Trichomonas vaginalis detection using real-time TaqMan PCR

Abstract

1978 women and 93 men, all suspected of having a Trichomonas vaginalis infection, were tested for the presence of T. vaginalis by real-time PCR using the T. vaginalis-specific 2-kb repeated sequence, and by direct microscopy and culture. 40 samples were positive by T. vaginalis real-time PCR and 27 were positive by wet mount microscopy, either direct or after culture. All samples positive by direct microscopy of culture were also positive by real-time PCR. Of the 13 samples which were real-time PCR positive but negative by direct microscopy and culture 11 were confirmed by another T. vaginalis real-time PCR based on the beta tubulin gene. Only 2 samples (0.1%) showed inhibition in the PCR. The prevalence of T. vaginalis infection in the female patients was 1.8%. The sensitivity, specificity, positive and negative predictive values of the real-time PCR were 100%, 99.9%, 95% and 100%, respectively. The same test characteristics for the combined conventional T. vaginalis detection methods (microscopy + culture) were 71%, 100%, 100% and 99%, respectively. Therefore, real-time PCR is the method of choice for the diagnosis of T. vaginalis infection.

Introduction

Trichomonas vaginalis infection is the most prevalent non-viral sexually transmitted disease (STD), with worldwide infections of an estimated 180 million occurring each year (World Health Organization, 2001). In men, infection with the T. vaginalis parasite is frequently asymptomatic, but can be responsible for prostatitis, urethritis and epididymitis (Jordan et al., 2001, Kengne et al., 1994). In women T. vaginalis is the main cause of vaginitis, cervicitis and urethritis. Vaginitis with a purulent discharge is the prominent symptom. T. vaginalis infections have been suggested also to play a role as cofactor in the transmission of human immunodeficiency virus (Laga et al., 1993, Sorvillo and Kerndt, 1998), to be a risk factor for developing cervical cancer (Zhang et al., 1995), and to be associated with poor outcome in pregnancy (Cotch et al., 1997, Pastorek et al., 1996). Still, it has been estimated that 10–50% of T. vaginalis infections in women are asymptomatic (Burstein and Zenilman, 1999).

Historically, T. vaginalis infections have been diagnosed using direct microscopic examination (“wet mount”) for the presence of pathogenic flagellates with characteristic active motility. Often the swab specimens are also used for T. vaginalis cultures which are usually examined by microscopic examination after 3–5 days. The most popular Trichomonas selective culture media, such as several modifications of Diamond's medium and the newer more practical pouch cultures, are about equally sensitive (Draper et al., 1993, Schwebke and Burgess, 2004). However, both culture and direct microscopy are inefficient in detecting low numbers of parasites, defective parasites or organisms not surviving the transfer to culture medium.

In 1992 the detection of T. vaginalis using the polymerase chain reaction (PCR) was first described (Riley et al., 1992). In various subsequent publications, T. vaginalis PCR was shown to be much more sensitive than culture (> 90% versus < 70%), whereas the detection rate of direct microscopic examination was usually < 50% (Kengne et al., 1994, Madico et al., 1998, Schee van der et al., 1999, Wendel et al., 2002, World Health Organization, 2001). As targets for T. vaginalis-specific PCR assays, different T. vaginalis genes were used, such as the ferredoxin gene (Jordan et al., 2001, Riley et al., 1992), the beta tubulin gene (Madico et al., 1998), a highly repeated 2-kb DNA sequence (Kengne et al., 1994), the18S ribosomal gene (Mayta et al., 2000) and the adhesion protein gene (Alderete et al., 1995). However, despite the fact that the T. vaginalis PCR has shown to be rapid, specific and very sensitive, T. vaginalis culture still remains the gold standard for most laboratories (Lawing et al., 2000, Schee van der et al., 1999, Schwebke and Burgess, 2004). Moreover, conventional protocols for the detection of specific PCR products are relatively labor intensive and, therefore, quite expensive. Some recent papers described the use of real-time PCR for the detection of T. vaginalis in limited numbers of genital samples of either women (Caliendo et al., 2005, Jordan et al., 2001, Smith et al., 2005) or men (Hardick et al., 2003).

The purpose of this study was to develop a sensitive real-time PCR for the detection of T. vaginalis in genital swabs of women and men and test the new method prospectively in about 2000 patients. We choose to use TaqMan technology since that is the standard method used in our routine molecular diagnostic laboratory. The new PCR was compared with direct microscopy and culture. Discordant results were confirmed by a T. vaginalis real-time PCR targeting another gene of T. vaginalis. Finally, the possible use of urine samples for the detection of T. vaginalis was briefly investigated.