Increasing evidence supports the need for multiplex testing of sexually transmitted infections

Feb. 22, 2016

An estimated one million individuals acquire a new sexually transmitted infection (STI) every day. According to the World Health Organization (WHO), each year an estimated 500 million people worldwide become ill with syphilis, gonorrhea, chlamydia, or trichomoniasis. The chronic sequelae of STIs include long-term disability and death but may also cause reduced fertility, increased infant and child mortality, and increased transmission rates of human immunodeficiency virus (HIV).

Since my article proposing more comprehensive multiplex testing for STIs was published in the December 2013 issue of this journal (, the evidence for the importance of such testing has become even more compelling.

A worsening problem

New data has highlighted the prevalence of Mycoplasma genitalium (MG), for example, which has caused surprise and concern among healthcare professionals. In the UK and elsewhere, infection rates have been found to be around one percent, similar to rates for Neisseria gonorrhoea (NG), which is often part of routine screening. As both infections can display common symptoms (or none) and similar long-term health effects, there is a very strong argument for routine testing for MG. This would facilitate the detection of otherwise overlooked MG infections.

In many countries, there is a minimum recommended number of STI tests to be performed, depending on symptoms. In the UK, for example, the British Association for Sexual Health and HIV (BASHH) includes Chlamydia trachomatis (CT), gonorrhea, and syphilis (Treponema pallidum) in its guidelines. Symptomatic women may also be tested for Trichomonas vaginalis (TV), which causes vaginitis and cervicitis in women and urethritis in men. However, Trichomonas infections are often asymptomatic and, because prevalence is assumed to be low, rarely tested for. In addition, wet mount microscopy, the routine diagnostic method for women, is insensitive, so TV infection remains underdiagnosed.

As with MG, new data suggests much higher incidences of TV than previously thought, and this is reflected in the new guidelines, which now recommend molecular approaches for TV detection. MG is now accepted as an STI, being implicated in urethritis and cervicitis, but the previous lack of recognition has led to inappropriate treatments and a significant rise in antimicrobial resistance.

In cases where individuals show overt physical symptoms, such as genital lesions or ulcers, herpes and syphilis testing is offered. Depending on the country, testing for tropical genital ulcerative diseases such as chancroid (Haemophilus ducreyi) is now advised according to patient history. Ureaplasma urealyticum is not routinely tested for, although there is a body of evidence implicating this bacteria in long-term health complications, similar to other bacterial STIs.

A multiplex solution

If you consider the bacterial, viral, and protozoa infections mentioned, all of which can justify routine testing, you build up a panel that could benefit from a multiplex approach. Thus a single patient sample, such as first-void urine or a genital swab, could be used to simultaneously detect infections, including those not anticipated.

Another major advantage of multiplex testing, as I noted in the 2013 article, is detection of the presence of co-infection. As many as 35 percent of individuals with STIs have been reported to have co-infections. Considering that many screening programs only look for CT and NG, there is the real possibility that this number would increase if a more comprehensive STI panel were used.

The increasing reports of antibiotic resistance among STI bacteria are also cause for concern and further highlight the need to accurately define the causal agent of infection, in order to allow tailored treatment. Widespread resistance of NG to penicillin-based and quinolone-based antibiotics has been compounded by the emerging resistance to cephalosporins, which has hindered the treatment and control of gonorrhea. This underlines the importance of accurate diagnosis and implementation of suitable treatment in the fight against the build-up of antibiotic resistance.

BASHH guidelines also recommend that people being tested for STIs should have the most accurate diagnostic test in its class for each infection for which they are being tested and that all diagnostic samples be processed in a timely fashion in order that results can be conveyed quickly and acted on appropriately. A multiplex molecular method for all key target infections fulfills these recommendations.

Common methods for diagnosis of STIs include microscopy and wet preparation, pathogen culture, or polymerase chain reaction (PCR) to correctly identify the causative agent. For some STIs, like syphilis, detection of the host response to infection (antibodies) is the routine diagnostic method. However, microscopy is insensitive, requires specialized equipment, and is dependent on the training and experience of the microscopist. Pathogen culture is lengthy, lacks sensitivity, and is hampered by difficulties in successfully culturing fastidious species. Serological tests are prone to false positive results and often require a second, confirmatory serologic test, which targets a different antigen. Another shortcoming of serological diagnosis is that antibodies may persist long after successful treatment of the infection. PCR is often preferred due to its increased sensitivity and shorter turnaround times.

For practical purposes, diagnosis of MG is limited to nucleic acid amplification testing (NAAT), as culture is extremely slow (up to six months), challenging, and insensitive. Improved sensitivity of molecular approaches over wet mount microscopy will also improve detection of Trichomonas vaginalis. Likewise, single or multiplexed NAAT are preferred to serology for the diagnosis of HSV infections.

The variety of PCR methods available for each STI makes the appropriate choice of diagnostic tests difficult. Most commercially available STI tests are uniplex or duplex assays. However, repeat PCR testing for multiple pathogens is time-consuming, expensive, and impractical, whereas a multiplex approach would provide a cost-effective alternative and improve patient outcomes by ensuring that more asymptomatic infections and co-infections are identified.


There are compelling justifications for a multiplex approach to STI detection. These include today’s greater awareness of the range of infectious agents, with higher infection rates than previously anticipated; increasing reports of antibiotic resistance; and higher co-infection rates. The increased sensitivity and specificity and reduced time to result offered by molecular technologies also are encouraging the adoption of this strategy. In combination, more rapid and accurate multiplex testing will improve detection rates, reduce risk of inadvertent spread of infection, and improve antibiotic stewardship. With increased antibiotic resistance of many of the bacterial STIs becoming a real concern, adoption of this approach is more important than ever.

As Molecular Diagnostics Manager at Randox Laboratories Ltd, Martin Crockard, PhD, works with a team of more than 20 molecular biologists in his molecular group and has developed multiplex infection and mutation profiling arrays for clinical use. Martin has more than 20 years’ experience leading molecular biology projects, over 14 at Randox. He is also working with Randox Engineering to develop fully automated analyzers, to bring the molecular tests to the point of clinical need.