first-line agent in the CDC, WHO or UK treatment guideline for gonorrhea, azithromycin 2 g as a single dose may be a useful agent in selected individual cases. Like spectinomycin, it may be given to pregnant women and also to patients with severe penicillin allergies in whom cephalosporins are contraindicated. It is not recommended as a first-line agent as resistance to azithromycin may develop on treatment and azithromycin-resistant N. gonorrhoeae isolates are already circulating. 3 In really difficult cases, use of gentamicin 240 mg as a single intramuscular dose may be used. Although there remains debate over the MIC breakpoints that determine clinical susceptibility and resistance, this agent has been used with success as a first-line agent in Malawi for treatment of the male urethritis syndrome for over 14 years, and clinical treatment failures appear to be rare. Given the increasing prevalence of multidrug resistant gonococci globally, it is likely that future therapy will require multidrug therapy and a move away from single dose regimens in an attempt to maintain the useful life of the few antibiotics currently available to reliably treat gonorrhea.

Molecular typing of gonococci
N. gonorrhoeae exhibit a high degree of genetic variation due to their competence for horizontal gene exchange and are non-clonal in nature. In vivo mixed infections are thought to occur frequently, particularly in individuals with multiple partners or a high rate of partner change, contributing to genetic variation. The rate of genetic variation is unknown but it is possible, depending on the gene and method chosen, that each isolate can appear distinct unless part of a short transmission chain. While this questions the validity of using typing for long-term studies it has the potential for a greater level of discrimination that can distinguish between isolates of the same serovar, determine the genetic relatedness of antibiotic-resistant isolates and can identify isolates from sexual contacts. A variety of genotyping methods have been used with differing levels of discrimination; restriction endonuclease fingerprinting, ribotyping and amplification by polymerase chain reaction using arbitrary or repetitive element sequence-based primers are the least discriminatory while pulsed field gel electrophoresis, opa-typing ( Fig. 2.6 ), NG-MAST ( Fig. 2.7 ) and por sequencing give the greatest discrimination. All of these methods show discrimination equal to or greater than phenotyping and therefore it is essential that the question to be addressed is considered carefully before the genotypic method is chosen. NG-MAST, which examines variation in two hypervariable genes ( Fig. 2.49 ) has become popular because it gives highly discriminatory, robust and shareable data and new genotypes can be assigned through the NG-MAST website ( ) 18 . The most informative data that has been obtained using molecular typing is when microbiological and epidemiological approaches are combined and this has been shown to identify clusters of isolates that are circulating within networks of differing sexual orientation. 19

Fig. 2.49 Molecular typing using NG-MAST by amplification of two alleles, por and tbp B, to provide a sequence type.

The authors acknowledge the contribution of David Martin in co-writing the previous edition of this chapter with Dr C. Ison.


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2 Zhao S, Duncan M, Tomberg J, et al. Genetics of chromosomally mediated intermediate resistance to ceftriaxone and cefixime in Neisseria gonorrhoeae . Antimicrob Agents Chemother . 2009;53:3744-3751.
3 Chisholm SA, Neal TJ, Alawattegama AB, et al. Emergence of high-level azithromycin resistance in Neisseria gonorrhoeae in England and Wales. J Antimicrob Chemother . 2009;64:353-358.
4 Workowski KA, Beran SM, Douglas JM. Emerging resistance in Neisseria gonorrhoeae : Urgent need to strengthen prevention strategies. Ann Inter Med . 2008;148:606-613.
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6 Liebling MR, Arkfeld DG, Michelini GA, et al. Identification of Neisseria gonorrhoeae in synovial fluid using the polymerase chain reaction. Arthritis and Rheum . 1994;37:702-709.
7 Department of Health & Human Services, Centers for Disease Control and Prevention. Identification of Neisseria and related species. . (reviewed 10/24/08, accessed 10.25.09)
8 Unemo M, Palmer HM, Blackmore T, Herrera G, Fredlund H, Limnios A, Nguyen N, Tapsall J. Global transmission of prolyliminopeptidase-negative Neisseria gonorrhoeae strains: implications for changes in diagnostic strategies. Sex Transm Infect . 2007 Feb;83:47-51.
9 Alexander S, Ison C. Evaluation of commercial kits for the identification of Neisseria gonorrhoeae . J Med Microbiol . 2005;54:827-831.
10 Whiley DM, Garland SM, Harnett G, Lum G, Smith DW, Tabrizi SN, Sloots TP, Tapsall JW. Exploring ‘best practice’ for nucleic acid detection of Neisseria gonorrhoeae . Sex Health . 2008;5:17-23.
11 Cook RL, Hutchinson SL, Østergaard L, et al. Systematic review: Non-invasive testing for Chlamydia trachomatis and Neisseria gonorrhoeae . Ann Intern Med . 2005;142:914-925.
12 Schachter J, Moncada J, Liska S, Shayevich C, Klausner JD. Nucleic acid amplification tests in the diagnosis of chlamydial and gonococcal infections of the oropharynx and rectum in men who have sex with men. Sex Transm Dis . 2008;35:637-642.
13 Clinical and Laboratory Standards Institute. M2-A9 Performance Standards for Antimicrobial Susceptibility Testing: Seventeenth Informational Supplement. CLSI document M100-MS17 2007.
14 Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines 2006. .
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16 Clinical Effectiveness Group, MSSVD. National guideline on the diagnosis and treatment of gonorrhoea in adults. , 2005. (accessed 31.08.09)
17 Bignell C, IUSTI/WHO. 2009 European (IUSTI/WHO) guideline on the diagnosis and treatment of gonorrhoea in adults. Int J STD AIDS . 2009;20:453-457.
18 . (accessed 6 July 2010)
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3 Infections Caused by Chlamydia Trachomatis

Magnus Unemo, John R. Papp

Chlamydia trachomatis is the causative agent of trachoma, which is the world`s leading cause of preventable blindness and is endemic in many developing countries. C. trachomatis is also the etiological agent of human chlamydial urogenital tract infection, which is the most common bacterial STD. It has a worldwide distribution and causes substantial morbidity and economical cost. Lymphogranuloma venereum (LGV) is endemic in East and West Africa, India, and parts of South-East Asia, South America and the Caribbean. Since 2003, outbreaks of LGV proctitis and proctocolitis have been documented among men who have sex with men (MSM) in Europe and North America. Previously only sporadic cases had been observed.
The etiological agent of trachoma was first visualized in 1907 by Halberstaedter and von Prowazek, who identified typical intracytoplasmic inclusions in stained conjunctival scrapings from orangutans that had been inoculated with human trachomatous material. In the following three years, the bacterium was also associated with neonatal inclusion conjunctivitis, cervicitis in women, and non-gonococcal urethritis (NGU) in both sexes. A relationship between Chlamydia and LGV, the more invasive and sometimes systemic infection that primarily affects lymphatic tissue, was noted in the 1930s. LGV was propagated first in monkey brain, mice and then eggs. The first trachoma agent was isolated in the yolk sac of embryonated hen’s egg in 1957. In 1959 and 1964, the first urogenital tract isolates of C. trachomatis were isolated from the cervix and male urethras, respectively. 1 - 3

In 1999, a new taxonomic classification of the order Chlamydiales at the family, genus and species level based primarily on 16S rRNA and 23S rRNA gene sequences as well as other genetic, phenotypic and ecological data, was introduced ( Fig. 3.1 ). The order Chlamydiales comprises four families, i.e. Chlamydiaceae (also referred to as chlamydiae in the text below), Parachlamydiaceae, Waddliaceae and Simkaniaceae, of obligate intracellular bacteria, with a unique bi-phasic developmental cycle and >80% sequence identity in the full-length 16S rRNA and/or 23S rRNA genes. The family Chlamydiaceae contains the genera Chlamydia (3 species) and Chlamydophila (6 species), which share >90% 16S rRNA and 23S rRNA sequence homology ( Fig. 3.1 ) and express the family-specific KDO-trisaccharide epitope (previously named the genus-specific epitope) in their lipopolysaccharide (LPS, endotoxin). Within the genera, the different species display ≥95% sequence identity in the 16S rRNA and 23S rRNA genes to a genus type strain. Within the genus Chlamydia , the only human pathogenic species is C. trachomatis . C. trachomatis contains two biovars, i.e. trachoma (primarily non-invasive, epitheliotrophic, and infecting the eye [‘pathovariant’ trachoma] and the urogenital tract [‘pathovariant’ STD]) and LGV (invasive, causing a disseminating infection of the draining regional lymph nodes), depending on the cell- or organotropism, and is restricted to human hosts. Within the genus Chlamydophila , there are two species typically causing infections in humans, i.e. Chlamydophila pneumoniae and Chlamydophila psittaci . Chlamydophila pneumoniae (’TWAR’ micro-organism described in 1986), is a common human respiratory tract pathogen worldwide, but has also been suggested to cause e.g. coronary artery disease. Chlamydophila pneumoniae has been recently shown to infect also other animals. Chlamydophila psittaci , described in 1930, has a broad host range among non-human vertebrates and causes the zoonotic disease ornithosis (‘psittacosis’), which is characterized by respiratory disease in humans. 1 - 3

Fig. 3.1 Simplified, schematic drawing of the new (A) and old (B) taxonomic classification of the order Chlamydiales.
Chlamydiae were, when discovered, suspected to be protozoa, and later they were considered viruses (due to their small size and obligate requirement for host cells for propagation). However, in 1966 with the advent of tissue culture and electron microscopy it became evident that they were bacteria, although with limited metabolic capacity. Chlamydiae share most of the common structural features of Gram-negative bacteria, including a cell wall with an outer membrane (OM) consisting of rough LPS, porins and some additional proteins, a periplasmic space, although normal peptidoglycan and muramic acid are not detectable in the elementary body (EB), typical inner membrane, as well as ribosomes, DNA, RNA, and metabolic functions ( Table 3.1 ). However, chlamydiae cannot be cultured on artificial cell-free medium and the propagation of chlamydiae only within embryonated chickens’ eggs, in cell or tissue culture, has impeded the study of both the biology and clinical manifestations of infection. 1 - 3
Table 3.1 Distinguishing characteristics of species belonging to the family Chlamydiaceae.
Obligate intracellular pathogens, with limited metabolic capability
Unique biphasic (dimorphic) developmental cycle within an intracellular cytoplasmic inclusion
Cell wall similar to other Gram-negative bacteria, although normal peptidoglycan is not detectable in the elementary body
Contain DNA, RNA, and typical prokaryotic ribosomes
Small genome (about 1.0–1.2 million nucleotides)
Genomes highly conserved (intra- and interspecies) in DNA sequence, gene content and gene synteny (order)
Minor genomic differences sufficient to define a wide host range, tissue tropism and disease presentation
Phylogenetically distantly related to bacteria outside the Chlamydiales order
Comprehensive knowledge of the unique, bi-phasic intracellular developmental cycle and the structure of chlamydiae is crucial in understanding the parasite–host interaction ( Fig. 3.2 ). 3 - 10 The infectious particle, the EB, is a rigid, condensed and spore-like spheroid with a diameter of 200–400 nm ( Figs 3.3 – 3.5 ). It is environmentally resistant, metabolically inactive, and contains an electron-dense chromosome or nucleoid (DNA tightly condensed by histone-like proteins) and an OM composed primarily of LPS and proteins that are highly disulfide cross-linked. After initial attachment to the host cell, the EB appears to induce its own rapid entry by a receptor-mediated endocytosis. However, the specific host receptor and cognate ligand have not been conclusively identified. These may also differ for divergent species, serovars (also referred to as serotypes), host cells, electrostatic conditions, etc., and multiple entry mechanisms may even be involved. The EB resides within a membrane-bound endosome/phagosome, which is able to inhibit phagolysosomal fusion and subsequent lysis of bacteria by lysosomes of the host cell. Following entry, the membrane of the endosome (inclusion) is modified by secreted chlamydial proteins, e.g. inclusion membrane proteins (Incs), that have a role in controlling interactions with the host cell, inclusion development and avoidance of lysosomal targeting. There are many potential virulence factors, one of the best studied is the type III secretion system. The type III secretion system can deliver chlamydial effector proteins, such as Incs, CopN, and Tarp (potentially involved in invasion), into inclusion vacuoles and membranes, or into host cytoplasm, which may subvert the host cell for the benefit of the bacterium. Furthermore, the chlamydial protein cHsp60 (GroEL), related to common heat-shock proteins (Hsps), is upregulated in response to cellular stress, it modulates the host immune response, and is considered to have an immunopathogenic role. Within 6–8 hours post-entry, the EB decondenses to form a reticulate body (RB). The RB is more permeable, allowing uptake of ATP and required nutrients; it is metabolically active, and synthesizes its own RNA, DNA, proteins, and LPS using some metabolic precursors from the host cell. It is pleomorphic, less electron-dense, fragile, non-infectious, and has a diameter of 500–1000 nm ( Fig. 3.5 ). The RB replicates by binary fission, following a typical sigmoidal curve. The growth phase usually involves eight to ten bacterial cell divisions (generation time about 2–3 hours). By 24 hours, the RBs can be seen by microscopy within characteristic intracytoplasmic inclusions ( Fig. 3.6 ). During the next 24–36 hours, the RBs divide but the developmental cycle slows and becomes asynchronous, and RBs start to condense and transform into EBs, which predominate in mature inclusions containing several hundreds or even thousands of chlamydial cells. After 30–60 hours following iodine staining glycogen accumulation within the inclusion can be seen for C. trachomatis , but not for Chlamydophila psittaci and Chlamydophila pneumoniae that do not produce and accumulate large quantities of glycogen or glycogen-like deposits. Other biologic and phenotypic characteristics that distinguish these human pathogenic species can be seen in Table 3.2 . After 48–72 hours, the inclusion is extruded and/or the host cell is lysed, and the infectious EBs can be extracellularly transmitted, while the RBs do not survive. The time to completion of the developmental cycle is dependent on species, strain and cell type and may vary from 36 to 72 hours. Importantly, most studies on chlamydial biology, including developmental cycle, have been performed in vitro using continuous cell culture (usually Hela cells) with LGV strains and these artificial systems do not necessarily reflect what occurs in vivo. The ‘normal’ lytic cycle described above can be affected by many factors including IFN-γ, nutrient (e.g. tryptophan) depletion, antimicrobial treatment, heat shock, cyclic AMP, hormones and bacteriophage infection. In these circumstances chlamydiae may lose their infectivity as RB to EB transition is retarded and the infection becomes persistent (‘arrest of the developmental cycle’), i.e. the bacteria remain as viable but aberrant and non-dividing RBs, however these can be reactivated.

Fig. 3.2 Development cycle of chlamydiae. The infectious metabolically inactive elementary body (EB) attaches to the host cell and appears to induce its own rapid entry by a receptor-mediated endocytosis. The EB resides within a membrane-bound endosome/phagosome, which is able to inhibit phagolysosomal fusion. Within 6–8 hours post entry, the EB decondenses to form a metabolically active and replicating reticulate body (RB). By 24 hours, the RBs can be seen by microscopy within characteristic inclusions. During the next 24–36 hours, the RBs divide but the developmental cycle slows and becomes asynchronous, and RBs start to condense and transform into EBs, which predominate in mature inclusions. After 48–72 hours, the inclusion is extruded and/or the host cell is lysed, and the infectious EBs can be extracellularly transmitted, while the RBs do not survive. The time to completion of the developmental cycle may vary dependent on species, strain and cell type.

Fig. 3.3 Transmission electron micrograph showing a single C. trachomatis elementary body; condensation of the DNA is in progress (×120 000).
Courtesy of Elizabeth H. White.

Fig. 3.4 Transmission electron micrograph of a mature inclusion immediately prior to cell lysis. The inclusion contains mainly elementary bodies (EBs) which are electron dense and approximately 0.4 µm in diameter. The cell nucleus has been displaced by the inclusion.
Courtesy of Ian N. Clarke.

Fig. 3.5 Higher magnification image of the inclusion shown in Fig 3.4 . Dark, electron-dense elementary bodies (EBs; 0.4 µm) predominate although some electron-lucent reticulate bodies (RBs; 1.0 µm) are present; intermediate forms showing condensation of the chromosomal DNA are also present. Approximately in the center of the field a pair of RBs are seen just before final separation following binary fission.
Courtesy of Ian N. Clarke.

Fig. 3.6 Chlamydial inclusions in cell monolayer stained with fluorescein-labeled C. trachomatis -specific monoclonal antibody.

Table 3.2 Distinguishing biologic and phenotypic features of the pathogenic Chlamydia and Chlamydophila species that can infect humans.
The OM of the C. trachomatis EB contains proteins and LPS. 3 - 10 The major OM protein (MOMP, discovered in 1981) is a surface-exposed, transmembrane, cysteine-rich porin, which represents about 60% of the OM’s weight and approximately 30% of the organism’s weight. The MOMP has an approximate subunit mass of 38–43 kDa (differs by serovar); it is involved in the initial interaction with host cells; it is also immunologically the dominant OM protein, and exposes species, subspecies, and serovar-specific epitopes. Structural rigidity (osmotic stability) of the EB is due to extensive disulfide bond crosslinking between MOMP and other OM proteins (Omp2 [OmcB], Omp3 [OmcA], and likely additional ones), which is reduced in the EB–RB transition. PorB is an additional immunogenic, surface-exposed OM porin, which is highly conserved, substrate-specific, and expressed at low level. Omp85 is a highly conserved OM protein with mainly unknown function. Furthermore, at least a subset of a family of nine different polymorphic membrane proteins (Pmps) are also surface-exposed. These Pmps are considered to be autotransporters, interact with the host cell, e.g. in attachment, and have high immunogenicity.
C. trachomatis can be characterized serologically, using polyclonal (PAbs) or monoclonal antibodies (MAbs), based on different antigenic epitopes on the MOMP and/or genotypically by examining the MOMP-encoding ompA gene (formerly named omp1 ) into at least 19 serovars (genovars). Serovars A to C are the agents of endemic trachoma, while serovars B and especially D to K cause oculogenital infections and neonatal pneumonia. The LGV biovar including serovars L1–L3 causes the more invasive LGV ( Table 3.3 ). Urogenital infection by multiple serovars has been reported, and has been observed in vitro ( Fig. 3.7 ).

Table 3.3 Distinguishing features and infections associated with different serovars (two biovars, including three ‘pathovariants’) of C. trachomatis .

Fig. 3.7 HeLa cells infected with two C. trachomatis serovars and stained with two serovar-specific monoclonal antibodies, each labeled with a different fluorochrome. The upper cell contains two inclusions, one illustrating that both serovars coexist in the same cell and inclusion.
Detailed studies of the biology of chlamydial infection have been hampered by the limited, unsynchronized and obligate intracellular growth, and the absence of feasible direct or indirect methods for making mutants, transforming and manipulating the chlamydial genome. However, recent whole-genome sequencing has revealed major insights into the genetics, molecular structure, physiology and metabolism, intracellular biology and pathogenicity of chlamydiae. The first complete genome to be sequenced, C. trachomatis serovar D, contained 1,042,519 bp and 894 predicted proteins ( Fig. 3.8 ). 11 The genome of several chlamydiae isolates have now been sequenced, and the general features of some of these genomes can be seen in Table 3.4 . The genome sequences of chlamydiae are small (approximately 1.0–1.2 Mb; larger for Chlamydophila species) relative to most other bacterial genomes, and have a high level of conservation in sequence, overall gene content and gene synteny (order). Remarkably, 80% to 98% of all genes and gene synteny are conserved, i.e. a ‘core genome’ defining the basic phenotypic and biological attributes that are common to the chlamydiae. The minor differences are obviously sufficient to define host range, tissue tropisms, and disease presentation. Important information from the whole-genome sequences includes the presence of genes for a nearly complete peptidoglycan biosynthesis and recycling, penicillin-binding proteins 2 and 3, energy generating system (ATP and NADH production; in addition to ADP/ATP translocases that import essential ATP from the host), type III secretion system, and the families of Pmps and Incs, as well as the absence of the highly conserved prokaryotic gene for the key septation protein FtsZ that has been thought to be essential for cell division in all bacteria, the underrepresentation of genes for amino acid biosynthesis and nucleotide metabolism, and the unexpectedly high number of chlamydial genes related to higher organisms, particularly plant chloroplasts. The genome sequences have also been used to study gene expression (transcriptional analysis) by microarray and proteomic analyses. Furthermore, based on the genomic comparisons of many members of the family Chlamydiaceae, the taxonomic classification dividing into two genera, Chlamydia and Chlamydophila , has again been questioned. 12

Fig. 3.8 Protein functional assignments deduced from the C. trachomatis genome sequence.

Table 3.4 General properties of Chlamydia and Chlamydophila genomes.
Recently, detailed genomic comparisons of the two biovars (three ‘pathovariants’; represented by serovar A, D, L2) of C. trachomatis have shown that the genomes are highly similar in size, nucleotide sequence (>99.5% median nucleotide identity of genes), and number of predicted proteins. These genomes encode 889–920 predicted proteins ( Table 3.4 ). Of the predicted and also functional genes, 846 were common to all three genomes ( Fig. 3.9 ), and the remaining genes could be entirely accounted for by either in silico differences in gene prediction, or as a consequence of functional gene loss (pseudogenes were excluded in the comparison). Consequently, only minor genomic differences, including functional gene loss, pseudogenes, single nucleotide polymorphisms (SNPs) impacting on protein function, and also altered levels of gene expression, must determine the disease phenotype. Most of the diversity (gene content and sequence) between chlamydiae genomes is located in the plasticity zone (PZ) located at the replication terminus region of the chromosome. For example, differences in the genes encoding tryptophan synthase, located in the PZ, correlate with ocular (inactived enzyme) and urogenital tract tropism (functional enzyme, which seems crucial for using indole for tryptophan biosynthesis) for C. trachomatis serovars causing trachoma vs. STD. Furthermore, the nature of differences in the PZ genes encoding chlamydial cytotoxins, which are almost entirely deleted in LGV isolates, correlate with epitheliotrophic vs. lymphotropic properties.

Fig. 3.9 Distribution of predicted and functional genes that are unique and shared between C. trachomatis strains of serovar L2 (434), D (UW-3), and A (Har-13). Pseudogenes were excluded in this analysis.
Nearly all C. trachomatis isolates possess cryptic plasmids of approximately 7.5 kb (4.4 kDa), containing eight open reading frames, that have co-evolved with their cognate chromosome, which suggests that the plasmid is not a highly mobile element ( Fig. 3.10 ). The plasmid is a common target for genetic diagnostic tests due to its ubiquitous, multicopy (usually 7–10 copies per cell) and highly conserved nature (<1.1% polymorphism in DNA sequence between different serovars). The function of the plasmid is not fully understood, however, recent studies have proposed that it plays a role in the replication and control of copy number as well as in virulence. Exceedingly rare plasmid-free isolates, which fail to accumulate glycogen in the inclusion, have been cultured from human clinical samples. However, any secondary person-to-person transmission has not been established, which suggests that these strains have a reduced biological fitness. It has not been possible to eliminate the plasmid from C. trachomatis using plasmid-curing agents in vitro.

Fig. 3.10 The multicopy cryptic plasmid of approximately 7.5 kb, containing eight CDSs, that is present in nearly all C. trachomatis isolates (outer circle). The cryptic plasmid of the Swedish new variant of C. trachomatis (nvCT), which caused thousands of false-negative results (due to the 377 bp deletion) using Roche and Abbott systems for diagnosis in Sweden, is illustrated in the inner circle. The direction of transcription for all the CDSs is shown.
Courtesy of Ian N. Clarke.

Epidemiological Characterization (Typing) of Chlamydia trachomatis
Serotyping using PAbs or MAbs against different antigenic epitopes on the MOMP was the mainstay of epidemiological typing for several decades. However, these methods are culture-dependent and following the rapid advances in molecular biology and development of diagnostic nucleic acid amplification tests (NAATs), many culture-independent genotyping methods such as PCR-RFLP and especially sequencing of the ompA gene, encoding MOMP, have replaced serotyping. A remarkably similar distribution of serovars (genovars) has been observed worldwide, with the serovars D, E and F being most prevalent. Among MSM, the D, G and J serovars are particularly prevalent. Several studies have been undertaken to correlate type and severity of clinical manifestation, and individual ‘oculogenital’ serovar. However, most evidence excluded any correlation, especially when analyzing ompA phylogeny. Importantly, in general disease pathogenesis mechanisms may also be determined by the host immune response and, accordingly, be affected by diversities of the host genome. Most identified genomic polymorphisms have been identified in immune defense loci such as the human leukocyte antigens, IFN-γ, IL-10, TNF-α, and matrix metalloproteinase 9. 13 The intragenovar diversity is also minor and, obviously, the low discriminatory ability of these methods make them inappropriate for most epidemiological considerations. Recently more highly discriminative typing methods have been developed, such as multiple loci variable number of tandem repeat (VNTR) analysis (MLVA; three loci plus ompA sequencing) and multilocus sequence typing (MLST), i.e. based on house-keeping genes (seven loci; reflecting evolutionary changes in the entire Chlamydiaceae family) or more variable genes (five loci plus ompA ). 14 Nevertheless, these methodological approaches to typing are still preliminary and given the very high level of conservation observed between C. trachomatis genomes they need to be further evaluated, in different populations and in large studies, for discriminatory value, reproducibility and genetic stability of targets over time (e.g. in a sexual transmission chain). In the future, highly discriminative typing of C. trachomatis may advantageously be based on high-technology methods such as nanotechnology, whole-genome DNA tiling microarrays, whole-genome sequencing, and whole- or part-genome SNP typing.
A highly discriminatory, objective, reproducible, high-throughput and culture-independent method for typing of C. trachomatis is the ultimate goal that will advance our knowledge with regard to chlamydial biology and pathogenesis, and a better understanding of the pathogen population will lead to improved control measures. For example, such an approach could be used to increase knowledge regarding strain populations in different communities, about temporal and geographic changes, elucidate transmission chains, core groups and patterns in sexual networks, and monitor evolutionary changes of specific clones during spread. The ultimate discriminatory test would be used for contract tracing, medico-legal cases, investigating suspected treatment failures, and enabling identification of association with tissue or organ affinity, pathogenicity, high-risk groups and sexual behavior, new or persistent infection and clinical manifestations.

Control programs for C. trachomatis infections have greatly enhanced the knowledge of distribution of infections throughout the world. In most developed countries worldwide, C. trachomatis infections are the most prevalent bacterial STD ( Fig. 3.11 ), e.g. in the USA 1,108,374 infections were reported to the Centers for Disease Control and Prevention (CDC) in 2007. These reported infections were three times greater than those for gonorrhea. Though C. trachomatis case rates were high among all races and ethnicities, rates in blacks were over eight times higher than in whites ( Fig. 3.12 ). Age-specific chlamydial rates were highest in women aged 15 to 24 ( Fig. 3.13 ). However, the reported chlamydial rates are an underestimate of the true incidence due to underreporting, lack of screening coverage and the majority of infections being asymptomatic. Using a calculation with assumptions of incidence being based on prevalence divided by duration of infection, it was estimated that approximately three million incident chlamydial infections occur each year in the USA. The incidences of chlamydial infections are estimated to be even higher outside North America ( Fig. 3.14 ). Furthermore, in many, especially low-resource, countries worldwide the data on morbidity due to genital chlamydial infection remain scarce owing to the use of suboptimal diagnostic methods, the low number of tests performed and incomplete reporting. Risk factors for acquiring urogenital chlamydial infections are displayed in Table 3.5 .

Fig. 3.11 Global estimates of new cases (millions) of non-viral STDs among adults between the ages of 15 and 49 years, 2005.
Source: WHO. Prevalence and Incidence in 2005 of Selected Sexually Transmitted Infections: Methods and Results. Geneva. World Health Organization 2010 (in print).

Fig. 3.12 Incidence rates of C. trachomatis in the United States by race/ethnicity, 1998–2007.
Courtesy of Centers for Disease Control and Prevention (CDC).

Fig. 3.13 Incidence rates of C. trachomatis in the United States by age and sex, 2007.
Courtesy of Centers for Disease Control and Prevention (CDC).

Fig. 3.14 Estimated new cases (millions) of urogenital C. trachomatis infections by WHO region among adults between the ages of 15 and 49 years, 2005.
Source: WHO. Prevalence and Incidence in 2005 of Selected Sexually Transmitted Infections: Methods and Results. Geneva. World Health Organization 2010 (in print).
Table 3.5 Risk factors for acquiring, and indications for testing for urogenital C. trachomatis infection.
Patients with other STDs
Patient with recent history of STDs
Patients with C. trachomatis -associated syndromes
Sexual partners of patients with gonorrhea or C. trachomatis -associated syndromes
Sexually active young people (<25 years of age)
Patients with multiple, especially new and/or casual, sexual partners
Patients engaged in commercial sex
Neonates born to infected mothers
The true epidemiologic profile of C. trachomatis remains elusive also due to an incomplete understanding of the natural history of the infection as well as the long-term effects of different types of Chlamydia control and screening program. In several countries, comprising broad Chlamydia control and screening programs, the estimated prevalence of C. trachomatis infection and especially of long-term sequelae due to chlamydial infection has declined. However, in some countries, chlamydial case reports and chlamydial sequelae rates declined in the first 10 years after the introduction of control programs but then case reports started to increase while the sequelae rates continued to decline 13 and, in other countries, even an initial decline of the case reports was not observed. Reinfection with C. trachomatis is common and may contribute to the increased trends in the number of case reports. The reasons for high rates of reinfection may include risk behavior and possibly the inability to develop a protective immune response following exposure. Further knowledge regarding the long-term effects and cost-effectiveness of different control programs and screening approaches, both proactive and the most commonly used opportunistic screening, on the prevalence of C. trachomatis infection and its severe sequelae would be beneficial.
LGV has worldwide distribution but is most prevalent in tropical and subtropical countries, and, for example, endemic in East and West Africa, India, and parts of South-East Asia, South America and the Caribbean. In most cases, these epidemiologic observations represent classic clinical presentation of LGV, which is highlighted by inguinal lymphadenopathy. These symptoms are more commonly reported in men than women. In 2003, a cluster of rectal LGV infections, with characteristic lymph node involvement and/or proctitis or proctocolitis, was reported in MSM in the Netherlands. Subsequent reports were made in many other countries in Europe, North America, and Australia, which previously only had sporadically imported LGV cases. The number of rectal C. trachomatis that belong to LGV or non-LGV biovars remains unknown due to a lack of systematic screening and use of a discriminatory test.

Clinical Manifestations 15 , 16

Urethritis and sequelae in men
Chlamydial infection of the urethra results in the development of an NGU. C. trachomatis is the causative agent in up to 50% of cases of NGU, while other possible causes include especially Mycoplasma genitalium but also e.g. Ureaplasma urealyticum among others. Clinically, C. trachomatis -positive and C. trachomatis -negative NGU cannot be differentiated on the basis of signs or symptoms. The incubation period of NGU (including chlamydial urethritis) is longer than that of gonorrhea, varying from 1 to 3 weeks in the majority of cases, and the onset of symptoms of NGU is more insidious. These symptoms include dysuria, urethral pruritis, appearance of a clear or white urethral discharge ( Fig. 3.15 ), and occasionally frequency of micturition. Although the majority of men have symptoms, many are asymptomatic or minimally symptomatic, which results in prolonged periods when they are infectious to their sexual partner. Nevertheless, most asymptomatic men exhibit inflammation, as defined by persistent urethral leukocytosis in urethral secretions and/or pyuria. Despite these commonly milder manifestations, it is often difficult to differentiate chlamydial from gonococcal infections on clinical grounds alone and, indeed, the two infections may coexist in up to 30% of cases of acute gonococcal urethritis. C. trachomatis is also a frequent cause of postgonococcal urethritis (PGU) which follows treatment (normally single dose) of mixed gonococcal-chlamydial infections with an antimicrobial that is active against N. gonorrhoeae but not against C. trachomatis . In industrialized societies PGU is usually symptomatic, but in the developing world, the majority of cases are apparently either asymptomatic or minimally symptomatic.

Fig. 3.15 Chlamydial non-gonococcal urethritis (NGU). Mucoid, scanty urethral discharge with meatal erythema.
Courtesy of Woodruff JD, Parmley TH, Atlas of Gynecologic Pathology. New York, Gower Medical Publishing, 1988.
C. trachomatis , along with N. gonorrhoeae , has emerged as a major cause of acute epididymitis ( Fig. 3.16 ), due to infection ascending from the urethra, in sexually active young men. Chlamydiae were initially isolated from epididymal aspirates obtained from patients in the USA who were all under 35 years of age and who presented with concomitant urethritis. This contrasted with the findings in older men who did not present with urethritis and from whom only Gram-negative bacilli could be isolated. Chlamydial epididymitis usually presents in young men as NGU with associated unilateral epididymal or testicular pain, swelling, tenderness and fever. Studies of the role of C. trachomatis in the etiology of acute epididymitis in mineworkers in South Africa have also indicated that chlamydial infection can be more important than gonococcal infection as a cause of this complication in developing societies. In contrast, a role for C. trachomatis in the etiology of chronic abacterial prostatitis remains speculative despite the proven link between N. gonorrhoeae and acute bacterial prostatitis.

Fig. 3.16 Red, swollen scrotum of a man with chlamydial epididymitis.
Courtesy of Richard E. Berger.

Cervicitis, urethritis and sequelae in women
The endocervix ( Fig. 3.17 ) is the most common site of infection with C. trachomatis in women. In the majority of cases the infection may be completely asymptomatic. However, those with symptoms, which are frequently non-specific, may complain of mucopurulent discharge, vaginal pruritis, dysuria, or lower abdominal pain. On examination, the cervix may appear normal or may be severely eroded with follicular hypertrophy and an associated mucopurulent endocervical discharge and/or easily induced cervical bleeding ( Figs 3.18 – 3.24 ). Likewise, chlamydial infection of the urethra, which may be the sole site of infection in some women, may either be asymptomatic or associated with urethral symptoms such as dysuria and frequency of micturition. C. trachomatis has been implicated in over 60% of cases of the so-called acute urethral syndrome in women. The infection is characterized by the presence of urethral symptoms together with a sterile pyuria.

Fig. 3.17 Normal nulliparous cervix of a postmenarchal female, showing no cervical ectopy.

Fig. 3.18 Beefy red mucosa of columnar epithelium in chlamydial infection.
Courtesy of Paul Weisner.

Fig. 3.19 Columnar epithelium cobblestoned by follicular changes of chlamydial infection.
Courtesy of Bingham JS, Pocket Picture Guide to Clinical Medicine. Sexually Transmitted Diseases. London, Gower Medical Publishing Ltd., 1984.

Fig. 3.20 Cervicitis showing purulent discharge from the os. Focal bleeding at areas touched during external cleansing of the cervix is evidence of friability.

Fig. 3.21 Mucopurulent discharge seen coming from the cervical os, following removal of ectocervical mucus, in a C. trachomatis- infected woman.
Courtesy of Lourdes Frau.

Fig. 3.22 Endocervical swab sampled from a C. trachomatis -infected woman (left) compared with fresh swab (right). The yellow-green exudate may reflect infection of the endocervix or endometrium.
Courtesy of George Schmid.

Fig. 3.23 Histologic image of C. trachomatis -infected human cervix on biopsy, showing an intense follicular inflammatory infiltrate.
Courtesy of Robert Brunham, (Kunimoto et al.), Rev Infect Dis 1985; 7:665–673.

Fig. 3.24 Transmission electron micrograph, showing both a chlamydial inclusion and microabscess.
Courtesy of John Swanson, J Infect Dis 1975; 3:678–687.
Ascending spread of C. trachomatis from the endocervical canal to the endometrium, fallopian tubes, and peritoneal cavity is the most serious complication of both asymptomatic and symptomatic chlamydial infection in women (see Chapter 6 ). The resulting infection of the upper reproductive tract may be severe and characterized by lower abdominal pain, adnexal tenderness, and perhaps fever. The organism is capable of inducing a plasma cell endometritis, acute and chronic salpingitis ( Figs 3.25 – 3.27 ), pelvic inflammatory disease (PID), and full-blown peritonitis with associated perihepatitis (Fitz-Hugh–Curtis syndrome) and periappendicitis. Infected pregnant women are at risk of developing late postpartum endometritis. Resolution of these upper genital tract infections may result in chronic pelvic pain, tubal damage and infertility, or ectopic pregnancy.

Fig. 3.25 Acute salpingitis. The fallopian tube is congested and swollen. A dense adhesion has formed between the ampulla of the tube and the pelvic sidewall.

Fig. 3.26 Acute salpingitis. Hydrosalpinx with adhesions. Dye has been instilled into the grossly swollen fallopian tube on the right. Dense adhesions obscure the ovary.

Fig. 3.27 Acute salpingitis. Laparoscopic view of ‘violin-string’ adhesions in a patient with perihepatitis (Fitz–Hugh–Curtis syndrome).
Courtesy of Richard Sweet.

Lymphogranuloma venereum
Classically, LGV presents as a transient, herpetiform primary lesion of the external genitalia ( Fig. 3.28 ), but in many cases the lesion may pass unnoticed or manifest as an acute NGU in men or be completely asymptomatic in women as a result of primary infection of the cervix. Most cases seek medical attention when the regional lymphatics become infected, due to systemic spread of the bacteria. In men, swelling of the inguinal and femoral glands often results in the formation of suppurating buboes on either or both sides of the inguinal ligament (the ‘groove-sign’, which may be seen also in a minority of chancroid patients) ( Fig. 3.29 ). In women, the perirectal and deep pelvic glands may become involved if the primary lesion is found on the cervix and the patient may present with symptoms consistent with severe PID.

Fig. 3.28 Transient herpetiform primary lesion on external genitalia caused by lymphogranuloma venereum (LGV).
Courtesy of Ronald C. Ballard.

Fig. 3.29 ‘Groove sign’ in a man with lymphogranuloma venereum (LGV). Although often said to be pathognomonic for LGV, this sign is seen infrequently in LGV patients and may be produced by other conditions.
Courtesy of Ronald C. Ballard.
MSM may present with a severe ulcerative proctitis or proctocolitis with rectal pain, blood-stained discharge, markedly abnormal anoscopy, fever, and lymphoadenopathy. In common with women, who may also present with such lesions, failure to treat the disease at this stage may result in the formation of perirectal abscesses, rectal strictures, fistulas, and chronic scarring. Apart from these complications, which arise as a result of acute inflammatory changes, chronic manifestations of the disease may result in blockage of the lymphatics draining of the genitalia or rectum, causing edema. When this lymphatic edema is severe it is termed ‘elephantiasis’.

Non-LGV rectal chlamydial infections
The majority of non-LGV rectal C. trachomatis infections are asymptomatic but some may cause acute proctitis, and hyperplasia ( Figs 3.30 and 3.31 ). The symptoms of the proctitis may include rectal pain, discharge, bleeding, and abnormal anoscopy. However, in most cases, the symptoms are non-specific and may be confused with Crohn’s disease or neoplasia, thus requiring diagnostic testing for C. trachomatis infection. One recent report described the development of a lymphoid polyp with lymphoid follicular proctitis. 17 Florid follicular lymphoid hyperplasia ( Fig. 3.32 ) or germinal centers from rectal biopsies are also seen in follicular cervicitis and can occur in response to C. trachomatis infection or Crohn’s disease. Mucosal ulceration ( Fig. 3.33 ), acute cryptitis ( Fig. 3.34 ), and crypt depletion ( Fig. 3.35 ), which are idiopathic for inflammatory bowel disease, were observed following a rectal C. trachomatis infection. However, the absence of mucus in this case was more suggestive of infection highlighting the need for specific laboratory diagnostics of rectal specimens.

Fig. 3.30 Anoscopic view of rectal mucosa with area of focal purulence in a man with chlamydial proctitis.
Courtesy of Walter E. Stamm.

Fig. 3.31 Lymphoid follicular proctitis seen in a case of C. trachomatis rectal infection.
Courtesy of Bingham J.S., Pocket Picture Guide to Clinical Medicine. Sexually Transmitted Diseases. London, Gower Medical Publishing Ltd., 1984.

Fig. 3.32 Florid follicular hyperplasia seen on a rectal biopsy from a woman with a non-LGV rectal C. trachomatis infection.
Courtesy of Stewart Cramer.

Fig. 3.33 Mucosal ulceration seen on a rectal biopsy from a woman with a non-LGV rectal C. trachomatis infection.
Courtesy of Stewart Cramer.

Fig. 3.34 Acute cryptitis seen on a rectal biopsy from a woman with a non-LGV rectal C. trachomatis infection.
Courtesy of Stewart Cramer.

Fig. 3.35 Crypt cell depletion seen on a rectal biopsy from a woman with a non-LGV rectal C. trachomatis infection.
Courtesy of Stewart Cramer.

Other chlamydial infections in adults
Endemic trachoma, caused by C. trachomatis serovars (genovars) A to C, is the leading cause of preventive blindness ( Fig. 3.36 ) in the world. Blindness follows primary untreated infection that progresses to the development of destructive lesions and follicular conjunctivitis, which can also be seen after infection with oculogenital isolates ( Fig. 3.37 ). Ocular infections with sexually transmitted isolates in both men and women may occur as a result of accidental inoculation causing adult inclusion conjunctivitis. C. trachomatis may be associated with 50% of cases of sexually acquired reactive arthritis ( Fig. 3.38 ) by acting as a trigger to precipitate the disease in especially genetically predisposed (HLA-B27–haplotype) persons. In a few cases the urethritis and arthritis may be accompanied by both the conjunctivitis and the mucocutaneous lesions classically associated with Reiter’s syndrome ( Figs 3.39 and 3.40 ). Pharyngeal infection with C. trachomatis , which usually is asymptomatic, may also be rarely identified in both men and women.

Fig. 3.36 Inflammatory infiltration and neovascularization of the cornea (pannus). Similar destructive lesions are infrequently observed in ocular infection by urogenital strains of C. trachomatis (paratrachoma).
Courtesy of Spalton D.J., Hitchings R.A., Hunter P.A., Atlas of Clinical Ophthalmology. London, Gower Medical Publishing Ltd, 1984.

Fig. 3.37 Follicular conjunctivitis. Infection of the palpebral conjunctiva with lymphocytic follicle formation by C. trachomatis .
Courtesy of George Waring.

Fig. 3.38 A red and swollen third toe of a man presenting with Reiter’s syndrome arthritis.
Courtesy of Robert Wilkens.

Fig. 3.39 Scaling erythematous plaques on the penis. Circinate balanitis of Reiter’s syndrome. This is one of the infrequent but distinctive cutaneous findings associated with this syndrome.
Courtesy of Robert Wilkens.

Fig. 3.40 Keratoderma blenorrhagica in Reiter’s syndrome. Note the thick scales and crusts on the feet of this patient.
Courtesy of Dorothy Patton.

Perinatal infections
In neonates, chlamydial infection results from perinatal exposure to the infected cervix of the mother, i.e. mainly during passage through the birth canal (see Chapter 16 ). The most frequent symptomatic infection, developed in about 20–30% of these children, is a self-limited purulent conjunctivitis ( Fig. 3.41 ) that usually develops 5–12 days following birth. Within 1 to 3 months, some of the exposed children (approximately 10–20%) may also develop a subacute, afebrile pneumonia characterized by an insidious onset and a repetitive non-productive staccato cough with tachypnea. A chest radiograph frequently shows hyperinflation and generalized bilateral diffuse infiltrates ( Fig. 3.42 ).

Fig. 3.41 Chlamydial ophthalmia neonatorum. Erythematous conjunctiva is seen in this infant.

Fig. 3.42 Chlamydial infant pneumonia. Chest X-ray of a neonate showing the generalized diffuse infiltrates.
Courtesy of E.R. Alexander.

Laboratory Tests
Endemic trachoma can usually be diagnosed by an experienced physician on the basis of patient anamnesis and clinical examination. However, urogenital chlamydial infection is asymptomatic in the majority of women, and also in many men. Furthermore, in the symptomatic cases the symptoms and signs of infection diverge, and may be caused by other pathogens or may even be of non-infectious origin. Consequently, for diagnosis of urogenital C. trachomatis infection sensitive and specific laboratory diagnostics are crucial.
Until the early 1980s, isolation of C. trachomatis in tissue or cell culture was the main method for diagnosis of chlamydial infection. Thereafter, increased knowledge regarding the molecular structure of the OM of EBs and RBs, and the development of MAbs against different antigens of the organism resulted in the introduction of divergent non-culture antigen detection tests for diagnosis. This was followed by development of nucleic acid hybridization tests in the late 1980s. In the mid 1990s, commercially available NAATs were introduced. The NAATs are substantially more sensitive than the older diagnostic tests and have made it possible to expand screening to asymptomatic individuals, due to use of non-invasive specimens such as first catch urine and vaginal swabs. Where available and affordable, use of NAATs for diagnosis of C. trachomatis infections are strongly recommended. Appropriate specimen collection, transport and storage are crucial for a high sensitivity and specificity of all diagnostic methods. Furthermore, for all diagnostic methods strict concordance with the instructions of the manufacturer of commercial assays and comprehensive quality assurance and control are essential ( Table 3.6 ). 10, 18, 19 For further information regarding performance characteristics of diagnostic tests for STDs, see also Appendix 1 .

Table 3.6 Diagnostic tests for the detection of Chlamydia trachomatis .

Gram staining
The diagnosis of NGU in men is commonly based on presentation of scanty urethral discharge ( Fig. 3.15 ) and microscopic examination of Gram-stained urethral exudate. In symptomatic men, the Gram stain is sensitive and specific for the diagnosis of gonorrhea ( Fig. 3.43 ). Only the absence of intracellular Gram-negative diplococci in the presence of an inflammatory exudate ( Fig. 3.44 ) is useful in diagnosing NGU.

Fig. 3.43 Gonorrhea. A Gram stain of penile urethral discharge displays typical intracellular diplococci within polymorphonuclear leukocytes (inflammatory cells).

Fig. 3.44 Non-gonococcal urethritis (NGU). A Gram stain of penile urethral discharge displays inflammatory cells without visible diplococci.
Courtesy of Francisco J. Candal.

Papanicolaou smear
Laboratory diagnosis of C. trachomatis infection based on detection of intact chlamydial inclusions in Papanicolaou-stained cervical smears (Pap smear) has a low sensitivity, and cytologic changes accompanying C. trachomatis cervical infection are non-specific. Consequently, the Pap smear should not be used for etiological diagnosis, but findings of inflammatory cells may necessitate the application of specific STD laboratory diagnostics.

Cell culture
The first cell culture method for isolation of C. trachomatis was described in 1965. Until the early 1980s, the main and gold standard method for diagnosis of C. trachomatis infection was the centrifuge-assisted inoculation of clinical specimens onto susceptible viable cells in tissue culture followed by the demonstration of characteristic chlamydial inclusions after incubation.
An example of C. trachomatis isolation by cell culture is illustrated in Fig. 3.45 . Briefly, the specimen is collected using non-toxic swabs or cytobrushes and placed into a transport medium, e.g. sucrose phosphate (2SP) buffer containing fetal bovine serum and antimicrobials such as vancomycin, gentamicin, and nystatin, to inhibit other bacteria and fungi. In the laboratory, the specimen is inoculated onto confluent monolayers of DEAE-dextran treated HeLa 229 or cycloheximide-treated McCoy or BGMK (buffalo green monkey kidney) cell lines. The inoculated cells are centrifuged to enhance the attachment and ingestion of bacteria. DEAE-dextran increases the uptake of chlamydiae by the HeLa 229 cells while cycloheximide inhibits host cell protein synthesis and metabolism, resulting in an increased number and size of chlamydial inclusions. After incubation, characteristic inclusions can be observed microscopically. However, the inclusions are more easily visualized by staining with Giemsa, iodine, acridine orange, immunochemical stains using enzyme-conjugated MAbs, or most commonly, fluorescein-conjugated MAbs or PAbs towards chlamydiae-specific LPS or species-specific MOMP. Characteristic intracellular inclusions using different stains are illustrated in Figs 3.46 – 3.51 . Notably, inclusions of plasmid-free C. trachomatis strains do not stain with iodine ( Fig. 3.46B ). Clinical specimens can display cytotoxicity of the cell monolayers ( Fig. 3.52 ), due to collection or transport medium components, semen components, inflammatory cells or other micro-organisms.

Fig. 3.45 Isolation of C. trachomatis using cell culture method.

Fig. 3.46 BGMK cell monolayer infected with C. trachomatis L2. (A) The inclusions are dark-red after iodine staining. (B) The inclusions of a plasmid-free C. trachomatis serovar L2 are not stained.
Courtesy of Ian N. Clarke.

Fig. 3.47 Giemsa-stained inclusions (bright-field) displaying the open, vacuolar and granular nature of a C. trachomatis inclusion.
Courtesy of Billie R. Bird.

Fig. 3.48 Giemsa-stained inclusions (dark-field) displaying the open, vacuolar and granular nature of a C. trachomatis inclusion.
Courtesy of Billie R. Bird.

Fig. 3.49 C. trachomatis , serovar D, inclusions in cycloheximide-treated McCoy cell monolayers stained with a fluorescein-labeled species-specific monoclonal antibody.
Courtesy of Ian N. Clarke.

Fig. 3.50 Distinct red chlamydial inclusions detected using an alkaline-phosphatase monoclonal antibody method with naphthol-AS chromogenic substrate (original magnification × 250).
Courtesy of James Mahoney.

Fig. 3.51 Black, granular C. trachomatis inclusions produced using peroxidase-labeled monoclonal antibody and 1-chloro-4-naphthol chromogenic substrate (original magnification × 400).
Courtesy of Robert Suchland.

Fig. 3.52 Cytotoxicity in cell culture. Cells exhibit a round morphology and have sloughed from the cover slip, resulting in a diffuse cytopathic effect.
Compared to NAATs, culture of C. trachomatis typically only has a sensitivity of 50% to 80%. The sensitivity can be higher in experienced laboratories using comprehensively optimized and quality assured procedures with regard to collection, transportation, storage, and culture of specimens. Culture has been considered to be 100% specific. However, cultures can be cross-contaminated, all stains are not equally sensitive and specific, and inexperienced microscopists may misidentify artifacts as chlamydial inclusions. Furthermore, culture requires a rapid cold chain transport system to preserve the viability of C. trachomatis , it is technically complex, laborious and time-consuming and lacks internationally standardized and quality assured methods.
Culture for diagnosis of C. trachomatis is nowadays rarely performed in middle- or high-resource countries. However, maintaining the ability to perform chlamydial cell culture in reference laboratories is important, e.g. for investigation of medico-legal cases (ideally combined with NAATs for increased sensitivity), for extragenital samples such as rectal, pharyngeal, conjunctival, tracheal or lung specimens (suspected chlamydial pneumonia), for test-of-cure that is requested less than 2–3 weeks following treatment, in emergent situations, e.g. mutants that are undetected using NAATs such as the Swedish new variant of C. trachomatis (nvCT) 20 ( Fig. 3.10 ), or when viable bacteria are necessary for research purposes, such as antimicrobial resistance testing or sophisticated genetic analysis.

Antigen detection methods
In antigen detection methods, C. trachomatis antigen in clinical specimens is detected either directly by microscopy using fluorescent antibody (DFA) or, indirectly, by an enzyme immunoassay (EIA) or an optical immunoassay (OIA). As with nucleic acid detection methods (see below), chlamydial viability is not an issue, so specimen transport and storage conditions are less stringent than those required for chlamydial culture. Nevertheless, proper specimen collection remains crucial.
The sensitivity of antigen detection tests is substantially lower than that of NAATs. However, in settings where NAATs are not accessible and/or not affordable, the new generation EIAs (using signal amplification), including confirmatory testing of all positive samples, may be the best alternative for C. trachomatis diagnostics. This could be in resource-poor settings and for screening of high-risk or high-prevalence populations.

Direct fluorescent antibody detection
DFA tests have mostly used MAbs conjugated with fluorescent molecules for direct detection of C. trachomatis in cellular smears. MAbs against MOMP (species-specific) or LPS (may cross-react with other chlamydiae and also non-chlamydial bacterial species) are used. The MOMP MAbs display superior staining, more characteristic morphology of EBs, and less background fluorescence. Commonly used, currently available DFA tests such as MicroTrak C. trachomatis direct specimen test and Pathfinder Chlamydia DFA employ MOMP MAbs, conjugated with fluorescein isothiocyanate (FITC). Appropriate collection, application (by rolling of the swab) and fixation of the clinical specimen to the slide are necessary to ensure presence of columnar cells and non-distorted cell morphology. After application of the conjugated MAbs, these bind to chlamydial EBs and eventual RBs. A rinse step removes unbound antibody. Under UV fluorescence microscopic examination, C. trachomatis positive smears display apple-green fluorescence and morphologically characteristic EBs (examples of DFAs are illustrated in Figs 3.53 and 3.54 ). At least 10 fields of the slide, under magnification ×400, should be examined. If the preparation contains enough columnar epithelial cells and ideally ≥10 typical EBs are found, the result is considered positive.

Fig. 3.53 Direct fluorescent antibody (DFA) detection of C. trachomatis .

Fig. 3.54 Direct fluorescent antibody (DFA) stain of a smear of cervical exudate displaying apple-green fluorescing and morphological characteristic chlamydial elementary bodies (×630).
Courtesy of Howard Soule.
The sensitivity of DFA tests when compared to cell culture varies between studies, but averages ≥80% in women and symptomatic men. The test specificity can be high, i.e. ≥97%, using MOMP MAbs in experienced laboratories.
DFA tests do not require cold chain transportation of specimens, and the slides can be applied, fixed and mailed to the laboratory for staining and examination. Moreover, the DFA test can be used to assess the adequacy of the clinical samples ( Figs 3.55 – 3.57 ), i.e. slides displaying insufficient columnar epithelial cells or disrupted cells should be discarded, the size and morphology of the fluorescing organisms can be determined visually, and the test is rapid. Disadvantages of DFA tests include subjectiveness (experienced microscopists are required), and the labor-intensive and time-consuming evaluation of each slide, which makes the method unsuitable for high-throughput diagnostics and screening of specimens. Artifacts may be noted due to cross-reactions with other micro-organisms ( Figs 3.58 and 3.59 ).

Fig. 3.55 Normal endocervical epithelial columnar cells stained with a commercial chlamydial direct fluorescent antibody (DFA) reagent. The presence of such cells indicates adequate quality of the sample (×630).
Courtesy of Janice C. Bullard.

Fig. 3.56 An abundance of squamous cells in a urogenital specimen from a woman stained with a commercial chlamydial direct fluorescent antibody (DFA) reagent. This abundance of squamous cells and lack of sufficient number of columnar epithelial cells indicate that the specimen collection has been unsatisfactory (×630).
Courtesy of Janice C. Bullard.

Fig. 3.57 Inflammatory cells stained with a commercial chlamydial direct fluorescent antibody (DFA) reagent (×630).

Fig. 3.58 An example of cross-reacting micro-organisms rarely seen in a clinical specimen stained with a chlamydial MOMP-specific monoclonal antibody. These artifacts are seldom confusing to an experienced microscopist.
Courtesy of Janice C. Bullard.

Fig. 3.59 Another example of cross-reacting micro-organisms in a clinical specimen stained with a chlamydial MOMP-specific monoclonal antibody.
Courtesy of Linda Cles.

Enzyme immunoassays
The first chlamydial EIA, i.e. Chlamydiazyme, was introduced in 1984. There are currently several commercially available EIAs for detection of C. trachomatis in clinical specimens, such as the MicroTrak II Chlamydia EIA, Pathfinder Chlamydia Microplate (EIA microplate), IDEIA Chlamydia, and IDEIA PCE Chlamydia. The EIA methods mostly capture chlamydial LPS with MAbs or PAbs in a microtiter plate. Chlamydia-specific or secondary antibodies (against the detector antibody), conjugated with an enzyme such as horseradish peroxidase (HRP), are added following washing. These convert the color of a substrate, such as TMB and hydrogen peroxide to a colored product and the results can be detected visually or in a spectrophotometer (some EIAs are illustrated in Figs 3.60 and 3.61 ).

Fig. 3.60 Detection of C. trachomatis using enzyme immunoassay (EIA), by direct antigen capture technique.

Fig. 3.61 Detection of C. trachomatis using enzyme immunoassay (EIA), by monoclonal antibody (MAb) sandwich antigen capture technique.
The advantages of EIAs include use with invasive as well as non-invasive specimens such as urine, specimens can be stored and transported without a cold chain, opportunities for automation permitting high-throughput diagnostics, rapidness, ease of performance and objective interpretation of results. However, the older EIAs are clearly less sensitive than culture (65–85%). EIAs are also commonly less specific than the DFA tests and the antibodies can cross-react with LPS from other chlamydiae, gastrointestinal bacteria and other urogenital pathogens. However, the specificity has been substantially improved by the introduction of confirmatory testing using blocking antibody. It is crucial to verify all EIA positives by repeat testing using a blocking reaction with an unlabeled competing additional antibody, or by another method targeting a different antigen or molecule (such as DFA, NAATs).
The new generation IDEIA, named IDEIA PCE Chlamydia, has a significantly enhanced sensitivity. This method uses a polymer conjugate containing multiple copies of antibody and enzyme molecules that result in an amplification of the colorimetric output. This assay has not been comprehensively evaluated but the sensitivity can be comparable to cell culture and the specificity, after confirmatory testing, is high.

Rapid or ‘point-of-care’ tests
Adequate management of C. trachomatis in many settings worldwide is constrained by a lack of accessible, affordable, and easily performed diagnostic tests.
Several rapid qualitative point-of-care (POC) tests for C. trachomatis , commonly based on lateral flow and antigen membrane capture on immunochromatographic strips (ICS), have been developed (an example is illustrated in Fig. 3.62 ). These tests include the most commonly used Clearview Chlamydia, QuickVue Chlamydia, and Biostar OIA Chlamydia. Thus, these rapid and easily performed POC tests can be performed at site, e.g. at the clinic or in the field, they do not require expensive equipment or skilled personnel, and within 30 minutes the patients can receive results, and treatment if needed, before leaving the clinic. The modern POC tests have a relatively high specificity, however, they suffer from a low sensitivity.

Fig. 3.62 Detection of C. trachomatis using a rapid, qualitative point-of-care (POC) test. As illustrated, these are commonly based on lateral flow and antigen membrane capture on immunochromatic strips (ICS).
A recently developed POC test, Chlamydia Rapid Test (CRT), has a substantially higher sensitivity. However, when compared to NAATs this test displays clearly insufficient sensitivity, and should only be used when adequate laboratory facilities are lacking. Further improvements and evaluations of this test are crucial. Methods using high technology such as DNA hybridization on nanoparticles may be next generation quantitative POC tests.
The main utility of the current POC tests is in field and resource-poor settings where return for test results, and treatment if needed, is unlikely. In these settings, the trade-off between testing increased numbers of patients coupled with rapid results, immediate treatment, and even contact tracing, and use of a test with moderate sensitivity may be acceptable. The POC tests could in these settings also be used to increase the specificity of the syndromic management algorithms, which will reduce overtreatment, and to screen for asymptomatic infections. Nevertheless, the POC tests need to be cheaper, and further improved and evaluated, using appropriate reference standards (at clinic and laboratory) and choice of study population, trial site, study design, specimens, and strict quality assurance.
A C. trachomatis home use diagnostic test is also currently accessible via the internet under various brand names, including HandiLab-C, SureScreen and SELFCheck. This test is based on the detection of a C. trachomatis -specific enzyme, i.e. peptidase 123A, and it is not recommended owing to both low sensitivity and poor specificity.

In an acute, primary chlamydial infection, specific IgM, but also IgG and IgA may be detected. However, antibodies are not detectable in all cases of uncomplicated urogenital infection, and antibodies to C. trachomatis can persist for years. Accordingly, serological testing should not be used for routine diagnosis of uncomplicated C. trachomatis urogenital infections.

Complement fixation
In the 1940s, the first widely used serological test, i.e. complement fixation (CF) test, was developed. The CF test detects antibodies against chlamydiae-specific antigen (LPS), which is problematic especially due to the exceedingly high Chlamydophila pneumoniae seroprevalence in the adult population (often >50%). The CF test is also insensitive in urogenital and ocular C. trachomatis infections.

The more sensitive and specific gold standard microimmunofluorescence (MIF) test developed in the 1970s allowed clinical seroepidemiologic studies of chlamydial infection. The MIF test uses fixed, purified chlamydial antigens, which are applied onto a glass slide. Patient serum is then added, followed by an FITC-conjugate and evaluation under UV fluorescence microscopy ( Fig. 3.63 ). The MIF test, initially developed for serotyping of chlamydial isolates, detects antibodies against the MOMP and can differentiate species- and serovar-specific antibodies. Moreover, IgM, IgG, and IgA antibodies can be measured separately and, ideally using sequential samples, MIF may distinguish recent infections from past infections. However, the test lacks a universally agreed and quality assured standard. It is also laborious, time consuming and technically demanding, and its interpretation requires extensive experience.

Fig. 3.63 Detection of antibodies to C. trachomatis using a microimmunofluoresence assay.
Courtesy of Janice C. Bullard.
Today, there are also several commercially available, standardized methods such as ELISAs, based on e.g. recombinant or peptide-based specific MOMP antigens, or LPS. Assays to determine antibodies to the cryptic plasmid protein pgp3, which is detected also in the cytosol of host cells, that has been suggested as a marker for chlamydial infection and cHsp60 (GroEL), associated with severe sequelae of chlamydial infection, have also been used. Additional potentially useful antigens for serology have also been identified based on the available whole-genome sequences. ELISAs are preferable to MIF due to their objective measurement, ease of performance, rapidness, and high-throughput. Nevertheless, the sensitivity, which commonly has been suboptimal, and specificity of all novel assays need to be critically evaluated in large and well-designed studies.
As mentioned previously, measurement of chlamydial antibody has limited value for diagnosis of C. trachomatis infection and should not be used for routine diagnosis of uncomplicated C. trachomatis urogenital tract infections. This is due to the high background prevalence of chlamydial infection, because high levels of chlamydial antibodies can persist long after infection has been cleared and, in contrast, after uncomplicated mucosal infection, systemic antibody response may be delayed or not measurable. Furthermore, testing of a single serum specimen can usually not provide conclusive evidence of current infection. However, serology can aid in the diagnosis and/or screening for complicated C. trachomatis infections (reactive arthritis, PID, ectopic pregnancy, tubal factor infertility), neonatal pneumonia, and LGV infections, as well as in research and epidemiological studies, e.g. for the cumulative history of exposure of a sample population to chlamydial infection. Nevertheless, the serological results should always be interpreted with caution and not out of context.

Nucleic acid probe hybridization and amplification tests
Molecular detection of specific nucleic acid sequences and subsequent commercialization of such assays have vastly improved laboratory detection of C. trachomatis . Non-amplified technologies that rely on the binding of specific complementary nucleic acid probes and subsequent signal amplification to detect binding are about 10% to 15% less sensitive than amplified technologies. NAATs are considered to have superior performance characteristics than any other test types and can be used with a variety of specimen types that are collected during either invasive or non-invasive procedures. The performance characteristics, range of specimen types, automation and independence from maintaining organism viability during transit are factors that promote NAATs as the strongly recommended technology to diagnose and screen populations for C. trachomatis infections ( Table 3.6 ). Specific DNA or RNA target sequences are amplified and detected, most commonly, using a complementary probe attached to some method of signal amplification. For basic information regarding nucleic acid probe hybridization and different NAAT technologies, see Appendix 1 . The genetic targets are chosen on the basis of being conserved in high copy numbers among all C. trachomatis isolates. However, the organism may undergo genetic mutation rendering some targets inappropriate for detection. This occurred in Sweden when a deletion in a specific region of the cryptic plasmid resulted in many thousands of false-negative results when specimens were tested using two commercial NAAT systems ( Fig. 3.10 ). There are currently four commercially available FDA approved NAATs for the detection of C. trachomatis in the USA, however, in Europe many additional commercially available C. trachomatis NAATs are in use ( Table 3.7 ). The use of all these NAATs should be limited to laboratories that can perform molecular amplification procedures according to good laboratory practice ( Table 3.8 ). The need to meet certain physical and personnel requirements restricts widespread deployment of NAATs and they may not be a suitable testing platform in some resource-challenged settings where C. trachomatis is highly prevalent.

Table 3.7 FDA cleared commercially available nucleic acid amplification tests for the detection of Chlamydia trachomatis .
Table 3.8 General requirements a for laboratories performing high-complexity tests such as nucleic acid amplification tests in the USA. Laboratory staff
Laboratory director must have a doctoral degree and appropriate state licensure
Technical staff must at least have an undergraduate science degree and have passed annual competency assessments to perform tests Laboratory protocols
Documented test procedures including a course of action for aberrant test results
Monitor for contamination Quality assurance
Subscribe to an external proficiency test program
Monitor prevalence and develop a plan to react if significant changes are noted Laboratory design/layout
Separate areas for reagent and specimen preparation, amplification and detection
Amplification not in a closed system must maintain a uni-directional workflow
a Detailed requirements are listed in the ‘Code of Federal Regulations, Title 42, Volume 3, Part 493’.

Minimally or non-invasive specimen types
The greatest advance in screening for C. trachomatis infections has been the development and marketing of highly sophisticated NAATs that can use minimally or non-invasive specimens such as urine or vaginal swabs. In women, the sensitivity of detecting C. trachomatis from vaginal swabs has been reported to be similar to the sensitivity of the detection from endocervical swabs and slightly better than first-catch urine specimens. The ability of women to self-collect a vaginal swab enhances outreach services for screening through non-traditional venues such as schools and homes. The stability of nucleic acids on vaginal swabs transported by regular mail delivery at temperatures up to 50°C facilitates program expansion to home-based specimen collection. First-catch urine specimens are considered the specimen of choice for men. There are studies indicating that the use of self-collected meatal swabs from men may be a viable alternative to first-catch urine specimens but further research is required to determine the full utility of these specimen types.

Antimicrobial Susceptibility and Treatment
C. trachomatis is susceptible to many antimicrobials ( Table 3.9 ). Macrolides (mainly azithromycin and erythromycin), tetracycline, i.e. especially its derivate doxycycline, the fluoroquinolones ofloxacin and levofloxacin, and the penicillin amoxycillin (pregnant women) are the most commonly recommended antimicrobials for treatment of C. trachomatis infection ( Table 3.10 ). No evidence of emergence of acquired resistance to recommended antimicrobials has yet been identified in clinical C. trachomatis isolates, although some case reports have suggested resistance as a cause of treatment failures. In vitro antimicrobial susceptibility testing of C. trachomatis is not routinely performed, due to the lack of i) a universally accepted, standardized, reproducible and quality assured method, ii) known correlates between in vitro activity and in vivo efficacy (clinical treatment outcome), and iii) any in vitro stable homotypic resistance (phenotypically or genetically) and/or clinical resistance in clinical isolates to recommended treatment regimens.
Table 3.9 Minimum inhibitory concentrations (MICs) of antimicrobials in C. trachomatis isolates. Antimicrobial MIC (µg/mL) Erythromycin 0.02–1.0 Azithromycin 0.02–1.0 Doxycycline 0.02–0.5 Tetracycline 0.03–1.0 Ofloxacin 0.25–4.0 Levofloxacin 0.25–1.0 Garenoxacin 0.01–0.03 Moxifloxacin 0.02–0.12 Ciprofloxacin 1.0–2.0 Ampicillin 0.5–10 Penicillin 1.0–10 Rifampin 0.005–0.25 Sulfamethoxazole 0.50–4.0 Clarithromycin 0.01–0.02 Roxithromycin 0.03–0.25 Clindamycin 2.0–16 Spectinomycin 32–100 Gentamicin 500 Vancomycin 1000
Variabilities in exact MICs are common, i.e. due to variation of the few isolates examined, cell lines and methods (including concentration of inoculum, time between infection and addition of antibiotic, passage strategy, and subjectiveness of interpretation of results) used. This limits the in vitro and in vivo usefulness of many of these data.
For further information regarding MICs of antimicrobials in C. trachomatis and discussions regarding antimicrobial resistance and treatment failures, see References 2 and 3; Bowie WR, et al: Antimicrob Agents Chemother 1987; 31:470–472; Walsh M, et al: Antimicrob Agents Chemother 1987; 31:811–812; Moroni A, et al: J Chemother 1991; 3(Suppl 1):28–29; Donati M, et al: J Antimicrob Chemother 1998; 48:670–671; Samra Z, et al: Diagn Microbiol Infect Dis 2001; 39:177–179; Donati M, et al: J Antimicrob Chemother 2002; 50:407–419; Wang SA, et al: J Infect Dis 2005; 191:917–923; Horner P: Sex Transm Infect 2006; 82:340–343; Bebear C, de Barbeyrac B: Clin Microbiol Infect 2009; 15:4–10; Bebear CM, et al: Clin Microbiol Infect 2008; 14:801–805.

Table 3.10 Recommended treatment regimens for C. trachomatis infections and C. trachomatis -associated conditions.

Nevertheless, mutants resistant to fluoroquinolones ( gyrA mutations) and rifampin ( rpoB mutations) have been easily selected under growth in subinhibitory concentrations of the antimicrobials in vitro. A few clinical isolates (n = 4) have also demonstrated in vitro resistance to macrolides, including specific 23S rRNA resistance mutations, but all these isolates displayed mixed populations, i.e. containing organisms both exhibiting and lacking the resistance phenotype and mutation. Furthermore, heterotypic resistance can be observed in vitro when cells are inoculated with a large number of organisms. This resistance affects a small proportion of the inoculum and this is manifested by persistent aberrant inclusions that may be seen on subsequent passages. These isolates have poor survival characteristics in vitro and ultimately they cannot be cultivated further, suggesting a lower biological fitness. Importantly, if a large enough inoculum is applied, heterotypic resistance can be observed with many isolates and antimicrobials. Accordingly, this heterotypic resistance does not represent any clinically relevant antimicrobial resistance, instead it is mainly associated with the assay and excessive size of inoculum, and is possibly reflecting persistence. Nevertheless, numerous strains of C. suis (the closely related pathogen of pigs) express a stable homotypic tetracycline resistance phenotype, associated with the resistance gene tet(C) and the widespread prophylactic use of tetracycline in pig husbandry . Emergence and spread of clinically relevant antimicrobial resistance in C. trachomatis in the future should consequently not be excluded. This highlights the need for an effective, standardized, objective and quality assured method for antimicrobial susceptibility testing as well as for appropriate correlates between in vitro activity and treatment outcome. This could also elucidate the reason for presently suggested treatment failures, which may include reinfection, non-compliance with treatment, poor absorption of drug, or, as also suggested, reactivation of persistent latent infection caused by heterotypic resistance (associated with high chlamydial loads) or caused by other stimuli such as host factors.
Recommended treatment regimens for C. trachomatis infections are presented in Table 3.10 . The patient and ideally his/her recent sexual contacts (at least after specific testing) should be treated, and presumptive treatment of concomitant gonorrhea might be considered. With the exception of pregnant women, test-of-cure is not recommended after using recommended regimens for treatment, i.e. unless therapeutic compliance is in question, symptoms persist, or reinfection is suspected.


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The web site The web site contains extensive reviews of chlamydial infections and their diagnosis, hyperlinked to the original literature.

Further Reading

Fig. 3.1 For further information, see Everett KDE et al: Int J Syst Evol Microbiol 1999; 49:415–440; Rurangirwa FR, et al: Int J Syst Bacteriol 1999; 49:577–581; Horn M, et al: Microbiology 2000; 146:1231–1239; Bush RM and Everett KDE: Int J Syst Evol Microbiol 2001; 51:203–220; Everett KDE and Andersen AA: Int J Syst Evol Microbiol 2001; 51:251–253
Fig. 3.8 For further information regarding chlamydial genome sequences and deduced proteins, see Stephens RS, et al: Science 1998; 282:754–759; Read TD, et al: Nucleic Acids Res 2000; 28:1397–1406; Read TD, et al: Nucleic Acids Res 2003; 31:2134–2147; Carlson JH, et al: Infect Immun 2005; 73:6407–6418; Thomson NR, et al: Genome Res 2005; 15:629–640; Caldwell HD, et al: In Proceedings of the 11th International Symposium of Human Chlamydial Infections 2006; Thomson NR, et al: Genome Res 2008; 18:161–171; Seth-Smith HMB, et al: BMC Genomics 2009; 10:239; Unemo M, et al: Microbiology 2010; 156(Pt 5):1394–1404
Fig. 3.9 For further information, see Thomson NR, et al: Genome Res 2008; 18:161–171
Fig. 3.10 For further information regarding the cryptic plasmid and/or the nvCT, see Thomas NS, et al: Microbiology 1997; 143:1847–1854; Miyashita N, et al: J Infect Chemother 2001; 7:113–116; Pickett MA, et al: Microbiology 2005; 151:893–903; Ripa T and Nilsson PA: Sex Transm Dis 2007; 34:255–256; Carlson JH, et al: Infect Immun 2008; 76:2273–2283; Seth-Smith HMB, et al: BMC Genomics 2009; 10:239; Unemo M, et al: Microbiology 2010; 156(Pt 5):1394–1404
Fig. 3.45 For further information, see e.g. Ripa KT and Mårdh PA: J Clin Microbiol 1977; 6:328–331
Fig. 3.53 For further information regarding DFAs and their performance characteristics, see Tam MR, et al: N Engl J Med 1984; 310:1146–1150; Uyeda CT, et al: J Clin Microbiol 1984; 20:948–950; Beebe JL, et al: Sex Trans Dis 1996; 23:465–470; Schachter J: Immunol Invest 1997; 26:157–161; Newhall WJ, et al: J Clin Microbiol 1999; 37:681–685; Østergaard L: Best Pract Res Clin Obstet Gynaecol 2002; 16:789–799
Fig. 3.60 For further information regarding different EIAs and their performance characteristics, see Jones MF, et al: J Clin Microbiol 1984; 20:465–467; Mahony J, et al: J Clin Microbiol 1989; 27:1934–1938; Beebe JL, et al: Sex Trans Dis 1996; 23:465–470; Schachter J: Immunol Invest 1997; 26:157–161; Dean D, et al: J Clin Microbiol 1998; 36:94–99; Hirose T, et al: Int J STD AIDS 1998; 9:414–417; Newhall WJ, et al: J Clin Microbiol 1999; 37:681–685; Okadome A, et al: Int J STD AIDS 2000; 10:460–463; Tanaka M, et al: J Clin Pathol 2000; 53:350–354; Van Dyck E, et al: J Clin Microbiol 2001; 39:1751–1756; Chernesky M, et al: J Clin Microbiol 2001; 39:2306–2307; Østergaard L: Best Pract Res Clin Obstet Gynaecol 2002; 16:789–799; Horner P, et al: J Clin Microbiol 2005; 43:2065–2069
Fig. 3.61 For further information regarding different EIAs and their performance characteristics, see references for Fig. 3.60
Fig. 3.62 For further information regarding POC and home diagnostic tests, and their performance characteristics, see Pate MS, et al: J Clin Microbiol 1998; 36:2183–2186; Rani R, et al: Int J STD AIDS 2002; 13:22–24; Herring A, et al: Nat Rev Microbiol 2006; 4(12 Suppl):S41–48; Michel CE, et al: Lancet 2006; 367:1585–1590; Yin YP, et al: Sex Transm Infect 2006; 82(Suppl 5):33–37; Moi H: Tidsskr Nor Laegeforen 2007; 127:2083–2085 (In Norwegian, but English abstract); Mahilum-Tapey L, et al: Br Med J 2007; 335:1190–1194; Gaydos CA: Sex Transm Infect 2009; 85:158; Michel CE, et al: Sex Transm Infect 2009; 83:187–189
Fig. 3.63 For further information regarding serological assays for chlamydial antibody detection, and their performance characteristics and use, see Wang SP, et al: J Clin Microbiol 1975; 1:250–255; Chernesky M, et al: Sex Transm Dis 1998; 25:14–19; Tuuminen T, et al: J Clin Microbiol Methods 2000; 42:265–279; Bas S, et al: J Clin Microbiol 2001; 39:1368–1377; Nikkari S, et al: J Rheumatol 2001; 28:2487–2493; Verkooyen RP, et al: Int J STD AIDS 2002; 13(Suppl 2):23–25; Mouton JW, et al: Int J STD AIDS 2002; 13(Suppl 2):26–29; Bas S, et al: Rheumatol 2002; 41:1017–1020; Morré S, et al: J Clin Microbiol 2002; 40:584–587; Persson K: Best Pract Res Clin Gynaecol 2002; 16:801–814; Akande V: Hum Fertil 2002; 5(1 Suppl):S15-S20; Jones CS, et al: J Clin Pathol 2003; 56:225–229; Bax CJ, et al: Clin Diagn Lab Immunol 2003; 10:174–176; Lyytikäinen E, et al: Sex Transm Infect 2008; 84:19–22; Lyytikäinen E, et al: BMC Infect Dis 2008; 8:169; Wills GS, et al: Clin Vaccine Immunol 2009; 16:835–843
4 Genital Mycoplasmas

D. Taylor-Robinson, J.S. Jensen

Mycoplasmas, the trivial name for members of the class Mollicutes (’soft skinned’) are the smallest known free-living micro-organisms, intermediate in size between bacteria and viruses ( Fig. 4.1 ). They are unique among prokaryotes, differing by one or more characteristics from all other major groups of human pathogens, including viruses ( Table 4.1 ). The absence of a rigid cell wall ( Fig. 4.2 ) is the single most distinguishing feature of mycoplasmas 1 and is responsible for their being in a separate class, the Mollicutes. Many of the biologic properties of mycoplasmas are due to the absence of a rigid cell wall, including resistance to β-lactam antibiotics and marked pleomorphism among individual cells. In contrast to L-phase variants of bacteria, mycoplasmas are unable to synthesize cell-wall precursors under any conditions. The mycoplasmal cell membrane contains phospholipids, glycolipids, cholesterol, and various proteins. The extremely small genome size of mycoplasmas (approximately one-sixth the size of that of Escherichia coli ) ( Table 4.2 ) severely limits their biosynthetic capabilities, helps to explain their complex nutritional requirements for cultivation, and necessitates a parasitic or saprophytic existence for most species.

Fig. 4.1 Relative size of mycoplasma in comparison with other sexually transmitted micro-organisms. An individual mycoplasmal cell may be as small as 300 nm in diameter.

Table 4.1 Comparison of mycoplasmas with other microbial agents.

Fig. 4.2 Transmission electron micrograph showing mycoplasmas between two epithelial cells. Unlike other bacteria, mycoplasmas lack a rigid cell wall.
Table 4.2 Mycoplasmas have small genomes. Organism Genome size (base pairs) Predicted protein coding genes Mycoplasma genitalium G37 580 076 482 Mycoplasma pneumoniae M129 816 394 688 Ureaplasma urealyticum serovar 5 (ATCC-27817) 851 487 662 Ureaplasma parvum serovar 3 (ATCC-27815) 751 979 609 Escherichia coli E24377A 5 249 288 5146
The human mycoplasmas usually reside on the mucosal surfaces of the respiratory and urogenital tracts and rarely penetrate the submucosa, although systemic spread seems to be a feature of several mycoplasma species (e.g. Mycoplasma aligatoris ) which infect animals . Mycoplasmas are primarily found extracellularly, but an increasing number of species, like M. pneumoniae , M. genitalium, M. hominis ( Fig. 4.3 ), M. fermentans and M. penetrans, 2 have also been demonstrated intracellularly.

Fig. 4.3 Electron micrograph of immunogold-labeled M. hominis organisms within a vacuole in the cytoplasm of a Hela cell.
The species of mycoplasmas isolated from the urogenital tract of humans 3 - 5 are listed in Table 4.3 . M. genitalium ( Fig. 4.4 ) has been detected in the urethra of a significantly larger proportion of men (about 25%) with acute non-chlamydial non-gonococcal urethritis (NCNGU) than in those without this disease (about 5%). This, together with the urethral inflammation occurring after intra-urethral inoculation of non-human primates, especially chimpanzees, with M. genitalium and the failure to cure urethritis with antimicrobials having poor activity, such as tetracyclines, indicate that it is a cause of urethritis in men. M. genitalium has also been associated with cervicitis in several studies, although the association is weaker than that for NGU. In addition, serologic studies in humans and experimental studies in non-human primates suggest that it may be a cause of PID. The role of M. genitalium in disease during pregnancy needs to be further evaluated, as does its potential impact on the outcome of pregnancy.

Table 4.3 Mycoplasmas isolated from or detected in the genitourinary tract of humans.

Fig. 4.4 Transmission electron micrograph of M. genitalium showing an intact mycoplasma cell negatively stained with ammonium molybdate. The terminus is covered with a nap extending peripherally to the tip.
For more than 50 years there have been sporadic reports of the isolation of M. fermentans from the upper as well as the lower urogenital and respiratory tracts, bone marrow, and other anatomic sites. For the last 20 years, there have been reports of M. fermentans in patients with AIDS, 2 although there is no substantial evidence to indicate that it contributes to the disease. However, it has been implicated in an acute fatal respiratory disease of non-AIDS patients.
M. fermentans has also been detected in amniotic fluid collected at the time of cesarean section from a small proportion of women with intact membranes. Some of the mycoplasma-positive women had chorioamnionitis, suggesting that M. fermentans should be investigated further as a cause of maternal and fetal infection. More recently, M. fermentans has been detected by PCR assays in the joints of about 20% of patients with idiopathic chronic inflammatory arthritides, including rheumatoid arthritis, but not in those with degenerative or metabolic arthritis, findings that need pursuing. Some of the studies employing PCR methods, however, suffer from the fact that the primers targeting an M. fermentans insertion sequence, appear to amplify a similar sequence in the common commensal M. orale .
Of the mycoplasmas occurring in the urogenital tract, ureaplasmas (in this chapter used to denote U. urealyticum and U. parvum where the species determination has not been given), M. hominis and M. genitalium are detected most frequently and will be discussed in more detail in this chapter.

The isolation rates of ureaplasmas, M. hominis and M. genitalium in various populations are given in Table 4.4 . Ureaplasmas can be found in the cervix or vagina of 40–80% of sexually mature, asymptomatic women, and M. hominis in 20–50%. The incidence of each is somewhat lower in the urethra of healthy males. In women, colonization is linked to younger age, lower socio-economic status, sexual activity with multiple partners, Black race, oral contraceptive use and, importantly, to bacterial vaginosis (BV). Ureaplasmas and M. hominis may be transmitted to about 40% of babies born to infected mothers. The colonization of most infants appears to be transient, with a sharp decline in the rate of isolation after 3 months of age. Less than 10% of older children and sexually inexperienced adults are colonized. Colonization after puberty increases with sexual activity. In contrast to the very frequent detection of ureaplasmas and M. hominis , M. genitalium is found in only 1–3% of sexually active men and women in population-based studies, whereas it can be detected in about 5% of asymptomatic sexually transmitted disease (STD)-clinic attendees. Detection is primarily associated with the classical STD-risk factors such as young age, multiple partners, recent partner change, and early sexual debut. Little is know about transmission during birth, although one example of vertical transmission has been recorded.

Table 4.4 Epidemiology of ureaplasmas, M. hominis and M. genitalium .

Clinical Manifestations
Ureaplasmas and M. hominis are most often commensals in the lower genital tract, whereas the link between detection of M. genitalium and disease is much stronger. Genitourinary diseases in which these micro-organisms are suspected of having an etiologic role are listed in Table 4.5 . Difficulty in accepting ureaplasmas and M. hominis as the cause of disease has usually arisen because they are sometimes recovered as often from asymptomatic as from symptomatic individuals, or because samples cannot be obtained easily from the affected site for testing. A major principle is that organisms only reach the upper tract in a subpopulation of individuals infected in the lower urogenital tract, and that disease then develops in only a few of these individuals. It seems that in this situation ureaplasmas and M. hominis behave primarily as opportunists. In contrast, these mycoplasmas are recognized as common causes of extragenital disease in immunocompromised patients and in newborn infants, particularly preterm infants.

Table 4.5 Relationship of ureaplasmas, M. hominis and M. genitalium to diseases of the genitourinary tract of humans.

Non-gonococcal urethritis in men
M. genitalium ( Fig. 4.4 ) has been strongly and uniformly associated with non-gonococcal urethritis (NGU) in a large number of studies ( Fig 4.5A ) and has been detected in the urethra of 15–25% of men with symptomatic NGU compared to about 5–10% of those without this disease. The association is even stronger in non-chlamydial NGU ( Fig 4.5 B ), suggesting that M. genitalium and C. trachomatis act as separate causes of the condition. In several studies, M. genitalium has been found in more than one third of men with NCNGU. Among STD clinic populations, approximately 90% of M. genitalium- infected men have microscopic evidence of urethritis and around 75% report symptoms. The development of quantitative PCR assays for M. genitalium has shown a dose/response relationship between signs and symptoms of urethritis and M. genitalium DNA load in urethral and urine specimens, further supporting the causal relationship of M. genitalium with urethritis. Finally, animal experiments ( Table 4.6 ) have demonstrated that intra-urethral inoculation of some male animals with M. genitalium results in the development of urethritis, shown most impressively in chimpanzees with shedding of the organism for up to 18 weeks after inoculation. Furthermore, an antibody response could be detected in most of the chimpanzees.

Fig. 4.5 Odds ratios and 95% confidence intervals in studies of M. genitalium PCR-positivity in men with (A) Non-gonococcal urethritis (NGU), and (B) Non-chlamydial non-gonococcal urethritis (NCNGU).

Table 4.6 Results of inoculating male and female animals with M. genitalium .
Several clinical studies have shown a strong correlation between M. genitalium and persistent or recurrent NGU, probably due to the poor microbiologic treatment efficacy of tetracyclines eradicating M. genitalium from less than one third of the patients. M. genitalium has been found in up to 41% of men with persistent or recurrent urethritis after treatment with doxycycline. More recently, azithromycin treatment failure after a 1 g single dose was reported among 28% of men with M. genitalium -positive NGU and was correlated with the development of macrolide resistance in M. genitalium after treatment with a single dose. Clearly, randomized controlled treatment trials are needed to establish the optimal treatment regimen for this important pathogen.
In contrast to the consistency of studies linking M. genitalium with NGU, the association of ureaplasmas with this disease has been controversial. The results of human and animal inoculation studies, together with those of controlled antibiotic and serologic investigations, support a causal role for ureaplasmas in NGU, particularly chronic disease, although they lag behind Chlamydia trachomatis and M. genitalium in importance. Furthermore, the exact proportion of cases for which ureaplasmas are responsible has not been established ( Fig. 4.6 ). A number of studies have suggested that U. urealyticum is more closely associated with NGU than U. parvum, but in only a few studies has there been control for infection with C. trachomatis or M. genitalium . Predisposing factors, such as a lack of mucosal immunity in individuals who develop disease is likely to play a significant role as indicated by a single study where repeated inoculation of a human subject led to a gradual decrease of the inflammatory response. Some patients with hypogammaglobulinemia develop a prolonged urethritis with persistent ureaplasmal infection. In such cases, treatment is often complicated by antimicrobial resistance and a combination of different classes of antibiotics is recommended.

Fig. 4.6 The causes of non-gonococcal urethritis (NGU). A. Approximate incidence of NGU versus gonococcal urethritis in USA. B. It is well established that up to approximately one-half of NGU cases are due to C. trachomatis , about 25% due to M. genitalium , with the remainder caused by either ureaplasmas or other micro-organisms (perhaps those involved in bacterial vaginosis), the precise contributions of which are not certain.
There is no evidence supporting a role for M. hominis as a cause of urethritis.

The results of a number of studies suggest that the prostate can be infected by ureaplasmas during the course of an acute ureaplasmal infection of the urethra. Ureaplasmas have been isolated more often and in greater numbers from specimens taken from patients with acute prostatitis than from controls, and men with more than 10 4 organisms have been reported to respond to tetracycline therapy, unlike those with fewer organisms. However, unequivocal evidence for a causal role in acute disease does not exist. M. genitalium has been detected by PCR analysis of prostatic biopsies from 5 of 135 men and in semen from 2 of 18 men with chronic abacterial inflammatory prostatitis compared to none of 20 controls. However, further studies are needed to confirm these findings. Ureaplasmas have not been found in prostatic biopsies from patients with chronic abacterial prostatitis, and M. hominis has not been associated with prostatitis of any kind in most studies.

Ureaplasmas have been recovered from the urethra and directly from epididymal aspirate fluid, accompanied by a specific antibody response, in a patient with acute non-gonococcal, non-chlamydial epididymitis. Clinical experience as well as the detection of M. genitalium in a few patients during a treatment trial indicates that M. genitalium may be a cause of acute epididymitis. However, further studies are required to establish a causal role.

Urinary calculi
Infection stones, composed of magnesium ammonium phosphate (struvite) and carbonate–apatite ( Fig. 4.7 ) are thought to be caused by urea-hydrolyzing bacteria, including Proteus and ureaplasmas. In several studies, ureaplasmas have been detected in 12–27% of infection stones, occasionally in pure culture and more frequently in the urine and stones of patients with infection stones, compared to those with metabolic stones.

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