The recommended specimen types for NAT testing for CT/NG include vaginal swabs from women and urine specimens from men; both specimens are collected non-invasively, which improves testing acceptability in the at-risk populations [2].

Amplification inhibition is common for urogenital specimens, with consequent negative effects on test results [23]. The percentage of specimens containing amplification inhibitors ranges from 1 – 5 % for urines and as much as 20 % for cervical swabs. Initial studies found that inhibition could be reduced or eliminated if specimens were first refrigerated overnight or frozen and thawed before testing, pointing to the labile nature of some inhibitors. However, other inhibitors are quite stable and thus more difficult to neutralize. Many of the commercially available platforms include an internal control (IC) to identify specimens containing inhibitors. Testing algorithms state that when the internal control fails, the results of a specimen without detectable CT and/or NG cannot be reported as negative due to the likelihood that amplification inhibitors are present in the specimen.

A new strain of C. trachomatis containing a 377 base pair deletion within its cryptic plasmid was described [24]. The deletion was found to be located within the target sequence of the Roche Cobas assay, resulting in false-negative results for this CT strain [24]. This would impact the epidemiologic surveillance data in areas where this strain circulated. Once identified, several manufacturers, including Roche, altered the design of their NAT, incorporating dual targets for CT detection.

The specificity for some NAT platforms is problematic due to cross-reactivity with certain nongonococcal species of Neisseria [21, 25, 26]. This problem was thought to arise from the intraspecies and interspecies genetic recombination that occurs between Neisseria species, which can result in false-positive NAT results with certain of the commensal Neisseria species [19]. Of eight nongonococcal species tested, false-positive results were seen with N. cinerea and N. lactamica using the BD ProbeTec assay, with N. subflava and N. cinerea using the Roche Cobas assay, and with N. lactamica, N. meningitis, N. cuniculi, and N. mucosa using the Qiagen assay. Since N. cinerea, N. lactamica, N. subflava, and N. sicca isolates have been recovered from genital mucosa, genital specimens may result in false-positive results as is also true for pharyngeal specimens. A recent study evaluated the cross-reactivity patterns of six NAT tests designed to detect N. gonorrhoeae to nongonococcal strains. The study clearly demonstrated cross-reactivity from all six NATs, with false-positive reactions for the 234 isolates tested ranging from 1–14.1 %. The Cobas amplicor and ProbeTec tests showed the highest number of false-positive results at 14.1 % and 11 %, respectively [21].

When selecting a testing platform, the prevalence of infection in the population being tested is of significance. The analytical performance of any test is dependent on the prevalence of infection, with the risk of generating false-positive results being inversely related to the prevalence. These issues have fueled the debate over the need for confirmatory testing for positive test results for CT and NG, as both are reportable infections, with the potential for psychosocial and/or medicolegal consequences for a false-positive or a true-positive result [27]. The newest 2010 CDC Guidelines for STI testing do not contain language recommending routine repeat testing of initial positive results [2]. NAT testing for CT/NG is the preferred diagnostic method of choice in evaluating adults and adolescents as victims of sexual assault [2].

The advent of NAT, with its exquisite sensitivity, has given birth to a whole new mind-set for cleanliness in the molecular testing laboratory. No longer is disinfection of the benchtop after a day’s work adequate. New standards strive to remove or prevent amplicon contamination in the laboratory environment. Strategies includes daily cleaning of laboratory surfaces with a dilute bleach solution, and frequently replacing disposable gloves and gowns while working in the pre- and post-amplification area(s). Create and enforce a regular schedule for performing wipe testing to monitor for amplicon contamination. For many of the current NAT platforms, pre- and post-amplification steps should be performed, if possible, in separate rooms with positive and negative airflow, respectively. Alternatively, contamination can be greatly reduced when using a fully automated, closed real-time NAT system that incorporates sample preparation, nucleic acid amplification, and detection in a single closed reaction.

In at-risk populations, it is important to be able to detect rectal and/or oropharyngeal CT/NG infections using a NAT platform. To date, manufacturers of NAT tests for CT/NG have not included rectal and/or oropharyngeal specimens in their evaluations or clinical trials. Therefore, these applications are not described in the manufacturer’s packet insert and must be validated by individual laboratories.

Reaching high-risk populations for STI screening will improve with policies that emphasize the use of noninvasively collected specimens, including self-collected vaginal swabs and first-void urine specimen [28]. This is especially true when attempting to reach the young adult patient population, age range from 14 – 24 years, where the prevalence of CT and NG infections is highest and the willingness to undergo a pelvic examination or urethral swab collection is lowest.

Testing for STIs using Pap smear specimens has become more common but only after critical issues were addressed that enabled liquid-based cytology samples to be used for STI testing [14]. One of the most important solutions developed was the concept of using a “pre-aliquot,” prior to handling of the specimen in cytology, which solved the issue of cross-contamination from other specimens, as well as the ethical issue of waiting to perform the infectious disease testing until after the cytology screening is completed. Successful implementation of this strategy was not easy and required dialogue and cooperation between laboratories, pathologists, manufacturers, and regulatory bodies.

Resistance to penicillin, tetracycline, and fluoroquinolones is now common among N. gonorrhoeae isolates [7, 8, 29]. Now emergence of cephalosporin-resistant N. gonorrhoeae has been described in several countries around the world. Being able to screen for antimicrobial resistance using a NAT platform would be very useful from a public health perspective.

Trichomonas vaginalis (TV) is a flagellated protozoan and the only species within its genus that can infect squamous epithelial cells of the human urogenital tract [30]. TV infection is considered to be a nonulcerative STI but is associated with severe local inflammation. In women, symptoms may include vulvar irritation and vaginal discharge, which appears frothy, mucopurulent, and yellow-green in color. During a TV infection, the vaginal pH is often abnormally elevated (pH > 4.5). Complications of TV infection in untreated women include endometritis, infertility, and cervical erosion. In men infected with TV, symptoms may include profuse purulent urethritis and a form of NGU, with complications including chronic prostatitis, urethral strictures, epididymitis, and/or infertility if the infection is untreated [31, 32].

Detection of TV infections in males has received far less attention than these infections in females. However, with the advent of NAT, this understudied infection in men has become more appreciated, and thus research studies have provided important information on its prevalence, clinical symptoms, sequelae, and the most appropriate specimen to collect for proper diagnosis. TV has a greater role in NGU than previously thought [33]. As with diagnosing TV infections in female patients, direct microscopic examination of urethral discharge has poor sensitivity for detecting TV in male patients.

Worldwide, TV infection accounts for approximately 276.4 million cases annually [34], which is greater than the number of cases of C. trachomatis, N. gonorrhoeae, and syphilis combined. In the USA, the annual incidence of TV infection is estimated to be approximately 8 million cases [2]. However, because TV infection is not a reportable disease, this number may be an underestimate. Unlike CT and NG, where prevalence is higher in adolescents and young adults, TV is more equally distributed among all age groups.

Detecting TV in an individual is considered by healthcare providers to be a red flag for high-risk sexual behavior and is frequently present along with other STIs in the same individual. Diagnosing TV is difficult, because 50–70 % of all infected individuals are asymptomatic. Without a sensitive assay, infected individuals left untreated continue to act as a reservoir for ongoing disease transmission within the community.

TV infection is associated with two important sequelae: (1) an increased risk of acquiring human immunodeficiency virus (HIV); and (2) an increased risk of perinatal morbidity and mortality [35–38]. HIV transmission is enhanced by the local inflammatory reaction containing CD4-positive T cells within the genital tract that is present with TV infections. In women, TV infection is strongly associated with an abnormal vaginal ecology. Harboring TV may contribute to the change in vaginal flora, which is associated with decreased lactic acid production and subsequent increase in vaginal pH. Lactic acid production and the normally low pH of the genital tract environment help to inactivate HIV. Therefore, a change in the vaginal environment to a less hostile environment promotes an increased survival of HIV. If this hypothesis is correct, then controlling TV infections could lower HIV acquisition.

The increased risk of perinatal morbidity and mortality with TV infection is associated with premature rupture of membranes, preterm delivery, and low-birth-weight infants in pregnant women infected with TV [39]. Although controversial, these associations suggest a need for increased efforts to detect and treat this infection in pregnant women [40].

Screening for TV infection is less common than screening for CT and NG infections; often the practice is limited to public health clinics and obstetrical practices. Successful control of TV infections would be aided greatly by increased screening of high-risk populations, performing contact follow-ups with sexual partners, and using a more sensitive diagnostic assay. Improved detection of TV by using NAT, as is the case for CT, would be predicted to reduce the incidence of TV infection and assist in reducing HIV transmission and possibly even poor pregnancy outcomes [41].

Historically, the most common diagnostic method for TV in urogenital discharge was direct microscopic examination, broth culturing, or both. Direct microscopic examination of genital discharge material on a slide is certainly the most rapid and inexpensive method to use, but lacks adequate sensitivity, which is reported to be approximately 40–70 % [42]. The low level of sensitivity with microscopic examination may be due in part to the rapid loss of the characteristic protozoan motility once the organism is removed from a 37 °C environment. Loss of motility is accompanied by change in morphology; non-motile TV organisms round up and are difficult to differentiate from leukocytes, being similar in size.

Currently, broth culturing is the gold standard for detecting TV [43]. Successful growth in culture can be achieved with as few as 300–500 TV organisms per milliliter of vaginal fluid, but culturing requires 2–7 days of incubation and daily microscopic examination. Culture methods have sensitivities that range between 50 – 80 % but require specialized medium such as Diamond’s broth, Tricosel medium, or the In-Pouch system (Biomed Diagnostics, Inc. White City, OR). These specialized media may not be available in the physician’s office. In addition, some TV isolates do not grow in culture due to strain requirements, low numbers of organisms, or damaged/nonviable organisms.