Rhein inhibits Chlamydia trachomatis infection by regulating pathogen-host cell.

The global incidence of genital Chlamydia trachomatis infection increased rapidly as the primary available treatment of C. trachomatis infection being the use of antibiotics. However, the development of antibiotics resistant stain and other treatment failures are often observed in patients. Consequently, novel therapeutics are urgently required. Rhein is a monomer derivative of anthraquinone compounds with an anti-infection activity. This study investigated the effects of rhein on treating C. trachomatis infection. Rhein showed significant inhibitory effects on the growth of C. trachomatis in multiple serovars of C. trachomatis, including D, E, F and L1, and in various host cells, including HeLa, McCoy and Vero. Rhein could not directly inactivate C. trachomatis but could inhibit the growth of C. trachomatis by regulating pathogen-host cell interactions. Combined with azithromycin, the inhibitory effect of rehin was synergistic both in vitro and in vivo. Together these findings suggest that rhein could be developed for the treatment of C. trachomatis infections.

A suite of PCR-LwCas13a assays for detection and genotyping of Treponema pallidum in clinical samples.

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.

Development and application of Cas13a-based diagnostic assay for Neisseria gonorrhoeae detection and azithromycin resistance identification.

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.

Determining the in vitro susceptibility of Neisseria gonorrhoeae isolates from 8 cities in Guangdong Province through an improved microdilution method.

A microdilution method for the antibiotic susceptibility testing of Neisseria gonorrhoeae was established and improved, and the antibiotic resistance of N. gonorrhoeae samples isolated from 8 cities of Guangdong in 2016 was determined. The improved microdilution method was compared with the agar dilution method recommend by the World Health Organization (WHO) Western Pacific Region by testing the susceptibility of 100 clinical N. gonorrhoeae isolates. The essential agreement (EA), categorical agreement (CA), very major error (VME), major error (ME), and minor error (MIE) levels of the two methods were analyzed; the acceptable performance rates were measured as follows: ≥90% for EA or CA, ≤3% for VME or ME, and ≤7% for MIE. The EA, CA, VME, ME, and MIE of each method for 7 antibiotics, penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone, cefixime, and azithromycin, were 96%-100%, 94%-100%, 0%-3%, 0%-2%, and 0%-6%, respectively. The Wilcoxon signed-rank test results indicated 94%-100% agreement between the 2 methods after excluding off-scale values (P > 0.05). The susceptibility of 634 N. gonorrhoeae strains to the 7 antibiotics above were tested through the microdilution method. The resistant rates of the isolates against ciprofloxacin, tetracycline, penicillin, and azithromycin were 99.8%, 88.3%, 53.8%, and 11%, and the percentages of the isolates with decreased susceptibility to ceftriaxone (minimum inhibitory concentration [MIC] ≥0.125 µg/mL) and cefixime (MIC ≥0.25 µg/mL) were 2.1% and 12%, respectively, in Guangdong. Among 8 cities, Shenzhen had the highest rates of resistance against penicillin (77.8%) and decreased susceptibility against ceftriaxone (5.6%). Zhuhai had the highest rates of decreased susceptibility against cefixime (30.1%), and Jiangmen had the highest azithromycin-resistant isolates (16.8%). The findings from this study indicated that the improved microdilution method is an alternative for testing the antimicrobial susceptibility of N. gonorrhoeae. The resistance rates of N. gonorrhoeae against penicillin, tetracycline, and ciprofloxacin were high. While ceftriaxone, cefixime, and spectinomycin remained effective against N. gonorrhoeae, their effectiveness seemed to be decreasing over time. Azithromycin therapy requires timely susceptibility test results.

Prevalence of Chlamydia trachomatis Genotypes in Men Who Have Sex with Men and Men Who Have Sex with Women Using Multilocus VNTR Analysis-ompA Typing in Guangzhou, China.

BACKGROUND: Chlamydia trachomatis is one of the most prevalent bacterial sexually transmitted infection in China. Although C. trachomatis genotypes can be discriminated by outer membrane protein gene (ompA) sequencing, currently available methods have limited resolutions. This study used a high-resolution genotyping method, namely, multilocus variable number tandem-repeat analysis with ompA sequencing (MLVA)-ompA, to investigate the local epidemiology of C. trachomatis infections among men who have sex with men (MSM) and men who have sex with women (MSW) attending a sexually transmitted diseases (STD) clinic in Guangzhou, China. METHODS: Rectal specimens from MSM and urethral specimens from MSW were collected between January 2013 and July 2014 at the Guangdong Provincial Center STD clinic. The specimens were sent to the laboratory for analyses. All specimens that were tested positive for C. trachomatis by the commercial nucleic acid amplification tests were genotyped by MLVA-ompA. RESULTS: Fifty-one rectal specimens from MSM and 96 urethral specimens from MSW were identified with C. trachomatis. One hundred and forty-four of the 147 specimens were fully genotyped by MLVA-ompA. Rectal specimens from MSM were divided into four ompA genotypes and urethral specimens from MSW into nine genotypes. No mixed infections were found among all specimens. The most frequent genotypes were D, G, J, E and F. All specimens were further divided into 46 types after ompA genotyping was combined with MLVA. Genotypes D-8.7.1 and G-3.4a.3 were the most frequent among MSM, whereas genotypes D-3.4a.4, E-8.5.1, F-8.5.1, and J-3.4a.2 were the most frequent subtypes among MSW. The discriminatory index D was 0.90 for MLVA, 0.85 for ompA, and 0.95 for MLVA-ompA. CONCLUSIONS: The most prevalent MLVA-ompA genotypes were significantly different between MSM and MSW from Guangzhou, China. Moreover, MLVA-ompA represented a more favorable degree of discrimination than ompA and could be a reliable complement for ompA for the routine subtypes of C. trachomatis.

The prevalence and epidemiology of plasmid-mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates in Guangzhou, China, 2002-2012.

BACKGROUND: Gonococcal antimicrobial resistance is a global problem. Different resistance plasmids have emerged and spread among the isolates of Neisseria gonorrhoeae worldwide and in China. We conducted this study to monitor the plasmid-mediated penicillin and tetracycline resistance among N. gonorrhoeae isolates in Guangzhou from 2002 to 2012. METHODS: Consecutive isolates of N. gonorrhoeae were collected from outpatients with gonorrhea attending the STD clinic in Guangdong Provincial Centre for Skin Diseases and STIs Control and Prevention. Penicillinase-producing N. gonorrhoeae (PPNG) isolates were analyzed by the paper acidometric method. Plasmid-mediated resistance to tetracycline in N. gonorrhoeae (TRNG) isolates was screened by the agar plate dilution method. Plasmid types were determined for TRNG and PPNG isolates using polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) to penicillin and tetracycline were detected by the agar plate dilution. RESULTS: Of 1378 consecutive N. gonorrhoeae isolates, 429 PPNG and 639 TRNG isolates were identified. The prevalence of PPNG, TRNG, and PPNG/TRNG increased from 18.3 to 47.1 % (χ (2) = 31.57, p < 0.001), from 29.4 to 52.1 % (χ (2) = 16.28, p < 0.001) and from 10.0 to 26.2 % (χ (2) = 10.46, p < 0.001) between 2002 and 2012, respectively. Genotyping of plasmids among PPNGs showed that the majority (93.7 %) of the isolates were the Asian type plasmids, while the African type plasmid emerged in 2008 and rapidly increased to 14.0 % in 2012 (χ (2) = 25.03, p < 0.001). For TRNGs, all 639 isolates carried the Dutch type plasmid. MICs of penicillin G and tetracycline persisted at high levels and the MIC90s were 32-fold higher than the resistant cutoff point over 11 years. The prevalence rates of penicillin- and tetracycline-resistant N. gonorrhoeae varied from 90.9 to 91.1 % and from 88.3 to 89.3 % during 2002 to 2012, respectively. CONCLUSIONS: Resistance to penicillin and tetracycline among N. gonorrhoeae isolates remained at high levels in Guangzhou. The Asian type PPNG continued to spread and Dutch type TRNG was still the dominant strain. The African type PPNG has emerged and is spreading rapidly.

Antibiotic susceptibility of Neisseria gonorrhoeae isolates from Guangzhou, China, during 2002-2011.

Antibiotic susceptibility of Neisseria gonorrhoeae in Guangzhou during 2002-2011 showed that resistance to penicillin and ciprofloxacin was high, while ceftriaxone remained effective although there was a trend towards reduced sensitivity.