Background: Chlamydia trachomatis(CT) infection is one of the most pervasive sexually transmitted infections and has high prevalence in urogenital and extra-urogenital sites among men who have sex with men (MSM). This study investigated anatomical site-specific prevalence and genotypes of CT among MSM recruited from three geographic areas in China.Methods: We collected urine specimens and anorectal, pharyngeal swab specimens from 379 MSM. CT infection was identified using polymerase chain reaction and CT genotyping was determined by sequences of the ompA gene.Results: The results indicated that the overall prevalence of CT infection was 18.2% (95% confidence intervals [CIs], 13.9–22.5%) and significantly different between the cities (p = 0.048). The infection was most common at the anorectal site (15.6, 95%CIs 11.6–19.5%) followed by urethral (3.2, 95%CIs 1.4–5.0%) and oropharyngeal sites (1.6, 95%CIs 0.3–2.9%). Genotypes D and G were the most common CT strains in this population but genotype D was significantly predominated in Nanjing while genotype G was in Wuhan. No genotype related to lymphogranuloma venereum was found. CT infection was significantly related to the infection ofNeisseria gonorrhoeae(adjusted odds ratio [aOR] 14.27, 95%CIs 6.02–33.83,p < 0.001) and age. Men older than 40 years old were less likely to have a CT infection as compared to men under 30 years old (aOR 0.37, 95% CIs 0.15–0.93,p = 0.03).Conclusion: The high CT infection prevalence, particularly in the anorectal site, among MSM suggests the necessity to development an integrated CT screening and treatment program specifically focusing on this high-risk population. Surveillance of CT infections should be improved by including both infection and genotype based surveys into the current surveillance programs in China.

Purpose: Sexually transmitted infections (STIs), representing a major global health problem, are caused by different microbes, including bacteria, viruses, and protozoa. Unfortunately, infections of different sexually transmitted pathogens often present similar clinical symptoms, so it is almost impossible to distinguish them clinically. Therefore, the aim of the current study was to develop a sensitive, multitarget, and high-throughput method that can detect various agents responsible for STIs. Methods: We developed and tested a 23-plex PCR coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) assay (sexually transmitted infection-mass spectrometry, STI-MS) that simultaneously targets 11 different agents, including 8 most common clinical pathogens related to STIs (HSV-1, HSV-2, Neisseria gonorrhoeae, Chlamydia trachomatis, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, and Haemophilus ducreyi) and 3 controversial microorganisms as pathogens (Mycoplasma hominis, Ureaplasma urealyticum, and Ureaplasma parvum). Results: The results showed that the STI-MS approach can accurately detect the expected agents, without cross-reaction with other organisms. The limit of detection of each STI-MS assay was ranged from 1.739 to 10.009 copies/reaction, using probit analyses. The verification rate for each target organism of the STI-MS ranged from a minimum of 89.3% to a maximum of 100%, using conventional assays and ultrasensitive digital PCR to confirm the STI-MS-positive results. To further evaluate the clinical performance of this assay, 241 clinical specimens (124 urethral/cervical swabs and 117 urine) were tested in parallel using the STI-MS assay and monoplex real-time PCR for each agent. The overall validation parameters of STI-MS were extremely high including sensitivity (from 85.7% to 100%), specificity (from 92.3% to 100%), PPV (from 50% to 100%), and NPV (from 99.1% to 100%) for each target. Conclusion: STI-MS is a useful high-throughput screening tool for detecting mixed infections of STIs and has great potential for application in large-scale epidemiological programs for specific microorganisms of STI.

The growing antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a serious global threat to gonococcal therapy. Molecular typing is an ideal tool to reveal the association between specific genotype and resistance phenotype that provides effective data for tracking the transmission of resistant clones of N. gonorrhoeae. In our study, we aimed to describe the molecular epidemiology of AMR and the distribution of resistance-associated genotypes in Shenzhen during 2014–2018. In total, 909 isolates were collected from Shenzhen from 2014–2018. Two typing schemes, multilocus sequence typing (MLST) and N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR), were performed for all isolates. The distribution of resistance-associated genotypes was described using goeBURST analysis combined with data of logistic regression. Among 909 isolates, ST₈₁₂₃, ST₇₃₆₃, ST₁₉₀₁, ST₇₃₆₅, and ST₇₃₆₀ were most the common MLST sequence types (STs), and ST₃₄₈, ST₂₄₇₃, ST₄₉₇, and ST₁₉₉ were the most prevalent NG-STAR STs. The logistic regression analysis showed that NG-STAR_ST497, MLST_ST7365, and MLST_ST7360 were typically associated with decreased susceptibility to ceftriaxone. Furthermore, the internationally spreading ESC-resistant clone MLST_ST1901 has been prevalent at least in 2014 in Shenzhen and showed a significant increase during 2014–2018. Additionally, MLST_ST7363 owns the potential to become the next internationally spreading ceftriaxone-resistant ST. In conclusions, we performed a comprehensive epidemiological study to explore the correlation between AMR and specific STs, which provided important data for future studies of the molecular epidemiology of AMR in N. gonorrhoeae. Besides, these findings provide insight for adjusting surveillance strategies and therapy management in Shenzhen.

The antimicrobial resistance (AMR) in gonorrhea poses global threat of increasing public health concern. In response to this concern, molecular surveillance has been widely utilized to detail the changes in the evolution and distribution of Neisseria gonorrhoeae during AMR transmission. In this study, we performed a comprehensive molecular surveillance of 664 N . gonorrhoeae isolates collected in Shenzhen, one of the cities with the largest mobile population in China, 2019–2020. In 2020, ceftriaxone showed an unprecedented high resistance rate of 24.87%, and 67.83% of the ceftriaxone-resistant (Cro-R) isolates harbored a nonmosaic penA allele. The Cro-R isolates with nonmosaic penA alleles showed a tremendous increasing trend from 0.00% in 2014 to 20.45% in 2020, which proves the need for monitoring nonmosaic penA -related resistance. Importantly, genotyping indicated that multilocus sequence typing ST11231 (35.71%) had a notable rate of ceftriaxone resistance, which might become the focus of future surveillance. Whole-genome sequencing analysis showed that the internationally spreading FC428 clones have circulated in Shenzhen region with typical ceftriaxone resistance (MIC ≥ 0.5 mg/L) maintained. Our surveillance combined with genomic analysis provides current information to update gonorrhea management guidelines and emphasizes that continuous AMR surveillance for N. gonorrhoeae is essential. IMPORTANCE: We conducted a comprehensive molecular epidemiology analysis for antimicrobial-resistant Neisseria gonorrhoeae in Shenzhen during 2019–2020, which provided important data for personalized treatment and adjustment of monitoring strategy. Briefly, the proportion of ceftriaxone-resistant (Cro-R) isolates reached a stunning prevalence rate of 24.87% in 2020. A typical increment of Cro-R isolates with nonmosaic penA alleles proves the necessity of monitoring nonmosaic AMR mechanism and involving it into developing molecular detection methods. Whole-genome sequencing analysis showed that the international spreading FC428 clone has been circulating in Shenzhen with typical ceftriaxone resistance (MIC ≥ 0.5 mg/L) maintained. In summary, we conducted a comprehensive epidemiology study, providing significant data for therapy management. Our results not only improve the understanding of the distribution and transmission of AMR in N. gonorrhoeae but also provide effective AMR data for improving surveillance strategies in China.

Introduction: Antimicrobial resistance (AMR) of Neisseria gonorrhoeae (N. gonorrhoeae) becomes a grave public health problem in the world. A strengthened Antimicrobial Resistance Surveillance Program is needed to track the trend of AMR development. However, the lack of a proper antimicrobial susceptibility test (AST) method is a barrier to expand the AMR surveillance in China. Traditional agar dilution (AD) method is laborious and E-test strips have no approval license for clinical use. Herein, a Chinese group modified the microdilution (MD) method for clinical ASTs. The objective of this study is to compare the MD method with the AD method for N. gonorrhoeae AST. Materials and Methods: A total of 166 clinical isolates were tested for antimicrobial susceptibility of ceftriaxone, spectinomycin, azithromycin, ciprofloxacin, tetracycline, and penicillin using MD and AD method simultaneously. Results of MD method were read manually or automatically. Rates of essential agreement (EA), category agreement (CA), minor error, and very major error were compared. Results: The total EAs (compared with results read manually) of penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone, and azithromycin were 90.4%, 97.0%, 85.5%, 100.0%, 94%, and 72.3%; and CAs were 82.5%, 94.0%, 100%, 100%, 95.2%, and 94%, respectively. Conclusion: We conclude that the MD method might be an alternative for clinical AST of N. gonorrhoeae in China. In particular, MD method has the potency of accurate differentiation of isolates resistant to ceftriaxone or azithromycin, which were empirically recommended for gonococcal treatment, but its quality remained suboptimal, and further improvement is needed for clinical use.

Background: Complicated mechanisms and variable determinants related to drug resistance pose a major challenge to obtain comprehensive antimicrobial resistance (AMR) profiles of Neisseria gonorrhoeae. Meanwhile, cephalosporin-resistant mosaic penA alleles have been reported worldwide. Therefore, it is urgent to monitor the expansion of cephalosporin-resistant mosaic penA alleles. Objectives: To develop a comprehensive high-throughput method to efficiently screen AMR determinants. Methods: We developed a method based on multiplex PCR with MALDI-TOF MS, which can simultaneously screen for 24 mutations associated with multiple antimicrobial agents in 19 gonococcal AMR loci (NG-AMR-MS). The performance of the NG-AMR-MS method was assessed by testing 454 N. gonorrhoeae isolates with known MICs of six antibiotics, eight non-gonococcal Neisseria strains, 214 clinical samples and three plasmids with a confirmed mosaic penA allele. Results: The results show that NG-AMR-MS had a specificity of 100% with a sensitivity as low as 10 copies per reaction (except for PorB A121D/N/G, 100 copies per reaction). For clinical samples with gonococcal load >5 copies/μL, the method can accurately identify 20 AMR mutations. In addition, the method successfully detected specific cephalosporin-resistant strains with the A311V mutation in the penA allele. Conclusions: Our high-throughput method can provide comprehensive AMR profiles within a multiplex format. Furthermore, the method can be directly applied to screening for AMR among clinical samples, serving as an effective tool for overall monitoring of N. gonorrhoeae AMR and also provides a powerful means to comprehensively improve the level of monitoring.