Advantages of Syndromic Diagnostics: Detection of the Pathogens Causing Urethritis/Cervicitis with the STI CNM Real-Time PCR Kit from Vitro S.A.

Two commercial RT-PCR kits were compared for the detection of C. trachomatis, N. gonorrhoeae, and M. genitalium, based on the criteria of the Clinical and Laboratory Standards Institute (CLSI 2008).

The study was conducted at the Infanta Elena Hospital in Huelva, Andalusia, Spain, in 2023. A total of 200 samples were collected from different anatomical sites from patients attending our hospital’s infectious diseases clinic for the determination of C. trachomatis, N. gonorrhoeae, and M. genitalium. The samples were tested simultaneously using the different methods described.

The determinations were performed with the Xpert^® CT/NG Kit; the commercial STI CNM Real-Time PCR Kit, determined on a QuantStudio^™ 5 Real-Time PCR System (Applied Biosystems^™, Thermo Fisher Scientific, Inc., USA) after extraction on a Nextractor^® NX-48S (Genolution Inc., Republic of Korea); and the EasyNAT^® MG on an EasyNAT^® 2.0 device. The primers and probes of the Xpert^® CT/NG Kit recognize chromosomal sequences of C. trachomatis (CT1: one target) and N. gonorrhoeae (NG2 and NG4: two different targets). The STI CNM Real-Time PCR Kit uses fluorescent primers and probes for the target genes Opal (N. gonorrhoeae), cryptic plasmid (C. trachomatis) and MgPa (M. genitalium). The Xpert^® CT/NG Kit measures C. trachomatis and N. gonor-rhoeae in a single step, while the STI CNM Real-Time PCR Kit also measures M. genitalium. The EasyNAT^® MG only measures M. genitalium in a single step.

All tests were performed and analyzed according to the manufacturer’s instructions. Relevant guidelines and regulations were followed for all methods. Hospital Infanta Elena Ethics Committee waived the need for ethical approval and informed consent.

Cohen’s kappa coefficient and Spearman’s correlation were used for the statistical analyses to assess the agreement level. Diagnostic test efficiency indicators (sensitivity, specificity, positive and negative predictive values) were also utilized. These indicators were introduced by Yerushalmy (1947) and are widely used today to evaluate the efficiency of a diagnostic test compared to a reference standard.

A total of 200 samples were collected from 90 patients (14 women and 76 men), whose ages ranged from 18 to 79, with a mean age of 35 and a median age of 37 (Table SI).

Below are the results obtained with the different diagnostic techniques. Table I shows the results obtained with the Xpert^® CT/NG system for C. trachomatis and N. gonorrhoeae, a method of proven reliability (Xie et al. 2020; Cordioli et al. 2024). We consider this method to be the reference method as it has been recommended by the WHO (Jacobsson et al. 2018). In addition, the results for M. genitalium obtained with the EasyNAT^® MG are shown.

A total of 41 samples (20.5%) tested positive. 8% were positive for C. trachomatis, 3.5% for N. gonorrhoeae and 9.5% for M. genitalium. One sample was positive for both C. trachomatis and M. genitalium. Regarding anatomical localization, C. trachomatis showed a higher prevalence in urethral and rectal swabs, while N. gonorrhoeae was more common in pharyngeal swabs. The remarkably high positivity rate (100%) for M. genitalium in urine is surprising and can be explained by the fact that in our hospital, this sample is only sent to the laboratory in patients with a high suspicion of persistent urethritis after C. trachomatis/N. gonorrhoeae had previously been excluded. Only one sample (C. trachomatis) tested positive in the endocervical smear, although the small number of samples of this type analyzed does not allow for conclusive results.

Comparative results (Xpert^® CT/NG vs. STI CNM Real-Time PCR Kit). Table II is a concordance table between the two techniques. As shown, the agreement between the two is almost complete, both for positive and negative samples. The only positive sample that the STI CNM Real Time PCR Kit failed to detect tested positive.

Comparative results (EasyNAT^® MG vs. STI CNM Real-Time PCR Kit). Table III shows the results of the comparison between the two techniques used to detect M. genitalium. As shown, there is complete agreement between the two.

Ever since the American CDC recommended it in 2014 (Papp et al. 2014), nucleic acid amplification techniques have gained significant popularity in STI diagnostics. These techniques are favored due to their high sensitivity, specificity, and quick turnaround time for results. Over the years, several diagnostic kits have been introduced, improving the accuracy of STI diagnosis, streamlining laboratory processes, and enhancing the management of these infections. Currently, numerous studies validate the diagnostic efficacy of many of these tools (Bristow et al. 2017; Levy et al. 2019; Herrmann and Malm 2021; Tuddenham et al. 2022. These studies consistently demonstrate sensitivity and specificity results that are sufficiently high to warrant their routine use, replacing traditional culture and serology-based systems.

The efficacy of molecular techniques for diagnosing CT and NG has been extensively tested, and numerous commercial solutions offer high sensitivity and specificity both in the genital (Papp et al. 2014; Caruso et al. 2021) and extragenital infection (Cosentino et al. 2017; Cordioli et al. 2024). Our study aligns with this growing trend by showcasing that the STI CNM Real-Time PCR Kit from Vitro S.A. exhibits comparable sensitivity and specificity to other widely used techniques, such as the Xpert^® CT/NG Kit, for diagnosing C. trachomatis and N. gonorrhoeae (Xie et al. 2020; Cordioli et al. 2024).

M. genitalium is accountable for a significant proportion of non-chlamydial and non-gonococcal urethritis in men, ranging from 10% to 35%, and cervicitis in women, ranging from 10% to 25% (Horner et al. 2019; Gnanadurai and Fifer 2020). In the past, identifying infections caused by this microorganism was challenging due to the difficulties associated with isolating it in culture. However, with the introduction of molecular techniques, the diagnosis of M. genitalium infections has improved. The European Academy of Dermatology and Venereology now recommends nucleic acid amplification techniques as the most effective diagnostic method and emphasizes the importance of considering this microorganism in the differential diagnosis of urethritis and cervicitis (Jensen et al. 2022). Despite this recommendation, limited commercial techniques are available that include M. genitalium in their diagnostic panels.

Nevertheless, the approval of the first molecular technique for its detection by the FDA (Shipitsyna and Unemo 2020) has led to several studies demonstrating the high sensitivity and specificity of these techniques (Lee et al. 2012; Le Roy et al. 2012; 2014; Vandepitte et al. 2014; Kirkconnell et al. 2019; Shipitsyna and Unemo 2020; Salazar and García 2022). In our study, we compared the STI CNM Real-Time PCR Kit from Vitro S.A. with the EasyNAT^® MG from Ustar Biotechnologies (Hangzhou) Ltd. and found a strong agreement between both techniques, as well as high sensitivity and specificity. Therefore, like the recommendations for C. trachomatis and N. gonorrhoeae, we support the routine use of the STI CNM Real-Time PCR Kit in clinical laboratories.

However, the leading interest for the clinical laboratory in the STI assay from Vitro S.A. is not solely based on its commendable sensitivity and specificity. Instead, its value lies in identifying the three primary causative agents of urethritis/cervicitis through a single multiplex PCR reaction. This feature makes it an ideal candidate for integration into syndromic diagnostic strategies, which have proven to be cost-effective by enhancing patient management, optimizing antibiotic usage, preventing new infections, and streamlining overall laboratory operations (Ramanan et al. 2017; Dumkow et al. 2021; Karellis et al. 2022). Furthermore, in our laboratory, the implementation of this kit has resulted in a remarkable 76.5% reduction in economic costs for these analyses, reducing the expense from €68 to a mere €16 per test, which underscores its potential for improving cost-effectiveness in STI diagnostics.

The main limitation of our study was that it was carried out on routine clinical samples received in our laboratory without any specific recruitment of patients beyond selecting those with a suspected diagnosis of STI. This also meant that we did not reach a sufficiently high number of samples of each type analyzed. Therefore, although our results are in line with those of previous studies, which indicate that there are no significant differences in sensitivity or specificity based on the anatomical location of the sample (Bristow et al. 2017; Jansen et al. 2020; Herrmann and Malm 2021), it is essential to exercise caution when concluding due to the limited number of positive cases in specific anatomical locations.

To summarize, the STI CNM Real-Time PCR Kit developed by Vitro S.A. proves to be extremely valuable in identifying N. gonorrhoeae, C. trachomatis, and M. genitalium in various clinical samples, including urine, urethral, rectal, vaginal, endocervical and pharyngeal swabs. One of its key advantages is adopting a syndromic diagnostic approach, streamlining the sample processing procedure in the laboratory, expediting result delivery, and optimizing resource utilization. With its remarkable sensitivity and specificity, this kit is highly recommended for routine use in clinical laboratories as it consistently provides reliable results, thereby facilitating the effective management of sexually transmitted infections. Moreover, the kit’s capability to simultaneously detect the three primary causative agents of ure-thritis/cervicitis through a single multiplex PCR reaction, coupled with the substantial reduction in economic costs observed in our laboratory, further underscores its practicality and convenience in the clinical setting.