M M B ™ ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Diagnosis of Sexually Transmitted Diseases Methods and Protocols Edited by Colin R. MacKenzie and Birgit Henrich Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany Editors Colin R. MacKenzie Birgit Henrich Institute of Medical Microbiology Institute of Medical Microbiology and Hospital Hygiene and Hospital Hygiene Heinrich-Heine-University Heinrich-Heine-University University Hospital University Hospital Düsseldorf, Germany Düsseldorf, Germany ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-61779-936-5 ISBN 978-1-61779-937-2 (eBook) DOI 10.1007/978-1-61779-937-2 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2012939965 © Springer Science+Business Media New York 2012 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Preface Sexually transmitted infections (STI) continue to be a major cause of morbidity and mortality in the twenty-fi rst century, both in developed industrial countries and in the developing world. The WHO estimates that there are 448 million new curable infections per year, many of which will result in infertility, perinatal morbidity, and death ( h ttp://who.int ) . Human immunodefi ciency virus infections and the ensuing opportunistic infections are a major drain on the human and fi nancial resources of many countries in the developing world, and even with the availability of effective treatment the epidemic is not yet con- tained. Many STI are inapparent or asymptomatic, which makes their control very diffi cult and places the emphasis on screening for infections among this group. In wealthy industri- alized countries most people have ready and regular access to health services, such as during school, at the work place, or during pregnancy, and therefore screening for asymptomatic STI poses no major problem other than in the disadvantaged. The vast majority of the population in the developing world has no regular access to health care, and services are often days away from the home. Screening in this group is diffi cult and there is a need for simple reliable cheap diagnostic methods that can be performed at the point of care, on the same day and by staff with limited training. To add to the already considerable burden of STI, an increase in the resistance of many of the treatable organisms causing infection has been observed recently, and thus a need for rapid detection of resistance is desirable. A number of organisms can cause STI, the most common of which are Treponema pallidum, Chlamydia trachomatis, Neisseria gonorrhoeae, Haemophilus ducreyi, Mycoplasma genitalium and M. hominis, Ureaplasma urealyticum, U. parvum , K lebsiella granulomatis, Candida , viruses such as HIV, HSV, HBV, HCV, CMV, HPV, parasites such as T richomonas vaginalis , scabies. Testing for this wide range of pathogens has not been useful due to technological limitations in regions where STI prevalence is highest, and therefore a syndromic approach has been propagated by the WHO ( h ttp://who.int ). This syndromic approach to therapy classifi es STI into seven syndrome complexes; urethral discharge, genital ulcer disease, inguinal swelling, scrotal swelling, vaginal discharge, lower abdominal pain, and neonatal eye infections. Application of this approach has probably led to an overtreatment as many patients with symptoms do not have an STI. Therefore newer better applicable diagnostic tools are urgently required and the term “ASSURED” tests was introduced to describe the ideal test: Affordable, Sensitive, Specifi c, User-friendly, Rapid and robust, Equipment-free, Delivered. Molecular biological methods are very attractive for the diagnosis of STI since a well- de fi ned range of pathogens is responsible for the infection. In particular, the screening of asymptomatic patients requires an affordable and reliable (sensitive and specifi c) test. In addition new advances in molecular biology methods and techniques will undoubtedly lead to simple robust and affordable tests. We only have to look at the advances in PCR technol- ogy to see how far we have come in a decade. Tests developed and applied in developing countries will more than likely lead to ASSURED tests in developing countries. This book strives to cover the full range of molecular testing for STI. Special attention was given to including a range of methods from the simple and inexpensive to complex v vi Preface sophisticated methods, thus hoping to provide scientists in many different situations with the information they seek. Aspects of DNA extraction from small-volume samples or dif fi cult tissues, simple, nested, or multiplex PCR, use of duplex primers or other modi fi cations of primers and PCR conditions, sequence analysis for genotyping, denaturing gel analysis, microarrays using liquid beads or microspheres, and silicon nanoparticle- enhanced microcantilever detection of DNA are dealt with in individual chapters. Due to the increasing concern for antibiotic resistance in modern medicine and the appearance of resistant neisseria, the rapid and simple testing for resistance genes is an important factor for molecular testing, and chapters addressing this and the expression of virulence and host factors have also been included. Due to the high prevalence of STI and the asymptomatic nature of many diseases, self-collection of specimens has become more common, and the issue concerning self-collected specimens is dealt with in Chapter 2 7 . Where appropriate, the authors have provided a set of guidelines to aid the reader in the process of establishing a method from scratch. We have also included chapters on the ethical issues of sexual abuse and molecular testing as well as those issues pertaining to STI-testing and close the book with a review about the “transparent patient.” Laboratory diagnostic tests must meet strict standards in order to be approved for use in the diagnosis of disease in patients. In many instances the application determines the standards to be met; for example, a test in a low-prevalence region requires a higher specifi city than would be required in a high-prevalence population. Redesigning molecular tests to suit point-of-care testing will be a challenge for the future; however, the market is huge and the need for inexpensive tests in middle-income regions such as China, India, South Africa, and South America is urgent considering the expanding burden of HIV and related diseases in these regions. Molecular testing will undergo the same evolution as other methods; the greater the usage the better the evaluation resulting in the truly robust meth- ods becoming established and refi ned and the less useful tests falling by the wayside. We hope this book will be a useful adjunct to the literature in order to help this process. The twenty-fi rst century will certainly witness an exciting expansion in the use of molecular methodologies for the diagnosis of a wide range of human (and animal) diseases, and this will be particularly relevant for the diagnosis of STI. We are confi dent that the cur- rent volume in the series of “Molecular Methods” will be a valuable addition to the refer- ence literature for the scientist looking to establish this kind of test in their own lab. Düsseldorf, Germany Colin R . MacKenzie Birgit Henrich Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi 1 Overview of Molecular Biological Methods for the Detection of Pathogens Causing Sexually Transmitted Infections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Fernando Vazquez, Luis Otero, Santiago Melón, and María de Oña 2 Guidelines for the Qualitative Detection of Viral Genomes in Dried Blood Spots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Davide Gibellini, Elisa De Crignis, and Maria Carla Re 3 Guidelines for the Quantification of HIV and HCV in Small Volume Whole Blood Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Tony Bruns and Katrin Steinmetzer 4 Guidelines for High-Resolution Genotyping of Chlamydia trachomatis Using Multilocus Sequence Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Linus Christerson and Björn Herrmann 5 Guidelines for the Use of Molecular Tests for the Detection and Genotyping of Human Papilloma Virus from Clinical Specimens . . . . . . . . . . . 65 Sin Hang Lee 6 The Molecular Diagnosis of Sexually Transmitted Genital Ulcer Disease. . . . . . . . . 103 Cheng-Yen Chen and Ronald C. Ballard 7 Validation of a Sensitive and Specific Real-Time PCR for Detection and Quantitation of Hepatitis B Virus Covalently Closed Circular DNA in Plasma of Chronic Hepatitis B Patients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 R.B. Takkenberg, S. Menting, and M.G.H.M. Beld 8 Protocol for the Detection of Mycoplasma genitalium by PCR from Clinical Specimens and Subsequent Detection of Macrolide Resistance-Mediating Mutations in Region V of the 23S rRNA Gene . . . . . . . . . . . 129 Jørgen Skov Jensen 9 Protocols for Detection and Typing of Treponema pallidum Using PCR Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 S.M. Bruisten 10 Extraction of DNA from Dried Blood in the Diagnosis of Congenital CMV Infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Jutte J.C. de Vries, Maria Barbi, Sandro Binda, and Eric C.J. Claas 11 Protocol for the Use of PCR-Denaturing Gradient Gel Electrophoresis and Quantitative PCR to Determine Vaginal Microflora Constitution and Pathogens in Bacterial Vaginosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Beatrice Vitali, Elena Biagi, and Patrizia Brigidi vii viii Contents 12 Protocol for the Use of a Bead Array for the Multiple Detection of Genotype of Chlamydia trachomatis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Chung-Te Huang and Shu-Ying Li 13 Protocol for the Detection and Genotyping of Human Papillomaviruses Using a Liquid Bead Microarray Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 Stephen Cherne, Viorica Popov, and Qinghua Feng 14 Protocol for the Use of Enzyme-Linked Hybridization Assays for Genital Ulcer Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 Seweryn Bialasiewicz, Ian M. Mackay, David M. Whiley, and Theo P. Sloots 15 Protocol for the Rapid Detection of the Urogential Tract Mollicutes and Chlamydia with Concomitant LGV-(sub)typing . . . . . . . . . . . . . . . . . . . . . . . . 235 Nadine Möbius, Wibke Brenneisen, Anke Schaeffer, and Birgit Henrich 16 Protocol for the Use of Self-Reporting Duplex Mutation Primers to Detect PCR Products in the Diagnosis of HBV . . . . . . . . . . . . . . . . . . . . . . . . . 255 Qian-Feng Xia 17 Protocol for the Use of a Rapid Real-Time PCR Method for the Detection of HIV-1 Proviral DNA Using Double-Stranded Primer. . . . . . . . . . . . . . . . . . . . . 263 Chou-Pong Pau, Susan K. Wells, and Timothy C. Granade 18 Protocol for the Use of Light Upon Extension Real-Time PCR for the Determination of Viral Load in HBV Infection . . . . . . . . . . . . . . . . . . . . . . 273 Guimin Li, Wangfeng Li, and Lixia Liu 19 Protocol for the Use of a Silica Nanoparticle-Enhanced Microcantilever Sensor-Based Method to Detect HBV at Femtomolar Concentrations. . . . . . . . . . . 283 Sang-Myung Lee, Kyo Seon Hwang, Sang Kyung Kim, and Tae Song Kim 20 Protocol for the Detection of Treponema pallidum in Paraffin-Embedded Specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 Cheng-Yen Chen and Allan Pillay 21 Guidelines for the Use of Molecular Biological Methods to Detect Sexually Transmitted Pathogens in Cases of Suspected Sexual Abuse in Children . . . . . . . . . 307 Margaret R. Hammerschlag and Charlotte A. Gaydos 22 Protocol for the Molecular Detection of Antibiotic Resistance Mechanisms in Neisseria gonorrhoeae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 Namraj Goire, Theo P. Sloots, Michael D. Nissen, and David M. Whiley 23 Protocol for a Facile Multiplex PCR for Multi-antimicrobial Resistance and Gonococcus Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 Ratana Lawung, Rungrot Cherdtrakulkiat, and Virapong Prachayasittikul 24 Protocol for Gene Expression Profiling Using DNA Microarrays in Neisseria gonorrhoeae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Lydgia A. Jackson and David W. Dyer 25 Protocol for Analyzing Human Leukocyte Antigen Variants and Sexually Transmitted Infections: From Genotyping to Immunoassays. . . . . . . . 359 Jianming Tang and Anju Bansal Contents ix 26 Protocol for the Clonal Analysis of NK Cell Effector Functions by Multi-parameter Flow Cytometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 Kathrin Schönberg, Maryam Hejazi, and Markus Uhrberg 27 Protocol for a Mammalian Cell-Based Assay for Monitoring the HIV-1 Protease Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Chitra Rajakuberan, Brett J. Hilton, and Roland Wolkowicz 28 Self-Collection of Specimens for Nucleic Acid-Based Diagnosis of Pharyngeal, Cervicovaginal, Urethral, and Rectal Neisseria gonorrhoeae and Chlamydia trachomatis Infections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 Vivian Levy, Craig S. Blackmore, and Jeffrey D. Klausner 29 Sexually Transmitted Diseases: Reflections on Metaphors and Ethics. . . . . . . . . . . . 419 Gisela Badura-Lotter Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437