Table Of ContentM 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
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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
