Abstract
Background
In the diagnosis of sexually transmitted infections, there has been a demand for multiple molecular assays to rapidly and simultaneously detect not only pathogens but also drug resistance-associated mutations.
Methods
In this study, we developed a new rapid simultaneous molecular assay for the detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium, and M. genitalium macrolide (23S rRNA gene, A2058/A2059) and fluoroquinolone (ParC gene, S83I) drug resistance-associated mutations in approximately 35 minutes. We evaluated the basic and prospective clinical performance of the newly developed assay.
Results
The newly developed assay showed sufficient sensitivity to detect N. gonorrhoeae, C. trachomatis, T. vaginalis, and M. genitalium relative to the reference method. In a prospective study comparing the reference method across 178 urine samples from men and women, the total concordance rate, sensitivity, and specificity of the two assays for N. gonorrhoeae detection were 98.9% (176/178), 97.9% (46/47), and 99.2% (130/131), respectively; for C. trachomatis detection, they were 98.3% (175/178), 96.4% (81/84), and 100% (94/94); and for M. genitalium detection, they were 100% (178/178), 100% (20/20), and 100% (158/158). All samples were negative for T. vaginalis. Of the 16 M. genitalium-positive samples analyzed for the GENECUBETM assay, 81.3% (13/16) had A2058/A2059 mutations, 31.3% (5/16) had S83I mutations, and 25.0% (4/16) had simultaneous mutations, which was highly correlated with the sequence analysis.
Conclusions
This study suggests that the recently developed assay performed similarly to existing nucleic acid amplification tests and enables rapid and simultaneous detection, including the detection of drug resistance-associated mutations.
Supplementary Information
The online version contains supplementary material available at 10.1007/s40291-024-00769-z.
Key Points
| We developed a new rapid simultaneous molecular assay for the detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, and Mycoplasma genitalium, including 23S rRNA gene (A2058/A2059) mutations related to macrolide resistance and a ParC gene (S83I) mutation related to fluoroquinolone resistance of M. genitalium. |
| The newly developed assay detected these pathogens at a total concordance rate of over 98% against a reference method and correctly identified macrolide and fluoroquinolone drug resistance-associated mutations in M. genitalium. |
Introduction
Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Trichomonas vaginalis, and Mycoplasma genitalium are the leading causes of sexually transmitted infections (STIs) worldwide, with more than 1 million infections occurring each day according to the World Health Organization [1]. Many STIs are generally asymptomatic in the early stages of infection, making it possible for people to be infected unknowingly, which contributes to the spread of infection [2]. In addition, some patients have been reported to be infected with multiple STIs simultaneously, such as N. gonorrhoeae and C. trachomatis co-infections, making appropriate treatment difficult [3]. Therefore, the accurate diagnosis of STIs is important to prevent their spread and to facilitate the administration of appropriate treatment.
Various methods are used for the diagnosis of STIs, including bacterial culture, direct microscope tests, serologic tests, direct immunofluorescence tests, and nucleic acid amplification tests (NAATs) [4]. Conventional methods, such as bacterial culture or direct immunofluorescence tests, are only applicable to the diagnosis of N. gonorrhoeae and C. trachomatis. Direct microscopic tests for T. vaginalis are not sensitive enough [5]. In comparison to these methods, NAATs have superior rapidity, sensitivity, and specificity, and have therefore become the new “gold standard” for the diagnosis of many STIs [5, 6]. In addition, in recent years, NAATs have evolved to be capable of detecting multiple STIs [7–9].
Despite advances in the diagnosis of STIs using NAATs, there is global concern that the prevalence of drug-resistant pathogens is increasing worldwide [5, 6]. Many of these drug resistances are related to genetic mutations and can be detected using molecular biology techniques [10–12]. However, as the diagnosis of drug-resistant STIs has not been adequately accomplished using NAATs, the examination of drug resistance-associated mutations relies on sequence analyses [13]. To determine appropriate treatment regimens, NAATs must be combined with sequence analyses, which makes the diagnosis of STIs a time-consuming process [6]. Therefore, for the diagnosis of STIs, there has been a demand for the development of molecular assays that can rapidly and simultaneously detect not only pathogens but also mutations associated with drug resistance.
GENECUBETM (TOYOBO Co., Ltd.) is an automated rapid molecular identification system based on a quenching probe (Q probe) polymerase chain reaction that can complete a measurement in approximately 25 minutes and analyze up to 24 samples simultaneously [14–21]. The Q probe method is known to be effective for detecting point mutations, and an assay approved as in vitro diagnostic has been developed using GENECUBETM to detect macrolide resistance mutations in Mycoplasma pneumonia. [17, 19]. In addition, GENECUBETM can simultaneously perform four assays including up to 16 targets. Thus, a GENECUBETM assay would be useful for comprehensive multiple testing to detect STI pathogens and drug resistance mutations.
In this study, we developed a new rapid simultaneous molecular assay for the detection of N. gonorrhoeae, C. trachomatis, T. vaginalis, and M. genitalium using GENECUBETM. In addition, in recent years, there has been concern about the increase in macrolide and fluoroquinolone antibiotic resistance in M. genitalium. [22, 23]. Therefore, we designed this assay to detect 23S rRNA gene (A2058/A2059) mutations related to macrolide resistance and a ParC gene (S83I) mutation related to fluoroquinolone resistance. The newly developed assay was evaluated using reference material, pooled negative urine samples, and synthetic oligonucleotides mimicking M. genitalium 23S rRNA and ParC genes. Furthermore, we evaluated the prospective clinical performance of the new rapid simultaneous molecular assay.
Methods
This study was performed at the University of Tsukuba Hospital, TOYOBO Co., Ltd., and LSI Medience in Japan. The study protocol was approved by the Ethics Committee of the University of Tsukuba Hospital (approval number: R03-229). All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained from the patients in the study.
Simultaneous Rapid Molecular Assay of Sexually Transmitted Infections
We developed a simultaneous rapid molecular assay for STIs using GENECUBETM including the C. trachomatis/N. gonorrhoeae simultaneous detection assay, the T. vaginalis/M. genitalium simultaneous detection assay, and the M. genitalium drug resistance-associated mutation detection assay. In GENECUBETM, these assays can be performed simultaneously and it takes approximately 25 minutes from the preparation of reaction mixtures to the detection of target genes [14–21]. The detection targets for each assay were as follows: multiple copies of C. trachomatis cryptic plasmid (accession no. X06707.3) and a single copy of the porB gene (accession no. DQ064317.1), multiple copies of N. gonorrhoeae opa genes (Accession No. X52365.1), multiple copies of T. vaginalis 5.8S rRNA genes (Accession No. EU215366.1), multiple copies of M. genitalium mgpa genes (Accession No. EF117298.1), and a single copy of the M. genitalium 23S rRNA gene (Accession No. NR_077054). The analysis of a single copy of the ParC (Accession No. PP910731.1) gene for M. genitalium drug resistance-associated mutations is shown in Fig. 1. The assay contained internal controls to confirm the success of the examination.
Fig. 1.
Diagram of the developed simultaneous rapid molecular assay for sexually transmitted infections
In the non-purified direct method, which was completed in approximately 5 minutes, the sample was concentrated by centrifugation at 13,000 ×g and resuspended in 50 μL of GENECUBETM lysis buffer (TOYOBO Co., Ltd.). For the automated purification method, samples were subjected to nucleic acid purification using magLEAD (Precision System Science Co., Ltd., Chiba, Japan) for approximately 10 minutes.
Estimation of the Limit of Detection for the GENECUBETM Assay with Reference Material and Pooled Negative Urine Samples
To estimate the limitation of detection (LOD) for the GENECUBETM assay, we prepared four different concentrations of samples (30 copies/mL, 10 copies/mL, 3 copies/mL, and 1 copies/mL) using reference materials for N. gonorrhoeae (ATCC Neisseria gonorrhoeae (Zopf) Trevisan 19424™), C. trachomatis (ZeptMetrix® NATtrol C. trachomatis serotype D, External Run Control Medium), T. vaginalis (ATCC Trichomonas vaginalis Donne 30238™), and M. genitalium (ATCC Mycoplasma genitalium Tully et al. 33530™), and matrix (purified water and three negative pooled urine samples). The GENECUBETM assay was performed 20 times, with five replicates for each matrix. Similarly, each matrix was evaluated for the cobas® 6800/8800 System CT/NG (Roche Diagnostics, Indianapolis, IN, USA) and cobas® TV/MG. The LOD was determined as the concentration at which the detection rate was 95%.
Evaluation of M. genitalium Drug Resistance-Associated Mutation Detection Assay with GENECUBETM for the Detection of Synthetic Oligonucleotides
Synthetic oligonucleotides mimicking the domain V region of the M. genitalium 23S rRNA gene and M. genitalium ParC gene were obtained from Thermo Fisher Scientific (Tokyo, Japan). The oligonucleotide for the wild type was designed according to the sequence of M. genitalium G37 (accession no. L43967). The 23S rRNA gene mutations were made to include A2058G, A2058C, A2058T, A2059G, A2059C, and A2059T. ParC was designed to include G241T, A247C, A247T, G248T (S83I), G248A, T249A, G259A, G259C, and G259T mutations.
Study Design for the Analysis of Clinical Samples
Urine samples were prospectively collected from patients with suspected STIs from 20 March, 2023 to 17 May, 2023, and from 3 October, 2023 to 9 February, 2024. The clinical samples were transported under cool conditions (2–8 ℃) to the University of Tsukuba Hospital on the same day or the next business day for the evaluation of simultaneous rapid molecular assays using GENECUBETM. Afterwards, all samples were temporarily stored at approximately −80 °C and tested using the cobas® 6800/8800 System CT/NG (Roche Diagnostics) and cobas® 6800/8800 System TV/MG (Roche Diagnostics). The total concordance rate, sensitivity, and specificity were calculated from the results of the GENECUBETM and cobas® assays. Positive and negative controls were examined for each assay. Mycoplasma genitalium-positive samples were transferred to TOYOBO Co., Ltd. for sequencing.
Sequence Analysis of the MG 23S rRNA Gene and ParC Gene
For the M. genitalium-positive samples, amplification for the sequence analysis of the 23S rRNA gene and ParC gene of M. genitalium was performed at TOYOBO Co., Ltd. After amplification, the samples were transported to Eurofins Genomics KK (Tokyo, Japan) at approximately −20 °C for the sequence analysis. The obtained results were analyzed using the CLC Genomics Workbench version 12.0.1 (Qiagen NV, Venlo, Netherlands) and compared to the registered sequence of M. genitalium G37 (GenBank: L43967). The details of the sequence analysis conditions and polymerase chain reaction primers are described in the Electronic Supplementary Material (ESM) [24].
Statistical Analysis
Detection rates with 95% confidence intervals were calculated using the Clopper and Pearson method. Calculations were conducted using IBM SPSS Statistics version 29.0.2.0 (IBM Corp., Armonk, NY, USA).
Results
Evaluation of the Limit of Detection of the GENECUBETM Assay with Pooled Urine Samples Containing Each Reference Material (N. gonorrhoeae, C. trachomatis, T. vaginalis, M. genitalium)
A summary of the LOD evaluation for the GENECUBETM assay with pooled urine samples containing each reference material is shown in Table 1, and the details are shown in Table 1 of the ESM. The LOD of the newly developed GENECUBETM assay for pathogen detection was 3 colony-forming units/mL for N. gonorrhoeae, 10 copies/mL for C. trachomatis, 3 cells/mL for T. vaginalis, and 3 bacteria/mL for M. genitalium. Furthermore, the LOD of the GENECUBETM assay for the detection of M. genitalium mutations was 30 bacteria/mL for M. genitalium. In comparison, the LOD of the reference cobas® 6800/8800 system CT/NG and cobas® TV/MG were 3 colony-forming units/mL for N. gonorrhoeae, 3 copies/mL for C. trachomatis, 10 cells/mL for T. vaginalis, and 3 bacteria/mL for M. genitalium.
Table 1.
Summary of the estimated limit of detection test results
| (a) Summary of the estimated limit of detection test results for Neisseria gonorrhoeae | |||
|---|---|---|---|
| Concentration of sample (colony-forming unit/mL) | GENECUBETM | cobas® 6800/8800 system CT/NG | |
| (non-purified direct method) | (automated purification method) | ||
| N of detection/N of test, detection rate, % (95% confidence interval) | |||
| 30 | 20/20, 100 (83–100) | 20/20, 100 (83–100) | 19/20, 95 (75–100) |
| 10 | 20/20, 100 (83–100) | 20/20, 100 (83–100) | 20/20, 100 (83–100) |
| 3 | 20/20, 100 (83–100) | 19/20, 95 (75–100) | 20/20, 100 (83–100) |
| 1 | 4/20, 20 (6–44) | 2/20, 20 (1–32) | 5/20, 25 (9–49) |
| (b) Summary of the estimated limit of detection test results for Chlamydia trachomatis | |||
|---|---|---|---|
| Concentration of sample (copies/mL) | GENECUBETM | cobas® 6800/8800 system CT/NG | |
| (non-purified direct method) | (automated purification method) | ||
| N of detection/N of test, detection rate, % (95% confidence interval) | |||
| 30 | 20/20, 100 (83–100) | 20/20, 100 (83–100) | 20/20, 100 (83–100) |
| 10 | 19/20, 95 (75–100) | 12/20, 60 (36–81) | 20/20, 100 (83–100) |
| 3 | 12/20, 60 (36–81) | 9/20, 45 (23–69) | 20/20, 100 (83–100) |
| 1 | 5/20, 25 (9–49) | 5/20, 25 (9–49) | 18/20, 90 (68–99) |
| (c) Summary of the estimated limit of detection test results for Trichomonas vaginalis | |||
|---|---|---|---|
| Concentration of sample (cells/mL) | GENECUBETM | cobas® TV/MG | |
| (non-purified direct method) | (automated purification method) | ||
| N of detection/N of test, detection rate, % (95% confidence interval) | |||
| 30 | 20/20, 100 (83–100) | 20/20, 100 (83–100) | 20/20, 100 (83–100) |
| 10 | 20/20, 100 (83–100) | 19/20, 95 (75–100) | 20/20, 100 (83–100) |
| 3 | 20/20, 100 (83–100) | 18/20, 90 (68–99) | 18/20, 90 (68–99) |
| 1 | 18/20, 90 (68–99) | 11/20, 55 (32–77) | 15/20, 65 (51–91) |
| (d) Summary of the estimated limit of detection test results for Mycoplasma genitalium | |||
|---|---|---|---|
| Concentration of sample (bacteria/mL) | GENECUBETM | cobas® TV/MG | |
| (non-purified direct method) | (automated purification method) | ||
| N of detection/N of test, detection rate, % (95% confidence interval) | |||
| 30 | 20/20, 100 (83–100) | 20/20, 100 (83–100) | 20/20, 100 (83–100) |
| 10 | 19/20, 95 (75–100) | 20/20, 100 (83–100) | 20/20, 100 (83–100) |
| 3 | 20/20, 100 (83–100) | 20/20, 100 (83–100) | 20/20, 100 (83–100) |
| 1 | 9/20, 45 (23–69) | 14/20, 70 (46–88) | 16/20, 80 (56–94) |
| (e) Summary of the estimated limit of detection test results for the detection of Mycoplasma genitalium drug-resistance mutations | ||||
|---|---|---|---|---|
| 23S rRNA gene, A2058/A2059 | ParC gene, S83I | |||
| Concentration of sample (bacteria/mL) | GENECUBETM (non-purified direct method) |
(automated purification method) | GENECUBETM (non-purified direct method) |
(automated purification method) |
| N of detection/N of test, detection rate, % (95% confidence interval) | ||||
| 30 | 20/20, 100 (83–100) | 20/20, 100 (83–100) | 20/20, 100 (83–100) | 20/20, 100 (83–100) |
| 10 | 14/20, 70 (46–88) | 20/20, 100 (83–100) | 17/20, 85 (62–97) | 20/20, 100 (83–100) |
| 3 | 18/20, 90 (68–99) | 18/20, 90 (68–99) | 18/20, 90 (68–-99) | 20/20, 100 (83–100) |
| 1 | 4/20, 20 (6–44) | 2/20, 10 (1–32) | 4/20, 20 (6–44) | 4/20, 20 (6–44) |
A wild-type reference material was used. GENECUBETM indicated detection of M. genitalium drug resistance-associated mutations. The 95% confidence intervals were calculated using the Clopper and Pearson methods
GENECUBETM indicated simultaneous detection of Chlamydia trachomatis/N. gonorrhoeae. The 95% confidence intervals were calculated using the Clopper and Pearson methods
GENECUBETM indicated simultaneous detection of C. trachomatis/Neisseria gonorrhoeae. The 95% confidence intervals were calculated using the Clopper and Pearson methods
GENECUBETM indicated simultaneous detection of T. vaginalis and Mycoplasma genitalium. The 95% confidence intervals were calculated using the Clopper and Pearson methods
GENECUBETM indicated simultaneous detection of Trichomonas vaginalis and M. genitalium. The 95% confidence intervals were calculated using the Clopper and Pearson methods
Identification of M. genitalium Macrolide and Fluoroquinolone Drug Resistance-Associated Mutation Wild Type and Mutant Types of Synthetic Oligonucleotides with GENECUBETM
In the GENECUBETM assay for the detection of M. genitalium drug resistance-associated mutations, the synthetic oligonucleotides mimicking the 23S rRNA wild type and the ParC wild type were all judged to have no mutations, and the synthetic oligos containing the 23S rRNA A2058/A2059 mutant and ParC S83I were all judged to be mutant type. All synthetic oligonucleotides mimicking other ParC mutations were found to have no mutations. The fluorescence peak temperatures of the wild type and mutant types of 23S rRNA and ParC genes are shown in Fig. 2a, b. For the 23S rRNA gene, the fluorescence peak detection temperature of the mutant ranged from 45.2 to 52.5 °C, while that of the wild type was 58.3 °C (Table 2a of the ESM). For the ParC gene, the fluorescence peak detection temperature for the S83I mutation (G248T) was 61.2 °C, while those of the others ranged from 47.2 °C to 55.8 °C (Table 2b of the ESM).
Fig. 2.
a Representative fluorescence curves of the 23S rRNA gene (Mycoplasma genitalium macrolide resistance-associated mutation) wild-type and mutant-type oligonucleotides. Synthetic oligonucleotides mimicking the Mycoplasma genitalium 23S rRNA gene were analyzed for M. genitalium resistance-associated mutations using GENECUBETM. Amplified products were judged as wild-type (54.4–65.0 °C) or mutant-type (42.0–54.3 °C) according to the fluorescence peak temperature. b Representative fluorescence curves of the ParC gene S83I (G248T) mutation (Mycoplasma genitalium fluoroquinolone resistance-associated mutation) and other oligonucleotides. Synthetic oligonucleotides mimicking Mycoplasma genitalium ParC gene were analyzed for M. genitalium resistance-associated mutations using GENECUBETM. Amplified products were judged as S83I mutations (57.1–66.0 °C) or others (44.0–57.0 °C) according to the fluorescence peak temperature
Comparison Between the GENECUBETM Assay and the cobas® Assay Using Clinical Urine Samples
In this study, we prospectively evaluated the GENECUBETM assay using 178 fresh urine samples from patients with suspected STIs. Of the 178 samples, 47 were positive for N. gonorrhoeae, 84 were positive for C. trachomatis, 20 were positive for M. genitalium, and all samples were negative for T. vaginalis in the cobas® assay. For comparison of the GENECUBETM assay for pathogen detection with the cobas® assay, the total concordance, sensitivity, and specificity of the two assays were 98.9% (176/178), 97.9% (46/47), and 99.2% (130/131), respectively, for N. gonorrhoeae, 98.3% (175/178), 96.4% (81/84), and 100% (94/94) for C. trachomatis, 100% (178/178), 100% (20/20), and 100% (158/158) for M. genitalium (Table 2). All samples were negative for T. vaginalis in both assays.
Table 2.
Concordance rate of the GENECUBETM assay and cobas® CT/NG for urine samples obtained from patients with suspected sexually transmitted infections
| (a) Concordance rate of the GENECUBETM assay and cobas® CT/NG for Neisseria gonorrhoeae | |||
|---|---|---|---|
| Positive | Negative | ||
| Non-purified direct method GENECUBETM (urine sample) | |||
| Positive | 46 | 1 | |
| Negative | 1 | 130 | |
| Sensitivity (%) | 97.9 (88.7–99.9) | ||
| Specificity (%) | 99.2 (95.8–100) | ||
| Total concordance rate (%) | 98.9 (96.0–99.9) | ||
| (b) Concordance rate of the GENECUBETM assay and cobas® CT/NG for Chlamydia trachomatis | ||
|---|---|---|
| cobas® 6800/8800 system CT/NG | ||
| Positive | Negative | |
| Non-purified direct method GENECUBETM (urine sample) | ||
| Positive | 81 | 0 |
| Negative | 3 | 94 |
| Sensitivity (%) | 96.4 (89.9–99.3) | |
| Specificity (%) | 100 (96.2–100) | |
| Total concordance rate (%) | 98.3 (95.2–99.7) | |
| (c) Concordance rate of the GENECUBETM assay and cobas® TV/MG for Mycoplasma genitalium | ||
|---|---|---|
| cobas® TV/MG | ||
| Positive | Negative | |
| Non-purified direct method GENECUBETM (urine sample) | ||
| Positive | 20 | 0 |
| Negative | 0 | 158 |
| Sensitivity (%) | 100 (83.2–100) | |
| Specificity (%) | 100 (97.7–100) | |
| Total concordance rate (%) | 100 (97.9–100) | |
The data in parentheses are 95% confidence intervals. GENECUBETM indicated the simultaneous detection of Chlamydia trachomatis/N. gonorrhoeae
The data in parentheses are 95% confidence intervals. GENECUBETM indicated the simultaneous detection of C. trachomatis/Neisseria gonorrhoeae
The data in parentheses are 95% confidence intervals. GENECUBETM indicated the simultaneous detection of Trichomonas vaginalis and M. genitalium
Comparison of the GENECUBETM Assay and Sequence Analysis in Detecting Drug Resistance-Associated Mutations for M. genitalium
In the 20 M. genitalium-positive samples in the GENECUBETM assay for pathogen detection and the cobas® assay, 80% (16/20) were analyzed by the GENECUBETM assay for detection of M. genitalium mutations and 70% (14/20) by a sequence analysis. Of the 16 samples in the GENECUBETM assay for the detection of M. genitalium mutations, A2058/A2059 mutations were observed in 81.3% (13/16), S83I mutations were observed in 31.3% (5/16), and both mutations were observed in 25.0% (4/16), as shown in Fig. 3. In the comparison of the GENECUBETM assay and the sequence analysis, 15% (3/20) could only be analyzed by the GENECUBETM assay, 5% (1/20) could only be analyzed by the sequence analysis, and 70% (14/20) could be analyzed by both assays. Of the 14 samples analyzed by both assays, the results of the GENECUBETM assay and sequence analysis for mutations were concordant, except for one sample (Table 3 of the ESM). Samples in which mutations other than the S83I mutation were detected by the sequence analysis were detected as “other” by the GENECUBE™ assay. In the discordant sample, the GENECUBETM assay detected both wild-type and A2058/A2059 mutations, although the sequence analysis detected only wild-type A2058/A2059. Further analysis by DNA cloning showed that the discordant sample contained a mixture of wild-type and mutant types.
Fig. 3.
Results of Mycoplasma genitalium resistance-associated mutation detection assays using GENECUBETM. 23S rRNA A2058/A2059 mutations are related to macrolide resistance and ParC gene (S83I) mutations are related to fluoroquinolone resistance. ※In one of the 13 samples, A2058/A2059 wild type and mutant type were detected by the GENECUBETM assay
Discussion
This is the first study to evaluate the performance of a new rapid simultaneous molecular assay for the detection of N. gonorrhoeae, C. trachomatis, T. vaginalis, M. genitalium, and M. genitalium drug resistance-associated mutations using GENECUBETM. In this study, the GENECUBETM assay had equivalent performance to current NAATs and could simultaneously detect these targets in approximately 30 minutes with a non-purified direct method or 35 minutes with automated purification methods. In clinical practice, NAATs are considered as the gold standard for diagnosing STIs, and the cobas® assay, Aptima Combo 2® assay (Hologic Inc., San Diego, CA, USA), and Alinity® m STI assay (Abbott Laboratories, Des Plaines, IL, USA) are approved in vitro diagnostics and commercially available worldwide [5, 6]. The cobas® assay can detect N. gonorrhoeae and C. trachomatis, T. vaginalis, and M. genitalium simultaneously; however, separate assays are required. The Aptima Combo 2® assay (Hologic Inc., San Diego, CA, USA) and the Alinity® m STI assay (Abbott Laboratories) can simultaneously detect these four pathogens [9, 25]. These NAATs cannot detect drug resistance-associated mutations simultaneously, and additional examinations such as sequence analyses are required to evaluate drug resistance. In addition, these assays are not sufficiently rapid because the turnaround time is approximately 3 hours to detect each for N. gonorrhoeae/C. trachomatis or 3 hours for T. vaginalis/M. genitalium, with the cobas® assay, approximately 3.5 hours with the Aptima Combo 2® assay, and approximately 2 hours with the Alinity® m STI assay [9, 26].
In this study, the GENECUBETM assay showed a high correlation with the approved in vitro diagnostic, the cobas® assay for N. gonorrhoeae, C. trachomatis, and M. genitalium in the prospective study and sufficient detection sensitivity to the cobas® assay for T. vaginalis in the LOD evaluation. Although the LOD of the GENECUBETM assay for C. trachomatis (10 copies/mL) appeared slightly inferior to that of the cobas® assay (3 copies/mL), the GENECUBETM assay demonstrated a sensitivity of 96.4% (81/84) in detecting C. trachomatis compared to the cobas® assays in the prospective study. In addition, it has been reported that >99% of clinical urine samples contain C. trachomatis at more than 10 copies/mL [27]. Therefore, the difference in sensitivity between the GENECUBETM assay and cobas® assay for detecting C. trachomatis is not expected to be a clinical problem.
Recently, drug resistance rate against macrolide antibiotics in M. genitalium was reported to be 40–90% and has become a clinical concern. The US Centers for Disease Control and Prevention recommended sequencing of the A2058/A2059 mutation of the 23S rRNA gene, which contributes to macrolide resistance [6, 22, 23, 28–37]. Furthermore, drug resistance against fluoroquinolone antibiotics in M. genitalium is increasing and is reported to be 10–40%, while the S83I mutation of ParC has been reported to be the main cause of drug resistance [6, 22, 23, 28–38]. It has also been reported that the success rate of moxifloxacin treatment in patients with M. genitalium without the ParC S83I mutation is >95%, but while it is <50% in patients with M. genitalium with the ParC S83I mutation [35]. In a prospective study, the results of the GENECUBETM assay for 23S rRNA A2058/A2059 and ParC S83I mutations were highly correlated with the sequence analysis and were consistent with the mutation rates shown in previous studies. The results of this prospective study suggest that the GENECUBETM assay may be clinically useful for detecting drug resistance-associated mutations in M. genitalium.
The present study was associated with several limitations. First, because this prospective study did not include T. vaginalis-positive samples, further evaluation for detecting T. vaginalis in clinical samples is required. In addition, this prospective study was limited to urine samples; evaluation using samples such as cervical, vaginal, and oropharyngeal swabs is necessary. Second, because the sample size was limited for detecting M. genitalium drug resistance-associated mutations, further studies using larger sample sizes are required to evaluate the accuracy of the GENECUBETM assay. This was a single-center study, and the universality of M. genitalium drug resistance-associated mutations needs to be evaluated in a multicenter study. Third, the clinical efficacy of the GENECUBETM assay has not been verified, and further evaluation of its clinical efficacy and the performance is needed.
Conclusions
Our evaluation indicated that the new rapid simultaneous molecular assay using non-purified direct samples has sufficient performance to detect N. gonorrhoeae, C. trachomatis, T. vaginalis, and M. genitalium including the detection of 23S rRNA A2058/A2059 mutations related to macrolide resistance and ParC gene (S83I) mutations related to fluoroquinolone resistance in M. genitalium.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
We are very grateful to Ms. Asami Nakayama, Ms. Yoshiko Uekura, Dr. Yosuke Kawashima, and Mr. Hiroaki Nishiguchi for their support in this study.
Declarations
Funding
This study was financially supported by TOYOBO Co., Ltd.
Conflicts of Interest/Competing Interests
Masashi Michibuchi, Yoshikane Takafumi, Yuma Matsuba, and Tomomi Yamazaki are salaried employees of TOYOBO Co., Ltd. Hiromichi Suzuki received a lecture and advisory fee from TOYOBO Co., Ltd.
Ethics Approval
This study was performed in accordance with the relevant guidelines and regulations. The study protocol was approved by the Ethics Committee of the University of Tsukuba Hospital (approval number: R03-229).
Consent to Participate
Written informed consent was obtained from the patients in the study.
Consent for Publication
Not applicable.
Availability of Data and Material
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available because they contain information that could compromise research participant. privacy/consent. Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. All data generated or analyzed during this study are included in this published article.
Code Availability
Not applicable.
Authors’ Contributions
MM, YT, TY, MT, TO, and HS conceived of and designed the study. MM, YT, YM, and SH performed the experiments. MM and SH analyzed the data. MM, YT, TY, MT, TO, and HS wrote the manuscript.
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