Whole-genome sequencing (WGS) of Treponema pallidum subsp. pallidum (TPA) has been constrained by the lack of in vitro cultivation methods for isolating spirochetes from patient samples. We built upon recently developed enrichment methods to sequence TPA directly from primary syphilis chancre swabs collected in Guangzhou, China. By combining parallel, pooled whole-genome amplification (ppWGA) with hybrid selection, we generated high quality genomes from four of eight chancre-swab samples and two of two rabbit-passaged isolates, all subjected to challenging storage conditions. This approach enabled the first WGS of Chinese samples without rabbit passage and provided insights into TPA genetic diversity in China.

The performance of commonly used assays for diagnosis of syphilis varies considerably depending on stage of infection and sample type. In response to the need for improved syphilis diagnostics, we develop assays that pair PCR pre-amplification of the tpp47 gene of Treponema pallidum subsp. pallidum with CRISPR-LwCas13a. The PCR-LwCas13a assay achieves an order of magnitude better analytical sensitivity than real-time PCR with equivalent specificity. When applied to a panel of 216 biological specimens, including 135 clinically confirmed primary and secondary syphilis samples, the PCR-LwCas13a assay demonstrates 93.3% clinical sensitivity and 100% specificity, outperforming tpp47 real-time PCR and rabbit-infectivity testing. We further adapt this approach to distinguish Treponema pallidum subsp. pallidum lineages and identify genetic markers of macrolide resistance. Our study demonstrates the potential of CRISPR-based approaches to improve diagnosis and epidemiological surveillance of syphilis.

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.

Background: Treponema pallidum (T. pallidum) infection evokes significant immune responses, resulting in tissue damage. The immune mechanism underlying T. pallidum infection is still unclear, although microRNAs (miRNAs) have been shown to influence immune cell function and, consequently, the generation of antibody responses during other microbe infections. However, these mechanisms are unknown for T. pallidum. Methods: In this study, we performed a comprehensive analysis of differentially expressed miRNAs in healthy individuals, untreated patients with syphilis, patients in the serofast state, and serologically cured patients. miRNAs were profiled from the peripheral blood of patients obtained at the time of serological diagnosis. Then, both the target sequence analysis of these different miRNAs and pathway analysis were performed to identify important immune and cell signaling pathways. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed for microRNA analysis. Results: A total of 74 differentially regulated miRNAs were identified. Following RT-qPCR confirmation, three miRNAs (hsa-miR-195-5p, hsa-miR-223-3p, hsa-miR-589-3p) showed significant differences in the serofast and serologically cured states (P < 0.05). One miRNA (hsa-miR-195-5p) showed significant differences between untreated patients and healthy individuals. Conclusions: This is the first study of miRNA expression differences in peripheral blood mononuclear cells (PBMCs) in different stages of T. pallium infection. Our study suggests that the combination of three miRNAs has great potential to serve as a non-invasive biomarker of T. pallium infections, which will facilitate better diagnosis and treatment of T. pallium infections.

Background: Chlamydia trachomatis detection plays a crucial role in early diagnosis and treatment of C. trachomatis infection. In the current study, the capability of sexually transmitted disease (STD) laboratories to detect C. trachomatis was investigated in Guangdong, China.Methods: An external quality assessment panel, including 5 positive samples with different C. trachomatis loads and 2 negative samples was distributed to 654 participating laboratories in October 2019, and the test results were analyzed by Guangdong Central STD Laboratory. The use of various C. trachomatis detection methods in Guangdong from 2015 to 2019 was also retrospectively investigated.Results: Of the 654 participating STD laboratories, 559 (85.47%) used immune chromatographic-rapid diagnostic tests (IC-RDTs) to detect C. trachomatis in 2019, and 95 (14.53%) used nucleic acid amplification tests (NAATs). The rate of NAATs use increased approximately 4-fold from 2015 to 2019. The sensitivity of IC-RDTs decreased markedly from 97.32% to 30.89% with decreasing C. trachomatis load, whereas that of NAATs was 97.62% to 100% in all positive samples. With respect to negative samples the specificity of IC-RDTs was 97.13% to 97.30% and that of NAATs was 98.95% to 100%. Laboratories using IC-RDTs were less likely to detect C. trachomatis than those using NAATs in samples with C. trachomatis loads of 20000 copies/mL or less (P < 0.0001). Further analysis indicated no significant difference (P > 0.05) in detection rate among the 4 IC-RDT assays commonly used by the participating laboratories.