2.1 Ethical approval
The study protocol was established according to the ethical guidelines of the Helsinki Declaration and approved by the Human Ethics Committee of the People's Hospital of Guangxi Autonomous Region (V4.1 20190813). Written informed consent was obtained from each individual in this study.
2.2 Clinical samples and sample size estimation
Vaginal, cervical, urethral, and other genital tract swabs (e.g., prostatic secretions and warts) were collected consecutively from the People's Hospital of Guangxi Autonomous Region between 2019 and 2022. Individuals were not exposed to urogenital medication for 24 hours before collection. Specimen collection was carried out according to genital specimen collection procedures. The aim of the sample selection process was to enroll: (a) specimens from asymptomatic cases who came for a check-up, or for personal motivation, or had no symptoms of sexually transmitted diseases (STDs); (b) specimens from symptomatic patients who had suspected symptoms. The detailed clinical diagnosis and positive findings for other STI pathogens were recorded (Table 1). A comparative study flowchart was available in Figure 1.
Assuming a sensitivity (Se) of 85% and specificity (Sp) of 95%, we required 215 definite Ureaplasma positive cases to yield 90% power with a set to 5%. Estimating a 45% prevalence of confirmed Ureaplasma, derived from preliminary investigation, an aim to collect roughly 500 samples was conceived.
2.3 DNA extraction
DNA was extracted using DNA extraction kit (Repodx, Jiangsu, China). 50 µL of nucleic acid extraction solution was added to the 1.5 ml tube containing the precipitate, mixed vigorously on a vortex shaker, then placed in a dry block thermostat at 85±5°C for 10 minutes, followed by centrifugation at 12,000rpm for 2 minutes. The supernatant could be used directly for the PCR reaction or stored frozen at -20°C until testing (frozen for no more than 3 months; thawed only once and did not refreeze). For use, the frozen DNA must be thawed completely, mixed and centrifuged immediately, and the supernatant could be used for the PCR reaction. Prepared DNA samples were stored at -80 °C.
2.4 Clinical specimens detected by test system
All enrolled samples were tested for UU and UP species using evaluated commercial UU/UP detection PCR kit (Repodx, Jiangsu, China), with a multiplex fluorescent probe-based TaqMan real-time PCR assay system. Specific UU genes (serovars 2, 4, 5, and 7-13), UP genes (serovars 1, 3, 6, and 14), and endogenous internal control (IC, a highly conserved region of the human GAPDH gene) were detected in one reaction. The IC was used to control the whole assay workflow efficiency from specimen collection, DNA extraction, and amplification, to data analysis. Positive control (recombinant plasmid DNA of UU, UP, and human-GAPDH, 5×103 copies/μl, each), and negative control (5×103 copies/μl human-GAPDH plasmid DNA) for specimen processing were included in each run to monitor DNA extraction and PCR inhibition. PCR reactions were performed in a total volume of 50 μL (45 μL PCR reaction mixture and 5 μL template DNA). Cycle parameters were set as follows: 37°C for 5 min → 95°C for 5 min → (95°C for 10 s, 60°C for 60 s, 45 cycles).
2.5 Clinical specimens detected by reference system
Clinical diagnosis of UU and UP infection was not relied on the non-specific symptoms, underscoring the necessity for diagnostic accuracy criteria. According to EP12-A2 [21], the sequencing method known as the "gold standard" for genetic diagnosis is employed as a reference system (comparator assay) for diagnostic accuracy. Concurrently, an agreement measurement between the sequencing method and Repodx PCR kit was performed in line with EP12-A2.
The conserved regions within the UUR10_0680 gene and UP063 gene were used as target sequences to design primers for PCR [22]. The genetic targets shared similarity with 10 UU and 4 UP serovars, respectively, with 100% identity.
All primers were synthesized by Shanghai Generay Biotech Co., Ltd (Shanghai, China), respectively (UU-F1 5’-ATGAAGAATCTGTGGGAGGTGG-3’, UU-R1 5’-AATCGTCGCATTCCCTATGCT-3’; UP-F2 5’-ATTTAGCTGGTCCTTTATTCAC-3’, UP-R2 5’-GAAGTGCTACAAAATAACCCAT-3’). PCR products were purified and sequenced on ABI 3730XL DNA Analyzers by BGI (Beijing Liuhe Huada Gene Technology Co., Ltd, Shanghai, China). The sequencing results were compared with reported sequences in the GenBank database by using BLAST (http://ncbi.nlm.nhi.gov/BLAST).
2.6 Statistical analysis
2.6.1 UU/UP combination analysis
Ureaplasma has a unique position of being a commensal infection. Given that, we compared the performance between the two detection assays according to the four possible infection statuses (i.e., UU-, UP-; UU+, UP-; UU-, UP+; UU+, UP+).
PPA, NPA, overall agreement with 95% confidence intervals (95%CIs), specificity (Sp), and sensitivity (Se) were calculated using SPSS 26.0 (SPSS, Chicago, USA). kappa values with 95%CIs were calculated using the Clopper-Pearson method [BETAINV(alpha/2, x, n-x+1) < theta < BETAINV(1-alpha/2, x+1, n-x)]. A kappa value of at least 0.75 indicated good agreement. However, larger kappa values, such as 0.90, were preferred [23].
2.6.2 Stratified analysis of clinical factors
After deducting the random effects, the kappa value was also used to assess the overall agreement. Firstly, the kappa values of some clinical factors were calculated. Secondly, factors with large variation in kappa value were selected as possible interfering factors in the Repodx PCR kit (Table 3). If available, a stratified analysis was performed to compare the variation in the agreement statistics (PPA, NPA, and overall agreement) using the interfering factor as a stratified factor.