Single-dose azithromycin is recommended for treating Chlamydia trachomatis infections. Here, we established an in vitro cell model of azithromycin-induced persistent infection. Azithromycin inhibited the replication of C. trachomatis in a dose-time-dependent manner. Electron microscopy indicated that small inclusions in the induced model contained enlarged, aberrant and non-infectious reticulate bodies. RT-PCR showed that C. trachomatis still has the ability to express the unprocessed 16S rRNA gene in the model and that C. trachomatis recovered after the removal of azithromycin with a peak recovery time of 24 h. The mutations in 23S rRNA, L4 and L22 genes were not found in persistent infection, and qRT-PCR analysis showed that the relative expression level of euo in azithromycin treated infection was upregulated while omcB was downregulated. In summary, this study provides a novel in vitro cell model to examine the characteristics of azithromycin-induced persistent infection and contribute to the development of treatments for C. trachomatis infection.

Background Gonorrhoea, caused by Neisseria gonorrhoeae, has spread worldwide. Strains resistant to most antibiotics, including ceftriaxone and azithromycin, have emerged to an alarming level. Rapid testing for N. gonorrhoeae and its antimicrobial resistance will therefore contribute to clinical decision making for early diagnosis and rational drug use. Methods A Cas13a-based assay (specific high-sensitivity enzymatic reporter unlocking; SHERLOCK) was developed for N. gonorrhoeae detection (porA gene) and azithromycin resistance identification (A2059G, C2611T). Assays were evaluated for sensitivity with purified dsDNA and specificity with 17 non-gonococcal strains. Performance of SHERLOCK (porA) was compared with Roche Cobas 4800 using 43 urine samples. Identification of azithromycin resistance mutations (A2059G, C2611T) was evaluated using a total of 84 clinical isolates and 18 urine samples. Lateral flow was tested for this assay as a readout tool. Moreover, we directly assayed 27 urethral swabs from patients with urethritis to evaluate their status in terms of N. gonorrhoeae infection and azithromycin resistance. Results The SHERLOCK assay was successfully developed with a sensitivity of 10 copies/reaction, except 100 copies/reaction for A2059G, and no cross-reaction with other species. Comparison of the SHERLOCK assay with the Cobas 4800 revealed 100% concordance within 18 positive and 25 negative urine samples. Of the 84 isolates, 21 strains with azithromycin resistance mutations were distinguished and further verified by sequencing and MIC determination. In addition, 62.96% (17/27) strains from swab samples were detected with no mutant strains confirmed by sequencing. Conclusions The SHERLOCK assay for rapid N. gonorrhoeae detection combined with azithromycin resistance testing is a promising method for application in clinical practice.

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Studies have rarely assessed the genotype distribution of Chlamydia trachomatis (CT) in urine among men attending sexually transmitted disease clinics (MSCs) in China. This study was aimed at investigating the prevalence and molecular epidemiology of CT infection by examining urine samples among MSCs from different geographic areas of Guangdong Province, China. A cross-sectional study was conducted among MSCs from 10 human immunodeficiency virus sentinel surveillance sites in Guangdong Province. CT DNA was extracted from male urine samples and analyzed using a Roche cobas 4800 CT/NG. The ompA genes were amplified by nested PCR and sequenced. The leukocyte esterase test was performed by routine urine analysis at local clinics. Of the 1,903 samples, 163 (8.6%, 95% confidence interval [CI] 3.8-16.3%) tested positive for CT. The highest prevalence (10.5%) of CT infection was observed among participants aged between 21 and 30 years. A total of 130 CT-positive samples (79.8%, 130/163) were successfully genotyped by nested PCR, resulting in 8 genotypes. The most prevalent genotypes were D, E, F, and J, with proportions of 20.8%, 20.0%, 17.7%, and 16.9%, respectively. There were no significant correlations between the geographical areas, leukocyte esterase test results and genotype distribution. Promotion of detection and molecular epidemiology research is needed for effective and comprehensive prevention and control programs.

Gonorrhea caused by Neisseria gonorrhoeae has spread world-wide. Antimicrobial-resistant strains have emerged to an alarming level to most antibiotics, including to the ceftriaxone-azithromycin combination, currently recommended as first-line dual therapy. Rapid testing for antimicrobial resistance will contribute to clinical decision-making for rational drug use and will slow this trend. Herein, we developed a Cas13a-based assay for N. gonorrhoeae detection (porA target) and azithromycin resistance identification (A2059G and C2611T point mutations). We evaluated the sensitivity and specificity of this method, and 10 copies per reaction can be achieved in porA detection and C2611T identification, with no cross-reactions. Comparison of the Cas13a-based assay (porA target) with Roche Cobas 4800 assay (n=23 urine samples) revealed 100% concordance. Isolated N. gonorrhoeae strains were used to validate the identification of A2059G and C2611T resistance mutations. All tested strains (8 A2059G strains, 8 C2611T strains, and 8 wild-type strains) were successfully distinguished by our assay and verified by testing MIC for azithromycin and sequencing the 23S rRNA gene. We adopted lateral flow for the SHERLOCK assay readout, which showed a visible difference between test group and NC group results. To further evaluate the capability of our assay, we tested 27 urethral swabs from patients with urethritis for N. gonorrhoeae detection and azithromycin-resistance identification. Of these, 62.96% (17/27) strains were detected with no mutant strains and confirmed by sequencing. In conclusion, the novel Cas13a-based assay for rapid and accurate N. gonorrhoeae detection combined with azithromycin drug resistance testing is a promising assay for application in clinical practice.