Bacteriology
Evaluation of a new multiplex polymerase chain reaction assay STDFinder for the simultaneous detection of 7 sexually transmitted disease pathogens

https://doi.org/10.1016/j.diagmicrobio.2011.06.005Get rights and content

Abstract

We evaluated a new multiplex polymerase chain reaction (mPCR), “STDFinder assay”, a novel multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous detection of 7 clinically relevant pathogens of STDs, i.e., Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium, Treponema pallidum, and herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). An internal amplification control was included in the mPCR reaction. The limits of detection for the STDFinder assay varied among the 7 target organisms from 1 to 20 copies per MLPA assay. There were no cross-reactions among any of the probes. Two hundred and forty-two vaginal swabs and an additional 80 specimens with known results for N. gonorrhoeae and C. trachomatis, obtained from infertile women seen at an infertility research clinic at the Kigali Teaching Hospital in Rwanda, were tested by STDFinder assay and the results were confirmed by single real-time PCR using different species-specific targets. Compared to the reference standard, the STDFinder assay showed specificities and sensitivities of 100% and 100%, respectively, for N. gonorrhoeae, C. trachomatis, and M. genitalium; 90.2% and 100%, respectively, for Trichomonas vaginalis; and 96.1% and 100%, respectively, for HSV-2. No specimen was found to be positive for HSV-1 by either the STDFinder assay or the comparator method. Similarly, the sensitivity for Treponema pallidum could not be calculated due to the absence of any Treponema pallidum-positive samples. In conclusion, the STDFinder assays have comparable clinical sensitivity to the conventional mono and duplex real-time PCR assay and are suitable for the routine detection of a broad spectrum of these STDs at relatively low cost due to multiplexing.

Introduction

Sexually transmitted diseases (STDs) are a major cause of morbidity in sexually active individuals and continue to pose major medical, social, and economic burden worldwide. According to the World Health Organization, 340 million new cases of gonorrhoea, chlamydia, syphilis, and trichomoniasis occurred throughout the world in 1999 in men and women aged 15–49 years (http://www.who.int/mediacentre/factsheets/fs110/en/). Untreated STDs, particularly gonorrhoea and chlamydial infection which cause pelvic inflammatory disease in women, can lead to infertility in both men and women. Other sequelae of some STDs include ectopic pregnancy and risk of developing genital cancers, which cost the individuals and health care systems billions of dollars annually. Apart from causing serious sequelae, STDs increase the risk of both transmission and acquisition of HIV (http://www.who.int/mediacentre/factsheets/fs110/en/). The development of fast laboratory diagnostic screening method for STDs is therefore an imperative tool to minimize damage to the reproductive tract and to simultaneously improve women's health worldwide.

Several methods are available for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, and Mycoplasma genitalium, including bacterial culture, enzyme-linked immunosorbent assay for antigen or antibody detection (Clad et al., 2000), strand displacement amplification (Chan et al., 2000), and polymerase chain reaction (PCR), performed in monoplex or multiplex (Chernesky et al., 2005, Eastick et al., 2003, Geraats-Peters et al., 2005, Kawada et al., 2004, Ryu et al., 1999). Of these, PCR has been found to be a highly sensitive method for detecting these sexually transmitted pathogens. A multiplex assay has an additional advantage in screening since it involves the simultaneous detection of multiple pathogens. Moreover, the incorporation of an internal control reaction in a multiplex assay identifies the possible presence of PCR inhibition. A multiplex test is a prerequisite to reduce the costs of an assay as well as hands-on time. However, simultaneous molecular amplification and detection of multiple targets are technologically challenging as they may result in the reduction of the ability to amplify by competition between the amplification of the different mix reactions or to detect individual targets through nonspecific interactions between primers and probes (Markoulatos et al., 2002).

The multiplex ligation-dependent probe amplification (MLPA) technology, which uses a single easy-to-perform assay, is able to amplify up to 45 different targets simultaneously using one universal primer set in the final amplification (Schouten et al., 2002). Previously developed MLPA applications include the detection of changes in the copy numbers of specific chromosomal regions (Schouten et al., 2002), detection of CpG methylation of genes (Nygren et al., 2005), detection of recombination events (Langerak et al., 2005), and expression profiling studies (Eldering et al., 2003). Most recently, MLPA has successfully evaluated the identification of a wide range of viruses causing respiratory tract and central nervous system (CNS) infections (Reijans et al., 2008, Wolffs et al., 2009). Our aim was to evaluate and demonstrate the utility of the STDFinder assay (Fig. 1), a novel MLPA technology for the simultaneous detection of 7 clinically relevant pathogens of STDs, i.e., N. gonorrhoeae, C. trachomatis, Trichomonas vaginalis, M. genitalium, Treponema pallidum, and herpes simplex virus type 1 and 2 (HSV-1 and HSV-2).

Section snippets

Study population and clinical specimens

A total of 242 vaginal swabs taken from a case group consisting of infertile women, seen at an infertility research clinic at the Kigali Teaching Hospital in Rwanda (the largest public hospital in Rwanda) between November 2007 and March 2010, who were all eligible for enrollment in a case-control study investigating the aetiology and risk factors of infertility and its link with HIV/STDs were tested in this evaluation (Dhont et al., 2010). During speculum examination, 2 swabs of upper vaginal

Results

The analytical detection limit of the STDFinder assay was evaluated for C. trachomatis, N. gonorrhoeae, and HSV-1 and HSV-2 using external quality control samples (Quality Control for Molecular Diagnostics, Glasgow, Scotland, UK) and was found to be 1 copy/MLPA assay for C. trachomatis, 20 copies/MLPA assay for N. gonorrhoeae, 3 copies/MLPA assay for HSV-1, and 5 copies/MLPA assay for HSV-2. Previous evaluations of multiplex MLPA assays which detect respiratory viruses and viruses causing CNS

Discussion

The current study describes the evaluation of a new multiplex PCR assay for the simultaneous detection in clinical specimens of 7 potential STD pathogens including C. trachomatis, N. gonorrhoeae, M. genitalium, Trichomonas vaginalis, Treponema pallidum, HSV-1 and HSV-2 which are difficult to identify by other methods. Laboratory diagnosis of most STD pathogens based on culture, for example, is costly because it requires stringent specimen collection and special media suitable for a fastidious

Acknowledgments

The authors would like to thank the staff at the Department of Clinical Chemistry, Microbiology and Immunology, Center for Molecular Diagnostics, Ghent University Hospital for their technical assistance. They also thank Yvonne Hong from Emory University, Atlanta, GA, for her careful English editing of the manuscript. This study was approved by the National Ethics Committee of Rwanda and the University of Ghent Ethics Committee in Ghent, Belgium, and financially supported by a PhD grant from

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    Conflict of interest disclaimer: STDFinder assay reagents and ESwab and transport medium were provided free of charge by PathoFinder (Maastricht, The Netherlands) and Copan Italia (Italy), respectively.

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